JOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2010
CORNELIA BRAICU
ABSTRACT The present study aims to investigate the cytotoxic effect of the major aflatoxins (B1, B2, G2 and G2) and also aflatoxin combination, using a simple, rapid and cheap cytotoxicity test like MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay in three in vitro models (human umbilical vein endothelial cells [HUVEC], human lung fibroblasts [HFL] and A2780 cell line) and to extrapolate the data to in vivo situation using a prediction model. A difference in cell sensitivity has been observed for B1 and B1 + B2, in the following order A2789 > HFL > HUVEC, while for B2, G1, G2, Mix (B1 + B2 + G1 + G2) the order was HFL > A2789 > HUVEC when comparing the IC50 (half maximal inhibitory concentration) values. We confirm that in vitro cytotoxicity test MTT assay is able to predict in vivo toxicity, at least for aflatoxins using the prediction model. The values of LD50 (lethal dose 50%) calculated from experiments are different for each cell line. This fact may indicate that some species are more resistant than other and target organs are not necessarily those predicted, because the A2780 ovarian cancer cells seem to be more sensitive to B1 than cells of endothelial or fibroblasts origin. PRACTICAL APPLICATIONS This study is in concordance with the international tendency that refined the current techniques to lessen pain or distress, to reduce the number of animals necessary for a particular test or to replace animals with non-whole-animal models, such as in vitro cell cultures. The practical application of such methodologies may help solve the economic problem related to very expensive in vivo toxicology studies and implement preventive methods based on the calculated data and known mechanism of action of individual or combined toxins easily studied in vitro. The nature of coexistence of many types of mycotoxins in complex environmental samples, such as food and water, has been reported worldwide. How these mycotoxins might affect human health in combination is largely unknown. This study had, as a goal, to test the toxicity of the four aflatoxins and aflatoxin combination on human cells. Due to the lack of aflatoxins mixture data regarding the human cytotoxicity, the aim of this study was to specify, evaluate and predict the combined effects of mycotoxin mixtures. [source]

THE FREE RADICAL-SCAVENGING PROPERTY OF CHONDROITIN SULFATE FROM PIG LARYNGEAL CARTILAGE IN VITRO

JOURNAL OF FOOD BIOCHEMISTRY, Issue 1 2007
SHUANG-LI XIONG
ABSTRACT This study compared the free radical-scavenging properties of chondroitin sulfate (ChS) from pig laryngeal cartilage and its reduced or sulfonated derivatives. The binding behavior between Cu2+ and ChS and its derivatives, and the interaction between superoxide radical and ChS were studied by fluorescence quenching, equilibrium dialysis, infrared spectra and thermal analysis. Purified ChS inhibited the generation of hydroxyl radical and scavenged superoxide radical in a concentration-dependent manner. Reduced ChS did not scavenge hydroxyl radical and superoxide radical. Sulfonated ChS had no hydroxyl radical scavenging activity but scavenged superoxide radical as strongly as purified ChS. ChS showed strong binding activity with Cu2+ in deionized water but not in 0.01-M HCl. Both reduced ChS and sulfonated ChS did not exhibit such chelating behavior. The structural basis of hydroxyl radical inhibiting of ChS was attributed to a complex of the Cu2+ with the carboxyl group of glucuronic acid residue. The reaction of superoxide radical with the sulfate ester and the carboxyl group may be the basis of superoxide radical scavenging activity of ChS. [source]

LISTERIA MONOCYTOGENES AND ESCHERICHIA COLI O157:H7 INHIBITION IN VITRO BY LIPOSOME-ENCAPSULATED NISIN AND ETHYLENE DIAMINETETRAACETIC ACID

JOURNAL OF FOOD SAFETY, Issue 2 2008
T. MATTHEW TAYLOR
ABSTRACT Encapsulation technologies that effectively reduce antimicrobial interaction with food components or protect antimicrobial compounds from food processing measures have the potential to improve the microbiological safety of ready-to-eat foods. Recent application of liposomes for the preservation of cheese has spurred research into their utility in other food matrices. To ascertain the feasibility of encapsulated antimicrobial for the control of Listeria monocytogenes and Escherichia coli O157:H7 growth in a model system, nisin (5.0 and 10.0 µg/mL) and the chelator ethylene diaminetetraacetic acid were entrapped in phospholipid liposomes. While phosphatidylcholine (PC) liposomes did not produce significant inhibition of target pathogens, PC/phosphatidylglycerol 8/2 and 6/4 (mol%) produced significant inhibition of pathogens. Near-complete inhibition of E. coli O157:H7 with liposomal antimicrobials at concentrations below those reported necessary for unencapsulated antimicrobial and chelator suggests that liposomes may represent a powerful technology for the encapsulation of antimicrobials and the control of foodborne pathogens. PRACTICAL APPLICATIONS The activity of many antimicrobials is abolished in many food products for a variety of reasons. Interference and cross-reactions of the antimicrobial and various food constituents, such as protein and fat, are difficult to overcome and often require large amounts of antimicrobial in order to gain significant reductions in the pathogen load in a product. Loss of solubility of some antimicrobials based on pH or ionic strength will negatively affect the antimicrobial potential of a compound like nisin. Liposome encapsulation technologies, such as that reported here, may allow for the maintenance of antimicrobial activity by protecting the antimicrobial against cross-reactions with food components. Additionally, the liposome core represents a microenvironment which can be manipulated by the manufacturer in order to preserve optimal antimicrobial solubility and stability conditions until the time of release. [source]

