Assuntos
Antígenos CD28/imunologia , Antígenos CD40/antagonistas & inibidores , Sobrevivência de Enxerto/imunologia , Transplante de Coração/imunologia , Transplante de Pele/imunologia , Animais , Antígenos CD28/fisiologia , Antígenos CD40/fisiologia , Células Cultivadas , Facilitação Imunológica de Enxerto/métodos , Facilitação Imunológica de Enxerto/tendências , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Rejeição de Enxerto/terapia , Transplante de Coração/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Transdução de Sinais/imunologia , Transplante de Pele/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Subpopulações de Linfócitos T/transplanteRESUMO
This unit details the use of transient expression in mammalian cells to screen cDNA libraries with monoclonal antibodies (MAb) to isolate cDNA clones encoding cell-surface and intracellular proteins. The first protocol in this unit describes the cloning of cDNAs encoding cell-surface antigens. Several steps in this protocol involve transfection procedures that are described in greater detail elsewhere in this volume. The second protocol is a modification that facilitates isolation of cDNAs encoding antigens that are expressed intracellularly. Both protocols are designed for use with the expression vector CDM8, which contains a polylinker for subcloning double-stranded cDNA.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Superfície/genética , Antígenos/genética , Clonagem Molecular/métodos , Expressão Gênica , Biblioteca Gênica , Animais , Antígenos/imunologia , Antígenos de Superfície/imunologia , Células COS , Chlorocebus aethiops , DEAE-Dextrano , DNA Complementar/genética , Escherichia coli/genética , Vetores Genéticos/genética , Técnicas de Imunoadsorção/instrumentação , Espaço Intracelular , Mamíferos , Cloreto de Polivinila/análogos & derivados , Esferoplastos/metabolismo , Transfecção/métodosRESUMO
BACKGROUND: Organ transplant recipients currently require lifetime immunosuppressive therapy, with its accompanying side effects. Biological agents that block T-cell costimulatory pathways are important components of strategies being developed to induce transplantation tolerance. The aim of this study was to test the effect of a novel chimeric anti-human CD40 monoclonal antibody (Chi 220), either alone or in combination with CTLA4-Ig, on the survival of renal allografts in a nonhuman primate model. METHODS: Captive-bred adolescent male rhesus monkeys (Macaca mulatta) (4-10 kg) were used as recipients and donors. Four treatment protocols were tested: Chi220 monotherapy, CTLA4-Ig monotherapy, Chi220 combined with CTLA4-Ig, and H106 (anti-CD40L) combined with CTLA4-Ig. Control animals received human albumin. Recipients were followed for survival, renal allograft function as determined by measurement of serum blood urea nitrogen (BUN) and creatinine, chemistries (sodium, potassium, chloride, and bicarbonate), complete blood cell count (CBC) with differential, and the development of donor-specific alloantibody. RESULTS: Treatment with Chi220 for 14 days prolonged renal allograft survival (MST 38.5 vs. 7 days in untreated controls). Notably, simultaneous blockade of the CD28/B7 pathway did not further augment graft survival but did suppress the development of donor-specific antibodies, an effect not achieved with Chi220 alone, despite peripheral B cell depletion. Finally, treatment with Chi220 suppressed the primary immune response to cytomegalovirus, resulting in severe systemic manifestations. CONCLUSIONS: Blockade of the CD40 pathway with anti-CD40 mAb is immunosuppressive in a large animal, preclinical renal transplant model. The potential effect of this therapy on viral immune responses will be important to consider for the design of safe clinical trials.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos de Diferenciação/uso terapêutico , Antígenos CD40/imunologia , Sobrevivência de Enxerto/efeitos dos fármacos , Imunoconjugados , Transplante de Rim , Abatacepte , Animais , Anticorpos/uso terapêutico , Anticorpos Monoclonais/efeitos adversos , Formação de Anticorpos/efeitos dos fármacos , Antígenos CD , Antígenos de Diferenciação/efeitos adversos , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Antígenos CD28/efeitos dos fármacos , Ligante de CD40/imunologia , Antígeno CTLA-4 , Infecções por Citomegalovirus/induzido quimicamente , Infecções por Citomegalovirus/patologia , Quimioterapia Combinada , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Isoanticorpos/imunologia , Rim/patologia , Macaca mulatta , Masculino , Doadores de Tecidos , Transplante HomólogoRESUMO
This unit describes the use of COS cells to efficiently produce a desired protein in a short period of time. These cells express high levels of the SV40 large tumor (T) antigen, which is necessary to initiate viral DNA replication at the SV40 origin. Each COS cell transfected with DNA encoding a cell-surface antigen (in the appropriate vector) or cytoplasmic protein will express several thousand to several hundred thousand copies of the protein 72 hr posttransfection. If the transfected DNA encodes a secreted protein, up to 10 mg of protein can be recovered from the supernatant of the transfected COS cells 1 week posttransfection. COS cell transient expression systems have also been used to screen cDNA libraries, to isolate cDNAs encoding cell-surface proteins, secreted proteins, and DNA binding proteins, and to test protein expression vectors rapidly prior to the preparation of stable cell lines.
Assuntos
Células COS/metabolismo , Clonagem Molecular/métodos , Proteínas Recombinantes de Fusão/biossíntese , Animais , Antígenos Transformantes de Poliomavirus/biossíntese , Chlorocebus aethiops , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Vírus 40 dos Símios/genética , Transdução Genética/métodos , Replicação ViralRESUMO
Recombinant DNA technology has allowed the preparation of chimeric genes encoding proteins with novel properties. This unit describes the construction and subsequent testing of genes encoding immunoglobulin chimeras. The first protocol details fusion of a protein (or protein fragment) of interest onto an immunoglobulin constant region using a modified version of the expression vector pCDM8. The resulting fusion protein generally retains the functional properties of both the protein of interest and the immunoglobulin constant region; this can be demonstrated as described here.