Inhibition of endogenous nitric oxide activity impairs the colonic sparing effect of rofecoxib, the cyclooxygenase-2 inhibitor and resveratrol, the preferential cyclooxygenase-1 inhibitor in the course of experimental colitis. Role of oxidative stress biomarkers and proinflammatory cytokines.

The gut mucosal barrier plays a key role in the physiology of gastrointestinal (GI) tract, preventing under homeostatic conditions, the epithelial cells of the gastric mucosa from hydrochloric acid and intestinal mucosa from alkaline secretion, food toxins and pathogenic microbiota. Previous studies have documented that blockade of both isoforms of cyclooxygenase (COX): constitutive (COX-1) and inducible (COX-2), as well NO synthase in the stomach exacerbated the gastric damage induced by various ulcerogens, however, such as effects of non-selective and selective inhibition of COX-1, COX-2 and NOS enzymes on colonic damage have been little studied. The supplementation of NO by intragastric (i.g.) treatment with NO-releasing compound NO-aspirin (NO-ASA) or substrate for NO synthase L-arginine ameliorated the damage of upper GI-tract, but whether similar effect can be observed in colonic mucosa associated with the experimental colitis, and if above mentioned compounds can be effective in aggravation or protection of experimental colitis remains less recognized. In this study rats with experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzosulphonic acid (TNBS) were daily treated for 7 days with: 1) vehicle (i.g.), 2) ASA 40 mg/kg (i.g.), 3) rofecoxib 10 mg/kg (i.g.), 4) resveratrol 10 mg/kg (i.g.), 5) NO-ASA 40 mg/kg (i.g.), 6) L-arginine 200 mg/kg (i.g.) with or without of L-NNA 20 mg/kg (i.p.). The macroscopic and microscopic area of colonic damage was determined planimetrically, the colonic blood flow (CBF) was assessed by Laser flowmetry, and the oxidative stress biomarkers malondialdehyde and 4-hydroxynonenal (MDA+4-HNE), the antioxidative factors superoxide dismutase (SOD) and glutathione (GSH), as well as proinflammatory cytokines in the colonic mucosa (tumor necrosis factor alpha (TNF-α) and interleukin-1beta (IL-1ß)) were measured. We have documented that administration of TNBS produced gross and microscopic colonic damage and significantly decreased CBF (p<0.05). Treatment with ASA significantly increased the area of colonic damage (p<0.05), an effect accompanied by a significant decrease in the CBF, the significant increment of MDA+4-HNE, and the attenuation of the antioxidative properties in colonic mucosa, documented by a significant decrease of SOD activity and GSH concentration, and elevation of the colonic tissue levels of TNF-α and IL-1ß comparing to control Veh-treated TNBS rats. Administration of rofecoxib or resveratrol also significantly increased the colonic damage and significantly decreased the CBF, causing an increase in MDA+4-HNE and mucosal content of TNF-α and IL-1α and a significant decrease of the SOD activity and GSH content (p<0.05), however, these changes were significantly less pronounced as compared with ASA. On the contrary, the treatment with NO-ASA, or L-arginine, significantly diminished the area of colonic lesions, the MDA+4-HNE concentration, attenuated the TNF-α and IL-1ß levels, while increasing the CBF, SOD activity and GSH content (p<0.05). The concomitant treatment of L-NNA with rofecoxib or resveratrol reversed an increase in area of colonic damage and accompanying changes in CBF, colonic mucosa TNF-α and IL-1ß levels, the MDA+4-HNE concentration, and SOD activity and GSH content comparing to those observed in TNBS rats treated with these COX-inhibitors alone (p<0.05). In contrast, co-treatment with L-NNA and NO-ASA or L-arginine failed to significantly affect the decrease of colonic lesions accompanied by the rise in CBF, the attenuation of MDA+4-HNE concentration, TNF-α and IL-1ß levels, SOD activity and GSH content exerted by NO-ASA- or L-arginine treatment of the respective control TNBS-rats without L-NNA administration. These observations suggest that 1) the increase of NO availability either from NO-releasing donors such as NO-ASA or NO precursors such as L-arginine, can inhibit the inflammatory and microvasculature alterations, as well as increase in lipid peroxidation due to the enhanced efficacy of these compounds to increase the antioxidative properties of colonic mucosa, 2) unlike ASA which exacerbated the severity of colitis, the treatment with rofecoxib, the specific 'safer' COX-2 inhibitor or resveratrol, the polyphenolic compound known to act as the dual COX-1 and COX-2 inhibitor, can attenuate the colonic damage during course of TNBS colitis possibly via anti-inflammatory and antioxidative properties, and 3) the blockade of endogenous NO activity by L-NNA which also exacerbated the severity of mucosal damage in colitis, can abolish the sparing effect of rofecoxib and resveratrol indicating the NO bioavailability plays an important role in enhanced efficacy of both specific and dual COX inhibitors to ameliorate the experimental colitis.

The relationship between the presence of Helicobacter pylori and the composition of ionic and organic microelements in drinking water from Cracow.

