RESUMEN
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS, and CD8 T cells are the predominant T cell population in MS lesions. Given that transfer of CNS-specific CD8 T cells results in an attenuated clinical demyelinating disease in C57BL/6 mice with immunization-induced experimental autoimmune encephalomyelitis (EAE), we investigated the cellular targets and mechanisms of autoreactive regulatory CD8 T cells. In this study we report that myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced CD8 T cells could also attenuate adoptively transferred, CD4 T cell-mediated EAE. Whereas CD8(-/-) mice exhibited more severe EAE associated with increased autoreactivity and inflammatory cytokine production by myelin-specific CD4 T cells, this was reversed by adoptive transfer of MOG-specific CD8 T cells. These autoregulatory CD8 T cells required in vivo MHC class Ia (K(b)D(b)) presentation. Interestingly, MOG-specific CD8 T cells could also suppress adoptively induced disease using wild-type MOG35-55-specific CD4 T cells transferred into K(b)D(b-/-) recipient mice, suggesting direct targeting of encephalitogenic CD4 T cells. In vivo trafficking analysis revealed that autoregulatory CD8 T cells are dependent on neuroinflammation for CNS infiltration, and their suppression/cytotoxicity of MOG-specific CD4 T cells is observed both in the periphery and in the CNS. These studies provide important insights into the mechanism of disease suppression mediated by autoreactive CD8 T cells in EAE.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/prevención & control , Linfocitos T CD8-positivos/trasplante , Células Cultivadas , Citotoxicidad Inmunológica , Encefalomielitis Autoinmune Experimental/patología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia MolecularRESUMEN
Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). Although its etiology remains unknown, pathogenic T cells are thought to underlie MS immune pathology. We recently showed that MS patients harbor CNS-specific CD8+ Tregs that are deficient during disease relapse. We now demonstrate that CNS-specific CD8+ Tregs were cytolytic and could eliminate pathogenic CD4+ T cells. These CD8+ Tregs were present primarily in terminally differentiated (CD27-, CD45RO-) subset and their suppression was IFNγ, perforin and granzyme B-dependent. Interestingly, MS patients with acute relapse displayed a significant loss in terminally differentiated CD8+ T cells, with a concurrent loss in expression of perforin and granzyme B. Pre-treatment of exacerbation-derived CD8+ T cells with IL-12 significantly restored suppressive capability of these cells through upregulation of granzyme B. Our studies uncover immune-suppressive mechanisms of CNS-specific CD8+ Tregs, and may contribute to design of novel immune therapies for MS.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Esclerosis Múltiple/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Anciano , Diferenciación Celular/inmunología , Progresión de la Enfermedad , Femenino , Granzimas/biosíntesis , Granzimas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Interferón gamma/inmunología , Interleucina-12/farmacología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/patología , Perforina/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Regulación hacia Arriba , Adulto JovenRESUMEN
Daily administration of FDA-approved glatiramer acetate (GA) has beneficial effects on clinical course of relapsing remitting multiple sclerosis (RRMS). Although mechanisms of GA-action have been widely investigated and partially understood, immediate immune dynamics following GA-therapy are unknown. In the present study, we characterized the immediate effects of GA on phenotype, quantity and function of immune cells in MS patients. Prominent changes in immune cells were detected within 4-12h post-first GA-injection. T-cell modulation included significantly decreased CD4/CD8 ratio, perturbed homeostasis of predominantly CD8+ T-cells, significant enhancement in CD8+ T-cell mediated suppression and inhibitory potential of induced CD4-suppressors. Changes in APC were restricted to monocytes and included reduced stimulatory capacity in MLR and significantly increased IL-10 and TNF-α production. Our study provides the first evidence that GA treatment induces rapid immunologic changes within hours of first dose. Interestingly, these responses are not only restricted to innate immune cells but also include complex modulation of T-cell functionality.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Factores Inmunológicos/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Péptidos/uso terapéutico , Adulto , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Femenino , Acetato de Glatiramer , Humanos , Interleucina-10/inmunología , Prueba de Cultivo Mixto de Linfocitos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Adulto JovenRESUMEN
IL-17A is a proinflammatory cytokine that has received attention for its role in the pathogenesis of several autoimmune diseases. IL-17A has also been implicated in cardiac and renal allograft rejection. Accordingly, we hypothesized that depletion of IL-17A would enhance corneal allograft survival. Instead, our results demonstrate that blocking IL-17A in a mouse model of keratoplasty accelerated the tempo and increased the incidence of allograft rejection from 50 to 90%. We describe a novel mechanism by which CD4(+)CD25(+) regulatory T cells (Tregs) respond to IL-17A and enhance corneal allograft survival. Our findings suggest the following: 1) IL-17A is necessary for ocular immune privilege; 2) IL-17A is not required for the induction of anterior chamber-associated immune deviation; 3) Tregs require IL-17A to mediate a contact-dependent suppression; 4) corneal allograft Tregs suppress the efferent arm of the immune response and are Ag specific; 5) Tregs are not required for corneal allograft survival beyond day 30; and 6) corneal allograft-induced Treg-mediated suppression is transient. Our findings identify IL-17A as a cytokine essential for the maintenance of corneal immune privilege and establish a new paradigm whereby interplay between IL-17A and CD4(+)CD25(+) Tregs is necessary for survival of corneal allografts.
