Enhanced phenotype of calcipotriol-induced atopic dermatitis in filaggrin-deficient mice.

Impaired function of filaggrin (FLG) is a major predisposing factor for atopic dermatitis (AD). Several studies on FLG-deficient (Flg-/- ) mice have indicated an essential role for FLG in the skin barrier and the development of AD, but none of the studies have described the characteristics on Flg-/- mice with calcipotriol (CPT)-induced atopic dermatitis, which restricts the comprehensive understanding of functions of FLG. The present study sought to generate Flg-/- mice and applied CPT to produce AD-like dermatitis for in vivo analysis of the FLG functions. CPT was applied on the skin of Flg-/- mice to establish the AD-like dermatitis mouse model. The lesion inflammation was evaluated by gross ear thickness, histopathology, immunofluorescence, and cytokine production. Also, mucopolysaccharide polysulfate (MPS) and ceramide were used to observe the therapeutic function in this model. The results showed that the inflammation of CPT-induced dermatitis in Flg-/- mice was more severer than that of wild-type (WT) mice, as evident by the increased level of gross appearance, ear thickness, inflammatory cell infiltration (mast cells and CD3+ T cells), and inflammatory cytokine expression (interleukin (IL)-4, IL-6, IL-13, and thymic stromal lymphopoietin (TSLP)). The emollients MPS and ceramide partially restored the epidermal function and alleviated the skin inflammation in Flg-/- mice with CPT-induced AD-like dermatitis. The current study demonstrated that skin barrier protein FLG is critical in the pathogenesis of AD. Also, the AD mouse model induced by CPT in Flg-/- mice could be utilized to search for drug targets in AD.

Trogocytosis of peptide-MHC class II complexes from dendritic cells confers antigen-presenting ability on basophils.

Th2 immunity plays important roles in both protective and allergic responses. Nevertheless, the nature of antigen-presenting cells responsible for Th2 cell differentiation remains ill-defined compared with the nature of the cells responsible for Th1 and Th17 cell differentiation. Basophils have attracted attention as a producer of Th2-inducing cytokine IL-4, whereas their MHC class II (MHC-II) expression and function as antigen-presenting cells are matters of considerable controversy. Here we revisited the MHC-II expression on basophils and explored its functional relevance in Th2 cell differentiation. Basophils generated in vitro from bone marrow cells in culture with IL-3 plus GM-CSF displayed MHC-II on the cell surface, whereas those generated in culture with IL-3 alone did not. Of note, these MHC-II-expressing basophils showed little or no transcription of the corresponding MHC-II gene. The GM-CSF addition to culture expanded dendritic cells (DCs) other than basophils. Coculture of basophils and DCs revealed that basophils acquired peptide-MHC-II complexes from DCs via cell contact-dependent trogocytosis. The acquired complexes, together with CD86, enabled basophils to stimulate peptide-specific T cells, leading to their proliferation and IL-4 production, indicating that basophils can function as antigen-presenting cells for Th2 cell differentiation. Transfer of MHC-II from DCs to basophils was also detected in draining lymph nodes of mice with atopic dermatitis-like skin inflammation. Thus, the present study defined the mechanism by which basophils display MHC-II on the cell surface and appears to reconcile some discrepancies observed in previous studies.

Mast Cells Limit Ear Swelling Independently of the Chymase Mouse Mast Cell Protease 4 in an MC903-Induced Atopic Dermatitis-Like Mouse Model.

