Helicobacter pylori non-cytotoxic genotype enhances mucosal gastrin and mast cell tryptase.

AIMS: To determine the association, if any, between H pylori genotype and the gastric mucosal variations in the levels of gastrin, somatostatin, tryptase, and histamine. METHODS: 49 patients affected by duodenal ulcer and 48 by non-ulcer dyspepsia were studied. To identify the H pylori genotype, the presence of the cagA gene and vacA alleles m1, m2, s1, and s2 were analysed by polymerase chain reaction. Gastrin, somatostatin, tryptase, and histamine were measured in antral mucosal biopsies. RESULTS: 57 patients were infected with H pylori (30 with duodenal ulcer and 27 with non-ulcer dyspepsia). Gastrin and tryptase were increased in patients with H pylori infection, although the variations were statistically significant only for gastrin; somatostatin and histamine were not influenced by H pylori infection. In patients with non-ulcer dyspepsia the absence of the cagA gene and the presence of vacA alleles s2 and m2 were associated with higher values of tryptase and to a lesser extent of gastrin. These associations were not found in patients with duodenal ulcer. CONCLUSIONS: The cagA negative s2m2 strain of H pylori may be less dangerous for the gastric mucosa than other H pylori strains since it enhances tryptase production by gastric mucosal mast cells; this enzyme is thought to stimulate tissue turnover and favour wound healing.

[Biochemical markers of gastric functioning]. / Marcatori biochimici di funzionalità gastrica.

The serum determination of pepsinogen A (PGA) and pepsinogen C (PGC) might indicate gastric mucosal inflammation and atrophy. Body gastric mucosa produces both PGA and PGC, while antral mucosa produces only PGC. Therefore, diseases involving mainly the antrum, such as H. pylori infection, are mainly indicated by the variations in serum PGC than in serum PGA. In agreement, when the antral mucosa is infected by the more virulent cagA positive H. pylori strains, which cause severe inflammation, serum PGC significantly increases. Another indirect indicator of gastric inflammation is polymorphonuclear (PMN) oxidative burst after the stimulation with water extracts from H. pylori culture: this parameter is significantly increased in infected if compared to non-infected subjects. The higher oxidative burst response of peripheral PMN in infected patients, possibly consequent to the release of specific cytokines able to prime PMN towards H. pylori products, is unable to eliminate the infection, but it might concur in damaging the gastric mucosa.

Serum antibodies anti-H. pylori and anti-CagA: a comparison between four different assays.

The authors compare efficacy of two ELISA assays (one supplied by DIAMEDIX [Delta Biological s.r.l.], and the other by RADIM [RADIM I]) in detecting total anti-H. pylori antibodies, and of two further ELISA methods (one supplied by EUROSPITAL [Helori CTX IgG] and the other by RADIM [RADIM 2]) in identifying anti-CagA antibodies, using sera from 69 controls (20 adults and 49 children) and from 96 patients, obtained before endoscopy. Seventy-three of the patients had H. pylori infection, while the remaining 23 were H. pylori negative (histology and polymerase chain reaction [PCR]). Fifty-two of the H. pylori positive patients, had cagA-positive strain infection, identified by PCR. The DIAMEDIX assay was found to be more sensitive (92%) than RADIM 1 (79%) in identifying H. pylori positive patients, irrespective of the infecting strain. On the other hand, the DIAMEDIX assay was less specific than RADIM 1 for H. pylori-negative patients (43% vs. 83%). However, when patients already treated for H. pylori infection were excluded from the group of H. pylori-negative patients, the DIAMEDIX assay had a specificity of 89%. In identifying anti-CagA antibodies, the kit supplied by RADIM (RADIM 2) had a sensitivity of 90% and a specificity of 94%, whereas that supplied by EUROSPITAL had a sensitivity of 100% and a specificity of 76%. The performances of the two methods in the identification of anti-CagA antibodies were found to be similar. The authors conclude that, in view of its high sensitivity, the DIAMEDIX assay may be useful in screening for H. pylori infection.

Polymorphonuclear oxidative burst after Helicobacter pylori water extract stimulation is not influenced by the cytotoxic genotype but indicates infection and gastritis grade.

