Biologically Active α-Amino Amide Analogs and γδ T Cells-A Unique Anticancer Approach for Leukemia.

Advanced stage cancers are aggressive and difficult to treat with mono-therapeutics, substantially decreasing patient survival rates. Hence, there is an urgent need to develop unique therapeutic approaches to treat cancer with superior potency and efficacy. This study investigates a new approach to develop a potent combinational therapy to treat advanced stage leukemia. Biologically active α-amino amide analogs (RS)-N-(2-(cyclohexylamino)-2-oxo-1-phenylethyl)-N-phenylpropiolamide (α-AAA-A) and (RS)-N-(2-(cyclohexylamino)-2-oxo-1-phenylethyl)-N-phenylbut2-enamide (α-AAA-B) were synthesized using linear Ugi multicomponent reaction. Cytotoxicities and IC50 values of α-AAA-A and α-AAA-B against leukemia cancer cell lines (HL-60 and K562) were analyzed though MTT assay. Cytotoxic assay analyzed percent killing of leukemia cell lines due to the effect of γδ T cells alone or in combination with α-AAA-A or α-AAA-B. Synthesized biologically active molecule α-AAA-A exhibited increased cytotoxicity of HL-60 (54%) and K562 (44%) compared with α-AAA-B (44% and 36% respectively). Similarly, α-AAA-A showed low IC50 values for HL-60 (1.61 ± 0.11 µM) and K562 (3.01 ± 0.14 µM) compared to α-AAA-B (3.12 ± 0.15 µM and 6.21 ± 0.17 µM respectively). Additive effect of amide analogs and γδ T cells showed significantly high leukemia cancer cell killing as compared to γδ T cells alone. A unique combinational therapy with γδ T cells and biologically active anti-cancer molecules (α-AAA-A/B), concomitantly may be a promising cancer therapy.

Optimization of methods for peripheral blood mononuclear cells isolation and expansion of human gamma delta T cells.

Human Vg9/Vδ2 T cells (γδ T cells) are immune surveillance cells both in innate and adaptive immunity and are a possible target for anticancer therapies, which can induce immune responses in a variety of cancers. Small non-peptide antigens such as zoledronate can do activation and expansion of T cells in vitro. It is evident that for adoptive cancer therapies, large numbers of functional cells are needed into cancer patients. Hence, optimization of methods needs to be carried out for the efficient expansion of these T cells. Standardization of peripheral blood mononuclear cells (PBMCs) isolation was devised. Cytokines (interleukin 2 (IL-2) and interleukin 15 (IL-15)) and zoledronate were also standardized for different concentrations. It was found that an increased number of PBMCs were recovered when washing was done at 1100 revolution per minute (rpm). Significantly high expansion fold was (2524 ± 787 expansion fold) achieved when stimulation of PBMCs was done with 1 µM of zoledronate and both cytokines IL-2 and IL-15 supported the expansion and survival of cells at the concentrations of 100 IU/ml and 10 ng/ml respectively. 14-day cultures showed highly pure (91.6 ± 5.1%) and live (96.5 ± 2.5%) expanded γδ T cells. This study aimed to standardize an easy to manipulate technique for the expansion of γδ T cells, giving a higher yield.

Combinational therapeutics to combat cancer.

Mono-therapeutics is rarely effective as a treatment option, which limits the survival of patients in advanced grade aggressive cancers. Combinational therapeutics (multiple drugs for multiple targets) to combat cancer is gaining momentum in recent years. Hence, it is of interest to document known data for combinational therapeutics in cancer treatment. An amalgamation of therapeutic agents enhances the efficacy and potency of the therapy. Combinational therapy can potentially target multiple pathways that are necessary for the cancer cells to proliferate, and/or target molecules, which may help cancer to become more aggressive and metastasize. In this review, we discuss combinational therapeutics, which include human γδ T cells in combinations with biologically active anti-cancer molecules, which synergistically may produce promising combinational therapeutics.

Endometrial cancer patients have high affinity antibodies for estrogen metabolite-receptor aggregate: A potential biomarker for EC.

