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1.
Sci Rep ; 11(1): 8712, 2021 04 22.
Article in English | MEDLINE | ID: mdl-33888788

ABSTRACT

Obesity is associated with both chronic and acute respiratory illnesses, such as asthma, chronic obstructive pulmonary disease (COPD) or increased susceptibility to infectious diseases. Anatomical but also systemic and local metabolic alterations are proposed contributors to the pathophysiology of lung diseases in the context of obesity. To bring perspective to this discussion, we used NMR to compare the obesity-associated metabolomic profiles of the lung with those of the liver, heart, skeletal muscles, kidneys, brain and serum from male C57Bl/6J mice fed with a high-fat and high-sucrose (HFHSD) diet vs. standard (SD) chow for 14 weeks. Our results showed that the lung was the second most affected organ after the liver, and that the two organs shared reduced one-carbon (1C) metabolism and increased lipid accumulation. Altered 1C metabolism was found in all organs and in the serum, but serine levels were increased only in the lung of HFHSD compared to SD. Lastly, tricarboxylic acid (TCA)-derived metabolites were specifically and oppositely regulated in the serum and kidneys but not in other organs. Collectively, our data highlighted that HFHSD induced specific metabolic changes in all organs, the lung being the second most affected organ, the main alterations affecting metabolite concentrations of the 1C pathway and, to a minor extend, TCA. The absolute metabolite quantification performed in this study reveals some metabolic specificities affecting both the liver and the lung, that may reveal common metabolic determinants to the ongoing pathological process.


Subject(s)
Diet, High-Fat , Dietary Sucrose/administration & dosage , Lipid Metabolism , Liver/metabolism , Lung/metabolism , Obesity/metabolism , Animals , Magnetic Resonance Spectroscopy , Male , Mice , Mice, Inbred BALB C
2.
PLoS One ; 11(2): e0148686, 2016.
Article in English | MEDLINE | ID: mdl-26862742

ABSTRACT

Type 2 diabetes is characterized by peripheral insulin resistance and pancreatic beta cell dysfunction. Elevated free fatty acids (FFAs) may impair beta cell function and mass (lipotoxicity). Altered calcium homeostasis may be involved in defective insulin release. The endoplasmic reticulum (ER) is the major intracellular calcium store. Lipotoxicity induces ER stress and in parallel an ER calcium depletion through unknown ER calcium leak channels. The main purposes of this study is first to identify one of these channels and secondly, to check the opportunity to restore beta cells function (i.e., insulin secretion) after pharmacological inhibition of ER calcium store depletion. We investigated the functionality of translocon, an ER calcium leak channel and its involvement on FFAs-induced alterations in MIN6B1 cells and in human pancreatic islets. We evidenced that translocon acts as a functional ER calcium leak channel in human beta cells using anisomycin and puromycin (antibiotics), respectively blocker and opener of this channel. Puromycin induced a significant ER calcium release, inhibited by anisomycin pretreatment. Palmitate treatment was used as FFA model to induce a mild lipotoxic effect: ER calcium content was reduced, ER stress but not apoptosis were induced and glucose induced insulin secretion was decreased in our beta cells. Interestingly, translocon inhibition by chronic anisomycin treatment prevented dysfunctions induced by palmitate, avoiding reticular calcium depletion, ER stress and restoring insulin secretion. Our results provide for the first time compelling evidence that translocon actively participates to the palmitate-induced ER calcium leak and insulin secretion decrease in beta cells. Its inhibition reduces these lipotoxic effects. Taken together, our data indicate that TLC may be a new potential target for the treatment of type 2 diabetes.


Subject(s)
Insulin-Secreting Cells/drug effects , Palmitates/toxicity , Protein Translocation Systems/physiology , Animals , Anisomycin/pharmacology , Apoptosis/drug effects , Calcium/metabolism , Calcium Signaling/drug effects , Caspases/metabolism , Cells, Cultured , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/drug effects , Enzyme Activation/drug effects , Genes, Reporter , Heat-Shock Proteins/genetics , Heat-Shock Proteins/physiology , Homeostasis , Humans , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/metabolism , Ion Transport/drug effects , Mice , Protein Transport/drug effects , Puromycin/pharmacology , RNA Interference , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Recombinant Fusion Proteins/metabolism , Transfection
3.
J Mol Endocrinol ; 45(2): 99-105, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20505014

