Several clones of independently established somatic cell hybrids between two human lymphoid cell lines, Raji and Namalwa, were examined for surface immunoglobulin expression. Double-antibody radioimmunoassays were established for kappa and lambda light chains. Immunoglobulins were detergent-extrated by Triton X-100 and quantified by radioimmunoassay. The Raji parent expressed small amounts of kappa chains on its surface, and the Namalwa parent a 10 fold greater amount of lambda chains. We show that the majority of the hybrid clones co-express both parental phenotypes.

Burkitt Lymphoma↗

PubMed1977

Differences in the ConA-induced redistribution and agglutination patterns of EBV genome-free and EBV-carrying human lymphoma lines.

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Agglutination↗

PubMed1977

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.

Antigens, Viral↗

PubMed1977

Solubilisation of Epstein-Barr virus (EBV)-associated nuclear antigen from Raji cells and chromatin by treatment with various molarities of NaCl.

The solubilisation of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) by treatment with various molarities of NaCl was investigated using the 125I--IgG absorption assay. Ninety percent of the antigenic activity detected using the 125I--IgG absorption assay was insoluble at 0.15 M NaCl. It could be rendered soluble by treatment with 2.0 M NaCl, but reprecipitated upon return to 0.15 M NaCl. EBNA was partially extracted from Raji chromatin by treatment with 0.35 M NaCl. The efficiency of extraction was increased by homogenisation in 2.0 M NaCl followed by dialysis to 0.35 M NaCl. The data demonstrate the close association of EBNA with Raji chromatin and suggest that it may be a chromatin-associated non-histone protein.

Antigens, Viral↗

PubMed1977

[Quantitative EEG analysis of the effects of carbocromen, pentetrazole, beta-methyldigoxin, and strophantin-K in normal human volunteers (author's transl)].

With EEG-interval analysis we studied the influence on C.N.S. of some drugs normally used for cardiovascular diseases: carbochromen, pentetrazole, beta-methyldigoxin and Strophantin-K. All investigated drugs induced activation of alpha-waves, especially in frontal areas with primary small alpha-activity. Dominant alpha-frequency was accelerated. These results confirm the direct and stimulating effects of these drugs on C.N.S. in therapeutic doses. Alpha-acceleration may not only indicate psychostimulation but also indicate epileptogenic effects. All these drugs may provoke epileptic seizures in toxic doses.

Adult↗

PubMed1977

Moloney leukemia virus-induced cell surface antigen: detection and characterization in sodium dodecyl sulfate gels.

An experimental procedure for detecting and characterizing tumor-associated, virion, and histocompatibility antigens has been developed. The method takes advantage of the high resolution that proteins, solubilized by Triton X-100 and reduced, display after sodium dodecyl sulfate gel electrophoresis. The antigens can be detected as distinct molecular weight species by a highly sensitive inhibition of cytotoxic reaction. When coupled to the lactoperoxidase-catalyzed iodination of intact cells, the procedure permits the determination of externally exposed antigens. In the present study, the method has been applied to the Moloney leukemia virus-induced YAC lymphoma cells of strain A mice, which express a Moloney leukemia virus-determined cell surface antigen (MCSA) in addition to the type C viral proteins gp71, p30, p15, p15(E), p12, and p10. MCSA was identified as an exposed surface protein distinct in size and antigenic determinants from the major envelope and core protein of Moloney leukemia virus and the histocompatibility antigens. Multiple molecular weight species possessing antigenic determinants for MCSA, gp71, and H-2(a) have been detected. These results provide direct confirmation that MCSA is unrelated to the known virion structural proteins or to the H-2(a) antigen. This method should permit the direct identification and molecular weight characterization of any antigen whose determinants are not solely dependent on a complex quaternary structure and for which serological reagents are available.

Animals↗

PubMed1977

Identification of a purified complement-fixing antigen as the Epstein-Barr-virus determined nuclear antigen (EBNA) by its binding to metaphase chromosomes.

A soluble complement-fixing antigen carried by Epstein-Barr virus (EBV)-transformed human cells has been previously extracted from cell nuclei and purified by DNA-cellulose chromatography [Luka, J., Siegert, W. & Klein, G. (1977) J. Virol., in press]. On addition of this antigen to methanol/acetic acid-fixed metaphase chrmosomes, followed by exposure to human sera containing antibodies against the EBV-determined nuclear antigen (EBNA), brilliant positive staining was obtained by anti-complement immunofluorescence. There was no staining after exposure to EBV-negative sera. Moreover, a nuclear protein fraction, prepared from an EBV-negative cell line in an analogous fashion, failed to induce the staining reaction. These data identify the soluble purified antigen as the EBV-determined nuclear antigen. The purified antigen has a molecular weight of 174,000 +/- 15,000, as determined by sucrose gradient centrifugation and gel filtration experiments. In neutral buffers containing 0.5-1.0 M NaCl, the antigen dissociates into a form of approximately one-half the original molecular weight with retained complement-fixing activity. This "monomer" has a molecular weight of 98,000 +/- 8,000.

