RESUMEN
A missense mutation (R620W) of protein tyrosine phosphatase nonreceptor type 22 (PTPN22), which encodes lymphoid-tyrosine phosphatase (LYP), confers genetic risk for multiple autoimmune diseases including type 1 diabetes. LYP has been putatively demonstrated to attenuate proximal T and BCR signaling. However, limited data exist regarding PTPN22 expression within primary T cell subsets and the impact of the type 1 diabetes risk variant on human T cell activity. In this study, we demonstrate endogenous PTPN22 is differentially expressed and dynamically controlled following activation. From control subjects homozygous for the nonrisk allele, we observed 2.1- (p < 0.05) and 3.6-fold (p < 0.001) more PTPN22 transcripts in resting CD4+ memory and regulatory T cells (Tregs), respectively, over naive CD4+ T cells, with expression peaking 24 h postactivation. When LYP was overexpressed in conventional CD4+ T cells, TCR signaling and activation were blunted by LYP-620R (p < 0.001) but only modestly affected by the LYP-620W risk variant versus mock-transfected control, with similar results observed in Tregs. LYP overexpression only impacted proliferation following activation by APCs but not anti-CD3- and anti-CD28-coated microbeads, suggesting LYP modulation of pathways other than TCR. Notably, proliferation was significantly lower with LYP-620R than with LYP-620W overexpression in conventional CD4+ T cells but was similar in Treg. These data indicate that the LYP-620W variant is hypomorphic in the context of human CD4+ T cell activation and may have important implications for therapies seeking to restore immunological tolerance in autoimmune disorders.
Asunto(s)
Proteína Tirosina Fosfatasa no Receptora Tipo 22/metabolismo , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Autoinmunidad , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Regulación de la Expresión Génica , Variación Genética , Humanos , Tolerancia Inmunológica , Memoria Inmunológica , Activación de Linfocitos/genética , Mutación/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/genéticaRESUMEN
Emerging data suggest that type 1 diabetes affects not only the ß-cell-containing islets of Langerhans, but also the surrounding exocrine compartment. Using digital pathology, machine learning algorithms were applied to high-resolution, whole-slide images of human pancreata to determine whether the tissue composition in individuals with or at risk for type 1 diabetes differs from those without diabetes. Transplant-grade pancreata from organ donors were evaluated from 16 nondiabetic autoantibody-negative controls, 8 nondiabetic autoantibody-positive subjects with increased type 1 diabetes risk, and 19 persons with type 1 diabetes (0 to 12 years' duration). HALO image analysis algorithms were implemented to compare architecture of the main pancreatic duct as well as cell size, density, and area of acinar, endocrine, ductal, and other nonendocrine, nonexocrine tissues. Type 1 diabetes was found to affect exocrine area, acinar cell density, and size, whereas the type of difference correlated with the presence or absence of insulin-positive cells remaining in the pancreas. These changes were not observed before disease onset, as indicated by modeling cross-sectional data from pancreata of autoantibody-positive subjects and those diagnosed with type 1 diabetes. These data provide novel insights into anatomic differences in type 1 diabetes pancreata and demonstrate that machine learning can be adapted for the evaluation of disease processes from cross-sectional data sets.
