Effects of alveolar surfactant aggregates on T-lymphocyte proliferation.

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.

Rapid small-animal dual-energy X-ray absorptiometry using digital radiography.

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.

Histomorphometric evidence for increased bone turnover without change in cortical thickness or porosity after 2 years of cyclical hPTH(1-34) therapy in women with severe osteoporosis.

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.

Nuclear localization of the type 1 PTH/PTHrP receptor in rat tissues.

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.

Nuclear localization of the type 1 parathyroid hormone/parathyroid hormone-related peptide receptor in MC3T3-E1 cells: association with serum-induced cell proliferation.

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.

Serum osteocalcin and procollagen as markers for the risk of osteoporotic fracture in corticosteroid-treated asthmatic adults.

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.

Comparison of the biochemical responses to human parathyroid hormone-(1-31)NH2 and hPTH-(1-34) in healthy humans.

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.

The addition of a raloxifene analog (LY117018) allows for reduced PTH(1-34) dosing during reversal of osteopenia in ovariectomized rats.

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.

Assessment of maintenance therapy with reduced doses of PTH(1-34) in combination with a raloxifene analogue (LY117018) following anabolic therapy in the ovariectomized rat.

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.

Enhanced osteoblast development after continuous infusion of hPTH(1-84) in the rat.

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.

Recombinant rat surfactant-associated protein D inhibits human T lymphocyte proliferation and IL-2 production.

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.

Surfactant protein A inhibits T cell proliferation via its collagen-like tail and a 210-kDa receptor.

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.

Comparison of the response of pelvic and proximal tibial cancellous bone in rat to ovariectomy with estrogen replacement.

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.

Comparison of the antiasthmatic, oropharyngeal, and systemic glucocorticoid effects of budesonide administered through a pressurized aerosol plus spacer or the Turbuhaler dry powder inhaler.

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.

A randomized controlled trial to compare the efficacy of cyclical parathyroid hormone versus cyclical parathyroid hormone and sequential calcitonin to improve bone mass in postmenopausal women with osteoporosis.

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.

Chronic fetal placental embolization and hypoxemia cause hypertension and myocardial hypertrophy in fetal sheep.

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.

Surfactant associated protein-A inhibits human lymphocyte proliferation and IL-2 production.

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.

Immunoreactive parathyroid hormone-related protein: its association with preterm labor.

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.

Bone mineral density and the risk of fracture in patients receiving long-term inhaled steroid therapy for asthma.

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.

Calcitriol and its synthetic analogue MC 903 inhibit the interleukin-2-induced migration of human lymphocytes.

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.