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At least 1,189 records · Page 66Linked to original sources
PubMed2003

An improved in vitro model for testing the pulmonary toxicity of complex mixtures such as cigarette smoke.

Numerous approaches have been employed for testing the biological activity of cigarette smoke in vitro. None of them has managed to expose cultured lung cells in a realistic manner to the complex gaseous and particulate mixture that constitutes cigarette smoke. We have devised a system that makes this possible. The system presented here enables the direct exposure of human lung cells to native, unmodified cigarette mainstream smoke. It consists of a smoking machine, a dilution device for the smoke, analytical devices for online monitoring and a specially adapted exposure module based on the Cultex** cell cultivation system that is equipped with a gas-exposure top. Due to the special design of the exposure device and the optimised exposure conditions, this equipment allows cultured human lung cells to be exposed to freshly generated cigarette mainstream smoke. Exploratory experiments revealed that the smoke could be diluted over a wide concentration range in a reproducible way with respect to gas and particulate phases, and also demonstrated reproducible particle deposition depending on smoke concentration. Furthermore, it was shown that the exposed cells maintained their viability. Native cigarette mainstream smoke induced dose-dependent cellular effects in exposed cells with respect to cellular viability (viable cell number monitored by tetrazolium salt cleavage) and intracellular parameters (ATP and glutathione content). Therefore, fresh, physically and chemically unmodified cigarette mainstream smoke can be tested using this novel system.

Aerosols↗
PubMed1992

Rejuvenation of Sequoia sempervirens by Repeated Grafting of Shoot Tips onto Juvenile Rootstocks in Vitro: Model for Phase Reversal of Trees.

Repeated grafting of 1.5-centimeter long shoot tips from an adult Sequoia sempervirens tree onto fresh, rooted juvenile stem cuttings in vitro resulted in progressive restoration of juvenile traits. After four successive grafts, stem cuttings of previously adult shoots rooted as well, branched as profusely, and grew with as much or more vigor as those of seedling shoots. Reassays disclosed retention for 3 years of rooting competence at similar levels as originally restored. Adventitious shoot formation was remanifested and callus development was depressed in stem segments from the repeatedly grafted adult. The reversion was associated with appearance and disappearance of distinctive leaf proteins. Neither gibberellic acid nor N(6)-beneyladenine as nutrient supplements duplicated the graft effects.

Journal Article↗
PubMed1989

Effect of viral infection of Fc receptors and immunologically nonspecific receptors of blood polymorphonuclear leukocytes: an in vitro model.

I describe the effect on phagocytic activity of the migration of bovine blood polymorphonuclear leukocytes (PMNL) through collagen and a monolayer of PK-15 cells in vitro infected with rotaviruses (strain OSU, RFC-17, SA-11), a picornavirus (strain SVDV), bovine herpesvirus (BHV-1) and equine herpesvirus (EHV-1). The PMNL were examined before and after their migration as follows: (1) for EA rosette formation with sensitized sheep erythrocytes, (2) for phagocytic activity mediated through Fc receptors (FcRs), (3) for phagocytic activity mediated through immunologically nonspecific receptors (INsRs). Random migration of PMNL through the infected monolayer to RPMI-1640 medium containing or lacking chemotactic agents (zymosan activated serum--Act. serum) and/or containing control agents (lipopolysaccharide--LPS or formyl-L-methionyl-L-leucyl-L-phenylalanine--fMLP) decreased the percentage of rosettes forming PMNL and the phagocytosis mediated by FcRs and INsRs. The results obtained suggest that the viruses used in this study independently of their biological properties exerted a suppressive action on the phagocytic activity mediated by FcRs and INsRs.

Cell Movement↗
PubMed1998

Steroid production, cell proliferation, and apoptosis in cultured bovine antral and mural granulosa cells: development of an in vitro model to study estradiol production.

