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1.  Dietary Vitamin D Deficiency in Rats from Middle- to Old-age Leads to Elevated Tyrosine Nitration and Proteomics Changes in Levels of Key Proteins in Brain: Implications for Low Vitamin D-dependent Age-Related Cognitive Decline 
Free radical biology & medicine  2013;65:10.1016/j.freeradbiomed.2013.07.019.
In addition to the well-known effects of vitamin D (VitD) in maintaining bone health, there is increasing appreciation that this vitamin may serve important roles in other organs and tissues, including the brain. Given that VitD deficiency is especially widespread among the elderly, it is important to understand how the range of serum VitD levels that mimic those found in humans (from low to high) affects the brain during aging from middle-age to old-age. To address this issue, twenty-seven male F344 rats were split into three groups and fed isocaloric diets containing low (100 IU/kg food), control (1000 IU/kg food), or high (10000 IU/kg food) VitD beginning at middle-age (12 months) and continued for a period of 4–5 months. We compared the effects of these dietary VitD manipulations on oxidative and nitrosative stress measures in posterior brain cortices. The low VitD group showed global elevation of 3-nitrotyrosine (3-NT) compared to control and high VitD treated groups. Further investigation showed that this elevation may involve dysregulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and NF-κB mediated transcription of inducible nitric oxide synthase (iNOS) as indicated by translocation of NF-κB to the nucleus and elevation of iNOS levels. Proteomic techniques were used to provide insights into potential mechanisms underlying these effects. Several brain proteins were found at significantly elevated levels in low VitD group compared to the control and high VitD groups. Three of these proteins, 6-phosphofructokinase, triosephosphate isomerase, and pyruvate kinase, are involved directly in glycolysis. Two others, peroxiredoxin-3 and DJ-1/PARK7, have peroxidase activity and are found in mitochondria. Peptidyl-prolyl cis-trans isomerase A (PPIA or cyclophilin A) has been shown to have multiple roles including protein folding, regulation of protein kinases and phosphatases, immunoregulation, cell signaling, and redox status. Together, these results suggest that dietary VitD deficiency contributes to significant nitrosative stress in brain and may promote cognitive decline in middle-aged and elderly adults.
PMCID: PMC3859828  PMID: 23872023
Vitamin D; nitrosative stress; 3-nitrotyrosine; proteomics; metabolism; cognitive decline
2.  Alveolar Macrophage Cathelicidin Deficiency in Severe Sarcoidosis 
Journal of innate immunity  2012;4(5-6):569-578.
Dysfunctional immune responses characterize sarcoidosis but the status of cathelicidin, a potent immunoregulatory and anti-microbial molecule has not been established in clinical disease activity. Alveolar macrophage cathelicidin expression was determined in biopsy-proven sarcoidosis patients classified clinically as “severe” (requiring systemic treatment) or “non-severe” (never requiring treatment). Sarcoidosis and healthy control bronchoalveolar lavage (BAL) cells were analyzed for mRNA expression of cathelicidin, vitamin D receptor (VDR), and the VDR co-activator, steroid receptor co-activator-3 (SRC3) by quantitative PCR. Cathelicidin-derived peptide LL-37 was determined by immunocytochemistry. Serum calcidiol [25(OH)vitD2] (vitD2) and calcitriol [1,25(OH)2vitD3] (vitD3) were quantified. Results indicated reduced BAL cell expression of cathelicidin and SRC3 in severe but not non-severe sarcoidosis compared to controls. Serum levels of biologically active vitD3 in both severe and non-severe patients were within control range even though vitD2 levels in both groups were below recommended level (30ng/ml). Sarcoidosis and control alveolar macrophages were studied in vitro to determine cathelicidin responses to vitD3 and tumor necrosis factor-alpha (TNFα), a vitD3 antagonist elevated in active sarcoidosis. Alveolar macrophage cathelicidin was stimulated by vitD3 but repressed by TNFα which also repressed SRC3. Findings suggest that TNFα-mediated repression of SRC3 contributes to alveolar macrophage cathelicidin deficiency in severe sarcoidosis despite healthy vitD3 levels. Deficiency of cathelicidin, a multifunctional regulator of immune cells and pro-inflammatory cytokines, may impede resolution of inflammation in severe sarcoidosis lung.
PMCID: PMC3472801  PMID: 22759465
alveolar macrophage; sarcoidosis; cathelicidin; vitamin D; cytokines; steroid receptor co-activators
3.  Decreased Serum Vitamin D Levels in Children with Asthma are Associated with Increased Corticosteroid Usage 
There is little knowledge about clinical variables associated with vitamin D (vitD) insufficiency in asthmatic children.
To investigate disease variables associated with vitD insufficiency in childhood asthma and interaction of vitD with corticosteroid-mediated anti-inflammatory responses.
We analyzed 25-hydroxyvitamin D serum levels in 100 asthmatic children to investigate relationships between 25-hydroxyvitamin D levels and patient characteristics. We determined vitD effects on dexamethasone (DEX) induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) and IL-10 in peripheral blood mononuclear cells (PBMC).
The median 25-hydroxyvitamin D serum level was 31 ng/mL. 47% of subjects had vitD levels in the insufficient range (<30 ng/mL), while 17% were vitD deficient (<20 ng/mL). Log10 IgE (p=0.01, ρ=−0.25) and the number of positive aeroallergen skin prick tests (p=0.02, ρ=−0.23) showed a significant inverse correlation with vitD, whereas FEV1% predicted (p =0.004, ρ=0.34) and FEV1/FVC ratio (p=0.01, ρ=0.30) showed a significant positive correlation with vitD. The use of inhaled steroids (p=0.0475), oral steroids (p=0.02), and total steroid dose (p=0.001), all showed significant inverse correlations with vitD. The amount of MKP-1 and IL-10 mRNA induced by vitD plus DEX was significantly greater than that induced by DEX alone (p<0.01). In an experimental model of steroid resistance where DEX alone did not inhibit T cell proliferation, addition of vitD to DEX resulted in significant dose dependent suppression of cell proliferation.
Corticosteroid use and worsening airflow limitation is associated with lower vitD serum levels in asthmatics. VitD enhances glucocorticoid action in asthmatic PBMC and enhances the immunosuppressive function of DEX in vitro.
