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Crohn Disease (CD) is a complex genetic disorder for which more than 140 genes have been identified using genome wide association studies (GWAS). However, the genetic architecture of the trait remains largely unknown. The recent development of machine learning (ML) approaches incited us to apply them to classify healthy and diseased people according to their genomic information. The Immunochip dataset containing 18,227 CD patients and 34,050 healthy controls enrolled and genotyped by the international Inflammatory Bowel Disease genetic consortium (IIBDGC) has been re-analyzed using a set of ML methods: penalized logistic regression (LR), gradient boosted trees (GBT) and artificial neural networks (NN). The main score used to compare the methods was the Area Under the ROC Curve (AUC) statistics. The impact of quality control (QC), imputing and coding methods on LR results showed that QC methods and imputation of missing genotypes may artificially increase the scores. At the opposite, neither the patient/control ratio nor marker preselection or coding strategies significantly affected the results. LR methods, including Lasso, Ridge and ElasticNet provided similar results with a maximum AUC of 0.80. GBT methods like XGBoost, LightGBM and CatBoost, together with dense NN with one or more hidden layers, provided similar AUC values, suggesting limited epistatic effects in the genetic architecture of the trait. ML methods detected near all the genetic variants previously identified by GWAS among the best predictors plus additional predictors with lower effects. The robustness and complementarity of the different methods are also studied. Compared to LR, non-linear models such as GBT or NN may provide robust complementary approaches to identify and classify genetic markers. Tis work was supported by Fondation pour la Recherche Médical (ref DEI20151234405) and Investissements d’Avenir programme ANR-11-IDEX-0005-02, Sorbonne Paris Cite, Laboratoire d’excellence INFLAMEX. Te authors thank the students that participated to the wisdom of the crowd exercise.

Genetic roots of multiple sclerosis The genetics underlying who develops multiple sclerosis (MS) have been difficult to work out. Examining more than 47,000 cases and 68,000 controls with multiple genome-wide association studies, the International Multiple Sclerosis Genetics Consortium identified more than 200 risk loci in MS (see the Perspective by Briggs). Focusing on the best candidate genes, including a model of the major histocompatibility complex region, the authors identified statistically independent effects at the genome level. Gene expression studies detected that every major immune cell type is enriched for MS susceptibility genes and that MS risk variants are enriched in brain-resident immune cells, especially microglia. Up to 48% of the genetic contribution of MS can be explained through this analysis. Science , this issue p. eaav7188 ; see also p. 1383

Anna Negroni,1 Maria Pierdomenico,2 Salvatore Cucchiara,2 Laura Stronati3 1Division of Health Protection Technologies, Territorial and Production Systems Sustainability Department, ENEA, Rome, Italy; 2Department of Pediatrics and Infantile Neuropsychiatry, Pediatric Gastroenterology and Liver Unit, Sapienza University of Rome, Rome, Italy; 3Department of Cellular Biotechnology and Hematology, Sapienza University of Rome, Rome, Italy Abstract: The nucleotide-binding oligomerization domain (NOD) protein, NOD2, belonging to the intracellular NOD-like receptor family, detects conserved motifs in bacterial peptidoglycan and promotes their clearance through activation of a proinflammatory transcriptional program and other innate immune pathways, including autophagy and endoplasmic reticulum stress. An inactive form due to mutations or a constitutive high expression of NOD2 is associated with several inflammatory diseases, suggesting that balanced NOD2 signaling is critical for the maintenance of immune homeostasis. In this review, we discuss recent developments about the pathway and mechanisms of regulation of NOD2 and illustrate the principal functions of the gene, with particular emphasis on its central role in maintaining the equilibrium between intestinal microbiota and host immune responses to control inflammation. Furthermore, we survey recent studies illustrating the role of NOD2 in several inflammatory diseases, in particular, inflammatory bowel disease, of which it is the main susceptibility gene. Keywords: innate immunity, intestinal homeostasis, ER stress, autophagy, inflammatory bowel disease, extraintestinal disease

GWAS have identified >200 risk loci for Inflammatory Bowel Disease (IBD). The majority of disease associations are known to be driven by regulatory variants. To identify the putative causative genes that are perturbed by these variants, we generate a large transcriptome data set (nine disease-relevant cell types) and identify 23,650 cis-eQTL. We show that these are determined by ∼9720 regulatory modules, of which ∼3000 operate in multiple tissues and ∼970 on multiple genes. We identify regulatory modules that drive the disease association for 63 of the 200 risk loci, and show that these are enriched in multigenic modules. Based on these analyses, we resequence 45 of the corresponding 100 candidate genes in 6600 Crohn disease (CD) cases and 5500 controls, and show with burden tests that they include likely causative genes. Our analyses indicate that ≥10-fold larger sample sizes will be required to demonstrate the causality of individual genes using this approach., Most of the more than 200 known genetic risk loci for inflammatory bowel disease (IBD) reside in regulatory regions. Here, the authors provide eQTL datasets for six circulating immune cell types and ileal, colonic and rectal biopsies to map regulatory modules and identify potential causative genes for IBD.

