RESUMEN
With improved HCV therapy, challenges regarding HCV diagnosis, such as seronegative window period, false positive readings, and differentiation between recent, chronic, and resolved infections, are of increasing importance. To address these challenges an innovative device--SMARTube HIV & HCV--was used. Blood samples were tested for anti-HCV antibodies before and after incubation in the SMARTube, which promotes the in vitro stimulation of in vivo HCV primed lymphocytes, thus enhancing levels of anti-HCV antibodies. Comparing antibody levels, in concordant samples before and after SMARTube, yielded the Stimulation Index (SI). Among 5888 fresh blood samples, from various populations and regions worldwide, 641 were seropositive using plasma, while SMARTube processing (yielding enriched plasma, termed SMARTplasma) enabled diagnosis of 10 additional carriers in high-risk cohorts, that is, earlier detection. Using SMARTplasma eliminated all false positive results, using the current assays. In addition we show that SI calculation may serve as an important tool for differentiating between those who recently seroconverted, carriers of long-term infection, and those who have cleared the virus. SMARTube and the SI could lead to better, more informative diagnosis of HCV infections and play an important role in changing the way we treat both the infected individuals and the epidemic as a whole.
Asunto(s)
Anticuerpos contra la Hepatitis C/sangre , Hepatitis C Crónica/diagnóstico , Pruebas Inmunológicas/métodos , Formación de Anticuerpos , Antivirales/uso terapéutico , Portador Sano/diagnóstico , Portador Sano/virología , Diagnóstico Precoz , Reacciones Falso Positivas , Hepacivirus , Humanos , Pruebas Inmunológicas/instrumentación , Linfocitos/inmunología , Linfocitos/virología , Sensibilidad y Especificidad , Índice de Severidad de la EnfermedadRESUMEN
Hypoxia is an important factor that elicits numerous physiological and pathological responses. One of the major gene expression programs triggered by hypoxia is mediated through hypoxia-responsive transcription factor hypoxia-inducible factor 1 (HIF-1). Here, we report the identification and cloning of a novel HIF-1-responsive gene, designated RTP801. Its strong up-regulation by hypoxia was detected both in vitro and in vivo in an animal model of ischemic stroke. When induced from a tetracycline-repressible promoter, RTP801 protected MCF7 and PC12 cells from hypoxia in glucose-free medium and from H(2)O(2)-triggered apoptosis via a dramatic reduction in the generation of reactive oxygen species. However, expression of RTP801 appeared toxic for nondividing neuron-like PC12 cells and increased their sensitivity to ischemic injury and oxidative stress. Liposomal delivery of RTP801 cDNA to mouse lungs also resulted in massive cell death. Thus, the biological effect of RTP801 overexpression depends on the cell context and may be either protecting or detrimental for cells under conditions of oxidative or ischemic stresses. Altogether, the data suggest a complex type of involvement of RTP801 in the pathogenesis of ischemic diseases.
Asunto(s)
Apoptosis/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Diferenciación Celular , Clonación Molecular , Proteínas de Unión al ADN/química , Humanos , Peróxido de Hidrógeno/farmacología , Hipoxia/genética , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Hibridación in Situ , Liposomas/metabolismo , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Datos de Secuencia Molecular , Células PC12 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras , Homología de Secuencia de Aminoácido , Accidente Cerebrovascular/genética , Factores de Transcripción/química , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
cDNA microarray hybridization was used in an attempt to identify novel genes participating in cellular responses to prolonged hypoxia. One of the identified novel genes, designated Hi95 shared significant homology to a p53-regulated GADD family member PA26. In addition to its induction in response to prolonged hypoxia, the increased Hi95 transcription was observed following DNA damage or oxidative stress, but not following hyperthermia or serum starvation. Whereas induction of Hi95 by prolonged hypoxia or by oxidative stress is most likely p53-independent, its induction in response to DNA damaging treatments (gamma- or UV-irradiation, or doxorubicin) occurs in a p53-dependent manner. Overexpression of Hi95 full-length cDNA was found toxic for many types of cultured cells directly leading either to their apoptotic death or to sensitization to serum starvation and DNA damaging treatments. Unexpectedly, conditional overexpression of the Hi95 cDNA in MCF7-tet-off cells resulted in their protection against cell death induced by hypoxia/glucose deprivation or H(2)O(2). Thus, Hi95 gene seems to be involved in complex regulation of cell viability in response to different stress conditions.