The active form of vitamin D is the steroid hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Central to the mechanism of action of 1,25(OH)₂D₃ is its specific, high affinity intracellular receptor. This research focused on the participation of this receptor in the biology, biochemistry, and molecular biology of the vitamin D regulatory system. The effects of 1,25(OH)₂D₃ on the differentiation of hematopoietic cells were investigated using the cultured human promyelocytic leukemia cell line, HL-60, as a model. It was observed that 1,25(OH)₂D₃ induced macrophage differentiation in HL-60 cells and that a direct biochemical correlation existed between 1,25(OH)₂D₃ receptor saturation and a 1,25(OH)₂D₃-stimulated bioresponse. These data implicate 1,25(OH)₂D₃ as a natural cell differentiating agent and the 1,25(OH)₂D₃ receptor as the mediator of this hormone's action. Since the most fundamental level of control occurs by the regulation of gene expression, studies were undertaken to define the transcriptional control by 1,25(OH)₂D₃ over a known vitamin D-regulated endpoint protein. This work resulted in the molecular cloning of cDNAs to two avian intestinal calcium binding proteins, vitamin D-dependent calcium binding protein and a novel calmodulin-like protein. To gain further insight into the role of the 1,25(OH)₂D₃ receptor as a transcriptional regulator, avian and mammalian 1,25(OH)₂D₃ receptor mRNAs were characterized extensively by the techniques of in vitro translation and immunoprecipitation. These mRNAs were then utilized to construct cDNA libraries from which avian and human intestinal 1,25(OH)₂D₃ receptor cDNAs were isolated and their identity verified by hybrid-selected translation, sequencing, and Northern analysis. It was concluded that demonstrated 1,25(OH)₂D₃ receptors are polypeptides of 52-60 kDa whose activity is regulated by 1,25(OH)₂D₃ at both an mRNA and posttranslational level. Furthermore, the deduced amino acid sequence of receptor mRNA included a highly conserved cysteine, lysine, and arginine rich region that is homologous to other steroid receptors and the oncogene product v- erbA. Thus, the vitamin D receptor to be a specific trans -acting factor, modulating the pleiotropic effects of vitamin D including calcium homeostasis, and cellular differentiation.

There is little information available about the relationship of newborn vitamin D status with pregnancy outcome and infant health. The purpose of this cross-sectional study was to estimate the prevalence of vitamin D deficiency and insufficiency in newborns in the Saskatoon Health Region, identify risk factors for low neonatal levels of vitamin D, and determine whether any association exists between low levels of vitamin D and adverse pregnancy and neonatal outcomes. The Newborn Vitamin D Study was conducted between December, 2011 and February, 2012. Sixty-five maternal-fetal dyads delivering in the Saskatoon Health Region were included in the study. Mean cord blood vitamin D level was 64.1 nmol/L (standard deviation = 19.8 nmol/L), which is in the insufficient range. Cord blood vitamin D level was deficient (<50 nmol/L) in 22% and insufficient (50-75 nmol/L) in 48% of the 65 newborns studied. Simple linear regression indicated that low weight gain during pregnancy is significantly associated with low vitamin D levels (p = 0.04). However, younger maternal age (p < 0.01) and urban area of residence (p = 0.09) were the strongest predictors of low cord blood vitamin D levels in a multiple linear regression model (R2 of 0.519, p = 0.003). Cord blood vitamin D levels were not significantly associated with any pregnancy or neonatal outcomes. Despite 85% of mothers reporting having taken a daily prenatal supplement, 70% of newborns in our study population had either an insufficient or deficient cord blood vitamin D status. This suggests that prenatal supplements, which typically contain 400 IU of vitamin D, contain an inadequate dose of vitamin D to produce sufficient cord blood vitamin D levels in most newborns. Further research is necessary to inform maternal vitamin D supplementation guidelines and to investigate the role of vitamin D in pregnancy outcomes and infant health.

