Current Challenges to Align Inflammatory Key Events in Animals and Lung Cell Models In Vitro.

With numerous novel and innovative in vitro models emerging every year to reduce or replace animal testing, there is an urgent need to align the design, harmonization, and validation of such systems using in vitro-in vivo extrapolation (IVIVE) approaches. In particular, in inhalation toxicology, there is a lack of predictive and prevalidated in vitro lung models that can be considered a valid alternative for animal testing. The predictive power of such models can be enhanced by applying the Adverse Outcome Pathways (AOP) framework, which casually links key events (KE) relevant to IVIVE. However, one of the difficulties identified is that the endpoint analysis and readouts of specific assays in in vitro and animal models for specific toxicants are currently not harmonized, making the alignment challenging. We summarize the current state of the art in endpoint analysis in the two systems, focusing on inflammatory-induced effects and providing guidance for future research directions to improve the alignment.

EpCAM+CD73+ mark epithelial progenitor cells in postnatal human lung and are associated with pathogenesis of pulmonary disease including lung adenocarcinoma.

Lung injury in mice induces mobilization of discrete subsets of epithelial progenitor cells to promote new airway and alveolar structures. However, whether similar cell types exist in human lung remains unresolved. Using flow cytometry, we identified a distinct cluster of cells expressing the epithelial cell adhesion molecule (EpCAM), a cell surface marker expressed on epithelial progenitor cells, enriched in the ecto-5'-nucleotidase CD73 in unaffected postnatal human lungs resected from pediatric patients with congenital lung lesions. Within the EpCAM+CD73+ population, a small subset coexpresses integrin ß4 and HTII-280. This population remained stable with age. Spatially, EpCAM+CD73+ cells were positioned along the basal membrane of respiratory epithelium and alveolus next to CD73+ cells lacking EpCAM. Expanded EpCAM+CD73+ cells give rise to a pseudostratified epithelium in a two-dimensional air-liquid interface or a clonal three-dimensional organoid assay. Organoids generated under alveolar differentiation conditions were cystic-like and lacked robust alveolar mature cell types. Compared with unaffected postnatal lung, congenital lung lesions were marked by clusters of EpCAM+CD73+ cells in airway and cystic distal lung structures lined by simple epithelium composed of EpCAM+SCGB1A1+ cells and hyperplastic EpCAM+proSPC+ cells. In non-small-cell lung cancer (NSCLC), there was a marked increase in EpCAM+CD73+ tumor cells enriched in inhibitory immune checkpoint molecules CD47 and programmed death-ligand 1 (PD-L1), which was associated with poor survival in lung adenocarcinoma (LUAD). In conclusion, EpCAM+CD73+ cells are rare novel epithelial progenitor cells in the human lung. Importantly, reemergence of CD73 in lung adenocarcinoma enriched in negative immune checkpoint molecules may serve as a novel therapeutic target.

CD90+CD146+ identifies a pulmonary mesenchymal cell subtype with both immune modulatory and perivascular-like function in postnatal human lung.

Our understanding of mesenchymal cell subsets and their function in human lung affected by aging and in certain disease settings remains poorly described. We use a combination of flow cytometry, prospective cell-sorting strategies, confocal imaging, and modeling of microvessel formation using advanced microfluidic chip technology to characterize mesenchymal cell subtypes in human postnatal and adult lung. Tissue was obtained from patients undergoing elective surgery for congenital pulmonary airway malformations (CPAM) and other airway abnormalities including chronic obstructive pulmonary disease (COPD). In microscopically normal postnatal human lung, there was a fivefold higher mesenchymal compared with epithelial (EpCAM+) fraction, which diminished with age. The mesenchymal fraction composed of CD90+ and CD90+CD73+ cells was enriched in CXCL12 and platelet-derived growth factor receptor-α (PDGFRα) and located in close proximity to EpCAM+ cells in the alveolar region. Surprisingly, alveolar organoids generated from EpCAM+ cells supported by CD90+ subset were immature and displayed dysplastic features. In congenital lung lesions, cystic air spaces and dysplastic alveolar regions were marked with an underlying thick interstitium composed of CD90+ and CD90+PDGFRα+ cells. In postnatal lung, a subset of CD90+ cells coexpresses the pericyte marker CD146 and supports self-assembly of perfusable microvessels. CD90+CD146+ cells from COPD patients fail to support microvessel formation due to fibrinolysis. Targeting the plasmin-plasminogen system during microvessel self-assembly prevented fibrin gel degradation, but microvessels were narrower and excessive contraction blocked perfusion. These data provide important new information regarding the immunophenotypic identity of key mesenchymal lineages and their change in a diverse setting of congenital lung lesions and COPD.

