Medizin - Open Access LMU - Teil 05/22 Archiv

Long-term cytolytic T-lymphocyte (CTL) lines that are specific for distinct antigens associated with different phases of the replicative cycle of the murine cytomegalovirus (MCMV) were established by cloning of CTL lines derived from lymph nodes of latently infected BALB/c mice. Two CTL clones were characterized in detail. Both displayed the Lyt-2+, L3T4- surface phenotype, and the recognition of their respective target antigens was class I (DLd) major histocompatibility complex antigen restricted. Clone S1 was specific for a structural antigen of MCMV, and clone IE1 detected an MCMV-specified immediate-early (IE) membrane antigen. Clone IE1 retained lytic activity, antigen specificity, and self-restriction after prolonged propagation in the presence of recombinant human interleukin-2 without restimulation by antigen. This interleukin-2-dependent line of the clone IE1, line IE1-IL, can serve as a reference line for the definition of the antigenic determinant IE1 of an IE membrane antigen.

To study trans-activation of gene expression by murine cytomegalovirus (MCMV) immediate-early (IE) proteins, the IE coding region 1 (ie1), which encodes the 89,000-Mr IE phosphoprotein (pp89), was stably introduced into L cells. A cell line was selected and characterized that efficiently expressed the authentic viral protein. The pp89 that was constitutively expressed in L cells stimulated the expression of transfected recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of viral promoters. The regulatory function of the ie1 product was confirmed by transient expression assays in which MCMV IE genes were cotransfected into L cells together with recombinant constructs of the CAT gene. For CAT activation by the ie1 product, a promoter region was required, but there was no preferential activation of a herpes simplex virus type 1 delayed-early promoter. All plasmid constructs that contained the intact coding sequences for pp89 induced gene expression in trans. The MCMV enhancer region was not essential for the expression of a functional IE gene product, and testing of the cis-regulatory activity of the MCMV enhancer revealed a low activity in L cells. Another region transcribed at IE times of infection, IE coding region 2, was unable to induce CAT expression and also did not augment the functional activity of ie1 after cotransfection.

The cloned murine cytolytic T-lymphocyte line IE1-IL and several sublines detect a murine cytomegalovirus immediate-early (IE) membrane determinant in conjunction with Ld class I major histocompatibility glycoprotein. The lines retained cytolytic activity, strict antigen specificity, and self-restriction even when adapted to long-term, antigen-independent growth in the presence of interleukin-2 only (M. J. Reddehase, H.-J. Bühring, and U. H. Koszinowski, J. Virol. 57:408-412). These attributes allowed us to use IE1-IL as a stable, monospecific probe for tracing the expression of the IE membrane antigen throughout the viral replication cycle. Presentation of the antigen at the cell membrane proved to be most effective when expression of IE genes in infected mouse embryo fibroblasts was selectively enhanced by consecutive cycloheximide-actinomycin D treatment, whereas without enhancement high numbers of IE1-IL cytolytic T lymphocytes were required to demonstrate the antigen in the IE phase. In the early phase of infection when IE genes were no longer transcribed, cytolysis was not observed, although IE proteins were detectable in the nuclei of the infected cells. Without application of inhibitors IE membrane antigen expression was most prominent during the late phase of infection. Reinitiation of transcription from the genomic region encoding the major IE protein (pp89) and de novo synthesis of pp89 correlated with this reexpression of the IE membrane antigen.

High concentrations of lipids in ascites fluid caused by peritoneal carcinomatosis have been described recently. Since their nature has not yet been clarified, we isolated ascitic lipids from 25 patients with various neoplasms for further characterization. After chromatography on Sephadex G-100 gels, the ascitic lipids were fractionated on a Biogel A-5m column in three peaks. The second and third peaks were identified as low and high density lipoproteins, which were most likely of plasmatic origin, and represented the major amounts of ascitic lipids. The first peak was eluted in the void volume, indicating a molecular weight over 5 million. It consisted, on the average, of 65.3% protein, 16.2% triglycerides, 7.4% phospholipids, and 7.0% cholesterol. In a CsCl gradient, this protein-lipid complex floated in the density range from 1.128 to 1.181 g/ml. Sodium dodecyl sulfate: polyacrylamide gel electrophoresis separated up to 11 protein subunits (Mr 29,000 to 97,000), and electron microscopy revealed globular particles of 36 to 64 nm in diameter. The macromolecular complex showed no immunological reaction against anti-{alpha}- and anti-ß-lipoproteins, but a single precipitation line against anti-liver-specific lipoprotein was seen. The biochemical characteristics of this protein-lipid complex proved to have a close relationship to liver-specific lipoprotein. It is most likely derived from cell membranes of the peritoneum detached by carcinomatosis.