Andrew Draghi

Characterization of "Candidatus Piscichlamydia salmonis" (Order Chlamydiales), a Chlamydia-Like Bacterium Associated With Epitheliocystis in Farmed Atlantic Salmon (Salmo salar)

ABSTRACT To characterize intracellular gram-negative bacteria associated with epitheliocystis in farmed Atlantic salmon (Salmo salar), gills with proliferative lesions were collected for histopathology, conventional transmission and immunoelectron microscopy, in situ hybridization, and DNA extraction during epitheliocystis outbreaks in Ireland and Norway in 1999 and 2000, respectively, and compared by ultrastructure and immunoreactivity to nonproliferative gills from Ireland archived in 1995. Genomic DNA from proliferative gills was used to amplify 16S ribosomal DNA (rDNA) for molecular phylogenetic analyses. Epitheliocystis inclusions from proliferative gills possessed variably elongate reticulate bodies, examples of binary fission, and vacuolated and nonvacuolated intermediate bodies, whereas inclusions in nonproliferative gills had typical chlamydial developmental stages plus distinctive head-and-tail cells. Immunogold processing using anti-chlamydial lipopolysaccharide antibody labeled reticulate bodies from proliferative and nonproliferative gills. 16S rDNA amplified directly from Irish (1999) and Norwegian (2000) gill samples demonstrated 99% nucleotide identity, and riboprobes transcribed from cloned near-full-length 16S rDNA amplicons from Norwegian gills hybridized with inclusions in proliferative lesions from Irish (1999) and Norwegian (2000) sections. A 1,487-bp consensus 16S rRNA gene sequence representing the chlamydia-like bacterium (CLB) from proliferative gills had the highest percent nucleotide identity with endosymbionts of Acanthamoeba spp. (order Chlamydiales). Molecular phylogenetic relationships inferred from 16S rRNA gene sequences using distance and parsimony indicated that the CLB from proliferative gills branched with members of the order Chlamydiales. “Candidatus Piscichlamydia salmonis” is proposed for the CLB associated with epitheliocystis from proliferative gills of Atlantic salmon, which exhibits developmental stages different from those identified in nonproliferative gills.

Fecal osteoprotegerin may guide the introduction of second-line therapy in hospitalized children with ulcerative colitis.

Background: Osteoprotegerin (OPG) is increased in inflamed colonic mucosa and has a role in immune regulation and apoptosis resistance. Fecal OPG may be useful in predicting corticosteroid resistance in hospitalized children with severe ulcerative colitis (UC). We aimed to determine whether fecal OPG predicts the need for second‐line therapies in children hospitalized for UC. Methods: We included 83 children with UC admitted for intravenous corticosteroid treatment. Children were classified as responders/nonresponders based on the need for therapy escalation. Fecal OPG results were compared with those of four other fecal markers. Results: Of the enrolled children, seven had day 1 samples only, 53 children had day 3 samples only, and 23 had both. Twenty‐two children failed corticosteroid therapy and required infliximab (n = 20) or colectomy (n = 2). On the third treatment day the median fecal OPG levels were significantly higher in the nonresponders group compared with the responders: 77 pmol/L (interquartile range [IQR] 27–137) versus 13 pmol/L (3–109); P = 0.007. The best day 3 fecal OPG cutoff to predict second‐line therapy was >50 pmol/L with a sensitivity of 71% and specificity of 69% (area under the receiver operator curve [ROC] of 0.70%–95% confidence interval [CI] 0.57–0.82). Fecal OPG was superior to day 3 fecal calprotectin, lactoferrin, and S100A12 as a predictor of corticosteroid nonresponse, but equivalent to the less commonly used M2‐pyruvate kinase. Conclusions: Day 3 fecal OPG may guide the decision to institute second‐line therapy in children with severe UC. The role of OPG in the inflammatory response in pediatric UC deserves further study. (Inflamm Bowel Dis 2010;)

Polymorphisms in the Genes for Coagulation Factors II, V, and VII in Patients With Ischemic Heart Disease

