Diversity of Helicobacter pylori vacA and cagA Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases

The vacuolating cytotoxin and the cytotoxin-associated protein, encoded by vacA and cagA, respectively, are important virulence determinants of Helicobacter pylori. Sixty-five H. pylori strains were isolated from dyspeptic patients (19 with peptic ulcer disease, 43 with chronic gastritis, and 3 with gastric cancer) and studied for differences in the vacA and cagA genes and their relationship to VacA and CagA expression, cytotoxin activity, and the clinical outcome of infection. By PCR, fifty-four (83.1%) of 65 strains had the vacA signal sequence genotype s1 and only 10 (15.4%) had the type s2. After primer modification, the vacA middle-region types m1 and m2 were detected in 24 (36.9%) and 41 (63.1%) strains, respectively. The combinations s1-m2 (31 [47.7%]) and s1-m1 (23 [35.4%]) occurred more frequently than s2-m2 (10 [15.4%]) (P = 0.01). No strain with the combination s2-m1 was found. All 19 patients with peptic ulcers harbored type s1 strains, in contrast to 32 (74.4%) of 43 patients with gastritis (P = 0.02). The vacA genotype s1 was associated with the presence of cagA (P < 0.0001), VacA expression (P < 0.0001), and cytotoxin activity (P = 0.003). The cagA gene was detectable in 48 (73.8%) of 65 isolates and present in 16 (84.2%) of 19 ulcer patients and 29 (67.4%) of 43 patients with gastritis (P = 0.17). The vacA genotypes of German H. pylori isolates are identical to those previously reported. H. pylori strains of vacA type s1 are associated with the occurrence of peptic ulceration and the presence of cagA, cytotoxin activity, and VacA expression.     

Determination of Helicobacter pylori Virulence by Simple Gene Analysis of the cag Pathogenicity Island

Nucleic acid amplification was performed for five loci in the cag pathogenicity island (PAI) of Helicobacter pylori (comprising cagA, the cagA promoter region, cagE, cagT, and the left end of cagII [LEC]), and gastric inflammation in patients was evaluated. Of 204 H. pylori isolates from Japanese patients (53 with peptic ulcer, 55 with gastric cancer, and 96 with chronic gastritis), 197 (96.6%) were positive for all five loci. Two isolates (1%) were negative for all five loci, and five isolates (2.4%) were positive for only cagA and LEC. These latter seven isolates were all from patients with mild chronic gastritis. Neutrophil infiltration in gastric mucosa was significantly milder in patients infected with partially or totally deleted-PAI strains than in those with intact-PAI strains. The cagE gene was a more accurate marker of an intact cag PAI than the cagA gene, and cagE seemed to be more useful in discriminating between H. pylori strains causing different rates of disease progression.     

Typing of Helicobacter pylori vacA Gene and Detection of cagA Gene by PCR and Reverse Hybridization

The present report describes an analysis of two virulence genes of Helicobacter pylori. Parts of the cagA gene, as well as parts from the signal (s) and middle (m) regions of the mosaic vacA gene, were amplified with biotin-labelled PCR primers and the products were subsequently analyzed by a single-step reverse hybridization line probe assay (LiPA). This assay comprises a strip containing multiple specific probes for the vacA s region (s1a, s1b, and s2 alleles), the vacA m region (m1 and m2 alleles), and the cagA gene. A total of 103 H. pylori-positive materials, including cultured isolates, gastric biopsy specimens, and surgical specimens from patients living in Portugal (n = 55) and The Netherlands (n = 48) were tested by the PCR-LiPA. cagA was detected in 84 and 73% of the Portuguese and Dutch patients, respectively. vacA typing results, as determined by reverse hybridization, were completely concordant with those of sequence analysis. Most Portuguese patients (72%) contained type s1b, whereas most Dutch patients (61%) contained type s1a (P < 0.001). The method is also very effective at detecting the presence of multiple genotypes in a single biopsy specimen. The prevalence of multiple strains in Portuguese patient samples was significantly higher (29%) than that in Dutch patient samples (8%) (P = 0.001). There was a significant association between the presence of ulcers or gastric carcinoma and the presence of vacA type s1 (s1a or s1b; P = 0.008) and cagA (P = 0.003) genes.     