Asunto(s)
Trasplante de Córnea/métodos , Supervivencia de Injerto/inmunología , Inmunosupresores , Interleucina-17/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos , Rechazo de Injerto/inmunología , Fenómenos del Sistema Inmunológico , Interleucina-17/farmacología , Subunidad alfa del Receptor de Interleucina-2/inmunología , RatonesRESUMEN
Chlamydomonas reinhardtii possesses a CO(2)-concentrating mechanism (CCM) that allows the alga to grow at low CO(2) concentrations. One common feature seen in photosynthetic organisms possessing a CCM is the tight packaging of Rubisco within the cell. In many eukaryotic algae, Rubisco is localized to the pyrenoid, an electron-dense structure within the chloroplast. In order to identify genes required for a functional CCM, insertional Bleomycin resistance (Ble(R)) mutants were generated and screened for growth on minimal medium under high CO(2) conditions (5% CO(2) in air) but only slow or no growth under very low CO(2) conditions (0.01% CO(2) in air). One mutant identified from this screen was named cia6. Physiological studies established that cia6 grows poorly on low levels of CO(2) and has an impaired ability to accumulate inorganic carbon. The inserted Ble(R) disrupted a gene encoding a protein with sequence similarity to proteins containing SET domain methyltransferase, although experiments using overexpressed CIA6 failed to demonstrate the methyltransferase activity. Electron microscopy revealed that the pyrenoid of cia6 mutant cells is highly disorganized. Complementation of the mutant restored the pyrenoid, the ability to grow under low-CO(2) conditions, and the ability to concentrate inorganic carbon. Quantitative reverse transcription-polymerase chain reaction data from a low-CO(2) induction time-course experiment demonstrated that the up-regulation of several CCM components is slower in cia6 compared with the wild type. This slow induction was further confirmed at the protein level using western blots. These results indicated that CIA6 is required for the formation of the pyrenoid and further supported the notion that the pyrenoid is required for a functional CCM in C. reinhardtii.
Asunto(s)
Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , Genes de Plantas/genética , Proteínas de Plantas/genética , Aclimatación/efectos de los fármacos , Carbono/metabolismo , Dióxido de Carbono/farmacología , Chlamydomonas reinhardtii/efectos de los fármacos , Chlamydomonas reinhardtii/crecimiento & desarrollo , Chlamydomonas reinhardtii/ultraestructura , Clorofila/metabolismo , Cloroplastos/efectos de los fármacos , Cloroplastos/ultraestructura , Evolución Molecular , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Prueba de Complementación Genética , Sitios Genéticos/genética , Cinética , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Insercional/efectos de los fármacos , Mutación/genética , Fenotipo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estructura Terciaria de Proteína , Ribulosa-Bifosfato Carboxilasa/metabolismo , Factores de TiempoRESUMEN
Corneal allograft rejection has been described as a Th1-mediated process involving IFN-γ production. However, it has been reported that corneal allograft rejection soars in IFN-γ(-/-) mice or mice treated with anti-IFN-γ mAb. Th17 is a recently described IL-17A-producing Th cell population that has been linked to renal and cardiac graft rejection, which was originally thought to be Th1-mediated. We tested the hypothesis that Th17 cells mediate corneal allograft rejection in an IL-17A-dependent fashion and unexpectedly found that depletion of IL-17A increased the incidence of rejection to 90%. We demonstrate that the exacerbated rejection following depletion of IL-17A did not result from a loss of cross-regulation of Th1 cells or exaggerated delayed-type hypersensitivity responses. Instead, inhibition of the Th1 or Th17 cell lineages promoted the emergence of a Th2 cell subset that independently mediated allograft rejection. These findings demonstrate that IL-17A is not required for corneal allograft rejection and may instead contribute to the immune privilege of corneal allografts.
