EBV-specific Antigens Research Articles

Induction of Epstein-Barr virus nuclear antigen and DNA synthesis in a human epithelial cell line after Epstein-Barr virus infection.

The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma is supported by the presence of EBV genomes in the epithelial elements of the tumor and by elevated antibody titers to EBV-specific antigens in the patients; the levels of these titers are related to the clinical course of the disease. However, since most laboratory data suggest that EBV is a B-lymphotropic virus, it is unclear how the virus becomes associated with the epithelial elements of the nasopharynx. The purpose of the present work was to find a human model system to study this association. A human epithelial line (U) was found that could be directly infected by EBV, and viral functions, the induction of EBV nuclear antigen and cellular DNA synthesis, were demonstrated. The U line was established in 1957 by the late H. J. Van Kooten (Kok-Doorschodt at the University of Utrecht), and although it is no longer diploid, it exhibits density inhibition. When U cells were infected with EBV, EBV nuclear antigen was expressed in 6 to 16% of the cells, 1 and 2 days after infection with B95-8 virus, but not with the P3HR-1 strain. No evidence for virus replication was obtained; immunofluorescence staining for early antigens and virus capsid antigens gave negative results. Quantitative adsorption experiments for EBV indicated that the adsorption capacity of U cells is significant (60% of Raji cells). The present results also demonstrated that infection with the virus overcomes block(s) in cellular DNA synthesis caused by 5-fluorodeoxyuridine. The induction of DNA synthesis was determined by increased incorporation of [3H]thymidine into the cells. The highest level of isotope incorporation was observed at about 15 h after infection and thereafter decreased. Analysis of the induced DNA indicated that it was of cellular origin.

Hodgkin's disease: establishment and characterization of four in vitro cell lies.

Four in vitro cell lines (L 428, L 439, L 538, and L 540) were established from different materials of three patients with Hodgkin's disease: pleural effusions, peripheral blood, and bone marrow. The histological diagnosis was confirmed in all cases by several independent histologists. All four cell lines have been in culture for over 6 months up to over 3 years. The neoplastic nature of the culture cells is indicated by the demonstration of several structural and numeric chromosome abnormalities associated with a monoclonal pattern of marker chromosomes. EBV-specific antigens (EBNA, VCA) were not detected in either cell line. Ia-like antigens, receptors for human T cells, acid phosphatase, and acid esterase were showen to be present in the cultured cells. All cell lines lacked surface or cytoplasmic Ig, HTLA, receptors for C3b, C3d, IgG-Fc, mouse E or sheep E, and were devoid of lysozyme, peroxidase, and chloracetate esterase. The described features do not represent B cells, T cells, myeloid cells, monocytes, or macrophages. The morphology and the marker pattern of the culture cells, however, is identical with that of freshly obtained Hodgkin's (H)- and Sternberg-Reed (SR)-cells, except for the lack of CIg in the in vitro cells, which is explained by the culture conditions. Heterotransplantation in nude mice was achieved by intracranial inoculation and by s.c. transplantation of cultured cells embedded in a plasma clot. The described findings suggest that these cultured Hodgkin's cell lines are indeed derived from H and SR cells. The cellular origin of these cells is not clear, the loss of cellular differential markers during the process of possible dedifferentiation is discussed.

Epstein-Barr virus-induced proteins. IV. Characterization of an EBV-associated phosphopolypeptide.

Cells of the Epstein--Barr virus (EBV) non-producing lines NC37 and Raji were induced by the tumour promoter TPA and were labelled with 32P. The analysis by immunoprecipitation with human VCA+EA+ sera revealed that a major phosphopolypeptide with a mol. wt. of 58 000 was specifically precipitated. In addition, a phosphopolypeptide of the same size was the dominant phosphopolypeptide detected in EBV-producing B95-8 and P3HR-1 cells as well as in P3HR-1-superinfected NC37 cells. This phosphopolypeptide seemed to be EBV-specific, because it was not detectable in TPA-treated, but EBV genome-negative, Ramos and BJAB cells. Synthesis of this phosphopolypeptide was inhibited in TPA-treated NC37 cells by treatment with Ara-C. VCA+EA+ sera were found to be more immunoreactive with this phosphopolypeptide than VCA+EA- or EBNA+ sera. Based on these results this phosphopolypeptide may have some immunological relatedness to the EBV-specific early antigen (EA) complex defined by immunofluorescence.

Two neoplastic cell lines with unique features derived from Hodgkin's disease

Two in vitro cell lines (L428, L439) were established from pleural effusions of two patients with Hodgkin's disease. The histological diagnosis was ascertained in both cases by two independent pathologists. The neoplastic nature of the culture cells is indicated by the demonstration of several structural and numerical chromosome abnormalities associated with a monoclonal pattern of marker chromosomes. Heterotransplantation in nude mice was achieved by intracranial inoculation and by subcutaneous transplantation of cultured cells embedded in a plasma clot. EBV-specific antigens (EBNA, VCA) were not detectable in either cell line. Ia-like antigens, receptors for T cells, acid phosphatase and acid esterase were shown to be present in the cultured cells. The L428 and L439 cell line lacked surface- or cytoplasmic Ig, HTLA, receptors for C3b, C3d, IgG-Fc, mouse E or sheep E, and were devoid of lysozyme, peroxidase and chloracetate esterase. These features do not correspond to those of B cells, T cells, myeloid cells, monocytes or macrophages; the morphology and the marker pattern of the culture cells, however, is identical with that of freshly obtained Hodgkin (H)- and Sternberg-Reed (SR)- cells, except for the lack of Clg in the in vitro cells, which is explained by the culture conditions. These findings suggest that the L428 and L439 cell lines are indeed derived from H- and SR-cells and offer the possibility of gaining new information upon the nature of Hodgkin's disease.

