RESUMO
The adipokine, leptin, regulates blood glucose and the insulin secretory function of beta cells, while also modulating immune cell function. We hypothesized that the dual effects of leptin may prevent or suppress the autoreactive destruction of beta cells in a virally induced rodent model of type 1 diabetes. Nearly 100% of weanling BBDR rats treated with the combination of an innate immune system activator, polyinosinic:polycytidylic acid (pIC), and Kilham rat virus (KRV) become diabetic within a predictable time frame. We utilized this model to test the efficacy of leptin in preventing diabetes onset, remitting new onset disease, and preventing autoimmune recurrence in diabetic rats transplanted with syngeneic islet grafts. High doses of leptin delivered via an adenovirus vector (AdLeptin) or alzet pump prevented diabetes in>90% of rats treated with pIC+KRV. The serum hyperleptinemia generated by this treatment was associated with decreased body weight, decreased non-fasting serum insulin levels, and lack of islet insulitis in leptin-treated rats. In new onset diabetics, hyperleptinemia prevented rapid weight loss and diabetic ketoacidosis, and temporarily restored euglycemia. Leptin treatment also prolonged the survival of syngeneic islets transplanted into diabetic BBDR rats. In diverse therapeutic settings, we found leptin treatment to have significant beneficial effects in modulating virally induced diabetes. These findings merit further evaluation of leptin as a potential adjunct therapeutic agent for treatment of human type 1 diabetes.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Leptina/uso terapêutico , Infecções por Parvoviridae/imunologia , Parvovirus/imunologia , Animais , Glicemia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/virologia , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/virologia , Cetoacidose Diabética/prevenção & controle , Humanos , Transplante das Ilhotas Pancreáticas , Leptina/administração & dosagem , Leptina/imunologia , Infecções por Parvoviridae/virologia , Poli I-C/administração & dosagem , Poli I-C/imunologia , Ratos , Ratos Endogâmicos BB , Resultado do TratamentoRESUMO
Zebrafish embryos are emerging as models of glucose metabolism. However, patterns of endogenous glucose levels, and the role of the islet in glucoregulation, are unknown. We measured absolute glucose levels in zebrafish and mouse embryos, and demonstrate similar, dynamic glucose fluctuations in both species. Further, we show that chemical and genetic perturbations elicit mammalian-like glycemic responses in zebrafish embryos. We show that glucose is undetectable in early zebrafish and mouse embryos, but increases in parallel with pancreatic islet formation in both species. In zebrafish, increasing glucose is associated with activation of gluconeogenic phosphoenolpyruvate carboxykinase1 (pck1) transcription. Non-hepatic Pck1 protein is expressed in mouse embryos. We show using RNA in situ hybridization, that zebrafish pck1 mRNA is similarly expressed in multiple cell types prior to hepatogenesis. Further, we demonstrate that the Pck1 inhibitor 3-mercaptopicolinic acid suppresses normal glucose accumulation in early zebrafish embryos. This shows that pre- and extra-hepatic pck1 is functional, and provides glucose locally to rapidly developing tissues. To determine if the primary islet is glucoregulatory in early fish embryos, we injected pdx1-specific morpholinos into transgenic embryos expressing GFP in beta cells. Most morphant islets were hypomorphic, not a genetic, but embryos still exhibited persistent hyperglycemia. We conclude from these data that the early zebrafish islet is functional, and regulates endogenous glucose. In summary, we identify mechanisms of glucoregulation in zebrafish embryos that are conserved with embryonic and adult mammals. These observations justify use of this model in mechanistic studies of human metabolic disease.
