Disturbed calcium metabolism in subjects with elevated diastolic blood pressure

Summary Essential hypertension has been associated with disturbed calcium metabolism, but the available data are controversial. We measured parameters of calcium metabolism in groups of untreated male subjects (n = 78) with elevated diastolic blood pressure (101 ± 6 mmHg, mean ± SD) and age-matched male subjects (n=79) with low diastolic blood pressure (62 ± 4 mmHg). The participants of the study were drawn from a random population sample. Subjects with high diastolic blood pressure had significantly higher carboxy-terminal parathyroid hormone (PTH) plasma concentrations than controls with low diastolic blood pressure (median 114 vs. 43 pmol/l, P < 0.01). The 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D concentrations were comparable in both groups. Individuals with high diastolic blood pressure had significantly lower total serum calcium (2.41 ± 0.10 vs. 2.47 ± 0.10 mmol/l, mean ± SD; P < 0.01). PTH concentrations were correlated with diastolic pressure (r = –0.39, P < 0.001). The data are compatible with increased parathyroid activity despite unchanged concentrations of vitamin D metabolites in human hypertension.Abbreviations PTH parathyroid hormone - C-PTH carboxy-terminal parathyroid hormone - 1,25(OH)₂D 1,25-di-hydroxyvitamin D - 25(OH)D 25-hydroxyvitamin D     

Rapid stimulation of Na+/H+ exchange by 1,25-dihydroxyvitamin D3; interaction with parathyroid-hormone-dependent inhibition

We have examined the rapid effect of 1,25-dihydroxyvitamin-D₃ [1,25(OH)₂D₃] on apical Na⁺/H⁺ exchange activity in opossum kidney (OK) cells and in MCT cells (a culture of simian-virus-40-immortalized mouse cortical tubule cells) grown on filter support. Addition of 1,25(OH)₂D₃ (10 nM) for 1 min increased apical Na⁺/H⁺ exchange activity [recovery from an acid load; measured by 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein] in OK cells (by 56%) and in MCT cells (by 36%). The cellular mechanisms involved in 1,25(OH)₂D₃-dependent stimulation of Na⁺/H⁺ exchange were analysed in OK cells; stimulation of Na⁺/ H⁺ exchange by 1,25(OH)₂D₃ was not prevented by actinomycin D. Applying parathyroid hormone (PTH) reduced Na⁺/H⁺ exchange activity in OK cells (by 34% at 10 nM, 5 min); 1,25(OH)₂D₃ reversed PTH-induced inhibition, either when PTH was added prior to 1,25(OH)₂D₃ or when the two agonists were applied together. 1,25(OH)₂D₃ had no effect on basal OK cell cAMP content or on [Ca²⁺]_i (fura-2). 1,25(OH)₂D₃ attenuated PTH-induced cAMP accumulation and had no effect on the PTH-dependent increase in [Ca²⁺]_i. These data suggest a regulatory control (stimulation) of proximal tubular brush-border Na⁺/H⁺ exchange by 1,25(OH)₂D₃. This effect is non-genomic and might in part be explained by a release from cAMP-dependent control of transport activity.     

1α,25-Dihydroxyvitamin D3 inhibits the invasive potential of human breast cancer cellsin vitro

Using the Boyden chamber invasion assay, the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on the invasiveness of the highly invasive, oestrogen receptor-negative human breast cancer cell line MDA-MB-231 was examined. The MDA-MB-231 cells were shown to contain high-affinity receptors for 1,25(OH)₂D₃ with a K_d of 1.5 × 10^–11 M. When the cells were treated with 1,25(OH)₂D₃ for 4 days before the assay was performed, a dose-dependent inhibition of their invasive potential was demonstrated. Fifty per cent inhibition of invasion was obtained with a concentration of 13 pM of 1,25(OH)₂D₃. However, when the cells were treated for only 6 h during the assay, no inhibitory effect was seen. The process of migration was also affected by treatment with 1,25(OH)₂D₃ for 4 days, although the inhibition was not of the same magnitude as seen for the invasion. Fifty per cent inhibition of migration occurred at a concentration of 3.2 nM of 1,25(OH)₂D₃ (250 times higher than in the invasion assay). Inhibition of invasion and migration was not due to the known anti-proliferative effect of 1,25(OH)₂D₃, as no growth reduction could be demonstrated with treatment up to 5 days. Based on the present investigation it can therefore be concluded that 1,25(OH)₂D₃ is able to inhibit tumour cell invasiveness by a mechanism which is not exclusively based on its anti-proliferative and anti-migrative effects.     

