Latent Class Analysis for the Diagnosis of Clostridioides difficile Infection.

BACKGROUND: Clostridioides difficile infection (CDI) is an opportunistic disease that lacks a gold-standard test. Nucleic acid amplification tests such as real-time polymerase chain reaction (PCR) demonstrate an excellent limit of detection (LOD), whereas antigenic methods are able to detect protein toxin. Latent class analysis (LCA) provides an unbiased statistical approach to resolving true disease. METHODS: A cross-sectional study was conducted in patients with suspected CDI (N = 96). Four commercial real-time PCR tests, toxin antigen detection by enzyme immunoassay (EIA), toxigenic culture, and fecal calprotectin were performed. CDI clinical diagnosis was determined by consensus majority of 3 experts. LCA was performed using laboratory and clinical variables independent of any gold standard. RESULTS: Six LCA models were generated to determine CDI probability using 4 variables including toxin EIA, toxigenic culture, clinical diagnosis, and fecal calprotectin levels. Three defined zones as a function of real-time PCR cycle threshold (Ct) were identified using LCA: CDI likely (>90% probability), CDI equivocal (<90% and >10%), CDI unlikely (<10%). A single model comprising toxigenic culture, clinical diagnosis, and toxin EIA showed the best fitness. The following Ct cutoffs for 4 commercial test platforms were obtained using this model to delineate 3 CDI probability zones: GeneXpert®: 24.00, 33.61; Simplexa®: 28.97, 36.85; Elite MGB®: 30.18, 37.43; and BD Max™: 27.60, 34.26. CONCLUSIONS: The clinical implication of applying LCA to CDI is to report Ct values assigned to probability zones based on the commercial real-time PCR platform. A broad range of equivocation suggests clinical judgment is essential to the confirmation of CDI.

Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Macrophages play central roles in immunity as early effectors and modulating adaptive immune reponses; we implicated macrophages in the anticolitic effect of infection with the tapeworm Hymenolepis diminuta. Here, gene arrays revealed that H. diminuta antigen (HdAg) evoked a program in murine macrophages distinct from that elicited by IL-4. Further, HdAg suppressed LPS-evoked release of TNF-α and IL-1ß from macrophages via autocrine IL-10 signaling. In assessing the ability of macrophages treated in vitro with an extract of H. diminuta [M(HdAg)] to affect disease, intravenous, but not peritoneal, injection of M(HdAg) protected wild-type but not RAG1-/- mice from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Administration of splenic CD4+ T cells from in vitro cocultures with M(HdAg), but not those cocultured with M(IL-4) cells, inhibited DNBS-induced colitis; fractionation of the T-cell population indicated that the CD4+CD25+ T cells from cocultures with M(HdAg) drove the suppression of DNBS-induced colitis. Use of IL-4-/- or IL-10-/- CD4+ T cells revealed that neither cytokine alone from the donor cells was essential for the anticolitic effect. These data illustrate that HdAg evokes a unique regulatory program in macrophages, identifies HdAg-evoked IL-10 suppression of macrophage activation, and reveals the ability of HdAg-treated macrophages to educate ( i.e., condition) and mobilize CD4+CD25+ T cells, which could be deployed to treat colonic inflammation.-Reyes, J. L., Lopes, F., Leung, G., Jayme, T. S., Matisz, C. E., Shute, A., Burkhard, R., Carneiro, M., Workentine, M. L., Wang, A., Petri, B., Beck, P. L., Geuking, M. B., McKay, D. M., Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Crohn's-like disease in a patient exposed to anti-Interleukin-17 blockade (Ixekizumab) for the treatment of chronic plaque psoriasis: a case report.

