• Don't forget to check out Neurotransmitter.net's research index for a list of dozens of other resources created by Shawn Thomas.
• The articles are retrieved from PubMed Central and PubMed on a daily basis.
• Free full text articles are also available for the following subjects: biology, molecular and cellular biology, neuroscience, genetics and proteomics, psychiatry, pharmacology, endocrinology, cancer, biotechnology, informatics, and miscellaneous.
  
• You can jump directly to articles from Microbiology and Molecular Biology Reviews, Applied and Environmental Microbiology, Applied Microbiology, Bacteriological Reviews, BMC Immunology, BMC Infectious Diseases, BMC Microbiology, Clinical and Diagnostic Laboratory Immunology, Clinical and Molecular Allergy, Clinical Microbiology Reviews, Infection and Immunity, Journal of Bacteriology, Journal of Clinical Microbiology, Journal of Immune Based Therapies and Vaccines, Journal of Virology, Medical Immunology, Microbiology and Immunology, International Microbiology, Retrovirology, Virology Journal, Microbial Cell Factories, Antimicrobial Agents and Chemotherapy, and Malaria Journal.
• You can view earlier articles from these journals as well: skip 30, skip 60, skip 90, or skip 120 of the most recent articles.
• Confused by the terminology? Try searching The On-Line Medical Dictionary (OMD).

Web www.neurotransmitter.net

---

Recent Articles in Microbiology and Molecular Biology Reviews

---

Bogoyevitch MA, Kobe B
Uses for JNK: the many and varied substrates of the c-Jun N-terminal kinases.
Microbiol Mol Biol Rev. 2006 Dec;70(4):1061-95.
The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK. There are now more than 50 proteins shown to be substrates for JNK. These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K, and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself. [Abstract/Link to Full Text]

García MA, Gil J, Ventoso I, Guerra S, Domingo E, Rivas C, Esteban M
Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action.
Microbiol Mol Biol Rev. 2006 Dec;70(4):1032-60.
The double-stranded RNA-dependent protein kinase PKR is a critical mediator of the antiproliferative and antiviral effects exerted by interferons. Not only is PKR an effector molecule on the cellular response to double-stranded RNA, but it also integrates signals in response to Toll-like receptor activation, growth factors, and diverse cellular stresses. In this review, we provide a detailed picture on how signaling downstream of PKR unfolds and what are the ultimate consequences for the cell fate. PKR activation affects both transcription and translation. PKR phosphorylation of the alpha subunit of eukaryotic initiation factor 2 results in a blockade on translation initiation. However, PKR cannot avoid the translation of some cellular and viral mRNAs bearing special features in their 5' untranslated regions. In addition, PKR affects diverse transcriptional factors such as interferon regulatory factor 1, STATs, p53, activating transcription factor 3, and NF-kappaB. In particular, how PKR triggers a cascade of events involving IKK phosphorylation of IkappaB and NF-kappaB nuclear translocation has been intensively studied. At the cellular and organism levels PKR exerts antiproliferative effects, and it is a key antiviral agent. A point of convergence in both effects is that PKR activation results in apoptosis induction. The extent and strength of the antiviral action of PKR are clearly understood by the findings that unrelated viral proteins of animal viruses have evolved to inhibit PKR action by using diverse strategies. The case for the pathological consequences of the antiproliferative action of PKR is less understood, but therapeutic strategies aimed at targeting PKR are beginning to offer promising results. [Abstract/Link to Full Text]

Deutscher J, Francke C, Postma PW
How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria.
Microbiol Mol Biol Rev. 2006 Dec;70(4):939-1031.
The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens. [Abstract/Link to Full Text]

Mascher T, Helmann JD, Unden G
Stimulus perception in bacterial signal-transducing histidine kinases.
Microbiol Mol Biol Rev. 2006 Dec;70(4):910-38.
Two-component signal-transducing systems are ubiquitously distributed communication interfaces in bacteria. They consist of a histidine kinase that senses a specific environmental stimulus and a cognate response regulator that mediates the cellular response, mostly through differential expression of target genes. Histidine kinases are typically transmembrane proteins harboring at least two domains: an input (or sensor) domain and a cytoplasmic transmitter (or kinase) domain. They can be identified and classified by virtue of their conserved cytoplasmic kinase domains. In contrast, the sensor domains are highly variable, reflecting the plethora of different signals and modes of sensing. In order to gain insight into the mechanisms of stimulus perception by bacterial histidine kinases, we here survey sensor domain architecture and topology within the bacterial membrane, functional aspects related to this topology, and sequence and phylogenetic conservation. Based on these criteria, three groups of histidine kinases can be differentiated. (i) Periplasmic-sensing histidine kinases detect their stimuli (often small solutes) through an extracellular input domain. (ii) Histidine kinases with sensing mechanisms linked to the transmembrane regions detect stimuli (usually membrane-associated stimuli, such as ionic strength, osmolarity, turgor, or functional state of the cell envelope) via their membrane-spanning segments and sometimes via additional short extracellular loops. (iii) Cytoplasmic-sensing histidine kinases (either membrane anchored or soluble) detect cellular or diffusible signals reporting the metabolic or developmental state of the cell. This review provides an overview of mechanisms of stimulus perception for members of all three groups of bacterial signal-transducing histidine kinases. [Abstract/Link to Full Text]

Carballido-López R
The bacterial actin-like cytoskeleton.
Microbiol Mol Biol Rev. 2006 Dec;70(4):888-909.
Recent advances have shown conclusively that bacterial cells possess distant but true homologues of actin (MreB, ParM, and the recently uncovered MamK protein). Despite weak amino acid sequence similarity, MreB and ParM exhibit high structural homology to actin. Just like F-actin in eukaryotes, MreB and ParM assemble into highly dynamic filamentous structures in vivo and in vitro. MreB-like proteins are essential for cell viability and have been implicated in major cellular processes, including cell morphogenesis, chromosome segregation, and cell polarity. ParM (a plasmid-encoded actin homologue) is responsible for driving plasmid-DNA partitioning. The dynamic prokaryotic actin-like cytoskeleton is thought to serve as a central organizer for the targeting and accurate positioning of proteins and nucleoprotein complexes, thereby (and by analogy to the eukaryotic cytoskeleton) spatially and temporally controlling macromolecular trafficking in bacterial cells. In this paper, the general properties and known functions of the actin orthologues in bacteria are reviewed. [Abstract/Link to Full Text]

Barry ER, Bell SD
DNA replication in the archaea.
Microbiol Mol Biol Rev. 2006 Dec;70(4):876-87.
The archaeal DNA replication machinery bears striking similarity to that of eukaryotes and is clearly distinct from the bacterial apparatus. In recent years, considerable advances have been made in understanding the biochemistry of the archaeal replication proteins. Furthermore, a number of structures have now been obtained for individual components and higher-order assemblies of archaeal replication factors, yielding important insights into the mechanisms of DNA replication in both archaea and eukaryotes. [Abstract/Link to Full Text]

González JE, Keshavan ND
Messing with bacterial quorum sensing.
Microbiol Mol Biol Rev. 2006 Dec;70(4):859-75.
Quorum sensing is widely recognized as an efficient mechanism to regulate expression of specific genes responsible for communal behavior in bacteria. Several bacterial phenotypes essential for the successful establishment of symbiotic, pathogenic, or commensal relationships with eukaryotic hosts, including motility, exopolysaccharide production, biofilm formation, and toxin production, are often regulated by quorum sensing. Interestingly, eukaryotes produce quorum-sensing-interfering (QSI) compounds that have a positive or negative influence on the bacterial signaling network. This eukaryotic interference could result in further fine-tuning of bacterial quorum sensing. Furthermore, recent work involving the synthesis of structural homologs to the various quorum-sensing signal molecules has resulted in the development of additional QSI compounds that could be used to control pathogenic bacteria. The creation of transgenic plants that express bacterial quorum-sensing genes is yet another strategy to interfere with bacterial behavior. Further investigation on the manipulation of quorum-sensing systems could provide us with powerful tools against harmful bacteria. [Abstract/Link to Full Text]

Casadesús J, Low D
Epigenetic gene regulation in the bacterial world.
Microbiol Mol Biol Rev. 2006 Sep;70(3):830-56.
Like many eukaryotes, bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Unlike eukaryotes, however, bacteria use DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation plays roles in the virulence of diverse pathogens of humans and livestock animals, including pathogenic Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine at GANTC sites by the CcrM methylase regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation at GATC sites by the Dam methylase provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage genomes, transposase activity, and regulation of gene expression. Transcriptional repression by Dam methylation appears to be more common than transcriptional activation. Certain promoters are active only during the hemimethylation interval that follows DNA replication; repression is restored when the newly synthesized DNA strand is methylated. In the E. coli genome, however, methylation of specific GATC sites can be blocked by cognate DNA binding proteins. Blockage of GATC methylation beyond cell division permits transmission of DNA methylation patterns to daughter cells and can give rise to distinct epigenetic states, each propagated by a positive feedback loop. Switching between alternative DNA methylation patterns can split clonal bacterial populations into epigenetic lineages in a manner reminiscent of eukaryotic cell differentiation. Inheritance of self-propagating DNA methylation patterns governs phase variation in the E. coli pap operon, the agn43 gene, and other loci encoding virulence-related cell surface functions. [Abstract/Link to Full Text]

Hassa PO, Haenni SS, Elser M, Hottiger MO
Nuclear ADP-ribosylation reactions in mammalian cells: where are we today and where are we going?
Microbiol Mol Biol Rev. 2006 Sep;70(3):789-829.
Since poly-ADP ribose was discovered over 40 years ago, there has been significant progress in research into the biology of mono- and poly-ADP-ribosylation reactions. During the last decade, it became clear that ADP-ribosylation reactions play important roles in a wide range of physiological and pathophysiological processes, including inter- and intracellular signaling, transcriptional regulation, DNA repair pathways and maintenance of genomic stability, telomere dynamics, cell differentiation and proliferation, and necrosis and apoptosis. ADP-ribosylation reactions are phylogenetically ancient and can be classified into four major groups: mono-ADP-ribosylation, poly-ADP-ribosylation, ADP-ribose cyclization, and formation of O-acetyl-ADP-ribose. In the human genome, more than 30 different genes coding for enzymes associated with distinct ADP-ribosylation activities have been identified. This review highlights the recent advances in the rapidly growing field of nuclear mono-ADP-ribosylation and poly-ADP-ribosylation reactions and the distinct ADP-ribosylating enzyme families involved in these processes, including the proposed family of novel poly-ADP-ribose polymerase-like mono-ADP-ribose transferases and the potential mono-ADP-ribosylation activities of the sirtuin family of NAD(+)-dependent histone deacetylases. A special focus is placed on the known roles of distinct mono- and poly-ADP-ribosylation reactions in physiological processes, such as mitosis, cellular differentiation and proliferation, telomere dynamics, and aging, as well as "programmed necrosis" (i.e., high-mobility-group protein B1 release) and apoptosis (i.e., apoptosis-inducing factor shuttling). The proposed molecular mechanisms involved in these processes, such as signaling, chromatin modification (i.e., "histone code"), and remodeling of chromatin structure (i.e., DNA damage response, transcriptional regulation, and insulator function), are described. A potential cross talk between nuclear ADP-ribosylation processes and other NAD(+)-dependent pathways is discussed. [Abstract/Link to Full Text]

Sibbald MJ, Ziebandt AK, Engelmann S, Hecker M, de Jong A, Harmsen HJ, Raangs GC, Stokroos I, Arends JP, Dubois JY, van Dijl JM
Mapping the pathways to staphylococcal pathogenesis by comparative secretomics.
Microbiol Mol Biol Rev. 2006 Sep;70(3):755-88.
The gram-positive bacterium Staphylococcus aureus is a frequent component of the human microbial flora that can turn into a dangerous pathogen. As such, this organism is capable of infecting almost every tissue and organ system in the human body. It does so by actively exporting a variety of virulence factors to the cell surface and extracellular milieu. Upon reaching their respective destinations, these virulence factors have pivotal roles in the colonization and subversion of the human host. It is therefore of major importance to obtain a clear understanding of the protein transport pathways that are active in S. aureus. The present review aims to provide a state-of-the-art roadmap of staphylococcal secretomes, which include both protein transport pathways and the extracytoplasmic proteins of these organisms. Specifically, an overview is presented of the exported virulence factors, pathways for protein transport, signals for cellular protein retention or secretion, and the exoproteomes of different S. aureus isolates. The focus is on S. aureus, but comparisons with Staphylococcus epidermidis and other gram-positive bacteria, such as Bacillus subtilis, are included where appropriate. Importantly, the results of genomic and proteomic studies on S. aureus secretomes are integrated through a comparative "secretomics" approach, resulting in the first definition of the core and variant secretomes of this bacterium. While the core secretome seems to be largely employed for general housekeeping functions which are necessary to thrive in particular niches provided by the human host, the variant secretome seems to contain the "gadgets" that S. aureus needs to conquer these well-protected niches. [Abstract/Link to Full Text]

Shih YL, Rothfield L
The bacterial cytoskeleton.
Microbiol Mol Biol Rev. 2006 Sep;70(3):729-54.
In recent years it has been shown that bacteria contain a number of cytoskeletal structures. The bacterial cytoplasmic elements include homologs of the three major types of eukaryotic cytoskeletal proteins (actin, tubulin, and intermediate filament proteins) and a fourth group, the MinD-ParA group, that appears to be unique to bacteria. The cytoskeletal structures play important roles in cell division, cell polarity, cell shape regulation, plasmid partition, and other functions. The proteins self-assemble into filamentous structures in vitro and form intracellular ordered structures in vivo. In addition, there are a number of filamentous bacterial elements that may turn out to be cytoskeletal in nature. This review attempts to summarize and integrate the in vivo and in vitro aspects of these systems and to evaluate the probable future directions of this active research field. [Abstract/Link to Full Text]

Petkovi? H, Cullum J, Hranueli D, Hunter IS, Peri?-Concha N, Pigac J, Thamchaipenet A, Vujaklija D, Long PF
Genetics of Streptomyces rimosus, the oxytetracycline producer.
Microbiol Mol Biol Rev. 2006 Sep;70(3):704-28.
From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term. [Abstract/Link to Full Text]

Young KD
The selective value of bacterial shape.
Microbiol Mol Biol Rev. 2006 Sep;70(3):660-703.
Why do bacteria have shape? Is morphology valuable or just a trivial secondary characteristic? Why should bacteria have one shape instead of another? Three broad considerations suggest that bacterial shapes are not accidental but are biologically important: cells adopt uniform morphologies from among a wide variety of possibilities, some cells modify their shape as conditions demand, and morphology can be tracked through evolutionary lineages. All of these imply that shape is a selectable feature that aids survival. The aim of this review is to spell out the physical, environmental, and biological forces that favor different bacterial morphologies and which, therefore, contribute to natural selection. Specifically, cell shape is driven by eight general considerations: nutrient access, cell division and segregation, attachment to surfaces, passive dispersal, active motility, polar differentiation, the need to escape predators, and the advantages of cellular differentiation. Bacteria respond to these forces by performing a type of calculus, integrating over a number of environmental and behavioral factors to produce a size and shape that are optimal for the circumstances in which they live. Just as we are beginning to answer how bacteria create their shapes, it seems reasonable and essential that we expand our efforts to understand why they do so. [Abstract/Link to Full Text]

Zhou Q, Yik JH
The Yin and Yang of P-TEFb regulation: implications for human immunodeficiency virus gene expression and global control of cell growth and differentiation.
Microbiol Mol Biol Rev. 2006 Sep;70(3):646-59.
The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and antagonizing the effects of negative elongation factors. Not only is P-TEFb essential for transcription of the vast majority of cellular genes, but it is also a critical host cellular cofactor for the expression of the human immunodeficiency virus (HIV) type 1 genome. Given its important role in globally affecting transcription, P-TEFb's activity is dynamically controlled by both positive and negative regulators in order to achieve a functional equilibrium in sync with the overall transcriptional demand as well as the proliferative state of cells. Notably, this equilibrium can be shifted toward either the active or inactive state in response to diverse physiological stimuli that can ultimately affect the cellular decision between growth and differentiation. In this review, we examine the mechanisms by which the recently identified positive (the bromodomain protein Brd4) and negative (the noncoding 7SK small nuclear RNA and the HEXIM1 protein) regulators of P-TEFb affect the P-TEFb-dependent transcriptional elongation. We also discuss the consequences of perturbations of the dynamic associations of these regulators with P-TEFb in relation to the pathogenesis and progression of several major human diseases, such as cardiac hypertrophy, breast cancer, and HIV infection. [Abstract/Link to Full Text]

Moseley JB, Goode BL
The yeast actin cytoskeleton: from cellular function to biochemical mechanism.
Microbiol Mol Biol Rev. 2006 Sep;70(3):605-45.
All cells undergo rapid remodeling of their actin networks to regulate such critical processes as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. These events are driven by the coordinated activities of a set of 20 to 30 highly conserved actin-associated proteins, in addition to many cell-specific actin-associated proteins and numerous upstream signaling molecules. The combined activities of these factors control with exquisite precision the spatial and temporal assembly of actin structures and ensure dynamic turnover of actin structures such that cells can rapidly alter their cytoskeletons in response to internal and external cues. One of the most exciting principles to emerge from the last decade of research on actin is that the assembly of architecturally diverse actin structures is governed by highly conserved machinery and mechanisms. With this realization, it has become apparent that pioneering efforts in budding yeast have contributed substantially to defining the universal mechanisms regulating actin dynamics in eukaryotes. In this review, we first describe the filamentous actin structures found in Saccharomyces cerevisiae (patches, cables, and rings) and their physiological functions, and then we discuss in detail the specific roles of actin-associated proteins and their biochemical mechanisms of action. [Abstract/Link to Full Text]

MacPherson S, Larochelle M, Turcotte B
A fungal family of transcriptional regulators: the zinc cluster proteins.
Microbiol Mol Biol Rev. 2006 Sep;70(3):583-604.
The trace element zinc is required for proper functioning of a large number of proteins, including various enzymes. However, most zinc-containing proteins are transcription factors capable of binding DNA and are named zinc finger proteins. They form one of the largest families of transcriptional regulators and are categorized into various classes according to zinc-binding motifs. This review focuses on one class of zinc finger proteins called zinc cluster (or binuclear) proteins. Members of this family are exclusively fungal and possess the well-conserved motif CysX(2)CysX(6)CysX(5-12)CysX(2)CysX(6-8)Cys. The cysteine residues bind to two zinc atoms, which coordinate folding of the domain involved in DNA recognition. The first- and best-studied zinc cluster protein is Gal4p, a transcriptional activator of genes involved in the catabolism of galactose in the budding yeast Saccharomyces cerevisiae. Since the discovery of Gal4p, many other zinc cluster proteins have been characterized; they function in a wide range of processes, including primary and secondary metabolism and meiosis. Other roles include regulation of genes involved in the stress response as well as pleiotropic drug resistance, as demonstrated in budding yeast and in human fungal pathogens. With the number of characterized zinc cluster proteins growing rapidly, it is becoming more and more apparent that they are important regulators of fungal physiology. [Abstract/Link to Full Text]

Drider D, Fimland G, Héchard Y, McMullen LM, Prévost H
The continuing story of class IIa bacteriocins.
Microbiol Mol Biol Rev. 2006 Jun;70(2):564-82.
Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against. [Abstract/Link to Full Text]

Roeth JF, Collins KL
Human immunodeficiency virus type 1 Nef: adapting to intracellular trafficking pathways.
Microbiol Mol Biol Rev. 2006 Jun;70(2):548-63.
The Nef protein of primate lentiviruses is a unique protein that has evolved in several ways to manipulate the biology of an infected cell to support viral replication, immune evasion, pathogenesis, and viral spread. Nef is a small (25- to 34-kDa), myristoylated protein that binds to a collection of cellular factors and acts as an adaptor to generate novel protein interactions to accomplish specific functions. Of the many biological activities attributed to Nef, the reduction of surface levels of the viral receptor (CD4) and antigen-presenting molecules (major histocompatibility complex class I) has been intensely examined; recent evidence demonstrates that Nef utilizes multiple, distinct pathways to affect these proteins. To accomplish this, Nef promotes the formation of multiprotein complexes, recruiting host adaptor proteins to commandeer intracellular vesicular trafficking routes. The altered trafficking of several other host molecules has also been reported, and an emerging theory suggests that Nef generates pleiotrophic effects in the secretory and endocytic pathways that reprogram intracellular protein trafficking and may ultimately provide an efficient platform for viral assembly. This review critically discusses some of the major findings regarding the impact of human immunodeficiency virus type 1 Nef on host protein transport and addresses some emerging directions in this area of human immunodeficiency virus biology. [Abstract/Link to Full Text]

Lalucat J, Bennasar A, Bosch R, García-Valdés E, Palleroni NJ
Biology of Pseudomonas stutzeri.
Microbiol Mol Biol Rev. 2006 Jun;70(2):510-47.
Pseudomonas stutzeri is a nonfluorescent denitrifying bacterium widely distributed in the environment, and it has also been isolated as an opportunistic pathogen from humans. Over the past 15 years, much progress has been made in elucidating the taxonomy of this diverse taxonomical group, demonstrating the clonality of its populations. The species has received much attention because of its particular metabolic properties: it has been proposed as a model organism for denitrification studies; many strains have natural transformation properties, making it relevant for study of the transfer of genes in the environment; several strains are able to fix dinitrogen; and others participate in the degradation of pollutants or interact with toxic metals. This review considers the history of the discovery, nomenclatural changes, and early studies, together with the relevant biological and ecological properties, of P. stutzeri. [Abstract/Link to Full Text]

Ashby MK, Houmard J
Cyanobacterial two-component proteins: structure, diversity, distribution, and evolution.
Microbiol Mol Biol Rev. 2006 Jun;70(2):472-509.
A survey of the already characterized and potential two-component protein sequences that exist in the nine complete and seven partially annotated cyanobacterial genome sequences available (as of May 2005) showed that the cyanobacteria possess a much larger repertoire of such proteins than most other bacteria. By analysis of the domain structure of the 1,171 potential histidine kinases, response regulators, and hybrid kinases, many various arrangements of about thirty different modules could be distinguished. The number of two-component proteins is related in part to genome size but also to the variety of physiological properties and ecophysiologies of the different strains. Groups of orthologues were defined, only a few of which have representatives with known physiological functions. Based on comparisons with the proposed phylogenetic relationships between the strains, the orthology groups show that (i) a few genes, some of them clustered on the genome, have been conserved by all species, suggesting their very ancient origin and an essential role for the corresponding proteins, and (ii) duplications, fusions, gene losses, insertions, and deletions, as well as domain shuffling, occurred during evolution, leading to the extant repertoire. These mechanisms are put in perspective with the different genetic properties that cyanobacteria have to achieve genome plasticity. This review is designed to serve as a basis for orienting further research aimed at defining the most ancient regulatory mechanisms and understanding how evolution worked to select and keep the most appropriate systems for cyanobacteria to develop in the quite different environments that they have successfully colonized. [Abstract/Link to Full Text]

Fletcher J, Bender C, Budowle B, Cobb WT, Gold SE, Ishimaru CA, Luster D, Melcher U, Murch R, Scherm H, Seem RC, Sherwood JL, Sobral BW, Tolin SA
Plant pathogen forensics: capabilities, needs, and recommendations.
Microbiol Mol Biol Rev. 2006 Jun;70(2):450-71.
A biological attack on U.S. crops, rangelands, or forests could reduce yield and quality, erode consumer confidence, affect economic health and the environment, and possibly impact human nutrition and international relations. Preparedness for a crop bioterror event requires a strong national security plan that includes steps for microbial forensics and criminal attribution. However, U.S. crop producers, consultants, and agricultural scientists have traditionally focused primarily on strategies for prevention and management of diseases introduced naturally or unintentionally rather than on responding appropriately to an intentional pathogen introduction. We assess currently available information, technologies, and resources that were developed originally to ensure plant health but also could be utilized for postintroduction plant pathogen forensics. Recommendations for prioritization of efforts and resource expenditures needed to enhance our plant pathogen forensics capabilities are presented. [Abstract/Link to Full Text]

Jiang Y
Regulation of the cell cycle by protein phosphatase 2A in Saccharomyces cerevisiae.
Microbiol Mol Biol Rev. 2006 Jun;70(2):440-9.
Protein phosphatase 2A (PP2A) has long been implicated in cell cycle regulation in many different organisms. In the yeast Saccharomyces cerevisiae, PP2A controls cell cycle progression mainly through modulation of cyclin-dependent kinase (CDK) at the G(2)/M transition. However, CDK does not appear to be a direct target of PP2A. PP2A affects CDK activity through its roles in checkpoint controls. Inactivation of PP2A downregulates CDK by activating the morphogenesis checkpoint and, consequently, delays mitotic entry. Defects in PP2A also compromise the spindle checkpoint and predispose the cell to an error-prone mitotic exit. In addition, PP2A is involved in controlling the G(1)/S transition and cytokinesis. These findings suggest that PP2A functions in many stages of the cell cycle and its effect on cell cycle progression is pleiotropic. [Abstract/Link to Full Text]

Han MJ, Lee SY
The Escherichia coli proteome: past, present, and future prospects.
Microbiol Mol Biol Rev. 2006 Jun;70(2):362-439.
Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects. [Abstract/Link to Full Text]

Seib KL, Wu HJ, Kidd SP, Apicella MA, Jennings MP, McEwan AG
Defenses against oxidative stress in Neisseria gonorrhoeae: a system tailored for a challenging environment.
Microbiol Mol Biol Rev. 2006 Jun;70(2):344-61.
Neisseria gonorrhoeae is a host-adapted pathogen that colonizes primarily the human genitourinary tract. This bacterium encounters reactive oxygen and reactive nitrogen species as a consequence of localized inflammatory responses in the urethra of males and endocervix of females and also of the activity of commensal lactobacilli in the vaginal flora. This review describes recent advances in the understanding of defense systems against oxidative stress in N. gonorrhoeae and shows that while some of its defenses have similarities to the paradigm established with Escherichia coli, there are also some key differences. These differences include the presence of a defense system against superoxide based on manganese ions and a glutathione-dependent system for defense against nitric oxide which is under the control of a novel MerR-like transcriptional regulator. An understanding of the defenses against oxidative stress in N. gonorrhoeae and their regulation may provide new insights into the ways in which this bacterium survives challenges from polymorphonuclear leukocytes and urogenital epithelial cells. [Abstract/Link to Full Text]

Lesage G, Bussey H
Cell wall assembly in Saccharomyces cerevisiae.
Microbiol Mol Biol Rev. 2006 Jun;70(2):317-43.
An extracellular matrix composed of a layered meshwork of beta-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and its remodeling in S. cerevisiae. We then review the regulatory dynamics of cell wall assembly, an area where functional genomics offers new insights into the integration of cell wall growth and morphogenesis with a polarized secretory system that is under cell cycle and cell type program controls. [Abstract/Link to Full Text]

Toleman MA, Bennett PM, Walsh TR
ISCR elements: novel gene-capturing systems of the 21st century?
Microbiol Mol Biol Rev. 2006 Jun;70(2):296-316.
"Common regions" (CRs), such as Orf513, are being increasingly linked to mega-antibiotic-resistant regions. While their overall nucleotide sequences show little identity to other mobile elements, amino acid alignments indicate that they possess the key motifs of IS91-like elements, which have been linked to the mobility ent plasmids in pathogenic Escherichia coli. Further inspection reveals that they possess an IS91-like origin of replication and termination sites (terIS), and therefore CRs probably transpose via a rolling-circle replication mechanism. Accordingly, in this review we have renamed CRs as ISCRs to give a more accurate reflection of their functional properties. The genetic context surrounding ISCRs indicates that they can procure 5' sequences via misreading of the cognate terIS, i.e., "unchecked transposition." Clinically, the most worrying aspect of ISCRs is that they are increasingly being linked with more potent examples of resistance, i.e., metallo-beta-lactamases in Pseudomonas aeruginosa and co-trimoxazole resistance in Stenotrophomonas maltophilia. Furthermore, if ISCR elements do move via "unchecked RC transposition," as has been speculated for ISCR1, then this mechanism provides antibiotic resistance genes with a highly mobile genetic vehicle that could greatly exceed the effects of previously reported mobile genetic mechanisms. It has been hypothesized that bacteria will surprise us by extending their "genetic construction kit" to procure and evince additional DNA and, therefore, antibiotic resistance genes. It appears that ISCR elements have now firmly established themselves within that regimen. [Abstract/Link to Full Text]

Shoseyov O, Shani Z, Levy I
Carbohydrate binding modules: biochemical properties and novel applications.
Microbiol Mol Biol Rev. 2006 Jun;70(2):283-95.
Polysaccharide-degrading microorganisms express a repertoire of hydrolytic enzymes that act in synergy on plant cell wall and other natural polysaccharides to elicit the degradation of often-recalcitrant substrates. These enzymes, particularly those that hydrolyze cellulose and hemicellulose, have a complex molecular architecture comprising discrete modules which are normally joined by relatively unstructured linker sequences. This structure is typically comprised of a catalytic module and one or more carbohydrate binding modules (CBMs) that bind to the polysaccharide. CBMs, by bringing the biocatalyst into intimate and prolonged association with its substrate, allow and promote catalysis. Based on their properties, CBMs are grouped into 43 families that display substantial variation in substrate specificity, along with other properties that make them a gold mine for biotechnologists who seek natural molecular "Velcro" for diverse and unusual applications. In this article, we review recent progress in the field of CBMs and provide an up-to-date summary of the latest developments in CBM applications. [Abstract/Link to Full Text]

Santangelo GM
Glucose signaling in Saccharomyces cerevisiae.
Microbiol Mol Biol Rev. 2006 Mar;70(1):253-82.
Eukaryotic cells possess an exquisitely interwoven and fine-tuned series of signal transduction mechanisms with which to sense and respond to the ubiquitous fermentable carbon source glucose. The budding yeast Saccharomyces cerevisiae has proven to be a fertile model system with which to identify glucose signaling factors, determine the relevant functional and physical interrelationships, and characterize the corresponding metabolic, transcriptomic, and proteomic readouts. The early events in glucose signaling appear to require both extracellular sensing by transmembrane proteins and intracellular sensing by G proteins. Intermediate steps involve cAMP-dependent stimulation of protein kinase A (PKA) as well as one or more redundant PKA-independent pathways. The final steps are mediated by a relatively small collection of transcriptional regulators that collaborate closely to maximize the cellular rates of energy generation and growth. Understanding the nuclear events in this process may necessitate the further elaboration of a new model for eukaryotic gene regulation, called "reverse recruitment." An essential feature of this idea is that fine-structure mapping of nuclear architecture will be required to understand the reception of regulatory signals that emanate from the plasma membrane and cytoplasm. Completion of this task should result in a much improved understanding of eukaryotic growth, differentiation, and carcinogenesis. [Abstract/Link to Full Text]

Morgan-Kiss RM, Priscu JC, Pocock T, Gudynaite-Savitch L, Huner NP
Adaptation and acclimation of photosynthetic microorganisms to permanently cold environments.
Microbiol Mol Biol Rev. 2006 Mar;70(1):222-52.
Persistently cold environments constitute one of our world's largest ecosystems, and microorganisms dominate the biomass and metabolic activity in these extreme environments. The stress of low temperatures on life is exacerbated in organisms that rely on photoautrophic production of organic carbon and energy sources. Phototrophic organisms must coordinate temperature-independent reactions of light absorption and photochemistry with temperature-dependent processes of electron transport and utilization of energy sources through growth and metabolism. Despite this conundrum, phototrophic microorganisms thrive in all cold ecosystems described and (together with chemoautrophs) provide the base of autotrophic production in low-temperature food webs. Psychrophilic (organisms with a requirement for low growth temperatures) and psychrotolerant (organisms tolerant of low growth temperatures) photoautotrophs rely on low-temperature acclimative and adaptive strategies that have been described for other low-temperature-adapted heterotrophic organisms, such as cold-active proteins and maintenance of membrane fluidity. In addition, photoautrophic organisms possess other strategies to balance the absorption of light and the transduction of light energy to stored chemical energy products (NADPH and ATP) with downstream consumption of photosynthetically derived energy products at low temperatures. Lastly, differential adaptive and acclimative mechanisms exist in phototrophic microorganisms residing in low-temperature environments that are exposed to constant low-light environments versus high-light- and high-UV-exposed phototrophic assemblages. [Abstract/Link to Full Text]

Marraffini LA, Dedent AC, Schneewind O
Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria.
Microbiol Mol Biol Rev. 2006 Mar;70(1):192-221.
The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis. [Abstract/Link to Full Text]

---

Recent Articles in Applied and Environmental Microbiology

---

Mereish KA
Unsupported conclusions on the Bacillus anthracis spores.
Appl Environ Microbiol. 2007 Aug;73(15):5074. [Abstract/Link to Full Text]

Beecher DJ
Forensic application of microbiological culture analysis to identify mail intentionally contaminated with Bacillus anthracis spores.
Appl Environ Microbiol. 2006 Aug;72(8):5304-10.
The discovery of a letter intentionally filled with dried Bacillus anthracis spores in the office of a United States senator prompted the collection and quarantine of all mail in congressional buildings. This mail was subsequently searched for additional intentionally contaminated letters. A microbiological sampling strategy was used to locate heavy contamination within the 642 separate plastic bags containing the mail. Swab sampling identified 20 bags for manual and visual examination. Air sampling within the 20 bags indicated that one bag was orders of magnitude more contaminated than all the others. This bag contained a letter addressed to Senator Patrick Leahy that had been loaded with dried B. anthracis spores. Microbiological sampling of compartmentalized batches of mail proved to be efficient and relatively safe. Efficiency was increased by inoculating culture media in the hot zone rather than transferring swab samples to a laboratory for inoculation. All mail sampling was complete within 4 days with minimal contamination of the sampling environment or personnel. However, physically handling the intentionally contaminated letter proved to be exceptionally hazardous, as did sorting of cross-contaminated mail, which resulted in generation of hazardous aerosol and extensive contamination of protective clothing. Nearly 8 x 10(6) CFU was removed from the most highly cross-contaminated piece of mail found. Tracking data indicated that this and other heavily contaminated envelopes had been processed through the same mail sorting equipment as, and within 1 s of, two intentionally contaminated letters. [Abstract/Link to Full Text]

Costantini VP, Azevedo AC, Li X, Williams MC, Michel FC, Saif LJ
Effects of different animal waste treatment technologies on detection and viability of porcine enteric viruses.
Appl Environ Microbiol. 2007 Aug;73(16):5284-91.
Enteric pathogens in animal waste that is not properly processed can contaminate the environment and food. The persistence of pathogens in animal waste depends upon the waste treatment technology, but little is known about persistence of porcine viruses. Our objectives were to characterize the porcine enteric viruses (porcine noroviruses [PoNoVs], porcine sapoviruses [PoSaVs], rotavirus A [RV-A], RV-B, and RV-C) in fresh feces or manure and to evaluate the effects of different candidate environmentally superior technologies (ESTs) for animal waste treatment on the detection of these viruses. Untreated manure and samples collected at different stages during and after treatment were obtained from swine farms that used conventional waste management (CWM) and five different candidate ESTs. The RNA from porcine enteric viruses was detected by reverse transcription-PCR and/or seminested PCR; PoSaV and RV-A were also detected by enzyme-linked immunosorbent assay. Cell culture immunofluorescence (CCIF) and experimental inoculation of gnotobiotic (Gn) pigs were used to determine RV-A/C infectivity in posttreatment samples. The PoSaV and RV-A were detected in pretreatment samples from each farm, whereas PoNoV and RV-C were detected in pretreatment feces from three of five and four of five farms using the candidate ESTs, respectively. After treatment, PoSaV RNA was detected only in the samples from the farm using CWM and not from the farms using the candidate ESTs. RV-A and RV-C RNAs were detected in four of five and three of four candidate ESTs, respectively, after treatment, but infectious particles were not detected by CCIF, nor were clinical signs or seroconversion detected in inoculated Gn pigs. These results indicate that only RV-A/C RNA, but no viral infectivity, was detected after treatment. Our findings address a public health concern regarding environmental quality surrounding swine production units. [Abstract/Link to Full Text]

Blas-Galindo E, Cava F, López-Viñas E, Mendieta J, Berenguer J
Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus.
Appl Environ Microbiol. 2007 Aug;73(16):5138-45.
A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium. [Abstract/Link to Full Text]

Tominaga T, Hatakeyama Y
Development of innovative pediocin PA-1 by DNA shuffling among class IIa bacteriocins.
Appl Environ Microbiol. 2007 Aug;73(16):5292-9.
Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of Leuconostoc lactis isolated from a sour-spoiled dairy product. To develop an even more effective version of this chimera, a DNA-shuffling library was constructed, wherein four specific regions within the N-terminal half of pediocin PA-1 were shuffled with the corresponding sequences from 10 other class IIa bacteriocins. Activity screening indicated that 63 out of 280 shuffled mutants had antimicrobial activity. A colony overlay activity assay showed that one of the mutants (designated B1) produced a >7.8-mm growth inhibition circle on L. lactis, whereas the parent pediocin PA-1 did not produce any circle. Furthermore, the active shuffled mutants showed increased activity against various species of Lactobacillus, Pediococcus, and Carnobacterium. Sequence analysis revealed that the active mutants had novel N-terminal sequences; in active mutant B1, for example, the parental pediocin PA-1 sequence (KYYGNGVTCGKHSC) was changed to TKYYGNGVSCTKSGC. These new and improved DNA-shuffled bacteriocins could prove useful as food additives for inhibiting sour spoilage of dairy products. [Abstract/Link to Full Text]

Myers JL, Sekar R, Richardson LL
Molecular detection and ecological significance of the cyanobacterial genera Geitlerinema and Leptolyngbya in black band disease of corals.
Appl Environ Microbiol. 2007 Aug;73(16):5173-82.
Black band disease (BBD) is a pathogenic, sulfide-rich microbial mat dominated by filamentous cyanobacteria that infect corals worldwide. We isolated cyanobacteria from BBD into culture, confirmed their presence in the BBD community by using denaturing gradient gel electrophoresis (DGGE), and demonstrated their ecological significance in terms of physiological sulfide tolerance and photosynthesis-versus-irradiance values. Twenty-nine BBD samples were collected from nine host coral species, four of which have not previously been investigated, from reefs of the Florida Keys, the Bahamas, St. Croix, and the Philippines. From these samples, seven cyanobacteria were isolated into culture. Cloning and sequencing of the 16S rRNA gene using universal primers indicated that four isolates were related to the genus Geitlerinema and three to the genus Leptolyngbya. DGGE results, obtained using Cyanobacteria-specific 16S rRNA primers, revealed that the most common BBD cyanobacterial sequence, detected in 26 BBD field samples, was related to that of an Oscillatoria sp. The next most common sequence, 99% similar to that of the Geitlerinema BBD isolate, was present in three samples. One Leptolyngbya- and one Phormidium-related sequence were also found. Laboratory experiments using isolates of BBD Geitlerinema and Leptolyngbya revealed that they could carry out sulfide-resistant oxygenic photosynthesis, a relatively rare characteristic among cyanobacteria, and that they are adapted to the sulfide-rich, low-light BBD environment. The presence of the cyanotoxin microcystin in these cultures and in BBD suggests a role in BBD pathogenicity. Our results confirm the presence of Geitlerinema in the BBD microbial community and its ecological significance, which have been challenged, and provide evidence of a second ecologically significant BBD cyanobacterium, Leptolyngbya. [Abstract/Link to Full Text]

Caballero-Mellado J, Onofre-Lemus J, Estrada-de Los Santos P, Martínez-Aguilar L
The tomato rhizosphere, an environment rich in nitrogen-fixing Burkholderia species with capabilities of interest for agriculture and bioremediation.
Appl Environ Microbiol. 2007 Aug;73(16):5308-19.
Burkholderia strains are promising candidates for biotechnological applications. Unfortunately, most of these strains belong to species of the Burkholderia cepacia complex (Bcc) involved in human infections, hampering potential applications. Novel diazotrophic Burkholderia species, phylogenetically distant from the Bcc species, have been discovered recently, but their environmental distribution and relevant features for agro-biotechnological applications are little known. In this work, the occurrence of N2-fixing Burkholderia species in the rhizospheres and rhizoplanes of tomato plants field grown in Mexico was assessed. The results revealed a high level of diversity of diazotrophic Burkholderia species, including B. unamae, B. xenovorans, B. tropica, and two other unknown species, one of them phylogenetically closely related to B. kururiensis. These N2-fixing Burkholderia species exhibited activities involved in bioremediation, plant growth promotion, or biological control in vitro. Remarkably, B. unamae and B. kururiensis grew with aromatic compounds (phenol and benzene) as carbon sources, and the presence of aromatic oxygenase genes was confirmed in both species. The rhizospheric and endophyte nature of B. unamae and its ability to degrade aromatic compounds suggest that it could be used in rhizoremediation and for improvement of phytoremediation. B. kururiensis and other Burkholderia sp. strains grew with toluene. B. unamae and B. xenovorans exhibited ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity, and the occurrence of acdS genes encoding ACC deaminase was confirmed. Mineral phosphate solubilization through organic acid production appears to be the mechanism used by most diazotrophic Burkholderia species, but in B. tropica, there presumably exists an additional unknown mechanism. Most of the diazotrophic Burkholderia species produced hydroxamate-type siderophores. Certainly, the N2-fixing Burkholderia species associated with plants have great potential for agro-biotechnological applications. [Abstract/Link to Full Text]

Bermúdez-Humarán LG, Nouaille S, Zilberfarb V, Corthier G, Gruss A, Langella P, Issad T
Effects of intranasal administration of a leptin-secreting Lactococcus lactis recombinant on food intake, body weight, and immune response of mice.
Appl Environ Microbiol. 2007 Aug;73(16):5300-7.
Leptin is an adipocyte-derived pleiotropic hormone that modulates a large number of physiological functions, including control of body weight and regulation of the immune system. In this work, we show that a recombinant strain of the food-grade lactic acid bacterium Lactococcus lactis (LL-lep) can produce and efficiently secrete human leptin. The secreted leptin is a fully biologically active hormone, as demonstrated by its capacity to stimulate a STAT3 reporter gene in HEK293 cells transfected with the Ob-Rb leptin receptor. The immunomodulatory activity of leptin-secreting L. lactis was evaluated in vivo by coexpression with the human papillomavirus type 16 E7 protein. In C57BL/6 mice immunized intranasally with a recombinant L. lactis strain coproducing leptin and E7 antigen, the adaptive immune response was significantly higher than in mice immunized with recombinant L. lactis producing only E7 antigen, demonstrating adjuvanticity of leptin. We then analyzed the effects of intranasally administered LL-lep in obese ob/ob mice. We observed that daily administration of LL-lep to these mice significantly reduced body weight gain and food intake. These results demonstrate that leptin can be produced and secreted in an active form by L. lactis and that leptin-producing L. lactis regulates in vivo antigen-specific immune responses, as well as body weight and food consumption. [Abstract/Link to Full Text]

Blackwood CB, Hudleston D, Zak DR, Buyer JS
Interpreting ecological diversity indices applied to terminal restriction fragment length polymorphism data: insights from simulated microbial communities.
Appl Environ Microbiol. 2007 Aug;73(16):5276-83.
Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-E(var)) and true Shannon diversity (H'), with correlations between 0.71 and 0.81. TRF-H' and true H' were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H' cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-E(var)). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity. [Abstract/Link to Full Text]

Zhang J, Fu RY, Hugenholtz J, Li Y, Chen J
Glutathione protects Lactococcus lactis against acid stress.
Appl Environ Microbiol. 2007 Aug;73(16):5268-75.
Previously we showed that glutathione (GSH) can protect Lactococcus lactis against oxidative stress (Y. Li et al., Appl. Environ. Microbiol. 69:5739-5745, 2003). In the present study, we show that the GSH imported by L. lactis subsp. cremoris SK11 or produced by engineered L. lactis subsp. cremoris NZ9000 can protect both strains against a long-term mild acid challenge (pH 4.0) and a short-term severe acid challenge (pH 2.5). This shows for the first time that GSH can protect a gram-positive bacterium against acid stress. During acid challenge, strain SK11 containing imported GSH and strain NZ9000 containing self-produced GSH exhibited significantly higher intracellular pHs than the control. Furthermore, strain SK11 containing imported GSH had a significantly higher activity of glyceraldehyde-3-phosphate dehydrogenase than the control. These results suggest that the acid stress resistance of starter culture can be improved by selecting L. lactis strains capable of producing or importing GSH. [Abstract/Link to Full Text]

Mendes-Ferreira A, del Olmo M, García-Martínez J, Jiménez-Martí E, Leão C, Mendes-Faia A, Pérez-Ortín JE
Saccharomyces cerevisiae signature genes for predicting nitrogen deficiency during alcoholic fermentation.
Appl Environ Microbiol. 2007 Aug;73(16):5363-9.
Genome-wide analysis of the wine yeast strain Saccharomyces cerevisiae PYCC4072 identified 36 genes highly expressed under conditions of low or absent nitrogen in comparison with a nitrogen-replete condition. Reverse transcription-PCR analysis for four of these transcripts with this strain and its validation with another wine yeast strain underlines the usefulness of these signature genes for predicting nitrogen deficiency and therefore the diagnosis of wine stuck/sluggish fermentations. [Abstract/Link to Full Text]

Ragull S, Garcia-Nuñez M, Pedro-Botet ML, Sopena N, Esteve M, Montenegro R, Sabrià M
Legionella pneumophila in cooling towers: fluctuations in counts, determination of genetic variability by pulsed-field gel electrophoresis (PFGE), and persistence of PFGE patterns.
Appl Environ Microbiol. 2007 Aug;73(16):5382-4.
The concentrations of Legionella pneumophila in cooling towers may vary considerably over short periods of time, producing significant fluctuations throughout the year. Despite genetic variability, in small geographical areas the same indistinguishable pulsed-field gel electrophoresis patterns may be shared among different cooling towers and persist over time. [Abstract/Link to Full Text]

Kulp TR, Han S, Saltikov CW, Lanoil BD, Zargar K, Oremland RS
Effects of imposed salinity gradients on dissimilatory arsenate reduction, sulfate reduction, and other microbial processes in sediments from two California soda lakes.
Appl Environ Microbiol. 2007 Aug;73(16):5130-7.
Salinity effects on microbial community structure and on potential rates of arsenate reduction, arsenite oxidation, sulfate reduction, denitrification, and methanogenesis were examined in sediment slurries from two California soda lakes. We conducted experiments with Mono Lake and Searles Lake sediments over a wide range of salt concentrations (25 to 346 g liter(-1)). With the exception of sulfate reduction, rates of all processes demonstrated an inverse relationship to total salinity. However, each of these processes persisted at low but detectable rates at salt saturation. Denaturing gradient gel electrophoresis analysis of partial 16S rRNA genes amplified from As(V) reduction slurries revealed that distinct microbial populations grew at low (25 to 50 g liter(-1)), intermediate (100 to 200 g liter(-1)), and high (>300 g liter(-1)) salinity. At intermediate and high salinities, a close relative of a cultivated As-respiring halophile was present. These results suggest that organisms adapted to more dilute conditions can remain viable at high salinity and rapidly repopulate the lake during periods of rising lake level. In contrast to As reduction, sulfate reduction in Mono Lake slurries was undetectable at salt saturation. Furthermore, sulfate reduction was excluded from Searles Lake sediments at any salinity despite the presence of abundant sulfate. Sulfate reduction occurred in Searles Lake sediment slurries only following inoculation with Mono Lake sediment, indicating the absence of sulfate-reducing flora. Experiments with borate-amended Mono Lake slurries suggest that the notably high (0.46 molal) concentration of borate in the Searles Lake brine was responsible for the exclusion of sulfate reducers from that ecosystem. [Abstract/Link to Full Text]

El Karkouri K, Murat C, Zampieri E, Bonfante P
Identification of internal transcribed spacer sequence motifs in truffles: a first step toward their DNA bar coding.
Appl Environ Microbiol. 2007 Aug;73(16):5320-30.
This work presents DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat unit which are useful for the identification of five European and Asiatic truffles (Tuber magnatum, T. melanosporum, T. indicum, T. aestivum, and T. mesentericum). Truffles are edible mycorrhizal ascomycetes that show similar morphological characteristics but that have distinct organoleptic and economic values. A total of 36 out of 46 ITS1 or ITS2 sequence motifs have allowed an accurate in silico distinction of the five truffles to be made (i.e., by pattern matching and/or BLAST analysis on downloaded GenBank sequences and directly against GenBank databases). The motifs considered the intraspecific genetic variability of each species, including rare haplotypes, and assigned their respective species from either the ascocarps or ectomycorrhizas. The data indicate that short ITS1 or ITS2 motifs (< or = 50 bp in size) can be considered promising tools for truffle species identification. A dot blot hybridization analysis of T. magnatum and T. melanosporum compared with other close relatives or distant lineages allowed at least one highly specific motif to be identified for each species. These results were confirmed in a blind test which included new field isolates. The current work has provided a reliable new tool for a truffle oligonucleotide bar code and identification in ecological and evolutionary studies. [Abstract/Link to Full Text]

Mari X, Kerros ME, Weinbauer MG
Virus attachment to transparent exopolymeric particles along trophic gradients in the southwestern lagoon of New Caledonia.
Appl Environ Microbiol. 2007 Aug;73(16):5245-52.
Viruses on organic aggregates such as transparent exopolymeric particles (TEP) are not well investigated. The number of TEP-attached viruses was assessed along trophic gradients in the southwestern lagoon of New Caledonia by determining the fraction of viruses removed after magnetic isolation of TEP. In order to isolate TEP magnetically, TEP were formed in the presence of magnetic beads from submicrometer precursors collected along the trophic gradients. The mixed aggregates of TEP-beads-viruses were removed from solution with a magnetic field. The percentage of viruses associated with newly formed TEP averaged 8% (range, 3 to 13%) for most of the stations but was higher (ca. 30%) in one bay characterized by the low renewal rate of its water mass. The number of viruses (N) attached to TEP varied as a function of TEP size (d [in micrometers]) according to the formulas N = 100d(1.60) and N = 230d(1.75), respectively, for TEP occurring in water masses with short (i.e., <40 days) and long (i.e., >40 days) residence times. These two relationships imply that viral abundance decreases with TEP size, and they indicate that water residence time influences viral density and virus-bacterium interactions within aggregates. Our data suggest that the fraction of viruses attached to TEP is highest in areas characterized by a low renewal rate of the water mass and can constitute at times a significant fraction of total virus abundance. Due to the small distance between viruses and hosts on TEP, these particles may be hot spots for viral infection. [Abstract/Link to Full Text]

Read JD, Colussi PA, Ganatra MB, Taron CH
Acetamide selection of Kluyveromyces lactis cells transformed with an integrative vector leads to high-frequency formation of multicopy strains.
Appl Environ Microbiol. 2007 Aug;73(16):5088-96.
The yeast Kluyveromyces lactis has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into K. lactis cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields K. lactis transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the LAC4 chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies. [Abstract/Link to Full Text]

Yamaguchi S, Komeda H, Asano Y
New enzymatic method of chiral amino acid synthesis by dynamic kinetic resolution of amino acid amides: use of stereoselective amino acid amidases in the presence of alpha-amino-epsilon-caprolactam racemase.
Appl Environ Microbiol. 2007 Aug;73(16):5370-3.
D- and L-amino acids were produced from L- and D-amino acid amides by D-aminopeptidase from Ochrobactrum anthropi C1-38 and L-amino acid amidase from Pseudomonas azotoformans IAM 1603, respectively, in the presence of alpha-amino-epsilon-caprolactam racemase from Achromobacter obae as the catalyst by dynamic kinetic resolution of amino acid amides. [Abstract/Link to Full Text]

Gamble R, Muriana PM
Microplate fluorescence assay for measurement of the ability of strains of Listeria monocytogenes from meat and meat-processing plants to adhere to abiotic surfaces.
Appl Environ Microbiol. 2007 Aug;73(16):5235-44.
Listeria monocytogenes is a significant food-borne pathogen that is capable of adhering to and producing biofilms on processing equipment, making it difficult to eliminate from meat-processing environments and allowing potential contamination of ready-to-eat (RTE) products. We devised a fluorescence-based microplate method for screening isolates of L. monocytogenes for the ability to adhere to abiotic surfaces. Strains of L. monocytogenes were incubated for 2 days at 30 degrees C in 96-well microplates, and the plates were washed in a plate washer. The retained cells were incubated for 15 min at 25 degrees C with 5,6-carboxyfluorescein diacetate and washed again, and then the fluorescence was read with a plate reader. Several enzymatic treatments (protease, lipase, and cellulase) were effective in releasing adherent cells from the microplates, and this process was used for quantitation on microbiological media. Strongly adherent strains of L. monocytogenes were identified that had 15,000-fold-higher levels of fluorescence and 100,000-fold-higher plate counts in attachment assays than weakly adherent strains. Strongly adherent strains of L. monocytogenes adhered equally well to four different substrates (glass, plastic, rubber, and stainless steel); showed high-level attachment on microplates at 10, 20, 30, and 40 degrees C; and showed significant differences from weakly adherent strains when examined by scanning electron microscopy. A greater incidence of strong adherence was observed for strains isolated from RTE meats than for those isolated from environmental surfaces. Analysis of surface adherence among Listeria isolates from processing environments may provide a better understanding of the molecular mechanisms involved in attachment and suggest solutions to eliminate them from food-processing environments. [Abstract/Link to Full Text]

Lagaert S, Van Campenhout S, Pollet A, Bourgois TM, Delcour JA, Courtin CM, Volckaert G
Recombinant expression and characterization of a reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium adolescentis.
Appl Environ Microbiol. 2007 Aug;73(16):5374-7.
The family 8 glycoside hydrolase (RexA) from Bifidobacterium adolescentis was expressed in Escherichia coli. The recombinant enzyme was characterized as a reducing-end xylose-releasing exo-oligoxylanase. Apart from giving insights into this new class of enzymes, knowledge of the RexA enzyme helps to postulate a mechanism for the B. adolescentis breakdown of prebiotic xylooligosaccharides. [Abstract/Link to Full Text]

Torres-Bacete J, Hormigo D, Stuart M, Arroyo M, Torres P, Castillón MP, Acebal C, García JL, de la Mata I
Newly discovered penicillin acylase activity of aculeacin A acylase from Actinoplanes utahensis.
Appl Environ Microbiol. 2007 Aug;73(16):5378-81.
Aculeacin A acylase from Actinoplanes utahensis produced by Streptomyces lividans revealed acylase activities that are able to hydrolyze penicillin V and several natural aliphatic penicillins. Penicillin K was the best substrate, showing a catalytic efficiency of 34.79 mM(-1) s(-1). Furthermore, aculeacin A acylase was highly thermostable, with a midpoint transition temperature of 81.5 degrees C. [Abstract/Link to Full Text]

Shimizu T, Kinoshita H, Nihira T
Identification and in vivo functional analysis by gene disruption of ctnA, an activator gene involved in citrinin biosynthesis in Monascus purpureus.
Appl Environ Microbiol. 2007 Aug;73(16):5097-103.
Citrinin, a secondary fungal metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. From the red-pigment producer Monascus purpureus, a 21-kbp region flanking pksCT, which encodes citrinin polyketide synthase, was cloned. Four open reading frames (ORFs) (orf1, orf2, orf3, and orf4) in the 5'-flanking region and one ORF (orf5) in the 3'-flanking region were identified in the vicinity of pksCT. orf1 to orf5 encode a homolog of a dehydrogenase (similarity, 46%), a regulator (similarity, 38%), an oxygenase (similarity, 41%), an oxidoreductase (similarity, 26%), and a transporter (similarity, 58%), respectively. orf2 (2,006 bp with four introns) encodes a 576-amino-acid protein containing a typical Zn(II)2Cys6 DNA binding motif at the N terminus and was designated ctnA. Although reverse transcriptase PCR analysis revealed that all of these ORFs, except for orf1, were transcribed with pksCT under citrinin production conditions, the disruption of ctnA caused large decreases in the transcription of pksCT and orf5, together with reduction of citrinin production to barely detectable levels, suggesting that these two genes are under control of the ctnA product. Complementation of the ctnA disruptant with intact ctnA on an autonomously replicating plasmid restored both transcription and citrinin production, indicating that CtnA is a major activator of citrinin biosynthesis. [Abstract/Link to Full Text]

Chen WJ, Delmotte F, Richard-Cervera S, Douence L, Greif C, Corio-Costet MF
At least two origins of fungicide resistance in grapevine downy mildew populations.
Appl Environ Microbiol. 2007 Aug;73(16):5162-72.
Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc1 complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure. [Abstract/Link to Full Text]

Lulko AT, Veening JW, Buist G, Smits WK, Blom EJ, Beekman AC, Bron S, Kuipers OP
Production and secretion stress caused by overexpression of heterologous alpha-amylase leads to inhibition of sporulation and a prolonged motile phase in Bacillus subtilis.
Appl Environ Microbiol. 2007 Aug;73(16):5354-62.
Transcriptome analysis was used to investigate the global stress response of the gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ alpha-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon, which responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example, citM, ylxF, yloA, ykoJ, and several genes of the flgB operon. However, high-affinity CssR binding was observed only for htrA, htrB, and, possibly, citM. In addition, the DNA macroarray approach revealed that several genes of the sporulation pathway are downregulated by AmyQ overexpression and that a group of motility-specific (sigmaD-dependent) transcripts were clearly upregulated. Subsequent flow-cytometric analyses demonstrate that, upon overproduction of AmyQ as well as of a nonsecretable variant of the alpha-amylase, the process of sporulation is severely inhibited. Similar experiments were performed to investigate the expression levels of the hag promoter, a well-established reporter for sigmaD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of alpha-amylase overproduction. [Abstract/Link to Full Text]

Romero S, Merino E, Bolívar F, Gosset G, Martinez A
Metabolic engineering of Bacillus subtilis for ethanol production: lactate dehydrogenase plays a key role in fermentative metabolism.
Appl Environ Microbiol. 2007 Aug;73(16):5190-8.
Wild-type Bacillus subtilis ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 delta alsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 delta alsS udhA+). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain. [Abstract/Link to Full Text]

Ufnar JA, Ufnar DF, Wang SY, Ellender RD
Development of a swine-specific fecal pollution marker based on host differences in methanogen mcrA genes.
Appl Environ Microbiol. 2007 Aug;73(16):5209-17.
The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10(-6) g of wet pig feces in 500 ml of phosphate-buffered saline and 10(-4) g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker. [Abstract/Link to Full Text]

Butot S, Putallaz T, Croquet C, Lamothe G, Meyer R, Joosten H, Sánchez G
Attachment of enteric viruses to bottles.
Appl Environ Microbiol. 2007 Aug;73(16):5104-10.
Storage of water that was deliberately contaminated with enteric viruses in polyethylene terephthalate (PET) bottles led to a rapid decrease of the apparent viral load, thereby hampering the development of samples for a collaborative evaluation of viral detection methods for bottled water. To determine if this decrease was due to spontaneous inactivation or to adhesion, an elution protocol was developed and combined with a rapid and sensitive real-time reverse transcription-PCR-based method to quantify adsorbed norovirus (NV), hepatitis A virus (HAV), and rotavirus (RV) on bottle walls. The NV retention on PET bottle walls after 20 and 62 days reached an average level of 85% and 95% of the recovered inoculum, respectively. HAV and RV also showed adsorption onto PET bottles, reaching 90% and 80%, respectively, after 20 days of storage. NV and RV attachment was demonstrated to be dependent on the presence of autochthonous flora, whereas HAV adsorption was independent of it. Application of the elution and viral detection protocol to 294 commercially available water bottles obtained from 25 different countries did not give any positive result, thereby providing further evidence that the sources used for this product are free from enteric viruses and support for the theory that bottled water is not a vehicle for viral diseases. [Abstract/Link to Full Text]

Nocker A, Sossa-Fernandez P, Burr MD, Camper AK
Use of propidium monoazide for live/dead distinction in microbial ecology.
Appl Environ Microbiol. 2007 Aug;73(16):5111-7.
One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration. [Abstract/Link to Full Text]

Park M, Jeon Y, Jang HH, Ro HS, Park W, Madsen EL, Jeon CO
Molecular and biochemical characterization of 3-hydroxybenzoate 6-hydroxylase from Polaromonas naphthalenivorans CJ2.
Appl Environ Microbiol. 2007 Aug;73(16):5146-52.
Prior research revealed that Polaromonas naphthalenivorans CJ2 carries and expresses genes encoding the gentisate metabolic pathway for naphthalene. These metabolic genes are split into two clusters, comprising nagRAaGHAbAcAdBFCQEDJI'-orf1-tnpA and nagR2-orf2I''KL (C. O. Jeon, M. Park, H. Ro, W. Park, and E. L. Madsen, Appl. Environ. Microbiol. 72:1086-1095, 2006). BLAST homology searches of sequences in GenBank indicated that the orf2 gene from the small cluster likely encoded a salicylate 5-hydroxylase, presumed to catalyze the conversion of salicylate into gentisate. Here, we report physiological and genetic evidence that orf2 does not encode salicylate 5-hydroxylase. Instead, we have found that orf2 encodes 3-hydroxybenzoate 6-hydroxylase, the enzyme which catalyzes the NADH-dependent conversion of 3-hydroxybenzoate into gentisate. Accordingly, we have renamed orf2 nagX. After expression in Escherichia coli, the NagX enzyme had an approximate molecular mass of 43 kDa, as estimated by gel filtration, and was probably a monomeric protein. The enzyme was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity. Like other 3-hydroxybenzoate 6-hydroxylases, NagX utilized both NADH and NADPH as electron donors and exhibited a yellowish color, indicative of a bound flavin adenine dinucleotide. An engineered mutant of P. naphthalenivorans CJ2 defective in nagX failed to grow on 3-hydroxybenzoate but grew normally on naphthalene. These results indicate that the previously described small catabolic cluster in strain CJ2 may be multifunctional and is essential for the degradation of 3-hydroxybenzoate. Because nagX and an adjacent MarR-type regulatory gene are both closely related to homologues in Azoarcus species, this study raises questions about horizontal gene transfer events that contribute to operon evolution. [Abstract/Link to Full Text]

Ogden ID, MacRae M, Johnston M, Strachan NJ, Cody AJ, Dingle KE, Newell DG
Use of multilocus sequence typing to investigate the association between the presence of Campylobacter spp. in broiler drinking water and Campylobacter colonization in broilers.
Appl Environ Microbiol. 2007 Aug;73(16):5125-9.
The presence of campylobacters in broiler chickens and throughout the broiler water delivery systems of 12 farms in northeastern Scotland was investigated by sensitive enrichment methods and large-volume filtration. Campylobacter presence was independent of the water source and whether the water was treated. The genotypes of Campylobacter jejuni isolates recovered from chickens and various locations within the water delivery systems were compared by multilocus sequence typing. Matching strains in shed header tanks and birds were found at 1 of the 12 farms investigated. However, the sequence of contamination or whether the source was within or outside the shed was not determined. Nevertheless, these data provide evidence that drinking water could be associated with broiler infection by campylobacters. [Abstract/Link to Full Text]

Wang Q, Garrity GM, Tiedje JM, Cole JR
Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.
Appl Environ Microbiol. 2007 Aug;73(16):5261-7.
The Ribosomal Database Project (RDP) Classifier, a naïve Bayesian classifier, can rapidly and accurately classify bacterial 16S rRNA sequences into the new higher-order taxonomy proposed in Bergey's Taxonomic Outline of the Prokaryotes (2nd ed., release 5.0, Springer-Verlag, New York, NY, 2004). It provides taxonomic assignments from domain to genus, with confidence estimates for each assignment. The majority of classifications (98%) were of high estimated confidence (> or = 95%) and high accuracy (98%). In addition to being tested with the corpus of 5,014 type strain sequences from Bergey's outline, the RDP Classifier was tested with a corpus of 23,095 rRNA sequences as assigned by the NCBI into their alternative higher-order taxonomy. The results from leave-one-out testing on both corpora show that the overall accuracies at all levels of confidence for near-full-length and 400-base segments were 89% or above down to the genus level, and the majority of the classification errors appear to be due to anomalies in the current taxonomies. For shorter rRNA segments, such as those that might be generated by pyrosequencing, the error rate varied greatly over the length of the 16S rRNA gene, with segments around the V2 and V4 variable regions giving the lowest error rates. The RDP Classifier is suitable both for the analysis of single rRNA sequences and for the analysis of libraries of thousands of sequences. Another related tool, RDP Library Compare, was developed to facilitate microbial-community comparison based on 16S rRNA gene sequence libraries. It combines the RDP Classifier with a statistical test to flag taxa differentially represented between samples. The RDP Classifier and RDP Library Compare are available online at http://rdp.cme.msu.edu/. [Abstract/Link to Full Text]

---

Recent Articles in Applied Microbiology

---

Sharpe ES, Nickerson KW, Bulla LA, Aronson JN
Separation of spores and parasporal crystals of Bacillus thuringiensis in gradients of certain x-ray contrasting agents.
Appl Microbiol. 1975 Dec;30(6):1052-3.
Spores and parasporal crystals of Bacillus thuringiensis can be separated at moderate centrifugation speeds (10,000 to 12,000 rpm) in gradients of Renografin or sodium diatrizoate. [Abstract/Link to Full Text]

Hood MA, Bishop WS, Bishop FW, Meyers SP, Whelan T
Microbial Indicators of Oil-Rich Salt Marsh Sediments.
Appl Microbiol. 1975 Dec;30(6):982-987.
Selected microbial parameters were monitored in sediments from a pristine and an oil-field salt marsh. Although numbers of hydrocarbonoclastic bacteria and fungi were significantly greater in the oil field, the values did not show a strong correlation with levels of hydrocarbons (r = 0.43 and r = 0.49, respectively). However, a high correlation was noted between ratios of hydrocarbonoclastic and total aerobic heterotrophic bacteria and levels of hydrocarbons as well as the relative concentration of hydrocarbons (ratio of hydrocarbons to chloroform extractables) (r = 0.87 and r = 0.77, respectively). Data suggest that this first ratio is a more valid microbial indicator of hydrocarbon abundance than other factors examined. Significant differences in the ratio of pigmented to total colony-forming units, the ratio of different to total colony-forming units, and the diversity index were noted between the natural and oil-field marsh. It is suggested that the presence of hydrocarbons alters the relative abundance of the most predominant aerobic heterotrophic bacteria. [Abstract/Link to Full Text]

Porter SC
Accelerated Fermentation of Brewer's Wort by Saccharomyces carlsbergensis.
Appl Microbiol. 1975 Dec;30(6):970-974.
A rapid procedure for wort fermentation with Saccharomyces carlsbergensis at 12 C is described. Fermentation time was reduced from 7 to 4 days with normal inoculum by shaking. Increasing the inoculation to 5 to 10 times normal and shaking resulted in complete fermentation in 3 days. Maximum yeast population was reached rapidly with the large inocula, but fermentation proceeded at approximately the same rate when inoculations in excess of four times the normal were used. Similar results were obtained with both small-scale (100 ml) and microbrew (2.4 liters) fermentations. [Abstract/Link to Full Text]

Han YW, Anderson AW
Semisolid Fermentation of Ryegrass Straw.
Appl Microbiol. 1975 Dec;30(6):930-934.
Candida utilis, Aureobasidium pullulans, and Trichoderma viride were grown on pretreated ryegrass straw. The pretreatment consisted of hydrolysis of straw with 0.5 N H(2)SO(4) (water-substrate, 3:1) at 121 C, 100 C, and room temperature and adjustment of the hydrolysate to pH 4.5 to 5.0 with 5 N NH(4)OH. The 121 C pretreatment yielded a material containing 30% sugar and 2.3% N. The fermentation was carried on semisolid substrate (moisture level, 75%) in rotating jars for 2 to 3 days at room temperature. The organisms grew rapidly during the period from 18 to 42 h of incubation. During this period the number of microbial cells increased by 20- to 200-fold, and the level of NH(3)-N decreased from 1.3 to 0.9%. The fermentation resulted in a fourfold increase in protein, fivefold increase in crude fat, and 40% increase in the digestibility of straw. The best result in terms of increasing protein and digestibility of straw was obtained when C. utilis was grown on straw preheated at 121 C. [Abstract/Link to Full Text]

Ruiz-Argueso T, Rodriguez-Navarro A
Microbiology of Ripening Honey.
Appl Microbiol. 1975 Dec;30(6):893-896.
Two main groups of bacteria, classified as Gluconobacter and Lactobacillus, are present in ripening honey. A third bacterial group, classified as Zymomonas, and several types of yeast are occasionally isolated. Both in natural honey and in synthetic syrup the bacterial population decreases in the course of the ripening process. Lactobacillus and Gluconobacter disappear after minimum moisture (about 18%) is reached, but the former does so sooner than the latter. The presence of these bacteria in different parts of the bee has been also investigated. [Abstract/Link to Full Text]

Fleming HP, Etchells JL, Costilow RN
Microbial Inhibition by an Isolate of Pediococcus from Cucumber Brines.
Appl Microbiol. 1975 Dec;30(6):1040-1042.
We reported earlier that Pediococcus cerevisiae FBB-61 inhibited Lactobacillus plantarum FBB-67 in mixed species inoculation used for the fermentation of brined cucumbers. Herein, 16 isolates of the Pediococcus genus from various sources were tested for inhibitory activity against L. plantarum and other microorganisms by a seeded-agar screening technique. Only two of the 16 isolates gave consistent and distinctive zones of inhibition, and both were isolated from fermenting cucumber brines on separate occasions. These two isolates did not inhibit each other but did inhibit the other 14 Pediococcus isolates in addition to L. plantarum. They also inhibited several other gram-positive bacteria, but not four species each of gram-negative bacteria and yeasts tested. Inoculation of cucumber juice broth with P. cerevisiae FBB-61 and L. plantarum WSO resulted in a drastic reduction in the plate count of L. plantarum WSO during day 1, but counts increased rapidly thereafter. Consequently, acid production by L. plantarum WSO was delayed. Noninhibitory isolates of Pediococcus had no appreciable effect on growth and acid production by L. plantarum WSO. [Abstract/Link to Full Text]

Yadav NK, Gupta KG
Effect of substrates on acetoin production by Torulopsis colliculosa and Enterobacter species.
Appl Microbiol. 1975 Dec;30(6):889-92.
Under optimal conditions, Torulopsis colliculosa NRRL 172 and Enterobacter B-87 (ATCC 27613) produced 50 to 500 mg of acetoin per g of substrate. Whereas cane molasses, gur, glucose, and sucrose were suitable substrates for acetoin production, lactose and mannitol supported very good growth but yielded little or no acetoin. Production of acetoin increased with increases in the concentration of glucose, yeast extract, and peptone. Combination of substrates and intermittent feeding of substrate failed to increase the yields. [Abstract/Link to Full Text]

Lee TC, Rodriguez DB, Karasawa I, Lee TH, Simpson KL, Chichester CO
Chemical alteration of carotene biosynthesis in Phycomyces blakesleeanus and mutants.
Appl Microbiol. 1975 Dec;30(6):988-93.
The effects of diphenylamine, dimethyl sulfoxide, streptomycin, AMO-1618, and beta-ionone on the carotene composition of a wild-type and three mutant strains of Phycomyces blakesleeanus have been examined. Diphenylamine increased the phytoene and phytofluene concentrations of all strains while reducing the levels of the color carotenes. Dimethyl sulfoxide reduced the concentration of both cyclic and acyclic carotenes, whereas AMO-1618 increased the levels of all carotenes in all the strains. The wild type and mutants responded differently to the presence of streptomycin and beta-ionone. The possible mode of action of the above agents on carotenoid biosynthesis is discussed. [Abstract/Link to Full Text]

Chou CC, Marth EH
Incorporation of (2-14C)acetate into lipids of mink (Mustela vison) liver and intestine during in vitro and in vivo treatment with aflatoxin B1.
Appl Microbiol. 1975 Dec;30(6):946-50.
The in vitro and in vivo incorporation of (2-14C)acetate into lipids of mink (Mustela vison) liver and intestines was studied. In vitro, a dose of aflatoxin B1 as small as 7.5 mug/ml of medium reduced by 20% the amount of (2-14C)acetate incorporated into lipids of mink liver slices, whereas 180 mug caused 76% reduction in the synthesis of lipids from the radioactive precusor. Similar inhibition of lipid synthesis by aflatoxin also was observed with tissues from mink intestines and fatty liver. The degree of inhibition (19 to 84% for tissue from intestines and 19 to 64% for tissue from fatty livers) depended on the amount of aflatoxin B1 (7.5 TO 180 MUG) present in the medium. In vivo, a substantially increased amount of 14C-labeled lipids was found in the livers of mink injected with 600 mug of aflatoxin B1 per kg of body weight 20, 28, and 40 h earlier. However, no appreciable difference in incorporation of (2-14C)acetate into lipids was observed between toxin-treated and control animals when these animals were sacrificed and examined for 14C-labeled lipids at 4 and 10 h after toxin was administered. [Abstract/Link to Full Text]

Gledhill WE
Screening test for assessment of ultimate biodegradability: linear alkylbenzene sulfonates.
Appl Microbiol. 1975 Dec;30(6):922-9.
A relatively simple shake-flask system for determining CO2 evolution was developed to assess the ultimate biodegradability by soil and sewage micro-organisms of chemicals which enter the environment. Linear alkylbenzene sulfonates (LAS) were used as model compounds to evaluate the method and were found to undergo substantial biodegradation in this dilute system. At the 30 mg/liter test concentration, higher-molecular-weight LAS compounds were biodegraded at a slower rate and to a lesser extent than lower-molecular-weight LAS, an effect which was eliminated or greatly reduced upon incremental addition of the LAS to the test medium during the first week of incubation. LA35S was used to demonstrate rapid LAS desulfonation, and 14CO2 evolution studies with (14C) benzene ring-labeled LAS indicated concomitant biodegradation of the entire LAS molecule as well as the LAS aromatic component. The test can be employed to examine numerous compounds at the same time and is readily adapted to studies of the effect of variation in temperature and oxygen concentration on biodegradation. [Abstract/Link to Full Text]

Ray AC, Eakin RE
Studies on the biosynthesis of aspergillin by Aspergillus niger.
Appl Microbiol. 1975 Dec;30(6):909-15.
Two inhibitors of the biosynthesis of aspergillin, the black spore pigment of Aspergillus niger, have been investigated. 2,4-Dithiopyrimidine exerted its inhibitory effect by intracellularly chelating cupric ion required for normal pigmentation. Dimethylsulfoxide prevented the synthesis of certain phenolic precursors of the native pigment. Partial purification and characterization of pigments from mature cultures revealed the presence of at least three components: (i) a high-molecular-weight (approximately 20,000) native pigment fraction in untreated mold cultures, (ii) a lower-molecular-weight (approximately 5,000) melanin pigment found in both types of inhibited cultures, and (iii) a low-molecular-weight (368) green pigment found only in the 2,4-dithiopyrimidine-inhibited cultures and proposed to be a pentacyclic quinonoid derivative. A pathway for aspergillin biosynthesis is suggested based on these results. [Abstract/Link to Full Text]

Nip WK, Chang FC, Chu FS, Prentice N
Fate of ochratoxin A in brewing.
Appl Microbiol. 1975 Dec;30(6):1048-9.
The fate of ochratoxin A in brewing was investigated by adding (3H)ochratoxin A to the raw materials at 1- and 10-mug/g levels during mashing in a conventional microbrewing process. The results indicated that large portions (28 to 39%) of the added toxin were recovered in spent grains, with less recovery in the yeast (8 to 20%) and beer (14 to 18%). About 38 and 12% of the added toxin at levels of 1 and 10 mug/g, respectively, were degraded during brewing. [Abstract/Link to Full Text]

Chet I, Asketh P, Mitchell R
Repulsion of bacteria from marine surfaces.
Appl Microbiol. 1975 Dec;30(6):1043-5.
Organic compounds are capable of repelling motile bacteria from marine surfaces. The most effective compounds were acrylamide and benzoic and tannic acids. These were active at concentrations that were not toxic to the bacteria. Repellents were incorporated in nontoxic paints and applied to metal panels. Treated panels immersed in seawater developed a bacterial film of only 10(6) bacteria per cm6 after 12 days compared with untreated panels, which had 5 times 10(12) bacteria per cm2 after the same period. Field studies confirmed the effectiveness of these repellents. The use of biological repellents provides a new approach to the control of marine fouling. [Abstract/Link to Full Text]

Walker JD, Colwell RR, Petrakis L
Evaluation of petroleum-degrading potential of bacteria from water and sediment.
Appl Microbiol. 1975 Dec;30(6):1036-9.
Bacteria from water and sediment of an oil-polluted harbor were examined for ability to degrade petroleum. Water samples contained a greater variety of bacterial species capable of degrading petroleum than sediment. Cultures from both water and sediment contained Pseudomonas and Acinetobacter spp. Bacteria present in the water samples produced significantly greater degradation of 2-,3-,4-, and 5-ring cycloalkanes and mono-, di-, tri-, tetra-, and pentaaromatics compared with bacteria in sediment samples. [Abstract/Link to Full Text]

Napoli CA, Hubbell DH
Ultrastructure of Rhizobium-induced infection threads in clover root hairs.
Appl Microbiol. 1975 Dec;30(6):1003-9.
Ultrastructural studies of Rhizobium-induced infection threads in clover root hairs show that the infection thread is initiated by an invagination process. Root hair wall growth is redirected at a localized point, resulting in the formation of an open pore. There is no direct penetration through the wall, and the bacteria remain extracellular within the root hair. [Abstract/Link to Full Text]

Sugiyama H, Yang KH
Growth potential of Clostridium botulinum in fresh mushrooms packaged in semipermeable plastic film.
Appl Microbiol. 1975 Dec;30(6):964-9.
Fresh mushrooms (Agaricus bisporus) were inoculated in the stem, gill, or cap with Clostridium botulinum spores. They were placed with uninoculated mushrooms in paper board trays, which were then covered and sealed in a polyvinyl chloride stretch film to simulate prepackaged mushrooms available at retail stores. When incubated at 20 C, botulinum toxin could be detected as early as day 3, or 4, when the mushrooms still appear edible. Mushrooms inoculated in the stem with 1,000 type A spores frequently became botulinogenic; higher spore levels were needed if gills or caps were inoculation sites. Type B spores were less apt to produce toxic mushrooms. Respiration of the fresh mushrooms used up O2 more rapidly than could enter through the semipermeable wrapping film, so that the equilibrium O2 concentration became low enough for growth of C. botulinum. Inoculated mushrooms did not become botulinogenic when held at 4 C. [Abstract/Link to Full Text]

Carhart G, Hegeman G
Improved method of selection for mutants of Pseudomonas putida.
Appl Microbiol. 1975 Dec;30(6):1046-7.
Optimum conditions for enrichment of mutants of Pseudomonas putida in liquid culture were established using a procedure which combines N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis with an improved D-cycloserine selection. [Abstract/Link to Full Text]

Monheimer RH
Effects of three environmental variables on sulfate uptake by aerobic bacteria.
Appl Microbiol. 1975 Dec;30(6):975-81.
The effects of various concentrations of sulfate, organic sulfur, and organic carbon on sulfate uptake by aerobic bacteria were studied using pure cultures growing in a defined medium. Cultures of Pseudomonas fluorescens and Corynebacterium striatum took up sulfate faster when young, but sulfate uptake by Serratia marcescens was faster in older cultures. Organic sulfur was found to decrease sulfate uptake, but at concentrations somewhat higher than occurs in most natural freshwater ecosystems. Low levels of sulfate can theoretically directly limit bacterial biomass production but such limitation probably does not occur in natural systems. Evidence is presented which indirectly links the uptake of sulfate and organic carbon, adding credibility to the proposal that sulfate uptake can be used as an indicator of microbial biomass production in freshwater ecosystems. [Abstract/Link to Full Text]

Hicks GF, Corner TR
Distribution and effects of 2,4,5-trichlorophenoxyacetic acid in cells of Bacillus megaterium.
Appl Microbiol. 1975 Dec;30(6):959-63.
Cell death in a resting population of an asporogenous Bacillus megaterium was accelerated by ambient concentrations of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) equal to or greater than 10 mug/ml or 5 mug/mg of cells (dry weight), but only after prolonged exposure. Conversely, populations of growing cells were not markedly influenced even at 100 mug/ml. Effects on cell respiration were not manifest until the ambient concentration reached 1,000 mug of 2,4,5-T/ml, or 500 mug/mg. Cells of B. megaterium did, however, accumulate 2,4,5-T passively to a level approximately twofold above the ambient concentration. Most of the accumulated compound was easily washed from the cells, but, of the firmly bound herbicide, about 0.5 mug/mg of cells (dry weight), nearly 60% by weight, was localized in the protoplast membrane. The foregoing results, obtained with a purified preparation of 2,4,5-T were also elicited by 2,4,5-T analytical standards. The extracted contaminants did not produce the results alone nor did they influence the results when present in combination with 2,4,5-T. [Abstract/Link to Full Text]

Ohtomo T, Murakoshi S, Sugiyama J, Kurata H
Detection of aflatoxin B1 in silkworm larvae attacked by an Aspergillus flavus isolate from a sericultural farm.
Appl Microbiol. 1975 Dec;30(6):1034-5.
Aflatoxin B(1) (0.05 muM per larvae) was detected in silkworm larvae artificially attacked by an Aspergillus flavus isolate from a sericultural farm in Japan. [Abstract/Link to Full Text]

Jurtshuk P, Marcucci OM, McQuitty DN
Tetramethyl-p-phenylenediamine oxidase reaction in Azotobacter vinelandii.
Appl Microbiol. 1975 Dec;30(6):951-8.
It was possible to quantitate the tetramethyl-p-phenylenediamine (TMPD) oxidase reaction in Azotobacter vinelandii strain O using turbidimetrically standarized resting cell suspensions. The Q(O2) value obtained for whole cell oxidation of ascorbate-TMPD appeared to reflect the full measure of the high respiratory oxidative capability usually exhibited by this genera of organisms. The Q(O2) value for the TMPD oxidase reaction ranged from 1,700 to 2,000 and this value was equivalent to that obtained for the oxidation of the growth substrate, e.g., acetate. The kinetic analyses for TMPD oxidation by whole cells was similar to that obtained for the "particulate" A. vinelandii electron transport particle, that fraction which TMPD oxidase activity is exclusively associated with. Under the conditions used, there appeared to be no permeability problems; TMPD (reduced by ascorbate) readily penetrated the cell and oxidized at a rate comparable to that of the growth substrate. This, however, was not true for the oxidation of another electron donor, 2,6-dichloroindophenol, whose whole cell Q(O2) values, under comparable conditions, were twofold lower. The TMPD oxidase activity in A. vinelandii whole cells was found to be affected by the physiological growth conditions, and resting cells obtained from cells grown on sucrose, either under nitrogen-fixing conditions or on nitrate as the combined nitrogen source, exhibited low TMPD oxidase rates. Such low TMPD oxidase rates were also noted for chemically induced pleomorphic A. vinelandii cells, which suggests that modified growth conditions can (i) alter the nature of the intracellular terminal oxidase formed (or induced), or (ii) alter surface permeability, depending upon the growth conditions used. Preliminary studies on the quantitative TMPD oxidation reaction in mutant whole cells of both Azotobacter and a well-known Mucor bacilliformis strain AY1, deficient in cytochrome oxidase activity, showed this assay can be very useful for detecting respiratory deficiencies in the metabolism of whole cells. [Abstract/Link to Full Text]


Relationship of lactate dehydrogenase specificity and growth rate to lactate metabolism by Selenomonas ruminantium.
Appl Microbiol. 1975 Dec;30(6):916-21.
A lactate-fermenting strain of Selenomonas ruminantium (HD4) and a lactatenonfermenting strain (GA192) were examined with respect to the stereoisomers of lactate formed during glucose fermentation, the stereoisomers of lactate fermented by HD4, and the characteristics of the lactate dehydrogenases of the strains. GA192 formed L-lactate and HD4 formed L-lactate and small amounts of D-lactate from glucose. HD4 fermended L- but not D-lactate. Both strains contain nicotinamide adenine dinucleotide (NAD)-specific lactate dehydrogenases, and no NAD-independent lactate oxidation was detected. Continuous cultures of both strains grown with limiting glucose produced mainly propionate and acetate and little lactate at dilution rates less than 0.4/h, with shifts to increasing amounts of lactate and less acetate and propionate as the dilution rate was increased from 0.4/h to approximately 1/h. [Abstract/Link to Full Text]

Dazzo FB, Hubbell DH
Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.
Appl Microbiol. 1975 Dec;30(6):1017-33.
Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin. [Abstract/Link to Full Text]

Marchand A, Dulos E
Kinetics of a bacterial culture growth: validity of the affinity rule in biological systems.
Appl Microbiol. 1975 Dec;30(6):994-1002.
The kinetic study of a process is usually performed by measuring a convenient intensive property, P, as a function of time. The "affinity rule" states that, when a given process takes place under different external constraints (e.g., different temperatures, pressures, pH values, etc.), the various P versus time curves are related by an affinity transformation parallel to the time axis: in other words the P versus log time curves are parallel and can be superimposed by translation. The validity of the rule has been extensively tested in chemical and physiochemical processes, but there is no evidence as yet that it extends to biological systems. The present paper shows that the rule is indeed valid for the kinetics of growth of an Escherichia coli culture at various temperatures and pH values. More extended experiments are necessary to prove or disprove the general validity of the rule in biological systems, but its practical interest is evident: whenever it is valid it will be possible, from a very small number of measurements, to predict the complete behavior of the system in a number of various external conditions [Abstract/Link to Full Text]

Bradshaw JG, Peeler JT, Twedt RM
Heat resistance of ileal loop reactive Bacillus cereus strains isolated from commercially canned food.
Appl Microbiol. 1975 Dec;30(6):943-5.
Sporeformers isolated from a commercially canned food were identified as Bacillus cereus, lactose-positive variants. The thermal resistance of spore crops produced from each of two representative cultures was determined in 0.067 M phosphate buffer at pH 7.0. The D121.1 values for one isolate were approximately 0.03 min (z = 9.9C), whereas the D121.1 values for the other isolate were 2.35 min (z = 7.9 C). Thermal inactivation results for heat-stressed isolates from each strain showed no significant alteration in heat resistance from that of the two parent spore crops. Both isolates were reactive when injected into the ligated rabbit ileum. [Abstract/Link to Full Text]


Factors influencing detection and enumeration of Pseudomonas aeruginosa by most-probable-number and membrane filtration techniques.
Appl Microbiol. 1975 Dec;30(6):935-42.
Most-probable-number (MPN) and membrane filtration (mF) techniques were evaluated with respect to selectivity, sensitivity, and efficiency in recovering Pseudomonas aeruginosa strains in hospital fluids and extramural water environments. Known numbers of cells of a naturally occurring strain of P. aeruginosa maintained in distilled water or cells subcultured on Standard Methods agar were added to test samples containing various types and levels of background microbial contamiants. Environmental samples containing unknown numbers of P. aeruginosa strains also were tested. Asparagine and acetamide broths were employed as presumptive media in MPN tests, and mPA and Pseudosel agars were used in mF assays. Statistical analyses of data showed the superiority and comparability of the asparagine-MPN and mPA-mF systems. Greater precision and accuracy were consistently obtained in either assay technique by the use of naturally occurring cells as test organisms. The type of filter and nature of diluents employed, as well as pH of assay media, were found to greatly influence both recovery and developemnt of characteristic colonial morphology in the mPA-mF system. [Abstract/Link to Full Text]

Bitton G, Fox JL, Strickland HG
Removal of algae from Florida lakes by magnetic filtration.
Appl Microbiol. 1975 Dec;30(6):905-8.
Magnetic filtration was used for the removal of algal populations present in five lakes located in the vicinity of Gainesville, Fla. It was found that the use of this technique enabled a good removal (94%) of algal cells from three lakes where the pH was around 7. The other two lakes, with a higher pH, displayed a lower removal. However, the treatment was greatly improved by lowering the pH from 9.5 to 6.5. [Abstract/Link to Full Text]

Reade AE, Gregory KF
High-temperature production of protein-enriched feed from cassava by fungi.
Appl Microbiol. 1975 Dec;30(6):897-904.
A simple, nonaseptic, low-cast process for the conversion of cassava, a starchy tropical root crop, into microbial protein for use as animal feed was sought. Screening tests culminated in the isolation of a thermotolerant, amylase-producing mold, designated I-21, which was identified as Aspergillus fumigatus. The optimum pH for protein synthesis was 3-5, but the optimum temperature was less than the desired temperature (larger than or equal to 45 C) required for a nonaseptic fermentation. A. fumigatus I-21 and its asporogenous mutant I-21A grew equally well in a medium prepared from whole cassava roots with a mean protein doubling time at 45 C and pH 3.5 of 3.5 h. In batch culture, approximately 4% carbohydrate, supplied as whole cassava, could be feremented in 20 h, giving a final yield of 24 g of dry product, containing 36.9% crude protein, per liter. The conversion of carbohydrate used to crude protein was 22.1%. When determined as amino acids, the protein content of the product, which contained cassava bark and other unfermented residues, was 27.1%. With urea as the nitrogen source, no pH control was necessary. Preliminary data indicated that medium prepared from whole cassava roots was inhibitory to the mold unless the cassava pulp was heated to 70 C immediately after being ground. Heating to 70 C was required to gelatinize the starch and permit its complete utilization. [Abstract/Link to Full Text]

Buchanan RL, Ayres JC
Effect of initial pH on aflatoxin production.
Appl Microbiol. 1975 Dec;30(6):1050-1.
The effect of initial pH on aflatoxin production by Aspergillus parasiticus NRRL 2999 was examined in a semisynthetic medium. Maximal growth, aflatoxin production, and aflatoxin production per unit of growth occurred at initial pH levels of 5.0, 6.0, and 7.0 respectively. Initial pH levels less than pH 6.0 favored production of the B toxins, whereas levels greater than pH 6.0 favored production of the G toxins. [Abstract/Link to Full Text]

Manning HL
New medium for isolating iron-oxidizing and heterotrophic acidophilic bacteria from acid mine drainage.
Appl Microbiol. 1975 Dec;30(6):1010-6.
A new solid medium is described for growing iron and heterotrophic bacteria from acid mine drainage (AMD). Examination of AMD from five states revealed several kinds of colonies of iron-oxidizing bacteria: (i) smooth, (ii) smooth with secondary growth sectors or branching, (iii) star-shaped, (iv) radiating lobe, and (v) flat-rough. All AMD samples yielded whitish colonies that could not use ferrous iron, sulfur, or hydrogen, nor could they grow on nutrient agar, brain heart infusion agar, or Trypticase soy agar. Glucose and sucrose supported growth if the sugar-salts medium was at pH 3.0. The new iron medium has several advantages over others: (i) easy preparation, (ii) rapid growth, (iii) larger colonies, (iv) differentiation of colony morphology, and (v) detection of a new group of heterotrophic acidophilic bacteria. [Abstract/Link to Full Text]

---

Recent Articles in Bacteriological Reviews

---

Schwesinger MD
Additive recombination in bacteria.
Bacteriol Rev. 1977 Dec;41(4):872-902. [Abstract/Link to Full Text]

Saier MH
Bacterial phosphoenolpyruvate: sugar phosphotransferase systems: structural, functional, and evolutionary interrelationships.
Bacteriol Rev. 1977 Dec;41(4):856-71. [Abstract/Link to Full Text]

Huang AS
Viral pathogenesis and molecular biology.
Bacteriol Rev. 1977 Dec;41(4):811-21. [Abstract/Link to Full Text]

Maggon KK, Gupta SK, Venkitasubramanian TA
Biosynthesis of aflatoxins.
Bacteriol Rev. 1977 Dec;41(4):822-55. [Abstract/Link to Full Text]


Energy Conservation in Chemotrophic Anaerobic Bacteria.
Bacteriol Rev. 1977 Sep;41(3):809.
[This corrects the article on p. 100 in vol. 41.]. [Abstract/Link to Full Text]

Doi RH
Role of ribonucleic acid polymerase in gene selection in procaryotes.
Bacteriol Rev. 1977 Sep;41(3):568-94. [Abstract/Link to Full Text]

Whittenbury R, Dow CS
Morphogenesis and differentiation in Rhodomicrobium vannielii and other budding and prosthecate bacteria.
Bacteriol Rev. 1977 Sep;41(3):754-808. [Abstract/Link to Full Text]

Priest FG
Extracellular enzyme synthesis in the genus Bacillus.
Bacteriol Rev. 1977 Sep;41(3):711-53. [Abstract/Link to Full Text]

Orskov I, Orskov F, Jann B, Jann K
Serology, chemistry, and genetics of O and K antigens of Escherichia coli.
Bacteriol Rev. 1977 Sep;41(3):667-710. [Abstract/Link to Full Text]

Morgan EM, Rapp F
Measles virus and its associated diseases.
Bacteriol Rev. 1977 Sep;41(3):636-66. [Abstract/Link to Full Text]

Hopwood DA, Merrick MJ
Genetics of antibiotic production.
Bacteriol Rev. 1977 Sep;41(3):595-635. [Abstract/Link to Full Text]

Friedman RM
Antiviral activity of interferons.
Bacteriol Rev. 1977 Sep;41(3):543-67. [Abstract/Link to Full Text]

Donaldson DM, Tew JG
beta-Lysin of platelet origin.
Bacteriol Rev. 1977 Jun;41(2):501-13. [Abstract/Link to Full Text]

Smith H
Microbial surfaces in relation to pathogenicity.
Bacteriol Rev. 1977 Jun;41(2):475-500. [Abstract/Link to Full Text]

Zeikus JG
The biology of methanogenic bacteria.
Bacteriol Rev. 1977 Jun;41(2):514-41. [Abstract/Link to Full Text]

Smith AJ, Hoare DS
Specialist phototrophs, lithotrophs, and methylotrophs: a unity among a diversity of procaryotes?
Bacteriol Rev. 1977 Jun;41(2):419-48. [Abstract/Link to Full Text]

Kaneda T
Fatty acids of the genus Bacillus: an example of branched-chain preference.
Bacteriol Rev. 1977 Jun;41(2):391-418. [Abstract/Link to Full Text]

Langworth BF
Fusobacterium necrophorum: its characteristics and role as an animal pathogen.
Bacteriol Rev. 1977 Jun;41(2):373-90. [Abstract/Link to Full Text]

Katz E, Demain AL
The peptide antibiotics of Bacillus: chemistry, biogenesis, and possible functions.
Bacteriol Rev. 1977 Jun;41(2):449-74. [Abstract/Link to Full Text]

Thauer RK, Jungermann K, Decker K
Energy conservation in chemotrophic anaerobic bacteria.
Bacteriol Rev. 1977 Mar;41(1):100-80. [Abstract/Link to Full Text]

Holt PG, Keast D
Environmentally induced changes in immunological function: acute and chronic effects of inhalation of tobacco smoke and other atmospheric contaminants in man and experimental animals.
Bacteriol Rev. 1977 Mar;41(1):205-16. [Abstract/Link to Full Text]

Canale-Parola E
Physiology and evolution of spirochetes.
Bacteriol Rev. 1977 Mar;41(1):181-204. [Abstract/Link to Full Text]

Haddock BA, Jones CW
Bacterial respiration.
Bacteriol Rev. 1977 Mar;41(1):47-99. [Abstract/Link to Full Text]

Barksdale L, Kim KS
Mycobacterium.
Bacteriol Rev. 1977 Mar;41(1):217-372. [Abstract/Link to Full Text]

Swings J, De Ley J
The biology of Zymomonas.
Bacteriol Rev. 1977 Mar;41(1):1-46. [Abstract/Link to Full Text]


Degradation of Purines and Pyrimidines by Microorganisms.
Bacteriol Rev. 1976 Dec;40(4):963.
[This corrects the article on p. 403 in vol. 40.]. [Abstract/Link to Full Text]

Brown AD
Microbial water stress.
Bacteriol Rev. 1976 Dec;40(4):803-46. [Abstract/Link to Full Text]

Witkin EM
Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli.
Bacteriol Rev. 1976 Dec;40(4):869-907. [Abstract/Link to Full Text]

Wood WB, Revel HR
The genome of bacteriophage T4.
Bacteriol Rev. 1976 Dec;40(4):847-68. [Abstract/Link to Full Text]

Duckworth DH
"Who discovered bacteriophage?".
Bacteriol Rev. 1976 Dec;40(4):793-802. [Abstract/Link to Full Text]

---

Recent Articles in BMC Immunology

---

Frommhold D, Mannigel I, Schymeinsky J, Mocsai A, Poeschl J, Walzog B, Sperandio M
Spleen tyrosine kinase Syk is critical for sustained leukocyte adhesion during inflammation in vivo.
BMC Immunol. 2007 Nov 28;8(1):31.
ABSTRACT: BACKGROUND: During inflammation, beta2-integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk. RESULTS: We investigated leukocyte adhesion and rolling in cremaster muscle venules before and during stimulation with fMLP using mice with a Syk-/- hematopoietic system. In unstimulated venules, Syk-/- leukocytes adhered less efficiently than control leukocytes while rolling was similar between Syk-/- and control leukocytes. During fMLP superfusion, control mice showed significantly increased adhesion accompanied by reduced rolling. For Syk-/- leukocytes, an increase in adhesion with a concomitant decrease in rolling was only observed during the first three minutes during fMLP stimulation, but not at later time points. We also investigated leukocyte spreading against the vessel wall during fMLP stimulation and found a significant impairment of spreading for Syk-/- leukocytes. Additional in vitro experiments revealed that the adhesion and spreading defect seen in Syk-/- chimeric mice was due to compromised beta2-integrin-mediated outside-in signaling. CONCLUSIONS: We provide substantial evidence for an important role of Syk in mediating beta2-integrin dependent outside-in signaling leading to sustained leukocyte adhesion and spreading during the inflammatory response in vivo. [Abstract/Link to Full Text]

Ramirez-Pliego O, Escobar-Zarate DL, Rivera-Martinez GM, Cervantes-Badillo MG, Esquivel-Guadarrama FR, Rosas-Salgado G, Rosenstein Y, Santana MA
CD43 signals induce Type One lineage commitment of human CD4+ T cells.
BMC Immunol. 2007 Nov 23;8(1):30.
ABSTRACT: BACKGROUND: The activation and effector phenotype of T cells depend on the strength of the interaction of the TcR with its cognate antigen and additional signals provided by cytokines and by co-receptors. Lymphocytes sense both the presence of an antigen and also clues from antigen-presenting cells, which dictate the requisite response. CD43 is one of the most abundant molecules on the surface of T cells; it mediates its own signalling events and cooperates with those mediated by the T cell receptor in T cell priming. We have examined the role of CD43 signals on the effector phenotype of adult CD4+ and CD8+ human T cells, both alone and in the presence of signals from the TcR. RESULTS: CD43 signals direct the expression of IFNg in human T cells. In freshly isolated CD4+ T cells, CD43 signals potentiated expression of the IFNg gene induced by TcR activation; this was not seen in CD8+ T cells. In effector cells, CD43 signals alone induced the expression of the IFNg gene in CD4+ T cells and to a lesser extent in CD8+ cells. The combined signals from CD43 and the TcR increased the transcription of the T-bet gene in CD4+ T cells and inhibited the transcription of the GATA-3 gene in both populations of T cells, thus predisposing CD4+ T cells to commitment to the T1 lineage. In support of this, CD43 signals induced a transient membrane expression of the high-affinity chains of the receptors for IL-12 and IFNg in CD4+ T cells. CD43 and TcR signals also cooperated with those of IL-12 in the induction of IFNg expression. Moreover, CD43 signals induced the co-clustering of IFNgR and the TcR and cooperated with TcR and IL-12 signals, triggering a co-capping of both receptors in CD4+ populations, a phenomenon that has been associated with a T1 commitment. CONCLUSIONS: Our results suggest a key role for CD43 signals in the differentiation of human CD4+ T cells into a T1 pattern. [Abstract/Link to Full Text]

Elshal MF, Khan SS, Raghavachari N, Takahashi Y, Barb J, Bailey JJ, Munson PJ, Solomon MA, Danner RL, McCoy JP
A unique population of effector memory lymphocytes identified by CD146 having a distinct immunophenotypic and genomic profile.
BMC Immunol. 2007 Nov 13;8(1):29.
ABSTRACT: BACKGROUND: CD146 is a well described homotypic adhesion molecule found on endothelial cells and a limited number of other cell types. In cells from the peripheral circulation, CD146 has also been reported to be on activated lymphocytes in vitro and in vivo. The function associated with CD146 expression on lymphoid cells is unknown and very little information is available concerning the nature of CD146+ lymphocytes. In the current study, lymphocytes from healthy donors were characterized based upon the presence or absence of CD146 expression. RESULTS: CD146 was expressed on a low percentage of circulating T lymphocytes, B lymphocytes, and NK cells in healthy individuals. CD146 expression can be induced and upregulated in vitro on both B cells and T cells , but does not correlate with the expression of other markers of T cell activation. CD146 positive T cells do not represent clonal expansions as determined with the use of antiVbeta reagents. Data suggest that CD146 positive cells have enhanced adherence to endothelial monolayers in vitro. Gene profiling and immunophenotyping studies between CD146+ and CD146- T cells revealed several striking genotypic distinctions such as the upregulation of IL-8 and phenotypic differences including the paucity of CCR7 and CD45RA among CD146 positive T cells, consistent with effector memory function. A number of genes involved in cell adhesion, signal transduction, and cell communication are dramatically upregulated in CD146+ T cells compared to CD146- T cells. CONCLUSIONS: CD146 appears to identify small, unique populations of T as well as B lymphocytes in the circulation. The T cells have immunophenotypic characteristics of effector memory lymphocytes. The characteristics of these CD146+ lymphocytes in the circulation, together with the known functions in cell adhesion of CD146 on endothelial cells, suggests that these lymphocytes may represent a small subpopulation of cells primed to adhere to the endothelium and possibly extravasate to sites of inflammation. [Abstract/Link to Full Text]

Cerutti E, Campagnoli MF, Ferretti M, Garelli E, Crescenzio N, Rosolen A, Chiocchetti A, Lenardo MJ, Ramenghi U, Dianzani U
Co-inherited mutations of Fas and caspase-10 in development of the autoimmune lymphoproliferative syndrome.
BMC Immunol. 2007 Nov 13;8(1):28.
ABSTRACT: BACKGROUND: Autoimmune lymphoproliferative syndrome (ALPS) is a rare inherited disorder characterized by defective function of Fas, autoimmune manifestations that predominantly involve blood cells, polyclonal accumulation of lymphocytes in the spleen and lymph nodes with lymphoadenomegaly and/or splenomegaly, and expansion of TCRalphabeta+ CD4/CD8 double-negative (DN) T cells in the peripheral blood. Most frequently, it is due to Fas gene mutations, causing ALPS type Ia (ALPS-Ia). However, other mutations, namely of the FasL gene (ALPS-Ib) and the caspase-10 gene (ALPS-II) are occasionally detected, whereas some patients do not present any known mutations (ALPS-III). Recently, mutations of the NRAS gene have been suggested to cause ALPS-IV. RESULTS: This work reports two patients that are combined heterozygous for single nucleotide substitutions in the Fas and caspase-10 genes. The first patient carried a splice site defect suppressing allele expression in the Fas gene and the P501L substitution in caspase-10. The second had a mutation causing a premature stop codon (Q47X) in the Fas gene and the Y446C substitution in caspase-10. Fas expression was reduced and caspase-10 activity was decreased in both patients. In both patients, the mutations were inherited from distinct healthy parents. CONCLUSIONS: These data strongly suggest that co-transmission of these mutation was responsible for ALPS. [Abstract/Link to Full Text]

Genser B, Cooper PJ, Yazdanbaksh M, Barreto ML, Rodrigues LC
A guide to modern statistical analysis of immunological data.
BMC Immunol. 2007 Oct 26;8(1):27.
ABSTRACT: BACKGROUND: The number of subjects that can be recruited in immunological studies and the number of immunological parameters that can be measured has increased rapidly over the past decade and is likely to continue to expand. Large and complex immunological datasets can now be used to investigate complex scientific questions but to make the most of the potential in such data and to get the right answers- sophisticated statistical approaches are necessary. Such approaches are used in many other scientific disciplines, but immunological studies on the whole still use simple statistical techniques for data analysis. RESULTS: The paper provides an overview of the range of statistical methods that can be used to answer different immunological study questions. We discuss specific aspects of immunological studies and give examples of typical scientific questions related to immunological data. We review classical bivariate and multivariate statistical techniques (factor analysis, cluster analysis, discriminant analysis) and more advanced methods aimed to explore causal relationships (path analysis/structural equation modelling) and illustrate their application to immunological data. We show the main features of each method, the type of study question they can answer, the type of data they can be applied to, the assumptions required for each method and the software that can be used. CONCLUSIONS: This paper will help the immunologist to choose the correct statistical approach for a particular research question. [Abstract/Link to Full Text]

Birmachu W, Gleason RM, Bulbulian BJ, Riter CL, Vasilakos JP, Lipson KE, Nikolsky Y
Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists.
BMC Immunol. 2007 Oct 12;8(1):26.
ABSTRACT: BACKGROUND: Plasmacytoid Dendritic Cells (pDC) comprise approximately 0.2 to 0.8% of the blood mononuclear cells and are the primary type 1 interferon (IFN), producing cells, secreting high levels of IFN in response to viral infections. Plasmacytoid dendritic cells express predominantly TLRs 7 & 9, making them responsive to ssRNA and CpG DNA. The objective of this study was to evaluate the molecular and cellular processes altered upon stimulation of pDC with synthetic TLR 7 and TLR 7/8 agonists. To this end, we evaluated changes in global gene expression upon stimulation of 99.9% pure human pDC with the TLR7 selective agonists 3M-852A, and the TLR7/8 agonist 3M-011. RESULTS: Global gene expression was evaluated using the Affymetrix U133A GeneChip(R) and selected genes were confirmed using real time TaqMan(R) RTPCR. The gene expression profiles of the two agonists were similar indicating that changes in gene expression were solely due to stimulation through TLR7. Type 1 interferons were among the highest induced genes and included IFNB and multiple IFNa subtypes, IFNA2, A5, A6, A8, A1/13, A10, A14, A16, A17, A21. A large number of chemokines and co-stimulatory molecules as well as the chemokine receptor CCR7 were increased in expression indicating maturation and change in the migratory ability of pDC. Induction of an antiviral state was shown by the expression of several IFN-inducible genes with known anti-viral activity. Further analysis of the data using the pathway analysis tool MetaCore gave insight into molecular and cellular processes impacted. The analysis revealed transcription networks that show increased expression of signaling components in TLR7 and TLR3 pathways, and the cytosolic anti-viral pathway regulated by RIG1 and MDA5, suggestive of optimization of an antiviral state targeted towards RNA viruses. The analysis also revealed increased expression of a network of genes important for protein ISGylation as well as an anti-apoptotic and pro-survival gene expression program. CONCLUSION: Thus this study demonstrates that as early as 4 hr post stimulation, synthetic TLR7 agonists induce a complex transcription network responsible for activating pDC for innate anti-viral immune responses with optimized responses towards RNA viruses, increased co-stimulatory capacity, and increased survival. [Abstract/Link to Full Text]

Costa IG, Roepcke S, Schliep A
Gene expression trees in lymphoid development.
BMC Immunol. 2007 Oct 9;8(1):25.
ABSTRACT: BACKGROUND: The regulatory processes that govern cell proliferation and differentiation are central to developmental biology. Particularly well studied in this respect is the lymphoid system due to its importance for basic biology and for clinical applications. Gene expression measured in lymphoid cells in several distinguishable developmental stages helps in the elucidation of underlying molecular processes, which change gradually over time and lock cells in either the B cell, T cell or Natural Killer cell lineages. Large-scale analysis of these gene expression trees requires computational support for tasks ranging from visualization, querying, and finding clusters of similar genes, to answering detailed questions about the functional roles of individual genes. RESULTS: We present the first statistical framework designed to analyze gene expression data as it is collected in the course of lymphoid development through clusters of co-expressed genes and additional heterogeneous data. We introduce dependence trees for continuous variates, which model the inherent dependencies during the differentiation process naturally as gene expression trees. Several trees are combined in a mixture model to allow inference of potentially overlapping clusters of co-expressed genes. Additionally, we predict microRNA targets. CONCLUSIONS: Computational results for several data sets from the lymphoid system demonstrate the relevance of our framework. We recover well-known biological facts and identify promising novel regulatory elements of genes and their functional assignments. The implementation of our method (licensed under the GPL) is available at http://algorithmics.molgen.mpg.de/Supplements/ExpLym/. [Abstract/Link to Full Text]

Ganguly N, Giang PH, Basu SK, Mir FA, Siddiqui I, Sharma P
Mycobacterium tuberculosis 6-kDa early secreted antigenic target (ESAT-6) protein downregulates lipopolysaccharide induced c-myc expression by modulating the extracellular signal regulated kinases 1/2.
BMC Immunol. 2007;824.
BACKGROUND: Mycobacterium tuberculosis (Mtb) causes death of 2-3 million people every year. The persistence of the pathogenic mycobacteria inside the macrophage occurs through modulation of host cell signaling which allows them, unlike the other non-pathogenic species, to survive inside the host. The secretory proteins of M. tuberculosis have gained attention in recent years both as vaccine candidates and diagnostic tools; they target the immune system and trigger a putatively protective response; however, they may also be involved in the clinical symptoms of the disease. RESULTS: Our studies showed that RD-1-encoded secretory protein ESAT-6 is involved in modulation of the mitogen-activated protein (MAP) kinase-signaling pathway inside the macrophage. ESAT-6 induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the cytoplasm but not in the nucleus, which normally is the case for MAP kinases. ESAT-6 also antagonized LPS-induced ERK1/2 phosphorylation in the nucleus. Stimulation of cells by ESAT-6 along with sodium orthovanadate (a tyrosine phosphatase inhibitor) restored phosphorylation of ERK1/2 in the nucleus, suggesting active dephosphorylation of ERK1/2 by some putative phosphatase(s) in the nucleus. Further, ESAT-6 was found to down regulate the expression of LPS-inducible gene c-myc in an ERK1/2-dependent manner. CONCLUSION: This study showed the effect of secretory proteins of M. tuberculosis in the modulation of macrophage signaling pathways particularly ERK1/2 MAP kinase pathway. This modulation appears to be achieved by limiting the ERK1/2 activation in the nucleus which ultimately affects the macrophage gene expression. This could be a mechanism by which secretory proteins of Mtb might modulate gene expression inside the macrophages. [Abstract/Link to Full Text]

Sansome GM, Young AR, Meeusen EN, Bischof RJ
Production of monoclonal antibodies reactive with ovine eosinophils.
BMC Immunol. 2007;823.
BACKGROUND: There is strong evidence implicating eosinophils in host defence against parasites as well as allergic disease pathologies. However, a lack of reagents such as monoclonal antibodies (mAbs) specific for eosinophils has made it difficult to confirm the functional role of eosinophils in such disease conditions. Using an established mammary model of allergic inflammation in sheep, large numbers of inflammatory cells enriched for eosinophils were collected from parasite-stimulated mammary glands and used for the generation of mAbs against ovine eosinophils. RESULTS: A panel of mAbs was raised against ovine eosinophils of which two were shown to be highly specific for eosinophils. The reactivity of mAbs 3.252 and 1.2 identified eosinophils from various cell and tissue preparations with no detectable reactivity on cells of myeloid or lymphoid lineage, tissue mast cells, dendritic cells, epithelial cells or other connective tissues. Two other mAbs generated in this study (mAbs 4.4 and 4.10) were found to have reactivity for both eosinophils and neutrophils. CONCLUSION: This study describes the production of new reagents to identify eosinophils (as well as granulocytes) in sheep that will be useful in studying the role of eosinophils in disease pathologies in parasite and allergy models. [Abstract/Link to Full Text]

