Methylation of the Vitamin D Receptor (VDR) Gene, Together with Genetic Variation, Race, and Environment Influence the Signaling Efficacy of the Toll-Like Receptor 2/1-VDR Pathway.

BACKGROUND: The disparity in prevalence of infectious diseases across the globe is common knowledge. Vitamin D receptor (VDR)-mediated toll-like receptor (TLR) 2/1 signaling produces antimicrobial peptides, which is critical as a first line of defense in innate immunity. Numerous studies disclosed the independent role of genetic polymorphisms in this pathway, vitamin D status or season and more recently epigenetics, as factors contributing to infectious disease predisposition. Few studies address the interaction between environment, genetics, and epigenetics. Here, we hypothesized that VDR-mediated TLR2/1 signaling is influenced by a combination of environment, epigenetics and genetics, collectively influencing differential innate immunity. METHODS: Healthy Black and White South Africans (n = 100) donated blood, while ultraviolet index (UVI) was recorded for the duration of the study. LC-MS/MS supported 25(OH)D3 quantification. Monocyte/macrophage cultures, supplemented with/without 1,25(OH)2D3, were activated with the TLR2/1 elicitor, Pam3CSK4. VDR, cathelicidin antimicrobial peptide, hCAP-18, and 25-hydroxyvitamin D3-24-hydroxylase expression were quantified by RT-qPCR or flow cytometry. Pyrosequencing facilitated VDR methylation analysis and single-nucleotide polymorphism (SNP) genotyping in regions pinpointed through a bioinformatics workflow. RESULTS: Season interacted with race showing 25(OH)D3 deficiency in Blacks. UVI correlated with 25(OH)D3 and VDR methylation, likely influencing race differences in the latter. Regarding the TLR2/1 pathway, race differences in SNP genotype distribution were confirmed and functional analysis of VDR-mediated signaling showed interaction between race, season, and 25(OH)D3 status. Multivariate OPLS-DA mirrored several interactions between UVI, 25(OH)D3 status, DNA sequence, and methylation variants. Methylation of the third cytosine-phosphate-guanine dinucleotide (CpG) in the promoter CpG island (CGI) 1062, CGI 1062 CpG 3, significantly discriminated a 5.7-fold above average mean in VDR protein level upon TLR2/1 elicitation, the variation of which was further influenced by 25(OH)D3 status and the VDR SNP TaqI. CONCLUSION: Regulation of VDR-mediated TLR2/1 signaling is multifactorial, involving interaction between environment [UVI and consequent 25(OH)D3 status], epigenetics (VDR methylation at key regulatory sites), and genetics (TLR1, TIRAP, and VDR SNPs).

Efficacy of methylene blue in an experimental model of calcium channel blocker-induced shock.

STUDY OBJECTIVE: Calcium channel blocker poisonings account for a substantial number of reported deaths from cardiovascular drugs. Although supportive care is the mainstay of treatment, experimental therapies such as high-dose insulin-euglycemia and lipid emulsion have been studied in animal models and used in humans. In the most severe cases, even aggressive care is inadequate and deaths occur. In both experimental models and clinical cases of vasodilatory shock, methylene blue improves hemodynamic measures. It acts as a nitric oxide scavenger and inhibits guanylate cyclase that is responsible for the production of cyclic guanosine monophosphate (cGMP). Excessive cGMP production is associated with refractory vasodilatory shock in sepsis and anaphylaxis. The aim of this study is to determine the efficacy of methylene blue in an animal model of amlodipine-induced shock. METHODS: Sprague-Dawley rats were anesthetized, ventilated, and instrumented for continuous blood pressure and pulse rate monitoring. The dose of amlodipine that produced death within 60 minutes was 17 mg/kg per hour (LD50). Rats were divided into 2 groups: amlodipine followed by methylene blue or amlodipine followed by normal saline solution, with 15 rats in each group. Rats received methylene blue at 2 mg/kg during 5 minutes or an equivalent amount of normal saline solution in 3 intervals from the start of the protocol: minutes 5, 30, and 60. The animals were observed for a total of 2 hours after the start of the protocol. Mortality risk and survival time were analyzed with Fisher's exact test and Kaplan-Meier survival analysis with the log rank test. RESULTS: Overall, 1 of 15 rats (7%) in the saline solution-treated group survived to 120 minutes compared with 5 of 15 (33%) in the methylene blue-treated group (difference -26%; 95% confidence interval [CI] -54% to 0.3%). The median survival time for the normal saline solution group was 42 minutes (95% CI 28.1 to 55.9 minutes); for the methylene blue group, 109 minutes (95% CI 93.9 to 124.1 minutes). Pulse rate and mean arterial pressure (MAP) differences between groups were analyzed until 60 minutes. Pulse rate was significantly higher in the methylene blue-treated group beginning 25 minutes after the start of the amlodipine infusion (95% CI 30 to 113 minutes) that was analyzed until 60 minutes. MAP was significantly higher in the methylene blue-treated group starting 25 minutes after the amlodipine infusion (95% CI 2 to 30 minutes) that was analyzed until 60 minutes. CONCLUSION: Methylene blue did not result in a significant difference in mortality risk. There was an increased pulse rate, MAP, and median survival time in the methylene blue group.

Vitamin D Receptor Gene Expression and Function in a South African Population: Ethnicity, Vitamin D and FokI.

Polymorphisms of the vitamin D receptor gene (VDR) have been associated inconsistently with various diseases, across populations of diverse origin. The T(f) allele of the functional SNP FokI, in exon 2 of VDR, results in a longer vitamin D receptor protein (VDR) isoform, proposed to be less active. Genetic association of VDR with disease is likely confounded by ethnicity and environmental factors such as plasma 25(OH)D3 status. We hypothesized that VDR expression, VDR level and transactivation of target genes, CAMP and CYP24A1, depend on vitamin D, ethnicity and FokI genotype. Healthy volunteers participated in the study (African, n = 40 and White, n = 20). Plasma 25(OH)D3 levels were quantified by LC-MS and monocytes cultured, with or without 1,25(OH)2D3. Gene expression and protein level was quantified using qRT-PCR and flow cytometry, respectively. Mean plasma 25(OH)D3 status was normal and not significantly different between ethnicities. Neither 25(OH)D3 status nor 1,25(OH)2D3 supplementation significantly influenced expression or level of VDR. Africans had significantly higher mean VDR protein levels (P<0.050), nonetheless transactivated less CAMP expression than Whites. Genotyping the FokI polymorphism by pyrosequencing together with HapMap data, showed a significantly higher (P<0.050) frequency of the CC genotype in Africans than in Whites. FokI genotype, however, did not influence VDR expression or VDR level, but influenced overall transactivation of CAMP and 1,25(OH)2D3-elicited CYP24A1 induction; the latter, interacting with ethnicity. In conclusion, differential VDR expression relates to ethnicity, rather than 25(OH)D3 status and FokI genotype. Instead, VDR transactivation of CAMP is influenced by FokI genotype and, together with ethnicity, influence 1,25(OH)2D3-elicited CYP24A1 expression. Thus, the expression and role of VDR to transactivate target genes is determined not only by genetics, but also by ethnicity and environment involving complex interactions which may confound disease association.