Effect of maternal immunopotentiation on apoptosis-associated molecules expression in teratogen-treated embryos.

PROBLEM: Potentiation of the maternal immune system was shown by us to affect the embryonic response to teratogenic insults. In order to understand better the mechanisms underlying that phenomenon, we explored the effect of maternal immunopotentiation by rat splenocytes on the early stages of the embryonic response to cyclophosphamide (CP). METHOD OF STUDY: Immunopotentiated CP-treated embryos were analysed for cell cycle changes by flow cytometry, while cell proliferation and apoptosis were assessed by 5'-bromo-2'-deoxyuridine (BrdU) incorporation and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) respectively. The expression of the p65 subunit of NF-kappaB, IkappaBalpha, Bax, bcl-2 and p53 was assessed by flow cytometry. RESULTS: Exposure to CP resulted in significant growth retardation and in the appearance of cellular damage, a reduction in cell proliferation and the appearance of apoptotic cells, which were all found to be delayed in immunopotentiated embryos. In parallel, CP-treated embryos demonstrated a reduction in the percentage of p65- or IkappaBalpha-positive cells, while the percentage of bcl-2- or p53-positive cells increased initially and decreased later. Those changes were normalized by maternal immunopotentiation when tested at 24 hrs after exposure to the teratogen. CONCLUSION: Our data implicate maternal immunopotentiation to protect the embryo against teratogenic insults, possibly through its effect on the expression of p65, bcl-2 or p53.

Prebreeding maternal immunostimulation with Freund's complete adjuvant reduces placental damage and distal limb defects caused by methylnitrosourea.

PROBLEM: Immunostimulation reduces murine teratogen-induced birth defects. It is unclear if placental improvement contributes to this outcome. The current study examined murine placental ultrastructure and fetal limb development following maternal methylnitrosourea (MNU) exposure, +/-Freund's complete adjuvant (FCA) immunostimulation. METHOD OF STUDY: Two murine strains (CD-1, C57BL/6N) were administered MNU on gestation day 9 (GD9), FCA pre-breeding, or FCA + MNU. Fetal limb and placental development were examined on GD14. RESULTS: MNU decreased placental weight and reduced placental cellular viability; FCA reversed these effects. MNU shortened fetal limbs and increased digital defects in both strains. Placentas were less damaged in C57BL/6N versus CD-1 mice, and distal limb malformations improved only in CD-1 mice. FCA immunostimulation also increased pregnancy rate. CONCLUSION: Improved fetal outcome from immune-stimulated mice may not be dependent on improved placental morphology. However, placental function and morphology in immune-stimulated mice may not directly correlate, thus functional improvements should be examined for possible relationship to reduced birth defects.

Placental improvement and reduced distal limb defects by maternal interferon-gamma injection in methylnitrosourea-exposed mice.

BACKGROUND: Methylnitrosourea (MNU), an alkylating agent derived from creatinine metabolism, is cytotoxic, genotoxic, and mutagenic. Mid-gestational exposure to MNU leads to distal limb defects in mice. Previous studies have shown that nonspecific maternal immune stimulation protects against MNU-induced teratogenesis. A role for immune-mediated placental improvement in this effect remains uncertain. METHODS: The immune system of timed-pregnant C57BL/6N and CD-1 mice was stimulated by GD 7 intraperitoneal (IP) injection with the cytokine interferon-gamma (IFN-gamma). A teratogenic dose of MNU was then administered by IP injection on the morning of GD 9 to disrupt distal limb formation. Fetal limb length, body length, digital deformities, and placental integrity were evaluated on GD 14. RESULTS: The incidence of syndactyly, polydactyly, and interdigital webbing in MNU-exposed mice was decreased by maternal IFN-gamma treatment. In C57BL/6N mice, these defects were reduced by 47, 100, and 63%, respectively, as compared to previous reports on CD-1 mice, by 39, 71, and 20%, respectively. Administration of IFN-gamma significantly diminished MNU-induced endothelial and trophoblast placental damage in both strains of mice. CONCLUSIONS: These findings support a possible link between maternal immunity, placental integrity, and fetal distal limb development. Further, these results suggest that IFN-gamma might act through placental improvement to indirectly protect against MNU-induced fetal limb malformations.

Reduced birth defects caused by maternal immune stimulation in methylnitrosourea-exposed mice: association with placental improvement.

