Pharmacogenomics of mycophenolic acid in kidney transplantation: Contribution of immune response-related genes

Abstract Mycophenolic acid (MPA) inhibits IMPDH, involved in the guanosine nucleotides synthesis, and prevents DNA replication in immune cells. The repression of cell and humoral immunity by MPA induces allograft tolerance preventing acute rejection in solid organ transplantation. MPA is an effective and safe drug, but genetic and non-genetic factors have been implicated in the interindividual variability of drug response. Several studies have shown the impact of variants of pharmacokinetics or pharmacodynamics-related genes on MPA response in kidney transplantation. This review explored further the influence of genes involved in the immune response on clinical outcomes of kidney recipients on short- or long-term MPA treatment. Variants in genes related to T cell activation (CD28, CTL4, ICOS, PDPC1), pro-inflammatory cytokines (IL2, IL6, IL12A, IL12B, TNF, IFNG), immunomodulatory cytokines (IL4, IL10, TGFB1), and innate immune response (CD14, TLR2, TLR4) were shown to be associated with increased risk of acute rejection, graft function or survival, chronic graft nephropathy, viral infections or MPA-induced myelotoxicity. Some of the significant pharmacogenetic associations were confirmed by meta-analyses of kidney transplantation. These findings are suggestive that variants in immune response-related genes contribute to the variability of MPA response, and have potential application as biomarkers of acute rejection in kidney transplantation.

The low-density lipoprotein receptor-related protein 5 (LRP5) 4037C>T polymorphism: candidate for susceptibility to type 1 diabetes mellitus

ABSTRACT Objective: The present study has investigated the association between low-density lipoprotein receptor-related protein 5 (LRP5) 4037C>T polymorphism and type 1 diabetes mellitus (T1DM) susceptibility in a Brazilian population. Subjects and methods: A total number of 134 T1DM patients and 180 normoglycemic individuals (NG) aged 6-20 years were studied. Glycated hemoglobin and glucose levels were determined. Genotyping of LRP5 4037C>T (rs3736228) was performed. Results: T1DM patients showed poor glycemic control. Genotypes in the codominant (CT: OR = 2.99 [CI 95%: 1.71-5.24], p < 0.001; TT: OR = 5.34 [CI 95%: 1.05-2702], p < 0.001), dominant (CT + TT: OR = 3.16 [CI 95%: 1.84-5.43], p < 0.001) and log-additive (OR = 2.78 [CI 95%: 1.70-4.52], p < 0.001) models, and LRP5 4037T allele (OR = 2.88, [CI 95%: 1.78-4.77], p < 0.001) were associated with an increased risk of developing T1DM. LRP5 4037CT and CT+TT carriers in T1DM group showed higher concentrations of serum glucose and glycated hemoglobin when compared with CC carriers. Conclusion: The LRP5 4037C>T may represent a candidate for T1DM susceptibility, as well as poor glycemic control.

Circulating cell-free DNA as a biomarker in the diagnosis and prognosis of colorectal cancer

Abstract Colorectal cancer (CRC) is a disease without evident clinical symptoms in early stages, leading to late diagnosis and disease management. Current diagnostic and prognostic tools require invasive procedures and circulating molecular biomarkers fail to have optimal sensitivity and specificity. Circulating biomarkers with high clinical performance may be valuable for early diagnosis and prognosis of CRC. The purpose of this review was to investigate the application of circulating cell-free DNA (ccfDNA) in CRC diagnosis and prognosis and the analytical methods used in blood samples in articles published between 2005 and 2016. Based on specific inclusion and exclusion criteria, 26 articles were selected. Most studies used ccfDNA quantification as the molecular biomarker. The analytical method was mainly based on the quantitative polymerase chain reaction (qPCR). Biomarkers based on aberrantly methylated genes (n=6) and ccfDNA integrity/fragmentation (n=2) were also used for the CRC diagnosis. The CRC prognosis used the detection of oncogene mutations, such as KRAS and BRAF, in ccfDNA. Significant differences were found in variables among the studies revealing potential bias. ccfDNA quantification as a diagnostic biomarker for CRC has promising results but it lacks clinical specificity since other diseases present a similar increase in ccfDNA content. However, increasing research in the epigenomic field can lead the way to a clinically specific biomarker for the CRC early diagnosis. As for the analytical method, qPCR and derivatives seem to be a perfectly valid technique. The use of ccfDNA quantification in CRC prognosis seems promising. The attempt to use the ccfDNA quantification in clinical practice may reside in the prognosis using a qPCR technique.