Regulation of Matrix Vesicle Metabolism by Vitamin D Metabolites

Matrix vesicles are membrane organelles found in the extracellular matrix of calcifying cells. Vitamin D-responsive alkaline phosphatase specific activity has been localized to matrix vesicles in chondrocyte and osteoblast cultures. The effect of hormone is both metabolite and cell specific. Alkaline phosphatase in matrix vesicles produced by resting zone chondrocytes is stimulated by 24, 25(OH)₂D₃ whereas alkaline phosphatase in matrix vesicles produced by growth zone chondrocytes is responsive to 1,25(OH)₂D₃. However, mesenchymal cell cultures, which exhibit a chondrogenic phenotype when exposed to bone inductive proteins in vitro, produce vesicles with alkaline phosphatase activity that is unaffected by either 1,25(OH)₂D₃ or 24, 25(OH)₂D₃. Incorporation and release of arachidonic acid into phosphatidylethanolamine is also differentially regulated by 1,25(OH)₂D₃ and 24, 25(OH)₂D₃ in chondrocytes. These data suggest that vitamin D metabolites may regulate endochondral ossification by altering matrix vesicle enzyme activities, perhaps through changes in membrane phospholipid metabolism.     

Role of 1,25-Dihydroxy Vitamin D3 and Parathyroid Hormone in Urinary Calcium Excretion in Calcium Stone Formers

Purpose

To find out the possible role of 1,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] and parathyroid hormone (PTH) as intrinsic factors in urinary calcium stone formers (SFs), we investigated their relationship with serum and urinary biochemical parameters.

Materials and Methods

A total of 326 calcium SFs (male: 204, female: 122) were enrolled and underwent outpatient metabolic evaluations including 1,25(OH)₂D₃ and PTH as well as serum and 24-hour urinary biochemical parameters. As control, 163 age- and sex-matched (2:1) individuals (non-SFs) who have never urinary stone episode were included.

Results

1,25(OH)₂D₃ level was positively correlated with urinary calcium excretion (r=0.347, p<0.001). The hypercalciuric group and recurrent SFs had higher serum 1,25(OH)₂D₃ levels than the normocalciuric group (p<0.001) and first SFs (p=0.050). In the adjusted multiple linear regression analysis, serum 1,25(OH)₂D₃ level (β=0.259, p<0.001) and serum PTH level (β=-0.160, p<0.001) were significantly correlated with urinary calcium excretion. The patients in highest tertile of 1,25(OH)₂D₃ had a more than 3.1 fold risk of hypercalciuria than those in the lowest tertile (odds ratio=3.14, 95% confidence interval: 1.431-6.888, p=0.004). No correlation was observed between PTH and 1,25(OH)₂D₃ (R=0.005, p=0.929) in calcium SFs, while a negative correlation was found in controls (R=-0.269, p=0.001).

Conclusion

1,25(OH)₂D₃ was closely correlated with urinary calcium excretion, and high 1,25(OH)₂D₃ levels were detected in the hypercalciuric group and in recurrent SFs. However, 1,25(OH)₂D₃ was not correlated with PTH in calcium SFs. These findings suggest that 1,25(OH)₂D₃ might be important intrinsic factor for altered calcium regulation in SFs.     

Surface roughness mediates its effects on osteoblasts via protein kinase A and phospholipase A2

