RESUMO
Stem cell therapy using islet-like insulin-producing cells derived from human pluripotent stem cells has the potential to allow patients with type 1 diabetes to withdraw from insulin therapy. However, several issues exist regarding the use of stem cell therapy to treat type 1 diabetes. In this review, we will focus on the following topics: (1) autoimmune responses during the autologous transplantation of stem cell-derived islet cells, (2) a comparison of stem cell therapy with insulin injection therapy, (3) the impact of the islet microenvironment on stem cell-derived islet cells, and (4) the cost-effectiveness of stem cell-derived islet cell transplantation. Based on these various viewpoints, we will discuss what is required to perform stem cell therapy for patients with type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/terapia , Transplante de Células-Tronco , Animais , Autoimunidade , Microambiente Celular , Análise Custo-Benefício , Diabetes Mellitus Tipo 1/economia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Estudos de Viabilidade , Humanos , Transplante de Células-Tronco/economiaRESUMO
Type 1 Diabetes (T1D) is an autoimmune disease marked by direct elimination of insulin-producing ß cells by autoreactive T effectors. Recent T1D clinical trials utilizing autologous Tregs transfers to restore immune balance and improve disease has prompted us to design a novel Tregs-based antigen-specific T1D immunotherapy. We engineered a Chimeric Antigen Receptor (CAR) expressing a single-chain Fv recognizing the human pancreatic endocrine marker, HPi2. Human T cells, transduced with the resultant HPi2-CAR, proliferated and amplified Granzyme B accumulation when co-cultured with human, but not mouse ß cells. Furthermore, following exposure of HPi2-CAR transduced cells to islets, CD8+ lymphocytes demonstrated enhanced CD107a (LAMP-1) expression, while CD4+ cells produced increased levels of IL-2. HPi2-CAR Tregs failed to maintain expansion due to a persistent tonic signaling from the CAR engagement to unexpectantly HPi2 antigen present on Tregs. Overall, we show lack of functionality of HPi2-CAR and highlight the importance of careful selection of CAR recognition driver for the sustainable activity and expandability of engineered T cells.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Protaminas/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Humanos , Tolerância Imunológica/imunologia , Imunoterapia Adotiva/métodos , Ilhotas Pancreáticas , Pâncreas/citologia , Pâncreas/metabolismo , Protaminas/metabolismo , Engenharia de Proteínas/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismoRESUMO
We previously assessed the kinetics of T cell turnover in vivo by labeling cells with 2 H-H2 O over 42 days in individuals with type 1 diabetes (T1D) and demonstrated an increased turnover of CD4 memory T cells. We have now tested T cell turnover in individuals at risk for T1D using a 3-4-day labeling protocol with 2 H-glucose. We studied 30 relatives with T1D with and without autoantibodies, and 10 healthy controls. Peripheral blood mononuclear cells (PBMC) were flow-sorted into T cell subsets of interest; 2 H-DNA enrichment was measured by mass spectrometry and in-vivo turnover was calculated as maximum fractional enrichment of deuterated adenosine (Fmax ). Among CD4+ cells, Fmax was highest in regulatory T cells (Treg ), followed by effector and central memory T cells and lowest in naive cells. Similarly, CD8+ central and effector memory T cells had a higher turnover than CD8+ terminally differentiated effector memory T cells (TEMRA) and CD8+ -naive T cells. Relatives as a group showed significantly increased Treg turnover by Fmax compared to controls (1·733 ± 0·6784% versus 1·062 ± 0·3787%, P = 0·004), suggesting pre-existing immune dysfunction within families with T1D. However, there was no significant difference in Fmax between groups according to autoantibody or glucose tolerance status. Repeat testing in 20 subjects 1 year later demonstrated relatively higher within-subject compared to between-subject variability for the measurement of Fmax in various T cell subsets. The short labeling protocol with 2 H-glucose should be applied in the context of a clinical trial in which the therapy is expected to have large effects on T cell turnover.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Memória Imunológica , Linfócitos T Reguladores/imunologia , Adulto , Linfócitos T CD8-Positivos/patologia , Diabetes Mellitus Tipo 1/patologia , Feminino , Humanos , Cinética , Masculino , Fatores de Risco , Linfócitos T Reguladores/patologiaRESUMO
