Introduction Autoimmune diseases are often associated with increased production of reactive oxygen species (ROS). ROS may post-translationally modify proteins generating neoepitopes which may be a key aspect in the development of autoimmunity. Type 1 diabetes (T1D) and rheumatoid arthritis (RA) are autoimmune diseases that share some factors, such as association with the HLA-DRB1*04 and increased oxidative stress. My group has previously reported the presence of insulin oxidized by ROS (oxPTM-INS) in T1D patients and that antibodies to oxPTM-INS (oxPTM-INS-Ab) are detected in newly diagnosed T1D patients. My group has also reported an autoimmune reactivity against ROS modified type II collagen in rheumatoid arthritis patients (oxPTM-CII). Hypothesis This study is based on the hypothesis that ROS released during chronic inflammation are able to post-translationally modify proteins leading to the formation of neoepitopes. The main hypothesis is that these oxPTM play a key role in autoimmune response and thus influence the diseases pathobiology. Objectives The main objective of this study was to increase extend the understanding of oxPTM in autoimmunity and in arthritis. Specific aims: 1 - Testing the reactivity to oxPTM-INS in a longitudinal cohort of patients prior to diagnosis of T1D. 2 - Mapping oxPTM-INS neoepitopes. 3 - Testing reactivity to oxPTM-CII in large cohorts of patients with inflammatory arthritis. 4 - Mapping the oxPTM-CII neoepitopes. Methods Modification of CII and insulin were confirmed by PAGE and, in the case of insulin, mass spectrometry. Serum samples from subjects with inflammatory arthritis (including RA and spondyloarthritis) and T1D were used to evaluate the presence of oxPTM-CII-Ab and oxPTM-INS-Ab. The correlation between these antibodies with clinical, biochemical and HLA genotype was assessed. The epitope mapping for oxPTM-CII and oxPTM-CII was studied. Results 1 - Antibodies to at least one oxPTM-INS were present in over the 90% of children progressing to T1D (progr-T1D). •OH-INS-Ab were more common in progr-T1D children than in children non progressing to T1D positive for standard diabetes antibody marker (NP-AAB+) (82.6% vs 19%; p <0.001) and allowed discrimination between progr-T1D and NP-AAB+ children with 74% sensitivity and 91% specificity. None of the children non progressing to T1D negative for standard diabetes antibody marker NP-AAB- children were positive for oxPTM-INS-Ab. The comparison of plasma and serum from the same patients has confirmed all the previous data. In addition, we have shown that the correlation between plasma and serum analysis is very high. 2 - Mass spectrometry analysis confirmed previous data and has identified more amino acids modifications: cysteine oxidation and amino acid conversion. Comparing 2D gel-western blot analysis of insulin and oxPTM-INS performed using the most reactive serum samples from T1D patients was leading to the identifications of some interesting protein spots. 3 - In cohort 3 a stronger binding to oxPTM-CII was observed in serum samples from axSpA (74%) compare to PsA (33%), UA (35%) and FM (16%), (p <0.0001). Interestingly, binding of axSpA samples was similar to binding of serum samples from ERA (91.7%). Binding to ROS-CII was directed to a range of ROS-CII fragments between 25 and 150 kDa. In cohort 4 binding of AS serum samples to oxPTM-CII was significantly higher than binding to native CII (p <0.0001) with a percentage of autoreactivity of 30% to CII and 67.5% to at least one oxPTM-CII. 4 - Comparing 2D gel-western blot analysis of CII and oxPTM-CII performed using the most reactive serum samples from ERA patients was leading to the identifications of some interesting protein spots. Conclusions For the first time I have demonstrated reactivity to oxPTM-CII in axSpA patients and these data may support a further understanding on axSpA pathogenesis. Moreover, oxPTM-CII might mark diseased-tissue specific targets. I have shown that oxPTM-INS auto-reactivity is present before the diagnosis of T1D in over 90% of cases, for this reason oxPTM-INS-Ab may become a biomarker to predict children progressing to T1D and for early diagnosis of T1D.

