Mucin-microbiome signatures shape the tumor microenvironment in gastric cancer.

BACKGROUND AND AIMS: We aimed to identify mucin-microbiome signatures shaping the tumor microenvironment in gastric adenocarcinomas and clinical outcomes. METHODS: We performed high-throughput profiling of the mucin phenotypes present in 108 gastric adenocarcinomas and 20 functional dyspepsia cases using validated mucin-based RT-qPCRs with subsequent immunohistochemistry validation and correlated the data with clinical outcome parameters. The gastric microbiota was assessed by 16S rRNA gene sequencing, taxonomy, and community composition determined, microbial networks analyzed, and the metagenome inferred in association with mucin phenotypes and expression. RESULTS: Gastric adenocarcinomas with an intestinal mucin environment or high-level MUC13 expression are associated with poor survival. On the contrary, gastric MUC5AC or MUC6 abundance was associated with a more favorable outcome. The oral taxa Neisseria, Prevotella, and Veillonella had centralities in tumors with intestinal and mixed phenotypes and were associated with MUC13 overexpression, highlighting their role as potential drivers in MUC13 signaling in GC. Furthermore, dense bacterial networks were observed in intestinal and mixed mucin phenotype tumors whereas the lowest community complexity was shown in null mucin phenotype tumors due to higher Helicobacter abundance resulting in a more decreased diversity. Enrichment of oral or intestinal microbes was mucin phenotype dependent. More specifically, intestinal mucin phenotype tumors favored the establishment of pro-inflammatory oral taxa forming strong co-occurrence networks. CONCLUSIONS: Our results emphasize key roles for mucins in gastric cancer prognosis and shaping microbial networks in the tumor microenvironment. Specifically, the enriched oral taxa associated with aberrant MUC13 expression can be potential biomarkers in predicting disease outcomes. Video Abstract.

Proteolytic Cleavage of Bioactive Peptides and Protease-Activated Receptors in Acute and Post-Colitis.

The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been studied extensively using synthetic fluorogenic substrates targeting specific sets of proteases. We explored activities in colonic tissue from a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model by investigating the cleavage of bioactive peptides. Pure trypsin- and elastase-like proteases on the one hand and colonic tissue from rats with TNBS-induced colitis in the acute or post-inflammatory phase on the other, were incubated with relevant peptides to identify their cleavage pattern by mass spectrometry. An increased cleavage of several peptides was observed in the colon from acute colitis rats. The tethered ligand (TL) sequences of peptides mimicking the N-terminus of protease-activated receptors (PAR) 1 and 4 were significantly unmasked by acute colitis samples and these cleavages were positively correlated with thrombin activity. Increased cleavage of ß-endorphin and disarming of the TL-sequence of the PAR3-based peptide were observed in acute colitis and linked to chymotrypsin-like activity. Increased processing of the enkephalins points to the involvement of proteases with specificities different from trypsin- or chymotrypsin-like enzymes. In conclusion, our results suggest thrombin, chymotrypsin-like proteases and a set of proteases with different specificities as potential therapeutic targets in IBD.

The Effect of a Novel Serine Protease Inhibitor on Inflammation and Intestinal Permeability in a Murine Colitis Transfer Model.

Background: A protease/antiprotease disbalance is observed in inflammatory bowel diseases (IBD). We therefore studied the effect of the novel serine protease inhibitor UAMC-00050 on intestinal inflammation and permeability in a chronic colitis T cell transfer mouse model to get further insight into the regulation of T cell-mediated immunopathology. Methods: Colitis was induced in severe combined immunodeficient (SCID) mice, by the adoptive transfer of CD4+CD25-CD62L+ T cells. Animals were treated intraperitoneally (i.p.) 2x/day with vehicle or UAMC-00050 (5 mg/kg) from week 2 onwards. Colonic inflammation was assessed by clinical parameters, colonoscopy, macroscopy, microscopy, myeloperoxidase activity and cytokine expression levels. At week 4, 4 kDa FITC-dextran intestinal permeability was evaluated and T helper transcription factors, protease-activated receptors and junctional proteins were quantified by RT-qPCR. Results: Adoptive transfer of CD4+CD25-CD62L+ T cells resulted in colonic inflammation and an altered intestinal permeability. The serine protease inhibitor UAMC-00050 ameliorated both the inflammatory parameters and the intestinal barrier function. Furthermore, a decrease in colonic mRNA expression of Tbet and PAR4 was observed in colitis mice after UAMC-00050 treatment. Conclusion: The beneficial effect of UAMC-00050 on inflammation was apparent via a reduction of Tbet, IFN-γ, TNF-α, IL-1ß and IL-6. Based on these results, we hypothesize a pivotal effect of serine protease inhibition on the Th1 inflammatory profile potentially mediated via PAR4.

