Conditioned polarized Caco-2 cell monolayers allow to discriminate for the ability of gut-derived microorganisms to modulate permeability and antigen-induced basophil degranulation.

BACKGROUND: Food allergy is a common allergic disorder--especially in early childhood. The avoidance of the allergenic food is the only available method to prevent further reactions in sensitized patients. A better understanding of the immunologic mechanisms involved in this reaction would help to develop therapeutic approaches applicable to the prevention of food allergy. OBJECTIVE: To establish a multi-cell in vitro model of sensitized intestinal epithelium that mimics the intestinal epithelial barrier to study the capacity of probiotic microorganisms to modulate permeability, translocation and immunoreactivity of ovalbumin (OVA) used as a model antigen. METHODS: Polarized Caco-2 cell monolayers were conditioned by basolateral basophils and used to examine apical to basolateral transport of OVA by ELISA. Activation of basophils with translocated OVA was measured by beta-hexosaminidase release assay. This experimental setting was used to assess how microorganisms added apically affected these parameters. Basolateral secretion of cytokine/chemokines by polarized Caco-2 cell monolayers was analysed by ELISA. RESULTS: Basophils loaded with OVA-specific IgE responded to OVA in a dose-dependent manner. OVA transported across polarized Caco-2 cell monolayers was found to trigger basolateral basophil activation. Microorganisms including lactobacilli and Escherichia coli increased transepithelial electrical resistance while promoting OVA passage capable to trigger basophil activation. Non-inflammatory levels of IL-8 and thymic stromal lymphopoietin were produced basolaterally by Caco-2 cells exposed to microorganisms. CONCLUSION: The complex model designed in here is adequate to learn about the consequence of the interaction between microorganisms and epithelial cells vis-a-vis the barrier function and antigen translocation, two parameters essential to mucosal homeostasis. It can further serve as a direct tool to search for microorganisms with anti-allergic and anti-inflammatory properties.

Neuronal in vitro models for the estimation of acute systemic toxicity.

The objective of the EU funded integrated project "ACuteTox" is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABA(A) receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro-in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.

Stereospecificity of the siderophore pyochelin outer membrane transporters in fluorescent pseudomonads.

Pyochelin (Pch) and enantio-pyochelin (EPch) are enantiomer siderophores that are produced by Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, under iron limitation. Pch promotes growth of P. aeruginosa when iron is scarce, and EPch carries out the same biological function in P. fluorescens. However, the two siderophores are unable to promote growth in the heterologous species, indicating that siderophore-mediated iron uptake is highly stereospecific. In the present work, using binding and iron uptake assays, we found that FptA, the Fe-Pch outer membrane transporter of P. aeruginosa, recognized (K(d) = 2.5 +/- 1.1 nm) and transported Fe-Pch but did not interact with Fe-EPch. Likewise, FetA, the Fe-EPch receptor of P. fluorescens, was specific for Fe-EPch (K(d) = 3.7 +/- 2.1 nm) but did not bind and transport Fe-Pch. Growth promotion experiments performed under iron-limiting conditions confirmed that FptA and FetA are highly specific for Pch and EPch, respectively. When fptA and fetA along with adjacent transport genes involved in siderophore uptake were swapped between the two bacterial species, P. aeruginosa became able to utilize Fe-EPch as an iron source, and P. fluorescens was able to grow with Fe-Pch. Docking experiments using the FptA structure and binding assays showed that the stereospecificity of Pch recognition by FptA was mostly due to the configuration of the siderophore chiral centers C4'' and C2'' and was only weakly dependent on the configuration of the C4' carbon atom. Together, these findings increase our understanding of the stereospecific interaction between Pch and its outer membrane receptor FptA.

Fission yeast Sec3 and Exo70 are transported on actin cables and localize the exocyst complex to cell poles.

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP(2) and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk.

Defining new criteria for selection of cell-based intestinal models using publicly available databases.

BACKGROUND: The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies. RESULTS: We made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics. CONCLUSIONS: This study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question.

Integration of steroid hormone initiated membrane action to genomic function in the brain

Estrogen is a ligand for the estrogen receptor (ER), which on binding 17beta-estradiol, functions as a ligand-activated transcription factor and regulates the transcription of target genes. This is the slow genomic mode of action. However, rapid non-genomic actions of estrogen also exist at the cell membrane. Using a novel two-pulse paradigm in which the first pulse rapidly initiates non-genomic actions using a membrane-limited estrogen conjugate (E-BSA), while the second pulse promotes genomic transcription from a consensus estrogen response element (ERE), we have demonstrated that rapid actions of estrogen potentiate the slower transcriptional response from an ERE-reporter in neuroblastoma cells. Since rapid actions of estrogen activate kinases, we used selective inhibitors in the two-pulse paradigm to determine the intracellular signaling cascades important in such potentiation. Inhibition of protein kinase A (PKA), PKC, mitogen activated protein kinase (MAPK) or phosphatidylinositol 3-OH kinase (PI-3K) in the first pulse decreases potentiation of transcription. Also, our data with both dominant negative and constitutive mutants of Galpha subunits show that Galpha(q) initiates the rapid signaling cascade at the membrane in SK-N-BE(2)C neuroblastoma cells. We discuss two models of multiple kinase activation at the membrane Pulses of estrogen induce lordosis behavior in female rats. Infusion of E-BSA into the ventromedial hypothalamus followed by 17beta-estradiol in the second pulse could induce lordosis behavior, demonstrating the applicability of this paradigm in vivo. A model where non-genomic actions of estrogen couple to genomic actions unites both aspects of hormone action.