Tulone C, Uchiyama Y, Novelli M, Grosvenor N, Saftig P, Chain BM
Haematopoietic development and immunological function in the absence of cathepsin D.
BMC Immunol. 2007;822.
BACKGROUND: Cathepsin D is a well-characterized aspartic protease expressed ubiquitously in lysosomes. Cathepsin D deficiency is associated with a spectrum of pathologies leading ultimately to death. Cathepsin D is expressed at high levels in many cells of the immune system, but its role in immune function is not well understood. This study examines the reconstitution and function of the immune system in the absence of cathepsin D, using bone marrow radiation chimaeras in which all haematopoietic cells are derived from cathepsin D deficient mice. RESULTS: Cathepsin D deficient bone marrow cells fully reconstitute the major cellular components of both the adaptive and innate immune systems. Spleen cells from cathepsin D deficient chimaeric mice contained an increased number of autofluorescent granules characteristic of lipofuscin positive lysosomal storage diseases. Biochemical and ultrastructural changes in cathepsin D deficient spleen are consistent with increased autolysosomal activity. Chimaeric mice were immunised with either soluble (dinitrophenylated bovine gamma globulin) or particulate (sheep red blood cells) antigens. Both antigens induced equivalent immune responses in wild type or cathepsin D deficient chimaeras. CONCLUSION: All the parameters of haematopoietic reconstitution and adaptive immunity which were measured in this study were found to be normal in the absence of cathepsin D, even though cathepsin D deficiency leads to dysregulation of lysosomal function. [Abstract/Link to Full Text]

Goodell V, dela Rosa C, Slota M, MacLeod B, Disis ML
Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses.
BMC Immunol. 2007;821.
BACKGROUND: Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation) and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt), and cytomegalovirus (CMV) antigens. These antigens were determined to be low (HER-2/neu), moderate (tt), and robustly (CMV) immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response. RESULTS: Linear regression analysis indicates that both stimulation index (SI) (p = 0.011) and IFN-gamma secreting precursor frequency (p < 0.001) are significant indicators of antigen specific immunity. ROC curves plotted to assess the performance of tritiated thymidine incorporation and the ELISPOT assay indicate that SI is a significant indicator of low T cell response to the HER-2/neu vaccine (p = 0.05), and of moderate and robust responses to tt (p = 0.01) and CMV (p = 0.016), respectively. IFN-gamma precursor frequency is a significant indicator of a robust T cell response to CMV (p = 0.03), but not of moderate tt (p = 0.09), or low HER-2/neu (p = 0.09) T cell responses. CONCLUSION: These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients. [Abstract/Link to Full Text]

Carrol ED, Salway F, Pepper SD, Saunders E, Mankhambo LA, Ollier WE, Hart CA, Day P
Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis.
BMC Immunol. 2007;820.
BACKGROUND: The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use.The aim of this study was to modify the PAXgene Blood RNA System kit protocol for application to small, sick children, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression.Aliquots of 0.86 mL PAXgene reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an in-house TaqMan assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes (HPRT1, YWHAZ and GAPDH) were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease. RESULTS: Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors (ICAM: 3.56 vs 4.41, p = 0.04, and IL-6: 2.16 vs 6.73, p = 0.02). CONCLUSION: We have successfully modified the PAXgene blood collection system for use in small children and demonstrated preservation of RNA integrity and successful quantitative real-time PCR analysis. [Abstract/Link to Full Text]

Vinderola G, Matar C, Perdigón G
Milk fermentation products of L. helveticus R389 activate calcineurin as a signal to promote gut mucosal immunity.
BMC Immunol. 2007;819.
BACKGROUND: Fermented milks containing probiotic bacteria are a way of delivering bioactive constituents to targets in the gastrointestinal tract. We reported previously that the fermentation of milk at constant pH 6 by L. helveticus R389 increased its content of peptide fractions, and the oral administration of the non-bacterial fraction (FMSpH6) to mice increased total secretory IgA in the intestinal lumen and enhanced the number of IgA and various cytokines producing cells as well as the secretion of IL-6 by small intestine epithelial cells. We also demonstrated that this FMSpH6 was effective for the prevention of Salmonella typhimurium infection in mice. In this work, we studied in mice the impact of the oral administration of the supernatant of milk fermented by L. helveticus R389 on the gut physiology by measuring parameters such as calcium channels and E-cadherin expression, the activation of the biological signal calcineurin and mast and goblet cells, as a way to determine some mechanisms involved in the immunomodulating effects of the milk fermentation products, observed in previous studies. We analyzed the impact of the supernatant of milk fermented by L. helveticus R389 at pH6-controlled on the expression of calcineurin and on the reinforcement of the ephitelial barrier, measuring parameters such as calcium channels and E-cadherin expression and in the reinforcement of the non-specific immunity determining mast cells and goblet cells associated to the gut. RESULTS: We observed an enhanced expression of TRPV6 channels in the duodenum, indicating an improved capacity for dietary Ca2+ uptake. We demonstrated an enhanced expression of calcineurin in the small intestine, able to upregulate immune parameters such as IL-2 and TNF production, with an increase in the number of these cytokines secreting cells. We determined an increase in the number of mucosal mast cells and goblet cells, which would mean an improved state of mucosal surveillance at sites of infection. CONCLUSION: The oral administration of the supernatant of milk fermented by L. helveticus R389 enhanced the gut mucosal immunity by improving the mechanisms that reinforce the epithelial and non-specific barriers and the gut functioning at sites of infection, with an improvement in the expression of the enzyme calcineurin, an important signal in the network that activates the gut immune system. The results of this work contribute to revealing the mechanisms underlying the immunomodulation of the gut immune function by fermented milks with probiotic bacteria. [Abstract/Link to Full Text]

Nitsche A, Zhang M, Clauss T, Siegert W, Brune K, Pahl A
Cytokine profiles of cord and adult blood leukocytes: differences in expression are due to differences in expression and activation of transcription factors.
BMC Immunol. 2007;818.
BACKGROUND: Stem cell transplantation as therapy for hematological disorders is often hampered by severe graft-versus-host-disease. This may be reduced by umbilical cord blood transplantation, an effect that has been attributed to qualitative differences between neonatal and adult T cells. We compared levels of secreted proteins and cytokine mRNA induced in cord blood leukocytes (CBL) and adult blood leukocytes (ABL) by various stimuli. RESULTS: While interleukin-2 (IL-2) levels were similar in CBL and ABL, there was less induction of the Th1 cytokine interferon-gamma in CBL. Production of the Th2 cytokines IL-4, IL-5, and IL-13 and the hematopoietic cytokine IL-3 was much lower in CBL versus ABL after T-cell receptor-mediated stimulation, whereas production of GM-CSF was comparable in the 2 cell types. The lower levels of Th1 and Th2 cytokines were maintained in CBL during a 4-day time-course study, while after 12 hours IL-3 and GM-CSF reached in CBL levels similar to those in ABL. For all cytokines except IFN gamma, the IC50 values for inhibition by cyclosporin A were similar in ABL and CBL. In contrast, there was less expression and activation of transcription factors in CBL. Activation of NF-kappaB by TPA/ionomycin was detected in ABL but not CBL. Furthermore, there was less expression of the Th subset-specific transcription factors T-bet and c-maf in CBL versus ABL, whereas GATA-3 expression was similar. Expression of T-bet and c-maf correlated with expression of the Th1 and Th2 cytokines, respectively. Time course experiments revealed that T-bet expression was stimulated in both cell types, whereas c-maf and GATA-3 were induced only in ABL. CONCLUSION: The diminished capability of CBL to synthesize cytokines is probably due to decreased activation of NF-kappaB, whereas differences in Th subsets are due to differences in regulation of Th lineage-specific transcriptions factors. We propose that the reduced incidence and severity of GvHD after allogeneic transplantation of umbilical CB cells is due to lesser activation of specific transcription factors and a subsequent reduction in production of certain cytokines. [Abstract/Link to Full Text]

Saban R, Simpson C, Davis CA, Dozmorov I, Maier J, Fowler B, Ihnat MA, Hurst RE, Wershil BK, Saban MR
Transcription factor network downstream of protease activated receptors (PARs) modulating mouse bladder inflammation.
BMC Immunol. 2007;817.
BACKGROUND: All four PARs are present in the urinary bladder, and their expression is altered during inflammation. In order to search for therapeutic targets other than the receptors themselves, we set forth to determine TFs downstream of PAR activation in the C57BL/6 urinary bladders. METHODS: For this purpose, we used a protein/DNA combo array containing 345 different TF consensus sequences. Next, the TF selected was validated by EMSA and IHC. As mast cells seem to play a fundamental role in bladder inflammation, we determined whether c-kit receptor deficient (Kit w/Kit w-v) mice have an abrogated response to PAR stimulation. Finally, TFEB antibody was used for CHIP/Q-PCR assay and revealed up-regulation of genes known to be downstream of TFEB. RESULTS: TFEB, a member of the MiTF family of basic helix-loop-helix leucine zipper, was the only TF commonly up-regulated by all PAR-APs. IHC results confirm a correlation between inflammation and TFEB expression in C57BL/6 mice. In contrast, Kit w/Kit w-v mice did not exhibit inflammation in response to PAR activation. EMSA results confirmed the increased TFEB binding activity in C57BL/6 but not in Kit w/Kit w-v mice. CONCLUSION: This is the first report describing the increased expression of TFEB in bladder inflammation in response to PAR activation. As TFEB belongs to a family of TFs essential for mast cell survival, our findings suggest that this molecule may influence the participation of mast cells in PAR-mediated inflammation and that targeting TFEB/MiTF activity may be a novel approach for the treatment of bladder inflammatory disorders. [Abstract/Link to Full Text]

Alvarez M, Casadevall A
Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages.
BMC Immunol. 2007;816.
BACKGROUND: The interaction between macrophages and Cryptococcus neoformans (Cn) is critical for containing dissemination of this pathogenic yeast. However, Cn can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. Both events result in live extracellular yeasts capable of reproducing and disseminating in the extracellular milieu. Another method of exiting the intracellular confines of cells is through host cell-to-cell transfer of the pathogen, and this commonly occurs with the human immuno-deficiency virus (HIV) and CD4+ T cells and macrophages. In this report we have used time-lapse imaging to determine if this occurs with Cn. RESULTS: Live imaging of Cryptococcus neoformans interactions with murine macrophages revealed cell-to-cell spread of yeast cells from infected donor cells to uninfected cells. Although this phenomenon was relatively rare its occurrence documents a new capacity for this pathogen to infect adjacent cells without exiting the intracellular space. Cell-to-cell spread appeared to be an actin-dependent process. In addition, we noted that cryptococcal phagosomal extrusion was followed by the formation of massive vacuoles suggesting that intracellular residence is accompanied by long lasting damage to host cells. CONCLUSION: C. neoformans can escape the intracellular confines of macrophages in an actin dependent manner by cell-to-cell transfer of the yeast leading to infection of adjacent cells. In addition, complete extrusion of internalized Cn cells can lead to the formation of a massive vacuole which may be a sign of damage to the host macrophage. These observations document new outcomes for the interaction of C. neoformans with host cells that provide precedents for cell biological effects that may contribute to the pathogenesis of cryptococcal infections. [Abstract/Link to Full Text]

Ma H, Croudace JE, Lammas DA, May RC
Direct cell-to-cell spread of a pathogenic yeast.
BMC Immunol. 2007;815.
BACKGROUND: Cryptococcosis, a fatal fungal infection of the central nervous system, is one of the major killers of AIDS patients and other immunocompromised hosts. The causative agent, Cryptococcus neoformans, has a remarkable ability to 'hide' and proliferate within phagocytic cells of the human immune system. This intracellular phase is thought to underlie the ability of the pathogen to remain latent for long periods of time within infected individuals. RESULTS: We now report that Cryptococcus is able to undergo 'lateral transfer' between phagocytes, moving directly from infected to uninfected macrophages. This novel process was observed in both C. neoformans serotypes (A and D) and occurs in both immortalised cell lines and in primary human macrophages. Lateral transfer is independent of the initial route of uptake, since both serum-opsonised and antibody-opsonised C. neoformans are able to undergo direct cell-to-cell transfer. CONCLUSION: We provide the first evidence for lateral transfer of a human fungal pathogen. This rare event may occur repeatedly during latent cryptococcal infections, thereby allowing the pathogen to remain concealed from the immune system and protecting it from exposure to antifungal agents. [Abstract/Link to Full Text]

Walch M, Latinovic-Golic S, Velic A, Sundstrom H, Dumrese C, Wagner CA, Groscurth P, Ziegler U
Perforin enhances the granulysin-induced lysis of Listeria innocua in human dendritic cells.
BMC Immunol. 2007;814.
BACKGROUND: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play an essential role in the host defence against intracellular pathogens such as Listeria, and Mycobacteria. The key mediator of bacteria-directed cytotoxicity is granulysin, a 9 kDa protein stored in cytolytic granules together with perforin and granzymes. Granulysin binds to cell membranes and is subsequently taken up via a lipid raft-associated mechanism. In dendritic cells (DC) granulysin is further transferred via early endosomes to L. innocua-containing phagosomes were bacteriolysis is induced. In the present study we analysed the role of perforin in granulysin-induced intracellular bacteriolysis in DC. RESULTS: We found granulysin-induced lysis of intracellular Listeria significantly increased when perforin was simultaneously present. In pulse-chase experiments enhanced bacteriolysis was observed when perforin was added up to 25 minutes after loading the cells with granulysin demonstrating no ultimate need for simultaneous uptake of granulysin and perforin. The perforin concentration sufficient to enhance granulysin-induced intracellular bacteriolysis did not cause permanent membrane pores in Listeria-challenged DC as shown by dye exclusion test and LDH release. This was in contrast to non challenged DC that were more susceptible to perforin lysis. For Listeria-challenged DC, there was clear evidence for an Ca2+ influx in response to sublytic perforin demonstrating a short-lived change in the plasma membrane permeability. Perforin treatment did not affect granulysin binding, initial uptake or intracellular trafficking to early endosomes. However, enhanced colocalization of granulysin with listerial DNA in presence of perforin was found by confocal laser scanning microscopy. CONCLUSION: The results provide evidence that perforin increases granulysin-mediated killing of intracellular Listeria by enhanced phagosome-endosome fusion triggered by a transient Ca2+ flux. [Abstract/Link to Full Text]

Moebius JM, Widera D, Schmitz J, Kaltschmidt C, Piechaczek C
Impact of polysialylated CD56 on natural killer cell cytotoxicity.
BMC Immunol. 2007;813.
BACKGROUND: Siglec-7, a sialic acid binding inhibitory receptor expressed by NK cells is masked in vivo by a so far unknown ligand. It shows a strong binding prevalence for alpha-2,8-linked disialic acids in vitro. RESULTS: Here we describe the expression of PSA-NCAM (alpha-2,8-linked polysialic acid modified NCAM) on functional adult peripheral blood natural killer cells and examine its possible role in masking Siglec-7. Unmasking of Siglec-7 using Clostridium perfringens neuraminidase massively reduces NK cell cytotoxicity. By contrast a specific removal of PSA using Endo-NF does not lead to a reduction of NK cell cytotoxicity. CONCLUSION: The results presented here therefore indicate that PSA-NCAM is not involved in masking Siglec-7. [Abstract/Link to Full Text]

Gibbings DJ, Marcet-Palacios M, Sekar Y, Ng MC, Befus AD
CD8 alpha is expressed by human monocytes and enhances Fc gamma R-dependent responses.
BMC Immunol. 2007;812.
BACKGROUND: CD8 alpha enhances the responses of antigen-specific CTL activated through TCR through binding MHC class I, favoring lipid raft partitioning of TCR, and inducing intracellular signaling. CD8 alpha is also found on dendritic cells and rat macrophages, but whether CD8 alpha enhances responses of a partner receptor, like TCR, to activate these cells is not known. TCR and FcR, use analogous or occasionally interchangeable signaling mechanisms suggesting the possibility that CD8 alpha co-activates FcR responses. Interestingly, CD8 alpha+ monocytes are often associated with rat models of disease involving immune-complex deposition and FcR-mediated pathology, such as arthritis, glomerulonephritis, ischaemia, and tumors. While rat macrophages have been shown to express CD8 alpha evidence for CD8 alpha expression by mouse or human monocytes or macrophages was incomplete. RESULTS: We detected CD8 alpha, but not CD8 beta on human monocytes and the monocytic cell line THP-1 by flow cytometry. Reactivity of anti-CD8 alpha mAb with monocytes is at least partly independent of FcR as anti-CD8 alpha mAb detect CD8 alpha by western blot and inhibit binding of MHC class I tetramers. CD8 alpha mRNA is also found in monocytes and THP-1 suggesting CD8 alpha is synthesized by monocytes and not acquired from other CD8 alpha+ cell types. Interestingly, CD8 alpha from monocytes and blood T cells presented distinguishable patterns by 2-D electrophoresis. Anti-CD8 alpha mAb alone did not activate monocyte TNF release. In comparison, TNF release by human monocytes stimulated in a FcR-dependent manner with immune-complexes was enhanced by inclusion of anti-CD8 alpha mAb in immune-complexes. CONCLUSION: Human monocytes express CD8 alpha. Co-engagement of CD8 alpha and FcR enhances monocyte TNF release, suggesting FcR may be a novel partner receptor for CD8 alpha on innate immune cells. [Abstract/Link to Full Text]

Jiang Q, Coffield VM, Kondo M, Su L
TSLP is involved in expansion of early thymocyte progenitors.
BMC Immunol. 2007;811.
BACKGROUND: Thymic stromal derived lymphopoietin (TSLP) is preferentially and highly expressed in the thymus, but its function in T cell development is not clear. RESULTS: We report here that TSLP, independently or in combination with IL-7, enhances thymopoiesis in the murine fetal thymic organ culture (FTOC) model. Furthermore, TSLP preferentially increases the number and proliferation of the (DN1 and DN2) pro-T progenitor cells, and FTOC lobes from TSLP receptor-null mice show a decreased number of these cells. Finally, DN1-DN2 cells expanded with TSLP in vitro are functional T progenitors that are able to differentiate into mature T cells in fetal or adult thymus organs. CONCLUSION: Together, these data suggest that TSLP plays an important role in expansion of thymocyte progenitors and may be of value for expanding T progenitor cells in vitro. [Abstract/Link to Full Text]

Bhasin M, Wu M, Tsirka SE
Modulation of microglial/macrophage activation by macrophage inhibitory factor (TKP) or tuftsin (TKPR) attenuates the disease course of experimental autoimmune encephalomyelitis.
BMC Immunol. 2007;810.
BACKGROUND: Myelin Oligodendrocyte Glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) is the most commonly used mouse model for multiple sclerosis (MS). During the of progression of EAE, microglia, the immunocompetent cells of the brain, become activated and accumulate around demyelinated lesions. Microglial activation is mediated by the extracellular protease tissue Plasminogen Activator (tPA), and mice lacking tPA display altered EAE progression. In this study, we have used pharmacological inhibitors and stimulators of microglial/macrophage activation to examine the temporal requirement for microglial activation in EAE progression and to determine whether such approaches might potentially be of therapeutic value. RESULTS: Intervention using the tripeptide macrophage/microglia inhibitory factor MIF (TKP) and the tetrapeptide macrophage/microglial stimulator tuftsin (TKPR) attenuated EAE symptoms and revealed that the timing of macrophage/microglial activation is critical for the clinical outcome of EAE. We show that the disease progression can potentially be manipulated favorably at early stages by altering the timing of microglial activation, which in turn alters the systemic immune response to favor upregulation of T helper cell 2 genes that promote recovery from EAE. CONCLUSION: Preventative and therapeutic modulation of macrophage/microglial activity significantly alters the outcome of EAE at symptomatic stages. Specific molecular targets have been identified that represent potential avenues of exploration for the treatment and prevention of MS. [Abstract/Link to Full Text]

Latinovic-Golic S, Walch M, Sundstrom H, Dumrese C, Groscurth P, Ziegler U
Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes.
BMC Immunol. 2007;89.
BACKGROUND: Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. RESULTS: In this study we investigated generation of granulysin in lymphokine activated killer (LAK) cells and antigen (Listeria) specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation.Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. CONCLUSION: This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing. [Abstract/Link to Full Text]

Maris CH, Chappell CP, Jacob J
Interleukin-10 plays an early role in generating virus-specific T cell anergy.
BMC Immunol. 2007;88.
BACKGROUND: Infection of mice with the Armstrong strain of lymphocytic choriomeningitis virus (LCMVARM) leads to a robust immune response and efficient viral clearance. This is in contrast to infection with the variant strain LCMVClone13, which causes functional inactivation of effector T cells and viral persistence. The mechanism by which LCMVClone13 suppresses the antiviral immune response and persists in its host is unknown. RESULTS: Here we demonstrate that infection with LCMVClone13, but not with LCMVARM, resulted in a steady increase in the serum levels of the immuno-inhibitory cytokine, IL-10. Blockade of IL-10 using neutralizing monoclonal antibody injections in LCMVClone13-infected mice led to dramatically enhanced effector T cell responses at 8 days post-infection. Even though IL-10 blockade resulted in decreased viral titers, the generation and maintenance of memory T cells was still compromised. The functional inactivation of CD8+ T cells in IL-10-blocked, chronically infected mice 30 days post-infection was incomplete as potent CTL (cytotoxic T lymphocytes) could be generated by in vitro re-stimulation. IL-10 knockout mice showed a similar pattern of antiviral CD8 T cell responses: early antiviral T cells were dramatically increased and viral levels were decreased; however, CD8 T cells in IL-10 knockout mice were also eventually anergized and these mice became persistently infected. CONCLUSION: Our data suggest that IL-10 plays an early role in LCMVClone13-induced tolerance, although other factors collaborate with IL-10 to induce virus-specific tolerance. [Abstract/Link to Full Text]

Crucian B, Lee P, Stowe R, Jones J, Effenhauser R, Widen R, Sams C
Immune system changes during simulated planetary exploration on Devon Island, high arctic.
BMC Immunol. 2007;87.
BACKGROUND: Dysregulation of the immune system has been shown to occur during spaceflight, although the detailed nature of the phenomenon and the clinical risks for exploration class missions have yet to be established. Also, the growing clinical significance of immune system evaluation combined with epidemic infectious disease rates in third world countries provides a strong rationale for the development of field-compatible clinical immunology techniques and equipment. In July 2002 NASA performed a comprehensive immune assessment on field team members participating in the Haughton-Mars Project (HMP) on Devon Island in the high Canadian Arctic. The purpose of the study was to evaluate the effect of mission-associated stressors on the human immune system. To perform the study, the development of techniques for processing immune samples in remote field locations was required. Ten HMP-2002 participants volunteered for the study. A field protocol was developed at NASA-JSC for performing sample collection, blood staining/processing for immunophenotype analysis, whole-blood mitogenic culture for functional assessments and cell-sample preservation on-location at Devon Island. Specific assays included peripheral leukocyte distribution; constitutively activated T cells, intracellular cytokine profiles, plasma cortisol and EBV viral antibody levels. Study timepoints were 30 days prior to mission start, mid-mission and 60 days after mission completion. RESULTS: The protocol developed for immune sample processing in remote field locations functioned properly. Samples were processed on Devon Island, and stabilized for subsequent analysis at the Johnson Space Center in Houston. The data indicated that some phenotype, immune function and stress hormone changes occurred in the HMP field participants that were largely distinct from pre-mission baseline and post-mission recovery data. These immune changes appear similar to those observed in astronauts following spaceflight. CONCLUSION: The immune system changes described during the HMP field deployment validate the use of the HMP as a ground-based spaceflight/planetary exploration analog for some aspects of human physiology. The sample processing protocol developed for this study may have applications for immune studies in remote terrestrial field locations. Elements of this protocol could possibly be adapted for future in-flight immunology studies conducted during space missions. [Abstract/Link to Full Text]

Saban MR, Simpson C, Davis C, Wallis G, Knowlton N, Frank MB, Centola M, Gallucci RM, Saban R
Discriminators of mouse bladder response to intravesical Bacillus Calmette-Guerin (BCG).
BMC Immunol. 2007;86.
BACKGROUND: Intravesical Bacillus Calmette-Guerin (BCG) is an effective treatment for bladder superficial carcinoma and it is being tested in interstitial cystitis patients, but its precise mechanism of action remains poorly understood. It is not clear whether BCG induces the release of a unique set of cytokines apart from its pro-inflammatory effects. Therefore, we quantified bladder inflammatory responses and alterations in urinary cytokine protein induced by intravesical BCG and compared the results to non-specific pro-inflammatory stimuli (LPS and TNF-alpha). We went further to determine whether BCG treatment alters cytokine gene expression in the urinary bladder. METHODS: C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. Morphometric analyses were conducted in bladders isolated from all groups and urine was collected for multiplex analysis of 18 cytokines. In addition, chromatin immune precipitation combined with real-time polymerase chain reaction assay (CHIP/Q-PCR) was used to test whether intravesical BCG would alter bladder cytokine gene expression. RESULTS: Acute BCG instillation induced edema which was progressively replaced by an inflammatory infiltrate, composed primarily of neutrophils, in response to weekly administrations. Our morphological analysis suggests that these polymorphonuclear neutrophils are of prime importance for the bladder responses to BCG. Overall, the inflammation induced by BCG was higher than LPS or TNF-alpha treatment but the major difference observed was the unique granuloma formation in response to BCG. Among the cytokines measured, this study highlighted the importance of IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-10, IL-17, GM-CSF, KC, and Rantes as discriminators between generalized inflammation and BCG-specific inflammatory responses. CHIP/Q-PCR indicates that acute BCG instillation induced an up-regulation of IL-17A, IL-17B, and IL-17RA, whereas chronic BCG induced IL-17B, IL-17RA, and IL-17RB. CONCLUSION: To the best of our knowledge, the present work is the first to report that BCG induces an increase in the IL-17 family genes. In addition, BCG induces a unique type of persisting bladder inflammation different from TNF-alpha, LPS, and, most likely, other classical pro-inflammatory stimuli. [Abstract/Link to Full Text]

Froicu M, Cantorna MT
Vitamin D and the vitamin D receptor are critical for control of the innate immune response to colonic injury.
BMC Immunol. 2007;85.
BACKGROUND: The active form of vitamin D (1,25(OH)2D3) has been shown to inhibit development of inflammatory bowel disease (IBD) in IL-10 KO mice. Here, the role of the vitamin D receptor (VDR) and 1,25(OH)2D3 in acute experimental IBD was probed. RESULTS: VDR KO mice were extremely sensitive to dextran sodium sulfate (DSS) and there was increased mortality of the VDR KO mice at doses of DSS that only caused a mild form of colitis in wildtype (WT) mice. DSS colitis in the VDR KO mice was accompanied by high colonic expression of TNF-alpha, IL-1 alpha, IL-1beta, IL-12, IFN-gamma, IL-10, MIP-1alpha and KC. DSS concentrations as low as 0.5% were enough to induce bleeding, ulceration and weight loss in VDR KO mice. VDR KO mice failed to recover following the removal of DSS, while WT mice showed signs of recovery within 5 days of DSS removal. The early mortality of DSS treated VDR KO mice was likely due to perforation of the bowel and resulting endotoxemia. VDR KO mice were hyper-responsive to exogenously injected LPS and cultures of the peritoneal exudates of moribund DSS treated VDR KO mice were positive for bacterial growth. 1,25(OH)2D3 in the diet or rectally decreased the severity and extent of DSS-induced inflammation in WT mice. CONCLUSION: The data point to a critical role for the VDR and 1,25(OH)2D3 in control of innate immunity and the response of the colon to chemical injury. [Abstract/Link to Full Text]

Zheng J, Kohler ME, Chen Q, Weber J, Khan J, Johnson BD, Orentas RJ
Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen.
BMC Immunol. 2007;84.
BACKGROUND: We have developed a cell-based vaccine that features the expression of both CD80 and CD86 on the surface of a murine neuroblastoma cell line. The cellular immunity induced by this vaccine is enhanced by treatment with antibody that interferes with T-regulatory cell (Treg) function and we report here that immunization combined with interfering with Treg function also produces a profound serological effect. Serum from mice immunized with our cell-based vaccine in the context of Treg blockade was used to screen a cDNA expression library constructed from the parental neuroblastoma tumor cell line, AGN2a. RESULTS: Serum from mice vaccinated in the context of Treg blockade identified a number of potentially oncogenic transcripts that may serve as important immune targets in a tumor-derived cDNA library screen. This novel approach identified far more candidates than could be seen with serum derived from vaccine-treated only, Treg-depleted only, or tumor-bearing mice. The most commonly identified tumor-associated antigen, using serum from immunized and Treg-depleted mice, was the DEK oncogene. Altered expression of the DEK oncogene has been implicated in a number of human cancers. Importantly, we were able to demonstrate that the DEK oncogene also induces a T cell response. CONCLUSION: The use of post-vaccine immune serum in this report differs from previous approaches where serum collected at the time of cancer onset or diagnosis and was used for tumor antigen identification. We hypothesize that the use of diagnostic serum samples may be inadequate for the clinical translation of this approach, and that identification of protective immunogenic tumor antigens may require the use of serum from post-treatment or vaccinated subjects. The identification of DEK as a tumor-associated antigen capable of eliciting a T cell response validates our experimental approach and argues for the antigens we have identified here to be evaluated as targets of effector immunity and as vaccine candidates. [Abstract/Link to Full Text]

Kearns-Jonker M, Barteneva N, Mencel R, Hussain N, Shulkin I, Xu A, Yew M, Cramer DV
Use of molecular modeling and site-directed mutagenesis to define the structural basis for the immune response to carbohydrate xenoantigens.
BMC Immunol. 2007;83.
BACKGROUND: Natural antibodies directed at carbohydrates reject porcine xenografts. They are initially expressed in germline configuration and are encoded by a small number of structurally-related germline progenitors. The transplantation of genetically-modified pig organs prevents hyperacute rejection, but delayed graft rejection still occurs, partly due to humoral responses. IgVH genes encoding induced xenoantibodies are predominantly, not exclusively, derived from germline progenitors in the VH3 family. We have previously identified the immunoglobulin heavy chain genes encoding VH3 xenoantibodies in patients and primates. In this manuscript, we complete the structural analysis of induced xenoantibodies by identifying the IgVH genes encoding the small proportion of VH4 xenoantibodies and the germline progenitors encoding xenoantibody light chains. This information has been used to define the xenoantibody/carbohydrate binding site using computer-simulated modeling. RESULTS: The VH4-59 gene encodes antibodies in the VH4 family that are induced in human patients mounting active xenoantibody responses. The light chain of xenoantibodies is encoded by DPK5 and HSIGKV134. The structural information obtained by sequencing analysis was used to create computer-simulated models. Key contact sites for xenoantibody/carbohydrate interaction for VH3 family xenoantibodies include amino acids in sites 31, 33, 50, 57, 58 and the CDR3 region of the IgVH gene. Site-directed mutagenesis indicates that mutations in predicted contact sites alter binding to carbohydrate xenoantigens. Computer-simulated modeling suggests that the CDR3 region directly influences binding. CONCLUSION: Xenoantibodies induced during early and delayed xenograft responses are predominantly encoded by genes in the VH3 family, with a small proportion encoded by VH4 germline progenitors. This restricted group can be identified by the unique canonical structure of the light chain, heavy chain and CDR3. Computer-simulated models depict this structure with accuracy, as confirmed by site-directed mutagenesis. Computer-simulated drug design using computer-simulated models may now be applied to develop new drugs that may enhance the survival of xenografted organs. [Abstract/Link to Full Text]

Perez OD, Mitchell D, Nolan GP
Differential role of ICAM ligands in determination of human memory T cell differentiation.
BMC Immunol. 2007;82.
BACKGROUND: Leukocyte Function Antigen-1 (LFA-1) is a primary adhesion molecule that plays important roles in T cell activation, leukocyte recirculation, and trans-endothelial migration. By applying a multivariate intracellular phospho-proteomic analysis, we demonstrate that LFA-1 differentially activates signaling molecules. RESULTS: Signal intensity was dependent on both ICAM ligand and LFA-1 concentration. In the presence of CD3 and CD28 stimulation, ICAM-2 and ICAM-3 decreased TGFbeta1 production more than ICAM-1. In long-term differentiation experiments, stimulation with ICAM-3, CD3, and CD28 generated IFNgamma producing CD4+CD45RO+CD62L-CD11aBrightCD27- cells that had increased expression of intracellular BCL2, displayed distinct chemokine receptor profiles, and exhibited distinct migratory characteristics. Only CD3/CD28 with ICAM-3 generated CD4+CD45RO+CD62L-CD11aBrightCD27- cells that were functionally responsive to chemotaxis and exhibited higher frequencies of cells that signaled to JNK and ERK1/2 upon stimulation with MIP3alpha. Furthermore, these reports identify that the LFA-1 receptor, when presented with multiple ligands, can result in distinct T cell differentiation states and suggest that the combinatorial integration of ICAM ligand interactions with LFA-1 have functional consequences for T cell biology. CONCLUSION: Thus, the ICAM ligands, differentially modulate LFA-1 signaling in T cells and potentiate the development of memory human T cells in vitro. These findings are of importance in a mechanistic understanding of memory cell differentiation and ex vivo generation of memory cell subsets for therapeutic applications. [Abstract/Link to Full Text]

---

Recent Articles in BMC Infectious Diseases

---

Bacchetti P, Tien PC, Seaberg EC, O'Brien TR, Augenbraun MH, Kral AH, Busch MP, Edlin BR
Estimating past hepatitis C infection risk from reported risk factor histories: implications for imputing age of infection and modeling fibrosis progression.
BMC Infect Dis. 2007 Dec 10;7(1):145.
ABSTRACT: BACKGROUND: Chronic hepatitis C virus infection is prevalent and often causes hepatic fibrosis, which can progress to cirrhosis and cause liver cancer or liver failure. Study of fibrosis progression often relies on imputing the time of infection, often as the reported age of first injection drug use. We sought to examine the accuracy of such imputation and implications for modeling factors that influence progression rates. METHODS: We analyzed cross-sectional data on hepatitis C antibody status and reported risk factor histories from two large studies, the Women's Interagency HIV Study and the Urban Health Study, using modern survival analysis methods for current status data to model past infection risk year by year. We compared fitted distributions of past infection risk to reported age of first injection drug use. RESULTS: Although injection drug use appeared to be a very strong risk factor, models for both studies showed that many subjects had considerable probability of having been infected substantially before or after their reported age of first injection drug use. Persons reporting younger age of first injection drug use were more likely to have been infected after, and persons reporting older age of first injection drug use were more likely to have been infected before. CONCLUSIONS: In cross-sectional studies of fibrosis progression where date of HCV infection is estimated from risk factor histories, modern methods such as multiple imputation should be used to account for the substantial uncertainty about when infection occurred. The models presented here can provide the inputs needed by such methods. Using reported age of first injection drug use as the time of infection in studies of fibrosis progression is likely to produce a spuriously strong association of younger age of infection with slower rate of progression. [Abstract/Link to Full Text]

Holm MV, Blank PR, Szucs TD
Trends in influenza vaccination coverage rates in Germany over five seasons from 2001 to 2006.
BMC Infect Dis. 2007 Dec 10;7(1):144.
ABSTRACT: BACKGROUND: To assess influenza vaccination coverage from 2001 to 2006 in Germany, to understand drivers and barriers to vaccination and to identify vaccination intentions for season 2006/07. METHODS: 9 990 telephone-based household surveys from age 14 were conducted between 2001 and 2006. Essentially, the same questionnaire was used in all seasons. RESULTS: The influenza vaccination coverage rate reached 32.5% in 2005/06. In the elderly ([greater than or equal to] 60 years), the vaccination rate reached 58.9% in 2005/06. In those aged 65 years and older, it was 63.4%. Perceiving influenza as a serious illness was the most frequent reason for getting vaccinated. Thirteen percent of those vaccinated in 2005/06 indicated the threat of avian flu as a reason. The main reason for not getting vaccinated was thinking about it without putting it into practice. The major encouraging factor to vaccination was a recommendation by the family doctor. 49.6% of the respondents intend to get vaccinated against influenza in season 2006/07. CONCLUSIONS: Increasing vaccination rates were observed from 2001 to 2006 in Germany. The threat of avian influenza and the extended reimbursement programs may have contributed to the recent increase. [Abstract/Link to Full Text]

Giuliani M, Cordiali Fei P, Castilletti C, Di Carlo A, Palamara G, Boros S, Rezza G
Incidence of Human Herpesvirus 8 (HHV-8) Infection among HIV-uninfected individuals at high risk for sexually transmitted infections.
BMC Infect Dis. 2007 Dec 5;7(1):143.
ABSTRACT: BACKGROUND: The occurrence of,and risk factors for, HHV-8 infection have yet to be definitively determined, particularly among heterosexual individuals with at-risk behavior for sexually transmitted infections (STI). The objective of this study was to estimate the incidence and determinants of HHV-8 infection among HIV-uninfected individuals repeatedly attending an urban STI clinic. METHODS: Sera from consecutive HIV-uninfected individuals repeatedly tested for HIV-1 antibodies were additionally tested for HHV-8 antibodies using an immunofluorescence assay. To identify determinants of HHV-8 infection, a nested case-control study and multivariate logistic regression analysis were performed. RESULTS: Sera from 456 HIV-uninfected individuals (224 multiple-partner heterosexuals and 232 men who have sex with men [MSM]) were identified for inclusion in the study. The HHV-8 seroprevalence at enrollment was 9.4% (21/224; 95% C.I.: 6.0-14.2%) among heterosexuals with multiple partners and 22.0% (51/232; 95% C.I.: 16.9-28.0%) among MSM. Among the 203 multiple-partner heterosexuals and 181 MSM who were initially HHV-8-negative, 17 (IR=3.0/100 p-y, 95% C.I.: 1.9 - 4.8) and 21 (IR=3.3/100 p-y, 95% C.I:.2.1 - 5.1) seroconversions occurred, respectively. HHV-8 seroconversion tended to be associated with a high number of sexual partners during the follow-up among MSM (>10 partners: AOR=3.32 95% CI:0.89-12.46) and among the multiple-partner heterosexuals (>10 partner; AOR=3.46, 95% CI:0.42-28.2). Moreover, among MSM, HHV-8 seroconversion tended to be associated with STI (AOR=1.80 95%CI: 0.52-7.96). During the study period the HIV-1 incidence was lower than that of HHV-8 among both groups (0.89 per 100 p-y among MSM and 0.95 per 100 p-y among multiple-partner heterosexuals). CONCLUSIONS: The large difference between the incidence of HHV-8 and the incidence of HIV-1 and other STIs may suggest that the circulation of HHV-8 is sustained by practices other than classical at-risk sexual behavior. [Abstract/Link to Full Text]

Ben Abdelmoumen Mardassi B, Ayari H, Bejaoui-Khiari A, Mlik B, Moalla I, Amouna F
Genetic variability of the P120' surface protein gene of Mycoplasma hominis isolates recovered from Tunisian patients with uro-genital and infertility disorders.
BMC Infect Dis. 2007 Dec 5;7(1):142.
ABSTRACT: BACKGROUND: Among the surface antigens of Mycoplasma hominis, the P120' protein was previously shown to elicit a subtle antibody response and appears to be relatively conserved. To get better insight into the evolution of this protein, we analysed the genetic variability of its surface exposed region in 27 M. hominis isolates recovered from the genital tract of Tunisian patients with fertility disorder. METHODS: All specimens were processed for culture and PCR amplification of the N-terminal surface exposed region of p120' gene. PCR products were sequenced to evaluate the genetic variability, to test for adaptive selection, and to infer the phylogenetic relationship of the M. hominis isolates. RESULTS: Sequence analysis showed a total of 25 single nucleotide polymorphisms distributed through 23 polymorphic sites, yielding 13 haplotypes. All but one mutation were confined within three distinct regions. Analysis of the amino acid-based phylogenetic tree showed a predominant group of 17 closely related isolates while the remaining appear to have significantly diverged. CONCLUSIONS: By analysing a larger sample of M. hominis recovered from patients with urogenital infections, we show here that the P120' protein undergoes substantial level of genetic variability at its surface exposed region. [Abstract/Link to Full Text]

Paget J, Marquet R, Meijer A, van der Velden K
Influenza activity in Europe during eight seasons (1999-2007): an evaluation of the indicators used to measure activity and an assessment of the timing, length and course of peak activity (aspreada) across Europe.
BMC Infect Dis. 2007 Nov 30;7(1):141.
ABSTRACT: BACKGROUND: The European Influenza Surveillance Scheme (EISS) has collected clinical and virological data on influenza since 1996 in an increasing number of countries. The EISS dataset was used to characterise important epidemiological features of influenza activity in Europe during eight winters (1999-2007). The following questions were addressed: 1) are the sentinel clinical reports a good measure of influenza activity?; 2) how long is a typical influenza season in Europe?; 3) is there a westaeast and/or south-north course of peak activity (aspreada) of influenza in Europe? METHODS: Influenza activity was measured by collecting data from sentinel general practitioners (GPs) and reports by national reference laboratories. The sentinel reports were first evaluated by comparing them to the laboratory reports and were then used to assess the timing and spread of influenza activity across Europe during eight seasons. RESULTS: We found a good match between the clinical sentinel data and laboratory reports of influenza collected by sentinel physicians (overall match of 72% for +/- 1 week difference). We also found a moderate to good match between the clinical sentinel data and laboratory reports of influenza from non-sentinel sources (overall match of 60% for +/- 1 week). There were no statistically significant differences between countries using ILI (influenza-like illness) or ARI (acute respiratory disease) as case definition. When looking at the peak-weeks of clinical activity, the average length of an influenza season in Europe was 15,6 weeks (median 15 weeks; range 12-19 weeks). Plotting the peak weeks of clinical influenza activity reported by sentinel GPs against the longitude or latitude of each country indicated that there was a west-east spread of peak activity (spread) of influenza across Europe in four winters (2001-2002, 2002-2003, 2003-2004 and 2004-2005) and a south-north spread in three winters (2001-2002, 2004-2005 and 2006-2007). CONCLUSIONS: We found that: 1) the clinical data reported by sentinel physicians is a valid indicator of influenza activity; 2) the length of influenza activity across the whole of Europe was surprisingly long, ranging from 12-19 weeks; 3) in 4 out of the 8 seasons, there was a west-east spread of influenza, in 3 seasons a south-north spread; not associated with type of dominant virus in those seasons. [Abstract/Link to Full Text]

Schaaf HS, Marais BJ, Whitelaw A, Hesseling AC, Eley B, Hussey GD, Donald PR
Culture-confirmed childhood tuberculosis in Cape Town, South Africa: a review of 596 cases.
BMC Infect Dis. 2007 Nov 29;7(1):140.
ABSTRACT: BACKGROUND: The clinical, radiological and microbiological features of culture-confirmed childhood tuberculosis diagnosed at two referral hospitals are described. METHODS: Cultures of Mycobacterium tuberculosis from children less than 13 years of age at Tygerberg and Red Cross Children's Hospitals, Cape Town, South Africa, were collected from March 2003 through February 2005. Folder review and chest radiography were performed and drug susceptibility tests done. RESULTS: Of 596 children (median age 31 months), 330 (55.4%) were males. Of all children, 281 (47.1%) were HIV-uninfected, 133 (22.3%) HIV-infected and 182 (30.5%) not tested. Contact with infectious tuberculosis adults was recorded in 295 (49.5%) children. Missed opportunities for chemoprophylaxis were present in 117/182 (64.3%) children less than 5 years of age. Extrathoracic TB was less common in HIV-infected than in HIV-uninfected children (49/133 vs. 156/281; odds ratio 0.50, 95% confidence interval 0.32-0.78). Alveolar opacification (84/126 vs. 128/274; OR 1.85, 95%CI 1.08-3.19) and cavitation (33/126 vs. 44/274; OR 2.28, 95%CI 1.44-3.63) were more common in HIV-infected than in HIV-uninfected children. Microscopy for acid-fast bacilli on gastric aspirates and sputum was positive in 29/142 (20.4%) and 40/125 (32.0%) children, respectively. Sixty-seven of 592 (11.3%) children's isolates showed resistance to isoniazid and/or rifampicin; 43 (7.3%) were isoniazid-monoresistant, 2 (0.3%) rifampicin-monoresistant and 22 (3.7%) multidrug-resistant. Death in 41 children (6.9%) was more common in HIV-infected children and very young infants. CONCLUSION: HIV infection and missed opportunities for chemoprophylaxis were common in children with culture-confirmed TB. With cavitating disease and sputum or gastric aspirates positive for acid-fast bacilli, children may be infectious. Transmission of drug-resistant TB is high in this setting. [Abstract/Link to Full Text]

Mutapi F, Winborn G, Midzi N, Taylor M, Mduluza T, Maizels RM
Cytokine responses to Schistosoma haematobium in a Zimbabwean population: contrasting profiles for IFN-gamma, IL-4, IL-5 and IL-10 with age.
BMC Infect Dis. 2007 Nov 28;7(1):139.
ABSTRACT: BACKGROUND: The rate of development of parasite-specific immune responses can be studied by following their age profiles in exposed and infected hosts. This study determined the cytokine-age profiles of Zimbabweans resident in a Schistosoma haematobium endemic area and further investigated the relationship between the cytokine responses and infection intensity. METHODS: Schistosome adult worm antigen-specific IFN-gamma, IL-4, IL-5 and IL-10 cytokine responses elicited from whole blood cultures were studied in 190 Zimbabweans exposed to S. haematobium infection (aged 6 to 40 years old). The cytokines were measured using capture ELISAs and the data thus obtained together with S. haematobium egg count data from urine assays were analysed using a combination of parametric and nonparametric statistical approaches. RESULTS: Age profiles of schistosome infection in the study population showed that infection rose to peak in childhood (11-12 years) followed by a sharp decline in infection intensity while prevalence fell more gradually. Mean infection intensity was 37 eggs/10ml urine (SE 6.19 eggs/ 10ml urine) while infection prevalence was 54.7%. Measurements of parasite-specific cytokine responses showed that IL-4, IL-5 and IL-10 but not IFN-gamma followed distinct age-profiles. Parasite-specific IL-10 production developed early, peaking in the youngest age group and declining thereafter; while IL-4 and IL-5 responses were slower to develop with a later peak. High IL-10 producers were likely to be egg positive with infection intensity increasing with increasing IL-10 production. Furthermore people producing high levels of IL-10 produced little or no IL-5, suggesting that IL-10 may be involved in the regulation of IL-5 levels. IL-4 and IFN-gamma did not show a significant relationship with infection status or intensity and were positively associated with each other. CONCLUSIONS: Taken together, these results show that the IL-10 responses develop early compared to the IL-5 response and may be down-modulating immunopathological responses that occur during the early phase of infection. The results further support current suggestions that the Th1/Th2 dichotomy does not sufficiently explain susceptibility or resistance to schistosome infection. [Abstract/Link to Full Text]

Cariani E, Pollara CP, Valloncini B, Perandin F, Bonfanti C, Manca N
Relationship between pp65 antigenemia levels and real-time quantitative DNA PCR for Human Cytomegalovirus (HCMV) management in immunocompromised patients.
BMC Infect Dis. 2007 Nov 23;7(1):138.
ABSTRACT: BACKGROUND: Quantitative real-time PCR assays, which are more rapid and practical than pp65 antigenemia determination, are progressively becoming the preferred method for monitoring Human Cytomegalovirus (HCMV) reactivation. However, the relationship between HCMV DNA and antigenemia levels is still under investigation. The aim of this study was to analyse the relationship between HCMV DNA and pp65 antigenemia levels in order to identify clinically useful threshold values for the management of patients. METHODS: 475 consecutive samples from 156 immunosuppressed patients were tested for HCMV by pp65 antigenemia and Real-time PCR assay. RESULTS: 136 out of 475 consecutive samples derived from 48 patients showed evidence of HCMV infection. HCMV DNA was detected in 106 samples, pp65 antigen in 3, and both markers in 27. pp65 antigen detection was associated with higher HCMV DNA levels. The cut-off HCMV DNA level that best predicted pp65 antigenemia in this series of samples was 1.15 x 104, but different threshold levels could be observed for specific groups of patients. Symptomatic HCMV infection was observed in 5 out of 48 patients with HCMV infection. Detection of HCMV DNA and pp65 levels above 13 positive cells/200,000 were both associated with symptomatic infection. HCMV DNA load higher than 7,880 copies/ml at first detection could predict the onset of clinical symptoms. CONCLUSIONS: Both pp65 antigenemia and HCMV DNA load can be useful for the prospective monitoring of immunocompromised subjects. Specific cut-off levels capable of triggering pre-emptive antiviral treatment should be determined in accordance to the type of test used and the characteristics of patients and prospectively validated. [Abstract/Link to Full Text]

Massaro KS, Costa SF, Leone C, Chamone DA
Procalcitonin (PCT) and C-reactive Protein (CRP) as severe systemic infection markers in febrile neutropenic adults.
BMC Infect Dis. 2007 Nov 22;7(1):137.
ABSTRACT: BACKGROUND: Procalcitonin (PCT) is an inflammatory marker that has been used as indicator of severe bacterial infection. We evaluated the concentrations of PCT as a marker for systemic infection compared to C-reactive protein (CRP) in patients neutropenic febrile. METHODS: 52 adult patients were enrolled in the study. Blood sample was collected in order to determine the serum concentrations of PCT, CRP and other hematological parameters at the onset of fever. The patients were divided into 2 groups, one with severe infection (n=26) and the other in which the patients did not present such an infection (n=26). Then PCT and CRP concentrations at the fever onset were compared between groups using non parametric statistical tests, ROC curve, sensitivity, specificity, likelihood ratio, and Spearman's correlation coefficient. RESULTS: The mean of PCT was significantly higher in the group with severe infection (6.7 ng/mL versus 0.6 ng/mL - p=0.0075) comparing with CRP. Serum concentrations of 0.245 ng/mL of PCT displayed 100% de sensitivity and 69.2% specificity. PCT concentrations of 2,145 ng/mL presented a likelihood ratio of 13, which was not observed for any concentration of CRP. CONCLUSION: PCT seems to be an useful marker for the diagnosis of systemic infection in febrile neutropenic patients, probably better than CRP. [Abstract/Link to Full Text]

Atencia R, Bustamante FJ, Valdivieso A, Arrieta A, Rinon M, Prada A, Maruri N
Differential expression of viral PAMP receptors mRNA in peripheral blood of patients with chronic hepatitis C infection.
BMC Infect Dis. 2007 Nov 19;7(1):136.
ABSTRACT: BACKGROUND: Pathogen-associated molecular patterns (PAMP) receptors play a key role in the early host response to viruses. In this work, we determined mRNA levels of two members of the Toll-like Receptors family, (TLR3 and TLR7) and the helicase RIG-I, all of three recognizing viral RNA products, in peripheral blood of healthy donors and hepatitis C virus (HCV) patients, to observe if their transcripts are altered in this disease. METHODS: IFN-alpha, TLR3, TLR7 and RIG-I levels in peripheral blood from healthy controls (n=18) and chronic HCV patients (n=18) were quantified by real-time polymerase chain reaction. RESULTS: Our results show that IFN-alpha, TLR3, TLR7 and RIG-I mRNA levels are significantly down-regulated in patients with chronic HCV infection when compared with healthy controls. We also found that the measured levels of TLR3 and TLR7, but not RIG-I, correlated significantly with those of IFN-alpha CONCLUSIONS: Measuring the expression of RNA-sensing receptors like TLR3, TLR7 and RIG-I could provide a new set of molecular markers for the prognosis of the HCV infection. [Abstract/Link to Full Text]

Rizzo C, Di Bartolo I, Santantonio M, Coscia MF, Monno R, De Vito D, Ruggeri FM, Rizzo G
Epidemiological and virological investigation of a Norovirus outbreak in a resort in Puglia, Italy.
BMC Infect Dis. 2007 Nov 19;7(1):135.
ABSTRACT: BACKGROUND: This paper describes the third large outbreak of Norovirus (NoV) gastroenteritis reported in the Southern Italy region of Puglia. METHODS: A matched case control study was conducted, on 19 July 2005, for investigating risk factors, using a structured questionnaire on food consumption. A multivariate analysis was conducted to estimate the adjusted Odds Ratios. Laboratory and environmental investigation were also performed. RESULTS: On the day of the study 41 cases were identified and 41 controls were enrolled. Controls were matched for age and gender. The mean age of the cases was 26 years old, and 58% were female. The clinical pattern of the disease was characterised by the presence of diarrhoea (95%), vomiting (70%), abdominal pain (51%) and fever (32%). Of the 41 cases included in the study, the majority (65%) were residents of Northern Italian regions. No food samples were available for testing. The matched univariate analysis revealed that cases were more likely to have consumed raw mussels, eggs or ice cubes made of tap water than controls. In the multivariate conditional logistic regression analysis, having eaten raw mussels or ice made of tap water became more strongly associated with illness. All of the 20 faecal samples collected were tested for NoVs. Eighteen stools (90% of total examined) were positive by RT-PCR, and sequence analysis performed onto 3 samples confirmed the presence of a GGII NoV. No test specific for NoV was performed on water or food samples. CONCLUSIONS: The most likely hypothesis supported by the findings of the epidemiological investigation was that illness was associated with raw mussels and ice, made with tap water. These hypothesis could not be confirmed by specific microbiologic testing for NoV in food or ice. The lack of clear knowledge of NoV as a major causative agent of epidemic outbreaks of gastroenteritis in Italy is due to the absence of timely reporting of the cases to the local public health offices and the uncommon practice of saving clinical samples for virological analysis after bacteriological testing. [Abstract/Link to Full Text]

Schneider UV, Nielsen RL, Pedersen C, Eugen-Olsen J
The prognostic value of the suPARnosticTM ELISA assay in HIV-1 infected individuals is not affected by uPAR promoter polymorphisms.
BMC Infect Dis. 2007 Nov 16;7(1):134.
ABSTRACT: BACKGROUND: High blood levels of soluble urokinase Plasminogen Activator Receptor (suPAR) are associated with poor outcomes in human immunodeficiency-1 (HIV-1) infected individuals. Research on the clinical value of suPAR in HIV-1 infection led to the development of the suPARnosticTM assay for commercial use in 2006. The aim of this study was to: 1) Evaluate the prognostic value of the new suPARnosticTM assay and 2) Determine whether polymorphisms in the active promoter of uPAR influences survival and/or suPAR values in HIV-1 patients who are antiretroviral therapy (ART) naive. METHODS: DNA samples were collected retrospectively from 145 Danes infected with HIV-1 with known seroconversion times. In addition, plasma was collected retrospectively from 81 of these participants for use in the suPAR analysis. Survival was analysed using Kaplan Meier analysis. RESULTS: Survival was strongly correlated to suPAR levels (p<0.001). Levels at or above 6 ng/ml were associated with death in 13 of 27 patients within a two-year period; whereas only one of 54 patients with suPAR levels below 6 ng/ml died during this period. We identified two common uPAR promoter polymorphisms: a G to A transition at -118 and an A to G transition at -465 comparative to the transcription start site. These promoter transitions did not influence neither the suPAR levels nor patient survival. CONCLUSION: Plasma suPAR levels, as measured by the suPARnosticTM assay, were strongly predictive of survival in ART-naive HIV-1 infected patients. Furthermore, plasma suPAR levels were not influenced by uPAR promoter polymorphisms. [Abstract/Link to Full Text]

Gupta S, Gupta R, Singh S
Seroprevalence of HIV in pregnant women in North India: a tertiary care hospital based study.
BMC Infect Dis. 2007 Nov 15;7(1):133.
ABSTRACT: BACKGROUND: Estimating the seroprevalence of HIV in a low risk population such as pregnant women provides essential information for an effective implementation of AIDS control programmes, and also for the monitoring of HIV spread within a country. Very few studies are available from north India showing the current trend in HIV prevalence in the antenatal population; which led us to carry out this study at a tertiary care hospital in north India METHODS: Blood samples from pregnant women attending antenatal clinics at the All India Institute of Medical Sciences, New Delhi were collected after informed consent and pre-test counseling. The samples were tested for HIV antibodies as per the WHO guidelines, over a period of four years from January 2003 to December 2006. RESULTS: Of the 3529 pregnant women tested in four years, 0.88% (CI 0.5 - 1.24) women were found to be HIV seroreactive. Majority of the seroreactive pregnant women (41.9%) were in the age group of 20-24 years followed by the 30-34 yrs (25.8%) and 25-29 years (22.6%) age group. The mean age of the HIV positive women was 24.9 years (SD +/- 1.49 yrs). The HIV seroprevalence rates showed an increasing trend from 0.7% (CI 0.14 - 2.04) in 2003-2004 to 0.9% (CI 0.49 - 1.5) in 2005-2006. This prevalence rate indicates concern, as Delhi and its adjoining states are otherwise considered as 'low prevalence states'. CONCLUSION: Seroprevalence of HIV infection was found to be increasing in the last four years amongst pregnant women of North India. These findings are in contrast to the national projections. [Abstract/Link to Full Text]

Guan Y, Chen H, Li KS, Riley S, Leung GM, Webster R, Peiris M, Yuen KY
A model to control the epidemic of H5N1 influenza at the source.
BMC Infect Dis. 2007 Nov 13;7(1):132.
ABSTRACT: BACKGROUND: No country is fully prepared for a 1918-like pandemic influenza. Averting a pandemic of H5N1 influenza virus depends on the successful control of its endemicity, outbreaks in poultry and occasional spillage into human which carries a case-fatality rate of over 50%. The use of perimetric depopulation and vaccination has failed to halt the spread of the epidemic. Blanket vaccination for all poultry over a large geographical area is difficult. A combination of moratorium, segregation of water fowls from chickens and vaccination have been proved to be effective in the Hong Kong Special Administrative Region (HKSAR) since 2002 despite endemicity and outbreaks in neighbouring regions. Systematic surveillance in southern China showed that ducks and geese are the primary reservoirs which transmit the virus to chickens, minor poultry and even migratory birds. PRESENTATION OF THE HYPOTHESIS: We hypothesize that this combination of moratorium, poultry segregation and targeted vaccination if successfully adapted to an affected district or province in any geographical region with high endemicity would set an example for the control in other regions. TESTING THE HYPOTHESIS: A planned one-off moratorium of 3 weeks at the hottest month of the year should decrease the environmental burden as a source of re-infection. Backyard farms will then be re-populated by hatchlings from virus-free chickens and minor poultry only. Targeted immunization of the ducks and geese present only in the industrial farms and also the chickens would be strictly implemented as blanket immunization of all backyard poultry is almost impossible. Freely grazing ducks and geese would not be allowed until neutralizing antibodies of H5 subtype virus is achieved. As a proof of concept, a simple mathematical model with susceptible-infected-recovered (SIR) structure of coupled epidemics between aquatic birds (mainly ducks and geese) and chickens was used to estimate transmissibility within and between these two poultry populations. In the field the hypothesis is tested by prospective surveillance of poultry and immunocompetent patients hospitalized for severe pneumonia for the virus before and after the institution of these measures. Implications of the Hypothesis: A combination of targeted immunization with the correct vaccine, segregation of poultry species and moratorium of poultry in addition to the present surveillance, biosecurity and hygienic measures at the farm, market and personal levels could be important in the successful control of the H5N1 virus in poultry and human for an extensive geographical region with continuing outbreaks. . Alternatively a lesser scale of intervention at the district level can be considered if there is virus detection without evidence of excess poultry deaths since asymptomatic shedding is common in waterfowls. [Abstract/Link to Full Text]

Goos M, Zech WD, Jaiswal MK, Balakrishnan S, Ebert S, Mitchell T, Carri MT, Keller BU, Nau R
Expression of a Cu,Zn superoxide dismutase typical for familial amyotrophic lateral sclerosis increases the vulnerability of neuroblastoma cells to infectious injury.
BMC Infect Dis. 2007 Nov 12;7(1):131.
ABSTRACT: BACKGROUND: Infections can aggravate the course of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Mutations in the anti-oxidant enzyme Cu,Zn superoxide dismutase (EC 1.15.1.1, SOD1) are associated with familial ALS. Streptococcus pneumoniae, the most frequent respiratory pathogen, causes damage by the action of the cholesterol-binding virulence factor pneumolysin and by stimulation of the innate immune system, particularly via Toll-like-receptor 2. METHODS: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) and SH-SY5Y neuroblastoma cells transfected with wildtype SOD1 were both exposed to pneumolysin and in co-cultures with cultured human macrophages treated with the Toll like receptor 2 agonist N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl- [S]-lysyl-[S]-lysine x 3 HCl (Pam3CSK4). Cell viability and apoptotic cell death were compared morphologically and by in-situ tailing. With the help of the WST-1 test, cell viability was quantified, and by measurement of neuron-specific enolase in the culture supernatant neuronal damage in co-cultures was investigated. Intracellular calcium levels were measured by fluorescence analysis using fura-2 AM. RESULTS: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) were more vulnerable to the neurotoxic action of pneumolysin and to the attack of monocytes stimulated by Pam3CSK4 than SH-SY5Y cells transfected with wild-type human SOD1. The enhanced pneumolysin toxicity in G93A-SOD1 neuronal cells depended on the inability of these cells to cope with an increased calcium influx caused by pores formed by pneumolysin. This inability was caused by an impaired capacity of the mitochondria to remove cytoplasmic calcium. Treatment of G93A-SOD1 SH-SY5Y neuroblastoma cells with the antioxidant N-acetylcysteine reduced the toxicity of pneumolysin. CONCLUSIONS: The particular vulnerability of G93A-SOD1 neuronal cells to hemolysins and inflammation may be partly responsible for the clinical deterioration of ALS patients during infections. These findings link infection and motor neuron disease and suggest early treatment of respiratory infections in ALS patients. [Abstract/Link to Full Text]

de Arazoza H, Joanes J, Loumes R, Legeai C, Clemencon S, Perez J, Auvert B
The HIV/AIDS epidemic in Cuba: description and tentative explanation of its low HIV prevalence.
BMC Infect Dis. 2007 Nov 9;7(1):130.
ABSTRACT: BACKGROUND: The Cuban HIV/AIDS epidemic has the lowest prevalence rate of the Caribbean region. The objective of this paper is to give an overview of the HIV/AIDS epidemic in Cuba and to explore the reasons for this low prevalence. METHODS: Data were obtained from the Cuban HIV/AIDS programme established in 1983. This programme has an extensive adult HIV testing policy, including testing of all pregnant women. HIV and AIDS cases have been recorded since 1986. Persons found to be HIV-positive are interviewed on their sexual behaviour and partners. Tracing and voluntary testing of these partners are organised. Epidemiological description of this epidemic was obtained from analysis of this data set. Using elementary mathematical analyses, we estimated the coverage of the detection system (percentage of HIV-positive adults detected) and the average period between HIV infection and detection. Estimated HIV prevalence rates were corrected to account for the coverage. RESULTS: HIV prevalence has increased since 1996. In 2005, the prevalence among pregnant women was 0.017%. Estimated HIV prevalence among 15- to 49-year-olds was 0.081% (95%CI: 0.078- 0.083). Most (77%) of the HIV-positive adults were men, most (85.1%) of the detected HIV-positive men were reported as having sex with men (MSM), and most of the HIV positive women reported having had sex with MSM. The average period between HIV infection and detection was estimated to be 2.1 years (IQR = 1.7 - 2.2 years). We estimated that, for the year 2005, 79.6% (IQR: 77.3 - 81.4%) of the HIV-positive persons were detected. CONCLUSION: MSM drives the HIV epidemic in Cuba. The extensive HIV testing policy may be an important factor in explaining the low HIV prevalence. To reduce the HIV epidemic in Cuba, the epidemic among MSM should be addressed. To understand this epidemic further, data on sexual behaviour should be collected. Now that antiretroviral therapy is more widely available, the Cuban policy, based on intensive HIV testing and tracing of partners may be considered as a possible policy to control HIV/AIDS epidemics in other countries. [Abstract/Link to Full Text]

Gdoura R, Kchaou W, Chaari C, Znazen A, Keskes L, Rebai T, Hammami A
Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis and Mycoplasma genitalium infections and semen quality of infertile men.
BMC Infect Dis. 2007 Nov 8;7(1):129.
ABSTRACT: BACKGROUND: Genital ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum) and mycoplasmas (Mycoplasma genitalium and Mycoplasma hominis) are potentially pathogenic species playing an etiologic role in both genital infections and male infertility. Reports are, however, controversial regarding the effects of these microorganisms infections in the sperm seminological variables. This study aimed at determining the frequency of genital ureaplasmas and mycoplasmas in semen specimens collected from infertile men, and at comparing the seminological variables of semen from infected and non-infected men with these microorganisms. METHODS: A total of 120 semen samples collected from infertile men were investigated. Semen specimens were examined by in-house PCR-microtiter plate hybridization assay for the presence of genital ureaplasmas and mycoplasmas DNA. Semen analysis was assessed according to the guidelines of the World Health Organization. Standard parametric techniques (t-tests) and nonparametric techniques (Wilcoxon tests) were used for statistical analysis. RESULTS: The frequency of genital ureaplasmas and mycoplasmas detected in semen samples of infertile men was respectively 19.2 % (23/120) and 15.8 % (19/120). The frequency of Ureaplasma urealyticum (15 %) was higher than that of Mycoplasma hominis (10.8 %), Ureaplasma parvum (4.2 %) and Mycoplasma genitalium (5 %). Mixed species of mycoplasmas and ureaplasmas were detected in 6.7 % of semen samples. Comparison of the parameters of the standard semen analysis between the male partners of the infertile couples with and without genital ureaplasmas and mycoplasmas infection showed that the presence of Mycoplasma hominis DNA in semen samples is associated with low sperm concentration (p = 0.007) and abnormal sperm morphology (p = 0.03) and a negative correlation between sperm concentration and the detection of Mycoplasma genitalium in semen samples of infertile men (p = 0.05). The mean values of seminal volume, pH, vitality, motility and leukocyte count were not significantly related either to the detection of genital mycoplasmas DNA or to the detection of ureaplasmas DNA in semen specimens. CONCLUSION: Our results demonstrate that genital mycoplasmas and ureaplasmas seem to be widespread among the male partners of infertile couples in Tunisia. Genital mycoplasmas infections of the male genital tract could negatively influence semen quality. Our results also indicate that PCR-microtiter plate hybridization assay method provides a rapid and effective technique to detect human genital mycoplasmas and ureaplasmas which is useful for etiological and epidemiological studies of these pathogens. [Abstract/Link to Full Text]

Tamrakar R, Yamada T, Furuta I, Cho K, Morikawa M, Yamada H, Sakuragi N, Minakami H
Association between Lactobacillus species and bacterial vaginosis-related bacteria, and bacterial vaginosis scores in pregnant Japanese women.
BMC Infect Dis. 2007 Nov 7;7(1):128.
ABSTRACT: BACKGROUND: Bacterial vaginosis (BV), the etiology of which is still uncertain, increases the risk of preterm birth. Recent PCR-based studies suggested that BV is associated with complex vaginal bacterial communities, including many newly recognized bacterial species in non-pregnant women. METHODS: To examine whether these bacteria are also involved in BV in pregnant Japanese women, vaginal fluid samples were taken from 132 women, classified as normal (n=98), intermediate (n=21), or BV (n=13) using the Nugent gram stain criteria, and studied. DNA extracted from these samples was analyzed for bacterial sequences of any Lactobacillus, four Lactobacillus species, and four BV-related bacteria by PCR with primers for 16S ribosomal DNA including a universal Lactobacillus primer, Lactobacillus species-specific primers for L. crispatus, L. jensenii, L. gasseri, and L. iners, and BV-related bacterium-specific primers for BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium. RESULTS: The prevalences of L. crispatus, L. jensenii, and L. gasseri were significantly higher, while those of BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium were significantly lower in the normal group than in the BV group. Unlike other Lactobacillus species, the prevalence of L. iners did not differ between the three groups and women with L. iners were significantly more likely to have BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium. Linear regression analysis revealed associations of BVAB2 and Megasphaera with Nugent score, and multivariate regression analyses suggested a close relationship between Eggerthella-like bacterium and BV. CONCLUSIONS: The BV-related bacteria, including BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium, are common in the vagina of pregnant Japanese women with BV. The presence of L. iners may be correlated with vaginal colonization by these BV-related bacteria. [Abstract/Link to Full Text]

Brantsaeter AB, Holberg-Petersen M, Jeansson S, Goplen AK, Bruun JN
CMV quantitative PCR in the diagnosis of CMV disease in patients with HIV-infection - a retrospective autopsy based study.
BMC Infect Dis. 2007 Nov 6;7(1):127.
ABSTRACT: BACKGROUND: Patients with advanced HIV infection at the time of diagnosis and patients not responding to antiretroviral therapy are at risk of cytomegalovirus (CMV) disease. Earlier studies of patients with HIV infection have demonstrated that the diagnosis is often first made post-mortem. In recent years new molecular biological tests have become available for diagnosis of CMV disease. Although clinical evaluation of tests for diagnosis of CMV disease in HIV-infected individuals is suboptimal without autopsy, no results from such studies have been published. The aim of this study was to explore the diagnostic utility of CMV quantitative polymerase chain reaction (PCR) in plasma from HIV and CMV seropositive patients who died during the period 1991-2002 and in whom autopsy was performed. METHODS: Autopsy was performed in all cases, as part of routine evaluation of HIV-infected cases followed at Ullevaal University Hospital. Of 125 patients included, 53 had CMV disease, 37 of whom were first diagnosed at autopsy. CMV disease was diagnosed either by ophthalmoscopic findings typical of CMV retinitis, biopsy or autopsy. One or two plasma samples taken prior to the first diagnosis of CMV disease (alive or at autopsy) or death without CMV disease were analysed by CMV quantitative PCR. Sensitivity, specificity, positive and negative predictive values were calculated for different CMV viral load cut-offs and according to detection of viraemia in one versus two samples. RESULTS: Twenty-seven of 53 patients with CMV disease (51%) and 10 of 72 patients without CMV disease (14%) had detectable viraemia in at least one sample. Sensitivity and negative predictive value (NPV) of the test, maximised with a cut-off at the test's limit of detection of CMV viraemia (400 copies/mL), were 47% and 70%, respectively. With cut-off at 10 000 copies/mL, specificity and positive predictive value (PPV) were 100%. With a requirement for CMV viraemia in two samples, specificity and PPV were 100% in patients with CMV viraemia above the limit of detection. CONCLUSIONS: Our results indicate that quantitative CMV PCR is best used to rule in, rather than to rule out CMV disease in HIV-infected individuals at high risk. [Abstract/Link to Full Text]

Northey G, Evans MR, Sarvotham TS, Thomas DR, Howard TJ
Sentinel surveillance for travellers diarrhoea in primary care.
BMC Infect Dis. 2007 Nov 6;7(1):126.
ABSTRACT: BACKGROUND: Travellers diarrhoea is the most common health problem among international travellers and much of the burden falls on general practitioners. We assessed whether sentinel surveillance based in primary care could be used to monitor changes in the epidemiology of travellers diarrhoea. METHODS: A sentinel surveillance scheme of 30 volunteer general practices distributed throughout Wales provides weekly reports of consultations for eight infectious diseases to the national Communicable Disease Surveillance Centre. Travellers diarrhoea was introduced as a new reportable infection in July 2002. RESULTS: Between 1 July 2002 and 31 March 2005 there were 90 reports of travellers diarrhoea. The mean annual consultation rate was 15.2 per 100,000 population (95% confidence interval: 12.2-18.7), with the highest rates in summer, in people aged 15-24 years, and in travellers to Southern Europe. A higher proportion of travellers than expected had visited destinations outside Europe and North America when compared to the proportion of all United Kingdom travellers visiting these destinations (38% vs. 11%; Chi2 =53.3, p<0.0001). CONCLUSIONS: Sentinel surveillance has the potential to monitor secular trends in travellers diarrhoea and to help characterise population groups or travel destinations associated with higher risk. [Abstract/Link to Full Text]

Lloyd-Smith JO, Greig DJ, Hietala S, Ghneim GS, Palmer L, St Leger J, Grenfell BT, Gulland FM
Cyclical changes in seroprevalence of leptospirosis in California sea lions: endemic and epidemic disease in one host species?
BMC Infect Dis. 2007 Nov 6;7(1):125.
ABSTRACT: BACKGROUND: Leptospirosis is a zoonotic disease infecting a broad range of mammalian hosts, and is re-emerging globally. California sea lions (Zalophus californianus) have experienced recurrent outbreaks of leptospirosis since 1970, but it is unknown whether the pathogen persists in the sea lion population or is introduced repeatedly from external reservoirs. METHODS: We analyzed serum samples collected over an 11-year period from 1344 California sea lions that stranded alive on the California coast, using the microscopic agglutination test (MAT) for antibodies to Leptospira interrogans serovar Pomona. We evaluated seroprevalence among yearlings as a measure of incidence in the population, and characterized antibody persistence times based on temporal changes in the distribution of titer scores. We conducted multinomial logistic regression to determine individual risk factors for seropositivity with high and low titers. RESULTS: The serosurvey revealed cyclical patterns in seroprevalence to L. interrogans serovar Pomona, with 4-5 year periodicity and peak seroprevalence above 50%. Seroprevalence in yearling sea lions was an accurate index of exposure among all age classses, and indicated on-going exposure to leptospires in non-outbreak years. Analysis of titer decay rates showed that some individuals probably maintain high titers for more than a year following exposure. CONCLUSIONS: This study presents results of an unprecedented long-term serosurveillance program in marine mammals. Our results suggest that leptospirosis is endemic in California sea lions, but also causes periodic epidemics of acute disease. The findings call into question the classical dichotomy between maintenance hosts of leptospirosis, which experience chronic but largely asymptomatic infections, and accidental hosts, which suffer acute illness or death as a result of disease spillover from reservoir species. [Abstract/Link to Full Text]

Kupek EJ, de Souza DE, Petry A
Effectiveness of DNA-recombinant anti-hepatitis B vaccines in blood donors: a cohort study.
BMC Infect Dis. 2007 Nov 6;7(1):124.
ABSTRACT: BACKGROUND: Although various studies have demonstrated efficacy of DNA-recombinant anti-hepatitis B vaccines, their effectiveness in health care settings has not been researched adequately. This gap is particularly visible for blood donors, a group of significant importance in the reduction of transfusion-transmitted hepatitis B. METHODS: This is a double cohort study of 1411 repeat blood donors during the period 1998-2002, involving a vaccinated and an unvaccinated cohort, with matching of the two in terms of sex, age and residence. Average follow-up was 3.17 person-years. The outcome measure was infection with hepatitis B virus (HBV), defined by testing positive on serologic markers HBsAg or anti-HBC. All blood donors were from the blood bank in Joacaba, federal state of Santa Catarina, Brazil. RESULTS: The cohorts did not differ significantly regarding sex, age and marital status but the vaccinated cohort had higher mean number of blood donations and higher proportion of those residing in the county capital Joacaba. Hepatitis B incidences per 1000 person-years were zero among vaccinated and 2,33 among non-vaccinated, resulting in 100% vaccine effectiveness with 95% confidence interval from 30,1% to 100%. The number of vaccinated persons necessary to avoid one HBV infection in blood donors was estimated at 429 with 95% confidence interval from 217 to 21422. CONCLUSIONS: The results showed very high effectiveness of DNA-recombinant anti-HBV vaccines in blood donors. Its considerable variation in this study is likely due to the limited follow-up and the influence of confounding factors normally balanced out in efficacy clinical trials. [Abstract/Link to Full Text]

Regaya F, Oussaief L, Bejaoui M, Karoui M, Zili M, Khelifa R
Parvovirus B19 infection in Tunisian patients with sickle-cell anemia and acute erythroblastopenia.
BMC Infect Dis. 2007 Oct 25;7(1):123.
ABSTRACT: BACKGROUND: Human parvovirus B19 is the etiologic agent of erythema infectiosum in children. It is also associated with other clinical manifestations in different target groups. Patients with chronic hemolytic anemia are at high risk of developing acute erythroblastopenia following infection by the virus. They usually become highly viremic and pose an increased risk of virus transmission. Close monitoring of such high risk groups is required for epidemiologic surveillance and disease prevention activities. Here we report a molecular epidemiological study on B19 virus infection in Tunisian patients with chronic hemolytic anemia. METHODS: This study was conducted on 92 young chronic hemolytic anemia patients who attended the same ward at the National Bone Marrow Transplantation Center of Tunis and 46 controls from a different hospital. Screening for IgM and IgG anti-B19 antibodies was performed using commercially available enzyme immunoassays and B19 DNA was detected by nested PCR in the overlapping VP1/VP2 region. DNA was sequenced using dideoxy-terminator cycle sequencing technology. Results Anti-parvovirus B19 IgG antibodies were detected in 26 of 46 sickle-cell anemia patients, 18 of 46 beta-thalassemia and 7 of 46 controls. Anti-parvovirus B19 IgM antibodies were detected only in 4 of the sickle-cell anemia patients: two siblings and two unrelated who presented with acute erythroblastopenia at the time of blood collection for this study and had no history of past transfusion. B19 DNA was detected only in sera of these four patients and the corresponding 288 bp nested DNA amplicons were sequenced. The sequences obtained were all identical and phylogenetic analysis showed that they belonged to a new B19 virus strain of Genotype1. Conclusions A new parvovirus B19 strain of genotype1 was detected in four Tunisian patients with sickle-cell anemia. Virus transmission appeared to be nosocomial and resulted in acute erythroblastopenia in the four patients. The possibility of independent transmission of this B19 variant to the patients is unlikely in light of the present epidemiological data. However this possibility cannot be ruled out because of the low genetic variability of the virus. [Abstract/Link to Full Text]

Adetifa IM, Lugos MD, Hammond A, Jeffries D, Donkor S, Adegbola RA, Hill PC
Comparison of Two Interferon Gamma Release Assays in the diagnosis of Mycobacterium tuberculosis infection and disease in The Gambia.
BMC Infect Dis. 2007 Oct 25;7(1):122.
ABSTRACT: BACKGROUND: IFN-gamma Release Assays (IGRAs) have been licensed for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Their performance may depend on assay format and may vary across populations and settings. We compared the diagnostic performance of an in-house T -cell and commercial whole blood-based IGRAs for the diagnosis of LTBI and TB disease in The Gambia. METHODS: Newly diagnosed sputum smear positive cases and their household contacts were recruited. Cases and contacts were bled for IGRA and contacts had a Mantoux skin test. We assessed agreement and discordance between the tests and categorized a contact's level of M.tuberculosis exposure according to where s/he slept relative to a case: the same room, same house or a different house. We assessed the relationship between exposure and test results by multiple logistic regression. RESULTS: In 80 newly diagnosed TB cases, the sensitivity of ELISPOT was 78.7% and for QFT-GIT was 64.0% (p=0.047). Of 194 household contacts 57.1% and 58.8% were positive for ELISPOT and QFT-GIT respectively. The overall agreement between both IGRAs for LTBI in contacts was 71.4% and there was no significant discordance (p=0.29). There was significant discordance between the IGRAs and TST. Neither IGRA nor TST had evidence of false positive results because of Bacille Calmette Guerin (BCG) vaccination. However, agreement between QFT-GIT and TST as well as discordance between both IGRAs and TST were associated with BCG vaccination. Both IGRAs responded to the M.tuberculosis exposure gradient and were positively associated with increasing TST induration (p=0.003 for ELISPOT and p=0.001 for QFT-GIT). CONCLUSIONS: The ELISPOT test is more sensitive than the QFT-GIT for diagnosing TB disease. The two tests perform similarly in the diagnosis of LTBI in TB contacts. Significant discordance between the two IGRAs and between each and the TST remains largely unexplained. [Abstract/Link to Full Text]

Killeen GF, Tami A, Kihonda J, Okumu FO, Kotas ME, Grundmann H, Kasigudi N, Ngonyani H, Mayagaya V, Nathan R, Abdulla S, Charlwood JD, Smith TA, Lengeler C
Cost-sharing strategies combining targeted public subsidies with private-sector delivery achieve high bednet coverage and reduced malaria transmission in Kilombero Valley, southern Tanzania.
BMC Infect Dis. 2007 Oct 25;7(1):121.
ABSTRACT: BACKGROUND: Cost-sharing schemes incorporating modest targeted subsidies have promoted insecticide-treated nets (ITNs) for malaria prevention in the Kilombero Valley, southern Tanzania, since 1996. Here we evaluate resulting changes in bednet coverage and malaria transmission. METHODS: Bednets were sold through local agents at fixed prices representing a 34% subsidy relative to full delivery cost. A further targeted subsidy of 15% was provided to vulnerable groups through discount vouchers delivered through antenatal clinics and regular immunizations. Continuous entomological surveys (2,376 trap nights) were conducted from October 2001 to September 2003 in 25 randomly-selected population clusters of a demographic surveillance system which monitored net coverage. RESULTS: Mean net usage of 75 % (11,982/16,086) across all age groups was achieved but now-obsolete technologies available at the time resulted in low insecticide treatment rates. Malaria transmission remained intense but was substantially reduced: Compared with an exceptionally high historical mean EIR of 1481, even non-users of nets were protected (EIR [fold reduction] = 349 infectious bites per person per year [x4]), while the average resident (244 [x6]), users of typical nets (210 [x7]) and users of insecticidal nets (105 [x14]) enjoyed increasing benefits. CONCLUSION: Despite low net treatment levels, community-level protection was equivalent to the personal protection of an ITN. Greater gains for net users and non-users are predicted if more expensive long-lasting ITN technologies can be similarly promoted with correspondingly augmented subsidies. Cost sharing strategies represent an important option for national programmes lacking adequate financing to fully subsidize comprehensive ITN coverage. [Abstract/Link to Full Text]

Zeeshan M, Jabeen K, Akbar AN, Ali AW, Farooqui SZ, Mehraj V, Zafar A
Evaluation of immune response to Hepatitis B Vaccine in health care workers at a tertiary care hospital in Pakistan: an observational prospective study.
BMC Infect Dis. 2007 Oct 25;7(1):120.
ABSTRACT: BACKGROUND: Seroconversion rates reported after Hepatitis B virus (HBV) vaccination globally ranges from 85-90%. Health care workers (HCWs) are at high risk of acquiring HBV and non responder's rate after HBV vaccination were not reported previously in Pakistani HCWs. Therefore we evaluated immune response to HBV vaccine in HCWs at a tertiary care hospital in Karachi, Pakistan. METHOD: Descriptive observational study conducted at Aga Khan University Hospital (AKUH) from April 2003 to July 2004. Newly HBV vaccinated HCWs were evaluated for immune response by measuring serum Hepatitis B surface antibody (HBsAb) levels, 6 weeks post vaccination. RESULTS: Initially 666 employees were included in the study. 14 participants were excluded due to incomplete records. 271 (41%) participants were females and 381(59%) were males. Majority of the participants were young (<25-39 years old), regardless of gender. Out of 652 HCWs, 90 (14%) remained seronegative after six weeks of post vaccination. The percentage of non responders increased gradually from 9% in participants of <25, 13% in 25-34, 26% in 35-49, and 63% in >50 years of age. Male non responders were more frequent (18%) than female (8%). Male gender and older age were significantly associated with non-response. CONCLUSION: Seroconversion rate after HBV vaccination in Pakistani HCWs was similar to that reported in western and neighboring population. HCWs with reduced immune response to HBV vaccine in a high disease prevalent population are at great risk. Therefore, it is crucial to check post vaccination HBsAb in all HCWs. This strategy will ensure safety at work by reducing nosocomial transmission and will have a cost effective impact at an individual as well as at national level, which is very much desired in a resource limited country. [Abstract/Link to Full Text]

Veras MA, Enanoria WT, Castilho EA, Reingold AL
Effectiveness of the polysaccharide pneumococcal vaccine among HIV-infected persons in Brazil: a case control study.
BMC Infect Dis. 2007 Oct 23;7(1):119.
ABSTRACT: BACKGROUND: Polysaccharide pneumococcal vaccine is recommended for use in HIV-infected adults in Brazil but there is uncertainty about its effectiveness in this patient population. The main objective of this study was to assess the effectiveness of the 23-valent polysaccharide pneumococcal vaccine against invasive pneumococcal infection among HIV-infected adult patients in Sao Paulo, Brazil. METHODS: A case-control study of 79 cases and 242 controls matched on CD4+ cell count and health care setting was conducted. Among HIV-infected adults in Sao Paulo, Brazil, with and without S. pneumoniae recovered from a normally sterile site; prior receipt of 23 valent polysaccharide pneumococcal vaccine was determined by review of medical records and patient interview. RESULTS: After adjustment for confounding factors, the point estimate for the effectiveness of 23 valent polysaccharide vaccine among HIV-infected adults against all invasive pneumococcal infection was 18% (95% CI: <0 to 62%). CONCLUSIONS: We were unable to demonstrate a statistically significant protective effect of 23 valent polysaccharide against invasive pneumococcal infection vaccine among HIV-infected adults in Brazil. While the vaccine is relatively inexpensive and safe, its effectiveness among HIV-infected adults in Brazil is uncertain. [Abstract/Link to Full Text]

Chen J, Zhang F, Fang F, Chang H, Chen Z
Vaccination with hemagglutinin or neuraminidase DNA protects BALB/c mice against influenza virus infection in presence of maternal antibody.
BMC Infect Dis. 2007 Oct 16;7(1):118.
ABSTRACT: BACKGROUND: Maternal antibody is the major form of protection against disease in early life; however, its presence interferes with active immunization of offspring. In order to overcome the immunosuppression caused by maternal antibody, several immune strategies were explored in this paper using mouse model and influenza vaccines. RESULTS: The results showed that: i) when the offspring were immunized with the same vaccine as their mothers, whether inactivated or DNA vaccine, the presence of maternal antibody inhibited offspring immune response and the offspring could not be protected from a lethal influenza virus infection; ii) when the offspring, born to mothers immunized with inactivated vaccine, were immunized with NA DNA vaccine, the interference of maternal antibody were overcome and the offspring could survive a lethal virus challenge; iii) when the offspring were immunized with different DNA vaccine from that for their mothers , the interference of maternal antibody were also overcome. In addition, high-dose inactivated vaccine in maternal immunization caused partial inhibition in offspring when the offspring were immunized with HA DNA vaccine, while lower dose caused no significant immunosuppression. CONCLUSIONS: To avoid the interference of maternal antibody in influenza vaccination of offspring, mothers and their offspring shall not be immunized with the same vaccine. If mothers are immunized with inactivated vaccine, NA DNA vaccine for the offspring shall be effective; and if mothers are immunized with HA (NA) DNA, NA (HA) DNA for the offspring shall be effective. [Abstract/Link to Full Text]

Kramer A, Below H, Bieber N, Kampf G, Toma CD, Huebner NO, Assadian O
Quantity of ethanol absorption after excessive hand disinfection using three commercially available hand rubs is minimal and below toxic levels for humans.
BMC Infect Dis. 2007;7117.
BACKGROUND: Despite the increasing promotion of alcohol-based hand rubs and the worldwide use of ethanol-based hand rubs in hospitals only few studies have specifically addressed the issue of ethanol absorption when repeatedly applied to human skin. The aim of this study was to assess if ethanol absorption occurs during hygienic and surgical hand disinfection using three different alcohol-based hand-rubs, and to quantify absorption levels in humans. METHODS: Twelve volunteers applied three hand-rubs containing 95% (hand-rub A), 85% (hand-rub B) and 55% ethanol (hand-rub C; all w/w). For hygienic hand disinfection, 4 mL were applied 20 times for 30 s, with 1 minute break between applications. For surgical hand disinfection, 20 mL of each hand rub was applied to hands and arms up to the level of the elbow 10 times for 3 minutes, with a break of 5 minutes between applications. Blood concentrations of ethanol and acetaldehyde were determined immediately prior and up to 90 minutes after application using head space gas chromatography. RESULTS: The median of absorbed ethanol after hygienic hand disinfection was 1365 mg (A), 630 mg (B), and 358 mg (C). The proportion of absorbed ethanol was 2.3% (A), 1.1% (B), and 0.9% (C). After surgical hand disinfection, the median of absorbed ethanol was 1067 mg (A), 1542 mg (B), and 477 mg (C). The proportion of absorbed ethanol was 0.7% (A), 1.1% (B), and 0.5% (C). The highest median acetaldehyde concentration after 20 hygienic hand disinfections was 0.57 mg/L (hand-rub C, after 30 min), after 10 surgical hand disinfections 3.99 mg/L (hand-rub A, after 20 minutes). CONCLUSION: The overall dermal and pulmonary absorption of ethanol was below toxic levels in humans and allows the conclusion that the use of the evaluated ethanol-based hand-rubs is safe. [Abstract/Link to Full Text]

Trampuz A, Steinhuber A, Wittwer M, Leib SL
Rapid Diagnosis of experimental meningitis by bacterial heat production in cerebrospinal fluid.
BMC Infect Dis. 2007 Oct 10;7(1):116.
ABSTRACT: BACKGROUND: Calorimetry is a nonspecific technique which allows direct measurement of heat generated by biological processes in the living cell. We evaluated the potential of calorimetry for rapid detection of bacterial growth in cerebrospinal fluid (CSF) in a rat model of bacterial meningitis. METHODS: Infant rats were infected on postnatal day 11 by direct intracisternal injection with either Streptococcus pneumoniae, Neisseria meningitidis or Listeria monocytogenes. Control animals were injected with sterile saline or heat-inactivated S. pneumoniae. CSF was obtained at 18 hours after infection for quantitative cultures and heat flow measurement. For calorimetry, 10 microL and 1 microL CSF were inoculated in calorimetry ampoules containing 3 mL trypticase soy broth (TSB). RESULTS: The mean bacterial titer (+/- SD) in CSF was 1.5 +/- 0.6 x 10E8 for S. pneumoniae, 1.3 +/- 0.3 x 10E6 for N. meningitidis and 3.5 +/- 2.2 x 10E4 for L. monocytogenes. Calorimetric detection time was defined as the time until heat flow signal exceeded 10 microW. Heat signal was detected in 10 microL CSF samples from all infected animals with a mean (+/- SD) detection time of 1.5 +/- 0.2 hours for S. pneumoniae, 3.9 +/- 0.7 hours for N. meningitidis and 9.1 +/- 0.5 hours for L. monocytogenes. CSF samples from non-infected animals generated no detectable heat (<10 microW). The total heat was the highest in S. pneumoniae ranging from 6.7 to 7.5 Joules, followed by L. monocytogenes (5.6 to 6.1 Joules) and N. meningitidis (3.5 to 4.4 Joules). The lowest detectable bacterial titer by calorimetry was 2 cfu for S. pneumoniae, 4 cfu for N. meningitidis and 7 cfu for L. monocytogenes. CONCLUSIONS: By means of calorimetry, detection times of <4 hours for S. pneumoniae and N. meningitidis and <10 hours for Listeria monocytogenes using as little as 10 micoL CSF were achieved. Calorimetry is a new diagnostic method allowing rapid and accurate diagnosis of bacterial meningitis from a small volume of CSF. [Abstract/Link to Full Text]

---

Recent Articles in BMC Microbiology

---

Yean Yean C, Su Yin L, Lalitha P, Ravichandran M
A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species.
BMC Microbiol. 2007 Dec 11;7(1):112.
ABSTRACT: BACKGROUND: Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE). This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene. RESULTS: Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to E. faecalis, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases. CONCLUSION: The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common Enterococcus spp. and their antibiotic sensitivity pattern. The PCR assay developed in this study can be used as an effective surveillance tool to study the prevalence of enterococci and their antibiotic resistance pattern in hospitals and farm animals. [Abstract/Link to Full Text]

Kloppenburg GT, de Graaf R, Grauls GE, Bruggeman CA, Stassen FR
Chlamydia pneumoniae aggravates vein graft intimal hyperplasia in a rat model.
BMC Microbiol. 2007 Dec 6;7(1):111.
ABSTRACT: BACKGROUND: Along with angioplasty, autologus vein grafts are commonly used for artery bypass grafting in patients with advanced arterial stenosis and drug-resistant angina pectoris. Although initially a successful procedure, long-term functionality is limited due to proliferation and migration of smooth muscle cells. Like in atherosclerosis, common chronic infections caused by viruses and bacteria may contribute to this process of vein graft failure. Here we investigated the possible role of Chlamydia pneumoniae (Cpn) in the pathogenesis of venous graft failure in an experimental animal model. In 2 groups (n=10 rats/group), an epigastric vein-to-common femoral artery interposition graft was placed. Immediately thereafter, rats were infected with Cpn (5*108 IFU) or injected with control solutions. Rats were sacrificed three weeks after surgery and the grafts were harvested for morphometrical and immunohistochemical analysis. RESULTS: Cpn administration immediately after vein grafting resulted in a significant increase in medial cross-sectional area, wall thickness and total wall area. There were no significant differences in T-cell or macrophage influx. Likewise, although positive immunostaining for both HSP60 and CRP could be detected, no differences were found between groups. Based on the observation that the number of cells/mm2 was also not altered, we conclude that Cpn infection stimulates smooth muscle cell proliferation by hereunto unknown molecular mechanisms, resulting in a significant increase in intimal hyperplasia. CONCLUSION: In conclusion, in a well defined animal model we present here for the first time evidence for a role of Chlamydia pneumoniae in the process of venous graft failure. [Abstract/Link to Full Text]

Bouzidi MF, Parlange F, Nicolas P, Mouzeyar S
Expressed Sequence Tags from the oomycete Plasmopara halstedii, an obligate parasite of the sunflower.
BMC Microbiol. 2007 Dec 6;7(1):110.
ABSTRACT: BACKGROUND: Sunflower downy mildew is a major disease caused by the obligatory biotrophic oomycete Plasmopara halstedii. Little is known about the molecular mechanisms underlying its pathogenicity. In this study we used a genomics approach to gain a first insight into the transcriptome of P. halstedii. RESULTS: To identify genes from the obligatory biotrophic oomycete Plasmopara halstedii that are expressed during infection in sunflower (Helianthus annuus L.) we employed the suppression subtraction hybridization (SSH) method from sunflower seedlings infected by P. halstedii. Using this method and random sequencing of clones, a total of 602 expressed sequence tags (ESTs) corresponding to 230 unique sequence sets were identified. To determine the origin of the unisequences, PCR primers were designed to amplify these gene fragments from genomic DNA isolated either from P. halstedii sporangia or from Helianthus annuus. Only 145 nonredundant ESTs which correspond to a total of 373 ESTs (67.7%) proved to be derived from P. halstedii genes and that are expressed during infection in sunflower. A set of 87 nonredundant sequences were identified as showing matches to sequences deposited in public databases. Nevertheless, about 7% of the ESTs seem to be unique to P. halstedii without any homolog in any public database. CONCLUSIONS: A summary of the assignment of nonredundant ESTs to functional categories as well as their relative abundance is listed and discussed. Annotation of the ESTs revealed a number of genes that could function in virulence. We provide a first glimpse into the gene content of P. halstedii. These resources should accelerate research on this important pathogen. [Abstract/Link to Full Text]

Lee JE, Reed J, Shields MS, Spiegel KM, Farrell LD, Sheridan PP
Phylogenetic analysis of Shiga toxin 1 and Shiga toxin 2 genes associated with disease outbreaks.
BMC Microbiol. 2007 Dec 4;7(1):109.
ABSTRACT: BACKGROUND: Shiga toxins 1 and 2 (Stx1 and Stx2) are bacteriophage-encoded proteins that have been associated with hemorrhagic colitis, hemolytic uremic syndrome and other severe disease conditions. Stx1 and Stx2 are genetically and immunologically distinct but share the same compound toxin structure, method of entry and enzymatic function. RESULTS: Phylogenetic analysis was performed using Stx1 and Stx2 amino acid and nucleotide sequences from 41 strains of Escherichia coli, along with known stx sequences available from GenBank. The analysis confirmed the Stx1 and Stx2 divergence, and showed that there is generally more sequence variation among stx2 genes than stx1. The phylograms showed generally flat topologies among our strains' stx1 and stx2 genes. In the stx2 gene, 39.5% of the amino acid sites display very low nonsynonymous to synonymous substitution ratios. CONCLUSIONS: The stx1 and stx2 genes used in this phylogenetic study show sequence conservation with no significant divergence with respect to place or time. These data could indicate that Shiga toxins are experiencing purifying selection. [Abstract/Link to Full Text]

Sundquist A, Bigdeli S, Jalili R, Druzin ML, Waller S, Pullen KM, El-Sayed YY, Taslimi MM, Batzoglou S, Ronaghi M
Bacterial flora typing with deep, targeted, chip-based Pyrosequencing.
BMC Microbiol. 2007 Nov 30;7(1):108.
ABSTRACT: BACKGROUND: The metagenomic analysis of microbial communities holds the potential to improve our understanding of the role of microbes in clinical conditions. Recent, dramatic improvements in DNA sequencing throughput and cost will enable such analyses on individuals. However, such advances in throughput generally come at the cost of shorter read-lengths, limiting the discriminatory power of each read. In particular, classifying the microbial content of samples by sequencing the 1,600 bp 16S rRNA gene will be affected by such limitations. RESULTS: We describe a method for identifying the phylogenetic content of bacterial samples using deep, high-throughput Pyrosequencing targeted at the 16S rRNA gene. Our analysis is adapted to the shorter read-lengths of such technology and uses a database of 16S rDNA to determine the most specific phylogenetic classification for reads, resulting in a weighted phylogenetic tree characterizing the content of the sample. We present results for six samples obtained from the human vagina during pregnancy that corroborates previous studies using conventional techniques. Next, we analyze the power of our method to classify reads at each level of the phylogeny using simulation experiments. We assess the impacts of read-length and database completeness on our method, and predict how we do as technology improves and more bacteria are sequenced. Finally, we study the utility of targeting specific 16S variable regions and show that such an approach considerably improves results for certain types of microbial samples. Using simulation, our method can be used to determine the most informative variable region. CONCLUSIONS: This study provides positive validation of the effectiveness of targeting 16S metagenomes using short-read sequencing technology. Our methodology allows us to infer the most specific assignment of the sequence reads within the phylogeny, and to identify the most discriminative variable region to target. The analysis of deep, high-throughput Pyrosequencing on human flora samples will accelerate the study of the relationship between the microbial world and ourselves. [Abstract/Link to Full Text]

Valle-Aviles L, Valentin-Berrios S, Gonzalez-Mendez RR, Rodriguez-Del Valle N
Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii.
BMC Microbiol. 2007 Nov 29;7(1):107.
ABSTRACT: BACKGROUND: Sporothrix schenckii is pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in S. schenckii responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in S. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle. RESULTS: Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of sscmk1 revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of sscmk1 revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 - 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition. CONCLUSIONS: This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in S. schenckii and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus. The results suggest that the calcium/calmodulin kinases of yeasts are evolutionarily distinct from those in filamentous fungi. [Abstract/Link to Full Text]

Gupta RS, Mok A
Phylogenomics and signature proteins for the alpha Proteobacteria and its main groups.
BMC Microbiol. 2007 Nov 28;7(1):106.
ABSTRACT: BACKGROUND: Alpha proteobacteria are one of the largest and most extensively studied groups within bacteria. However, for these bacteria as a whole and for all of its major subgroups (viz. Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales, Sphingomonadales and Caulobacterales), very few or no distinctive molecular or biochemical characteristics are known. RESULTS: We have carried out comprehensive phylogenomic analyses by means of Blastp and PSI-Blast searches on the open reading frames in the genomes of several a-proteobacteria (viz. Bradyrhizobium japonicum, Brucella suis, Caulobacter crescentus, Gluconobacter oxydans, Mesorhizobium loti, Nitrobacter winogradskyi, Novosphingobium aromaticivorans, Rhodobacter sphaeroides 2.4.1, Silicibacter sp. TM1040, Rhodospirillum rubrum and Wolbachia (Drosophila) endosymbiont). These studies have identified several proteins that are distinctive characteristics of all a-proteobacteria, as well as numerous proteins that are unique repertoires of all of its main orders (viz. Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales, Sphingomonadales and Caulobacterales) and many families (viz. Rickettsiaceae, Anaplasmataceae, Rhodospirillaceae, Acetobacteraceae, Bradyrhiozobiaceae, Brucellaceae and Bartonellaceae). Many other proteins that are present at different phylogenetic depths in a-proteobacteria provide important information regarding their evolution. The evolutionary relationships among a-proteobacteria as deduced from these studies are in excellent agreement with their branching pattern in phylogenetic trees and character compatibility cliques based on concatenated sequences for many conserved proteins. These studies provide evidence that the major groups within a-proteobacteria have diverged in the following order: (Rickettsiales(Rhodospirillales(Sphingomonadales (Rhodobacterales(Caulobacterales-Parvularculales(Rhizobiales)))))). We also describe two conserved inserts in DNA Gyrase B and RNA polymerase beta subunit that are distinctive characteristics of the Sphingomonadales and Rhodosprilllales species, respectively. The results presented here also provide support for the grouping of Hyphomonadaceae and Parvularcula species with the Caulobacterales and the placement of Stappia aggregata with the Rhizobiaceae group. CONCLUSIONS: The a-proteobacteria-specific proteins and indels described here provide valuable novel tools for the taxonomic, biochemical and molecular biological studies on these bacteria. Their functional studies should prove helpful in identifying novel biochemical and physiological characteristics that are unique to these bacteria. [Abstract/Link to Full Text]

Tadesse S, Graumann PL
DprA/Smf protein localizes at the DNA uptake machinery in competent Bacillus subtilis cells.
BMC Microbiol. 2007 Nov 28;7(1):105.
ABSTRACT: BACKGROUND: DprA is a widely conserved bacterial protein and has been shown to confer an important function during transformation in competent cells, possibly through protection of incoming DNA. B. subtilis DprA (called Smf) has been shown to play an important role during transformation with chromosomal DNA, but its mode of action is unknown. RESULTS: We show that B. subtilis DprA/Smf is more important for transformation with plasmid DNA than with chromosomal DNA. A functional Smf-YFP fusion localized as discrete foci to the cell pole in a subset of cells grown to competence, dependent on the ComK master transcription factor. Smf-YFP foci colocalized with ComGA-CFP. However, a considerable number of cells having high ComK activity contained Smf dispersed throughout the cytosol and lacked a polar Smf assembly. The absence of polar Smf-YFP foci in these cells strongly correlated with the absence of ComGA-CFP foci, and comGA mutant cells mostly lacked polar Smf-YFP foci. Smf formed polar assemblies in the absence of RecA, and RecA formed dynamic threads after addition of DNA in a smf deletion strain. Upon addition of DNA, Smf-YFP foci relocalized from the poles to the cell centre, dependent on the presence of RecA protein. CONCLUSIONS: Our data show that Smf is recruited to the polar competence machinery, and that polar Smf assembly requires a functional DNA uptake complex. High ComK levels drive expression of Smf in 20% of all cells grown to competence, but not all competent cells contain a polar DNA uptake machinery, showing that ComK activity is necessary but not sufficient to achieve assembly of the uptake machinery in all cells. Smf and RecA localize independently of each other, in agreement with our finding that Smf is much more important for plasmid transformation than RecA, but RecA influences the dynamic localization pattern of Smf. Our data show that DprA/Smf acts downstream of the DNA uptake machinery, and support the idea that Smf protects incoming ssDNA, possibly in conjunction with RecA. [Abstract/Link to Full Text]

He ZM, Price MS, Obrian GR, Georgianna DR, Payne GA
Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus.
BMC Microbiol. 2007 Nov 26;7(1):104.
ABSTRACT: BACKGROUND: An available whole genome sequence for Aspergillus flavus provides the opportunity to characterize factors involved in pathogenicity and to elucidate the regulatory networks involved in aflatoxin biosynthesis. Functional analysis of genes within the genome is greatly facilitated by the ability to disrupt or mis-express target genes and then evaluate their result on the phenotype of the fungus. Large-scale functional analysis requires an efficient genetic transformation system and the ability to readily select transformants with altered expression, and usually requires generation of double (or multi) gene deletion strains or the use of prototrophic strains. However, dominant selectable markers, an efficient transformation system and an efficient screening system for transformants in A. flavus are absent. RESULTS: The efficiency of the genetic transformation system for A. flavus based on uracil auxotrophy was improved. In addition, A. flavus was shown to be sensitive to the antibiotic, phleomycin. Transformation of A. flavus with the ble gene for resistance to phleomycin resulted in stable transformants when selected on 100 ug/ml phleomycin. We also compared the phleomycin system with one based on complementation for uracil auxotrophy which was confirmed by uracil and 5-fluoroorotic acid selection and via transformation with the pyr4 gene from Neurospora crassa and pyrG gene from A. nidulans in A. flavus NRRL 3357. A transformation protocol using pyr4 as a selectable marker resulted in site specific disruption of a target gene. A rapid and convenient colony PCR method for screening genetically altered transformants was also developed in this study. CONCLUSIONS: We employed phleomycin resistance as a new positive selectable marker for genetic transformation of A. flavus. The experiments outlined herein constitute the first report of the use of the antibiotic phleomycin for transformation of A. flavus. Further, we demonstrated that this transformation protocol can be used for directed gene disruption in A. flavus. The significance of this is twofold. First, it allows strains to be transformed without having to generate an auxotrophic mutation, which is time consuming and may result in undesirable mutations. Second, this protocol allows for double gene knockouts when used in conjunction with existing strains with auxotrophic mutations. To further facilitate functional analysis in this strain we developed a colony PCR-based method that is a rapid and convenient method for screening genetically altered transformants. This work will be of interest to those working on molecular biology of aflatoxin metabolism in A. flavus, especially for functional analysis using gene deletion. [Abstract/Link to Full Text]

Mello FC, Souto FJ, Nabuco LC, Villela-Nogueira CA, Coelho HS, Franz HC, Saraiva JC, Virgolino HA, Motta-Castro AR, Melo MM, Martins RM, Gomes SA
Hepatitis B virus genotypes circulating in Brazil: molecular characterization of genotype F isolates.
BMC Microbiol. 2007 Nov 23;7(1):103.
ABSTRACT: BACKGROUND: Hepatitis B virus (HBV) isolates have been classified in eight genotypes, A to H, which exhibit distinct geographical distributions. Genotypes A, D and F are predominant in Brazil, a country formed by a miscegenated population, where the proportion of individuals from Caucasian, Amerindian and African origins varies by region. Genotype F, which is the most divergent, is considered indigenous to the Americas. A systematic molecular characterization of HBV isolates from different parts of the world would be invaluable in establishing HBV evolutionary origins and dispersion patterns. A large-scale study is needed to map the region-by-region distribution of the HBV genotypes in Brazil. RESULTS: Genotyping by PCR-RFLP of 303 HBV isolates from HBsAg-positive blood donors showed that at least two of the three genotypes, A, D, and F, co-circulate in each of the five geographic regions of Brazil. No other genotypes were identified. Overall, genotype A was most prevalent (48.5%), and most of these isolates were classified as subgenotype A1 (138/153; 90.2%). Genotype D was the most common genotype in the South (84.2%) and Central (47.6%) regions. The prevalence of genotype F was low (13%) countrywide. Nucleotide sequencing of the S gene and a phylogenetic analysis of 32 HBV genotype F isolates showed that a great majority (28/32; 87.5%) belonged to subgenotype F2, cluster II. The deduced serotype of 31 of 32 F isolates was adw4. The remaining isolate showed a leucine-to-isoleucine substitution at position 127. CONCLUSIONS: The presence of genotypes A, D and F, and the absence of other genotypes in a large cohort of HBV infected individuals may reflect the ethnic origins of the Brazilian population. The high prevalence of isolates from subgenotype A1 (of African origin) indicates that the African influx during the colonial slavery period had a major impact on the circulation of HBV genotype A currently found in Brazil. Although most genotype F isolates belonged to cluster II, the presence of some isolates belonging to clusters I (subgroup Ib) and IV suggests the existence of two or more founder viral populations of genotype F in Brazil. [Abstract/Link to Full Text]

Teruya H, Higa F, Akamine M, Ishikawa C, Okudaira T, Tomimori K, Mukaida N, Tateyama M, Heuner K, Fujita J, Mori N
Mechanisms of Legionella pneumophila-induced interleukin-8 expression in human lung epithelial cells.
BMC Microbiol. 2007 Nov 22;7(1):102.
ABSTRACT: BACKGROUND: Legionella pneumophila is a facultative intracellular bacterium, capable of replicating within the phagosomes of macrophages and monocytes, but little is known about its interaction with human lung epithelial cells. We investigated the effect of L. pneumophila on the expression of interleukin-8 (IL-8) in human A549 alveolar and NCI-H292 tracheal epithelial cell lines. RESULTS: Infection of L. pneumophila strain, but not heat-killed strain, resulted in upregulation of IL-8. IL-8 mRNA expression was induced immediately after the infection and its signal became gradually stronger until 24 h after infection. On the other hand, IL-8 expression in A549 cells infected with L. pneumophila lacking a functional type IV secretion system was transient. The IL-8 expression was slightly induced at 16 h and increased at 24 h after infection with flagellin-deficient Legionella. Activation of the IL-8 promoter by L. pneumophila infection occurred through the action of nuclear factor-kappa B (NF-kappa B). Transfection of dominant negative mutants of NF-kappa B-inducing kinase, I kappa B kinase and I kappa B inhibited L. pneumophila-mediated activation of IL-8 promoter. Treatment with hsp90 inhibitor suppressed L. pneumophila-induced IL-8 mRNA due to deactivation of NF-kappa B. CONCLUSIONS: Collectively, these results suggest that L. pneumophila induces activation of NF-kappa B through an intracellular signaling pathway that involves NF-kappa B-inducing kinase and I kappa B kinase, leading to IL-8 gene transcription, and that hsp90 acts as a crucial regulator in L. pneumophila-induced IL-8 expression, presumably contributing to immune response in L. pneumophila. The presence of flagellin and a type IV secretion system are critical for Legionella to induce IL-8 expression in lung epithelial cells. [Abstract/Link to Full Text]

Cleusix V, Lacroix C, Vollenweider S, Duboux M, Le Blay G
Inhibitory activity spectrum of reuterin produced by Lactobacillus reuteri against intestinal bacteria.
BMC Microbiol. 2007 Nov 12;7(1):101.
ABSTRACT: BACKGROUND: Reuterin produced from glycerol by Lactobacillus reuteri, a normal inhabitant of the human intestine, is a broad-spectrum antimicrobial agent. It has been postulated that reuterin could play a role in the probiotic effects of Lb. reuteri. Reuterin is active toward enteropathogens, yeasts, fungi, protozoa and viruses, but its effect on commensal intestinal bacteria is unknown. Moreover reuterin's mode of action has not yet been elucidated. Glutathione, a powerful antioxidant, which also plays a key role in detoxifying reactive aldehydes, protects certain bacteria from oxidative stress, and could also be implicated in resistance to reuterin. The aim of this work was to test the activity of reuterin against a representative panel of intestinal bacteria and to study a possible correlation between intracellular low molecular weight thiols (LMW-SH) such as glutathione, hydrogen peroxide and/or reuterin sensitivity. Reuterin was produced by Lb. reuteri SD2112 in pure glycerol solution, purified and used to test the minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC). Hydrogen peroxide sensitivity and intracellular LMW-SH concentration were also analysed. RESULTS: Our data showed that most tested intestinal bacteria showed MIC below that for a sensitive indicator Escherichia coli (7.5-15 mM). Lactobacilli and Clostridium clostridioforme were more resistant with MIC ranging from 15 to 50 mM. No correlation between bacterial intracellular concentrations of LMW-SH, including glutathione, and reuterin or hydrogen peroxide sensitivities were found. CONCLUSIONS: Our data showed that intestinal bacteria were very sensitive to reuterin and that their intracellular concentration of LMW-SH was not directly linked to their capacity to resist reuterin or hydrogen peroxide. This suggests that detoxification by LMW-SH such as glutathione is not a general mechanism and that other mechanisms are probably involved in bacterial tolerance to reuterin and hydrogene peroxide. [Abstract/Link to Full Text]