AIM: Elevated levels of 16α-hydroxyestrone (16α-OHE1 ) have been described in endometrial cancer (EC) and estrogen receptors (ER) expressed in endometrial tissue, but research on their combined role is lacking. We aimed to investigate the affinity and binding specificity of EC antibodies against the 16α-OHE1 -ERα aggregate in the serum of EC patients. Specificities of EC antibodies were also evaluated according to various clinical characteristics found in these cancer patients. METHODS: The binding specificity and affinity of EC antibodies against 16α-OHE1 -ERα in the serum of 120 EC patients were evaluated by direct binding and competition ELISA and quantitative precipitation titration. Binding of EC antibodies was also determined according to various clinical characteristics in EC patients through competition ELISA. RESULTS: Antibodies from EC patients demonstrated high recognition of 16α-OHE1 -ERα compared to ERα (P < 0.05) or 16α-OHE1 (P < 0.001). The relative affinity of EC IgG was 1.49 × 10-7 M, 1.34 × 10-6 M and 1.13 × 10-6 M for 16α-OHE1 -ERα, ERα and 16α-OHE1 , respectively. Several factors, such as obesity, postmenopausal status, use of hormonal therapy, ER and progesterone receptor (PR) status, low 2-OHE1 /16α-OHE1 ratio, chemotherapy and hypertension, augment the production of antibodies against 16α-OHE1 -ERα in EC patients. CONCLUSION: 16α-OHE1 -ERα is a high-affinity antigen for EC antibodies in the serum of EC patients and might function as a biomarker for this disease. Furthermore, several factors enhanced the production of antibodies against 16α-OHE1 -ERα in the sera of these EC patients.

Estrogenization of insulin by catecholestrogen produced high affinity autoantibodies and altered the normal function of insulin in type 1 diabetes.

AIMS: Insulin (Ins) covalently modified by catecholestrogens (CEs) was commonly found in diabetic patients who have developed insulin resistance. Estrogenization of insulin altered its molecular function and effect carbohydrates metabolisms in these patients. Insulin resistance is a common phenomenon in diabetes but the exact mechanism remains unknown. In this study, binding specificity and affinity of autoantibodies against estrogenized insulin (4-hydroxyestradiol-insulin; 4-OHE2-Ins) were assayed in the serum of type 1 diabetes (T1D) patients in order to explain the phenomena behind insulin resistance. MATERIALS AND METHODS: Specificity and affinity of autoantibodies from the sera of 66 T1D patients and 41 controls were analyzed by direct binding, competition ELISA and quantitative precipitin titration. Insulin was also estimated in the serum of T1D patients by ELISA. KEY FINDING: Estrogenized insulin (4-OHE2-Ins) exhibited high affinity and specificity to T1D autoantibodies in comparison to Ins (p < .05) or 4-OHE2 (p < .001). Estrogenization of insulin alters its interaction with the insulin receptor (IR). The affinity constant of 4-OHE2-Ins with the T1D autoantibodies was found to be 1.41 × 10-7 M. SIGNIFICANCE: Estrogenization of insulin by catecholestrogen makes these molecules highly antigenic and produced high-affinity autoantibodies in T1D patients. As a result, patients develop insulin resistance and presented this molecule as a potential biomarker for T1D.

Depression linked to higher antibodies production against estrogenized insulin in type 1 diabetes.

Depression has been commonly associated with type 1 diabetes (T1D) and insulin covalently modified with catecholestrogens (CEs) was found in serum of these T1D patients. This study aimed to know whether depression link to higher antibodies against estrogenized insulin in T1D. ELISA (direct binding and competition) and quantitative precipitin titration were used to detect antibodies and their affinities against estrogenized insulin in the serum of 66 depressed T1D (DT1D) patients (out of 110 T1D) and 41 control subjects. Antibodies from DT1D patients showed high binding specificity to estrogenized insulin (2-hydroestradiol-insulin; 2-OHE2-Ins) in comparison to overall T1D patients (p < 0.05) or control subjects (p < 0.001). However, T1D sera demonstrate high recognition to 2-OHE2-Ins as compared to Ins (p < 0.05) or 2-OHE2 (p < 0.001). The affinity of antibodies from DT1D and T1D patients was 1.32 × 10-7 M and 1.43 × 10-7 M, respectively. Depression linked to higher antibodies production against estrogenized insulin in T1D. Furthermore, depression in T1D generates inflammatory conditions that further increased antibodies production in T1D patients.

