Immunolocalisation of ghrelin and obestatin in human testis, seminal vesicles, prostate and spermatozoa.

The role of ghrelin and obestatin in male reproduction has not completely been clarified. We explored ghrelin and obestatin localisation in the male reproductive system. Polyclonal antibodies anti-ghrelin and anti-obestatin were used to detect the expression of these hormones in human testis, prostate and seminal vesicles by immunocytochemistry, while in ejaculated and swim up selected spermatozoa by immunofluorescence. Sertoli cells were positive for both peptides and Leydig cells for ghrelin; germ cells were negative for both hormones. Mild signals for ghrelin and obestatin were observed in rete testis; efferent ductules were the most immune reactive region for both peptides. Epididymis was moderately positive for ghrelin; vas deferens and seminal vesicles showed intense obestatin and moderate ghrelin labelling; prostate tissue expressed obestatin alone. Ejaculated and selected spermatozoa were positive for both peptides in different head and tail regions. This study confirms ghrelin localisation in Leydig and Sertoli cells; the finding that ghrelin is expressed in rete testis, epididymis, vas deferens and seminal vesicles is novel, as well as the localisation of obestatin in almost all tracts of the male reproductive system. This research could offer insights for stimulating other studies, particularly on the role of obestatin in sperm physiology, which is still obscure.

Development of a mouse model of Helicobacter pylori infection that mimics human disease.

The human pathogen Helicobacter pylori is associated with gastritis, peptic ulcer disease, and gastric cancer. The pathogenesis of H. pylori infection in vivo was studied by adapting fresh clinical isolates of bacteria to colonize the stomachs of mice. A gastric pathology resembling human disease was observed in infections with cytotoxin-producing strains but not with noncytotoxic strains. Oral immunization with purified H. pylori antigens protected mice from bacterial infection. This mouse model will allow the development of therapeutic agents and vaccines against H. pylori infection in humans.

Increased phospholipase activity in Helicobacter pylori strains isolated from patients with gastric carcinoma.

PURPOSE: Phospholipase activity, one of Helicobacter pylori pathogenicity factors, has not been investigated enough, so far, although it may induce a remarkable damage to the gastric mucosa. In the present work, we have compared the whole phospholipase activity of H. pylori strains isolated from patients with gastric carcinoma with that of strains isolated from dyspeptic patients without gastric carcinoma. METHODS: We measured the phospholipase activity of one distinct H. pylori colony isolated from each of 10 patients with gastric carcinoma and 10 controls, dyspeptic patients without endoscopic and histological signs of gastric carcinoma. We also determined the phospholipase activity of 20 additional strains isolated from different areas of neoplastic and non-neoplastic tissue of two patients with gastric carcinoma, the cagA and vacA positive G27 and 328 wild strains and their respective vacA and cagA negative isogenic mutants. The whole phospholipase activity of strains was determined by measuring the release of (14)C-labeled palmitic acid from the radioactive l-3-phosphatidylcholine, 1,2-di[1-(14)C]palmiloyl substrate; results were expressed in pmol of palmitic acid per mg of protein. RESULTS: H. pylori strains isolated from patients with gastric carcinoma had levels of phospholipase activity significantly higher than those of strains isolated from controls (99.37 [S.D. 40.45] versus 34.46 [S.D. 16.46], P<0.001). In patients with gastric carcinoma, the mean phospholipase activity of strains isolated from neoplastic tissue was similar to that of strains isolated from non-neoplastic tissues (123.02 [S.D. 44.36] and 115.77 [S.D. 81.48], respectively. Interruption of cagA gene caused a ca. 20% reduction of phospholipase activity (36.38 versus 45.22 of the wild strain); that of vacA caused no reduction of phospholipase activity (26.53 and 25.37 of the wild strain). CONCLUSIONS: The infection by H. pylori strains that produce high levels of phospholipase may increase the risk of developing gastric carcinoma. We hypothesise that indirect products of phospholipase activity, such as prostaglandins, leukotrienes and lysophospholipids, may mediate carcinogenesis.

Helicobacter pylori exotoxins and gastroduodenal diseases associated with cytotoxic strain infection.

