Maternal LPS Exposure during Pregnancy Impairs Testicular Development,Steroidogenesis and Spermatogenesis in Male Offspring

Lipopolysaccharide (LPS) is associated with adverse developmental outcomes including embryonic resorption, fetal death, congenital teratogenesis and fetal growth retardation. Here, we explored the effects of maternal LPS exposure during pregnancy on testicular development, steroidogenesis and spermatogenesis in male offspring. The pregnant mice were intraperitoneally injected with LPS (50 µg/kg) daily from gestational day (GD) 13 to GD 17. At fetal period, a significant decrease in body weight and abnormal Leydig cell aggregations were observed in males whose mothers were exposed to LPS during pregnancy. At postnatal day (PND) 26, anogenital distance (AGD), a sensitive index of altered androgen action, was markedly reduced in male pups whose mothers were exposed to LPS daily from GD13 to GD 17. At PND35, the weight of testes, prostates and seminal vesicles, and serum testosterone (T) level were significantly decreased in LPS-treated male pups. At adulthood, the number of sperm was significantly decreased in male offspring whose mothers were exposed to LPS on GD 13–17. Maternal LPS exposure during gestation obviously diminished the percent of seminiferous tubules in stages I–VI, increased the percent of seminiferous tubules in stages IX–XII, and caused massive sloughing of germ cells in seminiferous tubules in mouse testes. Moreover, maternal LPS exposure significantly reduced serum T level in male mice whose mothers were exposed to LPS challenge during pregnancy. Taken together, these results suggest that maternal LPS exposure during pregnancy disrupts T production. The decreased T synthesis might be associated with LPS-induced impairments for spermatogenesis in male offspring.     

Ontogeny and postnatal persistence of a strong suppressor activity in man

We report here on the ontogeny and postnatal persistence of an inhibited human immune response in which lymphocytes from human newborns strongly suppress the proliferation of adults' lymphocytes stimulated by phytohemagglutinin (PHA) or alloantigens in vitro. For this research we used a 2-way mixed lymphocyte culture (MLC) supplemented with PHA, with sex chromosomes acting as markers for dividing male and female cells, or alternatively a double chamber system. The proliferation of maternal lymphocytes was significantly suppressed by fetal lymphoid cells from the liver as early as the 8th week of gestation and by those from fetal blood at the 14th week or later during gestation. This strong suppressor activity persisted in 11-mo-old infants but usually disappeared after that time.     

The Influence of a High‐Fat Dietary Environment in the Fetal Period on Postnatal Metabolic and Immune Function

Few reports show whether a high‐fat (HF) dietary environment in the fetal period affects immune function or the development of lifestyle‐related disease at maturity. We examined the influence of an HF dietary environment in the fetal period on postnatal metabolic and immune function. A total of 16 pregnant mice were given control (CON) diet and 16 were given HF diet in the gestational period, from mating to delivery. After delivery lactating mice were given either CON or HF diet, resulting in four groups. After weaning, the offspring mice were given the same diet that their mothers received during lactation. HF dietary intake in the postnatal period increased fat pad weights, serum glucose, and leptin levels. An HF diet in the fetal period resulted in fewer splenic lymphocytes, a thinner thymic cortex, and impaired antigen‐specific immune reactions. Furthermore, tumor necrosis factor (TNF)‐α production and serum triglyceride levels were elevated in the fetal HF group. In addition, the HF‐HF group showed a consistent decrease in ovalbumin (OVA)‐specific IgG and elevation of IgE, associated with advanced fatty changes in the liver. Results from this study suggest that HF environment during the fetal period induces epigenetic propensity toward obesity and immunological burden in part due to increased adipose tissue mass, significant reduction in the number of immune cells and decreased activities of immune cells.     

