Expressão gênica de interferon-gama, fator de necrose tumoral alfa e interleucinas 2 e 5 no baço de gatos naturalmente infectados por Leishmania spp: Karina Yukie Hirata. -

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Intron 4 polymorphism of the endothelial nitric oxide synthase (eNOS) gene is associated with decreased NO production in a mercury-exposed population

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is a potent vasodilator and plays a prominent role in regulating the cardiovascular system. Decreased basal NO release may predispose to cardiovascular diseases. Evidence suggests that the 27 nt repeat polymorphism of the intron 4 in the eNOS gene may regulate eNOS expression. On the other hand, some recent reports strongly suggest an association between methylmercury (MeHg) exposures and altered NO synthesis. In the present study, we investigate the contribution of the 27-pb tandem repeat polymorphism on nitric oxide production, which could enhance susceptibility to cardiovascular disease in the MeHg-exposed study population. Two-hundred-two participants (98 men and 104 women), all chronically exposed to MeHg through fish consumption were examined. Mean blood Hg concentration and nitrite plasma concentration were 50.5 +/- 35.4 mu g/L and 251.4 +/- 106.3 nM, respectively. Mean systolic and diastolic blood pressure were 120.1 +/- 19.4 mm Hg and 72.0 +/- 10.6 mm Hg, respectively. Mean body mass index was 24.5 +/- 4.3 kg/m(2) and the mean heart rate was 69.8 +/- 11.8 bpm. There were no significant differences in age, arterial blood pressure, body mass index or cardiac frequency between genotype groups (all P>0.05). However, we observed different nitrite concentrations in the genotypes groups, with lower nitrite levels for the 4a4a genotype carriers. Age, gender and the presence of intron 4 polymorphism contributed to nitrite reduction as a result of blood Hg concentration. Taken together, our results show that the 27 nt repeat polymorphism of the intron 4 in the eNOS gene increases susceptibility to cardiovascular diseases after MeHg exposure by modulating nitric oxide levels. (C) 2011 Elsevier B.V. All rights reserved.

Pretherapeutic Expression of the hOCT1 Gene Predicts a Complete Molecular Response to Imatinib Mesylate in Chronic-Phase Chronic Myeloid Leukemia

In this retrospective study we evaluated the pretherapeutic mRNA expression of the hOCT1 (human organic cation transporter 1) gene in patients with chronic-phase (CP) chronic myeloid leukemia (CML) who varied in terms of their response to imatinib (IM). hOCT1 mRNA was quantified by real-time PCR. Patients were classified as expressing either high (n = 44) or low hOCT1 mRNA (n = 44). The complete cytogenetic response rates observed at 6, 12 and 18 months were 47.7, 84.1 and 91%, respectively, in patients with high hOCT1 mRNA and 47.5, 81.8 and 86.3%, respectively, in patients with low hOCT1 transcripts. The major molecular response rates were not significantly different between patients with high and low hOCT1 mRNA after 6 months of therapy (22.7 vs. 9.1%; p = 0.07), but they were significantly different after 12 months (54.5 vs. 31.8%; p = 0.026) and 18 months (77.2 vs. 56.8%; p = 0.034). Complete molecular responses were observed in 5 patients with low and 17 patients with high hOCT1 mRNA (p = 0.003). The 5-year event-free and overall survival analyses revealed no significant differences between the groups. These data imply that knowledge of the pretherapeutic level of hOCT1 could be a useful marker to predict IM therapy outcome in treatment-naive CP CML patients. Copyright (C) 2012 S. Karger AG, Basel

Expressão do gene da leptina e seu receptor Ob-Rb no parênquima mamário de novilhas leiteiras

