Vitamin D regulates microbiome-dependent cancer immunity.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

Loss of the glycosyltransferase Galnt11 affects vitamin D homeostasis and bone composition.

O-glycosylation is a conserved posttranslational modification that impacts many aspects of organismal viability and function. Recent studies examining the glycosyltransferase Galnt11 demonstrated that it glycosylates the endocytic receptor megalin in the kidneys, enabling proper binding and reabsorption of ligands, including vitamin D-binding protein (DBP). Galnt11-deficient mice were unable to properly reabsorb DBP from the urine. Vitamin D plays an essential role in mineral homeostasis and its deficiency is associated with bone diseases such as rickets, osteomalacia, and osteoporosis. We therefore set out to examine the effects of the loss of Galnt11 on vitamin D homeostasis and bone composition. We found significantly decreased levels of serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, consistent with decreased reabsorption of DBP. This was accompanied by a significant reduction in blood calcium levels and a physiologic increase in parathyroid hormone (PTH) in Galnt11-deficient mice. Bones in Galnt11-deficient mice were smaller and displayed a decrease in cortical bone accompanied by an increase in trabecular bone and an increase in a marker of bone formation, consistent with PTH-mediated effects on bone. These results support a unified model for the role of Galnt11 in bone and mineral homeostasis, wherein loss of Galnt11 leads to decreased reabsorption of DBP by megalin, resulting in a cascade of disrupted mineral and bone homeostasis including decreased circulating vitamin D and calcium levels, a physiological increase in PTH, an overall loss of cortical bone, and an increase in trabecular bone. Our study elucidates how defects in O-glycosylation can influence vitamin D and mineral homeostasis and the integrity of the skeletal system.

The Molecular Aspects of Functional Activity of Macrophage-Activating Factor GcMAF.

Group-specific component macrophage-activating factor (GcMAF) is the vitamin D3-binding protein (DBP) deglycosylated at Thr420. The protein is believed to exhibit a wide range of therapeutic properties associated with the activation of macrophagal immunity. An original method for GcMAF production, DBP conversion to GcMAF, and the analysis of the activating potency of GcMAF was developed in this study. Data unveiling the molecular causes of macrophage activation were obtained. GcMAF was found to interact with three CLEC10A derivatives having molecular weights of 29 kDa, 63 kDa, and 65 kDa. GcMAF interacts with high-molecular-weight derivatives via Ca2+-dependent receptor engagement. Binding to the 65 kDa or 63 kDa derivative determines the pro- and anti-inflammatory direction of cytokine mRNA expression: 65 kDa-pro-inflammatory (TNF-α, IL-1ß) and 63 kDa-anti-inflammatory (TGF-ß, IL-10). No Ca2+ ions are required for the interaction with the canonical 29 kDa CLEC10A. Both forms, DBP protein and GcMAF, bind to the 29 kDa CLEC10A. This interaction is characterized by the stochastic mRNA synthesis of the analyzed cytokines. Ex vivo experiments have demonstrated that when there is an excess of GcMAF ligand, CLEC10A forms aggregate, and the mRNA synthesis of analyzed cytokines is inhibited. A schematic diagram of the presumable mechanism of interaction between the CLEC10A derivatives and GcMAF is provided. The principles and elements of standardizing the GcMAF preparation are elaborated.

Vitamin D Binding Protein and Postsurgical Outcomes and Tissue Injury Markers After Hip Fracture: A Prospective Study.

