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PubMed2002

Cestode parasites: application of in vivo and in vitro models for studies on the host-parasite relationship.

Cestode worms, commonly also known as 'flat' worms or tapeworms, are an important class of endoparasitic organisms. In order to complete their life cycle, they infect intermediate and definitive hosts in succession, through oral ingestion of eggs or larvae, respectively. Serious disease in humans or other mammalian hosts is mostly caused by the larval stages. Echinococcus spp. and Taenia spp. have been extensively investigated in the laboratory due to the fact that they represent important veterinary medical challenges and also cause grave diseases in humans. In contrast, Hymenolepis spp. and Mesocestoides spp. infections are relatively rare in humans, but these parasites have been extensively studied because their life cycle stages can be easily cultured in vitro, and can also be conveniently maintained in laboratory animal hosts. Thus they are more easily experimentally accessible, and represent important models for investigating the various aspects of cestode biology. This review will focus on in vitro and in vivo models which have been developed for studies on the host-parasite relationship during infection with Echinococcus, Taenia, Hymenolepis, Mesocestoides and Spirometra, and will cover the use of these models to investigate the morphology and ultrastructure of respective genera, the immunological relationship with the host and the development of vaccination approaches, as well as applications of these models for studies on parasite metabolism, physiology and gene expression. In addition, the use of these models in the development of chemotherapeutic measures against cestode infections is reviewed.

Animals↗
PubMed1990

Cell locomotion forces versus cell contraction forces for collagen lattice contraction: an in vitro model of wound contraction.

Cultured human dermal fibroblasts suspended in a rapidly polymerizing collagen matrix produce a fibroblast-populated collagen lattice. With time, this lattice will undergo a reduction in size referred to as lattice contraction. During this process, two distinct cell populations develop. At the periphery of the lattice, highly oriented sheets of cells, morphologically identifiable as myofibroblasts, show cell-to-cell contacts and thick, actin-rich staining cytoplasmic stress fibers. It is proposed that these cells undergoing cell contraction produce a multicellular contractile unit which reorients the collagen fibrils associated with them. The cells in the central region, referred to as fibroblasts, are randomly oriented, with few cell-to-cell contacts and faintly staining actin cytoplasmic filaments. In contrast it is proposed that cells working as single units use cell locomotion forces to reorient the collagen fibrils associated with them. Using this model, we sought to determine which of these two mechanisms, cell contraction or cell locomotion, is responsible for the force that contracts collagen lattices. Our experiments showed that fibroblasts produce this contractile force, and that the mechanism for lattice contraction appears to be related to cell locomotion. This is in contrast to a myofibroblast; where the mechanism for contraction is based upon cell contractions. Fibroblasts attempting to move within the collagen matrix reorganize the surrounding collagen fibrils; when these collagen fibrils can be organized no further and cell-to-cell contacts develop, which occurs at the periphery of the lattice first, these cells can no longer participate in the dynamic aspects of lattice contraction.

Adenosine Triphosphate↗
PubMed2004

Apoptosis is not an invariable component of in vitro models of cortical cerebral ischaemia.

Characterising the mechanisms of cell death following focal cerebral ischaemia has been hampered by a lack of an in vitro assay emulating both the apoptotic and necrotic features observed in vivo. The present study systematically characterised oxygen-glucose-deprivation (OGD) in primary rat cortical neurones to establish a reproducible model with components of both cell-death endpoints. OGD induced a time-dependent reduction in cell viability, with 80% cell death occurring 24 h after 3 h exposure to 0% O2 and 0.5 mM glucose. Indicative of a necrotic component to OGD-induced cell death, N-methyl-D-aspartate (NMDA) receptor inhibition with MK-801 attenuated neuronal loss by 60%. The lack of protection by the caspase inhibitors DEVD-CHO and z-VAD-fmk suggested that under these conditions neurones did not die by an apoptotic mechanism. Moderating the severity of the insult by decreasing OGD exposure to 60 min did not reduce the amount of necrosis, but did induce a small degree of apoptosis (a slight reduction in cell death was observed in the presence of 10 uM DEVD-CHO). In separate experiments purported to enhance the apoptotic component, cells were gradually deprived of O2, exposed to 4% O2 (as opposed to 0%) during the OGD period, or maintained in serum-containing media throughout. While NMDA receptor antagonism significantly reduced cortical cell death under all conditions, a caspase-inhibitor sensitive component of cell death was not uncovered. These studies suggest that OGD of cultured cortical cells models the excitotoxic, but not the apoptotic component of cell death observed in vivo.

