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PubMed1985

In vitro models of mutagenesis.

The bypass of lesions in DNA with insertion of nucleotides opposite damaged bases has been studied as a model for mutagenesis in an in vitro system. Lesions introduced by dimethyl sulfate at adenines and by ultraviolet light at pyrimidine dimers act as termination sites on both double- and single-stranded DNA templates. Base selection opposite noninformational lesions is, in part, a property of the polymerases: different polymerases have different selectivities although all polymerases tested seem to prefer purines. The ability to insert "incorrect" bases is determined in part by the sequence 5' to the lesion on the template strand. The hypothesis that damaged purines tend to result in transversions can be applied to published data on activation of the c-ras oncogene.

Base Sequence↗
PubMed2004

The role of endogenous opioids in the control of gastrointestinal motility: predictions from in vitro modelling.

Gastrointestinal motility can be assessed in vitro by investigating the effects of drugs or gene knockouts on intestinal propulsion, and on neurone-mediated responses evoked by electrical field stimulation (EFS). The latter predominantly measure enteric motor activity and can detect prokinetic activity of exogenous agents. Some evidence suggests that naloxone has prokinetic activity when evaluated for an ability to modulate responses to EFS, but the effects are inconsistent across different species or intestinal regions. Models of intestinal peristalsis measure an integrated sensory-motor nerve function and possess more intact neuro-neuronal connections. In such preparations, the effects of naloxone also suggest a prokinetic property but again, this is inconsistent. By contrast, consistent prokinetic activity of naloxone is apparent in models where peristalsis is compromised by drug-induced suppression of motor nerve activity or by modulation of endogenous processes using receptor antagonists or inappropriate intraluminal distension. These data suggest that endogenous opioids play little or no role in normal intestinal physiology, but suppress intestinal motility when motor function is compromised. Consequently, drugs that antagonize opioid receptors may exert prokinetic activity in conditions where intestinal motility is reduced, such as constipation. Further work is required to elucidate the opiate receptor(s) involved.

Animals↗
PubMed1991

An in vitro model showing different rates of substrate cycle for phosphofructokinases of Escherichia coli with different kinetic properties.

An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fructose-bisphosphatase. The model is applied to the study of three PFK of Escherichia coli: two isoenzymes, phosphofructokinase-1 (PFK-1) and phosphofructokinase-2 (PFK-2), and a mutant form of phosphofructokinase-2 (PFK-2*). Results show that for a variety of conditions the PFK-1/fructose-bisphosphatase pair gives the lowest and the PFK-2*/fructose-bisphosphatase pair the highest rates of substrate cycle, with the PFK-2/fructose-bisphosphatase pair in an intermediate position. The effects of variables such as maximum activity ratios and MgATP concentration were explored. The possible role of MgATP in decreasing the futile cycle of the PFK-2/fructose-bisphosphatase pair is described. The results are discussed in terms of possible metabolic consequences of PFK-2* and of predictions of the model to be tested in vivo.

Adenosine Triphosphate↗
PubMed2003

New approaches to in vitro models of blood-brain barrier drug transport.

The pharmaceutical industry has been searching for an in vitro blood-brain barrier (BBB) model that preserves in vivo transporter functions in CNS drug discovery and development. The application of conditionally immortalized cell lines derived from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene, is a rational and promising approach to such a workable in vitro BBB model. The established brain capillary endothelial cell lines retain the in vivo transport rate of several compounds and various forms of gene expression. Furthermore, this new approach has enabled the development of stable and reproducible co-culture models with a pericyte cell line and/or an astrocyte cell line.

Animals↗
PubMed2000

Gamma and beta frequency oscillations in response to novel auditory stimuli: A comparison of human electroencephalogram (EEG) data with in vitro models.

Investigations using hippocampal slices maintained in vitro have demonstrated that bursts of oscillatory field potentials in the gamma frequency range (30-80 Hz) are followed by a slower oscillation in the beta 1 range (12-20 Hz). In this study, we demonstrate that a comparable gamma-to-beta transition is seen in the human electroencephalogram (EEG) in response to novel auditory stimuli. Correlations between gamma and beta 1 activity revealed a high degree of interdependence of synchronized oscillations in these bands in the human EEG. Evoked (stimulus-locked) gamma oscillations preceded beta 1 oscillations in response to novel stimuli, suggesting that this may be analogous to the gamma-to-beta shift observed in vitro. Beta 1 oscillations were the earliest discriminatory responses to show enhancement to novel stimuli, preceding changes in the broad-band event-related potential (mismatch negativity). Later peaks of induced beta activity over the parietal cortex were always accompanied by an underlying gamma frequency oscillation as seen in vitro. A further analogy between in vitro and human recordings was that both gamma and beta oscillations habituated markedly after the initial novel stimulus presentation.

