Asunto(s)
Linfocitos T CD8-positivos/inmunología , Ácidos Grasos/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/inmunología , Activación de Linfocitos/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Autoantígenos/metabolismo , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/químicaRESUMEN
DNA vaccines against autoimmune type 1 diabetes (T1D) contain a nonpredictable risk to induce autoreactive T cell responses rather than a protective immunity. Little is known if (and how) antigen expression and processing requirements favor the induction of autoreactive or protective immune responses by DNA immunization. Here, we analyzed whether structural properties of preproinsulin (ppins) variants and/or subcellular targeting of ppins designer antigens influence the priming of effector CD8+ T cell responses by DNA immunization. Primarily, we used H-2b RIP-B7.1 tg mice, expressing the co-stimulator molecule B7.1 in beta cells, to identify antigens that induce or fail to induce autoreactive ppins-specific (Kb/A12-21 and/or Kb/B22-29) CD8+ T cell responses. Female NOD mice, expressing the diabetes-susceptible H-2g7 haplotype, were used to test ppins variants for their potential to suppress spontaneous diabetes development. We showed that ppins antigens excluded from expression in the endoplasmic reticulum (ER) did not induce CD8+ T cells or autoimmune diabetes in RIP-B7.1 tg mice, but efficiently suppressed spontaneous diabetes development in NOD mice as well as ppins-induced CD8+ T cell-mediated autoimmune diabetes in PD-L1 -/- mice. The induction of a ppins-specific therapeutic immunity in mice has practical implications for the design of immune therapies against T1D in individuals expressing different major histocompatibility complex (MHC) I and II molecules.
RESUMEN
AIMS/HYPOTHESIS: A strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM. METHODS: Two groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements. RESULTS: PAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death. CONCLUSIONS/INTERPRETATION: The coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/metabolismo , Factores de Transcripción Paired Box/metabolismo , Animales , Apoptosis/fisiología , Proliferación Celular/fisiología , Diabetes Mellitus Tipo 1/patología , Femenino , Células Secretoras de Insulina/patología , Masculino , Ratones , Ratones MutantesRESUMEN
BACKGROUND: Elevated levels of C-peptide have been found in patients with insulin resistance and early type 2 diabetes. These patients are at greater risk to develop micro- and macrovascular complications. Since diabetic nephropathy involves glomerular hyperproliferation, the present study evaluates the role of C-peptide on human renal mesangial cell proliferation. METHODS AND RESULTS: C-peptide induces proliferation of human renal mesangial cells in a concentration-dependent manner with a maximal 2.6±0.4-fold induction at 10 nmol/L (P<0.05 compared with unstimulated cells; n=6), as revealed by [3H]-thymidine incorporation experiments. The proliferative effect of C-peptide is prevented by Src-kinase inhibitor-PP2, PI-3 kinase inhibitor-LY294002, and the ERK1/2 inhibitor-U126. Moreover, C-peptide induces phosphorylation of Src, as well as activation of PI-3 kinase and ERK1/2. Furthermore, C-peptide induces cyclin D1 expression as well as phosphorylation of retinoblastoma protein (Rb). CONCLUSIONS: These results demonstrate an active role of C-peptide on the proliferation of human renal mesangial cells in vitro involving PI-3 kinase and MAP kinase signaling pathways, suggesting a possible role of C-peptide in glomerular hyperproliferation in patients with diabetic nephropathy.
Asunto(s)
Péptido C/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Mesangiales/citología , Células Mesangiales/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Familia-src Quinasas/metabolismo , Butadienos/farmacología , Péptido C/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Ciclina D1/biosíntesis , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Mesangio Glomerular/efectos de los fármacos , Mesangio Glomerular/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Morfolinas/farmacología , Nitrilos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Pirimidinas/farmacología , Proteína de Retinoblastoma/metabolismo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
BACKGROUND: Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca(2+)-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage. METHODS AND RESULTS: We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone-induced effects. CONCLUSIONS: SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.
