A viral antigen-bearing cell line derived from culture of Paget's bone cells.

This study documents the characteristics of a viral antigen-bearing cell line derived from co-culture of the bone from a 71-year-old woman with Paget's disease of bone and HEp-2 cells. The cell line has survived in continuous culture for 3 1/2 years and 185 subcultures. The cells are epithelioid in appearance, produce alkaline and acid phosphatase, increase alkaline phosphatase activity in response to 1,25-(OH)2-D3 and contain receptors for 1,25-(OH)2-D3. Immunofluorescent studies utilizing antisera to respiratory syncytial virus and measles virus reveal antigens of both viruses in the cells. These cells do not produce bone in culture and the adenylate cyclase activity found in their plasma membrane does not increase significantly in response to parathyroid hormone or calcitonin. The cells are not contact inhibited and form spherical colonies in agarose. They are aneuploid and have a modal number of 62-74 as well as HeLa markers. When injected into athymic mice, osteosarcomas are produced. These tumors continue to bear viral antigens. The availability of this cell line should aid in further studies of the viral antigens associated with Paget's disease of bone.

Presence of brain-derived neurotrophic factor in brain and human and rat but not mouse serum detected by a sensitive and specific immunoassay.

Brain-derived neurotrophic factor (BDNF) is one of several endogenous proteins that play key roles in neuronal development and homeostasis. We describe here the characterization and use of a sensitive and specific enzyme-linked immunoassay (EIA) for BDNF protein. Recombinant BDNF was detected at concentrations as low as 10 pg/ml, whereas the EIA did not detect NT-3, NT-4/5, or NGF at concentrations as high as 100 ng/ml. Because BDNF protein sequences are identical among humans, mice, and rats, we utilized the BDNF EIA to detect BDNF in the circulation or brain regions of these species. High concentrations of BDNF were detected in human and rat serum, and up to 50-fold lower BDNF levels were present in citrated human or rat plasma. The BDNF signal (66-141 pg/ml) in 20% human plasma was completely blocked by pre-exposure of plasma to a monoclonal antibody (Mab) specific for BDNF but not by exposure to 5-fold greater concentrations of an irrelevant Mab of the same isotype (IgG1). There was a significant and positive correlation (r = +0.86) between plasma levels of BDNF and serotonin, an indoleamine that is specifically released from activated platelets. These results are consistent with the view that the BDNF detected in human and rat plasma is derived from platelet degranulation, and that circulating levels of BDNF are negligible. In contrast to human or rat serum, mouse serum contained no detectable BDNF. However, BDNF protein was readily detectable at 108-256 ng/g of tissue in hippocampus, frontal cortex, and neostriatum of mice and rats. Thus, the failure to detect BDNF in murine serum was not due to an assay defect but highlights a significant species difference in the tissue-specific expression of BDNF that may be of biological importance. The presence of BDNF protein in blood and brain regions at quantities which greatly exceed those described for NGF confirm the abundant distribution of this broadly-acting neurotrophic factor.

Temporal sequence of events in the estrous cycle of the bitch.

Serum concentrations of luteinizing hormone (LH), estradiol-17 beta, and progesteron were compared with the onset of diestrus in Beagle bitches. The LH peak occurred 8.0 (standard error (SE), 0.3) days before the onset of diestrus. Vaginal cytologic characteristics during proestrus were studied, and vaginal cornification was complete 12.1 (SE,0.2) days before the onset of diestrus. A time sequence of hormonal, vaginal cytologic, and developmental events was outlined during proestrus, estrus, and early diestrus.

For debate: pet birds as an independent risk factor for lung cancer.

To find out whether keeping birds in the home is an independent risk factor for lung cancer a case-control study was carried out in four main hospitals in The Hague, The Netherlands. Forty nine patients under 65 years of age with lung cancer each were matched for age and sex with two control subjects who attended the same general practice. Data were collected on social class, cigarette smoking, intake of beta carotene and vitamin C, and alcohol consumption. It was found that smoking, birdkeeping, and a low intake of vitamin C were significantly and independently related to the incidence of lung cancer. The odds ratio for lung cancer among people who keep birds as pets was estimated to be 6.7 after adjusting for smoking and vitamin C intake. The results of this study suggest that keeping pet birds is an independent risk factor for lung cancer.

