Sarahann Bradley

Region-specific growth effects in the developing rat prostate following fetal exposure to estrogenic ultraviolet filters.

Background and objectives Exposure to environmental endocrine disruptors is a potential risk factor for humans. Many of these chemicals have been shown to exhibit disruption of normal cellular and developmental processes in animal models. Ultraviolet (UV) filters used as sunscreens in cosmetics have previously been shown to exhibit estrogenic activity in in vitro and in vivo assays. We examined the effects of two UV filters, 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor (3-BC), in the developing prostate of the fetal rat. Methods Pregnant Long Evans rats were fed diets containing doses of 4-MBC and 3-BC that resulted in average daily intakes of these chemicals corresponding to the lowest observed adverse effects level (LOAEL) and the no observed adverse effects level (NOAEL) doses in prior developmental toxicity studies. Using digital photographs of serial sections from postnatal day 1 animals, we identified, contoured, and aligned the epithelial ducts from specific regions of the developing prostate, plus the accessory sex glands and calculated the total volume for each region from three-dimensional, surface-rendered models. Results Fetal exposure to 4-MBC (7.0 mg/kg body weight/day) resulted in a significant increase (p < 0.05) in tissue volume in the prostate and accessory sex glands. Treated males exhibited a 62% increase in the number of ducts in the caudal dorsal prostate. Increased distal branching morphogenesis appears to be a consequence of exposure in the ventral region, resulting in a 106% increase in ductal volume. Conclusions 4-MBC exposure during development of the male reproductive accessory sex glands exhibited classical growth effects associated with estrogenic endocrine disruptors. The different regional responses suggest that the two developmental processes of ductal outgrowth and branching morphogenesis are affected independently by exposure to the environmental chemicals.

Characterization of a Cytolytic Strain of Equine Infectious Anemia Virus

ABSTRACT A novel strain of equine infectious anemia virus (EIAV) called vMA-1c that rapidly and specifically killed infected equine fibroblasts (ED cells) but not other infectible cell lines was established. This strain was generated from an avirulent, noncytopathic strain of EIAV, MA-1. Studies with this new cytolytic strain of virus have permitted us to define viral parameters associated with EIAV-induced cell killing and begin to explore the mechanism. vMA-1c infection resulted in induction of rapid cell death, enhanced fusogenic activity, and increased rates of spread in equine fibroblasts compared to other strains of EIAV. The highly cytolytic nature of vMA-1c suggested that this strain might be superinfecting equine fibroblasts. Receptor interference studies demonstrated that prior infection of equine fibroblasts with EIAV did not alter the ability of vMA-1c to infect and kill these cells. In similar studies in a canine fibroblast cell line, receptor interference did occur. vMA-1c infection of equine fibroblasts was also associated with large quantities of unintegrated viral DNA, a well-established hallmark of retroviral superinfection. Cloning of the vMA-1c genome identified nucleotide changes that would result in at least one amino acid change in all viral proteins. A chimeric infectious molecular clone containing the vMA-1c tat, S2, and env open reading frames recapitulated most of the characteristics of vMA-1c, including superinfection, fibroblast killing, and fusogenic activity. In summary, in vitro selection for a strain of EIAV that rapidly killed cells resulted in the generation of a virus that was able to superinfect these cells, presumably by the use of a novel mechanism of cell entry. This phenotype mapped to the 3′ half of the genome.

Evolution of the equine infectious anemia virus long terminal repeat during the alteration of cell tropism.

ABSTRACT Equine infectious anemia virus (EIAV) is a lentivirus with in vivo cell tropism primarily for tissue macrophages; however, in vitro the virus can be adapted to fibroblasts and other cell types. Tropism adaptation is associated with both envelope and long terminal repeat (LTR) changes, and findings strongly suggest that these regions of the genome influence cell tropism and virulence. Furthermore, high levels of genetic variation have been well documented in both of these genomic regions. However, specific EIAV nucleotide or amino acid changes that are responsible for cell tropism changes have not been identified. A study was undertaken with the highly virulent, macrophage-tropic strain of virus EIAVwyo to identify LTR changes associated with alterations in cell tropism. We found the stepwise generation of a new transcription factor binding motif within the enhancer that was associated with adaptation of EIAV to endothelial cells and fibroblasts. An LTR that contained the new motif had enhanced transcriptional activity in fibroblasts, whereas the new site did not alter LTR activity in a macrophage cell line. This finding supports a previous prediction that selection for new LTR genetic variants may be a consequence of cell-specific selective pressures. Additional investigations of the EIAVwyo LTR were performed in vivo to determine if LTR evolution could be detected over the course of a 3-year infection. Consistent with previous in vivo findings, we observed no changes in the enhancer region of the LTR over that time period, indicating that the EIAVwyo LTR was evolutionarily stable in vivo.