Asunto(s)
Diferenciación Celular/fisiología , Sistema de Conducción Cardíaco/citología , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Canales de Potasio Calcio-Activados/fisiología , Animales , Bencimidazoles/farmacología , Agonistas de los Canales de Calcio/farmacología , Línea Celular , Proliferación Celular , Citoesqueleto/fisiología , Sistema de Conducción Cardíaco/fisiología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Miocitos Cardíacos/fisiología , Células Madre Pluripotentes/fisiología , Canales de Potasio Calcio-Activados/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVE: RIP-B7.1 mice expressing the costimulator molecule B7.1 (CD80) on pancreatic beta-cells are a well established model to characterize preproinsulin-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD). Different immunization strategies could prime preproinsulin-specific CD8 T-cells in wild-type C57BL/6 (B6) mice, but did not induce diabetes. We tested whether altering the B7-H1 (PD-L1) coinhibition on pancreatic beta-cells can reveal a diabetogenic potential of preproinsulin-specific CD8 T-cells. RESEARCH DESIGN AND METHODS: DNA-based immunization and adoptive T-cell transfers were used to characterize the induction of preproinsulin-specific CD8 T-cell responses and EAD in RIP-B7.1, B6, B7-H1(-/-), PD-1(-/-) or bone marrow chimeric mice. RESULTS: Preproinsulin-specific CD8 T-cells primed in B6 mice revealed their diabetogenic potential after adoptive transfer into congenic RIP-B7.1 hosts. Furthermore, preproinsulin-specific CD8 T-cells primed in anti-B7-H1 antibody-treated B6 mice, or primed in B7-H1(-/-) or PD-1(-/-) mice induced EAD. Immunization of bone marrow chimeric mice showed that deficiency of either B7-H.1 in pancreatic beta-cells or of PD-1 in autoreactive CD8 T-cells induced EAD. CONCLUSIONS: An imbalance between costimulator (B7.1) and coinhibitor (B7-H1) signals on pancreatic beta-cells can trigger pancreatic beta-cell-destruction by preproinsulin-specific CD8 T-cells. Hence, regulation of the susceptibility of the beta-cells for a preproinsulin-specific CD8 T-cell attack can allow or suppress EAD.
Asunto(s)
Antígeno B7-1/genética , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/patología , Glicoproteínas de Membrana/genética , Péptidos/genética , Animales , Antígeno B7-H1 , Humanos , Insulina/genética , Interferón gamma/deficiencia , Glicoproteínas de Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Péptidos/deficiencia , Precursores de Proteínas/genéticaRESUMEN
Proinsulin is a major diabetes-associated autoantigen. APL have been shown to manipulate the immune response of T cells. Here, we generated a lysosomal protease-resistant proinsulin 74-90-derived APL using a CS-directed amino acid modification approach. These prAPL activated TGF-beta 1 secretion in proinsulin-reactive T cells from PBMC of patients with T1D. We provide evidence that proinsulin-derived prAPL modulate the cytokine signature of proinsulin-reactive T cells at a micromolar range by increasing anti-inflammatory cytokines, including TGF-beta 1. Thus, the use of prAPL is a promising tool to mitigate autoaggressive T cells.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Leucocitos Mononucleares/metabolismo , Proinsulina/inmunología , Factor de Crecimiento Transformador beta1/metabolismo , Secuencia de Aminoácidos , Autoantígenos/inmunología , Separación Celular , Diabetes Mellitus Tipo 1/inmunología , Citometría de Flujo , Humanos , Leucocitos Mononucleares/inmunología , Ligandos , Activación de Linfocitos/inmunología , Datos de Secuencia Molecular , Proinsulina/química , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta1/inmunologíaRESUMEN
RIP-B7.1 mice express the costimulator molecule B7.1 (CD80) on pancreatic beta cells and are a well-established model for studying de novo induction of diabetogenic CD8 T cells. Immunization of RIP-B7.1 mice with preproinsulin (ppins)-encoding plasmid DNA efficiently induces experimental autoimmune diabetes (EAD). EAD is associated with an influx of CD8 T cells specific for the K(b)/A(12-21) epitope into the pancreatic islets and the subsequent destruction of beta cells. In this study, we used this model to investigate how ppins-derived Ags are expressed and processed to prime diabetogenic, K(b)/A(12-21)-specific CD8 T cells. Targeting the K(b)/A(12-21) epitope, the insulin A chain, or the ppins to the endoplasmic reticulum (ER) (but not to the cytosol and/or nucleus) efficiently elicited K(b)/A(12-21)-specific CD8 T cell responses. The K(b)/A(12-21) epitope represents the COOH terminus of the ppins molecule and, hence, did not require COOH-terminal processing before binding its restriction element in the ER. However, K(b)/A(12-21)-specific CD8 T cells were also induced by COOH-terminally extended ppins-specific polypeptides expressed in the ER, indicating that the epitope position at the COOH terminus is less important for its diabetogenicity than is targeting the Ag to the ER. The K(b)/A(12-21) epitope had a low avidity for K(b) molecules. When epitopes of unrelated Ags were coprimed at the same site of Ag delivery, "strong" K(b)-restricted (but not D(b)-restricted) CD8 T cell responses led to the suppression of K(b)/A(12-21)-specific CD8 T cell priming and reduced EAD. Thus, direct expression and processing of the "weak" K(b)/A(12-21) epitope in the ER favor priming of autoreactive CD8 T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Experimental/metabolismo , Retículo Endoplásmico/metabolismo , Epítopos de Linfocito T/metabolismo , Insulina/metabolismo , Animales , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Western Blotting , Diabetes Mellitus Experimental/inmunología , Retículo Endoplásmico/inmunología , Epítopos de Linfocito T/inmunología , Técnica del Anticuerpo Fluorescente , Insulina/inmunología , Ratones , Precursores de Proteínas/inmunología , Precursores de Proteínas/metabolismoRESUMEN