H. pylori-associated gastric mucosal inflammation is characterized by the presence of polymorphonuclear (PMN) leukocyte infiltrate, which is more severe when the infecting strain is cagA positive. After appropriate stimuli, such as bacterial products, PMN release large amounts of oxygen derived free radicals and proteases, to kill the bacterium. H. pylori seems to be particularly resistant to the oxidative machinery of PMN, which can in turn damage the host gastric mucosa. We evaluated peripheral PMN oxidative burst response after stimulation with water extracts from cagA positive (WEcagA+) or negative (WEcagA-) H. pylori strains in infected (n=31) and non-infected patients (n=32) in comparison with healthy controls (n=16); the influence of gastric mucosal inflammatory infiltrate and activity grade on PMN oxidative burst were also assessed. PMN oxidative burst was measured by FACS analysis. H. pylori water extracts were obtained from bacterial culture. H. pylori genotype was determined by means of the polymerase chain reaction. The PMN oxidative burst in H. pylori infected patients was significantly higher than that in H. pylori negative or healthy controls, no differences being found when the results following WEcagA+ and WEcagA- stimulation were compared. The difference in PMN oxidative burst obtained after WEcagA- and E. coli (standard stimulus for PMN oxidative burst) stimulation discriminated H. pylori infected from non-infected patients with a sensitivity of 90% and a specificity of 97%. The grade of PMN oxidative burst correlated with PMN infiltration grade of the gastric mucosa. Our findings allow to conclude that PMN oxidative burst activation by H. pyloriWE is species- but not strain-correlated. PMN priming, probably consequent to the action of soluble mediators released by mononuclear cells, makes PMN hyper-responsive to H. pylori products, thus favoring the release in the gastric mucosa of infected patients of large amounts of oxygen-derived free radicals, which are not enough to eliminate the infection, but may contribute to damaging the gastric mucosa itself. Peripheral PMN oxidative burst response to H. pyloriWE might furthermore be of help in diagnosing H. pylori infection.

Glucose metabolic alterations in isolated and perfused rat hepatocytes induced by pancreatic cancer conditioned medium: a low molecular weight factor possibly involved.

A serious insulin resistance characterizes pancreatic cancer-associated diabetes mellitus. Elsewhere, we demonstrated that MIA PaCa2 cultured cells secrete a soluble factor responsible for reduced glucose tolerance induced in SCID mice. The intracellular mechanism of insulin resistance was investigated in isolated and perfused rat hepatocytes incubated with MIA PaCa2 conditioned medium. Lactate production was reduced compared to hepatocytes incubated with control medium while 1,2-DAG was increased and PKC was activated in the hepatocytes incubated with MIA PaCa2 conditioned medium. This behavior was not reproduced treating the hepatocytes with the growth factors EGF, interleukin Ibeta, interleukin-6, and TGF-beta1. In an attempt to make a biochemical identification of the hypothesized tumor associated-diabetogenic factors we observed a low molecular weight protein in the conditioned medium, absent in the nonconditioned one, that may be responsible for the described behaviors.

Portal but not peripheral serum levels of interleukin 6 could interfere with glucose metabolism in patients with pancreatic cancer.

UNLABELLED: Interleukin 6 (IL-6), an autocrine growth factor for many tumors, seems to favour tumor spread to the liver. Our aims were first to evaluate the pattern of portal and systemic IL-6 levels in patients with pancreatic cancer (PC, n = 18) and chronic pancreatitis (CP, n = 22) compared with controls (CS, n = 20); and second, to ascertain whether there was any relation between IL-6 levels and tumor spread or PC-associated Diabetes mellitus. For all subjects, a fasting serum sample was obtained from a cubital vein; a portal serum sample was obtained from nine PC and three CP patients. In cubital and portal sera we measured IL-6, interleukin 1 beta (IL-1b), CA 19-9, c-reactive protein (CRP) and amylase. Systemic IL-6 levels were significantly higher in PC patients than in CS. In PC, portal IL-6 levels were significantly higher than the corresponding systemic values. The same pattern was found in the three CP patients, whereas IL-1b, CA 19-9, CRP and amylase portal levels were the same as systemic values. No correlation was found between PC stage and systemic or portal IL-6 levels. Portal IL-6 levels were correlated with the corresponding fasting serum glucose values. A significant correlation was found between IL-6 values and CRP, ALT, total bilirubin, GGT and creatinine, but not amylase. IN CONCLUSION: (1) Portal IL-6, which is partly of pancreatic origin, is first metabolised in the liver; (2) Systemic IL-6 reflects hepatic and renal functions rather than local conditions in the pancreas; (3) IL-6 does not appear to influence PC spread; (4) IL-6, which is released in large amounts by the inflamed pancreas, may contribute to determining diabetes, thus interfering with the signal transducing pathways involved in glucose metabolism in liver cells.

