Carbon cloth-based immunosensor for detection of 25-hydroxy vitamin D3.

Vitamin D (VD) deficiency is a global health concern due to its serious health impacts, and at present, the monitoring of VD status is expensive. Here, a novel immunosensor for sensitive and label-free detection of 25-hydroxy vitamin D3 (25VD3) is reported. Nanostructured cerium(IV) oxide (nCeO2) was anchored onto carbon cloth (CC) via electrophoretic deposition to fabricate a nanoplatform (nCeO2/CC). Subsequently, bioactive molecules (anti-25VD3 and BSA) were introduced to fabricate the nanobioplatform BSA/anti-25VD3/nCeO2/CC as an immunosensor. The analytical performance of the developed immunosensor was studied towards 25VD3 detection. The immunosensor provides a broad linear range of 1-200 ng mL-1, high sensitivity of 2.08 µA ng-1 mL cm-2, a detection limit of 4.63 ng mL-1, and a response time of 15 min, which is better than that of previous reports. The biosensor exhibited high selectivity, good reproducibility, and excellent stability for about 45 days. The potential application of the proposed immunosensor was observed for real serum samples towards 25VD3 detection that demonstrated a high correlation with the conventional enzyme-linked immunosorbent assay. Graphical abstract.

Maternal and Cord Blood 25-Hydroxyvitamin D3 Are Associated with Increased Cord Blood and Naive and Activated Regulatory T Cells: The Barwon Infant Study.

Vitamin D has shown immune-modulatory effects but mostly in in vitro and animal studies. Regulatory T cells (Treg) are important for a balanced immune system. The relationship between vitamin D on the number of circulating neonatal Treg is unclear. We sought to investigate the association between maternal and neonatal vitamin D metabolites and cord blood (CB) Treg subsets. In a cohort of Australian infants (n = 1074), recruited using an unselected antenatal sampling frame, 158 mother-infant pairs had data on the following: 1) 25-hydroxyvitamin D3 (25(OH)D3) measures in both maternal peripheral blood (28- to 32-wk gestation) and infant CB; 2) proportions (percentage of CD4+ T cells) of CB Treg subsets (CD4+CD45RA+ FOXP3low naive Treg, and CD4+CD45RA- FOXP3high activated Treg [aTreg]); and 3) possible confounders, including maternal personal UV radiation. Multiple regression analyses were used. The median 25(OH)D3 was 85.4 and 50.7 nmol/l for maternal and CB samples, respectively. Higher maternal 25(OH)D3 levels were associated with increased CB naive Treg (relative adjusted mean difference [AMD] per 25 nmol/l increase: 5%; 95% confidence interval [CI]: 1-9%), and aTreg (AMD per 25 nmol/l increase: 17%; 95% CI: 6-28%). Furthermore, a positive association between CB 25(OH)D3 levels and CB aTreg (AMD per 25 nmol/l increase: 29%; 95% CI: 13-48%) was also evident. These results persisted after adjustment for other factors such as maternal personal UV radiation and season of birth. 25(OH)D3, may play a role in the adaptive neonatal immune system via induction of FOXP3+ Tregs. Further studies of immune priming actions of antenatal 25(OH)D3 are warranted.

The Impact of Serum 25-Hydroxyvitamin D3 Levels on Allergic Rhinitis.

We aimed to clarify the relation between allergic rhinitis and the serum levels of 25-hydroxivitamin D in the adult population. The study group consisted of 86 patients with allergic rhinitis who were diagnosed with the help of history of allergy, positive signs for allergy, blood samples, and positive skin prick tests; while the control group included 43 age- and sex-matched healthy volunteers with negative skin prick tests. The demographic data, medical history, findings in the physical examinations, serum levels of total immunoglobulin E (IgE) and 25-hydroxyvitamin D, and skin prick test results of the groups were noted. A total of 129 patients fulfilling the necessary criteria were enrolled. The median serum 25-hydroxyvitamin D levels in the study group were significantly lower compared to the control group (P = .014). In the study group, median serum vitamin D levels were significantly higher in men, compared to women (P = .03). There was a significant negative correlation between IgE and vitamin D levels in the allergic rhinitis group (P = .028, r = -0.246). This study showed that patients with allergic rhinitis might be more vulnerable to have lower serum levels of vitamin D. Thus, vitamin D supplementation as an adjunctive therapy may be considered in those patients.

