Mycobacterium genavense granulomatous typhlocolitis in a horse.

A 23-y-old gelding was presented to a veterinary teaching hospital with a history of chronic, refractory diarrhea. Clinically, the horse was in poor body condition, with a thickened and corrugated large intestine identified by transcutaneous abdominal ultrasonography. At postmortem examination following euthanasia, the large colon and cecum had segmental thickening of the intestinal wall with innumerable mucosal ulcers and prominent polypoid mucosal masses. Many mesenteric and hepatic lymph nodes were enlarged. Histology revealed granulomatous and ulcerative typhlocolitis and granulomatous lymphadenitis with myriad acid-fast, variably gram-positive, intrahistiocytic bacilli that stained by immunohistochemistry for mycobacteria. Molecular testing by PCR and sequencing identified the causative agent as Mycobacterium genavense, which is an unusual presentation of infection in a horse.

Evaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis.

Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced goblet cell (GBC) numbers and/or mucin 2 (MUC2) expression, which are responsible for mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is undetermined if Ki-67 immunoreactivity, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to histologic severity in GC and LPC. Study objectives included comparing Ki-67 immunoreactivity, GBC population and MUC2 expression in dogs with GC, LPC and non-inflamed colon; and exploring the use of ribonucleic acid (RNAscope®) in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs over an eight-year period. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colitis (NC) (n=10) group based on final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope® ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per dog were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using image analysis software. Spearman's correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic score (WHS) and measured variables. Linear regression models were used to compare relationships between WHS with KI67%, GBC%, and MUC2%; and between GBC% and MUC2%. Median WHS was highest in dogs with GC. Median KI67% normalised to WHS was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation overall. Median MUC2% normalised to WHS in the NC group (10.02%; range, 3.05-39.09%) was two- and three-fold higher than in the GC and LPC groups respectively. With increased colonic inflammation, despite minimal changes in GBC% overall, MUC2 expression markedly declined in the LPC group (-27.4%; 95%-CI, -49.8, 5.9%) and mildly declined in the GC and NC groups. Granulomatous colitis and LPC likely involve different pathways regulating MUC2 expression. Decreased MUC2 gene expression is observed in dogs with chronic colitis compared to dogs without colonic signs. Changes in MUC2 expression appear influenced by GBC activity rather than quantity in GC and LPC.

Nonsteroidal antiinflammatory drugs reduce anion secretion and increase apoptosis in equine colonic mucosa ex vivo.

OBJECTIVE: Right dorsal colitis causes chronic colic associated with long-term treatment with nonsteroidal antiinflammatory drugs (NSAIDs). This study was designed to determine if NSAIDs could inhibit anion transporters that protect against intestinal mucosal injury in other species. ANIMALS: 20 healthy horses. METHODS: The effects of indomethacin (INDO) and firocoxib (FIR), on short-circuit current (Isc) in mucosa from the right dorsal colon (RDC) and right ventral colon (RVC) were measured in Ussing chambers by standard electrophysiological techniques. Immunohistochemical methods were used to detect apoptosis (caspase-3) with these NSAIDs and phenylbutazone (PBZ) and to locate the NKCC1 transporter. RESULTS: The Isc in RDC and RVC incubated with INDO or FIR was increased almost 3-fold (P < .0001) by prostaglandin E2 (PGE2) through a system inhibited by loop diuretics (P < .0001). Although these findings and anion replacement studies were consistent with anion secretion, the RDC also displayed an Isc response suggestive of a unique transporter apparently absent in RVC or NSAID-free solutions. In RDC, FIR, INDO, and PBZ induced apoptosis in the lower half of crypts. However, significant differences in apoptotic index were recorded in the RDC between NSAID-treated and control tissues (no NSAID). CLINICAL RELEVANCE: The effects of NSAIDs on Isc were consistent with reduced anion secretion, which could represent the pharmacological equivalent of the transport failure responsible for Cystic Fibrosis (CF) in other species. Failure of anion secretion could interfere with buffering acid from intraluminal fermentation, which could suggest a treatment target for right dorsal colitis.