CONTEXT: Hip fracture is a serious injury that can lead to increased morbidity and mortality. Vitamin D binding protein (DBP) is a potential prognostic indicator of outcomes since it is important for actin scavenging and inflammation after tissue injury. OBJECTIVE: To determine whether circulating DBP is associated with mobility or mortality after hip fracture and its association with acute tissue injury markers. METHODS: Post hoc analysis of a multisite North American prospective study of 260 patients with hip fracture; mobility assessed at 30 and 60 days and mortality at 60 days after repair surgery. Biochemical markers were measured before, and 2 to 4 days after surgery. Tissue injury markers were measured in 100 randomly selected patients and controls. The primary outcome was mobility and mortality by DBP tertiles. Secondary outcomes were assessment of pre- and postoperative biomarkers. RESULTS: Among all patients (81 ± 9 years, BMI 25 ± 4 kg/m2; 72% female), the highest DBP tertile had greater mobility at 30 (OR: 2.66; 95% CI: 1.43, 4.92; P = .002) and 60 days (OR: 2.31; 95% CI: 1.17, 4.54; P = .014) and reduced mortality (OR: 0.18; 95% CI: 0.04, 0.86; P = .032) compared with the lowest DBP tertile (<28.0 mg/dL). Total 25-hydroxyvitamin-D did not differ between tertiles (22.0 ± 9.5 ng/mL). Circulating DBP and gelsolin were lower and interleukin-6, C-reactive protein, and F-actin were higher (P < .01) in patients vs controls, and worsened (P < .01) after surgery. CONCLUSION: High circulating DBP concentrations are associated with better mobility and reduced mortality after hip fracture surgery. The role of DBP as an acute phase reactant to tissue injury and clinical outcomes should be addressed in future study.

Association of functional, inherited vitamin D-binding protein variants with melanoma-specific death.

BACKGROUND: It is unclear whether genetic variants affecting vitamin D metabolism are associated with melanoma prognosis. Two functional missense variants in the vitamin D-binding protein gene (GC), rs7041 and rs4588, determine 3 common haplotypes, Gc1s, Gc1f, and Gc2, of which Gc1f may be associated with decreased all-cause death among melanoma patients based on results of a prior study, but the association of Gc1f with melanoma-specific death is unclear. METHODS: We investigated the association of the Gc1s, Gc1f, and Gc2 haplotypes with melanoma-specific and all-cause death among 4490 individuals with incident, invasive primary melanoma in 2 population-based studies using multivariable Cox-proportional hazards regression. RESULTS: In the pooled analysis of both datasets, the patients with the Gc1f haplotype had a 37% lower risk of melanoma-specific death than the patients without Gc1f (hazard ratio [HR] = 0.63, 95% confidence interval [CI] = 0.47 to 0.83, P = .001), with adjustments for age, sex, study center, first- or higher-order primary melanoma, tumor site, pigmentary phenotypes, and Breslow thickness. Associations were similar in both studies. In pooled analyses stratified by Breslow thickness, the corresponding melanoma-specific death HRs for those patients with the Gc1f haplotype compared with those without Gc1f were 0.89 (95% CI = 0.63 to 1.27) among participants with tumor Breslow thickness equal to or less than 2.0 mm and 0.40 (95% CI = 0.25 to 0.63) among participants with tumor Breslow thickness greater than 2.0 mm (Pinteraction = .003). CONCLUSIONS: Our findings suggest that individuals with the GC haplotype Gc1f may have a lower risk of dying from melanoma-specifically from thicker, higher-risk melanoma-than individuals without this Gc1f haplotype.

Investigation of the Changes in Concentrations of Vitamin D-Binding Protein and Lactoferin in Plasma and Peritoneal Fluid of Patients with Endometriosis.

An evaluation of the association between the concentrations of vitamin D-binding protein and lactoferrin in the plasma and peritoneal fluid may facilitate the elucidation of molecular mechanisms in endometriosis. Vitamin D-binding protein and lactoferrin concentrations were measured by ELISA in plasma and peritoneal fluid samples from 95 women with suspected endometriosis as classified by laparoscopy into groups with (n = 59) and without endometriosis (n = 36). There were no differences (p > 0.05) in the plasma and peritoneal fluid concentrations of vitamin D-binding protein and lactoferrin between women with and without endometriosis. In women with endometriosis, there was a significant correlation between plasma and peritoneal fluid vitamin D-binding protein concentrations (r = 0.821; p = 0.000), but there was no correlation between lactoferrin concentrations in those compartments (r = 0.049; p > 0.05). Furthermore, in endometriosis, lactoferrin was found to correlate poorly with vitamin D-binding protein (r= -0.236; p > 0.05) in plasma, while in the peritoneal fluid, the correlation between those proteins was significant (r = 0.399; p = 0.002). The characteristic properties of vitamin D-binding protein and lactoferrin and the associations between their plasma and peritoneal fluid concentrations found in women with endometriosis may provide a novel panel of markers to identify high-risk patients in need of further diagnostic measures.

