Amit Assa

Vitamin D Deficiency Promotes Epithelial Barrier Dysfunction and Intestinal Inflammation

BACKGROUNDnVitamin D, an important modulator of the immune system, has been shown to protect mucosal barrier homeostasis. This study investigates the effects of vitamin D deficiency on infection-induced changes in intestinal epithelial barrier function in vitro and on Citrobacter rodentium-induced colitis in mice.nnnMETHODSnPolarized epithelial Caco2-bbe cells were grown in medium with or without vitamin D and challenged with enterohemorrhagic Escherichia coli O157:H7. Barrier function and tight junction protein expression were assessed. Weaned C57BL/6 mice were fed either a vitamin D-sufficient or vitamin D-deficient diet and then infected with C. rodentium. Disease severity was assessed by histological analysis, intestinal permeability assay, measurement of inflammatory cytokine levels, and microbiome analysis.nnnRESULTSn1,25(OH)2D3 altered E. coli O157:H7-induced reductions in transepithelial electrical resistance (P < .01), decreased permeability (P < .05), and preserved barrier integrity. Vitamin D-deficient mice challenged with C. rodentium demonstrated increased colonic hyperplasia and epithelial barrier dysfunction (P < .0001 and P < .05, respectively). Vitamin D deficiency resulted in an altered composition of the fecal microbiome both in the absence and presence of C. rodentium infection.nnnCONCLUSIONSnThis study demonstrates that vitamin D is an important mediator of intestinal epithelial defenses against infectious agents. Vitamin D deficiency predisposes to more-severe intestinal injury in an infectious model of colitis.

Probiotic Lactobacillus rhamnosus Inhibits the Formation of Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) are an essential component of the antimicrobial repertoire and represent an effective means by which neutrophils capture, contain, and kill microorganisms. However, the uncontrolled or excessive liberation of NETs also damages surrounding cells and can contribute to disease pathophysiology. Alterations in the gut microbiota, as well as the presence of local and systemic markers of inflammation, are strongly associated with the manifestation of a spectrum of intestinal disorders, including chronic inflammatory bowel disease. Although probiotics exert beneficial effects on gut homeostasis, their direct effect on neutrophils, which are abundant in the setting of intestinal inflammation, remains unclear. In this study, we investigated the effects of nonpathogenic, enteropathogenic, and probiotic bacteria on the dynamics of NET formation. Using murine bone marrow–derived neutrophils and the neutrophil-differentiated human myeloid cell line d.HL-60, we demonstrate for the first time, to our knowledge, that probiotic Lactobacillus rhamnosus strain GG inhibits both PMA- and Staphylococcus aureus–induced formation of NETs. Moreover, probiotic L. rhamnosus strain GG had potent antioxidative activity: dampening reactive oxygen species production and phagocytic capacity of the neutrophils while protecting against cell cytotoxicity. Within the milieu of the gut, this represents a novel mechanism by which probiotics can locally dampen innate immune responses and confer desensitization toward luminal Ags.

Infliximab Maintains Durable Response and Facilitates Catch-up Growth in Luminal Pediatric Crohn's Disease

Background:Infliximab induces and maintains clinical remission in children with Crohns disease (CD), but specifically pediatric long-term data remain sparse. Methods:Patients (N = 195) who received infliximab ± immunomodulator for luminal CD were retrospectively reviewed. Outcomes included clinical response, linear growth, and mucosal healing. Durability of response was assessed using Cox proportional hazards models. Levels of infliximab and antibodies (antibodies to infliximab) were measured when response was lost. Results:Among 195 patients (median age, 13.9 yr; median CD duration, 1.6 yr), 81% experienced complete response (judged by physician global assessment and pediatric Crohns disease activity index ⩽10). Longer duration of diagnosed CD and female gender were associated with lower response. During first year of follow-up, 35% of subjects had regimen individualized through dose escalation/interval shortening. Despite regimen optimization, 16/157 complete responders experienced loss of response at a rate of 2% to 6% per year over 5 years, associated with development of antibodies to infliximab. Concurrent immunomodulation for ≥30 weeks significantly decreased loss of response (hazard ratio = 0.25, 95% confidence interval, 0.08–0.76; P = 0.014). Follow-up endoscopic examination was performed in 40 responders, of whom 22 (73%) demonstrated complete resolution of mucosal ulceration. Patients with growth potential (Tanner 1/2 at induction) demonstrated significant improvements in mean height z-score from induction to years 1 and 2 of follow-up (P < 0.001). With infliximab initiation within the first 18 months after diagnosis, mean height z-score normalized to 0 after 3 years. Conclusions:These data demonstrate sustained effectiveness of infliximab in children and adolescents with luminal CD. Durability of response is increased by concomitant immunomodulation. Clinical response is associated with enhanced linear growth, particularly when therapy is initiated early.

