Shuji Mitsuhashi

Low Serum Vitamin D During Remission Increases Risk of Clinical Relapse in Patients With Ulcerative Colitis

BACKGROUND & AIMS: Vitamin D levels have been associated with disease activity in patients with ulcerative colitis (UC), but it is unclear whether they affect the risk of disease relapse. We sought to determine the association between baseline vitamin D levels during a period of clinical remission and risk of subsequent UC relapse. METHODS: We performed a physician‐blinded prospective study of 70 patients with UC in clinical remission followed up after a surveillance colonoscopy at a tertiary academic medical center. Serum samples were collected at the time of colonoscopy and baseline endoscopic and histologic activity were determined. Levels of 25‐hydroxy‐vitamin D were measured using an enzyme‐linked immunosorbent assay. The primary outcome was rate of clinical relapse, determined over 12 months. RESULTS: The mean baseline vitamin D level was lower among patients with relapse (29.5 ng/mL) than without (50.3 ng/mL) (P = .001). Remission vitamin D level (≤35 ng/mL) was associated with a risk of clinical relapse (odds ratio, 1.25; 95% confidence interval [CI], 1.01–1.56; P = .044) over 12 months, independent of endoscopic or histologic grade at enrollment. A receiver operating characteristic curve of vitamin D levels for the outcome of relapse had an area under the curve of 0.72; and a serum level of 35 ng/mL or less had a sensitivity of 70% (95% CI, 46%–88%) and a specificity of 74% (95% CI 57%–83%) for predicting risk of clinical relapse. CONCLUSIONS: Serum levels of vitamin D of 35 ng/mL or less during periods of clinical remission increase the risk of UC relapse. Clinical trials to obtain vitamin D levels higher than this threshold should be considered.

Luminal Extracellular Vesicles (EVs) in Inflammatory Bowel Disease (IBD) Exhibit Proinflammatory Effects on Epithelial Cells and Macrophages.

Background:Extracellular vesicles (EVs) are membrane-enclosed particles released by cells as a means of intercellular communication. They are potential novel biomarkers, as they are readily isolated from body fluids, and their composition reflects disease pathways. Whether these particles are released from sites of intestinal inflammation in inflammatory bowel disease (IBD) has not previously been determined. Methods:EVs were isolated by ultracentrifugation of colonic luminal fluid aspirates and characterized according to surface proteins, and constituent mRNA and proteins. The effects of EVs on colonic epithelial cells and macrophages in culture were assessed at the transcriptional, translational, and functional levels. Results:Intestinal luminal aspirates contained abundant EVs, at a mean concentration of 4.3 × 1011 particles/mL and with a mean diameter of 146 nm. EVs from patients with IBD with a high endoscopic score (≥1) contained significantly higher mRNA and protein levels of interleukin 6 (IL-6), IL-8, IL-10, and tumor necrosis factor &agr; than EVs from healthy controls. EVs were absorbed by cultured colonic epithelial cells, leading to an increased translation of IL-8 protein by recipient cells when treated with EVs from patients with IBD. EVs and EV-treated epithelial cells induced migration of a significantly greater number of macrophages than epithelial cells alone. Conclusions:EVs shed from sites of intestinal inflammation in patients with IBD have a distinct mRNA and protein profile from those of healthy individuals. These EVs have proinflammatory effects on the colonic epithelium, in vitro. Their stability in luminal samples and their mRNA and protein content identify them as a potential fecal biomarker that reflects mucosal inflammatory pathways.

