A Humanized Diet Profile May Facilitate Colonization and Immune Stimulation in Human Microbiota-Colonized Mice.

BACKGROUND: In spite of the importance of the use of gnotobiotic mice for human fecal transfer, colonization efficiency and immune stimulation after human microbiota inoculation in mice are poorly studied compared to mouse microbiota inoculation. We tested the colonization efficiency and immune responses in mice bred for one additional generation after inoculating the parent generation with either a human (HM) or a mouse microbiota (MM). Furthermore, we tested if colonization efficiency and immune stimulation could be improved in HM-colonized mice by dietary approaches: if these were fed a diet closer to the human diet either in its sources of animal fat and protein [the "animal source" (AS) diet] or in its proportions of macronutrients from the normal sources of a mouse diet [the "human profile" (HP) diet]. RESULTS: Although significantly lower in mice with a human microbiota (30-40% vs. 61-70%) the colonization efficiency was significantly higher in HM mice fed the HP diet (40%), and in MM mice fed AS (70%). The microbiota of mice fed HP was comparable to the microbiota of mice fed a standard rodent chow, while the microbiota of mice fed the animal source diet (AS) clustered separately. Mice inoculated with mouse fecal matter had significantly more CD4+ T cells and Cd4 expression and significantly fewer regulatory T cells (Tregs) and FoxP3 expression than human microbiota inoculated mice, but cell proportions differences were mostly apparent between mice fed the AS diet. Mice fed the HP diet had significantly higher expression of Cd8a. CONCLUSION: It is concluded that a diet with a humanized profile could support the establishment of a human microbiota in mice, which will, however, still elicit a lower colonization efficiency compared to mice inoculated with a mouse microbiota.

Winter cholecalciferol supplementation at 55°N has little effect on markers of innate immune defense in healthy children aged 4-8 years: a secondary analysis from a randomized controlled trial.

PURPOSE: We explored the effect of winter cholecalciferol (vitamin D3) supplementation on innate immune markers in healthy Danish children (55°N). METHODS: In the double-blind, placebo-controlled trial, ODIN Junior, 119 healthy, white, 4-8 year-olds were randomized to 0 (placebo), 10 or 20 µg/day of vitamin D3 for 20 weeks (October-March). Cheek mucosal swabs, blood samples, and questionnaires on acute respiratory infections the previous month were collected at baseline and endpoint. Innate immune markers were measured as secondary outcomes including in vivo oral mucosal gene expression of calprotectin (S100A9), lipocalin-2 (LCN2), beta-defensin-4 (DEFB4), interleukin-8 (IL-8), viperin (RSAD2), and the cathelicidin-antimicrobial-peptide (CAMP); ex vivo whole-blood lipopolysaccharide (LPS)-induced cathelicidin, IL-8, and IL-6; and plasma cathelicidin, together with serum 25-hydroxyvitamin D [25(OH)D]. RESULTS: Serum 25(OH)D was 56.7 ± 12.3 nmol/L at baseline and 31.1 ± 7.5, 61.8 ± 10.6, and 75.8 ± 11.5 nmol/L at endpoint after placebo, 10 and 20 µg/day of vitamin D3 (P < 0.0001), respectively. A decreased oral mucosal S100A9 expression with placebo [- 18 (95% CI - 1; - 32)%] was marginally avoided with 20 µg/day [6 (- 13; 28)%] (P = 0.06). Likewise, a decreased LPS-induced IL-8 with placebo [- 438 (95% CI - 693; - 184) ng/L] was marginally avoided with 20 µg/day [- 109 (- 374; 157) ng/L] (P = 0.07). All other immune markers and respiratory infection episodes were unaffected by vitamin D3 supplementation (all P > 0.11). CONCLUSIONS: Winter vitamin D3 supplementation of 10 µg/day did not affect innate immune markers, whereas 20 µg/day tended to maintain the capacity to produce a few markers in healthy children.

In Vivo and Ex Vivo Inflammatory Markers of Common Metabolic Phenotypes in Humans.

