RESUMO
The central dogma of gene therapy relies on the application of novel therapeutic genes to treat or prevent diseases. The main types of vectors used for gene transfer are adenovirus, retrovirus, lentivirus, liposome, and adeno-associated virus vectors. Gene therapy has emerged as a promising alternative for the treatment of inflammatory diseases. The main targets are cytokines, co-stimulatory molecules, and different types of cells from hematological and mesenchymal sources. In this review, we focus on molecules with anti-inflammatory effects used for in vivo gene therapy mediated by adenoviral gene transfer in the treatment of immune-mediated inflammatory diseases, with particular emphasis on autoinflammatory and autoimmune diseases.
Assuntos
Adenoviridae/imunologia , Anti-Inflamatórios/uso terapêutico , Doenças Autoimunes/terapia , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos , Inflamação/terapia , Animais , Doenças Autoimunes/imunologia , Produtos Biológicos/uso terapêutico , Inflamação/imunologia , Camundongos , Coelhos , RatosRESUMO
BACKGROUND: Ro60 ribonucleoprotein is a conserved molecule belonging to the family of Ro ribonucleoproteins and targeted by autoantibodies produced in patients with systemic lupus erythematosus or Sjogren's syndrome. Ro60 plays a role in postranscription events, as well as in the nucleocytoplasmic shuttling of RNA polymerase III transcripts. OBJECTIVE: To evaluate the in vitro effects of Ro60 on T3 RNA polymerase transcription. METHODS: Ro60 ribonucleoprotein was affinity-purified from human spleen extracts using 4B-Sepharose linked to anti-Ro60 monoclonal antibodies. Purified Ro60 was incorporated into the T3 RNA polymerase transcription reaction system using pTRI-beta-Actin-human DNA as a template. RESULTS: Ro60 inhibited initiation of the transcription process in a dose-dependent manner; neither elongation nor termination was affected by Ro. CONCLUSION: In vitro, Ro60 appears to inhibit the transcription of a T3 RNA polymerase-dependent template.
Assuntos
Autoantígenos/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Citoplasmático Pequeno , Ribonucleoproteínas/metabolismo , Transcrição Gênica/fisiologia , Adulto , Autoantígenos/isolamento & purificação , Autoantígenos/farmacologia , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Masculino , Valores de Referência , Ribonucleoproteínas/isolamento & purificação , Ribonucleoproteínas/farmacologia , Sensibilidade e Especificidade , Baço/química , Baço/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Serum antibodies in scleroderma patients are generally directed against the nucleolus and centromeres. A small proportion of patients have serum antibodies to the centrioles and mitotic apparatus. OBJECTIVE: To determine the prevalence of serum autoantibodies against the mitotic apparatus in scleroderma patients. MATERIAL AND METHODS: Sera from 113 patients with various forms of scleroderma were tested for antinuclear antibodies by indirect immunofluorescence on HEp-2 cells. The specificity of the antibodies was determined by Western blot. RESULTS: Only two scleroderma sera recognized the mitotic apparatus. Western blot results showed that in both cases the target was an about 235 kDa protein corresponding to the NuMA determinant. Affinity-purified anti-NuMa antibodies were used to perform immunolocalization in synchronized HEp-2 cells using scanning laser confocal microscopy. The anti-NuMA autoantibodies recognized the mitotic asters but neither the centrioles nor the microtubules. CONCLUSION: Our data suggest that anti-NuMA autoantibodies may be devoid of clinical significance in scleroderma. However, they remain useful as probes in cell biology studies.
