Associação entre o polimorfismo VKORC1: C. 174-136G>A (rs9934438) e a dose semanal de varfarina em pacientes idosos ­ estudo piloto / Association between VKORC1: C.174-136G> A (rs9934438) polymorphism and weekly warfarin dose in elderly patients - pilot study

A varfarina é um anticoagulante utilizado na prevenção e no tratamento de doenças tromboembólicas, com janela terapêutica estreita e, elevado risco de hemorragias. Nos idosos, a principal indicação para tratamento anticoagulante oral é fibrilação atrial, cuja prevalência aumenta com a idade, atingindo 8% após os 80 anos. O exame utilizado para controle da anticoagulação oral é o tempo de protrombina, através do cálculo da razão normalizada internacional (RNI), visando o ajuste de dose e manutenção da faixa terapêutica (RNI= 2 a 3). Os idosos requerem um monitoramento efetivo devido a fatores inerentes da idade. Embora seja conhecido que os fatores genéticos influenciam na resposta terapêutica à varfarina, na rotina da maioria dos hospitais a farmacogenética ainda não é considerada no ajuste de dose. Visando estabelecer uma conduta terapêutica personalizada, o presente estudo tem como objetivo avaliar a associação entre o polimorfismo rs9934438 do gene VKORC1, que codifica a enzima vitamina K epóxido redutase, e a dose semanal de varfarina necessária para atingir o RNI adequado. Até o momento, foram incluídos 52 pacientes com idade superior a 70 anos, de ambos os sexos e em uso de varfarina. A análise do polimorfismofoi realizada através da PCR em tempo real utilizando os reagentes TaqMan™ Sample-to-SNP™ e o sistema de detecção TaqMan® SNP Genotyping Assay. As análises estatísticas foram realizadas utilizando o pacote SPSS v. 16.0 e nível de significância adotado foi de 5%. Dos 52 pacientes incluídos até o momento, 37 (71%) permaneceram na faixa terapêutica (Time in Therapeutic Range, TTR) em pelo menos 50% do tempo de anticoagulação e, para eles a dose semanal de varfarina variou de 15mg a 62,5mg. Apesar de ser um estudo piloto, a distribuição dos genótipos está em equilíbrio gênico, segundo Hardy-Weinberg (AA=19,2%, AG=34,6%, GG= 46,2%, χ2= 3,34 e p=0,067). Para os idosos com TTR ≥50%, a frequência do alelo A foi significantemente maior entre os pacientes que utilizaram doses menores de varfarina (Exato de Fisher, p=0,005). Adicionalmente, os portadores do genótipo AA necessitaram, em média, de aproximadamente metade da dose para atingir a faixa terapêutica quando comparados aos portadores do genótipo GG, 20,5 versus 36,5 mg/semana, respectivamente (ANOVA, p=0,006/ Pós-teste Bonferroni, p=0,039). Os resultados permitem concluir que portadores de alelo A são mais responsivos ao tratamento com varfarina, sugerindo que o perfil genotípico pode ser de grande valor para o direcionamento da dose terapêutica em idosos. (AU)

On-site detection of equid alphaherpesvirus 3 in perineal and genital swabs of mares and stallions.

Equine coital exanthema (ECE) is an infectious, venereally transmitted muco-cutaneous disease affecting mares and stallions, caused by equid alphaherpesvirus 3 (EHV3). Diagnostic tools for rapid identification of EHV3 are of primary importance to diminish the risk of EHV3 dissemination at the time of breeding. In the last years, it has been shown that the performance of the insulated-isothermal polymerase chain reaction (iiPCR) is comparable to virus isolation, nested PCR and real-time PCR (qPCR) in detecting pathogens of various animal species. Analytical sensitivity and specificity of the iiPCR were compared with a qPCR, using a plasmid containing the target region of the EHV3 glycoprotein G gene and an Argentinian EHV3 isolate (E/9283/07 C3A). In order to evaluate the diagnostic performance of the iiPCR, nucleic acids of 85 perineal and genital swabs (PGS) of mares and stallions were extracted by tacoTM mini and tested by both techniques. EHV3 was detected in 46 and 45 of the 85 PGS by the iiPCR and qPCR, respectively. There was almost perfect agreement between the two diagnostic methods (98.82%; 95% CI: 95.03-100%; κ = 0.98). The iiPCR had a limit of detection of 95.00% at 6 genome equivalents per reaction and a detection endpoint for viral DNA comparable to that of the qPCR, and did not react with six non-targeted equine pathogens. The iiPCR represents a sensitive and specific method for the rapid on-site diagnosis of EHV3 infection. Its routinely implementation in breeding facilities, and artificial insemination and embryo transfer centers, will contribute to prevent the dissemination of this venereal, highly contagious disease in horses.