Atopic dermatitis (AD) is a complex, often lifelong allergic disease with severe pruritus affecting around 10% of both humans and dogs. To investigate the role of mast cells (MCs) and MC-specific proteases on the immunopathogenesis of AD, a vitamin D3-analog (MC903) was used to induce clinical AD-like symptoms in c-kit-dependent MC-deficient Wsh-/- and the MC protease-deficient mMCP-4-/-, mMCP-6-/-, and CPA3-/- mouse strains. MC903-treatment on the ear lobe increased clinical scores and ear-thickening, along with increased MC and granulocyte infiltration and activity, as well as increased levels of interleukin 33 (IL-33) locally and thymic stromal lymphopoietin (TSLP) both locally and systemically. The MC-deficient Wsh-/- mice showed significantly increased clinical score and ear thickening albeit having lower ear tissue levels of IL-33 and TSLP as well as lower serum levels of TSLP as compared to the WT mice. In contrast, although having significantly increased IL-33 ear tissue levels the chymase-deficient mMCP-4-/- mice showed similar clinical score, ear thickening, and TSLP levels in ear tissue and serum as the WT mice, whereas mMCP-6 and CPA3 -deficient mice showed a slightly reduced ear thickening and granulocyte infiltration. Our results suggest that MCs promote and control the level of MC903-induced AD-like inflammation.

Role of local versus systemic vitamin D receptors in vascular calcification.

OBJECTIVE: Calcitriol and various analogs are commonly used to suppress secondary hyperparathyroidism in chronic kidney disease but may also exacerbate vascular calcification. Although this could be because of increased intestinal calcium and phosphate absorption, direct effects through vitamin D receptors (VDRs) on vascular smooth muscle have also been proposed. APPROACH AND RESULTS: The role of these receptors was investigated by examining gene regulation in rat aortas treated with calcitriol ex vivo and in vivo and by transplanting aortas from VDR-null (VDR(-/-)) mice into wild-type mice before induction of uremia and treatment with calcitriol. In cultured rat aortas, calcitriol increased the expression of mRNA for CYP24A1 but not mRNA for any bone-related or calcification-related genes. Gene expression in aortas in vivo was not altered by doses of calcitriol that promote calcification. Calcitriol markedly increased aortic calcification in uremic mice and this did not differ between VDR(-/-) aortic allografts and VDR(+/+) recipient aortas. CONCLUSIONS: Calcitriol promotes vascular calcification through a systemic action rather than through a direct vascular action.

A role of 1,25(OH)2D3 supplementation in rats with nonalcoholic steatohepatitis induced by choline-deficient diet.

BACKGROUND AND AIMS: It has been reported that 1,25(OH)2D3 (1,25-VD3) ameliorates the progression of nonalcoholic steatohepatitis (NASH). However, it is unclear whether 1,25-VD3 plays a role in NASH induced by a choline-deficient (CD) diet. In this study, we investigated the roles of 1,25-VD3 in the development and progression of NASH in rats induced by a CD diet. METHODS AND RESULTS: Wistar rats with NASH induced by a CD diet were subjected to intraperitoneal injections of 1, 5, or 10 µg/kg of 1,25-VD3 twice weekly for 12 weeks. The administration of 1,25-VD3 decreased free fatty acids (FFAs), triglycerides (TGs), thiobarbituric acid-reactive substances (TBARS), the number of apoptotic cells, and the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in the liver, and it improved liver histology, but it did not change the total antioxidant capacity (TAOC) in the liver. Interestingly, the level of CK18-M30 was decreased in the liver of model animals. Treatment with 1,25-VD3 may restrain the downregulation of CK18-M30 in the liver and its release into the bloodstream, thus decreasing the level of serum CK18-M30. 1,25-VD3 supplementation elevated the serum level of 25(OH)D3 and the expression of VDR in the liver. The dose-effect relationship of 1,25-VD3 indicated that 1,25-VD3 slows down the development and progression of NASH induced by a CD diet, but higher doses of 1,25-VD3 may lead to adverse effects. CONCLUSION: The results suggest the presence of both antagonistic and adverse dose-dependent effects of the long-term supplementation of 1,25-VD3 on NASH induced by a CD diet.

1,25-Dihydroxyvitamin D is not responsible for toxicity caused by vitamin D or 25-hydroxyvitamin D.

