Antibiotic treatment with ampicillin accelerates the healing of colonic damage impaired by aspirin and coxib in the experimental colitis. Importance of intestinal bacteria, colonic microcirculation and proinflammatory cytokines.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for their anti-inflammatory, analgesic and antipyretic effects, however their use is associated with the broad spectrum of side effects observed in human as well as the experimental animals. Despite damaging activity of NSAIDs in upper gastrointestinal (GI) tract, these drugs exert deleterious influence in lower GI tract, including colon. The role of GI microflora in the pathogenesis of NSAIDs-induced experimental colonic damage is not completely understood. The aim of this study was 1) to evaluate the relative importance of the GI microflora on the experimental colonic damage in the presence of caused by NSAID, and 2) to assess the efficacy of antibiotic treatment with ampicillin on the process of healing of colitis. We compared the effect of vehicle, ASA applied 40 mg/kg intragastrically (i.g.) or the selective cyclooxygenase (COX)-2 inhibitor, celecoxib (25 mg/kg i.g.) without or with ampicillin treatment (800 mg/kg i.g.) administered throughout the period of 10 days, on the intensity of TNBS-induced colitis in rats. The severity of colonic damage, the alterations in the colonic blood flow (CBF) and myeloperoxidase (MPO) activity, the mucosal expression of TNF-α, IL-1ß, COX-2, VEGF and iNOS and the plasma concentration of TNF-α and IL-1ß were assessed. In all rats, the faeces samples as well as those from the colonic mucosa, blood, liver and spleen underwent microbiological evaluation for intestinal bacterial species including Escherichia coli and Enterococcus spp. The administration of TNBS resulted in macroscopic and microscopic lesions accompanied by the significant fall in the CBF, an increase in tissue weight and 4-5-fold rise in the MPO activity and a significant increase in the plasma IL-1ß and TNF-α levels. ASA or celecoxib significantly increased the area of colonic lesions, enhanced MPO activity and caused the marked increase in colonic tissue weight and plasma IL-1ß and TNF-α levels, as well as an overexpression of mRNA for IL-1ß and TNF-α, COX-2, VEGF and iNOS in the colonic tissue. ASA and coxib also resulted also in a significant increase of E. coli counts in the stool at day 3 and day 10 day of the observation compared with the intact rats. Moreover, E. coli translocation from the colon to the blood and extraintestinal organs such as liver and spleen in the group of rats treated without or with ASA and coxib. E. coli was the most common bacteria isolated from these organs. Treatment with ampicillin significantly attenuated the ASA- or celecoxib-induced increase in plasma levels of IL-1ß and TNF-α and suppressed the mucosal mRNA expression for IL-1ß and TNF-ß, COX-2, iNOS and VEGF in the colonic mucosa. Ampicillin administration caused a significant fall in the number of E. coli in the faeces at day 3 and day 10 of observation in ASA- and coxib-treated rats with colitis. Antibiotic therapy markedly reduced bacterial translocation to the colonic tissue and the extraintestinal organs such as the liver and spleen. We conclude that administration of ASA and to lesser extent of celecoxib, delays the healing of experimental colitis and enhances the alterations in colonic blood flow, proinflammatory markers such as IL-1ß, TNF-α, COX-2, iNOS and VEGF and increased intestinal mucosal permeability resulting in the intestinal bacterial translocation to the blood, spleen and liver. Antibiotic treatment with ampicillin is effective in the diminishing of the severity of colonic damage, counteracts both the NSAID-induced fall in colonic microcirculation and bacterial E.coli translocation to the extraintestinal organs.

Prevalence and characterization of Helicobacter pylori (H. pylori) infection and colonization in dentists.

H. pylori is an important factor in the pathogenesis of numerous diseases including gastro-intestinal, metabolic and vascular disorders. Therefore, identification of individuals at risk of this infection remains of critical importance. Dentists and dental professionals may be at increased risk due to the contact with oral cavity of patients with the presence of H. pylori in the oral cavity where it may serve as reservoir for gastric infections and participate in the pathogenesis oral mucosal lesions and ulceration. However, evidence regarding the occurrence of H. pylori infections and colonization in dentists is conflicting, but has been based mainly on serological studies, which carry significant limitations. Therefore, we attempted to characterize H. pylori infection in practising dentists in relation to the duration of their work as dental professionals. Moreover, apart from seropositivity, which was used by majority of previous studies, we have performed urea-breath test (UBT), which has been shown to represent active H. pylori infection in stomach as well as the H. pylori culture from the oral cavity. We found that while the occurrence of either gastric or oral H. pylori in dentists is not greater than in general population, it seems that in male dentists there is a greater risk of gastric H. pylori infection. Moreover, we found a relationship between the length of dentist occupation with the presence of H. pylori in gingival sulcus. In conclusion, while overall occurrence of H. pylori in dentists did not differ from that reported for stomach or oral cavity in general population, there was an increased occurrence of H. pylori in male dentists and the presence of this germ in the oral cavity appears to be related to the length of professional exposure.

Assessment of co-existence of Helicobacter pylori and Candida fungi in diseases of the upper gastrointestinal tract.

