Aluminum Accelerates Osteoblastic Differentiation But is Cytotoxic in Long-Term Rat Calvaria Cell Cultures

We have examined the effects of aluminum (Al) on osteoprogenitor proliferation and differentiation, cell survival, and bone formation in long-term rat calvaria (RC) cell cultures. RC cells were grown in α minimal essential medium containing 10% fetal bovine serum, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate with or without Al added to final concentrations of 1 μM—1 mM. Al caused a dose-dependent increase in the number of bone nodules present at early times (day 11) but had no significant effect on nodule numbers at later times (day 17). Time course experiments showed that Al increased nodule number beginning from day 7. Alkaline phosphatase activity, assessed at four stages during the differentiation sequence of RC cell cultures (from 4 to 13 days) was stimulated by Al at all times. However, Al decreased colony formation, inhibited cell growth in late log phase, and decreased saturation density of the treated cultures. Al concentrations of 30 μM and above resulted in degeneration of the cell layer and an increasing fibrillar appearance of the matrix present in between or adjacent to nodules when cultures were maintained for more than 15 days. The presence of Al significantly decreased the viability of cells obtained from 13–17 days cultures, as determined by plating efficiency and trypan blue exclusion. We frequently observed cellular toxicity (in 8 of 10 experiments) in cultures containing 300 μM Al, and by days 17–19, cells, nodules, and matrix were disintegrating in these cultures. We conclude that Al accelerates the rate of osteoprogenitor cell differentiation and the formation of bone nodules while concomitantly inhibiting nodule mineralization. However, concentrations that accelerate differentiation appear to be cytotoxic in long-term cultures. Received: 29 April 1997 / Accepted: 9 December 1998     

Thiamine (Vitamin B1) Protects against Glucose- and Insulin-Mediated Proliferation of Human Infragenicular Arterial Smooth Muscle Cells

2 . Cells were identified as ASMC by immunohistochemical analysis. Cells from passages 3-5 were exposed to glucose concentrations of 0.1 and 0.2% with and without insulin concentrations of 100 ng/mL and 1000 ng/mL, in the presence or absence of 200 μM of thiamine. Standard hemocytometry and ³H-thymidine incorporation quantified cell proliferation after incubation for 6 days and 24 hr, respectively. The data suggest that thiamine inhibits human infragenicular ASMC proliferation induced by high glucose and insulin. Vitamin B1 intake may prove important in delaying the atherosclerotic complications of diabetes.     

Studies on the biocompatibility and the interaction of silver nanoparticles with human mesenchymal stem cells (hMSCs)

Purpose  Silver nanoparticles (Ag-NPs) are widely used in different areas, e.g., in the food, electronic, or clothing industry due to well-known slow-release antiseptic activities. Despite the widespread use of nanosilver, there is a serious lack of information concerning the biological activities of nanosilver on human tissue cells. Materials and methods  In this study, the influence of spherical Ag-NPs (diameter about 100 nm) on the biological functions (proliferation, cytokine release, and chemotaxis) of human mesenchymal stem cells (hMSCs) was analyzed. Results  The results showed a concentration-dependent activation of hMSCs at nanosilver levels of 2.5 μg mL⁻¹, and cytotoxic cell reactions occurred at Ag-NPs concentrations above 5 μg mL⁻¹. Cell proliferation and the chemotaxis of hMSC both decreased with increasing Ag-NPs concentrations. Different effects on the cytokine release from hMSCs were observed in the presence of Ag-NPs and Ag⁺ ions. The release of IL-8 was significantly increased at high but noncytotoxic concentrations of Ag-NPs (2.5 μg mL⁻¹). In contrast, the levels of IL-6 and VEGF were concomitantly decreased compared to the control group. The synthesis of IL-11 was not affected at different Ag-NP concentrations. The agglomeration tendency of Ag-NPs in different biological media increased with a high electrolyte content, e.g., in RPMI. However, complexation with fetal calf serum in the cell culture media stabilized the Ag-NPs against agglomeration. Conclusion  In summary, the results showed that Ag-NPs exert cytotoxic effects on hMSCs at high concentrations but also induce cell activation (as analyzed by the release of IL-8) at high but nontoxic concentrations of nanosilver. “Best of Abstracts – Chirurgisches Forum 2009, Deutsche Gesellschaft für Chirurgie”     

Suppression of cyclic guanosine monophosphate formation in rat cerebellar slices by propofol, ketamine and midazolam