Link S, Schmitt K, Beier D, Gross R
Identification and regulation of expression of a gene encoding a filamentous hemagglutinin-related protein in Bordetella holmesii.
BMC Microbiol. 2007 Nov 7;7(1):100.
ABSTRACT: BACKGROUND: Bordetella holmesii is a human pathogen closely related to B. pertussis, the etiological agent of whooping cough. It is able to cause disease in immunocompromised patients, but also whooping cough-like symptoms in otherwise healthy individuals. However, virtually nothing was known so far about the underlying virulence mechanisms and previous attempts to identify virulence factors related to those of B. pertussis were not successful. RESULTS: By use of a PCR approach we were able to identify a B. holmesii gene encoding a protein with significant sequence similarities to the filamentous hemagglutinin (FHA) of B. avium and to a lesser extent to the FHA proteins of B. pertussis, B. parapertussis, and B. bronchiseptica. For these human and animal pathogens FHA is a crucial virulence factor required for successful colonization of the host. Interestingly, the B. holmesii protein shows a relatively high overall sequence similarity with the B. avium protein, while sequence conservation with the FHA proteins of the human and mammalian pathogens is quite limited and is most prominent in signal sequences required for their export to the cell surface. In the other Bordetellae expression of the fhaB gene encoding FHA was shown to be regulated by the master regulator of virulence, the BvgAS two-component system. Recently, we identified orthologs of BvgAS in B. holmesii, and here we show that this system also contributes to regulation of fhaB expression in B. holmesii. Accordingly, the purified BvgA response regulator of B. holmesii was shown to bind specifically in the upstream region of the fhaB promoter in vitro in a manner similar to that previously described for the BvgA protein of B. pertussis. Moreover, by deletion analysis of the fhaB promoter region we show that the BvgA binding sites are relevant for in vivo transcription from this promoter in B. holmesii. CONCLUSION: The data reported here show that B. holmesii is endowed with a factor highly related to filamentous hemagglutinin (FHA), a prominent virulence factor of the well characterized pathogenic Bordetellae. We show that like in the other Bordetellae the virulence regulatory BvgAS system is also involved in the regulation of fhaB expression in B. holmesii. Taken together these data indicate that in contrast to previous notions B. holmesii may in fact make use of virulence mechanisms related to those described for the other Bordetellae. [Abstract/Link to Full Text]

Highlander SK, Hulten KG, Qin X, Jiang H, Yerrapragada S, Mason EO, Shang Y, Williams TM, Fortunov RM, Liu Y, Igboeli O, Petrosino J, Tirumalai M, Uzman A, Fox GE, Cardenas AM, Muzny DM, Hemphill L, Ding Y, Dugan S, Blyth PR, Buhay CJ, Dinh HH, Hawes AC, Holder M, Kovar CL, Lee SL, Liu W, Nazareth LV, Wang Q, Zhou J, Kaplan SL, Weinstock GM
Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus.
BMC Microbiol. 2007 Nov 6;7(1):99.
ABSTRACT: BACKGROUND: Community acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA) increasingly causes disease worldwide. USA300 has emerged as the predominant clone causing superficial and invasive infections in children and adults in the USA. Epidemiological studies suggest that USA300 is more virulent than other CA-MRSA. The genetic determinants that render virulence and dominance to USA300 remain unclear. RESULTS: We sequenced the genomes of two pediatric USA300 isolates: one CA-MRSA and one CA-methicillin susceptible (MSSA), isolated at Texas Children's Hospital in Houston. DNA sequencing was performed by Sanger dideoxy whole genome shotgun (WGS) and 454 Life Sciences pyrosequencing strategies. The sequence of the USA300 MRSA strain was rigorously annotated. In USA300, MRSA 2685 chromosomal open reading frames were predicted and 3.1 and 27 kilobase (kb) plasmids were identified. USA300 MSSA contained a 20 kb plasmid with some homology to the 27 kb plasmid. Two regions found in US300 MRSA were absent in USA300 MSSA. The USA300 sequence was aligned with other sequenced S. aureus genomes and regions unique to USA300 MRSA were identified. CONCLUSIONS: USA300-MRSA is highly similar to other MRSA strains based on whole genome alignments and gene content, indicating that the differences in pathogenesis are due to subtle changes rather than to large-scale acquisition of virulence factor genes. The USA300 Houston isolate differs from another sequenced USA300 strain isolate, derived from a patient in San Francisco, in plasmid content and a number of sequence polymorphisms. Such differences will provide new insights into the evolution of pathogens. [Abstract/Link to Full Text]

Mellmann A, Weniger T, Berssenbrugge C, Rothganger J, Sammeth M, Stoye J, Harmsen D
Based Upon Repeat Pattern (BURP): an algorithm to characterize the long-term evolution of Staphylococcus aureus populations based on spa polymorphisms.
BMC Microbiol. 2007 Oct 29;7(1):98.
ABSTRACT: BACKGROUND: For typing of Staphylococcus aureus, DNA sequencing of the repeat region of the protein A (spa) gene is a well established discriminatory method for outbreak investigations. Recently, it was hypothesized that this region reflects also long-term epidemiology. However, no automated and objective algorithm existed to cluster different repeat regions. In this study, the Based Upon Repeat Pattern (BURP) implementation that is a heuristic variant of the newly described EDSI algorithm was investigated to infer the clonal relatedness of different spa types. For calibration of BURP parameters, 400 representative S. aureus strains with different spa types were characterized by MLST and clustered using eBURST as "gold standard" for their phylogeny. Typing concordance analysis between eBURST and BURP clustering (spa-CC) were performed using all possible BURP parameters to determine their optimal combination. BURP was subsequently evaluated with a strain collection reflecting the natural population of S. aureus. RESULTS: In total, the 400 strains exhibited 122 different MLST types. eBURST grouped them into 23 clonal complexes (CC; 354 isolates) and 33 singletons (46 isolates). BURP clustering of spa types using all possible parameter combinations and subsequent comparison with eBURST CCs resulted in concordances ranging from 8.2 to 96.2%. However, 96.2% concordance was reached only if spa types shorter than 8 repeats were excluded, which resulted in 37% excluded spa types. Therefore, the optimal combination of the BURP parameters was "exclude spa types shorter than 5 repeats, cluster spa types into spa-CC if cost distances are less than 4" exhibiting 95.3% concordance to eBURST, 24 spa-CCs, 40 singletons, and exclusion of 7.8% spa types only. Analyzing the natural population with these parameters, the comparison of whole-genome micro-array groupings (at the level of 0.31 Pearson correlation index) and spa-CCs gave a concordance of 87.1%; BURP spa-CCs vs. manually grouped spa types resulted in 95.7% concordance. CONCLUSIONS: BURP is the first automated and objective tool to infer clonal relatedness from spa repeat regions. It is able to extract an evolutionary signal rather congruent to MLST and micro-array data. [Abstract/Link to Full Text]

Bergholz TM, Wick LM, Qi W, Riordan JT, Ouellette LM, Whittam TS
Global transcriptional response of Escherichia coli O157:H7 to growth transitions in glucose minimal medium.
BMC Microbiol. 2007 Oct 29;7(1):97.
ABSTRACT: BACKGROUND: Global patterns of gene expression of Escherichia coli K-12 during growth transitions have been deeply investigated, however, comparable studies of E. coli O157:H7 have not been explored, particularly with respect to factors regulating virulence genes and genomic islands specific to this pathogen. To examine the impact of growth phase on the dynamics of the transcriptome, O157:H7 Sakai strain was cultured in MOPS minimal media (0.1% glucose), RNA harvested at 10 time points from early exponential to full stationary phase, and relative gene expression was measured by co-hybridization on high-density DNA microarrays. Expression levels of 14 genes, including those encoding Shiga toxins and other virulence factors associated with the locus of enterocyte effacement (LEE), were confirmed by Q-PCR. RESULTS: Analysis of variance (R/MAANOVA, Fs test) identified 442 (36%) of 1239 O157-specific ORFs and 2110 (59%) of 3647 backbone ORFs that changed in expression significantly over time. QT cluster analysis placed 2468 of the 2552 significant ORFs into 12 groups; each group representing a distinct expression pattern. ORFs from the largest cluster (n=1078) decreased in expression from late exponential to early stationary phase: most of these ORFs are involved in functions associated with steady state growth. Also represented in this cluster are ORFs of the TAI island, encoding tellurite resistance and urease activity, which decreased ~4-fold. Most ORFs of the LEE pathogenicity island also decreased ~2-fold by early stationary phase. The ORFs encoding proteins secreted via the LEE encoded type III secretion system, such as tccP and espJ, also decreased in expression from exponential to stationary phase. Three of the clusters (n=154) comprised genes that are transiently upregulated at the transition into stationary phase and included genes involved in nutrient scavenging. Upregulated genes with an increase in mRNA levels from late exponential to early stationary phase belonged to one cluster (n=923) which includes genes involved in stress responses (e.g. gadAB, osmBC, and dps). These transcript levels remained relatively high for >3h in stationary phase. The Shiga toxin genes (stx1AB and stx2B) were significantly induced after transition into stationary phase. CONCLUSIONS: Expression of more than 300 O157-specific ORFs, many implicated in virulence of the O157 pathogen, was modulated in a growth dependent manner. These results provide a baseline transcriptional profile that can be compared to patterns of gene expression of this important foodborne pathogen under adverse environmental conditions. [Abstract/Link to Full Text]

Han Y, Qiu J, Guo Z, Gao H, Song Y, Zhou D, Yang R
Comparative transcriptomics in Yersinia pestis: a global view of environmental modulation of gene expression.
BMC Microbiol. 2007 Oct 29;7(1):96.
ABSTRACT: BACKGROUND: Environmental modulation of gene expression in Yersinia pestis is critical for its life style and pathogenesis. Using cDNA microarray technology, we have analyzed the global gene expression of this deadly pathogen when grown under different stress conditions in vitro. RESULTS: To provide us with a comprehensive view of environmental modulation of global gene expression in Y. pestis, we have analyzed the gene expression profiles of 25 different stress conditions. Almost all known virulence genes of Y. pestis were differentially regulated under multiple environmental perturbations. Clustering enabled us to functionally classify co-expressed genes, including some uncharacterized genes. Collections of operons were predicted from the microarray data, and some of these were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Several regulatory DNA motifs, probably recognized by the regulatory protein Fur, PurR, or Fnr, were predicted from the clustered genes, and a Fur binding site in the corresponding promoter regions was verified by electrophoretic mobility shift assay (EMSA). CONCLUSION: The comparative transcriptomics analysis we present here not only benefits our understanding of the molecular determinants of pathogenesis and cellular regulatory circuits in Y. pestis, it also serves as a basis for integrating increasing volumes of microarray data using existing methods. [Abstract/Link to Full Text]

Oloya J, Kazwala R, Lund A, Opuda-Asibo J, Demelash B, Skjerve E, Johansen TB, Djonne B
Characterization of mycobacteria isolated from slaughter cattle in pastoral regions of Uganda.
BMC Microbiol. 2007 Oct 25;7(1):95.
ABSTRACT: BACKGROUND: Bovine tuberculosis is a zoonotic problem in pastoral cattle and communities in Uganda. Tuberculin tests in pastoral cattle had shown a high herd but low animal prevalence, with a high proportion of avian reactors. No work had been done to identify the mycobacterial species involved. The objective of the study was to isolate and characterise Mycobacterial species causing tuberculous lesions in slaughtered animals. Lesioned organs compatible with bovine tuberculosis in slaughtered cattle from pastoral areas in Uganda were collected and cultured to isolate mycobacteria. AccuProbe culture identification kits for the Mycobacterium tuberculosis complex, M. avium complex and M. avium were used to identify the isolates. Spoligotyping and Insertion Sequence (IS) 1311 and IS1245 Restriction Fragment Length Polymorphism analysis (RFLP) were used to further characterise the isolates. RESULTS: Of the 61 lesioned organs and tissues cultured, 19 isolates were identified as M. bovis, 3 as M. avium subsp. hominissuis, 1 as M. intracellulare, 1 as a mixed culture of M. bovis and M. avium sp. and 1 as M. avium sp. and unidentified mycobacteria. Eleven other mycobacteria outside the tuberculosis and avium complex groups were also isolated. Ten new spoligopatterns grouped into three clusters were identified from M.bovis isolates. Two of the three M. avium subsp. hominissuis isolates showed similar patterns on the IS1311 RFLP but all were different on the IS1245 RFLP. CONCLUSION: The isolation of M. bovis confirms the ongoing infection with spoligotypes unique to Uganda. Isolation of environmental mycobacteria could explain the high avian or non specific tuberculin reactor patterns commonly observed in pastoral cattle and suggests their pathogenic or opportunistic role in the infection of cattle with disseminated bovine tuberculous lesions. [Abstract/Link to Full Text]

Propst-Graham KL, Preheim LC, Vander Top EA, Snitily MU, Gentry-Nielsen MJ
Cirrhosis-induced defects in innate pulmonary defenses against Streptococcus pneumoniae.
BMC Microbiol. 2007 Oct 23;7(1):94.
ABSTRACT: BACKGROUND: The risk of mortality from pneumonia caused by Streptococcus pneumoniae is increased in patients with cirrhosis. However, the specific pneumococcal virulence factors and host immune defects responsible for this finding have not been clearly established. This study used a cirrhotic rat model of pneumococcal pneumonia to identify defect(s) in innate pulmonary defenses in the cirrhotic host and to determine the impact of the pneumococcal toxin pneumolysin on these defenses in the setting of severe cirrhosis. RESULTS: No cirrhosis-associated defects in mucociliary clearance of pneumococci were found in these studies, but early intrapulmonary killing of the organisms before the arrival of neutrophils was significantly impaired. This defect was exacerbated by pneumolysin production in cirrhotic but not in control rats. Neutrophil-mediated killing of a particularly virulent type 3 pneumococcal strain also was significantly diminished within the lungs of cirrhotic rats with ascites. Levels of lysozyme and complement component C3 were both significantly reduced in bronchoalveolar lavage fluid from cirrhotic rats. Finally, complement deposition was reduced on the surface of pneumococci recovered from the lungs of cirrhotic rats in comparison to organisms recovered from the lungs of control animals. CONCLUSIONS: Increased mortality from pneumococcal pneumonia in this cirrhotic host is related to defects in both early pre-neutrophil- and later neutrophil-mediated pulmonary killing of the organisms. The fact that pneumolysin production impaired pre-neutrophil-mediated pneumococcal killing in cirrhotic but not control rats suggests that pneumolysin may be particularly detrimental to this defense mechanism in the severely cirrhotic host. The decrease in neutrophil-mediated killing of pneumococci within the lungs of the cirrhotic host is related to insufficient deposition of host proteins such as complement C3 on their surfaces. Pneumolysin likely plays a role in complement consumption within the lungs. Our studies, however, were unable to determine whether pneumolysin more negatively impacted this defense mechanism in cirrhotic than in control rats. These findings contribute to our understanding of the defects in innate pulmonary defenses that lead to increased mortality from pneumococcal pneumonia in the severely cirrhotic host. They also suggest that pneumolysin may be a particularly potent pneumococcal virulence factor in the setting of cirrhosis. [Abstract/Link to Full Text]

Jangid K, Kong R, Patole MS, Shouche YS
luxRI homologs are universally present in the genus Aeromonas.
BMC Microbiol. 2007 Oct 23;7(1):93.
ABSTRACT: BACKGROUND: Aeromonas spp. have been regarded as "emerging pathogens". Aeromonads possess multifactorial virulence and the production of many of these virulence determinants is associated with high cell density, a phenomenon that might be regulated by quorum sensing. However, only two species of the genus are reported to possess the luxRI quorum sensing gene homologs. The purpose of this study was to investigate if the luxRI homologs are universally present in the Aeromonas strains collected from various culture collections, clinical laboratories and field studies. RESULTS: Of all the 73 Aeromonas strains used in the study, seventy-one strains elicited acyl-homoserine lactone-mediated response in multiple biosensor strains. However, dot blot hybridization revealed that the luxRI homologs are present in all the strains. PCR amplification and sequencing revealed that the luxRI homologs shared a very high percentage sequence similarity. No evidence for lateral gene transfer of the luxRI homologs between aeromonads and other genera was noted. CONCLUSIONS: We propose that the luxRI quorum sensing gene homologs are universally present in the genus Aeromonas independently from their origin. This study is the first genus-wide report of the taxonomic distribution of the luxRI homologs. [Abstract/Link to Full Text]

Tercarioli GR, Bagagli E, Reis GM, Theodoro RC, Bosco SM, Macoris SA, Richini-Pereira VB
Ecological study of Paracoccidioides brasiliensis in soil: growth ability, conidia production and molecular detection.
BMC Microbiol. 2007 Oct 22;7(1):92.
ABSTRACT: BACKGROUND: Paracoccidioides brasiliensis ecology is not completely understood, although several pieces of evidence point to the soil as its most probable habitat. The present study aimed to investigate the fungal growth, conidia production and molecular pathogen detection in different soil conditions. METHODS: Soils samples of clayey, sandy and medium textures were collected from ground surface and the interior of armadillo burrows in a hyperendemic area of Paracoccidioidomycosis. P. brasiliensis was inoculated in soil with controlled humidity and in culture medium containing soil extracts. The molecular detection was carried out by Nested PCR, using panfungal and species specific primers from the ITS-5.8S rDNA region. RESULTS: The soil texture does not affect fungus development and the growth is more abundant on / in soil saturated with water. Some soil samples inhibited the development of P. brasiliensis, especially those that contain high values of Exchangeable Aluminum (H+Al) in their composition. Some isolates produced a large number of conidia, mainly in soil-extract agar medium. The molecular detection was positive only in samples collected from armadillo burrows, both in sandy and clayey soil. CONCLUSIONS: P.brasiliensis may grow and produce the infectious conidia in sandy and clayey soil, containing high water content, mainly in wild animal burrows, but without high values of H+Al. [Abstract/Link to Full Text]

Denison AM, Thompson HA, Massung RF
IS1111 insertion sequences of Coxiella burnetii: characterization and use for repetitive element PCR-based differentiation of Coxiella burnetii isolates.
BMC Microbiol. 2007;791.
BACKGROUND: Coxiella burnetii contains the IS1111 transposase which is present 20 times in the Nine Mile phase I (9Mi/I) genome. A single PCR primer that binds to each IS element, and primers specific to a region approximately 500-bp upstream of each of the 20 IS1111 elements were designed. The amplified products were characterized and used to develop a repetitive element PCR genotyping method. RESULTS: Isolates Nine Mile phase II, Nine Mile RSA 514, Nine Mile Baca, Scottish, Ohio, Australian QD, Henzerling phase I, Henzerling phase II, M44, KAV, PAV, Q238, Q195 and WAV were tested by PCR and compared to 9Mi/I. Sequencing was used to determine the exact differences in isolates which lacked specific IS elements or produced PCR products of differing size. From this data, an algorithm was created utilizing four primer pairs that allows for differentiation of unknown isolates into five genomic groups. Additional isolates (Priscilla Q177, Idaho Q, Qiyi, Poker Cat, Q229 and Q172) and nine veterinary samples were characterized using the algorithm which resulted in their placement into three distinct genomic groups. CONCLUSION: Through this study significant differences, including missing elements and sequence alterations within and near IS element coding regions, were found between the isolates tested. Further, a method for differentiation of C. burnetii isolates into one of five genomic groups was created. This algorithm may ultimately help to determine the relatedness between known and unknown isolates of C. burnetii. [Abstract/Link to Full Text]

Hansen VM, Rosenquist H, Baggersen DL, Brown S, Christensen BB
Characterization of Campylobacter phages including analysis of host range by selected Campylobacter Penner serotypes.
BMC Microbiol. 2007 Oct 18;7(1):90.
ABSTRACT: BACKGROUND: The predominant food borne pathogen in the western world today is Campylobacter. Campylobacter specific bacteriophages (phages) have been proposed as an alternative agent for reducing the burden of Campylobacter in broilers. One concern in relation to phage biocontrol is the narrow host range often displayed by phages. To identify the potential of phages as a Campylobacter reducing agent we needed to determine their infectivity on a panel of isolates representing the Campylobacter strains found in broilers as well as humans. RESULTS: In this study, Campylobacter phages were isolated from the intestines of broilers and ducks and from abattoir sewage. Twelve phages were investigated to determine their ability to infect the Campylobacter Penner serotypes commonly present in Danish poultry and patients with campylobacteriosis. A total of 89% of the Campylobacter jejuni strains and 14% of the Campylobacter coli strains could be infected by at least one of the bacteriophages. The majority of the phages infected the most common serotypes in Danish broilers (O:1,44; O:2; O:4-complex), but showed limited ability to infect 21 of the less frequent Campylobacter serotypes. Pulse field gel electrophoresis (PFGE) and restriction endonuclease analysis (REA) were used to characterize the phage genomes. Three categories of bacteriophages were observed. I: a genome size of 194 kb and refractory to digestion with HhaI; II: a genome size of 140 kb and digestible by HhaI; and III: a genome size undeterminable in PFGE. The categorization of the phages correlated with the host range patterns displayed by the phages. Six phages were subjected to transmission electron microscopy (TEM). They all belonged to the family of myoviriedae. CONCLUSION: We have characterized and identified the host range of 12 Danish Campylobacter phages. Due to their ability to infect the common serotypes in Denmark we suggest the phages can become an effective agent in the effort to reduce the incidence of campylobacteriosis in Denmark. This study provides the basis for future experiments in Campylobacter phages and knowledge for the selection of Campylobacter phages for biocontrol in broilers. [Abstract/Link to Full Text]

Hammami R, Zouhir A, Ben Hamida J, Fliss I
BACTIBASE: a new web-accessible database for bacteriocin characterization.
BMC Microbiol. 2007 Oct 17;7(1):89.
ABSTRACT: BACKGROUND: Bacteriocins are very diverse group of antimicrobial peptides produced by a wide range of bacteria and known for their inhibitory activity against various human and animal pathogens. Although many bacteriocins are now well characterized, much information is still missing or is unavailable to potential users. The assembly of such information in one central resource such as a database would therefore be of great benefit to the exploitation of these bioactive molecules in the present context of increasing antibiotic resistance and natural bio-preservation need. Description: In the present paper, we present the development of a new and original database BACTIBASE that contains calculated or predicted structural and functional properties of 123 bacteriocins produced by both Gram-positive and Gram-negative bacteria. The information in this database is very easy to extract and allows rapid and precise prediction of relationships structure / function and antimicrobial activities of these peptides and therefore better exploitation of their biological activity in both the medical and food sectors. CONCLUSION: The BACTIBASE database is freely available at http://bactibase.pfba-lab.org, web-based platform enabling easy retrieval, via various filters, of sets of bacteriocins that will enable detailed analysis of a number of microbiological and physicochemical data. [Abstract/Link to Full Text]

Banus S, Vandebriel RJ, Pennings JL, Gremmer ER, Wester PW, van Kranen HJ, Breit TM, Demant P, Mooi FR, Hoebee B, Kimman TG
Comparative gene expression profiling in two congenic mouse strains following Bordetella pertussis infection.
BMC Microbiol. 2007 Oct 12;7(1):88.
ABSTRACT: BACKGROUND: Susceptibility to Bordetella pertussis infection varies widely. These differences can partly be explained by genetic host factors. HcB-28 mice are more resistant to B. pertussis infection than C3H mice, which could partially be ascribed to the B. pertussis susceptibility locus-1 (Bps1) on chromosome 12. The presence of C57BL/10 genome on this locus instead of C3H genome resulted in a decreased number of bacteria in the lung. To further elucidate the role of host genetic factors, in particular in the Bps1 locus, in B. pertussis infection, and to identify candidate genes within in this region, we compared expression profiles in the lungs of the C3H and HcB-28 mouse strains following B. pertussis inoculation. Twelve and a half percent of the genomes of these mice are from a different genetic background. RESULTS: Upon B. pertussis inoculation 2,353 genes were differentially expressed in the lungs of both mouse strains. Two hundred and six genes were differentially expressed between the two mouse strains, but, remarkably, none of these were up- or down-regulated upon B. pertussis infection. Of these 206 genes, 17 were located in the Bps1 region. Eight of these genes, which showed a strong difference in gene expression between the two mouse strains, map to the immunoglobulin heavy chain complex (Igh). CONCLUSIONS: Gene expression changes upon B. pertussis infection are highly identical between the two mouse strains despite the differences in the course of B. pertussis infection. Because the genes that were differentially regulated between the mouse strains only showed differences in expression before infection, it appears likely that such intrinsic differences in gene regulation are involved in determining differences in susceptibility to B. pertussis infection. Alternatively, such genetic differences in susceptibility may be explained by genes that are not differentially regulated between these two mouse strains. Genes in the Igh complex, among which Igh-1a/b, are likely candidates to explain differences in susceptibility to B. pertussis. Thus, by microarray analysis we significantly reduced the number of candidate susceptibility genes within the Bps1 locus. Further work should establish the role of the Igh complex in B. pertussis infection. [Abstract/Link to Full Text]

Olson AB, Andrysiak AK, Tracz DM, Guard-Bouldin J, Demczuk W, Ng LK, Maki A, Jamieson F, Gilmour MW
Limited genetic diversity in Salmonella enterica Serovar Enteritidis PT13.
BMC Microbiol. 2007 Oct 1;7(1):87.
ABSTRACT: BACKGROUND: Salmonella enterica serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping S. Enteritidis PT13. RESULTS: CGH using an oligonucleotide array based upon chromosomal coding sequences of S. enterica serovar Typhimurium strain LT2 and the Salmonella genomic island 1 successfully determined major genetic differences between S. Typhimurium and S. Enteritidis PT13, but no significant strain-to-strain differences were observed between S. Enteritidis PT13 isolates. Individual loci (safA and fliC) that were identified as potentially divergent in the CGH data set were sequenced in a panel of S. Enteritidis strains, and no differences were detected between the PT13 strains. Additional sequence-based typing was performed at the fimA, mdh, manB, cyaA, citT, caiC, dmsA, ratA and STM0660 loci. Similarly, no diversity was observed amongst PT13 strains. Variation in plasmid content between PT13 strains was observed, but macrorestriction with BglII did not identify further differences. Automated rep-PCR patterns were variable between serovars, but S. Enteritidis PT13 strains could not be differentiated. CONCLUSIONS: None of the methods identified any significant variation between PT13 strains. Greater than 11,300 base pairs of sequence for each of seven S. Enteritidis PT13 strains were analyzed without detecting a single polymorphic site, although diversity between different phage types of S. Enteritidis was observed. These data suggest that Canadian S. Enteritidis PT13 strains are highly related genetically. [Abstract/Link to Full Text]

Pant N, Marcotte H, Bruessow H, Svensson L, Hammarstrom L
Effective prophylaxis against rotavirus diarrhea using a combination of Lactobacillus rhamnosus GG and antibodies.
BMC Microbiol. 2007 Sep 27;7(1):86.
ABSTRACT: BACKGROUND: Rotavirus is a worldwide cause of infectious infantile diarrhea that claims over 600,000 lives annually. Recently, two new vaccine candidates have been developed but their efficacy in developing countries, still remains to be proven. Oral delivery of specific immunoglobulins provides passive immunity and is a fast acting treatment for rotavirus diarrhea. Probiotic bacteria have also gained considerable attention lately as treatment for rotavirus diarrhea. Here we report an evaluation of the therapeutic potential of different probiotics and their combination with anti - rotavirus antibodies in a mouse model of rotavirus diarrhea. RESULTS: Of the six probiotic bacteria tested, Lactobacillus rhamnosus strain GG had the strongest influence in reducing prevalence, duration and severity of diarrhea and was therefore chosen for combination treatment with immunoglobulins. The combination treatment reduced the diarrhea outcome measures significantly, prevented histopathological changes and reduced the virus load in the intestines. CONCLUSION: The advantages associated with immunoglobulins and probiotics based therapy is that the treatment provides a rapid therapeutic effect and is cost efficient. These components do not require special storage conditions and could potentially complement the rehydration therapy that is currently used. [Abstract/Link to Full Text]

Kampf G, Pitten FA, Heeg P, Christiansen B
Efficacy of two ethanol-based skin antiseptics on the forehead at shorter application times.
BMC Microbiol. 2007;785.
BACKGROUND: Recent research suggests that alcohol-based skin antiseptics exhibit their efficacy on the resident skin flora of the forehead in less than 10 minutes. That is why we have looked at the efficacy of two ethanol-based skin antiseptics applied for 10, 2.5 and 2 minutes on skin with a high density of sebaceous glands. Each experiment was performed in a reference-controlled cross-over design with at least 20 participants. Application of isopropanol (70%, v/v) for 10 minutes to the forehead served as the reference treatment. The clear (skin antiseptic A) and coloured preparations (skin antiseptic B) contain 85% ethanol (w/w). Pre-values and post-values (immediately after the application and after 30 min) were obtained by swabbing a marked area of 5 cm2 for about 10 s. Swabs were vortexed in tryptic soy broth containing valid neutralizing agents. After serial dilution aliquots were spread on tryptic soy agar. Colonies were counted after incubation of plates at 36 degrees C for 48 h. The mean log10 reduction of bacteria was calculated. The Wilcoxon matched-pairs signed-ranks test was used for a comparison of treatments. RESULTS: Skin antiseptic A applied for 10 min was significantly more effective than the reference treatment. When applied for 2.5 min (three experiments) it was significantly more effective than the reference treatment immediately after application (2.7 versus 2.2 log10 reduction; p < 0.001) and equally effective after 30 min (2.8 versus 2.6 log10 reduction; p = 0.053). Skin antiseptic B applied for 2.5 min (three experiments) was significantly more effective than the reference treatment both immediately after application (2.3 versus 1.9 log10 reduction; p < 0.001) and after 30 min (2.5 versus 2.1 log10 reduction; p = 0.002). CONCLUSION: The clear and coloured skin antiseptics applied for 2.5 min on the skin of the forehead fulfilled the efficacy requirements for skin antisepsis. The shorter application time on skin with a high density of sebaceous glands will allow to act more efficiently in clinical practice. [Abstract/Link to Full Text]

Rodenburg W, Keijer J, Kramer E, Roosing S, Vink C, Katan MB, van der Meer R, Bovee-Oudenhoven IM
Salmonella induces prominent gene expression in the rat colon.
BMC Microbiol. 2007;784.
BACKGROUND: Salmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyer's patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point). As fructo-oligosaccharides (FOS) affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection. RESULTS: Salmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase), antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2), inflammation (e.g. calprotectin), oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2) and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9). Furthermore, Salmonella translocation increased serum IFN gamma and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap), showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes. CONCLUSION: We conclude that the colon is a target tissue for Salmonella, considering the abundant changes in mucosal gene expression. [Abstract/Link to Full Text]

Pathak D, Chakravorty S, Hanif M, Tyagi JS
Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR.
BMC Microbiol. 2007;783.
BACKGROUND: Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage. RESULTS: Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20 degrees C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples. CONCLUSION: We conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment. [Abstract/Link to Full Text]

---

Recent Articles in Clinical and Diagnostic Laboratory Immunology

---

Chang SC, Wang JT, Huang LM, Chen YC, Fang CT, Sheng WH, Wang JL, Yu CJ, Yang PC
Longitudinal analysis of Severe Acute Respiratory Syndrome (SARS) coronavirus-specific antibody in SARS patients.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1455-7.
The serum antibodies to severe acute respiratory syndrome (SARS) coronavirus of 18 SARS patients were checked at 1 month and every 3 months after disease onset. All of them except one, who missed blood sampling at 1 month, tested positive for the immunoglobulin G (IgG) antibody at 1 month. Fifteen out of 17 tested positive for the IgM antibody at 1 month. The serum IgM antibody of most patients became undetectable within 6 months after the onset of SARS. The IgG antibody of all 17 patients, whose serum was checked 1 year after disease onset, remained positive. [Abstract/Link to Full Text]

Jeong KY, Lee H, Lee JS, Lee J, Lee IY, Ree HI, Hong CS, Park JW, Yong TS
Immunoglobulin E binding reactivity of a recombinant allergen homologous to alpha-Tubulin from Tyrophagus putrescentiae.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1451-4.
Storage mites may cause allergic respiratory diseases in urban areas as well as pose an occupational hazard in rural areas. Characterization of storage mite allergens is important for the development of diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report on the cloning and expression of alpha-tubulin from the storage mite (Tyrophagus putrescentiae). The deduced amino acid sequence of the alpha-tubulin from the storage mite showed as much as 97.3% identity to the alpha-tubulin sequences from other organisms. The highly conserved amino acid sequences of alpha-tubulins across different species of mites may indicate that cross-reactivity for this potential allergen exists. The frequency of immunoglobulin E reactivity of this recombinant protein is 29.3% in sera from storage mite-allergic subjects. [Abstract/Link to Full Text]

Stuen S, Dahl H, Bergström K, Moum T
Unidirectional suppression of Anaplasma phagocytophilum genotypes in infected lambs.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1448-50.
Five-month-old lambs were simultaneously infected with different doses of two 16S rRNA genetic variants of Anaplasma phagocytophilum and thereafter followed for clinical observation and blood sampling. The result of the study indicates a unidirectional suppression of genotypes in infected lambs, at least during a certain period of an A. phagocytophilum infection. [Abstract/Link to Full Text]

Wang CW, Wang LC, Chang MH, Ni YH, Chen HL, Hsu HY, Chen DS
Long-term follow-up of Hepatitis B Surface antibody levels in subjects receiving universal Hepatitis B vaccination in infancy in an area of hyperendemicity: correlation between radioimmunoassay and enzyme immunoassay.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1442-7.
The aims of the present study were to determine (i) the long-term immunogenicity and the decay rate of hepatitis B virus (HBV) surface antibody (anti-HBs) from universal hepatitis B vaccination at infancy for a healthy population in an area of hyperendemicity and (ii) whether the anti-HBs levels measured by enzyme immunoassay (EIA) were closely correlated with those assayed by radioimmunoassay (RIA) methods during long-term monitoring. A total of 1,337 apparently healthy children (696 boys and 641 girls) who were vaccinated against HBV at infancy and monitored for anti-HBs annually from 7 to 16 years of age entered the study. Serum samples were analyzed for anti-HBs by RIA at 7 to 15 years of age and were also analyzed by EIA at 13 to 16 years of age. Antibody titers were quantified in mIU/ml by EIA as well as by the ratio of the count in the sample to the count for a negative control (S/N) by RIA. In non-boosted children, the average decay of anti-HBs from 7 to 16 years of ages indicated that approximately 20% of the geometric mean titer decays per year. There was a good correlation between serum anti-HBs levels measured by the RIA and the EIA methods (r=0.91; P<0.0001). An equation for RIA to EIA level conversion was established: log EIA titer=-0.12+ (1.31 . log RIA S/N). The anti-HBs titers measured by EIA correlate well with the S/N assayed by RIA. The annual decay rate of the log anti-HBs level may help in planning booster immunizations for hypo-responders or individuals at risk in adolescence. [Abstract/Link to Full Text]

Sghiri R, Feinberg J, Thabet F, Dellagi K, Boukadida J, Ben Abdelaziz A, Casanova JL, Barbouche MR
Gamma interferon is dispensable for neopterin production in vivo.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1437-41.
Previous studies have indicated that neopterin is synthesized in vitro by human monocyte-derived macrophages and dendritic cells upon stimulation with gamma interferon (IFN-gamma). Neopterin production under specific conditions in vitro has also been obtained upon stimulation with IFN-alpha and/or IFN-beta. However, it is unknown if any IFN-gamma-independent neopterin synthesis is possible in vivo. In the present study we investigated the serum neopterin concentrations in patients affected by the syndrome of Mendelian susceptibility to mycobacterial disease (MSMD). Indeed, this syndrome is characterized by deeply impaired or absent IFN-gamma production or function due to severe mutations in molecules involved in IFN-gamma/interleukin-12 (IL-12)/IL-23-dependent pathway. Serum neopterin levels were measured by an enzyme-linked immunosorbent assay in 27 patients with MSMD. We found that serum neopterin levels are elevated in the complete absence of IFN-gamma activity due either to a complete deficiency of its receptor or to deleterious mutations of IL-12 or its receptor. These data clearly indicate that, as reported from in vitro studies, other stimuli are able to induce neopterin synthesis in vivo. Consequently, neopterin cannot be used as means of diagnosis of MSMD due to IFN-gamma-, IL-12-, and IL-23-dependent pathway defects. [Abstract/Link to Full Text]

Sanakkayala N, Sokolovska A, Gulani J, Hogenesch H, Sriranganathan N, Boyle SM, Schurig GG, Vemulapalli R
Induction of antigen-specific Th1-type immune responses by gamma-irradiated recombinant Brucella abortus RB51.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1429-36.
Brucella abortus strain RB51 is an attenuated rough mutant used as the live vaccine against bovine brucellosis in the United States and other countries. We previously reported the development of strain RB51 as a bacterial vaccine vector for inducing Th1-type immune responses against heterologous proteins. Because safety concerns may preclude the use of strain RB51-based recombinant live vaccines, we explored the ability of a gamma-irradiated recombinant RB51 strain to induce heterologous antigen-specific immune responses in BALB/c mice. Exposure of strain RB51G/LacZ expressing Escherichia coli beta-galactosidase to a minimum of 300 kilorads of gamma radiation resulted in complete loss of replicative ability. These bacteria, however, remained metabolically active and continued to synthesize beta-galactosidase. A single intraperitoneal inoculation of mice with 10(9) CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a beta-galactosidase-specific Th1-type immune response. Though no obvious differences were detected in immune responses to B. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulent B. abortus. In vitro experiments indicated that gamma-irradiated and heat-killed RB51G/LacZ induced maturation of dendritic cells; however, stimulation with gamma-irradiated bacteria resulted in more interleukin-12 secretion. These results suggest that recombinant RB51 strains exposed to an appropriate minimum dose of gamma radiation are unable to replicate but retain their ability to stimulate Th1 immune responses against the heterologous antigens and confer protection against B. abortus challenge in mice. [Abstract/Link to Full Text]

Iqbal HS, Solomon S, Murugavel KG, Solomon SS, Balakrishnan P
Evaluation and diagnostic usefulness of domestic and imported enzyme-linked immunosorbent assays for detection of human immunodeficiency virus type 1 antibody in India.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1425-8.
Diagnosis of human immunodeficiency virus (HIV) infection is important for patient management and prevention of new infections. The number of test kits available for the detection of HIV antibodies is unprecedented. In order to identify appropriate test kits, we evaluated a variety of commercial kits manufactured abroad as well as in India. The plasma and serum specimens (n=264) were collected from individuals attending the Voluntary Counseling and Testing Centre at the YRG Centre for AIDS and Education. The specimens were used to evaluate six commercially available HIV test kits: Enzaids HIV 1+2, HIV-CheX, Murex HIV-1.2.0, Genscreen HIV 1/2 version 2, Vironostika HIV Uni-Form II Ag/Ab, and CombAids RS Advantage. High sensitivities and specificities (>or=99%) were observed for the Enzaids, Murex, Vironostika, and CombAids assays. HIV-CheX showed the highest number of false-positive and false-negative results. The Genscreen test also gave many false positives. The study indicated that the Enzaids, Murex, and Vironostika enzyme-linked immunosorbent assay kits and the CombAids RS Advantage rapid assay could be used to achieve acceptable results for the detection of HIV antibodies. A combination of two tests is recommended to optimize the efficiency of HIV antibody testing algorithms, especially when evaluation with an HIV Western blot confirmatory test is not possible. [Abstract/Link to Full Text]

Pattanapanyasat K, Lerdwana S, Noulsri E, Chaowanachan T, Wasinrapee P, Sakulploy N, Pobkeeree V, Suksripanich O, Thanprasertsuk S, Spira TJ, Tappero JW, Levine WC
Evaluation of a new single-parameter volumetric flow cytometer (CyFlow(green)) for enumeration of absolute CD4+ T lymphocytes in human immunodeficiency virus type 1-infected Thai patients.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1416-24.
Use of the standard dual-platform flow cytometric method for determination of CD4(+) T-lymphocyte counts, which needs both a flow cytometer (FCM) and hematological analyzer, would inevitably lead to increased variability. The development of new single-platform (SP) FCMs that provide direct CD4(+) T-lymphocyte counts for improved assay precision and accuracy have recently attracted attention. This study evaluated one of those systems, CyFlow(green) (Partec), a single-parameter SP volumetric FCM. The performance of CyFlow(green) was compared with those of two reference standard SP microbead-based technologies of the three-color TruCOUNT tube with the FACScan FCM and a two-color FACSCount system (Becton Dickinson Biosciences). Absolute CD4(+) and CD8(+) T-lymphocyte counts in 200 human immunodeficiency virus type 1-seropositive blood specimens were determined. Statistical analysis for correlation and agreement were performed. A high correlation of absolute CD4 counts was shown when those obtained with CyFlow(green) were compared with those obtained with the bead-based three-color TruCOUNT system (R(2)=0.96; mean bias, -69.1 cells/microl; 95% confidence interval [CI], -225.7 to+87.5 cells/microl) and the FACSCount system (R(2)=0.97; mean bias, -40.0 cells/microl; 95% CI, -165.1 to+85.1 cells/microl). The correlation of the CD4(+) T-lymphocyte counts obtained by the two bead-based systems was high (R(2)=0.98). Interestingly, CyFlow(green) yielded CD4(+) T-lymphocyte counts that were 21.8 and 7.2 cells/microl lower than those obtained with the TruCOUNT and the FACSCount systems, respectively, when CD4(+) T-lymphocyte counts were <250 CD4(+) T-lymphocyte counts/microl range or 17.3 and 5.8 cells/microl less, respectively, when CD4(+) T-lymphocyte counts were <200 cells/microl. The single-parameter CyFlow(green) volumetric technology performed well in comparison with the performance of the standard SP bead-based FCM system. However, a multicenter comparative study is needed before this FCM machine is implemented in resource-limited settings. [Abstract/Link to Full Text]

Kurkjian KM, Vaz LE, Haque R, Cetre-Sossah C, Akhter S, Roy S, Steurer F, Amann J, Ali M, Chowdhury R, Wagatsuma Y, Williamson J, Crawford S, Breiman RF, Maguire JH, Bern C, Secor WE
Application of an improved method for the recombinant k 39 enzyme-linked immunosorbent assay to detect visceral leishmaniasis disease and infection in Bangladesh.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1410-5.
Several serology-based immunoassays are used to diagnose visceral leishmaniasis (VL), a chronic protozoan parasitic disease caused by the Leishmania donovani complex. These tests are primarily designed to diagnose the most severe clinical form of VL, known as kala-azar. However, leishmanial infection is frequently asymptomatic and may manifest only as a positive serologic response or positive leishmanin skin test. We modified a previously described enzyme-linked immunosorbent assay (ELISA) that detects patient antibodies reactive with the recombinant Leishmania protein K39 (rK39) to confirm suspected kala-azar and to detect asymptomatic infection in a community study in Bangladesh. With the inclusion of a standard curve on each ELISA plate, the rK39 ELISA was more repeatable (kappa coefficient of agreement=0.970) and more reliable compared to the original method (kappa=0.587, P<0.001). The cutoff point for a positive antibody response was chosen based on the 99th percentile of the ELISA distribution for the negative-control sera. However, we found that sera from all patients with active kala-azar yielded values more than twice the magnitude of this cutoff. Using receiver-operator characteristic curves, we determined a second cutoff value predictive of kala-azar. Using these criteria, the sensitivity and specificity of the modified ELISA for kala-azar were 97.0% and 98.9%, respectively, for sera from our study population. We hypothesize that individuals with antibody levels greater than the 99th percentile of the negative controls but less than the cutoff point for kala-azar have asymptomatic leishmanial infections. [Abstract/Link to Full Text]

Griffin JF, Spittle E, Rodgers CR, Liggett S, Cooper M, Bakker D, Bannantine JP
Immunoglobulin G1 enzyme-linked immunosorbent assay for diagnosis of Johne's Disease in red deer (Cervus elaphus).
Clin Diagn Lab Immunol. 2005 Dec;12(12):1401-9.
This study was designed to develop a customized enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Johne's disease (JD) in farmed deer. Two antigens were selected on the basis of their superior diagnostic readouts: denatured purified protein derivative (PPDj) and undenatured protoplasmic antigen (PpAg). ELISA development was based on the antigen reactivity of the immunoglobulin G1 (IgG1) isotype, which is a highly specific marker for mycobacterial disease seroreactivity in deer. Sensitivity estimates and test parameters were established using 102 Mycobacterium paratuberculosis-infected animals from more than 10 deer herds, and specificity estimates were determined using 508 uninfected animals from 5 known disease-free herds. A receiver-operated characteristic analysis determined that at a cut point of 50 ELISA units, there was a specificity of 99.5% and sensitivities of 84.0% with PPDj antigen, 88.0% with PpAg, and 91.0% when the antigens were used serially in a composite test. Estimated sensitivity was further improved using recombinant protein antigens unique for M. paratuberculosis, which identified infected animals that were unreactive to PPDj or PpAg. While 80% of animals that were seropositive in the IgG1 ELISA had detectable histopathology, the assay could also detect animals with subclinical disease. The test was significantly less sensitive (75%) for animals that were culture positive for M. paratuberculosis but with no detectable pathology than for those with pathological evidence of JD (>90%). When the IgG1 ELISA was used annually over a 4-year period in a deer herd with high levels of clinical JD, it eliminated clinical disease, increased production levels, and reduced JD-related mortality. [Abstract/Link to Full Text]

Bhat N, Gaensbauer J, Peek RM, Bloch K, Tham KT, Blaser MJ, Perez-Perez G
Local and systemic immune and inflammatory responses to Helicobacter pylori strains.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1393-400.
Colonization with Helicobacter pylori eventuates in varied clinical outcomes, which relate to both bacterial and host factors. Here we examine the relationships between cagA status, serum and gastric juice antibody responses, and gastric inflammation in dyspeptic patients. Serum, gastric juice, and gastric biopsy specimens were obtained from 89 patients undergoing endoscopy. H. pylori colonization and cagA status were determined by histology, culture, and PCR methods, and acute inflammation and chronic inflammation in the gastric mucosa were scored by a single pathologist. Serum and gastric juice antibodies to H. pylori whole-cell and CagA antigens were determined by enzyme-linked immunosorbent assay. Relationships between variables were sequentially analyzed using univariate and multivariate statistical methods. Of the 89 subjects, 62 were colonized by H. pylori. By univariate analyses, levels of serum immunoglobulin G (IgG) and IgA and gastric juice IgA antibodies against whole-cell and CagA antigens each were significantly higher in the H. pylori-positive group than in the H. pylori-negative group (P<0.001). H. pylori and CagA sero-positivities were both significantly associated with enhanced inflammation in gastric antrum and body (P<0.02). The presence of gastric juice antibodies to H. pylori antigens was associated with more severe gastric inflammation. However, in multivariate analyses, only the presence of serum antibodies against CagA and, to a lesser extent, whole-cell antigens remained significantly associated with acute and chronic inflammation in antrum and body (P<0.05). Thus, serum antibody response to CagA correlates with severity of gastric inflammation. Furthermore, given the relationships demonstrated by multivariate analysis, determination of gastric juice antibodies may provide a better representation of serum, rather than secretory, immune response. [Abstract/Link to Full Text]

Haghighi HR, Gong J, Gyles CL, Hayes MA, Sanei B, Parvizi P, Gisavi H, Chambers JR, Sharif S
Modulation of antibody-mediated immune response by probiotics in chickens.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1387-92.
Probiotic bacteria, including Lactobacillus acidophilus and Bifidobacterium bifidum, have been shown to enhance antibody responses in mammals. The objective of this study was to examine the effects of a probiotic product containing the above bacteria in addition to Streptococcus faecalis on the induction of the chicken antibody response to various antigens, both systemically and in the gut. The birds received probiotics via oral gavage and subsequently were immunized with sheep red blood cells (SRBC) and bovine serum albumin (BSA) to evaluate antibody responses in serum or with tetanus toxoid (TT) to measure the mucosal antibody response in gut contents. Control groups received phosphate-buffered saline. Overall, BSA and SRBC induced a detectable antibody response as early as week 1 postimmunization (p.i.), which lasted until week 3 p.i. Probiotic-treated birds had significantly (P <or= 0.001) more serum antibody (predominantly immunoglobulin M [IgM]) to SRBC than the birds that were not treated with probiotics. However, treatment with probiotics did not enhance the serum IgM and IgG antibody responses to BSA. Immunization with TT resulted in the presence of specific IgA and IgG antibody responses in the gut. Again, treatment with probiotics did not change the level or duration of the antibody response in the gut. In conclusion, probiotics enhance the systemic antibody response to some antigens in chickens, but it remains to be seen whether probiotics have an effect on the generation of the mucosal antibody response. [Abstract/Link to Full Text]

Sgouras DN, Panayotopoulou EG, Martinez-Gonzalez B, Petraki K, Michopoulos S, Mentis A
Lactobacillus johnsonii La1 attenuates Helicobacter pylori-associated gastritis and reduces levels of proinflammatory chemokines in C57BL/6 mice.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1378-86.
In clinical settings, Lactobacillus johnsonii La1 administration has been reported to have a favorable effect on Helicobacter pylori-associated gastritis, although the mechanism remains unclear. We administered, continuously through the water supply, live La1 to H. pylori-infected C57BL/6 mice and followed colonization, the development of H. pylori-associated gastritis in the lamina propria, and the levels of proinflammatory chemokines macrophage inflammatory protein 2 (MIP-2) and keratinocyte-derived cytokine (KC) in the serum and gastric tissue over a period of 3 months. We documented a significant attenuation in both lymphocytic (P=0.038) and neutrophilic (P=0.003) inflammatory infiltration in the lamina propria as well as in the circulating levels of anti-H. pylori immunoglobulin G antibodies (P=0.003), although we did not observe a suppressive effect of La1 on H. pylori colonizing numbers. Other lactobacilli, such as L. amylovorus DCE 471 and L. acidophilus IBB 801, did not attenuate H. pylori-associated gastritis to the same extent. MIP-2 serum levels were distinctly reduced during the early stages of H. pylori infection in the La1-treated animals, as were gastric mucosal levels of MIP-2 and KC. Finally, we also observed a significant reduction (P=0.046) in H. pylori-induced interleukin-8 secretion by human adenocarcinoma AGS cells in vitro in the presence of neutralized (pH 6.8) La1 spent culture supernatants, without concomitant loss of H. pylori viability. These observations suggest that during the early infection stages, administration of La1 can attenuate H. pylori-induced gastritis in vivo, possibly by reducing proinflammatory chemotactic signals responsible for the recruitment of lymphocytes and neutrophils in the lamina propria. [Abstract/Link to Full Text]

Hunt ME, Brown DR
Mycoplasma alligatoris infection promotes CD95 (FasR) expression and apoptosis of primary cardiac fibroblasts.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1370-7.
Mycoplasma alligatoris causes acute lethal primary infection of susceptible hosts. A genome survey implicated sialidase and hyaluronidase, potential promoters of CD95-mediated eukaryotic cell death, as virulence factors of M. alligatoris. We used immunofluorescence imaging and flow cytometry to examine the effects of M. alligatoris infection in vitro on CD95 expression and apoptosis by alligator cardiac fibroblasts, a major cell type of a target organ of M. alligatoris infection in vivo. A uniform distribution of CD95 in primary cultured cardiac, skeletal muscle, and embryonic fibroblasts was demonstrated by using polyclonal antibodies against the N or C terminus of mouse or human CD95. Anti-CD95 antibodies reacted on Western blots of fibroblast lysates with a band with the predicted apparent molecular weight of CD95, but soluble CD95 was not detected in plasma from control or M. alligatoris-infected alligators. The proportion of CD95-gated cardiac fibroblasts increased threefold (P<0.01) 48 h after inoculation with M. alligatoris. Infection induced morphological changes in cardiac fibroblasts, including translocation of CD95 characteristic of apoptosis and an eightfold increase (P<0.16) in 5-bromo-2'-deoxyuridine (BrdU) incorporation measured in a terminal deoxynucleotide transferase dUTP nick end-labeling apoptosis assay. The proportion of BrdU-gated controls activated with agonistic immunoglobulin M against human CD95 also increased threefold (P<0.03 for muscle). Heat-inactivated M. alligatoris and sterile M. alligatoris-conditioned culture supernatant had no effect. This is the first report of a CD95 homolog in the class Reptilia and establishes a new model that can be used to test the direct bacterial interaction with upstream components of the CD95 signal transduction pathway. [Abstract/Link to Full Text]

Jin Y, Cao C, Li P, Liu X, Huang W, Li C, Ma Q
Boosting immune response to hepatitis B DNA vaccine by coadministration of Prothymosin alpha-expressing plasmid.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1364-9.
DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. In this study, we report that coadministration of a hepatitis B virus (HBV) DNA vaccine with prothymosin alpha as an adjuvant improves antibody responses to HBV S antigen. We also observed higher seroconversion rates and higher antibody titers. Prothymosin alpha appears to increase the number and affinity of hepatitis B surface antigen-specific, gamma interferon-secreting T cells and to enhance cellular immune response to the PreS2S DNA vaccine. Interestingly, administering the DNA separately from the prothymosin alpha plasmid abrogated the enhancement of DNA vaccine potency. The results suggest that prothymosin alpha may be a promising adjuvant for DNA vaccines. [Abstract/Link to Full Text]

Smithson A, Sarrias MR, Barcelo J, Suarez B, Horcajada JP, Soto SM, Soriano A, Vila J, Martinez JA, Vives J, Mensa J, Lozano F
Expression of interleukin-8 receptors (CXCR1 and CXCR2) in premenopausal women with recurrent urinary tract infections.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1358-63.
The migration of neutrophils through infected tissues is mediated by the CXC chemokines and its receptors (CXCR1 and CXCR2). It has been proposed that a CXCR1 deficiency could confer susceptibility to acute pyelonephritis in children. The objective of the study is to assess the surface expression of CXCR1 and CXCR2 and the existence of polymorphisms in the CXCR1 gene in premenopausal women with recurrent urinary tract infections. The study included 20 premenopausal women with recurrent urinary infections, with normal urinary tracts, and without diseases potentially associated with relapsing urinary infections and 30 controls without previous urinary infections. The levels of CXCR1 and CXCR2 expression on neutrophils were measured and analyzed by flow cytometry by measuring the mean fluorescence intensity (MFI) channel. The promoter and coding regions of the CXCR1 gene were analyzed for the presence of polymorphisms by a sequence-based typing method. Patients with recurrent urinary tract infections exhibited median levels of CXCR1 expression, determined from MFI values, similar to those of the controls. The analysis of CXCR2 showed that patients with recurrent urinary infections had lower median levels of expression, determined from the MFI values, than the controls (P = 0.002, Mann-Whitney U test). No polymorphisms were detected at the promoter or at the exon 1 region of the CXCR1 gene either in the patients or in the controls. Polymorphisms were detected at the exon 2 of CXCR1, but their frequencies did not differ between patients and controls. We have found a low level of CXCR2 expression in patients with recurrent urinary tract infections. These results suggest that a low level of CXCR2 expression may increase the susceptibilities of premenopausal women to urinary tract infections. [Abstract/Link to Full Text]

Binder SR, Genovese MC, Merrill JT, Morris RI, Metzger AL
Computer-assisted pattern recognition of autoantibody results.
Clin Diagn Lab Immunol. 2005 Dec;12(12):1353-7.
Immunoassay-based anti-nuclear antibody (ANA) screens are increasingly used in the initial evaluation of autoimmune disorders, but these tests offer no "pattern information" comparable to the information from indirect fluorescence assay-based screens. Thus, there is no indication of "next steps" when a positive result is obtained. To improve the utility of immunoassay-based ANA screening, we evaluated a new method that combines a multiplex immunoassay with a k nearest neighbor (kNN) algorithm for computer-assisted pattern recognition. We assembled a training set, consisting of 1,152 sera from patients with various rheumatic diseases and non-diseased patients. The clinical sensitivity and specificity of the multiplex method and algorithm were evaluated with a test set that consisted of 173 sera collected at a rheumatology clinic from patients diagnosed by using standard criteria, as well as 152 age- and sex-matched sera from presumably healthy individuals (sera collected at a blood bank). The test set was also evaluated with a HEp-2 cell-based enzyme-linked immunosorbent assay (ELISA). Both the ELISA and multiplex immunoassay results were positive for 94% of the systemic lupus erythematosus (SLE) patients. The kNN algorithm correctly proposed an SLE pattern for 84% of the antibody-positive SLE patients. For patients with no connective tissue disease, the multiplex method found fewer positive results than the ELISA screen, and no disease was proposed by the kNN algorithm for most of these patients. In conclusion, the automated algorithm could identify SLE patterns and may be useful in the identification of patients who would benefit from early referral to a specialist, as well as patients who do not require further evaluation. [Abstract/Link to Full Text]

McKeown NE, Opriessnig T, Thomas P, Guenette DK, Elvinger F, Fenaux M, Halbur PG, Meng XJ
Effects of porcine circovirus type 2 (PCV2) maternal antibodies on experimental infection of piglets with PCV2.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1347-51.
To determine the effects of porcine circovirus type 2 (PCV2) maternal antibodies on and response to experimental PCV2 infection, 24 piglets were divided into four groups on the basis of the enzyme-linked immunosorbent assay titers of PCV2 maternal antibodies: group A (n = 6; sample/positive [S/P] ratio, <0.2), group B (n = 5; S/P ratio, >0.2 to <0.5), and groups C (n = 8) and D (n = 5) (S/P ratio, >0.5). Piglets in groups A, B, and C were inoculated with PCV2 at day 0 and challenged with PCV2 at day 42. Group D piglets were not exposed to PCV2 at day 0 but were challenged at day 42. Before challenge, seroconversion to PCV2 antibodies occurred in five of six group A piglets, and the antibody level rose above the cutoff level in one of five group B piglets. Viremia was detected in five of six, four of five, and two of eight pigs in groups A, B, and C, respectively. After challenge, PCV2 DNA was detectable from 7 to 21 days postchallenge in the sera from six of six, four of five, three of eight, and five of five pigs in groups A, B, C, and D, respectively. The results indicated that protection against PCV2 infection conferred by maternal antibodies is titer dependent: higher titers are generally protective, but low titers are not. [Abstract/Link to Full Text]

Oyamada M, Davoust B, Boni M, Dereure J, Bucheton B, Hammad A, Itamoto K, Okuda M, Inokuma H
Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and sequencing methodologies.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1343-6.
Babesia and Hepatozoon infections of dogs in a village of eastern Sudan were analyzed by using a single PCR and sequencing. Among 78 dogs, 5 were infected with Babesia canis rossi and 2 others were infected with B. canis vogeli. Thirty-three dogs were positive for Hepatozoon. Hepatozoon canis was detected by sequence analysis. [Abstract/Link to Full Text]

Karaca K, Swayne DE, Grosenbaugh D, Bublot M, Robles A, Spackman E, Nordgren R
Immunogenicity of fowlpox virus expressing the avian influenza virus H5 gene (TROVAC AIV-H5) in cats.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1340-2.
Vaccination of cats with fowlpox virus expressing the avian influenza (AI) virus H5 hemagglutinin gene (TROVAC AI) resulted in detectable hemagglutination inhibition (HI) antibody responses to the homologous A/Turkey/Ireland/1378/83 (H5N8) (A/tky/Ire/83) AI virus antigen. The HI antibody responses to heterologous A/Chicken/Indonesia/7/03 (H5N1) (A/ck/Indonesia/03) AI virus antigen were also detected in all vaccinated cats, but only after booster vaccinations. The vaccine described in this study and other poxvirus-vectored vaccines may be of value for the prophylaxis of AI virus-associated morbidity and mortality in mammals. [Abstract/Link to Full Text]

Harraghy N, Mitchell TJ
Isolation and characterization of the promoter and partial enhancer region of the porcine inter-alpha-trypsin inhibitor heavy chain 4 gene.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1336-9.
A porcine genomic library was screened for clones containing the promoter of the major acute-phase protein in pigs, inter-alpha-trypsin heavy chain 4 (ITIH4). Following isolation of the promoter, a functional analysis was performed with Hep3B cells. The promoter was induced by interleukin-6 (IL-6) but not by IL-1beta. However, IL-1beta was shown to inhibit the IL-6-induced activation of the porcine ITIH4 promoter. [Abstract/Link to Full Text]

Araj GF, Kattar MM, Fattouh LG, Bajakian KO, Kobeissi SA
Evaluation of the PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays for diagnosis of human brucellosis.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1334-5.
PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis. [Abstract/Link to Full Text]

Seeborg FO, Gay H, Schmiege LM, Bernard D, Shearer WT
Immunoglobulin G kappa [IgG kappa] and IgG lambda paraproteinemia in a child with AIDS and response to highly active antiretroviral therapy.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1331-3.
We report an 8-year-old boy with AIDS, extremely elevated serum immunoglobulin G (IgG) concentration and IgG kappa [IgG(kappa)] and IgG lambda [IgG(lambda)] paraproteinemia. This paraproteinemia partially responded to highly active antiretroviral therapy. This case emphasizes the importance of controlling B-cell activation. [Abstract/Link to Full Text]

Buckland MS, Mylonaki M, Rampton D, Longhurst HJ
Serological markers (anti-Saccharomyces cerevisiae mannan antibodies and antineutrophil cytoplasmic antibodies) in inflammatory bowel disease: diagnostic utility and phenotypic correlation.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1328-30.
We have evaluated the utility of antineutrophil cytoplasmic antibodies and anti-Saccharomyces cerevisiae mannan antibodies for distinguishing Crohn's disease from ulcerative colitis and other diarrheal illnesses by evaluating sera from 396 patients. Sensitivity, specificity, and phenotypic correlations were investigated. The implications of our findings for implementing these tests in routine clinical testing are discussed. [Abstract/Link to Full Text]

Scotter JM, Chambers ST
Comparison of galactomannan detection, PCR-enzyme-linked immunosorbent assay, and real-time PCR for diagnosis of invasive aspergillosis in a neutropenic rat model and effect of caspofungin acetate.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1322-7.
The performance of different in vitro diagnostic tests for the diagnosis of invasive aspergillosis (IA) was investigated in a transiently neutropenic rat model. Rats were immunosuppressed with cyclophosphamide and then inoculated intravenously with 1.5 x 10(4) CFU Aspergillus fumigatus spores. Animals were then either treated with caspofungin acetate, 1 mg/kg/day for 7 days, or not treated. PCR-enzyme-linked immunosorbent assay (ELISA), real-time PCR, and galactomannan (GM) detection were performed on postmortem blood samples, along with culture of liver, lung, and kidney homogenate. Caspofungin-treated animals showed a decrease in residual tissue burden of A. fumigatus from organ homogenate compared to untreated animals (P < 0.002). PCR-ELISA returned positive results for 11/17 animals treated with antifungal agents and for 10/17 untreated animals. Galactomannan was positive in 8/17 caspofungin-treated animals and 4/17 untreated animals. Real-time PCR was positive in 2/17 treated and 3/17 untreated animals. This study demonstrates that PCR-ELISA is a more sensitive test than either GM detection (P = 0.052) or real-time PCR (P < 0.01) for diagnosis of IA but that any of the three tests may return false-negative results in cases of histologically proven disease. Galactomannan indices from animals treated with antifungal agents showed a trend (P = 0.1) towards higher levels than those of untreated animals, but no effect was observed with PCR-ELISA indices (P = 0.29). GM detection, as previously described, may be enhanced by the administration of caspofungin, but PCR-ELISA appears not to be affected in the same way. We conclude that PCR-ELISA is a more sensitive and reliable method for laboratory diagnosis of IA. [Abstract/Link to Full Text]

Chan KH, Cheng VC, Woo PC, Lau SK, Poon LL, Guan Y, Seto WH, Yuen KY, Peiris JS
Serological responses in patients with severe acute respiratory syndrome coronavirus infection and cross-reactivity with human coronaviruses 229E, OC43, and NL63.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1317-21.
The serological response profile of severe acute respiratory syndrome (SARS) coronavirus (CoV) infection was defined by neutralization tests and subclass-specific immunofluorescent (IF) tests using serial sera from 20 patients. SARS CoV total immunoglobulin (Ig) (IgG, IgA, and IgM [IgGAM]) was the first antibody to be detectable. There was no difference in time to seroconversion between the patients who survived (n = 14) and those who died (n = 6). Although SARS CoV IgM was still detectable by IF tests with 8 of 11 patients at 7 months postinfection, the geometric mean titers dropped from 282 at 1 month postinfection to 19 at 7 months (P = 0.001). In contrast, neutralizing antibody and SARS CoV IgGAM and IgG antibody titers remained stable over this period. The SARS CoV antibody response was sometimes associated with an increase in preexisting IF IgG antibody titers for human coronaviruses OC43, 229E, and NL63. There was no change in IF IgG titer for virus capsid antigen from the herpesvirus that was used as an unrelated control, Epstein-Barr virus. In contrast, patients who had OC43 infections, and probably also 229E infections, without prior exposure to SARS CoV had increases of antibodies specific for the infecting virus but not for SARS CoV. There is a need for awareness of cross-reactive antibody responses between coronaviruses when interpreting IF serology. [Abstract/Link to Full Text]

Goletti D, Vincenti D, Carrara S, Butera O, Bizzoni F, Bernardini G, Amicosante M, Girardi E
Selected RD1 peptides for active tuberculosis diagnosis: comparison of a gamma interferon whole-blood enzyme-linked immunosorbent assay and an enzyme-linked immunospot assay.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1311-6.
We recently set up a gamma interferon (IFN-gamma) enzyme-linked immunospot assay (ELISPOT), using selected early secreted antigenic target 6 (ESAT-6) peptides, that appears specific for active tuberculosis (A-TB). However, ELISPOT is difficult to automate. Thus, the objective of this study was to determine if the same selected peptides may be used in a technique more suitable for routine work in clinical laboratories, such as whole-blood enzyme-linked immunosorbent assay (WBE). For this purpose, 27 patients with A-TB and 41 control patients were enrolled. Our WBE, using the already described selected peptides from ESAT-6 plus three new ones from culture filtrate protein 10, was performed, and data were compared with those obtained by ELISPOT. Using our selected peptides, IFN-gamma production, evaluated by both WBE and ELISPOT, was significantly higher in patients with A-TB than in controls (P < 0.0001). Statistical analysis showed a good correlation between the results obtained by WBE and ELISPOT (r = 0.80, P < 0.001). To substantiate our data, we compared our WBE results with those obtained by QuantiFERON-TB Gold, a whole-blood assay based on region of difference 1 (RD1) overlapping peptides approved for TB infection diagnosis. We observed a slightly higher sensitivity with QuantiFERON-TB Gold than with our WBE (89% versus 81%); however, our test provided a better specificity result (90% versus 68%). In conclusion, results obtained by WBE based on selected RD1 peptides significantly correlate with those generated by ELISPOT. Moreover, our assay appears more specific for A-TB diagnosis than QuantiFERON-TB Gold, and thus it may represent a complementary tool for A-TB diagnosis for routine use in clinical laboratories. [Abstract/Link to Full Text]

Maeland JA, Bevanger L, Lyng RV
Immunological markers of the R4 protein of Streptococcus agalactiae.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1305-10.
This study focuses on immunological markers of R4, an important Streptococcus group B (GBS) protein. The results obtained by using rabbit antisera and purified proteins for antigens in enzyme-linked immunosorbent assay-based experiments provided evidence that R4 possesses two antigenic determinants. One of the determinants is shared with the alpha-like protein 3 (Alp3) of GBS, was named R4/Alp3 common, and was expressed by GBS, which possessed the Alp3-encoding gene alp3 or the R4-encoding gene rib. The other antigenic determinant was detected only in rib-positive GBS organisms and was named R4 specific. This determinant probably is an immunological marker unique to the R4 protein. Neither of the antigenic R4 determinants showed serological cross-reactivity with the GBS proteins Calpha, Cbeta, and R3 or with alpha-like protein 2. Of 60 clinical serotype III GBS strains, 56 (93%) isolates possessed the rib gene and 50 (89%) of the rib-positive isolates expressed levels of R4 detectable by antibody-based tests, consistent with R4 expression failure or low-level expression in approximately 10% of rib-positive GBS. alp3 was not detected in type III GBS but was possessed by six of eight type V strains and six of six type VIII strains. All alp3-positive strains were recognized by the R4/Alp3 common antibodies, but none of them were recognized by the R4-specific antibodies. NCTC 9828, a reference strain for R3 and R4, expressed the determinant R4/Alp3 common but not R4 specific. A monoclonal R4 antibody, previously considered to be R4 specific and used in GBS serotyping, targeted R4/Alp3 common and is thus not R4 specific. The results show that failure to discriminate between R4 specific and R4/Alp3 common by antisera designed for GBS serotyping can result in the false identification of Alp3 as R4 or vice versa, whereas anti-R4 antibodies targeting only the determinant R4 specific will detect only R4. Both R4 and Alp3 need further evaluation with respect to the immunobiological function of each distinct antigenic determinant, for instance, with regard to their potential as GBS vaccine components. [Abstract/Link to Full Text]

Kizza HM, Rodriguez B, Quinones-Mateu M, Mirza M, Aung H, Yen-Lieberman B, Starkey C, Horter L, Peters P, Baseke J, Johnson JL, Toossi Z
Persistent replication of human immunodeficiency virus type 1 despite treatment of pulmonary tuberculosis in dually infected subjects.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1298-304.
Tuberculosis (TB) is the most common life-threatening infection in human immunodeficiency virus (HIV)-infected persons and frequently occurs before the onset of severe immunodeficiency. Development of TB is associated with increased HIV type 1 (HIV-1) viral load, a fall in CD4 lymphocyte counts, and increased mortality. The aim of this study was to examine how treatment of pulmonary TB affected HIV-1 activity in HIV-1/TB-coinfected subjects with CD4 cell counts of >100 cells/mul. HIV-1/TB-coinfected subjects were recruited in Kampala, Uganda, and were monitored over time. Based upon a significant (0.5 log10 copies/ml) decrease in viral load by the end of treatment, two patient groups could be distinguished. Responders (n = 17) had more rapid resolution of anemia and pulmonary lesions on chest radiography during TB treatment. This group had a significant increase in viral load to levels not different from those at baseline 6 months after completion of TB treatment. HIV-1 viral load in nonresponders (n = 10) with TB treatment increased and at the 6 month follow-up was significantly higher than that at the time of diagnosis of TB. Compared to baseline levels, serum markers of macrophage activation including soluble CD14 decreased significantly by the end of TB treatment in responders but not in nonresponders. These data further define the impact of pulmonary TB on HIV-1 disease. HIV-1 replication during dual HIV-1/TB infection is not amenable to virologic control by treatment of TB alone. Concurrent institution of highly active antiretroviral treatment needs to be evaluated in patients dually infected with pulmonary TB and HIV-1. [Abstract/Link to Full Text]

Koraha J, Tsuneyoshi N, Kimoto M, Gauchat JF, Nakatake H, Fukudome K
Comparison of lipopolysaccharide-binding functions of CD14 and MD-2.
Clin Diagn Lab Immunol. 2005 Nov;12(11):1292-7.
Prior to being recognized by the cell surface Toll-like receptor 4/MD-2 complex, lipopolysaccharide (LPS) in the bacterial outer membrane has to be processed by LPS-binding protein and CD14. CD14 forms a complex with monomeric LPS extracted by LPS-binding protein and transfers LPS to the cell surface signaling complex. In a previous study, we prepared a functional recombinant MD-2 using a bacterial expression system. We expressed the recombinant protein in Escherichia coli as a fusion protein with thioredoxin and demonstrated specific binding to LPS. In this study, we prepared recombinant CD14 fusion proteins using the same approach. Specific binding of LPS was demonstrated with a recombinant protein containing 151 amino-terminal residues. The region contained a hydrophilic region and the first three leucine-rich repeats (LRRs). The LRRs appeared to contribute to the binding because removal of the region resulted in a reduction in the binding function. LPS binding to the recombinant MD-2 was resistant to detergents. On the other hand, the binding to CD14 was prevented in the presence of low concentrations of detergents. In the case of human MD-2, the secondary myristoyl chain of LPS added by LpxM was required for the binding. A nonpathogenic penta-acyl LPS mutant lacking the myristoyl chain did not bind to MD-2 but did so normally to CD14. The broader LPS-binding spectrum of CD14 may allow recognition of multiple pathogens, and the lower affinity for LPS binding of CD14 allows transmission of captured materials to MD-2. [Abstract/Link to Full Text]

---

Recent Articles in Clinical and Molecular Allergy

---

Hsieh CJ, Hall K, Ha T, Li C, Krishnaswamy G, Chi DS
Baicalein inhibits IL-1beta- and TNF-alpha-induced inflammatory cytokine production from human mast cells via regulation of the NF-kapaB pathway.
Clin Mol Allergy. 2007 Nov 26;5(1):5.
ABSTRACT: BACKGROUND: Human mast cells are multifunctional cells capable of a wide variety of inflammatory responses. Baicalein (BAI), isolated from the traditional Chinese herbal medicine Huangqin (Scutellaria baicalensis Georgi), has been shown to have anti-inflammatory effects. We examined its effects and mechanisms on the expression of inflammatory cytokines in an IL-1beta- and TNF-alpha-activated human mast cell line, HMC-1. METHODS: HMC-1 cells were stimulated either with IL-1beta (10 ng/ml) or TNF-alpha (100 U/ml) in the presence or absence of BAI. We assessed the expression of IL-6, IL-8, and MCP-1 by ELISA and RT-PCR, NF-kapaB activation by electrophoretic mobility shift assay (EMSA), and IkapaBalpha activation by Western blot. RESULTS: BAI (1.8 to 30 microM) significantly inhibited production of IL-6, IL-8, and MCP-1 in a dose-dependent manner in IL-1beta-activated HMC-1. BAI (30 microM) also significantly inhibited production of IL-6, IL-8, and MCP-1 in TNF-alpha-activated HMC-1. Inhibitory effects appear to involve the NF-kapaB pathway. BAI inhibited NF-kapaB activation in IL-1beta- and TNF-alpha-activated HMC-1. Furthermore, BAI increased cytoplasmic IkapaBalpha proteins in IL-1beta- and TNF-alpha-activated HMC-1. CONCLUSIONS: Our results showed that BAI inhibited the production of inflammatory cytokines through inhibition of NF-kapaB activation and IkapaBalpha phosphorylation and degradation in human mast cells. This inhibitory effect of BAI on the expression of inflammatory cytokines suggests its usefulness in the development of novel anti-inflammatory therapies. [Abstract/Link to Full Text]

Kampe M, Stahlenheim G, Janson C, Stolt I, Carlson M
Systemic and local eosinophil inflammation during the birch pollen season in allergic patients with predominant rhinitis or asthma.
Clin Mol Allergy. 2007 Oct 29;5(1):4.
ABSTRACT: BACKGROUND: The aim of the study was to investigate the inflammatory reaction during the birch pollen season in birch-allergic patients with rhinitis or asthma as the predominant symptom. METHOD: Subjects with birch pollen asthma (n=7) or rhinitis (n=9) and a control group (n=5) were studied before and during birch pollen season. Blood tests, spirometry, nasal lavage and induced sputum were performed at both visits. Eosinophil cationic protein (ECP) and human neutrophil lipocalin were analysed in blood, nasal lavage and induced sputum. Eosinophil number (B-Eos) was measured in blood and nasal lavage. Symptoms, medication and Peak Expiratory Flow were recorded during the pollen season in a diary. RESULTS: Patients with allergic asthma had a larger decline in FEV1 after inhaling hypertonic saline solution outside the pollen season than patients with rhinitis (median) (-7.0 vs.-0.4%, p=0.02). The asthmatics also had a significantly lower Peak Expiratory Flow. The seasonal increase in B-Eos was higher among patients with asthma (+0.17 x 109/L) and rhinitis (+0.27 x 109/L) than among controls (+0.01 x 109/L, p=0.01). Allergic asthmatics and patients with rhinitis also had a larger seasonal increase in sputum ECP (+2180 and +310 mug/L) than the controls (-146 mug/L, p=0.02). No significant differences in inflammatory parameters were found between the two groups of allergic patients. CONCLUSION: Our study indicates that patients with allergic asthma and allergic rhinitis have the same degree of eosinophil inflammation. Despite this, only the asthmatic group experienced an impairment in lung function during the pollen season. This indicated that factors other than the eosinophil inflammation determine whether or not allergic patients develop asthma. [Abstract/Link to Full Text]

Behrmann J
Looking ahead at the potential benefits of biotechnology-derived allergen therapeutics.
Clin Mol Allergy. 2007;53.
While biotechnology-derived allergen therapeutics show promise in improving the safety of immunotherapy, they may prove to have additional benefits in comparison to conventional allergenic extracts that deserve commentary. These issues range from product stability and compatibility to medical practice issues, which will be the focus of this article. [Abstract/Link to Full Text]

Aalberse RC
Assessment of allergen cross-reactivity.
Clin Mol Allergy. 2007;52.
The prediction of allergen cross-reactivity is currently largely based on linear sequence data, but will soon include 3D information on homology among surface exposed residues. To evaluate procedures for these predictions, we need ways to quantitatively assess actual cross-reactivity between two allergens. Three parameters are mentioned: 1) the fraction of the epitopes that is cross-reactive; 2) the fraction of IgE that is cross-reactive; 3) the relative affinity of the interaction between IgE and the two allergens. This editorial briefly compares direct binding protocols with the often more appropriate reciprocal inhibition protocols. The latter type of protocol provides information on symmetric versus asymmetric cross-reactivity, and thus on the distinction between complete (= sensitising) allergens versus incomplete, cross-reacting allergens. The need to define the affinity threshold of the assay and a caveat on the use of serum pools are also discussed. [Abstract/Link to Full Text]

Kurup VP, Barrios CS, Raju R, Johnson BD, Levy MB, Fink JN
Immune response modulation by curcumin in a latex allergy model.
Clin Mol Allergy. 2007;51.
BACKGROUND: There has been a worldwide increase in allergy and asthma over the last few decades, particularly in industrially developed nations. This resulted in a renewed interest to understand the pathogenesis of allergy in recent years. The progress made in the pathogenesis of allergic disease has led to the exploration of novel alternative therapies, which include herbal medicines as well. Curcumin, present in turmeric, a frequently used spice in Asia has been shown to have anti-allergic and inflammatory potential. METHODS: We used a murine model of latex allergy to investigate the role of curcumin as an immunomodulator. BALB/c mice were exposed to latex allergens and developed latex allergy with a Th2 type of immune response. These animals were treated with curcumin and the immunological and inflammatory responses were evaluated. RESULTS: Animals exposed to latex showed enhanced serum IgE, latex specific IgG1, IL-4, IL-5, IL-13, eosinophils and inflammation in the lungs. Intragastric treatment of latex-sensitized mice with curcumin demonstrated a diminished Th2 response with a concurrent reduction in lung inflammation. Eosinophilia in curcumin-treated mice was markedly reduced, co-stimulatory molecule expression (CD80, CD86, and OX40L) on antigen-presenting cells was decreased, and expression of MMP-9, OAT, and TSLP genes was also attenuated. CONCLUSION: These results suggest that curcumin has potential therapeutic value for controlling allergic responses resulting from exposure to allergens. [Abstract/Link to Full Text]

Ly NP, Ruiz-Pérez B, Onderdonk AB, Tzianabos AO, Litonjua AA, Liang C, Laskey D, Delaney ML, DuBois AM, Levy H, Gold DR, Ryan LM, Weiss ST, Celedón JC
Mode of delivery and cord blood cytokines: a birth cohort study.
Clin Mol Allergy. 2006;413.
BACKGROUND: The mechanisms for the association between birth by cesarean section and atopy and asthma are largely unknown. OBJECTIVE: To examine whether cesarean section results in neonatal secretion of cytokines that are associated with increased risk of atopy and/or asthma in childhood. To examine whether the association between mode of delivery and neonatal immune responses is explained by exposure to the maternal gut flora (a marker of the vaginal flora). METHODS: CBMCs were isolated from 37 neonates at delivery, and secretion of IL-13, IFN-gamma, and IL-10 (at baseline and after stimulation with antigens [dust mite and cat dander allergens, phytohemagglutinin, and lipopolysaccharide]) was quantified by ELISA. Total and specific microbes were quantified in maternal stool. The relation between mode of delivery and cord blood cytokines was examined by linear regression. The relation between maternal stool microbes and cord blood cytokines was examined by Spearman's correlation coefficients. RESULTS: Cesarean section was associated with increased levels of IL-13 and IFN-gamma. In multivariate analyses, cesarean section was associated with an increment of 79.4 pg/ml in secretion of IL-13 by CBMCs after stimulation with dust mite allergen (P < 0.001). Among children born by vaginal delivery, gram-positive anaerobes and total anaerobes in maternal stool were positively correlated with levels of IL-10, and gram-negative aerobic bacteria in maternal stool were negatively correlated with levels of IL-13 and IFN-gamma. CONCLUSION: Cesarean section is associated with increased levels of IL-13 and IFN-gamma, perhaps because of lack of labor and/or reduced exposure to specific microbes (e.g., gram-positive anaerobes) at birth. [Abstract/Link to Full Text]

Westwood GS, Huang SW, Keyhani NO
Molecular and immunological characterization of allergens from the entomopathogenic fungus Beauveria bassiana.
Clin Mol Allergy. 2006;412.
BACKGROUND: Entomopathogenic fungi such as Beauveria bassiana are considered promising biological control agents for a variety of arthropod pests. Beauveria species, however, have the potential to elicit allergenic reactions in humans, although no specific allergens have been characterized to date. METHODS: Four putative allergens were identified within B. bassiana expressed sequence tag (EST) datasets. IgE-reactivity studies were performed using sera from patients displaying mold allergies against recombinant B. bassiana proteins expressed in E. coli. RESULTS: Full length cDNA and genomic nucleotide sequences of four potential B. bassiana allergens were isolated. BLASTX search results led to their putative designation as follows; Bb-Eno1, with similarity to fungal enolases; Bb-f2, similar to the Aspergillus fumigatus major allergen, Asp f2 and to a fibrinogen binding mannoprotein; Bb-Ald, similar to aldehyde dehydrogenases; and Bb-Hex, similar to N-acetyl-hexosaminadases. All four genes were cloned into E. coli expression systems and recombinant proteins were produced. Immunoblots of E. coli extracts probed with pooled as well as individual human sera from patients displaying mould allergies demonstrated IgE reactivity versus recombinant Bb-Eno1 and Bb-Ald. CONCLUSION: Four putative Beauveria bassiana allergens were identified. Recombinant proteins corresponding to two of the four, Bb-Eno1 and Bb-Ald were bound by sera IgEs derived from patients with fungal allergies. These data confirm the potential allergenicity of B. bassiana by identification of specific human IgE reactive epitopes. [Abstract/Link to Full Text]

Kurup VP, Knutsen AP, Moss RB, Bansal NK
Specific antibodies to recombinant allergens of Aspergillus fumigatus in cystic fibrosis patients with ABPA.
Clin Mol Allergy. 2006;411.
BACKGROUND: Aspergillus fumigatus, a widely distributed fungus, has been implicated in causing life threatening infections as well as severe asthma and allergic diseases in man. Allergic affliction like allergic bronchopulmonary aspergillosis (ABPA) is a disabling lung disease frequently seen in patients with asthma and cystic fibrosis. Immunodiagnosis of the former is comparatively easier due to the availability of purified antigens and sensitive methods. However, this is not true with cystic fibrosis patients where the prevalence of ABPA is fairly high and the morbidity and mortality are significant. METHODS: In the present study, we have evaluated purified recombinant allergens from A. fumigatus, namely Asp f 1, f 2, f 3, f 4, and f 6 using ELISA and a semi-automated method (ImmunoCAP). We studied 17 patients each from cystic fibrosis with ABPA, and cystic fibrosis with asthma, 22 cystic fibrosis with no ABPA or asthma, and 11 age matched controls. RESULTS: The results indicate that no antigen, antibody or method is capable of differentiating cystic fibrosis (CF) with ABPA from other CF patients, although some allergens showed strong reaction or showed more prevalence among the patients studied. CONCLUSION: When results of several allergens such as Asp f 1, f 2, f 3, f 4, and f 6 in their binding to IgA, IgG, and IgE antibodies were analyzed, a more strong discrimination of CF patients with ABPA was possible from the other groups studied. [Abstract/Link to Full Text]

Lee SK, Ye YM, Yoon SH, Lee BO, Kim SH, Park HS
Evaluation of the sensitization rates and identification of IgE-binding components in wild and genetically modified potatoes in patients with allergic disorders.
Clin Mol Allergy. 2006;410.
BACKGROUND: The potato is one of the most common types of genetically modified (GM) food. However, there are no published data evaluating the impact of genetic manipulations on the allergenicity of GM potatoes. To compare the allergenicity of GM potatoes with that of wild-type potatoes using in vivo and in vitro methods in adult allergy patients sensitized to potatoes. METHODS: A total of 1886 patients with various allergic diseases and 38 healthy controls participated in the study. Skin-prick testing and IgE-ELISA were carried out with extracts prepared from wild-type and GM potatoes. An ELISA inhibition test was used to confirm the binding specificity. IgE-binding components in extracts from the two types of potato were identified by SDS-PAGE and IgE-immunoblotting. The effects of digestive enzymes and heat on the allergenicity of the extracts was evaluated by preincubating the potatoes with or without simulated gastric and intestinal fluids in the absence or presence of heat. RESULTS: Positive responses (ratio of the wheal size induced by the allergen to that induced by histamine (A/H) > or = 2+) to wild-type or GM potato extracts, as demonstrated by the skin-prick test, were observed in 108 patients (5.7%). Serum-specific IgE was detected in 0-88% of subjects who tested positively. ELISA inhibition tests indicated significant inhibition when extract from each type of potato was added. IgE-immunoblot analysis demonstrated the presence of 14 IgE-binding components within the wild-type potato and 9 within the GM potato. Furthermore, a common 45-kDa binding component that yielded similar IgE-binding patterns was noted in more than 80% of the reactions using sera from patients sensitized to wild-type or GM potato. Exposure to simulated gastric fluid and heat treatment similarly inhibited IgE binding by extracts from wild-type and GM potatoes, whereas minimal changes were obtained following exposure of the extracts to simulated intestinal fluid. CONCLUSION: Our results strongly suggest that genetic manipulation of potatoes does not increase their allergenic risk. The sensitization rate of adult allergy patients to both types of extract was 5.7%, and a common major allergen (45 kDa) was identified. [Abstract/Link to Full Text]

Imboden M, Nieters A, Bircher AJ, Brutsche M, Becker N, Wjst M, Ackermann-Liebrich U, Berger W, Probst-Hensch NM
Cytokine gene polymorphisms and atopic disease in two European cohorts. (ECRHS-Basel and SAPALDIA).
Clin Mol Allergy. 2006;49.
BACKGROUND: Atopy and allergic phenotypes are biologically characterized by an imbalanced T helper cell response skewed towards a type 2 (TH2) immune response associated with elevated serum immunoglobulin E (IgE) levels. Polymorphisms in cytokine genes might modulate regulation of the TH1/TH2 balance. We thus aimed at reproducing our previous findings from a European study population on the association of various cytokine polymorphisms with self-reported hay fever as well as increased total and specific IgE levels in two comparable study populations. METHODS: Two prospective Caucasian cohorts were used. In the Basel center of the European Community Respiratory Health Survey (ECRHS, n = 418) ten distinct cytokine polymorphisms of putative functional relevance were genotyped. In the Swiss cohort Study on Air Pollution And Lung Disease In Adults (SAPALDIA, n = 6003) two cytokine polymorphisms were genotyped. The associations of these polymorphisms with atopy were estimated by covariance and logistic regression analysis. RESULTS: We confirmed IL4, IL10, IL6 and IL18 as candidate genes for atopic health outcomes. In the large, well-characterized SAPALDIA cohort the IL6(-174G>C) and IL18(-137G>C) polymorphisms were associated with circulating total IgE concentrations in subjects with hay fever. The IL18(-137G>C) polymorphism was also associated with the prevalence of hay fever. CONCLUSION: Comprehensive characterization of genetic variation in extended cytokine candidate gene regions is now needed. Large study networks must follow to investigate the association of risk patterns defined by genetic predisposing and environmental risk factors with specific atopic phenotypes. [Abstract/Link to Full Text]

Vadlamudi RS, Chi DS, Krishnaswamy G
Intestinal strongyloidiasis and hyperinfection syndrome.
Clin Mol Allergy. 2006;48.
In spite of recent advances with experiments on animal models, strongyloidiasis, an infection caused by the nematode parasite Strongyloides stercoralis, has still been an elusive disease. Though endemic in some developing countries, strongyloidiasis still poses a threat to the developed world. Due to the peculiar but characteristic features of autoinfection, hyperinfection syndrome involving only pulmonary and gastrointestinal systems, and disseminated infection with involvement of other organs, strongyloidiasis needs special attention by the physician, especially one serving patients in areas endemic for strongyloidiasis. Strongyloidiasis can occur without any symptoms, or as a potentially fatal hyperinfection or disseminated infection. Th2 cell-mediated immunity, humoral immunity and mucosal immunity have been shown to have protective effects against this parasitic infection especially in animal models. Any factors that suppress these mechanisms (such as intercurrent immune suppression or glucocorticoid therapy) could potentially trigger hyperinfection or disseminated infection which could be fatal. Even with the recent advances in laboratory tests, strongyloidiasis is still difficult to diagnose. But once diagnosed, the disease can be treated effectively with antihelminthic drugs like Ivermectin. This review article summarizes a case of strongyloidiasis and various aspects of strongyloidiasis, with emphasis on epidemiology, life cycle of Strongyloides stercoralis, clinical manifestations of the disease, corticosteroids and strongyloidiasis, diagnostic aspects of the disease, various host defense pathways against strongyloidiasis, and available treatment options. [Abstract/Link to Full Text]

Liu T, Wang BQ, Yang PC
A possible link between sinusitis and lower airway hypersensitivity: the role of Staphylococcal enterotoxin B.
Clin Mol Allergy. 2006;47.
BACKGROUND AND AIMS: The prevalence of asthma has been keeping arising with unknown etiology. The cumulative evidence indicates that chronic rhinosinusitis (CRS) closely relates to asthma, but the detailed mechanisms remain unclear. The present study aimed to take insight into the role of Staphylococcus enterotoxin B (SEB) in a possible association between CRS and asthma. METHODS: 38 patients with both CRS and asthma underwent functional endoscopic sinus surgery. Serum specific IgE and cytokines, clinical symptoms of CRS and asthma were evaluated before and after the surgery. Peripheral blood mononuclear cells (PBMCs) were separated from the patients and cultured. Th2 response of the cultured PBMCs in the presence or absence of specific antigens and SEB was evaluated. RESULTS: Besides the improvement of CRS symptoms, amelioration of asthma was also observed in the patients with both CRS and asthma after the sinus surgery. The preoperatively elevated Th2 cytokines, IL-4 and IL-5, normalized postoperatively. Th2 response was generated with separated PBMCs in the presence of specific antigens. SEB was required for maintaining Th2 response in these separated PBMCs. CONCLUSION: The present results indicate that a possible link exists between CRS and lower airway hypersensitivity. Sinusitis derived SEB may play a role in sustaining Th2 responses in the low airway hypersensitivity related to sinusitis. [Abstract/Link to Full Text]

Holse M, Assing K, Poulsen LK
CCR3, CCR5, CCR8 and CXCR3 expression in memory T helper cells from allergic rhinitis patients, asymptomatically sensitized and healthy individuals.
Clin Mol Allergy. 2006;46.
BACKGROUND: Chemokine receptors have been suggested to be preferentially expressed on CD4+ T cells with CCR3 and CCR8 linked to the T helper (Th) 2 subset and CCR5 and CXCR3 to the Th1 subset, however this remains controversial. OBJECTIVE: Our aim was to compare the CCR3, CCR5, CCR8 and CXCR3 expression in memory Th cells from allergic, asymptomatically sensitized and healthy individuals. METHODS: Peripheral blood mononuclear cells from 8 pollen allergic rhinitis patients, 10 asymptomatically sensitized and 10 healthy individuals were stimulated for 7 days with allergen or tetanus toxoid. CCR3, CCR5, CCR8, CXCR3, CD4 and CD45RO were detected by flow cytometry. RESULTS: No differences in chemokine receptor expression were observed between the three groups on day 0, and seven days of unstimulated culture did not change the expression. Both antigenic stimuli increased the chemokine receptor expression, tetanus toxoid being the most potent. No differences in percentage chemokine receptor positive memory Th cells were observed between the three groups on day 7. Only a change in MFI for CCR5 was significantly different between the three groups after allergen stimulation of the Th cells. CONCLUSION: We conclude that even though allergen and antigen induced increased chemokine receptor expression, no differences in profiles were identified in memory Th cells from patient groups with different atopic status. [Abstract/Link to Full Text]

Kauffman HF, Tamm M, Timmerman JA, Borger P
House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms.
Clin Mol Allergy. 2006;45.
House dust mite allergens (HDM) cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1) and unknown enzymatic activity (Der p 2, Der p 5) induce biological responses in a human airway-derived epithelial cell line (A549), and if so, to elucidate the underlying mechanism(s) of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca2+ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR)1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca2+ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca2+ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells. [Abstract/Link to Full Text]

Kim N, Kwon SS, Lee J, Kim S, Yoo TJ
Protective effect of the DNA vaccine encoding the major house dust mite allergens on allergic inflammation in the murine model of house dust mite allergy.
Clin Mol Allergy. 2006;44.
BACKGROUND: Vaccination with naked DNA encoding antigen induces cellular and humoral immunity characterized by the activation of specific Th1 cells. OBJECTIVE: To evaluate the effects of vaccination with mixed naked DNA plasmids encoding Der p 1, Der p 2, Der p 3, Der f 1, Der f 2, and Der f 3, the major house dust mite allergens on the allergic inflammation to the whole house dust mites (HDM) crude extract. METHODS: Three hundred micrograms of these gene mixtures were injected into muscle of BALB/c mice. Control mice were injected with the pcDNA 3.1 blank vector. After 3 weeks, the mice were actively sensitized and inhaled with the whole house dust mite extract intranasally. RESULTS : The vaccinated mice showed a significantly decreased synthesis of total and HDM-specific IgE compared with controls. Analysis of the cytokine profile of lymphocytes after challenge with HDM crude extract revealed that mRNA expression of interferon-gamma was higher in the vaccinated mice than in the controls. Reduced infiltration of inflammatory cells and the prominent infiltration of CD8+ T cells were observed in histology of lung tissue from the vaccinated mice. CONCLUSION: Vaccination with DNA encoding the major house dust mite allergens provides a promising approach for treating allergic responses to whole house dust mite allergens. [Abstract/Link to Full Text]

Puthothu B, Krueger M, Heinze J, Forster J, Heinzmann A
Impact of IL8 and IL8-receptor alpha polymorphisms on the genetics of bronchial asthma and severe RSV infections.
Clin Mol Allergy. 2006;42.
BACKGROUND: Interleukin 8 (IL8) belongs to the family of chemokines. It mediates the activation and migration of neutrophils from peripheral blood into tissue and hereby plays a pivotal role in the initiation of inflammation. Thus it is important in inflammatory lung diseases like bronchial asthma or severe infections by Respiratory Syncytial Virus (RSV). IL8 acts through binding to the IL8-Receptor alpha (IL8RA). For both genes association with asthma has been described. In addition, IL8 has been found in association with RSV bronchiolitis. The aim of our study was to test both genes for association with asthma and severe RSV infections. In addition we were interested in whether a common genetic background of both diseases exists in regards to these genes. METHODS: We genotyped the two IL8 promotor polymorphisms -251A/T and -781C/T and the three amino acid variants M31R, S276T and R335C in IL8RA on 322 children with asthma, 131 infants with severe RSV associated diseases and 270 controls. Statistical analyses made use of the Armitage's trend test for single polymorphisms and FAMHAP for calculations of haplotypes. RESULTS: We found association of the IL8 polymorphism -781C/T as well as IL8 haplotypes with asthma (p = 0.011 and p = 0.036, respectively). In addition, direct comparison of the asthmatic population with the RSV population revealed significant differences, both for -781C/T alone (p = 0.034) and IL8 haplotypes (p = 0.005). The amino acid variants in IL8RA were evenly distributed in between all three populations. CONCLUSION: We conclude from our data that IL8 might play a role in the genetic predisposition to asthma and that these effects are different or even opposite to the effects on severe RSV diseases. Furthermore, IL8RA is unlikely to play a major role in the genetics of either disease. [Abstract/Link to Full Text]

Knutsen AP, Kariuki B, Consolino JD, Warrier MR
IL-4 alpha chain receptor (IL-4Ralpha) polymorphisms in allergic bronchopulmonary sspergillosis.
Clin Mol Allergy. 2006;43.
BACKGROUND: Allergic bronchopulmonary aspergillosis occurs in 7-10% of cystic fibrosis (CF) and 1-2% of asthmatic patients. HLA-DR restriction and increased sensitivity to IL-4 stimulation have been proposed as risk factors in these populations. OBJECTIVE: We examined for the presence of IL-4 receptor alpha chain (IL-4Ralpha) single nucleotide polymorphisms (SNPs) in ABPA and whether these accounted for increased sensitivity to IL-4 stimulation. METHODS: One extracellular (ile75val) and four cytoplasmic IL-4Ralpha SNPs were analyzed in 40 CF and 22 asthmatic patients and in 56 non-ABPA CF and asthmatic patients. Sensitivity to IL-4 stimulation was measured by induction of CD23 expression on B cells. RESULTS: IL-4Ralpha SNPs were observed in 95% of ABPA patients. The predominant IL-4Ralpha SNP was the extracellular IL-4Ralpha SNP, ile75val, observed in 80% of ABPA patients. CONCLUSION: The presence of IL-4Ralpha SNPs, principally ile75val, appears to be a genetic risk for the development of ABPA. [Abstract/Link to Full Text]

Smith CS, Smith SA, Grier TJ, Justus DE
Aluminum sulfate significantly reduces the skin test response to common allergens in sensitized patients.
Clin Mol Allergy. 2006;41.
BACKGROUND: Avoidance of allergens is still recommended as the first and best way to prevent allergic illnesses and their comorbid diseases. Despite a variety of attempts there has been very limited success in the area of environmental control of allergic disease. Our objective was to identify a non-invasive, non-pharmacological method to reduce indoor allergen loads in atopic persons' homes and public environments. We employed a novel in vivo approach to examine the possibility of using aluminum sulfate to control environmental allergens. METHODS: Fifty skin test reactive patients were simultaneously skin tested with conventional test materials and the actions of the protein/glycoprotein modifier, aluminum sulfate. Common allergens, dog, cat, dust mite, Alternaria, and cockroach were used in the study. RESULTS: Skin test reactivity was significantly reduced by the modifier aluminum sulfate. Our studies demonstrate that the effects of histamine were not affected by the presence of aluminum sulfate. In fact, skin test reactivity was reduced independent of whether aluminum sulfate was present in the allergen test material or removed prior to testing, indicating that the allergens had in some way been inactivated. CONCLUSION: Aluminum sulfate was found to reduce the in vivo allergic reaction cascade induced by skin testing with common allergens. The exact mechanism is not clear but appears to involve the alteration of IgE-binding epitopes on the allergen. Our results indicate that it may be possible to diminish the allergenicity of an environment by application of the active agent aluminum sulfate, thus producing environmental control without complete removal of the allergen. [Abstract/Link to Full Text]

Matheu V, Perez E, Hernández M, Díaz E, Darias R, González A, García JC, Sánchez I, Feliciano L, Caballero A, de la Torre F
Insulin allergy and resistance successfully treated by desensitisation with Aspart insulin.
Clin Mol Allergy. 2005;316.
A 25-year-old, with type I Diabetes Mellitus with a previous diagnosis of Protamine Allergy but not to human Insulin, started to notice anaphylactic reactions immediately after bolus with Insulin. Skin prick and intradermal test were positive to all insulins. Skin tests to other potential allergens resulted negative. Examination after bolus of Human Insulin revealed urticaria. Daily insulin requirement were around 2-2,4 U/Kg/day. Slow desensitisation with Aspart insulin, the insulin with lowest size of skin test, was performed using subcutaneous insulin pump. Six months after the end of desensitisation his daily insulin requirement decreased to 0.8 U/Kg/day and oral corticosteroids are being reduced with no symptoms. [Abstract/Link to Full Text]

Isidoro-García M, Dávila I, Laffond E, Moreno E, Lorente F, González-Sarmiento R
Interleukin-4 (IL4) and Interleukin-4 receptor (IL4RA) polymorphisms in asthma: a case control study.
Clin Mol Allergy. 2005 Nov 29;315.
BACKGROUND: IL4/IL4RA pathway plays an important role in atopy and asthma. Different polymorphisms in IL4 and IL4RA genes have been described. Particularly, -33C>TIL4 and 576Q>RIL4RA SNPs have been independently associated to atopy and asthma. The purpose of this study was to analyse these polymorphisms in a population of patients with a well-characterized asthma phenotype. METHODS: A total of 212 unrelated Caucasian individuals, 133 patients with asthma and 79 healthy subjects without symptoms or history of asthma or atopy and with negative skin prick tests were recruited. Lung function was measured by spirometry and asthma was specialist physician-diagnosed according to the ATS (American Thoracic Society) criteria and classified following the GINA (Global Initiative for Asthma) guidelines. Skin prick tests were performed according to EAACI recommendations. -33C>TIL4 was studied with TaqMan assay and 576Q>RIL4RA by PCR-RFLP technique. Hardy-Weinberg equilibrium was analysed in all groups. Dichotomous variables were analysed using chi2, Fisher exact test, Monte Carlo simulation test and odds ratio test. To model the effects of multiple covariates logistic regression was used. RESULTS: No statistically significant differences between the group of patients with asthma and the controls were found when the allele and genotype distribution of -33C>TIL4 and 576Q>RIL4RA polymorphisms were compared. However, the T allele of the -33C>TIL4 SNP was more frequent in patients with persistent asthma. Multivariate analysis adjusted for age and sex confirmed that carriers of allele T had an increased risk of persistent asthma (OR: 2.77, 95%CI: 1.18-6.49; p = 0.019). Analysis of combination of polymorphisms showed that patients carrying both the T allele of -33C>TIL4 and the A allele of 576Q>RIL4RA had an increased risk of asthma. This association was particularly observed in persistent asthma [Fisher's p value = 0.0021, Monte Carlo p value (after 10(4) simulations) = 0.0016, OR:3.39; 95% CI:1.50-7.66]. CONCLUSION: Our results show a trend of association between the genetic combination of the T allele of -33C>TIL4 and the A allele of 576Q>RIL4RA with asthma. This genetic variant was more frequently observed in patients with persistent asthma. As long as this study was performed in a small population, further studies in other populations are needed to confirm these results. [Abstract/Link to Full Text]

Zacharisen MC, Levy MB, Shaw JL, Kurup VP
Severe allergic reactions to guinea pig.
Clin Mol Allergy. 2005 Oct 27;314.
BACKGROUND: Allergic sensitization and reactions to guinea pig (Cavia porcellus) have been well documented in laboratory animal handlers, primarily manifesting as rhinitis, conjunctivitis, and asthma. Severe allergic reactions, however, are rare. METHODS: We report two patients with severe allergic reactions following non-occupational exposure to guinea pigs. The first patient, an 11-year-old female, developed ocular, nasal, skin and laryngeal edema symptoms immediately after handling a guinea pig. The second patient, a 24-year-old female, developed symptoms of isolated laryngeal edema after cleaning a guinea pig cage. Percutaneous skin testing, RAST, ELISA and ELISA inhibition testing with guinea pig extract were performed. RESULTS: Both patients had IgE-mediated allergy to guinea pig confirmed by ELISA and either RAST or skin testing. ELISA inhibition studies confirmed the specificity of the IgE reactivity to guinea pig. CONCLUSION: Severe IgE-mediated reactions can occur following non-occupational guinea pig exposure. Physicians should be aware of this possibility. [Abstract/Link to Full Text]

Sankian M, Varasteh A, Pazouki N, Mahmoudi M
Sequence homology: a poor predictive value for profilins cross-reactivity.
Clin Mol Allergy. 2005 Sep 10;313.
BACKGROUND: Profilins are highly cross-reactive allergens which bind IgE antibodies of almost 20% of plant-allergic patients. This study is aimed at investigating cross-reactivity of melon profilin with other plant profilins and the role of the linear and conformational epitopes in human IgE cross-reactivity. METHODS: Seventeen patients with melon allergy were selected based on clinical history and a positive skin prick test to melon extract. Melon profilin has been cloned and expressed in E. coli. The IgE binding and cross-reactivity of the recombinant profilin were measured by ELISA and inhibition ELISA. The amino acid sequence of melon profilin was compared with other profilin sequences. A combination of chemical cleavage and immunoblotting techniques were used to define the role of conformational and linear epitopes in IgE binding. Comparative modeling was used to construct three-dimensional models of profilins and to assess theoretical impact of amino acid differences on conformational structure. RESULTS: Profilin was identified as a major IgE-binding component of melon. Alignment of amino acid sequences of melon profilin with other profilins showed the most identity with watermelon profilin. This melon profilin showed substantial cross-reactivity with the tomato, peach, grape and Cynodon dactylon (Bermuda grass) pollen profilins. Cantaloupe, watermelon, banana and Poa pratensis (Kentucky blue grass) displayed no notable inhibition. Our experiments also indicated human IgE only react with complete melon profilin. Immunoblotting analysis with rabbit polyclonal antibody shows the reaction of the antibody to the fragmented and complete melon profilin. Although, the well-known linear epitope of profilins were identical in melon and watermelon, comparison of three-dimensional models of watermelon and melon profilins indicated amino acid differences influence the electric potential and accessibility of the solvent-accessible surface of profilins that may markedly affect conformational epitopes. CONCLUSION: Human IgE reactivity to melon profilin strongly depends on the highly conserved conformational structure, rather than a high degree of amino acid sequence identity or even linear epitopes identity. [Abstract/Link to Full Text]

Haye R, Døsen LK
Insect sting allergy. A study from 1980 to 2003 of patients who started treatment with venom immunotherapy between 1980 and 1998.
Clin Mol Allergy. 2005 Aug 19;312.
BACKGROUND: Previously we treated patients with insect sting allergy with venom immunotherapy (IT) using whole body insect extracts. From 1980 we changed to insect venoms. The purpose of this study was to analyse data from the patients in order to improve our treatment. METHODS: This is an open, single centre study on patients treated with venom IT 14 years or older with a history of a systemic allergic reaction to an insect sting, a positive skin prick test (SPT) or a positive RAST and willingness to comply with five years of IT. Clinical and laboratory data were registered prospectively at the start of IT and after five years of treatment until 2003 on patients who started IT between 1980 and 1998. Questionnaires were answered in 1989, 1993 and 2003. Statistical analysis was done with Pearson's chi square, Fisher's exact or the t-test. RESULTS: Of 315 patients treated, 44 were given bee, 248 common wasp and 23 both venoms. Of the common wasp sting incidents 5.5 % resulted in a severe allergic reaction (SAR) during adequate IT and 22% after cessation. Seventy-one per cent of the patients carried epinephrine. Precautionary steps were taken by 77% of the patients during or after inadequate IT. On or after adequate IT 83% felt completely or substantially safe. Surprisingly 29 % of those inadequately treated felt safer and 50% were satisfied with having had the opportunity to be treated. The SPT became negative in 68% of the wasp allergic patients after five years of adequate IT. Increased risk of experiencing SAR to a future sting in wasp allergic patients after cessation of adequate IT was significantly associated with a SAR due to IT during the rush regimen. SAR due to IT occurred very rarely during maintenance dosing. CONCLUSION: Adequate venom IT is very effective while ongoing but somewhat less effective after cessation, while inadequate treatment gives poor results. More of our patients should complete five years of IT and some should continue IT. The type of reaction to IT during incremental dosing may be of help in deciding who should continue beyond five years. Maintenance IT may be taken over by the general physician. [Abstract/Link to Full Text]

Kurup VP, Sussman GL, Yeang HY, Elms N, Breiteneder H, Arif SA, Kelly KJ, Bansal NK, Fink JN
Specific IgE response to purified and recombinant allergens in latex allergy.
Clin Mol Allergy. 2005 Aug 10;311.
BACKGROUND: In recent years, allergy to natural rubber latex has emerged as a major allergy among certain occupational groups and patients with underlying diseases. The sensitization and development of latex allergy has been attributed to exposure to products containing residual latex proteins. Although improved manufacturing procedures resulted in a considerable reduction of new cases, the potential risk for some patient groups is still great. In addition the prevalent cross-reactivity of latex proteins with other food allergens poses a major concern. A number of purified allergens and a few commercial kits are currently available, but no concerted effort was undertaken to evaluate them. METHODS: We studied 11 purified latex allergens, Hev b 1 to Hev b 10, and Hev b 13 along with several crude allergen extracts and two commercial ImmunoCAP assays to evaluate specific IgE antibody in the sera from latex allergic patients and controls. Health care workers and spina bifida patients with clinical symptoms of latex allergy, spina bifida patients without latex allergy, and non-atopic health care workers have been studied. RESULTS: The results suggest that Hev b 2, 5, 6, and 13 together identified over 80 percent health care workers with latex allergy, while Hev b 6 along with Hev b 1 or 3 detected specific IgE antibody in all sera studied from patients with spina bifida and latex allergy. The ImmunoCAP results using both Hev b 5 amplified and non-amplified closely agreed with the clinical diagnosis of latex allergy in health care workers and in spina bifida. CONCLUSION: Although the purified allergens and crude extracts reacted diversely with IgE from different patient groups, the results indicated that use of certain combinations of purified recombinant antigens will be useful in commercial kits or in in-house assays for detecting specific IgE antibody in the sera. The results suggest that a combination of Hev b 2, 3, 5, 6, and 13 together detected specific IgE in 80% of the sera from latex allergic patients. Both ImmunoCAPs correctly identified over 95% of latex allergic patients, however, showed reactivity with a few normal control subjects. [Abstract/Link to Full Text]

Hoffmann HJ, Bøgebjerg M, Nielsen LP, Dahl R
Lysis with Saponin improves detection of the response through CD203c and CD63 in the basophil activation test after crosslinking of the high affinity IgE receptor FcepsilonRI.
Clin Mol Allergy. 2005 Jul 4;310.
BACKGROUND: The basophil activation test (BAT), in which translocation of markers to the surface of blood basophils is measured in response to allergen by flow cytometry, is a rapid assay that is gaining popularity. Two markers are currently being evaluated for the BAT; CD63 and the lineage-specific CD203c. In a recent report, detection of CD203c after lysis with Saponin was shown to be superior to detection of CD63 after lysis with formic acid. We wanted to compare a) lysis with formic acid and lysis with Saponin, b) the response through CD203c and CD63, and c) the definition 10% activated cells above background with the probability binning metric T(chi) > 4, on sets of data generated with blood basophils stimulated with varying concentrations of anti-FcepsilonRI antibody. METHODS: Blood from volunteers was incubated with serial logarithmic dilutions of anti-FcepsilonRI and subsequently with antibodies to CD203c PE and CD63 FITC. Sets of samples set up in parallel were lysed with either Saponin based Whole Blood Lysing reagent or with formic acid based Immunoprep/Q-prep. Samples were acquired on a FACS Calibur, but were compensated and analysed offline. Responders were defined as persons who had 10% or more activated basophils above background, or a T(chi) > 4, for two consecutive dilutions of anti-FcepsilonRI antibody. RESULTS: More basophils (median 1164 vs. median 397) and better discrimination of upregulated CD203c and CD63 amongst responders were obtained after lysis with Saponin than after lysis with formic acid. We suggest that CD203c may be a more sensitive marker for the BAT than CD63, as 6/11 responders were found with CD203c, compared with 3/11 with CD63. Most responders (7/11) were identified with probability binning. CONCLUSION: A combination of lysis with Saponin and the markers CD203c and CD63 computed by probability binning may be the most sensitive method of detecting activation of basophils after stimulation through FcepsilonRI. [Abstract/Link to Full Text]