Asunto(s)
Trasplante de Córnea , Rechazo de Injerto/inmunología , Interleucina-17/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Inmunología del Trasplante , Traslado Adoptivo , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Supervivencia de Injerto , Inmunohistoquímica , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Trasplante HomólogoRESUMEN
Aquatic photosynthetic organisms such as the green alga Chlamydomonas reinhardtii respond to low-CO(2) conditions by inducing a CO(2) concentrating mechanism (CCM). Important components of the CCM are the carbonic anhydrases (CAs), zinc metalloenzymes that catalyze the interconversion of CO(2) and HCO(-)(3). Six CAs have previously been identified in C. reinhardtii. Here, we identify and characterize two additional beta-type CAs. These two CAs are closely related beta-type CAs and have been designated as CAH7 and CAH8. Conceptual translation shows that CAH7 and CAH8 encode proteins of 399 and 333 amino acids, respectively, and they contain targeting sequences. An unusual characteristic of these two CAs is that they have carboxy-terminal extensions containing a hydrophobic sequence. Both these CAs are constitutively expressed at the transcript and protein level. The CAH7 and CAH8 open reading frames were cloned in the overexpression vector pMal-c2x and expressed as recombinant proteins. Activity assays showed that CAH7 and CAH8 are both active CAs. Antibodies were raised against both CAH7 and CAH8, and immunolocalization studies showed that CAH8 was localized in the periplasmic space. A possible role for CAH8 in the inorganic carbon acquisition by C. reinhardtii is discussed.
Asunto(s)
Proteínas Algáceas/metabolismo , Anhidrasas Carbónicas/metabolismo , Chlamydomonas reinhardtii/enzimología , Proteínas Algáceas/genética , Secuencia de Aminoácidos , Animales , Western Blotting , Dióxido de Carbono/metabolismo , Anhidrasas Carbónicas/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/ultraestructura , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
PURPOSE: To describe 2 cases of congenital corneal endothelial edema resulting from novel de novo mutations. METHODS: Case A patient was a 15-month-old white child and case B patient was a 3-year-old Hispanic child presenting with bilateral cloudy corneas since birth. Clinicopathologic findings are presented. DNA samples were screened for mutations in candidate genes by Sanger sequencing. RESULTS: Slit-lamp examination of case A patient revealed stromal edema and haze. Histology of the keratoplasty button showed stromal thickening with loss of endothelium and thin Descemet membrane. Sanger sequencing established the diagnosis of congenital hereditary endothelial dystrophy by detection of a compound heterozygous mutation in SLC4A11. The proband displayed a novel de novo frameshift mutation in one SLC4A11 allele, p.(Pro817Argfs*32), in conjunction with a maternally inherited missense mutation in SLC4A11, p.(Arg869His). Case B patient similarly presented with stromal edema and stromal haze. Histopathologic analysis revealed a spongy epithelium, focal discontinuities in Bowman layer, stromal thickening with areas of compacted posterior stroma, variable thickness of Descemet membrane, and regional multilayered endothelium. Sanger sequencing found a novel de novo nonsense mutation in the first exon of ZEB1, p.(Cys7*). CONCLUSIONS: To the authors' knowledge, we report the earliest clinical presentation of posterior polymorphous corneal dystrophy resulting from a de novo mutation in ZEB1. Additionally, we present a congenital hereditary endothelial dystrophy case with a thin Descemet membrane with a novel compound heterozygous SLC4A11 mutation. In the absence of a family history or consanguinity, de novo mutations may result in congenital corneal endothelial dystrophies.
