C3H Strain Research Articles

Response of C3H-A vy fB Female Mice to a Low Oncogenic, Milk-Borne Mammary Tumor Virus

When low oncogenic milk-borne mammary tumor virus (MTV) of strain DD was tested in C3H-AvyfB mice, as compared with the similar strain C3HfB, mammary tumors occurred with a higher incidence and at lower age in C3H-AvyfBfDD (with DD-MTV) than in C3HfBfDD (with DD-MTV), as observed in virgin females. Since the effect of breeding tended to mask any difference between the breeders, mammary tumor incidences were similar with a slightly higher age at tumor occurrence in the C3H-AvyfBfDD females. Thus the C3H-AvyfB virgins (with the Avy gene) were almost as susceptible to mammary tumors with the less potent DD-MTV as were C3HfB virgins with the more virulent C3H-MTV of strain C3H.

EXPERIMENTAL MURINE LEPROSY.

Mice with the inbred strains C57/BL and C3H were inoculated subcutaneously on the thorax with M. lepraemurium. In C57/BL mice a firm, raised, sharply defined nodular infiltrate developed 4 weeks afterwards, while in the C3H strain of the infection produced a soft, flattened infiltrate with ill-defined margins, which did not become palpable until 10 weeks after inoculation. A limited spread of the infection occurred early in both strains, but apparently multiplication of the microorganisms was very restricted in C57/BL mice; progressive, disseminated growth of the bacilli was observed in the C3H strain only. In C57/BL mice the granulomatous reaction, developing 4 weeks after inoculation and leading to abcess formation, ulceration and scar formation, apparently inhibited both local multiplication and further spread of the bacilli. In C3H mice no host reaction was detected and the bacilli appeared to grow unrestrictedly. In some C57/BL animals, decrease in host resistance occurred during the infection, causing reactivation of the local lesion and an apparently rapid proliferation of bacilli. Observations regarding the lesions in superinfected animals indicated that a systemic immune reaction develops in the C57/BL strain about 4 weeks after inoculation, whereas this does not occur in the C3H strain.

EXPERIMENTAL MURINE LEPROSY.

M. lepraemurium was injected subcutaneously into two inbred strains of mice, C3H and C57/BL, in order to study the local reaction at various time intervals. Within 6 hours an acute inflammatory reaction developed at the site of injection. In the course of the following days it was replaced by a mononuclear infiltrate. The influx of mononuclear cells appeared to be somewhat greater in C57/BL than in C3H mice. Apart from this, little difference was observed between the two strains until at 4 weeks when a vigorous granulomatous reaction developed in the C57/BL strain. This reaction apparently arrested further local spread of the infection. The histological appearance of the inflitrate indicated that a delayed hypersensitivity reaction was taking place. No signs of such reaction was observed in the C3H strain.

Reduced Capacity to Produce Specific ‘Effector’ Cells after Injection of CBA Mice with C3H Cells

Lymphocytes from mice of strain CBA are strongly MLC-responsive to lymphocytes from the H-2-compatible but M-antigen-incompatible strain C3H. This strong reactivity disappears after infusion of CBA mice with C3H lymphocytes. This study shows that the host-versus-graft reactivity (swelling of local lymph node after antigen injection) is specifically reduced after injection of CBA mice with C3H times CBA spleen cells. However, lymphocytes from such mice showed a specifically increased GVH reactivity (inhibition of erythroid cell growth) compared with lymphocytes from unimmunized mice. Lymphocytes from normal CBA mice showed a high proliferative rate in the spleens of irradiated C3H times CBA mice. Such 'educated' cells showed strongly increased specific GVH reactivity. Lymphocytes from CBA mice previously injected with C3H times CBA cells showed reduced capacity to proliferate when injected into irradiated C3H times CBA hybrids and a poor capacity to develop new 'effector' cells reactive against C3H times CBA bone marrow target cells. The results indicate that the presence of specifically 'MLC-responsive' lymphocytes in a lymphoid cell population is a prerequisite of its production of 'effector' cells able to respond in this GVH assay.

