DBA/2 Research Articles

Genetic analysis of serum thyroxine content and audiogenic seizures in recombinant inbred and congenic strains of mice

We previously proposed that the audiogenic seizure (AGS) susceptibility of 21 ± 1-day-old DBA/2 (D2) mice may result from an early postnatal elevation in serum thyroxine (T4) concentration. In the present study we used seven C57 × DBA (BXD) recombinant inbred strains and the D2.B6-Iasb congenic strain to study the association between serum T4 content and susceptibility to AGS. The D2.B6-Iasb congenic mice are genetically similar to the D2 mice except for the Iasb gene, which inhibits AGS susceptibility. The total and estimated free serum T4 concentrations in these strains at 14 ± 1 days of age were compared with the previously determined AGS susceptibilities of these strains at 21 ± 1 days of age. We found no significant correlations between serum T4 concentration and AGS susceptibility in these strains. It is unlikely, therefore, that inherited differences in neonatal serum T4 content are directly responsible for differences in susceptibility to AGS in 21 ± 1-day-old mice. The mechanisms by which the experimental manipulation of serum T4 content influences AGS susceptibility are discussed.

Alloimmunization-activated suppressor cells. II. In vitro activity of suppressor cells implicated in the abrogation of lethal graft-versus-host reaction.

Incompatibility for DBA/2 (D2) minor histocompatibility antigens (MiHA) alone leads to a severe lethal graft-versus-host reaction (GVHR) in irradiated (D2 X B10.D2)F1 mice receiving a bone-marrow-plus-spleen-cell graft from B10.D2 donors. This mortality is abrogated when the donors are preimmunized simultaneously against specific D2 MiHA and unrelated H-2 antigens three days before grafting. Results presented here demonstrate that spleen cells from such preimmunized donors, which are able to diminish the intensity of the GVHR developed by normal cells, exhibit the following properties: (1) reduced proliferative response to D2 MiHA in one-way MLC; (2) radioresistant suppressive effect on the proliferative responses of both normal and specifically primed B10.D2 cells; (3) inability to lyse D2 target cells even after in vitro boosting, implying that the suppressive effect detected in vitro is not due to removal of the stimulating antigens from the culture; and (4) inability to suppress the development of an anti-MiHA cytolytic response by specifically primed B10.D2 cells, despite a pronounced suppressive effect on their proliferation. Taken together with the GVHR observations, these results suggest that simultaneous immunization against specific MiHA and unrelated H-2 antigens activates, in the spleens of treated donors, suppressor cells that can inhibit the immune response at the recognition phase, and probably also at an afferent, but not efferent, stage of cytotoxic effector-cell differentiation.

Genetic heterogeneity for the development of audiogenic seizures in mice

Susceptibility to audiogenic seizures (AS) in mice is age dependent. In the prototype DBA/2 (D2) strain, AS susceptibility is greatest around 21 days of age (juvenile-onset), but then gradually subsides. By the time D2 mice reach adulthood (60–80 days) they become completely resistant to AS. Mice of the C5BL/6 (B6) strain are resistant to AS at all ages. Even though the D2 and B6 strains are AS-resistant at adult ages, several B6 × D2 (BXD) recombinant inbred (RI) strains are highly AS-susceptible as adults. Previous studies showed that AS susceptibility in 21-day-old mice was strongly influenced by a major gene, Ias, closely linked to the Ah locus. It appears, however, that Ias does not influence AS susceptibility in adult mice since no association was found between the Ah locus and AS susceptibility in adult BXD RI strains. Furthermore, the developmental profile of AS susceptibility in one RI strain, BXD-15, was biphasic. The incidence of clonic-tonic seizures in these mice at 21, 42 and 60–80 days of age was 82.2%, 14.8%, and 66.6%, respectively. The inheritance of AS susceptibility in the F 1 hybrids from crosses between the B6, D2, and BXD-15 strains was also different at young and adult ages. The incidence of clonic-tonic seizures in the B6BXD-15F 1 hybrids went from 0% at 21 days of age to 24% at 60–80 days; whereas the incidence of clonic-tonic seizures in the D2BXD-15F 1 hybrids went from 100% at 21 days to 0% at 60–80 days. Subtle differences were also observed between young and adult mice for the latency period (in seconds) to seizure onset, for the type of seizures, and for the incidence of seizures in males and females. Taken together, these findings suggest that the genetic mechanism responsible for juvenile-onset AS susceptibility is different from that responsible for adult-onset AS susceptibility in BXD-15 mice. The implications of these findings to the study of certain human idiopathic epilepsies are mentioned.

