Infection with CagA-positive Helicobacter pylori strain containing three EPIYA C phosphorylation sites is associated with more severe gastric lesions in experimentally infected Mongolian gerbils (Meriones unguiculatus).

Infection with Helicobacter pylori strains containing high number of EPIYA-C phosphorylation sites in the CagA is associated with significant gastritis and increased risk of developing pre-malignant gastric lesions and gastric carcinoma. However, these findings have not been reproduced in animal models yet. Therefore, we investigated the effect on the gastric mucosa of Mongolian gerbil (Meriones unguiculatus) infected with CagA-positive H. pylori strains exhibiting one or three EPIYA-C phosphorilation sites. Mongolian gerbils were inoculated with H. pylori clonal isolates containing one or three EPIYA-C phosphorylation sites. Control group was composed by uninfected animals challenged with Brucella broth alone. Gastric fragments were evaluated by the modified Sydney System and digital morphometry. Clonal relatedness between the isolates was considered by the identical RAPD-PCR profiles and sequencing of five housekeeping genes, vacA i/d region and of oipA. The other virulence markers were present in both isolates (vacA s1i1d1m1, iceA2, and intact dupA). CagA of both isolates was translocated and phosphorylated in AGS cells. After 45 days of infection, there was a significant increase in the number of inflammatory cells and in the area of the lamina propria in the infected animals, notably in those infected by the CagA-positive strain with three EPIYA-C phosphorylation sites. After six months of infection, a high number of EPIYA-C phosphorylation sites was associated with progressive increase in the intensity of gastritis and in the area of the lamina propria. Atrophy, intestinal metaplasia, and dysplasia were also observed more frequently in animals infected with the CagA-positive isolate with three EPIYA-C sites.  We conclude that infection with H. pylori strain carrying a high number of CagA EPIYA-C phosphorylation sites is associated with more severe gastric lesions in an animal model of H. pylori infection.

Effect of eugenol exposure time and post-removal delay on the bond strength of a self-etching adhesive to dentin.

OBJECTIVES: This study evaluated the effects of the exposure time of eugenol-based provisional restorative material and the time elapsed between the provisional material removal and the adhesive procedure on the bond strength of the composite to dentin. MATERIALS AND METHODS: Human third molars were sectioned into two halves that were enclosed in resin cylinders. The cavities were prepared over the buccal/lingual faces with diamond burs. Zinc oxide and eugenol (ZOE) provisional material was inserted into cavities and left for 24 hours, 7 days or 14 days. The cavities not restored with ZOE were used as controls. After ZOE removal or over fresh dentin (control), self-etching Adper SE Plus was applied immediately, after a 7- or 14-day delay. The cavity was restored with non-eugenol provisional material during this delay period. Cylinders of resin cement were built-up over the hybridized dentin. A shear load was applied to the cylinders at a crosshead speed of 0.5 mm/minute until failure. The data were statistically analyzed using two-way ANOVA and Tukey's tests (α=0.05). RESULTS: Using IRM as a provisional restoration for 24 hours followed by its removal and immediate adhesive application resulted in the lowest values of shear bond strength. There was no significant difference between the other experimental conditions. CONCLUSIONS: The use of IRM for 24 hours adversely affected the shear bond strength of a self-etching adhesive to dentin. The recovery of the proper bond strength occurred one week after IRM removal.

Differences in peripheral blood lymphocyte phenotypes between Helicobacter pylori-positive children and adults with duodenal ulcer.

