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The parathyroid hormone receptor type 1 (PTHR1) is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP) and primarily signals via intracellular pathways involving adenylyl cyclase and phospholipase C. The intracellular tail domain of the PTHR1 contributes to G protein subunit coupling that is important for second messenger signalling. In addition, the intracellular domain has a potential nuclear localization sequence (NLS) that, if functional, could point to an intracrine role for the receptor. In the present study, we have utilized 2 sets of constructs that employ either a [KRK(484-486)AAA](3Ala) mutation in the putative NLS or the non-mutant counterpart and included (a) the full-length rat PTHR1 with FLAG and c-myc epitope tags at the N-terminus and C-terminus, respectively (designated as PTHR1(3Ala)-TAG and PTHR1-TAG); and (b) only the putative NLS-containing intracellular domain (471-488), with green fluorescent protein (GFP) fused to the C-terminus (designated as GFP-(3Ala)471-488 or GFP-471-488). Porcine kidney LLC-PK1 cells stably expressing the PTHR1(3Ala)-TAG exhibited reduced signalling via both cAMP and cytosolic calcium transients in spite of greater cell surface expression relative to cells expressing PTHR1-TAG. We also examined the ability of the intracellular tail to influence the cellular localization of a heterologous protein. LLC-PK1 cells transiently transfected with GFP-471-488, exhibited increased fluorescence within the nucleus, relative to cells transfected with GFP alone that was not observed when cells were transiently transfected with the mutated construct, GFP-(3Ala)471-488. However, LLC-PK1 cells transiently transfected with either the full-length PTHR1-TAG or the PTHR1(3Ala)-TAG constructs did not exhibit nuclear localization of these receptors. Moreover, mouse osteoblast-like cells (MC3T3-E1) transiently expressing PTHR1-TAG also failed to demonstrate nuclear localization, although both full-length PTHR1 constructs exhibited plasma membrane immunofluorescence in both cell lines. Thus, the 484-486 sequence is critical for the full signalling responsiveness of the intact PTHR1, but the putative nuclear localization signal may not function as such within the intact receptor., (Copyright 2010 Elsevier Inc. All rights reserved.)

This study demonstrates that the PTH1R NLS can target a fusion protein to the nucleus, and that this is blocked by sequences downstream of the NLS. GFP fused to the NLS showed a significant increase in nuclear targeting compared to GFP alone or GFP fused to a peptide of the same length. In previous studies, we demonstrated that the type I PTH/PTHrP receptor (PTH1R) localizes to the nucleus of cells within rat liver, kidney, uterus, ovary and gut. Similarly, nuclear localization of the PTH1R was observed in the cultured osteoblast-like cells MC3T3-E1, UMR106, ROS 17/2.8 and SaOS-2. We have identified a putative bipartite nuclear localization signal (NLS), from residues 471-488 in the protein sequence of the PTH1R. In this study, several PTH1R constructs were made in the Enhanced Green Fluorescent Protein (EGFP) expression vector (Clontech), transiently transfected into LLC-PK1 Clone 46 cells, and the resultant fusion protein expression followed by fluorescence microscopy. This particular clone of LLC-PK1 shows no biochemical response in vitro to parathyroid hormone. Constructs included the entire PTH1R sequence (PTH1R-GFP), the putative NLS fused to the C-terminus of GFP (GFP-NLS) or the NLS through to the C-terminus of the PTH1R fused to GFP (GFP-NLSCT). Deconvolution fluorescence microscopy of cells transfected with PTH1R-GFP showed abundant fluorescent signal throughout the cells with distinctly fluorescing plasma membranes. These cells also exhibited an increase in cAMP production in response to (0-10(-8) M) hPTH(1-34), with an increase in cAMP from 11 fmol/mug of protein to 101 fmol/microg. In contrast, cells transfected with the GFP-NLS construct showed significant nuclear sequestration of fluorescence as compared to GFP alone, GFP-NLSCT, or a short amino acid sequence fused to GFP (GFP-FFVAIYCFCNGEVQAEI). These results indicate that the NLS at residues 471-488 of the mature rat PTH1R is functional and plays a role in targeting the PTH1R the nucleus, also the addition of GFP to the C-terminus of the PTH1R still allows cAMP generation which will be useful for further studies.

The type 1 PTH/PTH-related peptide receptor (PTH1R) is a class B G protein-coupled receptor that demonstrates immunoreactivity in the nucleus as well as cytoplasm of target cells. Our previous studies on the PTH1R have shown that it associates with the importin family of transport regulatory proteins. To investigate the role of the importins in PTH1R nuclear import, we used small interfering (si)RNA technology to knock down the expression of importin-beta in the mouse osteoblast-like cell line, MC3T3-E1. Immunofluorescence microscopy as well as ligand blotting for PTH1R in nuclear fractions of importin-beta siRNA-treated cells demonstrated a decrease in nuclear localization of the PTH1R in comparison with control cells. Under normal culture conditions, PTH1R is present in both the nucleus and cytoplasm of cells. Serum starvation favors nuclear localization of PTH1R, whereas returning cells to serum or treatment with PTH-related peptide induced its cytoplasmic localization. To address the nuclear export of PTH1R, interactions between PTH1R and chromosomal region maintenance 1 (CRM1) were investigated. PTH1R and CRM1 coimmunoprecipitated from MC3T3-E1 cells, suggesting that CRM1 and PTH1R form a complex in vivo. After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, PTH1R accumulated in the nucleus. Taken together, our studies show that PTH1R shuttles from the nucleus to the cytoplasm under normal physiological conditions and that this nuclear-cytoplasmic transport is dependent upon importin-alpha/beta and CRM1.

Extremely low frequency magnetic fields (ELF MF) have been reported to alter a number of cell signaling pathways, including those involved in proliferation, differentiation and apoptosis where cytosolic free calcium ([Ca(2+)](c)) plays an important role. To better understand the biological conditions under which ELF MF exposure might alter [Ca(2+)](c), we measured [Ca(2+)](c) by ratiometric fluorescence spectrophotometry during exposure to ELF MF in Jurkat E6.1 cells synchronized to different phases of the cell cycle. Suspensions of cells were exposed either to a near zero MF (Null) or a 60 Hz, 100 microT sinusoidal MF superimposed upon a collinear 78.1 microT static MF (AC + DC). An initial series of experiments indicated that the maximum increase in [Ca(2+)](c) above baseline after stimulation with anti-CD3 was significantly higher in samples exposed to AC + DC (n = 30) compared to Null (n = 30) with the largest difference in G2-M enriched samples. However, in a second study with G2-M enriched cells, samples treated with AC + DC (n = 17) were not statistically different from Null-treated samples (n = 27). Detailed analysis revealed that the dynamics in [Ca(2+)](c) before and after stimulation with anti-CD3 were dissimilar between Null samples from each study. From the results, we concluded (i) that the ELF MF increased [Ca(2+)](c) during an antibody-induced signaling event, (ii) that the ELF MF effect did not depend to a large degree on cell cycle, and (iii) that a field-related change in [Ca(2+)](c) signaling appeared to correlate with features in the [Ca(2+)](c) dynamics. Future work could evaluate [Ca(2+)](c) dynamics in relation to the phase of the cell cycle and inter-study variation, which may reveal factors important for the observation of real-time effects of ELF MF on [Ca(2+)](c).

Previous studies have shown that the type 1 PTH receptor (PTH1R), a class B G protein-coupled receptor, appears in the nucleus of target cells. Through immunofluorescence and deconvolution microscopy, we demonstrate that PTH1R, importin alpha(1), and importin beta are present within the nucleus and cytoplasm of osteoblast-like cell lines with the nuclear PTH1R being restricted to the nucleoplasm. Immunofluorescence studies showed that nuclear accumulation of PTH1R was associated with specific stages of the cell cycle. Using immunoprecipitation and affinity chromatography, we show that the PTH1R forms a complex with the importin family of transport molecules. Total cell protein from osteoblast-like cells was immunoprecipitated with antibodies for PTH1R, importin alpha(1), or importin beta. When the immunoprecipitates were separated and subsequently exposed to biotinylated PTH (1-84) a single band was present on the gel at 66.3 kDa, corresponding to the PTH1R. To confirm the interaction between PTH1R and both importin alpha(1) and beta, the complex was purified from total cell protein of osteoblast-like cells using a PTH-linked affinity chromatography column. Using an anti-importin alpha(1) antibody, Western blots detected importin alpha(1) at 58 kDa in the purified sample. Also, using an anti-importin beta antibody, Western blots detected importin beta at 94 kDa. These results indicate that the importins were associated with the PTH1R at the time of the purification. In conclusion, we show that the PTH1R forms a complex with the transport regulatory proteins, importin alpha(1) and importin beta, and that nuclear PTH1R is associated with the nucleoplasm.

The effects of alveolar large aggregate (LA) and small aggregate (SA) surfactant subfractions isolated from healthy adult rats on mitogen-stimulated proliferative responses of human peripheral blood mononuclear cells (PBMC) was examined. Various concentrations of total surfactant suppressed proliferation of stimulated lymphocytes by up to 95% of mitogen-stimulated cells alone. LA subfractions of total surfactant had no effect on proliferation, whereas SA significantly enhanced the lymphocyte proliferation at lower concentrations (7.8 microg/ml) compared to mitogen-stimulated cells alone. Higher concentrations of SA (62.5 microg/ml) inhibited lymphocyte proliferation. This concentration-dependent effect of SA on proliferation of PBMC was also present when cells were stimulated with various lectins including anti-CD3, concanavalin A and phytohemagglutinin. Analysis of the supernatant of mitogen-stimulated cell cultures treated with inhibitory concentrations of SA showed decreased amounts of interleukin (IL)-2, compared to cells alone, which could be reversed by adding exogenous IL-2 to the cell cultures with the SA. These results suggest that alveolar surfactant subfractions have distinct functions within the alveoli, both biophysically and with respect to their effects on the host's immunomodulatory responses.

