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At least 487 records · Page 27Linked to original sources
PubMed1995

Characterization of LLC-PK1 kidney epithelial cells as an in vitro model for studying renal tubular reabsorption of protein drugs.

PURPOSE: The purpose of this study was to assess whether LLC-PK1 renal epithelial cells could serve as an in vitro model for studying the renal tubular reabsorption of protein drugs. METHODS: The association of 111In-labeled model protein drugs, bovine serum albumin (BSA), superoxide dismutase (SOD), soybean trypsin inhibitor (STI), and [Asu1.7]-eel calcitonin (Asu-ECT), with the monolayers of LLC-PK1 renal epithelial cells was characterized under various conditions. RESULTS: The cellular association of these proteins was temperature-dependent and varied according to the protein. Saturation kinetics were observed for STI association, with the apparent Km and Vmax values determined to be 66.3 micrograms/ml and 250 ng/mg protein/min, respectively. The association of STI decreased with increases in medium pH from 5.4 to 8.4 and was inhibited significantly by 2,4-dinitrophenol, sodium azide, cytochalasin B, and colchicine, suggesting that the cellular association involved endocytosis. Mutual inhibition was observed in competitive binding experiments with the four protein drugs, suggesting that they shared a common binding site on the luminal membrane of LLC-PK1 cells. Taken together, these findings show that a variety of protein drugs bind to LLC-PK1 cells in a non-specific manner and possibly undergo endocytosis, a phenomenon that is similar to in vivo proximal tubular reabsorption. CONCLUSIONS: LLC-PK1 renal epithelial cells would be a suitable model system for the study of the renal proximal tubular reabsorption of protein drugs.

Absorption↗
PubMed1982

An in vitro model for studying the effects of pharmacological agents on human ciliary beat frequency: effects of lignocaine.

1 The effects of lignocaine hydrochloride on ciliary beat frequency (CBF), measured photometrically in vitro, using human epithelium obtained by nasal brushing was studied in 20 healthy subjects. Above lignocaine concentrations of 2.5 x 10(-3) g/ml cilio-inhibition occurred in a dose-dependent manner. Concentrations greater than 2 x 10(-2) g/ml caused ciliostasis. 2 Lignocaine aerosol was sprayed in vivo on the mucosa of one nasal cavity in five healthy subjects and five patients premedicated for bronchoscopy. CBF, measured in vitro in nasal brushings, was not different for treated and untreated sides. 3 Variation of intrinsic CBF with time after nasal brushings were taken was studied in ten normal subjects. No significant change in CBF was found until after 24 h. 4 We conclude that lignocaine hydrochloride, at concentrations three orders of magnitude above drug levels encountered in clinical practice, inhibits human CBF in a dose-dependent manner. Aerosolised lignocaine hydrochloride in doses which produce local nasal mucosal anaesthesia, does not inhibit CBF subsequently measured in vitro. Lignocaine is a suitable local anaesthetic for use at fibreoptic bronchoscopy when samples are being obtained for study of ciliary function. 5 The measurement of CBF in nasal mucosal brushings provides an in vitro model suitable for studying the effects of pharmacological agents on human ciliary activity.

Adult↗
PubMed1988

[Studies on KU-1 and KU-7 cells as an in vitro model of human transitional cell carcinoma of urinary bladder].

Cellular characteristics of KU-1 and KU-7 cells after a long term in culture were evaluated for eligibility of in vitro model of human transitional cell carcinoma of the urinary bladder. The KU-7 cells derived from superficial papillary tumor showed small, polygonal and homogeneous cells, while the KU-1 cells derived from a broad basic invasive carcinoma showed a variety in size as well as piling up tendency. The adhesiveness of these cells in culture was examined and a significant difference in their cellular structures, a thick multilayered mass in KU-1 while a thin flat spreading in KU-7, was found by each rotating culture and culture on collagen sponge matrix. KU-7 cells cultured without sera responded to transforming growth factor (TGF-alpha) but KU-1 cells did not show any response to the factor. These findings indicate that KU-7 and KU-1 cells have maintained some basic characteristics of papillary tumors and infiltrating carcinomas, respectively.

