Effect of a single early-gestational alcohol consumption on the insulin binding by immune cells of adult rats

Summary. Rat dams were exposed to 15% ethanol containing water ad libitum for 24 h at the 3rd day of gestation. Adult progenies’ immune cells (peritoneal, blood and thymic lymphocytes, monocyte-macrophage-granulocyte group and mast cells) were studied by flow cytometry for binding of FITC-labelled insulin (compared to the appropriate cells of untreated animals). All of the cell types studied bound significantly less insulin, except mast cells. The results call attention to the life-long effect of occasional alcohol consumption in early pregnancy manifested in the immune system. Received 27 June 2005; returned for revision 23 August 2005; accepted by I. Ahnfelt-R?nne 29 August 2005     

Examining maternal influence on OLETF rats' early overweight: Insights from a cross‐fostering study

Obese female Otsuka Long Evans Tokushima Fatty (OLETF) rats display increased nursing time and frequency compared to lean LETO controls, suggesting a maternal contribution to pup preobesity. In previous studies, OLETF pups presented high adiposity, showed greater suckling efficiency, initiative and weight gain from nursing than controls throughout lactation. To further elucidate maternal–infant interactions contributing to pup preobesity, we cross‐fostered pups a day after birth and examined maternal behavior. Nursing frequency decreased in OLETF dams raising LETO pups (OdLp) in the third postnatal week, while LETO dams raising OLETF pups showed no significant changes. Fat % was greater in the milk of OLETF versus LETO dams. OdLp pups showed long‐term body weight (BW) increase, suggesting that maternal environment can induce BW increases even in the absence of a genetic tendency. Additionally, interaction between OLETF dams and pups produces high nursing frequency, exposing the pups to abundant high‐fat milk, thus strengthening their preobese phenotype. © 2009 Wiley Periodicals, Inc. Dev. Psychobiol 51: 358–366, 2009.     

Developmental delays associated with prenatal alcohol exposure are reversed by thyroid hormone treatment

Exposure to alcohol in utero has been associated with hypothyroidism and a variety of developmental defects characteristic of thyroid dysfunction. The present work examined whether these abnormalities could be reversed in infant rats treated with thyroid hormones. Subjects were offspring of dams which were on the following diet regimen during gestation: (1) free access to liquid diet containing ethanol (alcohol pups); (2) an equal volume of isocaloric liquid diet (pair-fed pups); or (3) ad libitum control diet (control pups). Neonates from each group were foster-nursed by control dams, and received triiodothyronine (T3; 0.1 mg/kg/day; s.c.) or saline treatments on postnatal days 1 to 10. The alcohol neonates displayed reduced serum thyroxine which was restored to normal by postnatal day 14. In addition, these pups showed a delayed appearance of developmental landmarks, including righting reflex, dental eruption, auditory startle response and eye opening. The retarded incisor eruption and eye opening were reversed in alcohol pups by T3 treatments. The present data suggest that at least some of the developmental abnormalities associated with prenatal alcohol exposure are attributable to perinatal hypothyroidism and can be restored by early hormone replacement therapy.     

Effects of stress during pregnancy on maternal behavior in mice

The effects of stress experienced during pregnancy and raising stressed offspring on maternal behavior were investigated in Swiss-Webster mice. Dams were either stressed or not stressed during pregnancy, and raised either prenatally stressed or nonstressed cross-fostered pups. Maternal behaviors such as grooming, nursing, pup retrieval and maternal aggression were assessed during the first 4 days after birth. Nonstressed dams raising stressed pups and stressed dams raising nonstressed pups groomed and nursed their pups significantly less than did control dams (stressed dams raising stressed pups and nonstressed dams raising nonstressed pups). Nonstressed dams raising stressed pups were also the slowest to retrieve both the first and last pup in retrieval tests. Nonstressed dams raising nonstressed pups were significantly less aggressive than other dams. In contrast, stressed dams raising stressed pups exhibited high levels of nursing and grooming, retrieved their pups rapidly and were very aggressive towards an intruder. These results indicate that raising stressed pups, or experiencing stress during a pregnancy can have significant effects on maternal behaviors. Stressed dams raising stressed pups exhibit maternal care comparable to that of nonstressed dams raising nonstressed pups at least for nesting/nurturing behaviors, and show increased levels of aggression and pup retrieval.     

