Showing papers in "Microbiology and Immunology in 2007"

Interleukin-17 as an effector molecule of innate and acquired immunity against infections.

Abstract: Interleukin (IL)-17 is a proinflammatory cytokine which induces differentiation and migration of neutrophils through induction of cytokines and chemokines including granulocyte-colony stimulating factor and CXCL8/IL-8. IL-17-producing CD4(+) T cells (Th17) have pivotal role in pathogenesis of autoimmune diseases. IL-17 is also involved in protective immunity against various infections. IL-17 has important role in induction of neutrophil-mediated protective immune response against extracellular bacterial or fungal pathogens such as Klebsiella pneumoniae and Candida albicans. Importance of IL-17 in protection against intracellular pathogens including Mycobacterium has also been reported. Interestingly, not only CD4(+) T cells but atypical CD4(-)CD8(-) T cells expressing T cell receptor (TCR) gammadelta produce IL-17, and IL-17 producing cells participate in both innate and acquired immune response to infections. Furthermore, neutrophil induction may not be the only mechanism of IL-17-mediated protective immunity. IL-17 seems to participate in host defense through regulation of cell-mediated immunity or induction of antimicrobial peptides such as beta-defensins. In this review, we summarize recent progress on the role of IL-17 in immune response against infections, and discuss possible application of IL-17 in prevention and treatment of infectious diseases.

Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: Epidemiological and molecular aspects

Abstract: Infections caused by the genus Staphylococcus are of great importance for human health. Staphylococcus species are divided into coagulase-positive staphylococci, represented by S. aureus, a pathogen that can cause infections of the skin and other organs in immunocompetent patients, and coagulase-negative staphylococci (CNS) which comprise different species normally involved in infectious processes in immunocompromised patients or patients using catheters. Oxacillin has been one of the main drugs used for the treatment of staphylococcal infections; however, a large number of S. aureus and CNS isolates of nosocomial origin are resistant to this drug. Methicillin resistance is encoded by the mecA gene which is inserted in the SCC mec cassette. This cassette is a mobile genetic element consisting of five different types and several subtypes. Oxacillin-resistant strains are detected by phenotypic and genotypic methods. Epidemiologically, methicillin-resistant S. aureus strains can be divided into five large pandemic clones, called Brazilian, Hungarian, Iberian, New York/Japan and Pediatric. The objective of the present review was to discuss aspects of resistance, epidemiology, genetics and detection of oxacillin resistance in Staphylococcus spp., since these microorganisms are increasingly more frequent in Brazil.

Dectin-1 is not required for the host defense to Cryptococcus neoformans.

Abstract: Dectin-1 is known as a sole receptor for β-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.

Photoactivated Ethidium Monoazide Directly Cleaves Bacterial DNA and Is Applied to PCR for Discrimination of Live and Dead Bacteria

Abstract: Ethidium monoazide (EMA) is a DNA intercalating agent and a eukaryotic topoisomerase II poison. We found that EMA treatment and subsequent visible light irradiation (photoactivation or photolysis) shows a bactericidal effect, hence the mechanism was analyzed. When bacterial cells were treated with more than 10 μg/ml of EMA for 1 hr plus photoactivation for 20 min, cleavage of bacterial DNA was confirmed by agarose gel electrophoresis and electron microscopic studies. The cleavage of chromosomal DNA was seen when it was treated in vitro with EMA and photolysis, which showed that the cleavage directly took place without the assistance of DNA gyrase/topoisomerase IV and the DNA repair enzymes of bacteria. It was also verified, by using negatively supercoiled pBR322 DNA, that medium/high concentrations of EMA (1 to 100 μg/ml) led to breaks of double-stranded DNA and that low concentrations of EMA (10 to 100 ng/ml) generated a single-stranded break. EMA is known to easily penetrate dead but not live bacteria. After treatment of 10 μg/ml of EMA for 30 min and photoactivation for 5 min, EMA cleaved the DNA of dead but not live Klebsiella oxytoca. When the cleaved DNA was used for templates in PCR targeting 16S rDNA, PCR product from the dead bacteria was completely suppressed. We demonstrated that EMA and photolysis directly cleaved bacterial DNA and are effective tools for discriminating live from dead bacteria by PCR.

