Shoshana Savion

TNF‐α Protects Embryos Exposed to Developmental Toxicants

BACKGROUND:  Tumor necrosis factor α (ΤNF‐α) has been implicated in mediating post‐implantation embryo loss or the embryonic maldevelopment induced by development toxicants or maternal metabolic imbalances. In order to clarify the role of TNF‐α further, a comparative study was performed in TNF‐α knockout and TNF‐α positive mice, exposed to a reference teratogen, cyclophosphamide (CP).

Apoptosis in the Uterus of Mice with Pregnancy Loss

PROBLEM: The mechanisms mediating pregnancy loss induced by various agents are far from being understood. Thus, we investigated the possible involvement of one such mechanism, the apoptotic process, in pregnancy loss induced by lipopolysaccharide (LPS) or cyclophosphamide (CP) as well as the associated changes in the apoptosis‐regulating gene products p53 and bcl‐2.

Ciprofloxacin immunomodulation of experimental antiphospholipid syndrome associated with elevation of interleukin‐3 and granulocyte‐macrophage colony‐stimulating factor expression

OBJECTIVE To evaluate the immunomodulatory potential of ciprofloxacin in mice with experimental antiphospholipid syndrome (APS). METHODS Ciprofloxacin or ceftazidime (control antibiotic) was given to mice with experimentally induced APS. The titers of autoantibodies, levels of cytokines, and number of cytokine-producing cells were determined by enzyme-linked immunosorbent assay. Myeloid progenitor cells were determined by granulocyte-macrophage colony-forming unit, and interleukin-3 (IL-3) messenger RNA (mRNA) was tested by Northern analysis. RESULTS A decrease in the incidence of pregnancy loss and an improvement in the clinical manifestations of APS were noted in the mice treated with ciprofloxacin, compared with the mice given ceftazidime. The effect of ciprofloxacin was found to be associated with increased serum levels of IL-3 and with increased IL-3 mRNA transcription in the splenocytes. Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was documented by elevated titers in the sera and elevated numbers of colony-forming cells in the bone marrow. CONCLUSION Ciprofloxacin prevents the manifestations of experimental APS. This effect may be associated with increased IL-3 levels and GM-CSF expression.

TGFβ2 mRNA expression and pregnancy failure in mice

PROBLEM: We describe here a pattern of transforming growth factor (TGF) β2 mRNA expression at the fetomaternal interface in mice with high rate of resorptions as well as its expression following maternal immunopotentiation.

Immunostimulation increases the resistance of mouse embryos to the teratogenic effect of diabetes mellitus

Summary The present work was aimed to assess the possible effect of stimulation of the maternal immune system on the teratogenic potential of diabetes mellitus. ICR female mice were immunized with splenocytes of male rats 3 weeks before the beginning of mating and were injected with 240 mg/kg streptozocin (STZ) 10 days after immunization. Females with blood glucose levels over 27.8 mmol/l and HbA1 c levels over 6 standard deviations (SD) above the mean of intact animals were used for teratological studies. The rate of malformed fetuses, resorptions and fetal weights were evaluated for animals killed on day 19 of pregnancy using routine teratological methods. Also, phenotyping of spleen cells of these females was performed by fluorescein activated cell sorter analysis. Two main effects possibly due to immunostimulation of ICR females were observed: 1) immunostimulated females had significantly fewer litters with malformed fetuses than non-immunized females: only 4 litters out of 22 (18 %) compared to 10 out of 16 (63 %). Correspondingly, the incidence of malformed fetuses was also decreased: 2.1 compared to 8.9 %; 2) a significant increase in the pregnancy rate in immunized diabetic ICR mice: 69 % as compared to 44 % in non-immunized diabetic females. Also, immunostimulation resulted in a visible increase in spleen cellularity and a certain increase in the number of cells with mature T-cell and macrophage surface markers. These results strongly suggest that immunostimulation increases the tolerance of ICR females to the teratogenic effect of STZ-induced diabetes. [Diabetologia (1997) 40: 635–640]

NF-κB DNA-binding activity in embryos responding to a teratogen, cyclophosphamide

BackgroundThe Rel/NF-κB transcription factors have been shown to regulate apoptosis in different cell types, acting as inducers or blockers in a stimuli- and cell type-dependent fashion. One of the Rel/NF-κB subunits, RelA, has been shown to be crucial for normal embryonic development, in which it functions in the embryonic liver as a protector against TNFα-induced physiological apoptosis. This study assesses whether NF-κB may be involved in the embryos response to teratogens. Fot this, we evaluated how NF-KappaB DNA binding activity in embryonic organs demonstraiting differential sensitivity to a reference teratogen, cyclophosphamide, correlates with dysmorphic events induced by the teratogen at the cellular level (excessive apoptosis) and at the organ level (structural anomalies).ResultsThe embryonic brain and liver were used as target organs. We observed that the Cyclophosphamide-induced excessive apoptosis in the brain, followed by the formation of severe craniofacial structural anomalies, was accompanied by suppression of NF-κB DNA-binding activity as well as by a significant and lasting increase in the activity of caspases 3 and 8. However, in the liver, in which cyclophosphamide induced transient apoptosis was not followed by dysmorphogenesis, no suppression of NF-κB DNA-binding activity was registered and the level of active caspases 3 and 8 was significantly lower than in the brain. It has also been observed that both the brain and liver became much more sensitive to the CP-induced teratogenic insult if the embryos were exposed to a combined treatment with the teratogen and sodium salicylate that suppressed NF-κB DNA-binding activity in these organs.ConclusionThe results of this study demonstrate that suppression of NF-κB DNA-binding activity in embryos responding to the teratogenic insult may be associated with their decreased resistance to this insult. They also suggest that teratogens may suppress NF-κB DNA-binding activity in the embryonic tissues in an organ type- and dose-dependent fashion.

