Developmental Immunotoxicology of Di-(2-Ethylhexyl)phthalate (DEHP): Age-Based Assessment in the Female Rat.

Di-(2-ethylhexyl)phthalate (DEHP) helps provide flexibility to plastic products including food containers and medical IV devices. Age-based sensitivity to DEHP-induced immunotoxicity was compared in female CD strain rats following administration for 16 consecutive days in utero vs. adult exposure. Pregnant and non-pregnant rats received 0, 37.5, 75, 150, or 300 mg/kg/day of DEHP in soybean oil daily by oral gavage. In pregnant animals, this corresponded to days 6-21 of gestation. In exposed offspring, anogenital distance (AGD) was measured at one and three weeks of age. For a subset of offspring, immune assessment was conducted at 5 weeks of age following KLH immunization at 3 and 4 weeks. KLH immunizations occurred in remaining offspring at 11 and 12 weeks with immune assessment at 13 weeks. Nonpregnant adults were immunized concurrently with adult offspring and underwent immune assessment 13 weeks post-DEHP exposure. Litter size, sex ratio, and 1-week body weights were unaffected by treatment. However, AGD was altered at both ages assessed. Three-week (but not 5-week) body weights were elevated in exposed offspring. Immune organ weights, thymus histology, antibody levels (IgG and IgE), DTH reaction to KLH, total and differential leukocyte counts, ex vivo cytokine production (IL-2, -4, -10, -12, interferon-gamma), as well as TNF-alpha and nitric oxide production by macrophages were not altered by treatment at any age. Flow cytometry analysis of splenocytes showed no differences during the juvenile assessment. In conclusion, in utero exposure of female rats to DEHP alters some developmental parameters (i.e., AGD) but has no persistent effect on the immune system based on the assessment parameters. Adult female rats also had no detectable immune alteration following similar exposures.

The effects of dietary vitamin E and selenium deficiencies on plasma thyroid and thymic hormone concentrations in the chicken.

Beginning at hatching, male Cornell K strain single comb white leghorn chickens were fed a basal diet, with or without vitamin E (100 IU/kg) and/or selenium (Se, 0.2 ppm). After 3 weeks of treatment, animals fed either the Se-deficient or basal diet had significantly reduced plasma Se-dependent glutathione peroxidase activities when compared to those fed a vitamin E and Se-supplemented diet. Similarly, animals fed the vitamin E-deficient or basal diet had significantly reduced plasma alpha-tocopherol levels. The effect of these treatments on plasma concentrations of thyroid hormones (T(3)/T(4)), growth hormone (GH), and thymic hormone (thymulin) was determined using radioimmunoassay and ELISA. A deficiency in Se, but not in vitamin E, resulted in an increase in plasma T(4) concentrations while plasma T(3) concentrations were decreased. Plasma GH levels showed some fluctuation as a result of the dietary treatments but there was no significant correlation between plasma GH levels and any of the other variables. A significant decrease in plasma thymulin levels was observed in Se-deficient birds compared to those receiving adequate Se in the diet. A vitamin E deficiency had no measurable effect on plasma thymulin levels. From these studies, we conclude that plasma thymulin concentrations directly correlate with plasma T(3) concentrations which are negatively affected by a Se deficiency.

Developmental immunotoxicity of cyclosporin-A in rats: age-associated differential effects.

Cyclosporin-A (CYP-A) is a widely used immunosuppressive drug. Yet, information on the long-term impact of embryonic exposure is relatively scarce. The effects of CYP-A on reproductive and immunologic parameters in CD strain female offspring exposed in utero at doses of 0, 0.2, 2, 10, or 20 mg/kg/day (from gestational day 6 to 21) were compared against identically dosed CD adult rats. Embryotoxicity was seen at the two highest doses. CYP-A was acutely immunotoxic in adults (tested at 20 mg/kg/day dose) but with minimum long-term effects. In contrast, the offspring experienced relatively persistent alterations. CYP-A exposure increased ano-genital distance in the neonates. In the 5-week-old offspring, the delayed type hypersensitivity (DTH) response and splenic B cell number (determined by flow cytometry) were both decreased at the 2 mg dose level. IL-4 level was reduced and blood monocytes were increased at both exposure doses. All other parameters were unchanged. In the adult offspring (13-week-old), no difference was seen in either the DTH response or B cell ratios, but IL-4 level was increased at 2 mg/kg/day, and anti-KLH IgG titer decreased at both doses. In exposed non-pregnant adults, changes were minimal following a 13-week recovery period. Blood neutrophils were increased at all doses of the drug and flow cytometry data suggested some perturbation in CD4(+)CD8(+) cells, macrophages, and B-cells. All other parameters were unchanged. In conclusion, the adult rodent immune system largely recovers from CYP-A exposure given sufficient time. However, embryonic exposure appears to produce a series of immune perturbations including functional impairment during postnatal maturation.

Developmental immunotoxicity of dexamethasone: comparison of fetal versus adult exposures.