Purpose  The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) system is involved in glutamatergic neurotransmission. The current study determined the effects of propofol, ketamine and midazolam on rat cerebellar cGMP formation, attempting to clarify whether the effect was due to suppression of NO-cGMP system or to direct interaction with glutamatergic receptors. Methods  Cerebellar slices, obtained from six-to eight-day-old Wistar rats, were pretreated with propofol (10 μM–1 mM), ketamine (10–100 μM) or midazolam (1–100 μM) for 30 min. and then stimulated with L-glutamate (3 mM), N-methyl-D-aspartate (NMDA, 0.1 mM), kainate (0.1 mM) or sodium nitroprusside (SNP, 0.3 mM) (n = 5–11 for each group). The levels of cGMP were determined by radioimmunoassay. Results  None of the anaesthetics studied altered cGMP levels when no stimulant was given. Propofol (10 μM–1 mM) suppressed L-glutamate-, NMDA-, kainate- and SNP stimulated cGMP formation in a concentrationdependent manner, the sensitivity to propofol was in the order of NMDA > kainate > L-glutamate, SNP Ketamine (10–100 μM) suppressed L-glutamate- and NMDA-stimulated cGMP formation, but did not suppress kainate-or SNP-stimulated cGMP formation. Midazolam (10–100 μM) did not affect NMDA-, L-glutamate-or SNP-stimulated cGMP formation, but suppressed kainate-induced formation. Conclusion  The inhibitory effects of propofol, ketamine and midazolam on cGMP formation in rat cerebellar slices are due mainly to interaction with receptors for excitatory amines, and not due to the suppression of nitric oxide synthase or guanylate cyclase activities.

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Résumé Objectif  Le système monoxyde d’azote (NO)/monophosphate de guanosine cyclique (cGMP) participe à la neurotransmission glutamatergique. La présente étude recherchait l’influence du propofol, de la kétamine et du midazolam sur la formation cérébelleuse de cGMP en essayant de préciser si cette influence résultait de la suppression du système NO-cGMP ou d’une interaction directe avec les récepteurs glutamatergiques. Méthodes  Des tranches de cerveau prélevées sur des rats Wistar agés de six à huit jours ont été prétraitées avec du propofol (10 μm–1 mM), de la kétamine (10–100 μM) ou du midazolam (1–100 μM) et ensuite stimulées avec du L-glutamate (3 mM), du N-méthyl-D-aspartate (NMDA, 0,1 mM), du kainate (0,1 mM) ou du nitroprussiate de sodium (SNP 0,3 mM) (n = 5 pour chaque groupe). Le radio-immunodosage a servi à déterminer le niveau de cGMP Résultats  En absence de stimulus, aucun des anesthésiques n’a modifié les niveaux de cGMP Le propofol (10 μM-mM) supprimait la formation de cGMP stimulée par le L-glutamate, le NMDA, le kainate et le SNP proportionnellement à la concentration. La sensibilité au propofol s’établissait dans l’ordre suivant NMDA > kainate > L-glutamate > SNP La kétamine (10–100 μM) supprimait la formation stimulée par le NMDA de L-glutamate et de cGMP mais ne supprimait pas la formation de cGMP stimulée par le kainate et le SNP Le midazolam (10–100 μM) n’affectait pas la formation de cGMP stimulée par le NMDA, le L-glutamate et le SNP mais supprimait la formation induite par le kainate. Conclusion  Linfluence inhibitrice du propofol, de la kétamine et du midazolam sur la formation de cGMP dans les tranches cérébelleuses de rats est principalement causée par l’interaction des récepteurs des amines excitateurs et non par la suppression de la synthase du monoxyde d’azote ou à l’action de la guanylate cyclase.

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Supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, (No. 07457355) and Research Grant for Women from ESSO-Sckiyu, Japan. Presented in part at the Annual Meeting of the ASA, Atlanta, October 1995.     

Can intravenous atropine prevent bradycardia and hypotension during induction of total intravenous anesthesia with propofol and remifentanil?

This study was conducted to examine whether pretreatment with intravenous atropine could prevent bradycardia and hypotension during induction of total intravenous anesthesia with propofol and remifentanil in a prospective randomized placebo-controlled manner. Seventy patients, aged 24–78 years, were randomly divided into two groups, and received 0.5 mg atropine or placebo saline 1 min before induction of intravenous anesthesia with remifentanil at 0.4 μg/kg/min, propofol at a target blood concentration of 3 μg/ml, and vecuronium 1.5 mg/kg. Immediately after tracheal intubation, the infusion rate of remfentanil and the target concentration of propofol were reduced to and kept at 0.1 μg/kg/min and 2 μg/ml, respectively, for 10 min. Noninvasive blood pressure (BP) and heartrate (HR) were measured and recorded every minute. Intravenous atropine could prevent a fall in HR, but not a fall in BP, during induction of intravenous anesthesia with propofol and remifentanil of our dosing regimen. Our data suggested that a fall in HR induced by propofol–remifentanil anesthesia was mainly caused by centrally mediated sympatholytic and/or vagotonic actions of propofol and remifentanil, whereas a fall in BP was mainly the result of their direct vasodilating actions.     