Association of XRCC1 gene polymorphisms and pancreatic cancer risk in a Chinese population.

We conducted a case-control study to assess the role of the XRCC1 Arg399Gln, Arg280His, and Arg194Trp gene polymorphisms in pancreatic cancer susceptibility in a Chinese population. A total of 152 patients diagnosed with pancreatic cancer and 264 control subjects were enrolled in this study between March 2012 and October 2014. XRCC1 Arg399Gln, Arg280His, and Arg194Trp were genotyped using the polymerase chain reaction-restriction fragment length polymorphism method. As determined by the chi-squared test, a statistically significant difference was observed between pancreatic cancer patients and control subjects in regard to the genetic distribution of XRCC1 Arg399Gln (χ(2) = 16.13, P < 0.001). Using an unconditional regression analysis, we found that the TT genotype of Arg399Gln was associated with a significantly increased risk of pancreatic cancer (OR = 2.33, 95%CI = 1.20-4.51), and that the CT+TT genotype also significantly increased pancreatic cancer risk (OR = 1.58, 95%CI = 1.04-2.41), compared to the wild-type genotype. In conclusion, we found that XRCC1 Arg399Gln genetic variations are associated with pancreatic cancer development, whereas the XRCC1 Arg280His and Arg194Trp polymorphisms did not affect pancreatic cancer risk.

Thermotherapy-induced reduction in glioma invasiveness is mediated by tumor necrosis factor-alpha.

Thermotherapy has been proven to be effective for the treatment of various tumors, including glioma. We determined whether tumor necrosis factor-alpha (TNF-α) is involved in the regulation of the biological processes of glioma development. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were used to investigate the levels of TNF-α mRNA and heat shock factor-1 (HSF1) protein, respectively, in glioma cells. Radioimmunoassay was used to dynamically monitor the contents of TNF-α in the nutrient fluid of C6 cells after thermotherapy treatment. Crystal violet staining was used to determine glioma invasiveness. The most obvious increases in HSF1 protein and TNF-α mRNA in C6 cells were observed at 30 and 60 min after thermotherapy, respectively. In addition, the radioactivity of TNF-α in the culture fluid of the C6 cells reached a peak after 120 min of thermotherapy. In addition, glioma invasiveness decreased and the concentration of TNF-α reached a maximum after 120 min of thermotherapy. Our results show that the decrease in thermotherapy-mediated glioma invasiveness is due to the accelerated release of TNF-α, which could promote the release of HSF1 from neurospongioma cells.

Effect of transvaginal ultrasound on human chorionic villus cell apoptosis during pregnancy.

With the advancement of ultrasonic technology in recent years, sonography has become a common medical diagnostic tool, as it has elevated output sonic intensity and elongated exposure time. This study investigates the effect of ultrasound on human chorionic villus cell apoptosis during early pregnancy. Transvaginal ultrasound was performed for a total of 60 women who had undergone induced abortion at our hospital. They were randomly divided into the control, short ultrasound (10 min), and long ultrasound (20 min) groups (N = 20 each). Twenty-four hours after ultrasonic exposure, chorionic villus tissues were extracted during induced abortion, and were tested for cell apoptosis using flow cytometry. Bax and B cell lymphoma-2 (Bcl-2) protein levels were also quantified by immunohistochemistry. We found that the long ultrasound group had significantly higher cell apoptosis rates compared to the short ultrasound group, which in turn had higher rates compared to the control group (P < 0.05 in both cases). Bax protein levels were elevated in both the long and short ultrasound groups (P < 0.05). Bcl-2 proteins in two ultrasound groups, however, were downregulated as compared to those in the control group (P < 0.05). It is therefore possible that transvaginal sonography can potentiate the apoptosis of human chorionic villus cells by increasing the Bax/Bcl-2 protein ratio.

