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Treating adverse risk myelodysplastic syndromes with azacitidine exacerbates thrombocytopenia. We report a study of eltrombopag in combination with azacitidine using a 3 + 3 cohort design. Patients with baseline platelets of

Archive contatins all the data acquired with the respect to the paper mentioned in the title. It is split in three folders by data type: "Fabrication" folder contains all of the details related to the deposition, fabrication and testing of FeAl hall bars. "Measurements" folder contains all experimental results. They are grouped by type (as subfolders) and then again by sample codes. Typically structure of the measurement subfolders are the following: "Raw_data" - contains data in a form as it was acquired from the machine or very close to that. "Cleaned_data" - contains processed raw_data according to the need for the specific plot. For example removal of extra columns, normalizations, unit conversions, fittings etc. Specific operations performed depend on the measurement type and described in "README.md" files. "Plotting" - contatins Veusz (Free and Open source plotting software, https://veusz.github.io/) project files linked to the "Cleaned_data". Beware that moving the data on disk relative to the Veusz project file would destroy the links and the data files will have to be relinked. In order to avoid this created a copy of Veusz project and unlink all the datasets. This will embed the data within a project file and will make it independent from "Cleaned_data" file "Modeling" contains results of auxilary calculations and numerical modeling.

The variation of transport behaviour in a mesoscopic Fe60Al40 wire, initially possessing the ordered B2-phase structure, has been observed while inducing a phase transition to the disordered A2 structure. Gradual disordering was achieved using a highly focused beam of Ne+-ions. Both electrical resistance and anomalous Hall effect were measured in parallel with the local ion irradiation. Both the normal and Hall resistivity show a peak as a function of fluence. Moreover, the relationship between Hall resistivity and normal resistivity reconfirms the presence of two distinct regimes in the transition. Furthermore, field-dependence and temperature-dependence measurements were used to identify that it is necessary to consider the effect of scattering from magnetic clusters to understand these different regimes in transport properties.

MAX phases are nano-lamellar composite materials of the form Mn+1AXn, where n is 1, 2 or 3; M an early transition metal; A is an A-group element and X is carbon or nitrogen [1,2]. An interesting combination of metallic and ceramic properties as well as potential applications in spintronics [1,3] led to significant research interest in MAX phases. Literature on the effect of systematic disordering of the nano-laminar structure on the magnetic and transport properties is still limited. In particular, MAX phase systems doped with magnetic ions via ion-irradiation may result in large variations of the magneto-transport properties. Here we observe the magneto-transport properties and attempt to separate the contributions of structural changes due to the irradiation and magnetic effects due to the doping on the magneto-transport. A prototype material is Cr2AlC, formed from a unit cell of Cr2C sandwiched between atomic planes of Al. In this work, we study 50 nm and 500 nm thick thin films of Cr2AlC grown on Si (111) by sputtering and subsequent annealing. Structural characterization using X-ray Diffraction in Bragg-Brentano geometry shows a pronounced MAX phase, confirmed by the occurrence of the 002 superlattice reflection. The films were irradiated with Co+ at 450 (50) keV for the 500 (50) nm thick films. The Co+ fluence varied between 1×10^12 - 1×10^15 ions.cm^-2, in full order steps. The Co+ irradiation led to a gradual suppression of the 0002 superstructure reflection, while preserving the fundamental peaks, implying the intermixing of the nano-laminar MAX phase structure. The magnetic properties are characterized using vibrating sample magnetometry at low temperatures, showing an increasing paramagnetic behavior as the Co+-fluence increases. In comparison, magneto-resistance measurements show that for the 500 nm film thickness, the magnetoresistance reaches up to 3 % (10 T) for 100 K, at an optimized Co+-fluence of 5×10^13 ions.cm^-2. The above results suggest that in the low-fluence regime, the irradiation-induced disorder remains sufficiently low to obtain pronounced magneto-resistance values. Understanding the defect state in the optimized MAX phase films will shed light on the magneto-transport mechanisms in these nano-laminated materials.

MAX phases are nano-lamellar composite materials of the form Mn+1AXn, where n is 1, 2 or 3; M an early transition metal; A is an A-group element and X is carbon or nitrogen[1,2]. An interesting combination of metallic and ceramic properties as well as potential applications in spintronics [1,3] led to significant research interest in MAX phases. Literature on the effect of systematic disordering of the nano-laminar structure on the magnetic and transport properties is still limited. In particular, MAX phase systems doped with magnetic ions via ion-irradiation may result in large variations of the magneto-transport properties.Here we observe the magneto-transport properties and attempt to separate the contributions of structural changes due to the irradiation and magnetic effects due to the doping on the magneto-transport. A prototype material is Cr2AlC, formed from a unit cell of Cr2C sandwiched between atomic planes of Al. In this work, we study 50 nm and 500 nm thick thin films of Cr2AlC grown on Si(111) by sputtering and subsequent annealing.Structural characterization using X-ray Diffraction in Bragg-Brentano geometry shows a pronounced MAX phase, confirmed by the occurrence of the 002 superstructure reflection. The films were irradiated with Co+ at 450 (50) keV for the 500 (50) nm thick films. The Co+ fluence varied between 1E12-1E15 ions.cm-2, in full order steps. The Co+ irradiation led to a gradual suppression of the 002 superstructure reflection, while preserving the fundamental peaks, implying the intermixing of the nano-laminar MAX phase structure. The magnetic properties are characterized using vibrating sample magnetometry at low temperatures (Fig.1a), showing an increasing paramagnetic behavior as the Co+ fluence increases. In comparison, magneto-resistance measurements (Fig.1b-c) show that for the 500 nm film thickness, the magnetoresistance reaches up to 3 % (10 T) for 100 K, at an optimized Co+ fluence of 5E13 ions.cm-2. The above results suggest that in the low-fluence regime, the irradiation induced disorder remains sufficiently low to obtain pronounced magneto-resistance values.Understanding the defect state in the optimized MAX phase films will shed light on the magneto-transport mechanisms in these nano-laminated materials.

Point 1: The study of animals in nature is essential for developing an ecologically valid understanding of behavior. Small mammals, however, are often fossorial and exceedingly difficult to monitor in the wild. This limits both the taxonomic scope of field observation, and excludes species that are powerful models for the study of behavioral mechanisms.Point 2: Here, we implement an automated radio telemetry system (ARTS) designed to track small fossorial mammals. Our ARTS uses an isotropic antenna array coupled with broadband receivers. We characterized transmission at our study site and tested the ARTS’ ability to track 48 prairie voles.Point 3: We compared position estimates from nonlinear least squares, nonparameteric, and Bayesian trilateration methods and found Bayesian trilateration to have the smallest error. To examine the ability of the system to track biologically significant behavior we used ARTS data to investigate circadian rhythms of freely behaving prairie voles. We used Lomb-Scargle analysis to estimate periodic patterns from irregularly sampled time series of speed. Prairie voles demonstrated ultradian movement at periods of approximately 45 and 90 min, observations on a time scale not possible using data from traditional methods.Point 4: This ARTS offers a new tool to observe rodent field behavior at time scales and in environments which have not been previously possible, such as investigating social and spatial behaviors on the scale of minutes, hours, and days in natural environments.

