Diagnosis of 5α-reductase type 2 deficiency: Contribution of anti-Müllerian hormone evaluation

Aim: To evaluate anti-Müllerian hormone (AMH) levels in patients with clinical and molecular diagnosis of 5α-reductase 2 deficiency. Patients and methods: Data from 14 patients whose age ranged from 21 days to 29 years were analyzed according to age and pubertal stage. Sexual ambiguity was rated as Prader III in 11 patients. LH, FSH, testosterone (T), dihydrotestosterone (DHT) and AMH serum levels were measured in all but two patients, who had been previously submitted to gonadectomy; T and DHT were also measured in 20 age-matched controls. Results: Gonadotropin levels were normal in all but one patient who retained gonads (six of whom had reached puberty) and T/DHT ratio was elevated in all patients when compared to controls. All prepubertal patients had AMH levels < -1 SD for age, while most pubertal patients had AMH levels compatible with pubertal stage. Conclusions: Prepubertal patients with 5α-reductase 2 deficiency have AMH values in the lower part of the normal range. These data indicate that T does not need to be converted to DHT to inhibit AMH secretion by Sertoli cells. © Freund Publishing House Ltd., London.

Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos

The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.

Effects of 12-week overground walking training at ventilatory threshold velocity in type 2 diabetic women

This study analyzed the effects of overground walking training at ventilatory threshold (VT) velocity on glycaemic control, body composition, physical fitness and lipid profile in DM2 women. Nineteen sedentary patients were randomly assigned to a control group (CG; n=10, 55.9±2.2 years) or a trained group (TG; n=9, 53.4±2.3 years). Both groups were subjected to anthropometric measures, a 12-h fasting blood sampling and a graded treadmill exercise test at baseline and after a 12-week period, during which TG followed a training program involving overground walking at VT velocity for 20-60min/session three times/week. Significant group×time interactions (P<0.05) in glycated hemoglobin (HbA1c), body mass, body mass index (BMI), peak oxygen uptake (VO 2peak) and exercise duration were observed as effects of training exercise, whereas intervention did not induced significant changes (P>0.05) in fasting blood glucose, submaximal fitness parameters and lipid profile. Our results suggest that overground walking training at VT velocity improves long term glycaemic control, body composition and exercise capacity, attesting for the relevance of this parameter as an effective strategy for the exercise intensity prescription in DM2 population. © 2011 Elsevier B.V.

Determinants of blood pressure in type 2 diabetic subjects with high occurrence of inadequate glycemic control

Study design: Association study Objective: To analyze the association between different biological/behavioral risk factors and blood pressure in a sample of type 2 diabetes mellitus patients with poor glycemic control. Methods: A sample of 121 type 2 diabetic patients was selected in the Public Healthcare System in a middle size Brazilian city. Blood pressure was measured using an aneroid device, previously calibrated. Six determinants of blood pressure were taken into count: age, hypoglycemic agents, general obesity, abdominal obesity, eating behaviors and physical activity level. Results: The type 2 diabetic patients presented mean age of 60.1±8.9 years-old and, at least, one risk factor. Eating behaviors (OR adj= 0.31 [0.12-0.75]) and sports practice (OR adj= 0.12 [0.02-0.75]) constituted protective factors associated with lower systolic blood pressure. On the other hand, age was positively associated with high systolic blood pressure (OR adj= 3.81 [1.39-10.38]). Patients with 5-6 risk factors, presented higher values of systolic and (F= 3.857; p= 0.011 [post hoc with p= 0.039]), diastolic blood pressure (F= 4.158; p= 0.008 [post hoc with p= 0.036]) and increased occurrence of hypertension (p= 0.010). Conclusion: Our findings indicate that, behavioral variables were important determinants of blood pressure in type 2 diabetic patients with poor glycemic control and clustering of behavioral and biological risk factors increase the hypertension occurrence.

