RESUMEN

Podocytes are essential to the structure and function of the glomerular filtration barrier; however, they also exhibit increased expression of MHC class II molecules under inflammatory conditions, and they remove Ig and immune complexes from the glomerular basement membrane (GBM). This finding suggests that podocytes may act as antigen-presenting cells, taking up and processing antigens to initiate specific T cell responses, similar to professional hematopoietic cells such as dendritic cells or macrophages. Here, MHC-antigen complexes expressed exclusively on podocytes of transgenic mice were sufficient to activate CD8+ T cells in vivo. In addition, deleting MHC class II exclusively on podocytes prevented the induction of experimental anti-GBM nephritis. Podocytes ingested soluble and particulate antigens, activated CD4+ T cells, and crosspresented exogenous antigen on MHC class I molecules to CD8+ T cells. In conclusion, podocytes participate in the antigen-specific activation of adaptive immune responses, providing a potential target for immunotherapies of inflammatory kidney diseases and transplant rejection.

Asunto(s)

Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Barrera de Filtración Glomerular/inmunología , Podocitos/citología , Podocitos/inmunología , Inmunidad Adaptativa/inmunología , Animales , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/patología , Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Comunicación Celular/inmunología , Femenino , Membrana Basal Glomerular/citología , Membrana Basal Glomerular/inmunología , Membrana Basal Glomerular/metabolismo , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunoglobulina G/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Microesferas , Ovalbúmina/inmunología , Ovalbúmina/farmacocinética , Fagocitosis/inmunología , Podocitos/metabolismo

RESUMEN

Diphtheria toxin (DTx) receptor (DTR)-mediated conditional cell ablation in transgenic mice is a powerful tool to analyze cell function in vivo. Transgenic mice with cell-specific expression of the human DTR have been developed that allow conditional depletion of these cells in vivo through administration of the toxin. We have performed a careful analysis of mice after DTx injection and found an unexpected side effect. Treatment of wild-type C57BL/6 mice with DTx leads to a marked transient and completely reversible proteinuria, as a consequence of podocyte dysfunction that is morphologically characterized by foot process fusion and detachment from the glomerular basal membrane. In vitro analysis displayed that DTx-treated podocytes show diminished attachment to basal membrane proteins. Five to 9 days after DTx application the mice recover completely. Glomerular proteinuria is a hallmark of glomerular disease due to dysfunction of the filtration barrier. Rodents have been extensively used experimentally to better define the mechanisms of disease induction and progression. However, nongenetic mouse models of proteinuric glomerular damage are limited and display various shortcomings. We suggest DTx-induced transient kidney dysfunction as a new reversible model of experimental podocyte injury, which could be used as an additional approach to complement studies in human.

Asunto(s)

Toxina Diftérica/toxicidad , Podocitos/efectos de los fármacos , Proteinuria/inducido químicamente , Análisis de Varianza , Animales , Peso Corporal/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCID , Ratones Transgénicos , Podocitos/fisiología , Proteinuria/patología , Proteinuria/fisiopatología

RESUMEN

There is still a vital need for new therapies in order to prevent or treat type I diabetes. In this respect, we report that MCS-18 a novel natural product isolated from the plant Helleborus purpurascens (i.e. Christmas rose) is able to increase diabetes free survival using the NOD-mouse model, which is accompanied with a diminished IFN-γ secretion of splenocytes. In the animal group which has been treated with MCS-18 during week 8 and week 12 of age 70% of the animals showed a diabetes free survival at week 30, whereas in contrast in the untreated animals less than 10% were free of diabetes. MCS-18 treatment significantly reduced islet T-cell infiltrates as well as the rate of T-cell proliferation. Periinsular infiltrates in the MCS-18 treated animals showed a significantly enhanced number of Foxp3(+) CD25(+) T cells, indicating the increased presence of regulatory T cells. These studies show that MCS-18 exerts an efficient immunosuppressive activity with remarkable potential for the therapy of diseases characterized by pathological over-activation of the immune system.