Immune analytes have been widely tested in efforts to understand the heterogeneity of disease progression, risk, and therapeutic responses in type 1 diabetes (T1D). The future clinical utility of such analytes as biomarkers depends on their technical and biological variability, as well as their correlation with clinical outcomes. To assess the variability of a panel of 91 immune analytes, we conducted a prospective study of adults with T1D (<3 years from diagnosis), at 9-10 visits over 1 year. Autoantibodies and frequencies of T-cell, natural killer cell, and myeloid subsets were evaluated; autoreactive T-cell frequencies and function were also measured. We calculated an intraclass correlation coefficient (ICC) for each marker, which is a relative measure of between- and within-subject variability. Of the 91 analytes tested, we identified 35 with high between- and low within-subject variability, indicating their potential ability to be used to stratify subjects. We also provide extensive data regarding technical variability for 64 of the 91 analytes. To pilot the concept that ICC can be used to identify analytes that reflect biological outcomes, the association between each immune analyte and C-peptide was also evaluated using partial least squares modeling. CD8 effector memory T-cell (CD8 EM) frequency exhibited a high ICC and a positive correlation with C-peptide, which was also seen in an independent dataset of recent-onset T1D subjects. More work is needed to better understand the mechanisms underlying this relationship. Here we find that there are a limited number of technically reproducible immune analytes that also have a high ICC. We propose the use of ICC to define within- and between-subject variability and measurement of technical variability for future biomarker identification studies. Employing such a method is critical for selection of analytes to be tested in the context of future clinical trials aiming to understand heterogeneity in disease progression and response to therapy.
Assuntos
Autoanticorpos/imunologia , Biomarcadores/análise , Diabetes Mellitus Tipo 1/imunologia , Células Matadoras Naturais/imunologia , Células Mieloides/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Autoanticorpos/metabolismo , Peptídeo C/análise , Peptídeo C/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Humanos , Memória Imunológica/imunologia , Células Matadoras Naturais/metabolismo , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Células Mieloides/metabolismo , Estudos Prospectivos , Linfócitos T/metabolismo , Adulto JovemRESUMO
OBJECTIVE: We aimed to investigate T-cell receptor (TCR) repertoires in type 1 diabetes (T1D) patients receiving autologous hematopoietic stem cell transplantation (AHSCT) treatment. METHODS: High-throughput deep TCR beta (TCRB) chain sequencing was performed to assess millions of individual TCRs in five T1D patients receiving AHSCT treatment and another five patients receiving insulin treatment during 12 months of follow-up. RESULTS: No significant changes in TCRB sequence reads, complementarity-determining region 3 (CDR3) sequences, or the usage of TCRB VJ gene-segments were observed at 12 months after AHSCT. Compared with the baseline, the usage of TCRB VJ gene-segments at 12 months decreased in the insulin treatment group (1836.4 ± 437.7 vs 2763.6 ± 390.6, P = 0.015), and the change rates were larger than those undergoing AHSCT (-0.62 ± 0.16 vs 0.06 ± 0.45, P = 0.002). Changes in the TCR repertoire were smaller after AHSCT than those with insulin treatment (P = 2.2*10-32 ). TCRBV 7-7/TCRBJ 2-5 was depleted after AHSCT while expanded with insulin treatment. TCRBV 12-4, TCRBV 10-3, TCRBV 12-3/TCRBJ 1-2 were expanded after AHSCT while ablated with insulin treatment. CONCLUSIONS: We found that AHSCT is safe without reduction in the diversity of TCR repertoires and TCR repertoires tend to be more stable after AHSCT. Furthermore, these four candidate TCRBV/TCRBJ gene usages on CDR3 regions may act as therapeutic targets and biomarkers.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/imunologia , Transplante de Células-Tronco Hematopoéticas , Linfócitos T/imunologia , Adolescente , Regiões Determinantes de Complementaridade , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Masculino , Transplante Autólogo , Adulto JovemRESUMO
BACKGROUND: In the present work we described the recombinant production and characterization of heterodimeric construction ZnT8-Arg-Trp325 fused to thioredoxin using a high-performance expression system such as Escherichia coli. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs. RESULTS: ZnT8 was expressed in E. coli as a fusion protein with thioredoxin (TrxZnT8). After 3 h for induction, recombinant protein was obtained from the intracellular soluble fraction and from inclusion bodies and purified by affinity chromatography. The expression and purification steps, analyzed by SDS-PAGE and western blot, revealed a band compatible with TrxZnT8 expected theoretical molecular weight (≈ 36.8 kDa). The immunochemical ability of TrxZnT8 to compete with [35S]ZnT8 (synthesized with rabbit reticulocyte lysate system) was assessed qualitatively by incubating ZnT8A positive patient sera in the presence of 0.2-0.3 µM TrxZnT8. Results were expressed as standard deviation scores (SDs). All sera became virtually negative under antigen excess (19.26-1.29 for TrxZnT8). Also, radiometric quantitative competition assays with ZnT8A positive patient sera were performed by adding TrxZnT8 (37.0 pM-2.2 µM), using [35S]ZnT8. All dose-response curves showed similar protein concentration that caused 50% inhibition (14.9-0.15 nM for TrxZnT8). On the other hand, preincubated bridge ELISA for ZnT8A detection was developed. This assay showed 51.7% of sensitivity and 97.1% of specificity. CONCLUSIONS: It was possible to obtain with high-yield purified heterodimeric construction of ZnT8 in E. coli and it was applied in cost-effective immunoassay for ZnT8A detection.
Assuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/imunologia , Escherichia coli/genética , Transportador 8 de Zinco/genética , Transportador 8 de Zinco/imunologia , Animais , Antígenos/genética , Antígenos/imunologia , Autoanticorpos/imunologia , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Tiorredoxinas/química , Tiorredoxinas/genéticaAssuntos
Autoanticorpos , Glutamato Descarboxilase/imunologia , Células Secretoras de Insulina/imunologia , Diabetes Autoimune Latente em Adultos , Adulto , Autoanticorpos/sangue , Autoanticorpos/imunologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Feminino , Humanos , Diabetes Autoimune Latente em Adultos/sangue , Diabetes Autoimune Latente em Adultos/diagnóstico , Diabetes Autoimune Latente em Adultos/economia , Diabetes Autoimune Latente em Adultos/imunologia , MasculinoRESUMO
Beta-cell replacement is the only physiologically relevant alternative to insulin injections in patients with type 1 diabetes (T1D). Pancreas and islet transplantation from deceased organ donors can provide a new beta-cell pool to produce insulin, help blood glucose management, and delay secondary diabetes complications. For children and adolescents with T1D, whole pancreas transplantation is not a viable option because of surgical complications, whereas islet transplantation, even if it is procedurally simpler, must still overcome the burden of immunosuppression to become a routine therapy for children in the future.
Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Terapia de Imunossupressão/efeitos adversos , Transplante das Ilhotas Pancreáticas/efeitos adversos , Fatores Etários , Animais , Complicações do Diabetes/prevenção & controle , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/economia , Diabetes Mellitus Tipo 1/imunologia , Custos de Cuidados de Saúde , Humanos , Hiperglicemia/prevenção & controle , Hipoglicemia/prevenção & controle , Terapia de Imunossupressão/economia , Transplante das Ilhotas Pancreáticas/economia , Transplante das Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/normas , Transplante de Pâncreas/efeitos adversos , Guias de Prática Clínica como Assunto , Doadores de Tecidos/provisão & distribuição , Transplante Autólogo/efeitos adversos , Transplante Autólogo/economia , Transplante Autólogo/normas , Transplante Heterólogo/efeitos adversos , Transplante Heterólogo/economia , Transplante Heterólogo/normas , Transplante Homólogo/efeitos adversos , Transplante Homólogo/economia , Transplante Homólogo/normas , Estados Unidos , United States Food and Drug AdministrationRESUMO
Susceptibility to type 1 diabetes (T1D) is strongly associated with MHC class II molecules, particularly HLA-DQ8 (DQ8: DQA1*03:01/DQB1*03:02). Monitoring T1D-specific T cell responses to DQ8-restricted epitopes may be key to understanding the immunopathology of the disease. In this study, we examined DQ8-restricted T cell responses to glutamic acid decarboxylase 65 (GAD65) using DQ8 tetramers. We demonstrated that GAD65 121-140 and GAD65 250-266 elicited responses from DQ8+ subjects. Circulating CD4+ T cells specific for these epitopes were detected significantly more often in T1D patients than in healthy individuals after in vitro expansion. T cell clones specific for GAD65 121-140 and GAD65 250-266 carried a Th1-dominant phenotype, with some of the GAD65 121-140-specific T cell clones producing IL-17. GAD65 250-266-specific CD4+ T cells could also be detected by direct ex vivo staining. Analysis of unmanipulated peripheral blood mononuclear cells (PBMCs) revealed that GAD65 250-266-specific T cells could be found in both healthy and diabetic individuals but the frequencies of specific T cells were higher in subjects with type 1 diabetes. Taken together, our results suggest a proinflammatory role for T cells specific for DQ8-restricted GAD65 121-140 and GAD65 250-266 epitopes and implicate their possible contribution to the progression of T1D.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/fisiopatologia , Glutamato Descarboxilase/efeitos adversos , Antígenos HLA-DQ/genética , Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/metabolismo , Humanos , Estatísticas não Paramétricas , WashingtonRESUMO
