Sidra Islam

Immunochemical studies on HNE-modified HSA: Anti-HNE-HSA antibodies as a probe for HNE damaged albumin in SLE.

Non-enzymatic lipid peroxidation of cellular membranes occurs during periods of sustained oxidative stress. 4-Hydroxynonenal (HNE), the most reactive lipid peroxidation product, is capable of modifying and/or cross-linking proteins leading to impaired physiological functions. The formation of protein adducts produce structural modifications which generate neo-antigens and induce auto-antibodies. Enhanced oxidative stress and accumulation of HNE-modified proteins are associated with systemic lupus erythematosus (SLE) and other autoimmune diseases. This study has probed the role of lipid peroxidation derived aldehydes in SLE. We report the structural perturbations in human serum albumin (HSA) upon modification with HNE and the consequential enhanced immunogenicity. The induced antibodies were found to be highly specific for the immunogen and exhibited cross-reactivity with HNE-modified epitopes on proteins, amino acids and nucleic acid. The experimentally induced anti-HNE-HSA antibodies appreciably recognized HNE modified epitopes on the HSA obtained from SLE patients. These antibodies, therefore, form a good immunochemical probe to detect such damages in lupus patients. Possible role of anti-HNE-HSA antibodies as a marker for detection/progression of SLE has been discussed.

Circulating autoantibodies in cancer patients have high specificity for glycoxidation modified histone H2A.

BACKGROUND Novel immunological epitopes upon glyoxidation modified proteins have been discovered and multi-specific natural antibodies against them have been identified. The association of glyoxidation with cancer has also been reported. We probed the link of glyoxidation of histone H2A with autoimmune response in cancer. METHODS We report the formation of thermo stable amorphous aggregate formation in histone H2A upon methylglyoxal modification using circular dichroism (CD) analysis and transmission electron microscopy. RESULTS The modified histone was found to be highly immunogenic that generates specific immune response in rabbits as analysed by cross reaction studies by competitive ELISA and Western blotting technique. The anti-methylglyoxal modified H2A antibodies were found in the circulating autoantibodies in various types of cancer patients by immunosorbent assay and gel retardation studies. The results clearly indicate the formation of highly specific antibodies in cancer patients against glyoxidation modified histone H2A with cross reactive tendencies with other glycated proteins and nucleic acids. CONCLUSIONS The results are important because a link between AGE-RAGE axis (Advanced glycation end products and Receptors for AGEs) and carcinogenesis is emerging and the role of glyoxidation of proteins is expected in the development of biomarkers for the early detection of cancer.

Studies on glycoxidatively modified human IgG: Implications in immuno-pathology of type 2 diabetes mellitus

Structural rearrangements and condensations of proteins under hyperglycemic stress have been implicated in various pathological disorders. This study aims to probe the role of methylglyoxal (MG) modified human immunoglobulin G (MG-IgG) in immuno-pathology of type 2 diabetes mellitus (T2DM). MG was found to perturb the structural integrity of IgG, affect its aromatic micro-environment and cause the generation of advanced glycation end products (AGEs) and aggregate adducts. It liberated the hydrophobic pockets of the protein, reduced its β pleated sheet structure and affected its tertiary conformation. Transition from β sheet to α helix and random coil was also observed in IgG upon modification by MG. It acted with strong oxidative potential and caused oligomerisation and disordered or amorphous type aggregation in the modified protein. Modified IgG had a cytotoxic and genotoxic impact. The MG modified IgG presented novel antigenic determinants that lead to an aggressive immune response. The antibodies had high affinity towards the immunogen. Auto-antibodies derived from T2DM patients exhibited strong affinity towards the modified IgG in comparison to the unmodified protein. Specificity of serum antibodies from T2DM patients was further confirmed by competitive-inhibition ELISA. The potential role of MG-IgG in the immunopathogenesis of T2DM has been discussed.