Objetivou-se com este trabalho avaliar os efeitos de uma dieta de alto nível de energia e proteína combinada com a aplicação de bST no perfil de expressão dos genes da leptina e de seu receptor Ob-Rb no parênquima mamário de novilhas leiteiras. Foram utilizadas amostras de parênquima mamário de 32 novilhas holandesas distribuídas aleatoriamente em quatro tratamentos (n=8): dieta com alto ou baixo teor de energia e proteína combinada ou não com a aplicação de bST. O delineamento utilizado foi em blocos casualizados com arranjo de tratamentos em esquema fatorial 2 × 2. A extração do RNA total das amostras de tecido foi feita e o nível de expressão gênica foi analisado por qRT-PCR utilizando-se o gene da glicuronidase β como controle, pelo método 2-ΔΔCt. Animais que receberam a dieta com alto conteúdo de energia e proteína apresentaram maior expressão de mRNA de leptina, com aumento de 56%, e menor expressão de mRNA do receptor Ob-Rb, com redução de 18%. Por outro lado, a aplicação de bST resultou em diminuição da expressão do mRNA de leptina e do receptor Ob-Rb em 74% e 23%, respectivamente. Não houve interação entre dieta e aplicação de bST. O aumento na expressão de leptina pode explicar, ao menos em parte, os efeitos negativos da dieta de alta energia e proteína, oferecida no período pré-púbere, sobre a produção de leite de novilhas leiteiras.

Allelic variations in the vitamin D receptor gene, insulin secretion and parents' heights are independently associated with height in obese children and adolescents

Polymorphisms in the VDR gene were reported to be associated with variations in intrauterine and postnatal growth and with adult height, but also with other traits that are strongly correlated such as the BMI, insulin sensitivity, insulin secretion and hyperglycemia. Here, we assessed the impact of VDR polymorphisms on body height and its interactions with obesity- and glucose tolerance-related traits in obese children and adolescents. We studied 173 prepubertal (Tanner's stage 1) and 146 pubertal (Tanner's stages 2-5) obese children who were referred for a weight-loss program. Three single nucleotide polymorphisms were genotyped: rs1544410 (BsmI), rs7975232 (ApaI) and rs731236 (TaqI). BsmI and TaqI genotypes were significantly associated with height in pubertal children, but the associations did not reach statistical significance in prepubertal children. In stepwise regression analyses, the lean body mass, insulin secretion, BsmI or TaqI genotypes and the father's and the mother's height were independently and positively associated with height in pubertal children. These covariables accounted for 46% of the trait variance. The height of homozygous carriers of the minor allele of BsmI was 0.65 z-scores (4 cm) higher than the height of homozygous carriers of the major allele (P=.0006). Haplotype analyses confirmed the associations of the minor alleles of BsmI and TaqI with increased height. In conclusion, VDR genotypes were significantly associated with height in pubertal obese children. The associations were independent from the effects of confounding traits, such as the body fat mass, insulin secretion, insulin sensitivity and glucose tolerance. (C) 2012 Elsevier Inc. All rights reserved.

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.

Functional single-nucleotide polymorphisms in the DEFB1 gene are associated with systemic lupus erythematosus in Southern Brazilians

Systemic lupus erythematosus (SLE) is an autoimmune disease that results in inflammation and tissue damage. The etiology of SLE remains unknown, but recent studies have shown that the innate immune system may have a role in SLE pathogenesis through the secretion of small cationic peptides named defensins. The aim of the study was to determine the possible involvement in SLE of three functional single nucleotide polymorphisms (SNPs) (c.-52G>A, c.-44C>G and c.-20G>A) in the 5'UTR region of DEFB1 gene, by analyzing them in a population of 139 SLE patients and 288 healthy controls. The c.-52G>A SNP showed significant differences in allele and genotype frequency distribution between SLE patients and controls (p = 0.01 and p = 0.02 respectively) indicating protection against SLE (A allele, OR = 0.68, AA genotype OR = 0.51). Significant differences were also observed for c.-44C>G SNP, the C/G genotype being associated with susceptibility to SLE (OR = 1.60, p = 0.04). Moreover, statistically significant differences between patients and controls were found for two DEFB1 haplotypes (GCA and GGG, p = 0.01 and p = 0.02 respectively). When considering DEFB1 SNPs and SLE clinical and laboratory manifestations, significant association was found with neuropsychiatric disorders, immunological alterations and anti-DNA antibodies. In conclusion, our results evidence a possible role for the c.-52G>A and c.-44C>G DEFB1 polymorphisms in SLE pathogenesis, that can be considered as possible risk factors for development of disease and disease-related clinical manifestations. Additional studies are needed, to corroborate these results as well as functional studies to understand the biological role of these SNPs in the pathogenesis of SLE. Lupus (2012) 21, 625-631.