Dissection of the role of pili and type 2 and 3 secretion systems in adherence and biofilm formation of an atypical enteropathogenic escherichia coli strain

Atypical enteropathogenic Escherichia coli (aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2 eae mutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion of fimA in 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2 sslE mutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a double eae espA mutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2 eae mutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains. ©2013, American Society for Microbiology.

Polymorphism in the promoter region of the mannose-binding lectin gene among human T-cell lymphotropic virus infected subjects

ABSTRACT: The present study investigated the frequency of the mutations at positions -550 and -221 of the mannose-binding lectin (MBL) gene in a sample of 75 human T-cell lymphotropic virus (HTLV) infected patients and 96 HTLV seronegative controls, in order to evaluate the occurrence of a possible association between the polymorphism and HTLV infection. A sequence specific primer-polymerase chain reaction was used for discrimination of the polymorphism. The analysis of allele frequencies at position -550 did not show any significant differences between HTLV infected group and controls, but there was a significant difference at position -221. The comparative analysis of haplotypes frequencies were not significant, but the genotype frequencies between the two groups, revealed a higher prevalence of genotype LYLX (25.3%), associated with medium and low MBL serum levels among HTLV infected subjects. The odds ratio estimation demonstrated that the presence of genotype LYLX was associated with an increased risk of HTLV infection (p = 0.0096; 1.38 < IC95% < 7.7605). There was no association between proviral load and the promoter polymorphism, but when promoter and exon 1 mutations were matched, it was possible to identify a significant higher proviral load among HTLV infected individuals carrying haplotypes correlated to low serum levels of MBL. The present study shows that the polymorphism in the promoter region of the MBL gene may be a genetic marker associated with HTLV infection, and emphasizes the need for further studies to determinate if the present polymorphism have any impact on diseases linked to HTLV infection.

Caracterização sorológica e detecção molecular do HTLV em amostras de pacientes com distúrbios neurológicos no Estado do Pará, Brasil (1996-2005)

ABSTRACT: Human T-lymphotropic virus tipe 1 is recognized as the etiologic agent of tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). A very similar clinical disease has been increasingly associated to HTLV-2, whose pathogenicity still requires further assessments. This transversal, retrospective epidemiological survey aimed to determine the prevalence of HTLV among individuals with neurological disturbances and further evaluate cases of inconclusive serology using molecular biology methods. The present study involved patients inhabitants of Pará State and/or admitted at health institutions of the and who were referred to the Virology Section of Instituto Evandro Chagas (IEC) by local doctors between January of 1996 and December 2005, to search for the presence of HTLV-1/2 serum antibodies. Of these patients 353 were selected, with age between 9 months and 79 years, who presented at least one signal or symptom of the Marsh’s Complex (1996), as well as had HTLV-1/2 positive serology at screening and confirmatory ELISA. The overall prevalence of HTLV antibodies by ELISA as 8,8% (31/353), with rates of 10,6% (19/179) and 6,9% (12/174) for female and male patients, respectively. Among HTLV-1/2 the 31 ELISA-positive patients it was noted that 15 (48.4%) of 31 had paresis (n = 8), parestesis (n = 5), and paraplegia (n = 3). Of these 31 HTLV ELISA positive patients, 25 could be submitted to WB for assessment of viral types, which were distributed as follow: 80% (20/25) were HTLV-1, 12% (3/25) were HTLV-2, one case was of HTLV-1+HTLV-2 infection (4%), and serum from one patient yielded an indeterminate profile (4%). Only 14 of these 25 patients could be re-localised for collection of an additional sample for molecular analysis. It was observed that 78.6% of samples typed by WB had the proviral TAX region successfully amplified by nested-PCR. In addition, types were confirmed as based on results obtained from the amplification of the POL region using real-time PCR; this denoted good specificity and sensitivity of the WB used in this study. The sample defined as HTLV-1+HTLV-2 infection by WB was amplified in its TAX region but real time PCR confirmed HTLV-1 infection only. The patient with WB indeterminate profile and one of samples typed as HTLV-2 by WB were amplified by nested-PCR but the real time PCR was negative for HTLV-1 and HTLV-2 in both samples. One patient presenting clinical manifestations of crural myalgia and parestesia with duration of about 7 years reacted HTLV-2-positive by both WB and real-time PCR, a denoting a clear HTLV-2- related chronic myelopathy. This study has identified a case of possible vertical transmission in two distinct situations: a patient whose mother presented antibodies for HTLV-1 by WB and two sisters who reacted HTLV-1-positive by WB and real-time PCR. Although of epidemiological relevance, results from this study warrant further and broader analyses concerning the molecular epidemiology of HTLV types and subtypes HTLV. In addition, a more complete clinical assessment of neurological symptoms should be further performed, in order to better characterise cases of HTLV-related chronic myelopathy in our region.

