Role of TNF-alpha in prenatal alterations in dams of mice under thermal stress

The possible involvement of cytokines such as tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) that are suspected of causing pregnancy loss and miscarriage has been investigated in dams of mice subjected to hyperthermia. Thermal stress was induced by exposing mice dams at 40+/-2 degrees C for 4 h every day during the different phases of the gestation period whereas the normothermic animals were housed at 22+/-2 degrees C. The effect of maternal thermal stress was measured in pregnant mice at different phases of the gestation period namely, blastogenesis-implantation phase (days 0-5 postconceptionem [p.c.]), organogenesis or embryogenesis phase (days 6-15 p.c.) and fetogenesis phase (days 16-20 p.c.). Uterine examination of dams subjected to hyperthermia on days 6-15 p.c. showed maximum reduction in live fetus number, gestational index and maximum pre and postimplantation loss in comparison with dams housed in normothermic environment and dams exposed to thermal stress between days 0-5 and 16-20 p.c. Maximum resorption rate and number of non-viable fetuses were observed in dams exposed to hyperthermia during days 6-15 p.c. Elevated levels of TNF-alpha and IL-1 beta were observed in the amniotic fluid of dams subjected to hyperthermia during days 6-15 p.c. but IFN-gamma levels remained unaltered. Single intraperitoneal (i.p.) administration of recombinant mouse TNF-alpha at a dose of 1 and 0.5 ng/mice in dams on day 6 in normothermic condition resulted in a reduced number of live fetuses. Administration of anti-TNF-alpha antibody i.p. at a dose of 10 microg/dam on day 6 p.c. and subjected to thermal stress between days 6-15 p.c. increased marginally the number of fetuses but failed to attain statistical significance in comparison with days 6-15 p.c. thermally stressed dams without antibody treatment. It is concluded that the induction of TNF-alpha, in the amniotic fluid is associated with thermal stress during pregnancy and may be linked to the reproductive performances of dams. This study will help in understanding the mechanism of thermal injury in pregnant subjects.     

The natural history of Down syndrome conceptuses diagnosed prenatally that are not electively terminated.

The pregnancy outcomes on cases of Down syndrome diagnosed prenatally in which the mother did not elect termination were evaluated in data reported to a comprehensive Register of Down syndrome for England and Wales for 1989-94. In the 168 cases in which placental biopsy was not used, the overall rate of spontaneous loss was 35%, but this figure masks considerable heterogeneity by gestational stage at ascertainment. Data on ages at diagnostic procedure and on pregnancy termination enabled a more precise survival analysis. The loss rates were approximately 50% for those fetuses ascertained at 15-17 completed wk, 43% at 18 wk, 31% at 19 wk, 25% at 20 wk, and then a leveling off at approximately 20%-25% for fetuses ascertained at 21-28 completed wk. For fetuses ascertained prior to 18 wk, there was no evidence that maternal age was associated with fetal loss, consistent with earlier reports. At 18 wk and after, however, maternal age was on the average approximately 3 years greater in fetuses that were lost. Comparison of successive gestational birth cohorts provided no evidence in these 168 cases that the diagnostic procedure itself had any effect on loss or that selective ascertainment of mothers in risk of loss had any effect on the results. In contrast, in the 21 cases in which placental biopsy had been undertaken, the overall loss rates were not only higher when appropriate comparisons could be made, but there was some evidence for selective ascertainment and/or procedure-associated losses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transforming growth factor beta 1 acts as an autocrine-negative growth regulator in colon enterocytic differentiation but not in goblet cell maturation