Maternal and Pup Genotype Contribution to Growth in Wild-Type and tau Mutant Syrian Hamsters

The single gene mutation tau in the Syrian hamster—apart from its effect on the circadian organization of locomotor activity—has a pronounced influence on body weight. In this study we investigate the impact of maternal and pup genotypes at the tau-locus on the growth rate of pups. Homozygous tau mutant hamsters (circadian period of 20 hours) had lower growth rates and adult body weights than wild-type hamsters, whereas heterozygous tau mutants (circadian period of 22 hours) were intermediate. In addition, heterozygous pups from heterozygous dams grew heavier than those from wild-type and homozygous dams. The effect of maternal genotype was further evaluated in a cross-foster design, where wild-type and homozygous mutant pups were fostered at birth to either wild-type or homozygous mutant dams. At all ages, the maternal tau genotype had a negative effect on body weight, whereas the pup tau genotype had a positive effect during the preweaning period and a negative effect afterward.     

Ultrasonic vocalizations and maternal-infant interactions in a rat model of fetal alcohol syndrome

When isolated from their dams and littermates, rat pups emit ultrasonic vocalizations to elicit attention and retrieval from their dams. This study examined the effects of perinatal alcohol exposure on ultrasonic vocalizations and maternal-infant interactions. Alcohol was administered throughout gestation to the dams and during the early postnatal period to the pups. Control groups consisted of a nontreated control and an intubated, pair-fed control. Ultrasonic vocalizations were measured on postnatal day (PD) 5 under varying conditions of isolation. Maternal behaviors were examined on PD2, 4, 6, 8, and 10. Maternal behaviors were not significantly affected by prior alcohol administration to either the dams or the pups. However, ethanol-exposed rat pups vocalized more on PD5 than controls regardless of condition. The heightened vocalization response of the ethanol-exposed pups might be an underlying factor in the persistent effects of perinatal ethanol exposure on social behavior.     

ORIGINAL ARTICLE: The Persistence of Paternal Antigens in the Maternal Body is Involved in Regulatory T‐Cell Expansion and Fetal‐Maternal Tolerance in Murine Pregnancy

Citation Zenclussen ML, Thuere C, Ahmad N, Wafula PO, Fest S, Teles A, Leber A, Casalis PA, Bechmann I, Priller J, Volk H‐D, Zenclussen AC. The persistence of paternal antigens in the maternal body is involved in regulatory T‐cell expansion and fetal‐maternal tolerance in murine pregnancy. Am J Reprod Immunol 2010; 63: 200–208 Problem Mammalian pregnancy is a state of immunological tolerance and CD4⁺ CD25⁺ regulatory T cells (Treg) contribute to its maintenance. Knowing that Treg act in an antigen‐specific way during pregnancy, we hypothesized that they are generated after maternal immune cells encounter paternal antigens. Method of study We mated wild type females with transgenic green fluorescent protein (GFP) males in an allogenic setting and killed them on different days of pregnancy. Results Presence of paternal and maternal MHC class II⁺ cells in vaginal lavage on day 0.5 of pregnancy was confirmed. Thus, antigen presentation may take place early during pregnancy in the periphery either by the direct or indirect pathways. Foxp3⁺ cells known to have regulatory activity could be detected on day 2 of pregnancy in lymph nodes and shortly after implantation at the fetal‐maternal interface. Conclusion Our data suggest that paternal antigens are processed early during pregnancy, which leads to the generation of Treg. The continuous release of placental antigens into the maternal circulation allows the maintenance of a Treg population which is specific for paternal antigens and mediates tolerance toward the semi‐allogeneic fetus until the time point of birth.     