Telepresence interface with applications to microsurgery and surgical simulation.

To address the needs for performing microsurgical procedures, the SRI telepresence surgery workstation has been combined with a pair of micromanipulator arms. The prototype microsurgery system has been tested with ex-vivo tasks similar to those required for surgical procedures, such as cutting, grasping, suturing, and knot tying. Initial animal testing has been done on a rat model in which end-to-end anastomosis of the femoral artery (approximately 1 millimeter in diameter) was completed with ten rats, and 100% patency was obtained. To address the needs of surgical training, SRI has begun to develop a system that uses a 6-DOF telepresence workstation. A computer-generated stereo image is reflected in a mirror and appears to be superimposed on the surgeon's hands, creating an immersive and realistic environment. Tools held in the surgeon's hands are connected to left- and right-hand manipulators that both continuously measure tool position/orientation and apply force/torque to the tools. Furthermore, the visual image and tool locations are registered, so that the user perceives that he or she is looking at and moving the simulated tools in the visual image.

Tissue phosphatase changes following triamcinolone associated with cleft palate in rats.

One theory of the development of cleft palate in rats involves the action of lysosomal enzymes secreted by epithelial cells at the time of fusion of the palatal shelves. To test this theory we studied the biochemistry of the palates of fetal rats daily between days 14 and 19 (from 3 days before to 3 days after palate closure). Triamcinolone was administered once im on gestation day 14 to Wistar rats; 0.5 mg/kg body weight produced approximately 50% cleft palates. Pooled control palatal tissue was compared with pooled experimental tissue; that from fetuses with clefts being pooled separately from those not affected. Acid phosphatase and beta-glucuronidase were assayed. Concentration vs. time curves for both enzymes were very similar. Prior to the time of palate closure both enzymes were present in low concentration. Between days 16 and 17, the normal time of closure, there was an abrupt increased in enzyme concentration, with experimental tissue showing a significant elevation over control tissue on days 17 and 18. Alkaline phosphatase was also present in small amounts before closure and significantly higher in control tissue on day 17. Protein was depressed in palates having clefts on day 17; thus the ratio of enzyme activities to protein synthesis was significantly elevated at a critical time. Unaffected experimental palates had a normal ratio. These results suggest imbalanced acid phosphatase, beta-glucuronidase, and alkaline phosphatase activity compared with protein synthesis at the time of palate closure following triamcinolone in rats.

Long-term culture of cells from bone affected by Paget's disease.

Cells obtained from surgical bone specimens of eight patients with Paget's disease of bone were maintained in culture for up to 8 months and seven passages. The doubling time during the period of maximal cell growth ranged from 4 to 12 days. Evidence consistent with the hypothesis that many of the cells were bone cells included the following: (a) histochemical techniques demonstrated staining of some cells for alkaline phosphatase or acid phosphatase and succinic dehydrogenase; (b) parathyroid extract stimulated increased uptake of 3H-thymidine and 3H-uridine; (c) parathyroid extract suppressed and salmon calcitonin stimulated uptake of 3H-proline; and (d) crystalline calcium deposits were found within cells and extracellularly. Ultrastructural analysis revealed that three of the eight cultures contained cells whose nuclei had inclusions which were almost identical to those found in the osteoclast nuclei of all patients with Paget's disease. The maintenance of cells derived from pagetic bone in long-term culture should aid in testing the hypothesis that Paget's disease represents a slow virus infection of bone.

Cell cultures from bone affected by Paget's disease.

Cells obtained from Paget's bone specimens were maintained in culture for up to 8 1/2 months. The cells had several characteristics of bone cells including the presence of alkaline phosphatase, acid phosphatase, or succinic dehydrogenase as demonstrated histochemically. Electron microscopy revealed nuclear inclusions similar to those found in the osterclasts of Paget's disease in 4 or 11 cultures. Immunohistology utilizing specific antisera demonstrated the presence of respiratory syncytial virus antigen(s) in 7 or 7 patients. Negative results were obtained with antisera to a variety of paramyxoviruses.

Immunohistological demonstration of respiratory syncytial virus antigens in Paget disease of bone.

