Andrea Carla Bauer

Polymorphisms in the TLR3 gene are associated with risk for type 1 diabetes mellitus.

INTRODUCTION Viral pathogens seem to play a role in triggering the autoimmune destruction that leads to the development of type 1 diabetes mellitus (T1DM). Toll-like receptor 3 (TLR3) has been shown to recognize double-stranded RNA, a molecular signature of most viruses. It is expressed at high levels in pancreatic β-cells and immune cells, suggesting a role for it in the pathogenesis of T1DM. Therefore, the aim of this study was to investigate whether TLR3 polymorphisms are associated with T1DM. METHODS Frequencies of the TLR3 rs11721827, rs13126816, rs5743313, rs7668666, and rs3775291 polymorphisms were analyzed in 449 T1DM patients and in 507 nondiabetic subjects. Haplotypes constructed from the combination of these polymorphisms were inferred using a Bayesian statistical method. RESULTS The rs3775291 and rs13126816 polymorphisms were associated with T1DM, and the strongest association was observed for the additive model (odds ratio (OR)=2.3, 95% CI 1.3-4.2 and OR=2.1, 95% CI 1.3-3.1 respectively). In the same way, the frequency of T1DM was higher as more risk alleles of the five polymorphisms were present (P-trend=0.001). Moreover, in T1DM patients, the minor alleles of the rs5743313 and rs117221827 polymorphisms were associated with an early age at diagnosis and worse glycemic control. CONCLUSION The TLR3 rs3775291 and rs13126816 polymorphisms are associated with risk for T1DM, while the rs5743313 and rs11721827 polymorphisms are associated with age at T1DM diagnosis and poor glycemic control. The number of risk alleles of the five TLR3 polymorphisms in the haplotypes seems to influence the risk for T1DM, suggesting that these polymorphisms might interact in the susceptibility for the disease.

Current role of the NLRP3 inflammasome on obesity and insulin resistance: A systematic review

NLRP3 inflammasome activation seems to be a culprit behind the chronic inflammation characteristic of obesity and insulin resistance (IR). Nutrient excess generates danger-associated molecules that activate NLRP3 inflammasome-caspase 1, leading to maturation of IL-1β and IL-18, which are proinflammatory cytokines released by immune cells infiltrating the adipose tissue (AT) from obese subjects. Although several studies have reported an association of the NLRP3 inflammasome with obesity and/or IR; contradictory results were also reported by other studies. Therefore, we conducted a systematic review to summarize results of studies that evaluated the association of the NLRP3 with obesity and IR. Nineteen studies were included in the review. These studies focused on NLRP3 expression/polymorphism analyses in AT. Overall, human studies indicate that obesity and IR are associated with increased NLRP3 expression in AT. Studies in obese mice corroborate this association. Moreover, high fat diet (HFD) increases Nlrp3 expression in murine AT while calorie-restricted diet decreases its expression. Hence, Nlrp3 blockade in mice protects against HFD-induced obesity and IR. NLRP3 rs10754558 polymorphism is associated with risk for T2DM in Chinese Han populations. In conclusion, available studies strongly points for an association between NLRP3 inflammasome and obesity/IR.

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.

Different digestion enzymes used for human pancreatic islet isolation: A mixed treatment comparison (MTC) meta-analysis

Collagenases are critical reagents determining yield and quality of isolated human pancreatic islets and may affect islet transplantation outcome. Some islet transplantation centers have compared 2 or more collagenase blends; however, the results regarding differences in quantity and quality of islets are conflicting. Thus, for the first time, a mixed treatment comparison (MTC) meta-analysis was carried out to compile data about the effect of different collagenases used for human pancreas digestion on islet yield, purity, viability and stimulation index (SI). Pubmed, Embase and Cochrane libraries were searched. Of 755 articles retrieved, a total of 15 articles fulfilled the eligibility criteria and were included in the MTC meta-analysis. Our results revealed that Vitacyte and Liberase MTF were associated with a small increase in islet yield (islet equivalent number/g pancreas) when compared with Sevac enzyme [standardized mean difference (95% credible interval – CrI) = −2.19 (−4.25 to −0.21) and −2.28 (−4.49 to −0.23), respectively]. However, all other enzyme comparisons did not show any significant difference regarding islet yield. Purity and viability percentages were not significantly different among any of the analyzed digestion enzymes. Interestingly, Vitacyte and Serva NB1 were associated with increased SI when compared with Liberase MTF enzyme [unstandardized weighted mean difference (95% CrI) = −1.69 (−2.87 to −0.51) and −1.07 (−1.79 to −0.39), respectively]. In conclusion, our MTC meta-analysis suggests that the digestion enzymes currently being used for islet isolation works with similar efficiency regarding islet yield, purity and viability; however, Vitacyte and Serva NB1 enzymes seem to be associated with an improved SI as compared with Liberase MTF.

Effect of co-culture of mesenchymal stem/stromal cells with pancreatic islets on viability and function outcomes: a systematic review and meta-analysis

ABSTRACT The maintenance of viable and functional pancreatic islets is crucial for successful islet transplantation from brain-dead donors. To overcome islet quality loss during culture, some studies have co-cultured islets with mesenchymal stem/stromal cells (MSC). However, it is still uncertain if MSC-secreted factors are enough to improve islet quality or if a physical contact between MSCs and islets is needed. Therefore, we performed a systematic review and meta-analysis to clarify the effect of different culture contact systems of islets with MSCs on viability and insulin secretion outcomes. Pubmed and Embase were searched. Twenty studies fulfilled the eligibility criteria and were included in the qualitative synthesis and/or meta-analysis. For both outcomes, pooled weighted mean differences (WMD) between islet cultured alone (control group) and the co-culture condition were calculated. Viability mean was higher in islets co-cultured with MSCs compared with islet cultured alone [WMD = 18.08 (95% CI 12.59–23.57)]. The improvement in viability was higher in islets co-cultured in indirect or mixed contact with MSCs than in direct physical contact (P <0.001). Moreover, the mean of insulin stimulation index (ISI) was higher in islets from co-culture condition compared with islet cultured alone [WMD = 0.83 (95% CI 0.54–1.13)], independently of contact system. Results from the studies that were analyzed only qualitatively are in accordance with meta-analysis data. Co-culture of islets with MSCs has the potential for protecting islets from injury during culture period. Moreover, culture time appears to influence the beneficial effect of different methods of co-culture on viability and function of islets.