GWAS have identified >200 risk loci for Inflammatory Bowel Disease (IBD). The majority of disease associations are known to be driven by regulatory variants. To identify the putative causative genes that are perturbed by these variants, we generate a large transcriptome data set (nine disease-relevant cell types) and identify 23,650 cis-eQTL. We show that these are determined by ∼9720 regulatory modules, of which ∼3000 operate in multiple tissues and ∼970 on multiple genes. We identify regulatory modules that drive the disease association for 63 of the 200 risk loci, and show that these are enriched in multigenic modules. Based on these analyses, we resequence 45 of the corresponding 100 candidate genes in 6600 Crohn disease (CD) cases and 5500 controls, and show with burden tests that they include likely causative genes. Our analyses indicate that ≥10-fold larger sample sizes will be required to demonstrate the causality of individual genes using this approach. © 2018 The Author(s).

Background: In most European countries advertisements of infant formula are forbidden, while those of follow-on formula are allowed. Recent researches carried out in Australia and Great Britain have suggested that advertisement of toddler formula is used by the producing industry as a line extension to promote infant and follow-on formulas. Objectives: The objective of the study is to assess how advertisements of follow-on formulas are perceived by pregnant women and mothers in Italy. Materials and Methods: The study was carried out in 8 cities of the North, Centre and South of Italy and had two components: 1) a quantitative analysis of 562 self-administered questionnaires for mothers of children under 3 years of age, to explore their exposure to and perception of formula advertisements; 2) a qualitative analysis of 80 in-depth semi-structured interviews to 80 pregnant women, in their 32-36 weeks of gestation with no other children, on their understanding and perception of two advertisements for follow-on formulas. Results: Asked in the self-administered questionnaires whether they had ever come across advertisements of infant formulas, 81% of mothers reported that they had, despite the fact that such advertisements are prohibited by law. The qualitative interviews to pregnant women showed their inability to identify the advertised products at first glance due to the ambiguity of the numeral 2 and the presumed age of the portrayed babies; this confusion did not disappear after careful observation of the advertisements and reading of the text. Conclusions: Advertisements of follow-on formulas are perceived by many pregnant women and mothers as promoting infant formulas.

We assembled and analyzed genetic data of 47,351 multiple sclerosis (MS) subjects and 68,284 control subjects and establish a reference map of the genetic architecture of MS that includes 200 autosomal susceptibility variants outside the major histocompatibility complex (MHC), one chromosome X variant, and 32 independent associations within the extended MHC. We used an ensemble of methods to prioritize up to 551 potentially associated MS susceptibility genes, that implicate multiple innate and adaptive pathways distributed across the cellular components of the immune system. Using expression profiles from purified human microglia, we do find enrichment for MS genes in these brain - resident immune cells. Thus, while MS is most likely initially triggered by perturbation of peripheral immune responses the functional responses of microglia and other brain cells are also altered and may have a role in targeting an autoimmune process to the central nervous system.One Sentence Summary:We report a detailed genetic and genomic map of multiple sclerosis, and describe the role of putatively affected genes in the peripheral immune system and brain resident microglia.

Dose-response curves were measured for the induction of chromosomal aberrations in peripheral blood lymphocytes after acute exposure in vitro to 60Co gamma rays. Blood was obtained from four different healthy donors, and chromosomes were either observed at metaphase, following colcemid accumulation, or prematurely condensed by calyculin A. Cells were analysed in three different Italian laboratories. Chromosomes 1, 2, and 4 were painted, and simple-type interchanges between painted and non-painted chromosomes were scored in cells exposed in the dose range 0.1-3.0 Gy. The chemical-induced premature chromosome condensation method was also used combined with chromosome painting (chromosome 4 only) to determine calibration curves for high dose exposures (up to 20 Gy X rays). Calibration curves described in this paper will be used in our laboratories for biological dosimetry by fluorescence in situ hybridisation.