BACKGROUND: Low-grade systemic inflammation (LGSI) is often characterized by elevated levels of interleukin (IL)6, tumor necrosis factor (TNF)α, and C-reactive protein (CRP). Other serum proteins, ex vivo-stimulated cytokine production, and leukocyte count have, however, also been suggested LGSI-markers, but their associations with the metabolic syndrome (MS) are less clear. We aimed to evaluate mutual relationships between in vivo and ex vivo inflammatory markers and their association with MS and its subcomponents. METHODS: A cross-sectional study of 118 overweight adults with one or several features of MS. Inflammatory markers included fasting serum levels of IL6, TNFα, CRP, and pentraxin-3 (PTX3), IL1-receptors, leukocytes, and whole-blood ex vivo-produced IL1ß, IL6, TNFα, and IL8 after lipopolysaccharide stimulation. RESULTS: All classical serum LGSI-markers correlated with each other, and IL6 and CRP were also correlated with leukocyte count. Ex vivo-produced cytokines were intercorrelated and correlated with leukocyte count, but did not correlate with the serum immune markers. MS score, body mass index, and glycated hemoglobin (HbA1c) were associated with 8%-16% higher inflammatory score per standard deviation increment (P = 0.030, 0.001, and 0.034, respectively), primarily driven by higher serum IL6. Serum PTX3 was only significantly associated with high-density lipoprotein cholesterol (1.19[1.04; 1.37], P = 0.013). HbA1c was inversely associated with surface expression of IL1R1 on monocytes and IL1R2 on granulocytes (P < 0.01) and with a 3%-9% lower ex vivo production of cytokines when adjusting for leukocyte count, as were plasma triacylglycerol (9%-10% lower IL1ß and IL6). Leukocyte count was most consistently associated with MS and its subcomponents, although not with HbA1. CONCLUSIONS: The classical fasting serum markers of LGSI and leukocyte counts associated best with measures of MS-associated LGSI, whereas ex vivo cytokine production was only associated with prevailing glycemia and dyslipidemia. Taken together, this indicates that the relationship between in vivo and ex vivo inflammatory markers is complex and may depend on the MS phenotype.

Postnatal Hematopoiesis and Gut Microbiota in NOD Mice Deviate from C57BL/6 Mice.

Neonatal studies in different mouse strains reveal that early life colonization affects the development of adaptive immunity in mice. The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes, but neonatal studies of NOD mice are lacking. We hypothesized that NOD mice deviate from another much used mouse strain, C57BL/6, with respect to postnatal microbiota and/or hematopoiesis and compared this in newborn mice of dams housed under the same conditions. A distinct bacteria profile rich in staphylococci was found at postnatal days (PND) 1-4 in NOD mice. Furthermore, a distinct splenic cell profile high in a granulocytic phenotype was evident in the neonatal NOD mice whereas neonatal C57BL/6 mice showed a profile rich in monocytes. Neonatal expression of Reg3g and Muc2 in the gut was deviating in NOD mice and coincided with fewer bacteria attaching to the Mucosal surface in NOD compared to C57BL/6 mice.

Astragalus root and elderberry fruit extracts enhance the IFN-ß stimulatory effects of Lactobacillus acidophilus in murine-derived dendritic cells.

Many foods and food components boost the immune system, but little data are available regarding the mechanisms by which they do. Bacterial strains have disparate effects in stimulating the immune system. In dendritic cells, the gram-negative bacteria Escherichia coli upregulates proinflammatory cytokines, whereas gram-positive Lactobacillus acidophilus induces a robust interferon (IFN)-ß response. The immune-modulating effects of astragalus root and elderberry fruit extracts were examined in bone marrow-derived murine dendritic cells that were stimulated with L. acidophilus or E. coli. IFN-ß and other cytokines were measured by ELISA and RT-PCR. Endocytosis of fluorescence-labeled dextran and L. acidophilus in the presence of elderberry fruit or astragalus root extract was evaluated in dendritic cells. Our results show that both extracts enhanced L. acidophilus-induced IFN-ß production and slightly decreased the proinflammatory response to E. coli. The enhanced IFN-ß production was associated with upregulation of toll-like receptor 3 and to a varying degree, the cytokines IL-12, IL-6, IL-1ß and TNF-α. Both extracts increased endocytosis in immature dendritic cells, and only slightly influenced the viability of the cells. In conclusion, astragalus root and elderberry fruit extracts increase the IFN-ß inducing activity of L. acidophilus in dendritic cells, suggesting that they may exert antiviral and immune-enhancing activity.

Low-dose perinatal exposure to di(2-ethylhexyl) phthalate induces anti-androgenic effects in male rats.

