Utilização da vitamina D3 e seus metabólitos na alimentação de frangos de corte sobre parâmetros imunológicos e morfometria intestinal

Os efeitos da suplementação da vitamina D3 e de seus diferentes metabólitos foram avaliados na resposta imune e na morfometria intestinal de frangos de corte. Foram utilizados 952 frangos de corte de um dia de idade, distribuídos em um delineamento inteiramente casualizado, com quatro tratamentos, sete repetições e 34 aves por unidade experimental. Os tratamentos foram constituídos por quatros diferentes fontes de vitamina D3: colecalciferol (D3), 25-hidroxicolecalciferol (25(OH)D3), 1,25-dihidroxicolecalciferol (1,25(OH)2D3) e 1α-hidroxicolecalciferol (1α(OH)D3). As diferentes fontes foram incluídas na dieta, fornecendo 2000 e 1600 UI de vitamina D, nas fases pré-inicial, inicial e de crescimento, respectivamente. O peso relativo do intestino delgado diferiu entre os tratamentos aos 7, 21 e 42 dias e o peso relativo do fígado somente aos 42 dias de idade. Os demais órgãos e parâmetros imunológicos avaliados (peso dos órgãos linfóides, reação de hipersensibilidade cutânea basofílica, avaliação da atividade de macrófagos, dosagem de nitrito e perfil heterofilo: linfócito foram similares entre os animais alimentados com os diferentes metabólitos. Houve efeito (P<0,05) dos diferentes metabólitos da vitamina D3 sobre o comprimento dos vilos de jejuno e íleo aos sete dias, diferindo entre os animais alimentados com 1,25(OH)2D3 e 1α(OH)D3 para jejuno e 1,25(OH)2D3 e vitamina D3 para íleo (P<0,05). Para as demais fases não foi observado influência (P>0,05) dos tratamentos. Os parâmetros imunológicos não foram afetados pelos diferentes metabólitos de vitamina D. Os diferentes metabólitos de vitamina D afetaram positivamente a morfometria intestinal na altura de vilo na fase inicial, sendo os melhores resultados obtido pelos animais alimentados com 1,25(OH)2D3, contudo os parâmetros imunológicos foram similares entre os metabólitos estudados.

Response to an oral calcium load in nephrolithiasis patients with fluctuating parathyroid hormone and ionized calcium levels

the response to an oral calcium load test was assessed in 17 hypercalciuric nephrolithiasis patients who presented elevated parathyroid hormone (PTH) irrespective of the ionized calcium (sCa2+) levels. Blood samples were collected at baseline (0 min) and at 60 and 180 min after 1 g calcium load for serum PTH, total calcium, sCa2+, and 1.25(OH)2D3 determinations. According to the sCa2+ level at baseline, patients were classified as normocalcemic (N = 9) or hypercalcemic (N = 8). Six healthy subjects were also evaluated as controls. Bone mineral density was reduced in 14/17 patients. In the normocalcemic group, mean PTH levels at 0, 60 and 180 min (95 ± 76, 56 ± 40, 57 ± 45 pg/ml, respectively) did not differ from the hypercalcemic group (130 ± 75, 68 ± 35, 80 ± 33 pg/ml) but were significantly higher compared to healthy subjects despite a similar elevation in sCa2+ after 60 and 180 min vs baseline in all 3 groups. Mean total calcium and 1.25(OH)2D3 were similar in the 3 groups. Additionally, we observed that 5 of 9 normocalcemic patients presented a significantly higher concentration-time curve for serum PTH (AUC0',60',180') than the other 4 patients and the healthy subjects, suggesting a primary parathyroid dysfunction. These data suggest that the individual response to an oral calcium load test may be a valuable dynamic tool to disclose a subtle primary hyperparathyroidism in patients with high PTH and fluctuating sCa2+ levels, avoiding repeated measurements of both parameters.

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

Affiliation: Unité de recherche en Arthrose, Centre de recherche du Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame

Inflammation intestinale associée à la fibrose kystique : rôle du CFTR et propriétés anti-inflammatoires de la vitamine D

