Biomedical subjects

H Kurazono

Publications and source records attributed to H Kurazono.

At least 19 recordsLinked to original sources

PubMed2004

Genotypes and phenotypes of Shiga toxin producing-Escherichia coli isolated from healthy cattle in Thailand.

Shiga toxin producing-Escherichia coli (STEC) has not yet been identified as an important aetiologic agent of human disease in Thailand. To evaluate the potential for STEC to contribute to human disease in Thailand, 139 fecal samples were collected from healthy cattle from five different provinces and analysed by genotypic and phenotypic methods for STEC. Of 139 samples, 27 (19.4%) were positive for stx1 and/or stx2 by multiplex polymerase chain reaction, or for O157 lipopolysaccharide (LPS) by immunoassay. Isolates positive for stx and/or O157 were subdivided into 49 strains that varied in the presence of the virulence determinants stx1+/stx2+ (22 strains), stx2+ (22 strains), stx1+ (4 strains), and O157 LPS (1 strain). Within these 49 distinguishable strains, other virulence determinants varied as follows: hlyA+ (77.6%), eae+ and tir+ (4.1%), and katP+ (6.12%). The most predominant profile (22 isolates) was stx1+/stx2+, eae-, tir-, etpD-, hlyA+, katP-. For further characterization of the isolated strains by two molecular typing assays, plasmid profiles and ERIC PCR were performed. The results suggest that the genetic and phenotypic profiles of STEC associated with human disease are not prevalent at this time in cattle in Thailand.

Animals↗

PubMed2001

Structural and sequence diversity of the pathogenicity island of uropathogenic Escherichia coli which encodes the USP protein.

A total of 321 uropathogenic Escherichia coli (UPEC) strains and 12 strains of E. coli isolated from stool samples of healthy individuals, which were previously shown to be positive in colony hybridization test using the usp (encoding for the uropathogenic-specific protein) DNA probe, were examined by PCR amplification to determine the size of the usp gene and the pathogenicity island (PI). Three types of size variation were observed for the usp gene and four types for the PI. Sequencing analysis of the PIs from seven representative strains (six UPEC and one from a normal healthy individual) revealed that the usp genes can be classified into two groups, each having different sequences in the 3'-terminal region. The peptides encoded by the three open reading frames (ORFs) downstream of usp had identical 23 amino acid residues in the C-terminal region. The subregion encoding these small ORFs has a mosaic structure constituted of six segments. The positions of these segments vary from strain to strain, and in some strains, two to four segments are deleted. This indicates that rearrangements occur frequently in this region and the mosaic arrangement apparently contributes to the size variation observed in the PCR examination of the usp genes and PIs.

Amino Acid Sequence↗

PubMed2001

Production of beta-defensin-2 by human colonic epithelial cells induced by Salmonella enteritidis flagella filament structural protein.

We recently showed that FliC of Salmonella enteritidis increased human beta-defensin-2 (hBD-2) expression, and now describe the signaling responsible pathway. FliC increased the intracellular Ca(2+) concentration ([Ca(2+)](in)) in Caco-2 cells. The [Ca(2+)](in) increase induced by FliC was prevented by U73122 and heparin, but not by chelating extracellular Ca(2+) or pertussis toxin. The FliC-induced increase in hBD-2 promoter activity via nuclear factor kappaB (NF-kappaB) was also inhibited by chelation of intracellular Ca(2+) or by U73122. We conclude that FliC increased [Ca(2+)](in) via inositol 1,4,5-trisphosphate, which was followed by up-regulating hBD-2 mRNA expression via an NF-kappaB-dependent pathway.

Caco-2 Cells↗

PubMed2001

Salmonella enteritidis FliC (flagella filament protein) induces human beta-defensin-2 mRNA production by Caco-2 cells.