Regulation of transplacental virus infection by developmental and immunological factors: studies with lactate dehydrogenase-elevating virus.

Placental and fetal infections with lactate dehydrogenase-elevating virus (LDV) were determined by virus titration, indirect fluorescence antibody (IFA), and in situ hybridization with cDNA probes. Experiments were designed to determine the effects of gestational age, timing of maternal LDV infection, and immunological (antibody and cytokine) factors on mouse placental and fetal LDV infection. Virus infection of the placenta was detected at high levels (almost all placentas infected) within 24 h post-maternal infection (p.m.i.), whereas fetal LDV infection was detected only at a low level by 24 h p.m.i. The percentage of fetuses becoming LDV infected progressively increased between 24 and 72 h p.m.i. When fetal infection was studied at 72 h p.m.i., earlier gestational ages (9-11 days) were associated with fetal resistance to infection, whereas between 12.5 and 15 days of gestation, virus infection was detected in 50-71% of fetuses. Maternal treatment with interferon-gamma (IFN-gamma) or anti-LDV monoclonal antibodies was associated with reduced rates of fetal, but not placental, LDV infection. These results demonstrate that both developmental and immunological factors are important in the regulation of transplacental LDV infection.

Cytokine regulation of lactate dehydrogenase-elevating virus : inhibition of viral replication by interferon-γ

The mechanisms which regulate the replication of lactate dehydrogenase-elevating virus (LDV), a persistent murine model virus which infects macrophages, are unclear. For this study, the effects of murine recombinant interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on LDV replication were examined. LDV permissiveness was reduced in macrophages obtained from uninfected mice treated with IFN-gamma prior to cell harvest and in vitro LDV infection. Virus inhibition by IFN-gamma was also observed when neonatal LDV-infected mice were injected with this cytokine prior to macrophage harvest and analysis of LDV replication-positive cells. Persistently LDV-infected mice demonstrated an increase in viremia levels following treatment with TNF-alpha. Neither IFN-gamma nor TNF-alpha had any direct in vitro effect on LDV replication in cultured macrophages, suggesting that the actions of these cytokines required secondary or accessory in vivo events. These results provide evidence for cytokine-mediated regulation of LDV infection and support a role for the immune system in the LDV-host relationship.

Atypical fetal prostate development is associated with ipsilateral hypoplasia of the wolffian ducts in the ACI rat.

For over a half century, the ACI (August × Copenhagen) rat has been a primary model for studying renal agenesis and ipsilateral hypoplasia (IHP) of the Wolffian‐derived structures (WDS). Because the ACI rat is also used as a model for prostate research, it is important to examine the relationship of IHP and urogenital sinus (UGS) development. The prostate is dependent on androgens for proper growth and differentiation. Alteration in androgen production and/or delivery to the UGS has the potential to perturbate normal development. In this study, we investigate whether the ipsilateral loss of the WDS is associated with altered prostate development. Digital images of serial‐sectioned fetal ACI rat UGS were used to create three‐dimensional (3‐D) surface‐rendered models of the developing prostate, seminal vesicle, vas deferens, and utricle on gestational day 21. The number and volume of prostate ducts developing from the UGS were calculated from the 3‐D model data. Animals exhibiting IHP had a significant decrease in total fetal prostate volume (40%; P < 0.005) with significant regional specific differences when compared with normal male ACI rats. Anatomical and histological differences in the utricle, abnormal histology of the ipsilateral testes, and a truncation of the ipsilateral Wolffian ductal mesenchyme were also seen in the animals with IHP. Additional research is needed to further understand the mechanisms and consequences of IHP on prostate growth and development. Alterations to normal prenatal development of the male accessory sex organs can have important consequences for the growth and morphology of the adult gland. Anat Rec, 2010.

Determination of the viremia threshold for dental cross-infection in a mouse model.

An animal model of dental virus transmission was developed using the lactate dehydrogenase-elevating virus (LDV) of mice to study cross infection. Mouse-to-mouse cross-infection was carried out by scaling the teeth of LDV-infected donor mice with dental instruments, immediately prior to using the contaminated instruments on the teeth of recipient indicator mice. The level of donor viremia was found to correlate with the rate of virus cross-infection, with a viremia threshold level of 10(7.5) ID50/ml observed for dental cross-infection. The blood volume transferred during dental cross-infection was approximately 10(-4) to 10(-5) ml, demonstrating the inefficiency of virus cross-infection, since deposition of about 1000 virions on dental instruments was associated with the threshold limit. Virus transferred during dental cross-infection rapidly entered the blood circulation, showing that dental cross-infection was not dependent on an oral infection. The results from these model studies predict the general inefficiency of dental instrument virus cross-infection, and a further reduced likelihood of dental cross-infection with appropriately cleaned instruments.