Mind the gap between feeling bad and feeling dead: Stress but not death reminders elicit endocrine responses.

Stressors and mortality salience share considerable conceptual overlap. Thus, we examined the impact of a standard mortality salience and a standard stress manipulation on the activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis via endocrine measures of stress; a neutral control condition completed the design. The results revealed that stress elicits increased salivary α-amylase and salivary cortisol reactions; however, no endocrine reactions were found in the mortality salience and the control conditions. To the contrary, we did not find any differences regarding positive and negative affect between any conditions. Implications for social and health psychology are being discussed.

Nonstructural Nipah virus C protein regulates both the early host proinflammatory response and viral virulence.

Nipah virus (NiV) is a highly pathogenic, negative-strand RNA paramyxovirus that has recently emerged from flying foxes to cause serious human disease. We have analyzed the role of the nonstructural NiV C protein in viral immunopathogenesis using recombinant virus lacking the expression of NiV C (NiVΔC). While wild-type NiV was highly pathogenic in the hamster animal model, NiVΔC was strongly attenuated. Replication of NiVΔC was followed by the production of NiV-specific antibodies and associated with higher recruitment of inflammatory cells and less intensive histopathological lesions in different organs than in wild-type-NiV-infected animals. To analyze the molecular basis of NiVΔC attenuation, we studied early changes in gene expression in infected primary human endothelial cells, a major cellular target of NiV infection. The transcriptomic approach revealed the striking difference between wild-type and mutant NiV in the expression of genes involved in immunity, with the particularly interesting differential patterns of proinflammatory cytokines. Compared to wild-type virus, NiVΔC induced increased expression of interleukin 1 beta (IL-1ß), IL-8, CXCL2, CXCL3, CXCL6, CCL20, and beta interferon. Furthermore, the expression of NiV C in stably transfected cells decreased the production of the same panel of cytokines, revealing a role of the C protein in the regulation of cytokine balance. Together, these results suggest that NiV C regulates expression of proinflammatory cytokines, therefore providing a signal responsible for the coordination of leukocyte recruitment and the chemokine-induced immune response and controlling the lethal outcome of the infection.

Interactions of silica nanoparticles with lung epithelial cells and the association to flotillins.

Amorphous silica nanoparticles (aSNPs) gain increasing popularity for industrial and therapeutic claims. The lung with its surface area of 100-140 m(2) displays an ideal target for therapeutic approaches, but it represents also a serious area of attack for harmful nanomaterials. The exact nature of the cytotoxic effects of NPs is still unknown. Furthermore, cellular pathways and the destiny of internalized NPs are still poorly understood. Therefore, we examined the cytotoxicity (MTS, LDH) and inflammatory responses (IL-8) for different-sized aSNPs (30, 70, 300 nm) on our lung epithelial cells line NCI H441 and endothelial cell line ISO-HAS-1. Additionally, colocalization studies have been conducted via immunofluorescence staining for flotillin-1- and flotillin-2-bearing endocytic vesicles. Subsequently, the relevance of flotillins concerning the viability of aSNP-exposed epithelial cells has been evaluated using flotillin-1/2 depleted cells (siRNA). This study reveals the relevance of the nanoparticle size regarding cytotoxicity (MTS, LDH) and inflammatory responses (IL-8), whereat the smaller the size of the nanoparticle is, the more harmful are the effects. All different aSNP sizes have been incorporated in flotillin-1- and flotillin-2-labelled vesicles in lung epithelial and endothelial cells, which display a marker for late endosomal or lysosomal structures and appear to exhibit a clathrin- or caveolae-independent mode of endocytosis. Flotillin-depleted H441 showed a clearly decreased uptake of aSNPs. Additionally, the viability of aSNP-exposed cells was reduced in these cells. These findings indicate a contribution of flotillins in as yet unknown (clathrin or caveolae-independent) endocytosis mechanisms and (or) endosomal storage.

Nipah virus uses leukocytes for efficient dissemination within a host.

