RESUMEN
PURPOSE: Previous studies have demonstrated that adeno-associated virus (AAV) efficiently transduced retinal pigmented epithelial (RPE) cells. The goal of this study was to further evaluate and characterize transgene expression within the RPE cells over time in vivo. METHODS: Adeno-associated virus-mediated gene transfer was monitored and quantified by retinal photography following subretinal injection of a recombinant AAV encoding the green fluorescent protein gene (rAAVCMV-gfp) into rat eyes. Retinal function of transduced rat eyes was measured by electroretinography. RESULTS: The maximum level of transgene expression was reached at 8 weeks postinjection followed by a gradual decrease throughout the experimental period. Interestingly, it was observed that while gfp expression was stable in some RPE cells, gfp fluorescence completely disappeared in other cells over the duration of the experiment. The expression of AAV-mediated gfp in RPE cells did not alter the retinal function for over 1 year CONCLUSIONS: These results confirm the importance of this direct visualization system to study vector transgene expression in vivo and support the use of AAV for diseases treatable by targeting RPE cells.
Asunto(s)
Dependovirus/genética , Expresión Génica , Proteínas Luminiscentes/genética , Retina/metabolismo , Animales , Electrorretinografía , Fluorofotometría , Estudios de Seguimiento , Vectores Genéticos , Proteínas Fluorescentes Verdes , Inyecciones , Monitoreo Fisiológico , Fotograbar , Ratas , Proteínas Recombinantes de Fusión , TransfecciónRESUMEN
The purpose of this study was to evaluate recombinant adeno-associated virus (AAV) as an in vivo gene transfer vector for the retina and to explore the possibility of monitoring the expression of green fluorescent protein (GFP) using a noninvasive method. Rats were injected subretinally with rAAV-gfp or rAAV-lacZ. Strong expression of the reporter gene in a circular area surrounding the injection site was observed in retinal whole mounts and tissue sections. Higher magnification revealed that cells demonstrating high levels of green fluorescence were hexagonal in shape, indicating they were retinal pigment epithelium (RPE) cells. Histological observation of retinal sections demonstrated that recombinant AAV specifically transduced RPE cells. Ten animals were injected with rAAV-gfp for longitudinal studies and the fluorescence was monitored by retinal fluorescence photography. The GFP signal was detected in 100% of the animals as early as 2 weeks postinjection and remained present throughout the experimental period of 4 months. After 2 weeks, a gradual increase in the number of transduced cells occurred before reaching maximal levels of GFP expression at 8 weeks. This was followed by a small decrease over 4 weeks before reaching stable expression at 16 weeks. Our results demonstrated that rAAV efficiently transduces rat RPE cells and that retinal fluorescence photography is suitable for monitoring GFP expression. By using this noninvasive technique, we demonstrated that repetitive measurements of GFP expression in vivo in the rAAV-gfp-transduced retina are possible. This study demonstrated that retinal fluorescence photography is a potent tool for studying AAV-mediated gene delivery in the retina.
Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen/normas , Vectores Genéticos , Retina/metabolismo , Animales , Resistencia a Medicamentos/genética , Estudios de Evaluación como Asunto , Fluorescencia , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Neomicina/farmacología , Fotograbar , Ratas , Recombinación Genética , Transducción GenéticaRESUMEN
Polymyositis is regarded as an autoimmune inflammatory muscle disease. A major subgroup of patients have autoantibodies to cellular histidyl-transfer RNA synthetase (HRS). We have analyzed the role of the autoantigen HRS in the induction of murine myositis in a comparative study of inoculation of BALB/c mice with recombinant HRS protein versus naked DNA coding for HRS. Adult BALB/c mice produced antibodies to human HRS following inoculation with HRS protein and adjuvant, but myositis was not observed. Alternatively, expression plasmid DNA constructs encoding full-length and truncated human HRS were inoculated intramuscularly in gene transfer studies. DNA-inoculated mice produced relatively low anti-HRS antibody titers. However, in contrast to recombinant HRS protein-inoculated mice, HRS gene transfer induced pathology with evidence of cellular infiltration of perivascular and endomysial regions of the inoculated muscle. Multiple inoculations of a plasmid construct encoding a hybrid molecule consisting of HRS and the transferrin receptor cytoplasmic tail induced the highest levels of antibodies and persisting cellular infiltration. Unlike HRS, expression of influenza virus hemagglutinin (HA) following inoculation of an HA plasmid did not induce myositis. Transfer of naked DNA constructs expressing HRS is likely to provide valuable information on the autoimmune response to this protein and its role in the development of myositis.