Respiratory syncytial virus antisera have been found to produce a positive immunohistologic response in osteoclasts in bone sections or in cells cultured from Paget disease lesions in 12 out of 12 patients tested. These experiments were carefully controlled by several means. Use of experimentally infected cells served as positive controls. Adsorption of antisera on human bone powder and KB cells did not remove the specific immunologic stain, but adsorption of the antisera by the virus did. Negative results were also obtained in osteoclasts of patients with primary or secondary hyperparathyroidism. In addition, negative results in specimens of Paget disease were found with antisera to measles; parainfluenza 1, 2, and 3; influenza A, B and C; rubella; and herpes simplex. These results are consistent with the hypothesis that the nuclear and cytoplasmic inclusions in the osteoclasts of Paget disease are a result of viral activity.

Levels of lutenizing hormone, estradiol and progesterone in serum during the estrous cycle and pregnancy in the beagle bitch (38491).

Levels of luteinizing hormone (LH), estradiol-17 beta and progesterone were determined by specific radioimmunoassays in sera obtained from Beagle bitches during proestrus, estrus and diestrus. Concentrations of LH (expressed as NIH-LH-SI equivalents) were 2.8 plus or minus 0.1 ng/ml in proestrus, 35.5 plus or minus 10.0 ng/ml during early estrus and 2.2 plus or minus 0.1 ng/ml in early diestrus. Peak levels of estradiol-17beta (68.9 plus or minus 11.0 ng/ml) were detected 24 hr prior to the LH peak, declined rapidly and reached basal levels (17.8 plus or minus 6.3 ng/ml) by five days following the LH peak. Levels of progesterone were 1.7 plus or minus 0.3 ng/ml during proestrus, 3.5 plus or minus 0.3 ng/ml during early estrus and 23.3 plus or minus 2.8 ng/ml on day 5 after the LH peak . Progesterone levels remained elevated through day 28 of diestrus and pregnancy. A significant decrease (p smaller than 0.05) in levels of prosgesterone occurred between day 28 of pregnancy and one day prior to shelping (3.3 plus or minus 1.2 ng/ml, with a further decrease on the day of whelping (1.1 plus or minus 0.2 ng/ml). Levels of estradiol-17beta and LH did not change significantly (p smaller than 0.0k) during diestrus or pregnancy.

Characterization and enrichment of fetal rat hepatoblasts by immunoadsorption ("panning") and fluorescence-activated cell sorting.

We developed methods for enriching fetal hepatoblasts by combining panning and multiparametric fluorescence-activated cell sorting. In unpurified, dissociated fetal liver cell suspensions of embryonic age day 15, 3.2% +/- 1.3% and 2.5% +/- 0.7% cells expressed albumin and alpha-fetoprotein, respectively. The remainder exhibited a hemopoietic, endothelial or stromal cell phenotype. Cells were panned first with an antibody to red blood cells to remove erythroid cells and then with monoclonal antibodies OX-43/OX-44 to remove hemopoietic and endothelial cells. This procedure eliminated 84% of fetal hepatic cells, with enrichment of the remainder for albumin or alpha-fetoprotein expression (up to sixfold increase). Flow cytometric analysis of unlabeled cells revealed two populations, which differed in granularity and autofluorescence. After panning, fluorescence-activated cell sorting for agranular cells yielded OX-43/44-positive cells that were essentially all hemopoietic precursor cells or OX-43/44-negative cells that were mostly hemopoietic precursor cells, along with 3.0% +/- 0.7% alpha-fetoprotein-positive cells. In contrast, sorting for granular, OX-43/44-negative cells enriched for predominantly alpha-fetoprotein-positive, parenchymal precursor cells (75.1% +/- 4.7%). Multiparametric flow cytometric analysis of the expression of an oval cell antigen, OC.3, which is a bile duct and putative liver stem cell marker, showed that most OC.3-positive cells coexpressed OX-43/OX-44 and morphologically were hemopoietic precursor cells. However, approximately 30% of the OX-43/44-negative, granular cells expressed OC.3. Although the physiological significance of OC.3-positive hepatoblasts remains to be determined, the ability to isolate distinct liver cell populations by means of fluorescence-activated cell sorting should facilitate further studies.(ABSTRACT TRUNCATED AT 250 WORDS)