ABSTRACT

Owing to strong interactions between pancreatic islets and the surrounding capillary network, we hypothesized that high glucose concentrations might affect key angiogenesis factors from isolated human islets, thus contributing to beta-cell failure in diabetes. Human islets from eight distinct donors were studied following 96 h in culture in the presence of normal (5.5 mmol/l) or high (16.7 mmol/l) glucose concentrations. Similar studies were performed with HUVECs. Human angiogenesis-related genes and corresponding proteins were studied by real-time quantitative PCR (RT-qPCR) and protein arrays respectively. Angiogenesis and proliferation assays were also performed with HUVECs under the same culture conditions. RT-qPCR and proteome analysis of human islets incubated with 16.7 mM/l glucose revealed a significant decrease in pro-angiogenic factors including vascular endothelial growth factor (VEGF) mRNA by 20% and VEGF protein levels by 42% as well as additional proteins such as fibroblast growth factor-4 by 41%, MMP9 by 18%, monocyte chemoattractant protein-1 by 21%, and prolactin by 25%. In contrast, we observed a 17% increase in thrombospondin-1 (TSP-1, listed as THBS1 in the HUGO database) and a 37% increase in angiotensinogen gene expression levels, but neither angiotensin-converting enzyme nor angiotensin II type 1 receptor gene expression was affected. The amounts of anti-angiogenic proteins such as TSP-1 and serpin B5/maspin were also increased by 70 and 98% respectively as well as endostatin by 63%. Angiogenesis assays of HUVECS in the presence of high glucose concentrations revealed a 30% decrease in tree-like tubular network formation. These data suggest that glucose reduces key factors of islet angiogenesis, which might exacerbate beta-cell failure.


Subject(s)
Glucose/pharmacology , Islets of Langerhans/physiology , Neovascularization, Physiologic/drug effects , Angiotensinogen/genetics , Angiotensinogen/metabolism , Animals , Cell Proliferation/drug effects , Cells, Cultured , Humans , Insulin/genetics , Insulin/metabolism , Islets of Langerhans/cytology , Islets of Langerhans/drug effects , Middle Aged , Thrombospondin 1/genetics , Thrombospondin 1/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism
4.
Diabetologia ; 52(7): 1391-9, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19421731

ABSTRACT

AIMS/HYPOTHESIS: Displaying immunomodulatory capacities, mesenchymal stem cells (MSCs) are considered as beneficial agents for autoimmune diseases. The aim of this study was to examine the ability of MSCs to prevent autoimmune diabetes in the NOD mouse model. METHODS: Prevention of spontaneous insulitis or of diabetes was evaluated after a single i.v. injection of MSCs in 4-week-old female NOD mice, or following the co-injection of MSCs and diabetogenic T cells in irradiated male NOD recipients, respectively. The frequency of CD4(+)FOXP3(+) cells and Foxp3 mRNA levels in the spleen of male NOD recipients were also quantified. In vivo cell homing was assessed by monitoring 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled T cells or MSCs. In vitro, cell proliferation and cytokine production were assessed by adding graded doses of irradiated MSCs to insulin B9-23 peptide-specific T cell lines in the presence of irradiated splenocytes pulsed with the peptide. RESULTS: MSCs reduced the capacity of diabetogenic T cells to infiltrate pancreatic islets and to transfer diabetes. This protective effect was not associated with the modification of diabetogenic T cell homing, but correlated with a preferential migration of MSCs to pancreatic lymph nodes. While injection of diabetogenic T cells resulted in a decrease in levels of FOXP3(+) regulatory T cells, this decrease was inhibited by MSC co-transfer. Moreover, MSCs were able to suppress both allogeneic and insulin-specific proliferative responses in vitro. This suppressive effect was associated with the induction of IL10-secreting FOXP3(+) T cells. CONCLUSIONS/INTERPRETATION: MSCs prevent autoimmune beta cell destruction and subsequent diabetes by inducing regulatory T cells. MSCs may thus offer a novel cell-based approach for the prevention of autoimmune diabetes and for islet cell transplantation.


Subject(s)
Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/prevention & control , Mesenchymal Stem Cell Transplantation , Prediabetic State/therapy , T-Lymphocytes, Regulatory/cytology , Animals , Cell Movement/immunology , Cells, Cultured , Disease Models, Animal , Female , Flow Cytometry , Forkhead Transcription Factors/metabolism , Insulin-Secreting Cells/immunology , Interleukin-10/metabolism , Lymphocyte Culture Test, Mixed , Male , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Prediabetic State/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism
5.
Eur J Endocrinol ; 157(1): 53-61, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17609402

ABSTRACT

OBJECTIVE: Diabetes is clinically classified into two types: type 1 (T1D) and type 2 diabetes (T2D). Nevertheless, intermediate forms of diabetes are frequent and difficult to recognize and manage appropriately. In this study, we investigated whether patients with intermediate form of diabetes, here called unclassified diabetes (UD), have beta-cell autoimmune markers. RESEARCH DESIGN AND METHODS: beta-cell autoimmune markers (beta-cell autoantibodies (aAb), peripheral blood mononuclear cells (PBMC) responsive to five islet proteins, cytokine secretion, and human leukocyte antigen (HLA)-DQB1 genotypes) were analyzed in 50 UD patients, 23 age- and HLA-matched normal control subjects, and 23 classic T2D patients. RESULTS: We observed that 16 out of 50 (32%) UD patients demonstrated responsive PBMCs, as opposed to 1 out of 23 (5%) age- and HLA-matched normal control subjects, and 0 out of 23 classic T2D patients. Overall, 29 (58%) UD patients had at least one marker of beta-cell autoimmunity (beta-cell aAb and/or PBMC autoreactivity), in association with high-risk HLA genotypes DQB1*0201 and/or DQB1*0302. Moreover, the 13 (26%) UD patients who had beta-cell aAb were not the same as those with PBMC autoreactivity, except for one patient. Patients with PBMC autoreactivity were older at the onset of the disease and had a better residual beta-cell function than those with beta-cell aAb. CONCLUSIONS: Our data confirm that T-cell autoimmunity can be detected in latent autoimmune diabetes in adults patients. We show an inverse correlation between humoral and cellular beta-cell autoimmunities. Possible protective cellular responses in the patients with beta-cell PBMC autoreactivity could have potential therapeutic implications.