Functional differences in airway dendritic cells determine susceptibility to IgE-sensitization.

Respiratory IgE-sensitization to innocuous antigens increases the risk for developing diseases such as allergic asthma. Dendritic cells (DC) residing in the airways orchestrate the immune response following antigen exposure and their ability to sample and present antigens to naïve T cells in airway draining lymph nodes contributes to allergen-specific IgE-sensitization. In order to characterize inhaled antigen capture and presentation by DC subtypes in vivo, we used an adjuvant-free respiratory sensitization model using two genetically distinct rat strains, one of which is naturally resistant and the other naturally susceptible to allergic sensitization. Upon multiple exposures to ovalbumin (OVA), the susceptible strain developed OVA-specific IgE and airway inflammation, whereas the resistant strain did not. Using fluorescently tagged OVA and flow cytometry, we demonstrated significant differences in antigen uptake efficiency and presentation associated with either IgE-sensitization or resistance to allergen exposures in respective strains. We further identified CD4+ conventional DC (cDC) as the subset involved in airway antigen sampling in both strains, however, CD4+ cDC in the susceptible strain were less efficient in OVA sampling and displayed increased MHC-II expression compared with the resistant strain. This was associated with generation of an exaggerated Th2 response and a deficiency of airway regulatory T cells in the susceptible strain. These data suggest that subsets of cDC are able to induce either sensitization or resistance to inhaled antigens as determined by genetic background, which may provide an underlying basis for genetically determined susceptibility to respiratory allergic sensitization and IgE production in susceptible individuals.

Identification and Characterization of a Dendritic Cell Precursor in Parenchymal Lung Tissue.

The pulmonary parenchymal and mucosal microenvironments are constantly exposed to the external environment and thus require continuous surveillance to maintain steady-state immunological homeostasis. This is achieved by a mobile network of pulmonary dendritic cells (DC) and macrophages (mø) that constantly sample and process microenvironmental antigens into signals that can initiate or dampen inflammation, either locally or after onward migration to draining lymph nodes. The constant steady-state turnover of pulmonary DC and mø requires replenishment from bone marrow precursors; however, the nature of the pulmonary precursor cell (PC) remains unclear, although recent studies suggest that subsets of pulmonary DC may derive from circulating monocytic precursors. In the current study, we describe a population of cells in steady-state mouse lung tissue that has the surface phenotypic and ultrastructural characteristics of a common DC progenitor. Irradiation and reconstitution studies confirmed the bone marrow origins of this PC and showed that it had rapid depletion and reconstitution kinetics that were similar to those of DC, with a 50% repopulation by donor-derived cells by Days 7-9 after reconstitution. This was significantly faster than the rates observed for mø, which showed 50% repopulation by donor-derived cells beyond Days 16-21 after reconstitution. Purified PC gained antigen-presenting function and a cell surface phenotype similar to that of pulmonary DC after maturation in vitro, with light and electron microscopy confirming a myeloid DC morphology. To the best of our knowledge, this is the first study to describe a PC for DC in lung tissue; the findings have implications for the restoration of pulmonary immunological homeostasis after bone marrow transplant.

