Natália Emerim Lemos

The A Allele of the rs1990760 Polymorphism in the IFIH1 Gene Is Associated with Protection for Arterial Hypertension in Type 1 Diabetic Patients and with Expression of This Gene in Human Mononuclear Cells

Background The rs1990760 polymorphism of interferon induced with helicase C domain 1 (IFIH1) has been associated with type 1 diabetes mellitus (T1DM). Here, we investigated whether this polymorphism is associated with T1DM or its clinical characteristics in a Brazilian population, and if IFIH1 gene expression in mononuclear cells from T1DM patients differs according to the genotypes of this polymorphism. A meta-analysis was also conducted to evaluate if the rs1990760 polymorphism is associated with T1DM. Methods Frequencies of the rs1990760 polymorphism were analyzed in 527 T1DM patients and in 517 healthy subjects. IFIH1 gene expressions according to genotypes were measured in a sub-sample of 26 T1DM patients by quantitative real-time PCR. Results Our data show the association of the A allele with risk to T1DM under a dominant model of inheritance [odds ratio (OR) = 1.421, P = 0.037], adjusting for ethnicity. The meta-analysis revealed significant association between the rs199760A allele and risk for T1DM for all analyzed inheritance models. Surprisingly, T1DM patients carrying the A allele showed lower levels of systolic (P = 0.001) and diastolic (P = 1×10−10) blood pressures as compared to G/G carriers. Furthermore, the A/A genotype seems to be associated with protection to arterial hypertension (AH) after adjustment for covariates (OR = 0.339, P = 0.019). IFIH1 gene expression in mononuclear cells from 26 T1DM patients did not differ among genotypes (P = 0.274). Nevertheless, IFIH1 gene expression was increased in mononuclear cells from T1DM patients with AH as compared with T1DM patients without AH [6.7 (1.7–2.0) vs. 1.8 (1.3–7.1) arbitrary units; P = 0.036]. The association with blood pressures and AH was not observed in patients with type 2 diabetes mellitus. Conclusions Our results indicate that the rs1990760 polymorphism is associated with T1DM. Interestingly, the rs1990760 A allele seems to be associated with protection for AH in T1DM patients. Further studies are needed to confirm the association with AH.

Exendin-4 protects rat islets against loss of viability and function induced by brain death

Islet quality loss after isolation from brain-dead donors still hinders the implementation of human islet transplantation for treatment of type 1 diabetes. In this scenario, systemic inflammation elicited by donor brain death (BD) is among the main factors influencing islet viability and functional impairment. Exendin-4 is largely recognized to promote anti-inflammatory and cytoprotective effects on β-cells. Therefore, we hypothesized that administration of exendin-4 to brain-dead donors might improve islet survival and insulin secretory capabilities. Here, using a rat model of BD, we demonstrate that exendin-4 administration to the brain-dead donors increases both islet viability and glucose-stimulated insulin secretion. In this model, exendin-4 treatment produced a significant decrease in interleukin-1β expression in the pancreas. Furthermore, exendin-4 treatment increased the expression of superoxide dismutase-2 and prevented BD-induced elevation in uncoupling protein-2 expression. Such observations were accompanied by a reduction in gene expression of two genes often associated with endoplasmic reticulum (ER) stress response in freshly isolated islets from treated animals, C/EBP homologous protein and immunoglobulin heavy-chain binding protein. As ER stress response has been shown to be triggered by and to participate in cytokine-induced β-cell death, we suggest that exendin-4 might exert its beneficial effects through alleviation of pancreatic inflammation and oxidative stress, which in turn could prevent islet ER stress and β-cell death. Our findings might unveil a novel strategy to preserve islet quality from brain-dead donors. After testing in the human pancreatic islet transplantation setting, this approach might sum to the ongoing effort to achieve consistent and successful single-donor islet transplantation.

Exendin-4 attenuates brain death-induced liver damage in the rat.