Although the natural niche for H. pylori (Hp) is the human stomach, for widespread infection to occur this microorganism may need to survive in the external environment. Molecular techniques such as polymerase (PCR) have revealed the presence of Hp DNA in water, indicating that this environment could act as a reservoir for this bacterium. The aim of this study was to analyse the occurrence of Hp in tap water from Cracow and to examine the relationship between 26 parameters and the presence of Hp DNA due to the lack of such information related to this issue in Poland. Additional aim of this study was to determine whether the correlation between Hp DNA detection and seasonal changes of water quality in 379 water samples collected from various water treatment plants (WTPs), could be found. Water samples were subjected to PCR for glmM and cagA genes. Ionic and organic composition of microelements were determined in accordance to Polish and ISO standards. The data obtained from tests show that 212 (55.96%) objects were Hp DNA (glmM) positive and among them 145 (68.40%) waters samples revealed expression of cagA. Linear Discriminant Analysis and Principal Component Analysis were used and provided that the selected variables (p < 0.05): colour, pH, conductivity at 25°C, chlorides, nitrites, nitrates, phosphates, chlorates, chlorites, sulphates, free chlorine, sodium, magnesium, potassium, calcium, organic carbon, trichloromethane, bromodichloromethane, dibromochloroethane, total iron, ammonium ion, and Æ©TMHs distinguished the water samples that contain Hp DNA and do not contain Hp DNA. We conclude that the ionic and organic composition of microelements in water might influence the presence of Hp. Thus, determination of the selected microelements may indirectly indicate or sometimes predict the presence of Hp in drinking water.

Participation of the intestinal microbiota in the mechanism of beneficial effect of treatment with synbiotic Syngut on experimental colitis under stress conditions.

Gut-brain axis plays a central role in the regulation of stress related diseases such as irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD). It is increasingly recognized that stress modulates gut microbiota community structure and activity and represents an important causal factor in dysbiosis. This study was designed to determine the effect of daily treatment with synbiotic (Syngut) containing inulin, Lactobacillus acidophilus, Bifidobacterium lactis W51, Lactobacillus plantarum W21 and Lactococcus lactis applied i.g. at a dose of 50 mg/kg i.g. on the colonic damage and colonic mucosal blood flow in rats with experimentally induced TNBS-colitis that were additionally exposed or not to acute stress (episodes of cold restraint stress every other day before colitis induction). Control rats received daily treatment with vehicle (saline, i.g.) or mesalazine (50 mg/kg-d i.g.), the standard drug recommended in therapy of IBD. At the termination of TNBS colitis, the histologic evaluation of colonic mucosa, mucosal malonyldialdehyde (MDA) level and plasma concentrations of proinflammatory cytokines (TNF-α, IL-1ß) and adipokine adiponectin were assessed. the samples of colonic mucosa not involving colonic lesions and surrounding the flared mucosa were excised for the determination of mRNA expression for proinflammatory biomarkers TNF-α, IL-1ß, IL-10 and COX-2 as well as antioxidazing factors SOD-1 and SOD-2. Finally, the gut microbial profiles were analyzed by 16S rRNA sequencing at phylum, family and genus level. Episodes of cold stress significantly aggravated the course of TNBS colitis, and significantly increased the release of proinflammatory cytokines as well as the significant increase in the MDA concentration has been observed as compared with non-stressed TNBS rats. These changes were followed by the significant fall in the CBF and plasma adiponectin levels and by the overexpression of mRNA of proinflammatory biomarkers. Synbiotic treatment with Syngut significantly reduced the area of colonic lesions observed macroscopically and microscopically in rats with TNBS colitis with or without exposure to cold stress, significantly increased the CBF, normalized plasma adiponectin levels and significantly attenuated the release and colonic expression of proinflammatory cytokines and biomarkers. the analysis of the gut microbiota showed a significant reduction of microbial diversity (Shannon index) in rats with TNBS colitis with or without exposure to stress. The therapy with Syngut failed to significantly affect the alpha diversity. At the phylum level, the significant rise in Proteobacteria has been observed in stressed rats with TNBS colitis and this effects was attenuated by treatment with Syngut. At family level, TNBS colitis alone or in combination with stress led to a significant decrease of SCFA producing bacterial taxa such as Ruminococaceae and Lachnospiraceae and Syngut counteracted this effect. We conclude that: 1) cold stress exacerbates the gastrointestinal inflammation in experimental colitis; 2) the synbiotic therapy with Syngut ameliorates the gut inflammation in rats with TNBS colitis combined with cold stress; 3) the beneficial effect of Syngut is accompanied by increase of anti-inflammatory taxa such as Ruminococaceae and Lachnospiraceae, and 4) the modulation of gut microbiota with Syngut alleviates stress-related intestinal inflammation suggesting a potential usefulness of synbiotic therapy in intestinal disorders accompanied by stress in patients with IBD.

Involvement of epithelial-mesenchymal transition-inducing transcription factors in the mechanism of Helicobacter pylori-induced fibroblasts activation.