Assuntos
Embrião não Mamífero/metabolismo , Glucose/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Desenvolvimento Embrionário/efeitos dos fármacos , Proteínas de Fluorescência Verde/análise , Hibridização In Situ , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/fisiologia , Filogenia , Ácidos Picolínicos/farmacologia , RNA Mensageiro/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Proteomic profiling of serum is a powerful technique to identify differentially expressed proteins that can serve as biomarkers predictive of disease onset. In this study, we utilized two-dimensional (2D) gel analysis followed by matrix-assisted-laser desorption/ionization time-of-flight mass spectrometry analysis to identify putative serum biomarkers for autoimmune type 1 diabetes (T1D) in biobreeding diabetes resistant (BBDR) rats induced to express the disease. Treatment with toll-like receptor 3 ligand, polyinosinic:polycytidilic acid (pIC), plus infection with Kilham rat virus (KRV), a rat parvovirus, results in nearly 100% of young BBDR rats becoming diabetic within 11-21 d. Sera collected from prediabetic rats at early time points following treatment with pIC + KRV were analyzed by 2D gel electrophoresis and compared with sera from control rats treated with phosphate-buffered saline, pIC alone or pIC + H1, a non-diabetogenic parvovirus. None of the latter three control treatments precipitates T1D. 2D gel analysis revealed that haptoglobin, an acute phase and hemoglobin scavenger protein, was differentially expressed in the sera of rats treated with pIC + KRV relative to control groups. These results were confirmed by Western blot and enzyme-linked immunosorbent assay studies, which further validated haptoglobin levels as being differentially increased in the sera of pIC + KRV-treated rats relative to controls during the first week following infection. Early elevations in serum haptoglobin were also observed in LEW1.WR1 rats that became diabetic following infection with rat cytomegalovirus. The identification and validation of haptoglobin as a putative serum biomarker for autoimmune T1D in rats now affords us the opportunity to test the validity of this protein as a biomarker for human T1D, particularly in those situations where viral infection is believed to precede the onset of disease.
Assuntos
Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/virologia , Haptoglobinas/análise , Animais , Biomarcadores/sangue , Western Blotting , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Experimental/virologia , Diabetes Mellitus Tipo 1/sangue , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Masculino , Parvovirus , Poli I-C , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The centrosome is important for microtubule organization and cell cycle progression in animal cells. Recently, mutations in the centrosomal protein, pericentrin, have been linked to human microcephalic osteodysplastic primordial dwarfism (MOPD II), a rare genetic disease characterized by severe growth retardation and early onset of type 2 diabetes among other clinical manifestations. While the link between centrosomal and cell cycle defects may account for growth deficiencies, the mechanism linking pericentrin mutations with dysregulated glucose homeostasis and pre-pubertal onset of diabetes is unknown. In this report we observed abundant expression of pericentrin in quiescent pancreatic beta-cells of normal animals which led us to hypothesize that pericentrin may have a critical function in beta-cells distinct from its known role in regulating cell cycle progression. In addition to the typical centrosome localization, pericentrin was also enriched with secretory vesicles in the cytoplasm. Pericentrin overexpression in beta-cells resulted in aggregation of insulin-containing secretory vesicles with cytoplasmic, but not centrosomal, pericentriolar material and an increase in total levels of intracellular insulin. RNAi- mediated silencing of pericentrin in secretory beta-cells caused dysregulated secretory vesicle hypersecretion of insulin into the media. Together, these data suggest that pericentrin may regulate the intracellular distribution and secretion of insulin. Mice transplanted with pericentrin-depleted islets exhibited abnormal fasting hypoglycemia and inability to regulate blood glucose normally during a glucose challenge, which is consistent with our in vitro data. This previously unrecognized function for a centrosomal protein to mediate vesicle docking in secretory endocrine cells emphasizes the adaptability of these scaffolding proteins to regulate diverse cellular processes and identifies a novel target for modulating regulated protein secretion in disorders such as diabetes.
Assuntos
Antígenos/metabolismo , Centrossomo/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Vesículas Secretórias/metabolismo , Animais , Antígenos/genética , Linhagem Celular Tumoral , Feminino , Imunofluorescência , Células Secretoras de Insulina/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , RNA Interferente Pequeno/genética , Radioimunoensaio , Vesículas Secretórias/ultraestruturaRESUMO
BACKGROUND: Transplantation of human skin on immunodeficient mice that support engraftment with functional human immune systems would be an invaluable tool for investigating mechanisms involved in wound healing and transplantation. Nonobese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) readily engraft with human immune systems, but human skin graft integrity is poor. In contrast, human skin graft integrity is excellent on CB17-scid bg (SCID.bg) mice, but they engraft poorly with human immune systems. METHODS: Human skin grafts transplanted onto immunodeficient NSG, SCID.bg, and other immunodeficient strains were evaluated for graft integrity, preservation of graft endothelium, and their ability to be rejected after engraftment of allogeneic peripheral blood mononuclear cells. RESULTS: Human skin transplanted onto NSG mice develops an inflammatory infiltrate, consisting predominately of host Gr1(+) cells, that is detrimental to the survival of human endothelium in the graft. Treatment of graft recipients with anti-Gr1 antibody reduces this cellular infiltrate, preserves graft endothelium, and promotes wound healing, tissue development, and graft remodeling. Excellent graft integrity of the transplanted skin includes multilayered stratified human epidermis, well-developed human vasculature, human fibroblasts, and passenger leukocytes. Injection of unfractionated, CD4 or CD8 allogeneic human peripheral blood mononuclear cell induces a rapid destruction of the transplanted skin graft. CONCLUSIONS: NSG mice treated with anti-Gr1 antibody provide a model optimized for both human skin graft integrity and engraftment of a functional human immune system. This model provides the opportunity to investigate mechanisms orchestrating inflammation, wound healing, revascularization, tissue remodeling, and allograft rejection and can provide guidance for improving outcomes after clinical transplantation.
