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Abstract: Aerolized cyclosporine A (CsA) has been successfully used for prevention of organ rejection in lung transplant recipients. Various formulations of CsA are available and so far no direct comparison of their pharmacokinetics has been performed. Since clinical studies are elaborate, we sought a way to predict the kinetic behaviour of a propylene glycol solution of CsA (CsA-PG) and a liposomal formulation (L-CsA). The permeability across the human bronchial cell line Calu-3 revealed low permeability for CsA with the apparent permeability for CsA-PG being twice as high as for L-CsA. Employing a previously described dialysis model, the diffusion of CsA from human lung tissue into human blood was determined ex vivo. Consistent with the cell culture model results, we observed that the degree and rate of drug transfer into human blood was more pronounced for CsA-PG than for L-CsA with the area under the curve (AUC) of CsA-PG being about 1.6 times higher than for the L-CsA formulation. The diffusion rate was more than 50% higher from CsA-PG than from the liposomes. To conclude, both model systems consistently revealed that L-CsA displayed clearly a prolonged release effect and favourable longer tissue retention than CsA-PG. [Copyright &y& Elsevier]

Abstract: The primary targets for SARS-CoV infection are the epithelial cells in the respiratory and intestinal tract. The angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor for SARS-CoV. ACE-2 has been shown to be expressed at the apical domain of polarized Calu-3 cells. In this report, interferon alfacon 1 was examined for inhibitory activities against SARS-CoV on human lung carcinoma epithelial Calu-3 cell line and the other three African green monkey kidney epithelial cell lines. Interferon alfacon 1 demonstrated significant antiviral activity in neutral red uptake assay and virus yield reduction assay. The data might provide an important insight into the mechanism of pathogenesis of SARS-CoV allowing further development of antiviral therapies for treating SARS infections. [Copyright &y& Elsevier]

The CFTR protein, encoded by the gene whose mutations induce Cystic Fibrosis, is an anion channel devoted mainly to chloride and bicarbonate transmembrane transport, but which also regulates transport of several other ions. Moreover, it is implicated in the cell response to inflammation, and, reciprocally, cftr gene expression is modulated by inflammatory stimuli and transduction pathways. Looking for a control of CFTR expression by ionic conditions, we investigated the effect of altered extracellular bicarbonate ion concentration on CFTR expression in human pulmonary Calu-3 cells. We found that basal cftr gene transcription is enhanced when extracellular HCO3- concentration increases from 0 to 25 mmol/l. The transduction pathway controlled by these extracellular [HCO3-] variations includes cAMP production linked to the stimulation of soluble adenylyl cyclase (sAC), and nuclear accumulation of the transcription factor, CREB. Basal membrane content in CFTR protein exhibits the same variations as cftr mRNA in cells incubated in the presence of extracellular [HCO3-] between 0 and 25 mmol/l, and is also decreased by inhibiting sAC in the presence of HCO3-. These results show that bicarbonate-controlled sAC stimulation must be taken into account in cell physiology and that basal CFTR expression depends on an ionic parameter. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Permeability estimates using Caco-2 cells do not accurately predict the absorption of hydrophilic drugs that are primarily absorbed via the paracellular pathway. The objective of this study was to investigate whether modulation of tight junctions would help differentiation of paracellularly absorbed compounds. Tight junctions in Caco-2 cell monolayers were manipulated using calcium depletion approaches to decrease the transepithelial electrical resistance (TEER) of the monolayers, and permeability of hydrophilic compounds were measured under these conditions. Permeability of these compounds were also measured in Calu-3 cells, which have tighter junctions than Caco-2 cells. Calcium depletion loosened the tight junctions of Caco-2 cells to varying levels as measured by the decrease in TEER values of the monolayers. While the absolute permeability of all the model compounds increased as the tight junctions were loosened, the ratios of their permeability relative to mannitol permeability were similar. The permeability of these compounds in the tighter Calu-3 cells were also found to be similar to each other. Altering the tight junctions of Caco-2 cells to obtain leakier cell monolayers, or using a cell line with tighter junctions like Calu-3 cells, did not improve differentiation between well absorbed and poorly absorbed hydrophilic drugs. Mere manipulation of the tight junctions to increase or decrease transepithelial electrical resistance does not appear to be a viable approach to predict human absorption for hydrophilic compounds that are primarily absorbed via the paracellular pathway. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this work was to evaluate the biocompatibility of novel respirable powder formulations of nanoparticles (NP) entrapped in mannitol microspheres using human respiratory epithelial cell lines. Microspheres formulated at NP:mannitol ratios of 10:90, 20:80 and 40:60 were evaluated using the Calu-3 and A549 cell lines. The MTT cell viability assay revealed an absence of overt toxicity to Calu-3 or A549 cells following exposure to the formulations containing <1.3mg NP/ml (equivalent to 0.87mg NP/cm2) for up to 48h. Transepithelial electrical resistance (TER) and solute permeability in Calu-3 cell layers were determined following exposure of the cells to the NP:mannitol 20:80 formulation. After administration of the formulation dissolved in serum-free cell culture medium (1.3mg/ml NP suspension) to the cells, neither TER nor permeability were altered compared to untreated cell layers. Confocal microscopy did not reveal any NP internalisation under the conditions used in this study, although evidence of mucoadhesion was observed. All the data presented are encouraging with respect to the development of chitosan NP-containing microspheres for the pulmonary administration of therapeutic macromolecules. Not only do the formulations possess suitable aerodynamic characteristics and the capacity to encapsulate proteins as shown previously; they have now been shown to exhibit in vitro biocompatibility. [Copyright &y& Elsevier]

