Effects of inositol, LiCl, and heparin on the antibody responses to SRBC by normal and immunodeficient XID mice.

Spleen cells from naïve adult immunocompetent and immunodeficient XID mice were cultured on agar containing sheep red blood cells (SRBC) with and without myo-inositol, scyllo-inositol, lithium chloride, or heparin, and after 1 or 2 days the number of colonies of antiSRBC antibody-forming cells (PFC) were determined. It was found that myo-inositol and scyllo-inositol at one-tenth the concentration were equally effective in increasing the number of specific PFC. Myo-inositol, scyllo-inositol, and lithium chloride accelerated the appearance of direct foci in cultures of spleen cells from normal and XID mice. When heparin was added to cultures of XID spleen cells, PFC were found to be increased on Day 1; however, PFC and foci were not increased in cultures of spleen cells from competent mice until 1 day later. The addition of combinations of these agents to cultures of spleen cells had no positive or negative effect on the generation of foci or PFC. Normal mice given heparin intraperitoneally with SRBC had increased splenic PFC on Days 3 and 4 but not on Day 7. The results suggest that these agents modulate B-cell responses by increasing the rate of proliferation and/or secretion through a signaling pathway(s) distal to, or more likely, independent of Bruton's tyrosine Kinase (BTK). It is not clear that the mechanism is the same with each agent.

Modulation of the antibody response to sheep red blood cells in normal and immunodeficient XID mice by myo-inositol.

Over the dose ranges tested, dietary vitamin A and myo-inositol induce similar changes in murine fetal growth and development. Because vitamin A is also known to affect the immune response, studies were conducted to determine if dietary myo-inositol might have an effect on antibody production. It was found that in vivo in inbred mice myo-inositol (4 mg/g of diet) accelerated the rate of appearance of plaqueforming cells (PFC) in a primary response to sheep red blood cells (SRBC). In vitro, myo-inositol accelerated the rate of appearance of colonies of anti-SRBC PFC (foci) and significantly increased the number of PFC per colony, but did not affect the number of foci per culture noted at the end of the culture period, myo-inositol had no effect on the PFC IgM:IgG ratio following a single exposure to the agent, but exposure to myo-inositol in vivo and in vitro resulted in a decrease in the number of IgM PFC per focus in a primary response and IgM and IgG PFC per focus in a secondary response. Based on studies suggesting that myo-inositol or a phosphorylated metabolite might act downstream from Bruton's tyrosine kinase (Btk) in a signal transduction pathway in B cells, immunodeficient CBA/CaHN-XID/J mice were fed a standard diet or the same diet supplemented with 0.4% myo-inositol. Mice given the supplemented diet produced significantly more IgM anti-SRBC antibody than did XID mice given the control diet (4.3 +/- 2.5 vs 1.7 +/- 2.8, 1/log2), and produced approximately the same amount as immunocompetent controls (2.9 +/- 0.9). When rechallenged with SRBC, XID mice given supplemental inositol produced significantly more IgM antibody than did the XID and immunocompetent controls (3.6 +/- 0.5 vs 1.8 +/- 1.1 and 1.5 +/- 0.7, respectively). Added dietary inositol did not have a significant effect on primary or secondary IgG responses to SRBC, which remained impaired. These results suggest that dietary myo-inositol or a derivative may be able to modulate B-cell IgM responses by interacting within the inositol second messenger system downstream from Bruton's tyrosine kinase.

Effects of vitamin A and inositol on term weights of H-2 congenic mice.

Pregnant H-2 congenic mice, C57BL/10, B10.A, B10.A(15R), and B10.A(18R) were fed Purina Laboratory Chow 5001 or the same diet supplemented with 400 IU vitamin A dissolved in corn oil or 0.4% (w/w) myo-inositol. On the 18th day of gestation, the dams were sacrificed, and the fetuses were weighted, sexed, and examined for developmental abnormalities. Term fetal weight was found to be significantly reduced in progeny of dams bearing d alleles distal to Ea in the H-2 complex when the diet was supplemented with vitamin A or myo-inositol (B10.A and B10.A [18R]). Fetuses from dams of all strains fed the diet supplemented with vitamin A had significantly increased frequencies of microphthalmia; the frequency of microphthalmia was moderately but not significantly increased in one of the two strains fed the diet supplemented with myo-inositol (B10.A[15R] but not B10.A[18R]).

