Emanuela Del Giudice

Fecal HMGB1 is a novel marker of intestinal mucosal inflammation in pediatric inflammatory bowel disease.

OBJECTIVES:High-mobility group box 1 (HMGB1) is a nuclear protein with functions in the regulation of transcription. In inflammatory conditions, HMGB1 is actively secreted from immune cells in the extracellular matrix, where it behaves as a proinflammatory cytokine. The aim of the present study was to investigate the role of HMGB1 in pediatric inflammatory bowel disease (IBD).METHODS:We analyzed the stools of 19 children with Crohns disease (CD), 21 with ulcerative colitis (UC), and 13 controls. The gene/protein expression levels of HMGB1 were assessed in bioptic specimens of all children using real-time PCR and western blot assay. Finally, intracellular localization of the protein was analyzed by western blot, after separation of nuclear and cytoplasmic extracts, and by immunohistochemistry.RESULTS:HMGB1 protein levels were significantly increased (P<0.001) in the stools of patients, but were undetectable in the controls; fecal HMGB1 correlated well with fecal calprotectin levels (r: 0.77 in CD, r: 0.70 in UC; P<0.01); and mRNA and protein expression were unchanged in inflamed bioptic tissues compared with controls. However, by separately analyzing the nuclear and cytoplasmic fraction, we detected the cytoplasmic HMGB1 expression to be significantly enhanced (P<0.01) in the inflamed tissues of the patients. In addition, HMGB1 was significantly detected in 16 patients with inactive disease, whose endoscopic scores showed persisting inflammation, suggesting that it may be a sensitive marker of mucosal inflammation, although the disease is clinically inactive.CONCLUSIONS:It was shown for the first time in our study that HMGB1 is secreted by human inflamed intestinal tissues and abundantly found in the stools of IBD patients. Hence, it can be considered as a novel marker for intestinal inflammation. We can also suggest that the presence of HMGB1 in large amounts in the fecal stream of IBD patients is mainly due to active secretion of the protein stored in the nucleus rather than a “de novo” synthesis.

Premature Subclinical Atherosclerosis in Pediatric Inflammatory Bowel Disease

OBJECTIVES To investigate the risk for developing an early endothelial dysfunction based on increased intima media thickness (IMT) and reduced flow-mediated dilation (FMD) in children with inflammatory bowel disease (IBD), and to evaluate the role of traditional and nontraditional risk factors in determining premature atherosclerosis. STUDY DESIGN We studied 27 patients with Crohns disease (CD) and 25 patients with ulcerative colitis (UC) (mean age, 15.2 years; mean duration of disease, 48.05 months); 31 subjects served as controls. Demographic data (age, sex, family history of diabetes, cardiovascular disease, hypertension, hypercholesterolemia), traditional risk factors for atherosclerosis (blood pressure, body mass index, active and passive smoking, dyslipidemia), and UC and CD activity indexes (Pediatric Ulcerative Colitis Activity Index and Pediatric Crohns Disease Activity Index, respectively) were collected. The IMT of the carotid arteries was measured by high-resolution B-mode ultrasound, and endothelial function was evaluated by FMD in the brachial artery in response to reactive hyperemia. RESULTS Compared with controls, patients with CD had significantly greater exposure to passive smoking and had lower body mass index and high-density lipoprotein cholesterol values. IMT was significantly higher in patients than controls (P < .0001), and the percentage of FMD was significantly lower in both patients with CD (P < .0001) and patients with UC (P < .01) versus controls. In multivariate analysis, diagnosis of IBD was an independent risk factor for atherosclerosis. CONCLUSION Premature endothelial dysfunction occurs in pediatric IBD. This represents a new challenge in the management of pediatric IBD, leading to prevention strategies of cardiovascular disease.