Candida spp. were found in the gastric mucosa of 27 (17%) patients, out of whom 18 (11%) showed co-existence of the fungi with H. pylori. Analysis of relationship between selected disorders of the upper gastrointestinal tract (non ulcer dyspepsia NUD, gastric ulcer, duodenal ulcer) and infection with H. pylori and/or Candida revealed a link between co-existence of H. pylori with Candida and gastric ulcers suggesting synergism of those microorganism in pathogenesis of the disease. On the contrary, according to quantitative studies performed, the fungi alone do not play a significant role in pathogenesis of the above mentioned disorders as they colonize only epithelium to the extent that is not pathologically significant (<10(3) CFU/ml). Genetical study was carried out on 57 Helicobacter pylori strains isolated from bioptates of the gastric mucosa. The genotypes of the strains (gene cagA and alleles of gene vacA - m1, m2, s1, s2) were determined using the PCR technique. As it was shown, the patients infected with H. pylori strains of genotype cagA+, vacA s1 are exposed to higher risk of peptic ulcer disease (PUD) as compared to the patients infected with cagA-, vacA s2 strains. In the case of the NUD patients a correlation with allele m2 was found only (p<0.001). This may suggest that in future some of the NUD patients infected with cagA+, vacA s1 strains will fall into the group at higher risk for PUD.

Association of the presence of Helicobacter pylori in the oral cavity and in the stomach.

UNLABELLED: Helicobacter pylori is a gram-negative, microaerophilic rod-shaped bacteria that lives beneath the gastric mucous layer, on the surface of epithelial cells. Stomach infection with this organism causes inflammation of the gastric mucosa, which can lead to gastritis, duodenal or gastric ulcer and even in rare cases to gastric carcinoma or MALT lymphoma. Approximately 50% of the world's population is believed to be infected with H. pylori. Most infections is probably acquired in childhood, but the exact route of transmission is unknown. It has been speculated that dental plaque might harbour Helicobacter pylori and, therefore, might be a source of gastric infection. In order to address this issue we studied the relationships between oral and gastric infections with H. pylori in 100 subjects. METHODS: Gastric H. pylori infection was determined by (13)C-urea breath test (UBT) and the presence of the bacteria in oral cavity was monitored by the culture from the saliva and from dental plaque. RESULTS: H. pylori was found in the stomach in 51% of studied individuals, while oral H. pylori was found in 54% (in saliva) and in 48.3% (in gingival pockets), the difference was not statistical significant (p=NS). Interestingly, anti-Hp IgA was found in 84% of studied individuals. No relationship was found between the presence of the bacteria in the oral cavity and the H. pylori gastric infection. 54.9% of subjects with stomach infection showed concomitant presence of H. pylori in saliva. 52.3% of examined subjects with negative UBT-test revealed the presence of H. pylori in culture from the saliva. The X(2) value of relationship between UBT and culture H pylori in saliva was 0.029 (p=0.9). Similarly, no relationship was found between the presence of H. pylori in the stomach and in the dental plaque (X2=0.6); p=0.4). As expected, the presence of H. pylori in the dental plaque was significantly correlated with the presence of bacteria in the saliva (X2=18.4; p=0.0002). We also compared the presence of H. pylori in the saliva of patients with and without teeth. The cultured H. pylori was found in 63.7% of patients without teeth and in 52.9% of patients with teeth. This indicates that the presence of teeth does not seem to affect the occurrence of H. pylori in saliva. We conclude that oral activity contamination with of H. pylori occurs at similar degree to that in the stomach. However, there was no significant correlation between the occurrence of H. pylori in the stomach and in the oral cavity indicating that other factors, like susceptibility to infection due to acid environment in the stomach may be the major factor in gastric infection with that bacteria, while oral cavity may serve only as transient food-related contamination without clear relation to gastric infection.

Helicobacter pylori and CagA status, serum gastrin, interleukin-8 and gastric acid secretion in gastric cancer.

BACKGROUND: Despite numerous epidemiological studies, the association between Helicobacter pylori infection and gastric cancer (GC) remains unexplained. This study was designed to determine the seropositivity of H. pylori and cytotoxin-associated gene A (CagA), serum gastrin and interleukin-8 (IL-8) levels as well as basal intragastric pH and maximal histamine-induced gastric acid outputs (MAO) in a large series of GC patients and controls. METHODS: 337 GC patients (118 men and 219 women; median age 59.4; range 21-87) and 337 controls randomized for sex and age entered the study. Serum IgG antibodies to H. pylori and CagA and serum levels of IL-8 were measured by enzyme-linked immunosorbent assay, while serum-amidated gastrin was determined by specific radioimmunoassay and correlated with gastric luminal pH. RESULTS: The numbers of GC patients and controls involved in the study in various age groups, ranging from 20 to > 70 years, were similar, but overall H. pylori IgG seropositivity in GC patients was significantly higher (90.8%) than in controls (79.2%). The overall CagA seropositivity in GC patients was about double (58.2%) that in controls (25.2%). Serum gastrin levels over the calculated cut-off value (38.88 pM/L) were found in several-fold larger number in GC patients (48%) than in controls (8.3%) and. similarly, serum IL-8 values over the cut-off point (1.77 pg/mL) occurred in almost all (99.7%) GC patients but in only a few controls (0.3%). Basal intragastric pH above the cut-off point (pH = 4.50) was observed in about 58.2% of GC patients compared to 15.1% in controls, and strong correlation between the serum gastrin and gastric pH was found in GC but weak in controls. The cut-off value for MAO was 12.3 mml/h; MAO below this cut-off value occurred in 89.9% of GC patients and in only 4.7% of controls. A summary odds ratio (SOR) in GC for H. pylori IgG was 2.59 (95% Cl: 1.61-4.22) for CagA - 4.12 (95% Cl; 2.93-5.8), for serum gastrin - 10.25 (95%; 6.47-16.47) and for MAO - 15.2 (95% Cl; 9.45-39.82). Multivariable analysis of serum gastrin, IgG and CagA, and luminal pH and MAO values revealed that only gastrin and CagA have significant influence on GC formation (OR > 1 in logistic regression). CONCLUSIONS: 1. CG patients show significantly higher H. pylori IgG and CagA seropositivity than dyspeptic age- and gender-matched controls, confirming that gastric infection with CagA expressing H. pylori greatly increases the risk of GC. 2. Serum gastrin levels in GC but not in controls are correlated with the rise in intragastric pH, indicating that excessive gastrin release in GC is affected by lower intragastric pH. 3. Serum gastrin level and CagA seropositivity are significantly increased in the majority of GC patients, and are the only variables in multivariable analysis to have a predominant influence on GC formation, which suggests that both these parameters may be implicated in H. pylori-related gastric carcinogenesis. 4. H. pylori-infected GC patients produce significantly more IL-8 than do non-GC controls, probably reflecting CagA-positive H. pylori-associated gastritis.

