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1.
Australas J Dermatol ; 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31944277

RESUMO

BACKGROUND/OBJECTIVE: Ingenol mebutate gel is approved for actinic keratosis field therapy, but little has been published as a treatment of basal cell carcinoma (BCC). Our objective is to characterise the histopathological changes and the infiltrating cell populations to better understand its mechanism of action. METHODS: Sixteen patients with various BCC subtypes were prospectively evaluated and treated once daily for two consecutive days with ingenol mebutate gel 0.05% under occlusion. Patients were randomised to two arms: the first arm was biopsied between the third and the tenth day after treatment initiation ('early immune response'), and the second arm was biopsied at day 30 after treatment initiation ('late immune response'). The immunopathology was evaluated by immunohistochemistry: anti-CD3, anti-CD4, anti-CD8, anti-CD20, anti-CD56, anti-CD68, anti-Bcl-2, anti-CASP3, anti-FoxP3, anti-GrzB and anti-TIA-1. RESULTS: Ten BCCs were in complete remission after 2 years of follow-up. The early immune response was characterised by a quick recruitment of T lymphocytes, macrophages and natural killer cells. At later time-points, T-regulatory cells and some pro-apoptotic markers were detected. Treatment-related adverse events were described. CONCLUSION: Ingenol mebutate gel produces a transient immuno-inflammatory response and an important necrosis reaction in BCCs. Larger studies will be required to determine the maximum effective tolerated dose of ingenol mebutate gel for BCC.

2.
Front Immunol ; 10: 1732, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428087

RESUMO

Previous studies indicate that B-lymphocytes play a key role activating diabetogenic T-lymphocytes during the development of autoimmune diabetes. Recently, two transgenic NOD mouse models were generated: the NOD-PerIg and the 116C-NOD mice. In NOD-PerIg mice, B-lymphocytes acquire an activated proliferative phenotype and support accelerated autoimmune diabetes development. In contrast, in 116C-NOD mice, B-lymphocytes display an anergic-like phenotype delaying autoimmune diabetes onset and decreasing disease incidence. The present study further evaluates the T- and B-lymphocyte phenotype in both models. In islet-infiltrating B-lymphocytes (IIBLs) from 116C-NOD mice, the expression of H2-Kd and H2-Ag7 is decreased, whereas that of BAFF, BAFF-R, and TACI is increased. In contrast, IIBLs from NOD-PerIg show an increase in CD86 and FAS expression. In addition, islet-infiltrating T-lymphocytes (IITLs) from NOD-PerIg mice exhibit an increase in PD-1 expression. Moreover, proliferation assays indicate a high capacity of B-lymphocytes from NOD-PerIg mice to secrete high amounts of cytokines and induce T-lymphocyte activation compared to 116C B-lymphocytes. This functional variability between 116C and PerIg B-lymphocytes ultimately results in differences in the ability to shape T-lymphocyte phenotype. These results support the role of B-lymphocytes as key regulators of T-lymphocytes in autoimmune diabetes and provide essential information on the phenotypic characteristics of the T- and B-lymphocytes involved in the autoimmune response in autoimmune diabetes.

3.
Sci Rep ; 8(1): 8106, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29802270

RESUMO

Type 1 diabetes can be overcome by regulatory T cells (Treg) in NOD mice yet an efficient method to generate and maintain antigen-specific Treg is difficult to come by. Here, we devised a combination therapy of peptide/MHC tetramers and IL-2/anti-IL-2 monoclonal antibody complexes to generate antigen-specific Treg and maintain them over extended time periods. We first optimized treatment protocols conceived to obtain an improved islet-specific Treg/effector T cell ratio that led to the in vivo expansion and activation of these Treg as well as to an improved suppressor function. Optimized protocols were applied to treatment for testing diabetes prevention in NOD mice as well as in an accelerated T cell transfer model of T1D. The combined treatment led to robust protection against diabetes, and in the NOD model, to a close to complete prevention of insulitis. Treatment was accompanied with increased secretion of IL-10, detectable in total splenocytes and in Foxp3- CD4 T cells. Our data suggest that a dual protection mechanism takes place by the collaboration of Foxp3+ and Foxp3- regulatory cells. We conclude that antigen-specific Treg are an important target to improve current clinical interventions against this disease.