The relationship between microbiota and polyamine concentration in the human intestine: a pilot study.

Abstract: The fecal microbiota of 10 hospitalized elderly subjects and 14 healthy adults were analyzed by terminal-restriction fragment length polymorphism (T-RFLP) analysis using Hha I, Msp I, Hae III, and Alu I, as well as fecal polyamine (PA) concentration. The T-RFLP profiles of the fecal microbiota of the subjects were roughly divided into 2 clusters-I (9 out of 11 were derived from hospitalized elderly subjects) and II (12 out of 13 were derived from healthy adults). The average concentration of putrescine in Cluster II was 5.8 times higher than that of putrescine in Cluster I (P=0.0015). Using a phylogenetic assignment database for T-RFLP analysis of human colonic microbiota, the terminal-restriction fragments (T-RFs) characteristically detected in the case of subjects with high fecal PA concentration were predicted to be derived from bacterial species and phylotypes belonging to Clostridium subcluster XIVa, particularly including Clostridium xylanolyticum, Clostridium saccharolyticum, the uncultured human intestinal bacterium clone JW1H4 (a relative of Desulfotomaculum guttoideum), Roseburia intestinalis, the uncultured bacterium clone 41F10 (a relative of Eubacterium ramulus), Roseburia cecicola, Ruminococcus obeum and its relatives. From these results, we concluded that fecal microbiota may be linked with fecal PA concentration and that some bacterial species belonging to Clostridium subcluster XIVa may play a major role in the control of intestinal PA concentration in humans.

Detection of Enterotoxin and Toxic Shock Syndrome Toxin 1 Genes in Staphylococcus, with Emphasis on Coagulase-Negative Staphylococci

Abstract: The detection of staphylococcal enterotoxins is decisive for the confirmation of an outbreak and for the determination of the enterotoxigenicity of strains. Since the recognition of their antigenicity, a large number of serological methods for the detection of enterotoxins in food and culture media have been proposed. Since immunological methods require detectable amounts of toxin, molecular biology techniques represent important tools in the microbiology laboratory. In the present study, polymerase chain reaction (PCR) was used to identify genes responsible for the production of enterotoxins and toxic shock syndrome toxin 1 (TSST-1) in S. aureus and coagulase-negative staphylococci (CNS) isolated from patients and the results were compared with those obtained by the reverse passive latex agglutination (RPLA) assay. PCR detection of toxin genes revealed a higher percentage of toxigenic S. aureus strains (46.7%) than the RPLA method (38.3%). Analysis of the toxigenic profile of CNS strains showed that 26.7% of the isolates produced some type of toxin, and one or more toxin-specific genes were detected in 40% of the isolates. These results suggests the need for further studies in order to better characterize the pathogenic potential of CNS and indicate that attention should be paid to the toxigenic capacity of this group of microorganisms.

Genotyping and quantitation of noroviruses in oysters from two distinct sea areas in Japan.

Abstract: Norovirus (NV) is a causative agent of acute gastroenteritis in humans, and shellfishes including oysters act as major vehicles of the virus. To investigate the genetic characteristics of NVs, we collected 1,512 oysters for raw consumption between October 2002 and March 2005 from two distinct areas (area A: the Sanriku Sea area; area B: the Setouchi Sea area). We detected the capsid gene and subjected it to phylogenetic analysis. By further quantification of the copy number of the genome by using real-time PCR, the NV capcid gene was detected in approximately 5% of the oysters, and they showed wide diversity. Two percent of the oysters from area B showed relatively large number of NVs, i.e., over 100 copies of capsid gene/oyster, whereas this was not observed in area A. Most of the detected NVs from oysters and humans were genetically related when the capsid region was compared. These results suggested that NVs obtained from humans and those obtained from oysters showed a potential relationship to each other and that some populations of Japanese oysters accumulated a relatively large number of NVs.