Subject(s)
Antibody Formation/physiology , Autoimmunity/physiology , Diabetes Mellitus, Type 1/immunology , Immunity, Cellular/physiology , Adult , Aged , Autoantibodies/blood , Biomarkers/blood , Case-Control Studies , Diabetes Mellitus, Type 1/pathology , Diabetes Mellitus, Type 2/immunology , Disease Progression , Female , Humans , Insulin-Secreting Cells/immunology , Male , Middle Aged , T-Lymphocytes/immunology
6.
Clin Exp Immunol ; 148(3): 432-9, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17374136

ABSTRACT

Chemokines and their receptors are part of polarized T helper 1 (Th1)- and Th2-mediated immune responses which control trafficking of immunogenic cells to sites of inflammation. The chemokine stromal cell-derived factor-1 CXCL-12 (SDF-1) and its ligand the CXCR4 chemokine receptor are important regulatory elements. CXCR4 is expressed on the surface of CD4(+) T cells, dendritic cells and B lymphocytes. Levels of CXCR4 mRNA were increased in pancreatic lymph nodes (PLNs) of 4-week-old non-obese diabetic (NOD) mice in comparison to Balb/C mice. However, a significant reduction of CXCR4 was noticed at 12 weeks both at the mRNA and protein levels while expression increased in the inflamed islets. The percentage of SDF-1 attracted splenocytes in a transwell chemotaxis assay was significantly increased in NOD versus Balb/c mice. SDF-1 attracted T cells completely abolished the capacity of diabetogenic T cells to transfer diabetes in the recipients of an adoptive cell co-transfer. When T splenocytes from NOD females treated with AMD3100, a specific CXCR4 antagonist, were mixed with diabetogenic T cells during adoptive cell co-transfer experiments, prevalence of diabetes in the recipients rose from 33% to 75% (P < 0.001). This effect was associated with an increase of interferon (IFN)-gamma mRNA and a reduction of interleukin (IL)-4 mRNA levels both in PLNs and isolated islets. AMD3100 also reduced IL-4 and IL-10 production of plate-bound anti-CD3 and anti-CD28-stimulated splenocytes. Immunofluorescence studies indicated that AMD3100 reduced the number of CXCR4(+) and SDF-1 positive cells in the inflamed islets. We can conclude that the CXCL-12/CXCR4 pathway has protective effects against autoimmune diabetes.


Subject(s)
Autoimmune Diseases/immunology , Chemokines, CXC/immunology , Diabetes Mellitus, Experimental/immunology , Diabetes Mellitus, Type 1/immunology , Receptors, CXCR4/immunology , Adoptive Transfer , Animals , Autoimmune Diseases/prevention & control , Benzylamines , Cells, Cultured , Chemokine CXCL12 , Chemokines, CXC/biosynthesis , Chemokines, CXC/genetics , Cyclams , Cytokines/biosynthesis , Diabetes Mellitus, Experimental/prevention & control , Diabetes Mellitus, Type 1/prevention & control , Female , Heterocyclic Compounds/pharmacology , Islets of Langerhans/immunology , Lymph Nodes/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , RNA, Messenger/genetics , Receptors, CXCR4/antagonists & inhibitors , Receptors, CXCR4/biosynthesis , Receptors, CXCR4/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Signal Transduction/drug effects , Signal Transduction/immunology , Th2 Cells/drug effects , Th2 Cells/immunology
7.
Diabetologia ; 48(5): 922-30, 2005 May.
Article in English | MEDLINE | ID: mdl-15834701

ABSTRACT

AIMS/HYPOTHESIS: Progression to type 1 diabetes is associated with intramolecular epitope spreading to disease-specific antibody epitopes located in the middle region of glutamic acid decarboxylase 65 (GAD65). METHODS: The relationship between intramolecular epitope spreading of autoantibodies specific to GAD65 in relation to the risk of developing type 1 diabetes was tested in 22 high-risk individuals and 38 low-risk individuals. We determined the conformational epitopes in this longitudinal study by means of competition experiments using recombinant Fab of four GAD65-specific monoclonal antibodies. RESULTS: Sera from high-risk children in the preclinical stage recognise a specific combination of GAD65 antibody epitopes located in the middle and the C-terminus of GAD65. High risk of progressing to disease is associated with the emergence of antibodies specific for conformational epitopes at the N-terminus and the middle region. Binding to already established antibody epitopes located in the middle and at the N-terminus increases and shows a significant relation (p=0.005) with HLA, which confers risk of developing diabetes. CONCLUSIONS/INTERPRETATION: In type 1 diabetes, GAD65 antibodies are initially generated against the middle and C-terminal regions of GAD65. In genetically predisposed subjects the autoimmune response may then undergo intramolecular epitope spreading towards epitopes on the N-terminus and further epitopes located in the middle. These findings clearly demonstrate that the GAD65 autoantibody response in the preclinical stage of type 1 diabetes is dynamic and related to the HLA genotypes that confer risk of diabetes. GAD65-specific Fab should prove useful in predicting progression from islet autoimmunity to clinical onset of type 1 diabetes.