Gastric cancer (GC) originating from the lining of the stomach is one of the most frequent malignancies in humans. The most efficient method giving hope of full recovery from GC is gastric resection combined with adjuvant chemotherapy and radiotherapy. However, over 50% of patients after treatment suffer from recurrence and peritoneal metastasis. The bacteria Helicobacter pylori (Hp) is nowadays considered as the major pathogen capable of colonizing gastric mucosa. This bug causes deregulation of multiple signaling pathways including the activation of nuclear factor kappaB (NFκB) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) responsible for inflammation and development of carcinogenic cascade. The pathomechanism of these changes remains little understood, but the Hp virulence factors affecting mainly gastric epithelium have been postulated. Nevertheless, the changes associated with inflammation progressing to cancer are not only limited to epithelial cells. The cells surrounding the tumor, mainly activated fibroblasts (CAFs - cancer-associated fibroblasts) create molecular microenvironment promoting tumorigenesis and cancer invasion. The downstream targets of STAT3, epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) are expressed in activated fibroblasts providing them with additional properties. Thus, our aim was to determine if the Hp strain expressing CagA and VacA cytotoxins may result in the activation/differentiation of rat gastric fibroblasts resulting in NFκB and STAT3 signaling, which could lead to EMT-TFs expression and secretome responsible for inflammatory and EMT inducing microenvironment. In this study, gastric samples were harvested from 8-week-old Spraque-Dowley rats and the primary and secondary fibroblast cultures were established. The 70% confluent secondary fibroblast cultures were infected with 1 x 109 of live Hp expressing cytotoxins CagA VacA per dish and incubated in humidified atmosphere for 3, 24, 48 and 72 hours, before the conditioned media or the cells were used for endpoint experiments. As the control, fibroblast culture in DMEM with 10% FBS and antibiotics, free from Hp infection was used. The expression of mRNA for 18S (control), toll-like receptors: TLR2 and TLR4, STAT3, NFκB p65/Rel A, inhibitor of NF-κB (Iκß), Snail and Twist was determined by RT-PCR. The protein expression of Snail and Twist was assessed by Western blot technique. The fibroblast supernatant was collected at 72 hours from non-infected and Hp (cagA+ vacA+)-infected culture and the concentrations of interleukin 8 (IL-8), hepatocyte growth factor (HGF) and stromal derived factor-1 (SDF-1) were measured by ELISA. In fibroblasts infected with Hp (cagA+ vacA+), the significant increase of mRNA expression for both, TLR2 and TLR4, as well as STAT3, NFκB/RelA subunit was observed already after 3 hours of fibroblasts infection with Hp strain compared with control non-infected fibroblasts. Simultaneously, the significant decrease of Iκß mRNA has been noticed starting from 48 hours after the Hp infection of fibroblasts was carried out. The strong increase in the expression of Snail1 and Twist mRNA was recorded already at 3 hours in Hp-infected fibroblasts comparing to control non-infected fibroblasts and this increase persisted at 24 and 48 hours being the most pronounced at 72 hours post incubation with Hp. The expression of Snail1 protein was observed after 3 hours post Hp infection and this increase persisted throughout entire time periods upon Hp infection. In contrast, no detectable level of Twist protein expression was observed up to 72 hours post-infection neither in control conditions nor in fibroblasts co-infected with Hp. These changes in fibroblasts were accompanied by a significant increase in the release of HGF, SDF-1 and IL-8 determined in cell supernatants collected from Hp-infected fibroblasts. These data indicate that the activation/differentiation of rat gastric fibroblasts can occur directly by Hp releasing CagA and indirectly through TLR2 and TLR4 and these effects can be mediated by transcription factors NFκB and STAT3 signaling leading to rapid Snail1 protein expression. We conclude that NFκB and STAT3 signaling together with Snail1 protein expression may activate the secretome responsible for fibroblasts inflammatory and EMT-inducing microenvironment likely serving as prerequisite for GC development.

Role of curcumin in protection of gastric mucosa against stress-induced gastric mucosal damage. Involvement of hypoacidity, vasoactive mediators and sensory neuropeptides.

