RESUMEN
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.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Memoria Inmunológica , Linfocitos T Reguladores/inmunología , Adulto , Linfocitos T CD8-positivos/patología , Diabetes Mellitus Tipo 1/patología , Femenino , Humanos , Cinética , Masculino , Factores de Riesgo , Linfocitos T Reguladores/patologíaRESUMEN
Blood transcriptional profiles could serve as biomarkers of clinical changes in subjects at-risk for or diagnosed with diabetes. However, transcriptional variation over time is poorly understood due to the impracticality of frequent longitudinal phlebotomy in large patient cohorts. We have developed a novel transcriptome assessment method that could be applied to fingerstick blood samples self-collected by study volunteers. Fifteen µL of blood from a fingerstick yielded sufficient RNA to analyse > 176 transcripts by high-throughput quantitative polymerase chain reaction (PCR). We enrolled 13 subjects with type 1 diabetes and 14 controls to perform weekly collections at home for a period of 6 months. Subjects returned an average of 24 of 26 total weekly samples, and transcript data were obtained successfully for > 99% of samples returned. A high degree of correlation between fingerstick data and data from a standard 3 mL venipuncture sample was observed. Increases in interferon-stimulated gene expression were associated with self-reported respiratory infections, indicating that real-world transcriptional changes can be detected using this assay. In summary, we show that longitudinal monitoring of gene expression is feasible using ultra-low-volume blood samples self-collected by study participants at home, and can be used to monitor changes in gene expression frequently over extended periods.
Asunto(s)
Recolección de Muestras de Sangre , Volumen Sanguíneo , Diabetes Mellitus Tipo 1/genética , Perfilación de la Expresión Génica/métodos , Adolescente , Adulto , Biomarcadores , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/diagnóstico , Femenino , Humanos , Estudios Longitudinales , Masculino , Flebotomía , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Autocuidado , Adulto JovenRESUMEN
As the immune pathways involved in the pathogenesis of type 1 diabetes (T1D) are not fully understood, biomarkers implicating novel mechanisms of disease are of great interest and call for independent evaluation. Recently, it was reported that individuals with T1D display dramatic elevations in circulating components of neutrophil extracellular traps (NETs), indicating a potential role for NETosis in T1D. Our aim was to evaluate further the potential of NET-associated proteins as novel circulating biomarkers in T1D. We tested serum from subjects with T1D (n = 44) with a median age of 26·5 years and a median duration of 2·2 years, along with 38 age-matched controls. T1D subjects did not show elevations in either neutrophil elastase (NE) or proteinase 3 (PR3), as reported previously. In fact, both NE and PR3 levels were reduced significantly in T1D subjects, particularly in subjects within 3 years of diagnosis, consistent with the known reduction in neutrophil counts in recent-onset T1D. Indeed, levels of both NE and PR3 correlated with absolute neutrophil counts. Therefore, while not ruling out potential local or transient spikes in NETosis activity, the levels of these serum markers do not support a role for systemically elevated NETosis in the T1D population we studied. Rather, a modest reduction in these markers may reflect other important aspects of disease activity associated with reduced neutrophil numbers.
Asunto(s)
Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/diagnóstico , Elastasa de Leucocito/sangre , Mieloblastina/sangre , Neutrófilos/inmunología , Adulto , Edad de Inicio , Biomarcadores/sangre , Estudios de Casos y Controles , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Trampas Extracelulares/inmunología , Femenino , Expresión Génica , Humanos , Recuento de Leucocitos , Elastasa de Leucocito/genética , Elastasa de Leucocito/inmunología , Masculino , Mieloblastina/genética , Mieloblastina/inmunología , Neutrófilos/patologíaRESUMEN