Amino Acid Chloromethyl Ketones↗
PubMed1994

Programmed cell death of the normal human neutrophil: an in vitro model of senescence.

The present study provides experimental data which indicate that the neutrophil is ideal for studying programmed cell death or apoptosis in vitro. Neutrophils can be obtained from human peripheral blood in large numbers with minimal experimental manipulation and are easily separated from other leukocytes, providing nearly pure cell suspensions. The neutrophil life span in vitro is sufficiently short to allow observations to be made within eight hours after experimental manipulation. Neutrophils can also be easily maintained in serum-free, chemically defined media which can be systematically altered, thereby defining specific variables that influence the apoptotic process. Since the neutrophils do not need an exogenous trigger to undergo programmed cell death, it is also an excellent model to study senescence. It was determined from this study that neutrophils undergo apoptosis most efficiently at 37 degrees C, a temperature requirement for physiologic cell death. Neutrophils undergo apoptosis at a slightly faster rate and maintain membrane integrity better when incubated in a tissue culture medium (e.g., RPMI 1640) compared with a balanced salt solution (e.g., HBBB). Cycloheximide, an inhibitor of protein synthesis, was shown to accelerate apoptosis in a dose-dependent manner. The presence of Zn++ significantly decreased the rate of apoptosis, whereas the presence of Ca++ and Mg++ had no apparent effect. These studies indicate that the process of senescence, culminating in cell death, is subject to modulation by a variety of agents and experimental conditions. In addition, the ultrastructural features of neutrophils undergoing programmed cell death in vitro were compared in detail to those occurring in vivo and were found to be comparable.

Apoptosis↗
PubMed2004

An in vitro model to evaluate the cytokine response in Echinococcus infections.

With the aim of proposing an alternative model to animal experimentation, we investigated cytokine production in response to antigens in an in vitro system. This is a co-culture system of healthy human leukocytes and enterocyte-like Caco-2 cells. The antigens tested, EgA31, EgTrp, and FABP1, are candidates for vaccines in infections caused by Echinococcus spp. in the gut. All three have previously been described in the protoscolex stage and belong to protein families which confer protective immunity against several helminths. In this study, we evaluate the Th1/Th2 profile (Th1: IL-12, IFN-gamma; Th2: IL-6, IL-10) in response to protoscoleces, EgA31 and the mixture of EgA31, EgTrp and FABP1. No cytokine production was detected in response to protoscoleces. Neither IFN-gamma nor IL-6, but a significant IL-10 and IL-12 concentration was detected in response to both types of antigens. These findings suggest that EgA31 and the mixture EgA31/EgTrp/FABP1 generated an immunogenic response associated with a mixed Th1/Th2 cytokine.

Animals↗
PubMed2004

Rabbit retinal organ culture as an in-vitro model of hepatic retinopathy.