Objectives The gut-liver axis has been recently investigated in depth in relation to intestinal and hepatic diseases. Key actors are bile acid (BA) receptors, as farnesoid-X-receptor (FXR), pregnane-X-receptor (PXR), and G-protein-coupled-receptor (GPCR; TGR5), that control a broad range of metabolic processes as well as inflammation and fibrosis. The present study aims to investigate the impact of intestinal inflammation on liver health with a focus on FXR, PXR, and TGR5 expression. The strategy to improve liver health by reducing gut inflammation is also considered. Modulation of BA receptors in the inflamed colonic tissues of inflammatory bowel disease (IBD) pediatric patients is analyzed. Methods A dextran sodium sulphate (DSS) colitis animal model was built. Co-cultures with Caco2 and HepG2 cell lines were set up. Modulation of BA receptors in biopsies of IBD pediatric patients was assessed by real-time PCR and immunohistochemistry. Results Histology showed inflammatory cell infiltration in the liver of DSS mice, where FXR and PXR were significantly decreased and oxidative stress was increased. Exposure of Caco2 to inflammatory stimuli resulted in the reduction of BA receptor expression in HepG2. Caco2 treatment with dipotassium glycyrrhizate (DPG) reduced these effects on liver cells. Inflamed colon of patients showed altered FXR, PXR, and TGR5 expression. Conclusions This study strongly suggests that gut inflammation affects hepatic cells by altering BA receptor levels as well as increasing the production of pro-inflammatory cytokines and oxidative stress. Hence, reducing gut inflammation is needed not only to improve the intestinal disease but also to protect the liver.

Gut mucosal healing (MH) is considered a key therapeutic target and prognostic parameter in the management of inflammatory bowel disease (IBD). The dipotassium glycyrrhizate (DPG), a salt of the glycoconjugated triterpene glycyrrhizin, has been shown to inhibit the High Mobility Group Box 1 (HMGB1) protein, an allarmin strongly implicated in the pathogenesis of most inflammatory and auto-immune disorders. Here we discuss new insights on how DPG acts on MH comparing the acute phase and the recovery phase from experimental colitis in mice. We found that DPG strongly accelerates MH by differently regulating pro-inflammatory (CXCL1, CXCL3, CXCL5, PTGS2, IL-1β, IL-6, CCL12, CCL7) and wound healing (COL3A1, MMP9, VTN, PLAUR, SERPINE, CSF3, FGF2, FGF7, PLAT, TIMP1) genes as observed only during the recovery phase of colitis. Relevant issue is the identification of extracellular matrix (ECM) remodeling genes, VTN, and PLAUR, as crucial genes to achieve MH during DPG treatment. Furthermore, a noticeable recovery of intestinal epithelial barrier structural organization, wound repair ability, and functionality is observed in two human colorectal adenocarcinoma cell lines exposed to DPG during inflammation. Thus, our study identifies DPG as a potent tool for controlling intestinal inflammation and improving MH.

Background and Aims Recent evidence implicates gut microbiota (GM) and immune alterations in autism spectrum disorders (ASD). We assess GM profile and peripheral levels of immunological, neuronal and bacterial molecules in ASD children and controls. Alarmin HMGB1 was explored as a non-invasive biomarker to monitor gastrointestinal (GI) symptoms. Methods Thirty ASD children and 14 controls entered into the study. GM metagenomic analysis was performed for 16 ASD patients and 7 controls. GM functional profile was assessed by GO term analysis. Blood levels of IL-1β, TNFα, TGFβ, IL-10, INFγ, IL-8, lipopolysaccharide, Neurotensin, Sortilin1 and GSSG/GSH ratio were analyzed in all subjects by ELISA. Fecal HMGB1 was analyzed by Western blot. Results We observed a significant decrease in bacterial diversity. Furthermore, 82 GO terms underrepresented in ASD. Four of them pointed at 3,3 phenylpropionate catabolism and were imputable to Escherichia coli (E. coli) group. Serum levels of TNFα, TGFβ, NT, and SORT-1 increased in ASD patients. Fecal levels of HMGB1 correlated with GI sign severity in ASD children. Conclusions We suggest that a decrease of E. coli might affect the propionate catabolism in ASD. We report occurrence of peripheral inflammation in ASD children. We propose fecal HMGB1 as a non-invasive biomarker to detect GI symptoms.

Non-alcoholic fatty liver disease (NAFLD) is currently considered the main cause of chronic liver disease worldwide. Mechanisms leading to the development and progression of this disease are topics of great interest for researchers and clinicians. The current multi-hit hypothesis has thrown the crosstalk between liver and adipose tissue into sharp focus. It is well known that adipose tissue produces circulating factors, known as adipocytokines, which exert several effects on liver cells, promoting the onset of NAFLD and its progression to non-alcoholic steatohepatitis in obese subjects. In a similar way, hepatocytes may also respond to obesogenic stimuli by producing and releasing hepatokines into the circulation. Here, the authors provide an overview of recent advances in our understanding of the role of the most relevant adipocytokines and hepatokines in NAFLD pathogenesis, highlighting their possible molecular and functional interactions. © 2015 Taylor & Francis.

