Genetic control of the antibody response in inbred mice. Transfer of response by spleen cells and linkage to the major histocompatibility (H-2) locus.

The transfer of spleen cells from (C3H x C57Bl/6) F(1) mice, capable of responding to (T,G)-A--L, into irradiated C3H parental recipients, normally incapable of responding to (T,G)-A--L, transfers the ability to make either a primary or secondary immune response to this synthetic polypeptide antigen. This localizes the genetic control of the ability to respond to the spleen cell population and indicates that the genetic control is exerted upon a process directly related to antibody formation. Studies with congenic strains of mice and linkage studies in segregating backcross populations show that the ability to respond to (T,G)-A--L and (H,G)-A--L is linked to the H-2 locus and can thus be localized to the IXth mouse linkage group. Note Added in Proof: Of the three possible recombinant animals noted in Tables IV and V, two were infertile. The third animal was not a recombinant, since progeny testing and reimmunization showed that this animal was an H-2(2)/H-2(k) heterozygote capable of responding well to (T,G)-A--L.

Glucocorticoid-induced cleft palate in the mouse: two major histocompatibility complex, H-2, loci with different mechanisms.

Isolated cleft palate is induced in the progeny of pregnant mice that are given glucocorticoids. The incidence varies among inbred strains and with dose and stage of gestation when the drug is given. One chromosomal region responsible for strain-associated differences in sensitivity is the major histocompatibility complex, H-2. H-2a is associated with susceptibility, H-2b with resistance. There appear to be both maternal and embryonic genetic factors affecting the sensitivity to glucocorticoids. In experiments reported here congenic strains of mice with H-2a, H-2d and H-2k haplotypes on a C57BL/10 genomic background were used. This allowed the determination of the effect on sensitivity by two H-2 subregions; the subregions are H-2K to I-E and I-C to H-2D. Methods included dose-response analysis and reciprocal cross analysis using dexamethasone given on day 12 of pregnancy. Results show that each subregion affects the strain's sensitivity to dexamethasone-induced cleft palate. The regression coefficients for B10.A-H-2a (45.4 +/- 4.13) were different from those for B10.BR-H-2k (67.2 +/- 10.8) and B10.D2-H-2d (70.5 +/- 9.74). The estimated mean arcsine % cleft palate at 160 mg/kg was different for each strain: B10.A-H-2a, 53.1 +/- 2.19; B10.BR-H-2k, 33.1 +/- 2.27; B10.D2-H-2d, 25.0 +/- 2.75. Different patterns of change in sensitivity were observed among the reciprocal crosses. In summary, the H-2K to I-E subregion seemed to influence both maternal and embryonic factors, whereas only embryonic factors were influenced by the I-C to H-2D subregion. These data suggest that the mechanisms affecting glucocorticoid sensitivity which are genetically encoded within each H-2 subregion are different, and there is an interaction between the alleles. The mode of interaction can be either complementation or epistasis.

Effect on bone ash weights of long-term treatment of mice with oral promethazine HCl.

Ten and 18-month-old female B6D2F1 mice were given promethazine HCl in their drinking water (2.0-4.0 mg/dl and 1.0 mg/dl, respectively) and age, sex and weight matched controls were given acidified tap water. The surviving mice were killed when they were 30.5 months old and femur, ilium and sacrum ash weights were determined. It was found that promethazine HCl effectively prevented age-related mineral loss at the higher does level. There was no evidence of excess morbidity or mortality among the mice given promethazine.

Effect of promethazine on double tetracycline bone labeling in old mice.