Previous studies from this laboratory (Schroy, P., Rifkin, J., Coffey, R.J., Winawer, S., and Friedman, E. (Cancer Res., 50: 261-265, 1990; Schroy, P.C., Winawer, S., and Friedman, E. Cancer Lett., 48: 53-58, 1989) found that a 7-day treatment of the human colon carcinoma cell line HT29 with the differentiation agent hexamethylene bisacetamide (HMBA) induces both a 4-5-fold increase in transforming growth factor beta 1 (TGF beta 1) mRNA levels and reduced tumorigenicity in vivo. A series of 15 cloned lines with different commitments to differentiation has been isolated from 20-day HMBA-treated HT29 cells, maintained without HMBA, and utilized to study the role of TGF beta 1 in colon carcinoma differentiation. Two such lines, HD6 and HD8, differentiate to 97 and 76% mucus-secreting goblet cells, respectively, in columnar monolayers in postconfluent culture. Both HD6 and HD8 cells exhibit low TGF beta 1 mRNA levels, little different from the undifferentiated HT29 parental line, and exhibit no growth modulation in response to exogenous TGF beta 1. In contrast, two other lines, HD3 and HD4, differentiate to fluid-transporting enterocytic cells with functional brush borders and exhibit autocrine-negative growth response to TGF beta 1. Both lines express TGF beta 1 mRNA at levels 11-12-fold higher than the parental line and respond to exogenous TGF beta 1 by growth inhibition. HD3 cells secrete biologically active TGF beta 1 into conditioned media, which inhibited growth of a TGF beta 1-sensitive mink cell line. This inhibition was blocked by antisera to TGF beta 1, proving the specificity of the inhibition. A range of concentrations of this TGF beta 1 antiserum stimulated HD3 cell growth in a dose-dependent manner, further documenting the autocrine-negative response of the cells to TGF beta 1. Another cell line, HI1, was blocked in enterocytic differentiation. HI1 cells synthesized as much TGF beta 1 mRNA as HD3 and HD4 cells, yet they responded to exogenous TGF beta 1 with less growth inhibition, suggesting some impairment in their response to TGF beta 1. A third class of response to TGF beta 1 was exhibited by the HP1 cell line, which was resistant to HMBA-induced differentiation, remaining undifferentiated with a multilayered growth pattern. HP1 cells synthesized TGF beta 1 mRNA at levels over 20 times the parental level but were stimulated to divide by TGF beta 1, exhibiting autocrine-positive response to this growth factor.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of donor age on the sensitivity of osteoblasts to the proliferative effects of TGF(beta) and 1,25(OH(2)) vitamin D(3)

The loss of osteoblast function in aging bone is one of the major causes of osteopenia, or loss of bone mass. In this study, this loss of function was investigated by examining the proliferative response of rat long bone periosteal osteoblasts to TGF(beta1) and 1,25-dihydroxy vitamin D(3) (1,25-D(3)) as a function of donor age. Using a DNA binding fluorescent dye, DNA levels were measured in osteoblast cultures derived from either young adult (3-4 months) or old (14-15 months) rats following treatment with two concentrations (10(-9) M or 10(-12) M) of either 1,25-D(3) or TGF(beta1) or with vehicle. Cells from young rat bone, when treated with 1, 25-D(3), showed a dose-dependent increase in proliferation when treated with the higher dose and a decrease in proliferation when treated with the lower dose. Osteoblasts isolated from old rats did not respond to 1, 25-D(3) treatment. A similar pattern of response to TGF(beta1) was found. When treated with 10(-9) M TGF(beta1), the rate of proliferation increased for young rat osteoblasts, but the old rat derived cells were unresponsive. The 10(-12) M dose of TGF(beta1) was ineffective for both young and old cells. This study has shown that osteoblasts derived from old donors are impaired in their ability to respond to vitamin D and TGF(beta), two of the major controlling factors of skeletal development and maintenance.     

A pivotal role for galectin-1 in fetomaternal tolerance

A successful pregnancy requires synchronized adaptation of maternal immune-endocrine mechanisms to the fetus. Here we show that galectin-1 (Gal-1), an immunoregulatory glycan-binding protein, has a pivotal role in conferring fetomaternal tolerance. Consistently with a marked decrease in Gal-1 expression during failing pregnancies, Gal-1-deficient (Lgals1-/-) mice showed higher rates of fetal loss compared to wild-type mice in allogeneic matings, whereas fetal survival was unaffected in syngeneic matings. Treatment with recombinant Gal-1 prevented fetal loss and restored tolerance through multiple mechanisms, including the induction of tolerogenic dendritic cells, which in turn promoted the expansion of interleukin-10 (IL-10)-secreting regulatory T cells in vivo. Accordingly, Gal-1's protective effects were abrogated in mice depleted of regulatory T cells or deficient in IL-10. In addition, we provide evidence for synergy between Gal-1 and progesterone in the maintenance of pregnancy. Thus, Gal-1 is a pivotal regulator of fetomaternal tolerance that has potential therapeutic implications in threatened pregnancies.     