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks.

Nano-lamellar composite materials, known as MAX-phases, can possess a combination of ceramic and metallic properties. A prototype compound is Cr2AlC, formed from a unit cell of Cr2C sandwiched between atomic planes of Al, thereby imparting a good electrical conductivity, as well as mechanical stability, radiation and oxidation resistance [1, 2]. These properties rely on the lamellar structure of the compound, and systematic introduction of defects, such as displacing or doping atoms within the layers, has the potential to tune electron transport and modify magnetic properties [3]. An ideal tool for defect implantation is ion-irradiation, available both in the form of a broad-beam for wafer-scale processing as well as focused ion-beams for device prototyping. Here we observe the modifications to the structural, transport and magnetic behavior of 500 nm thick Cr2AlC after irradiation with Co+ ions, and Ar+ noble gas ions as control. The films were irradiated with 450 keV of Co+ ions at fluences varying from 5E12 to 5E15 ions.cm-2, and the control samples with 400 keV Ar+ ions keeping the sample fluences. Structural analysis using XRD shows that ion-irradiation induces a suppression of the 0002 reflection, indicating a gradual decay of the nano-lamellar structure. Increasing ion-fluence also leads to an increase of the saturation magnetization at 1.5 K, whereby both Ar+ and Co+ cause an increased magnetization, respectively to 150 and 190 kA.m-1, for the highest fluences used. Large variations of the transport properties are observed. Magnetoresistance (MR) in the non-irradiated sample shows a classical B2 dependency, even up to high temperatures. At Co+ fluences of 5E13 ions.cm-2 the MR shows a two orders of magnitude increase, up to 3% (10 T) at 100 K. A similar effect also occurs for 5E12 ions.cm-2 Ar+ irradiated films, however with a smaller MR-increase. It appears that resistivity increases and the residual resistance ratio reduces with increasing fluence due to the introduction of disorder. These results show that ion irradiation induces significant changes in the transport properties of MAX phase materials, that will be further investigated. The systematic disordering of nano-laminated MAX phase films may therefore reveal interesting disorder and spin-related transport phenomena. Funding by the Deutsche Forschungsgemeinschaft (DFG) - Project number 456078299 is acknowledged. Ion-irradiation has been performed at the Ion Beam Centre of the HZDR. [1] A. S. Ingason, M. Dahlqvist, J. Rosen, Magnetic MAX phases from theory and experiments; a review; J. Phys.: Condens. Matter 28, 433003, (2016). [2] M. W. Barsoum, MAX Phases: Properties of Machinable Ternary Carbides and Nitrides; Weinheim: Willey-VCH (2013). [3] C. Wang, T. Yang, C. L. Tracy, C. Lu, H. Zhang, Y.-J. Hu, L. Wang, L. Qi, L. Gu, Q. Huang, J.Zhang, J. Wang, J. Xue, R. C. Ewing, Y. Wang, Disorder in Mn+1AXn phases at the atomic scale, Nature Communications 10, 622 (2019).

The atomic arrangement in certain B2-ordered alloys, such as Fe60Al40, determines intrinsic material properties like magnetism. Here we have investigated the influence of open-volume defects on the atomic ordering process at elevated temperatures in Fe60Al40 thin films. A dependence of the ordering process on the type and concentration of defects is observed by positron annihilation spectroscopy combined with ab-initio calculations. Comparing the lifetimes of positrons in the alloy for different annealing and irradiation treatments reveals the role of mono-vacancies, triple defects as well as large vacancy clusters: The rate of atomic ordering to the ordered B2 state is increased in the presence of mono-vacancies whereas triple defects and vacancy complexes decrease the ordering rate. Furthermore, an agglomeration of vacancies during annealing to di-vacancies and larger vacancy clusters is observed. The distribution of open-volume defects can be modified in such a way as to control the thermal stability via ion-irradiation and thermal pre-treatments.

Phenotypic similarities between the pulmonary manifestations of the rare conditions cystic fibrosis (CF) and alpha-1 antitrypsin deficiency (AATD) on the one hand and common chronic obstructive pulmonary disease (COPD) on the other have long been recognized.

A focus point in this work was the study of the influence of a low substrate temperature, as well as the minor addition of Y (0.1-0.3 at.%), on the formation of the stable Cr2AlC-MAX (ternary alloy with general formula Mn + 1AXn: M = early transition metal, A = A-Group element, mostly IIIA or IVA, X = C or N, n = 1-3) phase. The coatings, deposited by High Power Impulse Magnetron Sputtering, consisted of a mixture of disordered solid solution (Cr,Al)2Cx and ordered Cr2AlC-MAX phase. All deposited coatings without and with 0.1-0.3 at.% Y addition were polycrystalline, and showed (110) texture and a columnar morphology. The measured strong lattice distortions along with the existence of the texture in the as-deposited samples indicate that compressive stress acts in the a-b plane and tensile perpendicular to this. A schematic model of the structural and chemical changes in the as-deposited layers due to deposition inhomogeneity and low deposition temperature, based on the X-ray diffraction, energy dispersive X-ray, scanning electron microscopy and magnetic measurements has been developed.

Cystic Fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). Newly developed “correctors” such as lumacaftor (VX-809) that improve CFTR maturation and trafficking and “potentiators” such as ivacaftor (VX-770) that enhance channel activity may provide important advances in CF therapy. Although VX-770 has demonstrated substantial clinical efficacy in the small subset of patients with a mutation (G551D) that affects only channel activity, a single compound is not sufficient to treat patients with the more common CFTR mutation, ΔF508. Thus, patients with ΔF508 will likely require treatment with both correctors and potentiators to achieve clinical benefit. However, whereas the effectiveness of acute treatment with this drug combination has been demonstrated in vitro, the impact of chronic therapy has not been established. In studies of human primary airway epithelial cells, we found that both acute and chronic treatment with VX-770 improved CFTR function in cells with the G551D mutation, consistent with clinical studies. In contrast, chronic VX-770 administration caused a dose-dependent reversal of VX-809-mediated CFTR correction in ΔF508 homozygous cultures. This result reflected the destabilization of corrected ΔF508 CFTR by VX-770, dramatically increasing its turnover rate. Chronic VX-770 treatment also reduced mature wild-type CFTR levels and function. These findings demonstrate that chronic treatment with CFTR potentiators and correctors may have unexpected effects that cannot be predicted from short-term studies. Combining of these drugs to maximize rescue of ΔF508 CFTR may require changes in dosing and/or development of new potentiator compounds that do not interfere with CFTR stability.