Clinical Implications
Our study suggests that vitD supplementation may potentiate anti-inflammatory function of corticosteroids in asthmatics and thereby improve asthma control.
PMCID: PMC2866800  PMID: 20381849
vitamin D; children; asthma
4.  Prognostic Role of Vitamin D Status and Efficacy of Vitamin D Supplementation in Cancer Patients: A Systematic Review 
The Oncologist  2011;16(9):1215-1227.
Whether or not hypovitaminosis D can influence the prognosis of cancer patients and whether or not vitamin D supplementation improves outcome were examined through a literature review. It was concluded that the currently available evidence is insufficient to recommend vitamin D supplementation in cancer patients in clinical practice.
Whether or not hypovitaminosis D can influence the prognosis of cancer patients and whether or not vitamin D (vitD) supplementation improves outcome remain controversial.
Studies evaluating the prognostic role of vitD and vitD receptor (VDR) in cancer patients and trials evaluating the efficacy of vitD administration on patient outcome were identified by a search of MEDLINE, EMBASE, ISI Web of Knowledge, and the Cochrane Library through June 2010.
Twenty-five studies were included. A negative prognostic role for low serum vitD level was observed in five cohort studies including patients with breast cancer (one study), colon cancer (two studies), prostate cancer (one study), and melanoma (one study), but not in two studies on non-small cell lung cancer and one study on breast cancer. Three of four studies showed that VDR+ tumors carry a better prognosis than VDR− tumors, whereas VDR polymorphisms were significantly associated with prognosis in five of 10 studies. A significant interaction between serum vitD level and VDR polymorphism was observed in one study. Three randomized trials involving advanced prostate cancer patients explored the prognostic role of vitD supplementation. A meta-analysis of these trials showed no effect on survival (pooled risk ratio, 1.07; 95% confidence interval, CI, 0.93–1.23), with strong heterogeneity among studies.
Hypovitaminosis D seems to be associated with a worse prognosis in some cancers, but vitD supplementation failed to demonstrate a benefit in prostate cancer patients. The currently available evidence is insufficient to recommend vitD supplementation in cancer patients in clinical practice.
PMCID: PMC3228169  PMID: 21835895
Vitamin D; Vitamin D receptor; Neoplasm; Prognosis
5.  Seasonal variation of serum 25-hydroxyvitamin D levels in adult patients with inflammatory bowel disease 
Osteoporosis International  2010;22(11):2857-2867.
Patients with inflammatory bowel disease (IBD) are at risk of osteoporosis. Vitamin D (vitD) deficiency is known as a risk factor of osteoporosis. We observed low vitD blood levels in adult IBD patients both at the end of summer and winter. Furthermore, effects of oral vitD supplementation in (generally low) daily dosages were poor.
Patients with IBD are at risk of osteoporosis. This study evaluates seasonal vitD status, determinants of vitD deficiency and effects of vitD supplementation in adult IBD patients.
Patients were screened for vitD deficiency at the end of summer and winter using serum 25OHD3 (cut-off point, <50 nmol/L) combined with routine laboratory tests. A standardized questionnaire was used for demographic/lifestyle data i.e. IBD activity, health behaviour and vitD intake through diet and ultraviolet light.
Late-summer, 39% of the included 316 patients were vitD deficient. Late-winter, 57% of the follow-up patients (n = 281) were deficient. Independent protective determinants of vitD deficiency were oral vitD supplementation (summer/winter: odds ratio [OR], 0.52 [95% confidence interval [CI], 0.29–0.94]/OR, 0.44 [95% CI, 0.26–0.75]), recent sun holiday (summer: OR, 0.42 [95% CI, 0.24–0.74]) and regular solarium visits (summer/winter: OR, 0.28 [95% CI, 0.13–0.63]/OR, 0.17 [0.06–0.50]). IBD activity (p = 0.031), red blood cell distribution width (RDW; p = 0.04) and erythrocyte sedimentation rate (p = 0.03) were associated with low vitD levels using univariate analyses of the extreme 25OHD quartiles. In a subgroup with vitD supplementation, still 30% (late-summer) and 44% (late-winter) were vitD deficient.
VitD deficiency is common in IBD patients, but prevalence might be comparable with the general population. Ultraviolet light is essential for adequate vitD levels. Effects of oral vitD supplementation in (generally low) daily dosages are poor. Determinants for low vitD levels were IBD activity and elevated inflammatory markers, suggesting that increased risk of osteoporosis in IBD might be more related to the inflammation than to vitD deficiency.
PMCID: PMC3186887  PMID: 21113577
Inflammatory bowel disease; Osteoporosis; Pathophysiology; Prevalence; Seasonal variation; Serum 25-hydroxyvitamin D
6.  Ribavirin induced anaemia: the effect of vitamin D supplementation on erythropoietin and erythrocyte indices in normal Wistar rat 
Objectives: To measure the effect of vitamin D3 (VitD) supplementation on erythrocyte indices, serum and kidney erythropoietin (EPO) in normal rats treated with Pegylated interferon-α (Peg-INF-α) and ribavirin (RBV). Materials and methods: Eighty male Wistar rats were divided equally into 8 groups. ‘Control’; ‘P’: only received Peg-INF-α; ‘PD’: Peg-INF-α/VitD; ‘PR’: Peg-INF-α/RBV; ‘PRD’: Peg-INF-α/RBV/VitD; ‘R’: only received RBV; ‘RD’: RBV/VitD and ‘VitD’: only received vitamin D3. Peg-INF-α-2a was injected subcutaneously (6 µg/rat/week) for 4 weeks. RBV (4 mg/rat/day) and VitD (500 IU/rat/day) were given orally for 5 weeks. Blood samples were collected to measure erythrocyte indices and serum 25(OH) vitamin D. EPO was measured in serum samples and kidney specimens by ELISA. Results: Peg-INF-α alone did not affect the RBCs count, haemoglobin, serum and kidney EPO compared to control (P > 0.05). RBV significantly decreased (P < 0.05) the erythrocyte count, haemoglobin and EPO levels in kidney and serum, either individually (R group) or combined with Peg-INF-α (PR group), compared to ‘Control’ and ‘P’ groups. VitD prevented the development of anaemia and significantly increased the concentrations of EPO at serum and kidney levels in the ‘RD’ and ‘PRD’ groups compared to ‘R’ and ‘PR’ groups. There was a significant positive correlation between blood levels of VitD with serum and kidney EPO, Red cell count and haemoglobin concentrations. Conclusion: VitD could have a potential beneficial role in the prevention of ribavirin-induced anaemia by promoting endogenous EPO. Further studies are needed to explore the role of vitamin D in the prevention of ribavirin associated anaemia.