Polymorphisms of the UCP2 Gene Are Associated with Glomerular Filtration Rate in Type 2 Diabetic Patients and with Decreased UCP2 Gene Expression in Human Kidney

Introduction Uncoupling protein 2 (UCP2) reduces production of reactive oxygen species (ROS) by mitochondria. ROS overproduction is one of the major contributors to the pathogenesis of chronic diabetic complications, such as diabetic kidney disease (DKD). Thus, deleterious polymorphisms in the UCP2 gene are candidate risk factors for DKD. In this study, we investigated whether UCP2 -866G/A, Ala55Val and Ins/Del polymorphisms were associated with DKD in patients with type 2 diabetes mellitus (T2DM), and whether they had an effect on UCP2 gene expression in human kidney tissue biopsies. Materials and Methods In a case-control study, frequencies of the UCP2 -866G/A, Ala55Val and Ins/Del polymorphisms as well as frequencies of the haplotypes constituted by them were analyzed in 287 T2DM patients with DKD and 281 T2DM patients without this complication. In a cross-sectional study, UCP2 gene expression was evaluated in 42 kidney biopsy samples stratified according to the presence of the UCP2 mutated -866A/55Val/Ins haplotype. Results In the T2DM group, multivariate logistic regression analysis showed that the -866A/55Val/Ins haplotype was an independent risk factor for DKD (OR = 2.136, 95% CI 1.036–4.404), although neither genotype nor allele frequencies of the individual polymorphisms differed between case and control groups. Interestingly, T2DM patients carrying the mutated haplotype showed decreased estimated glomerular filtration rate (eGFR) when compared to subjects with the reference haplotype (adjusted P= 0.035). In kidney biopsy samples, UCP2 expression was significantly decreased in UCP2 mutated haplotype carriers when compared to kidneys from patients with the reference haplotype (0.32 ± 1.20 vs. 1.85 ± 1.16 n fold change; adjusted P< 0.000001). Discussion Data reported here suggest that the UCP2 -866A/55Val/Ins haplotype is associated with an increased risk for DKD and with a lower eGFR in T2DM patients. Furthermore, this mutated haplotype was associated with decreased UCP2 gene expression in human kidneys.

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.

MicroRNAs and diabetic kidney disease: Systematic review and bioinformatic analysis

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Emerging evidence has suggested a role for miRNAs in the development of diabetic kidney disease (DKD), indicating that miRNAs may represent potential biomarkers of this disease. However, results are still inconclusive. Therefore, we performed a systematic review of the literature on the subject, followed by bioinformatic analysis. PubMed and EMBASE were searched to identify all studies that compared miRNA expressions between patients with DKD and diabetic patients without this complication or healthy subjects. MiRNA expressions were analyzed in kidney biopsies, urine/urinary exosomes or total blood/plasma/serum. MiRNAs consistently dysregulated in DKD patients were submitted to bioinformatic analysis to retrieve their putative target genes and identify potentially affected pathways under their regulation. As result, twenty-seven studies were included in the systematic review. Among 151 dysregulated miRNAs reported in these studies, 6 miRNAs were consistently dysregulated in DKD patients compared to controls: miR-21-5p, miR-29a-3p, miR-126-3p, miR-192-5p, miR-214-3p, and miR-342-3p. Bioinformatic analysis indicated that these 6 miRNAs are involved in pathways related to DKD pathogenesis, such as apoptosis, fibrosis, and extracellular matrix accumulation. In conclusion, six miRNAs seem to be dysregulated in patients with different stages of DKD, constituting potential biomarkers of this disease.

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.

Role of Innate Immunity in Preeclampsia: A Systematic Review:

Innate immune system dysfunction has been known to be a key player in preeclampsia (PE). Activation of the maternal innate immunity may be triggered by invading microorganisms or endogenous ligands, which are detected by different pattern recognition receptors (PRRs). Although some studies have linked PRR activation to PE, it is still unclear if dysregulated PRR expression is associated with the development of this complication. Therefore, we conducted a systematic review of the literature, searching articles that evaluated associations of PRRs with PE. Twenty-six studies met the inclusion criteria: 20 of them analyzed PRR expressions and 6 studies investigated the association between PRR polymorphisms and PE. Among the PRRs, only few studies analyzed retinoic acid–inducible gene I-like helicase (RLH) and/or toll-like receptor (TLR)-1, 5, 6, 7, 8, and 9 expressions in immune cells or placentas from women with PE and controls; thus, it is inconclusive if these PRRs are involved in PE. Results from the 10 studies that analyzed TLR-2 expressions in women with PE and controls are also contradictory. The majority of the studies that investigated TLR-3 and -4 expressions indicate that these PRRs are increased in placenta or immune cells from women with PE compared to pregnant control woman. To date, polymorphisms in TLR-2, -3, and -4 and nucleotide-binding oligomerization domain-like receptor 2 genes do not seem to be associated with PE development. No study has evaluated the association between polymorphisms in genes codifying other TLRs or RLHs genes. In conclusion, available data in literature support a role for TLR-3 and TLR-4 in the pathogenesis of PE.