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.

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.

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.

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.

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.

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.

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.

Compound screening and transcriptional profiling in human primary keratinocytes: a brief guideline.

Cultured human primary keratinocytes constitute suitable targets for in-depth evaluation of the proliferative or differentiative potential of compounds. There is, however, a double-edged and intrinsically inseparable transition from biological activity to cytotoxicity for any agent under investigation. For that reason, we here first of all present an established protocol for the isolation, cultivation, and analysis of primary foreskin-derived keratinocytes. Taking calcitriol as example, we then reveal how a straightforward photometric cell culture assay can be exploited to assess overall cell viability in response to increasing compound doses. With predetermined cellular cytotoxicity at hand, physiologically meaningful (sub-toxic) compound concentrations for subsequent stimulation of cells can be readily selected, and, in doing so, differentially expressed genes with biological significance can be reliably identified.

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.

Selective cytotoxic action and DNA damage by calcitriol-Cu(II) interaction: putative mechanism of cancer prevention.

BACKGROUND: Vitamin D is known to play an important role in cancer-prevention. One of the features associated with the onset of malignancy is the elevation of Cu (II) levels. The mode of cancer-prevention mediated by calcitriol, the biologically active form of vitamin D, remain largely unknown. METHODS: Using exogenously added Cu (II) to stimulate a malignancy like condition in a novel cellular system of rabbit calcitriol overloaded lymphocytes, we assessed lipid peroxidation, protein carbonylation, DNA damage and consequent apoptosis. Free radical mediators were identified using free radical scavengers and the role of Cu (II) in the reaction was elucidated using chelators of redox active cellular metal ions. RESULTS: Lipid peroxidation and protein carbonylation (markers of oxidative stress), consequent DNA fragmentation and apoptosis were observed due to calcitriol-Cu (II) interaction. Hydroxyl radicals, hydrogen peroxide and superoxide anions mediate oxidative stress produced during this interaction. Amongst cellular redox active metals, copper was found to be responsible for this reaction. CONCLUSION: This is the first report implicating Cu (II) and calcitriol interaction as the cause of selective cytotoxic action of calcitriol against malignant cells. We show that this interaction leads to the production of oxidative stress due to free radical production and consequent DNA fragmentation, which leads to apoptosis. A putative mechanism is presented to explain this biological effect.

The atopic dermatitis-like symptoms induced by MC903 were alleviated in JNK1 knockout mice.

Atopic dermatitis (AD) is a common allergic disease, imposing large social and economic burdens worldwide. Atopic dermatitis is characterized by eczematous skin lesions and immunoglobulin E (IgE) hypersecretion. We investigated the role of JNK1 on the development of AD in mice. The vitamin D3 analogue MC903, a psoriasis therapeutic drug, was used to induce AD-like symptoms in wild-type (WT) and JNK1-/- mice. The symptoms of AD were less severe in JNK1-/- mice compared with WT mice. JNK1-/- mice showed less ear thickening and infiltration of eosinophils and mast cells in AD-like lesions than did WT mice when treated with MC903. MC903-treated JNK1-/- mice also showed significantly lower level of serum IgE, which was elevated in MC903-treated WT mice. Splenocytes isolated from MC903-treated WT and JNK1-/- mice were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. Splenocytes from JNK1-/- mice produced lower levels of T-helper (Th2) cytokines (interleukin-4 and -13) and transcription factor GATA-binding protein 3, and produced increased levels of the Th1 cytokines interferon-γ and transcription factor T-box expressed in T cells. Our results indicate that JNK1 plays an important role in the pathogenesis of AD and may be a useful target for therapies to ameliorate AD.

The mechanism of calcitriol in cancer prevention and treatment.