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Algoritmos , Autoanticuerpos/inmunología , Diabetes Mellitus Tipo 1/patología , Procesamiento de Imagen Asistido por Computador/métodos , Aprendizaje Automático , Páncreas/patología , Adolescente , Autoanticuerpos/sangre , Estudios de Casos y Controles , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Humanos , Insulina/análisis , Páncreas/inmunología , Páncreas/metabolismo , Donantes de TejidosRESUMEN
AIMS/HYPOTHESIS: The circadian clock influences both diabetes and immunity. Our goal in this study was to characterise more thoroughly the circadian patterns of immune cell populations and cytokines that are particularly relevant to the immune pathology of type 1 diabetes and thus fill in a current gap in our understanding of this disease. METHODS: Ten individuals with established type 1 diabetes (mean disease duration 11 years, age 18-40 years, six female) participated in a circadian sampling protocol, each providing six blood samples over a 24 h period. RESULTS: Daily ranges of population frequencies were sometimes large and possibly clinically significant. Several immune populations, such as dendritic cells, CD4 and CD8 T cells and their effector memory subpopulations, CD4 regulatory T cells, B cells and cytokine IL-6, exhibited statistically significant circadian rhythmicity. In a comparison with historical healthy control individuals, but using shipped samples, we observed that participants with type 1 diabetes had statistically significant phase shifts occurring in the time of peak occurrence of B cells (+4.8 h), CD4 and CD8 T cells (~ +5 h) and their naive and effector memory subsets (~ +3.3 to +4.5 h), and regulatory T cells (+4.1 h). An independent streptozotocin murine experiment confirmed the phase shifting of CD8 T cells and suggests that circadian dysrhythmia in type 1 diabetes might be an effect and not a cause of the disease. CONCLUSIONS/INTERPRETATION: Future efforts investigating this newly described aspect of type 1 diabetes in human participants are warranted. Peripheral immune populations should be measured near the same time of day in order to reduce circadian-related variation.
Asunto(s)
Trastornos Cronobiológicos/inmunología , Ritmo Circadiano/inmunología , Diabetes Mellitus Tipo 1/inmunología , Sistema Inmunológico/fisiología , Adolescente , Adulto , Animales , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Relojes Circadianos/genética , Células Dendríticas/inmunología , Femenino , Citometría de Flujo , Humanos , Interleucina-6/sangre , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T Reguladores/inmunología , Adulto JovenRESUMEN
AIMS/HYPOTHESIS: Normal cellular prion protein (PrPC) is a conserved mammalian glycoprotein found on the outer plasma membrane leaflet through a glycophosphatidylinositol anchor. Although PrPC is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. The misfolded pathogenic isoform PrPSc (the scrapie form of PrP) is a causative agent of neurodegenerative prion diseases. The aim of this study is to evaluate PrPC localisation, expression and trafficking in pancreases from organ donors with and without type 1 diabetes and to infer PrPC function through studies on interacting protein partners. METHODS: In order to evaluate localisation and trafficking of PrPC in the human pancreas, 12 non-diabetic, 12 type 1 diabetic and 12 autoantibody-positive organ donor tissue samples were analysed using immunofluorescence analysis. Furthermore, total RNA was isolated from 29 non-diabetic, 29 type 1 diabetic and 24 autoantibody-positive donors to estimate PrPC expression in the human pancreas. Additionally, we performed PrPC-specific immunoblot analysis on total pancreatic protein from non-diabetic and type 1 diabetic organ donors to test whether changes in PrPC mRNA levels leads to a concomitant increase in PrPC protein levels in human pancreases. RESULTS: In non-diabetic and type 1 diabetic pancreases (the latter displaying both insulin-positive [INS(+)] and -negative [INS(-)] islets), we found PrPC in islets co-registering with beta cells in all INS(+) islets and, strikingly, unexpected activation of PrPC in alpha cells within diabetic INS(-) islets. We found PrPC localised to the plasma membrane and endoplasmic reticulum (ER) but not the Golgi, defining two cellular pools and an unconventional protein trafficking mechanism bypassing the Golgi. We demonstrate PrPC co-registration with established protein partners, neural cell adhesion molecule 1 (NCAM1) and stress-inducible phosphoprotein 1 (STI1; encoded by STIP1) on the plasma membrane and ER, respectively, linking PrPC function with cyto-protection, signalling, differentiation and morphogenesis. We demonstrate that both PRNP (encoding PrPC) and STIP1 gene expression are dramatically altered in type 1 diabetic and autoantibody-positive pancreases. CONCLUSIONS/INTERPRETATION: As the first study to address PrPC expression in non-diabetic and type 1 diabetic human pancreas, we provide new insights for PrPC in the pathogenesis of type 1 diabetes. We evaluated the cell-type specific expression of PrPC in the human pancreas and discovered possible connections with potential interacting proteins that we speculate might address mechanisms relevant to the role of PrPC in the human pancreas.