Depression and its related parameters increased the production of autoantibodies against 16α-hydroxyestrone-albumin complex in systemic lupus erythematosus.

Depression is the common and early symptoms associated with early onset of SLE, 16α-hydroxyestrone (16α-OHE1) levels were found to be significantly higher in serum and urine in patients with SLE. This study was carried out in order to know whether depression and its related parameters in the SLE patients enhanced the production of autoantibodies against 16α-OHE1-albumin (A) complexes. The autoantibodies in the serum of 100 SLE [including 65 depressed SLE (DSLE)] patients and 37 control subjects were detected by using direct binding, inhibition ELISA and quantitative precipitin titration. Autoantibodies from DSLE patients (and also the patients who were taken anti-depressant and with neurological symptoms) showed high binding to 16α-OHE1-A in contrast to SLE (p < 0.05) and control subjects (p < 0.001). Although, SLE sera showed high recognition to 16α-OHE1-A in comparison to A (p < 0.05) or 16α-OHE1 (p < 0.001). The affinity of autoantibodies for 16α-OHE1-A was found to be high for DSLE (1.16 × 10-7 M) and SLE (1.24 × 10-7 M) patients as detected by Langmuir plot. The concentration of 16α-OHE1 (p < 0.05) and inflammatory cytokines (IL-6, p < 0.05 and IL-17, p < 0.001) in the serum of SLE patients was found to be significantly higher than controls. Depression and its related parameters in SLE enhanced the production of autoantibodies against 16α-OHE1-A through the generation of inflammatory conditions. Depression in SLE patients increased the release of pro-inflammatory cytokine (IL-6 and IL-17) that in turn generating more autoantibodies and showed strong recognition to 16α-OHE1-A.

Depression enhanced the production of autoantibodies against 16α­hydroxyestrone-estrogen receptor adduct in breast cancer.

Mentally depressed breast cancer (MDBC) patients expressed estrogen receptor (ER) and 16α­hydroxyestrone (16α­OHE1) is directly responsible for causing breast cancer (BC). This study aimed to identify whether depression in breast cancer patients enhanced the production of autoantibodies against 16α­OHE1-ER adduct in breast cancer patients. The antibodies in the serum of 65 breast cancer patients (including 35 MDBC) and 40 control subjects were screened by direct binding, inhibition enzyme-linked immunosorbent assay (ELISA), and quantitative precipitin titration. Competition ELISA was also utilized for the estimation of 16α­OHE1 in the serum of 30 cancer patients. Autoantibodies from MDBC showed strong recognition to 16α­OHE1-ER in comparison to overall breast cancer patients (p < 0.05) and control subjects (p < 0.001). Although breast cancer sera showed high binding to 16α­OHE1-ER in comparison to ER (p < 0.05) or 16α­OHE1 (p < 0.001), the relative affinities of autoantibodies for 16α­OHE1-ER were found to be 1.38 × 10-7 and 1.23 × 10-7 for breast cancer and MDBC patients respectively. No significant difference, either in the level of 16α­OHE1 or 2­hydroxyestrone/16α­OHE1 ratio, was observed in the serum of cancer patients compared with controls, although inflammatory cytokines (IL-6, TNF-α) were significantly high in these patients. Depression in breast cancer patients augments the production of autoantibodies against 16α­OHE1-ER through the generation of inflammatory conditions. Depression in these patients increased the release of pro-inflammatory cytokines that generate more autoantibodies and show strong binding with 16α­OHE1-ER.

