RESUMEN
Despite significant investment and technical efforts, veterinary vaccine manufacturers continue to experience challenges with the transition from historic animal-based potency methods to in vitro potency assays. These challenges have a number of contributing factors, including an inadequate understanding of protective antigens and epitopes, a lack of ruggedness and discriminating capabilities in evolving immunologically-based methods, inconsistencies between methods used for in-process antigen measurement and finished product potency, and a lack of clear methods to characterize the finished formulation (including complex adjuvants). A lack of harmonized guidelines and consistent regulatory expectations further complicates these efforts. There is room for optimism, however. There are numerous examples of successful in vitro potency test implementations. Titrations of modified live viral and bacterial vaccines, immune-based quantitative assays, and the recent application of direct physicochemical methods have allowed the transition from animal testing in many applications globally. Specific challenges for assay development and implementation are discussed in the areas of 1) target antigen selection, 2) complexity of finished product formulation, 3) potency discrimination, and 4) stability-indicating relevance.
Asunto(s)
Antígenos/inmunología , Vacunación/veterinaria , Vacunas/inmunología , Alternativas a las Pruebas en Animales/métodos , Alternativas a las Pruebas en Animales/normas , Animales , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Reproducibilidad de los Resultados , Vacunas/administración & dosificación , Drogas Veterinarias/normas , Medicina Veterinaria/métodos , Medicina Veterinaria/normasRESUMEN
Type I diabetes and multiple sclerosis (MS) are distinct autoimmune diseases where T cells target either islet or CNS self-proteins. Unexpectedly, we found that autoreactive T cells in diabetic patients, relatives with high diabetes risk, nonobese diabetic (NOD) mice, and MS patients routinely target classical islet as well as CNS autoantigens. The pathogenic potential of CNS autoreactivity was testable in NOD mice. Pertussis holotoxin, without additional Ags or adjuvants, allowed development of an NOD mouse-specific, autoimmune encephalitis with variable primary-progressive, monophasic, and relapsing-remitting courses. T cells from diabetic donors transferred CNS disease to pertussis toxin-pretreated NOD.scid mice, with accumulation of CD3/IFN-gamma transcripts in the brain. Diabetes and MS appear more closely related than previously perceived. NOD mouse-specific, autoimmune encephalitis provides a new MS model to identify factors that determine alternative disease outcomes in hosts with similar autoreactive T cell repertoires.
Asunto(s)
Autoantígenos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Islotes Pancreáticos/inmunología , Esclerosis Múltiple/inmunología , Proteína Básica de Mielina/inmunología , Enfermedad Aguda , Traslado Adoptivo , Adulto , Secuencia de Aminoácidos , Animales , División Celular/inmunología , Citocinas/biosíntesis , Citocinas/genética , Diabetes Mellitus Tipo 1/etiología , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Estudios de Seguimiento , Humanos , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Endogámicos NZB , Ratones SCID , Datos de Secuencia Molecular , Especificidad de Órganos/inmunología , Estudios Prospectivos , Recurrencia , Especificidad de la Especie , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Cross-reactive T cells that recognize both Tep69 (dominant nonobese diabetic (NOD) T cell epitope in ICA69 (islet cell autoantigen of 69 kDa)) and ABBOS (dominant NOD T cell epitope in BSA) are routinely generated during human and NOD mouse prediabetes. Here we analyzed how systemic administration of these mimicry peptides affects progressive autoimmunity in adoptively transferred and cyclophosphamide-accelerated NOD mouse diabetes. These models were chosen to approximate mid to late stage prediabetes, the typical status of probands in human intervention trials. Unexpectedly, high dose (100 microg) i.v. ABBOS prevented, while Tep69 exacerbated, disease in both study models. Peptide effects required cognate recognition of endogenous self-Ag, because both treatments were ineffective in ICA69null NOD congenic mice adoptively transferred with wild-type, diabetic splenocytes. The affinity of ABBOS for NOD I-A(g7) was orders of magnitude higher than that of Tep69. This explained 1) the expansion of the mimicry T cell pool following i.v. Tep69, 2) the long-term unresponsiveness of these cells after i.v. ABBOS, and 3) precipitation of the disease after low dose i.v. ABBOS. Disease precipitation and prevention in mid to late stage prediabetes are thus governed by affinity profiles and doses of therapeutic peptides. ABBOS or ABBOS analogues with even higher MHC affinity may be candidates for experimental intervention strategies in human prediabetes, but the dose translation from NOD mice to humans requires caution.
Asunto(s)
Autoantígenos/biosíntesis , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/terapia , Antígenos de Histocompatibilidad Clase II/metabolismo , Péptidos/administración & dosificación , Péptidos/inmunología , Estado Prediabético/inmunología , Estado Prediabético/terapia , Traslado Adoptivo/métodos , Secuencia de Aminoácidos , Animales , Autoantígenos/administración & dosificación , Autoantígenos/inmunología , Autoantígenos/metabolismo , Epítopos de Linfocito T/administración & dosificación , Epítopos de Linfocito T/inmunología , Femenino , Tolerancia Inmunológica , Inyecciones Intravenosas , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Imitación Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Unión Proteica/inmunología , Albúmina Sérica Bovina/administración & dosificación , Albúmina Sérica Bovina/inmunología , Albúmina Sérica Bovina/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
A previously reported monitoring system provided continuous direct measurements of oxygen consumption and intravascular pressures. These data were combined with interval measurements of cardiac output and blood oxygen saturations to derive various hemodynamic and oxygen transport variables. This system has now been modified so that cardiac output is measured continuously in real-time.