Asunto(s)

Productos Biológicos/uso terapéutico , Diabetes Mellitus Tipo 1/prevención & control , Inmunosupresores/uso terapéutico , Animales , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/efectos de los fármacos , Insulina/metabolismo , Secreción de Insulina , Interferón gamma/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Estimación de Kaplan-Meier , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , ARN Mensajero/genética , Bazo/inmunología , Bazo/metabolismo , Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo

RESUMEN

HSV-1 is a very successful representative of the α-herpesvirus family, and ∼ 90% of the population is seropositive for this particular virus. Although the pathogen usually causes the well-known mild lesions on the lips, also, severe infections of the eye or the brain can be observed in rare cases. It is well known, that this virus can efficiently infect the most potent APCs, i.e., the DCs, in their immature and mature state. Although the infection of the iDC has been shown to be productive, infection of mMDDCs is believed to be abortive in the early phase of the viral replication cycle. In line with these findings, no virus particles can be detected in the supernatant of HSV-1-infected mMDDC. In this study, however, we show for the first time that this pathogen completes its replication cycle in mMDDCs. We detected the presence of viral gene transcripts of all three phases of the replication cycle, as well as of late viral proteins, and even the generation of small amounts of progeny virus. Although we could confirm the findings that these particles are not released into the supernatant, surprisingly, the newly generated viral particles can be passed on to Vero cells, as well as to primary keratinocytes in a cell-cell contact-dependent manner. Finally, we provide evidence that the viral gE is involved in the transfer of infectious virus from mMDDCs to other permissive cells.

Asunto(s)

Células Dendríticas/inmunología , Células Dendríticas/virología , Herpes Simple/transmisión , Herpesvirus Humano 1/fisiología , Virión/fisiología , Replicación Viral , Animales , Western Blotting , Adhesión Celular , Comunicación Celular , Movimiento Celular , Chlorocebus aethiops , Células Dendríticas/metabolismo , Herpes Simple/inmunología , Herpes Simple/metabolismo , ARN Mensajero/genética , ARN Viral/genética , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismo

RESUMEN

Salmonella enterica is a versatile vaccine carrier for heterologous antigens. One strategy for vaccine antigen delivery is the use of live attenuated S. enterica strains that translocate heterologous antigens into antigen-presenting cells by means of type III secretion systems (T3SS). The feasibility of this approach has been demonstrated in various experimental vaccination studies. The efficacy of recombinant live vaccines is critically influenced by the optimal level of attenuation and many other factors. For the rational design of approaches involving translocation by T3SS, additional parameters are the level of expression of the heterologous antigens and the selection of carrier proteins for the delivery of antigens to desirable subcellular compartments of the target cell. We deployed the Salmonella pathogenicity island 2 (SPI2)-encoded T3SS for antigen delivery. The SPI2-T3SS and effector proteins are encoded by members of the large SsrAB regulon, including promoters with highly variable strength of expression. We investigated the effect of various in vivo-activated promoters of the SsrAB regulon on the efficacy of recombinant Salmonella vaccines. We observed that the use of promoters with higher strength results in greater synthesis of recombinant antigens and greater stimulation of T-cell responses in cell culture assays for the stimulation of T cells by the model antigen ovalbumin. In contrast, in vaccination experiments, promoters with a low level of expression resulted in the induction of higher amounts of T cells reactive to the model antigen listeriolysin. These results demonstrate that high-level expression of heterologous antigens does not necessarily result in optimal stimulation of immune responses.

