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In situ lymphoid cells of mouse mammary tumors. II. The characterization of lymphoid cells separated from mouse mammary tumors.

Lymphoid cells were isolated from mouse mammary tumors by isokinetic gradient centrifugation. theta-bearing and ALS-sensitive cells were always the largest subpopulations found in these tumors. In fractions 4 to 6 from the isokinetic gradient, theta-positive cells ranged from 8 to 46% in autochthonous tumors and 14 to 47% in passaged tumors, and ALS-positive cells ranged from 34 to 46% in autochthonous tumors and 14 to 100% in passaged tumors. SIg-positive cells were only found in occasional tumors and, where present, averaged 2% of the separated population. Fc receptor-bearing cells averaged 15% and phagocytic cells averaged 8% of the cells found in fractions 4 to 6 of the gradient. Occasional ALS-positive and one-third of the Fc receptor-bearing and phagocytic cells also separated in fractions 7 to 10 of the isokinetic gradient. Although the individual tumors were very variable in the proportions of the various lymphoid subpopulations that they contained, this variability was a stable characteristic for at least one passage generation.

Animals↗

Inhibition of interleukin-2-induced proliferation of mouse T cells by normal mouse serum.

The effects of serum or plasma from both normal mice and nude mice on the proliferation of cultured T lymphocytes were studied. Normal mouse serum or heparinized plasma was found to significantly inhibit the proliferation of interleukin-2 (IL-2) stimulated cultured T cells, which were either autologous or allogeneic with the serum source. This inhibitory effect was either markedly or completely reversed by adding increased amounts of IL-2 to the cultures. Serum or plasma from nude mice was found to be as inhibitory to the proliferation as serum or plasma from normal mice. Absorption of the plasma with cultured T cells prior to assay did not eliminate or abrogate the plasma's inhibitory effect, but instead slightly increased its inhibitory activity. These studies indicate that serum or plasma from either normal mice or nude mice possesses a factor that can inhibit the proliferation of IL-2-stimulated cultured T cells. This factor probably acts either by interacting directly with the IL-2 or interfering with its stimulatory activity. The fact that adding excess IL-2 can reverse this effect may have important implications in the area of immune modulation and immunotherapy.

Animals↗

Metabolic conversion of 12-O-tetradecanoylphorbol-13-acetate in adult and newborn mouse skin and mouse liver microsomes.

Tritiated 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied to adult mouse skin; at specified time intervals the mice were killed, and the labeled phorbol was extracted and subjected to separation and quantitation by high-pressure liquid chromatography. After 24 hr, TPA comprised greater than 96% of the recovered label from the skin, and its apparent half-life was 17.8 hr. Pretreatment of adult skin with TPA for 4 weeks before treatment with labeled TPA resulted in an increase in the clearance rate of TPA from the skin. Skin from newborn mice was capable of converting TPA into monoesters and phorbol, but the clearance rate in the adult was about 12 times more rapid than it was in the newborn. Epidermal homogenates converted TPA into 12-O-tetradecanoylphorbol, phorbol-13-acetate, and phorbol. Hepatic homogenates were able to convert TPA to monoesters and phorbol at rates 14 to 15 times faster than were epidermal homogenates. Attempts to isolate any previously undescribed metabolites of TPA by use of liver homogenates were unsuccessful, and mixed-function oxidation did not contribute to the metabolism of TPA. From inhibitor studies it was judged that esterases were implicated in the conversion of TPA to monoesters and phorbol. The results support the hypothesis that the tumor-promoting activity of TPA is directly related to its concentration in a specific tissue and that conversion of TPA to an active metabolite probably does not occur.

Animals↗

Natural cytotoxicity in the mouse embryo. I. Evidence for the presence of natural cytotoxic function in the yolk sac of the 10-day mouse embryo.

Yolk sac cells from 10-day mouse embryos have natural cytotoxic activity directed against YAC-1 tumor targets but not against P815 tumor targets. Other embryonic tissues tested, including TG-16 embryonal carcinoma, fetal thymus, and fetal liver, do not display natural cytotoxic activity comparable to the yolk sac activity. The correlations between yolk sac natural cytotoxic function, and adult NK and promonocyte cytotoxic functions are briefly discussed.

