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PubMed1991

Effect of subinhibitory concentrations of clindamycin and trospectomycin on the adherence of Staphylococcus epidermidis in an in vitro model of vascular catheter colonization.

Septicemia, often due to Staphylococcus epidermidis, is a life-threatening complication associated with indwelling vascular catheters. An important factor in the development of such infections is glycocalix, or slime. An in vitro model that mimics intravenous delivery systems in humans was developed. It consisted of a modified Robbins device containing slices of silicone catheters in the removable ports, through which S. epidermidis diluted in 5% dextrose-normal saline with 10% heat-inactivated normal human serum was run, with and without clindamycin and trospectomycin. S. epidermidis was recovered from all catheters in the absence of antibiotics; no growth was detected with antibiotics. Scanning electron microscopy demonstrated significant reduction in glycocalix and no visible organisms with all concentrations except 0.5 micrograms/ml trospectomycin and 1 microgram/ml clindamycin; for those, a moderate amount of glycocalix and a few bacteria were seen. Thus, subinhibitory levels of trospectomycin and clindamycin may have a role in the prevention of microbial adherence to vascular catheters.

Anti-Bacterial Agents↗
PubMed2006

Effects of chloride flux modulators in an in vitro model of brain edema formation.

Brain edema is a serious consequence of hemispheric stroke and traumatic brain injury and contributes significantly to patient mortality. In the present study, we measured water contents in hippocampal slices as an in vitro model of edema formation. Excitotoxic conditions induced by N-methyl-D-aspartate (NMDA, 300 microM), as well as ischemia induced by oxygen-glucose deprivation (OGD), caused cellular edema formation as indicated by an increase of slice water contents. In the presence of furosemide, an inhibitor of the Na,K,Cl-cotransporter, NMDA-induced edema were reduced by 64% while OGD-induced edema were unaffected. The same observation, i.e., reduction of excitotoxic edema formation but no effect on ischemia-induced edema, was made with chloride transport inhibitors such as DIDS and niflumic acid. Under ischemic conditions, modulation of GABAA receptors by bicuculline, a GABA antagonist, or by diazepam, a GABAergic agonist, did not significantly affect edema formation. Further experiments demonstrated that low chloride conditions prevented NMDA-induced, but not OGD-induced, water influx. Omission of calcium ions had no effect. Our results show that NMDA-induced edema formation is highly dependent on chloride influx as it was prevented by low-chloride conditions and by various compounds that interfere with chloride influx. In contrast, OGD-induced edema observed in brain slices was not affected by modulators of chloride fluxes. The results are discussed with reference to ionic changes occurring during tissue ischemia.

Animals↗
PubMedDate not supplied

Isolation, immortalization, and initial characterization of uterine cell lines: an in vitro model system for the porcine uterus.

The aim of this study was to develop immortalized cell lines from porcine uterus. Endometrial cells including luminal epithelium (LE), glandular epithelium (GE), stroma (ST), and myometrium (MYO) were enzymatically isolated from the uterus of a day 12 pregnant gilt. Primary cultures were immortalized by transduction with a retroviral vector containing the E6 and E7 open reading frames of human papillomavirus type 16 (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA-317. Cells having integrated the vector were selected by resistance to the neomycin analog G418 (0.4-1.5 mg/ml). Surviving cells were maintained in complete culture medium containing G418 (0.1 mg/ml) and subcultured for 1 yr. Expression of the E7 protein was confirmed in all cell lines by Western blotting. Phase contrast microscopy revealed that LE and GE cells exhibited cobblestone morphology, whereas ST and MYO cells exhibited spindle-shaped morphology. The epithelial origin of LE and GE was confirmed by positive immunostaining for cytokeratin. Stromal and MYO cells were vimentin-positive, but cytokeratin-negative. The MYO cell lines were positive for smooth muscle alpha-actin staining, whereas LE, GE, and ST cell lines were negative for alpha-actin. Western blotting indicated that all cell lines expressed both estrogen and progesterone receptors, but only GE cells secreted uteroferrin (UF). Collectively, these porcine uterine cell lines provide an in vitro model for studying cell type-specific actions of hormones and cytokines, signal transduction pathways, cell-cell interactions, and gene expression.