Impact of Glucocorticoid Receptor Gene Polymorphisms on the Metabolic Profile of Adult Patients with the Classical Form of 21-Hydroxylase Deficiency

Background: CAH patients have an increased risk of cardiovascular disease, and it remains unknown if lifelong glucocorticoid (GC) treatment is a contributing factor. In the general population, glucocorticoid receptor gene (NR3C1) polymorphisms are associated with an adverse metabolic profile. Our aim was to analyze the association between the NR3C1 polymorphisms and the metabolic profile of CAH patients. Methodology: Sixty-eight adult patients (34SV/34SW) with a mean age of 28.4 +/- 9 years received dexamethasone (mean 0.27 +/- 0.11 mg/day) to obtain normal androgen levels. SW patients also received fludrocortisone (50 mu g/day). Metabolic syndrome (MetS) was defined by the NCEP ATPIII criteria and obesity by BMI >= 30 kg/m(2). NR3C1 alleles were genotyped, and association analyses with phenotype were carried out with Chi-square, t-test and regression analysis. Results: Obesity and MetS were observed in 23.5% and 7.3% of patients, respectively, and were not correlated with GC doses and treatment duration. BMI was positively correlated with blood pressure (BP), triglycerides (TG), LDL-c levels and HOMA-IR and inversely correlated with HDL-c levels. BclI and A3669G variants were found in 26.4% and 9.6% of alleles, respectively. Heterozygotes for the BclI polymorphism presented with higher BMI (29 kg/m(2) +/- 5.3 vs. 26 kg/m(2) +/- 5.3, respectively) and waist circumference (89 cm +/- 12.7 vs. 81 cm +/- 13, respectively) compared to wild-type subjects. Hypertension was found in 12% of patients and heterozygotes for the BclI polymorphism presented higher systolic BP than wild type subjects. Low HDL-c and high TG levels were identified in 30% and 10% of patients, respectively, and were not associated with the NR3C1 polymorphisms. A3669G carriers and non-carriers did not differ. Conclusion: In addition to GC therapy, the BclI GR variant might play an important role in obesity susceptibility in CAH patients. Genotyping of GR polymorphisms could result in the identification of a subgroup at risk patients, allowing for the establishment of personalized treatment and the avoidance of long-term adverse consequences.

Aberrant transcript produced by a splice donor site deletion in the TECTA gene is associated with autosomal dominant deafness in a Brazilian family

We ascertained a Brazilian family with nine individuals affected by autosomal dominant nonsyndromic sensorineural hearing loss. The bilateral hearing loss affected mainly mid-high frequencies, was apparently stable with an early onset. Microsatellites close to the DFNA8/DFNA12 locus, which harbors the TECTA gene, showed significant multipoint lod scores (32) close to marker D11S4107. Sequencing of the exons and exon-intron boundaries of the TECTA gene in one affected subject revealed the deletion c.5383 + 5delGTGA in the 5' end of intron 16, that includes the last two bases of the donor splice site consensus sequence. This mutation segregates with deafness within the family. To date, 33 different TECTA mutations associated with autossomal dominant hearing loss have been described. Among them is the mutation reported herein, first described by Hildebrand et al. (2011) in a UK family. The audioprofiles from the UK and Brazilian families were similar. In order to investigate the transcripts produced by the mutated allele, we performed cDNA analysis of a lymphoblastoid cell line from an affected heterozygote with the c.5383 + 5delGTGA and a noncarrier from the same family. The analysis allowed us to identify an aberrant transcript with skipping of exon 16, without affecting the reading frame. One of the dominant TECTA mutations already described, a synonymous substitution in exon 16 (c.5331 G<A), was also shown to affect splicing resulting in an aberrant transcript lacking exon 16. Despite the difference in the DNA level, both the synonymous substitution in exon 16 (c.5331 G<A) and the mutation described herein affect splicing of exon 16, leading to its skipping. At the protein level they would have the same effect, an in-frame deletion of 37 amino-acids (p.S1758Y/G1759_N1795del) probably leading to an impaired function of the ZP domain. Thus, like the TECTA missense mutations associated with dominant hearing loss, the c5383 + 5delGTGA mutation does not have an inactivating effect on the protein. (C) 2012 Elsevier B.V. All rights reserved.