Developmental vitamin D (DVD) deficiency alters pup-retrieval but not isolation-induced pup ultrasonic vocalizations in the rat

Evidence from animal experiments now demonstrates that prenatal vitamin D levels influence brain development. The aims of this study were to examine isolation-induced pup ultrasonic vocalizations and maternal-infant interactions using a pup-retrieval test in developmental vitamin D (DVD) deficient and control rats. Sprague-Dawley rats were fed a vitamin D deficient diet or control diet six weeks prior to mating until birth and housed under UVB-free lighting conditions. In two separate experiments we recorded ultrasonic vocalizations at 46 KHz in isolated pups and we performed a pup-retrieval test on the day of birth. There was no significant effect of maternal diet on the calling rate of isolation-induced ultrasonic vocalizations by pups. We found that DVD-deficient dams retrieved their pups sooner than control dams and engaged in more pup directed activities (sniffing and carrying pups) and had a longer latency for self-grooming and rearing than control dams. We also assessed vitamin D related measures from a terminal blood sample immediately after the pup-retrieval test and found that DVD-deficient dams and pups had significantly lower levels of 25 OH D₃, 1,25 (OH)₂D₃ and phosphate, elevated levels of parathyroid hormone (PTH) but there was no significant effect of maternal diet on calcium levels. We speculate that the altered maternal-pup interactions identified in the DVD model may impact on early periods of brain development and behaviour.     

Effects of prenatal alcohol exposure on rat pups' ability to elicit retrieval behavior from dams

Research on fetal alcohol syndrome (FAS) has historically held that postnatal deficits result directly from prenatal alcohol exposure. Such exposure may alter infant behavior, and this in turn may affect maternal responsiveness and consequently increase the infant's risk for postnatal deficits. This study examined the effect of prenatal alcohol exposure on postnatal blood ethanol concentrations, pup development, and the ability of pups to elicit retrieval behaviors from the dam. Dams given access to a 15% ethanol solution throughout gestation and lactation showed blood ethanol concentrations of 0.1%, whereas their pups had concentrations below the sensitivity of the test. Prenatal alcohol exposure was shown to have pharmacological effects on the pup's righting reflex and nutritional effects on its rate of weight gain. Control pups were better able to elicit retrieval behavior from control and alcoholic dams than were fetally alcoholic pups. These results indicate that the condition of the pup can influence maternal responsiveness which, in turn, can influence pup development.     

Correlation of maternal and pup NK-like activity and TNF responses against cytomegalovirus to pregnancy outcome in inbred guinea pigs

Immunologic causes for poor outcome of pregnancy complicated by primary cytomegalovirus (CMV) infection are only partially understood. Maternal and pup tumor necrosis factor (TNF) and natural killer cell (NK)-like activity associated with primary gestational CMV infection initiated in either the first or third trimester equivalent in the inbred guinea pig model were investigated. Poor pregnancy outcome defined as fetal resorptions, premature delivery, stillbirths, and intrauterine growth retardation occurred with infection at either gestational time. Induction of TNF and NK activity by CMV infection during pregnancy correlated with resorptions in early pregnancy infection and with premature labor in late pregnancy infection. Stillbirths occurred with CMV infection at either time. Regardless of the gestational time of CMV acquisition, poor outcome correlated with higher maternal NK and TNF responses during the first weeks after maternal virus acquisition. Furthermore, CMV infected dams with loss of >/= 50% of conceptus had higher TNF responses than infected dams with < 50% conceptus loss. Likewise, pups born in litters from CMV-infected dams with resorptions and/or premature labor also had enhanced NK activity and TNF response to CMV compared with pups born to dams not having resorptions or premature labor. TNF responses in the delivered pups of infected dams were higher than from pups of uninfected dams regardless of litter outcome, whereas pup NK responses were enhanced only in pups from litters of dams with premature labor or resorptions. Enhanced NK and TNF activity appear to be associated with premature delivery and other poor outcomes of pregnancy.     

Effect of exogenous thyroxine on the development of the Purkinje cell in fetal alcohol effects in the rat