This paper describes the characteristics of exotoxins produced by Helicobacter pylori, and in particular the vacuolating toxin (VacA) and the cytotoxin-associated protein (CagA). The possible association between infection by strains of certain phenotypic or genomic types and the seriousness of gastroduodenal diseases is discussed. Helicobacter pylori induces various morphological changes in cells in vitro, but only infection by strains which induce cytovacuolation has been studied at present. In its native form, VacA is a protein aggregate made of subunits with a mass of 95 kDa. In vitro it stimulates a cellular v-type ATPase present on the endosomes and creates an acidic environment inside the vacuoles. It also alters in vitro a K(+)-dependent phosphatase activity and could impair the flux of sodium through the cells. Purified VacA causes ulceration in mice; experimental infection in mice with strains which also express the CagA protein causes gastric erosions, vacuolation and epithelial and stromal polymorphonuclear (PMN) cell infiltration. In vivo vacuolation can be observed in gastric cells from patients infected with type I (VacA-CagA positive) H. pylori. CagA is a protein of 128-140 kDa molecular weight, noncytotoxic and highly immunogenic. It is coexpressed in approximately 70% of cytotoxin-producing strains. In CagA positive strain infection, increased levels of interleukin-8 (IL-8) are secreted by the colonized gastric mucosa. Patients infected by cytotoxic strains and/or patients with anti-CagA antibodies are more likely to have active gastritis, and are more likely to develop peptic ulcer or gastric cancer. The different outcomes of infection could be determined by host factors, diet, or by the age at which infection is acquired.

CagA/cytotoxic strains of Helicobacter pylori and interleukin-8 in gastric epithelial cell lines.

AIMS: To investigate: (1) whether Helicobacter pylori directly induces interleukin-8 (IL-8) message expression and protein secretion in established gastric epithelial cell lines; and (2) if CagA/cytotoxin positive and negative strains of H pylori differ in their ability to induce epithelial IL-8. METHODS: Gastric epithelial cell lines were co-cultured with H pylori NCTC 11637 and 10 clinical isolates (four cytotoxic, six non-cytotoxic) and secreted IL-8 was measured by enzyme linked immunosorbent assay (ELISA). Specific induction of gastric epithelial IL-8 mRNA was examined by reverse transcription and polymerase chain reaction (RT-PCR) amplification. RESULTS: H pylori (NCTC 11637) induced IL-8 secretion from three gastric epithelial cell lines (KATO-3, ST42, AGS) but not from MKN 45 (gastric) or intestinal (SW480, HT29) cell lines. H mustelae did not stimulate IL-8 secretion from KATO-3, ST42, and AGS cells. H pylori induced IL-8 secretion was reduced by heat killing, sonication, freeze thawing or formalin fixation of the bacteria. CagA/cytotoxin positive strains of H pylori induced significantly higher IL-8 secretion than CagA/cytotoxin negative strains in the three positive gastric epithelial cell lines (KATO-3, ST42: p < 0.01; AGS: p < 0.02). A significant increase (p < 0.01) in the expression of IL-8 mRNA relative to G3PDH mRNA was observed in KATO-3 cells after three hours of co-culture with CagA/cytotoxin positive strains. CONCLUSIONS: H pylori directly increases gastric epithelial IL-8 mRNA expression and IL-8 protein secretion in a strain specific manner. Induction of epithelial IL-8 by CagA/cytotoxin positive strains is likely to result in neutrophil chemotaxis and activation and thus mucosal damage. These observations on epithelial IL-8 may explain the association between CagA/cytotoxin positive strains and gastroduodenal disease.

Activity of omeprazole on Helicobacter pylori and relation to toxicity of strains.

AIMS: To see whether the activity of omeprazole on Helicobacter pylori is associated with toxicity of strains; to determine whether omeprazole inhibited vacuolisation of cells in culture induced by H pylori cytotoxin and by ureas, and if omeprazole prevented H pylori motility. METHODS: Minimal inhibitory concentrations (MICs) of omeprazole were determined for seven cytotoxic and five non-cytotoxic H pylori strains. Omeprazole at different concentrations was incubated with cytotoxic and non-cytotoxic extracts of H pylori, or with purified H pylori urease, and added to cells in culture. Inhibition of motility by omeprazole was tested in semi-solid medium. RESULTS: MIC90 of omeprazole was 40 micrograms/ml. MICs for cytotoxic and noncytotoxic organisms were similar. Omeprazole did not prevent vacuolisation induced by the cytotoxic extract, but at high concentrations it inhibited the formation of vacuoles induced by urease. Motility was not inhibited by the drug. CONCLUSIONS: H pylori cytotoxin is not the target of the antimicrobial activity of omeprazole. Should the drug reach clinically effective concentrations in vivo, it could potentially prevent the mucosal damage caused by the vacuolising activity of urease.

Expression of 120 kilodalton protein and cytotoxicity in Helicobacter pylori.