Amelioration of DSS-induced colitis in mice by TNF-α-stimulated mesenchymal stem cells derived from feline adipose tissue via COX-2/PGE2 activation.

BACKGROUND: Mesenchymal stem cells (MSCs) have been investigated as therapeutic agents for inflammatory bowel disease (IBD). Stimulation of MSCs with pro-inflammatory cytokines is an approach to enhance their immunomodulatory effects. However, further investigation is required to support their application in immune-mediated disorders and companion animals. OBJECTIVES: This study aimed to assess the therapeutic effect of tumor necrosis factor (TNF)-α-stimulated feline adipose tissue-derived MSCs (fAT-MSCs) in a dextran sulfate sodium (DSS)-induced colitis mouse model. METHODS: Colitis mice was made by drinking water with 3% DSS and fAT-MSCs were injected intraperitoneally. Colons were collected on day 10. The severity of the disease was evaluated and compared. Raw 264.7 cells were cultured with the conditioned medium to determine the mechanism, using quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: TNF-α-stimulated fAT-MSCs more improved severity of DSS-induced colitis in disease activity, colon length, histologic score, and inflammatory cytokine. In sectionized colon tissues, the group comprising TNF-α-stimulated fAT-MSCs had higher proportion of CD11b+CD206+ macrophages than in the other groups. In vitro, TNF-α-stimulation increased cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) secretion from fAT-MSCs. The conditioned medium from TNF-α-stimulated fAT-MSCs enhanced the expression of interleukin-10 and arginase-1 in LPS-activated Raw 264.7 cells. CONCLUSIONS: These results represent that TNF-α-stimulated fat-mscs ameliorate the inflamed colon more effectively. Furthermore, we demonstrated that the effectiveness was interlinked with the COX-2/PGE2 pathway.

The synbiotic mixture of Bacillus licheniformis and Saccharomyces cerevisiae extract aggravates dextran sulfate sodium induced colitis in rats.

BACKGROUND: Uncertain effects of probiotics and/or prebiotics have been reported in experimental and clinical colitis. This study aims to examine the effects of a synbiotic combination comprising Bacillus licheniformis DSM 17236 and Saccharomyces cerevisiae cell wall extract on dextran sulfate sodium (DSS)-induced colitis in Sprague Dawley rats. METHODS: Acute colitis was induced in rats by oral administration of DSS 3.5% for 7 days. Fifty rats were divided equally into five groups; one control group and the other groups were induced with colitis and treated with or without the tested synbiotic, mixed with diet, for 28 days and sulfasalazine (100 mg/kg) via intragastric tube once daily for 14 days. RESULTS: Symptomatically, the synbiotic administration raised the disease activity index (DAI) to comparable scores of the DSS group, specially from the 2nd to 7th days post DSS intoxication. It also induced a significant (p < 0.05) amplification of WBCs, myeloperoxidase (MPO), malondialdehyde (MDA), nuclear factor kappa B (NF-kB) expression and proinflammatory cytokines tumor necrosis factor alpha (TNFα), interferon gamma (INFγ), and interleukin-1 beta (IL-1ß) while depressed the antioxidant enzymes glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) when compared with the DSS and control groups. The DSS intoxicated and Synbiotic+DSS groups showed desquamations of the covering epithelium, noticeable diffuse leukocytic infiltrations, sever catarrhal enteritis, ischemic colitis with diffuse coagulative necrosis of the entire colonic mucosa. Contrarily, sulfasalazine proved to be effective in the reduction of the tested inflammatory markers and the pathological degenerative changes of the DSS ulcerative colitis. CONCLUSION: The examined synbiotic did not ameliorate but aggravated the DSS-induced colitis, so it should be subjected to intensive experimental and clinical testing before their use in animals and human.

Anti-inflammation and gut microbiota regulation properties of fatty acids derived from fermented milk in mice with dextran sulfate sodium-induced colitis.