This study was undertaken to characterize the relationship between changes in steroid production, cell cycle activity (ie, cell proliferation) and apoptosis in antral and mural bovine granulosa cells cultured in vitro. This was done to select conditions promoting optimal estradiol production by bovine granulosa cells cultured in completely defined conditions. In the first experiment, antral granulosa cells were cultured over the entire 4 days of the culture period in the presence of either 0, 2, or 10 ng/ml of FSH (chronic conditions) or were maintained under minimal FSH support (0.5 ng/ml FSH) for the first 3 days of culture and then were challenged over the fourth day of culture with either 0, 2, or 10 ng/ml FSH (challenged conditions). Compared with cells exposed to constant FSH levels (chronic conditions), the FSH-induced production of estradiol was higher (P < 0.006) and that of progesterone was lower (P < 0.02) over the last 24 h of culture, when antral granulosa cells were maintained under minimal FSH support during the first 3 days of culture (challenged conditions). In the second experiment, dynamics of estradiol and progesterone productions, conversion of [14C]androstenedione into subsequent steroid metabolites, DNA content, cell cycle activity, and apoptosis (as assessed by flow cytometry) of antral and mural granulosa cells over the first 3 days of culture under minimal FSH support and in response to a challenge with FSH during the last 24 h of culture were evaluated. Estradiol production as well as the conversion of androstenedione into testosterone and estradiol were greater (P < 0.01) in antral than in mural granulosa cells cultured under challenged conditions. A higher proportion of mural than antral granulosa cells were in the proliferative state at the end of culture (P < 0.03). This may be related to the decreased ability of mural cells to produce estradiol. FSH suppressed (P < 0.05) the spontaneous onset of apoptosis in both cell types. These results suggest that functional differences between these two cell compartments need to be considered in studying bovine granulosa cells in vitro. Because of their large (400 to 600%) FSH-induced estradiol production, antral granulosa cells cultured under challenged conditions provide a model that can be used to examine substances for their ability to alter estradiol production and apoptosis in bovine granulosa cells.

Animals↗
PubMed1999

In vivo mitochondrial import. A comparison of leader sequence charge and structural relationships with the in vitro model resulting in evidence for co-translational import.

The positive charges and structural properties of the mitochondrial leader sequence of aldehyde dehydrogenase have been extensively studied in vitro. The results of these studies showed that increasing the helicity of this leader would compensate for reduced import from positive charge substitutions of arginine with glutamine or the insertion of negative charged residues made in the native leader. In this in vivo study, utilizing the green fluorescent protein (GFP) as a passenger protein, import results showed the opposite effect with respect to helicity, but the results from mutations made within the native leader sequence were consistent between the in vitro and in vivo experiments. Leader mutations that reduced the efficiency of import resulted in a cytosolic accumulation of a truncated GFP chimera that was fluorescent but devoid of a mitochondrial leader. The native leader efficiently imported before GFP could achieve a stable, import-incompetent structure, suggesting that import was coupled with translation. As a test for a co-translational mechanism, a chimera of GFP that contained the native leader of aldehyde dehydrogenase attached at the N terminus and a C-terminal endoplasmic reticulum targeting signal attached to the C terminus of GFP was constructed. This chimera was localized exclusively to mitochondria. The import result with the dual signal chimera provides support for a co-translational mitochondrial import pathway.

Aldehyde Dehydrogenase↗
PubMed2004

Origins of mammalian hematopoiesis: in vivo paradigms and in vitro models.

Though a topic of medical interest for centuries, our understanding of vertebrate hematopoietic or "blood-forming" tissue development has improved greatly only in recent years and given a series of scientific and technical milestones. Key among these observations was the description of procedures that allowed the transplantation of blood-forming activity. Beyond this, other advances include the creation of a variety of knock-out animals (mice and more recently zebrafish), microdissection of embryonic and fetal blood-forming tissues, hematopoietic stem (HSC) and progenitor cell (HPC) colony-forming assays, the discovery of cytokines with defined hematopoietic activities, gene transfer technologies, and the description of lineage-specific surface antigens for the identification and purification of pluripotent and differentiated blood cells. The availability of both murine and human embryonic stem cells (ESC) and the delineation of in vitro systems to direct their differentiation have now been added to this analytical arsenal. Such tools have allowed researchers to interrogate the complex developmental processes behind both primitive (yolk sac or extraembryonic) and definitive (intraembryonic) hematopoietic tissue formation. Using ES cells, we hope to not only gain additional basic insights into hematopoietic development but also to develop platforms for therapeutic use in patients suffering from hematological disease. In this review, we will focus on points of convergence and divergence between murine and human hematopoiesis in vivo and in vitro, and use these observations to evaluate the literature regarding attempts to create hematopoietic tissue from embryonic stem cells, the pitfalls encountered therein, and what challenges remain.