Nipah virus (NiV) is a recently emerged zoonotic paramyxovirus whose natural reservoirs are several species of Pteropus fruit bats. NiV provokes a widespread vasculitis often associated with severe encephalitis, with up to 75% mortality in humans. We have analyzed the pathogenesis of NiV infection, using human leukocyte cultures and the hamster animal model, which closely reproduces human NiV infection. We report that human lymphocytes and monocytes are not permissive for NiV and a low level of virus replication is detected only in dendritic cells. Interestingly, despite the absence of infection, lymphocytes could efficiently bind NiV and transfer infection to endothelial and Vero cells. This lymphocyte-mediated transinfection was inhibited after proteolytic digestion and neutralization by NiV-specific antibodies, suggesting that cells could transfer infectious virus to other permissive cells without the requirement for NiV internalization. In NiV-infected hamsters, leukocytes captured and carried NiV after intraperitoneal infection without themselves being productively infected. Such NiV-loaded mononuclear leukocytes transfer lethal NiV infection into naïve animals, demonstrating efficient virus transinfection in vivo. Altogether, these results reveal a remarkable capacity of NiV to hijack leukocytes as vehicles to transinfect host cells and spread the virus throughout the organism. This mode of virus transmission represents a rapid and potent method of NiV dissemination, which may contribute to its high pathogenicity.

Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: comparison with conventional monocultures.

BACKGROUND: To date silica nanoparticles (SNPs) play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities). They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs) of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures. METHODS: Thus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release), and TER (Transepithelial Electrical Resistance) measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release) and apoptosis markers were investigated. RESULTS: Regarding toxic effects (viability, membrane integrity, TER) the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment. Analysis of the individual markers indicated that the cells suffered from DNA damage, hypoxia and ER-stress. CONCLUSION: We present evidence that our in vitro coculture model of the alveolar-capillary barrier is clearly advantageous compared to conventional monocultures in evaluating the extent of damage caused by hazardous material encountering the principle biological barrier in the lower respiratory tract.

Measles virus M protein-driven particle production does not involve the endosomal sorting complex required for transport (ESCRT) system.

Assembly and budding of enveloped RNA viruses rely on viral matrix (M) proteins and host proteins involved in sorting and vesiculation of cellular cargoes, such as the endosomal sorting complex required for transport (ESCRT). The measles virus (MV) M protein promotes virus-like particle (VLP) production, and we now show that it shares association with detergent-resistant or tetraspanin-enriched membrane microdomains with ebolavirus VP40 protein, yet accumulates less efficiently at the plasma membrane. Unlike VP40, which recruits ESCRT components via its N-terminal late (L) domain and exploits them for particle production, the M protein does this independently of this pathway, as (i) ablation of motifs bearing similarity to canonical L domains did not affect VLP production, (ii) it did not redistribute Tsg101, AIP-1 or Vps4A to the plasma membrane, and (iii) neither VLP nor infectious virus production was sensitive to inhibition by dominant-negative Vps4A. Importantly, transfer of the VP40 L domain into the MV M protein did not cause recruitment of ESCRT proteins or confer sensitivity of VLP release to Vps4A, indicating that MV particle production occurs independently of and cannot be routed into an ESCRT-dependent pathway.

Side-specific effects by cadmium exposure: apical and basolateral treatment in a coculture model of the blood-air barrier.

