Marina Aloi

Necroptosis Is Active in Children With Inflammatory Bowel Disease and Contributes to Heighten Intestinal Inflammation

OBJECTIVES:A new caspase-independent mode of programmed cell death, termed necroptosis, has recently been identified. Altered expression of molecules involved in the necroptosis pathway has been shown to trigger intestinal inflammation. The initiation of necroptosis is principally mediated by the release of receptor interacting protein 3 (RIP3) from suppression by caspase-8. Furthermore, it has been suggested that the mixed lineage kinase domain-like (MLKL) factor is an interacting target of RIP3 in active necroptosis. This study aims at investigating the occurrence of necroptosis in children with inflammatory bowel disease (IBD) and its contribution to human intestinal inflammation.METHODS:Biopsy samples were collected from the ileum and colon of 33 children with Crohns disease, 30 with ulcerative colitis, and 20 healthy controls. Ten children with allergic colitis (AC) were used as non-IBD comparators. RIP3, caspase-8, and MLKL protein expression levels were evaluated by western blotting. The adenocarcinoma cell line HT29 was used for in vitro experiments.RESULTS:RIP3 and MLKL increased (P<0.01) in inflamed tissues of IBD and AC patients, whereas caspase-8 was reduced. No variations were observed in uninflamed tissues of patients. The relationship between RIP3 increase, active necroptosis, and intestinal inflammation was confirmed by in vitro analyses.CONCLUSIONS:We show for the first time that necroptosis is strongly associated with intestinal inflammation in children with IBD and contributes to strengthen the inflammatory process. We believe that RIP3 and MLKL could represent attractive targets for the management of human IBD.

Phenotype and disease course of early-onset pediatric inflammatory bowel disease.

Background:Early-onset (EO) pediatric inflammatory bowel diseases (IBD) seem to be more extensive than those with a later onset. To test this hypothesis, we examined the phenotype and disease course of patients with IBD diagnosis at 0 to 5 years, compared with the ranges 6 to 11 and 12 to 18 years. Methods:Anatomic locations and behaviors were assessed according to Paris classification in 506 consecutive patients: 224 Crohns disease, 245 ulcerative colitis, and 37 IBD-unclassified. Results:Eleven percent of patients were in the range 0 to 5 years, 39% in 6 to 11 years, and 50% in 12 to 18 years. Ulcerative colitis was the most frequent diagnosis in EO-IBD and in 6- to 11-year-old group, whereas Crohns disease was predominant in older children. A classification as IBD-unclassified was more common in the range 0 to 5 years compared with the other groups (P < 0.005). EO Crohns disease showed a more frequent isolated colonic (P < 0.005) and upper gastrointestinal involvement than later-onset disease. Sixty-two percent of the patients in the 0 to 5 years range had pancolonic ulcerative colitis, compared with 38% of 6 to 11 years (P = 0.02) and 31% of 12–18 years (P = 0.002) range. No statistical difference for family history for IBD was found in the 3-year age groups. Therapies at the diagnosis were similar for all children. However, at latest follow-up, a significantly higher proportion of younger children were under steroids compared with older groups (P < 0.05). Surgical risk did not differ according to age. Conclusions:EO-IBD exhibits an extensive phenotype and benefit from aggressive treatment strategies, although surgical risk is similar to later-onset disease. A family history for IBD is not common in EO disease.

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.

