Zdenka Turk

Detection of autoantibodies against advanced glycation endproducts and AGE-immune complexes in serum of patients with diabetes mellitus

Advanced glycation of protein causes their immunogenicity. The evidence that advanced glycation endproducts (AGEs) have antigenic properties has led to a hypothesis that the AGE structure found in vivo may exert an autoimmune response. In the present study, we showed the sera of diabetic patients as well as of nondiabetic individuals to contain autoantibodies to epitopes of AGE structures. Contrary to what might be expected, we observed lower AGE antibody titers in diabetic subjects, and postulated that the antibodies against AGEs form immune complexes in vivo, hampering their determination. The existence of immune complexes containing AGE moiety was established by two independent criteria: (a) serum AGE-immune complexes (AGE-IC) were detected by enzyme-linked immunosorbent assay (ELISA) using an immunochemical bridge; and (b) soluble AGE-IC were precipitated from serum by polyethylene glycol and analyzed. We demonstrated the presence of circulating AGE-IC in sera, predominantly in the sera of diabetic subjects. We also found an inverse correlation between serum AGE level and AGE-IC (r=-0.8, P<0.000), indicating the serum level of AGEs to decline with an increasing presence of AGE-IC. The content of AGE in soluble immune complexes was significantly higher in diabetic patients than in control subjects (3.51+/-1.9 vs. 1.89+/-1.0 microgEq/ml (P<0.00004), and correlated inversely with free antibodies (r=-0.26, P<0.01). Interactions of AGE autoantibodies with AGE as a continuously produced antigen result in the formation of AGE-immune complexes that may play a role in the atherogenic processes.

Rat tissue collagen modified by advanced glycation: correlation with duration of diabetes and glycemic control.

Abstract Collagenous proteins are especially prone to nonenzymatic glycation, because they contain several dibasic amino acid residues with free amino groups, have a very slow turnover rate, and are exposed to ambient levels of glucose. The aim of this study was to determine the time-dependent course of advanced glycation process in diabetic rats in relation to glycemic control and duration of diabetes, compared to age-matched controls. Immunochemical assay with antibodies to advanced glycation end products (AGE) was first developed to qualitatively detect and quantify the AGE formed in rat tendon and aortic collagen. Individual collagen samples were extracted by extensive pepsin and collagenase digestion. The amount of AGE was measured by competitive ELISA and results were expressed as AGE U/mg collagen. Diabetic rats showed a significant increase in AGE content in aortic collagen at 20 weeks (n=6, 206.6 ± 16.7 U/mg collagen) compared with that measured at 4 and 12 weeks (n=6, 110 ± 12.8 U/mg collagen, and n=13, 184.9 ± 12.3 U/mg collagen at 4 and 12 weeks, respectively; p < 0.001 between 20 weeks and 4 weeks; p < 0.01 between 20 weeks and 12 weeks). The amount of AGE in tendon collagen of diabetic rats increased from 1.9 ± 0.38 U/mg at 4 weeks to 11.2 ± 6.1 U/mg collagen at 20 weeks, p < 0.001. Considerable disparity was observed in the intensity of glycation between aortic and tendon collagen. AGE-content per mg of aortic collagen was several-fold to that found in tendon collagen (p < 0.001). To investigate the effect of glycemic control on the advanced glycation process, total aortic AGE-collagen content was compared between untreated diabetic rats (D; n=13, 184.9 ± 12.3 U/mg) and diabetic rats treated for 12 weeks with insulin (DI; n=6, 133.9 ± 10.7 U/mg), or phlorizin (DP; n=6, 132.4 ± 8.9 U/mg), or by a combination of insulin/phlorizin (DIP; n=6, 124.3 ± 6.5 U/mg). In spite of therapy used, all groups of diabetic animals had a significantly higher aortic AGE-collagen content than those in the nondiabetic control group (C: n=8, 104.6 ± 14.9 U/mg) of the same age (D, DI, DP, DIP vs. C, p < 0.001). Comparison between the mean levels of glycated hemoglobin (D: 5.62 ± 0.38 % vs.C: 1.7 ± 0.05 %) and mean AGE levels in the studied group of animals yielded a very good exponential correlation (r = 0.89, p < 0.001). Glycation-derived late-stage collagen modification was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and by immunoblotting confirmed to contain (an) AGE-structure(s). Our study provides strong immunochemical evidence of AGE formation in vivo during hyperglycemia, and of their temporal association with structural alterations of extracellular matrix proteins. The advanced glycation process is retarded and reduced in intensity, but not completely abolished, by glycemia regulation with, or independently of, insulin.

