Biomedical subjects
Publications and source records attributed to J Hilgers.
36 genetic markers were examined in 14 inbred strains of mice maintained at the Cancer Research Institute, Bombay. The genetic marker profiles of these strains when compared with the profiles of similar strains maintained elsewhere revealed discrepancies in 4 of them which are reported and discussed. The results emphasize the importance of genetic monitoring of inbred strains.
The SHN mouse strain, which has a high incidence of mammary cancer, developed by inbreeding and selection from Swiss stock mice by Dr. H. Nagasawa and co-workers (Meiji University, Tokyo, Japan), harbored an endogenous mammary tumor virus (MTV) responsible for a high frequency of mammary tumors early in life. The locus was called "Mtv-4" and was only comparable with Mtv-2 of the GR mouse strain in its inducing capacity of mammary cancer. Molecular hybridization with 32P-labeled MTV complementary DNA showed that the characteristic Mtv-2 bands of the GR strain were absent in the SHN strain.
The murine macrophage-like cells NCTC 1469, J774A, WEHI-3, IC21, and P388D1, were compared with respect to their peroxidatic activity, display of cell surface antigens, and production of colony-stimulating factor. Peroxidatic activity was demonstrated in the nuclear envelope and in the cisternae of the rough endoplasmic reticulum of NCTC 1469 cells, J774A cells, and P388D1 cells, and in granules of WEHI-3 cells and IC21 cells. Colony-stimulating factor was produced only by WEHI-3 cells. NCTC 1469 and IC21 cells had a weak expression of M1/69 and Mac-1 antigens, whereas P388D1 cells expressed these antigens at a high level. WEHI-3 cells expressed M1/69 at a high level and Mac-1 at a low level, whereas J774A cells had an opposite expression T 200, Pgp-1, and ThB antigens were expressed at different levels by the various cell lines, without overt correlation among themselves or with the expression of M1/69 or Mac-1 antigens. These data suggest that macrophage-like cell lines cannot be placed in a maturation/differentiation lineage.
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Urethane administrated in the drinking water of breeding female mice of five inbred strains (DDf, KF, C3Hf, ddY and C57BL) leads to a higher incidence of mammary tumors at an extremely early age in four of these strains (DDf, KF, C3Hf and ddY). In the case of the DDf strain the tumors are adenoacanthomas, but in the case of the other strains adenocarcinomas are also observed. The effect of urethane in the drinking water on expression of endogenous retroviral structural proteins of the B and C type (MTV and MuLV) was studied in the milk by an immunodiffusion test and in organ extracts by radioimmunoassays (for MTVp27, MTVgp52 and MuLVp30). Organs tested include salivary glands, thymus and spleen from female and male animals. In addition, prostate and seminal vesicles were tested in male animals and uterus and mammary glands in females. Mammary tumors induced by urethane were also treated. Urethane does not induce or enhance endogenous retroviral antigen expression, either in these organs or in the mammary gland, one of the target organs for tumorigenesis by this agent.
To test whether alloimmunization with H-2 or/and non-H-2 different normal tissues may increase the immunity to syngeneic tumors, groups of BALB/c (H-2d) mice were immunized with a series of allogeneic lymphoid cells and then challenged i.p. with syngeneic lymphoma cells. The outgrowth of otherwise lethal doses of the Moloney virus-induced lymphoma YC8 and of its clones was inhibited in BALB/c mice immune to DBA/2 (H-2d), C3Hf (H-2k), C3H.SW (H-2b), C3H.OH (H-2o2) and to B10 background tissues but not in mice immunized to A/He, BALB.K (H-2k) or BALB.B (H-2b) normal tissues. Anti-YC8 effect was also induced by immunizing BALB/c recipients with a pool of five different allogeneic cell lines which included C3Hf, C57BL/6J (H-2b), N:NIH (H-2q), B10.M (H-2f), and DBA/2 lymphoid cells. No growth inhibition of other BALB/c lymphomas induced by Moloney virus (LSTRA), X-rays (RL male I) or urethane (UR-1) was evident in alloimmune mice. In vivo transfer of growth inhibition of YC8 was obtained with BALB/c anti-B10.D2 peritoneal exudate cells in a Winn assay. The ability of these alloimmune lymphoid cells to delay significantly the survival time of BALB/c mice injected with the mixture of immune cell and YC8 cells was abrogated by anti-Thy 1.2 plus C' treatment. In addition, nu/nu BALB/c mice were unable to develop resistance to YC8 outgrowth after alloimmunization. The results of this study show that: (1) syngeneic growth of a lymphoma can be prevented by alloimmunization with normal cells; (2) this cross-reaction involved non-H-2 antigens; (3) the phenomenon appeared to be mediated by T cells.
