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1.
Artif Cells Nanomed Biotechnol ; 48(1): 77-83, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31852325

RESUMO

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of insulin-producing cells. Due to the ability of apoptotic cells clearance to induce tolerance, we previously generated liposomes rich in phophatidylserine (PS) -a feature of apoptotic cells- loaded with insulin peptides to mimic apoptotic beta-cells. PS-liposomes arrested autoimmunity in experimental T1D through the induction of tolerance. The aim of this study was to investigate the potential of several peptides from different T1D autoantigens encapsulated in (PS)-liposomes for T1D prevention and to assess its safety. T1D autoantigens (Insulin, C-peptide, GAD65 and IA2) were encapsulated in PS-liposomes. Liposomes were administered to the 'gold-standard' model for the study of autoimmune T1D, the Non-Obese Diabetic mouse, that spontaneously develop the disease. Safety and toxicity of liposomes were also determined. Only PS-liposomes encapsulating insulin peptides decrease T1D incidence in the Non-Obese Diabetic mouse model. Disease prevention correlates with a decrease in the severity of the autoimmune islet destruction driven by leukocytes. PS-liposomes neither showed toxic effect nor secondary complications. Among the here referred autoantigens, insulin peptides are the best candidates to be encapsulated in liposomes, like an artificial apoptotic cell, for the arrest of autoimmunity in T1D in a safe manner.

2.
Transl Res ; 210: 8-25, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30953609

RESUMO

Type 1 diabetes (T1D) is a chronic metabolic disease of unknown etiology that results from ß-cell destruction. The onset of the disease, which arises after a long asymptomatic period of autoimmune attack, may be followed by a relapsing and remitting progression, a phenomenon that is most evident during the partial remission phase (PR). This stage lasts for a few months, shows minor requirements of exogenous insulin and could be explained by a recovery of immunological tolerance. This study aims to identify new biomarkers at early stages of pediatric T1D that reflect immunoregulatory changes. To that end, pediatric patients with T1D (n = 52) and age-related control subjects (n = 30) were recruited. Immune response-related molecules and lymphocyte subsets were determined starting at T1D onset and until the second year of progression. Results showed that circulating TGF-ß levels decreased during PR, and that betatrophin concentration was increased in all the considered stages without differing among studied checkpoints. Moreover, an increase of regulatory T, B and NK subsets was found during T1D progression, probably reflecting an attempt to restore self-tolerance. By contrast, a reduction in monocyte levels was observed at the early stages of diabetes. The results reveal significant changes in immunological parameters during the different early stages of T1D in children, which could ultimately serve as potential biomarkers to characterize the progression of T1D.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Proteínas Semelhantes a Angiopoietina/sangue , Biomarcadores/sangue , Índice de Massa Corporal , Estudos de Casos e Controles , Criança , Diabetes Mellitus Tipo 1/sangue , Progressão da Doença , Feminino , Humanos , Memória Imunológica , Subpopulações de Linfócitos/metabolismo , Masculino , Monócitos/metabolismo , Hormônios Peptídicos/sangue , Projetos Piloto , Indução de Remissão , Fator de Crescimento Transformador beta/sangue
3.
Nanomedicine (Lond) ; 12(11): 1231-1242, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28593827

RESUMO

AIM: Based on the ability of apoptosis to induce immunological tolerance, liposomes were generated mimicking apoptotic cells, and they arrest autoimmunity in Type 1 diabetes. Our aim was to validate the immunotherapy in other autoimmune disease: multiple sclerosis. MATERIALS & METHODS: Phosphatidylserine-rich liposomes were loaded with disease-specific autoantigen. Therapeutic capability of liposomes was assessed in vitro and in vivo. RESULTS: Liposomes induced a tolerogenic phenotype in dendritic cells, and arrested autoimmunity, thus decreasing the incidence, delaying the onset and reducing the severity of experimental disease, correlating with an increase in a probably regulatory CD25+ FoxP3- CD4+ T-cell subset. CONCLUSION: This is the first work that confirms phosphatidylserine-liposomes as a powerful tool to arrest multiple sclerosis, demonstrating its relevance for clinical application.


