Sections Of Mucosa Research Articles

Study of biofilm formation in C57Bl/6J mice by clinical isolates of Helicobacter pylori.

Background/Aim:Despite the significant number of studies on H. pylori pathogenesis, not much data has been published concerning its ability to form biofilm in the host stomach. This study aims to evaluate the potential of clinical isolates of H. pylori to form biofilm in C57BL/6J mice model.Materials and Methods:Two strains of H. pylori were selected from a collection of clinical isolates; one (19B), an efficient biofilm producer and the other (4B), with weak biofilm-forming ability. Mice infected through gastric avages were examined after one and two weeks. Colonization was determined by CFU and urease activity; the anti-H. pylori IgA was measured by ELISA, and chronic infections were evaluated by histopathology. Bacterial communities within mucosal sections were studied by immunofluorescence and scanning electron microscopy (SEM).Results:Successful infection was obtained by both test strains. Strain 19B with higher ability to form biofilm in vitro also showed a higher colonization rate in the mice stomach one week after infection. Difference (P < 0.05) in IgA titers was observed between the infected mice and the controls as well as between 19B and 4B infected mice, two weeks after the last challenge. Immunofluorescence and SEM results showed tightly colonizing H. pylori in stomach mucosal sections and in squamous and glandular epithelium.Conclusion:H. pylori is able to form biofilm in the mouse stomach and induce IgA production, reflecting the same potential as in humans. Firm attachment of coccoid form bacteria to host cells suggests the importance of this state in biofilm formation by H. pylori. Occurrence of biofilm in squamous and glandular epithelium of the mouse stomach proposes that H. pylori can all parts of the upper gastrointestinal tract.

Immunohistochemical evaluation of hedgehog signalling in epithelial/mesenchymal interactions in squamous cell carcinoma transformation: a pilot study.

Precancerous lesions have been studied because of their carcinogenic potential and their association with squamous cell carcinoma (SCC) has been reported. In the tumour microenvironment, the processes of angiogenesis and tissue remodelling are regulated by a family of proteins (Hedgehog) described as being able to modulate epithelial/mesenchymal interactions. The objective of this study was to perform a comparative study of precancerous lesions and SCCs by immunohistochemistry for the presence of Sonic, Gli2, SMO and Patched proteins, members of the Hedgehog pathway. Sixteen cases diagnosed as actinic cheilitis associated with SCC were compared to normal oral mucosa. The sections were subjected to immunohistochemistry and the positively stained cells were counted by morphometric analysis. There was a significant progressive increase in expression of all proteins of the Hedgehog pathway, both in the epithelium and in the connective tissue, when sections of normal mucosa, dysplasia and carcinoma were compared (P < 0.05). Thus, one may suggest that the Hedgehog pathway in tumour transformation influences SCC, and more studies should be conducted to expand the understanding of the role of these proteins in neoplastic transformation.

C-MET expression in colorectal adenomas and primary carcinomas with its corresponding metastases.

c-MET plays an important role in tumor proliferation, invasion and metastasis. In this study we examined the expression of c-MET in colorectal adenomas, primary adenocarcinomas and their corresponding lymph node, peritoneal and liver metastases. We correlated our findings with clinicopathological features. Twenty three cases of colorectal adenoma and 102 cases of primary colorectal carcinoma and their corresponding metastases (44 lymph nodes, 21 peritoneal deposits and 16 liver metastases) were studied to evaluate c-MET expression by immunohistochemistry. For comparison, 12 sections of adjacent healthy colorectal mucosa were examined. Statistically significant differences were present among normal tissues, colorectal adenomas and primary colorectal carcinomas (P=0.011). Normal tissues showed a negative or weak reaction in 66.67% and 33.33% of cases respectively. Expression of c-MET was positive in 47.8% of adenomas. A significant positive association was identified between c-MET high expression and degree of dysplasia (P=0.024). c-MET was highly expressed in 66.7% of primary colorectal carcinoma. Significant positive correlations were detected between c-MET expression and TNM stage (P=0.036), lymph node metastasis (LNM), peritoneal deposits and liver metastasis (P=0.038, P=0.094 and P=0.045, respectively). c-MET expression in metastatic tissues was significantly higher than that of the primary tumor. c-MET expression is gradually up-regulated in the development and progression of colorectal cancer (CRC) from normal epithelium to adenoma to colorectal carcinoma to metastases.

