Biomedical subjects
Publications and source records attributed to G Klein.
The patterns of long-chain neutral glycolipids and gangliosides of established, L-cell-derived murine fibroblast lines and their low- and high-tumorigenic hybrids were studied.
A culture of mouse A9 cells yielded clones of both high and low malignancy. Confirmation of the differences in malignancy of the clones was obtained after their hybridization with subclones of YACIR a lymphoma induced by Moloney leukemia virus. Low malignant A9 clones suppressed the malignancy of the YACIR partner in the hybrids. Hybrids between a highly malignant A9 cline and two different clones of YACIR "complemented" to yield hybrids of reduced malignancy. No correlation was formed between the malignancy of a particular A9 clone and its ability to produce a focus-forming mouse C-type virus.
Electron microscopy examination of nasopharyngeal carcinoma grafts in nude mice showed that the tumor cells retained their epithelial characteristics, as betrayed by the presence of keratin fibrils and of functional complexes at the cell membrane. Thus the cells positive for Epstein-Barr virus-specific nuclear antigen present in such grafts do represent the progeny of human epithelial tumor cells. C-type virus particles were observed in one tumor graft, indicating that epithelial human cells could pick up a mouse virus.
An association has been found between the Epstein-Barr virus (EBV) and complement (C3d) receptors on human lymphoid cells. The evidence was four-fold: there was a correlation between the expression of these two receptors; inhibition experiments showed that the binding sites probably are close to each other in the cell membrane, although not identical; EBV and complement receptors have been found to co-cap in either order; and lymphoid cell lines lacking complement receptors could not be superinfected with EBV.
Antibodies purified from sera of patients with Burkitt's lymphoma by absorption onto and elution from cultured lymphoblastoid cells, were labeled with 125I and repurified by repeating the absorption-elution procedure. The doubly purified antibodies could bound with a high degree of efficiency to immunoadsorbents prepared by entrapping in polyacrylamide gel normal human serum or exudate fluids from patients with ovarian or breast cancer. Binding was specific, as it could be inhibited by high dilutions of human sera but not by animal sera. Globulin fractions of human sera were strongly inhibitory. Their capacity to inhibit was lost after treatment with rabbit anti-human IgG serum, indicating that at least part of the inhibition could be ascribed to antibodies. Some inhibitory activity was also found, however, in the "albumin" fractions of sera, suggesting that an antigen was also involved. It appears, therefore, that practically all human sera contain antibodies reacting with an antigen(s) which is present on human cells and is also circulating freely in serum and other body fluids. Sera of patients with Burkitt's lymphoma contained, on the average higher concentrations of these antibodies, as compared to sera of individuals not suffering from this disease. This antigen is thought to be associated with the cell membrane and has been identified in cells of widely differing origin.
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Growth of lymphoblastoid cells (B95-8, Raji) is not inhibited by the presence of 0.4 mM phosphonoacetic acid. The synthesis of Epstein-Barr virus (EBV) in the producer line B95-8 is completely inhibited, as shown by the total inhibition of viral capsid antigen synthesis. Early viral antigens are made normally in the presence of phosphonoacetic acid, but EBV DNA synthesis is blocked in cells entering the productive cycle. Nonproducer cells in the population replicate the resident EBV DNA by a mechanism that is resistant to phosphonoacetic acid. These results are consistant with the hypotheses that EBV DNA is replicated by two mechanisms, one in the noninduced cell and a different mechanism in the producer cell, and that prior replication of EBV DNA, probably by the second mode, is a prerequisite for late gene expression.
Viral DNA molecules were purified from a nontransforming and a transforming strain of Epstein-Barr virus. Each viral DNA was labeled in vitro and renatured in the presence of an excess of either one or the other unlabeled viral DNA. Both viral DNAs were also digested with the Eco R1 restriction endonuclease and subsequently labeled by using avian myeloblastosis virus DNA polymerase to repair either the EcoR1 nuclease-generated single-stranded ends of the DNAs or their single-stranded ends produced by a second digestion with exonuclease III after the first EcoR1 nuclease digestion. The results of these experiments support three general conclusions: (i) the DNAs of these two strains of Epstein-Barr virus share approximately 90% of their nucleotide sequences; (ii) both viral DNA populations are reasonably homogenous; and (iii) both DNAs contain repetitions or inverted repetitions of some of their nucleotide sequences.
