The Epstein-Barr virus-encoded glycoprotein gp 110 (BALF 4) can serve as a target for antibody-dependent cell-mediated cytotoxicity (ADCC).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is thought to play a major role in controlling the spread of the Epstein-Barr virus (EBV) in an infected individual. Recently, the viral membrane protein gp 350/220, which is also expressed at the surface of the virus producing cell, was identified as a target for ADCC reactions. Due to its glycoprotein nature, the EBV protein gp 110 is another possible ADCC target. It is one of the most abundant proteins found during the late phase of viral replication; until now, however, researchers have not been able to localize it on the surface of EBV positive cells. By means of recombinant vaccinia viruses containing the genes for gp 350/220 and gp 110, respectively, we expressed these proteins in lymphoblastoid cells, which were then used as targets in ADCC studies with sera from EBV-positive and -negative individuals. In these experiments we were able to demonstrate the feasibility of our approach for the investigation of EBV-specific ADCC reactions and could confirm the role of gp 350/220 as an ADCC target. Furthermore, we were able to show that gp 110 can also be recognized in an ADCC reaction, proving that at least some gp 110 molecules must be expressed at the cell surface.

The BI'LF4 trans-activator of Epstein-Barr virus is modulated by type and differentiation of the host cell.

We have analyzed the activity and regulated expression of a new Epstein-Barr virus (EBV) trans-activator (I'ta) encoded by left reading frame 4 (BI'LF4) of the BamHI I'fragment. The gene was detected in all genomes of established EBV strains and individual isolates, with the exception of B95-8, where the type-specific deletion of this open reading frame is tolerated in vitro. Specific trans-activation of two EBV promoters (early MS and I'ta promoter) could be shown in cotransfection assays. The I'ta product affected autoactivation but had no influence on heterologous target promoters. The I'ta promoter segment was shown to be costimulated in the process of host cell differentiation in the absence of other EBV gene products. Expression of the reading frame in bacteria identified a 48-kDa protein as a stable gene product. I'ta-specific antibodies were detected in sera from EBV-positive persons (nasopharyngeal carcinoma). When expressed with suitable eucaryotic vectors, a nuclear protein could be immunostained in transfected cells. Our experiments suggest a cell type-specific requirement for I'ta in the lytic cycle of EBV at a determined differentiation stage of the host cell.

Expression of class I major histocompatibility complex antigens in Epstein-Barr virus-carrying lymphoblastoid cell lines and Burkitt lymphoma cells.

Epstein-Barr virus (EBV) carrying lymphoblastoid cell lines (LCLs) and EBV-positive Burkitt lymphoma (BL) cells were compared for their expression of class I antigens of the major histocompatibility complex. Five common BL lines, LCLs, pokeweed mitogen-stimulated blasts and resting B-cells from healthy donors, and eight pairs of BL cells and LCLs, each pair originating from one patient, were tested. Quantitative analysis was performed using a radioimmunoassay; qualitative aspects were studied by one- and two-dimensional gel electrophoresis. In general, LCLs expressed significantly higher amounts of class I antigens than BL cells, the latter showing class I densities similar to or lower than peripheral resting B-cells. From analysis of the expression of class I-specific RNA, there is some evidence that class I antigen expression is regulated on the transcriptional level. In two BL cells studied, class I expression could be enhanced by gamma-interferon, whereas the corresponding LCLs seemed to be refractory to this treatment. One- and two-dimensional gel electrophoresis showed that in some BL lines, in addition to the generally lower class I expression, distinct class I specificities were down-regulated. None of these alterations in class I expression was EBV specific; however, they may well play a role in the recognition of BL cells and LCLs by cellular immune mechanisms. Thus, down-regulation of class I antigens may contribute to the resistance of BL cells to cytotoxic T-lymphocytes, whereas their enhanced expression may improve the recognition of EBV-infected LCLs.

Distribution pattern of alpha and beta myosin in normal and diseased human ventricular myocardium.

