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1.
Transplant Proc ; 52(3): 982-986, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32113692

RESUMO

Immunologic and nonimmunologic mechanisms contribute to islet loss on transplantation in the liver. A site outside of the vascular bed may act as a sanctuary site avoiding immediate inflammatory response. We developed a unique approach by using a human amniotic membrane (HAM) for islet transplant onto the liver surface by combining it with human amniotic epithelial cells (HAECs). Decellularized 1.0 × 1.0-cm HAM was placed on the surface of the liver; 2000 islet equivalent of human islets mixed with 0.4 × 106 HAECs were infused into the space between the membrane and surface. Two pieces of the decellularized HAM were placed under the subcutaneous space to study the angiogenic potential. After cotransplant 57.1% of recipient mice became normoglycemic by the third day after transplant, and 100% attained euglycemia 2 weeks post-transplant. Bodyweight of 85.7% of mice gradually increased over time. The HAM and islets were identified in histologic section in all. Neovascularization was observed to be dramatically increased. In conclusion, HAM is a very versatile biological membrane; HAECs help angiogenesis and better engraftment of islets on the liver surface, eliminating the need for vascular deployment of islet cells and avoiding the risk of portal vein thrombosis and instant blood-mediated inflammatory reaction-related islet loss in the liver.


Assuntos
Âmnio , Células Epiteliais , Transplante das Ilhotas Pancreáticas/métodos , Animais , Sobrevivência de Enxerto , Humanos , Transplante das Ilhotas Pancreáticas/imunologia , Fígado , Camundongos
2.
J Clin Invest ; 130(1): 287-294, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31763998

RESUMO

Activation of host T cells that mediate allograft rejection is a 2-step process. The first occurs in secondary lymphoid organs where T cells encounter alloantigens presented by host DCs and differentiate to effectors. Antigen presentation at these sites occurs principally via transfer of intact, donor MHC-peptide complexes from graft cells to host DCs (cross-dressing) or by uptake and processing of donor antigens into allopeptides bound to self-MHC molecules (indirect presentation). The second step takes place in the graft, where effector T cells reengage with host DCs before causing rejection. How host DCs present alloantigens to T cells in the graft is not known. Using mouse islet and kidney transplantation models, imaging cytometry, and 2-photon intravital microscopy, we demonstrate extensive cross-dressing of intragraft host DCs with donor MHC-peptide complexes that occurred early after transplantation, whereas host DCs presenting donor antigen via the indirect pathway were rare. Cross-dressed DCs stably engaged TCR-transgenic effector CD8+ T cells that recognized donor antigen and were sufficient for sustaining acute rejection. In the chronic kidney rejection model, cross-dressing declined over time but was still conspicuous 8 weeks after transplantation. We conclude that cross-dressing of host DCs with donor MHC molecules is a major antigen presentation pathway driving effector T cell responses within allografts.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Rejeição de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Transplante de Rim , Ativação Linfocitária , Aloenxertos , Animais , Linfócitos T CD8-Positivos/patologia , Células Dendríticas/patologia , Rejeição de Enxerto/patologia , Camundongos , Camundongos Knockout , Imunologia de Transplantes
3.
Islets ; 11(5): 119-128, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31483188

RESUMO

Islet transplantation is efficacious to prevent severe hypoglycemia and glycemic liability of selected patients of type 1 diabetes. However, since calcineurin inhibitor (CNI) causes ß-cell and nephrotoxicity, alternative drug(s) with similar potency and safety profile to CNI will be highly desirable. Here we tested whether JAK3 inhibitor, tofacitinib could be used instead of tacrolimus in CIT07 immunosuppression regimen in cynomolgus nonhuman primate (NHP) model. Five independent streptozotocin (STZ)-induced diabetic monkeys were transplanted with MHC-mismatched allogeneic islets and three animals were further re-transplanted upon insufficient glycemic control or early islet graft rejection. After islet transplantation, blood glucose levels were quickly stabilized and maximal islet graft survival as measured by serum C-peptide concentration was >330, 98, >134, 31, or 22 days, respectively, after transplantation (median survival day; 98 days). Cellular and humoral immune responses were efficiently suppressed by JAK3 inhibitor-based immunosuppression during the follow-up periods. Although intermittent increases of the genome copy number of cynomolgus cytomegalovirus (CMV) were detected by quantitative real-time PCR analyses, serious infections or posttransplant lymphoproliferative disease (PTLD) was not found in all animals. Taken together, we have shown that JAK3 inhibitor could be used in replacement of tacrolimus in a highly translatable NHP islet transplantation model and these results suggest that JAK3 inhibitor will be potentially incorporated in human allogeneic islet transplantation.