Perinatal di(2-ethylhexyl) phthalate (DEHP) exposure was examined in time-mated Wistar rats gavaged from gestation day 7 to postnatal day 16 with doses from 3 to 900 mg/kg-d. These doses covered the whole dose-response curve for the demasculinizing effects of DEHP including low-dose effects. At a relatively low dose of 10 mg/kg-d, DEHP caused adverse anti-androgenic effects on male rat development as male anogenital distance was decreased, the incidence of nipple retention was increased, weight of levator ani/bulbocavernosus muscles and prostate was reduced and mild external genitalia dysgenesis was observed. Higher doses of DEHP induced histopathological effects on the testes, reduced testis weight, and expression of androgen-regulated genes in the prostate. The results provide new evidence of low-dose effects of DEHP and are consistent with the EU NOAEL of 5 mg/kg for DEHP. Our results also indicate a reason for concern about human exposure to DEHP.

Dysgenesis and histological changes of genitals and perturbations of gene expression in male rats after in utero exposure to antiandrogen mixtures.

We investigated the ability of a mixture of three androgen receptor antagonists to induce disruption of male sexual differentiation after perinatal exposure. The aim was to assess whether the joint effects of vinclozolin, flutamide, and procymidone can be predicted based on dose-response data of the individual chemicals. Chemicals were administered orally to pregnant Wistar rats from gestational day 7 to postnatal day 16. Changes in reproductive organ weights and of androgen-regulated gene expression in prostates from male rat pups were chosen as end points for extensive dose-response studies. With all end points, the joint effects of the three antiandrogens were dose additive. Histological evaluations showed that dysgenesis and hypoplasia of prostates, seminal vesicles, and epididymis were seen with the highest mixture doses. No changes were observed in any single-compound low-dose group for these lesions, nor were there histopathological changes in the testes. Pronounced dysgenesis of external genitals was observed with all doses of the mixture, and severe dysgenesis was seen with a mixture for which the individual compounds caused no effects. A combination of doses of each chemical that on its own did not produce significant reductions in the weights of seminal vesicles and PBP C3 expression induced a marked mixture effect. Thus, antiandrogens cause additive effects on end points of various molecular complexities such as alterations at the morphological and the molecular level. Exposure to antiandrogens, which appears to exert only small effects when judged on a chemical-by-chemical basis, may induce marked responses in concert with, possibly unrecognized, similarly acting chemicals.

Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat.

OBJECTIVE: The aim of this study was to assess whether the joint effects of three androgen receptor antagonists (vinclozolin, flutamide, procymidone) on male sexual differentiation after in utero and postnatal exposures can be predicted based on dose-response data of the individual chemicals. METHODS: Test chemicals and mixtures were administered by gavage to time-mated nulliparous, young adult Wistar rats from gestational day 7 to the day before expected birth, and from postnatal days 1-16. Changes in anogenital distance (AGD) and nipple retention (NR) in male offspring rats were chosen as end points for extensive dose-response studies. Vinclozolin, flutamide, and procymidone were combined at a mixture ratio proportional to their individual potencies for causing retention of six nipples in male offspring. RESULTS: With AGD as the end point, the joint effects of the three anti-androgens were essentially dose additive. The observed responses for NR were slightly higher than those expected on the basis of dose addition. A combination of doses of each chemical, which on its own did not produce statistically significant AGD alterations, induced half-maximal mixture effects. At individual doses associated with only modest effects on NR, the mixture induced NR approaching female values in the males. CONCLUSIONS: Effects of a mixture of similarly acting anti-androgens can be predicted fairly accurately on the basis of the potency of the individual mixture components by using the dose addition concept. Exposure to anti-androgens, which individually appears to exert only small effects, may induce marked responses in concert with, possibly unrecognized, similarly acting chemicals.

Mechanisms underlying the anti-androgenic effects of diethylhexyl phthalate in fetal rat testis.