Production of factors regulating macrophage motility in the early stages of mixed lymphocyte culture

The macrophage migration stimulation factor (MSF) is produced during the first hours of mixed lymphocyte culture (MLC) in the H-2 system. The activity of MSF is characterized by sharp peaks of migration stimulation in the culture fluids of allogeneic MLC. The peaks appeared every 5 hours. After 12--16 hours in the culture fluids of allogeneic MLC, there has been demonstrated macrophage migration inhibition factor whose activity rose by 48 hours. Optimal concentrations were revealed for stimulation and inhibition of macrophage migrations on the dilution of 1--2-day MLC culture fluids. The existence of the biorhythmical mechanism that controls the macrophage mobility through the balance of soluble mediators with alternative activity is suggested.     

Gestational stage sensitivity to ultrasound effect on postnatal growth and development of mice

BACKGROUND: An experiment was conducted to find out whether ultrasound exposure leads to changes in postnatal growth and development in the mouse. METHODS: A total of 15 pregnant Swiss albino mice were exposed to diagnostic levels of ultrasound (3.5 MHz, 65 mW/cm2, I(SPTP) = 1 mW/cm2 Intensity(Spatial Peak-Temporal Peak), I(SATA) = 240 mW/cm2 Intensity(Spatial Average-Temporal Average)) for 30 min for a single day between days 10 and 18 of gestation (GD 10-18). Virgin female mice were placed with same age group males for mating in the ratio 2 females : 1 male and examined the next morning for the presence of vaginal plug, a sign of successful copulation. The females with vaginal plugs were separated and labeled as 0-day pregnant. Maternal vaginal temperature was also measured. A sham exposed control group of 15 pregnant mice was maintained for comparison. All exposed as well as control animals were left to complete gestation and parturition. Their offspring were used in our further studies. They were monitored during early postnatal life for standard developmental markers, postnatal mortality, body weight, body length, head length, and head width, and growth restriction was recorded up to 6 weeks of age. RESULTS: An exposure to ultrasound induced nonsignificant deviations in the maternal vaginal temperature or developmental markers. Significant low birth weight was observed in the present study, after exposure at GD 11, 12, 14, and 16. However, 14 and 16 days postcoitus during the fetal period appears to be the most sensitive to the ultrasound effect, in view of the number of different effects as well as severity of most of the observed effects when exposed on these gestation days. CONCLUSIONS: The results indicate that diagnostic ultrasound can induce harmful effects on mouse growth and development when given at certain critical periods of gestation.     

Developmental Sensitivity to the Induction of Great Vessel Malformations by N‐(2‐Aminoethyl)ethanolamine

N‐(2‐Aminoethyl)ethanolamine (AEEA) induced malformations of the great vessels in the offspring of rats treated during gestation and early lactation (Schneider et al., 2012. Birth Defects Res B Dev Reprod Toxicol [in press]). The aim of this study was to determine if in utero exposure alone was sufficient to induce these malformations or whether a peri‐postnatal exposure or physiological component was required. Three groups of five time‐mated female Wistar Han rats were administered AEEA (250 mg/kg/day) by gavage from gestation day (GD) 6 to GD 19 (groups 1 and 2) or from GD 6 to postnatal day 3 (group 3). Animals were euthanized on GD 21 (group 1) or postnatal day 4 (groups 2 and 3), and the hearts of the offspring were examined for changes to the great vessels. The incidence of malformations in group 1 was 91.1%, and primarily consisted of high aortic arch and abnormal carotid course. One fetus had an aortic aneurysm. All fetuses in groups 2 and 3 were malformed, primarily exhibiting abnormal carotid course and aneurysms, which mainly affected the aorta, ductus arteriosus, and pulmonary trunk. The incidence of high aortic arch was lower relative to group 1. Aneurysms were more prevalent in group 3 compared to group 2. These findings indicate that exposure to AEEA during gestation alone was sufficient to induce malformations of the great vessels and aneurysms, which may be triggered by physiological changes that occur during or after birth, but that the critical period of susceptibility to AEEA‐induced aneurysms in the rat extends beyond gestation into the early postnatal period.     

Teratologic studies on rat perinates and offspring from dams treated with ethylnitrosourea (ENU).