Assuntos
Anticorpos Monoclonais/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Antígenos de Histocompatibilidade/química , Interleucina-2/imunologia , Peptídeos/química , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos , Animais , Diabetes Mellitus Tipo 1/tratamento farmacológico , Feminino , Camundongos , Peptídeos/farmacologia , Fenótipo , Multimerização Proteica , Estrutura Quaternária de Proteína , Linfócitos T Reguladores/efeitos dos fármacos
4.
Front Immunol ; 9: 253, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29491866

RESUMO

Type 1 diabetes (T1D) is a metabolic disease caused by the autoimmune destruction of insulin-producing ß-cells. With its incidence increasing worldwide, to find a safe approach to permanently cease autoimmunity and allow ß-cell recovery has become vital. Relying on the inherent ability of apoptotic cells to induce immunological tolerance, we demonstrated that liposomes mimicking apoptotic ß-cells arrested autoimmunity to ß-cells and prevented experimental T1D through tolerogenic dendritic cell (DC) generation. These liposomes contained phosphatidylserine (PS)-the main signal of the apoptotic cell membrane-and ß-cell autoantigens. To move toward a clinical application, PS-liposomes with optimum size and composition for phagocytosis were loaded with human insulin peptides and tested on DCs from patients with T1D and control age-related subjects. PS accelerated phagocytosis of liposomes with a dynamic typical of apoptotic cell clearance, preserving DCs viability. After PS-liposomes phagocytosis, the expression pattern of molecules involved in efferocytosis, antigen presentation, immunoregulation, and activation in DCs concurred with a tolerogenic functionality, both in patients and control subjects. Furthermore, DCs exposed to PS-liposomes displayed decreased ability to stimulate autologous T cell proliferation. Moreover, transcriptional changes in DCs from patients with T1D after PS-liposomes phagocytosis pointed to an immunoregulatory prolife. Bioinformatics analysis showed 233 differentially expressed genes. Genes involved in antigen presentation were downregulated, whereas genes pertaining to tolerogenic/anti-inflammatory pathways were mostly upregulated. In conclusion, PS-liposomes phagocytosis mimics efferocytosis and leads to phenotypic and functional changes in human DCs, which are accountable for tolerance induction. The herein reported results reinforce the potential of this novel immunotherapy to re-establish immunological tolerance, opening the door to new therapeutic approaches in the field of autoimmunity.


Assuntos
Apoptose/imunologia , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Tolerância Imunológica/imunologia , Fosfatidilserinas/imunologia , Adolescente , Adulto , Autoantígenos/imunologia , Células Cultivadas , Feminino , Humanos , Imunoterapia/métodos , Lipossomos , Masculino , Pessoa de Meia-Idade , Mimetismo Molecular/imunologia , Fagocitose , Adulto Jovem
7.
Diabetes ; 65(7): 1977-1987, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26961115

RESUMO

While the autoimmune destruction of pancreatic ß-cells underlying type 1 diabetes (1D) development is ultimately mediated by T-cells in NOD mice and also likely humans, B-lymphocytes play an additional key pathogenic role. It appears expression of plasma membrane bound immunoglobulin (Ig) molecules that efficiently capture ß-cell antigens allows autoreactive B-lymphocytes bypassing normal tolerance induction processes to be the subset of antigen presenting cells most efficiently activating diabetogenic T-cells. NOD mice transgenically expressing Ig molecules recognizing antigens that are (insulin) or not (hen egg lysozyme; HEL) expressed by ß-cells have proven useful in dissecting the developmental basis of diabetogenic B-lymphocytes. However, these transgenic Ig specificities were originally selected for their ability to recognize insulin or HEL as foreign, rather than autoantigens. Thus, we generated and characterized NOD mice transgenically expressing an Ig molecule representative of a large proportion of naturally occurring islet-infiltrating B-lymphocytes in NOD mice recognizing the neuronal antigen peripherin. Transgenic peripherin autoreactive B-lymphocytes infiltrate NOD pancreatic islets, acquire an activated proliferative phenotype, and potently support accelerated T1D development. These results support the concept of neuronal autoimmunity as a pathogenic feature of T1D, and targeting such responses could ultimately provide an effective disease intervention approach.

8.
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
9.
PLoS One ; 10(6): e0127057, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26039878

RESUMO

INTRODUCTION: The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow ß-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. OBJECTIVE: To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to ß-cells in type 1 diabetes. METHODS: A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides. RESULTS: We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4+ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4+ T cell expansion. CONCLUSIONS: We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases.