Biological therapy in a pediatric crohn disease population at a referral center

Objective: The antitumor necrosis factor &agr; (TNF&agr;) antibodies infliximab and adalimumab are effective in inducing and maintaining remission in pediatric patients with Crohn disease (CD). The aim of the study was to evaluate the long-term efficacy and safety of biological therapy in pediatric patients with CD followed at a referral center. Methods: This work is a retrospective observational study enrolling patients with CD treated with infliximab or adalimumab beyond the induction protocol. The patients’ data were collected from the units IBD database (maximum follow-up evaluation after 36 months of treatment). The efficacy was evaluated by the Pediatric Crohn Disease Activity Index score and by analysis of the cumulative probability of continuing therapy; the safety was assessed in terms of adverse events. Results: We enrolled 78 patients; the mean therapy duration was 27.2 ± 16.7 months, and the mean age at enrollment was 15 ± 3.1 years. The Kaplan-Meier analysis showed a cumulative probability of continuing therapy of 81%, 54%, and 33% at 1, 2, and 3 years, respectively, from the introduction of therapy. No association between the patients’ baseline characteristics and the long-term outcome was found. The evaluation of the concomitant therapy with immunomodulators and anti-TNF&agr; therapy versus anti-TNF&agr; alone did not show a different outcome. No serious adverse events were recorded. Conclusions: The study indicates that biological therapy is effective and safe in pediatric patients with CD in a longer follow-up period. The response to treatment was not influenced by the patients’ baseline characteristics or by the immunomodulator association.

The Nuclear Protein HMGB1 is Increased in the Stools of IBD Patients and Appears to Be a Novel Marker of Intestinal Inflammation

Background The balance between T regulatory cells (Treg) and T helper type 17 (Th17) cells appears important in modulating immune responses in inflammatory bowel disease. CD39 is an immune cell ectonucleotidase, expressed in lipid rafts, which generates immune suppressive adenosine. Genetic polymorphisms of CD39 are noted in IBD and the ectoenzyme has been validated as a marker of memory type Treg. CD161 (killer cell lectin subfamily B, member 1 or NKR-P1A) is a relatively specific surface marker for human Th17 cells and is linked to activation of acid sphingomyelinase that might in turn impact CD39 bioactivity. It is unknown whether there are T cell populations in IBD that express both markers and the putative relevance is unexplored. Aims: To identify dual expression of CD39 and CD161 in blood T cells from patients with IBD, and explore the role of CD161 in regulating immune deviation of CD4+CD39+ T cell populations. Methods The percentage of CD39+CD161+ CD4 T cells in peripheral blood of patients with active or inactive Crohns disease was determined by FACS. Four phenotypic subtypes of peripheral blood CD4 T cells including CD39+CD161+, CD39+CD161-, CD39-CD161+, and CD39-CD161from healthy donors were sorted by FACS, and stimulated respectively by anti-CD3/CD28 antibodies with with TGF-beta and IL-2 (for Treg cell expansion) or IL-6, IL-23, IL-1beta and TGFbeta (for Th17 cell expansion) for 3 days, and intracellular IL-17 and INF-gamma or FOXP3 were detected by FACS. These four subgroups were treated by ATP or NAD+ for 1 hour, and stained by Annexin V to detect early events of P2X7-mediated apoptosis. Result The percentage of CD39+CD161+ CD4 T cells in peripheral blood of normal controls, active and inactive Crohns disease patients was 1.26±0.73, 4.55±2.39 and 0.95±0.52%, respectively (p=0.01, for active disease vs. both controls and inactive Crohns disease). After 3 days, Treg expansion, of FOXP3-positive cells within the CD39+CD161+ group, was less than from the CD39+CD161group (p=0.01). After 3 days In Vitro expansion, the percentages of intracellular IL-17-positive and/or IFN-gamma-positive cells in CD4+CD39+CD161+ populations were higher than other three groups (p=0.01). However, CD39+CD161+ CD4 T cells also exhibited highest levels of NAD+-induced apoptosis (p=0.05 vs. other groups). Conclusions Peripheral blood CD4+CD39+CD161+ T cells are increased in active Crohns disease. CD161 expression impacts the reciprocal balance of Treg and Th17 cells generated from CD4+CD39+ cells.

Outcome of Biological Therapy in Pediatric Inflammatory Bowel Disease at a Single Tertiary Center