Subject(s)
Autoantibodies/blood , Diabetes Mellitus, Type 1/epidemiology , Diabetes Mellitus, Type 1/immunology , Glutamate Decarboxylase/immunology , Isoenzymes/immunology , Adolescent , Child , Epitopes/analysis , Female , Genotype , HLA Antigens , Humans , Immunoglobulin Fab Fragments/immunology , Major Histocompatibility Complex , Male , Risk Factors
8.
Clin Exp Immunol ; 138(2): 337-41, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15498046

ABSTRACT

Type I diabetes (TID) is an autoimmune disease characterized in part by the presence of autoantibodies directed against glutamic acid decarboxylase 65 (GAD65), among other pancreatic islet antigens. We investigated the independent epitope specificities of these GAD65 antibodies (GAD65Ab) and their combinations in the sera of new onset TID patients and first-degree relatives positive for GAD65Ab. For our analysis, we used four GAD65-specific recombinant Fabs (rFabs) that recognize different conformational determinants of GAD65 located throughout the molecule, including the N-terminal, the middle and the C-terminal regions. We used these epitope-specific rFabs in competition assays to determine the binding specificity of the autoantibodies found in patient sera. Among the 61 sera from newly diagnosed GAD65Ab-positive TID patients GAD65 binding was competed for 23 sera by all four rFabs, 29 by at least two rFabs, and in nine sera were displaced by one or no rFab. In contrast, none of the 24 sera from GAD65Ab-positive first-degree relatives of TID patients were displaced by all four rFabs. When using all four rFabs simultaneously to compete with GAD65Ab binding, binding of sera from TID patients was reduced by an average of 70%. A significantly weaker competition was observed when evaluating sera of GAD65Ab-positive first-degree relatives (P < 0.0001).


Subject(s)
Autoantibodies/biosynthesis , Diabetes Mellitus, Type 1/immunology , Epitopes/immunology , Glutamate Decarboxylase/immunology , Isoenzymes/immunology , Adolescent , Adult , Aged , Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , Child , Child, Preschool , Female , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin G/immunology , Infant , Male , Middle Aged , Recombinant Proteins/immunology
9.
Best Pract Res Clin Endocrinol Metab ; 18(2): 289-302, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15157841

ABSTRACT

The abundance of published data on the neonatal effects of maternal Graves' disease (GD) contrasts with the paucity of information on fetal effects. In our yet unpublished study, we prospectively studied 72 pregnant women with a history of Graves' disease. Fetal ultrasonography was done at 22 and 32 weeks of gestational age. Fetal goiter was found at 32 weeks in 11 of the fetuses of the 41 mothers with positive TSH-receptor antibodies and/or antithyroid treatment and in none of the fetuses of the 31 other mothers. In the 11 fetuses with goiter, ultrasound findings (thyroid Doppler and bone maturation), fetal heart rate, and maternal antibody and antithyroid drug status effectively discriminated between hypothyroidism (n=7) and hyperthyroidism (n=4). One fetus with hyperthyroidism died in utero at 35 weeks from heart failure. Treatment was successful in the ten other fetuses. One fetus without goiter had moderate hypothyroidism at birth. This study showed that it is of the utmost importance to have the fetal thyroid scrutinized by an expert ultrasonographist and to have team work with obstetricians and paediatric endocrinologists in pregnant mothers with GD. This allowed us to accurately determine fetal thyroid status and to adapt the treatment in mothers successfully. Fetal hyperthyroidism does exist and needs an appropriate aggressive treatment.


Subject(s)
Fetal Diseases/physiopathology , Goiter/physiopathology , Graves Disease/physiopathology , Infant, Newborn, Diseases/physiopathology , Pregnancy Complications/physiopathology , Female , Fetal Diseases/diagnostic imaging , Fetal Diseases/therapy , Goiter/diagnostic imaging , Goiter/therapy , Graves Disease/diagnosis , Graves Disease/therapy , Humans , Infant, Newborn , Infant, Newborn, Diseases/diagnosis , Infant, Newborn, Diseases/therapy , Pregnancy , Pregnancy Complications/diagnosis , Pregnancy Complications/therapy , Thyroid Gland/physiology , Ultrasonography
10.
Clin Exp Immunol ; 135(1): 74-84, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14678267