Les patients fibrose kystique (FK) souffrent de complications digestives qui incluent une inflammation intestinale modérée dont l’étiologie est méconnue. Les personnes atteintes de FK présentent également une malabsorption des vitamines liposolubles, telles la vitamine D. Or, la vitamine D possède des propriétés immunomodulatrices et anti-inflammatoires. Le présent projet vise à investiguer le rôle du CFTR, dont le gène est muté dans la FK, dans l’étiologie de cette inflammation intestinale et à étudier le potentiel anti-inflammatoire de la vitamine D sur celle-ci. Le CFTR a été invalidé génétiquement par la méthode des ARN interférents (shRNAi) et/ou inhibé pharmacologiquement par l’utilisation d’un antagoniste inhibiteur spécifique (CFTRinh-172). Un état inflammatoire a été induit par les cytokines pro-inflammatoires TNF-α et IL-1β. Afin d’évaluer le rôle anti-inflammatoire de la vitamine D, les cellules ont été pré-traitées avec la forme bioactive de la vitamine D, la 1,25(OH)2D3. La sécrétion et l’expression génique d’interleukine-8, ainsi que l’activation de la voie de signalisation p38MAPK et du facteur de transcription NFκB ont été évaluées. Pour explorer la voie par laquelle la vitamine D exerce ses actions anti-inflammatoires, les cellules ont été pré-incubées avec le BIRB796 pour inhiber la voie p38MAPK. Finalement l’expression génique du récepteur nucléaire de la vitamine D et des hydroxylases intestinales impliquées dans son métabolisme a été déterminée. Nos résultats suggèrent que le CFTR a un rôle dans l’étiologie de l’inflammation intestinale associée à la FK. De plus, la vitamine D semble moduler à la baisse la réponse inflammatoire de la cellule intestinale dont le CFTR a été génétiquement invalidé.

Arterial diffuse intimal thickening associated with enzootic calcinosis of sheep

Foram feitos estudos morfométrico, imunoistoquímico e ultra-estrutural do espessamento intimal difuso (DIT) das artérias de 7 ovinos com sinais clínicos de calcinose enzoótica espontânea causada pela ingestão da planta Nierembergia veitchii. As lesões caracterizavam-se por deposição de sais de cálcio na média como placas e estrias que, com frequência faziam saliência para a luz, criando rugosidades e irregularidades da íntima. Não foram observadas calcificações na artéria pulmonar e no sistema venoso. Microscopicamente, as calcificações das artérias restringiam-se quase que exclusivamente à média. Na imunoistoquimica, foi verificada a-actina nas células da média e nas do espessamento intimal. Receptores para 1,25(OH)2D3 foram evidenciados nos núcleos das células musculares da média, da íntima e das células endoteliais. As análises morfométricas em microscopia ótica revelaram, nas artérias, DIT irregularmente distribuido sem relação com a intensidade dos processos de calcificação subjacente nem com a espessura da média remanescente. A morfometria das alterações ultra-estruturais das células musculares lisas da média e da íntima espessada, mostrou que nessas últimas foi verificado aumento de até 318% na fração volumétrica das organelas de síntese em detrimento dos elementos contráteis, quando comparados com os valores das células da média. Essas modificações indicam a transformação das células musculares lisas de um tipo contrátil para células de tipo sintético. Entre essses dois extremos foram evidenciadas diversas formas intermediárias. Foram observadas evidências histológicas e ultra-estruturais da transformação e migração de células da média através das fenestras da lâmina elástica interna, para a íntima, onde proliferam e formam o DIT. Foi concluído que o DIT é um componente constante nas lesões arterioscleróticas nas calcinoses dos ovinos induzidas pela planta N. veitchii e que as células predominantes são células musculares lisas provenientes de predecessoras da média. Foi constatado que o DIT só excepcionalmente apresenta calcificação, vascularização, presença de células espumosas e fendas de colesterol. Não foram vistas áreas de necrose. Sugere-se que o fator indutor das alterações arteriais é o 1,25(OH)2D3 contido em N. veitchii.

Avaliação do efeito da vitamina D3 na expressão dos genes, VDR, GR e PPARy em adipócitos humanos

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Análise de pathway e network em tecido adiposo in vivo e o papel da vitamina D na adipogênese in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A genetic validation study reveals a role of vitamin D metabolism in the response to interferon-alfa-based therapy of chronic hepatitis C

Background To perform a comprehensive study on the relationship between vitamin D metabolism and the response to interferon-α-based therapy of chronic hepatitis C. Methodology/Principal Findings Associations between a functionally relevant polymorphism in the gene encoding the vitamin D 1α-hydroxylase (CYP27B1-1260 rs10877012) and the response to treatment with pegylated interferon-α (PEG-IFN-α) and ribavirin were determined in 701 patients with chronic hepatitis C. In addition, associations between serum concentrations of 25-hydroxyvitamin D3 (25[OH]D3) and treatment outcome were analysed. CYP27B1-1260 rs10877012 was found to be an independent predictor of sustained virologic response (SVR) in patients with poor-response IL28B genotypes (15% difference in SVR for rs10877012 genotype AA vs. CC, p = 0.02, OR = 1.52, 95% CI = 1.061–2.188), but not in patients with favourable IL28B genotype. Patients with chronic hepatitis C showed a high prevalence of vitamin D insufficiency (25[OH]D3<20 ng/mL) during all seasons, but 25(OH)D3 serum levels were not associated with treatment outcome. Conclusions/Significance Our study suggests a role of bioactive vitamin D (1,25[OH]2D3, calcitriol) in the response to treatment of chronic hepatitis C. However, serum concentration of the calcitriol precursor 25(OH)D3 is not a suitable predictor of treatment outcome.

