Medizin - Open Access LMU - Teil 08/22 Archiv

Processing of endogenously synthesized proteins generates short peptides that are presented by MHC class I molecules to CD8 T lymphocytes. Here it is documented that not only the sequence of the presented peptide but also the residues by which it is flanked in the protein determine the efficiency of processing and presentation. This became evident when a viral sequence of proven antigenicity was inserted at different positions into an unrelated carrier protein. Not different peptides, but different amounts of the antigenic insert itself were retrieved by isolation of naturally processed peptides from cells expressing the different chimeric proteins. Low yield of antigenic peptide from an unfavorable integration site could be overcome by flanking the insert with oligo-alanine to space it from disruptive neighboring sequences. Notably, the degree of protection against lethal virus disease related directly to the amount of naturally processed antigenic peptide.

The gene regulatory immediate early protein, pp89, of murine cytomegalovirus interacts with both DNA-associated and isolated histones in vitro. We characterized the histone-binding region of pp89 and its cellular localization during cell division to examine the possible interaction between pp89 and chromatin. pp89 expressed constitutively in cell line BALB/c 3T3 IE1 does not interact with condensed chromatin. As observed in infected cells, pp89 is localized within the nucleus of cells during interphase but spreads throughout the cell plasma following degradation of the nuclear membrane during early mitosis. In late telophase, pp89 is reorganized within the nucleus. Analysis of pp89 deletion mutants and of fragments generated by cleavage at pH 2·5 revealed that the regions responsible for association with histone are located between amino acids 71 and 415, and are not identical with the domain that shows homology to histone H2B or the highly acidic carboxy-terminal region. A potential gene-activating role of the high affinity of pp89 for isolated histones and the low affinity for DNA-associated histones is discussed.

The effect of murine cytomegalovirus on myelopoiesis was studied in long-term bone marrow culture to find an in vitro correlate for the lethal virus interference with bone marrow reconstitution (W. Mutter, M. J. Reddehase, F. W. Busch, H.-J. Bühring, and U. H. Koszinowski, J. Exp. Med. 167:1645-1658, 1988). The in vitro generation of granulocyte-monocyte progenitors (CFU-GM) discontinued after infection of the stromal cell layer, whereas the proliferation and differentiation of CFU-GM to granulocyte-monocyte colonies remained unaffected. A protocol was established to probe the functional integrity of earlier hematopoietic cells. Pre-CFU-GM (the progenitors of the CFU-GM) could be recovered from an infected bone marrow donor culture by transfer onto an inductive recipient stromal cell layer. Thus, at least in vitro, infection of bone marrow stroma appears to be the only cause of the defect in myelopoiesis.

The immediate-early gene ie2 of murine cytomegalovirus was characterized. The 1.75-kb ie2 transcript is spliced from three exons, of 78, 124, and 1,283 nucleotides, which are separated by introns of 1,245 and 364 nucleotides. An ATG codon located in the third exon leads into an open reading frame of 391 codons. Immediate-early expression of the predicted polypeptide was confirmed by immunoprecipitation of a 43-kDa protein by using an antiserum raised against a bacterial fusion protein. The predicted IE2 amino acid sequence has regions with similarity to amino acid sequences of members of the human cytomegalovirus US22 family.

The regulatory immediate-early (IE) protein pp89 of murine cytomegalovirus induces CD8+ T lymphocytes that protect against lethal murine cytomegalovirus infection. The IE1 epitope is the only epitope of pp89 that is recognized by BALB/c cytolytic T lymphocytes (CTL). Using synthetic peptides, the optimal and minimal antigenic sequences of the IE1 epitope have been defined. To evaluate the predictive value of data obtained with synthetic peptides, recombinant vaccines encoding this single T-cell epitope were constructed using as a vector the hepatitis B virus core antigen encoded in recombinant vaccinia virus. In infected cells expressing the chimeric proteins, only IE1 epitope sequences that were recognized as synthetic peptides at concentrations lower than 10(-6) M were presented to CTL. Vaccination of mice with the recombinant vaccinia virus that encoded a chimeric protein carrying the optimal 9-amino-acid IE1 epitope sequence elicited CD8+ T lymphocytes with antiviral activity and, furthermore, protected against lethal disease. The results thus show for the first time that recombinant vaccines containing a single foreign nonameric CTL epitope can induce T-lymphocyte-mediated protective immunity.

While it is generally accepted that cholesterol supersaturation of bile is of key importance in the rapid formation of cholesterol crystals, the role of total biliary protein and pH in the pathogenesis of cholesterol gallstones is less well understood. The relation of cholesterol saturation, total protein, and pH was studied in 73 gallbladder bile samples with and 35 gallbladder bile samples without cholesterol crystals. In samples containing crystals, a trend to higher values of cholesterol and to a higher cholesterol saturation index was observed. However, significantly (P = 0.02) higher concentrations of total protein were found in samples with crystals [0.80 +/- 0.40 g/dL (8.0 +/- 4.0 g/L)] than in samples without crystals [0.63 +/- 0.26 g/dL (6.3 +/- 2.6 g/L)]. Moreover, of 22 bile samples with total protein concentrations greater than 10.0 g/L, cholesterol crystals were detected in all but 2. Total lipids, bile acids, phospholipids, and pH values were not significantly different in the two groups of bile samples. It was concluded that high biliary protein concentrations are frequently associated with cholesterol crystals and may, therefore, be a possible risk factor in the pathogenesis of cholesterol gallstones.