Vitamin D intoxication was produced with oral doses of either vitamin D3 or 25-hydroxyvitamin D3 in CYP27B1 -/- (1α-hydroxylase knockout) and wild-type mice. These compounds were equally toxic in wild-type and the mutant mice. Since the null mutant mice are unable to produce 1,25-dihydroxyvitamin D, it is clear 1,25-dihydroxyvitamin D is not responsible for vitamin D intoxication. On the other hand, 25-hydroxyvitamin D rises to levels of 400-700 ng/ml or 1000-1750 nM in the serum of both groups of mice. Toxicity was evidenced by severe hypercalcemia and weight loss. Measurement of 1,25-dihydroxyvitamin D3 in serum confirmed its absence from serum of the CYP27B1 -/- mice given 25-hydroxyvitamin D3. Since high concentrations of 25-hydroxyvitamin D can bind the vitamin D receptor and can induce transcription, 25-hydroxyvitamin D is likely responsible for toxicity of vitamin D excess.

ACC1-expressing pathogenic T helper 2 cell populations facilitate lung and skin inflammation in mice.

T cells possess distinguishing effector functions and drive inflammatory disorders. We have previously identified IL-5-producing Th2 cells as the pathogenic population predominantly involved in the pathology of allergic inflammation. However, the cell-intrinsic signaling pathways that control the pathogenic Th2 cell function are still unclear. We herein report the high expression of acetyl-CoA carboxylase 1 (ACC1) in the pathogenic CD4+ T cell population in the lung and skin. The genetic deletion of CD4+ T cell-intrinsic ACC1 dampened eosinophilic and basophilic inflammation in the lung and skin by constraining IL-5 or IL-3 production. Mechanistically, ACC1-dependent fatty acid biosynthesis induces the pathogenic cytokine production of CD4+ T cells via metabolic reprogramming and the availability of acetyl-CoA for epigenetic regulation. We thus identified a distinct phenotype of the pathogenic T cell population in the lung and skin, and ACC1 was shown to be an essential regulator controlling the pathogenic function of these populations to promote type 2 inflammation.

9-Cis retinoic acid reduces 1alpha,25-dihydroxycholecalciferol-induced renal calcification by altering vitamin K-dependent gamma-carboxylation of matrix gamma-carboxyglutamic acid protein in A/J male mice.

Matrix gamma-carboxyglutamic acid protein (MGP), a vitamin K-dependent protein, is involved in regulation of tissue calcification. We previously reported that 9-cis retinoic acid (RA) mitigates 1alpha,25-dihydroxycholecalciferol [1,25(OH)(2)D3]-induced renal calcification in a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung cancer A/J male mouse model. This raised the question if the mechanism(s) underlying this calcification involves vitamin K. We assessed expression and vitamin K dependent gamma-carboxylation of MGP and vitamin K concentrations [phylloquinone (PK), as well as its conversion product, menaquinone-4 (MK-4)] in tissues obtained from NNK-injected A/J male mice fed 1,25(OH)(2)D3 (2.5 microg/kg diet; D group) +/- RA (15 mg/kg diet) for 20 wk. Renal calcification was only observed in the D group (2/10; 20% of the group). Renal MGP mRNA and uncarboxylated MGP (ucMGP) increased in response to D (P < 0.05) but not in response to RA or RA + D. In contrast, gamma-carboxylated MGP increased to 2.2-fold of the control in response to D+RA (P < 0.05) but not in response to RA or D alone. Although all diets contained equal amounts of PK, the kidney MK-4 concentration was higher in the D group (P < 0.05) and lower in the RA group (P < 0.05) compared with the RA+D or control groups. Renal PK concentrations were lower in the RA and RA+D groups than in the control and D groups (P < 0.05). These data suggest that 9-cis RA mitigated 1,25(OH)(2)D3-induced renal calcification by modifying the 1,25(OH)(2)D3-induced increase in ucMGP. The mechanisms by which 9-cis RA and 1,25(OH)(2)D3 alter vitamin K concentrations warrant further investigation.

Inter-species differences in sensitivity to the calcemic activity of the novel 1,25-dihydroxyvitamin D3 analog BXL746.