PTH-related protein is released into the mother's bloodstream during location: evidence for beneficial effects on maternal calcium-phosphate metabolism

Recent studies have indicated that parathyroid hormone-related protein (PTHrP) may have important actions in lactation, affecting the mammary gland, and also calcium metabolism in the newborn and the mother. However, there are as yet no longitudinal studies to support the notion of an endocrine role of this peptide during nursing. We studied a group of 12 nursing mothers, mean age 32 years, after they had been nursing for an average of 7 weeks (B) and also 4 months after stopping nursing (A). It was assumed that changes occurring between A and B correspond to the effect of lactation. Blood was assayed for prolactin (PRL), PTHrP (two-site immunoradiometric assay with sheep antibody against PTHrP(1-40), and goat antibody against PTHrP(60-72), detection limit 0.3 pmol/l), intact PTH (iPTH), ionized calcium (Ca2+), 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), alkaline phosphatase (alkP), as well as for creatinine (Cr), protein, phosphorus (P), and total calcium (Ca). Fasting 2-h urine samples were analyzed for Ca excretion (CaE) and renal phosphate threshold (TmP/GFR). PRL was significantly higher during lactation than after weaning (39 +/- 10 vs. 13 +/- 9 micrograms/l; p = 0.018) and so was PTHrP (2.8 +/- 0.35 vs. 0.52 +/- 0.04 pmol/l; p = 0.002), values during lactation being above the normal limit (1.3 pmol/l) in all 12 mothers. There was a significant correlation between PRL and PTHrP during lactation (r = 0.8, p = 0.002). Whole blood Ca2+ did not significantly change from A (1.20 +/- 0.02 mmol/l) to B (1.22 +/- 0.02, mmol/l), whereas total Ca corrected for protein (2.18 +/- 0.02 mmol/l) or uncorrected (2.18 +/- 0.02 mmol/l) significantly rose during lactation (2.31 +/- 0.02 mmol/l, p = 0.003 and 2.37 +/- 0.03 mmol/l, p = 0.002, respectively). Conversely, iPTH decreased during lactation (3.47 +/- 0.38 vs. 2.11 +/- 0.35 pmol/l, A vs. B, p = 0.02). Serum-levels of 25(OH)D3 and 1,25(OH)2D3 did not significantly change from A to B (23 +/- 2.3 vs. 24 +/- 1.9 ng/ml and 29.5 +/- 6.0 vs. 21.9 +/- 1.8 pg/ml, respectively). Both TmP/GFR and P were higher during lactation than after weaning (1.15 +/- 0.03 vs. 0.86 +/- 0.05 mmol/l GF, p = 0.003 and 1.25 +/- 0.03 vs. 0.96 +/- 0.05 mmol/l, p = 0.002, respectively) as was alkP (74.0 +/- 7.1 vs. 52.6 +/- 6.9 U/l, p = 0.003). CaE did not differ between A and B (0.015 +/- 0.003 vs. 0.017 +/- 0.003 mmol/l GF, A vs. B, NS). We conclude that lactation is accompanied by an increase in serum PRL. This is associated with a release of PTHrP into the maternal blood circulation. A rise in total plasma Ca ensues, probably in part by increased bone turnover as suggested by the elevation of alkP. PTH secretion falls, with a subsequent rise of TmP/GFR and plasma P despite high plasma levels of PTHrP.

