miRNAs and NFKB1 and TRAF6 target genes: The initial functional study in CD14+ monocytes in rheumatoid arthritis patients.

We predicted miRNAs with regulatory impact on NFKB1 and TRAF6 gene expression and selected the miR-194-5p, miR-124-3p, miR-9-5p, and miR-340-5p and their target genes for expression analyses on CD14+ monocytes from rheumatoid arthritis (RA) patients and healthy controls. Additionally, we evaluated the influence of genes and miRNA expression on RA patients' cytokine levels. No difference was observed in genes or miRNAs expression when compared to healthy controls and RA patients or clinical parameters. However, we found a significant difference between miR-194-5p and miR-9-5p levels (FC=-2.31; p=0.031; FC=-3.05;p=0.031, respectively) and non-prednisone users as compared to prednisone using patients. We conducted correlation analyses to identify the strength of the relationship between expression data and cytokine plasma levels. We observed a moderate positive correlation between miR-124-3p expression and IL-6 plasma levels (r=0.46; p=0.033). In addition, overexpression of miRNAs was concomitant to TRAF6 and NFKB1 genes as indicated by correlation analyses: TRAF6 and miR-194-5p (r=0.60;p<0.001) and miR-9-5p (r=0.63;p<0.001) and NFKB1 and miR-194-5p (r=0.72;p<0.001), miR-9-5p (r=0.72;p<0.001) and miR-340-5p (r=0.61;p<0.001). NFKB1 and TRAF6 genes and miRNAs monocyte expression do not appear to be related to RA but showed a significant difference in different groups of RA therapy. In addition, increased levels of miRNAs can be linked to concomitant overexpression of TRAF6 and NFKB1 in monocytes and act as its regulators.

IL1ß, IL18, NFKB1 and IFNG gene interactions are associated with severity of rheumatoid arthritis: A pilot study.

Rheumatoid arthritis (RA) is an autoimmune disease which can lead to progressive and functional disability. Literature data suggest that some inflammatory proteins are dysregulated in RA patients and its genetic polymorphisms may contribute to the aetiology and pathogenesis of disease in different ethnic groups. Polymorphisms in IL1ß, IL18, NFKB1 and IFNG genes were studied in different populations with RA, but the analysis indicated contradictory results. Thereby, we hypothesised that polymorphisms in these genes could have a combined effect on susceptibility to and severity of disease. We evaluated the +3953 C/T IL1ß (rs1143634), -137 G/C IL18 (rs187238), -94 ins/del ATTG NFKB1 (rs28362491) and +874 T/A IFNG (rs2430561) polymorphisms in the northeastern Brazilian population. Peripheral blood samples were collected and DNA extraction was conducted. The polymorphisms were evaluated by RFLP and ARMS-PCR. An association was observed in rs1143634 which showed a protective effect against development of RA in carriers of the T allele (OR = 0.58; 95% CI 0.36-0.92; p = .020). In addition, we found an association among genotypes of the rs1143634 with the HAQ index (p = .021) and rs2430561 with DAS28 (p = .029) and CDAI (p = .029). In relation to combined effects of these SNPs (C/C to rs1143634, G/G to rs187238, I/I to rs28362491 and AA to rs2430561) we found a significant association with decreased functional disability (HAQ index p < .001) and ESR (p = .034), indicating a lower disease activity in carriers of these genotypes. GLM analysis confirmed these associations (HAQ (F = 5.497; p < .001) and ESR (F = 2.727; p = .032)). Our analysis indicated that in the studied population +3953 C/T IL-1ß (rs1143634), -137 G/C IL-18 (rs187238), -94 ins/del ATTG NFKB1 (rs28362491) and +874 T/A IFNG (rs2430561) polymorphisms can together contribute to RA severity although they do not individually influence the disease.

Influence of DRD1 and DRD3 Polymorphisms in the Occurrence of Motor Effects in Patients with Sporadic Parkinson's Disease.