The activities of 1,25-dihydroxyvitamin D3 and its synthetic analogs have been extensively studied in humans as well as in preclinical species, and recent data show potential therapeutic utility in cancer treatment. However, their chronic administration leads to changes in blood mineral ion concentrations, and at high doses can result in symptomatic hypercalcemia limiting therapeutic applicability. To overcome this issue, a therapeutic approach based on administration of intermittent, high doses of 1,25(OH)2D3 has been explored in prostate cancer patients. Despite these and other investigations, limited information is available on the effects of acute systemic administration of high doses of 1,25(OH)2D3 or its analogs. Here, we report a comparative analysis of the pro-calcemic effects of the novel 1,25(OH)2D3 analog BXL746 following acute or chronic administration in animals and humans. While chronic administration of BXL746 to rats, dogs and humans leads to similar modulation of calcemia in these species, single dose administration reveals >1000-fold higher sensitivity of dog compared to rat and human in induction of hypercalcemia and consequent systemic toxicity. Our data indicate that the rat is a more relevant species than the dog for the prediction of human results when acute administration of a 1,25(OH)2D3 analog is envisaged.

Acute hepatologic and nephrologic effects of calcitriol in Syrian golden hamster (Mesocricetus auratus).

Although vitamin D is included in the group of fat-soluble vitamins, it must be considered as a prohormone. Its active forms, including calcitriol, have pleiotropic effects and play an important role in the regulation of cell proliferation, differentiation and apoptosis, as well as in hormone secretion, and they demonstrate anti-cancer properties. Since calcitriol delivery can be beneficial for the organism, and Syrian golden hamsters represent a unique experimental model, we decided to investigate its toxicity in this species. In this study, we injected calcitriol intraperitoneally at doses 0 (control), 0.180±0.009 µg/kg and 0.717±0.032 µg/kg. Animal behavior was observed for 72 hrs after injection, and afterwards blood, liver and kidneys were collected for post-mortem examination, electron microscopy, and hematology analyses. The highest dose of calcitriol induced a change in animal behavior from calm to aggressive, and the liver surface showed morphological signs of damage. Following injection of calcitriol, ultrastructural changes were also observed in the liver and kidneys, e.g. vacuolization and increased number of mitochondria. There was also a trend for increased serum levels of aspartate aminotransferase (AST), but not of alanine aminotransferase (ALT) or GGTP (gamma-glutamyl transpeptidase). There was no change in Ca, Mg and P levels, as well as in blood morphology between experimental and control groups. These results indicate that calcitriol at 0.717, but not at 0.180 µg/kg, may induce acute damage to the liver and kidneys, without inducing calcemia. We propose that the hepatotoxic effect of calcitriol in hamster constitutes the primary cause of behavioral changes.

Dose-dependent effects of vitamin 1,25(OH)2D3 on oxidative stress and apoptosis.

Background The purpose of this study is to examine the dose-dependent effects of vitamin 1,25(OH)2D3 on apoptosis and oxidative stress. Methods In this study, 50 male Balb/c mice were used as control and experiment groups. The mice were divided into 5 groups each consisting of 10 mice. Calcitriol was intraperitoneally administered as low dose, medium dose, medium-high dose and high dose vitamin D groups (at 0.5, 1, 5 and 10 µg/kg, respectively), for three times a week during 14 days. At the end of the study, annexin V was measured by enzyme-linked immunosorbent assay method, and total antioxidant capacity and total oxidant status values were measured by colorimetric method in serum. Hematoxylin eosin staining was performed in liver tissues and periodic acid schiff staining was performed in kidney tissues. Results While comparing the results of medium-high dose (5 µg/kg) and high dose (10 µg/kg) vitamin D administration to that of the control group, it was observed that serum antioxidant status and annexin V levels decreased and glomerular mesenchial matrix ratio increased in kidney (p<0.05). In addition to these findings, in the group receiving high dose vitamin D (10 µg/kg), it was observed that the damage to the liver increased together with the the oxidative stress index values (p<0.05). Conclusions As a result, this study was the first in the literature to report that use of high-dose vitamin D (10 µg/kg) results in oxidant effect, rather than being an antioxidant, and causes severe histopathological toxicity in the liver and kidney.

Reversibility of calcitriol-induced medial artery calcification in rats with intact renal function.