FLUORESCENCE ACTIVATED CELL SORTING OF TRANSIENTLY TRANSFECTED As4.1 CELLS SHOWS RENIN ENHANCER DIRECTS ON/OFF SWITCHING OF RENIN PROMOTER IN VITRO

CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 4 2008
Brian J MorrisArticle first published online: 27 FEB 200
SUMMARY 1The proximal promoter of the renin gene is weak and its activity is influenced by a strong, far-upstream enhancer. This and the ability of renin expression in renal afferent arteriolar cells to be ,recruited' under chronic stimulation is consistent with the on/off switching (variegation) model of gene expression. If true, this would provide an example in which variegation controls a physiologically regulable gene. 2The present study tested the hypothesis that renin promoter activity may accord with the variegation model, at least in individual juxtaglomerular (mouse As4.1) cells in vitro. 3As4.1 cells were transiently transfected with constructs containing the mouse renin (Ren-1c) enhancer adjacent to the Ren-1c promoter and a linked reporter gene encoding enhanced green fluorescent protein (EGFP). The EGFP signal from individual cells was monitored by fluorescence activated cell sorting. 4In the presence of the renin enhancer there was 10-fold higher EGFP expression in transfected cells compared with cells transfected with EGFP constructs containing the promoter alone. There was, moreover, an 8-fold increase in the number of EGFP expressing cells. However, EGFP expression in individual transfected cells was similar in the presence or absence of the enhancer. 5Results from the in vitro system used suggest that the Ren-1c enhancer does not regulate the rate of promoter activity, but rather increases the probability of achieving an active transcriptional state. Limitations of these findings are discussed. [source]

Viability of Preadipocytes In Vitro: The Influence of Local Anesthetics and pH

DERMATOLOGIC SURGERY, Issue 8 2009
MAIKE KECK MD
BACKGROUND Autogenous fat transfer with lipoinjection for soft tissue augmentation is a commonly used surgical technique. Abundant donor tissue availability and relative ease of harvesting have made autologous fat an attractive soft tissue filler. The overall reliability of this technique is often disputed, and different authors describe different results after autologous fat transplantation despite using similar techniques. In this study, we examined the influence of different local anesthetics commonly used in fat harvest and the pH of the anesthetic solution on the viability of harvested preadipocytes. METHODS AND MATERIALS Preadipocytes were incubated with 1% lidocaine, 1% articaine plus epinephrine 1:200,000, 0.75% ropivacaine, and 1% prilocaine or our standardized tumescent solution (1 L of 0.9% sodium chloride solution plus 25 mL of 1% articaine plus epinephrine 1:200,000 plus 25 mL of bicarbonate) for 30 minutes. Additionally, we incubated cells with the local anesthetics as described above but diluted 1:2 with phosphate buffered saline (pH 7.4). Viability was measured using trypan blue dying as well as propidium iodine staining and fluorescence-activated cell sorting analysis. RESULTS There are significant differences in the viability of preadipocytes under the influence of various local anesthetics. DISCUSSION Our data could partially explain the varying results after autogenous fat transfer. [source]

Influence of the Orifice Inlet Angle on the Velocity Profile Across a Flow Convergence Region by Color Doppler In Vitro

ECHOCARDIOGRAPHY, Issue 5 2000
Martin Giesler M.D.
The converging flow field proximal to a leaking valve is determined among other things by the orifice inlet angle formed by the leaflets. Thus, the inlet angle affects the determination of regurgitant flow rate by the flow convergence method. Based on the hypothesis of spheric isovelocity surfaces, others had postulated that a local velocity within the flow convergence should change inversely proportional to changes in the three-dimensional inlet angle. This concept would allow correction of the determination of regurgitant flow for nonplanar orifice inlet angles. We tested this concept in vitro. In a flow model, the flow convergence region proximal to different orifice plates was imaged by color Doppler: funnel-shaped, planar and tip-shaped (inverted funnels) orifice plates, with circular orifices of 2- and 7-mm diameter. Velocity profiles across the flow convergence along the flow centerline were read from the color maps. As predicted, the local velocities were inversely related to the inlet angle, but only at the 2-mm funnel orifices, this effect was inversely proportional to the three-dimensional inlet angle (i.e., in agreement with the mentioned concept). However, for any 7-mm orifice and/or inlet angle of > 180°, the effect of the inlet angle was considerably less than predicted by the aforementioned concept. With increasing orifice diameter and with decreasing distance to the orifice, the effect of the orifice inlet angle was reduced. The effect of the orifice inlet angle on the flow convergence region is modulated by orifice size and the distance to the orifice. Therefore, correction of flow estimates in proportion to the three-dimensional inlet angle will lead to considerable errors in most situations of clinical relevance, namely to massive overcorrection when analyzing velocities located close to wide orifices. [source]

Epileptiform Activity Induced by Pharmacologic Reduction of M-Current in the Developing Hippocampus in Vitro