The antioxidizing properties of curcumin, a highly pleiotropic substance used for centuries in traditional medicine has been confirmed by numerous experimental and clinical studies. Curcumin exhibits anti-inflammatory, antiproliferative and anti-angiogenic actions inhibiting the development and progression of tumors but the efficacy of this compound to influence gastric acid secretion n in the stomach and to affect the gastric mucosal damage induced by non-topical ulcerogenes such as stress has been little studied. We determined the effect of curcumin on basal and pentagastrin- or histamine-stimulated gastric secretion, in rats with surgically implemented gastric fistulas and we assessed the contribution of gastric secretion, endogenous prostaglandin (PG), endogenous nitric oxide (NO), as well as sensory afferent nerves in the mechanisms underlying the potential gastroprotective effects of curcumin against stress-induced gastric mucosal lesions. Rats exposed to water immersion and restraint stress (WRS) for 3.5 h were pretreated either with: 1) vehicle (saline); 2) curcumin (2.5 - 100 mg/kg i.g.) or 3) curcumin (50 mg/kg i.g.) combined with or without indomethacin (5 mg/kg i.p.), SC-560 (5 mg/kg i.g.) or rofecoxib (10 mg/kg i.g.); 4) curcumin (50 mg/kg i.g.) co-administered with (L-NNA (20 mg/kg i.p.) with or without L-arginine (200 mg/kg i.g.), a substrate for NO-synthase; 5) curcumin (50 mg/kg i.g.) administered in rats with intact or capsaicin-induced functional ablation of sensory nerve fibers, and 6) curcumin (50 mg/kg i.g.) administered with capsazepine (5 mg/kg i.g.), the antagonist of vanilloid TRPV1 receptor. The number of gastric lesions was determined by planimetry, the gastric blood flow (GBF) was assessed by H2-gas clearance technique, the plasma gastrin concentrations were measured using the radioimmunoassay (RIA) and the expression of mRNA for tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in gastric mucosa was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Curcumin dose-dependently reduced the WRS-induced gastric lesions, the dose inhibiting these lesions by 50% being about 50 mg/kg. These effects of curcumin were accompanied by an increase in GBF and the reduction in basal and histamine- or pentagastrin-stimulated gastric acid secretion. The protective and hyperemic activities of curcumin (50 mg/kg i.g.) against WRS lesions were significantly attenuated (P < 0.05) in rats pretreated with rofecoxib and SC-560 and completely reversed (P < 0.01) by indomethacin. L-NNA significantly reduced (P < 0.05) the decrease in WRS-induced lesions and the accompanying rise in GBF caused by curcumin and these effects were restored by concurrent treatment with L-arginine (200 mg/kg i.g.). The curcumin-induced decrease in the number of WRS-induced gastric lesions and accompanying increase in the GBF were significantly attenuated (P < 0.05) in capsaicin-denervated rats and in those pretreated with capsazepine. These effects of curcumin in rats with capsaicin denervation were restored by concomitant treatment with exogenous calcitonin gene related pepetide (CGRP) combined with curcumin and subsequently exposed to WRS. The expression of mRNA for TNF-α, COX-2 and iNOS was significantly increased (P < 0.05) in vehicle-pretreated control rats exposed to WRS and significantly attenuated (P < 0.05) by curcumin administered in graded dosages. We conclude that curcumin exerts gastroprotective and hyperemic activities against experimental stress-induced gastric lesions by mechanism involving endogenous prostaglandins, NO, the neuropeptides such as CGRP released from capsaicin-sensitive afferent nerves and the activation of vanilloid TRPV1 receptors located on these sensory nerve terminals.

The renin-angiotensin system and its vasoactive metabolite angiotensin-(1-7) in the mechanism of the healing of preexisting gastric ulcers. The involvement of Mas receptors, nitric oxide, prostaglandins and proinflammatory cytokines.

The inhibition of angiotensin-converting enzyme (ACE) or the blockade of angiotensin (Ang) AT-1 receptors affords protection against acute gastric mucosal injury, but whether the major metabolite of renin-angiotensin system (RAS), Ang-(1-7), accelerates the healing process of preexisting gastric ulcers remains unknown. Previous studies documented that Ang-(1-7) acting via its own Mas receptor exerts vascular responses opposing those of Ang II. We studied the effects of the Ang-(1-7)/Mas receptor axis on the healing rate of acetic-acid-induced gastric ulcers with or without the blockade of Mas receptors by A 779 and compared it with the effects of activation and blockade of the AT-1 receptor by the treatment with Ang II and losartan, respectively, the inhibition of ACE by lisinopril, the NO/cNOS inhibition by L-NAME and inhibition of prostaglandin/COX system by indomethacin in the presence of Ang-(1-7). Additionally, ex vivo metabolism of Ang I in gastric tissue was assessed by LC/MS method. At day 9 after ulcer induction, the area of these ulcers and the accompanying changes in total gastric blood flow (GBF) were determined as were gastric mucosal blood flow (GMBF) at ulcer margin and gastric oxygen uptake (GVO2). The gastric mucosal expression of mRNAs for constitutive nitric oxide synthase (cNOS), superoxide dismutase (SOD), and pro-inflammatory cytokines interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α) and plasma level of both cytokines were determined by RT-PCR and ELISA. The 9 days treatment with Ang II dose-dependently increased the area of gastric ulcers and this effect was accompanied by a significant fall in the GBF, GVO2 and GMBF at ulcer margin. In contrast, treatment with Ang-(1-7) which produced a significant rise in the luminal content of NO significantly reduced the area of gastric ulcer and significantly increased the GBF, GVO2 and the GMBF at ulcer margin. Similar GMBF changes and significant reduction the area of gastric ulcer was observed in rats with gastric ulcers treated with the agonist of Mas receptor, AVE 0991. These effects of Ang-(1-7) and AVE 0991 were eliminated by blockade of the Mas receptor with A779. Similarly to Ang-(1-7), treatment with losartan or lisinopril significantly reduced the area of gastric ulcers and the accompanying increase in the GMBF at ulcer margin and these effects were significantly attenuated by a concomitant administration of L-NAME and indomethacin. The rate of healing of ulcers was associated with a decrease in ex vivo Ang-(1-7) formation and this effect was attenuated by lisinopril. The treatment with Ang-(1- 7) or AVE 0991 increased the expression of mRNA for cNOS and SOD and downregulated that of IL-1ß and TNF-α followed by the decrease in the plasma IL-1ß and TNF-α levels. We conclude that the Ang-(1-7)/Mas receptor system accelerates the healing of preexisting gastric ulcers via an increase in the gastric macro- and microcirculations, and an increase in gastric tissue oxygenation. These effects are mediated by PG and NO derived from overexpression of cNOS, an increase in the expression of antioxidizing enzyme SOD 2 and an anti-inflammatory action involving the inhibition of expression and release of pro-inflammatory cytokines IL-1ß and TNF-α. Our results seem to underlie the importance of the Ang-(1-7), AT-1 and Mas receptors in the regulation of local vascular and metabolic effects associated with mechanism of gastric ulcer healing.