Local Colonic Administration of a Serine Protease Inhibitor Improves Post-Inflammatory Visceral Hypersensitivity in Rats.

Dysregulation of the protease-antiprotease balance in the gastrointestinal tract has been suggested as a mechanism underlying visceral hypersensitivity in conditions such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). We aimed to study the potential therapeutic role of an intracolonically administered serine protease inhibitor for the treatment of abdominal pain in a post-inflammatory rat model for IBS. An enema containing 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to induce colitis in male Sprague-Dawley rats, whereas controls received a saline solution. Colonoscopies were performed to confirm colitis and follow-up mucosal healing. In the post-inflammatory phase, the serine protease inhibitor UAMC-00050 (0.1-5 mg/kg) or its vehicle alone (5% DMSO in H2O) was administered in the colon. Thirty minutes later, visceral mechanosensitivity to colorectal distensions was quantified by visceromotor responses (VMRs) and local effects on colonic compliance and inflammatory parameters were assessed. Specific proteolytic activities in fecal and colonic samples were measured using fluorogenic substrates. Pharmacokinetic parameters were evaluated using bioanalytical measurements with liquid chromatography-tandem mass spectrometry. Post-inflammatory rats had increased trypsin-like activity in colonic tissue and elevated elastase-like activity in fecal samples compared to controls. Treatment with UAMC-00050 decreased trypsin-like activity in colonic tissue of post-colitis animals. Pharmacokinetic experiments revealed that UAMC-00050 acted locally, being taken up in the bloodstream only minimally after administration. Local administration of UAMC-00050 normalized visceral hypersensitivity. These results support the role of serine proteases in the pathophysiology of visceral pain and the potential of locally administered serine protease inhibitors as clinically relevant therapeutics for the treatment of IBS patients with abdominal pain.

Review, Performance Comparison, and Validation of Models Predicting Type 2 Diabetes Remission After Bariatric Surgery in a Western European Population.

PURPOSE: The majority of patients with type 2 diabetes (T2DM) achieve remission after bariatric surgery. Several models are available to preoperatively predict T2DM remission. This study compares the performance of these models in a Western population one year after surgery and explores their predictive value in comparison to a model specifically designed for our study population. MATERIALS AND METHODS: Prediction models were retrieved using a literature search. Patients were retrospectively selected from a database of the Antwerp University Hospital. Performance of the models was assessed by determining the area under the receiver operating characteristic curve (AUROC), the accuracy, and the goodness of fit, and by comparing them to a custom-made logistic model. RESULTS: The probability of T2DM remission was calculated using 11 predictive scoring models and 8 regression models in a cohort of 250 patients. Complete T2DM remission occurred in 64.0% of patients. The IMS score (AUROC = 0.912; accuracy = 83.6%), DiaBetter score (0.907; 82.0%), and Ad-DiaRem score (0.903; 82.8%) best predicted T2DM remission and closely approached the performance of the custom-constructed model (0.917; 84.0%). The model by Ioffe et al. (0.630; 69.2%), Umemura et al. (0.692; 71.4%), and the ABCD score (0.757; 72.8%) were the least accurate. CONCLUSION: Most T2DM remission models reliably predicted one-year T2DM remission, with limited inter-model differences. The accuracy of most models approached that of the custom-constructed model, indicating a high predictive capability and performance in our patient cohort. To date, most models are only validated to estimate T2DM remission one year after surgery and they do not predict long-term remission.

In-Depth Study of Transmembrane Mucins in Association with Intestinal Barrier Dysfunction During the Course of T Cell Transfer and DSS-Induced Colitis.