Assuntos
Autoantígenos/imunologia , Autoimunidade , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/imunologia , Fosfatidilserinas/uso terapêutico , Animais , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Citocinas/metabolismo , Células Dendríticas/imunologia , Imunoterapia , Injeções Intraperitoneais , Insulina/uso terapêutico , Lipossomos , Camundongos Endogâmicos NOD , Fenótipo
10.
Proc Natl Acad Sci U S A ; 111(33): E3405-14, 2014 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-25092329

RESUMO

Type 1 diabetes is an autoimmune condition caused by the lymphocyte-mediated destruction of the insulin-producing ß cells in pancreatic islets. We aimed to identify final molecular entities targeted by the autoimmune assault on pancreatic ß cells that are causally related to ß cell viability. Here, we show that cyclin D3 is targeted by the autoimmune attack on pancreatic ß cells in vivo. Cyclin D3 is down-regulated in a dose-dependent manner in ß cells by leukocyte infiltration into the islets of the nonobese diabetic (NOD) type 1 diabetes-prone mouse model. Furthermore, we established a direct in vivo causal link between cyclin D3 expression levels and ß-cell fitness and viability in the NOD mice. We found that changes in cyclin D3 expression levels in vivo altered the ß-cell apoptosis rates, ß-cell area homeostasis, and ß-cell sensitivity to glucose without affecting ß-cell proliferation in the NOD mice. Cyclin D3-deficient NOD mice exhibited exacerbated diabetes and impaired glucose responsiveness; conversely, transgenic NOD mice overexpressing cyclin D3 in ß cells exhibited mild diabetes and improved glucose responsiveness. Overexpression of cyclin D3 in ß cells of cyclin D3-deficient mice rescued them from the exacerbated diabetes observed in transgene-negative littermates. Moreover, cyclin D3 overexpression protected the NOD-derived insulinoma NIT-1 cell line from cytokine-induced apoptosis. Here, for the first time to our knowledge, cyclin D3 is identified as a key molecule targeted by autoimmunity that plays a nonredundant, protective, and cell cycle-independent role in ß cells against inflammation-induced apoptosis and confers metabolic fitness to these cells.


Assuntos
Ciclo Celular , Ciclina D3/fisiologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Ilhotas Pancreáticas/patologia , Animais , Apoptose , Citocinas/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Camundongos , Camundongos Endogâmicos NOD
11.
J Immunol ; 192(7): 3080-90, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24610011

RESUMO

Autoreactive B cells are essential for the pathogenesis of type 1 diabetes. The genesis and dynamics of autoreactive B cells remain unknown. In this study, we analyzed the immune response in the NOD mouse model to the neuronal protein peripherin (PRPH), a target Ag of islet-infiltrating B cells. PRPH autoreactive B cells recognized a single linear epitope of this protein, in contrast to the multiple epitope recognition commonly observed during autoreactive B cell responses. Autoantibodies to this epitope were also detected in the disease-resistant NOR and C57BL/6 strains. To specifically detect the accumulation of these B cells, we developed a novel approach, octameric peptide display, to follow the dynamics and localization of anti-PRPH B cells during disease progression. Before extended insulitis was established, anti-PRPH B cells preferentially accumulated in the peritoneum. Anti-PRPH B cells were likewise detected in C57BL/6 mice, albeit at lower frequencies. As disease unfolded in NOD mice, anti-PRPH B cells invaded the islets and increased in number at the peritoneum of diabetic but not prediabetic mice. Isotype-switched B cells were only detected in the peritoneum. Anti-PRPH B cells represent a heterogeneous population composed of both B1 and B2 subsets. In the spleen, anti-PRPH B cell were predominantly in the follicular subset. Therefore, anti-PRPH B cells represent a heterogeneous population that is generated early in life but proliferates as diabetes is established. These findings on the temporal and spatial progression of autoreactive B cells should be relevant for our understanding of B cell function in diabetes pathogenesis.


Assuntos
Linfócitos B/imunologia , Diabetes Mellitus Tipo 1/imunologia , Ilhotas Pancreáticas/imunologia , Periferinas/imunologia , Sequência de Aminoácidos , Animais , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Western Blotting , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Progressão da Doença , Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Feminino , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Microscopia de Fluorescência , Dados de Sequência Molecular , Periferinas/genética , Periferinas/metabolismo , Peritônio/imunologia , Peritônio/metabolismo , Isoformas de Proteínas/imunologia , Baço/imunologia , Baço/metabolismo
12.
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
13.
ISRN Endocrinol ; 2013: 346987, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23555060

RESUMO

Type 1 diabetes is a metabolic disease caused by autoimmunity towards ß -cells. Different strategies have been developed to restore ß -cell function and to reestablish immune tolerance to prevent and cure the disease. Currently, there is no effective treatment strategy to restore endogenous insulin secretion in patients with type 1 diabetes. This study aims to restore insulin secretion in diabetic mice with experimental antigen-specific immunotherapy alone or in combination with rapamycin, a compound well known for its immunomodulatory effect. Nonobese diabetic (NOD) mice develop spontaneous type 1 diabetes after 12 weeks of age. Autologous tolerogenic dendritic cells-consisting in dendritic cells pulsed with islet apoptotic cells-were administered to diabetic NOD mice alone or in combination with rapamycin. The ability of this therapy to revert type 1 diabetes was determined by assessing the insulitis score and by measuring both blood glucose levels and C-peptide concentration. Our findings indicate that tolerogenic dendritic cells alone or in combination with rapamycin do not ameliorate diabetes in NOD mice. These results suggest that alternative strategies may be considered for the cure of type 1 diabetes.