Pharmacogenetic implications in the management of metabolic diseases in Brazilian populations

Dyslipidemia, diabetes, obesity and hypertension are common metabolic diseases. In the last decades, unhealthy lifestyle and aging have leads to an increased incidence of these diseases, increasing morbidity and mortality by cardiovascular causes. The treatment of metabolic diseases includes life-style interventions as healthy diet and physical exercise, as well as pharmacological interventions. Several drugs are available for the management of metabolic diseases including among others lipid-lowering antidiabetics and antihypertensive drugs. Variability in response to these drugs is influenced by both genetic and non-genetic factors. Polymorphisms in genes related to drug pharmacokinetics and pharmacodynamics have been shown to influence drug efficacy and safety. This review is focused on pharmacogenetic studies related to the management of metabolic diseases in samples of the Brazilian population. Associations of variants in drug metabolizing enzymes and transporters, drug target and metabolism-related genes with the efficacy and safety of lipid-lowering, antidiabetic and antihypertensive drugs are described. Most pharmacogenetic studies in Brazil have focused in pharmacological response to a small group of drugs, as statins and some antihypertensives, while there are almost no studies on antidiabetic and antiobesity drugs. Some studies reported significant associations of gene polymorphisms with drug response confirming previous data from other populations, whereas other works did not replicate, which may relay on the genetic admixture of our population. In conclusion, further studies are necessary considering larger sample sizes, new unexplored drugs and more genetic variants to obtain stronger conclusions to explore clinical applications of pharmacogenetic studies in our population.

Relationship between circulating VCAM-1, ICAM-1, E-selectin and MMP9 and the extent of coronary lesions

OBJECTIVES: Inflammatory molecules play a role in the development of atherosclerosis, which is the primary origin of cardiovascular disorders. However, to the best of our knowledge, no study has attempted to investigate the relationship between these circulating molecules and the prediction of cardiovascular risk. The present study aimed to investigate the relationships of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 serum concentrations with the extent of coronary lesions. METHODS: Seventy-four individuals who were undergoing coronary angiography for the first time for diagnostic purposes were enrolled in this study. The extent of the coronary lesion was assessed using the Friesinger Index, and subjects were classified into four groups: no lesions, minor lesions, intermediate lesions and major lesions. Serum biochemical parameters and serum concentrations of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 were analyzed. RESULTS: The vascular cell adhesion molecule-1 concentration was higher than 876 ng/mL in individuals with intermediate and major lesions (p<0.001 and p=0.020, respectively). Moreover, logistic regression analysis showed that these patients had an increased risk of having an intermediate lesion (p=0.007). Interestingly, all individuals with major lesions had vascular cell adhesion molecule-1 concentrations higher than 876 ng/mL. No association was found between the concentrations of the other proteins and the Friesinger Index. CONCLUSIONS: Serum vascular cell adhesion molecule-1 may be associated with the extent of coronary lesions. Moreover, it may represent an alternative to improve the cardiovascular risk classification in patients without acute coronary syndrome.