Diethylhexyl phthalate (DEHP) is widely used as a plasticizer in consumer products and is known to disturb the development of the male reproductive system in rats. The mechanisms by which DEHP exerts these effects are not yet fully elucidated, though some of the effects are related to reduced fetal testosterone production. The present study investigated the effects of four different doses of DEHP on fetal testicular histopathology, testosterone production and expression of proteins and genes involved in steroid synthesis in fetal testes. Pregnant Wistar rats were gavaged from GD 7 to 21 with vehicle, 10, 30, 100 or 300 mg/kg bw/day of DEHP. In male fetuses examined at GD 21, testicular testosterone production ex vivo and testicular testosterone levels were reduced significantly at the highest dose. Histopathological effects on gonocytes were observed at 100 and 300 mg/kg bw/day, whereas Leydig cell effects were mainly seen at 300 mg/kg bw/day. Quantitative RT-PCR revealed reduced testicular mRNA expression of the steroidogenesis related factors SR-B1, StAR, PBR and P450scc. Additionally, we observed reduced mRNA expression of the nuclear receptor SF-1, which regulates certain steps in steroid synthesis, and reduced expression of the cryptorchidism-associated Insl-3. Immunohistochemistry showed clear reductions of StAR, PBR, P450scc and PPARgamma protein levels in fetal Leydig cells, indicating that DEHP affects regulation of certain steps in cholesterol transport and steroid synthesis. The suppression of testosterone levels observed in phthalate-exposed fetal rats was likely caused by the low expression of these receptors and enzymes involved in steroidogenesis. It is conceivable that the observed effects of DEHP on the expression of nuclear receptors SF-1 and PPARgamma are involved in the downregulation of steroidogenic factors and testosterone levels and thereby underlie the disturbed development of the male reproductive system.

Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz.

The fungicide prochloraz has got multiple mechanisms of action that may influence the demasculinizing and reproductive toxic effects of the compound. In the present study, Wistar rats were dosed perinatally with prochloraz (50 and 150 mg/kg/day) from gestational day (GD) 7 to postnatal day (PND) 16. Caesarian sections were performed on selected dams at GD 21, while others were allowed to give birth to pups that were followed until PND 16. Prochloraz caused mild dysgenesis of the male external genitalia as well as reduced anogenital distance and retention of nipples in male pups. An increased anogenital distance indicated virilization of female pups. Effects on steroidogenesis in male fetuses became evident as decreased testicular and plasma levels of testosterone and increased levels of progesterone. Ex vivo synthesis of both steroid hormones was qualitatively similarly affected by prochloraz. Immunohistochemistry of fetal testes showed increased expression of 17alpha-hydroxylase/17,20-lyase (P450c17) and a reduction in 17beta-hydroxysteroid dehydrogenase (type 10) expression, whereas no changes in expression of genes involved in testicular steroidogenesis were observed. Increased expression of P450c17 mRNA was observed in fetal male adrenals, and the androgen-regulated genes ornithine decarboxylase, prostatic binding protein C3 as well as insulin-like growth factor I mRNA were reduced in ventral prostates PND 16. These results indicate that reduced activity of P450c17 may be a primary cause of the disrupted fetal steroidogenesis and that an altered androgen metabolism may play a role as well. In vitro studies on human adrenocortical carcinoma cells supported the findings in vivo as reduced testosterone and increased progesterone levels were observed. Overall, these results together indicate that prochloraz acts directly on the fetal testis to inhibit steroidogenesis and that this effect is exhibited at protein, and not at genomic, level.

Antiandrogenic effects in short-term in vivo studies of the fungicide fenarimol.

The fungicide fenarimol has estrogenic and antiandrogenic activity and inhibits aromatase activity in vitro. We tested, whether fenarimol had antiandrogenic effects in vivo. In a Hershberger assay, fenarimol given orally to castrated testosterone-treated male rats caused markedly reduced weights of ventral prostate, seminal vesicles, musc. levator ani/bulbocavernosus, and bulbourethral glands. Qualitatively similar, but weaker, effects were also evident in intact fenarimol-exposed young adult males, except that prostates were not significantly affected. Changes in androgen-regulated gene expression were determined by real-time RT-PCR in ventral prostates and fenarimol caused a pronounced decrease of prostate binding protein C3 (PBP C3), ornithin decarboxylase (ODC), and insulin-like-growth factor 1 (IGF-1) mRNA levels. The antiandogenic drug flutamide, included as a positive control, caused down-regulation of PBP C3 mRNA and up-regulation of TRPM-2 mRNA levels. Serum T4 levels were reduced after fenarimol treatment and a tendency towards increased LH levels was seen. However, no effects on testosterone levels or testosterone production ex vivo could be revealed. Taken together these results indicate that fenarimol acts as an antiandrogen in vivo having effects qualitatively comparable to those of flutamide on organ level, whereas differential effects on gene expression were observed. In an additional Hershberger test, the effects of fenarimol were compared to those of estradiol benzoate, prochloraz and the aromatase inhibitor fadrozole. The data indicate a similar mode of action of fenarimol and prochloraz in the males, whereas no indications were found that the estrogenic or aromatase inhibitory properties had important impact on the effects observed in the males. Thus, it is suggested that fenarimol mediates its antiandrogenic effects at least partly via antagonism of androgen receptors.