BACKGROUND: Methylnitrosourea (MNU) is a potent carcinogen and teratogen that is associated with central nervous system, craniofacial, skeletal, ocular, and appendicular birth defects following transplacental exposure at critical time points during development, and preliminary studies have suggested that nonspecific maternal immunostimulation may offer protection against development of these birth defects. METHODS: Our study examined morphologic alterations in fetal limb and digital development and placental integrity following maternal exposure to MNU on GD 9 in CD-1 mice, and characterized the improvement in placental integrity and abrogation of fetal defects following maternal immune stimulation with interferon-gamma (IFN-gamma) on GD 7. RESULTS: Fetal limbs were significantly shortened (p < 0.0001) and incidence of limb and digital defects (syndactyly, polydactyly, oligodactyly, clubbing, and webbing) was dramatically increased following mid-gestational maternal MNU exposure. Maternal immune stimulation with IFN-gamma on GD 7 lessened incidence of fetal limb shortening and maldevelopment on GD 12 and 14. Further, disruption of placental spongiotrophoblast integrity, increased cell death in placental trophoblasts with increased intercellular spaces in the spongiotrophoblast layer and minimal inflammation, and increased loss of fetal labyrinthine endothelial cells from MNU-exposed dams suggested that MNU-induced placental breakdown may contribute to fetal limb and digital maldevelopment. MNU + IFN-gamma was associated with diminished cell death within all layers of the placenta, especially in the labyrinthine layer. CONCLUSIONS: These data verify improved distal limb development in MNU-exposed mice as a result of maternal IFN-gamma administration, and suggest a link between placental integrity and proper fetal development.

Increased TNF-alpha expression in cultured mouse embryos exposed to teratogenic concentrations of glucose.

Diabetes-induced early embryonic death is accompanied by an increased expression of tumour necrosis factor alpha (TNF-alpha) in the embryonic microenvironment. The aim of the present study was to evaluate whether diabetes-induced embryopathic stress may also alter the expression of TNF-alpha produced by the embryo itself. As a model, whole postimplantation embryos were cultured for 24 h in a medium with high concentrations of glucose, one of the main diabetes-associated teratogenic metabolites. An anomaly such as an open neural tube was used as an end-point characterizing the glucose-induced teratogenic effect and the number of somites was counted to evaluate growth retardation induced by glucose. The expression of TNF-alpha (by immunohistochemistry), apoptosis (by TdT-mediated dUTP nick-end labelling; TUNEL) and the activity of caspases 3 and 8 (by a fluorometric assay) were evaluated in normal and malformed embryos. Ninety-seven per cent of the embryos exposed to 1300 mg glucose dl(-1) exhibited an open neural tube. The percentage of malformed embryos was smaller in media containing 800 and 500 mg glucose dl(-1) (68 and 37%, respectively) but it still exceeded significantly the value registered in embryos developing in a normoglycaemic medium (12%). In addition, a significant decrease in the number of somites was observed in embryos developing in media containing 1300 and 800 mg glucose dl(-1). Malformed embryos exhibited a greater number of nuclei that were positive in the TUNEL assay as well as a higher amount of active caspase 8 compared with normal embryos (with closed neural folds). TNF-alpha expression was detected in the neuroepithelial layer of the neural tube of the malformed embryos, whereas the expression of this cytokine was weak, if detectable, in normal embryos. Together, these findings indicate that TNF-alpha produced by the embryo may be involved in regulating the response of embryos to diabetes-generated embryopathic stress.

Reproductive/developmental toxicity and immunotoxicity assessment in the nonhuman primate model.

Nonhuman primates are being used increasingly as a non-rodent animal model during preclinical toxicology and safety assessment on the basis of proven similarity and comparability between nonhuman primates and humans. The validity of the nonhuman primate models applies to many aspects of toxicological testing and holds particularly true for the evaluation of reproductive toxicology and developmental toxicology. More recently, the advent of humanized antibodies and vaccines imposed further demand on nonhuman primate models since many immunotherapeutics do not interact with rodent receptors but frequently only cross-react with primate tissue. In this paper we discuss the suitability of primate models for reproductive, developmental and immunotoxicology testing, and present our initial data on the development of lymphatic organs and immune system in a nonhuman primate model.

Maternal immune stimulation in mice decreases fetal malformations caused by teratogens.

For unknown reasons, non-specific stimulation of the maternal immune system in pregnant mice has what appears to be a broad-spectrum efficacy for reducing birth defects. Immune stimulation by diverse procedures has proven effective, including footpad injection with Freund's complete adjuvant (FCA), intraperitoneal (IP) injection with inert particles to activate resident macrophages, IP injection with attenuated Bacillus Calmette-Guerin (BCG), and intrauterine injection with allogeneic or zenogeneic lymphocytes. Morphologic lesions that were significantly reduced included cleft palate and associated craniofacial defects, digit and limb defects, tail malformations, and neural tube defect (NTD). Teratogenic stimuli to induce these lesions included chemical agents (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], cyclophosphamide [CP], and valproic acid [VA]), physical agents (X-rays, hyperthermia), and streptozocin (STZ)-induced diabetes mellitus. Limited information is available regarding mechanisms by which such immune stimulation reduced fetal dysmorphogenesis. The collective literature suggests the possibility that immunoregulatory cytokines of maternal origin may be the effector molecules in this phenomenon.

Immunoteratology: I. MHC involvement in the embryo response to teratogens in mice.