Serum Vitamin D Levels Explored in the Latvian Cohort of Patients with Basal Cell Carcinoma Linked to the Sonic Hedgehog and Vitamin D Binding Protein Cutaneous Tissue Indices.

Ultraviolet radiation is known as one of the major contributors to skin malignancies, including basal cell carcinoma (BCC), which is the most common type of skin cancer. It is a heterogeneous tumor, which presents with various types that are stratified into low- and high-risk tumors. Sunlight is important for overall health and vitamin D synthesis in the skin, whereas deviations from the optimal level of vitamin D are shown to be associated with the risk of the development of BCC. The accumulating evidence suggests the ability of vitamin D to antagonize the Sonic Hedgehog (SHH) signaling, the key tumor pathway, and play a protective role in the development of BCC. Additionally, a vitamin D binding protein (DBP) is shown to be implicated in the complex regulation of vitamin D. Here, we aimed to explore serum vitamin D in patients with different primary and recurrent BCC of the head and neck and investigate cutaneous DBP and SHH indices, confirmed immunohistochemically in these subjects. According to the results, 94.9% of the Latvian cohort of BCC patients were found to be deficient in vitamin D. No significant differences in serum vitamin D levels were found between genders, primary and recurrent tumors, and different types of BCC. Serum vitamin D was inversely associated with tumor size. Susceptible male individuals with low blood vitamin D levels were recognized at risk of developing aggressive and recurrent BCC confirmed by the use of hierarchical clustering analysis. In smaller tumors with a favorable course, such as superficial and nodular BCC, the association between high DBP and low SHH tissue expression was found, providing supportive evidence of the existence of a link between vitamin D, proteins involved in its metabolism, as exemplified by the DBP and SHH signaling pathway. The assumption of a deficiency in the protective effect of vitamin D in patients with high-risk BCCs was proposed in low DBP and high SHH tissue indices. New extensions to existing knowledge and characterization of the BCC signaling pathways and their cross-talk with vitamin D are warranted when searching for a preferential effect of vitamin D on skin cancer.

Analysis of the Biological Properties of Blood Plasma Protein with GcMAF Functional Activity.

The main problem related to the studies focusing on group-specific component protein-derived macrophage-activating factor (GcMAF) is the lack of clarity about changes occurring in different types of macrophages and related changes in their properties under the effect of GcMAF in various clinical conditions. We analyzed the antitumor therapeutic properties of GcMAF in a Lewis carcinoma model in two clinical conditions: untreated tumor lesion and tumor resorption after exposure to Karanahan therapy. GcMAF is formed during site-specific deglycosylation of vitamin D3 binding protein (DBP). DBP was obtained from the blood of healthy donors using affinity chromatography on a column with covalently bound actin. GcMAF-related factor (GcMAF-RF) was converted in a mixture with induced lymphocytes through the cellular enzymatic pathway. The obtained GcMAF-RF activates murine peritoneal macrophages (p < 0.05), induces functional properties of dendritic cells (p < 0.05) and promotes in vitro polarization of human M0 macrophages to M1 macrophages (p < 0.01). Treatment of whole blood cells with GcMAF-RF results in active production of both pro- and anti-inflammatory cytokines. It is shown that macrophage activation by GcMAF-RF is inhibited by tumor-secreted factors. In order to identify the specific antitumor effect of GcMAF-RF-activated macrophages, an approach to primary reduction of humoral suppressor activity of the tumor using the Karanahan therapy followed by macrophage activation in the tumor-associated stroma (TAS) was proposed. A prominent additive effect of GcMAF-RF, which enhances the primary immune response activation by the Karanahan therapy, was shown in the model of murine Lewis carcinoma. Inhibition of the suppressive effect of TAS is the main condition required for the manifestation of the antitumor effect of GcMAF-RF. When properly applied in combination with any chemotherapy, significantly reducing the humoral immune response at the advanced tumor site, GcMAF-RF is a promising antitumor therapeutic agent that additively destroys the pro-tumor properties of macrophages of the tumor stroma.