We report on the influence of chemical disorder in Fe60Al40 thin films on their ferromagnetic resonance. Chemical disorder leads to increased nearest neighbour Fe-Fe magnetic interactions and plays a crucial role in inducing ferromagnetism. The saturation magnetization increases from 20 kA/m-1 for the chemically ordered film to 780 kA/m for disordered films. Disorder was induced by irradiation of Ne+ ions, and the depth-distribution of disorder was controlled by adjusting the ion-energy and -fluence. For moments aligned within the film plane, the resonant linewidth decreases with increasing ion-energy in the range from 2.5 to 30 keV, for a fixed ion-fluence. In-plane magnetic anisotropy is negligible in all cases. The linewidths for in-plane moment alignment are much larger than in materials that do not exhibit disorder induced ferromagnetism. These results may be explained by enhanced two-magnon scattering due to the presence of random defects, and help in preparing thin films with tailored spin-wave dynamic properties.

We show that sub-50 nm ion-induced lateral patterning of magnetic structures can be enabled by disorder induced ferromagnetism.[1] Disorder is induced through exposure of the chemically ordered alloy to energetic ions; collision cascades formed by the ions knock atoms from their ordered sites and the concomitant vacancies are filled randomly via thermal diffusion of atoms at room temperature. Here we consider the case of Fe60Al40 wherein the chemically ordered B2 structure is paramagnetic, and chemical disordering leads to the formation of the A2 structure which is ferromagnetic.[2] This ion-induced transition can be exploited to induce ferromagnetism in localized regions by ion-irradiation through lithographed shadow masks. We show that this technique may be useful for fabricating novel spin-transport devices. First we demonstrate the disorder-induced increase in saturation magnetization, Ms, in continuous films. Fe60Al40 films of 40 nm thickness were deposited on SiO2(150 nm)/Si(001) substrates by magnetron sputtering. The films were annealed at 773 K in vacuum to form the chemically ordered B2 phase structure. Hysteresis loops were measured using a vibrating sample magnetometer (VSM). Figure 1a shows that the B2 film is weakly ferromagnetic with a saturation magnetization of Ms = 20 kA m-1. The chemically ordered films were irradiated with 6 x 1014 ions cm-2 of Ne+-ions at 10 and 30 keV respectively. After irradiation, the Ms increases to 480 and 780 kA m-1, respectively for the 10 and 30 keV samples. The Ms of the 30 keV Ne+ sample is a factor of 40 larger than that of the annealed sample. Figure 1b shows X-ray Diffraction measurements around the 100 reflection for the sample after annealing, and after subsequent 10 keV Ne+-irradiation. The 100 reflection is allowed for the B2 superstructure but vanishes for the disordered A2 phase and this transition is clearly observed since the reflection initially present in the B2 film vanishes for the 10 keV sample. Magnetic patterning was performed on a 10 μm wide and 400 μm long wire of chemically ordered Fe60Al40 (Figure 2a). The wire was covered with a 150 nm thick resist layer, and patterned using e-beam lithography. Simulations based on the binary collision approximation (TRIM) showed that the 150 nm thick resist layer is sufficient to block impinging 10 keV Ne+-ions.[3] Lithography was used to carve out stripe like openings of 0.5 and 2 μm widths respectively. As shown in the micrograph in Figure 2b, the stripe-openings were separated by ~ 40 nm wide (and 150 nm high) resist walls, thereby stopping the impinging Ne+ ions reaching the areas directly underneath the resist. These areas can be expected to retain chemical order after exposure to Ne+, however, only if the lateral scattering of ions is restricted. The wire covered by the shadow-mask was exposed to 10 keV Ne+-ions at a fluence of 6 x 1014 ion cm-2. Figure 2c shows the magnetic contrast image, obtained using Kerr Microscopy on the sample prior to application of saturating magnetic fields. Striped magnetic regions are clearly observed possessing random magnetic orientations. Magnetic contrast was captured whilst sweeping the field to obtain hysteresis loops shown in Figure 2d, on a set of 32 (0.5 μm/spacer/2 μm/spacer) stripe-pairs. Magnetization reversal occurs via a two-staged process; the first reversal step occurs at ≈ ±3 mT and the second step at ≈ ±7 mT. The magneto-optic intensity changes by 80% in the first reversal step, indicating that the 2 μm wide stripes reverse collectively at the smaller field. In the stripe geometry, the internal demagnetizing field increases with the stripe width. Reversal of the 2 μm wide stripes therefore occurs at smaller externally applied field as compared with the 0.5 μm stripes, resulting in selective reversal. Magnetic contrast images were captured at remnant points of the above hysteresis loops and are shown in Figure 2e – h. Stripes with magnetization pointing towards the left or right appear as dark and bright contrast, respectively. Images captured after applying the saturating field of +/-18 mT followed by reduction to zero field show that the parallel magnetization configuration is preserved in remanence (Figure 2e and f). The antiparallel configuration is obtained after applying a reverse field of -/+5 mT to the saturated stripes and returning to zero field. Figure 2g and h show alternating light and dark contrast of the antiparallel state in remnant fields. Spin-resolved Photo-Emission Electron Microscopy (SPEEM) measurements were performed to observe magnetic contrast with high spatial resolution of ~ 50 nm. The SPEEM micrographs are shown in Figure 2e and g as magnified equivalent regions of the Kerr-images. The color scale varies from red to blue for magnetic moments pointing left or right respectively. Straight, low-contrast regions are found to separate the 500 nm and 2 μm wide high-contrast regions that correspond to the magnetic stripes. The straight low-contrast regions are unresponsive to the magnetic field and occur for both parallel and anti-parallel states. Since the straight low-contrast lines also exactly follow the pattern of the shadow mask, it can be concluded that these lines correspond to the 40 nm wide un-irradiated regions. Neighboring 500 nm and 2 μm magnetic stripes are therefore isolated by a continuous weakly magnetic spacer of 40 nm nominal width. Thus, parallel and antiparallel magnetization configurations can be programmed using the magnetic field history and are non-volatile i.e., stable when the field is switched off. Selective reversal and the existence of binary magnetic states namely is a prerequisite for spin-valves and advantageous in devices for storing data bits. In particular with respect to spin-transport devices, it is also necessary to ensure that the magnetic regions are separated by narrow spacers of zero or low magnetization. Showing that discrete magnetic nanostructures can be prepared by ion-irradiation has important consequences not previously considered in literature, such as the possibility of laterally patterned spin-transport devices – our results are a step in this direction. [1] R. Bali et al., Nano Letters 2013 (accepted). [2] J. Fassbender, et al., Phys. Rev. B 2008, 77, 174430. [3] J. F. Ziegler et al., Nucl. Instrum. Methods B 2010, 268, 11-12, 1818.