Incidence of Chlamydia pneumoniae infection in patients with coronary artery disease subjected to angioplasty or bypass surgery.

BACKGROUND: Seroepidemiological, pathological and animal studies suggest that chronic infection with Chlamydia pneumoniae (Cp) may directly impact the development or progression of atherosclerosis and coronary heart disease. The aim of the present study was to determine the seroprevalence of Cp infection and markers of systemic inflammation in patients undergoing routine coronary artery examination and prior to heart revascularization. MATERIAL AND METHODS: The research involved 76 patients with severe CAD and 81 control patients with normal coronary circulation confirmed by coronary angiography. The presence of serum IgG and IgA antibodies to Cp and plasma interleukin-8 (IL-8) levels was measured by EIA test. Furthermore, the levels of plasma C-reactive protein, fibrinogen, total cholesterol, and triglycerides were measured in all patients. RESULTS: Seropositivity to Cp was found in 60.5% for IgG and in 61.8% of cases for IgA with CAD patients, as compared to 26.0% and 29.5% in the controls (p<0.001), respectively. The levels of Interleukin-8, plasma fibrinogen, total cholesterol and triglycerides were significantly higher (p<0.001) in the CAD group, while C-reactive protein tended to have a higher value in patients with atherosclerosis than in the control group, although the difference was not significant. CONCLUSIONS: Cp infection significantly increases the risk of CAD, usually requiring coronary bypass surgery or percutaneous coronary intervention as effective measures. It may also modify the levels of serum lipids, CRP and fibrinogen, increasing the risk of atherosclerosis. The strong correlation between the elevated IgG and IgA titers of Cp in patients treated with angioplasty or surgery may impact their follow-up; this issue requires further investigation.

Helicobacter pylori infection, gastrin, cyclooxygenase-2, and apoptosis in colorectal cancer.

Helicobacter pylori (HP) infection is usually accompanied by an increased plasma level of gastrin, a potent mitogen able to induce cyclooxygenase (COX)-2. This study examined (a) the seroprevalence of HP, its cytotoxic protein, CagA, and cytokines (tumor necrosis factor alpha, interleukins 1beta and 8) in 80 patients with colorectal cancers, before and after the removal of tumor, compared with 160 age- and gender-matched controls; (b) the gene expression of gastrin and its receptors (CCKB-R) in the cancer tissue, (c) the plasma levels and tumor tissue contents of gastrin, and (d) the mRNA expression of COX-1, COX-2, and apoptotic proteins (Bax and Bcl2) in cancer tissue and intact colonic mucosa. Anti-HP IgG, anti-CagA IgG seroprevalence, and cytokine levels were analyzed by enzyme-linked immunosorbent assay tests; gene expressions of gastrin, CCKB-R, COX-1, COX-2, Bax, and Bcl2 by reverse transcriptase polymerase chain reaction; and gastrin by radioimmunoassay. The seroprevalence of HP, especially that expressing CagA, was significantly higher in cancer patients than in controls and did not change 1 week after tumor resection while plasma cytokines were significantly reduced after this operation. Both gastrin and CCKB-R mRNA were detected in the cancer tissue and the resection margin; similarly, COX-2 mRNA was expressed in most of cancers and their resection margin but not in intact colonic mucosa, where only COX-1 was detected. The colorectal cancer tissue contained several folds more immunoreactive gastrin than cancer resection margin and many folds more than the intact colonic mucosa. We conclude that colon adenocarcinoma and its resection margin overexpress gastrin, its receptors, CCKB-R, and COX-2, and that HP infection may contribute to colonic cancerogenesis via overexpression of gastrin and COX-2, which may account for the stimulation of the tumor growth and the reduction in apoptosis as documented by enhanced mRNA expression of anti-apoptotic Bcl2 over proapoptotic Bax proteins.

Expression of hepatocyte growth factor, transforming growth factor alpha, apoptosis related proteins Bax and Bcl-2, and gastrin in human gastric cancer.