Advanced glycation endproducts in peripheral nerve in type 2 diabetes with neuropathy

Advanced glycation endproducts (AGE) accumulate over proteins as a consequence of diabetic hyperglycemia, and thus contribute to the pathogenesis of diabetic complications. To improve the understanding of the pathology of diabetic neuropathy, AGE accumulation was analyzed in sural and/or femoral nerves obtained under spinal anesthesia from 8 type 2 diabetic patients with both distal symmetrical polyneuropathy and proximal neuropathy. Pronounced AGE immunoreactivity was detected on axons and myelin sheaths in 90% of diabetic peripheral nerves but not in the control specimen. The intensity of axonal AGE immunopositivity significantly correlated with the severity of morphological alterations (p<0.005). AGE localization, demonstrated by immunohistochemical methods, was also present in the endoneurium, perineurium and microvessels. Morphometric analysis of the diabetic peripheral nerve showed perineurial thickening (diabetic vs. control, 15.5±4.9 vs. 6.6±2.1 µm, p<0.001), narrowing of the microvessel lumina (66.6±50.5 vs. 579.5±38.4 x103 µm2, p<0.001) and significant reduction in the number of preserved axons (3.6±3 vs. 8.9±2.3 per 105 µm2 per area, p<0.037). The sera of diabetic patients contained epitope(s) of AGE structure and soluble immune complexes containing AGE moiety. In conclusion, to the best of our knowledge, this is the first study providing evidence for excessive AGE formation on peripheral nerve components, primarily axons, and a significantly higher level of circulating AGE-immune complexes in patients with both distal diabetic polyneuropathy and proximal neuropathy. Humoral immune mechanisms, including the production of anti-AGE autoantibody, may potentially be involved in the development of structural abnormalities described in this report.

Proinflammatory cytokines and receptor activator of nuclear factor kappaB-ligand/osteoprotegerin associated with bone deterioration in patients with Crohn´s disease

Objectives The high incidence of bone disease and the increasing evidence of Crohns disease (CD) bone decline in corticosteroid users and nonusers suggest that bone metabolism is affected by inflammatory process. The aim of the study was to compare serum levels of proinflammatory cytokines, markers of bone turnover and regulatory molecules of osteoclast biogenesis, receptor activator of nuclear factor κB-ligand (RANKL) and osteoprotegerin (OPG), between naïve and long-standing CD patients. Methods The study included 95 CD patients, 15 of them with newly diagnosed and previously untreated CD. The spine and hip bone mineral density was measured by dual-energy X-ray absorptiometry. Biochemical markers were determined by immunoassay. Results Osteopenia was recorded at diagnosis in 53% of naïve patients and osteoporosis was found in 26% of long-standing CD patients. The newly diagnosed patients showed correlation between TNF-&agr; and soluble RANKL (sRANKL) (r=0.5; P=0.04), and this positive relationship characterized the study population as a whole (r=0.3; P=0.003). Analysis of the OPG and sRANKL relationship showed absence of correlation in patients with healthy skeleton, whereas an inverse correlation was found in those with osteopenia (r=−0.31; P=0.033) and osteoporosis (r=−0.48; P=0.028). In naïve patients with reduced T score, the correlation between sRANKL and OPG was highly inverse (r=−0.8; P=0.02) and these patients were characterized by lower BMI, significantly higher level of proinflammatory cytokines, elevated C-reactive protein, and increased activity of free sRANKL and OPG. Conclusion Bone disease that accompanies CD at diagnosis suggests that bone metabolism is affected by the underlying inflammatory process per se, as probably confirmed by our finding of the central proinflammatory cytokine TNF-&agr; being strongly associated with the osteoclastogenic mediator RANKL, and inversely with bone density.