Abstract: Neltenexine has been applied to human lung diseases such as chronic obstructive pulmonary disease (COPD) as a mucolytic agent. However, we have no information on the neltenexine action in bronchial epithelial cells. We studied the neltenexine action on the ion transport in human submucosal serous Calu-3 cells. Under a hyper-secreting condition caused by terbutaline (a β2-adrenergic agonist), neltenexine diminished anion secretion by inhibiting the Cl− and uptake via Na+/K+/2Cl− cotransporter and cotransporter without blockade of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, and also diminished anion secretion via stimulation of exchanger, which facilitates the extrusion of more CFTR-permeant anion, Cl−, with the uptake of less CFTR-permeant anion, . Thus, neltenexine reduced the hyper-secretion to keep an appropriate fluid level in the airway, providing a possibility that neltenexine can be an effective drug in airway obstructive diseases by decreasing the airway resistance under a hyper-secreting condition. [Copyright &y& Elsevier]

Background and purpose:Calu-3 cells are derived from serous cells of human lung submucosal glands, a prime target for therapy in cystic fibrosis (CF). Calu-3 cells can be cultured to form epithelia capable of transepithelial transport of chloride. A CF Calu-3 cell is not available.Experimental approach:A retroviral vector was used to cause persistent down regulation of CFTR using siRNA methodology, in Calu-3 cells. A Calu-3 cell line with CFTR content less than 5% of the original line has been established. Epithelia grown using the modified cells have been used in comparative studies of transporting capability.Key results:All aspects of cAMP activated chloride secretion were attenuated in the epithelia with reduced CFTR content. However transporting capability was reduced less than the CFTR content. From studies with the CFTR channel inhibitor, GlyH-101, it was concluded that wild type Calu-3 cells have a reserve of CFTR channels not located in the membrane, but available for replacement, while in the modified Calu-3 cell line there was little or no reserve. Lubiprostone, a putative ClC-2 activator, increased transepithelial chloride secretion in both modified and wild type Calu-3 epithelia. Modified Calu-3 epithelia with the residual CFTR currents blocked with GlyH-101 responded equally well to lubiprostone as those without the blocking agent.Conclusions and implications:It appears that lubiprostone is capable of stimulating a non-CFTR dependent transepithelial chloride secretion in Calu-3 monolayers, with obvious implications for CF therapy. Cell lines, however, do not always reflect the behaviour of the native tissue with integrity.British Journal of Pharmacology (2007) 150, 1055–1065. doi:10.1038/sj.bjp.0707175; published online 5 March 2007 [ABSTRACT FROM AUTHOR]

This study was performed to evaluate insulin permeability across human bronchial epithelial cell lines and investigate if insulin is transported via the paracellular or transcellular pathway. The movement of insulin across two bronchial epithelial cells, 16HBE14o- and Calu-3, was studied in the presence or absence of octylmaltoside. Mannitol and propanolol have been used as paracellular and transcellular marker, respectively, and transepithelial electrical resistance (TEER) was determined to investigate the tight junctional integrity of the monolayers. The possible endocytotic mechanism of insulin across these two cell lines was studied by confocal laser scanning microscopy after incubating the cells with fluorescent-labeled insulin. The TEER values for both cell monolayers were >400 Ω cm 2 at confluency. There was a decrease in the TEER values when octylmaltoside was added to the apical side of transwells. Similarly, the apparent permeability coefficient ( P app ) values of insulin, mannitol and propanolol, showed an increase with the rise in the concentration of octylmaltoside. In the absence of octylmaltoside, the P app values for insulin and the markers were in the following order: propanolol > mannitol > insulin. Confocal microscopic studies revealed that the uptake of insulin by the bronchial epithelial cells perhaps occurs via translocation across the cell. The data presented in this study demonstrate that insulin perhaps moves across the bronchial cells via both paracellular and transcellular pathways. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of proteins was investigated. TMC nanoparticles were prepared by ionic crosslinking of TMC solution (with or without ovalbumin) with tripolyphosphate, at ambient temperature while stirring. The size, zeta-potential and morphology of the nanoparticles were investigated as a function of the preparation conditions. Protein loading, protein integrity and protein release were studied. The toxicity of the TMC nanoparticles was tested by ciliary beat frequency measurements of chicken embryo trachea and in vitro cytotoxicity assays. The in vivo uptake of FITC-albumin-loaded TMC nanoparticles by nasal epithelia tissue in rats was studied by confocal laser scanning microscopy. The nanoparticles had an average size of about 350 nm and a positive zeta-potential. They showed a loading efficiency up to 95% and a loading capacity up to 50% (w/w). The integrity of the entrapped ovalbumin was preserved. Release studies showed that more than 70% of the protein remained associated with the TMC nanoparticles for at least 3 h on incubation in PBS (pH 7.4) at 37 °C. Cytotoxicity tests with Calu-3 cells showed no toxic effects of the nanoparticles, whereas a partially reversible cilio-inhibiting effect on the ciliary beat frequency of chicken trachea was observed. In vivo uptake studies indicated the transport of FITC-albumin-associated TMC nanoparticles across the nasal mucosa. In conclusion, TMC nanoparticles are a potential new delivery system for transport of proteins through the nasal mucosa. [Copyright &y& Elsevier]