16 α-Hydroxyestrone induced adduct generate high affinity autoantibodies in SLE.

PURPOSE: Increased 16 α-hydroxyestrone (16 α-OHE1) and autoantibodies against histone H1 (H1) have been well described in systemic lupus erythematosus (SLE), but the combination effects of 16 α-OHE1 and H1 remains unclear. Here, we tried to assess the affinity and binding specificity of SLE autoantibodies against the 16 α-OHE1-H1 adduct. IgG was induced against this adduct and was also used as immunochemical probe for the estimation of 16 α-OHE1 in the serum of SLE patients. MATERIALS AND METHODS: The affinity and binding specificities of SLE autoantibodies against 16 α-OHE1-H1 were determined by direct binding and inhibition ELISA as well as quantitative precipitation titration in 60 patients and 30 control subjects. RESULTS: Purified SLE autoantibodies showed greater recognition to 16 α-OHE1-H1 over H1 (p < 0.05) or 16 α-OHE1 (p < 0.001). The relative affinity of SLE autoantibodies for 16 α-OHE1-H1, H1 and 16 α-OHE1 was 1.41 × 10-7, 1.31 × 10-6, and 1.03 × 10-6, respectively. The concentration of 16 α-OHE1 in the sera of SLE patients was significantly higher than controls (p < 0.05) as estimated by anti-16 α-OHE1-H1 antibodies. CONCLUSIONS: High affinity of 16 α-OHE1-H1 with SLE autoantibodies might suggest an antigenic role of this adduct in the production of these autoantibodies. The anti-16 α-OHE1-H1 antibody is a good immunochemical probe to measure 16 α-OHE1 in different SLE sera.

Detection of 16α-Hydroxyestrone-histone 1 Adduct as High-Affinity Antigen for Rheumatoid Arthritis Autoantibodies.

Increased concentrations of 16α-hydroxyestrone (16α-OHE1) have been observed in rheumatoid arthritis (RA), but the underlying mechanism of this remains elusive. Here we aimed to identify the role played by 16α-OHE1 in RA. In 40 RA patients, the specificities of antibodies from the sera of these patients were checked by direct binding, inhibition ELISA, and quantitative precipitation titration. Competition ELISA was also used for the estimation of 16α-OHE1 in the serum of different RA patients. RA IgG from a patient's sera showed strong recognition to 16α-OHE1-H1 (histone 1) adduct in comparison to control subjects (p < 0.001), as the formation of this adduct brings out various biochemical changes that might generate neo-epitopes, which have been well-recognized by these antibodies. The affinity of RA antibodies for 16α-OHE1-H1 (1.10 × 10- 7 M) was high, as detected by the Langmuir plot. Comparing RA patients to the controls, no significant differences were detected in the level of 16α-OHE1 or 2-hydroxyestrone/16α-OHE1 ratio. 16α-OHE1-H1 might have an antigenic role and function as a high-affinity antigen for RA autoantibodies and, therefore, could be used as a biomarker for this disease.

Recombinant Interferon Alpha-2b is a High-Affinity Antigen for Type 1 Diabetes Autoantibodies.

OBJECTIVES: Type 1 diabetes results from T-cell-mediated destruction of the beta cells of the pancreas and is associated with several autoimmune phenomena. Many studies have suggested the involvement of interferon alpha (IFN α) in the development of type 1 diabetes, but the exact mechanism remains unclear. In this study, the binding of type 1 diabetes antibodies with recombinant interferon alpha-2b (hrIFN α-2b), their gene (cIFN α-2b gene) and commercially available interferon α-2b (IFN α-2b) were assessed. Furthermore, we also sought to use anti-hrIFN α-2b antibodies as a probe for the estimation of plasma IFN α in patients with type 1 diabetes. METHODS: The binding specificity of antibodies was analyzed by direct binding, inhibition ELISA and quantitative precipitin titration in 45 patients with type 1 diabetes and 30 control subjects. Competition ELISA was also used to estimate INF α in the serum of patients with type 1 diabetes. RESULTS: Antibodies from type 1 diabetes sera, purified in a protein A-agarose matrix, exhibited greater recognition of hrIFN α-2b than IFN α-2b (p<0.05) and cIFN α-2b gene (p<0.001). The relative affinity of type 1 diabetes antibodies for the hrIFN α-2b, IFN α-2b and cIFN α-2b genes was found to be 1.34×10-7, 1.28×10-6 and 1.13×10-6, respectively. The concentration of plasma INF α evaluated by induced antibodies was found to be significantly higher than in controls (p<0.05). CONCLUSIONS: High binding of hrIFN α-2b with IgG from patients with type 1 diabetes might suggest involvement of hrIFN α-2b in type 1 diabetes, especially as an antigenic agent. Anti-hrIFN α-2b antibodies were shown to be good probes for estimation of plasma INF α in patients with type 1 diabetes.