ABSTRACT

We used a GAD65-specific human B-T cell line cognate system in vitro to investigate the modulation of GAD65 presentation by autoantibody, assessed in a proliferation assay. Generally, if the T cell determinant overlaps or resides within the antibody epitope, effects of presentation are blunted while if they are distant can lead to potent presentation. For three different autoreactive B-T cell line cognate pairs, the modulation of GAD65 presentation followed the mode of overlapping or distant epitopes with resultant potent or undetectable presentation. However, other cognate pairs elicited variability in this pattern of presentation. Notably, one B cell line, DPC, whose antibody epitope did not overlap with the T cell determinants, was consistently poor in presenting GAD65. Using the fluorescent dye Alexa Fluor 647 conjugated to GAD65 to study receptor-mediated antigen endocytosis showed that all the antigen-specific B cell clones were efficient in intracellular accumulation of the antigen. Additionally, multicolour immunofluorescence microscopy showed that the internalized GAD65/surface IgG complexes were rapidly targeted to a perinuclear compartment in all GAD-specific B cell clones. This analysis also demonstrated that HLA-DM expression was reduced strongly in DPC compared to the stimulatory B cell clones. Thus the capability of antigen-specific B cells to capture and present antigen to human T cell lines is dependent on the spatial relationship of B and T cell epitopes as well other factors which contribute to the efficiency of presentation.


Subject(s)
Antigen Presentation/immunology , B-Lymphocytes/immunology , Diabetes Mellitus, Type 1/immunology , Glutamate Decarboxylase/immunology , Isoenzymes/immunology , Antibodies, Monoclonal/immunology , Antibody Affinity , Antigens, Surface/analysis , Autoimmunity , Cell Line, Transformed , Dose-Response Relationship, Immunologic , Endocytosis/immunology , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Flow Cytometry , Glutamate Decarboxylase/analysis , HLA-D Antigens/analysis , Herpesvirus 4, Human , Humans , Immunoglobulin G/analysis , Immunoglobulin G/immunology , Isoenzymes/analysis , Microscopy, Fluorescence
12.
Prenat Diagn ; 20(10): 799-806, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11038457

ABSTRACT

Fetal thyroid goitres may reveal hormonal imbalance. This can jeopardize neurological development and fetal outcome even when early postnatal treatment is provided. We report a series of 11 goitres diagnosed antenatally in women with past or present thyroid disorders or discovered fortuitously on ultrasound scan. Fetuses presented with hyperthyroidism in three cases and hypothyroidism in eight. Hypothyroidism was iatrogenic in five cases, due to maternal anti-thyroid drugs. Hyperthyroidism was induced by transplacental transfer of thyroid stimulating antibodies (TSHrab). Accurate diagnosis of fetal thyroid status was obtained by fetal blood sampling but this invasive method was deemed necessary only in four cases as maternal clinical and biological data and ultrasound signs provided sufficient information to infer the type of thyroid disorder in the remaining patients. Fetal therapy relied on reduction of maternal antithyroid medication and, in selected cases, intra-amniotic injection of levothyroxin in hypothyroidism, and on administration of antithyroid drugs in hyperthyroidism. All newborns were healthy and none displayed consequences of severe thyroid imbalance. No caesarean section was performed for dystocia. Fetal thyroid goitres can be managed successfully with selected use of invasive diagnostic and therapeutic techniques.


Subject(s)
Fetal Diseases/diagnostic imaging , Fetal Diseases/therapy , Goiter/embryology , Goiter/therapy , Ultrasonography, Prenatal , Female , Hospital Units , Humans , Hyperthyroidism/embryology , Hyperthyroidism/etiology , Hyperthyroidism/therapy , Hypothyroidism/embryology , Hypothyroidism/etiology , Hypothyroidism/therapy , Infant, Newborn , Pregnancy , Pregnancy Outcome , Pregnancy Trimester, Third
13.
Diabetologia ; 43(7): 881-9, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10952461

ABSTRACT

AIMS/HYPOTHESIS: Glutamic acid decarboxylase 65 is a major autoantigen in Type I (insulin-dependent) diabetes mellitus, autoimmune polyendocrine syndrome and stiff-man syndrome. These disorders are characterised by the presence of multiple autoantibodies to the autoantigen which can be distinguished in a variety of different ways. We have investigated the role of single amino-acid mutations in glutamic acid decarboxylase 65 in distinguishing the binding of serum antibodies and a variety of patient-derived human IgG monoclonal antibodies directed to different determinants of the autoantigen. METHODS: We identified a mutant of glutamic acid decarboxylase 65 that contained four single amino-acid mutations from the wild-type molecule. The role of these mutations was investigated by site-directed mutagenesis. We investigated the binding of patient-derived serum antibodies to glutamic acid decarboxylase 65 to a number of single and double amino-acid mutants using immunoprecipitation with labelled, recombinant antigen. To overcome the heterogeneity of different anti-glutamic acid decarboxylase 65 antibodies present in a patient's serum, the binding of a panel of eleven patient-derived human monoclonal antibodies recognising different determinants on the autoantigen was also studied. RESULTS: Two replacements in glutamic acid decarboxylase 65 at Asn247Ser and Leu574Pro were identified that preferentially influence the anti-glutamic acid decarboxylase 65 serum antibodies of Type I diabetic patients, without statistically significantly effecting those recognised in other disorders. Single or double amino-acid replacements Asn247Ser and Leu574Pro in the autoantigen showed differential affects on expression of epitopes recognised by the human monoclonals. The double replacement of Asn247Ser and Leu574Pro in glutamic acid decarboxylase 65 resulted in the loss of binding of all eleven human monoclonal antibodies, irrespective of their epitope recognition. In contrast, single replacement of Leu574Pro statistically significantly reduced the binding of some carboxyl terminal-directed antibodies such as MICA 1, MICA 3 and DP-A without influencing the binding of other monoclonals. Replacement of Asn247Ser did not, however, influence the binding of any patients serum or human monoclonal antibodies. CONCLUSION/INTERPRETATION: Two distantly spaced amino acids, Asn247 and Leu574 in glutamic acid decarboxylase 65 were identified that act in concert to greatly influence the conformational structure of the autoantigen and statistically significantly influence the binding of antibodies present in Type I diabetic sera. The single or double amino-acid mutants can be used to distinguish some anti-glutamic acid decarboxylase-65 autoantibodies and could prove useful in distinguishing Type I diabetic from autoimmune polyendocrine syndrome and stiff-man syndrome patients' sera as well as to study changes in antibody patterns during disease progression.