EPILEPSIA, Issue 1 2006
Fernando Peña
Summary:,Purpose: Benign familial neonatal convulsions (BFNCs), an inheritable epilepsy that occurs in neonates but not in adults, is caused by hypofunctional mutations in genes codifying for the M-type K+ current. In an attempt to develop an in vitro model of this disease, we tested whether blocking M-current with linopirdine induces epileptiform activity in brain slices from animals of different ages. Methods: Horizontal hippocampus,entorhinal cortex slices were obtained from neonatal (1,2 weeks after birth) and adult (8,9 weeks after birth) rats. Extracellular field recordings of the CA1 region were performed. After recording control conditions, linopirdine was added to the bath, and field activity was recorded continuously for 3 h. 4-Aminopyridine, a drug commonly used to induce epileptiform activity in vitro, was used as a control for our experimental conditions. Results: Bath perfusion of linopirdine induced epileptiform activity only in slices from neonatal rats. Epileptiform activity consisted of interictal-like and ictal-like activity. In slices from adult rats, linopirdine induced erratic interictal-like activity. In contrast, 4-aminopyridine was able to induce epileptiform activity in slices from both neonatal and adult rats. Conclusions: We demonstrated that blockade of M-current in vitro produces epileptiform activity with a developmental pattern similar to that observed in BNFCs. This could be an in vitro model that can be used to study the cellular mechanisms of epileptogenesis and the developmental features of BFNCs, as well as to develop some therapeutic strategies. [source]

Effects of Antiepileptic Drugs on Refractory Seizures in the Intact Immature Corticohippocampal Formation In Vitro

EPILEPSIA, Issue 11 2003
Pascale Paule Quilichini
Summary:,Purpose: We developed a new in vitro preparation of immature rats, in which intact corticohippocampal formations (CHFs) depleted in magnesium ions become progressively epileptic. The better to characterize this model, we examined the effects of 14 antiepileptic drugs (AEDs) currently used in clinical practice. Methods: Recurrent ictal-like seizures (ILEs, four per hour) were generated in intact CHFs of P7,8 rats, and extracellular recordings were performed in the hippocampus and neocortex. AEDs were applied at clinically relevant concentrations (at least two), during 30 min after the third ILE. Their ability to prevent or to delay the next ILE was examined. Results: Valproic acid and benzodiazepines (clobazam and midazolam) but also phenobarbital and levetiracetam prevent the occurrence of seizures. In contrast, usual concentrations of carbamazepine (CBZ), phenytoin, vigabatrin, tiagabine, gabapentin, lamotrigine (LTG), topiramate, felbamate, and ethosuximide did not suppress ILEs. In addition, LTG and CBZ aggravate seizures in one third of the cases. Conclusions: This intact in vitro preparation in immature animals appears to be quite resistant to most AEDs. Blockade of seizures was achieved with drugs acting mainly at the ,-aminobutyric acid (GABA)A -receptor site but not with those that increase the amount of GABA. Drugs with a broad spectrum of activity are efficient but not those preferentially used in partial seizures or absences. We suggest that this preparation may correspond to a model of epilepsy with generalized convulsive seizures and could be helpful to develop new AEDs for refractory infantile epilepsies. [source]

Amiodarone Attenuates Fluoride-induced Hyperkalemia in Vitro

ACADEMIC EMERGENCY MEDICINE, Issue 2 2003
Mark Su MD
Abstract Poisoning by hydrofluoric acid or fluoride salts results in hypocalcemia, hypomagnesemia, and hyperkalemia with subsequent cardiac dysrhythmias. In previous studies, quinidine attenuated fluoride-induced hyperkalemia in vitro, and enhanced survival in animals. Like quinidine, amiodarone is a potassium channel blocker, although amiodarone is more familiar to clinicians due to its recent inclusion in advanced cardiac life support (ACLS) protocols. Objectives: This in-vitro study of human erythrocytes was designed to determine whether amiodarone could attenuate fluoride-induced hyperkalemia. Methods: Six healthy volunteers each donated 60 mL of blood on three occasions. Each specimen was divided into 12 tubes, incubated at 37°C, and oxygenated with room air. An aqueous sodium fluoride (F,) solution was added to tubes 1,9. Incremental amounts of quinidine were added to tubes 1,4 (Q1,Q4) to attain calculated concentrations of 0.73 ,g/mL, 1.45 ,g/mL, 2.9 ,g/mL, and 5.8 ,g/mL, respectively. Incremental amounts of amiodarone were added to tubes 5,8 (A1,A4) to attain calculated concentrations of 0.38 ,g/mL, 0.75 ,g/mL, 1.5 ,g/mL, and 3.0 ,g/mL, respectively. Tubes 9,12 were controls for each of F,, amiodarone, quinidine alone, and no additive, respectively. Extracellular potassium concentration ([K+]) was followed, and an objective endpoint was defined as the rise in potassium concentration at 6 hours. Results: Fluoride produced a significant change in [K+] by 6 hours in all samples. Quinidine produced a J-shaped curve in its ability to attenuate the rise in [K+], with only one concentration, Q3, demonstrating significance versus tube 9 (control). Amiodarone also demonstrated a J-shaped dose,response effect, with statistical significance at A1, A2, and A3 versus tube 9 (control). There was no significant difference among the effective concentrations (Q3, A1, A2, and A3) of both drugs. Conclusions: In this in-vitro model using human blood, amiodarone and quinidine both attenuated F, -induced hyperkalemia. Further study is indicated to determine whether amiodarone enhances survival in F, -poisoned animals. [source]

Fibronectin Functionalized Hydroxyapatite Coatings: Improving Dermal Fibroblast Adhesion In Vitro and In Vivo,

