Detection of elastin derived peptides in cerebrospinal fluid of patients with first ever ischaemic stroke.

OBJECTIVE: We have previously reported the optimized methods for the detection of elastin derived peptides (EDP) in the serum, synovial fluid, and bronchoalveolar lavage. The aim of the present study was to investigate whether EDP are detectable in cerebrospinal fluid (CSF) of patients with acute brain ischaemia. PATIENTS AND METHODS: Twenty-seven first ever ischaemic stroke patients (mean age 61.5+/-10.8 years; age range 47-70 years; 12 women) were studied in acute phase (1-15 days after the onset) with clinical evaluations, radiological assessments, and the analysis of serum and CSF based on Western blot and ELISA for the detection and quantification of EDP. RESULTS: None of the serum EDP concentrations are significantly higher in stroke patients compared with 25 healthy control individuals. However, EDP levels in CSF are strongly (p<0.0001) elevated compared with healthy subjects. They correlated with total cholesterol (r=0.53; p=0.02), triglycerides (r=0.67; p=0.004) and retinopathy (r=0.24; p=0.03), and with the interval between the stroke onset and the time of lumbar puncture (r=0.35; p=0.02). CONCLUSION: EDPs are detectable in CSF of healthy subjects and patients with ischaemic stroke. Acute brain infarction is followed by increased levels of EDP in CSF.

Serum antibodies to collagen type IV and development of diabetic vascular complications in children with type 1 (insulin-dependent) diabetes mellitus. A longitudinal study.

Thickening of basement membrane in capillaries and small vessels is a well-known finding and important in the progression of diabetic microangiopathy. To monitor the metabolism of the basement membrane protein collagen type IV (CIV) in diabetes mellitus, serum levels of IgG, IgM and IgA to CIV were measured using an ELISA method in 28 children with Type 1 diabetes mellitus over a period of 6 years. These values were compared to serum antibodies to CIV in 24 age- and sex-matched controls. At the end of the study, 11 children had diabetic microangiopathy. IgG to CIV was associated with age (r = .33, P = .026), diabetes duration (r = .32, P = .021), HbA1c (r = .31, P = .019), microalbuminuria (r = .32, P = .022) and anti-AGE antibodies (r = .47, P = .0007). IgM to CIV correlated with age (r = .46, P = .001), diabetes duration (r = .45, P = .001), HbA1c (r = .26, P = .038) and anti-AGE antibodies (r = .26, P = .038) and IgA to CIV with triglycerides (r = .29, P = .038) and anti-AGE antibodies (r = .44, P = .0025). We suggest that serum levels of IgG to CIV can be used as a marker for the development of diabetic microalbuminuria.

Antibodies to advanced glycation end products in children with diabetes mellitus.

The tissue accumulation of advanced glycation end products (AGE) alters the structure and function of long-lived proteins. A number of studies have shown that tissue accumulation of AGE correlates with the severity of diabetic complications. Proteins containing AGE are highly immunogenic and anti-AGE antibodies were found in sera of diabetic rats and human. Considering the potential use of anti-AGE antibodies as a marker of AGE deposition during diabetes, we have investigated, by competitive ELISA, the presence of anti-AGE antibodies in sera of 58 children with Type 1 diabetes mellitus. The patients were studied for the period of 5 years. Positive for anti-AGE antibodies were 19 children with diabetes. Fourteen of them showed initial data for vascular complications. Anti-AGE antibodies were related to age (r = .25, P = .024), duration of diabetes (r = .41, P = .0001), HbA1c (r = .27, P = .016), microalbuminuria (r = .41, P = .0001), retinopathy (r = .35, P = .001), triglycerides (r = .27, P = .016), and total cholesterol (r = .19, P = .05). In conclusion, our study showed that the investigation of the levels and dynamics of anti-AGE antibodies might give the possibility for early diagnosis and prognosis of the severity of diabetic late complications.

Detection of serum collagen type IV in children with type 1 (insulin-dependent) diabetes mellitus--a longitudinal study.