Assuntos
Imunossupressão/métodos , Imunossupressores/uso terapêutico , Transplante das Ilhotas Pancreáticas , Janus Quinase 3/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/terapia , Avaliação Pré-Clínica de Medicamentos , Feminino , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/efeitos dos fármacos , Imunossupressão/veterinária , Transplante das Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/métodos , Macaca fascicularis , Masculino , Condicionamento Pré-Transplante/métodos , Condicionamento Pré-Transplante/veterinária , Imunologia de Transplantes/efeitos dos fármacos , Transplante Heterólogo
5.
Transpl Int ; 32(9): 903-912, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31033036

RESUMO

Pancreatic islet allotransplantation is a treatment for patients with severe forms of type 1 diabetes. As long-term graft function and survival are not yet optimal, additional studies are warranted in order to continue improving transplant outcomes. The mechanisms of islet graft loss and tolerance induction are often studied in murine diabetes models. Despite numerous islet transplantation studies successfully performed over recent years, translation from experimental mouse models to human clinical application remains elusive. This review aims at critically discussing the strengths and limitations of current mouse models of diabetes and experimental islet transplantation. In particular, we will analyze the causes leading to diabetes and compare the immunological mechanisms responsible for rejection between mouse and human. A better understanding of the experimental mouse models should facilitate translation to human clinical application.


Assuntos
Transplante das Ilhotas Pancreáticas/imunologia , Animais , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Humanos , Camundongos , Transplante Homólogo
6.
Front Immunol ; 10: 742, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024566

RESUMO

The IL-7/IL-7R pathway is essential for lymphocyte development and disturbances in the pathway can lead to immune deficiency or T cell mediated destruction. Here, the effect of transient hyperexpression of IL-7 was investigated on immune regulation and allograft rejection under immunosuppression. An experimental in vivo immunosuppressive mouse model of IL-7 hyperexpression was developed using transgenic mice (C57BL/6 background) carrying a tetracycline inducible IL-7 expression cassette, which allowed the temporally controlled induction of IL-7 hyperexpression by Dexamethasone and Doxycycline treatment. Upon induction of IL-7, the B220+ c-kit+ Pro/Pre-B I compartment in the bone marrow increased as compared to control mice in a serum IL-7 concentration-correlated manner. IL-7 hyperexpression also preferentially increased the population size of memory CD8+ T cells in secondary lymphoid organs, and reduced the proportion of CD4+Foxp3+ T regulatory cells. Of relevance to disease, conventional CD4+ T cells from an IL-7-rich milieu escaped T regulatory cell-mediated suppression in vitro and in a model of autoimmune diabetes in vivo. These findings were validated using an IL-7/anti-IL7 complex treatment mouse model to create an IL-7 rich environment. To study the effect of IL-7 on islet graft survival in a mismatched allograft model, BALB/c mice were rendered diabetic by streptozotocin und transplanted with IL-7-inducible or control islets from C57BL/6 mice. As expected, Dexamethasone and Doxycycline treatment prolonged graft median survival as compared to the untreated control group in this transplantation mouse model. However, upon induction of local IL-7 hyperexpression in the transplanted islets, graft survival time was decreased and this was accompanied by an increased CD4+ and CD8+ T cell infiltration in the islets. Altogether, the findings show that transient elevations of IL-7 can impair immune regulation and lead to graft loss also under immune suppression.


Assuntos
Rejeição de Enxerto/imunologia , Interleucina-7/biossíntese , Linfócitos T/imunologia , Animais , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/terapia , Feminino , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Homeostase/imunologia , Memória Imunológica , Imunossupressores/farmacologia , Interleucina-7/genética , Transplante das Ilhotas Pancreáticas/efeitos adversos , Transplante das Ilhotas Pancreáticas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Imunológicos , Células Precursoras de Linfócitos B/imunologia , Subpopulações de Linfócitos T/imunologia , Tolerância ao Transplante/imunologia , Transplante Homólogo , Regulação para Cima
7.
J Control Release ; 305: 176-193, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31029742