Identification of a Porphyromonas gingivalis Novel Protein Sov Required for the Secretion of Gingipains

Abstract: Gingipains are extracellular proteases important for the virulence of Porphyromonas gingivalis; however, the mechanism for the secretion of gingipains is poorly understood. In this report, we found that insertion mutants for PG0809 (83K1 and 83K2) were defective in black pigmentation and hemolysis. We cloned and sequenced PG0809 and found that PG0809 contains two additional nucleotides that are not deposited in the W83 genome database. The revised sequence reveals an in-frame fusion of PG0810 and PG0809 and is designated the sov gene. We constructed a sov deletion mutant (83K3) and showed that 83K3 was defective in the activities of black pigmentation, hemolysis, and hemagglutination. Furthermore, in 83K3, the activities of gingipains were severely reduced whereas those of other secreted proteases DPPIV, DPP-7, and PtpA were not affected. Immunoblot analysis using anti-RgpB antiserum showed that Arg-gingipains were poorly secreted in an outer membrane or into an extracellular portion but accumulated within the cells of 83K3, suggesting the secretion of gingipains is defected in 83K3. Taken together, our findings indicated that Sov is a novel protein required for the secretion of gingipains and suggested that the secretion system for gingipains is different from the conserved secretion systems.

WT1 (Wilms' Tumor 1) Peptide Immunotherapy for Renal Cell Carcinoma

Abstract: Tumor-specific immunotherapy with a Wilms' tumor 1 (WT1) peptide has been on clinical trial for leukemia, myelodysplastic syndrome, breast and lung cancers and is producing promising results. In this study, we treated three patients with renal cell carcinoma with an anchor modified, HLA-A*2402 binding WT1 peptide which was emulsified in Freund's incomplete adjuvant. In two patients tumor growth was suppressed and clinical response was evaluated as stable disease by the RECIST criteria after 3 months of weekly immunizations. Notably, development of new metastases has stopped in these patients for a prolonged period. No deleterious side effects were observed. Peptide-specific T cells were expanded in PBMCs of the patients and a substantial fraction of them bore the surface phenotype consistent with a CD8+ cytotoxic effector population. Although established tumors did not regress further, considering the component of the vaccine, i.e. peptide alone, the stabilization effect suggested the potential of WT1 peptide to develop into a more effective vaccine. To our knowledge, this is the first report of WT1 immunotherapy for renal cell carcinoma. Hopefully, the results will stimulate more extensive clinical studies.

Biofilm Acts as a Microenvironment for Plankton-Associated Vibrio cholerae in the Aquatic Environment of Bangladesh

Abstract: The role of biofilm as a microenvironment of plankton-associated Vibrio cholerae was investigated using plexiglass as a bait A total of 72 biofilm samples were tested using culture, direct fluorescent antibody (DFA) and molecular techniques following standard procedures. Culturable V. cholerae (smooth and rugose variants) were isolated from 33% of the samples. V. cholerae O1 were detected by FA technique throughout the year except April and June. All V. cholerae O1 isolates were positive for tcpA, ctxA and ace genes while V. cholerae non-O1, non-O139 isolates lacked these genes. V. cholerae O1 (both Inaba and Ogawa) strains had identical ribotype pattern (R1), but V. cholerae non-O1, non-O139 had different ribotype patterns. All V. cholerae O1 strains were resistant to vibrio-static compound (O/129). All V. cholerae O1 except one were resistant to trimethoprime-sulphamethoxazole, streptomycin, nalidixic acid and furazolidone but sensitive to ciprofloxacin, and tetracycline. This study indicates that plexiglass can act as a bait to form biofilm, a microenvironment that provides shelter for plankton containing V. cholerae in the aquatic environment of Bangladesh.