Assuntos
Autoantígenos/administração & dosagem , Imunoterapia/métodos , Lipossomos/química , Esclerose Múltipla/terapia , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Peptídeos/administração & dosagem , Fosfatidilserinas/química , Animais , Autoantígenos/imunologia , Autoantígenos/uso terapêutico , Feminino , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/uso terapêutico , Peptídeos/imunologia , Peptídeos/uso terapêutico , Linfócitos T Reguladores/imunologia
4.
Mol Cell Endocrinol ; 426: 101-12, 2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-26911933

RESUMO

The transmembrane glycoprotein CD26 or dipeptidyl peptidase IV (DPPIV) is a multifunctional protein. In immune system, CD26 plays a role in T-cell function and is also involved in thymic maturation and emigration patterns. In preclinical studies, treatment with DPPIV inhibitors reduces insulitis and delays or even reverses the new -onset of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, the specific mechanisms involved in these effects remain unknown. The aim of the present study was to investigate how DPPIV inhibition modifies the expression of genes in the thymus of NOD mice by microarray analysis. Changes in the gene expression of ß-cell autoantigens and Aire in thymic epithelial cells (TECs) were also evaluated by using qRT-PCR. A DPPIV inhibitor, MK626, was orally administered in the diet for 4 and 6 weeks starting at 6-8 weeks of age. Thymic glands from treated and control mice were obtained for each study checkpoint. Thymus transcriptome analysis revealed that 58 genes were significantly over-expressed in MK626-treated mice after 6 weeks of treatment. Changes in gene expression in the thymus were confined mainly to the immune system, including innate immunity, chemotaxis, antigen presentation and immunoregulation. Most of the genes are implicated in central tolerance mechanisms through several pathways. No differences were observed in the expression of Aire and ß-cell autoantigens in TECs. In the current study, we demonstrate that treatment with the DPPIV inhibitor MK626 in NOD mice alters the expression of the immune response-related genes in the thymus, especially those related to immunological central tolerance, and may contribute to the prevention of T1D.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Indinavir/farmacologia , Animais , Apresentação do Antígeno/genética , Feminino , Regulação da Expressão Gênica/imunologia , Redes Reguladoras de Genes , Imunomodulação/genética , Camundongos Endogâmicos NOD , Timo/efeitos dos fármacos , Timo/metabolismo , Transcriptoma
5.
Eur J Immunol ; 46(3): 593-608, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26639224

RESUMO

Autoreactive B lymphocytes play a key role as APCs in diaebetogenesis. However, it remains unclear whether B-cell tolerance is compromised in NOD mice. Here, we describe a new B lymphocyte transgenic NOD mouse model, the 116C-NOD mouse, where the transgenes derive from an islet-infiltrating B lymphocyte of a (8.3-NODxNOR) F1 mouse. The 116C-NOD mouse produces clonal B lymphocytes with pancreatic islet beta cell specificity. The incidence of T1D in 116C-NOD mice is decreased in both genders when compared with NOD mice. Moreover, several immune selection mechanisms (including clonal deletion and anergy) acting on the development, phenotype, and function of autoreactive B lymphocytes during T1D development have been identified in the 116C-NOD mouse. Surprisingly, a more accurate analysis revealed that, despite their anergic phenotype, 116C B cells express some costimulatory molecules after activation, and induce a T-cell shift toward a Th17 phenotype. Furthermore, this shift on T lymphocytes seems to occur not only when both T and B cells contact, but also when helper T (Th) lineage is established. The 116C-NOD mouse model could be useful to elucidate the mechanisms involved in the generation of Th-cell lineages.


Assuntos
Linfócitos B/imunologia , Anergia Clonal , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Tolerância Imunológica/genética , Ativação Linfocitária , Células Th17/imunologia , Animais , Deleção Clonal , Citocinas/genética , Citocinas/imunologia , Tolerância Imunológica/imunologia , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Fenótipo , Baço/anatomia & histologia , Baço/citologia , Baço/imunologia , Transgenes
6.
PLoS One ; 8(5): e63296, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23691013

