RESUMEN
The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. Biochemical studies have demonstrated that NS5A interacts in vitro with and inhibits the IFN-induced, RNA-dependent protein kinase, PKR, and that NS5A interacts with at least one other cellular kinase. The present study describes the establishment and characterization of various stable NS5A-expressing human cell lines, and the development of a cell culture-based assay for determining the inherent IFN resistance of clinical NS5A isolates. Human epithelioid (Hela) and osteosarcoma (U2-OS) cell lines were generated that express NS5A under tight regulation by the tetracycline-dependent promoter. Maximal expression of NS5A occurred at 48 hours following the removal of tetracycline from the culture medium. The half-life of NS5A in these cell lines was between 4 to 6 hours. NS5A protein expression was localized cytoplasmically, with a staining pattern consistent with the location of the Golgi apparatus and endoplasmic reticulum. In the majority of cell lines, no obvious phenotypic changes were observed. However, three genotype 1b NS5A-expressing osteosarcoma cell lines exhibited cytopathic effect and severely reduced proliferation as a result of high-level NS5A expression. Full-length NS5A protein isolated from a genotype 1b IFN-nonresponsive patient (NS5A-1b) was capable of rescuing encephalomyocardititis virus replication during IFN challenge up to 40-fold, whereas a full-length NS5A-1a and an interferon sensitivity determining region (ISDR) deletion mutant (NS5A-1a-triangle upISDR) isolated from a genotype 1a IFN-nonresponsive patient showed no rescue activity. The NS5A-1b and NS5A-1a proteins also rescued vesicular stomatitis virus replication during IFN treatment by two- to threefold. These data cummulatively suggest that NS5A expression alone can render cells partially resistant to the effects of IFN against IFN-sensitive viruses, and that in some systems, these effects may be independent of the putative ISDR. A scenario is discussed in which the NS5A protein may employ multiple strategies contributing to IFN resistance during HCV infection.
Asunto(s)
Interferones/farmacología , ARN Polimerasa Dependiente del ARN/inmunología , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Secuencia de Aminoácidos , Western Blotting , División Celular , Farmacorresistencia Microbiana , Virus de la Encefalomiocarditis/efectos de los fármacos , Virus de la Encefalomiocarditis/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Viral de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Datos de Secuencia Molecular , Tetraciclina/farmacología , Transfección , Células Tumorales Cultivadas , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Virus de la Estomatitis Vesicular Indiana/fisiología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Ensayo de Placa Viral/métodosRESUMEN
BACKGROUND: Chronic infection with hepatitis C virus (HCV) is associated with progressive liver damage, including the development of cirrhosis and hepatocellular carcinoma, and HCV is a leading cause of liver dysfunction worldwide. The current therapy for chronic HCV infection, interferon-alpha (IFN), is effective in a minority of HCV-infected patients. Several studies have demonstrated a correlation between therapeutic outcome and the amino acid sequence of a small region of the HCV non-structural 5A (NS5A) gene product. It has been suggested that this region, termed the interferon sensitivity-determining region (ISDR), may mediate IFN resistance by directly interacting with one or more cellular proteins associated with the IFN-mediated antiviral response. OBJECTIVES: In an attempt to define the molecular mechanism by which the NS5A protein and the ISDR might contribute to HCV resistance to IFN, we examined whether NS5A could regulate the IFN-induced protein kinase, PKR, a primary mediator of the IFN-induced antiviral response. STUDY DESIGN: Multiple approaches, including in vitro assays using recombinant proteins, the transfection of recombinant clones into cultured cells, and in vivo studies in yeast, were used to examine the interaction of NS5A with PKR, as well as the functional significance of the interaction. An ISDR deletion mutant was prepared to evaluate the importance of the ISDR in mediating the NS5A-PKR interaction and the requirement of this region for PKR inhibition. RESULTS: NS5A repressed PKR activity through a direct interaction with the protein kinase catalytic domain. Both PKR repression and interaction required the presence of the ISDR. CONCLUSIONS: Inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN. Thus,therapeutic strategies designed to block the NS5A-PKR interaction may increase the efficacy of IFN therapy in HCV-infected individuals.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Microbiana/genética , Hepacivirus/efectos de los fármacos , Interferón-alfa/farmacología , Proteínas no Estructurales Virales/farmacología , eIF-2 Quinasa/antagonistas & inhibidores , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatitis C/tratamiento farmacológico , Humanos , Proteínas no Estructurales Virales/genética , eIF-2 Quinasa/genéticaRESUMEN
The interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) mediates the antiviral and antiproliferative actions of IFN, in part, via its translational inhibitory properties. Previous studies have demonstrated that PKR forms dimers and that dimerization is likely to be required for activation and/or function. In the present study we used multiple approaches to examine the modulation of PKR dimerization. Deletion analysis with the lambda repressor fusion system identified a previously unrecognized site involved in PKR dimerization. This site comprised amino acids (aa) 244 to 296, which span part of the third basic region of PKR and the catalytic subdomains I and II. Using the yeast two-hybrid system and far-Western analysis, we verified the importance of this region for dimerization. Furthermore, coexpression of the 52-aa region alone inhibited the formation of full-length PKR dimers in the lambda repressor fusion and two-hybrid systems. Importantly, coexpression of aa 244 to 296 exerted a dominant-negative effect on wild-type kinase activity in a functional assay. Due to its role as a mediator of IFN-induced antiviral resistance, PKR is a target of viral and cellular inhibitors. Curiously, PKR aa 244 to 296 contain the binding site for a select group of specific inhibitors, including the cellular protein P58IPK. We demonstrated, utilizing both the yeast and lambda systems, that P58IPK, a member of the tetratricopeptide repeat protein family, can block kinase activity by preventing PKR dimerization. In contrast, a nonfunctional form of P58IPK lacking a TPR motif did not inhibit kinase activity or perturb PKR dimers. These results highlight a potential mechanism of PKR inhibition and define a novel class of PKR inhibitors. Finally, the data document the first known example of inhibition of protein kinase dimerization by a cellular protein inhibitor. On the basis of these results we propose a model for the regulation of PKR dimerization.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Represoras/farmacología , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/metabolismo , Sitios de Unión , Análisis Mutacional de ADN , Dimerización , Interferones/farmacología , Modelos Biológicos , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , ARN Bicatenario/metabolismo , Proteínas Recombinantes de Fusión , Proteínas Represoras/genética , Eliminación de Secuencia , Proteínas Virales , Proteínas Reguladoras y Accesorias Virales , eIF-2 Quinasa/genéticaRESUMEN
Hepatitis C virus (HCV) is the major cause of non-A non-B hepatitis and a leading cause of liver dysfunction worldwide. While the current therapy for chronic HCV infection is parenteral administration of type 1 interferon (IFN), only a fraction of HCV-infected individuals completely respond to treatment. Previous studies have correlated the IFN sensitivity of strain HCV-1b with mutations within a discrete region of the viral nonstructural 5A protein (NS5A), termed the interferon sensitivity determining region (ISDR), suggesting that NS5A may contribute to the IFN-resistant phenotype of HCV. To determine the importance of HCV NS5A and the NS5A ISDR in mediating HCV IFN resistance, we tested whether the NS5A protein could regulate the IFN-induced protein kinase, PKR, a mediator of IFN-induced antiviral resistance and a target of viral and cellular inhibitors. Using multiple approaches, including biochemical, transfection, and yeast genetics analyses, we can now report that NS5A represses PKR through a direct interaction with the protein kinase catalytic domain and that both PKR repression and interaction requires the ISDR. Thus, inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN. Finally the inhibition of the PKR protein kinase, by NS5A is the first described function for this HCV protein.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Interferón Tipo I/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas no Estructurales Virales/fisiología , Sitios de Unión , Catálisis , Farmacorresistencia Microbiana , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas no Estructurales Virales/genética , eIF-2 QuinasaRESUMEN