Background and Aims. Anti-TNFα antibodies Infliximab (IFX) and Adalimumab (ADA) have been shown to be effective agents in inducing and maintaining remission in pediatric patients (pts) suffering from inflammatory bowel disease (IBD). However, data relating to long term outcomes of maintenance therapy are scarce. The aim of our study was to evaluate the long term efficacy and safety of biological therapy in pediatric IBD pts followed at a single center. Methods. All IBD treated with IFX or ADA with a maximum follow-up period up to 36 months for Crohns disease (CD) and 12 months for ulcerative colitis (UC) were enrolled. Pts data were analyzed using the database for IBD pts of the Unit. Efficacy was evaluated by PCDAI and PUCAI clinical scores for CD and UC respectively. Safety was evaluated in terms of side effects and adverse events leading to discontinuation. Seventy two pts (69 CD, 13 UC) were enrolled. Results (mean±SD). Characteristics of pts at the enrollment phase are in Table 1. The main reasons for starting IFX were unresponsiveness to conventional therapies (44%) and perianal disease (37.2%) for CD, unresponsiveness to other therapies (30.8%) and steroid dependency or resistance (53.8%) for UC. ADA was started for disease relapse (34.6%) and unresponsiveness to other therapies (42.2%, 11.5 % unresponsive to IFX). Therapy duration was 14.2 ± 11.4 and 6.8 ± 8.3 months (mts) for CD and UC pts on IFX respectively and 18.6 ± 11.6 mts for ADA pts. Immunomodulator therapy was stopped in 18% of CD pts and in no UC pts on IFX and in 92.3% on ADA during the follow-up period. Corticosteroid free remission at 6 and 12 mts was 71% and 65 % for CD IFX pts, 50% and 66% for UC IFX pts and 69% and 78% for ADA pts. PCDAI at 0, 3, 12, 24, 36 mts and PUCAI at 0, 3 and 12 mts are in Table 2. IFX was stopped in 29 CD pts and 10 UC pts, and ADA was stopped in 10 pts because of: long term remission (45% for CD IFX pts and 27% for UC IFX pts, 11.5% ADA pts), unresponsiveness (3% for CD IFX pts, 36% for UC IFX pts), loss of response (27.6% for CD IFX pts, 16% for UC IFX pts, 11.5% ADA pts) or infections (11.5% ADA pts). Five UC pts required colectomy. At 12 mts, of the 13 CD pts stopping IFX for remission 7 were still in remission, 5 restarted biological therapy (3 ADA, 2 IFX), 1 underwent ileal resection. The 3 pts stopping ADA for remission were inactive at 12 mts. Side effects were reported in 22 pts, of these 7 discontinued therapy. Conclusions. In children with IBD, IFX and ADA are an effective and durable treatment over a 3-year period; biological therapy can also postpone colectomy in UC pts. The safety profile was acceptable for both agents with no serious adverse events or malignancies reported. Table 1

150 Intralesional Corticosteroid Injection Following Endoscopic Balloon Dilation in Stricturing Pediatric Crohn's Disease: A Prospective Randomized, DoubleBlind, Controlled Trial

Aim: 30% of patients hospitalized with severe UC prove steroid-refractory. We aimed to evaluate outcomes and predictors of response to infliximab as rescue therapy in severe pediatric UC. Methods: As part of a prospective multicenter study, we evaluated factors associated with immediate and 1-year response to infliximab in steroid-refractory severe pediatric UC. Data were recorded at admission, days 3 and 5, at introduction of infliximab, at discharge and 1-year thereafter, using standardized data collection forms. Disease activity was determined using the validated Pediatric UC Activity Index (PUCAI). Serum TNF alpha level was determined before infliximab treatment using a cytokine antibody panel (TransSignal, CA). Concurrently, fecal calprotectin and lactoferrin levels were ascertained using standard assays in a central laboratory. Results: Of 128 children admitted, 33 failed steroids and treated with infliximab within 10.5±6 days. Mean PUCAI score at introduction of infliximab was 66±13 points, indicating persistence of severe colitis. 25/33 children (76%) responded and were discharged within 5±4 days of infliximab therapy; 7 in complete clinical remission (PUCAI 0.2). CRP, ESR, albumin and hemoglobin were not predictive of response to infliximab. Neither fecal calprotectin nor lactoferrin values were predictive of response (area under ROC curve 0.61 and 0.63, respectively; P>0.2). Serum TNF-alpha level was similar between responders and non-responders (10.6pg/ml (IQR 4-30) vs. 8.3pg/ml (5.7-11); P=0.4). 8 of the 25 responders received only 3-dose induction, and the others continued maintenance therapy without concomitant immunomodulation. Cumulative 1-year sustained response rate was 55% (18/33). There were no deaths and only 1 patient stopped treatment due to infusion reaction. Conclusion: Infliximab is safe and effective in inducing and maintaining clinical remission in steroid-refractory pediatric UC. Serum TNF-alpha level and fecal biomarkers are not useful in predicting outcome, but higher disease severity, judged clinically, and new onset disease are associated with reduced response.