BACKGROUND: Gastric cancer is one of the most frequent neoplasms and a leading cause of the death world-wide. In recent years, epidemiological and animal studies demonstrated a link between gastric cancer and chronic infection with H. pylori. The exact mechanism responsible for the development of gastric cancer in H. pylori-infected patients still remains unclear. There is evidence that the up-regulation of certain growth factors could play an important role in the promotion of the gastric carcinogenesis. AIMS: The present study was designed to determine the gene expression of major known growth factors such as transforming growth factor alpha (TGFalpha), hepatocyte growth factor (HGF) and gastrin in the gastric cancer tissue, the surrounding mucosa and, for comparison, in the normal gastric mucosa. Furthermore, the luminal and plasma levels of gastrin in patients with gastric cancer were determined. In addition, the gene and protein expressions of apoptosis-related proteins such as Bax and Bcl-2 were investigated by reverse transcription-polymerase chain reaction and Western blot. Twenty-five gastric cancer patients and 40 age- and gender-matched control subjects hospitalized with non-ulcer dyspepsia were included into this study. RESULTS: An overall H. pylori-seropositivity among gastric cancer patients was about 72% and was significantly higher than in the controls (56%). The prevalence of CagA-positive strains was also significantly higher among gastric cancer patients than in controls (56% vs. 32%). The gene expression of HGF and TGFalpha was detected more frequently in gastric cancer tissue samples than in normal gastric mucosa (52% vs. 12% for HGF and 48% vs. 24% for TGFalpha). The extent of protein expression in Western blotting analysis for HGF and TGFalpha correlated with the mRNA expression of these factors. Gene expression of gastrin was detected in the antrum of all tested patients and in the majority (84%) of gastric cancer patients. The median plasma and luminal concentrations of gastrin in gastric cancer patients were significantly higher than in controls. The gene expression of bcl-2 was detected in all (100%) and that of proapoptotic bax only in 56% of gastric cancer samples. In comparison to the surrounding non-tumorous tisssue, the gene expression of bax was significantly down-regulated and the gene expression of bcl-2 was up-regulated in gastric cancer tissue. At the protein level, Bax was not detectable and Bcl-2 was seen in 80% of gastric cancer samples. CONCLUSIONS: It is concluded that the patients infected with H. pylori, especially with CagA-positive strains, are at a higher risk of developing a gastric cancer. An increased production and release of gastrin, as well as an over-expression of growth factors such as HGF and TGFalpha, might contribute to the gastric carcinogenesis. In addition, a dysregulation of the Bax/Bcl-2 system with significant up-regulation of Bcl-2 is observed in gastric cancer.

Prevalence of Helicobacter pylori infection in coronary artery disease and effect of its eradication on coronary lumen reduction after percutaneous coronary angioplasty.

BACKGROUND: Gastric infection caused by Helicobacter pylori has recently been associated with increased risk of coronary artery disease. AIM: To: 1) determine seroprevalence of Helicobacter pylori and its cytotoxin associated gene A in patients with/without coronary artery disease (group A), 2) assess influence of Helicobacter pylori eradication on coronary artery lumen reduction after percutaneous coronary angioplasty (group B) and 3) determine influence of Helicobacter pylori eradication on plasma cytokines, lipids and coagulation factors in patients subjected to percutaneous coronary angioplasty (group B). PATIENTS AND METHODS: Group A included 100 patients with coronary artery disease (subgroup 1) and 100 patients without (subgroup II). For Helicobacter pylori seroprevalence, plasma anti-Helicobacter pylori and anti-cytotoxin associated gene A IgG were examined. Group B included 40 patients with significant single-vessel coronary arterial disease and Helicobacter pylori infection confirmed by 13C-urea breath test and serologically using anti-Helicobacter pylori and anti-cytotoxin associated gene A IgG. Six months after percutaneous coronary angioplasty and triple anti-Helicobacter pylori therapy, the Helicobacter pylori status reassessed by urea breath test was negative in all but two patients of subgroup I subjected to Helicobacter pylori therapy. Coronary angiography and laboratory tests were repeated in both subgroups of group B included in the trial and reduction in coronary artery lumen in these subgroups was compared to baseline after percutaneous coronary angioplasty considered as 100%. RESULTS: Helicobacter pylori seropositivity reached 81.5% of coronary artery disease (subgroup I) and was significantly higher than that in controls without coronary artery disease (subgroup II) (51%), the odds ratio being 4.3 for Helicobacter pylori in coronary artery disease. Cytotoxin associated gene A IgG detection was also significantly higher (47.3%) in coronary artery disease than in controls (28%) giving the odds ratio about 2.3. Mean coronary artery lumen reduction in patients undergoing percutaneous coronary angioplasty + Helicobacter pylori eradication therapy (subgroup I) was significantly (p<0.05) smaller compared to percutaneous coronary angioplasty + placebo-treated subgroup II (22% vs 41%). CONCLUSIONS: 1) There is a significant link between coronary artery disease and infection with Helicobacter pylori, especially expressing CagA proteins, 2) Helicobacter pylori eradication significantly attenuates reduction in coronary artery lumen in coronary artery disease patients after percutaneous coronary angioplasty possibly by elimination of chronic inflammation and decline in proinflammatory cytokine release, and 3) Infection of Chlamydia pneumoniae in these percutaneous coronary angioplasty patients is not affected by eradication therapy.

Water extracts of Helicobacter pylori suppress the expression of histidine decarboxylase and reduce histamine content in the rat gastric mucosa.

BACKGROUND: Acute Helicobacter pylori (Hp) infection in humans may be associated with markedly reduced gastric acid secretion, but the mechanism of this hypochlorhydria has not been fully explained. AIMS: This study was designed to investigate how water extracts (WE) of Hp applied on rat gastric mucosa affect gastric secretion and mucosal histamine concentration as well as the gene expression for histamine decarboxylase (HDC), the key enzyme converting histidine to histamine and for interleukin-1beta (IL-1beta), the important proinflammatory cytokine. MATERIALS AND METHODS: Wistar rats were surgically equipped with small cannulas to form gastric fistulas (GF). Four weeks after formation of GF, rats received either saline (control group) or WE obtained from type I Hp strain expressing CagA/VacA proteins and from type II Hp strain negative for CagA/VacA. Hp-WE was applied intragastrically (i.g.) in a volume of 1 ml at days 0, 2, 4 and 6 (total 4 times). At days 7 and 14, the secretory tests were performed during which basal gastric acid and pepsin secretion was examined and acid and pepsin outputs were measured. After secretory tests, the rats were sacrificed, the stomachs removed and the damage to the gastric mucosa was assessed by measuring the lesion area planimetrically and by histology, the gene expression in gastric mucosa for HDC and interleukin-1beta (IL-1beta) was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot. Additionally, somatostatin concentration in gastric juice, gastric mucosal histamine content and plasma gastrin and IL-1beta levels were determined using radioimmunoassay (RIA). RESULTS: Administration of Hp-WE failed to induce gross mucosal damage but microscopic examination revealed partial denudation of gastric surface epithelium without causing deep necrosis. In secretory tests, Hp-WE produced marked hypochlorhydria but type I Hp-WE induced significantly stronger inhibition of acid and pepsin secretion than type II Hp-WE, both at days 7 and 14. Both, type I and type II Hp-WE suppressed significantly the gene expression for HDC mRNA and lowered significantly gastric mucosal histamine content as compared to respective values in vehicle-treated control gastric mucosa. Furthermore, Hp-WE, resulted in a significant increase in expression of IL-1beta mRNA and a significant fall in luminal somatostatin concentration as well as a insignificant elevation of plasma gastrin level, the type I Hp-WE being more effective in these alterations than type II Hp-WE. CONCLUSIONS: (1) Ability of Hp-WE to induce superficial damage, the reduction in HDC mRNA and accompanying fall in gastric histamine release, contribute, at least in part, to marked hypochlorhydria observed in the stomach exposed to repeated Hp-WE treatments, and (2) the deleterious effect of Hp-WE on the gastric mucosa involves an impairment of gastrin-somatostatin link possibly resulting from the action of Hp-derived toxins and the induction in mucosal cells of proinflammatory cytokine such as IL-1beta.

