RESUMEN
Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP(C), such as PrP(BSE) in bovine spongiform encephalopathy (BSE) in cattle and PrP(CJD) in Creutzfeldt-Jakob disease (CJD) in humans. Disruption of PrP(C) expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities. However, the impact of ablating PrP(C) function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP(C)-deficient cattle produced by a sequential gene-targeting system. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification. PrP(C)-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.
Asunto(s)
Animales Modificados Genéticamente/genética , Silenciador del Gen , Ingeniería Genética/métodos , Proteínas PrPC/genética , Animales , BovinosRESUMEN
Antigen-specific human polyclonal antibodies (hpAbs), produced by hyperimmunization, could be useful for treating many human diseases. However, yields from available transgenic mice and transchromosomic (Tc) cattle carrying human immunoglobulin loci are too low for therapeutic applications. We report a Tc bovine system that produces large yields of hpAbs. Tc cattle were generated by transferring a human artificial chromosome vector carrying the entire unrearranged, human immunoglobulin heavy (hIGH) and kappa-light (hIGK) chain loci to bovine fibroblasts in which two endogenous bovine IgH chain loci were inactivated. Plasma from the oldest animal contained >2 g/l of hIgG, paired with either human kappa-light chain (up to approximately 650 microg/ml, fully human) or with bovine kappa- or lambda-light chain (chimeric), with a normal hIgG subclass distribution. Hyperimmunization with anthrax protective antigen triggered a hIgG-mediated humoral immune response comprising a high proportion of antigen-specific hIgG. Purified, fully human and chimeric hIgGs were highly active in an in vitro toxin neutralization assay and protective in an in vivo mouse challenge assay.
Asunto(s)
Animales Modificados Genéticamente , Inmunoglobulina G/biosíntesis , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/biosíntesis , Animales , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Células CHO , Bovinos/genética , Cromosomas Artificiales Humanos/genética , Cricetinae , Cricetulus , Técnicas de Inactivación de Genes , Glicosilación , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/inmunología , Ratones , Pruebas de Neutralización , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Brucella spp. cause disease in humans and livestock and are potential biowarfare agents. Defining the protective immune response is necessary to design vaccines. This has largely been done with mice, brucella-susceptible BALB/c and resistant C57BL strains. Since interferon-gamma is key to brucella resistance, contrary to expectations, we found that ex vivo splenocytes from naïve BALB/c mice produced IL-12 and interferon-gamma in cultures with brucellae at levels comparable to those of splenocytes from the more resistant C57BL/10 mice. Moreover, both IL-12 and interferon-gamma were produced in the first week following infection of BALB/c mice. However, by the third week of infection we found decreased IL-12Rbeta2 expression by BABL/c splenocytes, corresponding to their inability to produce interferon-gamma in Brucella recall responses at this time as reported previously. Administering recombinant IL-12 to these mice ameliorated the interferon-gamma hiatus, resulted in a 1000-fold reduction in CFU during primary infection and increased survival following secondary challenge.