Coeliac disease (CD) is a malabsorptive enteropathy resulting from intolerance to gluten. Environmental factors and the microbiota are suggested to have critical roles in the onset of CD. The CD71 IgA receptor on epithelial cells is responsible for abnormal retrotranscytosis of IgA-gluten peptide complexes from the intestinal lumen into the lamina propria, inducing intestinal inflammation. However, understanding the role of gluten in the CD physiopathology has been hindered by the absence of relevant animal models. Here, we generated a mouse model for CD to study the factors controlling its pathogenesis as well as to investigate the influence of oral delivery of probiotics on disease development. Gluten sensitivity was established by feeding three generations of BALB/c mice a gluten-free diet (GÀ) followed by gluten challenge (G þ ) for 30 days. The G þ mice developed villous atrophy, crypt hyperplasia and infiltration of T cells and macrophages in the small intestine. Inflammation was associated with an overexpression of CD71 on the apical side of enterocytes and an increase of plasma cells producing IgA, which colocalised with the CD71. Moreover, IgA colocalised with the transglutaminase 2 (TG2), the production of which was increased in the lamina propria of G þ mice. These mice displayed increased production of cyclooxygenase-2 (COX-2), pro-inflammatory cytokines and IL-15, as well as anti-gliadin and anti-TG2 autoantibodies. The commensal flora-isolated presumptive probiotic Saccharomyces boulardii KK1 strain hydrolysed the 28-kDa a-gliadin fraction, and its oral delivery in G þ mice improved enteropathy development in association with decrease of epithelial cell CD71 expression and local cytokine production. In conclusion, the G þ BALB/c mouse represents a new mouse model for human CD based on histopathological features and expression of common biomarkers. The selected probiotic treatment reversing disease development will allow the study of the role of probiotics as a new therapeutic approach of CD.

Indoleamine 2,3‑dioxygenase (IDO), through L‑tryptophan depletion, activates general control non‑derepressible (GCN) 2 kinase and suppresses T‑cell proliferation, in addition to suppressing aerobic glycolysis and glutaminolysis, which are required for these rapidly proliferating cells. A number of, however not all of these alterations, are partially mediated through IDO‑induced p53 upregulation. In two‑way mixed lymphocyte reactions (MLRs), IDO reduced cellular proliferation. In MLR‑derived T‑cells, IDO induced the expression levels of p53 and p21, however concurrently reduced the levels of ζ‑chain, c‑Myc, lactate dehydrogenase A (LDH‑A) and glutaminase (GLS)2. However, p53 had no effect on the expression of the above proteins. These results were recapitulated in T‑cells activated with anti‑CD2, anti‑CD3 and anti‑CD28 by direct activation of the GCN2 kinase with tryptophanol. In conclusion, IDO, through GCN2 kinase activation, downregulates the levels of TCR‑complex ζ‑chain and c‑Myc, resulting in the suppression of T‑cell proliferation and a reduction in the levels of LDH‑A and GLS2, which are key enzymes involved in aerobic glycolysis and glutaminolysis, respectively.

