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Vitro Models (vitro + models)
Selected AbstractsEVALUATION OF RED CURRANTS (RIBES RUBRUM L.), BLACK CURRANTS (RIBES NIGRUM L.), RED AND GREEN GOOSEBERRIES (RIBES UVA-CRISPA) FOR POTENTIAL MANAGEMENT OF TYPE 2 DIABETES AND HYPERTENSION USING IN VITRO MODELSJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2010MARCIA DA SILVA PINTO ABSTRACT Red currants (Ribes rubrum L.), black currants (Ribes nigrum L.), red and green gooseberries (Ribes uva-crispa) were evaluated for the total phenolics, antioxidant capacity based on 2, 2-diphenyl-1-picrylhydrazyl radical scavenging assay and functionality such as in vitro inhibition of ,-amylase, ,-glucosidase and angiotensin I-converting enzyme (ACE) relevant for potential management of hyperglycemia and hypertension. The total phenolics content ranged from 3.2 (green gooseberries) to 13.5 (black currants) mg/g fruit fresh weight. No correlation was found between total phenolics and antioxidant activity. The major phenolic compounds were quercetin derivatives (black currants and green gooseberries) and chlorogenic acid (red currants and red gooseberries). Red currants had the highest ,-glucosidase, ,-amylase and ACE inhibitory activities. Therefore red currants could be good dietary sources with potential antidiabetes and antihypertension functionality to compliment overall dietary management of early stages of type 2 diabetes. [source] HEALTH BENEFITS OF APPLE PHENOLICS FROM POSTHARVEST STAGES FOR POTENTIAL TYPE 2 DIABETES MANAGEMENT USING IN VITRO MODELSJOURNAL OF FOOD BIOCHEMISTRY, Issue 1 2010I. ADYANTHAYA ABSTRACT An increasing number of studies indicate that regular intake of fruits and vegetables have clear links to reduced risk of chronic diseases like diabetes and cardiovascular disease. The beneficial effects in many cases have been attributed to the phenolic and antioxidant content of the fruits and vegetables. Apples are a major source of fiber and contain good dietary phenolics with antioxidant function. Previous epidemiological studies have indicated that intake of apples reduces the risk of developing type 2 diabetes. Our studies indicate that this reduced risk is potentially because of the modulation of postprandial glucose increase by phenolics present in apples via inhibition of, -glucosidase. Phenolic content was evaluated during 3 months of postharvest storage of four varieties of apples and results indicated positive linkage to enhanced postharvest preservation and, -glucosidase inhibition. These in vitro results along with existing epidemiological studies provide strong biochemical rationale for further animal or human clinical studies. PRACTICAL APPLICATIONS The understanding of phenolic-linked antioxidant enzyme responses during postharvest storage of apples has implications for using the same phenolic functional ingredients toward health benefits such as ,-glucosidase inhibition linked to glycemic index control associated with type 2 diabetes. Therefore strategies to understand phenolic-linked postharvest preservation and natural treatments to extend this preservation in selected varieties, such as McIntosh and Cortland in this study, can be basis for food ingredient design for health benefits. These strategies can then be extended to prolong postharvest preservation and enhance phenolic linked human health benefits of a wide variety of fruits and vegetables. [source] Validity and ethics of the human 4-h patch test as an alternative method to assess acute skin irritation potentialCONTACT DERMATITIS, Issue 1 2001Michael K. Robinson For more than 50 years, the Draize rabbit skin irritation test has reigned supreme as the regulatory method of choice for the identification of skin irritant chemicals. To date no in vitro alternative test has been validated as an adequate replacement. However, one potential option, to test the endpoint of concern (skin irritation) in the species of concern (man) has been overlooked. The advent of predictive in vitro tools for the identification of substances corrosive to the skin has opened up the practical possibility of carrying out safe and ethical studies on small panels of humans. The human 4-h patch test has been developed to meet the needs of identifying chemical skin irritation potential, providing data which is inherently superior to that given by a surrogate model, such as the rabbit. This paper reviews in detail the present state of the human 4-h patch test, highlighting its advantages and noting its utility as the ,gold standard' on which to build future in vitro models. [source] The role of autophagy in , -cell lipotoxicity and type 2 diabetesDIABETES OBESITY & METABOLISM, Issue 2010G. Las Autophagy, a ubiquitous catabolic pathway involved in both cell survival and cell death, has been implicated in many age-associated diseases. Recent findings have shown autophagy to be crucial for proper insulin secretion and , -cell viability. Transgenic mice lacking autophagy in their , -cells showed decreased , -cell mass and suppressed glucose-stimulated insulin secretion. Several studies showed that stress can stimulate autophagy in , -cells: the number of autophagosomes is increased in different in vivo models for diabetes, such as db/db mice, mice fed high-fat diet, pdx-1 knockout mice, as well as in in vitro models of glucotoxicity and lipotoxicity. Pharmacological and molecular inhibition of autophagy increases the susceptibility to cell stress, suggesting that autophagy protects against diabetes-relevant stresses. Recent findings, however, question these conclusions. Pancreases of diabetics and , -cells exposed to fatty acids show accumulation of abnormal autophagosome morphology and suppression of lysosomal gene expression suggesting impairment in autophagic turnover. In this review we attempt to give an overview of the data generated by others and by us in view of the possible role of autophagy in diabetes, a role which depending on the conditions, could be beneficial or detrimental in coping with stress. [source] Are CB1 receptor antagonists nootropic or cognitive impairing agents?DRUG DEVELOPMENT RESEARCH, Issue 8 2009Stephen A. Varvel Abstract For more than a decade, a considerable amount of research has examined the effects of rimonabant (SR 141716) and other