393 Helicobacter pylori Infection Causes Insulin Resistance Through the C-Jun/miR-203/SOCS3 Pathway

of humanized rats (HR) collected 1, 2 and 30 days after inoculationanalyzed by 454sequencing and 16sRNA-qPCR. Fermentative activity was investigated by quantification of short chain fatty acids by chromatography and enzymatic reaction of D and L-lactate. HR were sacrificed at d30 after inoculation and compared to Conventional (CV) and GF rats. Ki67 and GLP-1 immunohistochemistry were performed on paraformaldehyde-fixed colon mucosa sections. Results are expressed as mean ± SEM and statistical analysed used non parametric tests. Results: The fecal sample from the SBS patient used as inoculum, contained high amount of Lactobacillus (1.5x1010bacteria/g of feces) and no C. Leptum consistent with our previous studies. Transfer of this lactobiota in GF rats reached a steady state consortium after 3 days. At that time, bacteria population shifted from enriched population of lactates producers to enriched population of lactates consumers. Although the lactobiota inoculum was modified in the environment of GF rats, the sequencing highlighted that HR microbiota conserved characteristics of the inoculum at the bacteria gender level (6.45x108 Lactobacillus bacteria/g of feces and lack C. Leptum). After inoculum transfer, the lactates production significantly dropped the first 3 days (from 155mM to undetectable levels) as propionate, and butyrate (from 5.34mM to 1.87mM and from 1.84mM to 0.55mM respectively) while acetate was not modify (form 11.62mM to 13.99mM). Analysis of HR colon mucosa, revealed an increase in Ki-67 proliferating cells in the crypt (+131%, p<0.05 vs GF) while no difference in crypt depth (HR vs GF) and a significant increase in the number of GLP1 positive cells (+50%, p<0.05 vs CV). Conclusion: These data indicate for the first time that microbiota from SBS patients can significantly affect the phenotype of colonic mucosa. They suggest that this lactobiota has an active role in the adaptative mechanisms following extensive small bowel resection.

Effect of Cannabis sativa extract on gastric acid secretion, oxidative stress and gastric mucosal integrity in rats

Studies were conducted in pylorus-ligated rats to investigate the effect of Cannabis sativa extract on gastric acid secretion, experimental gastric ulcer and on oxidative stress and inflammatory markers in the gastric mucosa. C. sativa (5, 10 and 20 mg/kg, expressed as Δ9-tetrahydrocannabinol) was administered subcutaneously daily for 4 weeks prior to pylorus ligation and different treatments. Under basal conditions, pretreatment with cannabis extract at doses of 5 and 10 mg/kg increased gastric acid secretion and induced minimally visible gastric mucosal lesions in the 4 h pylorus-ligated rat. Malondialdehyde and nitric acid concentration increased, while reduced glutathione decreased by cannabis at doses of 5 and 10 mg/kg in gastric mucosa. TNF-α increased by cannabis extract at doses of 5 and 10 mg/kg but decreased following the high dose of 20 mg/kg. On the other hand, the gastric acid secretory responses stimulated by pentagastrin or carbachol (but not histamine) were inhibited in rats pretreated with cannabis extract. Under these conditions, cannabis decreased pepsin content after pentagastrin and carbachol but not histamine stimulation. Cannabis also decreased lipid peroxidation and nitric oxide content, and increased both reduced glutathione and catalase activity in mucosa. Moreover, cannabis decreased mucosal inflammation (level of TNF-α) and the development of gastric mucosal lesions. Cannabis administered for 1 month prior to pylorus-ligation and either acidified aspirin or ethanol (96 %) decreased the development of gastric mucosal damage in a dose-dependent manner, along with reduction in gastric acid output, gastric mucosal oxidative stress and inflammation (TNF-α). Sections of gastric mucosa stained with periodic acid Schiff showed increased mucus secretion by cannabis in basal conditions and after treatment with aspirin or ethanol. Results indicate that: (1) the effect of cannabis differs in basal conditions and after exposure of the gastric mucosa to high acid concentrations or other chemical noxious agents; (2) cannabis administered systemically exerts gastric mucosal protective effects against mucosal damage evoked by stimulation of gastric acid secretion, acidified aspirin or ethanol. These effects of cannabis are likely to involve inhibition of gastric acid and pepsin secretion, increased mucus, decreased oxidative stress and inflammation in gastric mucosa.