Bone tissue concentrations of ciprofloxacin released from biodegradable screws implanted in rabbits skull

Previously, ciprofloxacin-releasing polylactide/polyglycolide 80/20 (SR-PLGA) screws have proven to be biocompatible with sufficient strength. However, there has been no information about the local concentrations of ciprofloxacin in bone tissue after their implantation. To measure bone concentrations of ciprofloxacin, two screws were implanted in each rabbit, one on either side of the sagittal suture (n = 28 rabbits). Follow-up periods were 2, 4, 8, 16, 24, 52 and 78 weeks. From each rabbit, bone blocks containing one screw were retrieved and used to measure drug concentration. Ciprofloxacin concentration at 2 weeks follow-up was 4.4 ± 4.2 μg/g, 14.1 ± 2.7 μg/g at 4 weeks and 7.6 ± 4.5 μg/g at 8 weeks. Measured concentrations were very low at 16 weeks (mean 0.09 ± 0.04 μg/g) and 24 weeks (mean 0.04 ± 0.02 μg/g). Surprisingly, the concentration was higher again at 1 year (mean 1.3 ± 1.0 μg/g). At 1.5 years, concentration had decreased again (mean 0.8 ± 0.6 μg/g). The drug concentration in bone tissue was higher than minimal inhibitory concentration of ciprofloxacin (Staphylococcus aureus 0.1–1.0 μg/g) at 2, 4, and 8 weeks. Ciprofloxacin-releasing SR-PLGA 80/20 screws could be used clinically for osteofixation and infection treatment in cranial bone.     

Effect of a newly synthesized Zn sulfophthalocyanine derivative on cell morphology,viability, proliferation,and cytotoxicity in a human lung cancer cell line (A549)

Photodynamic therapy (PDT) is a photochemotherapeutic process that is used for the treatment of cancer. Photofrin is the most widely used photosensitizer, however, the chemical composition of Photofrin is unclear and it has a low absorption in the therapeutic wavelength (600–900 nm). This factor has stimulated research in synthesis and testing of new photosensitizers. This in vitro study evaluated the effectiveness of a Zn sulfophthalocyanine (ZnPcS_mix) as a potential photosensitizer in the treatment of human lung cancer. Lung cancer cells (A549) were divided into four groups: group 1 was control cells receiving neither light nor drug; group 2 was light control for cells exposed to laser irradiation at a fluence of 4.98 J/cm²; group 3 was drug control for cells incubated with 15.8 μM photosensitizer and not exposed to laser irradiation, while group 4 was cells receiving the experimental treatment with 15.8 μM photosensitizer and irradiation with 4.98 J/cm². Laser irradiations were performed using a 636-nm diode laser with an output power of 110 mW at 4.98 J/cm². Changes in cellular responses were evaluated by cell morphology, viability, proliferation, and cytotoxicity. While control groups 1, 2, and 3 showed no changes in cell morphology, viability, proliferation, or cytotoxicity, group 4 receiving both photosensitizer and irradiation showed changes in cell morphology, a decrease in cell viability and proliferation, and an increase in cytotoxicity, cell death, and cell membrane damage. Irradiation or photosensitizer alone had no effect on the lung cancer cells since the cells remained viable and showed no evidence of damage. However, irradiation in the presence of a photosensitizer induced cell death.     

Microdialysis of paraspinal muscle in healthy volunteers and patients underwent posterior lumbar fusion surgery

Paraspinal muscle damage is inevitable during conventional posterior lumbar fusion surgery. Minimal invasive surgery is postulated to result in less muscle damage and better outcome. The aim of this study was to monitor metabolic changes of the paraspinal muscle and to evaluate paraspinal muscle damage during surgery using microdialysis (MD). The basic interstitial metabolisms of the paraspinal muscle and the deltoid muscle were monitored using the MD technique in eight patients, who underwent posterior lumbar fusion surgery (six male and two female, median age 57.7 years, range 37–74) and eight healthy individuals for different positions (five male and three female, age 24.1 ± 0.8 years). Concentrations of glucose, glycerol, and lactate pyruvate ratio (L/P) in both tissues were compared. In the healthy group, the glucose and glycerol concentrations and L/P were unchanged in the paraspinal muscle when the body position changed from prone to supine. The glucose concentration and L/P were stable in the paraspinal muscle during the surgery. Glycerol concentrations increased significantly to 243.0 ± 144.1 μM in the paraspinal muscle and 118.9 ± 79.8 μM in the deltoid muscle in the surgery group. Mean glycerol concentration difference (GCD) between the paraspinal muscle and the deltoid tissue was 124.1 μM (P = 0.003, with 95% confidence interval 83.4–164.9 μM). The key metabolism of paraspinal muscle can be monitored by MD during the conventional posterior lumbar fusion surgery. The glycerol concentration in the paraspinal muscle is markedly increased compared with the deltoid muscle during the surgery. It is proposed that GCD can be used to evaluate surgery related paraspinal muscle damage. Changing body position did not affect the paraspinal muscle metabolism in the healthy subjects.     