The electrochemical oxidation of five germole derivatives: 1,1-bis(trimethylsilyl)-2,3,4,5-tetraphenyl-1-germacyclopentadiene (I), 1,1-dimethyl-2,3,4,5-tetraphenyl-1-germacyclopentadiene (II), 1-methyl-1-chloro-2,3,4,5-tetraphenyl-1-germacyclopentadiene (III), 1,1-dichloro-2,3,4,5-tetraphenyl-1-germacyclopentadiene (IV) and a bicyclic compound (V) 1,1′-dimethyl-2,2′,3,3′,4,4′,5,5′-octaphenyl bisilole, bonded through a Si–Si bond, has been investigated, both by cyclic voltammetry and controlled-potential electrolysis. The effects of the nature of electrolyte, atmosphere and substituents attached to the germanium atom in the germacyclopentadiene ring on the outcome of the electrochemical oxidation are discussed. [Copyright &y& Elsevier]

A microinterface between two immiscible phases (micro ITIES), established by a microchannel (internal diameter: 50 μm, length: 15 μm) drilled through a polyimide thin plate, gives reproducible voltammograms for the transfer of charges. As expected for such a microinterface, the regime for mass transport depends on the scan rate of voltammetry, low scan rates being characterized by steady-state currents corresponding to radial diffusion; faster sweeps give rise to conventional cyclic voltammograms, from linear diffusion. This has been used as an electrochemical method for the study of tetraalkylammonium picrates at interfaces between water and immiscible solvents (nitrobenzene, 1,2-dichloroethane). The partition of Me4NPi, Et4NPi, Pr4NPi, and Bu4NPi at the W∣NB and W∣DCE interfaces is clearly illustrated by voltammetry under steady-state conditions, allowing the measurement of Gibbs energies and interfacial potentials for the transfer of the ions from water to NB. When the organic solvent is DCE, the estimation of ionic interactions is risky in such a poorly dissociating medium; the microhole interface works well however, showing the influence of the medium on solvation of the cations and on the extraction of the picrates. [Copyright &y& Elsevier]

Conductive sulfur-containing cathode materials for high-capacity lithium secondary batteries have been synthesized by heating a mixture of polyacrylonitrile and elemental sulfur. The structures and electrochemical properties of the materials depend on the reaction conditions, particularly synthesis temperatures. FTIR, Raman and XPS measurements show that the fundamental chemical structure of the materials is composed of a dehydropyridine type matrix with S–S bonds in the side-chain. The cycle performances of the materials synthesized at different temperatures have been examined as active cathode materials in lithium cells. Due to the existence of π-conjugation and the C–S–S–C covalent bond, significantly improved redox rates and cycleability at room temperature are achieved. A stable discharge capacity for the material synthesized at 450 °C is maintained at about 480 mAh/g and the capacity retention remains at ca. 92% (referred to the second cycle) after about 240 cycles. The excellent cycle characteristics supply a good foundation for practical application of this material in rechargeable lithium batteries. [Copyright &y& Elsevier]

In this paper we report the development of an electrochemical in situ rapid-scan time-resolved microscope infrared reflection spectroscopy (RS-TR-MFTIRS), which consists of a FTIR spectrometer with rapid-scan facility, an infrared microscope in combination with a nanostructured Pt microelectrode, and a versatile external reflection (or thin-layer) spectroelectrochemical flow cell. It has been demonstrated that the RS-TR-MFTIRS can successfully overcome the disadvantages in mass transport, transient response and uniformity of current flow that arise from a conventional external reflection infrared cell. The dynamic process of methanol oxidation on a nanostructured platinum microelectrode in alkaline solution was employed as a test reaction to characterize the performance of the newly developed technique. The results illustrate that cyclic voltammograms (CVs) and infrared spectra within a potential interval of 2.6 mV can be simultaneously recorded at a potential scan rate up to 200 mVs−1. The dynamic processes of the poisoning intermediate (adsorbed CO species) and reactive intermediate (formate ions) involved in methanol oxidation in alkaline solutions were studied intensively. It has been revealed that the bridge-bonded CO (COB) species and formate ions are generated preferentially at the onset potential (−0.54 V vs SCE) of methanol oxidation, while the linearly bonded CO (COL) species originate at more positive potentials that are close to the anodic current peak potential (−0.17 V vs SCE) of methanol oxidation. [Copyright &y& Elsevier]

The ac-impedance spectroscopy of the electro-oxidation of 2-propanol and methanol was investigated in a H2SO4 solution using a carbon-supported platinum catalyst as a function of potential between 350 and 750 mV (vs. SHE) at a variety of temperatures between 303 and 353 K. It is found that the impedance spectrum of 2-propanol is very similar to that of methanol. Semicircles that have induction loops at the low frequency ends were observed in complex plane plots at high potentials between 600 and 750 mV in both the cases of 2-propanol and methanol, which suggests that the rate-determining steps of the electro-oxidation of intermediates adsorbed on platinum should be involved in their reaction processes. Assuming appropriate models involving the adsorbed intermediates for 2-propanol and methanol, the impedance spectra were explained successfully. The impedance analysis provides the values of reaction parameters concerning a coverage relaxation time for an intermediate and a charge-transfer resistance, which make it possible to compare the reaction rates of the intermediates between 2-propanol and methanol. The reaction parameters suggest that a consecutive reaction involving the intermediate for 2-propanol proceeds more slowly than that for methanol, and also suggest that a direct reaction pathway from 2-propanol to acetone, which does not go through the intermediate, exists in parallel. Additionally, the influences of acetone concentration on the coverage relaxation time and the charge-transfer resistance for the electro-oxidation of 2-propanol were also discussed. [Copyright &y& Elsevier]