The amelioration of fetal alcohol effects on the postnatal development of the Purkinje cell by exogenous L-thyroxine was investigated in the neonatal rat. Time-pregnant rats were divided into three groups. Group A (n = 6) received 35% liquid ethanol diet; Group B (n = 6) was fed a liquid diet in which maltose dextrins replaced alcohol isocalorically, constituting the pair-fed group; Group C (n = 6) received the 35% liquid ethanol diet and, in addition, received exogenous thyroxine (5 microg/kg/day) subcutaneously. After the pups were born, the mothers were removed and the pups of each were surrogate fostered by dams who were fed normal rat chow and water ad libitum. An average of six pups, one from each litter, were killed at days 7, 14, 21, and 28 for each of the above three groups. Light and electron microscopic observations of lobule II/III revealed a delayed alignment of Purkinje cells (Pc) in alcohol-exposed pups compared to pair-fed pups. The Pc of the pair-fed group showed a single-layer arrangement at 7 days which was seen only at day 14 in the alcohol group. However, in the alcohol + T(4)-exposed pups a single-layer arrangement was quite often seen at 7 days. Morphological observations showed impaired evidence of protein synthesis at all time sequences in the pups of Group A compared to Group B. A most interesting finding was the morphological evidence of greater protein synthesis in the Pc of the alcohol + T(4) group at all times as indicated by a hypertrophied nucleus, abundant ribosomal collection, and numerous Nissl bodies.     

Thyrotropic hormone (TSH) regulation of triiodothyronine (T3) concentration in immune cells

Objective:  Cells of the immune system (peritoneal lymphocytes, monocytes, granulocytes and mast cells as well as thymocytes) contain triiodothyronine (T₃). The aim of the present experiments was to study whether thyrotropic hormone (TSH) regulates or not the T₃ concentration of these cells. Methods:  Peritoneal fluid and thymus cells of adult rats were studied by immunocytochemistry, combined with flow cytometry for triiodothyronine content with or without in-vitro TSH treatment. In addition, adult female CD1 mice were treated in vivo with 10 or 40 mU TSH and after 1 hour peritoneal immune cells were studied using the above mentioned method. Results:  Both in vitro (in rat) and in vivo (in mice) TSH treatments significantly elevated the T₃ content in each cell type. In vitro TSH 0.1 mU/ml cell suspension was enough to provoke about 50 % increase in T₃ production. Conclusion:  T₃ concentration in immune cells seems to be regulated by TSH, similarly to the T₃ in the thyroid. Considering the large number of immune cells in an organism, TSH regulation of their T₃ content could have an important physiological and pathological role, both in and beyond the immune system. Received 15 April 2008; returned for revision 19 May 2008; received from final revision 20 May 2008; accepted by I. Ahnfeld-R?nne 23 June 2008     

Reduced maternal regulatory T cell numbers and increased T helper type 2 cytokine production are associated with elevated levels of immunoglobulin E in cord blood

Background There is evidence that the basis of an atopic‐skewed immune response is acquired early in life, perhaps at the fetal stage. Thus, we hypothesized that the development of the fetal immune system might be influenced by maternal regulatory T cells (Treg) and maternal T cell cytokine production during pregnancy. The aim of the present study was to assess the influence of maternal Treg and cytokine production during pregnancy on Treg and atopy at birth. Methods Within the mother–child study LINA (Lifestyle and Environmental factors and their Influence on Newborns Allergy risk), we determined the frequency and function of Treg and the total IgE concentration in pregnant women in the 34th week of gestation and in corresponding cord bloods at birth (n=24). Furthermore, we assessed how maternal mitogen‐induced T‐helper type 1/T‐helper type 2 and inflammatory cytokines influence the level of cord blood Treg and IgE. Results Frequencies of CD4⁺CD25^high T cells were higher (P=0.001), whereas percentages of FOXP3⁺ T cells were lower (P<0.001) in cord blood cells compared with maternal blood. Reduced maternal CD4⁺CD25^high Treg frequencies correlated with increased total IgE concentrations at the 34th week of gestation (r=?0.32, P=0.028) and with increased IgE concentrations in cord blood (r=?0.50, P<0.001). Elevated maternal mitogen‐induced Th2 cytokine production was related to increased total IgE levels in the serum of corresponding cord bloods (IL‐4, r=0.53; IL‐5, r=0.43; IL‐13, r=0.52). Conclusions Because cord blood IgE has been shown to be predictive for allergic diseases in early childhood, our results indicate that reduced maternal Treg numbers and increased Th2 cytokine production during pregnancy might influence the allergy risk of the child. Cite this as: D. Hinz, J. C. Simon, C. Maier‐Simon, L. Milkova, S. Röder, U. Sack, M. Borte, I. Lehmann and G. Herberth, Clinical & Experimental Allergy, 2010 (40) 419–426.     

