In vitro toxicity screening of polyglycerol esters of fatty acids as excipients for pulmonary formulations.

One of the main problems for the development of pulmonary formulations is the low availability of approved excipients. Polyglycerol esters of fatty acids (PGFA) are promising molecules for acting as excipient for formulation development and drug delivery to the lung. However, their biocompatibility in the deep lung has not been studied so far. Main exposed cells include alveolar epithelial cells and alveolar macrophages. Due to the poor water-solubility of PGFAs, the exposure of alveolar macrophages is expected to be much higher than that of epithelial cells. In this study, two PGFAs and their mixture were tested regarding cytotoxicity to epithelial cells and cytotoxicity and functional impairment of macrophages. Cytotoxicity was assessed by dehydrogenase activity and lactate dehydrogenase release. Lysosome function, phospholipid accumulation, phagocytosis, nitric oxide production, and cytokine release were used to evaluate macrophage function. Cytotoxicity was increased with the increased polarity of PGFA molecules. At concentrations above 1 mg/ml accumulation in lysosomes, impairment of phagocytosis, secretion of nitric oxide, and increased release of cytokines were noted. The investigated PGFAs in concentrations up to 1 mg/ml can be considered as uncritical and are promising for advanced pulmonary delivery of high powder doses and drug targeting to alveolar macrophages.

Different Sensitivity of Macrophages to Phospholipidosis Induction by Amphiphilic Cationic Drugs.

Phospholipidosis (PLD), the intracellular accumulation of phospholipids, is an adaptive response to toxic stimuli and serves as an important parameter in the biological assessment of compounds. Cationic amphiphilic drugs are the main inducers of PLD and may impair the function of alveolar macrophages. In vivo and in vitro models are used for PLD screening but the choice of the cellular model may be important because PLD develops in a cell- and species-specific manner. In this study, a panel of different staining (LysoSensor, Acridine Orange, Nile Red, HCS LipidTOX, LysoID) was evaluated in murine (DMBM-2, J774, RAW264.7) and human (THP-1, monocyte-derived macrophages from peripheral blood) cells to identify the most sensitive and easy to analyze staining method and to detect species-specific differences in the reaction pattern. Amiodarone and chloroquine served as inducers of PLD. High content screening was used to compare number, area, and intensity of the staining. Due to the fast staining protocol and the sensitivity of the detection, LysoID proved to be the most suitable dye of the testing. The lower induction of PLD by chloroquine reported in vivo was also seen in this study. THP-1 macrophages, followed by DMBM-2 cells, produced the most similar reaction pattern to human monocyte-derived macrophages.

The Development of Indicator Cotton Swabs for the Detection of pH in Wounds.

Indicator cotton swabs have been developed in order to enable faster, less expensive, and simpler information gathering of a wound status. Swabs are normally used for cleaning the wound, but here, they were covalently functionalized with indicator chemistry. Thus, they in principle enable simultaneous wound cleaning and wound pH detection. Using an indicator dye with a color change from yellow to red, combined with an inert dye of blue color, a traffic light color change from green to red is induced when pH increases. The indicator cotton swabs (ICSs) show a color change from green (appropriate wound pH) to red (elevated wound pH). This color change can be interpreted by the naked eye as well as by an optical color measurement device in order to obtain quantitative data based on the CIE L*a*b* color space. Two types of swabs have been developed-indicator cotton swabs ICS1 with a sensitive range from pH 5 to 7 and swabs ICS2 with a sensitive range from 6.5 to 8.5. The swabs are gamma-sterilized and the effect of sterilization on performance was found to be negligible. Furthermore, cytotoxicity testing shows cell viability and endotoxin levels to be within the allowable range.

Peptides at the Interface: Self-Assembly of Amphiphilic Designer Peptides and Their Membrane Interaction Propensity.