BACKGROUND: Plaque psoriasis and inflammatory bowel disease (IBD) are both chronic immune-mediated inflammatory diseases with an overlapping genetic profile and have been linked in epidemiological studies. Psoriasis and IBD share similar components in their inflammatory pathways and animal and human studies have suggested a potential role for targeting interleukin (IL)-17 with novel antibody therapies in the treatment of these diseases. These studies, while promising for psoriasis, have been associated with deterioration in patients with IBD. Post-hoc analyses of clinical trials involving Ixekizumab revealed adverse outcomes in a small cluster of patients with IBD, prompting recommendations to monitor this population with the use of this drug. CASE PRESENTATION: Forty-two year old Caucasian male with treatment-refractory chronic plaque psoriasis who developed new onset diarrheal illness and rectal bleeding following a 12 week induction period with Ixekizumab (anti-IL-17 neutralizing antibody). Colonoscopy revealed severe ulceration throughout the ascending and transcending colon. Histopathology, combined with endoscopic findings, led to a diagnosis of Crohn's-like colitis. The patient's anti-IL-17 medication was discontinued and endoscopic remission was induced with the use of corticosteroids, escalated anti-TNF therapy and eventually anti IL-12/23 neutralizing antibody (ustekinumab). CONCLUSION: Murine studies implicate IL-17 and the downstream effects of its inhibition, in the breakdown of the gut epithelial layer, the disruption of normal host immune responses and the propagation of intestinal inflammation. The increasing use of IL-17 inhibitors has led to reports of exacerbation and potential development of inflammatory bowel disease. While clinical trials have revealed clusters of new inflammatory bowel disease cases amongst psoriasis patients using an IL-17 inhibitor, there remains a lack of evidence to suggest a causal relationship. This is the first case report of de-novo severe Crohn's-like IBD in association with the use of Ixekizumab requiring rescue with escalated dosing of anti-TNF therapy and highlights the importance of close monitoring in patients being treated with IL-17 inhibitors, especially in those patients with known risk factors for inflammatory bowel disease.

Another Whipple's triad? Pericardial, myocardial and valvular disease in an unusual case presentation from a Canadian perspective.

BACKGROUND: Whipple's disease is a clinically relevant multi-system disorder that is often undiagnosed given its elusive nature. We present an atypical case of Whipple's disease involving pan-valvular endocarditis and constrictive pericarditis, requiring cardiac intervention. A literature review was also performed assessing the prevalence of atypical cases of Whipple's disease. CASE PRESENTATION: A previously healthy 56-year-old male presented with a four-year history of congestive heart failure with weight loss and fatigue. Notably, he had absent gastrointestinal symptoms. He went on to develop pan-valvular endocarditis and constrictive pericarditis requiring urgent cardiac surgery. A clinical diagnosis of Whipple's disease was suspected, prompting duodenal biopsy sampling which was unremarkable, Subsequently, Tropheryma whipplei was identified by 16S rDNA PCR on the cardiac valvular tissue. He underwent prolonged antibiotic therapy with recovery of symptoms. CONCLUSIONS: Our study reports the first known case of Whipple's disease involving pan-valvular endocarditis and constrictive pericarditis. A literature review also highlights this presentation of atypical Whipple's with limited gastrointestinal manifestations. Duodenal involvement was limited and the gold standard of biopsy was not contributory. We also highlight the Canadian epidemiology of the disease from 2012 to 2016 with an approximate 4% prevalence rate amongst submitted samples. Routine investigations for Whipple's disease, including duodenal biopsy, in this case may have missed the diagnosis. A high degree of suspicion was critical for diagnosis of unusual manifestations of Whipple's disease.

Proceedings from the 8th Annual University of Calgary Leaders in Medicine Research Symposium.

On November 14, 2016, the Leaders in Medicine (LIM) program at the Cumming School of Medicine, University of Calgary hosted its 8th Annual Research Symposium. Professor Stephen Sawcer, Professor of Neurological Genetics at the University of Cambridge and an Honorary Consultant Neurologist at Addenbrooke's Hospital, was the keynote speaker and presented a lecture entitled, "Multiple sclerosis genetics - prospects and pitfalls". This was not only a cutting edge address on genetics but also a thoughtful overview on Dr. Sawcer's career and career choices. We were extremely grateful for the opportunity to have Dr. Sawcer participate in our annual symposium.

Macrophage Uptake of Necrotic Cell DNA Activates the AIM2 Inflammasome to Regulate a Proinflammatory Phenotype in CKD.