CB1 receptor antagonists in both in vivo and in vitro models of learning and memory. In addition to its utility in determining whether the effects of drugs are mediated though a CB1 receptor mechanism of action, these antagonists are useful in providing insight into the physiological function of the endogenous cannabinoid system. Several groups have reported that CB1 receptor antagonists enhance memory duration in a variety of spatial and operant paradigms, but not in all paradigms. Conversely, disruption of CB1 receptor signaling also impairs extinction learning in which the animal actively suppresses a learned response when reinforcement has been withheld. These extinction deficits occur in aversively motivated tasks, such as in fear conditioning or escape behavior in the Morris water maze task, but not in appetitively motivated tasks. Similarly, in electrophysiological models, CB1 receptor antagonists elicit a variety of effects, including enhancement of long-term potentiation (LTP), while disrupting long-term depression (LTD) and interfering with transient forms of plasticity, including depolarization-induced suppression of inhibition (DSI) and depolarization-induced suppression of excitation (DSE). The collective results of the in vivo and in vitro studies employing CB1 receptor antagonists, demonstrate that these receptors play integral roles in different components of cognitive processing. Functionally, pharmacological blockade of CB1 receptors may strengthen memory duration, but interferes with extinction of learned behaviors that are associated with traumatic or aversive memories. Drug Dev Res 70:555,565, 2009. © 2009 Wiley-Liss, Inc. [source] Benzo[a]pyrene bioavailability from pristine soil and contaminated sediment assessed using two in vitro modelsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2007Luba Vasiluk Abstract A major route of exposure to hydrophobic organic contaminants (HOCs), such as benzo[a]pyrene (BaP), is ingestion. Matrix-bound HOCs may become bioavailable after mobilization by the gastrointestinal fluids followed by sorption to the intestinal epithelium. The purpose of this research was to measure the bioavailability of [14C]-BaP bound to pristine soils or field-contaminated sediment using an in vitro model of gastrointestinal digestion followed by sorption to human enterocytes (Caco-2 cells) or to a surrogate membrane, ethylene vinyl acetate (EVA) thin film. Although Caco-2 cells had a twofold higher lipid-normalized fugacity capacity than EVA, [14C]-BaP uptake by Caco-2 lipids and EVA thin film demonstrated a linear relationship within the range of BaP concentrations tested. These results suggest that EVA thin film is a good membrane surrogate for passive uptake of BaP. The in vitro system provided enough sensitivity to detect matrix effects on bioavailability; after 5 h, significantly lower concentrations of [14C]-BaP were sorbed into Caco-2 cells from soil containing a higher percentage of organic matter compared to soil with a lower percentage of organic matter. The [14C]-BaP desorption rate from Caco-2 lipids consistently was twofold higher than from EVA thin film for all matrices tested. The more rapid kinetics observed with Caco-2 cells probably were due to the greater surface area available for absorption/desorption in the cells. After 5 h, the uptake of BaP into Caco-2 lipid was similar in live and metabolically inert Caco-2 cells, suggesting that the primary route of BaP uptake is by passive diffusion. Moreover, the driving force for uptake is the fugacity gradient that exists between the gastrointestinal fluid and the membrane. [source] Polycyclic aromatic hydrocarbons as inducers of cytochrome P4501A enzyme activity in the rainbow trout liver cell line, RTL-W1, and in primary cultures of rainbow trout hepatocytesENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2001Anja Behrens Abstract In order to investigate cell-specific differences in the response of in vitro models to environmental toxicants, we compared the capacity of nine polycyclic aromatic hydrocarbons (PAHs) to induce cytochrome P4501A (CYP1A) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes and a rainbow trout liver cell line, RTL-W1. Induction of CYP1A was estimated from the catalytic activity of 7-ethoxyresorufin- O -deethylase (EROD) and compared by median effective concentration (EC50) values, induction spans, and benzo[a]pyrene induction equivalency factors for inducing PAHs. The influence of culture conditions was investigated with respect to the presence or absence of serum and varying exposure times. Both in vitro systems lead to an identical classification of the PAHs in noninducing (anthracene, fluoranthene, phenanthrene, and pyrene) and inducing compounds with a similar ranking of inducing PAHs. Mean EC50 values in RTL-W1 cells were, respectively, 343 and 266 nM for benzo[a]anthracene, 57 and 92 nM for BaP, 134 and 283 nM for benzo[b]fluoranthene, 455 and 270 nM for chrysene, and 98 and 116 nM for 3-methylcholanthrene. Compared to primary hepatocytes, the RTL-W1 cell line was more sensitive in its EROD response to the presence or absence of serum and to the increase in exposure time, which led to higher EC50 values. [source] Comparison of Intrinsic Optical Signals Associated with Low Mg2+, and 4-Aminopyridine,Induced Seizure-Like Events Reveals Characteristic Features in Adult Rat Limbic SystemEPILEPSIA, Issue 6 2000Katharina Buchheim Summary: Purpose: To analyze the intrinsic optical signal change associated with seizure-like events in two frequently used in vitro models,the low-Mg2+ and the 4-aminopyridine (4-AP) models,and to monitor regions of onset and spread patterns of these discharges by using imaging of intrinsic optical signals (IOS). Methods: Combined hippocampal,entorhinal,cortex slices of adult rats were exposed to two different treatments: lowering extracellular Mg2+ concentrations or application of 100 ,M 4-AP. The electrographic features of the discharges were monitored using extracellular microelectrodes. Optical imaging was achieved by infrared transillumination of the slice and analysis of changes in light transmission using a subtraction approach. The electrographic features were compared with the optical changes. Regions of onset and spread patterns were analyzed in