UNLABELLED: VC is an important clinical entity; however, very little information is available on its resolution. Induction and regression of calcitriol-induced VC was studied in 47 rats. After calcitriol withdrawal, there was a relatively rapid regression of VC mediated by an active cellular process. INTRODUCTION: Vascular calcifications (VCs) represent an important risk factor for cardiovascular death. Although VCs are prevalent in relevant diseases (e.g., chronic kidney disease, osteoporosis, diabetes), the reversibility of extraskeletal calcifications is an unresolved issue. This study was conducted to investigate (1) the in vivo effect of calcitriol on VC and (2) whether calcitriol-induced VC would regress after suppression of calcitriol treatment. MATERIALS AND METHODS: The calcifying effect of calcitriol was studied in four groups of rats (n = 8) that received calcitriol (1 mug/kg, IP) for 2, 4, 6, and 8 days. The reversibility of VC was studied in three additional groups (n = 5) treated with 1 mug/kg of calcitriol for 8 days that were subsequently killed 1, 2, and 9 weeks after the last calcitriol dose. Aortic VC was assessed by histology and by quantification of aortic calcium and phosphorus content. The aortic wall was studied by histology and immunohistochemistry. Statistical analysis was performed by ANOVA and t-tests. RESULTS: Calcitriol administration resulted in a time-dependent induction of VC, with aortic calcium and phosphorus being significantly increased at 6 and 8 days. Treatment with calcitriol for 8 days resulted in massive medial calcification of the aorta with a 10- to 30-fold increase in the aortic Ca and P content. After suppressing calcitriol administration, a progressive decrease in von Kossa staining and aortic Ca (from 32.8 +/- 2.5 to 9.3 +/- 1.8 mg/g of tissue, p < 0.001) and P (from 11.9 +/- 1.2 to 2.7 +/- 1.8 mg/g of tissue, p = 0.001) content was evidenced. Histology of the aortic wall showed monocytes adhered to the aortic endothelium and macrophages involved in the reabsorption of calcium deposits. CONCLUSIONS: Our results show that calcitriol treatment induces time-dependent VC. After calcitriol withdrawal, VC regress rapidly with aortic calcium and phosphorus decreasing by 75% in the course of 9 weeks. An active cellular process seems to be involved in regression of VC.

1,25-Dihydroxyvitamin D3 and all-trans-retinoic acid sensitize breast cancer cells to chemotherapy-induced cell death.

We investigated the capacity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans-retinoic acid (ATRA) to sensitize three breast cancer cell lines to the cell killing effects of paclitaxel (Taxol) and Adriamycin, two chemotherapeutic agents commonly used in the treatment of breast cancer. In tissue culture colony assays, 1,25(OH)2D3 and ATRA were synergistic in inhibiting the clonogenicity of MCF-7 and T-47D cells that expressed estrogen receptor; vitamin D receptor; retinoic acid receptors (RARs) alpha, beta, and gamma; and retinoid X receptors alpha, beta, and gamma but were not additive in MDA-MB-231 cells that lacked expression of estrogen receptor, RARalpha, and RARbeta. The hormones used individually or in combination induced up to 40-50% cell death by a trypan blue exclusion assay in a dose-dependent manner up to concentrations of 10(-7) M in MCF-7 and T-47D cells, more modestly in MDA-MB-231 cells, and not at all in MCF-10 and MCF-12 nontransformed mammary epithelial cells. Pretreating the cancer cell lines with 1,25(OH)2D3 and ATRA individually or in combination for 3 days prior to a 1-h incubation with paclitaxel or Adriamycin decreased the ED50 for inhibition of colony formation or for cell death by trypan blue by up to 2 logs for paclitaxel and up to 1 log for Adriamycin in all three cell lines but had no effect on chemotherapy-induced MCF-12 cell death. The effects of the hormones were synergistic with those of the chemotherapy agents in all of the breast cancer cell lines, generally at the higher concentrations. Cell death took place by apoptosis. To determine one potential reason for the greater potentiation of the effects of paclitaxel than those of Adriamycin, we determined the effects of preincubation of MCF-7 cells on paclitaxel-induced phosphorylation of Bcl-2. Pretreatment of MCF-7 cells with either 1,25(OH)2D3 or ATRA increased the phosphorylation of Bcl-2 by variable concentrations of paclitaxel. These data suggest that pretreatment of breast cancer with 1,25(OH)2D3 or ATRA lowers the threshold for cell killing by chemotherapy agents and may provide a novel treatment option for this disease.