Earlier studies have shown that implant surface roughness influences osteoblast proliferation, differentiation, matrix synthesis and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones, such as 1,25-(OH)₂D₃, at the implant surface is also influenced by surface roughness and this effect is mediated by changes in prostaglandins. At present, it is not known which signaling pathways are involved in mediating cell response to surface roughness and how 1,25-(OH)₂D₃ treatment alters the activation of these pathways. This paper reviews a series of studies that have addressed this question. MG63 osteoblast-like cells were cultured on commercially pure titanium (cpTi) surfaces of two different roughnesses (R_a 0.54 and 4.92 μm) in the presence of control media or media containing 1,25-(OH)₂D₃ or 1,25-(OH)₂D₃ plus H8 (a protein kinase A inhibitor) or quinacrine (a phospholipase A₂ inhibitor). At harvest, the effect of these treatments on cell number and alkaline phosphatase specific activity was measured. Compared to cultures grown on the smooth surface, cell number was reduced on the rough surface. 1,25-(OH)₂D₃ inhibited cell number on both surfaces and inhibition of protein kinase A in the presence of 1,25-(OH)₂D₃ restored cell number to that seen in the control cultures. Inhibition of phospholipase A₂ in the presence of 1,25-(OH)₂D₃ caused a further reduction in cell number on the smooth surface, and partially reversed the inhibitory effects of 1,25-(OH)₂D₃ on the rough surface. Alkaline phosphatase specific activity was increased in cultures grown on the rough surface compared with those grown on the smooth surface; 1,25-(OH)₂D₃ treatment increased enzyme specific activity on both surfaces. Cultures treated with H8 and 1,25-(OH)₂D₃ displayed enzyme specific activity that approximated that seen in control cultures. Inhibition of phospholipase A₂ also inhibited the 1,25-(OH)₂D₃-dependent effect on the smooth surface, but on the rough surface there was an inhibition of the 1,25-(OH)₂D₃ effect as well as a partial inhibition of the surface roughness-dependent effect. The results indicate that surface roughness and 1,25-(OH)₂D₃ mediate their effects through phospholipase A₂, which catalyzes one of the rate-limiting steps in prostaglandin E₂ production. Further downstream, prostaglandin E₂ activates protein kinase A.     

Blockade of the renal tubular effects of vitamin D by cycloheximide in the rat

To further characterize the mechanisms by which 25(OH)vitamin D₃ (25(OH)D₃) and 1.25(OH)₂ vitamin D₃ (1,25(OH)₂D₃) suppress the phosphaturic action of parathyroid hormone (PTH) we have studied the effects of cycloheximide (cyclohex), a protein synthesis inhibitor, on the interaction between PTH and vitamin D metabolites in parathyroidectomized (PTX) rats, both in vivo and in vitro experiments. In clearance studies PTX PTH-infused rats were pretreated with cyclohex 2 h before the administration of vitamin D. In control, PTX PTH-infused rats not pretreated with cyclohex, the administration of 25(OH)D₃ and 1,25(OH)₂D₃ was associated with a fall in fractional excretion of phosphate (CP/CIN) from 0.30±0.05 to 0.16±0.02 and from 0.31±0.05 to 0.13±0.01 (P<0.005) respectively. Cyclohex-pretreated PTX PTH-infused rats failed to respond to both 25(OH)D₃ and 1,25(OH)₂D₃, and CP/CIN, which rose after PTH, remained 0.32±0.05 and 0.29±0.03 respectively. In vitro, both 25(OH)D₃ and 1,25(OH)₂D₃ inhibited the PTH-induced activation of adenylate cyclase in the renal isolated membrane fractions. Pretreatment with cyclohex abolished this effect of vitamin D metabolites. These results show that cyclohex blocks the antiphosphaturic effects of both 25(OH)D₃ and 1,25(OH)₂D₃ but does not alter the response to PTH. These findings are consistent with the possibility that the acute renal action of vitamin D depends on de novo synthesis of protein.An abstract of this work appeared in Clinical Research, 28 (2) A 387, 1980.     

Regulation of mineral metabolism by lithium

Lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), is widely used for the treatment of mood disorders. Side effects of lithium include nephrogenic diabetes insipidus, leading to renal water loss. Dehydration has in turn been shown to downregulate Klotho, which is required as co-receptor for the downregulation of 1,25(OH)₂D₃ formation by fibroblast growth factor 23 (FGF23). FGF23 decreases and 1,25(OH)₂D₃ stimulates renal tubular phosphate reabsorption. The present study explored whether lithium influences renal Klotho expression, FGF23 serum levels, 1,25(OH)₂D₃ formation, and renal phosphate excretion. To this end, mice were analyzed after a 14-day period of sham treatment or of treatment with lithium (200 mg/kg/day subcutaneously). Serum antidiuretic hormone (ADH), FGF23, and 1,25(OH)₂D₃ concentrations were determined by ELISA or EIA, renal Klotho protein abundance and GSK3 phosphorylation were analyzed by Western blotting, and serum phosphate and calcium concentration by photometry. Lithium treatment significantly increased renal GSK3 phosphorylation, enhanced serum ADH and FGF23 concentrations, downregulated renal Klotho expression, stimulated renal calcium and phosphate excretion, and decreased serum 1,25(OH)₂D₃ and phosphate concentrations. In conclusion, lithium treatment upregulates FGF23 formation, an effect paralleled by substantial decrease of serum 1,25(OH)₂D₃, and phosphate concentrations and thus possibly affecting tissue calcification.     