Self-assembling amphiphilic designer peptides have been successfully applied as nanomaterials in biomedical applications. Understanding molecular interactions at the peptide-membrane interface is crucial, since interactions at this site often determine (in)compatibility. The present study aims to elucidate how model membrane systems of different complexity (in particular single-component phospholipid bilayers and lipoproteins) respond to the presence of amphiphilic designer peptides. We focused on two short anionic peptides, V4WD2 and A6YD, which are structurally similar but showed a different self-assembly behavior. A6YD self-assembled into high aspect ratio nanofibers at low peptide concentrations, as evidenced by synchrotron small-angle X-ray scattering and electron microscopy. These supramolecular assemblies coexisted with membranes without remarkable interference. In contrast, V4WD2 formed only loosely associated assemblies over a large concentration regime, and the peptide promoted concentration-dependent disorder on the membrane arrangement. Perturbation effects were observed on both membrane systems although most likely induced by different modes of action. These results suggest that membrane activity critically depends on the peptide's inherent ability to form highly cohesive supramolecular structures.

Intracellular calcium levels as screening tool for nanoparticle toxicity.

The use of engineered nano-sized materials led to revolutionary developments in many industrial applications and in the medical field. These materials, however, also may cause cytotoxicity. In addition to size, surface properties and shape were identified as relevant parameters for cell damage. Cell damage may occur as disruption of membrane integrity, induction of apoptosis and by organelle damage. Generation of oxidative stress may serve as an indicator for cytotoxicity. Effects occurring upon short contact of particles with cells, for instance in the systemic blood circulation, could be identified according to increases of intracellular [Ca(2+) ] levels, which are caused by variety of toxic stimuli. Negatively charged, neutral and positively charged polystyrene particles of different sizes were used to study the role of size and surface properties on viability, membrane disruption, apoptosis, lysosome function, intracellular [Ca(2+) ] levels and generation of oxidative stress. Silica particles served to test this hypothesis. Twenty nm polystyrene particles as well as 12 nm and 40 nm silica particles caused membrane damage and apoptosis with no preference of the surface charge. Only 20 nm plain and amine functionalized polystyrene particles cause oxidative stress and only the plain particles lysosomal damage. A potential role of surface charge was identified for 200 nm polystyrene particles, where only the amidine particles caused lysosomal damage. Increases in intracellular [Ca(2+) ] levels and cytotoxicity after 24 h was often linked but determination of intracellular [Ca(2+) ] levels could serve to characterize further the type of membrane damage.

Use of whole genome expression analysis in the toxicity screening of nanoparticles.

The use of nanoparticles (NPs) offers exciting new options in technical and medical applications provided they do not cause adverse cellular effects. Cellular effects of NPs depend on particle parameters and exposure conditions. In this study, whole genome expression arrays were employed to identify the influence of particle size, cytotoxicity, protein coating, and surface functionalization of polystyrene particles as model particles and for short carbon nanotubes (CNTs) as particles with potential interest in medical treatment. Another aim of the study was to find out whether screening by microarray would identify other or additional targets than commonly used cell-based assays for NP action. Whole genome expression analysis and assays for cell viability, interleukin secretion, oxidative stress, and apoptosis were employed. Similar to conventional assays, microarray data identified inflammation, oxidative stress, and apoptosis as affected by NP treatment. Application of lower particle doses and presence of protein decreased the total number of regulated genes but did not markedly influence the top regulated genes. Cellular effects of CNTs were small; only carboxyl-functionalized single-walled CNTs caused appreciable regulation of genes. It can be concluded that regulated functions correlated well with results in cell-based assays. Presence of protein mitigated cytotoxicity but did not cause a different pattern of regulated processes.

Development of an advanced intestinal in vitro triple culture permeability model to study transport of nanoparticles.