EXPERIMENTAL MURINE LEPROSY

Mice of the inbred strains C57BL and C3H were inoculated in the foot-pads with Mycobacterium lepraemurium (MLM) and the inflammatory reaction was studied using light and electron microscopy. In C57BL mice a granulomatous reaction developed 3-4 weeks after inoculation. The inflammatory exudate at this stage showed numerous lymphocytes, monocytes and macrophages. The latter cell type often contained many lysosomes and appeared activated. The bacilli which were all within phagosomes showed extensive electron dense aggregates of the cytoplasm suggesting severe damage. Lymphocytes and macrophages in close contact with each other were often observed. In macrophages which contained damaged bacilli, spherical lipid-like bodies surrounded by granular endoplasmic reticulum were observed. It is suggested that this cell product could be of some significance for the bactericidal function of the macrophage. Contrary to these findings, the cellular infiltrate developing in C3H mice showed no lymphocytes and consisted exclusively of macrophages. These were all heavily loaded with bacilli. The vast majority of bacilli encountered in this strain was morphologically intact and presumably viable. Lipid-like bodies similar to those observed in infected C57BL macrophages were not encountered in C3H mice. It is concluded that unless the infected macrophages become immunologically activited they are unable to cause bacterial damage or to inhibit the growth of MLM.

Normal Tissue Alloantigens and Genetic Control of Susceptibility to Tumors: Microcytotoxicity Studies on Resistant C3Hf and Susceptible (A × C3Hf)F1 Mice Inoculated with Transplacentally Induced C3Hf Lung Tumor

Abstract The immune response of mice to a transplacentally induced lung tumor was investigated with the microcytotoxicity (MC) assay. The tumor, originally induced in C3Hf mice, does not grow readily when transplanted to normal syngeneic C3Hf recipients. It grows readily, however, in (A C3Hf)F1 hybrids and in strain C3H mice, which express in their normal lung tissue a component which constitutes a strong lung tumor-associated transplantation antigen (TATA) in C3Hf mice. Both lung tumor-immunized C3Hf and tumor-bearing (A × C3Hf)F1 and C3H mice possessed lymphoid cells reactive against cultured lung tumor cells in the MC assay. Reactivity was also observed against cells cultured from normal lungs of (A × C3Hf)F1 and C3H mice, but not against cells similarly cultured from C3Hf or C57BL/6 mice. Anti-tumor MC was inhibited by serum-blocking factors present in some but not all tumor-bearing and tumor-immunized mice. The MC assay and detection by it of serum-blocking factors does not distinguish the effective anti-C3Hf lung tumor immune response of immunized C3Hf mice from the ineffective immune response of tumor-bearing (A × C3Hf)F1 and C3H mice. Furthermore, in lung tumor-bearing mice cells reactive in the MC assay may be directed against a normal tissue antigen rather than a tumor-associated antigen.

Lysis mediated by T cells and restricted by H-2 antigen of target cells infected with vaccinia virus.

VARIOUS virus infections lead to the formation of cytotoxic lymphocytes (CL), which are capable of killing virus-infected target cells1−4. Specific lysis of target cells infected with 51Cr-labelled vaccinia virus could be observed when investigating the cell-mediated cytotoxic reaction to vaccinia virus5; the CL could be characterised as a T cell. The sensitised lymphocytes from C3H mice could only kill syngeneic L929 cells infected with vaccinia virus, whereas lysis by sensitised lymphocytes derived from DBA/2 mice was restricted to the syngeneic infected mastocytoma P815X2 cells. In the lymphocytic choriomeningitis infection the target cell lysis was shown to be restricted by H-2 antigen6. We report here experiments with primary fibroblasts of the mouse strains C3H, DBA/2 and the (C3H DBA/2)F1 generation were designed to affirm that the effector phase of virus-specific lysis of target cells mediated by T cells is restricted by H-2 antigen even in the vaccinia virus infection. Further experiments with H-2 alloantisera were performed to indicate the close local relationship between H-2 antigens and viral surface antigens.

