Serum Of Diabetic Patients Research Articles

Serum soluble α-Klotho levels in patients with diabetic nephropathy.

The study aimed to determine the variability in the stages of diabetic nephropathy by examining specific biochemical functions associated with the target organ. As a result, various biochemical parameters were assessed in all of the groups under investigation. These parameters encompassed soluble α-Klotho and serum insulin, which were determined through ELISA. Additionally, spectrophotometric methods were employed to assess other parameters such as blood levels of urea in all groups. Instead of using HPLC method, HbA1c levels were determined. Blood and urine samples were obtained from a total of 90 participants, who were aged between 37 and 70 years. A total of 70 patients were categorized into three groups according to their ACR. The first group consisted of patients with an ACR value of less than 30 mg/g. The second group included patients with an ACR value ranging from 30 to 300 mg/g. The third group comprised patients with an ACR value greater than 300 mg. Additionally, the study also involved 20 healthy individuals. The serum soluble α-Klotho in the patient group was significantly lower than that of the healthy subjects. There were strong negative correlations between serum soluble α-Klotho and both ACR and HOMA-IR. The AUC value was excellent, measuring at 0.93 with a p < 0.0001. Soluble α-Klotho levels in the sera of diabetic patients were shown to be lower and significantly linked to patients with diabetic nephropathy. This implies that klotho levels may be influenced by ACR in addition to playing a significant role in insulin resistance.

Association of N-Acetyl Asparagine with QTc in Diabetes: A Metabolomics Study.

Changes in the cardio-metabolomics profile and hormonal status have been associated with long QT syndrome, sudden cardiac death and increased mortality. The mechanisms underlying QTc duration are not fully understood. Therefore, an identification of novel markers that complement the diagnosis in these patients is needed. In the present study, we performed untargeted metabolomics on the sera of diabetic patients at a high risk of cardiovascular disease, followed up for 2.55 [2.34–2.88] years (NCT02431234), with the aim of identifying the metabolomic changes associated with QTc. We used independent weighted gene correlation network analysis (WGCNA) to explore the association between metabolites clusters and QTc at T1 (baseline) and T2 (follow up). The overlap of the highly correlated modules at T1 and T2 identified N-Acetyl asparagine as the only metabolite in common, which was involved with the urea cycle and metabolism of arginine, proline, glutamate, aspartate and asparagine. This analysis was confirmed by applying mixed models, further highlighting its association with QTc. In the current study, we were able to identify a metabolite associated with QTc in diabetic patients at two chronological time points, suggesting a previously unrecognized potential role of N-Acetyl asparagine in diabetic patients suffering from long QTc.

Nesfatin-1 - as a diagnosis regulatory peptide in type 2 diabetes mellitus.

One of the most common metabolic diseases, Type 2 Diabetes Mellitus (T2DM), is caused by a combination of two basic factors: insufficient insulin secretion by pancreatic -cells or a failure of insulin-sensitive tissues to respond adequately to insulin. The aim of this paper was to assess the diagnostic accuracy of serum nesfatin-1 in type 2 diabetes mellitus (T2DM). Sixty patients with T2DM were recruited from (Baquba Teaching Hospital) in Iraq during the period (from November 2021 - March 2022). T2DM group was classified into 30 newly diagnosis patients (without treatment) and 30 ongoing diabetic patients (with treatment) for comparison, as well 30 healthy individuals were included as a control. The ELISA Kit was used to measure serum nesfatin-1 and serum insulin, fasting serum glucose, and lipid profile test were measured through spectrophotometric, instead of HbA1c was determined using HPLC method. The concentration of serum nesfatin-1 in the T2DM group was significantly lower than that of the healthy subjects (p < 0.05). There was a significant difference in the serum nestafin-1 concentrations between newly diagnosis and ongoing T2DM patients. There were substantial negative connections between serum Nesfatin-1 concentration and HOMA-IR, as well as strong negative correlations between serum nesfatin-1 and serum insulin level. The concentration of serum Nesfatin-1, on the other hand, had no significant association with the anthropometries measurements and biochemical parameters. The area under the curve was excellent (AUC = 0.827, p = 0.0001), with high diagnostic accuracy (86.2) in differentiating newly diagnosis T2DM from the healthy subject group. Nesfatin-1 levels in the sera of diabetic patients was shown to be lower and nesfatin-1 levels were shown to be significantly linked to newly diagnosed patients.