Subject(s)
Autoantibodies/blood , Diabetes Mellitus, Type 2/immunology , Glutamate Decarboxylase/chemistry , Glutamate Decarboxylase/immunology , Isoenzymes/chemistry , Isoenzymes/immunology , Amino Acid Sequence , Amino Acid Substitution , Antibodies, Monoclonal/chemistry , Autoantibodies/chemistry , Base Sequence , Binding Sites, Antibody , DNA Primers , Diabetes Mellitus, Type 2/blood , Glutamate Decarboxylase/genetics , Humans , Immunoglobulin G/blood , Immunoglobulin G/chemistry , Isoenzymes/genetics , Mutagenesis, Site-Directed , Point Mutation , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/immunology
14.
J Clin Endocrinol Metab ; 84(7): 2419-24, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10404814

ABSTRACT

Antigenic proliferative responses of peripheral blood mononuclear cells (PBMC) to insulin were studied in 44 type 1 new-onset diabetic subjects. Of them, 14 (32%) had a stimulation index (> or =3) above the mean + 3 SD of 39 healthy controls and of 7 of 15 (47%) diabetic patients of long duration (P = 0.001). Responses to insulin were not dictated by specific major histocompatibility complex class II association and were not observed in normal subjects with diabetes-associated human leukocyte antigen-DR/DQ alleles. Whereas no relation of PBMC reactivity with insulin autoantibodies was found, there was a positive correlation with the presence of at least one of the four autoantibodies tested and with IA-2 antibody. An interesting finding was that the proportion of patients with subsequent low insulin requirement, up to 24 months, was significantly higher in patients who showed PBMC reactivity to insulin (8 of 8) than in those who did not (10 of 24, 42%; P = 0.004). The former had a higher mean stimulation index than the latter (3.3+/-2.6 vs. 1.5+/-0.6; P = 0.006). Furthermore, interleukin-4 (IL-4) production was lower in type 1 diabetic patients who proliferated to insulin than in those who did not (23+/-15 vs. 64+/-47 pg/mL; P = 0.04), but interferon-gamma, IL-2, and IL-10 productions were similar. In conclusion, these results suggest that proliferation to insulin may reflect the presence of an higher residual beta-cell mass.


Subject(s)
Cytokines/biosynthesis , Diabetes Mellitus, Type 1/blood , Insulin/pharmacology , Remission Induction , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Adolescent , Adult , Autoantibodies/blood , Cells, Cultured , Child , Diabetes Mellitus, Type 1/drug therapy , Diabetes Mellitus, Type 1/immunology , Female , HLA-DR3 Antigen/analysis , HLA-DR4 Antigen/analysis , Humans , Insulin/administration & dosage , Insulin/therapeutic use , Lymphocyte Activation/drug effects , Male
15.
Clin Exp Immunol ; 117(1): 30-7, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10403912

ABSTRACT

Autoantibodies and autoreactive T lymphocytes directed against several pancreatic beta cell proteins such as GAD65 have been identified in the circulation before and at the onset of clinical type 1 (insulin-dependent) diabetes. Using GAD65 synthetic peptides, we studied the proliferative response of peripheral blood mononuclear cells (PBMC) either from recently diagnosed type 1 diabetic patients, of whom the majority share the disease-associated HLA class II haplotype (DR4-DQB1*0201 or DR3-DQB1*0302), or from HLA-matched control subjects. We found that 67% (14/21) of the type 1 diabetic patients and 39% (9/23) of the control subjects exhibited a positive proliferative response. Compared with control subjects, however, PBMC from diabetic patients proliferated more frequently (P < 0.05) in the presence of peptide pools from the C-terminal region of GAD65 (amino acids 379-585). Diabetic patients with the same HLA-DQ or HLA-DR alleles showed partially identical T cell reactivity, but no clear correlation could be made between MHC class II specificity and T cell epitopes because of multiple combinations of class II alleles. In addition, by flow cytometry, we studied the direct binding of GAD65 peptides to MHC class II molecules of Epstein-Barr virus (EBV)-transformed B (EBV-B) cells obtained from a diabetic patient. We found that 11 GAD peptides were able to bind to the highly susceptible haplotype DRB1*0301/0401-DQA1*0301/0501-DQB1*0302/0201 on the surface of EBV-B cells in partial correlation with the results obtained in the proliferation assays.


