Hien Q. Huynh

Helicobacter pylori Activates Toll-Like Receptor 4 Expression in Gastrointestinal Epithelial Cells

ABSTRACT Helicobacter pylori activates the transcription factor NF-κB, leading to proinflammatory cytokine production by gastric epithelial cells. However, the receptors for the initial bacterial interaction with host cells which activate downstream signaling events have not been completely defined. Recently, it has been shown that microbial components activate Toll-like receptors (TLRs), thereby leading to AP-1- and NF-κB-dependent transcription and resulting in the production of proinflammatory cytokines. The aim of this study was to determine whether H. pylori activates TLR4. Reverse transcription-PCR showed that both type I and type II H. pylori clinical isolates induced TLR4 mRNA expression in AGS cells compared with that by uninfected controls. H. pylori upregulated TLR4 protein expression in two gastric epithelial cell lines (AGS and MKN45) and one intestinal epithelial cell line (T84). Monoclonal TLR4 antibody inhibited lipopolysaccharide-induced interleukin-8 secretion from THP-1 macrophages but not from gastric epithelial cells infected with H. pylori. H. pylori demonstrated increased adherence to CHO TLR4-transfected cells compared with that to both CHO TLR2-transfected and nontransfected CHO cells (P < 0.01). These results indicate that H. pylori activates TLR4 expression in epithelial cells and that TLR4 can serve as a receptor for H. pylori binding.

Probiotic preparation VSL#3 induces remission in children with mild to moderate acute ulcerative colitis: A pilot study

Background: Ulcerative colitis (UC) is a form of inflammatory bowel disease (IBD) that has periods of exacerbated symptoms and periods that are symptom‐free. The treatment of active UC with probiotic bacteria could possibly induce remission. We evaluated the clinical efficacy and safety profile of probiotic preparation VSL#3 in the treatment of mild to moderate acute UC in the pediatric population. Methods: Eighteen eligible patients between the ages of 3–17 with mild to moderate acute UC received open‐label VSL#3 daily in 2 divided doses for 8 weeks. The disease activity pre‐ and post‐VSL#3 therapy was assessed by the simple clinical colitis activity index (SCCAI); Mayo ulcerative colitis endoscopic score; inflammatory markers: erythrocyte sedimentation rate (ESR) and C‐reactive protein (CRP); serum cytokine profiling; and rectal tissue microbial profiling done at baseline and at week 8. Results: Thirteen patients completed 8 weeks of VSL#3 treatment and 5 patients were withdrawn due to lack of improvement. Remission (defined as SCCAI ≤3) was achieved in 56% of children (n = 10); response (decrease in SCCAI ≥2, but final score ≤5) in 6% (n = 1); and no change or worsening in 39% (n = 7). Post‐VSL#3 treatments demonstrated a bacterial taxonomy change in rectal biopsy. The VSL#3 was well tolerated in clinical trials and no biochemical and clinical adverse effects attributed to VSL#3 were identified. Conclusions: Treatment of pediatric patients diagnosed with mild to moderate UC with VSL#3 resulted in a remission rate of 56% and a combined remission/response rate of 61%.

Mucosal Barrier Depletion And Loss Of Bacterial Diversity Are Primary Abnormalities In Paediatric Ulcerative Colitis

BACKGROUND AND AIMS Ulcerative colitis [UC] is associated with colonic mucosa barrier defects and bacterial dysbiosis, but these features may simply be the result of inflammation. Therefore, we sought to assess whether these features are inherently abrogated in the terminal ileum [TI] of UC patients, where inflammation is absent. METHODS TI biopsies from paediatric inflammatory bowel disease [IBD] subsets [Crohns disease [CD; n = 13] and UC [n = 10]], and non-IBD disease controls [n = 12] were histologically graded, and alcian blue/periodic acid-Schiff stained biopsies were quantified. The mucosal barrier was assessed for mucin [MUC2], immunoglobulin [Ig]A, IgG, and total bacteria (fluorescence in-situ hybridisation [FISH probe EUB338]) by immunofluorescence. The regulation of mucin secretion was investigated by NLRP6 gene expression and immunofluorescence. The composition of the active mucosa-associated microbiota was explored by sequencing the 16S rRNA amplicon generated from total RNA. RESULTS Despite the absence of ileitis, UC patients displayed ileal barrier depletion illustrated by reductions in mucin-containing goblet cells and mucin production and altered epithelial NLRP6 expression. In both CD patients with ileitis and UC patients with normal histology, bacteria coated with IgA and IgG penetrated the TI mucin layer. Biopsy 16S rRNA sequencing revealed a reduction in α-diversity by three methods [Shannon, Simpson, and Equitability indices] between UC and non-IBD paediatric patients. CONCLUSIONS These findings suggest an underlying defect in the UC-afflicted intestinal tract even in the absence of inflammation, implicating barrier and microbial changes as primary abnormalities in UC that may play a causative role in disease development.