Glycation, oxidation and glycoxidation of IgG: a biophysical, biochemical, immunological and hematological study

Glycation and oxidation induce structural alterations in the proteins in an interdependent manner with consequent pathological implications. The published literature presents wide range of modifications in conformational characteristics of proteins by glycation and oxidation; however, there is little data that could elaborate the cumulative effect of both the processes. This study has analysed the modifications in IgG by methylglyoxal (MG) (glycative stress), hydroxyl radical () (oxidative stress) and by their combined action i.e. treatment of MG glycated IgG (glycoxidation). It further addresses the implications of the altered structural integrity of IgG on its immunological characteristics and impact on haematological parameters in rabbits. Using circular dichroism, FTIR, SDS-PAGE analysis, thioflavin-T fluorescence assay, congo red absorbance analysis, dynamic light scattering, transmission electron microscopy, ELISA, blood cell counts and rectal temperature studies, we report that the glycoxidative modification caused maximum alteration in the IgG as compared to the glycatively and oxidatively modified protein. Far-UV CD results confirmed the highest decline in the beta-pleated sheet content of the protein by glycoxidation. The damage led to the reduced flexibility and enhanced electronic interactions in IgG as observed by near-UV CD. Modifications caused cross-linking and adduct formation in the serum protein. The electron micrograph confirmed amorphous aggregation in modified IgG. The modifications increased the hydrodynamic radius of IgG by allowing the attachment of and MG residues. The glycoxidatively modified IgG induced the maximum antibody titres that showed high specificity towards the altered IgG. The glycoxidation of IgG leads to activation of inflammatory pathways.

Neo-Epitopes Generated on Hydroxyl Radical Modified GlycatedIgG Have Role in Immunopathology of Diabetes Type 2

Glycoxidation plays a crucial role in diabetes and its associated complications. Among the glycoxidation agents, methylglyoxal (MG) is known to have very highglycationpotential witha concomitant generation of reactive oxygen species (ROS) during its synthesis and degradation. The presentstudy probes the MG and ROSinduced structural damage to immunoglobulin G (IgG) and alterations in its immunogenicity in diabetes type 2 patients (T2DM). Human IgG was first glycated with MG followed by hydroxyl radical (OH•) modification. Glycoxidation mediated effects on IgG were evaluated by various physicochemical techniques likeultraviolet (UV) and fluorescence spectroscopy, 8-anilinonaphthalene-1-sulfonic acid (ANS) binding studies, carbonyl andfree sulfhydryl groups assay, matrix assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF), red blood cell (RBC) haemolysis assay, Congored (CR) staining analysis and scanning electron microscopy (SEM). The results revealed hyperchromicityin UV, advanced glycation end product (AGE)specific and ANS fluorescence, quenching in tyrosine and tryptophan fluorescence intensity,enhanced carbonyl content,reduction in free sulfhydryl groups,pronounced shift in m/z value of IgGand decrease in antioxidant activity in RBC induced haemolysis assayupon glycoxidation. SEM and CRstaining assay showed highly altered surface morphology in glycoxidised sample as compared to the native. Enzyme linked immunosorbent assay (ELISA) and band shift assay were performed to assess the changes in immunogenicity of IgG upon glyoxidation and its role in T2DM. The serum antibodies derived from T2DM patients demonstrated strong affinity towards OH• treated MG glycatedIgG (OH•-MG-IgG) when compared to native IgG (N-IgG) or IgGs treated with MG alone (MG-IgG) or OH• alone (OH•-IgG). This study shows the cumulating effect of OH• on the glycation potential of MG. The results point towards the modification of IgG in diabetes patients under the effect of glycoxidative stress, leading to the generation of neo-epitopes on theIgG molecule and rendering it immunogenic.