Molecular Survey of Babesia microti, Ehrlichia Species and Candidatus Neoehrlichia mikurensis in Wild Rodents from Shimane Prefecture, Japan

Abstract: A significant number of patients are diagnosed with "fevers of unknown origin" (FUO) in Shimane Prefecture in Japan where tick-borne diseases are endemic. We conducted molecular surveys for Babesia microti, Ehrlichia species, and Candidatus Neoehrlichia mikurensis in 62 FUO cases and 62 wild rodents from Shimane Prefecture, Japan. PCR using primers specific for the Babesia 18S small-subunit rRNA (rDNA) gene and Anaplasmataceae groESL amplified products from 45% (28/62) and 25.8% (16/62) of captured mice, respectively. Of the 28 18S rDNA PCR positives, 23 and five samples were positive for Hobetsu- and Kobe-type B. microti, respectively. In contrast, of the 16 groESL PCR positives, eight, one and seven samples were positive for Ehrlichia muris, Ehrlichia sp. HF565 and Candidatus N. mikurensis, respectively. Inoculation of selected blood samples into Golden Syrian hamsters indicated the presence of Hobetsu- and Kobe-type B. microti in four and one sample, respectively. Isolation of the latter strain was considered important as previous studies suggested that the distribution of this type was so far confined to Awaji Island in Hyogo Prefecture, where the first case of transfusion-associated human babesiosis originated. DNA samples from 62 FUO human cases tested negative for B. microti 18S rDNA gene, Anaplasmataceae groESL gene, Rickettsia japonica 17K genus-common antigen gene and Orientia tsutsugamushi 56K antigen gene by PCRs. We also conducted seroepidemiological surveys on 62 human sera collected in Shimane Prefecture from the FUO patients who were suspected of carrying tick-borne diseases. However, indirect immunofluorescent antibody tests using B. microti- and E. muris-infected cells detected IgG against E. muris in only a single positive sample. This study demonstrates the presence of several potentially important tick-borne pathogens in Shimane Prefecture and suggests the need for further study on the causative agents of FUOs.

Immunological Actions of Sophy β-Glucan (β-1,3-1,6 Glucan), Currently Available Commercially as a Health Food Supplement

Abstract: We examined the immunological actions of Sophy β-glucan (Ikewaki N., et al. United States Patent 6956120 and Japan Patent 2004-329077), a type of β-1,3–1,6 glucan produced by the black yeast Aureobasidium pullulans (A. pullulans) strain AFO-202, currently available commercially as a health food supplement, using different human in vitro experimental systems. Sophy β-glucan significantly (P<0.01) stimulated the 3H-thymidine incorporation rates (marker of DNA synthesis) in human peripheral blood mononuclear cells (PBMCs) obtained from normal adult donors, in vitro. Enzyme-linked immunoassays (EIAs) revealed that Sophy β-glucan stimulated the production of interleukin-8 (IL-8) or soluble Fas (sFas), but not that of IL-1β, IL-2, IL-6, IL-12 (p70+40), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) or soluble Fas ligand (sFasL), in either cultured PBMCs or cells of the human monocyte-like cell line, U937. The induction by Sophy β-glucan of DNA synthesis in PBMCs was completely blocked by the addition of monoclonal antibodies (mAbs) to CD11a, CD54, human leukocyte antigen-class II (HLA-class II), Toll-like receptor-2 (TLR-2), and Toll-like receptor-4 (TLR-4). In these blocking experiments using the mAbs, the main differences in the results between PBMCs and U937 cells were that the mAbs against TLR-2 and TLR-4 did not block the Sophy β-glucan -induced production of IL-8 in the U937 cells. Furthermore, a mAb to the β-glucan receptor, Dectin-1, significantly (P<0.05) blocked the Sophy β-glucan-induced DNA synthesis in the PBMCs, and Sophy β-glucan -induced production of IL-8 in the U937 cells. The Sophy β-glucan-induced production of IL-8 in the U937 cells was significantly (P<0.01) blocked by the conventional protein kinase C (PKC) inhibitor Go6976, the novel PKC inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89, and the protein tyrosine kinase (PTK) inhibitor herbimycin A. Among these, the blocking effect of the novel PKC (PKC delta isoenzyme) inhibitor Rottlerin was the most pronounced. Studies employing reverse transcriptase-polymerase chain reaction (RT-PCR) showed that Sophy β-glucan stimulated the expression of IL-8 mRNA in the U937 cells, and that this induction was inhibited by Rottlerin. Sophy β-glucan also blocked the stimulator cell induction of DNA synthesis and IFN-γ production in the responder cells in a one-way mixed lymphocyte reaction (MLR) using allogenic PBMCs. Interestingly, immunoglobulin G (IgG), but not IgM to Sophy β-glucan was detected in the sera derived from normal adult donors and from the umbilical cord blood of neonates. Taken together, these findings strongly suggest that the Sophy β-glucan may have unique immune regulatory or enhancing properties that could be exploited by the health food, medical and pharmaceutical industries.