Although dual-energy X-ray absorptiometry (DEXA) is an established technique for clinical assessment of areal bone mineral density (BMD), the spatial resolution, signal-to-noise ratio, scan time, and availability of clinical DEXA systems may be limiting factors for small-animal investigations using a large number of specimens. To avoid these limitations, we have implemented a clinical digital radiography system to perform rapid area DEXA analysis on in vitro rat bone specimens. A crossed step-wedge (comprised of epoxy-based materials that mimic the radiographic properties of tissue and bone) was used to calibrate the system. Digital radiographs of bone specimens (pelvis, spine, femur, and tibia from sham-ovariectomized [SHAM] and ovariectomized [OVX] rats) were obtained at 40 kilovolt peak (kVp) and 125 kVp, and the resulting areal BMD values were compared with those obtained with a clinical fan-beam DEXA system (Hologics QDR 4500). Our investigation indicates that the cross-wedge calibrated (CWC) DEXA technique provides high-precision measurements of bone mineral content (BMC; CV = 0.6%) and BMD (CV = 0.8%) within a short acquisition time (<30 s). Areal BMD measurements reported by the CWC-DEXA system are within 8.5% of those reported by a clinical fan-beam scanner, and BMC values are within 5% of the known value of test specimens. In an in vivo application, the CWC-DEXA system is capable of reporting significant differences between study groups (SHAM and OVX) that are not reported by a clinical fan-beam DEXA system, because of the reduced variance and improved object segmentation provided by the CWC-DEXA system.

Parathyroid hormone (PTH) increases trabecular but may decrease cortical bone mass during treatment of postmenopausal osteoporosis. In a 2-year trial, PTH, with or without sequential calcitonin (CT), was given to 29 osteoporotic women (mean age 67 +/- 7 years), in 3-month cycles [28 days hPTH(1-34), 50 microg/day, +/-42 days CT, 75 units/day, 20 days "free"]. Over 2 years, lumbar spine bone mineral density measurements increased an average of 10%. Paired iliac crest biopsies were obtained 28 days and 2 years after starting the trial. The addition of CT made no difference to changes seen with cyclical PTH alone. Thus, the histomorphometric analyses for all 29 treated patients were compared with a separate group of biopsies from untreated osteoporotic control patients (n = 15). No significant increments in total bone volume or trabecular architecture were seen over 2 years of cyclical PTH treatment, although the light microscopic appearance of bone was normal. At the level of the bone remodeling unit, a twofold increase in total trabecular erosion surface over the control measurements was observed within the first 28 days of PTH treatment (10 +/- 5 vs. 5 +/- 3% trabecular surface, p < 0.01), which was sustained over 2 years. Trabecular bone formation rates (surface referent) were 11 +/- 7 microm(3)/microm(2)/year in control patients and threefold higher in treated patients both acutely (31 +/- 31 microm(3)/microm(2)/year, p < 0.01) and after 2 years (33 +/- 43 microm(3)/microm(2)/year, p < 0. 05). The activation frequency of trabecular remodeling was threefold higher than controls through 2 years of treatment (p < 0.05). The mean wall thickness of completed osteons after 2 years of treatment was significantly larger than controls (28 +/- 7 vs. 22 +/- 5 microm, p < 0.01), suggesting a positive remodeling balance, as well as the histomorphometric evidence of increased bone turnover and the increased resorption surfaces. Over 2 years of cyclical PTH therapy, cortical thickness remained significantly higher than controls (680 +/- 202 vs 552 +/- 218 microm, p < 0.05), without significant changes in cortical porosity. Thus, the histomorphometric changes during cyclical PTH therapy in patients with severe osteoporosis are consistent with increased trabecular bone turnover and a positive remodeling balance, with no evidence for detrimental changes in cortical bone.

The localization of PTH/PTH-related peptide (PTHrP) receptor (PTHR) has traditionally been performed by autoradiography. Specific polyclonal antibodies to peptides unique to the PTHR are now available, which allow a more precise localization of the receptor in cells and tissues. We optimized the IHC procedure for the rat PTHR using 5-microm sections of paraffin-embedded rat kidney, liver, small intestine, uterus, and ovary. Adjacent sections were analyzed for the presence of PTHR mRNA (by in situ hybridization) and PTHrP peptide. A typical pattern of staining for both receptor protein and mRNA was observed in kidney in cells lining the proximal tubules and collecting ducts. In uterus and gut, the receptor and its mRNA are present in smooth muscle layers (PTHrP target) and in glandular cuboidal cells and surface columnar epithelium. This suggests that PTH, or more likely PTHrP, plays a role in surface/secretory epithelia that is as yet undefined. In the ovary, PTHR was readily detectable in the thecal layer of large antral follicles and oocytes, and was present in the cytoplasm and/or nucleus of granulosa cells, regions that also contained receptor transcripts. PTHR protein and mRNA were found in the liver in large hepatocytes radiating outward from central veins. Immunoreactive cells were also present around the periphery of the liver but not within two or three cell layers of the surface. Clear nuclear localization of the receptor protein was present in liver cells in addition to the expected cytoplasmic/peripheral staining. PTHR immunoreactivity was present in the nucleus of some cells in every tissue examined. RT-PCR confirmed the presence of PTHR transcripts in these same tissues. Examination of the hindlimbs of PTHR gene-ablated mice showed no reaction to this antibody, whereas hindlimbs from their wild-type littermates stained positively. The results emphasize that the PTHR is highly expressed in diverse tissues and, in addition, show that the receptor protein itself can be localized to the cell nucleus. Nuclear localization of the receptor suggests that there is a role for PTH and/or PTHrP in the regulation of nuclear events, either on the physical environment (nucleoskeleton) or directly on gene expression.

We have recently demonstrated that the receptor for parathyroid hormone (PTH) and PTH-related peptide (PTHrP), PTHR, can be localized to the nucleus of cells within the liver, kidney, uterus, gut, and ovary of the rat. We set out to determine the localization of the PTHR in cultured osteoblast-like cells. MC3T3-E1, ROS 17/2.8, UMR106, and SaOS-2 cells were cultured in alpha-modified eagle medium containing 15% fetal calf serum under standard conditions. Untreated cells were grown on glass coverslips to 75-95% confluence and fixed in 1% paraformaldehyde. For experiments designed to examine cells synchronized by serum starvation, cells were grown on glass coverslips, starved of serum for 46 h, and then fixed at 2-h intervals for a total of 26 h after the addition of serum to the medium. Parallel sets of cells were pulsed with [3H]thymidine to track the DNA duplication interval. The PTHR was localized by immunocytochemistry using a primary antibody raised against a portion of the N-terminal extracellular domain of the PTHR. The results presented herein indicate that the PTHR attains a nuclear localization in each cell line examined. In UMR106 cells, PTHR immunoreactivity was restricted to the nucleolus. After cell synchronization, MC3T3-E1 cells double approximately 24 h after the addition of serum. Immunocytochemistry for the PTHR in these cells showed that the receptor staining is initially diffuse for the first 6 h, then becomes more perinuclear in distribution by 12-16 h. Nuclear localization of the receptor is achieved approximately 16-20 h after the addition of serum and remains there throughout the mitotic phase. Intense staining of mitotic and postmitotic cells was observed. No change in cell proliferation kinetics was observed in MC3T3-E1 cells cultured in the presence of 25 nM PTH(1-34). These data suggest an important role for the PTHR in the nucleus of MC3T3-E1 cells at the time of DNA synthesis and mitosis.

Background: Dual energy x-ray absorptiometry provides the definitive measure of osteoporotic fracture risk., Objective: We sought to determine whether metabolic measures of bone formation and/or common features of clinical hypercortisonism provide a useful guide in selecting corticosteroid-treated asthmatic patients for referral for bone densitometry., Methods: We measured bone density and 8 AM serum osteocalcin, procollagen, and cortisol levels in 52 asthmatic adults aged 60.7 +/- 12.6 years (mean +/- SD). Years of steroid exposure for these patients was 11.8 +/- 10.7 (prednisone) and 11.78 +/- 4.98 (inhaled steroid). Using stepwise logistic regression, we assessed the capacity of the osteocalcin and procollagen levels, with or without the cortisol level, age, clinical features of hypercortisonism, and different lifetime exposures to inhaled and oral steroids for distinguishing between patients with greater or lesser risk of fracture., Results: Osteoporosis, defined as a bone density T score below -2.5, affected 26% of the group at the spine and 63% at the hip. At the spine, greater risk was associated only with lower cortisol levels (P =.003). Diagnostic accuracy was 71%, the false-positive rate was 26%, and the false-negative rate was 31%. At the hip, greater risk was associated with lower cortisol levels (P =.002), longer prednisone exposure, (P =.003), lower current doses of prednisone (P =.01) and inhaled steroid (P =.02), and older age (P =.01). Diagnostic accuracy was 83%, the false-positive rate was 13%, and the false-negative rate was 21%., Conclusions: Neither osteocalcin nor procollagen nor any of the clinical criteria analyzed proved sufficiently accurate to be reliable as indicators of the risk of fracture in these elderly, corticosteroid-treated asthmatic adults. They are therefore not useful for selecting such patients for diagnostic densitometry.