Antibody response in C3H mice injected with CBA lymphoid cells as detected by membrane immunofluorescent staining.

Lymphocytes from CBA mice are strongly responsive to cells from the H-2-identical strain C3H in vitro (MLC), whereas C3H lymphocytes are poorly reactive against CBA cells. Immunization of CBA mice with C3H lymphocytes did not yield any detectable specific antibodies. On the other hand, C3H mice immunized with CBA cells produced specific antibodies as detected by membrane immunofluorescence. Mice of the strains AKR and DBA/2 also possess this specific membrane alloantigen. We thus conclude that it is now possible to discriminate among lymphocytes of CBA and C3H origin.

The effects of active immunization of the decidual cell reaction and ectopic blastocyst development in mice.

One of the most striking features of early pregnancy in the mouse is the development of the decidual cell reaction (DCR). This cellular proliferation is induced in the uterine stroma by the presence of a blastocyst on the 4th day of pregnancy (the day of finding a vaginal plug was designated the 1st day of pregnancy). The significance of the DCR and the interactions between blastocyst and uterus, which result in its induction, are poorly understood. Kirby, Billington & James (1966) postulated that the DCR might be instrumental in preventing the rejection of the conceptus as foreign tissue. The extent of the DCR on the 7th day of pregnancy is dependent on the genotype of the mother and the genotype of the conceptus (Hetherington, 1971). Antigenic differences between mother and conceptus can apparently reduce the extent of the DCR. It is unlikely, however, that either the H-2 or H-3 locus is involved (Hetherington, 1973). The extent of the DCR can also be reduced by immunizing female mice to the antigens of the mating male (Clarke & Hetherington, 1971). In order to understand the mechanisms involved at the time of implan¬ tation which affect the development of the DCR, it is important to determine the genetic locus or loci involved. In the present study, CBA/CafTOf/Crc (CBA/Ca) and C3H/He-mgfTOf/Crc (C3H/He-mg) mice maintained at the Clinical Research Centre and C57BL/10ScSnfNMRI/Lac (BIO), BlO.LpofLACAf/Lac (BlO.Lp) and B10.AfC3H/Hef/Lac (BIO.A) mice maintained at the Laboratory Animal Centre, Carshalton, were used. Both the CBA and C3H strains were derived from a cross between Bagg Albino and DBA (Strong, 1942). After a few generations of inbreeding, a series of lines was produced which included the CBA and C3H strains. As a consequence, these strains are closely related and their H-2 loci are identical. They differ, how¬ ever, at the H-3 locus (for a comparison of the two genotypes, see Graff, 1970; Taylor, 1972). The effect of immunizing CBA mice to C3H and vice versa on the development of the DCR after inter-strain matings and on the growth of inbred blastocysts transferred under the kidney capsule of mice of the opposite strain was studied. The congenie strains BIO (H-2b, H-3a), B10.A (H-2a, H-3a) and BlO.Lp-a (H-2b, H-3b) were used to investigate the effect of specific immunization to the antigens of the H-2 and H-3 loci

Inheritance of antibody specificity. II. Anti-(4-hydroxy-5-bromo-3-nitrophenyl) acetyl in the mouse.

Mice of 17 inbred strains produced anti-(4-hydroxy-5-bromo-3-nitrophenyl)-acetyl (NBrP) of three different fine specificity types. Anti-NBrP antibodies of all allotype b mice (five strains tested) had a high relative affinity for (4-hydroxy-3.5-dinitrophenyl) acetyl (NNP) but low for (4-hydroxy-5-cloro-3-nitrophenyl) acetyl (NCP). Another category was characterized by high relative affinity for NCP but low for NNP. This category included most of the tested strains. The third category (CBA and C3H strains) had an intermediate fine specificity. Associated with fine specificity characteristics were anti-NBrP titers, mice of allotype b had lower titers than the other mice. Studies of congenic, recombinant inbred, F1 and backcross mice showed that both fine specificity and the magnitude of the anti-NBrP response of tbalb/C MICE WERE CONTROLLED BY AN ALLOTYPE-LINKED GENE. This gene was dominant over the C57BL/6 ALLELE. Lack of recombinant mice in the backcross generatioterns on the other suggest close linkage between the two genes.