Endogenous Life Cycle of Eimeria marmosopos (Apicomplexa: Eimeriidae) from the Opossum, Didelphis marsupialis (Didelphimorphia: Didelphidae) in Costa Rica.

The endogenous life cycle of Eimeria marmosopos was studied in experimentally infected young opossums, Didelphis marsupialis . All the endogenous stages were located in the epithelial cells of villi in the small intestine. Giemsa-stained mucosal scrapings and histological sections were studied for the diagnosis of all the life cycle stages. Eimeria marmosopos has 3 generations of meronts (M) that differ by size, shape, and number of merozoites (m), which also differ in their size, shape, and location of their nuclei within the cytoplasm of the meronts. The 3 meront types, M(1)-M(3), respectively, had 8-15 (m(1)), 4-9 (m(2)), and 22-30 (m(3)) merozoites. Macrogametocytes and microgametocytes, as well as macrogametes and microgametes, completed the sexual cycle, finishing with the formation of unsporulated oocysts. This parasite's endogenous development produced severe intestinal lesions in experimentally infected opossums. There are 56 Eimeria species known from all marsupials worldwide, but this is the first complete life cycle in which both the asexual and sexual stages have been documented.

Selective Cyclooxygenase-1 Inhibition by P6 and Gastrotoxicity: Preliminary Investigation

Background/Aims: Gastrointestinal damage (GD) is commonly associated with the inhibition of cyclooxygenase (COX)-1, one of the two known COXs, by traditional non-steroidal anti-inflammatory drugs. More recent evidences have proven that GD is caused by the simultaneous inhibition of the two COXs. This study was designed to evaluate the effect of the selective COX-1 inhibition on gastric integrity. Methods: GD was evaluated in male CD1 mice. Drugs were administered by gastric gavage at a dose of 50 mg/kg (injection volume of 100 µl). Control mice received an equal volume of the vehicle (10% ethanol). Each mouse, in groups of at least 6 mice, received one dose/day for 5 days. Results: In Western blot analysis, COX-1 expression levels were found to be significantly reduced in mice treated with 3-(5-chlorofuran-2-yl)-5-methyl-4-phenylisoxazole (P6) in comparison to mice pretreated with aspirin (ASA), which exhibited higher levels of COX-1, thus confirming the high selectivity of P6 towards COX-1 enzyme inhibition. Mucosal sections obtained from ASA-treated mice showed breaks in the epithelial barrier and a marked alteration of foveolae and gastric glands, whereas stomachs isolated from mice sacrificed after 5 days of chronic administration of P6 (at a dose of up to 50 mg/kg/day) showed sporadic transient mucosal hyperemia and did not seem to display any significant gastric damage. Conclusions: The selective COX-1 inhibition by P6 does not cause gastric damage in mice but preserves mucosal integrity.

Gastroprotective effect of alpha-pinene and its correlation with antiulcerogenic activity of essential oils obtained from Hyptis species.

Background:Alpha-pinene (α-pinene) is a monoterpene commonly found in essential oils with gastroprotective activity obtained from diverse medicinal plants, including Hyptis species. The genus Hyptis (lamiaceae) consists of almost 400 species widespread in tropical and temperate regions of America. In the north and northeastern Brazil, some Hyptis species are used in traditional medicine to treat gastrointestinal disturbances.Objective:The present study has investigated the gastoprotective effect of purified α-pinene in experimental gastric ulcer induced by ethanol and indomethacin in mice.Materials and Methods:Gastric ulcers were induced in male Swiss mice (20-30 g) by oral administration of absolute ethanol or indomethacin 45 min after oral pretreatment with vehicle, standard control drugs or α-pinene (10, 30, and 100 mg/kg). One hour after the ulcerative challenges, the stomach were removed, and gastric lesions areas measured. The effects of α-pinene on the gastric juice acidity were determined by pylorus ligation model. The gastrointestinal motility and mucus depletion were determined by measuring the gastric levels of phenol red and alcian blue, respectively. Hematoxylin and eosin stained sections of gastric mucosa of the experimental groups were used for histology analysis.Results:α-pinene pretreatment inhibited ethanol-induced gastric lesions, reduced volume and acidity of the gastric juice and increased gastric wall mucus (P < 0.05). Furthermore, we showed an interesting correlation between concentration of α-pinene and gastroprotective effect of Hyptis species (P Pearson = 0.98).Conclusion:Our data showed that the α-pinene exhibited significant antiulcerogenic activity and a great correlation between concentration of α-pinene and gastroprotective effect of Hyptis species was also observed.