In order to characterize macaque T-lymphocyte subsets, we used a chromophore from a dinoflagellate, peridinin chlorophyll A protein (PerCP), which, like fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE), can be excited by a 488-nm laser and emits light at 670 nm without spectral overlap with FITC and PE. Mouse monoclonal antibodies were conjugated with FITC, PE, and PerCP to detect CD4+ and CD8+ cells in macaque peripheral blood lymphocytes (PBL) subsets before and after activation and in nonactivated thymocytes. Resting and activated macaque blood CD4+ T-cells could be clearly delineated into discrete subsets with either CD28, CD45RA, or CD45RO as a second marker and CD26, CD29, CD44, or CD69 as a third marker. CD8+ cells were further subdivided by expression of similar combinations of markers. A subset of CD8+ CD28- T-cells in blood expressed the activation marker CD69, suggesting that they were already activated. Virtually all CD4+CD8+, CD4+CD8-, and CD4-CD8+ macaque thymocytes expressed CD2, CD3, and CD18 and not CD25, CD44, or CD45O, but macaque thymocyte subpopulations did differ in their expression of CD28 and CD29. The expression of T-cell receptor (TCR) subgroups on macaque PBL and thymocytes was analyzed before and after activation with staphylococcal enterotoxins (superantigens). The pattern of T-cell variable-region expression in macaques was similar to that seen in humans, with a high frequency of T cells expressing V beta 8. After superantigen stimulation, only minor changes in TCR V beta expression were detectable in PBL. A dramatic increase in V beta 8 expression was seen after stimulation of macaque thymus with staphylococcal enterotoxin D (SE-D), a minor increase after toxic shock syndrome toxin 1 (TSST-1) stimulation, and a simultaneous decrease in V beta 6 levels.
Asunto(s)
Receptores de Antígenos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Relación CD4-CD8 , Linfocitos T CD4-Positivos/inmunología , Separación Celular , Enterotoxinas/farmacología , Citometría de Flujo , Colorantes Fluorescentes , Genes de Inmunoglobulinas/inmunología , Humanos , Activación de Linfocitos/inmunología , Macaca nemestrina , Staphylococcus aureus , Linfocitos T Reguladores/inmunologíaRESUMEN
Our laboratory has undertaken an analysis of cellular and viral gene expression in CD4+ human lymphoid cell lines infected by the human and simian immunodeficiency viruses, HIV-1 and SIV/Mne, respectively. The purpose of the current study was to: (i) examine the effects of SIV/Mne infection on host macromolecular synthesis and compare the results to those in the HIV-1 system; and (ii) investigate the mechanisms responsible for the restriction of SIV/Mne infection in CD4 positive lymphoid cells which are readily infected by HIV-1. First we determined that SIV does not impose selective blocks on host macromolecular synthesis, unlike HIV-1, which induces both the selective inhibition of cellular protein synthesis and the degradation of cellular mRNAs (Agy, M., Wambach, M., Foy, K., and Katze, M. G., 1990, Virology 177, 251-258). No such selective reduction in cellular mRNA stability or protein synthesis was observed in cells infected by SIV/Mne. Additional differences between SIV and HIV-1 were observed using a panel of CD4+ human cell lines. While HIV-1-infected all cell lines. SIV/Mne efficiently infected only the MT-4, C8166, and 174 x CEM cell lines. Repeated efforts to infect CEM or Jurkat cells were unsuccessful as determined by PCR analysis of viral DNA. HUT 78 cells supported a limited infection detectable only by PCR analysis. These data suggest the block in viral replication in the nonsusceptible cell lines is at an early step. Interestingly, all the SIV susceptible cells were virally transformed, C8166 and MT-4 by HTLV-1, and 174 x CEM by Epstein-Barr virus. Furthermore FACS analysis revealed that all susceptible cells expressed two B cell associated markers, B7/BB1 and CD40. These observations taken together highlight differences between the HIV and SIV viruses, and suggest that for efficient replication, SIV/Mne may require an additional cell surface molecule, cofactors provided by transforming viruses, or a complex interplay between the two.