Helicobacter pylori cytotoxic genotype is associated with peptic ulcer and influences serology.

OBJECTIVE: We studied 146 patients with peptic ulcer disease (n = 72), antral gastritis (n = 58), or duodenitis (n = 16) to ascertain whether the cytotoxic genotype of Helicobacter pylori (Hp) is associated with peptic ulcer disease and/or antral gastritis and whether it influences the circulating levels of total anti-Hp antibodies, anti-cagA antibodies, and pepsinogens. METHODS: A gastric juice sample was obtained from each patient. After DNA extraction, polymerase chain reaction was used to amplify the genes urease A (ureA), cagA, and vacA of Hp. RESULTS: A significant association was found between peptic ulcer disease and the cytotoxic genotypes, characterized by the presence of s1 and m1 alleles of vacA and by cagA. Patients with a cagA-positive genotype showed a significant increase in anti-cagA antibodies and also had significantly increased circulating levels of pepsinogen C. CONCLUSIONS: Cytotoxic Hp strains are mainly involved in determining peptic ulcer disease, but not antral gastritis. The higher levels of circulating pepsinogen C found in patients infected with cytotoxic genotypes may reflect the higher degree of inflammation sustained by these strains.

Analysis of Helicobacter pylori vacA and cagA genotypes and serum antibody profile in benign and malignant gastroduodenal diseases.

BACKGROUND: Helicobacter pylori species comprise different strains, cytotoxic and non-cytotoxic, which can be identified on the basis of their genomic pattern. AIMS: (1) To evaluate the polymorphism of the vacA gene and to ascertain whether the cagA gene is present in patients with gastric adenocarcinoma. (2) To study the anti-H pylori antibody profile using western blotting. PATIENTS: Twenty one patients with gastric adenocarcinoma and 71 with H pylori associated benign disease (nine gastric ulcer, 29 duodenal ulcer, 25 antral gastritis, and eight duodenitis). METHODS: The polymerase chain reaction was used to verify the presence or absence of cagA and to study the polymorphism of vacA in gastric mucosal samples obtained during endoscopy for patients with benign diseases and at surgery for patients with gastric adenocarcinoma. Fasting sera were used to assess anti-H pylori antibodies against different H pylori antigens by western blotting. RESULTS: CagA gene and the allele s1 of vacA were significantly less frequent in patients with antral gastritis (60% and 60%) compared with patients with gastric adenocarcinoma (94% and 100%) and with other non-malignant gastroduodenal diseases (93% and 87%) (chi 2 = 16.01, p < 0.001; and chi 2 = 13.97, p < 0.01). In patients with gastric adenocarcinoma, antibodies against a 74 kDa H pylori antigen were less frequently found than in patients with benign diseases. CONCLUSIONS: H pylori infection caused by cagA positive/vacA s1 strains is a frequent finding in patients with gastric adenocarcinoma. Prospective studies are needed to confirm whether the low incidence of positive serological response to the 74 kDa H pylori antigen in patients with gastric adenocarcinoma is important.

[Premature membrane rupture. Comparison of diagnostic tests]. / Rottura intempestiva delle membrane. Test diagnostici a confronto.

BACKGROUND: Objective of this study was to evaluate the accuracy of the vaginal pH-test, the Fern-test, the research of foetal cells and of foetal fibronectin in vaginal discharge, which are used to diagnose premature rupture of membranes. METHODS: To this aim 40 pregnant patients between 24th and 37th weeks gestation have been examined, considered at risk for sub-clinic loss of aminiotic fluid: 23 were affected by preterm labour and 17 by suspected rupture of membranes. RESULTS: Subsequently amniotic sac was confirmed to be ripped in 10 cases (25%): 2 (8.7%) in the 23 patients with preterm labour, and 8 (47%) in the 17 patients with suspected PROM. Sensibility, specificity and accuracy were respectively: 70, 97 and 90% for pH-test; 70, 100 and 93% for Fern-test; 50, 93 and 82% for foetal cells; 100, 90 and 93% for fibronectin test. CONCLUSIONS: In personal experience fibronectin test appeared to be the most sensible and accurate marker. Fern-test was the most specific, while the research of foetal cells appeared to be the least reliable.