Induction of macrophage differentiation of human normal and leukemic myeloid stem cells by 1,25-dihydroxyvitamin D3 and its fluorinated analogues.

This study shows for the first time that 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] and two fluorinated analogues of 1,25-(OH)2D3 [24,24-F2-1,25-(OH)2D3 and 26, 26, 26, 27, 27, 27-F6-1,25-(OH)2D3] induce macrophage differentiation of human normal and leukemic myeloid stem cells. The addition of either 1,25-(OH)2D3 or one of the two fluorinated analogues of 1,25-(OH)2D3 at concentrations as low as 10(-9) M to culture plates containing normal human marrow cells stimulated myeloid stem cells to preferentially differentiate to colonies of monocytes and macrophages. Over 80% of the normal human myeloid colonies were composed of only monocytes and macrophages in culture plates containing 10(-7) M 1,25-(OH)2D3 or one of the fluorinated analogues. In contrast, control plates not containing 1,25-(OH)2D3 had less than 35% macrophage colonies. Likewise, 1,25-(OH)2D3 and the two vitamin D analogues induced macrophage differentiation of leukemic colony-forming cells taken from patients. In plates containing 10(-7) M 1,25-(OH)2D3 or one of the analogues at 10(-8) M, 80% of chronic myelogenous leukemia and approximately 50% of acute nonlymphocytic leukemia colony-forming cells differentiated to macrophage-like cells. In contrast, control plates had about 30 and 20% macrophage colonies in cultures from chronic myelogenous leukemia and acute nonlymphocytic leukemia patients, respectively. Our findings suggest that 1,25-(OH)2D3 may play a role in hematopoiesis and that the compound or a related analogue may possibly have a therapeutic role in some leukemias.

Inhibition of human cancer cell growth by 1,25-dihydroxyvitamin D3 metabolites.

Specific high-affinity 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors, which can undergo hormone-dependent activation and nuclear localization, have been demonstrated in a wide variety of established human cancer cell lines and surgically obtained human cancer tissues. 1,25-(OH)2D3 has been reported by some workers to stimulate cancer cell replication at low "physiological" concentrations and by ourselves and others to inhibit at higher concentrations. We report here that 1,25-(OH)2D3 had a biphasic effect on the replication of two distinct human cancer cell lines, i.e., the breast cancer T-47D and the malignant melanoma MM96, an effect analogous to that of estrogens on the breast cancer cell line MCF-7. These inhibitory effects were accompanied by marked morphological changes. Furthermore, two known metabolites of 1,25-(OH)2D3, i.e., 1,24,25-trihydroxyvitamin D3 and 1,25,26-trihydroxyvitamin D3, which compete for binding to the 1,25-(OH)2D3 receptor, did not stimulate but were almost equipotent with 1,25-(OH)2D3 in inhibiting the replication of both cell lines. The stimulatory but not the inhibitory effect of 1,25-(OH)2D3 was abolished by cortisone. These 1,25-dihydroxyvitamin D3 metabolites show promise for the inhibition of cancer growth, analogous to the effect of estrogens and antiestrogens in breast cancer but with potential application in a much wider range of human cancers.

1 alpha,25-Dihydroxyvitamin D3 enhances 12-O-tetradecanoylphorbol-13-acetate- induced tumorigenic transformation and osteopontin expression in mouse JB6 epidermal cells.