The clearance of mucus from the airways protects the lungs from inhaled noxious and infectious materials. Proper hydration of the mucus layer enables efficient mucus clearance through beating of cilia on airway epithelial cells, and reduced clearance of excessively concentrated mucus occurs in patients with chronic obstructive pulmonary disease and cystic fibrosis. Key steps in the mucus transport process are airway epithelia sensing and responding to changes in mucus hydration. We reported that extracellular adenosine triphosphate (ATP) and adenosine were important luminal auto-crine and paracrine signals that regulated the hydration of the surface of human airway epithelial cultures through their action on apical membrane purinoceptors. Mucus hydration in human airway epithelial cultures was sensed by an interaction between cilia and the overlying mucus layer: Changes in mechanical strain, proportional to mucus hydration, regulated ATP release rates, adjusting fluid secretion to optimize mucus layer hydration. This system provided a feedback mechanism by which airways maintained mucus hydration in an optimum range for cilia propulsion. Understanding how airway epithelia can sense and respond to changes in mucus properties helps us to understand how the mucus clearance system protects the airways in health and how it fails in lung diseases such as cystic fibrosis.

MUC5AC, a major gel-forming mucin expressed in the lungs, is secreted at increased rates in response to infectious agents, implying that mucins exert a protective role against inhaled pathogens. However, epidemiological and pathological studies suggest that excessive mucin secretion causes airways obstruction and inflammation. To determine whether increased MUC5AC secretion alone produces airway obstruction and/or inflammation, we generated a mouse model overexpressing Muc5ac mRNA ∼20-fold in the lungs, using the rCCSP promoter. The Muc5ac cDNA was cloned from mouse lungs and tagged internally with GFP. Bronchoalveolar lavage fluid (BALF) analysis demonstrated an approximate 18-fold increase in Muc5ac protein, which formed high-molecular-weight polymers. Histopathological studies and cell counts revealed no airway mucus obstruction or inflammation in the lungs of Muc5ac-transgenic (Muc5ac-Tg) mice. Mucus clearance was preserved, implying that the excess Muc5ac secretion produced an “expanded” rather than more concentrated mucus layer, a prediction confirmed by electron microscopy. To test whether the larger mucus barrier conferred increased protection against pathogens, Muc5ac-Tg animals were challenged with PR8/H1N1 influenza viruses and showed significant decreases in infection and neutrophilic responses. Plaque assay experiments demonstrated that Muc5ac-Tg BALF and purified Muc5ac reduced infection, likely via binding to α2,3-linked sialic acids, consistent with influenza protection in vivo. In conclusion, the normal mucus transport and absence of a pulmonary phenotype in Muc5ac-Tg mice suggests that mucin hypersecretion alone is not sufficient to trigger luminal mucus plugging or airways inflammation/goblet cell hyperplasia. In contrast, increased Muc5ac secretion appears to exhibit a protective role against influenza infection.

The infiltration of dissolved dies into vertically aligned carbon nanotube arrays (VA-CNT) is reported. The ultra hydrophobic surface of a CNT array can be completely infiltrated on a surface area of 48 mm2 by an appropriate choice of solvent. This is so far not possible by evaporation techniques. Depending on the solvent type, the CNT alignment remains unaltered, which is strongly modified, or even lost. The infiltrated CNT array provides a well ordered electrode structure for organic solar cells. The CNT can simultaneously serve as electron acceptor in combination with an appropriate donor dye. Sensors and organic LED are among further applications.

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-27632, conditionally induces an indefinite proliferative state in primary mammalian epithelial cells. These conditionally reprogrammed cells (CRCs) are karyotype-stable and nontumorigenic. Because self-renewal is a recognized property of stem cells, we investigated whether Y-27632 and feeder cells induced a stem-like phenotype. We found that CRCs share characteristics of adult stem cells and exhibit up-regulated expression of α6 and β1 integrins, ΔNp63α, CD44, and telomerase reverse transcriptase, as well as decreased Notch signaling and an increased level of nuclear β-catenin. The induction of CRCs is rapid (occurs within 2 d) and results from reprogramming of the entire cell population rather than the selection of a minor subpopulation. CRCs do not overexpress the transcription factor sets characteristic of embryonic or induced pluripotent stem cells (e.g., Sox2, Oct4, Nanog, or Klf4). The induction of CRCs is also reversible, and removal of Y-27632 and feeders allows the cells to differentiate normally. Thus, when CRCs from ectocervical epithelium or tracheal epithelium are placed in an air–liquid interface culture system, the cervical cells form a well differentiated stratified squamous epithelium, whereas the tracheal cells form a ciliated airway epithelium. We discuss the diagnostic and therapeutic opportunities afforded by a method that can generate adult stem-like cells in vitro without genetic manipulation.

Mucus clearance is the primary defense mechanism that protects airways from inhaled infectious and toxic agents. In the current Gel-on-Liquid mucus clearance model mucus gel is propelled on top of a “watery” periciliary layer surrounding the cilia. However, this model fails to explain the formation of distinct mucus layer in health or why mucus clearance fails in disease. We propose a Gel-on-Brush model in which the periciliary layer is occupied by membrane spanning mucins and mucopolysaccharides densely tethered to the airway surface. This brush prevents mucus penetration into the periciliary space and causes mucus to form a distinct layer. The relative osmotic moduli of the mucus and periciliary brush layers explain both the stability of mucus clearance in health and its failure in airway disease.

The aim of this study was to determine whether geographical differences impact the composition of bacterial communities present in the airways of cystic fibrosis (CF) patients attending CF centers in the United States or United Kingdom. Thirty-eight patients were matched on the basis of clinical parameters into 19 pairs comprised of one U.S. and one United Kingdom patient. Analysis was performed to determine what, if any, bacterial correlates could be identified. Two culture-independent strategies were used: terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA clone sequencing. Overall, 73 different terminal restriction fragment lengths were detected, ranging from 2 to 10 for U.S. and 2 to 15 for United Kingdom patients. The statistical analysis of T-RFLP data indicated that patient pairing was successful and revealed substantial transatlantic similarities in the bacterial communities. A small number of bands was present in the vast majority of patients in both locations, indicating that these are species common to the CF lung. Clone sequence analysis also revealed that a number of species not traditionally associated with the CF lung were present in both sample groups. The species number per sample was similar, but differences in species presence were observed between sample groups. Cluster analysis revealed geographical differences in bacterial presence and relative species abundance. Overall, the U.S. samples showed tighter clustering with each other compared to that of United Kingdom samples, which may reflect the lower diversity detected in the U.S. sample group. The impact of cross-infection and biogeography is considered, and the implications for treating CF lung infections also are discussed.