Antimicrobial peptides are crucial for host defense at mucosal surfaces. Bacterial factors responsible for induction of human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined. Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells. Using luciferase as a reporter gene for a approximately 2.1-kilobase pair hBD-2 promoter, the hBD-2-inducing factor in culture supernatant of S. enteritidis was isolated. The supernatant factor was heat-stable and proteinase-sensitive. After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis. Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells. In agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody. In gel retardation analyses, FliC increased binding of NF-kappaB (p65 homodimer) to hBD-2 gene promoter sequences. We conclude that S. enteritidis FliC induces hBD-2 expression in Caco-2 cells via NF-kappaB activation and thus plays an important role in up-regulation of the innate immune response.

Amino Acid Sequence↗

PubMed2001

Localization of Shiga toxins of enterohaemorrhagic Escherichia coli in kidneys of paediatric and geriatric patients with fatal haemolytic uraemic syndrome.

Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopenia and renal failure. Infection with enterohaemorrhagic Escherichia coli (EHEC), mainly O157:H7, has been strongly implicated as the major cause of HUS in children. The pathogenesis of HUS caused by the infection is not well understood and the defined sites of Stx in kidney of EHEC-infected humans has not been clearly demonstrated. The aim of this study was to investigate and compare the locations of Stx deposition in kidneys of paediatric and geriatric patients who died from enterohaemorrhagic E. coli O157 (EHEC) associated HUS, using an immunoperoxidase staining of the tissues. The study revealed that binding of Stx was relatively less and limited only to the renal tubules of an adult case (81 years old), while more binding was found at both renal tubules and glomeruli of an infant case (21 months old). The Stx binding in the infant's glomeruli was at podocytes, mesangial and endothelial cells. It has been known that young children are more susceptible than adults to HUS. One possibility for this is that the more extensive binding of the Stx to the kidney tissue of the paediatric patient might be due to the higher synthesis and expression of Stx receptors, i.e. Gb(3), in infants and less so in the aged individuals. However, other alternatives are possible, for example, the difference in stage of HUS in individual patients. Thus it is too early to draw any conclusion on this enigma and further investigation is required.

Aged↗

PubMed2001

Expression of human beta-defensin 2 (hBD-2) in Helicobacter pylori induced gastritis: antibacterial effect of hBD-2 against Helicobacter pylori.

BACKGROUND: Human beta-defensin 2 (hBD-2) plays a role in the innate defence system at mucosal surfaces. Colonisation of Helicobacter pylori in the stomach is an important pathological factor in gastrointestinal illnesses, including gastritis, peptic ulcer, and gastric adenocarcinoma. AIMS: To evaluate the antibacterial role of hBD-2 against H pylori infection in the gastric mucosa. SUBJECTS: Biopsied gastric mucosa specimens from H pylori positive (n=6) and H pylori negative (n=6) individuals were used. H pylori was determined by the presence of urease activity and microscopic examination. METHODS: The specimens were examined for hBD-2 expression by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridisation. The antibacterial effect of hBD-2 against H pylori was evaluated by the number of colony forming units of H pylori after incubation with 0, 10(-9), 10(-8), 10(-7), 10(-6), or 10(-5) M of hBD-2 peptide. RESULTS: All six H pylori positive specimens expressed a high level of hBD-2 mRNA while hBD-2 mRNA was not detected in the H pylori negative specimens by RT-PCR. Immunohistochemistry using anti-hBD-2 antiserum revealed that hBD-2 was expressed in the surface epithelium of H pylori infected specimens. In gastric specimens obtained after H pylori eradication, hBD-2 immunoreactivity had dramatically decreased. In situ hybridisation confirmed that hBD-2 transcripts were localised in the epithelium of H pylori infected gastric specimens. Incubation with hBD-2 reduced the growth rate of cultured H pylori in a dose dependent manner, and incubation with 10(-5) M hBD-2 completely inhibited the proliferation of H pylori. CONCLUSIONS: H pylori infection induces hBD-2 expression in the human gastric epithelium. hBD-2 inhibited the growth of H pylori in vitro, suggesting that hBD-2 plays an antibacterial role in H pylori induced gastritis.

Aged↗

PubMed2001

Effects of rearing conditions on the colonization of Salmonella enteritidis in the cecum of chicks.