Characterizing Normal Bowel Frequency and Consistency in a Representative Sample of Adults in the United States (NHANES)

Objectives:Our current understanding of normal bowel patterns in the United States (US) is limited. Available studies have included individuals with both normal and abnormal bowel patterns, making it difficult to characterize normal bowel patterns in the US. The current study aims to (1) examine frequency and consistency in individuals with self-reported normal bowel habits and (2) determine demographic factors associated with self-reported normalcy.Methods:This study used data from adult participants who completed bowel health questions as part of the National Health and Nutrition Examination Survey (NHANES) in 2009–2010 and who reported normal bowel patterns (N=4,775). Data regarding self-perceived bowel health; stool frequency; stool consistency (using the Bristol Stool Form Scale (BSFS)); and demographic factors were analyzed.Results:95.9% of the sample reported between 3 and 21 BMs per week. Among men, 90% reported a BSFS between 3 and 5, while for women it was 2–6. After controlling for age, the following demographic variables were associated with normalcy: male sex, higher education, higher income, <2 daily medications, and high daily fiber intake. Hispanic ethnicity was significantly associated with abnormal self-reported bowel habits.Conclusions:This is the first study to evaluate normal bowel frequency and consistency in a representative sample of adults in the US. The current findings bolster the common “3 and 3” metric of normal frequency (3 BMs/day to 3 BMs/week) while also suggesting different criteria for normal consistency for men and women. Finally, this study provides novel information about demographic factors associated with normal frequency and consistency.

Bilirubin suppresses Th17 immunity in colitis by upregulating CD39

Unconjugated bilirubin (UCB), a product of heme oxidation, has known immunosuppressant properties but the molecular mechanisms, other than antioxidant effects, remain largely unexplored. We note that UCB modulates T helper type 17 (Th17) immune responses, in a manner dependent upon heightened expression of CD39 ectonucleotidase. UCB has protective effects in experimental colitis, where it enhances recovery after injury and preferentially boosts IL-10 production by colonic intraepithelial CD4+ cells. In vitro, UCB confers immunoregulatory properties on human control Th17 cells, as reflected by increased levels of FOXP3 and CD39 with heightened cellular suppressor ability. Upregulation of CD39 by Th17 cells is dependent upon ligation of the aryl hydrocarbon receptor (AHR) by UCB. Genetic deletion of CD39, as in Entpd1-/- mice, or dysfunction of AHR, as in Ahrd mice, abrogates these UCB salutary effects in experimental colitis. However, in inflammatory bowel disease (IBD) samples, UCB fails to confer substantive immunosuppressive properties upon Th17 cells, because of decreased AHR levels under the conditions tested in vitro. Immunosuppressive effects of UCB are mediated by AHR resulting in CD39 upregulation by Th17. Boosting downstream effects of AHR via UCB or enhancing CD39-mediated ectoenzymatic activity might provide therapeutic options to address development of Th17 dysfunction in IBD.

Higher serum vitamin D levels are associated with protective serum cytokine profiles in patients with ulcerative colitis

Background & Aims Vitamin D has immune modulating effects on cytokines. Serum vitamin D levels are associated with the risk of relapse in patients with ulcerative colitis (UC), through unknown mechanisms. We tested the hypothesis that this beneficial role of vitamin D on UC is mediated through anti‐inflammatory serum cytokine profiles. Methods Serum samples from a prospective cohort of seventy UC patients in clinical remission were collected and baseline histological and endoscopic scores were recorded at enrollment. Clinical relapse events were recorded over the 12‐month follow‐up period. Serum vitamin D and cytokines levels (IL‐6, IL‐8, IL‐17A, TNF‐&agr;, IFN‐&ggr;, IL‐4, IL‐10) were quantified using ELISA. Linear regression was used to determine correlation between vitamin D and cytokine profiles. Logistic regression models were used to determine the association between serum cytokine profiles and baseline histologic mucosal healing and clinical relapse. Results Higher serum vitamin D levels positively correlated with higher ratios of IL‐4 + IL‐10/IL‐17A + TNF‐&agr; (r = 0.37, P < .01), and IL‐4 + IL‐10/IL‐6 + TNF‐&agr; (r = 0.32, P < .01). In multivariate analysis, IL‐4 + IL‐10/IL‐17A + TNF‐&agr; ratios at baseline were associated with the presence of histologic mucosal healing (O.R. 1.29, 95% CI 1.02–1.62, P = .03). A higher ratio of serum IL‐4 + IL‐10 to IL‐6 + TNF‐&agr; was associated with a reduced risk of clinical relapse (O.R. 0.72, 95% CI 0.58–0.89, P = .003), and longer time to relapse (p = .03), over the 12‐month follow‐up period. This ratio during remission had an AUC of 0.7 in predicting later clinical relapse. Conclusions Vitamin D is associated with anti‐inflammatory serum cytokine profiles. Anti‐inflammatory cytokine patterns may mediate the protective effects of higher serum vitamin D levels in patients with ulcerative colitis. HighlightsPositive correlation between serum vitamin D (25(OH)D) levels and anti‐inflammatory serum cytokine profiles.Serum cytokine profile IL‐4 + IL‐10/IL‐17A + TNF‐&agr; is associated with presence of histologic mucosal healing among ulcerative colitis patients in clinical remission.Baseline serum cytokine profile IL‐4 + IL‐10/IL‐6 + TNF‐&agr; during periods of clinical remission predicts longitudinal risk of relapse in ulcerative colitis patients.