CD4(+) memory cell development is dependent upon T cell receptor (TCR) signal strength, antigen dose and the cytokine milieu, all of which are altered in type 1 diabetes (T1D). We hypothesized that CD4(+) T cell turnover would be greater in type 1 diabetes subjects compared to controls. In vitro studies of T cell function are unable to evaluate dynamic aspects of immune cell homoeostasis. Therefore, we used deuterium oxide ((2) H(2)O) to assess in vivo turnover of CD4(+) T cell subsets in T1D (n = 10) and control subjects (n = 10). Serial samples of naive, memory and regulatory (T(reg)) CD4(+) T cell subsets were collected and enrichment of deoxyribose was determined by gas chromatography-mass spectrometry (GC-MS). Quantification of T cell turnover was performed using mathematical models to estimate fractional enrichment (f, n = 20), turnover rate (k, n = 20), proliferation (p, n = 10) and disappearance (d*, n = 10). Although turnover of T(regs) was greater than memory and naive cells in both controls and T1D subjects, no differences were seen between T1D and controls in T(reg) or naive kinetics. However, turnover of CD4(+) memory T cells was faster in those with T1D compared to control subjects. Measurement and modelling of incorporated deuterium is useful for evaluating the in vivo kinetics of immune cells in T1D and could be incorporated into studies of the natural history of disease or clinical trials designed to alter the disease course. The enhanced CD4(+) memory T cell turnover in T1D may be important in understanding the pathophysiology and potential treatments of autoimmune diabetes.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Adolescente , Adulto , Linfocitos T CD4-Positivos/patología , Estudios de Casos y Controles , Proliferación Celular , Desoxirribosa/metabolismo , Óxido de Deuterio/metabolismo , Diabetes Mellitus Tipo 1/patología , Femenino , Humanos , Memoria Inmunológica , Cinética , Masculino , Persona de Mediana Edad , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Adulto JovenRESUMEN
Inhibition of pancreatic glucagon secretion during hyperglycemia could be mediated by (a) glucose, (b) insulin, (c) somatostatin, or (d) glucose in conjunction with insulin. To determine the role of these factors in the mediation of glucagon suppression, we injected alloxan while clamping the arterial supply of the pancreatic splenic lobe of dogs, thus inducing insulin deficiency localized to the ventral lobe and avoiding hyperglycemia. Ventral lobe insulin, glucagon, and somatostatin outputs were then measured in response to a stepped IV glucose infusion. In control dogs glucagon suppression occurred at a glucose level of 150 mg/dl and somatostatin output increased at glucose greater than 250 mg/dl. In alloxan-treated dogs glucagon output was not suppressed nor did somatostatin output increase. We concluded that insulin was required in the mediation of glucagon suppression and somatostatin stimulation. Subsequently, we infused insulin at high rates directly into the artery that supplied the beta cell-deficient lobe in six alloxan-treated dogs. Insulin infusion alone did not cause suppression of glucagon or stimulation of somatostatin; however, insulin repletion during glucose infusions did restore the ability of hyperglycemia to suppress glucagon and stimulate somatostatin. We conclude that intra-islet insulin permits glucose to suppress glucagon secretion and stimulate somatostatin during hyperglycemia.
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Glucosa/farmacología , Hiperglucemia/fisiopatología , Insulina/fisiología , Islotes Pancreáticos/fisiología , Aloxano/farmacología , Animales , Arginina/farmacología , Perros , Glucagón/metabolismo , Insulina/deficiencia , Insulina/metabolismo , Secreción de Insulina , Somatostatina/metabolismoRESUMEN
The clinical presentation of type 1 diabetes usually involves symptoms such as polyuria and polydipsia. However, investigators in the Diabetes Prevention Trial of Type 1 Diabetes (DPT-1) have detected a group of subjects with type 1 diabetes who have a different phenotype. These subjects are asymptomatic, have normal (<6.1 mmol/l) (group A) or impaired (6.1- <7.0 mmol/l) (group B) fasting glucose, but have 2-h glucose values >11.1 mmol/l on their oral glucose tolerance tests (OGTT). Of the 585 OGTTs performed on islet cell antibody (ICA)-positive relatives with insulin autoantibodies (IAA) or low first-phase insulin response (FPIR), normal glucose tolerance (NGT) was found in 427 subjects; impaired glucose tolerance (IGT) was found in 87 subjects, and diabetes was found by 2-h OGTT criteria alone in 61 subjects. Despite marked differences in 2-h glucose values (NGT 5.8 +/- 1.1 mmol/l, IGT 8.9 +/- 0.9 mmol/l, and group A 13.5 +/- 2.5 mmol/l), there were no significant differences in fasting glucose values among NGT (4.8 +/- 0.5 mmol/l), IGT (5.03 +/- 0.5 mmol/l), and group A (4.99 +/- 0.7 mmol/l) categories. Mean FPIR was higher in subjects with NGT compared with subjects with IGT and subjects diagnosed by 2-h OGTT criteria alone. However, the correlation between FPIR and 2-h glucose value was low (r2 = 0.114). Multivariate analysis demonstrated that additional independent variables provide smaller contributions to the 2-h glucose value. In conclusion, there are asymptomatic type 1 diabetic subjects whose diabetes was diagnosed by the 2-h criteria on OGTT alone. Despite the importance of beta-cell dysfunction in the pathogenesis of type I diabetes, factors other than impaired FPIR must also contribute to postprandial glucose tolerance in these subjects.