The process of hydrogen electrosorption in thin Pd–Pt–Rh alloys has been investigated mainly in acidic solution using cyclic voltammetry and chronoamperometry. The influence of electrode potential on the amount of electrosorbed hydrogen is presented. On the plot of H/M (hydrogen atoms/metal atoms) vs. potential the regions of pure α- and β-phase existence as well as the region of α–β phase transition can be distinguished. The maximum amount of absorbed hydrogen depends on the bulk composition being relatively high for Pd-rich alloys, although smaller than for pure Pd. The α–β transition is shifted to lower potentials in comparison with pure Pd. The process of phase transition seems to control the rate of hydrogen absorption and desorption into/from thin Pd-based electrodes. The hysteresis effect is observed in chronoamperometric absorption and desorption experiments. Potential cycling of Pd–Pt–Rh alloys through the oxygen region leads to significant changes in hydrogen absorption properties with the appearance of an additional bulk phase. [Copyright &y& Elsevier]

The conducting polymer poly (3,4-ethylenedioxythiophene) (PEDOT) doped with its derivative sulfonatoalkoxy EDOT (S-EDOT) was electrochemically polymerized on the surface of electrodes along the shank of microfabricated neural probes. Reflective Fourier transform infrared spectroscopy demonstrated the composition of the resultant polymer. Examinations of scanning electron microscope images of the films indicated that the surface morphology was rough and the thickness varied with deposition time. The magnitude of the impedance of the PEDOT/S-EDOT coated electrode was lower than that of bare gold over a wide range of frequencies from 1 to 105 Hz. The roughness of the electrode surface resulted in a resistive component being added to the double layer capacitance. Cyclic voltammetry demonstrated the intrinsic redox reaction and the increased charge capacity of the PEDOT/S-EDOT coated electrodes. [Copyright &y& Elsevier]

In this work, we give three examples of interfacial reactions in Al/transition metal (TM)- or Si/TM systems in which the specificities of the reactive diffusion (selectivity, nucleation threshold) on the nanoscale are emphasized. In particular, this allows the stabilisation of phases and interfaces, which are not expected from the equilibrium phase diagram, and a morphological development of the phases formed which can induce nanostructuring. [Copyright &y& Elsevier]

In this work, we investigate transport and reaction kinetics in agglomerates of cathode catalyst layers used in proton-exchange membrane fuel cells. Two types of spherical agglomerates are evaluated, which represent limiting structures that can be obtained by distinct synthetic procedures. One type consists of a mixture of carbon/catalyst particles and a proton conducting perfluorosulfonated ionomer (PFSI). The other type consists of carbon/catalyst particles and water-filled pores. The performance of the former type is rationalized on the basis of the well-known Thiele-modulus. Characteristics of the latter type are studied using Nernst–Planck and Poisson equations. Aspects of current conversion, reactant and current distributions and catalyst utilization are explored. In general, the PFSI-filled agglomerates exhibit more homogeneous distributions of reaction rates. Effectiveness factors for these are close to one. However, it was found that proton penetration depths in water-flooded agglomerates could be quite substantial under certain conditions, resulting in unexpectedly high catalyst utilization. The effects of agglomerate radius and of boundary conditions on the agglomerate surface are studied. An approximate analytical solution was obtained for a planar geometry of agglomerates. The significance of these results for the optimization catalyst layers in the light of operation conditions and synthesis methods is discussed. [Copyright &y& Elsevier]

The aluminumphosphate designated as meso-ALPO was efficiently coated with 3-n-propylpyridiniumchloride silsesquioxqne polymer, SiPy+Cl−, forming a stable thin film attached to the surface by the Si–O–Al bond. The electroactive species cobalt (II) tetrasulphophthalocyanine complex, CoTsPc4−, was adsorbed on the matrix modified surface, ALPO/SiPy+Cl−, by an ion exchange reaction resulting in a solid designated as ALPO/SiPy+/CoTsPc4−. The complex showed high affinity of the solid phase and this was not leached off even in contact with 1.0 moll−1 KCl supporting electrolyte solution, presumably because it was strongly bound to the aluminumphosphate. The experiments revealed that, in the electrooxidation of oxalic acid, electron transfer processes with the metallic center are very fast and that the diffusion process of the acid into the electrode surface is slower. The ALPO/SiPy+/CoTsPc4− system is very stable and sensitive to oxalic acid oxidation. Its linear peak current density changes with concentration change of the oxalic acid up to a detection limit of 1×10−5 moll−1. The characteristics of the system are suitable for its use as an electrochemical sensor for oxalic acid. [Copyright &y& Elsevier]

The electrode potentials of some quinone derivatives in dimethylsulfoxide have been calculated. The calculations are carried out at the level of density functional theory with the inclusion of entropic and thermochemical corrections to yield Gibbs energies of redox reactions. The polarizable continuum model (PCM) is used to calculate solvation energies. The model furnishes the calculated electrode potentials in relatively good agreement with the experimental values. The results show that the method is likely to be useful in the prediction of electrode potentials of organic molecules in different aprotic solvents. [Copyright &y& Elsevier]