Cadmium (Cd(2+)) is a widespread environmental pollutant, which is associated with a wide variety of cytotoxic and metabolic effects. Recent studies showed that intoxication with the heavy metal most importantly targets the integrity of the epithelial barrier. In our study, the lung epithelial cell line, NCI H441, was cultured with the endothelial cell line, ISO-HAS-1, as a bilayer on a 24-well HTS-Transwell filter plate. This coculture model was exposed to various concentrations of CdCl(2). The transepithelial electrical resistance decreased on the apical side only after treatment with high Cd(2+) concentrations after 48 h. By contrast, a breakdown of TER to less than 5% of baseline could be observed much earlier (after 24 h) when Cd(2+) was administered from the basal side. Observations of cell layer fragmentation and widening of intercellular spaces confirmed the barrier breakdown only for the basolaterally treated samples. Furthermore, the cytotoxicity and release of proinflammatory markers was enhanced if samples were exposed to Cd(2+) from the basal side compared to treatment from the apical side. Moreover, we could demonstrate that a high concentration of Ca(2+) could prevent the barrier-disrupting effect of Cd(2+). In conclusion, the exposure of Cd(2+) to cocultures of lung cells caused a decrease in TER, major morphological changes, a reduction of cell viability and an increase of cytokine release, but the effects markedly differed between the two modes of exposure. Therefore, our results suggest that intact epithelial TJs may play a major role in protecting the air-blood barrier from inhaled Cd(2+).

Gold nanoparticles induce cytotoxicity in the alveolar type-II cell lines A549 and NCIH441.

BACKGROUND: During the last years engineered nanoparticles (NPs) have been extensively used in different technologies and consequently many questions have arisen about the risk and the impact on human health following exposure to nanoparticles. Nevertheless, at present knowledge about the cytotoxicity induced by NPs is still largely incomplete. In this context, we have investigated the cytotoxicity induced by gold nanoparticles (AuNPs), which differed in size and purification grade (presence or absence of sodium citrate residues on the particle surface) in vitro, in the human alveolar type-II (ATII)-like cell lines A549 and NCIH441. RESULTS: We found that the presence of sodium citrate residues on AuNPs impaired the viability of the ATII-like cell lines A549 and NCIH441. Interestingly, the presence of an excess of sodium citrate on the surface of NPs not only reduced the in vitro viability of the cell lines A549 and NCIH441, as shown by MTT assay, but also affected cellular proliferation and increased the release of lactate dehydrogenase (LDH), as demonstrated by Ki-67 and LDH-release assays respectively. Furthermore, we investigated the internalization of AuNPs by transmission electron microscopy (TEM) and we observed that particles were internalized by active endocytosis in the cell lines A549 and NCIH441 within 3 hr. In addition, gold particles accumulated in membrane-bound vesicles and were not found freely dispersed in the cytoplasm. CONCLUSION: Our data suggest that the presence of contaminants, such as sodium citrate, on the surface of gold nanoparticles might play a pivotal role in inducing cytotoxicity in vitro, but does not influence the uptake of the particles in human ATII-like cell lines.

Cytochrome P450 1A1 expression and activity in Caco-2 cells: modulation by apple juice extract and certain apple polyphenols.

Cytochrome P450 (CYP) 1A1 plays a role in drug metabolism of intestinal cells (e.g., by activating certain chemical carcinogens such as polycyclic aromatic hydrocarbons into carcinogenic metabolites). In the human colon carcinoma cell line Caco-2, we investigated the effects of a defined polyphenolic apple juice extract (AJE), the major principle flavonoid/dihydrochalkone constituents quercetin and phloretin, and the corresponding prototype glycosides rutin and phlorizin on CYP1A1 expression and activity. Incubations were carried out with or without the potent aryl hydrocarbon receptor agonist/CYP1A1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AJE and quercetin acted as weak inducers of CYP1A1 mRNA and protein, and AJE, quercetin, and phlorizin led to a slight induction of CYP1A1-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity. However, AJE, quercetin, and phloretin were highly effective in suppressing CYP1A1 induction in co-incubations of the cells with 1 nM TCDD. The antagonistic effects were seen on the levels of mRNA, enzyme protein, and catalytic activity. In contrast, the related glycosides rutin and phlorizin were inactive as inducers or inhibitors. Inhibition of CYP1A1 induction was not related to general cytotoxicity, which could be completely abolished by the addition of ascorbic acid/alpha-tocopherol. AJE, quercetin, and phloretin also antagonized the TCDD-mediated induction of a reporter gene driven by a regulatory sequence of the human CYP1A1 gene promoter. Our findings suggest that apple juice extract can antagonize TCDD-mediated CYP1A1 induction by interfering with AhR-dependent gene transcription and by inhibiting the catalytic activity of CYP1A1. These effects may result in reduced metabolic activation of certain chemical carcinogens, in particular, under conditions of sustained AhR activation.