BACKGROUND AND AIMS: There is evidence for a disturbed intestinal barrier function in inflammatory bowel diseases [IBD] but the underlying mechanisms are unclear. Because mucins represent the major components of the mucus barrier and disturbed mucin expression is reported in the colon of IBD patients, we studied the association between mucin expression, inflammation and intestinal permeability in experimental colitis. METHODS: We quantified 4-kDa FITC-dextran intestinal permeability and the expression of cytokines, mucins, junctional and polarity proteins at dedicated time points in the adoptive T cell transfer and dextran sodium sulfate [DSS]-induced colitis models. Mucin expression was also validated in biopsies from IBD patients. RESULTS: In both animal models, the course of colitis was associated with increased interleukin-1ß [IL-1ß] and tumour necrosis factor-α [TNF-α] expression and increased Muc1 and Muc13 expression. In the T cell transfer model, a gradually increasing Muc1 expression coincided with gradually increasing 4-kDa FITC-dextran intestinal permeability and correlated with enhanced IL-1ß expression. In the DSS model, Muc13 expression coincided with rapidly increased 4-kDa FITC-dextran intestinal permeability and correlated with TNF-α and Muc1 overexpression. Moreover, a significant association was observed between Muc1, Cldn1, Ocln, Par3 and aPKCζ expression in the T cell transfer model and between Muc13, Cldn1, Jam2, Tjp2, aPkcζ, Crb3 and Scrib expression in the DSS model. Additionally, MUC1 and MUC13 expression was upregulated in inflamed mucosa of IBD patients. CONCLUSIONS: Aberrantly expressed MUC1 and MUC13 might be involved in intestinal barrier dysfunction upon inflammation by affecting junctional and cell polarity proteins, indicating their potential as therapeutic targets in IBD.

Clinical- and surgery-specific risk factors for post-operative sepsis: a systematic review and meta-analysis of over 30 million patients.

Post-operative sepsis is a severe complication of surgery, which often worsens the clinical outcomes. While several risk factors have been identified, the importance of others remains uncertain. This systematic review and meta-analysis aimed to determine patient and surgery-related risk factors for post-operative sepsis. We reviewed Medline, the Web of Science, and the Cochrane library, systematically, for articles describing risk factors for sepsis. The role of eligible risk factors was investigated using a random-effects model, while analyzing univariate and multivariate data separately. Among 193 pro- and retrospective articles, comprising over 30 million patients, 38 eligible risk factors were selected for this meta-analysis. The patient-related risk factors associated with post-operative sepsis included male gender (odds ratio, OR 1.41), pre-existing heart failure (OR 2.53), diabetes (OR 1.41), and chronic kidney disease (OR 1.26). The surgery-related risk factors identified included emergency surgery (OR 3.38), peri-operative blood transfusion (OR 1.90), inpatient hospital stay (OR 2.31), and open surgery (OR 1.80). The adjusted overall incidence of surgical sepsis was 1.84%. In conclusion, multiple-patient and surgery-related risk factors are associated with the development of post-operative sepsis. Recognizing these risk factors could assist in the pre-operative identification of patients at risk of post-operative sepsis.

Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post-inflammatory rat model for irritable bowel syndrome.

BACKGROUND AND PURPOSE: Serine proteases have been re suggested as important mediators of visceral pain. We investigated their effect by using newly developed serine protease inhibitors with a well-characterized inhibitory profile in a rat model of post-inflammatory irritable bowel syndrome (IBS). EXPERIMENTAL APPROACH: Colitis was induced in rats receiving intrarectal trinitrobenzenesulphonic acid; controls received 0.9% NaCl. Colonoscopies were performed on day 3, to confirm colitis, and later until mucosal healing. Visceral hypersensitivity was quantified by visceromotor responses (VMRs) to colorectal distension, 30 min after i.p. injection of the serine protease inhibitors nafamostat, UAMC-00050 or UAMC-01162. Serine proteases, protease-activated receptors (PARs) and TRP channels were quantified by qPCR and immunohistochemistry. Proteolytic activity was characterized using fluorogenic substrates. KEY RESULTS: VMR was significantly elevated in post-colitis rats. Nafamostat normalized VMRs at the lowest dose tested. UAMC-00050 and UAMC-01162 significantly decreased VMR dose-dependently. Expression of mRNA for tryptase-αß-1and PAR4, and tryptase immunoreactivity was significantly increased in the colon of post-colitis animals. Trypsin-like activity was also significantly increased in the colon but not in the faeces. PAR2 and TRPA1 immunoreactivity co-localized with CGRP-positive nerve fibres in control and post-colitis animals. CONCLUSIONS AND IMPLICATIONS: Increased expression of serine proteases and activity together with increased expression of downstream molecules at the colonic and DRG level and in CGRP-positive sensory nerve fibres imply a role for serine proteases in post-inflammatory visceral hypersensitivity. Our results support further investigation of serine protease inhibitors as an interesting treatment strategy for IBS-related visceral pain.