The basement membrane is a major focus of scientific interest because of its role in a variety of diseases. In diabetes mellitus, a thickening of the capillary basement membrane results in microangiopathic lesions. To monitor the metabolism of the basement membrane protein collagen type IV (CIV) in diabetes mellitus, serum levels of CIV were measured using an enzyme-linked immunosorbent assay (ELISA) method in 28 children with type 1 diabetes mellitus over a period of 6 years. These values were compared to serum CIV levels in 24 age and sex matched controls. In the first 3 years, serum CIV levels were normal. In the 4th year, 1 patient and in years 5 and 6, 4 patients had increased CIV serum levels. At the end of the investigation, 3 children had developed retinopathy, 6 microalbuminuria, and 2 both microalbuminuria and retinopathy. Only those patients with microalbuminuria had increased CIV serum levels. In conclusion, we suggest that CIV serum levels can be used as a marker for the development of diabetic microalbuminuria.

Relationship between elastin-derived peptides and the development of microvascular complications: a longitudinal study in children with Type 1 (insulin-dependent) diabetes mellitus.

Levels of elastin-derived peptides (EDP) were determined by enzyme-linked immunosorbent assay (ELISA) in sera of 28 children with Type 1 (insulin-dependent) diabetes mellitus (mean age 11.6+/-2.8 years, diabetes duration 5.1+/-2.5 years). None of the children had clinical or laboratory evidence of vascular complications. The children were followed over a period of 6 years, and 24 healthy children of similar age and sex served as a control group. During the investigative period, 10 diabetic patients had increased EDP levels, with 9 having been diabetic for more than 5 years and 1 patient less than 5 years. Seven of these patients developed diabetic microvascular complications. In this group, EDP were independently associated with age (r=.39, P=.047), retinopathy (r=.48, P=.034), and antibodies to advanced glycation endproducts (AGE) (r=.52, P=.018). The data of this pilot study are not strong enough to appear that EDP are a useful predictor of subsequent development of microvascular complications. This may be due to the small number of subjects, short duration of the study, manner in which EDP or the endpoints were measured, or frequency of which EDP measurements were made. Further prospective and longer studies of larger populations are needed to identify the role of EDP as an early marker for the development of diabetic microvascular complications.

An association of anti-elastin IgA antibodies with development of retinopathy in diabetic children.

An important factor in the development of vascular wall alterations is degradation of the elastic fiber major protein-elastin. Elastin peptides derived from this degradation are present in the circulating blood and they are a stimulus for increased production of anti-elastin antibodies (AEAb). The aim of the present study was to examine the possible association between serum elastin AEAb and the development of diabetic vascular complications. Levels of AEAb (IgG, IgM and IgA) were determined by ELISA in sera of 28 children with Type 1 (insulin-dependent) diabetes mellitus (mean age 11.6+/-2.8 years, diabetes duration 5.1+/-2.5 years). None of the children had clinical or laboratory evidence of vascular complications. The children were followed over a period of 7 years, and 24 healthy children of similar age and sex served as a control group. During the study, four diabetics developed retinopathy, six microalbuminuria and two both retinopathy and microalbuminuria. Anti-elastin IgG showed correlation with diabetes duration (r=.48, P=.0007), HbA1c (r=.28, P=.05), triglycerides (r=.28, P=.05) and antibodies to advanced glycation endproducts (AGE) (r=.41, P=.005). Anti-elastin IgM correlated with HbA1c (r=.26, P=.038) and IgA with retinopathy (r=.32, P=.017). Our results suggest an association between the level of anti-elastin IgA antibodies and the development of diabetic retinopathy.

Humoral and cellular immune response to elastin in patients with systemic sclerosis.