Use of the Cultex® Radial Flow System as an in vitro exposure method to assess acute pulmonary toxicity of fine dusts and nanoparticles with special focus on the intra- and inter-laboratory reproducibility.

Exposure of the respiratory tract to airborne particles (including metal-dusts and nano-particles) is considered as a serious health hazard. For a wide range of substances basic knowledge about the toxic properties and the underlying pathomechanisms is lacking or even completely missing. Legislation demands the toxicological characterization of all chemicals placed on the market until 2018 (REACH). As toxicological in vivo data are rare with regard to acute lung toxicity or exhibit distinct limitations (e.g. inter-species differences) and legislation claims the reduction of animal experiments in general ("3R" principle), profound in vitro models have to be established and characterized to meet these requirements. In this paper we characterize a recently introduced advanced in vitro exposure system (Cultex® RFS) showing a great similarity to the physiological in vivo exposure situation for the assessment of acute pulmonary toxicity of airborne materials. Using the Cultex® RFS, human lung epithelial cells (A549 cells) were exposed to different concentrations of airborne metal dusts (nano- and microscale particles) at the air-liquid-interface (ALI). Cell viability (WST-1 assay) as a parameter of toxicity was assessed 24h after exposure with special focus on the intra- and inter-laboratory (three independent laboratories) reproducibility. Our results show the general applicability of the Cultex® RFS with regard to the requirements of the ECVAM (European Centre for the Validation of Alternative Methods) principles on test validity underlining its robustness and stability. Intra- and inter-laboratory reproducibility can be considered as sufficient if predefined quality criteria are respected. Special attention must be paid to the pure air controls that turned out to be a critical parameter for a rational interpretation of the results. Our results are encouraging and future work is planned to improve the inter-laboratory reproducibility, to consolidate the results so far and to develop a valid prediction model.

Influence of organophosphate poisoning on human dendritic cells.

Organophosphourus compounds (OPC, including nerve agents and pesticides) exhibit acute toxicity by inhibition of acetylcholinesterase. Lung affections are frequent complications and a risk factor for death. In addition, epidemiological studies reported immunological alterations after OPC exposure. In our experiments we investigated the effects of organophosphourus pesticides dimethoate and chlorpyrifos on dendritic cells (DC) that are essential for the initial immune response, especially in the pulmonary system. DC, differentiated from the monocyte cell line THP-1 by using various cytokines (IL-4, GM-CSF, TNF-α, Ionomycin), were exposed to organophosphourus compounds at different concentrations for a 24h time period. DC were characterized by flow cytometry and immunofluorescence using typical dendritic cell markers (e.g., CD11c, CD209 and CD83). After OPC exposure we investigated cell death, the secretion profile of inflammatory mediators, changes of DC morphology, and the effect on protein kinase signalling pathways. Our results revealed a successful differentiation of THP-1 into DC. OPC exposure caused a significant concentration-dependent influence on DC: Dendrites of the DC were shortened and damaged, DC-specific cell surface markers (i.e., CD83and CD209) decreased dramatically after chlorpyrifos exposure. Interestingly, the effects caused by dimethoate were in general less pronounced. The organophosphourus compounds affected the release of inflammatory cytokines, such as IL-1ß and IL-8. The anti-inflammatory cytokine IL-10 was significantly down regulated. Protein kinases like the Akt family or ERK, which are essential for cell survival and proliferation, were inhibited by both OPC. These findings indicate that the tested organophosphourus compounds induced significant changes in cell morphology, inhibited anti-inflammatory cytokines and influenced important protein signalling pathways which are involved in regulation of apoptosis. Thus our results highlight novel aspects -apparently independent of AChE inhibition- of OPC poisoning with regard to lung toxicity. Our findings contribute to the basic understanding of pulmonary complications caused by OPC poisoning.

