RESUMEN
A chronically immunosuppressed sheep model was established using a regimen of cyclosporin A (CsA; 2-3mg/kg twice daily) and ketoconazole (10mg/kg twice daily). Blood CsA concentrations reached a steady-state after 17 days of treatment. The clearance of CsA decreased from a mean (95% CI) of 9.47 (6.2-12.7)ml/min/kg after a single (first) dose (3mg/kg i.v.) to 1.62 (1.38-1.86)ml/min/kg after 18 days of CsA (3mg/kg i.v. twice daily) co-administration with ketoconazole. These data indicated that the combination of CsA and ketoconazole could be used to give stable high concentrations of CsA in the sheep. Using this regimen in the sheep, the long-term survival of skin allografts was monitored as an indicator of effective immunosuppression. CsA in blood was measured daily and CsA dose adjusted to various target concentration ranges. Provided that the trough concentration of blood CsA was maintained between 1500-2500 mg/l, long-term healthy skin allografts were maintained on the sheep without significant adverse effects on haematological or biochemical parameters.
Asunto(s)
Ciclosporina/farmacocinética , Supervivencia de Injerto , Inmunosupresores/farmacocinética , Trasplante de Piel/inmunología , Animales , Cromatografía Líquida de Alta Presión , Ciclosporina/administración & dosificación , Ciclosporina/análisis , Femenino , Cetoconazol/farmacología , Ovinos , Trasplante HomólogoRESUMEN
1. The pharmacokinetics of a single dose of Cyclosporine A (CsA) administered to sheep by intravenous (i.v.) route were examined. 2. Concomitant administration of ketoconazole was found to increase the area under the blood CsA concentration-time curve (AUC) and was effective when administered by the oral or intraperitoneal route. 3. The effects of CsA and ketoconazole on the immune system of sheep were also assessed. 4. A single dose of CsA 5 mg/kg resulted in abrogation of in vitro lymphocyte function manifest at 24 h after injection of CsA. Normal responsiveness recovered in 48-72 h. Numbers of T lymphocytes in peripheral blood were elevated transiently at 48 h although no other significant alteration in lymphocyte subsets was observed with this treatment. 5. Concomitant ketoconazole administration enhanced the CsA-induced suppression of in vitro lymphocyte responses. Blood levels of CsA (AUC values to 24 h) were significantly elevated with concomitant ketoconazole administration and depression of lymphocyte responses to mitogens were also significantly enhanced. An increase in the proportion of T4 positive cells in the blood was observed at 48 h and at 7 days after administration of CsA with ketoconazole. 6. These findings indicate that CsA effectively abrogates immunocompetence in the sheep and this immunosuppressive effect is enhanced by concomitant administration of ketoconazole.
Asunto(s)
Antifúngicos/farmacología , Ciclosporina/farmacocinética , Inmunosupresores/farmacocinética , Cetoconazol/farmacología , Animales , Antifúngicos/administración & dosificación , Ciclosporina/sangre , Inmunosupresores/sangre , Cetoconazol/administración & dosificación , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Mitógenos/fisiología , Análisis de Regresión , OvinosRESUMEN
BACKGROUND: Total-body irradiation, followed by hematopoietic system rescue by bone marrow transplantation (BMT), has been found to improve the response of patients with multiple myeloma to treatment with melphalan. The problems of nonhematopoietic toxicity from whole-body irradiation might be circumvented by using a bone-seeking radiopharmaceutical, such as samarium-153 ethylenediaminetetramethylene phosphonate (153Sm-EDTMP), to ablate the bone marrow. PURPOSE: A mouse model system for multiple myeloma was used to evaluate the potential therapeutic efficacy of sequential therapy with 153Sm-EDTMP, melphalan, and BMT. METHODS: Female C57BL/KaLwRij mice were inoculated with 8 x 10(5) 5T33 murine myeloma cells. Treatment protocols were begun 3 or 10 days later, when the myeloma was either confined to bone marrow or disseminated in liver, spleen, and lymph nodes, simulating human multiple myeloma. 