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Diabetes Mellitus Tipo 1/metabolismo , Páncreas/metabolismo , Proteínas PrPC/metabolismo , Adolescente , Adulto , Autoanticuerpos/sangre , Antígeno CD56/metabolismo , Membrana Celular/metabolismo , Niño , Retículo Endoplásmico/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Proteínas de Choque Térmico/metabolismo , Humanos , Inmunohistoquímica , Anticuerpos Insulínicos/inmunología , Masculino , Proteínas PrPC/genética , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Transporte de Proteínas , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Donantes de Tejidos , Adulto JovenRESUMEN
Identified as a major biomarker for type 1 diabetes (T1D) diagnosis, zinc transporter 8 autoantibody (ZnT8A) has shown promise for staging disease risk and disease diagnosis. However, existing assays for ZnT8 autoantibody (ZnT8A) are limited to detection by soluble domains of ZnT8, owing to difficulties in maintaining proper folding of a full-length ZnT8 protein outside its native membrane environment. Through a combined bioengineering and nanotechnology approach, we have developed a proteoliposome-based full-length ZnT8 self-antigen (full-length ZnT8 proteoliposomes; PLR-ZnT8) for efficient detection of ZnT8A on a plasmonic gold chip (pGOLD). The protective lipid matrix of proteoliposomes improved the proper folding and structural stability of full-length ZnT8, helping PLR-ZnT8 immobilized on pGOLD (PLR-ZnT8/pGOLD) achieve high-affinity capture of ZnT8A from T1D sera. Our PLR-ZnT8/pGOLD exhibited efficient ZnT8A detection for T1D diagnosis with â¼76% sensitivity and â¼97% specificity (n = 307), superior to assays based on detergent-solubilized full-length ZnT8 and the C-terminal domain of ZnT8. Multiplexed assays using pGOLD were also developed for simultaneous detection of ZnT8A, islet antigen 2 autoantibody, and glutamic acid decarboxylase autoantibody for diagnosing T1D.
Asunto(s)
Diabetes Mellitus Tipo 1/diagnóstico , Transportador 8 de Zinc/sangre , Células HEK293 , Humanos , Análisis por Matrices de Proteínas , Proteolípidos , Transportador 8 de Zinc/inmunologíaRESUMEN
Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic ß-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.
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Metilación de ADN , ADN/sangre , Células Secretoras de Insulina/patología , Oligodendroglía/patología , Adolescente , Adulto , Anciano , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Estudios de Casos y Controles , Muerte Celular , Niño , Preescolar , ADN/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Femenino , Marcadores Genéticos , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/genética , Esclerosis Múltiple Recurrente-Remitente/patología , Especificidad de Órganos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Pancreatitis Crónica/genética , Pancreatitis Crónica/patología , Regiones Promotoras Genéticas , Sensibilidad y Especificidad , Adulto JovenRESUMEN
During progression to type 1 diabetes, insulin-producing ß-cells are lost through an autoimmune attack resulting in unrestrained glucagon expression and secretion, activation of glycogenolysis, and escalating hyperglycemia. We recently identified a protein, designated islet homeostasis protein (IHoP), which specifically co-localizes within glucagon-positive α-cells and is overexpressed in the islets of both post-onset non-obese diabetic (NOD) mice and type 1 diabetes patients. Here we report that in the αTC1.9 mouse α-cell line, IHoP was released in response to high-glucose challenge and was found to regulate secretion of glucagon. We also show that in NOD mice with diabetes, major histocompatibility complex class II was upregulated in islets. In addition hyperglycemia was modulated in NOD mice via suppression of IHoP utilizing small interfering RNA (IHoP-siRNA) constructs/approaches. Suppression of IHoP in the pre-diabetes setting maintained normoglycemia, glyconeolysis, and fostered ß-cell restoration in NOD mice 35 weeks post treatment. Furthermore, we performed adoptive transfer experiments using splenocytes from IHoP-siRNA-treated NOD/ShiLtJ mice, which thwarted the development of hyperglycemia and the extent of insulitis seen in recipient mice. Last, IHoP can be detected in the serum of human type 1 diabetes patients and could potentially serve as an early novel biomarker for type 1 diabetes in patients.