Characterization of osteopontin charge forms produced by osteoblasts

Osteopontin (OPN) is a highly-phosphorylated extracellular matrix protein localized in bone, kidney, placenta, T-lymphocytes, macrophages, smooth muscle of the vascular system, milk, urine, and plasma. In ROS 17/2.8 osteoblast-like osteosarcoma cells, 1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] regulates OPN at the transcriptional level resulting in increased steady state mRNA levels and increased production of OPN protein, maximal at 48 hours. Using ROS 17/2.8 cells as an osteoblast model, OPN was purified from culture medium after three hour treatments of either vehicle (ethanol) or 1,25(OH)2D3 via barium citrate precipitation followed by immunoaffinity chromatography. ^ Here, further evidence of regulation of OPN by 1,25(OH)2D 3 at the posttranslational level is presented. Prior to the up-regulation of OPN at the transcriptional level, 1,25(OH)2D3 induces a shift in OPN isoelectric point (pI) detected on two-dimensional gels from pI 4.6 to pI 5.1. Loading equal amounts of [32P]-labeled OPN recovered from ROS 17/2.8 cells exposed to 1,25(OH)2D3 or vehicle alone for three hours reveals that the shift from pI 4.6 to 5.1 is the result of reduced phosphorylation. Using structural analogs to 1,25(OH) 2D3, analog AT [25-(OH)-16-ene-23-yne-D3], which triggers Ca2+ influx through voltage sensitive Ca2+ channels but does not bind to the vitamin D receptor, mimicked the OPN pI shift while analog BT [1,25(OH)2-22-ene-24-cyclopropyl-D 3], which binds to the vitamin D receptor but does not allow Ca 2+ influx, did not. Inclusion of the Ca2+ channel blocker nifedipine also blocks the charge shift conversion of OPN. Further analysis of the signaling pathway initiated by 1,25(OH)2D3 reveals that inhibition of the cyclic 3′,5′ -adenosine monophosphate-dependent kinase, protein kinase A, or inhibition of the cyclic 3′,5′-guanine monophosphate-dependent kinase, protein kinase G, also prevents the charge shift conversion. ^ Isolation of OPN from rat femurs and tibiae provides evidence for the existence of these two OPN charge forms in vivo, evidenced by differential migration on isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. Peptide sequencing of rat long bone fractions revealed the presence of a presumed dentin specific protein, dentin matrix protein-1 (DMP-1). Western blot analysis confirmed the existence of DMP-1 in these fractions. ^ Using the OPN charge forms in functional assays, it was determined that the charge forms have differential roles in both cell surface and mineralization functions. In cell attachment assays and Ca2+ influx assays using PC-3 prostate cancer cells, the pI 5.1 charge form of OPN was found to permit binding and increase intracellular Ca2+ concentrations of PC-3 cells. The increase in intracellular Ca2+ concentration was found to be integrin αvβ3-dependent. In mineralization assays, the pI 4.6 charge form of OPN promoted hydroxyapatite formation, while the pI 5.1 charge form had improved Ca2+ binding ability. ^ In conclusion, these findings suggest that 1,25(OH) 2D3 regulates OPN not only at the transcriptional level, but also plays a role in determination of the OPN phosphorylation state. The latter involves a short term (less than three hours) treatment and is associated with membrane-initiated Ca2+ influx. Functional assays utilizing the two OPN charge forms reveal the dependence of OPN post-translational state on its function. ^

New insights into the role of the duodenal vitamin D receptor in calcium homeostasis

It is generally believed that 1,25(OH)2D3, bound to its receptor (VDR) contributes to calcium homeostasis by regulating active calcium absorption in the proximal small intestine. However, studying patients with hereditary vitamin D-resistant rickets (HVDRR) provided investigators with a better understanding of VDR's role in calcium homeostasis. HVDRR patients have inactivating mutations in the VDR, and as a consequence they develop hypocalcemia, hyperparathyroidism and severe rickets. However, these phenotypes can be corrected if the patients are given IV infusions of calcium or dietary calcium. This raises the question of what is the physiological significance of VDR-regulated active calcium absorption if calcium homeostasis can be restored independently of the VDR. ^ In order to distinguish the contribution of VDR in the proximal small intestine to overall calcium homeostasis, I generated transgenic mice expressing the human VDR (hVDR) exclusively in the proximal small intestine of mVDR-/- mice by using an hVDR-expressing transgene driven by the duodenal-specific adenosine deaminase enhancer (hVDR+/mVDR-/-). hVDR+/mVDR-/- mice expressed transcriptionally active hVDR only in the proximal small intestine and responded to 1,25(OH)2D3 by up-regulating expression of TRPV6 and calbindin D9K, genes involved in calcium absorption. Furthermore, ligated duodenal loop assays determined that calcium absorption in hVDR+/mVDR-/- mice was as responsive to 1,25(OH)2D3 as in WT mice. Despite having a functional hVDR in the proximal small intestine, hVDR+/mVDR-/- mice were hypocalcemic, had hyperparathyroidism, and were rachitic when fed a normal rodent diet at weaning, as were the mVDR-/- mice. However, when fed a high calcium, phosphorus, and lactose diet (rescue diet), the hVDR+/mVDR-/- mice responded more effectively than the mVDR-/- mice by down-regulation of parathyroid hormone production and by a greater increase in bone mineralization. Furthermore, when three-month-old rachitic mice were fed a rescue diet for 3 weeks, serum calcium and bone mineral content were normalized in hVDR+/mVDR-/- mice, but not in mVDR-/- mice. ^ In conclusion, hVDR expression enabled young mice to better use the rescue diet than mVDR-/- mice. Expression of transgenic hVDR also protected the ability of older mice to respond to the rescue diet despite the absence of the VDR elsewhere in the intestinal tract. I propose that because hVDR+/mVDR-/- mice responded better than mVDR-/- mice to the rescue diet, it is likely that VDR expression in the proximal small intestine is necessary in nutritional (insufficient dietary calcium) and physiological (age) conditions when passive calcium absorption is inadequate. ^