Total, bioavailable and free 25-hydroxyvitamin D are associated with the prognosis of patients with non-small cell lung cancer.

PURPOSE: To analyze the prognostic value of total, bioavailable and free 25-hydroxyvitamin D [25(OH)D] as well as vitamin D-binding protein (VDBP) in patients with non-small cell lung cancer (NSCLC). METHODS: We prospectively collected and analyzed data for 395 patients diagnosed with NSCLC between January 2016 and December 2018 in two university-affiliated hospitals. Total and free 25(OH)D and VDBP were measured directly, and bioavailable 25(OH)D was calculated using a validated formula. Their prognostic values were evaluated by Cox proportional hazards model, and hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated. RESULTS: Patients with NSCLC had significantly lower levels of total, bioavailable, and free 25(OH)D and higher VDBP levels in comparison to healthy controls (all p < 0.001). In multivariate analyses, higher levels of total, bioavailable, and free 25(OH)D were independently associated better overall survival (OS) and progression-free survival (PFS). For OS, the adjusted HRs were 0.58 (95% CI, 0.40-0.87; p for trend = 0.008), 0.45 (95% CI, 0.30-0.67; p for trend < 0.001) and 0.49 (95% CI, 0.33-0.73; p for trend < 0.001) for the highest versus the lowest tertile of total, bioavailable and free 25(OH)D, respectively. The corresponding adjusted HRs for PFS were 0.61 (95% CI, 0.43-0.86; p for trend = 0.006), 0.56 (95% CI, 0.40-0.80; p for trend = 0.001) and 0.60 (95% CI, 0.42-0.85; p for trend = 0.004), respectively. However, VDBP was not associated with either OS or PFS. CONCLUSION: The current study suggested that total, bioavailable and free 25(OH)D may be reliable prognosis indicators in NSCLC patients, though the optimal 25(OH)D form for NSCLC prognosis remains to be assessed in future studies.

Potential roles of vitamin D binding protein in attenuating liver injury in sepsis.

BACKGROUND: In sepsis, vitamin D binding protein (VDBP) has been shown to be low-expressed. The current study examined the relationship between serum VDBP level and liver injury in sepsis patients, as well as in a mouse model for sepsis and in cultured liver epithelial cell line exposed to lipopolysaccharide (LPS). METHODS: The human study included 78 sepsis patients and 50 healthy volunteers. Sepsis patients were categorized into sepsis survivor group (n = 43) and sepsis non-survivor group (n = 35) based on 28-day mortality for data analysis. Adult male C57BL/6 mice were subjected to cecal ligation and puncture (CLP). Serum samples were collected on day 1, 3, 5 and 7 to determine the levels of VDBP, 25-hydroxyvitamin D [25(OH)D3], 1,25-dihydroxyvitamin D [1,25(OH)2D3], interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α). Potential protective effects of VDBP overexpression against LPS-induced liver damage were examined in cultured THLE2 cells. RESULTS: Serum levels of VDBP, 25(OH)D3, and 1,25(OH)2D3 were significantly lower in sepsis patients vs. the healthy control (P < 0.001), as well as in the sepsis non-survivor group vs. the sepsis survivor group (P < 0.001, P = 0.0338, or P = 0.0013, respectively). Lower serum VDBP level was associated with higher Acute Physiology and Chronic Health Evaluation (APACHE) II score (r = - 0.2565, P = 0.0234) and Sequential Organ Failure Assessment score (r = - 0.3522, P = 0.0016), but lower serum albumin (ALB, r = 0.4628, P < 0.001) and total protein (TP, r = 0.263, P = 0.02). In CLP mice, there was a 5-day period of serum VDBP reduction, followed by return towards the baseline on day 7. VDBP was also decreased in LPS-treated THLE2 cells (P < 0.001). VDBP overexpression reduced LPS-induced THLE2 damage. Reduced damage was associated with decreased oxidative stress and inactivation of the c-Jun N-terminal kinase signaling pathway. CONCLUSION: VDBP may be protective against sepsis-induced liver injury.