PMCID: PMC4211774  PMID: 25356124
Anaemia; erythropoietin hormone; pegylated interferon-α; ribavirin and vitamin D
7.  The role of 1α,25-dihydroxyvitamin D3 and cytokines in the promotion of distinct Foxp3+ and IL-10+ CD4+ T cells 
European journal of immunology  2012;42(10):2697-2708.
1α,25-Dihydroxyvitamin D3 (1α25VitD3) has potent immunomodulatory properties. We have previously demonstrated that 1α25VitD3 promotes human and murine IL-10-secreting CD4+ T cells. Because of the clinical relevance of this observation, we characterized these cells further and investigated their relationship with Foxp3+ regulatory T (Treg) cells. 1α25VitD3 increased the frequency of both Foxp3+ and IL-10+ CD4+T cells in vitro. However, Foxp3 was increased at high concentrations of 1α25VitD3 and IL-10 at more moderate levels, with little coexpression of these molecules. The Foxp3+ and IL-10+ T-cell populations showed comparable suppressive activity. We demonstrate that the enhancement of Foxp3 expression by 1α25VitD3 is impaired by IL-10. 1α25VitD3 enables the selective expansion of Foxp3+ Treg cells over their Foxp3− T-cell counterparts. Equally, 1α25VitD3 maintains Foxp3+ expression by sorted populations of human and murine Treg cells upon in vitro culture. A positive in vivo correlation between vitamin D status and CD4+Foxp3+ T cells in the airways was observed in a severe pediatric asthma cohort, supporting the in vitro observations. In summary, we provide evidence that 1α25VitD3 enhances the frequency of both IL-10+ and Foxp3+ Treg cells. In a translational setting, these data suggest that 1α25VitD3, over a broad concentration range, will be effective in enhancing the frequency of Treg cells.
PMCID: PMC3471131  PMID: 22903229
1α,25-Dihydroxyvitamin D3; Asthma; Immune regulation; Regulatory T cells
8.  Association of vitamin D and vitamin D receptor gene polymorphisms with chronic inflammation, insulin resistance and metabolic syndrome components in type 2 diabetic Egyptian patients 
Meta Gene  2014;2:540-556.
To date the published data concerning the possible interplay between vitamin D (VitD) and Vit D receptor (VDR) gene polymorphism with the immune/inflammatory mediators in type 2 diabetes mellitus (DM) is insufficient. Some of the immune non-classical actions of vitamin D may point to its role in the pathogenesis of type 2 DM through down-regulation of cytokines (IL-6). Although there is evidence to support a relationship among vitamin D status, chronic inflammation and insulin resistance, the underlying mechanism requires further exploration. We aimed to investigate the role of vitamin D in chronic inflammation and insulin resistance in type 2 DM. Moreover, to examine the association of VDR gene polymorphisms [VDR 2228570 C > T (FokI); VDR 1544410 A > G (BsmI)] with the components of metabolic syndrome (MetSyn) in type 2 diabetic Egyptian patients .
Subjects and methods
A total of 190 subjects were enrolled in this study, 60 controls and 130 type 2 diabetic patients (Group II). Group II was subdivided into 63 patients without MetSyn (subgroup IIa) and 67 patients with MetSyn (subgroup IIb). Genetic analysis for VDR gene polymorphisms was done in all subjects. VitD and IL-6 plasma levels were estimated.
The TT genotype for the VDR FokI was significantly more frequent in subgroup IIb than in subgroup IIa and controls (X2 = 6.83, P = 0.03 and X2 = 16.592, P = 0.000) respectively. The T allele was more frequent in the MetSyn group as compared to diabetics without MetSyn (p = 0.001), odds ratio (OR) and 95% CI for the T allele of C > T (FokI) = 2.30 (1.37–3.86). We did not detect any significant difference in VDR BsmI genotypes between patients and control groups (P = 0.947). FokI VDR was significantly associated with the lipid profile parameters, VitD and IL-6 plasma levels in subgroup IIa and associated with HOMA-IR, insulin, VitD, IL-6 levels, waist circumference (WC) and body mass index (BMI) in subgroup IIb while BsmI VDR variant was associated only with VitD values in both subgroups.
The present study suggests an interaction between VDR polymorphisms and important components of MetSyn, VitD and pro-inflammatory cytokines (IL-6). FokI VDR polymorphisms may be linked to mild inflammation and insulin resistance and might represent a genetic determinant for developing MetSyn in type 2 diabetic Egyptian patients. The challenge is determining the mechanisms of VitD action for recommendation of VitD supplementation that reduces the risks of MetSyn, insulin resistance and progression to type 2 diabetes.
PMCID: PMC4287888  PMID: 25606437
VitD, Vitamin D; DM, diabetes mellitus; VDR, Vit D receptor; MetSyn, metabolic syndrome; HOMA, Homeostasis of Metabolic Assessment; WC, waist circumference; OR, odds ratio; BMI, body mass index; IL-6, interleukin -6; SOCS, suppressors of cytokine signaling; IRS, insulin receptor substrates; CRP, C-reactive protein; FBG, fasting blood glucose; SBP, systolic blood pressure; DBP, diastolic blood pressure; HbA1c, glycated hemoglobin; FPI, fasting plasma insulin; TC, total cholesterol; TG, triglyceride; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; HPLC, High performance liquid chromatography; SD, standard deviation; X2, Chi-square; CI, confidence intervals; PGs, pro-inflammatory prostaglandins; NHANES III, National Health and Examination Survey; PTH, parathyroid hormone; Insulin resistance; Type 2 diabetes mellitus (DM); Metabolic syndrome; Vitamin D; Vitamin D Receptor gene; Polymorphisms; Pro-inflammatory cytokines; Interleukin-6 (IL-6)
9.  L-arginine and Vitamin D Adjunctive Therapies in Pulmonary Tuberculosis: A Randomised, Double-Blind, Placebo-Controlled Trial 
PLoS ONE  2013;8(8):e70032.