RESUMO

Islet transplantation is an alternative method of replacing exogenous insulin to treat type 1 diabetes. However, transplantation of allo- or xenograft islets causes the activation of host's immune reaction, which leads to the failure of the transplanted grafts. Immunosuppressive-sparing strategies have been introduced to avoid adverse effects associated with a long-term use of the immunosuppressive drugs. In this regard, macro/microencapsulation, surface camouflage, and surface modification with immune-privileged cells have been performed to protect the transplanted islets against instant blood-mediated inflammatory reactions or immune reactions. However, the increased size of the encapsulated islets after transplantation leads to insufficient oxygen and nutrients for the islets, causing most of them to undergo apoptosis. Therefore, recent studies have aimed at reducing the capsule thickness while maintaining immunoprotective ability of encapsulated islets. In this review, we discuss several techniques of thin-layer surface coating of pancreatic islets using a variety of polymers, therapeutic agents (TA), TA-loaded nano or microparticles, and living cells.


Assuntos
Diabetes Mellitus Tipo 1/terapia , Rejeição de Enxerto/prevenção & controle , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/imunologia , Animais , Anticoagulantes/uso terapêutico , Células Imobilizadas/citologia , Células Imobilizadas/imunologia , Células Imobilizadas/transplante , Diabetes Mellitus Tipo 1/imunologia , Rejeição de Enxerto/imunologia , Humanos , Ilhotas Pancreáticas/citologia , Transplante das Ilhotas Pancreáticas/imunologia , Polietilenoglicóis/uso terapêutico
8.
Sci Rep ; 9(1): 3918, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850640

RESUMO

The anterior chamber of the eye (ACE) has emerged as a promising clinical islet transplantation site because of its multiple advantages over the conventional intra-hepatic portal site. This includes reduced surgical invasiveness and increased islet graft survival rate. It also allows for enhanced accessibility and monitoring of the islets. Although the ACE is initially an immuno-privileged site, this privilege is disrupted once the islet grafts are re-vascularized. Given that the ACE is a confined space, achieving graft immune tolerance through local immunosuppressive drug delivery is therefore feasible. Here, we show that islet rejection in the ACE of mice can be significantly suppressed through local delivery of rapamycin by carefully designed sustained-release microparticles. In this 30-day study, allogeneic islet grafts with blank microparticles were completely rejected 18 days post-transplantation into mice. Importantly, allogeneic islet grafts co-injected with rapamycin releasing microparticles into a different eye of the same recipient were preserved much longer, with some grafts surviving for more than 30 days. Hence, islet allograft survival was enhanced by a localized and prolonged delivery of an immunosuppressive drug. We envisage that this procedure will relieve diabetic transplant recipients from harsh systemic immune suppression, while achieving improved glycemic control and reduced insulin dependence.


Assuntos
Câmara Anterior , Rejeição de Enxerto/prevenção & controle , Imunossupressores/administração & dosagem , Transplante das Ilhotas Pancreáticas/métodos , Sirolimo/administração & dosagem , Aloenxertos , Animais , Câmara Anterior/efeitos dos fármacos , Câmara Anterior/imunologia , Câmara Anterior/cirurgia , Preparações de Ação Retardada , Diabetes Mellitus/imunologia , Diabetes Mellitus/cirurgia , Sistemas de Liberação de Medicamentos , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Transplante das Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Modelos Animais , Tolerância ao Transplante/efeitos dos fármacos
9.
Xenotransplantation ; 26(2): e12461, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30230032

RESUMO

Encapsulated porcine islets could be used to treat type I diabetes without necessitating severe immunosuppression. Islet survival and secretory function in the encapsulation device need to be preserved to ensure efficient insulin output in response to surrounding stimuli. In the present study, we evaluated stimulated insulin secretion from adult and neonatal pig islets seeded on an acellular collagen matrix and encapsulated in alginate during long-term culture. Pig islets survived longer and secreted more insulin when cultured on acellular porcine dermis compared to human fascia. Islets from neonatal pigs could survive up to 33 weeks in vitro, and their insulin secretion increased during the first 5 weeks of culture in a beta-cell maturation medium. In fact, by the 4th week of culture, insulin secretion from neonatal islets attained the same level as adult islets and even surpassed it by the 18th week. Our results show that in vitro maturation of encapsulated neonatal porcine islets is possible and can actually compensate the initial low insulin secretion from these islets while allowing enough time to perform complete functional and biosafety characterization of islets before transplantation.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Insulina/metabolismo , Transplante das Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/citologia , Tempo , Alginatos , Animais , Colágeno/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Insulina/imunologia , Secreção de Insulina/imunologia , Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/métodos , Suínos , Transplante Heterólogo/métodos
10.
Xenotransplantation ; 26(2): e12476, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30552781