The immunological mechanisms involved in the development of duodenal ulcer, especially in childhood, are unclear. Helicobacter pylori-positive children and adults, with and without duodenal ulcer, were therefore compared with respect to CD4(+) T-cells, and CD8(+) T-cells, B-cells and B1a-cells, as well as cell activation (CD4(+)/HLA-DR(+) and CD8(+)/HLA-DR(+)) and co-stimulatory (CD4(+)/CD28(+) and CD8(+)/CD28(+)) markers, in peripheral blood. Children with and without duodenal ulcer differed significantly. In particular, there was a phenotypic change in CD8(+) T-cells from children with ulcer that involved a 200% increase in the number of CD8(+)/HLA-DR(+) cells/mm(3) and a decrease of 34.2% in the number of CD8(+)/CD28(+) cells/mm(3). This phenotype of chronically activated memory CD8(+) T-cells, which has also been observed in patients with AIDS and tuberculosis, is associated with disease severity and progression. A lower frequency of B1a-cells was also observed in the group of children with ulcer. Conversely, no difference between infected adults with and without ulcer was observed, but the percentage of CD4(+)/HLA-DR(+) cells was lower in adults with ulcer, suggesting that a down-regulated immune response may play a role in the development of duodenal ulcer in adults. Gastric inflammation correlated positively with CD4(+) and chronically activated CD4(+) T-cells in children and adults without duodenal ulcer, respectively. These results suggest that there are differences in the immunophenotyping profile between H. pylori-positive children and adults with duodenal ulcer, indicating the possibility of distinct immune mechanisms in the development of the disease according to age.

The role of IFN-gamma and IL-4 in gastric mucosa inflammation associated with Helicobacter heilmannii type 1 infection.

Although Helicobacter heilmannii infection is less common than H. pylori infection in humans, it is considered to be of medical importance because of its association with gastritis, gastric ulcer, carcinoma, and mucosa-associated lymphoid tissue lymphoma of the stomach. However, there have been no studies evaluating the role of the Th cell response in H. heilmannii gastric infection. We evaluated the participation of pro-inflammatory and anti-inflammatory cytokines, IFN-gamma and IL-4, in H. heilmannii gastric infection in genetically IFN-gamma- or IL-4-deficient mice. The serum IFN-gamma and IL-4 concentrations were determined by ELISA. The gastric polymorphonuclear infiltrate was higher (P = 0.007) in H. heilmannii-positive than in H. heilmannii-negative wild-type (WT) C57BL/6 mice, whereas no significant inflammation was demonstrable in the stomach of H. heilmannii-positive IFN-gamma(-/-) C57BL/6 mice. The degree of gastric inflammatory cells, especially in oxyntic mucosa, was also higher (P = 0.007) in infected IL-4(-/-) than in WT BALB/c mice. Serum IFN-gamma levels were significantly higher in IL-4(-/-) than in WT BALB/c mice, independently of H. heilmannii-positive or -negative status. Although no difference in serum IFN-gamma levels was seen between H. heilmannii-positive (11.3 +/- 3.07 pg/mL, mean +/- SD) and -negative (11.07 +/- 3.5 pg/mL) WT BALB/c mice, in the group of IL-4(-/-) animals, the serum concentration of IFN-gamma was significantly higher in the infected ones (38.16 +/- 10.5 pg/mL, P = 0.04). In contrast, serum IL-4 levels were significantly decreased in H. heilmannii-positive (N = 10) WT BALB/c animals compared to the negative (N = 10) animals. In conclusion, H. heilmannii infection induces a predominantly Th1 immune response, with IFN-gamma playing a central role in gastric inflammation.

The role of IFN-gamma and IL-4 in gastric mucosa inflammation associated with Helicobacter heilmannii type 1 infection

Although Helicobacter heilmannii infection is less common than H. pylori infection in humans, it is considered to be of medical importance because of its association with gastritis, gastric ulcer, carcinoma, and mucosa-associated lymphoid tissue lymphoma of the stomach. However, there have been no studies evaluating the role of the Th cell response in H. heilmannii gastric infection. We evaluated the participation of pro-inflammatory and anti-inflammatory cytokines, IFN-gamma and IL-4, in H. heilmannii gastric infection in genetically IFN-gamma- or IL-4-deficient mice. The serum IFN-gamma and IL-4 concentrations were determined by ELISA. The gastric polymorphonuclear infiltrate was higher (P = 0.007) in H. heilmannii-positive than in H. heilmannii-negative wild-type (WT) C57BL/6 mice, whereas no significant inflammation was demonstrable in the stomach of H. heilmannii-positive IFN-gamma-/- C57BL/6 mice. The degree of gastric inflammatory cells, especially in oxyntic mucosa, was also higher (P = 0.007) in infected IL-4-/- than in WT BALB/c mice. Serum IFN-gamma levels were significantly higher in IL-4-/- than in WT BALB/c mice, independently of H. heilmannii-positive or -negative status. Although no difference in serum IFN-gamma levels was seen between H. heilmannii-positive (11.3 ± 3.07 pg/mL, mean ± SD) and -negative (11.07 ± 3.5 pg/mL) WT BALB/c mice, in the group of IL-4-/- animals, the serum concentration of IFN-g was significantly higher in the infected ones (38.16 ± 10.5 pg/mL, P = 0.04). In contrast, serum IL-4 levels were significantly decreased in H. heilmannii-positive (N = 10) WT BALB/c animals compared to the negative (N = 10) animals. In conclusion, H. heilmannii infection induces a predominantly Th1 immune response, with IFN-gamma playing a central role in gastric inflammation.

