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.

Inflammatory mediators and the destruction of bone.

Bone is remodelled by the coordinated actions of osteoclasts and osteoblasts. Cellular remodelling occurs in discrete packets of bone, and is regulated by local cytokines produced in the environment of the remodelling cells. These cytokines are secreted by immune cells and by bone cells. In addition, some growth regulatory factors are incorporated into the noncollagenous bone matrix and are released in an active form when bone is stimulated to resorb. Complex interactions between these cytokines and their target cells are responsible for the normal delicate balance between bone resorption and bone formation, and disorders of bone loss are due to imbalances between the rates of resorption and formation.

A sensitive and selective radioimmunoassay for serum 24,25-dihydroxycholecalciferol in man.

We have developed a sensitive and selective radioimmunoassay for 24,25-dihydroxycholecalciferol (24,25(OH)2D3). Antisera with a high titre and affinity to 24,25(OH)2D3 were raised in rabbits immunized with a protein conjugate of the 3-hemisuccinate derivative of 24,25(OH)2D3. Serum samples were extracted by methanol/dichloromethane and the lipid extracts purified on Sephadex LH 20 and chromatographed by single step HPLC on a straight phase silica column. The radioimmunoassay is capable of measuring 24,25(OH)2D3 in the '24,25(OH)2D complex' isolated by HPLC and containing the comigrating metabolites: 24,25(OH)2D3, 24,25(OH)2D2, 25(OH)D3-26,23 lactone and 25,26(OH)2D2. The detection limit of 2 pg/assay tube is a marked improvement, compared to the competitive protein binding assay using rachitic rat serum. Measured by the radioimmunoassay 24,25(OH)2D3 ranged from 0.05 to 1.96 ng/ml with a mean of 0.85 ng/ml in 34 healthy adults. For comparison 24,25(OH)2D, measured simultaneously by competitive protein binding with rachitic rat serum, ranged from 0.1 to 4.0 ng/ml with a mean of 1.76 ng/ml.

Plasma 1,25(OH)2 vitamin D measured by radioimmunoassay and cytosol radioreceptor assay in normal subjects and patients with primary hyperparathyroidism and renal failure.

Antibodies to 1 alpha, 25-dihydroxy vitamin D3 25-hemisuccinate linked to albumin were produced and an immunoassay for 1,25-dihydroxy vitamin D developed. Plasma 1,25-dihydroxy vitamin D concentrations were compared using an immunoassay and cytosol radioreceptor assay. Both assays gave comparable results but the immunoassay was more reproducible, slightly more sensitive and had a lower detection limit. Using the immunoassay the plasma 1,25-dihydroxy vitamin D was 110.5 pmol/l (S.D. 29.4) in normal subjects; there was no difference between males and premenopausal females. It was negatively related to plasma phosphate. In renal failure and primary hyperparathyroidism plasma 1,25(OH)2D was positively related to radiocalcium absorption. Following 1 and 2 microgram of 1,25-dihydroxy vitamin D3 given orally the peak plasma concentration occurs within 12 h.

Measurement of 1,25-dihydroxycholecalciferol in man by radioimmunoassay.

1 alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3) has been measured in human serum by radioimmunoassay. The assay uses a high titre antiserum raised in sheep against 1,25-(OH)2D3-25-hemisuccinate, conjugated to bovine serum albumin. The sensitivity of the assay is 10 pg/tube. Other hydroxylated forms of vitamin D3 cross react with the antiserum and are therefore removed from serum extracts by chromatography on Sephadex LH 20 followed by high pressure liquid chromatography. The mean (+/- SEM) serum 1,25-(OH)2D3 concentration for a group of healthy adult subjects was 41 +/- 2.5 pg/ml. None was detected in anephric patients and the concentration was low or undetectable in patients with chronic renal failure.