RESUMO

BACKGROUND: Our previous in vitro demonstration of the immunoregulatory effects of microencapsulated hUCMS on human peripheral blood mononuclear cells (PBMCs) extracted from patients with recent onset, type 1 diabetes mellitus (DM), prompted us to test our product for xenograft (TX) in non obese diabetic (NOD) mice with spontaneous DM. METHODS: We transplanted microencapsulated hUCMS into the peritoneal cavity of NOD mice with either severe or mild DM. Blood glucose (BG) levels were monitored following TX, in either basal or upon glucose stimulation. RESULTS: Only the NODs with mild DM showed full and sustained remission of hyperglycemia throughout 216 days post-TX, unlike recipients with severe DM, where no remission of hyperglycemia was attained, as reflected by erratic BG levels at all times. CONCLUSIONS: These data suggest that the stage of DM disease process in NOD mice, reflecting steady decline of residual b-cell mass, plays a pivotal role in determining the success of this cell therapy approach for treatment of DM.


Assuntos
Hiperglicemia/terapia , Transplante das Ilhotas Pancreáticas/imunologia , Células-Tronco Mesenquimais/citologia , Transplante Heterólogo , Animais , Diabetes Mellitus Experimental/imunologia , Sobrevivência de Enxerto/imunologia , Humanos , Hiperglicemia/imunologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Células-Tronco Mesenquimais/imunologia , Camundongos Endogâmicos NOD , Transplante Heterólogo/métodos
11.
Biomaterials ; 192: 271-281, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30458362

RESUMO

Intraportal allogeneic islet transplantation has been demonstrated as a potential therapy for type 1 diabetes (T1D). The placement of islets into the liver and chronic immunosuppression to control rejection are two major limitations of islet transplantation. We hypothesize that localized immunomodulation with a novel form of FasL chimeric with streptavidin, SA-FasL, can provide protection and long-term function of islets at an extrahepatic site in the absence of chronic immunosuppression. Allogeneic islets modified with biotin and engineered to transiently display SA-FasL on their surface showed sustained survival following transplantation on microporous scaffolds into the peritoneal fat in combination with a short course (15 days) of rapamycin treatment. The challenges with modifying islets for clinical translation motivated the modification of scaffolds with SA-FasL as an off-the-shelf product. Poly (lactide-co-glycolide) (PLG) was conjugated with biotin and fabricated into particles and subsequently formed into microporous scaffolds to allow for rapid and efficient conjugation with SA-FasL. Biotinylated particles and scaffolds efficiently bound SA-FasL and induced apoptosis in cells expressing Fas receptor (FasR). Scaffolds functionalized with SA-FasL were subsequently seeded with allogeneic islets and transplanted into the peritoneal fat under the short-course of rapamycin treatment. Scaffolds modified with SA-FasL had robust engraftment of the transplanted islets that restored normoglycemia for 200 days. Transplantation without rapamycin or without SA-FasL did not support long-term survival and function. This work demonstrates that scaffolds functionalized with SA-FasL support allogeneic islet engraftment and long-term survival and function in an extrahepatic site in the absence of chronic immunosuppression with significant potential for clinical translation.


Assuntos
Proteína Ligante Fas/imunologia , Proteínas Imobilizadas/imunologia , Tolerância Imunológica , Transplante das Ilhotas Pancreáticas/métodos , Tecidos Suporte , Animais , Sobrevivência de Enxerto , Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Proteínas Recombinantes/imunologia , Estreptavidina/imunologia , Tecidos Suporte/química
12.
Xenotransplantation ; 26(2): e12474, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30461074