Indoleamine 2,3-dioxygenase (IDO) is expressed in antigen-presenting cells and by degrading L-tryptophan along the kynurenine pathway suppresses CD4+ T-cell proliferation, induces apoptosis and promotes differentiation towards a regulatory as opposed to an effector phenotype. Recent findings revealed that the above effects may be mediated through alterations in T-cell metabolism. In this study, the effect of IDO on fatty acid β-oxidation in CD4+ T-cells was evaluated in human mixed lymphocyte reactions (MLRs) using the IDO inhibitor, 1-DL-methyl-tryptophan. Protein analysis of CD4+ T-cells isolated from the MLR showed that L-tryptophan degradation acts by activating the general control non‑derepressible 2 kinase and aryl-hydrocarbon receptor in T-cells. In the absence of IDO inhibition, fatty acid oxidation increased along with increased activity of carnitine palmitoyltransferase I (CPT1), the latter due to the increased expression of CPT1 isoenzymes and alterations in acetyl-CoA carboxylase 2, the enzyme that controls CPT1 activity. Increased fatty acid oxidation due to the action of IDO was accompanied by an increased expression of forkhead box P3 (FoxP3) and a decreased expression of related orphan receptor γt (RORγt), the signature transcription factors of regulatory T-cells and T helper 17 cells, respectively. However, in MLR and in the presence of fatty acid in the culture medium, IDO did not inhibit proliferation. Additionally, fatty acid protected the CD4+ T-cells against apoptosis. Thus, IDO, by degrading L-tryptophan, enhances CPT1 activity and fatty acid oxidation, and exerts fatty acid-dependent effects in human alloreactive CD4+ T-cells.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Original Highlights of the studyWe evaluated the relationship of RLS and mortality in chronic hemodialysis patients For the diagnosis of RLS we implemented standard methodology, i.e. the essential clinical criteria of the International RLS Study Group. This is the first time this issue is investigated in an epidemiological survey in Greece.In contrast to previous reports the diagnosis of RLS does not influence the 3-year mortality in HD patients. ABSTRACTObjective: In chronic hemodialysis (HD) patients, the prevalence of restless legs syndrome (RLS) is significantly higher than in the general population. Uremic RLS has been related to an enhanced mortality of HD patients. In the general population, however, studies of this association have yielded inconsistent results. The aim of the present study was to re-evaluate the relationship of RLS and mortality in HD patients. Methods:We recorded the 3-year mortality rate in a population of 579 HD patients after assessment for RLS symptoms. In addition we analyzed the 5-year mortality rate after end-stage renal disease onset (i.e., commencement of HD). This HD patient population has been previously included in a cross-sectional survey of RLS prevalence in which RLS diagnosis was based on the essential clinical criteria of the International RLS Study Group. Mortality data were acquired from the national endstage renal disease registry. Survival probability was calculated by means of the Kaplan-Meier method and analyzed by the log-rank (Mantel-Cox) test. For multivariate survival analysis, we implemented a proportional hazards regression Conclusion: Diagnosis of RLS according to the essential clinical criteria of theInternational RLS Study Group does not seem to influence the 3-year mortality in HD patients. Our findings are in contrast to those in some previous reports, and reinforce the need for further studies of RLS and mortality in HD patients.

Amino acid deprivation activates general control nonderepressible 2 (GCN2) kinase and inhibits mammalian target of rapamycin (mTOR), affecting the immune response. In this study, the effects of GCN2 kinase activation or mTOR inhibition on human alloreactive CD4+ T-cells were evaluated. The mixed lymphocyte reaction, as a model of alloreactivity, the GCN2 kinase activator, tryptophanol (TRP), and the mTOR complex 1 inhibitor, rapamycin (RAP), were used. Both TRP and RAP suppressed cell proliferation and induced cell apoptosis. These events were p53-independent in the case of RAP, but were accompanied by an increase in p53 levels in the case of TRP. TRP decreased the levels of the Th2 signature transcription factor, GATA-3, as RAP did, yet the latter also decreased the levels of the Th1 and Th17 signature transcription factors, T-bet and RORγt, whereas it increased the levels of the Treg signature transcription factor, FoxP3. Accordingly, TRP decreased the production of interleukin (IL)-4, as RAP did, but RAP also decreased the levels of interferon-γ (IFN-γ) and IL-17. Both TRP and RAP increased the levels of IL-10. As regards hypoxia-inducible factor-1α (HIF-1α), which upregulates the Th17/Treg ratio, its levels were decreased by RAP. TRP increased the HIF-1α levels, which however, remained inactive. In conclusion, our findings indicate that, in primary human alloreactive CD4+ T-cells, the two systems that sense amino acid deprivation affect cell proliferation, apoptosis and differentiation in different ways or through different mechanisms. Both mTOR inhibition and GCN2 kinase activation exert immunosuppressive effects, since they inhibit cell proliferation and induce apoptosis. As regards CD4+ T-cell differentiation, mTOR inhibition exerted a more profound effect, since it suppressed differentiation into the Th1, Th2 and Th17 lineages, while it induced Treg differentiation. On the contrary, the activation of GCN2 kinase suppressed only Th2 differentiation.