Higher activity of the inducible nitric oxide synthase contributes to very early onset inflammatory bowel disease.

OBJECTIVES:The NOS2 gene encodes for the inducible nitric oxide synthase (iNOS), responsible for nitric oxide (NO) production, which contributes to antimicrobial and antipathogenic activities. Higher levels of both iNOS and NO-induced damage have been observed in inflammatory bowel disease (IBD) patients. NOS2 may have a role in a specific subset of IBD patients with severe and/or extensive colitis. Therefore, the aim of this study is to examine the role of NOS2 in such a subset, very early onset IBD (VEO-IBD).METHODS:Seventeen tag single nucleotide polymorphisms (SNPs) in the NOS2 gene were successfully genotyped in VEO-IBD patients. Genetic associations were replicated in an independent VEO-IBD cohort. Functional analysis for iNOS activity was performed on the most significantly associated functional variant.RESULTS:The NOS2 rs2297518 SNP was found to be associated in VEO-IBD in two independent cohorts. Upon combined analysis, a coding variant (S608L) showed the strongest association with VEO-IBD (Pcombined=1.13 × 10−6, OR (odds ratio)=3.398 (95% CI (confidence interval) 2.02–5.717)) as well as associations with VEO-Crohn’s disease and VEO-ulcerative colitis (UC). This variant also showed an association with UC diagnosed between 11 and 17 years of age but not with adult-onset IBD (>17 years). B-cell lymphoblastoid cell lines genotyped for the risk variant as well as Henle-407 cells transfected with a plasmid construct with the risk variant showed higher NO production. Colonic biopsies of VEO-IBD patients showed higher immunohistochemical staining of nitrotyrosine, indicating more nitrosative stress and tissue damage.CONCLUSIONS:These studies suggest the importance of iNOS in genetic susceptibility to younger IBD presentation due to higher NO production.

Diagnostic criteria for eosinophilic esophagitis: a 5-year retrospective review in a pediatric population.

Background: The diagnostic criterion based on the number of eosinophils (Eos) per high-power field (HPF) does not appear to capture all patients with eosinophilic esophagitis (EE). Objectives: To determine whether EE has been underrecognized at our institution, clinical variables predicting EE, and whether the Luna eosinophil granule (LEG) stain detects eosinophils better than hematoxylin and eosin (HE). Materials and Methods: Esophageal biopsies of 202 children younger than 18 years old from 2000 to 2004 were reviewed and Eos/HPF was recorded. Clinical variables from charts were reviewed and a marginal logit model was used to determine significance. LEG stains for 60 randomly selected patients were prepared and compared to HE originals. Results: EE diagnoses have risen from none in 2000 to 23 in 2004. The clinically significant variables that predicted EE were improvement from EE treatment (160 times more likely to have EE; P < 0.0005), final endoscopic diagnosis of EE (31 times; P = 0.004), absence of vascular pattern on endoscopy (20 times; P = 0.008), and vertical furrows on endoscopy (29 times; P = 0.039). LEG stain appeared to be superior to HE in detecting low Eos/HPF (mean 24.82 and 38.53, respectively). Peak counts of eosinophils in the most involved HPF significantly correlated with highest average count of eosinophils per HPF in the most involved specimen (Pearson correlation 0.958). Conclusions: Misdiagnosed EE cases decreased but prevalence appeared to increase. LEG potentially can be a more sensitive stain. The key variables that predict EE were typical endoscopic findings and improvement from specific EE treatment.

