Background and aims: Despite the progress made in understanding the immunological aspects of the pathogenesis of coeliac disease (CD), the early steps that allow gliadin to cross the intestinal barrier are still largely unknown. The aim of this study was to establish whether gliadin activates a zonulin dependent enterocyte intracellular signalling pathway(s) leading to increased intestinal permeability. Methods: The effect of gliadin on the enterocyte actin cytoskeleton was studied on rat intestinal epithelial (IEC-6) cell cultures by fluorescence microscopy and spectrofluorimetry. Zonulin concentration was measured on cell culture supernatants by enzyme linked immunosorbent assay. Transepithelial intestinal resistance (Rt) was measured on ex vivo intestinal tissues mounted in Ussing chambers. Results: Incubation of cells with gliadin led to a reversible protein kinase C (PKC) mediated actin polymerisation temporarily coincident with zonulin release. A significant reduction in Rt was observed after gliadin addition on rabbit intestinal mucosa mounted in Ussing chambers. Pretreatment with the zonulin inhibitor FZI/0 abolished the gliadin induced actin polymerisation and Rt reduction but not zonulin release. Conclusions: Gliadin induces zonulin release in intestinal epithelial cells in vitro. Activation of the zonulin pathway by PKC mediated cytoskeleton reorganisation and tight junction opening leads to a rapid increase in intestinal permeability.

Repeated injections of adult mice with recombinant murine TNF prolong the survival of NZB/W F1 mice, and suppress type I insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. To determine whether repeated TNF injections suppress T cell function in adult mice, we studied the responses of influenza hemagglutinin-specific T cells derived from T cell receptor (HNT-TCR) transgenic mice. Treatment of adult mice with murine TNF for 3 wk suppressed a broad range of T cell responses, including proliferation and cytokine production. Furthermore, T cell responses of HNT-TCR transgenic mice also expressing the human TNF-globin transgene were markedly reduced compared to HNT-TCR single transgenic littermates, indicating that sustained p55 TNF-R signaling is sufficient to suppress T cell function in vivo. Using a model of chronic TNF exposure in vitro, we demonstrate that (a) chronic TNF effects are dose and time dependent, (b) TNF suppresses the responses of both Th1 and Th2 T helper subsets, (c) the suppressive effects of endogenous TNF produced in T cell cultures could be reversed with neutralizing monoclonal antibodies to TNF, and (d) prolonged TNF exposure attenuates T cell receptor signaling. The finding that anti-TNF treatment in vivo enhances T cell proliferative responses and cytokine production provides evidence for a novel regulatory effect of TNF on T cells in healthy laboratory mice. These effects are more pronounced in chronic inflammatory disease. In addition, our data provide a mechanism through which prolonged TNF exposure suppresses disease in animal models of autoimmunity.

Context: Autoimmune polyendocrine syndrome type 1 (APS1) is a childhood-onset monogenic disorder caused by mutations in the autoimmune regulator (AIRE) gene, including the distinctive R139X in Sardinia. Its rarity and great variability in manifestations/onset ages make early diagnosis difficult. To date, very few longitudinal studies of APS1 patients have been reported. Objective: The aim of this study was to describe the features and clinical course of APS1 and correlate them with AIRE and HLA class II genotypes in a large homogeneous cohort of Sardinian patients followed for up to 25 yr. Patients: Twenty-two pediatric APS1 patients were studied prospectively. Results: This Sardinian series (female/male ratio, 1.44; median current age, 30.7 yr; range, 1.8–46 yr) showed early disease onset (age range, 0.3–10 yr; median, 3.5 yr) and severe phenotype (on average, seven manifestations per patient). Besides the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease, autoimmune hepatitis was a serious and surprisingly common/early/presenting feature (27%; two deaths), with a 5:1 female bias (median age, 6 yr; range, 2.5–11 yr). By contrast, type 1 diabetes was rare (one patient), and hypothyroidism was not seen. Additional disease components (several of them potentially life-threatening) appeared in adulthood. The major nonsense mutation, R139X, was found in 93% of the mutant AIRE alleles. High-titer interferon (IFN)-ω and IFN-α autoantibodies were detected in all patients tested, even preclinically at 4 months of age in one sibling. HLA alleles appear to influence the exact phenotype—the most interesting apparent association being between HLA-DRB1*0301-DQB1*0201, liver-kidney microsome autoantibodies (anti-CYP1A2), and autoimmune hepatitis. Conclusion: APS1 in Sardinia is characterized by severe phenotype, marked clinical heterogeneity, and relative genetic homogeneity. The single AIRE mutation, R139X, and the anti-IFN-ω and IFN-α autoantibodies are helpful for earlier diagnosis, especially when APS1 presents unusually. HLA genotypes can modify the phenotype.