Temporal association between lens protein glycation and cataract development in diabetic rats

Abstract In an attempt to shed more light on the relation between the glycation process and structural protein alterations, we followed the formation of glycated products in the lenses of hyperglycaemic Wistar rats during a period of 5 months following alloxan diabetes inducement. The study groups included non-diabetic (control), untreated diabetic rats (D), and diabetic rats receiving insulin alone or in combination with phlorizin, an inhibitor of renal tubular glucose transport. Lenses were removed at 4 and 20 weeks, and advanced glycation products in alkali-soluble lens proteins were determined by their characteristic spectrofluorescence (emission at 385 nm with excitation of 335 nm). In 20-week untreated diabetic rats as compared to control rats, a significant increase was observed in the fluorescence level (3.25±1.02 vs 1.61±0.17 FU/mg, P<0.001), while in 4-week animals the increase was from 1.26±0.11 FU/mg in controls to 1.80±0.25 FU/mg in diabetics (P<0.001). Daily treatment of 20-week diabetic rats with insulin alone (2.46±0.48 FU/mg) or in combination with phlorizin (2.30±0.26 FU/mg) did not significantly influence lens fluorescence level. The amount of glucose-bound ketoamine linkage was estimated after acid hydrolysis as released 5-hydroxymethylfurfural (HMF). In 20-week controls, it was slightly higher than in 4-week controls (0.57±0.31 vs 0.41±0.20 nmol HMF/mg, respectively). The diabetic group showed a significant increase, however. In 4-week diabetics, a level of 1.07±0.36 nmol HMF/mg was found, while in 20-week animals the glycated protein amount rose to 2.46±0.79 nmol HMF/mg. In addition to the increases in glycated content with continuing diabetic hyperglycaemia, significant changes in protein composition of alkali-soluble lenses developed. The SDS-PAGE pattern showed an appearance of protein polymers of heterogeneous size (C 4 weeks 3.0±1.1% vs C 20 weeks 17.9±2.9%; D 4 weeks 7.3±2.1% vs D 20 weeks 19.8±3.6%) and the proteins of high molecular weight (HMW) failed to penetrate into the gel. Only a small amount of these HMW proteins was present in controls (C 20 weeks 2.5±1.2%) and short-term diabetes (D 4 weeks 0.8±0.2%), whereas in long-term untreated diabetes there was a dramatic increase (D 20 weeks 30.5±3.2%) with a corresponding decrease in other peaks. All diabetic animals from this group had macroscopically detectable cataractous lenses. Treatment with insulin or insulin/phlorizin followed the HMW protein level of the untreated animals (28.2±4.0% or 27.08±3.3% vs 30.52±3.32%)

Products of advanced glycation in patients with type 2 diabetes and vascular disease.

Background: Non-enzymatic glycation leading to advanced glycation endproduct (AGE) formation is thought to contribute to vascular pathology. In the present study, AGEs and anti-AGE antibodies in free and immune complex-bound form were assayed in the serum of diabetic (DMCAD) (n = 69) and nondiabetic (n = 78) patients with coronary artery disease (CAD) and in control subjects (n = 47) free from vascular disease. Methods: A blocking enzyme-linked immunosorbent assay (ELISA) was used to test immunoreactivity against AGE epitope(s) and a competitive ELISA was used to measure total AGE content. Results: Anti-AGE immunoreactivity was significantly higher in diabetic than in control subjects (P = 0.045). Although a wide range of anti-AGE antibody titres were observed in nondiabetic CAD patients, there was no significant difference from those of control subjects. Both diabetic and nondiabetic CAD patients had a higher concentration of circulating immune complexes containing the AGE moiety as antigen than did control subjects (DMCAD versus control, P = 0.041; CAD versus control, P = 0.047). Study patients showed a positive correlation between serum AGE and AGE-immune complexes (DM, r = 0.29, P = 0.014; CAD, r = 0.26, P = 0.019), whereas no such correlation was recorded in controls (r = 0.08, P = 0.89). Conclusion: To our knowledge, this is the first study demonstrating increased AGE-immune complexes in patients with CAD, either with or without diabetes, suggesting that AGE-immune complexes might be involved in the atherosclerotic process, either as the result of it or as part of the pathophysiologic process.