Animals↗
PubMed2003

Role of carbohydrates in repair of human respiratory epithelium using an in vitro model.

BACKGROUND: The epithelial layer in the conducting airway provides a primary protective barrier. Repair of this barrier normally occurs rapidly after damage, but is compromised in diseases such as asthma. OBJECTIVE: We have developed a human in vitro model system to test our hypothesis that cell surface glycoconjugate-based interactions are required for the normal repair of damaged epithelium. METHODS: Lectins having narrow carbohydrate specificities were used to identify and block specific carbohydrate moieties on human airway-derived epithelial cells in culture. RESULTS: The lectin wheat germ agglutinin bound to N-acetyl glucosamine and inhibited the repair of epithelial damage while having little effect on cell viability. In contrast, other N-acetyl glucosamine binding lectins had no effect even when bound to the cell surface. The involvement of glycoconjugates was confirmed by pre-incubating the lectin with its specific sugar, preventing the inhibition of repair. CONCLUSION: These results indicate that lectin-binding sites are involved in epithelial repair and may be important in the repetitive cycles of injury and repair seen in asthma. This model system provides an insight into the role of glycoconjugates and will help to determine the function of specific carbohydrate groups in epithelial repair. These may present a target for therapeutic intervention in respiratory and other diseases.

Asthma↗
PubMed1985

In-vitro model for simultaneous simulation of the serum kinetics of two drugs with different half-lives.

Simultaneous administration of two drugs is frequently practised in clinical chemotherapy because of the synergistic potential of drug combinations. During in-vitro testing of the effect of such drug combinations, differences in the pharmacokinetic properties of the two drugs should be considered. This paper presents the mathematical background and the technical components of an in-vitro model that allows the simultaneous simulation of first order elimination kinetics of two drugs with different half-lives. The model allows simulations of multiple dose regimens of either bolus injections or continuous infusions of each drug.

Anti-Bacterial Agents↗
PubMedDate not supplied

In vitro model of endothelialization at anastomotic sites.

Endothelial cell (EC) migration from native arteries to vascular prostheses is one of the most important healing processes of implanted artificial grafts, which results in endothelial cell coverage of the luminal surface of the graft. This article presents an in vitro model of endothelialization at the anastomotic site, which can provide cellular level information on morphologic reconstruction, and its substrate dependency. Test materials included polyethylene terephthalate (PET), segmented polyurethanes (SPUs), and glass with or without protein precoating (collagen, fibronectin, and albumin). In vitro endothelialization on these substrates was quantitatively determined by phase contrast microscopy. The results obtained show: 1) The fastest endothelialization rate was found on PET, followed by relatively hydrophobic SPU, and glass--the slowest was on hydrophilic SPU; and 2) Precoating with proteins drastically altered the cellular behaviors; collagen greatly enhanced endothelialization in vitro, followed by fibronectin. Precoating by albumin, however, greatly retarded endothelialization. The present study strongly suggests that unidirectional endothelialization at the anastomotic sites is controlled by the surface characteristics of graft materials.

Anastomosis, Surgical↗
PubMed1987

An in vitro model for studying the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to human thymus.