RESUMO

BACKGROUND: Aberrant microRNA (miRNA) expression levels are associated with various graft rejections. We used our humanized mouse model with transplanted human islets to identify miRNAs in islet grafts related to xenograft rejection and circulating miRNAs associated with xenograft rejection-mediated ß-cell loss. METHODS: Diabetic immunodeficient NOD.scid mice were transplanted with human islets and subsequently achieved stable normoglycemia. Lymphocytes from NOD mice were then adoptively transferred to the humanized mice to induce human ß-cell destruction. Islet graft and plasma were collected immediately once blood glucose reached >200 mg/dL. miRNAs in the islet grafts and in the plasma with or without adoptive lymphocyte transfer (ALT) were measured using NanoString nCounter® miRNA Expression Assay and qPCR. RESULTS: A set of immune-related miRNAs was significantly increased in human islet grafts of ALT-treated mice compared to control mice. Of these miRNAs, miR-150-5p was significantly increased in the circulation of ALT-treated mice at tissue collection and the increase was a result of immune activation rather than simply the presence of lymphocytes in circulation. Furthermore, miR-150-5p was significantly increased in human islet graft and circulation prior to the development of hyperglycemia in the ALT-treated mice. CONCLUSIONS: Our data demonstrated that immune-related miRNAs are associated with human islet xenograft rejection in mice. miR-150-5p is increased in human islet graft and in the circulation during islet xenograft rejection and ß-cell destruction prior to hyperglycemia and may be an early biomarker for islet xenograft rejection.


Assuntos
Transplante das Ilhotas Pancreáticas/imunologia , Linfócitos/imunologia , MicroRNAs/genética , Transplante Heterólogo , Animais , Modelos Animais de Doenças , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Xenoenxertos/imunologia , Humanos , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , MicroRNAs/imunologia , Transplante Heterólogo/métodos
13.
FASEB J ; 33(3): 3137-3151, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30383447

RESUMO

Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) support preclinical studies of human pathogens, allograft rejection, and human T-cell function. However, a major limitation of PBMC engraftment is development of acute xenogeneic graft- versus-host disease (GVHD) due to human T-cell recognition of murine major histocompatibility complex (MHC). To address this, we created 2 NOD- scid IL-2 receptor subunit γ ( IL2rg) null (NSG) strains that lack murine MHC class I and II [NSG-ß-2-microglobulin ( B2M) null ( IA IE)null and NSG -( Kb Db) null ( IAnull)]. We observed rapid human IgG clearance in NSG- B2Mnull ( IA IE) null mice whereas clearance in NSG -( Kb Db) null ( IAnull) mice and NSG mice was comparable. Injection of human PBMCs into both strains enabled long-term engraftment of human CD4+ and CD8+ T cells without acute GVHD. Engrafted human T-cell function was documented by rejection of human islet allografts. Administration of human IL-2 to NSG -( Kb Db) null ( IAnull) mice via adeno-associated virus vector increased human CD45+ cell engraftment, including an increase in human regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data document that NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD and enable evaluation of human antibody therapeutics targeting human T cells.-Brehm, M. A., Kenney, L. L., Wiles, M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.


Assuntos
Doença Enxerto-Hospedeiro/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/transplante , Linfócitos T/imunologia , Animais , Feminino , Genes MHC Classe I , Genes MHC da Classe II , Sobrevivência de Enxerto/imunologia , Xenoenxertos , Humanos , Transplante das Ilhotas Pancreáticas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Fenótipo
14.
Transplant Proc ; 50(10): 3895-3899, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30577283

RESUMO

OBJECTIVE: The aim of this work was to investigate the transplantation efficacy of microencapsulated young market pig islets in a diabetic rat model. METHODS: Islets were isolated and purified from young market pigs obtained from a local slaughterhouse. The islets were encapsulated in barium alginate and subjected to a glucose-induced insulin release functional assay in culture. Microencapsulated islets were transplanted into diabetic Sprague-Dawley rats and removed after 30 days for histologic examination. RESULTS: The mean islet equivalent (IEQ) yield per gram of digested tissue was 3,125 ± 617 IEQ/g after isolation and 2,618 ± 917 IEQ/g after purification, respectively. Host rats' blood glucose concentrations normalized (from 22.3 ± 2.7 mmol/L to 5.1 ± 0.67 mmol/L) following encapsulated islet transplantation. After graft removal, hyperglycemia recurred in the rats, indicating that the grafts were responsible for maintaining euglycemia. Histology revealed viable islets in the capsules 30 days after graft removal. Immunolabeling of insulin verified that ß-cells within the capsules remained well granulated. No fibrosis or immune cells were found in histopathology. CONCLUSIONS: Barium alginate encapsulation of young market pig islets can normalize glucose regulation in diabetic rats without fibrosis or an immunologic response.