Twenty-five-month-old female B6AF1 mice were injected intramuscularly (i.m.) with declomycin (75 mg/kg) 50 days and 2 days, or 20 days and 2 days, before sacrifice, and cross-sections of their femoral shafts were examined quantitatively for areas of tetracycline fluorescence. Two groups of mice received promethazine HCl in their drinking water (12 mg/dl) for 1 year, and the control groups were untreated. It was found that: the number of discrete areas of cortical and endosteal tetracycline deposition was increased slightly in the groups given promethazine; the length of the endosteal and cortical tetracycline deposits were 2-3 times greater, respectively, in the promethazine treated groups; and the distance between the cortical tetracycline deposits and the endosteum was 2.5 times greater in the promethazine groups. These results support the view that net bone deposition in osteopenic old mice is enhanced by promethazine.

Age-related increase in erythrocyte oxidant sensitivity.

Unlike erythrocytes from elderly humans, red blood cells from old mice are not more sensitive than are cells from young animals to lysis in hypotonic solutions, probably because the mean corpuscular volume decreases rather than increases with age in this species. However, when subjected to an oxidant stress (sodium ascorbate) red blood cells from old animals accumulate more methemoglobin and fewer remain intact than is the case with red blood cells from young mice. The data suggest that this increased vulnerability to oxidative damage is manifest relatively early in the lifespan of red blood cells from old animals and is not solely a property of the older cells. The pathogenesis of the decreased resistance to peroxidation is not known, but it does not appear to be the result of changes in reduced glutathione, NADH: methemoglobin reductase, superoxide dismutase, glutathione reductase, glutamic-oxaloacetic transaminase, or glucose 6-phosphodehydrogenase.

Age-related changes in in vitro and in vivo survival of murine CFU-S and CFU-c.

Present evidence suggests that the proliferative potential of most cell types declines with age. An apparent exception to this is the erythroid and myeloid colony-forming cell (CFU-S) of the marrow. In an effort to reveal a latent proliferative defect in old marrow, CFU-S from the marrow of young and old B6AF1 mice were serially transplanted every 12 days into lethally irradiated young syngeneic hosts. Contrary to expectation, it was found that the number of CFU-S declined more slowly if the marrow had been obtained from old donors. Similarly, it was found that CFU-S, but not CFU-c, from old marrow replicated or were recruited more vigorously in long-term culture than CFU-S from young donors. The results obtained with anti-Thy 1.2 serum and complement treatment of old marrow cells suggest that CFU-S from old marrow replicate more vigorously because old marrow has more theta + helper cells or the old CFU-S is more responsive to theta + helper stimuli.

Effect of age on the intrinsic regulation of murine hemopoiesis.

As a part of studies conducted to determine the cause(s) of the decline in hemoglobin and peripheral blood lymphocytes observed in old mice, young mice were lethally irradiated and protected with bone marrow cells from young and old donors. Four months later the recipients of marrow from old mice had depressed hemoglobin and absolute lymphocyte levels in 4 of 6 and 5 of 6 experiments, respectively. Six months after lethally irradiated 20-month-old hosts were given marrow from young or 20-month-old donors, the recipients of old marrow had decreased lymphocyte counts but their hemoglobin levels were not different from those observed in mice given marrow from young donors. These results suggest that peripheral blood lymphocytes counts and to a lesser degree hemoglobin levels are at least partially controlled by mechanisms intrinsic to the marrow itself.

Development of immune competence.

Present evidence indicates that the precursors of B- and T-cells can be found in the extra-embryonic tissues four days after implantation (day 5) and that by the following day (day 10) certain of the B-cell precursors have differentiated to the stage of the antigen-binding cell. Cells able to secrete antibody are not detected, however, until the later stages of pregnancy. B-cell differentiation has been shown to advance in a stepwise manner through several compartments, and the early stages of maturation are independent of thymic or T-cell regulation. The thymic rudiment appears by the 12th day of pregnancy and the reticuloepithelial tissue is quickly colonized by T-stem cells which migrate from the fetal liver. Within 4 days these cells respond to PHA, recognize and respond to allogeneic antigens, and begin to seed to the peripheral lymphoid tissues. During the latter stages of pregnancy and until about the sixth week after birth T-cell mediated suppressor activity predominates. T-cell killer function can be detected in the neonatal thymus shortly after birth, but this activity increases slowly in the peripheral lymphoid tissues. T-cell helper activity increases slowly after birth as suppressor activity declines. The adult levels of helper-suppressor function are approached about six to eight weeks after birth.