Vitamin D (vitD) and L-arginine have important antimycobacterial effects in humans. Adjunctive therapy with these agents has the potential to improve outcomes in active tuberculosis (TB).
In a 4-arm randomised, double-blind, placebo-controlled factorial trial in adults with smear-positive pulmonary tuberculosis (PTB) in Timika, Indonesia, we tested the effect of oral adjunctive vitD 50,000 IU 4-weekly or matching placebo, and L-arginine 6.0 g daily or matching placebo, for 8 weeks, on proportions of participants with negative 4-week sputum culture, and on an 8-week clinical score (weight, FEV1, cough, sputum, haemoptysis). All participants with available endpoints were included in analyses according to the study arm to which they were originally assigned. Adults with new smear-positive PTB were eligible. The trial was registered at NCT00677339.
200 participants were enrolled, less than the intended sample size: 50 received L-arginine + active vitD, 49 received L-arginine + placebo vit D, 51 received placebo L-arginine + active vitD and 50 received placebo L-arginine + placebo vitD. According to the factorial model, 99 people received arginine, 101 placebo arginine, 101 vitamin D, 99 placebo vitamin D. Results for the primary endpoints were available in 155 (4-week culture) and 167 (clinical score) participants. Sputum culture conversion was achieved by week 4 in 48/76 (63%) participants in the active L-arginine versus 48/79 (61%) in placebo L-arginine arms (risk difference −3%, 95% CI −19 to 13%), and in 44/75 (59%) in the active vitD versus 52/80 (65%) in the placebo vitD arms (risk difference 7%, 95% CI −9 to 22%). The mean clinical outcome score also did not differ between study arms. There were no effects of the interventions on adverse event rates including hypercalcaemia, or other secondary outcomes.
Neither vitD nor L-arginine supplementation, at the doses administered and with the power attained, affected TB outcomes.
Registry Registry number: NCT00677339
PMCID: PMC3743888  PMID: 23967066
10.  Inflammatory cytokines, goblet cell hyperplasia and altered lung mechanics in Lgl1+/- mice 
Respiratory Research  2009;10(1):83.
Neonatal lung injury, a leading cause of morbidity in prematurely born infants, has been associated with arrested alveolar development and is often accompanied by goblet cell hyperplasia. Genes that regulate alveolarization and inflammation are likely to contribute to susceptibility to neonatal lung injury. We previously cloned Lgl1, a developmentally regulated secreted glycoprotein in the lung. In rat, O2 toxicity caused reduced levels of Lgl1, which normalized during recovery. We report here on the generation of an Lgl1 knockout mouse in order to determine whether deficiency of Lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury.
An Lgl1 knockout mouse was generated by introduction of a neomycin cassette in exon 2 of the Lgl1 gene. To evaluate the pulmonary phenotype of Lgl1+/- mice, we assessed lung morphology, Lgl1 RNA and protein, elastin fibers and lung function. We also analyzed tracheal goblet cells, and expression of mucin, interleukin (IL)-4 and IL-13 as markers of inflammation.
Absence of Lgl1 was lethal prior to lung formation. Postnatal Lgl1+/- lungs displayed delayed histological maturation, goblet cell hyperplasia, fragmented elastin fibers, and elevated expression of TH2 cytokines (IL-4 and IL-13). At one month of age, reduced expression of Lgl1 was associated with elevated tropoelastin expression and altered pulmonary mechanics.
Our findings confirm that Lgl1 is essential for viability and is required for developmental processes that precede lung formation. Lgl1+/- mice display a complex phenotype characterized by delayed histological maturation, features of inflammation in the post-natal period and altered lung mechanics at maturity. Lgl1 haploinsufficiency may contribute to lung disease in prematurity and to increased risk for late-onset respiratory disease.
PMCID: PMC2760518  PMID: 19772569
11.  1α,25-Dihydroxyvitamin D3 promotes CD200 expression by human peripheral and airway-resident T cells 
Thorax  2012;67(7):574-581.
CD200, a cell-surface immunoglobulin-like molecule expressed by immune and stromal cells, dampens the pro-inflammatory activity of tissue-resident innate cells via its receptor, CD200R. This interaction appears critical for peripheral immune tolerance, particularly in the airways where excessive inflammation is undesirable. Vitamin D contributes to pulmonary health and promotes regulatory immune pathways, therefore its influence on CD200 and CD200R was investigated.
CD200 and CD200R expression were assessed by qPCR and immunoreactivity of human lymphoid, myeloid and epithelial cells following 1α,25-dihydroxyvitamin D3 (1α,25VitD3) exposure in vitro and in peripheral T cells following 1α,25VitD3 oral ingestion in vivo. The effect of 1α25VitD3 was also assessed in human airway-resident cells.
1α25VitD3 potently upregulated CD200 on peripheral human CD4+ T cells in vitro, and in vivo there was a trend towards upregulation in healthy, but not asthmatic individuals. CD200R expression was not modulated in any cells studied. CD200 induction was observed to a lesser extent in CD8+ T cells and not in B cells or airway epithelium. T cells isolated from the human airway also responded strongly to 1α25VitD3 to upregulate CD200.
The capacity of 1α,25-dihydroxyvitamin D3 to induce CD200 expression by peripheral and respiratory tract T cells identifies an additional pathway via which vitamin D can restrain inflammation in the airways to maintain respiratory health.
PMCID: PMC3471129  PMID: 22334534
12.  Hypoxia and vitamin D differently contribute to leptin and dickkopf-related protein 2 production in human osteoarthritic subchondral bone osteoblasts 
Bone remodelling and increased subchondral densification are important in osteoarthritis (OA). Modifications of bone vascularization parameters, which lead to ischemic episodes associated with hypoxic conditions, have been suspected in OA. Among several factors potentially involved, leptin and dickkopf-related protein 2 (DKK2) are good candidates because they are upregulated in OA osteoblasts (Obs). Therefore, in the present study, we investigated the hypothesis that hypoxia may drive the expression of leptin and DKK2 in OA Obs.