Inflammatory bowel disease is a disorder of ganglioside metabolism (959.4)

AIM To demonstrate that the metabolism of ganglioside is altered in inflammatory bowel disease (IBD). METHODS Sections of intestinal mucosa from terminal ileum and/or colon were obtained from subjects with ulcerative colitis or inflammatory Crohn’s disease undergoing surgical resection for complications related to IBD. Control groups consisted of tissue samples of apparently normal intestinal mucosa from subjects with colorectal cancer and apparently normal intestinal mucosa from subjects with benign adenoma polyposis. Gangliosides of the intestinal mucosa were characterized using LC‐QQQ‐MS and compared among subject groups. Ganglioside catabolism enzymes HEXA and NEU3 were measured in the intestinal mucosa by western blot. RESULTS GM3 was significantly higher (P&lt;0.01) by 2.1 fold in IBD intestine compared to apparently normal intestinal mucosa. IBD intestine exhibited 2.7 fold lower (P&lt;0.001) GD1a content than control intestine. The mean relative expression of HEXA in IBD intestine was increased 1.7‐fold (P&lt;0.08) and the mean relative expression of NEU3 in IBD intestine was increased 8.3‐fold (P&lt; 0.001) compared to normal intestine. CONCLUSION Metabolism of gangliosides is altered in the intestinal mucosa of subjects with IBD. Investigation into utility of dietary ganglioside as a potential treatment of IBD is warranted.Grant Funding Source: Supported by Broad Foundation and CIHR

Chemical mapping of the colorectal cancer microenvironment via MALDI imaging mass spectrometry (MALDI-MSI) reveals novel cancer-associated field effects

Matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-MSI) is a rapidly advancing technique for intact tissue analysis that allows simultaneous localisation and quantification of biomolecules in different histological regions of interest. This approach can potentially offer novel insights into tumour microenvironmental (TME) biochemistry. In this study we employed MALDI-MSI to evaluate fresh frozen sections of colorectal cancer (CRC) tissue and adjacent healthy mucosa obtained from 12 consenting patients undergoing surgery for confirmed CRC. Specifically, we sought to address three objectives: (1) To identify biochemical differences between different morphological regions within the CRC TME; (2) To characterise the biochemical differences between cancerous and healthy colorectal tissue using MALDI-MSI; (3) To determine whether MALDI-MSI profiling of tumour-adjacent tissue can identify novel metabolic ‘field effects’ associated with cancer. Our results demonstrate that CRC tissue harbours characteristic phospholipid signatures compared with healthy tissue and additionally, different tissue regions within the CRC TME reveal distinct biochemical profiles. Furthermore we observed biochemical differences between tumour-adjacent and tumour-remote healthy mucosa. We have referred to this ‘field effect’, exhibited by the tumour locale, as cancer-adjacent metaboplasia (CAM) and this finding builds on the established concept of field cancerisation.

Aberrant, ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy. Material and methodsWe examined and quantified expression of VEGF, VEGF-R1 and VEGF-R2 in three different human colonic tissue arrays containing sections of adenocarcinoma (n=43) and normal mucosa (n = 41). In human colon cancer cell lines HCT116 and HT29 and normal colon cell lines NCM356 and NCM460, we examined expression of VEGF, VEGF-R1 and VEGF-R2 mRNA and protein, VEGF production and secretion into the culture medium; and, the effect of a potent, selective inhibitor of VEGF receptors, AL-993, on cell proliferation. ResultsHuman colorectal cancer specimens had strong expression of VEGF in cancer cells and also expressed VEGF-R1 and VEGF-R2.In vitro studies showed that human colon cancer cell lines, HCT116 and HT29, but not normal colonic cell lines, express VEGF, VEGF-R1 and VEGF-R2 and secrete VEGF into the medium up to a concentration 2000pg/ml within 48h. Furthermore, we showed that inhibition of VEGF receptors using a specific VEGF-R inhibitor significantly reduced proliferation (by >50%) of cultured colon cancer cell lines. ConclusionsOur findings support the contention that VEGF generated by colon cancer cells stimulates their growth directly through an autocrine mechanism that is independent of its primary function in the induction of angiogenesis.

Abstract 3879: Osteopontin (SPP1/OPN) alternative splice variants and metastatic potential in esophageal adenocarcinoma.