An unidentified pancreatic cancer cell product alters some intracellular pathways of glucose metabolism in isolated rat hepatocytes.

In this study we assessed whether conditioned media from a human pancreatic cancer cell line (MIA PaCa 2) can interfere with some intracellular pathways involved in glucose metabolism in isolated rat hepatocytes. The hepatocytes, isolated from Male Wistar rats, were incubated with MIA PaCa 2-conditioned or nonconditioned media. Conditioned and nonconditioned hepatocytes were run for 120 min in the presence or absence of insulin (100 mM) and were sampled at fixed time intervals. Supernatant glucose levels decreased to a similar extent over time in both conditioned and nonconditioned hepatocytes, while lactate levels significantly increased in nonconditioned hepatocytes with respect to conditioned hepatocytes. A pyruvate kinase activity increase was observed only in nonconditioned hepatocytes and was biphasic in nature, since this increased activity was detected both after a few and after 30 min following insulin stimulation. The cyclic AMP level increase was significantly higher in conditioned than in nonconditioned hepatocytes. It appears that MIA PaCa 2 cells produce a factor(s) that may interfere with one of the insulin-mediated intracellular pathways of glucose metabolism, namely, glycolysis. This detrimental effect on glycolysis is supported by the blunted rise in lactate concentration in the medium after the glucose challenge. This substance(s) probably transfers its signal inside the target cells, activating the adenylate cyclase pathway. These results support the hypothesis that pancreatic cancer is the cause rather than the consequence of diabetes mellitus.

[Silent celiac disease: results of a study in secondary schools of Padua]. / La celiachia silente: risultati di uno studio nelle scuole medie di Padova.

Aim of this study was the assessment of the prevalence of coeliac disease (CD) in children attending the secondary school in the city of Padua. 939 students, aged 10-15 years (mean age: 12 years, 7 months), 35% eligible population, were accepted to undergo a study process which included three stages: a) in all students venous sample was taken for measurement of the IgG and IgA anti-gliadin antibodies (AGA); b) measurement of serum immunoglobulins and anti-endomysium antibodies (AEA) if AGA IgA was resulted positive; c) intestinal biopsy was performed in 3 students; two of them had pathologic levels of AGA IgG and IgA and AEA. These patients were females and had decreased rates of statural growth, anemia with iron deficiency, anorexia, abdominal pain, asthenia. The third girl had positive AGA IgG and IgA but absence of AEA and normal biopsy. She also had symptoms of abdominal pain, reduced height. Follow-up studies have been planned to establish a latent phase of CD. In conclusion, the prevalence of CD was 2.13/1000 (0.37-8.55, 95% CI), if we consider the patients with established diagnosis of CD in the same urban area and of the same age, the overall incidence increases to 2.6/1000. This prevalence, therefore, is higher, than that of 0.5/1000 previously reported in the general population, with a ratio of 1/4 between patients already known and the cases detected in this study.

Helicobacter pylori serology in patients with chronic gastritis.

OBJECTIVES: Helicobacter pylori (Hp) infection is known to cause several gastroduodenal diseases. In patients with non-ulcer dyspepsia, we assessed the link between Hp infection and gastric mucosal inflammation, as well as the influence of Hp and inflammation on the serum levels of anti-Hp antibodies (IgG), pepsinogen A (PGA), pepsinogen C (PGC), and gastrin. METHODS: Entering the study were 221 patients with non-ulcer dyspepsia, all of whom underwent upper gastrointestinal endoscopy. RESULTS: Of the 221 patients investigated, 135 (61%) were Hp positive. The higher the bacterial load, the worse the associated gastritis, the gastric antrum and body being considered. All of the serological indices studied were found to be influenced by gastritis. Serum IgG satisfactorily discriminated between Hp-positive and Hp-negative subjects, with a sensitivity of 84% and a specificity of 86%. PGC, PGA, and gastrin were less accurate. Only PGC only found to be correlated with Hp load. The product of IgG and PGC improved the diagnostic accuracy of IgG alone. CONCLUSIONS: Hp infection, frequently found in patients with non-ulcer dyspepsia, is associated with gastric mucosal inflammation; of the indices studied, serum IgG and PGC most accurately indicated Hp infection, and their product may be proposed as an aid in diagnosing Hp infection in dyspeptic patients.