Nonmicrobial inflammation contributes to CKD progression and fibrosis. Absent in melanoma 2 (AIM2) is an inflammasome-forming receptor for double-stranded DNA. AIM2 is expressed in the kidney and activated mainly by macrophages. We investigated the potential pathogenic role of the AIM2 inflammasome in kidney disease. In kidneys from patients with diabetic or nondiabetic CKD, immunofluorescence showed AIM2 expression in glomeruli, tubules, and infiltrating leukocytes. In a mouse model of unilateral ureteral obstruction (UUO), Aim2 deficiency attenuated the renal injury, fibrosis, and inflammation observed in wild-type (WT) littermates. In bone marrow chimera studies, UUO induced substantially more tubular injury and IL-1ß cleavage in Aim2-/- or WT mice that received WT bone marrow than in WT mice that received Aim2-/- bone marrow. Intravital microscopy of the kidney in LysM(gfp/gfp) mice 5-6 days after UUO demonstrated the significant recruitment of GFP+ proinflammatory macrophages that crawled along injured tubules, engulfed DNA from necrotic cells, and expressed active caspase-1. DNA uptake occurred in large vacuolar structures within recruited macrophages but not resident CX3CR1+ renal phagocytes. In vitro, macrophages that engulfed necrotic debris showed AIM2-dependent activation of caspase-1 and IL-1ß, as well as the formation of AIM2+ ASC specks. ASC specks are a hallmark of inflammasome activation. Cotreatment with DNaseI attenuated the increase in IL-1ß levels, confirming that DNA was the principal damage-associated molecular pattern in this process. Therefore, the activation of the AIM2 inflammasome by DNA from necrotic cells drives a proinflammatory phenotype that contributes to chronic injury in the kidney.

Inhibition of Intestinal Epithelial Wound Healing through Protease-Activated Receptor-2 Activation in Caco2 Cells.

The mechanisms of epithelial wound healing are not completely understood, especially in the context of proteases and their receptors. It was recently shown that activation of protease-activated receptor-2 (PAR2) on intestinal epithelial cells induced the expression of cyclooxygenase-2 (COX-2), which has protective functions in the gastrointestinal tract. It was hypothesized that PAR2-induced COX-2 could enhance wound healing in intestinal epithelial cells. Caco2 cells were used to model epithelial wound healing of circular wounds. Cellular proliferation was studied with a 5-ethynyl-2'-deoxyuridine assay, and migration was studied during wound healing in the absence of proliferation. Immunofluorescence was used to visualize E-cadherin and F-actin, and the cellular transcription profile during wound healing and PAR2 activation was explored with RNA sequencing. PAR2 activation inhibited Caco2 wound healing by reducing cell migration, independently of COX-2 activity. Interestingly, even though migration was reduced, proliferation was increased. When the actin dynamics and cell-cell junctions were investigated, PAR2 activation was found to induce actin cabling and prevent the internalization of E-cadherin. To further investigate the effect of PAR2 on transcriptionally dependent wound healing, RNA sequencing was performed. This analysis revealed that PAR2 activation, in the absence of wounding, induced a similar transcriptional profile compared with wounding alone. These findings represent a novel effect of PAR2 activation on the mechanisms of epithelial cell wound healing that could influence the resolution of intestinal inflammation.

The Need for an Executive Leadership Curriculum in Scientist-Clinician Training Programs.

The health of Canadians depends on effective leadership among health care providers to facilitate the translation of new health discoveries into clinical practice. Clinician-scientists play an important role in bridging the gap between research and clinical practice, and require effective leadership skills to advance clinical practice successfully. To accelerate the leadership development in clinician scientist trainees, with the aim of developing strong leaders in administration and health advocacy, the Leaders in Medicine (LIM) training program at the University of Calgary created an Executive Leadership Coaching Program involving three phases: 1) an evidence-based evaluation tool, the Core Values IndexTM (CVI), that was used to identify the key drivers behind how individuals can be most effective in making their contribution; 2) small group workshops to debrief the results of the CVI assessment; and 3) one-on-one executive coaching sessions to facilitate the discovery, development and deployment of individual leadership capabilities. Coaching in leadership strategies enables clinician-scientist trainees to lead, influence, manage and deliver science-based improvements into the practice of medicine. We strongly recommend that other Canadian scientist-clinician training programs consider opportunities like the ones we offer to our LIM trainees. This training has important implications for the delivery of healthcare in Canada.