Target cell-dependent T cell-mediated lysis of vaccinia virus-infected cells.

Vaccinia virus specific cytotoxicity against infected target cells was observed in vitro. Spleen lymphocytes from normal and immunized mice of the inbred strains C3H and DBA/2 were incubated with vaccinia virus-infected and non-infected 51 Cr-labeled mastocytoma P-815-X2 cells and L-929 fibroblasts, which were used as targets. Cytotoxic lymphocytes could be isolated from the mice as early as 2 days after infection with vaccinia virus. The highest cytotoxic effect was obtained with lymphocytes taken 6 days after infection. The degree of lysis was correlated with the ratio of immune lymphocytes to target cells. Specific blocking of target cell lysis resulted after addition of anti-vaccinia antibody from different sources. The effector cells could be characterized as T cells by elimination of macrophages and B cells. Target cell killing was only possible in a syngeneic system; allogeneic infected target cells were not lysed significantly.

Disappearance of specifically mlc-responsive lymphocytes in cba mice injected with cells from the h-2-compatible, m-antigen-incompatible strain c3h.

The mixed lymphocyte culture (MLC) response of lymphocytes from CBA mice injected with spleen cells from the H-2-compatible strain C3H was studied. The response of CBA lymphocytes against C3H cells was higher than that obtained against H-2-disparate strains. Intravenous injection of C3H cells resulted in a markedly increased MLC response in lymph nodes but not in the thymus during the first 1 to 3 days. Thereafter, the specific MLC response decreased drastically, far below that of nonimmunized animals. Reactivity was back to normal in the thymus after approximately 4 weeks but remained suppressed in lymph nodes for more than half a year. A decreased response was also noted in spleen, Peyer's patches, and the peripheral blood lymphocyte population. Such a state of relative unresponsiveness was achieved by injecting as few as 10-4 C3H spleen cells. This exhaustion of the specific MLC response could not be explained by production of blocking serum factors or of cells that can inhibit the MLC response. The strong MLC response obtained by lymphocytes from nonimmunized animals may be due to disparity at the newly detected M locus. This antigenic system is characterized by strong MLC stimulatory capacity and no detectable production of humoral antibodies or development of effector cells capable of killing M-antigen-bearing cells. A possible explanation of the results is that the CBA mice become chimaeric for a long time after injection of C3H cells. This prolonged exposure to a foreign transplantation antigen may lead to exhaustion of the specifically responsive lymphocytes.

Receptor Sites for Antigen-Antibody Complexes on Cells Derived from Solid Tumors: Detection by Means of Antibody Sensitized Sheep Erythrocytes Labeled with Technetium-99m

Surface receptor sites for the Fc portion of antigen-antibody complexes were demonstrated on cells derived from three methylcholanthrene-induced fibrosarcomas, one of strain C3H and two of strain BALB/c origin, two spontaneously occurring malignant melanomas (B16 in strain C57BL/6 and Harding-Passey in strain BALB/c mice), a Moloney sarcoma virus-induced tumor of strain BALB/c origin and the Walker 256 carcinosarcoma of Holtzman rats. Primary cell cultures derived from these tumors adsorbed technetium-99m labeled, antibody-sensitized sheep erythrocytes (99mTc EA) as determined either by visual scoring of adherence or radioisotopic quantitation. Depending upon the tumor tested, from 20% to greater than 95% of the target cells absorbed 99mTc EA. All cells lost their reactivity after 1 or 2 passages in vitro, but this was regained after a single passage in vivo. Indicator erythrocytes coated with F(ab')2 fragments of the sensitizing sheep erythrocytes (SRBC) antiserum did not adhere thereby demonstrating that the hemadsorption required an intact Fc portion of the antibody molecule. Adherence of 99mTc EA was blocked by soluble immune complexes prepared with ovalbumin and rabbit antibody directed against it and Escherichia coli 055:B5 lipopolysaccharide and mouse antibody directed against it. Normal rabbit or mouse serum, immune serum, or antigen alone did not block adherence of 99mTc EA thereby demonstrating that the receptors had greater affinity for immune complexes than for either antigen or antibody alone. The existence of membrane receptors on tumor-derived cells which react with the Fc portion of antigen-antibody complexes may provide an explanation for the mechanism by which immune complexes are capable of blocking cell-mediated tumor cell destruction irrespective of whether the receptors are on the tumor cells themselves or on admixed lymphocytes and macrophages.