The humoral immune response against elastin and collagen was studied in parallel with the delayed type hypersensitivity (DTH) reaction to elastin and the percentage of lymphocyte subpopulations in peripheral blood in 20 patients with systemic sclerosis (SSc). An increase of anti-elastin antibodies of all subclasses was found with a significant prevalence of IgE and IgA antibodies. The profile of anti-collagen type I and type IV antibodies showed an increase of IgE isotypes. In 25% of the patients (5 out of 20) positive DTH reactions to elastin were observed as compared to the negative skin reactions in all control individuals. At the same time a significant hyporeactivity to common bacterial and mould antigens was found in 40% of the patients (versus 16% in the control group) which could be an explanation for the low incidence of positive anti-elastin DTH reaction. The DTH hyporeactivity in SSc cases was in contrast with the increased percentage of CD4 T cells (58.4 vs. 42.0) and increased CD4/CD8 ratio (2.5 vs. 1.5) in the peripheral blood of the patients. This finding together with the increased IgE antibodies to elastin and collagen type I and type IV might suggest a possible shift of the immune balance towards the Th2 type of immune response. This is in line with the increased CD8+CD57+ cells which correlated with the highest number of other parameters studied - disease duration, total skin score, IgE anti-elastin antibodies, IgG anti-collagen type I antibodies, CD4/CD8 ratio and CD19 B cells. The results of this study demonstrated the existence of both humoral and cell-mediated immune response against elastin in SSc patients. However, we could not define whether this was an essential part of pathogenetic mechanisms or a secondary phenomenon reflecting the extent of the damage of connective tissue.

Determination of anti-AGE antibodies in human serum.

The aim of this study was to develop an immunoenzyme method for the determination of anti-AGE antibodies in human serum. Human aortic elastin glycated in vitro (AGE-elastin) was used as an antigen, expressing AGE-epitopes, common to all glycated proteins. Polyclonal serum from guinea-pig against AGE-Hemocyanin was obtained according to Nakayama et al. [(1989) Biochem Biophys Res Commun 162: 740-45] and its specificity was tested via direct and competitive ELISA. Sera of 20 type 1 diabetic patients and 20 healthy subjects were tested using the method described. Seventeen patients had elevated levels of competing factors that may be anti-AGE antibodies, compared with the healthy group. The method could be used for investigation of different clinical groups of type 1 diabetic patients. Such a study would help in understanding the pathogenic role of autoantibodies against advanced glycation end products of proteins for the development of long-term diabetic complications.

Age-related changes in the levels of IgG, IgM and IgA antibodies to type I collagen in the sera of normal human subjects.

Serum samples from healthy subjects of different ages (within the age range of 1-75 years) were tested for the presence of anti-type I collagen (anti-CI) IgG, IgM and IgA by enzyme-linked immunosorbent assay (ELISA). All the tested sera showed anti-CI antibodies from the three immunoglobulin classes with the following age-related regularities: 1. Anti-CI IgG and IgM showed statistically non-significant changes up to the age of 60 decreasing thereafter. 2. Anti-Ci IgA changed negligibly up to the age of 40 increasing with age thereafter. The established age-related changes in the levels of anti-CI antibodies may serve as a basis for future studies of normal and pathological turnover of type I collagen.

Age-dependent changes in the level of antielastin antibodies of different immunoglobulin classes (IgG, IgM, IgA and IgD) in the human serum.

Serum samples from healthy subjects of different ages (within the age range 1-75 years) were tested for the presence of antielastin IgG, IgM, IgA and IgD by an enzyme-linked immunosorbent assay, utilizing insoluble human aortic elastin. All the tested sera showed detectable levels of antielastin antibodies of the four classes with the following regularities in changing their level with age. Antielastin IgG and IgM showed relatively high levels in the serum of children, growing even higher in the serum of subjects 18-20 years old. Then their levels were stabilized in the serum of 30-60 year olds for IgG and of subjects 30-50 years old for IgM and gradually decreased thereafter. The antielastin IgA showed non-significant changes with age up to the age of 40 and then its level gradually increased. The antielastin IgD showed statistically non-significant changes with age and a tendency to decrease after the age of 60.