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues, limbs and male reproductive organs. Recently we clarified that excess cell death caused by apoptosis occurred in these organs and tissues of rat fetuses from dams treated with ENU at day 13 of gestation (GD13). In this study, we examined fetuses at GD21 and offspring at 10 weeks of age after ENU administration to pregnant rats at GD13 in order to clarify the relationship between ENU-induced apoptosis in the fetal tissues and teratogenicity of ENU. Severe intrauterine growth retardation was observed in the ENU group, and the body weight of the offspring in the ENU group was significantly lower than that of the control group throughout the experiment. In addition, a high incidence of microencephaly, ectrodactyly and curved caudal vertebrae was observed in the offspring from dams treated with ENU at GD13. Judging from the results of our previous and present studies, it was strongly suggested that ENU-induced apoptosis in rat fetal tissues may play an important role in the induction of anomalies in the corresponding tissues.     

Interferon production by human and murine lymphocytes in response to alloantigens

The production of interferon (IF) by human and mouse lymphocytes sensitized to alloantigens in mixed lymphocyte cultures (MLC) was analyzed. During primary MLC, IF appeared in the culture fluid on day 2 and was maximal on day 5. Based on several biologic criteria, the IF produced is of the "immune" type. When lymphocytes sensitized to alloantigens were reestimulated in vitro, IF was produced within a few hours of culture. In all stimulated cultures, cell proliferation was observed in spite of the high concentrations of IF. The IF-producing cells in human MLC were identified as T lymphocytes lacking the receptor for the Fc fragment of IgG molecules (Fc gamma R(-)). Human MLC supernatants containing immune type IF mediate the enhancement of natural killer (NK) cell activity and protect NK target cells from lysis.     

Neoplastic cells obtained from Hodgkin's disease are potent stimulators of human primary mixed lymphocyte cultures

Neoplastic cells obtained from the pleural effusion of a patient with Hodgkin's disease have been maintained in culture since 1978. These tumor cells have been shown to have the cytologic features, cytochemical staining, and cell surface markers of Reed-Sternberg cells. In this study we demonstrate that the cell line termed L428 is a potent stimulator of the primary human mixed lymphocyte reaction. Significant proliferation occurred when mononuclear leukocytes obtained from normal donors were stimulated with radiated L428 cells at responder:stimulator ratios varying from 200:1 to 20:1. Proliferative responses occurred between days 3 and 6 of the cultures with maximal proliferation on day 5. Under optimal culture conditions, mean net proliferative response of 14 normal donors was 51,000 +/- 10,600 dpm. The mixed lymphocyte response was totally blocked by concentrations of monoclonal anti-Ia antibody that had no effect on concanavalin A-induced proliferation. However, the mixed lymphocyte response was not blocked by an anti-K562 cell monoclonal antibody of the same immunoglobulin subclass that binds to the L428 cells. Antigen processing by responder monocytes or Ia-positive cells was not required for the MLC. When responder T cells from two normals were depleted of Ia-bearing cells and monocytes, the mixed lymphocyte reaction between the two normals was eliminated, yet the stimulation of each normal by the L428 cells was not reduced. The cells that proliferated in response to stimulation by the L428 cells were T cells, primarily of the helper subset. No IL 1 activity could be detected in concentrated supernatants of L428 cultures after stimulation of L428 cells by mitogens, phorbol esters, or muramyl dipeptide, or in the MLC. All of these cultures contain fetal calf serum. However, the L428 cells are capable of producing IL 1, because IL 1 was detected when the L428 cells were stimulated with LPS in the absence of fetal calf serum. These neoplastic cells, obtained from Hodgkin's disease, have many similarities to the murine as well as human dendritic cells.     