The 1-31 fragment of human PTH [hPTH-(1-31)NH2] has been shown, like hPTH-(1-34), to have anabolic effects on the skeletons of ovariectomized rats when given intermittently, but, unlike hPTH-(1-34), it does so without affecting serum calcium concentrations and does not activate the protein kinase C second messenger pathway in some target cells. To investigate the biochemical responses to hPTH-(1-31) in humans, we have directly compared it to hPTH-(1-34) during the course of slow infusions of each. Ten healthy adults, five men and five women, aged 26+/-5 yr (range, 22-37), each received 8-h continuous infusions of 8 pmol/kg.h hPTH-(1-34) and hPTH-(1-31) given in random order at least 2 weeks apart. During the infusions there were significant increases in both plasma and urinary cAMP (P < 0.05), but there were no differences in the responses between the two peptides (P = 0.362 for plasma; P = 0.987 for urine). There were also significant phosphaturic and natriuretic responses to the two peptides, which again were not different between peptides. During the infusion of hPTH-(1-34) serum ionized calcium (Ca2+) increased from 1.21+/-0.033 to 1.29+/-0.046 mmol/L (P < 0.01), and endogenous hPTH-(1-84) decreased from 29.6+/-9 to 15.0+/-5.7 pg/mL (P < 0.01), such that there was a negative correlation between them (r2 = 0.45). However, when hPTH-(1-31) was infused, neither serum Ca2+ (1.24+/-0.03 vs. 1.25+/-0.03) nor hPTH-(1-84) (26.8+/-5 vs. 30.7+/-12 pg/mL) was affected. Circulating concentrations of 1,25-dihydroxyvitamin D3 increased from 92+/-42 to 131+/-63 pmol/L (P < 0.05) during infusion of hPTH-(1-34) and from 92+/-27 to 110+/-42 pmol/L (P = NS) during hPTH-(1-31) infusion. There was also a significant increase in the urinary measure of type I collagen degradation of aminoterminal telopeptides from 78+/-45 to 101+/-51 nmol/mmol creatinine (P < 0.05) when hPTH-(1-34) was infused, but it was not affected (68+/-30 vs. 66+/-24 nmol/mmol creatinine) by hPTH-(1-31). Therefore, hPTH-(1-31) appears to be equivalent and equipotent to hPTH-(1-34) in the release of cAMP from target tissues and the renal handling of phosphate and sodium. However, at the doses employed, it does not increase serum calcium, is a weaker stimulator of the 25-hydroxyvitamin D-1alpha-hydroxylase, and does not induce rapid bone resorption.

To test the hypothesis that an antiresorptive agent might reduce the dosing requirement for an anabolic drug during reversal of osteopenia due to estrogen deficiency, the following experiment was conducted in 6-month-old female rats. Ovariectomy or sham surgery was performed and the following six experimental groups were studied. Untreated (SHAM) or ovariectomized (OVX) animals served as control groups. Four weeks post-OVX, osteopenic rats (now 7 months old), were treated in one of four experimental protocols: human parathyroid hormone (hPTH(1-34)), 80 microg/kg/day, given by subcutaneous injection 5 days/week; a selective estrogen receptor modulator (SERM), raloxifene analog LY117018 (RA), 3 mg/kg/day, given by gavage 5 days/week; and two combinations of LY117018 at the same dose and frequency with hPTH(1-34) (same dose, 5 times/week) and a reduced dosing interval of hPTH(1-34) (same dose, 2 times/week). After 12 weeks of treatment, the four experimental groups were sacrificed at age 10 months. SHAM and OVX controls were also studied at 7 and 10 months of age. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry at four skeletal sites: two mixed cortical/trabecular sites (femur and tibia) and two predominantly trabecular sites (lumbar spine and pelvis). The differences in BMD were consistent at all four sites. RA alone maintained BMD at all skeletal sites, but the results were not significantly improved over OVX controls, at age 10 months. hPTH(1-34) injections given 5 days/week resulted in BMD increments significantly higher than in either OVX or SHAM controls (p < 0.001). While the RA did not enhance the anabolic effects of full doses of hPTH(1-34), the addition of RA treatment to twice-weekly hPTH(1-34) dosing resulted in BMD increments at all four skeletal sites that were similar to the more intensive anabolic regimen of hPTH(1-34) therapy given 5 times/week. Therefore, an antiresorptive agent such as SERMs may potentially reduce the pharmacologic doses of PTH needed to reverse estrogen deficiency-induced osteopenia.

This experiment was designed to evaluate the ability of a raloxifene analogue (RA), LY117018, with or without reduced dosing of human parathyroid hormone (hPTH)(1-34) to maintain gains in bone mass after a fully anabolic treatment regimen given to aging osteopenic rats. Six-month-old rats were ovariectomized (ovx) or sham-operated (sham). After 1 month, ovx rats were treated with an anabolic regimen consisting of subcutaneous hPTH(1-34) 80 microg/kg/day and oral raloxifene 3 mg/kg/day, each given 5 days/week for 3 months. Thereafter, the treated ovx rats went on to an 8 week maintenance phase of treatment with either RA alone at the same dose, hPTH(1-34) at a reduced dosing interval (twice a week), or a combination of the two. Bone mineral density (BMD) was measured ex vivo at four skeletal sites, lumbar spine (L2-4), proximal hemipelvis, whole femur, and tibia, by dual-energy X-ray densitometry. All four sites showed a similar pattern of response. After the 3 month anabolic phase, the sham group had significantly higher BMD values than ovx rats at all skeletal sites (p < or = 0.002). The ovx rats treated with PTH + RA during the anabolic phase of the protocol had significantly higher BMD than the sham group in the femur, tibia, and spine (p < or = 0.02) and higher but not significantly different values in the pelvis. Following the 2 month maintenance phase, comparisons were made with the PTH-RA group at the end of the anabolic phase. Decrements in BMD were seen in all three maintenance therapy groups, but they were not statistically significant in the RA plus reduced PTH dose group. However, reduced hPTH(1-34) dosing and RA alone resulted in significant reductions of bone mass measurements at several skeletal sites during the maintenance phase. We conclude that the raloxifene analogue LY117018 may be useful in maintaining bone mass in aging ovx rats following anabolic therapy with hPTH(1-34) and raloxifene analogue, but that this strategy only allows for dose reduction of hPTH(1-34) rather than its discontinuation.

Rats and humans respond to intermittent treatment with parathyroid hormone (PTH) with increased bone density and cancellous bone volume. In the rat, osteoblast expression of insulin-like growth factor-I (IGF-I) is elevated by intermittent PTH. We examined the effect of continuous infusion of rhPTH(1-84), a bone catabolic regime, on the IGF system in rat pelvis. Female Sprague-Dawley rats (12 weeks, 250 g) were randomly assigned to receive 0, 0.1, 1, or 5 microg/100 g body weight (b.w.) rhPTH(1-84) (0, 0.106, 1.06, or 5.305 nmol/kg) in vehicle (1% normal rat serum in saline) delivered by subcutaneous Alzet minipump. After 7 days, blood was taken for serum chemistry and pelvises were processed for immunocytochemistry. Sections of pelvis from rats continuously infused with 0.1 or 1 microg/100 g b.w. rhPTH(1-84) for 7 days did not differ significantly from those of the vehicle-treated controls. However, continuous infusion of 5 microg/100 g b.w. rhPTH(1-84) resulted in a dramatic increase in cellular development, with trabeculae surrounded by many layers of large, plump osteoblasts. All pelvis osteoblasts expressed osteocalcin, but only those from rats that received 0, 0.1, or 1 microg/100 g b.w. rhPTH(1-84) showed positive staining for IGF-I. The extra-abundant osteoblasts from rats that received 5 microg/100 g b.w. rhPTH(1-84) did not stain for IGF-I. However, although all osteoblasts stained positively for IGF binding proteins (IGFBPs)-3, -4, and -5, staining for these IGFBPs increased as the dose of rhPTH(1-84) (and osteoblast number) increased. These results suggest that continuous infusion of PTH has a direct effect on osteoblast development (either recruitment or proliferation), decreases the expression of IGF-I, and enhances the expression of IGFBPs in pelvis, factors which may interact to bring about negative bone balance.

Components of the airspace-lining material may contribute to the local regulation of immune function within the lung. We report here that recombinant rat pulmonary surfactant-associated protein D (SP-D) inhibits the lectin- and anti-CD3-stimulated proliferation of human PBMCs. Inhibition was associated with a decreased production of IL-2, and the addition of human rIL-2 blocked the inhibitory action of SP-D. These effects were not inhibited by maltose, indicating that the inhibitory activity was not dependent upon the lectin activity of SP-D. Studies employing mutant SP-D lacking N-linked sugars or defective in multimerization further indicated that inhibition was not dependent upon cellular interactions with the N-linked oligosaccharide on SP-D or the oligomerization of trimeric SP-D subunits. Although a peptide containing an inverted DGR showed similar IL-2-dependent effects on anti-CD3-stimulated proliferation, deletion of the conserved DGRDGR sequence near the amino-terminal end of the collagen domain did not decrease the suppressive activity of SP-D. We hypothesize that SP-D can dampen lymphocyte responses to exogenous stimuli and protect the lung against collateral immune-mediated damage.