153Sm, a potent beta particle-emitting radioisotope of short half-life (46.7 hours), was linked to the bone-seeking chelate EDTMP. Animals in the first treatment group were each given 22.5 MBq 153Sm-EDTMP via the jugular vein (day 3 or 10), followed by 18.5 mg/kg melphalan (maximum tolerated dose) given intraperitoneally 5 days later (day 8 or 15) and syngeneic BMT another 2 days later (day 10 or 17). Survival in groups of six to 10 animals for each time series was compared with that in mice left untreated (control cohort), in mice treated with 153Sm-EDTMP alone (day 3 or 10), or in mice treated with melphalan alone (day 8 or 15). The hematopoietic systems of animals in the latter two treatment groups recovered full function, obviating the necessity of BMT. The end point was onset of paraparesis, at which time the animals were immediately killed by carbon dioxide asphyxiation. RESULTS: Median survival in untreated control animals was 23 days in those with localized disease and 24 days in those with disseminated myeloma. Treatment with 153Sm-EDTMP alone improved survival to a median of 29 days when commenced on day 3 and 30 days when begun on day 10. Melphalan treatment alone improved the median survival to 31 days for animals with localized myeloma and 34 days in animals with disseminated disease. Additional improvement in survival to a median of 42 days was achieved in animals treated 3 days after tumor inoculation with sequential 153Sm-EDTMP, melphalan, and BMT; median survival was 40 days using this regimen in animals with disseminated myeloma. CONCLUSIONS: Animals in all three treatment protocols survived longer than those left untreated after inoculation with myeloma cells (P < .001). Sequential treatment with 153Sm-EDTMP, melphalan, and BMT was significantly more effective than single-agent treatment (P < .01). No evidence of radiotoxicity was detected in nonhematopoietic organs. IMPLICATIONS: The survival advantage conferred by our sequential treatment protocol suggests its potential clinical usefulness in the treatment of multiple myeloma and other hematologic malignancies in humans.
Asunto(s)
Trasplante de Médula Ósea/métodos , Mieloma Múltiple/terapia , Compuestos Organometálicos/uso terapéutico , Compuestos Organofosforados/uso terapéutico , Animales , Terapia Combinada , Femenino , Técnicas In Vitro , Masculino , Melfalán/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Samario/uso terapéutico , Análisis de Supervivencia , Células Tumorales CultivadasRESUMEN
The 5T33 multiple myeloma is one of a series of transplantable murine myelomas arising spontaneously in C57BL/KaLwRij mice. This study describes the establishment and characterisation of the 5T33 murine myeloma in vitro as a cultured cell line in terms of its morphology, growth rate, expression of paraprotein (IgG2b) and tumorigenicity in syngeneic animals. The 5T33 cell line has been in continuous culture for over 10 months and has achieved more than passage 34. In culture, 5T33 myeloma grows as single cells or in small clusters of loosely adherent cells on an adherent stromal cell layer. Maximum doubling time is approximately 25 h, and over 90% of the cells express cytoplasmic IgG2b paraprotein. The cultured 5T33 myeloma cells are highly tumorigenic in C57BL/KaLwRij mice with as few as 500 cells inducing paralysis and death as early as day 36 post-tumour inoculation. Kinetics of tumour development and detection of IgG2b paraprotein are dose dependent. Two weeks following intravenous inoculation of 5 x 10(5) cultured 5T33 myeloma cells, tumour cells were readily identified in the bone marrow. By 3 weeks post-tumour inoculation, 5T33 myeloma cells were found in various tissues throughout the animal. Studies are now underway to determine the sensitivity of this cell line to various therapeutic modalities.
