C3H/HeN mammary tumor-bearing mice develop type-specific neutralizing antibodies and group-specific precipitating antibodies for the mouse mammary tumor virus.

The development of mouse mammary tumor virus (MMTV)-neutralizing antibodies in various strains of mice was measured by their ability to neutralize the focus-forming capacity of a Kirsten sarcoma virus (C3H MMTV) pseudotype containing the MMTV envelope glycoprotein gp52. C3H/HeN, but not GR/N and RIII, mammary tumor-bearing mice were found to develop neutralizing antibodies to this pseudotype. In addition, non-tumor-bearing C3H/HeN, GR/N, RIII, NIH Swiss, C57BL/6, and BALB/c mice and 13 feral mice were also negative for neutralizing antibodies. The neutralization was immunoglobulin G mediated, and the antibodies of C3H/HeN mammary tumor-bearing mice were type specific and capable of distinguishing C3H and GR/N MMTVs from RIII and C3H/HeNf MMTVs. Precipitating antibodies were detected in sera from RIII and GR/N tumor-bearing mice, GR/N non-tumor-bearing mice, and six of the feral mice, although these same sera did not neutralize the Kirsten sarcoma virus (C3H MMTV) pseudotype. The results of this study and of a previous study demonstrate that C3H/HeN mammary tumor-bearing mice develop three functionally distinct antibody populations: (i) group-specific virus-precipitating antibodies; (ii) type-specific virus-neutralizing antibodies; and (iii) type-specific cytotoxic antibodies.     

Biochemical comparison of H-2K antigen isolated from C3HfB/HeN and C3H/HeN mice

The H-2K glycoproteins were isolated from spleen cells of C3H/HeN and C3HfB/HeN mice and compared by tryptic peptide mapping techniques. The two antigens were found to be very similar in that more than 90 percent of detectable peptides appeared identical. However, two lysine-containing peptide present in tryptic digests of H-2K antigens isolated from C3H mice were absent from tryptic digests of H-2K antigens isolated from C3Hf mice. This was probably not the result of altered glycosylation since neuramindase digestion demonstrated that the disparate peptides were not glycopeptides but most probably resulted from substitution of one or two amino acids in the H-2K molecule of C3HfB/HeN mice. These differences were small but significant and demonstrated that H-2K^k (C3H) and H-2K^kv1 (C3Hf) antigens are structurally distinct. This is compatible with the observed reciprocal skin-graft rejection, MLR, and generation of cytotoxic T lymphocytes between the two strains. The significance of this finding in conjunction with what is known about properties of 1-ethyl-1-nitrosourea-induced tumors of C3Hf mice is discussed.     

Dietary manipulation of mammary tumor development in adult C3H/Bi mice

Chronic energy intake restriction (CEIR) in virgin female mice is one of the most effective ways of reducing significantly mammary adenocarcinoma in C3H/Bi mice, a strain which develops mammary adenocarcinoma associated with the murine mammary tumor virus spontaneously and at high incidence. In this study, the influence of chronic energy intake restriction imposed on fully mature (4- to 5-month-old), breeding female C3H/Bi mice was addressed, and the influence of energy intake where energy was derived largely from fat versus diets in which energy was derived largely from carbohydrates on tumor development and survival rate was investigated. The results show that chronic energy intake restriction can be delayed until full maturation and successful reproduction and still reduce significantly the incidence of mammary tumor development in this relatively short-lived strain of mice. Our findings demonstrate that the overriding dietary factor controlling mammary tumor development in these experiments in C3H/Bi mice was the level of energy intake, regardless of the primary source of energy (fat or carbohydrates).     

Antileishmanial activities of macrophages from C3H/HeN and C3H/HeJ mice treated with Mycobacterium bovis strain BCG

C3H/HeN and C3H/HeJ mice were infected ip with viable BCG, a macrophage-activating agent, and their peritoneal exudate macrophages exposed to Leishmania tropica amastigotes. Macrophages from BCG-infected C3H/HeN mice had both leishmanicidal activities described for lymphokine activation of C3H/HeN macrophages in vitro: increased resistance to L. tropica infection, followed by intracellular killing of the parasite. Macrophages from BCG-infected C3H/HeN mice were also activated to kill tumor cells in vitro. In contrast, macrophages from BCG-treated C3H/HeJ mice were not resistant to L. tropica infection, did not kill intracellular amastigotes over 72 hr in culture, and were not cytotoxic to tumor cells.     