BACKGROUND: In patients with liver insufficiency the blood ammonia concentration is elevated, which induces structural and functional retinal alterations referred to as "hepatic retinopathy". The fact that some of these alterations are reversible after timely liver transplantation rationalizes therapeutic/preventive approaches to maintain the retina before a donor organ is available. Here, we describe further elaboration and characterization of our retinal organ culture model. METHODS: Retinal pieces of neonatal rabbits were explanted and cultured for up to 22 days. After 7 days in vitro (DIV7), some of the cultures were exposed to 0.25, 0.5, 1.0, 3.0, or 7.0 mM ammonia for varying periods of time and then studied by light and electron microscopy. RESULTS: Untreated (control) cultures underwent several changes during prolonged culturing; these included increased expression of GFAP and of Bcl-2, and decreased levels of glutamine synthetase, in Müller (glial) cells. However, the retinal layering remained intact and even some retinal ganglion cells survived although their axons had been cut. Exposure to elevated levels of ammonia caused morphologic alterations in the explants compatible with those noted in hepatic retinopathy in vivo. Specifically, we observed neuronal cellular degeneration with destruction of retinal layers and formation of rosettes, as well as decreased expression of intermediate filament proteins, migration of cell nuclei, cell swelling, and up-regulation of glutamine synthetase immunoreactivity in (some) Müller cells. CONCLUSION: Our findings indicate that the organ culture model may be a useful tool for detailed studies on the cellular and subcellular mechanisms underlying hepatic retinopathy and for in vitro testing of measures to alleviate HR symptoms.

Ammonia↗
PubMed1990

In vitro model of far-field stationary potentials: boundary effects on propagated potentials.

Far-field stationary potentials have been said to result from several factors such as changes in the anatomical orientation of the direction of a propagating nerve action potential (AP) or local modifications of the impedance in the external volume conductor by changes in its geometry or resistivity. In the case of an impedance variation due to the presence of boundaries in the geometry of the medium, the findings reported in this paper showed that it is possible to record simultaneously AP and stationary potentials which we have called boundary potentials (BPs). The BP amplitude depended on the number of active axons at the boundary, on the distance between the boundary and one of the recording electrodes, and on the local impedance variation. Its polarity depended only on the direction of the AP propagation relative to the sign of the local impedance modification, the recording electrodes being in the same relative positions with respect to the direction of the propagating impulse. Finally, the recording of a BP remained possible whatever the relative position of the boundary and of the recording electrodes. These findings are in accordance with some clinical observations not yet explained.

Action Potentials↗
PubMed2002

Concentrates containing factor IX could improve haemostasis under conditions of thrombocytopenia: studies in an in vitro model.

BACKGROUND AND OBJECTIVES: We explored the effect on haemostasis of different factor IX (FIX) concentrates under thrombocytopenic conditions using an in vitro perfusion technique. MATERIALS AND METHODS: A moderate experimental thrombocytopenia (25 000-30 000 platelets/microl) was induced by means of a filtration procedure in blood anticoagulated with low-molecular-weight heparin. The effects of three different FIX concentrates - a prothrombin complex concentrate (PCC), an intermediate-purity concentrate (FIX/X), and a high-purity concentrate (HPFIX) - on platelet deposition and fibrin formation on subendothelium were assessed at two different shear rates (600/second and 1200/second). Activation of the coagulation system was monitored through assessment of prothrombin activation fragment 1 + 2 (F1 + 2). RESULTS: Fibrin deposition increased after addition of FIX concentrates, but only showed a significant increase in experiments performed after incubation of PCC at the lower shear rate (600/second) (64.25 +/- 9.61% vs. control 31.22 +/- 8.02%; P < 0.05). Addition of FIX concentrates caused a small increase in the percentage of platelet deposition and area of those aggregates. These differences reached levels of statistical significance in the presence of FIX/X and HPFIX in experiments performed at a shear rate of 600/second. F1 + 2 baseline values in anticoagulated thrombocytopenic blood were 1.15 +/- 0.13 nm and reached levels of 2.49 +/- 0.24 and 3.60 +/- 0.33 nm at shear rates of 600 and 1200/second, respectively. Increments in F1 + 2 observed after addition of different FIX concentrates always remained in the previous ranges. CONCLUSIONS: Data from the present study provide experimental support favouring the concept that FIX concentrates containing other activated factors could improve haemostasis under conditions of moderate thrombocytopenia.