The role of local and systemic cytokines in patients infected with Clostridium difficile.

It is widely accepted that the pathogenesis of Clostridium difficile infection (CDI) is multifactorial, dependent on pathogen virulence factors produced by the organism as well as disorders of the gastrointestinal tract, the alteration in intestinal flora and the immune response of the host. In particular, the immune response in the course of CDI and the involvement of cytokines in the pathogenesis of CDI is not fully understood. The aim of our study was to evaluate the relationship between proinflammatory and anti-inflammatory cytokines and the course of CDI in vivo. We prospectively studied 80 patients. Our study group included 40 patients aged 30-87 years (mean age 66.9 years) with CDI hospitalized at Infectious Diseases Department and Gastroenterology and Hepatology Clinic, University Hospital in Cracow, and 40 healthy volunteers aged 24-62 years (mean age 51.1 years). The serum concentrations of cytokines IL-1ß, IL-6, IL-8, IL-10, tumor necrosis factor (TNF-α), myeloperoxidase (MPO), and prostaglandin E2 (PGE2) were measured using ELISA assays. Additionally, the routine biochemical parameters were assessed including the following: white blood cells with differential leukocyte count, platelets counts, and blood plasma levels of creatinine, alanine transaminase, and C-reactive protein were determined. We noted a significant increase in the concentration of the following cytokines in the CDI group when compared to the control group: IL-1b (4.7 vs. 3.6 pg/ml), IL-6 (21.0 vs. 0.04 pg/ml), IL-10 (8.5 vs. 0.5 pg/ml), TNF-α (7.1 vs. 0.09 pg/ml). In addition the serum concentration of MPO (1056.0 vs. 498.0 pg/ml), and PGE2 (2036.7 vs. 1492.0 pg/ml) showed a significant increase in CDI patients as compared with control subjects. Most CDI patients did not show any increase in the concentration of IL-8. We did observe a direct relationship between TNF-α and creatinine. The course of CDI is characterized by an initial local inflammatory process followed by a systemic inflammatory response, which manifests clinically as fever, and includes leukocytosis, an increase in the level of neutrophils in the blood, and an increase in the serum concentrations of TNF-α, IL-1ß, IL-6, IL-10, MPO and PGE2. Despite the leading role of IL-8 in the local inflammatory process, we postulate TNF-α and IL-6 play a key role in the systemic inflammatory response in CDI, and the plasma TNF-α level seems to act as a major factor of poor prognosis in patients with CDI.

Melatonin and omentin: influence factors in the obstructive sleep apnoea syndrome?

The incidence of the obstructive sleep apnoea syndrome (OSAS) is rising as it is often associated with obesity. Actually, the adipose tissue is working as an endocrine organ with complex interactions. Recently, many adipokines such as omentin-1 were discovered. The role of omentin-1 in the pathogenesis of OSAS has not been clearly determined. Melatonin has a known influence on the sleep and wake rhythm. The data on the involvement of melatonin in OSAS are rare. Therefore we evaluated the changes in plasma levels of omentin-1 and melatonin before and after continuous positive airway pressure (CPAP) therapy in OSA patients. 10 patients with newly diagnosed OSAS were included in the study. They underwent diagnostic polysomnography with blood drawings in a 2 hour interval for 24 hours. In the second night sufficient CPAP therapy was established. After three months of CPAP therapy the measurements were repeated. As controls 10 healthy volunteers were recruited. The same blood analysis and a polygraphic measurement were made and compared with the patients. OSA patients showed significantly higher omentin-1 plasma levels (17.22±13.94 versus 9.24±4.85 ng/ml, p<0.05). After three months of therapy the plasma levels of omentin-1 decreased toward the values observed in the controls at 8.00 a.m. Melatonin showed the usual peak at 2.00 a.m. in the volunteer group. OSA patients showed a later peak of melatonin at 6.00 a.m. which returned to 2.00 a.m. after CPAP therapy. We conclude that omentin as well as melatonin seem to be involved in pathogenesis of OSAS. To what exent, further studies will have to face that question.

Molecular alterations in fibroblasts exposed to Helicobacter pylori: a missing link in bacterial inflammation progressing into gastric carcinogenesis?