Development in the cynomolgus macaque following administration of ustekinumab,a human anti‐il‐12/23p40 monoclonal antibody,during pregnancy and lactation

BACKGROUND: Ustekinumab is a human monoclonal antibody that binds to the p40 subunit of interleukin (IL) 12 and IL‐23 and inhibits their pharmacological activity. To evaluate potential effects of ustekinumab treatment during pregnancy, developmental studies were conducted in cynomolgus macaques. METHODS: Ustekinumab was tested in two embryo/fetal development (EFD) studies and in a combined EFD/pre and postnatal development (PPND) study. In the EFD studies, pregnant macaques (12/group) were dosed with saline or ustekinumab (9 mg/kg IV, 22.5 mg/kg SC, or 45 mg/kg IV or SC during the period of major organogenesis, gestation day [GD] 20–50). Fetuses were harvested on GD100–102 and examined for any effects on development. In the EFD/PPND study, pregnant macaques were injected with saline or ustekinumab (22.5 or 45 mg/kg SC) from GD20 through lactation day 33. Infants were examined from birth through 6 months of age for morphological and functional development. Potential effects on the immune system were evaluated by immunophenotyping of peripheral blood lymphocytes and immunohistopathology of lymphoid tissues in fetuses and infants and by T‐dependent antibody response (TDAR) to KLH and TTX and by DTH response in infants. Ustekinumab concentrations were measured in serum from dams, fetus, and infants and in breast milk. RESULTS: Ustekinumab treatment produced no maternal toxicity and no toxicity in the fetuses or infants, including no effects on the TDAR or DTH responses. Ustekinumab was present in serum from GD100 fetuses and was present in infant serum through day 120 post‐birth. Low levels of ustekinumab were present in breast milk. CONCLUSIONS: Exposure of macaque fetuses and infants to ustekinumab had no adverse effects on pre‐ and postnatal development. Birth Defects Res (Part B) 89:351–363, 2010. © 2010 Wiley‐Liss, Inc.     

The postnatal development of the genital system in male rats following the prenatal administration of corticosterone

Corticosterone administration to pregnant Wistar rats on days 16 and 18 of pregnancy leads to changes in genital system of male offspring during postnatal ontogenesis: reduction of ano-genital distance in two days old rats, increase of preputial glands' weight in 35 and 70 day old embryos, changes in nature of puberal increase in testosterone blood level from day 35 to day 70 of life. The obtained data suggest that the increase in the corticosteroid level in blood of pregnant females owing to any stress factor can affect the postnatal development of genital system of male offspring.     

Adrenergic modulation of cardiac development in the rat: effects of prenatal exposure to propranolol via continuous maternal infusion

During early postnatal development, catecholamines are thought to modulate cardiac cell replication and differentiation, and to program future beta-adrenergic sensitivity. To determine if the sensitive period for these events extends to prenatal ages, pregnant rats were infused with propranolol continuously via osmotic minipumps from gestational day 7 through parturition and the offspring were examined for markers of cardiac cellular development (basal ornithine decarboxylase activity and levels of DNA and protein) and for reactivity to acute beta-adrenergic challenge (heart rate responses and stimulation of ornithine decarboxylase). During the propranolol infusion, fetal cardiac responses to terbutaline, a beta-adrenergic agonist, were completely blocked; after discontinuation of beta-blockade at birth, responses became normal and remained unaffected into young adulthood. Biochemical markers indicated a delay in cellular development caused by propranolol: basal ornithine decarboxylase activity was elevated in the fetus and DNA was subnormal for the first week after birth. Cardiac growth was maintained in the face of DNA deficits by cell enlargement (elevated protein/DNA) which persisted through weaning. By young adulthood, all markers were within normal limits. These data suggest that fetal catecholamines, acting on beta-receptors, do play an initial role in cardiac cellular development, but that the critical period for programming of beta-adrenergic responsiveness occurs later in maturation.     