Combining the Love of Art, Science, Business and Medicine to Innovate and Enhance Patient Health. Highlights from the 7th Annual Leaders in Medicine Symposium of the Cummings School of Medicine, Calgary, AB.

The Leader in Medicine (LIM) Program of the Cumming School of Medicine, University of Calgary, hosted its 7th Annual LIM Research Symposium on October 30, 2015 and participation grew once again, with a total of six oral and 99 posters presentations! Over 45 of our Faculty members also participated in the symposium. This year's LIM Symposium theme was "Innovations in Medicine" and the invited guest speaker was our own Dr. Breanne Everett (MD/MBA). She completed her residency in plastic surgery at University of Calgary and holds both a medical degree and an MBA from the University of Calgary. In her inspiring talk, entitled "Marrying Business and Medicine: Toe-ing a Fine Line", she described how she dealt with a clinical problem (diabetic foot ulcers), came up with an innovation that optimized patient care, started her own company and delivered her product to market to enhance the health of the community. She clearly illustrated how to complete the full circle, from identifying a clinical problem to developing and providing a solution that both enhances clinical care and patient health as well as reduces health care costs and hospital admissions. The research symposium was an outstanding success and the abstracts are included in companion article in CIM.

Proceedings from the 7th Annual University of Calgary Leaders in Medicine Research Symposium.

TOn October 30th, 2015, the Leaders in Medicine (LIM) program at the Cumming School of Medicine, University of Calgary hosted its 7th Annual Research Symposium. Dr. Breanne Everett, President and CEO of Orpyx Medical Technologies and holder both of medical and MBA degrees from the University of Calgary, presented a lecture entitled "Marrying Business and Medicine: Toe-ing a Fine Line". The LIM symposium also provides a forum for both LIM and non-LIM medical students to present their research work in oral and poster presentation formats. This year over 100 students submitted their work and six oral presentations and 99 posters were presented. The oral presentations were as follows: Ryan Lewinson, Prediction of wedged insole-induced changes to knee joint moments during walkingLindsey Logan, Robotic measures provide insight on sensorimotor and cognitive impairments following traumatic brain injury Jackie Mann, What medication information do community doctors want to receive in discharge summaries for safer transfers? Ashley Jensen, Increased mortality associated with resident handoff periods at ten veterans administration medical centers Jason Bau, Keratinocyte growth factor protects against C. difficile-induced cell injury and death Michael Keough, A novel drug class promotes regeneration of central nervous system myelin by overcoming inhibitory scar molecules in vitro and in vivo For further details on the University of Calgary Leaders in Medicine Program see "A Prescription that Addresses the Decline of Basic Science Education in Medical School" (Clinical and Investigative Medicine. 2014;37(5):E29). The LIM Symposium has the following objectives: (1) to showcase the variety of projects undertaken by students in the LIM Program as well as University of Calgary medical students; (2) to encourage medical student participation in research and special projects; (3) to inform students and faculty about the diversity of opportunities available for research and special projects during medical school and beyond; and, (4) to enhance student and staff interactions, with the ultimate goal being to enhance translational medicine improve health.

Differential expression of LEF1/TCFs family members in colonic carcinogenesis.