ALLOIMMUNIZATION-ACTIVATED SUPPRESSOR CELLS I

Incompatibility for DBA/2 (D2) minor histocompatibility antigens (MiHA) alone leads to a severe lethal graft-versus-host reaction (GVHR) in adult (D2 X B10.D2)F1 recipients of B10.D2 bone marrow and spleen cells. In this genetic combination, mortality is completely abrogated by preimmunization of the graft donor against unrelated H-2b antigens and specific D2 MiHA a short time before grafting. Protection against GVHR mortality is elicited by immunizing the donors with a single transfusion of incompatible spleen cells or whole blood. Abrogation of GVHR mortality is due mainly to the immunization with specific MiHA, and only to a much lesser degree to the immunization with unrelated H-2 antigens; the protective effect induced by association of these two types of immunization, however, is significantly better than that elicited by either type of immunization alone. It is unlikely that this abrogation of GVHR mortality results from "dilution" of specifically reactive cells; rather, suppressor cells appear to be a contributing factor, because the preimmunization activates, in the donor spleen, suppressor cells capable of decreasing the severity of the GVHR developed against MiHA by normal cells. Histopathologic observations indicated that the lesions induced in various tissues after grafting of preimmunized donor cells were considerably less severe than those induced by grafting of normal donor cells. However, simultaneous immunization with specific MiHA and unrelated H-2 antigens may also exacerbate the GVHR, depending upon the conditions used for preimmunization; abrogation of GVHR mortality is favored by a single immunization with unrelated H-2b antigens and specific MiHA administered simultaneously a short time before grafting, whereas an acceleration of GVHR mortality is favored by long intervals and multiple immunizations. It is suggested that, depending upon the conditions used for the preimmunization, the allogeneic effect produced by stimulation with unrelated H-2 antigens may augment the response to MiHA or it may enhance the activation of suppressor cells that contribute to the abrogation of GVHR mortality.

Induction of liver microsomal cytochrome P-450 And associated monooxygenases by octachlorostyrene in inbred strains of mice: Lack of correlation with the murinE Ah locus

Single intraperitoneal injections of octachlorostyrene (OCS) and hexachlorobenzene in genetically polycyclic aromatic hydrocarbon ‘responsive’ C57/BL/6 (B6) mice led to a time- and dose-dependent increase in the levels of liver microsomal cytochromes P-450 and b 5 as well as in the activities of NADPH cytochrome P-450 (cytochrome c) reductase, ethylmorphine (EM) N-demethylase, 4-nitroanisole (PNA) O-demethylase and acetanilide 4-hydroxylase (AcA hydroxylase). No, or only a very moderate, increase in the activity of aryl hydrocarbon hydroxylase was seen after OCS and HCB, respectively. Pretreatments with phenobarbital (PB) or 3-methylcholanthrene (MC) both increased AcA hydroxylase activity to a similar degree, whereas pretreatment with polychlorinated biphenyls (Aroclor 1254) had an effect equal to the sum of PB and MC. Judged from sodium dodecylsulfate polyacrylamide gel electrophoresis studies, OCS and HCB predominantly increased a microsomal polypeptide of apparent mol. wt 52,000, similar to PB. A reduced response was seen after OCS or HCB treatment of aromatic hydrocarbon ‘non-responsive’ DBA/2 (D2) mice compared to B6 mice, both with respect to AcA hydroxylase as well as EM demethylase and PNA demethylase activities. OCS treatment of B6D2F1 mice resulted in a doubling of AcA hydroxylase activity, but in mice of the (B6D2)D2 backcross no distinct subgroupings of individual AcA hydroxylase activities were apparent. These results demonstrate that OCS is an inducer of the PB-type in mice and that induction of AcA hydroxylase by OCS is not regulated by the Ah locus.

Genetic linkage between the AH locus and a major gene that inhibits susceptibility to audiogenic seizures in mice.

Mice of the DBA/2 (D2) strain are highly susceptible to sound-induced seizures at 21 days of age; whereas, mice of the C57BL/6 (B6) strain are resistant to these seizures. Although the difference in susceptibility to audiogenic seizures (ASs) between these two strains is inherited as a multiple-factor trait, an association was observed between susceptibility to ASs and the Ah locus. The Ah locus controls the inducibility of aryl hydrocarbon hydroxylase (AHH) activity by a number of aromatic hydrocarbons. B6 mice carry the Ahb allele and have inducible AHH activity; whereas, D2 mice carry the Ahd allele and have noninducible activity. Inducibility is inherited as a Mendelian dominant trait in crosses between these strains. Mice carrying the Ahb allele are generally less susceptible to ASs sat 21 days of age than are mice carrying the Ahd allele. The combined results from B6 X D2 recombinant inbred strains, congenic strains (where the Ahb allele was placed into the D2 genome and the Ahd allele placed into the B6 genome), the B6D2F1 X D2 backcross generation, and a random survey of various inbred strains, suggest that the association between these two traits is due to genetic linkage, rather than to pleiotrophy or to chance. A major gene that inhibits susceptibility to ASs appears to be closely linked to the Ah locus. This gene has been designated Ias, for inhibition of ASs. A large portion of the genetic variability of AS susceptibility may be due to the segregation of Ias.