Animals↗
PubMed2006

Class III effects of dofetilide and arrhythmias are modulated by [K+]o in an in vitro model of simulated-ischemia and reperfusion in guinea-pig ventricular myocardium.

To evaluate class III effects of clinically relevant concentrations of dofetilide (5 and 10 nmol/l) and the effects of extracellular potassium [K+]o modulation of arrhythmias onset at the level of the "border zone," we used a previously reported in vitro model whereby normoxic and ischemic/reperfused zones were studied. Guinea-pig right ventricular strips (driven at 1 Hz at 36.5+/-0.5 degrees C) were superfused with Tyrode's solution in oxygenated (HCO3- 25 mmol/l, K+ 4 mmol/l, pH 7.35+/-0.05, glucose 5.5 mmol/l: normal zone) and ischemia-simulating conditions (HCO3- 9 mmol/l, pH 6.90+/-0.05, no oxygen and no glucose: altered zone) having either [K+]o 4 (n=20), 8 (n=20) or 12 (n=20) mmol/l. Action potentials in normal and altered zones were recorded simultaneously during 30 min of simulated-ischemia and after 30 min of reperfusion with oxygenated Tyrode's solution. Each preparation served as control for successive phases of dofetilide studies (at 5 and 10 nmol/l) and action potential values were normalized to those present at the beginning of the experiment. During simulated-ischemia, the higher the [K+]o the worse were action potential changes, although full recovery was seen upon 30 min of reperfusion in all [K+]o groups. A high incidence of ischemia/reperfusion arrhythmias was observed in 4 and 12 mmol/l [K+]o groups as opposed to a low incidence of arrhythmias in 8 mmol/l [K+]o group. Dofetilide at 5 and 10 nmol/l with all [K+]o explored: (i) exhibited class III effects, (ii) was effective (or neutral) against ventricular arrhythmias during both simulated-ischemia and reperfusion, and (iii) did not globally increase the dispersion of action potential durations between normal and altered zones. Different arrhythmogenic mechanisms are involved in this model at different [K+]o with 8 mmol/l providing relative protection. Class III effects of dofetilide are evident in the normal zone when in the ischemic-like zone [K+]o ranges from 4 to 12 mmol/l. Thus dofetilide did not increase dispersion of repolarization and had either an antiarrhythmic or a neutral effect during ischemia/reperfusion.

Action Potentials↗
PubMed1986

The mechanism of human bladder tumor implantation in an in vitro model.

Implantation of tumor cells in the bladder following transurethral resection of superficial bladder tumors is believed to be one factor in the etiology of bladder tumor recurrences. Using an in vitro model system we have studied the initial interaction between bladder carcinoma cells and a naturally produced basement membrane-like substrate. Minced explants of superficial low grade human bladder tumors from 10 patients were plated into culture dishes coated with a naturally produced extracellular matrix (ECM). This ECM has been shown to resemble the human urothelial basement membrane and submucosa in its macromolecular composition and ultrastructural appearance. It was found that a firm attachment of the human bladder tumor cells occurred within one hour, reached a maximal value within 24 hours and was followed by flattening and proliferation of the plated cells. These results indicate that prevention of tumor implantation should be initiated in the first hour after transurethral resection of the bladder tumors. This assay can be used for the investigation of various treatments to prevent tumor implantation.

Basement Membrane↗
PubMed2001

An in vitro model for the study of microglia-induced neurodegeneration: involvement of nitric oxide and tumor necrosis factor-alpha.