Investigation of possible mechanisms to describe the hyporesponsiveness of pulmonary leukocytes has led to the study of pulmonary surfactant and its constituents as immune suppressive agents. Pulmonary surfactant is a phospholipid-protein mixture that reduces surface tension in the lung and prevents collapse of the alveoli. The most abundant protein in this mixture is a hydrophilic molecule termed surfactant-associated protein A (SP-A). Previously, we showed that bovine (b) SP-A can inhibit human T lymphocyte proliferation and interleukin-2 production in vitro. Results presented in this investigation showed that different sources of human SP-A and bSP-A as well as recombinant rat SP-A inhibited human T lymphocyte proliferation in a dose-dependent manner. A structurally similar collagenous protein, C1q, did not block the in vitro inhibitory action of SP-A. The addition of large concentrations of mannan to SP-A-treated cultures also did not disrupt inhibition, suggesting that the effect is not mediated by the carbohydrate recognition domain of SP-A. Use of recombinant mutant SP-As revealed that a 36-amino acid Arg-Gly-Asp (RGD) motif-containing span of the collagen-like domain was responsible for the inhibition of T cell proliferation. A polyclonal antiserum directed against an SP-A receptor (SP-R210) completely blocked the inhibition of T cell proliferation by SP-A. These results emphasize a potential role for SP-A in dampening lymphocyte responses to exogenous stimuli. The data also provide further support for the concept that SP-A maintains a balance between the clearance of inhaled pathogens and protection against collateral immune-mediated damage.

In this study, we found that the trabecular architecture of the rat pelvis has similarities to that of human iliac crest. Although we made no direct comparisons between the estrogen deficiency-induced rat osteopenia model and postmenopausal histomorphometry of iliac crest, we attempted to determine whether the rat pelvis might be appropriate to study changes in bone modeling and in situ changes in osteoblast protein expression. Three groups of young, sexually mature rats (12 weeks of age, each group comprising six animals) were either ovariectomized (ovx) and treated with 17beta-estradiol (ovx + E), vehicle (ovx), or sham-operated (sham). Histomorphometric variables were quantitated in the pelvis and compared with proximal tibial metaphysis in the three groups. Immunocytochemical localization of osteocalcin was also evaluated in the two skeletal sites. There was a greater reduction in bone volume of the proximal tibial metaphysis of ovx rats than in the pelvis of ovx rats when compared with sham-operated animals (p < 0.01), although bone formation rates were significantly higher at the pelvic site than tibial metaphysis (p < 0.01). The more rapid loss of bone between the tibia and pelvis may reflect differences in longitudinal growth in young rats, but the other intersite differences in bone remodeling consequent to ovx were at least as well demonstrated in the pelvic trabecular structure. Because ex vivo removal of the rat pelvis is simple, and provides a larger histomorphometric section with which to evaluate dynamic changes in metabolic bone disease, we suggest that this site may be useful in studies of osteopenia in the sexually mature female rat. Immunocytochemical demonstration of osteocalcin in trabecular surface osteoblasts was excellent in both sites. These results suggest that the rat pelvis is as accessible for histological study as the more conventional appendicular sites. When compared with the proximal tibial metaphysis, the rat pelvis (1) has a more homogeneous trabecular structure; (2) has more than twice as much trabecular bone area to sample; (3) has no open epiphyseal growth cartilages; (4) loses trabecular bone half as rapidly after ovx; (5) displays a greater increase in bone turnover after ovx; and (6) is the same anatomic site that is sampled in humans. We have also shown that the pelvis is a suitable site to demonstrate immunocytochemistry for osteoblast-derived proteins.

To determine therapeutically and systemically equivalent dosages of budesonide inhaled through the Turbuhaler dry powder inhalation device (Astra Pharma Production AB, Södertälje, Sweden) or pressurized metered-dose inhaler (pMDI) plus Nebuhaler spacer (Astra Pharma Production AB), we compared these devices in a randomized, open, parallel-group trial. Adults with moderate to severe asthma inhaled budesonide (0.4, 0.8, 1.6, and 2.4 mg/day), for 2 weeks at each dose level, through the Turbuhaler (n = 30) or pMDI + Nebuhaler (n = 28). Dose-dependent effects were demonstrated on asthma symptoms (p = 0.0001), daily peak expiratory flow (p = 0.02), blood eosinophils (p = 0.0001), urinary cortisol output per day (p = 0.0001), serum cortisol (p = 0.006), serum osteocalcin (p = 0.0001), and the oropharyngeal Candida colony count (p = 0.0007. analysis of covariance). The ratio of the responses to the two inhalation devices approximated 1.0 for each index measured; that is, no significant between-device difference was found (p > or = 0.29). However, the 95% confidence limits for the ratio of their respective systemic effects on osteocalcin production were 0.83 to 1.48. Thus in adults who use inhalation devices efficiently and have optimally controlled asthma, conversions from the pMDI + Nebuhaler to the Turbuhaler may reasonably be made at milligram equivalent doses of budesonide, then down-titrated to minimize possible systemic effects. Because earlier studies have shown that the Turbuhaler can double intrapulmonary drug delivery in comparison with a pMDI without a spacer, a 50% dose reduction may be indicated when converting from a pMDI to the Turbuhaler.

Short cycles of human (h) PTH-(1-34) may have an anabolic effect to increase bone mass in patients with osteoporosis. As PTH also stimulates bone resorption, it is theoretically possible to enhance the anabolic effects of PTH by using a sequential antiresorptive agent in the treatment cycle. To test this hypothesis, 30 women with osteoporosis, aged 67 +/- 8 yr, completed a 2-yr protocol that comprised 28-day courses of hPTH-(1-34) (800 U) given by daily sc injections; each course was repeated at 3-month intervals. By random allocation, patients either received sequential calcitonin (CT) immediately following the cycle of hPTH-(1-34) (75 U/day, sc; PTH + CT; n = 16) or placebo CT (PTH alone; n = 14) for 42 days. Baseline bone mineral density (BMD) at the lumbar spine site revealed t scores of -3.7 +/- 1.2 (+/-SD) for the PTH alone group and -3.0 +/- 1.4 for the PTH + CT groups, who had 2.0 +/- 2.3 and 1.8 +/- 2.4 vertebral fractures, respectively, at entry to the study. At the end of the 2 yr, the lumbar spine BMD increased from 0.720 +/- 0.130 to 0.793 +/- 0.177 g/cm2 (10.2%) in the PTH group and from 0.760 +/- 0.168 to 0.820 +/- 0.149 g/cm2 (7.9%) in the PTH + CT group. These changes were significant over time in both groups (P < 0.001). Although the final 2-yr lumbar spine BMD was not significantly different between the two treatment groups, those patients receiving sequential CT injections gained bone mass at a consistently slower rate. Changes in BMD at the femoral neck averaged +2.4% and -1.8% in the PTH and PTH + CT groups, respectively, neither of which was significant. In the group receiving only cyclical hPTH-(1-34), the observed 2-yr vertebral fracture incidence was 4.5 compared to 23.0/100 patient yr in the PTH + CT group (P = 0.078). During the first two cycles, changes in biochemical markers of bone formation (serum total alkaline phosphatase, bone-specific alkaline phosphatase, and osteocalcin) and bone resorption (fasting urinary hydroxyproline and N-telopeptide excretion) were significantly increased over pretreatment values after 28 days of hPTH-(1-34) injections (P < 0.05 to P < 0.01 for both groups). Even end of cycle values remained elevated over the study baseline across time (P < 0.01). There were no significant differences for any outcome parameter between the two treatment groups. We conclude that short cycles (28 days) of daily hPTH-(1-34) injections result in significant increases in lumbar spine BMD, without significant changes in cortical bone mass at the femoral neck. Very low incident vertebral fracture rates were documented over 2 yr. However, there is no evidence that sequential antiresorptive therapy with CT is of any benefit over that conferred by cyclical PTH alone.

To examine the cardiovascular effects on the fetus of an elevated umbilical vascular resistance resulting in fetal hypoxemia, we embolized the fetal side of the placenta in pregnant sheep and measured cardiovascular and hormonal changes and cellular growth in fetal heart. Chronically catheterized fetal sheep were embolized (n = 6) for 21 days between 0.74 and 0.88 of gestation into the descending aorta until arterial oxygen content was decreased by 40-50% of the preembolization value. Control animals (n = 6) received saline only. During embolization, fetuses became chronically hypoxemic (P < 0.001) and hypertensive (P < 0.001), with a progressive increase in umbilical artery resistance index (P < 0.001). There was also an increase in fetal plasma norepinephrine throughout the study period (P < 0.05). On day 21 of embolization, fetuses showed asymmetrical growth restriction, increased heart weight (P < 0.01), and increase in right and left ventricular wall thickness (P < 0.05) compared with control animals. The protein-to-DNA ratio, an index of cell size, increased in the right ventricular myocardium in the embolized group (P < 0.001), suggesting myocardial cell hypertrophy. We conclude that, during chronic placental damage leading to fetal hypoxemia with an increase in umbilical artery resistance index, fetuses developed arterial hypertension and asymmetrical growth restriction and that increases in afterload to the heart and plasma norepinephrine likely caused fetal myocardial hypertrophy.

The hyporesponsive state of lung-derived mononuclear leukocytes has been, in part, attributed to the effects of the lipid rather than the protein components of pulmonary surfactant. In the present study, however, the results suggest that purified preparations of pulmonary surfactant-associated protein A (SP-A) suppress both phytohemagglutinin (PHA, 1 microgram/ml)- and anti-CD-3 (1 to 10 ng/ml) activated proliferation of human peripheral blood and tonsillar mononuclear cells in a dose-dependent manner at concentrations as low as 50 pM (6.25 micrograms/ml) when added at the initiation of cultures. Addition of SP-A to PHA-stimulated peripheral blood mononuclear cells (PBMC) as late as 24 to 36 h after PHA was also capable of suppressing [3H]thymidine incorporation measured at 72 h. In contrast, concanavalin A (Con A; 2 micrograms/ml)-stimulated PBMC proliferation was slightly augmented by the addition of SP-A. Analysis of the supernatants of PHA-stimulated cultures treated with SP-A revealed that accompanying the inhibition of proliferation was a corresponding decline in measurable interleukin-2 (IL-2) concentrations, from 154 pg/ml for the PHA-treated cells to 57.8, 28.4, 5.2, and less than 2 pg/ml of IL-2 when SP-A was added at 6.25, 12.5, 25, and 50 micrograms/ml, respectively. We suggest that the action of SP-A on PHA-stimulated human PBMC may involve the blocking of a costimulatory signal crucial for in vitro T-cell activation.