25-hydroxyvitamin D, 24, 25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D in human cerebrospinal fluid

Summary Samples of CSF and plasma were obtained simultaneously from 46 adult patients who had no endocrine disorders and were undergoing routine diagnostic lumbar puncture because of suspected or proved prolapse of a disc. Concentrations of 25-OHD, 24,25(OH)₂D and 1,25(OH)₂D were measured. The samples were purified by column chromatography and fractionated by HPLC. In the appropriate fractions the vitamin D metabolites were measured by PBA, and cytoreceptor assay. The results were as follows (median, range in brackets): 25-OHD in CSF 8.3 ng/ml (2.0–24.8), in plasma 14.5 ng/ml (7.0–36.0). 24,25(OH)₂D in CSF 1.8 ng/ml (0.3–4.6) and 2.5 ng/ml (0.4–4.7) in plasma. 1.25(OH)₂ D in CSF 25.0 pg/ml (2.2–39.0) and 31.0 pg/ml (10.1–55.0) in plasma. The correlations between plasma and CSF concentrations were as follows: 25-OHDr=0.479 (P<0.001); 24,25(OH)₂Dr=0.815 (P<0.001) and for 1.25(OH)₂Dr=0.497 (P<0.001).Our findings showed vitamin D metabolites to be present in human CSF.Abbreviations Ca Calcium - CSF Cerebrospinal fluid - Vitamin D₃ Cholecalciferol - CPM Counts per min - 24, 25 (OH)₂D 24, 25-dihydroxyvitamin D₃ - 1,25(OH)₂D 1,25-dihydroxyvitamin D₃ - Vitamin D₂ Ergocalciferol - HPLC High-pressure liquid chromatography - 25OHD 25-hydroxyvitamin D₃ - PTH Parathyroid hormone - PBA Protein binding assay - RIA Radioimmunoassay - D-CaBP Vitamin D dependent calcium-binding protein     

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 D₃ [25(OH)D₃] and its active metabolite 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] 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)D₃ and 1,25(OH)₂D₃ 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)₂D₃, but not 25(OH)D₃, were subject to significant seasonal variation, with lower levels in autumn and winter. 25(OH)D₃ and 1,25(OH)₂D₃ levels did not correlate with plasma cytokine responses. Furthermore, 25(OH)D₃ 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.     

Plasma 1,25 Dihydroxy Vitamin D<Subscript>3</Subscript> Level and Expression of Vitamin D Receptor and Cathelicidin in Pulmonary Tuberculosis

Introduction  Vitamin D₃, which exerts its effect through vitamin D receptor (VDR), is known for its potent immunomodulatory activities. Associations between low serum vitamin D₃ levels and increased risk of tuberculosis have been reported. Study Subjects and Methods  Plasma 1,25 dihydroxy vitamin D₃ levels (1,25(OH)₂ D₃) and ex vivo levels of VDR protein from peripheral blood mononuclear cells were studied in 65 pulmonary tuberculosis (PTB) patients and 60 normal healthy subjects (NHS) using enzyme-linked immunosorbent assay-based methods. Using real-time polymerase chain reaction (PCR), induction of VDR, cathelicidin, and CYP27B1 mRNA were studied in live Mycobacterium tuberculosis-stimulated macrophage cultures treated with or without 1,25 dihydroxy vitamin D₃. VDR and CYP27B1 (-1077 A/T) gene polymorphisms were studied using PCR-based methods. Results  1,25(OH)₂ D₃ were significantly increased (p = 0.0004), while ex vivo levels of VDR protein were significantly decreased in PTB patients (p = 0.017) as compared to NHS. 1,25(OH)₂ D₃ levels were not different between variant genotypes of CYP27B1. A trend towards decreased levels of VDR protein was observed among NHS with BsmI BB and TaqI tt genotypes compared to NHS with other genotypes. Relative quantification of mRNA using real-time PCR revealed increased VDR mRNA expression in live M. tuberculosis-stimulated culture in PTB patients (p < 0.01) than normal healthy subjects. Cathelicidin mRNA expression was significantly increased in vitamin D₃-treated cultures compared to unstimulated and M. tuberculosis-stimulated culture in both patients (p < 0.001) and NHS (p < 0.05). Conclusions  The present study suggests that PTB patients may have increased 1,25(OH)₂ D₃ levels, and this might lead to downregulation of VDR expression. Decreased VDR levels could result in defective VDR signaling. Moreover, addition of 1,25(OH)₂ D₃ might lead to increased expression of cathelicidin which could enhance the immunity against tuberculosis.     