Effects of phenobarbital, 3-methylcholanthrene and β-naphthoflavone pretreatment on mouse liver microsomal enzymes and on metabolite patterns of benzo[a]pyrene

C57B1/6 (B6), C3H, and DBA/2 (D2) strains of mice were pretreated with either a single dose of 3-methylcholanthrene (3-MC), β-naphthoflavone (β-NF) or α-naphthoflavone (α-NF), or with daily injections of phenobarbital (PB) for 4 days; control mice were injected with corn oil or saline only. Comparisons were made of the patterns of hepatic microsomal-protein bands on acrylamide gel electropherograms as well as the patterns of individual metabolites of benzo[ a]pyrene (BP) extracted by ethyl acetate from incubation systems using these microsomes. Hepatic microsomes from control mice of all three strains produced similar protein bands and BP metabolite profiles, with B6 mice having slightly higher yields of the 9,10-diol and phenols of BP. α-NF had no apparent effect on the protein bands or the yields of BP metabolites. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of hepatic microsomes revealed that PB pretreatment was followed by increases in the intensities of five protein bands with molecular weights between 43,000 and 68,000. The yields of BP metabolites were increased 1.5- to 3-fold for all three strains of mice after PB pretreatment. Pretreatment with 3-MC or β-NF had no apparent effect on the hepatic microsomal enzymes of D2 mice, either in terms of protein bands or metabolites of BP. For B6 and C3H mice, pretreatment with 3-MC or β-NF resulted in the appearance of a new protein band easily detectable in the range between molecular weights 53,000 and 58,000, as well as in increases of all BP metabolites. Pretreatment of B6 and C3H mice with 3-MC caused 10-fold increases in the microsomal yields of the 7,8-diol, and 5-fold increases in the yields of BP phenols. Pretreatment with β-NF caused a 7-fold increase of the 7,8-diol catalyzed by the hepatic microsomes of B6 mice and a 4-fold increase in the 7,8-diol in C3H mice; the yields of phenols were increased about 2.5-fold for both B6 and C3H mice. The results suggest that the induced microsomal proteins with molecular weights between 43,000 and 58,000 may contain induced forms of cytochromes(s) P-450 that activate different carbon atoms of the BP molecule and, therefore, lead to different metabolite profiles.

Expression of xenotropic murine leukemia viruses as cell-surface gp70 in genetic crosses between strains DBA/2 and C57BL/6.

Flow microfluorometry was used to assess levels of xenotropic murine leukemia virus envelope-related cell-surface antigens (XenCSA) expressed on lymphocytes of mice derived from crosses between C57BL/6 (B6) and DBA/2 (D2); 24 recombinant inbred strains (BXD RIs) and 62 backcross mice were studied. The results suggest that XenCSA expression is affected by more than one gene but that the predominant influence is exerted by a single semidominant gene apparently located on chromosome 4 at or in close proximity to the Fv-1 locus. Studies of spontaneous virus production in B6D2F1 X D2 mice suggest that this locus may also affect production by spleen cells of xenotropic MuLV registering in a fluorescent antibody assay of mink lung cells.

In vitro production of immune interferon by spleen cells of mice immunized with Herpes Simplex Virus

In Vitro production of Immune Interferon (IF) in response to Herpes Simplex Virus (HSV) antigen by sensitized spleen cells from C57B1/6 (B6) mice could be detected as early as 3 and for at least 20 days after ip infection of HSV. Maximal levels of IF were produced after 10 hr of culture, but there was no decay of activity when supernatants were sampled during the subsequent 3 days. The IF produced shared certain known properties of immune IF and was not neutralized by an antiserum against viral-induced (type I) IF. DBA/2 (D2) mice which are considerably more sensitive in vivo to HSV infection than B6 mice produced significantly lower amounts of immune IF in the in vitro test system regardless whether high or low doses of virus were injected. The same pattern of results was observed when resistant B6D2F1 hybrid mice were compared with A J and Balb/c mice which are about as sensible to ip infection with HSV as DBA/2 mice in our laboratory. These results demonstrate a remarkable defect of in vitro cellular immunity in mice susceptible to a virus infection when compared with resistant mice. Conceivably, a similar defect may be of in vivo relevance.

The Fv-2r resistance gene in mice: its effect on spleen colony formation by Friend virus-transformed cells.

To determine the mechanism by which the recessive Fv-2r gene confers complete resistance to spleen focus formation and to the induction of early splenomegaly characteristic of the Friend virus (FV) disease syndrome, we compared the parameters of FV infection in susceptible DBA/2 (D2) (Fv-1n, Fv-2s) and partially congenic D2.Fv-2r (Fv-1n, Fv-2r) mice. After infecting these mice with N-tropic FV complex, we followed the replication of spleen focus-forming virus (SFFV) and the generation of SFFV-transformed tumor colony-forming cells (CFC) in their spleens. By both parameters D2.Fv-2r mice were 20- to 100-fold less susceptible than DBA/2 controls, but the inhibition was only partial. Transplantation of washed spleen cells from SFFV-infected DBA/2 mice resulted in equal growth and recovery of tumor CFC from both D2.Fv-2r and Fv-2s mice. However, these tumor cells grew as colonies in Fv-2s mice, whereas they grew diffusely in the spleens of D2.Fv-2r hosts and did not develop into macroscopically or microscopically visible colonies. Thus the completeness of the resistance to spleen focus formation that defines the Fv-2r gene was reflected only in the complete suppression of the colonial growth of tumor cells, whereas the other parameters of infection showed no or only partial inhibition.