Phenotypic study of peripheral blood lymphocytes and humoral immune response in Helicobacter pylori infection according to age.

There are differences between children and adults in certain aspects of the Helicobacter pylori (HP) infection, among them the lower titre of IgG antibodies anti-HP in the former group. Thus, we investigated by means of flow cytometry CD4+/CD3+ (CD4+T), CD8+/CD3+ (CD8+T) and CD19+/CD3- (B) cells, activation/co-stimulatory markers (CD4+/HLA-DR+, CD4+/CD28+, CD8+/HLA-DR+ and CD8+/CD28+) and by means of ELISA IgG anti-HP antibodies in the peripheral blood from HP-positive and -negative children and adults. An increased CD4+/CD28+ and CD8+/CD28+ percentage and number of CD4+/CD3+ cells were seen in infected adults. Conversely, no difference was observed between infected and noninfected children, but when they were stratified by age, an increased CD4+/CD28+ cell percentage was seen in the HP-positive group older than 10 years. The mean level of IgG anti-HP was lower in younger infected children, increased with age and correlated with CD4+ cells. Our data suggest that the immune response to HP infection vary according to the age. Low percentage of activated CD4+ cell may contribute to the lower level of serum IgG anti-HP observed in younger infected children. In addition, the CD4+ cell participation during the infection seems to begin after 10 years old, when the immune response becomes similar to that seen in adults.

Association of Helicobacter species with hepatitis C cirrhosis with or without hepatocellular carcinoma.

BACKGROUND AND AIMS: Recent studies have suggested that bacterial coinfection with Helicobacter species in patients already infected with hepatitis C virus (HCV) could be involved in the development of cirrhosis and hepatocellular carcinoma (HCC). A retrospective cross sectional study was performed in order to explore the association between Helicobacter species and HCV associated liver diseases. METHODS: The presence of Helicobacter species was tested by polymerase chain reaction on liver samples from four groups of patients. RESULTS: Helicobacter 16S rDNA was found in only 4.2% of liver samples from control patients (n=24) and in 3.5% of liver samples from patients with non-cirrhotic chronic hepatitis C (n=29) while it was found in 68.0% of liver samples from patients with HCV positive cirrhosis without HCC (n=25) as well as in 61.3% of cirrhotic liver samples from patients with HCV positive cirrhosis and HCC (n=31). In addition, when the HCC tumour tissue was tested (n=21), 90.5% of samples were positive. DNA from Helicobacter pylori- and Helicobacter pullorum-like organisms was found. CONCLUSIONS: There is an association between the presence of Helicobacter species DNA in the liver and hepatitis C cirrhosis, with or without HCC. Indeed, the presence of these bacteria could be the result of structural changes in the liver. Alternatively, Helicobacter species could be a co-risk factor in HCV chronic liver diseases. This result warrants prospective studies to determine the possible causal role of these bacteria in the progression of chronic hepatitis C.