Asunto(s)
Proteínas de Transporte de Anión/genética , Antiportadores/genética , Distrofias Hereditarias de la Córnea/genética , Edema Corneal/genética , Mutación del Sistema de Lectura , Proteínas de Homeodominio/genética , Mutación Missense , Factores de Transcripción/genética , Preescolar , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/cirugía , Edema Corneal/diagnóstico , Edema Corneal/cirugía , Análisis Mutacional de ADN , Humanos , Lactante , Queratoplastia Penetrante , Reacción en Cadena de la Polimerasa , Agudeza Visual , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
OBJECTIVE: To determine the antigenic determinants and specific molecular requirements for the generation of autoregulatory neuroantigen-specific CD8(+) T cells in models of multiple sclerosis (MS). METHODS: We have previously shown that MOG35-55-specific CD8(+) T cells suppress experimental autoimmune encephalomyelitis (EAE) in the C57BL/6 model. In this study, we utilized multiple models of EAE to assess the ability to generate autoregulatory CD8(+) T cells. RESULTS: We demonstrate that alternative myelin peptides (PLP178-191) and other susceptible mouse strains (SJL) generated myelin-specific CD8(+) T cells, which were fully capable of suppressing disease. The disease-ameliorating function of these cells was dependent on the specific cognate myelin antigen. Generation of these autoregulatory CD8(+) T cells was not affected by thymic selection, but was dependent on the presence of both CD4(+) and CD8(+) T-cell epitopes in the immunizing encephalitogenic antigen. CONCLUSIONS: These studies show that the generation of autoregulatory CD8(+) T cells is a more generalized, antigen-specific phenomenon across multiple neuroantigens and mouse strains, with significant implications in understanding disease regulation.
RESUMEN
PURPOSE: Expansion of the intronic CTG18.1 triplet repeat locus within TCF4 contributes significant risk to the development of Fuchs' endothelial corneal dystrophy (FECD) in Eurasian populations, but the mechanisms by which the expanded repeats result in degeneration of the endothelium have been hitherto unknown. The purpose of this study was to examine FECD endothelial samples for the presence of RNA nuclear foci, the hallmark of toxic RNA, as well as evidence of haploinsufficiency of TCF4. METHODS: Using fluorescence in situ hybridization, we examined for the presence of nuclear RNA foci containing expanded CUG transcripts in corneal endothelial samples from FECD subjects with CTG18.1 expansion. We also examined for any changes in expression levels of TCF4 by quantitative real-time PCR. RESULTS: Numerous discrete nuclear RNA foci were identified in endothelial samples of FECD subjects (n = 8) harboring the CTG18.1 expansion, but not in controls lacking the expansion (n = 5) (P = 7.8 × 10(-4)). Percentage of cells with foci in expansion-positive endothelial samples ranged from 33% to 88%. RNA foci were absent in endothelial samples from an FECD subject without CTG18.1 expansion and a subject with endothelial dysfunction without FECD. Expression of the constitutive TCF4 exon encoding the basic helix-loop-helix domain was unaltered with CTG18.1 expansion. CONCLUSIONS: Our findings suggest that the RNA nuclear foci are pathognomonic for CTG18.1 expansion-mediated endothelial disease. The RNA nuclear foci have been previously found only in rare neurodegenerative disorders caused by repeat expansions. Our detection of abundant ribonuclear foci in FECD implicates a role for toxic RNA in this common disease.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Distrofia Endotelial de Fuchs/genética , Predisposición Genética a la Enfermedad , ARN Nuclear/genética , Factores de Transcripción/genética , Expansión de Repetición de Trinucleótido , Anciano , Anciano de 80 o más Años , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Endotelio Corneal/metabolismo , Femenino , Distrofia Endotelial de Fuchs/metabolismo , Genotipo , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Factor de Transcripción 4 , Factores de Transcripción/metabolismoRESUMEN
A pharmaceutical composition composed of several traditional Chinese medicines is claimed to treat age-related macular degeneration (AMD). This represents a novel and alternative therapeutic solution for wet AMD, with the potential advantage of treating both the symptoms and the underlying causes of this devastating degenerative retinal disease.