PROBLEM: The present study was carried out to evaluate an involvement of MHC-associated maternal immunoreactivity in response to environmental teratogens. METHODS: Two chemicals, cyclophosphamide (CP) and 2,3-quinoxalinedimetanol, 1,4-dioxide (QD) were used as the reference teratogens (RT). The response to these RT was investigated in syngeneically and allogeneically mated CBA/J and C57B1/6 mice. In part of C57B1/6 female mice, paraaortic lymph nodes were extirpated 14 days before mating to allogeneic or syngeneic males. Twenty or 40 mg/kg of CP or 300 or 600 mg/kg of QD were injected on day 12 and 9 of pregnancy, accordingly (vaginal plug indicates day 1 of pregnancy). On day 19 of pregnancy implantation sites, resorption, live and dead fetuses were recorded and live fetuses were examined with methods routinely used in applied teratology. RESULTS: Both mice strains showed equal response to teratogens but the RT-induced effect was significantly weaker in allogeneic than syngeneic mouse combinations. Extirpation of draining lymph nodes dramatically increased the sensitivity to RT in allogeneically mated females but failed to alter that of syngeneically mated ones. CONCLUSION: The results of this study suggest that fetomaternal MHC incompatibility exerts the favourable influence on teratological resistance of the embryo and MHC-associated immunoreactivity of "mother-fetus" axis is possibly responsible for this effect.

MHC-associated immunopotentiation affects the embryo response to teratogens.

The present study was performed to evaluate whether the effect of environmental teratogens can be modified by maternal immunostimulation. Two chemicals, cyclophosphamide (CP) and 2,3-quinoxalinedimetanol,1,4-dioxide (QD) were used as the reference teratogens (RT). The response to these RT was investigated in two animal models: (i) primigravid C57Bl/6 mice who underwent intrauterine immunization with allogeneic paternal (CBA/J), third-party (BALB/c) or syngeneic male splenocytes 21 days before mating; (ii) C57Bl/6 and CBA/J mice who were treated with RT during the second pregnancy only, after a different mating combination (syngeneic or allogeneic) in the first and the second pregnancy. Different doses of CP and QD were injected on days 12 and 9 of pregnancy, respectively. On day 19 of pregnancy implantation sites, resorptions, live and dead fetuses were recorded and live fetuses were examined for external and internal malformations with methods routinely used in teratological study. It was shown that intrauterine immunopotentiation with allogeneic paternal splenocytes clearly enhances the tolerance of F1 embryos to RT. Thus, in CP-treated females the resorption rate and the proportion of malformed fetuses were significantly reduced. It was followed by an almost two-fold increase in fetal weight. The protective effect of such immunization in QD-treated females was manifested as a dramatic decrease of the proportion of malformed fetuses and the resorption rate. Syngeneic splenocytes could not significantly influence an embryo's sensitivity to RT. The response to RT was also significantly weaker in the second pregnancy of female mice mated twice allogeneically than that observed in allogeneically mated primigravid mice. These results show that the embryo's response to environmental teratogens may be influenced by fetomaternal immune interactions.

Minimal immunological changes in structurally malformed rats after prenatal exposure to cyclophosphamide.

In order to compare the sensitivities of morphological and immunological parameters in a teratology study, effects in day 20 rat fetuses were studied after a single exposure to the immunosuppressive cytostatic agent cyclophosphamide (CP) on either day 11 or day 15 of gestation. Teratological methods included evaluation of external and skeletal morphology. Furthermore histology, immunohistochemistry and flow cytometry were performed on fetal thymus, liver and spleen. Immune function was assayed using the Trichinella spiralis infection model. Treatment resulted in dose-dependent gross morphological malformations, and in addition in overt skeletal anomalies such as brachygnathia, wavy ribs, and lordosis. In contrast, the immunological parameters tested revealed only minimal differences between treated and control groups. These results suggest either a remarkable recovery of the immune system after treatment, or a relatively high resistance of the immune system to the present treatment.

Phenytoin teratogenicity in the primary and secondary mouse embryonic palate is influenced by the H-2 histocompatibility locus.

Inbred and congenic strains of mice have been studied for susceptibility to phenytoin-induced cleft lip with or without cleft palate (CLP) and isolated cleft palate (CP). The role of genes linked to the H-2 complex on chromosome 17 has been confirmed. Congenic strains with the A background have identical levels of spontaneous CLP, whereas those strains having the A background with the H-2a haplotype have significantly higher rates of induced CLP than their congenic partners with the H-2b or H-2s haplotype. No such significant difference in the degree of CLP produced by phenytoin is demonstrable in strains with the B background. Rates of isolated CP produced by phenytoin are significantly higher in strains with H-2a than in their congenic partner strains with either H-2b or H-2s, whether the background is A or B.

Susceptibility to phenytoin-induced cleft palate in mice is influenced by genes linked to H-2 and H-3.

Pregnant female mice of various congenic strains were injected on days 11 through 14 of gestation with phenytoin, and the fetuses were examined for the occurrence of cleft palate. The pattern of susceptibility indicates that two genes in the H-2 complex and another gene linked to H-3 are involved in the determination of this trait.