Hepatic changes in mice chronically infected with Helicobacter trogontum

We infected NIH germ-free female mice with Helicobacter trogontum, a recently described intestinal bacterium of rats, in order to study the lesions it induced in the liver of this host. Fifteen mice were challenged with a single dose of H. trogontum (test group) and killed 6, 12 and 18 months after inoculation (5 animals/group). Nine animals were challenged with 0.85 percent saline alone (control group) and killed at the same times. Fragments from the liver, cecum and colon were obtained for microbiologic and histologic examination. Stool samples were also collected. H. trogontum was detected in the cecum, colon and/or stool samples of all test mice. As expected, the bacterium was not isolated from any specimen obtained from the control animals. On the other hand, although we could not cultivate the bacterium from the liver, 13 test animals (86.7 percent) presented histological changes in this organ. The 6-month group presented infiltration of mononuclear and polymorphonuclear cells in the hepatic parenchyma and the two other groups presented foci of mononuclear cells. The results suggest that H. trogontum can elicit a hepatic inflammatory response in mice since the only difference between control and test animals was the presence of H. trogontum in the latter. This result, together with the growing number of related reports in the literature, reinforces the possible role of Helicobacter infection in the pathogenesis of hepatobiliary diseases

Hepatic changes in mice chronically infected with Helicobacter trogontum.

We infected NIH germ-free female mice with Helicobacter trogontum, a recently described intestinal bacterium of rats, in order to study the lesions it induced in the liver of this host. Fifteen mice were challenged with a single dose of H. trogontum (test group) and killed 6, 12 and 18 months after inoculation (5 animals/group). Nine animals were challenged with 0.85% saline alone (control group) and killed at the same times. Fragments from the liver, cecum and colon were obtained for microbiologic and histologic examination. Stool samples were also collected. H. trogontum was detected in the cecum, colon and/or stool samples of all test mice. As expected, the bacterium was not isolated from any specimen obtained from the control animals. On the other hand, although we could not cultivate the bacterium from the liver, 13 test animals (86.7%) presented histological changes in this organ. The 6-month group presented infiltration of mononuclear and polymorphonuclear cells in the hepatic parenchyma and the two other groups presented foci of mononuclear cells. The results suggest that H. trogontum can elicit a hepatic inflammatory response in mice since the only difference between control and test animals was the presence of H. trogontum in the latter. This result, together with the growing number of related reports in the literature, reinforces the possible role of Helicobacter infection in the pathogenesis of hepatobiliary diseases.

Prevalence and risk factors associated with Helicobacter pylori infection in native populations from Brazilian Western Amazon.

We evaluated the prevalence of, and factors associated with, Helicobacter pylori infection in 222 subjects from 3 distinct communities of native populations (Uru-Eu-Wau-Wau Indians and 2 riverine communities living on the banks of the Machado river and in Portuchuelo) living in isolation in the rainforest of Brazilian Western Amazon. The overall prevalence was 78.8% (95% CI 72.7-83.9). The prevalence was higher in the Machado river community compared with Portuchuelo (chi2 = 3.84, P = 0.05), but no significant difference was observed between the Machado river community and the Uru-Eu-Wau-Wau Indians. Logistic regression showed that residential crowding and age were factors associated with the presence of H. pylori infection. Acquisition of the bacterium started early in life and by the age of 2 years 50% of children were infected. The prevalence increased with age, reaching near universal levels during adulthood (97.9%). Residential crowding was high with a global index of 3.3 persons/room (SD = 1.8), varying significantly between the 3 communities (P = 0.001). These data provide further evidence supporting direct person-to-person spread of the bacterium.

Cellular immune responses in Helicobacter heilmannii infection: evaluation of the role of the host and the bacterium.

We evaluated some aspects of the immune response to Helicobacter heilmannii in two mouse strains. Gastritis that was more severe in infected C57BL/6 mice. A proliferative response to H. pylori antigens was observed in splenocytes from H. heilmannii-positive and -negative mice, similar in the positive- and negative-BALB/c mice, but lower in the positive- than in the negative-C57BL/6 animals. A decrease in B cells and an increase in CD4+ cells after stimulation with type I H. pylori antigen and an increase in CD8+ cells after stimulation with type I and II antigens was observed in infected C57BL/6 mice. Conversely, the percentage of CD4+, CD8+, and B cells was similar in positive- and negative-BALB/c mice. These results demonstrated that the immune response is similar in H. heilmannii and H. pylori infection and strengthened the importance of host and bacterial virulence markers in the immune response to gastric Helicobacter infections.

Anti-CagA antibodies in Helicobacter pylori-positive patients and blood donors from Nigeria.