Gene copy number and differential gene expression in haploid and diploid males of the stingless bee, Melipona quadrifasciata

Complementary sex determination in Hymenoptera implies that heterozygosity at the sex locus leads to the development of diploid females, whereas hemizygosity results in haploid males. Diploid males can arise through inbreeding. In social species, these pose a double burden on colony fitness, from significant reduction in its worker force and through being less viable and fertile than haploid males. Apart from being "misfits", diploid males are of interest to assess molecular correlates for possibly ploidy-related bionomic differences. Herein, we generated suppression subtractive cDNA libraries from newly emerged haploid and diploid males of the stingless bee Melipona quadrifasciata to enrich for differentially expressed genes. Gene Ontology classification revealed that in haploid males more DEGs were related to stress responsiveness, biosynthetic processes, reproductive processes and spermatogenesis, whereas in diploid ones differentially expressed genes were associated with cellular organization, nervous system development and amino acid transport were prevalent. Furthermore, both libraries contained over 40 % ESTs representing possibly novel transcripts. Quantitative RT-PCR analyses confirmed the differential expression of a representative DEG set in newly emerged males. Several muscle formation and energy metabolism-related genes were under-expressed in diploid males. On including 5-day-old males in the analysis, changes in transcript abundance during sexual maturation were revealed.

Fc gamma Receptor Gene Polymorphisms in Childhood Immune Thrombocytopenic Purpura

Immune thrombocytopenic purpura (ITP) is a common hematological disorder in the childhood, and it is one of the most common forms of autoimmune disease in pediatric patients. The ITP basis is a primary dysfunction of the immune system. This study aimed to analyze the genetic polymorphisms of the Fc gamma receptors IIA and IIIA. The genetic polymorphisms of the Fc receptors gamma IIA (131H/R) and gamma RIIIA (158V/F) were analyzed by polymerase chain reaction-restriction fragment length polymorphism technique. Odds ratio and 95% confidence interval were calculated by chi(2) test. Homozygous polymorphic genotype for the Fc gamma RIIIA was significantly more frequent among patients compared with controls (odds ratio = 0.27; 95% confidence interval, 0.09-0.80; P = 0.03). There was no statistical difference between the ITP group and the controls in the analysis of combinations of alleles of the high-affinity Fc receptor, but the ITP individuals with this combination had a lower duration of disease (P = 0.01). Genetic polymorphisms in immune system genes can be important for ITP pathogenesis and disease outcome.

Tax Gene Characterization of Human T-Lymphotropic Virus Type 1 Strains from Brazilian HIV-Coinfected Patients

The tax gene of human T-lymphotropic virus type 1 (HTLV-1) diverges among isolates according to geographic regions and has been classified into two genotypes: taxA and taxB. In Brazil, taxA is the most prevalent genotype in symptomatic and asymptomatic carriers. Few studies have been conducted in HIV-infected patients. The present study characterized the tax gene (1059 bp) in 13 Brazilian HIV-1/HTLV-1-coinfected patients from the south and southeast regions. The results confirmed the transcontinental HTLV-1 subgroup A of the Cosmopolitan subtype and showed high nucleotide similarity both among Brazilian sequences and in relation to the ATK prototype (99.5% and 99.2%, respectively). Six nucleotide substitutions were highly conserved among isolates, ranging from 76.9% to 100%: C7401T, T7914C, C7920T, C7982T, G8231A, and A8367C. The presence of the Brazilian molecular signature of genotype taxA was confirmed in all of the isolates, and they clustered into two Latin American clusters, which confirms the double introduction of HTLV-1 in Brazil.

