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The repertoire of known auto-antigens is limited to a very small proportion of all human proteins, and the reason why only some proteins become auto-antigens is unclear. The 65 kDa isoform of the enzyme glutamic acid decarboxylase (GAD-65) is a major auto-antigen in type I diabetes, and in various neurological diseases. Most patients with type I diabetes (70-80%) have auto-antibodies against GAD-65, which often appear years before clinical onset of the autoimmune diabetes. Thus, the aim of the study is to focus on the immunogenicity of GAD65 and its reactive oxygen species (ROS) conformer in STZ-induced diabetic rats and on human diabetic patients. In the present study, GAD-65 was modified by hydroxyl radical following Fenton's reaction. The modifications in the structure of the GAD-65 are supported by UV-vis and fluorescence spectral studies. Immunogenicity of both native and hydroxyl radical modified GAD-65 (ROS-GAD-65) was studied in experimental rabbits and was confirmed by inducing type I diabetes in experimental male albino rats using streptozotocin (45 mg/kg). We found that ROS-GAD-65 was a better immunogen as compared to the native GAD-65. A considerable high binding to ROS-GAD-65 was observed as compared to native GAD-65 in both the serum antibodies from diabetes animal models and as well as in the serum samples of type I diabetes. Hydrogen peroxide under the exposure of UV light produces hydroxyl radical (·OH) which is most potent oxidant, and could cause protein damage (GAD-65) to the extent of generating neo-epitopes on the molecule, thus making it immunogenic. © 2015 IUBMB Life, 67(10):746-756, 2015 [ABSTRACT FROM AUTHOR]

4-Aminobiphenyl (4-ABP), an aromatic amine is a major environmental carcinogen found mainly in cigarette smoke. It has been vastly implicated in mutagenesis and cancer development. In this study, commercially available human placental DNA was exposed to 4-ABP (1.3 mM) in presence of sodium nitroprusside (SNP; 8 mM) at 37°C for 3 h. The 4-ABP + SNP-mediated structural changes in human DNA were studied by ultraviolet, circular dichroism and fluorescence spectroscopy, thermal melting profile, agarose gel electrophoresis, and nuclease S1 digestibility assay. Spectroscopical analysis and melting temperature studies suggest structural perturbations in the DNA as a result of modification. This might be due to generation of single-stranded regions and destabilization of hydrogen bonds. Modification was also visualized in agarose gel electrophoresis. Furthermore, nuclease S1 digestibility confirmed the generation of single strand breaks. Rabbits challenged with 4-ABP-SNP-modified human DNA-induced high-titer immunogen-specific antibodies, which showed Cross-reaction with modified/unmodified DNA bases and ss-DNA in competitive inhibition assay. The immunogen specificity of induced antibodies against 4-ABP-SNP-modified human DNA was further confirmed in gel retardation assay. It may be concluded that induction of anti-modified DNA antibodies could be due to perturbation in the DNA structure and its subsequent recognition by immunoregulatory cells as a foreign molecule. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 568-576, 2014. [ABSTRACT FROM AUTHOR]

Abstract: The highly reactive electrophile, methylglyoxal (MG), a break down product of carbohydrates, is a major environmental mutagen having potential genotoxic effects. Previous studies have suggested the reaction of MG with free amino groups of proteins forming advanced glycation end products (AGEs). This results in the generation of free radicals which play an important role in pathophysiology of aging and diabetic complications. MG also reacts with free amino group of nucleic acids resulting in the formation of DNA–AGEs. While the formation of nucleoside AGEs has been demonstrated previously, no extensive studies have been performed to assess the genotoxicity and immunogenicity of DNA–AGEs. In this study we report both the genotoxicity and immunogenicity of AGEs formed by MG–Lys–Cu2+ system. Genotoxicity of the experimentally generated AGEs was confirmed by comet-assay. Spectroscopical analysis and melting temperature studies suggest structural perturbations in the DNA as a result of modification. This might be due to generation of single-stranded regions and destabilization of hydrogen bonds. Immunogenicity of native and MG–Lys–Cu2+-DNA was probed in female rabbits. The modified DNA was highly immunogenic eliciting high titre immunogen specific antibodies, while the unmodified form was almost non-immunogenic. The results show structural perturbations in MG–Lys–Cu2+-DNA generating new epitopes that render the molecule immunogenic. [Copyright &y& Elsevier]