The prevalence of antibodies to CagA protein was evaluated in 174 Helicobacter pylori-positive. subjects: 110 patients submitted to upper gastrointestinal endoscopy and 64 male blood donors. The patients were from different regions of Nigeria: Jos, North, Benue,West and East and the blood donors were from Jos. Sera were assayed for anti-CagA antibodies using Helicobacter p120, CagA ELISA (Viva Diagnostika, Hürth, Germany). Anti-CagA antibodies were detected in 104 (94.5%) patients and in 61 (95.3%) blood donors (P=1.0). Patients from the Benue region presented a lower frequency of anti-CagA antibodies than patients from the other regions (P=0.0004). When the subjects were stratified by age, a significant increase in IgG concentration was observed among the men (P=0.03) but not among the women (P=0.57) or among the blood donors (P=0.83). In conclusion, similarly toAsian countries, CagA-positive H. pylori infection is highly frequent in Nigeria.

iceA genotypes of Helicobacter pylori strains isolated from Brazilian children and adults.

Data concerning the geographic distribution of iceA alleles are scarce, and information on the association of the gene with the disease is rare and still controversial. Furthermore, no such study has been developed in Brazil, where duodenal ulcer and gastric adenocarcinoma are very common. We investigated, by PCR, the frequency of iceA alleles and cagA status in Helicobacter pylori strains isolated from 142 patients (62 children and 80 adults; 66 female; mean age, 30.0 years; age range, 3 to 78 years) with gastritis, duodenal ulcer, or gastric adenocarcinoma. iceA was identified in bacterium samples obtained from all patients. Eleven (7.7%) of them were infected with multiple strains. Among the patients with nonmixed infection, iceA2 allele was detected in 118 (90.1%). iceA2 allele was associated with ulcer (P = 0.02) and with carcinoma (P = 0.001). iceA2 amplicons of 229, 334, or 549 bp were detected, but none of them was associated with the patient's disorder. iceA2 strains were more frequent in patients older than 7 years (P = 0.001). The gene was also more frequent in strains obtained from males (P = 0.02). cagA was more common in strains obtained from carcinoma (P = 0.0008) and ulcer patients (P < 0.006). cagA-positive strains were more frequent in children older than 7 years (P < 0.003). No association between cagA status and sex was found (P = 0.28). In conclusion, we think iceA should not be used as a reliable marker for predicting the clinical outcome of H. pylori infection.

Accurate prediction of macrolide resistance in Helicobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: multicenter validation study.

Helicobacter pylori strains from 299 patients were tested in six laboratories in different countries. Macrolide susceptibility of the strains was determined by agar dilution (17.4%) or the epsilometer test (82.6%). Mutations in the 23S ribosomal DNA (rDNA) that are associated with macrolide resistance were analyzed by PCR and reverse hybridization (PCR-line probe assay [LiPA]). This method identifies A2115G, G2141A, A2142G, A2142C, A2142T, A2143G, and A2143C mutations in the 23S rDNA. vacA s-region (s1a, s1b, s1c, and s2) and m-region (m1, m2a, and m2b) genotypes and cagA status were also determined using another PCR-LiPA system. Of the 299 strains investigated by MIC testing, 130 (43.5%) were resistant and 169 (56.5%) were susceptible to clarithromycin. Of the 130 resistant strains, 127 (97.7%) contained 23S rDNA mutations, whereas 167 (98.8%) of the 169 susceptible strains contained wild-type sequences. The predominant mutations were A2143G (45.2%) and A2142G (33.3%). Twenty-eight (19.8%) strains contained multiple 23S rDNA mutations. Only five resistant strains contained the A2142C mutation (three of these in combination with the A2142G mutation), and the A2115G, G2141A, A2142T, and A2143C mutations were not found. MICs of clarithromycin for the A2142G mutant strains were significantly higher than MICs for the A2143G strains. Although there was no significant association between 23S rDNA mutations and the vacA and cagA status, clarithromycin-susceptible strains more often contained mixed vacA genotypes, indicating the presence of multiple H. pylori strains. In conclusion, our data confirmed the very strong association between 23S rDNA mutations and macrolide resistance and showed that the PCR-LiPA permits accurate and reliable diagnosis of macrolide resistance in H. pylori.

vacA genotypes in Helicobacter pylori strains isolated from children with and without duodenal ulcer in Brazil.