Rationale: Pulmonary infection in cystic fibrosis (CF) is polymicrobial and it is possible that anaerobic bacteria, not detected by routine aerobic culture methods, reside within infected anaerobic airway mucus. Objectives: To determine whether anaerobic bacteria are present in the sputum of patients with CF. Methods: Sputumsamples were collected from clinically stable adults with CF and bronchoalveolar lavage fluid (BALF) samples from children with CF. Induced sputum samples were collected from healthy volunteers who did not have CF. All samples were processed using anaerobic bacteriologic techniques and bacteria within the samples were quantified and identified. Measurements and Main Results: Anaerobic species primarily within the genera Prevotella, Veillonella, Propionibacterium, and Actinomyces were isolated in high numbers from 42 of 66 (64%) sputum samples from adult patients with CF. Colonization with Pseudomonas aeruginosa significantly increased the likelihood that anaerobic bacteria would be present in the sputum. Similar anaerobic species were identified in BALF from pediatric patients with CF. Although anaerobes were detected in induced sputum samples from 16 of 20 volunteers, they were present in much lower numbers and were generally different species compared with those detected in CF sputum. Species-dependent differences in the susceptibility of the anaerobes to antibiotics with knownactivity against anaerobes were apparent with all isolates susceptible to meropenem. Conclusions: A range of anaerobic species are present in large numbers in the lungs of patients with CF. If these anaerobic bacteria are contributing significantly to infectionandinflammationin the CF lung, informed alterations to antibiotic treatment to target anaerobes, in addition to the primary infecting pathogens, may improve management.

A vexing problem in cystic fibrosis (CF) pathogenesis has been to explain the high prevalence of Pseudomonas aeruginosa biofilms in CF airways. We speculated that airway surface liquid (ASL) hyperabsorption generates a concentrated airway mucus that interacts with P. aeruginosa to promote biofilms. To model CF vs. normal airway infections, normal (2.5% solids) and CF-like concentrated (8% solids) mucus were prepared, placed in flat chambers, and infected with an ≈5 × 103 strain PAO1 P. aeruginosa. Although bacteria grew to 1010 cfu/ml in both mucus concentrations, macrocolony formation was detected only in the CF-like (8% solids) mucus. Biophysical and functional measurements revealed that concentrated mucus exhibited properties that restrict bacterial motility and small molecule diffusion, resulting in high local bacterial densities with high autoinducer concentrations. These properties also rendered secondary forms of antimicrobial defense, e.g., lactoferrin, ineffective in preventing biofilm formation in a CF-like mucus environment. These data link airway surface liquid hyperabsorption to the high incidence of P. aeruginosa biofilms in CF via changes in the hydration-dependent physical–chemical properties of mucus and suggest that the thickened mucus gel model will be useful to develop therapies of P. aeruginosa biofilms in CF airways.

Inefficient adenoviral vector (AdV)-mediated gene transfer to the ciliated respiratory epithelium has hindered gene transfer strategies for the treatment of cystic fibrosis lung disease. In part, the inefficiency is due to an absence of the coxsackie B and adenovirus type 2 and 5 receptor (CAR) from the apical membranes of polarized epithelia. In this study, using an in vitro model of human ciliated airway epithelium, we show that providing a glycosylphosphatidylinositol (GPI)-linked AdV receptor (GPI-CAR) at the apical surface did not significantly improve AdV gene transfer efficiency because the lumenal surface glycocalyx limited the access of AdV to apical GPI-CAR. The highly glycosylated tethered mucins were considered to be significant glycocalyx components that restricted AdV access because proteolytic digestion and inhibitors of O-linked glycosylation enhanced AdV gene transfer. To determine whether these in vitro observations are relevant to the in vivo situation, we generated transgenic mice expressing GPI-CAR at the surface of the airway epithelium, crossbred these mice with mice that were genetically devoid of tethered mucin type 1 (Muc1), and tested the efficiency of gene transfer to murine airways expressing apical GPI-human CAR (GPI-hCAR) in the presence and absence of Muc1. We determined that AdV gene transfer to the murine airway epithelium was inefficient even in GPI-hCAR transgenic mice but that the gene transfer efficiency improved in the absence of Muc1. However, the inability to achieve a high gene transfer efficiency, even in mice with a deletion of Muc1, suggested that other glycocalyx components, possibly other tethered mucin types, also provide a significant barrier to AdV interacting with the airway lumenal surface.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basal conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H+,K+-ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent secretory pathway that was defective in CF: (i) ASL [] was higher in NL than CF ASL; (ii) activating CFTR with forskolin/3-isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii) NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pHASL is abnormally regulated under basal conditions because of absent CFTR-dependent secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL.

The polarized distribution of HCO3− transport was investigated in human nasal epithelial cells from normal and cystic fibrosis (CF) tissues. To test for HCO3− transport via conductive versus electroneutral Cl−/HCO3− exchange (anion exchange, AE) pathways, nasal cells were loaded with the pH probe 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein and mounted in a bilateral perfusion chamber. In normal, but not CF, epithelia, replacing mucosal Cl− with gluconate caused intracellular pH (pHi) to increase, and the initial rates (ΔpH min−1) of this increase were modestly augmented (∼26 %) when normal cells were pretreated with forskolin (10 μm). Recovery from this alkaline shift was dependent on mucosal Cl−, was insensitive to the AE inhibitor 4,4′-diisothiocyanatodihydrostilbene-2,2′-disulfonic acid (H2DIDS; 1.5 mm), but was sensitive to the cystic fibrosis transmembrane conductance regulator (CFTR) channel inhibitor diphenylamine-2-carboxylate (DPC; 100 μm). In contrast, removal of serosal Cl− caused pHi to alkalinize in both normal and CF epithelia. Recovery from this alkaline challenge was dependent on serosal Cl− and blocked by H2DIDS. Additional studies showed that serosally applied Ba2+ (5.0 mm) in normal, but not CF, cells induced influx of HCO3− across the apical membrane that was reversibly blocked by mucosal DPC. In a final series of studies, normal and CF cells acutely alkaline loaded by replacing bilateral Krebs bicarbonate Ringer (KBR) with Hepes-buffered Ringer solution exhibited basolateral, but not apical, recovery from an alkaline challenge that was dependent on Cl−, independent of Na+ and blocked by H2DIDS. We conclude that: (1) normal, but not CF, nasal epithelia have a constitutively active DPC-sensitive HCO3− influx/efflux pathway across the apical membrane of cells, consistent with the movement of HCO3− via CFTR; and (2) both normal and CF nasal epithelia have Na+-independent, H2DIDS-sensitive AE at their basolateral domain.