Regulation of intestinal permeability: The role of proteases.

The gastrointestinal barrier is - with approximately 400 m2 - the human body's largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extra-intestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins via activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases.

Beneficial Effects of Anti-Interleukin-6 Antibodies on Impaired Gastrointestinal Motility, Inflammation and Increased Colonic Permeability in a Murine Model of Sepsis Are Most Pronounced When Administered in a Preventive Setup.

BACKGROUND AND OBJECTIVES: During sepsis, gastrointestinal ileus, mucosal barrier dysfunction and bacterial translocation are accepted to be important triggers that can maintain or exacerbate the septic state. In the caecal ligation and puncture animal model of sepsis, we demonstrated that systemic and colonic interleukin-6 levels are significantly increased coinciding with an impaired colonic barrier function. We therefore aimed to study the effect of therapeutic or curative administration of anti-IL6 antibodies on overall GI motility, colonic permeability and translocation of intestinal bacteria in blood and mesenteric lymph nodes in the mouse caecal ligation and puncture model. METHODS: OF-1 mice were randomized to either the preventive or curative protocol, in which they received 1 mg/kg of antibodies to interleukin-6, or its IgG isotype control solution. They subsequently underwent either the caecal ligation and puncture procedure, or sham-surgery. GI motility was assessed 48 h following the procedure, as well as colonic permeability, serum and colon cytokines, colonic tight junction proteins at the mRNA level; cultures of blood and mesenteric lymph nodes were performed. RESULTS: Preventive administration of anti-interleukin-6 antibodies successfully counteracted the gastrointestinal motility disturbances and impaired colonic barrier function that could be observed in vehicle-treated septic animals. Serum and colonic levels of proinflammatory cytokines were significantly lower when animals were preventively treated with anti-interleukin-6 antibodies. A repetitive injection 24 h later resulted in the most pronounced effects. Curative treatment significantly lowered systemic and colonic inflammation markers while the effects on transit and permeability were unfortunately no longer significant. CONCLUSIONS: Caecal ligation and puncture resulted in septic ileus with an increased colonic permeability. Antibodies to interleukin-6 were able to ameliorate gastro-intestinal motility, suppress inflammation and normalize the permeability of the colonic wall, with the preventive administration combined with a repeat injection being far more efficacious than the sole preventive or curative one.

Visceral hypersensitivity in inflammatory bowel diseases and irritable bowel syndrome: The role of proteases.

Proteases, enzymes catalyzing the hydrolysis of peptide bonds, are present at high concentrations in the gastrointestinal tract. Besides their well-known role in the digestive process, they also function as signaling molecules through the activation of protease-activated receptors (PARs). Based on their chemical mechanism for catalysis, proteases can be classified into several classes: serine, cysteine, aspartic, metallo- and threonine proteases represent the mammalian protease families. In particular, the class of serine proteases will play a significant role in this review. In the last decades, proteases have been suggested to play a key role in the pathogenesis of visceral hypersensitivity, which is a major factor contributing to abdominal pain in patients with inflammatory bowel diseases and/or irritable bowel syndrome. So far, only a few preclinical animal studies have investigated the effect of protease inhibitors specifically on visceral sensitivity while their effect on inflammation is described in more detail. In our accompanying review we describe their effect on gastrointestinal permeability. On account of their promising results in the field of visceral hypersensitivity, further research is warranted. The aim of this review is to give an overview on the concept of visceral hypersensitivity as well as on the physiological and pathophysiological functions of proteases herein.

P2X3 receptors mediate visceral hypersensitivity during acute chemically-induced colitis and in the post-inflammatory phase via different mechanisms of sensitization.