Data concerning the association between vacA genotypes and disease in children in both developed and developing countries are scarce, especially because of the small number of children with a duodenal ulcer studied. The vacA genotypes of Helicobacter pylori strains obtained from 65 children (24 with a duodenal ulcer and 41 without a duodenal ulcer; 33 girls; mean age, 10.2 years; age range, 1 to 17 years) were investigated as described by J. C. Atherton et al. (J. Clin. Microbiol. 37:2979-2982, 1999). Ten (15.4%) children were infected with more than one H. pylori strain. None of these patients were included in our analysis of the relationship between gastric disorders and specific vacA genotypes. The s1 allele was detected in all H. pylori strains isolated from patients with a duodenal ulcer and from 21 (58.3%) patients without a duodenal ulcer (P = 0.003). Strains with the s2 allele were found only in patients without ulcer (n = 15; 41.7%). Most s1 strains had the s1b allele (97.5%), a result similar to that reported for adults from the Iberian peninsula, which could reflect the Brazilian population origin. One untypeable s1 strain was isolated. The m1 allele was also more frequently found in strains obtained from duodenal ulcer patients (P = 0.028). The m2 allele was found in strains obtained from 20 (36. 4%) children, 3 (15.8%) with an ulcer and 17 (47.2%) without an ulcer. Only one m hybrid strain (m1 and m2 hybrid) was detected. It was demonstrated for the first time that the frequencies of colonization with strains with the s1 allele (14.3% in children up to 8 years of age and 85.7% in older patients; P = 0.012) and of strains with the m1 allele (11.1% in patients up to the age 8 years and 88.9% in older children; P = 0.013) increase with age.

Immunoblot analysis of humoral immune response to Helicobacter pylori in children with and without duodenal ulcer.

Several studies have demonstrated that enzyme-linked immunosorbent assay is not a sensitive and specific method to diagnose Helicobacter pylori infection in children, especially in the younger ones. Since serum immune response can also be determined by immunoblotting and it permits the detection of antibodies to virulence factors such as CagA and VacA, we evaluated the accuracy of a commercial immunoblotting test to diagnose H. pylori infection and to assess the humoral immune response to different H. pylori antigens in 122 children who underwent upper gastrointestinal endoscopy. The presence of H. pylori was determined in antral biopsy specimens by culture, preformed urease test, and histological analysis. H. pylori was identified by microbiological and histopathological methods in 66 children (including all of the 21 who had duodenal ulcer). Antibodies to H. pylori were detected in 63 infected children and in 8 noninfected ones. The sensitivity, specificity, and positive and negative predictive values of the immunoblotting test were 95.5, 85.7, 88.7, and 94.1%, respectively. The number of immunoreactive bands increased with age (P = 0.003), and the bands of 35 kDa (P = 0.013); 89 kDa, the VacA antigen (P = 0.001); and 116 kDa, the CagA antigen (P = 0.00004) were more frequently observed in older children. The frequency of the bands of 89 kDa (P = 0.001) and 116 kDa (P = 0.03) was higher in children with duodenal ulcer than in H. pylori-positive children without the disease. In conclusion, the immunoblotting test appears to be useful for the diagnosis of H. pylori infection in children, even in the younger ones.

Factors associated with Helicobacter pylori infection by a cagA-positive strain in children.

Although infection with a cagA-positive Helicobacter pylori strain is considered a risk factor for the development of duodenal peptic ulcer in adults, this association has not been demonstrated in children. The presence of cagA was investigated by polymerase chain reaction in H. pylori strains isolated from 27 children with duodenal ulcer and 53 without duodenal ulcer. All patients (100%) with duodenal ulcer and 33 (62.3%) without ulcer were colonized by a cagA-positive strain (P=.00007). A cagA-positive status was also associated with a more marked macroscopic gastritis, with a greater inflammatory infiltrate of both mononuclear and polymorphonuclear cells in the antral and oxyntic gastric mucosae and degenerative and regenerative changes of the gastric mucosa. Increased cagA positivity was also associated with increased age, but no association between cagA-positive status and sex was observed.