Intestinal epithelial cell culture models, such as Caco-2 cells, are commonly used to assess absorption of drug molecules and transcytosis of nanoparticles across the intestinal mucosa. However, it is known that mucus strongly impacts nanoparticle mobility and that specialized M cells are involved in particulate uptake. Thus, to get a clear understanding of how nanoparticles interact with the intestinal mucosa, in vitro models are necessary that integrate the main cell types. This work aimed at developing an alternative in vitro permeability model based on a triple culture: Caco-2 cells, mucus-secreting goblet cells and M cells. Therefore, Caco-2 cells and mucus-secreting goblet cells were cocultured on Transwells and Raji B cells were added to stimulate differentiation of M cells. The in vitro triple culture model was characterized regarding confluence, integrity, differentiation/expression of M cells and cell surface architecture. Permeability of model drugs and of 50 and 200 nm polystyrene nanoparticles was studied. Data from the in vitro model were compared with ex vivo permeability results (Ussing chambers and porcine intestine) and correlated well. Nanoparticle uptake was size-dependent and strongly impacted by the mucus layer. Moreover, nanoparticle permeability studies clearly demonstrated that particles were capable of penetrating the intestinal barrier mainly via specialized M cells. It can be concluded that goblet cells and M cells strongly impact nanoparticle uptake in the intestine and should thus be integrated in an in vitro permeability model. The presented model will be an efficient tool to study intestinal transcellular uptake of particulate systems.

Integration of mucus and its impact within in vitro setups for inhaled drugs and formulations: Identifying the limits of simple vs. complex methodologies when studying drug dissolution and permeability.

Traditionally, developing inhaled drug formulations relied on trial and error, yet recent technological advancements have deepened the understanding of 'inhalation biopharmaceutics' i.e. the processes that occur to influence the rate and extent of drug exposure in the lungs. This knowledge has led to the development of new in vitro models that predict the in vivo behavior of drugs, facilitating the enhancement of existing formulation and the development of novel ones. Our prior research examined how simulated lung fluid (SLF) affects the solubility of inhaled drugs. Building on this, we aimed to explore drug dissolution and permeability in lung mucosa models containing mucus. Thus, the permeation of four active pharmaceutical ingredients (APIs), salbutamol sulphate (SS), tiotropium bromide (TioBr), formoterol fumarate (FF) and budesonide (BUD), was assayed in porcine mucus covered Calu-3 cell layers, cultivated at an air liquid interface (ALI) or submerged in a liquid covered (LC) culture system. Further analysis on BUD and FF involved their transport in a mucus-covered PAMPA system. Finally, their dissolution post-aerosolization from Symbicort® was compared using 'simple' Transwell and complex DissolvIt® apparatuses, alone or in presence of porcine mucus or polymer-lipid mucus simulant. The presence of porcine mucus impacted both permeability and dissolution of inhaled drugs. For instance, permeability of SS was reduced by a factor of ten in the Calu-3 ALI model while the permeability of BUD was reduced by factor of two in LC and ALI setups. The comparison of dissolution methodologies indicated that drug dissolution performance was highly dependent on the setup, observing decreased release efficiency and higher variability in Transwell system compared to DissolvIt®. Overall, results demonstrate that relatively simple methodologies can be used to discriminate between formulations in early phase drug product development. However, for more advanced stages complex methods are required. Crucially, it was clear that the impact of mucus and selection of its composition in in vitro testing of dissolution and permeability should not be neglected when developing drugs and formulations intended for inhalation.

In-vitro permeability of neutral polystyrene particles via buccal mucosa.

Drugs can be absorbed well in the oral cavity, which eliminates problems related to intestinal and hepatic first-pass metabolism. Although it is well-established that nanoparticles are small enough to penetrate/permeate epithelial barriers, there is no clear understanding of how they interact with the buccal mucosa. This work provides useful information regarding particle properties with regard to mucosal uptake and can be used for the rational design of nanocarriers. In the buccal mucosa, the uptake of neutral polystyrene nanoparticles (PP) is size-dependent. Compared to 25 and 50 nm particles, 200 nm PP particles penetrate into deeper regions of the mucosa. This is attributed to the structure of the buccal mucosa, i.e., mucus layer and microplicae. The particles permeate the mucus layer and deposit in ridge-like folds of superficial buccal cells. Thus, the effects of thermodynamic driving forces and/or interparticle electrostatic repulsion are enhanced and cellular uptake might be reduced for smaller particle sizes.

