Adipose tissue is responsive to estrogen and expresses both estrogen receptor alpha and beta. To test the hypothesis that the estrogenic soy isoflavone genistein can have effects on adipose tissue, juvenile or adult C57/BL6 mice were ovariectomized and given daily injections of vehicle, 17beta-estradiol (5 microg/kg.d) or genistein (8-200 mg/kg.d) sc for 21-28 d. To test effects of dietary genistein, 25- to 27-d-old mice were fed diets containing 0-1500 parts per million (ppm) genistein for 12 d. Mice were killed and fat pads weighed. Parametrial fat pads were used for morphometric and Northern analysis. Genistein injections decreased adipose weight and adipocyte circumference at higher doses; effects in adult and juvenile mice were similar. Genistein decreased lipoprotein lipase mRNA, which may be a critical aspect of its adipose effects. Juveniles fed 500-1500 ppm dietary genistein had dose-responsive decreases in fat pad weights of 37-57%, compared with controls; 300 ppm genistein did not cause decreases. Genistein doses of 300, 500, 1000, and 1500 ppm produced serum genistein concentrations of 1.02 +/- 0.14 microM, 1.79 +/- 0.32 microM, 2.55 +/- 0.18 microM, and 3.81 +/- 0.39 microM, respectively. These results indicate dietary genistein at 500-1500 ppm produces antilipogenic effects in mice at serum levels that humans are realistically exposed to.

Use of soy-based infant formulas and soy͞isoflavone supplements has aroused concern because of potential estrogenic effects of the soy isoflavones genistein and daidzein. Here we show that s.c. genistein injections in ovariectomized adult mice produced doseresponsive decreases in thymic weight of up to 80%. Genistein's thymic effects occurred through both estrogen receptor (ER) and non-ER-mediated mechanisms, as the genistein effects on thymus were only partially blocked by the ER antagonist ICI 182,780. Genistein decreased thymocyte numbers up to 86% and doubled apoptosis, indicating that the mechanism of the genistein effect on loss of thymocytes is caused in part by increased apoptosis. Genistein injection caused decreases in relative percentages of thymic CD4 ؉ CD8 ؊ and double-positive CD4 ؉ CD8 ؉ thymocytes, providing evidence that genistein may affect early thymocyte maturation and the maturation of the CD4 ؉ CD8 ؊ helper T cell lineage. Decreases in the relative percentages of CD4 ؉ CD8 ؊ thymocytes were accompanied by decreases in relative percentages of splenic CD4 ؉ CD8 ؊ cells and a systemic lymphocytopenia. In addition, genistein produced suppression of humoral immunity. Genistein injected at 8 mg͞kg per day produced serum genistein levels comparable to those reported in soy-fed human infants, and this dose caused significant thymic and immune changes in mice. Critically, dietary genistein at concentrations that produced serum genistein levels substantially less than those in soy-fed infants produced marked thymic atrophy. These results raise the possibility that serum genistein concentrations found in soy-fed infants may be capable of producing thymic and immune abnormalities, as suggested by previous reports of immune impairments in soy-fed human infants. S oy-based formula for human infant nutrition is widely used, with approximately 25% of formula-fed infants in the U.S. consuming soy-based formula (1). This number represents 15% of all infants in the U.S., or about 750,000 infants͞year (1, 2). Infants consuming soy formula are exposed to high levels of genistein and daidzein, estrogenic isoflavones present in soybeans and soy products. On average, infants fed soy-based formula consume 6.0-11.9 mg of isoflavones͞kg per day (3, 4), an order of magnitude greater than adults eating high-soy diets. Total plasma levels of isoflavones and genistein in soy-fed infants range from 2.0 to 6.6 and 1.5 to 4.4 mol͞liter, respectively (3), 10-fold greater than levels in Japanese adults whose diets have historically included soy, and 200-fold greater than plasma levels in infants fed cow's milk formula or human breast milk (3, 5). Levels of the free genistein aglycone as a percent of total genistein are higher in rat pups than in adults (6), but have not been measured in human infants. If a similar phenomenon occurs in humans, relative levels of the biologically active free aglycones may be even greater than the 10-fold difference documented in total (free ϩ conjugated) serum isoflavone and genistein levels in...

