Durante la infección por el virus de la hepatitis C (VHC), los anticuerpos específicos aparecen varias semanas posterior a la exposición, van dirigidos contra las diversas proteínas del virus incluyendo anticuerpos contra la envoltura viral (E2)y la proteína no estructural 2 (NS2). En este trabajo se diseñó un ensayo casero de ELISA que incorpora, además de NS3, NS5a, NS5b y el core, a las proteínas E2 y NS2 del VHC en sustitución de NS4, con el fin de evaluar su capacidad diagnóstica en comparación con un estuche comercial de 4ta generación. La validación de la prueba casera demostró una especificidad y sensibilidad similar a las obtenidas con el estuche comercial de 4ta generación (Biokit©), con un índice kappa igual a 0,969, al compararse con el mismo. Esto sugiere que la prueba diseñada podría utilizarse de manera segura para la detección de anticuerpos VHC específicos de tipo IgG para el diagnóstico de la hepatitis C y constituirse como una alternativa de producción nacional más económica.

During hepatitis C (HCV) infection specific antibodies appear several weeks after exposure, including viral envelope (E2) and non-structural protein 2 ( NS2). In this work we designed an in-house ELISA assay, that incorporate beside NS3, NS5a, NS5b and core, the HCVE2 and NS2 proteins in substitution of NS4, in order to evaluate its diagnostic utility as compared to a fourth generation commercial kit. The in-house test demonstrated a specificity and sensitivity similar to those obtained with the commercial kit, with a kappa index equal to 0.969, when it was compared with the 4th generation commercial kit (BioelisaBiokit ©), suggesting that our test could be used for the diagnosis of specific IgG antibodies detection against hepatitis C and to become a more economic national alternative.

Foram estudadas as respostas cronotrópicas cardíacas à manobra de Valsalva e ao exercício dinâmico de vinte pacientes chagásicos com função ventricular esquerda normal e sem alterações da contractilidade segmentar por ecocardiografia bidimensional. O aumento absoluto da frequência cardíaca dos pacientes (Δ = 21,5 ± 10 bpm, M ± DP) durante a manobra de Valsalva foi significativamente menor quando se comparava ao grupo controle (Δ = 31,30 ± 70, p = 0,03). A frequência cardíaca mínima (58,24 ± 8,90 vs 62,80 ± 10, p = 0,68) e a diminuição da frequência cardíaca absoluta no final da manobra (Δ = 38,30 ± 13 vs Δ = 31,47 ± 17, p = 0,10) não foram diferentes em comparação com o grupo controle. A aceleração inicial da frequência cardíaca durante o exercício dinâmico (Δ = 12 ± 7,55 vs Δ = 19 ± 7,27, p = 0,01) também foi menor, mas a recuperação da frequência cardíaca, durante os primeiros dez segundos, foi maior no grupo sero-positivos [mediana:14 (intervalo interquartil: 9,75-17,50) vs 5 (0 - 8,75), p = 0,001]. Os níveis séricos de auto-anticorpos muscarínicos cardíacos foram significativamente maiores nos pacientes chagásicos do que no grupo controle [(mediana: 34,58 densidade óptica (intervalo interquartil 17 - 46,5) vs (mediana: 0, intervalo interquartil 0 - 22,25) p = 0,001] e a correlação é significativa e direta (r = 0,68, p = 0,002) com o início da recuperação da frequência cardíaca durante o exercício dinâmico. Os resultados desta investigação sugerem que indiretamente, os auto-anticorpos muscarínicos cardíacos, podem ter ação agonista positiva sobre o controle parassimpático da frequência cardíaca dos pacientes chagásicos.

