Triolo G

Glucose-induced loss of glycosyl-phosphatidylinositol-anchored membrane regulators of complement activation (CD59, CD55) by in vitro cultured human umbilical vein endothelial cells

Aims/hypothesis. This study examines whether increased glucose concentrations are responsible for a decreased expression of membrane regulators of complement activation molecules. The effect of high glucose in determining an increase in membrane attack complex deposition on endothelial cells was also investigated. Methods. Endothelial cells were isolated from umbilical cord tissue, cultured in the presence of increased concentrations of glucose, and the expression of CD46, CD55, and CD59 was detected by ELISA (enzyme-linked immunosorbent assay) and by flow cytometry. Glucose-treated endothelial cells were also incubated with antiendothelial cell antibodies and fresh complement to assess the amount of membrane attack complex formation. Results. High concentrations of glucose decreased the expression of CD59 and CD55 by endothelial cells in a time-dependent and glucose concentration-dependent manner without affecting CD46 expression. High concentrations of soluble CD59 were found in the supernatants of cells treated with high glucose. The decrease in CD59 expression induced by high glucose concentrations was reversed by coincubation of cells with a calcium channel blocking agent (Verapamil). All of these effects were not reproduced by osmotic control media. Cells treated with concentrations of high glucose were more susceptible to complement activation and membrane attack complex formation after exposure to antiendothelial cell antibodies. Conclusion/Interpretation. We speculate that hyperglycaemia could directly contribute to a loss of CD59 and CD55 molecules through a calcium-dependent phosphoinositol-specific phospholipase C activation and subsequent regulation of cell wall expression of GPI-anchored proteins. This phenomenon could facilitate the activation of a complement pathway and could play a part in the aetiology of endothelial dysfunction in diabetes. [Diabetologia (2000) 43: 1039–1047]

IgG anti‐endothelial cell antibodies (AECA) in type I diabetes mellitus; induction of adhesion molecule expression in cultured endothelial cells

AECA were detected in 25 of 71 patients with type 1 diabetes mellitus and in two of 33 healthy subjects. Patients with diabetes of < 1 year duration and those with long‐standing disease had the highest levels of these antibodies. Inhibition studies suggest that at least part of the AECA reactivity is due to cross‐reactive anti‐ssDNA antibodies. AECA‐positive sera were able to increase intercellular adhesion molecule‐1 (ICAM‐1) and E‐selectin on human umbilical vein endothelial cells (HUVEC). Increased binding of polymorphonuclear (PMN) cells was also found to accompany raised E‐selectin expression. Soluble ICAM‐1 and E‐selectin were also found to be increased in the sera of AECA‐positive patients. An effect of AECA on endothelial cell function is suggested in diabetes mellitus.

Detection of anti-myeloperoxidase antibodies in the serum of patients with type 1 diabetes mellitus

Anti-myeloperoxidase (anti-MPO) antibodies were detected in 34 of 88 (38%) patients with type 1 diabetes mellitus but in only 3 of 55 (5.7%) healthy subjects and in 4 of 20 patients with autoimmune disease. Specificity of anti-MPO antibodies was assessed by MPO inhibition studies. No relationship was found between the occurrence of anti-MPO and anti-thyroperoxidase antibodies. Levels of soluble intercellular adhesion molecule 1 (ICAM-1) were found to be higher in anti-MPO antibodypositive (n=28, 508±126 ng/ml) than in anti-MPO antibody-negative (n=58, 438±140 ng/ml;P<0.05) patients. A state of chronic neutrophil activation has been described in diabetes mellitus. As anti-MPO antibodies can stimulate neutrophils to damage endothelial cells in systemic vasculitis, this suggests that a similar mechanism may be operative in the development of diabetic angiopathy.

Anti-single-stranded DNA antibody in the sera of patients with type 2 diabetes mellitus

Abstract Anti-single-stranded(ss)DNA antibodies were searched for by enzyme-linked immunosorbent assay (ELISA) in the serum of 202 outpatients with non-insulin-dependent diabetes mellitus and 135 healthy subjects to investigate their prevalence in the serum of patients with type 2 diabetes and their relationship with the presence of vascular complications. Of the 202 patients 128 had vascular complications. Anti-ssDNA antibodies were observed to be significantly more frequent in the serum of patients with vascular complications (33.6%) and in particular in patients with overt nephropathy (50%) than in patients without complications (6.7%) or controls (6.7%). Anti-ssDNA antibodies have been previously described in patients with type 1 diabetes before clinical evidence of vascular disease, and their cross-reactivity with a variety of anionic biological molecules or cells, i.e. platelets and endothelial cells, assessed. It seems not unreasonable that these autoantibodies detected in patients with type 2 diabetes could be of importance in the pathogenesis or progression of angiopathy.

Two-site ELISA for quantification of the terminal C5b-9 complement complex in plasma: Use of monoclonal and polyclonal antibodies against a neoantigen of the complex

A quantitative ELISA procedure using monoclonal and polyclonal antibodies against neoantigens of the terminal C5b-9 complement complex has been developed. The ELISA was demonstrated to be both sensitive and reproducible. The normal range for C5b-9 determinations, defined as 2.5-97.5% interval of the values obtained in 76 healthy blood donors, was 3.12-10.3 AU/ml. The presence of rheumatoid factor did not affect the determination of C5b-9 as demonstrated by immunoabsorption studies.

The Terminal Complement Complex (TCC) in the Plasma of Patients with Type 1 Diabetes Mellitus. Relationship with Albumin Excretion Rate

Increased TCC levels were found to be present in the plasma of patients with type 1 diabetes associated with the presence of anti-heparan sulfate antibodies and increased urinary excretion of albumin. The authors suggest a role of terminal complement activation in the pathogenesis of diabetic nephropathy.