Obs from the sclerotic portion of OA tibial plateaus were cultured under either 20% or 2% oxygen tension in the presence or not of 50 nM 1,25-dihydroxyvitamin D3 (VitD3). The expression of leptin, osteocalcin, DKK2, hypoxia-inducible factor 1α (Hif-1α) and Hif-2α was measured by real-time polymerase chain reaction and leptin production was measured by enzyme-linked immunosorbent assay (ELISA). The expression of Hif-1α, Hif-2α, leptin and DKK2 was reduced using silencing RNAs (siRNAs). The signalling pathway of hypoxia-induced leptin was investigated by Western blot analysis and with mitogen-activated protein kinase (MAPK) inhibitors.
The expression of leptin and DKK2 in Obs was stimulated 7-fold and 1.8-fold, respectively (P <0.05) under hypoxia. Interestingly, whereas VitD3 stimulated leptin and DKK2 expression 2- and 4.2-fold, respectively, under normoxia, it stimulated their expression by 28- and 6.2-fold, respectively, under hypoxia (P <0.05). The hypoxia-induced leptin production was confirmed by ELISA, particularly in the presence of VitD3 (P <0.02). Compared to Obs incubated in the presence of scramble siRNAs, siHif-2α inhibited VitD3-stimulated leptin mRNA and protein levels by 70% (P =0.004) and 60% (P <0.02), respectively, whereas it failed to significantly alter the expression of DKK2. siHif-1α has no effect on these genes. Immunoblot analysis showed that VitD3 greatly stabilized Hif-2α under hypoxic conditions. The increase in leptin expression under hypoxia was also regulated, by p38 MAPK (P <0.03) and phosphoinositide 3-kinase (P <0.05). We found that the expression of leptin and DKK2 were not related to each other under hypoxia.
Hypoxic conditions via Hif-2 regulation trigger Obs to produce leptin, particularly under VitD3 stimulation, whereas DKK2 is regulated mainly by VitD3 rather than hypoxia.
PMCID: PMC4302570  PMID: 25312721
13.  Vitamin D Status and Related Factors in Newborns in Shanghai, China 
Nutrients  2014;6(12):5600-5610.
With the increasing recognition of the importance of the non-skeletal effects of vitamin D (VitD), more and more attention has been drawn to VitD status in early life. However, the VitD status of newborns and factors that influence VitD levels in Shanghai, China, remain unclear. A total of 1030 pregnant women were selected from two hospitals in Shanghai, one of the largest cities in China located at 31 degrees north latitude. Umbilical cord serum concentrations of 25-hydroxy vitamin D [25(OH)D] were measured by LC-MS-MS, and questionnaires were used to collect information. The median cord serum 25(OH)D concentration was 22.4 ng/mL; the concentration lower than 20 ng/mL accounted for 36.3% of the participants, and the concentration lower than 30 ng/mL for 84.1%. A multivariable logistic regression model showed that the determinants of low 25(OH)D status were being born during autumn or winter months and a lack of VitD-related multivitamin supplementation. The relative risk was 1.7 for both autumn (95% CI, 1.1–2.6) and winter (95% CI, 1.1–2.5) births (p < 0.05). VitD-related multivitamin supplementation more than once a day during pregnancy reduced the risk of VitD deficiency [adjusted OR (aOR) = 0.6, 95% CI (0.45–1.0) for VitD supplementation] (p < 0.05). VitD deficiency and insufficiency are common in newborns in Shanghai, China, and are independently associated with season and VitD supplementation. Our findings may assist future efforts to correct low levels of 25(OH)D in Shanghai mothers and their newborn children.
PMCID: PMC4276986  PMID: 25486368
vitamin D; related factors; newborn
14.  Intradermal application of vitamin D3 increases migration of CD14+ dermal dendritic cells and promotes the development of Foxp3+ regulatory T cells 
The active form of vitamin D3 (VitD) is a potent immunosuppressive drug. Its effects are mediated in part through dendritic cells (DCs) that promote the development of regulatory T cells (Tregs). However, it remains elusive how VitD would influence the different human skin DC subsets, e.g., CD1a+/langerin+ Langerhans cells, CD14+ DDCs and CD1a+ DDCs upon administration through the skin route in their natural environment. We addressed this issue by intradermal (ID) administration of VitD in a human skin explant system that closely resembles physiological conditions. ID injection of VitD selectively enhanced the migration of CD14+ DDCs, a subset known for the induction of tolerance. Moreover, ID injection of VitD repressed the LPS-induced T cell stimulatory capacity of migrating DCs. These migrating DCs collectively induced T cells with suppressive activity and abolished IFN-γ productivity. Those induced T cells were characterized by the expression of Foxp3. Thus, we report the novel finding that ID injection of VitD not only modifies skin DC migration, but also programs these DCs in their natural milieu to promote the development of Foxp3+ Tregs.
PMCID: PMC3859743  PMID: 23291929
vitamin D; skin dendritic cells; tolerance; regulatory T cells; intradermal injection
15.  The role of vitamin D in pulmonary disease: COPD, asthma, infection, and cancer 
Respiratory Research  2011;12(1):31.
The role of vitamin D (VitD) in calcium and bone homeostasis is well described. In the last years, it has been recognized that in addition to this classical function, VitD modulates a variety of processes and regulatory systems including host defense, inflammation, immunity, and repair. VitD deficiency appears to be frequent in industrialized countries. Especially patients with lung diseases have often low VitD serum levels. Epidemiological data indicate that low levels of serum VitD is associated with impaired pulmonary function, increased incidence of inflammatory, infectious or neoplastic diseases. Several lung diseases, all inflammatory in nature, may be related to activities of VitD including asthma, COPD and cancer. The exact mechanisms underlying these data are unknown, however, VitD appears to impact on the function of inflammatory and structural cells, including dendritic cells, lymphocytes, monocytes, and epithelial cells. This review summarizes the knowledge on the classical and newly discovered functions of VitD, the molecular and cellular mechanism of action and the available data on the relationship between lung disease and VitD status.
PMCID: PMC3071319  PMID: 21418564
Vitamin D; mortality; asthma; COPD; respiratory tract infection; immunity
16.  Pretreatment vitamin D level and response to neoadjuvant chemotherapy in women with breast cancer on the I-SPY trial (CALGB 150007/150015/ACRIN6657) 
Cancer Medicine  2014;3(3):693-701.