A structurally optimized celecoxib derivative inhibits human pancreatic cancer cell growth

Deregulation of the phosphatidylinositol 3-kinase (PI-3K)/PDK-l/Akt signaling cascade is associated with pancreatic cancer tumor invasion, angiogenesis, and tumor progression. As such, it has been postulated that PDK-1/Akt signaling inhibitors may hold promise as novel therapeutic agents for pancreatic cancer. Disadvantages of currently available Akt inhibitors include tumor resistance, poor specificity, potential toxicity, and poor bioavailability. Previous studies have demonstrated that OSU-03012, a celecoxib derivative, specifically inhibits PDK-1 mediated phosphorylation of Akt with IC₅₀ values in the low mM range. Human pancreatic cancer cell lines AsPC-1, BxPC-3, Mia-PaCa 2, and PANC-1 were cultured in media containing varying concentrations of OSU-03012, 5-fluorouracil (5-FU), and gemcitabine, and changes in Akt phosphorylation and cell viability were evaluated using western blotting and a 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, respectively. Treatment with OSU-03012 resulted in decreased PDK-1-mediated Akt phosphorylation and cell growth inhibition for all cell lines with IC₅₀ values ranging between 1.0 and 2.5 μM. Resistance to 5-FU and gemcitabine was observed in cell lines AsPC-1 and BxPC-3. Further analyses indicate that OSU-03012 induces both proapoptotic and antiproliferative effects in these cells. Taken together, these data suggest that OSU-03012 has potential value as a novel therapy for pancreatic cancer. Presented at the 2005 American Hepato-Pancreato-Biliary Association Congress, Hollywood Florida, April 14–17, 2005.     

Pharmacokinetics of Teriparatide (rhPTH[1–34]) and Calcium Pharmacodynamics in Postmenopausal Women with Osteoporosis

Teriparatide (rhPTH[1–34]) affects calcium metabolism in a pattern consistent with the known actions of endogenous parathyroid hormone (PTH). This report describes the pharmacokinetics and resulting serum calcium response to teriparatide in postmenopausal women with osteoporosis. Pharmacokinetic samples for this analysis were obtained from 360 women who participated in the Fracture Prevention Trial. Postmenopausal women with osteoporosis received daily subcutaneous injections of either teriparatide 20 μg (4.86 μmol) or placebo, median 21 months’ treatment. Serum teriparatide and calcium concentrations were measured throughout the study. An indirect-response model was developed to describe the pharmacokinetic–pharmacodynamic relationship between teriparatide concentrations and serum calcium response. The pharmacokinetics of teriparatide were characterized by rapid absorption (maximum concentration achieved within 30 min) and rapid elimination (half-life of 1 h), resulting in a total duration of exposure to the peptide of approximately 4 h. Teriparatide transiently increased serum calcium, with the maximum effect observed at approximately 4.25 h (median increase 0.4 mg/dl [0.1 mmol/l]). Calcium concentrations returned to predose levels by 16–24 h after each dose. Persistent hypercalcemia was not observed; one teriparatide 20 μg-treated patient had a predose serum calcium value above the normal range but <11.0 mg/dl (2.75 mmol/l). Following once-daily subcutaneous administration, teriparatide produces a modest but transient increase in serum calcium, consistent with the known effects of endogenous PTH on mineral metabolism. The excursion in serum calcium is brief, due to the short length of time that teriparatide concentrations are elevated.     

Actions and Comparative Efficacy of Phosphatidylcholine Formulation and Isolated Sodium Deoxycholate for Different Cell Types

