Laura Stronati

Randomised clinical trial: the effectiveness of Lactobacillus reuteri ATCC 55730 rectal enema in children with active distal ulcerative colitis

Background  Intestinal microbiota manipulation, one of the pathogenetic components of inflammatory bowel disease (IBD), has become an attractive therapy for ulcerative colitis (UC).

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.

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.

Characterization of adherent-invasive Escherichia coli isolated from pediatric patients with inflammatory bowel disease†

Background: Crohns disease (CD) and ulcerative colitis (UC), known as inflammatory bowel diseases (IBD), are characterized by an abnormal immunological response to commensal bacteria colonizing intestinal lumen and mucosa. Among the latter, strains of adherent‐invasive Escherichia coli (AIEC), capable of adhering to and invading epithelium, and to replicate in macrophages, have been described in CD adults. We aimed at identifying and characterizing AIEC strains in pediatric IBD. Methods: In all, 24 CD children, 10 UC, and 23 controls were investigated. Mucosal biopsies, taken during colonoscopy, were analyzed for the presence of AIEC strains by an adhesive‐invasive test. Protein expression of the specific AIEC receptor, the carcinoembryonic antigen‐related cell adhesion molecule 6 (CEACAM6), was evaluated by western blot and immunohistochemistry, while tumor necrosis factor alpha (TNF‐&agr;) and interleukin (IL)‐8 mRNA expression was detected by real‐time polymerase chain reaction (PCR), after bacterial infection. Transmission electron microscopy and trans‐epithelial electric resistance assays were performed on biopsies to assess bacteria‐induced morphological and functional epithelial alterations. Results: Two bacterial strains, EC15 and EC10, were found to adhere and invade the Caco2 cell line, similar to the well‐known AIEC strain LF82 (positive control): they upregulated CEACAM6, TNF‐&agr;, and IL‐8 gene/protein expression, in vitro and in cultured intestinal mucosa; they could also survive inside macrophages and damage the epithelial barrier integrity. Lesions in the inflamed tissues were associated with bacterial infection. Conclusions: This is the first study showing the presence of adhesive‐invasive bacteria strains in the inflamed tissues of children with IBD. Collective features of these strains indicate that they belong to the AIEC spectrum, suggesting their possible role in disease pathogenesis. (Inflamm Bowel Dis 2011)

Pediatric inflammatory bowel diseases and the risk of lymphoma: Should we revise our treatment strategies?

Inflammatory bowel diseases (IBDs) are lifelong inflammatory gastrointestinal diseases starting in about one third of patients during childhood. Treatment strategies aim to control this chronic inflammatory process. Owing to recent advances in the understanding of IBD, immunosuppressive agents (mainly against TNFα directed) as well as biological drugs are more and more often used. This therapeutic approach clearly improved the clinical condition of the majority of patients with IBD. However, with this more aggressive treatment strategy, safety concerns clearly arise. Recently, the description of a series of a particularly severe form of T cell lymphoma in pediatric and young adult patients with IBD under immunomodulator and biological combination therapy raised the question of the risks of treatment-induced side effects or complications. As reviewed in the present article, there is a slightly increased risk of not only lymphoma development in IBD patients, potentially related to the inflammatory process, but also to the use of immunosuppressive therapies. On the basis of the literature data, we reanalyzed current treatment strategies for children with moderate-to-severe IBD, who are candidates to receive immunomodulator and/or biological agents potentially accelerating the risk of lymphoma development. Comparative clinical studies in IBD are still missing; however, it is prudent to think about adapting immunosuppressive therapies to the inflammatory process of the underlying disorder and if possible to reduce them to monotherapy. Alternative treatment strategies for heavy immunosuppression exist (eg, enteral nutrition in Crohn disease or colectomy in patients with ulcerative colitis) and should be considered whenever appropriate. There is a major need for comparative studies before evidence-based guidelines can be established for safest and best treatment strategies of pediatric patients with IBD.

935 MHz cellular phone radiation. An in vitro study of genotoxicity in human lymphocytes.

Purpose: The possibility of genotoxicity of radiofrequency radiation (RFR) applied alone or in combination with x-rays was investigated in vitro using several assays on human lymphocytes. The chosen specific absorption rate (SAR) values are near the upper limit of actual energy absorption in localized tissue when persons use some cellular telephones. The purpose of the combined exposures was to examine whether RFR might act epigenetically by reducing the fidelity of repair of DNA damage caused by a well-characterized and established mutagen. Methods: Blood specimens from 14 donors were exposed continuously for 24 h to a Global System for Mobile Communications (GSM) basic 935 MHz signal. The signal was applied at two SAR; 1 and 2 W/Kg, alone or combined with a 1-min exposure to 1.0 Gy of 250 kVp x-rays given immediately before or after the RFR. The assays employed were the alkaline comet technique to detect DNA strand breakage, metaphase analyses to detect unstable chromosomal aberrations and sister chromatid exchanges, micronuclei in cytokinesis-blocked binucleate lymphocytes and the nuclear division index to detect alterations in the speed of in vitro cell cycling. Results: By comparison with appropriate sham-exposed and control samples, no effect of RFR alone could be found for any of the assay endpoints. In addition RFR did not modify any measured effects of the x-radiation. Conclusions: This study has used several standard in vitro tests for chromosomal and DNA damage in Go human lymphocytes exposed in vitro to a combination of x-rays and RFR. It has comprehensively examined whether a 24-h continuous exposure to a 935 MHz GSM basic signal delivering SAR of 1 or 2 W/Kg is genotoxic per se or whether, it can influence the genotoxicity of the well-established clastogenic agent; x-radiation. Within the experimental parameters of the study in all instances no effect from the RFR signal was observed.