Factors associated with statin-related adverse muscular events in adult dyslipidemic outpatients

ABSTRACT Statins are the most prescribed lowering-cholesterol drugs. They are well tolerated, however, some patients present muscular adverse symptoms. Clinical and laboratory data from 120 dyslipidemic patients prescribed with statins were obtained from January to December/2013 at a University Hospital in Sao Paulo city, Brazil, to study factors associated with statin-related adverse muscular events (AME). Pharmacotherapy and statin-related AME data (serum CK elevation and any degree of myopathy, myalgia, myositis or rhabdomyolysis) of the dyslipidemic patients were recorded. The study was approved by local Ethics Committees. Simvastatin (70%) and atorvastatin (25%) were the most prescribed statins. AME related to statin treatment were found in 17% of the patients. Mean age and use of simvastatin were lower in AME group than non-AME group (p<0.05). Simvastatin users were less likely to develop AME than atorvastatin users (OR=0.21; 95%CI=0.07-0.57; p<0.01). The use of P-glycoprotein (ABCB1) efflux pump inhibitors was associated with high risk for AME (OR=5.26; 95%CI=1.55-17.79; p<0.01). Serum liver enzymes were increased up to three-fold in 2.5% of the statin-treated patients. The results are suggestive that the type of statin prescribed and the concomitant use of ABCB1 inhibitors increase the susceptibility to adverse muscular events during statin therapy in dyslipidemic outpatients

Relationship between glycemic control and OPG gene polymorphisms with lower bone mineral density in patients with type 1 Diabetes mellitus

ABSTRACT The aim of the present study was to investigate the bone mineral density (BMD) of patients with type 1 Diabetes mellitus (T1DM). We also assessed the association between osteoprotegerin (OPG) genetic polymorphisms and BMD. Genotyping was performed for 1181G>C and 163A>G OPG polymorphisms by allelic discrimination in 119 patients with T1DM and 161 normoglycemic (NG) individuals, aged 6 to 20 years old. Glycemic control, serum parameters of bone metabolism and BMD were evaluated. T1DM patients showed low BMD, poor glycemic control and decreased total calcium values when compared to controls (p < 0.05). For all the polymorphisms studied, the genotype and allele frequencies in patients with T1DM were not significantly different from the controls. In patients with T1DM, carriers of OPG 1181CC showed higher concentrations of ionized calcium compared to patients with GG+GC genotypes. These results suggest that low BMD is associated with poor glycemic control in T1DM. Despite the lack of a detected association between OPG polymorphisms and BMD in these patients, the increased ionized calcium in those carrying OPG 1181CC suggests a possible increase in osteoclastogenesis, a conclusion that may be supported by the lower BMD observed in these subjects.

Exposure to potentially inappropriate medications in Brazilian elderly outpatients with metabolic diseases

ABSTRACT Management of pharmacotherapy in elderly with metabolic diseases is challenging and potentially inappropriate medications (PIMs) are risk factors for drug interactions and adverse events. The exposure to PIMs in elderly outpatients with metabolic diseases and its relationship with polypharmacy and other variables was investigated. PIMs prescribed to 207 elderly patients (aged 60 to 96 years) with metabolic diseases who attended a University Hospital of Sao Paulo city, Brazil, from April/2010 to January/2011, were evaluated. PIMs were detected using both 2003 Beers and 2008 STOPP criteria. The association between PIMs and age, gender and polypharmacy was also examined. 2008 STOPP criteria detected more PIMs (44.4 %) than 2003 Beers criteria (16.0%, p<0.001). Beers detected mainly PIMs antihypertensive (clonidine, 20.0%; doxazosin, 10.0%) and antidepressant (fluoxetine, 15.0%; amitriptyline, 10.0%) PIMs. Medicines used for cardiovascular (aspirin, 53.7%) and endocrine system (glibenclamide, 21.3%) were PIMs more frequently detected by 2008 STOPP. Unlike age and gender, polypharmacy increased the risk of PIMs by both 2003 Beers (OR: 4.0, CI95%: 1.2-13.8, p<0.031) and 2008 STOPP (OR: 6.8, CI95%: 3.0-15.3, p<0.001). Beers and STOPP criteria are important tools to evaluate the exposure to PIMs, which is strongly associated with polypharmacy in elderly outpatients with metabolic diseases.