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Diabetes Mellitus Tipo 1/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas/metabolismo , Animales , Línea Celular , Femenino , Glucagón/análisis , Glucagón/metabolismo , Antígenos HLA-D/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Hiperglucemia/metabolismo , Islotes Pancreáticos/química , Masculino , Ratones , Ratones Endogámicos NOD , Proteínas/análisis , Proteínas/antagonistas & inhibidores , Transactivadores/metabolismoRESUMEN
Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematologic malignancies, but the risk of graft-versus-host disease (GVHD) is a major limitation for wider application. Ideally, strategies to improve allo-HCT would involve suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-versus-malignancy (GVM). Recently, using a xenograft model, we serendipitously discovered that myxoma virus (MYXV) prevented GVHD while permitting GVM. In this study, we show that MYXV binds to resting, primary human T lymphocytes but will only proceed into active virus infection after the T cells receive activation signals. MYXV-infected T lymphocytes exhibited impaired proliferation after activation with reduced expression of interferon-γ, interleukin-2 (IL-2), and soluble IL-2Rα, but did not affect expression of IL-4 and IL-10. MYXV suppressed T-cell proliferation in 2 patterns (full vs partial) depending on the donor. In terms of GVM, we show that MYXV-infected activated human T lymphocytes effectively deliver live oncolytic virus to human multiple myeloma cells, thus augmenting GVM by transfer of active oncolytic virus to residual cancer cells. Given this dual capacity of reducing GVHD plus increasing the antineoplastic effectiveness of GVM, ex vivo virotherapy with MYXV may be a promising clinical adjunct to allo-HCT regimens.
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Mieloma Múltiple/terapia , Myxoma virus/inmunología , Viroterapia Oncolítica/métodos , Virus Oncolíticos/inmunología , Linfocitos T/inmunología , Linfocitos T/virología , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/terapia , Trasplante de Células Madre Hematopoyéticas , Humanos , Activación de Linfocitos , Infecciones por Poxviridae/inmunología , Linfocitos T/citología , Infecciones Tumorales por Virus/inmunologíaRESUMEN
Viral infections elicit antiviral antibodies and have been associated with various chronic diseases. Detection of these antibodies can facilitate diagnosis, treatment of infection, and understanding of the mechanisms of virus-associated diseases. In this work, we assayed antiviral antibodies using a novel high-density nucleic acid programmable protein array (HD-NAPPA) platform. Individual viral proteins were expressed in situ directly from plasmids encoding proteins in an array of microscopic reaction chambers. Quality of protein display and serum response was assured by comparing intra- and inter-array correlation within or between printing batches with average correlation coefficients of 0.91 and 0.96, respectively. HD-NAPPA showed higher signal-to-background ratio compared with standard NAPPA on planar glass slides and ELISA. Antibody responses to 761 antigens from 25 different viruses were profiled among patients with juvenile idiopathic arthritis and type 1 diabetes. Common and unique antibody reactivity patterns were detected between patients and healthy controls. We believe HD-viral-NAPPA will enable the study of host-pathogen interactions at unprecedented dimensions and elucidate the role of pathogen infections in disease development.
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Anticuerpos Antivirales/sangre , Artritis Juvenil/sangre , Autoanticuerpos/sangre , Biomarcadores/sangre , Diabetes Mellitus Tipo 1/sangre , Análisis por Matrices de Proteínas/métodos , Proteómica/métodos , Artritis Juvenil/inmunología , Estudios de Casos y Controles , Preescolar , Diabetes Mellitus Tipo 1/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Interacciones Huésped-Patógeno , Humanos , Inmunoprecipitación , Masculino , Ácidos Nucleicos/química , Proteínas Virales/metabolismoRESUMEN
We developed a novel poly(lactic-co-glycolic acid)-based, microparticle (MP) system providing concurrent delivery of multiple encapsulated immuno-suppressive factors and antigen, for in vivo conditioning of dendritic cells (DCs) toward a tolerance promoting pathway. Subcutaneous administration prevents onset of type 1 diabetes (T1D) in NOD mice. Two MP sizes were made: phagocytosable MPs were fabricated encapsulating vitamin D3 or insulin B(9-23) peptide, while unphagocytosable MPs were fabricated encapsulating TGF-ß1 or GM-CSF. The combination of Vit D3/TGF-ß1 MPs confers an immature and LPS activation-resistant phenotype to DCs, and MP-delivered antigen is efficiently and functionally presented. Notably, two subcutaneous injections into 4week old NOD mice using the combination of MPs encapsulating Vit D3, Ins B, TGF-ß1 and GM-CSF protected 40% of mice from T1D development, significant in comparison to the control. This work represents one of the first applications of a biomaterial-based, MP vaccine system to successfully prevent autoimmune diabetes.