A Novel Agar Medium to Detect Hydrogen Peroxide-Producing Bacteria Based on the Prussian Blue-Forming Reaction

Abstract: 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.

Shifting seroepidemiology of hepatitis A in Japan, 1973-2003

Abstract: Background Hepatitis A infection is caused by hepatitis A virus (HAV) contracted through fecal-oral transmission Life-long immunity is conferred after infection Improved sanitary conditions have generally resulted in a significant decline in the incidence of hepatitis A However, a low incidence of infection results in increased HAV susceptibility The present study investigates the prevalence of anti-HAV antibody and clarifies the current HAV status and HAV susceptibility in Japan at 2003 Methods A total of 2,430 serum specimens collected during 2003 from Japanese individuals ranging in age from 0–92 years, were tested for anti-HAV antibody using an inhibition enzyme linked immunosorbent assay All specimens were obtained from the WHO and the National Serum Reference Bank/National Institute of Infectious Diseases, Tokyo, Japan Results The overall seroprevalence was 122% Anti-HAV antibodies were rarely detected in individuals between 0–44 years of age Starting from the age of 45–49 years, seropositivity gradually increased through age 65 years and above Seroprevalence was not affected by gender, and geographic distribution did not affect age-specific seroprevalence until the age of 60 years Conclusions HAV susceptibility in Japan is increasing annually Particularly, the prevalence of anti-HAV antibody in individuals older than 50 years in 2003 was 503%, which is significantly lower than that of corresponding studies in 1994 (743%), 1984 (969%) and 1973 (969%) The growing susceptible population of advanced age results in more frequent HAV infection among them The surveillance of anti-HAV antibody prevalence is useful for implementing preventive measures and for controlling the spread of HAV

Molecular Characterization of Methicillin-Resistant Staphylococcus aureus in Hospitals in Niigata, Japan: Divergence and Transmission

Abstract: The major methicillin-resistant Staphylococcus aureus(MRSA) distributed among hospitals in Japan is New York/Japan clone [multilocus sequence type 5 (ST5), agr type 2 and methicillin resistance locus type (SCC mec) II] which possesses both the toxic shock syndrome toxin 1 gene (tst) and staphylococcal enterotoxin C gene (sec). In this study, we collected 245 MRSA strains from four hospitals during 2001 to 2005 in Niigata, Japan, and analyzed tst and sec genes and SCC mec type among them. A total of 13 strains were further examined for their genotypes, virulence gene patterns and drug resistance. Among the 245 strains four tst sec genes patterns were observed; tst(+) sec(+) strains represented a majority of 86.5% and 9.4% were tst(-) sec(-). SCCmec typing revealed that 91.4% had type II, 4.1% type IV and 4.1% type I. Multilocus sequence typing (MLST) revealed that 10 of the 13 typed strains belonged to clonal complex 5 (7 had ST5 while 3 were single locus variants of ST5) with similar characteristics to the New York/Japan clone and possessed multi-drug resistance with high virulence gene content. The remaining 3 strains were ST8 (n=2) and ST91 (n=1). The ST91 strain had SCC mec IV and seemed to originate in the community, while ST8 strains exhibited SCC mec type I, which is distinct from community type IV. The data suggest that MRSA in hospitals in Niigata now mainly includes the New York/Japan clone (undergoing genomic divergence and clonal expansion) and other minor types (e.g. ST8) as well as the community type.

