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Objective To study the reproductive history of patients with hereditary 1,25-dihydroxyvitamin D–resistant rickets (HVDRR) who have a nonfunctioning vitamin D receptor (VDR). Design Retrospective cohort study. Setting Tertiary university-affiliated medical center. Patient(s) Sixteen HVDRR patients and six spouses, four female and two male. Intervention(s) None. Main Outcome Measure(s) The data on age at menarche, time of conception, and number of pregnancies, abortions, and healthy newborns as reported by HVDRR women and partners of HVDRR men were analyzed, as were the results of semen sample analyses from HVDRR men. Result(s) All 16 patients had normal puberty. The mean age at menarche was 13.8 ± 0.8 years. Two married HVDRR women reported four normal pregnancies and four healthy newborns. Four married HVDRR men reported 15 pregnancies and nine healthy newborns. The wives of two of these men, who are brothers, gave birth to three healthy newborns and had six natural miscarriages during the second trimester of pregnancy. Time to conceive for all the female study patients was Conclusion(s) The VDR is expressed throughout the organs of reproduction, suggesting a role for vitamin D in reproduction. However, the reproductive potential of HVDRR patients with a mutant VDR gene with a nonfunctioning VDR appears to be normal.

Human osteoblasts (hOB) produce and respond to 1,25(OH)(2)D(3) (1,25D), suggesting an autocrine/paracrine system. We therefore examined hormonal modulation of the expression and activity of 25 hydroxy-vitamin D(3)-1alpha hydroxylase (1-Ohase) in hOB. Cells from pre- and post-menopausal women or men, were treated with estrogenic compounds and 1-OHase expression and activity were measured. 1-OHase mRNA expression was highest in pre-menopausal women hOB and was increased by all hormones tested. In post-menopausal hOB all hormones except biochainin A (BA) and genistein (G) increased 1-OHase mRNA expressions to less extent. In male-derived hOB only dihydrotestosterone (DHT) and carboxy BA (cBA) increased 1-OHase mRNA expression. 1,25D production from 25(OH)D(3) had a K(m) of approximately 769-400 ng/ml (1.92-1.07 microM) and V(max) of 31.3-17.4 ng/ml (0.078-0.044 microM/60 min/5 x 10(6)cells) respectively, and was increased by all hormones except raloxifene (Ral) with higher stimulation in pre- than in post-menopausal cells. Only BA was almost five times more potent in pre- rather than post-menopausal hOBs. In male hOB only DHT and cBA increased 1,25D production whereas estradiol-17beta (E(2)) had no effect and BA decreased it. These results provide evidence for the expression of 1-OHase mRNA and production of 1,25D in hOBs, which are age and sex dependent and are hormonally modulated. The role of this local autocrine/paracrine 1,25D system in bone physiology deserves further investigation.

We demonstrated previously that vitamin D metabolites modulate the response of bone and cartilage cells to 17β-estradiol (E2) and dihydrotestosterone (DHT) both in cell cultures and in vivo. In the present study, we investigated to what extent pretreatment with 1,25(OH)2D3 or 24,25(OH)2D3 would reduce the minimal effective dose of E2, DHT or progesterone (P) required for stimulation of DNA synthesis and creatine kinase specific activity in cultured osteoblast-like ROS 17 2.8 cells and in rat embryo epiphyseal cartilage cells, and to what extent such pretreatment would increase the maximal response. We measured responses to sex steroids after pretreatment of the cells for 5 days with 0.02% ethanol vehicle or with the vitamin D metabolites 1,25(OH)2D3 (0.12 nM), or 24,25(OH)2D3 (1.2 nM) singly or in combination. Pretreatment of ROS 17 2.8 cells with 1,25(OH)2D3, but not 24,25(OH)2D3, increased synergistically their response to E2 but not to P, and did not affect their lack of response to DHT. Pretreatment of epiphyseal cartilage cells with either 1,25(OH)2D3 or 24,25(OH)2D3 increased synergistically their DNA synthetic response to all three steroids, but their CK response only to E2 or DHT. The minimal dose for causing a significant response to E2 in ROS 17 2.8 cells or to either E2 or DHT in epiphyseal cartilage cells was reduced 10-fold after pretreatment with vitamin D metabolites. After pretreatment, the maximal response was more than doubled in ROS 17 2.8 cells; epiphyseal cartilage cells showed a similar 10-fold decrease in the dose required for maximal response to E2 or DHT; the improvement in the response to P was significant only for DNA synthesis. We conclude that pretreatment with the appropriate vitamin D metabolite(s) both reduces by an order of magnitude, or more, the amount of sex steroids needed to stimulate skeletal derived cells and increases synergistically the maximal response of the cells.

