An evaluation of several biochemical markers for bone formation and resorption in a protocol utilizing cyclical parathyroid hormone and calcitonin therapy for osteoporosis.

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.

Aluminum intoxication in vitamin D-deficient rats: studies of bone aluminum localization and histomorphometry before and after vitamin D repletion.

Aluminum accumulation by both dialysis patients and nonuremic patients, requiring chronic total parenteral nutrition, may be an etiological factor in the development of severe osteomalacia. To study the role of aluminum toxicity in bone, further experiments have been conducted in the nonuremic, vitamin D-deficient rat. Weanling rats were raised on vitamin D-deficient diets, and half received parenteral aluminum (5 mg/wk), for 30 days. In the first experiment low doses of 25-OH cholecalciferol (500 ng/week) were given subcutaneously for a further 30 days. Control rats were maintained on a similar protocol, but were supplemented with cholecalciferol (5 micrograms/week) from the outset until sacrifice at 60 days. In the second experiment a single bolus of cholecalciferol (5 micrograms) was given to study short-term changes in serum biochemistry and bone histology at 96 hr. Quantitative bone histomorphometric analyses of the proximal tibial metaphysis were made in all experimental groups. In the experimental vitamin D-deficient group, with the highest bone aluminum content (as assessed by extraction of whole bone aluminum), X-ray microanalysis was performed to determine the distribution of aluminum in bone tissue and bone cell organelles. The results showed that control rats treated with prolonged aluminum therapy (30 mg over 60 days) had evidence of both reduced osteoid matrix synthesis and mineralization. However, in vitamin D-deficient rats, there was no evidence that aluminum exacerbated the osteomalacic lesion, even though there was histochemical evidence of aluminum deposition at the bone-osteoid interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum aluminum levels as a reflection of renal osteodystrophy status and bone surface aluminum staining.

Twenty eight (14%) out of 196 patients in a regional dialysis population were found to have serum aluminum levels greater than or equal to 5 mumol/L or 135 micrograms/L; 21 consented to undergo a bone biopsy to identify the spectrum of renal osteodystrophy associated with this degree of hyperaluminemia. Both the Aluminon reagent and the acid solochrome azurine (ASA) stain were used to identify aluminum deposits. A control group of 13 patients with biochemical and histological evidence of severe secondary hyperparathyroidism was used to contrast the measured parameters of bone histology in the hyperaluminemic group. Al(OH)3 was used as the principal phosphate binder in all patients. In the hyperaluminemic group, 67% had either dialysis osteomalacia or aplastic bone lesions, and all except one aplastic lesion were positive for bone surface aluminum deposits by the Aluminon stain. The Aluminon stain was also positive in one of three cases of osteitis fibrosa and three of four mild lesions, whereas it was negative in all biopsies from the control group. However, the ASA stain was positive in all biopsies from the hyperaluminemic group and in 11 of 13 control biopsies from the patients with "pure" osteitis fibrosa. For all biopsy data from both groups, there were significant (P less than 0.01) negative correlations between the ASA-stained surface aluminum deposits and resorption indices (total eroded surface, r = -0.68; surface osteoclast counts, r = -0.53) and indices of bone formation (surface osteoblast counts, r = -0.61; mineral apposition rate, r = -0.63; bone formation rate, r = -0.69). These correlations were not significant for Aluminon-stained surface deposits with the exception of the bone formation indices, which had lower correlation coefficients (r = -0.44). These data suggest that hyperaluminemia greater than or equal to 5 mumol/L has a predictive value to identify impaired mineralization in dialysis patients that is high enough to affect clinical decision making. However, the more sensitive ASA stain identifies surface aluminum across the whole spectrum of renal osteodystrophy and is consistent with a toxic role for aluminum at any level of exposure.

Bone densitometric and histomorphometric responses to sequential human parathyroid hormone (1-38) and salmon calcitonin in osteoporotic patients.

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)

Targeting transgene expression to airway epithelia and submucosal glands, prominent sites of human CFTR expression.

Targeting therapeutic gene expression to disease-affected tissues is an essential component of effective and safe gene therapy. After birth, CFTR gene expression in human lungs is localized predominantly in the epithelial cells lining the upper airways, especially in the ducts and serous tubules of the submucosal glands. We have developed a K18 expression cassette, based on the DNA control elements of the human cytokeratin 18 gene. Temporal and spatial analyses of transgenic mice demonstrated that this expression cassette targets transgene expression to almost all cell types in which CFTR is expressed. Airway epithelium expression started as early as 11.5 days of gestational age and continued into the adulthood of the transgenic mice. In these adult mice, the pattern of the reporter expression strikingly matched that of the human cytokeratin 18 and human CFTR genes. The transgene expression was epithelium-specific and undetectable in connective tissue, muscle, bone, cartilage, blood, and endothelial cells. Significantly, high levels of expression were detected in tracheal submucosal glands. Together, these results suggest that our K18 expression cassette has a high potential for clinical application in gene therapy for patients with cystic fibrosis.