Boumiza R, Debard AL, Monneret G
The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives.
Clin Mol Allergy. 2005 Jun 30;39.
The diagnosis of immediate allergy is mainly based upon an evocative clinical history, positive skin tests (gold standard) and, if available, detection of specific IgE. In some complicated cases, functional in vitro tests are necessary. The general concept of those tests is to mimic in vitro the contact between allergens and circulating basophils. The first approach to basophil functional responses was the histamine release test but this has remained controversial due to insufficient sensitivity and specificity. During recent years an increasing number of studies have demonstrated that flow cytometry is a reliable tool for monitoring basophil activation upon allergen challenge by detecting surface expression of degranulation/activation markers (CD63 or CD203c). This article reviews the recent improvements to the basophil activation test made possible by flow cytometry, focusing on the use of anti-CRTH2/DP2 antibodies for basophil recognition. On the basis of a new triple staining protocol, the basophil activation test has become a standardized tool for in vitro diagnosis of immediate allergy. It is also suitable for pharmacological studies on non-purified human basophils. Multicenter studies are now required for its clinical assessment in large patient populations and to define the cut-off values for clinical decision-making. [Abstract/Link to Full Text]

Korinth G, Broding HC, Uter W, Drexler H
Secondary prevention of allergic symptoms in a dairy farmer by use of a milking robot.
Clin Mol Allergy. 2005 Jun 22;38.
BACKGROUND: Animal-derived allergens include lipocalins which play an increasing role in occupational respiratory sensitizations. The prevention of sensitization in stock farming is often difficult due to intense exposure, with traditional milking still requiring close animal contact. Complete avoidance of allergen exposure is only possible if stock farming is abandoned. This is, however, often not feasible in small dairy plants because of the resulting loss of income. CASE PRESENTATION: In a 37-year-old female farmer daily asthmatic complaints appeared, associated with cow dust-derived allergen exposure by milking with a conventional device. Respiratory symptoms increased during a period of 12 years. Allergic bronchial asthma was diagnosed, caused by sensitization against cow dust-derived allergens, as demonstrated by positive skin prick test and by detection of IgE antibodies. In a separate specific inhalation challenge test using a 10% extract of cow dust-derived allergens a 330% increase of airway resistance was detected. To enable further dairy farming, a milking robot was installed in 1999, i.e., an automatic milking system. The novel milking technique reduced the daily exposure from over 2 hours to approximately 10 min. The clinical course after the installation of the milking robot was favourable, with less frequent allergic and asthmatic symptoms. Furthermore, asthma medication could be reduced. Improvement was noted also in terms of lung-function and decreased total serum IgE. CONCLUSION: The case presented and the evidence from the literature indicates that the strategy of exposure minimization to allergens at workplaces can be an effective alternative to total elimination. In farmers with cow dust allergy a milking robot is an appropriate technical measure to minimize allergen-exposure. [Abstract/Link to Full Text]

Wjst M
Another explanation for the low allergy rate in the rural Alpine foothills.
Clin Mol Allergy. 2005 Jun 5;37.
A low allergy rate in coal and wood heated homes has been described in the small villages in the Alpine foothills and subsequently found to be associated with the farming environment. This was interpreted within the framework of the hygiene hypothesis but there are also alternative explanations. Lower air pollution could be one reason, which is, however, unlikely since the differences between the Bavarian countryside and the Munich municipal area were only weak. There could be genetic differences between the urban and rural population by previous isolation or by self-selection. The potential drop-out of allergy genes, however, will also not explain the absent increase of allergies in two generations. More likely, other lifestyle factors are important. Dietary habits are different in farmers and a less frequent vitamin D supplementation of newborns (otherwise expected to be allergy promoting) has been shown recently. The underlying cause for the "non-allergic farm child" remains speculative until the transfer of any farm-associated factor is leading to a similar risk reduction in the general population. [Abstract/Link to Full Text]

Belleau JT, Gandhi RK, McPherson HM, Lew DB
Research upregulation of CD23 (FcepsilonRII) expression in human airway smooth muscle cells (huASMC) in response to IL-4, GM-CSF, and IL-4/GM-CSF.
Clin Mol Allergy. 2005 May 20;36.
BACKGROUND: Airway smooth muscle cells play a key role in remodeling that contributes to airway hyperreactivity. Airway smooth muscle remodeling includes hypertrophy and hyperplasia. It has been previously shown that the expression of CD23 on ASMC in rabbits can be induced by the IgE component of the atopic serum. We examined if other components of atopic serum are capable of inducing CD23 expression independent of IgE. METHODS: Serum starved huASMC were stimulated with either IL-4, GM-CSF, IL-13, IL-5, PGD2, LTD4, tryptase or a combination of IL-4, IL-5, IL-13 each with GM-CSF for a period of 24 h. CD23 expression was analyzed by flow cytometry, western blot, and indirect immunofluorescence. RESULTS: The CD23 protein expression was upregulated in huASMC in response to IL-4, GM-CSF, and IL-4/GM-CSF. The percentage of cells with increased fluorescence intensity above the control was 25.1 +/- 4.2% (IL-4), 15.6 +/- 2.7% (GM-CSF) and 32.9 +/- 13.9% (IL-4/GMCSF combination)(n = 3). The protein content of IL-4/GMCSF stimulated cells was significantly elevated. Expression of CD23 in response to IL-4, GM-CSF, IL-4/GM-CSF was accompanied by changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling. Western blot revealed abundant expression of the IL-4Ralpha and a low level expression of IL-2Rgammac in huASMC. Stimulation with IL-4 resulted in the phosphorylation of STAT-6 and an increase in the expression of the IL-2Rgammac. CONCLUSION: CD23 on huASMC is upregulated by IL-4, GM-CSF, and IL-4/GM-CSF. The expression of CD23 is accompanied by an increase in cell volume and an increase in protein content per cell, suggesting hypertrophy. Upregulation of CD23 by IL-4/GM-CSF results in phenotypic changes in huASMC that could play a role in cell migration or a change in the synthetic function of the cells. Upregulation of CD23 in huASMC by IL-4 and GM-CSF can contribute to changes in huASMC and may provide an avenue for new therapeutic options in asthma targeting ASMC. [Abstract/Link to Full Text]

Uings I, Puxeddu I, Temkin V, Smith SJ, Fattah D, Ray KP, Levi-Schaffer F
Effects of dexamethasone on TNF-alpha-induced release of cytokines from purified human blood eosinophils.
Clin Mol Allergy. 2005 Apr 27;3(1):5.
SUMMARY: BACKGROUND: TNF-alpha is an important mediator in allergy also for its effects on eosinophils. METHODS: The effect of dexamethasone on TNF-alpha induced eosinophils survival, degranulation (ECP), cytokines release (IL-8, GM-CSF) and adhesion to VCAM-1, ICAM-1 and IgG coated wells (EPO release) were evaluated. RESULTS: The drug inhibited IL-8 and GM-CSF production, but not viability, degranulation or adhesion in human peripheral blood eosinophils. CONCLUSION: These results indicate that part of the activity of glucocorticosteroids on eosinophils may be mediated by their ability to inhibit cytokine secretion that in turn is important for the perpetuation of the allergic inflammation. [Abstract/Link to Full Text]

---

Recent Articles in Clinical Microbiology Reviews

---

Hall KK, Lyman JA
Updated review of blood culture contamination.
Clin Microbiol Rev. 2006 Oct;19(4):788-802.
Blood culture contamination represents an ongoing source of frustration for clinicians and microbiologists alike. Ambiguous culture results often lead to diagnostic uncertainty in clinical management and are associated with increased health care costs due to unnecessary treatment and testing. A variety of strategies have been investigated and employed to decrease contamination rates. In addition, numerous approaches to increase our ability to distinguish between clinically significant bacteremia and contamination have been explored. In recent years, there has been an increase in the application of computer-based tools to support infection control activities as well as provide clinical decision support related to the management of infectious diseases. Finally, new approaches for estimating bacteremia risk which have the potential to decrease unnecessary blood culture utilization have been developed and evaluated. In this review, we provide an overview of blood culture contamination and describe the potential utility of a variety of approaches to improve both detection and prevention. While it is clear that progress is being made, fundamental challenges remain. [Abstract/Link to Full Text]

Meletiadis J, Chanock S, Walsh TJ
Human pharmacogenomic variations and their implications for antifungal efficacy.
Clin Microbiol Rev. 2006 Oct;19(4):763-87.
Pharmacogenomics is defined as the study of the impacts of heritable traits on pharmacology and toxicology. Candidate genes with potential pharmacogenomic importance include drug transporters involved in absorption and excretion, phase I enzymes (e.g., cytochrome P450-dependent mixed-function oxidases) and phase II enzymes (e.g., glucuronosyltransferases) contributing to metabolism, and those molecules (e.g., albumin, A1-acid glycoprotein, and lipoproteins) involved in the distribution of antifungal compounds. By using the tools of population genetics to define interindividual differences in drug absorption, distribution, metabolism, and excretion, pharmacogenomic models for genetic variations in antifungal pharmacokinetics can be derived. Pharmacogenomic factors may become especially important in the treatment of immunocompromised patients or those with persistent or refractory mycoses that cannot be explained by elevated MICs and where rational dosage optimization of the antifungal agent may be particularly critical. Pharmacogenomics has the potential to shift the paradigm of therapy and to improve the selection of antifungal compounds and adjustment of dosage based upon individual variations in drug absorption, metabolism, and excretion. [Abstract/Link to Full Text]

VandeWoude S, Apetrei C
Going wild: lessons from naturally occurring T-lymphotropic lentiviruses.
Clin Microbiol Rev. 2006 Oct;19(4):728-62.
Over 40 nonhuman primate (NHP) species harbor species-specific simian immunodeficiency viruses (SIVs). Similarly, more than 20 species of nondomestic felids and African hyenids demonstrate seroreactivity against feline immunodeficiency virus (FIV) antigens. While it has been challenging to study the biological implications of nonfatal infections in natural populations, epidemiologic and clinical studies performed thus far have only rarely detected increased morbidity or impaired fecundity/survival of naturally infected SIV- or FIV-seropositive versus -seronegative animals. Cross-species transmissions of these agents are rare in nature but have been used to develop experimental systems to evaluate mechanisms of pathogenicity and to develop animal models of HIV/AIDS. Given that felids and primates are substantially evolutionarily removed yet demonstrate the same pattern of apparently nonpathogenic lentiviral infections, comparison of the biological behaviors of these viruses can yield important implications for host-lentiviral adaptation which are relevant to human HIV/AIDS infection. This review therefore evaluates similarities in epidemiology, lentiviral genotyping, pathogenicity, host immune responses, and cross-species transmission of FIVs and factors associated with the establishment of lentiviral infections in new species. This comparison of consistent patterns in lentivirus biology will expose new directions for scientific inquiry for understanding the basis for virulence versus avirulence. [Abstract/Link to Full Text]

Swanson SJ, Neitzel D, Reed KD, Belongia EA
Coinfections acquired from ixodes ticks.
Clin Microbiol Rev. 2006 Oct;19(4):708-27.
The pathogens that cause Lyme disease (LD), human anaplasmosis, and babesiosis can coexist in Ixodes ticks and cause human coinfections. Although the risk of human coinfection differs by geographic location, the true prevalence of coinfecting pathogens among Ixodes ticks remains largely unknown for the majority of geographic locations. The prevalence of dually infected Ixodes ticks appears highest among ticks from regions of North America and Europe where LD is endemic, with reported prevalences of < or =28%. In North America and Europe, the majority of tick-borne coinfections occur among humans with diagnosed LD. Humans coinfected with LD and babesiosis appear to have more intense, prolonged symptoms than those with LD alone. Coinfected persons can also manifest diverse, influenza-like symptoms, and abnormal laboratory test results are frequently observed. Coinfecting pathogens might alter the efficiency of transmission, cause cooperative or competitive pathogen interactions, and alter disease severity among hosts. No prospective studies to assess the immunologic effects of coinfection among humans have been conducted, but animal models demonstrate that certain coinfections can modulate the immune response. Clinicians should consider the likelihood of coinfection when pursuing laboratory testing or selecting therapy for patients with tick-borne illness. [Abstract/Link to Full Text]

Garcia JE, Puentes A, Patarroyo ME
Developmental biology of sporozoite-host interactions in Plasmodium falciparum malaria: implications for vaccine design.
Clin Microbiol Rev. 2006 Oct;19(4):686-707.
The Plasmodium falciparum sporozoite infects different types of cells in a mosquito's salivary glands and human epithelial and Kuppfer cells and hepatocytes. These become differentiated later on, transforming themselves into the invasive red blood cell form, the merozoite. The ability of sporozoites to interact with different types of cells requires a wide variety of mechanisms allowing them to survive in both hosts: mobility, receptor-ligand interactions with different cellular receptors, and transformation and development into other invasive parasite forms, which are vitally important for parasite survival. Sporozoite complexity is reflected in the large quantity of proteins that can be expressed. Some of them have been extensively studied, such as CSP, TRAP, STARP, LSA-1, LSA-3, SALSA, SPECT1, SPECT2, MAEBL, and SPATR, due to their importance in infection and their potential use as vaccines. Our work has been focused on the search for the molecular mechanisms of parasite-host cellular receptor-ligand interactions by identifying amino acid sequences and the critical binding residues from these proteins relevant to parasite invasion. Once such sequences have been identified, it will be possible to modify them to induce a strong immune response against P. falciparum in the experimental Aotus monkey model. This all leads towards developing multistage, multicomponent, subunit-based vaccines that will be effective in eradicating or controlling malaria caused by P. falciparum. [Abstract/Link to Full Text]

Mathema B, Kurepina NE, Bifani PJ, Kreiswirth BN
Molecular epidemiology of tuberculosis: current insights.
Clin Microbiol Rev. 2006 Oct;19(4):658-85.
Molecular epidemiologic studies of tuberculosis (TB) have focused largely on utilizing molecular techniques to address short- and long-term epidemiologic questions, such as in outbreak investigations and in assessing the global dissemination of strains, respectively. This is done primarily by examining the extent of genetic diversity of clinical strains of Mycobacterium tuberculosis. When molecular methods are used in conjunction with classical epidemiology, their utility for TB control has been realized. For instance, molecular epidemiologic studies have added much-needed accuracy and precision in describing transmission dynamics, and they have facilitated investigation of previously unresolved issues, such as estimates of recent-versus-reactive disease and the extent of exogenous reinfection. In addition, there is mounting evidence to suggest that specific strains of M. tuberculosis belonging to discrete phylogenetic clusters (lineages) may differ in virulence, pathogenesis, and epidemiologic characteristics, all of which may significantly impact TB control and vaccine development strategies. Here, we review the current methods, concepts, and applications of molecular approaches used to better understand the epidemiology of TB. [Abstract/Link to Full Text]

Koenig SM, Truwit JD
Ventilator-associated pneumonia: diagnosis, treatment, and prevention.
Clin Microbiol Rev. 2006 Oct;19(4):637-57.
While critically ill patients experience a life-threatening illness, they commonly contract ventilator-associated pneumonia. This nosocomial infection increases morbidity and likely mortality as well as the cost of health care. This article reviews the literature with regard to diagnosis, treatment, and prevention. It provides conclusions that can be implemented in practice as well as an algorithm for the bedside clinician and also focuses on the controversies with regard to diagnostic tools and approaches, treatment plans, and prevention strategies. [Abstract/Link to Full Text]

Gillim-Ross L, Subbarao K
Emerging respiratory viruses: challenges and vaccine strategies.
Clin Microbiol Rev. 2006 Oct;19(4):614-36.
The current threat of avian influenza to the human population, the potential for the reemergence of severe acute respiratory syndrome (SARS)-associated coronavirus, and the identification of multiple novel respiratory viruses underline the necessity for the development of therapeutic and preventive strategies to combat viral infection. Vaccine development is a key component in the prevention of widespread viral infection and in the reduction of morbidity and mortality associated with many viral infections. In this review we describe the different approaches currently being evaluated in the development of vaccines against SARS-associated coronavirus and avian influenza viruses and also highlight the many obstacles encountered in the development of these vaccines. Lessons learned from current vaccine studies, coupled with our increasing knowledge of the host and viral factors involved in viral pathogenesis, will help to increase the speed with which efficacious vaccines targeting newly emerging viral pathogens can be developed. [Abstract/Link to Full Text]

Algood HM, Cover TL
Helicobacter pylori persistence: an overview of interactions between H. pylori and host immune defenses.
Clin Microbiol Rev. 2006 Oct;19(4):597-613.
Helicobacter pylori is a gram-negative bacterium that persistently colonizes more than half of the global human population. In order to successfully colonize the human stomach, H. pylori must initially overcome multiple innate host defenses. Remarkably, H. pylori can persistently colonize the stomach for decades or an entire lifetime despite development of an acquired immune response. This review focuses on the immune response to H. pylori and the mechanisms by which H. pylori resists immune clearance. Three main sections of the review are devoted to (i) analysis of the immune response to H. pylori in humans, (ii) analysis of interactions of H. pylori with host immune defenses in animal models, and (iii) interactions of H. pylori with immune cells in vitro. The topics addressed in this review are important for understanding how H. pylori resists immune clearance and also are relevant for understanding the pathogenesis of diseases caused by H. pylori (peptic ulcer disease, gastric adenocarcinoma, and gastric lymphoma). [Abstract/Link to Full Text]

Diemert DJ
Prevention and self-treatment of traveler's diarrhea.
Clin Microbiol Rev. 2006 Jul;19(3):583-94.
Of the millions who travel from the industrialized world to developing countries every year, between 20% and 50% will develop at least one episode of diarrhea, making it the most common medical ailment afflicting travelers. Although usually a mild illness, traveler's diarrhea can result in significant morbidity and hardship overseas. Precautions can be taken to minimize the risk of developing traveler's diarrhea, either through avoidance of potentially contaminated food or drink or through various prophylactic measures, including both nonpharmacological and antimicrobial strategies. If diarrhea does develop despite the precautions taken, effective treatment-usually a combination of an antibiotic and an antimotility agent-can be brought by the traveler and initiated as soon as symptoms develop. In the future, vaccines-several of which are in the advanced stages of clinical testing-may be added to the list of prophylactic measures. [Abstract/Link to Full Text]

McCullers JA
Insights into the interaction between influenza virus and pneumococcus.
Clin Microbiol Rev. 2006 Jul;19(3):571-82.
Bacterial infections following influenza are an important cause of morbidity and mortality worldwide. Based on the historical importance of pneumonia as a cause of death during pandemic influenza, the increasingly likely possibility that highly pathogenic avian influenza viruses will trigger the next worldwide pandemic underscores the need to understand the multiple mechanisms underlying the interaction between influenza virus and bacterial pathogens such as Streptococcus pneumoniae. There is ample evidence to support the historical view that influenza virus alters the lungs in a way that predisposes to adherence, invasion, and induction of disease by pneumococcus. Access to receptors is a key factor and may be facilitated by the virus through epithelial damage, by exposure or up-regulation of receptors, or by provoking the epithelial regeneration response to cytotoxic damage. More recent data indicate that alteration of the immune response by diminishing the ability of the host to clear pneumococcus or by amplification of the inflammatory cascade is another key factor. Identification and exploration of the underlying mechanisms responsible for this synergism will provide targets for prevention and treatment using drugs and vaccines. [Abstract/Link to Full Text]

Warner DF, Mizrahi V
Tuberculosis chemotherapy: the influence of bacillary stress and damage response pathways on drug efficacy.
Clin Microbiol Rev. 2006 Jul;19(3):558-70.
The global tuberculosis (TB) control effort is focused on interrupting transmission of the causative agent, Mycobacterium tuberculosis, through chemotherapeutic intervention in active infectious disease. The insufficiency of this approach is manifest in the inexorable annual increase in TB infection and mortality rates and the emergence of multidrug-resistant isolates. Critically, the limited efficacy of the current frontline anti-TB drug combination suggests that heterogeneity of host and bacillary physiologies might impair drug activity. This review explores the possibility that strategies enabling adaptation of M. tuberculosis to hostile in vivo conditions might contribute to the subversion of anti-TB chemotherapy. In particular, evidence that infecting bacilli are exposed to environmental and host immune-mediated DNA-damaging insults suggests a role for error-prone DNA repair synthesis in the generation of chromosomally encoded antibiotic resistance mutations. The failure of frontline anti-TB drugs to sterilize a population of susceptible bacilli is independent of genetic resistance, however, and instead implies the operation of alternative tolerance mechanisms. Specifically, it is proposed that the emergence of persister subpopulations might depend on the switch to an altered metabolic state mediated by the stringent response alarmone, (p)ppGpp, possibly involving some or all of the many toxin-antitoxin modules identified in the M. tuberculosis genome. [Abstract/Link to Full Text]

Kahn JS
Epidemiology of human metapneumovirus.
Clin Microbiol Rev. 2006 Jul;19(3):546-57.
Since the discovery of human metapneumovirus (hMPV) in 2001, the virus has been identified worldwide. hMPV is a common respiratory pathogen, particularly in infants and young children. The virus is associated with both upper and lower respiratory tract infections and may be a trigger for asthma. At least two major genotypes of hMPV circulate during community outbreaks. Whether these genotypes represent distinct serotypes remains controversial. The major challenges faced by the medical and scientific communities are the understanding of the pathogenesis of hMPV disease and the development of a safe and effective vaccine to protect against infection and disease caused by this newly recognized respiratory virus. [Abstract/Link to Full Text]

Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T
Bats: important reservoir hosts of emerging viruses.
Clin Microbiol Rev. 2006 Jul;19(3):531-45.
Bats (order Chiroptera, suborders Megachiroptera ["flying foxes"] and Microchiroptera) are abundant, diverse, and geographically widespread. These mammals provide us with resources, but their importance is minimized and many of their populations and species are at risk, even threatened or endangered. Some of their characteristics (food choices, colonial or solitary nature, population structure, ability to fly, seasonal migration and daily movement patterns, torpor and hibernation, life span, roosting behaviors, ability to echolocate, virus susceptibility) make them exquisitely suitable hosts of viruses and other disease agents. Bats of certain species are well recognized as being capable of transmitting rabies virus, but recent observations of outbreaks and epidemics of newly recognized human and livestock diseases caused by viruses transmitted by various megachiropteran and microchiropteran bats have drawn attention anew to these remarkable mammals. This paper summarizes information regarding chiropteran characteristics and information regarding 66 viruses that have been isolated from bats. From these summaries, it is clear that we do not know enough about bat biology; we are doing too little in terms of bat conservation; and there remain a multitude of questions regarding the role of bats in disease emergence. [Abstract/Link to Full Text]

Singh A, Goering RV, Simjee S, Foley SL, Zervos MJ
Application of molecular techniques to the study of hospital infection.
Clin Microbiol Rev. 2006 Jul;19(3):512-30.
Nosocomial infections are an important source of morbidity and mortality in hospital settings, afflicting an estimated 2 million patients in United States each year. This number represents up to 5% of hospitalized patients and results in an estimated 88,000 deaths and 4.5 billion dollars in excess health care costs. Increasingly, hospital-acquired infections with multidrug-resistant pathogens represent a major problem in patients. Understanding pathogen relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. The role of pathogen typing is to determine whether epidemiologically related isolates are also genetically related. To determine molecular relatedness of isolates for epidemiologic investigation, new technologies based on DNA, or molecular analysis, are methods of choice. These DNA-based molecular methodologies include pulsed-field gel electrophoresis (PFGE), PCR-based typing methods, and multilocus sequence analysis. Establishing clonality of pathogens can aid in the identification of the source (environmental or personnel) of organisms, distinguish infectious from noninfectious strains, and distinguish relapse from reinfection. The integration of molecular typing with conventional hospital epidemiologic surveillance has been proven to be cost-effective due to the associated reduction in the number of nosocomial infections. Cost-effectiveness is maximized through the collaboration of the laboratory, through epidemiologic typing, and the infection control department during epidemiologic investigations. [Abstract/Link to Full Text]

Jenssen H, Hamill P, Hancock RE
Peptide antimicrobial agents.
Clin Microbiol Rev. 2006 Jul;19(3):491-511.
Antimicrobial host defense peptides are produced by all complex organisms as well as some microbes and have diverse and complex antimicrobial activities. Collectively these peptides demonstrate a broad range of antiviral and antibacterial activities and modes of action, and it is important to distinguish between direct microbicidal and indirect activities against such pathogens. The structural requirements of peptides for antiviral and antibacterial activities are evaluated in light of the diverse set of primary and secondary structures described for host defense peptides. Peptides with antifungal and antiparasitic activities are discussed in less detail, although the broad-spectrum activities of such peptides indicate that they are important host defense molecules. Knowledge regarding the relationship between peptide structure and function as well as their mechanism of action is being applied in the design of antimicrobial peptide variants as potential novel therapeutic agents. [Abstract/Link to Full Text]

Kusters JG, van Vliet AH, Kuipers EJ
Pathogenesis of Helicobacter pylori infection.
Clin Microbiol Rev. 2006 Jul;19(3):449-90.
Helicobacter pylori is the first formally recognized bacterial carcinogen and is one of the most successful human pathogens, as over half of the world's population is colonized with this gram-negative bacterium. Unless treated, colonization usually persists lifelong. H. pylori infection represents a key factor in the etiology of various gastrointestinal diseases, ranging from chronic active gastritis without clinical symptoms to peptic ulceration, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Disease outcome is the result of the complex interplay between the host and the bacterium. Host immune gene polymorphisms and gastric acid secretion largely determine the bacterium's ability to colonize a specific gastric niche. Bacterial virulence factors such as the cytotoxin-associated gene pathogenicity island-encoded protein CagA and the vacuolating cytotoxin VacA aid in this colonization of the gastric mucosa and subsequently seem to modulate the host's immune system. This review focuses on the microbiological, clinical, immunological, and biochemical aspects of the pathogenesis of H. pylori. [Abstract/Link to Full Text]

Pfaller MA, Diekema DJ, Sheehan DJ
Interpretive breakpoints for fluconazole and Candida revisited: a blueprint for the future of antifungal susceptibility testing.
Clin Microbiol Rev. 2006 Apr;19(2):435-47.
Developing interpretive breakpoints for any given organism-drug combination requires integration of the MIC distribution, pharmacokinetic and pharmacodynamic parameters, and the relationship between in vitro activity and outcome from both in vivo and clinical studies. Previously, the Subcommittee for Antifungal Testing of the Clinical and Laboratory Standards Institute (CLSI [formerly National Committee for Clinical Laboratory Standards]) proposed MIC interpretive breakpoints for fluconazole and Candida spp. These breakpoints were considered to be somewhat weak, because the clinical data supporting them came largely from mucosal infections and there were very few infections involving strains with elevated fluconazole MICs. We readdress the issue of fluconazole breakpoints for Candida by using published clinical and microbiologic data to provide further validation of the breakpoints proposed by the CLSI in 1997. We also address interpretive breakpoints for agar disk diffusion testing of fluconazole. The MIC distribution for fluconazole was determined with a collection of 13,338 clinical isolates. The overall MIC at which 90% of the isolates were inhibited was 8 microg/ml: 91% were susceptible (S) at a MIC of <or=8 microg/ml and 3% were resistant (R) (MIC >or= 64 microg/ml). Similar results were obtained for 2,190 isolates from randomized clinical trials. Analysis of available data for 1,295 patient-episode-isolate events (692 represented mucosal infections and 603 represented invasive infections) from 12 published clinical studies demonstrated an overall success rate of 77%, including 85% for those episodes in which the fluconazole MIC was <or=8 microg/ml, 67% for those episodes in which the MIC was 16 to 32 microg/ml, and 42% for those episodes with resistant (MIC >or= 64 microg/ml) isolates. Pharmacodynamic analysis demonstrated a strong relationship between MIC, fluconazole dose, and outcome. A dose/MIC ratio of approximately 25 was supportive of the following susceptibility breakpoints for fluconazole and Candida spp.: S, MIC <or= 8 microg/ml; susceptible-dose dependent (SDD), MIC = 16 to 32 microg/ml; R, MIC >or= 64 microg/ml. The corresponding disk test breakpoints are as follows: S, >or=19 mm; SDD, 15 to 18 mm; R, <or=14 mm. [Abstract/Link to Full Text]

Church D, Elsayed S, Reid O, Winston B, Lindsay R
Burn wound infections.
Clin Microbiol Rev. 2006 Apr;19(2):403-34.
Burns are one of the most common and devastating forms of trauma. Patients with serious thermal injury require immediate specialized care in order to minimize morbidity and mortality. Significant thermal injuries induce a state of immunosuppression that predisposes burn patients to infectious complications. A current summary of the classifications of burn wound infections, including their diagnosis, treatment, and prevention, is given. Early excision of the eschar has substantially decreased the incidence of invasive burn wound infection and secondary sepsis, but most deaths in severely burn-injured patients are still due to burn wound sepsis or complications due to inhalation injury. Burn patients are also at risk for developing sepsis secondary to pneumonia, catheter-related infections, and suppurative thrombophlebitis. The introduction of silver-impregnated devices (e.g., central lines and Foley urinary catheters) may reduce the incidence of nosocomial infections due to prolonged placement of these devices. Improved outcomes for severely burned patients have been attributed to medical advances in fluid resuscitation, nutritional support, pulmonary and burn wound care, and infection control practices. [Abstract/Link to Full Text]

Piddock LJ
Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria.
Clin Microbiol Rev. 2006 Apr;19(2):382-402.
Efflux pump genes and proteins are present in both antibiotic-susceptible and antibiotic-resistant bacteria. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug (antibiotic) resistance (MDR). However, the clinical relevance of efflux-mediated resistance is species, drug, and infection dependent. This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans. Recent structural data provide valuable insights into the mechanisms of drug transport. MDR efflux pumps contribute to antibiotic resistance in bacteria in several ways: (i) inherent resistance to an entire class of agents, (ii) inherent resistance to specific agents, and (iii) resistance conferred by overexpression of an efflux pump. Enhanced efflux can be mediated by mutations in (i) the local repressor gene, (ii) a global regulatory gene, (iii) the promoter region of the transporter gene, or (iv) insertion elements upstream of the transporter gene. Some data suggest that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche. Inhibitors of various efflux pump systems have been described; typically these are plant alkaloids, but as yet no product has been marketed. [Abstract/Link to Full Text]

Scollard DM, Adams LB, Gillis TP, Krahenbuhl JL, Truman RW, Williams DL
The continuing challenges of leprosy.
Clin Microbiol Rev. 2006 Apr;19(2):338-81.
Leprosy is best understood as two conjoined diseases. The first is a chronic mycobacterial infection that elicits an extraordinary range of cellular immune responses in humans. The second is a peripheral neuropathy that is initiated by the infection and the accompanying immunological events. The infection is curable but not preventable, and leprosy remains a major global health problem, especially in the developing world, publicity to the contrary notwithstanding. Mycobacterium leprae remains noncultivable, and for over a century leprosy has presented major challenges in the fields of microbiology, pathology, immunology, and genetics; it continues to do so today. This review focuses on recent advances in our understanding of M. leprae and the host response to it, especially concerning molecular identification of M. leprae, knowledge of its genome, transcriptome, and proteome, its mechanisms of microbial resistance, and recognition of strains by variable-number tandem repeat analysis. Advances in experimental models include studies in gene knockout mice and the development of molecular techniques to explore the armadillo model. In clinical studies, notable progress has been made concerning the immunology and immunopathology of leprosy, the genetics of human resistance, mechanisms of nerve injury, and chemotherapy. In nearly all of these areas, however, leprosy remains poorly understood compared to other major bacterial diseases. [Abstract/Link to Full Text]

Liévin-Le Moal V, Servin AL
The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota.
Clin Microbiol Rev. 2006 Apr;19(2):315-37.
The intestinal tract is a complex ecosystem that combines resident microbiota and the cells of various phenotypes with complex metabolic activities that line the epithelial wall. The intestinal cells that make up the epithelium provide physical and chemical barriers that protect the host against the unwanted intrusion of microorganisms that hijack the cellular molecules and signaling pathways of the host and become pathogenic. Some of the organisms making up the intestinal microbiota also have microbicidal effects that contribute to the barrier against enteric pathogens. This review describes the two cell lineages present in the intestinal epithelium: the goblet cells and the Paneth cells, both of which play a pivotal role in the first line of enteric defense by producing mucus and antimicrobial peptides, respectively. We also analyze recent insights into the intestinal microbiota and the mechanisms by which some resident species act as a barrier to enteric pathogens. Moreover, this review examines whether the cells producing mucins or antimicrobial peptides and the resident microbiota act in partnership and whether they function individually and/or synergistically to provide the host with an effective front line of defense against harmful enteric pathogens. [Abstract/Link to Full Text]

Lindström M, Korkeala H
Laboratory diagnostics of botulism.
Clin Microbiol Rev. 2006 Apr;19(2):298-314.
Botulism is a potentially lethal paralytic disease caused by botulinum neurotoxin. Human pathogenic neurotoxins of types A, B, E, and F are produced by a diverse group of anaerobic spore-forming bacteria, including Clostridium botulinum groups I and II, Clostridium butyricum, and Clostridium baratii. The routine laboratory diagnostics of botulism is based on the detection of botulinum neurotoxin in the patient. Detection of toxin-producing clostridia in the patient and/or the vehicle confirms the diagnosis. The neurotoxin detection is based on the mouse lethality assay. Sensitive and rapid in vitro assays have been developed, but they have not yet been appropriately validated on clinical and food matrices. Culture methods for C. botulinum are poorly developed, and efficient isolation and identification tools are lacking. Molecular techniques targeted to the neurotoxin genes are ideal for the detection and identification of C. botulinum, but they do not detect biologically active neurotoxin and should not be used alone. Apart from rapid diagnosis, the laboratory diagnostics of botulism should aim at increasing our understanding of the epidemiology and prevention of the disease. Therefore, the toxin-producing organisms should be routinely isolated from the patient and the vehicle. The physiological group and genetic traits of the isolates should be determined. [Abstract/Link to Full Text]

Corsaro D, Greub G
Pathogenic potential of novel Chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria.
Clin Microbiol Rev. 2006 Apr;19(2):283-97.
Novel chlamydiae are newly recognized members of the phylum Chlamydiales that are only distantly related to the classic Chlamydiaceae, i.e., Chlamydia and Chlamydophila species. They also exhibit an obligate biphasic intracellular life cycle within eukaryote host cells. Some of these new chlamydiae are currently considered potential emerging human and/or animal pathogens. Parachlamydia acanthamoebae and Simkania negevensis are both emerging respiratory human pathogens, Waddlia chondrophila could be a novel abortigenic bovine agent, and Piscichlamydia salmonis has recently been identified as an agent of the gill epitheliocystis in the Atlantic salmon. Fritschea spp. and Rhabdochlamydia spp. seem to be confined to arthropods, but some evidence for human exposure exists. In this review, we first summarize the data supporting a pathogenic potential of the novel chlamydiae for humans and other vertebrates and the interactions that most of these chlamydiae have with free-living amoebae. We then review the diagnostic approaches to infections potentially due to the novel chlamydiae, especially focusing on the currently available PCR-based protocols, mammalian cell culture, the amoebal coculture system, and serology. [Abstract/Link to Full Text]

Brown-Elliott BA, Brown JM, Conville PS, Wallace RJ
Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy.
Clin Microbiol Rev. 2006 Apr;19(2):259-82.
The recent explosion of newly described species of Nocardia results from the impact in the last decade of newer molecular technology, including PCR restriction enzyme analysis and 16S rRNA sequencing. These molecular techniques have revolutionized the identification of the nocardiae by providing rapid and accurate identification of recognized nocardiae and, at the same time, revealing new species and a number of yet-to-be-described species. There are currently more than 30 species of nocardiae of human clinical significance, with the majority of isolates being N. nova complex, N. abscessus, N. transvalensis complex, N. farcinica, N. asteroides type VI (N. cyriacigeorgica), and N. brasiliensis. These species cause a wide variety of diseases and have variable drug susceptibilities. Accurate identification often requires referral to a reference laboratory with molecular capabilities, as many newer species are genetically distinct from established species yet have few or no distinguishing phenotypic characteristics. Correct identification is important in deciding the clinical relevance of a species and in the clinical management and treatment of patients with nocardial disease. This review characterizes the currently known pathogenic species of Nocardia, including clinical disease, drug susceptibility, and methods of identification. [Abstract/Link to Full Text]

Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JD, Wengenack NL, Rosenblatt JE, Cockerill FR, Smith TF
Real-time PCR in clinical microbiology: applications for routine laboratory testing.
Clin Microbiol Rev. 2006 Jan;19(1):165-256.
Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory. [Abstract/Link to Full Text]

Harrison LH
Prospects for vaccine prevention of meningococcal infection.
Clin Microbiol Rev. 2006 Jan;19(1):142-64.
Neisseria meningitidis is the leading cause of bacterial meningitis in the United States and worldwide. A serogroup A/C/W-135/Y polysaccharide meningococcal vaccine has been licensed in the United States since 1981 but has not been used universally outside of the military. On 14 January 2005, a polysaccharide conjugate vaccine that covers meningococcal serogroups A, C, W-135, and Y was licensed in the United States for 11- to 55-year-olds and is now recommended for the routine immunization of adolescents and other high-risk groups. This review covers the changing epidemiology of meningococcal disease in the United States, issues related to vaccine prevention, and recommendations on the use of the new vaccine. [Abstract/Link to Full Text]

Busscher HJ, van der Mei HC
Microbial adhesion in flow displacement systems.
Clin Microbiol Rev. 2006 Jan;19(1):127-41.
Flow displacement systems are superior to many other (static) systems for studying microbial adhesion to surfaces because mass transport and prevailing shear conditions can be adequately controlled and notoriously ill-defined slight rinsing steps to remove so-called "loosely adhering organisms" can be avoided. In this review, we present the basic background required to calculate mass transport and shear rates in flow displacement systems, focusing on the parallel plate flow chamber as an example. Critical features in the design of flow displacement systems are discussed, as well as different strategies for data analysis. Finally, selected examples of working with flow displacement systems are given for diverse biomedical applications. [Abstract/Link to Full Text]

Croft SL, Sundar S, Fairlamb AH
Drug resistance in leishmaniasis.
Clin Microbiol Rev. 2006 Jan;19(1):111-26.
Leishmaniasis is a complex disease, with visceral and cutaneous manifestations, and is caused by over 15 different species of the protozoan parasite genus Leishmania. There are significant differences in the sensitivity of these species both to the standard drugs, for example, pentavalent antimonials and miltefosine, and those on clinical trial, for example, paromomycin. Over 60% of patients with visceral leishmaniasis in Bihar State, India, do not respond to treatment with pentavalent antimonials. This is now considered to be due to acquired resistance. Although this class of drugs has been used for over 60 years for leishmaniasis treatment, it is only in the past 2 years that the mechanisms of action and resistance have been identified, related to drug metabolism, thiol metabolism, and drug efflux. With the introduction of new therapies, including miltefosine in 2002 and paromomycin in 2005-2006, it is essential that there be a strategy to prevent the emergence of resistance to new drugs; combination therapy, monitoring of therapy, and improved diagnostics could play an essential role in this strategy. [Abstract/Link to Full Text]

Vanittanakom N, Cooper CR, Fisher MC, Sirisanthana T
Penicillium marneffei infection and recent advances in the epidemiology and molecular biology aspects.
Clin Microbiol Rev. 2006 Jan;19(1):95-110.
Penicillium marneffei infection is an important emerging public health problem, especially among patients infected with human immunodeficiency virus in the areas of endemicity in southeast Asia, India, and China. Within these regions, P. marneffei infection is regarded as an AIDS-defining illness, and the severity of the disease depends on the immunological status of the infected individual. Early diagnosis by serologic and molecular assay-based methods have been developed and are proving to be important in diagnosing infection. The occurrence of natural reservoirs and the molecular epidemiology of P. marneffei have been studied; however, the natural history and mode of transmission of the organism remain unclear. Soil exposure, especially during the rainy season, has been suggested to be a critical risk factor. Using a highly discriminatory molecular technique, multilocus microsatellite typing, to characterize this fungus, several isolates from bamboo rats and humans were shown to share identical multilocus genotypes. These data suggest either that transmission of P. marneffei may occur from rodents to humans or that rodents and humans are coinfected from common environmental sources. These putative natural cycles of P. marneffei infection need further investigation. Studies on the fungal genetics of P. marneffei have been focused on the characterization of genetic determinants that may play important roles in asexual development, mycelial-to-yeast phase transition, and the expression of antigenic determinants. Molecular studies have identified several genes involved in germination, hyphal development, conidiogenesis, and yeast cell polarity. A number of functionally important genes, such as the malate synthase- and catalase-peroxidase protein-encoding genes, have been identified as being upregulated in the yeast phase. Future investigations pertaining to the roles of these genes in host-fungus interactions may provide the key knowledge to understanding the pathogenicity of P. marneffei. [Abstract/Link to Full Text]

---

Recent Articles in Infection and Immunity

---

Ding SZ, Minohara Y, Fan XJ, Wang J, Reyes VE, Patel J, Dirden-Kramer B, Boldogh I, Ernst PB, Crowe SE
Helicobacter pylori infection induces oxidative stress and programmed cell death in human gastric epithelial cells.
Infect Immun. 2007 Aug;75(8):4030-9.
Helicobacter pylori infection is associated with altered gastric epithelial cell turnover. To evaluate the role of oxidative stress in cell death, gastric epithelial cells were exposed to various strains of H. pylori, inflammatory cytokines, and hydrogen peroxide in the absence or presence of antioxidant agents. Increased intracellular reactive oxygen species (ROS) were detected using a redox-sensitive fluorescent dye, a cytochrome c reduction assay, and measurements of glutathione. Apoptosis was evaluated by detecting DNA fragmentation and caspase activation. Infection with H. pylori or exposure of epithelial cells to hydrogen peroxide resulted in apoptosis and a dose-dependent increase in ROS generation that was enhanced by pretreatment with inflammatory cytokines. Basal levels of ROS were greater in epithelial cells isolated from gastric mucosal biopsy specimens from H. pylori-infected subjects than in cells from uninfected individuals. H. pylori strains bearing the cag pathogenicity island (PAI) induced higher levels of intracellular oxygen metabolites than isogenic cag PAI-deficient mutants. H. pylori infection and hydrogen peroxide exposure resulted in similar patterns of caspase 3 and 8 activation. Antioxidants inhibited both ROS generation and DNA fragmentation by H. pylori. These results indicate that bacterial factors and the host inflammatory response confer oxidative stress to the gastric epithelium during H. pylori infection that may lead to apoptosis. [Abstract/Link to Full Text]

Schneider OD, Weiss AA, Miller WE
Pertussis toxin utilizes proximal components of the T-cell receptor complex to initiate signal transduction events in T cells.
Infect Immun. 2007 Aug;75(8):4040-9.
Pertussis toxin (PTx) is an AB(5) toxin produced by the human pathogen Bordetella pertussis. Previous work demonstrates that the five binding (B) subunits of PTx can have profound effects on T lymphocytes independent of the enzymatic activity of the A subunit. Stimulation of T cells with holotoxin (PTx) or the B subunit alone (PTxB) rapidly induces signaling events resulting in inositol phosphate accumulation, Ca(2+) mobilization, interleukin-2 (IL-2) production, and mitogenic cell growth. Although previous reports suggest the presence of PTx signaling receptors expressed on T cells, to date, the receptor(s) and membrane proximal signaling events utilized by PTx remain unknown. Here we genetically and biochemically define the membrane proximal components utilized by PTx to initiate signal transduction in T cells. Using mutants of the Jurkat T-cell line deficient for key components of the T-cell receptor (TCR) pathway, we have compared stimulation with PTx to that of anti-CD3 monoclonal antibody (MAb), which directly interacts with and activates the TCR complex. Our genetic data in combination with biochemical analysis show that PTx (via the B subunit) activates TCR signaling similar to that of anti-CD3 MAb, including activation of key signaling intermediates such as Lck, ZAP-70, and phospholipase C-gamma1. Moreover, the data indicate that costimulatory activity, as provided by CD28 ligation, is required for PTx to fully stimulate downstream indicators of T-cell activation such as IL-2 gene expression. By illuminating the signaling pathways that PTx activates in T cells, we provide a mechanistic understanding for how these signals deregulate immune system functions during B. pertussis infection. [Abstract/Link to Full Text]

Hansen JK, Demick KP, Mansfield JM, Forest KT
Conserved regions from Neisseria gonorrhoeae pilin are immunosilent and not immunosuppressive.
Infect Immun. 2007 Aug;75(8):4138-47.
PilE is the primary subunit of type IV pili from Neisseria gonorrhoeae and contains a surface-exposed hypervariable region thought to be one feature of pili that has prevented development of a pilin-based vaccine. We have created a three-dimensional structure-based antigen by replacing the hypervariable region of PilE with an aspartate-glutamine linker chosen from the sequence of Pseudomonas aeruginosa PilA. We then characterized murine immune responses to this novel protein to determine if conserved PilE regions could serve as a vaccine candidate. The control PilE protein elicited strong T-cell-dependent B-cell responses that are specific to epitopes in both the hypervariable deletion and control proteins. In contrast, the hypervariable deletion protein was unable to elicit an immune response in mice, suggesting that in the absence of the hypervariable region, the conserved regions of PilE alone are not sufficient for antibody production. Further analysis of these PilE proteins with suppressor cell assays showed that neither suppresses T- or B-cell responses, and flow cytometry experiments suggested that they do not exert suppressor effects by activating T regulatory cells. Our results show that in the murine model, the hypervariable region of PilE is required to activate immune responses to pilin, whereas the conserved regions are unusually nonimmunogenic. In addition, we show that both hypervariable and conserved regions of pilin are not suppressive, suggesting that PilE does not cause the decrease in T-cell populations observed during gonococcal cervicitis. [Abstract/Link to Full Text]

Oellerich MF, Jacobi CA, Freund S, Niedung K, Bach A, Heesemann J, Trülzsch K
Yersinia enterocolitica infection of mice reveals clonal invasion and abscess formation.
Infect Immun. 2007 Aug;75(8):3802-11.
Yersinia enterocolitica is a common cause of food-borne gastrointestinal disease leading to self-limiting diarrhea and mesenteric lymphadenitis. Occasionally, focal abscess formation in the livers and spleens of certain predisposed patients (those with iron overload states such as hemochromatosis) is observed. In the mouse oral infection model, yersiniae produce a similar disease involving the replication of yersiniae in the small intestine, the invasion of Peyer's patches, and dissemination to the liver and spleen. In these tissues and organs, yersiniae are known to replicate predominantly extracellularly and to form microcolonies. By infecting mice orally with a mixture of equal amounts of green- and red-fluorescing yersiniae (yersiniae expressing green or red fluorescent protein), we were able to show for the first time that yersiniae produce exclusively monoclonal microcolonies in Peyer's patches, the liver, and the spleen, indicating that a single bacterium is sufficient to induce microcolony and microabscess formation in vivo. Furthermore, we present evidence for the clonal invasion of Peyer's patches from the small intestine. The finding that only very few yersiniae are required to establish microcolonies in Peyer's patches is due to both Yersinia-specific and host-specific factors. We demonstrate that yersiniae growing in the small intestinal lumen show strongly reduced levels of invasin, the most important factor for the early invasion of Peyer's patches. Furthermore, we show that the host severely restricts sequential microcolony formation in previously infected Peyer's patches. [Abstract/Link to Full Text]

Schaber JA, Triffo WJ, Suh SJ, Oliver JW, Hastert MC, Griswold JA, Auer M, Hamood AN, Rumbaugh KP
Pseudomonas aeruginosa forms biofilms in acute infection independent of cell-to-cell signaling.
Infect Immun. 2007 Aug;75(8):3715-21.
Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS. [Abstract/Link to Full Text]

Quin LR, Onwubiko C, Moore QC, Mills MF, McDaniel LS, Carmicle S
Factor H binding to PspC of Streptococcus pneumoniae increases adherence to human cell lines in vitro and enhances invasion of mouse lungs in vivo.
Infect Immun. 2007 Aug;75(8):4082-7.
Pneumococcal surface protein C (PspC) binds to both human secretory immunoglobulin A (sIgA) and complement factor H (FH). FH, a regulator of the alternative pathway of complement, can also mediate adherence of different host cells. Since PspC contributes to adherence and invasion of host cells, we hypothesized that the interaction of PspC with FH may also mediate adherence of pneumococci to human cells. In this study, we investigated FH- and sIgA-mediated pneumococcal adherence to human cell lines in vitro. Adherence assays demonstrated that preincubation of Streptococcus pneumoniae D39 with FH increased adherence to human umbilical vein endothelial cells (HUVEC) 5-fold and to lung epithelial cells (SK-MES-1) 18-fold, relative to that of D39 without FH on the surface. The presence of sIgA enhanced adherence to SK-MES-1 6-fold and to pharyngeal epithelial cells (Detroit 562) 14-fold. Furthermore, sIgA had an additive effect on adherence to HUVEC; specifically, preincubation of D39 with both FH and sIgA led to a 21-fold increase in adherence. Finally, using a mouse model, we examined the significance of the FH-PspC interaction in pneumococcal nasal colonization and lung invasion. Mice intranasally infected with D39 preincubated with FH had increased bacteremia and lung invasion, but they had similar levels of nasopharyngeal colonization compared to that of mice challenged with D39 without FH. [Abstract/Link to Full Text]

Caro-Hernández P, Fernández-Lago L, de Miguel MJ, Martín-Martín AI, Cloeckaert A, Grilló MJ, Vizcaíno N
Role of the Omp25/Omp31 family in outer membrane properties and virulence of Brucella ovis.
Infect Immun. 2007 Aug;75(8):4050-61.
The genes coding for the five outer membrane proteins (OMPs) of the Omp25/Omp31 family expected to be located in the outer membrane (OM) of rough virulent Brucella ovis PA were inactivated to evaluate their role in virulence and OM properties. The OM properties of the mutant strains and of the mutants complemented with the corresponding wild-type genes were analyzed, in comparison with the parental strain and rough B. abortus RB51, in several tests: (i) binding of anti-Omp25 and anti-Omp31 monoclonal antibodies, (ii) autoagglutination of bacterial suspensions, and (iii) assessment of susceptibility to polymyxin B, sodium deoxycholate, hydrogen peroxide, and nonimmune ram serum. A tight balance of the members of the Omp25/Omp31 family was seen to be essential for the stability of the B. ovis OM, and important differences between the OMs of B. ovis PA and B. abortus RB51 rough strains were observed. Regarding virulence, the absence of Omp25d and Omp22 from the OM of B. ovis PA led to a drastic reduction in spleen colonization in mice. While the greater susceptibility of the Deltaomp22 mutant to nonimmune serum and its difficulty in surviving in the stationary phase might be on the basis of its dramatic attenuation, no defects in the OM able to explain the attenuation of the Deltaomp25d mutant were found, especially considering that the fully virulent Deltaomp25c mutant displayed more important OM defects. Accordingly, Omp25d, and perhaps Omp22, could be directly involved in the penetration and/or survival of B. ovis inside host cells. This aspect, together with the role of Omp25d and Omp22 in the virulence both of B. ovis in rams and of other Brucella species, should be thoroughly evaluated in future studies. [Abstract/Link to Full Text]

Qadri F, Saha A, Ahmed T, Al Tarique A, Begum YA, Svennerholm AM
Disease burden due to enterotoxigenic Escherichia coli in the first 2 years of life in an urban community in Bangladesh.
Infect Immun. 2007 Aug;75(8):3961-8.
A cohort of 321 children was followed from birth up to 2 years of age to determine the incidence of enterotoxigenic Escherichia coli (ETEC) in Bangladesh. The average number of diarrheal days and incidence rates were 6.6 and 2.3/child/year, respectively. ETEC was the most common pathogen and was isolated in 19.5% cases, with an incidence of 0.5 episode/child/year. The prevalence of rotavirus diarrhea was lower (10%). ETEC expressing the heat-stable enterotoxin (ST) was predominant. Strains isolated from diarrheal cases were positive for colonization factors (CFs) in higher frequency (66%) than from healthy children (33%) (P < 0.001). The heat-labile toxin (LT)-positive strains from healthy children were more often CF negative (92%) than those isolated from children with diarrhea (73%) (P < 0.001). In children with symptomatic or asymptomatic infections by CFA/I, CS1 plus CS3, CS2 plus CS3, or CS5 plus CS6 strains, a repeat episode of diarrhea or infection by the homologous CF type was uncommon. Repeat symptomatic infections were noted mostly for LT- and ST-expressing ETEC. ETEC diarrhea was more prevalent in children in the A and AB groups than in those in the O blood group (P = 0.032 to 0.023). Children with ETEC diarrhea were underweight and growth stunted at the 2-year follow-up period, showing the importance of strategies to prevent and decrease ETEC diarrheal morbidity in children. [Abstract/Link to Full Text]

Paulin SM, Jagannathan A, Campbell J, Wallis TS, Stevens MP
Net replication of Salmonella enterica serovars Typhimurium and Choleraesuis in porcine intestinal mucosa and nodes is associated with their differential virulence.
Infect Immun. 2007 Aug;75(8):3950-60.
Salmonella enterica is a facultative intracellular pathogen of worldwide importance and causes a spectrum of diseases depending on serovar- and host-specific factors. Oral infection of pigs with S. enterica serovar Typhimurium strain 4/74 produces acute enteritis but is rarely fatal, whereas serovar Choleraesuis strain A50 causes systemic disease with a high mortality rate. With a porcine ligated ileal loop model, we observed that systemic virulence of serovar Choleraesuis A50 is not associated with enhanced intestinal invasion, secretory responses, or neutrophil recruitment compared to serovar Typhimurium 4/74. The net growth in vivo of serovar Choleraesuis A50 and serovar Typhimurium 4/74 was monitored following oral inoculation of pigs with strains harboring pHSG422, which exhibits temperature-sensitive replication. Analysis of plasmid partitioning revealed that the enteric virulence of serovar Typhimurium 4/74 relative to that of serovar Choleraesuis A50 is associated with rapid replication in the intestinal wall, whereas systemic virulence of serovar Choleraesuis A50 is associated with enhanced persistence in intestinal mesenteric lymph nodes. Faster replication of serovar Typhimurium, compared to that of serovar Choleraesuis, in the intestinal mucosa was associated with greater induction of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-8 (IL-8), and IL-18 as detected by reverse transcriptase PCR analysis of transcripts from infected mucosa. During replication in batch culture and porcine alveolar macrophages, transcription of genes encoding components of type III secretion systems 1 (sipC) and 2 (sseC) was observed to be significantly higher in serovar Typhimurium 4/74 than in serovar Choleraesuis A50, and this may contribute to the differences in epithelial invasion and intracellular proliferation. The rapid induction of proinflammatory responses by strain 4/74 may explain why pigs confine serovar Typhimurium infection to the intestines, whereas slow replication of serovar Choleraesuis may enable it to evade host innate immunity and thus disseminate by stealth. [Abstract/Link to Full Text]

Chatfield CH, Cianciotto NP
The secreted pyomelanin pigment of Legionella pneumophila confers ferric reductase activity.
Infect Immun. 2007 Aug;75(8):4062-70.
The virulence of Legionella pneumophila is dependent upon its capacity to acquire iron. To identify genes involved in expression of its siderophore, we screened a mutagenized population of L. pneumophila for strains that were no longer able to rescue the growth of a ferrous transport mutant. However, an unusual mutant was obtained that displayed a strong inhibitory effect on the feoB mutant. Due to an insertion in hmgA that encodes homogentisate 1,2-dioxygenase, the mutant secreted increased levels of pyomelanin, the L. pneumophila pigment that is derived from secreted homogentisic acid (HGA). Thus, we hypothesized that L. pneumophila-secreted HGA-melanin has intrinsic ferric reductase activity, converting Fe(3+) to Fe(2+), but that hyperpigmentation results in excessive reduction of iron that can, in the case of the feoB mutant, be inhibitory to growth. In support of this hypothesis, we demonstrated, for the first time, that wild-type L. pneumophila secretes ferric reductase activity. Moreover, whereas the hyperpigmented mutant had increased secreted activity, an lly mutant specifically impaired for pigment production lacked the activity. Compatible with the nature of HGA-melanins, the secreted ferric reductase activity was positively influenced by the amount of tyrosine in the growth medium, resistant to protease, acid precipitable, and heterogeneous in size. Together, these data represent the first demonstration of pyomelanin-mediated ferric reduction by a pathogenic bacterium. [Abstract/Link to Full Text]

Hollifield M, Bou Ghanem E, de Villiers WJ, Garvy BA
Scavenger receptor A dampens induction of inflammation in response to the fungal pathogen Pneumocystis carinii.
Infect Immun. 2007 Aug;75(8):3999-4005.
Alveolar macrophages are the effector cells largely responsible for clearance of Pneumocystis carinii from the lungs. Binding of organisms to beta-glucan and mannose receptors has been shown to stimulate phagocytosis of the organisms. To further define the mechanisms used by alveolar macrophages for clearance of P. carinii, mice deficient in the expression of scavenger receptor A (SRA) were infected with P. carinii, and clearance of organisms was monitored over time. SRA-deficient (SRAKO) mice consistently cleared P. carinii faster than did wild-type control mice. Expedited clearance corresponded to elevated numbers of activated CD4(+) T cells in the alveolar spaces of SRAKO mice compared to wild-type mice. Alveolar macrophages from SRAKO mice had increased expression of CD11b on their surfaces, consistent with an activated phenotype. However, they were not more phagocytic than macrophages expressing SRA, as measured by an in vivo phagocytosis assay. SRAKO alveolar macrophages produced significantly more tumor necrosis factor alpha (TNF-alpha) than wild-type macrophages when stimulated with lipopolysaccharide in vitro but less TNF-alpha in response to P. carinii in vitro. However, upon in vivo stimulation, SRAKO mice produced significantly more TNF-alpha, interleukin 12 (IL-12), and IL-18 in response to P. carinii infection than did wild-type mice. Together, these data indicate that SRA controls inflammatory cytokines produced by alveolar macrophages in the context of P. carinii infection. [Abstract/Link to Full Text]

Goldmann O, von Köckritz-Blickwede M, Höltje C, Chhatwal GS, Geffers R, Medina E
Transcriptome analysis of murine macrophages in response to infection with Streptococcus pyogenes reveals an unusual activation program.
Infect Immun. 2007 Aug;75(8):4148-57.
The complex response of murine macrophages to infection with Streptococcus pyogenes was investigated at the level of gene expression with a high-density oligomer microarray. More than 400 genes were identified as being differentially regulated. Many of the up-regulated genes encode molecules involved in the immune response and in inflammation, transcription, signaling, apoptosis, the cell cycle, electron transport, and cell adhesion. Of particular interest was the up-regulation of proinflammatory cytokines, typical of the classically activated macrophages (M1 phenotype), such as tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-6, and as well as the up-regulation of anti-inflammatory mediators, such as IL-1 decoy receptor and IL-10, associated with alternative macrophage activation (M2 phenotype). Furthermore, the gene encoding inducible nitric oxide synthase (iNOS), an enzyme typically implicated in classical activation, was not induced in infected macrophages. Instead, the gene encoding arginase, a competitor for the iNOS substrate arginine involved in the alternative activation pathway, was up-regulated in S. pyogenes-infected cells. Thus, the microarray-based gene expression analysis demonstrated that S. pyogenes induces an atypical activation program in macrophages, with some but not all features of the classical or alternative activation phenotypes. The microarray data also suggested that the bactericidal activity of macrophages against S. pyogenes is mediated by phagocyte oxidase, as p47phox was up-regulated in infected cells. Indeed, the in vivo and in vitro killing of S. pyogenes was markedly diminished in the absence of functional phagocyte (p47(phox-/-)) but not in the absence of iNOS (iNOS(-/-)). An understanding of how macrophages respond to S. pyogenes at the molecular level may facilitate the development of new therapeutic paradigms. [Abstract/Link to Full Text]

Radosevic K, Wieland CW, Rodriguez A, Weverling GJ, Mintardjo R, Gillissen G, Vogels R, Skeiky YA, Hone DM, Sadoff JC, van der Poll T, Havenga M, Goudsmit J
Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: CD4 and CD8 T-cell epitope mapping and role of gamma interferon.
Infect Immun. 2007 Aug;75(8):4105-15.
There is an urgent need for an efficacious vaccine against tuberculosis (TB). Cellular immune responses are key to an effective protective response against TB. Recombinant adenovirus (rAd) vectors are especially suited to the induction of strong T-cell immunity and thus represent promising vaccine vehicles for the prevention of TB. We have previously reported on rAd vector serotype 35, the serotype of choice due to low preexisting immunity worldwide, which expresses a unique fusion protein of Mycobacterium tuberculosis antigens Ag85A, Ag85B, and TB10.4 (Ad35-TBS). Here, we demonstrate that Ad35-TBS confers protection against M. tuberculosis when administered to mice through either an intranasal or an intramuscular route. Histological evaluation of lung tissue corroborated the protection and, in addition, demonstrated differences between two mouse strains, with diffuse inflammation in BALB/c mice and distinct granuloma formation in C57BL/6 mice. Epitope mapping analysis in these mouse strains showed that the major T-cell epitopes are conserved in the artificial fusion protein, while three novel CD8 peptides were discovered. Using a defined set of T-cell epitopes, we reveal differences between the two mouse strains in the type of protective immune response, demonstrating that different antigen-specific gamma interferon (IFN-gamma)-producing T cells can provide protection against M. tuberculosis challenge. While in BALB/c (H-2(d)) mice, a dominant CD8 T-cell response was detected, in C57BL/6 (H-2(b)) mice, more balanced CD4/CD8 T-cell responses were observed, with a more pronounced CD4 response in the lungs. These results unify conflicting reports on the relative importance of CD4 versus CD8 T-cell responses in protection and emphasize the key role of IFN-gamma. [Abstract/Link to Full Text]

Van Alst NE, Picardo KF, Iglewski BH, Haidaris CG
Nitrate sensing and metabolism modulate motility, biofilm formation, and virulence in Pseudomonas aeruginosa.
Infect Immun. 2007 Aug;75(8):3780-90.
Infection by the bacterial opportunist Pseudomonas aeruginosa frequently assumes the form of a biofilm, requiring motility for biofilm formation and dispersal and an ability to grow in nutrient- and oxygen-limited environments. Anaerobic growth by P. aeruginosa is accomplished through the denitrification enzyme pathway that catalyzes the sequential reduction of nitrate to nitrogen gas. Mutants mutated in the two-component nitrate sensor-response regulator and in membrane nitrate reductase displayed altered motility and biofilm formation compared to wild-type P. aeruginosa PAO1. Analysis of additional nitrate dissimilation mutants demonstrated a second level of regulation in P. aeruginosa motility that is independent of nitrate sensor-response regulator function and is associated with nitric oxide production. Because motility and biofilm formation are important for P. aeruginosa pathogenicity, we examined the virulence of selected regulatory and structural gene mutants in the surrogate model host Caenorhabditis elegans. Interestingly, the membrane nitrate reductase mutant was avirulent in C. elegans, while nitrate sensor-response regulator mutants were fully virulent. The data demonstrate that nitrate sensing, response regulation, and metabolism are linked directly to factors important in P. aeruginosa pathogenesis. [Abstract/Link to Full Text]

Herrmann JM, Bernardo J, Long HJ, Seetoo K, McMenamin ME, Batista EL, Van Dyke TE, Simons ER
Sequential chemotactic and phagocytic activation of human polymorphonuclear neutrophils.
Infect Immun. 2007 Aug;75(8):3989-98.
Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants' origins are reached, specific receptors bind to the invader's surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Delta[Ca(2+)](i) and DeltapH(i)) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca(2+)](i), mostly from intracellular stores, simultaneously with a drop in pH(i); these are followed by a drop in [Ca(2+)](i) and a rise in pH(i), with the latter being due to a Na(+)/H(+) antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca(2+)](i) is restored, 10(-7) M fMLP, previously shown to elicit maximal Delta[Ca(2+)](i) but no bactericidal functions, did not prevent the cells' responses with Delta[Ca(2+)](i) to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 mug/ml IC, previously shown to elicit maximal Delta[Ca(2+)](i) and bactericidal functions, exhibited no subsequent Delta[Ca(2+)](i) or DeltapH(i) to either stimulus. While exposure to 10(-7) M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10(-5) M fMLP did, presumably via the low-affinity receptor, using a different Ca(2+) source. [Abstract/Link to Full Text]

Ram S, Ngampasutadol J, Cox AD, Blom AM, Lewis LA, St Michael F, Stupak J, Gulati S, Rice PA
Heptose I glycan substitutions on Neisseria gonorrhoeae lipooligosaccharide influence C4b-binding protein binding and serum resistance.
Infect Immun. 2007 Aug;75(8):4071-81.
Lipooligosaccharide (LOS) heptose (Hep) glycan substitutions influence gonococcal serum resistance. Several gonococcal strains bind the classical complement pathway inhibitor, C4b-binding protein (C4BP), via their porin (Por) molecule to escape complement-dependent killing by normal human serum (NHS). We show that the proximal glucose (Glc) on HepI is required for C4BP binding to Por1B-bearing gonococcal strains MS11 and 1291 but not to FA19 (Por1A). The presence of only the proximal Glc on HepI (lgtE mutant) permitted maximal C4BP binding to MS11 but not to 1291. Replacing 1291 lgtE Por with MS11 Por increased C4BP binding to levels that paralleled MS11 lgtE, suggesting that replacement of the Por1B molecule dictated the effects of HepI glycans on C4BP binding. The remainder of the strain background did not affect C4BP binding; replacing the Por of strain F62 with MS11 Por (F62 PorMS11) and truncating HepI mirrored the findings in the MS11 background. C4BP binding correlated with resistance to killing by NHS in most instances. F62 PorMS11 and its lgtE mutant were sensitive to NHS despite binding C4BP, secondary to kinetically overwhelming classical pathway activation and possibly increased alternative pathway activation (measured by factor Bb binding) by the F62 background. FA19 lgtF (HepI unsubstituted) resisted killing by only 10% NHS, not 50% NHS, despite binding levels of C4BP similar to those of FA19 and FA19 lgtE (both resistant to 50% serum), suggesting a role for the proximal Glc in serum resistance independently of C4BP binding. This study provides mechanistic insights into how HepI LOS substitutions affect the serum resistance of N. gonorrhoeae. [Abstract/Link to Full Text]

Lundell AC, Andersson K, Josefsson E, Steinkasserer A, Rudin A
Soluble CD14 and CD83 from human neonatal antigen-presenting cells are inducible by commensal bacteria and suppress allergen-induced human neonatal Th2 differentiation.
Infect Immun. 2007 Aug;75(8):4097-104.
CD14 is expressed on the cell surface of various antigen-presenting cells, and CD83 is a maturation marker for dendritic cells (DC). CD14 and CD83 are also present as soluble proteins, and both have immunoregulatory functions. We examined whether neonatal cord blood monocytes or DC released soluble CD14 (sCD14) or sCD83 when exposed to the commensal intestinal bacteria Clostridium perfringens, Staphylococcus aureus, Lactobacillus rhamnosus, Escherichia coli, and Bacteroides fragilis. We found that the gram-positive bacteria C. perfringens and S. aureus, but not gram-negative bacteria, induced the release of sCD14 from monocytes. DC, on the other hand, released sCD14 in response to both gram-positive and gram-negative bacteria. Moreover, the expression of the virulence factor staphylococcal protein A seemed to be important for S. aureus-induced sCD14 production from both monocytes and DC. Soluble CD83 was released from DC, but not from monocytes, when exposed to both gram-positive and gram-negative bacteria. Finally, to investigate whether sCD14 or sCD83 could modulate neonatal allergen-induced T-cell differentiation, DC were exposed to birch allergen alone or in the presence of sCD14 or sCD83 and then cocultured with autologous T cells. We demonstrate that sCD14 and sCD83 inhibited the birch allergen-induced Th2 differentiation by suppressing interleukin 13 production. Together, these results suggest that the commensal intestinal flora may be an important stimulus for the developing immune system by inducing the immunoregulatory proteins sCD14 and sCD83, which may be involved in preventing T-cell sensitization to allergens in infants. [Abstract/Link to Full Text]

Abd Alla MD, White GL, Rogers TB, Cary ME, Carey DW, Ravdin JI
Adherence-inhibitory intestinal immunoglobulin a antibody response in baboons elicited by use of a synthetic intranasal lectin-based amebiasis subunit vaccine.
Infect Immun. 2007 Aug;75(8):3812-22.
We designed an amebiasis subunit vaccine that is constructed by using four peptide epitopes of the galactose-inhibitable lectin heavy subunit that were recognized by intestinal secretory immunoglobulin A (IgA) antibodies from immune human subjects. These epitopes are contained in the region encompassing amino acids 758 to 1134 of the lectin heavy subunit, designated LC3. Baboons (Papio anubis) are natural hosts for Entamoeba histolytica; naturally infected baboons raised in captivity possess serum IgA antibodies to the same four LC3 epitopes as humans. Uninfected, seronegative baboons received four intranasal immunizations at 7-day intervals with the synthetic peptide vaccine (400, 800, or 1,600 mug per nostril) with cholera toxin (20 mug) as the adjuvant. As determined by an enzyme-linked immunosorbent assay (ELISA), each dose of the peptide vaccine elicited antipeptide serum IgA and IgG and intestinal IgA antibody responses in all six immunized baboons by day 28, 7 days after the last immunization (P, <0.01 for each dose compared to the cholera toxin control). The peptide vaccine elicited serum IgG and intestinal IgA antibodies that recognized purified recombinant LC3 protein (P, <0.008 and 0.02, respectively) and native lectin protein (P < 0.01). In addition, an indirect immunofluorescence assay with whole trophozoites (P < 0.01) and Western blot analysis confirmed that serum IgG antibodies from vaccinated baboons recognized native lectin protein on the surfaces of axenic E. histolytica trophozoites or from solubilized amebae. All four synthetic peptides were immunogenic; the vaccine elicited dose- and time-dependent responses, as determined by ELISA optical density readings indicating the production of serum and intestinal antibodies (P, <0.02 for antipeptide and antilectin antibodies). As a positive control, intranasal immunization with purified recombinant LC3 protein with cholera toxin as the adjuvant elicited a serum anti-LC3 IgA and IgG antibody response (P, 0.05 and <0.0001, respectively); however, no intestinal anti-LC3 IgA antibody response was observed (P = 0.4). Of interest, serum IgA and IgG antibodies elicited by the recombinant LC3 vaccine did not recognize any of the four putatively protective LC3 peptide epitopes. Both serum and fecal antibodies elicited by the peptide vaccine exhibited neutralizing activity, as determined by their dose-dependent inhibition of the galactose-specific adherence of E. histolytica trophozoites to Chinese hamster ovary cells in vitro (P, <0.001 for each group of antibodies compared to the control). In summary, a lectin-based intranasal polylysine-linked synthetic peptide vaccine was effective in eliciting an adherence-inhibitory, intestinal antilectin IgA antibody response in baboons. Future studies with the baboon model will determine vaccine efficacy against asymptomatic E. histolytica intestinal infection. [Abstract/Link to Full Text]

Akman-Anderson L, Olivier M, Luckhart S
Induction of nitric oxide synthase and activation of signaling proteins in Anopheles mosquitoes by the malaria pigment, hemozoin.
Infect Immun. 2007 Aug;75(8):4012-9.
Anopheles stephensi, a major vector for malaria parasite transmission, responds to Plasmodium infection by synthesis of inflammatory levels of nitric oxide (NO), which can limit parasite development in the midgut. We have previously shown that Plasmodium falciparum glycosylphosphatidylinositols (PfGPIs) can induce A. stephensi NO synthase (AsNOS) expression in the midgut epithelium in vivo in a manner similar to the manner in which cytokines and NO are induced by PfGPIs in mammalian cells. In mosquito cells, signaling by PfGPIs and P. falciparum merozoites is mediated through Akt/protein kinase B (Akt/PKB), the mitogen-activated protein kinase kinase DSOR1, and extracellular signal-regulated kinase (ERK). In mammalian cells, a second parasite factor, malaria pigment or hemozoin (Hz), signals NOS induction through ERK- and nuclear factor kappa B-dependent pathways and has been demonstrated to be a novel proinflammatory ligand for Toll-like receptor 9. In this study, we demonstrate that Hz can also induce AsNOS gene expression in immortalized A. stephensi and Anopheles gambiae cell lines in vitro and in A. stephensi midgut tissue in vivo. In mosquito cells, Hz signaling is mediated through transforming growth factor beta-associated kinase 1, Akt/PKB, ERK, and atypical protein kinase C zeta/lambda. Our results show that Hz is a prominent parasite-derived signal for Anopheles and that signaling pathways activated by PfGPIs and Hz have both unique and shared components. Together with our previous findings, our data indicate that parasite signaling of innate immunity is conserved in mosquito and mammalian cells. [Abstract/Link to Full Text]

Kuenzle S, von Büdingen HC, Meier M, Harrer MD, Urich E, Becher B, Goebels N
Pathogen specificity and autoimmunity are distinct features of antigen-driven immune responses in neuroborreliosis.
Infect Immun. 2007 Aug;75(8):3842-7.
Neuroborreliosis (NB) is a chronic infectious disease of the central nervous system (CNS) caused by a tick-borne spirochete, Borrelia burgdorferi. In addition to direct effects of the causative infectious agent, additional immunity-mediated mechanisms are thought to play a role in the CNS pathology of NB. In order to further understand the involvement of humoral immune mechanisms in NB, we dissected the intrathecal antibody responses down to the single-plasma-cell level. Starting with single-cell reverse transcription-PCR of fluorescence-activated cell sorter-sorted cerebrospinal fluid plasma cells from an NB patient, we identified expanded clones and resurrected the antigen specificity of their secreted antibodies through recombinant expression of the correctly paired immunoglobulin heavy- and light-chain genes as monoclonal antibodies (MAbs). As expected, we found specificity for the causative infectious agent, B. burgdorferi, among the clonally expanded plasma cell (cePC)-derived MAbs. However, from an independent cePC of the same patient, we could derive MAbs specific for human CNS myelin, without detectable cross-reactivity with B. burgdorferi antigens. While reactivity against B. burgdorferi is a known feature of humoral immune responses in NB, we show (i) that immune responses specific for self antigens may be a distinct feature of CNS infections independent of pathogen reactivity and (ii) that humoral autoimmunity in NB (since found in cePC) is the result of a truly antigen-driven immune response. Our findings indicate that in NB mechanisms may be at play that induce distinct immune responses specific for pathogen and self antigens independent from "molecular mimicry." [Abstract/Link to Full Text]

Meyer GK, Neetz A, Brandes G, Tsikas D, Butterfield JH, Just I, Gerhard R
Clostridium difficile toxins A and B directly stimulate human mast cells.
Infect Immun. 2007 Aug;75(8):3868-76.
Clostridium difficile toxins A and B (TcdA and TcdB) are the causative agents of antibiotic-associated pseudomembranous colitis. Mucosal mast cells play a crucial role in the inflammatory processes underlying this disease. We studied the direct effects of TcdA and TcdB on the human mast cell line HMC-1 with respect to degranulation, cytokine release, and the activation of proinflammatory signal pathways. TcdA and TcdB inactivate Rho GTPases, the master regulators of the actin cytoskeleton. The inactivation of Rho GTPases induced a reorganization of the actin cytoskeleton accompanied by morphological changes of cells. The TcdB-induced reorganization of the actin cytoskeleton in HMC-1 cells reduced the number of electron-dense mast cell-specific granules. Accordingly, TcdB induced the release of hexosaminidase, a marker for degranulation, in HMC-1 cells. The actin rearrangement was found to be responsible for degranulation since latrunculin B induced a comparable hexosaminidase release. In addition, TcdB as well as latrunculin B induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 and also resulted in a p38 MAPK-dependent increased formation of prostaglandins D(2) and E(2). The autocrine stimulation of HMC-1 cells by prostaglandins partially contributed to the degranulation. Interestingly, TcdB-treated HMC-1 cells, but not latrunculin B-treated HMC-1 cells, showed a strong p38 MAPK-dependent increase in interleukin-8 release. Differences in the mast cell responses to TcdB and latrunculin B are probably due to the presence of functionally inactive Rho GTPases in toxin-treated cells. Thus, the HMC-1 cell line is a promising model for studying the direct effects of C. difficile toxins on mast cells independently of the tissue context. [Abstract/Link to Full Text]

Harper M, Boyce JD, Cox AD, St Michael F, Wilkie IW, Blackall PJ, Adler B
Pasteurella multocida expresses two lipopolysaccharide glycoforms simultaneously, but only a single form is required for virulence: identification of two acceptor-specific heptosyl I transferases.
Infect Immun. 2007 Aug;75(8):3885-93.
Lipopolysaccharide (LPS) is a critical virulence determinant in Pasteurella multocida and a major antigen responsible for host protective immunity. In other mucosal pathogens, variation in LPS or lipooligosaccharide structure typically occurs in the outer core oligosaccharide regions due to phase variation. P. multocida elaborates a conserved oligosaccharide extension attached to two different, simultaneously expressed inner core structures, one containing a single phosphorylated 3-deoxy-D-manno-octulosonic acid (Kdo) residue and the other containing two Kdo residues. We demonstrate that two heptosyltransferases, HptA and HptB, add the first heptose molecule to the Kdo(1) residue and that each exclusively recognizes different acceptor molecules. HptA is specific for the glycoform containing a single, phosphorylated Kdo residue (glycoform A), while HptB is specific for the glycoform containing two Kdo residues (glycoform B). In addition, KdkA was identified as a Kdo kinase, required for phosphorylation of the first Kdo molecule. Importantly, virulence data obtained from infected chickens showed that while wild-type P. multocida expresses both LPS glycoforms in vivo, bacterial mutants that produced only glycoform B were fully virulent, demonstrating for the first time that expression of a single LPS form is sufficient for P. multocida survival in vivo. We conclude that the ability of P. multocida to elaborate alternative inner core LPS structures is due to the simultaneous expression of two different heptosyltransferases that add the first heptose residue to the nascent LPS molecule and to the expression of both a bifunctional Kdo transferase and a Kdo kinase, which results in the initial assembly of two inner core structures. [Abstract/Link to Full Text]

Chakrabarty K, Wu W, Booth JL, Duggan ES, Nagle NN, Coggeshall KM, Metcalf JP
Human lung innate immune response to Bacillus anthracis spore infection.
Infect Immun. 2007 Aug;75(8):3729-38.
Bacillus anthracis, the causative agent of inhalational anthrax, enters a host through the pulmonary system before dissemination. We have previously shown that human alveolar macrophages participate in the initial innate immune response to B. anthracis spores through cell signal-mediated cytokine release. We proposed that the lung epithelia also participate in the innate immune response to this pathogen, and we have developed a human lung slice model to study this process. Exposure of our model to B. anthracis (Sterne) spores rapidly activated the mitogen-activated protein kinase signaling pathways ERK, p38, and JNK. In addition, an RNase protection assay showed induction of mRNA of several cytokines and chemokines. This finding was reflected at the translational level by protein peak increases of 3-, 25-, 9-, 34-, and 5-fold for interleukin-6 (IL-6), tumor necrosis factor alpha, IL-8, macrophage inflammatory protein 1alpha/beta, and monocyte chemoattractant protein 1, respectively, as determined by an enzyme-linked immunosorbent assay. Inhibition of individual pathways by UO126, SP600125, and SB0203580 decreased induction of chemokines and cytokines by spores, but this depended on the pathways inhibited and the cytokines and chemokines induced. Combining all three inhibitors reduced induction of all cytokines and chemokines tested to background levels. An immunohistochemistry analysis of IL-6 and IL-8 revealed that alveolar epithelial cells and macrophages and a few interstitial cells are the source of the cytokines and chemokines. Taken together, these data showed the activation of the pulmonary epithelium in response to B. anthracis spore exposure. Thus, the lung epithelia actively participate in the innate immune response to B. anthracis infection through cell signal-mediated elaboration of cytokines and chemokines. [Abstract/Link to Full Text]

Harland DN, Chu K, Haque A, Nelson M, Walker NJ, Sarkar-Tyson M, Atkins TP, Moore B, Brown KA, Bancroft G, Titball RW, Atkins HS
Identification of a LolC homologue in Burkholderia pseudomallei, a novel protective antigen for melioidosis.
Infect Immun. 2007 Aug;75(8):4173-80.
Melioidosis is an emerging disease of humans in Southeast Asia and tropical Australia. The bacterium causing this disease, Burkholderia pseudomallei, is also considered a bioterrorism agent, and as yet there is no licensed vaccine for preventing B. pseudomallei infection. In this study, we evaluated selected proteins (LolC, PotF, and OppA) of the ATP-binding cassette systems of B. pseudomallei as candidate vaccine antigens. Nonmembrane regions of the B. pseudomallei proteins were expressed and purified from Escherichia coli and then evaluated as vaccine candidates in an established mouse model of B. pseudomallei infection. When delivered with the monophosphoryl lipid A-trehalose dicorynomycolate adjuvant, the proteins stimulated antigen-specific humoral and cellular immune responses. Immunization with LolC or PotF protein domains afforded significant protection against a subsequent challenge with B. pseudomallei. The most promising vaccine candidate, LolC, provided a greater level of protection when it was administered with immune-stimulating complexes complexed with CpG oligodeoxynucleotide 10103. Immunization with LolC also protected against a subsequent challenge with a heterologous strain of B. pseudomallei, demonstrating the potential utility of this protein as a vaccine antigen for melioidosis. [Abstract/Link to Full Text]

Wiersinga WJ, Wieland CW, van der Windt GJ, de Boer A, Florquin S, Dondorp A, Day NP, Peacock SJ, van der Poll T
Endogenous interleukin-18 improves the early antimicrobial host response in severe melioidosis.
Infect Immun. 2007 Aug;75(8):3739-46.
Melioidosis is caused by the soil saprophyte Burkholderia pseudomallei and is endemic in Southeast Asia. The pathogenesis of melioidosis is still largely unknown, although gamma interferon (IFN-gamma) seems to play an obligatory role in host defense. Previously, we have shown that IFN-gamma production in melioidosis is controlled in part by interleukin-18 (IL-18). The aim of the present study was to determine the role of IL-18 in the immune response to B. pseudomallei. For this the following investigations were performed. (i) Plasma IL-18 and blood monocyte IL-18 mRNA levels were elevated in 34 patients with culture-proven melioidosis compared to the levels in 32 local healthy controls; in addition, IL-18 binding protein levels were markedly elevated in patients, strongly correlating with mortality. (ii) IL-18 gene-deficient (IL-18 knockout [KO]) mice showed accelerated mortality after intranasal infection with a lethal dose of B. pseudomallei, which was accompanied by enhanced bacterial growth in their lungs, livers, spleens, kidneys, and blood at 24 and 48 h postinfection, compared to wild-type mice. In addition, IL-18 KO mice displayed evidence of enhanced hepatocellular injury and renal insufficiency. Together, these data indicate that the enhanced production of IL-18 in melioidosis is an essential part of a protective immune response to this severe infection. [Abstract/Link to Full Text]

Williams SM, Chen YT, Andermann TM, Carter JE, McGee DJ, Ottemann KM
Helicobacter pylori chemotaxis modulates inflammation and bacterium-gastric epithelium interactions in infected mice.
Infect Immun. 2007 Aug;75(8):3747-57.
The ulcer-causing pathogen Helicobacter pylori uses directed motility, or chemotaxis, to both colonize the stomach and promote disease development. Previous work showed that mutants lacking the TlpB chemoreceptor, one of the receptors predicted to drive chemotaxis, led to less inflammation in the gerbil stomach than did the wild type. Here we expanded these findings and examined the effects on inflammation of completely nonchemotactic mutants and mutants lacking each chemoreceptor. Of note, all mutants colonized mice to the same levels as did wild-type H. pylori. Infection by completely nonchemotactic mutants (cheW or cheY) resulted in significantly less inflammation after both 3 and 6 months of infection. Mutants lacking either the TlpA or TlpB H. pylori chemotaxis receptors also had alterations in inflammation severity, while mutants lacking either of the other two chemoreceptors (TlpC and HylB) behaved like the wild type. Fully nonchemotactic and chemoreceptor mutants adhered to cultured gastric epithelial cells and caused cellular release of the chemokine interleukin-8 in vitro similar to the release caused by the wild type. The situation appeared to be different in the stomach. Using silver-stained histological sections, we found that nonchemotactic cheY or cheW mutants were less likely than the wild type to be intimately associated with the cells of the gastric mucosa, although there was not a strict correlation between intimate association and inflammation. Because others have shown that in vivo adherence promotes inflammation, we propose a model in which H. pylori uses chemotaxis to guide it to a productive interaction with the stomach epithelium. [Abstract/Link to Full Text]

Navarathna DH, Nickerson KW, Duhamel GE, Jerrels TR, Petro TM
Exogenous farnesol interferes with the normal progression of cytokine expression during candidiasis in a mouse model.
Infect Immun. 2007 Aug;75(8):4006-11.
Candida albicans, a dimorphic fungus composed of yeast and mycelial forms, is the most common human fungal pathogen. Th1 cytokines such as interleukin-2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), which are induced by macrophage IL-12, are critical to resistance against systemic candidiasis, while Th2 cytokines such as IL-4 and IL-5 are less critical. Farnesol is a quorum-sensing molecule produced by C. albicans that controls the formation of mycelia but is also a virulence factor. To determine whether farnesol enhances the virulence of C. albicans by modulating the production of Th1 and Th2 cytokines, mice were pretreated with farnesol prior to intravenous infection with a sublethal dose of farnesol-producing C. albicans. Production of IL-2, IL-4, IL-5, TNF-alpha, IFN-gamma, and IL-12 was evaluated by bead-array flow cytometry and enzyme-linked immunosorbent assay. Mice exhibited an elevation in serum TNF-alpha levels at 48 h and an elevation in IFN-gamma and IL-12 levels at 6 to 12 h after infection with C. albicans. Pretreatment with farnesol significantly reduced the elevation of both IFN-gamma and IL-12 but not TNF-alpha. In contrast, mice pretreated with farnesol exhibited an unexpected elevation in IL-5 levels. To determine whether farnesol has a direct effect on macrophage production of IL-12, peritoneal macrophages were pretreated with farnesol prior to stimulation with IFN-gamma plus lipopolysaccharide (LPS). Farnesol inhibited production of both IL-12 p40 and p70 from IFN-gamma/LPS-stimulated macrophages. Therefore, the role of farnesol in systemic candidiasis is likely due to its ability to inhibit the critical Th1 cytokines IFN-gamma and IL-12 and perhaps to enhance a Th2 cytokine, IL-5. [Abstract/Link to Full Text]

Schmitter T, Pils S, Weibel S, Agerer F, Peterson L, Buntru A, Kopp K, Hauck CR
Opa proteins of pathogenic neisseriae initiate Src kinase-dependent or lipid raft-mediated uptake via distinct human carcinoembryonic antigen-related cell adhesion molecule isoforms.
Infect Immun. 2007 Aug;75(8):4116-26.
Several pathogenic bacteria exploit human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) for adhesion to and invasion into their host cells. CEACAM isoforms have characteristic expression patterns on epithelial, endothelial, or hematopoietic cells, providing bacteria with distinct sets of receptors on particular tissues. For example, while CEACAM1 and CEACAM6 have a wide tissue distribution, CEACAM3, CEACAM4, and CEACAM8 are uniquely expressed on primary human granulocytes, whereas CEA and CEACAM7 are limited to epithelia. By reconstitution of a CEACAM-deficient cell line with individual CEACAMs, we have analyzed the requirements for CEACAM-mediated internalization of Neisseria gonorrhoeae. Our results point to two mechanistically different uptake pathways triggered by either epithelial CEACAMs (CEACAM1, CEA, and CEACAM6) or the granulocyte-specific CEACAM3. In particular, CEACAM3-mediated uptake critically depends on Src family protein tyrosine kinase (PTK) activity, and CEACAM3 associates with the SH2 domains of several Src PTKs. In contrast, epithelial CEACAMs require the integrity of cholesterol-rich membrane microdomains and are affected by cholesterol depletion, whereas CEACAM3-mediated uptake by transfected cells or the opsonin-independent phagocytosis by human granulocytes is not altered in the presence of cholesterol chelators. These results allow the subdivision of all human CEACAMs known to be utilized as pathogen receptors into functional groups and point to important consequences for bacterial engagement of distinct CEACAM isoforms. [Abstract/Link to Full Text]

Torrado E, Adusumilli S, Fraga AG, Small PL, Castro AG, Pedrosa J
Mycolactone-mediated inhibition of tumor necrosis factor production by macrophages infected with Mycobacterium ulcerans has implications for the control of infection.
Infect Immun. 2007 Aug;75(8):3979-88.
The pathogenicity of Mycobacterium ulcerans, the agent of Buruli ulcer, depends on the cytotoxic exotoxin mycolactone. Little is known about the immune response to this pathogen. Following the demonstration of an intracellular growth phase in the life cycle of M. ulcerans, we investigated the production of tumor necrosis factor (TNF) induced by intramacrophage bacilli of diverse toxigenesis/virulence, as well as the biological relevance of TNF during M. ulcerans experimental infections. Our data show that murine bone marrow-derived macrophages infected with mycolactone-negative strains of M. ulcerans (nonvirulent) produce high amounts of TNF, while macrophages infected with mycolactone-positive strains of intermediate or high virulence produce intermediate or low amounts of TNF, respectively. These results are in accordance with the finding that TNF receptor P55-deficient (TNF-P55 KO) mice are not more susceptible than wild-type mice to infection by the highly virulent strains but are more susceptible to nonvirulent and intermediately virulent strains, demonstrating that TNF is required to control the proliferation of these strains in animals experimentally infected by M. ulcerans. We also show that mycolactone produced by intramacrophage M. ulcerans bacilli inhibits, in a dose-dependent manner, but does not abrogate, the production of macrophage inflammatory protein 2, which is consistent with the persistent inflammatory responses observed in experimentally infected mice. [Abstract/Link to Full Text]

Labaied M, Harupa A, Dumpit RF, Coppens I, Mikolajczak SA, Kappe SH
Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection.
Infect Immun. 2007 Aug;75(8):3758-68.
Malaria infection starts when sporozoites are transmitted to the mammalian host during a mosquito bite. Sporozoites enter the blood circulation, reach the liver, and infect hepatocytes. The formation of a parasitophorous vacuole (PV) establishes their intracellular niche. Recently, two members of the 6-Cys domain protein family, P52 and P36, were each shown to play an important albeit nonessential role in Plasmodium berghei sporozoite infectivity for the rodent host. Here, we generated p52/p36-deficient Plasmodium yoelii parasites by the simultaneous deletion of both genes using a single genetic manipulation. p52/p36-deficient parasites exhibited normal progression through the life cycle during blood-stage infection, transmission to mosquitoes, mosquito-stage development, and sporozoite infection of the salivary glands. p52/p36-deficient sporozoites also showed normal motility and cell traversal activity. However, immunofluorescence analysis and electron microscopic observations revealed that p52/p36-deficient parasites did not form a PV within hepatocytes in vitro and in vivo. The p52/p36-deficient parasites localized as free entities in the host cell cytoplasm or the host cell nucleoplasm and did not develop as liver stages. Consequently, they did not cause blood-stage infections even at high sporozoite inoculation doses. Mice immunized with p52/p36-deficient sporozoites were completely protected against infectious sporozoite challenge. Our results demonstrate for the first time the generation of two-locus gene deletion-attenuated parasites that infect the liver but do not progress to blood-stage infection. The study will critically guide the design of Plasmodium falciparum live attenuated malaria vaccines. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Bacteriology

---

Hussa EA, O'Shea TM, Darnell CL, Ruby EG, Visick KL
Two-component response regulators of Vibrio fischeri: identification, mutagenesis, and characterization.
J Bacteriol. 2007 Aug;189(16):5825-38.
Two-component signal transduction systems are utilized by prokaryotic and eukaryotic cells to sense and respond to environmental stimuli, both to maintain homeostasis and to rapidly adapt to changing conditions. Studies have begun to emerge that utilize a large-scale mutagenesis approach to analyzing these systems in prokaryotic organisms. Due to the recent availability of its genome sequence, such a global approach is now possible for the marine bioluminescent bacterium Vibrio fischeri, which exists either in a free-living state or as a mutualistic symbiont within a host organism such as the Hawaiian squid species Euprymna scolopes. In this work, we identified 40 putative two-component response regulators encoded within the V. fischeri genome. Based on the type of effector domain present, we classified six as NarL type, 13 as OmpR type, and six as NtrC type; the remaining 15 lacked a predicted DNA-binding domain. We subsequently mutated 35 of these genes via a vector integration approach and analyzed the resulting mutants for roles in bioluminescence, motility, and competitive colonization of squid. Through these assays, we identified three novel regulators of V. fischeri luminescence and seven regulators that altered motility. Furthermore, we found 11 regulators with a previously undescribed effect on competitive colonization of the host squid. Interestingly, five of the newly characterized regulators each affected two or more of the phenotypes examined, strongly suggesting interconnectivity among systems. This work represents the first large-scale mutagenesis of a class of genes in V. fischeri using a genomic approach and emphasizes the importance of two-component signal transduction in bacterium-host interactions. [Abstract/Link to Full Text]

Chi F, Leider M, Leendertz F, Bergmann C, Boesch C, Schenk S, Pauli G, Ellerbrok H, Hakenbeck R
New Streptococcus pneumoniae clones in deceased wild chimpanzees.
J Bacteriol. 2007 Aug;189(16):6085-8.
In wild chimpanzees in the Taï National Park, Côte d'Ivoire, sudden deaths which were preceded by respiratory problems had been observed since 1999. Two new clones of Streptococcus pneumoniae were identified in deceased apes on the basis of multilocus sequence typing analysis and ply, lytA, and pbp2x sequences. The findings suggest that virulent S. pneumoniae occurs in populations of wild chimpanzees with the potential to cause infections similar to those observed in humans. [Abstract/Link to Full Text]

Zahrl D, Wagner A, Tscherner M, Koraimann G
GroEL plays a central role in stress-induced negative regulation of bacterial conjugation by promoting proteolytic degradation of the activator protein TraJ.
J Bacteriol. 2007 Aug;189(16):5885-94.
Transcription of DNA transfer genes is a prerequisite for conjugative DNA transfer of F-like plasmids. Transfer gene expression is sensed by the donor cell and is regulated by a complex network of plasmid- and host-encoded factors. In this study we analyzed the effect of induction of the heat shock regulon on transfer gene expression and DNA transfer in Escherichia coli. Raising the growth temperature from 22 degrees C to 43 degrees C transiently reduced transfer gene expression to undetectable levels and reduced conjugative transfer by 2 to 3 orders of magnitude. In contrast, when host cells carried the temperature-sensitive groEL44 allele, heat shock-mediated repression was alleviated. These data implied that the chaperonin GroEL was involved in negative regulation after heat shock. Investigation of the role of GroEL in this regulatory process revealed that, in groEL(Ts) cells, TraJ, the plasmid-encoded master activator of type IV secretion (T4S) system genes, was less susceptible to proteolysis and had a prolonged half-life compared to isogenic wild-type E. coli cells. This result suggested a direct role for GroEL in proteolysis of TraJ, down-regulation of T4S system gene expression, and conjugation after heat shock. Strong support for this novel role for GroEL in regulation of bacterial conjugation was the finding that GroEL specifically interacted with TraJ in vivo. Our results further suggested that in wild-type cells this interaction was followed by rapid degradation of TraJ whereas in groEL(Ts) cells TraJ remained trapped in the temperature-sensitive GroEL protein and thus was not amenable to proteolysis. [Abstract/Link to Full Text]

Tremblay PL, Drepper T, Masepohl B, Hallenbeck PC
Membrane sequestration of PII proteins and nitrogenase regulation in the photosynthetic bacterium Rhodobacter capsulatus.
J Bacteriol. 2007 Aug;189(16):5850-9.
Both Rhodobacter capsulatus PII homologs GlnB and GlnK were found to be necessary for the proper regulation of nitrogenase activity and modification in response to an ammonium shock. As previously reported for several other bacteria, ammonium addition triggered the AmtB-dependent association of GlnK with the R. capsulatus membrane. Native polyacrylamide gel electrophoresis analysis indicates that the modification/demodification of one PII homolog is aberrant in the absence of the other. In a glnK mutant, more GlnB was found to be membrane associated under these conditions. In a glnB mutant, GlnK fails to be significantly sequestered by AmtB, even though it appears to be fully deuridylylated. Additionally, the ammonium-induced enhanced sequestration by AmtB of the unmodifiable GlnK variant GlnK-Y51F follows the wild-type GlnK pattern with a high level in the cytoplasm without the addition of ammonium and an increased level in the membrane fraction after ammonium treatment. These results suggest that factors other than PII modification are driving its association with AmtB in the membrane in R. capsulatus. [Abstract/Link to Full Text]

Torres AG, López-Sánchez GN, Milflores-Flores L, Patel SD, Rojas-López M, Martínez de la Peña CF, Arenas-Hernández MM, Martínez-Laguna Y
Ler and H-NS, regulators controlling expression of the long polar fimbriae of Escherichia coli O157:H7.
J Bacteriol. 2007 Aug;189(16):5916-28.
Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 colonizes the human intestine and is responsible for diarrheal outbreaks worldwide. Previously we showed that EHEC produces long polar fimbriae (LPF) and that maximum expression is observed during the exponential phase of growth at 37 degrees C and pH 6.5. In this study, we analyzed the roles of several regulators in the expression of LPF using the beta-galactosidase reporter system, and we found that H-NS functions as a transcriptional silencer while Ler functions as an antisilencer of LPF expression. Interestingly, deletion of the hns and ler genes in EHEC caused constitutive expression of the fusion reporter protein. Semiquantitative reverse transcription (RT)-PCR was also used to analyze LPF expression in the EHEC ler or hns mutant strain. The hns mutant exhibited an increase in lpf mRNA expression, while expression in the ler mutant was decreased, compared to that in the wild-type strain. Using primer extension analysis, we identified two potential transcriptional start sites within the regulatory region of lpf and located consensus hexamers of -10 (CAAGAT) and -35 (TTCAAA), which are commonly found in sigma(70)-dependent promoters. Further, we determined whether H-NS and Ler interact directly with the lpf promoter region by using purified His-tagged proteins and electrophoretic mobility shift assays. Our data are the first to show direct binding interactions between the H-NS and Ler proteins within the regulatory sequence of the lpf operon. Based on the electrophoretic mobility shift assay, RT-PCR, primer extension, and beta-galactosidase assay results, we concluded that the E. coli O157:H7 lpf operon possesses a promoter dependent on sigma(70), that H-NS binds to the regulatory sequence of lpfA and "silences" the transcription of lpf, and that Ler binds to the regulatory sequence and inhibits the action of the H-NS protein. [Abstract/Link to Full Text]

Nwaneshiudu AI, Mucci T, Pickard DJ, Okeke IN
A second large plasmid encodes conjugative transfer and antimicrobial resistance in O119:H2 and some typical O111 enteropathogenic Escherichia coli strains.
J Bacteriol. 2007 Aug;189(16):6074-9.
A novel and functional conjugative transfer system identified in O119:H2 enteropathogenic Escherichia coli (EPEC) strain MB80 by subtractive hybridization is encoded on a large multidrug resistance plasmid, distinct from the well-described EPEC adherence factor (EAF) plasmid. Variants of the MB80 conjugative resistance plasmid were identified in other EPEC strains, including the prototypical O111:NM strain B171, from which the EAF plasmid has been sequenced. This separate large plasmid and the selective advantage that it confers in the antibiotic era have been overlooked because it comigrates with the virulence plasmid on conventional gels. [Abstract/Link to Full Text]

Condemine G, Ghazi A
Differential regulation of two oligogalacturonate outer membrane channels, KdgN and KdgM, of Dickeya dadantii (Erwinia chrysanthemi).
J Bacteriol. 2007 Aug;189(16):5955-62.
The entry of oligogalacturonates into Dickeya dadantii occurs through the specific channel KdgM. The genome of the bacterium encodes a second member of this family of outer membrane proteins, KdgN. We showed that this protein is also involved in the uptake of oligogalacturonates. When KdgN was reconstituted in proteoliposomes, it formed channels with a conductance of about 450 pS at a positive potential. These channels had weak anionic selectivity. The regulation of kdgN is complex, and five genes controlling the expression of kdgN have been identified: kdgR, pecS, ompR, hns, and crp. Moreover, kdgN was regulated by growth phase but only when bacteria were grown in rich medium. Most of these regulators of kdgN also control kdgM expression, but some of them regulate kdgM in the opposite manner: while PecS and OmpR are repressors of kdgM, they are activators of kdgN. This pattern resembles the regulation of the Escherichia coli general porins OmpF and OmpC, but such opposite regulation of two specific outer membrane channels has never been described before. KdgN may allow the bacteria to collect oligogalacturonates under saprophytic conditions, when virulence genes, including kdgM, are not expressed. [Abstract/Link to Full Text]

Lawrenz MB, Miller VL
Comparative analysis of the regulation of rovA from the pathogenic yersiniae.
J Bacteriol. 2007 Aug;189(16):5963-75.
RovA is a MarR/SlyA-type regulator that mediates the transcription of inv in Yersinia enterocolitica and Y. pseudotuberculosis. In Y. pseudotuberculosis, rovA transcription is controlled primarily by H-NS and RovA, which bind to similar regions within the rovA promoter. At 37 degrees C, rovA transcription is repressed by H-NS. Transcription of rovA results when RovA relieves H-NS-mediated repression. The region of the rovA promoter that H-NS and RovA bind is not conserved in the Y. enterocolitica promoter. Using green fluorescent protein reporters, we determined that the Y. enterocolitica rovA (rovA(Yent)) promoter is weaker than the Y. pseudotuberculosis promoter. However, despite the missing H-NS/RovA binding site in the rovA(Yent) promoter, H-NS and RovA are still involved in the regulation of rovA(Yent). DNA binding studies suggest that H-NS and RovA bind with a higher affinity to the Y. pseudotuberculosis/Y. pestis rovA (rovA(Ypstb/Ypestis)) promoter than to the rovA(Yent) promoter. Furthermore, H-NS appears to bind to two regions in a cooperative fashion within the rovA(Yent) promoter that is not observed with the rovA(Ypstb/Ypestis) promoter. Finally, using a transposon mutagenesis approach, we identified a new positive regulator of rovA in Y. enterocolitica, LeuO. In Escherichia coli, LeuO regulates gene expression via changes in levels of RpoS and H-NS, but LeuO-mediated regulation of rovA(Yent) appears to be independent of either of these two proteins. Together, these data demonstrate that while the rovA regulatory factors are conserved in Yersinia, divergence of Y. enterocolitica and Y. pseudotuberculosis/Y. pestis during evolution has resulted in modifications in the mechanisms that are responsible for controlling rovA transcription. [Abstract/Link to Full Text]

Alexander DC, Anders CL, Lee L, Jensen SE
pcd mutants of Streptomyces clavuligerus still produce cephamycin C.
J Bacteriol. 2007 Aug;189(16):5867-74.
Biosynthesis of cephamycin C in Streptomyces clavuligerus involves the initial conversion of lysine to alpha-aminoadipic acid. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase carry out this two-step reaction, and genes encoding each of these enzymes are found within the cephamycin C gene cluster. However, while mutation of the lat gene causes complete loss of cephamycin production, pcd mutants still produce cephamycin at 30% to 70% of wild-type levels. Cephamycin production by pcd mutants could be restored to wild-type levels either by supplementation of the growth medium with alpha-aminoadipic acid or by complementation of the mutation with an intact copy of the pcd gene. Neither heterologous PCR nor Southern analyses showed any evidence for the presence of a second pcd gene. Furthermore, cell extracts from pcd mutants lack detectable PCD activity. Cephamycin production in the absence of detectable PCD activity suggests that S. clavuligerus must have some alternate means of producing the aminoadipyl-cysteinyl-valine needed for cephamycin biosynthesis. [Abstract/Link to Full Text]

Nishimura K, Johansen SK, Inaoka T, Hosaka T, Tokuyama S, Tahara Y, Okamoto S, Kawamura F, Douthwaite S, Ochi K
Identification of the RsmG methyltransferase target as 16S rRNA nucleotide G527 and characterization of Bacillus subtilis rsmG mutants.
J Bacteriol. 2007 Aug;189(16):6068-73.
The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness between the rsmG mutant and parent strains under the various culture conditions examined. B. subtilis rsmG mutants emerged spontaneously at a relatively high frequency, 10(-6). Importantly, in the rsmG mutant background, high-level-streptomycin-resistant rpsL (encoding ribosomal protein S12) mutants emerged at a frequency 200 times greater than that seen for the wild-type strain. This elevated frequency in the emergence of high-level streptomycin resistance was facilitated by a mutation pattern in rpsL more varied than that obtained by selection of the wild-type strain. [Abstract/Link to Full Text]

Gao R, Lynn DG
Integration of rotation and piston motions in coiled-coil signal transduction.
J Bacteriol. 2007 Aug;189(16):6048-56.
A coordinated response to a complex and dynamic environment requires an organism to simultaneously monitor and interpret multiple signaling cues. In bacteria and some eukaryotes, environmental responses depend on the histidine autokinases (HKs). For example, VirA, a large integral membrane HK from Agrobacterium tumefaciens, regulates the expression of virulence genes in response to signals from multiple molecular classes (phenol, pH, and sugar). The ability of this pathogen to perceive inputs from different known host signals within a single protein receptor provides an opportunity to understand the mechanisms of signal integration. Here we exploited the conserved domain organization of the HKs and engineered chimeric kinases to explore the signaling mechanisms of phenol sensing and pH/sugar integration. Our data implicate a piston-assisted rotation of coiled coils for integration of multiple inputs and regulation of critical responses during pathogenesis. [Abstract/Link to Full Text]

Khodursky AB
Evolution, adaptation, and supercoiling.
J Bacteriol. 2007 Aug;189(16):5789-91. [Abstract/Link to Full Text]

Champion K, Higgins NP
Growth rate toxicity phenotypes and homeostatic supercoil control differentiate Escherichia coli from Salmonella enterica serovar Typhimurium.
J Bacteriol. 2007 Aug;189(16):5839-49.
Escherichia coli and Salmonella enterica serovar Typhimurium share high degrees of DNA and amino acid identity for 65% of the homologous genes shared by the two genomes. Yet, there are different phenotypes for null mutants in several genes that contribute to DNA condensation and nucleoid formation. The mutant R436-S form of the GyrB protein has a temperature-sensitive phenotype in Salmonella, showing disruption of supercoiling near the terminus and replicon failure at 42 degrees C. But this mutation in E. coli is lethal at the permissive temperature. A unifying hypothesis for why the same mutation in highly conserved homologous genes of different species leads to different physiologies focuses on homeotic supercoil control. During rapid growth in mid-log phase, E. coli generates 15% more negative supercoils in pBR322 DNA than Salmonella. Differences in compaction and torsional strain on chromosomal DNA explain a complex set of single-gene phenotypes and provide insight into how supercoiling may modulate epigenetic effects on chromosome structure and function and on prophage behavior in vivo. [Abstract/Link to Full Text]

Ikushiro H, Islam MM, Tojo H, Hayashi H
Molecular characterization of membrane-associated soluble serine palmitoyltransferases from Sphingobacterium multivorum and Bdellovibrio stolpii.
J Bacteriol. 2007 Aug;189(15):5749-61.
Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl coenzyme A (CoA) to form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated heterodimers belonging to the pyridoxal 5'-phosphate (PLP)-dependent alpha-oxamine synthase family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant water-soluble homodimeric SPT of the same family (H. Ikushiro et al., J. Biol. Chem. 276:18249-18256, 2001). This enzyme is suitable for the detailed mechanistic studies of SPT, although single crystals appropriate for high-resolution crystallography have not yet been obtained. We have now isolated three novel SPT genes from Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Bdellovibrio stolpii, respectively. Each gene product exhibits an approximately 30% sequence identity to both eukaryotic subunits, and the putative catalytic amino acid residues are conserved. All bacterial SPTs were successfully overproduced in Escherichia coli and purified as water-soluble active homodimers. The spectroscopic properties of the purified SPTs are characteristic of PLP-dependent enzymes. The KDS formation by the bacterial SPTs was confirmed by high-performance liquid chromatography/mass spectrometry. The Sphingobacterium SPTs obeyed normal steady-state ordered Bi-Bi kinetics, while the Bdellovibrio SPT underwent a remarkable substrate inhibition at palmitoyl CoA concentrations higher than 100 microM, as does the eukaryotic enzyme. Immunoelectron microscopy showed that unlike the cytosolic Sphingomonas SPT, S. multivorum and Bdellovibrio SPTs were bound to the inner membrane of cells as peripheral membrane proteins, indicating that these enzymes can be a prokaryotic model mimicking the membrane-associated eukaryotic SPT. [Abstract/Link to Full Text]

Kim YJ, Ko IJ, Lee JM, Kang HY, Kim YM, Kaplan S, Oh JI
Dominant role of the cbb3 oxidase in regulation of photosynthesis gene expression through the PrrBA system in Rhodobacter sphaeroides 2.4.1.
J Bacteriol. 2007 Aug;189(15):5617-25.
In this study, the H303A mutant form of the cbb(3) oxidase (H303A oxidase), which has the H303A mutation in its catalytic subunit (CcoN), was purified from Rhodobacter sphaeroides. The H303A oxidase showed the same catalytic activity as did the wild-type form of the oxidase (WT oxidase). The heme contents of the mutant and WT forms of the cbb(3) oxidase were also comparable. However, the puf and puc operons, which are under the control of the PrrBA two-component system, were shown to be derepressed aerobically in the R. sphaeroides strain expressing the H303A oxidase. Since the strain harboring the H303A oxidase exhibited the same cytochrome c oxidase activity as the stain harboring the WT oxidase did, the aerobic derepression of photosynthesis gene expression observed in the H303A mutant appears to be the result of a defective signaling function of the H303A oxidase rather than reflecting any redox changes in the ubiquinone/ubiquinol pool. It was also demonstrated that ubiquinone inhibits not only the autokinase activity of full-length PrrB but also that of the truncated form of PrrB lacking its transmembrane domain, including the proposed quinone binding sequence. These results imply that the suggested ubiquinone binding site within the PrrB transmembrane domain is not necessary for the inhibition of PrrB kinase activity by ubiquinone. Instead, it is probable that signaling through H303 of the CcoN subunit of the cbb(3) oxidase is part of the pathway through which the cbb(3) oxidase affects the relative kinase/phosphatase activity of the membrane-bound PrrB. [Abstract/Link to Full Text]

Bologna FP, Andreo CS, Drincovich MF
Escherichia coli malic enzymes: two isoforms with substantial differences in kinetic properties, metabolic regulation, and structure.
J Bacteriol. 2007 Aug;189(16):5937-46.
Malic enzymes (MEs) catalyze the oxidative decarboxylation of malate in the presence of a divalent metal ion. In eukaryotes, well-conserved cytoplasmic, mitochondrial, and plastidic MEs have been characterized. On the other hand, distinct groups can be detected among prokaryotic MEs, which are more diverse in structure and less well characterized than their eukaryotic counterparts. In Escherichia coli, two genes with a high degree of homology to ME can be detected: sfcA and maeB. MaeB possesses a multimodular structure: the N-terminal extension shows homology to ME, while the C-terminal extension shows homology to phosphotransacetylases (PTAs). In the present work, a detailed characterization of the products of E. coli sfcA and maeB was performed. The results indicate that the two MEs exhibit relevant kinetic, regulatory, and structural differences. SfcA is a NAD(P) ME, while MaeB is a NADP-specific ME highly regulated by key metabolites. Characterization of truncated versions of MaeB indicated that the PTA domain is not essential for the ME reaction. Nevertheless, truncated MaeB without the PTA domain loses most of its metabolic ME modulation and its native oligomeric state. Thus, the association of the two structural domains in MaeB seems to facilitate metabolic control of the enzyme. Although the PTA domain in MaeB is highly similar to the domains of proteins with PTA activity, MaeB and its PTA domain do not exhibit PTA activity. Determination of the distinct properties of recombinant products of sfcA and maeB performed in the present work will help to clarify the roles of MEs in prokaryotic metabolism. [Abstract/Link to Full Text]

