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PubMed1999

Transendothelial permeability changes induced by free radicals in an in vitro model of the blood-brain barrier.

In the present study, we investigated the changes in blood-brain barrier (BBB) permeability following brain endothelial cell exposure to different xenobiotics able to promote free radical generation during their metabolism. Our in vitro BBB model consisted of confluent monolayers of immortalized rat brain capillary endothelial cells (RBE4) grown on collagen-coated filters in the presence of C6 glioma cells grown in the lower compartment. We have recently shown that a range of xenobiotics, including menadione, nitrofurazone, and methylviologen (paraquat) may undergo monoelectronic redox cycling in isolated brain capillaries, giving rise to reactive oxygen species. In this study, addition of 100 microM menadione to the culture medium for 30 min significantly increased the permeability of endothelial cell monolayers to radiolabeled sucrose. The effect on endothelial permeability induced by menadione was dose-dependent and reversible. These permeability changes preceded the onset of cell death, as assessed by the Trypan blue exclusion method. Pre-incubation with superoxide dismutase and catalase blocked changes in sucrose permeability to control levels in a dose-dependent manner, suggesting the involvement of reactive oxygen species in menadione-induced BBB opening.

Animals↗
PubMed1999

In vitro models of T cell development.

A number of in vitro systems are currently being used to study both thymocyte development and thymic stromal cell function. However, the usefulness of dispersed culture systems is limited since they often involve disruption of interactions within the normal three-dimensional architecture of the thymus in vivo which are critical for normal development to proceed. In contrast, Fetal Thymus Organ Culture (FTOC) provides an experimental system where such interactions are maintained, thereby allowing in vitro access to key aspects of thymocyte development. More recently, Reaggregate Thymus Organ Cultures (RTOCs) have allowed detailed analysis of thymic stromal cell function, while retroviral transfection of thymocyte subsets under FTOC conditions provides a rapid means to investigate thymocyte development at the molecular level. Current use of the FTOC approach is summarised here, and where appropriate is compared to the use of dispersed culture systems.

Animals↗
PubMed1986

The platelet reactivity of vascular graft prostheses: an in vitro model to test the effect of preclotting.

An in vitro perfusion system was used to study the platelet reactivity of the following vascular graft materials when tested with human blood: expanded polytetrafluoroethylene (ePTFE), crimped Dacron Bionit (DB) and preclotted Dacron Bionit (DB/PC). These materials were simultaneously compared to silicone rubber (SR) using an identical perfusion circuit with the same donor's blood. All vascular graft materials tested in this in vitro perfusion system caused some degree of platelet activation as shown by a decrease in platelet count, an increase in platelet factor 3 activity, elevation of plasma levels of both platelet factor 4 and beta-thromboglobulin and decreased platelet aggregability. The observed platelet activation was striking for Dacron and especially preclotted Dacron, with ePTFE showing low levels of platelet activation. Platelet activation by Dacron was initially rapid and then levelled off, whereas the platelet activation with preclotted Dacron began more slowly, but reached much greater levels after three hours of in vitro perfusion.

Blood Coagulation↗
PubMed1984

Gentamicin-induced stimulation of DNA synthesis in rat kidney. Comparison between in vivo and in vitro models.

We have performed combined in vivo and in vitro measurements of thymidine uptake into kidney cortex DNA of animals treated with gentamicin for 7 days at 10 or 20 mg/kg daily (BID). Labelled thymidine is taken up by cortex fragments in vitro (90 min incubation) and incorporated into DNA; treatment of the animals with gentamicin results in a significant dose-dependent enhancement of this in vitro thymidine incorporation; labelled cells are found primarily in the proximal tubules and interstitium; there is an excellent correlation (r : 0.983, n = 15) between the changes of incorporation measured in vivo and in vitro as demonstrated by the sequential use of [3H]thymidine (in vivo) and [14C]thymidine (in vitro) within the same animals.

Animals↗
PubMed1991

Penetration power of antiseptic creams ("in vitro" model with pig skin).

By means of an "in vitro" method using pig skin, the authors determine the penetration power of some antiseptic creams in order out the most effective one from this point of view in the treatment of subscar-located infections. The following antiseptic creams were studied: 1% Silver Sulfadiazine, 1% Silver Sulfadiazine with 2.2% Cerium Nitrate, 2.2% Cerium Nitrate, 10% Iodine Povidone, 0.2% Nitrofurazone 0.1%, 0.5% and 1% Chlorhexidine. These products were faced with 17 microorganisms isolated from burn wounds and a control one. The minimal inhibitory concentrations (MIC) obtained after passing through the penetration power of some antiseptic creams in order to find out the most effective one from this point of view in the treatment of subscar-located infections.