The relationship between melatonin level and antioxidant enzymes in diabetic patients with and without nephropathy

Background and objective: Diabetes is the most common cause of chronic renal disease globally. Diabetic nephropathy (DN) is one of the most serious consequences of type 2 diabetes. Melatonin, a powerful antioxidant that has been shown to alleviate DN, deficiency and a functional relationship between melatonin and insulin have been linked to the etiology of type 2 diabetes mellitus. The purpose of this research is to assess the relationship between melatonin level and antioxidant enzyme activity (catalase, glutathione peroxidase, superoxide dismutase, paraoxonase 1, and glutathione-s-transferase) in diabetic patients with and without nephropathy. Methods: This case-control study was conducted on 45 healthy control subjects, 45 diabetic patients without nephropathy, and 45 diabetic patients with nephropathy. Serum samples of participants were used to evaluate antioxidant enzyme activities, melatonin levels, and MDA using specific assays. Results: The results showed that the concentration of melatonin is not affected in diabetic patients without nephropathy, but decreased significantly in diabetic patients with nephropathy when compared with healthy subjects. Antioxidant enzymes activity in sera of diabetic patients with and without nephropathy were significantly lower than that of healthy subject group. The superoxide dismutase enzyme has a specific exception because its activity is elevated, unlike other antioxidant enzymes. Conclusions: Melatonin decreased significantly in sera of diabetic patients with nephropathy. Diabetic nephropathy affects antioxidant enzymes activity and lipid peroxidation significantly compared with healthy controls.

Glycemic control and dyslipidemia in type 2 diabetic patients in an Indian rural tribal locality: A family medicine practitioner’s perspective

Background: Diabetes and dyslipidemia commonly coexist, frequently associated with various cardio-vascular (CV) risk factors and good glycemic control is key for prevention of long-term CV complications. Although diabetes and dyslipidemia commonly coexist in India, there is a lack of evidence on pattern of dyslipidemia and whether dyslipidemia is adequately managed or not, particularly in rural population in a real-world setting. Aims and Objectives: This study was conducted to assess present glycemic status and lipid profile of the population residing in a rural tribal locality of Jharkhand (India) as part of project for fellowship in diabetes course by Department of Endocrinology, DEDU, CMC, Vellore. Materials and Methods: This non-interventional cross-sectional study was conducted in a tribal locality of Jharkhand (India) after concept note approval for ethical clearance from CMC Vellore. Whole-blood and sera of diabetic patients were analyzed for fasting-blood-sugar, Glycated-hemoglobin (HbA1c), total-cholesterol (CH), triglycerides (TGs), high-density-lipoprotein-cholesterol, low-density-lipoprotein-cholesterol, and very-VLDL-C. Correlation test of HbA1c with lipid-ratios and individual lipid indexes was done. Results: Mean Hb1Ac level was 7.24 ± 1.80 and interestingly, was marginally higher (7.31 ± 1.92 vs. 6.92 ± 1.16) in patients with DM &lt;5 years as compared to those with DM &gt;5 years. Mixed dyslipidemias were common with abnormal TG, LDL, VLDL, High-density lipoprotein (HDL), and Total CH values. Hb1Ac-levels showed significant positive correlation with serum CH, TG, LDL, and VLDL levels while significant negative correlation with HDL levels in the study. Conclusion: Apart from a reliable indicator of long-term glycemic control, HbA1c can also be used as a predictor of dyslipidemia and thus early diagnosis of dyslipidemia can prevent life-threatening CV-complications.

