Annapaula Giulietti

Vitamin D and diabetes

Vitamin D deficiency predisposes individuals to type 1 and type 2 diabetes, and receptors for its activated form—1α,25-dihydroxyvitamin D3—have been identified in both beta cells and immune cells. Vitamin D deficiency has been shown to impair insulin synthesis and secretion in humans and in animal models of diabetes, suggesting a role in the development of type 2 diabetes. Furthermore, epidemiological studies suggest a link between vitamin D deficiency in early life and the later onset of type 1 diabetes. In some populations, type 1 diabetes is associated with certain polymorphisms within the vitamin D receptor gene. In studies in nonobese diabetic mice, pharmacological doses of 1α,25-dihydroxyvitamin D3, or its structural analogues, have been shown to delay the onset of diabetes, mainly through immune modulation. Vitamin D deficiency may, therefore, be involved in the pathogenesis of both forms of diabetes, and a better understanding of the mechanisms involved could lead to the development of preventive strategies.

Immune Regulation of 25‐Hydroxyvitamin‐D3‐1α‐Hydroxylase in Human Monocytes

Monocytes express 1α‐hydroxylase, the enzyme responsible for final hydroxylation of vitamin D3, in response to IFNγ and CD14/TLR4 activation. Cross‐talk between the JAK‐STAT, the NF‐κB, and the p38 MAPK pathways is necessary, and direct binding of C/EBPβ to its recognition sites in the promoter of the 1α‐hydroxylase gene is a prerequisite.

Polymorphisms in innate immunity genes predispose to bacteremia and death in the medical intensive care unit

Objective:Critically ill patients are at risk of sepsis, organ failure, and death. Studying the impact of genetic determinants may improve our understanding of the pathophysiology and allow identification of patients who would benefit from specific treatments. Our aim was to study the influence of single nucleotide polymorphisms in selected genes involved in innate immunity on the development of bacteremia or risk of death in patients admitted to a medical intensive care unit. Design, Setting, and Patients:DNA was available from 774 medical intensive care unit patients. We selected 31 single nucleotide polymorphisms in 14 genes involved in host innate immune defense. Serum levels of MASP2 and chemotactic capacity, phagocytosis, and killing capacity of monocytes at admission were quantified. Univariate Kaplan-Meier estimates with log-rank analysis and multivariate logistic regression were performed. Bootstrap resampling technique and ten-fold cross-validation were used to assess replication stability, prognostic importance of the variables, and repeatability of the final regression model. Main Results:Patients with at least one NOD2 variant were shown to have a reduced phagocytosis by monocytes (p = 0.03) and a higher risk of bacteremia than wild-type patients (p = 0.02). The NOD2/TLR4 combination was associated with bacteremia using survival analyses (time to bacteremia development, log-rank p < 0.0001), univariate regression (p = 0.0003), and multivariate regression analysis (odds ratio [OR] 4.26, 95% confidence interval [CI] 1.85–9.81; p = 0.0006). Similarly, the same combination was associated with hospital mortality using survival analysis (log-rank p = 0.03), univariate regression (p = 0.02), and multivariate regression analysis (OR 2.27, 95% CI 1.09–4.74; p = 0.03). Also variants in the MASP2 gene were significantly associated with hospital mortality (survival analysis log-rank-p = 0.003; univariate regression p = 0.02; multivariate regression analysis OR 2.35, 95% CI 1.38–3.99; p = 0.002). Conclusions:Functional polymorphisms in genes involved in innate immunity predispose to severe infections and death, and may become part of a risk model, allowing identification of patients at risk, who could benefit from early introduction of specific preventive or therapeutic interventions.

Unaltered Diabetes Presentation in NOD Mice Lacking the Vitamin D Receptor

OBJECTIVE— Vitamin D deficiency increases risk for type 1 diabetes in genetically predisposed individuals, while high doses of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] prevent insulitis and diabetes in NOD mice. RESEARCH DESIGN AND METHODS— Since 1,25(OH)2D3 regulates gene transcription through the vitamin D receptor (VDR), we investigated the role of VDR in diabetes development by creating NOD mice without functional VDR. RESULTS— VDR−/− NOD mice are rachitic and have lower numbers of putative regulator cells [TCR-α/β+CD4−CD8− (natural killer T-cells) and CD4+CD25+ T-cells [in central and peripheral immune organs compared with VDR+/+ NOD littermates. Lipopolysaccharide-stimulated VDR−/− NOD macrophages expressed lower interleukin (IL)-1, IL-6, and CC chemokine ligand 2 mRNA, correlating with less nuclear translocation of p65 nuclear factor-κB compared with VDR+/+ NOD macrophages. Thymic and lymph node dendritic cells from VDR−/− NOD mice displayed an even less mature CD11c+CD86+ phenotype than VDR+/+ NOD mice. Despite this immune phenotype linked to diabetes in NOD mice, VDR−/− NOD mice developed insulitis and diabetes at the same rate and incidence as VDR+/+ NOD littermates. CONCLUSIONS— Despite aggravating known immune abnormalities in NOD mice, disruption of VDR does not alter disease presentation in NOD mice in contrast to the more aggressive diabetes presentation in vitamin D–deficient NOD mice.