Animals↗

Complete ischaemia of the normal mouse, rat and rabbit testes and of mouse testes with SAI sarcoma.

After complete ischaemia of mouse, rat and rabbit left testes produced by ligating the funiculus spermaticus, a diminution and degenerative changes occurred in the testes following a temporary swelling. Degenerative changes were associated, in addition to necrosis of all cells in the testes, with an infiltration of leucocytes, mainly polymorphonuclear and mononuclear cells, and lymphocytes from the neighbourhood of the testis through the tunica albuginea into the surface parts of testicular tissue. The cells enumerated above migrated gradually from the periphery to the centre of the testis. First, they colonized the interstitial tissue and then perturbed the membrana propria of the seminiferous tubules and got inside the tubules. In the majority of animals, the appearance of connective tissue with collagen fibres was noted behind the zone of leucocytes and lymphocytes while the testis was shrinking. Ischaemic testes disappeared completely within 6 to 10 weeks after devitalization in most of the mice. Antibody production in the course of the degenerative process in the ischaemic testes was not demonstrated by indirect immunofluorescence nor was it detectable by spermagglutination test in rabbits, and the skin tests in rats and rabbits were also negative. The contralateral, non-ischaemic testes were not damaged in any of the examined males as shown by histological analysis. A similar degenerative process occurred in most of the testes of mice in which syngeneic SAI sarcoma cells grew and were implanted intracutaneously on the back of mice of the same inbred strain. Upon intraperitoneal inoculation of tumour cells the growth of ascites tumour appeared 5 to 8 weeks after implantation of the tumorous testis in about 30% of the mice, 70% of mice were resistant. Ascites tumours were demonstrated in control animals without previous tumour implantation and deaths were recorded in 95% of mice.

Animals↗

Enhancement by immunostimulants of the production by mouse spleen cells of factor(s) stimulating differentiation of mouse myeloid leukemic cells.

Mouse myeloid leukemic Ml cells can be induced to differentiate into macrophages and granulocytes in vitro by a factor(s) stimulating differentiation of the cells (D-factor) or by various chemical compounds. Spleen lymphocytes and spleen macrophages have been shown to produce D-factor when treated with various mitogens including lipopolysaccharide or synthetic double-stranded polyribonucleotide, poly(I) . poly(C). Several immunostimulants from microorganisms were tested for ability to induce differentiation of Ml cells and to stimulate production of D-factor by spleen cells. Mycobacterium bovis BCG (500 micrograms/ml) and the cell wall skeleton of Nocardia rubra (50 micrograms/ml) induced differentiation of Ml cells; but Corynebacterium parvum CN6134 and the cell wall skeleton of Propionibacterium acnes C7 had no effect on Ml cells. On the other hand, all immunostimulants tested at concentrations of more than 10 micrograms/ml were found to stimulate production of D-factor by both spleen lymphocytes and spleen macrophages. Synthetic derivatives of N-acetylmuramyl dipeptide(MDP), the minimal adjuvant-active subunit of the bacterial cell wall, had no direct effect on the differentiation of Ml cells and only slightly stimulated the production of D-factor by spleen cells. Therefore, the structure of microorganisms required for induction of differentiation of Ml cells and for stimulation of production of D-factor are suggested to be different from that of MDP.

Acetylmuramyl-Alanyl-Isoglutamine↗

Mouse spleen cell-derived toxoplasma growth inhibitory factor: its effect on toxoplasma multiplication in the mouse kidney cells.

When Toxoplasma tachyzoites were inoculated into normal kidney cell monolayers, they multiplied in the cells and further causing cell rupture. However, when Toxoplasma immune lymphokines (LKs) was added to normal kidney cells, the intracellular multiplication of the tachyzoites was inhibited remarkably; particularly, in the case in which the tachyzoites were exposed to immune fresh serum prior to inoculation into the cells. Toxoplasma growth inhibitory factor (Toxo-GIF) in LKs, with a m.w. of 30,000 to 40,000 was found to be contained in LKs-II or MIF-I fraction after Sephadex G-100 gel filtration. LKs collected from Balb/c mice inhibited tachyzoite multiplication in ICR-JCL normal mice kidney cells, suggesting that LKs activity did not show mouse-strain specificity. In contrast, the addition of Toxoplasma immune serum to infected cells had no effect on the intracellular multiplication of Toxoplasma and also did not prevent cell-to-cell spread. However, when extracellular tachyzoites were exposed to Toxoplasma immune fresh or heat-inactivated immune serum, especially to the former, at 37 degrees C for 30 min, their active penetration into the kidney cells were extremely inhibited and their subsequent multiplication in the cells were almost totally inhibited within 24 h as compared with those exposed to normal serum.