Objective: Parathyroid hormone-related protein (PTHrP) is a 141 amino acid protein which contains a 1-36 N-terminal domain resembling parathyroid hormone which has smooth muscle relaxant activity and a mid (67-86) domain which reportedly alters placental calcium transport. Using specific antibodies to these regions of PTHrP, the objective of this study was to determine changes in the levels and localization of the peptides in placenta and membranes that might be indicative of their biological activity and role during term and preterm labor., Study Design: Placenta and fetal membranes were collected from patients with preterm delivery (PTL) (n = 16), term cesarean section in the absence of labor (n = 10) and term vaginal delivery (n = 5). Immunohistochemistry was performed with specific antisera visualized by the avidin-biotin peroxidase method and the staining intensity was quantified with an image analysis system MCID., Results: Immunoreactive (ir)-PTHrP(1-34) and ir-PTHrP(67-86) were localized to the amnionic epithelium chorionic trophoblasts, decidual cells and placental syncytiotrophoblast. Intense immunostaining was observed for ir-PTHrP(67-86) but not for ir-PTHrP(1-34) in the endothelial lining of the villous capillaries. Ir-PTHrP(1-34) staining was lower in placenta and fetal membranes of PTL patients compared with term cesarean section in the absence of labor (P < 0.05 Mann-Whitney test). In contrast, there was no difference in ir-PTHrP [67-86] staining intensity between delivery categories., Conclusion: These results showing differential localization of PTHrP(1-34) and PTHrP(67-86) suggest cell specific processing of PTHrP precursor in the human placenta. Moreover, the changes in ir-PTHrP(1-34) but not ir-PTHr(67-86) with labor are indicative of a particular role for this peptide in the delivery process.

To determine whether high-dose or prolonged inhaled steroid therapy for asthma increases a patient's risk of osteoporosis and fracture, we measured bone density in 26 men and 43 women (41 postmenopausal, all of whom had received supplemental estrogen therapy) after treatment with an inhaled steroid for 10.1 +/- 5.5 years and oral prednisone for 10.7 +/- 9.7 years (mean +/- SD). Most had stopped receiving prednisone since commencing the inhaled steroid therapy. We found that bone densities (adjusted for age and sex to yield a z score) were lower in association with higher daily doses of inhaled steroid (p = 0.013 ANCOVA) and with the duration of past prednisone therapy (p = 0.032). Larger cumulative inhaled steroid doses were associated with higher bone densities (p = 0.002) and a reduction in the numbers of patients at risk of fracture. Bone density also increased with the amount of supplemental estrogen therapy (p = 0.058) and, at equivalent levels of inhaled and oral steroid use, women showed higher bone density z scores than did men. Women with a lifetime dose of inhaled steroid greater than 3 gm had normal bone density regardless of the amount of past or current prednisone use or the current dose of inhaled steroid. These data indicate that the daily dose, but not the duration, of inhaled steroid therapy may adversely affect bone density, and that estrogen therapy may offset this bone-depleting effect in postmenopausal women.

Sarcoidosis is characterized by the accumulation of activated lymphocytes in the lungs and other organs and by the spontaneous production of the active form of vitamin D, calcitriol. We hypothesized that calcitriol may modulate the responsiveness of human lymphocytes to the relevant biologic mediator, interleukin-2 (IL-2). After culture for 48 h with phytohemagglutinin, human peripheral blood lymphocytes migrated through nitrocellulose filters, secured in microchemotaxis chambers, in response to IL-2. When calcitriol at 1 nM was included in the cultures, the migratory response to IL-2 was completely abrogated. This inhibitory effect was seen despite the fact that cultured lymphocytes continued to express the IL-2 receptor and other activation markers. A similar but more rapid effect could be demonstrated by including calcitriol in the lower well during our 3-h chemokinesis assay. Calcitriol blocked IL-2-induced lymphocyte migration in a dose-dependent fashion. The synthetic noncalcemic vitamin D analogue MC 903 was equally effective in this assay. IL-2-induced migration could also be prevented by the protein kinase C inhibitor H-7, but calcitriol appeared to be at least 1,000 times more potent. Our studies suggest that calcitriol is a potent natural immunomodulator with rapid suppressive effects that may be mediated through protein kinase C. Synthetic analogues such as MC 903 may offer exciting therapeutic opportunities.

Background: There is growing evidence to suggest the importance of the lymphocyte in the pathogenesis of asthma, particularly in the late phase reactions and ongoing bronchial hyperreactivity. Platelet activating factor (PAF) has also been identified as a potentially important mediator in asthma., Methods: The migration of human peripheral blood lymphocytes obtained from normal volunteers in response to PAF and the effect of PAF antagonists was studied in a well standardised in vitro assay using nitrocellulose micropore filters in a microchemotaxis chamber., Results: PAF is a potent stimulus to in vitro human lymphocyte migration; at an optimal concentration of 1 nM it augmented lymphocyte chemokinesis to 310% (SE 33%) of control values. The response to PAF appears to be specific since lyso-PAF and other related membrane phospholipids had no effect. PAF-induced migration could be abrogated by specific PAF receptor antagonists such as WEB 2086 (100 nM), and was partially blocked by the cyclooxygenase inhibitor flurbiprofen at a concentration of 1 microM., Conclusions: PAF stimulates the in vitro migration of human lymphocytes through a specific PAF receptor. Part of the response may be due to the generation of cyclooxygenase products. PAF may play a part in the recruitment of lymphocytes to asthmatic airways.

The amino-terminal fragments of human PTH [hPTH-(1-34)] and PTH-related peptide [PTHrP-(1-34)] appear to be equipotent in several rodent models. However, continuous i.v. infusions of these peptides to young human volunteers suggested that a 10-fold higher molar dose of PTHrP was required to produce comparable circulating levels of the peptide and biochemical responses similar to PTH. As PTHrP has a wide variety of target tissues in mammalian species and may, therefore, play a paracrine, rather than an endocrine, hormonal role in vivo, we evaluated whether enhanced metabolic clearance of injected PTHrP might explain its apparently reduced potency as a PTH-like hormone. Ten healthy subjects [age, 25 +/- 9 (+/- SD) yr] received in random order either hPTH-(1-34) or hPTHrP-(1-34) given by bolus i.v. injections in a dose of 10.7 nmol. Measurements of plasma immunoreactive peptide indicated a comparable volume of distribution for each, but the apparent t1/2 (8.3 +/- 1.6 min) and plasma clearance (4.0 +/- 1.4 L/min) for hPTHrP were significantly (P < 0.05) accelerated compared to those of hPTH (t1/2, 10.2 +/- 0.5 min; clearance, 2.0 +/- 0.4 L/min). Peak plasma cAMP levels were 9-fold lower in response to hPTHrP (29.5 +/- 19 vs. 190 +/- 63 pmol/L; P < 0.01), and increases in urinary cAMP excretion were 5-fold lower (2.1 +/- 1.1 vs. 11.2 +/- 3.7 nmol/mmol creatinine; P < 0.01). No major differences were observed in the urinary excretion of phosphate, calcium, or sodium between the two peptides. Although hPTHrP-(1-34) has a 2-fold higher MCR than hPTH-(1-34), this may not explain the more than 5-fold lower plasma or urinary cAMP response to PTHrP in humans. The comparable effects of PTH and PTHrP on urinary phosphate, calcium, and sodium may indicate a non-cAMP-dependent pathway for these responses, although the intracellular pool of cAMP generated to either peptide, and thus the local target tissue response, could not be estimated in the present study.

Regulation of long bone growth by growth hormone and other endocrine factors is mediated by the local synthesis of IGF-I in the growth plate. Recent evidence suggests that different regions of the growth plate exhibit variable growth rates. To investigate whether IGF-I gene expression in the growth plate differs in relation to growth, we examined the distribution of IGF-I mRNA and peptide using in situ hybridization and immunohistochemistry, respectively, in the tibiae of 18-week-old rats (n = 6). Osteoblasts were identified by osteocalcin immunoreactivity, and osteoclasts by tartrate-resistant acid phosphatase (TRAP) histochemistry. The abundance of IGF-I mRNA in growth plate chondrocytes was quantified by counting the autoradiographic signal associated with each cell. IGF-I mRNA was identified in chondrocytes of both the proliferative and hypertrophic zones of the growth plate. Cells in the marginal regions of both zones contained significantly more IGF-I mRNA than those in the central region (p < 0.05). In addition, IGF-I mRNA levels were greater in the periphery of the growth plate on the medial side of the tibia (p < 0.05) in which there was more active growth than the lateral side. IGF-I immunoreactivity was present predominantly in the hypertrophic zone chondrocytes and no regional differences in its distribution were observed. IGF-I mRNA and peptide were also identified in periosteal fibroblasts, notably at sites of muscle attachment to bone, and in osteoblasts at active sites of bone remodelling in the periosteal, endocortical, and endosteal bone envelopes. In the TRAP-positive osteoclasts, IGF-I immunoreactivity, but not IGF-I mRNA, was detected. In addition, both IGF-I mRNA and peptide were identified in the hemopoietic cells of the metaphyseal bone marrow, whereas only IGF-I immunoreactivity was detectable in the diaphysis. We conclude that, in the tibiae of mature rats: (i) IGF-I gene expression in the growth plate is related to its growth and/or synthetic activity; and (ii) the presence of IGF-I in osteoblasts and osteoclasts suggests its involvement in active bone growth and remodeling.