Makhlin J, Kofman T, Borovok I, Kohler C, Engelmann S, Cohen G, Aharonowitz Y
Staphylococcus aureus ArcR controls expression of the arginine deiminase operon.
J Bacteriol. 2007 Aug;189(16):5976-86.
We identified a single open reading frame that is strongly similar to ArcR, a member of the Crp/Fnr family of bacterial transcriptional regulators, in all sequenced Staphylococcus aureus genomes. The arcR gene encoding ArcR forms an operon with the arginine deiminase (ADI) pathway genes arcABDC that enable the utilization of arginine as a source of energy for growth under anaerobic conditions. In this report, we show that under anaerobic conditions, S. aureus growth is subject to glucose catabolic repression and is enhanced by arginine. Likewise, glucose and arginine have reciprocal effects on the transcription of the arcABDCR genes. Furthermore, we show using a mutant deleted for arcR that the transcription of the arc operon under anaerobic conditions depends strictly on a functional ArcR. These findings are supported by proteome analyses, which showed that under anaerobic conditions the expression of the ADI catabolic proteins depends on ArcR. Bioinformatic analysis of S. aureus ArcR predicts an N-terminal nucleotide binding domain and a C-terminal helix-turn-helix DNA binding motif. ArcR binds to a conserved Crp-like sequence motif, TGTGA-N(6)-TCACA, present in the arc promoter region and thereby activates the expression of the ADI pathway genes. Crp-like sequence motifs were also found in the regulatory regions of some 30 other S. aureus genes mostly encoding anaerobic enzymatic systems, virulence factors, and regulatory systems. ArcR was tested and found to bind to the regulatory regions of four such genes, adh1, lctE, srrAB, and lukM. In one case, for lctE, encoding l-lactate dehydrogenase, ArcR was able to bind only in the presence of cyclic AMP. These observations suggest that ArcR is likely to play an important role in the expression of numerous genes required for anaerobic growth. [Abstract/Link to Full Text]

Li J, Attila C, Wang L, Wood TK, Valdes JJ, Bentley WE
Quorum sensing in Escherichia coli is signaled by AI-2/LsrR: effects on small RNA and biofilm architecture.
J Bacteriol. 2007 Aug;189(16):6011-20.
The regulatory network for the uptake of Escherichia coli autoinducer 2 (AI-2) is comprised of a transporter complex, LsrABCD; its repressor, LsrR; and a cognate signal kinase, LsrK. This network is an integral part of the AI-2 quorum-sensing (QS) system. Because LsrR and LsrK directly regulate AI-2 uptake, we hypothesized that they might play a wider role in regulating other QS-related cellular functions. In this study, we characterized physiological changes due to the genomic deletion of lsrR and lsrK. We discovered that many genes were coregulated by lsrK and lsrR but in a distinctly different manner than that for the lsr operon (where LsrR serves as a repressor that is derepressed by the binding of phospho-AI-2 to the LsrR protein). An extended model for AI-2 signaling that is consistent with all current data on AI-2, LuxS, and the LuxS regulon is proposed. Additionally, we found that both the quantity and architecture of biofilms were regulated by this distinct mechanism, as lsrK and lsrR knockouts behaved identically. Similar biofilm architectures probably resulted from the concerted response of a set of genes including flu and wza, the expression of which is influenced by lsrRK. We also found for the first time that the generation of several small RNAs (including DsrA, which was previously linked to QS systems in Vibrio harveyi) was affected by LsrR. Our results suggest that AI-2 is indeed a QS signal in E. coli, especially when it acts through the transcriptional regulator LsrR. [Abstract/Link to Full Text]

Campo N, Rudner DZ
SpoIVB and CtpB are both forespore signals in the activation of the sporulation transcription factor sigmaK in Bacillus subtilis.
J Bacteriol. 2007 Aug;189(16):6021-7.
The proteolytic activation of the mother cell transcription factor pro-sigma(K) is controlled by a signal transduction pathway during sporulation in the bacterium Bacillus subtilis. The pro-sigma(K) processing enzyme SpoIVFB, a membrane-embedded metalloprotease, is held inactive by two other integral membrane proteins, SpoIVFA and BofA, in the mother cell membrane that surrounds the forespore. Two signaling serine proteases, SpoIVB and CtpB, trigger pro-sigma(K) processing by cleaving the regulatory protein SpoIVFA. The SpoIVB signal is absolutely required to activate pro-sigma(K) processing and is derived from the forespore compartment. CtpB is necessary for the proper timing of sigma(K) activation and was thought to be a mother cell signal. Here, we show that the ctpB gene is expressed in both the mother cell and forespore compartments but that synthesis in the forespore under the control of sigma(G) is both necessary and sufficient for the proper timing of pro-sigma(K) processing. We further show that SpoIVB cleaves CtpB in vitro and in vivo but that this cleavage does not appear to be necessary for CtpB activation. Thus, both signaling proteins are made in the forespore and independently target the same regulatory protein. [Abstract/Link to Full Text]

Uzureau S, Godefroid M, Deschamps C, Lemaire J, De Bolle X, Letesson JJ
Mutations of the quorum sensing-dependent regulator VjbR lead to drastic surface modifications in Brucella melitensis.
J Bacteriol. 2007 Aug;189(16):6035-47.
Successful establishment of infection by bacterial pathogens requires fine-tuning of virulence-related genes. Quorum sensing (QS) is a global regulation process based on the synthesis of, detection of, and response to small diffusible molecules, called N-acyl-homoserine lactones (AHL), in gram-negative bacteria. In numerous species, QS has been shown to regulate genes involved in the establishment of pathogenic interactions with the host. Brucella melitensis produces N-dodecanoyl homoserine lactones (C(12)-HSL), which down regulate the expression of flagellar genes and of the virB operon (encoding a type IV secretion system), both of which encode surface virulence factors. A QS-related regulator, called VjbR, was identified as a transcriptional activator of these genes. We hypothesized that VjbR mediates the C(12)-HSL effects described above. vjbR alleles mutated in the region coding for the AHL binding domain were constructed to test this hypothesis. These alleles expressed in trans in a DeltavjbR background behave as constitutive regulators both in vitro and in a cellular model of infection. Interestingly, the resulting B. melitensis strains, unable to respond to AHLs, aggregate spontaneously in liquid culture. Preliminary characterization of these strains showed altered expression of some outer membrane proteins and overproduction of a matrix-forming exopolysaccharide, suggesting for the first time that B. melitensis could form biofilms. Together, these results indicate that QS through VjbR is a major regulatory system of important cell surface structures of Brucella and as such plays a key role in host-pathogen interactions. [Abstract/Link to Full Text]

Candela M, Bergmann S, Vici M, Vitali B, Turroni S, Eikmanns BJ, Hammerschmidt S, Brigidi P
Binding of human plasminogen to Bifidobacterium.
J Bacteriol. 2007 Aug;189(16):5929-36.
Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important health-promoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. By using flow cytometry, we demonstrated a dose-dependent human plasminogen-binding activity for four strains belonging to three bifidobacterial species: Bifidobacterium lactis, B. bifidum, and B. longum. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified five putative plasminogen-binding proteins in the cell wall fraction of the model strain B. lactis BI07. The data suggest that plasminogen binding to B. lactis is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction. [Abstract/Link to Full Text]

Paoletti L, Lu YJ, Schujman GE, de Mendoza D, Rock CO
Coupling of fatty acid and phospholipid synthesis in Bacillus subtilis.
J Bacteriol. 2007 Aug;189(16):5816-24.
plsX (acyl-acyl carrier protein [ACP]:phosphate acyltransferase), plsY (yneS) (acyl-phosphate:glycerol-phosphate acyltransferase), and plsC (yhdO) (acyl-ACP:1-acylglycerol-phosphate acyltransferase) function in phosphatidic acid formation, the precursor to membrane phospholipids. The physiological functions of these genes was inferred from their in vitro biochemical activities, and this study investigated their roles in gram-positive phospholipid metabolism through the analysis of conditional knockout strains in the Bacillus subtilis model system. The depletion of PlsX led to the cessation of both fatty acid synthesis and phospholipid synthesis. The inactivation of PlsY also blocked phospholipid synthesis, but fatty acid formation continued due to the appearance of acylphosphate intermediates and fatty acids arising from their hydrolysis. Phospholipid synthesis ceased following PlsC depletion, but fatty acid synthesis continued at a high rate, leading to the accumulation of fatty acids arising from the dephosphorylation of 1-acylglycerol-3-P followed by the deacylation of monoacylglycerol. Analysis of glycerol 3-P acylation in B. subtilis membranes showed that PlsY was an acylphosphate-specific acyltransferase, whereas PlsC used only acyl-ACP as an acyl donor. PlsX was found in the soluble fraction of disrupted cells but was associated with the cell membrane in intact organisms. These data establish that PlsX is a key enzyme that coordinates the production of fatty acids and membrane phospholipids in B. subtilis. [Abstract/Link to Full Text]

Yang J, Ogawa Y, Camakaris H, Shimada T, Ishihama A, Pittard AJ
folA, a new member of the TyrR regulon in Escherichia coli K-12.
J Bacteriol. 2007 Aug;189(16):6080-4.
The folA gene was identified as a new member of the TyrR regulon by genomic SELEX. Binding of TyrR to two sites in folA activated its transcription. Mutations in the N-terminal or central domain of TyrR, the alpha subunit of RNA polymerase, or integration host factor all abolished activation of the folA promoter. [Abstract/Link to Full Text]

Fujiwara K, Taguchi H
Filamentous morphology in GroE-depleted Escherichia coli induced by impaired folding of FtsE.
J Bacteriol. 2007 Aug;189(16):5860-6.
The chaperonin GroE (GroEL and the cochaperonin GroES) is the only chaperone system that is essential for the viability of Escherichia coli. It is known that GroE-depleted cells exhibit a filamentous morphology, suggesting that GroE is required for the folding of proteins involved in cell division. Although previous studies, including proteome-wide analyses of GroE substrates, have suggested several targets of GroE in cell division, there is no direct in vivo evidence to identify which substrates exhibit obligate dependence on GroE for folding. Among the candidate substrates, we found that prior excess production of FtsE, a protein engaged in cell division, completely suppressed the filamentation of GroE-depleted E. coli. The GroE depletion led to a drastic decrease in FtsE, and the cells exhibited a known phenotype associated with impaired FtsE function. In the GroE-depleted filamentous cells, the localizations of FtsA and ZipA, both of which assemble with the FtsZ septal ring before FtsE, were normal, whereas FtsX, the interaction partner of FtsE, and FtsQ, which is recruited after FtsE, did not localize to the ring, suggesting that the decrease in FtsE is a cause of the filamentous morphology. Finally, a reconstituted cell-free translation system revealed that the folding of newly translated FtsE was stringently dependent on GroEL/GroES. Based on these findings, we concluded that FtsE is a target substrate of the GroE system in E. coli cell division. [Abstract/Link to Full Text]

Awano N, Xu C, Ke H, Inoue K, Inouye M, Phadtare S
Complementation analysis of the cold-sensitive phenotype of the Escherichia coli csdA deletion strain.
J Bacteriol. 2007 Aug;189(16):5808-15.
The cold shock response of Escherichia coli is elicited by downshift of temperature from 37 degrees C to 15 degrees C and is characterized by induction of several cold shock proteins, including CsdA, during the acclimation phase. CsdA, a DEAD-box protein, has been proposed to participate in a variety of processes, such as ribosome biogenesis, mRNA decay, translation initiation, and gene regulation. It is not clear which of the functions of CsdA play a role in its essential cold shock function or whether all do, and so far no protein has been shown to complement its function in vivo. Our screening of an E. coli genomic library for an in vivo counterpart of CsdA that can compensate for its absence at low temperature revealed only one protein, RhlE, another DEAD-box RNA helicase. We also observed that although not detected in our genetic screening, two cold shock-inducible proteins, namely, CspA, an RNA chaperone, and RNase R, an exonuclease, can also complement the cold shock function of CsdA. Interestingly, the absence of CsdA and RNase R leads to increased sensitivity of the cells to even moderate temperature downshifts. The correlation between the helicase activity of CsdA and the stability of mRNAs of cold-inducible genes was shown using cspA mRNA, which was significantly stabilized in the DeltacsdA cells, an effect counteracted by overexpression of wild-type CsdA or RNase R but not by that of the helicase-deficient mutant of CsdA. These results suggest that the primary role of CsdA in cold acclimation of cells is in mRNA decay and that its helicase activity is pivotal for promoting degradation of mRNAs stabilized at low temperature. [Abstract/Link to Full Text]

Cecchini KR, Gorton TS, Geary SJ
Transcriptional responses of Mycoplasma gallisepticum strain R in association with eukaryotic cells.
J Bacteriol. 2007 Aug;189(16):5803-7.
Mycoplasma gallisepticum is an etiologic agent of chronic respiratory disease in chickens and infectious sinusitis in turkeys. Other than proteins important for cytadherence, few M. gallisepticum factors or pathways contributing to host cell interactions have been identified. In this study, an oligonucleotide-based microarray was utilized to investigate transcriptional changes in M. gallisepticum strain R(low) upon exposure to eukaryotic cells. Fifty-eight genes were either up- or downregulated upon exposure to MRC-5 lung fibroblasts grown in vitro, including genes encoding transport-, metabolism-, and translation-associated proteins. Twenty of the 58 regulated genes have no assigned function. These results indicate that M. gallisepticum regulates gene expression upon exposure to eukaryotic cells, revealing genes and pathways likely to be important for host-bacterium interaction. [Abstract/Link to Full Text]

Kawasaki T, Nagata S, Fujiwara A, Satsuma H, Fujie M, Usami S, Yamada T
Genomic characterization of the filamentous integrative bacteriophages {phi}RSS1 and {phi}RSM1, which infect Ralstonia solanacearum.
J Bacteriol. 2007 Aug;189(16):5792-802.
The genomic DNA sequences were determined for two filamentous integrative bacteriophages, phiRSS1 and phiRSM1, of the phytopathogen Ralstonia solanacearum. The 6,662-base sequence of phiRSS1 contained 11 open reading frames (ORFs). In the databases, this sequence showed high homology (95% identity) to the circular double-stranded DNA plasmid pJTPS1 (6,633 bp) isolated from a spontaneously occurring avirulent mutant of R. solanacearum. Two major differences between the two sequences were observed within phiRSS1 ORF7, corresponding to pIII, a minor coat protein required for host adsorption, and at the phiRSS1 intergenic (IG) region. The 9,004-base sequence of phiRSM1 showed 12 ORFs located on the same strand (plus strand) and 2 ORFs on the opposite strand. Compared with Ff-type phages, two insertions are obvious in the phiRSM1 replication module. Genomic DNA fragments containing the phiRSM integration junctions were cloned and sequenced from phiRSM lysogenic strain R. solanacearum MAFF211270. The att core sequence was identified as 5'-TGGCGGAGAGGGT-3', corresponding to the 3' end of the serine tRNA (UCG) gene. Interestingly, ORF14, located next to the attP site on the phiRSM1 genome, showed high amino acid sequence homology with bacterial DNA recombinases and resolvases, different from XerCD recombinases. attP of phiRSS1 is within a sequence element of the IG region. [Abstract/Link to Full Text]

de Jonge MI, Pehau-Arnaudet G, Fretz MM, Romain F, Bottai D, Brodin P, Honoré N, Marchal G, Jiskoot W, England P, Cole ST, Brosch R
ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity.
J Bacteriol. 2007 Aug;189(16):6028-34.
The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol. Using flotation gradient centrifugation, we demonstrate that ESAT-6 showed strong association with liposomes, and in particular with preparations containing DMPC and cholesterol, whereas the interaction of CFP-10 with membranes appeared to be weaker and less specific. Most importantly, binding to the biomembranes no longer occurred when the proteins were present as a 1:1 ESAT-6.CFP-10 complex. However, lowering of the pH resulted in dissociation of the protein complex and subsequent protein-liposome interaction. Finally, cryoelectron microscopy revealed that ESAT-6 destabilized and lysed liposomes, whereas CFP-10 did not. In conclusion, we propose that one of the main features of ESAT-6 in the infection process of M. tuberculosis is the interaction with biomembranes that occurs after dissociation from its putative chaperone CFP-10 under acidic conditions typically encountered in the phagosome. [Abstract/Link to Full Text]

Titgemeyer F, Amon J, Parche S, Mahfoud M, Bail J, Schlicht M, Rehm N, Hillmann D, Stephan J, Walter B, Burkovski A, Niederweis M
A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis.
J Bacteriol. 2007 Aug;189(16):5903-15.
We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively. [Abstract/Link to Full Text]

Barré O, Mourlane F, Solioz M
Copper induction of lactate oxidase of Lactococcus lactis: a novel metal stress response.
J Bacteriol. 2007 Aug;189(16):5947-54.
Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 muM copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5' region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Clinical Microbiology

---

Fritsche TR, McDermott PF, Shryock TR, Walker RD, Morishita TY
Agar dilution and disk diffusion susceptibility testing of Campylobacter spp.
J Clin Microbiol. 2007 Aug;45(8):2758-9; author reply 2759. [Abstract/Link to Full Text]

Luangtongkum T, Morishita TY, El-Tayeb AB, Ison AJ, Zhang Q
Comparison of antimicrobial susceptibility testing of Campylobacter spp. by the agar dilution and the agar disk diffusion methods.
J Clin Microbiol. 2007 Feb;45(2):590-4.
The correlation and the level of agreement between the standardized agar dilution and the agar disk diffusion methods for antimicrobial susceptibility testing of Campylobacter were investigated. A high-level agreement between the two methods was evident for aminoglycosides and fluoroquinolones, while a low-level agreement was observed for other antibiotics. [Abstract/Link to Full Text]

Makristathis A, Hirschl AM, Rüssmann H, Koletzko S
Detection and clarithromycin susceptibility testing of Helicobacter pylori in stool specimens by real-time PCR: how to get accurate test results.
J Clin Microbiol. 2007 Aug;45(8):2756; author reply 2756-7. [Abstract/Link to Full Text]

Lottspeich C, Schwarzer A, Panthel K, Koletzko S, Rüssmann H
Evaluation of the novel Helicobacter pylori ClariRes real-time PCR assay for detection and clarithromycin susceptibility testing of H. pylori in stool specimens from symptomatic children.
J Clin Microbiol. 2007 Jun;45(6):1718-22.
The aim of the present study was to evaluate the Helicobacter pylori ClariRes assay (Ingenetix, Vienna, Austria) for the detection of H. pylori infection and the simultaneous clarithromycin susceptibility testing of the H. pylori isolates in stool samples from 100 symptomatic children. The results obtained by this novel biprobe real-time PCR method were directly compared with the results obtained from histological examination of gastric biopsy specimens, culturing, the [13C]urea breath test, and a monoclonal antibody-based stool antigen enzyme immunoassay (EIA). Fecal specimens from all 54 children who were shown to be noninfected by "gold standard" tests gave true-negative PCR results (specificity, 100%). Of the remaining 46 individuals with a positive H. pylori status, 29 were found to be positive by real-time PCR (sensitivity, 63%). For these 29 cases, the H. pylori ClariRes assay confirmed all results from phenotypic clarithromycin susceptibility testing by Etest. In summary, this investigation demonstrates that detection of Helicobacter DNA in stool samples by real-time PCR is a difficult task and that this method cannot replace the stool antigen EIA (sensitivity, 95.7%) for the accurate diagnosis of H. pylori infection in children. [Abstract/Link to Full Text]

Gulati BR, Deepa R, Singh BK, Rao CD
Diversity in Indian equine rotaviruses: identification of genotype G10,P6[1] and G1 strains and a new VP7 genotype (G16) strain in specimens from diarrheic foals in India.
J Clin Microbiol. 2007 Jul;45(7):2354. [Abstract/Link to Full Text]

Gulati BR, Deepa R, Singh BK, Rao CD
Diversity in Indian equine rotaviruses: identification of genotype G10,P6[1] and G1 strains and a new VP7 genotype (G16) strain in specimens from diarrheic foals in India.
J Clin Microbiol. 2007 Mar;45(3):972-8.
Rotaviruses causing severe diarrhea in foals in two organized farms in northern India, during the period from 2003 to 2005, were characterized by electropherotyping, serotyping, and sequence analysis of the genes encoding the outer capsid proteins. Of 137 specimens, 47 (34.31%) were positive for rotavirus and exhibited at least five different electropherotypes (E), E1 to E5. Strains belonging to different electropherotypes exhibited either a different serotype/genotype specificity or a lack of reactivity to typing monoclonal antibodies (MAbs) used in this study. Strains belonging to E1, E2, and E5 exhibited genotype G10,P6[1], G3, and G1 specificities and accounted for 19.0, 42.9, and 9.5% of the isolates, respectively. Though they possessed G10-type VP7, the E1 strains exhibited high reactivity with the G6-specific MAb, suggesting that the uncommon combination of the outer capsid proteins altered the specificity of the conformation-dependent antigenic epitopes on VP7. E3 and E4 strains accounted for 28.6% of the isolates and were untypeable. Sequence analysis of VP7 from E4 strains (Erv92 and Erv99) revealed that they represent a new VP7 genotype, G16. The detection of unexpected bovine rotavirus-derived G10,P6[1] reassortants, G1 serotype strains, and a new genotype (G16) strain in two distant farms reveals an interesting epidemiological situation and diversity of equine rotaviruses in India. [Abstract/Link to Full Text]

Weitzel T, Reither K, Mockenhaupt FP, Stark K, Ignatius R, Saad E, Seidu-Korkor A, Bienzle U, Schreier E
Field evaluation of a rota- and adenovirus immunochromatographic assay using stool samples from children with acute diarrhea in Ghana.
J Clin Microbiol. 2007 Aug;45(8):2695-7.
We evaluated the Rida Quick rotavirus/adenovirus Combi rapid immunochromatographic test (ICT) under field conditions with Ghanaian children with acute diarrhea. Compared to PCR results, sensitivities and specificities were 75% and 95% for rotavirus and 22% and 84% for adenovirus. In resource-poor settings, ICTs may help to overcome difficulties in the diagnosis of rotavirus infection. [Abstract/Link to Full Text]

Su HP, Yang HW, Chen YL, Ferng TL, Chou YL, Chung TC, Chen CH, Chiang CS, Kuan MM, Lin HH, Chen YS
Prevalence of melioidosis in the Er-Ren River Basin, Taiwan: implications for transmission.
J Clin Microbiol. 2007 Aug;45(8):2599-603.
An increase in melioidosis cases compared to other areas in Taiwan was observed in the Er-Ren River Basin, southwestern Taiwan, from November 2001 to August 2006. The objective of this study was to determine the association between the level of exposure to Burkholderia pseudomallei and the incidence rate of melioidosis and to survey the transmission modes of B. pseudomallei in the Er-Ren River Basin. The serosurveillance of melioidosis gave seropositivity rates of 36.6%, 21.6%, and 10.9%, respectively, for residents in regions A, B, and C within the Er-Ren Basin area. Culture and PCR-based detection of B. pseudomallei from soil demonstrated that the geographical distribution of this bacterium was confined to a particular site in region B. The distribution of seropositive titers was significantly associated with the incidence rate of melioidosis (120, 68, or 36 incidence cases per 100,000 population in region A, B, or C in 2005), whereas it did not correlate with the geographical distribution of B. pseudomallei within the soil. A survey of transmission modes showed that residents with seropositivity were linked to factors such as having confronted flooding and having walked barefoot on soil, which are potential risk factors associated with exposure to B. pseudomallei. Our findings indicated that the Er-Ren River Basin in Taiwan has the potential to become a high-prevalence area for melioidosis. This is the first report that documents a high prevalence of melioidosis in an area north of latitude 20 degrees N. [Abstract/Link to Full Text]

Solnik-Isaac H, Weinberger M, Tabak M, Ben-David A, Shachar D, Yaron S
Quinolone resistance of Salmonella enterica serovar Virchow isolates from humans and poultry in Israel: evidence for clonal expansion.
J Clin Microbiol. 2007 Aug;45(8):2575-9.
Salmonella enterica serovar Virchow is highly prevalent in humans and farm animals in Israel. In addition to high rates of resistance to multiple antibiotics, this serovar exhibits a high incidence of resistance to nalidixic acid. More than 90% of Salmonella serovar Virchow isolates of human and poultry origin obtained from 1997 to 2004 were resistant to nalidixic acid (MIC > or = 128 microg/ml), with reduced susceptibility to ciprofloxacin (MIC between 0.125 and 0.250 microg/ml). Most isolates belonged to two predominant, closely related pulsed-field gel electrophoresis image types. Investigation of the mechanisms of quinolone resistance revealed that this pathogen probably emerged from a parental clone that overproduced the AcrAB efflux pump and had a single point mutation in gyrA leading to the Asp87Tyr substitution. The close resemblance between human and poultry isolates points to poultry as a likely source of Salmonella serovar Virchow in the food chain. [Abstract/Link to Full Text]

Thomson KS, Cornish NE, Hong SG, Hemrick K, Herdt C, Moland ES
Comparison of Phoenix and VITEK 2 extended-spectrum-beta-lactamase detection tests for analysis of Escherichia coli and Klebsiella isolates with well-characterized beta-lactamases.
J Clin Microbiol. 2007 Aug;45(8):2380-4.
The VITEK 2 and Phoenix extended-spectrum beta-lactamase (ESBL) detection systems, which comprise confirmatory tests and expert systems, were evaluated for their ability to discriminate between 102 well-characterized strains of ESBL-positive or -negative Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca. At least 38 distinct ESBLs were included. The strains were chosen to include some known to cause false-positive and false-negative CLSI ESBL confirmatory test results. Therefore, enzyme characterizations, rather than CLSI tests, were the reference methods for the Phoenix and VITEK 2 evaluations. A third arm of the study was conducted with the Phoenix test using two normally inactive expert rules intended to enhance ESBL detection, in addition to using the currently available software. The Phoenix ESBL confirmatory test and unmodified expert system exhibited 96% sensitivity and 81% specificity for ESBL detection. Activation of the two additional rules increased sensitivity to 99% but reduced the specificity to 58%. The VITEK 2 ESBL confirmatory test exhibited 91% sensitivity, which was reduced to 89% sensitivity by its expert system, while its specificity was 85%. Many of the expert system interpretations of both instruments were helpful, but some were suboptimal. The VITEK 2 expert system was potentially more frustrating because it provided more inconclusive interpretations of the results. Considering the high degree of diagnostic difficulty posed by the strains, both ESBL confirmatory tests were highly sensitive. The expert systems of both instruments require modification to update and enhance their utility. [Abstract/Link to Full Text]

Vinasco J, Li O, Alvarado A, Diaz D, Hoyos L, Tabachi L, Sirigireddy K, Ferguson C, Moro MH
Molecular evidence of a new strain of Ehrlichia canis from South America.
J Clin Microbiol. 2007 Aug;45(8):2716-9.
Blood samples from dogs with clinical signs compatible with ehrlichiosis were examined for infection of Ehrlichia canis using PCR, multiplex real-time PCR, and DNA sequencing analysis. Eleven of 25 samples were positive for a new strain of E. canis. This is the first molecular identification of E. canis infection in dogs from Peru. [Abstract/Link to Full Text]

Cai Y, Kong F, Gilbert GL
Three new macrolide efflux (mef) gene variants in Streptococcus agalactiae.
J Clin Microbiol. 2007 Aug;45(8):2754-5. [Abstract/Link to Full Text]

Grohs P, Mainardi JL, Podglajen I, Hanras X, Eckert C, Buu-Hoï A, Varon E, Gutmann L
Relevance of routine use of the anaerobic blood culture bottle.
J Clin Microbiol. 2007 Aug;45(8):2711-5.
Using the BacT/Alert automated system, we conducted a 1-year retrospective study on blood cultures, focusing on the relevance of routine use of the anaerobic bottle. The rate of patients with positive blood cultures was 19.7%. Among these, 13.5% had a positive anaerobic bottle in the absence of any aerobic bottle, and 2/3 of these grew with nonobligate anaerobes. These patients were hospitalized in 20 out of 26 wards of the hospital group. For 65.4% of the monomicrobial-positive blood cultures growing Enterobacteriaceae, the anaerobic bottle detected growth earlier than the corresponding aerobic bottle. These data suggest that, in our institution, the use of an anaerobic bottle is still relevant. [Abstract/Link to Full Text]

Anderson KF, Lonsway DR, Rasheed JK, Biddle J, Jensen B, McDougal LK, Carey RB, Thompson A, Stocker S, Limbago B, Patel JB
Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae.
J Clin Microbiol. 2007 Aug;45(8):2723-5.
The Klebsiella pneumoniae carbapenem (KPC) beta-lactamase occurs in Enterobacteriaceae and can confer resistance to all beta-lactam agents including carbapenems. The enzyme may confer low-level carbapenem resistance, and the failure of susceptibility methods to identify this resistance has been reported. Automated and nonautomated methods for carbapenem susceptibility were evaluated for identification of KPC-mediated resistance. Ertapenem was a more sensitive indicator of KPC resistance than meropenem and imipenem independently of the method used. Carbapenemase production could be confirmed with the modified Hodge test. [Abstract/Link to Full Text]

Gnanashanmugam D, Falkovitz-Halpern MS, Dodge A, Fang M, Wong LJ, Esparza M, Hammon R, Rivas-Merelles EE, Santos JI, Maldonado Y
Shedding and reversion of oral polio vaccine type 3 in Mexican vaccinees: comparison of mutant analysis by PCR and enzyme cleavage to a real-time PCR assay.
J Clin Microbiol. 2007 Aug;45(8):2419-25.
A uracil-to-cytosine mutation at nucleotide position 472 of oral poliovirus vaccine type 3 (OPV3) contributes to the development of vaccine-associated paralytic poliomyelitis (VAPP). To analyze OPV3 shedding patterns, we previously used the multistep method of mutant analysis by PCR and enzyme cleavage (MAPREC). This involves conventional reverse transcription-PCR to detect OPV3, followed by a restriction digest to quantify position 472 reversion. Real-time PCR detects and quantifies nucleic acid as PCR occurs and avoids postreaction processing. The goal of this study was to compare a real-time PCR method to MAPREC. Seventy-three stool samples from Mexican OPV recipients underwent the reverse transcription-PCR step of MAPREC and real-time PCR. Real-time PCR identified 23% more OPV3-positive samples than conventional reverse transcription-PCR. When reversion was compared, the revertant proportion (RP), defined as the percentage of revertants in a sample, differed by < or =10% in 21/25 (84%) samples. The four samples differing by >10% were obtained within 5 days of OPV administration. The real-time PCR assay identified samples with an RP of > or =85% with 94% sensitivity and 86% specificity compared to MAPREC. The mean difference in RP between the two methods was 3.6% (95% confidence interval, -0.3 to 7.5%). Real-time PCR methods reliably detect OPV3, and reversion estimates correlate more consistently with MAPREC when OPV3 reversion rates are high. Detecting VAPP-related mutations by real-time PCR is rapid and efficient and can be useful in monitoring ongoing global polio eradication efforts. [Abstract/Link to Full Text]

Grüner BM, Han SR, Meyer HG, Wulf U, Bhakdi S, Siegel EK
Characterization of a catalase-negative methicillin-resistant Staphylococcus aureus strain.
J Clin Microbiol. 2007 Aug;45(8):2684-5.
We describe an unusual clinical strain of catalase-negative methicillin-resistant Staphylococcus aureus sensu stricto. Sequence analysis of its catalase gene showed 99.60% identities to the catalase genes of the reference strains. A 5-base deletion, however, led to a shift of the nucleotide reading frame and a loss of the enzymatic activity. [Abstract/Link to Full Text]

Gheit T, Billoud G, de Koning MN, Gemignani F, Forslund O, Sylla BS, Vaccarella S, Franceschi S, Landi S, Quint WG, Canzian F, Tommasino M
Development of a sensitive and specific multiplex PCR method combined with DNA microarray primer extension to detect Betapapillomavirus types.
J Clin Microbiol. 2007 Aug;45(8):2537-44.
Emerging lines of evidence indicate that the cutaneous human papillomavirus (HPV) types that belong to the genus Betapapillomavirus (beta HPV) are involved in the development of nonmelanoma skin cancer. Unlike the situation for mucosal HPV types, highly sensitive and reliable methods to identify characterized cutaneous HPV types in a single assay are limited. Here, we describe a novel one-shot method for the detection of all characterized beta HPV types, namely, HPV type 5 (HPV5), 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96. This assay combines two different techniques: multiplex PCR using HPV type-specific primers for amplification of each E7 gene and array primer extension (APEX) for typing. This method has been validated using clinical samples which were analyzed simultaneously for the presence of cutaneous HPV types by two additional methods, i.e., the FAP59/64 PCR protocol and a commercially available PCR-reverse hybridization assay (PM-PCR RHA). Our data show good agreement between the results obtained with the multiplex PCR/APEX assay and the PM-PCR RHA method (overall HPV positivity of 92.2% for multiplex PCR/APEX assay versus 90.6% with the PM-PCR RHA) (kappa value, 50; 95% confidence interval, 13 to 88). In addition, the multiplex PCR/APEX assay showed higher sensitivity than the PM-PCR RHA did. This favorable feature and the high-throughput potential make this assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of cutaneous types in skin cancer. [Abstract/Link to Full Text]

Shin JH, Chae MJ, Song JW, Jung SI, Cho D, Kee SJ, Kim SH, Shin MG, Suh SP, Ryang DW
Changes in karyotype and azole susceptibility of sequential bloodstream isolates from patients with Candida glabrata candidemia.
J Clin Microbiol. 2007 Aug;45(8):2385-91.
We examined the changes in genotypes and azole susceptibilities among sequential bloodstream isolates of Candida glabrata during the course of fungemia and the relationship of these changes to antifungal therapy. Forty-one isolates were obtained from 15 patients (9 patients who received antifungal therapy and 6 patients who did not) over periods of up to 36 days. The isolates were analyzed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and tested for antifungal susceptibility to fluconazole, itraconazole, and voriconazole. PFGE typing consisted of electrophoretic karyotyping and restriction endonuclease analysis of genomic DNA by use of NotI (REAG-N). The 41 isolates yielded 23 different karyotypes and 11 different REAG-N patterns but only 3 MLST types. The sequential strains from each patient had identical or similar REAG-N patterns. However, they had two or three different karyotypes in 6 (40%) of 15 patients. The isolates from these six patients exhibited the same or similar azole susceptibilities, and five patients did not receive antifungal therapy. Development of acquired azole resistance in sequential isolates was detected for only one patient. For this patient, an isolate of the same genotype obtained after azole therapy showed three- or fourfold increases in the MICs of all three azole antifungals and exhibited increased expression of the CgCDR1 efflux pump. This study shows that karyotypic changes can develop rapidly among sequential bloodstream strains of C. glabrata from the same patient without antifungal therapy. In addition, we confirmed that C. glabrata could acquire azole resistance during the course of fungemia in association with azole therapy. [Abstract/Link to Full Text]

van Belkum A, Niesters HG, MacKay WG, van Leeuwen WB
Quality control of direct molecular diagnostics for methicillin-resistant Staphylococcus aureus.
J Clin Microbiol. 2007 Aug;45(8):2698-700.
Ten samples containing various amounts of methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S. aureus, methicillin-resistant Staphylococcus epidermidis (MRSE), and combinations thereof were distributed to 51 laboratories for molecular diagnostics testing. Samples containing 10(2) to 10(3) MRSA cells were frequently reported to be negative. MRSE samples were scored as negative by all commercial tests but by only two out of three in-house tests. [Abstract/Link to Full Text]

Rossney AS, Shore AC, Morgan PM, Fitzgibbon MM, O'Connell B, Coleman DC
The emergence and importation of diverse genotypes of methicillin-resistant Staphylococcus aureus (MRSA) harboring the Panton-Valentine leukocidin gene (pvl) reveal that pvl is a poor marker for community-acquired MRSA strains in Ireland.
J Clin Microbiol. 2007 Aug;45(8):2554-63.
Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) carrying pvl is an emerging problem worldwide. CA-MRSA tends to harbor staphylococcal cassette chromosome mec type IV (SCCmec IV), to be non-multiantibiotic resistant, and to have different genotypes from the local hospital-acquired MRSA (HA-MRSA). However, in Ireland, 80% of HA-MRSA isolates have the non-multiantibiotic-resistant genotype ST22-MRSA-IV. This study investigated MRSA isolates from Ireland (CA-MRSA, health care-associated MRSA, and HA-MRSA) for the carriage of pvl and determined the genotypic characteristics of all pvl-positive isolates identified. All 1,389 MRSA isolates were investigated by antibiogram-resistogram typing and SmaI DNA macrorestriction analysis. pvl-positive isolates were further characterized by multilocus sequence typing and SCCmec, agr, and toxin gene typing. Twenty-five (1.8%) MRSA isolates belonging to six genotypes (ST30, ST8, ST22, ST80, ST5, and ST154) harbored pvl. Nineteen of these (76%) were CA-MRSA isolates, but a prospective study of MRSA isolates from 401 patients showed that only 6.7% (2/30) of patients with CA-MRSA yielded pvl-positive isolates. Thus, pvl cannot be used as a sole marker for CA-MRSA. Fifty-two percent of pvl-positive MRSA isolates were recovered from patients with skin and soft tissue infections; thirty-six percent were from patients of non-Irish ethnic origin, reflecting the increasing heterogeneity of the Irish population due to immigration. All 25 pvl-positive isolates carried SCCmec IV; 14 (56%) harbored SCCmec IV.1 or IV.3, and the remaining 11 isolates could not be subtyped. This study demonstrates that pvl is not a reliable marker for CA-MRSA in Ireland and reveals the emergence and importation of diverse genotypes of pvl-positive MRSA in Ireland. [Abstract/Link to Full Text]

Lindh M, Alestig E, Arnholm B, Eilard A, Hellstrand K, Lagging M, Wahlberg T, Wejstål R, Westin J, Norkrans G
Response prediction and treatment tailoring for chronic hepatitis C virus genotype 1 infection.
J Clin Microbiol. 2007 Aug;45(8):2439-45.
We monitored early viral response during the treatment of hepatitis C virus (HCV) infection with the aim of identifying predictors of treatment outcome. We studied 53 patients with genotype 1 infection who received 180 microg/week pegylated interferon alfa-2a and 1,000 or 1,200 mg/day ribavirin depending on body weight and serially assessed HCV RNA in serum, using the Cobas TaqMan assay. Thirty-one patients (58%) achieved sustained viral response (SVR). SVR was obtained in 100% (10/10) of patients with pretreatment viremia concentrations below 400,000 IU/ml, in 100% (14/14) of patients with more than 1.5 log reduction of HCV RNA after 4 days of treatment, and in 95% (22/23) of patients with a rate of decline in viremia higher than 0.70 log units/week during the second phase. Non-SVR was seen in all patients with a second-phase decline rate lower than 0.35 log units/week. Patients with slopes between 0.50 and 0.80 log units/week achieved SVR (4/4) unless the treatment dose was modified (3/3). We conclude that the second-phase slope appears to be an accurate and useful predictor of treatment response. On the basis of these findings, we propose a model of tailored treatment which takes into account the second-phase slope and the amount of HCV RNA after 21 days of treatment. [Abstract/Link to Full Text]

Manzur A, Tubau F, Pujol M, Calatayud L, Dominguez MA, Peña C, Sora M, Gudiol F, Ariza J
Nosocomial outbreak due to extended-spectrum-beta-lactamase- producing Enterobacter cloacae in a cardiothoracic intensive care unit.
J Clin Microbiol. 2007 Aug;45(8):2365-9.
Enterobacter cloacae has been associated with several outbreaks, usually involving strains that overproduce chromosomal beta-lactamase or, uncommonly, strains expressing extended-spectrum beta-lactamases (ESBL). Only sporadic cases of ESBL-producing E. cloacae have been identified in our hospital in recent years. We describe the epidemiology and clinical and microbiological characteristics of an outbreak caused by ESBL-producing E. cloacae in a cardiothoracic intensive care unit (CT-ICU). Prospective surveillance of patients with infection or colonization by ESBL-producing E. cloacae among patients admitted to the CT-ICU was performed during the outbreak. Production of ESBL was determined by decreased susceptibility to expanded-spectrum cephalosporins and a positive double-disk test result. Clone relatedness was determined by pulsed-field gel electrophoresis (PFGE). From July to September 2005, seven patients in the CT-ICU with ESBL-producing E. cloacae were identified (four males; median age, 73 years; range, 45 to 76 years); six patients had cardiac surgery. Four patients developed infections; three had primary bacteremia, one had ventilator-associated pneumonia, and one had tracheobronchitis. ESBL-producing E. cloacae showed resistance to quinolones and aminoglycosides. PFGE revealed two patterns. Five isolates belonged to clone A; two carried a single ESBL (pI 8.2 and a positive PCR result for the SHV type), and three carried two ESBLs (pIs 8.1 and 8.2 and positive PCR results for the SHV and CTX-M-9 types). Isolates belonging to clone B carried a single ESBL (pI 5.4 and a positive PCR result for the TEM type). Review of antibiotic consumption showed increased use of cefepime and quinolones during June and July 2005. The outbreak was stopped by the implementation of barrier measures and cephalosporin restriction. ESBL production could be increasingly common in nosocomial pathogens other than Escherichia coli or Klebsiella pneumoniae. [Abstract/Link to Full Text]

Chernesky M, Freund GG, Hook E, Leone P, D'Ascoli P, Martens M
Detection of Chlamydia trachomatis and Neisseria gonorrhoeae infections in North American women by testing SurePath liquid-based Pap specimens in APTIMA assays.
J Clin Microbiol. 2007 Aug;45(8):2434-8.
The APTIMA COMBO 2 assay, which detects and amplifies rRNA from Chlamydia trachomatis and/or Neisseria gonorrhoeae, is approved for use on ThinPrep liquid-based Pap test specimens. The objective was to determine the clinical utility of the APTIMA assays (APTIMA COMBO 2 assay, APTIMA CT assay for Chlamydia trachomatis, and APTIMA GC assay for Neisseria gonorrhoeae) for screening women during their annual Pap exam, using SurePath liquid-based Pap test specimens. Two cervical samples were collected from 1,615 females attending six clinical sites in North America. A cervical broom sample was processed for cytology, with the residuum aliquoted into an APTIMA specimen transfer kit tube. The second cervical swab sample was put into APTIMA specimen transport medium, and both samples were tested with each APTIMA assay on a direct sampling system. Using a subject-infected status that utilized cervical-swab specimen results from two APTIMA assays, the prevalence was 7.9% for Chlamydia trachomatis and 2.5% for N. gonorrhoeae. For the liquid-based Pap samples, the sensitivities, specificities, positive predictive values, and negative predictive values for Chlamydia trachomatis detection were 85.2%, 99.5%, 93.2%, and 98.7%, respectively, for the APTIMA COMBO 2 assay and 89.1%, 98.7%, 85.7%, and 99.1%, respectively, for the APTIMA CT assay. For N. gonorrhoeae detection, the values were 92.5%, 100%, 100%, and 99.8%, respectively, for the APTIMA COMBO 2 assay and 92.5%, 99.9%, 97.4%, and 99.8%, respectively, for the APTIMA GC assay. The high predictive values support the use of the assays with SurePath liquid-based Pap specimens processed with the APTIMA specimen transfer kit. [Abstract/Link to Full Text]

Lass-Flörl C, Grif K, Kontoyiannis DP
Molecular typing of Aspergillus terreus isolates collected in Houston, Texas, and Innsbruck, Austria: evidence of great genetic diversity.
J Clin Microbiol. 2007 Aug;45(8):2686-90.
Aspergillus terreus isolates collected from patients at The M. D. Anderson Cancer Center in Houston, TX, and at The University Hospital of Innsbruck, Austria, were analyzed using random amplification of polymorphic DNA-PCR with three different primers. No strain similarity in either institution was detected, indicating great genetic diversity of A. terreus. [Abstract/Link to Full Text]

Han ET, Watanabe R, Sattabongkot J, Khuntirat B, Sirichaisinthop J, Iriko H, Jin L, Takeo S, Tsuboi T
Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis.
J Clin Microbiol. 2007 Aug;45(8):2521-8.
Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic. [Abstract/Link to Full Text]

Brady JM, Stemper ME, Weigel A, Chyou PH, Reed KD, Shukla SK
Sporadic "transitional" community-associated methicillin-resistant Staphylococcus aureus strains from health care facilities in the United States.
J Clin Microbiol. 2007 Aug;45(8):2654-61.
We describe phenotypic and genotypic traits of a group of methicillin-resistant Staphylococcus aureus (MRSA) clones that are either remnants of unsuccessful community-associated MRSA (CA-MRSA) clones or represent a transitional state with some yet-to-be-acquired characteristics of CA-MRSA. These rare strains (n = 20) were identified during a 10-year period (1990-1999) from 13 unrelated health care facilities in Wisconsin. The isolates were recovered from patients in nosocomial or long-term chronic care facilities (60%) and outpatient settings (40%). Sixty percent (n = 12) of the isolates were recovered from skin and soft tissue infections, whereas the remaining isolates (n = 8) were from invasive infections. Ninety percent of isolates were susceptible to all antibiotic classes tested or resistant to erythromycin and clindamycin. Pulsed-field gel electrophoresis, multilocus sequence typing, and spa typing clustered these isolates into 8, 8, and 14 clonal groups, respectively. Eight plasmid profiles were represented in these strains. All four agr types were represented, with type IV being predominant (40%). All strains harbored subtypes of type IV staphylococcal cassette chromosome mec but lacked genes for the virulence factor Panton-Valentine leukocidin (PVL). The strains harbored one or more of the following toxin genes: sea, seb, sec, sed, see, seh, sej, sek, sel, seg, sei, sem, sen, and seo. Individual clonal groups maintained the same set of enterotoxin genes even though they were isolated over extended time periods, suggesting significant genomic stability. The potential role of PVL-carrying phages and plasmids in the success of CA-MRSA clones has been discussed. [Abstract/Link to Full Text]

Pimentel JD, Chan RC
Desulfovibrio fairfieldensis bacteremia associated with choledocholithiasis and endoscopic retrograde cholangiopancreatography.
J Clin Microbiol. 2007 Aug;45(8):2747-50.
Desulfovibrio fairfieldensis is a gram-negative, curved, motile, anaerobic bacillus. D. fairfieldensis has been isolated only from human specimens and is considered a normal resident of the human gastrointestinal tract. We report the second case of Desulfovibrio bacteremia associated with choledocholithiasis and review the other reported cases of D. fairfieldensis bacteremia. [Abstract/Link to Full Text]

Musher DM, Manhas A, Jain P, Nuila F, Waqar A, Logan N, Marino B, Graviss EA
Detection of Clostridium difficile toxin: comparison of enzyme immunoassay results with results obtained by cytotoxicity assay.
J Clin Microbiol. 2007 Aug;45(8):2737-9.
Several kinds of laboratory techniques are available to detect Clostridium difficile toxin in fecal samples. Because questions have been raised about the reliability of immunoassays compared to the accepted standard, cytotoxicity assay, we studied three enzyme immunoassays (EIAs) and one rapid EIA, which demonstrated relatively good sensitivities and specificities compared to cytotoxicity assay. [Abstract/Link to Full Text]

Jalal H, Stephen H, Alexander S, Carne C, Sonnex C
Development of real-time PCR assays for genotyping of Chlamydia trachomatis.
J Clin Microbiol. 2007 Aug;45(8):2649-53.
We have developed and validated a nested real-time PCR (NRT-PCR) for the genotyping of Chlamydia trachomatis and used it specifically for the typing of either eight genovars from D to K or three genovars of lymphogranuloma venereum (LGV). The 11 probes used in the NRT-PCR correctly identified the DNA from D to K and LGV reference strains and did not cross-react with the DNA from 26 strains representing the bacterial pathogens and commensals of the oropharynx, genital tract, and rectum. The NRT-PCR had a 95% probability of detection at four genome copies (confidence interval, three to six copies) of C. trachomatis per reaction. One hundred cervical and urethral swab specimens containing C. trachomatis DNA from 63 women and 37 men were used to validate the method. The results from the NRT-PCR and the DNA sequencing of amplicons generated from the omp1 gene showed 100% correlation for these samples. The assay also identified the LGV-II genotype in 24 of 48 rectal swab specimens containing C. trachomatis DNA that were obtained from men having sex with men. The Sexually Transmitted Bacteria Reference Laboratory, London, independently confirmed these results using group-specific LGV real-time PCR and restriction fragment length polymorphism analysis. Compared with the NRT-PCR, non-NRT-PCR was found to be less sensitive: it typed C. trachomatis DNA in only 80% of the genital samples and 90% of the rectal swab samples. This is the first successful demonstration of the use of real-time PCR for the genotype-specific typing of C. trachomatis strains that cause sexually transmitted diseases. [Abstract/Link to Full Text]

Lalonde MS, Troyer RM, Syed AR, Bulime S, Demers K, Bajunirwe F, Arts EJ
Sensitive oligonucleotide ligation assay for low-level detection of nevirapine resistance mutations in human immunodeficiency virus type 1 quasispecies.
J Clin Microbiol. 2007 Aug;45(8):2604-15.
This study has adapted the oligonucleotide ligation assay (OLA) to probe for low-level nevirapine (NVP) resistance mutations K103N and Y181C in the human immunodeficiency virus type 1 (HIV-1) population of infected mother-infant pairs from Uganda. When NVP is used to prevent perinatal transmission, NVP-resistant HIV-1 clones may be rapidly selected due to a low barrier for mutation and a relatively high level of fitness (compared to that of other drug-resistant HIV-1 clones). Monitoring for even a low frequency of NVP resistance mutations may help predict the success of subsequent treatment or warrant the use of another regimen to prevent transmission in a subsequent pregnancy. The standard OLA was optimized by using nonstandard bases in oligonucleotides to allow promiscuous base pairing and accommodate significant HIV-1 heterogeneity. Radiolabeled as opposed to fluorescently tagged oligonucleotides increased the sensitivity, whereas alteration of the template, oligonucleotides, salt, and thermostable DNA ligase concentrations increased the specificity for the detection of minority codons. This modified OLA is now capable of detecting mutants with the K103N or the Y181C mutation present in an HIV-1 population at a frequency of approximately 0.4% and is at least 10- to 30-fold more sensitive than the original protocol. A cohort of 19 Ugandan mothers who received NVP treatment perinatally were sampled 6 weeks postdelivery. Ten of 19 HIV-1 DNA samples extracted from peripheral blood mononuclear cells had a detectable K103N (0.5 to 44%) or Y181C (0.8 to 92.5%) mutation, but only one plasma HIV-1 RNA sample had a viral population with the Y181C mutation. These findings suggest that OLA is a robust, sensitive, and specific method for the detection of low-frequency drug resistance mutations in an intrapatient HIV-1 population. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Immune Based Therapies and Vaccines

---

Kelly CD, O'Loughlin C, Gelder FB, Peterson JW, Sower LE, Cirino NM
Rapid generation of an anthrax immunotherapeutic from goats using a novel non-toxic muramyl dipeptide adjuvant.
J Immune Based Ther Vaccines. 2007;511.
ABSTRACT: BACKGROUND: There is a clear need for vaccines and therapeutics for potential biological weapons of mass destruction and emerging diseases. Anthrax, caused by the bacterium Bacillus anthracis, has been used as both a biological warfare agent and bioterrorist weapon previously. Although antibiotic therapy is effective in the early stages of anthrax infection, it does not have any effect once exposed individuals become symptomatic due to B. anthracis exotoxin accumulation. The bipartite exotoxins are the major contributing factors to the morbidity and mortality observed in acute anthrax infections. METHODS: Using recombinant B. anthracis protective antigen (PA83), covalently coupled to a novel non-toxic muramyl dipeptide (NT-MDP) derivative we hyper-immunized goats three times over the course of 14 weeks. Goats were plasmapheresed and the IgG fraction (not affinity purified) and F(ab')2 derivatives were characterized in vitro and in vivo for protection against lethal toxin mediated intoxication. RESULTS: Anti-PA83 IgG conferred 100% protection at 7.5 mug in a cell toxin neutralization assay. Mice exposed to 5 LD50 of Bacillus anthracis Ames spores by intranares inoculation demonstrated 60% survival 14 d post-infection when administered a single bolus dose (32 mg/kg body weight) of anti-PA83 IgG at 24 h post spore challenge. Anti-PA83 F(ab')2 fragments retained similar neutralization and protection levels both in vitro and in vivo. CONCLUSION: The protection afforded by these GMP-grade caprine immunotherapeutics post-exposure in the pilot murine model suggests they could be used effectively to treat post-exposure, symptomatic human anthrax patients following a bioterrorism event. These results also indicate that recombinant PA83 coupled to NT-MDP is a potent inducer of neutralizing antibodies and suggest it would be a promising vaccine candidate for anthrax. The ease of production, ease of covalent attachment, and immunostimulatory activity of the NT-MDP indicate it would be a superior adjuvant to alum or other traditional adjuvants in vaccine formulations. [Abstract/Link to Full Text]

Horthongkham N, Srihtrakul T, Athipanyasilp N, Siritantikorn S, Kantakamalakul W, Poovorawan Y, Sutthent R
Specific antibody response of mice after immunization with COS-7 cell derived avian influenza virus (H5N1) recombinant proteins.
J Immune Based Ther Vaccines. 2007 Oct 3;5(1):10.
ABSTRACT: To develop avian influenza H5N1 recombinant protein, the hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural (NS1) of avian influenza H5N1 isolates from Thailand were engineered to be expressed in prokaryotic (E. coli) and mammalian cell (COS-7) system. The plasmid pBAD-His and pSec-His were used as vectors for these inserted genes. Mice immunized with purified recombinant proteins at concentration 50-250 ug intramuscularly with Alum adjuvant at week 0, week 2, and week 3 showed a good immunogenicity measured by ELISA and neutralization assay. The HA and NS recombinant proteins produced in COS-7 cells can induce specific antibody titer detected by neutralization assay significantly higher than corresponding recombinant proteins produced in E.coli system. The antibody produced in immunized mice could neutralize heterologous avian influenza virus determined by micro-neutralization assay. This study shows that avian influenza virus H5N1 recombinant proteins produced in mammalian cell system were able to induce neutralizing antibody response. [Abstract/Link to Full Text]

Pedersen AE, Ronchese F
CTLA-4 blockade during dendritic cell based booster vaccination influences dendritic cell survival and CTL expansion.
J Immune Based Ther Vaccines. 2007;59.
Dendritic cells (DCs) are potent antigen-presenting cells and critical for the priming of CD8+ T cells. Therefore the use of these cells as adjuvant cells has been tested in a large number of experimental and clinical vaccination studies, in particular cancer vaccine studies. A number of protocols are emerging that combine vaccination with CTL expanding strategies, such as e.g. blockade of CTLA-4 signalling. On the other hand, the lifespan and in vivo survival of therapeutic DCs have only been addressed in a few studies, although this is of importance for the kinetics of CTL induction during vaccination. We have previously reported that DCs loaded with specific antigens are eliminated by antigen specific CTLs in vivo and that this elimination affects the potential for in vivo CTL generation. We now show that CTLA-4 blockade increases the number of DC vaccine induced LCMV gp33 specific CTLs and the lysis of relevant in vivo targets. However, the CTLA-4 blockage dependent expansion of CTLs also affect DC survival during booster DC injections and our data suggest that during a booster DC vaccine, the largest increase in CTL levels is already obtained during the first vaccination. [Abstract/Link to Full Text]

Anuradha B, Santosh CM, Hari Sai Priya V, Suman Latha G, Murthy KJ, Vijaya Lakshmi V
Age-related waning of in vitro Interferon-gamma levels against r32kDaBCG in BCG vaccinated children.
J Immune Based Ther Vaccines. 2007;58.
BACKGROUND: Mycobacterium bovis BCG vaccine has displayed inconsistent efficacy in different trials conducted in various geographical regions. Nevertheless, it significantly reduces the risk of severe childhood tuberculosis and continues to be used to prevent tuberculosis in many countries. Many studies revealed that efficacy of vaccine wanes with age. Most of the studies were based on in vivo and in vitro responses to tuberculin. With the advent of newer tests such as in vitro interferon-gamma assays and identification of potent immunogenic mycobacterial proteins there is a need to corroborate the observations. This study aims at ascertaining the need for a booster at a later age as indicated by in vitro release of IFN-gamma while evaluating Ag85A as an antigen. METHODS: Ninety healthy children who were without any clinical evidence of the disease, 45 with a BCG-scar and the remaining 45 without scar and 25 with tuberculosis were included in the study. The incidence of TB was analyzed in 216 children attending a DOTS clinic during 1996-2005. CD3+, CD4+ and CD8+ cell counts were measured by Flow cytometry. r32kDaBCG (Ag85A-BCG) protein was used to stimulate T cells in in vitro T cell responses and interferon-gamma (IFN-gamma) cytokine levels in the supernatants were measured by ELISA. RESULTS: High incidence of TB was observed in age group 13-14 years followed by children in the age group 10-12 years (Chi-square 242.22; p < 0.000). T cell subsets were within the normal range in all subjects. 79% of vaccinated children showed positive proliferative responses with a mean SI value of 4.98 +/- 1.99 while only 39% of the unvaccinated and 58% of the tuberculosis children showed positive responses with mean values of 2.9 +/- 1.6 (p < 0.001) and 2.9 +/- 1.7(p < 0.057), respectively. The stimulation indices in vaccinated children decreased in the older children concurring with an increase in the incidence of TB. CONCLUSION: Significantly high levels of in vitro IFN-gamma demonstrated in BCG vaccinated children in our study substantiate the observation that BCG is effective in children, but the effect may wane with age. The immunity could be boosted using modified r32kDa (Ag85A) of BCG. [Abstract/Link to Full Text]

Ghanekar SA, Bhatia S, Ruitenberg JJ, DeLa Rosa C, Disis ML, Maino VC, Maecker HT, Waters CA
Phenotype and in vitro function of mature MDDC generated from cryopreserved PBMC of cancer patients are equivalent to those from healthy donors.
J Immune Based Ther Vaccines. 2007;57.
BACKGROUND: Monocyte-derived-dendritic-cells (MDDC) are the major DC type used in vaccine-based clinical studies for a variety of cancers. In order to assess whether in vitro differentiated MDDC from cryopreserved PBMC of cancer patients are functionally distinct from those of healthy donors, we compared these cells for their expression of co-stimulatory and functional markers. In addition, the effect of cryopreservation of PBMC precursors on the quality of MDDC was also evaluated using samples from healthy donors. METHODS: Using flow cytometry, we compared normal donors and cancer patients MDDC grown in the presence of GM-CSF+IL-4 (immature MDDC), and GM-CSF+IL-4+TNFalpha+IL-1beta+IL-6+PGE-2 (mature MDDC) for (a) surface phenotype such as CD209, CD83 and CD86, (b) intracellular functional markers such as IL-12 and cyclooxygenase-2 (COX-2), (c) ability to secrete IL-8 and IL-12, and (d) ability to stimulate allogeneic and antigen-specific autologous T cells. RESULTS: Cryopreservation of precursors did affect MDDC marker expression, however, only two markers, CD86 and COX-2, were significantly affected. Mature MDDC from healthy donors and cancer patients up-regulated the expression of CD83, CD86, frequencies of IL-12+ and COX-2+ cells, and secretion of IL-8; and down-regulated CD209 expression relative to their immature counterparts. Compared to healthy donors, mature MDDC generated from cancer patients were equivalent in the expression of nearly all the markers studied and importantly, were equivalent in their ability to stimulate allogeneic and antigen-specific T cells in vitro. CONCLUSION: Our data show that cryopreservation of DC precursors does not significantly affect the majority of the MDDC markers, although the trends are towards reduced expression of co-stimulatory makers and cytokines. In addition, monocytes from cryopreserved PBMC of cancer patients can be fully differentiated into mature DC with phenotype and function equivalent to those derived from healthy donors. [Abstract/Link to Full Text]

Hardy GA, Imami N, Nelson MR, Sullivan AK, Moss R, Aasa-Chapman MM, Gazzard B, Gotch FM
A phase I, randomized study of combined IL-2 and therapeutic immunisation with antiretroviral therapy.
J Immune Based Ther Vaccines. 2007;56.
BACKGROUND: Fully functional HIV-1-specific CD8 and CD4 effector T-cell responses are vital to the containment of viral activity and disease progression. These responses are lacking in HIV-1-infected patients with progressive disease. We attempted to augment fully functional HIV-1-specific CD8 and CD4 effector T-cell responses in patients with advanced chronic HIV-1 infection. DESIGN: Chronically infected patients with low CD4 counts T-cell counts who commenced antiretroviral therapy (ART) were subsequently treated with combined interleukin-2 and therapeutic vaccination. METHODS: Thirty six anti-retroviral naive patients were recruited and initiated on combination ART for 17 weeks before randomization to: A) ongoing ART alone; B) ART with IL-2 twice daily for 5 days every four weeks starting at week 17 for 3 cycles; C) ART with IL-2 as in group B and Remune HIV-1 vaccine administered once every 3 months, starting at week 17; and D) ART with Remune vaccine as in group C. Patients were studied for 65 weeks following commencement of ART, with an additional prior 6 week lead-in observation period. CD4 and CD8 T-cell counts, evaluations of HIV-1 RNA levels and proliferative responses to recall and HIV-1 antigens were complemented with assessment of IL-4-secretion alongside quantification of anti-HIV-1 CD8 T-cell responses and neutralizing antibody titres. RESULTS: Neither IL-2 nor Remune vaccination induced sustained HIV-1-specific T-cell responses. However, we report an inverse relationship between HIV-1-specific proliferative responses and IL-4 production which continuously increased in patients receiving immunotherapy, but not patients receiving ART alone. CONCLUSION: Induction of HIV-1-specific cell-mediated responses is a major challenge in chronically HIV-1-infected patients even when combining immunisation with IL-2 therapy. An antigen-specific IL-4-associated suppressive response may play a role in attenuating HIV-specific responses. [Abstract/Link to Full Text]

Brignone C, Grygar C, Marcu M, Perrin G, Triebel F
IMP321 (sLAG-3), an immunopotentiator for T cell responses against a HBsAg antigen in healthy adults: a single blind randomised controlled phase I study.
J Immune Based Ther Vaccines. 2007;55.
BACKGROUND: LAG-3 (CD223) is a natural high affinity ligand for MHC class II. The soluble form (sLAG-3) induces maturation of monocyte-derived dendritic cells in vitro and is used as a potent Th1-like immune enhancer with many antigens in animal models. To extend this observation to human, a proof of concept study was conducted with a clinical-grade sLAG-3, termed IMP321, coinjected with alum-non-absorbed recombinant hepatitis B surface antigen. METHODS: In a randomised, single blind controlled phase I dose escalation study, 48 seronegative healthy volunteers aged 18-55 years were vaccinated at 0, 4 and 8 weeks by subcutaneous injection with 10 microg HBsAg mixed with saline (control) or with IMP321 at one of four doses (3, 10, 30 and 100 microg). To evaluate the efficacy of this three injections over 2 months immunization protocol, an additional control group was injected with the commercial vaccine Engerix-B. RESULTS: IMP321 was very well tolerated. Indeed, a lower incidence of adverse events was reported from the HBsAg plus IMP321 groups than from the Engerix-B group. HBsAg-specific antibody responses (anti-HBs) appeared sooner and were higher at 8 and 12 weeks in IMP321 recipients compared to HBsAg control subjects. More importantly, increased numbers of responders to HBsAg were found in IMP321 recipients compared HBsAg group, as revealed by higher post-vaccination frequencies of CD4 Th1 or CD8 Tc1 antigen specific T cells. IMP321 induced CD4 Th1 antigen-specific T cells in some of these naïve individuals after only one injection, especially in the 10 and 30 microg dose groups. CONCLUSION: IMP321 as an adjuvant to HBsAg was well-tolerated and enhanced T cell response vaccine immunogenicity (i.e. induced both CD4 Th1 and CD8 Tc1 antigen-specific T cells). This latter property has allowed the development of IMP321 as an immunopotentiator for therapeutic vaccines. [Abstract/Link to Full Text]

Liska V, Bigert SA, Bennett PS, Olsen D, Chang R, Burke CJ
Evaluation of a recombinant human gelatin as a substitute for a hydrolyzed porcine gelatin in a refrigerator-stable Oka/Merck live varicella vaccine.
J Immune Based Ther Vaccines. 2007;54.
BACKGROUND: The labile nature of live, attenuated varicella-zoster virus (Oka/Merck) requires robust stabilization during virus bulk preparation and vaccine manufacturing in order to preserve potency through storage and administration. One stabilizing ingredient used in a varicella-zoster virus (VZV) vaccine is hydrolyzed porcine gelatin which represents the major protein/peptide-based excipient in the vaccine formulation. METHODS: In this comparative study, a recombinant human gelatin fragment (8.5 kD) was assessed as a potential replacement for hydrolyzed porcine gelatin in an experimental live, attenuated VZV (Oka/Merck) vaccine. VZV (Oka/Merck) was harvested in two formulations prepared with either a hydrolyzed porcine gelatin or a recombinant human gelatin. Moreover, the viral stability in the experimental VZV (Oka/Merck) vaccines was evaluated under accelerated and real-time conditions in a comparative study. RESULTS AND DISCUSSION: The stabilizing effect of recombinant human gelatin on VZV (Oka/Merck) potency change during vaccine lyophilization was similar to the experimental vaccine containing porcine-derived gelatin. Vaccine viral potency changes were comparable in stabilized VZV (Oka/Merck) formulations containing either hydrolyzed porcine gelatin or recombinant human gelatin. No statistically significant difference in potency stability was observed between the vaccine formulations stored at any of the temperatures tested. CONCLUSION: The recombinant human gelatin demonstrated similar ability to stabilize the live attenuated VZV (Oka/Merck) in an experimental, refrigerator-stable varicella vaccine when compared to the vaccine preparation formulated with hydrolyzed porcine gelatin used in currently marketed varicella vaccine. [Abstract/Link to Full Text]

Shofner JD, Vasquez JG, Berger CL, Edelson RL
Improved generation of anti-tumor immunity by antigen dose limitation.
J Immune Based Ther Vaccines. 2007;52.
BACKGROUND: The malignant cells of cutaneous T cell lymphoma (CTCL) display immunogenic peptides derived from the clonal T cell receptor (TCR) providing an attractive model for refinement of anti-tumor immunization methodology. To produce a clinically meaningful anti-tumor response, induction of cytotoxic anti-CTCL cells must be maximized while suppressive T regulatory cells (Treg) should be minimized. We have demonstrated that engulfment of apoptotic CTCL cells by dendritic cells (DC) can lead to either CD8 anti-CTCL responses or immunosuppressive Treg induction. Treg generation is favored when the number of apoptotic cells available for ingestion is high. METHODS: In this study, we sought to determine whether the balance between immunity and immunosuppression could be shifted towards a CD8 anti-CTCL response by lowering the ratio of apoptotic CTCL cells available for DC ingestion. CTCL cell apoptosis was produced by engagement of the TCR by anti-CD3 antibody affixed to magnetic beads. RESULTS: The physical perturbation inherent in passage through a separation column induced monocytes to differentiate into DC, demonstrated by increased expression of class II and CD86 and decreased expression of the monocyte marker CD14. The immature DC internalized and processed apoptotic CTCL cells and could potentially present the tumor-derived peptides in the context of MHC class I and II. As the number of apoptotic cells increased, there was a dose-dependent increase in the expression of Treg markers CTLA-4, CD25, and FoxP3, with a ratio of apoptotic cell/DC loading of > 10:1 corresponding to the greatest Treg induction. These inducible phenotypic Treg also functionally inhibited CD8-mediated perforin expression in vitro. At lower levels of apoptotic cell/DC loading of < 5:1, there was an expansion of the CD8 T cell compartment with increased perforin expression and increased CTCL cell death, indicating anti-tumor activity. CONCLUSION: These findings demonstrate that the ratio of apoptotic cells supplied to DC is an important determinant of whether CD8 anti-tumor immunity or immunosuppression is generated. [Abstract/Link to Full Text]

Flaminio MJ, Borges AS, Nydam DV, Horohov DW, Hecker R, Matychak MB
The effect of CpG-ODN on antigen presenting cells of the foal.
J Immune Based Ther Vaccines. 2007;51.
BACKGROUND: Cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) has been used successfully to induce immune responses against viral and intracellular organisms in mammals. The main objective of this study was to test the effect of CpG-ODN on antigen presenting cells of young foals. METHODS: Peripheral blood monocytes of foals (n = 7) were isolated in the first day of life and monthly thereafter up to 3 months of life. Adult horse (n = 7) monocytes were isolated and tested once for comparison. Isolated monocytes were stimulated with IL-4 and GM-CSF (to obtain dendritic cells, DC) or not stimulated (to obtain macrophages). Macrophages and DCs were stimulated for 14-16 hours with either CpG-ODN, LPS or not stimulated. The stimulated and non-stimulated cells were tested for cell surface markers (CD86 and MHC class II) using flow cytometry, mRNA expression of cytokines (IL-12, IFNalpha, IL-10) and TLR-9 using real time quantitative RT-PCR, and for the activation of the transcription factor NF-kappaB p65 using a chemiluminescence assay. RESULTS: The median fluorescence of the MHC class II molecule in non-stimulated foal macrophages and DCs at birth were 12.5 times and 11.2 times inferior, respectively, than adult horse cells (p = 0.009). That difference subsided at 3 months of life (p = 0.3). The expression of the CD86 co-stimulatory molecule was comparable in adult horse and foal macrophages and DCs, independent of treatment. CpG-ODN stimulation induced IL-12p40 (53 times) and IFNalpha (23 times) mRNA expression in CpG-ODN-treated adult horse DCs (p = 0.078), but not macrophages, in comparison to non-stimulated cells. In contrast, foal APCs did not respond to CpG-ODN stimulation with increased cytokine mRNA expression up to 3 months of age. TLR-9 mRNA expression and NF-kB activation (NF-kB p65) in foal DCs and macrophages were comparable (p > 0.05) to adult horse cells. CONCLUSION: CpG-ODN treatment did not induce specific maturation and cytokine expression in foal macrophages and DCs. Nevertheless, adult horse DCs, but not macrophages, increased their expression of IL-12 and IFNalpha cytokines upon CpG-ODN stimulation. Importantly, foals presented an age-dependent limitation in the expression of MHC class II in macrophages and DCs, independent of treatment. [Abstract/Link to Full Text]

Huang KH, Boisvert MP, Chung F, Loignon M, Zarowny D, Cyr L, Toma E, Bernard NF
Longitudinal changes in HIV-specific IFN-gamma secretion in subjects who received Remune vaccination prior to treatment interruption.
J Immune Based Ther Vaccines. 2006;47.
BACKGROUND: Despite the benefits of highly active antiretroviral therapy (HAART) for suppressing viral replication in HIV infection, virus persists and rebounds during treatment interruption (TI). This study explored whether HAART intensification with Remune vaccination before TI can boost HIV-1-specific immunity, leading to improved control of viremia off HAART. METHODS: Ten chronically HIV-infected adults were enrolled in this proof of concept study. After a 6-month HAART intensification phase with didanosine, hydroxyurea, granulocyte-macrophage colony-stimulating factor, (GM-CSF), and a first dose of Remune (HIV-1 Immunogen), HAART was discontinued. Patients continued to receive Remune every 3 months until the end of study. HAART was restarted if viral load did not fall below 50,000 copies/ml of plasma within 3 months or if CD4+ counts decreased to <200 cells/mm3. HIV-specific immunity was monitored with the interferon-gamma (IFN-gamma) ELISPOT assay. RESULTS: All subjects experienced viral rebound during TIs. Although the magnitude and breadth of HIV-specific responses to HLA-restricted optimal peptide panels and Gag p55 peptide pools increased and viral load decreased by 0.44 log10 units from TI#1 to TI#2, no significant correlations between these parameters were observed. The patients spent 50.4% of their 36 months follow up off HAART. CONCLUSION: Stopping HAART in this vaccinated population induced immune responses that persisted after therapy was restarted. Induction of HIV-specific immunity beyond IFN-gamma secretion may be contributing to better control of viremia during subsequent TIs allowing for long periods off HAART. [Abstract/Link to Full Text]

Lim SY, Meyer M, Kjonaas RA, Ghosh SK
Phytol-based novel adjuvants in vaccine formulation: 1. assessment of safety and efficacy during stimulation of humoral and cell-mediated immune responses.
J Immune Based Ther Vaccines. 2006;46.
BACKGROUND: Vaccine efficacy depends significantly on the use of appropriate adjuvant(s) in the formulation. Phytol, a dietary diterpene alcohol, is similar in structure to naturally occurring isoprenoid adjuvants; but little is known of its adjuvanticity. In this report, we describe the relative safety and efficacy of phytol and its hydrogenated derivative PHIS-01 compared to commercial adjuvants. METHODS: We tested adjuvant properties using a formulation consisting of either a hapten, phthalate-conjugated to a protein, keyhole limpet hemocyanin (KLH), or ovalbumin (OVA) emulsified with the test adjuvants in mice without any surfactant. Humoral immunity was assessed in terms of titer, specificity, and isotypic profiles. The effect on cell-mediated immunity was studied by assaying the induction of either OVA- or B-lymphoma-specific cytotoxic T-lymphocyte (CTL) activity. RESULTS AND DISCUSSION: The phytol compounds, particularly PHIS-01, elicit increased titers of all major IgG subclasses, especially IgG2a. Unlike commercial adjuvants, both phytol compounds are capable of inducing specific cytotoxic effector T cell responses specific to both OVA and B-lymphoma tested. Phytols as adjuvants are also distinctive in that they provoke no adverse anti-DNA autoimmune response. Intraperitoneally administered phytol is comparable to complete Freund's adjuvant in toxicity in doses over 40 ug/mouse, but PHIS-01 has no such toxicity. CONCLUSION: These results and our ongoing studies on antibacterial immunity show that phytol and PHIS-01 are novel and effective adjuvants with little toxicity. [Abstract/Link to Full Text]

Lim SY, Bauermeister A, Kjonaas RA, Ghosh SK
Phytol-based novel adjuvants in vaccine formulation: 2. Assessment of efficacy in the induction of protective immune responses to lethal bacterial infections in mice.
J Immune Based Ther Vaccines. 2006;45.
BACKGROUND: Adjuvants are known to significantly enhance vaccine efficacy. However, commercial adjuvants often have limited use because of toxicity in humans. The objective of this study was to determine the comparative effectiveness of a diterpene alcohol, phytol and its hydrogenated derivative PHIS-01, relative to incomplete Freund's adjuvant (IFA), a commonly used adjuvant in augmenting protective immunity in mice against E. coli and S. aureus, and in terms of inflammatory cytokines. METHODS: Vaccines, consisting of heat-attenuated E. coli or S. aureus and either of the two phytol-based adjuvants or IFA, were tested in female BALB/c mice. The vaccines were administered intraperitoneally at 10-day intervals. The efficacy of the phytol and PHIS-01, as compared to IFA, was assessed by ELISA in terms of anti-bacterial antibody and inflammatory cytokines. We also examined the ability of the vaccines to induce specific protective immunity by challenging mice with different doses of live bacteria. RESULTS AND DISCUSSION: IFA, phytol, and PHIS-01 were equally efficient in evoking anti-E. coli antibody response and in providing protective immunity against live E. coli challenges. In contrast, the antibody response to S. aureus was significant when PHIS-01 was used as the adjuvant. However, in terms of the ability to induce protective immunity, phytol was most effective against S. aureus. Moreover, during challenges with live E. coli and S. aureus immune mice produced much less IL-6, the mediators of fatal septic shock syndromes. CONCLUSION: Our results show that vaccine formulations containing phytol and PHIS-01 as adjuvants confer a robust and protective immunity against both Gram-negative and Gram-positive bacteria without inducing adverse inflammatory cytokine due to IL-6. [Abstract/Link to Full Text]

Monteiro-Maia R, Ortigão-de-Sampaio MB, Pinho RT, Castello-Branco LR
Modulation of humoral immune response to oral BCG vaccination by Mycobacterium bovis BCG Moreau Rio de Janeiro (RDJ) in healthy adults.
J Immune Based Ther Vaccines. 2006;44.
BACKGROUND: Oral administration of BCG was the route initially used by Calmette and Guérin, but was replaced by intradermal administration in virtually all countries after the Lubeck accident. However, Brazil continued to administer oral BCG Moreau RDJ, which was maintained until the mid-1970s when it was substituted by the intradermal route. Although BCG vaccination has been used in humans since 1921, little is known of the induced immune response. The aim of this study was to analyse immunological responses after oral vaccination with M. bovis BCG Moreau RDJ. METHODS: This study in healthy volunteers has measured cellular and humoral aspects of the immunological response to oral M. bovis BCG Moreau RDJ in Rio de Janeiro, Brazil. T-cell trafficking and Th1 and Th2 cytokine responses are described, as well as isotype-specific antibody production using novel techniques. RESULTS: Oral immunisation has no adverse effects. We have shown that there are cellular and humoral immunological responses after oral immunisation. Oral revaccination does not induce a positive skin test in responsive individuals and multiple booster orally was able to induce modulation in humoral immunological responses (switch from IgG to IgA) in previously immunised subjects and incapable of inducing tolerance. In contrast, the cellular immune response does not differ between vaccinated individuals with positive and negative skin test reactions. CONCLUSION: All subjects, including those who did not respond to the skin test at study commencement, were capable of mounting humoral and cellular immune response to the antigens tested. [Abstract/Link to Full Text]

Argani H, Akhtarishojaie E
Levamizole enhances immune responsiveness to intra-dermal and intra-muscular hepatitis B vaccination in chronic hemodialysis patients.
J Immune Based Ther Vaccines. 2006;43.
BACKGROUND: Hemodialysis patient are at high risk for hepatitis B virus (HBV) infection. Although preventive vaccination is done routinely, the response to vaccination is low in this patient population. The aim of this study was to evaluate the effect of Levamizol, an enhancer of the immune responsiveness, on different routes of vaccination, i.e., intradermal (i.d.) versus intramuscular (i.m.), in stable chronic hemodialysis patients. MATERIALS AND METHODS: Forty four chronic hemodialyses patient were divided into four equal groups. The first group was received 40 microg HB vaccine intramuscularly. The second group was received 20 microg HB vaccine intradermally. The third and the fourth group received 20 microg vaccine i.m. or i.d., respectively, in three doses plus oral Levamisole (100 mg for 12 day). After one and six months from the last dose of vaccine, HBs antibody titers were measured. RESULTS: The response rate to vaccine (HBs Antibody>10 microg/L) in the routine i.m. HB vaccination was low (60%). It increased to 70% with i.d. route. Levamisole significantly raised the response rate to 90% (P < 0.01). Also in the Levamisole groups protective HB antibody titers were maintained until the end of six months. We conclude that HD patients must be vaccinated by i.d. route and addition of Levamisole. Levamisole also increases antibody maintenance. [Abstract/Link to Full Text]

Arnaiz B, Madrigal-Estebas L, Todryk S, James TC, Doherty DG, Bond U
A novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens.
J Immune Based Ther Vaccines. 2006;42.
The heat shock protein, Hsp70, has been shown to play an important role in tumour immunity. Vaccination with Hsp70-peptide complexes (Hsp70-PCs), isolated from autologous tumour cells, can induce protective immune responses. We have developed a novel method to identify synthetic mimic peptides of Hsp70-PCs and to test their ability to activate T-cells. Peptides (referred to as "recognisers") that bind to Hsp70-PCs from the human breast carcinoma cell line, MDA-MB-231, were identified by bio-panning a random peptide M13 phage display library. Synthetic recogniser peptides were subsequently used as bait in a reverse bio-panning experiment to identify potential Hsp70-PC mimic peptides. The ability of the recogniser and mimic peptides to prime human lymphocyte responses against tumour cell antigens was tested by stimulating lymphocytes with autologous peptide-loaded monocyte-derived dendritic cells (DCs). Priming and subsequent stimulation with either the recogniser or mimic peptide resulted in interferon-gamma (IFN-gamma) secretion by the lymphocytes. Furthermore, DCs loaded with Hsp70, Hsp70-PC or the recogniser or the mimic peptide primed the lymphocytes to respond to soluble extracts from breast cells. These results highlight the potential application of synthetic peptide-mimics of Hsp70-PCs, as modulators of the immune response against tumours. [Abstract/Link to Full Text]

Newell MK, Villalobos-Menuey E, Schweitzer SC, Harper ME, Camley RE
Cellular metabolism as a basis for immune privilege.
J Immune Based Ther Vaccines. 2006;41.
We hypothesize that the energy strategy of a cell is a key factor for determining how, or if, the immune system interacts with that cell. Cells have a limited number of metabolic states, in part, depending on the type of fuels the cell consumes. Cellular fuels include glucose (carbohydrates), lipids (fats), and proteins. We propose that the cell's ability to switch to, and efficiently use, fat for fuel confers immune privilege. Additionally, because uncoupling proteins are involved in the fat burning process and reportedly in protection from free radicals, we hypothesize that uncoupling proteins play an important role in immune privilege. Thus, changes in metabolism (caused by oxidative stresses, fuel availability, age, hormones, radiation, or drugs) will dictate and initiate changes in immune recognition and in the nature of the immune response. This has profound implications for controlling the symptoms of autoimmune diseases, for preventing graft rejection, and for targeting tumor cells for destruction. [Abstract/Link to Full Text]

Pires A, Nelson M, Pozniak AL, Fisher M, Gazzard B, Gotch F, Imami N
Mycobacterial immune reconstitution inflammatory syndrome in HIV-1 infection after antiretroviral therapy is associated with deregulated specific T-cell responses: beneficial effect of IL-2 and GM-CSF immunotherapy.
J Immune Based Ther Vaccines. 2005 Sep 25;37.
BACKGROUND: With the advent of antiretroviral therapy (ART) cases of immune reconstitution inflammatory syndrome (IRIS) have increasingly been reported. IRIS usually occurs in individuals with a rapidly rising CD4 T-cell count or percentage upon initiation of ART, who develop a deregulated immune response to infection with or without reactivation of opportunistic organisms. Here, we evaluated rises in absolute CD4 T-cells, and specific CD4 T-cell responses in 4 HIV-1+ individuals presenting with mycobacterial associated IRIS who received in conjunction with ART, IL-2 plus GM-CSF immunotherapy. METHODS: We assessed CD4 T-cell counts, HIV-1 RNA loads, phenotype for naïve and activation markers, and in vitro proliferative responses. Results were compared with those observed in 11 matched, successfully treated asymptomatic clinical progressors (CP) with no evidence of opportunistic infections, and uninfected controls. RESULTS: Median CD4 T-cell counts in IRIS patients rose from 22 cells/microl before initiation of ART, to 70 cells/microl after 8 months of therapy (median 6.5 fold increase). This coincided with IRIS diagnosis, lower levels of naïve CD4 T-cells, increased expression of immune activation markers, and weak CD4 T-cell responses. In contrast, CP had a median CD4 T-cell counts of 76 cells/microl at baseline, which rose to 249 cells/microl 6 months post ART, when strong T-cell responses were seen in > 80% of patients. Higher levels of expression of immune activation markers were seen in IRIS patients compared to CP and UC (IRIS > CP > UC). Immunotherapy with IL-2 and GM-CSF paralleled clinical recovery. CONCLUSION: These data suggest that mycobacterial IRIS is associated with inadequate immune reconstitution rather than vigorous specific T-cell responses, and concomitant administration of IL-2 and GM-CSF immunotherapy with effective ART may correct/augment T-cell immunity in such setting resulting in clinical benefit. [Abstract/Link to Full Text]

Cassell S, Kavanaugh A
Psoriatic arthritis: pathogenesis and novel immunomodulatory approaches to treatment.
J Immune Based Ther Vaccines. 2005 Sep 2;36.
Psoriatic arthritis (PsA) is a chronic inflammatory arthropathy characterized by the association of arthritis and psoriasis. PsA runs a variable course, from mild synovitis to severe, progressive, erosive arthropathy. The pathogenesis of PsA involves alteration in the components of the immune response, although the exact cause of PsA is unknown. A number of patients with severe peripheral arthritis fail to respond to standard conventional therapy. Advances in biotechnology and in our understanding of the immunopathogenesis of PsA have led to great interest and progress in regards to biologic treatments for PsA. Notable success achieved with recently introduced biologic therapies has paved the way for further research and development of additional therapies that should improve outcomes for affected patients. [Abstract/Link to Full Text]

Hill AF, Polvino WJ, Wilson DB
The significance of glucose, insulin and potassium for immunology and oncology: a new model of immunity.
J Immune Based Ther Vaccines. 2005 Aug 19;35.
BACKGROUND: A recent development in critical care medicine makes it urgent that research into the effect of hormones on immunity be pursued aggressively. Studies have demonstrated a large reduction in mortality as a result of infusion with glucose, insulin and potassium. Our work in the oncology setting has led us to propose that the principal reason for such an effect is that GIK stimulates lymphocytes to proliferate and attack pathogens, sparing the patient the stress of infection. That suggestion is based on a new model of immunity that describes the effect of hormones on lymphocytes. We hypothesized that the application of glucose, insulin, thyroid and potassium would awaken inert tumor infiltrating lymphocytes to destroy the tumor. METHODS: The antitumor effect of a thyroxine, glucose, insulin, and potassium (TGIK) combination was studied in a series of controlled experiments in murine models of tumor progression to assess the biologic activity of the formulation, the effect of route of administration, the effect on tumor type, and the requirement for insulin in the TGIK formulation. RESULTS: Melanoma and colon tumors inoculated with TGIK were significantly reduced in size or retarded in growth compared to controls injected with saline. I.P. and I.M. injections showed that the formulation had no effect systemically at the doses administered. CONCLUSION: We conclude that TGIK has anti-tumor activity when administered intratumorally, probably by stimulating lymphocytes to attack tumors. This is similar to the effect of GIK on reducing sepsis in critical care patients. We suggest that when GIK is administered exogenously, it restores immune competence to the critically ill or cancer patient and causes destruction of pathogens or tumors, while endogenous resources are devoted to repair. This implies that hormonal therapy may be useful in treating various other pathologies involving immune suppression, as well as malignancies. We also propose research that could bring resolution of the controversy over mechanism and point the way to new therapeutic strategies for numerous diseases including chronic infections and auto-immune diseases. [Abstract/Link to Full Text]

Salskov-Iversen M, Berger CL, Edelson RL
Rapid construction of a dendritic cell vaccine through physical perturbation and apoptotic malignant T cell loading.
J Immune Based Ther Vaccines. 2005 Jul 19;34.
We have demonstrated that adherence and release of monocytes from a plastic surface drives their differentiation into immature dendritic cells (DC,) that can mature further during overnight incubation in the presence of apoptotic malignant T cells. Based on these results, we sought to develop a clinically, practical, rapid means for producing DC loaded with malignant cells. A leukapheresis harvest containing the clonal, leukemic expansion of malignant CD4+ T cells was obtained from the blood of patients with cutaneous T cell lymphoma (CTCL). CTCL cells were purified with a CD3-magnetic bead column where CD3 engagement rendered the malignant T cells apoptotic. The monocyte fraction was simultaneously activated by column passage, re-added to the apoptotic CTCL cells and co-cultured overnight. CTCL cell apoptosis, DC differentiation and apoptotic malignant T cell ingestion were measured by immunostaining. The results demonstrate that as monocytes passed through the column matrix, they became activated and differentiated into semi-mature DC expressing significantly increased levels of class II, CD83 and CD86 (markers associated with maturing DC) and reduced expression of the monocyte markers CD14 and CD36. Apoptotic malignant T cells were avidly engulfed by the phagocytic transitioning DC. The addition of supportive cytokines further enhanced the number of DC that contained apoptotic malignant T cells. Functional studies confirmed that column passaged DC increased class II expression as shown by significantly enhanced stimulation in mixed leukocyte culture compared to control monocytes. In addition, DC loaded with apoptotic CTCL cells stimulated an increase in the percentage and absolute number of CD8 T cells compared to co-cultivation with non-loaded DC. After CD8 T cells were stimulated by DC loaded with malignant cells, they mediated increased apoptosis of residual CTCL cells and TNF-alpha secretion indicating development of enhanced cytolytic function. We report a simple one-step procedure where maturing DC containing apoptotic malignant T cells can be prepared rapidly for potential use in vaccine immunotherapy. Ready access to both the DC and apoptotic cells provided by this system will allow extension to other malignancies through the addition of a variety of apoptotic tumor cells and maturation stimuli. [Abstract/Link to Full Text]

Brown PA, Angel JB
Granulocyte-macrophage colony-stimulating factor as an immune-based therapy in HIV infection.
J Immune Based Ther Vaccines. 2005 May 18;3(1):3.
The HIV/AIDS epidemic continues to spread despite more than 20 years of significant research and major advances in its treatment. The introduction of highly active antiretroviral therapy in recent years has significantly improved disease treatment with a dramatic impact in HIV/AIDS associated morbidity and mortality in countries which have access to this therapy. Despite these advances, such therapies are imperfect and other therapeutic modalities, including immune-based therapies, are being actively sought. Potential benefits of immune-based therapies include: 1) the improvement of HIV-specific immunity to enhance control of viral replication, 2) the improvement of other aspects of host immunity in order to prevent or delay the development of opportunistic infections and 3) the potential to purge virus from cellular reservoirs which are sustained despite the effects of potent antiretroviral therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been studied as one of these immune-based therapies. Several randomized, controlled trials have demonstrated benefits of using GM-CSF as an adjunct to conventional anti-retroviral therapy, although such benefits have not been universally observed. Individual studies have shown that GM-CSF increases CD4+ T cells counts and may be associated with decreased plasma HIV RNA levels. There is limited evidence that GM-CSF may help prevent the emergence of antiretroviral drug resistant viruses and that it may decrease the risk of infection in advanced HIV disease. Despite its high costs and the need to be administered subcutaneously, encouraging results continue to emerge from further studies, suggesting that GM-CSF has the potential to become an effective agent in the treatment of HIV infection. [Abstract/Link to Full Text]

Ruiz W, McClements WL, Jansen KU, Esser MT
Kinetics and isotype profile of antibody responses in rhesus macaques induced following vaccination with HPV 6, 11, 16 and 18 L1-virus-like particles formulated with or without Merck aluminum adjuvant.
J Immune Based Ther Vaccines. 2005 Apr 20;3(1):2.
BACKGROUND: Human papillomaviruses (HPV) are the most common sexually transmitted viruses. Infection of the cervical epithelium by HPVs can lead to the development of cervical cancer. Recent advances in vaccine research have shown that immunization with papillomavirus-like particles (VLPs) containing the major structural viral protein, L1 from HPV 16 can provide protection from the establishment of a chronic HPV 16 infection and related cervical intraepithelial neoplasia (CIN) in baseline HPV 16 naive women. METHODS: To better understand the quantitative and qualitative effects of aluminum adjuvant on the immunogenic properties of an HPV 6, 11, 16 and 18L1 VLP vaccine, we used an HPV-specific, antibody isotyping assay and a competitive immunoassay that measures antibodies to neutralizing epitopes to profile sera from rhesus macaques immunized with the HPV L1 VLP vaccine formulated with or without aluminum adjuvant. RESULTS: Immunization with VLPs formulated with the aluminum adjuvant elicited a significantly stronger immune response with higher peak antibody titers both at four weeks post vaccination (12.7 to 41.9-fold higher) as well as in the persistent phase at week 52 (4.3 to 26.7-fold higher) than that of VLPs alone. Furthermore, the aluminum adjuvant formulated HPV VLP vaccine elicited a predominantly T helper type 2 response, with high levels of IgG1 and IgG4 and low levels of IgG2. The vaccine also elicited high levels of serum IgA, which may be important in providing mucosal immunity to impart protection in the anogenital tract. CONCLUSION: These results show that the HPV 6, 11, 16 and 18 L1-VLP vaccine formulated with Merck aluminum adjuvant elicits a robust and durable immune response and holds promise as a vaccine for preventing cervical cancer. [Abstract/Link to Full Text]

Briassoulis G, Karabatsou I, Gogoglou V, Tsorva A
BCG vaccination at three different age groups: response and effectiveness.
J Immune Based Ther Vaccines. 2005 Apr 1;3(1):1.
BACKGROUND: The protection, which some BCG vaccines could confer against the development of tuberculosis (TB) in childhood, might be indirectly reflected by the subsequent development of BCG immune response. The objectives of the study were to examine effectiveness and possible differences of post-vaccination reaction to a lyophilized BCG at different age groups and to evaluate its protection against TB in a decade's period. METHODS: We studied the post-vaccination PPD-skin reaction and scar formation at three different school levels, corresponding to ages of 6, 12 and 15 years old, vaccinated by a lyophilized BCG vaccine (Pasteur Institute), currently used in our country. During a 10-year follow up the reported TB cases in vaccinated and non-vaccinated adolescences up to 24-years old were analyzed and compared to the number of cumulative cases observed in the adult population of two neighboring territories (vaccinated and non-vaccinated). RESULTS AND DISCUSSION: There was a significant correlation (r2 = 0.87, p < 0.0001) between tuberculin induration and scar formation. There was no statistically significant difference between the three age groups (6, 12, and 15 year-old, respectively) in regard to the diameter of tuberculin induration or scar formation. Although 34% of 10-year later indurations were unpredictably related to the initial ones (increased or decreased), they were significantly correlated (r2 = 0.45, p = 0.009). The relative percentage of TB for the 14-24 years-age group to the adult studied population was significantly lower among the immunized children compared to the non-immunized population of the same age group (17/77, 22% vs. 71/101, 70%, p < .0001). CONCLUSION: Our data suggest that the lyophilized BCG vaccine used for BCG programs at different age groups is equally effective and may confer satisfactory protection against tuberculosis in puberty. [Abstract/Link to Full Text]

Musselli C, Daverio-Zanetti S, Zanetti M
Antigenized antibodies expressing Vbeta8.2 TCR peptides immunize against rat experimental allergic encephalomyelitis.
J Immune Based Ther Vaccines. 2004 Nov 12;2(1):9.
BACKGROUND: Immunity against the T cell receptor (TCR) is considered to play a central role in the regulation of experimental allergic encephalomyelitis (EAE), a model system of autoimmune disease characterized by a restricted usage of TCR genes. Methods of specific vaccination against the TCR of pathogenetic T cells have included attenuated T cells and synthetic peptides from the sequence of the TCR. These approaches have led to the concept that anti-idiotypic immunity against antigenic sites of the TCR, which are a key regulatory element in this disease. METHODS: The present study in the Lewis rat used a conventional idiotypic immunization based on antigenized antibodies expressing selected peptide sequences of the Vbeta8.2 TCR (93ASSDSSNTE101 and 39DMGHGLRLIHYSYDVNSTEKG59). RESULTS: The study demonstrates that vaccination with antigenized antibodies markedly attenuates, and in some instances, prevents clinical EAE induced with the encephalitogenic peptide 68GSLPQKSQRSQDENPVVHF88 in complete Freunds' adjuvant (CFA). Antigenized antibodies induced an anti-idiotypic response against the Vbeta8.2 TCR, which was detected by ELISA and flowcytometry. No evidence was obtained of a T cell response against the corresponding Vbeta8.2 TCR peptides. CONCLUSIONS: The results indicate that antigenized antibodies expressing conformationally-constrained TCR peptides are a simple means to induce humoral anti-idiotypic immunity against the TCR and to vaccinate against EAE. The study also suggests the possibility to target idiotypic determinants of TCR borne on pathogenetic T cells to vaccinate against disease. [Abstract/Link to Full Text]

Mohamadzadeh M, Mohamadzadeh H, Brammer M, Sestak K, Luftig RB
Identification of proteases employed by dendritic cells in the processing of protein purified derivative (PPD).
J Immune Based Ther Vaccines. 2004 Aug 2;2(1):8.
Dendritic cells (DC) are known to present exogenous protein Ag effectively to T cells. In this study we sought to identify the proteases that DC employ during antigen processing. The murine epidermal-derived DC line Xs52, when pulsed with PPD, optimally activated the PPD-reactive Th1 clone LNC.2F1 as well as the Th2 clone LNC.4k1, and this activation was completely blocked by chloroquine pretreatment. These results validate the capacity of XS52 DC to digest PPD into immunogenic peptides inducing antigen specific T cell immune responses. XS52 DC, as well as splenic DC and DCs derived from bone marrow degraded standard substrates for cathepsins B, C, D/E, H, J, and L, tryptase, and chymases, indicating that DC express a variety of protease activities. Treatment of XS52 DC with pepstatin A, an inhibitor of aspartic acid proteases, completely abrogated their capacity to present native PPD, but not trypsin-digested PPD fragments to Th1 and Th2 cell clones. Pepstatin A also inhibited cathepsin D/E activity selectively among the XS52 DC-associated protease activities. On the other hand, inhibitors of serine proteases (dichloroisocoumarin, DCI) or of cystein proteases (E-64) did not impair XS52 DC presentation of PPD, nor did they inhibit cathepsin D/E activity. Finally, all tested DC populations (XS52 DC, splenic DC, and bone marrow-derived DC) constitutively expressed cathepsin D mRNA. These results suggest that DC primarily employ cathepsin D (and perhaps E) to digest PPD into antigenic peptides. [Abstract/Link to Full Text]

Osada T, Nagawa H, Shibata Y
Tumor-infiltrating effector cells of alpha-galactosylceramide-induced antitumor immunity in metastatic liver tumor.
J Immune Based Ther Vaccines. 2004 Jul 13;2(1):7.
BACKGROUND: alpha-Galactosylceramide (alpha-GalCer) can be presented by CD1d molecules of antigen-presenting cells, and is known to induce a potent NKT cell-dependent cytotoxic response against tumor cells. However, the main effector cells in alpha-GalCer-induced antitumor immunity are still controversial. METHODS: In order to elucidate the cell phenotype that plays the most important role in alpha-GalCer-induced antitumor immunity, we purified and analyzed tumor-infiltrating leukocytes (TILs) from liver metastatic nodules of a colon cancer cell line (Colon26), comparing alpha-GalCer- and control vehicle-treated mice. Flow cytometry was performed to analyze cell phenotype in TILs and IFN-gamma ELISA was performed to detect antigen-specific immune response. RESULTS: Flow cytometry analysis showed a significantly higher infiltration of NK cells (DX5+, T cell receptor alphabeta (TCR)-) into tumors in alpha-GalCer-treated mice compared to vehicle-treated mice. The DX5+TCR+ cell population was not significantly different between these two groups, indicating that these cells were not the main effector cells. Interestingly, the CD8+ T cell population was increased in TILs of alpha-GalCer-treated mice, and the activation level of these cells based on CD69 expression was higher than that in vehicle-treated mice. Moreover, the number of tumor-infiltrating dendritic cells (DCs) was increased in alpha-GalCer-treated mice. IFN-gamma ELISA showed stronger antigen-specific response in TILs from alpha-GalCer-treated mice compared to those from vehicle-treated mice, although the difference between these two groups was not significant. CONCLUSIONS: In alpha-GalCer-induced antitumor immunity, NK cells seem to be some of the main effector cells and both CD8+ T cells and DCs, which are related to acquired immunity, might also play important roles in this antitumor immune response. These results suggest that alpha-GalCer has a multifunctional role in modulation of the immune response. [Abstract/Link to Full Text]

Zapata-Velandia A, Ng SS, Brennan RF, Simonsen NR, Gastanaduy M, Zabaleta J, Lentz JJ, Craver RD, Correa H, Delgado A, Pitts AL, Himel JR, Udall JN, Schmidt-Sommerfeld E, Brown RF, Athas GB, Keats BB, Mannick EE
Association of the T allele of an intronic single nucleotide polymorphism in the colony stimulating factor 1 receptor with Crohn's disease: a case-control study.
J Immune Based Ther Vaccines. 2004 May 14;2(1):6.
BACKGROUND: Polymorphisms in several genes (NOD2, MDR1, SLC22A4) have been associated with susceptibility to Crohn's disease. Identification of the remaining Crohn's susceptibility genes is essential for the development of disease-specific targets for immunotherapy. Using gene expression analysis, we identified a differentially expressed gene on 5q33, the colony stimulating factor 1 receptor (CSF1R) gene, and hypothesized that it is a Crohn's susceptibility gene. The CSF1R gene is involved in monocyte to macrophage differentiation and in innate immunity. METHODS: Patients provided informed consent prior to entry into the study as approved by the Institutional Review Board at LSU Health Sciences Center. We performed forward and reverse sequencing of genomic DNA from 111 unrelated patients with Crohn's disease and 108 controls. We also stained paraffin-embedded, ileal and colonic tissue sections from patients with Crohn's disease and controls with a polyclonal antibody raised against the human CSF1R protein. RESULTS: A single nucleotide polymorphism (A2033T) near a Runx1 binding site in the eleventh intron of the colony stimulating factor 1 receptor was identified. The T allele of this single nucleotide polymorphism occurred in 27% of patients with Crohn's disease but in only 13% of controls (X2 = 6.74, p < 0.01, odds ratio (O.R.) = 2.49, 1.23 < O.R. < 5.01). Using immunohistochemistry, positive staining with a polyclonal antibody to CSF1R was observed in the superficial epithelium of ileal and colonic tissue sections. CONCLUSIONS: We conclude that the colony stimulating factor receptor 1 gene may be a susceptibility gene for Crohn's disease. [Abstract/Link to Full Text]

Sawada-Hirai R, Jiang I, Wang F, Sun SM, Nedellec R, Ruther P, Alvarez A, Millis D, Morrow PR, Kang AS
Human anti-anthrax protective antigen neutralizing monoclonal antibodies derived from donors vaccinated with anthrax vaccine adsorbed.
J Immune Based Ther Vaccines. 2004 May 12;2(1):5.
BACKGROUND: Potent anthrax toxin neutralizing human monoclonal antibodies were generated from peripheral blood lymphocytes obtained from Anthrax Vaccine Adsorbed (AVA) immune donors. The anti-anthrax toxin human monoclonal antibodies were evaluated for neutralization of anthrax lethal toxin in vivo in the Fisher 344 rat bolus toxin challenge model. METHODS: Human peripheral blood lymphocytes from AVA immunized donors were engrafted into severe combined immunodeficient (SCID) mice. Vaccination with anthrax protective antigen and lethal factor produced a significant increase in antigen specific human IgG in the mouse serum. The antibody producing lymphocytes were immortalized by hybridoma formation. The genes encoding the protective antibodies were rescued and stable cell lines expressing full-length human immunoglobulin were established. The antibodies were characterized by; (1) surface plasmon resonance; (2) inhibition of toxin in an in vitro mouse macrophage cell line protection assay and (3) in vivo in a Fischer 344 bolus lethal toxin challenge model. RESULTS: The range of antibodies generated were diverse with evidence of extensive hyper mutation, and all were of very high affinity for PA83~1 x 10-10-11M. Moreover all the antibodies were potent inhibitors of anthrax lethal toxin in vitro. A single IV dose of AVP-21D9 or AVP-22G12 was found to confer full protection with as little as 0.5x (AVP-21D9) and 1x (AVP-22G12) molar equivalence relative to the anthrax toxin in the rat challenge prophylaxis model. CONCLUSION: Here we describe a powerful technology to capture the recall antibody response to AVA vaccination and provide detailed molecular characterization of the protective human monoclonal antibodies. AVP-21D9, AVP-22G12 and AVP-1C6 protect rats from anthrax lethal toxin at low dose. Aglycosylated versions of the most potent antibodies are also protective in vivo, suggesting that lethal toxin neutralization is not Fc effector mediated. The protective effect of AVP-21D9 persists for at least one week in rats. These potent fully human anti-PA toxin-neutralizing antibodies are attractive candidates for prophylaxis and/or treatment against Anthrax Class A bioterrorism toxins. [Abstract/Link to Full Text]

Numazaki K
Current problems of perinatal Chlamydia trachomatis infections.
J Immune Based Ther Vaccines. 2004 Feb 13;2(1):4.
Chlamydia trachomatis has been recognized as a pathogen of trachoma, nongonococcal urethritis, salpingitis, endocervicitis, pelvic inflammatory disease, inclusion conjunctivitis of neonates, follicular conjunctivitis of adults, infantile pneumonia and associated conditions. Chlamydial infections during pregnancy may also cause a variety of perinatal complications. Different antigenic strains of C. trachomatis from endocervical, nasopharyngeal and conjunctival origins have been associated with different clinical conditions. Control programs emphasizing early diagnosis, targeted screening, and effective treatment will lead to an eventual decline in the incidence of perinatal chlamydial infection. This review focuses on current problems of perinatal C. trachomatis infections in the aspects of microbiological and immunological pathogenesis. [Abstract/Link to Full Text]

---

Recent Articles in Journal of Virology

---

Sedlackova L, Rice SA
Herpes simplex virus type 1 immediate-early protein ICP27 is required for efficient incorporation of ICP0 and ICP4 into virions.
J Virol. 2008 Jan;82(1):268-77.
Early in infection, herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins ICP0 and ICP4 localize to the nucleus, where they stimulate viral transcription. Later in infection, ICP0 and to a lesser extent ICP4 accumulate in the cytoplasm, but their biological role there is unknown. Previously, it was shown that the cytoplasmic localization of ICP0/4 requires the multifunctional IE protein ICP27, which is itself an activator of viral gene expression. Here, we identify a viral ICP27 mutant, d3-4, which is unable to efficiently localize ICP0 and ICP4 to the cytoplasm but which otherwise resembles wild-type HSV-1 in its growth and viral gene expression phenotypes. These results genetically separate the function of ICP27 that affects ICP0/4 localization from its other functions, which affect viral growth and gene expression. As both ICP0 and ICP4 are known to be minor virion components, we used d3-4 to test the hypothesis that the cytoplasmic localization of these proteins is required for their incorporation into viral particles. Consistent with this conjecture, d3-4 virions were found to lack ICP0 in their tegument and to have greatly reduced levels of ICP4. Thus, the cytoplasmic localization of ICP0 and ICP4 appears to be a prerequisite for the assembly of these important transcriptional regulatory proteins into viral particles. Furthermore, our results show that ICP27 plays a previously unrecognized role in determining the composition of HSV-1 virions. [Abstract/Link to Full Text]

Gao Y, Colletti K, Pari GS
Identification of human cytomegalovirus UL84 virus- and cell-encoded binding partners by using proteomics analysis.
J Virol. 2008 Jan;82(1):96-104.
Human cytomegalovirus (HCMV) UL84 is a phosphoprotein that shuttles from the nucleus to the cytoplasm and is required for oriLyt-dependent DNA replication and viral growth. UL84 was previously shown to interact with IE2 (IE86) in infected cells, and this interaction down-regulates IE2-mediated transcriptional activation in transient assays. UL84 and IE2 were also shown to cooperatively activate a promoter within HCMV oriLyt. UL84 alone can interact with an RNA stem-loop within oriLyt and is bound to this structure within the virion. In an effort to investigate the binding partners for UL84 in infected cells, we pulled down UL84 from protein lysates prepared from HCMV-infected human fibroblasts by using a UL84-specific antibody and resolved the immunoprecipitated protein complexes by two-dimensional gel electrophoresis. We subsequently identified individual proteins by matrix-assisted laser desorption ionization-tandem time of flight analysis. This analysis revealed that UL84 interacts with viral proteins UL44, pp65, and IE2. In addition, a number of cell-encoded proteins were identified, including ubiquitin-conjugating enzyme E2, casein kinase II (CKII), and the multifunctional protein p32. We also confirmed the interaction between UL84 and IE2 as well as the interaction of UL84 with importin alpha. UL44, pp65, and CKII interactions were confirmed to occur in infected and cotransfected cells by coimmunoprecipitation assays followed by Western blotting. Ubiquitination of UL84 occurred in the presence and absence of the proteasome activity inhibitor MG132 in infected cells. The identification of UL84 binding partners is a significant step toward the understanding of the function of this significant replication protein. [Abstract/Link to Full Text]

Pacheco B, Basmaciogullari S, Labonte JA, Xiang SH, Sodroski J
Adaptation of the human immunodeficiency virus type 1 envelope glycoproteins to new world monkey receptors.
J Virol. 2008 Jan;82(1):346-57.
Human immunodeficiency virus type 1 (HIV-1) infection encounters an early block in the cells of New World monkeys because the CD4 receptor does not efficiently support HIV-1 entry. We adapted HIV-1(NL4-3) and HIV-1(KB9), two HIV-1 variants with different envelope glycoproteins, to replicate efficiently in cells expressing the CD4 and CXCR4 proteins of the common marmoset, a New World monkey. The HIV-1(NL4-3) adaptation involves three gp120 changes that result in a specific increase in affinity for the marmoset CD4 glycoprotein. The already high affinity of the HIV-1(KB9) envelope glycoproteins for marmoset CD4 did not significantly change as a result of the adaptation. Instead, changes in the gp120 variable loops and gp41 ectodomain resulted in improved replication in cells expressing the marmoset receptors. HIV-1(KB9) became relatively sensitive to neutralization by soluble CD4 and antibodies as a result of the adaptation. These results demonstrate the distinct mechanistic pathways by which the HIV-1 envelope glycoproteins can adapt to less-than-optimal CD4 molecules and provide HIV-1 variants that can overcome some of the early blocks in New World monkey cells. [Abstract/Link to Full Text]

Robertson SJ, Ammann CG, Messer RJ, Carmody AB, Myers L, Dittmer U, Nair S, Gerlach N, Evans LH, Cafruny WA, Hasenkrug KJ
Suppression of acute anti-friend virus CD8+ T-cell responses by coinfection with lactate dehydrogenase-elevating virus.
J Virol. 2008 Jan;82(1):408-18.
Friend virus (FV) and lactate dehydrogenase-elevating virus (LDV) are endemic mouse viruses that can cause long-term chronic infections in mice. We found that numerous mouse-passaged FV isolates also contained LDV and that coinfection with LDV delayed FV-specific CD8(+) T-cell responses during acute infection. While LDV did not alter the type of acute pathology induced by FV, which was severe splenomegaly caused by erythroproliferation, the immunosuppression mediated by LDV increased both the severity and the duration of FV infection. Compared to mice infected with FV alone, those coinfected with both FV and LDV had delayed CD8(+) T-cell responses, as measured by FV-specific tetramers. This delayed response accounted for the prolonged and exacerbated acute phase of FV infection. Suppression of FV-specific CD8(+) T-cell responses occurred not only in mice infected concomitantly with LDV but also in mice chronically infected with LDV 8 weeks prior to infection with FV. The LDV-induced suppression was not mediated by T regulatory cells, and no inhibition of the CD4(+) T-cell or antibody responses was observed. Considering that most human adults are carriers of chronically infectious viruses at the time of new virus insults and that coinfections with viruses such as human immunodeficiency virus and hepatitis C virus are currently epidemic, it is of great interest to determine how infection with one virus may impact host responses to a second infection. Coinfection of mice with LDV and FV provides a well-defined, natural host model for such studies. [Abstract/Link to Full Text]