Flotillin-involved uptake of silica nanoparticles and responses of an alveolar-capillary barrier in vitro.

Drug and gene delivery via nanoparticles across biological barriers such as the alveolar-capillary barrier of the lung constitutes an interesting and increasingly relevant field in nanomedicine. Nevertheless, potential hazardous effects of nanoparticles (NPs) as well as their cellular and systemic fate should be thoroughly examined. Hence, this study was designed to evaluate the effects of amorphous silica NPs (Sicastar) and (poly)organosiloxane NPs (AmOrSil) on the viability and the inflammatory response as well as on the cellular uptake mechanisms and fate in cells of the alveolar barrier. For this purpose, the alveolar epithelial cell line (NCI H441) and microvascular endothelial cell line (ISO-HAS-1) were used in an experimental set up resembling the alveolar-capillary barrier of the lung. In terms of IL-8 and sICAM Sicastar resulted in harmful effects at higher concentrations (60 µg/ml) in conventional monocultures but not in the coculture, whereas AmOrSil showed no significant effects. Immunofluorescence counterstaining of endosomal structures in NP-incubated cells showed no evidence for a clathrin- or caveolae-mediated uptake mechanism. However, NPs were enclosed in flotillin-1 and -2 marked vesicles in both cell types. Flotillins appear to play a role in cellular uptake or trafficking mechanisms of NPs and are discussed as indicators for clathrin- or caveolae-independent uptake mechanisms. In addition, we examined the transport of NPs across this in vitro model of the alveolar-capillary barrier forming a tight barrier with a transepithelial electrical resistance of 560±8 Ω cm(2). H441 in coculture with endothelial cells took up much less NPs compared to monocultures. Moreover, coculturing prevented the transport of NP from the epithelial compartment to the endothelial layer on the bottom of the filter insert. This supports the relevance of coculture models, which favour a differentiated and polarised epithelial layer as in vitro test systems for nanoparticle uptake.

Hospitality and the mental health of children and families.

In many ancient cultures, hospitality was a significant moral and religious practice in responding to the needs of strangers. Although largely trivialized in contemporary society, the practice of hospitality continues to create contexts that contribute to individual and communal flourishing and that resist several dehumanizing social trends. A recovery of the practice can help faith communities connect their moral and theological commitments to the needs of children, families, and neighborhoods.

Acute cytotoxicity and apoptotic effects after l-Pam exposure in different cocultures of the proximal and distal respiratory system.

Sulphur and nitrogen mustard are strong alkylating agents which can cause after inhalation acute lung injury in the larynx, trachea and large bronchi and can lead to alveolar edema. In our study we tested the N-Lost l-Phenylalanine Mustard (l-Pam). Therefore we seeded the alveolar type II cell line NCI H441 on the upper membrane of a Transwell filter plate and the endothelial cell line ISO-Has-1 on the lower side of the membrane for the alveolar model and combined the human bronchial explant-outgrowth cells and fibroblasts in the bronchial model and exposed both models with various concentrations of l-Pam. Treatment with l-Pam led to a concentration-dependent decrease of the transepithelial electrical resistance and therefore impairment of barrier function in both models. Changes in morphology could be observed. In the bronchial model damaged cell organelles whereas in the alveolar model a widening of intercellular spaces could be seen. Loss of cell-matrix adhesion as well as apoptotic and necrotic cell death could be demonstrated. In conclusion, treatment with the nitrogen mustard in the coculture models showed comparable results to sulphur mustard treatment and thus this model could be useful to explore similarities and differences in signal transduction pathways after treatment with both sulphur and nitrogen mustard.

An impaired alveolar-capillary barrier in vitro: effect of proinflammatory cytokines and consequences on nanocarrier interaction.