Parkinson's disease (PD) is a multisystem disorder that affects 2-3% of the population ≥ 65 years of age. The main pharmacologic agent use in the treatment of clinical symptoms of PD is levodopa (L-DOPA). However, the chronic use of L-DOPA might result in the emergence of motor complications such as motor fluctuation and dyskinesia. Previous studies have shown that the inter-individual variability and pharmacogenetic profile of PD patients seem to influence the occurrence of motor complications. For these reasons, the purpose of this study was to evaluate a possible relationship between DRD1 A48G and DRD3 Ser9Gly genetic variants with the occurrence of motor complications in PD patients in a Brazilian population. A total of 228 patients with idiopathic PD were enrolled. Patients were genotyped for DRD1 A48G and DRD3 Ser9Gly polymorphisms using PCR-RFLP. The univariate and multivariate analyses were performed to assess the association of these polymorphisms with the occurrence of motor fluctuation and dyskinesia in PD patients. Multiple Poisson regression analyses showed a protector effect to the occurrence of dyskinesia for individuals carrying of the DRD1 G/G genotype (PR 0.294; CI 0.09-0.87; p ≤ 0.020) after the threshold Bonferroni's. Besides, we verified risk increased to the occurrence of motor complications with daily L-DOPA dosage, disease duration, and users of rasagiline, selegiline, or entacapone (p < 0.05 for all). Our results suggest that the DRD1 A48G polymorphism and the presence of extrinsic and intrinsic factors may role an effect in the occurrence of dyskinesia in PD patients.

Pharmacogenetic Profile and the Occurrence of Visual Hallucinations in Patients With Sporadic Parkinson's Disease.

Visual hallucinations are significant nonmotor symptoms in the course of treatment of Parkinson's disease. Previous studies have shown that the interindividual variability and pharmacogenetic profile of Parkinson's disease patients seem to influence the occurrence of visual hallucinations. In our study, we investigated a possible relationship of sequence variants in DRD1, DRD2, DRD3, DAT1, and COMT genes with the presence of visual hallucinations in Parkinson's disease patients. A total of 224 Brazilian patients from the Pro-Parkinson service at the Clinical Hospital of the University of Pernambuco, diagnosed with sporadic Parkinson's disease, were enrolled. Parkinson's disease patients were divided into 2 groups based on the presence or absence of visual hallucinations. The sequence variants for DRD1, DRD2, DRD3, DAT1, and COMT were determined through the polymerase chain reaction-restriction fragment length polymorphism technique. Multiple Poisson regression analyses showed that individuals carrying the DRD3 Ser/Ser and Ser/Gly genotypes presented increased prevalence ratios of visual hallucinations (9.7-fold and 4.4-fold, respectively; P < .001). Regarding DAT1 rs28363170, there was a 9.82-fold increase in the prevalence ratio in patients with the 10/11 genotype, 8.78-fold for the 10/8 genotype, and 2.44-fold for the 9/8 genotypes (P < .001, for all). In addition, visual hallucinations were also associated with use of transdermal patches with rotigotine (PR, 3.7; 95%CI, 1.2-10.9; P = .017) and rasagiline (PR, 2.8; 95%CI, 1.3-6.0; P = .006). Our results suggest that the genetic variants DRD3 and DAT1, along with other therapeutic confounders, may influence the prevalence ratio of visual hallucinations.