Altered regulation of protein disulfide isomerase in cells resistant to the growth-inhibitory effects of transforming growth factor beta 1

A murine keratinocyte cell line that is resistant to the growth-inhibitory effects of transforming growth factor beta 1 (TGF beta 1) was examined for differential gene expression patterns that may be related to the mechanism of the loss of TGF beta 1 responsiveness. Cells that were resistant to the growth-inhibitory effects of TGF beta 1 (KCR cells) were derived from K-ras-transformed BALB/MK keratinocytes (KC cells). Using a subtractive hybridization procedure with KC and KCR mRNAs, we isolated a complementary DNA clone for murine protein disulfide isomerase (PDI). The mRNA for PDI is inhibited by TGF beta 1 treatment in the parental KC cells, but not in the TGF beta 1-resistant KCR cells. Similar PDI down-regulation also occurs in other TGF beta-sensitive cells, but not in a human pancreatic carcinoma cell line which is insensitive to the growth-inhibitory effects of TGF beta 1. The results suggest that misregulation of PDI, an important component of co- and posttranslational modification systems, may be involved in the mechanism by which some cells escape from the growth-inhibitory effects of TGF beta 1.     

Alterations in phosphoinositide metabolism associated with 17 beta-estradiol and growth factor treatment of MCF-7 breast cancer cells

Steady-state levels of phosphatidyl inositol (PtdIns) turnover are examined in MCF-7 human breast cancer cells in response to estradiol treatment. Elevated levels of PtdIns are observed 12-24 h after estradiol treatment, occur at estradiol concentrations as low as 10(-12) M, and are competitively blocked by the antiestrogen LY117018. MCF-7 cells secrete a transforming growth factor (TGF) alpha-like material which can partly replace estradiol in conferring tumorgenicity in nude mice. We show that acute or chronic treatment of MCF-7 cells with TGF alpha results in elevated PtdIns turnover and that chronic treatment increases growth rate. In contrast TGF beta is growth inhibitory and blocks estradiol-induced increases in PtdIns turnover. A phosphatidyl inositol 4,5-bisphosphate specific phospholipase-C activity has been identified and is elevated in association with estradiol treatment. These data are consistent with estradiol-induced autocrine growth factors, including TGF alpha, acting through the PtdIns turnover pathway as part of their mechanism of action.     

Fetal origins of adult vascular dysfunction in mice lacking endothelial nitric oxide synthase

Epidemiological studies have shown increased incidence of hypertension and coronary artery disease in growth-restricted fetuses during their adult life. A novel animal model was used to test the hypothesis regarding the role of an abnormal uterine environment in fetal programming of adult vascular dysfunction. Mice lacking a functional endothelial nitric oxide synthase (NOS3-/-KO, where KO is knockout) and wild-type (WT) mice (NOS3+/+WT) were crossbred to produce homozygous NOS3-/-KO, maternally derived heterozygous (NOS3+/-mat, mother with NOS3 deficiency), paternally derived heterozygous (NOS3+/-pat, normal mother), and NOS3+/+WT litters. Number of fetuses per litter was smaller in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Adult female mice from these litters (7-8 wk old) were killed, and ring preparations of carotid and mesenteric arteries were mounted in a wire myograph to evaluate the passive and reactive vascular characteristics. Slope of the length-tension plot (a measure of vascular compliance) was increased, and optimal diameter (as calculated by Laplace equation) was decreased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Acetylcholine caused vasorelaxation in NOS3+/-pat and NOS3+/+WT and contraction in NOS3-/-KO and NOS3+/-mat mice. Responses to phenylephrine and Ca2+ were increased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Relaxation to isoproterenol was decreased in NOS3-/-KO and NOS3+/-mat vs. NOS3+/-pat and NOS3+/+WT mice. Abnormalities in the passive and reactive in vitro vascular properties seen in NOS+/-mat that developed in a NOS3-deficient maternal/uterine environment compared with the genetically identical NOS3+/-pat mice that developed in a normal environment are the first direct evidence in support of a role for uterine environment in determining vascular function in later life.     