Risk Factors for Fecal Urgency Among Individuals With and Without Diarrhea, Based on Data From the National Health and Nutrition Examination Survey

Background & Aims Fecal urgency is a common symptom among patients with gastrointestinal disorders, but can also occur in healthy individuals with normal bowel habits. There have been few studies of fecal urgency in the general population. We performed a cross‐sectional analysis of data from the National Health and Nutrition Examination Survey (NHANES) to analyze the prevalence of and risk factors for this symptom. Methods We analyzed data from 4676 persons who completed the Bowel Health Questionnaire from the NHANES, from 2009 through 2010. The NHANES sampled a nationally representative group of adults in the United States and provides information on demographics, medical comorbidities, and dietary habits of survey participants. The Bowel Health Questionnaire provided additional information about bowel symptoms such as urgency, incontinence, constipation, and diarrhea. We identified individuals with fecal urgency and calculated differences in fecal urgency among subgroups using chi‐squared analysis. We used logistic regression to identify factors associated with urgency. Results In our study population, the prevalence of fecal urgency was 3.3%; 29.5% of individuals with fecal urgency had diarrhea. The prevalence of fecal urgency was significantly higher in individuals who had diarrhea (14.8%) than in individuals without diarrhea (3.1%). Older age, female sex, poverty, urinary urge incontinence, diarrhea, and increased stool frequency were all associated with fecal urgency on multivariable analysis. Decreased fiber intake and increased carbohydrate intake were associated with urgency among individuals with diarrhea. Conclusions In an analysis of data from 4676 individuals who completed a Bowel Health Questionnaire from the NHANES, we found a significantly higher proportion of individuals with diarrhea to have fecal urgency. However, most individuals with fecal urgency do not have diarrhea. Factors associated with fecal urgency vary among individuals with and without diarrhea.

Demographic and Dietary Associations of Chronic Diarrhea in a Representative Sample of Adults in the United States

Objectives:No studies to date estimate the prevalence of chronic diarrhea in the United States using the Bristol stool form scale (BSFS). This study aims to report the prevalence and associated factors of chronic diarrhea using BSFS scores in a nationally representative sample of US adults.Methods:We identified 5,246 adult participants (age ≥20 years) who completed the bowel health questionnaire in the National Health and Nutrition Examination Survey 2009–2010 data set. Chronic diarrhea was defined as type 6 or 7 rating on the BSFS (mushy or liquid consistency) as the “usual or the most common stool type.” Co-variables included age, race, education, poverty income ratio, body mass index, number of medications, feeling depressed, physical activity, and dietary intake. Prevalence estimates and prevalence odds ratios (PORs) were analyzed in adjusted multivariable models using appropriate sampling weights.Results:We found a prevalence of chronic diarrhea of 6.6% (95% confidence interval (CI) 5.8, 7.4) in the nationally representative data set. High daily carbohydrate intake (POR 1.56, 95% CI 1.02, 2.40), obesity (POR 2.04, 95% CI 1.44, 2.89), feeling depressed (POR 1.84, 95% CI 1.21, 2.80), older age (POR 1.02, 95% CI 1.01, 1.02), and female sex (POR 1.68, 95% CI 1.28, 2.21) were positively correlated with chronic diarrhea. Non-Hispanic White race (POR 0.49, 95% CI 0.29, 0.81) and higher education (POR 0.60, 95% CI 0.43, 0.83) were negatively correlated with chronic diarrhea.Conclusions:In a nationally representative sample of the US adults, the prevalence of chronic diarrhea was 6.6%. We identified demographic, lifestyle, and dietary factors associated with chronic diarrhea.