Host Cell Responses to Genotypically Similar Helicobacter pylori Isolates from United States and Japan

Associations of Helicobacter pylori genotypes with disease differ between Western countries and Asia. Therefore, we directly compared histopathological and in vitro responses to clinical isolates with similar genotypes. Sixty-three cagA⁺ vacAs1/m1 H. pylori isolates (United States, n = 24; Japan, n = 39) and eight cagA-negative vacAs2/m2 strains were incubated with AGS cells, and supernatants were assayed for interleukin-8 (IL-8) and for DNA fragmentation. CagA tyrosine phosphorylation in AGS cells and the sequence of the putative HP0638 (oipA) signal sequence region were determined for 22 representative strains. HP0638 and/or cag island mutant strains were created and examined in IL-8 and CagA tyrosine phosphorylation assays. Levels of IL-8 induction and DNA fragmentation were similar in the U.S. and Japanese cagA⁺ vacAs1/m1 isolates. All 10 of the isolates with the highest IL-8 induction and 8 of the 10 isolates with the lowest IL-8 induction had an in-frame oipA open reading frame, and all 10 of the isolates with the highest IL-8 induction and 7 of the 10 isolates with the lowest IL-8 induction induced CagA tyrosine phosphorylation in AGS cells. Eight isolates from gastric ulcer patients induced significantly more apoptosis in vitro, and more severe gastritis and atrophy in vivo, than other Japanese isolates. Disruption of HP0638 did not affect IL-8 induction or CagA tyrosine phosphorylation. Thus, H. pylori cagA⁺ vacAs1/m1 isolates from the United States and Japan induce similar IL-8 and apoptosis levels. Inactivation of HP0638 does not alter epithelial responses mediated by the cag island in vitro. Assessment of apoptosis in vitro identified a group of H. pylori isolates that induce more severe gastric inflammation and atrophy.     

Antrum- and Corpus Mucosa-Infiltrating CD4+ Lymphocytes in Helicobacter pylori Gastritis Display a Th1 Phenotype

In this study, cytokine patterns produced by CD4⁺ T cells isolated from antrum or corpus gastral biopsy specimens of 10 patients with Helicobacter pylori-positive gastritis were compared. To this end, expression of intracellular cytokines (interleukin-4 [IL-4] and gamma interferon) and of CD4 was assessed by flow cytometry. Ten to 60% of the isolated CD4⁺ T cells produced gamma interferon upon stimulation. With the exception of one patient, IL-4-positive CD4⁺ cells were not detected. Therefore, CD4⁺ cells infiltrating antrum and corpus stomach mucosa during H. pylori infection show a Th1 phenotype. This polarized Th1-type response may contribute to the inability of the immune system to eradicate H. pylori infection.     

Lack of correlation between vacuolating cytotoxin activity,cagA gene inHelicobacter pylori, and peptic ulcer disease in children

To determine the prevalence of thecagA gene and vacuolating cytotoxin inHelicobacter pylori isolates obtained from children and to characterize the relationship betweencagA, cytotoxin production, and ulcerogenesis, pediatricHelicobacter pylori isolates were tested forcagA by the polymerase chain reaction and for vacuolating cytotoxin by a cell culture assay.Helicobacter pylori isolates were obtained from 33 children referred for upper gastrointestinal endoscopy. Twenty-six of these isolates were tested forcagA by the polymerase chain reaction; all 26 (100%) were positive. Of the 26 children from whom these isolates were obtained, 26 (100%) had chronic gastritis and 12 (46%) had duodenal ulcers. Nine (30%) of 30 isolates tested showed expression of vacuolating cytotoxin, only three of which came from patients with duodenal ulceration (odds ratio 0.81, 95% confidence interval 0.1–5.3). Of the 23cagA-positive isolates tested for cytotoxin, only nine (39%) were positive. There was no association between vacuolating cytotoxin and clinical symptoms, nor was cytotoxicity associated with ulcerogenesis. In summary, the findings suggest thatcagA is not a marker of duodenal ulceration or of vacuolating cytotoxin production in children referred for endoscopy.     

Variants of the 3′ Region of the cagA Gene in Helicobacter pylori Isolates from Patients with Different H. pylori-Associated Diseases

The CagA protein of Helicobacter pylori is an immunogenic antigen of variable size and unknown function that has been associated with increased virulence as well as two mutually exclusive diseases, duodenal ulcer and gastric carcinoma. The 3′ region of the cagA gene contains repeated sequences. To determine whether there are structural changes in the 3′ region of cagA that predict outcome of H. pylori infection, we examined 155 cagA gene-positive H. pylori isolates from Japanese patients including 50 patients with simple gastritis, 40 with gastric ulcer, 35 with duodenal ulcer, and 30 with gastric cancer. The 3′ region of the cagA gene was amplified by PCR followed by sequencing. CagA proteins were detected by immunoblotting using a polyclonal antibody against recombinant CagA. One hundred forty-five strains yielded PCR products of 642 to 651 bp; 10 strains had products of 756 to 813 bp. The sequence of the 3′ region of the cagA gene in Japan differs markedly from the primary sequence of cagA genes from Western isolates. Sequence analysis of the PCR products showed four types of primary gene structure (designated types A, B, C, and D) depending on the type and number of repeats. Six of the seven type C strains were found in patients with gastric cancer (P < 0.01 in comparison to noncancer patients). Comparison of type A and type C strains from patients with gastric cancer showed that type C was associated with higher levels of CagA antibody and more severe degrees of atrophy. Differences in cagA genotype may be useful for molecular epidemiology and may provide a marker for differences in virulence among cagA-positive H. pylori strains.     