Animals↗

The mononuclear phagocyte system of the mouse as demonstrated by the immunoperoxidase technique using anti-mouse macrophage antiserum.

With an antibody raised to mouse peritoneal macrophages, it is possible to demonstrate macrophages in various murine tissues using an immunoperoxidase technique. The distribution of these cells is described and illustrated. The antigenic determinant(s) appears to reside on the macrophage surface and this has been confirmed by appropriate electron microscopic studies of stained Kupffer cells. With an antibody to human macrophages, the method may be useful in clinical histopathological practice.

Animals↗

Serological distinciton of heavy chain variable regions (VH subgroups) of mouse immunoglobulins. I. Common VH determinants on the heavy chains of mouse myeloma proteins having different binding sites.

16 of more than 100 mouse myeloma proteins, including 3 proteins of the IgG2a class and 13 of the IgA class, were shown to have a similar heavy chain variable region (VH) antigen(s) (U10-173). The proteins bearing these antigenic determinants (U10-173+ proteins) represented at least five different ligand-binding specificities. These findings, taken togeter with available sequence data for VH regions of U10-173+ proteins, have led us to conclude that U10-173 defines a small number of related VH subgroups. The ability to detect VH subgroups in mice by serological means, as has been done in humans also, promises new and useful kinds of VH markers for immunologic study.

Animals↗

In vitro responses of nude mouse-xenografted human colon carcinomas exposed to doxorubicin derivatives in tissue culture and in the mouse.

In vitro responses of 4 xenografted human colon tumors (T183, T219, T245, and T348) to various doses of 4'-deoxydoxorubicin have been investigated. The individual tumors showed marked differences in drug responsiveness, ranging from high sensitivity at low doses (T219; 125 ng/ml) to very low sensitivity at high doses (T245; 4000 ng/ml). The sensitivity ranking deduced from these in vitro experiments correlates well with the ranking deduced earlie (Guiliani et al., Int. J. Cancer, 27: 5-13, 1981) from in vivo drug treatments of transplants of these tumors in the nude mouse. The effect of in vitro drug treatment (4'-deoxydoxorubicin; 250 ng/ml; 1-hr incubation) on the in vivo growth of one of the tumors, T219, in nude mice was investigated. Growth of the tumor in nude mice was markedly delayed by pretreatments in vitro with 4'-deoxydoxorubicin. Furthermore, in vitro responsiveness of the T219 tumor was investigated following in vivo and in vitro treatment of the tumor with 4'-deoxydoxorubicin. Both of the pretreatments produced very similar decreases in drug responsiveness to all of the doxorubicin derivatives tested (4'-deoxydoxorubicin, 4'-O-methyldoxorubicin, 4'-epidoxorubicin, 4-demethoxydoxorubicin, and N-trifluoroacetyldoxorubicin-14-valeriate).

Animals↗

Some flow-cytofluorimetric studies of the nuclear ploidy of mouse hepatocytes: iii. further observations on early changes in nuclear ploidy of mouse hepatocytes following various experimental procedures.

Distribution of nuclear ploidy in female mouse hepatocytes was measured cytofluorimetrically using ethidium-bromide-stained hepatocyte nuclei liberated by in situ collagenase perfusion of the liver via the portal vein. After i.v. administration of lead acetate or an i.p. general anaesthetic (Valium and Hypnorm), rapid shifts of 8N and 4N nuclei to lower ploidy levels were recorded. It was possible to block the ploidy changes with i.p. colchicine, although no blocked metaphases were observed histologically. These observations were consistent with an earlier report on changes in nuclear ploidy induced by carbon tetrachloride (Steele et al., 1981b). It is speculated that these changes in ploidy may represent an early response of the hepatocytes to stimulation--an aspect of liver behaviour of which we were not previously aware.