Assuntos
Diabetes Mellitus Experimental/cirurgia , Composição de Medicamentos/métodos , Transplante das Ilhotas Pancreáticas/métodos , Transplante Heterólogo/métodos , Alginatos , Animais , Diabetes Mellitus Experimental/sangue , Hiperglicemia/etiologia , Ilhotas Pancreáticas/fisiologia , Transplante das Ilhotas Pancreáticas/imunologia , Masculino , Ratos , Ratos Sprague-Dawley , Sus scrofa
15.
Transplant Proc ; 50(10): 3900-3905, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30577284

RESUMO

BACKGROUND: Our previous study demonstrated that regulatory B cells (Bregs) play an indispensable role in dual anti-CD45RB/anti-TIM-1 antibody-induced transplantation tolerance through an IL-10-dependent pathway. However, the potentially specific subsets of Bregs have not been clearly demonstrated. In this study, we aimed to detect the subsets of the Bregs possessing the ability to produce IL-10 in the early stage and tolerance maintenance stage of allogeneic islet transplantation treated by dual anti-CD45RB/anti-TIM-1. METHODS: Isolated Balb/C islets were transplanted under the kidney capsule of C57BL/6J diabetic mice. The recipients were treated with anti-CD45RB and anti-TIM-1. Two major IL-10-producing Breg subsets of CD19+CD5+CD1d+ B10 cells and CD19+TIM-1+B cells, and the T cells of CD4+CD25+ (Tregs), CD4+IFN-γ+ Th1 cells, and CD4+IL-4+ Th2 cells in splenocytes of grafted recipients were detected by flow cytometry after 1 week, 2 weeks, and more than 100 days posttransplantation. RESULTS: On day 14 after islet transplantation, the frequency of CD19+CD5+CD1d+ B10 cells in recipient mice increased significantly, compared with day 0 and 7 (P = .001, P < .001, respectively), while it dropped to normal level in the recipients with long-term graft survival. In contrast, there was significant increase of CD19+TIM-1+B cells on day 14 (P = .003) and day 100 after transplant (P = .009). CD4+CD25+ Treg cells have similar increasing tendency with CD19+TIM-1+B cells, as they increased from normal 10% to almost 20% both on day 14 and day 90 after transplantation. Conversely, the expression of CD4+IFN-γ+ Th1 cells decreased significantly on day 14 (P = .004) and on day 100 after transplant (P = .002). As another kind of helper T cells, CD4+IL-4+ Th2 cells increased only on day 14 after transplant (P < .001). CONCLUSIONS: CD19+CD5+CD1d+ B10 cells may play an important role only in the early stage of transplantation tolerance induction by dual anti-CD45RB/anti-TIM-1 antibody treatment, whereas CD19+TIM-1+B cells may play crucial parts in the whole process of tolerance induction and maintenance.


Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B Reguladores/imunologia , Interleucina-10/biossíntese , Transplante das Ilhotas Pancreáticas/imunologia , Tolerância ao Transplante/imunologia , Aloenxertos , Animais , Diabetes Mellitus Experimental , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
16.
J Immunol Res ; 2018: 3861079, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30116750

RESUMO

Peripheral CD4/CD8 double-positive (DP) T cells are associated with autoimmune disorders, cancer, and viral infection. However, the relationship between organ transplantation and DP T cells is unclear. Here, we examined the functional characteristics of peripheral DP T cells and analyzed their significance with respect to islet graft rejection in a nonhuman primate model of islet transplantation. DP T cells were functionally equivalent to conventional CD4 and CD8 T cells in terms of helper and cytotoxic activity, respectively. DP T cells expressed high levels of CXCR5 and PD-1 and secreted IFN-γ, IL-4, and IL-21 in amounts equivalent to those secreted by CD4 or CD8 T cells; also, they produced large amounts of granzyme B and perforin. In addition, under steady-state conditions, DP T cells expressed eomesodermin (Eomes) and promyelocytic leukemia zinc finger (PLZF) proteins, both of which act as transcription factors in innate/memory-like T cells. The number of peripheral DP T cells in the islet transplantation model was high in the group that experienced graft rejection; this was not the case in the long-term survival group. Interestingly, numbers of effector memory T cells (TEM) within the DP T cell population increased significantly during islet graft rejection, as did those of TEM within the cytotoxic CD8 T cells. Furthermore, the most conspicuous of which was the increase of CD4hiCD8low T cell subpopulation at that point. Taken together, the data suggest that peripheral DP T cells showing an innate/memory-like phenotype have both helper and cytotoxic activity in vitro and that they may act as a novel biomarker for graft rejection after islet transplantation.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Rejeição de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Modelos Animais de Doenças , Feminino , Macaca mulatta , Masculino
17.
Biomaterials ; 182: 191-201, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30134210