Role of microRNAs 221/222 on Statin Induced Nitric Oxide Release in Human Endothelial Cells / Papel dos microRNAs 221/222 na Liberação de Óxido Nítrico Induzida por Estatinas em Células Endoteliais Humanas

Background: Nitric oxide (NO) has been largely associated with cardiovascular protection through improvement of endothelial function. Recently, new evidence about modulation of NO release by microRNAs (miRs) has been reported, which could be involved with statin-dependent pleiotropic effects, including anti-inflammatory properties related to vascular endothelium function. Objective: To evaluate the effects of cholesterol-lowering drugs including the inhibitors of cholesterol synthesis, atorvastatin and simvastatin, and the inhibitor of cholesterol absorption ezetimibe on NO release, NOS3 mRNA expression and miRs potentially involved in NO bioavailability. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to atorvastatin, simvastatin or ezetimibe (0 to 5.0 μM). Cells were submitted to total RNA extraction and relative quantification of NOS3 mRNA and miRs -221, -222 and -1303 by qPCR. NO release was measured in supernatants by ozone-chemiluminescence. Results: Both statins increased NO levels and NOS3 mRNA expression but no influence was observed for ezetimibe treatment. Atorvastatin, simvastatin and ezetimibe down-regulated the expression of miR-221, whereas miR-222 was reduced only after the atorvastatin treatment. The magnitude of the reduction of miR-221 and miR-222 after treatment with statins correlated with the increment in NOS3 mRNA levels. No influence was observed on the miR-1303 expression after treatments. Conclusion: NO release in endothelial cells is increased by statins but not by the inhibitor of cholesterol absorption, ezetimibe. Our results provide new evidence about the participation of regulatory miRs 221/222 on NO release induction mediated by statins. Although ezetimibe did not modulate NO levels, the down-regulation of miR-221 could involve potential effects on endothelial function. .

Fundamento: O óxido nítrico (NO) tem sido amplamente associado com proteção cardiovascular através de melhoria da função endotelial. Recentemente, novas evidências sobre a modulação do NO na liberação de microRNAs (miRs) têm sido relatadas, o que poderia estar envolvido com efeitos pleiotrópicos dependentes de estatinas, incluindo propriedades anti-inflamatórias relacionadas com a função do endotélio vascular. Objetivo: Avaliar os efeitos dos medicamentos redutores de colesterol, incluindo os inibidores da síntese de colesterol, atorvastatina e sinvastatina, e o inibidor da absorção de colesterol, ezetimiba, na liberação de NO, expressão do mRNA do NOS3 e miRs potencialmente envolvidos na biodisponibilidade do NO. Métodos: Células endoteliais da veia umbilical humana (HUVEC) foram expostas à atorvastatina, sinvastatina ou ezetimiba (0 a 5,0 μM). As células foram submetidas à extração do RNA total e quantificação relativa de mRNA do NOS3 e dos miRs-221,-222 e -1303 por qPCR. A liberação de NO foi medida em sobrenadantes por ozônio-quimioluminescência. Resultados: Ambas as estatinas aumentaram os níveis de NO e a expressão do mRNA do NOS3, mas nenhum efeito foi observado em relação ao tratamento com ezetimiba. A atorvastatina, sinvastatina e ezetimiba regularam negativamente a expressão do miR-221, enquanto que o miR-222 reduziu somente após o tratamento com atorvastatina. A magnitude da redução de miR-221 e miR-222 após tratamento com estatinas correlacionou com o incremento nos níveis de mRNA do NOS3. Nenhuma influência foi observada sobre a expressão do miR-1303 após os tratamentos. Conclusão: A liberação de NO pelas células endoteliais é aumentada por estatinas, mas não pelo inibidor da absorção de colesterol ezetimiba. Nossos resultados fornecem novas evidências sobre a participação dos miRs regulatórios 221/222 na liberação de NO mediada por estatinas. Embora a ezetimiba não tenha modulado os níveis de NO, a regulação negativa ...

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5-10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil.