Age-related osteopenia in the mouse: effects of an H1 blocker, a phenothiazine, and promethazine.

Mature and old B6AF1 and B6D2F1 mice were given acidified tap water or promethazine HCl (a phenothiazine with H1 receptor blocking activity), chlorpheniramine (an H1 blocker) or trifluoperazine (a phenothiazine with no H1 blocking activity) in their drinking water, and the effects of these agents on bone mineral content were assessed by intermittently measuring the 24-h whole body retention of Tc 99m methylene diphosphonate (Tc 99m MDP, an indicator of bone metabolism) and at the end of the studies by determining ash weights of femur, ilium and sacrum. It was found that 24-h retention of Tc 99m MDP was elevated in old mice as it is in old osteopenic humans, that promethazine but not chlorpheniramine or trifluoperazine inhibited bone loss in aging mice, and that there was a correlation between decrease in retention of Tc 99m MDP and decreased bone loss. These preliminary results suggest that the ability of promethazine to inhibit age-related bone loss may not be mediated through its action as an H1 blocker or as a phenothiazine. However, more agents of each type need to be tested before this point can be established.

Effects of H1 blocking agents on age-related osteopenia in the mouse.

Old female B6AF1 mice were given acidified tap water, distilled water, one of five H1 blockers or chlorpheniramine (an H1 blocker) and trifluoperazine (a phenothiazine with no H1 blocking activity) in their drinking water for 5 months, and the effects of these agents on bone mineral metabolism were assessed by determining ash weights of femur, ilium and sacrum at the end of the study. In one experiment 24 h whole-body retention (WBR) of Tc 99m methylene diphosphonate (Tc 99m MDP, an indicator of bone metabolism) was measured at the beginning of the study and 40 days later. It was found that: promethazine and dimenhydrinate were the most effective of the H1 blockers in preventing age-related loss of bone mass; distilled water, chlorpheniramine, and chlorpheniramine plus trifluoperazine had no effect on the loss of bone mass; mean bone mass in the groups given meclizine and pyrilamine were greater than but not significantly different from that in the control group given acidified tap water; and only promethazine induced a significant reduction in the WBR of Tc 99m (the other H1 blockers induced small but not significant reductions).

Allogeneic radiation chimeras. Long-term studies.

Lethally irradiated mice protected with allogeneic fetal liver cells or with syngeneic or allogeneic marrow and spleen cells treated with antisera to mouse immunoglobulins or to the T cell-associated theta antigen and their controls were observed for up to 750 days. The best survival rates were found in the large groups given syngeneic marrow and spleen or allogeneic fetal liver cells (70-85% 700-day survival); in contrast, 43% of the group injected with allogeneic cells treated with anti-theta serum and 19% of those given antiimmunoglobulin-treated cells were alive 700 days postradiation. Pulmonary infection was the most frequent cause of death of long-term survivors in all groups. Tumor incidence was increased in recipients of allogeneic cells (13% versus 4% among syngeneic chimeras), but the renal pathology seen in these groups was no greater than that noted in the syngeneic controls. Beginning 600 days after irradiation, mice from experimental and control groups were killed and their spleens were cultured with thymus-dependent antigens and the mitogens concanavalin A and lipopolysaccharide, Escherichia coli. The most frequent finding in all groups was mild to moderate impairment of T cell-dependent responses.

Evidence for clustering of H-2K, H-2D, and the Fc receptor on the membranes of B cells.