Background  Phosphatidylcholine formulation has been used to dissolve local fat deposits. This study aimed to evaluate and compare the effects of phosphatidylcholine formulation and its vehicle sodium deoxycholate alone on different cell lines to understand better its mechanism of action. Methods  Cells and media including 3T3-L1 preadipocytes, normal foreskin fibroblasts, neonatal human dermal microvascular endothelial cells (CADMEC), and fetal human skeletal muscle cells (HSkMC) were used. After 24 h, cells were exposed in 3-4, 5-dimethylthiazol-2-yl-2, 3-diphenyl tetrazolium bromide reagent (MTT assays) to increasing dosages of phosphatidylcholine formulation (0.0156–0.5 mg/ml) or an equivalent vehicle, sodium deoxycholate solution, pH 9.0 (0.0066–0.210 mg/ml). Viability was assessed after 1, 2, and 3 days of treatment. Fat tissue (4 × 4 cm) obtained ex vivo from the dorsal fat pads of five rabbits was injected with 2 ml of phosphatidylcholine formulation (50 mg/ml), sodium deoxycholate (21 mg/ml), or normal saline and incubated for 24 h. These were examined histologically to identify cell lysis and morphologic changes. Results  At 0.125- and 0.25-mg/ml doses of phosphatidylcholine solution, CADMEC and HSkMC were more sensitive (P < 0.001, one-way ANOVA) than adipocytes at all time points examined. Phosphatidylcholine formulation at a dose of 0.5 mg/ml and the equivalent vehicle, sodium deoxycholate, at a dose of 0.21-mg/ml both induced nearly 100% fat cell lysis after 24 h, and evidence of cell lysis as early as 6 h after exposure. After incubation of fat tissue for 24 h with phosphatidylcholine formulation, loss of intracellular lipid staining with an increase in extracellular lipids was seen. Conclusions  Isolated sodium deoxycholate was almost as effective as the phosphatidylcholine formulation, at clinical concentrations, in reducing the viability of mature adipocytes over time. Similar cytotoxic effects of phosphatidylcholine formulation on normal foreskin fibroblasts, endothelial cells, and human skeletal muscle cells also were observed. The data prove that the formulation acts in a nonspecific manner and that its unintentional administration to other tissues causes cell death.     

Ciliogenesis in normal human kidney development and post-natal life

Ciliogenesis in developing and post-natal human kidneys appears to influence cell proliferation and differentiation, apico–basal cell polarity, and tubular lumen formation. We have analyzed the appearance of primary cilia and differentiation of kidney cells in ten human conceptuses aged 6–22 weeks and in one 5-year-old kidney, using a double immunofluorescence labeling technique for α-tubulin, γ-tubulin, Oct-4, and Ki-67 and by electron microscopy. Immature forms of nephrons and ampullae were characterized by intense cell proliferation, which subsequently decreased during development. Primary cilia appeared on the surfaces of non-proliferating cells in developing nephrons, gradually increasing in length from 0.59 μm in renal vesicles to 0.81 μm in the S-forms of nephrons, ultimately reaching 3.04 μm in length in mature fetal and post-natal nephrons. Ciliary length increased from 0.59 μm in ampullae to 1.28 μm in post-natal collecting tubules. Mesenchymal to epithelial transformation of kidney cells coincided with the appearance of apico–basal polarity, both gap and tight junctions, and lumen formation. Up-regulation of Oct-4 expression correlated with the onset of kidney cell differentiation. Our results demonstrate the importance of proper primary cilia lengthening and Oct-4 expression for the normal development of fetal and post-natal kidneys and of apico–basal polarity for normal tubular lumen formation. Disturbances in these processes are associated with ciliopathies.     

Diclofenac and NS-398, a selective cyclooxygenase-2 inhibitor, decrease agonist-induced contractions of the pig isolated ureter

Non-steroidal anti-inflammatory drugs (NSAIDs) are currently considered a first-line treatment of renal colic. Their action has been ascribed to the inhibition of renal prostaglandin synthesis, which decreases renal blood flow and diuresis, and consequently lowers the pressure in the renal pelvis and ureter. However, the effects of NSAIDs on induced contractions of ureteral smooth muscle have received little attention. Also, there is a lack of clinically relevant spasmolytic drugs for the ureter. Therefore, we studied the influence of the non-selective cyclooxygenase (COX) inhibitor diclofenac, a NSAID drug customarily used in the treatment of renal colic, and of NS-398, a selective COX-2 inhibitor, on induced contractions of the pig ureter. Serotonin (0.1–30 μM), norepinephrine (0.1–30 μM) and neurokinin A (0.03–10 μM) induced reproducible concentration-dependent contractions, which were inhibited by diclofenac and NS-398 (10–300 μM) in a concentration-dependent manner. The sensitivity of neurokinin A-induced contractions to diclofenac was 3–4 times greater than that of the amines. Depending on the concentration, inhibition ranged between 25 and 96% of the initially induced contractile activity. In the presence of inhibitors, supramaximal concentrations of agonists were unable to trigger recuperation of the initially induced contractions. Prostaglandin F_2α did not reverse the effect of diclofenac on agonist-induced contractions. Removal of diclofenac or NS-398 from the organ baths showed that the inhibition was totally reversible. Thus, the non-selective COX inhibitor diclofenac and the selective COX-2 inhibitor NS-398 are almost equipotent in reducing agonist-induced contractions in the isolated porcine ureter. Although the clinical relevance of this spasmolytic effect remains to be demonstrated, the data suggest that patients suffering from renal colic may benefit not only from the anti-diuretic and analgesic effects of diclofenac, but also from its potential spasmolytic properties. Moreover, selective COX-2 inhibitors may have clinical potential, as they may cause fewer side effects. Received: 21 February 2000 / Accepted: 27 July 2000     