Leptin receptor gene polymorphisms are associated with adiposity and metabolic alterations in Brazilian individuals / Polimorfismos no gene do receptor de leptina são associados com adiposidade e alterações metabólicas em indivíduos brasileiros

OBJECTIVE: The aim of the study was to investigate whether adiposity and metabolic markers, such as leptin, glucose, and lipids, are influenced by leptin (LEP) and leptin receptor (LEPR) gene polymorphisms in a sample of our population. SUBJECTS AND METHODS: A group of 326 individuals of Caucasian-European descent, aged 30 to 80 years, 87 men and 239 women, 148 obese and 178 non-obese, was randomly selected at two clinical hospitals in the city of Sao Paulo, Brazil. All individuals declared their ethnic group as white during the initial interview. Anthropometric measurements, body mass index (BMI), and fat mass were evaluated. Blood samples were drawn for DNA extraction and measurements of leptin, soluble leptin receptor, glucose, and lipids. LEP -2548G>A and LEPR Lys109Arg (c.326A>G), Gln233Arg (c.668A>G) and Lys656Asn (c.1968G>C) polymorphisms were detected by PCR-RFLP. RESULTS: Increased leptin and serum lipids, and LEPR Arg223Arg (GG genotype) were associated with higher risk for obesity (p < 0.05), while reduced risk was found in LEPR Arg109Arg (GG genotype) carriers (OR: 0.38, 95%CI: 0.19-0.77, p = 0.007). Multiple linear regression analysis showed a relationship between LEPR 223Arg, increased waist circumference, and leptinemia (p < 0.05), while LEPR 109Arg was associated with high total cholesterol and triglycerides (p < 0.05). LEPR haplotype 3 (AGG: 109Lys/233Arg/656Lys) carriers have increased risk for obesity (OR: 2.56, 95% CI: 1.19-5.49, p = 0.017). Moreover, this haplotype was associated with increased BMI, waist circumference, and leptinemia (p < 0.05). CONCLUSIONS: LEPR polymorphisms are associated with obesity, hyperleptinemia, and atherogenic lipid profile, suggesting their potential role for leptin resistance and cardiovascular risk. Moreover, LEPR haplotype 3 confers susceptibility to adiposity and hyperleptinemia in our population.

OBJETIVO: O estudo teve por objetivo investigar a influência de polimorfismos nos genes da leptina (LEP) e do receptor de leptina (LEPR) na adiposidade e em marcadores metabólicos, como leptina, glicose e lipí­deos, em uma amostra de nossa população. SUJEITOS E MÉTODOS: Um grupo de 326 indivíduos com idade de 30 a 80 anos, 87 homens e 239 mulheres, 148 obesos e 178 não obesos, e de etnia branca foi selecionado aleatoriamente em dois hospitais clínicos da cidade de São Paulo, Brasil. Medidas antropométricas, índice de massa corporal (IMC) e gordura corporal foram avaliados. Amostras de sangue foram obtidas para extração de DNA e determinações de leptina, receptor de leptina solúvel, glicose e lipídeos. Os polimorfismos LEP -2548G>A e LEPR Lys109Arg (c.326A>G), Gln233Arg (c.668A>G) e Lys656Asn (c.1968G>C) foram detectados por PCR-RFLP. RESULTADOS: Leptina e lipídeos séricos aumentados e LEPR Arg223Arg (genótipo GG) foram associados com maior risco de obesidade (p < 0,05), enquanto foi encontrado risco reduzido de obesidade, em portadores de LEPR Arg109Arg (genótipo GG) (OR: 0,38, 95%CI: 0,19-0,77, p = 0,007). A análise de regressão linear múltipla mostrou relação entre o alelo LEPR 223Arg e circunferência abdominal e leptinemia aumentadas (p < 0,05), enquanto o alelo LEPR 109Arg foi associado com aumento de colesterol total e triglicerídeos (p < 0,05). Os portadores do haplotipo 3 do LEPR (AGG: 109Lys/233Arg/656Lys) tiveram maior risco aumentado para obesidade (OR: 2.56, 95% CI: 1.19-5.49, p = 0,017). Além disso, esse haplótipo foi associado com IMC, circunferência abdominal e leptinemia aumentados (p < 0,05). CONCLUSÕES: Polimorfismos de LEPR são associados com obesidade, hiperleptinemia e perfil lipídico aterogênico sugerindo seu papel potencial para a resistência à leptina e risco cardiovascular.