Lef1/Tcfs family, which includes Lef1, Tcf1, Tcf3, and Tcf4, is required for the transcriptional activation induced by ß-catenin. However, whether all the members play the same role in colon carcinogenesis is not clear. We found that Lef1 and Tcf1, but not Tcf3 and Tcf4, were upregulated at both mRNA and protein level with the formation of colon tumor in AOM-DSS mouse model. The same profiles were seen in human specimens with the evolvement from adenoma to adenocarcinoma. Additionally, Lef1 and Tcf1 were correlated with a subgroup of Wnt target genes, including Lgr5, a key gene of intestinal stem cell. Further studies supported the role of Tcf1 on sphere formation and transcriptional regulation of Lgr5 in vitro. Interestingly, 3' UTR of each Lef1/Tcfs member were targeted by diverse miRNAs, which were negatively correlated with respective member in human colon cancer specimens. Furthermore, these miRNAs were verified to repress Tcf1 and Lef1 in vitro. Taken together, Lef1 and Tcf1 showed oncogenic effect in colonic carcinogenesis. Cellular context of miRNAs might play important roles in carcinogenesis by altering the expression pattern of Lef/Tcfs members. © 2017 Wiley Periodicals, Inc.

Fast core rotation in red-giant stars as revealed by gravity-dominated mixed modes.

When the core hydrogen is exhausted during stellar evolution, the central region of a star contracts and the outer envelope expands and cools, giving rise to a red giant. Convection takes place over much of the star's radius. Conservation of angular momentum requires that the cores of these stars rotate faster than their envelopes; indirect evidence supports this. Information about the angular-momentum distribution is inaccessible to direct observations, but it can be extracted from the effect of rotation on oscillation modes that probe the stellar interior. Here we report an increasing rotation rate from the surface of the star to the stellar core in the interiors of red giants, obtained using the rotational frequency splitting of recently detected 'mixed modes'. By comparison with theoretical stellar models, we conclude that the core must rotate at least ten times faster than the surface. This observational result confirms the theoretical prediction of a steep gradient in the rotation profile towards the deep stellar interior.

Gravity modes as a way to distinguish between hydrogen- and helium-burning red giant stars.

Red giants are evolved stars that have exhausted the supply of hydrogen in their cores and instead burn hydrogen in a surrounding shell. Once a red giant is sufficiently evolved, the helium in the core also undergoes fusion. Outstanding issues in our understanding of red giants include uncertainties in the amount of mass lost at the surface before helium ignition and the amount of internal mixing from rotation and other processes. Progress is hampered by our inability to distinguish between red giants burning helium in the core and those still only burning hydrogen in a shell. Asteroseismology offers a way forward, being a powerful tool for probing the internal structures of stars using their natural oscillation frequencies. Here we report observations of gravity-mode period spacings in red giants that permit a distinction between evolutionary stages to be made. We use high-precision photometry obtained by the Kepler spacecraft over more than a year to measure oscillations in several hundred red giants. We find many stars whose dipole modes show sequences with approximately regular period spacings. These stars fall into two clear groups, allowing us to distinguish unambiguously between hydrogen-shell-burning stars (period spacing mostly ∼ 50 seconds) and those that are also burning helium (period spacing ∼ 100 to 300 seconds).

Microscopic Colitis Evolved Into Inflammatory Bowel Diseases Is Characterized by Increased Th1/Tc1 Cells in Colonic Mucosal Lamina Propria.

BACKGROUND: An association between microscopic colitis (MC), i.e., lymphocytic colitis (LC) and collagenous colitis (CC), and inflammatory bowel diseases (IBD) has been noticed. A subset of MC cases may evolve into IBD, and IBD in remission may present as MC in a histologic pattern. Moreover, MC and IBD may coexist in different regions of the bowel. A link between MC and IBD in their pathogenesis is, therefore, suggested. Abnormal mucosal immunity is likely the key. METHODS: We reviewed 2324 MC cases in Calgary over 14 years and identified 20 cases evolved into IBD (IBD transformers). 13 of them were further investigated for colonic mucosal lamina propria mononuclear cells (LPMNCs), as opposed to 22 cases whose MC resolved. On their index colonic biopsy immunohistochemistry was performed to detect major T cell subsets characterized by key cytokines and master transcription factors (IFNγ and T-bet for Th1/Tc1, GATA-3 for Th2/Tc2, IL-17 and RORc for Th17/Tc17, FoxP3 for Treg/Tcreg) as well as TNFα+ cells (partly representing Th1). LPMNCs positive for each marker were counted (average number per high-power field). RESULTS: IBD transformers had increased IFNγ+, T-bet+, TNF-α+, and GATA-3+ LPMNCs compared to the MC-resolved cases. The LC-to-IBD subgroup had increased IFNγ+ and GATA-3+ cells compared to the LC-resolved subgroup. The CC-to-IBD subgroup had increased T-bet+, TNF-α+, and GATA-3+ cells compared to the CC-resolved subgroup. Among MC-resolved patients, more TNF-α+ and RORc+ cells were seen in LC than in CC. CONCLUSION: Th1/Tc1- and TNFα-producing cells, and likely a subset of Th2/Tc2 cells as well, may be involved in the MC-to-IBD transformation.

