RESUMEN
Solid phase immunoassays (SPI) are now routinely used to detect HLA antibodies. However, the flow cytometric crossmatch (FCXM) remains the established method for assessing final donor-recipient compatibility. Since 2005 we have followed a protocol whereby the final allocation decision for renal transplantation is based on SPI (not the FCXM). Here we report long-term graft outcomes for 508 consecutive kidney transplants using this protocol. All recipients were negative for donor-specific antibody by SPI. Primary outcomes are graft survival and incidence of acute rejection within 1 year (AR <1 year) for FCXM+ (n = 54) and FCXM- (n = 454) recipients. Median follow-up is 7.1 years. FCXM+ recipients were significantly different from FCXM- recipients for the following risk factors: living donor (24% vs. 39%, p = 0.03), duration of dialysis (31.0 months vs. 13.5 months, p = 0.008), retransplants (17% vs. 7.3%, p = 0.04), % sensitized (63% vs. 19%, p = 0.001), and PRA >80% (20% vs. 4.8%, p = 0.001). Despite these differences, 5-year actual graft survival rates are 87% and 84%, respectively. AR <1 year occurred in 13% FCXM+ and 12% FCXM- recipients. Crossmatch status was not associated with graft outcomes in any univariate or multivariate model. Renal transplantation can be performed successfully, using SPI as the definitive test for donor-recipient compatibility.
Asunto(s)
Tipificación y Pruebas Cruzadas Sanguíneas , Rechazo de Injerto/diagnóstico , Asignación de Recursos para la Atención de Salud/métodos , Prueba de Histocompatibilidad/métodos , Isoanticuerpos/inmunología , Trasplante de Riñón , Obtención de Tejidos y Órganos , Linfocitos B/inmunología , Femenino , Citometría de Flujo/métodos , Estudios de Seguimiento , Rechazo de Injerto/prevención & control , Supervivencia de Injerto , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Factores de Riesgo , Donantes de TejidosRESUMEN
A novel HLA allele, DRB3*01:15, was likely derived via cis-gene conversion.
Asunto(s)
Conversión Génica , Cadenas HLA-DRB1/genética , Cadenas HLA-DRB3/genética , Alelos , Población Negra , HumanosAsunto(s)
Anticuerpos Antivirales/sangre , Inmunización/métodos , Virus de la Hepatitis Murina/inmunología , ARN Polimerasa Dependiente del ARN/inmunología , Vacunas de ADN/inmunología , Proteínas Virales/inmunología , Animales , Antígenos Virales/inmunología , Línea Celular , Células HeLa , Humanos , Virus de la Hepatitis Murina/enzimología , Virus de la Hepatitis Murina/genética , Plásmidos/genética , ARN Polimerasa Dependiente del ARN/genética , Conejos , Vacunas de ADN/administración & dosificación , Proteínas Virales/genéticaRESUMEN
Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Membranas Intracelulares/metabolismo , Virus de la Hepatitis Murina/enzimología , ARN Viral/biosíntesis , Proteínas Virales/metabolismo , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Cloruros/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , ARN Polimerasas Dirigidas por ADN/genética , Células HeLa , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Leupeptinas/farmacología , Ratones , Conejos , Coloración y Etiquetado , Factores de Tiempo , Proteínas Virales/genética , Compuestos de Zinc/farmacologíaRESUMEN
The RNA polymerase gene of the murine coronavirus mouse hepatitis virus (MHV) encodes a polyprotein of greater than 750 kDa. The amino-terminal cleavage product of the MHV polymerase polyprotein, p28, has been shown to be cleaved from the polyprotein by the virus-encoded protease PCP-1. We aim to identify the MHV-JHM proteolytic products downstream of p28 and to determine which viral proteinase domains are responsible for generating each of them. To this end, we have generated antisera directed at specific MHV-JHM ORF1a regions and have used these antisera to identify six viral proteins, representing a large portion of ORF1a, from MHV-JHM-infected cells. These proteins include p28, p72, p65, p250, p210, and p27.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Virus de la Hepatitis Murina/enzimología , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Endopeptidasas/metabolismo , Ratones , Precursores de Proteínas/metabolismo , ConejosRESUMEN
The replicase of mouse hepatitis virus strain JHM (MHV-JHM) is encoded by two overlapping open reading frames, ORF1a and ORF1b, which are translated to produce a 750-kDa precursor polyprotein. The polyprotein is proposed to be processed by viral proteinases to generate the functional replicase complex. To date, only the MHV-JHM amino-terminal proteins p28 and p72, which is processed to p65, have been identified. To further elucidate the biogenesis of the MHV-JHM replicase, we cloned and expressed five regions of ORF1a in bacteria and prepared rabbit antisera to each region. Using the immune sera to immunoprecipitate radiolabeled proteins from MHV-JHM infected cells, we determined that the MHV-JHM ORF1a is initially processed to generate p28, p72, p250, and p150. Pulse-chase analysis revealed that these intermediates are further processed to generate p65, p210, p40, p27, the MHV 3C-like proteinase, and p15. A putative replicase complex consisting of p250, p210, p40, p150, and a large protein (> 300 kDa) coprecipitate from infected cells disrupted with NP-40, indicating that these proteins are closely associated even after initial proteolytic processing. Immunofluorescence studies revealed punctate labeling of ORF1a proteins in the perinuclear region of infected cells, consistent with a membrane-association of the replicase complex. Furthermore, in vitro transcription/translation studies of the MHV-JHM 3Cpro and flanking hydrophobic domains confirm that 3C protease activity is significantly enhanced in the presence of canine microsomal membranes. Overall, our results demonstrate that the MHV-JHM ORF1a polyprotein: (1) is processed into more than 10 protein intermediates and products, (2) requires membranes for efficient biogenesis, and (3) is detected in discrete membranous regions in the cytoplasm of infected cells.
Asunto(s)
Virus de la Hepatitis Murina/metabolismo , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Animales , Anticuerpos , Células Cultivadas , Citoplasma/metabolismo , Perros , Técnica del Anticuerpo Fluorescente Indirecta , Membranas Intracelulares/metabolismo , Ratones , Microsomas/metabolismo , Virus de la Hepatitis Murina/química , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina/métodos , Transcripción Genética , Proteínas Virales/aislamiento & purificaciónRESUMEN
The RNA polymerase gene of murine coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. The amino-terminal product of the MHV polymerase polyprotein, p28, is generated by cleavage of the polyprotein by PCP-1. To identify the viral products downstream of p28, we generated a fusion-protein specific antiserum directed against the region adjacent to p28 and used the antiserum to detect virus-specific proteins from MHV-JHM infected cells. When this antiserum was used to immunoprecipitate radiolabeled proteins from MHV-JHM infected cell lysates, virus-specific proteins of 72 and 65 kDa were detected. Furthermore, pulse and chase experiments demonstrated that p72 is likely a precursor to the mature protein product, p65. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5'-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. We also cloned and expressed the 72 kDa region immediately downstream from p28, and tested the ability of in vitro translated PCP-1 and PCP-2 to cleave p72 to p65 in trans. Our results indicate that neither viral proteinase domain PCP-1 nor PCP-2 is capable of cleavage of p72 to produce p65 in vitro. The role of MHV proteinases in the processing of p72 and p65 is discussed.