Chromium tetraaminophthalocyanine (CrIIITAPc) has been synthesized and its electrochemical and spectroelectrochemical properties reported. The first reduction and oxidation in this complex occur at the central metal, giving CrIITAPc and CrIVTAPc complexes, respectively, the latter having been observed for the first time. The CrIIITAPc species was polymerized onto glassy carbon (GCE) or indium tin oxide (ITO) electrodes and the polymer evolution confirmed by UV/Visible spectroscopy and by scanning electron microscopy. The polymers were employed for the catalytic oxidation of nitrite or nitric oxide, and the catalysis involved the CrIVTAPc−1 species. [Copyright &y& Elsevier]

A comparison between the analytical performance of a hanging mercury drop electrode (HMDE) and a boron-doped diamond (BDD) electrode for the quantification of 4-nitrophenol (4-NP) in spiked pure and natural waters is reported in this work. Square wave voltammetry (SWV) was chosen as the electroanalytical technique and Britton–Robinson buffer as the electrolyte. For the reduction process, the quantification limits varied between 5.7 and 66.0 μgl−1 for the HMDE and between 14.1 and 61.3 μgl−1 for the BDD electrode for water samples with increasing degree of contamination. The oxidation of 4-NP on BDD was also used for analytical purposes and the quantification limits in this case varied from 9.4 to 53.1 μgl−1. In all cases, the detection limits followed the same trend. These latter results illustrate the advantages arising from the possibility of using an oxidation process on BDD electrodes for analytical purposes in contaminated matrices. The recovery experiments showed values above 95% for spiked samples thus indicating the feasibility of the electroanalytical methodology to quantify 4-NP in pure or natural waters. HPLC measurements were also performed for comparison and confirmed the values measured by SWV. [Copyright &y& Elsevier]

Pulmonary immunization against inhaled pathogens such as Mycobacterium tuberculosis would induce local and systemic immune responses and protect from entry and dissemination of the pathogen. The aim of this study was to evaluate cationic submicron emulsion as a potential carrier for DNA vaccines to the lung. DNA loaded emulsions were 128–152 nm in size and retained positive zeta potential above +40 mV during 3 months of storage. Loading efficiency was above 99%, DNA was protected from DNase I degradation up to 60 min and was stable in presence of 75% fetal calf serum (FCS). The plasmid DNA was detected in the endo-lysosomal compartment of the human bronchial cell line, Calu-3, 6 h after application. No cytotoxic effect on these cells was observed. Human dendritic cells were matured in presence of DNA loaded emulsion, although to a lesser extent than DNA solution indicating slower release and lower exposure to unmethylated CpG sequences. These results indicate that cationic submicron emulsions are potential DNA vaccine carriers to the lung since they are able to transfect pulmonary epithelial cells, which possibly induce cross priming of antigen presenting cells and directly activate dendritic cells, resulting in stimulation of antigen specific T-cells. [Copyright &y& Elsevier]

Background and objective: The combined application of cytokines on embryonic fibroblasts and dermal substitute were studied for optimal skin defect coverage. The mechanism of combined treatment of leukemia inhibitory factor (LIF)-transfected embryonic fibroblasts and vascular endothelial growth factor (VEGF) were elucidated and subsequently the in vivo applications of both were tested in an artificial dermal substitute.Methods: Mouse embryonic fibroblast cells, BALB-3T3, were stably transfected with mouse full-length LIF cDNA and added to various doses of VEGF for detection of signaling interaction. LIF-transfected cells and VEGF treatment were tested with pig-tendon derived collagen dermal substitute in the backs of BALB/c male mice up to for 14 days.Results: LIF-transfected cells as well as vector-transfected fibroblasts significantly proliferated by 1, 10, or 100 ng VEGF on days 3 and 5. Erk mitogen-activated protein (MAP) kinase phosphorylation was observed from 1 to 30 min in LIF-transfected and 10 ng of VEFG, and 1 to 60 min in LIF-transfected and 100 ng VEFG treatments. The cellular fibronectin levels also increased in LIF-transfected cells with 10 and 100 ng VEGF additions. In in vivo analyses, LIF-transfected embryonic fibroblasts with 50 μg of VEGF markedly enhanced collagen I expression and CD34 angiogenic marker on days 7 and 14.Conclusion: LIF transfection and VEGF treatment enhanced phosphorylated-Erk-MAP kinase in vitro. In vivo study revealed that the combined application of LIF transfection of embryonic fibroblasts with an angiogenic factor such as VEGF in the template of a dermal substitute induced greater skin collagen production and angiogenesis in the dermal substitute. [Copyright &y& Elsevier]