ADVANCED ENGINEERING MATERIALS, Issue 8 2010
Catherine J. Pendegrass
Skin-penetrating devices including intraosseous transcutaneous amputation prostheses (ITAP) and external fixator pins rely on a skin-implant seal to prevent infection. In this study, we assess the effectiveness of fibronectin (Fn) functionalized hydroxyapatite (HA) coatings for promoting dermal fibroblast and dermal tissue attachment and ingrowth in vitro and in vivo. By measuring the number of focal adhesions per unit cell area we have demonstrated that HA significantly promotes dermal fibroblast attachment compared with titanium alloy. Dermal fibroblast attachment is promoted further using Fn functionalized HA coatings incorporated into an implant design with 700,µm pores, which significantly increased dermal tissue ingrowth and attachment compared with non-functionalized HA and titanium alloy controls incorporating 500 or 1000,µm pores. We postulate that Fn functionalized HA coatings applied to transdermal implants may promote and sustain the skin-implant interface and assist in preventing infection long term. [source]

Influence of TiO2 Nanoparticles Incorporated into Elastomeric Polyesters on their Biocompatibility In Vitro and In Vivo

ADVANCED ENGINEERING MATERIALS, Issue 11 2009
Miroslawa El-Fray
Abstract Fibroblasts proliferation and apoptosis as well as tissue response after implantation of elastomers containing nanocrystalline TiO2 were investigated in the present in vitro and in vivo study. Materials investigated were soft poly(aliphatic/aromatic-ester) multiblock thermoplastic elastomers with poly(ethylene terephthalate) (PET) hard segments and dimerized linoleic acid (DLA) soft segments, respectively, containing 0.2,wt% TiO2 nanoparticles. An investigation of the influence of TiO2 nanoparticles incorporated into polymeric material on in vitro biocompatibility revealed enhanced cell proliferation and diminished number of necrotic and apoptotic cells as compared to nanoparticles-free polymer. Implantation tests indicated that the observed tissue changes were similar to those observed with medical-grade silicone elastomer, no evidence of contact necrosis being observed. The unchanged morphology of rat liver hepatocytes and the lack of parenchymal necrosis also indicated that exposure to the material containing TiO2 nanoparticles, did not cause any cytotoxic reactions. The present study, thus, showed that elastomeric polyester containing TiO2 nanoparticles are interesting biomimetic constructs for improved tissue regeneration. [source]

Production of oxalates In Vitro by Microbes Isolated from Rock Surfaces with prehistoric paints in the Lower Pecos Region, Texas

GEOARCHAEOLOGY: AN INTERNATIONAL JOURNAL, Issue 1 2008
Darren Hess
Calcium oxalate-rich rock coatings are ubiquitous on limestone inside dry rock shelters and under bluff overhangs along canyon walls in southwestern Texas. Prehistoric pictographs occur in more than 250 such sites, and the ancient paints are encapsulated within the natural rock coating. Previous studies suggest lichens were the source of the oxalate; however, we report here that microbes cultured and isolated from samples of the coating produce oxalate in vitro. Twenty different bacteria species have been identified in samples from eight different sites, with Bacillus the most common genus, represented by five species. HPLC analyses of inoculated R2B medium after eight months of bacterial growth revealed the presence of oxalate ions in the solid phase of the growth medium. © 2008 Wiley Periodicals, Inc. [source]

Controlled Degradability of Polysaccharide Multilayer Films In Vitro and In Vivo,

ADVANCED FUNCTIONAL MATERIALS, Issue 11 2005
C. Picart
Abstract This article demonstrates the possibility of tuning the degradability of polysaccharide multilayer films in vitro and in vivo. Chitosan and hyaluronan multilayer films (CHI/HA) were either native or crosslinked using a water soluble carbodiimide, 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) at various concentrations in combination with N-hydroxysulfosuccinimide. The in-vitro degradation of the films in contact with lysozyme and hyaluronidase was followed by quartz crystal microbalance measurements, fluorimetry, and confocal laser scanning microscopy after labeling of the chitosan with fluorescein isothiocyanate (CHIFITC). The native films were subjected to degradation by these enzymes, and the crosslinked films were more resistant to enzymatic degradation. Films made of chitosan of medium molecular weight were more resistant than films made of chitosan-oligosaccharides. The films were also brought in contact with plasma, which induced a change in film structure for the native film but did not have any effect on the crosslinked film. The in-vitro study shows that macrophages can degrade all types of films and internalize the chitosan. The in-vivo degradation of the films implanted in mouse peritoneal cavity for a week again showed an almost complete degradation of the native films, whereas the crosslinked films were only partially degraded. Taken together, these results suggest that polysaccharide multilayer films are of potential interest for in-vivo applications as biodegradable coatings, and that degradation can be tuned by using chitosan of different molecular weights and by controlling film crosslinking. [source]

In Vitro and In Vivo Complementation of the Helicobacter pylori Arginase Mutant Using an Intergenic Chromosomal Site

HELICOBACTER, Issue 5 2006
Melanie L. Langford
Abstract Background:, Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. Materials and methods:, A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203,hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). Results:, A rocF mutant unable to hydrolyze or consume l -arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3, ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. Conclusions:, This complementation strategy should greatly facilitate genetic experiments in H. pylori. [source]

Analysis of Volatile Compounds Emitted by the Helicobacter pylori Reference Strain NCTC 11637 In Vitro

HELICOBACTER, Issue 1 2006
Matthias Lechner
No abstract is available for this article. [source]

HCV796: A selective nonstructural protein 5B polymerase inhibitor with potent anti-hepatitis C virus activity In Vitro, in mice with chimeric human livers, and in humans infected with hepatitis C virus,