Macrophages Control the Bioavailability of Vitamin D and Vitamin D-Regulated T Cell Responses.

The active form of vitamin D3 (1,25(OH)2D3) has a great impact on T cell effector function. Thus, 1,25(OH)2D3 promotes T helper 2 (Th2) and regulatory T (Treg) cell function and concomitantly inhibits Th1 and Th17 cell function. Thus, it is believed that vitamin D exerts anti-inflammatory effects. However, vitamin D binding protein (DBP) strongly binds both 1,25(OH)2D3 and the precursor 25(OH)D3, leaving only a minor fraction of vitamin D in the free, bioavailable form. Accordingly, DBP in physiological concentrations would be expected to block the effect of vitamin D on T cells and dendritic cells. In the present study, we show that pro-inflammatory, monocyte-derived M1 macrophages express very high levels of the 25(OH)D-1α-hydroxylase CYP27B1 that enables them to convert 25(OH)D3 into 1,25(OH)2D3 even in the presence of physiological concentrations of DBP. Co-cultivation of M1 macrophages with T cells allows them to overcome the sequestering of 25(OH)D3 by DBP and to produce sufficient levels of 1,25(OH)2D3 to affect T cell effector function. This study suggests that in highly inflammatory conditions, M1 macrophages can produce sufficient levels of 1,25(OH)2D3 to modify T cell responses and thereby reduce T cell-mediated inflammation via a vitamin D-mediated negative feed-back loop.

Targeting a cell surface vitamin D receptor on tumor-associated macrophages in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive tumor with limited treatment options and poor prognosis. We applied the in vivo phage display technology to isolate peptides homing to the immunosuppressive cellular microenvironment of TNBC as a strategy for non-malignant target discovery. We identified a cyclic peptide (CSSTRESAC) that specifically binds to a vitamin D receptor, protein disulfide-isomerase A3 (PDIA3) expressed on the cell surface of tumor-associated macrophages (TAM), and targets breast cancer in syngeneic TNBC, non-TNBC xenograft, and transgenic mouse models. Systemic administration of CSSTRESAC to TNBC-bearing mice shifted the cytokine profile toward an antitumor immune response and delayed tumor growth. Moreover, CSSTRESAC enabled ligand-directed theranostic delivery to tumors and a mathematical model confirmed our experimental findings. Finally, in silico analysis showed PDIA3-expressing TAM in TNBC patients. This work uncovers a functional interplay between a cell surface vitamin D receptor in TAM and antitumor immune response that could be therapeutically exploited.

Association of Vitamin D Receptor and Vitamin D-Binding Protein Polymorphisms with Familial Breast Cancer Prognosis in a Mono-Institutional Cohort.