The majority of liver grafts destined for transplantation originate from brain dead donors. However, significantly better posttransplantation outcomes are achieved when organs from living donors are used, suggesting that brain death (BD) causes irreversible damage to the liver tissue. Recently, glucagon‐like peptide‐1 (GLP1) analogues were shown to possess interesting hepatic protection effects in different liver disease models. We hypothesized that donor treatment with the GLP1 analogue exendin‐4 (Ex‐4) could alleviate BD‐induced liver damage. A rat model of BD was employed in order to estimate BD‐induced liver damage and Ex‐4s potential protective effects. Liver damage was assessed by biochemical determination of circulating hepatic markers. Apoptosis in the hepatic tissue was assessed by immunoblot and immunohistochemistry using an antibody that only recognizes the active form of caspase‐3. Gene expression changes in inflammation and stress response genes were monitored by quantitative real‐time polymerase chain reaction. Here, we show that Ex‐4 administration to the brain dead liver donors significantly reduces levels of circulating aspartate aminotransferase and lactate dehydrogenase. This was accompanied by a remarkable reduction in hepatocyte apoptosis. In this model, BD caused up‐regulation of tumor necrosis factor and stress‐related genes, confirming previous findings in clinical and animal studies. In conclusion, treatment of brain dead rats with Ex‐4 reduced BD‐induced liver damage. Further investigation is needed to determine the molecular basis of the observed liver protection. After testing in a randomized clinical trial, the inclusion of GLP1 analogues in organ donor management might help to improve organ quality, maximize organ donation, and possibly increase liver transplantation success rates. Liver Transpl 21:1410‐1418, 2015.

Association between Asp299Gly and Thr399Ile Polymorphisms in Toll-LikeReceptor 4 Gene and Type 2 Diabetes Mellitus: Case-Control Study and Meta-Analysis

Objective: This paper describes a case-control study and a meta-analysis conducted to determine whether the TLR4 Asp299Gly (rs4986790) and Thr399Ile (rs4986791) polymorphisms are associated with type 2 diabetes mellitus (T2DM). Methods: In the case-control study were enrolled 1683 T2DM patients and 584 nondiabetic subjects from Brazil. A literature search was conducted in order to identify studies that investigated associations between the referred TLR4 polymorphisms and T2DM. Pooled odds ratios (OR) were calculated for allele contrast and dominant inheritance models. Results: In the case-control study, genotype and allele frequencies of the Asp299Gly and Thr399Ile polymorphisms differed between T2DM patients and nondiabetic subjects (P<0.05). Moreover, the presence of the minor alleles of these polymorphisms were significantly associated with protection for T2DM, after adjusting for ethnicity, under a dominant model [Asp299Gly: OR=0.68 (95% CI 0.49-0.94); Thr399Ile: OR=0.65 (95% CI 0.46-0.90)]. Seven studies were eligible for inclusion in the meta-analysis. Meta-analysis results showed that the Asp299Gly polymorphism was associated with T2DM protection [OR=0.68 (95% CI 0.46-1.00), allele contrast model]. Stratification by ethnicity revealed that both polymorphisms were associated with T2DM protection under allele contrast and dominant models in Brazilian population but not in Europeans. Conclusions: In our case-control study, we were able to demonstrate a possible association between the TLR4 Asp299Gly and Thr399Ile polymorphisms and protection for T2DM. In agreement, the meta-analysis results showed an association of the Asp299Gly polymorphism with T2DM protection in the whole group, and associations of the Asp299Gly and Thr399Ile polymorphisms with T2DM protection in the Brazilian group but not in European descendent. This is the largest TLR4 meta-analysis performed so far. In other ethnicities further studies with large sample size are necessary to confirm these associations in different ethnicities as well as to elucidate the roles possibly played by these polymorphisms in the pathogenesis of T2DM.

Assessment of Regulatory T Cells in Childhood Immune Thrombocytopenic Purpura

This study had the objective to assess the frequency of Tregs in children newly diagnosed with ITP and ascertain whether an association exists between Tregs and platelet counts, by means of a comparison with healthy controls. This case-control study included 19 patients newly diagnosed with ITP—whose blood samples were collected at four points in time: before any therapy and 1, 3, and 6 months after diagnosis—and 19 healthy controls. Tregs (CD4+ CD25+Foxp3 T cells) were evaluated by flow cytometry. There was a statistically significant difference in platelet count between the case and control groups. There were no significant differences in Treg counts between cases and controls at any point during the course of the study and no difference in Treg counts between the chronic and nonchronic groups and no significant correlation between Tregs and platelet counts in the case and control groups. The findings of this study did not show any statistically significant correlation between Tregs and number of platelets in the case and control groups. Treg cells did not play a role in the regulation of autoimmunity in children with ITP.

