Redução da resistência à insulina após resposta virológica sustentada com agentes antivirais diretos: nem toda população melhora / Insulin resistance reduction after sustained virological response with direct acting antiviral: not every population improves

ABSTRACT BACKGROUND: Hepatitis C virus (HCV) infection is a serious public health problem, that affects approximately 170 million people worldwide. Chronic HCV infection is associated with hepatic insulin resistance and an increased risk of diabetes HCV-infected patients has been well documented. OBJECTIVE: To assess the homeostasis model assessment of insulin resistance (HOMA-IR) index in patients treated with direct acting antiviral (DAAs) medication in the sustained virological response (SVR), categorized by the presence or absence of cirrhosis. METHODS: A prospective study was conducted. Data were collected at the beginning of treatment (t-base) and in the twelfth week after the completion of treatment (t-SVR12). The inclusion criteria were presence of: HCV infection (RNA-HCV positive), age ≥18 years, completion of DAAs' therapy, and presence of diabetes with use of oral hypoglycemic agents. All samples were collected during the study period. The exclusion criteria were: presence of HBV/HIV co-infection, hepatocellular carcinoma at baseline, diabetic patients taking insulin and transplanted patients (liver/kidney). Fibrosis was assessed by hepatic elastography or biopsy (METAVIR). Cirrhosis was determined by clinical results or imaging. HOMA-IR was calculated as fasting insulin (μU/mL) × fasting glucose (mmol/L)/22.5) The patients were divided into two groups: the general study population (all patients, including the diabetic patients) and the special population (patients with normal values of HOMA-IR, which is >2.5, and without diabetes). The delta HOMA-IR value was calculated as: HOMA-IR at t-base - HOMA-IR at t-SVR12. For the descriptive statistical analysis, the paired t-test and generalized linear model assuming the log binding function were performed. A P value of < 0.05 was considered significant. RESULTS: We included 150 patients, and 75 were cirrhotic. The mean age was 55.3±9.97 and body mass index was 27.4±5.18. Twenty-two (14.67%) were diabetic patients using oral hypoglycemic agents, and 17 (11%) were cirrhotic. In the general study population, the mean glucose and HOMA-IR values increased at t-SVR12, but insulin decreased. Delta HOMA-IR was negative at t-SVR12, but there was no significant difference. Excluding diabetic patients and those with normal HOMA-IR values (<2.5), mean glucose, insulin and HOMA-IR decreased at t-SVR12. Delta HOMA-IR decreased significantly at t-SVR12 (P: 0.02). CONCLUSION: In the general population, glucose and HOMA-IR values increased at t-SVR12, but insulin decreased. In the special population, glucose, insulin, HOMA-IR and Delta HOMA-IR decreased at t-SVR12.