Animals↗
PubMed1997

In-vitro modelling of the bactericidal activity of teicoplanin versus flucloxacillin as used in surgical prophylaxis, against Staphylococcus aureus.

The bactericidal activities of teicoplanin and flucloxacillin in a 50:50 mixture of human serum and Iso-sensitest broth were compared in an in-vitro pharmacokinetic model, at serum concentrations present during surgical prophylaxis. The bactericidal activity of teicoplanin with and without serum was also compared. Six strains of Staphylococcus aureus were tested. The bactericidal rate of teicoplanin in serum was significantly lower than the rate in broth alone. However, there was no significant difference in the bactericidal rates in serum of teicoplanin compared with flucloxacillin, an antibiotic which is commonly used as prophylaxis for certain surgical procedures.

Anti-Bacterial Agents↗
PubMed2002

Basal glycogenolysis in mouse skeletal muscle: in vitro model predicts in vivo fluxes.

A previously published mammalian kinetic model of skeletal muscle glycogenolysis, consisting of literature in vitro parameters, was modified by substituting mouse specific Vmax values. The model demonstrates that glycogen breakdown to lactate is under ATPase control. Our criteria to test whether in vitro parameters could reproduce in vivo dynamics was the ability of the model to fit phosphocreatine (PCr) and inorganic phosphate (Pi) dynamic NMR data from ischemic basal mouse hindlimbs and predict biochemically-assayed lactate concentrations. Fitting was accomplished by optimizing four parameters--the ATPase rate coefficient, fraction of activated glycogen phosphorylase, and the equilibrium constants of creatine kinase and adenylate kinase (due to the absence of pH in the model). The optimized parameter values were physiologically reasonable, the resultant model fit the [PCr] and [Pi] timecourses well, and the model predicted the final measured lactate concentration. This result demonstrates that additional features of in vivo enzyme binding are not necessary for quantitative description of glycogenolytic dynamics.

Animals↗
PubMed2004

Usefulness and limitation of primary cultured porcine choroid plexus epithelial cells as an in vitro model to study drug transport at the blood-CSF barrier.

The epithelial cells of the choroid plexus form the anatomical structure responsible for the blood-cerebrospinal fluid (CSF) barrier. Here we present our recent progress in the application of porcine choroid plexus epithelial cells in the investigation of permeability and transport properties of this tissue in vitro. Isolated cells are seeded on permeable supports where they grow to confluent monolayers. The cell differentiation is significantly increased under serum-free culture conditions, verifiable by an improvement of barrier properties and enhanced characteristics of the epithelial phenotype. We underline the importance of a tight model system for the investigation of transport processes by showing that permeability and transport properties critically depend on the electrical tightness of the monolayer. The mechanisms of vectorial transfer of micronutrients across the epithelial layer have been investigated in detail for ascorbic acid and myo-inositol transport. Additionally, we describe the transfer of organic anions and the expression of the corresponding transport proteins in vitro. The model system was applied to determine permeation rates of various drugs into the CSF. In conclusion our porcine in vitro model of the blood-CSF barrier provides a reliable system to study the transport characteristics of the choroid plexus epithelium and to probe the passage of drugs.

Animals↗
PubMed1991

Expression of retroviral transduced human CD18 in murine cells: an in vitro model of gene therapy for leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease caused by a defective CD18 gene. The cell-surface glycoprotein encoded by this gene CD18 is normally expressed in cells of the hematopoietic system. An in vitro murine model of CD18 gene replacement therapy was developed to investigate the feasibility of an in vivo murine hematopoietic stem cell gene therapy model. Human CD18-transducing retroviruses were used to transfer a functional human CD18 gene into a variety of cells including (i) murine lymphoblasts (which express murine CD11a and murine CD18), (ii) murine fibroblasts (which have no endogenous murine CD11a/CD18 expression), and (iii) murine fibroblasts, which have been stably transfected with a human CD11a gene. In murine lymphoblasts, human CD18 was expressed on the cell surface as a heterodimer with murine CD11a. Cell-surface expression of human CD18 had no apparent effect on the level of endogenous murine CD11a/CD18 expression. Immunoprecipitation of cell-surface labeled proteins in murine lymphoblasts with a human CD18 specific antibody co-precipitated murine CD11a. Human CD18 can be detected by immunochemistry in the cytoplasm of fibroblasts infected with CD18 encoding retrovirus, but coexpression with CD11a is required for cell-surface expression of either subunit in fibroblasts. These studies suggest that human CD18 will form a heterodimer with murine CD11a and that human CD18 is not expressed on the cell surface of cells not expressing CD11. This provides the basis for the development of a murine hematopoietic stem cell gene replacement therapy model for the treatment of LAD.

3T3 Cells↗
PubMedDate not supplied

Characterization of an in vitro model for evaluating the interface between skin and percutaneous biomaterials.