Vitamin D: Immunomodulatory Aspects.

Vitamin D is a group of liposoluble prohormones consisting of 5 different vitamins, the most important forms being vitamin D2 and vitamin D3. The ergocalciferol (vitamin D2) is less efficacious and derives from irradiated fungi, while colecalciferol (vitamin D3), derived from cholesterol, is synthesized via ultraviolet B rays in animal organisms. Only the ultraviolet B rays (290 to 315 nm) portion of the solar ray photolyzes 7-dehydrocholesterol in the skin to previtamin D3, which is converted subsequently to vitamin D3. Moreover, the skin makes little vitamin D from the sun at latitudes above 37 degrees north or below 37 degrees south of the equator. Calcidiol [25(OH)D] is the more stable metabolite of vitamin D in serum and the best indicator of the vitamin D status. Optimal values range are >30 ng/mL. Calcitriol [1,25(OH)2D] is the active hormone form of vitamin D. The 1,25(OH)2D binds to its nuclear receptor (vitamin D receptor), expressed in many tissues, regulating the expression of genes involved in calcium metabolism, cell differentiation, apoptosis, and immunity. About immunity, calcitriol stimulates innate immune responses by enhancing the chemotactic and phagocytotic responses of macrophages as well as the production of antimicrobial peptides. 1,25(OH)2D strongly enhances production of interleukine-10 by stimulating T regulatory cells and inhibiting Th1 and Th17 cell differentiation. Furthermore, several studies suggest that lower 25(OH)D serum levels are associated with an increased risk of respiratory infection at all ages in a dose-response manner.

Anti-annexin A5 antibodies and 25-hydroxy-cholecalciferol in female patients with primary antiphospholipid syndrome.

Vascular antiphospholipid syndrome (VAPS) and obstetric (OAPS) are different entities because some patients only develop thrombosis (without recurrent pregnancy losses) and vice versa. Only two articles have reported that low 25-hydroxy-cholecalciferol (vitamin D3, VD3) levels were not correlated with the presence of conventional antiphospholipid antibodies (aPL Abs: anticardiolipin (aCL), anti-beta2glycoprotein I (aß2gpI), and lupus anticoagulant (LA)), but no article analyzed the association of VD3 and anti-annexin A5 (aanxA5) Abs. The aim of our study was to investigate the association between VD3, multiple positivity of conventional aPL and aanxA5 Abs levels only in female OAPS vs. VAPS. Our study included 62 consecutive female PAPS patients. Concentrations of Abs were measured by ELISA, while VD3 levels were determined by immunochemiluminescence. Only 10/62 (16.13%) had sufficient (≥ 30 ng/ml) VD3 levels, while 48/62 (77.42%) and 4/62 (6.45%) had insufficiency and VD3 deficiency, respectively. Statistically significant VD3 deficiency was noticed in VAPS (vs. OAPS, P = 0.013). A negative correlation between VD3 levels and the age of patients was noticed (r = - 0.493, P = 0.032) only in VAPS subgroup. Multiple positivity of aPL and aanxA5 Abs was not associated with VD3 deficiency. Newly emerging aPL Abs, such as aanxA5 Abs, or their combinations with classical aPL Abs are not associated with VD3 deficiency in neither OAPS nor VAPS patients. Due to its immunomodulatory roles in B-Ly homeostasis, supplementation with VD3 should be considered in APS, at least in subgroup with severe form of the disease, i.e., VAPS.

Immunoreactivity to CYP24A1, but not vitamin D receptor, is increased in mast cells of keratinocyte skin cancers.