Relationship of methylglyoxal-adduct biogenesis to LDL and triglyceride levels in diabetics

AIMS Protein glycation leading to advanced glycation-endproducts (AGE) is enhanced in diabetes by increased blood glucose and collateral endogenous production of reactive α-dicarbonyls. Among AGE precursors, methylglyoxal (MG) is considered as one of the key intermediates. We hypothesized it to be a common product of both carbonyl and oxidative stress, and investigated its biogenesis in relation to glycemic and lipid status in diabetic patients. METHODS Serum and urine MG-adducts were measured by competitive immunofluorometric assay in 83 diabetic and 20 healthy subjects. KEY FINDINGS A significant association of MG-adducts serum level with LDL (r=0.31;p=0.003) was observed. A correlation between LDL-c, HDL-C and PPG as independent variables and serum MG-adducts as a dependent variable was found (p<0.014) using multiple stepwise regression, whereas urine albumin/creatinine ratio was independently associated with urine MG-adducts. LDL cut-off >3.0mmol/l discriminated patients with higher serum MG-adducts (p=0.0052), although there was no between-subgroup difference in glycemic control. Patients on statin therapy had a lower MG-adduct level. The positive relationship between LDL-c and MG-adducts (r=0.38;p=0.042) was noted in patients free of statin treatment, whereas an inverse tendency was found in the statin-treated subgroup. SIGNIFICANCE Significant relationship between LDL and MG-adduct production, as well as tight correlation between triglycerides and urinary MG-adduct excretion suggest that the lipoxidation and glyceraldehyde-3-phosphate route, along with the glycolytic pathway, might be an important source of MG generation. The glycotoxin methylglyoxal seems to be a common factor linking hyperglycemia and intensive lipolysis, two dominant metabolic changes in diabetes.

Humoral SPARC/osteonectin protein in plasma cell dyscrasias

The matricellular protein SPARC (secreted protein acidic and rich in cysteine)/osteonectin was determined in patients with multiple myeloma and related disease to assess the hypothesized role of SPARC as a possible marker of tumor burden and disease progression. Soluble SPARC was measured by competitive enzyme-linked immunosorbent assay (ELISA) in plasma of 42 patients, including sequential measurements in individual patients, and in 20 healthy controls. SPARC values were heterogeneous in multiple myeloma patients showing a decline from baseline levels recorded in controls (456±195 vs 600±63 ng/ml, p=0.00023). A SPARC showed a significant positive correlation with platelet count (r=0.72, p=0.000000, n=42), hemoglobin (r=0.52, p=0.00037, n=42), and IgG level (r=0.43, p=0.0085, n=42) and negative correlation with β2-microglobulin (r=−0.46, p=0.0023, n=42), aspartate aminotransferase (AST) (r=−0.42, p=0.0061, n=41), interleukin (IL)-6 (r=−0.41, p=0.008, n=42), lactate dehydrogenase (LDH) (r=−0.36, p=0.02, n=41), and percentage of plasma cells in bone marrow aspirate (r=−0.34, p=0.029, n=42). No correlation was found between SPARC and “M” component or disease stage. Investigations performed during the course of disease, including sequential measurements in individual patients, showed a trend to downregulation by disease progression, with the lowest level recorded in the terminal stage (217±107 ng/ml, n=11). Patients with established osteolytic lesions had lower plasma SPARC at diagnosis (309±197 vs 581±293, p=0.021), which correlated with osteocalcin by disease progression (r=0.31, p=0.026). The results of this pilot study revealed abnormalities in the level of humoral SPARC in multiple myeloma and an overall trend to downregulation in the advanced stage of the disease. The regulation of SPARC seems to be opposite to the markers of tumor burden and of aggressive multiple myeloma phenotype.