Crossover Subsets of CD4+ T Lymphocytes in the Intestinal Lamina Propria of Patients with Crohn's Disease and Ulcerative Colitis.

BACKGROUND: Hovhannisyan et al. first showed evidence of plasticity between Treg and Th17 in the inflamed intestine of Crohn's disease (CD) patients. Our previous report suggests that the inflammatory cytokine milieu generates IL-17+ Foxp3+ CD4+ T lymphocytes which is a crossover population converting Treg subset to Th17 in the peripheral blood of IBD patients. This is considered as an evidence of Treg/Th17 plasticity. AIM: The aim of this study was to characterize a variety of helper T cell crossover population, not limited to IL-17+ Foxp3+ CD4+ T lymphocytes, in the lamina propria (LP) of IBD patients. METHODS: Fresh colonoscopic biopsies were obtained from patients with CD (n = 50) and ulcerative colitis (UC, n = 32) and from healthy controls (HC, n = 25). LP mononuclear cells were assessed for intracellular cytokines and transcription factors such as IFNγ, IL-13, IL-17, IL-22, T-bet, Gata-3, RORγt, and Foxp3 using multicolor flow cytometry to detect subsets of LP CD4+ T lymphocytes. RESULTS: Patients with IBD demonstrated increased crossover populations in IL-17+ Foxp3+, T-bet+ Foxp3+, Gata3+ Foxp3+, RORγt+ Foxp3+ populations compared to HC. There was an inverse correlation of Harvey-Bradshaw index with Gata3+ Foxp3+ population in CD patients, while IL-13+ Foxp3+ population was directly correlated with Mayo clinical scores in UC patients. Furthermore, total IL-22 expressing cells as well as Th22 and IL-22+ Th1 populations were decreased in UC compared to CD and HC. CONCLUSION: IBD patients exhibit the increased crossover populations in LP Treg cells toward Th2 and Th17 compared to HC. The prevalence of Treg/Th2 crossover populations is associated with clinical disease score of IBD.

Effect of Oral Capsule- vs Colonoscopy-Delivered Fecal Microbiota Transplantation on Recurrent Clostridium difficile Infection: A Randomized Clinical Trial.

Importance: Fecal microbiota transplantation (FMT) is effective in preventing recurrent Clostridium difficile infection (RCDI). However, it is not known whether clinical efficacy differs by route of delivery. Objective: To determine whether FMT by oral capsule is noninferior to colonoscopy delivery in efficacy. Design, Setting, and Participants: Noninferiority, unblinded, randomized trial conducted in 3 academic centers in Alberta, Canada. A total of 116 adult patients with RCDI were enrolled between October 2014 and September 2016, with follow-up to December 2016. The noninferiority margin was 15%. Interventions: Participants were randomly assigned to FMT by capsule or by colonoscopy at a 1:1 ratio. Main Outcomes and Measures: The primary outcome was the proportion of patients without RCDI 12 weeks after FMT. Secondary outcomes included (1) serious and minor adverse events, (2) changes in quality of life by the 36-Item Short Form Survey on a scale of 0 (worst possible quality of life) to 100 (best quality of life), and (3) patient perception on a scale of 1 (not at all unpleasant) to 10 (extremely unpleasant) and satisfaction on a scale of 1 (best) to 10 (worst). Results: Among 116 patients randomized (mean [SD] age, 58 [19] years; 79 women [68%]), 105 (91%) completed the trial, with 57 patients randomized to the capsule group and 59 to the colonoscopy group. In per-protocol analysis, prevention of RCDI after a single treatment was achieved in 96.2% in both the capsule group (51/53) and the colonoscopy group (50/52) (difference, 0%; 1-sided 95% CI, -6.1% to infinity; P < .001), meeting the criterion for noninferiority. One patient in each group died of underlying cardiopulmonary illness unrelated to FMT. Rates of minor adverse events were 5.4% for the capsule group vs 12.5% for the colonoscopy group. There was no significant between-group difference in improvement in quality of life. A significantly greater proportion of participants receiving capsules rated their experience as "not at all unpleasant" (66% vs 44%; difference, 22% [95% CI, 3%-40%]; P = .01). Conclusions and Relevance: Among adults with RCDI, FMT via oral capsules was not inferior to delivery by colonoscopy for preventing recurrent infection over 12 weeks. Treatment with oral capsules may be an effective approach to treating RCDI. Trial Registration: clinicaltrials.gov Identifier: NCT02254811.