Asunto(s)

Antígenos/metabolismo , Células Dendríticas/microbiología , Regulación de la Expresión Génica , Macrófagos/microbiología , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/genética , Vacunas Sintéticas/metabolismo , Animales , Antígenos/genética , Antígenos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células de la Médula Ósea , Células Cultivadas , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ovalbúmina/genética , Ovalbúmina/inmunología , Plásmidos , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Regulón , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad , Linfocitos T/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vacunación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología

RESUMEN

Myocilin, a secreted glycoprotein of the olfactomedin family, is constitutively expressed in podocytes of the rat kidney and induced in mesangial cells during mesangioproliferative glomerulonephritis. As myocilin has been found to be associated with fibrillar components of the extracellular matrix, and adhesive properties have been shown for other members of the olfactomedin family, we hypothesized that myocilin might play a role in cell-matrix interactions in the glomerulus. To elucidate functional properties of myocilin, recombinant myocilin was expressed in 293 EBNA cells and purified by Ni-chelate and heparin chromatography. Culture plates were coated with myocilin, and primary rat mesangial cells and cells from an immortal murine podocyte cell line were seeded onto the plates in serum free conditions. Both cell types showed concentration-dependant attachment to myocilin, an effect that was statistically significant and could be blocked with specific antibodies. When compared to equal amounts of fibronectin or collagen 1, myocilin was less effective in promoting substrate adhesion. Synergistic effects in substrate adhesion were observed when myocilin was added to low concentrations of fibronectin. Twenty-five percent of cells that had attached to myocilin substrates showed spreading and expressed focal contacts which were labeled by vinculin/phalloidin staining. Comparable findings were observed when human or murine trabecular meshwork cells were seeded on myocilin substrates. Adhesive properties of myocilin required multimer formation, and were not observed when culture plates were coated with a C-terminal fragment of myocilin, containing the olfactomedin domain. We conclude that myocilin promotes substrate adhesion of podocytes and mesangial cells, and might contribute to cell-matrix adhesion of both cell types in vivo.

Asunto(s)

Adhesión Celular/efectos de los fármacos , Proteínas del Citoesqueleto/farmacología , Proteínas del Ojo/farmacología , Glicoproteínas/farmacología , Células Mesangiales/efectos de los fármacos , Podocitos/efectos de los fármacos , Animales , Línea Celular , Fibronectinas/farmacología , Adhesiones Focales/ultraestructura , Humanos , Células Mesangiales/citología , Ratones , Podocitos/citología , Ratas , Transfección

RESUMEN

OBJECTIVES: To study the role of cytotoxic T-lymphocyte (CTL) escape for disease progression in HIV-1 infection, we analyzed the CTL response to the dominant human leukocyte antigen (HLA)-B8-restricted CTL epitope FLKEKGGL (FL8) in HIV-1 Nef. METHODS: HIV-1 nef genes derived from 56 patients were analyzed by polymerase chain reaction (PCR)-based sequencing. T-cell responses against FL8 and mutated FL8 variants were detected by gamma-interferon (gamma-IFN) enzyme linked immunospot (ELISPOT) assay. RESULTS: The longitudinal analysis of an HIV-1-infected patient with good control of HIV-1 viremia for several years demonstrated an association of rising viremia with the emergence of CTL escape mutations within the HLA-B8-restricted Nef-specific CTL epitopes FLKEKGGL and WPAIRERM. Analysis of nef genes in 56 HIV-1-infected patients demonstrated a significant correlation between the occurrence of mutations in the FL8 epitope and the presence of HLA-B8. The mutations within the FL8 epitope could decrease CTL recognition; however, there was strong variation regarding the recognition of viral variants between individual donors. The presence of FL8 mutations was associated with lower CD4 cell counts and higher viral loads. CONCLUSIONS: Our data demonstrate a strong CTL selection pressure on the immunodominant HLA-B8-restricted CTL epitope FL8 in HIV-1 Nef. The association of FL8 mutations with lower CD4 cell counts indicates an important role of CTL escape mutations for disease progression.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/inmunología , Epítopos de Linfocito T , Antígeno HLA-B8/fisiología , Epítopos Inmunodominantes , Linfocitos T Citotóxicos/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Síndrome de Inmunodeficiencia Adquirida/virología , Secuencia de Bases , Recuento de Linfocito CD4 , Femenino , Antígeno HLA-B8/análisis , Humanos , Estudios Longitudinales , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Carga Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química