Terapia personalizada: Contribuição da farmacogenômica cardiiovascular / Persolnalized therapy: contribution of the cardiovascular pharmacogenomics

A indicação terapêutica para prevenção e tratamentode doenças cardiovasculares apresenta uma variedadeampla de classes de fármacos com mecanismos deação (farmacodinâmica) e vias de biotransformação(farmacocinética) distintos. Estudos que investigaram arelação da genômica de várias populações com respostaterapêutica têm demonstrado perfis farmacogenômicosdiferenciais que tem contribuído de forma interessantetanto na terapia personalizada como em pesquisas clínicasmais direcionadas e melhor caracterizadas em diferentespopulações. Nesta revisão, discute-se o estado da arteda farmacogenômica e farmacogenética dos principaisfármacos utilizados no tratamento da doença cardiovascularcomo anticoagulantes, antiagregantes plaquetários,betabloqueadores, anti-hipertensivos e estatinas...

Therapeutic indication in the prevention and treatment ofcardiovascular diseases presents a variety of drug classeswith different mechanisms of action (pharmacodinamics)and metabolism pathways (pharmacokinetics).Studies thathave investigated the relationship of genomic data of severalpopulations with therapeutic response have demonstrated differentialpharmacogenornics profiles that have contributed inan interesting way to the personalized medicine as well as tonew drug discovery protocols in different populations. In thisreview we discuss the state of the art of pharmacogenomicsand pharmacogenetics of most drugs used in the treatment ofcardiovascular disease such as anticoagulants, antiplatelets,beta blockers, antihypertensives and statins...

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Análise da expressão do mRNA da proteína S100β em adipócitos de pacientes com diabetes melito tipo 2 / Analysis of the mRNA expression of the S100β protein in adipocytes of patients with diabetes mellitus, type 2

OBJETIVO: O presente trabalho objetiva compreender a possível relação do nível de expressão gênica do mRNA da proteína S100β em adipócitos com o diabetes melito do tipo 2, pela comparação de dados de portadores dessa doença com os de indivíduos normoglicêmicos. MATERIAIS E MÉTODOS: Foram selecionadas amostras de tecido adiposo de oito pacientes da Seção de Coronárias do Instituto Dante Pazzanese de Cardiologia (IDPC), sendo quatro do grupo diabetes e quatro do grupo de normoglicêmicos. Essas amostras foram submetidas à técnica de RT-PCR em tempo real. RESULTADOS: Por meio do Test-t de Student para os valores de diferença entre os ciclos threshold (ΔCt), observou-se que houve aumento de aproximadamente 15 vezes (p = 0,015) da expressão do mRNA da proteína S100β nos adipócitos dos indivíduos do grupo diabetes quando comparado aos do grupo controle. CONCLUSÃO: Nossos resultados evidenciam, de forma inédita, coexistência entre o aumento da expressão do gene S100β e a patologia do diabetes melito do tipo 2.

OBJECTIVE: This study aims to explore the possible relationship between the expression level of S100β protein mRNA with diabetes mellitus type 2 in adipocytes from patients with this disease in comparison with normoglycemic individuals. MATERIALS AND METHODS: Samples of adipose tissue of eight patients from the coronary section of the Institute Dante Pazzanese of Cardiology (IDPC), four in Group Diabetes and four of Normoglycemic group, were evaluated by RT-PCR real time. RESULTS: An increase around 15 times values, between the threshold cycle (ΔCt), of mRNA expression of S100β protein in adipocytes of the diabetes group was observed in comparison to the control group (p = 0.015). CONCLUSION: Our results indicate, for the first time, that there is coexistence of increased expression of the S100β and the type 2 diabetes mellitus gene.