Percutaneous devices play an essential role in medicine; however, they are often associated with a significant risk of infection. One approach to circumvent infection would be to heal the wound around the devices by promoting skin cell attachment. We used two in vitro assay models to evaluate cutaneous response to poly(2-hydoxyethyl methacrylate) (poly(HEMA)). One approach was to use a cell adhesion assay to test the effects of surface modification of poly(HEMA), and the second used an organ culture system of newborn foreskin biopsies implanted with porous poly(HEMA) rods (20 microm pores) to evaluate the skin/poly(HEMA) interface. Surface modification of poly(HEMA) using 1,1'-carbonyldiimidazole (CDI) enhanced keratinocyte, fibroblast, and endothelial cell adhesion. Keratinocytes in the organ culture model not only remained functionally and structurally viable as observed by immunohistochemistry and electron microscopy, but migrated into the pores of CDI-modified poly(HEMA) rods. No biointegration was seen in the non-CDI-modified poly(HEMA). Laminin 5 immunostaining was seen along the poly(HEMA)/skin interface in a pattern resembling the junctional epithelium of the tooth, the unique natural interface between the skin and tooth that serves as a barrier to bacteria. In vitro systematic evaluation of biomaterials for use in animal implant studies is both cost effective and time efficient.

Aged↗
PubMed1991

Combination therapy with ciprofloxacin plus azlocillin against Pseudomonas aeruginosa: effect of simultaneous versus staggered administration in an in vitro model of infection.

The effect of dose scheduling on the pharmacodynamics of simulated human doses of ciprofloxacin (200 mg intravenously [iv] every 12 h) and azlocillin (4 g iv every 12 h) alone or in combination against Pseudomonas aeruginosa was studied in a two-compartment in vitro kinetic model of infection. Studies with the two drugs in combination were compared using simultaneous or staggered (first doses of each drug were administered 6 h apart) dosing schedules. Bacterial regrowth and resistance were prevented by all combination dosing schedules; however, the simultaneous regimen consistently provided the greatest extent of killing for all strains, particularly in those initially resistant to ciprofloxacin. These enhanced effects of the combination were corroborated by an increase in the peak and duration of bactericidal activity in the analogous "serum" compartment of the model. These data show the potential usefulness of simultaneous dosing of an antipseudomonal beta-lactam with ciprofloxacin against P. aeruginosa.

Azlocillin↗
PubMedDate not supplied

Proposal for a new UVA protection factor: use of an in vitro model of immediate pigment darkening.

In order to define a new method for measuring UVA photoprotection, we built an in vitro immediate pigment darkening model (IPD). IPD is a photochemical reversible reaction induced by UVA on the skin. Our model consists of aqueous solutions of melanocytic compounds (dihydroxyphenylalanine/pheomelanins). Irradiation of these solutions with UVA induces an increase in their absorbance. Oxygen deprivation inhibits the solution darkening and light turn-off induces a decrease in the absorbance as observed in vivo. A UVA photoprotection parameter (PUVA) was defined using the ability of a sunscreen to inhibit the model reaction. A calibration of the reaction inhibition is realised using neutral beam attenuators. PUVA is defined as the percentage absorbance of a beam attenuator which would have the same inhibitory effect as the sunscreen tested. A correlation between PUVA and Diffey-Robson parameter is presented. The method developed here could be use as a indicative tool before human experiments.

Humans↗
PubMed2002

A new in vitro model to evaluate differential responses of endothelial cells to simulated arterial shear stress waveforms.

In the circulation, flow-responsive endothelial cells (ECs) lining the lumen of blood vessels are continuously exposed to complex hemodynamic forces. To increase our understanding of EC response to these dynamic shearing forces, a novel in vitro flow model was developed to simulate pulsatile shear stress waveforms encountered by the endothelium in the arterial circulation. A modified waveform modeled after flow patterns in the human abdominal aorta was used to evaluate the biological responsiveness of human umbilical vein ECs to this new type of stimulus. Arterial pulsatile flow for 24 hours was compared to an equivalent time-average steady laminar shear stress, using no flow (static) culture conditions as a baseline. While both flow stimuli induced comparable changes in cell shape and alignment, distinct patterns of responses were observed in the distribution of actin stress fibers and vinculin-associated adhesion complexes, intrinsic migratory characteristics, and the expression of eNOS mRNA and protein. These results thus reveal a unique responsiveness of ECs to an arterial waveform and begin to elucidate the complex sensing capabilities of the endothelium to the dynamic characteristics of flows throughout the human vascular tree.

Adaptation, Physiological↗
PubMed1996

Protection of porcine endothelial cells from complement-mediated cytotoxicity by the human complement regulators CD59, C1 inhibitor, and soluble complement receptor type 1. Analysis in a pig-to-human in vitro model relevant to hyperacute xenograft rejection.