An association study was conducted to investigate the relation between 14 variants of glucose transporter 1 gene (SLC2A1) and the risk of type 2 diabetes (T2DM) leading to nephropathy. We also performed a meta-analysis of 11 studies investigating association between diabetic nephropathy (DN) and SLC2A1 variants. The cohort included 197 cases (T2DM with nephropathy), 155 diseased controls (T2DM without nephropathy) and 246 healthy controls. The association of variants with disease progression was tested using generalized odds ratio (ORG). The risk of type 2 diabetes leading to nephropathy was estimated by the OR of additive and co-dominant models. The mode of inheritance was assessed using the degree of dominance index (h-index). We synthesized results of 11 studies examining association between 5 SLC2A1 variants and DN. ORG was used to assess the association between variants and DN using random effects models. Significant results were derived for co-dominant model of rs12407920 [OR = 2.01 (1.17–3.45)], rs841847 [OR = 1.73 (1.17–2.56)] and rs841853 [OR = 1.74 (1.18–2.55)] and for additive model of rs3729548 [OR = 0.52 (0.29–0.90)]. The mode of inheritance for rs12407920, rs841847 and rs841853 was ‘dominance of each minor allele’ and for rs3729548 ‘non-dominance’. Frequency of one haplotype (C-G-G-A-T-C-C-T-G-T-C-C-A-G) differed significantly between cases and healthy controls [p = .014]. Regarding meta-analysis, rs841853 contributed to an increased risk of DN [(ORG = 1.43 (1.09–1.88); ORG = 1.58 (1.01–2.48)] between diseased controls versus cases and healthy controls versus cases, respectively. Further studies confirm the association of rs12407920, rs841847, rs841853, as well as rs3729548 and the risk of T2DM leading to nephropathy.

Although kidney transplantation is the best therapy for end-stage renal disease, rejection remains a concern, and currently available immunosuppressive agents contribute to morbidity and mortality. Thus, novel immunosuppressive drugs are required. Dichloroacetate (DCA) is already used in the treatment of congenital lactic acidosis and characterized by limited toxicity. As DCA inhibits aerobic glycolysis, which is a prerequisite for CD4+ T-cell proliferation and differentiation into effector T-cells, its possible immunosuppressive role in mixed lymphocyte reaction (MLR), a model of alloreactivity, was investigated. Glucose and lactate concentrations were measured in the supernatants, and cell proliferation was assessed immunoenzymatically. CD4+ T‑cells were then isolated from the MLRs and the expression of cleaved caspase‑3, various enzymes involved in glycolysis, and the signature transcription factors of CD4+ T‑cell subsets were evaluated by western blotting. In MLRs, DCA decreased glucose consumption and aerobic glycolysis, while it exerted a negligible effect on cell proliferation. In CD4+ T‑cells, DCA induced apoptosis, and decreased the expression of glucose trasporter‑1, hexokinase II, lactate dehydrogenase‑A and phosphorylated pyruvate dehydrogenase, while it increased total pyruvate dehydrogenase. In addition, DCA increased the expression of transcription factor forkhead box P3, whereas it decreased the expression of T‑box transcription factor TBX21, trans‑acting T-cell-specific transcription factor GATA‑3 and retinoic acid receptor related orphan receptor‑γt. In conclusion, in alloreactive CD4+ T‑cells, DCA inhibits aerobic glycolysis, induces apoptosis and favors differentiation towards the regulatory T‑cell subset. These characteristics render it a promising immunosuppressive agent in the field of transplantation.

Preconditioning of primary human RPTECs in a medium without tryptophan increases their survival under hypoxia by inducing autophagy. Identifying new molecular mechanisms that protect renal tissue from hypoxia could be proved clinically important in the prevention of AKI.