Glycation initiates with the non-enzymatic reaction of amino group of proteins and lipoproteins by carbonyl group of sugar moiety and intermediates of glycative stress such as methylglyoxal (MG). The initial glycation leads to the formation of early glycation products (Amadori products) which undergo rearrangement, cyclization and dehydration to form advanced glycation end products (AGEs). The main objective of the present study is to investigate the non-enzymatic glycation of low density lipoprotein (LDL) by MG at different concentration and at increasing incubation time period in vitro . This modification may increase the formation of Amadori products and AGEs which are physico-chemically characterized with respect to the extent of LDL modification. Additionally, immunogenicity of native and MG modified LDL (MG-LDL) was probed in female rabbits in vivo . Immunogenicity of MG-LDL was found to be highly immunogenic, eliciting high titer immunogen-specific antibodies while native form of LDL is less immunogenic. Furthermore, the histopathology and immune-fluorescence studies suggest that the kidney section of immunized rabbits exhibit the presence of immune complex (MG-LDL-IgG) deposition in the glomerular basement membrane (GBM). [ABSTRACT FROM AUTHOR]

Glycation of biologically important macromolecules leads to the establishment of advanced glycation end products (AGEs) having significant role in the pathophysiology of various diseases. d -Ribose, is a highly reactive pentose sugar resulting in the rapid formation of AGEs. Formation of d -ribose derived glycated DNA and LDL has been previously demonstrated; however no comparative, extensive studies have been performed to assess the immunogenicity of d -ribose glycated calf thymus DNA (CT-DNA) and LDL. In the present study, the results showed that animals immunized with d -ribose modified CT-DNA and LDL induced antibodies as detected by direct binding and competition ELISA. The modified CT-DNA and LDL were found to be highly immunogenic, eliciting high titer immunogen-specific antibodies, while the native forms of DNA was almost non-immunogenic. The induced antibodies from modified CT-DNA and LDL exhibited wide range of heterogeneity in recognizing various nucleic acid conformers, DNA bases and amino acids. Furthermore, Serum antibodies from diabetes and diabetes atherosclerosis patients were screened for their binding to native CT-DNA, LDL and glycated CT-DNA, LDL. Glycated CT-DNA showed almost equivalent binding to both diabetes and diabetic atherosclerosis group while high recognition was observed when glycated LDL was used as an antigen. [ABSTRACT FROM AUTHOR]

In diabetes mellitus, hyperglycemia mediated non-enzymatic glycosylation of proteins results in the pathogenesis of diabetes-associated secondary complications via the generation of advanced glycation end products (AGEs). The focus of this study is to reveal the immunological aspects of methylglyoxal (MG) mediated glycation of fibrinogen protein. The induced immunogenicity of modified fibrinogen is analyzed by direct binding and inhibition ELISA. Direct binding ELISA confirmed that MG glycated fibrinogen (MG-Fib) is highly immunogenic and induces a high titer of antibodies in comparison to its native analog. Cross-reactivity and antigen-binding specificity of induced antibodies were confirmed by inhibition ELISA. The enhanced affinity of immunoglobulin G (IgG) from immunized rabbits' sera and MG glycated fibrinogen is probably the aftermath of neo-epitopes generation in the native structure of protein upon modification. Thus, we deduce that under the glycative stress, MG-mediated structural alterations in fibrinogen could induce the generation of antibodies which might serve as a potential biomarker in diabetes mellitus and its associated secondary disorders. • Non-enzymatic glycation is associated with the pathogenesis of diabetes mellitus. • The immunogenicity of modified fibrinogen is analyzed by direct binding and competitive/inhibition ELISA. • Direct binding ELISA infers that MG glycated fibrinogen is highly immunogenic and induces high titer of antibodies. • Cross reactivity and antigen binding specificity of induced antibodies were confirmed by competitive ELISA. [ABSTRACT FROM AUTHOR]