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Células Dendríticas/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Portadores de Fármacos , Ácido Láctico , Ácido Poliglicólico , Vacunas/administración & dosificación , Animales , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Colecalciferol/farmacología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Tolerancia Inmunológica/inmunología , Insulina/farmacología , Lipopolisacáridos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Tamaño de la Partícula , Fragmentos de Péptidos/farmacología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Factor de Crecimiento Transformador beta1/farmacología , Vacunas/inmunologíaRESUMEN
BACKGROUND: Minimal Change Disease (MCD) is associated with CD80 expression in podocytes and elevated urinary CD80 excretion during active renal disease. We have evaluated the urinary excretion of CTLA-4 and CD80 during different stages of the nephrotic syndrome in patients with MCD to test the hypothesis that persistent increased urinary CD80 excretion in patients with MCD in relapse is due to an ineffectual CTLA-4 response of the host to curtail the activation of CD80. METHODS: Thirty-two children with biopsy-proven MCD were studied during relapse and/or remission. Eleven healthy subjects served as controls. RESULTS: Urinary CD80 excretion was significantly increased in MCD patients in relapse relative to that in MCD patients in remission (p < 0.001) and controls (p < 0.001). Although urinary CTLA-4 excretion was higher in MCD patients in relapse than in MCD patients in remission (p = 0.01) and controls (p = 0.03), no significant correlation was observed between urinary CD80 excretion and urinary CTLA-4 level in MCD patients at the time of relapse (p = 0.06). At the time of remission, CD80 had decreased significantly in all patients, but CTLA-4 levels either decreased or remained unchanged in all but five patients, and no correlation was observed between urinary CD80 excretion and CTLA-4 level (p = 0.7). CONCLUSIONS: Urinary CTLA-4 levels do not correlate with urinary CD80 excretion, suggesting the possibility that the CTLA4 response may be suboptimal in this disease during relapse.
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Antígeno B7-1/orina , Antígeno CTLA-4/análisis , Nefrosis Lipoidea/orina , Podocitos/metabolismo , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Recurrencia , Adulto JovenRESUMEN
Histopathological heterogeneity in the human pancreas is well documented; however, functional evidence at the tissue level is scarce. Herein, we investigate in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in donors without diabetes (ND; n = 15), positive for one islet autoantibody (1AAb+; n = 7), and with type 1 diabetes (T1D; <14 months duration, n = 5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features are comparable across regions in ND. In T1D, insulin secretion and beta-cell volume are significantly reduced within all regions, while glucagon and enzymes are unaltered. Beta-cell volume is lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ are consistent across the PH, PB, and PT. This study supports low inter-regional variation in pancreas slice function and, potentially, increased metabolic demand in 1AAb+.
Asunto(s)
Diabetes Mellitus Tipo 1 , Insulina , Islotes Pancreáticos , Humanos , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Masculino , Insulina/metabolismo , Femenino , Secreción de Insulina/efectos de los fármacos , Adulto , Persona de Mediana Edad , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Células Acinares/metabolismo , Células Acinares/patología , Glucagón/metabolismo , Glucosa/metabolismo , Autoanticuerpos/inmunología , Amilasas/metabolismoRESUMEN
Histopathological heterogeneity in human pancreas has been well documented; however, functional evidence at the tissue level is scarce. Herein we investigated in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in no diabetes (ND, n=15), single islet autoantibody-positive (1AAb+, n=7), and type 1 diabetes donors (T1D, <14 months duration, n=5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features were comparable across the regions in ND. In T1D, insulin secretion and beta-cell volume were significantly reduced within all regions, while glucagon and enzymes were unaltered. Beta-cell volume was lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ were consistent across PH, PB and PT. This study supports low inter-regional variation in pancreas slice function and potentially, increased metabolic demand in 1AAb+.