HEPATOLOGY, Issue 3 2009
Norman M. Kneteman
Anti-hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration (IC50) values of 0.01 to 0.14 ,M for genotype 1, with half maximal effective concentration (EC50s) of 5 nM and 9 nM against genotype 1a and 1b replicons. In the chimeric mouse model, a 2.02 ± 0.55 log reduction in HCV titer was seen with monotherapy, whereas a suboptimal dose of 30 mg/kg three times per day in combination with interferon demonstrated a 2.44 log reduction (P = 0.001 versus interferon alone) Clinical outcomes in combination with pegylated interferon and ribavirin have revealed additive efficacy in treatment naïve patients. Abnormal liver function test results were observed in 8% of HCV-796 patients treated for over 8 weeks, resulting in suspension of further trial activity. Conclusion: The RNA-dependent RNA polymerase inhibitor HCV796 demonstrated potent anti-HCV activity consistently through enzyme inhibition assays, subgenomic replicon, and chimeric mouse studies. Strong correlations of outcomes in the mouse model were seen with subsequent clinical trials, including a plateau in dose-related antiviral activity and additive impact from combination therapy with interferon. These outcomes demonstrate the utility of the range of in vitro and in vivo models now available for anti-HCV drug development and support the potential utility of polymerase inhibitors in future combination therapies for HCV treatment. (HEPATOLOGY 2009.) [source]

A new strategy for studying In Vitro the drug susceptibility of clinical isolates of human hepatitis B virus

HEPATOLOGY, Issue 4 2004
David Durantel
Resistance of hepatitis B virus (HBV) to antivirals has become a major clinical problem. Our objective was to develop a new method for the cloning of naturally occurring HBV genomes and a phenotypic assay capable of assessing HBV drug susceptibility and DNA synthesis capacity in vitro. Viral DNA was extracted from sera and was amplified by polymerase chain reaction, and amplicons were cloned into vectors that enable, after cell transfection, the initiation of the intracellular HBV replication cycle. Single or multiple clones were used to transfect Huh7 cells. The viral DNA synthesis capacity and drug susceptibility were determined by measuring the level of intracellular DNA intermediate, synthesized in absence or presence of antiviral, using Southern blot analysis. We have developed, calibrated, then used this phenotypic assay to determine the drug susceptibility of HBV quasispecies isolated throughout the course of therapy from patients selected according to their mutation profile. A multiclonal and longitudinal analysis enabled us to measure the variation of drug susceptibility of different viral quasispecies by comparison of IC50/IC90s with standards. The presence of famciclovir- or lamivudine-induced mutations in the viral population caused a change in viral DNA synthesis capacity and drug susceptibility in vitro, demonstrating the clinical relevance of the assay. In conclusion, our phenotypic assay enables the in vitro characterization of DNA synthesis capacity and drug susceptibility of HBV quasispecies isolated from patients. This assay should allow a better monitoring of patients undergoing antiviral therapy, as well as the screening of novel drugs on emerging resistant strains. (Hepatology 2004;40:855,864). [source]

The Effect of NaF In Vitro on the Mechanical and Material Properties of Trabecular and Cortical Bone

ADVANCED MATERIALS, Issue 4 2009
Philipp J. Thurner
High doses of sodium fluoride in bones lead to severe softening, by weakening interfacial properties between the inorganic minerals and the organic components, while leaving mineralization unchanged. This leads to reduction of microdamage and associated stress-whitening pointing to a change in failure mode. Accordingly, elastic modulus, failure stress, and indentation-distance increase are decreased, whereas failure strain is increased. [source]

Bone Morphogenetic Protein 2 Induces Cyclo-oxygenase 2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of Bone Morphogenetic Protein 2 In Vitro and In Vivo

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2005
Daichi Chikazu
Abstract We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfa1) consensus sequence (5,-AACCACA-3,) at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2+/+ mice but not in cells from COX-2,/, mice. In vivo, BMP-2 (10 ,g/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (,CT), was decreased by 78% in COX-2,/, mice compared with COX-2+/+ mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfa1 binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo. [source]

Strategies for Directing the Differentiation of Stem Cells Into the Osteogenic Lineage In Vitro,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2004
Boon Chin Heng
Abstract A major area in regenerative medicine is the application of stem cells in bone reconstruction and bone tissue engineering. This will require well-defined and efficient protocols for directing the differentiation of stem cells into the osteogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages on transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying osteogenesis and bone development, and facilitate the genetic manipulation of stem cells for therapeutic applications. The development of pharmokinetic and cytotoxicity/genotoxicity screening tests for bone-related biomaterials and drugs could also use protocols developed for the osteogenic differentiation of stem cells. This review critically examines the various strategies that could be used to direct the differentiation of stem cells into the osteogenic lineage in vitro. [source]