A coculture system of human thymic epithelial (HuTE) cells and thymocytes (T lymphocyte precursors) has been established and characterized as an in vitro model for assessing the potential toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to human thymus. HuTE cells in culture were responsive to TCDD as judged by induction of the cytochrome P1-450 monooxygenase activities, 7-ethoxycoumarin O-deethylase (ECOD) and 7-ethoxyresorufin O-deethylase (EROD). Measurement of the responsiveness of thymocytes cocultured on TCDD-pretreated HuTE monolayers to the mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) indicated that TCDD can act directly on HuTE cells to suppress thymocyte maturation (at a concentration of 10 nM, TCDD produced a 25 to 50% inhibition of thymocyte responsiveness to Con A and PHA). Both the induction of cytochrome P1-450 monooxygenase activity (EC50 values approximately 1 nM) and immunosuppressive responses elicited by TCDD in HuTE cells were concentration-dependent and stereospecific (as judged by the relative activities of chlorinated dibenzo-p-dioxin and dibenzofuran isomers), indicating involvement of the Ah receptor which was detected in all HuTE strains examined. Initial characterization of these Ah receptor-mediated responses in several strains of HuTE cells indicated marked interstrain differences in maximally inducible ECOD and EROD activities which did not appear to directly correlate with measured concentrations of the cytosolic Ah receptor, and in certain strains examined, differences in sensitivity and magnitude were observed for TCDD-evoked immunotoxic responses but not always for the induction response. These data on the actions of TCDD on cultured HuTE cells suggest that human thymus is a target for TCDD and related halogenated aromatic compounds. In HuTE cells, measurement of either the Ah receptor concentration or of marker responses such as the induction of cytochrome P1-450 alone cannot provide an accurate quantitative assessment of susceptibility to TCDD-induced thymus toxicity.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1987

Primary cultures of rat aortic endothelial and smooth muscle cells: I. An in vitro model to study xenobiotic-induced vascular cytotoxicity.

Primary cultures of rat vascular endothelial and smooth muscle cells were developed as models to study xenobiotic-induced cytotoxicity. Endothelial and smooth muscle cells were isolated by enzymatic digestion and mechanical dissociation of rat thoracic aortae. Optimal cell growth and minimal fibroblast contamination in cultures of both cell types were obtained in Medium 199 supplemented with 10% fetal bovine serum. Cultured cells were characterized by distinctive morphologic features and growth patterns. Intercellular endothelial cell junctions were selectively stained with silver nitrate. Endothelial cells also exhibited a nonthrombogenic surface, as reflected by platelet-binding studies. Confluent cultures of smooth muscle cells, but not endothelial cells, contracted in response to norepinephrine (10 microM). Cultures of both cell types were exposed to acrolein (2, 5 or 50 ppm), an environmental pollutant, for 4 and 24 h. Morphologic damage, lactate dehydrogenase release, and cellular thiol content were used as indices of cytotoxicity. Acrolein-induced enzyme leakage and morphologic alterations were dose- and time-dependent and more pronounced in cultures of smooth muscle cells than in endothelial cells. The total thiol content of endothelial cells exposed to acrolein (50 ppm) for 24 h was not significantly different from that of respective controls. In contrast, the content of treated smooth muscle cells was higher than that of controls. These observations show that primary cultures of vascular cells provide a useful model to evaluate xenobiotic-induced cytotoxicity. The information obtained using a cell culture system may be complemented by the use of other in vivo and in vitro models to determine the mechanisms by which xenobiotics cause vascular cell injury.

Acrolein↗
PubMed2003

In vitro model for the study of the dissociation of increasing antigenemia and decreasing DNAemia and viremia during treatment of human cytomegalovirus infection with ganciclovir in transplant recipients.

The paradox phenomenon (i.e., the dissociation of increasing antigenemia and decreasing DNAemia and viremia) that occurs during treatment of human cytomegalovirus (HCMV) infections with ganciclovir (Gcv), in transplant recipients, was investigated by use of an in vitro model for the study of interactions between polymorphonuclear leukocytes and endothelial cells. The paradox phenomenon was reproduced in vitro in the presence of Gcv and, to a much lesser extent, in the presence of cidofovir, but not in the presence of foscarnet. The pathogenetic basis for such a paradox response was found, by use of drug concentrations in the range of 90%-99% of the inhibitory dose, to rely on the partial synthesis of HCMV phosphoprotein 65. The opposite situation (i.e., the simultaneous increase of antigenemia, viremia, and DNAemia), which is observed in clinical conditions associated with inefficacy of treatment due to drug-resistant strains, was also reproduced in vitro by use of drug-resistant HCMV strains. The conclusion for clinicians is that antiviral therapy must be changed only in the latter case.

Antigens, Viral↗
PubMed2007

MAPK signaling pathways mediate AMPA receptor trafficking in an in vitro model of classical conditioning.