A case-control study to determination FBXW7 and Fetuin-A levels in patients with type 2 diabetes in Iraq.

diabetes mellitus (type 2) is a chronic disease characterized by hyperglycemia resulting either from a deficiency of insulin, or decrease insulin action or both. This study focused on determining the new marker F- box and WD repeat domain containing protein 7 (FBXW7) as well as Fetuin -A and their correlation with other parameters in Diabetes mellitus type 2. Sixty diabetes type2 patients were recruited from (National diabetes Center for treatment and Research) at Al Mustansriya University in Iraq during the period (from November 2018-March 2019) with an age range (37-69) years. The patient group was subdivided into two groups: group I (30) newly diagnosed T2DM and group II (30) T2DM (on metformin monotherapy), for comparison, 30 healthy individuals were included as a control. The protein levels (FBXW7, and Fetuin-A) were estimated using quantitative enzyme-linked immunesorbent assay (ELISA). Biochemical parameters; fasting serum glucose (FSG), and lipid profile were measured through spectrophotometrically. Insulin was determined using ELISA kit finally, HbA1c was determined using HPLC. Serum levels of FBXW7 protein were significantly higher (p-value = 0.01), whereas, there was no significant difference (p ˃ 0.05) in the level of Fetuin-A in group I as compared to control group. There was no significant difference (p ˃ 0.05) in the levels of both proteins who group II was compared with the control group. On the other hand, FBXW7 level were significantly lower in group II as compared to group I. Fasting blood sugar FSG, glycated hemoglobin HbA1c, body mass index BMI, waist- to-height ratio WHtR, and lipid profile were significantly elevated, except for high density lipoproteins, HDL was decreased in Group I as compared to control group, whereas insulin appeared non-significant (P > 0.05). FBXW7 Protein was negatively correlated to Fetuin -A, HOMA-IR, Insulin concentration, and BMI, whereas positive correlate to FSG, HbA1c, lipid profile, and atherogenic index. According to ROC analysis, the optimal cutoff value for FBXW7 protein was (1.455) ng/ml. Levels of FBXW7 and Fetuin-A proteins in sera of Diabetic patients were elevated. FBXW7 was significantly correlated with newly diagnosed patients. Metformin reduces FBXW7 and Fetuin-A levels.

Prevalence of auto-antibodies against D-ribose-glycated-hemoglobin in diabetes mellitus.

Glycation of biological macromolecules, due to hyperglycemia, promotes the formation of advanced glycation end products (AGEs). It is accelerated in diabetic patients and is responsible for the pathophysiology and progression of diabetes. Previous reports have shown that amount of AGEs formation and glycation-induced structural damage is higher in hemoglobin (Hb) than other proteins present in blood. In our previous study, we have shown structural changes in Hb by D-ribose which may result into the generation of immunogenic neo-epitopes. Thus, we hypothesized that D-ribose induced structural perturbations in Hb, could result in the formation of neo-epitopes which may provoke an auto-immune response and may also be involved in the immuno-pathogenesis of diabetes type-2 associated complications. Therefore, in the current study, we analyzed the prevalence of autoantibodies in diabetic patient's sera against D-ribose glycated-Hb by direct binding and competitive ELISA. Direct binding ELISA confirmed that autoantibodies in diabetic patients exhibit significantly high binding with D-ribose glycated-Hb as compared to its native form. The antigen binding specificity of these antibodies was also screened by competitive inhibition ELISA. We also used D-glucose glycated-Hb as a positive control to detect the presence of auto-antibodies by direct binding and inhibiton ELISA. We found that D-glucose glycated-Hb binds with T2DM samples but the affinity to binding is lower than D-ribose glycated-Hb. The overall findings of this study suggest the prevalence of circulating autoantibodies against D-ribose glycated-Hb in diabetic patients and thus, the level of these autoantibodies may be used as biomarker for progression of diabetes.

