In vitro bone-cell response to a capacitively coupled electrical field. The role of field strength, pulse pattern, and duty cycle.

Newborn rat calvarial bone cells were grown to confluence and subjected to a matrix of sine wave 60-kHz capacitively coupled electrical signals of various field strengths, pulse-burst patterns, and duty cycles. Both [3H] thymidine incorporation into DNA and alkaline phosphatase activity were evaluated in field strengths ranging from 0.0001 to 20 mV/cm, with pulse-burst patterns ranging from continuous to 5 milliseconds ON/495 milliseconds OFF, with daily duty cycles ranging from 0.25% to 25%. A significant increase in proliferation occurred in field strengths of 0.1, 1, and 20 mV/cm when the signal was applied continuously for six hours. Significant proliferation also occurred when the 20-mV/cm field was pulsed for six hours at 5 milliseconds ON/495 milliseconds OFF and at 5 milliseconds ON/245 milliseconds OFF. No change in alkaline phosphatase activity occurred in the 20-mV/cm field with any signal. At 1 mV/cm, there was a significant decrease in alkaline phosphatase activity in the continuous signal and in the 5 milliseconds ON/62 milliseconds OFF signal; in the lower fields evaluated, there was an actual decrease in alkaline phosphatase activity with some of the signals. The field strength plays a dominant role in determining the bone-cell's proliferative response, and to a lesser extent the alkaline phosphatase activity response, to a capacitively coupled electric field. The pulse configuration and the duty cycle are also important, but only if the proper field strength is being applied to the cell.     

Adenylate cyclase activity as a predictor of thyroid tumor aggressiveness

Prior studies in our laboratory have shown that human thyroid neoplasms have a greater adenylate cyclase activity in response to thyroid stimulating hormone (TSH) than does the adjacent histologically normal thyroid tissue. However, there is little information relating activity of the TSH receptor-adenylate cyclase system to the type of thyroid neoplasm. Thyroid tissue from 67 patients was divided by clinical and histological criteria into 6 categories: normal (59), benign tumors (20), stage 1 carcinoma—intrathyroidal involvement only (25), stage 2 carcinomaregional lymph node involvement (6), stage 3 and 4 carcinoma—tissue invasion or distant metastasis (11), and medullary carcinoma (3). Adenylate cyclase activity in an 8,000 x g thyroid membrane preparation was determined in the basal state and when maximally stimulated with 300 mU/ml TSH. The cyclase responsiveness was the ratio of TSH stimulated adenylate cyclase activity compared to basal adenylate cyclase activity. The cyclase responsiveness by category is: normal, 2.8±0.2 (mean ± SEM); benign, 17.9±2.4; stage 1 carcinoma, 9.2±1.9; stage 2 carcinoma, 4.0±1.0; stage 3 and 4 carcinoma, 1.6±0.4; and medullary carcinoma, 1.05±0.04 (for the neoplasms,p <0.02 by ANOVA). Tumor stage was the only correlate with this trend as other prognostic risk factors (age, sex, a history of neck irradiation, or papillary versus follicular histology) showed no difference in cyclase responsiveness. These studies demonstrate a consistent inverse correlation between adenylate cyclase responsiveness and tumor stage or aggressiveness. Cyclase responsiveness appears to have clinical application for predicting which thyroid tumors will behave aggressively.