Production of the mouse mammary tumor virus in cultures of BALB/cfC3H strain

The mouse mammary tumor virus (MTV) reproduces by a budding mechanism at the cell membrane of mouse mammary epithelial cells. In tissue culture, the tumor cells release their virions in the culture supernatant from which they can be removed by high speed centrifugation. Mammary tumor cells from the RIII, GR, and A strains of mice generally produce yields of virus which decrease after a few months. Cells derived from a spontaneous mammary tumor in a BALB/cfC3H mouse have shown the capability to shed relatively large amounts of virus continuously. A quantitative estimation by membrane immunofluorescence of the number of virus producing cells in one-year-old cultures revealed the presence of viral antigen on 80 to 90% of the cells; by comparison, cultures from other mouse strains had a ratio of only 10 to 15% virus producing cells. High speed centrifugation pellets obtained from 50 ml culture supernatant provided large amounts of mature virus particles which have been characterized by electron microscopy.     

Direct detection of exogenous mouse mammary tumor virus sequences in lymphoid cells of BALB/cfC3H female mice.

The presence of exogenous mouse mammary tumor virus (MMTV) (C3H) DNA sequences in lymphoid tissue (spleen, bone marrow, and thymus) and nonlymphoid tissue (liver and kidney) of BALB/cfC3H female mice was directly assessed by DNA hybridization methods. Lymphoid tissues were found positive for integrated MMTV(C3H) sequences in females as young as 4 weeks. In most samples, the level of splenic MMTV(C3H) infection was low (2 to 5%). Infection remained throughout the life of the animal. The percentage of spleen samples found positive for exogenous viral infection was significantly higher in females bearing mammary tumors, whether virgin or multiparous. Liver and kidney DNAs were negative for exogenous MMTV sequences, suggesting tissue type selectivity in MMTV infection.     

Comparison of mammary tumorigenesis between litters in relation to mammary tumor virus antigenic expression in the milk of C3H/He mice

As a possible step to estimate the relationship between mammary tumor virus (MTV) and mammary tumorigenesis in mice, MTV antigenic expression in mother's milk and spontaneous mammary tumorigenesis in their daughters were compared between the 1st, the 2nd and the 3rd litters of the highly inbred strains of C3H/He mice with low mammary tumor incidence. While MTV antigenic expression was detected in all undiluted samples at each litter by immunodiffusion test, the amount of antigen as measured by the single radial immunodiffusion method was increased with increasing litter numbers. On the other hand, the development of preneoplastic mammary hyperplastic alveolar nodules was different little between litters and mammary tumor incidence by 13 months of age was very low with no difference in all litters. The pattern of estrous cycles and plasma prolactin level were also similar in each litter. The results suggest that spontaneous mammary tumorigenesis in mice is not always affected quantitatively by the amount of MTV when endocrine and genetical conditions are similar.     

Growth and development of Gymnophalloides seoi in immunocompetent and immunosuppressed C3H/HeN mice

The growth and development of Gymnophalloides seoi were studied in C3H/HeN mice and effects of immunosuppression of the host on the worm development were observed. Two hundred metacercariae of G. seoi were orally administered to each mouse, and worms were recovered on days 1, 3, 5, 7, 14 and 21 post-infection (PI). The worm recovery rate was significantly higher in immunosuppressed (ImSP) mice than in immunocompetent (ImCT) mice except on days 1 and 3 PI. The worms attained sexual maturity by day 3 PI with eggs in the uterus, and worm dimensions and the number of uterine eggs continuously increased until day 14 PI in ImSP mice. Worms recovered from ImSP mice were significantly larger in size than those from ImCT mice on days 1 and 3 PI, and the number of uterine eggs was significantly larger in ImSP mice on days 5 and 7 PI. Genital organs such as the ovary, testes, and vitellaria, that were already developed in the metacercarial stage, grew a little in size until day 14 PI. The results show that the C3H/HeN mouse is, though not excellent, a suitable laboratory host for G. seoi.     

Morphologic analysis of enteric lesions in conventional and streptomycin-treated inbred C3H/HeN mice infected with Serpulina (Treponema) hyodysenteriae.