Strong Stimulation of CBA Lymphocytes in the Mixed Lymphocyte Interaction with Cells from the H‐2‐Identical Strain C3H

Lymphocyt e! from the H‐2‐identical mouse strains CBA and C3H were tested for mixed lymphocyte culture (MLC) responsiveness in vitro. It was observed that CBA lymphocytes were stimulated by C3H cells to the same extent as or stronger than cells from H‐2‐disparate donors. C3H lymphocytes, however, were stimulated poorly by CBA cells. The stimulatory effect by C3Hcells of CBA lymphocytes was observed to be positively correlated with the age of the C3H donor. A similar but less pronounced difference was found by varying the age of donors for H‐2 disparate cells. Spleen cells from normal and thymectomized, irradiated, and bone marrow‐reconstituted C3H × CBA mice stimulated CBA lymphocytes to approximately the same extent, indicating that the stimulatory antigen is present on both T and B cells or possibly other cell types However, they arc poorly expressed or absent on thymic cells. It is concluded that certain non‐H‐2‐determined transplantation antigens may be strongly stimulatory in MLC tests.

Induction of cleft palate in mice by tranquilizers and barbiturates.

AbstractSeveral barbiturates and tranquilizers, individually or in combination, were injected into pregnant mice of the A/J, CD1 and C3H strains in doses sufficient to induce sedation during the expected time of palate closure. Similar treatments were given during periods of fetal development 1 or more days prior to the time for normal palate closure. Among 2074 fetuses from treated pregnant females, 308 had isolated cleft palates. The most susceptible strains were A/J and CD1; the more teratogenic drugs were phenobarbital and hydroxyzine; the most critical treatment periods were at least 1 day prior to the time the palate would normally have closed. Thus, barbiturates and tranquilizers were shown to be significant cleft palate teratogens in mice although their mechanism of action was not investigated.

Weak graft-versus-host response of CBA lymphocytes against the H-2-identical strain C3H.

Lymphocytes from CBA and C3H mice were tested for graft‐versus‐host (GVH) reactivity in C3H × CBA recipients. Three different GVH assays were used, namely, the spleen enlargement test, the popliteal lymph node assay, and inhibition of allogeneic bone marrow proliferation CBA lymphocytes showed only weak reactivity against C3H X CBA tissues in all these tests, while there was strong reactivity in H‐2‐different F1 hybrids. The capacity of C3H and CBA cells in producing a host‐versus‐graft (HVG) reaction was also tested. It was observed that CBA cells injected into the foot pads of C3H mice induced an enlargement of the local popliteal lymph node that was as great as when C3H cells were injected into CBA mice. On the other hand, cells from the H‐2‐different strain 57B1 induced a considerably smaller HVG reaition when injected into CBA mice. It is concluded that antigens that strongly stimulate mixed lymphocyte cultures do not always stimulate allogeneic lymphocytes to strong GVH reactions in vivo.

Variants of globin chains identified by carboxymethyl-cellulose chromatography among different strains of mice

1. 1. Whole hemolysate globin of C3H strain mice shows a β-chain variant located in the slow component, which is more basic than the β-chains of the fast component. 2. 2. By comparing the β-like chains between C3H and Japanese Dancing mice, the existence of a different elution volume has been proved which indicates a different structure. 3. 3. C3H mice globin consists of a kind of β-chain which is eluted earlier than the β-chains of C57 BL mice.