Low 25-hydroxyvitamin D (25OHD) has been associated with an increased cancer incidence and poorer prognosis. Single nucleotide polymorphisms (SNPs) of vitamin D receptor (VDR) and vitamin D binding protein (GC gene) may interfere with vitamin D activity. This study assesses the role of VDR and GC SNPs on breast cancer (BC) recurrence and survival in a cohort of patients with a family history of breast cancer, without the pathogenic variant for BRCA1 and BRCA2. A consecutive series of patients who underwent genetic testing were genotyped for VDR and GC genes. Specifically, ApaI, FokI, TaqI, BsmI and rs2282679, rs4588, rs7041 SNPs were determined. A total of 368 wild type (WT) patients with BC were analyzed for VDR and GC SNPs. The GC rs2282679 minor allele was significantly associated with luminal subtype of the primary tumor compared to Her2+/TN breast cancer (p = 0.007). Multivariate Cox models showed that BmsI and TaqI are significantly associated with BC outcome. Patients with the major alleles showed more than 30% lower hazard of relapse (BsmI p = 0.02 and TaqI p = 0.03). Our study supports the evidence for a pivotal role of 25OHD metabolism in BC. GC SNPs may influence the hormone tumor responsiveness and VDR may affect tumor prognosis.

Prediagnostic Circulating Concentrations of Vitamin D Binding Protein and Survival among Patients with Colorectal Cancer.

BACKGROUND: Higher total 25-hydroxyvitamin D [25(OH)D] levels are associated with improved survival among patients with colorectal cancer, but the relationships between circulating vitamin D binding protein (VDBP), and bioavailable or free 25(OH)D, and colorectal cancer survival remain unknown. METHODS: We examined the associations between prediagnostic plasma levels of vitamin D-related markers and survival among 603 White participants diagnosed with colorectal cancer from two prospective U.S. cohorts. Plasma VDBP and total 25(OH)D were directly measured, while bioavailable and free 25(OH)D was calculated using a validated formula on the basis of total 25(OH)D, VDBP, and albumin levels. Cox proportional hazards regression was used to estimate HRs for overall and colorectal cancer-specific mortality, with adjustment for other prognostic markers and potential confounders. RESULTS: Higher VDBP levels were associated with improved overall (P trend = 0.001) and colorectal cancer-specific survival (P trend = 0.02). Compared with patients in the lowest quartile, those in the highest quartile of VDBP had a multivariate HR of 0.58 [95% confidence interval (CI), 0.41-0.80] for overall mortality and 0.58 (95% CI, 0.37-0.91) for colorectal cancer-specific mortality. The results remained similar after further adjustment for total 25(OH)D levels. In contrast, neither bioavailable nor free 25(OH)D levels were associated with overall or colorectal cancer-specific mortality (all P trend > 0.15). CONCLUSIONS: Prediagnostic circulating concentrations of VDBP were positively associated with survival among patients with colorectal cancer. IMPACT: The clinical utility of VDBP as a prognostic marker warrants further exploration, as well as research into underlying mechanisms of action.

Vitamin D-Binding Protein in Pregnancy and Reproductive Health.

Vitamin D-binding protein (VDBP), the main carrier of vitamin D, has recently been implicated in reproductive health and pregnancy outcomes including endometriosis, polycystic ovary syndrome (PCOS), pre-eclampsia, and gestational diabetes mellitus (GDM). Improved methods for measuring VDBP and an increased understanding of its role in biological processes have led to a number of newly published studies exploring VDBP in the context of pregnancy. Here, we synthesize the available evidence regarding the role of VDBP in reproductive health and pregnancy, and we highlight areas requiring further study. Overall, low levels of maternal serum VDBP concentrations have been associated with infertility, endometriosis, PCOS and spontaneous miscarriage, as well as adverse pregnancy outcomes including GDM, pre-eclampsia, preterm birth and fetal growth restriction. However, increased VDBP concentration in cervicovaginal fluid has been linked to unexplained recurrent pregnancy loss and premature rupture of membranes. Some genetic variants of VDBP have also been associated with these adverse outcomes. Further studies using more accurate VDBP assays and accounting for ethnic variation and potential confounders are needed to clarify whether VDBP is associated with reproductive health and pregnancy outcomes, and the mechanisms underlying these relationships.