Effects of Secreted Frizzled-Related Protein 3 on Osteoblasts In Vitro,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2004
Yoon-Sok Chung
Abstract To examine if sFRP3s act as decoy receptors for Wnt, we examined the effects of recombinant sFRP3 on mouse osteoblast proliferation and differentiation. We found that sFRP3 unexpectedly increased osteoblast differentiation, suggesting it may act through other mechanisms besides acting as a decoy receptor for Wnt's. Introduction: Secreted frizzled-related proteins (sFRPs) are a truncated form of frizzled receptor, missing both the transmembrane and cytosolic domains. Because previous studies have shown that sFRPs bind and act as decoy receptors for Wnt proteins that promote osteoblast differentiation, we postulated that sFRP3 acts as an inhibitor of osteoblast differentiation. Materials and Methods: We examined the effects of mouse recombinant sFRP3 and/or Wnt-3A on cell proliferation and differentiation using MC3T3-E1 mouse osteoblasts and primary cultures of mouse bone marrow stromal cells. We evaluated the effects of sFRP3 on ,-catenin levels using Western immunoblot analyses. Results: We found that sFRP3 suppressed osteoblast cell number in a dose-dependent manner that was the result of a decrease in proliferation and not because of an increase in apoptosis. Surprisingly, sFRP3 increased osteoblast differentiation, which could not be explained based on sFRP3 acting as a decoy receptor for stimulatory Wnt's. Furthermore, sFRP3 did not inhibit Wnt3A-induced increase in alkaline phosphatase (ALP) activity. Wnt3A, but not sFRP3 treatment, increased cellular ,-catenin levels, and sFRP3 failed to block Wnt3A-induced increase in cellular ,-catenin levels. Treatment with endostatin, an agent known to degrade ,-catenin, did not inhibit sFRP3-induced increase in ALP activity. sFRP1, like sFRP3, inhibited proliferation and stimulated ALP activity in MC3T3-E1 mouse osteoblasts. Conclusions: Based on our findings, we conclude that sFRP3 decreased osteoblast proliferation and unexpectedly increased parameters of osteoblast differentiation. Based on our findings, we propose that sFRP3 may stimulate differentiation through a ,-catenin-independent pathway in addition to its previously known function as a decoy receptor for Wnt's. [source]

Cytokines, Osteoprotegerin, and RANKL In Vitro and Histomorphometric Indices of Bone Turnover in Patients With Different Bone Diseases,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2003
Heide Siggelkow
Abstract Cytokines are supposed to play an essential role in the regulation of the bone metabolic unit. However, information on cytokine production of primary human osteoblasts from patients with metabolic bone disease is scarce, and few attempts have been made to correlate such data to histomorphometric parameters of individual patients. We investigated 11 patients with metabolic bone disease referred to our outpatient department for bone biopsy and analyzed interleukin (IL)-1, IL-6, and TNF-, protein release and gene expression in primary osteoblast cultures. Compared with four controls, five patients showed normal cytokine protein release, whereas six patients showed much higher levels of interleukin-6 (26-fold) and TNF-, (84-fold). All three cytokines were strongly correlated concerning gene expression and/or protein levels (r = 0.72,0.96). Histomorphometric analysis of the bone samples showed that eroded surface (ES/BS) as a parameter of bone resorption was significantly associated with TNF-,. In addition, RANKL gene expression was positively associated with ES/BS and osteoclast surface (Oc.S/BS). Finally, the formation parameters osteoid volume and osteoid surface were negatively associated with TNF-,. In conclusion, in an in vitro-ex vivo model of bone cells obtained from a group of 11 patients with different forms of metabolic bone disease, cytokine release in conditioned medium was significantly associated with bone resorption and bone formation, as quantified by histomorphometry. TNF-, seemed to be the more important cytokine; its effect on bone resorption could be mediated by RANKL. [source]

Strong Static Magnetic Field Stimulates Bone Formation to a Definite Orientation In Vitro and In Vivo,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2002
Hiroko Kotani Ph.D.
Abstract The induction of bone formation to an intentional orientation is a potentially viable clinical treatment for bone disorders. Among the many chemical and physical factors, a static magnetic field (SMF) of tesla order can regulate the shapes of blood cells and matrix fibers. This study investigated the effects of a strong SMF (8 T) on bone formation in both in vivo and in vitro systems. After 60 h of exposure to the SMF, cultured mouse osteoblastic MC3T3-E1 cells were transformed to rodlike shapes and were orientated in the direction parallel to the magnetic field. Although this strong SMF exposure did not affect cell proliferation, it up-regulated cell differentiation and matrix synthesis as determined by ALP and alizarin red stainings, respectively. The SMF also stimulated ectopic bone formation in and around subcutaneously implanted bone morphogenetic protein (BMP) 2-containing pellets in mice, in which the orientation of bone formation was parallel to the magnetic field. It is concluded that a strong SMF has the potency not only to stimulate bone formation, but also to regulate its orientation in both in vitro and in vivo models. This is the first study to show the regulation of the orientation of adherent cells by a magnetic field. We propose that the combination of a strong SMF and a potent osteogenic agent such as BMP possibly may lead to an effective treatment of bone fractures and defects. [source]

Midregion Parathyroid Hormone-Related Protein Inhibits Growth and Invasion In Vitro and Tumorigenesis In Vivo of Human Breast Cancer Cells

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2001
Claudio Luparello
Abstract Parathyroid hormone-related protein (PTHrP) is critical for normal mammary development and is overexpressed by breast cancers. PTHrP is a peptide hormone that undergoes extensive post-translational processing, and PTHrP(38,94)-amide is one of the mature secretory forms of the peptide. In this study, we explored the effect of PTHrP(38,94)-amide in a panel of six breast cancer cell lines "in vitro" and in MDA-MB231 cells "in vivo" specifically examining cell viability, proliferation, invasiveness, and growth in nude mice. PTHrP(38,94)-amide markedly inhibited proliferation and also caused striking toxicity and accelerated cell death in breast cancer cells. In addition, direct injection of PTHrP(38,94)-amide into MDA-MB231 breast cancer cells passaged in immunodeficient mice produced a marked reduction in tumor growth. These studies (i) indicate breast cancer cells are one of the few tissues in which specific effects of midregion PTHrP have been established to date, (ii) support a role for midregion secretory forms of PTHrP in modulating not only normal but also pathological mammary growth and differentiation, (iii) add further evidence for the existence of a specific midregion PTHrP receptor, and (iv) provide a novel molecule for modeling of small molecule analogues that may have anti-breast cancer effects. [source]