Effects of cyclosporine a on chick osteoclastsIn vitro

Summary We have studied the effects of cyclosporine A (CsA) on basal and bovine parathyroid hormone (1–34) (bPTH)-stimulated bone resorption by osteoclasts in 24-hour cultures of chick long bone cells. At a high concentration (10 μg/ml), CsA had a cytotoxic effect on both osteoclasts and mononuclear cells in the culture. At 1 μg/ml, CsA inhibited basal and bPTH-stimulated bone resorption but was not cytotoxic over 24 hours. We also studied the binding of bPTH to the osteoblastic cell line, Saos-2, and chick long bone cells in suspension culture. CsA inhibited bPTH binding in Saos-2 in a dose-dependent manner; inhibition of binding was also observed in chick bone cells. The effects of CsA on osteoclast viability and resorptive function may be due to a direct effect on the osteoclasts and/or to an interaction with the nonosteoclastic cell population in the culture.     

Inhibitory effect of fluoride on the secretion of insulin

Summary The oral administration of sodium fluoride (NaF) (40 μmol/100 body weight [bw]) to fasting rats produced an immediate fall in insulin levels and the consequent increase in glycemia. These phenomena were observed with plasma fluoride concentrations 5–15 μM. Glycemia and insulin returned to normal levels within 4–5 hours, together with the washing out of fluoride from plasma and soft tissues. The insulin secretion of isolated Langerhans islets, perifused with solutions containing 5, 10, or 20 μM fluoride, was found to be significantly inhibited as a function of fluoride levels, both with basal and stimulatory concentrations of glucose. One hour after the intake of 60 mg of NaF, fasting human volunteers showed increased fluoride (5–15 μM) together with a significant fall of plasma insulin levels.     

Gentamicin negatively influenced osteogenic function in vitro

Local delivery of gentamicin is an accepted method of infection prophylaxis in the surgery of open fractures. However, the few reports of studies into the effect of locally applied gentamicin on osteoblasts used inadequate methods. In our study, we used the well-characterised C2C12 cell line with reproducible differentiation pathway into the osteoblast lineage. We investigated the viability, cell number, alkaline phosphatase activity, and the expression of osteogenic genes of C2C12 cells after exposure to gentamicin at concentrations of 12.5–800 μg/ml for 48 h. Exposure of C2C12 cells to gentamicin (12.5–800 mg/ml) for 48 h showed no significant changes in the cell number, but cell viability was decreased by one-third at the tested concentrations of 200–800 μg/ml. The alkaline phosphatase activity was significantly decreased by one-third to one-half at any tested concentration (12.5–800 μg/ml) of gentamicin. Any tested concentration of gentamicin up to 800 μg/ml for 48 h did not inhibit or decrease the osteogenic gene expression of osterix and alkaline phosphatase of the C2C12 cells. In conclusion, gentamicin at high concentrations as achieved by local application reduced cellular viability and alkaline phosphatase activity in vitro and therefore may be detrimental for bone healing and repair in vivo.

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Résumé La délivrance locale de gentamycine est une méthode usuelle de prophylaxie de l’infection dans la chirurgie des fractures ouvertes. Il y a peu d’études fiables sur les effets de la gentamycine sur les ostéoblastes. Dans cette étude est utilisée la lignée C2C12 avec reproductibilité des voies de différenciation dans la lignée ostéoblastique. Nous avons étudié la viabilité, le nombre de cellules, l’activité phosphatase alcaline et l’expression des gènes ostéogèniques des cellules C2C12 après exposition pendant 48 heures à des concentrations de gentamycine de 12,5 μg/ml à 800 μg/ml. Il n’y avait pas de modification significative du nombre de cellules mais la viabilité était diminuée d’un tiers pour les concentrations de 200 à 800 μg/ml. L’activité phosphatase alcaline était diminuée d’un tiers à la moitié pour toutes les concentrations étudiées. Dans aucun cas il n’y avait diminution ou inhibition de l’expression génique de l’osterix ou de la phosphatase alcaline des cellules C1C12. En conclusion, la gentamycine à haute concentration réduit la viabilité cellulaire et l’activité phosphatase alcaline in vitro, ce qui est peut-être néfaste pour la cicatrisation osseuse in vivo.