Necroptosis is a caspases-independent form of programmed cell death exhibiting intermediate features between necrosis and apoptosis. Albeit some physiological roles during embryonic development, tissue homeostasis and innate immune response are documented, necroptosis is mainly considered a pro-inflammatory cell death. Key actors of necroptosis are the receptor-interacting-protein-kinases, RIPK1 and RIPK3, and their target, the mixed-lineage-kinase-domain-like protein, MLKL. The intestinal epithelium has one of the highest rates of cellular turnover in a process that is tightly regulated. Altered necroptosis at the intestinal epithelium leads to uncontrolled microbial translocation and deleterious inflammation. Indeed, necroptosis has been associated to chronic inflammatory diseases and cancer. Drugs that inhibit necroptosis could, therefore, be used therapeutically for the treatment of these diseases, and researches to develop such inhibitors are already underway. In this Review, we outline pathways for necroptosis and its role in chronic inflammation and cancer. We also discuss current and developing therapies that target necroptosis machinery.

Lipopolysaccharide (LPS) is currently considered one of the major players in non-alcoholic fatty liver disease (NAFLD) pathogenesis and progression. Here, we aim to investigate the possible role of LPS-induced TNF-α factor (LITAF) in inducing a pro-inflammatory and pro-fibrogenic phenotype of non-alcoholic steatohepatitis (NASH). We found that children with NAFLD displayed, in different liver-resident cells, an increased expression of LITAF which correlated with histological traits of hepatic inflammation and fibrosis. Total and nuclear LITAF expression increased in mouse and human hepatic stellate cells (HSCs). Moreover, LPS induced LITAF-dependent transcription of IL-1β, IL-6 and TNF-α in the clonal myofibroblastic HSC LX-2 cell line, and this effect was hampered by LITAF silencing. We showed, for the first time in HSCs, that LITAF recruitment to these cytokine promoters is LPS dependent. However, preventing LITAF nuclear translocation by p38MAPK inhibitor, the expression of IL-6 and TNF-α was significantly reduced with the aid of p65NF-ĸB, while IL-1β transcription exclusively required LITAF expression/activity. Finally, IL-1β levels in plasma mirrored those in the liver and correlated with LPS levels and LITAF-positive HSCs in children with NASH. In conclusion, a more severe histological profile in paediatric NAFLD is associated with LITAF over-expression in HSCs, which in turn correlates with hepatic and circulating IL-1β levels outlining a panel of potential biomarkers of NASH-related liver damage. The in vitro study highlights the role of LITAF as a key regulator of the LPS-induced pro-inflammatory pattern in HSCs and suggests p38MAPK inhibitors as a possible therapeutic approach against hepatic inflammation in NASH.

IBD includes two classic entities, Crohn's disease and ulcerative colitis, and a third undetermined form (IBD-U), characterized by a chronic relapsing course resulting in a high rate of morbidity and impaired quality of life. Children with IBD are vulnerable in terms of growth failure, malnutrition and emotional effects. The aims of therapy have now transitioned from symptomatic control to the achievement of mucosal healing and deep remission. This type of therapy has been made possible by the advent of disease-modifying drugs, such as biologic agents, which are capable of interrupting the inflammatory cascade underlying IBD. Biologic agents are generally administered in patients who are refractory to conventional therapies. However, there is growing support that such agents could be used in the initial phases of the disease, typically in paediatric patients, to interrupt and cease the inflammatory process. Until several years ago, most therapeutic programmes in paediatric patients with IBD were borrowed from adult trials, whereas paediatric studies were often retrospective and uncontrolled. However, guidelines on therapeutic management of paediatric IBD and controlled, prospective, randomized trials including children with IBD have now been published. Here, the current knowledge concerning treatment options for children with IBD are reported. We also highlight the effectiveness and safety of new therapeutic advances in these paediatric patients.© 2014 Macmillan Publishers Limited. All rights reserved.