Background: Retinol deficiency may contribute toward the development of chronic lung disease in very low birth weight (VLBW) infants. We examined the retinol status during early infancy in VLBW infants from birth to 6 weeks 'post-term'., Methods: Concentrations of serum retinol (SR) and its carrier proteins, retinol-binding protein (RBP), and transthyretin (TTR), were determined at birth, then weekly for 8 weeks, and at 4-6 weeks 'post-term' in preterm infants of less than 34 weeks gestation. The SR values of umbilical cord blood at birth from the preterm infants were compared to the maternal SR levels as well as to cord SR levels of term infants., Results: From 24 through 33 weeks gestation, umbilical cord SR at birth was significantly lower than, but did not correlate with, maternal SR (P < 0.01). The cord SR in term infants was also higher than that in preterm infants (262 +/- 68 vs. 183 +/- 67 micrograms/l, P < 0.01). Longitudinal profiles of SR in 18 VLBW infants showed that, despite regular retinol supplementation, there was a decline in SR after birth, reaching a nadir of 128 +/- 40 microgram/l at 5-7 weeks (P < 0.001), followed by an increase toward levels comparable to those seen in full term infants. At follow-up at the corrected age of 4-6 weeks 'post-term', SR levels in VLBW infant (222 +/- 74 micrograms/l) had returned to within the normal range for term cord SR values. The concentrations of RBP also showed a similar biphasic pattern. Transthyretin levels did not change for 8 weeks but increased significantly at 4-6 weeks 'post-term'., Conclusions: Current practices of retinol supplementation in VLBW infants fail to maintain adequate retinol status in those infants during the neonatal period. Further efforts to improve the retinol status in these infants should be explored.

In fetal sheep, prolonged hypoxia (for 24 h) induced by a reduction in maternal uterine artery blood flow, increases insulin-like growth factor binding protein-1 (IGFBP-1) levels and decreases IGFBP-2 levels in the plasma, with corresponding changes in messenger RNA (mRNA) levels in the liver. Since IGFBP-1 synthesis in liver cells in vitro is stimulated by compounds that increase intracellular cAMP concentrations, we hypothesized that the increased IGFBP-1 synthesis during prolonged hypoxemia may be induced by circulating catecholamines, that are released during hypoxia, and that elevate fetal liver cAMP levels. Our aim was to determine the effect of 24-h catecholamine infusions on the synthesis and release of IGFBP-1 and IGFBP-2 in fetal sheep. Vascular catheters were implanted into fetuses at 110-115 days gestation in 14 pregnant ewes. After a 5-day recovery period, fetuses received a 24-h infusion of either norepinephrine (1 micrograms/kg.min, n = 5), epinephrine (0.25 micrograms/kg.min, n = 5), or vehicle (normal saline, n = 4). Fetal carotid arterial samples were collected at specified intervals throughout the infusion for the determination of blood glucose concentrations, plasma catecholamine concentrations by HPLC, insulin, and glucagon concentrations by RIA, and IGFBP levels by Western ligand blotting. After 24 h, the ewe and fetus were killed and selected fetal tissues (liver and kidney) were collected, and analyzed for IGFBP mRNA levels by northern blotting followed by laser densitometric quantification. Plasma catecholamine concentrations were increased in treated fetuses to levels that may be expected in fetuses subjected to prolonged hypoxia. In epinephrine and norepinephrine infused fetuses, blood glucose and plasma glucagon concentrations were increased significantly, whereas plasma insulin concentrations were decreased significantly. Norepinephrine and epinephrine infusions increased IGFBP-1 levels significantly (2- to 5-fold) in fetal plasma within 8-12 h, and the time course pattern of elevation of plasma IGFBP-1 levels was similar to that observed in prolonged hypoxia. After 24 h of either norepinephrine or epinephrine infusion, IGFBP-1 mRNA levels in the liver of fetuses were increased significantly (5- to 7-fold) compared to those of vehicle infused fetuses. IGFBP-2, -3, and -4 levels in fetal plasma were not affected by either infusion, nor were IGFBP-2 mRNA levels in fetal liver and kidney.(ABSTRACT TRUNCATED AT 400 WORDS)

Prostaglandins (PGs) have been implicated as stimulants to myometrial contractility at parturition in many species. To determine whether the increased production of PGs at parturition reflects a general increase in the metabolism of arachidonic acid or a specific increase in PG endoperoxide H synthase (PGHS) compared with lipoxygenase activities, and to determine intrauterine sites of these activities, we examined the metabolism of [3H]arachidonic acid by homogenates of placenta, amnion and chorion from sheep at days 78-80, 100-105, 135-140 of pregnancy and at term (day 145). Tissues were also obtained from fetuses at day 125; four of these were infused for 84 h with cortisol and four were used as saline-treated controls. The endogenous arachidonic acid content at the start of incubation was measured by capillary gas chromatography. Radioactive metabolites were separated and quantified by reverse-phase high-pressure liquid chromatography. At each gestational age arachidonic acid was converted to PGs, leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). Conversion to PGs was greater in amnion than in chorion or placenta between days 78 and 140. The formation of PGs rose in placenta at term to a mean value twice that of amnion and ten times that of chorion. In amnion, the ratio of PG:LT rose significantly at term relative to 100-140 days of gestation. In placenta, the ratio of PG:LT produced from arachidonic acid and the ratio of total PGHS:lipoxygenase products rose significantly at term. In the day-125 fetuses treated with cortisol there was a significant increase in PG production relative to that in control fetuses infused with saline in placenta, amnion and chorion; the placenta and amnion being the major sites of PG production. Production of LTs and HETEs also rose significantly in the chorion and the placenta relative to controls. In both the placenta and the amnion there was a significant increase in the ratio of total PGHS to lipoxygenase products formed. We conclude that at term labour and in labour induced by intrafetal cortisol infusion, the placenta is the major site of arachidonic acid metabolism, and that there is a preferential increase in the formation of PGs over lipoxygenase products. These results are consistent with the suggestion that there is an increase in the expression or activity of PGHS in the placenta of sheep in late pregnancy.

Female patients (n = 20) with osteoporosis, aged 66 +/- 5 yr were studied during a 24-h infusion of parathyroid hormone (PTH [1-34]) at a rate of 0.5 IU equivalents/kg.h, and then during a 28-d period of subcutaneous injections, at a dose of 800 IU equivalents per day. Thereafter half the patients received subcutaneous injections of calcitonin, 75 U/d for 42 d, and all patients were followed to the end of a 90-d cycle. Biochemical markers of bone formation (serum alkaline phosphatase, osteocalcin, and the carboxy-terminal extension peptide of pro-collagen 1) and bone resorption (fasting urine calcium, hydroxyproline, and deoxypyridinoline) were compared during treatment by the intravenous and subcutaneous route of PTH administration, and subsequently during calcitonin therapy. During intravenous PTH infusion there were significant reductions in all three bone formation markers, despite expected rises in urinary calcium and hydroxyproline. By contrast, the circulating markers of bone formation increased rapidly by > 100% of baseline values during daily PTH injections (P < 0.001). Significant increases in bone resorption markers were only seen at the end of the 28 d of injections, but were < 100% over baseline values, (P < 0.05). Quantitative bone histomorphometry from biopsies obtained after 28 d of PTH treatment confirmed that bone formation at both the cellular and tissue levels were two to five times higher than similar indices measured in a control group of biopsies from untreated osteoporotic women. Subsequent treatment of these patients with calcitonin showed no significant changes in the biochemical markers of bone formation and only a modest attenuation of bone resorption. Thus, PTH infusion may inhibit bone formation, as judged by circulating biochemical markers, whereas daily injections confirm the potent anabolic actions of the hormone. Sequential calcitonin therapy does not appear to act synergistically with PTH in cyclical therapeutic protocols.

We have examined the effects of reduced uterine blood flow and prolonged fetal hypoxemia on the temporal relationship between changes in hormones associated with the activity of the pituitary-adrenal axis (corticotrophin-releasing hormone (CRH), adrenocorticotrophin (ACTH), cortisol, and prostaglandin E2 (PGE2) in the ovine fetus at 120-125 days of pregnancy, and we sought evidence for placental secretion of CRH and ACTH during prolonged hypoxemia. Uterine blood flow was reduced by placing an adjustable Teflon clamp around the maternal common internal iliac artery to decrease fetal arterial oxygen saturation from mean values of 59.1 +/- 3.3 to 25.7 +/- 4.6% (+/- SEM, n = 10). There was a transient peak in immunoreactive (IR-) CRH at 1-2 h after reducing uterine blood flow. IR-ACTH rose to peak values at +2 h, then gradually decreased to control level by +12 h. Fetal plasma cortisol and PGE2 concentrations were elevated significantly by +2 and +4 h, respectively, and at 20-24 h. The identity of IR-CRH in fetal plasma and in ovine placental extracts was confirmed by HPLC, but there was no consistent umbilical vein--femoral arterial concentration difference for either IR-CRH or IR-ACTH during normoxemia or hypoxemia. We conclude that a sequence of endocrine changes involving CRH, ACTH, PGE2, and cortisol occurs in the fetus during a prolonged reduction in uterine blood flow. However, we did not obtain evidence, for placental secretion of either CRH or ACTH in response to this manipulation.