Dexamethasone-21 phosphate was administered (s.c.) to pregnant CD rats at days 6-21 of gestation (0, 0.0625, 0.125, 0.25, and 0.5 mg/kg/day) with identical exposure of non-pregnant adult females. Some reproductive (anogenital distance) and growth (body weight) measures of pups were altered. In the juvenile (5 weeks), the delayed type hypersensitivity response to KLH was significantly reduced at all doses examined and this pattern continued into adulthood (13 weeks). In contrast, the DTH response of adults exposed to DEX was unaltered even at the highest dose. Few DEX-induced changes were seen in offspring or adult blood parameters or in splenocytes analyzed for cell surface makers (by flow cytometry). The thymus of both exposed pups (both ages) and adults showed a marked reduction in the medulla/lobe area beginning with the 0.125 mg/kg/day DEX exposure level. Macrophage production of TNF and NO was only marginally affected as was splenocyte production of IL-4 and IFN-gamma. In contrast, pups assessed as juveniles were significantly depressed in splenic IL-2 and IL-10 production. DEX exposure altered serum antibody levels across age groups with an increase of KLH-specific IgG (beginning with the 0.0125 mg/kg/day dose) while total IgE was reduced. These results suggest that while DEX exposure produces some common alterations following in utero versus adult exposure, fetal exposure (even at the lowest doses tested) produces marked and persistent functional loss (DTH) not evident in exposed adults. Furthermore, there was no apparent advantage in delaying immune assessment until the offspring reached adulthood.

Functional interactions between Hsp90 and the co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae.

Hsp90 complexes contain a class of co-chaperones characterized by a tetratricopeptide repeat (TPR) domain, which mediates binding to a carboxyl-terminal EEVD region in Hsp90. Among Hsp90 TPR co-chaperones in Saccharomyces cerevisiae, only Cns1 is essential. The amino terminus of Cns1, which harbors the TPR domain, is sufficient for viability when overexpressed. In a screen for temperature-sensitive alleles of CNS1, we identified mutations resulting in substitutions of conserved residues in the TPR domain. Mutations in CNS1 disrupt in vitro and in vivo interaction with Hsp90 and reduce Hsp90 function, indicating that Cns1 is a bona fide co-chaperone. Genetic interactions between CNS1 and another Hsp90 co-chaperone, CPR7, suggest that the two co-chaperones share an essential role in the cell. Although both the TPR and the isomerase domains of the cyclophilin Cpr7 are required for viability of cns1 mutant cells, this requirement does not depend on the catalytic function of the isomerase domain. Instead, hydrophilic residues on the surface of this domain appear to be important for the common Cns1.Cpr7 function. Although both co-chaperones interact with Hsp90 primarily through the carboxyl terminus (EEVD), Cns1 and Cpr7 are mostly found in complexes distinct from Hsp90. EEVD is required for normal growth in cns1 mutant cells, demonstrating for the first time in vivo requirement for this conserved region of Hsp90. Overall, our findings reveal a considerable degree of complexity in the interactions not only between Hsp90 and its co-chaperones, but also among the co-chaperones themselves.

In vivo thymulin treatments enhance avian lung natural killer cell cytotoxicity in response to infectious bronchitis virus.

Previous work has shown that in vitro thymulin treatments have the ability to enhance natural killer (NK) cell cytotoxicity. The purpose of the experiments presented here was to examine the in vivo effects of thymulin on avian NK cell activity in response to a viral infection. Five and a half-week-old K-strain chickens infected with avian infectious bronchitis virus (IBV) served as the model for these experiments. Daily thymulin injections began at varying time points prior to or post-infection. The controls received daily injections of the ZnCl(2)-containing carboxymethyl-cellulose (CMC) diluent. A 51Cr-cytolytic release assay was used to determine the activity of the NK cells harvested via lung lavage from the respiratory tracts of infected chickens. The results of these experiments showed that in vivo thymulin treatments enhance NK cytotoxicity. The greatest enhancement of NK cytotoxicity was observed at 10 days post-infection in those chickens that began receiving thymulin after infection. These results suggest that thymulin may not only have a role in enhancing immunosurveillance but also in enhancing the response of the innate immune system following infection. Dose-response experiments found that the 50 ng/100 g body weight (Bwt) dose significantly depressed the cytolytic activity of the NK cells in comparison to either the 10 ng/100 g Bwt dose or the control.

The effects of thymulin on macrophage responsiveness to interferon-gamma.

The effect of in vivo and in vitro thymulin treatments on macrophage responsiveness to interferon-gamma was evaluated in chickens. Seven-week-old chickens were treated with 0, 1, 10, or 100ng thymulin per 100g body weight. Abdominal exudate cells (AEC), a source of macrophages, were harvested and cultured in the presence of graded levels of recombinant chicken interferon-gamma (ChIFN-gamma). Responsiveness to ChIFN-gamma was determined by measuring the induction of nitric oxide production. One and 2-day thymulin treatment at 10 and 100ng per 100g body weight doses significantly increased responsiveness to ChIFN-gamma while 1ng per 100g body weight had no effect. Other experiments compared the effect of thymulin treatments in Cornell K strain chickens, having normal serum thymulin levels with sex-linked dwarf (SLD) chickens which are deficient in serum thymulin. The dose of thymulin treatment required to significantly increase responsiveness to ChIFN-gamma differed between strains. Finally, the effect of direct in vitro thymulin treatments on macrophage responsiveness to ChIFN-gamma was evaluated. There were no significant increases in responsiveness to ChIFN-gamma between treatment groups within the macrophage cell line, HD-11, when cultured in the presence of 0-200pg thymulin/ml. These data suggest that the effect of thymulin on AEC responsiveness to ChIFN-gamma is indirectly mediated.