Preferential recognition of advanced glycation end products by serum antibodies and low-grade systemic inflammation in diabetes mellitus and its complications

BackgroundAdvanced glycation end products (AGEs) have shown to possess antigenicity. This study analyzes the detrimental effect of non-enzymatic glycation on human serum albumin (HSA) leading to the production of antibodies. MethodsHSA (20 μM) incubated with d-glucose formed AGEs confirmed by scanning electron microscopy (SEM). DNA-damage was assessed with comet assay. Antibodies against in-vitro formed AGEs was evaluated in the sera of diabetic patients by enzyme-linked immunosorbent assay. Molecular docking was performed to demonstrate affinity of native and glycated-HSA with IgG. Low-grade systemic inflammation was quantified with IL-4, IL-6, TNF-α and NF-кβ in serum and mRNA expression. ResultsScanning Electron Microscopy showed the formation of aggregates in glycated-HSA. Comet assay showed DNA damage T2DM with CKD. Serum auto-antibodies in diabetes patients with chronic kidney disease (CKD) showed appreciably high recognition with glycated-HSA compared to native HSA. Molecular docking showed less affinity of glycated-HSA with IgG. Serum IL-4, IL-6, and TNF-α were found significantly higher in T2DM with CKD compared to T2DM and healthy ones. mRNA expression of IL-4, IL-6 and NF-кβ are also found significantly higher in T2DM with CKD. ConclusionThe non-enzymatic glycation-induced damage to the HSA generate neo-epitopes that possess immunogenic response and low-grade systemic inflammation.

Biophysical and immunological characterization of 2-dRib modified HSA and its implications in diabetes mellitus

Glycoxidation of protein may lead to develop diabetes. In the present study, different concentrations of 2-deoxy d-ribose (2-dRib) were used to modify human serum albumin (HSA). Nitro Blue Tetrazolium (NBT) assay results showed that yield of the fructosamine content was directly proportional to the concentration of 2-dRib. UV and fluorescence spectroscopy results showed an increment in hyperchromicity and decrease in fluorescence intensity of 2-dRib modified HSA as compared to native HSA. Further secondary structural changes were confirmed by UV-circular dichroism (UV-CD) and Fourier transform infrared spectroscopy (FT-IR). To evaluate the immunogenicity of 2-dRib modified HSA, rabbits were immunized with native and 2-dRib modified HSA. Modified HSA sera showed high antibodies titre as compared to native HSA. Moreover, the binding affinity of native and modified HSA with diabetic patient's sera has been evaluated by direct binding ELISA. It was found that diabetic patient's sera showed high binding affinity with the modified HSA as compared to native HSA. On the basis of above findings, it can be concluded that 2-dRib is a potential glycating agent that can cause alteration in HSA structure and make HSA more immunogenic that might play a role in onset and progression of diabetes mellitus and its complications.

Glycated LDL increase VCAM-1 expression and secretion in endothelial cells and promote monocyte adhesion through mechanisms involving endoplasmic reticulum stress.

Type 2 Diabetes Mellitus is a worldwide epidemic, and its atherosclerotic complications produce morbidity and mortality in affected patients. It is known that the vascular cell adhesion molecule-1 (VCAM-1) levels are increased in the sera of diabetic patients. Our aim was to investigate the impact of the endoplasmic reticulum stress (ERS) in VCAM-1 expression and secretion in human endothelial cells (HEC) exposed to glycated low-density lipoproteins (gLDL). The results showed that 24h incubation of HEC with gLDL induces (i) stimulation of VCAM-1 expression and secretion, determining increased monocyte adhesion to HEC; (ii) RAGE up-regulation and free cholesterol loading; (iii) ERS activation (increased eIF2α phosphorylation and CHOP mRNA levels, and decreased GRP78 protein expression); and (iv) oxidative stress [increased levels of reactive oxygen species (ROS) and glutamate cysteine ligase catalytic unit gene expression]. Treatment of gLDL-exposed HEC with ERS inhibitors, salubrinal (Sal) and sodium phenylbutyrate (PBA), decreased intracellular ROS. Incubation of gLDL-exposed cells with the anti-oxidant N-acetyl-cysteine (NAC)reduced ERS, revealed by decreased eIF2α phosphorylation and CHOP gene expression and increased GRP78 expression, thus validating the interconnection between ERS and oxidative stress. Sal, PBA, NAC and inhibitors of p38MAP kinase and NF-kB induced the decrease of VCAM-1 expression and of the ensuing monocyte adhesion induced by gLDL. In conclusion, in HEC, gLDL stimulate the expression of cellular VCAM-1, the secretion of soluble VCAM-1, and the adhesion of monocytes through mechanisms involving p38MAP kinase and NF-kB signalling pathways activated by RAGE, ERS and oxidative stress, thus contributing to diabetic atherosclerosis.

