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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.

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., (Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.)

During red tide bloom events, the marine diatom Pseudo-nitzschia produces the toxin domoic acid (DA), which has been associated with stranding and mortality events involving California sea lions (Zalophus californianus) and southern sea otters (Enhydra lutris). In addition to these well-documented DA-induced neurotoxic events, there is increasing concern that DA may exert chronic effects, such as immunomodulation, which may potentially increase an individual's susceptibility to a number of opportunistic infections following nonlethal exposure. We investigated the effects of DA on innate (phagocytosis and respiratory burst) and adaptive (mitogen-induced lymphocyte proliferation) immune functions with the use of peripheral blood leukocytes collected from healthy California sea lions and southern sea otters upon in vitro exposure to 0 (unexposed control), 0.0001, 0.001, 0.01, 0.1, 1.0, 10, and 100 microM DA. Domoic acid did not significantly modulate phagocytosis or respiratory burst in either species. For California sea lions, DA significantly increased ConA-induced T-lymphocyte proliferation upon exposure to DA concentrations ranging from 0.0001 to 10 microM, resulting in a nonlinear dose-response curve. There was no effect on lymphocyte proliferation at the highest concentration of DA tested. No effects on lymphocyte proliferation were observed in southern sea otters. Importantly, the in vitro DA concentrations affecting T-cell proliferation were within or below the range of DA in serum measured in free-ranging California sea lions following natural exposure, suggesting a risk for immunomodulation in free-ranging animals. Understanding the risk for immunomodulation upon DA exposure will contribute in the health assessment and management of California sea lions and southern sea otters, as well as guide veterinarians and wildlife rehabilitators in caring for and treating afflicted animals.

Arctic charr Salvelinus alpinus production facilities, nonproduction water sources and effluents in the United States and Canada were sampled to determine if chlamydiae associated with epitheliocystis were present in water and were associated with inclusions of epitheliocystis in gill tissue. Gills from 607 fish from 13 sites were processed for histopathologic examination and DNA extraction. Water was collected from 21 locations for DNA testing. Eighteen fish from one location had inclusions of epitheliocystis with proliferative and inflammatory gill lesions. Inclusions were stained using the Gimenez technique and, at the ultrastructural level, consisted of intracytoplasmic membrane-bound vacuoles containing reticulate and intermediate bodies in a fibrillar matrix. PCR using Order Chlamydiales-specific primers performed on DNA extracts from 12 of 13 infected fish yielded amplicons that were identical to (GQ302988) or differed at one base from (GQ302987) the 16S ribosomal RNA gene signature sequence of 'Candidatus Piscichlamydia salmonis', which is the chlamydia that was previously identified in epitheliocystis inclusions of farmed Atlantic salmon. In situ hybridization using a approximately 1.5 kb riboprobe corresponding to the 'Candidatus Piscichlamydia salmonis' 16S rRNA genetic sequence (AY462244) confirmed its presence within Arctic charr gill inclusions. DNA isolated from water samples was tested by Chlamydiales-specific PCR and yielded 54 partial 16S rRNA genetic sequences spanning the signature region; however, no 16S rRNA genetic sequences associated with epitheliocystis were identified. This is the first report of 'Candidatus Piscichlamydia salmonis' associated with epitheliocystis in Arctic charr, the first identification of 'Candidatus Piscichlamydia salmonis' from a freshwater production location, and the first reported occurrence in North America.

Infections of branchial epithelium by intracellular gram-negative bacteria, termed epitheliocystis, have limited culture of Arctic charr Salvelinus alpinus. To characterize a bacterium associated with epitheliocystis in cultured charr, gills were sampled for histopathologic examination, conventional and immunoelectron microscopy, in situ hybridization, 16S ribosomal DNA (rDNA) amplification, sequence analysis and phylogenetic inference. Sampling was conducted at the Freshwater Institute (Shepherdstown, West Virginia, USA) during outbreaks of epitheliocystis in April and May 2002. Granular, basophilic, cytoplasmic inclusions in charr gill were shown to stain with Macchiavello, Lendrum's phloxine-tartrazine and Gimenez histochemical techniques. Ultrastructurally, inclusions were membrane-bound and contained round to elongate reticulate bodies that were immunoreactive to an antibody against chlamydial lipopolysaccharide, suggesting the presence of similar epitopes. DNA extracted from gills supported amplification of the most polymorphic and phylogenetically relevant region of the 16S rRNA gene, which had 97 to 100% identity with several uncultured clinical Neochlamydia spp. (order Chlamydiales) Clones WB13 (AY225593.1) and WB258 (AY225594.1). Sequence-specific riboprobes localized to inclusions during in situ hybridization experiments. Taxonomic affiliation was inferred by distance- and parsimony-based phylogenetic analyses of the 16S sequence, which branched with Neochlamydia hartmannellae in the order Chlamydiales with high confidence. This is the first molecular characterization of a chlamydia associated with epitheliocystis in Arctic charr and the fourth Neochlamydia spp. sequence to be associated with epitheliocystis. Presence of a clinical neochlamydial sequence, first identified from a cat, in Arctic charr suggests a possible mammalian and piscine host range for some environmental chlamydiae.

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.

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.