Salmonella enteritidis is the cause of human salmonellosis associated with contaminated eggs. In this study, we artificially challenged S. enteritidis to chicks just after hatching, and the effects of breeding conditions on the intestinal carriage of S. enteritidis were examined. S. enteritidis was not directly detected from spleen, liver and blood, but were constantly isolated from the cecal contents throughout the experiment. When chicks were reared in the unsanitary conditions and in the high housing density, the numbers of S. enteritidis increased. The subsequent experiment was undertaken to examine whether the antibacterial additive in a feed would have any impact on S. enteritidis colonization in chicks. Some antibiotic effective on the growth promotion had an influence on S. enteritidis colonization.

Animal Husbandry↗

PubMed2001

The presence of the virulence island containing the usp gene in uropathogenic Escherichia coli is associated with urinary tract infection in an experimental mouse model.

PURPOSE: A putative virulence island commonly noted in the genome of uropathogenic Escherichia coli strains has recently been reported. We have observed that the island includes a gene consisting of a protein designated uropathogenic specific protein (usp) and 3 small open reading frames (orfU1-3). In our current study we assessed the importance of the genes located in the putative virulence island in the pathogenesis of urinary tract infection using a mouse pyelonephritis model. MATERIALS AND METHODS: A total of 427 E. coli strains isolated from the urine of 194, 76 and 107 subjects suffering from cystitis, pyelonephritis and prostatitis, respectively, and 50 isolates from the feces of healthy individuals were examined for genotypes and serotypes. In addition, several recombinant E. coli strains possessing usp and/or orfU1 to 3 were constructed for evaluating the significance of these genes using an experimental pyelonephritis mouse model. RESULTS: The usp was significantly more often associated with uropathogenic E. coli strains (79.4% from cystitis, 93.4% from pyelonephritis and 88.8% from prostatitis) than with fecal E. coli strains from healthy individuals (24%). Furthermore, usp was frequently associated with all common serotypes of uropathogenic E. coli (71.7% to 100%). In challenge experiments using the mouse urinary tract infection model the vector possessing usp significantly enhanced the infectibility of the E. coli host cell, whereas the 3 small proteins at the downstream of usp failed to show the effect. CONCLUSION: Our results indicate that usp may contribute to the causation of urinary tract infection and may be considered a major virulence determinant of uropathogenic E. coli.

Animals↗

PubMed2001

Validation of salmonellosis and shigellosis diagnostic test kits at a provincial hospital in Thailand.

Rapid Diagnosis of salmonellosis and shigellosis was performed using six different diagnostic test kits which recently have been made available commercially. They were Salmo-Dot, Typhi-Dot, Shigel Dot A, B, C, and D test kits for detection of Salmonella spp., group D salmonellae, and groups A, B, C, and D Shigella spp., respectively. The principle of all test kits is a membrane (dot) ELISA using specific monoclonal antibodies to the respective pathogens as the detection reagents. The present study was designed to validate the accuracy of the test kits, at a laboratory in a provincial hospital in Thailand, in comparison with the conventional bacterial culture method alone or with the combined results of the culture and the Western blot analysis (WB) for detecting the respective bacterial lipopolysacchharides (LPS) in specimens. Five hundred rectal swab samples of patients with diarrhea who seeked treatment at the hospital, were evaluated. The diagnostic accuracy of the Salmo-Dot was 91.0% when compared with the conventional bacterial culture method alone but was 100.0% in comparison with the combined results of the culture and the WB. The Typhi-Dot and the Shigel-Dot A, B, C, and D showed 100%, 99.2%, 95.0%, 94.0% and 96.4%, respectively when compared with the culture alone and all were 100% in comparison with the combination of the results of the bacterial culture and the WB. The Shigel-Dot A revealed antigen of type 1 Shigella dysenteriae in several specimens in which the bacteria could not be recovered by the culture method. This difference is important as type 1 Shigella dysenteriae have high epidemic potential and often cause severe morbidity. Unawareness of their presence by the conventional culture may have great impact on disease surveillance for public health. The pathogen detection using the six diagnostic test kits is sensitive, specific, rapid, and relatively simple and less expensive. Several specimens can be tested at the same time without much increase in turn around time. Moreover, these kits produce no contaminated waste as compared with the bacterial culture method. The test kits should be used for rapid screening of specimens of patients with diarrhea especially in areas where culture facilities are inadequate.