Fetal weight at term influenced by H-2-associated loci.

Pregnant mice which in theory differ only in the region of the major histocompatibility complex (MHC) on chromosome 17 (C57BL/10, the inbred partner [host strain], and B10.D2, B10.BR, B10.A, B10.A[2R], B10.A[5R], B10.A[15R] and B10.A[18R]) were sacrificed on the 11th and 18th days of gestation, and the fetuses were sexed and weighed. Fetuses from reciprocal crosses between B10.A and B10.BR, B10.D2 and C57BL/10 were weighed and sexed on the 18th day of gestation. It was found that (i) fetal weights were not significantly different among the strains examined on day 11 (B10.BR, B10.A[15R] and B10.A[18R]), (ii) B10.BR fetuses of both sexes weighed significantly less than fetuses from the other strains on day 18, (iii) B10.D2 18-day-old male but not female fetuses were heavier than the males from the other strains (this difference was not present when corrections for litter size were made), (iv) the fetuses from the B10.A x B10.BR cross were the smallest, those from the B10.D2 x B10.A cross the largest, and those from the B10.A x C57BL/10 crosses intermediate, and (v) maternal effects were noted in the B10.A x B10.BR and B10.A x B10.D2 but not the B10.A x C57BL/10 crosses. The results suggest that there are two or more MHC associated loci that influence growth rate in late gestation. Among the candidate genes are Ped and Igfr II.

Murine fetal development enhanced by dietary vitamin A, corn oil, and inositol.

Previous work on the effects of dietary vitamin A on craniofacial anomalies in mice revealed that 18-day-old fetuses from dams given 200 IU of vitamin A in corn oil daily in their diet weighed approximately 10% more than fetuses from mothers fed the unsupplemented standard mouse diet, Purina 5001. In the experiments reported here, it has been found that water-soluble vitamin A (200 IU/day) and myo-inositol (5 mg/day) added separately to the diets of pregnant mice increased the weight of 11-day gestations by approximately 25% and enhanced development of the eyes by the equivalent of 0.5-1.0 day without significantly affecting development of the liver or hind limbs. Corn oil alone (0.2 ml/day) had a similar effect on weight and eye development of 11-day fetuses and, in addition, growth of the hind limbs was enhanced modestly. The addition to the diet of vitamin A (200 IU/day) dissolved in corn oil (0.2 ml/day) resulted in a 25% increase in the weight of the 11-day fetuses and enhanced development of the eyes and hind limbs by the equivalent of about 1 day of gestation, suggesting that corn oil contains a factor(s) that interacts with vitamin A to accelerate limb development. Corn oil contains very small quantities of beta-carotene or retinol (< 1.0 IU vitamin A/ml); however, it is a rich source of the essential growth factors linoleic and linolenic acids and of inositol esters. The data suggest that the growth-promoting actions of dietary corn oil are due in part to the inositol esters.

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.

Effects of H-2 and dietary vitamin A on the frequency of dorsoventral vaginal septum.

The frequency of dorsoventral vaginal septum (DVS) in mice is determined in part by genes associated with the major histocompatibility complex H-2. Data presented here confirm that one locus (DVS-1) maps centromeric to E alpha and that the second (DVS-2), previously shown to be associated with the S-to-D region, maps to the C4:B144 interval, most likely between Dcp-2 which contributes to glucocorticoid-induced cleft palate susceptibility and Acp which enhances cleft palate susceptibility through the action of vitamin A. Comparisons of data obtained in this laboratory in the periods 1981-1983 and 1985-1990 and observations from the production colony from which many of the strains were purchased revealed minor variations in frequencies of DVS within strains which may be due to differences in ascertainment and/or to environmental factors. The addition of vitamin A to the diet of pregnant mice at a dose that increases the frequency of cleft palate in susceptible strains had no effect on the incidence of DVS.