Small Intestine Contrast Ultrasonography in Pediatric Crohn's Disease

OBJECTIVE To evaluate the diagnostic accuracy of small intestine contrast ultrasonography (SICUS) in pediatric Crohns disease (CD). STUDY DESIGN A total of 51 consecutive patients (median age 15 years; range 3-20, 31 male patients), 21 with suspected and 30 with proven CD, were studied. All patients underwent standard ultrasonography (ie, transabdominal ultrasonography [TUS]), SICUS, small bowel follow-through, and upper and lower endoscopy. SICUS was performed in patients after they ingested an oral contrast solution. TUS and SICUS were compared with small bowel follow-through and endoscopy via use of the final diagnosis as reference standard. RESULTS In undiagnosed patients, the sensitivity and specificity of TUS and SICUS in detecting CD small bowel lesions were 75% and 100% and 100% and 100%, respectively. In patients with proven CD, the sensitivity and specificity of TUS and SICUS were 76% and 100% and 96% and 100%, respectively. The agreement (k) with radiology for site of lesions was almost perfect for SICUS (0.93), both for jejunal and ileal lesions, and it was fair (0.40) for jejunal and substantial (0.68) for ileal lesions for TUS. Compared with radiology SICUS correctly assessed the length of lesions, whereas TUS underestimated it (P = .0001). CONCLUSIONS The radiation-free technique SICUS is comparable with radiology and more accurate than TUS in assessing small bowel lesions in pediatric CD, mainly in the detection of proximal small bowel disease.

Usefulness of single-balloon enteroscopy in pediatric Crohn's disease

BACKGROUND Single-balloon enteroscopy (SBE) has not been reported in pediatric Crohns disease (CD). OBJECTIVE To determine technical performance, yield, safety, and clinical impact of SBE in pediatric patients with suspected and established CD. DESIGN Prospective, cohort study. SETTING Academic tertiary-care referral center. PATIENTS This study involved 16 patients (group A) with suspected CD and unspecific upper and lower GI endoscopy results and 14 patients (group B) with longstanding CD with previous surgery and showing signs unaccountable by conventional endoscopy. All underwent magnetic resonance imaging, and 14 patients in group A also underwent wireless capsule endoscopy. INTERVENTION SBE. MAIN OUTCOME MEASUREMENTS SBE diagnostic and therapeutic yield, technical performance, clinical impact, and safety. RESULTS In group A, SBE aided diagnosis of CD in 12 patients and eosinophilic enteropathy in 2 patients, whereas no lesions were found in 2 patients. WCE was diagnostic of CD in 3 patients, suggestive of CD in 7 patients, and unspecific in the remaining patients. In group B, SBE revealed moderate-to-severe disease activity in most patients, leading to the introduction of or change in biological therapy, with a marked decrease in the pediatric Crohns disease activity index scores. SBE allowed successful dilation of small-bowel strictures in 2 patients in group A and 3 in group B. No complications occurred. LIMITATIONS Small sample size, no direct comparison with imaging or other endoscopic techniques. CONCLUSION SBE is a useful and safe endoscopic procedure for evaluating the small bowel in pediatric patients with suspected or established CD. Not only does it allow a definite diagnosis of CD when the latter is uncertain, but it is also very effective in the management of small-bowel strictures, thus avoiding surgery. It may be helpful in redirecting therapy in selected CD patients.

Malignancy and mortality in pediatric patients with inflammatory bowel disease: a multinational study from the porto pediatric IBD group.

Background:The combination of the severity of pediatric-onset inflammatory bowel disease (IBD) phenotypes and the need for intense medical treatment may increase the risk of malignancy and mortality, but evidence regarding the extent of the problem is scarce. Therefore, the Porto Pediatric IBD working group of ESPGHAN conducted a multinational-based survey of cancer and mortality in pediatric IBD. Methods:A survey among pediatric gastroenterologists of 20 European countries and Israel on cancer and/or mortality in the pediatric patient population with IBD was undertaken. One representative from each country repeatedly contacted all pediatric gastroenterologists from each country for reporting retrospectively cancer and/or mortality of pediatric patients with IBD after IBD onset, during 2006–2011. Results:We identified 18 cases of cancers and/or 31 deaths in 44 children (26 males) who were diagnosed with IBD (ulcerative colitis, n = 21) at a median age of 10.0 years (inter quartile range, 3.0–14.0). Causes of mortality were infectious (n = 14), cancer (n = 5), uncontrolled disease activity of IBD (n = 4), procedure-related (n = 3), other non-IBD related diseases (n = 3), and unknown (n = 2). The most common malignancies were hematopoietic tumors (n = 11), of which 3 were hepatosplenic T-cell lymphoma and 3 Ebstein–Barr virus–associated lymphomas. Conclusions:Cancer and mortality in pediatric IBD are rare, but cumulative rates are not insignificant. Mortality is primarily related to infections, particularly in patients with 2 or more immunosuppressive agents, followed by cancer and uncontrolled disease. At least 6 lymphomas were likely treatment-associated by virtue of their phenotype.