Changes in Soluble CD4 and CD8 Proteins in Healthy Pregnant and Postpartum Women

PROBLEM: We examined physiological changes in serum levels of soluble CD4 (sCD4) and soluble CD8 (sCD8) during pregnancy and 1–12 months postpartum to study changes in the maternal immune system during and after pregnancy. METHOD: The serum concentrations of sCD4 and sCD8 were measured by enzyme immunoassay in the sera separated from blood samples withdrawn from healthy women in the 1st, 2nd, and 3rd trimesters of pregnancy and 1,4,7, and 10–12 months postpartum (n=182) and healthy non-pregnant women (n=25), and in 90 of the women, the changes in sCD4 and sCD8 were compared with changes in the number of peripheral CD4⁺ and CD8⁺ cells measured by flow cytometry. RESULTS: The serum concentration of sCD4 decreased throughout pregnancy, from the first trimester, and recovered gradually after delivery. The serum concentration of sCD8 did not change significantly during or after pregnancy compared to the concentration in the nonpregnant controls, but the concentration 1 month postpartum was significantly higher than that in the 3rd trimester. The numbers of CD4⁺ and CD8⁺ cells decreased during pregnancy but did not change significantly after delivery. Interestingly, the ratio of the serum sCD4 level to the number of CD4⁺ cells decreased and the ratio of the sCD8 level to the number of CD8⁺ cells increased in the first and second trimesters of pregnancy, but these ratios were within the normal range from the third trimester of pregnancy to 10–12 months postpartum. CONCLUSIONS: Decreases in serum sCD4 concentration and in the ratio sCD4/CD4⁺ cells, and an increase in the ratio sCD8/CD8⁺ cells may be important factors in the immunological changes that occur during pregnancy.     

Moderate prenatal ethanol exposure in the rat promotes kidney cell apoptosis,nephron deficits,and sex-specific kidney dysfunction in adult offspring

Alcohol during pregnancy can impair fetal development and result in offspring with neurodevelopmental deficits. Less is known about how low to moderate alcohol exposure can affect other organs, such as the kidney. Here, the effects of moderate ethanol exposure throughout pregnancy on kidney development were examined using a rat model. Rats were fed a liquid diet containing 6% ethanol (vol/vol) or control (0% ethanol) throughout pregnancy. Kidneys were collected at embryonic day (E) 20 or postnatal day (PN) 30 and total glomerular (nephron) number determined using unbiased stereology. Kidney function was examined in offspring at 8 and 19 months. At E20, fetuses exposed to ethanol had fewer nephrons with increased apoptosis. Alcohol exposure caused kidney dysregulation of pro- (Bax) and anti- (Bcl-2) apoptotic factors, and reduced expression of the cell proliferation marker, Ki67. Prenatal alcohol decreased expression of Gdnf and Tgfb1, important regulators of branching morphogenesis, in male fetuses. At PN30, kidney volume and nephron number were lower in offspring exposed to prenatal alcohol. Urine flow and osmolality were normal in offspring exposed to alcohol however sodium excretion tended to be lower in females prenatally exposed to alcohol. Findings suggest exposure to moderate levels of alcohol during pregnancy results in impaired kidney development and leads to a permanent nephron deficit. Although the impact on adult kidney function was relatively minor, these data highlight that even at moderate levels, alcohol consumption during pregnancy can have deleterious long-term outcomes and should be avoided.     

Adverse effects of melatonin on rat pups of Wistar–Kyoto dams receiving melatonin supplementation during pregnancy

This report documents an incidental finding during a study investigating the effects of melatonin supplementation on the development of blood pressure in SHR. Administration of 10 mg/kg/day of melatonin in drinking water during pregnancy to Wistar-Kyoto (WKY) dams caused a loss of more than 50% of the pups by the age of three weeks and 95% by the age of 6 weeks. There was no maternal morbidity or mortality in the two strains or death of any of the SHR pups. No obvious physical defects were present but mean body weight was lower in the surviving WKY rats when compared to that of melatonin supplemented SHR or non-supplemented WKY pups. The reason for the high mortality in WKY pups is uncertain and appears to be strain if not batch specific. There is a need for caution in its use, particularly during pregnancy, and clearly necessitates more detailed studies.     