Vitamin D deficiency predisposes to adherent-invasive Escherichia coli-induced barrier dysfunction and experimental colonic injury.

Background:Adherent-invasive Escherichia coli (AIEC) colonization has been strongly implicated in the pathogenesis of Crohns disease. Environmental triggers such as vitamin D deficiency have emerged as key factors in the pathogenesis of inflammatory bowel diseases. The aim of this study was to investigate the effects of 1,25(OH)2D3 on AIEC infection-induced changes in vivo and in vitro. Methods:Barrier function was assessed in polarized epithelial Caco-2-bbe cells grown in medium with or without vitamin D and challenged with AIEC strain LF82. Weaned C57BL/6 mice were fed either a vitamin D–sufficient or –deficient diet for 5 weeks and then infected with AIEC, in the absence and presence of low-dose dextran sodium sulphate. Disease severity was assessed by histological analysis and in vivo intestinal permeability assay. Presence of invasive bacteria was assessed by transmission electron microscopy. Results:Caco-2-bbe cells incubated with 1,25(OH)2D3 were protected against AIEC-induced disruption of transepithelial electrical resistance and tight-junction protein redistribution. Vitamin D–deficient C57BL/6 mice given a course of 2% dextran sodium sulphate exhibited pronounced epithelial barrier dysfunction, were more susceptible to AIEC colonization, and showed exacerbated colonic injury. Transmission electron microscopy of colonic tissue from infected mice demonstrated invasion of AIEC and fecal microbiome analysis revealed shifts in microbial communities. Conclusions:These data show that vitamin D is able to mitigate the deleterious effects of AIEC on the intestinal mucosa, by maintaining intestinal epithelial barrier homeostasis and preserving tight-junction architecture. This study highlights the association between vitamin D status, dysbiosis, and Crohns disease.

Oral microbiome composition changes in mouse models of colitis.

Oral mucosal pathologies are frequent in inflammatory bowel disease (IBD). Since host–microbiome interactions are implicated in the pathogenesis of IBD, in this study the potential for changes affecting the oral microbiome was evaluated using two complementary mouse models of colitis: either chemically (dextran sulfate sodium) or with Citrobacter rodentium infection.

Mucosa-Associated Ileal Microbiota in New-Onset Pediatric Crohn's Disease.

Background:The composition of the intestinal microbiome seems relevant to the pathogenesis of Crohns disease (CD), with differences in both diversity and composition of the gut microbiota in patients with CD compared with healthy individuals. However, there are still conflicting reports on the importance of various bacterial taxa in the pathogenesis of CD. The aim of this study was to characterize the composition of mucosa-associated intestinal microbiota in newly diagnosed pediatric patients with CD. Methods:Mucosa-associated bacteria were identified from ileal biopsy specimens obtained at colonoscopy of 10 patients with either ileal or ileocolonic new-onset CD and 15 controls without mucosal inflammation. Microbial composition was performed by profiling the 16S rDNA V6 region using Illumina sequencing. Samples were analyzed for differences in alpha/beta diversity and also for differentially abundant taxa. Results:Alpha diversity did not differ between the controls and CD cases or between CD subjects with localized ileal disease compared with those with more extensive disease. Controls also did not clearly separate from patients with CD by principal coordinate analyses; however, 117 operational taxonomic units were found to be differentially abundant between the two groups. In particular, numerous operational taxonomic units associated with Faecalibacterium prausnitzii species were increased in children with CD. Conclusions:These findings contribute to emerging evidence regarding dysbiosis in pediatric CD, and provide additional evidence challenging the protective role of F. prausnitzii in CD.

Transforming growth factor-β1 protects against intestinal epithelial barrier dysfunction caused by hypoxia-reoxygenation.