The mitogen-activated protein kinase (MAPK) signal transduction pathways have been implicated in underlying mechanisms of synaptic plasticity and learning. However, the differential roles of the MAPK family members extracellular signal-regulated kinase (ERK) and p38 in learning remain to be clarified. Here, an in vitro model of classical conditioning was examined to assess the roles of ERK and p38 MAPK in this form of learning. Previous studies showed that NMDA-mediated trafficking of synaptic glutamate receptor 4 (GluR4)-containing AMPA receptors (AMPARs) underlies conditioning in this preparation and that this is accomplished through GluR4 interactions with the immediate-early gene protein Arc and the actin cytoskeleton. Here, it is shown that attenuation of conditioned responses (CRs) by ERK and p38 MAPK antagonists is associated with significantly reduced synaptic localization of GluR4 subunits. Western blotting reveals that p38 MAPK significantly increases its activation levels during late stages of conditioning during CR expression. In contrast, ERK MAPK activation is enhanced in early conditioning during CR acquisition. The results suggest that MAPKs have a central role in the synaptic delivery of GluR4-containing AMPARs during in vitro classical conditioning.

Animals↗
PubMedDate not supplied

Sulfonated dextran inhibits complement activation and complement-dependent cytotoxicity in an in vitro model of hyperacute xenograft rejection.

In the present study, we demonstrate that a substituted soluble dextran derivative bearing 73% carboxylic groups and 15% benzylamide sulfonate groups, termed CMDBS25, inhibits complement activation and complement-mediated damage in an in vitro model of xenogeneic rejection. Incubation of porcine aortic endothelial cells with normal human serum resulted in time-dependent complement consumption as assessed by C3a generation in the fluid phase and deposition of activated complement fragments C3, C5 and of C5b-9 on target cells. The presence of C5b-9 membrane attack complex was associated with 51Cr release from prelabelled endothelial cells. The addition of 5-25 mg of CMDBS25/ml under the experimental conditions used, inhibited complement activation and C3a generation in a dose-dependent fashion. CMDBS25 (25 mg/ml) totally suppressed iC3b, C5 and C5b-9 cytolytic complex deposition on cells and inhibits by 42% lysis of target endothelial cells. Native dextran had no effect. Our observations document the anti-complementary properties of sulfonated dextran derivatives and their potential as therapeutic agents for the prevention of complement-dependent hyperacute xenograft rejection.

Animals↗
PubMed1984

The assessment of antimicrobial activity in an in-vitro model of the treatment of bacterial cystitis.

In order to investigate the comparative activity of agents of the nalidixic acid series, cultures of nalidixic acid sensitive strains of E. coli were exposed to the drugs in an in-vitro model that simulates the hydrokinetic aspects of the treatment of bacterial cystitis. Intrinsic activity, as judged by the response to a single dose of drug and resistance as judged by response to a repeat dose, were investigated. All seven compounds tested in this way were able to inhibit bacterial growth for considerable periods of time even when the peak concentration achieved was as low as 10 mg/l. However, resistance emerged readily, particularly to nalidixic acid, pipemidic acid and piromidic acid. Norfloxacin was the most active of the seven compounds tested and was the only one to which resistance did not emerge at the concentrations tested. However, when two nalidixic acid resistant strains were tested in the bladder model, norfloxacin resistance was observed to emerge with one strain, but not with the other.

Anti-Infective Agents, Urinary↗
PubMed2002

Genome-wide allelotyping of a new in vitro model system reveals early events in breast cancer progression.