Interleukin (IL)-23 Receptor, IL-17A and IL-17F Gene Polymorphisms in Brazilian Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a progressive, autoimmune disease for which the previous studies have shown that some functional polymorphisms can influence its etiology. Knowing this, the aim of this study was to investigate the association of +2199 A/C IL-23R (rs10889677), -197 G/A IL-17A (rs2275913), and +7488 A/G IL-17F (rs763780) gene polymorphisms with RA susceptibility and clinical features in a Brazilian population. A total of 127 RA patients and 134 healthy controls were recruited for the analyses of polymorphic variants. Genotyping was performed using RFLP-PCR. Logistic regression was used to analyze the genotype distribution of the polymorphisms. Individuals carrying the homozygous CC genotype for the IL-23R polymorphism seem to be at lower risk for RA development (OR 0.22; p = 0.004), as well as those carrying the variant C allele (OR 0.56; p = 0.002). For the -197 G/A IL-17A polymorphism, the wild-type genotype (GG) was significantly associated with a 3.18-fold (OR 3.18; p = 0.033) increased risk for RA. In relation to the +7488 A/G IL-17F polymorphism, no significant difference was found between RA cases and control subjects (p > 0.05). Moreover, when investigating the relationship between polymorphisms and clinical features, no evidence of an association was found. Our findings suggest that the variants +2199 A/C IL-23R and -197 G/A IL-17A could contribute to RA development in the studied population. However, larger studies are needed to fully understand this genetic predisposition.

Influence of IL-6, IL-8, and TGF-β 1 gene polymorphisms on the risk of human papillomavirus-infection in women from Pernambuco, Brazil

Human papillomavirus (HPV) infections are strongly associated with the development of cervical intraepithelial neoplasias and invasive cervical cancer. Polymorphisms in cytokine-encoding genes and behavioural cofactors could play an important role in protecting an individual against viral infections and cancer. Here, we investigated whether IL-6 -174 G>C, IL-8 +396 G>T, and TGF-β1 +869 G>C and +915 G>C polymorphisms were associated with susceptibility to HPV infection in women from north-east (Pernambuco) Brazil. We analysed 108 healthy uninfected women (HC) and 108 HPV-positive women with cervical lesions. Genetic polymorphisms were assessed using Sanger sequencing and polymerase chain reaction-restriction fragment length polymorphism. Comparison of the distribution of the genotypic and allelic frequencies of the IL-18 +396 T>G polymorphism between HPV infected woman an uninfected controls showed that the GG genotype and G allele were both more frequent in the HC group, and were associated with protection from HPV infection (p = 0.0015; OR = 0.29 CI95% = 0.13-0.61; p = 0.0005; OR = 0.45 CI95% 0.29-0.7, respectively). Individuals from the control group could have previously had HPV infection that was spontaneously eliminated; however, it was undetectable at the time of sample collection. Based on our findings, we hypothesize that the IL-8 +396 G>T polymorphism could interfere with susceptibility to HPV infection, by modulating the ability of immune system to fight the virus.

Influence of IL-6, IL-8, and TGF-ß1 gene polymorphisms on the risk of human papillomavirus-infection in women from Pernambuco, Brazil.

Human papillomavirus (HPV) infections are strongly associated with the development of cervical intraepithelial neoplasias and invasive cervical cancer. Polymorphisms in cytokine-encoding genes and behavioural cofactors could play an important role in protecting an individual against viral infections and cancer. Here, we investigated whether IL-6 -174 G>C, IL-8 +396 G>T, and TGF-ß1 +869 G>C and +915 G>C polymorphisms were associated with susceptibility to HPV infection in women from north-east (Pernambuco) Brazil. We analysed 108 healthy uninfected women (HC) and 108 HPV-positive women with cervical lesions. Genetic polymorphisms were assessed using Sanger sequencing and polymerase chain reaction-restriction fragment length polymorphism. Comparison of the distribution of the genotypic and allelic frequencies of the IL-18 +396 T>G polymorphism between HPV infected woman an uninfected controls showed that the GG genotype and G allele were both more frequent in the HC group, and were associated with protection from HPV infection (p = 0.0015; OR = 0.29 CI95% = 0.13-0.61; p = 0.0005; OR = 0.45 CI95% 0.29-0.7, respectively). Individuals from the control group could have previously had HPV infection that was spontaneously eliminated; however, it was undetectable at the time of sample collection. Based on our findings, we hypothesize that the IL-8 +396 G>T polymorphism could interfere with susceptibility to HPV infection, by modulating the ability of immune system to fight the virus.