RESUMEN
Introduction: The antigen-presenting cell function of insulin-reactive B cells promotes type 1 diabetes (T1D) in non-obese diabetic (NOD) mice by stimulating pathogenic T cells leading to destruction of insulin-producing ß-cells of pancreatic islets. Methods/Results: To target insulin-reactive B cells, AKS-107, a human IgG1 Fc molecule fused with human insulin A and B chains, was engineered to retain conformational insulin epitopes that bound mouse and human B cell receptors but prevented binding to the insulin metabolic receptor. AKS-107 Fc-mediated deletion of insulin-reactive B cells was demonstrated via ex vivo and in vivo experiments with insulin-reactive B cell receptor transgenic mouse strains, VH125Tg/NOD and Tg125(H+L)/NOD. As an additional immune tolerance feature, the Y16A mutation of the insulin B(9-23) dominant T cell epitope was engineered into AKS-107 to suppress activation of insulin-specific T cells. In mice and non-human primates, AKS-107 was well-tolerated, non-immunogenic, did not cause hypoglycemia even at high doses, and showed an expectedly protracted pharmacokinetic profile. AKS-107 reproducibly prevented spontaneous diabetes from developing in NOD and VH125Tg/NOD mice that persisted for months after cessation of treatment, demonstrating durable immune tolerance. Discussion: These preclinical outcomes position AKS-107 for clinical development in T1D prevention settings.
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Diabetes Mellitus Tipo 1 , Islotes Pancreáticos , Ratones , Animales , Humanos , Ratones Endogámicos NOD , Linfocitos B , Ratones Transgénicos , Receptores de Antígenos de Linfocitos B , InmunoterapiaRESUMEN
Pancreatic beta cells are among the slowest replicating cells in the human body and have not been observed to increase in number except during the fetal and neonatal period, in cases of obesity, during puberty, as well as during pregnancy. Pregnancy is associated with increased beta cell mass to meet heightened insulin demands. This phenomenon raises the intriguing possibility that factors present in the serum of pregnant individuals may stimulate beta cell proliferation and offer insights into expansion of the beta cell mass for treatment and prevention of diabetes. The primary objective of this study was to test the hypothesis that serum from pregnant donors contains bioactive factors capable of inducing human beta cell proliferation. An immortalized human beta cell line with protracted replication (EndoC-ßH1) was cultured in media supplemented with serum from pregnant and non-pregnant female and male donors and assessed for differences in proliferation. This experiment was followed by assessment of proliferation of primary human beta cells. Sera from five out of six pregnant donors induced a significant increase in the proliferation rate of EndoC-ßH1 cells. Pooled serum from the cohort of pregnant donors also increased the rate of proliferation in primary human beta cells. This study demonstrates that serum from pregnant donors stimulates human beta cell proliferation. These findings suggest the existence of pregnancy-associated factors that can offer novel avenues for beta cell regeneration and diabetes prevention strategies. Further research is warranted to elucidate the specific factors responsible for this effect.
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Diabetes Mellitus , Células Secretoras de Insulina , Recién Nacido , Humanos , Masculino , Femenino , Embarazo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Línea Celular , Diabetes Mellitus/metabolismo , Proliferación CelularRESUMEN
We sought to determine if autologous umbilical cord blood (UCB) infusion followed by 1 year of supplementation with vitamin D and docosahexaenoic acid (DHA) can preserve C-peptide in children with type 1 diabetes. We conducted an open-label, 2:1 randomized study in which 15 type 1 diabetes subjects with stimulated C-peptide > .2 pmol/mL received either (1) autologous UCB infusion, 1 year of daily oral vitamin D (2000 IU), and DHA (38 mg/kg) and intensive diabetes management or (2) intensive diabetes management alone. Primary analyses were performed 1 year after UCB infusion. Treated (N = 10) and control (N = 5) subjects had median ages of 7.2 and 6.6 years, respectively. No severe adverse events were observed. Although the absolute rate of C-peptide decline was slower in treated versus control subjects, intergroup comparisons failed to reach significance (P = .29). Area under the curve C-peptide declined and insulin use increased in both groups (P < .01). Vitamin D levels remained stable in treated subjects but declined in control subjects (P = .01). DHA levels rose in treated subjects versus control subjects (P = .003). CD4/CD8 ratio remained stable in treated subjects but declined in control subjects (P = .03). No changes were seen in regulatory T cell frequency, total CD4 counts, or autoantibody titers. Autologous UCB infusion followed by daily supplementation with vitamin D and DHA was safe but failed to preserve C-peptide. Lack of significance may reflect small sample size. Future efforts will require expansion of specific immunoregulatory cell subsets, optimization of combined immunoregulatory and anti-inflammatory agents, and larger study cohorts.