Asunto(s)
Diabetes Mellitus Tipo 1/diagnóstico , Prueba de Tolerancia a la Glucosa , Adolescente , Adulto , Anticuerpos/análisis , Glucemia/análisis , Niño , Preescolar , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Intolerancia a la Glucosa/diagnóstico , Intolerancia a la Glucosa/etiología , Haplotipos , Humanos , Lactante , Insulina/metabolismo , Secreción de Insulina , Masculino , Valores de Referencia , Factores de TiempoRESUMEN
A total of 85 islet cell antibody (ICA)+ or insulin autoantibody (IAA)+ relatives of patients with type 1 diabetes have been followed as part of the Seattle Family Study for a mean of 2.8 years. Of the subjects followed, 10 developed diabetes during this time period. The presence of GAD antibodies was strongly associated with the development of diabetes. In contrast, the presence of IAAs did not influence the risk of diabetes among ICA+ GAD+ subjects. When either the initial absolute acute insulin response to glucose (AIRg) or the AIR percentile, which accounts for the individual's insulin sensitivity, was below the 10th percentile of normal subjects, the risk of diabetes approached 50% at 5 years. However, impaired beta-cell function did not influence the risk of diabetes among those who were GAD+. There were 13 subjects with low AIRg and 13 subjects with two or more antibodies who had not progressed to diabetes during the course of the study. Other measurements of beta-cell function or demographic characteristics were not different in this group of nonprogressors compared with those with low AIRg who did not progress to diabetes. We conclude that ICA+ relatives with GAD antibodies or low AIRg have a high risk for development of diabetes, but among ICA+ GAD+ relatives, the addition of IAA or a single determination of AIRg does not enhance the prediction of diabetes. We suggest that prediction of diabetes risk depends on both the type and the number of antibodies present. In addition, there are a group of ICA+ relatives with low AIRg and/or multiple antibodies who have not progressed to diabetes over the course of the study.
Asunto(s)
Autoanticuerpos/análisis , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/fisiopatología , Islotes Pancreáticos/fisiopatología , Adolescente , Adulto , Niño , Preescolar , Diabetes Mellitus Tipo 1/genética , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Glucosa/farmacología , Glutamato Descarboxilasa/inmunología , Humanos , Insulina/inmunología , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
Nicotinamide is being used in trials to prevent or delay the development of clinical IDDM. A related compound, niacin, has been shown to cause insulin resistance in normal subjects, resulting in increased insulin secretion. This study was designed to answer the question: Does the short-term administration of nicotinamide cause insulin resistance in subjects who have a high risk of developing IDDM? Eight islet cell antibody-positive (ICA+) relatives of IDDM patients were given nicotinamide at a dose of 2 g/day for 2 weeks. Measurements of first-phase insulin release, insulin sensitivity, glucose effectiveness, and the constant for glucose disappearance (Kg) were measured at baseline, at the end of 2 weeks of therapy, and after subjects had been off therapy for at least 2 weeks. Nicotinamide administration caused a 23.6% decrease in insulin sensitivity (P = 0.02). This decrease was associated with a fall in Kg despite increased insulin secretion. Our data suggest that the use of nicotinamide in subjects who are at risk of developing IDDM may be complicated by the drug's effects on insulin sensitivity. By inducing insulin resistance, a therapeutic effect of nicotinamide on the diabetes disease process may be missed, and the interpretation of insulin secretion measurements that are obtained during the intervention trials using nicotinamide may be complicated by the changes in insulin secretion that are caused by the increased insulin resistance. Therefore, we strongly support the recommendation that at least one subgroup of subjects enrolled in clinical trials to prevent IDDM have regular measurements of both insulin sensitivity and insulin secretion performed. This subgroup should be randomly assigned and large enough for statistical analysis to interpret properly the changes in insulin secretion that may occur.