Given the strong evidence for a beneficial role of vitamin D in diabetes and CVD pathogenesis, and the prevalence of vitamin D deficiency, vitamin D supplementation has been advocated for the prevention of cardiometabolic disease. To provide information on the effects of 5,000IU (125µg) vitamin D3 on cardiometabolic risk, a double blind, RCT in a cohort of overweight and obese UK adult males with plasma 25(OH)D concentration < 75nmol/L for a duration of 8 weeks was conducted. To the best of my knowledge, this is the first RCT to investigate the effect of 5,000IU (125µg) vitamin D3 on cardiometabolic markers in vitamin D insufficient, non-hypertensive and non-diabetic overweight and obese adult males.

Colorectal cancer (CRC) is common, with >1 million annual incidence worldwide and is associated with significant morbidity and mortality. Prevention is a particularly appealing strategy to combat CRC, but there is a paucity of well-founded mechanistic research in the area. CRC is a complex disorder, with both genetic and environmental factors influencing incidence. The heritable component of CRC variance is estimated to be ~35%, with ~5% due to highly penetrant mutations. Common genetic variance likely makes up the majority of the heritable component, yet a large proportion of heritability remains unexplained. One possible explanation is gene-environment interactions (GxE) where-by both genetic and environmental factors interact to influence risk. Observational data implicate vitamin D as an environmental risk factor in CRC aetiology and in-house data indicates that genotype at the GWAS identified CRC risk locus rs9929218 influences this association, i.e. a GxE. The rs9929218 locus is intronic within CDH1, a tumour suppressor gene, and present evidence suggests a gene-environment interaction model of vitamin D-induced CDH1 transcription dependent on genotype at the rs9929218 locus and mediated by VDR and FOXO transcription factors and SIRT1, a FOXO regulator. To test this model, two broad approaches were employed - an observational approach to assess the association between human plasma vitamin D status, rs9929218 genotype and normal colorectal mucosa CDH1 expression and an interventional approach treating cell lines, organoids and human subjects with vitamin D to assess genotype-specific effects. Observational analysis of vitamin D level (25OHD) in CRC cases identified a significant influence of age, gender, BMI and selected vitamin D pathway genetic variation, while analysis of 424 normal colorectal mucosa samples from CRC cases and cancer-free subjects demonstrated strong sampling, gender, age and site differences in gene expression. 25OHD was not significantly associated with mucosa gene expression at individual gene level. However, association with a number of pathways relevant to tumourigenesis, including 'cell adhesion', 'migration' and 'cell death' was seen, providing possible mechanism to the published observational data. Circulating 25OHD level was not associated with mucosa CDH1 expression, yet crucially, analysis demonstrated a strong additive gene-environment interaction effect between plasma 25OHD, the rs9929218 genotype and NM expression of VDR, FOXO and SIRT1 explaining ~70% of the variance of mucosal CDH1 expression. The interventional approach first investigated vitamin D effects on CRC cell lines and human colorectal mucosa organoids. Calcitriol, the active form of vitamin D, induced CDH1 expression in 4 CRC cell lines, with interaction effects explaining 66% of the variance of CDH1 expression. CDH1 induction was replicated in human colorectal epithelial organoids, a non-aberrant tissue model, while gene enrichment analysis from both cells and organoids implicate vitamin D in a number of processes relevant to CRC tumourigenesis including regulation of cell proliferation, differentiation, migration and apoptosis, consistent with the pleiotropic effects of vitamin D reported in the published literature. Finally, a novel human intervention study was undertaken to investigate the impact of vitamin D supplementation in human blood and rectal mucosa. Short-term high-dose supplementation of 50 participants significantly induced CDH1 expression in rectal mucosa, with significant gene interaction effects between 25OHD, rs992818 genotype and CDH1, VDR and FOXO expression, thus independently replicating the same gene interaction effects seen in the human observational study. Meanwhile, transcriptome profiling identified numerous pathways relevant to tumourigenesis significantly enriched after supplementation, validating several pathways also associated with vitamin D status in the observational study. In summary, the research presented in this thesis demonstrates that vitamin D treatment of cells, epithelial organoids and human subjects induces CDH1 expression, and that strong gene interaction effects involving the colorectal cancer risk locus rs9929218 modulate this effect. FOXO transcription factors influence the gene interaction effect, consistent with the proposed model of ligand dependent regulation of FOXO by VDR and transcription activation of the CDH1 gene by the FOXO complex dependent on rs9929218 genotype. These data provide support for rectal CDH1 expression as an intermediate biomarker for vitamin D chemopreventive studies and suggest that gene environment interactions underlie some of the missing heritability of CRC.