Role of HMGB1 as a suitable biomarker of subclinical intestinal inflammation and mucosal healing in patients with inflammatory bowel disease.

Background:Noninvasive biomarkers of high- and low-grade intestinal inflammation and of mucosal healing (MH) in patients with inflammatory bowel disease are currently lacking. We have recently shown that fecal high mobility group box 1 (HMGB1) protein is a novel biomarker of gut inflammation. We aimed at investigating in a mouse model if HMGB1 was able to foresee both a clinically evident and a subclinical gut inflammation and if its normalization indicated MH. We also aimed at confirming the results in patients with Crohns disease (CD) and ulcerative colitis. Methods:C57BL6/J mice were treated with increasing doses of dextran sodium sulphate to induce colitis of different severity degrees; 28 with CD, 23 with ulcerative colitis, and 17 controls were also enrolled. Fecal HMGB1 was analyzed by enzyme-linked immunosorbent assay and immunoblotting. Results:Fecal HMGB1 increased by 5-, 11-, 18-, and 24-folds with dextran sodium sulphate doses of 0.25%, 0.50%, 1%, and 4%, respectively, showing that the protein detected a high-grade and a subclinical inflammation. After a recovery time of 4-week posttreatment, HMGB1 returned to control levels, paralleling MH. In patients, fecal HMGB1 significantly correlated with endoscopic indexes (Simple Endoscopic Score for Crohns Disease [SES-CD], endoscopic Mayo subscore), but not with the disease activity indexes (Crohns disease Activity Index, partial Mayo score). Conclusions:Fecal HMGB1 is a robust noninvasive biomarker of clinically overt and subclinical gut inflammation; it can also be a surrogate marker of MH. We suggest the use of fecal HMGB1 to monitor the disease course and assess therapy outcomes in inflammatory bowel disease.

Second-generation colon capsule endoscopy vs. colonoscopy in pediatric ulcerative colitis: a pilot study

BACKGROUND AND STUDY AIMS Second-generation colon capsule endoscopy (CCE-2) may overcome the invasiveness of colonoscopy in the evaluation of mucosal inflammation, especially in pediatric ulcerative colitis. The aim of this pilot study was to determine the diagnostic accuracy of CCE-2 in evaluating disease activity, using colonoscopy as a gold standard. Disease extent, tolerability, interobserver agreement, and safety were also evaluated. METHODS A total of 30 consecutive pediatric patients with ulcerative colitis were prospectively enrolled (mean age 14.1 ± 3.2 years). Patients underwent CCE-2 followed by colonoscopy in the late afternoon or the following day. The blinded procedures were performed, and the diagnostic accuracy of CCE-2 to assess disease activity was determined using a modified Matts score, which classified patients as either normal (Matts score ≤ 6) or with active inflammation (Matts score > 6). Interobserver agreement was assessed using the kappa statistic. RESULTS One patient was excluded from the analysis because they were unable to swallow the capsule, leaving 29 patients available for analysis. The sensitivity of CCE-2 for disease activity was 96 % (95 % confidence interval [CI] 79 - 99) and specificity was 100 % (95 %CI 61 - 100). The positive and negative predictive values of CCE-2 were 100 % (95 %CI 85 - 100) and 85 % (95 %CI 49 - 97), respectively. No serious adverse events were reported. CCE-2 had a higher overall tolerability than colonoscopy (P < 0.05). Interobserver agreement was excellent in all cases (κ > 0.86). CONCLUSIONS Using a modified Matts score, CCE-2 was accurate in evaluating significant mucosal inflammation in children with ulcerative colitis. TRIAL REGISTRATION ClinicalTrials.gov--NCT01740349.