The precise function of activated microglia and their secretory products remains controversial. In order to assess the role of microglial secretion products, we established an in vitro model of an inflammatory reaction in the brain by co-culturing microglial and neuronal cell lines. Upon stimulation with interferon-gamma and lipopolysaccharides, the microglial cells adopted an activated phenotype and secreted tumor necrosis factor-alpha (TNF-alpha), prostaglandin E(2) and nitric oxide (NO). Neuronal degeneration was quantified by measuring the concentrations of microtubule associated protein tau and neuron specific enolase, which are also used as diagnostic tool in Alzheimer's disease, in supernatants. In activated contact co-cultures, the levels of these neuronal markers were significantly raised compared to non-activated co-cultures. NO-synthase inhibitors significantly diminished the rise of tau in activated co-cultures, while indomethacin, superoxide dismutase, or a neutralizing TNF-alpha antibody did not. When a chemical NO-donor or TNF-alpha were added to pure neuronal cultures, cell viability was significantly reduced. TNF-alpha increased neuronal sensitivity towards NO. There were indications that a part of the cells died by apoptosis. This model demonstrates a neurotoxic role for NO in microglia-induced neurodegeneration and provides a valuable in vitro tool for the study of microglia-neuron interactions during inflammation in the brain.

Animals↗
PubMed2005

An in vitro model for fluid pressurization of screw holes in metal-backed total joint components.

Fluid pressure may stimulate osteolysis near screw holes in joint arthroplasty components. We developed a generalized in vitro model of a polyethylene liner and metal backing with a screw hole to investigate whether implant design factors influence local fluid pressure. We observed an order of magnitude of variation in the peak screw hole pressure (from 16.0 and 163 kPa) under clinically relevant loading conditions. Of the implant factors investigated, the surface finish of the metallic base plate had the greatest effect on peak screw hole fluid pressures; the thickness of the polyethylene liner, as well as the gap between the liner and the base plate, were also significant design variables. Our data suggest that unpolished metal base plates, thick polyethylene liners, and tight conformity between the liner and the metal base plate will all contribute to significantly reduced peak screw hole fluid pressures in joint arthroplasty.

Bone Screws↗
PubMed2004

Neutrophil-mediated formation of carcinogenic N-nitroso compounds in an in vitro model for intestinal inflammation.

In order to study neutrophil-mediated formation of carcinogenic N-nitroso compounds as a mechanism of inflammation-related colon carcinogenesis, we designed an in vitro model for intestinal inflammation, consisting of a coincubation system with human colon cells (Caco-2 cells) and activated human neutrophils (PMN), as important immunoreactive cells. We investigated whether nitrosamines and nitrosamides could be formed upon addition of dimethylamine, morpholine and methylurea to the coincubations as nitrosatable precursors, which are known to produce carcinogenic N-nitroso compounds. Incubations of pure nitric oxide with dimethylamine and morpholine showed that NO-mediated formation of nitrosodimethylamine and nitrosomorpholine is possible under the incubation conditions. During the coincubations of activated PMN and Caco-2 cells, 0.34 nmol nitrite/10(6) PMN was produced. Dose-dependent formation of NMOR was observed in this PMN/Caco-2 system; addition of 5mM morpholine resulted in a significantly increased NMOR formation of 4.2 nM. However, no detectable NDMA and methylnitrosourea were formed in this coincubation system. These results suggest that activated human neutrophils are able to synthesize carcinogenic N-nitrosamines, e.g. NMOR, which implies a risk of colon carcinogenesis during chronic inflammation. However, the observed relatively low level of nitrosation suggests that also other risk factors are contributing to the association between chronic inflammation and colon cancer risk.

Caco-2 Cells↗
PubMedDate not supplied

Post-implantation differentiation and proliferation of cytotrophoblast cells: in vitro models--a review.

Cytotrophoblast cells, specialized placental cells, proliferate early in pregnancy and then differentiate into tumour-like cells that invade the uterus and its vasculature. We have established in vitro models of three-dimensional cultures for anchoring villi and cell islands on extracellular matrix in order to study regulation of cytotrophoblast cell differentiation and proliferation. It has been demonstrated that cytotrophoblast cells from cell islands and cell columns share the same characteristics and that their differentiation is triggered by interaction with the extracellular matrix. The fact that during much of the first trimester maternal blood flow to the placenta is at a minimum, suggests that oxygen tension might regulate cytotrophoblast proliferation and differentiation. Hypoxia, comparable to that encountered by early gestation cytotrophoblast cells in the intervillous space, stimulated the cells to enter the cell cycle and inhibited their differentiation along the invasive pathway. Thus, oxygen gradient and cell-matrix interactions at the maternal-fetal interface play an important role in the regulation of cytotrophoblast proliferation and differentiation.

