RESUMEN
BACKGROUND: No strawberry allergen has so far been identified and characterized. METHODS: Serum samples were collected from patients with a suggestive case history of adverse reactions to strawberry and other fruits. Extracts from fresh and frozen strawberries were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and mass spectrometry. Patient blood samples were analysed for inhibition of IgE binding and basophil degranulation. RESULTS: Several IgE-binding proteins could be detected. In more than half of the patient sera, a 20/18-kDa doublet band was observed in Western blotting. These two bands were excised and analysed by mass spectrometry showing the presence of proteins belonging to the Bet v 1 family of allergens. Inhibition of the IgE binding to the 20/18-kDa doublet was obtained by addition of two recombinantly expressed allergens belonging to the Bet v 1 family (Bet v 1 and Mal d 1) and strawberry protein extract. In a cell-based assay of patient blood samples, basophil degranulation could be induced by strawberry protein extract and by Bet v 1 and Mal d 1. CONCLUSIONS: We conclude that strawberry homologues to Bet v 1 may be allergens of importance for adverse reactions to strawberry.
Asunto(s)
Alérgenos/aislamiento & purificación , Hipersensibilidad a los Alimentos/inmunología , Fragaria/inmunología , Inmunoglobulina E/inmunología , Proteínas de Plantas/aislamiento & purificación , Adulto , Alérgenos/química , Alérgenos/inmunología , Secuencia de Aminoácidos , Antígenos de Plantas , Prueba de Desgranulación de los Basófilos , Western Blotting , Electroforesis en Gel de Poliacrilamida , Femenino , Hipersensibilidad a los Alimentos/sangre , Hipersensibilidad a los Alimentos/etiología , Fragaria/efectos adversos , Humanos , Inmunoglobulina E/sangre , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Peso Molecular , Extractos Vegetales/inmunología , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Alineación de SecuenciaRESUMEN
AIMS/HYPOTHESIS: Type 1 diabetes mellitus is a multifactorial autoimmune disease characterised by selective destruction of beta cells in the islets of Langerhans. We have previously shown that IL-1 beta modulates beta cell function, causes beta cell death and induces expression changes in 82 out of 1815 protein spots detected by two-dimensional gel electrophoresis (2-DGE) in diabetes-prone bio-breeding (BB-DP) rat islets in vitro. The aim of this study was to describe the relevance of these proteins in the development of diabetes in vivo. METHODS: Syngeneic neonatal islets ( n=200) were transplanted under the kidney capsule of 30-day-old BB-DP and control rats, removed to different time points after transplantation or at the onset of diabetes, and metabolically labelled with S(35)-methionine for 2-DGE. The 82 proteins were re-localised and followed. In addition, transplants were examined for expression of IL-1 beta mRNA by in situ hybridisation. RESULTS: All 82 proteins could be re-localised in all syngeneic transplants from BB-DP and control rats. A total of 60 of the 82 proteins were changed during development of diabetes. Of the 82 proteins, 32 were changed in expression at the onset of diabetes compared to non-diabetic BB-DP rats, and 25 of these were changed as by IL-1 beta in vitro. Highest expression of IL-1 beta mRNA was found at the onset of diabetes. CONCLUSIONS/INTERPRETATION: IL-1 beta-induced protein expression changes in islets in vitro also occur in vivo and change in a complex pattern during the development of diabetes in the BB-DP rat. No single protein seems to be responsible for the development of diabetes, but rather the cumulative numbers of changes seem to interfere with the intracellular stability of the beta cell.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Regulación de la Expresión Génica/inmunología , Interleucina-1/genética , Animales , Separación Celular/métodos , Hibridación in Situ , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/inmunología , ARN Mensajero/genética , Ratas , Ratas Endogámicas BBRESUMEN
The surface of Chlamydia pneumoniae is covered with proteins but their exact identification is not known probably because of the presence of conformational epitopes. A family of 21 pmp genes has been found by DNA sequencing. In common, these genes have the capacity to encode the amino acid motif GGAI. Several of the genes have the capacity to encode outer membrane proteins of about 100 kDa. Thus, they are candidate genes to encode the protein(s) present in the 98-kDa protein band of the C. pneumoniae outer membrane complex. The production of recombinant GGAI proteins is described as is the use of polyclonal antibodies raised against the recombinant GGAI proteins to determine their expression in C. pneumoniae elementary bodies. At least three of the proteins, Omp4, 5, and 11, are expressed.
Asunto(s)
Secuencias de Aminoácidos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Chlamydophila pneumoniae/inmunología , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Línea Celular , Infecciones por Chlamydia/inmunología , Chlamydophila pneumoniae/química , Chlamydophila pneumoniae/genética , Electroforesis en Gel Bidimensional , Humanos , Immunoblotting , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: The association of Chlamydia pneumoniae with the development of atherosclerosis is based on serology and on detection of C pneumoniae-specific DNA by polymerase chain reaction in the atheromas. METHODS AND RESULTS: Because the humoral immune response frequently recognizes epitopes present on the surface of the bacteria, we analyzed what components are present on the C pneumoniae surface. We identified a family of proteins, the GGAI or Omp4-15 proteins, of which at least 3 are present on the surface of C pneumoniae. We immunized rabbits with recombinant GGAI proteins and used these antibodies in immunofluorescence microscopy of experimentally infected mice. In lung sections, a massive infiltration with polymorph nuclear neutrophil cells was observed. In the bronchial epithelial cells, C pneumoniae inclusions were seen. Evidence was found of differential expression of the GGAI proteins. CONCLUSIONS: On the basis of surface localization, differential expression, and the fact that the proteins are recognized by the human humoral immune response, we speculate whether these proteins, in addition to the lipopolysaccharides, are of importance for the immunopathogenesis of C pneumoniae.