We have demonstrated previously that 17 beta-estradiol (E2) stimulates proliferation of skeletal tissues, both in vivo and in vitro, as measured by increased DNA synthesis and creatine kinase (CK) specific activity. The effect of E2 on bone is sex specific. E2 is active only in females and androgens only in males. By contrast, in cartilage of both sexes, dihydrotestosterone (DHT) as well as E2 stimulates CK specific activity and DNA synthesis. In bone, we find that sex steroids stimulate skeletal cell proliferation in gonadectomized as well as in immature rats. Ovariectomized (OVX) rats, between 1 and 4 weeks after surgery, show stimulation of CK by E2. The basal activity and response of CK changes with the varying endogenous levels of E2 in cycling rats, in which the highest basal activity is at proestrus and estrus and the highest response is in diestrus. In rats of all ages tested, both the basal and stimulated specific activity of CK is higher in diaphysis and epiphysis than in the uterus, or in the adipose tissue adjacent to the uterus, which has a response similar to that of the uterus itself. The effect of E2 in vivo, and in chrondroblasts and osteoblasts in vitro, is inhibited by high levels of the antiestrogen tamoxifen which, by itself, in similar high concentrations, shows stimulatory effects. In addition to the sex steroids, skeletal cells are also stimulated by secosteroid and peptide calciotrophic hormones. The interactions of the sex steroids with these hormones modulate the response of cartilage and bone cells to both sex steroids and the other calciotrophic hormones. These results provide the first steps towards understanding the regulation of bone cell proliferation and growth by the concerted action of a variety of hormones and growth factors.

Two unrelated kindreds with four affected children having 1,25-dihydroxyvitamin D resistance, rickets, and alopecia are described. The children exhibited early onset of severe rickets with hypocalcemia, hypophosphatemia, elevated serum alkaline phosphatase levels, and secondary hyperparathyroidism. Radiography showed diffuse demineralization and classic changes of rickets. All affected children had total-body alopecia. Serum levels of 1,25-dihydroxyvitamin D3 were elevated and rose to extremely high values during treatment, with no apparent change in the mineral disorder. However, secondary hyperparathyroidism and hypophosphatemia did remit during treatment despite persistently low calcium levels. Skin biopsy was performed in the parents and affected children in one kindred. Analysis of 1,25-dihydroxyvitamin D3 receptors in cultured fibroblasts indicated apparent normal receptors in the parents and undetectable receptors in both affected children. After long periods of treatment with vitamin D metabolites and mineral replacement, healing took place in the older child in each kindred. These data suggest that the healing occurred spontaneously as the children reached seven to nine years of age rather than as a result of the treatment. The biochemical lesion in these children appeared to be a genetically transmitted defect in the 1,25-dihydroxyvitamin D3 receptor. The mechanisms by which healing was initiated and maintained remain to be elucidated.

The concentrations of unconjugated 25-OHD, 24, 25(OH)2D, and 1,25(OH)2D were measured in human milk by competitive protein-binding radioassays following successive preparative Sephadex LH-20 chromatography and HPLC. The mean (+/- SE) concentration of 25-OHD was 0.37 +/- 0.03 ng/ml, of 24,25(OH)2D was 24.8 +/- 1.9 pg/ml, and of 1,25(OH)2D was 2.2 +/-0.1 pg/ml. The concentration of 25-OHD3 in milk as determined by HPLC and UV detection at 254 nm was 0.27 +/- 0.08 ng/ml. The milk concentrations of vitamin D metabolites did not correlate with the maternal serum 25-OHD levels. The total amounts of unconjugated vitamin D metabolites correspond to the known low bioassayable vitamin D antirachitic activity in human milk.