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Histidina-ARNt Ligasa/genética , Histidina-ARNt Ligasa/inmunología , Inmunización , Miositis/inmunología , Animales , Modelos Animales de Enfermedad , Esquema de Medicación , Femenino , Técnicas de Transferencia de Gen , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/farmacología , Histidina-ARNt Ligasa/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos , Datos de Secuencia Molecular , Músculo Esquelético/inmunología , Músculo Esquelético/patología , Miositis/inducido químicamente , Polimiositis/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacologíaRESUMEN
The genetic predisposition to inclusion body myositis (IBM) is probably multifactorial. The deposition of the beta-amyloid protein is a characteristic histological feature of both IBM and Alzheimer's disease (AD). The epsilon 4 allele of apolipoprotein E (APO E) has been strongly associated with familial and late-onset AD. We therefore compared the APO E allele frequencies in a group of 14 patients with IBM with those in a group of patients with other inflammatory muscle diseases and in the general population. The frequency of the epsilon 4 allele in IBM was increased (0.29) compared with that in patients with other inflammatory muscle diseases (0.15) and the general population (0.13) (p < 0.05). These data suggest that APO E genotype may be one of the factors involved in determining the predisposition to the development of IBM.
Asunto(s)
Apolipoproteínas E/genética , Miositis por Cuerpos de Inclusión/genética , Adulto , Edad de Inicio , Anciano , Alelos , Femenino , Genotipo , Humanos , Cuerpos de Inclusión Viral/genética , Masculino , Persona de Mediana Edad , Distribución por SexoRESUMEN
The ability of non-professional antigen-presenting cells (APC) to process and present antigen to the immune system has been the subject of debate in autoimmunity and tumour immunology. The role of muscle cells in the processing and presentation of antigen to T cells via class I and class II MHC pathways is of increasing interest. Muscle cells are the targets of autoimmune attack in the inflammatory muscle diseases, and direct intramuscular injection of antigen-expressing DNA constructs is under scrutiny as a means of vaccination. Furthermore, the immunological properties of muscle cells are of relevance in attempts to transfer myoblasts as replacement cells in dystrophic diseases or as depot cells for the secretion of certain molecules in deficiency states. Using class I and class II MHC transfectant clones of the C2C12 myoblast cell line, myoblasts have been shown to be capable of presenting antigen to, and stimulating secretion of IL-2 by, T cell hybridomas via both of these pathways. The epitopes which are dominantly presented by professional APC after processing of native antigens were also presented by the myoblast cell line after processing of either ovalbumin (class I) or hen egg lysozyme (class II). Further, antigen processing and presentation via the class II pathway were enhanced by pretreatment of the myoblasts with interferon-gamma (IFN-gamma). Up-regulation of invariant chain expression by this treatment may have contributed to this enhanced presentation, but an effect of IFN-gamma on the expression of other molecules such as H-2 DM may have also played a role. The demonstration of the antigen-presenting properties of these myoblasts is of relevance to all three areas mentioned above. In each situation myoblasts comprise a significant population within muscle. In the case of inflammatory muscle diseases the process of muscle degeneration and regeneration is on-going, while in the vaccination procedure some muscle damage occurs, and vaccination is more effective when muscle damage has preceded inoculation.