relevant anatomic regions of the slice. Results: Both lowering extracellular Mg2+ concentrations and application of 4-AP induced seizure-like events. The relative duration of the intrinsic optical signal change associated with seizure-like events in the low-Mg2+ model was significantly longer compared with that seen with those occurring in the 4-AP model, although duration of field potentials did not differ significantly in the two models. Seizure-like events of the low-Mg2+ model originated predominantly in the entorhinal cortex, with subsequent propagation toward the subiculum and neocortical structures. In contrast, no consistent region of onset or spread patterns were seen in the 4-AP model, indicating that the seizure initiation is not confined to a particular region in this model. Conclusions: We conclude that different forms of spontaneous epileptiform activity are associated with characteristic optical signal changes and that optical imaging represents an excellent method to assess regions of seizure onset and spread patterns. [source] Hypoxia-activated microglial mediators of neuronal survival are differentially regulated by tetracyclinesGLIA, Issue 8 2006Aaron Y. Lai Abstract The tetracycline derivatives minocycline (MINO) and doxycycline (DOXY) have been shown to be neuroprotective in in vivo and in vitro models of stroke. This neuroprotection is thought to be due to the suppression of microglial activation. However, the specific molecular parameters in microglia of the tetracyclines' effect are not understood. We subjected cultured rat microglial and neuronal cells to in vitro hypoxia and examined the effects of MINO and DOXY pre-treatments. Our data showed that MINO and DOXY protect against hypoxia-induced neuronal death by a mechanism dependent on regulation of microglial factors, but likely unrelated to regulation of microglial proliferation/viability. Both MINO and DOXY suppressed the hypoxic activation of ED-1, a marker for microglial activation. Morphological analyses of hypoxic microglia using the microglial marker Iba1 revealed that treatment with MINO and DOXY caused a higher percentage of microglia to remain in a non-activated state. MINO suppressed the hypoxic upregulation of pro-inflammatory agents nitric oxide (NO), interleukin-1 beta (IL-1,), and tumor necrosis factor alpha (TNF-,), while DOXY down-regulated only NO and IL-1,. In contrast, the hypoxic activation of pro-survival/neuroprotective microglial proteins, such as brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), were unaffected by tetracycline treatments. Taken together, these results suggest that MINO and DOXY may provide neuroprotection against stroke by selectively down-regulating microglial toxic factors while maintaining functional pro-survival factors. © 2006 Wiley-Liss, Inc. [source] Expression, regulation, and function of ,V integrins in hepatocellular carcinoma: An in vivo and in vitro studyHEPATOLOGY, Issue 2 2002Mimoun Nejjari The expression of ,V integrins by neoplastic cells contributes to the promotion of local invasion and metastasis. The most characteristic extracellular ligands of ,V integrins are vitronectin and fibronectin. Hepatocytes are the main source of vitronectin, and the capacity to synthesize and secrete vitronectin is usually retained in hepatocellular carcinoma. The aim of this study was to explore the expression, regulation, and functional role of ,V integrins in hepatocellular carcinoma. We first analyzed the expression of ,V integrins and their ligands fibronectin and vitronectin in 80 cases of hepatocellular carcinoma. ,V integrin chain was detected in 44 cases and vitronectin in 50. Twenty-four of the 44 ,V-positive tumors contained large amounts of vitronectin. These cases presented more frequently with adverse histoprognostic factors, including infiltrative growth pattern (62.5%), lack of capsule (71%), presence of capsular invasion (57%), and satellite nodules (50%). We then used HepG2 and Hep3B cell lines as in vitro models to study ,V integrin regulation and function. HepG2 and Hep3B cells expressed ,V integrin chain and used ,V,1 and ,V,5 for adhesion and migration on vitronectin. Tumor necrosis factor (TNF) , and transforming growth factor (TGF) , significantly increased the expression levels of ,V integrins and stimulated the adhesion and migration of both HepG2 and Hep3B cell lines on vitronectin. The effects of growth factors on cell adhesion and migration were reproduced by incubation with conditioned medium from rat liver myofibroblasts. In conclusion, our results support the existence of an ,V integrin/vitronectin connection in hepatocellular carcinoma and suggest that this connection may be an adverse prognostic factor. [source] The combination of epigallocatechin gallate and curcumin suppresses ER,-breast cancer cell growth in vitro and in vivoINTERNATIONAL JOURNAL OF CANCER, Issue 9 2008Tiffany J. Somers-Edgar Abstract Both epigallocatechin gallate (EGCG) and curcumin have shown efficacy in various in vivo and in vitro models of cancer. This study was designed to determine the efficacy of these naturally derived polyphenolic compounds in vitro and in vivo, when given in combination. Studies in MDA-MB-231 cells demonstrated that EGCG + curcumin was synergistically cytotoxic and that this correlated with G2/M-phase cell cycle arrest. After 12 hr, EGCG (25 ,M) + curcumin (3 ,M) increased the proportion of cells in G2/M-phase to 263 ± 16% of control and this correlated with a 50 ± 4% decrease in cell number compared to control. To determine if this in vitro result would translate in vivo, athymic nude female mice were implanted with MDA-MB-231 cells and treated with curcumin (200 mg/kg/day, po), EGCG (25 mg/kg/day, ip), EGCG + curcumin, or vehicle control (5 ml/kg/day, po) for 10 weeks. Tumor volume in the EGCG + curcumin treated mice decreased 49% compared to vehicle control mice (p < 0.05), which correlated with a 78 ± 6% decrease in levels of VEGFR-1 protein expression in the tumors. Curcumin treatment significantly decreased tumor protein levels of EGFR and Akt, however the expression of these proteins was not further decreased following combination treatment. Therefore, these results demonstrate that the combination of EGCG and curcumin is efficacious in both in vitro and in