Release, biological potency, and biochemical integrity of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) combined with Augment(TM) Bone Graft or GEM 21S beta-tricalcium phosphate (beta-TCP).

Over 10 million surgical procedures are performed annually in the United States to treat musculoskeletal injuries, and a significant portion of these involve orthopedic bone grafting. The goals of the study were to evaluate the in vitro and in vivo release kinetics, biological potency and biochemical integrity of rhPDGF-BB combined with large (1000-2000 microm) and small (250-1000 microm) beta-TCP particles. Recombinant human platelet-derived growth factor B homodimer (rhPDGF-BB) is a protein growth factor under development as a therapeutic for accelerating bone healing. Release of the protein was monitored in vitro by ELISA, and in vivo by measurement of radioactive rhPDGF-BB implanted in rat calvarial defects. Biological activity was measured using a cell-based bioassay, and biochemical integrity was determined by SDS-PAGE and high pressure size exclusion chromatography (HPSEC). Release of rhPDGF-BB occurred rapidly from beta-TCP both in vitro and in vivo. Almost 100% of the rhPDGF-BB was recovered from large and small beta-TCP after 90 min in vitro. Approximately 90% of the rhPDGF-BB was depleted from calvarial defect sites within 72 h of implantation. RhPDGF-BB retained 100% of its biological potency compared to reference standard rhPDGF-BB, manifested as a single band at ~30 kDa by SDS-PAGE and a single peak eluted after 13 min by HPSEC following release from beta-TCP. RhPDGF-BB is rapidly released from large and small beta-TCP particles and is biochemically unaltered following release.

Selenite suppression of cadmium-induced testicular apoptosis.

The characteristic apoptotic ladder-like patterns of rat testicular DNA on agarose gel electrophoresis which results from treatment with CdCl2 are suppressed by the administration of Na2SeO3. The examination of testicular tissue using an ELISA programmed cell death detection procedure confirmed this selenite suppression of cadmium-induced apoptosis. The administration of the Na2SeO3 at either 0.5, 1, 2 h prior to or 0.5, 1, 2 h after the administration of the CdCl2 appear to be almost equally effective at suppressing the apoptotic response. These results are in accord with previous studies on the Na2SeO3 suppression of cadmium induced necrotic changes in tissues and suggest that Na2SeO3 interferes with both necrosis and apoptosis.

Enzyme immunoassay to determine heavy metals using antibodies to specific metal-EDTA complexes: optimization and validation of an immunoassay for soluble indium.

An immunoassay that measures soluble indium at concentrations from 0.005 ppb to 320 ppm is described. The assay utilized a monoclonal antibody that binds specifically to indium-EDTA complexes in an antigen-inhibition format. The sensitivity of the assay could be modulated by changing the nature of the soluble inhibiting antigen. The range of the assay was from 0.6 to 320 ppm, 0.1 to 120 ppm, or 0.005 to 2000 ppb when indium-EDTA, indium-(p-nitrobenzyl)-EDTA, or indium-EDTA-bovine serum albumin, respectively was used as the soluble inhibiting antigen. The assay reliably monitored indium concentration in the presence of a 100-fold excess of manganese, magnesium, or copper ions and the quantitation of indium by immunoassay correlated closely with the values obtained using atomic absorption spectroscopy. This technology could be employed in immunoassays for other metals that are priority pollutants.

Localization and induced expression of fusion genes in the rat lung.

Liposome-mediated gene transfer is useful for DNA transfection into cells in culture. We wondered whether this method could be used to introduce new DNA into the intact lung. Fusion genes containing either the Rous sarcoma virus (RSV) promoter or the mouse mammary tumor virus (MMTV) promoter (which contains glucocorticoid response elements) were linked to the bacterial gene chloramphenicol acetyltransferase (CAT), an enzyme not present in mammalian cells. Plasmids containing the RSV-CAT fusion gene were mixed with cationic liposomes (Lipofectin; BRL, Inc., Grand Island, NY), and single doses were instilled into the cervical trachea of anesthetized rats. Control rats received either liposomes or plasmid. After 24, 48, and 72 h, lungs were perfused free of blood, homogenized, and analyzed for CAT enzyme activity. Liver and kidney tissue were also obtained. We found that rats given either intratracheal liposomes or plasmid had no detectable CAT activity. By contrast, 24 h after instillation of lipid:DNA complexes, lung CAT expression remained elevated for the next 48 h but was barely detectable in liver or kidney. In another group of rats, MMTV-CAT:liposome complexes were instilled intratracheally and then the rats were injected with either dexamethasone or saline. We found that the dexamethasone-treated rats had a 5- to 10-fold higher level of lung CAT expression at 24 and 48 h than the saline-treated controls had; liver and kidney CAT levels were negligible in both groups. Dexamethasone treatment did not increase RSV-CAT expression, indicating that the dexamethasone effect on MMTV-CAT expression was related to the presence of the MMTV promoter.(ABSTRACT TRUNCATED AT 250 WORDS)