Laboratory studies suggest that vitamin D (vitD) enhances chemotherapy-induced cell death. The objective of this study was to determine whether pretreatment vitD levels were associated with response to neoadjuvant chemotherapy (NACT) in women with breast cancer. Study patients (n = 82) were enrolled on the I-SPY TRIAL, had HER2-negative tumors, and available pretreatment serum. VitD levels were measured via DiaSorin radioimmunoassay. The primary outcome was pathologic residual cancer burden (RCB; dichotomized 0/1 vs. 2/3). Secondary outcomes included biomarkers of proliferation, differentiation, and apoptosis (Ki67, grade, Bcl2, respectively) and 3-year relapse-free survival (RFS). Mean and median vitD values were 22.7 ng/mL (SD 11.9) and 23.1 ng/mL, respectively; 72% of patients had levels deemed “insufficient” (<30 ng/mL) by the Institute of Medicine (IOM). VitD level was not associated with attaining RCB 0/1 after NACT (univariate odds ratio [OR], 1.01; 95% CI, 0.96–1.05) even after adjustment for hormone receptor status (HR), grade, Ki67, or body mass index (BMI). Lower vitD levels were associated with higher tumor Ki67 adjusting for race (OR, 0.95; 95% CI, 0.90–0.99). VitD level was not associated with 3-year RFS, either alone (hazard ratio [HzR], 0.98; 95% CI, 0.95–1.02) or after adjustment for HR, grade, Ki-67, BMI, or response. VitD insufficiency was common at the time of breast cancer diagnosis among women who were candidates for NACT and was associated with a more proliferative phenotype. However, vitD levels had no impact on tumor response to NACT or short-term prognosis.
PMCID: PMC4101761  PMID: 24719175
Breast cancer; neoadjuvant chemotherapy; response; vitamin D
17.  Cloning and functional characteristics of murine large granular lymphocyte-1: a member of the Ly-49 gene family (Ly-49G2)  
Large granular lymphocyte (LGL) 1 is a cell surface glycoprotein expressed on a subset (50%) of C57BL/6 natural killer (NK) cells. Immunoprecipitation experiments reveal that the LGL-1 protein exists as a disulfide-linked 40-kD homodimer. Functional studies of LGL-1+ cells indicate that selected H-2d target cells are not lysed efficiently by these interleukin (IL)-2-cultured NK cells. These findings suggested that LGL-1 may be a member of the Ly-49 gene family. Here we report the molecular cloning of the LGL-1 cDNA from a severe combined immunodeficient-adherent lymphokine-activated killer cell library transfected into Cos-7 cells and find LGL-1 to be homologous to the Ly- 49 gene at both the nucleotide (85%) and amino acid levels (73%). Sequencing of our LGL-1 cDNA has revealed it to be nearly identical to the Ly-49G2 cDNA recently isolated by cross-hybridization with an Ly-49 probe. LGL-1 represents a type II transmembrane protein of 267 amino acids with its carboxyl end exposed extracellularly. The LGL-1 protein contains 11 highly conserved cysteine residues and a 25-amino acid transmembrane region. Southern blot analysis demonstrates that there are a number of homologous genes in mouse DNA that hybridize strongly to LGL-1. Northern analyses using poly A+ RNA from LGL-1+ NK cells indicate that LGL-1 is expressed as a 1.4 kb mRNA. Two-color flow cytometry analysis (FCA) of C57BL/6 splenic NK cells demonstrates that LGL-1 and Ly-49 label overlapping subsets of cells. FCA identifies four subsets of NK cells as defined by LGL-1 versus Ly-49 staining. We have sorted these individual subsets, expanded them in IL-2, and performed cytotoxicity experiments to determine their target cell profiles in relation to class I expression. Results of these studies are complex, but indicate that Ly-49 may not be the only molecule that recognizes class I as an inhibitory signal for cytotoxicity. LGL-1+ cells also fail to lyse several H-2d-expressing tumor targets and concanavalin A lymphoblasts from BALB/c but not C57BL/6 mice. This inhibition of lysis by LGL-1+ NK cells is negated by addition of monoclonal antibody (mAb) 4D11 that recognizes the LGL-1 protein. When mAbs to the class I molecules H-2Dd and H-2Ld (alpha 1 alpha 2 domains only) are added to cytotoxicity assays, LGL-1+ cells lyse H-2d targets very effectively.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC2192132  PMID: 7629495
18.  Vitamin D status of early preterm infants and the effects of vitamin D intake during hospital stay 
To evaluate vitamin D (vitD) status in early preterm infants (EPTIs) at birth and during birth hospitalisation on current vitD intake.
Serum 25-hydroxyvitamin-D [25(OH)D] concentrations, vitD intake and risk factors for low vitD status were assessed in 120 infants born at ≤32 weeks gestation.
Mean (SD) serum 25(OH)D at birth was 46.2 (14.0) nmol/L with lower concentrations in infants born <28 weeks than at 28–32 weeks gestation, p=0.02. Serum 25(OH)D was <50 nmol/L in 63% of mothers, 64% of infants at birth and 35% of infants at discharge. Mean daily vitD intake was 289±96 IU at 4 weeks of age and 60% achieved 400 IU/day intake at discharge.
Serum 25(OH)D <50 nmol/L was widespread in parturient women and in EPTIs at birth and at discharge. Optimising maternal vitD status during pregnancy and improving postnatal vitD intake may enhance infant vitD status during hospitalisation.
PMCID: PMC3933171  PMID: 23852093
Neonatology; Nutrition
19.  Lipoic acid, but not tempol, preserves vascular compliance and decreases medial calcification in a model of elastocalcinosis 
Vascular calcification decreases compliance and increases morbidity. Mechanisms of this process are unclear. The role of oxidative stress and effects of antioxidants have been poorly explored. We investigated effects of the antioxidants lipoic acid (LA) and tempol in a model of atherosclerosis associated with elastocalcinosis. Male New Zealand white rabbits (2.5-3.0 kg) were fed regular chow (controls) or a 0.5% cholesterol (chol) diet+104 IU/day vitamin D2 (vitD) for 12 weeks, and assigned to treatment with water (vehicle, n=20), 0.12 mmol·kg-1·day-1 LA (n=11) or 0.1 mmol·kg-1·day-1 tempol (n=15). Chol+vitD-fed rabbits developed atherosclerotic plaques associated with expansive remodeling, elastic fiber disruption, medial calcification, and increased aortic stiffness. Histologically, LA prevented medial calcification by ∼60% and aortic stiffening by ∼60%. LA also preserved responsiveness to constrictor agents, while intima-media thickening was increased. In contrast to LA, tempol was associated with increased plaque collagen content, medial calcification and aortic stiffness, and produced differential changes in vasoactive responses in the chol+vitD group. Both LA and tempol prevented superoxide signals with chol+vitD. However, only LA prevented hydrogen peroxide-related signals with chol+vitD, while tempol enhanced them. These data suggest that LA, opposite to tempol, can minimize calcification and compliance loss in elastocalcionosis by inhibition of hydrogen peroxide generation.