BACKGROUND Cardiovascular disease remains the leading cause of mortality in the United States, accounting for approximately 33% of all deaths in this country. Of these deaths, most are due to acute myocardial infarctions (AMIs), which are associated with thrombotic coronary artery obstruction and/or occlusion. These events could potentially be due to alterations in genes coding for coagulation factors. Several polymorphisms have been described in the factor II, V, and VII genes, which may predispose one to increased risk for ischemic heart disease (IHD). OBJECTIVE To determine if mutations in 3 coagulation factor genes could predispose an individual to increased risk for arterial thrombosis as a mechanism for developing unstable angina (UA) or AMI. METHODS We examined 125 hospitalized patients (mean age, 53 +/- 6 years, 79 men and 46 women), including 32 with AMI, 68 with UA, and 25 noncardiac controls, for a genetic predisposition for increased risk of IHD. EDTA-anticoagulated whole blood was collected at the time of hospital admission. DNA was extracted, and the polymorphisms were detected by polymerase chain reaction amplification of these genes with subsequent restriction enzyme digestion and gel electrophoresis. RESULTS Our results showed that 3 (9.4%), 3 (4.4%), and 1 (4%) individuals were heterozygous for prothrombin G20210A and 3 (9.4%), 5 (7.4%), and 1 (4%) individuals were heterozygous for factor V Leiden in the AMI, UA, and control groups, respectively. The following genotype frequencies for the factor VII R353Q polymorphism were identified: 25 (78.1%), 56 (82.4%), and 18 (72%) with RR and 7 (21.9%), 12 (17. 6%), and 7 (28%) with RQ in the AMI, UA, and control groups, respectively. No QQ homozygotes were identified. For the HVR4 size polymorphism, the following genotypes were identified: 3 (9.4%), 4 (5.9%), and 5 (20%) individuals with H7H7; 11 (34.4%), 33 (48.5%), and 12 (48%) with H6H7; and 18 (56.2%), 31 (45.6%), and 8 (32%) with H6H6 genotypes in the AMI, UA, and control groups, respectively. There were no H7H5 and H6H5 genotypes found in this study. CONCLUSIONS Although the frequency differences of these polymorphisms in patients with AMI and UA were not statistically significant from those in controls, several trends are consistent with what has been reported in the literature. Although any of these or other undefined genetic abnormalities may result in IHD, it is possible that phenotypic predisposition to IHD initially presents as UA. A larger population study addressing the significance of these polymorphisms in the sequence of events that lead to IHD, including cases of UA, is warranted.

Mycobacterium Marinum Dermatitis and Panniculitis with Chronic Pleuritis in a Captive White Whale (Delphinapterus Leucas) with Aortic Rupture

A 16-year-old female white whale, Delphinapterus leucas, died after nearly 18 months of chronic lymphopenia and pyogranulomatous dermatitis. Necropsy revealed rupture of the aorta with hemorrhage into the cranial mediastinum and between fascial planes of the ventral neck musculature. Multiple foci of ulcerative dermatitis and panniculitis were present across the thorax and abdomen and surrounded the genital folds. In addition, there was a chronic proliferative pleuritis with over 20 liters of histiocytic exudate in the thoracic cavity. Acid-fast bacteria consistent with Mycobacterium sp. were identified in sections of skin lesions and in cytospins of pleural exudate. Cultures of pleura and 1 skin lesion collected at necropsy yielded sparse growth of an acid-fast bacillus with colony characteristics and morphology consistent with Mycobacterium marinum. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis confirmed the presence of M. marinum DNA in samples of skin. This is the first documented occurrence of mycobacteriosis in a white whale and is a unique presentation of mycobacterial dermatitis and panniculitis with chronic pleuritis in a cetacean. The improved PCR-RFLP protocol utilized in this case unifies techniques from several protocols to differentiate between species of Nocardia and rapidly growing mycobacteria clinically relevant to aquatic animals.

Proinflammatory fecal mRNA and childhood bacterial enteric infections

Introduction: Assessment of specific mRNAs in human samples is useful in characterizing disease. However, mRNA in human stool has been understudied. Methods: We purified fecal RNA from 46 children infected with Campylobacter jejuni, Escherichia coli O157:H7, Salmonella spp., or Shigella sonnei and 26 controls, and compared the proportions of IL-1β, IL-8, osteoprotegerin, and calprotectin mRNA between groups using qRT-PCR. We determined the concentrations of calprotectin, IL-8, and osteoprotegerin by enzyme immunoassays in cognate specimens. Results: Compared to controls, infected stools showed increased transcripts of IL-1β, IL-8, and calprotectin. mRNA and protein concentrations correlated for IL-8, but not for calprotectin. Discussion: Stool mRNA quantification offers a potentially useful, noninvasive way to assess inflammation in the gastrointestinal tract, and may be more sensitive than EIA.