Nonspecific suppressor T cells cause decreased mixed lymphocyte culture reactivity in bone marrow transplant patients

Decreased reactivity in mixed lymphocyte culture (MLC) was observed in patients within 1 yr after allogeneic and autologous bone marrow transplantation. Suppressor activity of peripheral blood mononuclear cells (PBMC) from transplant patients was studied by adding these cells as modulator cells to a bidirectional MLC with cells from normal individuals. PBMC from transplant patients markedly suppressed MLC reactivity in a dose-dependent manner. Suppressor activity was present in cells forming rosettes with sheep erythrocytes. Treatment of modulator cells with monoclonal antibodies against T cell differentiation antigens (OKT8, OKIa1) and complement completely abolished suppression of MLC. Suppressor activity was unaffected by 30 Gy irradiation. Suppressor activity declined gradually after transplantation and was inversely correlated with MLC reactivity of each patient at a significant level (p less than 0.01). These observations suggest that OKT8+ Ia+ radioresistant suppressor T cells play a role in the development of decreased MLC reactivity observed during the early post-transplant period.     

Severity and timing of early thyroid deficiency as factors in the induction of learning disorders in rats

In Experiment 1 (N = 277 rats), more extreme deficits in maze learning than heretofore shown appeared in the adult offspring of mothers exposed for 16 pre- and 16 postnatal days to thiouracil-treated mash diets in doses up to 0.3%; the same offspring also displayed deficits in single-alternation pattern learning and a modified operant discrimination task. Surprisingly small maze learning deficits, however, were found in the offspring of mothers which received thiouracil during the 16 postnatal days only, despite previous findings indicating that the postnatal half of the total 32-day perinatal period was the more critical in determining later learning impairments. Reconciliation was provided by Experiment 2 (N = 54), in which the manipulation of a 0.2% thiouracil diet starting at birth or 3, 6, 10, or 15 days before birth indicated a lower age boundary of the critical period for the induction of maze learning deficit by thyroid deficiency at approximately the fetal age at which thyroid tissue becomes functional-around the 18th day of gestation.     

Vitamin A Supplementation in Early Life Enhances the Intestinal Immune Response of Rats with Gestational Vitamin A Deficiency by Increasing the Number of Immune Cells

Vitamin A is a critical micronutrient for regulating immunity in many organisms. Our previous study demonstrated that gestational or early-life vitamin A deficiency decreases the number of immune cells in offspring. The present study aims to test whether vitamin A supplementation can restore lymphocyte pools in vitamin A-deficient rats and thereby improve the function of their intestinal mucosa; furthermore, the study aimed to identify the best time frame for vitamin A supplementation. Vitamin A-deficient pregnant rats or their offspring were administered a low-dose of vitamin A daily for 7 days starting on gestational day 14 or postnatal day 1, day 14 or day 28. Serum retinol concentrations increased significantly in all four groups that received vitamin A supplementation, as determined by high-performance liquid chromatography. The intestinal levels of secretory immunoglobulin A and polymeric immunoglobulin receptor increased significantly with lipopolysaccharide challenge in the rats that received vitamin A supplementation starting on postnatal day 1. The rats in this group had higher numbers of CD8⁺ intestinal intraepithelial lymphocytes, CD11_C ⁺ dendritic cells in the Peyer''s patches and CD4⁺CD25⁺ T cells in the spleen compared with the vitamin A-deficient rats; flow cytometric analysis also demonstrated that vitamin A supplementation decreased the number of B cells in the mesenteric lymph nodes. Additionally, vitamin A supplementation during late gestation increased the numbers of CD8⁺ intestinal intraepithelial lymphocytes and decreased the numbers of B lymphocytes in the mesenteric lymph nodes. However, no significant differences in lymphocyte levels were found between the rats in the other two vitamin A supplement groups and the vitamin A-deficient group. In conclusion, the best recovery of a subset of lymphocytes in the offspring of gestational vitamin A-deficient rats and the greatest improvement in the intestinal mucosal immune response are achieved when vitamin A supplementation occurs during the early postnatal period.     