Antral biopsy culture supernatants from 14 subjects with chronic gastritis, known to have IgA antibodies to the 120 kilodalton protein, showed positive recognition of this antigen in western blots against a cytotoxin positive strain of Helicobacter pylori but gave negative reactions with two cytotoxin negative strains. Control immunoblots with culture supernatants from 13 non-responders to the protein were all negative. This indicates a direct association between expression of the 120 kilodalton protein in H pylori strains and cytotoxicity.

The production of neuraminidase and fucosidase by Helicobacter pylori: their possible relationship to pathogenicity.

The pathogenicity of enterobacteria often correlates with their production of neuraminidase (sialidase). Forty-nine Helicobacter pylori isolates have therefore been examined for their production of neuraminidase and other glycosidases. All 49 isolates produced considerable neuraminidase (median 228 IU/microg protein, interquartile range 121-370), pH optimum 7.5. Nine of the 49 also produced fucosidase (median 23 IU/microg protein, interquartile range 12-39), pH optimum 7.0. Production of these enzymes did not correlate with bacterial Cag A expression or duodenal ulceration. Neutrophils exposed to neuraminidase show increased adherence to endothelium so the neuraminidase production by H. pylori could partly explain the predominant neutrophil inflammatory infiltrate seen in H. pylori-associated gastritis. Inhibition of this enzyme by use of neuraminidase-inhibitors could be a useful therapeutic approach.

Serologic detection of Helicobacter pylori infection with cagA-positive strains in duodenal ulcer, gastric cancer, and asymptomatic gastritis.

CagA has been suggested as a marker for more virulent strains of Helicobacter pylori. Studies using purified proteins and an enzyme-linked immunosorbent assay (ELISA) method for serological detection of antibodies against CagA reported considerable discordance between the results of the ELISA and molecular detection of the cagA gene, with a tendency for estimation of the prevalence of cagA-positive H. Pylori to be higher by ELISA than by colony hybridization. It is not clear whether the discordance was either due to simultaneous infections with both cagA-positive and -negative strains or because of false-positive ELISA results. We correlated the presence of cagA-positive H. pylori by Polymerase chain reaction (PCR) with the presence of serum antibodies against the CagA protein from denatured H. pylori lysates. Gastric biopsies and sera were obtained from 75 patients from Korea; 25 each with gastric carcinoma, duodenal ulcer, and simple gastritis. Seventy-four of 75 isolates (98.6%) were cagA-positive by PCR and 70 sera were CagA antibody-positive by Western blotting. The cagA gene is common in H. pylori isolates from Korea regardless of the underlying disease. The presence of cagA is almost always associated with antibody to the CagA protein as determined by Western blotting. Western blotting may be the preferred method for serological detection of infection with cagA-positive H. pylori.

Progress in defining the inflammatory cascade.

Helicobacter pylori infection is characterized by an inflammatory response in the gastric epithelium, the intensity of which appears to be type-strain specific. Infections caused by Type 1 H. pylori organisms, i.e., those expressing VacA (the cytotoxin) and CagA (the cytotoxin-associated protein), are associated with a strong polymorph mucosal infiltration in vivo, and with increased secretion of interleukin-8 by epithelial cells. The inflammatory potential of Type II strains (non-cytotoxic, VacA- and CagA-negative) is probably less pronounced. The small urease subunit, porins, and other substances produced by H. pylori show neutrophil chemotactic activities in vitro. These bacterial components promote the adhesion of polymorphs to endothelial cells and stimulate polymorphs to generate oxygen reactive metabolites. This can severely damage the gastroduodenal mucosa.

Helicobacter pylori infection and infertility.

OBJECTIVE: To determine (1) the prevalence of Helicobacter pylori infection in male and female patients with reproductive disorders and controls; (2) the presence of anti-H. pylori antibodies in samples of follicular fluid, vaginal secretions and sperm; and (3) the existence of a structural homology between a major spermatozoa protein, tubulin, and H. pylori proteins. PATIENTS AND METHODS: Serum samples from 167 patients with infertility and 837 age- and gender-matched controls (blood donors) were examined by enzyme-linked immunosorbent assay (ELISA) and Western blotting to determine the seropositivity for H. pylori infection. The presence of anti-H. pylori antibodies in samples of follicular fluid, vaginal secretions and sperm was determined using the same techniques. The possible cross-reactivity with spermatozoa of anti-H. pylori hyperimmune sera and human antibodies was studied by immunofluorescence. The N-acid homology of human tubulin with the principal H. pylori proteins was assayed by the WU-blastp program available on the Internet. RESULTS: The prevalence of infection was significantly higher in patients than controls (49.1% v. 33.5%, P < 0.001). Follicular fluids from infected patients contained specific antibodies in all cases, sperm samples in about 50% of cases, and vaginal secretions in a minority of cases. Sera to H. pylori whole antigens and VacA reacted with the tails and the pericentriolar area of human spermatozoa (which are rich in tubulin); sera to urease and heat-shock protein (Hsp) did not. Follicular fluids with anti-H. pylori antibodies immune reacted with spermatozoa. A linear homology was found between beta-tubulin and three H. pylori proteins, flagellin, VacA and CagA. CONCLUSIONS: H. pylori infection may increase the risk of developing reproductive disorders or worsen the clinical expression of this syndrome.