The Relationship between Vitamin D Metabolites and Androgens in Women with Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age, with hyperandrogenism present in up to 90% of affected women. Some evidence suggests a link between vitamin D deficiency and PCOS features via insulin resistance and inflammation. Our aim was to explore the relationship between biochemical markers of vitamin D status and androgens in women with PCOS. This cross-sectional study used bio-banked samples from 46 pre-menopausal women with PCOS (mean ± SD: age 30 ± 6 years; BMI 29 ± 6 kg/m2). We measured 25-hydroxyvitamin D (25[OH]D), vitamin D-binding protein (DBP), total testosterone, sex hormone-binding globulin (SHBG), and calculated the free androgen index (FAI) and bioavailable and free 25(OH)D. Fasting glucose and insulin were used to calculate the homeostatic model assessment of insulin resistance (HOMA-IR) and body fat percentage was determined via dual energy x-ray absorptiometry. High-sensitivity C-reactive protein (hs-CRP) was measured as a marker of inflammation. DBP was positively associated with total 25(OH)D and expectedly, negatively associated with free 25(OH)D. There were no associations between vitamin D metabolites and total testosterone, SHBG or FAI, even after adjusting for age, body fat percentage, HOMA-IR and hs-CRP. We found no associations between vitamin D metabolites and androgens in women with PCOS. Studies that have identified a vitamin D-androgen link have largely relied on methodology with numerous pitfalls; future studies should exclusively use gold-standard measures to confirm these findings in this population.

Vitamin D - Deglycosylated Vitamin D Binding Protein Dimer: Positive Synergistic Effects on Recognition, Activation, Phagocytosis and Oxidative Stress on Macrophages.

BACKGROUND: We have recently shown positive effects in the quality of life in autism and amyloid lateral sclerosis patients using a newly developed 25-OH vitamin D deglycosylated vitamin D binding protein complex (VitD~dgVDBP) by reducing oxidative stress. The question arises whether this reduction of oxidative stress was due to a synergistic effect of the dimer in the recognition and activation of phagocytosis on macrophages combined with a lower oxidative burst compared to the VitD free proteins, namely vitamin D binding protein (VDBP: Gc Protein) and deglycosylated dgVDBP (GcMAF). METHODS: VDBP sandwich ELISA of equal protein concentration of VDBP, dgVDBP, and VitD~dgVDBP (1 µg/ mL by BCA protein technique) was used to identify immune affinity to polyclonal antibodies raised against human VDBP. The 25(OH) vitamin D levels of VDBP, dgVDBP and VitD~dgVDBP were estimated by a competitive immune assay using a monoclonal antibody. Macrophage phagocytosis and oxidative burst in absence or presence of 400 pg/mL VDBP, 400 pg/mL dgVDBP, and 400 pg/mL VitD~dgVDBP was measured. RESULTS: The recognition of the antibody against VDBP protein was significantly more than 4-fold higher for VitD~dgVDBP (769.2 +/- 35.1%) compared to dgVDBP (186.5 +/- 16.8 %; p < 0.01) and 7-fold higher to VDBP (100 +/- 11.4 %; p < 0.001). 25(OH) vitamin D levels of VDBP (20.7 nmol/mg; p < 0.001) and dgVDBP (28.8 +/- 3.9 nmol/mL; p < 0.001) was significantly lower than of VitD~dgVDBP (324.0 +/- 12.8 nmol/mL). The calculated VitD/ protein ratio showed significantly higher results in favor of VitD~dgVDBP (1.01 +/- 0.12) compared to dgVDBP (0.06 +/- 0.03; p < 0.001) and VDBP (0.05 +/- 0.01; p < 0.001). The estimation of macrophage phagocytosis rate of VitD~dgVDBP (5,864.3 +/- 742.2 cps) was significantly higher compared to dgVDBP (2,789.6 +/- 102.7 cps; p < 0.01) and VDBP (1,134.3 +/- 135.9 cps) whereas the production of macrophage superoxide anion radicals showed significantly higher levels of dgVDBP (255.3 +/- 14.5 cps) in comparison to VDBP (148.6 +/- 24.7 cps, p < 0.01) and VitD~dgVDBP (142.3 +/- 20.0 cps; p < 0.001). Linear regression between VDBP antibody affinity and macrophage phagocytosis of VDBP, dgVDBP and VitD~dgVDBP resulted in a correlation coefficient of r = 0.95 in favor of VitD~dgVDBP. CONCLUSIONS: VitD~dgVDBP (Il-42) showed higher macrophage activation and lower oxidative burst than VitD free dgVDBP (GcMaf) and VDBP (Gc) which may result from a synergistic effect by presenting protein bound Vitamin D better to macrophages.

