Amador Goncalves-Primo

Inflammatory cytokine gene polymorphisms and spontaneous preterm birth

The objective was to search for an association between spontaneous preterm birth (sPTB) and single and/or combined polymorphisms in genes TNFA -308 G>A, IL10 -1082 G>A, IL10 -819 C>T, IL10 -592 C>A, IL6 -174 G>C, and IFNG +874 A>T. Genotyping was performed on 410 Brazilian ethnically matched women managed at two hospitals (two independent case-control sets). One set consisted of 122 cases and 101 controls, and the other set comprised 82 cases and 105 controls. We compared genotype and genotype-combination frequencies between cases and controls using Fishers exact or the chi(2) tests and confirmed results using logistic regression. Among the six SNPs studied, we found no independent association between any single SNP and sPTB risk. The multi-locus analysis revealed a significant association between sPTB and the TNFA(GG)/IL6(GG)/IFNG(AA) genotype combination (p=0.002), confirmed by logistic regression. Our data suggest that the combination of TNF-alpha, IFN-gamma, and IL-6 maternal gene polymorphisms might contribute to susceptibility to sPTB. This finding could be investigated as a possible genetic marker for the risk of spontaneous preterm birth.

New approach reveals CD28 and IFNG gene interaction in the susceptibility to cervical cancer

Cervical cancer is a complex disease with multiple environmental and genetic determinants. In this study, we sought an association between polymorphisms in immune response genes and cervical cancer using both single-locus and multi-locus analysis approaches. A total of 14 single nucleotide polymorphisms (SNPs) distributed in CD28, CTLA4, ICOS, PDCD1, FAS, TNFA, IL6, IFNG, TGFB1 and IL10 genes were determined in patients and healthy individuals from three independent case/control sets. The first two sets comprised White individuals (one group with 82 cases and 85 controls, the other with 83 cases and 85 controls) and the third was constituted by non-white individuals (64 cases and 75 controls). The multi-locus analysis revealed higher frequencies in cancer patients of three three-genotype combinations [CD28+17(TT)/IFNG+874(AA)/TNFA-308(GG), CD28+17(TT)/IFN+847(AA)/PDCD1+7785(CT), and CD28 +17(TT)/IFNG+874(AA)/ICOS+1564(TT)] (P < 0.01, Monte Carlo simulation). We hypothesized that this two-genotype [CD28(TT) and IFNG(AA)] combination could have a major contribution to the observed association. To address this question, we analyzed the frequency of the CD28(TT), IFNG(AA) genotype combination in the three groups combined, and observed its increase in patients (P = 0.0011 by Fishers exact test). The contribution of a third polymorphism did not reach statistical significance (P = 0.1). Further analysis suggested that gene-gene interaction between CD28 and IFNG might contribute to susceptibility to cervical cancer. Our results showed an epistatic effect between CD28 and IFNG genes in susceptibility to cervical cancer, a finding that might be relevant for a better understanding of the disease pathogenesis. In addition, the novel analytical approach herein proposed might be useful for increasing the statistical power of future genome-wide multi-locus studies.

TLR4 mRNA levels as tools to estimate risk for early posttransplantation kidney graft dysfunction.

Background The participation of Toll-like receptor (TLR) 4, an innate immunity receptor, has been previously demonstrated in the pathogenesis of acute renal injury. We aimed to investigate whether messenger RNA (mRNA) levels of TLR4 and its adapter molecule, myeloid differentiation primary response gene (MYD) 88, are associated with delayed graft function (DGF) and could be used as biomarkers of its occurrence. Methods TLR4 and MYD88 gene mRNA levels were evaluated with real-time polymerase chain reaction, in preimplantation biopsies (n=89) and first day posttransplantation samples of urine (n=67) and blood (n=80) from graft recipients and analyzed according to donor type (living or deceased) and DGF occurrence. Results Expression levels of both genes were higher in biopsies from deceased donors than from living donors (P<0.001 for both) but did not differ between deceased-donor kidney transplants with and without DGF; in urine, TLR4 expression levels were higher in patients with prolonged DGF (DGF lasting >14 days) (P=0.05, compared with cases without DGF); in blood, lower mRNA levels of TLR4 and MYD88 predicted pDGF occurrence with an accuracy of 86% and 87%, respectively. Conclusion The expression levels of TLR4 and MYD88 were higher in kidneys from deceased donors than from living donors. Lower levels of expression of both genes in blood were associated with DGF occurrence. The prediction of prolonged DGF by low TLR4 and MYD88 expression levels in blood with a greater the 85% accuracy was the most important finding of this study.