Vitamin E is essential for mouse placentation but not for embryonic development itself

Vitamin E (alpha-tocopherol) was discovered 80 years ago to be an indispensable nutrient for reproduction in the female. However, it has not been clarified when or where vitamin E is required during pregnancy. We examined the role of alpha-tocopherol in pregnancy using alpha-tocopherol transfer protein (Ttpa)-deficient mice fed specific alpha-tocopherol diets that led to daily, measurable change in plasma alpha-tocopherol levels from nearly normal to almost undetectable levels. A dietary supplement of alpha-tocopherol to pregnant Ttpa-/- (homozygous null) mice was shown to be essential for maintenance of pregnancy from 6.5 to 13.5 days postcoitum but found not to be crucial before or after this time span, which corresponds to initial development and maturation of the placenta. In addition, exposure to a low alpha-tocopherol environment after initiation of placental formation might result in necrosis of placental syncytiotrophoblast cells, followed by necrosis of fetal blood vessel endothelial cells. When Ttpa(-/-)-fertilized eggs were transferred into Ttpa+/+ (wild-type) recipients, plasma alpha-tocopherol concentrations in the Ttpa-/- fetuses were below the detection limit but the fetuses grew normally. These results indicate that alpha-tocopherol is indispensable for the proliferation and/or function of the placenta but not necessary for development of the embryo itself.     

Costs of gestation in an Arctic ruminant: copper reserves in muskoxen

The transfer of trace minerals between mother and fetus may be critical for survival of young ruminants especially among species at high latitudes, which gestate during a long winter and grow through a brief summer. We examined the distribution of copper and metalloproteins (ceruloplasmin and metallothionein) in muskoxen and their fetuses, three times during gestation. Hepatic levels of copper were high in mothers (179 microg g(-1) whole tissue) and did not change through gestation, whereas fetuses accumulated large reserves of Cu (>300 microg g(-1)), likely stored in proteins such as metallothionein, during the last third of gestation. The effect of fetal Cu demands on the pregnant female was tested by supplementation of Cu by subcutaneous injections of Cu gluconate (30 mg Cu/week) during pregnancy. Maternal copper supplementation did not significantly increase hepatic Cu in newborns (412 microg g(-1) for supplemented vs. 303 microg g(-1) for unsupplemented neonates), probably because the diet was already adequate in copper (14 microg g(-1) dry matter). Ceruloplasmin activity declined in pregnant muskoxen that had not received injections of Cu and suggested increased systemic demands for copper during late gestation. Supplies of Cu to the fetus could be limited either by low levels of Cu in the maternal liver, or in the maternal diet during late winter when fetal gains in mass and liver Cu are greatest.     

The relation between maternal restraint and food deprivation, plasma corticosterone, and induction of cleft palate in the offspring of mice.

The blood level of corticosterone was measured in mice following the injection on day 14 of pregnancy of a dose of corticosterone sufficient to cause a low frequency of cleft palate in the fetuses. This was compared with the blood levels present during maternal restraint and food deprivation that produced a similar frequency of cleft palate. The mean blood level over the 24 h following injection of corticosterone was 660 mug/100 ml, and during a similar period of restraint was 485 mug/100 ml. Other mice were subjected either to restraint or food deprivation for 24 h beginning day 14 of pregnancy, the plasma corticosterone levels measured during that time, and the frequency of cleft palate in late fetuses compared with the individual plasma corticosterone levels during treatment. There was a significant (P less than 0.025) correlation between high maternal corticosteroid levels and the frequency of cleft palate in the offspring of the restrained mice but not in the food-deprived animals. It is suggested that in some stressed mice endogenous plasma corticosterone can reach levels sufficient to account for the development of cleft palate.     

Effect of iron deficiency on placental cytokine expression and fetal growth in the pregnant rat.