The by-products of milk fermentation by lactic acid bacteria provide potential health benefits to the balance of host intestinal microflora. In this study, the anti-inflammatory properties of fatty acids from monoculture-strain (Lactiplantibacillusplantarum A3) and multiple-strain (Streptococcus thermophilus, Lactobacillus bulgaricus, and L. plantarum A3 1:1:2) fermented milk were evaluated in a mouse model of dextran sulfate sodium-induced colitis, and the gut microbiota regulation properties of the fatty acids were also investigated. Results showed that fatty acids can attenuate the inflammatory response by inhibiting the expression of inflammatory factors IL-6 and tumor necrosis factor-α, and blocking the phosphorylation of the JNK in MAPK signal pathway. In addition, the relative abundance of the taxa Akkermansia and Lactobacillus were both enriched after the fatty acid intervention. This finding suggests that fatty acids from the milk fermentation with mixed lactic acid bacteria starters can reduce the severity of dextran sulfate sodium-induced colitis and enhance the abundance of the probiotics in the mice intestinal tract.

A comprehensive and comparative proteomic analysis of horse serum proteins in colitis.

BACKGROUND: Equine colitis is a diarrhoeal disease caused by inflammation of the large bowel and can potentially be life-threatening due to its rapid progression. Pathogenesis is multifactorial and pathophysiology is highly complicated, therefore, reliable diagnostic biomarkers are needed in the veterinary field. OBJECTIVE: Serum is one of the most commonly used diagnostic tools in equine clinical investigation. To discover diagnostic or prognostic protein markers for colitis in horse serum, comprehensive and comparative proteomic analysis was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). STUDY DESIGN: Case-control study. METHODS: Serum samples were collected from 36 healthy Thoroughbreds and 12 Thoroughbreds with colitis. Serum from each horse suffering from colitis was collected daily until death or recovery. Collected sera were digested with trypsin. Peptides obtained from serum proteins were measured by Q-Exactive HF Orbitrap mass spectrometer. The identification and quantification of peptides were performed using Proteome Discoverer version 2.2. RESULTS: On day 1 of treatment, eight proteins in the colitis group were upregulated (P < .05, more than a twofold change) compared with the healthy group. Among the eight proteins, biliverdin reductase B was significantly upregulated (P < .05) in the non-survivor group (n = 5) compared with the survivor group (n = 7). On the last day of the treatment, haemoglobin subunit alpha, clusterin, glyceraldehyde-3-phosphate dehydrogenase, lipopolysaccharide-binding protein, and biliverdin reductase B showed significant increases (P < .05) in the non-survivor group. MAIN LIMITATIONS: The number of the identified proteins is limited due to the existence of abundant proteins. CONCLUSIONS: Measuring the changes of these proteins together may enable a potential prognosis or early diagnosis of horses suffering from colitis.

Hypoxic preconditioning promotes the immunosuppressive effects of mesenchymal stem cells in mice with colitis.

Mesenchymal stem cells are promising candidates for stem cell therapy in many diseases, especially in immune-associated diseases. Inflammatory bowel disease is a chronic autoimmune disease that can lead to colorectal cancer if it is not controlled. Mesenchymal stem cells are always under a hypoxic environment in vivo, whether in bone marrow or adipose tissue, whereas researchers always culture MSCs (mesenchymal stem cells) under normoxic conditions (21%). In this study, we aimed to investigate whether hypoxia (1%) affects the therapeutic effect of MSCs. We hypothesize that hypoxia may benefit the treatment efficacy of MSCs. We used DSS to induce IBD (inflammatory bowel disease) in mice and then injected MSCs that had been preconditioned under normoxic conditions (21%) and hypoxic conditions (1%). We found that compared with normoxic-preconditioned MSCs (n-MSCs), hypoxic-preconditioned MSCs (h-MSCs) could alleviate colon inflammation to a large extent, as determined by inflammatory cytokines and CD3+ T cell activation. Mechanistic studies showed that hypoxia could promote iNOS expression in MSCs. Therefore, our data suggest that hypoxia may be more appropriate than normoxia for facilitating MSCs exertion of therapeutic functions.