Pro- and Anti-Inflammatory Cytokines Produced by Human Monocytes Challenged In Vitro with Paracoccidioides brasiliensis

Abstract: Department of Pediatrics Botucatu Medical School Sao Paulo State University, 18618-970, Botucatu, Sao Paulo

In vitro Th1 cytokine-independent Th2 suppressive effects of bifidobacteria.

Abstract: A comparison between 17 strains of lactic acid bacteria and 15 strains of bifidobacteria indicated that bifidobacteria induced significantly lower levels of interleukin-12 (IL-12) in murine splenic cells. The present study aims to evaluate the effect and mechanism of Bifidobacterium longum BB536, a probiotic strain, in suppressing antigen-induced Th2 immune response in vitro. BB536 suppressed immunoglobulin (Ig) E and IL-4 production by ovalbumin-sensitized splenic cells, but induction of Th1-inducing cytokine production, such as IL-12 and gamma interferon (IFN-gamma) tended to be lower compared with lactic acid bacteria. Neutralization with antibodies to IL-12, IFN-gamma, IL-10 and transforming growth factor beta indicated negative involvement of Th1-inducing cytokines and regulatory cytokines in the suppression of Th2 immune response by BB536, especially when treated at higher doses of BB536 (>10 microg cells/ml). Furthermore, BB536 induced the maturation of immature bone marrow-derived dendritic cells (BM-DCs), and suppressed antigen-induced IL-4 production mediated by BM-DCs. These results suggested that BB536 suppressed Th2 immune responses, partially independent of Th1-inducing cytokines and independent of regulatory cytokines, mediated by antigen-presenting cells such as dendritic cells.

Sensitive Detection of Bacillus anthracis Using a Binding Protein Originating from γ‐Phage

Abstract: Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Therefore, there is a pressing need to develop novel methods for rapid, simple, and precise detection of B. anthracis. Here, we report that the C-terminal region of gamma-phage lysin protein (PlyG) binds specifically to the cell wall of B. anthracis and the recombinant protein corresponding to this region (positions, 156-233), PlyGB, is available as a bioprobe for detection of B. anthracis. Our detection method, based on a membrane direct blot assay using recombinant PlyGB, was more rapid and sensitive than the gamma-phage test and was simpler and more inexpensive than genetic methods such as PCR, or immunological methods using specific antibodies. Furthermore, its specificity was comparable to the gamma-phage test. PlyGB is applicable in conventional methods instead of antibodies and could be a potent tool for detection of B. anthracis.

Molecular detection of various rickettsiae in mites (acari: trombiculidae) in southern Jeolla Province, Korea.

Abstract: This study revealed the presence of various rickettsial agents in mites from wild rodents collected in Southern Jeolla Province, Korea, by nested polymerase chain reaction (PCR) and sequence analysis of a partial citrate synthase and rickettsia outer membrane protein B genes. Rickettsial agents closely related to the Rickettsia species TwKM02, R. australis, and the Rickettsia species Cf15 were successfully identified in this study, for the first time in Korea, and R. japonica, R. akari, R. conorii, R. felis, and R. typhi were also detected, as previously described. The data presented in this paper extend knowledge on the geographic distribution of SFG rickettsiae in eastern Asia and it may necessary to consider the role of mites in rickettsial transmission.

Proteomic characterization of enterohemorrhagic escherichia coli O157:H7 in the oxidation-induced viable but non-culturable state.

Abstract: Enterohemorrhagic Escherichia coli (EHEC) O157 strain F2, a food isolate of an outbreak, is resistant to oxidative stress, but has increased stress-sensitivity after passage through mice. The stress-sensitive variant of F2 (designated MP37) has decreased culturability, but retains membrane integrity under stress conditions, indicating that the cells enter a viable but non-culturable (VBNC) state. Proteomic analyses revealed that MP37 in the VBNC state had decreased levels of some oxidation-responsive factors (AhpCF, AceF), but it markedly increased levels of outer membrane protein W (OmpW). Because F2 expressed higher levels of some ribosome-associated proteins (RaiA, S6, Bcp) than MP37, the effect of animal passage on the induction of the VBNC state in the EHEC O157 cells might be due to ribosomal activity.