ABSTRACT Intestinal epithelia regulate barrier integrity when challenged by inflammation, oxidative stress, and microbes. Transforming growth factor-&bgr;1 (TGF-&bgr;1) is a cytokine with known beneficial effects on intestinal epithelia, including barrier enhancement, after exposure to proinflammatory cytokines and infectious agents. The aim of this study was to determine whether TGF-&bgr;1 directly protects intestinal epithelia during hypoxia-reoxygenation (HR). Intestinal epithelial monolayers (T84, Caco-2bbe) were exposed to either hypoxia (1% O2, 1 h) or oxidative stress (hydrogen peroxide, 1 mM), followed by normoxic atmosphere for different time points in the absence and presence of varying concentrations of TGF-&bgr;1. Transepithelial electrical resistance (TER) assessed barrier function, with RNA extracted for reverse transcription polymerase chain reaction analysis of GPx-1, HIF-1, heme-oxygenase-1 (HO-1), and NOX-1. In some experiments, intestinal epithelia were exposed to enterohemorrhagic Escherichia coli (EHEC) O157:H7 during the reoxygenation period and TER recorded 7 h after the infectious challenge. Hypoxia-reoxygenation significantly decreased TER in intestinal epithelia compared with normoxic controls. Transforming growth factor-&bgr;1 pretreatment ameliorated HR-induced epithelial barrier dysfunction in T84 (at 1 – 3 h) and Caco-2bbe (1 h) monolayers. Transforming growth factor-&bgr;1 preserved barrier integrity for up to 16 h after challenge with hydrogen peroxide. In TGF-&bgr;1–treated epithelial monolayers, only HO-1 mRNA significantly increased after HR (P < 0.05 vs. normoxic controls). The EHEC-induced epithelial barrier dysfunction was significantly worsened by intestinal HR (P < 0.05 vs. normoxia-EHEC–infected cells), but this was not protected by TGF-&bgr;1 pretreatment. Transforming growth factor-&bgr;1 preserves loss of epithelial barrier integrity caused by the stress of HR via a mechanism that may involve the upregulation of HO-1 transcription. Targeted treatment with TGF-&bgr; could lead to novel therapies in enteric diseases characterized by HR injury.

Perianal Pediatric Crohn Disease Is Associated With a Distinct Phenotype and Greater Inflammatory Burden

Objectives: Data on the outcomes of children with perianal Crohn disease (pCD) are limited, although its presence is often used for justifying early use of biologics. We aimed to assess whether pCD in children is associated with more severe outcomes as found in adults. Methods: Data were extracted from the ImageKids database, a prospective, multicenter, longitudinal cohort study. The study enrolled 246 children at disease onset or thereafter. All patients underwent comprehensive clinical, endoscopic, and radiologic evaluation at enrollment; 98 children had repeat evaluation at 18 months. Results: Of the 234 included patients (mean age 14.2u200a±u200a2.4 years; 131 [56%] boys), 57 (24%) had perianal findings, whereas only 21 (9%) had fistulizing perianal disease. Children with pCD had reduced weight and height z scores compared with non-pCD patients (−0.9 vs −0.35, Pu200a=u200a0.03 and −0.68 vs −0.23, respectively; Pu200a=u200a0.04), higher weighted pediatric CD activity index (32 [interquartile range 16–50] vs 20 [8–37]; Pu200a=u200a0.004), lower serum albumin (3.6u200a±u200a0.7 vs 4.5u200a±u200a0.8, Pu200a=u200a0.016), and higher magnetic resonance enterography global inflammatory score (Pu200a=u200a0.04). Children with pCD had more rectal (57% vs 38%, Pu200a=u200a0.04), and jejunal involvement (31% vs 11% Pu200a=u200a0.003) and a higher prevalence of granulomas (64% vs 23%, Pu200a=u200a0.0001). Magnetic resonance enterography–based damage scores did not differ between groups. Patients with skin tags/fissures only, had similar clinical, endoscopic, and radiologic characteristics as patients with no perianal findings. Conclusions: Pediatric patients with pCD with fistulizing disease have distinct phenotypic features and a predisposition to a greater inflammatory burden.

The Characteristics and Long-term Outcomes of Pediatric Crohnʼs Disease Patients with Perianal Disease