Major human pathogen Helicobacter pylori (Hp) can colonize the gastric mucosa causing inflammation and being of potential risk for gastric cancer development but the contribution of fibroblasts to the pathogenesis of Hp in the stomach has been little studied. Normal stroma contains few fibroblasts, especially myofibroblasts, but their number rapidly increases in the reactive stroma surrounding inflammatory region and neoplastic tissue. We determined the effect of coincubation of cultured rat gastric fibroblasts with alive Hp on the transdifferentiation of fibroblasts into myofibroblasts associated with Hp-induced inflammation and neoplasia. Gastric mucosal samples were harvested from 8-week-old Spraque-Dowley rats and cultured to obtain the sub-confluent fibroblasts. The isolated fibroblasts were infected with 1 x 10(9) of live Hp (ATCC 700824, cagA+, vacA+) per dish and incubated in humidified atmosphere for 3, 24 and 48 hours. At respective times, fibroblasts were harvested and the expression of mRNA for α-smooth muscle actin (SMA), hypoxia inducible factor (HIF)-1α, collagen I, heat shock protein (HSP)-70, heme oxygenase (HO)-1, Bax and Ki67 transcripts was determined by RT-PCR with specific primers. Hp increased the transdifferentiation of fibroblasts into myofibroblasts as reflected by the time-dependent overexpression of mRNA for α-SMA. The increased expression of HIF-1α and collagen I was observed in fibroblasts co-cultured with Hp. The expression of HSP70 which was negligible in isolated fibroblasts incubated with vehicle (saline) showed time-dependent 2-3 fold increase in those incubated with Hp. The HO-1 mRNA was strongly expressed in rat gastric fibroblasts without or with the co-incubation with Hp. The mRNA for Bax was progressively downregulated within the time of incubation while no significant changes in expression of proliferation marker Ki67 were recorded. We conclude that Hp-induced transdifferentiation of fibroblasts into myofibroblasts involves an increased expression of the early carcinogenic marker HIF-1α, and inhibition of proapoptotic Bax expression, and 2) the overexpression of HSP70 and the unchanged expression HO-1 and Ki67 probably represent the enhanced protective activity of Hp-infected fibroblasts to maintain their own integrity under inflammatory action of this bacteria and its cytotoxins.

Expression and release of leptin and proinflammatory cytokines in patients with ulcerative colitis and infectious diarrhea.

Leptin plays not only an important role in regulation of food intake, but also in the mechanism of inflammation. The universal presence of leptin in the cells of immune system and its secretion by these cells caused increasing interest in the role of this hormone in ulcerative colitis (UC). We determined the role of leptin in 80 patients, aged from 18 to 69 years, including 50 patients with active UC and 30 patients with infectious diarrhea. The tests were performed within 48 hours of the first symptoms, in the period of remission of UC and 8 weeks after resolution of infectious diarrhea. Endoscopy was performed in each patient, and the biopsy samples were taken for the assessments of expression of mRNA for leptin, IL-1ß, IL-6 and TNF-α by RT-PCR and Western blot. Blood tests included concentrations of leptin, IL-1ß, IL-6 and TNF-α. In addition, the plasma levels of leptin, IL-1ß, IL-6 and TNF-α were assessed by ELISA. Serum concentrations of leptin was significantly increased in patients with exacerbation of UC over that in patients with UC in remission. The serum leptin concentration was significantly higher in patients with infectious diarrhea, than the patients that recovered from infectious diarrhea. The leptin protein was overexpressed in the biopsy samples of the mucosa of large intestine compared to those with exacerbation of UC, and in patients after successful recovery from infectious diarrhea. The leptin mRNA was overexpressed in patients with infectious diarrhea compared with that in the group of patients after successful recovery from this condition. Serum concentrations of leptin failed to correlate with severity of exacerbation of UC and with extent of intestinal inflammatory lesions in patients with UC. However, the correlation was observed between serum concentrations of leptin in patients with exacerbation of UC and serum concentrations of proinflammatory cytokines IL-1ß and TNF-α. We conclude that 1) the increased leptin in exacerbated UC is related to the increased serum proinflammatory cytokines IL-1ß, TNF-α and IL-6 levels; 2) In patients with infectious diarrhea, the concentrations of leptin in intestinal mucosa correlates with serum concentrations of cytokines IL-1ß, IL-6 and TNF-α and with an increased expression of leptin mRNA in intestinal mucosa but not with alterations in serum levels of this hormone; 3) leptin may serve as useful predictive marker of inflammation in inflammatory bowel disease (IBD).

Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, interacts with gastric oxidative metabolism and enhances stress-induced gastric lesions.

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase known to exert vasoconstriction of vascular bed. The elevation of ADMA has been considered as the cardiovascular risk factor associated with hyperlipidemia, hypercholesterolemia and metabolic syndrome. ADMA is produced by the action of dimethylarginine dimethylaminohydrolase (DDAH), which hydrolyzes ADMA to L-citrulline and dimethylamine. Previous studies have shown that endogenous NO plays an important role in the mechanism of gastric mucosal defense, but the role of ADMA in the pathogenesis of serious clinical entity, such as the acute gastric mucosal injury induced by stress has been little studied. In present study, we determined the effect of intragastric (i.g.) pretreatment with ADMA applied in graded doses ranging from 0.1 up to 20 mg/kg on gastric mucosal lesions induced by 3.5 h of water immersion and restraint stress (WRS). The number of gastric lesions was determined by planimetry and the gastric blood flow (GBF) was assessed by laser Doppler technique. The malondialdehyde and 4-hydroxynonenal (MDA+4-HNE) concentration, as an index of oxygen radical-lipid peroxidation was assessed in the gastric mucosa in rats exposed to WRS with or without ADMA administration. Proinflammatory cytokines IL-1ß, TNF-α, superoxide dismutase (SOD) and glutathione peroxidase (GPx) mRNAs in the gastric mucosa and plasma levels of ADMA, IL-1ß and TNF-α were analyzed by RT-PCR and ELISA, respectively. The exposure of rats to WRS for 3.5 h produced acute gastric lesions accompanied by a significant rise in the plasma ADMA levels and a significant fall in the GBF, an increase in MDA+4-HNE concentrations and the significant increase in the expression and release of IL-1ß and TNF-α. The pretreatment with ADMA, applied i.g. 30 min before WRS dose-dependently, aggravated WRS damage and this effect was accompanied by a further significant fall in the GBF. The ADMA induced exacerbation of WRS lesions and the accompanying rise in the plasma ADMA levels and the fall in GBF were significantly attenuated by concurrent treatment with glyceryl trinitrate (GTN) (10 mg/kg i.g.) in the presence of ADMA. Administration of ADMA resulted in a significant decrease in the expression of SOD and GPx mRNAs and the up-regulation of mRNA for IL-1ß and TNF-α followed by an increase in these plasma cytokine levels as compared to respective values observed in vehicle-pretreated animals. We conclude that 1) ADMA could be implicated in the mechanism of WRS-induced ulcerogenesis, 2) ADMA exacerbates WRS-induced gastric lesions due to enhancement in neutrophil dependent lipid peroxidation and overexpression and release of proinflammatory cytokines IL-1ß and TNF-α and a potent depletion of antioxidative enzymes SOD and GPx expression and activity.