Association of genetic variations in the vitamin D pathway with susceptibility to tuberculosis in Kazakhstan.

Tuberculosis (TB) poses an important health challenge and a significant economic burden for Kazakhstan and in Central Asia. Recent findings show a number of immunological related processes and host Mycobacterium tuberculosis defense are impacted by a variety of genes of the human host including those that play a part in the vitamin D metabolism. We investigated the genetic variation of genes in the vitamin D metabolic pathway of a cohort 50 TB cases in Kazakhstan and compared them to 34 controls living in the same household with someone infected with TB. We specifically analyzed 11 SNPs belonging to the following genes: DHCR7, CYP2R1, GC-1, CYP24A1, CYP27A1, CYP27B1, VDR and TNFα. These genes play a number of different roles including synthesis, activation, delivery and binding of the activated vitamin D. Our preliminary results indicate significant association of VDR (vitamin D receptor) SNPs (rs1544410, BsmI, with OR = 0.425, CI 0.221-0.816, p = 0.009 and rs731236, TaqI with OR = 0.443, CI 0.228-0.859, p = 0.015) and CYP24A1 (rs6013897 with OR = 0.436, CI 0.191-0.996, p = 0.045) with TB. Interaction of genetic variation of VDR and CYP24A1 may impact susceptibility to TB. The findings provided initial clues to understand individual genetic differences in relation to susceptibility and protection to TB.

Characteristic Morphological Changes and Rapid Actin Accumulation in Serum-MAF-treated Macrophages.

BACKGROUND/AIM: Serum-derived macrophage activating factor, serum-MAF, is known to increase the phagocytic activity of macrophages by enhancing the engulfment efficiency. To elucidate the mechanisms underlying phagocytic activation, morphological changes were observed and analyzed. MATERIALS AND METHODS: Morphological changes in macrophages were observed and quantitatively analyzed using scanning electron microscope (SEM) and confocal microscope. RESULTS: SEM and confocal microscopy images revealed frill-like structures and active actin accumulations, respectively, in serum-MAF treated macrophages. Actin accumulation was induced within 5 min following serum-MAF treatment. CONCLUSION: Serum-MAF induced a rapid rearrangement of cytoskeletal actin and enhanced phagocytic activity. Findings of the current study may contribute to the development of techniques that facilitate activation of the human immune system, which in turn may be beneficial for cancer immunotherapy.

Induction of α cell-restricted Gc in dedifferentiating ß cells contributes to stress-induced ß-cell dysfunction.

Diabetic ß cell failure is associated with ß cell dedifferentiation. To identify effector genes of dedifferentiation, we integrated analyses of histone methylation as a surrogate of gene activation status and RNA expression in ß cells sorted from mice with multiparity-induced diabetes. Interestingly, only a narrow subset of genes demonstrated concordant changes to histone methylation and RNA levels in dedifferentiating ß cells. Notable among them was the α cell signature gene Gc, encoding a vitamin D-binding protein. While diabetes was associated with Gc induction, Gc-deficient islets did not induce ß cell dedifferentiation markers and maintained normal ex vivo insulin secretion in the face of metabolic challenge. Moreover, Gc-deficient mice exhibited a more robust insulin secretory response than normal controls during hyperglycemic clamps. The data are consistent with a functional role of Gc activation in ß cell dysfunction, and indicate that multiparity-induced diabetes is associated with altered ß cell fate.