Padronização da extração de DNA genômico a partir de diferentes fases do leite humano/ Standardization of genomic DNA extraction from different phases of human milk

O leite humano (LH) é um fluido fisiológico composto por eletrólitos, glicídios, glicoproteínas e células nucleadas, como leucócitos e células epiteliais mamárias. Posto isso, o LH pode ser uma fonte viável e segura para a obtenção de DNA comparado à extração pelo sangue periférico, visto que não é necessário realizar o acesso venoso periférico nas puérperas, o que seria incômodo para elas. Dessa forma, devido à escassez de informações na literatura, este trabalho propõe a padronização de técnicas para extração de DNA do LH em suas diferentes fases (colostro, transição e maduro) pelas seguintes metodologias: Salting out, Qiagen QIAamp DNA Mini Kit™ e Biopur®. Os resultados das três técnicas de extração produziram um DNA de maior pureza, porém em concentrações mais baixas, ainda que suficientes para processos de amplificação, comparado ao DNA extraído pelo sangue periférico

Genetic polymorphisms of human platelet antigens in Euro-African and Japanese descendants from Parana, Southern Brazil

The frequency distributions of HPA-1 to HPA-6 and HPA-15 were evaluated in two Brazilian populations from Parana: a mixed population of predominantly Caucasians and a population of Japanese descendants. Genotyping was performed by PCR-SSP in 364 unrelated individuals. Differences in the distribution of HPA highlight diversity in Brazilian miscegenation and the importance of formation of the HPA panel composed of regional blood donors

The Influence of Interleukin 17A and IL17F Polymorphisms on Chronic Periodontitis Disease in Brazilian Patients

A case-control study was conducted on patients with chronic periodontitis (CP) and healthy controls with the aim of evaluating possible association between interleukin 17A (IL17A) G197A (rs2275913) and IL17F T7488C (rs763780) polymorphisms and periodontitis. Genotypes were determined by PCR-RFLP method. Statistical analyses were conducted using the OpenEpi and SNPStas software to calculate Chi-square with Yates correction or Fisher’s exact tests, odds ratios (OR), and 95% confidence intervals (CIs). SNPStas software was used to calculate Hardy-Weinberg equilibrium. IL17A AA genotype was more frequent in patients with chronic periodontitis (CP) in the codominant and recessive models (P=0.09; OR = 2.53 and P=0.03; OR = 2.46, resp.), the females with CP (P=0.01, OR = 4.34), Caucasoid patients with CP (P=0.01, OR = 3.45), and nonsmoking Caucasian patients with CP (P=0.04, OR = 3.51). The IL17A A allele was also more frequent in Caucasians with CP (P=0.04, OR = 1.59). IL17F T7488C polymorphism was not associated with chronic periodontitis. In these patients from Southern Brazil, the IL17A rs2275913 polymorphisms, IL17A AA genotype, and the A allele were associated with a susceptibility to chronic periodontitis

IL18 Polymorphism and Periodontitis Susceptibility, Regardless of IL12B, MMP9, and Smoking Habits

Genetic variations contribute to the susceptibility in the development of periodontitis. The aim of this study was to investigate the influence of IL18, IL12, and MMP9 polymorphisms in the chronic periodontitis. This case-control study involved 381 individuals matched by gender and age. Genotyping of IL18 (rs187238 and rs1946518) and IL12B (rs3212227) was performed by PCR-SSP and PCR-RFLP was used for MMP9 (rs3918242). IL-18 and MMP-9 were quantified in the serum by ELISA. SNPStats and OpenEpi software were used for statistical analysis and, in order to eliminate smoking as a confounding factor, the analyses were also performed in nonsmoking subjects. The IL18-137G/C genotype was associated with the risk of chronic periodontitis in nonsmokers (Pc=0.03; OR=1.99; overdominant inherence model). In the multivariate analyses, homozygous IL18-137G/G and IL18-607C/C were more frequent in males compared to women with these same genotypes (OR=2.51 and OR=3.30, respectively). The serum levels of the IL-18 in patients were higher than those in healthy controls (P=0.005). IL12B and MMP9 polymorphisms and MMP-9 serum concentration were similar in patients and controls. In this study, IL18 was associated with chronic periodontitis susceptibility. Men had greater risk than women for developing the disease when IL18 polymorphism was considered and the susceptibility was independent of the smoking status