Animals↗
PubMed1996

In vitro modeling of the bone/implant interface.

BACKGROUND: The purpose of this review is to examine the usefulness of cell culture methods to model the mechanisms of bone formation on the surfaces of candidate implant materials. METHODS: The central objective is to show that in vitro methods are uniquely valuable in providing an understanding of how new bone is formed on solid surfaces. It should be emphasized, at the outset, that the use of cell culture studies as cytotoxicity assays will not be addressed, nor is it implied that cell cultures can model all the complexities of the in vivo environment. Nevertheless, by comparison with in vivo data, which are by nature retrospective, it is shown that primary differentiating osteogenic cell cultures, derived from bone marrow, illustrate a sequence of extracellular matrix elaboration events that characterize the establishment of the interface between newly formed bone and solid surfaces. These solid surfaces either may be implant materials, or indeed previously formed bone matrix, which has been resorbed during normal bone remodeling events. In each case the first biologically derived matrix at these sites is a morphologically distinct collagen fibre-free extracellular matrix, which, in bone histology has been referred to for > 100 years as a cement line. RESULTS: The sequence starts with secretion and adsorption to the substratum of organic components, of which the major proteins are osteopontin and bone sialoprotein. Mineralization of this matrix occurs by the seeding of nanocrystalline calcium phosphate, which precedes the appearance of morphologically identifiable collagen fibres. This is clearly contrary to the dogma that collagen is necessary for mineralization of bone, but is in agreement with specific cases of other, particularly dental, calcified connective tissues. Although collagen is synthesized by the differentiating osteogenic cells that elaborate the cement line interface, it is not adsorbed to the underlying solid surface. Following the elaboration of the cement line matrix, collagen fibre assembly occurs and is then mineralized to produce morphologically identifiable bone matrix. CONCLUSION: Key elements of this sequence of events can be seen at the interface of implants retrieved from in vivo experiments, which indicates that these in vitro methods not only mimic known in vivo phenomena, but also provide a mechanistic understanding of bone elaboration at implant surfaces. However, distinction is drawn between the events of new bone formation at implant surfaces and other bone/implant morphologies, which are unrelated to de novo bone formation at the implant surface. Finally, this new information emerging from bone marrow cell culture studies demands a re-examination of the concepts of bone-bonding and nonbonding implant materials.

Adult↗
PubMedDate not supplied

[Contribution of in vitro models to the management of hematologic risk in pharmacovigilance].

The decisions of health authorities concerning adverse effects of drugs are usually notified following clinical observations, but are rarely associated to experimental data. The haematopoietic tissue is one of the most sensitive to these effects. In order to anticipate and to explain adverse effects, it becomes necessary to carry out in vitro assays on normal human haematopoietic progenitors. We had the opportunity to use human cord blood progenitors which are able to repopulate allogeneic aplastic bone marrow. Many advantages are associated with this model: numerous samples, non-invasive, absence of species bias, possibilities of mechanistic approach. The clonogenic potential of progenitors in soft agar, as well as their ability to expand in liquid medium after stimulation with specific growth factors, have been used. Evidence of dose-related toxicity by inhibition of colony formation or proliferation was analysed in the presence of reference molecules. Results were reproducible despite an intrinsic variability of progenitor density between samples. They were comparable to assays on bone marrow progenitors reported by us and others. Comparison of toxicity thresholds with plasma therapeutic ranges showed the potential risk for some molecules tested.

Animals↗
PubMed1999

Bacillus-Calmette-Guérin (BCG) and 3D tumors: an in vitro model for the study of adhesion and invasion.