Toll-like receptor 4 activation attenuates profibrotic response in control lung fibroblasts but not in fibroblasts from patients with IPF.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a median survival of 3 yr. IPF deteriorates upon viral or bacterial lung infection although pulmonary infection (pneumonia) in healthy lungs rarely induces fibrosis. Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4), initiating proinflammatory pathways. As TLR4 has already been linked to hepatic fibrosis and scleroderma, we now investigated the role of TLR4 in IPF fibroblasts. Lung tissue sections from patients with IPF were analyzed for TLR4 expression. Isolated normal human lung fibroblasts (NL-FB) and IPF fibroblasts (IPF-FB) were exposed to LPS and transforming growth factor-ß (TGF-ß) before expression analysis of receptors, profibrotic mediators, and cytokines. TLR4 is expressed in fibroblast foci of IPF lungs as well as in primary NL-FB and IPF-FB. As a model for a gram-negative pneumonia in the nonfibrotic lung, NL-FB and IPF-FB were coexposed to LPS and TGF-ß. Whereas NL-FB produced significantly less connective tissue growth factor upon costimulation compared with TGF-ß stimulation alone, IPF-FB showed significantly increased profibrotic markers compared with control fibroblasts after costimulation. Although levels of antifibrotic prostaglandin E2 were elevated after costimulation, they were not responsible for this effect. However, significant downregulation of TGF-ß receptor type 1 in control fibroblasts seems to contribute to the reduced profibrotic response in our in vitro model. Normal and IPF fibroblasts thus differ in their profibrotic response upon LPS-induced TLR4 stimulation.

Characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling.

BACKGROUND: In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However, undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study, we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients. METHODS: Differentiated cell cultures were evaluated with immunolabelling of markers for ciliated, mucus-secreting and basal cells, and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally, epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay. RESULTS: Pediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also, immunofluorescence analysis revealed the presence of ciliated, mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight. CONCLUSION: In summary, primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated, mucus-producing and basal cells, which adequately reflect the in vivo properties of the human respiratory epithelium.

Interaction of biomedical nanoparticles with the pulmonary immune system.

Engineered nanoparticles (NPs) offer site-specific delivery, deposition and cellular uptake due to their unique physicochemical properties and were shown to modulate immune responses. The respiratory tract with its vast surface area is an attractive target organ for innovative immunomodulatory therapeutic applications by pulmonary administration of such NPs, enabling interactions with resident antigen-presenting cells (APCs), such as dendritic cells and macrophages. Depending on the respiratory tract compartment, e.g. conducting airways, lung parenchyma, or lung draining lymph nodes, APCs extensively vary in their number, morphology, phenotype, and function. Unique characteristics and plasticity render APC populations ideal targets for inhaled specific immunomodulators. Modulation of immune responses may operate in different steps of the immune cell-antigen interaction, i.e. antigen uptake, trafficking, processing, and presentation to T cells. Meticulous analysis of the immunomodulatory potential, as well as pharmacologic and biocompatibility testing of inhalable NPs is required to develop novel strategies for the treatment of respiratory disorders such as allergic asthma. The safe-by-design and characterization of such NPs requires well coordinated interdisciplinary research uniting engineers, chemists biologists and respiratory physicians. In this review we will focus on in vivo data available to facilitate the design of nanocarrier-based strategies using NPs to modulate pulmonary immune responses.

Virosome-bound antigen enhances DC-dependent specific CD4+ T cell stimulation, inducing a Th1 and Treg profile in vitro.

There is considerable interest to develop antigen-carriers for immune-modulatory clinical applications, but insufficient information is available on their effects on antigen-presenting cells. We employed virosomes coupled to ovalbumin (OVA) to study their interaction with murine bone marrow-derived dendritic cells (BMDCs) and modulation of downstream T cell responses. BMDCs were treated in vitro with virosomes or liposomes prior to determining BMDC phenotype, viability, and intracellular trafficking. Antigen-specific CD4+ T cell activation was measured by co-culture of BMDCs with DO11.10 CD4+ T cells. Compared to liposomes, virosomes were rapidly taken up. Neither nanocarrier type affected BMDC viability, nor did a moderate degree of activation differ for markers such as CD40, CD80, CD86. Virosome uptake occurred via clathrin-mediated endocytosis and phagocytosis, with co-localization in late endosomes. Only BMDCs treated with OVA-coupled virosomes induced enhanced OVA-specific CD4+ T cell proliferation. Antigen-coupled virosomes are endowed with an intrinsic ability to modulate DC-dependent adaptive immune responses.