In mouse skin models, mast cells have been shown to express vitamin D receptor (VDR) that can mediate the immunosuppressive effects of ultraviolet B radiation and vitamin D3. However, VDR activation leads to the expression of CYP24A1, a hydroxylase that can inactivate vitamin D3 metabolites. To examine immunoreactivity to VDR and CYP24A1 in mast cells from normal human skin, keratinocyte skin cancers, and disorders of chronic inflammation. Frozen biopsies were collected from the non-lesional and lesional skin of patients with actinic keratosis (AK), Bowen's disease/squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and psoriasis. The expression of VDR and CYP24A1 in tryptase-positive mast cells was analysed using double-staining methods. Less than 0.5% of the mast cells were immunoreactive to VDR in both the non-lesional and lesional skin for all disease groups. In non-lesional skin, only 0.5-2.9% of the mast cells were immunopositive for CYP24A1, however, the percentage of mast cells containing CYP24A1 was significantly increased in lesional skin of AK, SCC, and BCC. In contrast to human skin, LAD2 mast cells cultured from a patient with mast cell sarcoma/leukaemia revealed that about 34% and 6.5% of the cells were immunopositive for VDR and CYP24A1, respectively. Whereas a very small proportion of mast cells in human skin express VDR and CYP24A1, the proportion of mast cells expressing CYP24A1 in keratinocyte skin cancers is increased; the mechanism underlying this is unclear.

Low utility of serum 25-hydroxyvitamin D3 and 1, 25-dihydroxyvitamin D3 in predicting peripheral Treg and Th17 cell counts in ESRD and renal transplant patients.

BACKGROUND: Vitamin D has shown an immune-modulatory effect in different studies. Vitamin D stimulates Tregs and inhibits Th17 cells. The immune-modulatory role of vitamin D in chronic kidney disease (CKD) and renal transplant patients is unclear. We measured whether different serum levels of vitamin D were associated with an increased or decreased presence of lymphocyte subsets including Treg and Th17 cells in end-stage renal disease (ESRD) and renal transplant recipients. METHODS: Eighty-seven renal transplant recipients and 53 end-stage renal disease (ESRD) patients were enrolled in this study. The absolute counts of CD4+ and CD8+ T, CD16+ CD56+ NK, CD19+ B, CD4+ CD25+ CD127- Foxp3+ (Tregs), Helios+ Tregs, CD38+ Tregs, and CD4+ CD17+ (Th17) cells were analyzed in peripheral blood in both patient groups. In addition, serum 25 (OH) D3, 1, 25 (OH)2 D3, IL-6, IL-17, IL-23, and TGF-ß1 were measured. The association between lymphocyte subset counts and 1, 25 (OH)2 D3 or 25 (OH) D3 was studied, as was the association between serum IL-6, IL-17, IL-23, or TGF-ß1 and 1,25 (OH)2 D3 or 25 (OH) D3. RESULTS: Serum 25 (OH) D3 and 1,25 (OH)2 D3 levels were not independently associated with peripheral CD4+ T, CD19+ B, CD16+ CD56+ NK, Treg, or Th17 cell counts. In contrast to serum 25 (OH) D3, serum1, 25 (OH)2 D3 was positively associated with CD8+ T cells counts in renal transplant recipients. CONCLUSION: Our findings indicate low utility of serum 25 (OH) D3 and 1, 25 (OH)2 D3 levels in predicting a change in lymphocyte subset counts in ESRD and renal transplant patients.

Improved accuracy of an tandem liquid chromatography-mass spectrometry method measuring 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D metabolites in serum using unspiked controls and its application to determining cross-reactivity of a chemiluminescent microparticle immunoassay.