PURPOSE: To study adhesion, penetration and internalization of BCG and effector-cells to and into three-dimensional in vitro cell aggregates from benign and malignant urothelial origin mimicking small in vitro tumors. MATERIALS AND METHODS: Multicellular spheroids (MCS) were generated by "liquid-overlay" technique. Adhesion and penetration of viable FITC-labelled BCG into MCS from urothelial cancer cell lines and normal urothelial cells was studied by electron microscopy (TEM) and fluorescence microscopy. Spheroid growth during BCG-co-incubation was determined by light microscopy. Peripheral blood mononuclear cells (PBMC) were stimulated with BCG to generate BCG-activated-killer (BAK) cells. The infiltration of these effectors and of lymphokine-activated killer (LAK) cells into MCS was examined at different intervals by means of immunohistochemistry. The resulting cytotoxicity was judged in a 3H-l-methionine release assay. RESULTS: BCG adhered to MCS from tumor cells but not to benign cell MCS. Intracellular internalization of the bacteria was detectable in superficial tumor cell-layers (1-5) whereas BCG was not found in deeper layers. Proliferation of malignant MCS was reduced in the presence of BCG. Benign MCS showed contact inhibition growth arrest, which was not altered by BCG. BAK and LAK effector cells both infiltrated tumor cell MCS as opposed to unstimulated PBMC. In contrast to LAK cells, BAK cells did not infiltrate into benign cell MCS and were not cytotoxic towards them. CONCLUSION: With regard to the clinical situation the selective adhesion and internalization of BCG to malignant cells might explain why BCG has been rarely found in follow-up biopsies in tumor free patients. More interestingly, the selective adhesion of BCG to and infiltration of BAK effector cells into malignant cell spheroids suggests a selective mode of action of BCG.

Adjuvants, Immunologic↗
PubMed2000

Colophony: an in vitro model for the induction of sensitization.

BACKGROUND: The potential of colophony fumes from soldering flux to induce asthma has been known since the 1970s, however, no direct in vitro or in vivo evidence has been reported. The present study investigated the potential of colophony to stimulate human phagocytic cells to produce reactive oxygen species. METHODS: The human cell line HL-60 was differentiated to produce cells with a monocyte-like and a neutrophil-like phenotypes. A number of procedures were used to confirm the phenotype of these differentiated cells including morphology, esterase activity, flow cytometry and phagocytosis. The potential of colophony to stimulate human phagocytic cells to produce reactive oxygen species was monitored using flow cytoenzymology. RESULTS: We were able to show that intracellular peroxide levels were increased in both monocyte-like and neutrophil-like cells, but not in undifferentiated HL-60 cells following the addition of colophony. CONCLUSIONS: The resin acid epoxides and hydroperoxides which have been suggested to be sensitizers in contact allergy, are degraded during the soldering process. However, conditions for the oxidation of colophony may occur in vivo as a result of the colophony-induced oxidative burst from neutrophils and monocytes. These oxidation products may then interact with body proteins to further initiate immune responses. Therefore for the preparation of low molecular weight chemical (LMWC)-protein conjugates, consideration must be taken to determine whether the LMWC is undergoing a reaction in vivo before it is interacting with body proteins.

Cell Differentiation↗
PubMed1991

Development and application of an in vitro model for screening anti-hepatitis B virus therapeutics.

The development of effective anti-hepatitis B virus agents has been hampered by the lack of reliable in vitro systems for the screening of new therapeutics. In an effort to circumvent this problem, we have developed an in vitro system for screening anti-hepatitis B virus drugs using hepatitis B virus DNA-transfected Hep G2 cells. The cell line designated 2.2.15 produces replicative viral DNA intermediates, mature Dane particles and high levels of viral antigens. Subconfluent 2.2.15 cells were treated with a variety of commonly used anti-hepatitis B virus therapeutics, and their efficacy was determined by analyzing changes in the replicative cellular or extracellular hepatitis B virus DNA content by Southern blotting or slot-blot hybridization. The slot-blot method was sensitive, reproducible and rapid and correlated well with Southern blotting. Analysis of the media for hepatitis B virus DNA was indicative of changes in intracellular, replicative hepatitis B virus DNA, permitting sampling of the media. Therefore 2.2.15 cells may provide a valuable method for identifying and monitoring effective anti-hepatitis B virus therapeutics. Using this system to test various agents, we confirm that 2'-deoxyguanosine strongly inhibited viral replication, whereas others tested were less effective. Correlation with in vivo systems is now needed.

Antiviral Agents↗
PubMed1987

Is the guinea pig trachea a good in vitro model of human large and central airways? Comparison on leukotriene-, methacholine-, histamine- and antigen-induced contractions.