vivo models of ER,- breast cancer and that regulation of VEGFR-1 may play a key role in this effect. © 2007 Wiley-Liss, Inc. [source] Steroid hormone receptors and coregulators in endocrine-resistant and estrogen-independent breast cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 4 2006Nanna Sarvilinna Abstract Resistance to hormonal therapy is often a problem in the treatment of breast cancer patients. It has been suggested that resistance could be explained by altered nuclear hormone receptor or coregulator levels or inappropriately increased agonist activity of selective estrogen receptor modulator (SERM). To test these hypotheses, we have established novel MCF-7 cell line-derived in vitro models of anti-estrogen- and progestin-resistant and estrogen-independent breast cancer by long-term culture in the presence of toremifene and medroxyprogesterone acetate (MPA) and in the absence of estradiol, respectively. Using cell growth and multiprobe ribonuclease protection assays, the expression of 5 nuclear hormone receptors and 9 coregulators as well as the alterations in the cell proliferation and target gene transcription in response to hormonal treatments were studied. Progesterone receptor (PR) expression was decreased and silencing mediator for retinoid acid and thyroid hormone receptors (SMRT) and amplified in breast cancer-1 (AIB1) expression increased in anti-estrogen-resistant cells. Estrogen caused PR and ER, upregulation in all cell lines, but we did not observe increased agonist activity of anti-estrogen measured by regulation of these estrogen target genes. Basal ER, levels and estrogenic growth response were decreased and p300/CBP-associated factor (pCAF) and AIB1 upregulated by estrogen in progestin-resistant cells, but coregulator levels were unchanged. Estrogen-independent cells were still estrogen-responsive and PR, nuclear receptor corepressor (N-CoR) and SMRT expression was increased whereas steroid receptor coactivator-1 (SRC-1a) and CBP-related protein p300 (p300) expression decreased. Their growth was inhibited by toremifene, but estradiol was able to abrogate this effect, which might have interesting clinical implications concerning the use of postmenopausal hormone replacement therapy. © 2005 Wiley-Liss, Inc. [source] Two new lipoaminoacids with complementary modes of action: new prospects to fight out against skin agingINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 1 2010S. Dumont Synopsis The mode of action of two cosmetic active ingredients (AIs), palmitoyl glycine (PG) and cocoyl alanine (CA) was studied with cDNA array experiments and quantitative PCR confirmations, which were performed on experimentally aged human fibroblasts. These preliminary studies revealed complementary profiles. Thus, specific supplementary investigations were then carried out for each AI. Protocols used were based either on in vitro models: (i) biochemical assays, (ii) monolayer cell culture (primary human fibroblasts and keratinocytes) and (iii) the model of capillary-like tube formation by human endothelial cells or on ex vivo models, i.e. topically treated skin explants and both immunohistochemical and ChromameterTM investigations. New prospects are proposed to fight out against skin aging. Indeed, PG and CA showed complementary properties and thus enabled a regulation or a restoration effect on main aging-associated disorders. Thus, they can not only act on tissue architecture, cell,cell interactions and extracellular matrix protection but also on inflammation, cell longevity, skin immune system protection, skin radiance and stem cell survey. Finally, a clinical trial performed on Caucasian women confirmed AI anti-wrinkle efficacy, which was superior to that of a market reference ingredient. In the future, complementary experiments enabling a better understanding of the aging-induced decline of epidermal stem cells would be of a great interest. Résumé Le mode d'action de deux actifs cosmétiques, Palmitoyl glycine (PG) et Cocoyl Alanine (CA), a été déterminéà l'aide d'expériences de cDNA arrays et de confirmations par qPCR, réalisées sur des fibroblastes humains vieillis expérimentalement. Ces études préliminaires ont révélé des modes d'action complémentaires. Des expériences supplémentaires spécifiques ont donc ensuite été réalisées pour chaque actif. Les protocoles utilisés étaient basés sur des modèles in vitro: i) études biochimiques, ii) cultures cellulaires en monocouches (cultures primaires de fibroblastes et de kératinocytes humains) et iii) modèle de formation de pseudo-tubules par des cellules endothéliales humaines; ou sur des modèles ex-vivo, i.e. des explants de peau traités de manière topique et analysés à l'aide d'études immuno-histochimiques et d'un ChromamètreTM. De nouvelles perspectives s'ouvrent pour combattre le vieillissement cutané. En effet, PG et CA montrent des propriétés complémentaires et permettent ainsi une régulation ou une restauration des principaux dysfonctionnements liés à l'âge. Ainsi, ils peuvent agir non-seulement sur l'architecture des tissus, l'interaction entre les cellules et la protection de la matrice extracellulaire mais aussi sur l'inflammation, la longévité cellulaire, la survie des cellules souches, le système de protection immunitaire et l'éclat de la peau. Finalement, des essais cliniques réalisés sur des femmes de type Caucasien ont confirmé l'efficacité antirides des actifs, laquelle était supérieure à celle d'une référence anti-âge du marché. Dans un futur proche, des tests complémentaires pourraient permettre une meilleure compréhension de la dégradation des cellules souches épidermiques au cours du vieillissement. [source] Skin-lightening products revisitedINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 4 2003L. Petit Synopsis Skin colour typology depends on the amount and location of its chromophores. Among them, eumelanins derived from 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI), and phaeomelanins are of utmost importance. These biomolecules result from the multi-step enzymatic and non-enzymatic conversion of tyrosine into melanins. Pigmentation disorders are multiple and depend on alterations in the density in active melanocytes, and on specific abnormalities of any of the complex melanogenesis mechanisms. This review presents