Mesenchymal stem cells have ameliorative effect on the colitis model via Nrf2/HO-1 pathway

Purpose: To evaluate the ameliorative effect of mesenchymal stem cells (MSCs) on acetic acid colitis model via Nrf2/HO-1 pathway in rats. Methods: In this study, 30 rats were divided into three groups. Acute colitis was induced by rectal administration of 4% solution of acetic acid. MSCs were injected intraperitoneally in the treatment group. Results: Increased levels of tumor necrosis factor-α (TNF-α), pentraxin-3, and malondialdehyde (MDA) in colitis group were revealed biochemically. Increased level of TNF-α and decreased levels of Nrf2 and interleukin-10 (IL-10) were observed in rectum tissues. Increased fibrous tissue proliferation, vascularization and inflammatory cell infiltration were described in the colitis group. Significant improvement was observed in MSCs treated group histopathologically. Increased immunopositivity of TNF-α, vascular endothelial growth factor (VEGF) and CD68 markers was observed in the colitis group cells, and decreased level of this positivity was observed in MSCs treated group. Conclusions: Biochemical, histopathological and immunohistochemical results strongly support the ameliorative effect of MSCs against acetic induced colitis model via Nrf2/HO-1 pathway in rats.

Contrast-enhanced ultrasonography in dogs with inflammatory bowel disease.

BACKGROUND: Contrast-enhanced ultrasonography (CEUS) is used to evaluate vascularity of the gastrointestinal wall in neoplastic and inflammatory diseases. OBJECTIVE: To assess the feasibility of CEUS for the evaluation of duodenal perfusion in dogs with inflammatory bowel disease (IBD). ANIMALS: Forty-two dogs with IBD and 20 clinically healthy dogs. METHODS: All CEUS studies of the duodenum were analyzed to obtain time-intensity curves and perfusion parameters. The procedure was repeated in 12 IBD dogs 2 months after a standardized treatment. RESULTS: On CEUS, the duodenal wall showed a typical perfusion pattern characterized by a radial and simultaneous enhancement of the wall in all dogs. On qualitative assessment, no differences were observed in contrast medium distribution between healthy and affected dogs, or between dogs with IBD before and after treatment. Peak intensity (PI) and area under the curve (AUC) significantly differed between healthy (PI = 3.58 arbitrary units [au; 1.86-4.93 au] and AUC = 47.63 au seconds [aus, 22.68-62.15]) and affected dogs (PI = 5.10 au [0.63-15.16 au] and AUC = 63.62 aus [5.31-212.20 aus]; P = .03 and .03, respectively). No significant differences were found for the perfusion parameters before and after treatment. CONCLUSIONS AND CLINICAL IMPORTANCE: We showed that CEUS allows discrimination between IBD affected dogs and healthy dogs by evaluation of time-intensity curves, but did not provide useful information for monitoring therapeutic response. The qualitative assessment identified no significant differences between healthy and affected dogs, or between dogs before and after treatment.

Assessment of the impact of age and of blood-derived inflammatory markers in horses with colitis.

OBJECTIVES: To determine the impact of age on survival in horses with colitis and to elucidate whether a lower type-1/type-2 cytokine ratio or an exaggerated inflammatory state contribute to reduced survival in aged horses. DESIGN: Part 1: Retrospective cohort analysis. Part 2: Analytic observational study. ANIMALS: Part 1: One hundred twenty-four adult horses with colitis. Part 2: Twenty-nine adult horses with new diarrhea onset while hospitalized. MEASUREMENTS AND MAIN RESULTS: Part 1: Patient signalment, select clinicopathological data, diagnoses, treatment, hospitalization length, and invoice were compared between survivors (n = 101) and nonsurvivors (n = 23). Only age and plasma transfusion retained statistical significance in the final multivariate outcome model, with 8.5 times lower odds of survival in transfused horses (95% confidence interval [CI], 2.6-27.2%). Additionally, the likelihood of nonsurvival increased by 11.8% (95% CI, 4-20.2%) for every year the horse aged (P = 0.002). Similarly, geriatric horses (≥20 years) were 15.2 times more likely to die than young-adults (2-12 years, P = 0.03), independent of financial investment, documented comorbidities, and duration of hospitalization. Part 2: Select cytokine analyses were performed on serum collected from hospitalized horses within 1 hour of diarrhea onset (T0) and 6 hours later. At T0, all recorded clinicopathological variables were comparable between geriatric and young-adult horses, suggesting a similar degree of systemic illness. The median concentration of type-2 cytokines interleukin-4 and interleukin-10, and type-1 cytokine interferon-γ did not differ between age groups. Inflammatory cytokines interleukin-6 and tumor necrosis factor-α were significantly higher in geriatric compared to young-adult horses at both sampling time points. CONCLUSIONS: Outcome of colitis was less favorable in aging horses and patients receiving a plasma transfusion. Although an exaggerated inflammatory state, based on increased interleukin-6 and tumor necrosis factor-α concentrations, in geriatric horses may contribute to reduced survival, a lower type-1/type-2 cytokines ratio was not identified in our geriatric population.