Asunto(s)
Mieloma Múltiple/patología , Células Tumorales Cultivadas/patología , Animales , Anticuerpos Antineoplásicos/análisis , Femenino , Inmunoglobulina G/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Mieloma Múltiple/inmunología , Mieloma Múltiple/mortalidad , Células Tumorales Cultivadas/inmunologíaRESUMEN
Mouse cytomegalovirus (MCMV) infection induces persisting myocarditis in the susceptible BALB/c strain. Autoantibodies to cardiac myosin are produced in both susceptible BALB/c and resistant C57BL/6 mice following MCMV infection. These affinity-purified anti-cardiac myosin antibodies cross-react with MCMV protein(s). The polypeptides of CMV which share immunological cross-reactivity with the 200,000 MW polypeptide, the heavy chain of myosin, were viral polypeptides of 83,000, 94,000 and 116,000 MW recognized by BALB/c post-infection sera and polypeptides of 66,000 and 94,000 MW recognized by C57BL/6 post-infection sera. Passive transfer of anti-cardiac myosin antibodies from Day 56 post-infection sera of the BALB/c strain induced inflammation and necrosis of the myocardium of uninfected BALB/c recipients. This late immune sera contains autoantibodies specific for the cardiac isoform of myosin. Furthermore, immunization with cardiac myosin induced myocarditis and high titres of cardiac myosin antibodies in uninfected mice of the susceptible BALB/c strain only. However, antibodies to myosin elicited in cardiac myosin-immunized BALB/c mice did not cross-react with MCMV by ELISA. We suggest that virus infection may modulate the immune recognition of the common-epitope(s) shared between MCMV protein(s) and the heavy chain of myosin. Of particular interest is the possibility that molecular mimicry of CMV with cardiac myosin may contribute to the pathogenesis of autoimmune myocarditis following virus infection.
Asunto(s)
Autoanticuerpos/biosíntesis , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Miocarditis/inmunología , Miosinas/inmunología , Animales , Antígenos Virales/inmunología , Reacciones Cruzadas/inmunología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Miocardio/inmunologíaRESUMEN
Certain murine monoclonal antibodies (mAb) raised against structural proteins of mouse cytomegalovirus (MCMV) display distinct patterns of multiple organ-autoreactivity in addition to their viral specificities. We analysed the autoreactivity of five such mAb by immunoperoxidase histochemistry, western immunoblot and enzyme-linked immunosorbent assay (ELISA). Four mAb recognized cellular autoantigens in the salivary gland, lung, heart, liver, kidney, ileum, striated muscle and brain, as detected by immunoperoxidase histochemistry. However, the mAb showed different specificities for nuclear, cytoplasmic and surface membrane antigens on various cell types in addition to common autoreactivities. Immunoblot analyses showed that some of the mAb recognized polypeptides of various molecular weights obtained from 100,000 g supernatants of normal BALB/c liver, brain, striated and cardiac muscle homogenates. Reactivity of the mAb with a 200,000 molecular weight (MW) polypeptide was similar to our previous finding of the reaction of late immune polyclonal sera with a 200,000 MW polypeptide, the heavy chain of myosin. The mAb reacted to the cardiac isoform of myosin as determined by ELISA and immunoblot. Reactivity of mAb with cardiac myosin, as detected by immunoblot, was removed by absorption with cardiac myosin and recovered in the eluate. However, cardiac myosin used in a competitive inhibition ELISA did not abrogate the reactivity of the mAb with MCMV antigens. These anti-MCMV mAb appear to be multispecific for both virus and self-antigens, including cardiac myosin, and possibly recognize these different antigens through partly similar or distinct antigen-binding sites.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Autoanticuerpos/inmunología , Citomegalovirus/inmunología , Animales , Especificidad de Anticuerpos/inmunología , Autoinmunidad , Western Blotting , Electroforesis en Gel de Poliacrilamida , Femenino , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos BALB C , Miocardio/inmunología , Miosinas/inmunología , Péptidos/inmunologíaRESUMEN