Over the last decade, animal models of atopic dermatitis (AD) have received increasing attention. They include NC/Nga mice, a hapten-induced mouse model, and transgenic and knockout mouse models. Although the pathogenesis of skin inflammation elicited in these models and that in AD are not quite the same, it is pertinent to ask what these animal models really tell us about the pathogenesis and possible therapies for the disease. NC/Nga mice may yield information relevant to the dissection of the crucial components of the pathophysiology of AD rather than the assessment of potentially therapeutic agents for its treatment. A hapten-induced mouse model created by repeated application of 2,4,6-trinitrochlorobenzene (TNCB) is a simple and reproducible one. This model offers several advantages over others: by changing hapten and the mouse strain used, various types of chronic inflammation, probably reflecting heterogeneity in clinical presentation of AD, can be induced; this model is also of enormous value in its high reproducibility as well as the ease of quantitative assessment by measuring ear thickness. Among various transgenic and knockout mouse models, the IL-18-transgenic mouse is one of the closest available mouse models of human AD, although the onset of the AD-like lesions in the IL-18-transgenic mice is such a late event. Although these mice all have significant disadvantages, it is important to review the current literature on the models in the hope that one may identify useful areas for investigation. [Copyright &y& Elsevier]

Background: Androgenic alopecia (AGA) is the most common type of baldness in men. Although etiological studies have proved that androgen is one of the causes of this symptom, the defined molecular mechanism underlying androgen-related actions remains largely unknown.Objectives: To clarify the difference in the gene expression profile of dermal papilla cells (DPCs) in skin affected by baldness.Methods: DNA macroarray study was carried out on cultured DPCs from AGA skin comparing with DPCs from skin that is not affected by baldness.Results: From DNA macroarray analysis, we observed that 107 of the 1185 analyzed genes had differing expression levels. A marked difference was observed in the decreased gene expression of BMP2 and ephrin A3 and up-regulated in NT-4 gene. In order to clarify the roles of BMP2 and ephrin A3 in the hair follicles, we examined the proliferation of hair follicle keratinocyte and expression of a hair acidic keratin gene. Both BMP2 and ephrin A3 raised the proliferation rate of the outer root sheath cells (ORSCs) and induced gene expression in acidic hair keratin 3-II.Conclusion: These results lead us to the hypothesis that both BMP2 and ephrin A3 function as hair growth promoting factors in the hair cycle. [Copyright &y& Elsevier]

It has been proposed that transforming growth factor-β (TGF-β) is involved in the growth inhibition of normal human epidermal keratinocytes (NHEK) by 1α,25-dihydoxyvitamin D3 (1α,25(OH)2D3), although this is still controversial because of the difficulty in blocking TGF-β activity completely.To determine whether TGF-β is involved in early phase growth inhibition by 1α,25(OH)2D3.TGF-β mRNA was detected by ribonuclease protection assay (RPA), and biological active TGF-β was determined by a luciferase reporter assay. To block intrinsic TGF-β activity completely, we constructed an adenovirus vector expressing a truncated TGF-β type II receptor with a dominant negative effect (AdexTβTR) that blocks TGF-β signal transduction.1α,25(OH)2D3 slightly upregulated TGF-β1 and TGF-β2 after 24h according to an RPA and a luciferase reporter assay, however growth inhibition by 1α,25(OH)2D3 occurred at 6h. The addition of 10-6M of 1α,25(OH)2D3 to NHEK infected with AdexTβTR or AdexLacZ (control vector) reduced DNA synthesis to 59.3 and 62.2% at 6h, respectively. There was no significant difference in cell number after a 3-day incubation with AdexTβTR or AdexLacZ-infected cells treated with 1α,25(OH)2D3.Since 1α,25(OH)2D3 rapidly inhibits NHEK growth regardless of the prevention of TGF-β signal transduction, TGF-β is not involved in early phase growth inhibition by 1α,25(OH)2D3. [Copyright &y& Elsevier]

We evaluated the relationship between apical surface fluid (ASF) and protein secretion in Calu-3 cells grown at an air–liquid interface. Calu-3 monolayers responded to forskolin, a cystic fibrosis transmembrane regulator (CFTR) channel agonist, by secreting a significant amount of ASF. Such a response from Calu-3 monolayers was not observed with CFTR channel blockers glybenclamide and DPC. Other ion channel mediators, PGF-2α, PMA, DNDS, and DIDS, had no effect on Calu-3 ASF secretion. Forskolin decreased Calu-3 protein secretion and glybenclamide increased protein secretion. Similarly, forskolin decreased Calu-3 lysozyme secretion, whereas glybenclamide and DPC increased lysozyme secretion. We observed significant changes in Calu-3 fluid and protein secretions with ion channel mediators known to alter CFTR activity. Our results demonstrate a functional link between fluid and protein secretions in Calu-3 apical surface and suggested a possible involvement of CFTR in these processes. [Copyright &y& Elsevier]

Pulmonary gene delivery is thought to play an important role in treating genetically related diseases and may induce immunity towards pathogens entering the body via the airways. In this study we prepared poly (d,l-lactide-co-glycolide) (PLGA) nanoparticles bearing polyethyleneimine (PEI) on their surface and characterized them for their potential in serving as non-viral gene carriers to the pulmonary epithelium. Particles that were synthesized at different PLGA–PEI ratios and loaded with DNA in several PEI–DNA ratios, exhibited narrow size distribution in all formulations, with mean particle sizes ranging between 207 and 231 nm. Zeta potential was strongly positive (above 30 mV) for all the PEI–DNA ratios examined and the loading efficiency exceeded 99% for all formulations. Internalization of the DNA-loaded PLGA–PEI nanoparticles was studied in the human airway submucosal epithelial cell line, Calu-3, and DNA was detected in the endo-lysosomal compartment 6 h after particles were applied. Cytotoxicity of these nanoparticles was dependent on the PEI–DNA ratio and best cell viability was achieved by PEI–DNA ratios 1:1 and 0.5:1. These findings demonstrate that PLGA–PEI nanoparticles are a potential new delivery system to carry genes to the lung epithelium. [Copyright &y& Elsevier]

