RESUMEN
Self-specific B cells play a main role in the pathogenesis of lupus. This autoimmune disease is characterized by the generation of autoantibodies against self antigens, and the elimination of B and T cells involved in the pathological immune response is a logical approach for effective therapy. We have previously constructed a chimeric molecule by coupling a DNA-mimotope peptides to an anti-CD32 antibody. Using this protein molecule for the treatment of lupus-prone MRL/lpr mice, we suppressed selectively the autoreactive B-lymphocytes by cross-linking B cell receptors with the inhibitory FcγRIIb receptors. This approach was limited by the development of anti-chimeric antibodies in MRL mice. In order to avoid this problem, we established a murine severe combined immunodeficiency lupus model, allowing a long-term chimera therapy. Elimination of the double-stranded DNA-specific B cells by chimera therapy in MRL-transferred immunodeficient mice resulted in inhibition of T cell proliferation and prevented the appearance of IgG anti-DNA antibodies and of proteinuria.
Asunto(s)
Linfocitos B/inmunología , ADN/inmunología , Lupus Eritematoso Sistémico/inmunología , Traslado Adoptivo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Linfocitos B/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN/química , ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulinas Intravenosas/administración & dosificación , Glomérulos Renales/inmunología , Glomérulos Renales/patología , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/mortalidad , Lupus Eritematoso Sistémico/patología , Lupus Eritematoso Sistémico/terapia , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos MRL lpr , Ratones SCID , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Premedicación , Receptores de IgG/metabolismo , Transducción de Señal , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Fluorescent microscopy and fluorescent imaging by flow cytometry are two of the fastest growing areas in the medical and biological research. Innovations in fluorescent chemistry and synthesis of new dye probes are closely related to the development of service equipment such as light sources, and detection techniques. Among compounds known as fluorescent labels, the cyanine-based dyes have become widely used since they have high excitation coefficients, narrow emission bands and high fluorescence upon binding to nucleic acids. The key methods for evaluation of apoptosis and cell cycle allow measuring DNA content by several flow cytometric techniques. We have synthesized new monomethine cyanine dyes and have characterized their applicability for staining of live and/or apoptotic cells. Imaging experiments by flow cytometry and confocal laser scanning microscopy (CLSM) have been also performed. Two of the dyes have shown high-affinity binding to the nuclei at high dilutions, up to 10(-9)M. Flow cytometry and CLSM have confirmed that these dyes labeled selectively non-living, e.g. ethanol-fixed cells that makes them appropriate for estimations of cell viability and apoptosis. The novel structures proved to be appropriate also for analysis of the cell cycle.
Asunto(s)
Citometría de Flujo , Colorantes Fluorescentes/química , Microscopía Confocal , Bazo/metabolismo , Células 3T3 , Animales , Apoptosis , Carbocianinas/química , Supervivencia Celular , Células Cultivadas , Femenino , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Ratones , Ratones Endogámicos BALB C , Bazo/citologíaRESUMEN
Killed viral vaccines and bacterial toxoids are weakly immunogenic. Numerous compounds are under evaluation as immunological adjuvants and peptide-carriers to improve the immune response. The hemocyanins, giant extracellular copper proteins in the blood of many mollusks, are widely used as immune stimulants. In the present study we investigated the adjuvant properties of hemocyanins isolated from marine gastropods Rapana thomasiana and Megathura crenulata. An immunization with Influenza vaccine or tetanus toxoid combined with Rapana thomasiana hemocyanin (RtH) and Keyhole limpet hemocyanin (KLH) in mice induced an anti-influenza cytotoxic response lasting at least 5 months and an antibody response to viral proteins. The IgG antibody response to the tetanus toxoid (TT) combined with RtH or KLH was comparable to the response of the toxoid in complete Freund's adjuvant. The results obtained demonstrate that the both hemocyanins are acceptable as potential bio-adjuvants for subunit vaccines.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Gastrópodos/metabolismo , Hemocianinas/análogos & derivados , Hemocianinas/farmacología , Animales , Proteínas Bacterianas/inmunología , Línea Celular , Perros , Femenino , Hemocianinas/química , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Vacunas contra la Influenza/inmunología , Ratones , Ratones Endogámicos BALB C , Orthomyxoviridae , Toxoide Tetánico/inmunología , Proteínas Virales/inmunologíaRESUMEN
A novel approach for the selective silencing of targeted autoreactive B lymphocytes is reviewed that mimics the physiological mechanisms for suppressing B cell activity. It is based on the use of bi- or tri-specific chimeric antibodies that cross-link BCRs with a pre-selected antigen-binding specificity with one or more inhibitory types of receptors on the surface of the same disease-associated B lymphocyte. The effect of these engineered antibodies was proved to be specific as they only suppressed the production of the targeted pathological antibodies while sparing those with other specificities. The administration of the chimeric molecules to lupus-prone MRL/lpr mice resulted in decreased levels of disease-associated IgG autoantibodies and of proteinuria, in the prevention of cutaneous lesions, in decreased sizes of the lymphoid organs and in prolonged survival. These results prove that it is indeed possible to selectively silence unwanted B lymphocytes as well as to significantly delay the natural course of a spontaneous antibody-mediated autoimmune disease.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Enfermedades Autoinmunes/terapia , Autoinmunidad , Linfocitos B/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Autoanticuerpos , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Linfocitos B/patología , Inmunoglobulina G/inmunología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/terapia , Ratones , Ratones Endogámicos MRL lpr , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de IgG/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunologíaRESUMEN
DNA-specific B cells in SLE represent a logical target for therapeutic intervention. We hypothesize that it is possible to re-establish tolerance to native DNA in SCID mice with cells transferred from SLE patients or from lupus-prone MRL/lpr mice by administering chimeric molecules, containing a monoclonal antibody against inhibitory B cell receptors coupled to a peptide that antigenically mimics DNA. These protein-engineered molecules are able to co-crosslink selectively the antigen receptors of B cells possessing anti-native DNA specificity with the inhibitory surface receptors, thus delivering a strong suppressive signal.
Asunto(s)
Anticuerpos Antinucleares/genética , Anticuerpos Biespecíficos/uso terapéutico , Inmunoterapia , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/terapia , Animales , Anticuerpos Antinucleares/inmunología , Anticuerpos Biespecíficos/inmunología , Linfocitos B/inmunología , Biomimética , ADN/inmunología , Modelos Animales de Enfermedad , Retroalimentación Fisiológica , Humanos , Tolerancia Inmunológica , Inmunoterapia/tendencias , Ratones , Ingeniería de ProteínasRESUMEN
Intravenous immunoglobulin (IVIg) preparations are known to modulate autoimmune/inflammatory diseases through several F(ab')(2)- and Fc-dependent mechanisms. In this study, we show that the in vitro and the in vivo exposure of B lymphocytes from lupus-prone and from healthy mice to IVIg results in an increased expression of their surface inhibitory FcgammaIIB receptors. Further, this exposure enhanced the ability of a chimeric antibody, cross-linking FcgammaRIIB and immunoglobulin receptors on DNA-specific B lymphocytes, to suppress IgG anti-DNA antibody production. F(ab')(2) fragments of IVIg had a similar activity as the intact preparation, whereas Fc fragments had no effect. This study describes a novel approach with clinical relevance for modulating B lymphocyte activity.