Adherent-invasive Escherichia coli (AIEC) in pediatric Crohn’s disease patients: phenotypic and genetic pathogenic features

BackgroundAdherent-invasive Escherichia coli (AIEC) have been implicated in the ethiopathogenesis of Crohn’s disease (CD). In this study, we analyzed a collection of intestinal mucosa-associated E. coli isolates, presenting AIEC phenotypes, isolated from biopsies of CD pediatric patients and non-inflammatory bowel diseases (IBD) controls, in order to investigate their genetic and phenotypic pathogenic features.ResultsA total of 616 E. coli isolates from biopsies of four pediatric CD patients and of four non-IBD controls were collected and individually analyzed. For AIEC identification, adherent isolates were assayed for invasiveness, and the capacity of the adhesive-invasive isolates to survive and replicate intracellularly was determined over macrophages J774. In this way we identified 36 AIEC-like isolates. Interestingly, their relative abundance was significantly higher in CD patients (10%; 31/308) than in non-IBD controls (1%; 5/308) (χ 2 = 38.96 p < 0.001). Furthermore pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) techniques were applied to analyze the clonality of the 36 AIEC-like isolates. The results obtained allowed us to identify 27 distinct genotypes (22 from CD patients and 5 from non-IBD controls). As for the AIEC prototype strain LF82, all 27 AIEC genotypes presented an aggregative pattern of adherence (AA) that was inhibited by D-mannose, indicating that adhesiveness of AIEC is likely mediated by type 1 pili. PCR analisys was used to investigate presence of virulence genes. The results indicated that among the 27 AIEC isolates, the incidence of genes encoding virulence factors K1 (χ 2 = 6.167 P = 0.013), kps MT II (χ 2 = 6.167 P = 0.013), fyuA (χ 2 = 6.167 P = 0.013), and ibeA (χ 2 = 8.867 P = 0.003) was significantly higher among AIEC strains isolated from CD patients than non-IBD controls.ConclusionsThe identification of AIEC strains in both CD and non-IBD controls, confirmed the “pathobiont” nature of AIEC strains. The finding that AIEC-like isolates were more abundant in CD patients, indicates that a close association of these strains with CD may also exists in pediatric patients.

Interactions between intestinal microbiota and innate immune system in pediatric inflammatory bowel disease

Inflammatory bowel disease (IBD) is the result of an altered immune homeostasis within the intestinal mucosa against the gut microbiota, leading to chronic inflammation in genetically predisposed individuals. Under normal conditions, the immune system defends against pathogens and prevents the passage of excessive intestinal bacteria; regulatory pathways must maintain a low-grade, controlled inflammation in a healthy gut, but also induce a protective response against pathogens. The innate immune system is the first-line defense from microbes; dendritic cells, macrophages, and epithelial cells produce an initial, immediate response. The immune system constantly controls commensal bacteria and utilizes constitutive antimicrobial mechanisms to sustain immune homeostasis. The discovery that several genes linked to IBD modulate microbial recognition and innate immune pathways, such as nucleotide oligomerization domain 2 (Nod2), and genes that mediate autophagy (ie, ATG16L1, IRGM), has highlighted the critical role of host-microbe interactions in controlling intestinal immune homeostasis. Commensal microorganisms actively interact with the intestinal mucosa and influence the activity of the immune system as well as the amplitude of the immune response. In contrast, host factors can influence microbes, which in turn modulate disease susceptibility. In this paper, we focus on the mechanisms that mediate host-microbe interactions and how the disruption of this balance leads to chronic intestinal inflammation in IBD.