Distinctive colonic mucosal cytokine signature in new-onset, untreated pediatric Crohn disease

Objective: The aim of the study was to compare the colonic mucosal immune response in children with new, untreated Crohn disease (CD-New), CD in remission (CD-Remission), and unaffected children (CTRL [controls]). Methods: We performed flow cytometry of mitogen-stimulated colonic lamina propria mononuclear cells isolated from colonic biopsies and 72-hour biopsy explant cultures, and analyzed the supernatant by an unbiased multiplex cytokine array of 45 analytes. Results: Thirty-six children were studied (mean age 14 ± 3 years, 14 girls): 12 CD-New, 11 CD-Remission, and 13 CTRL. We found that stimulation of lamina propria mononuclear cells isolated from colonic biopsies induced comparable intracellular cytokine levels of interferon (IFN-&ggr;), interleukin (IL)-17, and tumor necrosis factor (TNF)-&agr; in T cells from CD-New, CD-Remission, and CTRL, suggesting that mucosal innate inflammation plays a larger role than activated T cells in CD-New. To measure factors released during the ongoing inflammatory response in CD-New, we cultured colonic biopsy explants and uncovered 13/45 factors that were significantly higher in CD-New versus CD-Remission, whereas 10 were increased in CD-New over CTRL. Ingenuity Pathway Analysis software revealed the anticipated interconnectivity of TNF-&agr;, IL-6, and CSF-2 in CD-New of the colon. A novel subnetwork of chemokines was, however, evident, whereas IL-17a appeared as a peripheral factor. Principal component analysis and hierarchal clustering showed that CD-New and CD-Remission separated into distinct subgroups based on the 13 factors. Conclusions: At diagnosis of inflammatory bowel disease, the colonic cytokine response contains a predominance of innate immune factors, with chemoattractants and vascular adhesion molecules playing a central role.

Targeting the PXR-TLR4 signaling pathway to reduce intestinal inflammation in an experimental model of necrotizing enterocolitis

BackgroundThere is substantial evidence that signaling through Toll-like receptor 4 (TLR4) contributes to the pathogenesis of necrotizing enterocolitis (NEC). Pregnane X receptor (PXR), a xenobiotic sensor and signaling intermediate for certain host-bacterial metabolites, has been shown to negatively regulate TLR4 signaling. Here we investigated the relationship between PXR and TLR4 in the developing murine intestine and explored the capacity of PXR to modulate inflammatory pathways involved in experimental NEC.MethodsWild-type and PXR−/− mice were studied at various time points of development in an experimental model of NEC. In addition, we studied the ability of the secondary bile acid lithocholic acid (LCA), a known PXR agonist in liver, to activate intestinal PXR and reduce NEC-related intestinal inflammation.ResultsWe found a reciprocal relationship between the developmental expression of PXR and TLR4 in wild-type murine intestine, with PXR acting to reduce TLR4 expression by decreasing TLR4 mRNA stability. In addition, PXR−/− mice exhibited a remarkably heightened severity of disease in experimental NEC. Moreover, LCA attenuated intestinal proinflammatory responses in the early stages of experimental NEC.ConclusionThese findings provide proactive insights into the regulation of TLR4 in the developing intestine. Targeting PXR may be a novel approach for NEC prevention.

Programmed cell death-1, PD-1, is dysregulated in T cells from children with new onset type 1 diabetes

Background Programmed death cell 1 (PD-1) is an inhibitor of T cell activation and is also functionally linked to glycolysis. We hypothesized that PD-1 expression is defective in activated T cells from children with type 1 diabetes (T1D), resulting in abnormal T cell glucose metabolism. Methods In this pilot study, we enrolled children with new onset T1D within 2 weeks of diagnosis (T1D), unaffected siblings of T1D (SIBS), unaffected, unrelated children (CTRL), children with new onset, and untreated Crohn disease (CD). We repeated the assays 4–6 months post-diagnosis in T1D (T1D follow up). We analyzed anti-CD3/-CD28-stimulated peripheral blood mononuclear cells (PBMC) subsets for PD-1 expression by flow cytometry at baseline and after 24 h in culture. We measured cytokines in the culture medium by multiplex ELISA and glycolytic capacity with a flux analyzer. Results We enrolled 37 children. T cells derived from subjects with T1D had decreased PD-1 expression compared to the other study groups. However, in T1D follow-up T cells expressed PD-1 similarly to controls, but had no differences in PBMC cytokine production. Nonetheless, T1D follow up PBMCs had enhanced glycolytic capacity compared to T1D. Conclusions Activated T cells from T1D fail to upregulate PD-1 upon T-cell receptor stimulation, which may contribute to the pathogenesis of T1D. T1D follow up PBMC expression of PD-1 normalizes, together with a significant increase in glycolysis compared to T1D. Thus, insulin therapy in T1D children is associated with normal PD1 expression and heightened glycolytic capacity in PBMC.