Assessment of Carbon Nanotubes on Barrier Function, Ciliary Beating Frequency and Cytokine Release in In Vitro Models of the Respiratory Tract.

The exposure to inhaled carbon nanotubes (CNT) may have adverse effects on workers upon chronic exposure. In order to assess the toxicity of inhaled nanoparticles in a physiologically relevant manner, an air-liquid interface culture of mono and cocultures of respiratory cells and assessment in reconstructed bronchial and alveolar tissues was used. The effect of CNT4003 reference particles applied in simulated lung fluid was studied in bronchial (Calu-3 cells, EpiAirway™ and MucilAir™ tissues) and alveolar (A549 +/-THP-1 and EpiAlveolar™ +/-THP-1) models. Cytotoxicity, transepithelial electrical resistance, interleukin 6 and 8 secretion, mucociliary clearance and ciliary beating frequency were used as readout parameters. With the exception of increased secretion of interleukin 6 in the EpiAlveolar™ tissues, no adverse effects of CNT4003 particles, applied at doses corresponding to the maximum estimated lifetime exposure of workers, in the bronchial and alveolar models were noted, suggesting no marked differences between the models. Since the doses for whole-life exposure were applied over a shorter time, it is not clear if the interleukin 6 increase in the EpiAlveolar™ tissues has physiological relevance.

Lipid-based particle engineering via spray-drying for targeted delivery of antibiotics to the lung.

Pulmonary delivery of antibiotics for the treatment of tuberculosis provides several benefits compared to conventional oral and parenteral administration. API-loaded particles delivered directly to alveolar macrophages, where Mycobacterium tuberculosis resides, can reduce the required dose and decrease the severe side effects of conventional treatment. In this work, lipid-microparticles loaded with rifampicin were engineered via spray-drying to be administered as a carrier-free dry powder for inhalation. Although, it is well-known that spray-drying of lipid-based excipients is strongly limited, a completely lipid-based formulation using diglycerol full ester of behenic acid was produced. The solid state of the lipid, providing high melting temperature, absence of polymorphism and monophasic crystallization, led to high yield of spray-dried particles (83%). Inhalable particles of mass median aerodynamic diameter of 2.36 µm, median geometric size of 2.05 µm, and negative surface (-50.03 mV) were engineered. Such attributes were defined for deep lung deposition and targeted delivery of antibiotics to alveolar macrophages. Superior aerodynamic performance as carrier-free DPI was associated to a high fine particle fraction of 79.5 %. No in vitro cytotoxic effects were found after exposing epithelial cell lines and alveolar macrophages. In vitro uptake of particles into alveolar macrophages indicated the efficiency of their targeted delivery. The use of highly processable and safe lipid-based excipients for particle engineering via spray-drying can extend the availability of materials for functionalized applications for pulmonary delivery.

Action of polystyrene nanoparticles of different sizes on lysosomal function and integrity.

BACKGROUND: Data from environmental exposure to nanoparticles (NPs) suggest that chronic exposure may increase the incidence of lung, cardiovascular and neurodegenerative diseases. Impairment of cell function by intracellular accumulation of NPs is also suspected. Many types of NPs have been detected in the endosomal-lysosomal system and, upon repeated exposure, alterations of the endosomal-lysosomal system may occur. To identify such effects we compared the effect of carboxyl polystyrene particles (CPS) of different sizes (20-500 nm) on lysosomes of the endothelial cell line EAhy926 after short (24h) and long (72h-96h) exposure times. Lysosomal localization of CPS, as well as lysosomal pH, lysosomal membrane integrity, morphology of the endosomal-lysosomal system and activities of the lysosomal enzymes,cathepsin B and sulfatases, upon exposure to CPS were recorded. RESULTS: CPS in sizes ≤100 nm showed high co-localization with lysosomes already after 4h, larger CPS after 24h. None of the particles at non-cytotoxic concentrations caused marked changes in lysosomal pH or destroyed lysosomal membrane integrity. At 24h of exposure, 20 nm CPS induced significant dilatation of the endosomal-lysosomal system and reduced activity of lysosomal sulfatases. After 72h, these alterations were less pronounced. CONCLUSIONS: Despite accumulation in lysosomes CPS induced only small changes in lysosomes. Upon longer contact, these changes are even less pronounced. The presented panel of assays may serve to identify effects on lysosomes also for other NPs.