Assuntos
Rejeição de Enxerto/patologia , Receptores de Interleucina-2/deficiência , Transplante de Pele/métodos , Animais , Antígenos CD/análise , Eritrócitos/fisiologia , Citometria de Fluxo , Humanos , Antígenos Comuns de Leucócito/análise , Transfusão de Leucócitos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Pele/patologia , Baço/patologia , Transplante Heterólogo , Transplante Homólogo , CicatrizaçãoRESUMO
"Humanized" mouse models created by engraftment of immunodeficient mice with human hematolymphoid cells or tissues are an emerging technology with broad appeal across multiple biomedical disciplines. However, investigators wishing to utilize humanized mice with engrafted functional human immune systems are faced with a myriad of variables to consider. In this study, we analyze HSC engraftment methodologies using three immunodeficient mouse strains harboring the IL2rgamma(null) mutation; NOD-scid IL2rgamma(null), NOD-Rag1(null) IL2rgamma(null), and BALB/c-Rag1(null) IL2rgamma(null) mice. Strategies compared engraftment of human HSC derived from umbilical cord blood following intravenous injection into adult mice and intracardiac and intrahepatic injection into newborn mice. We observed that newborn recipients exhibited enhanced engraftment as compared to adult recipients. Irrespective of the protocol or age of recipient, both immunodeficient NOD strains support enhanced hematopoietic cell engraftment as compared to the BALB/c strain. Our data define key parameters for establishing humanized mouse models to study human immunity.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/imunologia , Subunidade gama Comum de Receptores de Interleucina/imunologia , Animais , Animais Recém-Nascidos , Citometria de Fluxo , Histocitoquímica , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Modelos Animais , Organismos Livres de Patógenos Específicos , Estatísticas não ParamétricasRESUMO
BACKGROUND: Alpha-galactosylceramide (alpha-GalCer) is an invariant natural killer T (iNKT) cell ligand that prevents type 1 diabetes in NOD mice. However, alpha-GalCer can activate or suppress immune responses, raising concern about its potential use in human diabetes. MATERIALS AND METHODS: To evaluate this therapeutic issue further, BBDR and LEW.1WR1 rats were treated with Kilham rat virus (KRV) plus polyinosinic-polycytidylic acid, with or without alpha-GalCer, and followed for onset of diabetes. RESULTS: alpha-GalCer did not prevent diabetes in inducible rat models. To investigate this discrepancy, we analyzed iNKT cell function. Splenocytes stimulated with alpha-GalCer produced similar levels of IFNgamma in all rat strains, but less than mouse splenocytes. Rat splenocytes stimulated with alpha-GalCer preferentially produced IL-12, whereas mouse splenocytes preferentially produced IL-4. CONCLUSION: alpha-GalCer elicits species-specific cytokine responses in iNKT cells. In humans with type 1 diabetes, differences in iNKT cell responses to stimulation with alpha-GalCer due to age, genetic variability and other factors may influence its therapeutic potential.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Galactosilceramidas/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diabetes Mellitus Tipo 1/virologia , Modelos Animais de Doenças , Feminino , Galactosilceramidas/fisiologia , Interferon gama/metabolismo , Interleucina-12/metabolismo , Interleucina-4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Ratos , Fatores Sexuais , Baço/citologia , Baço/metabolismoRESUMO
Gimap5 (GTPase of the immunity-associated protein 5) has been linked to the regulation of T cell survival, and polymorphisms in the human GIMAP5 gene associate with autoimmune disorders. The BioBreeding diabetes-prone (BBDP) rat has a mutation in the Gimap5 gene that leads to spontaneous apoptosis of peripheral T cells by an unknown mechanism. Because Gimap5 localizes to the endoplasmic reticulum (ER), we hypothesized that absence of functional Gimap5 protein initiates T cell death through disruptions in ER homeostasis. We observed increases in ER stress-associated chaperones in T cells but not thymocytes or B cells from Gimap5(-/-) BBDP rats. We then discovered that ER stress-induced apoptotic signaling through C/EBP-homologous protein (CHOP) occurs in Gimap5(-/-) T cells. Knockdown of CHOP by siRNA protected Gimap5(-/-) T cells from ER stress-induced apoptosis, thereby identifying a role for this cellular pathway in the T cell lymphopenia of the BBDP rat. These findings indicate a direct relationship between Gimap5 and the maintenance of ER homeostasis in the survival of T cells.