OBJECTIVES: Experiments using P2X3 knock-out mice or more general P2X receptor antagonists suggest that P2X3 receptors contribute to visceral hypersensitivity. We aimed to investigate the effect of the selective P2X3 antagonist A-317491 on visceral sensitivity under physiological conditions, during acute colitis and in the post-inflammatory phase of colitis. METHODS: Trinitrobenzene sulphonic-acid colitis was monitored by colonoscopy: on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor convalescence and determine the exact timepoint of endoscopic healing in each rat. Visceral sensitivity was assessed by quantifying visceromotor responses to colorectal distension in controls, rats with acute colitis and post-colitis rats. A-317491 was administered 30 min prior to visceral sensitivity testing. Expression of P2X3 receptors (RT-PCR and immunohistochemistry) and the intracellular signalling molecules cdk5, csk and CASK (RT-PCR) were quantified in colonic tissue and dorsal root ganglia. ATP release in response to colorectal distension was measured by luminiscence. RESULTS: Rats with acute TNBS-colitis displayed significant visceral hypersensitivity that was dose-dependently, but not fully, reversed by A-317491. Hypersenstivity was accompanied by an increased colonic release of ATP. Post-colitis rats also displayed visceral hypersensitivity that was dose-dependently reduced and fully normalized by A-317491 without increased release of ATP. A-317491 did not modify visceral sensitivity in controls. P2X3 mRNA and protein expression in the colon and dorsal root ganglia were similar in control, acute colitis and post-colitis groups, while colonic mRNA expression of cdk5, csk and CASK was increased in the post-colitis group only. CONCLUSIONS: These findings indicate that P2X3 receptors are not involved in sensory signaling under physiological conditions whereas they modulate visceral hypersensitivity during acute TNBS-colitis and even more so in the post-inflammatory phase, albeit via different mechanisms of sensitization, validating P2X3 receptors as potential new targets in the treatment of abdominal pain syndromes.

Treatment with egg antigens of Schistosoma mansoni ameliorates experimental colitis in mice through a colonic T-cell-dependent mechanism.

BACKGROUND: Helminth-derived molecules are being identified as a new therapeutic approach for immune-mediated diseases. We investigated the anti-inflammatory effect and the immunological mechanisms of Schistosoma mansoni soluble egg antigens (SmSEA) in a mouse model of chronic colitis. METHODS: Colitis was induced in immunocompromised severe combined immunodeficiency mice by the adoptive transfer of CD4CD25CD62L T cells. Two weeks post-transfer, SmSEA treatments were started (study 1: 1 × 20 µg SmSEA per week 5 times; study 2: 2 × 20 µg SmSEA per week 3 times). From the start of the treatment (week 2), the clinical outcome and colonic inflammation were assessed at different time points by a clinical disease score and colonoscopy, respectively. At the end of the studies, the colons were harvested for macroscopic examination, and colonic lamina propria mononuclear cells were isolated for flow cytometric T-cell characterization. RESULTS: In both studies, administration of SmSEA in colitis mice improved all the inflammatory parameters studied. However in study 1, this beneficial effect on inflammation diminished with time, and the T-cell characterization of the lamina propria mononuclear cells, performed at week 6, revealed no immunological effects of the SmSEA treatment. In study 2, mice were killed earlier (week 4) and at that time point, we found a significant downregulation of the number of interleukin-17A-producing T cells and a significant upregulation of the number of interleukin-4-producing T cells in the colon of the SmSEA-treated colitis mice. CONCLUSIONS: Our results demonstrated that the administration of SmSEA reduces the severity of colitis in the adoptive transfer mouse model characterized by an increased Th2 response and a suppressed Th17 response in the colon.

Worm proteins of Schistosoma mansoni reduce the severity of experimental chronic colitis in mice by suppressing colonic proinflammatory immune responses.