Asunto(s)
Autoanticuerpos/sangre , Glucemia/metabolismo , Diabetes Mellitus Tipo 1/epidemiología , Resistencia a la Insulina , Insulina/sangre , Islotes Pancreáticos/efectos de los fármacos , Niacinamida/uso terapéutico , Adulto , Biomarcadores/sangre , Familia , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Niacinamida/farmacología , Factores de RiesgoRESUMEN
Type 1 diabetes is a cell-mediated autoimmune disease characterized by autoantibody and peripheral blood mononuclear cell (PBMC) reactivity to islet cell proteins. Type 2 diabetes is not an autoimmune disease but rather results from both insulin resistance and a nonautoimmune insulin secretory defect. There is, however, a group of phenotypic type 2 diabetic patients who have islet autoantibodies that are similar to those of type 1 diabetic patients. In this study, we investigated, using cellular immunoblotting, whether type 2 diabetic patients positive for islet autoantibodies have PBMC responses to islet proteins. We observed that autoantibody negative (Ab-) type 2 diabetic patients (n = 9) and normal control subjects (n = 12) demonstrated PBMCs responsive to 0-3 molecular weight regions. In contrast, autoantibody positive (Ab+) type 2 diabetic patients (n = 11) demonstrated PBMC responses to 3-18 molecular weight regions, similar to that of type 1 diabetic patients (responsive to 4-18 molecular weight regions). PBMCs from over 90% of the Ab+ type 2 and type 1 diabetic patients were observed to proliferate to islet proteins in the vicinity of 97 kDa. In contrast, 65-90% of type 1 diabetic patients had responsive PBMCs for islet proteins in most of the molecular weight regions, whereas <60% of the Ab+ type 2 diabetic patients had PBMCs responsive to the same molecular weight proteins. Ab+ type 2 diabetic patients appear to be heterogeneous with respect to cellular reactivity to islet proteins. Some subjects demonstrate PBMC responses similar to those of "classic" type 1 diabetic patients, whereas others have PBMC responses potentially distinct from type 1 diabetic patients.
Asunto(s)
Autoanticuerpos/sangre , Autoantígenos/inmunología , Diabetes Mellitus Tipo 2/inmunología , Inmunidad Celular , Islotes Pancreáticos/inmunología , Adolescente , Adulto , Anciano , Autoantígenos/química , Índice de Masa Corporal , Niño , Femenino , Humanos , Immunoblotting , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Peso MolecularRESUMEN
The goal of the Fourth International Workshop for Standardization of ICA Measurements was to determine the specificity of ICA assays and their ability to distinguish between control sera (n = 57) and sera from IDDM-related individuals--representing relatives of IDDM patients (n = 21), healthy individuals who later developed IDDM (n = 8), or newly diagnosed IDDM patients (n = 23). Results from 28 laboratories were analyzed. The mean specificity (percentage of control sera reported as negative) among 27 laboratories was 91%, including 6 laboratories with 100% specificity. Nevertheless, 78% of laboratories found at least one control sample > 0 JDF U. Among samples from first-degree relatives, the mean concordance was 86%, including three sera found negative (0 JDF U) by all laboratories. Among individuals who later developed diabetes, the mean concordance was 93%, with two sera found positive by 100% of laboratories. In sera from newly diagnosed IDDM patients, the mean concordance was 82%. Three sera were found positive and one serum negative by all laboratories. The JDF U of the sera considered to be positive were significantly greater than each laboratory's average for the controls. In conclusion, the results from laboratories participating in the Fourth International ICA Workshop demonstrated excellent specificity, good concordance, and an ability to separate control sera from defined, IDDM-related subjects.