     

Zoledronate reverses mandibular bone loss in osteoprotegerin-deficient mice

Summary  To characterize the changes in osteoprotegerin-deficient (OPG−/−) mice mandibles and the possible mandibular bone loss prevention by zoledronate. This preventive effect in the mandible differed from that in the proximal tibia and was independent of the OPG pathway. Introduction  The study aimed to characterize both the changes in the mandible in osteoprotegerin-deficient (OPG^−/−) mice and possible mandibular bone loss prevention by zoledronate. Methods  Twenty-eight 6-week-old female mice (C57BL/6J), including OPG^−/− (n = 21) and wild-type (WT) (n = 7) mice, were assigned to four groups after 2 weeks of acclimatization to local vivarium conditions: wild mice with vehicle (WT group); OPG^−/− mice with vehicle (OPG^−/− group); and OPG^−/− mice that were subcutaneously injected with either 50 or 150 μg/kg zoledronate (Zol-50 and Zol-150 groups, respectively). Mice were sacrificed at 4 weeks after these treatments and after fasting for 12 h. Sera were harvested for biochemical analyses. The right mandible and tibia of each mouse were selected for microCT analysis. Student’s t-test was performed for comparisons of bone parameters at different sites in the WT group. Analysis of variance (ANOVA) was used to compare the biomarkers and bone parameters in the different treatment groups. Results  Serum bone-specific alkaline phosphatase (B-ALP) and tartrate-resistant acid phosphatase 5b (TRACP-5b) were significantly decreased in WT mice as compared to the levels in the OPG^−/− mice (P < 0.05). Zoledronate treatment decreased the high serum B-ALP activity observed in OPG^−/− mice to the levels seen in WT mice, while serum TRACP-5b concentrations were decreased to levels even lower than those in WT mice. There were substantial variations in BMD and microstructure of the mandibular and proximal tibial trabeculae. Mandibular bone loss was less affected by OPG gene deprivation than the proximal tibia was. Both zoledronate groups showed greater BMD, trabecular BV/TV, Tb.Th, Tb.N, and Conn.D and a significant decrease in Tb.Sp and SMI as compared to the findings in OPG^−/− mice (P < 0.05). However, higher apparent BMD and more compact plate-like trabeculae were observed in the mandible after treatment with zoledronate as compared to the findings in the proximal tibia. No significant differences were found in any parameter in both zoledronate groups. Conclusions  The present study showed that zoledronate could reverse the significant bone loss in mice mandibles that was induced by OPG gene deficiency. This preventive effect, which was accompanied with considerable inhibition of bone turnover, differed in the mandible and in the proximal tibia and was independent of the OPG pathway. Drs. Sheng and Xu contributed equally to this work.     

Propofol enhances red cell antioxidant capacity in swine and humans

Purpose  To determine the effect of an anaesthetic with antioxidant potential, propofol, on red blood cell (RBC) antioxidant enzyme activities and RBC susceptibility to peroxidative challenge. Methods  Propofol was administered by intravenous bolus (2.5 mg·kg⁻¹) and continuous infusion (36 and 72 ml·hr⁻¹ in nine swine; 216 ml·hr⁻¹ in two swine), to achieve serum concentrations between 5 and 30μg·ml⁻¹ for two hours at each rate. Arterial blood sampling was at 0,10, 30, 60, and 120 min for each rate of infusion, for measurement of plasma propofol concentration, activities of plasma and RBC Superoxide dismutase, glutathione peroxidase, gluthathione reductase, RBC catalase, and RBC malondialdehyde (MDA) formation in response to exvivo oxidative challenge with t-butyl hydrogen peroxide (tBHP; 1.5mM). Antioxidant mechanisms were determined byin vitro study of MDA formation, GSH depletion, and oxidation of haemoglobin to methaemoglobin in human erythrocytes exposed to propofol 0–75 μM. The antioxidant potential of propofol was compared with that of alpha-tocopherol utilising the reaction with 2,4,6-tripyridyl-s-triazine (TPTZ). Results  Propofol had no effect on plasma or RBC antioxidant enzyme activities. It inhibited RBC MDA production over the range of 0–20 μg·ml⁻¹ (y = −18.683x + 85.431 ; R² = 0.8174). Effective propofol concentrations for 25% and 50% reductions in MDA levels were 7–12 and 12–20 μg·ml⁻¹, respectively. Propofol has a similar effect on human erythrocytesin vitro (R₂ = 0.98). Conclusion  Propofol antagonises the effects of forced peroxidation of red cells at anaesthetic and sub-anaesthetic concentrations in swine. Its actions include scavenging of oxygen derived free radicals in a tocopherol-like manner.