Non-alcoholic fatty liver disease is one of the most important causes of liver-related morbidity in children. In non-alcoholic fatty liver disease, the activation of liver resident macrophage pool is a central event in the progression of liver injury. The aims of the present study were to evaluate the polarization of liver macrophages and the possible role of Wnt3a production by macrophages in hepatic progenitor cell response in the progression of pediatric nonalcoholic fatty liver disease. 32 children with biopsy-proven non-alcoholic fatty liver disease were included. 20 out of 32 patients were treated with docosahexaenoic acid for 18 months and biopsies at the baseline and after 18 months were included. Hepatic progenitor cell activation, macrophage subsets and Wnt/β-catenin pathway were evaluated by immunohistochemistry and immunofluorescence. Our results indicated that in pediatric non-alcoholic fatty liver disease, pro-inflammatory macrophages were the predominant subset. Macrophage polarization was correlated with Non-alcoholic fatty liver disease Activity Score, ductular reaction, and portal fibrosis; docosahexaenoic acid treatment determined a macrophage polarization towards an anti-inflammatory phenotype in correlation with the reduction of serum inflammatory cytokines, with increased macrophage apoptosis, and with the upregulation of macrophage Wnt3a expression; macrophage Wnt3a expression was correlated with β-catenin phosphorylation in hepatic progenitor cells and signs of commitment towards hepatocyte fate. In conclusion, macrophage polarization seems to have a key role in the progression of pediatric non-alcoholic fatty liver disease; the modulation of macrophage polarization could drive hepatic progenitor cell response by Wnt3a production. © 2016 Carpino et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Background: Large evidence supports the role of microRNAs as new important inflammatory mediators by regulating both the adaptive and innate immunity. In the present study, we speculated that miR-320 controls NOD2 (nucleotide-binding oligomerization domain) expression, because it contains multiple binding sites in the 3′-untranslated region of the gene. NOD2, the first gene associated to increased susceptibility to Crohn's disease, is a cytosolic receptor that senses wall peptides of bacteria and promotes their clearance through initiation of a proinflammatory transcriptional program. This study aims at demonstrating that NOD2 is a target of miR-320 as well as investigating the role of inflammation in modulating the miR-320 control on NOD2 expression and analyzing miR-320 expression in intestinal biopsies of children with inflammatory bowel disease. Methods: The colonic adenocarcinoma cell line HT29 was used to assess the miR-320-mediated regulation of NOD2 expression. MiR-320 and NOD2 expression were analyzed in mucosal samples of 40 children with inflammatory bowel disease. Results: During inflammation, NOD2 expression is inversely correlated with miR-320 expression in vitro and ex vivo. Exogenous miR-320 transfection in HT29 cells leads to a significant decrease of NOD2 expression, whereas the miR-320 inhibitor transfection leads to increase of NOD2 expression, nuclear translocation of nuclear factor B, and activation of downstream cytokines. Conclusions: We show for the first time that NOD2 expression is under the control of miR-320. We also show in vitro and ex vivo that inflammation induces a decrease of miR-320 and the latter correlates negatively with NOD2 expression. © 2016 Crohn's & Colitis Foundation of America, Inc.

Background: Krill oil is a marine derived oil rich in phospholipids, astaxanthin and omega-3 fatty acids. Several studies have found benefits of krill oil against oxidative and inflammatory damage. Aims: We aimed at assessing the ability of krill oil to reduce intestinal inflammation by improving epithelial barrier integrity, increasing cell survival and reducing pathogenicity of adherent-invasive Escherichia coli. Methods: CACO2 and HT29 cells were exposed to cytomix (TNFα and IFNγ) to induce inflammation and co-exposed to cytomix and krill oil. E-cadherin, ZO-1 and F-actin levels were analyzed by immunofluorescence to assess barrier integrity. Scratch test was performed to measure wound healing. Cell survival was analyzed by flow cytometry. Adherent-invasive Escherichia coli LF82 was used for adhesion/invasion assay. Results: In inflamed cells E-cadherin and ZO-1 decreased, with loss of cell-cell adhesion, and F-actin polymerization increased stress fibres; krill oil restored initial conditions and improved wound healing, reduced bacterial adhesion/invasion in epithelial cells and survival within macrophages; krill oil reduced LF82-induced mRNA expression of pro-inflammatory cytokines. Conclusions: Krill oil improves intestinal barrier integrity and epithelial restitution during inflammation and controls bacterial adhesion and invasion to epithelial cells. Thus, krill oil may represent an innovative tool to reduce intestinal inflammation. © 2015 Editrice Gastroenterologica Italiana S.r.l.

Intestinal epithelial cells (IECs) form a physiochemical barrier that separates the intestinal lumen from the host's internal milieu and is critical for electrolyte passage, nutrient absorption, and interaction with commensal microbiota. Moreover, IECs are strongly involved in the intestinal mucosal inflammatory response as well as in mucosal innate and adaptive immune responses. Cell death in the intestinal barrier is finely controlled, since alterations may lead to severe disorders, including inflammatory diseases. The emerging picture indicates that intestinal epithelial cell death is strictly related to the maintenance of tissue homeostasis. This review is focused on previous reports on different forms of cell death in intestinal epithelium. © 2015 Anna Negroni et al.