Experimental Infection of Mongolian Gerbils with Wild-Type and Mutant Helicobacter pylori Strains

Experimental Helicobacter pylori infection was studied in Mongolian gerbils with fresh human isolates that carry or do not carry cagA (cagA-positive or cagA-negative, respectively), multiply passaged laboratory strains, wild-type strain G1.1, or isogenic ureA, cagA, or vacA mutants of G1.1. Animals were sacrificed 1 to 32 weeks after challenge, the stomach was removed from each animal for quantitative culture, urease test, and histologic testing, and blood was collected for antibody determinations. No colonization occurred after ≥20 in vitro passages of wild-type strain G1.1 or with the ureA mutant of G1.1. In contrast, infection occurred in animals challenged with wild-type G1.1 (99 of 101 animals) or the cagA (25 of 25) or vacA (25 of 29) mutant of G1.1. Infection with G1.1 persisted for at least 8 months. All 15 animals challenged with any of three fresh human cagA-positive isolates became infected, in contrast to only 6 (23%) of 26 animals challenged with one of four fresh human cagA-negative isolates (P < 0.001). Similar to infection in humans, H. pylori colonization of gerbils induced gastric inflammation and a systemic antibody response to H. pylori antigens. These data confirm the utility of gerbils as an animal model of H. pylori infection and indicate the importance of bacterial strain characteristics for successful infection.     

High Prevalence of Clarithromycin Resistance and cagA,vacA, iceA2, and babA2 Genotypes of Helicobacter pylori in Brazilian Children

