Vibrio cholerae-derived zonula occludins toxin (Zot) is a multifunctional protein that reversibly disassembles intestinal tight junctions (tjs). Zot structure-function analysis has mapped this activity to aa 288-293, named AT1002. AT1002 reduced transepithelial electrical resistance across rat small intestine, ex vivo, as did Zot and its processed mature form, ΔG. AT1002 increased in vivo permeability to sugar tracers, whereas scrambled control peptides did not. Binding and barrier assays in proteinase activated receptor (PAR)(2)-expressing and PAR(2)-null cells established AT1002 activity to be PAR(2) dependent. Coincident with the increased intestinal permeability, confocal microscopy of AT1002-exposed rat intestinal IEC6 cells revealed displacement of ZO-1 and occludin from intercellular boundaries. In coimmunoprecipitation assays, AT1002 decreased ZO-1-occludin and ZO-1-claudin 1 interactions coincident with PKCα-dependent ZO-1 serine/threonine phosphorylation. Further, AT1002 increased serine phosphorylation of myosin 1C and, at the same time, transiently diminished its association with ZO-1. The COOH-terminal domain of ZO-1 was required for its association with myosin 1C. These data indicate that the NH(2)-terminal portion of active Zot contains a PAR(2)-activating motif, FCIGRL, that increases PKCα-dependent ZO-1 and myosin 1C serine/threonine phosphorylation. These modifications provoke selective disengagement of ZO-1 from its binding partners, occludin, claudin 1, and myosin 1C, coincident with opening of tjs.

SummaryAnti-transglutaminase antibodies are the diagnostic markers of coeliac disease. A role is suggested for infectious agents in the production of antitransglutaminase antibodies. The aim was to measure positive antitransglutaminase antibody levels in children with infectious diseases and to compare immunological and biological characteristics of the antitransglutaminase antibodies derived from these children with that from coeliac patients. Two hundred and twenty-two children suffering from infectious diseases were enrolled prospectively along with seven biopsy-proven coeliacs. Serum samples were tested for anti-transglutaminase antibodies and anti-endomysium antibodies; positive samples were tested for coeliac-related human leucocyte antigen (HLA)-DQ2/8 and anti-viral antibodies. Purified anti-transglutaminase antibodies from the two study groups were tested for urea-dependent avidity, and their ability to induce cytoskeletal rearrangement and to modulate cell-cycle in Caco-2 cells, using phalloidin staining and bromodeoxyuridine incorporation assays, respectively. Nine of 222 children (4%) tested positive to anti-transglutaminase, one of whom also tested positive for anti-endomysium antibodies. This patient was positive for HLA-DQ2 and was diagnosed as coeliac following intestinal biopsy. Of the eight remaining children, two were positive for HLA-DQ8. Levels of anti-transglutaminase returned to normal in all subjects, despite a gluten-containing diet. Purified anti-transglutaminase of the two study groups induced actin rearrangements and cell-cycle progression. During an infectious disease, antitransglutaminase antibodies can be produced temporarily and independently of gluten. The infection-triggered anti-transglutaminase antibodies have the same biological properties as that of the coeliacs, with the same in-vivo potential for damage.

IntroductionThe use of anti-retroviral therapy (ART) has dramatically reduced HIV-1 associated morbidity and mortality. However, HIV-1 infected individuals have increased rates of morbidity and mortality compared to the non-HIV-1 infected population and this appears to be related to end-organ diseases collectively referred to as Serious Non-AIDS Events (SNAEs). Circulating miRNAs are reported as promising biomarkers for a number of human disease conditions including those that constitute SNAEs. Our study sought to investigate the potential of selected miRNAs in predicting mortality in HIV-1 infected ART treated individuals.Materials and MethodsA set of miRNAs was chosen based on published associations with human disease conditions that constitute SNAEs. This case: control study compared 126 cases (individuals who died whilst on therapy), and 247 matched controls (individuals who remained alive). Cases and controls were ART treated participants of two pivotal HIV-1 trials. The relative abundance of each miRNA in serum was measured, by RTqPCR. Associations with mortality (all-cause, cardiovascular and malignancy) were assessed by logistic regression analysis. Correlations between miRNAs and CD4+ T cell count, hs-CRP, IL-6 and D-dimer were also assessed.ResultsNone of the selected miRNAs was associated with all-cause, cardiovascular or malignancy mortality. The levels of three miRNAs (miRs -21, -122 and -200a) correlated with IL-6 while miR-21 also correlated with D-dimer. Additionally, the abundance of miRs -31, -150 and -223, correlated with baseline CD4+ T cell count while the same three miRNAs plus miR-145 correlated with nadir CD4+ T cell count.DiscussionNo associations with mortality were found with any circulating miRNA studied. These results cast doubt onto the effectiveness of circulating miRNA as early predictors of mortality or the major underlying diseases that contribute to mortality in participants treated for HIV-1 infection.

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BackgroundCereals of baking quality with absent or reduced toxicity are actively sought as alternative therapy to a gluten-free diet (GFD) for patients with coeliac disease (CD). Triticum monococcum, an ancient wheat, is a potential candidate having no toxicity in in-vitro and ex-vivo studies. The aim of our study was to investigate on the safety of administration of a single dose of gluten of Tm in patients with CD on GFD.MethodsWe performed a single blind, cross-over study involving 12 CD patients who had been on a GFD for at least 12 months, challenged on day 0, 14 and 28 with a single fixed dose of 2.5 grams of the following (random order): Tm, rice (as reference atoxic protein) and Amygluten (as reference toxic protein) dispersed in a gluten-free pudding. The primary end-point of the study was the change in intestinal permeability, as assessed by changes in the urinary lactulose/rhamnose ratio (L/R ratio) measured by High Pressure Liquid Chromatography. We also assessed the occurrence of adverse gastrointestinal events, graded for intensity and duration according to the WHO scale. Variables were expressed as mean ± SD; paired t-test and χ2 test were used as appropriate.ResultsThe urinary L/R ratio did not change significantly upon challenge with the 3 cereals, and was 0.055 ± 0.026 for Tm Vs 0.058 ± 0.035 for rice (p = 0.6736) and Vs 0.063 ± 0.054 with Amygluten (p = 0.6071). Adverse gastrointestinal events were 8 for Tm, Vs 11 for rice (p = 0.6321) and Vs 31 for Amygluten p = 0.0016), and, in all cases events were graded as “mild” or “moderate” with TM and rice, and as “severe” or “disabling” in 4 cases during Amygluten.ConclusionsNo definite conclusion can be drawn on the safety of Tm, based on no change in urinary L/R because even Amygluten, a toxic wheat protein, did not cause a significant change in urinary L/R indicating low sensitivity of this methodology in studies on acute toxicity. Tm was, however, well tolerated by all patients providing the rationale for further investigation on the safety of this cereal for CD patients.Trial registrationEudraCT-AIFA n2008-000697-20