Joyce I. Merryman

Studies on chicken polyclonal anti-peptide antibodies specific for parathyroid hormonerelated protein (1–36)

Chicken polyclonal antibodies were prepared against a synthetic peptide corresponding to the first 36 N-terminal amino acids of parathyroid hormone-related protein (PTHrP) by immunizing laying hens. Significant increases of antibodies to PTHrP were first detected after the second immunization. Production of anti-PTHrP egg yolk antibodies peaked 1-2 weeks after the second through sixth immunizations and declined over a period of 2-4 weeks. Polyclonal IgG (IgY) to PTHrP was purified from the egg yolks with high levels of PTHrP specific binding. The anti-PTHrP IgG was used to develop a radioimmunoassay for PTHrP that was able to detect 100 pg PTHrP ml-1 (23 pM) in conditioned cell culture medium. The anti-PTHrP IgG was bound to a solid phase and utilized to immunopurify iodinated [Tyr36]-PTHrP (1-36). Anti-PTHrP IgG inhibited the in vitro biologic activity of PTHrP as demonstrated by the inhibition of adenylate cyclase stimulation in a rat osteoblast-like cell line (ROS 17/2.8). The anti PTHrP IgG was immunopurified and utilized for immunohistochemical localization of PTHrP in canine skin. Chickens were advantageous in producing large amounts of high affinity, neutralizing antibodies to a highly conserved mammalian protein such as PTHrP. The antibodies will be useful to investigate the function and metabolism of PTHrP in vivo and in vitro.

Effects of transforming growth factor-α on parathyroid hormone- and parathyroid hormone-related protein- mediated bone resorption and adenylate cyclase stimulation in vitro☆

The effects of transforming growth factor-alpha (TGF alpha) were determined on the ability of parathyroid hormone (PTH) or parathyroid hormone-related protein (PTHrP) to stimulate bone resorption and adenylate cyclase in vitro. Bovine PTH-(1-34) and human PTHrP-(1-34) were equipotent in their ability to stimulate bone resorption in neonatal mouse calvaria with maximal stimulation (2.9 and 2.8-fold increases in 45Ca release, respectively) at a concentration of 10 nM. Combinations of TGF alpha with bPTH-(1-34) or hPTHrP-(1-34) had additive effects on their ability to stimulate bone resorption when submaximal concentrations of the agonists were used. There was no evidence of synergism between TGF alpha bPTH-(1-34) or hPTHrP-(1-34) in their ability to stimulate bone resorption in vitro, nor was TGF alpha able to increase bone resorption induced by maximal concentrations of bPTH-(1-34) or hPTHrP-(1-34). TGF alpha potentiated the effects of either bPTH-(1-34) or hPTHrP-(1-34) on the stimulation of adenylate cyclase in osteoblast-like ROS 17/2.8 cells. These data indicate that TGF alpha has additive effects with submaximal concentrations of PTH or PTHrP on their ability to stimulate bone resorption which may be important in the pathogenesis of humoral hypercalcemia of malignancy.

Effects of gallium nitrate in nude mice bearing a canine adenocarcinoma model of humoral hypercalcemia of malignancy: Biochemical, histomorphometric, and ultrastructural investigations

Hypercalcemic nude mice bearing a canine adenocarcinoma (CAC-8) model of humoral hypercalcemia of malignancy (HHM) were treated daily with gallium nitrate (60 mg/kg elemental gallium subcutaneously on day 0 followed by 20 mg/kg day for four days. Concentrations of gallium in bone were undetectable (<0.00005 µg/g bone) in vehicle-treated mice but markedly elevated in gallium-treated mice (>235 ± 6 µg/g bone). Gallium nitrate significantly decreased serum calcium and urinary calcium excretion in tumor-bearing mice compared with vehicle-treated controls. Histomorphometric evaluation of lumbar vertebrae revealed a significant decrease in the number of osteoclasts/mm trabecular bone in gallium-treated tumor-bearing mice compared with controls. Osteoclasts from tumor-bearing mice treated with gallium nitrate were significantly decreased in size, had reduced tartrate-resistant acid phosphatase staining intensity and ultrastructurally had fewer intracytoplasmic vesicles compared with vehicle-treated controls. Osteoclasts in gallium-treated mice were small and flattened with poorly developed cytoplasmic organelles. The findings of this investigation indicated that gallium nitrate reduced serum calcium in an animal model of HHM by inhibiting osteoclastic bone resorption.