Asunto(s)
Linfocitos T CD4-Positivos/microbiología , Genes Virales , VIH-1/genética , Virus de la Inmunodeficiencia de los Simios/genética , Proteínas Estructurales Virales/genética , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Secuencia de Bases , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Expresión Génica , VIH-1/crecimiento & desarrollo , Humanos , Macaca nemestrina , Datos de Secuencia Molecular , Virus de la Inmunodeficiencia de los Simios/crecimiento & desarrollo , Transformación Genética , Replicación ViralRESUMEN
HIV and the related simian immunodeficiency virus (SIV), which causes AIDS in macaques, infect only a small percentage of CD4+ lymphocytes at any point during the disease. We have identified three distinct cellular phenotypes within the CD4+ subpopulation in macaques, based on cell surface expression of CD44 and CD45R, which putatively represent successive stages of postthymic proliferation and functional maturation. Two of these subsets, CD44hi CD45R+, which contained virtually all circulating cells in cycle, and CD44hi CD45R-, which was noncycling and has been linked to immunologic memory, were selectively depleted in SIV-infected animals at an asymptomatic stage of disease. To test whether SIV infection was restricted to cells with this phenotype in vivo, we used the polymerase chain reaction to sensitively detect SIV DNA in purified subpopulations of CD4+ lymphocytes. We found that SIV exclusively infected blood lymphocytes expressing high levels of CD44. Within this subset infection occurred not only in the fraction containing actively proliferating cells (CD45R+), but also in resting, putative memory cells (CD45R-). These data directly demonstrate that cellular maturation stages of normal postthymic T lymphocyte differentiation are important factors in permitting lentivirus infection in vivo, and that noncycling, memory T cells may be a reservoir for SIV.
Asunto(s)
Linfocitos T CD4-Positivos/microbiología , Memoria Inmunológica , Virus de la Inmunodeficiencia de los Simios/crecimiento & desarrollo , Animales , Antígenos de Diferenciación/análisis , Linfocitos T CD4-Positivos/citología , ADN Viral/análisis , Genes gag/genética , Antígenos Comunes de Leucocito , Macaca nemestrina , Reacción en Cadena de la Polimerasa , Receptores Mensajeros de Linfocitos , Virus de la Inmunodeficiencia de los Simios/genéticaRESUMEN
Previous studies had tested the susceptibility of two macaque species, Macaca nemestrina and M. mulatta, to infection with the primate lymphotropic lentivirus SIVmne. In this report we describe the results obtained after infecting eleven M. fascicularis with SIVmne. Six of the animals had previously been immunized with a recombinant vaccinia virus expressing the envelope gene of HIV-1. All eleven animals became seropositive. To date ten animals have died 43 to 155 weeks post infection of an AIDS-like disease.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Immunoblotting , Macaca fascicularis , Serotipificación , Síndrome de Inmunodeficiencia Adquirida del Simio/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Subgrupos de Linfocitos T/inmunología , Vacunas Sintéticas/inmunología , Virus Vaccinia , Vacunas Virales/inmunologíaRESUMEN
Single-cell clones, designated E11S, C11R, and A1S, were obtained from the HuT-78 T cell line persistently infected with an isolate of Simian immunodeficiency virus (SIV), SIV/Mne. The infected clones, unlike uncloned uninfected HuT-78 cells, no longer expressed the CD4 marker and, after their CD3 receptors were cross-linked, had dramatically reduced intracellular free calcium ([Ca2+]i) responses. In one clone, E11S, the unresponsiveness was not limited to the inositol phospholipid pathway of signaling since a reduction in CD3-mediated activation of protein tyrosine kinase-dependent phosphorylation also was evident in this SIV-infected clone. These results led us to test whether T lymphocytes from animals infected with SIV had defective [Ca2+]i responses