To study the role of 1 alpha,25-dihydroxyvitamin D3 (calcitriol) in tumor promotion, we used JB6 C141.5a cells, a mouse epidermal cell model of tumor promotion. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) irreversibly induces anchorage-independent growth and tumorigenicity in these cells. Since we previously showed that calcitriol does not transform these cells but inhibits their proliferation, we hypothesized that calcitriol would inhibit TPA-induced transformation. Concurrent treatment of JB6 C141.5a cells with TPA and calcitriol revealed that calcitriol enhanced (1.7- to 10-fold, depending on dose) TPA-induced anchorage-independent growth without enhancing cell proliferation. Furthermore, a more than additive effect on osteopontin mRNA and protein levels was observed with concurrent drug treatment, which yielded a more highly phosphorylated form of osteopontin. These studies suggest coordinate regulation between the signaling pathways for calcitriol and TPA in JB6 C141.5a cells and further implicate expression of phosphorylated osteopontin in tumorigenesis.

1 alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol (Ro24-5531), a new deltanoid (vitamin D analogue) for prevention of breast cancer in the rat.

We have used the vitamin D analogue, 1 alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalcifero l (Ro24-5531), for inhibition of mammary carcinogenesis induced by N-nitroso-N-methylurea (NMU) in Sprague-Dawley rats. Rats were first treated with a single dose of either 15 or 50 mg/kg body weight NMU and then fed Ro24-5531 (2.5 or 1.25 nmol/kg of diet) for 5-7 months. Ro24-5531 significantly extended tumor latency and lessened tumor incidence as well as tumor number in rats treated with the lower dose of NMU. In rats treated with the higher dose of NMU, Ro24-5531 was fed in combination with tamoxifen; in these experiments, Ro24-5531 significantly enhanced the ability of tamoxifen to reduce total tumor burden, as well as to increase the probability that an animal would be tumor free at the end of the experiment. In vitro, Ro24-5531 was 10-100 times more potent than 1,25-dihydroxyvitamin D3 for inhibition of proliferation of human breast cancer cell lines as well as primary cultures of cells from 2 patients with acute myelogenous leukemia. When fed chronically, Ro24-5531 did not elevate serum calcium in the present studies. We propose the new term, "deltanoids," for the set of molecules composed of vitamin D and its synthetic analogues, in a manner similar to the naming of "retinoids" for the corresponding set of molecules related to vitamin A.

Topical Tetracycline Improves MC903-induced Atopic Dermatitis in Mice through Inhibition of Inflammatory Cytokines and Thymic Stromal Lymphopoietin Expression.

BACKGROUND: Tetracycline (TET) has been found to have both antibiotic and anti-inflammatory properties. The anti-inflammatory effect of topical TET on atopic dermatitis (AD) has not been reported. The purpose of this study was to explore the potential role of topical TET and its anti-inflammatory effects in a mouse model of AD. METHODS: The 2% TET was applied topically to ears of MC903-induced AD-like BALB/c mice once a day. AD-like symptoms and severity were evaluated by assessing skin scoring of dermatitis, ear thickness, and frequency of scratching. Serum IgE and thymic stromal lymphopoietin (TSLP) levels were measured by enzyme-linked immunosorbent assay. Western blot was used for analyzing the expressions of TSLP, protease-activated receptor 2 (PAR2), and nuclear factor-kappa B (NF-κB) in skin lesions. Real-time polymerase chain reaction was performed to assess the mRNA levels of TSLP and inflammatory cytokines including interleukin (IL)-4, IL-13, tumor necrosis factor (TNF)-α, and IL-1ß in skin lesions. RESULTS: Scoring of dermatitis (9.00 ± 0.63 vs. 6.67 ± 1.03, P = 0.001), ear thickness (0.44 ± 0.02 mm vs. 0.40 ± 0.03 mm, P = 0.018), and serum IgE level (421.06 ± 212.13 pg/ml vs. 244.15 ± 121.39 pg/ml, P = 0.047) were all improved in the 2% TET treatment group compared with AD group. Topical TET significantly reduced the serum level of TSLP (119.04 ± 38.92 pg/ml vs. 65.95 ± 54.61 pg/ml, P = 0.011) and both mRNA and protein expressions of TSLP in skin lesions compared with AD group (P = 0.003 and 0.011, respectively), and NF-κB and PAR2 expression in skin lesions were also suppressed (P = 0.016 and 0.040, respectively). Furthermore, expressions of inflammatory cytokines IL-4, IL-13, and TNF-α in skin lesions were down-regulated in 2% TET group compared with AD group (P = 0.035, 0.008, and 0.044, respectively). CONCLUSIONS: Topical TET exerted anti-inflammatory effects through suppression of TSLP and inflammatory cytokines in AD mouse model, suggesting TET as a potential agent for the topical treatment of AD in the future.