some of the main skin-lightening agents with respect to their mechanisms of action and side-effects. Some of the novel compounds may lead to new perspectives in the fields of dermatology and cosmetology. The methods commonly used to assess efficacy of skin-lightening products rely on in vitro models including cell-free enzymatic assays, melanocyte cultures and reconstructed epidermis bioassays. Animal models have little relevance. By contrast, human testing with the support of instrumental evaluations is the most informative. Résumé La couleur de la peau et la typologie dépendant de la quantité et de la localization de ses chromophores. Parmi ceux-ci, les eumélanines dérivées des 5.6-dihydroxyindole-2-carboxylic acide (DHICA) et 5.6-dihydroxyindole (DHI) et les phaeomélanines sont de la plus grande importance. Ces biomolécules résultent de la conversion enzymatique et non enzymatiques en plusieurs étapes de la tyrosine en mélanines. Les troubles de la pigmentation sont multiples et dépendent d'altérations dans la densité de mélanocytes actifs et d'anomalies spécifiques touchant l'un ou l'autre étape du processus complexe de la mélanogenèse. Cette revue présente quelques agents dépigmentants en considérant leurs mécnaismes d'action et leurs effets secondaires. Certains des nouveaux composés ouvrent de nouvelles perspectives dans les domaines de la dermatologie et de la cosmétologie. Les méthodes visant àévaluer l'efficacité de produits dépigmentants font appel à des modèles in vitro incluant des bioessais enzymatiques, des cultures de mélnaocytes et l'épiderme reconstruit. Les modèles animaux sont peu pertinants. En revanche, els tests sur volontaires humains s'appuyant sur des évaluations instrumentales sont les plus informatives. [source] High-phosphate-induced calcification is related to SM22, promoter methylation in vascular smooth muscle cellsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2010Addy Montes de Oca Abstract Hyperphosphatemia is closely related to vascular calcification in patients with chronic kidney disease. Vascular smooth muscle cells (VSMCs) exposed to high phosphate concentrations in vitro undergo phenotypic transition to osteoblast-like cells. Mechanisms underlying this transdifferentiation are not clear. In this study we used two in vitro models, human aortic smooth muscle cells and rat aortic rings, to investigate the phenotypic transition of VSMCs induced by high phosphate. We found that high phosphate concentration (3.3,mmol/L) in the medium was associated with increased DNA methyltransferase activity and methylation of the promoter region of SM22,. This was accompanied by loss of the smooth muscle cell,specific protein SM22,, gain of the osteoblast transcription factor Cbfa1, and increased alkaline phosphatase activity with the subsequent in vitro calcification. The addition of a demethylating agent (procaine) to the high-phosphate medium reduced DNA methyltransferase activity and prevented methylation of the SM22, promoter, which was accompanied by an increase in SM22, expression and less calcification. Additionally, downregulation of SM22,, either by siRNA or by a methyl group donor (S -adenosyl methionine), resulted in overexpression of Cbfa1. In conclusion, we demonstrate that methylation of SM22, promoter is an important event in vascular smooth muscle cell calcification and that high phosphate induces this epigenetic modification. These findings uncover a new insight into mechanisms by which high phosphate concentration promotes vascular calcification. © 2010 American Society for Bone and Mineral Research [source] Strategies for Directing the Differentiation of Stem Cells Into the Osteogenic Lineage In Vitro,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2004Boon 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] Phosphate regulates embryonic endochondral bone developmentJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2009Alena A. Zalutskaya Abstract Phosphate is required for terminal differentiation of hypertrophic chondrocytes during postnatal growth plate maturation. In vitro models of chondrocyte differentiation demonstrate that 7,mM phosphate, a concentration analogous to that of the late gestational fetus, activates the mitochondrial apoptotic pathway in hypertrophic chondrocytes. This raises the question as to whether extracellular phosphate modulates chondrocyte differentiation and apoptosis during embryonic endochondral bone formation. To address this question, we performed investigations in the mouse metatarsal culture model that recapitulates in vivo bone development. Metatarsals were cultured for 4, 8, and 12 days with 1.25 and 7,mM phosphate. Metatarsals cultured with 7,mM phosphate showed a decrease in proliferation compared to those cultured in 1.25,mM phosphate. This decrease in proliferation was accompanied by an early enhancement in hypertrophic chondrocyte differentiation, associated with an increase in FGF18 expression. By 8 days in culture, an increase caspase-9 activation and apoptosis of hypertrophic chondrocytes was observed in the metatarsals cultured in 7,mM phosphate. Immunohistochemical analyses of embryonic bones demonstrated activation of caspase-9 in hypertrophic chondrocytes, associated with vascular invasion. Thus, these investigations demonstrate that phosphate promotes chondrocyte differentiation during embryonic development and implicate a physiological role for phosphate activation of the mitochondrial apoptotic pathway during embryonic endochondral bone formation. J. Cell. Biochem. 