Oral administration of streptomycin is known to enhance the susceptibility of mice to enteric pathogens by altering the indigenous flora. We examined the effect of oral streptomycin treatment on the susceptibility of inbred C3H/HeN mice to infection with Serpulina (Treponema) hyodysenteriae. A total of 56 mice were randomly divided into four groups (A-D) of 14 each. From days 0 to 7, mice in groups A and B received streptomycin in their drinking water and mice in groups C and D served as controls. On day 7, mice in groups A and C were inoculated intragastrically with S. hyodysenteriae serotype 4, strain A1, and groups B and D served as uninoculated controls and received sterile trypticase soy broth. Clinical signs were monitored daily and body weights were recorded weekly. Mice were euthanized and necropsied for bacteriologic and histopathologic examinations on day 7 (2/group) and on days 14, 21, 28, and 35 (3/group) of the experiment. Soft fecal pellets were noticed in infected groups (A and C), but no significant differences in body weights were observed between groups (P greater than 0.05). Macroscopic changes were noted only in infected groups (A and C) beginning on day 21 of the experiment and consisted of catarrhal typhlitis, cecal emptiness, and atrophy. Histologically, the cecum and colon of mice in groups A and C had goblet cell hyperplasia, which preceded crypt epithelial cell hyperplasia, inflammatory cell infiltrates, and focal necrosis of mucosal epithelium. S. hyodysenteriae was reisolated from 10 of 12 mice in each infected group (A and C) from day 14 (7th day postinoculation) through day 35 (28th day postinoculation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Protective immunity against Rickettsia heilongjiangensis in a C3H/HeN mouse model mediated by outer membrane protein B-pulsed dendritic cells

Rickettsia heilongjiangensis is an obligate intracellular bacterium that causes Far-Eastern tick-borne spotted fever. Outer membrane protein B(Omp B) is an important surface protein antigen of rickettsiae. In the present study, the omp B gene of R. heilongjiangensis was divided into four fragments, resulting in four recombinant proteins(OmpB-p1, Omp B-p2, Omp B-p3, and Omp B-p4). Each Omp B was used in vitro to stimulate murine bone marrow-derived dendritic cells(BMDCs) of C3H/He N mice, and the Omp B-pulsed BMDCs were transferred to naive C3H/He N mice. On day 14 post-transfer of BMDCs, the mice were challenged with R. heilongjiangensis and the rickettsial loads in the mice were quantitatively determined on day 7 post-challenge. Mice receiving BMDCs pulsed with Omp B-p2, Omp B-p3, or Omp B-p4 exhibited significantly lower bacterial load compared with mice receiving Omp B-p1-pulsed BMDCs. CD4+ and CD8+ T cells isolated from the spleen of C3H/He N mice receiving BMDCs pulsed with each OmpB were co-cultured with BMDCs pulsed with the respective cognate protein. In flow cytometric analysis, the expression level of CD69 on CD4+ or CD8+ T cells from mice receiving BMDCs pulsed with Omp B-p2, OmpB-p3, or Omp B-p4 was higher than that on cells from mice receiving Omp B-p1-pulsed BMDCs, while the expression level of tumor necrosis factor(TNF)-α on CD8+ T cells and interferon(IFN)-γ on the CD4+ and CD8+ T cells from mice receiving Omp B-p2,-p3, or-p4 was significantly higher than on cells from mice receiving Omp B-p1-pulsed BMDCs. Our results suggest that the protective Omp Bs could activate CD4+ and CD8+ T cells and drive their differentiation toward CD4+ Th1 and CD8+ Tcl cells, respectively, which produce greater amounts of TNF-α and, in particular, IFN-γ, to enhance rickettsicidal activity of host cells.     

BALB/Mtv-null mice responding to strong mouse mammary tumor virus superantigens restrict mammary tumorigenesis

The absence of endogenous mouse mammary tumor viruses (MMTVs) in the congenic mouse strain, BALB/Mtv-null, restricts the early steps of exogenous C3H MMTV infection, preventing the superantigen (Sag) response and mammary tumorigenesis. Here we demonstrate that BALB/Mtv-null mice also resist tumor induction by FM MMTV, which encodes a stronger Sag compared to C3H MMTV. In contrast to infections with C3H MMTV, Mtv-null mice show FM-MMTV Sag-specific responses comparable to those observed in susceptible BALB/c mice. Neither virus shows significant replication in the spleen or mammary gland. Thus, Mtv-null mice restrict MMTV replication and mammary tumorigenesis even after a robust Sag response.     