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Péptido C/sangre , Trasplante de Células Madre de Sangre del Cordón Umbilical , Diabetes Mellitus Tipo 1/terapia , Ácidos Docosahexaenoicos/administración & dosificación , Vitamina D/administración & dosificación , Administración Oral , Área Bajo la Curva , Recuento de Linfocito CD4 , Estudios de Casos y Controles , Niño , Preescolar , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Femenino , Humanos , Lactante , Infusiones Intravenosas , Masculino , Subgrupos de Linfocitos T , Trasplante AutólogoRESUMEN
Type 1 Diabetes (T1D) is an autoimmune disorder characterized by aberrant T cell responses. Innate immune activation defects may facilitate a T helper 1 (Th1) phenotype. The cytokine IL-18 synergizes with IL-12 to induce IFNγ production and Th1 differentiation. The IL-18R subunit (IL18RAP) SNP rs917997 has been linked to decreased IL18RAP gene expression. Prior reports link rs917997 allele A with protection from T1D, and conversely with susceptibility to Celiac disease. However, few studies have investigated the IL-18 pathway in T1D. In this study, we analyzed responsiveness to IL-18 in T1D, and the effect of rs917997 genotype on IL18RAP gene expression post-activation. Upon IL-12 and IL-18 treatment, peripheral blood mononuclear cells from subjects carrying susceptibility alleles at rs917997 produced higher levels of IFNγ than those with protective genotypes. Additionally, the SNP modified IL18RAP surface protein expression by NK cells and gene expression in activated T cells. Taken together, these data suggest that the disease-associated rs917997 allele G permits hyperresponsiveness to IL-18, providing a novel target for therapeutic intervention in T1D.
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Enfermedades Autoinmunes/genética , Interferón gamma/biosíntesis , Subunidad beta del Receptor de Interleucina-18/genética , Leucocitos Mononucleares/inmunología , Alelos , Enfermedades Autoinmunes/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-12/biosíntesis , Interleucina-12/genética , Interleucina-12/inmunología , Interleucina-12/farmacología , Interleucina-18/biosíntesis , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-18/farmacología , Subunidad beta del Receptor de Interleucina-18/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Polimorfismo de Nucleótido Simple , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
BACKGROUND: Minimal change disease (MCD) is the most common cause of nephrotic syndrome in children and is associated with the expression of CD80 in podocytes and the increased excretion of CD80 in urine. We hypothesized that serum from patients with MCD might stimulate CD80 expression in cultured podocytes. METHODS: Sera and peripheral blood mononuclear cells (PBMCs) were collected from subjects with MCD in relapse and remission and from normal controls. Immortalized human podocytes were incubated with culture media containing patient sera or supernatants from patient and control PBMC cultures. CD80 expression was measured by quantitative PCR and western blot analysis. RESULTS: Sera collected from patients with MCD in relapse, but not in remission, significantly increased CD80 expression (mean ± standard deviation: 1.8 ± 0.7 vs. 0.8 ± 0.2; p < 0.004) and CD80 protein secretion by podocytes (p < 0.05 between relapse and normal controls). No such CD80 increase was observed when podocytes were incubated with supernatants of PBMC cultures from patients in relapse. CONCLUSIONS: Sera from MCD patients in relapse, but not in remission, stimulated CD80 expression in cultured podocytes. Identifying this factor in sera could provide insights into the pathogenesis of this disorder. No role in CD80 expression by podocytes was found for cytokines released by PBMCs.