Assuntos
Anticorpos Antinucleares/análise , Lúpus Eritematoso Sistêmico/imunologia , Escleroderma Sistêmico/imunologia , Fuso Acromático/imunologia , Anticorpos Antinucleares/imunologia , Western Blotting , Células Cultivadas , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Lúpus Eritematoso Sistêmico/sangue , Masculino , Microscopia Confocal , Escleroderma Sistêmico/sangue , Sensibilidade e EspecificidadeRESUMO
These studies were carried out to examine the presence of the inflammatory cytokines IL-6 and TNFalpha in kidneys of patients with lupus nephritis as an indicator of their possible role in its pathogenesis. A total of 19 kidney biopsies from patients with type III or IV lupus nephritis were processed by direct immunofluorescence using monoclonal anti-IL-6 and TNFalpha antibodies. Local expression of these genes was demonstrated both by in situ hybridization and by reverse transcriptase-PCR amplification of total RNA isolated from kidney tissue. Fifty-two percent of the biopsies exhibited IL-6 and TNFalpha deposited along the glomeruli and tubules; in situ expression of these cytokines was demonstrated in 6 biopsies with type IV, and 1 with type III nephritis. Inflammatory cytokines are actively synthesized in the kidneys of patients with lupus nephritis and therefore, may play a role in its pathogenesis.
Assuntos
Genes/genética , Interleucina-6/genética , Nefrite Lúpica/genética , Fator de Necrose Tumoral alfa/genética , Adolescente , Adulto , Feminino , Técnica Direta de Fluorescência para Anticorpo , Expressão Gênica/genética , Humanos , Hibridização in Situ Fluorescente , Rim/química , Rim/metabolismo , Masculino , RNA Mensageiro/análiseRESUMO
Estudiamos la prevalencia de anticuerpos contra la DNA-topoisomerasa y su restricción a isotipos, en 20 anticuerpos anti-nucleolares positivos.Los isotipos fueron determinados en pruebas de inmunofluorescencia indirecta con células HEp-2 y anticuerpos monoclonales anti-IgG1, IgG2, IgG3 e IgG4:La especialidad anti-DNA-topoisomerasa I se determinó por pruebas de doble difusión en agar y por Western blot,utilizando como antígeno, extracto de timo de ternera y un suero prototipo anti-Scl-70. Resultados: Los patrones de fluorescencia tuvieron una localización nucleolar en todos los casos y el 75 por ciento presentó conjuntamente patrón nucleosomal. En pruebas de doble difusión, de los 20 sueros, siete (35 por ciento)reconocieron los extractos de timo de ternera y de ellos, 4(20 por ciento) presentaron especificidad anti-DNA-Topoisomerasa I. Por Western blot, 15 sueros (75 por ciento) mostraton bandas reactivas a un antígeno de 70 kD (DNA-Topo I) y fueron de subclase IgG1.Estos resultados nos permiten concluir que los sueros de Esclerodermia con anticuerpos anti-nucleolares presentan , en gran proporción , especificidad anti- DNA- topoisomerasa I; no obstante, las pruebas de precipitación en agar, no son lo suficientemente sensibles para detectar su prevalencia real; para este propósito, se requiere de pruebas sensitivas como el Western blot
Assuntos
Anticorpos Antinucleares , Escleroderma Sistêmico/imunologiaRESUMO
Estudiamos la prevalencia de anticuerpos contra la DNA-topoisomerasa y su restricción a isotipos, en 20 anticuerpos anti-nucleolares positivos.Los isotipos fueron determinados en pruebas de inmunofluorescencia indirecta con células HEp-2 y anticuerpos monoclonales anti-IgG1, IgG2, IgG3 e IgG4:La especialidad anti-DNA-topoisomerasa I se determinó por pruebas de doble difusión en agar y por Western blot,utilizando como antígeno, extracto de timo de ternera y un suero prototipo anti-Scl-70. Resultados: Los patrones de fluorescencia tuvieron una localización nucleolar en todos los casos y el 75 por ciento presentó conjuntamente patrón nucleosomal. En pruebas de doble difusión, de los 20 sueros, siete (35 por ciento)reconocieron los extractos de timo de ternera y de ellos, 4(20 por ciento) presentaron especificidad anti-DNA-Topoisomerasa I. Por Western blot, 15 sueros (75 por ciento) mostraton bandas reactivas a un antígeno de 70 kD (DNA-Topo I) y fueron de subclase IgG1.Estos resultados nos permiten concluir que los sueros de Esclerodermia con anticuerpos anti-nucleolares presentan , en gran proporción , especificidad anti- DNA- topoisomerasa I; no obstante, las pruebas de precipitación en agar, no son lo suficientemente sensibles para detectar su prevalencia real; para este propósito, se requiere de pruebas sensitivas como el Western blot