T1232 Inflammation Is the Main Determinant of Bone Loss in Naïve Patients with Crohn Disease

Background/Aim The high incidence of bone disease and increasing evidence for Crohn disease affecting bone in corticosteroid users and non-users suggest that bone metabolism is affected by inflammatory process. As the biogenesis of receptor activator of nuclear factor κ B-ligand (RANKL) and its decoy receptor osteoprotegerin (OPG) may be a consequence of intestinal inflammation and because this signal pathway is shared between the immune and bone system, we hypothesized that the action of sRANKL, OPG and other inflammatory cytokines is not limited to the induction of local inflammation but might be directly or indirectly involved in the activation of bone metabolism. This study aimed to determine comparative serum levels of proinflammatory cytokines, markers of bone formation and resorption, and regulatory molecules of osteoclast biogenesis, in nai ; ve and long-standing patients with Crohn disease. Patients and Methods The study included 95 patients, 15 of them newly diagnosed and untreated. Serum sRANKL, OPG, TNF-α , IL-1β , IL-6, osteocalcin and C-telopeptide I were measured by immunoassay. The spine and total hip bone mineral density (BMD) was measured by DXA. Results Decreased BMD at diagnosis was found in 53% and low bone mass in 72% of the study population. The newly diagnosed, untreated patients showed correlation between TNF-α and sRANKL (r=0.6 ; p=0.027), and this positive relationship characterized the study population as a whole (r=0.5 ; p=0.002). Multiple regression identified TNF-α as the best predictor of sRANKL (p<0.001). Patients with increased parameter of bone resorption were characterized by elevation of TNF-α , IL-6, CRP and OPG in systemic circulation. Analysis of the OPG and sRANKL relationship according to subgroups showed absence of correlation in patients with healthy skeleton, and an inverse relationship in those with pathologic BMD (r=-0.36 ; p=0.003). In newly diagnosed patients with BMD t-score≤ -1.0, the correlation between sRANKL and OPG was highly inverse (r=-0.8 ; p=0.02). Conclusion Bone disease that accompanies Crohn disease at diagnosis in previously untreated patients suggests that bone metabolism is affected by the underlying inflammatory process per se, as probably confirmed by our finding that the central proinflammatory cytokine TNF-α was strongly associated with the osteoclastogenic mediator RANKL.

Decrease of oxidatively modified LDL during long-term LDL-apheresis treatment

The aim of the present study was to follow-up generation of autoantibodies against oxidatively modified LDL in a patient suffering from severe combined hyperlipidemia during long-term LDL apheresis alone or in combination with statin therapy. An ELISA using MDA-LDL as coating antigen measured anti-oxLDL titre. Baseline value of 82.8 2.1 U/ml was reduced only for 7.8% after 2-month of statin therapy, and decrease of 10.7% was achieved by LDL-apheresis treatment. Long-term LDL-apheresis combined with statin was more effective. Multiple comparison among the four 6-month periods showed significant reduction of mean values in II and III semester (58.0 13.8 vs 37.6 6.0 U/ml, p<0.00053) of the treatment. In parallel to the changes in this biochemical parameter, regression of numerous xanthomas was clinically observed. In spite of this, the presence of ox-LDL antibodies was enhanced in comparison to antibody titer detected in a group of age-matched normolipemic controls (n=15 ; 19.4 8.6 ; p<0.01). The significant reduction of baseline total cholesterol (58%), total triglycerides (80%), LDL cholesterol (48%), apoprotein B (50%) and apolipoprotein (a) (61%) may be considered as a good response to the treatment. LDL/HDL cholesterol ratio, an index of atherosclerosis, was significantly reduced from 13.0 to 2.6. Classical lipoprotein parameters showed low correlation coefficients with anti-oxLDL titer. The reason may lie in the possibility that the primary location of the analyzed parameter is not in the circulation. The production of autoantibodies against oxLDL was reduced during LDL-apheresis, indicating a lower level of the atherogenic antigen. The study showed an additional benefit of LDL-apheresis treatment.