Helicobacter pylori-gastrin link in MALT lymphoma.

BACKGROUND: There is accumulating evidence for the role of Helicobacter pylori in the development of gastric cancer as well as of lymphomas that arise in mucosa-associated lymphoid tissue (MALT). We reported recently that gastric cancer patients show high prevalence of cagA-positive H. pylori and express gastrin and gastrin receptors enabling them to stimulate tumour growth in autocrine fashion. AIMS: Since the H. pylori infection is considered to be more strongly associated with MALT lymphoma than with gastric cancer, we decided to determine the gastrin and its receptors' mRNA expression and gastrin content in this tumour as well as the release of this hormone both into plasma and gastric lumen. Twenty MALT lymphoma patients were compared with 100 age- and gender-matched controls with similar dyspeptic symptoms. RESULTS: The overall H. pylori seropositivity in MALT lymphoma was about 90% and CagA positivity was 70%, compared to 56% and 33%, respectively, in controls. The serum gastrin in MALT lymphoma was about sixfold higher than in controls while gastric luminal gastrin in these patients was over 70 times higher than in controls. Gastrin content in tumour was about 10-fold higher than in antral mucosa. Gastrin and gastrin-receptor (CCKB-receptor) mRNA were detected by reverse transcriptase-polymerase chain reaction in cancer tissue whilst in the fundic and antral mucosa, only enhanced expression of CCKB-receptor mRNA and gastrin mRNA was detected, respectively. Histamine stimulation in MALT lymphoma induced acid secretion that was only about 30% of control value due to atrophic gastritis. This study confirms an important role of CagA-positive H. pylori in the pathogenesis of MALT lymphoma and shows that this lymphoma is capable of synthesizing and releasing potent growth promoting gastrin, possibly due to the action on G-cells of H. pylori-originated Nalpha-methyl histamine and cytokines (tumour necrosis factor alpha and interleukin-8). CONCLUSIONS: Gastric MALT lymphoma is closely linked to CagA-positive H. pylori infection. Gastrin and its receptors may be implicated in the pathogenesis of gastric lymphoma.

Nucleotide-induced movements in the myosin head near the converter region.

Structural changes in subfragment 1 of skeletal muscle myosin were investigated by cross-linking trypsin-cleaved S1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In the absence of nucleotide the alkali light chains are cross-linked to the 27 kDa heavy chain fragment; the presence of MgATP reduces the efficiency of this reaction. On the other hand, MgATP promotes the cross-link formation between the N-terminal 27 kDa and C-terminal 20 kDa fragments of the heavy chain. The chemical cleavage of the cross-linked heavy chains fragments with N-chlorosuccinimide and hydroxylamine indicates that the cross-links are formed between the regions spanning residues 131-204 and 699-809. These results indicate that the two regions of the heavy chain that are relatively distant in nucleotide-free skeletal S1 [Rayment et al. (1993) Science 261, 50-58] can potentially interact upon addition of nucleotide.

Protective role of endogenous nitric oxide (NO) in lipopolysaccharide--induced pancreatic damage (a new experimental model of acute pancreatitis).

Lipopolysaccharide (LPS) derived from the bacterial cell wall activates the inflammatory response in the tissue but the role of LPS in the pathogenesis of pancreatic damage and in the activation of NO system in the pancreas has not been fully explained. The aim of this study was to investigate the effect of repeated administration of LPS to the rats on the integrity of the pancreas, on the ability of isolated pancreatic acini to secrete the amylase and on the plasma level of tumor necrosis factor alpha (TNFalpha). The role of NO in the pancreatic resistance to the damage was assessed in animals subjected to repeated administration of LPS. To induce pancreatic damage one group of rats received intraperitoneal (i.p.) injection of LPS (from E. coli) every day during 5 consecutive days (10 mg/kg--day). Another groups of animals were given N(G)-nitro-L-arginine (L-NNA), an inhibitor of NO synthase (NOS) (20 mg/kg i.p.) alone or in combination with L-arginine (100 mg/kg i.p.), 30 min prior to each LPS injection. Plasma level of TNFalpha was determined by ELISA kit. Repeated administration of LPS produced mild pancreatic inflammation that was most pronounced at day 5 of LPS treatment and manifested as edema, neutrophil infiltration and hemorrhage of the pancreas. The survival rate after 5 days treatment with LPS was 87.5%. Pancreatic weight, plasma levels of TNFalpha and amylase, pancreatic blood flow (PBF) and NO generation by pancreatic acini were markedly increased in rats subjected to repeated administration of LPS whereas the amylase response of isolated pancreatic acini to pancreatic secretagogues was significantly attenuated. Suppression of NOS by L-NNA resulted in a dramatic increase in the mortality of the animals reaching 50% and significantly increased inflammatory changes in the pancreatic tissue, decreased PBF, abolished the ability of pancreatic acini to release NO and to secrete amylase. Pancreatic weight and plasma levels of amylase and TNFalpha significantly increased in the group of rats treated with combination of LPS+L-NNA as compared to the animals received LPS alone. Addition of L-arginine to L-NNA+LPS administration reversed all harmful effects produced by L-NNA in the pancreas. We conclude that repeated administration of high doses of bacterial LPS to the rats could induce pancreatic tissue damage by itself, however, it is not able to produce severe pancreatitis. Suppression of NO generation significantly aggravates the pancreatic lesion produced by LPS leading to the dramatic mortality in treated rats. The rise of plasma level of TNFalpha corresponds to the severity of pancreatic inflammation.