14.
Diabetes Metab Res Rev ; 29(6): 446-51, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23483713

RESUMO

BACKGROUND: The aim of this study was to characterize the clinical characteristics and insulin secretion in adults with latent autoimmune diabetes in adults (LADA). We also compared these characteristics in subjects with antibody-negative type 2 diabetes (T2DM) or adult-onset type 1 diabetes (T1DM) to subjects with LADA. METHODS: In this cross-sectional study, 82 patients with LADA, 78 with T1DM and 485 with T2DM were studied. Clinical and metabolic data, in particular those that related to metabolic syndrome, fasting C-peptide and islet-cell autoantibodies [glutamic acid decarboxylase (GADAb) and IA2 (IA2Ab)] were measured. RESULTS: The frequency of metabolic syndrome in patients with LADA (37.3%) was higher than in those with T1DM (15.5%; p = 0.005) and lower than in patients with T2DM (67.2%; p < 0.001). During the first 36 months of the disease, the C-peptide concentration in LADA patients was higher than in subjects with T1DM but was lower than in T2DM patients (p < 0.01 for comparisons). Glycemic control in LADA patients (HbA1c 8.1%) was worse than in patients with T2DM (HbA1c 7.6%; p =0.007). An inverse association between GADAb titers and C-peptide concentrations was found in subjects with LADA (p < 0.001). Finally, LADA patients rapidly progressed to insulin treatment. CONCLUSIONS: As in other European populations, patients with LADA in Spain have a distinct metabolic profile compared with patients with T1DM or T2DM. LADA is also associated with higher impairment of beta-cell function and has worse glycemic control than in T2DM. Beta cell function is related to GADAb titers in patients with LADA.


Assuntos
Autoimunidade , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/imunologia , Adulto , Idoso , Autoanticorpos/sangue , Peptídeo C/sangue , Estudos Transversais , Feminino , Humanos , Insulina/metabolismo , Secreção de Insulina , Masculino , Síndrome Metabólica/diagnóstico , Síndrome Metabólica/imunologia , Pessoa de Meia-Idade , Espanha/epidemiologia
15.
Inmunología (1987) ; 30(1): 2-7, ene.-mar. 2011. ilus, tab
Artigo em Espanhol | IBECS | ID: ibc-109189

RESUMO

The Non-obese diabetic (NOD) mice exhibit a susceptibility to spontaneous development of autoimmune diabetes and is the most widely used experimental model for the study of the disease. The NOD strain was established by inbreeding in 1980. This model has a MHC-matched diabetes resistant homologous, NOR/Lt mice, an insulitis-resistant and diabetes-free strain produced from an isolated genetic contamination within a NOD/Lt line. To evaluate the role of transgenes, transgenic mice can be generated in CD-1 mice for technical advantages and then backcrossed to inbred strains. To obtain transgenic mice in NOD or NOR background starting from CD-1, at least 20 backcrosses are required, spending more than two years in the process.Nucleotide repeats (microsatellites) mapped to specific locations on each chromosome are used to evaluate genomic polymorphism. From 23 microsatellites we selected eleven that were variant in PCR amplimer size between CD-1 colony and NOD or NOR strains. We used these microsatellites to identify individuals that were used for backcrossing, thus accelerating the acquisition of a new genetic background. Results yield a defined analysis of the genome in question and profiles were compared to detect genetic variation among individuals. After the selection of mice for backcrossing at the third generation, the 11 specific markers were acquired at the 5th generation and maintained to the 10th generation. Diabetes incidence and insulitis score correlated with the acquisition of genetic background, demonstrating that using this strategy, 5-6 crosses are enough to obtain the genotype of interest, shortening the process in more than one year and a half (AU)


