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Abstract-Matrix Gla protein (MGP) is synthesized in a vitamin K-dependent way in smooth muscle cells of the healthy vessel wall, and its mRNA transcription is substantially upregulated in atherosclerotic lesions. Here we report the preparation of a monoclonal antibody against human MGP and its use in an enzyme-linked immunosorbent assay. The intra-assay and interassay coefficients of variation in serum samples were 5.4% and 12.6%, respectively, and the lower detection limit was 8.5% of the normal serum value. Individual within-day variations were Ͻ11% and did not show a distinct circadian pattern. Day-to-day variations in fasting morning samples were Ͻ8%. In a first explorative survey, serum MGP concentrations were found to be significantly increased in patients with severe atherosclerosis, whereas these values were normal in those with low bone mass and osteoporosis. This finding is consistent with the high MGP mRNA expression observed in atherosclerotic vessels and plaques. More elaborate studies are required to assess the potential clinical utility of this newly developed assay.

In this paper the development of a battery of approximately 70 mouse monoclonal antibodies (McAbs) to RCMV-induced antigens and their characterization is discussed. Their reactivity with the whole scala of ca. 30 virus specific proteins was tested in an enzyme linked immunoassay (ELISA) whereas their ability to detect RCMV-antigens at different locations of in vitro infected cell cultures and at different stages of infection was tested by immunofluorescence. In order to determine to what specific (viral) protein each of these McAbs is directed against we used an immunoprecipitation technique, followed by SDS-PAGE. Furthermore, neutralizing capacity of each McAb was tested, as well as the immunoglobulin class they belong to. In this manner we defined six categories of monoclonal antibodies on the basis of immunofluorescence aspect. The six categories identify most important viral structural proteins.

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Viruses may be important in atherosclerosis as inciters of arterial injury or as modulators of the metabolism of the infected cells. Cytomegalovirus (CMV) is one of the members of the herpesvirus family ;that infect human beings. Virus was detected in arterial walls of patients with atherosclerosis (1). The effect of CMV on endothelial cells and on the metabolism of these cells was subject of this study.Human endothelial cells monolayers were established from cells obtained by collagenase treatment of human umbilical cord veins and arteries. The cells were grown in medium 199 containing growth factor and 20% fetal calf serum. All monolayers were near confluency at the time of viral infection. The cells were purified by FACS cell sorting using dilacLDL as marker (2). Infection of endothelial cells was performed using CMV laboratory strains ADI69 and Kerr and wild strains isolated from immunosuppressed patients (obtained by Dr. H.T. Wei land, Dept. Virology, University of Leiden, The Netherlands). Intracytoplasmic and intra-nuclear viral antigens were detected in 10-20% of the endothelial cells by fluorescence microscppy using TRITC-labeled monoclonal antibodies against CMV. Centrifugation of the cell monolayer during virus incubation resulted in an increase in the percentage of virus positive cells. The effect of virus infection on presence ot Tacxor VIII in the cells was studied using fluorescence microscopic examination of cell monolayers using a double staining technique. The presence of factor VIII in the cytoplasm of the cells was demonstrated using FITC-labeled antibodies and viral antigens were demonstrated by TRITC-labeled antibodies The results of these experiments showed that HCMV-infection of endothelial cells resulted in the disappearance of factor VIII from the cytoplasm of the endothelial cells at 48-72 hrs post infection without affecting the viability of the infected cells. The importance of this finding in relation to the function of the endothelial cells will be discussed.References:(1) Melnick, J.L. et al. The Lancet (1983): 644-647.(2) Voyta, J.C. et al. J. Cell Biol. 99 (1984): 2034-2040.