Sankaran S, George MD, Reay E, Guadalupe M, Flamm J, Prindiville T, Dandekar S
Rapid onset of intestinal epithelial barrier dysfunction in primary human immunodeficiency virus infection is driven by an imbalance between immune response and mucosal repair and regeneration.
J Virol. 2008 Jan;82(1):538-45.
Gut-associated lymphoid tissue (GALT) is an early target for human immunodeficiency virus type 1 (HIV-1) infection and is a site for severe CD4(+) T-cell depletion. HIV-associated enteropathy is well-documented in chronic HIV-1 infection. However, the initial host responses to HIV infection in GALT and the early molecular correlates of HIV enteropathogenesis have not been characterized during primary HIV infection. In this study, we provide evidence of viral replication in GALT resident CD4(+) T cells and macrophages in primary-stage patients and identify early patterns of host mucosal responses and changes in the molecular microenvironment through gene expression profiling. High levels of viral replication in GALT and marked CD4(+) T-cell depletion correlated with decreased expression levels of genes regulating epithelial barrier maintenance and digestive/metabolic functions. These changes coincided with a marked increase in the transcription of immune activation-, inflammation-, and apoptosis-associated genes. Our findings indicate that HIV-induced pathogenesis in GALT emerges at both the molecular and cellular levels prior to seroconversion in primary HIV infection, potentially setting the stage for disease progression by impairing the ability to control viral replication and repair and regenerate intestinal mucosal tissues. [Abstract/Link to Full Text]

Sun M, Fuentes SM, Timani K, Sun D, Murphy C, Lin Y, August A, Teng MN, He B
Akt plays a critical role in replication of nonsegmented negative-stranded RNA viruses.
J Virol. 2008 Jan;82(1):105-14.
The order Mononegavirales (comprised of nonsegmented negative-stranded RNA viruses or NNSVs) contains many important pathogens. Parainfluenza virus 5 (PIV5), formerly known as simian virus 5, is a prototypical paramyxovirus and encodes a V protein, which has a cysteine-rich C terminus that is conserved among all paramyxoviruses. The V protein of PIV5, like that of many other paramyxoviruses, plays an important role in regulating viral RNA synthesis. In this work, we show that V interacts with Akt, a serine/threonine kinase, also known as protein kinase B. Both pharmacological inhibitors and small interfering RNA against Akt1 reduced PIV5 replication, indicating that Akt plays a critical role in PIV5 replication. Furthermore, treatment with Akt inhibitors also reduced the replication of several other paramyxoviruses, as well as vesicular stomatitis virus, the prototypical rhabdovirus, indicating that Akt may play a more universal role in NNSV replication. The phosphoproteins (P proteins) of NNSVs are essential cofactors for the viral RNA polymerase complex and require heavy phosphorylation for their activity. Inhibition of Akt activity reduced the level of P phosphorylation, suggesting that Akt is involved in regulating viral RNA synthesis. In addition, Akt1 phosphorylated a recombinant P protein of PIV5 purified from bacteria. The finding that Akt plays a critical role in replication of NNSV will lead to a better understanding of how these viruses replicate, as well as novel strategies to treat infectious diseases caused by NNSVs. [Abstract/Link to Full Text]

Slobodskaya O, Laarman A, Spaan WJ
Intracellular restriction of a productive noncytopathic coronavirus infection.
J Virol. 2008 Jan;82(1):451-60.
Virus infection in vitro can either result in a cytopathic effect (CPE) or proceed without visible changes in infected cells (noncytopathic infection). We are interested in understanding the mechanisms controlling the impact of coronavirus infection on host cells. To this end, we compared a productive, noncytopathic infection of murine hepatitis virus (MHV) strain A59 in the fibroblastlike cell line NIH 3T3 with cytopathic MHV infections. Infected NIH 3T3 cells could be cultured for up to 4 weeks without apparent CPE and yet produce virus at 10(7) to 10(8) PFU/ml. Using flow cytometry, we demonstrated that NIH 3T3 cells expressed as much MHV receptor CEACAM1 as other cell lines which die from MHV infection. In contrast, using quantitative reverse transcription-PCR and metabolic labeling of RNA, we found that the rate of viral RNA amplification in NIH 3T3 cells was lower than the rate in cells in which MHV induces a CPE. The rate of cellular RNA synthesis in contact-inhibited confluent NIH 3T3 cells was also lower than in cells permissive to cytopathic MHV infection. However, the induction of cellular RNA synthesis in growing NIH 3T3 cells did not result in an increase of either viral RNA amplification or CPE. Our results suggest that a specific, receptor CEACAM1-independent mechanism restricting coronaviral RNA synthesis and CPE is present in NIH 3T3 and, possibly, other cells with preserved contact inhibition. [Abstract/Link to Full Text]

Valentine LE, Piaskowski SM, Rakasz EG, Henry NL, Wilson NA, Watkins DI
Recognition of escape variants in ELISPOT does not always predict CD8+ T-cell recognition of simian immunodeficiency virus-infected cells expressing the same variant sequences.
J Virol. 2008 Jan;82(1):575-81.
Human immunodeficiency virus (HIV)'s tremendous sequence variability is a major obstacle for the development of cytotoxic-T-lymphocyte-based vaccines, especially since much of this variability is selected for by CD8(+) T cells. We investigated to what extent reactivity to escape variant peptides in standard enzyme-linked immunospot (ELISPOT) assays predicts the recognition of cells infected with corresponding escape variant viruses. Most of the variant peptides tested were recognized in standard ELISPOT and intracellular cytokine stain (ICS) assays. Functional avidity of epitope-specific T cells for some of the variants was, however, markedly reduced. These mutations which reduced avidity also abrogated recognition by epitope-specific CD8(+) T cells in a viral suppression assay. Our results indicate that "cross-reactive" CD8(+) T-cell responses identified in ELISPOT and ICS assays using a single high concentration of variant peptide often fail to predict the recognition of cells infected with variant viruses. [Abstract/Link to Full Text]

Guan H, Jiao J, Ricciardi RP
Tumorigenic adenovirus type 12 E1A inhibits phosphorylation of NF-kappaB by PKAc, causing loss of DNA binding and transactivation.
J Virol. 2008 Jan;82(1):40-8.
Human adenovirus type 12 (Ad12) E1A protein (E1A-12) is the key determinant of viral tumorigenesis. E1A-12 mediates major histocompatibility complex class I (MHC-I) shutoff by inhibiting the DNA binding of the transcriptional activator NF-kappaB (p50/p65) to the class I enhancer. This enables Ad12 tumorigenic cells to avoid class I recognition and lysis by cytotoxic T lymphocytes. In this study, we demonstrate that the phosphorylation of p50 and p65 by the catalytic subunit of protein kinase A (PKAc) is essential for NF-kappaB DNA binding and transactivation activity. Treatment with H89 and knockdown of PKAc in cells led to the inhibition of phosphorylation at p50 Ser(337) and p65 Ser(276) and loss of DNA binding by NF-kappaB. Importantly, NF-kappaB phosphorylation by PKAc was repressed by tumorigenic E1A-12, but not by nontumorigenic Ad5 E1A (E1A-5). The stable introduction of E1A-12 into Ad5 nontumorigenic cells resulted in a decrease in the phosphorylation of NF-kappaB, loss of NF-kappaB DNA binding, and the failure of NF-kappaB to activate a target promoter, as well as diminution of MHC-I transcription and cell surface expression. Significantly, the amount and enzymatic activity of PKAc were not altered in Ad12 tumorigenic cells relative to its amount and activity in nontumorigenic Ad5 cells. These results demonstrate that E1A-12 specifically prevents NF-kappaB from being phosphorylated by PKAc. [Abstract/Link to Full Text]

Mee CJ, Grove J, Harris HJ, Hu K, Balfe P, McKeating JA
Effect of cell polarization on hepatitis C virus entry.
J Virol. 2008 Jan;82(1):461-70.
The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains. [Abstract/Link to Full Text]

Fujiwara M, Tanuma J, Koizumi H, Kawashima Y, Honda K, Mastuoka-Aizawa S, Dohki S, Oka S, Takiguchi M
Different abilities of escape mutant-specific cytotoxic T cells to suppress replication of escape mutant and wild-type human immunodeficiency virus type 1 in new hosts.
J Virol. 2008 Jan;82(1):138-47.
There is much evidence that in human immunodeficiency virus type 1 (HIV-1)-infected individuals, strong cytotoxic T lymphocyte (CTL)-mediated immune pressure results in the selection of HIV-1 mutants that have escaped from wild-type-specific CTLs. If escape mutant-specific CTLs are not elicited in new hosts sharing donor HLA molecules, the transmission of these mutants results in the accumulation of escape mutants in the population. However, whether escape mutant-specific CTLs are definitively not elicited in new hosts sharing donor HLA molecules still remains unclear. A previous study showed that a Y-to-F substitution at the second position (2F) of the Nef138-10 epitope is significantly detected in HLA-A*2402(+) hemophilic donors. Presently, we confirmed that this 2F mutant was an escape mutant by demonstrating strong and weak abilities of Nef138-10-specific CTL clones to suppress replication of the wild-type and 2F mutant viruses, respectively. We demonstrated the existence of the 2F-specific CTLs in three new hosts who had been primarily infected with the 2F mutant. The 2F-specific CTL clones suppressed the replication of both wild-type and mutant viruses. However, the abilities of these clones to suppress replication of the 2F virus were much weaker than those of wild-type-specific and the 2F-specific ones to suppress replication of the wild-type virus. These findings indicate that the 2F mutant is conserved in HIV-1-infected donors having HLA-A*2402, because the 2F-specific CTLs failed to completely suppress the 2F mutant replication and effectively prevented viral reversion in new hosts carrying HLA-A*2402. [Abstract/Link to Full Text]

Lopes L, Dewannieux M, Gileadi U, Bailey R, Ikeda Y, Whittaker C, Collin MP, Cerundolo V, Tomihari M, Ariizumi K, Collins MK
Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses.
J Virol. 2008 Jan;82(1):86-95.
Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2(+) cells after intravenous injection and in CD11c(+) dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8(+) T-cell response in HLA-A2 transgenic mice and stimulated a CD4(+) T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A(b). As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine. [Abstract/Link to Full Text]

Bryant KF, Coen DM
Inhibition of translation by a short element in the 5' leader of the herpes simplex virus 1 DNA polymerase transcript.
J Virol. 2008 Jan;82(1):77-85.
Many viruses regulate gene expression, both globally and specifically, to achieve maximal rates of replication. During herpes simplex virus 1 infection, translation of the DNA polymerase (Pol) catalytic subunit is inefficient relative to other proteins of the same temporal class (D. R. Yager, A. I. Marcy, and D. M. Coen., J. Virol. 64:2217-2225, 1990). To investigate the mechanisms involved in the inefficient translation of Pol and to determine whether this inefficient translation could affect viral replication, we performed a mutagenic analysis of the 5' end of the pol transcript. We found that a short sequence ( approximately 55 bases) in the 5' leader of the transcript is both necessary and sufficient to inhibit translation in rabbit reticulocyte lysates and sufficient to inhibit reporter gene translation in transfected cells. RNase structure mapping experiments indicated that the inhibitory element adopts a structure that contains regions of a double-stranded nature, which may interfere with ribosomal loading and/or scanning. Pol accumulated to approximately 2- to 3-fold-higher levels per mRNA in cells infected with a mutant virus containing a deletion of the approximately 55-base inhibitory element than in cells infected with a control virus containing this element. Additionally, the mutant virus replicated less efficiently than the control virus. These results suggest that the inhibitory element regulates Pol translation during infection and that its inhibition of Pol translation is beneficial for viral replication. [Abstract/Link to Full Text]

Yang X, Thannhauser TW, Burrows M, Cox-Foster D, Gildow FE, Gray SM
Coupling genetics and proteomics to identify aphid proteins associated with vector-specific transmission of polerovirus (luteoviridae).
J Virol. 2008 Jan;82(1):291-9.
Cereal yellow dwarf virus-RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F(2) progeny from crosses of vector and nonvector genotypes of S. graminum showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F(2) genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of S. graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission. [Abstract/Link to Full Text]

Jelacic TM, Thompson D, Hanson C, Cmarik JL, Nishigaki K, Ruscetti S
The tyrosine kinase sf-Stk and its downstream signals are required for maintenance of friend spleen focus-forming virus-induced fibroblast transformation.
J Virol. 2008 Jan;82(1):419-27.
Infection of erythroid progenitor cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia and eventually to erythroleukemia in susceptible strains of mice. The viral envelope protein, SFFV gp55, forms a complex with the erythropoietin receptor (EpoR) and a short form of the receptor tyrosine kinase Stk (sf-Stk), activating both and inducing Epo-independent proliferation. Recently, we discovered that coexpression of SFFV gp55 and sf-Stk is sufficient to transform NIH 3T3 and primary fibroblasts. In the current study, we demonstrate that sf-Stk and its downstream effectors are critical to this transformation. Unlike SFFV-derived erythroleukemia cells, which depend on PU.1 expression for maintenance of the transformed phenotype, SFFV gp55-sf-Stk-transformed fibroblasts are negative for PU.1. Underscoring the importance of sf-Stk to fibroblast transformation, knockdown of sf-Stk abolished the ability of these cells to form anchorage-independent colonies. Like SFFV-infected erythroid cells, SFFV gp55-sf-Stk-transformed fibroblasts express high levels of phosphorylated MEK, ERK, phosphatidylinositol 3-kinase (PI3K), Gab1/2, Akt, Jun kinase (JNK), and STAT3, but unlike virus-infected erythroid cells they fail to express phosphorylated STATs 1 and 5, which may require involvement of the EpoR. In addition, the p38 mitogen-activated protein kinase (MAPK) stress response is suppressed in the transformed fibroblasts. Inhibition of either JNK or the PI3K pathway decreases both monolayer proliferation and anchorage-independent growth of the transformed fibroblasts as does the putative kinase inhibitor luteolin, but inhibition of p38 MAPK has no effect. Our results indicate that sf-Stk is a molecular endpoint of transformation that could be targeted directly or with agents against its downstream effectors. [Abstract/Link to Full Text]

Nazarian SH, Rahman MM, Werden SJ, Villeneuve D, Meng X, Brunetti C, Valeriano C, Wong C, Singh R, Barrett JW, Xiang Y, McFadden G
Yaba monkey tumor virus encodes a functional inhibitor of interleukin-18.
J Virol. 2008 Jan;82(1):522-8.
Interleukin-18 (IL-18) is a critical proinflammatory cytokine whose extracellular bioactivity is regulated by a cellular IL-18 binding protein (IL-18BP). Many poxviruses have acquired variants of this IL-18BP gene, some of which have been shown to act as viral virulence factors. Yaba monkey tumor virus (YMTV) encodes a related family member, 14L, which is similar to the orthopoxvirus IL-18BPs. YMTV 14L was expressed from a baculovirus system and tested for its ability to bind and inhibit IL-18. We found that YMTV 14L bound both human IL-18 (hIL-18) and murine IL-18 with high affinity, at 4.1 nM and 6.5 nM, respectively. YMTV 14L was able to fully sequester hIL-18 but could only partially inhibit the biological activity of hIL-18 as measured by gamma interferon secretion from KG-1 cells. Additionally, 17 hIL-18 point mutants were tested by surface plasmon resonance for their ability to bind to YMTV 14L. Two clusters of hIL-18 surface residues were found to be important for the hIL-18-YMTV 14L interaction, in contrast to results for the Variola virus IL-18BP, which has been shown to primarily interact with a single cluster of three amino acids. The altered binding specificity of YMTV 14L most likely represents an adaptation resulting in increased fitness of the virus and affirms the plasticity of poxviral inhibitor domains that target cytokines like IL-18. [Abstract/Link to Full Text]

Domingo-Gil E, Pérez-Jiménez E, Ventoso I, Nájera JL, Esteban M
Expression of the E3L gene of vaccinia virus in transgenic mice decreases host resistance to vaccinia virus and Leishmania major infections.
J Virol. 2008 Jan;82(1):254-67.
The E3L gene of vaccinia virus (VACV) encodes the E3 protein that in cultured cells inhibits the activation of interferon (IFN)-induced proteins, double-stranded RNA-dependent protein kinase (PKR), 2'-5'-oligoadenylate synthetase/RNase L (2-5A system) and adenosine deaminase (ADAR-1), thus helping the virus to evade host responses. Here, we have characterized the in vivo E3 functions in a murine inducible cell culture system (E3L-TetOFF) and in transgenic mice (TgE3L). Inducible E3 expression in cultured cells conferred on cells resistance to the antiviral action of IFN against different viruses, while expression of the E3L gene in TgE3L mice triggered enhanced sensitivity of the animals to pathogens. Virus infection monitored in TgE3L mice by different inoculation routes (intraperitoneal and tail scarification) showed that transgenic mice became more susceptible to VACV infection than control mice. TgE3L mice were also more susceptible to Leishmania major infection, leading to an increase in parasitemia compared to control mice. The enhanced sensitivity of TgE3L mice to VACV and L. major infections occurred together with alterations in the host immune system, as revealed by decreased T-cell responses to viral antigens in the spleen and lymph nodes and by differences in the levels of specific innate cell populations. These results demonstrate that expression of the E3L gene in transgenic mice partly reverses the resistance of the host to viral and parasitic infections and that these effects are associated with immune alterations. [Abstract/Link to Full Text]

Xiao R, Park K, Lee H, Kim J, Park C
Identification and classification of endogenous retroviruses in cattle.
J Virol. 2008 Jan;82(1):582-7.
The aim of this study was to identify the endogenous retrovirus (ERV) sequences in a bovine genome. We subjected bovine genomic DNA to PCR with degenerate or ovine ERV (OERV) family-specific primers that aimed to amplify the retroviral pro/pol region. Sequence analysis of 113 clones obtained by PCR revealed that 69 were of retroviral origin. On the basis of the OERV classification system, these clones from degenerate PCR could be divided into the beta3, gamma4, and gamma9 families. PCR with OERV family-specific primers revealed an additional ERV that was classified into the bovine endogenous retrovirus (BERV) gamma7 family. In conclusion, here we report the results of a genome scale study of the BERV. Our study shows that the ERV family expansion in cattle may be somewhat limited, while more diverse family members of ERVs have been reported from other artiodactyls, such as pigs and sheep. [Abstract/Link to Full Text]

Oh J, Chang KW, Wierzchoslawski R, Alvord WG, Hughes SH
Rous sarcoma virus (RSV) integration in vivo: a CA dinucleotide is not required in U3, and RSV linear DNA does not autointegrate.
J Virol. 2008 Jan;82(1):503-12.
The sequences required for integration of retroviral DNA have been analyzed in vitro. However, the in vitro experiments do not agree on which sequences are required for integration: for example, whether or not the conserved CA dinucleotide in the 3' end of the viral DNA is required for normal integration. At least a portion of the problem is due to differences in the experimental conditions used in the in vitro assays. To avoid the issue of what experimental conditions to use, we took an in vivo approach. We made mutations in the 5' end of the U3 sequence of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z. We present evidence that, in RSV, the CA dinucleotide in the 5' end of U3 is not essential for appropriate integration. This result differs from the results seen with mutations in the U5 end, where the CA appears to be essential for proper integration in vivo. In addition, based on the structure of circular viral DNAs smaller than the full-length viral genome, our results suggest that there is little, if any, integrase-mediated autointegration of RSV linear DNA in vivo. [Abstract/Link to Full Text]

Danthi P, Kobayashi T, Holm GH, Hansberger MW, Abel TW, Dermody TS
Reovirus apoptosis and virulence are regulated by host cell membrane penetration efficiency.
J Virol. 2008 Jan;82(1):161-72.
Apoptosis plays an important role in the pathogenesis of reovirus encephalitis and myocarditis in infected animals. Differences in apoptosis efficiency displayed by reovirus strains are linked to the viral mu1-encoding M2 gene segment. Studies using pharmacologic inhibitors of reovirus replication demonstrate that apoptosis induction by reovirus requires viral disassembly in cellular endosomes but not RNA synthesis. Since the mu1 protein functions to pierce endosomal membranes during this temporal window, these findings point to an important role for mu1 in activating signaling pathways that lead to apoptosis. To understand mechanisms used by mu1 to induce apoptosis, a panel of mu1 mutant viruses generated by reverse genetics was analyzed for the capacities to penetrate host cell membranes, activate proapoptotic signaling pathways, evoke cell death, and produce encephalitis in newborn mice. We found that single amino acid changes within the delta region of mu1 reduce the efficiency of membrane penetration. These mutations also diminish the capacities of reovirus to activate proapoptotic transcription factors NF-kappaB and IRF-3 and elicit apoptosis. Additionally, we observed that following intracranial inoculation, an apoptosis-deficient mu1 mutant is less virulent in newborn mice in comparison to the wild-type virus. These results indicate a critical function for the membrane penetration activity of mu1 in evoking prodeath signaling pathways that regulate reovirus pathogenesis. [Abstract/Link to Full Text]

Liang Y, Huang T, Ly H, Parslow TG, Liang Y
Mutational analyses of packaging signals in influenza virus PA, PB1, and PB2 genomic RNA segments.
J Virol. 2008 Jan;82(1):229-36.
The influenza A virus genome consists of eight negative-sense RNA segments that must each be packaged to produce an infectious virion. We have previously mapped the minimal cis-acting regions necessary for efficient packaging of the PA, PB1, and PB2 segments, which encode the three protein subunits of the viral RNA polymerase. The packaging signals in each of these RNAs lie within two separate regions at the 3' and 5' termini, each encompassing the untranslated region and extending up to 80 bases into the adjacent coding sequence. In this study, we introduced scanning mutations across the coding regions in each of these RNA segments in order to finely define the packaging signals. We found that mutations producing the most severe defects were confined to a few discrete 5' sites in the PA or PB1 coding regions but extended across the entire (80-base) 5' coding region of PB2. In sequence comparisons among more than 580 influenza A strains from diverse hosts, these highly deleterious mutations were each found to affect one or more conserved bases, though they did not all lie within the most broadly conserved portions of the regions that we interrogated. We have introduced silent and conserved mutations to the critical packaging sites, which did not affect protein function but impaired viral replication at levels roughly similar to those of their defects in RNA packaging. Interestingly, certain mutations showed strong tendencies to revert to wild-type sequences, which implies that these putative packaging signals are critical for the influenza life cycle. [Abstract/Link to Full Text]

Zhu Q, Yang H, Chen W, Cao W, Zhong G, Jiao P, Deng G, Yu K, Yang C, Bu Z, Kawaoka Y, Chen H
A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens.
J Virol. 2008 Jan;82(1):220-8.
In 2001 and 2003, we isolated two H5N1 viruses, A/swine/Fujian/1/01 (SW/FJ/01) and A/swine/Fujian/1/03 (SW/FJ/03), from pigs in Fujian Province, southern China. Genetically, these two viruses are similar, although the NS gene of the SW/FJ/03 virus has a 15-nucleotide deletion at coding positions 612 to 626. The SW/FJ/01 virus is highly lethal for chickens, whereas the SW/FJ/03 virus is nonpathogenic for chickens when administrated intravenously or intranasally. To understand the molecular basis for the difference in virulence, we used reverse genetics to create a series of single-gene recombinants of both viruses. We found that a recombinant virus containing the mutated NS gene from the SW/FJ/03 virus in the SW/FJ/01 virus background was completely attenuated in chickens. We also found that viruses expressing the mutant NS1 protein of SW/FJ/03 did not antagonize the induction of interferon (IFN) protein. Conversely, only the recombinant virus containing the wild-type SW/FJ/01 NS gene in the SW/FJ/03 background was lethal in chickens and antagonized IFN protein levels. Further, we proved that the NS1 genes of the two viruses differ in their stabilities in the host cells and in their abilities to interact with the chicken cleavage and polyadenylation specificity factor. These results indicate that the deletion of amino acids 191 to 195 of the NS1 protein is critical for the attenuation of the SW/FJ/03 virus in chickens and that this deletion affects the ability of the virus to antagonize IFN induction in host cells. [Abstract/Link to Full Text]

Sekaric P, Cherry JJ, Androphy EJ
Binding of human papillomavirus type 16 E6 to E6AP is not required for activation of hTERT.
J Virol. 2008 Jan;82(1):71-6.
The human papillomavirus (HPV) type 16 (HPV16) E6 protein stimulates transcription of the catalytic subunit of telomerase, hTERT, in epithelial cells. It has been reported that binding to the ubiquitin ligase E6AP is required for this E6 activity, with E6 directing E6AP to the hTERT promoter. We previously reported two E6AP binding-defective HPV16 E6 mutations that induced immortalization of human mammary epithelial cells. Because activation of hTERT is proposed to be necessary for epithelial cell immortalization, we sought to further characterize the relationship between E6/E6AP association and telomerase induction. We demonstrate that while these E6 mutants do not bind E6AP, they retain the capability to stimulate the expression of hTERT. Chromatin immunoprecipitation assays confirmed the presence of Myc, wild-type E6, and the E6AP binding-defective E6 mutants, but not E6AP itself, at the endogenous hTERT promoter. Interestingly, an immortalization-defective E6 mutant localized to the hTERT promoter but failed to increase transcription. We conclude that binding to E6AP is not necessary for E6 localization to or activation of the hTERT promoter and that another activity of E6 is involved in hTERT activation. [Abstract/Link to Full Text]

Kapoor A, Victoria J, Simmonds P, Wang C, Shafer RW, Nims R, Nielsen O, Delwart E
A highly divergent picornavirus in a marine mammal.
J Virol. 2008 Jan;82(1):311-20.
Nucleic acids from an unidentified virus from ringed seals (Phoca hispida) were amplified using sequence-independent PCR, subcloned, and then sequenced. The full genome of a novel RNA virus was derived, identifying the first sequence-confirmed picornavirus in a marine mammal. The phylogenetic position of the tentatively named seal picornavirus 1 (SePV-1) as an outlier to the grouping of parechoviruses was found consistently in alignable regions of the genome. A mean protein sequence identity of only 19.3 to 30.0% was found between the 3D polymerase gene sequence of SePV-1 and those of other picornaviruses. The predicted secondary structure of the short 506-base 5'-untranslated region showed some attributes of a type IVB internal ribosome entry site, and the polyprotein lacked an apparent L peptide, both properties associated with the Parechovirus genus. The presence of two SePV-1 2A genes and of the canonical sequence required for cotranslational cleavage resembled the genetic organization of Ljungan virus. Minor genetic variants were detected in culture supernatants derived from 8 of 108 (7.4%) seals collected in 2000 to 2002, indicating a high prevalence of SePV-1 in this hunted seal population. The high level of genetic divergence of SePV-1 compared to other picornaviruses and its mix of characteristics relative to its closest relatives support the provisional classification of SePV-1 as the prototype for a new genus in the family Picornaviridae. [Abstract/Link to Full Text]

Molina S, Castet V, Pichard-Garcia L, Wychowski C, Meurs E, Pascussi JM, Sureau C, Fabre JM, Sacunha A, Larrey D, Dubuisson J, Coste J, McKeating J, Maurel P, Fournier-Wirth C
Serum-derived hepatitis C virus infection of primary human hepatocytes is tetraspanin CD81 dependent.
J Virol. 2008 Jan;82(1):569-74.
Hepatitis C virus-positive serum (HCVser, genotypes 1a to 3a) or HCV cell culture (JFH1/HCVcc) infection of primary normal human hepatocytes was assessed by measuring intracellular HCV RNA strands. Anti-CD81 antibodies and siRNA-CD81 silencing markedly inhibited (>90%) HCVser infection irrespective of HCV genotype, viral load, or liver donor, while hCD81-large intracellular loop (LEL) had no effect. However, JFH1/HCVcc infection of hepatocytes was modestly inhibited (40 to 60%) by both hCD81-LEL and anti-CD81 antibodies. In conclusion, CD81 is involved in HCVser infection of human hepatocytes, and comparative studies of HCVser versus JFH1/HCVcc infection of human hepatocytes and Huh-7.5 cells revealed that the cell-virion combination is determinant of the entry process. [Abstract/Link to Full Text]

Luo D, Xu T, Hunke C, Grüber G, Vasudevan SG, Lescar J
Crystal structure of the NS3 protease-helicase from dengue virus.
J Virol. 2008 Jan;82(1):173-83.
Several flaviviruses are important human pathogens, including dengue virus, a disease against which neither a vaccine nor specific antiviral therapies currently exist. During infection, the flavivirus RNA genome is translated into a polyprotein, which is cleaved into several components. Nonstructural protein 3 (NS3) carries out enzymatic reactions essential for viral replication, including proteolysis of the polyprotein through its serine protease N-terminal domain, with a segment of 40 residues from the NS2B protein acting as a cofactor. The ATPase/helicase domain is located at the C terminus of NS3. Atomic structures are available for these domains separately, but a molecular view of the full-length flavivirus NS3 polypeptide is still lacking. We report a crystallographic structure of a complete NS3 molecule fused to 18 residues of the NS2B cofactor at a resolution of 3.15 A. The relative orientation between the protease and helicase domains is drastically different than the single-chain NS3-NS4A molecule from hepatitis C virus, which was caught in the act of cis cleavage at the NS3-NS4A junction. Here, the protease domain sits beneath the ATP binding site, giving the molecule an elongated shape. The domain arrangement found in the crystal structure fits nicely into an envelope determined ab initio using small-angle X-ray scattering experiments in solution, suggesting a stable molecular conformation. We propose that a basic patch located at the surface of the protease domain increases the affinity for nucleotides and could also participate in RNA binding, explaining the higher unwinding activity of the full-length enzyme compared to that of the isolated helicase domain. [Abstract/Link to Full Text]

Ujike M, Nishikawa H, Otaka A, Yamamoto N, Yamamoto N, Matsuoka M, Kodama E, Fujii N, Taguchi F
Heptad repeat-derived peptides block protease-mediated direct entry from the cell surface of severe acute respiratory syndrome coronavirus but not entry via the endosomal pathway.
J Virol. 2008 Jan;82(1):588-92.
The peptides derived from the heptad repeat (HRP) of severe acute respiratory syndrome coronavirus (SCoV) spike protein (sHRPs) are known to inhibit SCoV infection, yet their efficacies are fairly low. Recently our research showed that some proteases facilitated SCoV's direct entry from the cell surface, resulting in a more efficient infection than the previously known infection via endosomal entry. To compare the inhibitory effect of the sHRP in each pathway, we selected two sHRPs, which showed a strong inhibitory effect on the interaction of two heptad repeats in a rapid and virus-free in vitro assay system. We found that they efficiently inhibited SCoV infection of the protease-mediated cell surface pathway but had little effect on the endosomal pathway. This finding suggests that sHRPs may effectively prevent infection in the lungs, where SCoV infection could be enhanced by proteases produced in this organ. This is the first observation that HRP exhibits different effects on virus that takes the endosomal pathway and virus that enters directly from the cell surface. [Abstract/Link to Full Text]

Dasgupta A, Jung KJ, Jeong SJ, Brady JN
Inhibition of methyltransferases results in induction of g2/m checkpoint and programmed cell death in human T-lymphotropic virus type 1-transformed cells.
J Virol. 2008 Jan;82(1):49-59.
Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia. The HTLV-1-encoded protein Tax transactivates the viral long terminal repeat and plays a critical role in virus replication and transformation. Previous work from our laboratory demonstrated that coactivator-associated arginine methytransferase 1, a protein arginine methytransferase, was important for Tax-mediated transactivation. To further investigate the role of methyltransferases in viral transcription, we utilized adenosine-2,3-dialdehyde (AdOx), an adenosine analog and S-adenosylmethionine-dependent methyltransferase inhibitor. The addition of AdOx decreased Tax transactivation in C81, Hut102, and MT-2 cells. Unexpectedly, we found that AdOx potently inhibited the growth of HTLV-1-transformed cells. Further investigation revealed that AdOx inhibited the Tax-activated NF-kappaB pathway, resulting in reactivation of p53 and induction of p53 target genes. Analysis of the NF-kappaB pathway demonstrated that AdOx treatment resulted in degradation of the IkappaB kinase complex and inhibition of NF-kappaB through stabilization of the NF-kappaB inhibitor IkappaBalpha. Our data further demonstrated that AdOx induced G(2)/M cell cycle arrest and cell death in HTLV-1-transformed but not control lymphocytes. These studies demonstrate that protein methylation plays an important role in NF-kappaB activation and survival of HTLV-1-transformed cells. [Abstract/Link to Full Text]

Ryckman BJ, Rainish BL, Chase MC, Borton JA, Nelson JA, Jarvis MA, Johnson DC
Characterization of the human cytomegalovirus gH/gL/UL128-131 complex that mediates entry into epithelial and endothelial cells.
J Virol. 2008 Jan;82(1):60-70.
The entry of human cytomegalovirus (HCMV) into biologically relevant epithelial and endothelial cells involves endocytosis followed by low-pH-dependent fusion. This entry pathway is facilitated by the HCMV UL128, UL130, and UL131 proteins, which form one or more complexes with the virion envelope glycoprotein gH/gL. gH/gL/UL128-131 complexes appear to be distinct from the gH/gL/gO complex, which likely facilitates entry into fibroblasts. In order to better understand the assembly and protein-protein interactions of gH/gL/UL128-131 complexes, we generated HCMV mutants lacking UL128-131 proteins and nonreplicating adenovirus vectors expressing gH, gL, UL128, UL130, and UL131. Our results demonstrate that UL128, UL130, and UL131 can each independently assemble onto gH/gL scaffolds. However, the binding of individual UL128-131 proteins onto gH/gL can significantly affect the binding of other proteins; for example, UL128 increased the binding of both UL130 and UL131 to gH/gL. Direct interactions between gH/UL130, UL130/UL131, gL/UL128, and UL128/UL130 were also observed. The export of gH/gL complexes from the endoplasmic reticulum (ER) to the Golgi apparatus and cell surface was dramatically increased when all of UL128, UL130, and UL131 were coexpressed with gH/gL (with or without gO expression). Incorporation of gH/gL complexes into the virion envelope requires transport beyond the ER. Thus, we concluded that UL128, UL130, and UL131 must all bind simultaneously onto gH/gL for the production of complexes that can function in entry into epithelial and endothelial cells. [Abstract/Link to Full Text]

You S, Rice CM
3' RNA elements in hepatitis C virus replication: kissing partners and long poly(U).
J Virol. 2008 Jan;82(1):184-95.
The hepatitis C virus (HCV) genomic RNA possesses conserved structural elements that are essential for its replication. The 3' nontranslated region (NTR) contains several of these elements: a variable region, the poly(U/UC) tract, and a highly conserved 3' X tail, consisting of stem-loop 1 (SL1), SL2, and SL3. Studies of drug-selected, cell culture-adapted subgenomic replicons have indicated that an RNA element within the NS5B coding region, 5BSL3.2, forms a functional kissing-loop tertiary structure with part of the 3' NTR, 3' SL2. Recent advances now allow the efficient propagation of unadapted HCV genomes in the context of a complete infectious life cycle (HCV cell culture [HCVcc]). Using this system, we determine that the kissing-loop interaction between 5BSL3.2 and 3' SL2 is required for replication in the genotype 2a HCVcc context. Remarkably, the overall integrity of the 5BSL3 cruciform is not an absolute requirement for the kissing-loop interaction, suggesting a model in which trans-acting factor(s) that stabilize this interaction may interact initially with the 3' X tail rather than 5BSL3. The length and composition of the poly(U/UC) tract were also critical determinants of HCVcc replication, with a length of 33 consecutive U residues required for maximal RNA amplification. Interrupting the U homopolymer with C residues was deleterious, implicating a trans-acting factor with a preference for U over mixed pyrimidine nucleotides. Finally, we show that both the poly(U) and kissing-loop RNA elements can function outside of their normal genome contexts. This suggests that the poly(U/UC) tract does not function simply as an unstructured spacer to position the kissing-loop elements. [Abstract/Link to Full Text]

---

Recent Articles in Medical Immunology

---

Assari T
Chronic Granulomatous Disease; fundamental stages in our understanding of CGD.
Med Immunol. 2006;54.
It has been 50 years since chronic granulomatous disease was first reported as a disease which fatally affected the ability of children to survive infections. Various milestone discoveries from the insufficient ability of patients' leucocytes to destroy microbial particles to the underlying genetic predispositions through which the disease is inherited have had important consequences. Longterm antibiotic prophylaxis has helped to fight infections associated with chronic granulomatous disease while the steady progress in bone marrow transplantation and the prospect of gene therapy are hailed as long awaited permanent treatment options. This review unearths the important findings by scientists that have led to our current understanding of the disease. [Abstract/Link to Full Text]

Smith KA
The structure of IL2 bound to the three chains of the IL2 receptor and how signaling occurs.
Med Immunol. 2006;53.
The interleukin-2 molecule and receptor were the first of the interleukins to be discovered and characterized at the molecular level. Now after 20 years of effort, two groups have succeeded in determining the structure of IL2 bound to the external domains of the three receptor chains in a quaternary complex. What do we know now that we did not know before this structural information was available, and how do these new data help us to develop new therapies? [Abstract/Link to Full Text]

Smith KA
The continuing HIV vaccine saga: is a paradigm shift necessary?
Med Immunol. 2006;52.
As pointed out in previous editorials, the development of an effective vaccine for the Human Immunodeficiency Virus capable of preventing infection, or even one capable of preventing the Acquired Immunodeficiency Disease Syndrome, has eluded investigators for the past 20 years. Now Reche and Keskin and their co-workers have provided evidence that an entirely new approach, based upon modern bioinformatics methods and skillful in vitro immunological experiments, may result in an effective way to prime the T cell immune response of normal individuals against conserved peptide epitopes. [Abstract/Link to Full Text]

Reche PA, Keskin DB, Hussey RE, Ancuta P, Gabuzda D, Reinherz EL
Elicitation from virus-naive individuals of cytotoxic T lymphocytes directed against conserved HIV-1 epitopes.
Med Immunol. 2006;51.
Cytotoxic T lymphocytes (CTL) protect against viruses including HIV-1. To avoid viral escape mutants that thwart immunity, we chose 25 CTL epitopes defined in the context of natural infection with functional and/or structural constraints that maintain sequence conservation. By combining HLA binding predictions with knowledge concerning HLA allele frequencies, a metric estimating population protection coverage (PPC) was computed and epitope pools assembled. Strikingly, only a minority of immunocompetent HIV-1 infected individuals responds to pools with PPC >95%. In contrast, virus-naive individuals uniformly expand IFNgamma producing cells and mount anti-HIV-1 cytolytic activity. This disparity suggests a vaccine design paradigm shift from infected to normal subjects. [Abstract/Link to Full Text]

Smith KA
The continuing HIV vaccine saga: naked emperors alongside fairy godmothers.
Med Immunol. 2005 May 6;4(1):6.
The latest developments in the HIV vaccine field were aired at a Keystone Symposium recently. This Commentary summarizes some of the highlights from this meeting, and focuses on some of the developments that appeared particularly promising, as well as those that do not. Unfortunately, the "saga" continues. [Abstract/Link to Full Text]

Smith KA
Wanted, an Anthrax vaccine: Dead or Alive?
Med Immunol. 2005 Apr 18;4(1):5.
It has been more than 100 years since the realization that microbes are capable of causing disease. In that time, we have learned a great deal as to how each organism has adapted to the immune system so as to avoid elimination. As well, we have also learned an immense amount since Louis Pasteur first proposed that the solution to infectious diseases was to culture the microbes and attenuate their virulence, so as to use them as vaccines. From the optimism and promise of the 19th century and immunization as the ultimate answer to the invasion by the microbial world, to the scientific realities of the 21st century, it is of interest to retrace the steps of the earliest microbiologists cum immunologists, to realize how far we've come, as well as how far we yet have to go. This editorial focuses on the history of anthrax as a microbial disease, and the earliest efforts at producing a vaccine for its prevention. [Abstract/Link to Full Text]

Wang JY, Roehrl MH
Anthrax vaccine design: strategies to achieve comprehensive protection against spore, bacillus, and toxin.
Med Immunol. 2005 Mar 24;4(1):4.
The successful use of Bacillus anthracis as a lethal biological weapon has prompted renewed research interest in the development of more effective vaccines against anthrax. The disease consists of three critical components: spore, bacillus, and toxin, elimination of any of which confers at least partial protection against anthrax. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre- and postexposure vaccination to target primarily toxins. Vaccines effective against toxin have been licensed for human use, but need improvement. Vaccines against bacilli have recently been developed by us and others. Whether effective vaccines will be developed against spores is still an open question. An ideal vaccine would confer simultaneous protection against spores, bacilli, and toxins. One step towards this goal is our dually active vaccine, designed to destroy both bacilli and toxin. Existing and potential strategies towards potent and effective anthrax vaccines are discussed in this review. [Abstract/Link to Full Text]

Smith KA
The Classics of Immunology.
Med Immunol. 2005 Mar 10;4(1):3.
Medical Immunology will be publishing invited Reviews and Commentaries from investigators who are at the forefront of their fields, to up-date our readers as to the current state of their art. These Reviews and Commentaries will be accompanied by Editorials that place the current work into the perspective of the first contribution in an area, which resulted in a "Classic" paper. Where possible, links will be provided to the original publication, so that the modern student of immunology can read the original and draw their own conclusions as to the value of the "Classic" contribution, and its relationship to our contemporary views as to how the immune system functions. To begin this process at the very dawn of immunology, we highlight Sir Edward Jenner's first descriptions of the use of cowpox to immunize individuals against the dread disease smallpox. [Abstract/Link to Full Text]

Slifka MK
The Future of Smallpox Vaccination: is MVA the key?
Med Immunol. 2005 Mar 1;4(1):2.
Eradication of the smallpox virus through extensive global vaccination efforts has resulted in one of the most important breakthroughs in medical history, saving countless lives from the severe morbidity and mortality that is associated with this disease. Although smallpox is now extinct in nature, laboratory stocks of this virus still remain and the subject of smallpox vaccination has gained renewed attention due to the potential risk that smallpox may be used as a biological weapon by terrorists or rogue states. Despite having the longest history of any modern vaccine, there is still much to be learned about smallpox vaccination and the correlates of protection remain to be formally defined. This Commentary will discuss the strengths and weaknesses of traditional smallpox vaccination in comparison with immunization using modified vaccinia virus Ankura (MVA), a non-replicating virus with a strong safety record but weakened immunogenicity. [Abstract/Link to Full Text]

Tourneur L, Buzyn A, Chiocchia G
FADD adaptor in cancer.
Med Immunol. 2005 Feb 17;4(1):1.
FADD (Fas Associated protein with Death Domain) is a key adaptor molecule transmitting the death signal mediated by death receptors. In addition, this multiple functional protein is implicated in survival/proliferation and cell cycle progression. FADD functions are regulated via cellular sublocalization, protein phosphorylation, and inhibitory molecules. In the present review, we focus on the role of the FADD adaptor in cancer. Increasing evidence shows that defects in FADD protein expression are associated with tumor progression both in mice and humans. Better knowledge of the mechanisms leading to regulation of FADD functions will improve understanding of tumor growth and the immune escape mechanisms, and could open a new field for therapeutic interventions. [Abstract/Link to Full Text]

Smith KA
The quantal theory of how the immune system discriminates between "self and non-self"
Med Immunol. 2004 Dec 17;3(1):3.
In the past 50 years, immunologists have accumulated an amazing amount of information as to how the immune system functions. However, one of the most fundamental aspects of immunity, how the immune system discriminates between self vs. non-self, still remains an enigma. Any attempt to explain this most intriguing and fundamental characteristic must account for this decision at the level of the whole immune system, but as well, at the level of the individual cells making up the immune system. Moreover, it must provide for a molecular explanation as to how and why the cells behave as they do. The "Quantal Theory", proposed herein, is based upon the "Clonal Selection Theory", first proposed by Sir McFarland Burnet in 1955, in which he explained the remarkable specificity as well as diversity of recognition of everything foreign in the environment. The "Quantal Theory" is built upon Burnet's premise that after antigen selection of cell clones, a proliferative expansion of the selected cells ensues. Furthermore, it is derived from experiments which indicate that the proliferation of antigen-selected cell clones is determined by a quantal, "all-or-none", decision promulgated by a critical number of cellular receptors triggered by the T Cell Growth Factor (TCGF), interleukin 2 (IL2). An extraordinary number of experiments reported especially in the past 20 years, and detailed herein, indicate that the T cell Antigen Receptor (TCR) behaves similarly, and also that there are several critical numbers of triggered TCRs that determine different fates of the T cells. Moreover, the fates of the cells appear ultimately to be determined by the TCR triggering of the IL2 and IL2 receptor (IL2R) genes, which are also expressed in a very quantal fashion. The "Quantal Theory" states that the fundamental decisions of the T cell immune system are dependent upon the cells receiving a critical number of triggered TCRs and IL2Rs and that the cells respond in an all-or-none fashion. The "Quantal Theory" accounts fully for the development of T cells in the thymus, and such fundamental cellular fates as both "positive" and "negative" selection, as well as the decision to differentiate into a "Regulatory T cell" (T-Reg). In the periphery, the "Quantal Theory" accounts for the decision to proliferate or not in response to the presence of an antigen, either non-self or self, or to differentiate into a T-Reg. Since the immune system discriminates between self and non-self antigens by the accumulated number of triggered TCRs and IL2Rs, therapeutic manipulation of the determinants of these quantal decisions should permit new approaches to either enhance or dampen antigen-specific immune responses. [Abstract/Link to Full Text]

Fridkis-Hareli M, Reinherz EL
New approaches to eliciting protective immunity through T cell repertoire manipulation: the concept of thymic vaccination.
Med Immunol. 2004 Dec 8;3(1):2.
Conventional vaccines afford protection against infectious diseases by expanding existing pathogen-specific peripheral lymphocytes, both CD8 cytotoxic effector (CTL) and CD4 helper T cells. The latter induce B cell maturation and antibody production. As a consequence, lymphocytes within the memory pool are poised to rapidly proliferate at the time of a subsequent infection. The "thymic vaccination" concept offers a novel way to alter the primary T cell repertoire through exposure of thymocytes to altered peptide ligands (APL) with reduced T cell receptor (TCR) affinity relative to cognate antigens recognized by those same TCRs. Thymocyte maturation (i.e. positive selection) is enhanced by low affinity interaction between a TCR and an MHC-bound peptide in the thymus and subsequent emigration of mature cells into the peripheral T lymphocyte pool follows. In principal, such variants of antigens derived from infectious agents could be utilized for peptide-driven maturation of thymocytes bearing pathogen-specific TCRs. To test this idea, APLs of gp33-41, a Db-restricted peptide derived from the lymphocytic choriomeningitis virus (LCMV) glycoprotein, and of VSV8, a Kb-restricted peptide from the vesicular stomatitis virus (VSV) nucleoprotein, have been designed and their influence on thymic maturation of specific TCR-bearing transgenic thymocytes examined in vivo using irradiation chimeras. Injection of APL resulted in positive selection of CD8 T cells expressing the relevant viral specificity and in the export of those virus-specific CTL to lymph nodes without inducing T cell proliferation. Thus, exogenous APL administration offers the potential of expanding repertoires in vivo in a manner useful to the organism. To efficiently peripheralize antigen-specific T cells, concomitant enhancement of mechanisms promoting thymocyte migration appears to be required. This commentary describes the rationale for thymic vaccination and addresses the potential prophylactic and therapeutic applications of this approach for treatment of infectious diseases and cancer. Thymic vaccination-induced peptide-specific T cells might generate effective immune protection against disease-causing agents, including those for which no effective natural protection exists. [Abstract/Link to Full Text]

Feng B, Cheng S, Pear WS, Liou HC
NF-kB inhibitor blocks B cell development at two checkpoints.
Med Immunol. 2004 Mar 29;3(1):1.
Members of the NF-kB transcription factor family are differentially expressed in the B cell lineage. Disruption of individual or two NF-kB subunits exhibits distinct defects in B lymphocyte development, activation, and survival. However, the role each NF-kB plays during B cell development has been obscured by molecular compensation. To address this issue, a trans-dominant form of IkBalpha was transduced into bone marrow cells to act as a pan-inhibitor of NF-kB using a retroviral system. While the development of T-lymphocytes and myeloid cell lineages was not grossly affected by the transduced IkBalpha gene, a significant reduction in the number and percentage of B lineage cells was apparent in IkBalpha transduced chimeric mice. IkBalpha expression decreased the percentage of pre-B and immature B cell subsets in the bone marrow and further impaired the development of follicular mature B cells and marginal zone B cells in the periphery. Introduction of the Bcl-X transgene completely restored the pre-B and immature B cell pool in the bone marrow. However, despite a significant improvement of overall viability of the B cell lineage, Bcl-X expression was insufficient to overcome the maturation block resulting from NF-kB inhibition. Together, our study suggests that NF-kB activity is required for two distinct checkpoints during B cell development: one is for pre-B/immature B cell viability, the other is to provide both survival and maturation signals to ensure the proper development of follicular mature B cells. [Abstract/Link to Full Text]

Smith KA
The HIV vaccine saga.
Med Immunol. 2003 Feb 14;2(1):1.
The development of a vaccine that can prevent infection by the Human immunodeficiency virus or prevent the Acquired Immunodeficiency Syndrome has remained elusive despite 20 years of scientific effort. This "Commentary" analyzes the reasons that the development of a vaccine has been so difficult, and proposes a plan to work towards an immunological approach to investigate the best vaccine candidates in the first world in individuals who are already infected, before taking the most promising vaccines to the developing world to attempt to prevent infection and disease. SAGA: (Old Norse) "a long, continued heroic story that is action-packed, but not especially romantic, and that is historical or legendary or both". [Abstract/Link to Full Text]

Gorski A, Dabrowska K, Switala-Jele? K, Nowaczyk M, Weber-Dabrowska B, Boratynski J, Wietrzyk J, Opolski A
New insights into the possible role of bacteriophages in host defense and disease.
Med Immunol. 2003 Feb 14;2(1):2.
BACKGROUND: While the ability of bacteriophages to kill bacteria is well known and has been used in some centers to combat antibiotics - resistant infections, our knowledge about phage interactions with mammalian cells is very limited and phages have been believed to have no intrinsic tropism for those cells. PRESENTATION OF THE HYPOTHESIS: At least some phages (e.g., T4 coliphage) express Lys-Arg-Gly (KGD) sequence which binds beta3 integrins (primarily alphaIIbbeta3). Therefore, phages could bind beta3+ cells (platelets, monocytes, some lymphocytes and some neoplastic cells) and downregulate activities of those cells by inhibiting integrin functions. TESTING THE HYPOTHESIS: Binding of KGD+ phages to beta3 integrin+ cells may be detected using standard techniques involving phage - mediated bacterial lysis and plaque formation. Furthermore, the binding may be visualized by electron microscopy and fluorescence using labelled phages. Binding specificity can be confirmed with the aid of specific blocking peptides and monoclonal antibodies. In vivo effects of phage - cell interactions may be assessed by examining the possible biological effects of beta3 blockade (e.g., anti-metastatic activity). IMPLICATION OF THE HYPOTHESIS: If, indeed, phages can modify functions of beta3+ cells (platelets, monocytes, lymphocytes, cancer cells) they could be important biological response modifiers regulating migration and activities of those cells. Such novel understanding of their role could open novel perspectives in their potential use in treatment of cardiovascular and autoimmune disease, graft rejection and cancer. [Abstract/Link to Full Text]

Khanna A, Plummer M, Bromberek K, Woodliff J, Hariharan S
Immunomodulation in stable renal transplant recipients with concomitant tacrolimus and sirolimus therapy.
Med Immunol. 2002 Nov 19;1(1):3.
BACKGROUND: Long term treatment with immunosuppressive agents results in nephrotoxicity in renal transplant recipients. We explored the effect of combination of Tacrolimus (TAC) and Sirolimus (SRL) on the immune system in renal transplant recipients. METHODS: 10 stable renal transplant recipients were selected to participate in a pharmacokinetic study with a combination of TAC and SRL. Blood was drawn on day zero and 14 days post treatment. Lymphocyte proliferation was quantified by 3H-thymidine uptake assay (results expressed as counts per minute). The mRNA expression was studied by RT-PCR and serum levels of cytokines were quantified by ELISA and a cytokine bead array system. RESULTS: Lymphocyte proliferative response to PHA (p < 0.05), Con A (p < 0.006) and Anti-CD3 (p <0.005) were significantly decreased in patients who received both TAC and SRL compared to TAC alone. The mRNA expression of proinflammatory cytokines TNF-alpha (p < 0.05), cyclins G (p < 0.01) and E (p < 05) were decreased, and of TGF-beta (p < 0.03) and p21 (p < 0.05) were increased in patients treated with this combination. Circulating levels of IFN-gamma (p < 0.04), IL-4 (p < 0.02), and Il-2 (p < 0.03) were significantly inhibited and elevation of TGF-beta (p < 0.04) was observed in patients treated with TAC and SRL combination. CONCLUSION: These novel findings demonstrate that addition of SRL to TAC therapy enhances immuno modulation and causes increased immunosuppression providing a rationale for this concomitant therapy. [Abstract/Link to Full Text]

Smith KA
Optimal clinical trial designs for immune-based therapies in persistent viral infections.
Med Immunol. 2002 Nov 21;1(1):4.
There is now effective therapy for infection by the Human Immunodeficiency Virus (HIV), but there is no cure. Consequently, antiviral drugs must be administered continuously to suppress viral replication. Recently, a large phase III international immune-based therapy trial was discontinued because it is difficult to measure clinical endpoints while antivirals are administered. Since the immune system has evolved under the selective force of microbial infections, the immune reaction is antiviral. This commentary explores the rationale of using "Diagnostic Treatment Interruptions" of antiviral therapies to determine efficacies of immune-based therapies. [Abstract/Link to Full Text]

Beadling C, Smith KA
DNA array analysis of interleukin-2-regulated immediate/early genes.
Med Immunol. 2002 Nov 18;1(1):2.
BACKGROUND: Lymphocyte activation culminates in blastogenesis, cell cycle progression, DNA replication and mitosis. These complex cellular changes are programmed almost simultaneously by multiple ligands and receptors that trigger specific signal transduction pathways and transcription factors. Until now, the discovery of the genes regulated by each ligand/receptor pair has been hampered by the technologies available. RESULTS: To identify interleukin-2 (IL-2)-responsive genes, human peripheral blood mononuclear cells (PBMC) were pre-activated with anti-CD3, rested, and restimulated with IL-2 for 4 hr. Gene expression was analyzed using Affymetrix U95Av2 oligonucleotide arrays. To determine the most stringent parameters to score a gene as a bona fide IL-2 target, the expression of 19 known IL-2-regulated genes was examined first. All were induced at least 2-fold, with a difference in fluorescent intensity of >/= 100 at p < 0.05. An additional 53 unique genes met these criteria. To determine which of these were immediate/early IL-2 targets in T cells, purified T cells were stimulated with IL-2 for 4 hr in the presence of cycloheximide to prevent secondary gene expression. Of the 72 genes identified in PBMCs, 20 were detected as immediate/early IL-2-regulated genes in purified T cells. In addition, 27 unique genes were IL-2-regulated in T cells but not in PBMCs. CONCLUSIONS: For a successful reductionist approach to the analysis of gene expression in lymphocyte activation, it is necessary to examine purified cell populations and immediate/early gene expression regulated by each ligand/receptor pair involved. This approach should allow the discovery of genes regulated by all of the ligand/receptor pairs involved in lymphocyte activation. [Abstract/Link to Full Text]

Smith KA
Medical immunology: a new journal for a new subspecialty.
Med Immunol. 2002 Sep 30;1(1):1. [Abstract/Link to Full Text]

---

Recent Articles in Microbiology and Immunology

---

Horiuchi Y, Hanazawa A, Nakajima Y, Nariai Y, Asanuma H, Kuwabara M, Yukawa M, Ito H
T-helper (th)1/th2 imbalance in the peripheral blood of dogs with malignant tumor.
Microbiol Immunol. 2007;51(11):1135-8.
T helper type 1 cell (Th1)/Th2 imbalance has been observed in a variety of pathological conditions, including malignant diseases. We evaluated the Th1/Th2 in peripheral blood Th cells by means of intracytoplasmic cytokine analysis in 11 dogs with advanced malignant tumor; four of them showed metastatic tumor. The percentage of Th1 was significantly lower and the percentage of Th2 was significantly higher in diseased dogs compared to healthy dogs. The percentage of Th1 in three patients with metastatic tumor was significantly lower than that in the patients with non-metastatic tumor. We conclude that the Th1/Th2 balance was polarized to Th2 in dogs with cancer. [Abstract/Link to Full Text]

Fukui H, Mitsui S, Harima N, Nose M, Tsujimura K, Mizukami H, Morita A
Novel functions of herbal medicines in dendritic cells: role of amomi semen in tumor immunity.
Microbiol Immunol. 2007;51(11):1121-33.
Dendritic cells (DCs) have a major role in regulating immune responses, including tumor immunity and peripheral tolerance. In the present study, we identified novel functions of herbal medicines in DCs by screening 99 herbal medicines, most of which are among the 210 Chinese medicines approved by the Ministry of Health, Labour, and Welfare, Japan. Ethanol extracts were prepared, and a murine epidermal-derived Langerhans cell line, XS106, was used to screen the 99 extracts by analyzing major histocompatibility complex (MHC) class II expression. Amomi Semen (amomum seed), Polyporus (polyporus sclerotium), and Plantaginis Semen (plantago seed) potently activated XS106 and were selected for further analysis. The effects of these extracts on bone marrow-derived DCs (BM-DCs) generated in vitro were then analyzed using surface phenotype (MHC class II, CD80, and CD86) and interleukin (IL)-12p70 production as indicators. BM-DCs treated with Amomi Semen extract exhibited activated phenotypes and secreted IL-12p70. The activation level was similar to that induced by lipopolysaccharides. Finally, an E.G7-OVA tumor model (E.L4-OVA transfectant) was used to examine the anti-tumor effects of Amomi Semen extract. Vaccination of mice with a subcutaneous injection of BM-DCs treated with Amomi Semen extract and OVA peptide significantly inhibited the growth of tumor cells and prolonged survival time compared to controls. Furthermore, therapeutic effects were observed on established tumors. The inhibition rates for both the prophylactic and therapeutic protocols were comparable to those of lipopolysaccharides. These results indicate that Amomi Semen extract potently activate DCs and is potentially useful for DC vaccination. [Abstract/Link to Full Text]

Nakamura K, Kinjo T, Saijo S, Miyazato A, Adachi Y, Ohno N, Fujita J, Kaku M, Iwakura Y, Kawakami K
Dectin-1 Is Not Required for the Host Defense to Cryptococcus neoformans.
Microbiol Immunol. 2007;51(11):1115-9.
Dectin-1 is known as a sole receptor for beta-glucan, a major cell wall component of fungal microorganisms. In the current study, we examined the role of this molecule in the host defense to Cryptococcus neoformans, an opportunistic fungal pathogen in AIDS patients. There was no significant difference in the clinical course and cytokine production between dectin-1 gene-deficient and control mice. These results indicate that dectin-1 is not likely essential for the development of host protective responses to C. neoformans. [Abstract/Link to Full Text]

Kawase M, He F, Kubota A, Harata G, Hiramatsu M
Orally Administrated Lactobacillus gasseri TMC0356 and Lactobacillus GG Alleviated Nasal Blockage of Guinea Pig with Allergic Rhinitis.
Microbiol Immunol. 2007;51(11):1109-14.
Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC0356) were investigated for their ability to alleviate nasal blockage associated with allergic rhinitis using a guinea pig model. The increases in sRaw at 10 min and 5 hr after the exposure of the nasal mucosa to OVA were significantly alleviated in the guinea pigs orally administrated with LGG and TMC0356 compared with those of the control (P<0.05 and P<0.01). The total numbers of leukocytes, particularly eosinophils and neutrophils from the nasal cavity lavage fluid, and the OVA-specific IgE concentration in the serum were also decreased in the guinea pigs orally administrated with LGG and TMC0356, although the decreases were not statistically significant. These results suggest that LGG and TMC0356 can alleviate antigen-induced nasal blockage in earlyphase and late-phase inflammatory responses associated with allergic rhinitis. [Abstract/Link to Full Text]

Park SA, Song ES, Cho YJ, Ahn BY, Ha SH, Seong BL, Lee KH, Lee NG
Immune Responses of Mice to Influenza Subunit Vaccine in Combination with CIA07 as an Adjuvant.
Microbiol Immunol. 2007;51(11):1099-107.
CIA07 is an immunostimulatory agent composed of E. coli DNA fragments and modified LPS lacking the lipid A moiety. In this study, we investigated whether CIA07 promotes immune responses as an adjuvant to the influenza subunit vaccine. Balb/c mice were immunized intramuscularly once or twice at a 4-week interval with the trivalent influenza subunit vaccine antigen alone or in combination with CIA07 as adjuvant. Antigen-specific serum antibody titers and hemagglutination-inhibition (HI) antibody titers were assessed. At 4 weeks after each immunization, the antigen-specific total serum IgG antibody titer in mice receiving CIA07 was 2 to 3 times higher than that in animals administered antigen alone (P<0.05). The CIA07-treated group additionally displayed higher HI antibody titers against each of the 3 vaccine strains, compared to the antigen group. Animals receiving antigen alone displayed barely detectable antigen-specific serum IgG2a antibody titers. In contrast, coadministration of CIA07 with antigen led to significantly enhanced IgG2a antibody responses, suggesting that CIA07 stimulates a Th1-type immune response. Moreover, the CIA07-treated group displayed a marked increase in the number of interferon gamma-producing CD8(+) T cells in splenocytes. These data collectively demonstrate that CIA07 has the ability to induce both Th1-type cellular and Th2-type humoral immune responses to the influenza subunit vaccine, and support its potential as an effective adjuvant to the influenza vaccine. [Abstract/Link to Full Text]

Jeong SY, Ahn J, Cho YJ, Kim YJ, Kim DS, Jee Y, Lee H, Nam JH
Production of Cross-Reactive Peptide Antibodies against Viral Capsid Proteins of Human Enterovirus B to Apply Diagnostic Reagent.
Microbiol Immunol. 2007;51(11):1091-8.
The coxsackievirus group B (CVB) of the genus Enterovirus and the species human enterovirus B is a nonenveloped virus containing a single-stranded positive-sense RNA genome. Coxsackievirus has icosahedral symmetry and four capsid proteins, VP1, VP2, VP3, and VP4. Specific antibodies against each viral protein are prerequisites for various studies. In this study, we developed seven peptide-derived antibodies directed against coxsackievirus VP1 (NO1-NO5), VP2 (B3), and VP3 (GL3). We developed a type-specific antibody (NO1) and broadly cross-reactive antibodies (NO3 and NO5) to VP1. Anti-VP2 and anti-VP3 antibodies (B3 and GL3, respectively) are also cross-reactive to human enterovirus B such as CVB and echoviruses. Their sensitivities and reactivities are likely to be better than those of the commercial VP1 monoclonal antibody (MAb). The dot-blot analysis also showed that NO5 against VP1 is able to detect less than 1 microg [2x10(6) plaque-forming unit (pfu) of CVB3] of viruses, suggesting that it could be used to develop a diagnostic kit that can directly detect human enterovirus B. The antibodies produced here may allow us to undertake several studies, such as those involving viral trafficking, expression kinetics, and the roles of viral proteins in infection, and the development of diagnostic kits. [Abstract/Link to Full Text]

Daud NH, Kariwa H, Tanikawa Y, Nakamura I, Seto T, Miyashita D, Yoshii K, Nakauchi M, Yoshimatsu K, Arikawa J, Takashima I
Mode of Infection of Hokkaido Virus (Genus Hantavirus) among Grey Red-Backed Voles, Myodes rufocanus, in Hokkaido, Japan.
Microbiol Immunol. 2007;51(11):1081-90.
Hokkaido virus (HOKV) is a member of the genus Hantavirus, in the family Bunyaviridae. To investigate HOKV infection in the host Myodes rufocanus, the grey red-backed vole, 199 animals were captured at Tobetsu (October 2004 and July 2005) and Nakagawa (October 2004) in Hokkaido, Japan, for detection of antibody, antigen, and viral RNA. In the surveys in Tobetsu (2004) and Nakagawa (2004), seropositive animals were detected at a frequency of 6.0% (5/84) and 10.4% (5/48), respectively. No seropositive animals were detected in Tobetsu in 2005. Seroprevalence in males in Tobetsu and Nakagawa in 2004 was 25% (1/4) and 45.5% (5/11), respectively, which was higher than in females, at 5.0% (4/80) and 0% (0/37), respectively (P<0.01). These results suggest that male animals play an important role in the maintenance of HOKV in M. rufocanus. Two females were seronegative but viral RNA-positive, indicating that these animals had acute infections before antibody was produced. Another five infected animals in Nakagawa were all male and had high levels of antibodies and viral RNA, suggesting that they had persistent infections. Viral RNA copies in organs of infected animals in Nakagawa were quantified by real-time polymerase chain reaction. Two acutely infected animals had>/=10 times the number of RNA copies in their lungs compared to those of persistently infected animals. In most cases, lungs or spleen had the highest RNA copy number, regardless of infection status. [Abstract/Link to Full Text]

Kawai A, Fujita K
Small Red Bean (Azuki) Sheds Biologically Active Substances as a Prerequisite Step for Germination, One of Which Displays the Antiviral Activity against the Rabies Virus Infectivity and Infections in Culture.
Microbiol Immunol. 2007;51(11):1071-9.
When small red beans (azuki bean; Vigna angularis Ohwi et Ohashi) were soaked and warmed in water or saline, the beans began to absorb water to swell and exuded kinds of substances probably as a prerequisite step for seed germination. Such exudate fluids displayed strong antiviral activity against the rabies virus infections in culture. On the other hand, little anti-rabies activity was detected in the aqueous extracts from the red beans when tested soon after the extraction from powdered beans, while low titers of antiviral activity appeared gradually in the extracts during cold storage. In contrast, no antiviral activity was detected in the exudate fluids from non-colored azuki beans (white azuki), implicating that a certain anthocyanin-related substance is involved in the antiviral activity of red beans. Production of antiviral and cytotoxic activities were affected differently depending on the bean-soaking conditions. In addition, the antiviral activity resisted to 10 min-heating in boiling water, while the cytotoxicity was greatly weakened by the heating, suggesting that different substances are involved in the antiviral and cytotoxic activities. Further studies on the antiviral activity of the exudate fluids demonstrated that anti-rabies activity of the bean exudates affected not only the very early phase of infection cycle, but the viral infectivity was also affected similarly, implicating a possible application of azuki bean exudate fluids to post-exposure treatment of rabid dog-bite injuries in combination with vaccination. [Abstract/Link to Full Text]

Rahman MM, Matsuo T, Ogawa W, Koterasawa M, Kuroda T, Tsuchiya T
Molecular Cloning and Characterization of All RND-Type Efflux Transporters in Vibrio cholerae Non-O1.
Microbiol Immunol. 2007;51(11):1061-70.
Resistance Nodulation cell Division (RND) efflux transporters are thought to be involved in mediating multidrug resistance in Gram-negative bacteria, including Vibrio cholerae non-O1. There are six operons for putative RND-type efflux transporters present in the chromosome of V. cholerae O1 including two operons, vexAB and vexCD, which had already been identified. All of the six operons were cloned from V. cholerae non-O1, NCTC4716 by the PCR method, introduced, and expressed in cells of drug hypersusceptible Escherichia coli KAM33 (DeltaacrAB, DeltaydhE). Only vexEF conferred elevated minimum inhibitory concentrations (MICs) of some antimicrobial agents in the E. coli cells. However, VexEF did not confer increased MIC of any drug tested in tolC-deficient E. coli KAM43 cells. On the other hand, when E. coli KAM43 was transformed with vexAB, vexCD or vexEF together with tolC(Vc) of V. cholerae NCTC4716, we observed elevated MICs of various antimicrobial agents. Among them, E. coli KAM43 expressing both VexEF and TolC(Vc) showed much higher MICs and much broader substrate specificity than the other two. We also observed ethidium efflux activity via VexEF-TolC(Vc), and the activity required Na(+). Thus, VexEF-TolC (Vc) is either a Na(+)-activated or a Na(+)-coupled transporter. To our knowledge, this is the first report on the requirement of Na(+) for an RND-type efflux transporter. [Abstract/Link to Full Text]

Maki K, Holmes AR, Watabe E, Iguchi Y, Matsumoto S, Ikeda F, Tawara S, Mutoh S
Direct comparison of the pharmacodynamics of four antifungal drugs in a mouse model of disseminated candidiasis using microbiological assays of serum drug concentrations.
Microbiol Immunol. 2007;51(11):1053-9.
The aim of this study was to compare the pharmacodynamics of the azole antifungal drugs fluconazole, itraconazole and ketoconazole, and the polyene antifungal amphotericin B, in a mouse model of disseminated Candida albicans infection. In order to directly compare effective serum concentrations of these antifungals, drug concentrations were assayed microbiologically by measuring inhibition of C. albicans mycelial growth (mMIC) in a mouse serum-based assay (serum antifungal titer). Efficacy in the mouse infection model was determined using an organ-based (kidney burden) endpoint. For all four drugs, the serum antifungal titers, 8 hr after administration of single doses of drugs at a range of drug concentrations, correlated closely with C. albicans kidney fungal burden in the mouse model. The results showed that determining serum antifungal titer may be used to accurately represent kidney fungal burden in a mouse model of disseminated candidiasis and allowed direct comparison of the pharmacodynamics of differing classes of antifungal drugs. [Abstract/Link to Full Text]

Thong KL, Tang SS, Tan WS, Devi S
Peptide Mimotopes of Complex Carbohydrates in Salmonella enterica Serovar Typhi Which React with Both Carbohydrate-Specific Monoclonal Antibody and Polyclonal Sera from Typhoid Patients.
Microbiol Immunol. 2007;51(11):1045-52.
Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding. [Abstract/Link to Full Text]

Masujin K, Shimada K, Kimura KM, Imamura M, Yoshida A, Iwamaru Y, Mohri S, Yokoyama T
Applicability of current bovine spongiform encephalopathy (BSE) diagnostic procedures for chronic wasting disease (CWD).
Microbiol Immunol. 2007;51(10):1039-43.
Chronic wasting disease (CWD) in cervids is one of the transmissible spongiform encephalopathies ; however, its risk to humans is still obscure. An increase in number of diseased deer in North America has raised concerns regarding the CWD risk to humans. We demonstrated that the con-firmatory procedures and the commercial diagnostic kits for bovine spongiform encephalopathy (BSE) can be adopted for the diagnosis of CWD. No CWD case was confirmed in the surveillance of 558 cervids that were examined between 2003 and 2006 in Japan. [Abstract/Link to Full Text]

Nakayama J, Ohtsuki M, Oda T
Caspase-independent cell death by Fas ligation in human thymus-derived T cell line, HPB-ALL cells.
Microbiol Immunol. 2007;51(10):1029-37.
In HPB-ALL cells, a human thymus-derived T-cell line, Fas (CD95)-mediated cell death was inhibited by about only 50% as a result of treatment with an amount of benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-CH(2)F (zVAD-fmk) sufficient to block the caspase activity. Fas-mediated caspase-independent cell death was not observed in other lymphoblast cell lines or mouse activated splenocytes, but this type of cell death was observed in mouse and rat thymocytes, the same as for HPB-ALL cells. This suggests that Fas-mediated caspase-independent cell death is a common feature in thymocytes. The signaling pathway of caspase-independent cell death has not yet been fully elucidated. In HPB-ALL cells, DNA fragmentation, one of the features of apoptotic cells, did not occur in the caspase-independent cell death after Fas ligation. On the other hand, this type of cell death and the surface exposure of phosphatidylserine were recovered by pretreatment with geldanamycin, which brought about a decrease in receptor interacting protein (RIP) kinase expression. These results suggested that HPB-ALL cells have a caspase-independent RIP kinasedependent pathway for Fas ligation. [Abstract/Link to Full Text]

Tumitan AR, Monnazzi LG, Ghiraldi FR, Cilli EM, Machado de Medeiros BM
Pattern of macrophage activation in yersinia-resistant and yersinia-susceptible strains of mice.
Microbiol Immunol. 2007;51(10):1021-8.
Th1 cells, in cooperation with activated macrophages, are required to overcome Yersinia enterocolitica infection in mice. The pathway macrophages utilize to metabolize arginine can alter the outcome of inflammation in different ways. The objective of this study was to verify the pattern of macrophages activation in Y. enterocolitica infection of BALB/c (Yersinia-susceptible) and C57BL/6 (Yersinia-resistant) mice. Both strains of mice were infected with Y. enterocolitica O:8 WA 2707. Peritoneal macrophages and spleen cells were obtained on the 1st, 3rd and 5th day post-infection. The iNOS and the arginase activities were assayed in supernatants of macrophage cultures, by measuring their NO/citrulline and ornithine products, respectively. TGFbeta-1 production was also assayed. The Th1 and Th2 responses were evaluated in supernatants of lymphocyte cultures, by IFN-gamma and IL-4 production. Our results showed that in the early phase of Y. enterocolitica infection (1st and 3rd day), the macrophages from C57BL/6 mice produced higher levels of NO/citrulline and lower levels of ornithine than macrophages from BALB/c mice. The infection with Y. enterocolitica leads to an increase in the TGF-beta1 and IL-4 production by BALB/c mice and to an increase in the IFN-gamma levels produced by C57BL/6 mice. These results suggest that Y. enterocolitica infection leads to the modulation of M1 macrophages in C57Bl/6 mice, and M2 macrophages in BALB/c mice. The predominant macrophage population (M1 or M2) at the 1st and 3rd day of infection thus seems to be important in determining Y. enterocolitica susceptibility or resistance. [Abstract/Link to Full Text]

Tanikawa T, Kurohane K, Imai Y
Induction of preferential chemotaxis of unstimulated B-lymphocytes by 2-arachidonoylglycerol in immunized mice.
Microbiol Immunol. 2007;51(10):1013-9.
2-Arachidonoylglycerol (2-AG) is an endogenous ligand for cannabinoid receptors. There are two types of cannabinoid receptors, CB1 and CB2. We investigated the chemotactic activity of 2-AG using mouse lymphocytes because cells in the immune system are known to express CB2 . Spleen cell migration toward 2-AG was observed, which was completely inhibited by SR144528, a CB2-specific antagonist. 2-AG has been reported to induce a preferential B cell chemotaxis. We examined whether there is any difference in responsiveness during the activation of B cells. When spleen cells from immunized mice were tested, naive B cells but not germinal center B cells (GL7-positive) were increased in the fraction attracted by 2-AG. Furthermore, when Peyer's patch lymphocytes were tested after oral administration of cholera toxin, the number of IgA* B cells was increased in the fraction attracted by 2-AG. These results suggested that 2-AG preferentially attracts unstimulated naive B cells rather than activated and/or class-switched B cells. This property may influence the structure of B cell compartments in secondary lymphoid tissues. [Abstract/Link to Full Text]

Alimohammadian MH, Darabi H, Malekzadeh S, Mahmoodzadeh-Niknam H, Ajdary S, Khamesipour A, Bahonar A, Mofarrah A
Exposure to Leishmania major modulates the proportion of CD4+ T cells without affecting cellular immune responses.
Microbiol Immunol. 2007;51(10):1003-11.
The immune responses of individuals exposed to Leishmania major were evaluated and compared with those of non-exposed volunteers. Forty-one patients with active lesion(s), 43 healed individuals, 15 vaccinees 1 month or 1 year post vaccination, and 15 non-exposed volunteers were studied. Leishmanin skin test (LST) response, proliferative response of lymphocyte (PRL) to L. major antigen, IFN-gamma and IL-4 production, and percentage of L. major-specific CD4+, CD8+ and CD16+/CD56+ cells in peripheral blood mononuclear cells were assessed. Data showed positive LST (>5 mm) in 92% of patients, 98% of healed, and 80% or 43% of vaccinees 1 month and 1 year post vaccination, respectively. Positive PRL (SI>2.5) was displayed in 90%, 84%, 46% and 7% of patients, healed, vaccinated (post 1 year) and non-exposed donors, respectively. The mean +/-S.E. of IFN-gamma was 924 +/- 149, 1,278 +/- 185, 470 +/- 282 or 258 +/- 82 pg/ml in patients, healed cases and vaccinees after 1 month or 1 year, respectively. Positive IFN-gamma responders (>300 pg/ml) were shown in 72% of patients, 81% of healed cases, 31% or 39% of vaccinees and 0% of non-exposed donors. A reduced percentage of CD4+ T-cells and an increased percentage of NK cells were found in exposed individuals compared to non-exposed donors. The data indicated that exposure to L. major modulates the proportion of CD4+ T cells and increases NK cells percentage. However, the cellular immune responses including induction of LST, and IFN-gamma production are increased in exposed individuals. [Abstract/Link to Full Text]

Otani N, Okuno T
Human herpesvirus 6 infection of CD4+ T-cell subsets.
Microbiol Immunol. 2007;51(10):993-1001.
The immune system includes CD4+ regulatory T (T reg) cells that play a role in self-tolerance and demonstrate functional variations that govern immune responses. HHV-6 is an important immunosuppressive virus that completely replicates in vivo and in vitro in only CD4+ T cells. However, there have been no reports of the specific T-cell subpopulation that permits the replication of this virus. Here, we evaluated the infectivity of HHV-6 to specific T-cell populations such as CD4+CD25 high, which includes the majority of T reg cells, and CD4+CD25(-). These cells were isolated from peripheral blood and then expanded. The expanded cell fractions were then infected with the HHV-6 variant B strain, and the spreads of infected cells were evaluated by immunofluorescence. Viral growth was also quantified by real-time PCR. The effects of virus infection on cytokine production from these T-cell subsets were examined using ELISA. Our results revealed that both these fractions permitted complete HHV-6 replication. Virus infection enhanced the production of both Th1- and Th2-type cytokines from CD4+CD25(-) T cells; however, only Th2-type cytokine release was augmented from viral-infected CD4+CD25 high T cells. Further, while virusinfected CD4+CD25 high T cells shift their antiviral immunity toward Th2 dominance by producing IL-10, the role of virus-infected CD4+CD25(-) T cells remains obscure. [Abstract/Link to Full Text]

Otaki M, Jiang DP, Sasayama M, Nagano-Fujii M, Hotta H
Generation of recombinant adenovirus expressing siRNA against the L mRNA of measles virus and subacute sclerosing panencephalitis virus.
Microbiol Immunol. 2007;51(10):985-91.
Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by prolonged persistent infection of the central nervous system with a measles virus (MV) mutant called SSPE virus. At present, there is no effective treatment to completely cure SSPE and development of a new therapeutic measure(s) against this fatal slow virus infection is needed. We previously reported that replication of MV and SSPE virus was effectively inhibited by small interfering RNA (siRNA), either chemically synthetic or plasmid-driven ones, that were targeted against different sequences of the mRNA for the L protein of MV. In this study, we have generated recombinant adenovirus expressing the siRNAs (rAd-siRNA-MV-L2, -L4 and -L5) and demonstrated that these rAd-siRNAs efficiently inhibited replication of MV and SSPE virus in a dose-dependent manner. Due to their high capacity for gene delivery to nerve cells and the potential to inhibit SSPE virus replication, the rAd-siRNAs could be a good candidate for a novel therapeutic measure against SSPE. [Abstract/Link to Full Text]

Oyamada Y, Yamagishi J, Kihara T, Yoshida H, Wachi M, Ito H
Mechanism of inhibition of DNA gyrase by ES-1273, a novel DNA gyrase inhibitor.
Microbiol Immunol. 2007;51(10):977-84.
We investigated the mode of action of ES-1273, a novel DNA gyrase inhibitor obtained by optimization of ES-0615, which was found by screening our chemical library using anucleate cell blue assay. ES-1273 exhibited the same antibacterial activity against S. aureus strains with amino acid change(s) conferring quinolone- and coumarin-resistance as that against a susceptible strain. In addition, ES-1273 inhibited DNA gyrase supercoiling activity, but not ATPase activity of the GyrB subunit of DNA gyrase. Moreover, ES-1273 did not induce cleavable complex. These findings demonstrate that the mechanism by which ES-1273 inhibits DNA gyrase is different from that of the quinolones or the coumarins. Preincubation of DNA gyrase and substrate DNA prevented inhibition of DNA gyrase supercoiling activity by ES-1273. ES-1273 antagonized quinolone-induced cleavage. In electrophoretic mobility shift assay, no band representing DNA gyrase-DNA complex was observed in the presence of ES-1273. Taken together, these results indicate that ES-1273 prevents DNA from binding to DNA gyrase. Furthermore, our results from surface plasmon resonance experiments strongly suggest that ES-1273 interacts with DNA. Therefore, the interaction between ES-1273 and DNA prevents DNA from binding to DNA gyrase, resulting in inhibition of DNA gyrase supercoiling. Interestingly, we also found that ES-1273 inhibits topoisomerase IV and human topoisomerase IIalpha, but not human topoisomerase I. These findings indicate that ES-1273 is a type II topoisomerase specific inhibitor. [Abstract/Link to Full Text]

Khan R, Takahashi E, Ramamurthy T, Takeda Y, Okamoto K
Salt in surroundings influences the production of serine protease into milieu by Aeromonas sobria.
Microbiol Immunol. 2007;51(10):963-76.
Previously we have shown that the open reading frame 2 protein (ORF2 protein), which is encoded at the 3 ' end of serine protease of Aeromonas sobria (ASP), functions as a chaperone protein in periplasm in the production of ASP. Both proteins, ASP and ORF2 protein, associate in periplasm and ORF2 protein helps ASP to take an active form. ASP which is dissociated from ORF2 protein emerges in milieu . In this study, we examined the effect of sodium chloride (NaCl) in medium on ASP production by A. sobria. The ASP activity of culture supernatant was extremely decreased when A. sobria was cultured in medium containing 3.0% NaCl (concentration almost equivalent to sea water salinity). Our analysis showed that the transcription of asp by A. sobria is not inhibited by NaCl in medium and that A. sobria synthesizes and releases ASP in milieu even under the condition of 3.0% NaCl. However, these ASPs in milieu formed complex as with ORF2 proteins. This indicates that the maturation pathway of ASP is disturbed in A. sobria cultured in medium containing 3.0% NaCl. It is likely that ASP does not associate with ORF2 protein in the correct form in periplasam when A. sobria is cultured in medium containing 3.0% NaCl, though both proteins, ASP and ORF2 protein, make complexes and emerge outside of the cell. This idea suggests that the chaperone system of ASP possesses the ability to sense NaCl in surroundings and regulates the production of active ASP. [Abstract/Link to Full Text]

Chen X, Kodama T, Iida T, Honda T
Demonstration and characterization of manganese superoxide dismutase of Providencia alcalifaciens.
Microbiol Immunol. 2007;51(10):951-61.
Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and play a role in the pathogenesis of certain invasive bacteria. In this study, we reported for the first time here that Providencia alcalifaciens, a member of the family Enterobacteriaceae, produces a superoxide dismutase (SOD) as a major protein in culture supernatants. This protein was purified by a series of column chromatographic separations. The N-terminal amino acid sequence of the protein was determined to be highly homologous to manganese superoxide dismutase of Escherichia coli or Salmonella reported. The gene (sodA) encoding for SOD of P. alcalifaciens was cloned and sequenced. The sodA-encoded protein has a molecular weight of about 23.5 kDa, and the DNA sequence of P. alcalifaciens sodA gene (627 bp) has about 83% identity to the E. coli SOD gene. We constructed a sodA deletion mutant and its complemented strain of P. alcalifaciens. In J774, a macrophage cell line, the sodA deletion mutant was more susceptible to killing by macrophages than the wildtype strain and its complemented strain. When we injected the mutant strain, its complemented strain and wildtype strain intraperitoneally into DDY strain mice, we found that the sodA deletion mutant proved significantly less virulent while the complemented strain recovered the virulence to the same level of wildtype strain of P. alcalifaciens. These results suggested that manganese superoxide dismutase plays an important role in intracellular survival of P. alcalifaciens. [Abstract/Link to Full Text]

Kato H, Sugita T, Ishibashi Y, Nishikawa A
Evaluation of the levels of specific IgE against Cryptococcus diffluens and Cryptococcus liquefaciens in patients with atopic dermatitis.
Microbiol Immunol. 2007;51(10):945-50.
Cryptococcus diffluens and Cryptococcus liquefaciens, 2 basidiomycetous yeasts, frequently colonize the skin of patients with atopic dermatitis (AD). In this study, we investigated the presence of specific IgE antibodies against C. diffluens and C. liquefaciens in the sera of AD patients by using an enzyme immunoassay . Of the 122 AD serum samples tested, 43 (35.2%) and 50 (41.0%) were positive for specific IgE antibodies against C. diffluens and C. liquefaciens, respectively. The levels of specific IgE against the C. diffluens antigen and that against the C. liquefaciens antigen were strongly correlated (r=0.96). In contrast, no remarkable correlation was observed between the levels of specific IgE against the 2 Cryptococcus species and that of specific IgE against Malassezia restricta. Competitive enzyme-linked immunosorbent assay (ELISA) inhibition tests revealed that C. diffluens and C. liquefaciens shared common antigens. This finding was consistent with the IgE immunoblotting data which demonstrated that several IgE-binding proteins with molecular masses of 77, 54, and 30 kDa were recognized in both C. diffluens and C. liquefaciens antigens . These results suggest that fungal components from C. diffluens and C. liquefaciens may act as allergens and play a role in the pathogenesis of AD. [Abstract/Link to Full Text]

Rimbara E, Noguchi N, Kawai T, Sasatsu M
Correlation between substitutions in penicillin-binding protein 1 and amoxicillin resistance in Helicobacter pylori.
Microbiol Immunol. 2007;51(10):939-44.
The correlation between the substitutions of penicillin-binding protein 1 (PBP1) and amoxicillin resistance was studied for the determination of the substitutions in PBP1 which confer amoxicillin resistance in Helicobacter pylori. By the comparison of the amino acid sequences of PBP1 in the amoxicillinresistant (n=3), low-susceptible (n=3), and susceptible (n=13) H. pylori isolates, the substitution Asn562-->Tyr, which is adjacent to KTG motif (555-557), was common and specific to amoxicillin-resistant H. pylori. Additionally, all amoxicillin-resistant isolates had multiple substitutions such as Ser414-->Arg in the transpeptidase region of PBP1 of H. pylori. Furthermore all transformants obtained by the natural transformation using the pbp1 genes of amoxicillin-resistant H. pylori isolates had multiple substitutions including Asn562-->Tyr. These results suggest that multiple amino acid substitutions in the transpeptidase region of PBP1 are closely related to amoxicillin resistance in H. pylori. [Abstract/Link to Full Text]

Ikeno T, Fukuda K, Ogawa M, Honda M, Tanabe T, Taniguchi H
Small and rough colony pseudomonas aeruginosa with elevated biofilm formation ability isolated in hospitalized patients.
Microbiol Immunol. 2007;51(10):929-38.
Pseudomonas aeruginosa is a key pathogen of nosocomial infection, and causes persistent infection in patients with specific diseases like cystic fibrosis (CF). It has been reported that patients affected with CF discharge, at a high frequency, small colony variants with high adherence ability. In routine laboratory testing, we found atypical small and rough type (SR) colony variants of P. aeruginosa. The SRs and the counterpart wild type (WT) colonies showed similar biochemical features, antimicrobial susceptibilities, pulsed-field gel electrophoresis (PFGE) profiles, serotypes, and twitching motilities. The biofilm formation abilities of all the SR colonies, however, were extremely elevated as compared to those of the counterpart WT colonies. The frequency of SR-positive patients was 3.1% of the P. aeruginosa-positive inpatients (5/160), and that of the SR isolates was 0.6% of the P. aeruginosa strains (6/970) isolated in our laboratory over a period of 6 months. The SR-positive patients did not have any common disease or particular antibiotics treatment. The PFGE profiles showed that the SRs and the counterpart WTs were identical to each other, and also that three of the five SR/WT pairs were clonally similar. The three pairs were recovered from the feces, urine, and endotracheal secretion, respectively, of three patients hospitalized in two distinct wards. The results suggest that P. aeruginosa spontaneously produced highly adherent SR colonies in hospitalized patients, and these colonies may tend to spread in a hospital. [Abstract/Link to Full Text]

Kim SY, Adachi Y
Biological and genetic classification of canine intestinal lactic acid bacteria and bifidobacteria.
Microbiol Immunol. 2007;51(10):919-28.
To investigate the distribution of lactic acid bacteria (LAB) inhabiting canine intestines, a total of 374 gram-positive LAB and bifidobacteria (BF) isolated from large intestinal contents in 36 dogs were classified and identified by phenotypic and genetic analyses. Based on cell morphological sizes, these isolates were divided into seven biotypes containing the genera Lactobacillus, Bifidobacterium, Enterococcus, and Streptococcus. The LAB and BF isolates were classified into 38 chemotypes based on SDS-PAGE protein profile analysis of whole cells. Furthermore, partial 16S rDNA sequencing analysis demonstrated the presence of 24 bacterial species in the 38 chemotypes from 36 dogs. The identified species consisted of ten species belonging to the genus Lactobacillus (78.8%), seven species to the genus Bifidobacterium (6.8%), five species to the genus Enterococcus (11.6%), one species of Streptococcus bovis (2.0%), and one species of Pediococcus acidilactici (0.8%). In particular, the most predominant species in canine intestines were L. reuteri, L. animalis, and L. johnsonii and were found in the high frequency of occurrence of 77.8, 80.6, and 86.1%, respectively. Besides these, Enterococcus faecalis, Bifidobacterium animalis subsp. lactis, Pediococcus acidilactici, and Streptococcus bovis were also isolated in the present study. The sequences of the isolates also showed high levels of similarity to those of the reference strains registered previously in the DDBJ and the similarity was above 97.2%. Their partial 16S rRNA genes were registered in the DDBJ. [Abstract/Link to Full Text]

Samosornsuk W, Asakura M, Yoshida E, Taguchi T, Nishimura K, Eampokalap B, Phongsisay V, Chaicumpa W, Yamasaki S
Evaluation of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the identification of Campylobacter strains isolated from poultry in Thailand.
Microbiol Immunol. 2007;51(9):909-17.
We have recently developed a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for identifying Campylobacter jejuni, C. coli and C. fetus. In the present study, the applicability of this assay was evaluated with 34 Campylobacter-like organisms isolated from poultry in Thailand for species identification and was compared with other assays including API Campy, 16S rRNA gene sequence, and hippuricase (hipO) gene detection. Of the 34 strains analyzed, 20, 10 and 1 were identified as C. jejuni, C. coli, and Arcobacter cryaerophilus, respectively, and 3 could not be identified by API Campy. However, 16S rRNA gene analysis, showed that all 34 strains are C. jejuni/coli. To discriminate between these 2 species, the hipO gene, which is specifically present in C. jejuni, was examined by PCR and was detected in 20 strains, which were identified as C. jejuni by API Campy but not in the remaining 14 strains. Collective results indicated that 20 strains were C. jejuni whereas the 14 strains were C. coli. When the cdt gene-based multiplex PCR was employed, however, 19, 20 and 19 strains were identified as C. jejuni while 13, 14 and 13 were identified as C. coli by the cdtA, cdtB and cdtC gene-based multiplex PCR, respectively. Pulsed-field gel electrophoresis revealed that C. jejuni and C. coli strains analyzed are genetically diverse. Taken together, these data suggest that the cdt gene-based multiplex PCR, particularly cdtB gene-based multiplex PCR, is a simple, rapid and reliable method for identifying the species of Campylobacter strains. [Abstract/Link to Full Text]

Tsuda Y, Sakoda Y, Sakabe S, Mochizuki T, Namba Y, Kida H
Development of an immunochromatographic kit for rapid diagnosis of h5 avian influenza virus infection.
Microbiol Immunol. 2007;51(9):903-7.
Highly pathogenic avian influenza (HPAI) caused by the H5N1 subtype has given rise to serious damage in poultry industries in Asia. The virus has expanded its geographical range to Europe and Africa, posing a great risk to human health as well. For the control of avian influenza, a rapid diagnosis by detecting the causative virus and identifying its subtype is essential. In the present study, a rapid diagnosis kit combining immunochromatography with enzyme immunoassay which detects the H5 HA antigen of influenza A virus was developed using newly established anti-H5 HA monoclonal antibodies. The present kit specifically detected all of the H5 influenza viruses tested, and did not react with the other HA subtypes. H5 HA antigens were detected from swabs and tissue homogenates of chickens infected with HPAI virus strain A/chicken/Yamaguchi/7/04 (H5N1) from 2 days post inoculation. The kit showed enough sensitivity and specificity for the rapid diagnosis of HPAI. [Abstract/Link to Full Text]

Maruyama S, Shen H, Kanoh M, Matsumoto A, Asano Y
differential effect of Listeria monocytogenes infection on cytokine production and cytotoxicity of CD8 T cells.
Microbiol Immunol. 2007;51(9):893-901.
Bacterial infection induces a shift to type 1 CD4 T cell subset in an infected host and this shift is important for protection of the host from disease development. Many researchers think that the shift is antigen-dependent, but we previously demonstrated an initial induction step for CD4 T cell subsets during Listeria monocytogenes (Lm) infection is antigen-independent. Although Listeria is a TLR2 ligand, the immune system of the Lm-infected host responded to the pathogen to induce expression of CD69 but not CD25 on CD4 T cells, CD8 T cells and B cells even in the absence of TLR2 or MyD88. The antigen-independent activation of type 1 CD4 T cells accelerate the clearance of pathogens by activating innate immune cells with type 1 cytokines. Type 1 CD4 T cells and CD8 T cells also collaborate to protect the host from intracellular Lm infection. Since CD8 T cells function mainly as cytotoxic T cells and CD69-positive CD8 T cells increase during Lm-infection, cytotoxic activity of CD8 T cells was evaluated during Lm-infection. Although CD8 T cells were activated to produce IFN-gamma, the cytotoxic function of CD8 T cells in Lymphocytic choriomeningitis virus (LCMV) p14 TCR-transgenic mouse was not augmented by Lm-infection. Therefore, Lm-infection differentially influences on cytokine production and cytotoxicity of CD8 T cells. [Abstract/Link to Full Text]

Saito M, Seki M, Iida K, Nakayama H, Yoshida S
A novel agar medium to detect hydrogen peroxide-producing bacteria based on the prussian blue-forming reaction.
Microbiol Immunol. 2007;51(9):889-92.
The classic method for H(2)O(2) detection involving Prussian blue formation was adapted to formulate a novel agar medium that makes possible in situ detection of H(2)O(2) produced by bacteria. Using this medium, colonies of H(2)O(2)-producing species including Streptococcus pyogenes were easily identified by the appearance of blue halos. The utility of the medium was further illustrated by its successful application to the isolation of H(2)O(2)-producing mutants from a non-H(2)O(2)-producing stain of S. pyogenes. [Abstract/Link to Full Text]

Tokunaga A, Kawano M, Okura M, Iyoda S, Watanabe H, Osawa R
Identification of enterohemorrhagic Escherichia coli O157-Specific DNA sequence obtained from amplified fragment length polymorphism analysis.
Microbiol Immunol. 2007;51(9):883-8.
An approximately 1.1 kbp fragment that was commonly observed only in the enterohemorrhagic Escherichia coli (EHEC) O157 strains in an analysis of amplified fragment length polymorphism was found to be a partial gene sequence encoding the locus of toxB and a useful molecular marker for the identification of EHEC O157. [Abstract/Link to Full Text]

---

Recent Articles in International Microbiology

---

Martínez-Murcia AJ, Figueras MJ, Saavedra MJ, Stackebrandt E
The recently proposed species Aeromonas sharmana sp. nov., isolate GPTSA-6T, is not a member of the genus Aeromonas.
Int Microbiol. 2007 Mar;10(1):61-4.
A new species of the genus Aeromonas, Aeromonas sharmana sp. nov., was recently described on the basis of a single isolate, strain GPTSA-6T, obtained from a warm spring in India. The description of this new species included biochemical characterization, antibiotic susceptibility, cellular fatty-acid profiles, and 16S rRNA gene sequencing, but not DNADNA hybridization data. In the present article, phylogenetic analysis (branch distances in the tree and nucleotide signatures) of the 16S rRNA of isolate GPTSA-6T, together with certain phenotypic characteristics of the isolate reported in the earlier description, clearly indicate that this microorganism does not belong to the genus Aeromonas as known to date, although it falls within the radiation of the family Aeromonadaceae. Emendation from the List of Prokaryotic Names with Standing in Nomenclature is consequently proposed. [Abstract/Link to Full Text]

Cerdá P, Goñi P, Millán L, Rubio C, Gómez-Lus R
Detection of the aminoglycosidestreptothricin resistance gene cluster ant(6)-sat4-aph(3 ')-III in commensal viridans group streptococci.
Int Microbiol. 2007 Mar;10(1):57-60.
High-level aminoglycoside resistance was assessed in 190 commensal erythromycin-resistant alpha-hemolytic streptococcal strains. Of these, seven were also aminoglycoside-resistant: one Streptococcus mitis strain was resistant to high levels of kanamycin and carried the aph(3 ')-III gene, four S. mitis strains were resistant to high levels of streptomycin and lacked aminoglycoside-modifying enzymes, and two S. oralis strains that were resistant to high levels of kanamycin and streptomycin harbored both the aph(3 ')-III and the ant(6) genes. The two S. oralis strains also carried the ant(6)-sat4- aph(3 ' ')-III aminoglycoside-streptothricin resistance gene cluster, but it was not contained in a Tn5405-like structure. The presence of this resistance gene cluster in commensal streptococci suggests an exchange of resistance genes between these bacteria and enterococci or staphylococci. [Abstract/Link to Full Text]

Orden JA, Domínguez-Bernal G, Martínez-Pulgarín S, Blanco M, Blanco JE, Mora A, Blanco J, Blanco J, de la Fuente R
Necrotoxigenic Escherichia coli from sheep and goats produce a new type of cytotoxic necrotizing factor (CNF3) associated with the eae and ehxA genes.
Int Microbiol. 2007 Mar;10(1):47-55.
Fecal samples from sheep and goats were screened by tissue-culture assays and PCR for the presence of necrotoxigenic Escherichia coli (NTEC) producing cytotoxic necrotizing factors (CNFs). Of the 18 NTEC strains assayed, four were positive for the cnf1 gene while 14 strains were negative for the cnf1 and cnf2 genes. All of the NTEC strains had the eae gene and most of them also carried the ehxA gene. Moreover, all the cnf1- cnf2- NTEC strains were negative for several virulence markers associated with CNF1+ or CNF2+ strains. The cnf gene present in one of these strains was sequenced and analysis of the gene product revealed a new type of CNF, which was named CNF3 (and the coding gene cnf3). Oligonucleotide primers were designed to PCR-amplify a fragment of cnf3. The results showed that all strains examined in this study, except one cnf1+strain, were cnf3+. The association of cnf3 with eae and ehxA suggests that cnf3+ NTEC strains might be pathogenic for humans. [Abstract/Link to Full Text]

Moeller R, Douki T, Cadet J, Stackebrandt E, Nicholson WL, Rettberg P, Reitz G, Horneck G
UV-radiation-induced formation of DNA bipyrimidine photoproducts in Bacillus subtilis endospores and their repair during germination.
Int Microbiol. 2007 Mar;10(1):39-46.
The spore photoproduct (SP) is the main DNA lesion after UV-C irradiation, and its repair is crucial for the resistance of spores to UV. The aims of the present study were to assess the formation and repair of bipyrimidine photoproducts in spore DNA of various Bacillus subtilis strains using a sensitive HPLC tandem mass spectrometry assay. Strains deficient in nucleotide excision repair, spore photoproduct lyase, homologous recombination (recA), and with wild-type repair capability were investigated. Additionally, one strain deficient in the formation of major small, acid-soluble spore proteins (SASPs) was tested. In all SASP wild-type strains, UV-C irradiation generated almost exclusively SP (>95 %) but also a few by-photoproducts. In the major SASP-deficient strain, SP and by-photoproducts were generated in equal quantities. The status time of 60 min, >75% of the SP was repaired in wild-type strains and in the SASP-deficient strain, while half of the photoinduced SP was removed in the recA-deficient strain. SP-lyase-deficient spores repaired 20% of the SP produced. Thus, SP lyase, with respect to nucleotide excision repair, has a remarkable impact on the removal of SP upon spore germination. [Abstract/Link to Full Text]

Ferrera I, Sánchez O, Mas J
Characterization of a sulfide-oxidizing biofilm developed in a packed-column reactor.
Int Microbiol. 2007 Mar;10(1):29-37.
The potential of microbial mats to develop sulfide-oxidizing biofims was explored. A bioreactor specially designed for the treatment of sulfide-containing effluents was inoculated with a microbial-mat sample, and a complex microbial biofilm with sulfide-oxidation activity developed. The microbial composition of the biofilm was studied by pigment, microscopy, and 16S rRNA gene analyses. Purple sulfur bacteria and diatoms were observed by microscopy, chlorophyll a and bacteriochlorophyll a were detected in the pigment analysis, and high genetic diversity was found in the 16S rRNA gene library. Specialized anaerobic sulfur oxidizers (i.e., phototrophic purple and green sulfur bacteria) dominated the library. Aerobic phototrophs (diatoms) also developed and the oxygen produced allowed the growth of aerobic sulfide oxidizers, such as Thiomicrospira-like spp. Cyanobacteria, which are significant organisms in natural microbial mats, did not develop in the reactor but unexpected uncultured members from the Epsilonproteobacteria developed profusely. Moreover, a variety of more minor organisms, such as members of the Cytophaga-Flavobacterium-Bacteroides (CFB) and purple non-sulfur bacteria (Roseospirillum sp.), were also present. The results showed that a complex community with high genetic and metabolic diversity, including many uncultured organisms, can develop in a laboratory-scale reactor. [Abstract/Link to Full Text]

Chacón MR, Rodríguez-Galán O, Benítez T, Sousa S, Rey M, Llobell A, Delgado-Jarana J
Microscopic and transcriptome analyses of early colonization of tomato roots by Trichoderma harzianum.
Int Microbiol. 2007 Mar;10(1):19-27.
The capacity of the fungus Trichoderma harzianum CECT 2413 to colonize roots and stimulate plant growth was analyzed. Tobacco seedlings (Nicotiana benthamiana) transferred to Petri dishes inoculated with T. harzianum conidia showed increased plant fresh weight (140%) and foliar area (300%), as well as the proliferation of secondary roots (300%) and true leaves (140%). The interaction between strain CECT 2413 and the tomato-root system was also studied during the early stages of root colonization by the fungus. When T. harzianum conidia were inoculated into the liquid medium of hydroponically grown tomato plants (Lycopersicum esculentum), profuse adhesion of hyphae to the plant roots as well as colonization of the root epidermis and cortex were observed. Confocal microscopy of a T. harzianum transformant that expressed the green fluorescent protein (GFP) revealed intercellular hyphal growth and the formation of plant-induced papilla-like hyphal tips. Analysis of the T. harzianum-tomato interaction in soil indicated that the contact between T. harzianum and the roots persisted over a long period of time. This interaction was characterized by the presence of yeast-like cells, a novel and previously undescribed developmental change. To study the molecular mechanism underlying fungal ability to colonize the tomato-root system, the T. harzianum transcriptome was analyzed during the early stages of the plant-fungus interaction. The expression of fungal genes related to redox reactions, lipid metabolism, detoxification, and sugar or amino-acid transport increased when T. harzianum colonized tomato roots. These observations are similar to those regarding the interactions of mycorrhiza and pathogenic fungi with plants. [Abstract/Link to Full Text]

Azúa I, Unanue M, Ayo B, Artolozaga I, Iriberri J
Influence of age of aggregates and prokaryotic abundance on glucose and leucine uptake by heterotrophic marine prokaryotes.
Int Microbiol. 2007 Mar;10(1):13-8.
The kinetics of glucose and leucine uptake in attached and free-living prokaryotes in two types of microcosms with different nutrient qualities were compared. Microcosm type M1, derived from unaltered seawater, and microcosm type M2, from phytoplankton cultures, clearly expressed different kinetic parameters (Vmax/cell and K' m). In aggregates with low cell densities (M1 microcosm), the attached prokaryotes benefited from attachment as reflected in the higher potential uptake rates, while in aggregates with high cell densities (M2 microcosm) differences in the potential uptake rates of attached and free-living prokaryotes were not evident. The aging process and the chemical changes in aggregates of M2 microcosms were followed for 15-20 days. The results showed that as the aggregates aged and prokaryotic abundance increased, attached prokaryotes decreased their potential uptake rate and their K' m for substrate. This suggests an adaptive response by attached prokaryotes when aggregates undergo quantitative and qualitative impoverishment. [Abstract/Link to Full Text]

Abel A, Sánchez S, Arenas J, Criado MT, Ferreirós CM
Bioinformatic analysis of outer membrane proteome of Neisseria meningitidis and Neisseria lactamica.
Int Microbiol. 2007 Mar;10(1):5-11.
Two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE) and Western-blotting techniques were used to analyze and compare common and/or specific outer-membrane proteins and antigens from Neisseria meningitidis and Neisseria lactamica. Bioinformatic image analyses of proteome and immunoproteome maps indicated the presence of numerous proteins and several antigens shared by N. meningitidis and N. lactamica, although the inter-strain variation in the maps was of similar magnitude to the inter-species variation, and digital comparison of the maps did not reveal proteins found to be identical by MALDI-TOF fingerprinting analysis. PorA and RmpM, two relevant outer-membrane antigens, manifested as various spots at several different positions. While some of these were common to all the strains analyzed, others were exclusive to N. meningitidis and their electrophoretic mobilities were different than expected. One such spot, with a molecular mass of 19 kDa, may be the C-terminal fragment of RmpM (RmpM-Cter). The results demonstrate that computer-driven analysis based exclusively on spot positions in the proteome or immunoproteome maps is not a reliable approach to predict the identity of proteins or antigens; rather, other identification techniques are necessary to obtain accurate comparisons. [Abstract/Link to Full Text]

Garabal JI
Biodiversity and the survival of autochthonous fermented products.
Int Microbiol. 2007 Mar;10(1):1-3. [Abstract/Link to Full Text]

Cid VJ
Microbiology in the "-omics" era: a report on the 2nd FEMS Congress (Madrid, 4-8 July 2006).
Int Microbiol. 2006 Dec;9(4):303-8. [Abstract/Link to Full Text]

Bigas A, Garrido ME, Badiola I, Barbé J, Llagostera M
Colonization capacity and serum bactericidal activity of Haemophilus parasuis thy mutants.
Int Microbiol. 2006 Dec;9(4):297-301.
The bacterial thyA gene encodes the enzyme thymidylate synthase, which is essential for dTMP synthesis and, consequently, for DNA replication. In this work, a Haemophilus parasuis thyA mutant was constructed in order to analyze its colonization characteristics and its capacity to generate serum bactericidal activity in infected guinea pigs. The data showed that colonization by the H. parasuis thyA mutant was much less than that of the wild-type strain. Nevertheless, the mutant generated a strong immunogenic response in the host, as detected by measuring serum bactericidal activity. [Abstract/Link to Full Text]

Wierzchos J, Berlanga M, Ascaso C, Guerrero R
Micromorphological characterization and lithification of microbial mats from the Ebro Delta (Spain).
Int Microbiol. 2006 Dec;9(4):289-95.
The structural organization of microbial mats from the Ebro Delta (Spain) and their accretion and partial lithification processes were explored using scanning electron microscopy in back-scattered electron mode and low-temperature scanning electron microscopy. Two differentiated zones were distinguished in a transverse section of a fragment taken from the mat at a depth of 2.5 mm. The first consisted of an upper layer in which the dominant microorganisms, Microcoleus spp., actively grew in an embedded slack matrix of exopolysaccharides. Microcoleus filaments were oriented parallel to the surface and to each other, with filaments below arranged perpendicularly to one another but without crossing. Most of the minerals present were allochthonous grains of calcium phosphate biocorroded by cyanobacteria. The second zone was below a depth of 1 mm and made up of accretion layers with large deposits of calcium carbonate and smaller amounts of calcium phosphate of biological origin. The predominance of a particular type of mineral precipitation with a characteristic external shape and/or texture within a zone, e.g., sponge-like deposits of calcium phosphate, appears to depend on the taxa of the prevailing microorganisms. [Abstract/Link to Full Text]

Power T, Ortoneda M, Morrissey JP, Dobson AD
Differential expression of genes involved in iron metabolism in Aspergillus fumigatus.
Int Microbiol. 2006 Dec;9(4):281-7.
The ability of fungi to survive in many environments is linked to their capacity to acquire essential nutrients. Iron is generally complexed and available in very limited amounts. Like bacteria, fungi have evolved highly specific systems for iron acquisition. Production and uptake of iron-chelating siderophores has been shown to be important for certain human bacterial pathogens, as well as in fungal pathogens such as Cryptococcus neoformans and Fusarium graminearum. This system also enables the opportunistic fungal pathogen Aspergillus fumigatus to infect and subsequently colonize the human lung. In this study, advantage was taken of genome sequence data available for both Aspergillus nidulans and A. fumigatus either to partially clone or to design PCR primers for 10 genes putatively involved in siderophore biosynthesis or uptake in A. fumigatus. The expression of these genes was then monitored by semi-quantitative and quantitative real-time PCR over a range of iron concentrations. As expected, the putative biosynthetic genes sidA, sidC and sidD were all strongly up-regulated under iron starvation conditions, although the variable degree of induction indicates complex regulation by a number of transcriptional factors, including the GATA family protein SreA. In contrast, the gene sidE shows no iron-regulation, suggesting that SidE may not be involved in siderophore biosynthesis. The characterisation of the expression patterns of this subset of genes in the iron regulon facilitates further studies into the importance of iron acquisition for pathogenesis of A. fumigatus. [Abstract/Link to Full Text]

Arenas J, Abel A, Sánchez S, Alcalá B, Criado MT, Ferreirós CM
Locus NMB0035 codes for a 47-kDa surface-accessible conserved antigen in Neisseria.
Int Microbiol. 2006 Dec;9(4):273-80.
A47 kDa neisserial outer-membrane antigenic protein (P47) was purified to homogeneity and used to prepare polyclonal anti-P47 antisera. Protein P47 was identified by MALDI-TOF fingerprinting analysis as the hypothetical lipoprotein NMB0035. Two-dimensional diagonal SDS-PAGE results suggested that, contrary to previous findings, P47 is not strongly associated with other proteins in membrane complexes. Western blotting with the polyclonal monospecific serum showed that linear P47 epitopes were expressed in similar amounts in the 27 Neisseria meningitidis strains tested and, to a lesser extent, in commensal Neisseria, particularly N. lactamica. However, dot-blotting assays with the same serum demonstrated binding variability between meningococcal strains, indicating differences in surface accessibility or steric hindrance by other surface structures. Specific anti-P47 antibodies were bactericidal against the homologous strain but had variable activity against heterologous strains, consistent with the results from dot-blotting experiments. An in-depth study of P47 is necessary to evaluate its potential as a candidate for new vaccine designs. [Abstract/Link to Full Text]

Abreu F, Silva KT, Martins JL, Lins U
Cell viability in magnetotactic multicellular prokaryotes.
Int Microbiol. 2006 Dec;9(4):267-72.
A magnetotactic multicellular prokaryote (MMP) is an assembly of bacterial cells organized side by side in a hollow sphere in which each cell faces both the external environment and an internal acellular compartment in the center of the multicellular organism. MMPs swim as a unit propelled by the coordinated beating of the many flagella on the external surface of each cell. At every stage of its life cycle, MMPs are multicellular. Initially, a spherical MMP grows by enlarging the size of each of its cells, which then divide. Later, the cells separate into two identical spheres. Swimming individual cells of MMPs have never been observed. Here we have used fluorescent dyes and electron microscopy to study the viability of individual MMP cells. When separated from the MMP, the cells cease to move and they no longer respond to magnetic fields. Viability tests indicated that, although several cells could separate from a MMP before completely losing their motility and viability, all of the separated cells were dead. Our data show that the high level of cellular organization in MMPs is essential for their motility, magnetotactic behavior, and viability. [Abstract/Link to Full Text]