Iron deficiency anemia is the most common nutritional disorder in the world. Anemia is especially serious during pregnancy, with deleterious consequences for both the mother and her developing fetus. We have developed a model to investigate the mechanisms whereby fetal growth and development are affected by maternal anemia. Weanling rats were fed a control or iron-deficient diet before and throughout pregnancy and were killed at Day 21. Dams on the deficient diet had lower hematocrits, serum iron concentrations, and liver iron levels. Similar results were recorded in the fetus, except that the degree of deficiency was markedly less, indicating compensation by the placenta. No effect was observed on maternal weight or the number and viability of fetuses. The fetuses from iron-deficient dams, however, were smaller than controls, with higher placental:fetal ratios and relatively smaller livers. Iron deficiency increased levels of tumor necrosis factor alpha (TNFalpha) only in the trophoblast giant cells of the placenta. In contrast, levels of type 1 TNFalpha receptor increased significantly in giant cells, labyrinth, cytotrophoblast, and fetal vessels. Leptin levels increased significantly in labyrinth and marginally (P = 0.054) in trophoblast giant cells. No change was observed in leptin receptor levels in any region of the placentas from iron-deficient dams. The data show that iron deficiency not only has direct effects on iron levels and metabolism but also on other regulators of growth and development, such as placental cytokines, and that these changes may, in part at least, explain the deleterious consequences of maternal iron deficiency during pregnancy.     

Growth stimulation and differential regulation of transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 messenger RNA levels by norethindrone in MCF-7 human breast cancer cells.

Transforming growth factor-beta (TGF beta) is a potent growth inhibitor in most epithelial cells. We evaluated the effects of norethindrone (which in combination with estrogen is commonly used in oral contraceptives) and other progestins [medioxyprogesterone acetate (MPA) and R5020, which are not used in oral contraceptives] on cell growth and the expression of TGF beta 1, TGF beta 2, and TGF beta 3 mRNAs in MCF-7 human breast cancer cells. Growth of MCF-7 cells was stimulated by norethindrone (10(-8)-10(-5) M), with maximal growth stimulation at 10(-7) M norethindrone after 7 days of treatment. However, the growth of MCF-7 cells was not affected by MPA (10(-8) M) or R5020 (10(-8) M). Treatment with the antiestrogen 4-hydroxytamoxifen at a concentration of 10(-7) M blocked the growth stimulation induced by norethindrone. The norethindrone-induced growth stimulation was accompanied by a dramatic decrease in TGF beta 2 and TGF beta 3 mRNA levels, whereas the level of TGF beta 1 mRNA was not affected by any of the compounds tested. In addition, treatment with MPA or R5020 did not affect TGF beta 2 and TGF beta 3 mRNA levels. The inhibitory effect of norethindrone on TGF beta 2 and TGF beta 3 mRNA levels could be blocked by the addition of 10(-7) M 4-hydroxytamoxifen. Norethindrone as well as estradiol decreased estrogen receptor mRNA levels and increased progesterone receptor mRNA levels. This is the first report which demonstrates that norethindrone stimulates estrogen-responsive human breast cancer cell growth and inhibits the expression of TGF beta 2 and TGF beta 3 mRNAs. These results suggest that the differential regulation of TGF beta expression by norethindrone may be at least partly responsible for the growth stimulation induced by norethindrone. Thus, the norethindrone component of some oral contraceptives may be sufficiently estrogenic to facilitate the development of breast cancer.     

Pattern of compensatory expression of voltage-dependent Ca2+ channel alpha1 and beta subunits in brain of N-type Ca2+ channel alpha1B subunit gene-deficient mice with a CBA/JN genetic background.

The Ca(2+) channel alpha(1B) subunit is a pore-forming component capable of generating N-type Ca(2+) channel activity. Although the N-type Ca(2+) channel plays a role in a variety of neuronal functions, alpha(1B)-deficient mice with a CBA/JN genetic background show no apparent behavioral or anatomical-histological abnormality, presumably owing to compensation by other Ca(2+) channels. In this study, we examined the mRNA expression of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits in the olfactory bulb, cerebral cortex, hippocampus and cerebellum of alpha(1B)-deficient mice. We found that the mRNA expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits were the same in the olfactory bulbs of wild, heterozygous and homozygous alpha(1B)-deficient mice. In the cerebral cortex, alpha(1A) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits was found among wild, heterozygous and homozygous mice. In hippocampus and cerebellum, beta(4) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2) and beta(3) subunits was found among wild, heterozygous and homozygous mice. These results suggest that the compensatory mechanisms differ in different brain regions of alpha(1B)-deficient mice with a CBA/JN genetic background.