We isolated 45 Helicobacter pylori strains from 217 child patients. Resistance to clarithromycin, metronidazole, amoxicillin, and tetracycline was detected in 27%, 13%, 4%, and 0% of strains, respectively. The A2143G mutation was the most prevalent (67%) among clarithromycin-resistant strains. In addition, strain genotyping revealed a significant association between gastritis severity and the simultaneous presence of cagA, vacA s1m1, iceA2, and babA2 genes.Helicobacter pylori infection is found worldwide and constitutes a public health concern in many countries. Previous epidemiological studies have shown a high prevalence of H. pylori infection in Brazil (2, 20, 24). H. pylori infection, generally acquired in childhood, persists asymptomatically for decades in most individuals.Amoxicillin, tetracycline, metronidazole, and clarithromycin are frequently used, combined with proton pump inhibitors or bismuth salts, for the treatment of H. pylori infections (25). However, antibiotic resistance is frequently associated with eradication failure (3, 16). Resistance to metronidazole and clarithromycin is population dependent, and several studies suggest that clarithromycin resistance is higher in strains isolated from children than in strains isolated from adults (10). In Brazil, the prevalence of clarithromycin-resistant strains in adults is reported to be from 7 to 10% (15, 18). However, little is known about the prevalence of clarithromycin-resistant H. pylori infection in Brazilian children.The primary aims of this study were to determine the prevalence of clarithromycin-resistant H. pylori strains in children, to identify those isolates via rapid methodology, and to examine the severity of gastritis caused by the antibiotic-resistant H. pylori isolates. Metronidazole, amoxicillin, and tetracycline resistance was also studied. Furthermore, the study aimed to genotype the vacA and iceA genes and to detect the cagA gene in gastric biopsy specimens, since recent studies found a high frequency of cagA-positive and iceA2-positive strains as well as a strain with the vacA signal region genotype s1 and middle region sequence m1 among pediatric H. pylori isolates in Brazil (6, 7, 11, 23). This is also the first investigation of babA2 gene prevalence in Brazilian children.A total of 217 consecutive child patients, aged 1 to 18 years (mean age, 10 years) (105 girls and 112 boys), who underwent upper gastrointestinal endoscopy for the evaluation of dyspeptic symptoms at the outpatient clinic of Pediatric Gastroenterology at the Instituto da Criança, Faculdade de Medicina da Universidade de São Paulo, during 2008 and 2009 were included. The study was approved by the Ethics Committee of the University Hospital. Patients previously treated for H. pylori infections were not included.Gastric biopsy specimens were processed for histological examination and evaluated according to the updated Sydney system of classification and grading of gastritis (4).Antral gastric specimens were transported in sodium thioglycolate broth (Difco, Detroit, MI) in an ice bath and ground before submission to DNA extraction and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis with primers specific to the H. pylori 23S rRNA gene (17). The QIAmp tissue kit (Qiagen) was used for DNA extraction. Point mutations related to clarithromycin resistance in the 23S rRNA amplicon were investigated in all H. pylori isolates by PCR-RFLP using BsaI and MboII enzymes (27). The vacA, cagA, iceA, and babA2 genotypes were detected by PCR, as described elsewhere (1, 9, 21, 26, 28). In each experiment, H. pylori strain 26695 (ATCC 700392) was used as the positive-control strain.H. pylori strains were cultured on Belo Horizonte medium (22) under microaerophilic atmosphere at 37°C for 3 to 7 days, and the isolates were identified by Gram staining and biochemical tests for oxidase, catalase, and urease production. Resistance to clarithromycin, metronidazole, amoxicillin, and tetracycline was determined by the disc diffusion method (Oxoid), and MICs were determined by the Etest according to the manufacturer''s recommendations (AB Biodisk, Solna, Sweden). An isolate was considered resistant to clarithromycin or tetracycline if the MIC was >1 mg/liter and to metronidazole or amoxicillin if the MIC was >4 mg/liter (19).Data were analyzed by the two-tailed χ² test and Fisher exact test. P values of <0.05 were considered statistically significant.H. pylori was isolated in 45 (20.7%) of the 217 children; 12 (26.7%) of the 45 strains were clarithromycin resistant, 6 (13.3%) were metronidazole resistant, and 2 (4.4%) were amoxicillin resistant. All cultured H. pylori strains were susceptible to tetracycline (Fig. ​(Fig.1).1). No histological differences were observed between biopsy specimens with antibiotic-resistant strains and those with susceptible strains. PCR-RFLP was performed with all 12 clarithromycin-resistant isolates: 8 had the 23S rRNA A2143G point mutation, and 4 had the 23S rRNA A2142G mutation.Open in a separate windowFIG. 1.Distribution of MICs for the 45 H. pylori strains.Among the 45 H. pylori-infected children, 13 had mild chronic gastritis, 28 had moderate chronic gastritis, 2 had marked chronic gastritis, and 2 had normal gastric mucosa. The percentage of H. pylori-infected children with chronic gastritis was 95.5% (43 patients), while 4.4% of the children (2 patients) had normal mucosa (P < 0.001).vacA was detected in all 45 H. pylori-positive gastric biopsy specimens. The vacA genotypes s1m1, s2m2, and s1m2 or s2m1 were found in 57.7, 33.3, and 4.4% of the specimens, respectively. The iceA1 allele was detected in 9 (20%) and the iceA2 allele in 31 (68.9%) of the samples. Of the 45 H. pylori-positive biopsy specimens, 28 (62%) were cagA positive and 38 (84.4%) were babA2 positive. Correlation of histopathology results with vacA, cagA, and iceA genotypes showed that vacA s1m1-, cagA-, and iceA2-positive strains were more frequently found in patients with moderate and marked gastritis (77%) than in patients with mild gastritis (23%) (P < 0.001). Interestingly, in Slovenian children, vacA s1 and cagA were also shown to be associated with more pronounced chronic gastritis (12). In contrast, in Korean children, although vacA s1m1 cagA iceA1 was the predominant genotype, no association with gastritis severity was observed (14).In conclusion, we found a high incidence of clarithromycin-resistant H. pylori strains (27%) in Brazilian children. Furthermore, we found an association between clarithromycin resistance and either the vacA s1m1 (P = 0.007) or the iceA2 (P = 0.038) genotype. The high level of clarithromycin resistance among strains from children compared to adults (15, 18) suggests the importance of susceptibility testing, especially in Brazilian children. All together, these data stress the relevance of susceptibility testing and genotyping for establishing antibiotic treatment in pediatric H. pylori infection.In our study, PCR-RFLP proved to be a rapid and accurate method for the detection of clarithromycin resistance gene mutation directly in gastric biopsy samples. Only a few groups have studied mutations involved in clarithromycin resistance in strains obtained from children, and their results are similar to those obtained in our study (5, 13, 29).Our data also demonstrate an association between H. pylori infection and gastritis in Brazilian children. In addition, we confirmed the reported association of infection with vacA s1m1 cagA iceA2-positive H. pylori strains and gastritis severity (6, 11, 23). Furthermore, a high frequency of babA2 was found among H. pylori isolates. Previous studies of adults in Brazil reported a high prevalence of babA2-positive strains from patients with different upper gastrointestinal diseases (8). The high incidence of babA2 in H. pylori Brazilian isolates suggests that this gene could be a useful marker for identifying patients with a high risk of H. pylori infection in Brazil.     

cagA-Positive Helicobacter pylori Populations in China and The Netherlands Are Distinct

The aim of this research was to study whether and to what extent Chinese cagA-positive Helicobacter pylori isolates differ from those in The Netherlands. Analysis of random amplified polymorphic DNA (RAPD)-PCR-assessed DNA fingerprints of chromosomal DNA of 24 cagA-positive H. pylori isolates from Dutch (n = 12) and Chinese (n = 10) patients yielded the absence of clustering. Based on comparison of the sequence of a 243-nucleotide part of cagA, the Dutch (group I) and Chinese (group II) H. pylori isolates formed two separate branches with high confidence limits in the phylogenetic tree. These two clusters were not observed when the sequence of a 240-bp part of glmM was used in the comparison. The number of nonsynonymous substitutions was much higher in cagA than in glmM, indicating positive selection. The average levels of divergence of cagA at the nucleotide and protein levels between group I and II isolates were found to be high, 13.3 and 17.9%, respectively. Possibly, the pathogenicity island (PAI) that has been integrated into the chromosome of the ancestor of H. pylori now circulating in China contained a different cagA than the PAI that has been integrated into the chromosome of the ancestor of H. pylori now circulating in The Netherlands. We conclude that in China and The Netherlands, two distinct cagA-positive H. pylori populations are circulating.     