Animals↗
PubMed2002

Hepatic sinusoidal endothelium upregulates IL-1alpha, IFN-gamma, and iNOS in response to discordant xenogeneic islets in an in vitro model of xenoislet transplantation.

BACKGROUND: Data indicate that early islet graft failure is due to nonspecific inflammatory mechanisms that occur prior to T-cell-mediated rejection. The role of the host hepatic endothelium in mediating this immediate islet injury has not been elucidated. The endothelial cell may be important in this process because it is essentially the first cellular barrier encountered by intraportally introduced islets. We have characterized the initial response of hepatic endothelium to xenogeneic islets by measuring the expression of Il-1alpha, TNF-alpha, IFN-gamma, and iNOS in an in vitro dog-to-pig model of xenoislet transplantation. MATERIALS AND METHODS: Dog islets (500 islet equivalents) were cocultured with either porcine hepatic endothelium or porcine aortic endothelium over a 24-h period in serum-free medium. RNA was extracted at eight time points (0, 1, 2, 4, 6, 8, 12, and 24 h). Reverse-transcriptase polymerase chain reaction was performed on each sample. Polymerase chain reaction was done on the cDNA in order to visualize Il-1alpha, TNF-alpha, IFN-gamma, and iNOS expression. Bands were semiquantitated by comparison to an external standard (GAPDH) using band densitometry. RESULTS: Hepatic endothelium had early (1 h) expression of IL-1alpha, IFN-gamma, and iNOS. IL-1alpha peaked at 2 h, IFN-gamma at 12 h, and iNOS at 1 and 12 h. Aortic endothelium expressed low levels of IL-1alpha and TNF-alpha, but not IFN-gamma or iNOS. CONCLUSIONS: We have demonstrated that xenogeneic islets are able to activate host endothelial cells without serum or immune cells. The observed endothelial response corresponds with known islet toxic substances. Furthermore, the response differs between hepatic and aortic endothelial cells, suggesting that these differences may be important in choosing suitable implantation sites for islets. Our findings suggest that host endothelium may play an important part in early injury of islet xenotransplants.

Animals↗
PubMedDate not supplied

Induction of epileptiform activity by temperature elevation in hippocampal slices from young rats: an in vitro model for febrile seizures?

Extracellular field potential recordings were performed in the CA1 subfield of hippocampal slices obtained from Wistar rats aged 2-38 days. When the brain tissue was maintained at 35 degrees-36 degrees C (values obtained in the tissue chamber well), single-shock orthodromic stimuli elicited a response in the stratum pyramidale that consisted of a single population spike. In contrast, when the temperature in the well was increased to levels greater than 38.2 degrees C for periods of 5-15 min, the same type of stimuli elicited an epileptiform response characterized by a 250- to 600-ms-long, positive-going field potential with superimposed, multiple, negative-going population spikes. This potential resembled the epileptiform response recorded in the hippocampal slice in the presence of convulsants such as penicillin or bicuculline. Once the temperature was restored to control values (i.e., 35 degrees-36 degrees C) after induction of epileptiform activity, the abnormal response could be observed for less than or equal to 2 h. In some experiments (approximately one third of the successful trials), spontaneous epileptiform discharges appeared during and persisted after the increase in temperature. The ability of the hyperthermic period to induce epileptiform changes was age dependent: Epileptiform activity outlasting the period of temperature elevation was not observed in slices obtained from rats aged less than 4 days or greater than 28 days. Our data show that epileptiform activity can be induced by a transient increase in temperature and that the age of the animals from which slices are obtained plays an important role in the appearance of this phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed2005

Differentiation of epithelial Na+ channel function. An in vitro model.