TNFα-induced IKKß complex activation influences epithelial, but not stromal cell survival in endometriosis.

STUDY QUESTION: Can the activity of the IκB kinase (IKKß) complex in endometriotic cells contribute to endometriotic lesion survival? SUMMARY ANSWER: There is a constitutive activity of the IKKß catalytic complex in peritoneal and deeply infiltrating lesions that can influence epithelial, but not stromal cell viability. WHAT IS KNOWN ALREADY: Endometriotic lesions exist in an inflammatory microenvironment with higher local concentrations of cytokines, such as tumour necrosis factor α (TNFα). TNFα stimulates the activation of the IKKß complex, an important nodal point in multiple signalling pathways that influence gene transcription, proliferation and apoptosis. However, few data on the regulation of IKKß in endometriotic tissue are currently available. STUDY DESIGN, SIZE, DURATION: A retrospective analysis of endometriotic tissue from peritoneal, ovarian and deeply infiltrating lesions from 37 women. PARTICIPANTS/MATERIALS, SETTING, METHODS: Basal and activated (phosphorylated) IKKß concentrations were analysed by western blotting and immunohistochemistry. The relationship between the expression and activation of these proteins and peritoneal fluid (TNFα) concentrations, measured via ELISA, was examined. A subsequent in vitro analysis of TNFα treatment on the activation of IKKß and the effect on epithelial and stromal cell viability by its inhibition with PS1145 was also performed. MAIN RESULTS AND ROLE OF CHANCE: Levels of the phosphorylated IKKß complex in endometriotic lesions had a significant positive correlation with peritoneal fluid TNFα concentrations. Phosphorylated IKKß complex was more prevalent in peritoneal and deeply infiltrating endometriosis lesions compared with ovarian lesions. IKKß was present in both epithelial and stromal cells in all lesions but active IKKß was limited to epithelial cells. TNFα stimulated an increased expression of phosphorylated IKKß and the inhibition of this kinase with PS1145 significantly influenced ectopic epithelial cells viability but not eutopic epithelial cells, or endometrial stromal cells. LIMITATIONS, REASONS FOR CAUTION: In vitro analysis on epithelial cells was performed with immortalized cell lines and not primary cell cultures and only low sample numbers were available for the study. WIDER IMPLICATIONS OF THE FINDINGS: The regulation of aberrant signalling pathways represents a promising yet relatively unexplored area of endometriosis progression. The IKKß complex is activated by inflammation and is critical nodal point of numerous downstream kinase-signalling pathways, including NFκB (nuclear factor κB), mTOR (mammalian target of rapamycin) and BAD (Bcl2-antagonist of cell death). This study shows a significant relationship between peritoneal fluid TNFα and IKKß activation in epithelial cells that will have significant consequences for the continued survival of these cells at ectopic locations through the regulation of downstream pathways. LARGE SCALE DATA: None. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Swiss National Science Foundation (Grant Number 320030_140774). The authors have no conflict of interest to declare.

Pulmonary delivery of cationic gold nanoparticles boost antigen-specific CD4+ T Cell Proliferation.

To address how surface charge affects the fate of potential nanocarriers in the lung, gold nanoparticles (AuNPs) coated with polyvinyl alcohol containing either positively (NH2) or negatively (COOH) charged functional groups were intra-nasally instilled in mice, and their uptake by antigen presenting cell populations (APC) in broncho-alveolar lavage (BAL) fluid, trachea, and lung parenchyma, as well as trafficking to the lung draining lymph nodes (LDLNs) was assessed by flow cytometry. Furthermore, CD4+ T cell proliferation in LDLNs was investigated following instillation. All APC subpopulations preferentially captured positively-charged AuNPs compared to their negatively-charged counterparts. Uptake of AuNPs up-regulated expression of co-stimulatory molecules on all APC populations. Furthermore, positively-charged AuNPs induced enhanced OVA-specific CD4+ T cell stimulation in LDLNs compared to negatively-charged AuNPs, or polymer alone. Our findings demonstrate surface charge as a key parameter determining particle uptake by APC, and down-stream immune responses depend on the presence of particle core-bound polymer.