Alloantisera to H-2K, H-2D, and Ia antigens markedly inhibited the binding of EA but not FITC-IgG by the B cell Fc receptor. EA rosette formation approached normal levels when masked H-2 but not Ia specificities were allowed to cap on the membranes of B cells. beta2-mu coated SRBC were bound by the Fc receptor, and high concentrations of soluble beta2-mu were found to moderately inhibit EA rosette formation while lower concentrations enhanced binding. The data support the concept of Fc/Ia identity, and they suggest that H-2K, H-2D, and the Fc receptor may be closely grouped on the membranes of B cells. Further, these observations suggest that the beta2-microglobulin associated with H-2 could serve to link T cells with the Fc receptor of B cells during the inductive phase of antibody synthesis.

Age-related osteopenia: evidence for an intrinsic defect of bone resorbing cells and a possible treatment.

B6D2F1 and B6AF1 mice of various ages were given sublethal or lethal doses of X radiation and injected with marrow and/or spleen cells from young, mature, or old syngeneic donors. Four to five months later they were killed and ash weights were determined on femurs, sacrum, and ilium. It was found that large numbers of marrow cells (i.e., greater than 25 X 10(6] and/or spleen cells (greater than 50 X 10(6] from old mice retarded the growth of bone in young hosts and induce loss of bone mass in mature recipients, spleen cells from young donors consistently prevented the loss of bone mass normally seen in aging mice, and the thymus and T cells did not appear to play a significant role in bone resorption and remodeling. These observations suggested that in aging mice loss of bone mass is caused by an intrinsic defect in a hematopoietic cell population, perhaps the macrophage/osteoclast or their common precursor, which results directly or indirectly in increased bone resorption. On this basis, promethazine HCl, an inhibitor of macrophage metabolism and phagocytosis, was added to the drinking water (1.0 to 4.0 mg/dl) of aging mice. Four to five months later it was found that bone mass was significantly greater in the groups given promethazine than in the age and weight matched controls.

Effect of promethazine on lumbar vertebral bone mass in postmenopausal women.

OBJECTIVES: Work in mice suggests that the age-related loss of bone mineral noted in that species is caused by an intrinsic defect in a haematopoietic cell population which results directly or indirectly in increased bone resorption. This age-related loss of bone mineral is prevented or reversed by well-tolerated doses of promethazine HCL. The present study was undertaken to determine if promethazine would retard or reverse bone loss in postmenopausal women. DESIGN: Postmenopausal women whose spine (L2 to L4) bone mineral content (BMC) was two standard deviations below young normal values were assigned randomly to receive calcium or promethazine and calcium daily. Subjects who had been taking oral oestrogen for more than 4 years also were assigned randomly but independently to the calcium or promethazine groups. SETTING: All subjects were seen in the out-patient clinic of the Department of Medicine, School of Medicine, University of California, Los Angeles. SUBJECTS: Healthy, ambulatory postmenopausal females were recruited by word of mouth and by advertisement from the local community. Fifty-four subjects completed the first 6 months of the study and 43 completed 30 months. INTERVENTIONS: The subjects were assigned randomly to receive 1000 mg calcium daily or promethazine 50 mg and calcium 1000 mg daily throughout the period of the study. MAIN OUTCOME MEASURES: Bone mineral content of the lumbar vertebrae (L2 to L4) was determined by dual photon densitometry every 6 months. Dorsolumbar spine X-rays were obtained yearly and at the completion of the study to detect new compression fractures. RESULTS: In the groups not taking oestrogen, BMC decreased at the rate of 1.53% year-1 in the group given only calcium; in contrast, BMC increased at 3.22% year-1 in the group given promethazine and calcium (P < 0.001). Among the women taking oestrogen, increases in mean BMC were noted in both groups, but those taking promethazine and calcium had a greater rate of increase than observed in the group taking only calcium (5.62% vs. 1.97% per year-1, P < 0.001). CONCLUSIONS: These results suggest that promethazine can induce a modest increase in vertebral BMC in postmenopausal women who are not taking oestrogen and greater increases in those who are.