Confluent monolayers of epithelial cells grown on nonporous support form fluid-filled hemicysts called domes, which reflect active ion transport across the epithelium. Clara-like H441 lung adenocarcinoma cells grown on glass supports and exposed to 50 nM dexamethasone developed domes in a time-dependent fashion. Uplifting of small groups of cells occurred within 6-12 h, well formed domes appeared between 24 and 48 h, and after 7 days, individual domes started to merge. Cells inside of domes compared with those outside domes, or with monolayers not exposed to dexamethasone, differed by higher surfactant production, an increased cytokeratin expression, and the localization of claudin-4 proteins to the plasma membrane. In patch clamp studies, amiloride-blockable sodium currents were detected exclusively in cells inside domes, whereas in cells outside of domes, sodium crossed the membrane through La3+-sensitive nonspecific cation channels. Cells grown on permeable support without dexamethasone expressed amiloride-sensitive currents only after tight electrical coupling was achieved (transepithelial electrical resistance (R(t)) > 1 kilohm). In real-time quantitative PCR experiments, the addition of dexamethasone increased the content of claudin-4, occludin, and Na+ channel gamma-subunit (gamma-ENaC) mRNAs by 1.34-, 1.32-, and 1.80-fold, respectively, after 1 h and was followed by an increase at 6 h in the content of mRNA of alpha- and beta-ENaC and of alpha1- and beta1-Na,K-ATPase. In the absence of dexamethasone, neither change in gene expression nor cell uplifting was observed. Our data suggest that during epithelial differentiation, coordinated expression of tight junction proteins precedes the development of vectorial transport of sodium, which in turn leads to the fluid accumulation in basolateral spaces that is responsible for dome formation.

Base Sequence↗
PubMed2005

A three-dimensional in vitro model system to study the adaptation of craniofacial skeletal muscle following mechanostimulation.

The aim of this study was to investigate the in vitro response of human craniofacial muscle-derived myotubes (primitive/nascent muscle fibres), in three-dimensional constructs, to strain in vitro to mimic clinical scenarios, using expression of the mechanoresponsive gene gelatinase-A/matrix metalloproteinase-2 (MMP-2) as a marker of remodelling of muscle extracellular matrix. Three-dimensional (3D) constructs of cells derived from explants of human masseter muscle (human craniofacial muscle-derived cells; hCMDC) in collagen sponges were subjected to mechanical, uniaxial strain using the Bio-Stretch system. 3D myotube constructs were exposed to the strain regimes of rapid ramp stretch (RRS) or cyclical ramp strain (CRS) with 7.5% and 15% strain. The activity of MMP-2 was assessed by zymography of construct-conditioned medium, whilst lysates of the constructs were used to measure creatine phosphokinase (CPK) activity to confirm the presence of myotubes in the strained constructs. Scanning electron microscopy of the collagen sponges and the CPK assays confirmed the presence of myotubes. MMP-2 was expressed by all the samples and controls, but expression was found to be significantly higher in those cultures strained continuously (RRS), compared to cyclical strain (CRS), and in those strained at 15% compared to 7.5%. Thus, MMP-2 expression, and hence extracellular matrix remodelling, is up-regulated in response to strain and is dependent upon the amount and type of strain to which the muscle is subjected.

Adaptation, Physiological↗
PubMedDate not supplied

Inhibition of mouse bone marrow granulocyte-macrophage colony formation by factors derived from natural killer cells: an in vitro model for hybrid resistance.

Investigations were performed to study whether soluble factors produced by NK-cells could mediate "hybrid resistance" in vitro. NK-cells enriched from spleens of B6D2F1 hybrid mice were incubated with parental B6 bone marrow, and the effect of the derived supernatants on the development of granulocyte-macrophage colony forming cells (GM-CFC) was assessed. Cell free supernatants obtained from low density cells (LDC) of B6D2F1 hybrids stimulated with bone marrow cells (BMC) from B6 mice inhibited GM-CFC formation. The inhibition was similar using B6, D2 or B6D2F1 bone marrow cells as the targets for GM-CFC growth. Our findings suggest that NK cells from F1 hybrid mice when stimulated with BMC from B6 mice release inhibitory factors, different from IFN-gamma and that this production may represent a mechanism of natural resistance to parental H-2b bone marrow grafts.