CCR2 and CCR5 genes polymorphisms in women with cervical lesions from Pernambuco, Northeast Region of Brazil: a case-control study.

Polymorphisms in chemokine receptors play an important role in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer (CC). Our study examined the association of CCR2-64I (rs1799864) andCCR5-Δ32 (rs333) polymorphisms with susceptibility to develop cervical lesion (CIN and CC) in a Brazilian population. The genotyping of 139 women with cervical lesions and 151 women without cervical lesions for the CCR2-64I and CCR5-Δ32 polymorphisms were performed using polymerase chain reaction-restriction fragment length polymorphism. The individuals carrying heterozygous or homozygous genotypes (GA+AA) for CCR2-64I polymorphisms seem to be at lower risk for cervical lesion [odds ratio (OR) = 0.37, p = 0.0008)]. The same was observed for the A allele (OR = 0.39, p = 0.0002), while no association was detected (p > 0.05) with CCR5-Δ32 polymorphism. Regarding the human papillomavirus (HPV) type, patients carrying the CCR2-64Ipolymorphism were protected against infection by HPV type 16 (OR = 0.35, p = 0.0184). In summary, our study showed a protective effect ofCCR2-64I rs1799864 polymorphism against the development of cervical lesions (CIN and CC) and in the susceptibility of HPV 16 infection.

CCR2 and CCR5 genes polymorphisms in women with cervical lesions from Pernambuco, Northeast Region of Brazil: a case-control study

Polymorphisms in chemokine receptors play an important role in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer (CC). Our study examined the association of CCR2-64I (rs1799864) andCCR5-Δ32 (rs333) polymorphisms with susceptibility to develop cervical lesion (CIN and CC) in a Brazilian population. The genotyping of 139 women with cervical lesions and 151 women without cervical lesions for the CCR2-64I and CCR5-Δ32 polymorphisms were performed using polymerase chain reaction-restriction fragment length polymorphism. The individuals carrying heterozygous or homozygous genotypes (GA+AA) for CCR2-64I polymorphisms seem to be at lower risk for cervical lesion [odds ratio (OR) = 0.37, p = 0.0008)]. The same was observed for the A allele (OR = 0.39, p = 0.0002), while no association was detected (p > 0.05) with CCR5-Δ32 polymorphism. Regarding the human papillomavirus (HPV) type, patients carrying the CCR2-64Ipolymorphism were protected against infection by HPV type 16 (OR = 0.35, p = 0.0184). In summary, our study showed a protective effect ofCCR2-64I rs1799864 polymorphism against the development of cervical lesions (CIN and CC) and in the susceptibility of HPV 16 infection.

Interferon gamma and Interleukin 10 polymorphisms in Brazilian patients with systemic lupus erythematosus.

The pathogenesis of systemic lupus erythematosus (SLE) is complex, with several susceptibility genes and environmental factors involved in its development and clinical manifestation. Currently, there is a great amount of interest in the identification of biomarkers, as cytokines, that can quantify the susceptibility of SLE, the risk of future organ involvement, and association of their changes with disease activity. To investigate the associations between polymorphisms in the gene of Interferon gamma (IFN-γ) and in the promoter of the Interleukin-10 (IL-10) gene and SLE. The polymorphisms +874 T/A (rs2430561) in the IFN-γ gene and -1082G/A (rs1800896) in the IL-10 promoter were determined in 99 SLE patients and 100 healthy controls among women Brazilian using the refractory mutation system polymerase chain reaction method. Disease activity was assessed using the SLE activity index. There were significant differences in the distribution of the genotype T/A in IFN-γ gene polymorphism (+874) (χ (2) = 7.168; P = 0.0074) and the genotype G/A in IL-10 promoter polymorphism (-1082) (χ (2) = 4.654; P = 0.0310) between the SLE and control groups. However, no association was observed between clinical features and the polymorphisms studied. This study presents preliminary evidence for association between IL-10 and IFN-γ polymorphism and SLE susceptibility, but not with clinical features in a Northeast population from Brazil.