Although helminthic therapy as a possible new option to treat inflammatory bowel disease is a well-established concept by now, the search for immunomodulatory helminth-derived compounds and their mechanisms of action is still ongoing. We investigated the therapeutic potential and the underlying immunological mechanisms of Schistosoma mansoni soluble worm proteins (SmSWP) in an adoptive T cell transfer mouse model of chronic colitis. Both a curative and a preventive treatment protocol were included in this study. The curative administration of SmSWP (started when colitis was established), resulted in a significant improvement of the clinical disease score, colonoscopy, macroscopic and microscopic inflammation score, colon length and myeloperoxidase activity. The therapeutic potential of the preventive SmSWP treatment (started before colitis was established), was less pronounced compared with the curative SmSWP treatment but still resulted in an improved clinical disease score, body weight loss, colon length and microscopic inflammation score. Both the curative and preventive SmSWP treatment downregulated the mRNA expression of the proinflammatory cytokines IFN-γ and IL-17A and upregulated the mRNA expression of the anti-inflammatory cytokine IL-4 in the colon at the end of the experiment. This colonic immunomodulatory effect of SmSWP could not be confirmed at the protein level. Moreover, the effect of SmSWP appeared to be a local colonic phenomenon, since the flow cytometric T cell characterization of the mesenteric lymph nodes and the cytokine measurements in the serum did not reveal any effect of SmSWP treatment. In conclusion, SmSWP treatment reduced the severity of colitis in the adoptive transfer mouse model via the suppression of proinflammatory cytokines and the induction of an anti-inflammatory response in the colon.

Histamine H4 and H1 receptors contribute to postinflammatory visceral hypersensitivity.

OBJECTIVES: Substantial evidence implicates mast cells and their main constituent histamine in the pathogenesis of visceral hypersensitivity. We explored the specific contribution of histamine H4 (H4R) and H1 (H1R) receptors to visceral hypersensitivity in a postinflammatory rat model. DESIGN: Trinitrobenzenesulfonic acid (TNBS)-colitis was monitored individually by colonoscopy: first on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor convalescence and determine the exact timepoint of endoscopic healing in each rat. Experiments were performed 3 days after endoscopic resolution of colitis. Visceral sensitivity was assessed by quantifying visceromotor responses (VMRs) to colorectal distension. Colonic mast cell numbers, histamine release and H4R and H1R mRNA expression were quantified. JNJ7777120 (H4R antagonist) and/or levocetirizine (H1R antagonist) were administered 30 min prior to VMR assessment or histamine release assay. RESULTS: Postcolitis rats displayed a higher number of colonic mast cells, excessive histamine release and significantly enhanced VMRs. Heightened VMRs were dose-dependently reduced by JNJ7777120 and levocetirizine; combined administration of JNJ7777120 and levocetirizine potentiated the antinociceptive effect. In the colon, both H4R and H1R mRNA were present; in the dorsal root ganglia, only H1R mRNA was found. Only colonic H4R mRNA expression was increased in postcolitis rats. Excessive histamine release in postcolitis rats was attenuated by the highest dose of JNJ7777120. CONCLUSIONS: H4R and H1R antagonists dose-dependently reduce and even normalise postinflammatory visceral hypersensitivity via different underlying mechanisms but with a synergistic effect. Both receptor subtypes represent promising targets for the treatment of postinflammatory visceral hypersensitivity.

Colonoscopy and µPET/CT are valid techniques to monitor inflammation in the adoptive transfer colitis model in mice.

BACKGROUND: Preclinical in vivo research on inflammatory bowel diseases requires proper animal models and techniques allowing longitudinal monitoring of colonic inflammation without the need to kill animals. We evaluated colonoscopy and µ-positron emission tomography/computed tomography (µPET/CT) as monitoring tools in a model for chronic colitis in mice. METHODS: Colitis was induced by adoptive transfer of CD4(+)CD25(-)CD62L(+) T cells in immunocompromised severe combined immunodeficient mice. Three study protocols were designed. In study 1, colonoscopy and µPET/CT were performed once, 4 weeks after transfer. In study 2 and study 3, colitis was sequentially followed up through colonoscopy (study 2) or colonoscopy plus µPET/CT (study 3). Each study included postmortem evaluation of colonic inflammation (macroscopy, microscopy, and myeloperoxidase activity). RESULTS: In study 1, both colonoscopy and µPET/CT detected colitis 4 weeks after transfer. Study 2 showed a gradual increase in colonoscopic score from week 2 (1.4 ± 0.6) to week 8 (6.0 ± 1.1). In study 3, colitis was detected 2 weeks after transfer by µPET/CT (2.0 ± 0.4) but not by colonoscopy, whereas both techniques detected inflammation 4 and 6 weeks after transfer. Colonoscopy correlated with µPET/CT (r = 0.812, 0.884, and 0.781, respectively) and with postmortem analyses in all 3 studies. CONCLUSIONS: Adoptive transfer of CD4(+)CD25(-)CD62L(+) T cells in severe combined immunodeficient mice results in a moderate chronic colitis. Evolution of colitis could be monitored over time by both colonoscopy and µPET/CT. µPET/CT seems to detect inflammation at an earlier time point than colonoscopy. Both techniques represent reliable and safe methods without the need to kill animals.