Calcitriol (1, 25-dihydroxyvitamin D3) is the most biologically active metabolite derived from the secosteroid hormone vitamin D. Apart from its calcium homeostatic effects, epidemiological studies have shown that reduced serum calcitriol levels are associated with an increased risk of some types of cancer. Numerous recent epidemiological and experimental studies have reported that it elicits anti-proliferative, apoptotic and differentiation effects in several malignant cell types. The inhibition of calcitriol results in reduced effects of anticancer drugs. Results from a number of clinical trials revealed that sufficient dosing and exposure to calcitriol is critical for achieving antitumor effects during intermittent regimens. This review summarizes the role of calcitriol in anticancer therapy and the progress in understanding its mechanism.

Vitamin D less-calcemic analog modulates the expression of estrogen receptors, vitamin D receptor and 1α-hydroxylase 25-hydroxy vitamin D in human thyroid cancer cell lines.

Estrogen receptors (ERs) are expressed in various "non-reproductive" cancer cell types. Some cancer types express 1α-hydroxylase 25-hydroxy vitamin D (1OHase) whose product, 1,25(OH)2D3 can retard cancer cell proliferation. Thyroid carcinoma cell growth is apparently promoted by estrogens, but whether or not this interaction is modified by vitamin D metabolites/analogs is presently unknown. Here we assessed the effect of a less calcemic vitamin D analog [JK 1624 F2-2 (JKF)] in three human thyroid cancer cell lines: ARO (anaplastic carcinoma), NPA (papillary carcinoma) and MRO (follicular carcinoma). (1) All cell lines expressed both ERα and ERß, vitamin D receptor (VDR) and 1OHase mRNA quantified by Real Time PCR. There was a general abundance of ERß over ERα expression, such that the ratio of ERß to ERα mRNA was >1000:1, 228:1 and 7.7:1 in ARO, MRO and NPA cells, respectively. (2) JKF up regulated ERß expression in ARO (by 110±15%) and MRO (by 280±10%) but down regulated ERß in NPA cells (by 40±15%). The expression of VDR was up regulated by JKF in NPA (21±6%), down regulated in ARO (-24±7%) and not affected in MRO. (3) All three human thyroid cancer cell lines were found to express 1OHase, which was up regulated by JKF in MRO (350±25%) and NPA (35±8%) but down regulated in ARO (-20±5%). This is the first report to describe direct regulation of VDR and 1OHase expression by a vitamin D analog in human thyroid cancer cells. A functional role for the vitamin D system in human thyroid cancer is suggested by the finding that the vitamin D analog can affect ERs expression which is in turn involved in estrogen-induced cell growth in an ER-type specific manner in these cells.

1,25-Dihydroxyvitamin D3-glycoside of herbal origin exhibits delayed release pharmacokinetics when compared to its synthetic counterpart.

Vitamin D requires two metabolic steps to become biologically active. In a first step 25-hydroxyvitamin D3 is formed, which acts as storage form. After a tightly controlled step in kidney the active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is formed. Because kidney is the relevant metabolic organ for this conversion, 1,25(OH)2D3 needs to be supplemented in patients with kidney malfunction or kidney failure. Synthetic 1,25(OH)2D3 (calcitriol) has been available as a drug for decades. Due to its high potency and its kinetic profile (fast absorption and rapid elimination) its therapeutic windows has proven to be relatively narrow. A natural form of the active metabolite was identified in a few plants, such as Solanum glaucophyllum (SG) and suggested as alternative for animal and human health. An extract of a SG variety bred for high and uniform level of glycosylated 1,25(OH)2D3 was chemically characterized. Among the typical pharmaceutically inactive plant components (carbohydrates 54.3%, protein 24.9%, minerals 17.1% and water 4.1%) high levels of 1,25(OH)2D3 and a unique flavonoid content was found (1.11mg total quercetin/g extract) consisting exclusively of the quercetin glycosides hyperoside, isoquercetin, rutin and apinosylrutin. The molecular distribution of glycosyl moieties in 1,25(OH)2D3 extracted from SG as determined by gel permeation chromatography was found to be 1-10 hexose units per aglycone. 1,25(OH)2D3-1-ß-glucopyranoside was identified in the SG extract, while a di- and triglycoside have been identified in SG by other groups. The pharmacokinetic properties of synthetic 1,25(OH)2D3 and glycosylated 1,25(OH)2D3 extracted from SG were compared in male rats. When compared to synthetic 1,25(OH)2D3, SG-derived 1,25(OH)2D3 exhibited delayed absorption and elimination characteristics, resulting in delayed Tmax (6-12h vs. 1h) and increased T½ (approximately 30h vs. 23h). This putative modified release pattern may be attributed to the glycosylation of herbal 1,25(OH)2D3 because de-glycosylation by ubiquitous intestinal enzymes prior to intestinal uptake of the aglycone appears to be the rate limiting step. In effect, 1,25(OH)2D3 of herbal origin behaves like a precursor of calcitriol, resulting in a wider therapeutic window and thus better pharmacological tolerance. This article is part of a Special Issue entitled 'Vitamin D Workshop.'.