Diagnosis, Differential↗

PubMed2001

Estimation of vacuolating cytotoxin secreted by different strains of Helicobacter pylori using bead enzyme-linked immunosorbent assay & its correlation with bacterial genotype.

BACKGROUND & OBJECTIVES: A highly sensitive bead enzyme-linked immunosorbent assay was applied for the quantitative determination of vacuolating cytotoxin (VacA) released in the culture supernatant of 40 well characterized Helicobacter pylori strains in order to clarify the significance of allelic combination of the vacA gene as the predictor of the level of toxin secretion and also to determine the most appropriate genotype of H. pylori associated with high VacA release. Attempts were also made for the detection of VacA in the gastric juice of patients for the rapid diagnosis of H. pylori infection. METHODS: The genotypes of 40 H. pylori strains cultured from the gastric biopsy samples were determined by specific PCRs. The cell-free culture supernatant of the strains as well as the gastric juice of the patients were used for bead-ELISA and the purified VacA from the H. pylori strain ATCC49503 was used as positive control. RESULTS: Ninety per cent of the strains with vacAs1m1 allele combination secrete on an average 146.4 ng/ml of VacA while the corresponding value was 19.1 ng/ml for s1m2 strains. None of the s2m2 as well as the ice negative H. pylori strains produced detectable VacA in the medium while strains expressed the toxin irrespective of the presence or absence of cagA gene. Fifteen of 22 gastric juice samples yielded positive bead-ELISA results. INTERPRETATION & CONCLUSION: vacAs1, vacAm1 and iceA1 could be considered as the determinants of high VacA secretion. Also, the detection of VacA by bead-ELISA in the gastric juice could be considered as an alternative approach in the diagnosis of H. pylori infection.

Adult↗

PubMed2000

Enterotoxin adjuvants have direct effects on T cells and antigen-presenting cells that result in either interleukin-4-dependent or -independent immune responses.

In an in vitro study, Escherichia coli heat-labile toxin (LT) was shown to directly affect activated CD4(+) T cells and support interleukin (IL)-5 production in IL-4-deficient (IL-4(-/-)) mice, whereas cholera toxin (CT) did not. Both LT and CT enhanced B7-2 expression on B cells and macrophages. These effects were not influenced by CD40-CD40 ligand cosignaling. Addition of LT- or CT-treated antigen-presenting cells to anti-CD3-triggered CD4(+) T cells resulted in the induction of T cell proliferative responses. Further, these responses were inhibited by anti-B7-2 monoclonal antibody. Cocultivation of CD4(+) T cells with LT- or CT-treated antigen-presenting cells and anti-CD3 enhanced Th1- and IL-4-mediated Th2-type cytokine production. The results from in vitro studies were supported by in vivo studies in IL-4(-/-) mice, in which LT induced mucosal IgA responses but CT did not. Thus, although both LT and CT induce mucosal adjuvant responses via IL-4-dependent Th2-type responses, LT also elicits Th1- and IL-4-independent Th2-type responses.

Animals↗

PubMed2000

Characterization of a putative virulence island in the chromosome of uropathogenic Escherichia coli possessing a gene encoding a uropathogenic-specific protein.