Attempt To Infect Nonmalignant Nasopharyngeal Epithelial Cells From Humans and Squirrel Monkeys With Epstein-Barr Virus<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

With the use of Epstein-Barr virus (EBV), attempts were made to infect epithelial nasopharyngeal explant cell cultures prepared from nonmalignant human and squirrel monkey tissue. Explants were exposed to EBV from both HR-1 and B95-8 cells and examined for EBV-specific antigens by immunofluorescence and electron microscopy. Among the human specimens, no epithelial nasopharyngeal were found that could be infected with EBV. However, similar explants derived from squirrel monkey tissue were infected. The results were consistent with the hypothesis that EBV can infect at least certain explanted epithelial cells of the nasopharynx.

IgM antibodies to Epstein-Barr virus-associated membrane antigen in sera of infectious mononucleosis patients.

By the indirect immunofluorescence technique, IgM antibodies to the cell surface of an Epstein-Barr virus (EBV) producer cell line, P3HR-1, were detected in sera from infectious mononucleosis (IM) patients but not in sera from patients with Burkitt lymphoma or nasopharyngeal carcinoma nor in sera from healthy adult donors having antibodies to EBV-specific viral capsid antigen (VCA). Titers of the IgM antibodies were higher in the earlier stages of IM, a pattern similar to that for IgM antibodies to VCA. The IgM antibodies to the cell surface were identified as being those against the EBV-specific membrane antigen (MA) by the following criteria: (1) The antibodies were reactive to MA-positive cell preparations but to MA-negative cell preparations. (2) Titers of the IgM antibodies were not significantly affected after absorption of sera with sheep red blood cells which could completely eliminate heterophil antibodies in the same sera. Detection of the IgM antibodies to MA may have a particular diagnostic value for providing evidence of a recent EBV infection.

Spontaneous and induced patterns of the Epstein-Barr virus (ebv) cycle in a new set of somatic cell hybrids

A somatic cell hybrid line and its subclones obtained by fusing two Burkitt lymphoma lines (Raji and Namalwa) were examined for the expression of EBV-specific antigens both spontaneously and after induction. The hybrids retained spontaneous early antigen (EA) production at the level characteristic of Raji. Similarly the more permissive Raji pattern dominated the induction of EA by IUDR treatment or P3HR-1 virus superinfection. These findings accord with our previous results on independently derived Raji/Namalwa hybrids. Virus capsid antigen was induced in the hybrids by P3HR-1 virus superinfection though at a lower level than in the Raji parental cell.

Attempts to demonstrate virus‐specific sequences in human tumors. IV. EB viral DNA in European burkitt lymphoma and immunoblastic lymphadenopathy with excessive plasmacytosis

Various biopsies from different European malignant lymphomas, two biopsies from nasopharyngeal carcinomas, and material from non-neoplastic lymph nodes were assayed for the presence of Epstein-Barr virus (EBV) DNA by nucleic acid hybridization. Reassociation kinetics of in vitro-labelled EBV DNA were studied in the presence of tumor DNA. The lymphomas tested included among others follicular lymphomas, germinocytomas, immunoblastic lymphomas and lymphoplasmacytoid immunocytomas. Epstein-Barr viral DNA was demonstrated within the two nasopharyngeal carcinoma biopsies as expected. A histologically typical Burkitt lymphoma as well as an immunoblastic lymphadenopathy with excessive plasmacytosis also contained EBV-DNA. The Burkitt biopsy revealed about 15 EBV genome equivalents per cell. Antibodies against EBV-specific antigens were highly elevated in the serum of this patient. The material of the patient with immunoblastic lymphadenopathy contained 2-3 EBV genome equivalents per cell.

Demonstration of a cellular inhibitor of Epstein-Barr and cytomegalovirus synthesis

A particle free extract prepared from human D98 cells was tested for inhibitory activity against the Epstein-Barr Virus (EBV) and cytomegalovirus (CMV). The number of permissive human cells positive for EBV-specific antigens after infection with virus decreased by more than 90% when the cells were treated with D98 extract and poly- l-ornithine. Synthesis of CMV DNA was depressed in virus infected cells in the presence of the inhibitor. Replication of infectious CMV was reduced by approximately 99.9%. The data suggest that the D98 extract specifically depresses EBV and CMV replication.

Rescue of Epstein-Barr virus from somatic cell hybrids of Burkitt lymphoblastoid cells.

A Burkitt lymphoblastoid cell line, P3J-HR-1, was fused and hybridized to a human sternal marrow cell line. The somatic cell hybrids were negative when examined for Epstein-Barr virus (EBV) markers. When the hybrid cells were exposed to 5-iododeoxyuridine, both EBV-specific antigens and virus particles were induced as determined by the immunofluorescence test and by electron microscopy. The data presented suggest that the EBV genome can be transferred from a lymphoblastoid cell to another cell type during cell hybridization, that the EBV genome can persist in the hybrid cells for long periods of time, and that synthesis of the virus can be induced in the heterokaryons.

Density changes of cultured Burkitt lymphoma cells following EB viral synthesis.

Burkitt lymphoma cells harbouring EB virus can be separated from uninfected cells by using discontinuous gum acacia density gradients. It may be possible to use this method further to separate cells carrying different EBV-specific antigens.