The cyanobacterial lectin scytovirin (SVN) binds with high affinity to mannose-rich oligosaccharides on the envelope glycoprotein (GP) of a number of viruses, blocking entry into target cells. In this study, we assessed the ability of SVN to bind to the envelope GP of Zaire Ebola virus (ZEBOV) and inhibit its replication. SVN interacted specifically with the protein’s mucin-rich domain. In cell culture, it inhibited ZEBOV replication with a 50% virus-inhibitory concentration (EC50) of 50 nM, and was also active against the Angola strain of the related Marburg virus (MARV), with a similar EC50. Injected subcutaneously in mice, SVN reached a peak plasma level of 100 nm in 45 minutes, but was cleared within 4 hours. When ZEBOV-infected mice were given 30 mg/kg/day of SVN by subcutaneous injection every 6 hours, beginning the day before virus challenge, 9 of 10 animals survived the infection, while all infected, untreated mice died. When treatment was begun one hour or one day after challenge, 70-90% of mice survived. Quantitation of infectious virus and viral RNA in samples of serum, liver and spleen collected on days 2 and 5 postinfection showed a trend toward lower titers in treated than control mice, with a significant decrease in liver titers on day 2. Our findings provide further evidence of the potential of natural lectins as therapeutic agents for viral infections.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) continues to be a threat to human health in the Middle East. Development of countermeasures is ongoing; however, an animal model that faithfully recapitulates human disease has yet to be defined. A recent study indicated that inoculation of common marmosets resulted in inconsistent lethality. Based on these data we sought to compare two isolates of MERS-CoV. We followed disease progression in common marmosets after intratracheal exposure with: MERS-CoV-EMC/2012, MERS-CoV-Jordan-n3/2012, media, or inactivated virus. Our data suggest that common marmosets developed a mild to moderate non-lethal respiratory disease, which was quantifiable by computed tomography (CT), with limited other clinical signs. Based on CT data, clinical data, and virological data, MERS-CoV inoculation of common marmosets results in mild to moderate clinical signs of disease that are likely due to manipulations of the marmoset rather than as a result of robust viral replication.

The soy phytoestrogen, genistein, induces thymic atrophy when administered to ovariectomized mice by injection or in the diet. Injected genistein also causes decreased humoral immunity, but the effects of genistein on cellmediated immunity have not been addressed. Here we examined effects of injected and dietary genistein on cell-mediated immune responses. Female C57BL/6 mice (25-to 27-days-old) were ovariectomized, then placed on phytoestrogen-free feed 5 days later. Seven days after ovariectomy, they were given daily subcutaneous injections of either dimethylsulfoxide (DMSO) or genistein (8, 20, 80 mg/kg) for 28 days; some mice were given 80 mg/ kg genistein plus the anti-estrogen ICI 182,780 (5 mg/ kg/week). Cell-mediated immune response was tested by analyzing the delayed-type hypersensitivity (DTH) response to a hapten, 4-hydroxy-3-nitrophenyl acetyl succinimide (NP-O-SU), at the end of treatment. Reversibility of the effects of genistein was tested by measuring the DTH response in mice that were given genistein (20 or 80 mg/kg) for 28 days, then allowed to recover for 28 days. To determine if dietary genistein could affect cell-mediated immunity, mice ovariectomized as above were fed genistein at 0, 1000 or 1500 parts per million (ppm) for 28 days. There was a 46-67% decrease in the DTH response in the footpads of mice injected with 8-80 mg/kg genistein compared with controls (P<0·05 vs control for all treatment groups); these effects were reversible. On histopathological examination of the feet, there was decreased cell infiltration in genistein-treated animals compared with controls, and the numbers of CD4 + and CD8 + T cells in popliteal lymph nodes were reduced. The effects of genistein are mediated through both estrogen receptor (ER) and non-ER pathways, as the anti-estrogen ICI 182,780 only partially blocked the effects of genistein on the DTH response. Dietary genistein (1000 or 1500 ppm) decreased cellmediated immunity while producing serum genistein concentrations in the physiological range for humans under certain nutritional conditions. Further work is needed to determine if dietary genistein and phytoestrogen exposure can produce effects on cell-mediated immunity in humans or other animals under various nutritional conditions.

Simian Hemorrhagic Fever Virus (SHFV) has caused sporadic outbreaks of hemorrhagic fevers in macaques at primate research facilities. SHFV is a BSL-2 pathogen that has not been linked to human disease; as such, investigation of SHFV pathogenesis in non-human primates (NHPs) could serve as a model for hemorrhagic fever viruses such as Ebola, Marburg, and Lassa viruses. Here we describe the pathogenesis of SHFV in rhesus macaques inoculated with doses ranging from 50 PFU to 500,000 PFU. Disease severity was independent of dose with an overall mortality rate of 64% with signs of hemorrhagic fever and multiple organ system involvement. Analyses comparing survivors and non-survivors were performed to identify factors associated with survival revealing differences in the kinetics of viremia, immunosuppression, and regulation of hemostasis. Notable similarities between the pathogenesis of SHFV in NHPs and hemorrhagic fever viruses in humans suggest that SHFV may serve as a suitable model of BSL-4 pathogens.