Cannabidiol reduces intestinal inflammation through the control of neuroimmune axis.

Enteric glial cells (EGC) actively mediate acute and chronic inflammation in the gut; EGC proliferate and release neurotrophins, growth factors, and pro-inflammatory cytokines which, in turn, may amplify the immune response, representing a very important link between the nervous and immune systems in the intestine. Cannabidiol (CBD) is an interesting compound because of its ability to control reactive gliosis in the CNS, without any unwanted psychotropic effects. Therefore the rationale of our study was to investigate the effect of CBD on intestinal biopsies from patients with ulcerative colitis (UC) and from intestinal segments of mice with LPS-induced intestinal inflammation. CBD markedly counteracted reactive enteric gliosis in LPS-mice trough the massive reduction of astroglial signalling neurotrophin S100B. Histological, biochemical and immunohistochemical data demonstrated that S100B decrease was associated with a considerable decrease in mast cell and macrophages in the intestine of LPS-treated mice after CBD treatment. Moreover the treatment of LPS-mice with CBD reduced TNF-α expression and the presence of cleaved caspase-3. Similar results were obtained in ex vivo cultured human derived colonic biopsies. In biopsies of UC patients, both during active inflammation and in remission stimulated with LPS+INF-γ, an increased glial cell activation and intestinal damage were evidenced. CBD reduced the expression of S100B and iNOS proteins in the human biopsies confirming its well documented effect in septic mice. The activity of CBD is, at least partly, mediated via the selective PPAR-gamma receptor pathway. CBD targets enteric reactive gliosis, counteracts the inflammatory environment induced by LPS in mice and in human colonic cultures derived from UC patients. These actions lead to a reduction of intestinal damage mediated by PPARgamma receptor pathway. Our results therefore indicate that CBD indeed unravels a new therapeutic strategy to treat inflammatory bowel diseases.

Effect of genetic SSTR4 ablation on inflammatory peptide and receptor expression in the non-inflamed and inflamed murine intestine.

The recently suggested pivotal role of somatostatin (SOM) receptor 4 (SSTR4) in inflammation and nociception in several non-intestinal organs and in gastrointestinal (GI) physiology, necessitates exploration of the role of SSTR4 in GI pathophysiology. Therefore, the role of SSTR4 in GI activity was explored by investigating the effects of SSTR4 deficiency on intestinal motility, smooth muscle contractility and on the expression of SSTRs and neuropeptides in the healthy and Schistosoma mansoni-infected murine small intestine. Functional experiments revealed no differences in intestinal motility or smooth muscle cell contractility between wild-type and SSTR4 knockout (SSTR4(-/-)) mice in physiological conditions. As revealed by multiple immunofluorescent labellings, RT-PCR and quantitative real time RT-PCR (qPCR), genetic deficiency of SSTR4 considerably altered the expression of SOM and SSTRs in non-inflamed and inflamed conditions, affecting both extrinsic and intrinsic components of the intestinal innervation, along with SSTR expression in several non-neuronal cell types. Moreover, substance P and calcitonin gene-related peptide expression were significantly elevated in SSTR4(-/-) mice, confirming the modulatory role of SSTR4 on intestinal pro-inflammatory neuropeptide expression. These data suggest that SSTR4 plays a previously unexpected modulatory role in the regulation of intestinal SSTR expression. Moreover, in addition to the recently described inhibitory effects of SSTR4 on the neuronal release of pro-inflammatory peptides, SSTR4 appears also to be involved in the neuronal expression of both pro- and anti-inflammatory peptides in the murine small intestine.

Coexistence of non-adrenergic non-cholinergic inhibitory and excitatory neurotransmitters in a large neuronal subpopulation in the vaginal segment of the chicken oviduct.

