OBJECTIVETo investigate the effect of cytomegalovirus infection on expression of matrix metalloproteinase-2 in human endothelial cells.

METHODSHuman umbilical vein endothelial cells were cultured in vitro. Cells between 3-6 passages were infected with cytomegalovirus for different time. Expression of matrix metalloproteinase-2 mRNA was analyzed by reverse transcription-polymerase chain reaction, matrix metalloproteinase-2 activity was detected by gel zymography.

RESULTSExpression of matrix metalloproteinase-2 mRNA and its activity 6 hours after infection was almost equal to control, and was greatly enhanced 12 and 24 hours after infection.

CONCLUSIONCytomegalovirus infection up-regulates expression of matrix metalloproteinase-2 in human endothelial cells. It might be one of the mechanisms that cytomegalovirus is involved in atherosclerosis.

Cells, Cultured ; Cytomegalovirus ; physiology ; Endothelial Cells ; cytology ; metabolism ; virology ; Gene Expression Regulation, Enzymologic ; Host-Pathogen Interactions ; Humans ; Matrix Metalloproteinase 2 ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Umbilical Veins ; cytology

Diphtheria toxin A fragment (DTA) is an essential catalytic domain of diphtheria toxin (DT)-based immunotoxin. DTA protein and its antibodies play an important role in the studies on toxicology, purification and identification of DT-based immunotoxins. In this paper, DTA was expressed and purified from E. coli. After Q-Sepharose FF chromatography and (Ni+)-Sepharose affinity chromatography, 6 x His-DTA fusion protein with 90% purity was achieved. Using the purified DTA as antigen to immunize BalB/c mice, 2 hybridoma cell lines (designated as 3B6 and 3B9, respectively) secreting monoclonal antibodies (McAbs) against DTA were established. Investigations showed that both McAbs were characterized as IgG1 with titers of 1: 10(6). The binding of the McAbs to DTA was competitively inhibited by horse sera against DT. The fact that anti-DTA McAbs could be used in western blot analysis and affinity chromatography purification of DT-based immunotoxins implied that they will be useful agents in the studies on DT-based immunotoxins.
Animals ; Antibodies, Bacterial ; genetics ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Chromatography, Affinity ; Diphtheria Toxin ; immunology ; Escherichia coli ; genetics ; Female ; Immunotoxins ; isolation & purification ; Mice ; Mice, Inbred BALB C ; Peptide Fragments ; immunology ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification

Objective The goal of this study was to investigate whether murine cytomegalovirus (MCMV) is able to exacerbate the atherosclerotic process in apolipoprotein E knockout (apoE-/-) mice,and the effect of fluvastatin on the atherogenesis. Methods The apoE -/- mice kept on a west diet were given low dosage of MCMV. At 14,18 and 24 weeks post infection, AS lesion were measured on aorta. The fluvastatin was administered,and AS lesion were measured accordingly above. Results We observed that in the chronic phase of the infection, AS lesion area was significantly increased. MCMV gB mRNA was not amplified by real-time PCR from the arterial wall. The IgG antibody level of MCMV in blood plasma and the content of virus DNA in salivary gland were not correlated with AS lesions. After the administration of fluvastatin, there was no significant difference of AS lesions between MCMV infected group and mockinfected group. Conclusion MCMV may aggravate the AS lesion in apoE -/- mice in the chronic phase of infection, and promote more severe type of AS lesions. But it might not be the direct effects of mechanism of MCMV on the local lesion of AS. Fluvastatin could meliorate the progression of AS after MCMV infection,but this was not accomplished by decreasing MCMV duplication.