Vitamin A-enhanced cleft palate susceptibility gene maps between C4 and B144 within the H-2 complex.

Pregnant mice congenic with C57BL/10 in the region of H-2, the major histocompatibility complex on Chromosome 17 (B10.BR, B10.A, B10.A(1R), B10.A(2R), B10.A(15R), B10.A(18R), B10.OL, or crosses between them) were fed Purina Laboratory Chow or the same diet plus 400 IU of vitamin A daily from conception and given dexamethasone (80 or 160 mg/kg) intraperitoneally on the twelfth day of pregnancy. It was found that the added vitamin A increased the frequency of isolated cleft palate only in strains that had b alleles between C4 and B144. The enhancement of susceptibility to glucocorticosteroid-induced cleft palate by vitamin A appears to be a recessive trait and the locus, called Acp, maps centromeric to another corticosteroid-induced cleft palate gene (Dcp-2) that also is in the C4:B144 interval.

Effects of H-2 on neural tube defects in congenic mice.

Pregnant mice congenic with C57BL/10 (B10.A, B10.BR, B10.D2, B10.A[2R], B10.A[5R], B10.A[15Rd, B10.A[1R], B10.A[18R], and B10.0L) were fed Purina Mouse Chow or the same diet plus 200 IU of vitamin A daily. The pregnant dams were sacrificed on the eighteenth day of gestation, and the fetuses were sexed and examined for defects in neural tube development. The frequency of neural tube defects was low (mean frequency of all strains, 0.36%) and was not affected by the addition of vitamin A (200 IU/day) to the diet. Twenty-seven of the 29 defects observed occurred in the anterior tube (exencephaly); fourteen were identified in female fetuses, but the sex could not be determined in the other 15 cases because of fetal death and early autolysis. Variations in frequency among the strains suggest that a locus between E beta and H-2D has a moderate influence on the occurrence of neural tube defects. Strains that had H-2d alleles in this segment of the H-2 complex had relatively high frequencies, and those with H-2b or H-2k alleles had significantly lower frequencies.

Effects of H-2 and vitamin A on micrognathia in congenic mice.

Pregnant mice congenic with C57BL/10 (B10.A, B10.BR, B10.D2, B10.A(2R), B10.A(5R), B10.A(15R), B10.A(1R), B10.A(18R), and B10.OL) were fed Purina Mouse Chow or the same diet plus 200 IU of vitamin A daily. The pregnant dams were sacrificed on the eighteenth day of gestation, and the fetuses were sexed and examined for defects in mandibular development. On average, micrognathia occurred five times more frequently in female (1.5%) than male (0.3%) fetuses. The addition of vitamin A to the diet affected only females, reducing the frequency of this defect to that observed in males from dams fed the control diet. Micrognathia was strongly associated with micro- or anophthalmia, but not with defects of the palate. C57BL/10 fetuses had the highest frequency of micrognathia (3.2%) and B10.D2 and B10.A(5R) fetuses had the lowest (0.1%). The results suggest that a locus distal to C4 and perhaps proximal to Qa-1 may exert a moderate influence on mandibular development and a second locus proximal to E beta may have a weak effect.

Effects of H-2 and vitamin A on eye defects in congenic mice.

Pregnant mice congenic with C57BL/10 (B10.A, B10.BR, B10.D2, B10.A(2R), B10.A(5R), B10.A(15R), B10.A(1R), B10.A(18R), and B10.OL) were fed Purina Mouse Chow or the same diet plus 200 IU of vitamin A daily. The pregnant dams were sacrificed on the 18th day of gestation and the fetuses were sexed and examined for defects in eye development. It was found that the frequency of microphthalmia and anophthalmia in the female progeny of mice fed Mouse Chow was 7.4-9.2% in B10.A and B10.BR, 4.0-5.5% in B10.A(18R), B10, B10.A(5R), B10.A(1R), B10.A(15R), and B10.A(2R), and 0.8% and 1.4% in B10.D2 and B10.OL mice, respectively. On average, the frequency of these defects in the female progeny was 6.2 times greater than that in males (P less than 0.001). The right eye was 5.8 times more often affected than the left (P less than 0.001). The addition of vitamin A to the diet increased the frequency of these eye abnormalities in all strains, suggesting that this effect is not mediated by loci associated with H-2, as is the case with vitamin A-enhanced cleft palate. The addition of vitamin A to the diet did not affect the ratios of affected males to females, affected right to left eye, or microphthalmia to anophthalmia. The results suggest that there are two loci on chromosome 17, one centromeric to E beta and one telemeric to C4, that interact to determine to some degree the frequency of microphthalmia and anophthalmia.