Abstract Objective: The incidence of esophageal adenocarcinoma (EAC) has increased over 300% in the past decades in western countries. EAC is characterized by an early metastasis, with a 5-year survival rate of only 19%, making the identification of clinically-targetable proteins critical for improving patient outcome. Osteopontin (SPP1/OPN) is a secreted glycosylated phosphoprotein that is found overexpressed in many cancer types including EAC. OPN protein functions in cell adhesion and cell migration by regulating cell-matrix interactions and cellular signaling through binding with integrin and CD44 receptors. However, little is known about the role of OPN in EAC invasion and metastasis. Methods: We analyzed the OPN gene expression in a series of 46 esophageal samples including esophageal Barrett's, low-grade dysplasia, high-grade dysplasia and adenocarcinomas mRNA using Affymetrix U133A arrays. Real-time RT-PCR was performed to validate array results and examine a larger cohort of 130 EAC samples. Tissue microarrays (TMA) were made containing 64 EACs, eight lymph node metastases, eight dysplastic and 11 none dysplastic Barrett's mucosa sections from 59 patients. Primers flanking the potential gene alternative splice regions were designed and the in-frame transcript variants were sequenced. Expression of OPN in EAC cell lines was analyzed using RT-PCR and western blot analysis. Ectopic OPN expression of splice variants and OPN knockdown in EAC cell lines have been performed to examine its roles in tumor invasion and EMT assays. Results: OPN is highly overexpressed in EAC and overexpression of the gene is associated with poorer survival in patients. Three OPN splice variants were identified with their expression either proportionally increased or differentially expressed in the primary EAC and EAC-derived cells. Highly overexpressed OPN is a transcriptional event as no OPN gene amplification was found in the corresponding EACs. In addition, abundant OPN protein was found primarily in tumor cells in the tissue sections using immunohistochemical (IHC)-TMA assays. OPN variants differ in their N-terminal thrombin fragment with either exon 6 or 5 being absent in OPN-b or OPN-c, respectively. We found that EAC cells treated with the medium containing different OPN variants demonstrated distinctive morphological phenotypes. We further identified that OPN variants played different roles in tumor cell proliferation, epithelial-mesenchymal transition (EMT), and chemosensitivity. Conclusion: Upregulation of OPN is predominant event in primary EACs and associated with poorer survival in patients. The alternative splice variants demonstrate differential tumor invasion, EMT transition and chemosensitivity in EAC cells. Citation Format: Zhuwen Wang, Kimmy Leverenz, Dafydd G. Thomas, Amy L. Myers, Andrew C. Chang, Mark B. Orringer, Thomas J. Giordano, Jules Lin, David G. Beer, Lin Lin. Osteopontin (SPP1/OPN) alternative splice variants and metastatic potential in esophageal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3879. doi:10.1158/1538-7445.AM2013-3879

Ursodeoxycholic acid attenuates colonic epithelial secretory function

Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl(-) secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl(-) secretory responses to the Ca(2+) and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n = 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na(+)/K(+)-ATPase activity and basolateral K(+) channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg(-1)) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50-200 μm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea.

Expression profile of drosomycin-like defensin in oral epithelium and oral carcinoma cell lines

ObjectiveDrosomycin-like defensin (DLD) is a recently discovered antimicrobial peptide mainly active against filamentous fungi. The present study investigated the expression profile of DLD in oral epithelium and oral squamous cell carcinoma (SCC) cell lines. MethodsTissue sections of human oral mucosa, keratinocytes derived from oral mucosa (NOK) and eight kinds of SCC cell lines were used. In situ hybridization was performed on tissue sections of oral mucosa. Expression levels of DLD in the cells were observed by reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR assays. The cells were treated with IL-1β, IL-8 and TNF-α, and agonists for TLR2, TLR4 and β-glucan. DLD expression in cells was increased and decreased by the DLD gene and its siRNA transfection, respectively. The proliferation rates were assessed by cell counting. ResultsBy means of in situ hybridization, DLD mRNA positive staining was detected in the epithelial layer of the oral mucosa. An RT-PCR assay confirmed the expression of DLD mRNA in keratinocytes derived from oral epithelium. Expression of DLD in two out of eight cell lines was significantly lower than in NOK cells. The expression levels of DLD mRNA were not significantly changed in the cells stimulated with any cytokines or agonists. The cell proliferation rate where there was decreased expression of DLD was significantly lower than in the control. ConclusionDLD may be partially involved in the defence against filamentous fungal infection in the oral mucosa, and may also serve other functions, such as contribution to cell growth.