The pancreatic cancer cell line MIA PaCa2 produces one or more factors able to induce hyperglycemia in SCID mice.

A reduced glucose tolerance or frank diabetes mellitus is a frequent finding in patients with pancreatic cancer. The aim of this study was to verify whether the pancreatic cancer cell line MIA PaCa2 was able to produce any factor which could induce hyperglycemia in SCID (severe complete immunodeficient) mice. MIA PaCa2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) for 7 days. Twenty-five female SCID mice were used. They were daily i.p. injected with 300 ul of cell culture supernatants (Group T, n = 13) or with 300 ul of DMEM (Group C, n = 12) and followed up for 82 days. Blood glucose levels were significantly higher in Group T than in Group C on days 10 and 25. Intravenous glucose tolerance test, success-fully performed in 9 animals (4 controls and 5 treated), demonstrated a significantly reduced glucose tolerance in Group T compared to Group C mice. At sacrifice, plasma and pancreatic insulin and glucagon levels did not vary between groups. The ratio between pancreatic and plasma insulin was significantly lower in Group T than in Group C. We conclude that: 1. The pancreatic cancer cell line MIA PaCa2 produces one or more soluble factors able to cause hyperglycemia in vivo; 2. this effect is not immunologically mediated, and 3. this/these factor/s could both interfere with the pancreatic beta cells and/or with insulin peripheral action.

Gastrin stimulates gastric mast cells in rabbits.

In a previous study we demonstrated that in human gastric mucosa tryptase was localized only in mast cells and that its levels were correlated with serum gastrin, suggesting a link between gastrin action and mucosal mast cell function. The aim of the present study was to discover whether pentagastrin injection could stimulate gastric mucosal mast cells in rabbits. Ten female rabbits (group S) were injected s.c. with pentagastrin (10 mu g/kg); another group of ten animals (group C) was injected s.c. with an equal volume of saline solution. One hour after the injection the rabbits were sacrificed and their stomachs removed. Antrum (A), corpus (C) and fundus (F) mucosal homogenates were assayed for total protein, tryptase, pepsinogen A (PGA), histamine and gastrin. Histamine tissue levels were significantly lower in group S than in group C in the antrum (Mann-Whitney test: U = 82, P < 0.01) and in the corpus (U = 83, P < 0.005). Tryptase levels were significantly higher in group S than in group C in all gastric areas (antrum: U = 95, P < 0.001; corpus: U = 85, P < 0.005 and fundus: U = 75, P < 0.05). Total protein, PGA and gastrin did not vary significantly between groups. In group C, no significant correlations were found among the five parameters. In group S, corpus tryptase was correlated with fundus tryptase (Spearman's r = 0.831, P < 0.01). The same relationship was observed for histamine (r = 0.672, P < 0.05). In group S, antrum gastrin was inversely correlated with antrum tryptase (r = -0.903, P < 0.001), and with corpus PGA (r = -0.806, P < 0.05). This study demonstrates that bolus pentagastrin administration stimulates gastric mucosal mast cells in the rabbit.

A new method for detecting anti Helicobacter pylori antibodies: an analytical and clinical evaluation.

The diagnosis of Helicobacter pylori (Hp) infection is an important goal in clinical practice. In this paper we evaluated 1. the analytical reliability of a new second-generation antigen based enzyme immunoassay (Cobas Core Anti Helicobacter pylori EIA) in detecting anti-Hp IgG antibodies, and 2. the behaviour of anti-Hp IgG in patients with chronic atrophic and non-atrophic gastritis as compared to healthy controls. The findings from the dilution curve, the values of intra and inter assay coefficients of variations (never above 10%) and of the recovery test (between 96 and 109%), confirm that the method is reliable. Serum IgG anti-Hp levels were found to be significantly higher in patients with histologically identified Hp infection, than in those negative at histology. Furthermore, the grade of histological positivity was correlated with serum IgG levels. However, we found a discrepancy between a low prevalence of Hp staining and a high prevalence of Hp seropositivity in patients with chronic atrophic or non-atrophic gastritis, but not in controls. This suggests that IgG serum determination may be more useful than histology in determining a present or previous infection in patients with chronic atrophic or non-atrophic gastritis.