Acoustic Stimulation↗
PubMed1991

Epithelial-connective tissue interaction on the tooth surface: an in vitro model.

In the present study, and in vitro system was developed and designed to examine the interaction between gingival fibroblasts (GF) and epithelial cells (EC) on the tooth surface. Porcine roots were cut transversely into 300 microns-thick root slices (RS). Gingival explants were placed on the upper RS surface and cultured in a defined medium permissive for the growth of EC. After 4 or 6 days, RS yielding EC were transferred onto confluent cultures of GF and further co-cultured for either 4 or 8 d. Cultures were then fixed and examined by SEM. The upper RS surfaces and the upper half of their peripheral aspect were covered by EC. The lower half of the peripheral RS surfaces were populated by GF originating from the confluent culture of GF. EC and GF made contact at approximately the middle of the side of the root slice. In cultures of epithelial components grown in defined medium for either 4 or 6 d and harvested 4 d after assembling the system, the EC-GF junction was located 117 +/- 45 and 271 +/- 82 microns, respectively from the upper RS aspect. Extending the co-culture period did not affect the EC-GF junction location. These results indicate that GF-EC contact stops the migration of these cells on root surfaces in vitro. The described system should be valuable for studying cellular events that may affect the formation of a new dentogingival junction following surgical periodontal therapy.

Animals↗
PubMed1986

Effects of prednisolone on beta-adrenergic desensitization of normal peripheral lymphocytes: an in vitro model for steroid-controlled tachyphylaxis.

In vitro culture of human peripheral blood lymphocytes with the beta-adrenergic catecholamine isoproterenol for 24 hours, induced homologous beta-adrenergic desensitization, i.e. a large decrease in the number of beta-adrenergic binding sites and loss of the adenylate cyclase response to isoproterenol, without altering the effectiveness of prostaglandin E1 to stimulate the enzyme. Lymphocyte cultures pulsed for 24 hours with isoproterenol, washed free of the agent and cultured in hormone-free medium for 48 hours still showed marked suppression of the beta-adrenergic adenylate cyclase response and a lack of beta-receptors. When prednisolone was added to the isoproterenol-depleted resuspension medium of desensitized lymphocytes, however, the beta-adrenergic system recovered fully within 48 hours. Treatment of desensitized lymphocytes with prednisolone in the continuous presence of isoproterenol failed to restore beta-adrenergic responsiveness of the cells. The results are discussed with respect to the reconstituting effect of prednisolone on beta-adrenergic responsiveness in bronchial asthma after therapy induced tachyphylaxis.

Adenylyl Cyclases↗
PubMedDate not supplied

Round window membrane permeability. An in vitro model.

The round window membrane is regarded to be the main route for passage of potentially ototoxic substances from the middle ear cavity into the inner ear. This may be of clinical importance in acute otitis media and chronic otitis media, where sensorineural hearing impairment sometimes develops. The accuracy and reliability of an in vitro round window membrane permeability model was studied. The round window membrane of the mongolian gerbil was resected, together with its bony niche. The preparation was mounted between two glass chambers representing the middle ear and the inner ear. Passage through the round window membrane did not occur within 3 h for low density lipoprotein with a molecular weight of 2,300 kD. Only minute amounts of highly density lipoprotein, of molecular weight 115-350 kD, passed the round window membrane. The passage rate of horseradish peroxidase, which has previously been shown to pass the round window membrane in vivo, was estimated. The design of the present model is considered to make feasible controlled permeability studies on the round window membrane. Passage rates for different substances through the round window membrane can be calculated under controlled conditions by using this type of in vitro model.

Animals↗
PubMed2006

Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells.

The establishment of the primitive streak and its derivative germ layers, mesoderm and endoderm, are prerequisite steps in the formation of many tissues. To model these developmental stages in vitro, an ES cell line was established that expresses CD4 from the foxa2 locus in addition to GFP from the brachyury locus. A GFP-Bry(+) population expressing variable levels of CD4-Foxa2 developed upon differentiation of this ES cell line. Analysis of gene-expression patterns and developmental potential revealed that the CD4-Foxa2(hi)GFP-Bry(+) population displays characteristics of the anterior primitive streak, whereas the CD4-Foxa2(lo)GFP-Bry(+) cells resemble the posterior streak. Using this model, we were able to demonstrate that Wnt and TGF-beta/nodal/activin signaling simultaneously were required for the generation of the CD4-Foxa2(+)GFP-Bry(+) population. Wnt or low levels of activin-induced a posterior primitive streak population, whereas high levels of activin resulted in an anterior streak fate. Finally, sustained activin signaling was found to stimulate endoderm commitment from the CD4-Foxa2(+)GFP-Bry(+) ES cell population. These findings demonstrate that the early developmental events involved in germ-layer induction in the embryo are recapitulated in the ES cell model and uncover insights into the signaling pathways involved in the establishment of mesoderm and endoderm.