Asunto(s)
Autoanticuerpos/análisis , Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus/inmunología , Islotes Pancreáticos/inmunología , Animales , Diabetes Mellitus/sangre , Diabetes Mellitus Tipo 1/sangre , Técnica del Anticuerpo Fluorescente , Haplorrinos , Humanos , Técnicas para Inmunoenzimas , Islotes Pancreáticos/citología , Islotes Pancreáticos/patología , Papio , Ratas , Valores de ReferenciaRESUMEN
It is unknown among first-degree relatives of individuals with insulin-dependent diabetes mellitus (IDDM) whether the disease process occurs in relatively few but always progresses to clinical IDDM or whether subclinical disease is more common but remains nonprogressive in many cases. Islet cell antibodies (ICAs) were found in 21 of 724 (2.9%) first-degree relatives during screening in the greater Seattle area between 1983 and 1988. Measures of beta-cell function (glucose disappearance rate [Kg], fasting insulin, acute insulin response to intravenous arginine [AIRarg], acute insulin response to intravenous glucose [AIRgluc], slope of glucose potentiation of AIRarg) and insulin sensitivity were obtained. Twenty individuals, 9 ICA+ relatives and 11 ICA- relatives, were evaluated prospectively. When expressed in relation to the expected AIRgluc based on each subject's sensitivity index, AIRgluc in 18 of 20 relatives fell below 100%, indicating inappropriately low insulin secretion (subclinical beta-cell dysfunction). After a median follow-up of 42 mo, 10 of 11 ICA- relatives remained ICA-. None showed deteriorating beta-cell dysfunction, and none developed diabetes. Five ICA+ relatives showed persistent immunologic positivity. beta-Cell function remained remarkably stable in all except 2 relatives. One was a 15-yr-old boy who developed IDDM shortly after screening and before evaluation of beta-cell function could be carried out. The other was an 18-yr-old monozygotic twin who developed IDDM after 27 mo. Both of these individuals had ICAs of 80 Juvenile Diabetes Foundation U and had been discordant for less than 5 yr.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Diabetes Mellitus Tipo 1/fisiopatología , Familia , Islotes Pancreáticos/fisiopatología , Adulto , Anticuerpos/análisis , Diabetes Mellitus Tipo 1/epidemiología , Diabetes Mellitus Tipo 1/genética , Femenino , Estudios de Seguimiento , Glucosa/administración & dosificación , Antígenos HLA/análisis , Humanos , Insulina/metabolismo , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Masculino , Tamizaje Masivo , Persona de Mediana Edad , WashingtónRESUMEN
Insulin autoantibodies (IAA) have been identified in newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients and in individuals at high risk of developing the disease. However, the literature is not in agreement regarding the prevalence, significance, and predictive value of IAA. Previous workshops have shown that certain sera give markedly different results depending upon assay methodology and, therefore, have suggested that these discrepancies may be due to variations in the assay methodologies used: either the fluid phase RIA or the solid phase enzyme-linked immunosorbent assay (ELISA). Sera from controls (n = 61), newly diagnosed IDDM patients (n = 30), healthy subjects who later became diabetic (n = 8), and first degree relatives of diabetic probands (n = 22) were randomly numbered and sent without category identification to 19 RIA and 10 ELISA laboratories. Each laboratory's raw data from the control sera were used to determine the cut-off point for positive (3 SD above the mean of control sera) in the disease relevant categories. RIA and ELISA methods were comparable in obtaining a low frequency of IAA in control sera. However, the laboratories using RIA methods found a much higher percentage of sera to be IAA positive among both newly diagnosed patients and healthy individuals who later developed diabetes than laboratories using ELISA methods (P less than 0.005). In contrast, there was considerable overlap in the percentage of sera from first degree relatives found positive by both assays. These data strongly suggest that IAA measured by RIA methodology are more disease related than those measured by ELISA methods. Consequently, RIA assays or assays proven to perform as well should be used to measure IAA associated with IDDM.
Asunto(s)
Autoanticuerpos/análisis , Diabetes Mellitus Tipo 1/inmunología , Anticuerpos Insulínicos/análisis , Ensayo de Inmunoadsorción Enzimática , Humanos , RadioinmunoensayoRESUMEN
The presence of human leukocyte antigen (HLA) haplotype DQA1*0102, DQB1*0602 is associated with protection from type 1 diabetes. The Diabetes Prevention Trial-type 1 has identified 100 islet cell antibody (ICA)-positive relatives with this protective haplotype, far exceeding the number of such subjects reported in other studies worldwide. Comparisons between ICA+ relatives with and without DQB1*0602 demonstrated no differences in gender or age; however, among racial groups, African-American ICA+ relatives were more likely to carry this haplotype than others. The ICA+ DQB1*0602 individuals were less likely to have additional risk factors for diabetes [insulin autoantibody (IAA) positive or low first phase insulin release (FPIR)] than ICA+ relatives without DQB1*0602. However, 29% of the ICA+ DQB1*0602 relatives did have IAA or low FPIR. Although half of the ICA+ DQB1*0602 relatives had a high risk second haplotype, this was not associated with the additional risk factors for diabetes. Hispanic ICA+ individuals with DQB1*0602 were more likely to be IAA positive or to have low FPIR than other racial groups. In conclusion, the presence of ICA in the relatives described here suggests that whatever the mechanism that protects DQB1*0602 individuals from diabetes, it is likely to occur after the diabetes disease process has begun. In addition, there may be different effects of DQB1*0602 between ethnic groups.