Angiogenic remodeling in pediatric EoE is associated with increased levels of VEGF-A, angiogenin, IL-8, and activation of the TNF-α-NFκB pathway.

Objectives: Eosinophilic esophagitis (EoE) is a clinicopathologic diagnosis characterized by inflammation and infiltration of eosinophils at the esophageal mucosa. The underlying etiology of EoE remains elusive. Inflammatory diseases, such as asthma, are associated with structural remodeling of the airways, which includes angiogenesis. The aims of this study were to determine the angiogenic profile of esophageal mucosa in children presenting with EoE and to evaluate the putative mechanism(s) underlying the early inflammatory angiogenic response observed in EoE. Methods: Endoscopically obtained biopsy samples from 18 EoE and 18 control pediatric patients were analyzed for angiogenic markers (CD31, von Willebrand factor, vascular cell adhesion molecule-1) and tissue levels of angiogenic factors (vascular endothelial growth factor [VEGF]-A, VEGF-R2, angiogenin and interleukin [IL]-8). Expression levels of angiogenic factors and markers in EoE and control samples were characterized by immunofluorescence analysis and quantitative reverse transcriptase-polymerase chain reaction. Vascular density of biopsy samples was evaluated by immunofluorescence analysis. Results: Samples from patients with EoE exhibited higher levels of von Willebrand factor, CD31, and vascular cell adhesion molecule-1, which is suggestive of neovascularization and an activated endothelium. Moreover, EoE biopsies showed greater levels of the angiogenesis promoters VEGFA, angiogenin, and IL-8. Interestingly, there were greater cellular levels of tumor necrosis factor-&agr; in EoE samples compared with controls. Furthermore, there were higher nuclear levels of p50 and p65 subunits of NF&kgr;B and lower cellular levels of the inhibitor of NF&kgr;B, I&kgr;B-&agr;, in EoE samples compared with controls. Conclusions: We demonstrate increased angiogenesis in the esophageal mucosa of pediatric patients with EoE. The data also provided evidence that the angiogenic factors VEGF-A, angiogenin, and IL-8 were prominently involved in promoting angiogenic remodeling.

Diagnostic Characteristics of Given Video Capsule Endoscopy in Diagnosis of Celiac Disease: A Meta-Analysis

BACKGROUND AND AIMS In the view of small sample sizes of the studies published so far, the value of video capsule endoscopy (VCE) in diagnosing celiac disease (CD) is yet to be determined. The aim of this work was to systemically determine the overall diagnostic characteristics of VCE in diagnosing noncomplicated CD, compared to the gold standard, using meta-analysis. PATIENTS AND METHODS An extensive literature search was performed looking for prospective, controlled trials, with investigators blinded to results of the pathology of small-bowel biopsies. Two independent authors performed data extraction and assessment of the methodologic quality of each trial. Diagnostic characteristics of each trial were collected, and pooled sensitivity, specificity, likelihood ratios, and diagnostic odds ratios were computed. Description of complications and costs was included, if reported. RESULTS A total of three studies met the inclusion criteria (n = 107; 63 with CD and 44 without). The overall pooled VCE sensitivity was 83% (95% confidence interval [CI] = 71-90%) and specificity was 98% (95% CI = 88-99.6%). No major complications were reported. The costs were mentioned only in one study. CONCLUSIONS The overall diagnostic characteristics of VCE, when used to diagnose celiac disease, though good with an experienced eye, could not justify the routine use of VCE as an alternative to the pathology of small-bowel biopsies. More studies are needed with proper cost-benefit analysis.

10-year review of pediatric intestinal failure: clinical factors associated with outcome.