Cell Culture Techniques↗
PubMed1989

An in vitro model for the scintigraphic detection of thrombi using a 99Tcm-labelled antifibrin monoclonal antibody.

Because of their specific targeting properties, monoclonal antibodies have found widespread use in nuclear medicine. In this paper, a method is described for the evaluation of immunoscintigraphic parameters for the detection of thrombi, using a 99Tcm-labelled antifibrin monoclonal antibody (designated as Y22). An in vitro model was developed to evaluate the effects of various environmental conditions on uptake by plasma clots of 99Tcm-Tc-Y22 in circulating plasma on a gamma camera. The clots became visible as hotspots after approximately 1 h of circulation of 99Tcm-Y22 containing citrated plasma at 37 degrees C. Circulation of 99Tcm-fibrinogen, 99Tcm-HSA or 99Tcm-control MoAb did not show visible uptake by the clots under the same conditions. At 37 degrees C, 99Tcm-Y22 accumulated approximately four times faster than at 20 degrees C. Heparin did not affect binding of the antibody to clots. To assess the feasibility of thrombus detection in vivo, an extracorporeal rat thrombus model was used. A thrombus in a shunt between a carotid artery and a jugular vein became visible 1 h after injection of the labelled Y22 and, more clearly, after 3 h.

Animals↗
PubMed1985

Posterior capsule opacification. An in vitro model.

Posterior capsule opacification results from migration of lens epithelial cells onto the posterior capsule, where they produce collagen and contract to cause wrinkling of the capsule. We have designed an in vitro model that reproduces the process of opacification. Lens capsules removed from enucleated bovine eyes are placed within specially designed holders, creating a culture dish with the capsule as its bottom surface. Albino rabbit lens epithelial cells are then plated on the capsule. These capsules develop clinical and ultrastructural features of opacification very similar to those observed in humans. Membranes composed of confluent epithelial cells closely resemble the cellular membranes observed with specular microscopy in human patients. Transmission electron microscopy demonstrates that a membrane several cells thick forms on the capsules. This model allows careful morphologic, metabolic, and biochemical studies of the process of opacification and the investigation of therapies designed to prevent opacification of the posterior capsule.

Animals↗
PubMed1983

Heterogeneous responses of an in vitro model of human stomach cancer to anticancer drugs.

Four permanent clones of a human adenocarcinoma of the stomach and the parent line from which they were isolated were used as an in vitro model system to evaluate the effects of 8 anticancer agents on cell survival. The drugs tested were actinomycin D (Act-D), Bleomycin (Bleo), adriamycin (adria), melphalan, chlorambucil, 5 Fluorouracil (5FU), 1,2:5,6-Dianhydrogalactitol (DAG), and 1-(2-chloroethyl)-3-(4-methyl cyclohexyl-1-nitrosourea) (MeCCNU). Although the cell lines had similar growth properties, morphologies and modal chromosome numbers, the clones expressed heterogeneous survival responses to each of six drugs tested. A comparison of the doses lethal to 90% of a clonal population (LD90) for each drug indicated large differences between the most sensitive and least sensitive clones. For chlorambucil there was a 160% difference between the LD90 values of the most and least sensitive clones. For MeCCNU the difference was 200%; for adria, 230%; Bleo, 280%; 5FU, 360%; and melphalan, 600%. Despite the heterogeneity in response among the clones to these agents, no particular clone was always the most sensitive or resistant. Of particular interest was the finding that these stomach cancer clones demonstrated uniform responses to both Act-D and DAG. Since the differential drug sensitivities expressed by heterogeneous tumor populations could be a cause of treatment failure in the patient, the demonstration of uniform sensitivities to Act-D and DAG are encouraging and suggest that other anticancer drugs which produce uniform cell killing may be identified and tested. Act-D and adria were the most effective of the drugs tested when compared on a dose for dose basis. Both agents killed more than 99.9% of the parent cell line with doses below 3 micrograms/ml (1-h treatments). The cells were least sensitive to 5FU, with only 30% of the cells killed at 100 micrograms/ml. The studies reported here indicate that this human stomach cancer model can provide valuable insight into the design of clinical protocols for treatment of gastric carcinoma in man.