CD8+ T cells are associated with severe gastritis in Helicobacter pylori-infected mice in the absence of CD4+ T cells

Helicobacter pylori infection results in the development of chronic gastritis, and CD4⁺ T cells are a major component of the gastric cellular infiltrate. To examine whether CD4⁺ T cells are important in initiating and maintaining H. pylori-induced gastritis, mice deficient in CD4⁺ T cells (B6.BM1.GK 1.5 mice [GK 1.5 mice]) were infected with H. pylori. We found that as in normal mice, H. pylori-specific antibodies, mostly of the immunoglobulin M isotype, developed in GK 1.5 mice but were unable to cure H. pylori infection. Further, while the stomachs of H. pylori-infected GK 1.5 mice were more heavily infiltrated with CD8⁺ T cells and B cells, mice deficient in both CD4⁺ and CD8⁺ T cells developed mild inflammation comparable to the level observed for C57BL/6 mice. These observations suggest that CD4⁺ T cells may play an important role in regulating or suppressing gastric CD8⁺ T cells which, in the absence of CD4⁺ T cells, may mediate more-severe disease. These studies have revealed a potentially important role for CD8⁺ T cells in the gastric disease resulting from H. pylori infection.     

Comparison of Two Rapid Whole-Blood Tests for Helicobacter pylori Infection in Chinese Patients

Consecutive Chinese patients undergoing endoscopy for dyspepsia were tested for Helicobacter pylori infection by two rapid whole-blood tests: FlexPack HP (Abbott Laboratories) and Helisal One-Step (Cortecs Diagnostics). Biopsy-based tests (rapid urease test and histology) and the [¹³C]urea breath test were used as the “gold standard.” One hundred sixty-one consecutive patients were studied, and 88 (54.7%) were confirmed to have H. pylori infection. The sensitivities, specificities, and positive and negative predictive values were 81.8%, 83.6% (P = 0.008), 85.7% (P = 0.04), and 79.2% for FlexPack HP and 84.1%, 63.0% (P = 0.008), 73.3% (P = 0.047), and 76.7% for Helisal One-Step, respectively.     

Decreased Levels of CD54 (ICAM-1)-Positive Lymphocytes in the Peripheral Blood in Untreated Patients with Active Juvenile Dermatomyositis

Significant abnormalities are observed in the peripheral blood of juvenile dermatomyositis (JDM) patients with active disease. In this study, we confirm that there is a significant increase in the relative percentage of B lymphocytes in the peripheral blood of a group of untreated children with newly diagnosed active JDM compared to healthy children (P < 0.0001). In order to investigate if properties intrinsic to B cells contributed to their relative increase in JDM, the percentage of B cells expressing activation markers (CD23, CD25, CD54, and CD69) was measured and compared to pediatric controls. Compared to healthy children less than 10 years of age (not significantly different from the JDM group), the JDM patients had an increase in the proportion of lymphocytes expressing CD19 (B cells; P = 0.0017) and decreases in the percentage of lymphocytes that were CD3⁻ CD16⁺ and/or CD56⁺ (NK cells; P = 0.01) and CD3⁺ CD8⁺ (T suppressor/cytotoxic cells; P = 0.02). There were no significant differences in any of the B-cell activation markers assessed. Of note, the percentage of CD54⁺ non-B lymphocytes (i.e., T cells and NK cells expressing CD54) was significantly lower in the JDM patients (25% ± 5%) than in the “age-related” healthy control group (43% ± 4%; P = 0.013). These results suggest the following for untreated children with active JDM: (i) the increase in the percentage of peripheral blood B cells is not due to intrinsic B-cell activation, and (ii) CD54/ICAM-1⁺ non-B cells, CD8⁺ T cells, and NK cells are being removed from circulation and may be participating in the pathophysiology of the disease.     

Characterization of Clostridium difficile strains isolated from patients in Ontario, Canada, from 2004 to 2006

Clostridium difficile is the bacterium most commonly surmised to cause antimicrobial- and hospital-associated diarrhea in developed countries worldwide, and such infections are thought to be increasing in frequency and severity. A laboratory-based study was carried out to characterize C. difficile strains isolated from persons in Ontario, Canada, during 2004 to 2006 according to toxin type (enterotoxin A, cytotoxin B, and binary toxin [CDT]), tcdC gene characterization, ribotyping, pulsed-field gel electrophoresis, and toxinotyping. Clostridium difficile was isolated from 1,080/1,152 (94%) samples from 21 diagnostic laboratories. Isolates with toxin profiles A⁺ B⁺ CDT⁻, A⁺ B⁺ CDT⁺, A⁻ B⁺ CDT⁻, and A⁻ B⁺ CDT⁺ accounted for 63%, 34%, 2.4%, and 0.6% of isolates, respectively. Alterations in tcdC were detected in six different ribotypes, including ribotype 027. A total of 39 different ribotypes were identified, with ribotype 027/North American pulsotype 1 (NAP1), an internationally recognized outbreak strain associated with severe disease, being the second most common ribotype (19% of isolates). Transient resistance to metronidazole was identified in 19 (1.8%) isolates. While a large number of ribotypes were found, a few predominated across the province. The high prevalence and wide distribution of ribotype 027/NAP1 are disconcerting in view of the severity of disease associated with it.     