A number of cell surface markers (T200, ThB, Thy1, Lyt1 and Lyt2 and a glycolipid) and enzymes (ATP-ase, acid phosphatase, beta-glucuronidase, 5'-nucleotidase, non-specific esterase, ANAE and chloroacetate esterase) were determined for two murine T-cell lymphomas: the DBA/2-strain-derived SL2 with a phenotype close to that of a mature thymocyte and the GRS-strain-derived GSRL13 with a phenotype of a more primitive thymocyte. While the pattern of expression of the enzymes was similar for SL2 and GRSL13 and as such indistinguishable from that of the majority of thymus cells, the pattern of cell surface antigen expression was clearly different. GRSL12 cells express the ThB antigen and a glycolipid antigen detectable with monoclonal antibody 30-H11, but not Lyt1 and Lyt2 antigens. SL2 cells, however, do not express ThB and the glycolipid antigen, but do express Lyt1 and Lyt2.
A 'null' activity variant phenotype for sorbitol dehydrogenase (SDH) was observed in C57BL/LiA mice and used to examine the genetics of this enzyme. Linkage studies of the locus (Sdh-1) with non-agouti (a) and a biochemical locus encoding liver L-alpha-hydroxyacid oxidase (Hao-1) demonstrated that it is coincident with or closely linked to the structural locus, previously localized on chromosome 2. Alcohol dehydrogenase (ADH) isozymes were also examined, since the liver A2 isozyme exhibited some activity as a sorbitol dehydrogenase on cellulose acetate zymograms. It is apparent that SDH activity is not 'essential' in this mouse strain.
Male mice of strain GR develop T-cell leukemia at a low frequency late in life. These leukemia cells contain large amounts of mouse mammary tumor virus (MMTV) RNA and MMTV proteins in a precursor form (Nusse et al., J. Virol. 32:251-258, 1979). We used restriction enzyme analysis and molecular hybridization to identify MMTV proviruses in the DNA of these leukemia cells. GR leukemia cells contained additional integrated MMTV proviruses at various sites in the genome. This amplification of MMTV proviruses in GR leukemia cells is not restricted to one particular endogenous MMTV provirus of strain GR. The number and location of the extra MMTV proviruses present in transplants of GR leukemia cells did not change upon serial transplantation of the leukemia cells. Acquisition of MMTV proviruses was also found in a similar leukemia, L1210 of the DBA/2 mouse strain, but not in three other leukemias, SL2 of DBA/2, BW5147 of AKR, and a spontaneous thymoma of BALB/c. The two main classes of MMTV RNA, 35S and 24S, were present in the cytoplasmic RNA of GR leukemia cells, indicating that the aberrant processing of MMTV precursor proteins is not due to anomolously sized RNAs. We could not detect extra RNAs in GR leukemia cells which would represent read-through transcripts of cellular genes adjacent to the extra MMTV proviruses, initiated by a promoter signal in the right MMTV long terminal repeat sequence. These data suggest that acquisition of MMTV proviruses may coincide with the onset of leukemogenesis in GR male mice.
Cell surface markers, enzymatic patterns, proliferation characteristics and metastatic behaviour of the DSA/2 derived SL2 lymphoma were determined. SL2 cells are sensitive to a heterologous antiserum to murine T-cells and to allo-antisera for Thy 1.2 and TL 1.2.3. They show acid-phosphatase, betaglucuronidase, acid-alpha-naphthytesterase and non-specific esterase staining. The reactions for ATP-ase, and 5'nucleotidase were negative. The SL2 tumour cells can be transplanted in vivo, growing rapidly both as an ascites or a solid tumour, and can be grown in vitro as a suspension culture (doubling time about 18 hours). One hundred cells kill an animal after i.p. transplantation, while 1,000 cells kill an animal after s.c. transplantation. Histopathological examination combined with TEM shows that SL2 metastasizes rapidly, especially after i.p. injection. The metastasizing cells reach the blood vessels in the lung septa and extravascular positions in the liver.