Indapamide, a Thiazide-Like Diuretic, Decreases Bone Resorption In Vitro

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2001
Agnes Lalande
Abstract We recently showed that indapamide (IDP), a thiazide-related diuretic, increases bone mass and decreases bone resorption in spontaneously hypertensive rats supplemented with sodium. In the present study, we evaluated the in vitro effects of this diuretic on bone cells, as well as those of hydrochlorothiazide (HCTZ), the reference thiazide, and acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor. We showed that 10,4 M IDP and 10,4 M AZ, as well as 10,5 M pamidronate (APD), decreased bone resorption in organ cultures and in cocultures of osteoblast-like cells and bone marrow cells in the presence of 10,8 M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We investigated the mechanism of this antiresorptive effect of IDP; IDP decreased osteoclast differentiation as the number of osteoclasts developing in coculture of marrow and osteoblast-like cells was decreased markedly. We then investigated whether IDP affected osteoblast-like cells because these cells are involved in the osteoclast differentiation. Indeed, IDP increased osteoblast-like cell proliferation and alkaline phosphatase (ALP) expression. Nevertheless, it did not modify the colony-stimulating factor 1 (CSF-1) production by these cells. In addition, osteoblast-like cells expressed the Na+/Cl, cotransporter that is necessary for the renal action of thiazide diuretics, but IDP inhibited bone resorption in mice lacking this cotransporter, so the inhibition of bone resorption and osteoclast differentiation did not involve this pathway. Thus, we hypothesized that IDP may act directly on cells of the osteoclast lineage. We observed that resorption pits produced by spleen cells cultured in the presence of soluble osteoclast differentiation factor (sODF) and CSF-1 were decreased by 10,4 M IDP as well as 10,5 M APD. In conclusion, in vitro IDP increased osteoblast proliferation and decreased bone resorption at least in part by decreasing osteoclast differentiation via a direct effect on hematopoietic precursors. [source]

Role of the Latent Transforming Growth Factor ,,Binding Protein 1 in Fibrillin-Containing Microfibrils in Bone Cells In Vitro and In Vivo

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2000
Sarah L. Dallas
Abstract Latent transforming growth factor ,,binding proteins (LTBPs) are extracellular matrix (ECM) proteins that bind latent transforming growth factor , (TGF-,) and influence its availability in bone and other connective tissues. LTBPs have homology with fibrillins and may have related functions as microfibrillar proteins. However, at present little is known about their structural arrangement in the ECM. By using antibodies against purified LTBP1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 constructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibrillar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-nm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early colocalization of LTBP1 with fibronectin suggested a role for fibronectin in the initial assembly of LTBP1 into the matrix; however, in more differentiated osteoblast cultures, LTBP1 and fibronectin 1 were found in distinct fibrillar networks. Overexpression of LTBP1 deletion constructs in osteoblast-like cells showed that N-terminal amino acids 67,467 were sufficient for incorporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation. In embryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone. However, LTBP1-positive fibrils, which did not contain fibrillin 1, were present in cartilage matrix. These studies show that in addition to regulating TGF,1, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded. [source]

Spontaneous Pulmonary Vein Firing in Man: Relationship to Tachycardia-Pause Early Afterdepolarizations and Triggered Arrhythmia in Canine Pulmonary Veins In Vitro

JOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 10 2007
EUGENE PATTERSON Ph.D.
Introduction: Rapid firing originating within pulmonary veins (PVs) initiates atrial fibrillation (AF). The following studies were performed to evaluate spontaneous PV firing in patients with AF to distinguish focal versus reentrant mechanisms. Methods: Intracardiac recordings were obtained in 18 patients demonstrating paroxysmal AF. Microelectrode (ME) recordings were obtained from superfused canine PV sleeves (N = 48). Results: Spontaneous PV firing (566 ± 16 bpm; 127 ± 6 ms cycle length) giving rise to AF (52 episodes) was observed. Tachycardia-pause initiation was present in 132 of 200 episodes of rapid PV firing and 34 of 52 AF episodes. The pause cycle length preceding PV firing was 1,039 ± 86 ms following tachycardia (420 ± 40 ms cycle length). The remaining episodes were initiated following a 702 ± 32 ms pause during sinus rhythm (588 ± 63 ms). Spontaneous firing recorded with a multipolar mapping catheter did not detect electrical activity bridging the diastolic interval between the initial ectopic and preceding post-pause sinus beat. Tachycardia-pause initiated PV firing (138 ± 7 ms coupling interval) in patients correlated with tachycardia-pause enhanced isometric force, early afterdepolarization (EAD) amplitude, and triggered firing within canine PVs. Rapid firing (1,172 ± 134 bpm; 51 ± 8 ms cycle length) following an abbreviated coupling interval (69 ± 12 ms) was initiated in 13 of 18 canine PVs following tachycardia-pause pacing during norepinephrine + acetylcholine superfusion. Stimulation selectively activating local autonomic nerve terminals facilitated tachycardia-pause triggered firing in canine PVs (5 of 15 vs 0 of 15; P < 0.05). Conclusions: The studies demonstrate (1) tachycardia-pause initiation of rapid, short-coupled PV firing in AF patients and (2) tachycardia-pause facilitation of isometric force, EAD formation, and autonomic-dependent triggered firing within canine PVs, suggestive of a common arrhythmia mechanism. [source]