MicroRNAs and chronic inflammation contribution to gastrointestinal integrity.

Recent studies have revealed that chronic inflammation represents a major basis for different forms of human malignancies. Chronic inflammations are involved in the pathogenesis of 15-25% of human malignancies. Gastrointestinal (GI) cancer is one of the most common causes of mortality in the European Union. The mechanisms leading to cancer development and its progression are not completely understood. Advances are required both in early detection and therapy of GI cancers. There are many factors connecting inflammation and cancer. Cytokines that are small protein molecules regulating growth, differentiation, development and immune response mechanisms in cells. Overexpression of cyclooxynenase-2 is associated with decreased apoptosis, cell to cell adhesion, increased proliferation and angiogenesis contributes to the increased immunosuppresion and mediates carcinogenetic effects. MicroRNAs are regarded as a novel class of gene expression regulators. They are gene-silencing RNAs which negatively regulate gene expression. After binding to target mRNAs they lead either to mRNA destruction or inhibition of translation. Hence, they can play an important role in carcinogenesis. Currently, almost all of the miRNA-related studies on cancers based on the different expression profile of miRNAs in cancer cells compared to normal cells. In summary, miRNAs, proinflammatory cytokines and other factors, may be involved in cancer development based on chronic inflammation by controlling cell differentiation and apoptosis. Assessing the role of miRNAs will provide the new insights on their contribution to the link between chronic inflammation and subsequent cancer, and new markers for cancer diagnoses and cancer therapy.

Helicobacter pylori inhibits expression of heat shock protein 70 (HSP70) in human epithelial cell line. Importance of Cag A protein.

Heat shock proteins (HSP) are crucial for the maintenance of cell integrity under normal cell growth and at pathophysiological conditions such as colonization of gastric mucosa by Helicobacter pylori (Hp). The effect of Hp on mRNA expression for HSP70 in the gastric epithelial cells in vitro has been little studied and remains inconclusive. In this study we attempted to determine the alterations in gene expression for HSP70 induced by two live strains of Hp in the epithelial MKN7 cells. The following Hp strains were employed; 1) Hp strain expressing cagA and vacA, and 2) cagA and vacA negative Hp strain without or with addiction of exogenous recombinant protein CagA. MKN7 cells were incubated in a standard medium RPMI 1640 supplemented with 10% fetal bovine serum at 37 degrees C with 5% CO2 and humidified atmosphere under basal condition or in a presence of Hp (1 x 10(9) CFU per dish) without or with the recombinant CagA (10 microg/ml of RPMI 1640 medium). After 3 h, 24 h and 48 h of incubation with Hp and in some experiments with the prolonged incubation time up to 72 h, the cells were harvested, the total cellular RNA was isolated and the expression of mRNA for HSP70 was determined by RT-PCR. The incubation of the MKN cells with CagA protein alone failed to affect significantly the expression of HSP70. In contrast, the strain Hp (cagA+, vacA+) inhibited in time-dependent manner the expression of mRNA for HSP70. When the MKN7 cells were coincubated with Hp (cagA+, vacA+) and exogenous CagA, the significant inhibition of the signal intensity for HSP70 mRNA was observed at 3 h and 24 h of incubation and these effects were followed by complete disappearance of the signal for HSP70 mRNA at 48 h. The incubation of MKN7 with Hp (cagA-, vacA-) also significantly attenuated the expression of HSP70 mRNA with the most pronounced inhibitory effect observed at 72 h of incubation with this Hp strain. Addition of the recombinant CagA to Hp (cagA-, vacA-) completely suppressed the expression of HSP70 at 48 h and 72 h after the end of incubation periods. We conclude that 1) both, Hp (cagA+, vacA+) and Hp (cagA-, vacA-) inhibit expression of HSP70 in MKN7 human gastric epithelial cells independently of the presence or absence of cagA gene, and that 2) recombinant CagA protein may exert biological activity in vitro via acceleration of inhibitory effect of Hp negative for Cag A and VacA on HSP70 expression in epithelial cells infected with this bacteria.

Helicobacter pylori infection and serum gastrin, ghrelin and leptin in children of Polish shepherds.