RESUMEN

The main source for endogenous peptides presented by the MHC class I (MHC-I) pathway are de novo-synthesized proteins which are degraded via the ubiquitin proteasome pathway. Different MHC-I Ag pools can be distinguished: first, short-lived defective ribosomal products, which are degraded in concert with or shortly after their synthesis, and, second, functional proteins that enter the standard protein life cycle. To compare the contribution of these two Ag sources to the generation of MHC-I-presented peptides, we established murine cell lines which express as a model Ag the HIV-1 Gag polyprotein fused to ubiquitin (Ub) carrying the epitope SIINFEKL (SL). Gag was expressed either in its wild-type form (UbMGagSL) or as a variant UbRGagSL harboring an N-end rule degron signal. Although UbRGagSL displayed wild-type protein stability, its inherent defective ribosomal products rate observed after proteasome shutdown was increased concomitant with enhanced presentation of the SL epitope. In addition, UbRGagSL induces enhanced T cell stimulation of SL-specific B3Z hybridoma cells as measured in vitro and of adoptively transferred TCR-transgenic OT-1 T cells in vivo. Furthermore, an elevated frequency of SL-specific T cells was detected by IFN-gamma ELISPOT after immunization of naive C57BL/6 mice with UbRGagSL/EL4 cells. These results further underline the role of the defective ribosomal product pathway in adaptive immunity.

Asunto(s)

Presentación de Antígeno , Antígenos Virales/metabolismo , VIH-1 , Antígenos de Histocompatibilidad Clase I/metabolismo , Linfocitos T Citotóxicos/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Citotoxicidad Inmunológica , Epítopos/química , Epítopos/inmunología , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Hibridomas , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Ribosomas/metabolismo , Ubiquitina/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología

RESUMEN

Gene silencing by siRNAs offers the potential for reducing the expression of mutated genes that cause disease. It has been shown that the folding or secondary structure of a specific mRNA was significantly correlated to the silencing observed. Since this base pairing is dependent on free energy, the temperature of the cells may influence the effectiveness of a particular siRNA. The aqueous humor of the human eye has been measured to be around 34 degrees C with the lens acting as a thermal barrier in the eye. The trabecular meshwork, bathed by the aqueous humor, probably has a temperature lower than body temperature under normal conditions. Mutated myocilin, that is associated with primary open angle glaucoma, would appear to be a candidate for silencing by siRNAs. Silencing of the mutated myocilin might prevent additional accumulation of this protein in the rough endoplasmic reticulum. These experiments were undertaken to determine the influence of lowered temperatures on the silencing of myocilin by five siRNAs. Three different patterns of silencing emerged when the silencings were compared with cells grown at 33, 35, and 37 degrees C. For one of the siRNAs, the silencing was increased at lower temperatures. For two siRNAs, no significant changes in silencing were observed with different temperatures. Two of the siRNAs were significantly influenced by temperature with little if any silencing occurring at the lowest temperature. These data indicate that siRNAs directed to tissues in the anterior chamber of the eye should be checked at temperatures lower than 37 degrees C to determine their effectiveness.

Asunto(s)

Cámara Anterior/fisiología , Silenciador del Gen/fisiología , ARN Interferente Pequeño/genética , Temperatura , Células Cultivadas , Proteínas del Citoesqueleto/genética , Proteínas del Ojo/genética , Regulación de la Expresión Génica/genética , Glicoproteínas/genética , Humanos , ARN Mensajero/genética , Malla Trabecular/citología

RESUMEN

We report on the first HLA B13-restricted minimal cytotoxic T lymphocyte (CTL) epitope RQDILDLWI (RI9, amino acids 106-114 in HIV-1 Nef). In most patients the frequency of RI9-specific CTL exceeded the number of CTL against other epitopes, indicating that RI9 is a dominant epitope in HLA B13-positive patients. Targeting this conserved Nef epitope may be an important factor for the published association of HLA B13 with a favourable course of HIV-1 infection.