Inhibiting Inducible Nitric Oxide Synthase in Enteric Glia Restores Electrogenic Ion Transport in Mice With Colitis.

BACKGROUND & AIMS: Disturbances in the control of ion transport lead to epithelial barrier dysfunction in patients with colitis. Enteric glia regulate intestinal barrier function and colonic ion transport. However, it is not clear whether enteric glia are involved in epithelial hyporesponsiveness. We investigated enteric glial regulation of ion transport in mice with trinitrobenzene sulfonic acid- or dextran sodium sulfate-induced colitis and in Il10(-/-) mice. METHODS: Electrically evoked ion transport was measured in full-thickness segments of colon from CD1 and Il10(-/-) mice with or without colitis in Ussing chambers. Nitric oxide (NO) production was assessed using amperometry. Bacterial translocation was investigated in the liver, spleen, and blood of mice. RESULTS: Electrical stimulation of the colon evoked a tetrodotoxin-sensitive chloride secretion. In mice with colitis, ion transport almost completely disappeared. Inhibiting inducible NO synthase (NOS2), but not neuronal NOS (NOS1), partially restored the evoked secretory response. Blocking glial function with fluoroacetate, which is not a NOS2 inhibitor, also partially restored ion transport. Combined NOS2 inhibition and fluoroacetate administration fully restored secretion. Epithelial responsiveness to vasoactive intestinal peptide was increased after enteric glial function was blocked in mice with colitis. In colons of mice without colitis, NO was produced in the myenteric plexus almost completely via NOS1. NO production was increased in mice with colitis, compared with mice without colitis; a substantial proportion of NOS2 was blocked by fluoroacetate administration. Inhibition of enteric glial function in vivo reduced the severity of trinitrobenzene sulfonic acid-induced colitis and associated bacterial translocation. CONCLUSIONS: Increased production of NOS2 in enteric glia contributes to the dysregulation of intestinal ion transport in mice with colitis. Blocking enteric glial function in these mice restores epithelial barrier function and reduces bacterial translocation.

Ulcerative Colitis Patients With Clostridium difficile are at Increased Risk of Death, Colectomy, and Postoperative Complications: A Population-Based Inception Cohort Study.

OBJECTIVES: Clostridium difficile (C. difficile) may worsen the prognosis of ulcerative colitis (UC). The objectives of this study were to: (i) validate the International Classification of Diseases-10 (ICD-10) code for C. difficile; (ii) determine the risk of C. difficile infection after diagnosis of UC; (iii) evaluate the effect of C. difficile infection on the risk of colectomy; and (iv) assess the association between C. difficile and postoperative complications. METHODS: The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated by comparing ICD-10 codes for C. difficile with stool toxin tests. A population-based surveillance cohort of newly diagnosed UC patients living in Alberta, Canada were identified from 2003 to 2009 (n=1,754). The effect of a C. difficile infection on colectomy was modeled using competing risk survival regression after adjusting for covariates. The effect of a C. difficile infection on postoperative complications was assessed using a mixed effects logistic regression model. RESULTS: The sensitivity, specificity, PPV, and NPV of the ICD-10 code for C. difficile were 82.1%, 99.4%, 88.4%, and 99.1%, respectively. The risk of C. difficile infection within 5 years of diagnosis with UC was 3.4% (95% confidence interval (CI): 2.5-4.6%). The risk of colectomy was higher among UC patients diagnosed with C. difficile (sub-hazard ratio (sHR)=2.36; 95% CI: 1.47-3.80). C. difficile increased the risk of postoperative complications (odds ratio=4.84; 95% CI: 1.28-18.35). C. difficile was associated with mortality (sHR=2.56 times; 95% CI: 1.28-5.10). CONCLUSIONS: C. difficile diagnosis worsens the prognosis of newly diagnosed patients with UC by increasing the risk of colectomy, postoperative complications, and death.