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Some of the drugs, which are used in therapy of gout were tested with respect to their influence on semi-conservative DNA-synthesis and DNA-repair in vitro, using different cell systems. The agents (Benzbromarone, Allopurinol, Thiopurinol) were investigated in concentrations similar to blood-levels in normal therapy. The cells were irradiated with ultraviolet or ionizing radiation in order to damage their DNA and DNA-repair (unscheduled DNA-synthesis) was estimated by measuring the incorporation of radioactively labeled thymidine into acid insoluble material under appropriate conditions. The substances showed different effects on DNA-synthesis as well as on DNA-repair, even then, when their supposed influence on enzymes of nucleotide metabolism, was similar.
The ability of trinitrophenylated tumor cells to stimulate syngeneic antitumor response has been tested in 3 different tumor-host systems: A. Trinitrophenylated and inactivated Moloney induced YAC tumor cells (YAC-TNP) were able to induce the production of cytotoxic antibodies in low responding A/J mice, while inactivated YAC tumor cells (YAC-In) failed to induce such a response. Furthermore, A mice which were injected with YAC-TNP rejected 103 viable YAC tumor cells at a higher frequency than those injected with YAC-In. B. Trinitrophenylated and inactivated Gross virus induced G-35 tumor cells or Monoloney induced LSTRA cells (both syngeneic in BALB/c mice) were as immunogenic as nonmodified inactivated tumor cells. About 50% of the immunized mice survived indefinitely after injection of 103 viable tumors. Fruthermore, spleen cells from mice primed with either modified or nonomodified G-35 cells responded in vitro to G-35 in a mixed leukocyte tumor interaction and generated specific cell-mediated cytotoxic activity to 51Cr-G-35 syngeneic tumors. However, the donors of the primed cells did not produce detectable cytotoxic antibodies to G-35. C. In vitro sensitization of C57B1 spleen cells by trinitrophenylated Mitomycin C treated syngeneic EL-4 generated a stronger cytotoxic response to EL-4 cells than obtained by sensitization with Mitomycin C treated EL-4 cells alone, The superiority of the sensitizing capacity of trinitrophenylated EL-4 was readily demonstrated in conditions which were suboptimal for nonmodified Mitomycin C treated tumor. Both theoretical and practical implications of these results are discussed.
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There is excellent evidence for the belief that immune surveillance mechanisms prevent the outgrowth of potentially neoplastic cells induced by horizontally transmitted, ubiquitous, potentially oncogenic viruses, indigenous to natural populations. Polyoma virus in mice, Marek's disease in the chicken, Herpesvirus saimiri in the squirrel monkey, and the Epstein-Barr virus in man are examples. There is much less evidence for immune surveillance against chemically induced tumors. It is argued that surveillance against the virus-induced tumors may have evolved by the selective fixation of appropriate immune responsiveness (IR) genes. It is important to distinguish between the breakdown of an existing surveillance mechanism, e.g., by immunosuppresion, and the lack of immune recognition, due to the deficiency of the IR gene equipment. Presently available in vitro lymphocytotoxicity tests are not yet developed to the point where they can reliably distinguish between these alternatives.
The escape from immune rejection is, like the development of drug resistance in microorganisms, a multi-pathway process. Selection favors the phenotype, i.e., the cell that can grow in spite of the immune rejection process, not the underlying mechanism. Experimental evidence documents the roles of host immunosuppression (genetic or environmental), immunoresistance at the cellular level, "sneaking through" (i.e., growth of a tumor to irreversible size prior to the mobilization of an appropriate immune response), lack of antigenic recognition, and blocking enhancement type reactions.
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