Asunto(s)

Epítopos de Linfocito T , Productos del Gen nef , Infecciones por VIH/inmunología , VIH-1/inmunología , Antígenos HLA-B , Linfocitos T Citotóxicos/inmunología , Secuencias de Aminoácidos , Secuencia de Consenso , Citotoxicidad Inmunológica , Mapeo Epitopo , Infecciones por VIH/mortalidad , VIH-1/aislamiento & purificación , Antígeno HLA-B13 , Humanos , Epítopos Inmunodominantes , Tasa de Supervivencia , Productos del Gen nef del Virus de la Inmunodeficiencia Humana

RESUMEN

Norrie disease is an X-linked retinal dysplasia that presents with congenital blindness, sensorineural deafness, and mental retardation. Norrin, the protein product of the Norrie disease gene (NDP), is a secreted protein of unknown biochemical function. Norrie disease (Ndp(y/-)) mutant mice that are deficient in norrin develop blindness, show a distinct failure in retinal angiogenesis, and completely lack the deep capillary layers of the retina. We show here that the transgenic expression of ectopic norrin under control of a lens-specific promoter restores the formation of a normal retinal vascular network in Ndp(y/-) mutant mice. The improvement in structure correlates with restoration of neuronal function in the retina. In addition, lenses of transgenic mice with ectopic expression of norrin show significantly more capillaries in the hyaloid vasculature that surrounds the lens during development. In vitro, lenses of transgenic mice in coculture with microvascular endothelial cells induce proliferation of the cells. Transgenic mice with ectopic expression of norrin show more bromodeoxyuridine-labeled retinal progenitor cells at embryonic day 14.5 and thicker retinas at postnatal life than wild-type littermates, indicating a putative direct neurotrophic effect of norrin. These data provide direct evidence that norrin induces growth of ocular capillaries and that pharmacologic modulation of norrin might be used for treatment of the vascular abnormalities associated with Norrie disease or other vascular disorders of the retina.

Asunto(s)

Proteínas del Ojo/fisiología , Neovascularización Fisiológica/fisiología , Proteínas del Tejido Nervioso/fisiología , Vasos Retinianos/crecimiento & desarrollo , Animales , Capilares/citología , Capilares/crecimiento & desarrollo , Recuento de Células , División Celular , Células Cultivadas , Pollos , Técnicas de Cocultivo , Electrorretinografía , Células Endoteliales/citología , Endotelio Vascular/citología , Proteínas del Ojo/genética , Regulación de la Expresión Génica , Humanos , Cristalino/citología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Retina/fisiopatología , Retina/ultraestructura , Células Ganglionares de la Retina/patología , Piel/irrigación sanguínea , beta-Cristalinas/genética

RESUMEN

BACKGROUND: Myocilin is a 55 to 57 kD secreted glycoprotein and member of the olfactomedin protein family. It is expressed in high amounts in the outflow tissues of the aqueous humor in the eye where it is supposed to contribute to outflow resistance. Myocilin is mutated in some forms of primary open angle glaucoma and affected patients show very high intraocular pressures because of an increase in resistance to aqueous humor outflow. To obtain information, if myocilin may play a comparable role in other tissues with transendothelial fluid flow, we investigated its expression in the rat kidney. METHODS: The expression of myocilin in the normal rat kidney and its changes during mesangioproliferative glomerulonephritis were investigated by immunohistochemistry, one- and two-dimensional gel electrophoresis with Western blotting, and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Myocilin and its mRNA were detected in isolated glomeruli. Immunohistochemistry showed specific labeling of glomerular cells, while tubular and interstitial regions were essentially negative. Double staining with the podocyte-specific markers synaptopodin and ezrin indicated that myocilin-positive cells were predominately podocytes. During mesangioproliferative glomerulonephritis, an induction of myocilin immunoreactivity was observed. Labeling for myocilin was now observed in activated mesangial cells and areas of glomerular sclerosis. In parallel cell culture experiments, mRNA for myocilin was detected in cultured murine podocytes and rat mesangial cells. CONCLUSION: Myocilin is expressed in podocytes of the kidney and induced in mesangial cells during experimental mesangioproliferative glomerulonephritis. The specific function of myocilin in the kidney is not clear, but in a parallel to functions of other olfactomedin proteins, it might have a role in cell-cell adhesion and/or signaling processes.