In vivo effect of proton-pump inhibitors on gastric plasmin-dependent fibrinolysis: A study in a porcine model

IntroductionAlkalization of gastric fluids could inhibit plasmin-mediated and/or pepsin-mediated fibrinolysis. We evaluated the gastric antifibrinolytic effect of proton pump inhibitors in a live porcine model. Material and methodsSix pigs were randomly assigned to treatment with proton pump inhibitors vs no treatment. After endoscopic mucosal resection, 8μm sections were incubated on fibrin films. Fibrinolytic activity was assessed through focal lysis time. One-hundred-and-forty-two mucosal sections and 129 submucosal sections were analysed. Twenty-four additional sections were analysed on plates containing tranexamic acid to explore pepsin-mediated fibrinolysis. ResultsFocal lysis times in treated vs control groups were 21.0min vs 21.2min (p=0.39) in the mucosa, and 22.2min vs 20.2min (p=0.56) in the submucosa. No lysis could be seen on the plasmin-inhibited fibrin plates. ConclusionOnly plasmin-mediated fibrinolysis was observed. Proton pump inhibitors had no significant plasmin-dependant antifibrinolytic effect. They may enhance haemostasis through different pathways.

Ca2+ transients in submucous neurons during the colonic migrating motor complex in the isolated murine large intestine

The colonic migrating motor complex (CMMC) is a spontaneous, rhythmic, and neurally mediated motor pattern generated by myenteric neurons, which can propel fecal pellets in mice. Our aim was to determine whether submucous neurons were also activated during the CMMC. :The isolated murine colon was opened and sections of mucosa were removed to expose the submucous ganglia, which were then loaded with Fluo-4. Colonic migrating motor complexes, which occurred spontaneously or by mechanically stimulating the mucosa, were identified by displacement of the tissue (duration = 23.3 s). Between CMMCs, spontaneous Ca(2+) transients (frequency = 0.9 Hz) were observed in 55% (n = 8) of submucous neurons. During the CMMC, 98% (seven ganglia, n = 7) of submucous neurons within the same ganglion exhibited rapid Ca(2+) transients (1.6 Hz) superimposed on a sustained rise in Ca(2+) (duration ∼23 s) that occurred 1.7 s following the mucosal stimulus; whereas other neurons exhibited a similar, but delayed response that occurred either at 7 or 13 s following the stimulus. The activity in submucous neurons was correlated with activity in adjacent nerve varicosities. Ondansetron (1 mm; 5-HT(3) antagonist) significantly reduced the frequency and duration of the Ca(2+) transient responses. Activity in the submucous neurons appears to be secondary to that in the myenteric plexus and appears to be generated largely by activity in myenteric descending (serotonergic) interneurons. During the CMMC, there is likely to be an increase in secretion to lubricate and facilitate fecal pellet propulsion.

PWE-075 Lack of association between the PSCA rs2294008 polymorphism, or PSCA expression, and colorectal neoplasia

IntroductionProstate stem cell antigen (PSCA) has been implicated in the pathogenesis of several solid tumours, either due to changes in protein expression, or through association with the rs2294008 (C>T) polymorphism...

Simultaneous detection of gastric acid and histamine release to unravel the regulation of acid secretion from the guinea pig stomach

Gastric acid secretion is regulated by three primary components that activate the parietal cell: histamine, gastrin, and acetylcholine (ACh). Although much is known about these regulatory components individually, little is known on the interplay of these multiple activators and the degree of regulation they pose on the gastric acid secretion mechanism. We utilized a novel dual-sensing approach, where an iridium oxide sensor was used to monitor pH and a boron-doped diamond electrode was used for the detection of histamine from in vitro guinea pig stomach mucosal sections. Under basal conditions, gastrin was shown to be the main regulatory component of the total acid secretion and directly activated the parietal cell rather than by mediating gastric acid secretion through the release of histamine from the enterochromaffin-like cell, although both pathways were active. Under stimulated conditions with ACh, the gastrin and histamine components of the total acid secretion were not altered compared with levels observed under basal conditions, suggestive that ACh had no direct effect on the enterochromaffin-like cell and G cell. These data identify a new unique approach to investigate the regulation pathways active during acid secretion and the degree that they are utilized to drive total gastric acid secretion. The findings of this study will enhance our understanding on how these signaling mechanisms vary under pathophysiology or therapeutic management.