Development of an immunochromatographic kit for rapid diagnosis of H5 avian influenza virus infection.

Abstract: 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.

Comparative role of antibiotic and proton pump inhibitor in experimental Clostridium difficile infection in mice.

Abstract: Clostridium difficile inoculated BALB/c mice were investigated to assess the comparative role of antibiotic and proton pump inhibitor. They were examined for colonization and toxin production by C. difficile as well as myeloperoxidase activity and histopathological changes in the intestinal tract. The C. difficile count, toxin A and B titres and myeloperoxidase activity were significantly higher (P>0.05) in ampicillin and lansoprazole receiving groups as compared to the control and the C. difficile receiving groups. Similarly they showed significant difference (P>0.05) for epithelial damage, oedema and neutrophilic infiltrate in colons. In addition to antibiotic, PPI also acts as an independent risk factor for C. difficile infection in experimental studies.

Genetic characterization of thermal tolerance in Enterobacter sakazakii.

Abstract: Enterobacter sakazakii is an opportunistic pathogen that causes meningitis and necrotizing enterocolitis in neonates. Here we characterized the thermal tolerance of E. sakazakii isolates obtained from powdered infant formula and other food products in Japan. Isolates were categorized into three classes according to their thermal tolerance, and differential gene expression analysis showed that the heat-resistant clones expressed a higher level of infB (which encodes a translation initiation factor), than did the heat-sensitive isolates. Gene expression and DNA polymorphism analyses suggested that this gene target might be useful to unequivocally detect and identify heat-resistant clones, permitting epidemiological surveillance for this pathogen.

Prediction of Efficient Virological Response to Pegylated Interferon/Ribavirin Combination Therapy by NS5A Sequences of Hepatitis C Virus and Anti‐NS5A Antibodies in Pre‐Treatment Sera

Abstract: A considerable number of patients infected with Hepatitis C virus subtype 1b (HCV-1b) do not respond to pegylated interferon/ribavirin combination therapy. In this study we explored a useful factor(s) to predict treatment outcome. A total of 47 HCV-1b-infected patients were treated with pegylated interferon/ribavirin for 48 weeks. Sera of the patients were examined for the entire NS5A sequence of the HCV genome, HCV RNA titers and anti-NS5A antibodies. According to their responses, the patients were divided into two groups, early viral responders who cleared the virus by week 16 (EVR[16w]) and those who did not (Non-EVR[16w]). The mean number of mutations in the V3 region (aa 2356 to 2379) or that in the V3 region plus its N-terminally flanking region, which we refer to as interferon/ribavirin resistance-determining region (IRRDR; aa 2334 to 2379), of NS5A obtained from the pretreatment sera was significantly larger for EVR(16w) compared with Non-EVR(16w). Moreover, HCV-1b isolates with ≥5 mutations in V3 or those with ≥6 mutations in IRRDR were almost exclusively found in EVR(16w). Also, the presence of detectable levels of anti-NS5A antibodies in the pretreatment sera was closely associated with EVR(16w). In conclusion, a high degree of sequence variation in V3 (≥5) or IRRDR (≥6) and the presence of detectable levels of anti-NS5A antibodies in the pretreatment sera would be useful factors to predict EVR(16w). On the other hand, a less diverse sequence in V3 (≤4) or IRRDR (≤5) together with the absence of detectable anti-NS5A antibodies could be a predictive factor for Non-EVR(16w).