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Resumen Estudios previos en nuestro laboratorio han demostrado que los neoplasmas tiroideos humanos poseen una mayor actividad de adenilato ciclasa en respuesta a la administración de hormona estimulante de la tiroides (TSH) que el tejido tiroideo histológicamente normal adyacente. Sin embargo, existe muy poca información sobre la relation de la actividad del sistema receptor de TSH-adenilato ciclasa y el tipo del neoplasma tiroideo. Tejido tiroideo proveniente de 67 pacientes fue dividido mediante criterios chlínicos e histológicos en 6 categorias: normal (59), tumores benignos (20), extensión intratiroidea solamente en estado 1 (25), carcinoma-extensión ganglionar regional en estado 2 (6), carcinoma-invasión tisular o metástasis distantes en estados 3 y 4 (11), y carcinoma medular (3). La actividad de la adenilato ciclasa en una preparación de membrana tiroidea de 8,000 × g fue determinada en el estado basai y en estado de maxima estimulación con 300 mU/ml TSH. El grado de respuesta de la ciclasa fue la tasa de actividad de la adenilato ciclasa estimulada por TSH comparada con la actividad basai de la adenilato ciclasa. El grado de respuesta por categorías fue: normal, 2.8±0.2; tumor benigno, 17.9±2.4; carcinoma estado 1, 9.2±1.9; carcinoma estado 2, 4.0±1.0; carcinoma estados 3 y 4, 1.6±0.4; y carcinoma medular, 1.05±0.04 (para los neoplasmas,p < 0.02 por ANOVA). El estado del tumor apareció como el único factor de correlatión con esta gradación, ya que otros factures de pronóstico (edad, sexo, historia de irradiación cervical, histología papilar versus folicular) no demostraron diferencia en cuanto al grado de respuesta de la ciclasa. Estos estudios demuestran una consistente relación inversa entre el grado de respuesta de la adenilato ciclasa y el estado o agresividad tumoral. El grado de respuesta de la ciclasa parece tener aplicación clínica para predecir qué tumores tiroideos se habrán de comportar en forma agresiva.

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Résumé Les études antérieures provenant de nos laboratoires ont démontré une augmentation de l'activité enzymatique de l'adénylate-cyclase en réponse à la thyroïd stimulating hormone (TSH) dans le tissu thyroïdien humain tumoral, par rapport au tissu thyroïdien adjacent normal. Cependant il existe peu de données concernant l'activité du système récepteur TSH/adénylatecyclase par rapport au type de tumeur de la thyroïde. Les tissus thyroïdiens provenant de 67 patients différents ont été repartis en 6 groupes selon des critères cliniques et histologiques: normal (59), tumeur bénigne (20), cancer stade 1 (intrathyroïdien uniquement) (25), cancer stade 2 (envahissement ganglionnaire régional) (6), cancer stade 3 et 4 (envahissement tissulaire avoisinant ou métastases à distance (11), et cancer médullaire (3). A partir d'une préparation de membrane thyroïdienne centrifugée à 8,000 × g, l'activité de l'adénylate-cyclase a été déterminée en l'état basai et après stimulation maximale par 300 mU/ml de TSH. La réponse enzymatique a été mesurée comme étant le rapport de l'activité stimulée par la TSH/activité basale. Les résultats selon les 6 groupes étaient (moyen±ET): tissu normal, 2.8±0.2; tumeur bénigne, 17.9±2.4; cancer stade 1, 9.2±1.9; cancer stade 2, 4.0±1.0; cancers stade 3 et 4, 1.6±0.4; et cancer médullaire, 1.05±0.04 (p < 0.02 par l'analyse de variance pour les néoplasies). Le stade tumoral était la seule variable corrélée avec l'activité enzymatique. L'activité enzymatique n'était pas corrélée avec l'âge, le sexe, les antécédents d'irradiation cervicale antérieure ou l'histologie (papillaire vs. folliculaire). Ces études montrent un rapport inversement proportionnel entre l'activité d'adénylate-cyclase et le degré d'agressivité tumorale ou le stade. La réponse d l'adénylate-cyclase paraît avoir une application clinique: prévoir quelles tumeurs thyroïdiennes auront une évolution agressive.

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Presented at the International Association of Endocrine Surgeons in Sydney, Australia, September, 1987.

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Supported in part by the Medical Research Service of the Veterans Administration.     