Antigens, Viral↗

PubMed1977

Immune surveillance against virus-induced tumors and nonrejectability of spontaneous tumors: contrasting consequences of host versus tumor evolution.

Spontaneous tumours are defined as tumors that develop in the absence of all experimental interference. In contrast to the widely documented, strong rejection reactions against most virus-induced tumors, spontaneous tumors evoke little or no detectable rejection reaction in intact or preimmunized syngeneic hosts. The difference can be viewed in relation to the contrasting natural history of the two conditions. Spontaneous tumors evolve in several steps, as a fule. "Tumor progression" is a microevolutionary process at the level of the somatic tissue where successive clonal variants replace each other. Each new variant gains the upper hand due to its greater independence of some restricting host mechanism. Independence of immune restrictions must be part of this process. Host selection for immune resistance apparently plays no major role here, presumably because most of the naturally occurring tumors arise after the host has passed the peak of its reproductive period. Protection against the oncogenic effects of ubiquitous tumor viruses is, on the other hand, the result of host selection for immune mechanisms favoring prompt rejection of virus-transformed cells. This is neither synonymous with nor related to protection against the viral infection per se, which is frequently successful and usually quite harmless. A certain relationship can be perceived between the degree of viral ubiquity and the strength of immune protection against the corresponding tumor cells. Natural selection for host recognition of commonly occurring, virally induced changes in neoplastic cell membranes can be surmised to occur, at least in part, by the fixation of appropriate immune responsiveness (Ir) genes. The role of Ir genes for tumor recognition can be approached by the genetic analysis of the F1 hybrid resistance effect. Unresponsiveness to spontaneous tumors may be overcome by target-cell modification, e.g., by chemical coupling, somatic cell hybridization, or viral "xenogenization".

Animals↗

PubMed1977

Tumor-associated antigens in H-2 hemizygous isoantigenic variants of a somatic cell hybrid, derived from the fusion of a 3-methylcholanthrene-induced sarcoma and a mammary carcinoma.

To check the suggestion that 3-methylcholanthrene-induced, sarcoma-associated, tumor-specific transplantation antigens (TSTA) could be modified H-2 antigens, reciprocal isoantigenic variants derived from TA3Ha/MSWBS hybrid cells were examined. This hybrid was produced by the fusion of the TA3Ha ascites carcinoma (H-2a) and the 3-methylcholanthrene-induced MSWBS ascites sarcoma (H-2s). MSWBS expresses a strong TSTA capable of inducing a rejection reaction in the syngeneic A.SW host. The genetic determinants of the H-2 complex are known to be localized on chromosome No. 17. TA3Ha contributes two normal, telocentric chromosomes No. 17 to the hybrid. In contrast, both chromosomes No. 17 of MSWBS are localized on readily identifiable translocations (17/1 and 17/M1). We have previously shown that the chromosomes No. 17 of one parental strain or the other (but not both) can be eliminated from the hybrid upon selective passage in the opposite parental strain. The present studies showed that the two strain A-compatible variants that have lost the sarcoma-derived 17-chromosomes still contained the same TSTA as the two reciprocal strain A.SW-compatible variants that have lost the mammary carcinoma-derived H-2 chromosomes. These findings argue against the possibility that methylcholanthrene-induced TSTA is a modified form of H-2 or that its structural determinants(s) is localized on chromosome 17.

Animals↗

PubMed1977

Epstein-Barr virus genome studies in Burkitt's and non-Burkitt's lymphomas in Uganda.

Burkitt's lymphoma (BL) has been widely investigated and has attracted attention because of the possible etiologic role of the Epstein-Barr virus (EBV). To further determine the role of EBV in the causation of this tumor, we measured EBV-specific nuclear antigen (EBNA) and EBV DNA using immunofluorescence and nucleic acid hybridization techniques, respectively. Of 34 BL biopsies, 27 tissues (79%) were EBNA-positive, whereas none of the 25 non-BL biopsy tissues were EBNA-positive. Of 15 BL tumors tested, 14 (93%) were EBV DNA-positive with a mean of 39 (range, 8-86) EBV genome equivalents per cell. Each of the 15 non-BL biopsy specimens subjected to nucleic acid hybridization had less than two virus genome equivalents per cell, although all had serologic evidence of past EBV infection. The findings further supported the possible etiologic role of EBV in African BL and negated the passenger hypothesis. The EBV genome could, therefore, be used as a separating marker between African BL and non-BL lymphomas.