Background: Data on the outcomes of children with perianal Crohns disease are limited. We aimed to assess phenotypic features at diagnosis and long-term disease-specific outcomes of this phenotype. Methods: The medical records of 296 pediatric onset patients with Crohns disease, diagnosed from 2001 to 2015, were reviewed retrospectively. Baseline characteristics included age, sex, severity indices, laboratory data, endoscopic findings, and anthropometric measurements. Main outcome measures included time to first flare, hospitalization, surgery, and biological therapy. Results: Of the 296 included patients (median age 14.2 years), 70 (24%) had nonfistulizing perianal findings, whereas only 40 (13%) had fistulizing perianal disease at diagnosis. Perianal involvement was associated with female sex (P = 0.01), whereas fistulizing perianal disease resulted in a greater use of immunomodulators (P = 0.01). Time to hospitalization was shorter for both nonfistulizing and fistulizing perianal disease (hazard ratio [HR] 1.66 and 1.34, respectively, P = 0.027) and time to biological therapy (HR 2.1 and 1.7, respectively, P = 0.002). There were no differences in time to first flare or surgery. During a median follow-up of 8.5 years, additional 26 patients (10%) developed fistulizing perianal disease after a median time of 3.5 years. The presence of nonfistulizing disease at diagnosis was a significant risk factor for the development of fistulizing perianal disease (HR 3.4, P = 0.002). At the end of follow-up, complicated disease was more common in patients with any perianal involvement (P = 0.01). Conclusions: Pediatric patients with Crohns disease with both nonfistulizing and fistulizing disease have worse clinical outcomes. Nonfistulizing disease is a risk factor for the development of fistulizing disease over time.

Anti-TNFα Treatment After Surgical Resection for Crohnʼs Disease Is Effective Despite Previous Pharmacodynamic Failure

BACKGROUNDnThe outcome of patients with Crohns disease who failed anti-tumor necrosis factor alpha (anti-TNFα) therapy despite adequate serum drug levels (pharmacodynamic failure) is unclear. We aimed to assess such pediatric patients who underwent intestinal resection and were re-treated with the same anti-TNFα agent postoperatively.nnnMETHODSnPediatric patients with Crohns disease who underwent intestinal resection and were treated with anti-TNFα agents postoperatively were assessed retrospectively. Patients were stratified to those with preoperative anti-TNFα pharmacodynamic failure and those with no preoperative anti-TNFα treatment.nnnRESULTSnA total of 53 children were included, 18 with pharmacodynamic failure and 35 controls. Median age at intestinal resection was 14.8 years with 23 (43%) girls. The median time from intestinal resection to anti-TNFα initiation was 8 months (interquartile range 4-14 months). At the time of postoperative anti-TNFα initiation there were no differences in clinical, laboratory, and anthropometric measures between groups. Similar proportions of patients from both groups were in clinical remission on anti-TNFα treatment after 12 months and at the end of follow-up (1.8 years, interquartile range, 1-2.9 years): 89% versus 88.5% and 83% versus 80% for pharmacodynamic failure patients and controls, respectively; P = 0.9. No significant differences were observed at 14 weeks and 12 months of postoperative anti-TNFα treatment including endoscopic remission rate and fecal calprotectin. Both groups significantly improved all measures during postoperative anti-TNFα treatment.nnnCONCLUSIONSnPediatric patients with Crohns disease who failed anti-TNFα therapy despite adequate drug levels and underwent intestinal resection can be re-treated with the same agent for postoperative recurrence with high success rate similar to that of anti-TNFα naive patients.Background: The outcome of patients with Crohns disease who failed anti-tumor necrosis factor alpha (anti-TNF&agr;) therapy despite adequate serum drug levels (pharmacodynamic failure) is unclear. We aimed to assess such pediatric patients who underwent intestinal resection and were re-treated with the same anti-TNF&agr; agent postoperatively. Methods: Pediatric patients with Crohns disease who underwent intestinal resection and were treated with anti-TNF&agr; agents postoperatively were assessed retrospectively. Patients were stratified to those with preoperative anti-TNF&agr; pharmacodynamic failure and those with no preoperative anti-TNF&agr; treatment. Results: A total of 53 children were included, 18 with pharmacodynamic failure and 35 controls. Median age at intestinal resection was 14.8 years with 23 (43%) girls. The median time from intestinal resection to anti-TNF&agr; initiation was 8 months (interquartile range 4–14 months). At the time of postoperative anti-TNF&agr; initiation there were no differences in clinical, laboratory, and anthropometric measures between groups. Similar proportions of patients from both groups were in clinical remission on anti-TNF&agr; treatment after 12 months and at the end of follow-up (1.8 years, interquartile range, 1–2.9 years): 89% versus 88.5% and 83% versus 80% for pharmacodynamic failure patients and controls, respectively; P = 0.9. No significant differences were observed at 14 weeks and 12 months of postoperative anti-TNF&agr; treatment including endoscopic remission rate and fecal calprotectin. Both groups significantly improved all measures during postoperative anti-TNF&agr; treatment. Conclusions: Pediatric patients with Crohns disease who failed anti-TNF&agr; therapy despite adequate drug levels and underwent intestinal resection can be re-treated with the same agent for postoperative recurrence with high success rate similar to that of anti-TNF&agr; naive patients.