Animals↗

Monoclonal antibodies to mouse MHC antigens. II. Antibodies to the H-2Ld antigen, the products of a third polymorphic locus of the mouse major histocompatibility complex.

In order to study the fine structure of H-2L, the third H-2 locus of the mouse major histocompatibility complex, we have produced and characterized the monoclonal anti-Ld antibodies secreted by 3 different hybridoma cell lines. Two antibodies were obtained by a specific immunization directed to Ld determinants. Another antibody obtained from an immunization including, but not specific for, an L locus difference was found to detect Ld, and not Kd or Dd molecules. Although the fusions involved an H-2d myeloma variant line, no apparent adverse "autoreaction" was observed in these anti-H-2d antibody-producing hybridoma cells. All 3 antibodies exhibited cross-reactions with the antigens of certain haplotypes, and the reactivity patterns observed corresponded to the previously described alloantigenic specificities H-2.64 and H-2.65. This concordance with known H-2 specificities contrasts with the broad, unique specificity patterns exhibited by most monoclonal antibodies to H-2 antigens and may reflect a difference in the complexity of the polymorphism of the L locus relative to that of the K and D loci. Immunoprecipitation analyses using 1 of the monoclonal antibodies showed that the molecules precipitable by this reagent were indistinguishable from those precipitable by anti-Ld alloantisera. In addition, cross-reactions of Ld, Dq, and Lq molecules were revealed by sequential immunoprecipitation analyses, supporting a close evolutionary relationship of L and D loci.

Animals↗

An immunologic comparison between bone marrow and spleen-derived pluripotent hemopoietic stem cells (CFU-S) of mouse : effect of rabbit anti-mouse brain serum.

We used rabbit anti-mouse brain serum (RAMBS), with known activity against pluripotent hemopoietic stem cells (CFU-S) of mice, to compare the proportions of CFU-S from bone marrow and spleen which express the brain-associated CFU-S antigen(s). Eighty percent of marrow and 77% of spleen-derived CFU-S were inactivated by RAMBS. This suggests that these two populations of CFU-S are antigenically similar with respect to the brain-associated CFU-S antigen(s); this is in contrast to functional differences which are known to exist between the two populations of CFU-S.

Animals↗

Facial development in the mouse; a comparison between normal and mutant (amputated) mouse embryos.

This work extends previous investigations into cell interactions involved in specific morphogenetic events during the development of normal and mutant (amputated) mouse embryos. In the mutant mesenchyme, cells tend to clump together and form far more extensive areas of cell contact than are found in normal mesenchyme. This is confirmed for mutant facial mesenchyme. Facial outgrowth in the mutant is retarded. The first stages of this abnormality can be seen in the naso-frontal region at 10.5 days after conception. Neither the quantity of cells contributing to naso-frontal outgrowth nor cell proliferation in the naso-frontal region differ from normal in the mutant, and these factors can be eliminated as causes of the anomaly. Instead, cell clumping and increased areas of contact in the mutant arrest the normal expansion of the naso-frontal mesenchyme which presumably occurs as a result of increased secretion of intercellular matrix material between 9.5 and 10.5 days of development. The importance of this early expansion phase for facial development has not previously been recognized.

Animals↗

In vitro chronopharmacology of recombinant mouse IL-3, mouse GM-CSF, and human G-CSF on murine myeloid progenitor cells.

Circadian changes in in vitro pharmacodynamic effects of recombinant mouse interleukin-3 (rmIL-3), rm granulocyte-macrophage colony-stimulating factor (rmGM-CSF), and recombinant human G-CSF (rhG-CSF) were investigated in 418 male B6D2F1 mice. Seven distinct experiments were staggered from July to December 1991. All mice were standardized for 3 weeks with a lighting schedule consisting of 12 hours of light and 12 hours of dark (LD12:12). In each experiment, bone marrow was sampled from separate groups of nine to 10 mice each every 4 hours for 24 hours. Data were analyzed with analysis of variance (ANOVA) and Cosinor. This latter method computes the probability of rhythm detection and its parameters. Femoral myeloid progenitors were quantified using the colony-forming units granulocyte/macrophage (CFU-GM) assay in the presence or absence of recombinant CSFs. For each CSF, the number of colonies is a function of circadian time of bone marrow exposure (ANOVA and Cosinor; p < 0.0001) with the values at peak time being double those found at the trough. Peak CSF efficacy occurred at 3 hours after light onset (HALO, early rest span) irrespective of CSF type or dose. Furthermore, in the absence of any added CSF, the number of clusters varied significantly according to sampling time, with a similar peak at 3 HALO (ANOVA and Cosinor; p < 0.001). Further in vivo chronopharmacologic experiments are needed to assess the relevance of these in vitro rhythms in bone marrow responsiveness to hematopoietic growth factors.