Aims Oxidative stress and inflammation are always associated. Appropriate management of oxidative mediators may represent a therapeutic strategy to reduce inflammation, and use of antioxidant can be protective against inflammatory diseases. Glycyrrhizin (GL) plays an anti-inflammatory and antioxidant effect by inhibiting high mobility group box 1 (HMGB1) or 11-β-hydroxysteroid dehydrogenase type II (11βHSD2) enzyme. In this study, the potential role of dipotassium glycyrrhizate (DPG), a salt of GL, to reduce oxidative stress in intestinal inflammatory condition was investigated in vivo and the mechanism of action of DPG was studied in vitro. Results In a colitis mouse model DPG affected oxidative stress reducing iNOS and COX-2 expression, as well as NO and PGE2 levels. By means of LPS-stimulated macrophages we found that DPG inhibited the expression of pro-inflammatory cytokines and reduced iNOS and COX-2 expression in a time dependent manner, through two different ways of signal. DPG reduced, at a later time, both iNOS and COX-2, through a mechanism HMGB1-dependent, and at an earlier time only COX-2, through a mechanism AMP-activated kinase (AMPK)-phosphorylation-mediated. Conclusion DPG has a protective effect on colitis and inflammation through the inhibition of oxidative stress. This study clarifies the two-ways mechanism by which DPG inhibits iNOS and COX-2 during inflammation and demonstrates for the first time that AMPK is a target of DPG. Uncovering this mechanism is significant to clarify the relationship between energy homeostasis and anti-oxidative responses and suggests that DPG could play a relevant role in the development of new therapy against inflammatory diseases associated to oxidative stress. © 2015 Elsevier Inc. All rights reserved.

Objectives: Recently published works showed that occupational exposure to antineoplastic drugs (ANPD) is still frequent in hospital settings, despite significant safety policy improvements. The aim of this study was to assess the current level of occupational exposure to ANPD and any potentially associated cytogenetic damages in hospital nurses routinely handling ANPD. Methods: Occupationally ANPD-exposed (n = 71) and ANPD-unexposed (n = 77; control) nurses were recruited on a voluntary basis from five hospitals in Northern and Central Italy. Evaluation of surface contamination and dermal exposure to ANPD was assessed by determining cyclophosphamide (CP) on selected surfaces (wipes) and on exposed nurses’ clothes (pads). The concentration of unmetabolized CP—as a biomarker of internal dose—was measured in end-shift urine samples. Biomonitoring of genotoxic effects (i.e., biological effect monitoring) was conducted by analyzing micronuclei (MN) and chromosome aberrations (CA) in peripheral blood lymphocytes. Genetic polymorphisms for enzymes involved in metabolic detoxification (i.e., glutathione S-transferases) were analyzed as well. Results: We observed a significant increase in MN frequency (5.30 ± 2.99 and 3.29 ± 1.97; mean values ± standard deviation; p

Background: Endoplasmic reticulum stress and unfolded protein response have been recently associated with the development of inflammatory bowel diseases in adults. We aimed at assessing the involvement of these pathways also in paediatric inflammatory bowel disease by analysing the expression of the main genes involved in endoplasmic reticulum stress and correlating them with the degree of intestinal inflammation. Methods: Real-time PCR and Western blot analysis of the expression of the endoplasmic reticulum stress marker HSPA5 and of selected genes representing the three pathways of unfolded protein response (IRE-XBP1, PERK-ATF4, ATF6p90-p50) in inflamed and uninflamed biopsies from 28 inflammatory bowel disease paediatric patients and 10 controls. Results: HSPA5, PDIA4, as well as unspliced and spliced XBP1 mRNAs were significantly increased in patients' inflamed colonic mucosa compared to uninflamed mucosa and controls. HSPA5, PDIA4, ATF6, and phospho-IRE proteins were also upregulated, indicating the activation of the IRE-XBP1 and ATF6p90-p50 branches of unfolded protein response. A positive significant correlation between interleukin-8 levels, as a marker of inflammation, and upregulated genes was found in the inflamed colonic mucosa. Conclusion: A deregulation of the genes involved in the endoplasmic reticulum stress and unfolded protein response pathways may be a key component of the inflammatory response in paediatric patients with inflammatory bowel disease. © 2014 Editrice Gastroenterologica Italiana S.r.l.