Vitamin A-enhanced cleft palate susceptibility associated with H-2.

Pregnant mice from the congenic strains C57BL/10Sn, B10.BR, B10.A/SgSn, B10.A(5R)/SgSn, B10.A(2R)SgSn and B10.A(18R)Sg were fed Purina Laboratory Chow or the same diet plus approximately 400 IU vitamin A daily and given 80 mg/kg dexamethasone intra-peritoneally or a sham injection on the 12th day of pregnancy. It was found that only strains with b alleles between H-2S and H-2D had significantly higher frequencies of isolated cleft palate among their progeny when fed the supplemental vitamin A. The locus appears to be on the centromeric side of a dexamethasone-induced cleft palate gene which has been mapped to the same general area.

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).

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 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.

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.

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.

Sensitivity to corticosterone-induced cleft palate is not associated with H-2.

The frequency of cleft palate (CP) and corticosterone levels in maternal plasma and amniotic fluid were determined in pregnant C57BL/10 (H-2b) and congenic B10.A (H-2a) mice after the ip injection of repository ACTH, corticosterone acetate, or desoxycorticosterone acetate (DOCA) on the 11th through 14th day of gestation. It was found that ACTH (a) induces CP in B10.A but not C57BL/10 mice, (b) induces CP in B10.A mice at about the same frequency noted when diluent alone is injected, and (c) produces an elevation in maternal plasma corticosterone levels 1.5- to 2.0-fold lower and amniotic fluid levels 3- to 5-fold lower than those found after the injection of 5 mg corticosterone acetate, a dose which induces a comparable frequency of CP in B10.A mice. The injection of 5 mg corticosterone acetate produced CP frequencies in C57BL/10 and B10.A mice of 4.9 and 3.3%, respectively, and increasing the dose to 9.2 mg resulted in significant increases in CP to 23.8 and 24.7%, respectively. DOCA at two dose levels induced CP in B10.A fetuses at about the frequency noted when diluent alone has been given. These findings show that susceptibility to corticosterone induced CP is not associated with the major histocompatibility complex of the mouse, H-2, as is the case with glucocorticoids (e.g., cortisone, dexamethasone), and they raise the possibility that factors other than or in addition to corticosterone may be involved in spontaneous or ACTH- or stress-induced CP.

Glucocorticoid-induced cleft palate genes in chromosome 17: genetic linkage and mapping analyses.

Genes that influence susceptibility to dexamethasone-induced cleft palate and tentatively designated Dep are linked to the major histocompatibility complex H-2 in chromosome 17 of the mouse. Experiments presented refine the map of genes. The results show two or three Dep loci. The two-locus model maps Dep genes to the class II gene E beta and to the chromosomal region between the S and D genes. The three-locus model maps the Dep genes to the chromosomal regions from the centromere to E beta, from E beta to S, and from D to Pgk-2. Experiments were done by comparing the dexamethasone-induced cleft palate dose response of congenic strains with H-2 haplotypes that are recombinants of H-2a and H-2b. The analysis of genetic linkage between H-2 and Dep was expanded to include reciprocal backcrosses. A maternal factor was found to influence the frequency of dexamethasone-induced cleft palate in the backcross fetuses. The factor's origin is associated with the H-2 haplotype of the outcross mother, so the effect is actually a "grandmother effect" that probably is transmitted horizontally. Finally, the sexes were distributed unevenly between the fetuses with cleft palate in two of the congenic strains. This suggests interaction between the H-2-linked Dep genes and a Dep sex-associated gene that modulates susceptibility to dexamethasone-induced cleft palate.