Alexander B, Imhoff JF
Communities of green sulfur bacteria in marine and saline habitats analyzed by gene sequences of 16S rRNA and Fenna-Matthews-Olson protein.
Int Microbiol. 2006 Dec;9(4):259-66.
Communities of green sulfur bacteria were studied in selected marine and saline habitats on the basis of gene sequences of 16S rRNA and the Fenna- Matthews-Olson (FMO) protein. The availability of group-specific primers for both 16S rDNA and the fmoA gene, which is unique to green sulfur bacteria, has, for the first time, made it possible to analyze environmental communities of these bacteria by culture-independent methods using two independent genetic markers. Sequence results obtained with fmoA genes and with 16S rDNA were largely congruent to each other. All of the 16S rDNA and fmoA sequences from habitats of the Baltic Sea, the Mediterranean Sea, Sippewissett Salt Marsh (Massachusetts, USA), and Bad Water (Death Valley, California, USA) were found within salt-dependent phylogenetic lines of green sulfur bacteria established by pure culture studies. This strongly supports the existence of phylogenetic lineages of green sulfur bacteria specifically adapted to marine and saline environments and the exclusive occurrence of these bacteria in marine and saline habitats. The great majority of clone sequences belonged to different clusters of the Prosthecochloris genus and probably represent different species. Evidence for the occurrence of two new species of Prosthecochloris was also obtained. Different habitats were dominated by representatives from the Prosthecochloris group and different clusters or species of this genus were found either exclusively or as the clearly dominant green sulfur bacterium at different habitats. [Abstract/Link to Full Text]

Borkenstein CG, Fischer U
Sulfide removal and elemental sulfur recycling from a sulfide-polluted medium by Allochromatium vinosum strain 21D.
Int Microbiol. 2006 Dec;9(4):253-8.
Phototrophic purple sulfur bacteria oxidize sulfide to elemental sulfur, which is stored as intracellular sulfur globules. The mutant Allochromatium vinosum strain 21D, containing an inactivated dsrB gene, is unable to further oxidize intracellularly stored sulfur to sulfate. This mutant was used as a biocatalyst in a biotechnological process to eliminate sulfide from synthetic wastewater and to recycle elemental sulfur as a raw material. For this purpose, the mutant was grown in an illuminated 5-liter bioreactor (30 microE/m2/s PAR) at 30 degrees C for 61 days in anoxic phototrophic medium. The process of sulfide removal was semi-continuous and consisted of three consecutive fed-batch sections. Sulfide was repeatedly added into the bioreactor and oxidized by the cells to sulfur. In the presence of the mutant, no unwanted sulfate was produced during sulfide removal. A maximum sulfide removal rate of 49.3 microM/h, a maximum sulfide removal efficiency of 98.7%, and 60.4% sulfur recycling were achieved. [Abstract/Link to Full Text]

Vives-Flórez M, Garnica D
Comparison of virulence between clinical and environmental Pseudomonas aeruginosa isolates.
Int Microbiol. 2006 Dec;9(4):247-52.
New strains of Pseudomonas aeruginosa were isolated from clinical and environmental settings in order to characterize the virulence properties of this opportunistic pathogen. P. aeruginosa was frequently recovered from oil-contaminated samples but not from non-oil-contaminated soils. The virulence of five environmental and five clinical strains of P. aeruginosa was tested using two different models, Drosophila melanogaster and Lactuca sativa var. capitata L. There was no difference in the virulence between the two groups of isolates in either of the models. Since environmental P. aeruginosa strains are used for bioaugmentation in bioremediation programs, the results presented here should be taken into account in the future design of degradative consortia and/or in establishing containment measures. [Abstract/Link to Full Text]

Vázquez F, González EA, Garabal JI, Blanco J
Characterization of fimbriae extracts from porcine enterotoxigenic Escherichia coli strains carrying F6 (987P) antigen.
Int Microbiol. 2006 Dec;9(4):241-6.
Fimbrial extracts from porcine enterotoxigenic Escherichia coli (ETEC) strains carrying F6 (987P) intestinal colonization factor antigen wereobtained using the thermal shock method. The extracts were analyzed by SDSPAGE and immunoblotting using different fimbriae-specific antisera. Two major protein bands with molecular masses of 17.5 and 21.9 kDa were detected. The 21.9-kDa band was identified as the major subunit of F6 fimbrial antigen in strains of serogroups O9 and O141. The 17.5-kDa band was associated with porcine strains of serogroups O9 and O20. [Abstract/Link to Full Text]

Piqueras M
Year's comments for 2006.
Int Microbiol. 2006 Dec;9(4):237-40. [Abstract/Link to Full Text]

Guerrero R, Berlanga M
Life's unity and flexibility: the ecological link.
Int Microbiol. 2006 Sep;9(3):225-35.
The small size, ubiquity, metabolic versatility and flexibility, and genetic plasticity (horizontal transfer) of microbes allow them to tolerate and quickly adapt to unfavorable and/or changing environmental conditions. Prokaryotes are endowed with sophisticated cellular envelopes that contain molecules not found elsewhere in the biological world. Although prokaryotic cells lack the organelles that characterize their eukaryotic counterparts, their interiors are surprisingly complex. Prokaryotes sense their environment and respond as individual cells to specific environmental challenges; but prokaryotes also act cooperatively, displaying communal activities. In many microbial ecosystems, the functionally active unit is not a single species or population (clonal descendence of the same bacterium) but a consortium of two or more types of cells living in close symbiotic association. Only recently have we become aware that microbes are the basis for the functioning of the biosphere. Thus, we are at a unique time in the history of science, in which the interaction of technological advances and the exponential growth in our knowledge of the present microbial diversity will lead to significant advances not only in microbiology but also in biology and other sciences in general. [Abstract/Link to Full Text]

Schmidt TM
The maturing of microbial ecology.
Int Microbiol. 2006 Sep;9(3):217-23.
A.J. Kluyver and C.B. van Niel introduced many scientists to the exceptional metabolic capacity of microbes and their remarkable ability to adapt to changing environments in The Microbe's Contribution to Biology. Beyond providing an overview of the physiology and adaptability of microbes, the book outlined many of the basic principles for the emerging discipline of microbial ecology. While the study of pure cultures was highlighted, provided a unifying framework for understanding the vast metabolic potential of microbes and their roles in the global cycling of elements, extrapolation from pure cultures to natural environments has often been overshadowed by microbiologists inability to culture many of the microbes seen in natural environments. A combination of genomic approaches is now providing a culture-independent view of the microbial world, revealing a more diverse and dynamic community of microbes than originally anticipated. As methods for determining the diversity of microbial communities become increasingly accessible, a major challenge to microbial ecologists is to link the structure of natural microbial communities with their functions. This article presents several examples from studies of aquatic and terrestrial microbial communities in which culture and culture-independent methods are providing an enhanced appreciation for the microbe's contribution to the evolution and maintenance of life on Earth, and offers some thoughts about the graduate-level educational programs needed to enhance the maturing field of microbial ecology. [Abstract/Link to Full Text]

Mateos LM, Ordóñez E, Letek M, Gil JA
Corynebacterium glutamicum as a model bacterium for the bioremediation of arsenic.
Int Microbiol. 2006 Sep;9(3):207-15.
Arsenic is an extremely toxic metalloid that, when present in high concentrations, severely threatens the biota and human health. Arsenic contamination of soil, water, and air is a global growing environmental problem due to leaching from geological formations, the burning of fossil fuels, wastes generated by the gold mining industry present in uncontrolled landfills, and improper agriculture or medical uses. Unlike organic contaminants, which are degraded into harmless chemical species, metals and metalloids cannot be destroyed, but they can be immobilized or transformed into less toxic forms. The ubiquity of arsenic in the environment has led to the evolution in microbes of arsenic defense mechanisms. The most common of these mechanisms is based on the presence of the arsenic resistance operon (ars), which codes for: (i) a regulatory protein, ArsR; (ii) an arsenite permease, ArsB; and (iii) an enzyme involved in arsenate reduction, ArsC. Corynebacterium glutamicum, which is used for the industrial production of amino acids and nucleotides, is one of the most arsenic-resistant microorganisms described to date (up to 12 mM arsenite and >400 mM arseniate). Analysis of the C. glutamicum genome revealed the presence of two complete ars operons (ars1 and ars2) comprising the typical three-gene structure arsRBC, with an extra arsC1 located downstream from arsC1 (ars1 operon), and two orphan genes (arsB3 and arsC4). The involvement of both ars operons in arsenic resistance in C. glutamicum was confirmed by disruption and amplification of those genes. The strains obtained were resistant to up to 60 mM arsenite, one of the highest levels of bacterial resistance to arsenite so far described. Using tools for the genetic manipulation of C. glutamicum that were developed in our laboratory, we are attempting to obtain C. glutamicum mutant strains able to remove arsenic from contaminated water. [Abstract/Link to Full Text]

Empadinhas N, da Costa MS
Diversity and biosynthesis of compatible solutes in hyper/thermophiles.
Int Microbiol. 2006 Sep;9(3):199-206.
The accumulation of compatible solutes, either by uptake from the medium or by de novo synthesis, is a general response of microorganisms to osmotic stress. The diversity of compatible solutes is large but falls into a few major chemical categories, such as carbohydrates or their derivatives and amino acids or their derivatives. This review deals with compatible solutes found in thermophilic or hyperthermophilic bacteria and archaea that have not been commonly identified in microorganisms growing at low and moderate temperatures. The response to NaCl stress of Thermus thermophilus is an example of how a thermophilic bacterium responds to osmotic stress by compatible solute accumulation. Emphasis is made on the pathways leading to the synthesis of mannosylglycerate and glucosylglycerate that have been recently elucidated in several hyper/thermophilic microorganisms. The role of compatible solutes in the thermoprotection of these fascinating microorganisms is also discussed. [Abstract/Link to Full Text]

Pedrós-Alió C
Genomics and marine microbial ecology.
Int Microbiol. 2006 Sep;9(3):191-7.
Genomics has brought about a revolution in all fields of biology. Before the development of microbial ecology in the 1970s, microbes were not even considered in marine ecological studies. Today we know that half of the total primary production of the planet must be credited to microorganisms. This and other discoveries have changed dramatically the perspective and the focus of marine microbial ecology. The application of genomics-based approaches has provided new challenges and has allowed the discovery of novel functions, an appreciation of the great diversity of microorganisms, and the introduction of controversial ideas regarding the concepts of species, genome, and niche. Nevertheless, thorough knowledge of the traditional disciplines of biology is necessary to explore the possibilities arising from these new insights. This work reviews the different genomic techniques that can be applied to marine microbial ecology, including both sequencing of the complete genomes of microorganisms and metagenomics, which, in turn, can be complemented with the study of mRNAs (transcriptomics) and proteins (proteomics). The example of proteorhodopsin illustrates the type of information that can be gained from these approaches. A genomics perspective constitutes a map that will allow microbiologists to focus their research on potentially more productive aspects. [Abstract/Link to Full Text]

López R
Pneumococcus: the sugar-coated bacteria.
Int Microbiol. 2006 Sep;9(3):179-90.
The study of Streptococcus pneumoniae (the pneumococcus) had been a central issue in medicine for many decades until the use of antibiotics became generalized. Many fundamental contributions to the history of microbiology should credit this bacterium: the capsular precipitin reaction, the major role this reaction plays in the development of immunology through the identification of polysaccharides as antigens, and, mainly, the demonstration, by genetic transformation, that genes are composed of DNA-the finding from the study of bacteria that has had the greatest impact on biology. Currently, pneumococcus is the most common etiologic agent in acute otitis media, sinusitis, and pneumonia requiring the hospitalization of adults. Moreover, meningitis is the leading cause of death among children in developing countries. Here I discuss the contributions that led to the explosion of knowledge about pneumococcus and also report some of the contributions of our group to the understanding of the molecular basis of three important virulence factors: lytic enzymes, pneumococcal phages, and the genes coding for capsular polysaccharides. [Abstract/Link to Full Text]

Soyer-Gobillard MO
Edouard Chatton (1883-1947) and the dinoflagellate protists: concepts and models.
Int Microbiol. 2006 Sep;9(3):173-7.
Edouard Chatton contributed to our knowledge of single-celled protoctists, especially ciliates and dinoflagellates, free-living and/or symbiotic, in relation to the marine invertebrate animals in which they reside. More than the description of many new families, genera and species, and of their life cycles, he anticipated several major concepts of cell biology, including the fundamental difference between prokaryote and eukaryote protists, long time before the advent of electron microscopy. These concepts included: the reproductive ability of the kinetosome-centriole system; the homology of the kinetosome with the mitotic centriole of animal cells; and the different kinds of mitotic systems. Chatton trained more than thirty student collaborators, among them Andre Lwoff, who won the 1965 Nobel Prize in Physiology or Medicine. Later, the great cell biologist Hans Ris and I completed Chatton's light microscopy descriptions on syndinian mitosis dinoflagellate. We had at our disposal sophisticated electron microscopes as well as biochemical and molecular techniques and thus succeeded in corroborating the correct interpretation by Chatton of chromosome structure and mitotic cytology. [Abstract/Link to Full Text]

Sapp J
Two faces of the prokaryote concept.
Int Microbiol. 2006 Sep;9(3):163-72.
Bacteria had remained undefined when, in 1962, Roger Y. Stanier and C.B. van Niel published their famed paper ''The concept of a bacterium.'' The articulation of the prokaryote-eukaryote dichotomy was a vital moment in the history of biology. This article provides a brief overview of the context in which the prokaryote concept was successfully launched in the 1960s, and what it was meant to connote. Two concepts were initially distinguished within the prokaryote-eukaryote dichotomy at that time. One was organizational and referred to comparative cell structure; the other was phylogenetic and referred to a ''natural'' classification. Here, I examine how the two concepts became inseparable; how the prokaryotes came to signify a monophyletic group that preceded the eukaryotes, and how this view remained unquestioned for 15 years, until the birth of molecular evolutionary biology and coherent methods for bacteria phylogenetics based on 16S rRNA. Today, while microbial phylogeneticists generally agree that the prokaryote is a polyphyletic group, there is no agreement on whether the term should be maintained in an organizational sense. [Abstract/Link to Full Text]

Schaechter M
From growth physiology to systems biology.
Int Microbiol. 2006 Sep;9(3):157-61.
As it focuses on the integrated behavior of the entire cell, systems biology is a powerful extension of growth physiology. Here, I briefly trace some of the origins of modern-day bacterial growth physiology and its relevance to systems biology. I describe how growth physiology emerged from the foggy picture of the growth curve as a self-contained entity. For this insight, we can thank Henrici, Hershey, Monod, Maaløe, and others. As a result of their work, growth rate is understood to be the unitary manifestation of the response to nutritional conditions and to the control condition for studies on the effect of environmental stresses. For this response to be usefully reproducible, cultures must be in the steady state known as balanced growth. I point out that present-day experimenters are not always aware of this imperative and thus do not always use conditions that ensure the balanced growth of their control cultures. [Abstract/Link to Full Text]

Lederberg J
The microbe's contribution to biology--50 years after.
Int Microbiol. 2006 Sep;9(3):155-6. [Abstract/Link to Full Text]

---

Recent Articles in Retrovirology

---

Boris-Lawrie K
Bridging fundamental RNA biology, retroviral replication, and oncogenesis: Karen Beemon wins the 2007 Retrovirology Prize.
Retrovirology. 2007 Dec 10;4(1):88.
ABSTRACT: The 2007 M. Jeang Retrovirology Prize has been awarded to Dr. Karen L. Beemon. [Abstract/Link to Full Text]

van Marle G, Gill MJ, Kolodka D, McManus L, Grant T, Church DL
Compartmentalization of the gut viral reservoir in HIV-1 infected patients.
Retrovirology. 2007 Dec 4;4(1):87.
ABSTRACT: BACKGROUND: Recently there has been an increasing interest and appreciation for the gut as both a viral reservoir as well as an important host-pathogen interface in human immunodefiency virus type 1 (HIV-1) infection. The gut associated lymphoid tissue (GALT) is the largest lymphoid organ infected by HIV-1. In this study we examined if different HIV-1 quasispecies are found in different parts of the gut of HIV-1 infected individuals. RESULTS: Gut biopsies (esophagus, stomach, duodenum and colorectum) were obtained from eight HIV-1 infected preHAART (highly active antiretroviral therapy) patients. HIV-1 Nef and Reverse transcriptase (RT) encoding sequences were obtained through nested PCR amplification from DNA isolated from the gut biopsy tissues. The PCR fragments were cloned and sequenced. The resulting sequences were subjected to various phylogenetic analyses. Expression of the nef gene and viral RNA in the different gut tissues was determined using real-time RT-PCR. Phylogenetic analysis of the Nef protein-encoding region revealed compartmentalization of viral replication in the gut within patients. Viral diversity in both the Nef and RT encoding region varied in different parts of the gut. Moreover, increased nef gene expression (p<0.05) and higher levels of viral genome were observed in the colorectum (p<0.05). These differences could reflect an adaptation of HIV-1 to the various tissues. CONCLUSION: Our results indicated that different HIV-1 quasispecies populate different parts of the gut, and that viral replication in the gut is compartmentalized. These observations underscore the importance of the gut as a host-pathogen interface in HIV-1 infection. [Abstract/Link to Full Text]

Jeang KT
World AIDS Day 2007: AIDS at 26, are we there yet?
Retrovirology. 2007 Dec 1;4(1):86.
ABSTRACT: This editorial comments on selected progress made in combating the acquired immune deficiency syndrome (AIDS) after 26 years and some of the remaining challenges. [Abstract/Link to Full Text]

Holmgren B, da Silva Z, Vastrup P, Larsen O, Andersson S, Ravn H, Aaby P
Mortality associated with HIV-1, HIV-2, and HTLV-I single and dual infections in a middle-aged and older population in Guinea-Bissau.
Retrovirology. 2007 Nov 27;4(1):85.
ABSTRACT: BACKGROUND: In Guinea-Bissau HIV-1, HIV-2, and HTLV-I are prevalent in the general population. The natural history of HIV/HTLV-I single and dual infections has not been fully elucidated in this population. Previous studies have shown that combinations of these infections are more common in older women than in men. The present study compares mortality associated with HIV-1, HIV-2, and HTLV-I single and dual infections in individuals over 35 years of age within an urban community-based cohort in Guinea-Bissau. RESULTS: A total of 2,839 and 1,075 individuals were included in the HIV and HTLV-I mortality analyses respectively. Compared with HIV-negative individuals, adjusted mortality rate ratios (MRRs) were 4.9 (95 % confidence interval (CI): 2.3, 10.4) for HIV-1, 1.8 (95%CI: 1.5, 2.3) for HIV-2, and 5.9 (2.4, 14.3) for HIV-1/HIV-2 dual infections. MRR for HTLV-I-positive compared with HTLV-I-negative individuals was 1.7 (1.1, 2.7). Excluding all HIV-positive individuals from the analysis, the HTLV-I MRR was 2.3 (1.3, 3.8). The MRR of HTLV-I/HIV-2 dually infected individuals was 1.7 (0.7, 4.3), compared with HIV/HTLV-I-negative individuals. No statistically significant differences were found in retrovirus-associated mortality between men and women. CONCLUSION: HIV-1-associated excess mortality was low compared with community studies from other parts of Africa, presumably because this population was older and the introduction of HIV-1 into the community recent. HIV-2 and HTLV-I-associated mortality was 2-fold higher than the mortality in uninfected individuals. We found no significant differences between the mortality risk for HIV-2 and HTLV-I single infection, respectively, and HIV-2/HTLV-I dual infection. The higher prevalence of retroviral dual infections in older women is not explained by differential retrovirus-associated mortality for men and women. [Abstract/Link to Full Text]

Jacquot G, le Rouzic E, David A, Mazzolini J, Bouchet J, Bouaziz S, Niedergang F, Pancino G, Benichou S
Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages.
Retrovirology. 2007 Nov 26;4(1):84.
ABSTRACT: BACKGROUND: HIV-1 Vpr is a dynamic protein that primarily localizes in the nucleus, but a significant fraction is concentrated at the nuclear envelope (NE), supporting an interaction between Vpr and components of the nuclear pore complex, including the nucleoporin hCG1. In the present study, we have explored the contribution of Vpr accumulation at the NE to the Vpr functions, including G2-arrest and pro-apoptotic activities, and virus replication in primary macrophages. RESULTS: In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first alpha-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr. In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages. Other mutants with substitutions in the first alpha-helix (Vpr-A30L and Vpr-F34I) were similarly distributed between the nucleus and cytoplasm, demonstrating that this helix contains the determinants required for localization of Vpr at the NE. All these mutations also impaired the Vpr-mediated G2-arrest of the cell cycle and the subsequent cell death induction, indicating a functional link between these activities and the Vpr accumulation at the NE. However, this localization is not sufficient, since mutations within the C-terminal basic region of Vpr (Vpr-R80A and Vpr-R90K), disrupted the G2-arrest and apoptotic activities without altering NE localization. Finally, the replication of the Vpr-L23F and Vpr-K27M hCG1-binding deficient mutant viruses was also affected in primary macrophages from some but not all donors. CONCLUSIONS: These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages. [Abstract/Link to Full Text]

Wu JQ, Wang B, Belov L, Chrisp J, Learmont J, Dyer WB, Zaunders J, Cunninghama AL, Dwyer DE, Saksena NK
Antibody microarray analysis of cell surface antigens on CD4+ and CD8+ T cells from HIV+ individuals correlates with disease stages.
Retrovirology. 2007 Nov 26;4(1):83.
ABSTRACT: BACKGROUND: Expression levels of cell surface antigens such as CD38 and HLA-DR are related to HIV disease stages. To date, the immunophenotyping of cell surface antigens relies on flow cytometry, allowing estimation of 3-6 markers at a time. The recently described DotScan antibody microarray technology enables the simultaneous analysis of a large number of cell surface antigens. This new technology provides new opportunities to identify novel differential markers expressed or co-expressed on CD4+ and CD8+ T cells, which could aid in defining the stage of evolution of HIV infection and the immune status of the patient. RESULTS: Using this new technology, we compared cell surface antigen expression on purified CD4+ and CD8+ T cells between 3 HIV disease groups (long-term non-progressors controlling viremia naturally; HIV+ patients on highly active antiretroviral therapy (HAART) with HIV plasma viral loads <50 copies/ml; and HIV+ patients with viremia during HAART) and uninfected controls. Pairwise comparisons identified 17 statistically differential cell surface antigens including 5 novel ones (CD212b1, CD218a, CD183, CD3 epsilon and CD9), not previously reported. Notably, changes in activation marker expression were more pronounced in CD8+ T cells, whereas changes in the expression of cell membrane receptors for cytokines and chemokines were more pronounced in CD4+ T cells. CONCLUSIONS: Our study not only confirmed cell surface antigens previously reported to be related to HIV disease stages, but also identified 5 novel ones. Of these five, three markers point to major changes in responsiveness to certain cytokines, which are involved in Th1 responses. For the first time our study shows how density of cell surface antigens could be efficiently exploited in an array manner in relation to HIV disease stages. This new platform of identifying disease markers can be further extended to study other diseases. [Abstract/Link to Full Text]

Scaria V, Jadhav V
microRNAs in viral oncogenesis.
Retrovirology. 2007 Nov 24;4(1):82.
ABSTRACT: MicroRNAs are a recently discovered class of small noncoding functional RNAs. These molecules mediate post-transcriptional regulation of gene expression in a sequence specific manner. MicroRNAs are now known to be key players in a variety of biological processes and have been shown to be deregulated in a number of cancers. The discovery of viral encoded microRNAs, especially from a family of oncogenic viruses, has attracted immense attention towards the possibility of microRNAs as critical modulators of viral oncogenesis. The host-virus crosstalk mediated by microRNAs, messenger RNAs and proteins, is complex and involves the different cellular regulatory layers. In this commentary, we describe models of microRNA mediated viral oncogenesis. [Abstract/Link to Full Text]

Miller JH, Presnyak V, Smith HC
The dimerization domain of HIV-1 viral infectivity factor Vif is required to block APOBEC3G incorporation with virions.
Retrovirology. 2007 Nov 24;4(1):81.
ABSTRACT: BACKGROUND: The HIV-1 accessory protein known as viral infectivity factor or Vif binds to the host defence factor human APOBEC3G (hA3G) and prevents its assembly with viral particles and mediates its elimination through ubiquitination and degradation by the proteosomal pathway. In the absence of Vif, hA3G becomes incorporated within viral particles. During the post entry phase of infection, hA3G attenuates viral replication by binding to the viral RNA genome and deaminating deoxycytidines to form deoxyuridines within single stranded DNA regions of the replicated viral genome. Vif dimerization has been reported to be essential for viral infectivity but the mechanistic requirement for Vif multimerization is unknown. RESULTS: We demonstrate that a peptide antagonist of Vif dimerization fused to the cell transduction domain of HIV TAT suppresses live HIV-1 infectivity. We show rapid cellular uptake of the peptide and cytoplasmic distribution. Robust suppression of viral infectivity was dependent on the expression of Vif and hA3G. Disruption of Vif multimerization resulted in the production of virions with markedly increased hA3G content and reduced infectivity. CONCLUSIONS: The role of Vif multimerization in viral infectivity of nonpermissive cells has been validated with an antagonist of Vif dimerization. An important part of the mechanism for this antiretroviral effect is that blocking Vif dimerization enables hA3G incorporation within virions. We propose that Vif multimers are required to interact with hA3G to exclude it from viral particles during their assembly. Blocking Vif dimerization is an effective means of sustaining hA3G antiretroviral activity in HIV-1 infected cells. Vif dimerization is therefore a validated target for therapeutic HIV-1/AIDS drug development. [Abstract/Link to Full Text]

Naito Y, Nohtomi K, Onogi T, Uenishi R, Ui-Tei K, Saigo K, Takebe Y
Optimal design and validation of antiviral siRNA for targeting HIV-1.
Retrovirology. 2007 Nov 8;4(1):80.
ABSTRACT: We propose rational designing of antiviral short-interfering RNA (siRNA) targeting highly divergent HIV-1. In this study, conserved regions within HIV-1 genomes were identified through an exhaustive computational analysis, and the functionality of siRNAs targeting the highest possible conserved regions was validated. We present several promising antiviral siRNA candidates that effectively inhibited multiple subtypes of HIV-1 by targeting the best conserved regions in pandemic HIV-1 group M strains. [Abstract/Link to Full Text]

Savarino A, Pistello M, D'Ostilio D, Zabogli E, Taglia F, Mancini F, Ferro S, Matteucci D, De Luca L, Barreca ML, Ciervo A, Chimirri A, Ciccozzi M, Bendinelli M
Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS.
Retrovirology. 2007 Oct 30;4(1):79.
ABSTRACT: BACKGROUND: Treatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. METHODS: Phylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar Tn5 transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA were quantified by real-time PCR. RESULTS: The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. CONCLUSIONS: We report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in human clinical trials, as one of the most potent anti-FIV agent ever tested in vitro. This finding may provide new avenues for treating FIV infection and contribute to the development of a small animal model mimicking the effects of ART in humans. [Abstract/Link to Full Text]

Baldwin C, Berkhout B
HIV-1 drug-resistance and drug-dependence.
Retrovirology. 2007;478.
In this review, we will describe several recent HIV-1 studies in which a drug-dependent virus variant was selected. A common evolutionary route to the drug-dependence phenotype is proposed. First, the selection of a drug-resistance mutation that also affects the function of the targeted viral protein. Second, a compensatory mutation that repairs the protein function, but in the presence of the drug, which becomes an intrinsic part of the mechanism. The clinical relevance of drug-dependent HIV-1 variants is also discussed. [Abstract/Link to Full Text]

Warrilow D, Stenzel D, Harrich D
Isolated HIV-1 core is active for reverse transcription.
Retrovirology. 2007 Oct 24;4(1):77.
ABSTRACT: Whether purified HIV-1 virion cores are capable of reverse transcription or require uncoating to be activated is currently controversial. To address this question we purified cores from a virus culture and tested for the ability to generate authentic reverse transcription products. A dense fraction (approximately 1.28 g/ml) prepared without detergent, possibly derived from disrupted virions, was found to naturally occur as a minor sub-fraction in our preparations. Core-like particles were identified in this active fraction by electron microscopy. We are the first to report the detection of authentic strong-stop, first-strand transfer and full-length minus strand products in this core fraction without requirement for an uncoating activity. [Abstract/Link to Full Text]

Aquaro S, Muscoli C, Ranazzi A, Pollicita M, Granato T, Masuelli L, Modesti A, Perno CF, Mollace V
The contribution of peroxynitrite generation in HIV replication in human primary macrophages.
Retrovirology. 2007 Oct 21;4(1):76.
ABSTRACT: BACKGROUND: Monocytes/Macrophages (M/M) play a pivotal role as a source of virus during the whole course of HIV-1 infection. Enhanced oxidative stress is involved in the pathogenesis of HIV-1 infection. HIV-1 regulatory proteins induce a reduction of the expression and the activity of MnSOD, the mitochondrial isoform leading to a sustained generation of superoxide anions and peroxynitrite that represent important mediators of HIV-1 replication in M/M. MnTBAP (Mn(III)tetrakis(4-benzoic acid)porphrin chloride), a synthetic peroxynitrite decomposition catalyst, reduced oxidative stress subsequent to peroxynitrite generation. RESULTS: Virus production was assessed by p24 ELISA, western blot, and electron microscopy during treatment with MnTBAP. MnTBAP treatment showed a reduction of HIV-1 replication in both acutely and chronically infected M/M: 99% and 90% inhibition of p24 released in supernatants compared to controls, respectively. Maturation of p55 and p24 was strongly inhibited by MnTBAP in both acutely and chronically infected M/M. EC50 and EC90 are 3.7 (+/-0.05) M and 19.5 (+/-0.5) M, in acutely infected M/M; 6.3 (+/-0.003) M and 30 (+/-0.6) M, in chronically infected M/M. In acutely infected peripheral blood limphocytes (PBL), EC50 and EC90 are 7.4 (+/-0.06) muM and of 21.3 (+/-0.6) muM, respectively. Treatment of acutely-infected M/M with MnTBAP inhibited the elevated levels of malonildialdehyde (MDA) together with the nitrotyrosine staining observed during HIV-1 replication. MnTBAP strongly reduced HIV-1 particles in infected M/M, as shown by electron microscopy. Moreover, in presence of MnTBAP, HIV-1 infectivity was reduced of about 1 log compared to control. CONCLUSIONS: Results support the role of superoxide anions in HIV-1 replication in M/M and suggest that MnTBAP may counteract HIV-1 replication in combination with other antiretroviral treatments. [Abstract/Link to Full Text]

Binette J, Dube M, Mercier J, Cohen EA
Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.
Retrovirology. 2007 Oct 15;4(1):75.
ABSTRACT: BACKGROUND: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex. RESULTS: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. CONCLUSION: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97. [Abstract/Link to Full Text]

Yeung ML, Benkirane M, Jeang KT
Small non-coding RNAs, mammalian cells, and viruses: regulatory interactions?
Retrovirology. 2007 Oct 15;4(1):74.
ABSTRACT: Recent findings suggest that mammalian cells can use small non-coding RNAs (ncRNA) to regulate physiological viral infections. Here, we comment on several lines of evidence that support this concept. We discuss how viruses may in turn protect, suppress, evade, modulate, or adapt to the host cell's ncRNA regulatory schema. [Abstract/Link to Full Text]

Indik S, Gunzburg WH, Kulich P, Salmons B, Rouault F
Rapid spread of mouse mammary tumor virus in cultured human breast cells.
Retrovirology. 2007 Oct 11;4(1):73.
ABSTRACT: BACKGROUND: The role of mouse mammary tumor virus (MMTV) as a causative agent in human breast carcinogenesis has recently been the subject of renewed interest. The proposed model is based on the detection of MMTV sequences in human breast cancer but not in healthy breast tissue. One of the main drawbacks to this model, however, was that until now human cells had not been demonstrated to sustain productive MMTV infection. RESULTS: Here, we show for the first time the rapid spread of mouse mammary tumor virus, MMTV(GR), in cultured human mammary cells (Hs578T), ultimately leading to the infection of every cell in culture. The replication of the virus was monitored by quantitative PCR, quantitative RT-PCR and immunofluorescence imaging. The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody. Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3;-azido-3;-deoxythymidine. The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences. CONCLUSIONS: Taken together, our results show that human cells can support replication of mouse mammary tumor virus. [Abstract/Link to Full Text]

Zeddou M, Rahmouni S, Vandamme A, Jacobs N, Frippiat F, Leonard P, Schaaf-Lafontaine N, Vaira D, Boniver J, Moutschen M
Downregulation of CD94/NKG2A inhibitory receptors on CD8+ T cells in HIV infection is more pronounced in subjects with detected viral load than in their aviraemic counterparts.
Retrovirology. 2007;472.
The CD94/NKG2A heterodimer is a natural killer receptor (NKR), which inhibits cell-mediated cytotoxicity upon interaction with MHC class I gene products. It is expressed by NK cells and by a small fraction of activated CD8+ T lymphocytes. Abnormal upregulation of the CD94/NKG2A inhibitory NKR on cytotoxic T cells (CTLs) could be responsible for a failure of immunosurveillance in cancer or HIV infection. In this study, CD94/NKG2A receptor expression on CD8+ T lymphocytes and NK cells was assessed in 46 HIV-1-infected patients (24 viraemic, 22 aviraemic) and 10 healthy volunteers. The percentage of CD8+ T lymphocytes expressing the CD94/NKG2A inhibitory heterodimer was very significantly decreased in HIV-1-infected patients in comparison with non-infected controls. Within the HIV infected patients, the proportion of CD8+ T lymphocytes and NK cells expressing CD94/NKG2A was higher in subjects with undetectable viral loads in comparison with their viraemic counterparts. No significant difference was detected in the proportion of CD8+ T lymphocytes expressing the activatory CD94/NKG2C heterodimer between the HIV-1 infected patients and the healthy donors, nor between the vireamic and avireamic HIV-1 infected patients. In conclusion, chronic stimulation with HIV antigens in viraemic patients leads to a decreased rather than increased CD94/NKG2A expression on CD8+ T lymphocytes and NK cells. [Abstract/Link to Full Text]

Landry S, Halin M, Lefort S, Audet B, Vaquero C, Mesnard JM, Barbeau B
Detection, characterization and regulation of antisense transcripts in HIV-1.
Retrovirology. 2007;471.
BACKGROUND: We and others have recently demonstrated that the human retrovirus HTLV-I was producing a spliced antisense transcript, which led to the synthesis of the HBZ protein. The objective of the present study was to demonstrate the existence of antisense transcription in HIV-1 and to provide a better characterization of the transcript and its regulation. RESULTS: Initial experiments conducted by standard RT-PCR analysis in latently infected J1.1 cell line and pNL4.3-transfected 293T cells confirmed the existence of antisense transcription in HIV-1. A more adapted RT-PCR protocol with limited RT-PCR artefacts also led to a successful detection of antisense transcripts in several infected cell lines. RACE analyses demonstrated the existence of several transcription initiation sites mapping near the 5' border of the 3'LTR (in the antisense strand). Interestingly, a new polyA signal was identified on the antisense strand and harboured the polyA signal consensus sequence. Transfection experiments in 293T and Jurkat cells with an antisense luciferase-expressing NL4.3 proviral DNA showed luciferase reporter gene expression, which was further induced by various T-cell activators. In addition, the viral Tat protein was found to be a positive modulator of antisense transcription by transient and stable transfections of this proviral DNA construct. RT-PCR analyses in 293T cells stably transfected with a pNL4.3-derived construct further confirmed these results. Infection of 293T, Jurkat, SupT1, U937 and CEMT4 cells with pseudotyped virions produced from the antisense luciferase-expressing NL4.3 DNA clone led to the production of an AZT-sensitive luciferase signal, which was however less pronounced than the signal from NL4.3Luc-infected cells. CONCLUSION: These results demonstrate for the first time that antisense transcription exists in HIV-1 in the context of infection. Possible translation of the predicted antisense ORF in this transcript should thus be re-examined. [Abstract/Link to Full Text]

Brügger B, Krautkrämer E, Tibroni N, Munte CE, Rauch S, Leibrecht I, Glass B, Breuer S, Geyer M, Kräusslich HG, Kalbitzer HR, Wieland FT, Fackler OT
Human immunodeficiency virus type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains.
Retrovirology. 2007;470.
BACKGROUND: The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. RESULTS: Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. CONCLUSION: Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo. [Abstract/Link to Full Text]

Abdurahman S, Youssefi M, Höglund S, Vahlne A
Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity.
Retrovirology. 2007;469.
BACKGROUND: The mature HIV-1 conical core formation proceeds through highly regulated protease cleavage of the Gag precursor, which ultimately leads to substantial rearrangements of the capsid (CAp24) molecule involving both inter- and intra-molecular contacts of the CAp24 molecules. In this aspect, Asp51 which is located in the N-terminal domain of HIV-1 CAp24 plays an important role by forming a salt-bridge with the free imino terminus Pro1 following proteolytic cleavage and liberation of the CAp24 protein from the Pr55Gag precursor. Thus, previous substitution mutation of Asp51 to alanine (D51A) has shown to be lethal and that this invariable residue was found essential for tube formation in vitro, virus replication and virus capsid formation. RESULTS: We extended the above investigation by introducing three different D51 substitution mutations (D51N, D51E, and D51Q) into both prokaryotic and eukaryotic expression systems and studied their effects on in vitro capsid assembly and virus infectivity. Two substitution mutations (D51E and D51N) had no substantial effect on in vitro capsid assembly, yet they impaired viral infectivity and particle production. In contrast, the D51Q mutant was defective both for in vitro capsid assembly and for virus replication in cell culture. CONCLUSION: These results show that substitutions of D51 with glutamate, glutamine, or asparagine, three amino acid residues that are structurally related to aspartate, could partially rescue both in vitro capsid assembly and intra-cellular CAp24 production but not replication of the virus in cultured cells. [Abstract/Link to Full Text]

Bérubé J, Bouchard A, Berthoux L
Both TRIM5alpha and TRIMCyp have only weak antiviral activity in canine D17 cells.
Retrovirology. 2007;468.
BACKGROUND: TRIM5alpha, which is expressed in most primates and the related TRIMCyp, which has been found in one of the New World monkey species, are antiviral proteins of the TRIM5 family that are able to intercept incoming retroviruses early after their entry into cells. The mechanism of action has been partially elucidated for TRIM5alpha, which seems to promote premature decapsidation of the restricted retroviruses. In addition, through its N-terminal RING domain, TRIM5alpha may sensitize retroviruses to proteasome-mediated degradation. TRIM5alpha-mediated restriction requires a physical interaction with the capsid protein of targeted retroviruses. It is unclear whether other cellular proteins are involved in the inhibition mediated by TRIM5alpha and TRIMCyp. A previous report suggested that the inhibition of HIV-1 by the rhesus macaque orthologue of TRIM5alpha was inefficient in the D17a canine cell line, suggesting that the cellular environment was important for the restriction mechanism. Here we investigated further the behavior of TRIM5alpha and TRIMCyp in the D17 cells. RESULTS: We found that the various TRIM5alpha orthologues studied (human, rhesus macaque, African green monkey) as well as TRIMCyp had poor antiviral activity in the D17 cells, despite seemingly normal expression levels and subcellular distribution. Restriction of both HIV-1 and the distantly related N-tropic murine leukemia virus (N-MLV) was low in D17 cells. Both TRIM5alpharh and TRIMCyp promoted early HIV-1 decapsidation in murine cells, but weak levels of restriction in D17 cells correlated with the absence of accelerated decapsidation in these cells and also correlated with normal levels of cDNA synthesis. Fv1, a murine restriction factor structurally unrelated to TRIM5alpha, was fully functional in D17 cells, showing that the loss of activity was specific to TRIM5alpha/TRIMCyp. CONCLUSION: We show that D17 cells provide a poor environment for the inhibition of retroviral replication by proteins of the TRIM5 family. Because both TRIM5alpha and TRIMCyp are poorly active in these cells, despite having quite different viral target recognition domains, we conclude that a step either upstream or downstream of target recognition is impaired. We speculate that an unknown factor required for TRIM5alpha and TRIMCyp activity is missing or inadequately expressed in D17 cells. [Abstract/Link to Full Text]

van der Kuyl AC, Cornelissen M
Identifying HIV-1 dual infections.
Retrovirology. 2007;467.
Transmission of human immunodeficiency virus (HIV) is no exception to the phenomenon that a second, productive infection with another strain of the same virus is feasible. Experiments with RNA viruses have suggested that both coinfections (simultaneous infection with two strains of a virus) and superinfections (second infection after a specific immune response to the first infecting strain has developed) can result in increased fitness of the viral population. Concerns about dual infections with HIV are increasing. First, the frequent detection of superinfections seems to indicate that it will be difficult to develop a prophylactic vaccine. Second, HIV-1 superinfections have been associated with accelerated disease progression, although this is not true for all persons. In fact, superinfections have even been detected in persons controlling their HIV infections without antiretroviral therapy. Third, dual infections can give rise to recombinant viruses, which are increasingly found in the HIV-1 epidemic. Recombinants could have increased fitness over the parental strains, as in vitro models suggest, and could exhibit increased pathogenicity. Multiple drug resistant (MDR) strains could recombine to produce a pan-resistant, transmittable virus.We will describe in this review what is presently known about super- and re-infection among ambient viral infections, as well as the first cases of HIV-1 superinfection, including HIV-1 triple infections. The clinical implications, the impact of the immune system, and the effect of anti-retroviral therapy will be covered, as will as the timing of HIV superinfection. The methods used to detect HIV-1 dual infections will be discussed in detail. To increase the likelihood of detecting a dual HIV-1 infection, pre-selection of patients can be done by serotyping, heteroduplex mobility assays (HMA), counting the degenerate base codes in the HIV-1 genotyping sequence, or surveying unexpected increases in the viral load during follow-up. The actual demonstration of dual infections involves a great deal of additional research to completely characterize the patient's viral quasispecies. The identification of a source partner would of course confirm the authenticity of the second infection. [Abstract/Link to Full Text]

Gorry PR, McPhee DA, Verity E, Dyer WB, Wesselingh SL, Learmont J, Sullivan JS, Roche M, Zaunders JJ, Gabuzda D, Crowe SM, Mills J, Lewin SR, Brew BJ, Cunningham AL, Churchill MJ
Pathogenicity and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo.
Retrovirology. 2007;466.
In efforts to develop an effective vaccine, sterilizing immunity to primate lentiviruses has only been achieved by the use of live attenuated viruses carrying major deletions in nef and other accessory genes. Although live attenuated HIV vaccines are unlikely to be developed due to a myriad of safety concerns, opportunities exist to better understand the correlates of immune protection against HIV infection by studying rare cohorts of long-term survivors infected with attenuated, nef-deleted HIV strains such as the Sydney blood bank cohort (SBBC). Here, we review studies of viral evolution, pathogenicity, and immune responses to HIV infection in SBBC members. The studies show that potent, broadly neutralizing anti-HIV antibodies and robust CD8+ T-cell responses to HIV infection were not necessary for long-term control of HIV infection in a subset of SBBC members, and were not sufficient to prevent HIV sequence evolution, augmentation of pathogenicity and eventual progression of HIV infection in another subset. However, a persistent T-helper proliferative response to HIV p24 antigen was associated with long-term control of infection. Together, these results underscore the importance of the host in the eventual outcome of infection. Thus, whilst generating an effective antibody and CD8+ T-cell response are an essential component of vaccines aimed at preventing primary HIV infection, T-helper responses may be important in the generation of an effective therapeutic vaccine aimed at blunting chronic HIV infection. [Abstract/Link to Full Text]

Geskus RB, Prins M, Hubert JB, Miedema F, Berkhout B, Rouzioux C, Delfraissy JF, Meyer L
The HIV RNA setpoint theory revisited.
Retrovirology. 2007 Sep 21;4(1):65.
ABSTRACT: BACKGROUND: The evolution of plasma viral load after HIV infection has been described as reaching a setpoint, only to start rising again shortly before AIDS diagnosis. In contrast, CD4 T-cell count is considered to show a stable decrease. However, characteristics of marker evolution over time depend on the scale that is used to visualize trends. In reconsidering the setpoint theory for HIV RNA, we analyzed the evolution of CD4 T-cell count and HIV-1 RNA level from HIV seroconversion to AIDS diagnosis. Follow-up data were used from two cohort studies among homosexual men (N=400), restricting to the period before highly active antiretroviral therapy became widely available (1984 until 1996). Individual trajectories of both markers were fitted and averaged, both from seroconversion onwards and in the four years preceding AIDS diagnosis, using a bivariate random effects model. Both markers were evaluated on a scale that is directly related to AIDS risk. RESULTS: Individuals with faster AIDS progression had higher HIV RNA level six months after seroconversion. For CD4 T-cell count, this ordering was less clearly present. However, HIV RNA level and CD4 T-cell count showed qualitatively similar evolution over time after seroconversion, also when stratified by rate of progression to AIDS. In the four years preceding AIDS diagnosis, a non-significant change in HIV RNA increase was seen, whereas a significant biphasic pattern was present for CD4 T-cell decline. CONCLUSIONS: HIV RNA level has more setpoint behaviour than CD4 T-cell count as far as the level shortly after seroconversion is concerned. However, with respect to the, clinically more relevant, marker evolution over time after seroconversion, a setpoint theory holds as much for CD4 T-cell count as for HIV RNA level. [Abstract/Link to Full Text]

Jeang KT
The young, not-so-young, and the 2007 Retrovirology Prize: call for nominations.
Retrovirology. 2007;464.
Recent findings suggest an aging scientific work force and an almost static publishing productivity in the United States. The Retrovirology Prize seeks to recognize and encourage the work of a mid-career retrovirologist between the ages of 45 and 60. The 2006 Retrovirology Prize was awarded to Dr. Joseph G. Sodroski. [Abstract/Link to Full Text]

Zamborlini A, Lehmann-Che J, Clave E, Giron ML, Tobaly-Tapiero J, Roingeard P, Emiliani S, Toubert A, de Thé H, Saïb A
Centrosomal pre-integration latency of HIV-1 in quiescent cells.
Retrovirology. 2007;463.
Human immunodeficiency virus type 1 (HIV-1) efficiently replicates in dividing and non-dividing cells. However, HIV-1 infection is blocked at an early post-entry step in quiescent CD4+ T cells in vitro. The molecular basis of this restriction is still poorly understood. Here, we show that in quiescent cells, incoming HIV-1 sub-viral complexes concentrate and stably reside at the centrosome for several weeks. Upon cell activation, viral replication resumes leading to viral gene expression. Thus, HIV-1 can persist in quiescent cells as a stable, centrosome-associated, pre-integration intermediate. [Abstract/Link to Full Text]

Chen J, Hong K, Jia M, Liu H, Zhang Y, Liu S, Zhang X, Zhao H, Peng H, Ma P, Xing H, Ruan Y, Williams KL, Yu XG, Altfeld M, Walker BD, Shao Y
Human immunodeficiency virus type 1 specific cytotoxic T lymphocyte responses in Chinese infected with HIV-1 B'/C Recombinant (CRF07_BC).
Retrovirology. 2007;462.
BACKGROUND: The characterization of HIV-1-specific T cell responses in people infected with locally circulating HIV-1 strain will facilitate the development of HIV-1 vaccine. Sixty intravenous drug users infected with HIV-1 circulating recombinant form 07_BC (CRF07_BC), which has been spreading rapidly in western China from north to south, were recruited from Xinjiang, China to assess the HIV-1-specific T cell responses at single peptide level with overlapping peptides (OLP) covering the whole concensus clades B and C proteome. RESULTS: The median of the total magnitude and total number of OLPs recognized by CTL responses were 10925 SFC/million PBMC and 25 OLPs, respectively, when tested by clade C peptides, which was significantly higher than when tested by clade B peptides. The immunodominant regions, which cover 14% (58/413) of the HIV-1 proteome, are widely distributed throughout the HIV-1 proteome except in Tat, Vpu and Pol-PR, with Gag, Pol-RT, Pol-Int and Nef being most frequently targeted. The subdominant epitopes are mostly located in p24, Nef, integrase, Vpr and Vif. Of the responses directed to clade C OLPs, 61.75% (972/1574) can be observed when tested with corresponding clade B OLPs. However, Pol-PR and Vpu tend to be targeted in the clade B sequence rather than the clade C sequence, which is in line with the recombinant pattern of CRF07_BC. Stronger and broader CTL responses in subjects with CD4 cell counts ranging from 200 to 400/mm3 were observed when compared to those with less than 200/mm3 or more than 400/mm3, though there have been no significant correlations identified between the accumulative CTL responses or overall breadth and CD4 cell count or plasma viral load. CONCLUSION: This is the first study conducted to comprehensively address T cell responses in Chinese subjects infected with HIV-1 CRF07_BC in which subtle differences in cross-reactivity were observed, though similar patterns of overall immune responses were demonstrated with clade B infected populations. The immunodominant regions identified in this population can facilitate future HIV-1 vaccine development in China. [Abstract/Link to Full Text]

Goila-Gaur R, Khan MA, Miyagi E, Kao S, Strebel K
Targeting APOBEC3A to the viral nucleoprotein complex confers antiviral activity.
Retrovirology. 2007;461.
BACKGROUND: APOBEC3 (A3) proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and to transposition of endogenous retroelements. A3A has significant homology to the C-terminus of A3G but has only a single cytidine deaminase active site (CDA), unlike A3G, which has a second N-terminal CDA previously found to be important for Vif sensitivity and virus encapsidation. A3A is packaged into HIV-1 virions but, unlike A3G, does not have antiviral properties. Here, we investigated the reason for the lack of A3A antiviral activity. RESULTS: Sequence alignment of A3G and A3A revealed significant homology of A3A to the C-terminal region of A3G. However, while A3G co-purified with detergent-resistant viral nucleoprotein complexes (NPC), virus-associated A3A was highly detergent-sensitive leading us to speculate that the ability to assemble into NPC may be a property conveyed by the A3G N-terminus. To test this model, we constructed an A3G-3A chimeric protein, in which the N-terminal half of A3G was fused to A3A. Interestingly, the A3G-3A chimera was packaged into HIV-1 particles and, unlike A3A, associated with the viral NPC. Furthermore, the A3G-3A chimera displayed strong antiviral activity against HIV-1 and was sensitive to inhibition by HIV-1 Vif. CONCLUSION: Our results suggest that the A3G N-terminal domain carries determinants important for targeting the protein to viral NPCs. Transfer of this domain to A3A results in A3A targeting to viral NPCs and confers antiviral activity. [Abstract/Link to Full Text]

Petitjean G, Al Tabaa Y, Tuaillon E, Mettling C, Baillat V, Reynes J, Segondy M, Vendrell JP
Unintegrated HIV-1 provides an inducible and functional reservoir in untreated and highly active antiretroviral therapy-treated patients.
Retrovirology. 2007;460.
BACKGROUND: The presence of HIV-1 preintegration reservoir was assessed in an in vitro experimental model of latent HIV-1 infection, and in patients treated or not with highly active antiretroviral therapy (HAART). RESULTS: In resting CD4+ T lymphocytes latently infected in vitro with HIV-1, we demonstrated that the polyclonal activation induced a HIV-1 replication, which could be prevented by the use of an HIV-1 integrase inhibitor. We also showed that this reservoir was labile since the rescuable HIV-1-antigens production from unintegrated HIV-1 genomes declined over time. These data confirm that our experimental approach allows the characterization of a functional unintegrated HIV-1 reservoir. We then explored the preintegration reservoir in HIV-1-infected patients. This reservoir was detected in 11 of 12 untreated patients, in 4 of 10 sustained responders to HAART, and in one incomplete responder. This reservoir was also inducible, labile, and anti-HIV-1 integrase drug inhibited its induction. Finally, this reservoir was associated with the presence of spontaneous HIV-1 antigens producing CD4+ T cells in blood from 3 of 3 untreated patients and 2 of 2 sustained responders to HAART harboring a preintegration reservoir. CONCLUSION: This preintegration phase of HIV-1 latency could be a consequence of the ongoing viral replication in untreated patients and of a residual viral replication in treated patients. [Abstract/Link to Full Text]

Kozaczynska K, Cornelissen M, Reiss P, Zorgdrager F, van der Kuyl AC
HIV-1 sequence evolution in vivo after superinfection with three viral strains.
Retrovirology. 2007;459.
With millions of people infected worldwide, the evolution of HIV-1 in vivo has been the subject of much research. Although recombinant viruses were detected early in the epidemic, evidence that HIV-1 dual infections really occurred came much later. Dual infected patients, consisting of coinfected (second infection before seroconversion) and superinfected (second infection after seroconversion) individuals, opened up a new area of HIV-1 evolution studies. Here, we describe the in-depth analysis of HIV-1 over time in a patient twice superinfected with HIV-1, first with a subtype B (B2) strain and then with CRF01_AE after initial infection with a subtype B (B1) strain.The nucleotide evolution of gag and env-V3 of the three strains followed a similar pattern: a very low substitution rate in the first 2-3 years of infection, with an increase in synonymous substitutions thereafter. Convergent evolution at the protein level was rare: only a single amino acid in a gag p24 epitope showed convergence in the subtype B strains. Reversal of CTL-epitope mutations were also rare, and did not converge. Recombinant viruses were observed between the two subtype B strains. Luciferase-assays suggested that the CRF01_AE long terminal repeat (LTR) constituted the strongest promoter, but this was not reflected in the plasma viral load. Specific real-time PCR assays based upon the env gene showed that strain B2 and CRF01_AE RNA was present in equal amounts, while levels of strain B1 were 100-fold lower.All three strains were detected in seminal plasma, suggesting that simultaneous transmission is possible. [Abstract/Link to Full Text]

---

Recent Articles in Virology Journal

---

Hafez MM, Sharawy SM, Hassan AA, Salim RF, Abd El Salam FM, Ali AE
Prevalence of transfusion transmitted virus (TTV) genotypes among HCC patients in Qaluobia governorate.
Virol J. 2007 Dec 6;4(1):135.
ABSTRACT: BACKGROUND: transfusion transmitted virus (TTV) is a novel single-stranded DNA virus that was identified in patients with post-transfusion hepatitis of non-A-G type. Clinical significance of TTV infection was analyzed in Egyptian hepatocellular carcinoma (HCC) patients. The present study attempted to clarify these issues in Egypt, particularly in Qaluobia governorate, a country known for its high endemicity of liver disease and hepatotropic viruses. METHODS: TTV are determined in the serum of 60 samples obtained from HCC and liver cirrhosis (LC) patients and 30 healthy individuals. TTV DNA is amplified by nested-PCR with TTV-specific mixed primers derived from the conserved open reading frame 1 (ORF1) region followed by digestion with restriction enzyme. Using the enzymes HaeIII, DraI, EcoRI and PstI, we are able to distinguish between the four TTV genotypes. RESULTS: The positive rate of TTV detection was 46.7%, 40% and 36.7% among HCC, LC patients and healthy individuals respectively. The more prevalence genotype was detected in the positive serum samples was genotype 1 (35.7%) in HCC patients, (50%) in LC and (63.3%) in healthy individuals, Genotype 5 (21.4%), (25.5%) and (18.2%) in HCC, LC and healthy individuals respectively. DISCUSSION: This study indicates that TTV is commonly present in adult patients with HCC and LC as well as healthy individuals. The most prevalence TTV genotype is genotype 1. It seems that the infection neither contribute to the severity of liver disease no to the causation of HCC. [Abstract/Link to Full Text]

Marjuki H, Yen HL, Franks J, Webster RG, Pleschka S, Hoffmann E
Higher polymerase activity of a human influenza virus enhances activation of the hemagglutinin-induced Raf/MEK/ERK signal cascade.
Virol J. 2007 Dec 5;4(1):134.
ABSTRACT: Influenza viruses replicate within the nucleus of infected cells. Viral genomic RNA, three polymerase subunits (PB2, PB1, and PA), and the nucleoprotein (NP) form ribonucleoprotein complexes (RNPs) that are exported from the nucleus late during the infectious cycle. The virus-induced Raf/MEK/ERK (MAPK) signal cascade is crucial for efficient virus replication. Blockade of this pathway retards RNP export and reduces virus titers. Hemagglutinin (HA) accumulation and its tight association with lipid rafts activate ERK and enhance localization of cytoplasmic RNPs. We studied the induction of MAPK signal cascade by two seasonal human influenza A viruses A/HK/218449/06 (H3N2) and A/HK/218847/06 (H1N1) that differed substantially in their replication efficiency in tissue culture. Infection with H3N2 virus, which replicates efficiently, resulted in higher HA expression and its accumulation on the cell membrane, leading to substantially increased activation of MAPK signaling compared to that caused by H1N1 subtype. More H3N2-HAs were expressed and accumulated on the cell membrane than did H1N1-HAs. Viral polymerase genes, particularly H3N2-PB1 and H3N2-PB2, were observed to contribute to increased viral polymerase activity. Applying plasmid-based reverse genetics to analyze the role of PB1 protein in activating HA-induced MAPK cascade showed that recombinant H1N1 virus possessing the H3N2-PB1 (rgH1N1/H3N2-PB1) induced greater ERK activation, resulting in increased nuclear export of the viral genome and higher virus titers. We conclude that enhanced viral polymerase activity promotes the replication and transcription of viral RNA leading to increased accumulation of HA on the cell surface and thereby resulting in an upregulation of the MAPK cascade and more efficient nuclear RNP-export as well as virus production. [Abstract/Link to Full Text]

Harwani SC, Lurain NS, Zariffard MR, Spear GT
Differential inhibition of human cytomegalovirus (HCMV) by toll-like receptor ligands mediated by interferon-beta in human foreskin fibroblasts and cervical tissue.
Virol J. 2007 Dec 5;4(1):133.
ABSTRACT: Human cytomegalovirus (HCMV) can be acquired sexually and is shed from the genital tract. Cross-sectional studies in women show that changes in genital tract microbial flora affect HCMV infection and/or shedding. Since genital microbial flora may affect HCMV infection or replication by stimulating cells through Toll-like receptors (TLR), we assessed the effects of defined TLR-ligands on HCMV replication in foreskin fibroblasts and ectocervical tissue. Poly I:C (a TLR3-ligand) and lipopolysaccharide (LPS, a TLR4-ligand) inhibited HCMV and induced secretion of IL-8 and Interferon-beta (IFNbeta) in both foreskin fibroblasts and ectocervical tissue. The anti-HCMV effect was reversed by antibody to IFNbeta. CpG (TLR9 ligand) and lipoteichoic acid (LTA, TLR2 ligand) also inhibited HCMV infection in ectocervical tissue and this anti-HCMV effect was also reversed by anti-IFNbeta antibody. In contast, LTA and CpG did not inhibit HCMV infection in foreskin fibroblasts. This study shows that TLR ligands induce an HCMV-antiviral effect that is mediated by IFNbeta suggesting that changes in genital tract flora may affect HCMV infection or shedding by stimulating TLR. This study also contrasts the utility of two models that can be used for assessing the interaction of microbial flora with HCMV in the genital tract. Clear differences in the response to different TLR ligands suggests the explant model more closely reflects in vivo responses to genital infections. [Abstract/Link to Full Text]

Flint PL
Applying the scientific method when assessing the influence of migratory birds on the dispersal of H5N1.
Virol J. 2007 Dec 4;4(1):132.
ABSTRACT: BACKGROUND: The role of wild birds in the dispersal of highly pathogenic avian influenza virus H5N1 continues to be the subject of considerable debate. However, some researchers functionally examining the same question are applying opposing null hypotheses when examining this issue. DISCUSSION: I describe the correct method for establishing a null hypothesis under the scientific method. I suggest that the correct null hypothesis is that migratory birds can disperse this virus during migration and encourage researchers to design studies to falsify this null. Finally, I provide several examples where statements made during this debate, while strictly true, are not generally informative or are speculative. SUMMARY: By adhering to the scientific method, definitive answers regarding the role of wild birds in the dispersal of highly pathogenic viruses will be reached more effectively. [Abstract/Link to Full Text]

Serwer P, Hayes SJ, Lieman K
Aggregates of bacteriophage 0305phi8-36 seed future growth.
Virol J. 2007 Dec 4;4(1):131.
ABSTRACT: Lytic bacteriophage 0305phi8-36 forms visually observed aggregates during plaque formation. Aggregates intrinsically lower propagation potential. In the present study, the following observations indicate that lost propagation potential is regained with time: (1) Aggregates sometimes concentrate at the edge of clear plaques. (2) A semi-clear ring sometimes forms beyond the plaques. (3) Formation of a ring is completely correlated with the presence of aggregates at the same angular displacement along the plaque edge. To explain this aggregate-derived lowering/raising of propagation potential, the following hypothesis is presented: Aggregation/dissociation of bacteriophage of 0305phi8-36 is a selected phenomenon that evolved to maintain high host finding rate in a trade-off with maintaining high rate of bacteriophage progeny production. This hypothesis explains ringed plaque morphology observed for other bacteriophages and predicts that aggregates will undergo time-dependent change in structure as propagation potential increases. In support, fluorescence microscopy reveals time-dependent change in the distance between resolution-limited particles in aggregates. [Abstract/Link to Full Text]

Watson S, Mercier S, Bye C, Wilkinson J, Cunningham A, Harman A
Determination of Suitable Housekeeping Genes for Normalisation of Quantitative Real Time PCR Analysis of Cells Infected with Human Immunodeficiency Virus and Herpes Viruses.
Virol J. 2007 Dec 3;4(1):130.
ABSTRACT: The choice of an appropriate housekeeping gene for normalisation purposes has now become an essential requirement when designing QPCR experiments. This is of particular importance when using QPCR to measure viral and cellular gene transcription levels in the context of viral infections as viruses can significantly alter the host cell pathways that traditional housekeeping genes often encode components of. In this study we have determined the reliability of 10 housekeeping genes in context of four heavily studied viral infections; human immunodeficiency virus type 1, herpes simplex virus type 1, cytomegalovirus and varicella zoster virus infections using a variety of cell types and virus strains. This provides researchers of these viruses with a shortlist of potential housekeeping genes to use as normalisers for QPCR experiments. [Abstract/Link to Full Text]

Yassine HM, Al-Natour MQ, Lee CW, Saif YM
Interspecies and intraspecies transmission of triple reassortant H3N2 influenza A viruses.
Virol J. 2007 Nov 28;4(1):129.
ABSTRACT: The triple reassortant H3N2 viruses were isolated for the first time from pigs in 1998 and are known to be endemic in swine and turkeys populations in the United States. In 2004, we isolated two H3N2 triple reassortant viruses from two turkey breeder flocks in Ohio and Illinois. Infected hens showed no clinical signs, but experienced a complete cessation of egg production. In this study, we evaluated three triple reassortant H3N2 isolates of turkey origin and one isolate of swine origin for their transmission between swine and turkeys. Although all 4 viruses tested share high genetic similarity in all 8 genes, only the Ohio strain (A/turkey/Ohio/313053/04) was shown to transmit efficiently both ways between swine and turkeys. One isolate, A/turkey/North Carolina/03, was able to transmit from pigs to turkeys but not vice versa. Neither of the other two viruses transmitted either way. Sequence analysis of the HA1 gene of the Ohio strain showed one amino acid change (D to A) at residue 190 of the receptor binding domain upon transmission from turkeys to pigs. The Ohio virus was then tested for intraspecies transmission in three different avian species. The virus was shown to replicate and transmit among turkeys, replicate but does not transmit among chickens, and did not replicate in ducks. Identifying viruses with varying inter- and intra-species transmission potential should be useful for further studies on the molecular basis of interspecies transmission. [Abstract/Link to Full Text]

Baig S, Siddiqui A, Ahmed W, Qureshi H, Arif A
The association of complex liver disorders with HBV genotypes prevalent in Pakistan.
Virol J. 2007 Nov 27;4(1):128.
ABSTRACT: BACKGROUND: Genotyping of HBV is generally used for determining the epidemiological relationship between various virus strains and origin of infection mostly in research studies. The utility of genotyping for clinical applications is only beginning to gain importance. Whether HBV genotyping will constitute part of the clinical evaluation of Hepatitis B patients depend largely on the availability of the relevance of the evidence based information. Since Pakistan has a HBV genotype distribution which has been considered less virulent as investigated by earlier studies from South East Asian countries, a study on correlation between HBV genotypes and risk of progression to further complex hepatic infection was much needed. METHODS: A total of 295 patients with HBsAg positive were selected from the Pakistan Medical Research Council's ( PMRC), out patient clinics. Two hundred and twenty six (77%) were males, 69(23%) were females ( M to F ratio 3.3:1). RESULTS: Out of 295 hepatitis patients, 156(53.2%) had Acute(CAH), 71(24.2%) were HBV Carriers , 54(18.4%) had Chronic liver disease (CLD). Patients with Cirrhosis and HCC were 14(4.7%). Genotype D was the most prevalent genotype in all categories of HBV patients, Acute(108), Chronic(39),Carrier(53). Cirrhosis/HCC(7) were HBV/D positive. Genotype A was the second prevalent with 28(13%) in acute cases, 12(22.2%) in chronics ,14(19.7%) in carriers and 5(41.7)Cirrhosis/HCC patients. Mixed genotype (A/D)was found in 20(12.8%) of Acute patients, 3(5.6%) of Chronic and 4(5.6%) of carriers, none in case of severe liver conditions. CONCLUSION: Hepatitis B genotypes A, D and A/D combination were present in all categories of patients except that no A/D combination was detected in severe conditions. Genotype D was the dominant genotype. However, genotype A was found to be more strongly associated with severe liver disease. Mixed genotype (A/D) did not significantly appear to influence the clinical outcome. [Abstract/Link to Full Text]

Wang X, Li B, Zhao KN
Differential expression of papillomavirus L1 proteins encoded by authentic and codon modified L1 genes in methylcellulose-treated mouse keratinocytes.
Virol J. 2007 Nov 25;4(1):127.
ABSTRACT: Papillomaviruses (PVs) are double-stranded DNA viruses that infect keratinocytes in differentiating epithelia and induce hyperproliferative lesions. Here, we used methylcellulose to induce cell differentiation of primary mouse keratinocytes (KCs) in in vitro culture and assessed the expression of authentic and codon-modified version of L1 capsid genes from two PV types (HPV6b and BPV1). Based on the quantitative RT-PCR analysis, methylcellulose treatment did not influence the transcriptional expression of both authentic and codon-modified L1 genes in KCs. Western blot showed that methylcellulose significantly increased the levels of the L1 proteins expressed from two authentic L1 genes. Conversely, methylcellulose dramatically decreased L1 protein expression in KCs transfected with two codon-modified L1 expression constructs. These data suggest that L1 protein expression is associated with KC differentiation induced by methylcellulose treatment and regulated at the post-transcriptional level. [Abstract/Link to Full Text]

Sundaravaradan V, Das SR, Ramakrishnan R, Sehgal S, Gopalan S, Ahmad N, Jameel S
Role of HIV-1 subtype C envelope V3 to V5 regions in viral entry, coreceptor utilization and replication efficiency in primary T-lymphocytes and monocyte-derived macrophages.
Virol J. 2007 Nov 24;4(1):126.
ABSTRACT: BACKGROUND: Several subtypes of HIV-1 circulate in infected people worldwide, including subtype B in the United States and subtype C in Africa and India. To understand the biological properties of HIV-1 subtype C, including cellular tropism, virus entry, replication efficiency and cytopathic effects, we reciprocally inserted our previously characterized envelope V3-V5 regions derived from 9 subtype C infected patients from India into a subtype B molecular clone, pNL4-3. Equal amounts of the chimeric viruses were used to infect T-lymphocyte cell lines (A3.01 and MT-2), coreceptor cell lines (U373-MAGI- CCR5/CXCR4), primary blood T-lymphocytes (PBL) and monocyte-derived macrophages (MDM). RESULTS: We found that subtype C envelope V3-V5 region chimeras failed to replicate in T-lymphocyte cell lines but replicated in PBL and MDM. In addition, these chimeras were able to infect U373MAGI- CD4+-CCR5+ but not U373MAGI-CD4+-CXCR4+ cell line, suggesting CCR5 coreceptor utilization and R5 phenotypes. These subtype C chimeras were unable to induce syncytia in MT-2 cells, indicative of non-syncytium inducing (NSI) phenotypes. More importantly, the subtype C envelope chimeras replicated at higher levels in PBL and MDM compared with subtype B chimeras and isolates. Furthermore, the higher levels subtype C chimeras replication in PBL and MDM correlated with increased virus entry in U373MAGI-CD4+-CCR5+. CONCLUSIONS: Taken together, these results suggest that the envelope V3 to V5 regions of subtype C contributed to higher levels of HIV-1 replication compared with subtype B chimeras, which may contribute to higher viral loads and faster disease progression in subtype C infected individuals than other subtypes as well as rapid HIV-1 subtype C spread in India. [Abstract/Link to Full Text]

Garcia-Quintanilla A
Overcoming viral escape with vaccines that generate and display antigen diversity in vivo.
Virol J. 2007 Nov 22;4(1):125.
ABSTRACT: BACKGROUND: Viral diversity is a key problem for the design of effective and universal vaccines. Virtually, a vaccine candidate including most of the diversity for a given epitope would force the virus to create escape mutants above the viability threshold or with a high fitness cost. PRESENTATION OF THE HYPOTHESIS: Therefore, I hypothesize that priming the immune system with polyvalent vaccines where each single vehicle generates and displays multiple antigen variants in vivo, will elicit a broad and long-lasting immune response able to avoid viral escape. TESTING THE HYPOTHESIS: To this purpose, I propose the use of yeasts that carry virus-like particles designed to pack the antigen-coding RNA inside and replicate it via RNA-dependent RNA polymerase. This would produce diversity in vivo limited to the target of interest and without killing the vaccine vehicle. IMPLICATIONS OF THE HYPOTHESIS: This approach is in contrast with peptide cocktails synthesized in vitro and polyvalent strategies where every cell or vector displays a single or definite number of mutants; but similarly to all them, it should be able to overcome original antigenic sin, avoid major histocompatibility complex restriction, and elicit broad cross-reactive immune responses. Here I discuss additional advantages such as minimal global antagonism or those derived from using a yeast vehicle, and potential drawbacks like autoimmunity. Diversity generated by this method could be monitored both genotypically and phenotypically, and therefore selected or discarded before use if needed. [Abstract/Link to Full Text]

Moes L, Wirth M
The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon.
Virol J. 2007 Nov 22;4(1):124.
ABSTRACT: BACKGROUND: Bovine viral diarrhea virus (BVDV) is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped previously and it has been suggested that the IRES extends into the coding of the BVDV polyprotein. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESs and in the case of the closely related classical swine fever virus (CSFV) and the more distantly related Hepatitis C virus (HCV) pseudoknot function in translation has been demonstrated. RESULTS: To characterize the BVDV IRESs in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that an RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical swine fever virus, a pestivirus, and the hepatitis C viruses, another genus of the Flaviviridae. CONCLUSION: The IRES of the non-cytopathic BVDV SD1 strain displays features known from other pestivirus IRESes. The predicted pseudoknot in the 5'UTR of BVDV SD1 virus represents an important structural element in BVDV translation. [Abstract/Link to Full Text]

Costin JM, Rausch JM, Garry RF, Wimley WC
Viroporin potential of the lentivirus lytic peptide (LLP) domains of the HIV-1 gp41 protein.
Virol J. 2007 Nov 20;4(1):123.
ABSTRACT: BACKGROUND: Mechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development. Virally induced changes in membrane ionic permeability induced by lytic viruses of many families contribute to cytopathogenesis. HIV-1 induces disturbances in plasma membrane ion transport. The carboxyl terminus of TM (gp41) contains potential amphipathic alpha-helical motifs identified through their structural similarities to naturally occurring cytolytic peptides. These sequences have been dubbed lentiviral lytic peptides (LLP) -1, -2, and -3. RESULTS: Peptides corresponding to the LLP domains (from a clade B virus) partition into lipid membranes, fold into alpha-helices and disrupt model membrane permeability. A peptide corresponding to the LLP-1 domain of a clade D HIV-1 virus, LLP-1D displayed similar activity to the LLP-1 domain of the clade B virus in all assays, despite a lack of amino acid sequence identity. CONCLUSIONS: These results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin. Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs. [Abstract/Link to Full Text]

Klein J, Hussain M, Ahmad M, Normann P, Afzal M, Alexandersen S
Genetic characterisation of the recent foot-and-mouth disease virus subtype A/IRN/2005.
Virol J. 2007 Nov 15;4(1):122.
ABSTRACT: BACKGROUND: According to the World Reference Laboratory for FMD, a new subtype of FMDV serotype A was detected in Iran in 2005. This subtype was designated A/IRN/2005, and rapidly spread throughout Iran and moved westwards into Saudi Arabia and Turkey where it was initially detected from August 2005 and subsequently caused major disease problems in the spring of 2006. The same subtype reached Jordan in 2007. As part of an ongoing project we have also detected this subtype in Pakistan with the first positive samples detected in April 2006. To characterise this subtype in detail, we have sequenced and analysed the complete coding sequence of three subtype A/IRN/2005 isolates collected in Pakistan in 2006, the complete coding sequence of one subtype A/IRN/2005 isolate collected during the first outbreak in Turkey in 2005 and, in addition, the partial 1D coding sequence derived from 4 epithelium samples and 34 swab-samples from Asian buffaloes or cattle subsequently found to be infected with the A/IRN/2005 subtype. RESULTS: The phylogenies of the genome regions encoding for the structural proteins, displayed, with the exception of 1A, distinct, serotype-specific clustering and an evolutionary relationship of the A/IRN/2005 sublineage with the A22 sublineage. Potential recombination events have been detected in parts of the genome region coding for the non-structural proteins of FMDV. In addition, amino acid substitutions have been detected in the deduced VP1 protein sequence, potentially related to clinical or subclinical outcome of FMD. Indications of differential susceptibility for developing a subclinical course of disease between Asian buffaloes and cattle have been detected. Furthermore, hitherto unknown insertions of 2 amino acids before the second start codon, as well as sublineage specific amino acids have been detected in the genome region encoding for the leader proteinase of A/IRN/2005 sublineage. CONCLUSIONS: Our findings indicate that the A/IRN/2005 sublineage has undergone two different paths of evolution for the structural and non-structural genome regions. The structural genome regions have had their evolutionary starting point in the A22 sublineage. It can be assumed that, due to the quasispecies structure of FMDV populations and the error-prone replication process, advantageous mutations in a changed environment have been fixed and lead to the occurrence of the new A/IRN/2005 sublineage. Together with this mechanism, recombination within the non-structural genome regions, potentially modifying the virulence of the virus, may be involved in the success of this new sublineage. The possible origin of this recombinant virus may be a co-infection with Asia1 and a serotype A precursor of the A/IRN/2005 sublineage potentially within Asian Buffaloes, as these appears to relatively fairly become infected, but usually without developing clinical disease and consequently showing not a strong acute inflammatory immune response against a second FMDV infection. [Abstract/Link to Full Text]

Koch AL
Evolution of temperate pathogens: the bacteriophage/bacteria paradigm.
Virol J. 2007 Nov 9;4(1):121.
ABSTRACT: BACKGROUND: Taking as a pattern, how T4 and lambda viruses interacting with each other and with their Gram-negative host, Escherichia coli, a general model is constructed for the evolution of 'gentle' or temperate pathogens. This model is not simply either pure group or kin selection, but probably is common in a variety of host-parasite pairs in various taxonomic groups. The proposed mechanism is that for its own benefit the pathogen evolved ways to protect its host from attack by other pathogens and this has incidentally protected the host. Although appropriate mechanisms would have been developed and excluded related viral species and also other quite different pathogens, the important advance would have been when other individuals of the same species that arrive at the host subsequent to the first infecting one were excluded. RESULTS: Such a class of mechanisms would not compete one genotype with another, but simply would be of benefit to the first pathogen that had attacked a host organism. CONCLUSION: This would tend to protect and extend the life of the host against the detrimental effects of secondarily infecting pathogens. This leads to the pathogens becoming more temperate via the now favorable co-evolution with its host, which basically protects both host and virus against other pathogens although it may cause slowing of the growth of the primary infecting pathogen. Evolution by a 'gentle' strategy would be favored as long as the increased wellbeing of the host also favored the eventual transmission of the early infecting pathogen to other hosts. [Abstract/Link to Full Text]

Melancon JM, Fulmer PA, Kousoulas KG
The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment.
Virol J. 2007 Nov 8;4(1):120.
ABSTRACT: The HSV-1 UL20 protein (UL20p) and glycoprotein K (gK) are both important determinants of cytoplasmic virion morphogenesis and virus-induced cell fusion. In this manuscript, we examined the effect of UL20 mutations on the coordinate transport and Trans Golgi Network (TGN) localization of UL20p and gK, virus-induced cell fusion and infectious virus production. Deletion of 18 amino acids from the UL20p carboxyl terminus (UL20 mutant 204t) inhibited intracellular transport and cell-surface expression of both gK and UL20, resulting in accumulation of UL20p and gK in the endoplasmic reticulum (ER) in agreement with the inability of 204t to complement UL20-null virus replication and virus-induced cell fusion. In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids (UL20 mutants 211t and 216t, respectively) allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization. However, while both 211t and 216t failed to complement for infectious virus production, 216t complemented for virus-induced cell fusion, but 211t did not. These results indicated that the carboxyl terminal six amino acids of UL20p were crucial for infectious virus production, but not involved in intracellular localization of UL20p/gK and concomitant virus-induced cell fusion. In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites. UL20p tyrosine-modified mutants with both tyrosine residues changed enabled efficient intracellular transport and TGN localization of UL20p and gK, but failed to complement for either infectious virus production, or virus-induced cell fusion. These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion. [Abstract/Link to Full Text]

Zhang Q, Wu G, Richards E, Jia S, Zeng C
Retraction: Universal primers for HBV genome DNA amplification across subtypes: a case study for designing more effective viral primers.
Virol J. 2007;4119.
ABSTRACT: This is a retraction of the article submitted by Zhang et al. Virology J 2007, 4:92. [Abstract/Link to Full Text]

Mozdzanowska K, Zharikova D, Cudic M, Otvos L, Gerhard W
Roles of adjuvant and route of vaccination in antibody response and protection engendered by a synthetic matrix protein 2-based influenza A virus vaccine in the mouse.
Virol J. 2007 Oct 31;4(1):118.
ABSTRACT: BACKGROUND: The M2 ectodomain (M2e) of influenza A virus (IAV) strains that have circulated in humans during the past 90 years shows remarkably little structural diversity. Since M2e-specific antibodies (Abs) are capable of restricting IAV replication in vivo but are present only at minimal concentration in human sera, efforts are being made to develop a M2e-specific vaccine. We are exploring a synthetic multiple antigenic peptide (MAP) vaccine and here report on the role of adjuvants (cholera toxin and immunostimulatory oligodeoxynucleotide) and route of immunization on Ab response and strength of protection. RESULTS: Independent of adjuvants and immunization route, on average 87% of the M2e-MAP-induced Abs were specific for M2e peptide and a variable fraction of these M2e(pep)-specific Abs (average 15%) cross-reacted with presumably native M2e expressed by M2-transfected cells. The titer of these cross-reactive M2e(pep-nat)-specific Abs in sera of parenterally immunized mice displayed a sigmoidal relation to level of protection, with EC50 of ~20ug Ab/ml serum, though experiments with passive M2e(pep-nat) Abs indicated that serum Abs did not fully account for protection in parenterally vaccinated mice, particularly in upper airways. Intranasal vaccination engendered stronger protection and a higher proportion of G2a Abs than parenteral vaccination, and the strength of protection failed to correlate with M2e(pep-nat)-specific serum Ab titers, suggesting a role of airway-associated immunity in protection of intranasally vaccinated mice. Intranasal administration of M2e-MAP without adjuvant engendered no response but coadministration with infectious IAV slightly enhanced the M2e(pep-nat) Ab response and protection compared to vaccination with IAV or adjuvanted M2e-MAP alone. CONCLUSIONS: M2e-MAP is an effective immunogen as ~15% of the total M2e-MAP-induced Ab response is of desired specificity. While M2e(pep-nat)-specific serum Abs have an important role in restricting virus replication in trachea and lung, M2e-specific T cells and/or locally produced Abs contribute to protection in upper airways. Intranasal vaccination is preferable to parenteral vaccination, presumably because of induction of local protective immunity by the former route. Intranasal coadministration of M2e-MAP with infectious IAV merits further investigation in view of its potential applicability to human vaccination with live attenuated IAV. [Abstract/Link to Full Text]

Lu YW, Tan TL, Zhang J, Chen WN
Cellular apoptosis induced by replication of hepatitis B virus: possible link between viral genotype and clinical outcome.
Virol J. 2007 Oct 31;4(1):117.
ABSTRACT: HBV remains one of the major pathogens of liver diseases but the outcomes as inflammation, cirrhosis and cancer of the liver are greatly related to different viral genotypes. The aim of this study was to assess the pro-apoptotic effect of HBSP from three HBV genotypes on liver derived cells. HepG2 cells were applied in our system and transfected by HBV genotype A, B, and C. Cells were observed under phase contrast microscope, stained by apoptosis marker and analyzed by flow cytometre. HBSP expression was detected by western blot assay. BH3 sequences were aligned and analyzed by Vector NTI. HBV genotypes A, B, and C transfected cells displayed evidence of cell death which was further proved as apoptosis. Natural expression of a pro-apoptotic protein HBSP was detected during genomes transfection. The different apoptotic effects were correlated to the HBSP expression from each genome. Alignment and analysis of the BH3 domains from the three genomes revealed slight variance which might also contribute to the result. Our results suggested that variant HBSP expression and BH3 sequence of HBV genotypes may be involved in differential apoptotic effect in transfected cells. Detailed analysis of the role of HBV genotypes in cellular apoptotic process should provide molecular information on the reported clinical outcome of infection by different HBV genotypes. [Abstract/Link to Full Text]

Gruener M, Bravo IG, Momburg F, Alonso A, Tomakidi P
The E5 protein of the human papillomavirus type 16 down-regulates HLA-I surface expression in calnexin-expressing but not in calnexin-deficient cells.
Virol J. 2007 Oct 30;4(1):116.
ABSTRACT: The human papillomavirus type 16 E5 protein (HPV16 E5) down-regulates surface expression of HLA-I molecules. The molecular mechanisms underlying this effect are so far unknown. Here we show that HPV16 E5 down-regulates HLA-I surface expression in calnexin-containing but not in calnexin-deficient cells. Immunoprecipitation experiments reveal that calnexin and HPV16E5 can be co-precipitated and that this association depends on the presence of a wild-type first hydrophobic region of E5. When an E5 mutant (M1) in which the first putative transmembrane helix had been disrupted was used for the transfections calnexin-E5 co-precipitation was strongly impaired. In addition, we show that the M1 mutant is only able to marginally down-regulate HLA-I surface expression compared to the wild-type protein. Besides, we demonstrate that E5 forms a ternary complex with calnexin and the heavy chain of HLA-I, which is mediated by the first hydrophobic region of the E5 protein. On the basis of our results we conclude that formation of this complex is responsible for retention of HLA-I molecules in the ER of the cells. [Abstract/Link to Full Text]

Bonaldo MC, Mello SM, Trindade GF, Rangel AA, Duarte AS, Oliveira PJ, Freire MS, Kubelka CF, Galler R
Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes.
Virol J. 2007 Oct 30;4(1):115.
ABSTRACT: BACKGROUND: The yellow fever virus, a member of the genus Flavivirus, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor. RESULTS: YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 +/- 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus. CONCLUSIONS: This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection. [Abstract/Link to Full Text]

Song JW, Gu SH, Bennett SN, Arai S, Puorger M, Hilbe M, Yanagihara R
Seewis virus, a genetically distinct hantavirus in the Eurasian common shrew (Sorex araneus).
Virol J. 2007 Oct 30;4(1):114.
ABSTRACT: More than 20 years ago, hantaviral antigens were reported in tissues of the Eurasian common shrew (Sorex araneus), Eurasian water shrew (Neomys fodiens) and common mole (Talpa europea), suggesting that insectivores, or soricomorphs, might serve as reservoirs of unique hantaviruses. Using RT-PCR, sequences of a genetically distinct hantavirus, designated Seewis virus (SWSV), were amplified from lung tissue of a Eurasian common shrew, captured in October 2006 in Graubunden, Switzerland. Pair-wise analysis of the full-length S and partial M and L segments of SWSV indicated approximately 55%-72% similarity with hantaviruses harbored by Murinae, Arvicolinae, Neotominae and Sigmodontinae rodents. Phylogenetically, SWSV grouped with other recently identified shrew-borne hantaviruses. Intensified efforts are underway to clarify the genetic diversity of SWSV throughout the geographic range of the Eurasian common shrew, as well as to determine its relevance to human health. [Abstract/Link to Full Text]

Lang DM
Imperfect DNA mirror repeats in the gag gene of HIV-1 (HXB2) identify key functional domains and coincide with protein structural elements in each of the mature proteins.
Virol J. 2007 Oct 26;4(1):113.
ABSTRACT: BACKGROUND: A DNA mirror repeat is a sequence segment delimited on the basis of its containing a center of symmetry on a single strand, e.g. 5'-GCATGGTACG-3'. It is most frequently described in association with a functionally significant site in a genomic sequence, and its occurrence is regarded as noteworthy, if not unusual. However, imperfect mirror repeats (IMRs) having [greater than or equal to] 50% symmetry are common in the protein coding DNA of monomeric proteins and their distribution has been found to coincide with protein structural elements - helices, b_sheets and turns. In this study, the distribution of IMRs is evaluated in a polyprotein - to determine whether IMRs may be related to the position or order of protein cleavage or other hierarchal aspects of protein function. The gag gene of HIV-1 [GenBank:K03455] was selected for the study because its protein motifs and structural components are well documented. RESULTS: There is a highly specific relationship between IMRs and structural and functional aspects of the Gag polyprotein. The five longest IMRs in the polyprotein translate a key functional segment in each of the five cleavage products. Throughout the protein, IMRs coincide with functionally significant segments of the protein. A detailed annotation of the protein, which combines structural, functional and IMR data illustrates these associations. There is a significant statistical correlation between the ends of IMRs and the ends of PSEs in each of the mature proteins. Weakly symmetric IMRs ([greater than or equal to] 33%) are related to cleavage positions and processes. CONCLUSIONS: The frequency and distribution of IMRs in HIV-1 Gag indicates that DNA symmetry is a fundamental property of protein coding DNA and that different levels of symmetry are associated with different functional aspects of the gene and its protein. The interaction between IMRs and protein structure and function is precise and interwoven over the entire length of the polyprotein. The distribution of IMRs and their relationship to structural and functional motifs in the protein that they translate, suggest that DNA-driven processes, including the selection of mirror repeats, may be a constraining factor in molecular evolution. [Abstract/Link to Full Text]

Geiss BJ, Shimonkevitz LH, Sackal CI, Olson KE
Recombination-ready Sindbis replicon expression vectors for transgene expression.
Virol J. 2007 Oct 26;4(1):112.
ABSTRACT: BACKGROUND: Sindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation. RESULTS: Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis. CONCLUSIONS: Replication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function. [Abstract/Link to Full Text]

C PH, Rai M
Detection and frequency of recombination in tomato-infecting begomoviruses of South and Southeast Asia.
Virol J. 2007 Oct 26;4(1):111.
ABSTRACT: BACKGROUND: Tomato-infecting begomoviruses are widely distributed across the world and cause diseases of high economic impact on wide range of agriculturally important crops. Though recombination plays a pivotal role in diversification and evolution of these viruses, it is currently unknown whether there are differences in the number and quality of recombination events amongst different tomato-infecting begomovirus species. To examine this we sought to characterize the recombination events, estimate the frequency of recombination, and map recombination hotspots in tomato-infecting begomoviruses of South and Southeast Asia. RESULTS: Different methods used for recombination breakpoint analysis provided strong evidence for presence of recombination events in majority of the sequences analyzed. However, there was a clear evidence for absence or low Recombination events in viruses reported from North India. In addition, we provide evidence for non-random distribution of recombination events with the highest frequency of recombination being mapped in the portion of the N-terminal portion of Rep. CONCLUSIONS: The variable recombination observed in these viruses signified that all begomoviruses are not equally prone to recombination. Distribution of recombination hotspots was found to be reliant on the relatedness of the genomic region involved in the exchange. Overall the frequency of phylogenetic violations and number of recombination events decreased with increasing parental sequence diversity. These findings provide valuable new information for understanding the diversity and evolution of tomato-infecting begomoviruses in Asia. [Abstract/Link to Full Text]

Ishii Y, Kondo K, Matsumoto T, Tanaka K, Shinkai-Ouchi F, Hagiwara K, Kanda T
Thiol-reactive reagents inhibits intracellular trafficking of human papillomavirus type 16 pseudovirions by binding to cysteine residues of major capsid protein L1.
Virol J. 2007 Oct 26;4(1):110.
ABSTRACT: BACKGROUND: A human papillomavirus (HPV) virion is composed of capsid proteins L1 and L2. Several cysteine residues are located on L1 of various HPVs at markedly similar relative positions, suggesting their important functions. Although the authentic virions cannot be studied with cultured cells, surrogate pseudovirions consisting of capsid and reporter plasmid are available for studies dealing with infectivity. RESULTS: HPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)]. A labelled streptavidin was detected to bind to the complex of BPEOIA and L1 of the 16PVs incubated with BPEOIA. The analysis of molecular mass of trypsin-fragments derived from the complex of the BPEOIA and L1 indicated that BPEOIA bound to at least C146, C225, and C229. No appreciable change of the 16PVs carrying DTNB or NEM was detected by sedimentation analysis or electron microscopy. The 16PVs carrying DTNB or NEM were able to bind to and enter HeLa cells but degraded before they reached the perinuclear region. CONCLUSIONS: HPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET. Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs. [Abstract/Link to Full Text]

Mejias A, Chavez-Bueno S, Raynor MB, Connolly J, Kiener PA, Jafri HS, Ramilo O
Motavizumab, A Neutralizing Anti-Respiratory Syncytial Virus (RSV) Monoclonal Antibody Significantly Modifies The Local And Systemic Cytokine Responses Induced By RSV In The Mouse Model.
Virol J. 2007 Oct 25;4(1):109.
ABSTRACT: Motavizumab (MEDI-524) is a monoclonal antibody with enhanced neutralizing activity against RSV. In mice, motavizumab suppressed RSV replication which resulted in significant reduction of clinical parameters of disease severity. We evaluated the effect of motavizumab on the local and systemic immune response induced by RSV in the mouse model. Balb/c mice were intranasally inoculated with 106.5 PFU RSV A2 or medium. Motavizumab was given once intraperitoneally (1.25 mg/mouse) as prophylaxis, 24 h before virus inoculation. Bronchoalveolar lavage (BAL) and serum samples were obtained at days 1, 5 (acute) and 28 (long-term) post inoculation and analyzed with a multiplex assay (Beadlyte Upstate, NY) for simultaneous quantitation of 18 cytokines: IL-1 alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, KC (similar to human IL-8), IL-10, IL-12p40, IL-12p70, IL-13, IL-17, TNF-alpha, MCP-1, RANTES, IFN-gamma and GM-CSF. Overall, cytokine concentrations were lower in serum than in BAL samples. By day 28, only KC was detected in BAL specimens at low concentrations in all groups. Administration of motavizumab significantly reduced (p <0.05) BAL concentrations of IL-1alpha, IL-12p70 and TNF-alpha on day 1, and concentrations of IFN-gamma on days 1 and 5 compared with RSV-infected untreated controls. In the systemic compartment, the concentrations of IL-10, IFN-gamma and KC were significantly reduced in the motavizumab-treated mice compared with the untreated controls. In summary, prophylactic administration of motavizumab was associated with significant reductions on RSV replication and concentrations of cytokine and chemokines, which are likely related to the improvement observed in clinical markers of disease severity. [Abstract/Link to Full Text]

Warfield KL, Alves DA, Bradfute SB, Reed DK, Vantongeren S, Kalina WV, Olinger GG, Bavari S
Development of a model for marburgvirus based on severe-combined immunodeficiency mice.
Virol J. 2007 Oct 25;4(1):108.
ABSTRACT: The filoviruses, Ebola (EBOV) and Marburg (MARV), cause a lethal hemorrhagic fever. Human isolates of MARV are not lethal to immmunocompetent adult mice and, to date, there are no reports of a mouse-adapted MARV model. Previously, a uniformly lethal EBOV-Zaire mouse-adapted virus was developed by performing 9 sequential passages in progressively older mice (suckling to adult). Evaluation of this model identified many similarities between infection in mice and nonhuman primates, including viral tropism for antigen-presenting cells, high viral titers in the spleen and liver, and an equivalent mean time to death. Existence of the EBOV mouse model has increased our understanding of host responses to filovirus infections and likely has accelerated the development of countermeasures, as it is one of the only hemorrhagic fever viruses that has multiple candidate vaccines and therapeutics. Here, we demonstrate that serially passaging liver homogenates from MARV-infected severe combined immunodeficient (scid) mice was highly successful in reducing the time to death in scid mice from 50-70 days to 7-10 days after MARV-Ci67, -Musoke, or -Ravn challenge. We performed serial sampling studies to characterize the pathology of these scid mouse-adapted MARV strains. These scid mouse-adapted MARV models appear to have many similar properties as the MARV models previously developed in guinea pigs and nonhuman primates. Also, as shown here, the scid-adapted MARV mouse models can be used to evaluate the efficacy of candidate antiviral therapeutic molecules, such as phosphorodiamidate morpholino oligomers or antibodies. [Abstract/Link to Full Text]

Sivakumaran H, Wang B, Gill MJ, Beckholdt B, Saksena NK, Harrich D
Functional relevance of nonsynonymous mutations in the HIV-1 tat gene within an epidemiologically-linked transmission cohort.
Virol J. 2007 Oct 25;4(1):107.
ABSTRACT: Here we investigated the nature and functional consequences of mutations in the HIV-1 tat gene within an epidemiologically-linked AIDS transmission cohort consisting of a non-progressing donor (A) and two normal progressing recipients (B and C). Multiple nonsynonymous mutations in the tat first exon were observed across time in all individuals. Some mutations demonstrated striking host specificity despite the cohort being infected with a common virus. Phylogenetic segregation of the tat clones at the time of progression to AIDS was also observed especially in recipient C. Tat clones supporting high levels of transactivation were present at all time points in all individuals, although a number of clones defective for transactivation were observed for recipient C in later time points. Here we show that the tat quasispecies in a linked transmission cohort diversify and evolve independently between hosts following transmission. It supports the belief that quasispecies variation in HIV-1 is a mechanism for selection towards defining a fitter gene variant that is capable of resisting the human immune system. [Abstract/Link to Full Text]

Salahuddin SZ, Snyder KA, Godwin A, Grewal R, Prichard JG, Kelley AS, Revie D
The simultaneous presence and expression of human hepatitis C virus (HCV), human herpesvirus-6 (HHV-6), and human immunodeficiency virus-1 (HIV-1) in a single human T-cell.
Virol J. 2007 Oct 24;4(1):106.
ABSTRACT: We have developed a system that isolates and replicates HCV in vitro. These isolates are called CIMM-HCV. This system has made it possible to analyze the biology, nature, and extent of HCV variability, among other things. Individuals that are infected with HIV-1 are often also infected with HCV and HHV-6. In addition to HCV, our lab has systems for replicating HIV-1 and HHV-6. We asked whether all these viruses could infect the same cells. We report here the successful infection of a T-cell (CEM) by CIMM-HCV, HHV-6, and HIV-1. PCR analyses demonstrated that the CEM cells were productively infected by HHV-6A. RT-PCR showed that the same cell culture was positive for HCV and HIV-1. Co-infection of a T-cell by all three viruses was confirmed by transmission electron microscopy (TEM). All these viruses are highly cytolytic; therefore, triply-infected cells were short lived. However, HIV-1 and HCV co-infected cells unexpectedly lasted for several weeks. Viral replication was unhindered and the phenomenon of 'dominance' was not observed in our experiments. In addition, CIMM-HCV was present in the perinuclear space, suggesting their possible synthesis in the nucleus. This report is based entirely on viruses produced in vitro in our laboratories. As part of the determinations of host ranges of these viruses, studies were designed to demonstrate the infection of a single cell by these viruses and to study the consequences of this phenomenon. All measurements were made on cultured cells and cell culture supernatants. [Abstract/Link to Full Text]

---

Recent Articles in Microbial Cell Factories

---

Jordan E, Al-Halabi L, Schirrmann T, Hust M, Dubel S
Production of single chain Fab (scFab) fragments in Bacillus megaterium.
Microb Cell Fact. 2007 Nov 27;6(1):38.
ABSTRACT: BACKGROUND: The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli. RESULTS: The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli. CONCLUSION: B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli. [Abstract/Link to Full Text]

Peano C, Bicciato S, Corti G, Ferrari F, Rizzi E, Bonnal RJ, Bordoni R, Albertini A, Rossi Bernardi L, Donadio S, De Bellis G
Complete gene expression profiling of Saccharopolyspora erythraea using GeneChip DNA microarrays.
Microb Cell Fact. 2007 Nov 26;6(1):37.
ABSTRACT: BACKGROUND: The Saccharopolyspora erythraea genome sequence, recently published, presents considerable divergence from those of streptomycetes in gene organization and function, confirming the remarkable potential of S. erythraea for producing many other secondary metabolites in addition to erythromycin. In order to investigate, at whole transcriptome level, how S. erythraea genes are modulated, a DNA microarray was specifically designed and constructed on the S. erythraea strain NRRL 2338 genome sequence, and the expression profiles of 6494 ORFs were monitored during growth in complex liquid medium. RESULTS: The transcriptional analysis identified a set of 404 genes, whose transcriptional signals vary during growth and characterize three distinct phases: a rapid growth until 32 h (Phase A); a growth slowdown until 52 h (Phase B); and another rapid growth phase from 56 h to 72 h (Phase C) before the cells enter the stationary phase. A non-parametric statistical method, that identifies chromosomal regions with transcriptional imbalances, determined regional organization of transcription along the chromosome, highlighting differences between core and non-core regions, and strand specific patterns of expression. Microarray data were used to characterize the temporal behaviour of major functional classes and of all the gene clusters for secondary metabolism. The results confirmed that the ery cluster is up-regulated during Phase A and identified six additional clusters (for terpenes and non-ribosomal peptides) that are clearly regulated in later phases. CONCLUSIONS: The use of a S. erythraea DNA microarray improved specificity and sensitivity of gene expression analysis, allowing a global and at the same time detailed picture of how S. erythraea genes are modulated. This work underlines the importance of using DNA microarrays, coupled with an exhaustive statistical and bioinformatic analysis of the results, to understand the transcriptional organization of the chromosomes of micro-organisms producing natural products. [Abstract/Link to Full Text]

Liang L, Zhang J, Lin Z
Altering coenzyme specificity of Pichia stipitis xylose reductase by the semi-rational approach CASTing.
Microb Cell Fact. 2007 Nov 21;6(1):36.
ABSTRACT: BACKGROUND: The NAD(P)H-dependent Pichia stipitis xylose reductase (PsXR) is one of the key enzymes for xylose fermentation, and has been cloned into the commonly used ethanol-producing yeast Saccharomyces cerevisiae. In order to eliminate the redox imbalance resulting from the preference of this enzyme toward NADPH, efforts have been made to alter the coenzyme specificity of PsXR by site-directed mutagenesis, with limited success. Given the industrial importance of PsXR, it is of interest to investigate further ways to create mutants of PsXR that prefers NADH rather than NADPH, by the alternative directed evolution approach. RESULTS: Based on a homology model of PsXR, six residues were predicted to interact with the adenine ribose of NAD(P)H in PsXR and altered using a semi-rational mutagenesis approach (CASTing). Three rounds of saturation mutagenesis were carried to randomize these residues, and a microplate-based assay was applied in the screening. A best mutant 2-2C12, which carried four mutations K270S, N272P, S271G and R276F, was obtained. The mutant showed a preference toward NADH over NADPH by a factor of about 13-fold, or an improvement of about 42-fold, as measured by the ratio of the specificity constant kcat/Kmcoenzyme. Compared with the wild-type, the kcatNADH for the best mutant was only slightly lower, while the kcatNADPH decreased by a factor of about 10. Furthermore, the specific activity of 2-2C12 in the presence of NADH was 20.6 Umg-1, which is highest among PsXR mutants reported. CONCLUSIONS: A seemingly simplistic and yet very effective mutagenesis approach, CASTing, was applied successfully to alter the NAD(P)H preference for Pichia stipitis xylose reductase, an important enzyme for xylose-fermenting yeast. The observed change in the NAD(P)H preference for this enzyme seems to have resulted from the altered active site that is more unfavorable for NADPH than NADH in terms of both Km and kcat. There are potentials for application of our PsXR in constructing a more balanced XR-XDH pathway in recombinant xylose-fermenting S. cerevisiae strains. [Abstract/Link to Full Text]

Ferrer P
Systems biology and biological systems diversity for the engineering of microbial cell factories.
Microb Cell Fact. 2007 Nov 20;6(1):35.
ABSTRACT: - [Abstract/Link to Full Text]

Karnaukhova E, Ophir Y, Trinh L, Dalal N, Punt PJ, Golding B, Shiloach J
Expression of human alpha-1-proteinase inhibitor in Aspergillus niger.
Microb Cell Fact. 2007 Oct 29;6(1):34.
ABSTRACT: BACKGROUND: Human alpha-1-proteinase inhibitor (alpha1-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, alpha1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant alphaPI (r-alpha1-PI) could provide an attractive alternative. Although r-alpha1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation. RESULTS: We have explored the possibility of expressing the gene for human alpha1-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of alpha1-PI with a strongly expressed, secreted leader protein (glucoamylase G2), separated by dibasic processing site (N-V-I-S-K-R) that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and alpha1PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-alpha1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) analysis further confirmed that molecular mass of the r-alpha1-PI was similar to that of the pd-alpha1-PI. In vitro stability of the r-alpha1-PI from A. niger was tested in comparison with pd-alpha1-PI reference and non-glycosylated human r-alpha1-PI from E. coli. CONCLUSION: We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for alpha1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for alpha1-PI in A. niger was successfully achieved to produce the secreted mature human r-alpha1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth. [Abstract/Link to Full Text]

Villaverde A, Mattanovich D
Recombinant protein production in the new Millennium.
Microb Cell Fact. 2007;633. [Abstract/Link to Full Text]

Galao RP, Scheller N, Alves-Rodrigues I, Breinig T, Meyerhans A, Diez J
Saccharomyces cerevisiae: a versatile eukaryotic system in virology.
Microb Cell Fact. 2007 Oct 10;6(1):32.
ABSTRACT: The yeast Saccharomyces cerevisiae is a well-established model system for understanding fundamental cellular processes relevant to higher eukaryotic organisms. Less known is its value for virus research, an area in which Saccharomyces cerevisiae has proven to be very fruitful as well. The present review will discuss the main achievements of yeast-based studies in basic and applied virus research. These include the analysis of the function of individual proteins from important pathogenic viruses, the elucidation of key processes in viral replication through the development of systems that allow the replication of higher eukayotic viruses in yeast, and the use of yeast in antiviral drug development and vaccine production. [Abstract/Link to Full Text]

Bernal V, Sevilla A, Cánovas M, Iborra JL
Production of L-carnitine by secondary metabolism of bacteria.
Microb Cell Fact. 2007;631.
ABSTRACT: The increasing commercial demand for L-carnitine has led to a multiplication of efforts to improve its production with bacteria. The use of different cell environments, such as growing, resting, permeabilized, dried, osmotically stressed, freely suspended and immobilized cells, to maintain enzymes sufficiently active for L-carnitine production is discussed in the text. The different cell states of enterobacteria, such as Escherichia coli and Proteus sp., which can be used to produce L-carnitine from crotonobetaine or D-carnitine as substrate, are analyzed. Moreover, the combined application of both bioprocess and metabolic engineering has allowed a deeper understanding of the main factors controlling the production process, such as energy depletion and the alteration of the acetyl-CoA/CoA ratio which are coupled to the end of the biotransformation. Furthermore, the profiles of key central metabolic activities such as the TCA cycle, the glyoxylate shunt and the acetate metabolism are seen to be closely interrelated and affect the biotransformation efficiency. Although genetically modified strains have been obtained, new strain improvement strategies are still needed, especially in Escherichia coli as a model organism for molecular biology studies. This review aims to summarize and update the state of the art in L-carnitine production using E. coli and Proteus sp, emphasizing the importance of proper reactor design and operation strategies, together with metabolic engineering aspects and the need for feed-back between wet and in silico work to optimize this biotransformation. [Abstract/Link to Full Text]

Báez-Viveros JL, Flores N, Juárez K, Castillo-España P, Bolivar F, Gosset G
Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine.
Microb Cell Fact. 2007;630.
ABSTRACT: BACKGROUND: The rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed. RESULTS: MTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway. CONCLUSION: The identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains. [Abstract/Link to Full Text]

Serrano LM, Molenaar D, Wels M, Teusink B, Bron PA, de Vos WM, Smid EJ
Thioredoxin reductase is a key factor in the oxidative stress response of Lactobacillus plantarum WCFS1.
Microb Cell Fact. 2007 Aug 28;6(1):29.
ABSTRACT: BACKGROUND: Thioredoxin (TRX) is a powerful disulfide oxido-reductase that catalyzes a wide spectrum of redox reactions in the cell. The aim of this study is to elucidate the role of the TRX system in the oxidative stress response in Lactobacillus plantarum WCFS1. RESULTS: We have identified the trxB1-encoded thioredoxin reductase (TR) as a key enzyme in the oxidative stress response of Lactobacillus plantarum WCFS1. Overexpression of the trxB1 gene resulted in a 3-fold higher TR activity in comparison to the wild-type strain. Higher TR activity was associated with an increased resistance towards oxidative stress. Gene expression profiling showed significant differential expression of 27 genes in the trxB1-overexpression strain. We further determined the global transcriptional response to hydrogen peroxide stress in the trxB1-overexpression and wild-type strains grown in continuous cultures. Over expression of trxB1 was found to activate genes associated with DNA repair and stress mechanisms as well as genes associated with the activity of biosynthetic pathways for purine and sulfur-containing amino acids. Hydrogen peroxide stress and overproduction of TR collectively resulted in the up-regulation of 267 genes. A total of 16 genes showed a response to both TR overproduction and hydrogen peroxide stress. These genes are involved in the purine metabolism, central metabolism (gapB) as well as in stress-response (groEL, npr2), and transport (mntH2). CONCLUSIONS: Based on our findings we propose that the trxB1-encoded TR in L. plantarum is an important factor in the oxidative stress-associated signal transduction network containing at least 16 genes. This may explain the phenotype of the trxB1-overexpression strain, which appears to be prepared for encountering oxidative stress. This latter property can be used for engineering robustness towards oxidative stress in industrial strains of L. plantarum. [Abstract/Link to Full Text]

Glenting J, Poulsen LK, Kato K, Madsen SM, Frøkiær H, Wendt C, Sørensen HW
Production of Recombinant Peanut Allergen Ara h 2 using Lactococcus lactis.
Microb Cell Fact. 2007;6(1):28.
ABSTRACT: BACKGROUND: Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high-level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis. RESULTS: A synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N-terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2. CONCLUSION: Recombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics. [Abstract/Link to Full Text]

Faijes M, Mars AE, Smid EJ
Comparison of quenching and extraction methodologies for metabolome analysis of Lactobacillus plantarum.
Microb Cell Fact. 2007;627.
ABSTRACT: BACKGROUND: A reliable quenching and metabolite extraction method has been developed for Lactobacillus plantarum. The energy charge value was used as a critical indicator for fixation of metabolism. RESULTS: Four different aqueous quenching solutions, all containing 60% of methanol, were compared for their efficiency. Only the solutions containing either 70 mM HEPES or 0.85% (w/v) ammonium carbonate (pH 5.5) caused less than 10% cell leakage and the energy charge of the quenched cells was high, indicating rapid inactivation of the metabolism.The efficiency of extraction of intracellular metabolites from cell cultures depends on the extraction methods, and is expected to vary between micro-organisms. For L. plantarum, we have compared five different extraction methodologies based on (i) cold methanol, (ii) perchloric acid, (iii) boiling ethanol, (iv) chloroform/methanol (1:1) and (v) chloroform/water (1:1). Quantification of representative intracellular metabolites showed that the best extraction efficiencies were achieved with cold methanol, boiling ethanol and perchloric acid. CONCLUSION: The ammonium carbonate solution was selected as the most suitable quenching buffer for metabolomics studies in L. plantarum because (i) leakage is minimal, (ii) the energy charge indicates good fixation of metabolism, and (iii) all components are easily removed during freeze-drying. A modified procedure based on cold methanol extraction combined good extractability with mild extraction conditions and high enzymatic inactivation. These features make the combination of these quenching and extraction protocols very suitable for metabolomics studies with L. plantarum. [Abstract/Link to Full Text]

Graham RLj, Graham C, McMullan G
Microbial proteomics: a mass spectrometry primer for biologists.
Microb Cell Fact. 2007;626.
ABSTRACT: It is now more than 10 years since the publication of the first microbial genome sequence and science is now moving towards a post genomic era with transcriptomics and proteomics offering insights into cellular processes and function. The ability to assess the entire protein network of a cell at a given spatial or temporal point will have a profound effect upon microbial science as the function of proteins is inextricably linked to phenotype. Whilst such a situation is still beyond current technologies rapid advances in mass spectrometry, bioinformatics and protein separation technologies have produced a step change in our current proteomic capabilities. Subsequently a small, but steadily growing, number of groups are taking advantage of this cutting edge technology to discover more about the physiology and metabolism of microorganisms. From this research it will be possible to move towards a systems biology understanding of a microorganism. Where upon researchers can build a comprehensive cellular map for each microorganism that links an accurately annotated genome sequence to gene expression data, at a transcriptomic and proteomic level.In order for microbiologists to embrace the potential that proteomics offers, an understanding of a variety of analytical tools is required. The aim of this review is to provide a basic overview of mass spectrometry (MS) and its application to protein identification. In addition we will describe how the protein complexity of microbial samples can be reduced by gel-based and gel-free methodologies prior to analysis by MS. Finally in order to illustrate the power of microbial proteomics a case study of its current application within the Bacilliaceae is given together with a description of the emerging discipline of metaproteomics. [Abstract/Link to Full Text]

Salzano AM, Febbraio F, Farias T, Cetrangolo GP, Nucci R, Scaloni A, Manco G
Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge.
Microb Cell Fact. 2007;625.
ABSTRACT: BACKGROUND: Exposure to nickel (Ni) and its chemical derivatives has been associated with severe health effects in human. On the contrary, poor knowledge has been acquired on target physiological processes or molecular mechanisms of this metal in model organisms, including Bacteria and Archaea. In this study, we describe an analysis focused at identifying proteins involved in the recovery of the archaeon Sulfolobus solfataricus strain MT4 from Ni-induced stress. RESULTS: To this purpose, Sulfolobus solfataricus was grown in the presence of the highest nickel sulphate concentration still allowing cells to survive; crude extracts from treated and untreated cells were compared at the proteome level by using a bi-dimensional chromatography approach. We identified several proteins specifically repressed or induced as result of Ni treatment. Observed up-regulated proteins were largely endowed with the ability to trigger recovery from oxidative and osmotic stress in other biological systems. It is noteworthy that most of the proteins induced following Ni treatment perform similar functions and a few have eukaryal homologue counterparts. CONCLUSION: These findings suggest a series of preferential gene expression pathways activated in adaptation response to metal challenge. [Abstract/Link to Full Text]