The role of the actin-based cytoskeleton in the internalization process of Burkholderia multivorans by well-differentiated human airway epithelia was investigated by immunohistology and confocal microscopy. Our data suggest that an intact actin cytoskeleton is required for biofilm formation but not single cell entry or paracytosis.

Lumenal delivery of adenovirus vectors (AdV) results in inefficient gene transfer to human airway epithelium. The human coxsackievirus and adenovirus receptor (hCAR) was detected by immunofluorescence selectively at the basolateral surfaces of freshly excised human airway epithelial cells, suggesting that the absence of apical hCAR constitutes a barrier to adenovirus-mediated gene delivery in vivo. In transfected polarized Madin-Darby canine kidney cells, wild-type hCAR was expressed selectively at the basolateral membrane, whereas hCAR lacking the transmembrane and/or cytoplasmic domains was expressed on both the basolateral and apical membranes. Cells expressing apical hCAR still were not efficiently transduced by AdV applied to the apical surface. However, after the cells were treated with agents that remove components of the apical surface glycocalyx, AdV transduction occurred. These results indicate that adenovirus can infect via receptors located at the apical cell membrane but that the glycocalyx impedes interaction of AdV with apical receptors.

Current paradigms in cancer therapy suggest that activation of nuclear factor-κB (NF-κB) by a variety of stimuli, including some cytoreductive agents, may inhibit apoptosis. Thus, inhibiting NF-κB activation may sensitize cells to anticancer therapy, thereby providing a more effective treatment for certain cancers. E-1-deleted adenoviral (Ad) vectors encoding a "superrepressor" form of the NF-κB inhibitor IκBα (AdIκBαSR) or β-galactosidase (AdLacZ) were tested alone and in combination with tumor necrosis factor-α (TNF-α) in lung cancer cells for sensitization of the cells to death. Following transduction with AdIκBαSR, lung cancer cells expressed IκBαSR in a dose-dependent manner. Probing nuclear extracts of lung cancer cells with NF-κB-sequence-specific oligonucleotides indicated that there was a minimal amount of NF-κB in the nucleus at baseline and an expected and dramatic increase in nuclear NF-κB following exposure of cells to TNF-α. Control E-1-deleted AdLacZ did not promote NF-κB activation. Importantly, AdIκBαSR-mediated gene transfer resulted in the complete block of nuclear translocation of NF-κB by specific binding of its p65/relA component with transgenic IκBαSR. At the cellular level, transduction with AdIκBαSR resulted in increased cytotoxicity in lung cancer cells as opposed to transduction with equivalent doses of AdLacZ. In addition, whereas the parental cells were resistant to TNF-α-mediated cytotoxicity, IκBαSR-transduced cells could be sensitized to TNF-α. Consequently, AdIκBαSR transduction followed by exposure to TNF-α uniformly resulted in the death of non-small-cell lung cancer cells. These data suggest that novel approaches incorporating IκBα gene therapy may have a role in the treatment of lung cancer.

Airway surface liquid is comprised of mucus and an underlying, watery periciliary liquid (PCL). In contrast to the well-described axial transport of mucus along airway surfaces via ciliary action, theoretical analyses predict that the PCL is nearly stationary. Conventional and confocal microscopy of fluorescent microspheres and photoactivated fluorescent dyes were used with well-differentiated human tracheobronchial epithelial cell cultures exhibiting spontaneous, radial mucociliary transport to study the movements of mucus and PCL. These studies showed that the entire PCL is transported at approximately the same rate as mucus, 39.2+/-4.7 and 39.8+/-4.2 micrometer/sec, respectively. Removing the mucus layer reduced PCL transport by > 80%, to 4.8+/-0.6 micrometer/sec, a value close to that predicted from theoretical analyses of the ciliary beat cycle. Hence, the rapid movement of PCL is dependent upon the transport of mucus. Mucus-dependent PCL transport was spatially uniform and exceeded the rate expected for pure frictional coupling with the overlying mucus layer; hence, ciliary mixing most likely accelerates the diffusion of momentum from mucus into the PCL. The cephalad movement of PCL along airway epithelial surfaces makes this mucus-driven transport an important component of salt and water physiology in the lung in health and disease.

To test whether a major contribution of airways epithelial ion transport to lung defense reflects the regulation of airway surface liquid (ASL) ionic composition, we measured ASL composition using the filter paper technique. On nasal surfaces, the Cl- concentration (approximately 125 meq/liter) was similar to plasma, but the Na+ concentration (approximately 110 meq/liter) was below plasma, and K+ concentration (approximately 30 meq/liter) above plasma. The resting ASL osmolarity [2(Na+ + K+); 277 meq/liter] approximated isotonicity. There were no detectable differences between cystic fibrosis (CF) and normal subjects. In the lower airways, the Na+ concentrations were 80-85 meq/liter, K+ levels approximately 15 meq/liter, and Cl- concentrations 75-80 meq/liter. Measurements of Na+ activity with Na(+)-selective electrodes and osmolality with freezing point depression yielded values consistent with the monovalent cation measurements. Like the nasal surfaces, no differences in cations were detected between CF, normal, or chronic bronchitis subjects. The tracheobronchial ASL hypotonicity was hypothesized to reflect collection-induced gland secretion, a speculation consistent with observations in which induction of nasal gland secretion produced hypotonic secretions. We conclude that there are no significant differences in ASL ion concentrations between CF, normal, and chronic bronchitis subjects and, because ASL ion concentrations exceed values consistent with defensin activity, the failure of CF lung defense may reflect predominantly factors other than salt-dependent defensins.