Cytochrome P450-Mediated Estrogen Metabolites and Autoimmunity: Relationship and Link to Free Radicals.

Cytochrome P450 enzymes are responsible for the hydroxylation of various endogenous estrogens of the Phase I metabolic pathway. Cytochrome P450s produce hormonally active estrogen metabolites that are typically reactive and mutagenic. Although these metabolites are known to have important roles in autoimmunity, the underlying mechanism of this remains unknown. Here we report that cytochrome P450-mediated estrogen metabolites produce high ROS concentrations that can result in DNA damage. Such DNA damage can alter its immunogenicity, resulting in the induction and elevation of autoantibody concentrations, thus generating various autoimmune conditions. Here we focus on the mechanisms through which cytochrome P450-catalyzed estrogen metabolites induce immune responses and subsequently produce the autoimmune phenomenon.

Increased binding of circulating systemic lupus erythematosus autoantibodies to recombinant interferon alpha 2b.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by various types of immunological abnormalities including circulating and tissue-fixed autoantibodies reactive with autoantigens. The mechanism that can explain the production of these antibodies is unclear. Here we address the binding specificity of SLE autoantibodies with recombinant alpha interferon 2b (hrIFN α-2b), commercially available interferon (IFN α-2b), and the gene (cIFN α-2b) encoding this interferon. hrIFN α-2b showed higher binding with naturally occurring SLE autoantibodies as compared to IFN α-2b (p < 0.05) or cIFN α-2b gene (p < 0.001) as assessed by direct binding, inhibition ELISA, and quantitative precipitin titration. The relative affinity of SLE autoantibodies for hrIFN α-2b, IFN α-2b, and cIFN α-2b gene was in the order of 1.13 × 10(-7) , 1.38 × 10(-6) , and 1.22 × 10(-6) , respectively. hrIFN α-2b is shown to have unique epitopes that would explain the possible antigenic role of hrIFN α-2b in the generation of SLE autoantibodies. Anti-hrIFN α-2b antibodies have been shown to represent an alternative immunological probe for the estimation of interferon alpha 2b in the serum of SLE patients.

Cancer morbidity in rheumatoid arthritis: role of estrogen metabolites.

Estrogen metabolites have been implicated in rheumatoid arthritis (RA) and cancer, although the mechanism remains unestablished. Some estrogen metabolites, which are used for the assessment of cancer risk, play an important role in RA. The pathways by which malignancies associated with RA remain elusive. Possible mechanism involves enzymatic or nonenzymatic oxidation of estrogen into catecholestrogen metabolites through semiquinone and quinone redox cycle to produce free radicals that can cause DNA modifications. Modifications of DNA alter its immunogenicity and trigger various immune responses leading to elevated levels of cancer and RA antibodies. However, the role of different estrogen metabolites as a mediator of immune response cannot be ruled out in various immune-related diseases.

Preferential recognition of catechol-estrogen modified DNA by circulating autoantibodies in cancer patients.