Rasmussen LC, Sperling-Petersen HU, Mortensen KK
Hitting bacteria at the heart of the central dogma: sequence-specific inhibition.
Microb Cell Fact. 2007;624.
ABSTRACT: An important objective in developing new drugs is the achievement of high specificity to maximize curing effect and minimize side-effects, and high specificity is an integral part of the antisense approach. The antisense techniques have been extensively developed from the application of simple long, regular antisense RNA (asRNA) molecules to highly modified versions conferring resistance to nucleases, stability of hybrid formation and other beneficial characteristics, though still preserving the specificity of the original nucleic acids. These new and improved second- and third-generation antisense molecules have shown promising results. The first antisense drug has been approved and more are in clinical trials. However, these antisense drugs are mainly designed for the treatment of different human cancers and other human diseases. Applying antisense gene silencing and exploiting RNA interference (RNAi) are highly developed approaches in many eukaryotic systems. But in bacteria RNAi is absent, and gene silencing by antisense compounds is not nearly as well developed, despite its great potential and the intriguing possibility of applying antisense molecules in the fight against multiresistant bacteria. Recent breakthrough and current status on the development of antisense gene silencing in bacteria including especially phosphorothioate oligonucleotides (PS-ODNs), peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) will be presented in this review. [Abstract/Link to Full Text]

Patnaik PR
"Intelligent" descriptions of microbial kinetics in finitely dispersed bioreactors: neural and cybernetic models for PHB biosynthesis by Ralstonia eutropha.
Microb Cell Fact. 2007;623.
ABSTRACT: BACKGROUND: For many microbial processes, the complexity of the metabolisms and the responses to transient and realistic conditions are difficult to capture in mechanistic models. The cells seem to have an innate intelligence that enables them to respond optimally to environmental changes. Some "intelligent" models have therefore been proposed and compared with a mechanistic model for fed-batch cultures of Ralstonia eutropha. RESULTS: Two kinds of models have been proposed to describe such cellular behavior. Cybernetic models are derived through postulates of cellular intelligence and memory, and neural models use artificial intelligence through neural networks. Some competing models of both kinds have been compared for their ability to portray and optimize the synthesis of poly-beta-hydroxybutyrate by Ralstonia eutropha in fed-batch cultures with finite dispersion. Neural models enabled the formation of more of the polymer than cybernetic models, with lesser utilization of the carbon and nitrogen substrates. Both types of models were decidedly superior to a mechanistic model used as a reference, thus supporting the value of intelligent descriptions of microbial kinetics in incompletely dispersed bioreactors. CONCLUSION: Neural and cybernetic models describe and optimize unsteady state fed-batch microbial reactors with finite dispersion more effectively than mechanistic models. However, these "intelligent" models too have weaknesses, and hence a hybrid approach combining such models with some mechanistic features is suggested. [Abstract/Link to Full Text]

Rochat T, Bermúdez-Humarán L, Gratadoux JJ, Fourage C, Hoebler C, Corthier G, Langella P
Anti-inflammatory effects of Lactobacillus casei BL23 producing or not a manganese-dependant catalase on DSS-induced colitis in mice.
Microb Cell Fact. 2007;622.
BACKGROUND: Human immune cells generate large amounts of reactive oxygen species (ROS) throughout the respiratory burst that occurs during inflammation. In inflammatory bowel diseases, a sustained and abnormal activation of the immune system results in oxidative stress in the digestive tract and in a loss of intestinal homeostasis. We previously showed that the heterologous production of the Lactobacillus plantarum ATCC14431 manganese-dependant catalase (MnKat) in Lb. casei BL23 successfully enhances its survival when exposed to oxidative stress. In this study, we evaluated the preventive effects of this antioxidative Lb. casei strain in a murine model of dextran sodium sulfate (DSS)-induced moderate colitis. RESULTS: Either Lb. casei BL23 MnKat- or MnKat+ was administered daily to mice treated with DSS for 10 days. In contrast to control mice treated with PBS for which DSS induced bleeding diarrhea and mucosal lesions, mice treated with both Lb. casei strains presented a significant (p < 0.05) reduction of caecal and colonic inflammatory scores. CONCLUSION: No contribution of MnKat to the protective effect from epithelial damage has been observed in the tested conditions. In contrast, these results confirm the high interest of Lb. casei as an anti-inflammatory probiotic strain. [Abstract/Link to Full Text]

Resina D, Bollók M, Khatri NK, Valero F, Neubauer P, Ferrer P
Transcriptional response of P. pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction.
Microb Cell Fact. 2007;621.
BACKGROUND: The analysis of transcriptional levels of the genes involved in protein synthesis and secretion is a key factor to understand the host organism's responses to recombinant protein production, as well as their interaction with the cultivation conditions. Novel techniques such as the sandwich hybridization allow monitoring quantitatively the dynamic changes of specific RNAs. In this study, the transcriptional levels of some genes related to the unfolded protein response (UPR) and central metabolism of Pichia pastoris were analysed during batch and fed-batch cultivations using an X-33-derived strain expressing a Rhizopus oryzae lipase under control of the formaldehyde dehydrogenase promoter (FLD1), namely the alcohol oxidase gene AOX1, the formaldehyde dehydrogenase FLD1, the protein disulfide isomerase PDI, the KAR2 gene coding for the BiP chaperone, the 26S rRNA and the R. oryzae lipase gene ROL. RESULTS: The transcriptional levels of the selected set of genes were first analysed in P. pastoris cells growing in shake flask cultures containing different carbon and nitrogen sources combinations, glycerol + ammonium, methanol + methylamine and sorbitol + methylamine. The transcriptional levels of the AOX1 and FLD1 genes were coherent with the known regulatory mechanism of C1 substrates in P. pastoris, whereas ROL induction lead to the up-regulation of KAR2 and PDI transcriptional levels, thus suggesting that ROL overexpression triggers the UPR. This was further confirmed in fed-batch cultivations performed at different growth rates. Transcriptional levels of the analysed set of genes were generally higher at higher growth rates. Nevertheless, when ROL was overexpressed in a strain having the UPR constitutively activated, significantly lower relative induction levels of these marker genes were detected. CONCLUSION: The bead-based sandwich hybridization assay has shown its potential as a reliable instrument for quantification of specific mRNA species in P. pastoris cells grown in fed-batch cultures. As a proof-of-principle, the influence of the carbon and nitrogen sources, the specific growth rate, as well as the ROL overexpression on the transcriptional levels of a reduced set of bioprocess-relevant genes has been quantitatively studied, revealing that ROL overexpression and secretion seems to trigger the UPR in P. pastoris, resulting in a physiological bottleneck for the production process. [Abstract/Link to Full Text]

Pimienta E, Ayala JC, Rodríguez C, Ramos A, Van Mellaert L, Vallín C, Anné J
Recombinant production of Streptococcus equisimilis streptokinase by Streptomyces lividans.
Microb Cell Fact. 2007;620.
BACKGROUND: Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway. RESULTS: SK yield in the spent culture medium of S. lividans was higher when the Sec-dependent signal peptide mediates the SK translocation. Using a 1.5 L fermentor, the secretory production of the Vsi-SK fusion protein reached up to 15 mg SK/l. SK was partially purified from the culture supernatant by DEAE-Sephacel chromatography. A 44-kDa degradation product co-eluted with the 47-kDa mature SK. The first amino acid residues of the S. lividans-produced SK were identical with those of the expected N-terminal sequence. The Vsi signal peptide was thus correctly cleaved off and the N-terminus of mature Vsi-SK fusion protein released by S. lividans remained intact. This result also implicates that the processing of the recombinant SK secreted by Streptomyces probably occurred at its C-terminal end, as in its native host Streptococcus equisimilis. The specific activity of the partially purified Streptomyces-derived SK was determined at 2661 IU/mg protein. CONCLUSION: Heterologous expression of Streptococcus equisimilis ATCC9542 skc-2 in Streptomyces lividans was successfully achieved. SK can be translocated via both the Sec and the Tat pathway in S. lividans, but yield was about 30 times higher when the SK was fused to the Sec-dependent Vsi signal peptide compared to the fusion with the Tat-dependent signal peptide of S. lividans xylanase C. Small-scale fermentation led to a fourfold improvement of secretory SK yield in S. lividans compared to lab-scale conditions. The partially purified SK showed biological activity. Streptomyces lividans was shown to be a valuable host for the production of a world-wide important, biopharmaceutical product in a bio-active form. [Abstract/Link to Full Text]

Shirai T, Fujimura K, Furusawa C, Nagahisa K, Shioya S, Shimizu H
Study on roles of anaplerotic pathways in glutamate overproduction of Corynebacterium glutamicum by metabolic flux analysis.
Microb Cell Fact. 2007;619.
BACKGROUND: Corynebacterium glutamicum has several anaplerotic pathways (anaplerosis), which are essential for the productions of amino acids, such as lysine and glutamate. It is still not clear how flux changes in anaplerotic pathways happen when glutamate production is induced by triggers, such as biotin depletion and the addition of the detergent material, Tween 40. In this study, we quantitatively analyzed which anaplerotic pathway flux most markedly changes the glutamate overproduction induced by Tween 40 addition. RESULTS: We performed a metabolic flux analysis (MFA) with [1-13C]- and [U-13C]-labeled glucose in the glutamate production phase of C. glutamicum, based on the analysis of the time courses of 13C incorporation into proteinogenic amino acids by gas chromatography-mass spectrometry (GC-MS). The flux from phosphoenolpyruvate (PEP) to oxaloacetate (Oxa) catalyzed by phosphoenolpyruvate carboxylase (PEPc) was active in the growth phase not producing glutamate, whereas that from pyruvate to Oxa catalyzed by pyruvate carboxylase (Pc) was inactive. In the glutamate overproduction phase induced by the addition of the detergent material Tween 40, the reaction catalyzed by Pc also became active in addition to the reaction catalyzed by PEPc. CONCLUSION: It was clarified by a quantitative 13C MFA that the reaction catalyzed by Pc is most markedly increased, whereas other fluxes of PEPc and PEPck remain constant in the glutamate overproduction induced by Tween 40. This result is consistent with the previous results obtained in a comparative study on the glutamate productions of genetically recombinant Pc- and PEPc-overexpressing strains. The importance of a specific reaction in an anaplerotic pathway was elucidated at a metabolic level by MFA. [Abstract/Link to Full Text]

Ergin A, Büssow K, Sieper J, Thiel A, Duchmann R, Adam T
Homologous high-throughput expression and purification of highly conserved E coli proteins.
Microb Cell Fact. 2007;618.
BACKGROUND: Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD). Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s) involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies. RESULTS: As a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen) destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP), N-utilizing substance A (NusA), or glutathione S-transferase (GST) to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS). Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500 microg. CONCLUSION: Here, we report a cost-efficient procedure to produce around 200 soluble recombinant E. coli proteins in large-scale, including removal of LPS by polymyxin B coated beads for subsequent use of the proteins in downstream immunological studies. [Abstract/Link to Full Text]

Al-Ajlani MM, Sheikh MA, Ahmad Z, Hasnain S
Production of surfactin from Bacillus subtilis MZ-7 grown on pharmamedia commercial medium.
Microb Cell Fact. 2007;617.
BACKGROUND: Commercial medium (Pharmamedia) was investigated for the production of surfactin by Bacillus subtilis MZ-7. Different media (defined, semi-defined, and complex media) were compared for the production of surfactin after fixing the least influential variables in standardized fermentation conditions. Carbohydrate and nitrogen supplements were also tried to improve production in Pharmamedia. RESULTS: Surfactin production was confirmed using PCR along with other analytical techniques and monitored by RP-HPLC and MALDI-TOF-MS. We found that optimized and brain heart infusion media were best for production of surfactin (280 mg/L) and a relatively comparable production with Pharmamedia (220 mg/L), however, supplementing Pharmamedia with Fe+ (4.0 mM) and sucrose (2 g/L) leads to a maximum production of about (300 mg/L). CONCLUSION: Cottonseed-derived medium proved to be a suitable substrate for the production of bioactive substances including surfactin, a useful compound in both medical and biotechnological fields. The medium provided not only higher product accumulations but at considerably lower cost with potential for large scale industrial applications. [Abstract/Link to Full Text]

Papagianni M, Avramidis N, Filiousis G
Glycolysis and the regulation of glucose transport in Lactococcus lactis spp. lactis in batch and fed-batch culture.
Microb Cell Fact. 2007;616.
BACKGROUND: Despite the fact that many reports deal with glycolysis in Lactococcus lactis, there is not much information on the regulation of uptake of glucose itself. The aim of the present work was to investigate the effect of the glucose level on its specific uptake rate. RESULTS: Studies on aeration levels in pH controlled L. lactis spp. lactis batch cultures on glucose (55 mM) showed that product formation is extremely homolactic and the highest yield of lactate on glucose is obtained under microaerobic conditions (5% dissolved oxygen). Microaerobic conditions were therefore applied in experiments carried out to investigate the regulation of the uptake of glucose. The tool of glucostat fed-batch culture was employed, in which glucose was added at a rate suitable to maintain a stable concentration throughout the runs with glucose concentration ranging from 13.75 to 555 mM. The glucostat experiments showed that the concentration of glucose influences its specific uptake rate and consequently the glycolytic flux, as well as the fermentation pattern. The highest specific activities of the key glycolytic enzymes PFK, PYK and the LDH were obtained at 55 mM glucose, the area of the highest observed glycolytic flux. Reduction of the glycolytic flux by 55% in the 277 mM glucostat corresponded to an almost identical reduction in PFK activity, indicating a certain controlling influence of this enzyme on the flux, through the glucose effect. CONCLUSION: Determination of intracellular metabolites' pools showed that FBP cannot be regarded as a direct regulator of product formation, since almost identical concentrations were obtained at both low (13.75 mM) and high (138 mM) glucose levels, at which neither the glucose uptake rates and the glycolytic flux, nor the fermentation patterns were similar (mixed acids vs homolactic, respectively). Glucostat data showed instead that the control of the flux through the glycolytic pathway under the examined conditions, resides to a large extent in processes outside the pathway, like the ATP consuming reactions and glucose transport. A regulation mechanism is proposed governed by the energy state of the cell by which L. lactis can handle the glycolytic flux through the allosteric properties of key enzymes, with PFK having a significant influence on the control. [Abstract/Link to Full Text]

Surribas A, Resina D, Ferrer P, Valero F
Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in Pichia pastoris.
Microb Cell Fact. 2007;615.
BACKGROUND: Together with the development of optical sensors, fluorometry is becoming an increasingly attractive tool for the monitoring of cultivation processes. In this context, the green fluorescence protein (GFP) has been proposed as a molecular reporter when fused to target proteins to study their subcellular localization or secretion behaviour. The present work evaluates the use of the GFP fusion partner for monitoring extracellular production of a Rhizopus oryzae lipase (ROL) in Pichia pastoris by means of 2D-fluorimetric techniques RESULTS: In this study, the GFP-ROL fusion protein was successfully produced as a secreted fusion form in P. pastoris batch cultivations. Furthermore, both the fusion enzyme and the fluorescent protein (GFP S65T mutant) retained their biological activity. However, when multiwavelength spectrofluorometry was used for extracellular fusion protein monitoring, riboflavin appeared as a major interfering component with GFP signal. Only when riboflavin was removed by ultrafiltration from cultivation supernatants, GFP fluorescence signal linearly correlated to lipase activity CONCLUSION: P. pastoris appears to secrete/excrete significant amounts of riboflavin to the culture medium. When attempting to monitor extracellular protein production in P. pastoris using GFP fusions combined with multiwavelength spectrofluorimetric techniques, riboflavin may interfere with GFP fluorescence signal, thus limiting the application of some GFP variants for on-line extracellular recombinant protein quantification and monitoring purposes. [Abstract/Link to Full Text]

Walia R, Deb JK, Mukherjee KJ
Development of expression vectors for Escherichia coli based on the pCR2 replicon.
Microb Cell Fact. 2007;614.
BACKGROUND: Recent developments in metabolic engineering and the need for expanded compatibility required for co-expression studies, underscore the importance of developing new plasmid vectors with properties such as stability and compatibility. RESULTS: We utilized the pCR2 replicon of Corynebacterium renale, which harbours multiple plasmids, for constructing a range of expression vectors. Different antibiotic-resistance markers were introduced and the vectors were found to be 100% stable over a large number of generations in the absence of selection pressure. Compatibility of this plasmid was studied with different Escherichia coli plasmid replicons viz. pMB1 and p15A. It was observed that pCR2 was able to coexist with these E. coli plasmids for 60 generations in the absence of selection pressure. Soluble intracellular production was checked by expressing GFP under the lac promoter in an expression plasmid pCR2GFP. Also high level production of human IFNgamma was obtained by cloning the h-IFNgamma under a T7 promoter in the expression plasmid pCR2-IFNgamma and using a dual plasmid heat shock system for expression. Repeated sub-culturing in the absence of selection pressure for six days did not lead to any fall in the production levels post induction, for both GFP and h-IFNgamma, demonstrating that pCR2 is a useful plasmid in terms of stability and compatibility. CONCLUSION: We have constructed a series of expression vectors based on the pCR2 replicon and demonstrated its high stability and sustained expression capacity, in the absence of selection pressure which will make it an efficient tool for metabolic engineering and co-expression studies, as well as for scale up of expression. [Abstract/Link to Full Text]

Barghini P, Di Gioia D, Fava F, Ruzzi M
Vanillin production using metabolically engineered Escherichia coli under non-growing conditions.
Microb Cell Fact. 2007;613.
BACKGROUND: Vanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details. RESULTS: Effect of plasmid copy number in metabolic engineering of E. coli for the synthesis of vanillin has been evaluated by the use of genes encoding feruloyl-CoA synthetase and feruloyl hydratase/aldolase from Pseudomonas fluorescens BF13. The higher vanillin production yield was obtained using resting cells of E. coli strain JM109 harbouring a low-copy number vector and a promoter exhibiting a low activity to drive the expression of the catabolic genes. Optimization of the bioconversion of ferulic acid to vanillin was accomplished by a response surface methodology. The experimental conditions that allowed us to obtain high values for response functions were 3.3 mM ferulic acid and 4.5 g/L of biomass, with a yield of 70.6% and specific productivity of 5.9 micromoles/g x min after 3 hours of incubation. The final concentration of vanillin in the medium was increased up to 3.5 mM after a 6-hour incubation by sequential spiking of 1.1 mM ferulic acid. The resting cells could be reused up to four times maintaining the production yield levels over 50%, thus increasing three times the vanillin obtained per gram of biomass. CONCLUSION: Ferulic acid can be efficiently converted to vanillin, without accumulation of undesirable vanillin reduction/oxidation products, using E. coli JM109 cells expressing genes from the ferulic acid-degrader Pseudomonas fluorescens BF13. Optimization of culture conditions and bioconversion parameters, together with the reuse of the biomass, leaded to a final production of 2.52 g of vanillin per liter of culture, which is the highest found in the literature for recombinant strains and the highest achieved so far applying such strains under resting cells conditions. [Abstract/Link to Full Text]

Hazebrouck S, Pothelune L, Azevedo V, Corthier G, Wal JM, Langella P
Efficient production and secretion of bovine beta-lactoglobulin by Lactobacillus casei.
Microb Cell Fact. 2007;612.
BACKGROUND: Lactic acid bacteria (LAB) are attractive tools to deliver therapeutic molecules at the mucosal level. The model LAB Lactococcus lactis has been intensively used to produce and deliver such heterologous proteins. However, compared to recombinant lactococci, lactobacilli offer some advantages such as better survival in the digestive tract and immunomodulatory properties. Here, we compared different strategies to optimize the production of bovine beta-lactoglobulin (BLG), a major cow's milk allergen, in the probiotic strain Lactobacillus casei BL23. RESULTS: Using a nisin-inducible plasmid system, we first showed that L. casei BL23 strain could efficiently secrete a reporter protein, the staphylococcal nuclease (Nuc), with the lactococcal signal peptide SPUsp45 fused to its N-terminus. The fusion of SPUsp45 failed to drive BLG secretion but led to a 10-fold increase of intracellular BLG production. Secretion was significantly improved when the synthetic propeptide LEISSTCDA (hereafter called LEISS) was added to the N-terminus of the mature moiety of BLG. Secretion rate of LEISS-BLG was 6-fold higher than that of BLG alone while intracellular production reached then about 1 mg/L of culture. The highest yield of secretion was obtained by using Nuc as carrier protein. Insertion of Nuc between LEISS and BLG resulted in a 20-fold increase in BLG secretion, up to 27 microg/L of culture. Furthermore, the lactococcal nisRK regulatory genes were integrated into the BL23 chromosome. The nisRK insertion allowed a decrease of BLG synthesis in uninduced cultures while BLG production increased by 50% after nisin induction. Moreover, modification of the induction protocol led to increase the proportion of soluble BLG to around 74% of the total BLG production. CONCLUSION: BLG production and secretion in L. casei were significantly improved by fusions to a propeptide enhancer and a carrier protein. The resulting recombinant strains will be further tested for their ability to modulate the immune response against BLG via mucosal delivery in a cow's milk allergy model in mice. [Abstract/Link to Full Text]

Hanna ES, Roque-Barreira MC, Bernardes ES, Panunto-Castelo A, Sousa MV, Almeida IC, Brocchi M
Evidence for glycosylation on a DNA-binding protein of Salmonella enterica.
Microb Cell Fact. 2007;611.
BACKGROUND: All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column. RESULTS: The N-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps of S. enterica serovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD) of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period. CONCLUSION: Taken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein. [Abstract/Link to Full Text]

Quintero MJ, Maya D, Arévalo-Rodríguez M, Cebolla A, Chávez S
An improved system for estradiol-dependent regulation of gene expression in yeast.
Microb Cell Fact. 2007;610.
BACKGROUND: Saccharomyces cerevisiae is widely utilized in basic research as a model eukaryotic organism and in biotechnology as a host for heterologous protein production. Both activities demand the use of highly regulated systems, able to provide accurate control of gene expression in functional analysis, and timely recombinant protein synthesis during fermentative production. The tightly regulated GAL1-10 promoter is commonly used. However, induction of the GAL system requires the presence of the rather expensive inducer galactose and the absence of glucose in the culture media. An alternative to regulate transcription driven by GAL promoters, free of general metabolic changes, is the incorporation of the hybrid Gal4-ER-VP16 protein developed by D. Picard. This chimeric protein provides galactose-independent activation of transcription from GAL promoters in response to beta-estradiol, even in the presence of glucose. However, constitutive expression of this transactivator results in relatively high basal activity of the GAL promoters, therefore limiting the gene expression capacity that is required for a number of applications. RESULTS: In order to improve this expression tool, we have introduced additional regulatory elements allowing a simultaneous control of both the abundance and the intrinsic activity of the Gal4-ER-VP16 chimeric transactivator. The most efficient combination was obtained by placing the coding sequence of the hybrid activator under the control of the GAL1 promoter. This configuration results in an amplification feedback loop that is triggered by the hormone, and ultimately leads to the enhanced regulation of recombinant genes when these are also driven by a GAL1 promoter. The basal expression level of this system is as low as that of native GAL-driven genes in glucose-containing media. CONCLUSION: The feedback regulatory loop that we have engineered allows a 250-fold induction of the regulated gene, without increasing the basal activity of the target promoter, and achieving a 12-fold higher regulation efficiency than the previous configuration. [Abstract/Link to Full Text]

Turner P, Mamo G, Karlsson EN
Potential and utilization of thermophiles and thermostable enzymes in biorefining.
Microb Cell Fact. 2007;69.
In today's world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts. [Abstract/Link to Full Text]

---

Recent Articles in Antimicrobial Agents and Chemotherapy

---

Partridge SR
Genetic environment of ISEcp1 and blaACC-1.
Antimicrob Agents Chemother. 2007 Jul;51(7):2658-9; author reply 2659. [Abstract/Link to Full Text]

Papagiannitsis CC, Loli A, Tzouvelekis LS, Tzelepi E, Arlet G, Miriagou V
SCO-1, a novel plasmid-mediated class A beta-lactamase with carbenicillinase characteristics from Escherichia coli.
Antimicrob Agents Chemother. 2007 Jun;51(6):2185-8.
A novel class A beta-lactamase (SCO-1) encoded by an 80-kb self-transferable plasmid from Escherichia coli is described. The interaction of SCO-1 with beta-lactams was similar to that of the CARB-type enzymes. Also, SCO-1 exhibited a 51% amino acid sequence identity with the RTG subgroup of chromosomal carbenicillinases (RTG-1, CARB-5, and CARB-8). [Abstract/Link to Full Text]


New antimicrobial agents approved by the U.S. Food and Drug Administration in 2006 and new indications for previously approved agents.
Antimicrob Agents Chemother. 2007 Jul;51(7):2649. [Abstract/Link to Full Text]

Rossignol T, Logue ME, Reynolds K, Grenon M, Lowndes NF, Butler G
Transcriptional response of Candida parapsilosis following exposure to farnesol.
Antimicrob Agents Chemother. 2007 Jul;51(7):2304-12.
In Candida albicans, the quorum-sensing molecule farnesol inhibits the transition from yeast to hyphae but has no effect on cellular growth. We show that the addition of exogenous farnesol to cultures of Candida parapsilosis causes the cells to arrest, but not at a specific stage in the cell cycle. The cells are not susceptible to additional farnesol. However, the cells do eventually recover from arrest. Unlike in C. albicans, in C. parapsilosis sterols are localized to the tips of budding cells, and this polarization is disrupted by the addition of farnesol. We used the results of a genome sequence survey to design and manufacture partial genomic microarrays that were applied to determining the transcriptional response of C. parapsilosis to the presence of exogenous farnesol. In both C. albicans and C. parapsilosis, exposure to farnesol results in increased expression of the oxidoreductases GRP2 and ADH7 and altered expression of genes involved in sterol metabolism. There is no effect on expression of C. parapsilosis orthologs of genes involved in hyphal growth in C. albicans. Farnesol therefore differs significantly in its effects on C. parapsilosis and C. albicans. [Abstract/Link to Full Text]

Budd A, Alleva L, Alsharifi M, Koskinen A, Smythe V, Müllbacher A, Wood J, Clark I
Increased survival after gemfibrozil treatment of severe mouse influenza.
Antimicrob Agents Chemother. 2007 Aug;51(8):2965-8.
Gemfibrozil, an agent that inhibits production of proinflammatory cytokines in addition to its clinically useful lipid-lowering activity, increased survival in BALB/c mice that were already ill from infection by influenza virus A/Japan/305/57 (H2N2). Gemfibrozil was administered intraperitoneally once daily from days 4 to 10 after intranasal exposure to the virus. Survival increased from 26% in vehicle-treated mice (n = 50) to 52% in mice given gemfibrozil at 60 mg/kg/day (n = 46) (P = 0.0026). If this principle translates to patients, a drug already approved for human use, albeit by a different route for another purpose, might be adapted relatively fast for use against influenza, conceivably including human infection with a derivative of the avian H5N1 strain. [Abstract/Link to Full Text]

Adams DA, Riggs MM, Donskey CJ
Effect of fluoroquinolone treatment on growth of and toxin production by epidemic and nonepidemic clostridium difficile strains in the cecal contents of mice.
Antimicrob Agents Chemother. 2007 Aug;51(8):2674-8.
Several recent outbreaks of Clostridium difficile-associated disease (CDAD) have been attributed to the emergence of an epidemic strain with increased resistance to fluoroquinolone antibiotics. Some clinical studies have suggested that fluoroquinolones with enhanced antianaerobic activity (i.e., gatifloxacin and moxifloxacin) may have a greater propensity to induce CDAD than ciprofloxacin and levofloxacin do. We examined the effects of subcutaneous fluoroquinolone treatment on in vitro growth of and toxin production by epidemic and nonepidemic C. difficile isolates in cecal contents of mice and evaluated the potential for these agents to inhibit fluoroquinolone-susceptible isolates during treatment. When C. difficile isolates were inoculated into cecal contents collected 2 days after the final antibiotic dose, gatifloxacin and moxifloxacin promoted significantly more growth and toxin production than ciprofloxacin and levofloxacin did. During treatment, gatifloxacin and moxifloxacin inhibited growth of fluoroquinolone-susceptible but not fluoroquinolone-resistant isolates. Ciprofloxacin and levofloxacin promoted growth of C. difficile when administered at higher doses (i.e., 20 times the human dose in mg/kg of body weight), and levofloxacin inhibited growth of fluoroquinolone-susceptible, but not fluoroquinolone-resistant, C. difficile isolates when administered in combination with ceftriaxone. Thus, fluoroquinolones with enhanced antianaerobic activity (i.e., gatifloxacin and moxifloxacin) promoted C. difficile growth to a greater extent than did ciprofloxacin and levofloxacin in this model. However, our findings suggest that fluoroquinolones may exert selective pressure favoring the emergence of epidemic fluoroquinolone-resistant C. difficile strains by inhibiting fluoroquinolone-susceptible but not fluoroquinolone-resistant isolates during treatment and that agents such as levofloxacin or ciprofloxacin can exert such selective pressure when administered in combination with antibiotics that disrupt the anaerobic microflora. [Abstract/Link to Full Text]

Asif G, Hurwitz SJ, Shi J, Hernandez-Santiago BI, Schinazi RF
Pharmacokinetics of the antiviral agent beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine in rhesus monkeys.
Antimicrob Agents Chemother. 2007 Aug;51(8):2877-82.
beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is an effective inhibitor of hepatitis C virus (HCV) replication in vitro. The purpose of this study was to evaluate the single-dose pharmacokinetics of PSIota-6130 in rhesus monkeys following intravenous (i.v.) and oral administration. Noncompartmental analysis of the serum data obtained following oral and i.v. administration was performed. Pharmacokinetic studies with rhesus monkeys indicated slow and incomplete absorption with a mean absorption time (MAT) of 4.6 h and an oral bioavailability of 24.0% +/- 14.3% (mean +/- standard deviation), with comparable mean apparent half-lives following i.v. (4.54 +/- 3.98 h) and oral (5.64 +/- 1.13 h) administrations. The average percentages of the total dose recovered unchanged and in deaminated form in the urine were 32.9% +/- 12.6% and 18.9% +/- 6.6% (i.v.) and 6.0% +/- 3.9% and 3.9% +/- 1.0% (oral), respectively. The total bioavailability, taking into account the parent drug and its deaminated metabolite 2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206), was 64% +/- 26%. PSI-6130 was present in the cerebrospinal fluid after oral and i.v. dosing. However, no deamination of radiolabeled PSI-6130 was detected after 8 h of incubation in monkey and human whole blood. An N(4)-modified prodrug of PSI-6130 (PSI-6419) was orally administered to monkeys, but it failed to improve the oral bioavailability of PSI-6130. Further studies are warranted to improve the oral bioavailability and reduce the deamination of PSI-6130 in order to explore the potential of this drug for the treatment of HCV-infected individuals. [Abstract/Link to Full Text]

Li Q, Kozar MP, Shearer TW, Xie LH, Lin AJ, Smith KS, Si Y, Anova L, Zhang J, Milhous WK, Skillman DR
Pharmacokinetics, safety, and hydrolysis of oral pyrroloquinazolinediamines administered in single and multiple doses in rats.
Antimicrob Agents Chemother. 2007 Aug;51(8):2898-904.
Pyrroloquinazolinediamine (PQD) derivatives such as tetra-acetamide PQD (PQD-A4) and bis-ethylcarbamyl PQD (PQD-BE) were much safer (with therapeutic indices of 80 and 32, respectively) than their parent compound, PQD (therapeutic index, 10). Further evaluation of PQD-A4 and PQD-BE in single and multiple pharmacokinetic (PK) studies as well as corresponding toxicity studies was conducted with rats. PQD-A4 could be converted to two intermediate metabolites (monoacetamide PQD and bisacetamide PQD) first and then to the final metabolite, PQD, while PQD-BE was directly hydrolyzed to PQD without precursor and intermediate metabolites. Maximum tolerant doses showed that PQD-A4 and PQD-BE have only 1/12 and 1/6, respectively, of the toxicity of PQD after a single oral dose. Compared to the area under the concentration-time curve for PQD alone (2,965 ng.h/ml), values measured in animals treated with PQD-A4 and PQD-BE were one-third (1,047 ng.h/ml) and one-half (1,381 ng.h/ml) as high, respectively, after an equimolar dosage, suggesting that PQD was the only agent to induce the toxicity. Similar results were also shown in multiple treatments; PQD-A4 and PQD-BE generated two-fifths and three-fifths, respectively, of PQD concentrations, with 8.8-fold and 3.8-fold safety margins, respectively, over the parent drug. PK data indicated that the bioavailability of oral PQD-A4 was greatly limited at high dose levels, that PQD-A4 was slowly converted to PQD via a sequential three-step process of conversion, and that PQD-A4 was significantly less toxic than the one-step hydrolysis drug, PQD-BE. It was concluded that the slow and smaller release of PQD was the main reason for the reduction in toxicity and that the active intermediate metabolites can still maintain antimalarial potency. Therefore, the candidate with multiple-step hydrolysis of PQD could be developed as a safer potential agent for malaria treatment. [Abstract/Link to Full Text]

Jump RL, Pultz MJ, Donskey CJ
Vegetative Clostridium difficile survives in room air on moist surfaces and in gastric contents with reduced acidity: a potential mechanism to explain the association between proton pump inhibitors and C. difficile-associated diarrhea?
Antimicrob Agents Chemother. 2007 Aug;51(8):2883-7.
Proton pump inhibitors (PPIs) have been identified as a risk factor for Clostridium difficile-associated diarrhea (CDAD), though the mechanism is unclear because gastric acid does not kill C. difficile spores. We hypothesized that the vegetative form of C. difficile, which is killed by acid, could contribute to disease pathogenesis if it survives in room air and in gastric contents with elevated pH. We compared the numbers of C. difficile spores and vegetative cells in stools of patients prior to and during the treatment of CDAD. We assessed the survival of vegetative cells on moist or dry surfaces in room air versus anaerobic conditions and in human gastric contents, in pH-adjusted gastric contents, and in gastric contents from individuals receiving PPI therapy. Stool samples obtained from patients prior to the initiation of antibiotic treatment for C. difficile contained approximately 10-fold more vegetative cells than spores. On dry surfaces, vegetative C. difficile cells died rapidly, whereas they remained viable for up to 6 h on moist surfaces in room air. Vegetative C. difficile cells had only marginal survival in gastric contents at low pH; adjustment to a pH of >5 resulted in survival similar to that in the phosphate-buffered saline control. The survival of vegetative C. difficile in gastric contents obtained from patients receiving PPIs was also increased at a pH of >5. The ability of the vegetative form of C. difficile to survive on moist surfaces and in gastric contents with an elevated pH suggests a potential mechanism by which PPI therapy could increase the risk of acquiring C. difficile. [Abstract/Link to Full Text]

Pultz MJ, Donskey CJ
Effects of imipenem-cilastatin, ertapenem, piperacillin-tazobactam, and ceftriaxone treatments on persistence of intestinal colonization by extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae strains in mice.
Antimicrob Agents Chemother. 2007 Aug;51(8):3044-5. [Abstract/Link to Full Text]

Kaiser M, Wittlin S, Nehrbass-Stuedli A, Dong Y, Wang X, Hemphill A, Matile H, Brun R, Vennerstrom JL
Peroxide bond-dependent antiplasmodial specificity of artemisinin and OZ277 (RBx11160).
Antimicrob Agents Chemother. 2007 Aug;51(8):2991-3.
Using nonperoxidic analogs of artemisinin and OZ277 (RBx11160), the strong in vitro antiplasmodial activities of the latter two compounds were shown to be peroxide bond dependent. In contrast, the weak activities of artemisinin and OZ277 against six other protozoan parasites were peroxide bond independent. These data support the iron-dependent artemisinin alkylation hypothesis. [Abstract/Link to Full Text]

Leavitt A, Navon-Venezia S, Chmelnitsky I, Schwaber MJ, Carmeli Y
Emergence of KPC-2 and KPC-3 in carbapenem-resistant Klebsiella pneumoniae strains in an Israeli hospital.
Antimicrob Agents Chemother. 2007 Aug;51(8):3026-9.
Carbapenem resistance due to KPC has rarely been observed outside the United States. We noticed a sharp increase in carbapenem-resistant Klebsiella pneumoniae strains possessing KPC in Tel Aviv Medical Center from 2004 to 2006. Sixty percent of the isolates belonged to a single clone susceptible only to gentamicin and colistin and carried the bla(KPC-3) gene, while almost all other clones carried the bla(KPC-2) gene. This rapid dissemination of KPC outside the United States is worrisome. [Abstract/Link to Full Text]

Nsobya SL, Dokomajilar C, Joloba M, Dorsey G, Rosenthal PJ
Resistance-mediating Plasmodium falciparum pfcrt and pfmdr1 alleles after treatment with artesunate-amodiaquine in Uganda.
Antimicrob Agents Chemother. 2007 Aug;51(8):3023-5.
Key parasite polymorphisms were assessed in subjects treated for malaria with artesunate-amodiaquine in Tororo, Uganda. For pfcrt, all of the isolates tested had the CVIET haplotype. For pfmdr1, 86Y and 1246Y were common at baseline and their prevalences were significantly higher in new isolates after therapy, indicating that treatment selected for mutations associated with a decreased response to amodiaquine. [Abstract/Link to Full Text]

Kirkpatrick WR, Najvar LK, Bocanegra R, Patterson TF, Graybill JR
New guinea pig model of Cryptococcal meningitis.
Antimicrob Agents Chemother. 2007 Aug;51(8):3011-3.
We developed a guinea pig model of cryptococcal meningitis to evaluate antifungal agents. Immunosuppressed animals challenged intracranially with Cryptococcus neoformans responded to fluconazole and voriconazole. Disease was monitored by serial cerebrospinal fluid (CSF) cultures and quantitative organ cultures. Our model produces disseminating central nervous system disease and responds to antifungal therapy. [Abstract/Link to Full Text]

Huy NT, Mizunuma K, Kaur K, Nhien NT, Jain M, Uyen DT, Harada S, Jain R, Kamei K
2-tert-butyl-8-quinolinamines exhibit potent blood schizontocidal antimalarial activity via inhibition of heme crystallization.
Antimicrob Agents Chemother. 2007 Aug;51(8):2842-7.
We have recently reported that the attachment of a bulky metabolically stable tert-butyl group at the C-2 position of a quinoline ring in primaquine results in a tremendous improvement in the blood schizontocidal antimalarial activity of 8-quinolinamine. Because free heme released from hemoglobin catabolism in a malarial parasite is highly toxic, the parasite protects itself mainly by crystallization of heme into insoluble nontoxic hemozoin. We now demonstrate the ability of 2-tert-butylprimaquine to inhibit in vitro beta-hematin formation, to form a complex with heme with a stoichiometry of 1:1, and to enhance heme-induced hemolysis. The results described herein indicate that a major improvement in the blood-schizontocidal antimalarial activity of 2-tert-butylprimaquine might be due to a disturbance of heme catabolism pathway in the malarial parasite. [Abstract/Link to Full Text]

Bacciaglia A, Brenciani A, Varaldo PE, Giovanetti E
SmaI typeability and tetracycline susceptibility and resistance in Streptococcus pyogenes isolates with efflux-mediated erythromycin resistance.
Antimicrob Agents Chemother. 2007 Aug;51(8):3042-3. [Abstract/Link to Full Text]

Skiadas I, Pefanis A, Papalois A, Kyroudi A, Triantafyllidi H, Tsaganos T, Giamarellou H
Dexamethasone as adjuvant therapy to moxifloxacin attenuates valve destruction in experimental aortic valve endocarditis due to Staphylococcus aureus.
Antimicrob Agents Chemother. 2007 Aug;51(8):2848-54.
Although the beneficial effects of dexamethasone have frequently been investigated in various serious-infection settings, insufficient data on valve histology and cardiac function for infective endocarditis are available. The efficacy of moxifloxacin for the treatment of experimental aortic valve endocarditis due to methicillin-susceptible Staphylococcus aureus and the long-term effects of dexamethasone were evaluated in the current study. Sixty-eight rabbits were randomly assigned to four groups: A, B, C, and D. Group A consisted of 18 animals and functioned as a control group. Groups B and C consisted of 11 and 23 subjects, respectively, which received moxifloxacin for 5 days in a human-like pharmacokinetic simulation. Group D consisted of 16 animals that were administered moxifloxacin plus dexamethasone (0.25 mg/kg of body weight twice a day intravenously). The group B animals were sacrificed a day after the completion of treatment, and group C and D animals were sacrificed after 12 days in order to monitor any possible relapse and allow microbiological, histopathological, and echocardiographic evaluation of the long-term effects of glucocorticoids. No differences in survival, sterilization rates, or inflammatory infiltration and calcification of valve tissue were observed among the treated groups. However, the degrees of valve damage and collagenization were significantly worse, the fibroblast content was higher, and fractional shortening of the left ventricle fluctuated significantly in group C compared to group D (all groups, P < 0.05). We concluded that dexamethasone treatment for experimental S. aureus endocarditis attenuates valve destruction and preserves overall cardiac function without impeding the efficacy of moxifloxacin. [Abstract/Link to Full Text]

Korycka-Machala M, Rychta E, Brzostek A, Sayer HR, Rumijowska-Galewicz A, Bowater RP, Dziadek J
Evaluation of NAD(+) -dependent DNA ligase of mycobacteria as a potential target for antibiotics.
Antimicrob Agents Chemother. 2007 Aug;51(8):2888-97.
Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD(+)-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD(+)-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD(+)-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD(+)-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria. [Abstract/Link to Full Text]

Segal H, Jacobson RK, Garny S, Bamford CM, Elisha BG
Extended -10 promoter in ISAba-1 upstream of blaOXA-23 from Acinetobacter baumannii.
Antimicrob Agents Chemother. 2007 Aug;51(8):3040-1. [Abstract/Link to Full Text]

Nakata H, Amano M, Koh Y, Kodama E, Yang G, Bailey CM, Kohgo S, Hayakawa H, Matsuoka M, Anderson KS, Cheng YC, Mitsuya H
Activity against human immunodeficiency virus type 1, intracellular metabolism, and effects on human DNA polymerases of 4'-ethynyl-2-fluoro-2'-deoxyadenosine.
Antimicrob Agents Chemother. 2007 Aug;51(8):2701-8.
We examined the intracytoplasmic anabolism and kinetics of antiviral activity against human immunodeficiency virus type 1 (HIV-1) of a nucleoside reverse transcriptase inhibitor, 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), which has potent activity against wild-type and multidrug-resistant HIV-1 strains. When CEM cells were exposed to 0.1 microM [(3)H]EFdA or [(3)H]3'-azido-2',3'-dideoxythymidine (AZT) for 6 h, the intracellular EFdA-triphosphate (TP) level was 91.6 pmol/10(9) cells, while that of AZT was 396.5 pmol/10(9) cells. When CEM cells were exposed to 10 microM [(3)H]EFdA, the amount of EFdA-TP increased by 22-fold (2,090 pmol/10(9) cells), while the amount of [(3)H]AZT-TP increased only moderately by 2.4-fold (970 pmol/10(9) cells). The intracellular half-life values of EFdA-TP and AZT-TP were approximately 17 and approximately 3 h, respectively. When MT-4 cells were cultured with 0.01 microM EFdA for 24 h, thoroughly washed to remove EFdA, further cultured without EFdA for various periods of time, exposed to HIV-1(NL4-3), and cultured for an additional 5 days, the protection values were 75 and 47%, respectively, after 24 and 48 h with no drug incubation, while those with 1 microM AZT were 55 and 9.2%, respectively. The 50% inhibitory concentration values of EFdA-TP against human polymerases alpha, beta, and gamma were >100 microM, >100 microM, and 10 microM, respectively, while those of ddA-TP were >100 microM, 0.2 microM, and 0.2 microM, respectively. These data warrant further development of EFdA as a potential therapeutic agent for those patients who harbor wild-type HIV-1 and/or multidrug-resistant variants. [Abstract/Link to Full Text]

Thomas A, Laxton C, Rodman J, Myangar N, Horscroft N, Parkinson T
Investigating Toll-like receptor agonists for potential to treat hepatitis C virus infection.
Antimicrob Agents Chemother. 2007 Aug;51(8):2969-78.
Toll-like receptors (TLRs) are key mediators of innate immunity, and their activation by microbial components leads to the production of cytokines and interferons. Recombinant alpha interferon has been used to treat several viral diseases and is the current standard of care for hepatitis C virus (HCV) infection. Recently, agonists of TLR7 and TLR9 have been shown to have clinical efficacy in HCV patients, and this is correlated with their ability to induce endogenous type I interferon production. We have carried out a comprehensive study of agonists of TLRs 1 to 9 to determine if any additional TLRs can induce antiviral molecules from human peripheral blood mononuclear cells (PBMCs). The agonists were incubated with PBMCs, and the supernatant was then removed and added to HCV replicon cells to assess antiviral activity. Agonists of TLRs 3, 4, 7, 8, and 9 were found to be potent inducers of antiviral activity in PBMC supernatants, and the activity correlated with the induction of alpha interferon and the interferon-induced antiviral biomarker 2',5'-oligoadenylate synthase. Antiviral activity of TLR7 and TLR8 agonists was blocked by an antibody that binds to the type I interferon receptor, confirming that the antiviral activity results from type I interferon induction. TLR4 and TLR8 agonists were found to strongly induce the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha at concentrations similar to those inducing antiviral activity. This raises concerns about adverse side effects if these were to be used as antiviral agents. We therefore conclude that TLRs 3, 7, and 9 represent the most attractive targets for the development of new HCV therapies. [Abstract/Link to Full Text]

Negrea A, Bjur E, Ygberg SE, Elofsson M, Wolf-Watz H, Rhen M
Salicylidene acylhydrazides that affect type III protein secretion in Salmonella enterica serovar typhimurium.
Antimicrob Agents Chemother. 2007 Aug;51(8):2867-76.
A collection of nine salicylidene acylhydrazide compounds were tested for their ability to inhibit the activity of virulence-associated type III secretion systems (T3SSs) in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island 1 (SPI1) T3SS-mediated invasion of epithelial cells and in vitro secretion of SPI1 invasion-associated effector proteins. The use of a SPI1 effector beta-lactamase fusion protein implicated intracellular entrapment of the protein construct upon application of a salicylidene acylhydrazide, whereas the use of chromosomal transcriptional gene fusions revealed a compound-mediated transcriptional silencing of SPI1. Salicylidene acylhydrazides also affected intracellular bacterial replication in murine macrophage-like cells and blocked the transport of an epitope-tagged SPI2 effector protein. Two of the compounds significantly inhibited bacterial motility and expression of extracellular flagellin. We conclude that salicylidene acylhydrazides affect bacterial T3SS activity in S. enterica and hence could be used as lead substances when designing specific inhibitors of bacterial T3SSs in order to pharmaceutically intervene with bacterial virulence. [Abstract/Link to Full Text]

Fuchs BB, Tegos GP, Hamblin MR, Mylonakis E
Susceptibility of Cryptococcus neoformans to photodynamic inactivation is associated with cell wall integrity.
Antimicrob Agents Chemother. 2007 Aug;51(8):2929-36.
Photodynamic therapy is a rapidly developing antimicrobial technology which combines a nontoxic photoactivatable dye or photosensitizer with harmless visible light of the correct wavelength to excite the dye to its reactive triplet state to generate reactive oxygen species toxic to cells. In this report we present evidence that the fungal pathogen Cryptococcus neoformans is susceptible to photodynamic inactivation by use of a polycationic conjugate of polyethyleneimine and the photosensitizer chlorin(e6). A C. neoformans rom2 mutant, with a mutation involving a putative Rho1 guanyl nucleotide exchange factor that is part of the protein kinase C-cell wall integrity pathway, demonstrated a compromised cell wall and less (1,3)beta-d glucan than the wild-type strain and increased accumulation of PEI-ce6 as assessed by fluorescence uptake and confocal microscopy. Interestingly, C. neoformans rom2 was hypersusceptible to photodynamic inactivation and coincubation of wild-type C. neoformans strain KN99alpha with caspofungin-enhanced photoinactivation. These studies demonstrated that C. neoformans is sensitive to photodynamic therapy and illustrated the significance of cell wall integrity in microbial susceptibility to antimicrobial photodynamic inactivation. [Abstract/Link to Full Text]

Müller FM, Staudigel A, Salvenmoser S, Tredup A, Miltenberger R, Herrmann JV
Cross-resistance to medical and agricultural azole drugs in yeasts from the oropharynx of human immunodeficiency virus patients and from environmental Bavarian vine grapes.
Antimicrob Agents Chemother. 2007 Aug;51(8):3014-6.
Cross-resistance among Candida albicans isolates from the oropharynges of human immunodeficiency virus-infected patients (n = 16) and environmental yeast strains of various species (n = 54) to medical and agricultural azole drugs was observed. Precautions against the unnecessary widespread use of azoles in the environment and human medicine are strongly recommended to prevent patients from acquiring azole-resistant yeasts. [Abstract/Link to Full Text]

Lemaire S, Van Bambeke F, Mingeot-Leclercq MP, Tulkens PM
Modulation of the cellular accumulation and intracellular activity of daptomycin towards phagocytized Staphylococcus aureus by the P-glycoprotein (MDR1) efflux transporter in human THP-1 macrophages and madin-darby canine kidney cells.
Antimicrob Agents Chemother. 2007 Aug;51(8):2748-57.
P-glycoprotein (P-gp; MDR1), a major efflux transporter, recognizes various antibiotics and is present in macrophages. We have examined its effect on the modulation of the intracellular accumulation and activity of daptomycin towards phagocytized Staphylococcus aureus (ATCC 25923) in human THP-1 macrophages, in comparison with MDCK epithelial cells (wild type and MDCK-MDR1 overexpressing P-gp; the bulk of the protein was immunodetected at the surface of all three cell types). Daptomycin displayed concentration-dependent intracellular activity (Hill equation pattern) in THP-1 and MDCK cells with (i) 50% effective drug extracellular concentration (EC(50); relative potency) and static concentrations at 9 to 10 times the MIC and (ii) maximal efficacy (E(max); CFU decrease at infinite extracellular drug concentration) at 1.6 to 2 log compared to that of the postphagocytosis inoculum. Verapamil (100 microM) and elacridar (GF 120918; 0.5 microM), two known inhibitors of P-gp, decreased daptomycin EC(50) (about threefold) in THP-1 and MDCK cells without affecting E(max). Daptomycin EC(50) was about three- to fourfold higher and accumulation in MDCK-MDR1 commensurately lower than in wild-type cells. In THP-1 macrophages, (i) verapamil and ATP depletion increased, and ouabain (an inducer of mdr1 [the gene encoding P-gp] expression) decreased the accumulation of daptomycin in parallel with that of DiOC(2) (a known substrate of P-gp); (ii) silencing mdr1 with duplex human mdr1 siRNAs reduced the cell content in immunoreactive P-gp to 15 to 30% of controls and caused an eight- to 13-fold increase in daptomycin accumulation. We conclude that daptomycin is subject to efflux from THP-1 macrophages and MDCK cells by P-gp, which reduces its intracellular activity against phagocytized S. aureus. [Abstract/Link to Full Text]

Solon EG, Dowell JA, Lee J, King SP, Damle BD
Distribution of radioactivity in bone and related structures following administration of [14C]dalbavancin to New Zealand white rabbits.
Antimicrob Agents Chemother. 2007 Aug;51(8):3008-10.
Penetration of dalbavancin into noninfected bone and joint tissues was assessed after an intravenous dose of 20 mg/kg (of body weight) [(14)C]dalbavancin given to rabbits. Drug-derived radioactivity, determined over 14 days by either liquid scintillation counting or autoradiography, remained above the MIC for common gram-positive pathogens that cause bone and joint infections. [Abstract/Link to Full Text]

Donelli G, Francolini I, Romoli D, Guaglianone E, Piozzi A, Ragunath C, Kaplan JB
Synergistic activity of dispersin B and cefamandole nafate in inhibition of staphylococcal biofilm growth on polyurethanes.
Antimicrob Agents Chemother. 2007 Aug;51(8):2733-40.
Antibiotic therapies to eradicate medical device-associated infections often fail because of the ability of sessile bacteria, encased in their exopolysaccharide matrix, to be more drug resistant than planktonic organisms. In the last two decades, several strategies to prevent microbial adhesion and biofilm formation on the surfaces of medical devices, based mainly on the use of antiadhesive, antiseptic, and antibiotic coatings on polymer surfaces, have been developed. More recent alternative approaches are based on molecules able to interfere with quorum-sensing phenomena or to dissolve biofilms. Interestingly, a newly purified beta-N-acetylglucosaminidase, dispersin B, produced by the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans, is able to dissolve mature biofilms produced by Staphylococcus epidermidis as well as some other bacterial species. Therefore, in this study, we developed new polymeric matrices able to bind dispersin B either alone or in combination with an antibiotic molecule, cefamandole nafate (CEF). We showed that our functionalized polyurethanes could adsorb a significant amount of dispersin B, which was able to exert its hydrolytic activity against the exopolysaccharide matrix produced by staphylococcal strains. When microbial biofilms were exposed to both dispersin B and CEF, a synergistic action became evident, thus characterizing these polymer-dispersin B-antibiotic systems as promising, highly effective tools for preventing bacterial colonization of medical devices. [Abstract/Link to Full Text]

Pallecchi L, Bartoloni A, Fiorelli C, Mantella A, Di Maggio T, Gamboa H, Gotuzzo E, Kronvall G, Paradisi F, Rossolini GM
Rapid dissemination and diversity of CTX-M extended-spectrum beta-lactamase genes in commensal Escherichia coli isolates from healthy children from low-resource settings in Latin America.
Antimicrob Agents Chemother. 2007 Aug;51(8):2720-5.
A survey carried out in 2005 among members of a healthy population of children living in Bolivia and Peru revealed that fecal carriage of Escherichia coli strains resistant to expanded-spectrum cephalosporins was remarkably increased compared to that observed in the same settings in 2002 (1.7% in 2005 versus 0.1% in 2002). In this work, we demonstrated that this phenomenon was mainly related to the dissemination of CTX-M-type extended-spectrum beta-lactamase (ESBL) determinants among commensal E. coli strains. Of 50 ESBL-producing isolates collected in the 2005 survey, 44 harbored a CTX-M-type and 6 an SHV-type (SHV-2 or SHV-12) ESBL. Compared to 2002 results, an increased diversity of CTX-M-type ESBLs was also observed: members of the CTX-M-1 group (CTX-M-15) emerged in Bolivia (where only CTX-M-2 was observed in 2002), while members of the CTX-M-9 group (CTX-M-14 and CTX-M-24) emerged in Peru (where only CTX-M-15 and CTX-M-2 were observed in 2002). A new CTX-M-2 variant named CTX-M-56 was also detected. Molecular characterization of the CTX-M-producing isolates and gene transfer experiments suggested that different mechanisms could be involved in the spreading of different CTX-M group determinants and revealed that additional resistance determinants for non-beta-lactam antibiotics were preferentially carried by plasmids encoding certain CTX-M variants (CTX-M-15 and variants of the CTX-M-2 group). Three CTX-M-15-encoding conjugative plasmids from Peruvian isolates carried the new fluoroquinolone resistance gene aac(6')-Ib-cr. To our best knowledge, this is the first report of the detection of aac(6')-Ib-cr in Latin America. [Abstract/Link to Full Text]

Pai MP, Norenberg JP, Anderson T, Goade DW, Rodvold KA, Telepak RA, Mercier RC
Influence of morbid obesity on the single-dose pharmacokinetics of daptomycin.
Antimicrob Agents Chemother. 2007 Aug;51(8):2741-7.
The present study characterized the single-dose pharmacokinetics of daptomycin dosed as 4 mg/kg of total body weight (TBW) in seven morbidly obese and seven age-, sex-, race-, and serum creatinine-matched healthy subjects. The glomerular filtration rate (GFR) was measured for both groups following a single bolus injection of [(125)I]sodium iothalamate. Noncompartmental analysis was used to determine the pharmacokinetic parameters, and these values were normalized against TBW, ideal body weight (IBW), and fat-free weight (FFW) for comparison of the two groups. All subjects enrolled in this study were female, and the mean (+/-standard deviation) body mass index was 46.2 +/- 5.5 kg/m(2) or 21.8 +/- 1.9 kg/m(2) for the morbidly obese or normal-weight group, respectively. The maximum plasma concentration and area under the concentration-time curve from dosing to 24 h were approximately 60% higher (P < 0.05) in the morbidly obese group than in the normal-weight group, and these were a function of the higher total dose received in the morbidly obese group. No differences in daptomycin volume of distribution (V), total clearance, renal clearance, or protein binding were noted between the two groups. Of TBW, FFW, or IBW, TBW provided the best correlation to V. In contrast, TBW overestimated GFR through creatinine clearance calculations using the Cockcroft-Gault equation. Use of IBW in the Cockcroft-Gault equation or use of the four-variable modification of diet in renal disease equation best estimated GFR in morbidly obese subjects. Further studies of daptomycin pharmacokinetics in morbidly obese patients with acute bacterial infections and impaired renal function are necessary to better predict appropriate dosage intervals. [Abstract/Link to Full Text]

Sil D, Shrestha A, Kimbrell MR, Nguyen TB, Adisechan AK, Balakrishna R, Abbo BG, Malladi S, Miller KA, Short S, Cromer JR, Arora S, Datta A, David SA
Bound to shock: protection from lethal endotoxemic shock by a novel, nontoxic, alkylpolyamine lipopolysaccharide sequestrant.
Antimicrob Agents Chemother. 2007 Aug;51(8):2811-9.
Lipopolysaccharide (LPS), or endotoxin, a structural component of gram-negative bacterial outer membranes, plays a key role in the pathogenesis of septic shock, a syndrome of severe systemic inflammation which leads to multiple-system organ failure. Despite advances in antimicrobial chemotherapy, sepsis continues to be the commonest cause of death in the critically ill patient. This is attributable to the lack of therapeutic options that aim at limiting the exposure to the toxin and the prevention of subsequent downstream inflammatory processes. Polymyxin B (PMB), a peptide antibiotic, is a prototype small molecule that binds and neutralizes LPS toxicity. However, the antibiotic is too toxic for systemic use as an LPS sequestrant. Based on a nuclear magnetic resonance-derived model of polymyxin B-LPS complex, we had earlier identified the pharmacophore necessary for optimal recognition and neutralization of the toxin. Iterative cycles of pharmacophore-based ligand design and evaluation have yielded a synthetically easily accessible N(1),mono-alkyl-mono-homologated spermine derivative, DS-96. We have found that DS-96 binds LPS and neutralizes its toxicity with a potency indistinguishable from that of PMB in a wide range of in vitro assays, affords complete protection in a murine model of LPS-induced lethality, and is apparently nontoxic in vertebrate animal models. [Abstract/Link to Full Text]

---

Recent Articles in Malaria Journal

---

Kobbe R, Adjei S, Kreuzberg C, Kreuels B, Thompson B, Thompson PA, Marks F, Busch W, Tosun M, Schreiber N, Opoku E, Adjei O, Meyer CG, May J
Malaria incidence and efficacy of intermittent preventive treatment in infants (IPTi).
Malar J. 2007 Dec 9;6(1):163.
ABSTRACT: BACKGROUND: Intermittent preventive antimalarial treatment in infants (IPTi) is currently evaluated as a malaria control strategy. Among the factors influencing the extent of protection that is provided by IPTi are the transmission intensity, seasonality, drug resistance patterns, and the schedule of IPTi administrations. The aim of this study was to determine how far the protective efficacy of IPTi depends on spatio-temporal variations of the prevailing incidence of malaria. METHODS: One thousand seventy infants were enrolled in a registered controlled trial on the efficacy of IPTi with sulphadoxine-pyrimethamine (SP) in the Ashanti Region, Ghana, West Africa (ClinicalTrial.gov: NCT00206739). Stratification for the village of residence and the month of birth of study participants demonstrated that the malaria incidence was dependent on spatial (range of incidence rates in different villages 0.6-2.0 episodes/year) and temporal (range of incidence rates in children of different birth months 0.8-1.2 episodes/year) factors. The range of spatio-temporal variation allowed ecological analyses of the correlation between malaria incidence rates, anti-Plasmodium falciparum lysate IgG antibody levels and protective efficacies provided by IPTi. RESULTS: Protective efficacy of the first SP administration was positively correlated with malaria incidences in children living in a distinct village or born in a distinct month (R2 0.48, p<0.04 and R2 0.63, p<0.003, respectively). Corresponding trends were seen after the second and third study drug administration. Accordingly, IgG levels against parasite lysate increased with malaria incidence. This correlation was stronger in children who received IPTi, indicating an effect modification of the intervention. CONCLUSIONS: The spatial and temporal variations of malaria incidences in a geographically and meteorologically homogeneous study area exemplify the need for close monitoring of local incidence rates in all types of intervention studies. The increase of the protective efficacy of IPTi with malaria incidences may be relevant for IPTi implementation strategies and, possibly, for other malaria control measures. [Abstract/Link to Full Text]

Jones AE, Uddenfeldt Wort U, Morse AP, Hastings IM, Gagnon AS
Climate prediction of El Nino malaria epidemics in north-west Tanzania.
Malar J. 2007 Dec 6;6(1):162.
ABSTRACT: BACKGROUND: Malaria is a significant public health problem in Tanzania. Approximately 16 million malaria cases are reported every year and 100,000 to 125,000 deaths occur. Although most of Tanzania is endemic to malaria, epidemics occur in the highlands, notably in Kagera, a region that was subject to widespread malaria epidemics in 1997 and 1998. This study examined the relationship between climate and malaria incidence in Kagera with the aim of determining whether seasonal forecasts may assist in predicting malaria epidemics. METHODS: A regression analysis was performed on retrospective malaria and climatic data during each of the two annual malaria seasons to determine the climatic factors influencing malaria incidence. The ability of the DEMETER seasonal forecasting system in predicting the climatic anomalies associated with malaria epidemics was then assessed for each malaria season. RESULTS: It was found that malaria incidence is positively correlated with rainfall during the first season (Oct-Mar) (R-squared=0.73, p<0.01). For the second season (Apr-Sep), high malaria incidence was associated with increased rainfall, but also with high maximum temperature during the first rainy season (multiple R-squared=0.79, p<0.01). The robustness of these statistical models was tested by excluding the two epidemic years from the regression analysis. DEMETER would have been unable to predict the heavy El Nino rains associated with the 1998 epidemic. Nevertheless, this epidemic could still have been predicted using the temperature forecasts alone. The 1997 epidemic could have been predicted from observed temperatures in the preceding season, but the consideration of the rainfall forecasts would have improved the temperature-only forecasts over the remaining years. CONCLUSIONS: These results demonstrate the potential of a seasonal forecasting system in the development of a malaria early warning system in Kagera region. [Abstract/Link to Full Text]

Sutherland CJ, Drakeley CJ, Schellenberg D
How is childhood development of immunity to Plasmodium falciparum enhanced by certain antimalarial interventions ?
Malar J. 2007 Dec 4;6(1):161.
ABSTRACT: The development of acquired protective immunity to Plasmodium falciparum infection in young African children is considered in the context of three current strategies for malaria prevention: insecticide-impregnated bed nets or curtains, anti-sporozoite vaccines and intermittent preventive therapy. Evidence is presented that each of these measures may permit attenuated P. falciparum blood-stage infections, which do not cause clinical malaria but can act as an effective blood-stage "vaccine". It is proposed that the extended serum half-life, and rarely considered liver-stage prophylaxis provided by the anti-folate combination sulphadoxine-pyrimethamine frequently lead to such attenuated infections in high transmission areas, and thus contribute to the sustained protection from malaria observed among children receiving the combination as intermittent preventative therapy or for parasite clearance in vaccine trials. [Abstract/Link to Full Text]

Briand V, Cottrell G, Massougbodji A, Cot M
Intermittent preventive treatment for the prevention of malaria during pregnancy in high transmission areas.
Malar J. 2007 Dec 4;6(1):160.
ABSTRACT: Malaria in pregnancy is one of the major causes of maternal morbidity and adverse birth outcomes. In high transmission areas, its prevention has recently changed, moving from a weekly or bimonthly chemoprophylaxis to intermittent preventive treatment (IPTp). IPTp consists in the administration of a single curative dose of an efficacious anti-malarial drug at least twice during pregnancy - regardless of whether the woman is infected or not. The drug is administered under supervision during antenatal care visits. Sulphadoxine-pyrimethamine (SP) is the drug currently recommended by the WHO. While SP-IPTp seems an adequate strategy, there are many issues still to be explored to optimize it. This paper reviewed data on IPTp efficacy and discussed how to improve it. In particular, the determination of both the optimal number of doses and time of administration of the drug is essential, and this has not yet been done. As both foetal growth and deleterious effects of malaria are maximum in late pregnancy women should particularly be protected during this period. Monitoring of IPTp efficacy should be applied to all women, and not only to primi- and secondigravidae, as it has not been definitively established that multigravidae are not at risk for malaria morbidity and mortality. In HIV-positive women, there is an urgent need for specific information on drug administration patterns (need for higher doses, possible interference with sulpha-based prophylaxis of opportunistic infections). Because of the growing level of resistance of parasites to SP, alternative drugs for IPTp are urgently needed. Mefloquine is presently one of the most attractive options because of its long half-life, high efficacy in sub-Saharan Africa and safety during pregnancy. Also, efforts should be made to increase IPTp coverage by improving the practices of health care workers, the motivation of women and their perception of malaria complications in pregnancy. Because IPTp is not applicable in early pregnancy, which is a period when malaria may also be deleterious for women and their offspring, there is a necessity to integrate this strategy with other preventive measures which can be applied earlier in pregnancy such as insecticide-treated nets. [Abstract/Link to Full Text]

Djouaka RF, Bakare AA, Bankole HS, Doannio JM, Coulibaly ON, Kossou H, Tamo M, Basene HI, Popoola KK, Akogbeto MC
Does the spillage of petroleum products in Anopheles breeding sites have an impact on the pyrethroid resistance ?
Malar J. 2007 Dec 3;6(1):159.
ABSTRACT: BACKGROUND: The emergence of Anopheles populations capable of withstanding lethal doses of insecticides has weakened the efficacy of most insecticide based strategies of vector control and, has highlighted the need for further studies on the mechanisms of insecticide resistance and the various factors selecting resistant populations of mosquitoes. This research targeted the analysis of breeding sites and the oviposition behaviour of susceptible and resistant populations of Anopheles in localities of spilled petroleum products. The aim was to establish the possible contribution of oil spillage in the selection of pyrethroid resistance in malaria vectors. METHODS: Anopheles breeding sites were identified and the insecticide susceptibility of the Anopheles gambiae populations mapped in 15 localities of South Western Nigeria. The presence of oil particles as well as the turbidity, the dissolved oxygen and the pH of each identified breeding site was recorded. Data were cross-analysed to correlate the habitat types and the insecticide susceptibility status of emerging mosquitoes. The second phase of this study was basically a laboratory model to provide more information on the implication of the spillage of petroleum on the selection of pyrethroid resistance in An. gambiae. RESULTS: Moderate levels of resistance following exposure to permethrin-impregnated papers were recorded with the majority of An. gambiae samples collected in the South Western Nigeria. Data from this study established a link between the constituency of the breeding sites and the resistance status of the emerging Anopheles. CONCLUSION: This study has revealed the segregational occupation of breeding habitats by pyrethroid resistant and susceptible strains of An. gambiae in south-western Nigeria. Compiled results from field and laboratory research point out clear relationships between oil spillage and pyrethroid resistance in malaria vectors. The identification of this factor of resistance could serve as strong information in the management of insecticide resistance in some West African settings. [Abstract/Link to Full Text]

Protopopoff N, Van Bortel W, Marcotty T, Van Herp M, Maes P, Baza D, D'Alessandro U, Coosemans M
Spatial targeted vector control in the highlands of Burundi and its impact on malaria transmission.
Malar J. 2007 Dec 3;6(1):158.
ABSTRACT: BACKGROUND: Prevention of malaria epidemics is a priority for African countries. The 2000 malaria epidemic in Burundi prompted the government to implement measures for preventing future outbreaks. Case management with artemisinin-based combination therapy and malaria surveillance were nationally improved. A vector control programme was initiated in one of the most affected highland provinces. The focal distribution of malaria vectors in the highlands was the starting point for designing a targeted vector control strategy. The objective of this study was to present the results of this strategy on malaria transmission in an African highland region. METHODS: In Karuzi, in 2002-2005, vector control activities combining indoor residual spraying and long-lasting insecticidal nets were implemented. The interventions were done before the expected malaria transmission period and targeted the valleys between hills, with the expectation that this would also protect the populations living at higher altitudes. The impact on the Anopheles population and on malaria transmission was determined by nine cross-sectional surveys carried out at regular intervals throughout the study period. RESULTS: Anopheles gambiae s.l. and Anopheles funestus represented 95% of the collected anopheline species. In the valleys, where the vector control activities were implemented, Anopheles density was reduced by 82% (95% CI: 69-90). Similarly, transmission was decreased by 90% (95% CI: 63%-97%, p=0.001). In the sprayed valleys, Anopheles density was further reduced by 79.5% (95% CI: 51.7-91.3, p<0.001) in the houses with nets as compared to houses without them. No significant impact on vector density and malaria transmission was observed in the hill tops. However, the intervention focused on the high risk areas near the valley floor, where 93% of the vectors are found and 90% of the transmission occurs. CONCLUSIONS: Spatial targeted vector control effectively reduced Anopheles density and transmission in this highland district. Bed nets have an additional effect on Anopheles density though this did not translate in an additional impact on transmission. Though no impact was observed in the hilltops, the programme successfully covered the areas most at risk. Such a targeted strategy could prevent the emergence and spread of an epidemic from these high risk foci. [Abstract/Link to Full Text]

Duval L, Robert V, Csorba G, Hassanin A, Randrianarivelojosia M, Walston J, Nhim T, Goodman SM, Ariey F
Multiple host-switching of Haemosporidia parasites in bats.
Malar J. 2007 Nov 29;6(1):157.
ABSTRACT: BACKGROUND: There have been reported cases of host-switching in avian and lizard species of Plasmodium (Apicomplexa, Haemosporidia), as well as in those infecting different primate species. However, no evidence has previously been found for host-swapping between wild birds and mammals. METHODS: This paper presents the results of the sampling of blood parasites of wild-captured bats from Madagascar and Cambodia. The presence of Haemosporidia infection in these animals is confirmed and cytochrome b gene sequences were used to construct a phylogenetic analysis. RESULTS: Results reveal at least three different and independent Haemosporidia evolutionary histories in three different bat lineages from Madagascar and Cambodia. CONCLUSION: Phylogenetic analysis strongly suggests multiple host-switching of Haemosporidia parasites in bats with those from avian and primate hosts. [Abstract/Link to Full Text]

Vaillant M, Olliaro P
Geometric least squares means ratios for the analysis of Plasmodium falciparum in vitro susceptibility to antimalarial drugs.
Malar J. 2007 Nov 26;6(1):156.
ABSTRACT: BACKGROUND: The susceptibility of microbes such as Plasmodium falciparum to drugs is measured in vitro as the concentration of the drug achieving 50% of maximum effect (IC50); values from a population are summarized as geometric means. For antimalarial drugs, as well as for antibiotics, assessing changes in microbe susceptibility over time under drug pressure would help inform treatment policy decisions, but no standard statistical method exists as yet. METHODS: A mixed model was generated on log-transformed IC50 values and calculated geometric least squares means (GLSM) with 90% confidence intervals (CIs). In order to compare IC50s between years, GLSM ratios (GLSMR) with 90%CIs were calculated and, when both limits of the 90%CIs were below or above 100%, the difference was considered statistically significant. Results were compared to those obtained from ANOVA and a generalized linear model (GLM). RESULTS: GLSMRs were more conservative than ANOVA and resulted in lower levels of statistical significance. The GLSMRs approach allowed for random effect and adjustment for multiple comparisons. GLM was limited in the number of year-to-year comparisons by the need for a single reference year. The three analyses yielded generally consistent results. CONCLUSION: A robust analytical method can palliate inherent limitations of in vitro sensitivity testing. The random effects GLSMRs with adjustment for multiple comparisons and 90%CIs require only assumptions on the mixed model to be applied. Results are easy to display graphically and to interpret. The GLMSRs should be considered as an option for monitoring changes in drug susceptibility of P. falciparum malaria and other microbes. [Abstract/Link to Full Text]

Bass C, Williamson MS, Wilding CS, Donnelly MJ, Field LM
Identification of the main malaria vectors in the Anopheles gambiae species complex using a TaqMan real-time PCR assay.
Malar J. 2007 Nov 22;6(1):155.
ABSTRACT: BACKGROUND: The Anopheles gambiae sensu lato species complex comprises seven sibling species of mosquitoes that are morphologically indistinguishable. Rapid identification of the two main species which vector malaria, Anopheles arabiensis and An. gambiae sensu stricto, from the non-vector species Anopheles quadriannulatus is often required as part of vector control programmes. Currently the most widely used method for species identification is a multiplex PCR protocol that targets species specific differences in ribosomal DNA sequences. While this assay has proved to be reasonably robust in many studies, additional steps are required post-PCR making it time consuming. Recently, a high-throughput assay based on TaqMan single nucleotide polymorphism genotyping that detects and discriminates An. gambiae s.s and An. arabiensis has been reported. METHODS: A new TaqMan assay was developed that distinguishes between the main malaria vectors (An. arabiensis and An. gambiae s.s.) and the non-vector An. quadriannulatus after it was found that the existing TaqMan assay incorrectly identified An. quadriannulatus, An. merus and An. melas as An. gambiae s.s. The performance of this new TaqMan assay was compared against the existing TaqMan assay and the standard PCR method in a blind species identification trial of over 450 samples using field collected specimens from a total of 13 countries in Sub-Saharan Africa. RESULTS: The standard PCR method was found to be specific with a low number of incorrect scores (<1%), however when compared to the TaqMan assays it showed a significantly higher number of failed reactions (15%). Both the new vector-specific TaqMan assay and the exisiting TaqMan showed a very low number of incorrectly identified samples (0 and 0.54%) and failed reactions (1.25% and 2.96%). In tests of analytical sensitivity the new TaqMan assay showed a very low detection threshold and can consequently be used on a single leg from a fresh or silica-dried mosquito without the need to first extract DNA. CONCLUSION: This study describes a rapid and sensitive assay that very effectively identifies the two main malaria vectors of the An. gambiae species complex from the non-vector sibling species. The method is based on TaqMan SNP genotyping and can be used to screen single legs from dried specimens. In regions where An. merus/melas/bwambae, vectors with restricted distributions, are not present it can be used alone to discriminate vector from non-vector or in combination with the TaqMan assay to distinguish An. arabiensis and An. gambiae s.s. [Abstract/Link to Full Text]

Garg S, Alam MT, Das MK, Dev V, Kumar A, Dash AP, Sharma YD
Sequence diversity and natural selection at domain I of the apical membrane antigen 1 among Indian Plasmodium falciparum populations.
Malar J. 2007 Nov 22;6(1):154.
ABSTRACT: BACKGROUND: The Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate antigen. The complete AMA1 protein is comprised of three domains where domain I exhibits high sequence polymorphism and is thus named as the hyper-variable region (HVR). The present study describes the extent of genetic polymorphism and natural selection at domain I of the ama1 gene among Indian P. falciparum isolates. METHODS: The part of the ama1 gene covering domain I was PCR amplified and sequenced from 157 P. falciparum isolates collected from five different geographical regions of India. Statistical and phylogenetic analyses of the sequences were done using DnaSP ver. 4. 10. 9 and MEGA version 3.0 packages. RESULTS: A total of 57 AMA1 haplotypes were observed among 157 isolates sequenced. Forty-six of these 57 haplotypes are being reported here for the first time. The parasites collected from the high malaria transmission areas (Assam, Orissa, and Andaman and Nicobar Islands) showed more haplotypes (H) and nucleotide diversity p as compared to low malaria transmission areas (Uttar Pradesh and Goa). The comparison of all five Indian P. falciparum subpopulations indicated moderate level of genetic differentiation and limited gene flow (Fixation index ranging from 0.048 to 0.13) between populations. The difference between rates of non-synonymous and synonymous mutations, Tajima's D and McDonald-Kreitman test statistics suggested that the diversity at domain I of the AMA1 antigen is due to positive natural selection. The minimum recombination events were also high indicating the possible role of recombination in generating AMA1 allelic diversity. CONCLUSION: The level of genetic diversity and diversifying selection were higher in Assam, Orissa, and Andaman and Nicobar Islands populations as compared to Uttar Pradesh and Goa. The amounts of gene flow among these populations were moderate. The data reported here will be valuable for the development of AMA1-based malaria vaccine. [Abstract/Link to Full Text]

Enevold A, Nkya WM, Theisen M, Vestergaard LS, Jensen AT, Staalsoe T, Theander TG, Bygbjerg IC, Alifrangis M
Potential impact of host immunity on malaria treatment outcome in Tanzanian children infected with Plasmodium falciparum.
Malar J. 2007 Nov 16;6(1):153.
ABSTRACT: BACKGROUND: In malaria endemic areas children may recover from malaria after chemotherapy in spite of harbouring genotypically drug-resistant Plasmodium falciparum. This phenomenon suggests that there is a synergy between drug treatment and acquired immunity. This hypothesis was examined in an area of moderately intense transmission of P. falciparum in Tanzania during a drug trail with sulphadoxine-pyrimethamine (SP) or amodiaquine (AQ). METHODS: One hundred children with uncomplicated malaria were treated with either SP or AQ and followed for 28 days. Mutations in parasite genes related to SP and AQ-resistance as well as human sickle cell trait and alpha-thalassaemia were determined using PCR and sequence-specific oligonucleotide probes and enzyme-linked immunosorbent assay (SSOP-ELISA), and IgG antibody responses to a panel of P. falciparum antigens were assessed and related to treatment outcome. RESULTS: Parasitological or clinical treatment failure (TF) was observed in 68% and 38% of children receiving SP or AQ, respectively. In those with adequate clinical and parasitological response (ACPR) compared to children with TF, and for both treatment regimens, prevalence and levels of anti-Glutamate-rich Protein (GLURP)-specific IgG antibodies were significantly higher (P < 0.001), while prevalence of parasite haplotypes associated with SP and AQ resistance was lower (P = 0.02 and P = 0.07, respectively). Interestingly, anti-GLURP-IgG antibodies were more strongly associated with treatment outcome than parasite resistant haplotypes, while the IgG responses to none of the other 11 malaria antigens were not significantly associated with ACPR. CONCLUSIONS: These findings suggest that GLURP-specific IgG antibodies in this setting contribute to clearance of drug-resistant infections and support the hypothesis that acquired immunity enhances the clinical efficacy of drug therapy. The results should be confirmed in larger scale with greater sample size and with variation in transmission intensity. [Abstract/Link to Full Text]

Ndiaye JL, Tine RC, Faye B, Dieye EH, Diack PA, Lameyre V, Gaye O, Sow HD
Pilot feasibility study of an emergency paediatric kit for intra-rectal quinine administration used by the personnel of community-based health care units in Senegal.
Malar J. 2007 Nov 15;6(1):152.
ABSTRACT: BACKGROUND: Quinine injection is the reference treatment for malaria when oral administration is impossible. Quinine can also be administered by the intra-rectal route and, over the last ten years, a series of studies have been conducted in children to determine the ideal dose and dilution in the African situation. The aim of the present study was to evaluate the feasibility and usefulness of a kit for an immediate administration of quinine alkaloids (Quinimax(R)) by community health workers, prior to transfer of the child to a more sophisticated health care establishment. METHODS: A prospective, open, descriptive community intervention study conducted in northern Senegal at six village Health Units in children fewer than ten years of age with non-per-os malaria. Controls were given the routine care prior to transfer to a Health Center, and cases were in addition administered Quinimax(R) (20 mg/ml) via the intra-rectal route before transfer. Patients were followed through complete cure and parasitological tests were carried out on Days 0, 3 and 7. RESULTS: 134 patients (79 cases/55 controls) were recruited between November 2003 and May 2004 or October and November 2004. The two groups were comparable at inclusion. In the case group, oral drugs could be administered after a mean of 16.8 hours versus 33.6 hours in the control group. Time-to cure was shorter in cases than in controls. Complete parasite clearance was obtained in all patients by Day 7. The kit was well accepted by all concerned and more than 80% of community health workers judged the kit easy to use. CONCLUSION: The emergency paediatric kit is a useful tool in the management of malaria in children who cannot be treated orally. It is feasible and easy to use for health workers in community-based Health Units where, according to the WHO, nearly 80% of malarial morbidity and mortality occurs. [Abstract/Link to Full Text]

Okiro EA, Hay SI, Gikandi PW, Sharif SK, Noor AM, Peshu N, Marsh K, Snow RW
The decline in paediatric malaria admissions on the coast of Kenya.
Malar J. 2007 Nov 15;6(1):151.
ABSTRACT: BACKGROUND: There is only limited information on the health impact of expanded coverage of malaria control and preventative strategies in Africa. METHODS: Paediatric admission data were assembled over 8.25 years from three District Hospitals; Kilifi, Msambweni and Malindi, situated along the Kenyan Coast. Trends in monthly malaria admissions between January 1999 and March 2007 were analysed using several time-series models that adjusted for monthly non-malaria admission rates and the seasonality and trends in rainfall. RESULTS: Since January 1999, paediatric malaria admissions have significantly declined at all hospitals. This trend was observed against a background of rising or constant non-malaria admissions and unaffected by long-term rainfall throughout the surveillance period. By March 2007 the estimated proportional decline in malaria cases was 63% in Kilifi, 53% in Kwale and 28% in Malindi. Time-series models strongly suggest that the observed decline in malaria admissions was a result of malaria-specific control efforts in the hospital catchment areas. CONCLUSION: This study provides evidence of a changing disease burden on the Kenyan coast and that the most parsimonious explanation is an expansion in the coverage of interventions such as the use of insecticide-treated nets and the availability of anti-malarial medicines. While specific attribution to intervention coverage cannot be computed what is clear is that this area of Kenya is experiencing a malaria epidemiological transition. [Abstract/Link to Full Text]

Brasseur P, Agnamey P, Gaye O, Vaillant M, Taylor WR, Olliaro PL
Efficacy and safety of artesunate plus amodiaquine in routine use for the treatment of uncomplicated malaria in Casamance, southern Senegal.
Malar J. 2007 Nov 15;6(1):150.
ABSTRACT: BACKGROUND: There are no data on the long term use of an artemisinin combination treatment in moderate or high transmission areas of Africa. Methods and Findings Artesunate plus amodiaquine (AS+AQ) was used to treat slide-proven Plasmodium falciparum-infected patients of all ages in the Oussouye district, Casamance, Senegal, over a period of six years (2000 to 2005). Efficacy, by Kaplan Meier survival analysis (n=966), and safety (adverse event rates, n=752) were determined over 28 days. A weight-based dosing regimen was used for the loose tablets during 2000-2003 (n=731) and a commercially available co-blister was used during 2004-2005 (n=235). Annual crude (non PCR corrected) cure rates remained stable over the study period [range 88.5-96.7%; overall 94.6 (95% CI 92.9-95.9)]. Nine co-blister treated patients (0.9%) withdrew because of drug-related adverse events; seven had gastrointestinal complaints of whom two were hospitalized for vomiting. By Day 28, the mean total bilirubin (n=72), AST (n=94) and ALT (n=95) values decreased. Three patients had Day 28 AST/ALT values > 40 < 200 IU/L. Changes in white cell counts were unre-markable (n=87). CONCLUSION: AS+AQ in combination was highly efficacious and well-tolerated in this area and justi-fies the decision to use it as first line treatment. Long-term monitoring of safety and ef-ficacy should continue. [Abstract/Link to Full Text]

Luchavez J, Lintag ME, Coll-Black M, Baik F, Bell D
An assessment of various blood collection and transfer methods used for malaria rapid diagnostic tests.
Malar J. 2007 Nov 15;6(1):149.
ABSTRACT: BACKGROUND: Four blood collection and transfer devices commonly used for malaria rapid diagnostic tests (RDTs) were assessed for their consistency, accuracy and ease of use in the hands of laboratory technicians and village health workers. METHODS: Laboratory technicians and village health workers collected blood from a finger prick using each device in random order, and deposited the blood either on filter paper or into a suitable casette-type RDT. Consistency and accuracy of volume delivered was determined by comparing the measurements of the resulting blood spots/heights with the measurements of laboratory-prepared pipetted standard volumes. The effect of varying blood volumes on RDT sensitivity and ease of use was also observed. RESULTS: There was high variability in blood volume collected by the devices, with the straw and the loop, the most preferred devices, usually transferring volumes greater than intended, while the glass capillary tube and the plastic pipette transferring less volume than intended or none at all. Varying the blood volume delivered to RDTs indicated that this variation is critical to RDT sensitivity only when the transferred volume is very low. CONCLUSION: None of the blood transfer devices assessed performed consistently well. Adequate training on their use is clearly necessary, with more development efforts for improved designs to be used by remote health workers, in mind. [Abstract/Link to Full Text]

Zakeri S, Afsharpad M, Raeisi A, Djadid ND
Prevalence of mutations associated with antimalarial drugs in Plasmodium falciparum isolates prior to the introduction of sulphadoxine-pyrimethamine as first-line treatment in Iran.
Malar J. 2007 Nov 13;6(1):148.
ABSTRACT: BACKGROUND: This work was carried out to assess the patterns and prevalence of resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) in Iran. METHODS: The prevalence of pfcrt K76T, pfmdr1 N86Y, pfdhfr N51I, C59R, S108N/T and I164L and codons S436F/A, A437S, A437G, K540E, A581E, and A613S/T in pfdhps genes were genotyped by PCR/RFLP methods in 206 Plasmodium falciparum isolates from Chabahar and Sarbaz districts in Sistan and Baluchistan province, Iran, during 2003-2005. RESULTS: All P. falciparum isolates carried the 108N, while 98.5% parasite isolates carried the 59R mutation. 98.5% of patients carried both 108N and 59R. The prevalence of pfdhps 437G mutation was 17.1% (Chabahar) and 33% (Sarbaz) isolates. 20.4% of samples presented the pfdhfr 108N, 59R with pfdhps 437G mutations. The frequency of allele pfcrt 76T was 98%, while 41.4% (Chabahar) and 27.7% (Sarbaz) isolates carried pfmdr1 86Y allele. Eight distinct haplotypes were identified in all 206 samples, while the most prevalent haplotype was T76/N86/N51R59N108/A437 among both study areas. CONCLUSION: Finding the fixed level of CQ resistance polymorphisms (pfcrt 76T) suggests that CQ must be withdrawn from the current treatment strategy in Iran, while SP may remain the treatment of choice for uncomplicated malaria. [Abstract/Link to Full Text]

Armah HB, Wilson NO, Sarfo BY, Powell MD, Bond VC, Anderson W, Adjei AA, Gyasi RK, Tettey Y, Wiredu EK, Tongren JE, Udhayakumar V, Stiles JK
Cerebrospinal fluid and serum biomarkers of cerebral malaria mortality in Ghanaian children.
Malar J. 2007 Nov 12;6(1):147.
ABSTRACT: BACKGROUND: Plasmodium falciparum in a subset of patients can lead to a diffuse encephalopathy known as cerebral malaria (CM), a major contributor to malaria associated mortality in young children. This encephalopathy is associated with a 10-14% mortality rate. Despite treatment, mortality in CM can be as high as 30% while 10% of survivors of the disease may experience short- and long-term neurological complications. The pathogenesis of CM and other forms of severe malaria is multi-factorial and may involve cytokine and chemokine homeostasis, inflammation and vascular injury/repair, thus limiting the rate of discovery of prognostic predictors of severe malaria mortality. Identification of predictors of CM severity will enable the establishment of a prediction rule or severity index by clinicians to adjust this risk. METHODS: Postmortem serum and cerebrospinal fluid (CSF) samples were obtained within 2-4 hours of death in Ghanaian children dying of CM, severe malarial anemia (SMA), and non-malarial (NM) causes. Serum and CSF levels of 36 different biomarkers (IL-1beta, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, Eotaxin, FGF basic protein, CRP, G-CSF, GM-CSF, IFN-gamma, TNF-alpha, IP-10, MCP-1 (MCAF), MIP-1alpha, MIP-1beta, RANTES, SDF-1alpha, CXCL11 (I-TAC), Fas-ligand [Fas-L], soluble Fas [sFas], sTNF-R1 (p55), sTNF-R2 (p75), MMP-9, TGF-beta, PDGF bb and VEGF) were measured and the results compared between the three groups. RESULTS: After Bonferroni adjustment for other biomarkers, IP-10 was the only serum biomarker independently associated with CM mortality when compared to SMA and NM deaths. Eight CSF biomarkers (IL-1ra, IL-8, IP-10, PDGFbb, MIP-1beta, Fas-L, sTNF-R1, and sTNF-R2) were independently predictive of CM mortality when compared to SMA and NM deaths. Additionally, CSF IP-10/PDGFbb median ratio was statistically significantly higher in the CM group compared to the SMA and NM groups CONCLUSIONS: The parasite-induced local cerebral dysregulation in the production of IP-10, 1L-8, MIP-1beta, PDGFbb, IL-1ra, Fas-L, sTNF-R1, and sTNF-R2 may be involved in CM neuropathology, and their immunoassay may have potential utility in predicting mortality in CM. [Abstract/Link to Full Text]

Kabanywanyi AM, Mwita A, Sumari D, Mandike R, Mugittu K, Abdulla S
Efficacy and safety of artemisinin-based antimalarial in the treatment of uncomplicated malaria in children in southern Tanzania.
Malar J. 2007 Nov 11;6(1):146.
ABSTRACT: BACKGROUND: Tanzania switched the antimalarial first line to sulphadoxine-pyrimethamine (SP) in 2001 from ineffective chloroquine (CQ). By 2003 higher levels of SP resistance were recorded, prompting an urgent need for replacing the first line drug with ACT, as currently recommended by the World Health Organization. Despite this recommendation country-specific evidence-based data to support efficacy and safety profile of ACT is still limited. A study on the efficacy and safety of artesunate plus amodiaquine (AS+AQ) and artemether plus lumefantrine (AL)(Coartem(R)) was carried out in 2004 with the view of supporting the National Malaria Control Programme in the review of the policy in mainland Tanzania. METHODS: An in vivo efficacy study was conducted at Ipinda and Mlimba health facilities between May and November 2004. The study recruited children aged 6-59 months presenting with symptoms of uncomplicated malaria, history of fever or an axillary temperature [greater than or equal to]37.5oC; mono infection with Pasmodium falciparum (2,000-200,000 parasites/ul). Patients were randomized to received either SP or amodiaquine monotherapy or treated with standard doses of AS+AQ in Mlimba and Coartem in Kyela and followed-up for 28 days to assess treatment responses. This study reports results of the combination therapies. RESULTS: A total of 157 children (76 in Mlimba and 99 in Kyela) who were enrolled in to the study and treated with either AL or AS+AQ were successfully followed-up. Both combinations were tolerated and effected rapid fever and parasite clearance. The crude ACPRs were 80 (87 %) and 41 (63%) for AL and AS+AQ respectively. However, after PCR adjustments the corresponding figures raised to 100% (n=86) and 93.8% (n=45) in AL and AS+AQ groups, respectively. The mean haemoglobin improved moderately from day 0 to day 28 by 1g/dl in AL and 0.4g/dl in AS+AQ treatment group and was statistically significant (p<0.001 both). CONCLUSION: These findings provide substantial evidence that AL is highly efficacious in areas of high resistance of SP and supported the country's decision to switch from SP monotherapy to AL. [Abstract/Link to Full Text]

Molyneux S, Gikonyo C, Marsh V, Bejon P
Incorporating a quiz into informed consent processes: qualitative study of participants reactions.
Malar J. 2007 Nov 10;6(1):145.
ABSTRACT: BACKGROUND: Formal checks of participant understanding are now widely recommended to improve informed consent processes. However, the views of the participants these assessments are designed to protect are rarely considered. In this paper the findings of a qualitative study aimed at documenting community reactions to a semi-structured questionnaire ('quiz') are reported. The quiz was administered to 189 mothers after consenting for their children to participate in a malaria vaccine trial on the Kenyan Coast. METHODS: Once the malaria vaccine trial was underway, focus group discussions were held with some of these mothers (nine groups; 103 mothers), and with community-based field staff attached to the malaria vaccine trial (two groups of five workers). Individual interviews with other trial staff were also held. RESULTS: The quiz prompted community members to voice concerns about blood sampling and vaccine side-effects, thereby encouraging additional discussions and interactions between the research team and potential study participants. However, it also caused significant upset and concern. Some of the quiz questions, or the way in which they were asked, appeared to fuel misconceptions and fears, with potentially negative consequences for both the study and community members. CONCLUSION: Formal approaches to checking study understanding should be employed with sensitivity and caution. They are influenced by and impact upon complex social relationships between and among researchers and community members. Adequate consideration of these contexts in assessments of understanding, and in responding to the issues raised, requires strong social science capacity. [Abstract/Link to Full Text]

Hommerich L, von Oertzen C, Bedu-Addo G, Holmberg V, Acquah PA, Eggelte TA, Bienzle U, Mockenhaupt FP
Decline of placental malaria in southern Ghana after the implementation of intermittent preventive treatment in pregnancy.
Malar J. 2007 Nov 8;6(1):144.
ABSTRACT: BACKGROUND: Intermittent preventive treatment in pregnancy with sulphadoxine-pyrimethamine (IPTp-SP) has been adopted as policy by many countries in sub-Saharan Africa. However, data on the post-implementation effectiveness of this measure are scarce. METHODS: Clinical and parasitological parameters were assessed among women delivering at a district hospital in rural southern Ghana in the year 2000 when pyrimethamine chemoprophylaxis was recommended (n = 839) and in 2006 (n = 226), approximately one year after the implementation of IPTp-SP. Examinations were performed in an identical manner in 2000 and 2006 including the detection of placental Plasmodium falciparum infection by microscopy, histidine-rich protein 2, and PCR. RESULTS: In 2006, 77% of the women reported to have taken IPTp-SP at least once (26%, twice; 24%, thrice). In 2006 as compared to 2000, placental P. falciparum infection was reduced by 43-57% (P < 0.0001) and maternal anaemia by 33% (P = 0.0009), and median birth weight was 130 g higher (P = 0.02). In 2006, likewise, women who had taken [greater than or equal to]1 dose of IPTp-SP revealed less infection and anaemia and their children tended to have higher birth weights as compared to women who had not used IPTp-SP. However, placental P. falciparum infection was still observed in 11% (microscopy) to 26% (PCR) of those women who had taken three doses of IPTp-SP. CONCLUSIONS: In southern Ghana, placental malaria and maternal anaemia have declined substantially and birth weight has increased after the implementation of IPTp-SP. Likely, these effects can further be increased by improving IPTp-SP coverage and adherence. However, the remnant prevalence of infection in women having taken three doses of IPTp-SP suggests that additional antimalarial measures are needed to prevent malaria in pregnancy in this region. [Abstract/Link to Full Text]

Malamba S, Hladik W, Reingold A, Banage F, McFarland W, Rutherford G, Mimbe D, Nzaro E, Downing R, Mermin J
The effect of HIV on morbidity and mortality in children with severe malarial anaemia.
Malar J. 2007 Oct 31;6(1):143.
ABSTRACT: BACKGROUND: Malaria and HIV are common causes of mortality in sub-Saharan Africa. The effect of HIV infection on morbidity and mortality in children with severe malarial anaemia was assessed. METHODS: Children <5 years old were followed as part of a prospective cohort study to assess the transfusion-associated transmission of blood-borne pathogens at Mulago Hospital, Kampala, Uganda. All children were hospitalized with a diagnosis of severe malarial anaemia requiring blood transfusion. Survival to different time points post-transfusion was compared between HIV-infected and uninfected children. Generalized estimating equations were used to analyse repeated measurement outcomes of morbidity, adjusting for confounders. RESULTS: Of 847 children, 78 (9.2%) were HIV-infected. Median follow-up time was 162 days (inter-quartile range: 111, 169). HIV-infected children were more likely to die within 7 days (Hazard ratio [HR] = 2.86, 95% Confidence interval [CI] 1.30-6.29, P=0.009) and within 28 days (HR = 3.70, 95% CI 1.91-7.17, P<0.001) of an episode of severe malarial anaemia, and were more likely to die in the 6 months post-transfusion (HR = 5.70, 95% CI 3.54-9.16, P<0.001) compared to HIV-uninfected children. HIV-infected children had more frequent re-admissions due to malaria within 28 days (Incidence rate ratio (IRR) = 3.74, 95% CI 1.41-9.90, P = 0.008) and within 6 months (IRR = 2.66, 95% CI 1.17 - 6.07, P = 0.02) post-transfusion than HIV-uninfected children. CONCLUSION: HIV-infected children with severe malarial anaemia suffered higher all-cause mortality and malaria-related mortality than HIV-uninfected children. Children with HIV and malaria should receive aggressive treatment and further evaluation of their HIV disease, particularly with regard to cotrimoxazole prophylaxis and antiretroviral therapy. [Abstract/Link to Full Text]

Casimiro SL, Hemingway J, Sharp BL, Coleman M
Monitoring the operational impact of insecticide usage for malaria control on Anopheles funestus from Mozambique.
Malar J. 2007 Oct 31;6(1):142.
ABSTRACT: BACKGROUND: Indoor residual spraying (IRS) has again become popular for malaria control in Africa. This combined with the affirmation by WHO that DDT is appropriate for use in the absence of longer lasting insecticide formulations in some malaria endemic settings, has resulted in an increase in IRS with DDT as a major malaria vector control intervention in Africa. DDT was re-introduced into Mozambique's IRS programme in 2005 and is increasingly becoming the main insecticide used for malaria vector control in Mozambique. The selection of DDT as the insecticide of choice in Mozambique is evidence-based, taking account of the susceptibility of Anopheles funestus to all available insecticide choices, as well as operational costs of spraying. Previously lambda cyhalothrin had replaced DDT in Mozambique in 1993. However, resistance appeared quickly to this insecticide and, in 2000, the pyrethroid was phased out and the carbamate bendiocarb introduced. Low level resistance was detected by biochemical assay to bendiocarb in 1999 in both An. funestus and Anopheles arabiensis, although this was not evident in WHO bioassays of the same population. METHOD: Sentinel sites were established and monitored for insecticide resistance using WHO bioassays. These assays were conducted on 1-3 day old F1 offspring of field collected adult caught An. funestus females to determine levels of insecticide resistance in the malaria vector population. WHO biochemical assays were carried out to determine the frequency of insecticide resistance genes within the same population. RESULTS: In surveys conducted between 2002 and 2006, low levels of bendiocarb resistance were detected in An. funestus, populations using WHO bioassays. This is probably due to significantly elevated levels of acetylcholinesterase levels found in the same populations. Pyrethroid resistance was also detected in populations and linked to elevated levels of p450 monooxygenase activity. One site had shown reduction in pyrethroid resistance since the base line in 1999. [Abstract/Link to Full Text]

Cano J, Berzosa P, de Lucio A, Descalzo MA, Bobuakasi L, Nzambo S, Ondo M, Buatiche JN, Nseng G, Benito A
Transmission of malaria and genotypic variability of Plasmodium falciparum on the Island of Annobon (Equatorial Guinea).
Malar J. 2007 Oct 25;6(1):141.
ABSTRACT: BACKGROUND: Malaria transmission in Equatorial Guinea and its space-time variability has been widely studied, but there is not much information about the transmission of malaria on the small island of Annobon. In 2004, two transversal studies were carried out to establish the malaria transmission pattern on Annobon and analyse the circulating Plasmodium falciparum allelic forms. METHODS: A blood sample was taken from the selected children in order to determine Plasmodium infection by microscopical examination and by semi-nested multiplex PCR. The diversity of P. falciparum circulating alleles was studied on the basis of the genes encoding for the merozoite surface proteins, MSP-1 and MSP-2 of P. falciparum. RESULTS: The crude parasite rate was 17% during the dry season and 60% during the rainy season. The percentage of children sleeping under a bed net was over 80% in the two surveys. During the rainy season, 33.3% of the children surveyed were anaemic at the time of the study. No association was found between the crude parasite rate, the use of bed nets and gender, and anaemia. However, children between five and nine years of age were five times less at risk of being anaemic than those aged less than one year. A total of 28 populations of the three allelic families of the msp-1 gene were identified and 39 of the msp-2 gene. The variability of circulating allelic populations is significantly higher in the rainy than in the dry season, although the multiplicity of infections is similar in both, 2.2 and 1.9 respectively. CONCLUSIONS: Based on the high degree of geographical isolation of the Annobon population and the apparent marked seasonality of the transmission, it is feasible to believe that malaria can be well controlled from this small African island. [Abstract/Link to Full Text]

Talman AM, Duval L, Legrand E, Hubert V, Yen S, Bell D, Le Bras J, Ariey F, Houze S
Evaluation of the intra- and inter-specific genetic variability of Plasmodium lactate dehydrogenase.
Malar J. 2007 Oct 25;6(1):140.
ABSTRACT: BACKGROUND: Malaria diagnosis is vital to efficient control programmes and the recent advent of malaria rapid diagnostic tests (RDTs) provides a reliable and simple diagnostic method. However, a characterization of the efficiency of these tests and the proteins they detect is needed to maximize RDT sensitivity. METHODS: Plasmodial lactate dehydrogenase (pLDH) gene of wild isolates of the four human species of Plasmodium from a variety of malaria endemic settings were sequenced and analysed. RESULTS: No variation in nucleotide was found within Plasmodium falciparum, synonymous mutations were found for Plasmodium malariae and Plasmodium vivax; three different types of amino acid sequence were found for Plasmodium ovale. Conserved and variable regions were identified within each species. CONCLUSIONS: The results indicate that antigen variability is unlikely to explain variability in performance of RDTs detecting pLDH from cases of P. falciparum, P. vivax or P. malariae malaria, but may contribute to poor detection of P. ovale. [Abstract/Link to Full Text]

Hume JC, Tunnicliff M, Ranford-Cartwright LC, Day KP
Susceptibility of Anopheles gambiae and Anopheles stephensi to tropical isolates of Plasmodium falciparum.
Malar J. 2007 Oct 24;6(1):139.
ABSTRACT: BACKGROUND: The susceptibility of anopheline mosquito species to Plasmodium infection is known to be variable with some mosquitoes more permissive to infection than others. Little work, however, has been carried out investigating the susceptibility of major malaria vectors to geographically diverse tropical isolates of Plasmodium falciparum aside from examining the possibility of infection extending its range from tropical regions into more temperate zones. METHODS: This study investigates the susceptibility of two major tropical mosquito hosts (Anopheles gambiae and Anopheles stephensi) to P. falciparum isolates of different tropical geographical origins. Cultured parasite isolates were fed via membrane feeders simultaneously to both mosquito species and the resulting mosquito infections were compared. RESULTS: Infection prevalence was variable with African parasites equally successful in both mosquito species, Thai parasites significantly more successful in An. stephensi, and PNG parasites largely unsuccessful in both species. CONCLUSIONS: Infection success of P. falciparum was variable according to geographical origin of both the parasite and the mosquito. Data presented raise the possibility that local adaptation of tropical parasites and mosquitoes has a role to play in limiting gene flow between allopatric parasite populations. [Abstract/Link to Full Text]

Namutangula B, Ndeezi G, Byarugaba JS, Tumwine JK
Mannitol as adjunct therapy for childhood cerebral malaria in Uganda: a randomized clinical trial.
Malar J. 2007 Oct 24;6(1):138.
ABSTRACT: BACKGROUND: Several reports have suggested that raised intracranial pressure (ICP) is a major contributor to death among children with cerebral malaria. Mannitol, an osmotic diuretic, effectively lowers ICP and is used to treat post-traumatic raised ICP. It is not clear whether intravenous mannitol given to children with cerebral malaria improves clinical outcome. The objective of this study was to determine the effect of mannitol as adjunct therapy on the clinical outcome of children with cerebral malaria. METHODS: This randomized double-blind placebo controlled clinical trial was carried out at the Emergency Paediatric ward of Mulago Hospital, Uganda's national referral and teaching hospital. One hundred and fifty six children aged 6 to 60 months with cerebral malaria were randomized to either one dose of mannitol 1g/kg or placebo, in addition to intravenous quinine. Main outcome measures included coma recovery time; time to sit unsupported, begin oral intake; duration of hospitalization; death and adverse effects. RESULTS: Time to regain consciousness (p=0.11), sit unsupported (p=0.81), time to start oral intake (p=0.13) and total coma duration (p=0.07) were similar in both groups. There was no significant difference in the mortality between the placebo (13/80 or 16.3%) and mannitol (10/76 or 13.2%) groups: RR=1.2 (CI 0.5-2.7). No adverse effects were observed after administration of mannitol. CONCLUSIONS: Mannitol had no significant impact on clinical outcome of cerebral malaria. It is difficult to recommend intravenous mannitol as adjunct therapy for childhood cerebral malaria. Clinical registration number: ClinicalTrials.gov ID: NCT00113854. [Abstract/Link to Full Text]

Moreno M, Salgueiro P, Vicente JL, Cano J, Berzosa PJ, de Lucio A, Simard F, Caccone A, Do Rosario VE, Pinto J, Benito A
Genetic population structure of Anopheles gambiae in Equatorial Guinea.
Malar J. 2007;6137.
BACKGROUND: Patterns of genetic structure among mosquito vector populations in islands have received particular attention as these are considered potentially suitable sites for experimental trials on transgenic-based malaria control strategies. In this study, levels of genetic differentiation have been estimated between populations of Anopheles gambiae s.s. from the islands of Bioko and Annobón, and from continental Equatorial Guinea (EG) and Gabon. METHODS: Genotyping of 11 microsatellite loci located in chromosome 3 was performed in three island samples (two in Bioko and one in Annobón) and three mainland samples (two in EG and one in Gabon). Four samples belonged to the M molecular form and two to the S-form. Microsatellite data was used to estimate genetic diversity parameters, perform demographic equilibrium tests and analyse population differentiation. RESULTS: High levels of genetic differentiation were found between the more geographically remote island of Annobón and the continent, contrasting with the shallow differentiation between Bioko island, closest to mainland, and continental localities. In Bioko, differentiation between M and S forms was higher than that observed between island and mainland samples of the same molecular form. CONCLUSION: The observed patterns of population structure seem to be governed by the presence of both physical (the ocean) and biological (the M-S form discontinuity) barriers to gene flow. The significant degree of genetic isolation between M and S forms detected by microsatellite loci located outside the "genomic islands" of speciation identified in A. gambiae s.s. further supports the hypothesis of on-going incipient speciation within this species. The implications of these findings regarding vector control strategies are discussed. [Abstract/Link to Full Text]

Mozumder P, Marathe A
Role of information and communication networks in malaria survival.
Malar J. 2007;6136.
BACKGROUND: Quite often symptoms of malaria go unrecognized or untreated. According to the Multilateral Initiative on Malaria, 70% of the malaria cases that are treated at home are mismanaged. Up to 82% of all malaria episodes in sub-Saharan Africa are treated outside the formal health sector. Fast and appropriate diagnosis and treatment of malaria is extremely important in reducing morbidity and mortality. METHOD: Data from 70 different countries is pooled together to construct a panel dataset of health and socio-economic variables for a time span of (1960-2004). The generalized two-stage least squares and panel data models are used to investigate the impact of information and communication network (ICN) variables on malaria death probability. The intensity of ICN is represented by the number of telephone main lines per 1,000 people and the number of television sets per 1,000 people. RESULTS: The major finding is that the intensity of ICN is associated with reduced probability of deaths of people that are clinically identified as malaria infected. The results are robust for both indicators i.e. interpersonal and mass communication networks and for all model specifications examined. CONCLUSION: The results suggest that information and communication networks can substantially scale up the effectiveness of the existing resources for malaria prevention. Resources spent in preventing malaria are far less than needed. Expanded information and communication networks will widen the avenues for community based "participatory development", that encourages the use of local information, knowledge and decision making. Timely information, immediate care and collective knowledge based treatment can be extremely important in reducing child mortality and achieving the millennium development goal. [Abstract/Link to Full Text]

Jiang G, Charoenvit Y, Moreno A, Baraceros MF, Banania G, Richie N, Abot S, Ganeshan H, Fallarme V, Patterson NB, Geall A, Weiss WR, Strobert E, Caro-Aquilar I, Lanar DE, Saul A, Martin LB, Gowda K, Morrissette CR, Kaslow DC, Carucci DJ, Galinski MR, Doolan DL
Induction of multi-antigen multi-stage immune responses against Plasmodium falciparum in rhesus monkeys, in the absence of antigen interference, with heterologous DNA prime/poxvirus boost immunization.
Malar J. 2007 Oct 9;6(1):135.
ABSTRACT: The present study has evaluated the immunogenicity of single or multiple Plasmodium falciparum (Pf) antigens administered in a DNA prime/poxvirus boost regimen with or without the poloxamer CRL1005 in rhesus monkeys. Animals were primed with PfCSP plasmid DNA or a mixture of PfCSP, PfSSP2/TRAP, PfLSA1, PfAMA1 and PfMSP1-42 (CSLAM) DNA vaccines in PBS or formulated with CRL1005, and subsequently boosted with ALVAC-Pf7, a canarypox virus expressing the CSLAM antigens. Cell-mediated immune responses were evaluated by IFN-gamma ELIspot and intracellular cytokine staining, using recombinant proteins and overlapping synthetic peptides. Antigen-specific and parasite-specific antibody responses were evaluated by ELISA and IFAT, respectively. Immune responses to all components of the multi-antigen mixture were demonstrated following immunization with either DNA/PBS or DNA/CRL1005, and no antigen interference was observed in animals receiving CSLAM as compared to PfCSP alone. These data support the down-selection of the CSLAM antigen combination. CRL1005 formulation had no apparent effect on vaccine-induced T cell or antibody responses, either before or after viral boost. In high responder monkeys, CD4+IL-2+ responses were more predominant than CD8+ T cell responses. Furthermore, CD8+ IFN-gamma responses were detected only in the presence of detectable CD4+ T cell responses. Overall, this study demonstrates the potential for multivalent Pf vaccines based on rational antigen selection and combination, and suggests that further formulation development to increase the immunogenicity of DNA encoded antigens is warranted. [Abstract/Link to Full Text]

Hopkins H, Talisuna A, Whitty CJ, Staedke SG
Impact of home-based management of malaria on health outcomes in Africa: a systematic review of the evidence.
Malar J. 2007 Oct 8;6(1):134.
ABSTRACT: BACKGROUND: Home-based management of malaria (HMM) is promoted as a major strategy to improve prompt delivery of effective malaria treatment in Africa. HMM involves presumptively treating febrile children with pre-packaged antimalarial drugs distributed by members of the community. HMM has been implemented in several African countries, and artemisinin-based combination therapies (ACTs) will likely be introduced into these programmes on a wide scale. Case presentations: The published literature was searched for studies that evaluated the health impact of community- and home-based treatment for malaria in Africa. Criteria for inclusion were: 1) the intervention consisted of antimalarial treatment administered presumptively for febrile illness; 2) the treatment was administered by local community members who had no formal education in health care; 3) measured outcomes included specific health indicators such as malaria morbidity (incidence, severity, parasite rates) and/or mortality; and 4) the study was conducted in Africa. Of 1,069 potentially relevant publications identified, only six studies, carried out over 18 years, were identified as meeting inclusion criteria. Heterogeneity of the evaluations, including variability in study design, precluded meta-analysis. Discussion and evaluation: All trials evaluated presumptive treatment with chloroquine and were conducted in rural areas, and most were done in settings with seasonal malaria transmission. Conclusions regarding the impact of HMM on morbidity and mortality endpoints were mixed. Two studies showed no health impact, while another showed a decrease in malaria prevalence and incidence, but no impact on mortality. One study in Burkina Faso suggested that HMM decreased the proportion of severe malaria cases, while another study from the same country showed a decrease in the risk of progression to severe malaria. Of the four studies with mortality endpoints only one from Ethiopia showed a positive impact, with a reduction in the under-5 mortality rate of 40.6% (95% CI 29.2 - 50.6). CONCLUSIONS: Currently the evidence base for HMM in Africa, particularly regarding use of ACTs, is narrow and priorities for further research are discussed. To optimize treatment and maximize health benefits, drug regimens and delivery strategies in HMM programmes may need to be tailored to local conditions. Additional research could help guide programme development, policy decision-making, and implementation. [Abstract/Link to Full Text]