Assuntos
Apoptose , Retículo Endoplasmático/patologia , Proteínas de Ligação ao GTP/deficiência , Estresse Fisiológico , Linfócitos T/citologia , Linfócitos T/metabolismo , Fator de Transcrição CHOP/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Sobrevivência Celular , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico/metabolismo , Ativação Linfocitária , Chaperonas Moleculares/metabolismo , Ratos , Transdução de Sinais , Timo/metabolismoRESUMO
Activation of TLR4 by administration of LPS shortens the survival of skin allografts in mice treated with costimulation blockade through a CD8 T cell-dependent, MyD88-dependent, and type I IFN receptor-dependent pathway. The effect of TLR activation on the establishment of allogeneic hematopoietic chimerism in mice treated with costimulation blockade is not known. Using a costimulation blockade protocol based on a donor-specific transfusion (DST) and a short course of anti-CD154 mAb, we show that LPS administration at the time of DST matures host alloantigen-presenting dendritic cells, prevents the establishment of mixed allogeneic hematopoietic chimerism, and shortens survival of donor-specific skin allografts. LPS mediates its effects via a mechanism that involves both CD4(+) and CD8(+) T cells and results from signaling through either the MyD88 or the type I IFN receptor pathways. We also document that timing of LPS administration is critical, as injection of LPS 24 h before treatment with DST and anti-CD154 mAb does not prevent hematopoietic engraftment but administration the day after bone marrow transplantation does. We conclude that TLR4 activation prevents the induction of mixed allogeneic hematopoietic chimerism through type I IFN receptor and MyD88-dependent signaling, which leads to the up-regulation of costimulatory molecules on host APCs and the generation of alloreactive T cells. These data suggest that distinct but overlapping cellular and molecular mechanisms control the ability of TLR agonists to block tolerance induction to hematopoietic and skin allografts in mice treated with costimulation blockade.
Assuntos
Quimerismo , Transplante de Células-Tronco Hematopoéticas , Terapia de Imunossupressão , Isoantígenos/genética , Lipopolissacarídeos/administração & dosagem , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Receptores Toll-Like/agonistas , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Transplante de Medula Óssea/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas de Transporte/administração & dosagem , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Terapia de Imunossupressão/métodos , Interferon Tipo I/biossíntese , Interferon Tipo I/metabolismo , Interferon Tipo I/fisiologia , Isoantígenos/imunologia , Isoantígenos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/fisiologia , Poli I-C/administração & dosagem , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transplante de Pele/imunologia , Receptores Toll-Like/administração & dosagem , Receptores Toll-Like/metabolismoRESUMO
Recent studies have implicated the cell surface receptor Programmed Death-1 (PD-1) in numerous models of T cell anergy, though the specific mechanisms by which the PD-1 signal maintains tolerance is not clear. We demonstrate that the depletion of PD-1 with siRNA results in a complete reversal of clonal anergy in the A.E7 T cell model, suggesting that the mechanism by which PD-1 maintains the anergic phenotype is a T-cell-intrinsic phenomenon, and not one dependent on other cell populations in vivo. We have also shown that the neutralization of IL-2 during restimulation abrogates the effect of PD-1 depletion, suggesting that tolerance mediated by PD-1 is wholly IL-2 dependent, and likewise intrinsic to the tolerized cells.
Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Linfócitos T CD4-Positivos/imunologia , Anergia Clonal/imunologia , Animais , Antígenos/administração & dosagem , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sequência de Bases , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Proliferação de Células , Anergia Clonal/genética , Columbidae , Citocromos c/imunologia , Primers do DNA/genética , Interleucina-2/antagonistas & inibidores , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Receptor de Morte Celular Programada 1 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/imunologia , Transfecção , Tolerância ao Transplante/genética , Tolerância ao Transplante/imunologiaRESUMO
OBJECTIVE: NOD mice model human type 1 diabetes and are used to investigate tolerance induction protocols for islet transplantation in a setting of autoimmunity. However, costimulation blockade-based tolerance protocols have failed in prolonging islet allograft survival in NOD mice. RESEARCH DESIGN AND METHODS: To investigate the underlying mechanisms, we studied the ability of costimulation blockade to prolong islet allograft survival in congenic NOD mice bearing insulin-dependent diabetes (Idd) loci that reduce the frequency of diabetes. RESULTS: The frequency of diabetes is reduced in NOD.B6 Idd3 mice and is virtually absent in NOD.B6/B10 Idd3 Idd5 mice. Islet allograft survival in NOD.B6 Idd3 mice treated with costimulation blockade is prolonged compared with NOD mice, and in NOD.B6/B10 Idd3 Idd5, mice islet allograft survival is similar to that achieved in C57BL/6 mice. Conversely, some Idd loci were not beneficial for the induction of transplantation tolerance. Alloreactive CD8 T-cell depletion in (NOD x CBA)F1 mice treated with costimulation blockade was impaired compared with similarly treated (C57BL/6.H2(g7) x CBA)F1 mice. Injection of exogenous interleukin (IL)-2 into NOD mice treated with costimulation prolonged islet allograft survival. NOD.B6 Idd3 mice treated with costimulation blockade deleted alloreactive CD8 T-cells and exhibited prolonged islet allograft survival. CONCLUSIONS: Il2 is the Idd3 diabetes susceptibility gene and can influence the outcome of T-cell deletion and islet allograft survival in mice treated with costimulation blockade. These data suggest that Idd loci can facilitate induction of transplantation tolerance by costimulation blockade and that IL-2/Idd3 is a critical component in this process.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Sobrevivência de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Ligante de CD40/imunologia , Citotoxicidade Imunológica/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/cirurgia , Citometria de Fluxo , Sobrevivência de Enxerto/efeitos dos fármacos , Sobrevivência de Enxerto/genética , Transplante das Ilhotas Pancreáticas/métodos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos NOD , Transplante HomólogoRESUMO
Transplantation of allogeneic organs has proven to be an effective therapeutic for a large variety of disease states, but the chronic immunosuppression that is required for organ allograft survival increases the risk for infection and neoplasia and has direct organ toxicity. The establishment of transplantation tolerance, which obviates the need for chronic immunosuppression, is the ultimate goal in the field of transplantation. Many experimental approaches have been developed in animal models that permit long-term allograft survival in the absence of chronic immunosuppression. These approaches function by inducing peripheral or central tolerance to the allograft. Emerging as some of the most promising approaches for the induction of tolerance are protocols based on costimulation blockade. However, as these protocols move into the clinic, there is recognition that little is known as to their safety and efficacy when confronted with environmental perturbants such as virus infection. In animal models, it has been reported that virus infection can prevent the induction of tolerance by costimulation blockade and, in at least one experimental protocol, can lead to significant morbidity and mortality. In this review, we discuss how viruses modulate the induction and maintenance of transplantation tolerance.