Association between mannose-binding lectin and interleukin-1 receptor antagonist gene polymorphisms and recurrent vulvovaginal candidiasis

Objective The influence of functional polymorphisms in the genes coding for mannose-binding lectin (MBL) and interleukin-1 receptor antagonist (IL-1ra) on recurrent vulvovaginal candidiasis (RVVC) were examined in an urban Brazilian population. Methods DNA was isolated from buccal swabs of 100 women with RVVC and 100 control women and tested by gene amplification for a single nucleotide polymorphism in codon 54 of the MBL2 gene and for a length polymorphism in intron 2 of the IL1RN gene. Genotype and allele frequencies were compared between groups. Results The frequency of the variant MBL2 B allele, associated with reduced circulating and vaginal MBL concentrations, was 27.0% in RVVC and 8.5% in control women (p < .0001). The MBL2 B, B genotype was present in 12% of RVVC patients and 1% of controls (p = .0025). The IL1RN 2 allele frequency, associated with the highest level of unopposed IL-1 beta activity, was 24.0% in RVVC and 23.4% in controls. The IL1RN genotype distribution was also similar in both groups. Conclusion Carriage of the MBL2 codon 54 polymorphism, but not the IL1RN length polymorphism, predisposes to RVVC in Brazilian women.

Endostatin gene therapy stimulates upregulation of ICAM-1 and VCAM-1 in a metastatic renal cell carcinoma model

One of the greatest challenges in urological oncology is renal cell carcinoma (RCC), which is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and respond positively to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of ES-based antiangiogenic therapy to activate tumor-associated endothelial cells in metastatic RCC (mRCC). Balb/c-bearing Renca cells were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. The T-cell subsets and lymphocyte populations of tumors, mediastinal lymph nodes and the spleen were assessed by flow cytometry. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed by real-time PCR, flow cytometry and immunohistochemistry analysis. ES gene therapy led to an increase in the percentage of infiltrating CD4-interferon (IFN)-gamma cells (P<0.05), CD8-IFN-gamma cells (P<0.01) and CD49b-tumor necrosis factor-alpha cells (P<0.01). In addition, ES therapy caused an increase at the mRNA level of ICAM-1 (1.4-fold; P<0.01) and VCAM-1 (1.5-fold) (control vs treated group; P<0.001). Through flow cytometry, we found a significant increase in the CD34/ICAM-1 cells (8.1-fold; P<0.001) and CD34/VCAM-1 cells (1.6-fold; P<0.05). ES gene therapy induced a significant increase in both T CD4 and CD8 cells in the lymph nodes and the spleen, suggesting that ES therapy may facilitate cell survival or clonal expansion. CD49b cells were also present in increased quantities in all of these organs. In this study, we demonstrate an antitumor inflammatory effect of ES in an mRCC model, and this effect is mediated by an increase in ICAM-1 and VCAM-1 expression in tumor-associated endothelial cells.

Association of polymorphisms of endothelial nitric oxide synthase (eNOS) gene with the risk of primary open angle glaucoma in a Brazilian population

The present study aimed to investigate the association of endothelial nitric oxide synthase (eNOS) gene polymorphisms with primary open angle glaucoma (POAG). We conducted a case-control study that included 90 patients with POAG and 127 healthy controls whose blood samples were genotyped for the functional polymorphisms T-786C and Glu298Asp of the eNOS gene by Taqman fluorescent allelic discrimination assay. The T-786C polymorphism was significantly associated as a risk factor for POAG among women (OR: 228; 95% CI: 1.11 to 4.70, p = 0.024) and marginally associated to the risk of POAG in the patients >= 52 years of age at diagnosis (OR: 2.11; 95% CI: 0.98 to 4.55, p = 0,055). However, these results was not confirmed after adjustments for gender, age, self-declared skin color, tobacco smoking and eNOS genotypes by multivariate logistic regression model (OR: 2.08; 95% CI: 0.87 to 5.01, p = 0.101 and OR: 2.20; 95% CI: 0.95 to 5.12, p = 0.067, respectively). The haplotype CG of T-786C and Glu298Asp showed a borderline association with risk of POAG in the overall analysis (OR: 1.76; 95% CI: 0.98 to 3.14, p = 0.055) and among women (OR: 2.02; 95% CI: 0.98 to 4.16, p = 0.052). Furthermore, the CG haplotype was significantly associated with the development of POAG for the age at diagnosis group >= 52 years (OR: 3.48; 95% CI: 1.54 to 7.84, p = 0.002). We suggested that haplotypes of the polymorphisms T-786C and Glu298Asp of eNOS may interact with gender and age in modulating the risk of POAG. (C) 2012 Elsevier B.V. All rights reserved.