prior to detectable changes in CD4 levels or lymphadenopathy. The [Ca2+]i responses to both CD3 mAb and CD2 mAb were 10-50% less in T cells from Walter Reed stage 2 animals than in healthy controls. This anergy was more pronounced in chronically infected animals progressing to Walter Reed stage 3/4. The responses of these animals could not be augmented even when combinations of CD3 and CD4 mAb were used. Both CD4+CD44lo T cells, which are not infected with SIV, and the CD4+CD44hi T cell subset, previously shown to be the reservoir of SIV infection in blood, had pronounced defective responses to CD3 mAb. Similarly, both CD4+ and CD8+ T cells were consistently unresponsive in chronically infected animals, again implying that an indirect mechanism, rather than SIV infection per se, may be responsible for this immune dysfunction.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Antígenos CD2 , Complejo CD3 , Antígenos CD4 , Antígenos CD8 , Calcio/metabolismo , Línea Celular , Humanos , Macaca nemestrina , Receptores de Antígenos de Linfocitos T , Receptores Inmunológicos , Receptores Mensajeros de Linfocitos , Transducción de Señal/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismo , Subgrupos de Linfocitos T/metabolismoRESUMEN
Although all CD4+ cells theoretically are at risk for infection by human immunodeficiency viruses or the related simian immunodeficiency viruses found in Old World monkeys, only a small proportion of CD4+ lymphocytes from infected individuals have detectable virus. This suggests that immunodeficiency viruses may replicate predominantly in a minor subset or activated form of CD4+ T cells, a possibility we examined in macaques infected with a simian immunodeficiency virus isolate, SIV/Mne. Macaque CD4+ lymphocytes could be divided into two subtypes that differed in their level [high (hi) or low (lo)] of expression of a class of heterotypic adhesion receptors (HARs). In blood from animals infected with SIV/Mne, HARhi CD4+ T cells were lost selectively compared to HARlo CD4+ cells and, when cultured, exhibited 50-fold more recoverable reverse transcriptase activity. The HARhi CD4+ subset was also markedly more susceptible to productive infection following exposure to SIV/Mne in vitro. Both subsets are composed primarily of small resting lymphocytes. However, HARhi cells respond differentially to mitogenic stimulation and may thus be more likely to provide the cellular factors necessary to initiate or enhance virus replication. Thus, HAR expression may prove useful both as a prognostic indicator in immunodeficiency virus infection and as a tool to analyze pathogenesis of immunodeficiency viruses.
Asunto(s)
Receptores Inmunológicos/análisis , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T Colaboradores-Inductores/microbiología , Replicación Viral , Animales , Antígenos de Diferenciación de Linfocitos T/análisis , Adhesión Celular , Ciclo Celular , Supervivencia Celular , Células Cultivadas , ADN/análisis , Citometría de Flujo , Activación de Linfocitos , Macaca , Mitógenos/farmacología , Fenotipo , ADN Polimerasa Dirigida por ARN/metabolismo , Receptores Mensajeros de Linfocitos , Infecciones por Retroviridae/inmunología , Linfocitos T Colaboradores-Inductores/análisisRESUMEN
The immunologic status of patients with prior poliomyelitis was studied using two-color flow cytometric analyses. Ten lymphocyte subsets including subsets of CD4+ T cells, CD8+ T cells, B cells, and natural killer cells were examined. Eighteen patients presented with clinical symptoms compatible with the postpolio syndrome. This group was compared with 18 asymptomatic postpolio survivors and 22 age-matched healthy controls. The results demonstrated significant alterations in CD4+ subsets in the postpolio group as a whole when compared with normal controls. These findings reveal definite alterations in the immune status of postpolio survivors and raise the possibility that immunologic factors may contribute to late disease progression.