RESUMO

Although transplantation of pancreatic islets is a promising approach for treatment of type 1 diabetes mellitus, the engraftment efficiency of these islets is limited by host immune responses. Extensive efforts have been made to immunoisolate these islets by introducing barriers on the islet surface. To date, these barriers have not successfully protected islets from attack by the immune system. In addition, the inevitable permeability of an islet capsule cannot prevent filtration by proinflammatory cytokines and islet self-antigens. Thus, we have developed a surface engineering approach for localized immonumodulation of the islet microenvironment. Jagged-1 (JAG-1), as a potent immunomodulatory factor, was immobilized on the islet surface by mediation of a double-layer of heterobifunctional poly (ethylene glycol) (PEG). Immobilization and functionality of JAG-1 on PEGylated islet surfaces were established. When co-cultured with splenocytes, the JAG-1 conjugated islets induced a significant increase in regulatory T cells and regulated the cytokine levels produced by immune cells. The results demonstrated that JAG-1 immobilization could improve immunoprotection of pancreatic islets by localized modulation of the immune milieu from an inflammatory to an anti-inflammatory state. We also evaluated the effects of surface modification of these islets by JAG-1 in a xenotransplantation model. The transplanted JAG-1/PEG/islets group showed a significantly reduced blood glucose levels compared with the control group of diabetic mice during the acute phase of the immune response to the transplanted islets. Our results demonstrated that surface modification has the potential to shift the immune system from an inflammatory to anti-inflammatory milieu and may offer a new prospective for immunoprotection of pancreatic islets.


Assuntos
Diabetes Mellitus Experimental/terapia , Proteínas Imobilizadas/imunologia , Fatores Imunológicos/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/imunologia , Proteína Jagged-1/imunologia , Animais , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Células HEK293 , Humanos , Tolerância Imunológica , Transplante das Ilhotas Pancreáticas/métodos , Masculino , Camundongos , Ratos
18.
Xenotransplantation ; 25(6): e12450, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30117193

RESUMO

BACKGROUND: Xenogeneic donors would provide an unlimited source of islets for the treatment of type 1 diabetes (T1D). The goal of this study was to assess the function of microencapsulated adult porcine islets (APIs) transplanted ip in streptozotocin (STZ)-diabetic non-human primates (NHPs) given targeted immunosuppression. METHODS: APIs were encapsulated in: (a) single barium-gelled alginate capsules or (b) double alginate capsules with an inner, islet-containing compartment and a durable, biocompatible outer alginate layer. Immunosuppressed, streptozotocin-diabetic NHPs were transplanted ip with encapsulated APIs, and graft function was monitored by measuring blood glucose, %HbA1c, and porcine C-peptide. At graft failure, explanted capsules were assessed for biocompatibility and durability plus islet viability and functionality. Host immune responses were evaluated by phenotyping peritoneal cell populations, quantitation of peritoneal cytokines and chemokines, and measurement of anti-porcine IgG and IgM plus anti-Gal IgG. RESULTS: NHP recipients had reduced hyperglycemia, decreased exogenous insulin requirements, and lower percent hemoglobin A1c (%HbA1c) levels. Porcine C-peptide was detected in plasma of all recipients, but these levels diminished with time. However, relatively high levels of porcine C-peptide were detected locally in the peritoneal graft site of some recipients at sacrifice. IV glucose tolerance tests demonstrated metabolic function, but the grafts eventually failed in all diabetic NHPs regardless of the type of encapsulation or the host immunosuppression regimen. Explanted microcapsules were intact, "clean," and free-floating without evidence of fibrosis at graft failure, and some reversed diabetes when re-implanted ip in diabetic immunoincompetent mice. Histology of explanted capsules showed scant evidence of a host cellular response, and viable islets could be found. Flow cytometric analyses of peritoneal cells and peripheral blood showed similarly minimal evidence of a host immune response. Preformed anti-porcine IgG and IgM antibodies were present in recipient plasma, but these levels did not rise post-transplant. Peritoneal graft site cytokine or chemokine levels were equivalent to normal controls, with the exception of minimal elevation observed for IL-6 or IL-1ß, GRO-α, I-309, IP-10, and MCP-1. However, we found central necrosis in many of the encapsulated islets after graft failure, and explanted islets expressed endogenous markers of hypoxia (HIF-1α, osteopontin, and GLUT-1), suggesting a role for non-immunologic factors, likely hypoxia, in graft failure. CONCLUSIONS: With donor xenoislet microencapsulation and host immunosuppression, APIs corrected hyperglycemia after ip transplantation in STZ-diabetic NHPs in the short term. The islet xenografts lost efficacy gradually, but at graft failure, some viable islets remained, substantial porcine C-peptide was detected in the peritoneal graft site, and there was very little evidence of a host immune response. We postulate that chronic effects of non-immunologic factors, such as in vivo hypoxic and hyperglycemic conditions, damaged the encapsulated islet xenografts. To achieve long-term function, new approaches must be developed to prevent this damage, for example, by increasing the oxygen supply to microencapsulated islets in the ip space.