Genotyping of Dombrock and Lutheran blood group systems in blood donors from the southwestern region of the state of Paraná, Southern Brazil

Background: Lutheran and Dombrock are two blood group systems with low immunogenic antigens; they can cause mild-to-moderate transfusion reactions. For both, immunophenotyping is not performed in the pretransfusion routine in Brazil. In addition, the distribution of their antigenic frequencies is an important marker of ethnicity. Thus, the goal of this study was to carry out the genotyping of the LU*01, LU*02, DO*01 and DO*02 alleles of the Lutheran and Dombrock blood group systems in blood donors from the southwestern region of the state of Paraná, Southern Brazil. Method: Genotyping was performed for 251 blood donors by specific allele-polymerase chain reaction. The genotype and allele frequencies were obtained through direct counting and compared with other Brazilian populations using the chi-square test with Yates correction. Results: The distribution of genotype frequencies for LU were 0.4% for LU*01/LU*01, 6.8% for LU*01/LU*02 and 92.8% for LU*02/LU*02 and for DO, they were 19.9% for DO*01/DO*01, 44.6% for DO*01/DO*02 and 35.5% for DO*02/DO*02. The allele and genotype frequencies of LU and DO were similar to those expected for Caucasians, but the DO*01/DO*01 genotype frequency was different to other Brazilian populations. The rare LU*01/LU*01 genotype was found in a loyal blood donor. Conclusion: The genotyping techniques allowed the evaluation of the LU*01, LU*02, DO*01 and DO*02 alleles in blood donors registered in the Hemotherapy Center of the southwestern region of Paraná, Southern Brazil, and contributed to a genotyped blood donor database. Keywords: ART4 protein, BCAM human protein, Genotyping techniques, Blood group antigen

Influence of inflammasome NLRP3, and IL1B and IL2 gene polymorphisms in periodontitis susceptibility.

The pathogenesis of periodontitis (PD) involves several molecules of the immune system that interact in a network to eliminate the periodontopathogens, yet, they contribute to periodontal tissue destruction. The different mechanisms that lead to periodontal tissue damage are not clear. Despite this, immune response genes have been related to the development of PD previously, such as those involved in inflammasomes which are multiprotein complexes and cytokines including Interleukin-1. The aim of the study was to evaluate the polymorphisms in NLRP3 inflammasome, cytokine and receptor of cytokines genes in the development of periodontitis. This case-control study was conducted in 186 patients with PD (stage II and III and grade B) and 208 controls (localized gingivitis and periodontally healthy individuals). Genotyping was performed using PCR-RFLP for the SNP rs4612666 in NLRP3 and using PCR-SSP for IL1A, IL1B, IL1R, IL1RN, IL4RA, INFG, TGFB1, TNF, IL2, IL4, IL6, and IL10. Cytokine serum levels were measured using Luminex technology. SNPStats and OpenEpi software were used to perform statistical analysis. The higher frequencies of NLRP3 T/C and IL1B -511 T/T genotypes and IL2 (+166, -330) GT haplotype were observed in patients with PD compared to controls. The SNPs in NLRP3, IL1R +1970, IL6-174, TNF -308, IL2 +166 and -330, TGFB1 +869 and +915, IL4RA +1902, IL4-1098 and -590 were associated to PD in men. In conclusion, polymorphisms in NLRP3, IL1B and IL2 genes were associated to PD susceptibility. Men carrying the NLRP3, IL1R, IL6, TNF, IL2, TGFB1, IL4RA and IL4 polymorphisms had greater susceptibility than women for developing PD

IL17F: A Possible Risk Marker for Spondyloarthritis in HLA-B*27 Negative Brazilian Patients

