Introduction: Type 1 Diabetes is the autoimmune form of diabetes mellitus and accounts for about 5-10% of all cases of diabetes. It is one of the most common severe chronic childhood illnesses characterized by insulin deficiency as a result of the progressive T-cell mediated destruction of pancreatic Langerhans islet - cells. The factors initiating the destructive process are largely unknown but genetic and nongenetic factors are involved. It manifests a biphasic progression with a preclinical phase “insulitis” and a clinical phase “diabetes onset”. The preclinical phase is also characterized by the presence of circulating autoAbs targeted to a different, yet limited, series of molecules that are expressed specifically and unspecifically in the pancreatic islet -cells. To date, 5 are the known autoAbs: ICA (Islets Cell Ab), GADA (Glutammic Acid Decarboxylase 65kDa Ab), IA-2A (Tyrosin phosphatase-like insulinoma Ag 2 Ab), IAA (Insulin Ab), ZnT8A (Cation efflux Zinc Transporter 8 Ab). Search for T1D-autoAbs in patients with recent diagnosis of diabetes is utilized for diagnostic purposes, and in first-degree relatives as predictive markers of disease. The first type of autoAb, islet-cell autoantibody, “ICA” was described >35 years ago; however, the entire panel of T1D-autoAgs is not complete at the moment. For example, it has been recently (2007) identified a new major autoAg, ZnT8, that was found in 26% of T1D subjects previously classified as autoAbs-negative. Because up to 5% of children with the clinical features/diagnosis of T1D test negative for known autoAbs it is likely that other specific autoAbs/Ags remain to be identified. Aim: We set up a search for new autoantigens involved in T1D using a method that has to be considered innovative in its application to this organ-specific autoimmune disease, that we named immunoproteomic approach; in addition we developed a radioimmunobinding assay to the goal of detecting serum reactivity on a large-scale as validation of two new potential autoantigen molecules identified by immunoproteomic. Materials and Methods: We studied the autoantibody repertoire of subject with diabetes of early onset (1- 10 years of age), divided in different groups of sera: A1) Subjects with diabetes negative to all autoAbs (ICA, GADA, IA-2A, IAA, ZnT8A) and to the search for mutations in neonatal diabetes genes -INS, KCNJ11, ABCC8 and GCK- (5 sera); B) T1D subjects positive only to IAA (4 sera); C) T1D subjects positive only to ZnT8A (4sera) and D) T1D subjects positive only to GADA and IA-2A (8 sera). Sera from patients with diabetes due to insulin mutations were used as “negative” control (group E1). The reactivity of these sera was tested against cytoplasmic/membrane-enriched protein fraction from human pancreatic islets or from exocrine pancreas sera in order to exclude any source of contamination of pancreatic islets from this tissue. After bidimensional electrophoresis and classical Western Blot, images were acquired and spot detection/matching performed by Progenesis software. Spots revealed by control sera (group E1) were subtracted as “noise” in each categories of T1D-sera. For matching the reactivity between groups of sera we detected specific spots of each group and common spots to two or more groups of the type 1 diabetic patients. These spots were identified on the corresponding stain gel by Mass Spectrometry. Among identified protein spots of islets two molecules (IOH-X1 and IOH-X2) were chosen to be validated as new potential autoAgs with a “radioimmunobinding assay” in subjects with T1D developed early (≤10 years of age). We studied IOH-X1 in five constructs (ORF: aa 1-445; N-terminal: aa 1-125; Domain 2: aa 120-225; Central Fragment: aa 215-337 and C-terminal: aa 338-445), whereas IOH-X2 in three constructs (ORF: aa 1-165; N-terminal: aa1-80 and C-terminal: aa 75-165). ORF and selected constructs were amplified by PCR from corresponding human islet cDNA using specific coupled of primers. They were cloned in an eucariotic vector and then expressed in an in vitrocoupled transcription/translation reticulocyte lysate reaction in presence of a radioactive aminoacid (MetS35). The products purified on a sephadex column were used with human serum samples of diabetic and control patients in an immunoprecipitation assay and finally the radioactivity was valued by an appropriate instrument. Results: Matching revealed that among protein spots detected in pancreatic islet cytoplasmic/membraneenriched protein fraction, 10 were in common between negative to 5 autoAbs sera (group A1) and those IAA positive (group B), 24 were in common between group A1 and those ZnT8A positive sera (group C) and 14 spots were common to all three groups. We then proceeded to identifying these protein spots by MALDI MS/MS. Our initial analysis revealed the IOH-X1 protein among spots detected by all groups of T1D-sera and the IOH-X2 molecule among protein spots detected only by the negative to 5 autoAb sera group. Among potential autoantigens we also identified tubulins, a common spot by all three groups of sera, and the Protein disulfide isomerase/PDIA3, as a specific spot of ZnT8A positive sera group. Tubulins and PDI are known as uncommon antigens in T1D, already identified by others. To confirm protein identity of spots we performed a preliminary validation by Western Blot. We recognized with protein-specific primary antibodies the corresponding spots that were picked and identified on stain-gel by Maldi MS/MS as IOH-X1 and IOH-X2. At the same time, to confirm the robustness of our method, we set up the detection of a known autoAg spot by using GADA positive sera and subsequently a polyclonal anti-GAD65 antibody on the same nitrocellulose. In our preliminary study we tested only the ORF and the C-terminal construct of IOH-X1 protein whereas the others remain to be analysed. In the C-terminus assay, applied on 100 patients with type 1 diabetes, 100 controls (obese) and 100 children with coeliac disease, when the 99° percentile cut-off was utilized, we found that 24% patients with T1D and 9% patients with coeliac disease were positive to the assay. Conclusions: These results seem to indicate that our “immunoproteomic” method can detect known T1D autoantigens as well as it’s feasible for novel autoantigens identification. Further analysis remain to be performed to assess if these potential new biomarkers may be useful to improving the accuracy of T1D diagnosis in high risk patients and general population, and to provide new targets for tolerance induction strategies