Helicobacter pylori infection delays healing of ischaemia-reperfusion induced gastric ulcerations: new animal model for studying pathogenesis and therapy of H. pylori infection.

OBJECTIVE: Helicobacter pylori (Hp) infection is a major risk factor of peptic ulcerations but studies on its pathogenicity are limited due to the lack of an adequate animal model. In this study we developed the new model of gastric Hp infection in rat gastric mucosa, with acute gastric erosions progressing into ulcers in animals subjected initially to ischaemia-reperfusion (I/R). DESIGN: I/R lesions were produced in rats by clamping the coeliac artery for 0.5 h followed by 1 h reperfusion and gastric inoculation with type I Hp strain (CagA and VacA positive) or type II Hp strain (CagA and VacA negative), obtained from fresh clinical isolates, or with vehicle (saline). Gastric secretion during recovery from I/R lesions was determined in a separate group of rats equipped with chronic gastric fistula to inoculate the animals with Hp and then to collect gastric juice for determination of gastric acidity and pepsin outputs as well as luminal content of somatostatin. METHODS: The animals were killed at 0, 3, 12 or 24 h and 3, 5, 10 or 15 days after Hp inoculation and the area of gastric lesions was determined planimetrically and gastric blood flow (GBF) was measured by the H2-gas clearance technique. The venous blood was withdrawn for measurement of plasma interleukin (IL)-1beta and tumour necrosis factor alpha (TNFalpha) by ELISA and plasma gastrin, luminal somatostatin by RIA and the mucosal expression of transforming growth factor (TGFalpha) was analysed using RT-PCR with specific primers. Gastric Hp infection was assessed by histology, rapid urease test and Hp culture. The effect of triple therapy with omeprazole, amoxycillin and tinidazole on Hp infection and ulcer healing was also determined. RESULTS: Ischaemia alone resulted in an immediate fall in GBF and almost complete suppression of gastric secretion but without any gastric lesions. When ischaemia was followed by 1 h of reperfusion, acute gastric erosive lesions immediately occurred, reaching a maximum at 12 h after I/R and progressing after 3 days to deeper gastric ulcers that disappeared after 15 days. In Hp-inoculated rats, the number of viable Hp colonies gradually increased, reaching maximum at day 10 with infection with type I and at day 15 with infection with type II Hp. At day 15 the difference in Hp colonization was not significantly different between the stomachs infected by type I and type II Hp. Inoculation, especially with the type I Hp strain, significantly delayed healing of I/R-induced acute lesions and accelerated their progression into deeper chronic ulcers. This effect was accompanied by a significant fall in GBF and a higher increment in plasma IL-1beta and TNFalpha levels. Gastric acid secretion, which was completely inhibited up to 12 h after I/R, returned to the control value 24 h upon completion of the I/R procedure. This return was delayed in Hp-infected rats and accompanied by a significant elevation of plasma gastrin and a decrease in luminal somatostatin. The immunoreactivity of TGFalpha and expression of TGFalpha mRNA determined by RT-PCR were well defined in intact gastric mucosa but were significantly decreased, especially in mucosa infected with type I Hp strain, at day 15 after I/R. The triple therapy which cured Hp infection completely abolished the delay in ulcer healing caused by Hp. CONCLUSIONS: (1) Gastric infection with the Hp strain expressing cagA and vacA encoded cytotoxins delays healing of I/R-induced acute gastric lesions due to an impairment of gastric microcirculation, and excessive proinflammatory cytokine release and suppression of anti-ulcer TGFalpha expression. (2) The I/R induced suppression of gastric acid secretion may contribute to the hypergastrinaemia as a secondary phenomenon and may account for the spread of Hp infection observed during the progression of acute erosions into chronic ulcers. (3) I/R induced gastric ulcers may be a useful model for studying the action of Hp infection on gastric ulcerogenesis in ischaemic stomach and for testing anti-Hp therapy.

Adherence of bile-isolated bacteria to the bile ducts mucosa as a pathogenic factor in the development of inflammatory lesions.

Bacterial infection of the bile system appears to be an important factor in the formation of stones. In view of the hypothesis that strains of E. c. form an essential factor in infections of the bile ducts, an attempt has been made to determine the connection between infections of the bile ducts and the adherence of E. c. to the epithelium of the gallbladder. The research covered 148 patients operated electively for cholecystolithiasis (121), cholecystocholedocholithiasis (26) and recurrent lithiasis (1). In bile collected from the gallbladder in the course of the operation, E. coli strains were isolated. Cholangioscopy performed in 26 patients enabled the macroscopic evaluation and grading of inflammatory lesions of bile duct mucosa. The mucosa of the gallbladder was evaluated histologically. The adherence test was performed using homologous and heterologous strains of E. c. isolated from the bile of gallstone patients. The adherence occurred most frequently in the neck of the gallbladder (71-100%) in those patients in whom an infectious process of the bile ducts mucosa was endoscopically diagnosed. The adherence of bacteria to the epithelium of the gallbladder did not depend on the type of inflammation (acute, chronic).