To analyze comprehensively the relevance of the guinea pig trachea as a model of human large and central airways, the contractile effects of the peptidoleukotrienes (LTs), histamine, methacholine and antigen on guinea pig and human airways were compared in vitro. Although some differences were apparent, LTC4, LTD4, LTE4, histamine and methacholine had comparable EC50 values and elicited similar maximal responses in both guinea pig trachea and human bronchus (second-seventh generation). In the presence of l-serine borate (45 mM), LTC4 concentration-response curves were shifted significantly to the left in guinea pig trachea but not in human bronchus. Furthermore, the LT receptor antagonists (SK&F 102922 and FPL 55712) had similar potencies against LTC4- and LTD4-induced contractions of human bronchus, whereas, in the guinea pig trachea, they were much more effective antagonists of responses produced by LTD4 than those elicited by LTC4. These results provide further evidence that, unlike in human bronchus, LTC4- and LTD4-induced contractions in the guinea pig trachea are mediated via distinct leukotriene receptors. Ovalbumin-induced contractions of actively sensitized guinea pig tracheae exhibited the same profile as anti-immunoglobulin E-induced contractions of the passively sensitized human bronchus. Furthermore, antigen-induced contractions in both the guinea pig trachea and human bronchus possessed a similar sensitivity to inhibition by mepyramine (10 microM) and the LT antagonists (10 microM), added either alone or in combination. These results indicate that the isolated guinea pig trachea is a suitable model of human large and central airways.

Adult↗
PubMed1988

The rat hippocampal slice preparation as an in vitro model of ischemia.

In vivo models of cerebral ischemia do not fully control for the interacting effects of many variables (e.g., anesthesia, temperature, cerebrovascular changes) and often do not clearly define the region affected. Numerous in vivo studies have indicated that hyperglycemia augments ischemic brain damage; this effect is often attributed to lactic acidosis. To separate the effects on neuronal tissue of ischemia from those due to actions on the cerebrovascular system, we used an in vitro blood-free system as an ischemic model. In our study we evaluated the effects of various combinations of oxygen and glucose levels on evoked synaptic activity in the CA1 region of the rat hippocampal slice preparation. A 50% inhibitory dose for both oxygen and glucose on neuronal synaptic function was determined. It is our intention to use this model for preliminary screening of antihypoxic/anti-ischemic drugs.

Animals↗
PubMed1984

An in vitro model of ischemia: metabolic and electrical alterations in the hippocampal slice.

The transverse guinea pig hippocampal slice preparation was used to model the metabolic changes which occur in vivo during ischemia and recovery. Perfusing brain slices with medium devoid of glucose and oxygen elicits rapid decreases in phosphocreatine, ATP, intracellular pH, and in the evoked field potential recorded in the dentate gyrus. AMP and creatine rise during this period, while ADP and lactate levels remain unchanged. Cyclic AMP exhibits a transient increase in concentration. With the exception of ADP and lactate, these responses are very similar to those observed during in vivo ischemia. The return of glucose and oxygen to the incubation medium reverses these metabolic and electrophysiological effects and also leads to pronounced elevations in cyclic nucleotide concentrations. Metabolite concentrations approach, but do not reach, in vitro steady state levels during the first 30 min of recovery. Total adenylate and creatine steady state levels are approximately 50% of in vivo concentrations. The results suggest that, although hippocampal slices differ metabolically from in vivo tissue, they exhibit a similar pattern of metabolic responses to ischemic and reflow conditions.

Adenosine Monophosphate↗
PubMed2004

Involvement of OCTN2 and B0,+ in the transport of carnitine through an in vitro model of the blood-brain barrier.

Carnitine is known to accumulate in brain, therefore transport of carnitine through the blood-brain barrier was studied in an in vitro system using bovine brain capillary endothelial cells (BBCEC) grown on filter inserts in a co-culture system with glial cells. Long-term exposure of BBCEC to carnitine resulted in a high accumulation of long-chain acyl carnitines, which decreased dramatically upon removal of carnitine. Kinetic analysis of carnitine accumulation indicated a possibility of functioning of more than one transporter. BBCEC were incubated in the presence of substrates and inhibitors of known carnitine transporters added from either apical or basolateral side. Inhibition by replacement of sodium and expression of OCTN2 (RT-PCR) were in agreement with earlier reports on the functioning of OCTN2 in apical membrane. For the first time, functioning of OCTN2 was demonstrated in the basolateral membrane, as well as functioning in both membranes of a low affinity carnitine transporter B(0,+). Expression of B(0,+) in BBCEC was confirmed by RT-PCR. These results suggest that OCTN2 and B(0,+) could be involved in carnitine transport in both the apical and basolateral membrane.

Amino Acid Transport Systems, Neutral↗