Anti-CagA Immunoglobulin G Responses Correlate with Interleukin-8 Induction in Human Gastric Mucosal Biopsy Culture

Helicobacter pylori persists in the human stomach despite eliciting both cellular and humoral immune responses and inducing proinflammatory cytokines. To determine whether local humoral and cytokine responses are related to each other and to histologic responses, we studied 66 Japanese patients who underwent gastroscopy. Using specific enzyme-linked immunosorbent assays, we examined gastric antral mucosal-organ biopsy culture supernatants to assess interleukin-6 (IL-6) and interleukin-8 (IL-8) levels and antibody responses to H. pylori whole-cell antigens CagA, HspA, and HspB. Of the patients studied, 11 were H. pylori negative and 55 were H. pylori positive; by PCR, all strains were cagA⁺. As expected, compared to H. pylori-negative patients, H. pylori-positive patients had significantly higher humoral responses to all H. pylori antigens and had higher IL-8 (47.8 ± 3.5 versus 10.1 ± 4.3 ng/mg of biopsy protein; P < 0.001) and IL-6 levels (2.8 ± 0.3 versus 0.26 ± 0.2 ng/mg of protein; P < 0.001). Among the H. pylori-positive patients, supernatant anti-CagA immunoglobulin G (IgG) levels were significantly associated with H. pylori density (P < 0.005) and neutrophil infiltration (P < 0.005) scores. Anti-CagA immunoglobulin A levels were correlated with intestinal metaplasia (P < 0.05). Mononuclear cell infiltration scores were significantly associated with supernatant IL-6 levels (P < 0.005) and with IgG responses to whole-cell antigens (P < 0.05). Supernatant IL-8 levels were significantly associated with anti-CagA IgG (r = 0.75, P < 0.001). Anti-CagA responses correlated with neutrophil infiltration, intestinal metaplasia, H. pylori density, and IL-8 levels, suggesting that the absolute levels of these antibodies may be markers for gastric inflammation and premalignant changes in individual hosts.     

Cytotoxicity of Hemolytic, Cytotoxic Necrotizing Factor 1-Positive and -Negative Escherichia coli to Human T24 Bladder Cells

Approximately one-half of Escherichia coli isolates from patients with cystitis or pyelonephritis produce the pore-forming cytotoxin hemolysin, a molecule with the capacity to lyse erythrocytes and a range of nucleated cell types. A second toxin, cytotoxic necrotizing factor 1 (CNF1), is found in approximately 70% of hemolytic, but rarely in nonhemolytic, isolates. To evaluate the potential interplay of these two toxins, we used epidemiological and molecular biologic techniques to compare the cytotoxicity of hemolytic, CNF1⁺, and CNF1⁻ cystitis strains toward human T24 bladder epithelial cells in vitro. A total of 29 isolates from two collections of cystitis-associated E. coli were evaluated by using methylene blue staining of bladder monolayers at 1-h intervals after inoculation with each strain. Most (20 of 29) isolates damaged or destroyed the T24 monolayer (less than 50% remaining) within 4 h after inoculation. As a group, CNF1⁺ isolates from one collection (11 strains) were less cytotoxic at 4 h than the CNF1⁻ strains in that collection (P = 0.009), but this pattern was not observed among isolates from the second collection (18 strains). To directly evaluate the role of CNF1 in cytotoxicity of hemolytic E. coli without the variables present in multiple clinical isolates, we constructed mutants defective in production of CNF1. Compared to the CNF1⁺ parental isolates, no change in cytotoxicity was detected in these cnf1 mutants. Our results indicate that CNF1 does not have a detectable effect on the ability of hemolytic E. coli to damage human bladder cell monolayers in vitro.     