(2010). Ricerca di nuovi autoantigeni nel diabete di tipo 1:immunoproteomica delle isole pancreatiche umane.

Ricerca di nuovi autoantigeni nel diabete di tipo 1:immunoproteomica delle isole pancreatiche umane

RUSSO, LUCIA
2010-01-01

Abstract

Introduction: Type 1 Diabetes is the autoimmune form of diabetes mellitus and accounts for about 5-10% of all cases of diabetes. It is one of the most common severe chronic childhood illnesses characterized by insulin deficiency as a result of the progressive T-cell mediated destruction of pancreatic Langerhans islet - cells. The factors initiating the destructive process are largely unknown but genetic and nongenetic factors are involved. It manifests a biphasic progression with a preclinical phase “insulitis” and a clinical phase “diabetes onset”. The preclinical phase is also characterized by the presence of circulating autoAbs targeted to a different, yet limited, series of molecules that are expressed specifically and unspecifically in the pancreatic islet -cells. To date, 5 are the known autoAbs: ICA (Islets Cell Ab), GADA (Glutammic Acid Decarboxylase 65kDa Ab), IA-2A (Tyrosin phosphatase-like insulinoma Ag 2 Ab), IAA (Insulin Ab), ZnT8A (Cation efflux Zinc Transporter 8 Ab). Search for T1D-autoAbs in patients with recent diagnosis of diabetes is utilized for diagnostic purposes, and in first-degree relatives as predictive markers of disease. The first type of autoAb, islet-cell autoantibody, “ICA” was described >35 years ago; however, the entire panel of T1D-autoAgs is not complete at the moment. For example, it has been recently (2007) identified a new major autoAg, ZnT8, that was found in 26% of T1D subjects previously classified as autoAbs-negative. Because up to 5% of children with the clinical features/diagnosis of T1D test negative for known autoAbs it is likely that other specific autoAbs/Ags remain to be identified. Aim: We set up a search for new autoantigens involved in T1D using a method that has to be considered innovative in its application to this organ-specific autoimmune disease, that we named immunoproteomic approach; in addition we developed a radioimmunobinding assay to the goal of detecting serum reactivity on a large-scale as validation of two new potential autoantigen molecules identified by immunoproteomic. Materials and Methods: We studied the autoantibody repertoire of subject with diabetes of early onset (1- 10 years of age), divided in different groups of sera: A1) Subjects with diabetes negative to all autoAbs (ICA, GADA, IA-2A, IAA, ZnT8A) and to the search for mutations in neonatal diabetes genes -INS, KCNJ11, ABCC8 and GCK- (5 sera); B) T1D subjects positive only to IAA (4 sera); C) T1D subjects positive only to ZnT8A (4sera) and D) T1D subjects positive only to GADA and IA-2A (8 sera). Sera from patients with diabetes due to insulin mutations were used as “negative” control (group E1). The reactivity of these sera was tested against cytoplasmic/membrane-enriched protein fraction from human pancreatic islets or from exocrine pancreas sera in order to exclude any source of contamination of pancreatic islets from this tissue. After bidimensional electrophoresis and classical Western Blot, images were acquired and spot detection/matching performed by Progenesis software. Spots revealed by control sera (group E1) were subtracted as “noise” in each categories of T1D-sera. For matching the reactivity between groups of sera we detected specific spots of each group and common spots to two or more groups of the type 1 diabetic patients. These spots were