Multiple immune parameters such as frequencies of autoreactive CD4(+), CD8(+) T-cells and CD4(+)CD25(+)Foxp3(+) T-cells have been explored as biomarkers in human T1D. However, intra-individual temporal variation of these parameters has not been assessed systematically over time. We determined the variation in each of these parameters in a cohort of T1D and healthy donors (HDs), at monthly intervals for one year. Despite low intra- and inter-assay co-efficient of variation (CV), mean CVs for each of the immune parameters were 119.1% for CD4(+) T-cell-derived IFN-γ, 50.44% for autoreactive CD8(+) T-cells, and 31.24% for CD4(+)CD25(+)Foxp3(+) T-cells. Further, both HDs and T1D donors had similar CVs. The variation neither correlated with BMI, age, disease duration or insulin usage, nor were there detectable cyclical patterns of variation. However, averaging results from multiple visits for an individual provided a better estimate of the CV between visits. Based on our data we predict that by averaging values from three visits a treatment effect on these parameters with a 50% effect size could be detected with the same power using 1.8-4-fold fewer patients within a trial compared to using values from a single visit. Thus, our present data contribute to a more robust, accurate endpoint design for future clinical trials in T1D and aid in the identification of truly efficacious therapies.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Adulto , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Contagem de Células , Estudos Transversais , Diabetes Mellitus Tipo 1/sangue , Feminino , Humanos , Interferon gama/biossíntese , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fatores de TempoAssuntos
Medicamentos Biossimilares/uso terapêutico , Anticorpos Anti-Insulina/efeitos adversos , Insulina/análogos & derivados , Insulina/imunologia , Anticorpos Neutralizantes/efeitos adversos , Conscientização , Medicamentos Biossimilares/economia , Redução de Custos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/economia , Diabetes Mellitus Tipo 1/imunologia , Relação Dose-Resposta a Droga , Aprovação de Drogas , Substituição de Medicamentos/economia , Humanos , Insulina/economia , Insulina/uso terapêutico , Anticorpos Anti-Insulina/biossínteseRESUMO
Type 1 diabetes mellitus is an autoimmune disease that is characterized by the destruction of the islets of Langerhans cells which produce insulin. The current gold standard treatment is exogenous insulin injection, but this is onerous for the patients, and can lead to severe complications. Another approach involves transplanting pancreatic islet cells in order to restore endogenous insulin production under physiologic regulation. Although there has been some success with this treatment plan, there have been several hurdles. The largest hurdle is improving the 5 year survival of the graft, which is currently at 10%. In order to do so, there has been research into better locations for the graft, better isolation techniques, alternate immune suppression regimens, and novel transplantation methodologies utilizing encapsulated grafts. Another hurdle for pancreatic islet transplantation is that current methodologies require islets from several pancreata in order to create one successful graft, which leads to difficulties since there is a limited supply. However, there has been research looking into single donor transplants and porcine xenografts to increase the supply and address this problem. In this article, we review the current state of research regarding pancreatic islet transplantation.
Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Transplante das Ilhotas Pancreáticas/métodos , Transplante de Pâncreas/métodos , Transplante Heterólogo/métodos , Análise Custo-Benefício , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/mortalidade , Feminino , Rejeição de Enxerto , Sobrevivência de Enxerto/imunologia , Humanos , Imunidade Inata , Terapia de Imunossupressão , Transplante das Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/mortalidade , Masculino , Transplante de Pâncreas/mortalidade , Seleção de Pacientes , Medição de Risco , Coleta de Tecidos e Órgãos , Transplante Heterólogo/mortalidade , Resultado do TratamentoRESUMO
George S. Eisenbarth will remain in our memories as a brilliant scientist and great collaborator. His quest to discover the cause and prevention of type 1 (autoimmune) diabetes started from building predictive models based on immunogenetic markers. Despite his tremendous contributions to our understanding of the natural history of pre-type 1 diabetes and potential mechanisms, George left us with several big questions to answer before his quest is completed.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Imunogenética , Pesquisa , Diabetes Mellitus Tipo 1/história , Diabetes Mellitus Tipo 1/imunologia , Feminino , História do Século XX , História do Século XXI , Humanos , Imunogenética/história , Imunogenética/tendências , Masculino , Inovação Organizacional , Prevenção Primária , Pesquisa/história , Pesquisa/tendências , Apoio à Pesquisa como AssuntoRESUMO
George Eisenbarth's pioneering and visionary research has provided a critical foundation that will be built on in the years ahead as we progress toward prevention of type 1 diabetes. His almost 30-year old model that type 1 diabetes was a chronic and predictable autoimmune disease with multiple identifiable progressive stages with a potential for interventions to prevent progression to symptomatic diabetes has stood the test of time. To deliver on the Eisenbarth vision and his "unfinished journey," the field needs: (1) to improve detection of risk of type 1 diabetes, (2) to improve staging and prediction of progression, (3) to perform smaller, shorter, practical, and an increased number of prevention clinical trials, and (4) to increase awareness of the potential for risk detection, staging, and prevention of type 1 diabetes and benefit/risk of prevention.
Assuntos
Doenças Autoimunes/prevenção & controle , Diabetes Mellitus Tipo 1/prevenção & controle , Doenças Autoimunes/história , Doenças Autoimunes/imunologia , Doenças Autoimunes/fisiopatologia , Análise Custo-Benefício , Diabetes Mellitus Tipo 1/história , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/fisiopatologia , Suscetibilidade a Doenças/imunologia , Diagnóstico Precoce , Feminino , História do Século XX , História do Século XXI , Humanos , Masculino , Modelos ImunológicosRESUMO
In first-degree relatives of type 1 diabetic patients, we investigated whether diabetes risk assessment solely based on insulinoma antigen 2 (IA-2) and zinc transporter 8 (ZnT8) antibody status (IA-2A, respectively, ZnT8A) is as effective as screening for three or four autoantibodies [antibodies against insulin (IAA), glutamate decarboxylase 65 kDa (GAD) glutamate decarboxylase autoantibodies (GADA) and IA-2A with or without ZnT8A] in identifying children, adolescents and adults who progress rapidly to diabetes (within 5 years). Antibodies were determined by radiobinding assays during follow-up of 6444 siblings and offspring aged 0-39 years at inclusion and recruited consecutively by the Belgian Diabetes Registry. We identified 394 persistently IAA(+) , GADA(+) , IA-2A(+) and/or ZnT8A(+) relatives (6·1%). After a median follow-up time of 52 months, 132 relatives developed type 1 diabetes. In each age category tested (0-9, 10-19 and 20-39 years) progression to diabetes was significantly quicker in the presence of IA-2A and/or ZnT8A than in their joint absence (P < 0·001). Progression rate was age-independent in IA-2A(+) and/or ZnT8A(+) relatives but decreased with age if only GADA and/or IAA were present (P = 0·008). In the age group mainly considered for immune interventions until now (10-39 years), screening for IA-2A and ZnT8A alone identified 78% of the rapid progressors (versus 75% if positive for ≥ 2 antibodies among IAA, GADA, IA-2A and ZnT8A or versus 62% without testing for ZnT8A). Screening for IA-2A and ZnT8A alone allows identification of the majority of rapidly progressing prediabetic siblings and offspring regardless of age and is more cost-effective to select participants for intervention trials than conventional screening.