Abnormal levels of age-elastin derived peptides in sera of diabetic patients with arterial hypertension.

IntroductionAn important factor in vascular wall alterations is degradation of elastic fiber major protein – elastin. As a result, elastin derived peptides (EDP) are found in circulation. Advanced glycation might also involve elastin, because it is a protein with slow metabolism. The aim of our study was to measure serum levels of glycated elastin derived peptides (AGE-EDP) of elastin in patients with type 2 diabetes mellitus (T2DM) and arterial hypertension (AH).Material and methodsWe adapted an ELISA technique for the determination of AGE-EDP. Sera of 93 patients with T2DM and AH (mean age 61.4 ±11.3 years, diabetes duration 9.88 ±3.12 years; hypertension duration 9.28 ±4.98) were tested. These values were compared to 42 age- and sex-matched controls. Diabetics were divided in two groups according to presence – Group 1 (n = 67) or absence – Group 2 (n = 26) of microangiopathy.ResultsPatients with T2DM and AH showed statistically significantly higher levels of AGEEDP in comparison with healthy controls 0.060 (0.053÷0.065) vs. 0.039 (0.031÷0.044) (KW = 35.2; p < 0.0001). Group 1 showed significantly higher levels of AGE-EDP than the control group 0.069 (0.051÷0.070) vs. 0.039 (0.031÷0.044) (KW = 33.0; p < 0.0001). Group 2 also showed significantly higher levels of AGE-EDP than controls 0.058 (0.049÷0.064) vs. 0.039 (0.031÷0.044) (KW = 22.1; p < 0.0001). AGE-EDP showed a correlation with an insulin dose (r = –0.28; p = 0.05), systolic blood pressure (r = 0.25; p = 0.01), BMI (r = 0.39; p = 0.01) and retinopathy (r = 0.18; p = 0.05).ConclusionsThe measurement of non-invasive markers of elastin glycation may be useful in monitoring development of vascular wall alterations and therapeutic interventions.

Biochemical and Kinetic Studies on Alkaline Phosphatase and other Biochemical Features in Sera of Patients with type 2 Diabetes

Background :Alkaline phosphatase (ALP) was a widely used marker for skeletal and hepatobiliary disorders, but its activity was also increased in atherosclerosis and peripheral vascular disease. Several study has showed that ALP activity was increased in the sera of diabetic patients. The current study was conducted to evaluate ALP activity in type 2 diabetic patients and optimum conditions for enzyme activity in their sera.Methods: This study was carried out at in AL-Yarmok hospital(diabetic center) between February /2009 and April /2009. Fifty two patients with type 2 diabetes have been enrolled. Besides BMI, WHR, serum fasting blood glucose, ALP, HbA1C,uric acid and lipid profile levels have been performed .The relationship between ALP and other biochemical factors have been studied.Results: From a total 52 cases, FBG, HbA1C and ALP were significantly elevated P value &lt; 0.01 while Uric acid, Cholesterol, TG, HDL, LDL,VLDL and LDL/HDL were significantly different P value &lt; 0.05 in diabetic patients when compared with that found in control group . ALP was significantly associated with LDL (P &lt; 0.05) and significantly negative correlation with HbA1C (P

Biochemical and Kinetic Studies on Alkaline Phosphatase and other Biochemical Features in Sera of Patients with type 2 Diabetes