Catecholestrogens [4-hydroxyestradiol (4-OHE(2))] have been implicated in human carcinogenesis, although the mechanism remains unestablished. In this study pUC 18 plasmid DNA was modified with 4-OHE(2) and nitric oxide (NO). The modification induced in native DNA exhibited hyperchromicity, single strand breaks, damage to restriction sites, modification of bases, decrease in Tm and change in ellipticity. Modified DNA was found to be highly immunogenic in experimental animal, eliciting high titer antibodies. Circulating cancer autoantibodies showed preferable recognition of 4-OHE(2)-NO-DNA over native form (p < 0.001) and the oxidative epitopes on the DNA isolates from cancer patients were immunochemically detected by using experimentally induced anti-4-OHE(2)-NO-DNA antibodies as a probe. Preferential recognition of 4-OHE(2)-NO-DNA by cancer autoantibodies coupled with enhanced binding of induced antibodies to DNA isolated from cancer patients is an indicative of oxidative stress induced DNA damage in cancer. Possible involvement of unique epitopes on modified DNA in cancer autoantibody induction has been suggested.

Detection of autoantibodies against reactive oxygen species modified glutamic acid decarboxylase-65 in type 1 diabetes associated complications.

BACKGROUND: Autoantibodies against glutamate decarboxylase-65 (GAD65Abs) are thought to be a major immunological tool involved in pathogenic autoimmunity development in various diseases. GAD65Abs are a sensitive and specific marker for type 1 diabetes (T1D). These autoantibodies can also be found in 6-10% of patients classified with type 2 diabetes (T2D), as well as in 1-2% of the healthy population. The latter individuals are at low risk of developing T1D because the prevalence rate of GAD65Abs is only about 0.3%. It has, therefore, been suggested that the antibody binding to GAD65 in these three different GAD65Ab-positive phenotypes differ with respect to epitope specificity. The specificity of reactive oxygen species modified GAD65 (ROS-GAD65) is already well established in the T1D. However, its association in secondary complications of T1D has not yet been ascertained. Hence this study focuses on identification of autoantibodies against ROS-GAD65 (ROS-GAD65Abs) and quantitative assays in T1D associated complications. RESULTS: From the cohort of samples, serum autoantibodies from T1D retinopathic and nephropathic patients showed high recognition of ROS-GAD65 as compared to native GAD65 (N-GAD65). Uncomplicated T1D subjects also exhibited reactivity towards ROS-GAD65. However, this was found to be less as compared to the binding recorded from complicated subjects. These results were further proven by competitive ELISA estimations. The apparent association constants (AAC) indicate greater affinity of IgG from retinopathic T1D patients (1.90 x 10⁻6 M) followed by nephropathic (1.81 x 10⁻6 M) and uncomplicated (3.11 x 10⁻7 M) T1D patients for ROS-GAD65 compared to N-GAD65. CONCLUSION: Increased oxidative stress and blood glucose levels with extended duration of disease in complicated T1D could be responsible for the gradual formation and/or exposing cryptic epitopes on GAD65 that induce increased production of ROS-GAD65Abs. Hence regulation of ROS-GAD65Abs could offer novel tools for analysing and possibly treating T1D complications.

Immunochemical studies on catechol-estrogen modified plasmid: possible role in rheumatoid arthritis.

INTRODUCTION: Increased concentrations of estrogen metabolites (catecholestrogens) have been found in rheumatoid arthritis (RA) but the exact patho-etiology remains elusive. METHODS: The binding of antibodies from the sera of RA patients and control subjects to native and modified DNA was studied by direct binding and inhibition ELISA, quantitative precipitin titration. Experimentally induced antibodies were also checked to detect oxidative lesions in the DNA as well as for the estimation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in different fluids of RA. RESULTS: Anti-DNA IgG from RA sera, exhibited increased recognition of modified DNA than native DNA (nDNA; P < 0.001). The relative affinity of anti-DNA antibodies for modified and nDNA was in the order of 1.85 × 10(-7), 1.23 × 10(-7), and 1.2 × 10(-6). Samples of DNA from RA patients showed a significant inhibition in the induced antibody activity in comparison to DNA isolates from controls (P < 0.001). The concentration of 8-OHdG evaluated by induced antibody in RA patients was found to be significantly higher than controls ((P < 0.0001, P < 0.01, P < 0.05). CONCLUSION: High binding of modified DNA with the IgG from RA patient might explain possible antigenic role of 4-OHE(2)-modified DNA in the production of anti-DNA antibodies. In addition, the induced antibodies have been shown to represent an alternative immunochemical probe to detect oxidative lesions in DNA as well as for the estimation of 8-OHdG levels in different body fluid of RA patients, which may be used as marker in the diagnosis of the disease.