Eimeria ninakohlyakimovae casts NOX-independent NETosis and induces enhanced IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in caprine PMN.

Eimeria ninakohlyakimovae represents a highly pathogenic coccidian parasite causing severe haemorrhagic typhlocolitis in goat kids worldwide. NETosis was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites in vitro and in vivo. In vitro interactions of caprine PMN with parasitic stages of E. ninakohlyakimovae (i. e. oocysts and sporozoites) as well as soluble oocyst antigens (SOA) were analyzed at different ratios, concentrations and time spans. Extracellular DNA staining was used to illustrate classical molecules induced during caprine NETosis [i. e. histones (H3) and neutrophil elastase (NE)] via antibody-based immunofluorescence analyses. Functional inhibitor treatments with DPI and DNase I were applied to unveil role of NADPH oxidase (NOX) and characterize DNA-backbone composition of E. ninakohlyakimovae-triggered caprine NETosis. Scanning electron microscopy (SEM)- and immunofluorescence-analyses demonstrated that caprine PMN underwent NETosis upon contact with sporozoites and oocysts of E. ninakohlyakimovae, ensnaring filaments which firmly entrapped parasites. Detailed co-localization studies of E. ninakohlyakimovae-induced caprine NETosis revealed presence of PMN-derived DNA being adorned with nuclear H3 and NE corroborating molecular characteristics of NETosis. E. ninakohlyakoimovae-induced caprine NETosis was found to be NOX-independent since DPI inhibition led to a slight decrease of NETosis. Exposure of caprine PMN to vital E. ninakohlyakimovae sporozoites as well as SOA resulted in up-regulation of IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in stimulated PMN. Since vital E. ninakohlyakimovae-sporozoites induced caprine NETosis, this effective entrapment mechanism might reduce initial sporozoite epithelial host cell invasion during goat coccidiosis ultimately resulting in less macromeront formation and reduced merozoites I production.

Mortality, clinical findings, predisposing factors and treatment of Clostridioides difficile colitis in Japanese thoroughbred racehorses.

BACKGROUND: Although Clostridioides difficile-associated diseases (CDAD) is considered to be associated with colitis in horses, few studies have been performed with a focus on the characteristics of CDAD in thoroughbred racehorses. METHODS: Between 2010 and 2018, a test for C. difficile was performed using faecal samples from 137 thoroughbred racehorses with colitis presenting with diarrhoea and fever. The mortality rate, clinical findings, predisposing factors and the selected treatments were investigated in a retrospective manner. RESULTS: Twenty-four cases were diagnosed as CDAD and 113 as non-CDAD. The mortality rate was significantly higher in the CDAD group (83 per cent) than that in the non-CDAD group (34 per cent). The levels of serum amyloid A, blood urea nitrogen and packed cell volume at initial presentation were also significantly higher, and those of total protein and albumin were significantly lower in the CDAD group. The development of CDAD was associated with the administration of antimicrobials, surgery and hospitalisation. No significant improvement in mortality was observed for any of the selected treatment in both groups. CONCLUSION: CDAD in thoroughbred racehorses was identified as a high mortality disease with rapid progression of systemic inflammation and deterioration of the circulatory state. Further investigation is required to improve the treatment.

Detection of Leishmania spp. Infection by Immunohistochemistry in Archived Biopsy Samples from Dogs with Colitis in an Area Endemic for Leishmaniosis.