Background :Alkaline phosphatase (ALP) was a widely used marker for skeletal and hepatobiliary disorders, but its activity was also increased in atherosclerosis and peripheral vascular disease. Several study has showed that ALP activity was increased in the sera of diabetic patients. The current study was conducted to evaluate ALP activity in type 2 diabetic patients and optimum conditions for enzyme activity in their sera.Methods: This study was carried out at in AL-Yarmok hospital(diabetic center) between February /2009 and April /2009. Fifty two patients with type 2 diabetes have been enrolled. Besides BMI, WHR, serum fasting blood glucose, ALP, HbA1C,uric acid and lipid profile levels have been performed .The relationship between ALP and other biochemical factors have been studied.Results: From a total 52 cases, FBG, HbA1C and ALP were significantly elevated P value &lt; 0.01 while Uric acid, Cholesterol, TG, HDL, LDL,VLDL and LDL/HDL were significantly different P value &lt; 0.05 in diabetic patients when compared with that found in control group . ALP was significantly associated with LDL (P &lt; 0.05) and significantly negative correlation with HbA1C (P

Detection of autoantibodies against glycosylated-DNA in diabetic subjects: Its possible correlation with HbA(1C).

Aims: The vascular tissues have a long memory of their previous glycemic control and intervention studies have demonstrated that microvascular complications are highly correlated with mean glycemic control as measured by glycolated hemoglobin A1C (HbA1c). The present study was carried out to evaluate the autoantibodies against glycosylated DNA (DNAAGEs) in diabetic sera to see if the level of HbA1c has correlation with the activity of DNA-AGEs, another marker of chronic glycemia. Methods: Glucose-6-phosphate induced glycosylation of native DNA was studied in 150 diabetic sera (T1DM = 9, T2DM = 141) by spectroscopic techniques (UV and fluorescence) and agarose gel electrophoresis. Direct binding and inhibition enzyme immunoassays were carried out to evaluate binding and specificity of anti-glycated-DNA autoantibodies (anti-DNA-AGE autoantibodies) in sera of diabetes patients. A quantitative estimation of HbA1c was carried out in normal and diabetes sera.Results: Anti-DNA-AGEs autoantibodies in 55% of diabetic sera (85/150) showed more binding with glycated-DNA compared to native DNA which was subjected to inhibition ELISA indicating true interaction of these autoantibodies in diabetes with glycated DNA. Higher binding with glycosylated-DNA against native-DNA was observed in subjects with HbA1c of 9.8 ± 3.3% compared to those with HbA1c of 7.7 ± 1.7% (p < 0.001). Linear correlation analysis showed that mean absorbance difference was significantly related to HbA1c (r = 0.486, p < 0.001), nephropathy (r = 0.239, p < 0.003), retinopathy (r = 0.165, p < 0.05). Conclusions: Autoantibodies against glycosylated DNA were correlated with HbA1c and microvascular complications and may be useful as another biomarker for assessment of chronic glycemia.

Detection of Autoantibodies Against Glycosylated-DNA in Diabetic Subjects: Its Possible Correlation with HbA1C

Aims: The vascular tissues have a long memory of their previous glycemic control and intervention studies have demonstrated that microvascular complications are highly correlated with mean glycemic control as measured by glycolated hemoglobin A1C (HbA1c). The present study was carried out to evaluate the autoantibodies against glycosylated DNA (DNAAGEs) in diabetic sera to see if the level of HbA1c has correlation with the activity of DNA-AGEs, another marker of chronic glycemia.Methods: Glucose-6-phosphate induced glycosylation of native DNA was studied in 150 diabetic sera (T1DM = 9, T2DM = 141) by spectroscopic techniques (UV and fluorescence) and agarose gel electrophoresis. Direct binding and inhibition enzyme immunoassays were carried out to evaluate binding and specificity of anti-glycated-DNA autoantibodies (anti-DNA-AGE autoantibodies) in sera of diabetes patients. A quantitative estimation of HbA1cwas carried out in normal and diabetes sera.Results: Anti-DNA-AGEs autoantibodies in 55% of diabetic sera (85/150) showed more binding with glycated-DNA compared to native DNA which was subjected to inhibition ELISA indicating true interaction of these autoantibodies in diabetes with glycated DNA. Higher binding with glycosylated-DNA against native-DNA was observed in subjects with HbA1c of 9.8 ± 3.3% compared to those with HbA1c of 7.7 ± 1.7% (p &lt; 0.001). Linear correlation analysis showed that mean absorbance difference was significantly related to HbA1c (r= 0.486, p &lt; 0.001), nephropathy (r= 0.239, p &lt; 0.003), retinopathy (r= 0.165, p &lt; 0.05).Conclusions: Autoantibodies against glycosylated DNA were correlated with HbA1cand microvascular complications and may be useful as another biomarker for assessment of chronic glycemia.

