Asunto(s)
Ácidos y Sales Biliares/sangre , Animales , Cromatografía Líquida de Alta Presión , Perros , Haplorrinos , Humanos , Codorniz , Conejos , Ratas , Especificidad de la Especie , PorcinosRESUMEN
Antigenic variants of pseudorabies virus (PRV) containing mutations in a viral glycoprotein with a molecular weight of 82,000 (gIII) were isolated by selecting for resistance to a complement-dependent neutralizing monoclonal antibody (MCA82-2) directed against gIII. These mutants were completely resistant to neutralization with MCA82-2 in the presence of complement. Two mutants selected for further studies either did not express gIII or expressed an improperly processed form of the glycoprotein. The mutations were also associated with an altered plaque morphology (syncytium formation). The gIII gene was mapped by marker rescue of a gIII- mutant with cloned restriction enzyme fragments to the long unique region of the PRV genome between 0.376 and 0.383 map units. This corresponds to the map location of a glycoprotein described by Robbins et al. (J. Mol. Appl. Gen. 2:485-496, 1984). Since gIII is nonessential for viral replication in cell culture and has several other characteristics in common with the herpes simplex virus glycoprotein gC, gIII may represent the PRV equivalent to herpes simplex virus gC.
Asunto(s)
Herpesvirus Suido 1/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Células Cultivadas , Mapeo Cromosómico , Clonación Molecular , ADN Viral/genética , Prueba de Complementación Genética , Glicoproteínas/genética , Glicoproteínas/inmunología , Herpesvirus Suido 1/genética , Peso Molecular , Mutación , Pruebas de Neutralización , Simplexvirus/inmunología , Porcinos , Proteínas del Envoltorio Viral/genética , Ensayo de Placa ViralRESUMEN
Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Antígenos Virales/análisis , Herpesvirus Suido 1/inmunología , Animales , Fusión Celular , Línea Celular , Técnica del Anticuerpo Fluorescente , Hibridomas/inmunología , Inmunización Pasiva , Inmunodifusión , Riñón , Peso Molecular , Radioinmunoensayo , PorcinosRESUMEN
Glucan, a beta-1,3 polyglucose, was administered to mice either 1 h before or 1 h after a 650 rad exposure to cobalt-60 radiation. Compared to radiation controls, glucan-treated mice consistently exhibited a more rapid recovery of pluripotent stem cells and committed granulocyte, macrophage, and erythroid progenitor cells. This may partially explain the mechanism by which glucan also enhances survival in otherwise lethally irradiated mice.
Asunto(s)
Glucanos/farmacología , Células Madre Hematopoyéticas/efectos de la radiación , Células Madre/efectos de la radiación , Animales , Células de la Médula Ósea , Recuento de Células , Eritrocitos/citología , Femenino , Granulocitos/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Macrófagos/citología , Ratones , Bazo/citología , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
A pseudorabies virus variant ( mar197 -1) containing a mutation in a viral glycoprotein with a molecular weight of 50,000 ( gp50 ) was isolated by selecting for resistance to a neurtralizing monoclonal antibody ( MCA50 -1) directed against gp50 . This mutant was completely resistant to neutralization with MCA50 -1 in the presence or absence of complement, and was therefore defined as a mar (monoclonal-antibody-resistant) mutant. The mutation did not affect neutralization with polyvalent immune serum. The mar197 -1 mutant synthesized and processed gp50 normally, but the mutation prevented the binding and immunoprecipitation of gp50 by MCA50 -1. Thus, the mutation was within the structural portion of the gp50 gene affecting the epitope of the monoclonal antibody. The mutation was mapped by marker rescue with cloned pseudorabies restriction enzyme fragments to the short region of the pseudorabies genome between 0.813 and 0.832 map units. This is equivalent to a 2.1-kilobase-pair region.
Asunto(s)
Antígenos Virales/genética , Genes Virales , Glicoproteínas/genética , Herpesvirus Suido 1/genética , Proteínas Virales/genética , Clonación Molecular , Enzimas de Restricción del ADN , Desoxirribonucleasa BamHI , Epítopos/genética , Marcadores Genéticos , Glicoproteínas/inmunología , Herpesvirus Suido 1/inmunología , Herpesvirus Suido 1/aislamiento & purificación , Mutación , Pruebas de Neutralización , Proteínas Virales/inmunologíaRESUMEN
Our earlier studies in mice showed that sequential radiation and cyclophosphamide suppressed marrow stromal cells (MSC) and prevented local hemopoietic repopulation for several months. Because others have shown that busulfan administration caused marrow aplasia, we studied its ability, combined with radiation, to produce a persistent microenvironmental defect in mice. Intraperitoneal administration of four weekly doses of 20 mg/kg busulfan, starting one week after 1500 rad leg irradiation, produced a severe microenvironmental lesion for 6 months reflected by lack of repopulation in femoral marrow to greater than 50% of normal by MSC, hemopoietic stem cells (CFU-S), and granulocyte-macrophage precursors. Differential marrow cell counts revealed that precursors of hemopoietic cells were more profoundly affected than their progeny. Hemopoietic stem cells and MSC failed to recover in busulfan-treated mice at 6 months to the same extent as those treated with cyclophosphamide. In addition, the busulfan-treated mice had an excessive number of myeloid blast cells and a severe erythroid depletion suggesting that these animals were preleukemic. We conclude that: 1) sequential radiation and busulfan administration caused long-term microenvironmental damage reflected by incomplete repopulation of the femoral marrow and suppression of MSC, and 2) multiple courses of busulfan prevented hemopoietic repopulation longer than a similar regimen of cyclophosphamide.
Asunto(s)
Busulfano/efectos adversos , Sistema Hematopoyético/efectos de la radiación , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/efectos de la radiación , Ciclofosfamida/efectos adversos , Femenino , Células Madre Hematopoyéticas/efectos de la radiación , Sistema Hematopoyético/efectos de los fármacos , Ratones , Ratones EndogámicosRESUMEN
The studies reported in this paper evaluated one of the mechanisms by which extramedullary tumor caused anemia, neutrophilia, medullary erythroblastopenia, and suppression of marrow stromal cells (MSC) in tumor bearing mice. Since MSC have been shown to support hemopoiesis, we asked whether tumor released a suppressor which directly inhibited MSC colonies, and if it did, whether or not it was prostaglandin-E (PGE). We found that co-culture of Ehrlich ascites carcinoma (EAC) and Sarcoma (S-180) cells with normal mouse bone marrow cells profoundly suppressed formation of MSC colonies. At concentrations exceeding 15% of the medium, tumor-conditioned media not only suppressed MSC colonies but also enhanced the growth of granulocyte-macrophage colonies (CFUC) in vitro like Colony Stimulating Factor (CSF) from other sources. It did not suppress CFUE, nor did it inhibit growth or kill cells other than MSC in bone marrow cultures. Fibroblasts grew luxuriantly in 20% conditioned media from Ehrlich ascites carcinoma cells. Concentrations of PGE2 required to suppress MSC colonies greatly exceeded those detected in media conditioned by S-180. Production of PGE by S-180 cells was inhibited by growing the tumor cells in the presence of indomethacin, but the supernatant media, devoid of PGE, still markedly suppressed the growth of MSC from normal marrow. Because tumor produced CSF, we tested the suppressive effect of CSF in post-endotoxin serum to find that concentrations of 10 to 15% inhibited MSC colony growth. The results show that these tumors produced a substance, possibly CSF, that selectively inhibited MSC colony growth in vitro. It is conceivable that suppression of the supportive tissue (MSC) for erythropoiesis in the bone marrow by tumor led to diminished erythroblasts in that site.