Low vitamin D status has been consistently associated with an increased risk of multiple sclerosis (MS). Further, preclinical and in vitro data demonstrate immune regulatory properties of 1,25-dihydroxyvitamin D that may be beneficial for patients with MS. To date evidence of beneficial in vivo immunomodulation by supplementation with vitamin D3 in humans is lacking. In a one-year, open-label, phase I/II dose-escalation study of vitamin D3 (average ~14,000 IU/d over one year) with calcium (1,200mg/d) in patients with MS, we compared the effects of treatment on safety outcomes, clinical outcomes and selected biomarkers of immune system activity, relative to matched MS patients [age, sex, disease duration, disease modifying therapy, and expanded disability status scale (EDSS)] randomized to receive no supplementation. Mean serum 25(OH)D concentrations were 78.1±27.0 nmol/L at baseline and at one-year were 82.7±34.8 and 179.1±76.1 nmol/L in control and treated groups, respectively. Serum and urinary calcium and all other safety outcomes were unchanged throughout the trial. Compared to controls, treated patients tended to have fewer relapses (McNemar, p=0.09) and a greater proportion had a stable or improved EDSS at study end (p=0.018). We observed significantly reduced lymphocyte proliferative responses to antigenic challenge in the treatment group at one year, compared to baseline and control group responses. High serum 25(OH)D concentrations were not associated with short-term adverse effects in patients with MS, but with evidence of clinical improvement and beneficial immunomodulation.

Low vitamin D status has been consistently associated with an increased risk of multiple sclerosis (MS). Further, preclinical and in vitro data demonstrate immune regulatory properties of 1,25-dihydroxyvitamin D that may be beneficial for patients with MS. To date evidence of beneficial in vivo immunomodulation by supplementation with vitamin D3 in humans is lacking. In a one-year, open-label, phase I/II dose-escalation study of vitamin D3 (average ~14,000 IU/d over one year) with calcium (1,200mg/d) in patients with MS, we compared the effects of treatment on safety outcomes, clinical outcomes and selected biomarkers of immune system activity, relative to matched MS patients [age, sex, disease duration, disease modifying therapy, and expanded disability status scale (EDSS)] randomized to receive no supplementation. Mean serum 25(OH)D concentrations were 78.1±27.0 nmol/L at baseline and at one-year were 82.7±34.8 and 179.1±76.1 nmol/L in control and treated groups, respectively. Serum and urinary calcium and all other safety outcomes were unchanged throughout the trial. Compared to controls, treated patients tended to have fewer relapses (McNemar, p=0.09) and a greater proportion had a stable or improved EDSS at study end (p=0.018). We observed significantly reduced lymphocyte proliferative responses to antigenic challenge in the treatment group at one year, compared to baseline and control group responses. High serum 25(OH)D concentrations were not associated with short-term adverse effects in patients with MS, but with evidence of clinical improvement and beneficial immunomodulation.

No description available.