Screening for Effects of Inhaled Nanoparticles in Cell Culture Models for Prolonged Exposure.

Respiratory exposure of humans to environmental and therapeutic nanoparticles repeatedly occurs at relatively low concentrations. To identify adverse effects of particle accumulation under realistic conditions, monocultures of Calu-3 and A549 cells and co-cultures of A549 and THP-1 macrophages in the air-liquid interphase culture were exposed repeatedly to 2 µg/cm2 20 nm and 200 nm polystyrene particles with different functionalization. Particle accumulation, transepithelial electrical resistance, dextran (3-70 kDa) uptake and proinflammatory cytokine secretion were determined over 28 days. Calu-3 cells showed constant particle uptake without any change in barrier function and cytokine release. A549 cells preferentially ingested amino- and not-functionalized particles combined with decreased endocytosis. Cytokine release was transiently increased upon exposure to all particles. Carboxyl-functionalized demonstrated higher uptake and higher cytokine release than the other particles in the A549/THP-1 co-cultures. The evaluated respiratory cells and co-cultures ingested different amounts and types of particles and caused small (partly transient) effects. The data suggest that the healthy cells can adapt to low doses of non-cytotoxic particles.

Drug combination screening as a translational approach toward an improved drug therapy for chordoma.

PURPOSE: Drug screening programmes have revealed epidermal growth factor receptor inhibitors (EGFRis) as promising therapeutics for chordoma, an orphan malignant bone tumour, in the absence of a known genetic driver. Concurrently, the irreversible EGFRi afatinib (Giotrif®) is being evaluated in a multicentric Phase II trial. As tyrosine kinase inhibitor (TKI) monotherapies are invariably followed by resistance, we aimed to evaluate potential therapeutic combinations with EGFRis. METHODS: We screened 133 clinically approved anticancer drugs as single agents and in combination with two EGFRis (afatinib and erlotinib) in the clival chordoma cell line UM-Chor1. Synergistic combinations were analysed in a 7 × 7 matrix format. The most promising combination was further explored in clival (UM-Chor1, MUG-CC1) and sacral (MUG-Chor1, U-CH1) chordoma cell lines. Secretomes were analysed for receptor tyrosine kinase ligands (EGF, TGF-α, FGF-2 and VEGF-A) upon drug treatment. RESULTS: Drugs that were active as single agents (n = 45) included TKIs, HDAC and proteasome inhibitors, and cytostatic drugs. Six combinations were analysed in a matrix format: n = 4 resulted in a significantly increased cell killing (crizotinib, dabrafenib, panobinostat and doxorubicin), and n = 2 exhibited no or negligible effects (regorafenib, venetoclax). Clival chordoma cell lines were more responsive to combined EGFR-MET inhibition. EGFR-MET cross-talk (e.g. via TGF-α secretion) likely accounts for the synergistic effects of EGFR-MET inhibition. CONCLUSION: Our screen revealed promising combinations with EGFRis, such as the ALK/MET-inhibitor crizotinib, the HDAC-inhibitor panobinostat or the topoisomerase-II-inhibitor doxorubicin, which are part of standard chemotherapy regimens for various bone and soft-tissue sarcomas.

Cytokine-Mediated Inflammation in the Oral Cavity and Its Effect on Lipid Nanocarriers.