C3H mouse mammary tumor virus superantigen function requires a splice donor site in the envelope gene

Mouse mammary tumor virus (MMTV) encodes a superantigen (Sag) that is required for efficient milk-borne transmission of virus from mothers to offspring. The mRNA used for Sag expression is controversial, and at least four different promoters (two in the long terminal repeat and two in the envelope gene) for sag mRNA have been reported. To determine which RNA is responsible for Sag function during milk-borne MMTV transmission, we mutated a splice donor site unique to a spliced sag RNA from the 5' envelope promoter. The splice donor mutation in an infectious provirus was transfected into XC cells and injected into BALB/c mice. Mice injected with wild-type provirus showed Sag activity by the deletion of Sag-specific T cells and induction of mammary tumors in 100% of injected animals. However, mice injected with the splice donor mutant gave sporadic and delayed T-cell deletion and a low percentage of mammary tumors with a long latency, suggesting that the resulting tumors were due to the generation of recombinants with endogenous MMTVs. Third-litter offspring of mice injected with wild-type provirus showed Sag-specific T-cell deletion and developed mammary tumors with kinetics similar to those for mice infected by nursing on MMTV-infected mothers, whereas the third-litter offspring of the splice donor mutant-injected mice did not. One of the fifth-litter progeny of splice donor mutant-injected mice showed C3H Sag activity and had recombinants that repaired the splice donor mutation, thus confirming the necessity for the splice donor site for Sag function. These experiments are the first to show that the spliced sag mRNA from the 5' envelope promoter is required for efficient milk-borne transmission of C3H MMTV.     

Age-related changes in cell-mediated immunity in BALB/C mice.

The effect of increasing age on various tests of cell-mediated immunity was investigated in BALB/c mice both in vitro and in vivo with four different assay systems. The following results were obtained. 1) In contact sensitivity to DNFB, old mice (age 60 to 80 weeks) showed no differences in sensitization when compared to young adult mice (age 8 to 12 weeks). (In contrast, old NZB/W mice showed impaired contact sensitization when compared with young NZB/W MICE.)2) Unlike the reaction in contact sensitivity, cells from old BALB/c mice were defective in eliciting a graft-vs-host reaction. This was true also when a partially purified population of T cells was transferred. 3) In the mixed lymphocyte reaction, cells from old mice were as efficient or better than cells from young adult BALB/c mice in responding to or stimulating allogeneic cells. 4) Responses to PHA and Con A (Both T cell mitogens) were greatly reduced when old cells were cultured as compared with cells from young adult mice. Thus, we have found that within the same batch of mice, increasing age was associated with increased capabilities in some measures of cell-mediated immunologic function and decreased capabilities in other measures of the same.     

Distribution of mouse mammary tumor virus in Asian wild mice.

Several groups of wild mice (Mus musculus) were captured from eight different locations in Asia and bred for several generations in a facility free of any laboratory strains of mice carrying mouse mammary tumor virus (MMTV). The distribution of endogenous MMTV proviral sequences in the liver tissues of these mice was investigated by using Southern blot hybridizations. Four categories of mice were identified. Mice originating from Bogor, Indonesia (Cas-Bgr); He-mei, Taiwan (Cas-Hmi/1); and Malaysia (Cas-Mal) were found to carry an endogenous MMTV provirus consisting of the env, gag-pol, and long terminal repeat sequences. Mice captured from Kojuri, Republic of Korea (Sub-Kjr); Nagoya, Japan (Mol-nag); and three Chinese provinces, Shanghai (Sub-Shh), Beijing (Sub-Bjn), and Jiayuguang (Sub-Jyg/1), appeared to carry defective proviruses. Some mice originating from He-mei (Cas-Hmi/2) and Jiayuguang (Sub-Jyg/2) were found to be completely free of endogenous MMTV. Interestingly, however, the Sub-Jyg/2 mice, after several generations of inbreeding, were found, unlike all of the other subspecies that we examined in the present study, to develop mammary tumors at a high incidence (80 to 90%) with a short period of latency. Electron microscopic examination of the mammary glands and mammary tumors of these mice revealed the presence of numerous intracytoplasmic A, immature, budding, and mature B particles. Furthermore, the mammary tumors were found to contain MMTV proviral sequences. It seems, therefore, that Sub-Jyg/2 mice carry an exogenous MMTV which contributes to their developing mammary tumors.