Transmissão intrafamiliar do HTLV: investigação sorológica em familiares de pacientes acompanhados no ambulatório do Núcleo de Medicina Tropical da UFPA

O Vírus Linfotrópico Humano de Células T é um oncoretrovírus responsável por doenças linfoproliferativas, inflamatórias, degenerativas do Sistema Nervoso Central e por algumas alterações imunológicas do ser humano. Embora tenha associações com várias outras patologias, a Paraparesia Espástica Tropical ou Mielopatia Associada ao HTLV (PET/MAH), doença progressiva e incapacitante do Sistema Nervoso, e a Leucemia/Linfoma de Células T do Adulto (LLcTA), doença linfoproliferativa maligna e letal, são os principais agravos consistentemente definidos como provocados pelo HTLV-1. A propagação do vírus acontece de forma silenciosa, especialmente de mãe para filhos e pela via sexual. No Brasil, onde existem regiões de alta prevalência, ainda são escassas informações oficiais sobre essa transmissão. O objetivo do presente trabalho foi determinar a soroprevalência de anticorpos contra o Vírus Linfotrópico Humano de Células T – tipos 1 e 2 (HTLV-1/2) entre familiares de portadores confirmados do vírus, matriculados no ambulatório do Núcleo de Medicina Tropical (NMT), para estudar as características da transmissão do HTLV nos grupos familiares da região metropolitana de Belém do Pará. Foi realizado um estudo transversal, de base ambulatorial, envolvendo 82 pacientes matriculados no NMT e seus respectivos familiares, os quais foram submetidos à pesquisa de anticorpos anti-HTLV-1/2, utilizando-se o teste de ELISA (Ortho Diagnostic System Inc., US), no período entre junho de 2007 e novembro de 2009. A Soroprevalência da infecção pelo HTLV-1/2 foi observada em 40,2 % (33/82) das famílias e 24,0 % (50/208) no total de familiares pesquisados. A transmissão de mãe para filho(a) ocorreu em 23,2 % (19/82) das famílias, com taxas de soropositividade de 22,4 % (17/76) para filhas e 15,2 % (7/46) para filhos (p > 0.05). A transmissão sexual provável ocorreu em 25,6 % (21/82) das famílias e em 42,0 % (21/50) dos casais, com taxas de soropositividade de esposas e maridos de 53,1 % (18/34) e 18,8 % (3/16), respectivamente (p < 0.05). Não houve diferença significativa de soroprevalência entre familiares de portadores sintomáticos e assintomáticos e entre HTLV-1 e HTLV-2. Conclui-se que existe agregação da infecção nas famílias investigadas e que os dados obtidos estão em acordo com os previamente relatados na literatura. Os serviços de atendimento precisam realizar, rotineiramente, a educação dos indivíduos portadores de HTLV e manter ativas as medidas de controle dos comunicantes familiares, para evitar a propagação do vírus principalmente através do contacto sexual e amamentação.