Subject(s)
Autoantigens/immunology , Diabetes Mellitus, Type 1/immunology , Glutamate Decarboxylase/immunology , HLA Antigens/immunology , Islets of Langerhans/immunology , Lymphocyte Activation , Protein Isoforms/immunology , T-Lymphocytes/immunology , Adolescent , Adult , Amino Acid Sequence , Autoantibodies/immunology , Autoantigens/genetics , B-Lymphocytes/immunology , B-Lymphocytes/virology , Cell Division , Cell Line, Transformed , Child , Diabetes Mellitus, Type 1/genetics , Female , Genetic Predisposition to Disease , HLA Antigens/genetics , Haplotypes/genetics , Haplotypes/immunology , Herpesvirus 4, Human/immunology , Humans , Infant , Islets of Langerhans/enzymology , Male , Molecular Sequence Data , Peptide Fragments/genetics , Peptide Fragments/immunology
16.
J Mol Biol ; 287(5): 983-99, 1999 Apr 16.
Article in English | MEDLINE | ID: mdl-10222205

ABSTRACT

The smaller isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase 65 (GAD65), is unusually susceptible to becoming a target of autoimmunity affecting its major sites of expression, GABA-ergic neurons and pancreatic beta-cells. In contrast, a highly homologous isoform, GAD67, is not an autoantigen. We used homolog-scanning mutagenesis to identify GAD65-specific amino acid residues which form autoreactive B-cell epitopes in this molecule. Detailed mapping of 13 conformational epitopes, recognized by human monoclonal antibodies derived from patients, together with two and three-dimensional structure prediction led to a model of the GAD65 dimer. GAD65 has structural similarities to ornithine decarboxylase in the pyridoxal-5'-phosphate-binding middle domain (residues 201-460) and to dialkylglycine decarboxylase in the C-terminal domain (residues 461-585). Six distinct conformational and one linear epitopes cluster on the hydrophilic face of three amphipathic alpha-helices in exons 14-16 in the C-terminal domain. Two of those epitopes also require amino acids in exon 4 in the N-terminal domain. Two distinct epitopes reside entirely in the N-terminal domain. In the middle domain, four distinct conformational epitopes cluster on a charged patch formed by amino acids from three alpha-helices away from the active site, and a fifth epitope resides at the back of the pyridoxal 5'-phosphate binding site and involves amino acid residues in exons 6 and 11-12. The epitopes localize to multiple hydrophilic patches, several of which also harbor DR*0401-restricted T-cell epitopes, and cover most of the surface of the protein. The results reveal a remarkable spectrum of human autoreactivity to GAD65, targeting almost the entire surface, and suggest that native folded GAD65 is the immunogen for autoreactive B-cells.


Subject(s)
Epitope Mapping/methods , Glutamate Decarboxylase/chemistry , Glutamate Decarboxylase/immunology , Amino Acid Sequence , Antibodies, Monoclonal/metabolism , Binding Sites , Glutamate Decarboxylase/genetics , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/immunology , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid
17.
Immunol Lett ; 62(3): 123-30, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9698109

ABSTRACT

To study systematically the linear epitope specificity of anti-glutamic acid decarboxylase (GAD) autoantibodies associated with insulin-dependent diabetes mellitus (IDDM), we produced 93 overlapping 12-residue synthetic peptides derived from the sequence of the human GAD65 protein and covering the entire length of the protein. These peptides were used as antigens in an enzyme immunoassay to screen the sera from 10 IDDM patients, all of which contained at high level autoantibodies directed against GAD65. Three out of ten (30%) IDDM patients had antibodies that reacted with one or more of the synthetic peptides. Two of the peptide-reactive IDDM sera, which also bound denatured recombinant GAD65 on western blots, had the highest titers of anti-GAD antibodies in ELISA assay. Moreover, the anti-GAD antibodies-GAD complexes formed with these sera were characterized by low dissociation rates, indicative of their good stability. A fine specificity analysis, using analogs of antigen peptide 1 (residues 1-12), allowed us to identify the residues at positions 5-9 (GSGFW) as critical for antibody recognition.