Adenocarcinoma↗
PubMed1991

An in vitro model system to evaluate pulmonary macrophage, endothelial cell, and neutrophil interactions.

Pasteurella haemolytica, the cause of fibrinous pleuropneumonia in cattle, produces extensive microvascular endothelial cell (EC) damage. We have developed an in vitro model system to study the inflammatory process of this disease involving the interaction of pulmonary alveolar macrophages (AM), neutrophils (PMN), and EC. Bovine EC are grown to confluency in 24 well tissue culture plates. To mimic the vascular component, 10(6) PMN are later added to the EC monolayers. Bovine AM are plated onto millicell inserts and placed into the wells containing EC and PMN. The millicell insert serves as a semi-permeable barrier between AM and EC, allowing the exchange of only diffusible material. Preliminary work demonstrates that P. haemolytica stimulated AM resulted in EC damage presumably due to both soluble lipopolysaccharide and AM secreted products.

Animals↗
PubMed2002

Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models.

Antioxidant-rich fractions were extracted from pomegranate (Punica granatum) peels and seeds using ethyl acetate, methanol, and water. The extracts were screened for their potential as antioxidants using various in vitro models, such as beta-carotene-linoleate and 1,1-diphenyl-2-picryl hydrazyl (DPPH) model systems. The methanol extract of peels showed 83 and 81% antioxidant activity at 50 ppm using the beta-carotene-linoleate and DPPH model systems, respectively. Similarly, the methanol extract of seeds showed 22.6 and 23.2% antioxidant activity at 100 ppm using the beta-carotene-linoleate and DPPH model systems, respectively. As the methanol extract of pomegranate peel showed the highest antioxidant activity among all of the extracts, it was selected for testing of its effect on lipid peroxidation, hydroxyl radical scavenging activity, and human low-density lipoprotein (LDL) oxidation. The methanol extract showed 56, 58, and 93.7% inhibition using the thiobarbituric acid method, hydroxyl radical scavenging activity, and LDL oxidation, respectively, at 100 ppm. This is the first report on the antioxidant properties of the extracts from pomegranate peel and seeds. Owing to this property, the studies can be further extended to exploit them for their possible application for the preservation of food products as well as their use as health supplements and neutraceuticals.

Antioxidants↗
PubMed1998

A serum-free in vitro model of renal microvessel development.

The differentiation and organization of the embryonic renal vasculature is a crucial event in renal development. To study this process, we developed a serum-free in vitro model of renal microvessel development. Mouse embryonic kidney explants, when embedded specifically in type I collagen, demonstrate outgrowth of microvascular structures when stimulated by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA, 10-50 ng/ml). Other polypeptide growth factors stimulated little, if any, microvessel outgrowth from the explants. Similar outgrowths were not observed when other embryonic tissue explants were used. The number of microvessels observed depended on the gestational age of the explants. We hypothesize that TPA induces the in situ differentiation of metanephric mesenchymal cells into endothelial cell precursors and that specific matrix proteins and cell-matrix interactions are necessary for the organization of these precursors into microvessels. Our model will allow us to examine in detail the responsiveness of metanephric kidney cells to both growth factors and extracellular matrix molecules and to understand how they influence renal endothelial cell differentiation.

Animals↗
PubMed1998

Platelets and endothelial cells act in concert to delay thrombolysis--evidence from an in vitro model of the human occlusive thrombus.