Assuntos
Tolerância ao Transplante/imunologia , Viroses/imunologia , Animais , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/virologia , Humanos , Imunidade Inata/imunologia , Terapia de Imunossupressão/métodos , Transdução de Sinais/imunologia , Imunologia de TransplantesRESUMO
Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation, we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells, cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid, B-lymphoid, and erythroid lineages, but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization, which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Subunidade gama Comum de Receptores de Interleucina/deficiência , Animais , Antígenos CD34/sangue , Linfócitos B/citologia , Linfócitos B/imunologia , Técnicas de Cultura de Células/métodos , Feminino , Hematopoese , Humanos , Recém-Nascido , Subunidade gama Comum de Receptores de Interleucina/genética , Ativação Linfocitária , Linfopoese , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteínas Recombinantes/administração & dosagem , Linfócitos T/citologia , Linfócitos T/imunologia , Condicionamento Pré-Transplante , Transplante Heterólogo , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
T cell receptor (TCR) ligation (signal one) in the presence of co-stimulation (signal two) results in downstream signals that increase protein production enabling naïve T cells to fully activate and gain effector function. Enhanced production of proteins by a cell requires an increase in endoplasmic reticulum (ER) chaperone expression, which is accomplished through activation of a cellular mechanism known as the ER stress response. The ER stress response is initiated during the cascade of events that occur for the activation of many cells; however, this process has not been comprehensively studied for T cell function. In this study, we used primary T cells and mice circulating TCR transgenic CD8(+) T cells to investigate ER chaperone expression in which TCR signaling was initiated in the presence or absence of co-stimulation. In the presence of both signals, in vitro and in vivo analyses demonstrated induction of the ER stress response, as evidenced by elevated expression of GRP78 and other ER chaperones. Unexpectedly, ER chaperones were also increased in T cells exposed only to signal one, a treatment known to cause T cells to enter the 'nonresponsive' states of anergy and tolerance. Treatment of T cells with an inhibitor to protein kinase C (PKC), a serine/threonine protein kinase found downstream of TCR signaling, indicated PKC is involved in the induction of the ER stress response during the T cell activation process, thus revealing a previously unknown role for this signaling protein in T cells. Collectively, these data suggest that induction of the ER stress response through PKC signaling is an important component for the preparation of a T cell response to antigen.
Assuntos
Retículo Endoplasmático/enzimologia , Ativação Linfocitária/imunologia , Proteína Quinase C/metabolismo , Transdução de Sinais , Estresse Fisiológico , Linfócitos T/enzimologia , Linfócitos T/imunologia , Animais , Retículo Endoplasmático/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Interleucina-2/biossíntese , Ionomicina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Dibutirato de 12,13-Forbol/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Tapsigargina/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Immunodeficient NOD-scid mice bearing a targeted mutation in the IL2 receptor common gamma chain (Il2rgamma(null)) readily engraft with human stem cells. Here we analyzed human peripheral blood mononuclear cells (PBMC) for their ability to engraft NOD-scid Il2rgamma(null) mice and established engraftment kinetics, optimal cell dose, and the influence of injection route. Even at low PBMC input, NOD-scid Il2rgamma(null) mice reproducibly support high human PBMC engraftment that plateaus within 3-4 weeks. In contrast to previous stocks of immunodeficient mice, we observed low intra- and inter-donor variability of engraftment. NOD-scid Il2rgamma(null) mice rendered hyperglycemic by streptozotocin treatment return to normoglycemia following transplantation with human islets. Interestingly, these human islet grafts are rejected following injection of HLA-mismatched human PBMC as evidenced by return to hyperglycemia and loss of human C-peptide. These data suggest that humanized NOD-scid Il2rgamma(null) mice may represent an important surrogate for investigating in vivo mechanisms of human islet allograft rejection.
Assuntos
Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Ilhotas Pancreáticas/imunologia , Leucócitos Mononucleares/imunologia , Mutação/genética , Animais , Diabetes Mellitus/imunologia , Modelos Animais de Doenças , Rejeição de Enxerto/imunologia , Humanos , Transplante das Ilhotas Pancreáticas/imunologia , Leucócitos Mononucleares/transplante , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , FenótipoRESUMO
Allogeneic organ transplantation has proven to be an effective therapeutic strategy for patients with end-stage organ disease. However, the chronic immunosuppression that is required for the survival of the allograft increases the risk for infection and malignancy. The establishment of transplantation tolerance, defined functionally as the survival of a donor allograft in the absence of immunosuppression, is the ultimate goal in the field of transplantation. Transplantation tolerance can be achieved using approaches that induce peripheral and/or central tolerance to the allograft. Protocols based on costimulation blockade (CB) have emerged as some of the most promising protocols for inducing long-term allograft survival in the absence of chronic immunosuppression. Despite its potential, recent evidence suggests that the efficacy of costimulation blockade can be reduced by environmental perturbations such as infection or inflammation, which activate Toll-like receptors (TLR). In this review, we discuss how the activation of TLRs can affect the induction and maintenance of transplantation tolerance.