Determination of elastin-antielastin circulating immune complexes in serum of normal subjects and atherosclerotic patients.

Healthy subjects of different ages and a group of atherosclerotic patients were tested for the presence of elastin-antielastin circulating immune complexes (CIC) in their sera. For the purpose the authors used a method based on sequential precipitation with rising concentrations of polyethylene glycol (PEG), dissociation of the precipitated CIC and further analysis of the resuspended precipitates for the presence of elastin derived peptides by an enzyme-linked immunosorbent assay (ELISA). Elastin-antielastin CIC were detected only in the serum of atherosclerotic patients and that of healthy subjects over 60. The elastin-antielastin CIC obtained from the serum of atherosclerotic patients were precipitated by higher PEG concentrations mainly and had a higher elastin content in comparison with those isolated from the serum of healthy persons over 60.

Immunological investigations on the antigenic changes of human aortic elastin in aging and atherosclerosis.

Alpha-elastin of human aorta was investigated by an enzyme-linked immunosorbent assay (ELISA) on the material isolated from aborted human fetuses and healthy subjects killed by accident, assigned to 7 age groups. Samples up to the age of 55 were taken only from regions without detectable changes in the arterial wall, in the 60-75-year age group--both from normal areas and atherosclerotic plaques of the same aortas. An immune serum against alpha-elastin isolated from the normal aortic areas of a 61-year-old subject was produced in sheep. Testing with this serum showed the existence of some antigenic changes in the elastins from different age groups. A prevalence of the species-specific antigenic determinants was observed in alpha-elastin from fetal aorta while in alpha-elastin from the atherosclerotically altered human aorta the cross-reacting antigenic determinants prevailed in its antigenic structure.

Detection of elastin-antielastin circulating immune complexes (CIC) in diabetic patients with vascular damage.

Healthy subjects aged between 25 and 60 (20 cases) and between 61 and 65 (5 cases), and diabetic patients with vascular damage, aged between 24 and 62 (6 cases), were tested by a new method for the detection and identification of elastin-antielastin circulating immune complexes (CIC) in human sera. Such immune complexes were found in all patients' sera and only in one of the controls (at the age of 65). Among different patients, the elastin-antielastin CIC varied in size and elastin content, showing some correlation between these two characteristics and the existence of microvascular complications, as proved by the clinical and paraclinical investigation of the patients.

Investigation of the organ immunospecificity of human elastin by an enzyme-linked immunosorbent assay (ELISA).

The aim of the study was to investigate the organ immunospecificity of human elastin using an enzyme-linked immunosorbent assay (ELISA). Soluble alpha-elastin has been isolated from human aorta and lung. Antibodies against human lung alpha-elastin have been raised in young male sheep. No evidence for the existence of an organ immunospecificity of human elastin has been found.

Age-related changes in the level of circulating elastin-derived peptides in serum from normal and atherosclerotic subjects.

The level of the circulating elastin-derived peptides (CEDP) in the serum is believed to reflect the activity of the degradation of the elastic structures. This paper reports a new method, based on the 'sandwich' version of the enzyme-linked immunosorbent assay (ELISA), for the detection and quantification of CEDP in human serum. By this method we investigated the age-related changes in their level among healthy subjects within the age range of 1 and 75 years and among atherosclerotic subjects aged 50 to 75 years. The highest level of CEDP was found in the serum of the atherosclerotic patients, and the lowest, among the healthy subjects between 18 and 50 years of age.

Age-related changes in anti-elastin antibodies in serum from normal and atherosclerotic subjects.

A modified version of the enzyme-linked immunosorbent assay (ELISA), utilising human insoluble aortic elastin, was used for determination of anti-elastin antibodies in serum from normal and atherosclerotic subjects. The age-related changes in their level among healthy persons were investigated. Anti-elastin antibodies were found in all the tested human sera, showing the highest level at the age of 18-20 and the lowest at the age over 60 and especially among atherosclerotic patients. The possible role of the immune system in the turnover of elastin is discussed.