Antibody response to Helicobacter pylori CagA and heat-shock proteins in determining the risk of gastric cancer development.

OBJECTIVE: To investigate whether the systemic antibody response to Helicobacter pylori heat shock protein B can be considered, in addition to anti cytotoxin-associated protein [CagA) antibody determination, a further serological marker of increased risk of gastric cancer development. METHODS: A total of 98 Giemsa positive Helicobacter pylori patients (28 with gastric cancer, 30 with duodenal ulcer and 40 with nonulcer dyspepsia) were studied. Serum samples obtained from all patients were tested for IgG antibodies to CagA (116 kDa), VacA [89kDa) and heat skock protein B (54 kDa) antigens of Helicobacter pylori by the Western blot technique. RESULTS: 26/28 patients [(92.9% with gastric carcinoma, 29/30 patients [96.7%) with duodenal ulcer and 30/40 patients (75.0%) with non-ulcer dyspepsia were seropositive for CagA protein. The prevalence of serum IgG antibody to CagA in the cancer patients was not significantly higher than in duodenal ulcer and non-ulcer dyspepsia patients. The prevalence of antibodies to VacA was not significantly different between gastric carcinoma and non-ulcer dyspepsia patients. In contrast the prevalence of systemic antibodies to heat skock protein B was significantly higher in gastric cancer patients (78.6%) than in duodenal ulcer (36.7%, p=0.002) or nonulcer dyspepsia patients (52.5%, p=0.029). CONCLUSIONS: The detection of antibodies to heat shock protein B is proposed as an additional test which, in association with the determination of serum antibodies to CagA, could help in determining the risk of developing severe gastroduodenal disease, and gastric cancer, in particular.

CagA-positive Helicobacter pylori infection may increase the risk of food allergy development.

The aim of this study was to test whether patients with symptomatic food allergy and significant levels of immunoglobulin E (IgE) to alimentary antigens were more likely infected by H. pylori, especially by strains expressing the CagA protein, with respect to controls. A group of 38 patients with symptomatic food allergy and 53 age-matched controls were examined serologically for H. pylori infectious status, and for CagA seropositivity. IgE to alimentary allergens were measured by a commercial kit. The prevalence of H. pylori infection in patients with food allergy and controls was similar (42.1%, and 48.3%, respectively). However, anti-CagA antibodies in H. pylori-infected persons were detected in 62.5% of patients with food allergy, and 28% of controls (P = 0.030, odds ratio = 4.29). The mean level of IgE to the most common alimentary antigens in serum samples from infected patients with anti-CagA antibodies was significantly higher than in CagA-negative infected patients: 3.28 kU/L (SD 3.93), vs. 1.99 kU/L (SD 1.53), P = 0.002, 95% confidence interval = 0.61 to 2.53). Infection by CagA-positive H. pylori increases the risk of developing food allergy.

The infection by Helicobacter pylori strains expressing CagA is highly prevalent in women with autoimmune thyroid disorders.

UNLABELLED: H. pylori infection is putatively associated with extra-digestive disorders and may also play a role in the development of autoimmune thyroid diseases (ATD). It was recently found that monoclonal antibodies to an H. pylori strain with cagA-positivity reacted with follicular cells of the thyroid gland, and that an H. pylori organism possessing the cag pathogenicity island carried a gene encoding for an endogenous peroxidase. The aims of this study was (1); To ascertain whether the infection by strains endowed with an increased inflammatory potential (those expressing CagA) could further enhance the risk of developing ATD (2); To verify the possible existence of an immune cross-reactivity between autoantibodies to peroxidase and thyroglobulin and H. pylori antigens (3). To establish whether thyroid colloid antigens could cross-react with an anti-H. pylori serum. The study was partly designed retrospectively. We examined 41 consecutive women with ATD, and, as a control, 33 consecutive age- and socio-economic class-matched women without autoimmune thyroid disorders, living in the same area as patients, occurred at the same institution in the same period (six months). Both patients and controls were examined serologically for H. pylori infection and CagA status by Western blotting. Some serum samples were absorbed with H. pylori to determine whether the antibody levels decreased. Colloid proteins were resolved electrophoretically and matched with a hyperimmune serum raised in rabbits against a CagA-positive H. pylori. Thirty-two patients (78.0%) tested seropositive for H. pylori infection, vs. 16 controls (48.4%) (P = 0.008, OR = 3.78, RR = 1.61). The prevalence of anti-CagA antibodies was 71.8% in infected patients, and 50% in infected controls (P = 0.161, n.s.). The overall prevalence of infection by CagA-positive H. pylori was significantly higher in patients with ATD (23/41, or 56.0%) than that in controls (8/33, or 24.2%) (P = 0.006, OR = 3.99, RR = 2.31). The other tests gave negative or inexplicable results. IN CONCLUSION: CagA-positive H. pylori infection increases the risk of ATD development.