CASEIN‐INDUCED AMYLOIDOSIS IN THE NUDE MOUSE. I.

Amyloidosis was induced by repeated casein injections in a strain of mice with congenital aplasia of the thymus (nu/nu‐C3H). The development of amyloidosis in this strain was delayed compared with the development of amyloidosis in the strain of control mice C3H. Spleen grafts from casein sensitized non‐amyloidotic “nude” and C3H mice were transferred to mice from both strains. Only grafts from casein‐sensitized C3H mice could accelerate amyloid formation in recipients, whereas grafts from the casein‐sensitized “nude” mice had no such effect. Antibodies to casein could be detected neither in the donor mice nor in the recipients. The observed acceleration could be due to a transfer of casein stimulated T‐lymphocytes. Spleen cells from amyloidotic “nude” and C3H mice were transferred to both strains. Amyloid formation occurred only in recipients belonging to the C3H strain. As amyloid formation in the recipients—in this transfer model—is dependent on heavy cytotoxic treatment, it seems unlikely that amyloid formation in the recipients is due to immune reactions elicited by donor or recipient lymphoid cells. The reason why amyloidosis cannot be transferred by spleen cells to nude mice could be due to a poorer trapping of donor spleen cells in the nude spleens than in the normal C3H spleens.

EXPERIMENTAL MURINE LEPROSY

Female mice of the outbred strain NMRI, and the five inbred strains AKR, BALB/c, C3H, C57/BL, and White Label were infected with Mycobacterium lepraemurium. Infectious lesions in the liver were evaluated histologically with regard to the type of inflammatory reaction and content of bacilli. In the outbred strain the host response of individual mice varied considerably. This variation diminished if a large inoculum was used and the infection allowed to proceed for a long time. Within the inbred strains individual animals showed a more uniform type of reaction. However, clear differences in the host response to murine leprosy could be demonstrated in these strains, most markedly between the strains C3H and C57/BL. In C3H, the infection seemed to make uninhibited progress and the granulomas contained large numbers of bacilli with few or no surrounding lymphocytes. The granulomas of C57/BL mice contained fewer bacilli and regularly showed infiltration by lymphocytes. However, after a large inoculum of bacilli this difference in host response seemed less evident. The present study confirms our previous observation that outbred mice differ in their host response to murine leprosy. Evidence is presented that this variation is due to differences in the ability of individual mice to mount a cell mediated immune response against the mycobacterium. The capability to respond is shown to depend on genetic factors of the host.

Genetics of a new IgVH (T15 idiotype) marker in the mouse regulating natural antibody to phosphorylcholine.

The idiotype present on the Fab of a phosphorylcholine-binding IgA myeloma protein TEPC 15 (T15) of BALB/c origin was found in normal serum of BALB/c mice. Molecules carrying the T15 idiotype in normal serum could be adsorbed with Sepharose phosphorylcholine beads and R36A pneumococci. The T15 idiotype is absent in germ-free BALB/c but appears when the mice are conventionalized. A survey of normal sera of inbred strains for the T15 idiotype showed it to be present in BALB/c, 129, C57L, C58, and ST and absent or in low levels in CBA, C3H, C57BL/6, C57BL/Ka, C57BL/10, SJL, B10.D2, DBA/2, RIII, A, AL, AKR, NZB, and NH inbred strains of mice. The T15 idiotype is associated with some but not all strains carrying the IgC(H) allotypes found in BALB/c. Linkage of genes controlling the T15 idiotype in normal serum to the IgC(H) locus of BALB/c was demonstrated in F(2) progeny of a BALB/c and C57BL cross, Bailey's recombinant inbred strains, C x BD, C x BE, C x BG, C x BH, C x BI, C x BJ, C x BK, and CB20 congenic strains. Among these strains, only those possessing the IgC(H) locus of BALB/c including the F(2) progeny consisting of BALB/c homozygotes and BALB/c/C57BL heterozygotes and C x BG and C x BJ recombinants showed the T15 idiotype.