Prevalência dos genótipos do papilomavírus humano: comparação entre três métodos de detecção em pacientes de Pernambuco, Brasil / Prevalence of human papillomavirus genotypes: comparison between three detection methods in patients of Pernambuco, Brazil

OBJETIVO: comparar três métodos para detecção do HPV e determinar a prevalência dos genótipos encontrados. MÉTODOS: um total de 120 amostras de raspagem da região cervical de mulheres portadoras de neoplasia intraepitelial cervical foram analisadas pela reação em cadeia da polimerase convencional, usando os sistemas de primers MY09/11, GP05+/06+ e pela Nested-PCR. As amostras foram submetidas à extração de DNA e, logo após, amplificadas com os primers GH20 e PC04 (β-globina) para verificação da qualidade do DNA obtido e pela reação em cadeia da polimerase convencional e Nested-PCR. Os fragmentos amplificados foram visualizados em gel de agarose a 1,2 por cento, corados com Blue Green Loading Dye I. As amostras positivas foram sequenciadas usando o sequenciador automático de DNA "MegaBACE 1000". Para análise estatística foram utilizados os teste do Χ2 e o de Fisher com nível de significância de 5 por cento. RESULTADOS: quinze amostras não se amplificaram para os primers de β-globina, sendo eliminadas do estudo. Das amostras restantes, 40 por cento (42/105) foram positivas para os primers MY09/11, 98 por cento (103/105) para os primers GP05+/06+ e 92 por cento (97/105) para Nested-PCR. Considerado as técnicas MY09/11 e GP05+/06+, foi possível observar 100 por cento de amostras positivas para o HPV. Neste estudo, a prevalência dos genótipos foi de 58, 23, 5, 4 e 3 por cento para HPVs 16, 18, 31, 33 e 56, respectivamente. Os HPV 67 e 83 apresentaram 2 por cento e os HPV 6, 11, 58 e candHPV85, 1 por cento cada. A prevalência dos genótipos neste estudo está de acordo com o reportado em todo o mundo (IC95 por cento=0,4657-0,8976). CONCLUSÕES: para obter resultados mais confiáveis, é necessário o uso de mais que um sistema de primers para detecção do HPV. Acredita-se que as três técnicas estudadas são importantes e adequadas para o diagnóstico clínico do HPV quando apropriadamente combinadas.

PURPOSE: to compare three methods for the detection of HPV infection and to determine the prevalence of the genotypes found. METHODS: a total of 120 cervical scrape samples from patients with cervical intraepithelial neoplasia were analyzed by the conventional polymerase chain reaction using the MY09/11 and GP05+/06+ primers, and by the Nested polymerase chain reaction. The samples were subjected to DNA amplification with the GH20 and PC04 primers (β-globin) to verify DNA quality and also by polymerase chain reaction and Nested polymerase chain reaction. The amplicons were visualized in 1.2 percent agarose gel stained with Blue Green Loading Dye I. Positive samples also were sequenced using the automatic DNA sequencer "MegaBACE 1000". The Χ2 and Fisher tests were used for statistical analysis with the level of significance set at 5 percent. RESULTS: fifteen samples were eliminated from the study because they failed to amplify the β-globin gene. Of the remaining samples, 40 percent (42/105) were positive using primers MY09/11, 98 percent (103/105) using primers GP05+/06+, and 92 percent (97/105) using Nested-PCR. With the MY09/11 and GP05+/06+ techniques, it was possible to obtain 100 percent HPV-positive samples. In this study, the prevalence of the genotypes found was 57, 23, 5, 4 and 3 percent for HPV genotypes 16, 18, 31, 33 and 56, respectively. HPVs 67 and 83 were present in 2 percent, and genotypes 6, 11, 58 and candHPV85 were present in 1 percent each. The prevalence of the more common genotypes (HPV 16 and 18) in this study agrees with that reported worldwide (IC95 percent=0.4657-0.8976). CONCLUSIONS: to obtain more reliable results, it is necessary the use of more than one primer system to detect HPV infections. We believe that the three techniques studied are important and suitable for the clinical diagnosis of HPV, when they are appropriately combined.