Systemic cardiovascular disease in uremic rats induced by 1,25(OH)2D3.

OBJECTIVE: Vitamin D may contribute to cardiovascular disease in the absence of hypercalcemia in patients with chronic kidney disease. METHODS: We investigated the effects of long-term (6-week) treatment with 1,25(OH)2D3, at a non-hypercalcemic dosage (0.25 microg/kg per day per orally) in 5/6 nephrectomized rats: (i) vehicle-treated, sham-operated rats; (ii) 1,25(OH)2D3-treated, sham-operated rats; (iii) vehicle-treated, 5/6 nephrectomized rats; and (iv) 1,25(OH)2D3-treated, 5/6 nephrectomized rats. RESULTS: Creatinine clearance after 6 weeks was significantly lower and parathyroid hormone levels were significantly higher in 1,25(OH)2D3-treated uremic rats, compared with uremic controls (P < 0.01). Serum calcium levels, as well as the calcium-phosphorus product, did not differ between both groups. Mean systolic blood pressure in 1,25(OH)2D3-treated animals was significantly increased, compared with vehicle (each P < 0.01). In addition, 1,25(OH)2D3-treated uremic animals showed left ventricular hypertrophy. Diffuse aortic calcification involving the intima and media layer occurred in 1,25(OH)2D3-treated uremic animals, but not in other groups. The mean aortic wall area and lumen area were increased two-fold in 1,25(OH)2D3-treated uremic animals compared with vehicle (P < 0.01), whereas the wall/lumen ratio remained unchanged, indicating fusiform aneurysm formation. CONCLUSIONS: Hypertension, left ventricular hypertrophy, aortic calcification, and aneurysm, without hypercalcemia, occurred in 1,25(OH)2D3-treated, 5/6 nephrectomized rats. These data indicate a permissive effect of uremia for cardiovascular damage induced by non-hypercalcemic doses of 1,25(OH)2D3.

Toxicity and dose-response studies of 1,25-(OH)2-16-ene-23-yne vitamin D3 in transgenic mice.

The vitamin D3 analogue 1,25-(OH)2-16-ene-23-yne vitamin D3 (16,23-D3) in doses with low systemic toxicity has been demonstrated to inhibit retinoblastoma growth in transgenic mice. This study examines the dose-dependent response for inhibition of tumor growth in transgenic mice with retinoblastoma and evaluates the in vivo toxicity of 16,23-D3 in nontransgenic mice. Transgenic 8-10-week-old mice with retinoblastoma (n = 119) were randomly assigned to groups receiving 1.0, 0.75, 0.5, 0.35, 0.2, or 0.05 microg of 16,23-D3 and a vehicle alone (control) group i.p. five times a week for 5 weeks. An additional control group received no injection. Eyes were enucleated one week after the end of treatment, and tumor areas were measured. To determine the toxic dose, transgene-negative littermates received 0.5, 1.0, 1.5, 2.5, 3.5, 4.5, or 5.0 microg of 16,23-D3, and control groups received vehicle alone, 5 days a week for 5 weeks. Serum calcium levels were measured, and necropsies were performed on animals from each group. In the dose-response study, tumor growth inhibition was greatest in the group receiving 0.35 microg (55% inhibition; P = 0.0056) and was also significant in the group receiving 0.5 microg (42% inhibition; P = 0.036). The systemic toxic effects due to hypercalcemia occurred at doses of > or =1.0 microg. 16,23-D3 inhibits tumor growth at doses > or =0.35 microg and shows toxic effects at doses > or =1.0 microg related to hypercalcemia in mice fed an unrestricted diet. No toxicity was observed with lower doses.