Asunto(s)
Autoanticuerpos/genética , Antígenos HLA-DQ/análisis , Antígenos HLA-DQ/genética , Islotes Pancreáticos/inmunología , Adulto , Envejecimiento/fisiología , Autoanticuerpos/análisis , Diabetes Mellitus Tipo 1/genética , Familia , Femenino , Pruebas Genéticas , Cadenas alfa de HLA-DQ , Cadenas beta de HLA-DQ , Haplotipos , Humanos , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Masculino , Grupos Raciales , Medición de Riesgo , Caracteres SexualesRESUMEN
Animal serum is often used to generate human macrophages in vitro. Since fetal calf serum (FCS) may complicate antigen uptake, processing and presentation on HLA molecules, we tested the ability of M-CSF to generate macrophages at low fetal calf serum conditions. Peripheral blood monocytes from 12 individuals were cultured 1-4 days with 0-100 ng/ml macrophage colony stimulating factor (M-CSF) at either 1 (low) or 5% (v/v) FCS. Regardless of number of days in culture, maximal (50-100 ng/ml) M-CSF stimulation and low FCS induced 65+/-5% esterase positive cells in all individuals compared to 52+/-7% without M-CSF (P<0.001). M-CSF increased the mean proportion of esterase positive cells after 24 or 96 h by 13% (P<0.005) and 13% (P<0.005), respectively, in 1% FCS, and 8% (P<0.05) and 2% (NS), respectively, in 5% FCS, indicating a slight negative interaction between 5% FCS and M-CSF (P<0.05). All cells were positive for CD14 and HLA class II, but cell number did not increase, confirming that M-CSF promote macrophage differentiation also at low FCS. M-CSF increased the average cell size after 24 or 96 h by 5.9+/-1.0 (P<0.05) and 8.6+/-0.5 (P<0.001) microm, respectively, without an increase in 5% FCS, further demonstrating the efficiency of M-CSF to promote macrophage generation at low FCS. The culture supernatants were negative for IL-1beta and TNF-alpha, which demonstrates that M-CSF did not activate the macrophages. The generation of human macrophages by M-CSF at low FCS should prove useful in studies where higher FCS concentrations may interfere with the assay.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Medio de Cultivo Libre de Suero , HumanosRESUMEN
Insulin autoantibodies (IAA), a marker for insulin-dependent diabetes mellitus (IDDM), have been reported in other diseases such as thyroid disease and after treatment with sulfhydryl containing medications. Reported prevalences of IAA in non-diabetics vary widely, probably due in part to methodological differences between laboratories. In addition, certain sera may have a high non-specific binding to insulin. We compared a radioimmunoassay (RIA) for IAA which included non-specific binding with an RIA that incorporated a competitive displacement with cold insulin to remove non-specific binding. Using the RIA which measured specific plus non-specific binding, IAA positivity was found in 22/92 (23.9%) of sera from thyroid disease patients, 16/124 (12.9%) of random masked sera from a hospital laboratory, 27/335 (8.1%) of first degree relatives of IDDM patients, 63/178 (35.4%) of subjects with newly diagnosed IDDM, and 0/92 (0%) of normal controls. Insulin antibodies (IA) were found in 80/99 (80.8%) of insulin-treated diabetic subjects. In contrast, using the displacement assay which allowed measurement of specific binding, the frequency of IAA positivity was lower for subjects with thyroid disease (7/92 (7.6%)), random hospital sera (12/124 (9.8%)), and for first degree relatives of IDDM patients (8/335 (2.4%)), while higher for subjects with newly diagnosed IDDM (71/178 (39.9%)). Subjects with insulin-treated diabetes (78/99 (78.8%)) and normal subjects (1/92 (1.1%)) showed little change. Strikingly, three of the eight (37.5%) relatives of IDDM patients that were positive in the RIA measuring specific binding were detected only because cold displacement was utilized. We conclude: (1) subjects with thyroid disease and first degree relatives of IDDM patients frequently have high non-specific binding for IAA in an RIA not employing a cold displacement step, (2) in some newly diagnosed IDDM patients and first degree relatives of IDDM patients, IAA may be missed by an assay not optimized to measure specific binding, and (3) displacement with cold insulin increases both the specificity and sensitivity of RIAs measuring insulin autoantibodies.