We have studied the cardiac chronotropic responses to the Valsalva maneuver and to dynamic exercise of twenty chronic chagasic patients with normal left ventricular function and no segmental wall abnormalities by two-dimensional echocardiogram. The absolute increase in heart rate of the patients (Δ = 21.5 ± 10 bpm, M±SD) during the maneuver was significantly diminished when compared to controls (Δ = 31.30 ± 70, M±SD, p = 0.03). The minimum heart rate (58.24 ± 8.90 vs. 62.80 ± 10, p = 0.68) and the absolute decrease in heart rate at the end of the maneuver (Δ = 38.30 ± 13 vs. Δ = 31.47 ± 17, p = 0.10) were not different from controls. The initial heart rate acceleration during dynamic exercise (Δ = 12 ± 7.55 vs. Δ = 19 ± 7.27, M±SD, p = 0.01) was also diminished, but the heart rate recovery during the first ten seconds was more prominent in the sero-positive patients (Median: 14, Interquartile range: (9.75-17.50 vs. 5(0-8.75, p = 0.001). The serum levels of muscarinic cardiac auto-antibodies were significantly higher in the chagasic patients (Median: 34.58, Interquartile Range: 17-46.5, Optical Density) than in controls (Median: 0, Interquartile Range: 0-22.25, p = 0.001) and correlated significantly and directly (r = 0.68, p = 0.002) with early heart rate recovery during dynamic exercise. The results of this investigation indirectly suggest that, the cardiac muscarinic auto-antibodies may have positive agonist effects on parasympathetic heart rate control of chagasic patients.

La infección crónica por el virus de la Hepatitis C ocurre en 80% de los casos y conduce eventualmente a cirrosis y carcinoma hepatocelular. Se ha descrito que una respuesta inmunitaria adaptativa deficiente pudiera contribuir a la persistencia viral. Dicha alteración pudiera ser atribuida a defectos en señales de co-estimulación durante la activación de los linfocitos CD4+. En este estudio exploramos la respuesta de esta población linfoide en presencia del antígeno del core del VHC asociado con la co-estimulación vía CD28, midiendo la expresión de moléculas de superficie: CD69, CD25, CD40L y de citocinas intracitoplasmáticas tales como: IFN-γ, TGF-ß e IL-10. El análisis fue realizado en individuos infectados crónicos, familiares contactos y controles sanos. El antígeno del core del VHC indujo la expresión de CD40L en los linfocitos CD4+ de familiares contactos, a diferencia de lo observado en paciente infectados crónicos e individuos controles sanos. Al asociar la estimulación vía CD28, los pacientes infectados con VHC incrementaron la expresión de CD69 e IFN-γ, sugiriendo un déficit en la señalización a través de esta vía de coestimulación. Adicionalmente, se observó un incremento en la producción de TGF-ß en los pacientes infectados en respuesta al antígeno, el cual fue corregido al coestimular vía CD28. Nuestros resultados pueden ayudar a comprender los mecanismos de tolerancia generados durante la infección crónica por el VHC y ofrecer una aproximación que pudiera contribuir al diseño de estrategias terapéuticas para el control de la infección crónica y al desarrollo de inmunidad protectora contra el virus.

Hepatitis C chronic infection occurs in 80% of the cases and eventually leads to cirrhosis and hepatocellular carcinoma. A deficient adaptive immune response has been described during chronic infection which contributes to viral persistence. This altered T cell response could be associated to deficient costimulation signals during priming of T cells. We have conducted an in vitro study to explore the activation phenomenon of CD4+ T cells focusing on costimulation via the CD28 receptor, associated to stimulation with purified Hepatitis C (HCV) core antigen. Our study involved the induction of CD69, CD25 and CD40L activation receptors, along with detection of intracellular cytokines such as IFN-γ, TGF-ß and IL-10. Analysis was performed in chronically HCV infected patients, intrafamilial members of HCV-infected patients and healthy individuals. HCV core antigen induced CD40L expression in CD4+ cells from intrafamilial members, in contrast to chronically infected patients and control individuals. Association of CD28 crosslinking increased CD69 and IFN-γ expression in chronically infected patients, suggesting a detriment in this signaling pathway. Additionally, an increased TGF-ß expression was observed in CD4+ cells from HCV-infected patients, which was corrected by addition of CD28 crosslinking. Our results may contribute to understand the underlying mechanism of T cell tolerance against HCV during chronic infection, and to provide new targets for the designing of therapeutic strategies to control the infection and to offer protective immunity against the virus.