Neuroimmune interactions and inflammation have been proposed as factors involved in sensory-motor dysfunction and symptom generation in adult irritable bowel syndrome (IBS) patients. In children with IBS and healthy controls, we measured ileocolonic mast cell infiltration and fecal calprotectin and evaluated the relationships between these parameters and abdominal pain symptoms and stooling pattern.Irritable bowel syndrome patients diagnosed according to Pediatric Rome III criteria and healthy controls kept a 2-week pain/stooling diary. Ileocolonic mucosal mast cells (MC) and MC in close proximity to nerve fibers (MC-NF) were identified immunohistochemically and quantified. Fecal calprotectin concentration was measured.21 IBS patients and 10 controls were enrolled. The MC-NF count was significantly higher in the ileum (p = 0.01), right colon (p = 0.04), and left colon (p0.001) of IBS patients compared with controls. No differences in fecal calprotectin concentration were noted. Abdominal pain intensity score correlated with ileal MC count (r(s) = 0.47, p = 0.030) and right colon MC-NF count (r(s) = 0.52, p = 0.015). In addition, children with IBS with3 abdominal pain episodes/week had greater ileal (p = 0.002) and right colonic (p = 0.01) MC counts and greater ileal (p = 0.05) and right colonic (p = 0.016) MC-NF counts than children with less frequent pain. No relationship was found between MC and MC-NF and fecal calprotectin or stooling pattern.Mast cells-nerve fibers counts are increased in the ileocolonic mucosa of children with IBS. Mast cells and MC-NF counts are related to the intensity and frequency of abdominal pain.

Non-alcoholic fatty liver disease (NAFLD) affects 3-12% of the general pediatric population. HMGB1 protein is presently considered a potent inflammatory mediator in several liver diseases, even if its role in NAFLD is still unknown in clinical studies. Here we investigated the relationships between circulating HMGB1, TGF-β and MCP-1 and liver damage in pediatric NAFLD. HMGB1, TGF-β and MCP-1 plasma levels were measured in 110 obese children with biopsy-proven NAFLD and 40 age-matched obese controls. HMGB1, TGF-β and MCP-1, ALT, AST and cholesterol plasma levels were significantly higher in NAFLD than in control children. A significant association between increased levels of HMGB1, TGF-β and MCP-1 and high degrees of fibrosis was found. In this study, we showed for the first time that circulating levels of HMGB1 were raised in children with NAFLD and strongly correlated with fibrosis and systemic inflammation. © 2014 Informa UK, Ltd.

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 Crohn's 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 Crohn's Disease [SES-CD], endoscopic Mayo subscore), but not with the disease activity indexes (Crohn's 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. Copyright © 2014 Crohn's & Colitis Foundation of America, Inc.

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 (10 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

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 Crohn's 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

Background: Crohn's disease is a multifactorial disease in which an aberrant immune response to commensal intestinal microbiota leads to chronic inflammation. The small intestine of patients with Crohn's disease is colonized by a group of adherent-invasive Escherichia coli strongly able to adhere and invade intestinal epithelial cells lactoferrin is an iron-binding glycoprotein known to have anti-bacterial and anti-inflammatory activities. Aims: We explore the ability of bovine lactoferrin to modulate the interactions between the adherent-invasive E. coli strain LF82 and intestinal epithelial cells as well as the inflammatory response. Methods: Bacterial adhesion and invasion assays were used to assess the antimicrobial activity of lactoferrin. Electron microscopy was used to characterize bacteria-cell interactions. The mRNA expression of pro-inflammatory cytokines was measured both in cultured cells and in biopsies taken from intestine of patients affected by Crohn's disease. Results: Lactoferrin inhibited bacterial invasion through minimally affecting adhesion. This divergence was due to a mannose-dependent lactoferrin binding to the bacterial type 1 pili and consequent bacterial aggregation on the intestinal epithelial cell surface. Expression of pro-inflammatory cytokines, such as TNF-alpha, IL-8, and IL-6, was markedly inhibited by lactoferrin both in cultured and Crohn-derived intestinal cells. Conclusions: Bovine lactoferrin might function via an antibacterial and/or anti-inflammatory mechanism in the treatment of Crohn's disease. © 2014 Editrice Gastroenterologica Italiana S.r.l.

BackgroundRecent 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.MethodsWe 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.ResultsSignificant 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, PConclusionsCommon 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.