Genotyping of Mycobacterium tuberculosis isolates from alow-endemic setting in northwestern state of Paraná in Southern Brazil

The purpose of this study was to provide information about the genetic diversity and prevalent genotype of Mycobacterium tuberculosis in a low-endemic setting in northwestern state of Paraná in Southern Brazil. We employed spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) techniques to genotype M. tuberculosisisolates from patients with pulmonary tuberculosis (TB). The 93 isolates analyzed by spoligotyping were divided into 36 different patterns, 30 of which were described in the SITVIT database. Latin American and Mediterranean, Haarlem and T families were responsible for 26.9 percent, 17.2 percent and 11.8 percent of TB cases, respectively. From the 84 isolates analyzed by MIRU-VNTR, 58 shared a unique pattern and the remaining 26 belonged to nine clusters. The MIRU loci 40, 23, 10 and 16 were the most discriminatory. A combination of MIRU-VNTR and spoligotyping resulted in 85.7 percent discriminatory power (Hunter-Gaston index = 0.995). Thus, combining spoligotyping and MIRU-VNTR typing proved to be most useful for epidemiological study in this low-endemic setting in Southern Brazil. The current study demonstrated that there is significant diversity in circulating strains in the city of Maringá and the surrounding regions, with no single genotype of M. tuberculosispredominating.

Terapia gênica, doping genético e esporte: fundamentação e implicações para o futuro: [revisão] / Gene therapy, genetic doping and sport: fundaments and implications for the future: [review]

A busca pelo desempenho ótimo tem sido uma constante no esporte de alto rendimento. Para tanto, muitos atletas acabam utilizando drogas e métodos ilícitos, os quais podem ter importantes efeitos adversos. A terapia gênica é uma modalidade terapêutica bastante recente na medicina, cujos resultados têm, até o momento, indicado sua eficácia no tratamento de diversas doenças graves. O princípio da terapia gênica consiste na transferência vetorial de materiais genéticos para células-alvo, com o objetivo de suprir os produtos de um gene estruturalmente anormal no genoma do paciente. Recentemente, o potencial para uso indevido da terapia gênica entre atletas tem despertado a atenção de cientistas e de órgãos reguladores de esporte. A transferência de genes que poderiam melhorar o desempenho esportivo por atletas saudáveis, método proibido em 2003, foi denominado de doping genético. Os genes candidatos mais importantes para doping genético são os que codificam para GH, IGF-1, bloqueadores da miostatina, VEGF, endorfinas e encefalinas, eritropoetina, leptina e PPAR-delta. Uma vez inserido no genoma do atleta, o gene se expressaria gerando um produto endógeno capaz de melhorar o desempenho atlético. Assim, os métodos atuais de detecção de doping não são sensíveis a esse tipo de manipulação, o que poderia estimular seu uso indevido entre atletas. Além disso, a terapia gênica ainda apresenta problemas conhecidos de aplicação, como resposta inflamatória e falta de controle da ativação do gene. Em pessoas saudáveis, é provável que tais problemas sejam ainda mais importantes, já que haveria excesso do produto do gene transferido. Há também outros riscos ainda não conhecidos, específicos para cada tipo de gene. Em vista disso, debates sobre o doping genético devem ser iniciados no meio acadêmico e esportivo, para que sejam estudadas medidas de prevenção, controle e detecção do doping genético, evitando assim futuros problemas de uso indevido dessa promissora...

Optimal performance has been constantly sought for in high level competitive sport. To achieve this goal, many athletes use illicit drugs and methods, which could have important side effects. Gene therapy is a very recent therapeutic modality, whose results have shown to be efficient in the treatment of severe diseases so far. The basis of gene therapy is a vectorial transfer of genetic materials to target-cells in order to supply the products of an abnormal gene in the patient's genome. Recently, the potential for misuse of gene therapy among athletes has called attention of scientists and sports regulating organs. The transfer of genes that could improve athletic performance, a method prohibited by COI in 2003, was named gene doping. The most important candidate genes for gene doping are the ones which codify for the following proteins: GH, IGH-1, miostatin blockers, VEGF, endorfins and enkefalins, eritropoetin, leptin and PPAR-delta. Once inserted in the athlete genome, the gene would be expressed and produce an endogenous product capable of improving performance. Thus, current doping detection methods are not sensitive enough to detect gene doping, which in turn could stimulate its use among athletes. Moreover, gene therapy still presents known application problems, such as inflammatory response and lack of control of gene activation. It is probable that such problems would be even more important in healthy individuals, since there would be excessive product of the transferred gene. Moreover, other unknown risks specific for each gene are present. Therefore, debate on gene doping should be carried on in the academic as well as sports field, in order to study prevention, control and detection measures of gene doping, avoiding hence, future problems regarding the misuse of this promising therapy.