The presence, distribution and smooth muscle motor effects of galanin and pituitary adenylate cyclase activating peptide (PACAP) were studied in the nerves of the vaginal part of the oviduct of egg-laying hens. Galanin and PACAP immunoreactivity were found both in neuronal perikarya and nerve fibres within the wall of the vaginal segment. Both populations showed a similar distribution pattern. Particularly the circular muscle and the intramural vascular net were richly innervated. A few galanin- and PACAP-IR nerve fibres extended up to the mucosal folds. Multiple labelling showed galanin to be colocalised with PACAP as well as with vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS) in a large, partly intrinsic neuronal subpopulation innervating the smooth muscle wall. Pharmacological in vitro experiments showed that isolated vaginal muscle strips had a spontaneous basal activity that was not affected by the neuronal conductance blocker tetrodotoxin (TTX). Galanin induced concentration-dependent contractions that were TTX-insensitive. PACAP, VIP, nitric oxide (NO) and the NO donor nitroglycerin caused concentration-dependent relaxations that were TTX-insensitive. Electrical field stimulation of isolated muscle strips induced frequency-dependent relaxations that were blocked by TTX and reduced by the NOS blocker L-nitroarginine. These data provide evidence that the vaginal part of the oviduct contains a largely intrinsic, neuronal subpopulation, capable of releasing multiple non-adrenergic, non-cholinergic (NANC) motor agents for the control of local muscular activities. In addition, we provided pharmacological evidence that VIP, NO and PACAP exert an inhibitory and galanin an excitatory action on isolated muscle strips of the vaginal part of the chicken oviduct. Our results suggest that these NANC neurotransmitters play an important role in the regulation of neuromuscular activity in this region.

Functional evidence that ATP or a related purine is an inhibitory NANC neurotransmitter in the mouse jejunum: study on the identity of P2X and P2Y purinoceptors involved.

1. Conflicting views exist on whether ATP is a neurotransmitter in the enteric nervous system. We investigated the role of ATP in enteric transmission in circular muscle strips of the mouse jejunum. 2. On PGF2alpha-precontracted muscle strips and in the presence of atropine and guanethidine, electrical field stimulation (EFS, 1-8 Hz) of nonadrenergic noncholinergic (NANC) nerves induced transient relaxations that were abolished by the nerve-conductance blocker tetrodotoxin. The NO synthase blocker l-nitroarginine (l-NOARG) partially inhibited the NANC relaxations to EFS, but fast-twitch relaxations to EFS were still observed in the presence of l-NOARG. 3. In the presence of l-NOARG, ATP, the P2X receptor agonist alphabetaMeATP and the P2Y receptor agonist ADPbetaS relaxed jejunal muscle strips. Tetrodotoxin did not affect the relaxation to ATP and ADPbetaS, but inhibited that to alphabetaMeATP. 4. The l-NOARG-resistant NANC relaxations to EFS were almost abolished by apamin, a blocker of small-conductance Ca2+ activated K+ channels, and by suramin and PPADS, blockers of P2 purinoceptors. Relaxations to ATP were almost abolished by apamin and suramin but not affected by PPADS. 5. Desensitisation of alphabetaMeATP-sensitive P2X receptors, the P2X receptor blocker Evans blue and the P2X1,2,3 receptor blocker NF 279 inhibited the l-NOARG-resistant NANC relaxations to EFS and that to alphabetaMeATP without affecting the relaxation to ADPbetaS. Brilliant blue G, a P2X2,5,7 receptor blocker, did not affect the relaxations to EFS. 6. Desensitisation of P2Y receptors and MRS 2179, a P2Y1 receptor blocker, virtually abolished the l-NOARG-resistant NANC relaxations to EFS and the relaxation to ADPbetaS without affecting the relaxation to alphabetaMeATP. 7. Dipyridamole, an adenosine uptake inhibitor, or theophylline and 8-phenyltheophylline, blockers of P1 and A1 purinoceptors, respectively, did not affect the purinergic NANC relaxations to EFS. 8. Our results suggest that ATP or a related purine acts as an inhibitory NANC neurotransmitter in the mouse jejunum, activating P2 but not P1 purinoceptors. Relaxations to the purinergic NANC neurotransmitter mainly involve P2Y receptors of the P2Y1 subtype that are located postjunctionally. Purinergic NANC neurotransmission also involves P2X receptors, most likely of the P2X1 and P2X3 subtype, located pre- and/or postjunctionally.