Toward the goal of identifying early genetic losses, which mediate the release of human breast epithelium from replicative suppression leading to cellular immortalization, we have used a newly developed in vitro model system. This system consists of epithelial cultures derived from noncancerous breast tissue, treated with the chemical carcinogen N-ethyl-N-nitrosourea, and continuously passaged to yield cell populations culminating in the immortal phenotype. Genome-wide allelotyping of early passage N-ethyl-N-nitrosourea-exposed cell populations revealed aberrations at >10% (18 of 169) loci examined. Allelic losses encompassing chromosomes 6q24-6q27, implicating immortalization-associated candidate genes, hZAC and SEN6, occurred in two independently derived cell lines before the Hayflick limit. Additional LOH sites were present in one cell line at 3p11-3p26, 11p15, and 20p12-13. Allelic losses reported in this cell line preceded detectable levels of telomerase activity and the occurrence of p53-related aberrations. Information gained from the search for early immortalization-associated genetic deletions in cultured cells was applied in a novel approach toward the analysis of morphologically normal terminal ductal lobular units microdissected from 20 cases of ductal carcinoma in situ. Notably, clonal allelic losses at chromosome 3p24 and 6q24 were an early occurrence in adjoining terminal ductal lobular units of a proportion of primary tumors, which displayed loss of heterozygosity (3 of 11 and 3 of 6, respectively). The biological insights provided by the new model system reported here strongly suggest that early allelic losses delineated in immortalized cultures and validated in vivo could serve as surrogate endpoints to assist in the identification and intervention of high-risk benign breast tissue, which sustains the potential for continuous proliferation.

Alleles↗
PubMed2006

Feasibility of ultra-low-dose multi-detector-row CT-colonography: detection of artificial endoluminal lesions in an in-vitro-model with optimization of image quality using a noise reduction filter algorithm.

PURPOSE: To assess the most favorable slice thickness in Multi-Detector-Row CT-colonography (MDCTC), and the feasibility of dose reduction in an in-vitro-setting as well as the possibility of optimization of image quality using a noise reduction filter algorithm. - MATERIALS AND METHODS: 18 artificial lesions with sizes from 1 to 8 mm were randomly positioned in two cleansed pig colons. At a "Somatom Plus 4 Volume Zoom", six scanning protocols using a slice collimation of 2.5, 1, and 1 mm with a reconstructed slice thickness of 3, 3, and 1.25 mm were performed with tube currents of 100, and 10 mAs, respectively. Using a non-commercial software, a non-linear Gaussian filter was used to minimize image noise. Image noise was assessed before and after application of the filtering process. Using a threshold of -750 HU, two blinded readers analyzed the virtual colonography in respect to lesion location, size, and shape. Artifacts were noted. An automated detection system was evaluated. - RESULTS: Using 10 mAs, a ten-fold dose reduction was achieved. After application of the mathematical filter, image noise was reduced by 45-80% for 100 mAs, and by 50-70% for 10 mAs scans. Only with a slice thickness of 1.25 mm, all lesions could be detected. The definition of lesion size and shape was more accurate with higher mAs. Only minor noise artifacts were noted on low-dose images. The automated polyp detector marked not more than 60% of artificial lesions. - CONCLUSION: MDCTC benefits from narrow slice collimation. In an in-vitro-model, a significant dose reduction is achievable with preservation of a high lesion detection rate. The noise reduction filter algorithm improved image quality substantially.

Algorithms↗
PubMed1990

Effect of hexamethylene bisacetamide and cyclosporin A on recovery of herpes simplex virus type 2 from the in vitro model of latency in a human neuroblastoma cell line.

The goal of the present work was to examine whether hexamethylene bisacetamide (HMBA) and cyclosporin A affect the recovery of herpes simplex virus type 2 (HSV-2) from an in vitro model of HSV-2 latency in human neuroblastoma cell line IMR-32. IMR-32 cells were infected with HSV-2 at a multiplicity of infection of 0.1 plaque-forming units/cell and were cultured at 40 degrees C for 14 days, resulting in the establishment of a model of HSV-2 latency in IMR-32 cells. When the cultivation temperature was shifted down from 40 to 37 degrees C, recovery of virus growth began to occur after an incubation period of 2 days. During the time of shift-down of the incubation temperature, the latently infected cells were further cultured at 37 degrees C in the presence or absence of 5 mM HMBA or 0.5 micrograms/ml cyclosporin A, which does not affect stability of HSV-2 nor proliferation of IMR-32 cells. Consequently, the rate of HSV-2 recovery from the latently infected cells cultured in the presence of 5 mM HMBA was significantly increased, as compared with the untreated controls. In addition, the DNA methylation level of the latently infected IMR-32 cells cultured in the presence of HMBA was significantly decreased when compared to the level in the untreated controls. On the other hand, the cultivation of the latently infected cells in the presence of 0.5 micrograms/ml cyclosporin A resulted in a significant decrease in the rate of HSV-2 recovery. These findings indicate that the recovery of HSV-2 from the model of latency in IMR-32 cells is enhanced by HMBA treatment, which induces a significant decrease of total genomic DNA methylation level, and is inhibited by cyclosporin A treatment.