Uptake efficiency of surface modified gold nanoparticles does not correlate with functional changes and cytokine secretion in human dendritic cells in vitro.

Engineering nanoparticles (NPs) for immune modulation require a thorough understanding of their interaction(s) with cells. Gold NPs (AuNPs) were coated with polyethylene glycol (PEG), polyvinyl alcohol (PVA) or a mixture of both with either positive or negative surface charge to investigate uptake and cell response in monocyte-derived dendritic cells (MDDCs). Inductively coupled plasma optical emission spectrometry and transmission electron microscopy were used to confirm the presence of Au inside MDDCs. Cell viability, (pro-)inflammatory responses, MDDC phenotype, activation markers, antigen uptake and processing were analyzed. Cell death was only observed for PVA-NH2 AuNPs at the highest concentration. MDDCs internalize AuNPs, however, surface modification influenced uptake. Though limited uptake was observed for PEG-COOH AuNPs, a significant tumor necrosis factor-alpha release was induced. In contrast, (PEG+PVA)-NH2 and PVA-NH2 AuNPs were internalized to a higher extent and caused interleukin-1beta secretion. None of the AuNPs caused changes in MDDC phenotype, activation or immunological properties. From the clinical editor: This team of authors investigated the influence of gold nano-particles with different surface modifications on immunological properties in monocyte-derived dendritic cells. AuNPs triggered responses in these cells that has to be further investigated in terms of development of novel vaccine carriers.

Fluorescence-encoded gold nanoparticles: library design and modulation of cellular uptake into dendritic cells.

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo- and hetero-functional fluorescence-encoded gold nanoparticles (Au-NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)-based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV-Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence-activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero-functionalized Au-NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties.

Labeling of cancer cells with magnetic nanoparticles for magnetic resonance imaging.

PURPOSE: The process of invasion and metastasis formation of tumor cells can be studied by following the migration of labeled cells over prolonged time periods. This report investigates the applicability of iron oxide nanoparticles as a magnetic resonance imaging (MRI) contrast agent for cell labeling. METHODS: γFe2 O3 nanoparticles prepared with direct flame spray pyrolysis are biofunctionalized with poly-l-lysine (PLL). The nanoparticles within the cells were observed with transmission electron microscopy, bright-field microscopy, and magnetorelaxometry. MRI of labeled cells suspended in agarose was used to estimate the detection limit. RESULTS: PLL-coated particles are readily taken up, stored in intracellular clusters, and gradually degraded by the cells. During cell division, the nanoparticle clusters are divided and split between daughter cells. The MRI detection limit was found to be 25 cells/mm(3) for R2*, and 70 cells/mm(3) for R2. The iron specificity, however, was higher for R2 images. Due to the degradation of intracellular γFe2 O3 to paramagnetic iron ions within 13 days, the R1, R2, and R2* contrast gradually decreased over this time period to approximately 50% of its initial value. CONCLUSIONS: These results suggest that PLL-coated γFe2 O3 nanoparticles can be used as an MRI contrast agent for long-term studies of cell migration. Magn Reson Med 71:1896-1905, 2014. © 2013 Wiley Periodicals, Inc.

Size-dependent uptake of particles by pulmonary antigen-presenting cell populations and trafficking to regional lymph nodes.