Abstract Non-enzymatic glycation occurs rapidly which ultimately leads to the formation of advanced glycation endproducts (AGEs). These AGEs have shown to associated with the development of many diseases such as diabetes-mellitus. This study is focused on immunological characterization of glycated-Hb induced by d -ribose. Here, we analysed the immunogenicity of glycated-Hb by direct binding and competitive inhibition ELISA. Direct binding ELISA confirmed that glycated-Hb was highly immunogenic and induced high titre antibodies as compared to native-Hb. The antigen binding specificity and cross reactivity of these antibodies were also screened by competitive inhibition ELISA. The IgG from rabbit sera showed enhanced binding of glycated-Hb than native-Hb. Thus, it is possible that alterations in Hb induced by d -ribose could have generated highly immunogenic neoepitopes. Moreover, induced antibodies were also found to cross-react with other modified/native proteins. On the basis of the results of this study, we presume that this type of structural perturbations in Hb in vivo by d -ribose might take place in untreated diabetic condition that could induce such type of immunogenic auto-antibodies. Furthermore, increased level of these auto-antibodies could serve as a biomarker in diabetes and its progression. [ABSTRACT FROM AUTHOR]

A nonenzymatic reaction between reducing sugars and amino groups of proteins results in the formation of advanced glycation end products, which are linked to a number of chronic progressive diseases with macro- and microvascular complications. In this research, we sought to ascertain the immunological response to d -ibose-glycated fibrinogen. New Zealand White female rabbits were immunized with native and d -ribose-glycated (Rb-gly-Fb) fibrinogen and used for studying the immunological response. Serum from these rabbits analyzed using direct binding and competitive inhibition ELISA was found to contain a high titer of antibodies against Rb-gly-Fb; Rb-gly-Fb was much more immunogenic than its native form. The IgG against Rb-gly-Fb (Rb-gly-Fb-IgG) was highly specific against the immunogenic protein. Moreover, histopathology and immunofluorescence studies revealed the deposition of the Rb-gly-Fb-IgG immune complex in the glomerular basement membrane of the kidneys of immunized rabbits. Furthermore, immunization with Rb-gly-Fb increased the expression of genes encoding proinflammatory cytokines, tumour necrosis factor α, interleukin-6, interleukin-1β, and interferon-gamma, which is indicative of increased inflammation and the antigenic role of Rb-gly-Fb in provoking an immune response. • Fibrinogen because of glycation results in the structural perturbations. • Fibrinogen protein becomes highly immunogenic after the glycation reaction. • Immunogenicity of the Fibrinogen is due to the formation of neo-epitopes in its structure. [ABSTRACT FROM AUTHOR]

Glyoxal, methylglyoxal and 3-deoxyglucosones are reactive dicarbonyl compounds, which transform free amino groups of proteins and lipoproteins macromolecule into advanced glycation end-products (AGEs). AGEs play a significant role in the pathophysiology of aging and diabetic complications because of their genotoxic effect. Glyoxal also reacts with free amino group of nucleic acids resulting in the formation of DNA–AGEs. The present study reports the genotoxicity and immunogenicity of AGEs formed by Glyoxal–Arginine–Fe 3+ (G–Arg–Fe 3+ ) system as a glycating agent. Immunogenicity of native and G–Arg–Fe 3+ –DNA was probed in female rabbits. Immunofluorescence suggests the presence of immune complex deposition in the kidney section of immunized rabbits. Spectroscopic analysis and melting temperature indicates the structural modification in the human DNA. The modified human DNA is found to be highly immunogenic, whereas unmodified form was simply non-immunogenic. This study shows the presence of auto-antibodies against G–Arg–Fe 3+ modified human DNA in the sera of diabetes type 1 and in few cases type 2 patients due to secondary complications of nephropathy. The glyco-oxidative lesions have also been detected in the lymphocyte DNA isolated from patients having type 1 and type 2 diabetes. The results show structural perturbations generating new epitopes in G–Arg–Fe 3+ –DNA rendering it pretty immunogenic. [ABSTRACT FROM AUTHOR]