OBJECTIVE: Family unit is generally accepted as one of the contributors to Helicobacter pylori infection that is most frequently acquired in childhood, so it seems logical to diagnose and treat this infection in childhood. This study was designed to assess H. pylori prevalence in children from shepherd families having contacts with sheep. MATERIAL AND METHODS: This study involved 146 children (58 M/88 F, age 6-17 years; mean: 10.2 years) from families living in Polish Tatra Mountains with contact (group A, n=58) or without contact with sheep (group B, n=88). H. pylori status was determined by (13)C-urea breath test and was compared to 141 age- and gender-matched urban controls (group C). In both groups of mountain children, the anti-H. pylori and anti-CagA IgG were measured by ELISA and serum gastrin, ghrelin and leptin concentrations by RIA. RESULTS: The H. pylori prevalence in group A was significantly higher (58.6%) than that in group B (21.6%) and urban controls (26%). Serum gastrin concentrations were significantly higher in H. pylori-positive than in H. pylori-negative mountain children (52.2+/-5.8 pmol/L versus 22.7+/-2.1 pmol/L), while serum ghrelin and leptin concentrations were significantly lower in H. pylori-infected (741+/-112 pg/mL and 3.6+/-0.8 ng/mL) than in non-infected children (1323+/-104 pg/mL and 8.6+/-2.4 ng/mL). CONCLUSIONS: Children with sheep contact show about twice higher H. pylori prevalence and higher serum gastrin but lower ghrelin and leptin levels than those without H. pylori infection. Considering almost 100% positive 13C-urea breath test in sheep, it is reasonable to propose that H. pylori infection in shepherd children may originate from sheep and the infection might, therefore, be considered as zoonosis.

Healing of chronic gastric ulcers in diabetic rats treated with native aspirin, nitric oxide (NO)-derivative of aspirin and cyclooxygenase (COX)-2 inhibitor.

Previous studies have demonstrated that the gastric mucosa of diabetic rats is highly vulnerable to acute injury but the influence of nonsteroidal anti-inflammatory drugs (NSAID) and their new nitric oxide (NO) releasing derivatives of aspirin (NO-ASA) on the ulcer healing under diabetic conditions has been little studied. In this study streptozocin (STZ, 70 mg/kg injected intraperitoneally) was used to induce diabetes mellitus in rats. Four weeks after STZ injection, gastric ulcers were induced using the acetic acid method and rats with gastric ulcers received the treatment with 1) aspirin (ASA, 30 mg/kg-d i.g.), 2) NO-ASA applied in equimolar dose of 50 mg/kg-d i.g., 3) rofecoxib (5 mg/kg-d i.g.), the selective cyclooxygenase-(COX)-2 inhibitor and 4) SNAP (5 mg/kg-d i.g.), a donor of NO, combined with ASA (30 mg/kg-d i.g.). Ten days after the induction of the ulcers, the healing rate and the gastric blood flow (GBF) were measured by planimetry and hydrogen (H(2))-gas clearance method, respectively and the plasma cytokine such as IL-1beta, TNF-alpha and IL-10 were determined. In addition, the effect of insulin (4 IU/day/rat i.p.) with or without the blockade of NO-synthase by L-NNA (20 mg/kg-d i.p.) on the ulcer healing and the GBF in non-diabetic and diabetic rats was determined. In the diabetic rats, a significant delay in ulcer healing (approximately by 300%) was observed with an accompanied decrease in the GBF at ulcer margin. The prolongation of the healing in diabetic animals was associated with an increase in the plasma cytokine (IL-1beta, TNF-alpha and IL-10) levels. ASA and rofecoxib, that significantly suppressed the mucosal prostaglandin (PG) E(2) generation in ulcer area, delayed significantly the rate of ulcer healing and decreased the GBF at ulcer margin, while elevating plasma IL-1beta, TNF-alpha and IL-10 concentrations in non-diabetic rats and these alterations were significantly augmented in diabetic animals. In contrast to ASA, the treatment with NO-ASA failed to influence both, the ulcer healing and GBF at ulcer margin and significantly attenuated the plasma levels of IL-1beta, TNF-alpha and IL-10 as compared to those recorded in ASA- or rofecoxib-treated animals. Co-treatment of SNAP with native ASA abolished the deleterious effect of ASA on ulcer healing, GBF at ulcer margin and luminal NO release in diabetic rats. Administration of insulin in rats with diabetes, opposed the delay in ulcer healing, and the fall in the GBF at ulcer margin and these effects were counteracted by the concurrent treatment with L-NNA. We conclude that: 1) ulcer healing is dramatically impaired in experimental diabetes and this effect involves the fall in the gastric microcirculation at the ulcer margin and increased release of proinflammatory cytokines; 2) classic NSAID such as ASA and selective COX-2 inhibitors such as rofecoxib, prolong ulcer healing under diabetic conditions probably due to suppression of endogenous PG and the fall in the GBF at the ulcer margin suggesting that both COX isoforms, namely, COX-1 and COX-2, are important sources of PG during ulcer healing in diabetes; and 3) NO-ASA counteracts the impairment of ulcer healing in diabetic rats induced by ASA, mainly due to the release of NO that compensates for PG deficiency resulting in enhancement in the GBF at ulcer margin and suppression of cytokine release in the ulcer area.

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.