Stimulating effect of polyion on the production of cytolytic T lymphocytes in a cell culture in vitro

Introduction of polyion "vegetan" at the concentrations varying from 20 to 100 micrograms/ml into mixed lymphocyte culture (MLC) with insufficient antigenic stimulus, low immunogenic MLC, for the whole of the incubation period, leads to 30-70% increase in T-lymphocyte cytolytic activity. The effect of vegetan on the cytolytic activity of T-lymphocytes from normal MLC is less marked. In the in vivo model of primary synthesis of antibodies against heterologous erythrocyte antigens, vegetant causes 20-50-fold enhancement of a weak immune response compared to 1.5-2-fold when the latter approaches the highest levels. These findings indicate an inverse relationship between the immunostimulating activity of vegetan and the levels of the immune response, the latter being the target of activation. Vegetan was shown to induce more than 2-fold increase in the proliferation in both types of MLC as well as in mouse spleen lymphocyte monoculture. It is reasonable to propose that the preparation may stimulate the proliferation of both T-killers and other lymphocyte subpopulations.     

Leukocyte complement: a possible role for C5 in lymphocyte stimulation

The results presented here show that Fab' antibody fragments directed to complement proteins C5, C6, and C7 inhibit lymphocyte stimulation in mixed lymphocyte culture (MLC) by up to 65%, as determined by decreased incorporation of 3H-thymidine. Lymphocyte stimulation induced by PHA-mitogen was also inhibited up to 100% by anti-C5 Fab'. Specificity of these reactions was established by the findings that goat anti-C5 or murine hybridoma anti-C5 both inhibited MLC; the inhibitory activity of anti-C5 Fab' was absorbed with highly purified C5 (but not with C3), and antibody directed to C3 did not inhibit lymphocyte stimulation by MLC or PHA. The effects of anti-C5 were exerted in a nontoxic manner. Cleavage of lymphocyte associated C5 with factor B (Bb) or with trypsin resulted in stimulation of lymphocyte thymidine incorporation. Purified C5a was found to induce lymphocyte stimulation in serum-free medium in pulse-chase types of experiments. Anti-C6 and C7 Fab' also inhibited lymphocyte stimulation induced in one-way MLC. These results suggest that C5, C5a, and/or C6 and C7 may play a role in triggering of lymphocyte blastogenesis.     

Autoimmune processes after long-term low-level exposure to electromagnetic fields (experimental results) part 5. Study of the influence of blood serum from rats exposed to low-level electromagnetic fields on pregnancy and fetal and offspring development

The work evaluates the possible adverse effects on pregnancy and fetal and offspring development arising from the injection of blood serum from rats exposed to microwaves at a power density of 500 μW/cm² into intact female rats. The study is performed on 59 pregnant Wistar rats. Intrauterine mortality, embryo and fetal body weights, and placenta weight are used for the evaluation of embryo and fetal development. Generally accepted integral and specific parameters are used for the evaluation of the postnatal development of offspring during the first 30 days of life. It is shown that injection of blood serum from rats subjected to long-term RF exposure at 500 μW/cm² into intact rats on the tenth day of pregnancy results in adverse effects on fetal and offspring development. Higher total in utero and postnatal mortality, as well as delayed offspring development, are recorded.     

Zinc inhibits the mixed lymphocyte culture

The mixed lymphocyte culture (MLC) is an established clinical method for bone marrow transplantation, as it serves as an in vitro model for allogenic reaction and transplantation. We previously showed that cytokine release into the supernatant is a more specific and sensitive parameter for cross-reactivity in the MLC than the common measurement of cell proliferation. Therefore we tried to find an inhibitor of the MLC in vitro with the least side effects in vivo, measuring interferon (IFN)-γ as one of the most important cytokines in posttransplant medicine. Earlier studies showed that zinc is an important trace element for immune function with both stimulatory and inhibitory effects on immune cells. We found that slightly elevated zinc concentrations (three to four times the physiological level), which do not decrease T-cell proliferation in vitro nor produce immunosuppressive effects in vivo, suppress alloreactivity in the mixed lymphocyte culture. In this report we analyzed the mechanism whereby zinc influences the MLC to possibly find a nontoxic way of immunosuppression.