Estrogen regulates thymic development and immune function. Despite the critical role of estrogens in inducing thymic involution and modulating immune responses, the mechanism of this effect is unclear. Similarly, humans and animals are exposed to increasing amounts of the estrogenic soy isoflavone genistein in the diet, but whether genistein can induce immune changes has not been definitively established. We reported previously that genistein induces thymic atrophy in mice, and decreases both humoral and cell-mediated immunity. These thymic effects of genistein occur via estrogen receptor (ER)-mediated and non-ER-mediated pathways. Genistein injections produced the most pronounced effects, but dietary administration to mice that produced serum genistein concentrations similar to those reported in human infants consuming soy formula also had demonstrable effects. Microarray analysis of the effects of estradiol and genistein on neonatal thymus indicated that estradiol affected genes involved in transcription, apoptosis, cell cycle, and thymic development and function; genistein had similar effects on many estradiol target genes, but also had unique actions not replicated by estradiol. Despite extensive work showing inhibitory effects of genistein on immunity, other rodent studies reported that genistein or other phytoestrogens stimulate various aspects of immune function. Although the present data strongly indicate that genistein can regulate immune function, possibly at physiologic concentrations, further work is required to definitively establish overall thymic and immune effects of genistein and soy, which may vary with age, species, and specific end point. J. Nutr. 136: 704-708, 2006. KEY WORDS: immunity phytoestrogens isoflavones soy thymusThe possible effect on humans and animals of environmental chemicals that either mimic or inhibit the actions of endogenous hormones has become an area of great concern for both the scientific community and general public. Phytoestrogens represent a large group of estrogens derived from plant sources. These chemicals are ubiquitous in our food supply and these compounds may affect a variety of biological processes that have relevance for human and animal health. The immune system is a major estrogen target, and recent data indicated that one group of phytoestrogens, the isoflavones, can produce immune effects, although these studies have been inconsistent or even conflicting at times. Here, we summarize current knowledge related to the potential effects of isoflavones on the immune system.Human exposure to estrogenic soy isoflavones. Soybeans and foods containing soy have significant levels of isoflavones such as genistein and daidzein. Genistein, daidzein, and the daidzein metabolite equol are structurally similar to 17b-estradiol (E2) 4 and have estrogenic effects. Soy phytoestrogens bind to both the classical estrogen receptor, estrogen receptora, (ERa, and the more recently discovered second form of ER, ERb). In contrast to the most potent endogenous estrogen, ...

Estrogen receptors (ERs) are expressed in the thymus of both males and females, but their role in thymic development and function is unclear. To determine whether ERalpha plays a role in thymic function of either males or females, we compared thymuses of male and female wild-type (WT) and ERalpha knockout (alphaERKO) mice from birth to adulthood. Although thymic size was similar in both male and female WT and alphaERKO mice at birth (d 0), by postnatal d 5 and at all subsequent ages, both male and female alphaERKO mice had significant (30-55%) reductions in thymic weight. Morphometric analysis revealed a reduction in thymic medullary areas in adult alphaERKO mice compared with age-matched WT controls that paralleled thymic involution. There were changes in relative percentages of CD4+ and CD4+CD8+ T-cells, and large decreases (70-80%) in overall absolute numbers of CD4+ and CD4+CD8+ T-cells. Serum corticosterone and testosterone levels were not different in either neonatal or adult male WT or alphaERKO mice, and serum levels of 17beta-estradiol (E2) were similar in neonatal WT and alphaERKO males, indicating that increases in these thymolytic hormones are not responsible for the decreased thymic weight in alphaERKO males. Additionally, delayed-type hypersensitivity was significantly increased in male alphaERKO mice compared with WT mice. In summary, ERalpha deficiency does not inhibit initial differentiation or fetal thymic development, but the absence of ERalpha results in marked decreases in thymic size in both sexes during the postnatal period. These results are the first direct demonstration that the E2/ERalpha signaling system is necessary for maintenance of normal postnatal function of the female thymus gland. The similar results obtained in males demonstrate a role for the E2/ERalpha signaling system in the male thymus and emphasize that estrogens play a more critical role in the male than previously realized.