Asunto(s)
Medicamentos Herbarios Chinos/uso terapéutico , Degeneración Macular/tratamiento farmacológico , Animales , Neovascularización Coroidal , Combinación de Medicamentos , Humanos , Degeneración Macular/patología , Degeneración Macular/fisiopatología , Patentes como Asunto , Factores de Tiempo , Resultado del Tratamiento , Agudeza Visual/efectos de los fármacosRESUMEN
Age-related macular degeneration (AMD) is the primary cause of irreversible blindness among the elderly in the western world. To date, no cure is available and the current anti-VEGF therapy has only shown limited efficacy in improving visual acuity in neovascular AMD. The etiology of AMD remains elusive but research over the past decade has uncovered characteristic features of the disease. These features include: oxidative stress and retinal pigment epithelial cell cytotoxicity; loss of macromolecular permeability and hydraulic conductivity in Bruch's membrane; inflammation; choroidal neovascularization and vascular leakage; and loss of neuroprotection. Recent breakthroughs in understanding the pathogenesis of AMD have spawned an array of novel therapeutic agents designed to address these hallmarks. Here we review the features of AMD and highlight the most promising therapeutic and diagnostic approaches based on the patents published from 2008 to 2011.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Diseño de Fármacos , Degeneración Macular/tratamiento farmacológico , Anciano , Inhibidores de la Angiogénesis/farmacología , Animales , Ceguera/prevención & control , Humanos , Degeneración Macular/diagnóstico , Degeneración Macular/fisiopatología , Patentes como Asunto , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Corneal transplantation is the oldest, most common, and arguably the most successful form of organ transplantation. In uncomplicated first-time cases, corneal allografts enjoy a success rate of up to 90% even though the transplants are performed without HLA matching or the use of systemic immunosuppressive drugs. In rodents, corneal allografts transplanted across entire MHC and multiple minor histocompatibility barriers enjoy long-term survival in >50% of the hosts, while skin grafts invariably undergo immune rejection. These observations are the basis for "immune privilege" of corneal transplants. In spite of this immune privilege, immune rejection can occur and remains the leading cause of corneal graft failure. Rodent models of penetrating keratoplasty have facilitated studies that have challenged, and in some cases, refuted prevailing dogmas. The long-held belief that CD4+ T helper 1 (Th1) cells were the sole mediators of corneal allograft rejection has fallen to the wayside based on studies in interferon-gamma (IFN-γ)â»(/)â» mice. The dogma that biasing the alloimmune response down a Th2 pathway would enhance graft survival has also been disproven, and in fact, compelling evidence indicates that Th2-based immune rejection of corneal allografts is swifter and more intense than Th1-based rejection. Animal studies have also pre-empted emerging dogmas including the hypothesis that Th17 cells play a crucial role in allograft rejection. Instead, IL-17A appears to be necessary for corneal allograft survival. Finally, IFN-γ, and IL-17A, which were normally viewed as proinflammatory, exert the opposite effect in the context of corneal transplantation and are necessary for corneal allograft survival.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Trasplante de Córnea , Animales , Antígenos CD/inmunología , Rechazo de Injerto/inmunología , Humanos , Inmunidad Celular , Interleucina-17/inmunologíaRESUMEN
PURPOSE: To compare and contrast the T regulatory cells (Tregs) induced by anterior chamber (AC) injection of antigen with those induced by orthotopic corneal allografts. METHODS: Anterior chamber-associated immune deviation (ACAID) Tregs were induced by injecting C57BL/6 spleen cells into the AC of BALB/c mice. Delayed-type hypersensitivity responses to C57BL/6 alloantigens were evaluated by a conventional ear swelling assay. Corneal allograft Tregs were induced by applying orthotopic C57BL/6 corneal allografts onto BALB/c hosts. The effects of anti-CD25, anti-CD8, anti-interferon-γ (IFN-γ), anti-IL-17A, or cyclophosphamide treatments on corneal allograft survival and ACAID were evaluated. RESULTS: Administration of either anti-CD25 or anti-IFN-γ antibodies prevented the expression of ACAID and abolished the immune privilege of corneal allografts. By contrast, in vivo treatment with anti-CD8 antibody abrogated ACAID but had no effect on corneal allograft survival. Further discordance between ACAID and corneal allograft survival emerged in experiments in which the induction of allergic conjunctivitis or the administration of anti-IL-17A abolished the immune privilege of corneal allografts but had no effect on the induction or expression of ACAID. CONCLUSIONS: Although orthotopic corneal allografts are strategically located for the induction of ACAID by the sloughing of corneal cells into the AC, the results reported here indicate that the Tregs induced by orthotopic corneal allografts are remarkably different from the Tregs that are induced by AC injection of alloantigen. Although both of these Treg populations promote corneal allograft survival, they display distinctly different phenotypes.