Animals↗
PubMed2004

The development and characterization of an in vitro model of psoriasis.

In this study, the phenotype of psoriatic keratinocytes and fibroblasts in reconstructed skin models was compared to those constructed from normal cells. Characterization of this model by immunohistochemistry showed that classical markers of keratinocyte differentiation exhibited similar patterns of distribution in the psoriatic models to those derived from normal cells and generally reflected in vivo observations. Some crucial differences, however, were observed between normal and psoriatic models when pro-inflammatory gene expression and keratinocyte proliferation were investigated. Notably, the chemokine receptor CXCR2 was overexpressed in the psoriatic models, and, moreover, was localized to the granular layer of keratinocytes as seen in psoriasis in vivo. Pro-inflammatory genes (tumor necrosis factor alpha [TNF-alpha], interferon gamma [IFN-gamma], and interleukin 8 [IL-8]) were expressed at high levels in the psoriatic models, but were only minimally expressed in the normal models. Models derived from uninvolved psoriatic skin showed the same gene expression profile as those derived from involved skin along with an increased proliferation rate when compared to normal models. These results suggest that psoriatic individuals possess an inherent predisposition to develop the disease phenotype even in the absence of T cells. This study represents a comprehensive characterization of psoriatic human skin reconstructed in vitro, and demonstrates the potential of this model as a valuable tool in drug discovery.

Adult↗
PubMed1995

Making the in-vitro model closer to actual B lymphopoiesis in the bone marrow.

After more than a decade since Whitlock and Witte established an in-vitro long-term culture of bone marrow B lineage cells, c-kit+IL-7-receptor(R)+B precursor cells in fresh bone marrow are now able to be grown under a fully defined culture condition containing only BSA, transferrin, IL-7 and the ligand for c-kit(Kit-ligand;KL) as protein components. On the other hand, previous studies indicated that the actual intramarrow B-cell-genesis is a complex process involving multiple stromal cell-derived molecules. Thus, the next step for the culture of B-cell genesis is to develop this simple culture into a new defined culture of B-cell-genesis that is closer to the actual process. In this article, we will describe how this defined culture condition has developed from the original Whitlock-Witte type culture, how the B precursors under this culture-condition are different from that in the bone marrow, and finally our biased view on the future direction to which this defined culture should develop.

Animals↗
PubMed1988

An in vitro model of platelet aggregation in stenotic arteries.

Clinical and experimental evidence suggest a strong relationship between arterial stenosis, platelet aggregation, and subsequent thrombus formation. To facilitate the study of platelet accumulation in stenotic arteries, we developed an in vitro preparation. Arterial segments were perfused with whole citrated blood. A stenosis was created by applying an external plastic constrictor to the artery. Platelet accumulation within the stenosis was assessed by scanning electron microscopy and by radioactive counts from Indium-111 labeled platelets. Utilizing this preparation, 30 carotid arterial segments from 10 mongrel dogs were perfused at 100 mmHg for 15 min. In 10 arteries without a stenosis, scanning electron microscopy and radioactive counts demonstrated little platelet accumulation. In contrast, extensive platelet aggregation was observed in 10 arteries with stenoses. Moreover, in 10 stenotic arteries exposed to the thromboxane mimetic, U46619 (Upjohn Diagnostic Group), scanning electron microscopy and radioactive counts demonstrated a significant increase in platelet deposition. Conversely, we demonstrated a dimunition of platelet accumulation in stenosed arterial segments exposed to the prostacyclin analogue platelet inhibitor, Iloprost (Berlex Laboratories, Inc.). The in vitro preparation allows precise control of hemodynamic variables and makes it possible to perform multiple tests on segments of the same vessel from the same animal.

Animals↗