Cloning and expression analysis of a stress-induced GmIMT1 gene in soybean (Glycine max).

Here, we aimed to clone and identify the GmIMT1 gene related to the salt stress response in soybean. The full-length cDNA sequence of the GmIMT1 gene was amplified in soybean using degenerate primers of Mesembrythmum crystallium. To understand the stress response, the GmIMT1 gene was cloned and sequenced. Then, the expression vectors of the gene were constructed, and introduced into the model plant Arabidopsis thaliana through Agrobacterium mediated transformation, and the salt tolerance was analyzed in the transgenic plants. In addition, the expression patterns of GmIMT1 gene in soybean were analyzed. The expression was examined in different organs (roots, leaves, flower seeds, and stem) and under different stress conditions (drought, high salt, low temperature, salicylic acid, ethane, abscisic acid, and methyl jasmonate) by real-time fluorescent quantitative polymerase chain reaction analysis. The results showed that the root, leaves, and stems exhibited high level of GmIMT1 gene expression, whereas there was no expression in the seeds. In addition, the GmIMT1 gene expression was upregulated under all stress conditions. Overall, the results clearly indicate that GmIMT1 might be involved in multiple plant response pathways to the different environmental conditions. Furthermore transgenic plants exhibited higher salt-tolerance compared to wild type plants.

CD4+CD25+Foxp3+ indirect alloreactive T cells from renal transplant patients suppress both the direct and indirect pathways of allorecognition.

Alloreactive T cells recognize donor antigens by two routes: direct and indirect pathways of allorecognition. Although the direct pathway is reported to be dominant in allograft rejection, indirect allorecognition also plays an important role. Indirect alloreactivity is also observed in renal transplant patients irrespective of rejection. Previously we showed a predominance of interleukin (IL)-10 induced by indirect allorecognition of donor human leucocyte antigen (HLA)-DR peptides, suggesting the existence of indirect alloreactive T cells displaying regulatory activity. In the present work, our objective was to characterize these regulatory T cells. We detected indirect alloproliferation of peripheral blood mononuclear cells (PBMC) from renal transplant patients, induced by donor HLA-DR peptides, dependent on IL-4 or IL-10, suggesting regulatory activity as part of the alloreactive T-cell repertoire. PBMC-derived indirect alloreactive T-cell lines were established and produced both inflammatory and regulatory cytokines. We showed that two of these T-cell lines which were able to inhibit both direct and indirect alloproliferation of another T-cell line from the same patient presented a CD4(+)CD25(+)Foxp3(+) T-cell population. These data support the idea that indirect alloreactive T cells may also have regulatory activity and may contribute to the maintenance of the human renal allograft.

CD4+CD25+Foxp3+ indirect alloreactive T cells from renal transplant patients suppress both the direct and indirect pathways of allorecognition

Alloreactive T cells recognize donor antigens by two routes: direct and indirect pathways of allorecognition. Although the direct pathway is reported to be dominant in allograft rejection, indirect allorecognition also plays an important role. Indirect alloreactivity is also observed in renal transplant patients irrespective of rejection. Previously we showed a predominance of interleukin (IL)-10 induced by indirect allorecognition of donor human leucocyte antigen (HLA)-DR peptides, suggesting the existence of indirect alloreactive T cells displaying regulatory activity. In the present work, our objective was to characterize these regulatory T cells. We detected indirect alloproliferation of peripheral blood mononuclear cells (PBMC) from renal transplant patients, induced by donor HLA-DR peptides, dependent on IL-4 or IL-10, suggesting regulatory activity as part of the alloreactive T-cell repertoire. PBMC-derived indirect alloreactive T-cell lines were established and produced both inflammatory and regulatory cytokines. We showed that two of these T-cell lines which were able to inhibit both direct and indirect alloproliferation of another T-cell line from the same patient presented a CD4+CD25 +Foxp3+ T-cell population. These data support the idea that indirect alloreactive T cells may also have regulatory activity and may contribute to the maintenance of the human renal allograft.