Measurement of serum 25-hydroxyvitamin D [25(OH)D] is considered the best indicator of vitamin D status. Two minor vitamin D metabolites are common interferences encountered in 25(OH)D assays. The first is 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], which if not chromatographically resolved from 25-hydroxyvitamin D3 [25(OH)D3], can overestimate 25(OH)D concentrations. The second is 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3], which can cross-react with the antibodies in 25(OH)D immunoassays. Our aim was to develop an LC-MS/MS method capable of detecting both 3-epi-25(OH)D3 and 24R,25(OH)2D3 in serum without the use of a derivatization agent. We report an isotope dilution LC-MS/MS method, with electrospray ionization in the positive mode, that can simultaneously detect 24R,25(OH)2D3, 25(OH)D3, 3-epi-25(OH)D3, and 25-hydroxyvitamin D2. The method employs a cost-effective liquid-liquid extraction using only 150µL of sera and a total run time of 10min. Method performance was assessed by using quality controls made from pooled sera as an alternative to sera spiked with analytes. Biobanked samples, originally analyzed by chemiluminescent microparticle immunoassay (CMIA), were re-analyzed with this method to determine the contribution of 24R,25(OH)2D3 cross-reactivity to 25(OH)D measurement bias. The CMIA over-estimation of 25(OH)D measurements relative to LC-MS/MS was found to depend on both 25(OH)D and 24R,25(OH)2D3 concentrations.

The Active Metabolite of Vitamin D3 as a Potential Immunomodulator.

In the past, vitamin D was known for its classical, skeletal action as a regulator of calcium and bone homoeostasis. Currently, vitamin D was found to have a role in numerous physiological processes in the human body; thus, vitamin D has pleiotropic activity. The studies carried out in the past two decades showed the role of vitamin D in the regulation of immune system functions. Basically, these effects may be mediated not only via endocrine mechanism of circulating calcitriol but also via paracrine one (based on cell-cell communication that leads to production of signal inducing the changes in nearby/adjacent cells and modulating their differentiation or behaviour) and intracrine mechanism (the action of vitamin D inside a cell) of 1,25-dihydroxycholecalciferol (1,25(OH)2 D3 ) synthetized from its precursor 25-hydroxyvitamin D3 (25(OH)D3 ). Both vitamin D receptor (VDR) and 25-hydroxyvitamin D3 1-α-hydroxylase (CYP27B1) are expressed in several types of immune cells (i.e. antigen presenting cells, T and B cells), and thus, they are able to synthetize the bioactive form of vitamin D that modulates both the innate and adaptive immune system. This review discusses the role of vitamin D as regulator of immune system, and our understanding of how vitamin D regulates both adaptive and innate immunity as well as inflammatory cascade on the cellular level.

25-hydroxyvitamin D3 status is associated with developing adaptive and innate immune responses in the first 6 months of life.

BACKGROUND: Vitamin D (25[OH]D3) status in early life has been linked to the risk of allergic disease in multiple observational studies. While immunomodulating properties are well recognized, there are few longitudinal studies of 25(OH)D3 status, immune function and allergic disease in infants. OBJECTIVE: To investigate 25(OH)D3 levels at birth [cord blood (CB)] and at 6 months of age in relation to immune function at 6 months of age, and clinical outcomes up to 30 months of age in infants with a maternal history of atopy. METHODS: In a subset of infants (n = 225) enrolled in a RCT (ACTRN12606000281594), 25(OH)D3 levels were assessed in relation to peripheral blood mononuclear cell cytokine responses to house dust mite (HDM), ovalbumin (OVA) and ß-lactoglobulin allergens, or Toll-like receptor (TLR) ligands (lipopolysaccharide, lipoteichoic acid, polyinosinic : polycytidylic acid and CpG oligonucleotide) at 6 months of age, in addition to clinical outcomes (eczema, wheeze and allergen sensitisation) up to 30 months of age. RESULTS: Infants with higher 25(OH)D3 at birth (≥ 75 nmol/L, compared with < 50 nmol/L) had lower IL-5 and IL-13 responses to HDM by 6 months (P < 0.001 and P = 0.003, respectively). This was also reflected in strong inverse correlations between CB 25(OH)D3 levels and HDM IL-13 (ρ = -0.57; P = 0.0002) and IL-5 (ρ = -0.59, P = 0.0001) responses, with a similar trend for IL-5 (ρ = -0.29; P = 0.009) responses to OVA. For innate stimulations, higher 25(OH)D3 levels at 6 months were associated with greater responses to TLR ligands. Additionally, higher CB 25(OH)D3 was associated with reduced risk eczema at 6 months (P = 0.011) and 12 months (P = 0.034). CONCLUSION: This suggests that improving 25(OH)D3 status in pregnancy or early infancy may reduce the development of allergic disease in high-risk infants by inhibiting cytokine profiles associated with allergy. Results of clinical trials are awaited to determine the efficacy of vitamin D supplementation in allergy prevention.