Helicobacter pylori, gastrin and cyclooxygenases in gastric cancer.

BACKGROUND: Tumors arising in the stomach have worldwide distribution and the infection with Helicobacter pylori (HP) has been implicated in causation of this disease. The HP discovery, which is considered as the greatest advance of gastroenterology at the dawn of 3rd millennium, is accompanied by hypergastrinemia, which seems to play a key role in gastric cancerogenesis but no study was undertaken to assess the relationship between the HP infection and coexpression of gastrin and cyclooxygenases (COX), the rate limiting enzymes in the eicosanoids production. AIMS: Since gastrin is recognized as a effective gastric mitogen, it could be capable to induce COX-2, a potent tumor growth promoting and angiogenic factor, we decided 1) to compare the seroprevalence of HP and its cytotoxic protein, CagA, in gastric cancer patients with those in age- and gender-matched controls; 2) to determine the gene expression of gastrin and its receptors (CCK(B)-R) in gastric cancer, 3) to assess the plasma levels, gastric lumen and tumor tissue contents of gastrin and 4) to examine the mRNA and enzyme protein expression of COX-1 and COX-2 in cancer tissue and intact gastric mucosa before and after HP eradication. MATERIAL AND METHODS: The trial material included 20 patients with gastric cancers and 100 age- and gender-matched controls. Anti-HP and anti-CagA IgG seroprevalence was estimated by specific antisera using ELISA tests. Gene expressions of gastrin, CCK(B)-R, COX-1 and COX-2 was examined using RT-PCR with GAPDH as a reference and employing Western blot for COX-2 expression, while gastrin was measured by RIA. RESULTS: The seroprevalence of HP, especially that expressing CagA, was significantly higher in gastric cancers than in controls. Both gastrin and CCK(B)-R mRNA were detected by RT-PCR in the cancer tissue and similarly COX-2 mRNA and protein were found in most of cancers and in the HP infected antral mucosa but not in HP eradicated patients in whom only cancer tissue but not gastric mucosa expressed COX-2. The gastric cancer tissue contained 20 times more of immunoreactive gastrin than the HP infected antral gastric mucosa and following HP eradication the gastrin content in the tumor and antrum showed a marked and significant reduction. No significant change in CCK(B)-R expression was noticed before and after HP eradication in the tumor and the corpus mucosa. CONCLUSIONS: 1). Gastric carcinoma coexpresses gastrin, its receptors (CCK(B)-R), and COX-2; 2) HP infection may contribute to gastric cancerogenesis via gastrin andCOX-2 that may account for the stimulation of tumor growth, angiogenesis, and reduction in apoptosis 3) HP positive patients developing gastric cancer should be considered for HP eradication to reduce the HP provoked hypergastrinemia and COX-2 overexpression in the tumor tissue.

Mouse model of Helicobacter pylori infection: studies of gastric function and ulcer healing.

BACKGROUND: Helicobacter pylori infection in humans is a major risk factor for peptic ulcer, but studies on the relation between H. pylori infection and gastric pathology are limited due to a deficiency of convenient animal models resembling this infection in humans. METHODS: We studied the effects of inoculation of conventional BALB/c mice with CagA and VacA positive (type I) H. pylori or CagA and VacA negative H. pylori (type II) strains on gastric secretion and healing of chronic acetic acid-induced ulcers in mouse stomachs. The ulcer area, gastric blood flow, plasma interleukin (IL)-1beta and IL-12, as well as plasma gastrin and gastric luminal somatostatin were determined. Gastric mucosal biopsy samples were also taken for assessment of the presence of viable H. pylori using a rapid urease test, H. pylori-culture and the RT-PCR analysis of the signal for H. pylori CagA. RESULTS: Gastric acid and pepsin secretion was reduced by over 50% immediately after H. pylori inoculation and accompanied by a significant increment in plasma gastrin and fall in gastric luminal somatostatin content observed over all test days, particularly in mice infected with type I H. pylori. The area of ulcers in vehicle-treated controls decreased significantly starting from day 2 after ulcer induction and then continued to decline for a further 14 days to heal almost completely after 28 days. In contrast, the ulcers were present until day 28 in all mice infected with type I or type II H. pylori strains, being significantly larger, especially with type I H. pylori infection. The gastric blood flow at the ulcer margin and ulcer crater in vehicle-treated mice gradually increased with decreasing ulcer size, after 14 and 28 days reaching a value which was not significantly different from that in vehicle-administered mice. In contrast, the gastric blood flow in type I H. pylori and, to a lesser extent, in type II H. pylori infected mice was significantly lower than in vehicle controls, both at the margin and at the crater of ulcers at all tested days. Histological changes such as oedema or congestion of surface epithelium were found after 7 days whereas mucosal inflammatory infiltration appeared after 14 days with a further increase after 28 days, especially in type I H. pylori and to a lesser extent in type II H. pylori infected mice. Plasma IL-1beta and IL-12 were significantly elevated at all tested days of ulcer healing and their increments were significantly higher in type I than in type II H. pylori infection. CONCLUSIONS: Conventional mice with gastric ulcers can be successfully infected by both toxigenic and nontoxigenic H. pylori strains, and this infection causes an immediate suppression of gastric secretion and markedly delays the healing of ulcers due to the fall in mucosal microcirculation in the ulcer region, cytokine release and an impairment in the gastrin-somatostatin link that appears to be independent of gastritis and more pronounced with infection of toxigenic than nontoxigenic strains.