Asunto(s)
Autoanticuerpos/análisis , Enfermedades Autoinmunes/inmunología , Diabetes Mellitus Tipo 1/inmunología , Insulina/inmunología , Radioinmunoensayo/métodos , Enfermedades de la Tiroides/inmunología , Especificidad de Anticuerpos , Unión Competitiva , Frío , Humanos , Sensibilidad y EspecificidadRESUMEN
This study was undertaken to determine which type 1 diabetes-associated autoantibodies and what clinical characteristics are most useful to identify patients with type 1(1/2) diabetes. We studied 125 patients, recently diagnosed clinically with type 2 diabetes for the presence of islet cell antibodies (ICA), insulin autoantibodies (IAA), antibodies to glutamic acid decarboxylase(GADAb), and IA-2a (IA-2Ab). Patients with a diagnosis of type 2 diabetes who met all of the following criteria at diagnosis were studied: age > or = 30 years, no history of ketonuria or ketoacidosis, and not requiring insulin treatment. Thirty-six patients (29%) were positive for at least 1 antibody. Thirty-two (26%) were ICA positive and 20 (16%) GADAb positive. Insulin autoantibodies and IA-2Ab occurred less frequently in 2 (1.6%) and 8 (6.4%) patients, respectively. There was no significant difference in the ages at diagnosis between the Ab(+) and Ab(-) patients, age in years (range) 47.2 (32 to 64) versus 51.2 (31 to 77), respectively, P =.06. Body mass index (BMI) was different in the 2 groups, with Ab(+) patients being less obese, BMI (range) 28.3 kg/m(2) (17.6 to 54.9) versus 32.0 kg/m(2) (19.2 to 68.8), respectively, P =.01. Clinical presentation of diabetes was more commonly symptomatic with polyuria and polydipsia in Ab(+) patients, while in Ab(-) patients, diagnosis was more often incidental, P =.002. However, more than 95% of patients overlapped in both age and BMI irrespective of antibody status. Similarly, 42% of Ab(+) patients had their diabetes diagnosed incidentally, while 29% of Ab(-) patients presented with polyuria and polydipsia. We therefore conclude that screening with antibodies, mainly ICA and GAD, but not age, BMI, or clinical presentation should be used to identify type 1(1/2) diabetes.
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Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 2/diagnóstico , Glutamato Descarboxilasa/inmunología , Isoenzimas/inmunología , Adulto , Factores de Edad , Anciano , Pueblo Asiatico , Autoanticuerpos/clasificación , Población Negra , Índice de Masa Corporal , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/inmunología , Diagnóstico Diferencial , Femenino , Humanos , Hiperglucemia/etiología , Insulina/inmunología , Masculino , Persona de Mediana Edad , Fenotipo , Poliuria/etiología , Valor Predictivo de las Pruebas , Población BlancaRESUMEN
During the preclinical period of insulin-dependent diabetes mellitus (IDDM), progression to clinical IDDM is characterized by declining beta-cell function. Although the presence of insulin autoantibodies (IAA) improves the ability of islet cell antibodies (ICA) to predict subsequent clinical IDDM, few studies have examined the risk of developing IDDM in subjects positive for IAA but negative for both ICA and antibodies to glutamic acid decarboxylase (64kA). To investigate this question, detailed beta-cell function tests (acute insulin response to glucose [AIRgluc] and slope of glucose potentiation) were performed on eight IAA-positive first-degree relatives of insulin-dependent diabetics. All eight subjects were negative for ICA, and seven were tested for 64kA and were negative. Five subjects were studied prospectively for 22.4 +/- 9.4 months, while three subjects had only initial studies. Initial beta-cell function tests were normal in each subject. AIRgluc was 122.2% +/- 19.0% of the expected normal response, while slope was 168.6% +/- 20.6% of expected normal response. beta-cell function remained normal and remarkably stable in the five subjects followed prospectively. AIRgluc did not significantly change from an initial value of 147.9% +/- 23.1% of expected to 153.2% +/- 22.4% (NS). The slope of glucose potentiation varied little from 165.5% +/- 39.4% initially to 159.5% +/- 27.3% (NS) at the most recent determination. We conclude that among nondiabetic first-degree relatives of IDDM subjects, the presence of IAA in the absence of ICA and 64kA is not usually associated with and therefore does not reliably predict beta-cell dysfunction or progressive deterioration in beta-cell function.