RESUMO CONTEXTO: A infecção pelo vírus da hepatite C (VHC) é um grave problema de saúde pública, que afeta aproximadamente 170 milhões de pessoas no mundo. A infecção crônica pelo VHC está associada à resistência à insulina hepática e a um risco aumentado de diabetes. Os doentes infetados pelo VHC foram bem documentados. OBJETIVO: Avaliar o modelo de avaliação da homeostase do índice de resistência à insulina (HOMA-IR) em pacientes tratados com medicação antiviral de ação direta na resposta virológica sustentada (RVS), categorizada pela presença ou ausência de cirrose. MÉTODOS: Foi realizado um estudo prospectivo. Os dados foram coletados no início do tratamento (t-base) e na décima segunda semana após o término do tratamento (t-RVS12). Os critérios de inclusão foram presença de: infecção pelo VHC (RNA-VHC positivo), idade ≥18 anos, conclusão da terapia de antivirais de ação direta e presença de diabetes com uso de hipoglicemiantes orais. Todas as amostras foram coletadas durante o período do estudo. Os critérios de exclusão foram: presença de coinfecção VHB/HIV, carcinoma hepatocelular no início do estudo, pacientes diabéticos em uso de insulina e pacientes transplantados (fígado/rim). A fibrose foi avaliada por elastografia hepática ou biópsia (METAVIR). A cirrose foi determinada por resultados clínicos ou exames de imagem. O HOMA-IR foi calculado como insulinemia de jejum (μU/mL) x glicemia de jejum (mmol/L) /22,5). Os pacientes foram divididos em dois grupos: a população geral do estudo (todos os pacientes, incluindo os diabéticos) e a população especial (pacientes com valores normais de HOMA-IR, que é <2,5 e sem diabetes). O valor do delta HOMA-IR foi calculado como: HOMA-IR no t-base - HOMA-IR no t-RVS12. Para a análise estatística descritiva, foram utilizados o teste t pareado e o modelo linear generalizado, assumindo a função de ligação logarítmica. Um valor de P<0,05 foi considerado significativo. RESULTADOS: Foram incluídos 150 pacientes e 75 eram cirróticos. A idade média foi de 55,3±9,97 e o índice de massa corpórea foi de 27,4±5,18. Vinte e dois (14,67%) eram pacientes diabéticos em uso de hipoglicemiantes orais e 17 (11%) eram cirróticos. Na população geral do estudo, os valores médios de glicose e HOMA-IR aumentaram na t-SVR12, mas a insulina diminuiu. O delta HOMA-IR foi negativo em t-SVR12, mas não houve diferença significativa. Excluindo pacientes diabéticos e aqueles com valores normais de HOMA-IR (<2,5), a média de glicose, insulina e HOMA-IR diminuiu no t-RVS12. O delta HOMA-IR diminuiu significativamente em t-RVS12 (P: 0,02). CONCLUSÃO: Na população geral, os valores de glicose e HOMA-IR aumentaram no t-RVS12, mas a insulina diminuiu. Na população especial, glicose, insulina, HOMA-IR e delta HOMA-IR diminuíram no t-RVS12.

In vitro and in vivo inhibition of rabies virus replication by RNA interference

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

Apoptosis as pathogenic mechanism of infection with vesicular stomatitis virus: evidence in primary bovine fibroblast cultures

To determine whether fibroblasts from Blanco Orejinegro cattle, exhibit any level of resistance to infection against vesicular stomatitis virus (VSV) serotypes Indiana (VSV-I) or New Jersey (VSV-NJ), 30 fibroblast cultures were phenotyped to evaluate their resistance/susceptibility. Thirty three % of Blanco Orejinegro fibroblast cultures were classified as very resistant, 50% as resistant, and 17% as susceptible to VSV-I infection, whereas 20% were classified as very resistant, 50% as resistant and 30% as susceptible to VSV-NJ infection. Therefore, there appears to be a large variation in phenotypic polymorphism among the fibroblasts to infection by VSV. To elucidate the mechanisms responsible for this diversity, we searched for a possible relationship between resistance/ susceptibility and production of factors wi th antiviral activity; however fibroblasts did not secrete factors with antiviral activity. We examined also whether apoptosis where induced by infection and its correlation with the polymorphism of resistance/susceptibility to VSV. Using morphological analyses, hypoploidy measurements, and level of phosphatidyl serine expression, high levels of apoptosis were measured in VSV infected fibroblasts. However, no correlation exists between apoptosis and the category of resistance/susceptibility to infection, indicating that apoptosis is a pathogenic mechanism of VSV.

Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-alpha receptor

Abnormal production of interferon alpha (IFN-a) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-a. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-a inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-a receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-aB on Daudi cells, but did not block IFN-aB activity at higher concentrations (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-aB on Hep 2/c cells challenged with encephalomyocarditis virus.

Antivirugrama: ¿ciencia ficción o realidad? / Antivirugram: science fiction or reality?

El panorama creciente de infecciones virales severas, que amenazan la vida de los pacientes, la disponibilidad de técnicas de diagnósticos virológico rápido y los conocimientos patogénicos más acabados con biología molecular, han estimulado el desarrollo explosivo de fármacos antivirales. Como consecuencia nace la necesidad de incorporar a las decisiones clínicas la evaluación in vitro de algunos de estos medicamentos y la aparición de resistencia durante tratamientos prolongados, como ha sido el dramático caso de la infección por VIH/SIDA. Se revisan los antivirales licenciados para el uso médico y el grado de aplicación clínica de los estudios de susceptibilidad in vitro alcanzado en la actualidad