Previous studies have demonstrated the presence of Leishmania infantum amastigotes in the colonic mucosa of seropositive sick dogs. However, there are no studies that have investigated the presence of L. infantum infection in dogs diagnosed with inflammatory bowel disease (IBD). The aims of this study were: (1) to investigate retrospectively the presence of Leishmania spp. antigen by immunohistochemistry (IHC) in biopsy samples taken from the colon of dogs with IBD in an area endemic for leishmaniosis, and (2) to describe the main histopathological findings in these cases. Clinicopathological data and histopathological results were reviewed from 106 cases of canine colitis. IHC to detect Leishmania spp. antigen had been performed at the time of diagnosis in 13 cases and we performed IHC in 56 more cases. Five of the 69 cases (7.2%) were positive for Leishmania spp. antigen by IHC. Two positive biopsy samples had histiocytic inflammation and three had lymphoplasmacytic inflammation. The number of amastigotes was variable and independent of the type and grade of inflammatory infiltrate. The results suggest that Leishmania spp. infection is associated with chronic colitis in areas endemic for the infection. Therefore, Leishmania IHC should be used routinely as a diagnostic tool when evaluating colonic biopsy samples from dogs in endemic areas, to exclude or confirm an infection by this parasite in dogs with chronic colitis.

Deciphering the Enzymatic Function of the Bovine Enteric Infection-Related Protein YfeJ from Salmonella enterica Serotype Typhimurium.

Non-typhoidal Salmonella can colonize the gastrointestinal system of cattle and can also cause significant food-borne disease in humans. The use of a library of single-gene deletions in Salmonella enterica serotype Typhimurium allowed identification of several proteins that are under selection in the intestine of cattle. STM2437 ( yfeJ) encodes one of these proteins, and it is currently annotated as a type I glutamine amidotransferase. STM2437 was purified to homogeneity, and its catalytic properties with a wide range of γ-glutamyl derivatives were determined. The catalytic efficiency toward the hydrolysis of l-glutamine was extremely weak with a kcat/ Km value of 20 M-1 s-1. γ-l-Glutamyl hydroxamate was identified as the best substrate for STM2437, with a kcat/ Km value of 9.6 × 104 M-1 s-1. A homology model of STM2437 was constructed on the basis of the known crystal structure of a protein of unknown function (Protein Data Bank entry 3L7N ), and γ-l-glutamyl hydroxamate was docked into the active site based on the binding of l-glutamine in the active site of carbamoyl phosphate synthetase. Acivicin was shown to inactivate the enzyme by reaction with the active site cysteine residue and the subsequent loss of HCl. Mutation of Cys91 to serine completely abolished catalytic activity. Inactivation of STM2437 did not affect the ability of this strain to colonize mice, but it inhibited the growth of S. enterica Typhimurium in bacteriologic media containing γ-l-glutamyl hydroxamate.

Gamma-glutamyltranspeptidase expression by Helicobacter saguini, an enterohepatic Helicobacter species isolated from cotton top tamarins with chronic colitis.

BACKGROUND: Helicobacter saguini is a novel enterohepatic Helicobacter species isolated from captive cotton top tamarins with chronic colitis and colon cancer. Monoassociated H. saguini infection in gnotobiotic IL-10-/- mice causes typhlocolitis and dysplasia; however, the virulent mechanisms of this species are unknown. Gamma-glutamyltranspeptidase (GGT) is an enzymatic virulence factor expressed by pathogenic Helicobacter and Campylobacter species that inhibits host cellular proliferation and promotes inflammatory-mediated gastrointestinal pathology. The aim of this study was to determine if H. saguini expresses an enzymatically active GGT homologue with virulence properties. EXPERIMENTAL PROCEDURES: Two putative GGT paralogs (HSGGT1 and HSGGT2) identified in the H. saguini genome were bioinformatically analysed to predict enzymatic functionality and virulence potential. An isogenic knockout mutant strain and purified recombinant protein of HSGGT1 were created to study enzymatic activity and virulence properties by in vitro biochemical and cell culture experiments. RESULTS: Bioinformatic analysis predicted that HSGGT1 has enzymatic functionality and is most similar to the virulent homologue expressed by Helicobacter bilis, whereas HSGGT2 contains putatively inactivating mutations. An isogenic knockout mutant strain and recombinant HSGGT1 protein were successfully created and demonstrated that H. saguini has GGT enzymatic activity. Recombinant HSGGT1 protein and sonicate from wild-type but not mutant H. saguini inhibited gastrointestinal epithelial and lymphocyte cell proliferation without evidence of cell death. The antiproliferative effect by H. saguini sonicate or recombinant HSGGT1 protein could be significantly prevented with glutamine supplementation or the GGT-selective inhibitor acivicin. Recombinant HSGGT1 protein also induced proinflammatory gene expression in colon epithelial cells. CONCLUSIONS: This study shows that H. saguini may express GGT as a potential virulence factor and supports further in vitro and in vitro studies into how GGT expression by enterohepatic Helicobacter species influences the pathogenesis of gastrointestinal inflammatory diseases.