In the present study, we evaluated polyethylenimine (PEI) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting nondifferentiated COS-1 (green monkey fibroblasts) and well-differentiated human submucosal airway epithelial cells (Calu-3). Studying the effect of particle size, zeta potential, presence of serum proteins or chloroquine, it appeared that transfection efficiency depends on the experimental conditions and not on the MW of the PEI used. Comparing transfection efficiencies in both cell lines, we found that PEI was 3 orders of magnitude more effective in COS-1 than in Calu-3 cells, because Calu-3 cells are differentiated and secrete mucins, which impose an additional barrier to gene delivery. Transfection efficiency was strongly correlated to PEI cytotoxicity. Also, some evidence for PEI-induced apoptosis in both cell lines was found. In conclusion, our results indicate that PEI is a useful vector for nonviral transfection in undifferentiated cell lines. However, results from studies in differentiated bronchial epithelial cells suggest that PEI has yet to be optimized for successful gene therapy of cystic fibrosis (CF). [ABSTRACT FROM AUTHOR]

Lysozyme is secreted in large quantities in human airways (10–20 mg/day), where it helps to defend against bacterial and fungal infection. Lysozyme expression is restricted to the serous cells of the submucosal glands, which also express high levels of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. It is often assumed that mucus secretion in human airways is coupled to anion secretion through CFTR Cl– channels located in the apical membrane. Therefore, a defect in CFTR function could cause abnormal mucus secretion leading to persistent bacterial infection and inflammation of the airways. In this study we measured simultaneous secretion of lysozyme and Cl– from human airway epithelial serous cells. Secretion of lysozyme was measured by a turbidimetric assay that relies on the ability of lysozyme to disrupt the wall of the bacterium Micrococcus lysodeikticus, thus causing a fall in the optical density of the sample. Secretion of Cl– was measured as short-circuit current in a modified Ussing chamber. Activation of Cl– secretion by stimulation of cAMP- or Ca2+-dependent pathways caused comparable increases in lysozyme secretion. Similarly, blockers of Cl– secretion, such as diphenylamine-2-carboxylate (DPC), also reduced lysozyme secretion. However, while treatment of airway submucosal gland cells with antisense oligonucleotides directed against CFTR reduced Cl– secretion, it had no significant effect on the total amount of lysozyme secretion. These results suggest a role for functional CFTR in regulation of lysozyme secretion in human airways. [ABSTRACT FROM AUTHOR]

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) quickly spread worldwide following its emergence in Wuhan, China, and hit pandemic levels. Its tremendous incidence favoured the emergence of viral variants. The current genome diversity of SARS-CoV-2 has a clear impact on epidemiology and clinical practice, especially regarding transmission rates and the effectiveness of vaccines. In this study, we evaluated the replication of different SARS-CoV-2 isolates representing different virus genotypes which have been isolated throughout the pandemic. We used three distinct cell lines, including Vero E6 cells originating from monkeys; Caco-2 cells, an intestinal epithelium cell line originating from humans; and Calu-3 cells, a pulmonary epithelium cell line also originating from humans. We used RT-qPCR to replicate different SARS-CoV-2 genotypes by quantifying the virus released in the culture supernatant of infected cells. We found that the different viral isolates replicate similarly in Caco-2 cells, but show very different replicative capacities in Calu-3 cells. This was especially highlighted for the lineages B.1.1.7, B.1.351 and P.1, which are considered to be variants of concern. These results underscore the importance of the evaluation and characterisation of each SARS-CoV-2 isolate in order to establish the replication patterns before performing tests, and of the consideration of the ideal SARS-CoV-2 genotype–cell type pair for each assay. [ABSTRACT FROM AUTHOR]

Purpose. Nasal delivery of protein therapeutics can be compromised by the brief residence time at this mucosal surface. Some bioadhesive polymers have been suggested to extend residence time and improve protein uptake across the nasal mucosa. We examined several potential polymer platforms for their in vitro protein release, relative bioadhesive properties and induction of cytokine release from respiratory epithelium. Methods. Starch, alginate, chitosan or Carbopol® microparticles, containing the test protein bovine serum albumin (BSA), were prepared by spray-drying and characterized by laser diffraction and scanning electron microscopy. An open-membrane system was used to determine protein release profiles and confluent, polarized Calu-3 cell sheets were used to evaluate relative bioadhesion, enhancement of protein transport and induction of cytokine release in vitro. Results. All spray-dried microparticles averaged 2−4 μm in diameter. Loaded BSA was not covalently aggregated or degraded. Starch and alginate microparticles released protein more rapidly but were less adhesive to polarized Calu-3 cells than chitosan and Carbopol® microparticles. Protein transport across polarized Calu-3 cells was enhanced from Carbopol® gels and chitosan microparticles. A mixture of chitosan microparticles with lysophosphatidylcholine increased protein transport further. Microparticles prepared from either chitosan or starch microparticles, applied apically, induced the basolateral release of IL-6 and IL-8 from polarized Calu-3 cells. Release of other cytokines, such as IL-lβ, TNF-α, GM-CSF and TGF-β, were not affected by an apical exposure to polymer formulations. Conclusions. We have described two systems for the in vitro assessment of potential nasal platforms for protein delivery. Based upon these assessments, Carbopol® gels and chitosan microparticles provided the most desirable characteristics for protein therapeutic and protein antigen delivery, respectively, of the formulations examined. [ABSTRACT FROM AUTHOR]