Adolescent↗

PubMed1977

A solid-phase radioimmunoassay for Epstein-Barr virus-associated membrane antigen prepared from B95-8 cell culture supernatants.

Epstein-Barr virus(EBV)-associated membrane antigen (MA) was concentrated from B95-8 cell culture media by precipitation with polyethylene glycol followed by chromatography on Bio-Gel A-50m. In a RAJI cell-binding assay, MA-positive material could only be found in the void volume of the column. After ultracentrifugation all antigenic activity appeared in the pellet, which suggested that MA was present in aggregates, presumably fragments of cellular membranes and/or virus envelopes. The MA-containing preparation was photopolymerized in polyacrylamide gel. The homogenized gel was used in a solidphase radioimmunoassay with 125l-labeled IgG from an Anti-MA positive reference serum and an anti-MA negative control serum. The specificity of the reaction was confirmed in blocking tests with anti-EBV positive and negative sera. A good correlation was found between the results obtained in the radioimmunoassay and the results obtained in direct immunofluorescence tests for the detection of MA. The existence of at least two subspecificities of the MA complex could be confirmed by this radioimmunoassay.

Antibody Specificity↗

PubMed1977

Solubilization of the Epstein-Barr virus-determined nuclear antigen and its characterization as a DNA-binding protein.

The Eptstein-Barr virus (EBV)-determined nuclear antigen (EBNA) was solubilized from isolate nuclei of two EBV-transformed cell lines- Raji and AW-Ramos, by high-salt treatment. Its DNA-binding properties were studied by DNA-cellulose chromatography and a 51Cr release complement fixation assay. EBNA binds to both double-stranded and single-stranded calf thymus DNA, showing a higher affinity to double-stranded DNA. There was no detectable difference in the DNA binding of EBNA prepared from Raji and AW-Ramos cells.

Antigens, Viral↗

PubMed1977

Detection localization of Epstein-Barr virus-associated early antigens in single cells by autoradiography using 125I-labeled antibodies. (With 1 color plate).

A characteristic feature of virus-transformed cells is the expression of virus-specific antigens usually detectable by immunological or radioimmune procedures. We report here an autoradiographic method for the detection and localization of such antigens in individual cells which combines the cellular specificity and radiolabeling sensitivity of the above procedures. As a test system we have studied the reaction between virus antigen in some Epstein-Barr virus (EBV) DNA-containing cell lines and anti-virus antibody specificities in certain human sera. EBV DNA-containing cell lines express an EBV-determined nuclear antigen (EBNA) and some lines also express the EBV-associated antigens, early antigen (EA) and virus capsid antigen (VCA), in a minority of cells in the population. By employing appropriate 125I-IgG-labeled human sera, we clearly show that EA can be detected without difficulty in cell lines known to spontaneously express this antigen. Moreover, the specificity and sensitivity of the present method is such that low levels of EA cand be detected at cellular concentrations which remain undetectable by conventional immunofluorescence.

Animals↗

PubMed1977

Rejectability of virus-induced tumors and nonrejectability of spontaneous tumors: a lesson in contrasts.

In contrast to the excellent surveillance against tumors induced by the ubiquitous viruses in their natural host species, spontaneous tumors developing naturally, without experimental interference, evoke little or no rejection reaction. This may be viewed in relation to the natural history of tumors. In most cases, tumors evolve in several steps, with a successful of clonal variants following each other, characterized by increasing independence of host regulations (including immune restrictions). This process is commonly designated as tumor progression. In all probability, selection for nonrejectability is part of this process. Selection of the host for rejecting capacity is unlikely to play any major role, since the overwhelming majority of the naturally occurring tumors arise after the host has passed the peak of its reproductive period. The nonrejectability of spontaneous tumors may be overcome by target-cell modification, e.g., by chemical coupling, somatic cell hybridization, or viral xenogenization. Furthermore, genetic analysis of the F1 resistance effect in relation to seemingly nonrejectable tumors may reveal the existence of specific immune-response (Ir) genes that can influence the recognition of tumor-associated membrane changes by the host immune system.

Animals↗