Regulatory cell-mediated tolerance does not protect against chronic rejection

Background. Regulatory cells prevent graft loss to acute rejection and induce tolerance, possibly by promoting Th2 deviation. Th2 cytokines stimulate B cells, which cause alloantibody-mediated chronic rejection. We searched to determine whether regulatory cell-mediated tolerance protects or not against chronic rejection. Methods. Heart transplantation (Htx) was performed using RA (RT1P) and PVG (RT1c) rats as donor and recipients. Donor-specific blood transfusion (DSBT) was given on preTx day 12. Secondary grafts were implanted at day 100. Splenocytes were transferred from tolerant rats (and controls) into lightly irradiated (450 rad) naive PVG, which received RA Htx. Primary Htx were investigated for the development of vascular occlusion (VO), the production of Th1/Th2 intragraft cytokines, and for the nature of graft infiltrate as well as for endothelial deposition of immunoglobulin (Ig)G isotypes and complement (C3) binding. Results were compared with rejecting controls (no DSBT) and syngeneic Htx. Results. RA Htx were rejected within 10 days (8, 9, 10×4). PreTx DSBT prolonged primary Htx survival indefinitely (>140 days) with acceptance of secondary donor-specific (but not third-party) grafts (P <0.001). Naive irradiated PVG rats given splenocytes from tolerant rats but not from controls accepted RA Htx, showing the existence of regulatory cells in allograft acceptors. Despite being tolerant, DSBT-treated rats displayed typical features of chronic rejection at day 90 (VO=77%; P <0.001 vs. VO=4% in syngeneic rats). An overt Th2 deviation, particularly intragraft production of interleukin (IL)-4, was observed at day 30. Simultaneously to this Th2 deviation, B cells emerged in the grafts and endothelial deposition of IgG1 (Th2 dependent) and C3 binding were observed. Conclusions. Regulatory cells that prevent graft loss to acute rejection in primary and secondary grafts do not protect against the development of chronic rejection.

Monocytic Expression Behavior of Cytokines in Diabetic Patients upon Inflammatory Stimulation

Abstract: Cytokines are involved in the pathogenesis of type 1 diabetes. The disease is characterized by T cell‐mediated β cell destruction and a biased Th1 cytokine pattern. Type 2 diabetes also presents an inflammatory cytokine imbalance. In this study, mRNA expression of cytokines IL‐12, TNF‐α, IL‐1, and IL‐6 was studied in monocytes from diabetic patients after in vitro immune stimulation. Whereas IL‐12p40 was highly expressed in type 1 diabetic patients, TNF‐α, IL‐1, and IL‐6 transcripts were elevated in type 1 but especially type 2 diabetic patients compared with healthy controls, suggesting an important proinflammatory milieu. We conclude that circulating monocytes from type 1 as well as type 2 diabetic patients have an aberrant cytokine profile when stimulated by an immune stimulus such as IFNγ. This condition not only is likely to be involved in disease pathogenesis, but may contribute to its later complications.

Regulation of 25-hydroxyvitamin d-1α-hydroxylase by IFNγ in human monocytic THP1 cells☆

1,25-DihydroxyVitamin D 3 (1,25(OH) 2 D 3 ), a molecule with well-known actions in bone and mineral homeostasis, also plays a role in the immune system. Indeed, the receptor for 1,25(OH) 2 D 3 is found in most immune cells and important immunological effects have been described in vitro, reflected by its capacity to prevent autoimmunity and to prolong graft survival. The aim of this study was to elucidate the intracellular pathways used by the immune system to regulate 1,25(OH) 2 D 3 production. Therefore we studied the regulation of 25-hydroxyvitamin-D-1α-hydroxylase (1α hydroxylase) in THP 1 cells by IFNγ, demonstrating that its induction is highly dependent on the activation/differentiation by PMA and occurred at a late time point (140-fold at 72 h, P < 0.05). Complete inhibition with actinomycin D indicated that the observed induction was, at least in part, a transcriptional event. Dose-dependent inhibition with cycloheximide demonstrated that the induction was dependent on de novo protein synthesis, a finding that correlates with the late time point of up-regulation. The data presented indicate a role for 1,25(OH) 2 D 3 , activated by la hydroxylase, as a late down-tapering signal in the immune cascade.

Real-Time Polymerase Chain Reaction

Publisher Summary This chapter provides an overview of the real-time polymerase chain reaction (PCR) methodology and its applications. PCR is a technique based on the exponential amplification of DNA by the thermostable Thermus aquaticus (Taq) polymerase. The method uses a pair of synthetic oligonucleotide primers, each hybridizing to one strand of a double-stranded DNA (dsDNA) target, with the pair spanning a region that will be exponentially amplified. The annealed primers act as a substrate for the Taq DNA polymerase, creating a complementary DNA strand via sequential addition of deoxynucleotides. Numerous adaptations and applications to this classic end-point PCR are described, which include semiquantitative PCR, quantitative competitive PCR, and real-time PCR. Real-time PCR assays are characterized by high precision and reproducibility, and the accuracy of data obtained is largely dependent on several other factors. In order to design and analyze experiments using real-time PCR, factors such as sample preparation, quality of the standard, choice of a housekeeping gene, and normalization of samples need careful consideration and optimization. Furthermore, it is important that standardized criteria and international uniformity in experimental design and data analysis are reached, in order to be able to compare data between different laboratories. The introduction of this technology has revolutionarily simplified the quantification of DNA and RNA. This has had a great impact in the field of molecular research and diagnostics, since enormous amounts of data can be obtained within a very short research time.