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS-CoV-2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial–endothelial cell culture model based on Calu-3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu-3 cells, cultured for two weeks at an air–liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO-1). LMVECs cultured submerged for three weeks were CD31-positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu-3 cell cultures and the co-cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays. Importantly, the Calu-3/LMVEC co-cultures showed better cell viability and barrier function than mono-cultures. Moreover, there was no evidence for epithelial- and endothelial-to-mesenchymal transition (EMT and EndoMT, respectively) based on staining for the mesenchymal markers vimentin and α-SMA, respectively. These results indicate the potential of this new airway epithelial–endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial–endothelial and alveolar–endothelial models with primary lung epithelial cells. [ABSTRACT FROM AUTHOR]

Remdesivir (RDV; GS-5734) is currently the only FDA-approved antiviral drug for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The drug is approved for use in adults or children 12 years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2-infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed, and several, including molnupiravir and PF-07321332, are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of remdesivir nucleoside (RVn; GS-441524) that are processed to RVn monophosphate, the precursor of the active RVn triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma, and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types, including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells, and Huh7.5 cells. In Syrian hamsters, oral treatment with 1- O -octadecyl-2- O -benzyl-glycero-3-phosphate RVn (ODBG-P-RVn) was well tolerated and achieved therapeutic levels in plasma above the 90% effective concentration (EC 90 ) for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.

The development of inhaled drug products is expensive and involves time-consuming pharmacokinetic (PK) and pharmacodynamic (PD) studies. There are few in vitro cell-based assays to evaluate the disposition and action of orally inhaled drugs to guide early product development and minimise risk. The aim of the present study was to develop a co-culture bioassay, combining an airway epithelial cell line (Calu-3) with cultured human primary airway smooth muscle cells (ASM), integrated with apparatus to deliver pharmaceutical aerosols. An assay for measuring cyclic adenosine monophosphate (cAMP) in ASM derived from healthy donors was adapted to provide a biochemical surrogate for ASM relaxation. Concentration-response curves for cAMP were established for three drugs that elicit ASM relaxation: isoprenaline (ISO), forskolin (FOR) and salbutamol sulphate. The ASM bioassay was incorporated into a co-culture model in which air-interfaced Calu-3 cell layers, representing the permeability barrier of the airway epithelium, were grown on transwell inserts above ASM cells cultured in the well of the base-plate. The sensitivity of this bioassay to salbutamol delivered using different formulations and aerosol products was evaluated. ASM responded with concentration dependent increases in cAMP when exposed to 10-9 to 10-5 M ISO, FOR or salbutamol sulphate solutions for 15 or 30 min. Salbutamol formulated with different counter ions elicited differential cAMP responses in ASM (xinafoate > base = sulphate) suggesting that this bioassay could discriminate between formulations with different potency. A similar rank order of potency was observed for the different salbutamol salts when applied as aerosols to the co-culture model. We have developed a novel bioassay using human ASM in co-culture with human respiratory epithelial cells to better mimic various elements that contribute to the rate and extent of local drug availability in the lungs following topical administration. The bioassay offers an opportunity to investigate the factors determining the activity of inhaled bronchodilator drugs in a more biologically relevant system than that has previously been described and with further development and validation, this novel bioassay could provide a method to guide the more efficient development of inhaled bronchodilators, reducing the current reliance on in vivo studies. [ABSTRACT FROM AUTHOR]

Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within biofilm communities in vivo. Biofilm studies so far are mainly based on assays on plastic surfaces. The aim of this work was to investigate the capacity of clinical NTHi strains to form biofilm structures on polarized Calu-3 human airway epithelial cells and primary normal human bronchial epithelial cells and to characterize the biofilm architecture. Formation of adherent NTHi biofilms post colonization of host cells at multiple time-points was evaluated using confocal laser scanning microscopy and electron microscopy. NTHi biofilms were analyzed in terms of biofilm height and presence of extracellular matrix components, and their apoptotic effects on epithelial cells were measured by TUNEL assay. Strain Fi176 was observed to form robust biofilms on airway epithelia over time, while disrupting the integrity of Calu-3 monolayer by 72 h of co-culture. NTHi biofilms were observed to induce apoptotic DNA fragmentation in host cells at 24 h post infection. Biofilm formation on cell monolayers by Fi176ΔpilA strain was markedly reduced compared to WT strain. Biofilm inhibition and disruption assays by crystal violet staining indicated that DNA and proteins are part of NTHi biofilms in vitro. Our findings highlight critical stages of NTHi pathogenesis following host colonization and provide useful biofilm models for future antimicrobial drug discovery investigations. • NTHi forms adherent biofilms on Calu-3 and differentiated primary NHBE airway cells over time. • PilA plays a role in NTHi biofilm formation. • NTHi biofilm matrix consists of DNA, proteins and oligosaccharide structures with terminal N-acetylgalactosamine. • NTHi biofilms induce DNA fragmentation and loss of architectural integrity in host cells. • Physiologically-relevant biofilm models are proposed for future antimicrobial drug discovery research. [ABSTRACT FROM AUTHOR]