The respiratory tract is an attractive target organ for novel diagnostic and therapeutic applications with nano-sized carriers, but their immune effects and interactions with key resident antigen-presenting cells (APCs) such as dendritic cells (DCs) and alveolar macrophages (AMs) in different anatomical compartments remain poorly understood. Polystyrene particles ranging from 20 nm to 1,000 nm were instilled intranasally in BALB/c mice, and their interactions with APC populations in airways, lung parenchyma, and lung-draining lymph nodes (LDLNs) were examined after 2 and 24 hours by flow cytometry and confocal microscopy. In the main conducting airways and lung parenchyma, DC subpopulations preferentially captured 20-nm particles, compared with 1,000-nm particles that were transported to the LDLNs by migratory CD11blow DCs and that were observed in close proximity to CD3⁺ T cells. Generally, the uptake of particles increased the expression of CD40 and CD86 in all DC populations, independent of particle size, whereas 20-nm particles induced enhanced antigen presentation to CD4⁺ T cells in LDLNs in vivo. Despite measurable uptake by DCs, the majority of particles were taken up by AMs, irrespective of size. Confocal microscopy and FACS analysis showed few particles in the main conducting airways, but a homogeneous distribution of all particle sizes was evident in the lung parenchyma, mostly confined to AMs. Particulate size as a key parameter determining uptake and trafficking therefore determines the fate of inhaled particulates, and this may have important consequences in the development of novel carriers for pulmonary diagnostic or therapeutic applications.

Restricted aeroallergen access to airway mucosal dendritic cells in vivo limits allergen-specific CD4+ T cell proliferation during the induction of inhalation tolerance.

Chronic innocuous aeroallergen exposure attenuates CD4(+) T cell-mediated airways hyperresponsiveness in mice; however, the mechanism(s) remain unclear. We examined the role of airway mucosal dendritic cell (AMDC) subsets in this process using a multi-OVA aerosol-induced tolerance model in sensitized BALB/c mice. Aeroallergen capture by both CD11b(lo) and CD11b(hi) AMDC and the delivery of OVA to airway draining lymph nodes by CD8α(-) migratory dendritic cells (DC) were decreased in vivo (but not in vitro) when compared with sensitized but nontolerant mice. This was functionally significant, because in vivo proliferation of OVA-specific CD4(+) T cells was suppressed in airway draining lymph nodes of tolerized mice and could be restored by intranasal transfer of OVA-pulsed and activated exogenous DC, indicating a deficiency in Ag presentation by endogenous DC arriving from the airway mucosa. Bone marrow-derived DC Ag-presenting function was suppressed in multi-OVA tolerized mice, and allergen availability to airway APC populations was limited after multi-OVA exposure, as indicated by reduced OVA and dextran uptake by airway interstitial macrophages, with diffusion rather than localization of OVA across the airway mucosal surface. These data indicate that inhalation tolerance limits aeroallergen capture by AMDC subsets through a mechanism of bone marrow suppression of DC precursor function coupled with reduced Ag availability in vivo at the airway mucosa, resulting in limited Ag delivery to lymph nodes and hypoproliferation of allergen-specific CD4(+) T cells.

Surfactant Protein D modulates allergen particle uptake and inflammatory response in a human epithelial airway model.

BACKGROUND: Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated. METHODS: SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4+ T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array. RESULTS: SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and led to a decreased secretion of pro-inflammatory cytokines. CONCLUSION: These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen.

Pulmonary surfactant coating of multi-walled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro.

BACKGROUND: Increasing concern has been expressed regarding the potential adverse health effects that may be associated with human exposure to inhaled multi-walled carbon nanotubes (MWCNTs). Thus it is imperative that an understanding as to the underlying mechanisms and the identification of the key factors involved in adverse effects are gained. In the alveoli, MWCNTs first interact with the pulmonary surfactant. At this interface, proteins and lipids of the pulmonary surfactant bind to MWCNTs, affecting their surface characteristics. Aim of the present study was to investigate if the pre-coating of MWCNTs with pulmonary surfactant has an influence on potential adverse effects, upon both (i) human monocyte derived macrophages (MDM) monocultures, and (ii) a sophisticated in vitro model of the human epithelial airway barrier. Both in vitro systems were exposed to MWCNTs either pre-coated with a porcine pulmonary surfactant (Curosurf) or not. The effect of MWCNTs surface charge was also investigated in terms of amino (-NH2) and carboxyl (-COOH) surface modifications. RESULTS: Pre-coating of MWCNTs with Curosurf affects their oxidative potential by increasing the reactive oxygen species levels and decreasing intracellular glutathione depletion in MDM as well as decreases the release of Tumour necrosis factor alpha (TNF-α). In addition, an induction of apoptosis was observed after exposure to Curosurf pre-coated MWCNTs. In triple cell-co cultures the release of Interleukin-8 (IL-8) was increased after exposure to Curosurf pre-coated MWCNTs. Effects of the MWCNTs functionalizations were minor in both MDM and triple cell co-cultures. CONCLUSIONS: The present study clearly indicates that the pre-coating of MWCNTs with pulmonary surfactant more than the functionalization of the tubes is a key factor in determining their ability to cause oxidative stress, cytokine/chemokine release and apoptosis. Thus the coating of nano-objects with pulmonary surfactant should be considered for future lung in vitro risk assessment studies.