Assuntos
Autoanticorpos/sangue , Proteínas de Transporte de Cátions/imunologia , Diabetes Mellitus Tipo 1/imunologia , Progressão da Doença , Estado Pré-Diabético/sangue , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , Adolescente , Autoanticorpos/economia , Bélgica , Glicemia/imunologia , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/sangue , Família , Feminino , Glutamato Descarboxilase/imunologia , Humanos , Insulina/imunologia , Masculino , Estado Pré-Diabético/imunologia , Sistema de Registros , Risco , Transportador 8 de ZincoRESUMO
BACKGROUND: For cost-effective population-based diabetes prediction and confirmation, islet autoantibody assays must be made more economical. METHODS: We evaluated glutamic acid decarboxylase (GAD)-Ruc (renilla luciferase) and IA2ic (also known as ICA512ic)-Ruc (renilla luciferase) fusion protein constructs in high-throughput islet antibody assay formats. RESULTS: Antigen production via transfection onto COS cells in 100 mm culture dishes yielded sufficient antigen to assay 375 and 535 serum samples for GAD and IA2ic per dish, respectively. Antigen was usably stable after -80 °C storage for 40-80 days after which luciferase activity decreased. The mean signal-to-noise ratios for luciferase-based immunoprecitation system (LIPS) GAD and LIPS IA2ic were 88±24 and 219±89, respectively, comparing favourably to radio-binding assays (RBA) in the same format. However, the coefficient of variation among triplicate wells was higher for IA2ic than for GAD in LIPS, similar to findings in RBA format. Correlation coefficients between autoantibody indices determined from the RBA and LIPS methods were only R2=0.79 and R2=0.75 for GAD and IA2ic, respectively, raising the possibility that different epitopes were favoured in the two different assay formats. Nevertheless, overall concordance for the two assay types was high, at 228/240=95.0% for GAD and 494/521=94.8% for IA2ic. Using optimal cutoffs, Diabetes Autoantibody Standardization Program (DASP) 2010 sensitivity/specificity was 80/99% for GAD RBA, 80/99% for GAD LIPS, 70/98% for IA2 RBA, and 72/99% for IA2 LIPS. CONCLUSION: The LIPS assays for islet autoantibodies to GAD and IA2ic performed as well as RBA in DASP 2010. With further refinements in expression and storage, these assays may be more economical than current methods to measure islet autoantibodies in type 1 diabetes.
Assuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/imunologia , Imunoprecipitação/métodos , Ilhotas Pancreáticas/imunologia , Luciferases/imunologia , Animais , Células COS , Chlorocebus aethiops , Glutamato Descarboxilase/imunologia , Humanos , Imunoprecipitação/economia , Ensaio Radioligante , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/imunologia , Proteínas Recombinantes de Fusão/imunologia , Sensibilidade e EspecificidadeAssuntos
Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Glutamato Descarboxilase/imunologia , Animais , Criança , Ensaios Clínicos Fase III como Assunto , Diabetes Mellitus Tipo 1/imunologia , Progressão da Doença , Europa (Continente) , Apoio Financeiro , Humanos , Resultado do Tratamento , VacinaçãoRESUMO
Type 1 diabetes is characterized by near total absence of pancreatic b cells. Current treatments consisting of insulin injections and islet transplantation are clinically unsatisfactory. In order to develop a cure for type 1 diabetes, we must find a way to reverse autoimmunity, which underlies b cell destruction, as well as an effective strategy to generate new b cells. This article reviews the different approaches that are being taken to produce new b cells. Much emphasis has been placed on selecting the right non-b cell population, either in vivo or in vitro, as the starting material. Different cell types, including adult stem cells, other types of progenitor cells in situ, and even differentiated cell populations, as well as embryonic stem cells and induced pluripotent stem cells, will require different methods for islet and b cell induction. We discussed the pros and cons of the different strategies that are being used to re-invent the pancreatic b cell.
Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Transplante de Células-Tronco/métodos , Animais , Autoimunidade , Diferenciação Celular , Diabetes Mellitus/epidemiologia , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/epidemiologia , Células-Tronco Embrionárias/transplante , Endoderma/citologia , Gástrula/citologia , Células Secretoras de Glucagon/patologia , Humanos , Células Secretoras de Insulina/patologia , Transplante das Ilhotas Pancreáticas , Camundongos , Modelos Animais , Ductos Pancreáticos/patologia , Estado Pré-Diabético/economia , Estado Pré-Diabético/epidemiologia , Estados Unidos/epidemiologiaRESUMO
Like many other complex human disorders of unknown aetiology, autoimmune-mediated type 1 diabetes may ultimately be controlled via a therapeutic approach that combines multiple agents, each with differing modes of action. The numerous advantages of such a strategy include the ability to minimize toxicities and realize synergies to enhance and prolong efficacy. The recognition that combinations might offer far-reaching benefits, at a time when few single agents have yet proved themselves in well-powered trials, represents a significant challenge to our ability to conceive and implement rational treatment designs. As a first step in this process, the Immune Tolerance Network, in collaboration with the Juvenile Diabetes Research Foundation, convened a Type 1 Diabetes Combination Therapy Assessment Group, the recommendations of which are discussed in this Perspective paper.