Dengue Virus Type 3 Adaptive Changes during Epidemics in Sao Jose de Rio Preto, Brazil, 2006-2007

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Increased Expression of Angiotensin II Type 2 Receptors in the Solitary-Vagal Complex Blunts Renovascular Hypertension

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Gene expression profiles displayed by peripheral blood mononuclear cells from patients with type 2 diabetes mellitus focusing on biological processes implicated on the pathogenesis of the disease

Patients with type 2 diabetes mellitus (T2DM) exhibit insulin resistance associated with obesity and inflammatory response, besides an increased level of oxidative DNA damage as a consequence of the hyperglycemic condition and the generation of reactive oxygen species (ROS). In order to provide information on the mechanisms involved in the pathophysiology of T2DM, we analyzed the transcriptional expression patterns exhibited by peripheral blood mononuclear cells (PBMCs) from patients with T2DM compared to non-diabetic subjects, by investigating several biological processes: inflammatory and immune responses, responses to oxidative stress and hypoxia, fatty acid processing, and DNA repair. PBMCs were obtained from 20 T2DM patients and eight non-diabetic subjects. Total RNA was hybridized to Agilent whole human genome 4x44K one-color oligo-microarray. Microarray data were analyzed using the GeneSpring GX 11.0 software (Agilent). We used BRB-ArrayTools software (gene set analysis - GSA) to investigate significant gene sets and the Genomica tool to study a possible influence of clinical features on gene expression profiles. We showed that PBMCs from T2DM patients presented significant changes in gene expression, exhibiting 1320 differentially expressed genes compared to the control group. A great number of genes were involved in biological processes implicated in the pathogenesis of T2DM. Among the genes with high fold-change values, the up-regulated ones were associated with fatty acid metabolism and protection against lipid-induced oxidative stress, while the down-regulated ones were implicated in the suppression of pro-inflammatory cytokines production and DNA repair. Moreover, we identified two significant signaling pathways: adipocytokine, related to insulin resistance; and ceramide, related to oxidative stress and induction of apoptosis. In addition, expression profiles were not influenced by patient features, such as age, gender, obesity, pre/post-menopause age, neuropathy, glycemia, and HbA(1c) percentage. Hence, by studying expression profiles of PBMCs, we provided quantitative and qualitative differences and similarities between T2DM patients and non-diabetic individuals, contributing with new perspectives for a better understanding of the disease. (C) 2012 Elsevier B.V. All rights reserved.

LIRAGLUTIDE: NEW RESULTS IN THE TREATMENT OF TYPE 2 DIABETES MELLITUS

New drugs for type 2 diabetes that act on incretin metabolism have been shown to improve glycemic control, reduce body weight and have a low risk for hypoglycemia. Among these, liraglutide is the first glucagon-like peptide 1 (GLP-1) analogue approved for subcutaneous, once-daily administration. According to results from clinical trials, liraglutide is on attractive alternative for the early treatment of type 2 diabetes. The results of the LEAD (Liraglutide Effect and Action in Diabetes) study program demonstrated the efficacy and safety of liraglutide in terms of reduction of glycated hemoglobin (HbA(tc)) levels, significant loss of body weight that was maintained over the long term, better control of the lipid profile and systolic arterial pressure, reduction of the risk for hypoglycemia and reduction of cardiovascular risk. Moreover, the drug was demonstrated to be safe and can be co-administered with oral antidiabetic agents. The product's tolerability has been demonstrated, with nausea as the most common adverse event, which waned from the fourth week of treatment.