PMCID: PMC4051185  PMID: 24519127
Antioxidants; Vascular calcification; Atherosclerosis; Oxidative stress; Lipoic acid
20.  Vitamin C treatment attenuates hemorrhagic shock related multi-organ injuries through the induction of heme oxygenase-1 
Vitamin C (VitC) has recently been shown to exert beneficial effects, including protecting organ function and inhibiting inflammation, in various critical care conditions, but the specific mechanism remains unclear. Induction of heme oxygenase (HO)-1, a heat shock protein, has been shown to prevent organ injuries in hemorrhagic shock (HS) but the relationship between VitC and HO-1 are still ill-defined so far. Here we conducted a systemic in vivo study to investigate if VitC promoted HO-1 expression in multiple organs, and then tested if the HO-1 induction property of VitC was related to its organ protection and anti-inflammatory effect.
Firstly, to determine the HO-1 induction property of VitC, the HO-1 level were measured in tissues including kidney, liver and lung of the normal and HS model of Sprague–Dawley (SD) rats after VitC treatment (100 mg/kg body weight). Secondly, to testify if VitC prevented HS related organ injuries via inducing HO-1, the HS model of rats were separately pre- and post-treated with VitC, and some of them also received Zinc protoporphyrin (Znpp), a specific HO-1 inhibitor. The HO-1 activity in tissues was tested; the organ injuries (as judged by histological changes in tissues and the biochemical indicators level in serum) and inflammatory response in tissues (as judged by the level of pro-inflammatory cytokines Tumor necrosis factor-α and Interleukin-6 ) were analyzed.
The HO-1 mRNA and protein level in kidney, liver, and lung were highly induced by VitC treatement under normal and HS conditions. The HO-1 activity in tissues was enhanced by both VitC pre- and post-treatment, which was shown to improve the organ injuries and inhibit the inflammatory response in the HS model of rats. Of note, the beneficial effects of VitC were abolished after HO-1 activity was blocked by Znpp.
VitC led to a profound induction of HO-1 in multiple organs including the kidney, liver and lung, and this property might be responsible for the organ protection and inflammation inhibitory effects of both pre- and post-treatment with VitC in HS.
PMCID: PMC4246491  PMID: 25387896
21.  Comparative study of clinical grade human tolerogenic dendritic cells 
The use of tolerogenic DCs is a promising therapeutic strategy for transplantation and autoimmune disorders. Immunomodulatory DCs are primarily generated from monocytes (MDDCs) for in vitro experiments following protocols that fail to fulfil the strict regulatory rules of clinically applicable products. Here, we compared the efficacy of three different tolerance-inducing agents, dexamethasone, rapamycin and vitamin D3, on DC biology using GMP (Good Manufacturing Practice) or clinical grade reagents with the aim of defining their use for human cell therapy.
Tolerogenic MDDCs were generated by adding tolerogenic agents prior to the induction of maturation using TNF-α, IL-β and PGE2. We evaluated the effects of each agent on viability, efficiency of differentiation, phenotype, cytokine secretion and stability, the stimulatory capacity of tol-DCs and the T-cell profiles induced.
Differences relevant to therapeutic applicability were observed with the cellular products that were obtained. VitD3-induced tol-DCs exhibited a slightly reduced viability and yield compared to Dexa-and Rapa-tol-DCs. Phenotypically, while Dexa-and VitD3-tol-DCs were similar to immature DCs, Rapa-tol-DCs were not distinguishable from mature DCs. In addition, only Dexa-and moderately VitD3-tol-DCs exhibited IL-10 production. Interestingly, in all cases, the cytokine secretion profiles of tol-DCs were not modified by a subsequent TLR stimulation with LPS, indicating that all products had stable phenotypes. Functionally, clearly reduced alloantigen T cell proliferation was induced by tol-DCs obtained using any of these agent. Also, total interferon-gamma (IFN-γ) secretion by T cells stimulated with allogeneic tol-DCs was reduced in all three cases, but only T cells co-cultured with Rapa-tol-DCs showed impaired intracellular IFN-γ production. In addition, Rapa-DCs promoted CD4+ CD127 low/negative CD25high and Foxp3+ T cells.
Our results demonstrate contrasting influences of different clinical-grade pharmacological agents on human tol-DC generation. This should be taken into account for decisions on the use of a specific agent for the appropriate cellular therapy in the context of a particular disease.
PMCID: PMC3141500  PMID: 21658226
22.  Vitamin D deficiency reduces the benefits of progesterone treatment after brain injury in aged rats 
Neurobiology of aging  2009;32(5):864-874.
Administration of the neurosteroid progesterone (PROG) has been shown to be beneficial in a number of brain injury models and in two recent clinical trials. Given widespread vitamin D deficiency and increasing traumatic brain injuries (TBIs) in the elderly, we investigated the interaction of vitamin D deficiency and PROG with cortical contusion injury in aged rats. Vitamin D deficient (VitD-deficient) animals showed elevated inflammatory proteins (TNFα, IL-1β, IL-6, NFκB p65) in the brain even without injury. VitD-deficient rats with TBI, whether given PROG or vehicle, showed increased inflammation and greater open-field behavioral deficits compared to VitD-normal animals. Although PROG was beneficial in injured VitD-normal animals, in VitD-deficient subjects neurosteroid treatment conferred no improvement over vehicle. A supplemental dose of 1,25-dihydroxyvitamin D3 (VDH) given with the first PROG treatment dramatically improved results in VitD-deficient rats, but treatment with VDH alone did not. Our results suggest that VitD-deficiency can increase baseline brain inflammation, exacerbate the effects of TBI, and attenuate the benefits of PROG treatment; these effects may be reversed if the deficiency is corrected.