Serological Assessment of the Early Response to Eradication Therapy Using an Immunodominant Outer Membrane Protein of Helicobacter pylori

Eradication of Helicobacter pylori infection cures gastritis and prevents recurrence of peptic ulcers. Endoscopy is usually used to evaluate the effectiveness of eradication therapy. We designed a new noninvasive assay system for the early evaluation of eradication of H. pylori infection in which a crude H. pylori outer membrane protein preparation (HPOmp) is used as an antigen, and we determined the sensitivity and specificity of the serological assay system. Immunoblot analysis showed that anti-HPOmp antibodies reacted to a protein with a molecular mass of approximately 29 kDa. In those patients who responded to therapy, the anti-HPOmp immunoglobulin G (IgG) titers measured by enzyme-linked immunosorbent assay (ELISA) at 1 month after the end of therapy were significantly lower than those before treatment (34.8% reduction; P < 0.001), and the posttreatment reduction in the antibody titer was significantly greater than that of the titer measured with a commercially available anti-H. pylori IgG ELISA (34.8% versus 16.1%; P < 0.001). When a 25% reduction of anti-HPOmp IgG titer at 1 month after the end of treatment was taken as the cutoff value for H. pylori eradication, the sensitivity and specificity of our new assay were 75% (51 of 68 treatment responders) and 96% (22 of 23 nonresponders), respectively. Our results indicate that the novel serological test with HPOmp might be a clinically useful tool for assessment of eradication of H. pylori.     

Upregulation of CCL20 and recruitment of CCR6+ gastric infiltrating lymphocytes in Helicobacter pylori gastritis

Helicobacter pylori infection is associated with an inflammatory response in the gastric mucosa, leading to chronic gastritis, peptic ulcers, and gastric cancer. There is increased T-cell infiltration at the site of infection with H. pylori. CCR6, a specific β-chemokine receptor for CCL20 (MIP-3α/LARC/exodus), has recently been reported to mediate lymphocyte homeostasis and immune responses in mucosal tissue, and it may play a role in chemokine-mediated lymphocyte trafficking during gastric inflammation. In this study, we investigated the role of CCR6 and its ligand, CCL20, in inducing an inflammatory response in the gastric mucosa during H. pylori infection. Gastric infiltrating T lymphocytes were isolated from endoscopic biopsy specimens of H. pylori gastritis patients and analyzed for the expression of the CCR6 chemokine receptor. Our results demonstrated that there was significantly increased CCR6 expression in CD3⁺ T cells infiltrating the gastric mucosa, and the CCR6 ligand, the CCL20 chemokine, was selectively expressed in inflamed gastric tissues. The production of CCL20 was upregulated in response to H. pylori in gastric epithelial cells when there was stimulation by the proinflammatory cytokines interleukin-1β and tumor necrosis factor alpha. Furthermore, recombinant CCL20 induced lymphocyte chemotaxis migration in fresh gastric T cells ex vivo, indicating that the gastric T cells could migrate toward inflammatory sites via CCR6/CCL20 interaction. Our results suggest that the interaction between CCL20 and CCR6 may play a role in chemokine-mediated lymphocyte trafficking during gastric inflammation in Helicobacter infection.     

Lewis antigen expression by Helicobacter pylori strains colonizing different regions of the stomach of individual patients

The diversity in the expression of Lewis antigens (Le) of 226 single colonies of Helicobacter pylori isolated from four regions of the stomach of eight adults is shown. Le^y was expressed more in strains colonizing antrum than in strains colonizing fundus, whereas Le^x was more common in fundus strains. cagA⁺ strains were more associated with Le-negative strains.     

Analysis of Clarithromycin Resistance and CagA Status in Helicobacter pylori by Use of Feces from Children in Thailand