Filtration of fentanyl is not the cause of the elevation of arterial blood pressure associated with post-bypass ultrafiltration in children

Modified ultrafiltration after cardiopulmonary bypass in children has been shown to be associated with an increase in arterial blood pressure. As part of a series of studies to investigate the possible causes of this blood pressure elevation, the hypothesis that if filtration was removing a significant amount of fentanyl, then the increase in blood pressure might be due to pain was proposed. Ten children, aged between 0.5 and 9.3 years (median 3.8 years), weighing 5.9 to 25..5 kg (median 15.7 kg), underwent corrective cardiac surgery (incorporating modified ultrafiltration). A standard anesthetic protocol was followed, with up to 78 μg/kg of fentanyl given prebypass for analgesia. After completion of cardiopulmonary bypass, modified ultrafiltration was commenced at 100 mL/min until a hematocrit of 35% was reached. Samples were taken of arterial blood (prefiltration, 3, 10, and 20 minutes postfiltration), the venous reservoir blood (prefiltration) and the filtrate (5 and 10 minutes into filtration). Hemodynamic data were recorded both prefiltration and postfiltration. The hemodynamic data showed the expected rise in both systemic arterial pressure and cardiac index after ultrafiltration. The plasma fentanyl concentrations did not significantly change after ultrafiltration: 1.59 to 12.39 ng/mL (median 6.27 ng/mL) prefiltration and 2.05 to 15.59 ng/mL (6.29 ng/mL) at 3 minutes, 2.22 to 12.64 ng/mL (6.87 ng/mL) at 10 minutes, and 1.83 to 11.52 ng/mL (5.85 ng/mL) at 20 minutes postfiltration. The concentration of fentanyl in the venous reservoir, 2.06 to 11.64 ng/mL (7.04 ng/mL), was not significantly different from the plasma levels. The level of fentanyl in the filtrate was significantly less than the plasma levels, 0.243 to 1.87 ng/mL (0.894 ng/mL) at 5 minutes and 0.385 to 1.688 ng / mL (0.952 ng / mL) at 10 minutes into filtration; (P < 0.02 by the Wilcoxon signed-rank method). The data show that the plasma fentanyl concentration was not significantly reduced by modified ultrafiltration. The fentanyl levels found prefiltration were maintained postfiltration, and the observed changes in systemic arterial pressure were not due to an acute fall in the plasma concentration of analgesic drug.     

Expression profiling of metalloproteinases and inhibitors in cartilage

Objective   To profile the expression of all known members of the matrix metalloproteinase ( MMP ), a disintegrin and metalloproteinase with thrombospondin motifs ( ADAMTS ), and tissue inhibitor of metalloproteinases ( TIMP s) gene families in normal cartilage and that from patients with osteoarthritis (OA).
Methods   Human cartilage was obtained from femoral heads at joint replacement for either osteoarthritis or following fracture to the neck of femur. Total RNA was purified and expression of genes assayed using quantitative real-time PCR.
Results   Several members of the above gene families were regulated in OA. Genes increasing in expression in OA were: at P  < 0.001, MMP-13 , MMP-28 , ADAMTS-16 ; at P  < 0.01, MMP-9 , MMP-16 , ADAMTS-2 , ADAMTS-14 and at P  < 0.05, MMP-2 , TIMP-3 , ADAMTS-12 . Genes decreasing in expression in OA were: at P  < 0.001, MMP-1 , MMP-3 , ADAMTS-1 ; at P  < 0.01, MMP-10 , TIMP-1 , ADAMTS-9 and at P  < 0.05, TIMP-4 , ADAMTS-5 , ADAMTS-15 . Correlation analysis revealed that groups of genes across the gene families are co-expressed in cartilage.
Conclusion   This is the first comprehensive expression profile of all known MMP , ADAMTS and TIMP genes in cartilage. Patterns of expression provide a foundation on which to understand mechanisms of gene regulation in OA and potentially for refining the specificity of anti-proteolytic therapies.