Subject(s)
Antibody Specificity/immunology , Autoantibodies/immunology , Diabetes Mellitus, Type 1/immunology , Epitopes, B-Lymphocyte/immunology , Glutamate Decarboxylase/immunology , Diabetes Mellitus, Type 1/blood , Enzyme-Linked Immunosorbent Assay , Humans , Peptides/chemical synthesis , Peptides/immunology , Spectrum Analysis , Titrimetry
18.
J Clin Endocrinol Metab ; 81(7): 2595-600, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8675583

ABSTRACT

Thyroid antibody-dependent cell-mediated cytotoxicity (ADCC) has been reported in autoimmune thyroid disease, and its relationship with antithyroperoxidase antibodies (TPOAb) questioned. We studied the effect of highly purified human thyroperoxidase (TPO) on thyroid ADCC activity elicited by serum from patients with autoimmune thyroid disease. ADCC promoted by a pool of Graves' disease sera could be inhibited by the addition of TPO in a dose-dependent manner. TPO at 40 micrograms/mL decreased the ADCC observed in the presence of this serum pool by 50%. In the presence of 40 micrograms/mL TPO, ADCC was significantly reduced (P < 0.0005) from 39.6 +/- 10.6% (mean +/- SD) to 14.0 +/- 12.9% for the 18 Graves' disease sera tested and from 39.1 +/- 10.5% to 6.1 +/- 1.7% for the 16 thyroiditis sera tested. Purified thyroglobulin had no effect. Immunoaffinity-purified TPOAb could mediate ADCC in a dose-dependent manner, whereas purified antithyroglobulin antibodies could not. Three TPOAb-positive, but ADCC-negative, sera appear to contain an activity able to protect thyroid cells from ADCC. This protective effect is also observed on human fibroblasts. In conclusion, TPO is the major antigen involved in thyroid ADCC.


Subject(s)
Antibody-Dependent Cell Cytotoxicity , Autoantibodies/blood , Graves Disease/immunology , Iodide Peroxidase/immunology , Thyroiditis, Autoimmune/immunology , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged
19.
J Immunol ; 156(9): 3541-9, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8617984

ABSTRACT

The selective destruction by an autoimmune process of the beta cells in the pancreas is the hallmark of the type 1 insulin-dependent diabetes mellitus. What triggers islet cell-specific autoreactive T and B cells, however, remains unclear. Identification of the targets of the anti-islet cell autoantibodies frequently found in insulin-dependent diabetes mellitus patients and analysis of their sequences should provide some insights into the nature of this disease. We have combined EBV transformation with CD40 activation of peripheral B cells from one patient with insulin-dependent diabetes mellitus to isolate four B cell clones that secrete islet cell-specific autoantibodies, These four human monoclonal autoantibodies are of the IgG1 isotype, and they each recognize a different epitope of the glutamic acid decarboxylase enzyme. Analysis of their variable gene sequences shows that, while clonally unrelated, three of the four human monoclonal autoantibodies use a member of the VH4 family, and two have rearranged the same delta light chain variable gene. The IgG1 isotype of the four autoantibodies as well as the presence of somatic mutations in both heavy and light chain genes provide concrete evidence for their derivation by a T cell-dependent immune response.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Diabetes Mellitus, Type 1/immunology , Glutamate Decarboxylase/immunology , Immunodominant Epitopes/immunology , Immunoglobulin G/isolation & purification , Islets of Langerhans/immunology , Mutation/immunology , Adult , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Autoantigens/immunology , B-Lymphocytes/immunology , Base Sequence , Cell Line , Diabetes Mellitus, Type 1/genetics , Female , Humans , Immunoglobulin G/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/isolation & purification , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/isolation & purification , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/isolation & purification , Islets of Langerhans/metabolism , Molecular Sequence Data
20.
Rev Prat ; 44(9): 1184-91, 1994 May 01.
Article in French | MEDLINE | ID: mdl-7524135

ABSTRACT

Since the description of the structure of the TSH receptor using molecular biology techniques, it has become possible to analyse the role of anomalies of this receptor in thyroid disorders. Implicated in the pathophysiology of Graves' disease by indirect observations, the autoantigenic role of the TSH receptor has now been clearly confirmed. Nevertheless, the epitopes of the extracellular domain of the receptor corresponding to each type, stimulatory or epitopes of the extracellular domain of the receptor corresponding to each type, stimulatory or blocking, of anti-receptor toward activation or blocking. The events that induce and maintain autoimmunization to the receptor remain hypothetical, but the possible existence of soluble forms of the receptor opens new perspectives. In practice, however, assessment of TSH anti-receptor antibodies is useful in managing Graves' disease and in certain cases of primary myxoedema linked to the presence of blocking antibodies; it is mandatory in pregnant women for detection of foetal disease induced by maternal antibodies. The responsibility of the receptor is sought in other thyroid diseases such as toxic adenoma, rare forms of nonimmunologic, familial hyperthyroidism, simple goiter, nodules and lack of response to TSH. Recently, a mutation mapped into the 3rd intracellular loop has been shown in toxic adenoma. Such discoveries are as important for physicians (and patients) as for cellular biologists.


Subject(s)
Autoimmune Diseases/immunology , Receptors, Thyrotropin/physiology , Thyroid Diseases/immunology , Autoantibodies/immunology , Autoimmune Diseases/etiology , Epitopes/analysis , Humans , Receptors, Thyrotropin/immunology , Thyroid Diseases/etiology
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