Activins↗
PubMed1996

Isolated glomeruli and cultured mesangial cells as in vitro models to study immunosuppressive agents.

Immunosuppressive agents, such as cyclosporin A (CsA), by their vasoconstrictive properties, induce in vivo in patients and rodents a dramatic fall in renal hemodynamics. The aim of this study is to review the ability of some physiological and/or pharmacological agents which are supposed to be involved in the renal physiopathology of CsA to prevent the contraction induced by CsA in two in vitro glomerular models. Isolated glomeruli are obtained by a sieving method from male Sprague-Dawley rat superficial cortex. Mesangial cells from these isolated glomeruli are cultured in RPM1 1640 medium with 20% FCS in 5% CO2 atmosphere. The area of isolated glomeruli and cultured mesangial cells is assessed by an image analyzer with a video camera. Each glomerulus and cell is its own control and is photographed before incubation with any drug (T0) and then during incubation at 5, 10, 20, and 30 min. Incubations are performed during 30 min with 10(-6) mol/L CsA either with a 10 min pretreatment with the vasoactive agent or without pretreatment. CsA alone induces a time- and dose-dependent decrease in glomerular structure area (-4.7% at 10 min, -10.3% at 20 min, and -12.0% at 30 min for isolated glomeruli); Cremophore excipient or control solute does not induce any significant decrease in surface area. CsA with 10(-6) mol/L verapamil pretreatment induces only a slight decrease: -1.5% at 10 min, -3.0% at 20 min, and -4.8% at 30 min. Calcium blockers nifedipine and felodipine produce similar results. Likewise, with 10(-8) mol/L prostacyclin analog (iloprost), only a slight area decrease in mesangial cells is noted: -1.3% at 5 min, -1.8% at 10 min, and -3.3% at 20 min; with 10(-6) mol/L TXA2 synthesis inhibitor (CGS 12970) the results are -2.0% at 10 min, -3.6% at 20 min, and -4.3% at 30 min. Finally, a similar protective effect can be noted with 10(-5) mol/L theophylline: -0.4; -1.5 and -1.9% at 10, 20, and 30 min. In conclusion, CsA-induced contraction in two in vitro glomerular models can be partially or even totally prevented by pretreatment with various pharmacological agents.

Animals↗
PubMed1994

An in vitro model for T cell-independent induction of humoral immunity. A requirement for NK cells.

We previously established an in vitro polyclonal model for membrane Ig-mediated Ig secretion by B cells in response to T cell-independent type 2 (TI-2) Ags, by using dextran-conjugated anti-IgD Abs (alpha delta-dex). We demonstrated that resting B cells activated with alpha delta-dex plus IL-2 secreted large amounts of Ig only in the presence of NK cells. In this report we show that, in contrast to small B cell-enriched spleen cells that require activation with the combination of alpha delta-dex and IL-2 for induction of Ig secretion, large B cells were induced to secrete Ig in response to alpha delta-dex, alone. These responses were inhibited by previous depletion of asialo-Gm-1+ cells from the B cell-enriched population, and restored both by the addition of freshly explanted NK cells and by in vitro-activated NK cells, suggesting a requirement for NK cells. Small alpha delta-dex-activated B cells could, however, also be stimulated to secrete Ig in the absence of added cytokines but only after the addition of in vitro-activated NK cells and not freshly explanted splenic NK cells. When highly purified large B cells, obtained by electronic cell sorting, were cultured with in vitro-activated NK cells, Ig secretion was induced even in the absence of any other added B cell stimulus. Because the splenic marginal zone has been implicated in humoral immune responses to TI-2 Ags, we further fractionated large B cells into marginal zone (MZB) and follicular (FB) B cells by electronic cell sorting. In vitro-activated NK cells stimulated large MZB, but not FB, cells to secrete Ig in the absence of exogenous stimuli. These data establish a T cell-independent model for induction of Ig synthesis in the absence of any added cytokine and demonstrate a role for freshly explanted NK cells in stimulating Ab production in response to TI-2 Ags. Further, they show that pre-activated NK cells can induce Ig secretion from pre-activated B cells even in the absence of any added stimuli. These data also underscore a special role for the MZB cell in these responses.