This study was initiated to search for a homologue of the Vibrio cholerae zot gene in uropathogenic Escherichia coli (UPEC) using a specific DNA probe. The faint signal obtained at low stringency with some UPEC strains associated with prostatitis cases prompted us to examine UPEC strains by PCR using primers designed from the conserved regions of the proteins of the Zot group of putative NTPases containing the classical NTP binding motif. This led to the discovery of a DNA fragment in UPEC strains which hybridized with a probe designed from the PCR. Further analysis of this DNA fragment revealed an ORF which was designated as uropathogenic specific protein (Usp). The gene encoding Usp was 1038 bp long and codes for 346 amino acids with an appropriate SD sequence. Upstream and downstream analysis of usp revealed motifs of prokaryotic consensus promoters and three small ORFs with SDs and ribosome binding sites transcribed in the same direction of usp. The proximity of these set of genes in a specific area of the bacterial chromosome resembling a block of genes preferentially associated with UPEC coupled with the presence of a motif matching that of a Tn3 transposon family lead us to believe that this could be an hitherto unknown pathogenicity island.

Acid Anhydride Hydrolases↗

PubMed2000

Differential diagnosis of schistosomiasis mekongi and trichinellosis in human.

An indirect (plate) ELISA and, a more convenient version, a dot-blot (membrane) ELISA have been developed using haemocyanin of a mollusk, Megathura crenulata, i.e. keyhole limpet haemocyanin (KLH) and purified, specific antigen of Trichinella spiralis (APTsAg) obtained from a monoclonal antibody-affinity column chromatography, for differential diagnosis of schistosomiasis mekongi and trichinellosis. Serum samples of patients with parasitologically confirmed trichinellosis were reactive to both antigens in both versions of ELISA while sera of patients with schistosomiasis mekongi were positive only to the KLH. Both ELISA were negative when used to test sera of normal controls and patients with gnathostomiasis, paragonimiasis and opisthorchiasis.

Animals↗

PubMed2000

Analysis of Helicobacter pylori vacA gene and serum antibodies to VacA in Japan.

Vacuolating cytotoxin, VacA, is one of the most important pathogenetic factors produced by Helicobacter pylori. However, it is not clear whether the diversity in disease outcome may be ascribed to variations in strain and/or to the host responses to virulence factors. In this study, we analyzed the vacA middle region sequence among 65 Japanese isolates to clarify the variation in strain and assayed antibody titer to VacA by ELISA using purified VacA to evaluate the host response to cytotoxin. The nucleotide sequence identities compared among Japanese isolates were 92.8 +/- 3.56%, and compared to 88.3 +/- 2.89% in tox+ strains reported in GenBank. Positive correlation was found between the antibody titers and the severity of atrophic change of the stomach. In Japan the nucleotide sequences of the vacA middle region were highly homologous and genetically closer to tox+ strains. Antibody titers and host response to cytotoxin may be associated with atrophy of the stomach.

Adult↗

PubMed2000

Trichinella spiralis-specific monoclonal antibodies and affinity-purified antigen-based diagnosis.

Hybridomas secreting monoclonal antibodies (MAbs) to Trichinella spiralis were produced. Myeloma cells were fused with splenocytes of a mouse immunized with excretory-secretory (E-S) antigen of infective larvae. A large percentage of growing hybrids secreted antibodies cross-reactive to many of 23 heterologous parasites tested. Only 6 monoclones (designated 3F2, 5D1, 10F6, 11E4, 13D6 and 14D11) secreted MAbs specific to the E-S antigen and/or a crude extract (CE) of T. spiralis infective larvae. The 6 monoclones secreted IgM, IgG3, IgM, IgG3, IgG3 and IgG3, respectively. Clone 5D1 was selected to mass produce MAbs which were then coupled to CNBr-activated Sepharose CL-4B to prepare an affinity-purified antigen. Dot-blot ELISA with either purified antigen or CE was evaluated. There were 17 patients with acute trichinellosis and 76 individuals convalescing from T. spiralis infection (group 1). Controls were 170 patients with parasitic infections other than trichinellosis (group 2) and 35 healthy parasite-free controls (group 3). CE-ELISA was positive in all group 1 patients. However, sera from many group 2 patients also were reactive (opisthorchiasis-44.2%, schistosomiasis-44%, gnathostomiasis-30%, paragonimiasis-28.6%, taeniasis-27.3%, strongyloidiasis-23.1% and hookworm infections-20%). Affinity-purified antigen was 100% specific, all sera from group 2 and group 3 individuals tested negative. Although 74 of 76 patients (97.4%) with convalescing trichinellosis tested positive, sera from only 3 of 17 patients (17.6%) with acute T. spiralis were reactive. Thus, CE antigen is appropriate when sensitivity is needed, while purified antigen should be used when specificity is required. Dot-blot ELISA is easier to perform, more rapid and less expensive than indirect ELISA. Many samples can be assayed simultaneously, special equipment is not required, and results can be preserved for retrospective analysis. Dot-blot ELISA is therefore the method of choice for the rapid diagnosis of trichinellosis, particularly when more complex laboratory tests are unavailable.