Structural and immunological characterization of hydroxyl radical modified human IgG: Clinical correlation in rheumatoid arthritis

Structural alterations in proteins under oxidative stress have been widely implicated in the immuno-pathology of various disorders. This study has evaluated the extent of damage in the conformational characteristics of IgG by hydroxyl radical (OH) and studied its implications in the immuno-pathology of rheumatoid arthritis (RA). Using various biophysical and biochemical techniques, changes in aromatic microenvironment of the IgG and the protein aggregation became evident after treatment with OH. The SDS-PAGE study confirmed the protein aggregation while far ultraviolet circular dichroism spectroscopy (Far-UV CD) and fourier transform infrared spectroscopy (FTIR) inferred towards the alterations in secondary structure of IgG under OH stress. Dynamic light scattering showed that the modification increased the hydrodynamic radius and polydispersity of IgG. The free arginine and lysine content reduced upon modification. OH induced aggregation was confirmed by enhanced thioflavin-T (ThT) fluorescence and red shift in the congo red (CR) absorbance. The study on experimental animals reiterates the earlier findings of enhanced immunogenicity of OH treated IgG (OH-IgG) compared to that of native IgG. OH-IgG strongly interacted with the antibodies derived from the serum of 80 rheumatoid arthritis (RA) patients. The overwhelming and strong tendency of OH-IgG to bind the antibodies derived from the serum of RA patients points towards the modification of IgG under patho-physiological conditions in RA that generate neo-epitopes and eventually cause the generation of auto antibodies that circulate in the patient sera. Further studies on this aspect may possibly lead to the development of a biomarker for RA.

Methylglyoxal modified IgG generates autoimmune response in rheumatoid arthritis

The detection of autoantibodies generated against modified proteins that stimulate cellular and humoral immune response has developed a lot of interest in the recent years and a search for biomarkers for the early detection of diseases has increased. IgG protein has earned attention for its possible modifications under hyperglycaemic conditions in rheumatoid arthritis, wherein dicarbonyl stress has been reported to alter the structural integrity of the protein. This report suggests that the interaction of the methylglyoxal with the IgG has consequences in the autoimmunopathology of rheumatoid arthritis. Our molecular docking analysis of methylglyoxal and IgG revealed a close interaction between the two molecules. TNBS studies confirmed the interaction by showing a decline in free lysine-arginine content post-MG modifications in IgG. The modified IgG was thermally more stable and showed the generation of glycation adducts N-epsilon-carboxyethyllysine. Rheumatoid arthritis patients showed enhanced carbonyl stress which was expected to induce structural changes in the epitope makeup of IgG. The ELISA studies and gel retardation assay confirmed auto-antibodies against MG modified IgG (MG-IgG) pointing towards the generation of neoepitopes upon IgG after interaction with MG. This study establishes the IgG modification in RA patients under alter carbonyl concentrations.

Unsaturated aldehyde, 4-hydroxynonenal (HNE) alters the structural integrity of HSA with consequences in the immuno-pathology of rheumatoid arthritis

Human serum albumin (HSA) - the most abundant plasma protein plays an important role in the transport of endogenous and exogenous molecules in the body. Its modifications have been implicated in a variety of pathological disorders. We have studied the interaction of HNE with HSA at a molecular level by docking experiment and the results suggest a strong interaction between HNE and HSA. Immunological studies revealed that the circulating auto-antibodies in rheumatoid arthritis (RA) patients have a stronger affinity towards HNE-modified HSA. The HSA isolated from RA patients (RA-HSA) exhibited HNE mediated damage in its secondary and tertiary structure when compared to HSA derived from healthy human subjects (NH-HSA). RA patients presented a significant rise in carbonyls and a considerable decline in free thiol content. Preferential binding of experimentally induced anti-HNE-HSA antibodies to RA-HSA over NH-HSA was observed by ELISA. The results suggest HNE induced structural perturbations in HSA with neoepitopes that generate anti-HNE-HSA antibodies in RA. Hence, HNE-HSA may provide lead towards the development of a biomarker for the disease.

Physicochemical and immunological studies on hydroxylated IgG: Its possible role in the generation of autoantibodies in rheumatoid arthritis

Introduction & Aims: HPV testing is used as a means of triaging cervical smears with low grade squamous abnormalities or as part of co-testing with cytology. While HPV testing has a high sensitivity, it has a low specificity in detecting cervical intraepithelial neoplasia grade 2 and above (CIN 2+) leading to unnecessary colposcopy referrals. We investigate the accuracy of the p16/Ki-67 dual immunocytochemical stain in determining the presence of CIN 2+ lesions and its potential as a superior biomarker for triage.