Computer-Aided Histopathological Characterisation of Endometriosis Lesions.

Endometriosis is a common gynaecological condition characterised by the growth of endometrial tissue outside the uterus and is associated with pain and infertility. Currently, the gold standard for endometriosis diagnosis is laparoscopic excision and histological identification of endometrial epithelial and stromal cells. There is, however, currently no known association between the histological appearance, size, morphology, or subtype of endometriosis and disease prognosis. In this study, we used histopathological software to identify and quantify the number of endometrial epithelial and stromal cells within excised endometriotic lesions and assess the relationship between the cell contents and lesion subtypes. Prior to surgery for suspected endometriosis, patients provided menstrual and abdominal pain and dyspareunia scores. Endometriotic lesions removed during laparoscopic surgery were collected and prepared for immunohistochemistry from 26 patients. Endometrial epithelial and stromal cells were identified with Cytokeratin and CD10 antibodies, respectively. Whole slide sections were digitised and the QuPath software was trained to automatically detect and count epithelial and stromal cells across the whole section. Using this classifier, we identified a significantly larger number of strongly labelled CD10 stromal cells (p = 0.0477) in deeply infiltrating lesions (99,970 ± 2962) compared to superficial lesions (2456 ± 859). We found the ratio of epithelial to stromal cells was inverted in deeply infiltrating endometriosis lesions compared to superficial peritoneal and endometrioma lesions and we subsequently identified a correlation between total endometrial cells and abdominal pain (p = 0.0005) when counted via the automated software. Incorporating histological software into current standard diagnostic pipelines may improve endometriosis diagnosis and provide prognostic information in regards to severity and symptoms and eventually provide the potential to personalise adjuvant treatment decisions.

Efficacy of Combined in-vivo Electroporation-Mediated Gene Transfer of VEGF, HGF, and IL-10 on Skin Flap Survival, Monitored by Label-Free Optical Imaging: A Feasibility Study.

Preventing surgical flaps necrosis remains challenging. Laser Doppler imaging and ultrasound can monitor blood flow in flap regions, but they do not directly measure the cellular response to ischemia. The study aimed to investigate the efficacy of synergistic in-vivo electroporation-mediated gene transfer of interleukin 10 (IL-10) with either hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF) on the survival of a modified McFarlane flap, and to evaluate the effect of the treatment on cell metabolism, using label-free fluorescence lifetime imaging. Fifteen male Wistar rats (290-320 g) were randomly divided in three groups: group-A (control group) underwent surgery and received no gene transfer. Group-B received electroporation mediated hIL-10 gene delivery 24 h before and VEGF gene delivery 24 h after surgery. Group-C received electroporation mediated hIL-10 gene delivery 24 h before and hHGF gene delivery 24 h after surgery. The animals were assessed clinically and histologically. In addition, label-free fluorescence lifetime imaging was performed on the flap. Synergistic electroporation mediated gene delivery significantly decreased flap necrosis (P = 0.0079) and increased mean vessel density (P = 0.0079) in treatment groups B and C compared to control group-A. NADH fluorescence lifetime analysis indicated an increase in oxidative phosphorylation in the epidermis of the group-B (P = 0.039) relative to controls. These findings suggested synergistic in-vivo electroporation-mediated gene transfer as a promising therapeutic approach to enhance viability and vascularity of skin flap. Furthermore, the study showed that combinational gene therapy promoted an increase in tissue perfusion and a relative increase in oxidative metabolism within the epithelium.