[Prevalence of human papillomavirus genotypes: comparison between three detection methods in patients of Pernambuco, Brazil]. / Prevalência dos genótipos do papilomavírus humano: comparação entre três métodos de detecção em pacientes de Pernambuco, Brasil.

PURPOSE: to compare three methods for the detection of HPV infection and to determine the prevalence of the genotypes found. METHODS: a total of 120 cervical scrape samples from patients with cervical intraepithelial neoplasia were analyzed by the conventional polymerase chain reaction using the MY09/11 and GP05+/06+ primers, and by the Nested polymerase chain reaction. The samples were subjected to DNA amplification with the GH20 and PC04 primers (ß-globin) to verify DNA quality and also by polymerase chain reaction and Nested polymerase chain reaction. The amplicons were visualized in 1.2% agarose gel stained with Blue Green Loading Dye I. Positive samples also were sequenced using the automatic DNA sequencer "MegaBACE 1000". The Χ2 and Fisher tests were used for statistical analysis with the level of significance set at 5%. RESULTS: fifteen samples were eliminated from the study because they failed to amplify the ß-globin gene. Of the remaining samples, 40% (42/105) were positive using primers MY09/11, 98% (103/105) using primers GP05+/06+, and 92% (97/105) using Nested-PCR. With the MY09/11 and GP05+/06+ techniques, it was possible to obtain 100% HPV-positive samples. In this study, the prevalence of the genotypes found was 57, 23, 5, 4 and 3% for HPV genotypes 16, 18, 31, 33 and 56, respectively. HPVs 67 and 83 were present in 2%, and genotypes 6, 11, 58 and candHPV85 were present in 1% each. The prevalence of the more common genotypes (HPV 16 and 18) in this study agrees with that reported worldwide (IC95%=0.4657-0.8976). CONCLUSIONS: to obtain more reliable results, it is necessary the use of more than one primer system to detect HPV infections. We believe that the three techniques studied are important and suitable for the clinical diagnosis of HPV, when they are appropriately combined.

Immobilization of lipase from "Fusarium solani" FS1

Lipase from "Fusarium solani" FS1 was immobilized by covalent attachment to polyacrylamide beads and onto magnetized Dacron, retaining 12(per cent) and 97(per cent) of activity, respectively. Lipase was also entrapped within polyacrylamide beads, retaining 53(per cent) of activity. Investigations of the kinetic characteristics of the immobilized derivates using triolein as substrate showed that lipase immobilized onto polyacrilamide beads and Dracon did not follow Michaelis-Menten kinetics.

Production of extracellular lipase by the phytopathogenic fungus "Fusarium solani" FS1

A Brazilian strain of "Fusarium solani" was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.L(E-1) of lipase after 72 hours of cultivation at 25ºC in shake-flask at 120rpm in a medium containing 3(per cent) (w/v) peptone plus 0.5(per cent)(v/v) olive oil. Glucose (1(per cent) w/v) was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3(per cent)(w/v) resulted in a reduced lipase production while increased olive oil concentration (above o.5(per cent)) did not further stimulate lipase production. The optimum lipase activity was achieved at pH 8.6 and 30ºC and a good enzyme stability (80(per cent) activity retention) was observed at pH ranging from 7.6 to 8.6, and the activity rapidly dropped at temperatures above 50ºC. Lipase activity was stimulated by addition of n-hexane to the culture medium supernatatnts, in contrast to incubation with water-soluble solvents