Animals↗

Development of a nude mouse model of ras-mediated neoplasia using WR21 cells from a transgenic mouse salivary tumor.

A novel cell line (WR21) was derived from a salivary tumor in a male wap-ras transgenic mouse. Salivary tumors in wap-ras transgenic mice are extremely aggressive and express high levels of oncogenic ras protein from the activated, human Ha-ras transgene. WR21 cells also expressed high levels of oncogenic ras protein in vitro and in vivo. They gave rise to aggressive, highly anaplastic solid tumors when injected subcutaneously into athymic nude mice and approximately 90% of the mice had lung metastases by the fifth week of tumor growth. WR21 tumors were inhibited by cyclophosphamide, 5-fluorouracil, adriamycin, mitomycin C and actinomycin D, but not methotrexate. Our results suggest that the WR21/nude mice model will be useful for testing the efficacy of drug therapies against ras-mediated neoplasias.

Animals↗

An exon encoding the mouse growth hormone binding protein (mGHBP) carboxy terminus is located between exon 7 and 8 of the mouse growth hormone receptor gene.

A C57 black/6 mouse genomic library was screened for mGHR/BP using a mGHR cDNA hybridization probe. Two clones (mGHR-GA and mGHR-GB) were isolated. mGHR-GA contained an insert of 16 kb and hybridized only to exon 4 whereas mGHR-GB possessed an insert of 20 kb and hybridized to exons 6-10 of the mGHR cDNA. Oligonucleotide sequencing analysis confirmed that the mGHR-GA contained exon 4 and that the mGHR-GB possessed mGHR/BP genomic sequences from intron V through the 3' region of mGHR. In addition, mGHR-GB also contained a region, designated exon 8A, encoding the carboxy terminus of mGHBP. This exon is located between exons 7 and 8 of mGHR/BP gene. Two poly(A) additional signal sequences were found 54 bp downstream of the stop codon of mGHBP. Additionally, introns between exons 3/4 and 4/5 are > 12 and > 3 kb, respectively. Also, introns between exons 5/6, 6/7, 7/8A, 8A/8 (3' region of mGHBP), 8/9, 9/10, and the 3' region of mGHR are approx > 3 kb, 4 kb, 287 bp, 1569 bp, 6 kb, 265 bp, and > 3 kb, respectively. This study supports the hypothesis that mGHBP is generated by alternative splicing of a common primary mGHR transcript. It also demonstrates differences and similarities between mGHR/BP and hGHR genes.

Animals↗

High conservation of subunit composition of RNA polymerase I(A) between yeast and mouse and the molecular cloning of mouse RNA polymerase I 40-kDa subunit RPA40.

Mouse RNA polymerase I (or A) was purified from an ascites cell line MH134 to virtual homogeneity using a novel purification procedure and examined for subunit composition. In marked contrast to older purifications that reported 5-8 subunits, polymerase I was found to have 11 subunits with remarkable correspondence to those of yeasts. The cDNA encoding a 40-kDa subunit of this enzyme, designated RPA40, was isolated. It predicts a polypeptide of 355 amino acids (M(r) = 40,065) and is encoded by a single copy gene. Protein sequence analysis reveals that RPA40 is the homolog of yeast RPC40, having homology to alpha subunit of Escherichia coli RNA polymerase, yeast RPB3, and human RPB33 RNA polymerase II subunits. The high conservation of this subunit among distant eukaryotes and different RNA polymerases suggests functional importance of this protein as a core subunit.

Amino Acid Sequence↗