Post-translational modification of relevant proteins in tissue-specific autoimmunity / Chiara Vinci - : . , 2018 Jul 18. ((30. ciclo

Post-translational modification of relevant proteins in tissue-specific autoimmunity

2018-07-18

Abstract

Introduction Autoimmune diseases are often associated with increased production of reactive oxygen species (ROS). ROS may post-translationally modify proteins generating neoepitopes which may be a key aspect in the development of autoimmunity. Type 1 diabetes (T1D) and rheumatoid arthritis (RA) are autoimmune diseases that share some factors, such as association with the HLA-DRB1*04 and increased oxidative stress. My group has previously reported the presence of insulin oxidized by ROS (oxPTM-INS) in T1D patients and that antibodies to oxPTM-INS (oxPTM-INS-Ab) are detected in newly diagnosed T1D patients. My group has also reported an autoimmune reactivity against ROS modified type II collagen in rheumatoid arthritis patients (oxPTM-CII). Hypothesis This study is based on the hypothesis that ROS released during chronic inflammation are able to post-translationally modify proteins leading to the formation of neoepitopes. The main hypothesis is that these oxPTM play a key role in autoimmune response and thus influence the diseases pathobiology. Objectives The main objective of this study was to increase extend the understanding of oxPTM in autoimmunity and in arthritis. Specific aims: 1 - Testing the reactivity to oxPTM-INS in a longitudinal cohort of patients prior to diagnosis of T1D. 2 - Mapping oxPTM-INS neoepitopes. 3 - Testing reactivity to oxPTM-CII in large cohorts of patients with inflammatory arthritis. 4 - Mapping the oxPTM-CII neoepitopes. Methods Modification of CII and insulin were confirmed by PAGE and, in the case of insulin, mass spectrometry. Serum samples from subjects with inflammatory arthritis (including RA and spondyloarthritis) and T1D were used to evaluate the presence of oxPTM-CII-Ab and oxPTM-INS-Ab. The correlation between these antibodies with clinical, biochemical and HLA genotype was assessed. The epitope mapping for oxPTM-CII and oxPTM-CII was studied. Results 1 - Antibodies to at least one oxPTM-INS were present in over the 90% of children progressing to T1D (progr-T1D). •OH-INS-Ab were more common in progr-T1D children than in children non progressing to T1D positive for standard diabetes antibody marker (NP-AAB+) (82.6% vs 19%; p <0.001) and allowed discrimination between progr-T1D and NP-AAB+ children with 74% sensitivity and 91% specificity. None of the children non progressing to T1D negative for standard diabetes antibody marker NP-AAB- children were positive for oxPTM-INS-Ab. The comparison of plasma and serum from the same patients has confirmed all the previous data. In addition, we have shown that the correlation between plasma and serum analysis is very high. 2 - Mass spectrometry analysis confirmed previous data and has identified more amino acids modifications: cysteine oxidation and amino acid conversion. Comparing 2D gel-western blot analysis of insulin and oxPTM-INS performed using the most reactive serum samples from T1D patients was leading to the identifications of some interesting protein spots. 3 - In cohort 3 a stronger binding to oxPTM-CII was observed in serum samples from axSpA (74%) compare to PsA (33%), UA (35%) and FM (16%), (p <0.0001). Interestingly, binding of axSpA samples was similar to binding of serum samples from ERA (91.7%). Binding to ROS-CII was directed to a range of ROS-CII fragments between 25 and 150 kDa. In cohort 4 binding of AS serum samples to oxPTM-CII was significantly higher than binding to native CII (p <0.0001) with a percentage of autoreactivity of 30% to CII and 67.5% to at least one oxPTM-CII. 4 - Comparing 2D gel-western blot analysis of CII and oxPTM-CII performed using the most reactive serum samples from ERA patients was leading to the identifications of some interesting protein spots. Conclusions For the first time I have demonstrated reactivity to oxPTM-CII in axSpA patients and these data may support a further understanding on axSpA pathogenesis. Moreover, oxPTM-CII might mark diseased-tissue specific targets. I have shown that oxPTM-INS auto-reactivity is present before the diagnosis of T1D in over 90% of cases, for this reason oxPTM-INS-Ab may become a biomarker to predict children progressing to T1D and for early diagnosis of T1D.
post-translational modification; autoimmunity; type 1 diabetes; spondyloarthritis
Post-translational modification of relevant proteins in tissue-specific autoimmunity / Chiara Vinci - : . , 2018 Jul 18. ((30. ciclo
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12610/68732
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