Proteolytic degradation by cathepsin D of glycated hemoglobin from diabetes patients gives rise to hemorphin-7 peptides

Previous studies showed a significantly reduced level of hemorphins in the serum of diabetes patients. In order to elucidate the biochemical mechanisms responsible for this anomaly, the influence of hemoglobin glycation on hemorphin generation was studied. The glycation of hemoglobin occurs in the blood of diabetes patients and this could modify its enzymatic digestion and the resulting proteolytic products. Several samples of hemoglobin were obtained from the blood of type 1 diabetes patients ( n = 8) and normal healthy control subjects ( n = 2). The glycated hemoglobin samples were classified on the basis of their HbA1c values expressed as a percentage of total hemoglobin. Four solutions of glycated hemoglobin characterized by HbA1c values of 6%, 9.1%, 10.7% and 12.1% were treated with cathepsin D and the hemorphins obtained following the proteolysis were compared to controls. It was found that hemorphins were produced whatever the level of glycation of hemoglobin and also that the degree of glycation had no effect on the quantity of hemorphins released. Thus the alteration of hemoglobin does not seem to be the essential reason for the decrease in hemorphin concentrations in the sera of diabetic patients.

T cell reactivity to DR*0401- and DQ*0302-binding peptides of the putative autoantigen IA-2 in type 1 diabetes.

Type 1 diabetes is thought to be an autoimmune disease mediated by T lymphocytes recognizing critical islet cell antigens. Recently, the tyrosine phosphatase like protein IA-2 was suggested as a putative autoantigen in type 1 diabetes since autoantibodies are detected in sera of diabetic patients and prediabetic subjects. Similarly, T cell responses of peripheral blood lymphocytes of type 1 diabetic patients to this protein have been described. Only very few data is available about immunodominant epitopes of IA-2 recognized by T cells. We have studied T cell responses in type 1 diabetic patients and age and partly HLA matched controls to IA-2 peptides designed to bind HLA risk alleles of IDDM as DR*0401 and DQ*0302. Both diabetic patients and controls responded to IA-2ic and some of the peptides. Three peptides of the C-terminal region of IA-2 were recognised by T cells of a fraction of diabetic patients but at least two of these peptides triggered also T cell responses in DR*0401/DQ*0302-matched controls. Most peptides bound to different HLA alleles ("promiscous binders"). The identification of autoantigenic epitopes may offer clues to related sequences e.g. of viral origin what relates to models of diabetes pathogenesis ("molecular mimicry"). Secondly, the design of antigen- or even epitope-specific immune intervention strategies aiming at tolerization of disease specific T cells in type 1 diabetes may profit from the knowledge of immunodominant T cell epitopes of a putative autoantigen.

Preferential recognition of Amadori-rich lysine residues by serum antibodies in diabetes mellitus: Role of protein glycation in the disease process