Helicobacter pylori determinants of pathogenicity.

The bacterium Helicobacter pylori colonises the stomach of man and induces a strong inflammatory response. Differences in the possession of pathogenicity determinants by H. pylori isolates could account in part for the different clinical outcomes of infection. The main H. pylori pathogenic factors, i.e. urease, the cytotoxin VacA, and the genes involved in virulence contained in the pathogenicity island (PAI) cag, may promote tissue damage and ulceration, and could contribute to gastric cancer development. Strains with the mosaic vacA allelic type s1a/m1 and possessing the cag insertion are considered endowed with increased inflammatory potential, and are more likely to be isolated from patients with peptic ulcer and gastric cancer. The presence in H. pylori cag PAI of operons involved in the stimulation of gastric epithelial cells to secrete high levels of inflammatory cytokines, in mobilisation of DNA, and formation of secretory mechanisms and conjugation apparati, could contribute to increase the risk of gastric cancer development in patients infected by this microorganism.

Helicobacter pylori infection and cytotoxic antigen associated gene "A" status in short children.

BACKGROUND: Helicobacter pylori is now an accepted gastroduodenal pathogen and is being investigated for possible implications in nongastroenterological conditions such as growth impairment. Subjects infected by cytotoxic Cag-A positive strains seem more likely to develop serious gastroduodenal diseases but the possible role of Cag-A positive strains in non gastroenterological diseases has not been fully investigated. OBJECTIVE: 1) To evaluate the prevalence of Helicobacter pylori infection and Cag-A positivity in short children compared to auxologically normal children. All the subjects were without gastro-intestinal symptoms and were not obese or significantly underweight. 2) To verify the reliability of the ELISA assay for H. pylori. SUBJECTS: H. pylori infection was assessed in 338 children, 182 auxologically normal and 156 short children, with and without deficiency in growth hormone, by the determination of specific IgG antibody. In 79 subjects (all seropositive and a random sample of seronegative children), 13C-urea breath test and cytotoxic Cag-A positive strains were examined. RESULTS: The overall seroprevalence of H. pylori infection by IgG antibody was 18/156 (11.5%) and 13/182 (7.1%) in short and auxologically normal children respectively. The 13C-urea breath test was positive in 29 children: 17 (10.9%) short and 12 (6.6%) auxologically normal. Western blotting documented infection by cytotoxic Cag-A positive strains in 12/17 (70.6%) and 8/12 (66.6%) of short and auxologically normal children respectively. None of the differences between the two groups were significant. CONCLUSIONS: 1) We found a similar prevalence of H. pylori infection and Cag-A positivity in two large pediatric populations of short or auxologically normal children. Therefore: 1) Our data did not confirm a role of H. pylori infection in short stature in children. 2) We found a high reliability of ELISA assay for the detection of IgG antibodies compared to breath test.

Evaluation of a commercial ELISA kit for the serological diagnosis of Helicobacter pylori infection.

H. pylori infection can be diagnosed serologically. We evaluated an ELISA kit (Helicobacter pylori IgG, DIESSE) prepared using a glycine extract of an autoctonous H. pylori strain which produced the highest biologically active urease titre out of five strains tested. The kit was tested with serum samples from H. pylori-infected and uninfected adults and children. The Western Blot technique was used as reference method for the H. pylori infective status. Based on the results obtained by immunoblotting with serum samples from H. pylori negative subjects, two different cut-off values were considered for adults and children. Sensitivity and specificity were respectively 95% and 100% for adults, 95.6% and 97.8% for children. In conclusion, the clinical accuracy of this commercially available ELISA kit proved to be very good; the adoption of two different cut-off values for adults and children also improved its parameters of reliability.