Asunto(s)
Poliomielitis/inmunología , Linfocitos T/clasificación , Adulto , Antígenos de Diferenciación de Linfocitos T/análisis , Separación Celular , Citometría de Flujo/métodos , Humanos , Persona de Mediana Edad , Poliomielitis/complicaciones , Valores de Referencia , Linfocitos T/inmunologíaRESUMEN
A primate lymphotropic lentivirus was isolated on Hut 78 cells after cocultivation of a lymph node from a macaque that died with malignant lymphoma. In earlier studies SIV/Mne was inoculated into 17 macaques and two baboons. All of the macaques became viremic and seropositive. Fifteen of the macaques succumbed to a classic AIDS-like disease, whereas the baboons did not become viremic. The SIV/Mne virus has now been molecularly cloned and inoculated into Macaca nemestrina and baboons. A new transmission study has been initiated to test the effects of route and dosage on disease.
Asunto(s)
Macaca/microbiología , Enfermedades de los Monos/transmisión , Papio/microbiología , Infecciones por Retroviridae/veterinaria , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Anticuerpos Antivirales/biosíntesis , Western Blotting , Clonación Molecular , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Macaca mulatta/microbiología , Macaca nemestrina/microbiología , Enfermedades de los Monos/inmunología , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/transmisión , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Viremia/veterinariaRESUMEN
A primate lymphotropic lentivirus was isolated on the human T-cell line HuT 78 after cocultivation of a lymph node from a pig-tailed macaque (Macaca nemestrina) that had died with malignant lymphoma. This isolate, originally designated M. nemestrina immunodeficiency virus (MnIV) and now classified as simian immunodeficiency virus (SIV/Mne), was inoculated intravenously into three juvenile rhesus monkeys (Macaca mulatta), three juvenile pig-tailed macaques (M. nemestrina), and two juvenile baboons (Papio cynocephalus). All six macaques became viremic by 3 weeks after inoculation, whereas neither of the baboons developed viremia. One pig-tailed macaque died at 15 weeks with suppurative peritonitis secondary to ulcerative, necrotizing colitis. Immunologic abnormalities included a marked decrease in CD4+ peripheral blood lymphocytes. Although five macaques mounted an antibody response to SIV/Mne, the animal that died at 15 weeks remained antibody negative. Three other macaques (two rhesus and one pig-tailed) died 66 to 87 weeks after inoculation after exhibiting progressive weight loss, anemia, and diarrhea. Histopathologic findings at necropsy included various manifestations of immune deficiency, nephropathy, subacute encephalitis, pancreatitis, adenocarcinoma, and lymphoid atrophy. SIV/Mne could be readily isolated from the spleens and lymph nodes of all necropsied macaques, and from the cerebrospinal fluid, brains, bone marrow, livers, and pancreas of some of the animals. SIV antigens were localized by avidin-biotin immunohistochemistry to pancreatic islet cells and to bone marrow endothelial cells. The data suggest that African baboons may be resistant to infection by SIV/Mne, whereas Asian macaques are susceptible to infection with this pathogenic primate lentivirus.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/veterinaria , Macaca/microbiología , Papio/microbiología , Retroviridae/patogenicidad , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/microbiología , Animales , Anticuerpos Antivirales/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos Virales/análisis , Médula Ósea/inmunología , Glomerulonefritis/patología , Técnicas de Inmunoadsorción , Recuento de Leucocitos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/microbiología , Macaca/inmunología , Papio/inmunología , Retroviridae/inmunología , Especificidad de la Especie , Linfocitos T/inmunologíaRESUMEN
We describe a simple colorimetric method to measure 30 to 300 microM concentrations of sulfinic acids in biologic samples. The procedure employs the coupling reaction of an aromatic diazonium salt (Ar--N = N+) with the sulfinic acids (RSOOH) to produce a colored diazosulfone derivative (Ar-N = N-SOOR), which can be selectively extracted into an organic solvent. Linearity as well as noninterference by liver homogenate, phenols, amines, and thousandfold or greater excesses of sulfate, thiol, and dimethyl sulfoxide is demonstrated. Sensitivity of the method is about 10 nmol per sample. Because methanesulfinic acid is the principal product of the action of hydroxyl radicals upon dimethyl sulfoxide, and because intact animals can tolerate dimethyl sulfoxide in millimolar concentrations, the method may prove widely useful for detecting the involvement of hydroxyl radicals in pathologic processes in vivo.