Associations between Genetic Polymorphisms in IL-33, IL1R1 and Risk for Inflammatory Bowel Disease

Background Recent evidence suggests that the IL-33/IL1RL1 axis plays a critical role in several autoimmune and inflammatory disorders; however, its mechanistic role in inflammatory bowel disease (IBD) has not been clearly defined. We investigated the contribution of IL-33 and IL1RL1 polymorphisms to IBD risk, and possible correlations with phenotype in an Italian cohort of adult and pediatric patients. Methods We evaluated the association of six SNPs in IL-33 and IL1RL1 genes, in 805 Crohn’s disease (CD), 816 ulcerative colitis (UC), and 752 controls, using Taqman. IL-33 and IL1RL1 mRNA expression was also analyzed. Results Significant allele and genotype associations with IL-33 rs3939286 were found in CD (P = 0.004; P = 0.035) and UC patients (P = 0.002; P = 0.038). After stratifying the cohort for age at diagnosis, the differences remained significant only in the IBD adult-onset. Significant associations were also obtained in CD patients with two IL1RL1 polymorphisms (rs13015714 and rs2058660, P<0.015). By combining homo- and heterozygous carriers of the rs13015714 risk allele, differences were still significant for both CD adult- and pediatric-onset. Upon genotype-phenotype evaluation, an increased frequency of extensive colitis in adult UC (P = 0.019) and in steroid-responsive pediatric patients (P = 0.024) carrying the IL-33 rs3939286 risk genotype, was observed. mRNA expression of IL-33 and IL1RL1 in inflamed IBD biopsy samples was significantly increased. Conclusions Common IL-33 and IL1RL1 polymorphisms contribute to the risk of IBD in an Italian cohort of adult and pediatric patients, with some influence on sub-phenotypes.

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.

Activation of NOD2-mediated Intestinal Pathway in a Pediatric Population with Crohn's Disease

Background: NOD2 is an intracellular protein involved in host recognition of specific bacterial molecules and is genetically associated with several inflammatory diseases, including Crohns disease (CD). NOD2 stimulation activates the transcription factor, NF‐κB, through RIP2, a caspase‐recruitment domain‐containing kinase. NOD2/RIP2 signaling also mediates the activation of antimicrobial peptides such as human α‐defensin 5 (HD‐5) and human α‐defensin 6 (HD‐6), both produced by Paneth cells. The present study is aimed at describing the downstream events triggered specifically by NOD2 induction in order to demonstrate that the protein, other than overexpressed, is also physiologically associated with RIP2 and Erbin in the bioptic intestinal inflamed specimens of children affected by CD. Methods: Fifteen children with CD and 10 children used as controls were entered in the study. Mucosal biopsy specimens were taken during endoscopy and mRNA and protein expressions were detected by using real‐time polymerase chain reaction and Western blot. Results: NOD2 is able to form an immunocomplex with the kinase RIP2. As compared to controls, in the inflamed mucosa of patients both mRNA and protein expression levels of RIP2 are increased, and its active phosphorylated form is overexpressed. Conclusions: In this study we provide for the first time ex vivo evidence of physiologically relevant protein interactions with NOD2, which are able to trigger the innate immune response in intestinal mucosal specimens of children with CD. (Inflamm Bowel Dis 2009)

Lactobacillus reuteri ATCC55730 in cystic fibrosis.

Objectives: The aim of this study was to evaluate in patients with cystic fibrosis (CF) the effect of Lactobacillus reuteri (LR) on the rate of respiratory exacerbations and of the infections of both upper respiratory and gastrointestinal tracts. Methods: Prospective randomized, double-blind, placebo-controlled study enrolling 61 patients with CF with mild-to-moderate lung disease at the Regional Center for CF of the Department of Pediatrics, University of Rome “La Sapienza.” All of the patients were not hospital inpatients at the time of the enrollment. Inclusion criteria were forced expiratory volume in the first second (FEV1) >70% predicted; no inhaled or systemic steroids, no anti-inflammatory drugs, antileukotrienes, and mast cell membrane stabilizers; and no serious organ involvement. Exclusion criteria were a history of pulmonary exacerbation or upper respiratory infection in the previous 2 months; changes in medications in the last 2 months; a history of hemoptysis in the last 2 months; and colonization with Burkholderia cepacia or mycobacteria. Patients were randomly assigned to receive LR (30 patients) in 5 drops per day (1010 colony-forming units) or placebo (31 patients) for 6 months. Main outcomes were number of episodes of pulmonary exacerbations and hospital admissions for pulmonary exacerbations, number of gastrointestinal and upper respiratory tract infections. FEV1, fecal calprotectin, and cytokine profile in induced sputum and plasma were assessed at baseline and at the end of the trial. Results: Pulmonary exacerbations were significantly reduced in the LR group compared with the placebo group (P < 0.01; odds ratio 0.06 [95% confidence interval {CI} 0–0.40]; number needed to treat 3 [95% CI 2–7]). Similarly, the number of upper respiratory tract infections (in our series only otitis) was significantly reduced in the LR group compared with the placebo group (P < 0.05; odds ratio 0.14 [95% CI 0–0.96]; number needed to treat 6 [95% CI 3–102]). The 2 groups did not differ statistically in the mean number and duration of hospitalizations for pulmonary exacerbations and gastrointestinal infections. There was no significant statistical difference in the mean delta value of FEV1, fecal calprotectin concentration, and tested cytokines (tumor necrosis factor-&agr; and interleukin-8) between the 2 groups. Conclusions: LR reduces pulmonary exacerbations and upper respiratory tract infections in patients with CF with mild-to-moderate lung disease. LR administration may have a beneficial effect on the disease course of CF.