Asunto(s)

Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Glomerulonefritis Membranoproliferativa/genética , Glomerulonefritis Membranoproliferativa/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glomérulos Renales/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , ADN/genética , Expresión Génica , Glaucoma de Ángulo Abierto/genética , Glaucoma de Ángulo Abierto/metabolismo , Mesangio Glomerular/citología , Mesangio Glomerular/metabolismo , Glomerulonefritis Membranoproliferativa/patología , Humanos , Inmunohistoquímica , Glomérulos Renales/citología , Masculino , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

PURPOSE: To study the expression and localization of myocilin in the developing mouse eye. Myocilin is a 55- to 57-kDa secreted glycoprotein that is mutated in some forms of primary open-angle glaucoma. METHODS: The eyes of NMRI mice were studied from embryonic day (E) 14.5 to postnatal day (P) 21, and at 2-3 months of age. Immunohistochemistry was performed with antibodies against myocilin. The specificity of the antibodies was checked by two-dimensional gel electrophoresis. RNA was isolated from eyes at various ages, and the presence of myocilin mRNA was analyzed by northern blot hybridization. RESULTS: No immunostaining for myocilin was seen before E16.5. At around E17.5, a distinct positive immunoreactivity of optic nerve axons in the developing nerve fiber layer of the retina was observed. At P5-6, immunostaining appeared in perikarya of optic nerve ganglion cells. In the anterior eye, no immunoreactivity was observed until P10. At P12-14, the cells of the epithelial layers of ciliary body and iris, as well as the cells of the trabecular meshwork and iris stroma, became immunoreactive for myocilin. At that time, positive staining for myocilin was also seen in the corneal endothelium and in keratocytes of the corneal stroma. An essentially similar staining pattern was seen in adult eyes. Northern blot analysis for myocilin mRNA in RNA from developing mouse eyes was negative until P9. At P12, a distinct band was observed. A band with similar mobility, but somewhat more intense, was detected in mRNA from adult mouse eyes 2-3 months of age. CONCLUSIONS: The onset of immunoreactivity for myocilin in the retina occurs in parallel with the maturation of optic nerve ganglion cells. In the anterior eye, the expression of myocilin is associated with the final development of those tissues that are directly involved in aqueous humor dynamics. The presence of myocilin might be important for proper function and structure of mature optic nerve ganglion cells and aqueous humor outflow.

Asunto(s)

Proteínas del Ojo/metabolismo , Ojo/embriología , Glicoproteínas/metabolismo , Animales , Animales Recién Nacidos/metabolismo , Especificidad de Anticuerpos , Northern Blotting , Proteínas del Citoesqueleto , Electroforesis en Gel Bidimensional , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Proteínas del Ojo/genética , Técnica del Anticuerpo Fluorescente Indirecta , Glicoproteínas/genética , Ratones , ARN Mensajero/metabolismo

