Biomedical subjects
Publications and source records attributed to G Klein.
Karyoplasts and cytoplasts, obtained by enucleation of mouse L cell sublines, express H-2k and L virion antigens in amounts comparable to intact cells.
Epstein-Barr virus (EBV) carrying biopsies of Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC) were used to examine the question whether the EBV-associated nuclear antigen (EBNA) can be demonstrated by the acid fixed nuclear binding (AFNB) technique, developed previously for the demonstration of EBNA in cultured cell lines [11]. Extracts of 5 BL and 5 NPC biopsies gave a brilliant, EBNA specific fluorescence after binding to acid fixed chicken red cells. Similar extracts of 3 other African tumors that are not known to carry the EBV-genome were negative, in spite of the fact that they were derived from EBV-seropositive patients with relatively high anti-EBV (VCA) antibody titers. Crude extraction and DNA-cellulose purification gave equally active extracts, provided that incubation was carried out at 4 degrees C. These results show that the acid fixed nuclear binding technique can be applied to biopsy material. This may be helpful in searching for EBNA carrying cells in heterogeneous normal and tumor tissues in vivo where the direct in situ ACIF staining for EBNA is known to meet great difficulties.
Patients with Burkitt's lymphoma in chemotherapy-induced remission received through dermal scarifications one or two doses per week of approximately 3 X 10(8) living BCG organisms (Pasteur Institute vaccine). This treatment was always followed by usually rapid increases by 1--4 log2 steps in the antibody titers to Epstein-Barr virus (EBV)-associated cell membrane antigens. Titer increases of less than 2.5 log2 steps within the first month after the start of BCG treatment correlated with a significantly elevated frequency of extradural relapse as compared to that seen in patients with larger titer rises. During this time, antibodies to EBV-associated viral capsid antigens and early antigens of D and R specificity, as well as antibodies against herpes simplex, varicella, cytomegalovirus, measles, and respiratory syncytial virus antigens, did not show any consistent or impressive changes.
In a series of six TA3-HA/A.CA hybrid cell lines formed by the fusion of the TA3-HA mammary carcinoma of a strain A mouse with a normal embryonic fibroblast of an A.CA mouse and then converted to the ascites form in parental strain A, the capacity to grow in foreign strains was inversely related to the ability to absorb anti-H-2a antibody. The absorptive capacities of the hybrid cell lines were intermediate between the low absorptive capacity of the non-strain-specific parent TA3-HA ascites cell line and the much higher absorptive capacity of the strain-specific ascites line TA3-St of the same tumor. Each hybrid cell line possessed an abundance of large endogenous cell-surface glycoprotein molecules similar to epiglycanin, a glycoprotein detected at the surface of the parent TA3-HA cell. The results suggested that the amount of epiglycanin-like material at the hybrid cell surfaces, determined by chemical and immunochemical methods, may have been directly related to the capacities of the cells to grow in foreign mouse strains and inversely related to their capacities to absorb anti-H-2a antibody.
A human lymphoma cell line, positive for the Epstein-Barr virus (EBV)-associated nuclear antigen, was recently established from a North American Burkitt's lymphoma. This cell line, SU-AmB-2, contained EBV DNA both in the form of circular, nonintegrated DNA molecules of viral genome length, present in multiple copies per cell, and as integrated sequences. Having DNA present in both of these forms, it resembled cell lines established from African Burkitt's lymphomas. In studies on EBV strain differences, the episomal viral DNA in Burkitt's lymphoma cells may now be compared with viral DNA in nonmalignant cells with the use of cell lines from Burkitt's lymphoma patients of similar geographic origin.
An experimental system was developed that permitted nonrandom chromosome changes that occur in radiation leukemia virus (RadLV)-induced lymphomas to be followed during tumor progression. RadLV variant-induced preleukemia and leukemia cells originating from female inbred C57BL/6 mice were injected into male animals of the same strain. Since all donors were females and all recipients were males, the sex chromosome complements (XX and XY) were used to distinguish the preleukemia and leukemia cells from those of host origin. The G-banding analysis revealed that more than 50% of animals that were inoculated with preleukemia cells and that developed leukemia possessed tumor stem-lines of 41 chromosomes with a tristomy of chromosome #15. In animals inoculated with overt leukemia cells and in which tumor progression occurred, the G-banding an additional trisomy of chromosome #17. The cytogenic data strongly suggested that the trisomy of chromosome #15 was the first specific tumor-associated chromosome change that occurred in the process of conversion of RadLV-induced preleukemia cells to fully autonomous tumor cells.
A scaled-down version of a modified adult circle system capable of increasing normal humidity output 300% was constructed for pediatric use. The moisture content of the gases leaving the system was tested and found to be superior to that of any pediatric circle system commercially available that does not incorporate an electrically heated water vaporizer.
Cultured lines of three newly established methylcholanthrene-induced tumors, MBK and MBL in CBA and MC57M in C57BL mice, and two mammary tumors, SBfnHC in CBA and S3W in ASW mice, were superinfected in vitro with Moloney sarcoma or leukemia virus (MSV, MLV). After superinfection, they expressed the Moloney virus-determined cell surface antigen (MCSA) and murine C-type viral p30 antigen, and produced NB-tropic C-type virus. The virus-infected tumors became more rejectable in normally susceptible syngeneic mice compared with the original noninfected line. There was no difference in 400-rad irradiated hosts. Mice that have rejected the virus-infected tumors showed an increased resistance to the corresponding noninfected tumor. The protective effect was comparable, with only one exception, to the immunizing effect of irradiated, noninfected cells. In vitro tests showed that small numbers of viable MSV-infected MBL generated cytotoxic spleen cells against both uninfected and MSV-infected MBL in syngeneic mice, while the same numbers of viable noninfected MBL did not induce cytotoxic cells. Relatively large numbers of irradiated MBL and MSV-MBL had a similar activity in inducing cytotoxic spleen cells against MBL in syngeneic hosts.