All fibers in three normal, four dilated, and two ischemic human ventricles were classified according to their myosin content using three sets of monoclonal antibodies each specific for one myosin heavy chain isoform (alpha, beta and beta'). Numerous fibers contained only beta myosin heavy chain (denoted as beta fibers), others contained either alpha and beta, or beta and beta' myosin heavy chain (denoted as alpha beta and beta beta' fibers, respectively). The percentages of alpha beta fibers were systematically determined along the walls of seven homologous regions of the ventricular myocardium. In all ventricles, there was an alpha beta-fiber transmural gradient, with less alpha beta fiber in the subendocardium than in the subepicardium. More alpha beta fibers were found in the right than in the left ventricular wall but there was no difference between the mid-portion and the apex of the free wall of each ventricle. The diseased ventricles contained a lower alpha beta fiber percentage than the normal hearts. beta beta' fibers were very rare in the normal ventricles (less than 5%) and almost inexistent in pathological hearts. The correlation between the mean alpha beta fiber percentages of the diseased hearts and their cardiac indices (r = 0.88, P less than 0.05) suggests that the small amount of alpha myosin distributed in a large number of ventricular fibers could play a role in the contractile performance of the heart. In conclusion, this study provides evidence for 1) an alpha beta fiber transmural gradient, and 2) a lower alpha myosin ratio in diseased than in normal human ventricle.

Local diversity of myosin expression in mammalian atrial muscles. Variations depending on age and thyroid state in the rat and the rabbit.

Rat, rabbit, pig, and bovine atrial myocardia were investigated with anti-alpha and anti-beta myosin heavy chain monoclonal antibodies. Analysis of atrial fibers by indirect immunofluorescence and assay of myosin heavy chains in tissue micro samples by immunoaffinity chromatography revealed both heterogeneity and plasticity in the atrial myosin heavy chains, undetected by electrophoresis of native atrial myosins under nondenaturing conditions. We found both alpha- and beta-like myosin heavy chains to be expressed in rat and rabbit, as they are in pig and bovine, atrial myocardia. They were regionally distributed within atrial muscles. The beta-like myosin heavy chains were present at much lower levels in rat and rabbit atria than in pig and bovine atria. Young rat atrial myosin was composed of only alpha-like heavy chains. In the rat and the rabbit, hyperthyroidism induced a beta- to alpha-like myosin heavy chain transition, which was considerable in the right atria and complete in the left atria. In the rat, thyroidectomy induced a moderate alpha- to beta-like myosin heavy chain transition, visible in the left atria. The significance of this atrial myosin heavy chain polymorphism is discussed in relation to the existence of anatomical localizations of the two myosin variants.

Evidence that positional information is used to establish the prestalk-prespore pattern in Dictyostelium discoideum aggregates.

Two contrasting mechanisms have been proposed for the establishment of the prestalk-prespore pattern in the multicellular aggregate of the simple eukaryote Dictyostelium discoideum. One involves intermingled, non-position-dependent cell differentiation followed by sorting out which produces the pattern of prestalk cells in the anterior region and prespore cells posteriorly. The second mechanism involves patterning according to the position of cells within the aggregate, in which case intermingled cell types are not expected. Here we use a monoclonal antibody (MUD1), recognising a prespore cell surface antigen, to study the initial appearance of prespore cells in aggregates. Quantitative studies were made with a flow cytometer and frozen sections were used to localise the cells expressing the prespore antigen. This antigen first appeared at the onset of tip formation in the centre of aggregates in a position-dependent fashion. The prespore antigen was not detected in the tip region or in streams of cells entering the aggregate. We re-examined the evidence on which the non-position-dependent differentiation model is based. Our results support the positional model for pattern formation.

An assay for pattern formation in dictyostelium discoideum using monoclonal antibodies, flow cytometry, and subsequent data analysis.

An assay for determining the proportions of prespore cells in a simple multicellular organism, the slug stage of Dictyostelium discoideum, was established using a prespore-specific monoclonal antibody and a fluorescence-activated cell sorter. Appropriate techniques for data analysis were developed. The effects of slug size and age were determined. Small slugs have a lower percentage of prespore cells than large slugs. The percentage of prespore cells increases and then decreases in slugs aged between a few hours and 9 days. Pronounced effects were observed on the size of cells in aging slugs. In particular unlabelled (mostly prestalk) cells were larger than prespore cells in young slugs, but after 6 days migration they became considerably smaller than prespore cells. The fact that all unlabelled cells were coordinately shifted in size, suggests that these cells (which comprise prestalk, prestalklike, and predisc cells) are related to each other.

Analysis of proportion regulation in slugs of Dictyostelium discoideum using a monoclonal antibody and a FACS-IV.

A quantitative assay for estimating the proportion of prespore cells in D. discoideum slugs was established by labelling disaggregated slug cells with a prespore specific monoclonal antibody and analysing the cell population with a FACS-IV. The method is validated using a wild-type strain and its stalky mutant. "Wild-type" strains have different proportions of prespore cells and it is demonstrated that slugs of some strains have an increased percentage of prespore cells when migrated in the dark compared to the light and in the presence of EGTA. The technique is rapid and will make possible genetic analysis of proportion regulation in D. discoideum.