Animals↗
PubMed2003

Mass transport of macromolecules within an in vitro model of supragingival plaque.

The aim of this study was to examine the diffusion of macromolecules through an in vitro biofilm model of supragingival plaque. Polyspecies biofilms containing Actinomyces naeslundii, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, Veillonella dispar, and Candida albicans were formed on sintered hydroxyapatite disks and then incubated at room temperature for defined periods with fluorescent markers with molecular weights ranging from 3,000 to 900,000. Subsequent examination by confocal laser scanning microscopy revealed that the mean square penetration depths for all tested macromolecules except immunoglobulin M increased linearly with time, diffusion coefficients being linearly proportional to the cube roots of the molecular weights of the probes (range, 10,000 to 240,000). Compared to diffusion in bulk water, diffusion in the biofilms was markedly slower. The rate of diffusion for each probe appeared to be constant and not a function of biofilm depth. Analysis of diffusion phenomena through the biofilms suggested tortuosity as the most probable explanation for retarded diffusion. Selective binding of probes to receptors present in the biofilms could not explain the observed extent of retardation of diffusion. These results are relevant to oral health, as selective attenuated diffusion of fermentable carbohydrates and acids produced within dental plaque is thought to be essential for the development of carious lesions.

Actinomyces↗
PubMed2000

Toxicity of preserved and unpreserved antiglaucoma topical drugs in an in vitro model of conjunctival cells.

PURPOSE: To compare the toxicity of a short-time application of timolol with benzalkonium chloride (timolol-BAC+) and unpreserved timolol (timolol-BAC-) in a human conjunctival cell line. METHODS: Chang's conjunctival cell line (ATCC CCL 20.2) was treated for 15min. with 0.1%, 0.25% or 0.4% timolol-BAC(+) or BAC(-) and then examined immediately or 24h later. Cell viability, chromatin condensation and free radicals production were studied by microplate cold light cytometry. Moreover, relative cell number was evaluated by crystal violet colorimetric test. The comparison was done with an oxidative stress model of cells treated with 0.001-0.000001% hydrogen peroxide (H(2)O(2)). In addition, cell size and the expression of an apoptotic marker Apo2.7 were evaluated by flow cytometry. RESULTS: Timolol-BAC(+) induced a rapid decrease in cell viability ranging from 40% immediately after treatment to 85% 24h later. A small initial decrease in cell viability was also observed with all tested concentrations of timolol-BAC(-) but, 24h later, cell viability either tended to remain constant or cells completely recovered. Cell viability fell down after 24h exposure to 0.001% H(2)O( 2) whereas it was not modified at lower concentrations. 24h after treatment with 0.25% timolol-BAC(+), the relative cell number was reduced by 55% whereas it did not vary after 0.25% timolol-BAC(-) treatment. Only timolol-BAC(+) induced chromatin condensation and cell size reduction. Moreover, cells treated with timolol-BAC(+) overexpressed the apoptotic marker Apo2.7. Both timolol-BAC(+) and BAC(-) induced reactive oxygen species (ROS) production which was significantly more important when 0.25% or 0.4% timolol-BAC(+) were applied. Only 0.001% and 0.0001% H(2)O(2) generated a significant free radicals production. CONCLUSION: In our model of conjunctival cells in vitro timolol-BAC(+) induced irreversible cytotoxic damage with some characteristics of apoptosis. The active compound of timolol-BAC(-) could be responsible for reactive oxygen species production and for cell viability variations. The role of oxidative stress in timolol-BAC(+)-induced toxicity seems not to be predominant. in vitro toxic effects of antiglaucoma drugs could, in part, explain some ocular surface disorders in long-term treated patients.

Adrenergic beta-Antagonists↗
PubMed2002

Production of amines in equine cecal contents in an in vitro model of carbohydrate overload.

Acute laminitis can be induced experimentally in horses by the administration of carbohydrate, resulting in fermentation within the cecum and ischemia-reperfusion of the digits. The products of fermentation that trigger acute laminitis are as yet unknown; however, compounds such as amines might play a role due to their potential vasoactive properties. The objectives of this study were to quantify the amines present in equine cecal contents and to use a model of carbohydrate overload in vitro to test the hypothesis that carbohydrate fermentation is associated with increased amine production. Cecal contents from each horse were divided into aliquots and incubated anaerobically with either cornstarch or inulin (a form of fructan carbohydrate; both 1 g/100 mL). The pH was measured and samples were taken at the same time for amine measurement by HPLC at 2-h intervals over a 24-h period. In a second set of experiments, the effects of the antibiotic virginiamycin (1 mg/100 mL), calcium (CaPO4; 0.3 g/100 mL), and plant steroidal saponin (Yucca schidigera extract; 0.1 g/100 mL) were examined on pH and amine concentrations in cecal contents incubated with starch or inulin. Both starch and inulin caused significant time-dependent falls in pH, from 6.7 +/- 0.1 at 0 h to 5.2 +/- 0.1 (starch) and 5.0 +/- 0.1 (inulin) at 24 h. Fermentation of carbohydrate was also associated with increased production of phenylethylamine and isoamylamine (two- to threefold increases) as well as putrescine and cadaverine (1.5- to twofold increases). Virginiamycin inhibited the fall in pH and increases in production of phenylethylamine and isoamylamine, while calcium phosphate moderated the changes in pH only. Yucca schidigera extract was without effect. These data show that fermentation of carbohydrate by equine cecal microbiota may lead to increased production of amines.