108: 668,674, 2009. © 2009 Wiley-Liss, Inc. [source] INDIVIDUAL AND COMBINED CYTOTOXIC EFFECTS OF THE MAJOR FOUR AFLATOXINS IN DIFFERENT IN VITRO STABILIZED SYSTEMSJOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2010CORNELIA 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] Hypoxia-inducible factor and nuclear factor kappa-B activation in blood,brain barrier endothelium under hypoxic/reoxygenation stressJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Ken A. Witt Abstract This investigation focuses on transcription factor involvement in blood,brain barrier (BBB) endothelial cell-induced alterations under conditions of hypoxia and post-hypoxia/reoxygenation (H/R), using established in vivo/ex vivo and in vitro BBB models. Protein/DNA array analyses revealed a correlation in key transcription factor activation during hypoxia and H/R, including NF,B and hypoxia-inducible factor (HIF)1. Electrophoretic mobility shift assays confirmed NF,B and HIF1 binding activity ex vivo and in vitro, under conditions of hypoxia and H/R. Hypoxia- and H/R-treated BBB endothelium showed increased HIF1, protein expression in both cytoplasmic and nuclear fractions, in ex vivo and in vitro models. Co-immunoprecipitation of HIF1, and HIF1, was shown in the nuclear fraction under conditions of hypoxia and H/R in both models. Hypoxia- and H/R-treated BBB endothelium showed increased expression of NF,B-p65 protein in both cytoplasmic and nuclear fractions. Co-immunoprecipitation of NF,B-p65 with NF,B-p50 was shown in the nuclear fraction under conditions of hypoxia and H/R in the ex vivo model, and after H/R in the in vitro model. These data offer novel avenues in which to alter and/or investigate BBB activity across model systems and to further our understanding of upstream regulators during hypoxia and H/R. [source] Interaction between flavonoids and the blood,brain barrier: in vitro studiesJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Kuresh A. Youdim Abstract There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood,brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity. [source] In vitro differences of stress concentrations for internal and external hex implant,abutment connections: a short communicationJOURNAL OF ORAL REHABILITATION, Issue 1 2006Y. MAEDA summary, The aim of this study was to clarify the difference in the stress distribution patterns between implants with external-hex or internal-hex connection systems using in vitro models. Three 13 mm fixtures with external-hex and internal-hex connections were installed into an acrylic bone analogue. One piece abutments of 7 mm height was connected. Strain gauges were attached to the abutment surface, and the cervical and fixture tip areas of the bone analogue surface. Vertical and horizontal load applied was 30 N. Data were normalized for each model by obtaining values relative to the sum of the three values. Almost the same force distribution pattern was found under vertical load in both systems. Fixtures with external-hex showed an increase in strain at the cervical area under horizontal load, while in internal-hex fixtures the strain was at the fixture tip area. Within limitations of our model study, it was suggested that fixtures with internal-hex showed widely spread force distribution down to the fixture tip compared with external hex ones. [source] Putative signaling action of amelogenin utilizes the Wnt/,-catenin pathwayJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2009M. Matsuzawa Background and Objective:, While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells. Material and Methods:, To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a ,-galactosidase transgene under the control of a LEF/TCF and ,-catenin-inducible promoter. To investigate in greater detail the molecular mechanisms involved in the ,-catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full-length recombinant mouse amelogenin and were evaluated for phenotypic changes and ,-catenin signaling using a TOPFLASH construct and the LacZ reporter gene. Results:, In these in vitro models, we showed that amelogenin can activate ,-catenin signaling. Conclusion:, Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone. [source] Phenotypic comparison of periodontal ligament cells in vivo and in vitroJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2001P. Lekic The mammalian periodontal ligament contains heterogeneous populations of connective tissue cells, the precise function of which is poorly understood. Despite close proximity to bone and the application of high amplitude physical forces, cells in the periodontal ligament (PL) are capable of expressing regulatory factors that maintain PL width during adult life. The study of PL homeostasis and PL cell differentiation requires culture and phenotypic methods for precise characterization of PL cell populations, in particular those cells with an inherently osteogenic program. Currently it is unknown if cells cultured from the PL are phenotypically similar to the parental cells that are present in the tissues. We have compared the phenotype of cells in vivo with cells derived from the PL and expanded in vitro to assess the general validity of in vitro models for the study of phenotypic regulation in vivo. Rat PL cells were isolated by either scraping the root of the extracted first mandibular molars (Group A), or by scraping the alveolar socket following extraction of first mandibular molars (Group B), or by obtaining a mixture of cells after disaggregating a block of tissue consisting of first mandibular molar, PL and the surrounding alveolar bone (Group C). Cultured cells at confluence were fixed and immunostained for ,-smooth muscle actin (,-SMA), osteopontin (OPN), alkaline phosphatase (AP), or bone sialoprotein (BSP). For in vivo assessments, frontal sections of rat first mandibular molar were immunostained for ,-SMA, OPN, AP and BSP. We examined osteogenic differentiation of cultured PL cell cultures by bone nodule-forming assays. In vivo and at all examined sites, >68% of PL cells were immunostained for AP; ,50% and ,51% for OPN and ,-SMA (p=0.3), respectively, while only ,8% were positively stained for BSP (p<0.01). Analysis of cultured PL cells in Groups A, B and C showed 54%, 53% and 56% positive staining for ,-SMA respectively; 51%, 56%, 54% for OPN; 66%, 70%, 69% for AP and 2.2%, 1.4% and 2.8% for BSP. The mean percentage of PL cells in situ stained for the different markers was similar to that of cultured PL cells (Group A,Group B,Group C in situ for p>0.2) except for BSP which was 3 to 4 fold higher in vivo(p<0.01). PL cell cultures treated with dexamethasone showed mineralized tissue formation for all groups (A, B, C), but no mineralized tissue formation was detected in the absence of dexamethasone. As PL cells express quantitatively similar phenotypes in vitro and in vivo, we conclude that the in vitro models used here for assessment of PL cell differentiation appear to be appropriate and are independent of the cell sampling