Investigation of apoptosis-related gene expression levels in preimplantation biopsies as predictors of delayed kidney graft function.

Background The purpose of this study was to investigate the expression of the gene coding for the antiapoptotic molecule Bcl-2, the proapoptotic molecule Bax, and the apoptosis executor enzyme caspase-3 in preimplantation renal biopsies (PIB) as markers for delayed graft function. Methods In this prospective single-center study, gene expression levels were evaluated using real-time TaqMan polymerase chain reaction in PIB of kidneys from 72 deceased donors (DDs) and 18 living donors (LDs). Results CASP3 and BAX expression levels were higher, whereas those of BCL2 were lower, in DD than in LD PIB. In biopsies from DD, BCL2 levels were lower in cases with DGF, whereas no differences were observed concerning CASP3 and BAX. The BAX/BCL2 gene expression ratio greater than 2.29 associated with DGF with an odds ratio of 2.00. A multiple regression analysis including data of TLR4 expression in the first day posttransplant PB from a previous study of our group conducted in the same patients revealed a very strong association of the combination of BAX/BCL2 greater than 2.3 in PIB and TLR4 of 0.95 uRE or lesser in PB with the occurrence of DGF, with OR of 120 and positive and negative predictive values of 91% and 92%, respectively. Conclusions The power to predict DGF of the combination of high BAX/BCL2 expression in PIB and low TLR4 expression in the first day posttransplant peripheral blood observed in the present study is extremely high, in comparison to any other marker or combinations of markers so far published in the literature.

Failure to Up-Regulate BCL2 Gene Expression in Deceased Donors Kidneys Associated with Occurrence of Delayed Graft Function: 541

This study was designed to investigate apoptotic molecular pathways in the pathogenesis of delayed graft function (DGF) in human kidney transplantation (Tx). For this, we evaluated expression levels of the genes BCL2, BAX, and CASP3 in pre-implantation biopsies (PIB) from kidneys of 72 deceased donors (DD) and from 18 living donors (LD). DGF was defined as dialysis requirement within the first week after TX. Total RNA was extracted from PIB with RNeasy Mini Kit (Qiagen). Target and endogenous control (TATA-binding protein) genes expression levels were quantified by TaqMan® assays (Applied Biosystems) and results are presented as relative expression units (RU) calculated with the 2-(delta delta Ct) method. The reference group consisted of PIB from kidneys of 18 living donors (LD). Statistics: Mann-Whitney and Fisher ́s exact tests; significance was set at p< 0.05. Tx with DD kidneys that presented (n=27) or not (n=45) DGF did not differ in respect to donors‘ age, % of expanded criteria donors, recipients‘ age, and cold ischemia time (CIT). BCL2 expression in PIB was lower in kidneys from DD than from LD (median values: 0.6 vs 1.0 RU, p=0.007), did not differ between DD with and without expanded criteria nor between grafts with CIT < 30h and between 30 and 38h, and was lower in DD Tx with than without DGF (median values: 0.5 vs 0.8 RU, p=0.04). BAX expression in PIB was higher in kidneys from DD than from LD (median values: 2.0 vs 1.0 RU, p< 0.0001), did not differ between DD with and without expanded criteria, was higher in grafts with CIT between 30 and 38h (median values: 2.2 vs 1.7 RU, p=0.02), and did not differ between cases with than without DGF; BAX/BCL2 ratio was higher in kidneys from DD than from LD and in cases with DGF (median values: 3.0 vs 1.3, p< 0.0001); CASP3 expression was higher in kidneys from DD than from LD (median values: 1.2 vs 1.0 RU, p=0.005), did not differ between DD with and without expanded criteria, between grafts with CIT < 30h and from 30 to 38h, nor between cases with and without DGF. In conclusion, up-regulation of the anti-apoptotic gene BCL2 in PIB from DD conferred protection against DGF, whilst the expression of the executor caspase CASP3 and the pro-apoptotic BAX genes, although higher in DD than in LD, did not correlate with DGF. This finding is in line with experimental data (Gobe, 2000) showing that the protection of acute renal failure conferred by BCL2 probably results both from an anti-apoptotic effect and from a positive stimulus on the synthesis of several growth factors by cells of the distal tubule 561