Evaluation of 3-epi-25-hydroxyvitamin D3 cross-reactivity in the Roche Elecsys Vitamin D Total protein binding assay.

BACKGROUND: Presence of the 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3] metabolite affects accurate determination of 25(OH)D3 by most routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods and to an unknown extent in present immuno- and protein binding assays. We studied 3-epi-25(OH)D3 cross-reactivity in a competitive protein binding (CPB) assay (Roche Elecsys). METHODS: Neonatal samples, containing up to 58% of 3-epi-25(OH)D3 were used for measurement by the CPB assay and by an LC-MS/MS method separating 25(OH)D3 and 3-epi-25(OH)D3. Analytical recovery was also studied by addition of exogenous 3-epi-25(OH)D3. RESULTS: The CPB assay showed approximately 51% cross-reactivity to 3-epi-25(OH)D3 at exogenous addition. In contrast, there was minimal 3-epi-25(OH)D3 recognition by the CPB assay when present as the natural endogenous metabolite. CONCLUSIONS: The automated CPB assay displays minimal 3-epi-25(OH)D3 cross-reactivity in samples containing significant concentrations of endogenous 3-epi-25(OH)D3. Exogenous 3-epi-25(OH)D3 added to human serum or plasma seems to behave different from endogenous presence, and caution is warranted when using samples spiked with vitamin D metabolites for testing analytical specificity or external quality assurance in immuno- or protein binding assays.

Antibiotic-like actions of vitamin D.

Vitamin D is a secosteroid hormone that has expanding importance for a healthy lifestyle and disease prevention. A multitude of studies have highlighted that vitamin D acts not only in bone and calcium homeostasis but is critically important for human immunity. The discovery that the storage form of vitamin D (25-hydroxyvitamin D3) can be locally converted to the active form (1,25-hydroxyvitamin D3) in immune cells, epithelial cells and numerous other non-renal tissues highlights the importance of maintaining sufficient stores. When responding to a specific external stimulus, like bacterial invasion, intracrine synthesis of active vitamin D has the ability to regulate gene expression providing a specific response and directing cellular actions. These responses include the generation of antimicrobial peptides with production of these peptides dependent on vitamin D status. Vitamin D deficiency is associated with an increased rate of infection. This paper highlights the antibiotic like actions of vitamin D and importance of vitamin D sufficiency.

Vitamin D status is not associated with inflammatory cytokine levels during experimental human endotoxaemia.

Vitamin D has been shown to modulate innate immune responses in vitro and ex vivo; however, human in-vivo data are lacking. At high latitudes, seasonal vitamin D deficiency is common due to alternating ultraviolet (UV)-B radiation exposure. In the present study, we investigated whether levels of 25 hydroxyvitamin D(3) [25(OH)D(3) ] and its active metabolite 1,25 dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] are subject to seasonal variation and whether plasma levels of these vitamin D metabolites correlate with the in-vivo cytokine response during experimental human endotoxaemia [administration of lipopolysaccharide (LPS) in healthy volunteers]. Plasma levels of 25(OH)D(3) and 1,25(OH)(2) D(3) were determined in samples obtained just prior to administration of an intravenous bolus of 2 ng/kg LPS (derived from Escherichia coli O:113) in 112 healthy male volunteers. In the same subjects, plasma levels of the inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 were analysed serially after endotoxin administration. Plasma levels of 1,25(OH)(2) D(3) , but not 25(OH)D(3) , were subject to significant seasonal variation, with lower levels in autumn and winter. 25(OH)D(3) and 1,25(OH)(2) D(3) levels did not correlate with plasma cytokine responses. Furthermore, 25(OH)D(3) deficient subjects (< 50 nmol/l) displayed an identical cytokine response compared with sufficient subjects. In conclusion, plasma levels of vitamin D are not correlated with the LPS-induced TNF, IL-6 and IL-10 cytokine response in humans in vivo. These findings question the direct role of vitamin D in modulation of the innate immune response.

