Abstract 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. NFKB TRAF miR1945p, miR1945p miRp miR 194 5p, 5p p miR1243p, miR1243p 124 3p, 3p miR95p, miR95p 9 miR3405p 340 CD14 CD (RA Additionally parameters However FC=2.31 FC231 FC FC= 2.31 2 31 (FC=-2.31 p=0.031 p0031 0 031 FC=3.05p=0.031, FC305p0031 FCp 3.05 p=0.031, 3 05 FC=-3.05;p=0.031 respectively nonprednisone non IL6 IL 6 IL- r=0.46 r046 r 46 (r=0.46 p=0.033. p0033 p=0.033 . 033 p=0.033) addition r=0.60p<0.001 r060p0001 rp r=0.60 p<0.001 60 001 (r=0.60;p<0.001 r=0.63p<0.001 r063p0001 r=0.63 63 (r=0.63;p<0.001 r=0.72p<0.001, r072p0001 r=0.72 , 72 r=0.72p<0.001 (r=0.72;p<0.001 r=0.61p<0.001. r061p0001 r=0.61 61 (r=0.61;p<0.001) therapy regulators 19 12 34 CD1 FC=2.3 FC23 231 2.3 (FC=-2.3 p=0.03 p003 03 05p FC=3.05p=0.031 FC305p003 305 3.0 FC=-3.05;p=0.03 r=0.4 r04 4 (r=0.4 60p r=0.60p<0.00 r060p000 r060 r=0.6 p0001 p<0.00 00 (r=0.60;p<0.00 63p r=0.63p<0.00 r063p000 r063 (r=0.63;p<0.00 72p r072p000 r072 r=0.7 7 r=0.72p<0.00 (r=0.72;p<0.00 61p r=0.61p<0.001 r061p000 r061 (r=0.61;p<0.001 1 FC=2. FC2 23 2. (FC=-2. p=0.0 p00 FC=3.05p=0.03 FC305p00 30 3. FC=-3.05;p=0.0 r=0. r0 (r=0. r=0.60p<0.0 r060p00 r06 p000 p<0.0 (r=0.60;p<0.0 r=0.63p<0.0 r063p00 (r=0.63;p<0.0 r072p00 r07 r=0.72p<0.0 (r=0.72;p<0.0 r=0.61p<0.00 r061p00 (r=0.61;p<0.00 FC=2 (FC=-2 p=0. p0 FC=3.05p=0.0 FC305p0 FC=-3.05;p=0. r=0 (r=0 r=0.60p<0. r060p0 p<0. (r=0.60;p<0. r=0.63p<0. r063p0 (r=0.63;p<0. r072p0 r=0.72p<0. (r=0.72;p<0. r=0.61p<0.0 r061p0 (r=0.61;p<0.0 (FC=- p=0 FC=3.05p=0. FC305p FC=-3.05;p=0 r= (r= r=0.60p<0 r060p p<0 (r=0.60;p<0 r=0.63p<0 r063p (r=0.63;p<0 r072p r=0.72p<0 (r=0.72;p<0 r=0.61p<0. r061p (r=0.61;p<0. (FC= p= FC=3.05p=0 FC=-3.05;p= (r r=0.60p< p< (r=0.60;p< r=0.63p< (r=0.63;p< r=0.72p< (r=0.72;p< r=0.61p<0 (r=0.61;p<0 (FC FC=3.05p= FC=-3.05;p r=0.60p (r=0.60;p r=0.63p (r=0.63;p r=0.72p (r=0.72;p r=0.61p< (r=0.61;p< FC=3.05p r=0.61p (r=0.61;p

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.

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.

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%, 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%. RESULTADOS: quinze amostras não se amplificaram para os primers de β-globina, sendo eliminadas do estudo. Das amostras restantes, 40% (42/105) foram positivas para os primers MY09/11, 98% (103/105) para os primers GP05+/06+ e 92% (97/105) para Nested-PCR. Considerado as técnicas MY09/11 e GP05+/06+, foi possível observar 100% de amostras positivas para o HPV. Neste estudo, a prevalência dos genótipos foi de 58, 23, 5, 4 e 3% para HPVs 16, 18, 31, 33 e 56, respectivamente. Os HPV 67 e 83 apresentaram 2% e os HPV 6, 11, 58 e candHPV85, 1% cada. A prevalência dos genótipos neste estudo está de acordo com o reportado em todo o mundo (IC95%=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% 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 β-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.

Lipase de Fusarium solani FS1 foi imobilizada por ligação covalente usando esferas de poliacrilamida e Dacron magnetizado, retendo 12%, e 97% de atividade, respectivamente. A lipase foi também enclausurada em esferas de poliacrilamida e reteve 53% de sua atividade específica. Investigações sobre o comportamento cinético usando trioleína como substrato mostraram que as lipases imobilizadas não seguem a cinética de Michaelis-Menten.

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

A Brazilian strain of Fusarium solani was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.l-1 of lipase after 72 hours of cultivation at 25oC in shake-flask at 120 rpm in a medium containing 3% (w/v) peptone plus 0.5% (v/v) olive oil. Glucose (1% w/v) was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3% (w/v) resulted in a reduced lipase production while increased olive oil concentration (above 0.5%) did not further stimulate lipase production. The optimum lipase activity was achieved at pH 8.6 and 30oC and a good enzyme stability (80% 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 the addition of n-hexane to the culture medium supernatants, in contrast to incubation with water-soluble solvents.