Background Some industrial hygiene studies have assessed occupational exposure to antineoplastic drugs; other epidemiological investigations have detected various toxicological effects in exposure groups labeled with the job title. In no research has the same population been studied both environmentally and epidemiologically. The protocol of the epidemiological study presented here uses an integrated environmental and biological monitoring approach. The aim is to assess in hospital nurses preparing and/or administering therapy to cancer patients the current level of occupational exposure to antineoplastic drugs, DNA and chromosome damage as cancer predictive effects, and the association between the two. Methods/Design About 80 healthy non-smoking female nurses, who job it is to prepare or handle antineoplastic drugs, and a reference group of about 80 healthy non-smoking female nurses not occupationally exposed to chemicals will be examined simultaneously in a cross-sectional study. All the workers will be recruited from five hospitals in northern and central Italy after their informed consent has been obtained. Evaluation of surface contamination and dermal exposure to antineoplastic drugs will be assessed by determining cyclophosphamide on selected surfaces (wipes) and on the exposed nurses' clothes (pads). The concentration of unmetabolized cyclophosphamide as a biomarker of internal dose will be measured in end-shift urine samples from exposed nurses. Biomarkers of effect and susceptibility will be assessed in exposed and unexposed nurses: urinary concentration of 8-hydroxy-2-deoxyguanosine; DNA damage detected using the single-cell microgel electrophoresis (comet) assay in peripheral white blood cells; micronuclei and chromosome aberrations in peripheral blood lymphocytes. Genetic polymorphisms for enzymes involved in metabolic detoxification (i.e. glutathione S-transferases) will also be analysed. Using standardized questionnaires, occupational exposure will be determined in exposed nurses only, whereas potential confounders (medicine consumption, lifestyle habits, diet and other non-occupational exposures) will be assessed in both groups of hospital workers. Statistical analysis will be performed to ascertain the association between occupational exposure to antineoplastic drugs and biomarkers of DNA and chromosome damage, after taking into account the effects of individual genetic susceptibility, and the presence of confounding exposures. Discussion The findings of the study will be useful in updating prevention procedures for handling antineoplastic drugs.

We undertook a meta-analysis of six Crohn's disease genome-wide association studies (GWAS) comprising 6,333 affected individuals (cases) and 15,056 controls and followed up the top association signals in 15,694 cases, 14,026 controls and 414 parent-offspring trios. We identified 30 new susceptibility loci meeting genome-wide significance (P < 5 x 10(-8)). A series of in silico analyses highlighted particular genes within these loci and, together with manual curation, implicated functionally interesting candidate genes including SMAD3, ERAP2, IL10, IL2RA, TYK2, FUT2, DNMT3A, DENND1B, BACH2 and TAGAP. Combined with previously confirmed loci, these results identify 71 distinct loci with genome-wide significant evidence for association with Crohn's disease.

The p53 gene regulates cell cycle and apoptotic pathways after induction of DNA damage. Telomeres, capping chromosome ends, are involved in maintaining chromosome stability; alterations of their length have been related to increased levels of chromosomal aberrations. To study a possible interaction between chromosome aberrations, telomere dysfunction, and p53, we investigated via painting analysis the induction and persistence of chromosome aberrations in bone marrow and spleen cells of p53+/− (and wild type) mice exposed for 4, 13, or 26 weeks to 2 mg/kg melphalan (MLP), a chemotherapeutic agent with carcinogenic potential. In addition, telomere length was evaluated in bone marrow cells by quantitative fluorescence in situ hybridization (Q-FISH). Chromosome aberrations were significantly increased in both tissues after MLP treatment. The p53 genotype did not influence the response of spleen cells, whereas a slight but significant increase of the aberration frequency was measured in the bone marrow of p53+/− mice exposed to MLP for 13 weeks with respect to the level detected in the matched wild-type group. The main finding of our still preliminary results on telomere length modulation was again a difference between the two genotypes. In bone marrow cells of wild-type mice, MLP treatment was associated with telomere shortening, while in p53+/− mice telomere elongation was the prevalent response to MLP exposure. In agreement with previous literature data, our in vivo study suggests that even the lack of a single functional copy of the p53 gene might have an impact on the quantity and quality of chromosome alterations induced in cycling cells by a clastogenic exposure. Environ. Mol. Mutagen., 2008. © 2008 Wiley-Liss, Inc.

Chromosomal aberrations are a measure of genomic instability, which is known to play a key role in the initiation and promotion of carcinogenesis. Stable reciprocal translocations are of particular importance since they are often involved in neoplastic transformation and tumor cell clonal evolution. In this study, chromosome painting analysis was used to test for stable aberrations induced in the bone marrow of C57BL/6J and FVB mice exposed for 4 weeks to 2 or 4 mg/kg of melphalan (MLP), a chemotherapeutic agent with carcinogenic potential. To compare the chemical-induced damage in different tissues, chromosome aberrations were also analyzed by chromosome painting in the spleen of C57BL/6J mice. At the 2 mg/kg dose, MLP induced comparable levels of chromosome- type aberrations in bone marrow cells of both mouse strains and in splenocytes of C57BL/ 6J mice. At 4 mg/kg, no further increase in aberrations was detected in bone marrow, while a dose-effect relationship was found in spleen cells. This different response may result from a negative selection against highly damaged bone marrow cells during mitotic proliferation. The results indicate that chromosome painting is a useful tool for detecting stable chromosome aberrations in somatic cells exposed to MLP and possibly to other genotoxic chemical carcinogens.