Type 1 diabetes mellitus can result from the specific destruction of pancreatic beta cells by autoreactive T cells. As shown here, experimental autoimmune diabetes (EAD) is efficiently induced in RIP-B7.1 mice by preproinsulin (ppins)-encoding DNA vaccines. EAD develops in RIP-B7.1 mice within 3-4 wk after a single immunization with ppins-encoding plasmid DNA. RIP-B7.1 mice develop insulitis, insulin deficiency and hyperglycemia after vaccination with plasmids encoding murine ppins-I or murine ppins-II or human hu-ppins. EAD induction critically depends on CD8 T cells and is independent of CD4 T cells. To be diabetogenic, ppins-specific CD8 T cells had to express IFN-gamma. Neither expression of perforin nor signaling through the type I IFN receptor is an essential component of this pathogenic CD8 T cell phenotype. Using plasmids encoding truncated ppins variants, we show that EAD is only induced by DNA vaccines encoding the insulin A-chain. Diabetogenic CD8 T cells specifically recognize the Kb-restricted A12-21 epitope of the insulin A-chain. The RIP-B7.1 model hence represents an attractive model for the characterization of cellular and molecular events involved in the CD8 T cell-mediated immune pathogenesis of diabetes.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Experimental/inmunología , Insulina/inmunología , Proinsulina/inmunología , Precursores de Proteínas/inmunología , Secuencia de Aminoácidos , Animales , Enfermedades Autoinmunes/patología , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Experimental/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Plásmidos/genética , Proinsulina/genética , Precursores de Proteínas/genética , Vacunas de ADN/inmunologíaRESUMEN
PURPOSE: Primary chemotherapy is commonly used in patients with breast cancer to downstage the primary tumour prior to surgery. There is a need to establish, prior to commencement of chemotherapy, predictors of clinical and pathological response, which may then be surrogate markers for patient survival and thus allow identification of patients who are most likely to benefit from such treatment. PATIENTS AND METHODS: A total of 104 patients with large and locally advanced breast cancers received an anthracycline/docetaxel-based regimen prior to surgery. Immunohistochemistry was carried out on pre-treatment core biopsies of the tumour to detect hormone receptors (oestrogen-ER; progesterone-PR), a proliferation marker (MIB-1), the oncoprotein Bcl-2, an extracellular matrix degradation enzyme (cathepsin D), p53, and an oestrogen associated protein (pS2). Both clinical and pathological response were assessed following completion of chemotherapy. RESULTS: Patients whose tumours did not express oestrogen receptor (p = 0.02) or did not express Bcl-2 (p < 0.01) had a better pathological response in a univariate analysis. However, in a multivariate model, it was only the absence of detectable Bcl-2 protein that predicted a better pathological response (p = 0.001). CONCLUSIONS: This study has identified that patients whose breast cancers are most likely to experience the greatest degree of tumour destruction by primary chemotherapy do not express either oestrogen receptors or Bcl-2. This may have important implications in the selection of patients with breast cancer for primary chemotherapy who are most likely to gain a survival benefit.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/tratamiento farmacológico , Estadificación de Neoplasias , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Receptores de Estrógenos/biosíntesis , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/patología , Ciclofosfamida/administración & dosificación , Doxorrubicina/administración & dosificación , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Análisis Multivariante , Terapia Neoadyuvante , Valor Predictivo de las Pruebas , Prednisolona/administración & dosificación , Pronóstico , Receptores de Estrógenos/análisis , Vincristina/administración & dosificaciónRESUMEN
(Prepro)insulin is considered a central antigenic determinant in diabetic autoimmunity. Insulin has been used to modify diabetes development in NOD mice and prediabetic individuals. We have recently shown that (prepro)insulin can adversely promote diabetes development in murine type 1 diabetes. Based on these findings we have developed experimental autoimmune diabetes (EAD), a new mouse model characterized by (1) CD4(+)/CD8(+) insulitis, induced by (2) (prepro)insulin DNA vaccination, leading to (3) beta cell damage and insulin deficiency in (4) RIP-B7.1 transgenic mice (H-2(b)). EAD develops rapidly in 60-95% of mice after intramuscular, but not intradermal ("gene gun"), vaccination; and DNA plasmids expressing insulin or the insulin analogues glargine, aspart, and lispro are equally potent to induce EAD. Similar to NOD mice, diabetes is adoptively transferred into syngeneic recipients by spleen cell transplantation in a dose-dependent fashion. We have devised a two-stage concept of EAD in which T cell activation and expansion is driven by in vivo autoantigen expression, followed by islet damage that requires beta cell expression of costimulatory B7.1 for disease manifestation. Taken together, EAD is a novel, genetically defined animal model of type 1 diabetes suitable to analyze mechanisms and consequences of insulin-specific T cell autoimmunity.