1,25-dihydroxyvitamin D3 enhances the ability of transferred CD4+ CD25+ cells to modulate T helper type 2-driven asthmatic responses

The severity of allergic diseases may be modified by vitamin D. However, the immune pathways modulated by the active form of vitamin D, 1,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃], are yet to be fully elucidated. In this study, naturally occurring CD4⁺ CD25⁺ cells from the skin‐draining lymph nodes (SDLN) of mice treated with topical 1,25(OH)₂D₃ had an increased ability to suppress T helper type 2 (Th2) ‐skewed immune responses. CD4⁺ CD25⁺ cells transferred from mice treated with topical 1,25(OH)₂D₃ into ovalbumin (OVA) ‐sensitized mice challenged intranasally with OVA 18 hr later, significantly suppressed the capacity of airway‐draining lymph node (ADLN) cells to proliferate and secrete cytokines in response to further OVA stimulation ex vivo. The CD4⁺ CD25⁺ cells from 1,25(OH)₂D₃‐treated mice also reduced airway hyperresponsiveness and the proportions of neutrophils and eosinophils in bronchoalveolar lavage fluid (BALF). To test the effect of 1,25(OH)₂D₃ on cells able to respond to a specific antigen, CD4⁺ CD25⁺ cells were purified from the SDLN of OVA‐T‐cell receptor (TCR) transgenic mice treated 4 days earlier with topical 1,25(OH)₂D₃. CD4⁺ CD25⁺ cells from OVA‐TCR mice treated with 1,25(OH)₂D₃ were able to alter BALF cell content and suppress ADLN responses to a similar degree to those cells from non‐transgenic mice, suggesting that the effect of 1,25(OH)₂D₃ was not related to TCR signalling. In summary, topical 1,25(OH)₂D₃ increased the regulatory capacity of CD4⁺ CD25⁺ cells from the SDLN to suppress Th2‐mediated allergic airway disease. This work highlights how local 1,25(OH)₂D₃ production by lung epithelial cells may modulate the suppressive activity of local regulatory T cells.     

1,25-Dihydroxyvitamin D3 inhibits proliferation but not the suppressive function of regulatory T cells in the absence of antigen-presenting cells

Vitamin D₃ is known to induce regulatory T (Treg) cells by rendering antigen-presenting cells tolerogenic, its direct effect on human naturally occurring Treg cells is unclear. Here, we investigated if and how 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] can directly affect the proliferation and function of human naturally occurring Treg cells in vitro. First, we demonstrated that these Treg cells express vitamin D receptors that were up-regulated following anti-CD3/CD28-bead stimulation. 1,25(OH)₂D₃ inhibited proliferation of Treg cells even when exogenous interleukin-2 was provided. Treg cells were more susceptible to the inhibitory effect of 1,25(OH)₂D₃ than conventional T cells_. 1,25(OH)₂D₃ neither affected the anergic state nor the suppressive function of Treg cells but induced a subtle increase in interleukin-10-secreting cells. The cell-division-inhibiting effect of 1,25(OH)₂D₃ on Treg cells was also demonstrated in vivo by supplementing vitamin D-deficient HIV-1-infected patients with 2000 IU cholecalciferol (vitamin D₃). Increased serum 1,25(OH)₂D₃ levels were associated with a drop in the number and percentage of Treg cells, which may be attributed to a decrease in the proliferating Foxp3⁺ Treg cell population. In conclusion, 1,25(OH)₂D₃ directly affects Treg cell growth and promotes interleukin-10 production without apparent effects on activation status and suppressive phenotype whereas in vivo, high serum 1,25(OH)₂D₃ levels are associated with reduced Treg cell proliferation and a reduced number of Treg cells.     