The alveolar region of the lung is an important target for drug and gene delivery approaches. Treatment with drugs is often necessary under pathophysiological conditions, in which there is acute inflammation of the target organ. Therefore, in vitro models of the alveolar-capillary barrier, which mimic inflammatory conditions in the alveolar region, would be useful to analyse and predict effects of novel drugs on healthy or inflamed tissues. The epithelial cell line H441 was cultivated with primary isolated human pulmonary microvascular endothelial cells (HPMECs) or the endothelial cell line ISO-HAS-1 on opposite sides of a permeable filter support under physiological and inflammatory conditions. Both epithelial and endothelial cell types grew as polarized monolayers in bilayer coculture and were analysed in the presence and absence of the proinflammatory stimuli tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In addition, the nanocarrier polyethyleneimine (PEI) was chosen as a model compound to study cell uptake (Oregon Green (OG)-labelled PEI) and gene transfer (PEI-pDNA complex). Upon treatment with TNF-alpha and IFN-gamma, both cocultures exhibited comparable effects on the trans-bilayer electrical resistance, the transport of sodium fluorescein and the increase in secondary cytokine release. Basolateral (endothelial side) exposure to TNF-alpha or simultaneous exposure to TNF-alpha and IFN-gamma generated an alveolar-capillary barrier with inflammation-like characteristics, impaired barrier function and a local disruption of the continuous apical labelling of the tight junction plaque protein zonula occludens-1 (ZO-1). Although transfection rates of 8 per cent were obtained for H441 cells in non-polarized monocultures, apical-basolateral-differentiated (polarized) H441 in coculture could not be transfected. After basolateral cytokine exposure, uptake of fluorescently labelled PEI in polarized H441 was predominantly detected in those areas with a local disruption of ZO-1 expression. Accordingly, transfected cells were only sparsely found in coculture after basolateral costimulation with TNF-alpha and IFN-gamma. We designed a coculture model that mimics both the structural architecture of the alveolar-capillary barrier and inflammatory mechanisms with consequences on barrier characteristics, cytokine production and nanoparticle interaction. Our model will be suitable to systematically study adsorption, uptake and trafficking of newly synthesized nanosized carriers under different physiological conditions.

Nipah virus entry can occur by macropinocytosis.

Nipah virus (NiV) is a zoonotic biosafety level 4 paramyxovirus that emerged recently in Asia with high mortality in man. NiV is a member, with Hendra virus (HeV), of the Henipavirus genus in the Paramyxoviridae family. Although NiV entry, like that of other paramyxoviruses, is believed to occur via pH-independent fusion with the host cell's plasma membrane we present evidence that entry can occur by an endocytic pathway. The NiV receptor ephrinB2 has receptor kinase activity and we find that ephrinB2's cytoplasmic domain is required for entry but is dispensable for post-entry viral spread. The mutation of a single tyrosine residue (Y304F) in ephrinB2's cytoplasmic tail abrogates NiV entry. Moreover, our results show that NiV entry is inhibited by constructions and drugs specific for the endocytic pathway of macropinocytosis. Our findings could potentially permit the rapid development of novel low-cost antiviral treatments not only for NiV but also HeV.

Barrier functions and paracellular integrity in human cell culture models of the proximal respiratory unit.

Airway epithelial cells provide a barrier to the translocation of inhaled materials. Tight (TJ) and adherens junctions (AJ) play a key role in maintaining barrier functions, and are responsible for the selective transport of various substances through the paracellular pathway. In this study we compared a bronchial cell line (16HBE14o-) and primary bronchial cells (HBEC), both cocultivated with the fibroblast cell line Wi-38, with respect to their structural differentiation and their reaction to cytokine stimulation. HBEC formed a pseudostratified epithelial layer and expressed TJ and AJ proteins after 2 weeks in coculture. Mucus-producing and ciliated cells were found within 24 days. Additionally, a beating activity of the ciliated HBEC (14-19Hz) could be detected. 16HBE14o- in coculture showed a multilayered growth without differentiation to a pseudostratified airway epithelium. Simultaneous exposure to TNF-alpha- and IFN-gamma-induced significant changes in barrier function and paracellular permeability in the cocultures of HBEC/Wi-38 but not in the 16HBE14o-/Wi-38. In summary, HBEC in coculture mimic the structure of native polarized bronchial epithelium showing basal, mucus-producing and ciliated cells. Our system provides an opportunity to examine the factors that influence barrier and mucociliary function of bronchial epithelium within a time frame of 3 weeks up to 3 months in an in vivo-like differentiated model.