EBNA-containing chromatin from Raji cells was solubilized by treatment with high concentrations of urea and salt and fractionated by hydroxyapatite chromatography. Fractions eluting at different phosphate concentrations were analysed for the presence of EBNA by means of an anti-EBNA-specific 125I-labelled IgG absorption assay. The antigen was found in fractions containing non-histone chromatin proteins.
Con A-Sepharose affinity chromatography may be used in the analysis and classification of immune complexes. Experiments with model immune complexes suggest that the degree of affinity of an immune complex for Con A-Sepharose is determined by the antigen rather than the IgG antibody of the complex. It is possible that partial characterization of unknown antigens linked to IgG in immune complexes may be achieved in many diseases. Preliminary explorations with selected human sera indicate that the IgG containing immune complexes in Burkitt's lymphoma and nasopharyngeal carcinoma have affinity for Con A-Sepharose. By contrast IgG containing immune complexes in chronic hepatitis B seem to lack affinity for Con A-Sepharose.
Energy-transducing adenosine triphosphatase (ATPase) from Escherichia coli is inhibited by aurovertin. Aurovertin-resistant mutants were generated by nitrosoguanidine mutagenesis of E. coli AN180, whose growth on a nonfermentable carbon source was blocked by aurovertin. The ATPase activity of cell extracts from 15 different mutants (designated MA1, MA2, MA3, etc.) was found to be at least 20 times less sensitive to aurovertin than that from the parent strain. The aurovertin-resistant mutants did not show cross-resistance towards a number of ATPase inhibitors including azide, dicyclohexylcarbodiimide, quercetin, 7-chloro-4-nitrobenzofurazan, and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. Aurovertin inhibited the energization brought about by addition of ATP to E. coli AN180 membrane vesicles; it was without effect on MA1 and MA2 membrane vesicles energized by ATP. The mutation in MA1, like other mutations of the ATPase complex, maps in the unc region of the bacterial chromosome.
The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size.
By using autoradiography combined with 125I-labeled IgG prepared from human sera containing high anti-EA (early antigen) antibody titers, EA has been detected in a low fraction of cells in a variety of EBV DNA-containing cell lines previously designated as negative for this antigen. These positive cell lines are highly inducible for EA following treatment with iododeoxyuridine (IUDR) and also contain multiple copies of the virus genome on average. The correlation between IUDR inducibility and spontaneous expression of EA, in particular, suggests that the ability to express EA in both situations is interrelated.
Four tetraploid cell lines were derived from the Epstein-Barr virus (EBV)-carrying, nonproducer Burkitt lymphoma line Raji after colcemide treatment. EBV early antigen (EA) was induced by P3HR-1 virus superinfection and by iododeoxyuridine treatment. Inducibility was reduced in all tetraploid lines compared to the near-diploid counterpart. The average number of EBV genome equivalents per tetraploid cell was 60-70, compared to 50 in diploid cells. This contrasts with the EBV genome amplification previously observed in somatic cell hybrids.
Pretreatment with the P3HR-1 substrain of Epstein-Barr virus (EBV) inhibited approximately 85% of the DNA stimulation induced by Staphylococcus aureus in human B lymphocytes. In parallel experiments, the DNA stimulation induced by the transforming B95-8 substrain of EBV was almost completely inhibited by prior exposure to P3HR-1 virus. Phytohemagglutinin stimulation was only slightly inhibited, whereas the DNA synthesis of continuously growing Raji cells was inhibited to a considerable extent. The degree of DNA inhibition was correlated with the induction of the EBV-determined antigens (membrane, early, and nuclear antigens) in sensitive indicator cells.
Herpesvirus papio(HVP)-carrying baboon lymphoblastoid lines do not express a nuclear antigen like the Epstein-Barr virus(EBV)-determined nuclear antigen (EBNA), as judged by in situ anticomplement fluorescence staining, although the carry multiple viral genomes and, in the case of producerlines, early antigen (EA) and viral capsid antigen (VCA) that cross-react with the corresponding human EBV-determined antigens. To test whether the lack of in situ nuclear antigen expression is a property innate to the baboon virus or the baboon cell, nonproducer HVP-carrying baboon lymphoid cells of the 26 CB-1 line were superinfected with two human EBV strains. B95-8-derived EBV induced brilliant EBNA staining, proving that the baboon lymphoid cell was competent to synthesize EBNA. In the mirror experiment, HVP derived from the 9B or the 18C baboon line was added to the EBV-carrying Raji line, the EBV-negative Ramos and BJAB lines and the HVP-carrying nonproducer 26 CB-1 line, respectively. HVP induced EA and VCA in Raji, and EA in BJAB and 26 CB-1. EBNA was not induced in any of the three EBNA-negative lines, BJAB, Ramos and 26 CB-1. It is concluded that the lack of in situ nuclear staining in HVP-carrying baboon lines is a HVP-associated property and is not due to any innate inability of the baboon lymphoid cell to synthesize an antigen of the EBNA type.
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