Topical drug administration to the oral mucosa proves to be a promising treatment alternative for inflammatory diseases. However, disease-related changes in the cell barrier must be considered when developing such delivery systems. This study aimed at investigating the changes in the lining mucosa caused by inflammation and evaluating the consequences on drug delivery systems such as nanostructured lipid carriers (NLC). For this, TR146 cells were treated with inflammatory cytokines and bacterial components. Cell viability and integrity, reactive oxygen species (ROS), and interleukin (IL)-8 release were used as endpoints to assess inflammation. Translocation of phosphatidylserine, cytoskeletal arrangement, opening of desmosomes, and cell proliferation were examined. Transport studies with NLC were performed considering active and passive pathways. The results showed that IL-1ß and tumor necrosis factor α induced inflammation by increasing IL-8 and ROS production (22-fold and 2-fold). Morphologically, loss of cell-cell connections and formation of stress fibers and hyperplasia were observed. The charge of the cell membrane shifted from neutral to negative, which increased the absorption of NLC due to the repulsive interactions between the hydrophobic negative particles and the cell membrane on the one hand, and interactions with lipophilic membrane proteins such as caveolin on the other.

Novel approach for overcoming the stability challenges of lipid-based excipients. Part 3: Application of polyglycerol esters of fatty acids for the next generation of solid lipid nanoparticles.

Solid lipid nanoparticles (SLN) are an advantageous carrier system for the delivery of lipophilic active pharmaceutical ingredients (APIs). The use of SLN has been limited due to stability issues attributed to the unstable solid state of the lipid matrix. A novel approach for overcoming this problem is the application of polyglycerol esters of fatty acids (PGFAs) as lipid matrices with stable solid state. PG2-C18 full, a PGFA molecule, was used to develop SLN loaded with dexamethasone as a model API. Dexamethasone-loaded SLN were manufactured via melt-emulsification and high pressure homogenization in the dosage form of a lipid nanosuspension. SLN with median particle size of 242.1 ± 12.4 nm, zeta potential of -28.5 ± 7.8 mV, entrapment efficiency of 90.2 ± 0.7% and API released after 24 h of 81.7 ± 0.7%, were produced. Differential Scanning Calorimetry (DSC) and Small and Wide Angle X-Ray Scattering (SWAXS) analysis of the lipid nanosuspension evidenced the crystallization of PG2-C18 full in a monophasic system in α-form and absence of polymorphism and crystallite growth up to 6 months storage at room temperature. This resulted in stable performance of the SLN after storage: absence of particle agglomeration, no API expulsion, and stable release profile. The potential pulmonary administration of SLN was tested by the nebulization capacity of the lipid nanosuspension. Cellular exposures to SLN did not induce cytotoxicity or immune effect on pulmonary cells. The application of PGFAs as safe and stable lipid matrices provide a promising approach for the development of the next generation of SLN.

Novel approach for overcoming the stability challenges of lipid-based excipients. Part 2: Application of polyglycerol esters of fatty acids as hot melt coating excipients.

The application of hot melt coating (HMC) as an economic and solvent-free technology is restricted in pharmaceutical development, due to the instable solid-state of HMC excipients resulting in drug release instability. We have previously introduced polyglycerol esters of fatty acids (PGFAs) with stable solid-state (Part 1). In this work we showed a novel application of PGFAs as HMC excipients with stable performance. Three PGFA compounds with a HLB range of 5.1-6.2 were selected for developing immediate-release formulations. The HMC properties were investigated. The viscosity of molten lipids at 100 °C was suitable for atomizing. The DSC data showed the absence of low solidification fractions, thus reduced risk of agglomeration during the coating process. The driving force for crystallization of selected compounds was lower and the heat flow exotherms were broader compared to conventional HMC formulations, indicating a lower energy barrier for nucleation and lower crystallization rate. Lower spray rates and a process temperature close to solidification temperature were desired to provide homogeneous coating. DSC and X-ray diffraction data revealed stable solid state during 6 months storage at 40 °C. API release was directly proportional to HLB and indirectly proportional to crystalline network density and was stable during investigated 3 months. Cytotoxicity was assessed by dehydrogenase activity and no in vitro cytotoxic effect was observed.

Some Peculiarities in the Dose Dependence of Separate and Combined In Vitro Cardiotoxicity Effects Induced by CdS and PbS Nanoparticles With Special Attention to Hormesis Manifestations.