Antibodies against gluco-oxidatively modified human serum albumin detected in diabetes-associated complications.

BACKGROUND: Glycated proteins present new immunological epitopes on their surface against which autoantibodies are generated that have a possible role in immunopathogenesis in diabetic complications. METHODS: In the present study, in vitro glycation- and reactive oxygen species (ROS)-modified human serum albumin (HSA) has been studied by different spectroscopic techniques (UV and fluorescence) and thermal denaturation profiles. The binding characteristics of circulating autoantibodies in diabetic patients and diabetic patients with secondary complications against native HSA (N-HSA) and ROS-modified glycated HSA (RG-HSA) were assessed by direct and competition enzyme-linked immunosorbent assay (ELISA). In another approach, antibodies against RG-HSA (RG-HSA-Abs) induced in experimental animals were used as an immunochemical probe for the detection of gluco-oxidative lesions in blood proteins of patients (n = 8) with diabetic retinopathy. RESULTS: Modified RG-HSA showed marked structural changes. High recognition of RG-HSA was shown by diabetic serum autoantibodies. Diabetic patients with retinopathy, nephropathy and atherosclerosis showed significantly (p < 0.001) stronger binding to RG-HSA over N-HSA. Normal human sera exhibited negligible binding with either antigen. Competitive inhibition ELISA results show significantly high binding of RG-HSA-Abs to albumin, immunoglobulin G and red blood cell membrane isolated from diabetic retinopathic patients. CONCLUSION: In conclusion, these results suggest that hyperglycemia together with ROS may contribute to the immunopathogenesis of diabetes-associated complications.

Catecholoestrogens: possible role in systemic lupus erythematosus.

It is well established that risk of developing SLE is higher among women compared with men but only very little is understood about the underlying mechanisms. Oestrogen and their catechol metabolites seem to play an important role in SLE but the exact patho-aetiology remains elusive. The evidences concerning the possibility of catecholoestrogens (CEs) in the development of SLE are very limited and preliminary. The possible mechanism involves quinone-semiquinone redox cycling of CEs to generate the free radical that can cause DNA damage. This would probably alter its immunogenicity leading to the induction and elevated levels of SLE autoantibodies cross-reacting with native DNA. The data demonstrate the possible role of CE in presenting unique neo-epitopes that might form one of the factors in induction of SLE autoantibodies. However, the role of oestrogen in immune modulation cannot be rule out as a mediator of various immune-related diseases.

Enhanced binding of circulating SLE autoantibodies to catecholestrogen-copper-modified DNA.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by circulating and tissue fixed autoantibodies reactive with self-antigens, including nucleic acid and other nuclear components. The pathways by which these autoantibodies act as a pathogenic factor remain elusive. Present study has investigated the role of estrogens in SLE etiopathogenesis. Estrogen-modified DNA [4-OHE(2)-Cu(II)-DNA] showed single- and double-strand breaks, hyperchromicity, decrease in Tm, and modification of bases. The 4-OHE(2)-Cu(II)-DNA exhibited increased binding with naturally occurring anti-DNA autoantibodies as compared to the unmodified native form (P < 0.001) as assessed by ELISA, quantitative precipitin titration, and gel retardation assay. The relative affinity of anti-DNA antibodies for modified and native DNA was in the order of 2.1 x 10(-7) M and 1.3 x 10(-6) M, respectively. The data suggested that DNA modified with 4-OHE(2) and Cu(II) may be one of the factors for the induction of circulating anti-DNA autoantibodies in SLE.