Exaggerated IL-15 and Altered Expression of foxp3+ Cell-Derived Cytokines Contribute to Enhanced Colitis in Nlrp3-/- Mice.

The pathogenesis of Crohn's disease (CD) involves defects in the innate immune system, impairing responses to microbes. Studies have revealed that mutations NLRP3 are associated with CD. We reported previously that Nlrp3-/- mice were more susceptible to colitis and exhibited reduced colonic IL-10 expression. In the current study, we sought to determine how the loss of NLRP3 might be altering the function of regulatory T cells, a major source of IL-10. Colitis was induced in wild-type (WT) and Nlrp3-/- mice by treatment with dextran sulphate sodium (DSS). Lamina propria (LP) cells were assessed by flow cytometry and cytokine expression was assessed. DSS-treated Nlrp3-/- mice exhibited increased numbers of colonic foxp3+ T cells that expressed significantly lower levels of IL-10 but increased IL-17. This was associated with increased expression of colonic IL-15 and increased surface expression of IL-15 on LP dendritic cells. Neutralizing IL-15 in Nlrp3-/- mice attenuated the severity of colitis, decreased the number of colonic foxp3+ cells, and reduced the colonic expression of IL-12p40 and IL-17. These data suggest that the NLRP3 inflammasome can regulate intestinal inflammation through noncanonical mechanisms, providing additional insight as to how NLRP3 variants may contribute to the pathogenesis of CD.

TRPM8 activation attenuates inflammatory responses in mouse models of colitis.

Transient Receptor Potential Melastatin-8 (TRPM8), a recently identified member of the transient receptor potential (TRP) family of ion channels, is activated by mild cooling and by chemical compounds such as the supercooling agent, icilin. Since cooling, possibly involving TRPM8 stimulation, diminishes injury-induced peripheral inflammation, we hypothesized that TRPM8 activation may also attenuate systemic inflammation. We thus studied the involvement of TRPM8 in regulating colonic inflammation using two mouse models of chemically induced colitis. TRPM8 expression, localized immunohistochemically in transgenic TRPM8(GFP) mouse colon, was up-regulated in both human- and murine-inflamed colon samples, as measured by real-time PCR. Wild-type mice (but not TRPM8-nulls) treated systemically with the TRPM8 agonist, icilin showed an attenuation of chemically induced colitis, as reflected by a decrease in macroscopic and microscopic damage scores, bowel thickness, and myeloperoxidase activity compared with untreated animals. Furthermore, icilin treatment reduced the 2,4,6-trinitrobenzenesulfonic acid-induced increase in levels of inflammatory cytokines and chemokines in the colon. In comparison with wild-type mice, Dextran Sodium Sulfate (DSS)-treated TRPM8 knockout mice showed elevated colonic levels of the inflammatory neuropeptide calcitonin-gene-related peptide, although inflammatory indices were equivalent for both groups. Further, TRPM8 activation by icilin blocked capsaicin-triggered calcitonin-gene-related peptide release from colon tissue ex vivo and blocked capsaicin-triggered calcium signaling in Transient Receptor Potential Vaniloid-1 (TRPV1) and TRPM8 transfected HEK cells. Our data document an anti-inflammatory role for TRPM8 activation, in part due to an inhibiton of neuropeptide release, pointing to a novel therapeutic target for colitis and other inflammatory diseases.