Myocarditis accompanies sublethal mouse cytomegalovirus (MCMV) infection in susceptible BALB/c mice and persists beyond the acute phase of infection, in the absence of demonstrable virus antigen but in the continuing presence of autoantibodies to cardiac muscle. Heart tissue autoantibodies of the IgG class were first detected by ELISA in sera at Days 3-5 post-infection (PI) and persisted to Day 100, in two strains of MCMV-infected mice which are susceptible (BALB/c) and resistant (C57BL/10) to MCMV-induced myocarditis. Analysis by immunoblot showed that autoantibodies in early immune sera (Day 10) from both mouse strains reacted with the contractile proteins troponin, tropomyosin and myosin, as well as with other unidentified polypeptides within normal mouse organ homogenates. However, the dominant reactivity of late immune sera (Day 100) was to a 200,000 molecular weight (MW) polypeptide in muscle homogenates identified as the heavy chain of myosin. Autoantibodies reacting with the cardiac or striated muscle isoforms of myosin were assessed by ELISA in BALB/c and C57BL/10 mice. At Days 28, 56 and 100 PI only the susceptible BALB/c strain had high titres of autoantibodies reacting with the cardiac isoform of myosin. Increasing the virus dose given to C57BL/10 mice resulted in slight increases in titres of anti-myosin antibody; however, the peak antibody titres did not approach those of BALB/c mice and persisting myocarditis did not develop. Absorption experiments showed that cardiac myosin-specific antibodies were present in immune sera from susceptible BALB/c mice at Day 100 but not in resistant C57BL/10 mice by ELISA and immunoblot. These results demonstrate that autoimmunity to myosin is a prominent feature of the humoral autoimmune response following MCMV infection, and that there are differences both in fine isoform specificity and titre of anti-myosin antibodies between strains of mice that develop persisting myocarditis and strains that do not. Cardiac myosin-specific autoantibodies may play an immunopathogenic role in CMV-induced myocarditis.
Asunto(s)
Autoanticuerpos/análisis , Infecciones por Citomegalovirus/inmunología , Miocarditis/inmunología , Miocardio/inmunología , Miosinas/inmunología , Animales , Especificidad de Anticuerpos , Susceptibilidad a Enfermedades/inmunología , Femenino , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Músculos/inmunología , Miocarditis/etiologíaRESUMEN
Liver-specific lipoprotein (LSP) has been the subject of intense investigation as a candidate target antigen in chronic active hepatitis. Fundamental to the interest in LSP has been the belief that it is an antigen complex of hepatocyte plasma membrane origin. In this study the physical, biochemical and antigenic relationships between LSP and isolated hepatocyte plasma membrane (HPM) were investigated. Electron microscopic examination of LSP showed it to be devoid of plasmalemma sheets that were abundant in HPM. The plasma membrane marker enzyme 5'-nucleotidase was enriched 11-fold in HPM relative to liver homogenate, whereas the enzyme activity in LSP was 17% of that found in liver homogenate and only 1.5% of that found in HPM. The antigenic relationship between LSP and HPM was assessed using sera from rabbits immunized with either mouse LSP or mouse HPM. By filtration ELISA, antibody to LSP reacted poorly with entrapped HPM, relative to antibody to mouse HPM. Antisera to LSP and HPM were both effectively absorbed by the immunizing antigen, however antibody to LSP was not absorbed with HPM, and minimal cross-absorption of HPM antibody with LSP was found. By immunoblot of SDS-PAGE separated LSP and HPM, it was shown that antigenic cross-reactivity between LSP and HPM at the polypeptide level was rare. By immunofluorescence, antibody to LSP failed to react with the surface of viable mouse hepatocytes, whereas antibody to HPM showed linear fluorescence. The data show that the two preparations, LSP and HPM, are dissimilar antigen complexes. HPM may be a more appropriate preparation for the study of autoimmune liver disease than LSP.