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Studies on the mechanism of murine cytomegalovirus induced immunosuppression Whyte, Peter Frederick Muir


The present study was undertaken to elucidate the mechanism of murine cytomegalovirus-induced immunosuppression. Previous studies had established the immunosuppressive effect of murine cytomegalovirus (MCMV) in vivo, and in vitro, and had characterized a wide range of immunological function which were impaired during MCMV infection. Canditioned medium, prepared from the tissue culture medium of cultured spleen cells infected with MCMV, contained a soluble immunosuppressive factor which suppressed spleen cell response to stimulation by Con A. This virus induced immunosuppressive factor (VISF) was not present in conditioned media prepared from control cultures or from cultures of spleen cells inoculated with various other viruses or latex beads. VISF was produced within 24 hours after infection of the cells, and was not dependent upon the addition of exogenous fetal bovine serum to the culture medium. Attempts to extract VISF by lipid extraction techniques wer unsuccessful: no significant difference was observed between the capacities of control and MCMV conditioned media-extracted lipids to suppress Con A stimulation of spleen cells. Also, the production of VISF could not be abrogated by the addition of indomethacin, an inhibitor of prostaglandin synthesis, to the spleen cell cultures. From these studies, it was concluded that VISF was not a prostaglandin and was probably not any other type of lipid molecule. It was found that VISF could be concentrated in the residual volume of an ultrafiltration cell using an Amicon YM2 membrane filter (molecular exclusion limit of 1000 daltons), but VISF was not retained when an Amicon YM10 membrane filter (molecular exclusion limit of 10,000 daltons) was used. This indicated a molecular weight range for VISF of 1000-10,000 daltons. Gel filtration chromatography of concentrated samples of conditioned media on a Sephadex G-25 matrix revealed a low molecular weight fraction (MW 1400 daltons) of immunosuppressive activity present in the MCMV infected sample, but not in the control sample or in samples from spleen cells infected with other viruses or particles. When MCMV and control samples were digested with the proteolytic enzyme, proteinase K, prior to fractionation, this immunosuppressive activity was significantly diminished, but no significant change occurred in the corresponding control sample fraction. This suggested that VISF was a peptide or was, at least partly, peptide in nature. To correlate the above findings with immunosuppression in vivo, studies were performed on mice infected with a sublethal dose of MCMV. Virus-free serum collected four days after infection of the mice was found to contain immunosuppressive activity relative to control serum from uninfected mice. Fractionation of the serum by Sephadex G-25 column chromatography demonstrated several fractions in which the infected mouse serum was more suppressive than the fraction which contained the immunosuppressive activity from the MCMV conditioned medium sample, however the pattern was complicated by the number of other immunosuppressive substances present in both sera.

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