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Antígeno B7-1/biosíntesis , Nefrosis Lipoidea/metabolismo , Podocitos/metabolismo , Adolescente , Antiinflamatorios/uso terapéutico , Western Blotting , Células Cultivadas , Niño , Preescolar , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/metabolismo , Humanos , Pruebas de Función Renal , Masculino , Monocitos/metabolismo , Nefrosis Lipoidea/sangre , Nefrosis Lipoidea/tratamiento farmacológico , Prednisona/uso terapéutico , ARN/biosíntesis , ARN/genética , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Recurrencia , Suero , Adulto JovenRESUMEN
Type 1 diabetes (T1D) develops due to autoimmune targeting of the pancreatic islet ß-cells. Clinical symptoms arise from reduced insulin in circulation. The molecular events and interactions between discrete immune cell populations, infiltration of such leukocytes into pancreatic and islet tissue, and selective targeting of the islet ß-cells during autoimmunity and graft rejection are not entirely understood. One protein central to antigen presentation, priming of immune cells, trafficking of leukocytes, and vital for leukocyte effector function is the intercellular adhesion molecule-1 (ICAM-1). The gene encoding ICAM-1 is transcriptionally regulated and rapidly responsive (i.e., within hours) to pro-inflammatory cytokines. ICAM-1 is a transmembrane protein that can be glycosylated; its presence on the cell surface provides co-stimulatory functions for immune cell activation and stabilization of cell-cell contacts. ICAM-1 interacts with the ß2-integrins, CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1), which are present on discrete immune cell populations. A whole-body ICAM-1 deletion protects NOD mice from diabetes onset, strongly implicating this protein in autoimmune responses. Since several different cell types express ICAM-1, its biology is fundamentally essential for various physiological and pathological outcomes. Herein, we review the role of ICAM-1 during both autoimmunity and islet graft rejection to understand the mechanism(s) leading to islet ß-cell death and dysfunction that results in insufficient circulating quantities of insulin to control glucose homeostasis.
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Autoinmunidad , Rechazo de Injerto , Molécula 1 de Adhesión Intercelular , Islotes Pancreáticos , Animales , Humanos , Ratones , Insulinas , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Islotes Pancreáticos/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Antígeno de Macrófago-1/metabolismo , Ratones Endogámicos NODRESUMEN
Introduction: Type 1 diabetes (T1D) is defined by immune cell infiltration of the pancreas, in particular the islets of Langerhans, referred to as insulitis, which is especially prominent during the early disease stages in association with decreased beta cell mass. An in-depth understanding of the dynamics and phenotype of the immune cells infiltrating the pancreas and the accompanying changes in their profiles in peripheral blood during T1D development is critical to generate novel preventive and therapeutic approaches, as well as to find biomarkers for the disease process. Methods: Using multi-parameter flow cytometry, we explored the dynamic changes of immune cells infiltrating the pancreas and the pancreatic draining lymph nodes (PLN), compared to those in peripheral blood in female and male non-obese diabetic (NOD) mice during T1D progression. Results: The early stages of T1D development were characterized by an influx of innate dendritic cells and neutrophils in the pancreas. While dendritic cells seemed to move in and out (to the PLN), neutrophils accumulated during the pre-symptomatic phase and reached a maximum at 8 weeks of age, after which their numbers declined. During disease progression, CD4+ and CD8+ T cells appeared to continuously migrate from the PLN to the pancreas, which coincided with an increase in beta cell autoimmunity and insulitis severity, and a decline in insulin content. At 12 weeks of age, CD4+ and especially CD8+ T cells in the pancreas showed a dramatic shift from naïve to effector memory phenotype, in contrast to the PLN, where most of these cells remained naïve. A large proportion of pancreas infiltrating CD4+ T cells were naïve, indicating that antigenic stimulation was not necessary to traffic and invade the pancreas. Interestingly, a pre-effector-like T cell dominated the peripheral blood. These cells were intermediates between naïve and effector memory cells as identified by single cell RNA sequencing and might be a potential novel therapeutic target. Conclusion: These time- and tissue-dependent changes in the dynamics and functional states of CD4+ and CD8+ T cells are essential steps in our understanding of the disease process in NOD mice and need to be considered for the interpretation and design of disease-modifying therapies.