High vitamin D status in younger individuals is associated with low circulating thyrotropin.

BACKGROUND: Vitamin D is an immunomodulator and may affect autoimmune thyroid diseases. Vitamin D has also been shown to influence thyrocytes directly by attenuating thyrotropin (TSH)-stimulated iodide uptake and cell growth. However, it is unclear how vitamin D status is related to TSH at the population level. The goal of the present study was to investigate the relationship between vitamin D status and TSH levels according to thyroid autoantibodies in a population-based health survey in Thailand. METHODS: A total of 2582 adults, aged 15-98 years, were randomly selected according to the geographical region from the Thailand 4th National Health Examination Survey sample. By study design, the sexes were equally represented. Serum levels of 25-hydroxyvitamin D [25(OH)D], TSH, the thyroid peroxidase antibody (TPOAb), and the thyroglobulin antibody (TgAb) were measured in all subjects. RESULTS: The mean age was 55.0±0.4 (SE) years. In subjects positive for serum TgAb, serum TSH levels were higher, whereas total serum 25(OH)D levels were lower. In addition, the prevalence of vitamin D insufficiency in TgAb-positive subjects was significantly higher than that observed in TPOAb- and TgAb-negative subjects, whether based on cutoff values of 20 or 30 ng/mL: 8.3% vs. 5.6%, p<0.05; or 47.6% vs. 42.0%, p<0.05, respectively. However, vitamin D status was not associated with positive TPOAb and/or TgAb after controlling for sex and age. To explore the probable interaction between vitamin D status and age on serum TSH, analyses were performed according to age tertiles; it was found that higher 25(OH)D levels were independently associated with lower TSH, but only in subjects in the lowest age tertile. CONCLUSIONS: This population-based study showed that high vitamin D status in younger individuals is associated with low circulating TSH.

Availability of 25-hydroxyvitamin D(3) to APCs controls the balance between regulatory and inflammatory T cell responses.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], the active form of vitamin D, exerts potent effects on several tissues including cells of the immune system, where it affects T cell activation, differentiation and migration. The circulating, inactive form of vitamin D, 25(OH)D(3), is generally used as an indication of vitamin D status. However, use of this precursor depends on its uptake by cells and subsequent conversion by the enzyme 25(OH)D(3)-1α-hydroxylase (CYP27B1) into active 1,25(OH)(2)D(3). Using human T cells, we show in this study that addition of inactive 25(OH)D(3) is sufficient to alter T cell responses only when dendritic cells (DCs) are present. Mechanistically, CYP27B1 is induced in DCs upon maturation with LPS or upon T cell contact, resulting in the generation and release of 1,25(OH)(2)D(3), which subsequently affects T cell responses. In most tissues, vitamin D binding protein acts as a carrier to enhance the use of vitamin D. However, we show that vitamin D binding protein modulates T cell responses by restricting the availability of inactive 25(OH)D(3) to DC. These data indicate that the level of free 25(OH)D(3) available to DCs determines the inflammatory/regulatory balance of ensuing T cell responses.

[Immunomodulatory effects of vitamin D3 and bisphosphonates in nutritional osteoporosis in rats].

The aim of this study was to determine the effectiveness of separate and combined administration of vitamin D3 and different forms of bisphosphonate (disodium salt of methylenbisphosphonic acid dihydrate and alendronate) on the function of immune cells in rats with nutritional osteoporosis. It was shown that D-hypovitaminosis leads to reduced 25OHD3, which is a biomarker for vitamin D3 and disturbances of metabolic processes in bone tissue that correlated with osteoporosis manifestation. Immunologic disorders related to nutritional osteoporosis were accompanied by the decrease in phagocytic activity of granulocytes and impaired ability to produce bactericidal oxidants. Inhibition of B-cell immunity also occurred in patholgy. Thus, the present study revealed more pronounced immunomodulatory effects of vitamin D3 on phagocytic immunity, whereas bisphosphonates were effective in improving the humoral immune protection.