identified on the corresponding stain gel by Mass Spectrometry. Among identified protein spots of islets two molecules (IOH-X1 and IOH-X2) were chosen to be validated as new potential autoAgs with a “radioimmunobinding assay” in subjects with T1D developed early (≤10 years of age). We studied IOH-X1 in five constructs (ORF: aa 1-445; N-terminal: aa 1-125; Domain 2: aa 120-225; Central Fragment: aa 215-337 and C-terminal: aa 338-445), whereas IOH-X2 in three constructs (ORF: aa 1-165; N-terminal: aa1-80 and C-terminal: aa 75-165). ORF and selected constructs were amplified by PCR from corresponding human islet cDNA using specific coupled of primers. They were cloned in an eucariotic vector and then expressed in an in vitrocoupled transcription/translation reticulocyte lysate reaction in presence of a radioactive aminoacid (MetS35). The products purified on a sephadex column were used with human serum samples of diabetic and control patients in an immunoprecipitation assay and finally the radioactivity was valued by an appropriate instrument. Results: Matching revealed that among protein spots detected in pancreatic islet cytoplasmic/membraneenriched protein fraction, 10 were in common between negative to 5 autoAbs sera (group A1) and those IAA positive (group B), 24 were in common between group A1 and those ZnT8A positive sera (group C) and 14 spots were common to all three groups. We then proceeded to identifying these protein spots by MALDI MS/MS. Our initial analysis revealed the IOH-X1 protein among spots detected by all groups of T1D-sera and the IOH-X2 molecule among protein spots detected only by the negative to 5 autoAb sera group. Among potential autoantigens we also identified tubulins, a common spot by all three groups of sera, and the Protein disulfide isomerase/PDIA3, as a specific spot of ZnT8A positive sera group. Tubulins and PDI are known as uncommon antigens in T1D, already identified by others. To confirm protein identity of spots we performed a preliminary validation by Western Blot. We recognized with protein-specific primary antibodies the corresponding spots that were picked and identified on stain-gel by Maldi MS/MS as IOH-X1 and IOH-X2. At the same time, to confirm the robustness of our method, we set up the detection of a known autoAg spot by using GADA positive sera and subsequently a polyclonal anti-GAD65 antibody on the same nitrocellulose. In our preliminary study we tested only the ORF and the C-terminal construct of IOH-X1 protein whereas the others remain to be analysed. In the C-terminus assay, applied on 100 patients with type 1 diabetes, 100 controls (obese) and 100 children with coeliac disease, when the 99° percentile cut-off was utilized, we found that 24% patients with T1D and 9% patients with coeliac disease were positive to the assay. Conclusions: These results seem to indicate that our “immunoproteomic” method can detect known T1D autoantigens as well as it’s feasible for novel autoantigens identification. Further analysis remain to be performed to assess if these potential new biomarkers may be useful to improving the accuracy of T1D diagnosis in high risk patients and general population, and to provide new targets for tolerance induction strategies
2010
2010/2011
Immunologia e biotecnologie applicate
23.
Settore MED/05 - PATOLOGIA CLINICA
Italian
Tesi di dottorato
(2010). Ricerca di nuovi autoantigeni nel diabete di tipo 1:immunoproteomica delle isole pancreatiche umane.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/209587
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