The presence of the P2Y2 (P2U-purinergic) receptor on the apical surface of airway tissue raises the possibility that aerosolized UTP might be used therapeutically to induce Cl− secretion in individuals with cystic fibrosis. However, the duration of the effects of UTP may be limited by enzymatic degradation. We therefore have analyzed the metabolism of UTP and its metabolite UDP on polarized human nasal epithelium (HNE), and have compared the pharmacological activities of these two uridine nucleotides. HPLC analysis of medium bathing the mucosal surface of HNE cells revealed the presence of an ecto-nucleotidase(s) that hydrolyzed [3H]UTP and [3H]UDP with t½ values (at 1 μM nucleotide) of 14 and 27 min, respectively. An ecto-nucleoside diphosphokinase activity also was observed, which promoted conversion of [3H]UDP into [3H]UTP in the presence of ATP. The effects of UDP on [3H]inositol phosphate accumulation, intracellular calcium levels ([Ca2+]i), and Cl− secretory rates (ICl−) were quantitated in HNE cells in the presence of hexokinase and glucose to ensure that no UTP (or ATP) contaminated UDP solutions during the assays. Although UDP does not activate the human P2Y2 receptor, mucosal addition of UDP promoted [3H]inositol phosphate accumulation with an EC50 of 190 nM. Mucosal addition of UTP stimulated [3H]inositol phosphate accumulation with an EC50 of 280 nM. The maximal effects of mucosal UDP on [3H]inositol phosphate, [Ca2+]i, and ICl− responses were approximately one-half of those observed with mucosal UTP. Serosal application of UTP promoted a 50% greater [3H]inositol phosphate and calcium response than did mucosal application of UTP. In contrast, UDP had no effect when added to the serosal medium. Repetitive mucosal applications of UDP to HNE cells resulted in a progressive loss, i.e., desensitization, of the [Ca2+]i and ICl− response to UDP, whereas the corresponding responses to UTP remained unchanged. Our results provide evidence for the existence of a UDP receptor on HNE cells that is pharmacologically distinct from the P2Y2 receptor. The relative stability of UDP on the airway surface and the apparent predominant mucosal expression of this putative UDP receptor make it a potential target for cystic fibrosis treatment.

To the Editor: We remain enthusiastic about gene therapy, despite the slower-than-hoped-for rate of progress and the inefficiency of current adenoviral-mediated and liposomal-mediated CFTR gene transfer. One of the intents of our paper was to alert researchers in the field to problems with the efficiency of the adenovirus and consequently to stimulate new approaches to increase the efficiency of this vector by the use of other serotypes or new dosing strategies.

Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis.

We studied the consequences of cystic fibrosis transmembrane conductance regulator (CFTR) expression in NIH-3T3 fibroblasts as a model for the effects of virally transduced CFTR expression in non-epithelial cells. Fibroblasts were infected with a retrovirus vector that contained the human CFTR and neor cDNAs. We selected and expanded G418-resistant clones that encompassed a range of CFTR expression. CFTR-mediated Cl-conductance function was measured as whole cell current, and CFTR protein was quantitated by immunoblot analysis. Overall, there was a good relationship between CFTR protein levels and CFTR-mediated Cl- conductance. Some clones had consistently high basal levels of CFTR-mediated Cl- conductance. This variation in function was partially explained by CFTR protein levels and was not due to clonal variation in cAMP metabolism. High levels of CFTR expression were associated with depolarization of fibroblast membrane potential. The CFTR-expressing clones with the largest basally active CFTR Cl- conductances and the most depolarized membrane potentials also exhibited slower growth rates. These results suggest that potential side effects of gene replacement therapy for cystic fibrosis include functional consequences of CFTR expression in non-epithelial cells.

The rate of Cl- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl- conductance decreases the capability to secrete Cl-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl- secretion and apical membrane Cl- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl- conductance by external ATP is preserved in cystic fibrosis airway epithelia.

1. Intracellular pH (pHi) of cultured human airway epithelial cells from normal and cystic fibrosis (CF) subjects were measured with double-barrelled pH-sensitive liquid exchanger microelectrodes. The cells, which were grown to confluence on a permeable collagen matrix support, were mounted in a modified miniature Ussing chamber. All studies were conducted under open circuit conditions. Values are given as means +/- S.E.M. and n refers to the number of preparations. 2. Normal preparations (n = 15) were characterized by a transepithelial potential difference (Vt) of -18 +/- 2 mV, an apical membrane potential (Va) of -19 +/- 2 mV, a basolateral membrane potential (Vb) of -37 +/- 2 mV, a transepithelial resistance (Rt) of 253 +/- 15 omega cm2, a fractional apical membrane resistance (fRa) of 0.40 +/- 0.04 and an equivalent short circuit current (Ieq) of -73 +/- 7 microA cm-2. 3. CF preparations (n = 13) were characterized by a Vt of -46 +/- 7 mV, a Va of 3 +/- 5 mV, a Vb of -43 +/- 3 mV, Rt of 373 +/- 47 omega cm2, fRa of 0.44 +/- 0.04 and an Ieq of -130 +/- 16 microA cm-2. All parameters except Vb and fRa were significantly different (P < 0.025) from those of normal preparations. 4. Despite large differences in electrochemical driving force for proton flow across the apical cell membranes between normal and CF preparations (-4 +/- 3 mV and 20 +/- 7 mV, respectively), pHi was similar (7.15 +/- 0.02 and 7.11 +/- 0.05, respectively). The driving force across the basolateral membrane was similar in normal and CF preparations (22 +/- 3 and 26 +/- 3 mV, respectively). 5. Intracellular alkalinization achieved by removal of CO2 from the luminal Ringer solution or by luminal ammonium prepulse led to stimulation of Ieq in both normal (from -58 to -70 microA cm-2, n = 4; P < 0.05) and CF (from -144 to -163 microA cm-2, n = 4; P < 0.005) preparations. The increase in Ieq was associated with a reduction of Rt, increase in fRa, and hyperpolarization of Vb. All changes in bioelectric properties in response to intracellular alkalinization were fully reversible. 6. Intracellular acidification achieved by serosal ammonium prepulse led to marked reductions of Ieq in both normal (from -95 to -31 microA cm-2, n = 6; P < 0.05) and CF (from -111 to -67 microA cm-2, n = 7; P < 0.005) preparations.

A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistant cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possible passage number, influences the recovery of CFTR-targeted cells.

The common mutation causing cystic fibrosis is a deletion of phenylalanine 508 (delta F508), which occurs in a putative nucleotide-binding fold of the gene product. We report two additional mutations, substitution of cysteine for phenylalanine 508 (F508C) and substitution of valine for isoleucine 506 (I506V). Three compound heterozygous persons, two delta F508/F508C and one delta F508/I506V, had normal clinical and epithelial physiological studies indicating that the F508C and I506V mutations are benign. This opportunity to study the in vivo function of these mutations suggests that amino acid substitutions are more benign than changes in the length of this portion of the putative nucleotide-binding fold. These mutations must be taken into account when performing molecular diagnosis and carrier detection for cystic fibrosis.

The effects of culture conditions on growth and differentiation of human tracheobronchial epithelial (HTBE) cells have been defined. Epithelial cells were dissociated from tissues by protease treatment and were plated on tissue culture dishes in F12 medium supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, cholera toxin, bovine hypothalamus extract, and retinol. HTBE cells did not express any mucociliary function (ciliogenesis or mucin secretion) on tissue culture plastic, but they could be passaged 3 to 5 times with a total of 10 to 25 population doublings. Cells from early passages re-express both these functions when transplanted to tracheal grafts. When tissue culture plates were coated with collagen film or collagen gel substrata, cell attachment and proliferation were stimulated. However, the expression of mucous cell function in culture occurred only when cells were plated on collagen gel substrata and vitamin A (retinol) was present in the medium. Mucous cell differentiation under optimal conditions was defined by ultrastructural studies, by immunologic studies with mucin-specific monoclonal antibodies, and by carbohydrate and amino acid compositional analyses of mucin-like glycoproteins purified from culture medium. These results demonstrate for the first time that HTBE cells can express mucin synthesis and secretion under appropriate culture conditions.