RESUMEN

The structure of the myelin sheath in peripheral nerves requires the expression of a specific set of proteins. In the present study, we report that myocilin, a member of the olfactomedin protein family, is a component of the myelin sheath in peripheral nerves. Myocilin is a secreted glycoprotein that forms multimers and contains a leucine zipper and an olfactomedin domain. Mutations in myocilin are responsible for some forms of glaucoma, a neurodegenerative disease that is characterized by a continuous loss of optic nerve axons. Myocilin mRNA was detected by Northern blotting in RNA from the rat sciatic and ophthalmic nerves. By one- and two-dimensional gel electrophoresis of proteins from the rat and human sciatic nerves, myocilin was found to migrate at an isoelectric point (pI) of 5.2-5.3 and a molecular weight of 55-57 kDa. Immunohistochemistry showed immunoreactivity for myocilin in paranodal terminal loops of the nodes of Ranvier and outer mesaxons and basal/abaxonal regions of the myelin sheath. Double-labeling experiments with antibodies against myelin basic protein showed no overlapping, while overlapping immunoreactivity was observed with antibodies against myelin-associated glycoprotein. The expression of myocilin in the sciatic nerve became detectable at postnatal day (P) 15 and reached adult levels at P20. No or minor expression of myocilin mRNA was found in brain, spinal cord, and optic nerve. mRNA of myocilin was detected in schwannoma cells in situ, but at considerably lower levels than in myelinated nerves. Myocilin might significantly contribute to the structure of the myelin sheath in peripheral nerves.

Asunto(s)

Proteínas del Ojo/metabolismo , Glicoproteínas/metabolismo , Vaina de Mielina/química , Nervios Periféricos/citología , Envejecimiento , Animales , Northern Blotting/métodos , Western Blotting/métodos , Encéfalo/metabolismo , Células Cultivadas/metabolismo , Proteínas del Citoesqueleto , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunohistoquímica/métodos , Hígado/metabolismo , Proteína Básica de Mielina/metabolismo , Glicoproteína Asociada a Mielina/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neuroma Acústico/genética , Neuroma Acústico/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Médula Espinal/metabolismo , Factores de Tiempo

RESUMEN

PURPOSE: Mutations in the MYOC gene coding for myocilin are associated with elevated intraocular pressure (IOP), and recombinant myocilin, produced in a prokaryotic expression system, has been reported to affect aqueous outflow facility. This study was conducted to test whether perfusion with a fragment of recombinant myocilin (containing the full-length olfactomedin domain), produced in a eukaryotic expression system, affects facility. METHODS: 293 EBNA cells were transfected by a vector containing the BM40 signal peptide, a human cDNA coding for myocilin, and a polyhistidine tag (HisTag) sequence. Recombinant protein was isolated by Ni-chelate chromatography, and characterized, and perfused into cultured anterior segments of human and porcine eyes. RESULTS: Recombinant myocilin was secreted as a approximately 55-kDa intact protein and two fragments arising from cleavage of the recombinant protein at amino acid 215. The C-terminal fragment, containing the entire olfactomedin domain, was successfully isolated. When perfused into human and porcine eyes, this C-terminal fragment did not appreciably affect outflow facility. CONCLUSIONS: Although the olfactomedin domain appears to be important for the function of myocilin, perfusion with a recombinant myocilin fragment containing this domain does not change outflow facility. It is possible that both the olfactomedin and N-terminal domains (including the leucine zipper) must be present for myocilin to have full function. Alternatively, posttranslational modifications of myocilin may have a major impact on protein function.

Asunto(s)

Humor Acuoso/metabolismo , Proteínas de la Matriz Extracelular/fisiología , Proteínas del Ojo/fisiología , Glicoproteínas/fisiología , Malla Trabecular/metabolismo , Animales , Western Blotting , Técnicas de Cultivo de Célula , Proteínas del Citoesqueleto , Electroforesis en Gel Bidimensional , Células Epiteliales/virología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Proteínas de la Matriz Extracelular/administración & dosificación , Proteínas de la Matriz Extracelular/aislamiento & purificación , Proteínas del Ojo/administración & dosificación , Proteínas del Ojo/aislamiento & purificación , Glicoproteínas/administración & dosificación , Glicoproteínas/aislamiento & purificación , Herpesvirus Humano 4/fisiología , Humanos , Técnicas de Cultivo de Órganos , Perfusión , Plásmidos , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/aislamiento & purificación , Porcinos , Malla Trabecular/efectos de los fármacos , Malla Trabecular/patología , Transfección