Distinct roles of ecto-nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) in liver regeneration and fibrosis

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) are cell surface-located transmembrane ecto-enzymes of the CD39 superfamily which regulate inflammation and tissue repair by catalyzing the phosphohydrolysis of extracellular nucleotides and modulating purinergic signaling. In the liver, NTPDase2 is reportedly expressed on portal fibroblasts, but its functional role in regulating tissue regeneration and fibrosis is incompletely understood. Here, we studied the role of NTPDase2 in several models of liver injury using global knockout mice. Liver regeneration and severity of fibrosis were analyzed at different time points after exposure to carbon tetrachloride (CCl4) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or partial hepatectomy in C57BL/6 wild-type and globally NTPDase2-deficient (Entpd2 null) mice. After chronic CCl4 intoxication, Entpd2 null mice exhibit significantly more severe liver fibrosis, as assessed by collagen content and histology. In contrast, deletion of NTPDase2 does not have a substantial effect on biliary-type fibrosis in the setting of DDC feeding. In injured livers, NTPDase2 expression extends from the portal areas to fibrotic septae in pan-lobular (CCl4-induced) liver fibrosis; the same pattern was observed, albeit to a lesser extent in biliary-type (DDC-induced) fibrosis. Liver regeneration after partial hepatectomy is not substantively impaired in global Entpd2 null mice. NTPDase2 protects from liver fibrosis resulting from hepatocellular injury induced by CCl4. In contrast, Entpd2 deletion does not significantly impact fibrosis secondary to DDC injury or liver regeneration after partial hepatectomy. Our observations highlight mechanisms relating to purinergic signaling in the liver and indicate possible therapeutic avenues and new cellular targets to test in the management of hepatic fibrosis.

Complete deletion of CD39 is atheroprotective in apolipoprotein E-deficient mice

Cd39 scavenges extracellular ATP and ADP, ultimately generating adenosine, a nucleoside, which has anti-inflammatory effects in the vasculature. We have evaluated the role of Cd39 in the development of atherosclerosis in hyperlipidemic mice. ApoE KO (Cd39+/+/ApoE−/−) and Cd39/ApoE double KO (DKO) (Cd39−/−/ApoE−/−) mice were maintained on chow or Western diet for up to 20 weeks before evaluation of atherosclerotic lesions. We found that DKO mice exhibited significantly fewer atherosclerotic lesions than ApoE KO mice, irrespective of diet. Analyses of plaque composition revealed diminished foam cells in the fatty streaks and smaller necrotic cores in advanced lesions of DKO mice, when compared with those in ApoE KO mice. This atheroprotective phenotype was associated with impaired platelet reactivity to ADP in vitro and prolonged platelet survival, suggesting decreased platelet activation in vivo. Further studies with either genetic deletion or pharmacological inhibition of Cd39 in macrophages revealed increased cholesterol efflux mediated via ABCA1 to ApoA1. This phenomenon was associated with elevated plasma HDL levels in DKO mice. Our findings indicate that complete deletion of Cd39 paradoxically attenuates development of atherosclerosis in hyperlipidemic mice. We propose that this phenotype occurs, at least in part, from diminished platelet activation, increased plasma HDL levels, and enhanced cholesterol efflux and indicates the complexity of purinergic signaling in atherosclerosis.