This study analyzes effect of glycation on proteins rich in lysine residues as hyperglycemia induced protein glycation has been mainly reported in diabetes mellitus at the intrachain lysine residues leading to the formation of Amadori modified proteins. We have studied the effect of glucose on poly- l-lysine (PLL), a homopolymer of lysine residues. Levels of Amadori products in the glycated PLL were evaluated by fructosamine assay and the presence of 5-hydroxymethylfurfural (HMF) in the glycated PLL was analyzed by thiobarbituric acid assay. Fluorescence and FT-IR spectroscopy were applied to characterize the modified PLL. Binding characteristics of experimentally induced antibodies against glycated PLL and the presence of antibodies against glycated PLL in the sera of diabetes patients was evaluated by solid phase enzyme immunoassays. The fructosamine assay showed significantly high yield of early glycation (Amadori) products in the glycated PLL, which was confirmed by increased yield of HMF from Amadori products of glycated PLL. Loss in fluorescence intensity and appearance of a new band corresponding to Amadori products were observed in FT-IR spectrum of the glycated PLL. Glycated PLL was found to be highly immunogenic in rabbits as compared to the native form. Serum antibodies from diabetes patients showed appreciably high recognition of the glycated PLL. The results conclusively show the glycation induced damage to the lysine molecules and specific recognition of Amadori-lysine residues by serum antibodies from diabetes patients. The glycated lysine residues may serve as a diagnostic biomarker for early glycation process in diabetes mellitus.

Significant lower VVH7-like immunoreactivity serum level in diabetic patients: Evidence for independence from metabolic control and three key enzymes in hemorphin metabolism, cathepsin D, ACE and DPP-IV

Low circulating VVH7-like immunoreactivity (VVH7 i.r) level was amazingly observed in human diabetic sera. Here, we examined the impact of diabetes type, clinico-biological features and metabolic control on circulating VVH7 i.r level in this disease. ELISA test was used to measure VVH7 i.r in sera of 120 diabetic patients (type 1 diabetes in 64, type 2 diabetes in 56). Three enzymatic tests were also applied to determine serum cathepsin D (CD), dipeptidyl peptidase IV (DPP-IV) and angiotensin-converting enzyme (ACE) activities. A subgroup of 24 type 1 diabetic patients negative for microalbuminuria and hypertension were submitted to an ambulatory blood pressure monitoring to evaluate the relationship between VVH7 i.r level and blood pressure parameters. The mean serum concentration of VVH7 i.r was drastically reduced in diabetic patients (0.91 ± 0.93 μmol/l versus 5.63 ± 1.11 μmol/l in controls) ( p < 0.001). A negative correlation between VVH7 i.r level and daytime diastolic blood pressure existed in type 1 diabetic patients. There was no association of low VVH7 i.r with either type of diabetes or HbA1c level. An increase of cathepsin D activity was found in serum of diabetic patients compared to controls (0.47 U/ml versus 0.15 U/ml, respectively) whereas DPPIV activity was significantly decreased in diabetic sera (50.81 U/ml versus 282.10 U/l respectively). Diminution of VVH7 i.r in sera of diabetic patients was confirmed but still remained unexplained. Relationships between higher systolic blood pressure and decrease of VVH7 i.r reinforce the need to investigate this pathway in this disease to elucidate its role in macro- and micro-angiopathy.

Clinical Indications for Ophthalmic Corticosteroids

Advanced glycation end products (AGEs) have shown to possess antigenicity. This study analyzes the detrimental effect of non-enzymatic glycation on human serum albumin (HSA) leading to the production of antibodies.HSA (20 μM) incubated with d-glucose formed AGEs confirmed by scanning electron microscopy (SEM). DNA-damage was assessed with comet assay. Antibodies against in-vitro formed AGEs was evaluated in the sera of diabetic patients by enzyme-linked immunosorbent assay. Molecular docking was performed to demonstrate affinity of native and glycated-HSA with IgG. Low-grade systemic inflammation was quantified with IL-4, IL-6, TNF-α and NF-кβ in serum and mRNA expression.Scanning Electron Microscopy showed the formation of aggregates in glycated-HSA. Comet assay showed DNA damage T2DM with CKD. Serum auto-antibodies in diabetes patients with chronic kidney disease (CKD) showed appreciably high recognition with glycated-HSA compared to native HSA. Molecular docking showed less affinity of glycated-HSA with IgG. Serum IL-4, IL-6, and TNF-α were found significantly higher in T2DM with CKD compared to T2DM and healthy ones. mRNA expression of IL-4, IL-6 and NF-кβ are also found significantly higher in T2DM with CKD.The non-enzymatic glycation-induced damage to the HSA generate neo-epitopes that possess immunogenic response and low-grade systemic inflammation.