Assuntos
Diabetes Mellitus Experimental/induzido quimicamente , Composição de Medicamentos , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Transplante Heterólogo , Animais , Composição de Medicamentos/métodos , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Xenoenxertos/imunologia , Imunossupressão/métodos , Transplante das Ilhotas Pancreáticas/imunologia , Primatas , Estreptozocina/farmacologia , Suínos
19.
Nat Mater ; 17(8): 732-739, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29867165

RESUMO

Islet transplantation is a promising therapy for type 1 diabetes. However, chronic immunosuppression to control rejection of allogeneic islets induces morbidities and impairs islet function. T effector cells are responsible for islet allograft rejection and express Fas death receptors following activation, becoming sensitive to Fas-mediated apoptosis. Here, we report that localized immunomodulation using microgels presenting an apoptotic form of the Fas ligand with streptavidin (SA-FasL) results in prolonged survival of allogeneic islet grafts in diabetic mice. A short course of rapamycin treatment boosted the immunomodulatory efficacy of SA-FasL microgels, resulting in acceptance and function of allografts over 200 days. Survivors generated normal systemic responses to donor antigens, implying immune privilege of the graft, and had increased CD4+CD25+FoxP3+ T regulatory cells in the graft and draining lymph nodes. Deletion of T regulatory cells resulted in acute rejection of established islet allografts. This localized immunomodulatory biomaterial-enabled approach may provide an alternative to chronic immunosuppression for clinical islet transplantation.


Assuntos
Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/farmacologia , Proteína Ligante Fas/metabolismo , Proteína Ligante Fas/farmacologia , Imunomodulação/efeitos dos fármacos , Transplante das Ilhotas Pancreáticas/imunologia , Animais , Camundongos , Estreptavidina/metabolismo , Transplante Homólogo
20.
Proc Natl Acad Sci U S A ; 115(20): 5265-5270, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29712852

RESUMO

Regulatory T cells (Tregs) control organ-specific autoimmunity in a tissue antigen-specific manner, yet little is known about their specificity in a natural repertoire. In this study, we used the nonobese diabetic (NOD) mouse model of autoimmune diabetes to investigate the antigen specificity of Tregs present in the inflamed tissue, the islets of Langerhans. Compared with Tregs present in spleen and lymph node, Tregs in the islets showed evidence of antigen stimulation that correlated with higher proliferation and expression of activation markers CD103, ICOS, and TIGIT. T cell receptor (TCR) repertoire profiling demonstrated that islet Treg clonotypes are expanded in the islets, suggesting localized antigen-driven expansion in inflamed islets. To determine their specificity, we captured TCRαß pairs from islet Tregs using single-cell TCR sequencing and found direct evidence that some of these TCRs were specific for islet-derived antigens including insulin B:9-23 and proinsulin. Consistently, insulin B:9-23 tetramers readily detected insulin-specific Tregs in the islets of NOD mice. Lastly, islet Tregs from prediabetic NOD mice were effective at preventing diabetes in Treg-deficient NOD.CD28-/- recipients. These results provide a glimpse into the specificities of Tregs in a natural repertoire that are crucial for opposing the progression of autoimmune diabetes.


Assuntos
Doenças Autoimunes/imunologia , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Tolerância Imunológica/imunologia , Insulina/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Linfócitos T Reguladores/imunologia , Animais , Autoantígenos/imunologia , Doenças Autoimunes/terapia , Diabetes Mellitus Tipo 1/terapia , Camundongos Endogâmicos NOD , Camundongos SCID
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