Submaximal suppression of parathyroid hormone ameliorates calcitriol-induced aortic calcification and remodeling and myocardial fibrosis in uremic rats.

BACKGROUND AND OBJECTIVE: In subtotally nephrectomized rats, we studied to what extent high-dose calcitriol-induced cardiovascular disease can be modulated by almost complete suppression of parathyroid hormone (PTH), mediated by either cinacalcet (CINA) or parathyroidectomy (PTX). METHODS: Five groups were studied: sham-operated controls, uremic (U), uremic with calcitriol (U+1,25D), uremic and calcitriol with CINA (U+1,25D+CINA) and uremic and calcitriol with PTX (U+1,25D+PTX). Treatments lasted 14 weeks. RESULTS: Compared with U group animals, PTH was significantly lower with calcitriol treatment and almost completely suppressed in animals treated with either PTX or CINA. Serum calcium and phosphorus levels were similarly elevated in all groups receiving calcitriol. Renal function in uremic animals was significantly more impaired in the U+1,25D group. Aortic calcifications were pronounced in U+1,25D animals and reduced by more than 50% by concomittant treatment with CINA or PTX. Chondrocytes were observed near areas of calcification (>90%) and endochondral bone formation was confirmed by positive immunofluorescence for chondrocytic transcription factor sox9 and matrix protein collagen X. Altered arterial (aneurysmatic) geometry with a significant increase in wall/lumen and lumen/body weight ratio was found only in the U+1,25D group. Myocardial fibrosis was present in all uremic groups with a significant increase in the U+1,25D group. Connective tissue growth factor messenger RNA was significantly upregulated only in the U+1,25D group. CONCLUSION: Submaximal suppression of PTH by either CINA or PTX reduced vascular calcifications, arterial remodeling and myocardial fibrosis to a similar degree and independent of the serum calcium and phosphorus levels. These data do not indicate vasculotropic effects of calcimimetics independent of PTH suppression.

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.

Glycoconjugate histochemistry in the small and large intestine of normal and Solanum glaucophyllum-intoxicated rabbits.

Vitamin D participates in mineral homeostasis, immunomodulation, cell growth and differentiation. The leaves of Solanum glaucophyllum contain high levels of 1,25-dihydroxyvitamin D3 as glycoside derivatives and their chronic ingestion generates a hypervitaminosis D-like state. We analyzed changes on carbohydrate expression as a cell differentiation indicator on samples of the small and large intestine of S. glaucophyllum-intoxicated rabbits, using conventional and lectin histochemistry. Male New Zealand white rabbits were intoxicated with S. glaucophyllum during two or four weeks and killed the day after. A group of animals ("possibly recovered group") were intoxicated during 15 days and killed at day 45 of the beginning of the experiment. We found changes in the lectin binding pattern in the small and large intestine of the intoxicated rabbits. Some of these changes were reverted in the possibly recovered group. Vitamin D could be a new regulator factor of the intestinal glycosylation process.

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.