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Presentación de Antígeno , Músculos/inmunología , Animales , Antígenos de Diferenciación de Linfocitos B/análisis , Línea Celular , Antígenos de Histocompatibilidad Clase I/fisiología , Antígenos de Histocompatibilidad Clase II/análisis , Antígenos de Histocompatibilidad Clase II/fisiología , Interferón gamma/farmacología , Ratones , Ratones Endogámicos C3H , Ovalbúmina/inmunología , TransfecciónRESUMEN
The region between tumor necrosis factor (TNF) and HLA-B in the central major histocompatibility complex (MHC) is polymorphic and associated with several autoimmune diseases. The polymorphisms are haplospecific or haplotypic and retained within the same MHC ancestral haplotype (AH). We have cloned this region from four AHs into lambda bacteriophage and found that a highly polymorphic region in the TNF-HLA-B interval is duplicated. Clones from this region isolated from three MHC AHs show two populations. The regions, designated CL1 and CL2, have different sizes of Bam HI fragments carrying the duplicated sequences. These fragments correspond to those seen after Bam HI restriction fragment length polymorphism (RFLP) analysis of genomic DNA from the same cell lines. Pulsed field gel electrophoresis analysis shows that both CL1 and CL2 are in the central MHC and are about 16 kilobases apart. DNA cloning and RFLP analysis demonstrate that the CL region is highly polymorphic but retained within an MHC AH. Polymorphism and duplication are common characteristics of the genes found in the MHC and therefore the CL sequences have the potential to be interesting in this respect.
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Antígenos HLA-B/genética , Familia de Multigenes , Polimorfismo de Longitud del Fragmento de Restricción , Autorradiografía , Centrómero , Clonación Molecular , Electroforesis en Gel de Campo Pulsado , Complejo Mayor de Histocompatibilidad , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
A degree of conservation of the genes located between class II and class I [central major histocompatibility complex (MHC) genes] is apparent among mammalian species including primates and the mouse. Few others have been analyzed. The caprine MHC is of particular interest, since it has recently been observed that susceptibility to a lentivirus-induced polyarthritis (caprine arthritis) segregates with serologically defined MHC class I antigens. This arthritis resembles, in a number of respects, rheumatoid arthritis in man. Human cDNA probes were used to examine the caprine central MHC and class I and II genes by restriction fragment length polymorphism (RFLP) and by pulsed field gel electrophoresis (PFGE) in order to define the polymorphism and linkage of central MHC genes to class I and class II genes. An outbred population of dairy goats (Saanen, British Alpine, Anglo Nubian, and Toggenberg) was examined for class I and class II RFLPs. Both regions were found to be highly polymorphic. The number of fragments hybridizing to an HLA-B7 probe after Eco RI, Bam HI, Bgl II, or Hind III digestion suggests there may be 10-13 class I genes. The degree of polymorphism was comparable to that reported in the mouse. Limited polymorphism was found in the central MHC genes. The caprine C4 and CYP21 genes were duplicated and demonstrated RFLP with Bam HI, Hind III, Eco RV, and Taq I. An infrequent Taq I C2 polymorphism was found. PFGE revealed substantial conservation of both the order and linkage of the central MHC genes when compared with mouse and man. C4, C2, CYP21, HSP70, and tumor necrosis factor (TNF) genes are all located within 800 kilobase (kb) of the class I loci. Distant from the class I region, the C4, C2, and CYP21 genes are linked on a short genomic segment (180 kb Not I and 190 kb Pvu I fragments). HSP70 cohybridizes with the complement genes on a 380 kb Mlu I fragment. Linkage of HSP70, TNF, and class I genes was found on a single Not I fragment (610 kb). TNF and class I cohybridize on Pvu I (730 kb) and Not I (610 kb) fragments. Conservation of a similar central MHC genomic structure across species argues for functional interaction between the central MHC genes. We postulate selection for these central MHC genes through their role as non antigen-specific regulators of immune response.