BLOOD TLR-4 AND MYD88 MRNA LEVELS ARE ASSOCIATED WITH DELAYED GRAFT FUNCTION AFTER DECEASED DONOR KIDNEY TRANSPLANTATION: 1456

Introduction. The delay in kidney graft function (DGF) due to ischemia and reperfusion injury (IRI) occurs mainly in deceased-donor kidney transplant recipients (DD-R) and is associated with worst graft survival. There is evidence for a role of innate immunity in the pathogenesis of IRI. The purpose of this work was to evaluate TLR4 (Toll-Like Receptor 4) and its adaptor molecule MyD88 mRNA levels in pre-implantation biopsies (PIB) and in day 1 post-transplant (Tx) samples of urine and peripheral blood leukocytes (PBL), as predictors of DGF and/or of delay in graft function recovery (DGFR). Methods. We studied 69 DD-R and 18 recipients of living-donor Tx (LD-R) from a single center. DGF was defined as dialysis requirement within the first week after Tx, and DGFR was defined as ≥14 days needed for a 20% fall in serum creatinine in relation to the highest post-TX value. Total RNA was extracted from PIB, urine and PBL (buffy-coat) with RNeasy Mini Kit. After reverse transcription, Taqman assays were employed to quantify TLR4, MyD88 and TATA-binding protein (TBP, internal control) mRNA. TLR4 and MyD88 mRNA values (relative expression, 2-ΔΔCt method) were compared by the Mann-Whitney test and the significance was set at <0.05. The results are presented as median values. Results. TLR4 and MyD88 mRNA levels in PIB were higher in DD than in LD kidneys (1.32 vs 1.00, p<0.002, and 1.55 vs 1.00, p<0.001, respectively). No differences between DD-R and LD-R were observed regarding TLR4 and MyD88 expression in urine or PBL samples. Among the PIB of DD, no differences in TLR4 or MyD88 were observed between cases that presented or not DGF. The urinary mRNA levels of TLR4 and MyD88 were higher in DD-R with than without DGF (1.37 vs 1.06, NS, and 1.10 vs 0.65, p<0.05, respectively). On the other hand, the TLR4 and MyD88 mRNA levels in PBL were lower in DD-R with than without DGF (0.73 vs 1.28, p<0.001, and 0.88 vs 1.36, p<0.05, respectively). Regarding the occurrence or not of DGFR, similar results were observed: no differences in TLR4 or MyD88 expression levels in PIB; a trend for increased urinary TLR4 mRNA levels in cases with DGFR (1.45 vs 0.91, p=0.07); lower TLR4 and MyD88 mRNA levels in PBL of DD-R with than without DGFR (0.60 vs 1.00, p<0.003, and 0.68 vs 1.14, p<0.008, respectively). Conclusions. (1)TLR-4 and MyD88 mRNA levels were higher in PIB from DD than from LD; (2) there was no association between TLR-4 and MyD88 mRNA levels in PIB from DD and post-Tx graft function; (3) in day 1 urine, there was a trend for association of higher levels of MyD88 mRNA with DGF, and of TLR-4 with DGFR; (4) low TLR-4 and MyD88 mRNA levels in day 1 blood were associated with DGF and DGFR, representing a promising tool in the management of DD-R.