The molecular and cellular mechanisms that over a period of hours render a human thrombus progressively resistant to fibrinolysis have been probed with a novel in vitro model. The kinetics of clot formation and fibrinolysis were monitored by laser light scattering with platelet-rich model thrombi contained in cylindrical flow chambers. In selected experiments, human umbilical vein endothelial cells were also cultured to confluence on the inner walls of these "glass blood vessels". Following an "aging" period (0.5, 2 or 4 h), each thrombus was gently perfused with a bolus of plasminogen/recombinant tissue plasminogen activator to induce fibrinolysis. Platelets delayed lysis of 2 h-aged thrombi by approximately 70% and (non-stimulated) endothelial cells by approximately 30%, compared to cell-free control clots. However, even greater lytic delays (approximately 260%) resulted when both vascular cells were present in the same 2 h-aged thrombus. In contrast, rapid lysis was consistently achieved with R298E,R299E t-PA, a genetically engineered plasminogen activator that is insensitive to inhibition by plasminogen activator inhibitor type 1. These observations suggest platelets and endothelial cells act in concert to enrich the fibrin scaffold of an aging human thrombus in plasminogen activator inhibitor. We propose that the presence of both platelets and endothelial cells may contribute to progressive thrombolytic resistance.

Blood Platelets↗
PubMed2006

Pathogenesis of Aspergillus fumigatus and the kinetics of galactomannan in an in vitro model of early invasive pulmonary aspergillosis: implications for antifungal therapy.

BACKGROUND: Little is known about the pathogenesis of invasive pulmonary aspergillosis and the relationship between the kinetics of diagnostic markers and the outcome of antifungal therapy. METHODS: An in vitro model of the human alveolus, consisting of a bilayer of human alveolar epithelial and endothelial cells, was developed. An Aspergillus fumigatus strain expressing green fluorescent protein was used. Invasion of the cell bilayer was studied using confocal and electron microscopy. The kinetics of culture, polymerase chain reaction, and galactomannan were determined. Galactomannan was used to measure the antifungal effect of macrophages and amphotericin B. A mathematical model was developed, and results were bridged to humans. RESULTS: A. fumigatus penetrated the cellular bilayer 14-16 h after inoculation. Galactomannan levels were inextricably tied to fungal invasion and were a robust measure of the antifungal effect of macrophages and amphotericin B. Neither amphotericin nor macrophages alone was able to suppress the growth of A. fumigatus; rather, the combination was required. Monte Carlo simulations showed that human dosages of amphotericin B of at least 0.6 mg/kg were required to achieve adequate drug exposure. CONCLUSIONS: This model provides a strategy by which relationships among pathogenesis, immunological effectors, and antifungal drug therapy for invasive pulmonary aspergillosis may be further understood.

Amphotericin B↗
PubMed1998

In vitro model of arterial stenosis: correlation of MR signal dephasing and trans-stenotic pressure gradients.

PURPOSE: Turbulent flow just distal to stenoses causes signal loss (dephasing) on magnetic resonance angiography (MRA). This study correlates dephasing with trans-stenotic pressure gradients in an in vitro model of arterial stenosis. MATERIALS AND METHODS: Three-dimensional (3D) phase contrast, 2D time-of-flight, and 3D spoiled gradient echo MRA with/without gadolinium and varied echo time were performed for a system consisting of a peristaltic perfusion pump and a silastic vessel with stenoses of varying caliber. Length and diameter of dephasing jets were measured, and volumes calculated at varying pressure gradients and echo times, then correlated with percentage cross-sectional area stenosis as measured by conventional angiography. RESULTS: Dephasing occurred in all sequences at pressure gradients of > or =4 mmHg (1 mmHg = 133 Pa) and stenoses of greater than 70%, and varied directly with pressure gradient. The dephasing was greatest for 3D phase contrast (PC). Gadolinium did not diminish dephasing. CONCLUSIONS: MRA signal dephasing at stenoses varies directly with pressure gradient. MRA may provide a non-invasive means for determining the hemodynamic significance of arterial stenoses.

Arterial Occlusive Diseases↗