Maintaining reproductive health and diagnosing and treating conditions of infertility in stud dogs is important in canine theriogenology. However, there is still a great deal to be learned about reproductive physiology and factors that affect reproductive organs and semen quality in dogs. This thesis is an investigation of two factors in the male dog; serum 25-hydroxy Vitamin D (25OHVD) and prolactin (PRL) concentrations, and their possible associations with benign prostatic hyperplasia (BPH), prostate volume and/or sperm morphology and motility characteristics. 28 (Vitamin D Study) and 29 (28 plus one for the Prolactin study) client dogs of various breeds from the Ontario Veterinary College and Graham Animal Hospital in Southwestern Ontario, Canada were enrolled in the study from March to December 2009. Of these dogs 22 were successfully collected for semen. BPH was diagnosed using prostate volume measured by ultrasound, as well as clinical signs including blood in the ejaculate. Semen analysis was performed using manual microscopic techniques for morphology and computer assisted sperm analysis equipment for motility. In the vitamin D study, no associations were found between BPH and serum 25OHVD concentrations. In contrast, several sperm motility (motility, progressive motility, beat cross frequency (BCF), distance average path (DAP), curvilinear distance (DCL), linear distance (DSL), average path velocity (VAP), curvilinear velocity (VCL) and straight line velocity (VSL), amplitude lateral head displacement (ALH) and average orientation change (AOC)) and morphology characteristics (percentage normal sperm, head defects and detached heads) had desirable outcomes with 25OHVD concentrations between 120-180 nmol/l. Using bivariable analysis, positive associations were observed with 25OHVD and some semen quality characteristics from 4 to 8 years of age (motility, progressive motility, BCF, DCL, VCL, ALH, AOC) and at transformed prostate volumes smaller than or equal to 4.5 (motility, progressive motility, DCL, VCL, and normal morphology) while negative associations of these semen parameters were found at ages greater than 8 years and transformed prostate volumes greater than or equal to 5.5. Head defects were negatively associated with 25OHVD. Vitamin D may have an impact on spermatogenesis and normal sperm physiology that warrants further research. The prolactin study showed no statistically significant associations between serum PRL and BPH and serum PRL and sperm motility characteristics. However, two sperm morphology characteristics (percentage proximal droplets and percentage midpiece defects) had significant negative associations with PRL concentrations. Age interaction with PRL was also a factor in the percentage of midpiece defects with desirable outcomes associated at 4 years of age compared with older ages. Overall, undesirable outcomes occurred at PRL concentrations less than 2.5 ng/ml. In conclusion, both 25OHVD and PRL may have important roles in spermatogenesis and normal sperm physiology in the dog. / Ontario Veterinary College Pet Trust

Objective: Study the effects of vitamin D on viral driven innate immune responses, by looking at differences in cytokine production, receptor expression, and endogenous vitamin D levels. Methods: Primary peripheral blood mononuclear cells (PBMC) and epithelial cells (EC) were cultured in the presence of viral ligands and vitamin D. Enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) were used to determine cytokine production and mRNA expression. Results: PBMC stimulated with toll-like receptor 4 ligand (TLR4L), but not viral TLR8L, led to decreased pro- and anti-inflammatory cytokine production in the presence of 1,25(OH)2D3. RIG-like receptor (RLR) activation, on the other hand, in primary EC exhibited decreased pro-inflammatory cytokine production in the presence of vitamin D. Conclusions: Our findings are among the first to show differences between bacterial and viral driven innate immune responses in the presence of vitamin D. As responsiveness in RLR activated primary EC was altered in the presence of vitamin D, our data reveal the importance of studying the immune system as a whole.

Objective: Study the effects of vitamin D on viral driven innate immune responses, by looking at differences in cytokine production, receptor expression, and endogenous vitamin D levels. Methods: Primary peripheral blood mononuclear cells (PBMC) and epithelial cells (EC) were cultured in the presence of viral ligands and vitamin D. Enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) were used to determine cytokine production and mRNA expression. Results: PBMC stimulated with toll-like receptor 4 ligand (TLR4L), but not viral TLR8L, led to decreased pro- and anti-inflammatory cytokine production in the presence of 1,25(OH)2D3. RIG-like receptor (RLR) activation, on the other hand, in primary EC exhibited decreased pro-inflammatory cytokine production in the presence of vitamin D. Conclusions: Our findings are among the first to show differences between bacterial and viral driven innate immune responses in the presence of vitamin D. As responsiveness in RLR activated primary EC was altered in the presence of vitamin D, our data reveal the importance of studying the immune system as a whole.