RESUMO

INTRODUCTION: Efferocytosis is a crucial process by which apoptotic cells are cleared by phagocytes, maintaining immune tolerance to self in the absence of inflammation. Peripheral tolerance, lost in autoimmune processes, may be restored by the administration of autologous dendritic cells loaded with islet apoptotic cells in experimental type 1 diabetes. OBJECTIVE: To evaluate tolerogenic properties in dendritic cells induced by the clearance of apoptotic islet cells, thus explaining the re-establishment of tolerance in a context of autoimmunity. METHODS: Bone marrow derived dendritic cells from non-obese diabetic mice, a model of autoimmune diabetes, were generated and pulsed with islet apoptotic cells. The ability of these cells to induce autologous T cell proliferation and to suppress mature dendritic cell function was assessed, together with cytokine production. Microarray experiments were performed using dendritic cells to identify differentially expressed genes after efferocytosis. RESULTS: Molecular and functional changes in dendritic cells after the capture of apoptotic cells were observed. 1) Impaired ability of dendritic cells to stimulate autologous T cell proliferation after the capture of apoptotic cells even after proinflammatory stimuli, with a cytokine profile typical for immature dendritic cells. 2) Suppressive ability of mature dendritic cell function. 3) Microarray-based gene expression profiling of dendritic cells showed differential expression of genes involved in antigen processing and presentation after efferocytosis. 4) Prostaglandin E2 increased production was responsible for immunosuppressive mechanism of dendritic cells after the capture of apoptotic cells. CONCLUSIONS: The tolerogenic behaviour of dendritic cells after islet cells efferocytosis points to a mechanism of silencing potential autoreactive T cells in the microenvironment of autoimmunity. Our results suggest that dendritic cells may be programmed to induce specific immune tolerance using apoptotic cells; this is a viable strategy for a variety of autoimmune diseases.


Assuntos
Autoimunidade , Células Dendríticas/metabolismo , Dinoprostona/biossíntese , Fagocitose , Animais , Proliferação de Células , Células Dendríticas/citologia , Células Dendríticas/imunologia , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos NOD , Linfócitos T Reguladores/citologia
7.
Mol Immunol ; 45(11): 3152-62, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18433871

RESUMO

Rearrangement analysis of immunoglobulin genes is an exceptional opportunity to look back at the B lymphocyte differentiation during ontogeny and the subsequent immune response, and thus to study the selective pressures involved in autoimmune disorders. In a recent study to characterize the antigenic specificity of B lymphocytes during T1D progression, we generated hybridomas of islet-infiltrating B lymphocytes from NOD mice and other related strains developing insulitis, but with different degrees of susceptibility to T1D. We found that a sizable proportion of hybridomas produced monoclonal antibodies reactive to peripherin, an intermediate filament protein mainly found in the peripheral nervous system. Moreover, we found that anti-peripherin antibody-producing hybridomas originated from B lymphocytes that had undergone immunoglobulin class switch recombination, a characteristic of secondary immune response. Therefore, in the present study we performed immunoglobulin VL and VH analysis of these hybridomas to ascertain whether they were derived from B lymphocytes that had undergone antigen-driven selection. The results indicated that whereas some anti-peripherin hybridomas showed signs of oligoclonality, somatic hypermutation and/or secondary rearrangements (receptor edition and receptor revision), others seemed to directly derive from the preimmune repertoire. In view of these results, we conclude that anti-peripherin B lymphocytes are positively selected and primed in the course of T1D development in NOD mice, and reinforce the idea that peripherin is a relevant autoantigen targeted during T1D development in this animal model.


Assuntos
Linfócitos B/imunologia , Linfócitos B/patologia , Diabetes Mellitus/imunologia , Proteínas de Filamentos Intermediários/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Seleção Genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Células Clonais , Regiões Determinantes de Complementaridade/química , Diabetes Mellitus/patologia , Rearranjo Gênico do Linfócito B/imunologia , Hibridomas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Mutação/genética , Periferinas , Hipermutação Somática de Imunoglobulina/imunologia
8.
J Immunol ; 178(10): 6533-9, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17475883