Decreased immunosuppressive actions of 1α, 25-dihydroxyvitamin D3 in patients with immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease. 1α, 25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and vitamin D receptor (VDR) play important immune-suppressive roles in immune system. It has been reported that serum 1,25(OH)₂D₃ were lower in ITP patients. In this study, we evaluated local 1,25(OH)₂D₃ level and VDR mRNA expression further, and determined whether 1,25(OH)₂D₃/VDR were correlated with T cell dysfunction in ITP patients. We found that 1,25(OH)₂D₃/VDR levels were decreased in active ITP patients, and 1,25(OH)₂D₃ had significant anti-inflammatory effects on ITP patients, including both anti-proliferation of peripheral blood mononuclear cells (PBMCs) and reversing the abnormal T cells polarization. 1,25(OH)₂D₃ inhibited the differentiation of T helper (Th)1 and Tc1 cells but induced the differentiation of Th2, Tc2 and T regulatory (Treg) cells in ITP patients. However, the percentage of Th17 cells were not affected obviously with 1,25(OH)₂D₃. In addition, 1,25(OH)₂D₃ also suppressed pro-inflammatory cytokines (INF-γ and IL-17A) but promoted anti-inflammatory cytokine (IL-10) secretion in ITP patients. In conclusion, decreased 1,25(OH)₂D₃/VDR might participate in the pathogenesis of ITP, and appropriate supplement of 1,25(OH)₂D₃ may be a promising treatment.     

Protective effect of vitamin D3 analogues on endotoxin shock in mice

The effect of vitamin D₃ analogues on endotoxin shock in mice was investigated. Male ICR mice were orally administered vitamin D₃ analogues or vehicle, accompanied by an intraperitoneal injection of endotoxin (E. Coli lipopolysaccharide, LPS, 20 mg/kg). Endotoxin caused a decrease in survival rate in a time-dependent manner. Increases in plasma immunoreactive (i) eicosanoid and hepatic malondialdehyde (MDA) levels were also observed. Administration of 1-hydroxyvitamin D₃ (1-OH-D₃) improved the survival rate 24 to 48 h after endotoxin treatment. The effects were markedly observed at a dose of 20 ng/kg. In addition, 1-OH-D₃ restored the plasma iTXB₂ and hepatic MDA levels 8 h after endotoxin injection. However, it did not affect plasma iPGE₂, i6-keto-PGF₁ and blood iLTB₄ levels. At a dose of 20 ng/kg, both 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) and 1,24(R)-dihydroxyvitamin D₃ (1,24(R)-(OH)₂D₃) restored the survival rate, the plasma iTXB₂ and hepatic MDA levels. These results suggest that vitamin D₃ analogues may inhibit endotoxemia through regulation of the formation of TXA₂ and free radicals.     

Immunomodulatory effects of vitamin D in peripheral blood monocyte-derived macrophages from patients with rheumatoid arthritis

1,25-Dihydroxyvitamin D (1,25(OH)₂D₃), the active form of vitamin D, modulates both innate and adaptive immune responses. Emerging epidemiological data has also demonstrated disease-modifying and immunomodulatory effects of vitamin D in a wide range of human autoimmune diseases, including rheumatoid arthritis (RA). To evaluate in vitro effects of 1,25(OH) ₂D₃ in primary cultures of peripheral blood monocyte-derived macrophages of RA patients, monocyte/macrophages, isolated from peripheral blood mononuclear cells of RA patients and healthy subjects by exploiting their ability to adhere to plastic, were treated with increasing concentrations of 1,25(OH)₂D₃ for 48 h. TNF-α, IL-1 α, IL-1β, IL-6 and RANKL production was determined by ELISA and nitric oxide (NO) release using the Griess method. Immunocytochemistry analysis was also performed to evaluate alterations in transmembrane TNF-α expression after 1,25(OH) ₂D₃ treatment. A significant dose-dependent decrease in TNF-α and RANKL production by cultured RA macrophages after 1,25(OH)₂D₃ treatment was found, whereas a significant reduction in normal cells was observed only at higher concentrations. IL-1 α, IL-1β and IL-6 levels were reduced by 1,25(OH) ₂D₃ at higher concentrations in all cell populations. TNF-α immunostaining was less intense in treated cells compared with untreated. 1,25(OH) ₂D₃ significantly reduced NO levels regardless of the concentration used. Vitamin D downregulated proinflammatory mediators in monocyte-derived macrophages, and RA cells appeared more sensitive than normal cells. These effects further provide a rationale for the therapeutic value of vitamin D supplementation in the treatment for RA.     