PMCID: PMC3586224  PMID: 19482377
Aging; Inflammation; Progesterone; 1,25-Dihydroxyvitamin D3; Traumatic brain injury; Vitamin D deficiency
23.  Polymorphisms in GC and NADSYN1 Genes are associated with vitamin D status and metabolic profile in Non-diabetic adults 
Our aim was to assess the associations between vitamin D (vitD) status, metabolic profile and polymorphisms in genes involved in the transport (Group-Component: GC) and the hydroxylation (NAD synthetase 1: NADSYN1) of 25 hydroxyvitamin D (25(OH)D) in non-diabetic individuals.
We conducted a cross-sectional study with 323 individuals recruited from the Health Center of Guadeloupe, France. The rs2282679 T > G and rs2298849 T > C in GC and rs12785878 G > T in NADSYN1 were genotyped.
Mean age was 46(range 18–86) years. 57% of participants had vitD insufficiency, 8% had vitD deficiency, 61% were overweight and 58% had dyslipidemia. A higher frequency of overweight was noted in women carrying rs2298849T allele v CC carriers (71% v 50%; P = 0.035). The rs2282679G allele was associated with increased risks of vitD deficiency and vitD insufficiency (OR =3.53, P = 0.008, OR = 2.34, P = 0.02 respectively). The rs2298849 TT genotype was associated with vitD deficiency and overweight (OR =3.4, P = 0.004 and OR = 1.76, P = 0.04 respectively) and the rs12785878 GG genotype with vitD insufficiency and dyslipidemia (OR = 1.80, P = 0.01 and OR = 1.72, P = 0.03 respectively). Based on the number of risk alleles for rs2282679 and rs12785878 combined, a genotype score of 3 (vs. 0–1) was associated with a 5.5 ng/mL average reduction in serum 25(OH)D levels (P = 0.001).
The GC and NADSYN1 genes are associated with the vitamin D status and might contribute to dyslipidemia and overweight independently of 25(OH)D levels.
PMCID: PMC3849583  PMID: 24073860
Dyslipidemia; Overweight; Vitamin D; NAD synthetase 1; NADSYN; Group specific component; GC
24.  The Association of Concurrent Vitamin D and Sex Hormone Deficiency with Bone Loss and Fracture Risk in Older Men: The MrOS Study 
Low 25-hydroxyvitamin D (VitD), low sex hormones (SH), and high sex hormone binding globulin (SHBG) levels are common in older men. We tested the hypothesis that combinations of low VitD, low SH, and high SHBG would have a synergistic effect on bone mineral density (BMD), bone loss, and fracture risk in older men. Participants were a random subsample of 1468 men (mean age 74) from the Osteoporotic Fractures in Men Study (MrOS) plus 278 MrOS men with incident non-spine fractures studied in a case-cohort design. “Abnormal” was defined as lowest quartile for VitD (<20 ng/ml), bioavailable testosterone (BioT, <163 ng/dl), and bioavailable estradiol (BioE, <11 pg/ml); and highest quartile for SHBG (>59 nM).
Overall, 10% had isolated VitD deficiency; 40% had only low SH or high SHBG; 15% had both SH/SHBG and VitD abnormality, and 35% had no abnormality. Compared to men with all normal levels, those with both SH/SHBG and VitD abnormality tended to be older, more obese, and to report less physical activity. Isolated VitD deficiency, and low BioT with or without low VitD, was not significantly related to skeletal measures. The combination of VitD deficiency with low BioE and/or high SHBG was associated with significantly lower baseline BMD and higher annualized rates of hip bone loss than SH abnormalities alone or no abnormality. Compared to men with all normal levels, the multivariate-adjusted hazard ratio (95% CI) for incident non-spine fracture during 4.6 yr median follow-up was 1.2 (0.8–1.8) for low VitD alone; 1.3 (0.9–1.9) for low BioE and/or high SHBG alone; and 1.6 (1.1–2.5) for low BioE/high SHBG plus low VitD.
In summary, adverse skeletal effects of low sex steroid levels were most pronounced in older men with low VitD levels. The presence of low VitD in the presence of low BioE/high SHBG may contribute substantially to poor skeletal health.
PMCID: PMC3474871  PMID: 22777902
bone loss; fracture; older men; sex hormones; vitamin
25.  Prevalence of 25-OH Vitamin D Deficiency in a Population of Hemodialysis Patients and Efficacy of an Oral Ergocalciferol Supplementation Regimen 
American journal of nephrology  2013;37(6):568-574.
Optimal dosing regimens for 25-OH vitamin D (VitD) deficiency are unknown in hemodialysis (HD) patients. Our aim was to evaluate the efficacy of prescribing ergocalciferol supplementation based on KDOQI guidelines for chronic kidney disease (CKD) stages III–IV in HD patients.
We conducted a retrospective study of 96 urban, predominately African-American HD patients at a single-center dialysis unit with VitD insufficiency or deficiency treated with ergocalciferol. Patients were classified as either compliant or non-compliant with supplementation as determined by review of pharmacy records. The primary outcome was VitD levels 6 months after initiation of treatment and secondary outcomes were VitD levels at 11 months, bone/mineral and anemia parameters.
The population was predominately African-American (69%) and Hispanic (28%). There were 61 individuals in the compliant group and 35 individuals in the non-compliant group. The compliant group was older but otherwise similar in demographics and co-morbid conditions to the non-compliant group. After 6 months of treatment, the compliant group had a significant increase in VitD level (14.7 ± 6.0 to 28.7 ± 10.0 ng/ml, p < 0.0001) compared to the non-compliant group (14.7 ± 5.5 to 14.8 ± 7.1 ng/ml, p = 0.95). There were no differences in the incidence of hypercalcemia between the two groups. Except for a decrease in phosphorus in the compliant group (5.6 ± 1.6 to 4.9 ± 1.7 mg/dl, p = 0.004), there were no significant difference in bone/mineral or anemia parameters including dosing of darbepoetin.
An ergocalciferol-prescribing strategy using the KDOQI guidelines for stage III–IV kidney disease in HD patients with VitD deficiency or insufficiency is inadequate to achieve repletion or maintenance of normal VitD levels.
PMCID: PMC4109679  PMID: 23735861
Vitamin D deficiency; End-stage renal disease; Hemodialysis

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