Los ratones diabéticos no obesos (NOD) presentan susceptibilidad al desarrollo espontáneo de diabetes autoinmunitaria y son el modelo experimental más utilizado para el estudio de la enfermedad. La cepa NOD fue establecida por endogamia en 1980. Este modelo tiene un homólogo resistente a la diabetes con MHC compatible, el ratón NOR/Lt, una cepa sin diabetes y que no desarrolla insulitis producida a partir de una contaminación genética aislada en una línea de NOD/Lt. Para evaluar la función de los transgenes, los ratones transgénicos pueden generarse en ratones CD1 por sus ventajas técnicas y luego cruzarse con cepas consanguíneas. Para obtener ratones transgénicos con genotipo NOD o NOR a partir de CD1, se requieren como mínimo 20 retrocruzamientos, lo que supone un proceso de más de dos años.Las repeticiones de nucleótidos (microsatélites) identificadas en localizaciones específicas de cada cromosoma se utilizan para evaluar el polimorfismo genómico. De 23 microsatélites, seleccionamos 11 con tamaños de amplímero de PCR divergentes entre la colonia de CD1 y las cepas NOD o NOR. Utilizamos estos microsatélites para identificar individuos que se utilizaron para los retrocruzamientos, con lo que se aceleró la adquisición de unos nuevos genotipos. Los resultados produjeron un análisis definido del genoma en cuestión y se compararon los perfiles para detectar variaciones genéticas entre los individuos. Tras la selección de ratones para llevar a cabo el retrocruzamiento en la tercera generación, los 11 marcadores específicos se adquirieron en la 5.ª generación y se mantuvieron hasta la 10.ª generación. La puntuación de insulitis e incidencia de diabetes se correlacionó con la adquisición de genotipos, lo que demostró que mediante esta estrategia bastaban 5-6 retrocruzamientos para obtener el genotipo de interés, reduciendo así el proceso en más de un año y medio (AU)


Assuntos
Animais , Ratos , Diabetes Mellitus Tipo 1/genética , Repetições de Microssatélites/genética , Ratos Transgênicos/genética , Reação em Cadeia da Polimerase , Genótipo
16.
Diabetes ; 59(1): 272-81, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19833887

RESUMO

OBJECTIVE: The approximately 45-cM insulin-dependent diabetes 9 (Idd9) region on mouse chromosome 4 harbors several different type 1 diabetes-associated loci. Nonobese diabetic (NOD) mice congenic for the Idd9 region of C57BL/10 (B10) mice, carrying antidiabetogenic alleles in three different Idd9 subregions (Idd9.1, Idd9.2, and Idd9.3), are strongly resistant to type 1 diabetes. However, the mechanisms remain unclear. This study aimed to define mechanisms underlying the type 1 diabetes resistance afforded by B10 Idd9.1, Idd9.2, and/or Idd9.3. RESEARCH DESIGN AND METHODS: We used a reductionist approach that involves comparing the fate of a type 1 diabetes-relevant autoreactive CD8(+) T-cell population, specific for residues 206-214 of islet-specific glucose 6 phosphatase catalytic subunit-related protein (IGRP(206-214)), in noncongenic versus B10 Idd9-congenic (Idd9.1 + Idd9.2 + Idd9.3, Idd9.2 + Idd9.3, Idd9.1, Idd9.2, and Idd9.3) T-cell receptor (TCR)-transgenic (8.3) NOD mice. RESULTS: Most of the protective effect of Idd9 against 8.3-CD8(+) T-cell-enhanced type 1 diabetes was mediated by Idd9.1. Although Idd9.2 and Idd9.3 afforded some protection, the effects were small and did not enhance the greater protective effect of Idd9.1. B10 Idd9.1 afforded type 1 diabetes resistance without impairing the developmental biology or intrinsic diabetogenic potential of autoreactive CD8(+) T-cells. Studies in T- and B-cell-deficient 8.3-NOD.B10 Idd9.1 mice revealed that this antidiabetogenic effect was mediated by endogenous, nontransgenic T-cells in a B-cell-independent manner. Consistent with this, B10 Idd9.1 increased the suppressive function and antidiabetogenic activity of the FoxP3(+)CD4(+)CD25(+) T-cell subset in both TCR-transgenic and nontransgenic mice. CONCLUSIONS: A gene(s) within Idd9.1 regulates the development and function of FoxP3(+)CD4(+)CD25(+) regulatory T-cells and, in turn, the activation of CD8(+) effector T-cells in the pancreatic draining lymph nodes, without affecting their development or intrinsic diabetogenic potential.


Assuntos
Antígenos CD4/imunologia , Mapeamento Cromossômico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Fatores de Transcrição Forkhead/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD4/genética , Citocinas/metabolismo , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/genética , Glucose-6-Fosfatase/genética , Imunossupressão , Subunidade alfa de Receptor de Interleucina-2/genética , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL/genética , Camundongos Endogâmicos NOD/genética
17.
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
18.
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
19.
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
20.
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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