Water extracts of Helicobacter pylori delay healing of chronic gastric ulcers in rats: role of cytokines and gastrin-somatostatin link.

BACKGROUND: Helicobacter pylori (Hp) is considered as a major risk factor of peptic ulcer, but the pathogenic mechanism of its action has not been fully explained. AIMS: This study was designed: (1) to compare the ulcer healing effects of water extract (WE) obtained from type-I cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) expressing Hp and from type-II CagA- and VacA-negative Hp strain with those of vehicle (saline), and (2) to determine the alterations in gastric secretion, gastric blood flow (GBF) and expression of Hp-related cytokines during the ulcer healing in rats treated with toxigenic (type-I) and non-toxigenic (type-II) Hp-derived WE. METHODS: Gastric ulcers were produced by serosal application of acetic acid in rats with or without gastric fistula treated with vehicle (saline) or WE originating from type-I or type-II Hp administered intragastrically on days 1, 3, 5 and 7 upon ulcer induction. On days 3, 9 and 15, animals were lightly anesthetized with ether, the abdomen was opened and the GBF was measured by the H2-gas clearance technique in the ulcer area and non-ulcerated mucosa. Venous blood was withdrawn for the measurement of plasma cytokine (IL-1beta and TNFalpha) levels and plasma and gastric contents were also collected for gastrin and somatostatin determination by specific radioimmunoassay. RESULTS: Gastric ulcers healed gradually in vehicle-treated controls and the ulcer area on days 3, 9 and 15 was reduced by 12, 43 and 92%, respectively. In rats treated with WE of type-I Hp, ulcer healing was significantly delayed, and gastritis and infiltration of ulcerated gastric mucosa with inflammatory cells were observed histologically. The prolongation of ulcer healing by WE of both Hp strains was accompanied by a marked fall in the GBF at the ulcer margin and transient hyposecretion especially in rats given WE of type-I Hp strain. On day 15 of ulcer healing, the plasma concentration of IL-1beta and TNFalpha was negligible in vehicle control rats, but it was significantly elevated particularly in rats treated with WE of type-I Hp. RT-PCR analysis revealed that mucosal expression of IL-1beta and TNFalpha mRNA was significantly upregulated in the gastric mucosa of rats treated with either toxigenic or non-toxigenic Hp WE. The plasma gastrin level was significantly higher and the luminal concentration of somatostatin was significantly lower in rats treated with Hp-WE than in vehicle-treated controls and these alterations were more pronounced in rats treated with WE type-I than type-II Hp. CONCLUSIONS: WE of toxigenic Hp strain delays ulcer healing due to the reduction in the gastric microcirculation at the ulcer margin, the overexpression of inflammatory cytokines and the impairment of the gastrin-somatostatin link.

Helicobacter pylori and its eradication in rosacea.

Rosacea is a common condition of unknown etiology usually accompanied by gastrointestinal symptoms and favorably responding to the treatment with antibiotics. This study was designed to examine the prevalence of gastric Helicobacter pylori (Hp) infection verified by 13C-UTB-test, CLO, Hp culture and serology (IgG) in patients with rosacea. Gastroduodenoscopy was combined with pentagastrin secretory test and antral and fundic biopsy samples were taken for histological evaluation (the Sydney system). Blood samples were also taken for the determination of plasma gastrin using RIA and plasma interleukin (IL)-8 and tumor necrosis factor alpha (TNFalpha) using ELISA. This study was performed in 60 patients, 31-72 year old, with visible papules and pustules associated with erythema and flushing on the face and on 60 age- and gender-matched patients without any skin diseases but with similar as in rosacea gastrointestinal symptoms but without endoscopic changes in gastroduodenal mucosa (non-ulcer dyspepsia - NUD). The Hp prevalence in rosacea patients was about 88 % as compared to 65% in control NUD patients. Among rosacea patients, 67% were cytotoxin associated gene A (CagA) positive, while in NUD patients only 32% were CagA positive. Rosacea patients showed gastritis with activity of about 2.1 in antrum and 0.9 in the corpus of the stomach while those with NUD only mild gastritis with activity of approximately 1.0) confined to the antrum only. Following initial examination, typical 1 wk anti-Hp therapy including omeprazole (20 mg bd.), clarithromycin (500 mg bd.) and metronidazol (500 mg bd.) was carried out. After eradication, 51 out of 53 treated rosacea patients became Hp negative. Within 2-4 weeks, the symptoms of rosacea disappeared in 51 patients, markedly declined in 1 and remained unchanged in 1 other subject. A dramatic reduction in activity of gastritis (to 0.3 in antrum and to 0.1 in corpus) was observed. Basal plasma gastrin decreased from 48 +/- 5 pM before to 17+/-3 pM after eradication, while pentagastrin-induced maximal (MAO) declined, respectively, from about 16.6 +/- 4.2 to 8.5 +/- 1.8 mmol/h. Plasma TNFalpha and IL-8 were reduced after the therapy by 72% and 65%, respectively. We conclude that: 1) Rosacea is a disorder with various gastrointestinal symptoms closely related to gastritis, especially involving the antrum mucosa, with Hp expressing cagA in the majority of cases and elevated plasma levels of TNFalpha and IL-8; 2) The eradication of Hp leads to a dramatic improvement of symptoms of rosacea and reduction in related gastrointestinal symptoms, gastritis, hypergastrinemia and gastric acid secretion; and 3) Rosacea could be considered as one of the major extragastric symptoms of Hp infection probably mediated by Hp-related cytotoxins and cytokines.