An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.

A mouse model of Schwartz-Jampel syndrome reveals myelinating Schwann cell dysfunction with persistent axonal depolarization in vitro and distal peripheral nerve hyperexcitability when perlecan is lacking

Congenital peripheral nerve hyperexcitability (PNH) is usually associated with impaired function of voltage-gated K(+) channels (VGKCs) in neuromyotonia and demyelination in peripheral neuropathies. Schwartz-Jampel syndrome (SJS) is a form of PNH that is due to hypomorphic mutations of perlecan, the major proteoglycan of basement membranes. Schwann cell basement membrane and its cell receptors are critical for the myelination and organization of the nodes of Ranvier. We therefore studied a mouse model of SJS to determine whether a role for perlecan in these functions could account for PNH when perlecan is lacking. We revealed a role for perlecan in the longitudinal elongation and organization of myelinating Schwann cells because perlecan-deficient mice had shorter internodes, more numerous Schmidt-Lanterman incisures, and increased amounts of internodal fast VGKCs. Perlecan-deficient mice did not display demyelination events along the nerve trunk but developed dysmyelination of the preterminal segment associated with denervation processes at the neuromuscular junction. Investigating the excitability properties of the peripheral nerve suggested a persistent axonal depolarization during nerve firing in vitro, most likely due to defective K(+) homeostasis, and excluded the nerve trunk as the original site for PNH. Altogether, our data shed light on perlecan function by revealing critical roles in Schwann cell physiology and suggest that PNH in SJS originates distally from synergistic actions of peripheral nerve and neuromuscular junction changes.

Development And Use Of A Model To Predict Properties Of Infiltrated Solid Oxide Fuel Cell Anodes

Solid oxide fuel cells (SOFCs) provide a potentially clean way of using energy sources. One important aspect of a functioning fuel cell is the anode and its characteristics (e.g. conductivity). Using infiltration of conductor particles has been shown to be a method for production at lower cost with comparable functionality. While these methods have been demonstrated experimentally, there is a vast range of variables to consider. Because of the long time for manufacture, a model is desired to aid in the development of the desired anode formulation. This thesis aims to (1) use an idealized system to determine the appropriate size and aspect ratio to determine the percolation threshold and effective conductivity as well as to (2) simulate the infiltrated fabrication method to determine the effective conductivity and percolation threshold as a function of ceramic and pore former particle size, particle fraction and the cell¿s final porosity. The idealized system found that the aspect ratio of the cell does not affect the cells functionality and that an aspect ratio of 1 is the most efficient computationally to use. Additionally, at cell sizes greater than 50x50, the conductivity asymptotes to a constant value. Through the infiltrated model simulations, it was found that by increasing the size of the ceramic (YSZ) and pore former particles, the percolation threshold can be decreased and the effective conductivity at low loadings can be increased. Furthermore, by decreasing the porosity of the cell, the percolation threshold and effective conductivity at low loadings can also be increased

Annexins as cell-type-specific markers in the developing chicken chorionallantoic membrane

Between day E8 and E12 of embryonic development, the chicken chorioallantoic membrane (CAM) undergoes massive structural rearrangement enabling calcium-uptake from the eggshell to supply the growing embryo. However, the contribution of the various cell types of the chorionic epithelium including the capillary covering (CC) cells, villus cavity (VC) cells, endothelial-like cells, and basal cells to this developmental program is largely unknown. In order to obtain markers for the different cell types in the chorionic epithelium, we determined the expression patterns of various calcium-binding annexins in the developing chicken CAM. By reverse transcription/polymerase chain reaction with primers deduced from nucleotide sequences available in various databases, the presence of annexin (anx)-1, anx-2, anx-5, and anx-6 was demonstrated at days E8 and E12. Quantitative immunoblotting with novel antibodies raised against the recombinant proteins revealed that anx-1 and anx-5 were significantly up-regulated at day E12, whereas anx-2 and anx-6 expression remained almost unchanged in comparison to levels at day E8. Immunohistochemistry of paraffin-embedded sections of E12 CAM revealed anx-1 in CC cells and VC cells. Anx-2 was localized in capillaries in the chorionic epithelium and in basal cells of the allantoic epithelium, whereas anx-6 was detected in basal cells or endothelial-like cells of the chorionic epithelium and in the media of larger vessels in the mesenchyme. A 2-day exposure of the CAM to a tumor cell spheroid resulted in strong proliferation of anx-1-expressing CC cells suggesting that these cells participate in the embryonic response to experimental intervention. Thus, annexins exhibit complementary expression patterns and represent appropriate cell markers for the further characterization of CAM development and the interpretation of results obtained when using CAM as an experimental model.