PTH-related peptide (PTHrP) is one of the etiological factors associated with hypercalcemia of malignancy in humans and rodents. In both in vivo and in vitro animal systems its actions mimic those of PTH; however, its bioactivity in humans has not previously been assessed. Therefore, we compared the actions of the synthetic human (h) analogs hPTHrP-(1-34) and hPTH-(1-34) when given by iv infusion to 15 healthy subjects, aged 25 +/- 3 yr. Three 12-h test infusions were given to each subject in the order: hPTH-(1-34) at a dose of 8 pmol/kg.h, an equimolar dose (8 pmol/kg.h) of PTHrP-(1-34) (low dose), and a 10-fold higher dose (80 pmol/kg.h) of hPTHrP-(1-34) (high dose). PTH infusion resulted in significant increases from basal values in serum total ionized calcium, urinary phosphate and cAMP, and serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2d3]. No significant increases from basal values in any of these variables were observed during low dose PTHrP infusion. However, a 10-fold higher dose of PTHrP significantly increased serum calcium from 2.36 +/- 0.07 to 2.63 +/- 0.16 mmol/L (P less than 0.003), ionized calcium from 1.22 +/- 0.03 to 1.39 +/- 0.09 mmol/L (P less than 0.003), urinary phosphate from 0.21 +/- 0.19 to 0.31 +/- 0.16 mmol/L glomerular filtrate (P less than 0.05), urinary cAMP from 37 +/- 18 to 53 +/- 28 nmol/L glomerular filtrate (P less than 0.01), and serum 1,25-(OH)2D3 from 29.8 +/- 12.1 to 46.0 +/- 20.3 pmol/L (P less than 0.01). For each variable these changes were statistically equivalent to the increases observed during PTH infusion. The molar concentrations of circulating immunoreactive PTH-(1-34) and PTHrP-(1-34) (at the higher dose) achieved during infusion were at a ratio of 1:3. These results suggest that the in vivo actions of synthetic hPTHrP-(1-34) are comparable to those of hPTH-(1-34), but its biological activity after infusion may be less than that of hPTH-(1-34). Moreover, the increased concentrations of serum 1,25-(OH)2D3 observed with administration of hPTHrP-(1-34) are unlike the changes seen in hypercalcemia of malignancy in which levels of this vitamin D metabolite are frequently depressed.

To assess whether the use of larger than usual doses of inhaled steroid to treat severe asthma may adversely affect bone turnover and whether such an effect may be mitigated by altering the dose schedule, we investigated the effects of budesonide (BUD) on serum osteocalcin and the urinary output of hydroxyproline and calcium. Healthy adults were administered 1.2 or 2.4 mg of BUD per day (N = 40) or placebo (N = 8) in a crossover, double-blind comparison of morning versus diurnal dosing schedules for 1 month each. Both BUD doses reduced the 24-hour urinary free-cortisol output (p less than 0.001) and serum osteocalcin (p less than 0.001). The larger dose reduced the morning serum cortisol levels (p = 0.002). Neither dose increased the 8 AM urinary calcium or hydroxyproline output. Osteocalcin and plasma cortisol levels were higher on morning than on diurnal dosing (p = 0.01). The 24-hour urinary free-cortisol output was the same with either schedule (p = 0.96). Additional study is required to assess the clinical importance of the inhibitory effect of BUD on bone formation, as evidenced by the reduction in osteocalcin levels. Of concern is the possibility of serious bone complications resulting from the long-term use of inhaled steroid, particularly in growing children or patients in whom other risk factors for osteoporosis are present. The clinical advantage, if any, of morning dosing remains questionable.

Previous studies, in which adult rats were exposed to 1 ppm ozone for 2 weeks, demonstrated the appearance in plasma of separate factors that stimulated DNA synthesis by cultured pneumocytes and lung fibroblasts in a dose-dependent and cell-specific fashion. Both factors had isoelectric points of 6.45-6.75, but differed by molecular mass. The pneumocyte factor had an estimated weight of 38 +/- 3 kDa, while the fibroblast factor had an estimated molecular weight of 32 +/- 2 kDa. To determine whether the appearance of these factors in plasma is specific for ozone injury or whether they appear in response to other oxidant injuries, adult rats were exposed to 85% O2 or air for up to 2 weeks. Animals were sacrificed at 3, 5, 7, or 14 days after the onset of exposure. Plasma samples were subjected to sequential preparative electrofocusing and high-performance liquid chromatography (HPLC). Heat-inactivated plasma fractions, with an isoelectric point of 6.45-6.75, contained a factor of 32 +/- 2 kDa, which enhanced lung fibroblast DNA synthesis at a single time point on day 5 of 85% O2 exposure, and a factor of 38 +/- 3 kDa, which enhanced pneumocyte DNA synthesis on days 5, 7, and 14 of 85% O2 exposure. Of the known growth factors, those most likely to have these physical characteristics are platelet-derived growth factor (PDGF) and insulin-like growth factor-1. Additional groups of animals were exposed to air or 85% O2 for 5 days for plasma collection. Animals exposed to 85% O2 had a 60% increase of plasma immunoreactive PDGF and a 90% increase of plasma immunoreactive IGF-1, compared with values for control animals exposed to air.

Cyclical treatments of osteoporosis utilizing a skeletal Activator of bone remodelling, and sequential therapy with a Depressor to selectively block the resulting phase of osteoclastic resorption have been dubbed 'ADFR' therapy; there is usually a treatment Free interval while the activated bone multicellular units complete the remodelling cycle before the protocol is Repeated. In this report an ADFR protocol was developed in which all patients received synthetic hPTH (1-38) for the first 14 days of a 100 day cycle. Half the patients received no other therapy (Group 1), but were followed closely with repeated vertebral bone mineral measurements over two full cycles. The remaining patients (Group 2) were randomly allocated to receive salmon calcitonin, at an average dose of 79 units per day for a 56 day depressor period immediately following each phase of activation. Detailed bone histomorphometry was performed on iliac biopsies obtained before treatment and at the end of the second cycle (Day 200). In Group 1, the serum alkaline phosphatase (Alk. P'ase) increased by 23 +/- 12% (P less than 0.01) and by 18 +/- 16% (P less than 0.03) of the baseline values following PTH treatment during the first and second cycles, respectively. The overall changes in serum Alk. P'ase across time were significantly less (P less than 0.04) in Group 2; however this parameter also increased by 15 +/- 15% during the first cycle and 8 +/- 6% during the second cycle. Vertebral BMC increased by 13% in Group I (P less than 0.01), but forearm BMC decreased by 11% (P less than 0.05) over the two cycles of therapy. There were no significant changes in bone mineral measurements in Group 2, but the differences between the two groups were not significant. Eighteen paired biopsies were available for histomorphometric analysis. There were no significant changes in static parameters measuring total bone tissue, osteoclastic function or osteoid formation after two cycles of treatment. Individual bone formation rates (surface referent) were not significantly different between the two groups; the pooled data for all biopsies showed a small but insignificant increase from 0.030 +/- 0.018 to 0.035 +/- 0.028 mm3/mm2/day. However there was a significant increase in the activation frequency (the probability of a remodelling event occurring on queiscent cancellous surface) from 13 +/- 7 to 27 +/- 26/day x 10(-4) (P less than 0.05) when calculated for the pooled data from both groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.

Inhaled glucocorticosteroids have been developed for the treatment of asthma in an attempt to minimize the suppression of endogenous adrenal function that complicates oral or injected steroid usage, but it is unclear whether this strategy leads to reduced systemic complications in other areas, such as the skeleton. In this study we evaluated serum osteocalcin levels as a marker of skeletal metabolism in healthy volunteers treated with oral and inhaled steroids alone and in response to an oral calcitriol stimulation test. Forty subjects, aged 33 +/- 9 (mean +/- SD) yr were randomized to receive either high or low dose oral prednisolone (40 vs. 10 mg/day) or inhaled budesonide (3.2 vs. 0.8 mg/day). Each dose of budesonide is known to have a greater antiasthmatic potency than the dose of prednisolone with which it was compared. In addition 10 control subjects received placebos containing no active steroid drugs. During the second week of treatment, half of the subjects in each of the 4 steroid-treated groups and all subjects in the control group received oral calcitriol (2.0 micrograms/day). There was a marked dose-dependent reduction in serum cortisol levels, but this reduction was significantly less pronounced during budesonide treatment, such that low dose budesonide was without effect. During the first week of steroid therapy there were significant dose-dependent reductions in serum osteocalcin (P = 0.003), but this reduction was not significantly different between budesonide and prednisolone treatments. In response to calcitriol, serum osteocalcin increased by 35% in the control group (P = 0.06). Osteocalcin levels increased by 56% and 50% in the low dose budesonide and prednisolone groups and by 106% in the high dose budesonide group, but did not change in the high dose prednisolone group. The osteocalcin response to calcitriol was significantly higher in the budesonide groups (P = 0.03, by analysis of variance). High dose prednisolone caused increases in serum 1,25-dihydroxyvitamin D3 (P less than 0.02), urinary calcium excretion (P = 0.07), and urinary hydroxyproline (P less than 0.01). None of these changes was seen during budesonide therapy. There are as yet no data for these variables after long term use of inhaled budesonide in asthmatic patients, but our acute studies suggest that this potent topical glucocorticoid may have considerably less impact on the skeleton than oral prednisolone, even if used at doses high enough to suppress endogenous adrenal function.