Acute Disease↗
PubMed1986

The isolated perfused porcine skin flap (IPPSF). I. A novel in vitro model for percutaneous absorption and cutaneous toxicology studies.

This article describes the development of a novel in vitro alternative animal model for dermatology and cutaneous toxicology. A single-pedicle, axial-pattern, island-tubed skin flap was created in crossbred Yorkshire weanling pigs in one surgical procedure, then transferred 2 or 6 days later to a computer-controlled temperature-regulated perfusion chamber for 10-to 12-hr studies. Perfusate consisted of Krebs-Ringer bicarbonate buffer (pH 7.4) containing albumin and glucose. Viability was assessed by glucose utilization, lactate production, an absence of significant concentrations of the intracellular enzyme lactate dehydrogenase in the perfusate, and light and electron microscopy. A mean lactate to glucose ratio of 1.6 for flaps harvested 2 days after surgery and 1.8 for flaps taken 6 days after surgery suggested primarily anaerobic glycolysis. This preparation would be a humane alternative animal model for studies in cutaneous toxicology, physiology, oncology, and percutaneous drug absorption and metabolism.

Animals↗
PubMed1986

In vitro models in the study of antibiotic therapy of infections in neutropenic patients.

Most conventional methods for in vitro testing of antibiotics involve exposure of a bacterial inoculum to a constant, static concentration of drug. The in vivo concentrations of antibiotics change continually according to their pharmacokinetics. When two drugs are used, the ratios of their concentrations also change with time. The usual checkerboard tests for combined activity of two or more antibiotics do not consider the pharmacokinetic properties. An in vitro two-compartment pharmacokinetic model has been developed that presents changing concentrations of one or two antibiotics to isolated bacterial inocula. This model simulates the treatment of a bacterial infection in the absence of host defenses and thus mimics infection in a neutropenic patient. This model has been used to study the synergistic activity of beta-lactam/aminoglycoside combinations compared with conventional checkerboard and time-kill methods. Also, in this model, the addition of azlocillin or ceftazidime to netilmicin prevented the selection of resistant subpopulations of Pseudomonas aeruginosa that occurred with the aminoglycoside alone. In vitro pharmacokinetic models add kinetic parameters to conventional susceptibility testing and may prove useful in the design of trials of the optimal dosing and administration of antibiotics for infected neutropenic patients.

Agranulocytosis↗
PubMed2002

Comparative pharmacodynamics of three newer fluoroquinolones versus six strains of staphylococci in an in vitro model under aerobic and anaerobic conditions.

Six strains of staphylococci were exposed to levofloxacin, moxifloxacin, or trovafloxacin in an in vitro pharmacodynamic model under both aerobic and anaerobic conditions. Each agent demonstrated a rapid 3-log(10) kill versus susceptible isolates regardless of condition. Against clinical isolates with reduced susceptibility, regrowth occurred by 24 h and was frequently associated with further increases in MICs.

Aerobiosis↗
PubMed1993

In vitro model of hypoxia: basic fibroblast growth factor can rescue cultured CNS neurons from oxygen-deprived cell death.

We established an in vitro hypoxia model and investigated the protective effect of basic fibroblast growth factor (bFGF) against neuronal cell death caused by hypoxia. Hippocampal neurons obtained from rats on embryonic day (E) 17 and 20 and on postnatal day (P) 4 were cultured for 6-24 h in an oxygen-deprived state. This in vitro hypoxia study showed that the cultured neurons were sensitive to the oxygen deprivation. The cultured P4 rat hippocampal neurons seemed to be weaker in the hypoxia condition than those of E17 and E20 rats, suggesting that the cultured postnatal cells might be sensitive to hypoxia. bFGF, but not nerve growth factor, prevented the neuronal cell death caused by hypoxia in a dose-dependent manner.

Animals↗