TNF-α expression in embryos exposed to a teratogen

PROBLEM: The role of tumor necrosis factor (TNF)‐α produced by embryonic cells in normal and abnormal development is poorly understood. To assess to what extent TNF‐α may be involved in the process of induced dysmorphogenesis, the expression of TNF‐α and TNF‐α receptor (TNFRI) mRNA as well as TNF‐α protein was evaluated in embryos responding to a cyclophosphamide (CP)‐induced teratogenic insult. The effect of maternal immunostimulation increasing the embryos tolerance to CP on TNF‐α expression was also investigated.

A possible role for granulocyte macrophage‐colony stimulating factor in modulating teratogen‐induced effects

It was already shown that stimulation of the maternal immune system by allogeneic or xenogeneic leukocytes is capable of affecting embryonic responses to teratogenic insults and various cytokines, including granulocyte macrophage-colony stimulating factor (GM-CSF), were implicated as mediators of this effect. Therefore, in the present study we tried to assess the ability of GM-CSF to modulate teratogenic activity, along with possible changes in systemic as well as local maternal immune responses, that might be involved in the process. Thus, the percentage of cyclophosphamide (CP)-treated embryos exhibiting limb malformations was shown to decrease significantly following GM-CSF administration. This effect was found to be comparable to that demonstrated by intrauterine leukocytes administration. GM-CSF treatment resulted in a significant enhancement in maternal splenocytes Con-A-induced proliferation, as well as Interleukin-2 (IL-2) and IL-3 production. Examination of leukocyte cell surface antigens expressed by splenocytes revealed no statistically-significant changes in the level of the T lymphoid antigens Thy-1, CD5, CD4, CD8 and CD3, the macrophage antigen Mac-1, and the adhesion molecules LFA-1alpha, LFA-1beta and L-Selectin, following GM-CSF immunostimulation. In parallel, immunohistochemical analysis of the uteroplacental unit revealed Mac-1 and to a lesser extent LFA-1beta-positive cells localized to the myometrium and the placenta in both the control and the GM-CSF-treated groups, while no cells expressing Thy-1, CD3, CD4, CD8, or LFA-1alpha could be demonstrated. Our results suggest a possible role for GM-CSF in modulating teratogen-induced effects, a process in which maternal immune responses such as splenocytes proliferation and cytokine production might be involved.

Maternal immunopotentiation affects the teratogenic response to hyperthermia

Immune responses occurring between the embryo and mother have been shown to influence the embryos tolerance to teratogens, including chemical teratogens and diabetes-induced teratogenic insult. In this study, we tried to evaluate whether maternal immunostimulation alters the embryos response to heat shock, one of few teratogens which directly affect the embryo. In order to induce structural anomalies, both intact ICR female mice and mice which had been immunostimulated with xenogeneic rat splenocytes before mating, were exposed to two consecutive exposures to heat (43.6 +/- 0.2 degrees C) for 10 min on day 9 of pregnancy. The number of malformed fetuses, resorptions, and fetal weight were assessed on day 19 of pregnancy. Heat shock-induced apoptosis, and the level of heat shock protein (HSP) 60 expression, were examined in embryonic cells at different time points within 24 h after heating. All these indices differed dramatically in immunized and non-immunized heat shocked females. Heat shocked non-immunized females demonstrated an increased level of resorptions (approximately, 21% versus 8.6% in controls) and the proportion of fetuses with such anomalies as encephalocele and open eyes reached 28% and 21%, respectively. Maternal immunostimulation was associated with a significant decrease in the proportion of fetuses with encephalocele (12.8%), open eyes (8.9%), and resorptions (8%). The maximum level of heat shock-induced apoptosis in cell populations from the embryos of non-immunized females, was approximately, 30% versus 7% in cells of embryos of immunized mice. Heat shock was also followed by a significant increase in HSP60 expression, but only in the cells of embryos of non-immunized females. Together, these findings suggest that the tolerance of mouse embryos to a heat shock-induced teratogenic insult may, to some extent, depend on the character of the maternal immune responses.

Cellular events and the pattern of p53 protein expression following cyclophosphamide-initiated cell death in various organs of developing embryo.

This study was aimed at characterizing the temporal patterns of cell responses and p53 protein expression in the limbs, head, and liver of embryos responding to cyclophosphamide (CP)-induced teratogenic insult. ICR murine embryos were examined 24, 48, or 72 h after injection of 40 mg/kg CP on day 12 of pregnancy. The cellular events and temporal pattern of p53 protein expression were determined by FACS analysis and by TUNEL (apoptosis) in the head, limbs, and liver of the embryos. All tested organs showed apoptosis and a significantly decreased proportion of live cells after 24 h. Subsequent events were organ-dependent. In the liver, there were no dysmorphic events at any time and excessive cell death had been almost compensated for by 48 h. Compensation was preceded by G(1) arrest and accompanied by an increased level of p53 protein in surviving cells. Excessive cell death in the head and the limbs resulted in structural anomalies. In the head, there was an increased level of p53 protein and G(1) arrest after 24 h and the number of live cells at 48 h was equal to that seen in earlier samples, despite apoptosis. In the limbs, however, only isolated viable cells were seen by 48 h, but there was no increased level of p53 protein or G(1) arrest. Results of this study suggest that the differential sensitivity of tested organ systems to CP may be associated with differences in cellular events following CP-initiated cell death. They also suggest that the input of p53 in determining the response of these organ systems to CP-induced teratogenic insult may be different. Teratogenesis Carcinog. Mutagen. 19:353-367, 1999.