method. Further, dexamethasone-dependent progenitors are present both on the root and bone-related sides of the PL. [source] In vivo, in vitro and in silico methods for small molecule transfer across the BBBJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 12 2009Jurgen Mensch Abstract The inability of molecules to permeate the BBB is a significant source of attrition in Central Nervous System (CNS) drug discovery. Given the increasing medical drivers for new and improved CNS drugs, small molecule transfer across the BBB is attracting a heightened awareness within pharmaceutical industry and medical fields. In order to assess the potential for small CNS molecules to permeate the BBB, a variety of methods and models, from in silico to in vivo going through in vitro models are developed as predictive tools in drug discovery. This review gives a comprehensive overview of different approaches currently considered in drug discovery to circumvent the lack of small molecule transfer through the BBB, together with their inherent advantages and disadvantages. Particularly, special attention is drawn to in silico models, with a detailed and contemporary point of view on prediction tools and guidelines for rational design. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4429,4468, 2009 [source] Ethanol Treatment Reduces Bovine Bronchial Epithelial Cell MigrationALCOHOLISM, Issue 4 2005John R. Spurzem Background: Chronic ethanol abuse is associated with significant lung disease. Excessive alcohol intake increases risk for a variety of respiratory tract diseases, including pneumonia and bronchitis. Damage to airway epithelium is critical to the pathogenesis of airway disorders such as chronic bronchitis and chronic obstructive pulmonary disease. The ability of the airway epithelium to repair itself is an important step in the resolution of airway inflammation and disease. Ethanol exposure is known to modulate signaling systems in bronchial epithelial cells. We hypothesize that chronic ethanol exposure down-regulates the adenosine 3,:5,-cyclic monophosphate signaling cascade in airway epithelial cells, resulting in decreased epithelial cell migration and repair. Methods: We evaluated the effect of ethanol on primary cultures of bovine bronchial epithelial cells in in vitro models of cell migration, wound repair, cell attachment, and cell spreading. Results: Ethanol causes a concentration-dependent effect on closure of mechanical wounds in cell monolayers. Pretreatment of cells with 100 mm ethanol for 24 hr further slows wound closure. Ethanol pretreatment also reduced the protein kinase A response to wounding and made the cells unresponsive to stimuli of protein kinase A that accelerate wound closure. The effects of ethanol on cell migration in wound closure were confirmed in another assay of migration, the Boyden chamber cell migration assay. Prolonged treatment with ethanol also reduced other cell functions, such as spreading and attachment, which are necessary for epithelial repair. Conclusions: Ethanol modulates signaling systems that are relevant to airway injury and repair, suggesting that chronic, heavy ethanol ingestion has a detrimental impact on airway repair. Impaired response to inflammation and injury may contribute to chronic airway disease. [source] EDTA enhances high-throughput two-dimensional bioprinting by inhibiting salt scaling and cell aggregation at the nozzle surfaceJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 4 2009Cheryl A. Parzel Abstract Tissue-engineering strategies may be employed in the development of in vitro breast tissue models for use in testing regimens of drug therapies and vaccines. The physical and chemical interactions that occur among cells and extracellular matrix components can also be elucidated with these models to gain an understanding of the progression of transformed epithelial cells into tumours and the ultimate metastases of tumour cells. The modified inkjet printer may be a useful tool for creating three-dimensional (3D) in vitro models, because it offers an inexpensive and high-throughput solution to microfabrication, and because the printer can be easily manipulated to produce varying tissue attributes. We hypothesized, however, that when ink is replaced with a biologically based fluid (i.e. a ,bio-ink'), specifically a serum-free cell culture medium, printer nozzle failure can result from salt scale build-up as fluid evaporates on the printhead surface. In this study, ethylene diamine tetra-acetic acid (EDTA) was used as a culture medium additive to prevent salt scaling and cell aggregation during the bioprinting process. The results showed that EDTA, at a concentration typically found in commercially available trypsin solutions (0.53 mM), prevented nozzle failure when a serum-free culture medium was printed from a nozzle at 1000 drops/s. Furthermore, increasing concentrations of EDTA appeared to mildly decrease aggregation of 4T07 cells. Cell viability studies were performed to demonstrate that addition of EDTA did not result in significant cell death. In conclusion, it is recommended that EDTA be incorporated into bio-ink solutions containing salts that could lead to nozzle failure. Copyright © 2009 John Wiley & Sons, Ltd. [source] Genomics and systems biology , how relevant are the developments to veterinary pharmacology, toxicology and therapeutics?JOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 3 2005R. F. WITKAMP This review discusses some of the recent developments in genomics and its current and future relevance for veterinary pharmacology and toxicology. With the rapid progress made in this field several new approaches in pharmacological and toxicological research have developed and drug discovery and drug development strategies have changed dramatically. In this review, the term genomics is used to encompass the three sub-disciplines transcriptomics, proteomics and metabolomics (or metabonomics) to describe the formation and fate of mRNA, proteins and metabolites, respectively. The current status and methods of the technology and some applications are briefly described. Although the DNA sequencing programmes are receiving considerable attention, the real value of genomics for pharmacology and toxicology is brought by the parallel developments in