Assuntos
Imunidade Inata/imunologia , Tolerância ao Transplante/imunologia , Animais , Rejeição de Enxerto , Teste de Histocompatibilidade , Humanos , Ativação Linfocitária , Receptores de Reconhecimento de Padrão/fisiologia , Transdução de Sinais , Linfócitos T/imunologia , Receptores Toll-Like/fisiologia , Quimeras de Transplante , Transplante HomólogoRESUMO
Our understanding of the basic biology of diabetes has been guided by observations made using animal models, particularly rodents. However, humans are not mice, and outcomes predicted by murine studies are not always representative of actual outcomes in the clinic. In particular, investigators studying diabetes have relied heavily on mouse and rat models of autoimmune type 1-like diabetes, and experimental results using these models have not been representative of many of the clinical trials in type 1 diabetes. In this article, we describe the availability of new models of humanized mice for the study of three areas of diabetes. These include the use of humanized mice for the study of (1) human islet stem and progenitor cells, (2) human islet allograft rejection, and (3) human immunity and autoimmunity. These humanized mouse models provide an important preclinical bridge between in vitro studies and rodent models and the translation of discoveries in these model systems to the clinic.
Assuntos
Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/fisiopatologia , Modelos Animais de Doenças , Células Secretoras de Insulina/fisiologia , Camundongos , Animais , Autoimunidade/fisiologia , Diabetes Mellitus Tipo 1/imunologia , Humanos , Sistema Imunitário/fisiologia , Células Secretoras de Insulina/transplante , Camundongos SCID , Células-Tronco/fisiologia , Transplante Heterólogo/métodosRESUMO
Animal models provide many strategies to unravel the complex interplay of genetic, immunologic, and environmental factors involved in the pathogenesis of type 1A (autoimmune) diabetes. Diabetes can be studied at multiple levels, and new technological advancements provide insights into the functioning of organelle and cellular structures. The role of innate immunity in the response to environmental pathogens has provided possible biochemical and molecular mechanisms which can explain certain clinical events in diabetes. These investigations may uncover new therapies and strategies to prevent type 1A diabetes.
Assuntos
Pesquisa Biomédica/métodos , Diabetes Mellitus Experimental/etiologia , Modelos Animais de Doenças , Sistemas Automatizados de Assistência Junto ao Leito , Receptores Toll-Like/fisiologia , Viroses/complicações , Animais , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Experimental/virologia , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Diabetes Mellitus Tipo 1/virologia , Meio Ambiente , Predisposição Genética para Doença , Humanos , Imunidade Inata/genética , Imunidade Inata/fisiologia , Modelos Biológicos , RatosRESUMO
We investigated the mechanisms by which Toll-like receptor (TLR) agonists affect the induction of mixed chimerism and skin allograft survival in mice treated with co-stimulation blockade (CB). We report that TLR agonists prevent the generation of mixed chimerism by breaking tolerance in the alloreactive CD4(+) and CD8(+) T cell compartments, and that type I interferon (IFN) is important in this process. Understanding how environmental perturbations affect CB-induced transplantation tolerance may lead to more effective regimens that can be used as an approach for the treatment of type I diabetes, for which the transplantation of pancreatic islets is a promising therapy.
Assuntos
Quimerismo/efeitos dos fármacos , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Receptores Toll-Like/agonistas , Tolerância ao Transplante/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Poli I-C/farmacologia , Receptor de Interferon alfa e beta/genética , Transplante de Pele/imunologia , Transplante de Pele/veterinária , Receptor 3 Toll-Like/agonistas , Receptor 4 Toll-Like/agonistasRESUMO
TLR activation of innate immunity prevents the induction of transplantation tolerance and shortens skin allograft survival in mice treated with costimulation blockade. The mechanism by which TLR signaling mediates this effect has not been clear. We now report that administration of the TLR agonists LPS (TLR4) or polyinosinic:polycytidylic acid (TLR3) to mice treated with costimulation blockade prevents alloreactive CD8(+) T cell deletion, primes alloreactive CTLs, and shortens allograft survival. The TLR4- and MyD88-dependent pathways are required for LPS to shorten allograft survival, whereas polyinosinic:polycytidylic acid mediates its effects through a TLR3-independent pathway. These effects are all mediated by signaling through the type 1 IFN (IFN-alphabeta) receptor. Administration of IFN-beta recapitulates the detrimental effects of TLR agonists on transplantation tolerance. We conclude that the type 1 IFN generated as part of an innate immune response to TLR activation can in turn activate adaptive immune responses that abrogate transplantation tolerance. Blocking of type 1 IFN-dependent pathways in patients may improve allograft survival in the presence of exogenous TLR ligands.