Spherical nanoparticles (NPs) of cadmium and lead sulfides (diameter 37 ± 5 and 24 ± 4 nm, respectively) have been found to be cytotoxic for HL-1 cardiomyocytes as evidenced by decrease in adenosine triphosphate-dependent luminescence. Cadmium sulfide (CdS)-NPs were discovered to produce a much greater cytotoxic impact than lead sulphide (PbS)-NP. Given the same dose range, CdS-NP reduced the number of calcium spikes. A similar effect was observed for small doses of PbS-NP. In addition to cell hypertrophy under the impact of certain doses of CdS-NP and PbS-NP, doses causing cardiomyocyte size reduction were identified. For these 3 outcomes, we obtained both monotonic "dose-response" functions (well approximated by the hyperbolic function) and different variants of non-monotonic ones for which we found adequate mathematical expressions by modifying certain models of hormesis available in the literature. Data analysis using a response surface linear model with a cross-term provided new support to the previously established postulate that a diversity of types of joint action characteristic of one and the same pair of damaging agents is one of the important assertions of the general theory of combined toxicity.

Air-liquid interface culture changes surface properties of A549 cells.

A549 cells are common models in the assessment of respiratory cytotoxicity. To provide physiologically more representative exposure conditions and increase the differentiation state, respiratory cells, for instance Calu-3 bronchial epithelial cells, are cultured at an air-liquid interface (ALI). There are indications that A549 cells also change their phenotype upon culture in ALI. The influence of culture in two variations of transwell cultures compared to conventional culture in plastic wells on the phenotype of A549 cells was studied. Cells were characterized by morphology, proliferation and transepithelial electrical resistance, whole genome transcription analysis, Western blot and immunocytochemical detection of pro-surfactant proteins. Furthermore, lipid staining, surface morphology, cell elasticity, surface tension and reaction to quartz particles were performed. Relatively small changes were noted in the expression of differentiation markers for alveolar cells but A549 cells cultured in ALI showed marked differences in lipid staining and surface morphology, surface tension and cytotoxicity of quartz particles. Data show that changes in physiological reactions of A549 cells in ALI culture were rather caused by change of surface properties than by increased expression of surfactant proteins.

Prognostic value of B7-H1, B7-H3 and the stage, size, grade and necrosis (SSIGN) score in metastatic clear cell renal cell carcinoma.

INTRODUCTION: We compared the potential prognostic impact of B7-H1 and B7-H3 glycoprotein expressions with the Mayo Clinic Stage, Size, Grade, and Necrosis (SSIGN) score in metastatic clear cell renal cell carcinoma (mccRCC) during a long term follow-up. MATERIAL AND METHODS: We investigated 44 mccRCC patients, who underwent radical nephrectomy between 1995 and 2006 at a single tertiary academic center and received interferon therapy (IFNT) for at least three months. The SSIGN score was applied as a validated prediction outcome model. Representative tumor sections were immunostained with anti-B7-H3 and anti-B7-H1 antibodies. Hereafter, positive antigen-antibody reactions were measured using the Positive-Pixel-Count Algorithm of the Aperio-Technology Image Scope software. RESULTS: In total, 48% of patients were treated with cytoreductive nephrectomy and postoperative IFNT due to synchronous mccRCC, whereas 52% received IFNT after developing metachronous mccRCC. The SSIGN score was independently associated with a higher mortality risk. Patients with a SSIGN score ≤9 showed an extended 'nephrectomy to start of INFT'-interval (p = 0.02), less synchronous clinical metastases (p = 0.0002), as well as an increased median overall - (OS) or cancer-specific survival (CSS) (p = 0.01), respectively. Furthermore, B7-H3 expression levels of ≤16% were associated with an improved OS or CSS and correlated with a more frequent pathologic grade 1-2, as well as a longer 'nephrectomy to start of IFNT'-interval, respectively. B7-H1 expression patterns did not correlate with survival. CONCLUSIONS: The SSIGN score demonstrated the best prognostic performance. In contrast, B7-H3 expression patterns showed a low association with histopathological parameters, but predicted the cut-off-dependent impaired survival and in the future may define a cut-off to indicate checkpoint-inhibitor treatment.