Acid alpha-glucosidase purified from human placenta was used to immunize a mouse (strain Balb/cHeA) according to a procedure described earlier (Stähli, C., Staehlin, T., Miggiano, V., Schmidt, J. and Häring, P. (1980) J. Immunol. Methods 32, 297-304). After fusion of spleen cells with myeloma cells, about 10% of the hybrid clones obtained produced antibodies against acid alpha-glucosidase. Finally, eight stable clones producing antibodies against the enzyme were obtained. When purified acid alpha-glucosidase is analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, two major protein bands (mol. wt. 76000 and 70000,) a minor band of mol. wt. 9600 and several minor bands with a mol. wt. of 67000 or lower are seen. Since all these components react with the monoclonal antibodies, they must have at least one antigenic determinant in common.
Human B lymphocytes obtained from healthy donors were infected with Epstein-Barr virus in vitro. From the initiation of infection to the final establishment of a permanent lymphoblastoid cell line, fucosyl glycopeptides of the cell surface were investigated. In order of appearance the following events took place: expression of Epstein-Barr virus-determined nuclear antigen, mitotic activity of the cells and specific glycopeptide alterations on the cell surface. This specific alteration in glycopeptides, as determined by gel filtration, is manifested by the appearance of fast-eluting glycopeptides and was similar to that found on Burkitt lymphoma cells. Neither pokeweed-mitogen-stimulated B lymphocytes nor exponentially growing normal T lymphocytes exposed fast-eluting glycopeptides on their surfaces. Therefore, it is concluded that the appearance of these fast-eluting glycopeptides on the surface of lymphoblastoid cells after EBV infection is not the result of culture conditions or conditions of growth as such. The similarity with glycopeptides derived from Burkitt lymphoma cells, and the observation that a considerable proportion of the B cells becomes immortalized, are discussed.
A gene locus is described controlling liver activities in the house mouse of three glycosidases, i.e., beta-galactosidase, beta-glucuronidase, and N-acetyl-beta-hexosaminidase. An allele conferring low activity is present in the inbred strain LIS/A, and an allele for high activity is present in A/BrAf mice. The three enzyme activities are correlated with each other. The possible linkage between this gene and the Bgs locus on chromosome 9 is discussed.
Mammary tumor virus (MTV) antigen expression was tested in the male genital organs (testis, epididymis, prostate, seminal vesicle and coagulating gland) of DD/Tbr, KF and DDD strains (dd (German) mouse group), GR strain (Swiss origin), SHN and SLN strains (different Swiss origin), C3H and A strains (American origin), BALB/c and C57BL strains (low mammary tumor strains), and DDf and SHNf strains (MTV eliminated strains). Microimmunodiffusion analysis showed that expression of MTV antigen is tissue extracts was found most frequently in the prostate, sometimes in the seminal vesicle and coagulating gland but rarely in the epididymis and not at all in the testis of mammary cancer strains. No expression was found in these organs from BALB/c and C57BL strains. The earliest expression was found in GR, SHN and SLN strains of Swiss origin. Expression was not lost in SHNf strain, indicating that it is due to genetically transmitted MTV, rather than a milk-factor. Male genital organs from two mammary cancer strains of American origin became positive occasionally at rather higher age than in the strains of Swiss origin. DD/Tbr, KF and DDD strains expressed MTV antigen in the male genital organs at intermediate age between the strains of Swiss origin and American origin. Complete disappearance of MTV antigen expression in these organs of DDf strain indicates that the expression of MTV in DD/Tbr is due to a milk-factor.
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A solid-phase radioimmunoassay utilising bind of 125I-labelled protein A to antibodies bound to virus adsorbed onto microtitre plates was shown to be suitable for detection of humoral immunity to Gross murine leukaemia virus (MuLV). The specificity of the reaction was shown by the fact that only homologous or closely related viruses effectively inhibited binding of antibodies to adsorbed virus. With this method a low level of spontaneous humoral immunity was demonstrated in sera from AKR/Crc mice, a strain with high concentrations of endogenous virus, whereas little or no anti-viral activity was found inCBA/H-T6Crc, a subline that does not appear to express MuLV.