bio-informatics, bio-statistics and the integration of biology with mathematics and information technology. The ultimate level of integration is now mostly called systems biology, where mRNA, proteins and metabolites are being analysed in parallel, using a complete arsenal of analytical techniques (DNA-array, LC-MS/MS, GC-MS/MS, NMR, etc.). The information thus collected is analysed, integrated, linked to database information and translated to pathways and systems. This approach offers an enormous potential to study disease mechanisms and find new drug targets. Thus far, genomics and systems biology have not been introduced significantly in typical veterinary pharmacological and toxicological research programmes. The high costs and complexity connected to these large projects often form major obstacles for research groups with limited budgets. In other veterinary areas and disciplines, including infectious diseases, animal production and food-safety more examples of application are available. Genomics and bio-informatics provide outstanding opportunities to study pharmacology and toxicology in a more holistic way, taking into account the complexity of biological systems and based on the basic principles of physiology and the concept of homeostasis. Knowledge of biology, in vivo and in vitro models, and comparative pharmacology/toxicology is essential here, creating excellent opportunities for the veterinary trained scientist. [source] Liver fibrosis: searching for cell model answersLIVER INTERNATIONAL, Issue 4 2007Ma. Concepción Gutiérrez-Ruiz Abstract Hepatic stellate cells (HSC) are the principal fibrogenic cell type in the liver. Progress in understanding the cellular and molecular basis for the development and progression of liver fibrosis could be possible by the development of methods to isolate HSC from rodents and human liver. Growth of stellate cells on plastic led to a phenotypic response known as activation, which paralleled closely the response of these cells to injury in vivo. Actually, much of the current knowledge of stellate cell behaviour has been gained through primary culture studies, particularly from rats. Also, different laboratories that have established hepatic stellate cell lines from rats and humans have provided a stable and unlimited source of cells that express specific functions, making them suitable for culture-based studies of hepatic fibrosis. From these in vitro models grew a large body of information characterizing stellate cell activation, cytokine signalling, intracellular pathways regulating liver fibrogenesis, production of extracellular matrix proteins and development of antifibrotic drugs. [source] E2 quasispecies specificity of hepatitis C virus association with allografts immediately after liver transplantationLIVER TRANSPLANTATION, Issue 2 2004Michael G. Hughes Jr. It is unknown whether all hepatitis C virus (HCV) quasispecies variants found within patient serum have equal capacity to associate with the liver after transplantation; however, in vitro models of HCV infection suggest that variations in the hypervariable region 1 (HVR1) of the second envelope protein (E2) may be important in infectivity. The hypothesis of the current study is that the two hypervariable regions (HVR1 and HVR2) within E2 are important in the initial virus,liver interaction, and, therefore, certain HCV quasispecies variants will be isolated from the liver after reperfusion. In 8 patients with end-stage liver disease secondary to HCV infection, HCV envelope quasispecies were determined from intraoperative serum samples obtained before the anhepatic phase of transplantation and from liver biopsies 1.5 to 2.5 hours after the transplanted liver was perfused. Explanted (native) liver biopsies were taken as a control. Sequence analysis was performed on clones of specific HCV reverse transcriptase-polymerase chain reaction products spanning HVR1 and HVR2 of the E2 protein. HVR1 was more variable than HVR2 for all samples. Quasispecies isolated from postperfusion liver differed more from serum than did explanted liver quasispecies at HVR1 (P = 0.03) but not at HVR2 (P = 0.2). Comparison of HVR1 sequences from postperfusion liver versus serum revealed significantly less HVR1 genetic complexity and diversity (P = 0.02 and P = 0.04, respectively). Immediately after transplantation but before actual infection, liver allografts select out from the infecting serum inoculum a less heterogeneous, more closely related population of quasispecies variants. (Liver Transpl 2004;10:208,216.) [source] Inhibition of Canonical Wnt Signaling Increases Microvascular Hemorrhaging and Venular Remodeling in Adult RatsMICROCIRCULATION, Issue 5 2010JASON T. GLAW Microcirculation (2010) 17, 348,357. doi: 10.1111/j.1549-8719.2010.00036.x Abstract Objective:, The canonical Wnt signaling pathway, heavily studied in development and cancer, has recently been implicated in microvascular growth with the use of developmental and in vitro models. To date, however, no study exists showing the effects of perturbing the canonical Wnt pathway in a complete microvascular network undergoing physiological remodeling in vivo. Our objective was to investigate the effects of canonical Wnt inhibition on the microvascular remodeling of adult rats. Methods:, Canonical Wnt inhibitor DKK-1, Wnt inhibitor sFRP-1, BSA or saline was superfused onto the exteriorized mesenteric windows of 300 g adult female Sprague-Dawley rats for 20 minutes. Three days following surgery, mesenteric windows were imaged intravitally and harvested for immunofluorescence staining with smooth muscle alpha-actin and BRDU. Results:, We observed prominent differences in the response of the mesenteric microvasculature amongst the various treatment groups. Significant increases in hemorrhage area, vascular density, and draining vessel diameter were observed in windows treated with Wnt inhibitors as compared to control-treated windows. Additionally, confocal imaging analysis showed significant increases in proliferating cells as well as evidence of proliferating smooth muscle cells along venules. Conclusions:, Together, our results suggest that canonical Wnt inhibition plays an important role in microvascular remodeling, specifically venular remodeling. [source] | |||||||||||||||||||||||||||||||||||||