RESUMO

Most of our knowledge of the antigenic repertoire of autoreactive B lymphocytes in type 1 diabetes (T1D) comes from studies on the antigenic specificity of both circulating islet-reactive autoantibodies and peripheral B lymphocyte hybridomas generated from human blood or rodent spleen. In a recent study, we generated hybridoma cell lines of infiltrating B lymphocytes from different mouse strains developing insulitis, but with different degrees of susceptibility to T1D, to characterize the antigenic specificity of islet-infiltrating B lymphocytes during progression of the disease. We found that many hybridomas produced mAbs restricted to the peripheral nervous system (PNS), thus indicating an active B lymphocyte response against PNS elements in the pancreatic islet during disease development. The aim of this study was to identify the autoantigen recognized by these anti-PNS mAbs. Our results showed that peripherin is the autoantigen recognized by all anti-PNS mAbs, and, therefore, a relevant neuroendocrine autoantigen targeted by islet-infiltrating B lymphocytes. Moreover, we discovered that the immune dominant epitope of this B lymphocyte immune response is found at the C-terminal end of Per58 and Per61 isoforms. In conclusion, our study strongly suggests that peripherin is a major autoantigen targeted during T1D development and poses a new question on why peripherin-specific B lymphocytes are mainly attracted to the islet during disease.


Assuntos
Autoantígenos/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Movimento Celular/imunologia , Proteínas de Filamentos Intermediários/imunologia , Proteínas de Filamentos Intermediários/metabolismo , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Autoantígenos/imunologia , Subpopulações de Linfócitos B/patologia , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Feminino , Hibridomas , Insulinoma/imunologia , Insulinoma/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Neuroblastoma/imunologia , Neuroblastoma/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Periferinas , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo
9.
Diabetes ; 56(4): 940-9, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17395741

RESUMO

B-cells participate in the autoimmune response that precedes the onset of type 1 diabetes, but how these cells contribute to disease progression is unclear. In this study, we analyzed the phenotype and functional characteristics of islet-infiltrating B-cells in the diabetes-prone NOD mouse and in the insulitis-prone but diabetes-resistant (NOD x NOR)F1 mouse. The results indicate that B-cells accumulate in the islets of both mice influenced by sex traits. Phenotypically and functionally, these B-cells are highly affected by the islet inflammatory milieu, which may keep them in a silenced status. Moreover, although islet-infiltrating B-cells seem to be antigen experienced, they can only induce islet-infiltrating T-cell proliferation when they act as accessory cells. Thus, these results strongly suggest that islet-infiltrating B-cells do not activate islet-infiltrating T-cells in situ, although they may affect the progression of the disease otherwise.


Assuntos
Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/imunologia , Linfócitos T/imunologia , Animais , Técnicas de Cultura de Células , Citocinas/genética , Diabetes Mellitus Tipo 1/imunologia , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Ilhotas Pancreáticas/citologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD
10.
Diabetes ; 54(1): 69-77, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15616012

RESUMO

B-cells accumulate in pancreatic islets during the autoimmune response that precedes the onset of type 1 diabetes. However, the role and antigenic specificity of these cells remain a mystery. To elucidate the antigenic repertoire of islet-infiltrating B-cells in type 1 diabetes, we generated hybridoma cell lines of islet-infiltrating B-cells from nonobese diabetic (NOD) mice and NOD mice expressing a diabetogenic T-cell receptor (8.3-NOD). Surprisingly, characterization of the tissue specificity of the antibodies secreted by these cells revealed that a predominant fraction of these hybridomas produce antibodies specific for the pancreatic nervous system. Similar results were obtained with B-cell hybridomas derived from mild insulinic lesions of diabetes-resistant (NOD x NOR)F1 and 8.3-(NOD x NOR)F1 mice. Immunoglobulin class analyses further indicated that most islet-derived hybridomas had arisen from B-cells that had undergone immunoglobulin class switch recombination, suggesting that islet-associated B-cells are involved in active, T-helper-driven immune responses against local antigenic targets. This is the first evidence showing the existence of a predominant active B-cell response in situ against pancreatic nervous system elements in diabetogenesis. Our data are consistent with the idea that this B-cell response precedes the progression of insulitis to overt diabetes, thus strongly supporting the idea that pancreatic nervous system elements are early targets in type 1 diabetes.


Assuntos
Diabetes Mellitus Tipo 1/patologia , Ilhotas Pancreáticas/patologia , Sistema Nervoso/patologia , Animais , Cruzamentos Genéticos , Diabetes Mellitus Tipo 1/classificação , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Feminino , Hibridomas , Imunoglobulinas/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD
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