The rs2292239 polymorphism in ERBB3 gene is associated with risk for type 1 diabetes mellitus in a Brazilian population

The Erb-b2 receptor tyrosine kinase 3 (ERBB3) belongs to a family of epidermal growth factor receptors of protein tyrosine kinases, and regulates cell survival, differentiation and proliferation in several cell types. Previous studies have suggested that ERBB3 contributes to T1DM pathogenesis by modulating antigen presenting cell function, autoimmunity and cytokine-induced beta-cell apoptosis. Accordingly, some genome-wide association studies identified ERBB3 gene as a susceptibility locus for T1DM, with the strongest association signal being observed for the rs2292239 single nucleotide polymorphism (SNP) in intron 7 of the gene. Therefore, the aim of the present study was to replicate the association of the ERBB3 rs2292239 SNP with T1DM in a Brazilian population. We analyzed 421 T1DM patients (cases) and 510 nondiabetic subjects (controls). All subjects were self-declared as white. The ERBB3 rs2292239 (A/C) SNP was genotyped by real-time PCR using TaqMan MGB probes. Genotype (P=0.001) and allele (P=0.002) frequencies of the ERBB3 rs2292239 SNP were differently distributed between T1DM patients and nondiabetic controls. Moreover, the A allele was significantly associated with risk for T1DM when considering recessive (OR=1.58, 95% CI 1.11-2.27; P=0.015), additive (OR=1.78, 95% CI 1.21-2.62; P=0.004), and dominant (OR=1.39, 95% CI 1.07-1.81; P=0.016) models of inheritance. However, after adjustment for presence of high-risk HLA DR/DQ genotypes, the rs2292239 SNP remained independently associated with T1DM only for the additive model (OR=1.62, 95% CI 1.02-2.59; P=0.043). Our results suggest that the A/A genotype of the ERBB3 rs2292239 SNP is associated with risk for T1DM in a white Brazilian population.

Use of additives, scaffolds and extracellular matrix components for improvement of human pancreatic islet outcomes in vitro: A systematic review

ABSTRACT Pancreatic islet transplantation is an established treatment to restore insulin independence in type 1 diabetic patients. Its success rates have increased lately based on improvements in immunosuppressive therapies and on islet isolation and culture. It is known that the quality and quantity of viable transplanted islets are crucial for the achievement of insulin independence and some studies have shown that a significant number of islets are lost during culture time. Thus, in an effort to improve islet yield during culture period, researchers have tested a variety of additives in culture media as well as alternative culture devices, such as scaffolds. However, due to the use of different categories of additives or devices, it is difficult to draw a conclusion on the benefits of these strategies. Therefore, the aim of this systematic review was to summarize the results of studies that described the use of medium additives, scaffolds or extracellular matrix (ECM) components during human pancreatic islets culture. PubMed and Embase repositories were searched. Of 5083 articles retrieved, a total of 37 articles fulfilled the eligibility criteria and were included in the review. After data extraction, articles were grouped as follows: 1) “antiapoptotic/anti-inflammatory/antioxidant,” 2) “hormone,” 3) “sulphonylureas,” 4) “serum supplements,” and 5) “scaffolds or ECM components.” The effects of the reviewed additives, ECM or scaffolds on islet viability, apoptosis and function (glucose-stimulated insulin secretion - GSIS) were heterogeneous, making any major conclusion hard to sustain. Overall, some “antiapoptotic/anti-inflammatory/antioxidant” additives decreased apoptosis and improved GSIS. Moreover, islet culture with ECM components or scaffolds increased GSIS. More studies are needed to define the real impact of these strategies in improving islet transplantation outcomes.

Comparison of two techniques for evaluation of pancreatic islet viability: flow cytometry and FDA/PI staining

Background Type 1 diabetes (T1D) accounts for approximately 10% of all diabetes cases, and it is caused by autoimmune destruction of pancreatic beta-cells, which leads to insulin deficiency and fates individuals to require insulin treatment to survive. Although important advances in the treatment of T1D have been achieved in recent yrs., this disease is associated with chronic complications that lead to high morbidity and mortality rates in young adults of productive age. In patients with unstable T1D, pancreatic islet transplantation is a therapeutic option to restore insulin secretion and improve glycemic control. However, the success of islet transplantation is dependent, in part, on the number of isolated islets as well as factors associated with their quality, which is assessed by functional and viability tests. In this context, the method currently used for islet viability evaluation [fluorescein diacetate (FDA)/propidium iodide (PI) staining] is not accurate enough, and new methods have been researched, such as flow cytometry.