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Résumé Objectif  Déterminer l’effet d’un agent anesthésique possédant un potentiel antioxydant, le propofol, sur l’activité d’un enzyme antioxydant des globules rouges (GR) et sur la susceptibilité des GR à une provocation peroxydative. Méthodes  Le propofol a été administré en bolus intraveineux (2,5 mg·kg⁻¹) et en infusions continues (36 et 72 ml·h⁻¹ chez 9 porcs; 216 ml·h⁻¹ chez 2 porcs) pour obtenir des concentrations sériques entre 5 et 30 μg·ml⁻¹ durant deux heures à chaque vitesse d’infusion. Des prélèvements sanguins par voie artérielle ont été réalisés à 0, 10, 30, 60 et 120 min. pour chaque vitesse d’infusion; on a mesuré la concentration de propofol, l’activité de la superoxyde dismutase du plasma et des GR, de la peroxydase du glutathion, de la réductase du glutathion, de la catalase du GR, ainsi que de la formation dans le GR de la malondialdehyde (MDA) en réponse à une provocation oxydative exvivo avec le peroxyde d’hydrogène t-butylique (tBHP, 1,5 mM). Les mécanismes antioxydants ont été déterminés par l’étudein vitro de la formation de MDA, de la déplétion de GSH ainsi que de l’oxydation de l’hémoglobine en methémoglobine dans des GR humains exposés au propofol 0–75 μM. Le potentiel antioxydant du propofol a été comparé à celui de l’alpha-tocophérol en utilisant la réaction avec le 2,4,6-tripyridyl-s-triazine (TPTZ). Résultats  Le propofol n’a pas eu d’effet sur l’activité de l’enzyme antioxydant du plasma ou des GR. Il a inhibé la production de MDA par les GR pour tout le spectre de 0–20 μg·ml⁻¹ (y = −18.683x + 85.431 ; R² = 0,8174). Les concentrations de propofol efficaces pour obtenir une réduction des taux de MDA de 25 et de 50% étaient respectivement de 7–12 et de 12–20 μg·ml⁻¹. Le propofol a un effet analogue sur les globules rouges humainsin vitro (R₂ = 0,98). Conclusion  Le propofol, à des concentrations anesthésiques et subanesthésiques chez le porc, antagonise les effets d’une peroxydation forcée des globules rouges. Son mode d’action comporte l’épuration des radicaux libres provoqués par l’oxygène comme le fait le tocophérol.

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Supported in part by a research grant from Zeneca Pharma Inc (Canada Ltd.).

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Presented at the 71st Clinical and Scientific Congress of the International Anesthesia Research Society, March 14–18,1997 San Francisco, Ca, USA.     

Characterization of the Effects of Cyclooxygenase-2 Inhibition in the Regulation of Apoptosis in Human Small and Non–Small Cell Lung Cancer Cell Lines

Background Cyclooxygenase-2 enzyme (COX-2) is overexpressed in human non–small cell lung cancer (NSCLC) but is not expressed in small cell lung cancer. Selective COX-2 inhibitors have been shown to induce apoptosis in NSCLC cells, an effect which is associated with the regulation of intracellular MAP kinase (MAPK) signal pathways. Our aims were to characterize the effects of COX-2 inhibition by rofecoxib on apoptosis in human NSCLC and small cell lung cancer cell lines. Methods The human NSCLC cell line NCI-H2126 and small cell lung cancer cell line DMS-79 were used. Constitutive COX-2 protein levels were first determined by Western blot test. Levels of apoptosis were evaluated by using propidium iodide staining on FACScan analysis after incubation of NCI-H2126 and DMS-79 with p38 MAPK inhibitor SB202190 (25 μM), NF-κB inhibitor SN50 (75 μg/mL), and rofecoxib at 100 and 250 μM. All statistical analysis was performed by analysis of variance. Results Western blot test confirmed the presence of COX-2 enzyme in NCI-H2126 and absence in DMS-79. Interestingly, rofecoxib treatment demonstrated a dose-dependent increase in apoptosis in both cell lines. Given this finding, the effect of rofecoxib on NF-κB and p38 MAPK pathways was also examined. Apoptosis in both cell lines was unaltered by SN50, either alone or in combination with rofecoxib. A similar phenomenon was observed in NCI-H2126 cells treated with SB202190, either alone or in combination with rofecoxib. In contrast, p38 MAPK inhibition greatly upregulated DMS-79 apoptosis in a manner that was unaltered by the addition of rofecoxib. Conclusions Rofecoxib led to a dose-dependent increase in apoptosis in both tumor cell lines. This effect occurred independently of COX-2, NF-κB, and p38 MAPK pathways in DMS-79 cells. As such, rofecoxib must act on alternative pathways to regulate apoptosis in human small cell lung cancer cells. Presented at the Society of Surgical Oncology (SSO) Annual Meeting, New York, 18th-21st March 2004.