Microbiological and immunological features of oral candidiasis

Abstract: Candida albicans(C. albicans) is the major infectious agent of oral candidiasis, and both innate immunity and cell-mediated immune response participate in the control of the fungal infections. The aim of this study was to correlate the clinical forms of oral candidiasis with the number of colony forming units (CFU) of C. albicans in saliva and to characterize T cell response in patients with oral candidiasis. Participants included 75 subjects: 36 with lesions of candidiasis and 39 without lesions of oral candidiasis. A 2-ml sample of saliva was collected from all subjects for microbiological analysis. Cytokine levels were determined by ELISA in supernatants of peripheral blood mononuclear cells of 25 patients with oral candidiasis, after in vitro stimulation with C. albicans antigens. In 48% of patients, no association was observed with denture use. C. albicans was detected in the saliva of 91.7% of patients with oral candidiasis, and there was an association between the number of CFU and the presence of oral lesions. A type Th1 immune response was observed in supernatants of peripheral blood mononuclear cells stimulated with C. albicans antigens. In contrast, IL-5 and IL-10 levels were very low or undetectable. Together, this study shows an association between clinical forms of oral candidiasis and the number of colonies of C. albicans in saliva, and that a systemic immune response characterized by the production of TNF-alpha and IFN-gamma is observed in patients with oral candidiasis.

Analysis of risk epitopes of anti-neutrophil antibody MPO-ANCA in vasculitis in Japanese population.

Abstract: Autoantibodies to myeloperoxidase (MPO) are a subset of anti-neutrophil cytoplasmic antibody (ANCA, MPO-ANCA) detected in the sera of some patients with primary systemic vasculitis. The titer of MPO-ANCA does not always reflect disease activity and this inconsistency may be attributable to differences in epitopic specificity by MPO-ANCA among various patients with vasculitis. Epitope analysis may also explain the occurrence of MPO-ANCA in different vasculitic syndromes. We screened the sera of 148 MPO-ANCA positive patients from six vasculitic syndromes: rapidly progressive gromerulonephritis (RPGN), microscopic polyangiitis (MPA), idiopathic crescentic glomerulonephritis (I-CrGN), classic polyangiitis nodosa (cPAN), Churg-Strauss syndrome (CSS), Kawasaki disease (KD); and from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The sera were collected by the Intractable Vasculitis Research Project Group in Japan. No serum showed epitopes La and Lb of light chain of MPO, and sera with 68.6% of patients showed a positive reaction to one or more epitopes in heavy chain of MPO. Analysis of binding level showed that RPGN, I-CrGN and MPA sera mainly reacted to the Ha epitope at the N-termimus of the MPO heavy chain, CSS sera reacted to Ha and the Hf epitope close to the C-terminus of the MPO heavy chain, KD reacted mainly to Hf, while SLE and RA sera reacted to all epitopes. These results suggest that MPO-ANCA recognizing specific regions of the N-terminus of the MPO H-chain confer an increased risk of vasculitis RPGN, I-CrGN, MPA and CSS. Furthermore, the epitopic specificity of MPO-ANCA differentiates vasculitic from non-vasculitic syndromes associated with MPO-ANCA positivity and differentiates in the cirtain type of vasculitis from various vasculitic syndromes. In particular, vasculitic syndromes associated with kidney involvement had similar epitopic reactivity which suggests that this pattern confers an increased risk of vasculitis.

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

Abstract: 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.

Induction of preferential chemotaxis of unstimulated B-lymphocytes by 2-arachidonoylglycerol in immunized mice.

Abstract: 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.

Orally Administrated Lactobacillus gasseri TMC0356 and Lactobacillus GG Alleviated Nasal Blockage of Guinea Pig with Allergic Rhinitis

Abstract: 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.

Serotypes, Antimicrobial Susceptibility, and gyr A Gene Mutation of Campylobacter jejuni Isolates from Humans and Chickens in Thailand

Abstract: In Thailand, 51% (36/70) Campylobacter jejuni isolates from humans and 68% (47/69) isolates from poultry were classified into 10 Penner serotypes (serotype B, C, R, E, G, A, K, D, I, and L) and 9 serotypes (serotype A, C, I, K, B, E, S, D, and L), respectively. The rate of antimicrobial drug resistance to nalidixic acid, ciprofloxacin, ampicillin, tetracycline, and erythromycin shown by human isolates were 96%, 96%, 29%, 57%, and 14%, while that shown by poultry isolates were 77%, 77%, 22%, 26%, and 17%, respectively. All quinolone-resistant strains contained a mutation in the gyrA gene (T(86)-->I(86)), suggesting that the strains were already widespread in Thailand.