Spectrophotometric Analysis of Tooth Color Reproduction on Anterior All-Ceramic Crowns: Part 2: Color Reproduction and Its Transfer from In Vitro to In Vivo

JOURNAL OF ESTHETIC AND RESTORATIVE DENTISTRY, Issue 1 2010
AKI YOSHIDA RDT
ABSTRACT Color reproduction of an anterior tooth requires advanced laboratory techniques, talent, and artistic skills. Color matching in a laboratory requires the successful transfer from in vivo with careful considerations. The purpose of this study was to monitor and verify the color reproduction process for an anterior all-ceramic crown in a laboratory through spectrophotometric measurements. Furthermore, a crown insertion process using composite luting cements was assessed, and the final color match was measured and confirmed. An all-ceramic crown with a zirconia ceramic coping for the maxillary right central incisor was fabricated. There was a significant color difference between the prepared tooth and the die material. The die material selected was the closest match available. The ceramic coping filled with die material indicated a large color difference from the target tooth in both lightness and chromaticity. During the first bake, three different approaches were intentionally used corresponding with three different tooth regions (cervical, body, and incisal). The first bake created the fundamental color of the crown that allowed some color shifts in the enamel layer, which was added later. The color of the completed crown demonstrated an excellent color match, with ,E 1.27 in the incisal and 1.71 in the body. In the cervical area, color match with ,E 2.37 was fabricated with the expectation of a color effect from the underlying prepared tooth. The optimal use of composite luting cement adjusted the effect from the underlying prepared tooth color, and the color match fabricated at a laboratory was successfully transferred to the clinical setting. The precise color measurement system leads to an accurate verification of color reproduction and its transfer. CLINICAL SIGNIFICANCE The use of a dedicated dental spectrophotometer during the fabrication of an all-ceramic crown allows the dentist and the laboratory technician to accurately communicate important information to one another about the shade of the tooth preparation, the shade of the contralateral target tooth, and the influence of luting cement on the final restoration, thereby allowing the technician better control over the outcome of their tooth color matching efforts and the final color match of an all-ceramic restoration. (J Esthet Restor Dent 22:53,65, 2010) [source]

Antiangiogenetic Effects of 4 Varieties of Grapes,In Vitro

JOURNAL OF FOOD SCIENCE, Issue 6 2010
Ming Liu
Abstract:, The purpose of this study was to investigate the inhibitory effects of grapes on the human umbilical vein endothelial (HUVE) cells' capillary tube formation and matrix metalloproteinase-2 (MMP-2) expression secreted into the medium. Four different grape varieties (Concord, Niagara, Chardonnay, and Pinot Noir) were extracted using 80% acetone and the extracts were stored at ,80 °C. The total amount of phenolics and flavonoids for each of the 4 grape varieties were determined by spectrophotometry. Grape extracts were co-cultured with HUVE cells on Matrigel and inhibitory effects on tube formation were observed under a microscope. The inhibitory effects of grape extracts on MMP-2 expression were examined by zymogram. All 4 grape varieties inhibited the tube formation of HUVE cells in a dose-dependent manner on Matrigel. Except for Chardonnay, the other 3 grape varieties completely inhibited secretion of MMP-2 at 20 mg/mL. There was a significant positive relationship between the total phenolics and flavonoids and antiangiogenetic activities. The grapes tested have the potential to inhibit angiogenesis mainly by their phenolics and flavonoids contents, which partly contribute to their cancer chemopreventive efficacy. [source]

In Vitro,Potential of,Ascophyllum nodosum,Phenolic Antioxidant-Mediated ,-Glucosidase and ,-Amylase Inhibition

JOURNAL OF FOOD SCIENCE, Issue 3 2010
E. Apostolidis
ABSTRACT:,Ascophyllum nodosum,is a brown seaweed that grows abundantly in the Northeast coastal region. In this study, the potential of,A. nodosum,for type 2 diabetes management through antioxidant-mediated ,-glucosidase and ,-amylase inhibition was investigated. After the initial screening of 4 locally harvested seaweeds,,A. nodosum,was chosen for its highest phenolic content and was subjected to water extraction. Among extraction ratios of 50 g to 100 to 1000 mL at room temperature, 50 g/400 mL yielded the highest phenolic content of 4.5 mg/g wet weight. For evaluation of extraction temperature ranging from 20 to 80 °C, 50 g/400 mL was chosen as a minimum amount of extractant. Among temperatures studied, extraction at 80 °C resulted in the highest total phenolic contents (4.2 mg/g wet weight). All extracts had similar levels of antioxidant activity in the range of 60% to 70% in terms of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. The 80 °C extract had the highest ,-glucosidase and ,-amylase inhibitory activity with IC50 of 0.24 and 1.34 ,g phenolics, respectively, compared to the IC50 of acarbose, reference inhibitor, being 0.37 and 0.68 ,g. The results show that fresh,A. nodosum,has strong ,-glucosidase and mild ,-amylase inhibitory activities that correlated with phenolic contents. This study suggests a nutraceutical potential of,A. nodosum,based on phytochemical antioxidant and antihyperglycemia activities. [source]