Maternal diesel inhalation increases airway hyperreactivity in ozone-exposed offspring

Air pollutant exposure is linked with childhood asthma incidence and exacerbations, and maternal exposure to airborne pollutants during pregnancy increases airway hyperreactivity (AHR) in offspring. To determine if exposure to diesel exhaust (DE) during pregnancy worsened postnatal ozone-induced AHR, timed pregnant C57BL/6 mice were exposed to DE (0.5 or 2.0 mg/m(3)) 4 hours daily from Gestation Day 9-17, or received twice-weekly oropharyngeal aspirations of the collected DE particles (DEPs). Placentas and fetal lungs were harvested on Gestation Day 18 for cytokine analysis. In other litters, pups born to dams exposed to air or DE, or to dams treated with aspirated diesel particles, were exposed to filtered air or 1 ppm ozone beginning the day after birth, for 3 hours per day, 3 days per week for 4 weeks. Additional pups were monitored after a 4-week recovery period. Diesel inhalation or aspiration during pregnancy increased levels of placental and fetal lung cytokines. There were no significant effects on airway leukocytes, but prenatal diesel augmented ozone-induced elevations of bronchoalveolar lavage cytokines at 4 weeks. Mice born to the high-concentration diesel-exposed dams had worse ozone-induced AHR, which persisted in the 4-week recovery animals. Prenatal diesel exposure combined with postnatal ozone exposure also worsened secondary alveolar crest development. We conclude that maternal inhalation of DE in pregnancy provokes a fetal inflammatory response that, combined with postnatal ozone exposure, impairs alveolar development, and causes a more severe and long-lasting AHR to ozone exposure.     

Liaison between natural killer cells and dendritic cells in human gestation

A successful pregnancy relies on immunological adaptations that allow the fetus to grow and develop in the uterus, despite being recognized by maternal immune cells. Among several immunocompetent cell types present within the human maternal/fetal interface, DC-SIGN~ dendritic cells （DCs） and CD56＋ natural killer （NK） cells are of major importance for early pregnancy maintenance, not only generating maternal immunological tolerance but also regulating stromal cell differentiation. Previous reports show the presence of NK-DC cell conjugates in first trimester human decidua, suggesting that these cells may play a role in the modulation of the local immune response within the uterus. While effective immunity is necessary to protect the mother from harmful pathogens, some form of tolerance must be activated to avoid an immune response against fetal antigens. This review article discusses current evidence concerning the functions of DC and NK cells in pregnancy and their liaison in human decidua.     

Prenatal stress eliminates differential maternal attention to male offspring in Norway rats

Maternal licking behavior was observed in 20 Long-Evans rat dams on two consecutive days. Stimulus pups were male and female foster pups from dams that were either housed with 5 adult males during the last trimester of pregnancy (stressed) or housed alone (unstressed). Unstressed male pups received significantly more maternal licking than their female siblings, but prenatally stressed males and females received similar levels of maternal licking, comparable to that directed to unstressed females. In a second study, urine collected from prenatally stressed male pups elicited significantly less investigation from dams in a choice test than urine from age-matched unstressed males. It is concluded that the chemosignals which stimulate dams normally to provide more maternal attention to male than female neonates are deficient in prenatally stressed males. The results raise the possibility that differential maternal care may mediate some effects of prenatal stress on behavioral development in males.     

Selective breeding for infant vocal response: A role for postnatal maternal effects?

N:NIH rats were selectively bred on the basis of high or low rates of ultrasonic vocalization (USV) response to isolation at 10 days of age (Brunelli et al., 1997: Dev Psychobiol 31:255-265). To examine the possibility of postnatal maternal effects in the generational transmission of divergent traits, pups were cross-fostered shortly after birth between dams of the two lines (Low- and High-USV). Controls were fostered to dams of the same line (in-fostered). Additional (population) control data were obtained from the entire 13th generation of the selectively bred lines. USV rates of cross-fostered pups in each line were not significantly different from rates of in-fostered pups of the same line. High USV line pups cross-fostered to Low USV line dams weighed significantly less than in-fostered pups, on the day of testing. The results provide no evidence for a postnatal maternal contribution to the USV phenotype. Prenatal and/or perinatal maternal effects have not been ruled out.