Mice Deficient in Cyp4a14 Have An Increased Number of Goblet Cells and Attenuated Dextran Sulfate Sodium-Induced Colitis.

BACKGROUND/AIMS: Cyp4a14 is a member of cytochrome P450 (Cyp450) enzyme superfamily that possesses NADPH monooxygenase activity, which catalyzes omega-hydroxylation of medium-chain fatty acids and arachidonic acid. Study suggests that down-regulation of Cyp4a14 has an anti-inflammatory response in intestine. The present study was to test the function of Cyp4a14 in dextran sulfate sodium (DSS)-induced colitis. METHODS: Female Cyp4a14-knockout (KO) and wild-type (WT) mice were treated with DSS for 6 days to induce colitis. The colon of mice was histologically observed by hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) staining. The serum malondialdehyde (MDA), an endogenous indicator of oxidative stress, was chemically measured. Proinflammatory and NADPH oxidase genes were examined by quantitative polymerase chain reaction (qPCR). RESULTS: Cyp4a14-KO mice had a significantly higher number of goblet cells in the colon and were more resistant to DSS-induced colitis compared with the WT mice. The DSS-treated KO mice had lower levels of MDA. Consistent with the milder inflammatory pathological changes, DSS-treated KO mice had lower levels of IL-1ß, IL-6 and TNF-α mRNA in the liver and the colon. Moreover, the colon of DSS-treated Cyp4a14-KO and WT mice had higher mRNA levels of two members of NADPH oxidases, Nox2 and Nox4, suggesting that both Nox2 and Nox4 are inflammatory markers. By contrast, DSS-treated WT and KO mice had drastically decreased epithelium-localized Nox1 and dual oxidase (Duox) 2 mRNA levels, coinciding with the erosion of the mucosa induced by DSS. CONCLUSION: These results suggests a hypothesis that the increased goblet cell in the colon of Cyp4a14-KO mice provides protection from mucosal injury and Cyp4a14-increased oxidative stress exacerbates DSS-induced colitis. Therefore, Cyp4a14 may represent a potential target for treating colitis.

Intestinal Epithelial Cell-specific Deletion of α-Mannosidase II Ameliorates Experimental Colitis.

Inflammatory bowel disease (IBD) is a refractory disease of the gastrointestinal tract that is believed to develop in genetically susceptible individuals. Glycosylation, a type of post-translational modification, is involved in the development of a wide range of diseases, including IBD, by modulating the function of various glycoproteins. To identify novel genes contributing to the development of IBD, we analyzed single nucleotide polymorphisms (SNPs) of glycosylation-related genes in IBD patients and identified MAN2A1, encoding alpha-mannosidase II (α-MII), as a candidate gene. α-MII plays a crucial, but not exclusive, role in the maturation of N-glycans. We also observed that intestinal epithelial cells (IECs), which establish the first-line barrier and regulate gut immunity, selectively expressed α-MII with minimal expression of its isozyme, alpha-mannosidase IIx (α-MIIx). This led us to hypothesize that IEC-intrinsic α-MII is implicated in the pathogenesis of IBD. To test this hypothesis, we generated IEC-specific α-MII-deficient (α-MIIΔIEC) mice. Although α-MII deficiency has been shown to have a minimal effect on N-glycan maturation in most cell types due to the compensation by α-MIIx, ablation of α-MII impaired the maturation of N-glycans in IECs. α-MIIΔIEC mice were less susceptible to dextran sulfate sodium-induced colitis compared with control littermates. In accordance with this, neutrophil infiltration in the colonic mucosa was attenuated in α-MIIΔIEC mice. Furthermore, gene expression levels of neutrophil-attracting chemokines were downregulated in the colonic tissue. These results suggest that IEC-intrinsic α-MII promotes intestinal inflammation by facilitating chemokine expression. We propose SNPs in MAN2A1 as a novel genetic factor for IBD.Key words: inflammatory bowel disease, alpha-mannosidase II, intestinal epithelial cell, N-glycosylation.

