RESUMO
Lentiviral modification of hematopoietic stem cells (HSCs) paved the way for in vivo experimentation and therapeutic approaches in patients with genetic disease. A disadvantage of this method is the use of a ubiquitous promoter leads not only to genetic modification of the leukocyte subset of interest e.g. T-cells, but also all other subsequent leukocyte progeny of the parent HSCs. To overcome this limitation we tested a bicistronic lentivirus, enabling subset specific modifications. Designed novel lentiviral constructs harbor a global promoter (mPGK) regulating mCherry for HSCs selection and a T-cell specific promoter upstream of eGFP. Two T-cell specific promoters were assessed: the distal Lck-(dLck) and the CD3δ-promoter. Transduced HSCs were FACS sorted by mCherry expression and transferred into sublethally irradiated C57/BL6 mice. Successful transplantation and T-cell specific expression of eGFP was monitored by peripheral blood assessment. Furthermore, recruitment response of lentiviral engineered leukocytes to the site of inflammation was tested in a peritonitis model without functional impairment. Our constructed lentivirus enables fast generation of subset specific leukocyte transgenesis as shown in T-cells in vivo and opens new opportunities to modify other HSCs derived subsets in the future.
Assuntos
Células-Tronco Hematopoéticas/virologia , Infecções por Lentivirus/virologia , Lentivirus/genética , Subpopulações de Linfócitos T/fisiologia , Subpopulações de Linfócitos T/virologia , Animais , Complexo CD3/genética , Linhagem Celular Tumoral , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Inflamação/genética , Inflamação/virologia , Leucócitos/fisiologia , Leucócitos/virologia , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genéticaRESUMO
Human sera are the first choice as controls for diagnostic applications such as immunoassays, but are limited regarding availability, varying quality, and high costs. In this study, we aimed to circumvent these limitations by the use of a chimeric adaptor molecule comprising the extracellular domains of the human FcγRI (CD64) fused with human IgE Fc domains (CD64-IgE Fc). Allergen-specific antibodies were produced in rabbits using eight different allergens, extracts, and allergen mixtures including mites, pollen, drugs, and food. Preincubation of polyclonal IgG with CD64-IgE Fc established allergen-specific artificial sera that showed comparable results for more than 20 allergens and allergen extracts in three diagnostic systems for the determination of specific IgE. The agreement for these artificial sera is within ±1 radioallergosorbent test (RAST) class. Hence, rabbit IgG complexed with the IgG-specific CD64-IgE Fc adaptor molecule could provide a substitute for human reference sera with specificity for virtually any protein of interest.
Assuntos
Alérgenos/imunologia , Anticorpos/imunologia , Substitutos Sanguíneos/efeitos adversos , Imunoglobulina E/imunologia , Receptores de IgG/imunologia , Proteínas Recombinantes de Fusão/imunologia , Anticorpos/genética , Especificidade de Anticorpos/imunologia , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Imunização , Imunoglobulina E/química , Imunoglobulina E/genética , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Ligação Proteica/imunologia , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Receptores de IgG/química , Receptores de IgG/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: Analyses of the molecular basis underlying allergenicity and allergen cross-reactivity, as well as improvement of allergy diagnostics and therapeutics, are hampered by the lack of human monoclonal IgE antibodies and knowledge about their epitopes. Here, we addressed the consecutive generation and epitope delineation of a human monoclonal IgE against the prototypic allergen Bet v 1. METHODS: Phage-display scFv hybrid libraries of allergic donor-derived VH epsilon and synthetic VL were established from 107 mononuclear cells. An obtained scFv was converted into human immunoglobulin formats including IgE. Using variants of Bet v 1, the epitope of the antibody was mapped and extrapolated to other PR10 proteins. RESULTS: The obtained antibodies exhibited pronounced reactivity with Bet v 1, but were not reactive with the homologous PR10 protein Mal d 1. The epitope as defined by the IgE paratope and a set of chimeric Bet v 1 fusion proteins and fragments could be assigned to a C-terminal helix-structured motif comprised by amino acid residues 132-154, including the critical residue E149. Grafting this motif re-established the reactivity of the per se nonreactive Mal d 1 framework. Cross-reactivities predicted by primary structure analyses of different isoforms and PR10 proteins were verified by allergen chip-based analyses. CONCLUSIONS: The obtained results demonstrate that hybrid IgE repertoires represent a source for human antibodies with genuine paratopes. The IgE-derived information about the IgE epitope nature of Bet v 1 and homologues allows for detailed insights into molecular aspects of allergenicity and cross-reactivity within the PR10 protein family.
Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Epitopos/imunologia , Fagus/imunologia , Imunoglobulina E/imunologia , Proteínas de Plantas/imunologia , Alérgenos/química , Sequência de Aminoácidos , Especificidade de Anticorpos , Antígenos de Plantas/química , Reações Cruzadas/imunologia , Epitopos/química , Biblioteca Gênica , Humanos , Imunoglobulina E/química , Imunoglobulina E/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Proteínas de Plantas/química , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de SequênciaRESUMO
BACKGROUND: Generalized systemic reactions to stinging hymenoptera venom constitute a potentially fatal condition in venom-allergic individuals. Hence, the identification and characterization of all allergens is imperative for improvement of diagnosis and design of effective immunotherapeutic approaches. Our aim was the immunochemical characterization of the carbohydrate-rich protein Api m 10, an Apis mellifera venom component and putative allergen, with focus on the relevance of glycosylation. Furthermore, the presence of Api m 10 in honeybee venom (HBV) and licensed venom immunotherapy preparations was addressed. METHODS: Api m 10 was produced as soluble, aglycosylated protein in Escherichia coli and as differentially glycosylated protein providing a varying degree of fucosylation in insect cells. IgE reactivity and basophil activation of allergic patients were analyzed. For detection of Api m 10 in different venom preparations, a monoclonal human IgE antibody was generated. RESULTS: Both, the aglycosylated and the glycosylated variant of Api m 10 devoid of cross-reactive carbohydrate determinants (CCD), exhibited IgE reactivity with approximately 50% of HBV-sensitized patients. A corresponding reactivity could be documented for the activation of basophils. Although the detection of the native protein in crude HBV suggested content comparable to other relevant allergens, three therapeutical HBV extracts lacked detectable amounts of this component. CONCLUSION: Api m 10 is a genuine allergen of A. mellifera venom with IgE sensitizing potential in a significant fraction of allergic patients independent of CCD reactivity. Thus, Api m 10 could become a key element for component-resolved diagnostic tests and improved immunotherapeutic approaches in hymenoptera venom allergy.