Excessive active absorption of sodium is a unique abnormality of the airway epithelium in patients with cystic fibrosis. This defect is associated with thickened mucus and poor clearance of airway secretions and may contribute to the pulmonary disease in these patients. To study whether the inhibition of excessive absorption of sodium might affect the course of lung disease in cystic fibrosis, we performed a double-blind, crossover trial comparing aerosolized amiloride (5 mmol per liter; 3.5 ml four times daily), a sodium-channel blocker, with vehicle alone. Fourteen of the 18 adult patients initially enrolled in the study completed the one-year trial (25 weeks for each treatment). The mean (±SEM) loss of forced vital capacity (FVC) was reduced from 3.39±1.13 ml per day during treatment with vehicle alone to 1.44±0.67 ml per day during treatment with amiloride (P

1. Employing a primary cell culture system and intracellular microelectrodes, we quantitated and compared the Na+ and Cl- pathways in apical membranes of normal and cystic fibrosis (CF) human airway epithelia. 2. Like the transepithelial difference (PD) in situ, the PD of CF epithelia in culture (-27 +/- 4 mV, mean +/- S.E.M.; n = 28) exceeded the PD of normal epithelia (-10 +/- 1 mV; n = 22). The raised PD principally reflected an increase in the rate of active transport (equivalent short circuit, Ieq) for CF epithelia (61 +/- 9 microA cm-2) as compared with normal epithelia (23 +/- 3 microA cm-2). No significant differences in transepithelial resistance were detected. 3. As indicated by ion replacement studies (gluconate for Cl-), the apical membrane of normal cells exhibits an apical membrane Cl- conductance (GCl) that can be activated by isoprenaline. CF cells do not exhibit an apical membrane GCl, nor can a GCl be activated by isoprenaline. 4. CF cells exhibited a larger amiloride-sensitive Ieq and amiloride-sensitive apical membrane conductance (GNa) than normal cells. Further, the amiloride-sensitive Ieq was increased by isoprenaline in CF but not normal airway epithelia. 5. Equivalent circuit analysis yielded evidence for a more positive electromotive force (EMF) across the apical membrane and a more negative EMF across the basolateral membrane of CF cells as compared with normal cells. Baseline resistances of the apical (Ra) and basolateral (Rb) membranes did not differ for normal and CF cells. 6. Estimates of the resistance of the paracellular path to ion flow (Rs) by equivalent circuit analysis or ion substitution detected no differences in Rs between CF and normal cells. 7. We conclude that abnormalities in both cellular Cl- permeability (reduced) and Na+ permeability (increased) are characteristic of the cultured CF respiratory epithelial cell. These data suggest that a defect in the regulation of apical membrane permeabilities is a central feature of this disease.

After 30 years of continuous research into the mechanisms of human hypertension, we summarize the results obtained by the members of the multidisciplinary research group on hypertension of the Clinical Research Institute of Montreal on the disturbances of minerlocorticoid activity in a rigorously selected group of patients with early, mild essential hypertension. We attempt to integrate these findings with those of many other groups working on other aspects of hypertensive cardiovascular diseases. On the assumption that the increased peripheral resistance responsible for hypertension results from an imbalance or a disturbance of the equilibrium between the sympathetic nervous system and norepinephrine on one hand, and the vascular tone, sensitivity and responsiveness of the arterial smooth muscle to norepinephrine and to angiotensin II on the other hand, three models that fit the experimental and clinical facts as known at present are described.

MAJOR SCREENING PROGRAMS FOR HYPERTENSION ARE NECESSARY FOR THE FOLLOWING REASONS: (a) the 12 to 15% prevalence of hypertension in the North American population; (b) the high proportion of hypertensive patients who are inadequately treated or whose disease is not even recognized; (c) the fact that in too many medical centres hypertension is poorly investigated or considered an insignificant finding; (d) the demonstration by many groups, especially the Framingham study, of hypertension as the major factor in the occurrence of severe cardiovascular disease; and (e) the achievement of a marked decrease in severe cardiovascular complications following adequate treatment. But such screening programs are of little value if unaccompanied by a major educational effort directed not only to the public but also to the medical profession, and a parallel increase in the funding of biomedical research into the basic physiopathological mechanisms of hypertension.

Cystic fibrosis (CF) respiratory epithelia exhibit abnormal anion transport that may be linked to abnormal lung defense. In these studies, we investigated whether primary cultures of CF respiratory epithelial cells regulate abnormally the sulfate content of high molecular weight glycoconjugates (HMG) participating in airways' mucosal defense. HMG, including glycosaminoglycans and mucin-type glycoproteins released spontaneously into medium and HMG released from cell surfaces by trypsin, were metabolically labeled with 35SO4- and [6-3H]-glucosamine (GlcN) or 35SO4- and [3H]serine. All three classes of HMG from CF cells exhibited 35S/3H labeling ratios 1.5-4-fold greater than HMG from normal or disease control cells. Differences for labeling ratios of HMG from CF cells were shown to be the consequence of increased 35SO4- incorporation rather than decreased peptide synthesis and release or HMG glycosylation. The buoyant density of CF mucin-type HMG also was increased, consistent with increased sulfation. These observations suggest that oversulfation of a spectrum of HMG is a genetically determined characteristic of CF epithelial cells and may play an important pathophysiological role by altering the properties of mucous secretions and/or the interactions between selected bacteria and HMG at the airways' surface.

Extracorporeal pumping PA-LA perfusions were carried out for 24 hrs in 5 awake, alert lambs by cannulating the main PA and the LA with large bore polyurethane cannulae. A mean of 42% of the basal cardiac was pumped through the membrane lung. When the PA occluder cuff was progressively inflated, up to 80% of the basal cardiac output was diverted to the extracorporeal circuit for short periods. Hemothorax remained a significant problem. Three lambs were electively perfused 12 hrs, 36 hrs, and 8 days without a roller pump, using a low resistance spiral coil membrane lung pumped by the RV. By inflating the occluder cuff, a mean of 55% of basal cardiac output was pumped by the RV through the membrane lung. The 8 day bypass lamb remained healthy and active without any bleeding or gross pathology. We did not observe air emboli, thromboemboli or gross infections in either group. We believe that such a mode of cannulation will eventually allow long-term perfusion of a low resistance membrane oxygenator driven directly by the RV.