Asunto(s)
Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Insulina/inmunología , Islotes Pancreáticos/fisiopatología , Adolescente , Adulto , Arginina , Niño , Diabetes Mellitus Tipo 1/fisiopatología , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/sangre , Islotes Pancreáticos/inmunología , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
This investigation studied relative changes in periodontal conditions of 18 insulin-dependent diabetic patients. Measures of gingival inflammation, crevicular fluid aspartate aminotransferase (AST) levels, probing depth and attachment levels, the presence of three periodontal pathogens (Porphyromonas gingivalis, Bacteroides forsythus, and Actinobacillus actinomycetemcomitans) and serum antibody titers to these bacteria, and blood sugar levels (glycosylated hemoglobin, HbAlc) were studied before and 2 months after non-surgical debridement. Antibody titers to the same bacteria were also studied in sera from 18 sex- and age-matched periodontally healthy and non-diabetic subjects. Periodontal conditions showed significant improvement. The mean probing depth at 4 of the worst sites selected in each patient decreased from 5.7 mm to 4.8 mm (p < 0.0001). The mean full width probing depth changed from 2.9 mm (s.d. +/- 0.2) to 2.5 mm (s.d. +/- 0.3). A mean gain of 0.4 mm attachment level was recorded (P < 0.0001). The mean AST value decreased from 1009 microIU to 518 microIU (P < 0.006). Minimal differences in mean glycosylated hemoglobin values (HbAlc) were noticed before and after treatment. A. actinomycetemcomitans was never detected. P. gingivalis was present at 7% of the sites both before and after treatment. B. forsythus was found at 29% of sites (50% of patients) before and at 36% of sites (61% of patients) after treatment. Positive associations were found between the presence of B. forsythus and AST values, gingival index, probing depth, and attachment level (P < 0.05). Baseline serum IgG titers to P. gingivalis were significantly lower in the patients with diabetes (9.5 ELISA units vs. 28.5 ELISA units in the healthy controls). IgG titers to B. forsythus did not differ between diabetic and non-diabetic subjects. No changes in IgG titers occurred after treatment. Clinical improvements after mechanical non-surgical therapy in patients with insulin-dependent diabetes mellitus were modest after 2 months. Treatment did not eliminate B. forsythus and P. gingivalis and did not affect IgG titer responses. More intense therapy, and longer follow-up times, may be necessary to see more pronounced clinical and systemic effects.
Asunto(s)
Diabetes Mellitus Tipo 1/complicaciones , Enfermedades Periodontales/terapia , Adulto , Aggregatibacter actinomycetemcomitans/inmunología , Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Anticuerpos Antibacterianos/sangre , Aspartato Aminotransferasas/análisis , Bacteroides/inmunología , Bacteroides/aislamiento & purificación , Estudios de Casos y Controles , Raspado Dental , Diabetes Mellitus Tipo 1/sangre , Femenino , Estudios de Seguimiento , Líquido del Surco Gingival/enzimología , Gingivitis/complicaciones , Gingivitis/terapia , Hemoglobina Glucada/análisis , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Pérdida de la Inserción Periodontal/patología , Pérdida de la Inserción Periodontal/terapia , Enfermedades Periodontales/microbiología , Enfermedades Periodontales/patología , Índice Periodontal , Bolsa Periodontal/patología , Bolsa Periodontal/terapia , Porphyromonas gingivalis/inmunología , Porphyromonas gingivalis/aislamiento & purificación , Aplanamiento de la Raíz , Curetaje SubgingivalAsunto(s)
Tejido Adiposo/metabolismo , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Citocalasina B/metabolismo , Glicoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Proteínas Portadoras/aislamiento & purificación , Femenino , Glicoproteínas/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Peso Molecular , Polietilenglicoles , RatasRESUMEN
NBI-6024 is an altered peptide ligand (APL) corresponding to the 9-23 amino acid region of the insulin B chain (B(9-23)), an epitope recognized by inflammatory interferon-gamma-producing T helper (Th)1 lymphocytes in type 1 diabetic patients. Immunomodulatory effects of NBI-6024 administration in recent-onset diabetic patients in a phase I clinical trial (NBI-6024-0003) were measured in peripheral blood mononuclear cells using the enzyme-linked immunosorbent spot assay. Analysis of the mean magnitude of cytokine responses to B(9-23) and NBI-6024 for each cohort showed significant increases in interleukin-5 responses (a Th2 regulatory phenotype) in cohorts that received APL relative to those receiving placebo. A responder analysis showed that Th1 responses to B(9-23) and NBI-6024 were observed almost exclusively in the placebo-treated diabetic population but not in nondiabetic control subjects and that APL administration (five biweekly subcutaneous injections) significantly and dose-dependently reduced the percentage of patients with these Th1 responses. The results of this phase I clinical study strongly suggest that NBI-6024 treatment shifted the Th1 pathogenic responses in recent-onset type 1 diabetic patients to a protective Th2 regulatory phenotype. The significance of these findings on the clinical outcome of disease is currently under investigation in a phase II multidose study.