Efeitos do exercício físico sobre o estado redox cerebral / Effects of physical exercise over the redox brain state

A atividade física é conhecida por promover saúde e bem-estar. O exercício também é responsável por aumentar a produção de Espécies Reativas de Oxigênio (ERO) pelo acréscimo do consumo de oxigênio mitocondrial nos tecidos. O desequilíbrio entre a produção de EROs e as defesas oxidantes dos tecidos pode provocar danos oxidativos a proteínas, lipídios e DNA. O dano oxidativo cerebral é um mecanismo etiopatológico comum da apoptose e da neurodegeneração. O fator de crescimento cérebro-derivado desempenha um importante papel neste contexto. Nesta revisão, apresentamos os resultados de diferentes modelos de exercício físico no metabolismo oxidativo e neurotrófico do Sistema Nervoso Central (SNC). Também revisamos estudos que utilizaram suplementação antioxidante para prevenir danos oxidativos exercício-induzido ao SNC. Os modelos de exercício físico mais comuns foram as rodas de correr, a natação e a esteira com configurações de treinamento muito diferentes como a duração e a intensidade. Os resultados do treinamento físico no tecido cerebral são muito controversos, mas geralmente demonstram ganhos na plasticidade sináptica e na função cognitiva com exercícios de intensidade moderada e baixa.

Physical activity is known for promoting health and well-being. Exercise is also responsible for increasing the production of Oxygen Reactive Species (ORS) by increasing mitochondrial oxygen consumption causing tissue oxidative stress. The imbalance between ORS production and tissue antioxidant defenses can cause oxidative damage to proteins, lipids and DNA. Brain oxidative damage is a common etiopathology mechanism of apoptosis and neurodegeneration. The brain-derived neurotrophic factor plays an important role in this context. In this review, we showed the results of different models and configurations of physical exercise in oxidative and neurotrophic metabolism of the Central Nervous System (CNS). We also reviewed studies that utilized antioxidant supplementation to prevent exercise-induced oxidative damage to CNS. The commonest physical exercise models were running wheels, swimming and treadmill with very different configurations of physical training such as duration and intensity. The results of physical training on brain tissues are very controversial, but generally show improvement in synaptic plasticity and cognition function with low and moderate intensity exercises.

Characterization of ndh gene of isoniazid resistant and susceptible Mycobacterium tuberculosis isolates from Brazil

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG) , and within the inhA promoter and/or in structural gene. A small percentage (~ 10 percent) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh) . Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.

Da biologia molecular à medicina: métodos comumente utilizados em farmacogenética / From molecular biology to medicine: methods commonly used in pharmacogenetics

RESUMO: Nesta revisão, foram discutidos os procedimentos para obtenção, manuseio e armazenamento de amostras biológicas úteis em testes moleculares. Métodos para triagem e detecção direta de mutações e polimorfismos genéticos foram apresentados. Os princípios, estratégias, aplicações e limitações de variações da reação em cadeia pela polimerase (PCR), tais como eletroforese em gel com gradiente de desnaturação (DGGE), polimorfismo de conformaçãode fita simples (SSCP), polimorfismo de tamanhos de fragmentos de restrição (RFLP), amplificação alelo oligonucleotídeo-específica (PCR-ASO), PCR em tempo real PCR, arranjos de DNA e outras técnicas moleculares, foram comentados.