Inhibition of complement activation is considered a prerequisite to overcome hyperacute xenograft rejection. In the present study, we investigated the efficacy of C1 inhibitor (C1 inh) and recombinant soluble complement receptor type 1 (rsCR1) to protect xenogeneic cells against complement-mediated cytotoxicity in an in vitro xenotransplantation model. The addition of the soluble complement regulators to human serum led to a dose-dependent inhibition of complement-mediated destruction of aortic porcine endothelial cells (PEC). On a molar base, rsCR1 was more efficient than C1 inh. Transfection of PEC with cDNA of human CD59 resulted in several clones where protection against complement-mediated cell destruction correlated with the expression level of the inhibitor. Addition of low concentrations of C1 inh and rsCR1 to a CD59 (human)-positive PEC clone, expressing a suboptimal level of the membrane-bound regulator, resulted in a significant improvement of protection against complement-mediated cell destruction.

Animals↗
PubMed1992

A new in vitro model to test the effectiveness of topical antimicrobial agents. Use of an artificial eschar.

An in vitro method using a burn eschar 'equivalent' is described to compare the efficacy of several antimicrobial creams. Fragments of lyophilized pigskin were used as 'germ carriers' and after 24 h of treatment the effectiveness of the antimicrobial creams was tested through the evaluation of bacterial recovery both from the surface and from within the 'germ carriers'. The results showed a striking full skin thickness effectiveness of 0.5 per cent chlorhexidine, 10 per cent providone iodine and 2 per cent cerium nitrate in comparison with the weak activity demonstrated by 1 per cent silver sulphadiazine cream.

Administration, Topical↗
PubMed1994

An in vitro model for measuring tryptase release from nasal mucosa in response to allergen challenge.

A simple in vitro nasal mucosal culture model has been developed to measure release of the mast cell specific enzyme tryptase in response to allergen challenge. Patients who were undergoing inferior turbinectomy were skin-tested for commonly inhaled allergens. The mucosa from the inferior turbinates was kept viable using Minimal Essential Medium. Tryptase release into the medium was measured using the Pharmacia Riact Assay. There was a significant increase in tryptase release in response to allergen challenge from the mucosa harvested from skin-test positive patients. Mucosa from skin-test negative patients failed to demonstrate an increase in tryptase release. This could prove to be a useful research model for the study of nasal type I hypersensitivity and drugs that affect it.

Allergens↗
PubMed1993

In vitro models of thyroid cancer.

The well defined multiple steps and alternative pathways of thyroid follicular cell neoplasia in vivo provide a theoretically ideal basis for modelling the process of epithelial tumorigenesis in vitro. Much progress has already been made in studying the earliest stages. Pure cultures of normal human follicular cells representative of the differentiation and proliferative characteristics of the intact gland can be readily prepared and genetically manipulated with retroviral vectors, permitting direct testing of the causal role of oncogenes identified from analyses of clinical samples. Such studies have provided the clearest proof to date in a human system that ras mutation is both necessary and sufficient for induction of a benign epithelial neoplasm. The contrasting effects of ret activation promise to provide a model for studying the molecular basis for the divergent behaviour of papillary and follicular tumours derived from the same normal cell. For biochemical studies requiring larger cell numbers, conditionally immortalized normal cells have shown promising results, although instability of differentiation is still a problem. Spontaneously immortalized rat cells have also proved useful for this purpose, despite their uncertain relationship to in vivo states. The major challenge now is to overcome the poor in vitro growth of thyroid cancer cells to permit meaningful reconstruction of the later stages of tumorigenesis.

Cell Transformation, Neoplastic↗
PubMed1994

An in vitro model of fibroblast activity and adhesion formation during flexor tendon healing.

We studied fibroblast activity during tendon healing with an in vitro tendon culture model. Tendons were embedded in a translucent collagen gel matrix whose porous nature permitted free nutrient diffusion, fibroblast migration out of the tendon, and microphotographic documentation of fibroblast activity. Experiments were performed using one or more tendons cultured in the same collagen gel. We identified three zones of fibroblast activity in the gel. Zone I was an area of randomly dispersed cells directly adjacent to the tendon where collagen synthesis and remodeling were probably taking place. In zone II, spindle-shaped fibroblasts were aligned pointing away from the cut tendon end forming a sunburst-like aggregate of cells. Zone II fibroblasts were responsible for formation of migration trails by exerting a mechanical force on the collagen matrix, which was evident as a local gel contraction. Zone III was the leading edge of the sunburst populated by the fastest moving fibroblasts, which responded to guidance by other cut tendon ends. We speculate that the collagen gel used in the culture system may help maintain a chemotactic concentration gradient that allows fibroblasts to locate other distal cut tendon surfaces also embedded in the collagen gel.

Animals↗