Vitamin D binding protein and vitamin D in human allergen‐induced endobronchial inflammation

Allergic asthma is a chronic disease of the airways associated with airway hyperresponsiveness, a variable degree of airflow obstruction, airway remodelling and a characteristic airway inflammation. Factors of the vitamin D axis, which include vitamin D metabolites and vitamin D binding protein (VDBP), have been linked to asthma, but only few data exist about their regulation in the lung during acute allergen‐induced airway inflammation. Therefore, we analysed the regulation of factors of the vitamin D axis during the early‐ and late‐phase reaction of allergic asthma. Fifteen patients with mild allergic asthma underwent segmental allergen challenge. VDBP was analysed in bronchoalveolar lavage fluid (BALF) and serum using the enzyme‐linked immunosorbent assay (ELISA) technique. 25‐hydroxyvitamin D₃ [25(OH)D₃] and 1,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] were analysed by a commercial laboratory using the liquid chromatography–mass spectrometry (LC/MS) technique. VDBP (median 2·3, range 0·2–7·1 μg/ml), 25(OH)D₃ (median 0·060, range < 0·002–3·210 ng/ml) and 1,25(OH)₂D₃ (median < 0·1, range < 0·1–2·8 pg/ml) were significantly elevated in BALF 24 h but not 10 min after allergen challenge. After correction for plasma leakage using the plasma marker protein albumin, VDBP and 25(OH)D₃ were still increased significantly while 1,25(OH)₂D₃ was not. VDBP and 25(OH)D₃ were correlated with each other and with the inflammatory response 24 h after allergen challenge. Serum concentrations of all three factors were not influenced by allergen challenge. In conclusion, we report a significant increase in VDBP and 25(OH)D₃ in human BALF 24 h after allergen challenge, suggesting a role for these factors in the asthmatic late‐phase reaction.     

Effect of 1α-25-dihydroxyvitamin D3 on intimal hyperplasia developing in vascular anastomoses: a rabbit model

Introduction

A common problem encountered in routine daily practice of cardiovascular surgery is migration of smooth muscle cells leading to intimal hyperplasia developing at vascular anastomosis sites which then causes luminal narrowing. The aim of this study was to investigate the antiproliferative effect of 1,25 (OH)₂D₃ on intimal hyperplasia.

Material and methods

Twenty-one male white New Zealand rabbits weighing 2-3 kg were selected. There were 3 groups of animals each consisting of 7 rabbits. Group 1 was the control group. Group 2 was the sham group and group 3 consisted of rabbits receiving 1,25 (OH)₂D₃. The right carotid arteries of the subjects in groups 2 and 3 were transected and re-anastomosed. A daily dose of 25 ng 1,25 (OH)₂D₃ per 100 g body weight was administered for 14 days to rabbits in group 3. Rabbits in group 2 were not subject to any pharmaceutical agent. All the subjects were sacrificed at the end of the 28^th postoperative day. Their right carotid arteries were resected and then investigated histopathologically.

Results

Intimal thickness and intimal area were measured as significantly lower in group 1 when compared with the other groups (p = 0.004). In group 3, the ratios of thickness of tunica intima/thickness of tunica media and area of tunica intima/area of tunica media were significantly lower than those of group 2 (p = 0.015, p = 0.003).

Conclusions

1,25 (OH)₂D₃, the active metabolite of vitamin D, reduces the intimal hyperplasia developing after vascular anastomoses.     

Hypercalcemia and elevated serum 1,25-dihydroxyvitamin D3 in a patient with hodgkin's lymphoma

Summary A 74-year-old woman was hospitalized because of decreased appetite, fatigue, and weight loss. The laboratory examination revealed hypercalcemia, a slightly increased serum creatinine level, and a markedly elevated serum level of 1,25-dihydroxyvitamin D₃. The most important finding the physical examination revealed was enlarged inguinal lymph nodes. A biopsy disclosed lymphocyte-depleted Hodgkin's disease. After steroids, but not after calcitonin, both the elevated calcitriol concentration and serum calcium normalized. In spite of intensive chemotherapy, a further episode with hypercalcemia occurred and increased 1,25-dihydroxyvitamin D₃ serum levels were observed. According to the available evidence it seems probable that the humoral hypercalcemia in this patient resulted from production of 1,25-dihydroxyvitamin D₃ in the tumor.Abbreviations 1,25(OH)₂D₃ 1,25-dihydroxyvitamin D₃ - iPTH serum immunoreactive parathyroid hormone