Acetamides↗
PubMed1997

New in vitro model of traumatic neuronal injury: evaluation of secondary injury and glutamate receptor-mediated neurotoxicity.

The multiplicity and complexity of secondary injury processes following brain trauma in vivo make it difficult to elucidate the roles of specific injury mechanisms. As with other areas of CNS injury, such as ischemia, this has led to the development of in vitro models. Here we describe a new trauma model, in which standardized trauma is delivered to neuronal/glial cultures using a special mechanical device that produces concentric circular cuts in the cell layer. Changes in the number of circles (from 1 to 6) allows variation of injury severity. Comparison studies of cell death induced by such trauma in glial and neuronal/glial cultures demonstrated that glial cells are relatively resistant to this injury, and that the cell death after trauma to neuronal/glial cultures reflects primarily neuronal death. Consistent with other in vivo and in vitro studies, glutamate receptor antagonists MK 801 and MCPG were neuroprotective. Thus, this model appears useful for studying glutamatergic mechanisms involved in secondary injury, and may prove useful for evaluating certain pharmacological strategies for CNS trauma.

Animals↗
PubMed2000

Embryoid bodies: an in vitro model of mouse embryogenesis.

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm and ectoderm) of the organism, including the germline. To study early stages of development, as well as to investigate the impact of a gene knock-out in vitro, ES cells are differentiated into three-dimensional structures called embryoid bodies, because of their ability to mimick post-implantation embryonic tissues. This review summarises the work on ES cell differentiation into haematopoietic and vascular cells, neuronal and glial cells, myocytes, and adipocytes, using this in vitro model of early embryogenesis. We also present the potential of this method to analyse the impact of genetic alterations in vitro.

Animals↗
PubMed2005

Increased susceptibility of fat-laden Zucker-rat hepatocytes to bile acid-induced oncotic necrosis: an in vitro model of steatocholestasis.

UNLABELLED: Metabolic liver disorders cause chronic liver disease and liver failure in childhood. Many of these disorders share the histologic features of steatosis and cholestasis, or steatocholestasis. In this study we sought to (1) develop an in vitro model of steatocholestasis, (2) determine the mechanisms of cell death in this model, and (3) determine the role of mitochondrial disturbances in this model. METHODS: Hepatocytes were isolated from 8-week-old obese (fa/fa) and lean Zucker rats. Cell suspensions were treated with glycochenodeoxycholic acid (GCDC), after which reactive oxygen species (ROS) generation, oncotic necrosis, apoptosis, and ATP content were assessed. Isolated liver mitochondria were exposed to GCDC and analyzed for ROS generation, mitochondrial membrane-permeability transition (MPT), and cytochrome c release. Oncotic necrosis was significantly increased and apoptosis reduced in fa/fa hepatocytes exposed to GCDC compared with that in lean hepatocytes. Necrosis occurred by way of an ROS- and MPT-dependent pathway. Basal and dynamic ATP content did not differ between fa/fa and lean hepatocytes. GCDC stimulated ROS generation, MPT, and cytochrome c release to a similar extent in purified mitochondria from both fa/fa and lean rats. These findings suggest that fat-laden hepatocytes favor a necrotic rather than an apoptotic cell death when exposed to low concentrations of bile acids. The protective effects of antioxidants and MPT blockers suggest novel therapeutic strategies for the treatment of steatocholestatic metabolic liver diseases.

Adenosine Triphosphate↗