Animals↗

PubMed1999

Activation of Helicobacter pylori VacA toxin by alkaline or acid conditions increases its binding to a 250-kDa receptor protein-tyrosine phosphatase beta.

Helicobacter pylori, a Gram-negative gastric bacterium, secretes VacA, a cytotoxin that causes vacuolar degeneration of susceptible cells. Velocity sedimentation analysis showed that treatment of VacA at alkaline pH led to disassembly of VacA oligomers, an observation reported previously for acid-treated VacA. Exposure of VacA to acid or alkali increased its binding to AZ-521 cells, as shown by indirect immunofluorescence and flow cytometry. Moreover, immunoprecipitates with polyclonal antibodies against VacA from AZ-521 cells previously exposed to acid- or alkali-treated VacA had a 250-kDa glycoprotein containing galactose-beta(1-3)-N-acetylgalactosamine and galactose-beta(1-4)-N-acetylglucosamine. p250, purified by chromatography on peanut agglutinin affinity and Superose 6 columns, contained N-terminal and internal amino acid sequences of YRQQRKLVEEIGWSYT and LIIQDHILEATQDDY, respectively. These sequences are identical to those of a receptor protein-tyrosine phosphatase (RPTPbeta/PTPzeta); in agreement, p250 reacted with anti-human RPTPbeta monoclonal antibody. Immunoprecipitation with anti-human RPTPbeta antibody of solubilized membrane preparations previously incubated with VacA or heat-inactivated VacA demonstrated that RPTPbeta bound native, but not denatured, VacA. Acidic and alkaline treatments were associated with activation of VacA and increased binding to the cell surface RPTPbeta.

Amino Acid Sequence↗

PubMed1999

Application of Bead-ELISA method to detect Helicobacter pylori VacA.

Helicobacter pylori is an etiological agent of gastritis, gastric ulcer and gastric cancer. In order to clarify the significance of vacuolating cytotoxin (VacA) for the pathogenesis of Helicobacter pylori infection, we established and applied the sandwich bead enzyme-linked immunosorbent assay (Bead-ELISA) for quantitative determination of VacA in the culture mediums of H. pylori and other species of Helicobacter. The minimum concentration of VacA in culture medium detected by Bead-ELISA was 25 pg VacA/ml and its sensitivity was found to be quite high compared to vacuolation assay and Western blot analysis, e.g. the minimum concentrations of VacA in culture medium required for detection by vacuolation assay and Western blotting were 11 ng/ml and 38 ng/ml, respectively. All the H. pylori strains used were found to produce VacA in the culture medium by Bead ELISA, even though some strains were negative by Western blot and vacuolation assay. The results obtained by Bead-ELISA was consistent with those by PCR amplification of a 785 bp vacA fragments. A toxin immunologically similar to VacA produced by other strains of Helicobacter such as H. muridarum (ATCC49282), H. mustelae (F10) and H. felis (ATCC49179) could not be detected by Bead-ELISA as well as Western Blot.

Animals↗

PubMed1999

Establishment of the PCR system specific to Salmonella spp. and its application for the inspection of food and fecal samples.

We established the PCR detection system specific to Salmonella species using Salmonella enterotoxin gene (stn). The detection limit was one bacterial cell per one gram of fecal and minced-meat samples using enrichment procedure by Tripticase soy broth or Salmonella enrichment broth, respectively. We concluded that this PCR system is useful for the practical application in the field of the public hygiene.

Animals↗