Reduced muscle carnosine content in type 2, but not in type 1 diabetic patients

Carnosine is present in high concentrations in skeletal muscle where it contributes to acid buffering and functions also as a natural protector against oxidative and carbonyl stress. Animal studies have shown an anti-diabetic effect of carnosine supplementation. High carnosinase activity, the carnosine degrading enzyme in serum, is a risk factor for diabetic complications in humans. The aim of the present study was to compare the muscle carnosine concentration in diabetic subjects to the level in non-diabetics. Type 1 and 2 diabetic patients and matched healthy controls (total n = 58) were included in the study. Muscle carnosine content was evaluated by proton magnetic resonance spectroscopy (3 Tesla) in soleus and gastrocnemius. Significantly lower carnosine content (-45%) in gastrocnemius muscle, but not in soleus, was shown in type 2 diabetic patients compared with controls. No differences were observed in type 1 diabetic patients. Type II diabetic patients display a reduced muscular carnosine content. A reduction in muscle carnosine concentration may be partially associated with defective mechanisms against oxidative, glycative and carbonyl stress in muscle.

Lack of evidence to support the association of a single IL28B genotype SNP rs12979860 with the HTLV-1 clinical outcomes and proviral load

Background: The Interleukin 28B (IL28B) rs12979860 polymorphisms was recently reported to be associated with the human T-cell leukemia virus type 1 (HTLV-1) proviral load (PvL) and the development of the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Methods: In an attempt to examine this hypothesis, we assessed the association of the rs12979860 genotypes with HTLV-1 PvL levels and clinical status in 112 unrelated Brazilian subjects (81 HTLV-1 asymptomatic carriers, 24 individuals with HAM/TSP and 7 with Adult T cell Leukemia/Lymphoma (ATLL)). Results: All 112 samples were successfully genotyped and their PvLs compared. Neither the homozygote TT nor the heterozygote CT mutations nor the combination genotypes (TT/CT) were associated with a greater PvL. We also observed no significant difference in allele distribution between asymptomatic carriers and patients with HTLV-1 associated HAM/TSP. Conclusions: Our study failed to support the previously reported positive association between the IL28B rs12979860 polymorphisms and an increased risk of developing HAM/TSP in the Brazilian population.

Effect of Type 2 Diabetes Mellitus on the Pharmacokinetics of Metformin in Obese Pregnant Women

Background and Objective The use of metformin throughout gestation by women with polycystic ovary syndrome (PCOS) and type 2 diabetes mellitus (T2DM) significantly reduces the number of first-trimester spontaneous abortions and the rate of occurrence of gestational diabetes and hypertensive syndromes. Metformin is taken up into renal tubular cells by organic cation transport 2 (OCT2) and eliminated unchanged into the urine. The objective of this study was to analyse the influence of T2DM on the pharmacokinetics of metformin in obese pregnant women and in a control group of non-diabetic obese pregnant women with PCOS. Methods Eight non-diabetic obese pregnant women with PCOS and nine obese pregnant women with T2DM taking oral metformin 850 mg every 12 h were evaluated throughout gestation. Serial blood samples were collected over a 12-h period during the third trimester of pregnancy. Steady-state plasma concentrations of metformin were determined by high-performance liquid chromatography with a UV detector. The pharmacokinetic results of the two groups, reported as median and 25th and 75th percentile, were compared statistically using the Mann Whitney test, with the level of significance set at p < 0.05. Results The pharmacokinetic parameters detected for PCOS versus T2DM patients, reported as median, were, respectively: elimination half-life 3.75 versus 4.00 h; time to maximum concentration 2.00 versus 3.00 h; maximum concentration 1.42 versus 1.21 mu g/mL; mean concentration 0.53 versus 0.56 mu g/mL; area under the plasma concentration time curve from time zero to 12 h 6.42 versus 6.73 mu g.h/mL; apparent total oral clearance 105.39 versus 98.38 L/h; apparent volume of distribution after oral administration 550.51 versus 490.98 L; and fluctuation (maximum minimum concentration variation) of 179.56 versus 181.73%. No significant differences in pharmacokinetic parameters were observed between the groups. Conclusion T2DM in the presence of insulin use does not influence the pharmacokinetics of metformin in pregnant patients, demonstrating the absence of a need to increase the dose, and consequently does not influence the OCT2-mediated transport in pregnant women with PCOS.