We have demonstrated that epiphyseal chondroblasts contain specific receptors for 24R,25-dihydroxy vitamin D3(24,25(OH)2D3) while diaphyseal osteoblasts contain specific receptors for 1α25-dihydroxy vitamin D3(1,25(OH)2D3). Both metabolites induce DNA synthesis and creatine kinase (CKBB) activity. We have also found that the responsiveness of rat kidney to these metabolites changes during development. In embryonic and early postnatal stages, the kidney responds to 24,25(OH)2D3, later to both 24,25(OH)2D3 and 1,25(OH)2D3, and the mature kidney only to 1,25(OH)2D3. These responses correlate with changes in the specific receptors present in the kidney. Furthermore, we have compared developmental changes in skeletal (epiphysis, diaphysis and mandibular condyle) and non-skeletal (kidney, cerebellum, cerebrum, liver and pituitary) tissue in both rat (a postnatal developer) and rabbit (a perinatal developer). Epiphyseal or diaphyseal chondroblasts at any stage of development were predominantly responsive to 24,25(OH)2D3, whereas osteoblasts were responsive to 1,25(OH)2D3. In contrast, condylar chondroblasts, kidney, cerebellum and pituitary responded to 24,25(OH)2D3 during early development and subsequently developed responsiveness to 1,25(OH)2D3. Using primary cell cultures from kidneys at different stages of maturation, we showed the same developmental pattern as in vivo. Chronic treatment of the cells with 24,25(OH)2D3, but not 1,25(OH)2D3, caused precocious development of responsiveness to 1,25(OH)2D3 in culture. We suggest that 24,25(OH)2D3 acts as a maturation factor, during early development in kidney, and probably in other tissues, possibly by induction of receptor to 1,25(OH)2D3, accompanied by down-regulation of its own receptor.

There is emerging evidence for specific binding sites and biologic action for 24,25(OH)2D3 in the epiphyseal cartilage. The present study was undertaken to identify the target cells of 24,25(OH)2D3 in the epiphyses of rat bone using an autoradiographic technique. Pieces of epiphyseal cartilage obtained from 4-day-old vitamin D-deficient rats were incubated for 15 or 60 min with [3H]-24,25(OH)2D3 in the presence or absence of 100-fold excess of 25(OH)D3, 1,25(OH)2D3, or 24R,25(OH)2D3. The pieces were prepared for autoradiographic study by a new modified fixation method. Cytoplasmic and nuclear concentration of radioactivity was observed in all cell layers of the epiphyseal cartilage except for the hypertrophic cartilage zone. The highest concentration of radioactivity was seen in the proliferating chondroblasts of the columnar zone. After 15 min of incubation the radioactivity was seen mainly in the cell membrane and cytoplasm, whereas at 60 min radioactivity was also prominent in the nuclei. The competition with excess of cold metabolites revealed that only 24R,25(OH)2D3 caused a significant decrease in cytoplasmic and nuclear radioactivity. These data support the biochemical studies showing that the epiphyseal cartilage is a target tissue for 24,25(OH)2D3.

may signal the invasiveness of an amp-R strain that previously was carried only in the nasopharynx. Whether it would prove useful to monitor throat or nasopharyngeal cultures obtained from patients with H. influenzae type b meningius prior to initiating antibiotic therapy is unknown In the laboratory the presence of a heterogeneous population ofH. influenzae type b creates the potential for reports of false sensitivity to ampicillin. When the KirbyBauer disc method is utilized, agar plates must be inspected carefully for the presence of "breakthrough" colonies within the 20-mm zone of inhibition. 1 It also seems possible that. on rare occasions, only amp-S colonies could be subcultured from the original culture to sensitivity plates or utilized for the beta-lactamase assay. Furthermore. if the clinical isolate contains a small percentage of amp-R colonies, the color change may not be easily observed in the one-minute beta-lactamase test. since this reqmres greater than 97 units of penicillinase. 7 REFERENCES 1. Gray BM, Hubbell CA, and Dillon HD: Manner and meaning of susceptibility testing of ampicillin resistant