1149 Osteoprotegerin Expression is Upregulated in the Colon of Children With Active Inflammatory Bowel Disease

Background: Inflammatory Bowel Diseases (IBD) are chronic, relapsing conditions. Infections and other environmental exposures that vary by season have been postulated to trigger relapses; however, little information has been published on the seasonality of pediatric-onset IBD. Further understanding of this topic may provide valuable data on the epidemiology of Crohns Disease (CD) and Ulcerative Colitis (UC). Aim: To examine whether season is associated with disease activity in pediatric-onset IBD. Methods: ImproveCareNow (ICN) is a multicenter network of health care providers formed in 2007 to improve the quality of care of children with IBD. The ICN registry contains disease and treatment data, including physician global assessment (PGA), prospectively collected during outpatient encounters. This study analyzed data collected from December 2008-November 2010 at 12 centers across the USA. The percentage of visits during each season in which patients were classified as being in remission based on PGA was calculated. For patients with multiple visits per season, only the visit with the most severe PGA score was included. The distribution of sick visits (PGA of mild, moderate, or severe) and well visits (PGA of remission) across each season was also calculated. Seasons were defined as summer (Jun-Aug), fall (Sep-Nov), winter (Dec-Feb), and spring (Mar-May). Data was further divided into northern and southern regions (defined by 37° latitude). A generalized linear mixed model and the multinomial chi-square test were used for statistical analysis with p<0.05 considered significant. Results: The percentage of UC visits (n=1129)in remission was highest in summer and lowest in winter (summer 51.6%, fall 48.8%, spring 45.0%, winter 41.9%) with significant seasonal variation (p=0.01). A similar pattern was seen in CD (summer 51.4%, fall 47.5%, winter 46.9%, spring 45.8%, n=2937), though not statistically significant (p=0.09). The results were consistent across the northern and southern regions (UC p=0.58, CD p=0.08). The percentage of all sick visits was evenly distributed across seasons for UC (n=954, p=0.81) and CD (n=2414, p=0.07). However, the percentage of well visits was highest in the summer and lowest in the winter in both UC (28.7% vs. 20.5%, n=1440, p<0.001) and CD (28.1% vs. 22.6%, n=3688, p<0.001). Conclusion: In this large multicenter study, seasonal variation of disease activity was not found, nor was there seasonal variation when examined by region. Although the percentage of visits of UC patients in remission increased during the summer, this was due to a higher percentage of all well visits during this season. One potential explanation is that well visits may be intentionally scheduled during summer months to limit the amount of school missed by children.

Sa1158 Increased Serum Lipopolysaccharide in Crohn's Disease Is Associated With Alterations in Toll-Like Receptor Expression in Peripheral Blood

The incidence of VEO-IBD ranged from 0.8 to 3.3/100,000/year. The mean rate of increase was 4% per year (p=0.06; CI 0%, 8%). The rate of increase was significant in males (p= 0.04) compared to females (p=0.281). The incidence of UC increased strikingly from 0.6 to 2.3/100,000/year (CI 0.025, 0.164) at an average annual rate of 9%. Pancolonic UC increased 7-fold from 0.3 to 2.3/100,000/year (p=0.005). Using PUCAI scores, severe disease activity at presentation increased significantly from 0% to 36% (p=0.002). The incidence of CD increased at a slower rate of 0.02/100,000/year (CI -0.41, 0.079). Significantly more males (44%) than females (18%) had both upper and lower GI tract involvement (p=0.03). 76% of patients with CD had inflammatory disease behaviour while 24% had stricturing or penetrating disease. A total of 31% of children presented with perianal disease. At one year follow up 64% were in clinical remission, with 56% in steroid-free remission. Fifty two (35%) children commenced immunomodulators within 1 year of diagnosis while 6 (4%) underwent surgery. At maximum follow up (median 60 months), 24 (16%) children had commenced biologics, 76 (48%) immunomodulators and 20 (13%) had undergone surgery (12 UC; 8 CD). This is the first national population-based study describing the epidemiology and outcomes of VEO-IBD phenotypes. Boys hadmore extensive disease than girls. Substantial and sustained increases in the incidence and severity of UC in particular were observed. Prospective longitudinal studies are required to fully elucidate the factors underlying the aetiology and outcome of VEO-IBD.