HLA-B*27 is an important marker for spondyloarthritis (SpA), however, many SpA patients are HLA-B*27 negative. Thus, the aim of this study was to investigate the influence of IL17, TNF and VDR gene polymorphisms in SpA patients who were HLA-B*27 negative. This case-control study was conducted in 158 patients [102 patients with ankylosing spondylitis (AS) and 56 with psoriatic arthritis (PsA)] and 184 controls. HLA-B*27 genotyping was performed using PCR-SSP and IL17A (rs2275913), IL17F (rs763780), TNF-308 (rs1800629), TNF-238 (rs361525), FokI C>T (rs2228570), TaqI C>T (rs731236), ApaI A>C (rs7975232), and BsmI C>T (rs1544410) using PCR-RFLP. Statistical analyses were performed by Chi-square and logistic regression using OpenEpi and SNPStats software. The IL17F C allele frequency was higher in patients with SpA, AS and PsA compared to controls. The IL17F T/C genotype frequency was higher in SpA patients in an overdominant inheritance model and when men and women were separately analyzed. IL17A_IL17F AC haplotype was significantly associated to the risk for SpA patients. As for VDR, the ApaI a/a was a potential risk factor for SpA in men. In conclusion, IL17F C variant contributed to the risk of SpA in Brazilian patients who were HLA-B*27 negative and could be a potential marker for SpA

Association of IL16 polymorphisms with periodontitis in Brazilians: A case- control study.

Periodontitis (PD) is a chronic inflammatory process resulting from the relationship of the immune response with the components in dental plaque. Cytokines and their genetic polymorphisms seem to be involved in the immunopathogenesis of this disease. This study aimed to evaluate the correlation of IL16 polymorphism with PD. A case-control study was conducted in a sample of individuals from southern Brazil. The genotyping of IL16, rs11556218 T>G, rs4072111 C>T e rs4778889 T>C, was performed using the PCR-RFLP methodology. The serum level of IL-16 was determined using an IL-16 ELISA kit for humans. SNPStats and OpenEpi software and Wilcoxon's U test were used to perform statistical analysis. IL16 rs11556218 polymorphism was significantly associated to PD in nonsmoking patients: individuals with G/G genotype were less likely to develop PD compared to the T/T genotype (OR = 0.10; Pc = 0.019, codominant model). In addition, the TTT haplotype was associated with a high risk for PD (OR = 2.45; P = 0.01). A low IL-16 serum level was observed among individuals with PD when compared to controls (P = 0.027). Thus, the IL16 rs16556218 polymorphism and the serum levels of IL-16 were associated with periodontitis in a Brazilian sample, and this was influenced by environmental factors such as smoking

Vitamin D receptor gene polymorphisms are associated with leprosy in southern Brazil

Vitamin D, together with its nuclear receptor (VDR), plays an important role in modulating the immune response, decreasing the inflammatory process. Some polymorphisms of the VDR gene, such as BsmI (G>A rs1544410), ApaI (G>T rs7975232), and TaqI (T>C rs731236) could affect its stability and mRNA transcription activity, while FokI T>C (rs2228570) gives a truncated protein with three fewer amino acids and more efficiency in binding vitamin D. This study evaluated these four polymorphisms in the immunopathogenesis of leprosy in 404 patients and 432 control individuals without chronic or infectious disease in southern Brazil. When analyzing differences in the allele and genotype frequency of polymorphisms between patients (leprosy per se, multibacillary, and paucibacillary clinical forms) and controls, we found no statistically significant association. Regarding haplotype analysis, the bAt haplotype was associated with protection from leprosy per se (P = 0.004, OR = 0.34, CI = 0.16-0.71) and from the multibacillary clinical form (P = 0.005, OR = 0.30, CI = 0.13-0.70). In individuals aged 40 or more years, this haplotype has also showed protection against leprosy per se and multibacillary (OR = 0.26, CI = 0.09-0.76; OR = 0.26, CI = 0.07-0.78, respectively), while the BAt haplotype was a risk factor for leprosy per se in the same age group (OR = 1.34, CI = 1.04-1.73). In conclusion, despite having found no associations between the VDR gene polymorphisms with the development of leprosy, the haplotypes formed by the BsmI, ApaI, and TaqI polymorphisms were associated with leprosy per se and the multibacillary clinical form10CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO ARAUCÁRIA DE APOIO AO DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO DO ESTADO DO PARANÁ - FAsem informaçãosem informação1589/2017-CSDUE