Concentrations of PCBs, DDT and thyroid hormones in gray (Halichoerus grypus) and ringed (Phoca hispida) seals

The high levels of polychlorinated biphenyls (PCBs) and DDT in gray seal (Halichoerus grypus) and ringed seal (Phoca hispida botnica) in the Baltic Sea have been associated with pathological disruptions, including bone lesions and reproductive failures. The underlying environmental and toxicological mechanisms leading to these pathological changes are not yet fully understood. The present study investigated the relationship between the individual contaminant load and bone- and thyroid-related effects in adult gray seals (n = 30) and ringed seals (n = 46) in the highly contaminated Baltic Sea and in reference areas (Sable Island, Canada, and Svalbard, Norway). In the gray seals, multivariate and correlation analyses revealed a clear relationship between circulating 1,25-dihydroxyvitamin D3 (1,25(OH)2D), calcium, phosphate, and thyroid hormone (TH) levels and hepatic PCB and DDT load, which suggests contaminant-mediated disruption of the bone and thyroid homeostasis. Contaminants may depress 1,25(OH)2D levels or lead to hyperthyroidism, which may cause bone resorption. In the ringed seals, associations between circulating 1,25(OH)2D, THs, and hepatic contaminants were less prominent. These results suggest that bone lesions observed in the Baltic gray seals may be associated with contaminant-mediated vitamin D and thyroid disruption.

Multiple promoters direct the tissue-specific expression of novel N-terminal variant human vitamin D receptor gene transcripts

The effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are mediated by the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of transcriptional regulators. We have identified upstream exons of the human (h) VDR gene that are incorporated into variant transcripts, two of which encode N-terminal variant receptor proteins. Expression of the hVDR gene, which spans more than 60 kb and consists of at least 14 exons, is directed by two distinct promoters. A tissue-specific distal promoter generates unique transcripts in tissues involved in calcium regulation by 1,25-(OH)2D3 and can direct the expression of a luciferase reporter gene in a cell line-specific manner. These major N-terminal differences in hVDR transcripts, potentially resulting in structural differences in the expressed receptor, may contribute to cellular responsiveness to 1,25-(OH)2D3 through tissue differences in the regulation of VDR expression.

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1α,25(OH)2D3 and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17β-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.

Human vitamin D receptor phosphorylation by casein kinase II at Ser-208 potentiates transcriptional activation.

The potential functional significance of human 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor (hVDR) phosphorylation at Ser-208 was evaluated by cotransfecting COS-7 kidney cells with hVDR constructs and the catalytic subunit of human casein kinase 11 (CK-11). Under these conditions, hVDR is intensely phosphorylated in a reaction that depends on both CK-II and the presence of Ser-208. The resulting hyperphosphorylated receptor is unaltered in its kinetics for binding the 1,25(OH)2D3 ligand, its partitioning into the nucleus, and its ability to associate with a vitamin D responsive element. Replacement of Ser-208 with glycine or alanine indicates that phosphorylation of hVDR at Ser-208 is not obligatory for 1,25(OH)2D3 action, but coexpression of wild-type hVDR and CK-11 elicits a dose-dependent enhancement of 1,25(OH)2D3-stimulated transcription of a vitamin D responsive element reporter construct. This enhancement by CK-II is abolished by mutating Ser-208 to glycine or alanine and does not occur with glucocorticoid receptor-mediated transcription. Therefore, phosphorylation of hVDR by CK-11 at Ser-208 specifically modulates its transcriptional capacity, suggesting that this covalent modification alters the conformation of VDR to potentiate its interaction with the machinery for DNA transcription.