Prediction of outcomes in pediatric intestinal failure is challenging but essential to guide intestinal rehabilitation and transplantation decisions. This review of intestinal failure patients spanning 10 years examines clinical details in relation to outcome to identify factors that may refine predictive accuracy. A search was conducted to identify all children with intestinal failure managed at Stollery Childrens Hospital between January 1994 and December 2003. They were divided into 3 groups: early death occurring <or=30 days of age, parenteral nutrition dependence for 30-100 days, and parenteral nutrition dependence for >100 days. The long-term group was divided according to outcome: death or adaptation. Demographics, diagnosis, nutrition requirements, laboratory parameters, and clinical data were recorded. Groups were compared to identify factors associated with outcome. Necrotizing enterocolitis, gastroschisis, and intestinal atresias were the most common causes for intestinal failure; outcome was not related to diagnosis. Although withdrawal of therapy was common in the early death group, most babies had one or more additional significant comorbidity. Among the 29 babies requiring parenteral nutrition for >100 days with known outcomes, 12 died, 16 adapted fully, and 1 received a multivisceral transplant. Intestinal length >40 cm was associated with a significantly increased risk of mortality (P< .001). Abnormal laboratory values (bilirubin, aspartate aminotransferase, alanine aminotransferase, albumin, and platelet count) after 5 months of age were also significantly different between groups. This data, together with data from previous reviews, should be used to investigate potential predictive factors in prospective studies, particularly in the context of expert multidisciplinary care.

Helicobacter pylori Disrupts STAT1-Mediated Gamma Interferon-Induced Signal Transduction in Epithelial Cells

ABSTRACT Infection with Helicobacter pylori is chronic despite a vigorous mucosal immune response characterized by gastric T-helper type 1 cell expansion and gamma interferon (IFN-γ) production. IFN-γ signals by activation and nuclear translocation of signal transducer and activator of transcription 1 (STAT1); however, the effect of H. pylori infection on IFN-γ-STAT1 signaling is unknown. We infected human gastric (MKN45 and AGS) and laryngeal (HEp-2) epithelial cell lines with type 1 and type 2 H. pylori strains and then stimulated them with IFN-γ. Western blotting of whole-cell protein extracts revealed that infection with live, but not heat-killed, H. pylori time-dependently decreased IFN-γ-induced STAT1 tyrosine phosphorylation. Electrophoretic mobility shift assay of nuclear protein extracts demonstrated that H. pylori infection reduced IFN-γ-induced STAT1 DNA binding. STAT1 was unable to translocate from the cytoplasm to the nucleus in H. pylori-infected HEp-2 cells examined by immunofluorescence, and reverse transcription-PCR showed that IFN-γ-induced interferon regulatory factor 1 expression was inhibited. These effects were independent of the cagA, cagE, and VacA status of the infecting H. pylori strain. Furthermore, neither H. pylori culture supernatants nor conditioned medium from H. pylori-infected MKN45 cells inhibited IFN-γ-induced STAT1 tyrosine phosphorylation, suggesting that inhibition is independent of a soluble epithelial or bacterial factor but is dependent on bacterial contact with epithelial cells. H. pylori disruption of IFN-γ-STAT1 signaling in epithelial cells may represent a mechanism by which the bacterium modifies mucosal immune responses to promote its survival in the human host.

Patient and Parent Satisfaction with a Dietitian-and Nurse-Led Celiac Disease Clinic for Children at the Stollery Children’S Hospital, Edmonton, Alberta

OBJECTIVE To assess patient and parent satisfaction with a primarily nurse- and dietitian-led celiac disease clinic in a tertiary pediatric centre. METHODS An online survey was sent to families and patients attending the Stollery Childrens Hospitals Multidisciplinary Pediatric Celiac Clinic (Edmonton, Alberta) since 2007. The survey focused on clinic attendance, satisfaction with clinic structure, processes, and education and preference for alternatives to the current process. Respondents were asked to rank satisfaction or preference on a five-point Likert scale, with 1 being lowest and 5 being highest. RESULTS Most satisfaction related to follow-up with serology (4.6) and with a dietitian (4.3). The most preferred changes included either meeting the entire multidisciplinary team after the biopsy (4.7), or meeting with only the dietitian and nurse after the biopsy (4.4). The preferred education resources were the Internet (4.3) and the dietitian (4.2). The mean overall satisfaction score of the Multidisciplinary Pediatric Celiac Clinic was 4.0. CONCLUSIONS Results of the present survey suggested that patients and families value a multidisciplinary follow-up clinic for children with celiac disease. In particular, feedback based on repeat blood work and regular contact with a dietitian were highly valued. The present survey, outlining the most valued aspects of the clinic, may be useful for service delivery in other regions. In addition, it provides information on how to better support pediatric patients with celiac disease.