Acute morphological and toxicological effects in a human bronchial coculture model after sulfur mustard exposure.

Sulfur mustard (SM) is a strong alkylating agent. Inhalation of SM causes acute lung injury accompanied by severe disruption of the airway barrier. In our study, we tested the acute effects after mustard exposure in an in vitro coculture bronchial model of the proximal barrier. To achieve this, we seeded normal human bronchial epithelial explant-outgrowth cells (HBEC) together with lung fibroblasts as a bilayer on filter plates and exposed the bronchial model after 31 days of differentiation to various concentrations of SM (30, 100, 300, and 500 microM). The HBEC formed confluent layers, expressing functional tight junctions as measured by transepithelial electrical resistance (TER). Mucus production and cilia formation reappeared in the coculture model. TER was measured after 2 and 24 h following treatment. Depending on the different concentrations, TER decreased in the first 2 h up to 55% of the control at the highest concentration. After 24 h, TER seemed to recover because at concentrations up to 300 microM values were equal to the control. SM induced a widening of intercellular spaces and a loss in cell-matrix adhesion. Mucus production increased with the result that cilia ceased to beat. Changes in the proinflammatory cytokines interleukin (IL)-6 and IL-8 were also observed. Apoptotic markers such as cytochrome c, p53, Fas-associated protein with death domain, and procaspase-3 were significantly induced at concentrations of less than 100 microM. In summary, SM induces morphological and biochemical changes that reflect pathological effects of SM injury in vivo. It is hoped to use this coculture model to understand further the pathogenesis of SM-induced barrier injury and to search for novel approaches in SM therapy.

Viruses and dendritic cells: enemy mine.

Dendritic cells (DCs) act not only as sentinels for detection of, but also as target cells for viruses, and this can be important for viral transport and spread. All subsets of DCs are equipped with a battery of receptors recognizing virus-associated molecular signatures, and recognition of those launches a maturation programme that results in substantial alterations of morphology, motility and the DCs' interactive properties with the extracellular matrix and scanning T cells. In addition to being sensed, viruses are internalized into DCs and, for the major proportion, processed into peptides that are subsequently presented by major histocompatibility complex (MHC) molecules. Transmission of virus to T cells can occur after completion of their replication cycle if the intracellular milieu of the DC permits that. Alternatively, viruses can remain protected from degradation following entrapment by pattern recognition receptors in intracellular compartments, also referred to as virosomes, which translocate towards the DC/T cell interface. Most likely, transfer of virus to T cells occurs in these junctions, referred to as infectious synapses. In addition to promoting DC maturation, many viruses are able to downmodulate DC development and functions in order to evade immune recognition or to induce a generalized immunosuppression.

Measles virus nucleocapsid transport to the plasma membrane requires stable expression and surface accumulation of the viral matrix protein.

In measles virus (MV)-infected cells the matrix (M) protein plays a key role in virus assembly and budding processes at the plasma membrane because it mediates the contact between the viral surface glycoproteins and the nucleocapsids. By exchanging valine 101, a highly conserved residue among all paramyxoviral M proteins, we generated a recombinant MV (rMV) from cloned cDNA encoding for a M protein with an increased intracellular turnover. The mutant rMV was barely released from the infected cells. This assembly defect was not due to a defective M binding to other matrix- or nucleoproteins, but could rather be assigned to a reduced ability to associate with cellular membranes, and more importantly, to a defective accumulation at the plasma membrane which was accompanied by the deficient transport of nucleocapsids to the cell surface. Thus, we show for the first time that M stability and accumulation at intracellular membranes is a prerequisite for M and nucleocapsid co-transport to the plasma membrane and for subsequent virus assembly and budding processes.