An optimized in vitro model of the respiratory tract wall to study particle cell interactions

As a part of the respiratory tissue barrier, lung epithelial cells play an important role against the penetration of the body by inhaled particulate foreign materials. In most cell culture models, which are designed to study particle-cell interactions, the cells are immersed in medium. This does not reflect the physiological condition of lung epithelial cells which are exposed to air, separated from it only by a very thin liquid lining layer with a surfactant film at the air-liquid interface. In this study, A549 epithelial cells were grown on microporous membranes in a two chamber system. After the formation of a confluent monolayer the cells were exposed to air. The morphology of the cells and the expression of tight junction proteins were studied with confocal laser scanning and transmission electron microscopy. Air-exposed cells maintained monolayer structure for 2 days, expressed tight junctions and developed transepithelial electrical resistance. Surfactant was produced and released at the apical side of the air-exposed epithelial cells. In order to study particle-cell interactions fluorescent 1 microm polystyrene particles were sprayed over the epithelial surface. After 4 h, 8.8% of particles were found inside the epithelium. This fraction increased to 38% after 24 h. During all observations, particles were always found in the cells but never between them. In this study, we present an in vitro model of the respiratory tract wall consisting of air-exposed lung epithelial cells covered by a liquid lining layer with a surfactant film to study particle-cell interactions.

Gene expression profiling reveals consistent differences between clinical samples of human leukaemias and their model cell lines

Microarray gene expression profiles of fresh clinical samples of chronic myeloid leukaemia in chronic phase, acute promyelocytic leukaemia and acute monocytic leukaemia were compared with profiles from cell lines representing the corresponding types of leukaemia (K562, NB4, HL60). In a hierarchical clustering analysis, all clinical samples clustered separately from the cell lines, regardless of leukaemic subtype. Gene ontology analysis showed that cell lines chiefly overexpressed genes related to macromolecular metabolism, whereas in clinical samples genes related to the immune response were abundantly expressed. These findings must be taken into consideration when conclusions from cell line-based studies are extrapolated to patients.

Mechanisms of intrinsic beating variability in cardiac cell cultures and model pacemaker networks

Heart rate variability (HRV) exhibits fluctuations characterized by a power law behavior of its power spectrum. The interpretation of this nonlinear HRV behavior, resulting from interactions between extracardiac regulatory mechanisms, could be clinically useful. However, the involvement of intrinsic variations of pacemaker rate in HRV has scarcely been investigated. We examined beating variability in spontaneously active incubating cultures of neonatal rat ventricular myocytes using microelectrode arrays. In networks of mathematical model pacemaker cells, we evaluated the variability induced by the stochastic gating of transmembrane currents and of calcium release channels and by the dynamic turnover of ion channels. In the cultures, spontaneous activity originated from a mobile focus. Both the beat-to-beat movement of the focus and beat rate variability exhibited a power law behavior. In the model networks, stochastic fluctuations in transmembrane currents and stochastic gating of calcium release channels did not reproduce the spatiotemporal patterns observed in vitro. In contrast, long-term correlations produced by the turnover of ion channels induced variability patterns with a power law behavior similar to those observed experimentally. Therefore, phenomena leading to long-term correlated variations in pacemaker cellular function may, in conjunction with extracardiac regulatory mechanisms, contribute to the nonlinear characteristics of HRV.

Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine model

Mesenchymal stem cells (MSCs) provide an important source of pluripotent cells for musculoskeletal tissue repair. This study examined the impact of MSC implantation on cartilage healing characteristics in a large animal model. Twelve full-thickness 15-mm cartilage lesions in the femoropatellar articulations of six young mature horses were repaired by injection of a self-polymerizing autogenous fibrin vehicle containing mesenchymal stem cells, or autogenous fibrin alone in control joints. Arthroscopic second look and defect biopsy was obtained at 30 days, and all animals were euthanized 8 months after repair. Cartilage repair tissue and surrounding cartilage were assessed by histology, histochemistry, collagen type I and type II immunohistochemistry, collagen type II in situ hybridization, and matrix biochemical assays. Arthroscopic scores for MSC-implanted defects were significantly improved at the 30-day arthroscopic assessment. Biopsy showed MSC-implanted defects contained increased fibrous tissue with several defects containing predominantly type II collagen. Long-term assessment revealed repair tissue filled grafted and control lesions at 8 months, with no significant difference between stem cell-treated and control defects. Collagen type II and proteoglycan content in MSC-implanted and control defects were similar. Mesenchymal stem cell grafts improved the early healing response, but did not significantly enhance the long-term histologic appearance or biochemical composition of full-thickness cartilage lesions.

Orthotopic transplantation of v-src-expressing glioma cell lines into immunocompetent mice: establishment of a new transplantable in vivo model for malignant glioma

OBJECT: The aim of this study was to develop and characterize a new orthotopic, syngeneic, transplantable mouse brain tumor model by using the cell lines Tu-9648 and Tu-2449, which were previously isolated from tumors that arose spontaneously in glial fibrillary acidic protein (GFAP)-v-src transgenic mice. METHODS: Striatal implantation of a 1-microl suspension of 5000 to 10,000 cells from either clone into syngeneic B6C3F1 mice resulted in tumors that were histologically identified as malignant gliomas. Prior subcutaneous inoculations with irradiated autologous cells inhibited the otherwise robust development of a microscopically infiltrating malignant glioma. Untreated mice with implanted tumor cells were killed 12 days later, when the resultant gliomas were several millimeters in diameter. Immunohistochemically, the gliomas displayed both the astroglial marker GFAP and the oncogenic form of signal transducer and activator of transcription-3 (Stat3). This form is called tyrosine-705 phosphorylated Stat3, and is found in many malignant entities, including human gliomas. Phosphorylated Stat3 was particularly prominent, not only in the nucleus but also in the plasma membrane of peripherally infiltrating glioma cells, reflecting persistent overactivation of the Janus kinase/Stat3 signal transduction pathway. The Tu-2449 cells exhibited three non-random structural chromosomal aberrations, including a deletion of the long arm of chromosome 2 and an apparently balanced translocation between chromosomes 1 and 3. The GFAP-v-src transgene was mapped to the pericentromeric region of chromosome 18. CONCLUSIONS: The high rate of engraftment, the similarity to the high-grade malignant glioma of origin, and the rapid, locally invasive growth of these tumors should make this murine model useful in testing novel therapies for human malignant gliomas.

Agents used for chemoprevention of prostate cancer may influence PSA secretion independently of cell growth in the LNCaP model of human prostate cancer progression

BACKGROUND: The aim of this study was to evaluate the inhibitory growth effects of different potential chemopreventive agents in vitro and to determine their influence on PSA mRNA and protein expression with an established screening platform. METHODS: LNCaP and C4-2 cells were incubated with genistein, seleno-L-methionine, lycopene, DL-alpha-tocopherol, and trans-beta-carotene at three different concentrations and cell growth was determined by the MTT assay. PSA mRNA expression was assessed by quantitative real-time RT-PCR and secreted PSA protein levels were quantified by the microparticle enzyme immunoassay. RESULTS: Genistein, seleno-l-methionine and lycopene inhibited LNCaP cell growth, and the proliferation of C4-2 cells was suppressed by seleno-L-methionine and lycopene. PSA mRNA expression was downregulated by genistein in LNCaP but not C4-2 cells. No other compound tested altered PSA mRNA expression. PSA protein expression was downregulated by genistein, seleno-L-methionine, DL-alpha-tocopherol in LNCaP cells. In C4-2 cells only genistein significantly reduced the secretion of PSA protein. CONCLUSIONS: In the LNCaP progression model PSA expression depends on the compound, its concentration and on the hormonal dependence of the cell line used and does not necessarily reflect cell growth or death. Before potential substances are evaluated in clinical trials using PSA as a surrogate end point marker, their effect on PSA mRNA and protein expression has to be considered to correctly assess treatment response by PSA.