The protective effects of magnolol on acute trinitrobenzene sulfonic acid­induced colitis in rats.

The present study aimed to investigate the protective effects of magnolol on acute 2,4,6-trinitrobenzene sulfonic acid (TNBS)­induced colitis, and its underlying mechanisms. Experimental colitis was induced by intracolonic administration of TNBS/ethanol into rats. The model rats were randomly assigned into groups: TNBS, magnolol (high, medium and low doses), and salazosulfapyridine (positive control). All intervention regimens were administered by oral gavage, once a day for 7 consecutive days, 24 h after colitis induction. Histological and biochemical changes in colonic inflammation were evaluated by hematoxylin and eosin and immunohistochemistry, respectively. Rats treated with all doses of magnolol exhibited decreased colonic myeloperoxidase activity (P<0.05 vs. TNBS), reduced serum levels of proinflammatory cytokines [including interleukin (IL)­6 and IL­17], and downregulated Toll­like receptor-4 (TLR­4) mRNA expression. Histological analysis revealed that medium and high doses of magnolol conferred an anti­inflammatory effect, which was indicated by a decrease in disease activity index, an increase in thymus index, and downregulation of nuclear factor (NF)­κB p65 mRNA and TLR­4 protein expression. However, only high­dose magnolol significantly ameliorated the elevated colon weight/length ratio. The results of the present study indicate that magnolol exerts protective effects against acute TNBS­induced colitis in rats, and the TLR­4/NF­κB signaling pathway­mediated inhibitory effect on inflammatory cascades may contribute to the protective activity of magnolol.

Whole-Blood Taurine Concentrations in Cats With Intestinal Disease.

BACKGROUND: Increased delivery of taurine-conjugated bile acids to the distal bowel can lead to dysbiosis resulting in colitis in mouse models of inflammatory bowel disease. A similar situation also could occur in cats with intestinal disease and might therefore result in decreased whole-body taurine concentration. HYPOTHESIS/OBJECTIVES: To determine whether whole-blood taurine concentrations are decreased at the time of diagnosis in cats with intestinal disease and to correlate concentrations with clinical and laboratory variables. ANIMALS: Twenty-one cats with chronic inflammatory enteropathy and 7 cats with intestinal neoplasia from the University of Bristol. METHODS: Cats that had undergone a thorough investigation consisting of a CBC, serum biochemistry, serum cobalamin and folate concentrations, transabdominal ultrasound examination and histopathology of intestinal biopsy specimens, as well as additional testing if indicated, were included. Whole-blood from these cats collected at the time of histologic diagnosis and stored in ethylenediaminetetraacetic acid was retrospectively analyzed for taurine with an automated high-performance liquid chromatography amino acid analyzer. RESULTS: Although whole-blood taurine concentrations remained within the reference range, those cats with predominantly large intestinal clinical signs had significantly lower concentrations than did cats with small intestinal and mixed bowel clinical signs (P = 0.033) and this difference also was significant when assessed only in cats with chronic inflammatory enteropathy (P = 0.019). CONCLUSIONS AND CLINICAL IMPORTANCE: Additional studies are needed to determine whether large intestinal signs in cats with chronic inflammatory enteropathy are caused by alterations in the microbiota arising as a consequence of increased delivery of taurine-conjugated bile acids.