Although interleukin (IL)-2 may in part be responsible for lymphocyte accumulation to sites of active sarcoidosis, other cytokines that control such recruitment are not well characterized. Similarly, the pathogenic rationale for the ability of sarcoid macrophages to produce 1,25-dihydroxycholecalciferol (calcitriol) is not understood. We studied the release of chemokinetic lymphokines from human nylon wool-non-adherent tonsillar lymphocytes (HNTLs) employing a standard in vitro lymphocyte migration assay. If mitogen-stimulated HNTL supernatants were fractionated by high-performance liquid chromatography, five positive and one negative chemokinetic factors could be identified. The five lymphocyte chemoattractant factors (LCFs) ranged in mol wt from 5 to 35 kD and stimulated the in vitro migration of nonsensitized human lymphocytes by 200 to 500%. The LCFs appeared distinct from IL-2, IL-1, or gamma-interferon. Co-incubation of HNTLs with mitogen and 1 nM calcitriol prevented the production or release of two of the LCFs and significantly decreased the quantity of a third LCF. Calcitriol also resulted in the appearance of a second negative chemokinetic factor, lymphocyte migration inhibitory factor (LyMIF). Combined with our previous studies demonstrating that calcitriol interferes with IL-2-induced lymphocyte migration, these results provide a rationale for an anti-inflammatory role for calcitriol in sarcoidosis and other granulomatous disorders. These experiments also demonstrate that the control of lymphocyte recruitment to inflammatory foci is multifactorial.

The methylation of membrane phospholipids has been shown to occur following receptor-mediated activation of leukocytes. The present studies show that the human lymphocyte response to two positive chemokinetic signals (bradykinin and lymphocyte chemoattractant factor) can be interrupted by inhibitors of S-adenosyl-L-methionine-mediated transmethylation reactions. The chemokinetic response to the nonphysiologic stimulant colchicine is not affected. We speculate that phospholipid methylation accompanies receptor-mediated lymphocyte migration, and may facilitate activation of second messenger systems.

We have characterized an insulin-like growth factor (IGF)-binding protein present in ovine amniotic fluid. Using an activated charcoal-binding assay, whole amniotic fluid specifically bound approximately 20-30% of 125I-labelled human (h) IGF-II added, while the binding of 125I-labelled hIGF-I was minimal. Radioimmunoassay for IGF-I or -II in ovine biological fluids showed that values in amniotic fluid were 9- to 13-fold less than in fetal plasma, while gel filtration of amniotic fluid on Sephadex G-50 eluted with 1 mol acetic acid/l revealed no additional binding activity which had been complexed to IGFs at neutral pH. Together, these observations suggest that the binding activity in amniotic fluid is largely unsaturated. Competition studies for the displacement of 125I-labelled IGF-II binding to amniotic fluid by increasing amounts of unlabelled IGF-I or -II, using the charcoal assay, showed that IGF-II was 30-fold more potent than IGF-I. Scatchard analysis revealed a single class of binding site for IGF-II, with a binding affinity of 0.68 +/- 0.18 litres/nmol (mean +/- S.D., n = 3). Ligand blot analysis of amniotic fluid by separation on 8% SDS-PAGE, transfer to nitrocellulose membranes, incubation with 125I-labelled IGF-II and autoradiography revealed a single band of IGF-binding protein with approximate molecular size of 38 kDa. Additional IGF-binding species of 20, 28, 48 and greater than 180 kDa were present in ovine fetal plasma. Separation of amniotic fluid on Concanavalin A-Sepharose revealed that it had little carbohydrate content. These results show that ovine amniotic fluid contains an unsaturated, non-glycosylated IGF-binding protein with high affinity for IGF-II.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone (PTH) has been proposed as a skeletal activator for cyclical protocols of treatment for osteoporosis; among several potential drugs that might serve to depress the subsequent phase of osteoclastic bone resorption, calcitonin is the most selective. Twenty patients aged 50-78 years were enrolled in a study of their biochemical responses during a 14-day activation cycle with synthetic hPTH 1-38, given as a subcutaneous injection of 400 IU/day; half the patients were randomly allocated to receive a subsequent 56-day depressor cycle with calcitonin in a dose of 100 U/day, while the remainder received no further treatment. All patients received an initial 24-h intravenous infusion of hPTH 1-38 (0.5 U/kg/h) to evaluate the PTH-dependent renal synthesis of 1,25(OH)2D. Serum calcium increased from 2.20 +/- 0.07 mmol/l to 2.56 +/- 0.16 (P less than 0.005) during PTH infusion, but was not significantly different from baseline during intermittent treatment. Baseline concentrations of serum 1,25(OH)2D were 22.8 +/- 8.2 pg/ml, increased to 52.2 +/- 25.1 (P less than 0.005) during infusion and remained significantly higher than baseline after 14 days intermittent therapy (33.1 +/- 19.4, P less than 0.05). Gastrointestinal absorption of 45Ca, as represented by alpha (peak fractional absorption/h), increased from 0.397 +/- 0.173 to 0.552 +/- 0.210 (P less than 0.01) during hPTH 1-38 therapy and was moderately correlated with the increment in serum 1,25(OH)2D levels (r = 0.5, P less than 0.03). Daily calcium excretion was significantly increased above baseline during hPTH 1-38 therapy, but there were no correlations between changes in urinary calcium, alpha or serum 1,25(OH)2D levels. Baseline fasting urinary excretion of OH-proline increased during hPTH 1-38 treatment from 30.5 +/- 13.9 mol/mmol creatinine to 43.4 +/- 17.5 immediately after hPTH 1-38 infusion (P less than 0.025), and mean excretion was persistently higher than baseline during intermittent treatment; the increased urine calcium and OH-proline excretion are consistent with PTH-induced activation of bone resorption. Serum alkaline phosphatase and osteocalcin levels increased significantly during a 90-day period of observation after the hPTH 1-38 cycle, which is consistent with increased osteoblast activity in association with coupled bone formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult rats were exposed to 1 ppm (1.96 mg/m3) ozone or air for 2 wk. Animals were sacrificed at 3, 5, 7, or 14 d after the onset of exposure, and samples of plasma and lung lavage were obtained. Heat-inactivated plasma and lavage from animals exposed to ozone for 5 or 7 d significantly increased DNA synthesis by lung pneumocytes compared with plasma or lavage from air-exposed animals. Fractionation of plasma and lavage samples indicated that the factor responsible had an isoelectric point of 6.45-6.75, and a molecular weight of 38 +/- 3 kDa. This factor has a dose-dependent effect on lung pneumocyte DNA synthesis in culture. It has no effect on cultured fibroblast DNA synthesis, and is distinct from a previously described factor in the plasma of these ozone-exposed animals that enhances fibroblast DNA synthesis. The factor is detectable within 5 d of exposure, and may hold some promise as a marker of early oxidant lung injury.

Dendritic cells have been isolated from human tonsillar tissue and shown to act as accessory cells in a mitogenic response. The dendritic cells will induced receptors for the active metabolite of vitamin D3, 1,25(OH)2D3, in the responder E+ T cells. The dendritic cells themselves constitutively express receptors for the metabolite, and this distinguishes them from other non-T cells in lymphomedullary tissue. Expression of the 1,25(OH)2D3 receptor may be a dendritic cell property that facilitates their accessory cell role within the tissue microenvironment.

Vitamin D metabolism was studied in 65 patients with surgically proven primary hyperparathyroidism. The mean concentration of 1,25-dihydroxyvitamin D3 was 51.7 +/- 34 pg/ml (mean +/- SD) and was not significantly different from normal. Renal function was normal in 60 of these patients and in this group circulating 1,25-dihydroxyvitamin D3 was below the lower limit of normal in three and elevated in 17; it was related to the serum concentrations of amino-terminal parathyroid hormone, but was independent of serum calcium and the urinary excretion of calcium. The incidence of nephrolithiasis or hyperparathyroid bone disease or combined nephrolithiasis and bone disease in these patients was not related to the circulating concentration of 1,25-dihydroxyvitamin D3. In the remaining five patients, in whom renal impairment was present, circulating 1,25-dihydroxyvitamin D3 was below the lower limit of normal in four. Thus, in primary hyperparathyroidism the circulating concentration of 1,25-dihydroxyvitamin D3 is elevated in only a minority of patients and appears to be unrelated to the occurrence of nephrolithiasis or bone disease.

A 13-year-old girl with total alopecia who in infancy had rickets unresponsive to large doses of vitamin D2 is described. She had profound hypocalcaemia which was resistant to treatment with high doses of dihydrotachysterol, 1 alpha-hydroxycholecalciferol, and 1,25-dihydroxycholecalciferol. Serum concentrations of 25-hydroxyvitamin D were normal but those of 1,25-dihydroxycholecalciferol were markedly raised (674 and 745 pg/ml). In addition, 24,25-dihydroxyvitamin D was undetectable in serum. Administration of synthetic 24,25-dihydroxycholecalciferol was followed by normocalcaemia which persisted long after treatment was stopped. Her sister, who died at the age of 10 months, also had had total alopecia, rickets, and hypocalcaemia resistant to vitamin-D2 therapy. In this familial syndrome there seems to be end-organ resistance to the action of 1,25-dihydroxycholecalciferol, possibly as a result of changes at the receptor sites.

1. 1,25- Dihydroxycholecalciferol [1,25-(OH)2D3] was administered orally to four normal subjects and six patients with chronic renal failure not on dialysis. The serum concentration of 1,25-(OH)2D3 was measured by radioimmunoassay in both groups from samples taken before, and at regular intervals up to 48 h after, the oral dose. 2. The plasma half-time for the disappearance of the administered 1,25-(OH)2D3 was estimated by determining the time for a 50% reduction from the peak increment of the sterol. In normal subjects the calculated value ranged from 5 to 8 h compared with 18 to 44 h in uraemic patients. 3. It appears from our studies that in uraemic subjects there is impaired ability to metabolize or excrete 1,25-(OH)2D3.