Vitamin D status in systemic lupus erythematosus patients and its association with selected clinical and laboratory parameters.

OBJECTIVES: To identify relationships between vitamin D serum levels and the presence of autoantibodies directed against vitamin D and levels of interleukin(IL)-17 and IL-23 in patients with systemic lupus erythematosus (SLE). METHODS: The study included 49 patients with SLE. Serum concentrations of 25(OH)D(3) were measured with electrochemiluminescence immunoassay (ECLIA). Enzyme-linked immunosorbent assays (ELISA) were used to determine antibodies directed against 1,25(OH)(2)D(3) and levels of IL-17 and IL-23 in serum of SLE patients. In evaluation of vitamin D status, the control group consisted of 49 age and gender matched healthy individuals, whereas in assessment of anti-vitamin D antibodies the control group comprised 30 sera from blood donors. RESULTS: Serum concentration of 25(OH)D(3) in SLE patients during the warm season was 18.47 ± 9.14 ng/ml, which was significantly decreased as compared with that of the control group - 31.27 ± 12.65 ng/ml (p = 0.0005). During the cold season a trend toward lower concentration of 25(OH)D(3) in SLE patients was revealed; however, it did not reach statistical significance (11.71 ± 7.21 ng/ml vs. 16.01 ± 8.46 ng/ml; p = 0.054). Results within the recommended range for vitamin D (30-80 ng/ml; 70-200 nmol/l) were observed only in three patients. The 25(OH)D(3) concentration was decreased in SLE patients with renal disease or leucopenia as compared with the levels in patients who did not have either problem (p = 0.006 and p = 0.047, respectively). The cold season was found to be a risk factor for vitamin D deficiency (<20 ng/ml) (odds ratio = 9.25; p = 0.005). Autoantibodies directed against 1,25(OH)(2)D(3) were detected in three SLE patients. No significant difference in 25(OH)D(3) serum concentrations was found between SLE patients with and without these autoantibodies. No link was shown between the existence of autoantibodies against 1,25(OH)(2)D(3) and clinical or laboratory findings, including IL-17 and IL-23 levels. However, serum concentrations of IL-23 were lower in patients with vitamin D deficiency (p = 0.037). CONCLUSIONS: SLE patients, especially those with leucopenia or renal involvement, are at high risk of vitamin D deficiency and require vitamin D supplementation. Some SLE patient sera contained 1,25(OH)(2)D(3) antibodies, but these antibodies do not appear to affect vitamin D levels.

[Synthesis and identification of artificial complete antigen 25-hydroxyvitamin D(3);].

AIM: To synthesize the 25-hydroxyvitamin D(3); artificial complete antigen and to prepare the specific antibody against 25-hydroxyvitamin D(3);. METHODS: The active group carboxyl was introduced into 25-hydroxyvitamin D(3); and formed 25-hydroxyvitamin D(3);-hemisuccinate which possessed the structure of the hapten by chemical modification. The EDC method was applied to conjugate 25-hydroxyvitamin D(3);-hemisuccinate to bovine serum albumin as an artificial immunogen. The coating antigen 25-hydroxyvitamin D(3);-hemisuccinate-OVA was obtained in the same way. Ultraviolet, SDS-PAGE and MALDI-TOF were used to identify 25-hydroxyvitamin D(3);-hemisuccinate-BSA. RESULTS: BALB/c mice were immunized with 25-hydroxyvitamin D(3);-hemisuccinate-BSA to generate the polyclonal antibody of the 25-hydroxyvitamin D(3); worth high titer and the immunogen, 25-hydroxyvitamin D(3);-hemisuccinate-BSA, was successfully prepared with coupling ratio (12±0.16):1(N=3) coupling. CONCLUSION: The high titer and good sensitivity of anti-25-hydroxyvitamin D(3); antibody are produced in sera immunized BALB/c mice, which made it possible to develop a clinical diagnostics for illness.

Synthesis of two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3.

Hapten derivatives of 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) were synthesized using the Wittig-Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxylated vitamin D(3) analogues.