The clarithromycin resistance and CagA status of Helicobacter pylori in Thai children were investigated using fecal samples. Of the 284 samples, H. pylori was detected in 120 samples, and the clarithromycin resistance rate was 29.2%. The cagA gene was detected in 59 samples, and only 6.8% of these samples contained the East Asian CagA type.Helicobacter pylori is a pathogenic bacterium that colonizes the human stomach. The prevalence of antibiotic-resistant H. pylori, especially clarithromycin-resistant H. pylori, has been increasing worldwide and makes it difficult to successfully eradicate H. pylori. Clarithromycin resistance in H. pylori has been shown to be due to mutations at positions 2142 and 2143 of the 23S rRNA gene (7, 9).Although H. pylori is closely associated with gastric cancer, the rate of mortality due to gastric cancer is relatively low in Thailand, even though the rate of H. pylori infection in Thailand has been reported to be over 80% (6, 8). A difference in pathogenicity between H. pylori strains may explain the lack of the expected correlation between the rate of mortality due to gastric cancer and the rate of H. pylori infection. The CagA protein, which is one of the most important pathogenicity factors of H. pylori, has been classified into two types: the East Asian CagA type, found in H. pylori isolates from Japan, South Korea, and China, and the Western CagA type, found in H. pylori isolates from Europe, North America, and Australia. Each CagA type has tyrosine phosphorylation segments characterized by a Glu-Pro-Ile-Tyr-Ala (EPIYA) motif in the C-terminal region (3). However, the Western CagA type contains the EPIYA-A and EPIYA-B segments, followed by a variable number of EPIYA-C segments, while the East Asian CagA type contains the EPIYA-A, EPIYA-B, and EPIYA-D segments. Furthermore, the East Asian CagA type has been reported to induce more-severe cellular changes than the Western CagA type (2).Recently, we identified a noninvasive method for detecting clarithromycin-resistant H. pylori isolates from feces with high sensitivity and specificity (5). In this study, we used this previously developed method to investigate the clarithromycin resistance and CagA status of H. pylori in feces of Thai children.Fecal samples were obtained from 284 children (116 males/116 females; mean age, 6.60 years [range, 1 to 12 years]) from three schools in Chiang Mai in August 2006. The ages and genders of 52 children of ethnic minorities could not be obtained. The study protocol and the informed-consent document were reviewed and approved by Research Ethics Committee, Faculty of Medicine, Chiang Mai University.DNA was extracted from the feces, and the 23S rRNA gene of H. pylori was amplified as previously described (5). Samples were considered to contain clarithromycin-resistant H. pylori if mutations at positions 2142 and 2143 of the 23S rRNA gene were detected.For the amplification of the cagA gene, we designed new primers targeting the region containing the EPIYA-A and EPIYA-B segments by comparing 81 of the cagA genes registered in the DNA Data Bank of Japan (data not shown). Amplification was performed using primer pairs comprising primers 2553F (5′-AACCCTAGTCGGTAATGGGTTRTCT-3′) and 3222R (5′-ATTGCTATTAATGCGTGTGTGGC-3′) for the first-round PCR and 2612F (5′-CGGACATCAGGAAAGAATTGAA-3′), 2609F (5′-TTTCGGATATCAAGAAGAATTGAA-3′), and 2998R (5′-TTGAAAGCCCTACTTTACTGAGATCA-3′) for the second-round PCR.Samples were designated cagA positive when a PCR product of 180 bp was detected after the third-round PCR, which was performed using Go Taq Green master mix (Promega, Madison, WI) and the 2609F, 2612F, 2779R (5′-CACTCACCTTTTTTAGCAACTTGAG-3′), and 2780R (5′-GCTTTTACCTTTTTAGCAACTTGAG-3′) primers. The cagA-typing PCR was performed using an East Asian CagA-specific primer pair (East-Asian-F [5′-AAAGGAGTGGGCGGTTTCA-3′] and East-Asian-R [5′-CCTGCTTGATTTGCCTCATCA-3′]) and a Western CagA-specific primer pair (Western-F [5′-GGCATGATAAAGTTGATGATCTCAGT-3′] and Western-R [5′-AAAGGTCCGCCGAGATCAT-3′]), which targeted the EPIYA-D and EPIYA-C segments, respectively. Each typing PCR was performed using 1.5 μl of the second PCR product. For each PCR amplification, a PCR mixture that contained ultrapure water as the template was included to rule out false-positive results.Of the 284 fecal samples obtained from Thai children, H. pylori was detected in 120 (42.3%). Of the 120 H. pylori-positive samples, clarithromycin-resistant H. pylori was detected in 35 (29.2%) samples, and both clarithromycin-susceptible and -resistant H. pylori isolates were detected simultaneously in 5 samples. The incidence of clarithromycin-resistant H. pylori in this study was slightly higher than the rate reported for Thai adults in other studies (23.2%) (4). Because there have been few studies that focused on the rate of clarithromycin-resistant H. pylori in Thai children, the results of this study will be useful for estimating the rate of clarithromycin-resistant H. pylori infection in adults in Thailand in the future.The cagA gene was present in 59 (49.2%) of the H. pylori-positive samples. Of these samples, 20 (33.9%) samples contained the Western CagA type (containing the EPIYA-C segments) and 4 (6.8%) contained the East Asian CagA type (containing the EPIYA-D segments). The remaining 35 samples, which lacked any EPIYA-C or EPIYA-D motifs, were considered to contain the Western CagA type (1). To confirm the absence of the EPIYA-C and EPIYA-D segments in these 35 samples, DNA sequencing was performed on 14 cagA genes by using the second PCR product, and none of the cagA genes analyzed had an EPIYA-C or EPIYA-D segment. In summary, H. pylori isolates containing the East Asian CagA type (6.8%) were significantly less prevalent than H. pylori isolates containing the Western CagA type (93.2%). Most of the H. pylori strains isolated in Asian countries with high incidences of deaths from gastric cancer, such as Japan and China, have been reported to contain the East Asian CagA type (10). Since differences in the prevalences of the East Asian CagA type in Asian countries have been suggested to be one of the reasons underlying the differential mortality rates associated with gastric cancer (2), further investigation is needed to confirm this possibility.In this study, we detected a high proportion (59.3%) of CagA that contained neither the EPIYA-C nor the EPIYA-D segment. The low incidence of Western CagA containing the EPIYA-C segment in Thailand may be one of the reasons for the low mortality rate associated with gastric cancer in Thailand.In conclusion, we show that the prevalence of the CagA types of H. pylori in Thai children differs from that reported for other Asian countries. Furthermore, our study demonstrates the usefulness of this approach for detecting and typing CagA in H. pylori by using feces. This method may prove useful in further investigations of the prevalences of the CagA types in H. pylori isolates from infected individuals.