298 Regulation and Function of BMP-4 Expression in the Inflamed Gastric Mucosa

BACKGROUND/AIMS: We previously reported that inhibition of Bone Morphogenetic Protein (BMP) signaling In Vivo, leads to enhancement of Helicobacter pylori (HP)-induced gastric inflammation suggesting that the BMPs can exert novel anti-inflammatory actions in the stomach. The aim of this study was to examine the function and the regulation of BMP-4 expression in gastric inflammation. METHODS: Inflammation was induced by infection of 3 month-old C57BL/6-BMP-4-β-gal/+ mice with HP. Animals were analyzed 2 months after inoculation. Expression of BMP-4 was assessed by X-Gal staining and QRT-PCR. Morphology of the gastric mucosa was examined in sections stained with H&E. Phosphorylation of Smad1-5-8 was determined by immunohistochemistry with anti-phospho-Smad1-5-8 antibodies (abs). Expression of INF-γ was measured by QRT-PCR. Gastric mesenchymal and mucus cells were isolated from the canine gastric mucosa and characterized by staining with anti-vimentin abs and the lectin GSII, respectively. Phosphorylation of STAT1 and ERK2 and expression of BMP-4 in the mesenchymal cells were assessed by western blots. IL-8 expression in the mucus cells was measured by QRT-PCR. INF-γ, IL-17-α, IL-12 and IL23 content in supernatants of cultures of mouse splenocytes and dendritic cells was measured by ELISA. RESULTS: Analysis of mucosal sections of C57BL/6-BMP-4-β-gal/+ mice stained with X-gal, demonstrated that BMP-4 is expressed in the mesenchymal layers of the gastric mucosa. HP induced inflammatory changes in the gastric mucosa and it stimulated the expression of both INF-γ and BMP-4. Phospho-Smad1-5-8 staining of the inflamed gastric mucosa indicated the presence of positively stained nuclei in gastric epithelial cells, but not in mesenchymal cells, or in cells of the inflammatory infiltrate. INF-γ induced the phosphorylation of both STAT-1 and ERK2 and it induced BMP-4 protein expression in the mesenchymal cells. Incubation of the cells with inhibitors of both STAT-1 and ERK2 phosphorylation abrogated the stimulatory effect of INF-γ. BMP-4 failed to affect LPS-induced release of INF-γ, IL-17-α, IL-12 and IL-23 in cultures of mouse splenocytes and dendritic cells. In contrast, BMP-4 potently inhibited both basal and TNF-α stimulated IL-8 gene expression in cultures of isolated mucus cells. CONCLUSIONS: Inflammatory stimuli induce the expression of BMP-4 both In Vivo and In Vitro, through mechanisms that appear to involve the activation of both ERK2 and STAT-1. BMP-4, in turn, inhibits the expression of inflammatory molecules in epithelial, but not in immune cells, suggesting that BMP-4 is a novel anti-inflammatoy peptide released by the mesenchyme to modulate the inflammatory response of the gastric epithelium.

Expression of intestinal mucins may be affected by the dietary protein source

Mucin glycoproteins cover epithelial surfaces of ducts, including the digestive tract, in the body. The study investigated the effect of dietary protein on the expression of mucins in intestinal epithelia of mice. Male weanling mice were randomized into 4 groups of 8 mice each. The source of protein (plant or animal origin) and the level of each protein (10% or 20% crude protein) were arranged in a factorial manner to produce 4 diets which were randomly assigned to the four groups of mice. Mice were provided the diets for 14 days and euthanized on the fifteenth day. Mucosa and histological sections were prepared from the jejunum and colon of the intestines. Total RNA was prepared from the mucosa using Trizol®. cDNA was prepared using random hexamers and specific primers were used for real time PCR amplification of the message for Muc2 and Muc3. Expression of Muc2 in the jejunum was higher (1.45 fold) in mice fed plant protein compared to those fed animal protein. There was no difference in expression of Muc2 in the colon with the different protein sources. Expression of Muc2 did not vary with the level of dietary protein in both the jejunum and colon. Expression of Muc3 in the jejunum and colon did not vary with the level or source of dietary protein. It is concluded that expression of intestinal mucins may be affected by the source of protein in the diet.Research was supported by a RDC grant from East Tennessee State University.