A SequenceSpace analysis of Lys49 phospholipases A2: Clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity

'SequenceSpace' analysis is a novel approach which has been used to identify unique amino acids within a subfamily of phospholipases A2 (PLA2) in which the highly conserved active site residue Asp49 is substituted by Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor Ca2+ and possess extremely low catalytic activity, they demonstrate a Ca2+-independent membrane damaging activity through a poorly understood mechanism, which does not involve lipid hydrolysis. Additionally, Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic pharmacological activities, however the structural basis of these varied functions is largely unknown. Using the 'SequenceSpace' analysis we have identified nine residues highly unique to the Lys49-PLA2 sub-family, which are grouped in three amino acid clusters in the active site, hydrophobic substrate binding channel and homodimer interface regions. These three highly specific residue clusters may have relevance for the Ca2+-independent membrane damaging activity. Of a further 15 less stringently conserved residues, nine are located in two additional clusters which are well isolated from the active site region. The less strictly conserved clusters have been used in predictive sequence searches to correlate amino acid patterns in other venom PLA2s with their pharmacological activities, and motifs for presynaptic and combined toxicities are proposed.

Particles induce apical plasma membrane enlargement in epithelial lung cell line depending on particle surface area dose

Background Airborne particles entering the respiratory tract may interact with the apical plasma membrane (APM) of epithelial cells and enter them. Differences in the entering mechanisms of fine (between 0.1 μm and 2.5 μm) and ultrafine ( ≤ 0.1 μm) particles may be associated with different effects on the APM. Therefore, we studied particle-induced changes in APM surface area in relation to applied and intracellular particle size, surface and number. Methods Human pulmonary epithelial cells (A549 cell line) were incubated with various concentrations of different sized fluorescent polystyrene spheres without surface charge (∅ fine – 1.062 μm, ultrafine – 0.041 μm) by submersed exposure for 24 h. APM surface area of A549 cells was estimated by design-based stereology and transmission electron microscopy. Intracellular particles were visualized and quantified by confocal laser scanning microscopy. Results Particle exposure induced an increase in APM surface area compared to negative control (p < 0.01) at the same surface area concentration of fine and ultrafine particles a finding not observed at low particle concentrations. Ultrafine particle entering was less pronounced than fine particle entering into epithelial cells, however, at the same particle surface area dose, the number of intracellular ultrafine particles was higher than that of fine particles. The number of intracellular particles showed a stronger increase for fine than for ultrafine particles at rising particle concentrations. Conclusion This study demonstrates a particle-induced enlargement of the APM surface area of a pulmonary epithelial cell line, depending on particle surface area dose. Particle uptake by epithelial cells does not seem to be responsible for this effect. We propose that direct interactions between particle surface area and cell membrane cause the enlargement of the APM.

Lipid-protein interactions drive membrane protein topogenesis in accordance with the positive inside rule.

Transmembrane domain orientation within some membrane proteins is dependent on membrane lipid composition. Initial orientation occurs within the translocon, but final orientation is determined after membrane insertion by interactions within the protein and between lipid headgroups and protein extramembrane domains. Positively and negatively charged amino acids in extramembrane domains represent cytoplasmic retention and membrane translocation forces, respectively, which are determinants of protein orientation. Lipids with no net charge dampen the translocation potential of negative residues working in opposition to cytoplasmic retention of positive residues, thus allowing the functional presence of negative residues in cytoplasmic domains without affecting protein topology.

Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore.

Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 sec) and was seen as a 0.219 +/- 0.006 pF drop in capacitance, which corresponds well to the predicted surface area of the entire PVM (30-33 microns2). The formation and closure of a fission pore connecting the extracellular medium and the vacuolar space was detected as the PVM pinched off. This final stage of parasite entry was accomplished without any breach in cell membrane integrity.

T-cell epitope analysis using subtracted expression libraries (TEASEL): application to a 38-kDA autoantigen recognized by T cells from an insulin-dependent diabetic patient.

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases.

Two-Type Age-Dependent Branching Processes with Inhomogeneous Immigration as Models of Renewing Cell Population

2000 Mathematics Subject Classification: primary 60J80; secondary 60J85, 92C37.

Direct Visualization Of The Action Of Triton X-100 On Giant Vesicles Of Erythrocyte Membrane Lipids.

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.

Structural properties of lipid reconstructs and lipid composition of normotensive and hypertensive rat vascular smooth muscle cell membranes

Multiple cell membrane alterations have been reported to be the cause of various forms of hypertension. The present study focuses on the lipid portion of the membranes, characterizing the microviscosity of membranes reconstituted with lipids extracted from the aorta and mesenteric arteries of spontaneously hypertensive (SHR) and normotensive control rat strains (WKY and NWR). Membrane-incorporated phospholipid spin labels were used to monitor the bilayer structure at different depths. The packing of lipids extracted from both aorta and mesenteric arteries of normotensive and hypertensive rats was similar. Lipid extract analysis showed similar phospholipid composition for all membranes. However, cholesterol content was lower in SHR arteries than in normotensive animal arteries. These findings contrast with the fact that the SHR aorta is hyporeactive while the SHR mesenteric artery is hyperreactive to vasopressor agents when compared to the vessels of normotensive animal strains. Hence, factors other than microviscosity of bulk lipids contribute to the vascular smooth muscle reactivity and hypertension of SHR. The excess cholesterol in the arteries of normotensive animal strains apparently is not dissolved in bulk lipids and is not directly related to vascular reactivity since it is present in both the aorta and mesenteric arteries. The lower cholesterol concentrations in SHR arteries may in fact result from metabolic differences due to the hypertensive state or to genes that co-segregate with those that determine hypertension during the process of strain selection.