Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis

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1 - 1 - Supplementary Information Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis Volker Teichgräber 1, Martina Ulrich 2, Nicole Endlich 3, Joachim Riethmüller 4, Barbara Wilker 1, Cheyla Conceição De Oliveira Munding 2, Anna M van Heeckeren 5, Mark L Barr 6, Gabriele von Kürthy 7, Kurt W Schmid 8, Michael Weller 7, Burkhard Tümmler 9, Florian Lang 10, Heike Grassme 1, Gerd Döring 2 & Erich Gulbins 1 1 Department of Molecular Biology, Hufelandstrasse 55, University of Duisburg-Essen, Essen, Germany. 2 Institute of Medical Microbiology and Hygiene, Wilhelmstrasse 31, Tübingen, Germany. 3 Department of Anatomy, Friedrich-Loeffler-Str. 23c, University of Greifswald, Greifswald, Germany. 4 Children`s Clinic, Auf dem Schnarrenberg, University of Tübingen, Tübingen, Germany. 5 Case Western Reserve University, Biomedical Research Building 827, Euclid Avenue, Cleveland, Ohio , USA. 6 Department of Cardiothoracic Surgery, University of Southern California, 1520 San Pablo Street, Suite 4300, Los Angeles, California 90033, USA. 7 Department of Neurology, Auf dem Schnarrenberg, University of Tübingen, Tübingen, Germany. 8 Department of Pathology and Neuropathology, Hufelandstrasse 55, University of Duisburg-Essen, Essen, Germany. 9 Medical University School, Carl-Neuberg-Strasse, Hannover, Germany. 10 Department of Physiology, Gmelinstrasse 5, University of Tübingen, Tübingen, Germany. Address correspondence to: Dr. Erich Gulbins, Dept. of Molecular Biology, University of Duisburg Essen, Hufelandstrasse 55, Essen, Tel.: , Fax: , e mail: due.de

2 - 2 - Supplementary note 1 Sphingosine 1 phosphate and sphingosine levels in CF mice Sphingosine 1 phosphate levels were 2.6 ± 1.1 pmol/mg protein in Cftr MHH compared to 2.8 ± 1.1 pmol/mg protein in wild type mice. Sphingosine concentrations were 4.08 ± 0.25 pmol/mg protein in 24 weeks old Cftr MHH compared to 1.0 ± 0.11 pmol/mg protein in wild type mice (n = 5 each). We also tested whether the uptake of [ 3 H]sphingosine and [ 3 H]sphingosine 1 phosphate is affected by Cftr deficiency in tracheal epithelial cells. More than 95% of the added [ 3 H]sphingosine was converted to [ 3 H]sphingosine 1 phosphate within 5 min after addition to tracheal cells from wild type, Cftr KO and Cftr MHH mice. The uptake of [ 3 H]sphingosine 1 phosphate was reduced by approximately 50% in Cftr deficient epithelial cells (not shown).

3 - 3 - Supplementary Figure 1 Peptamen diet reduces accumulation of ceramide in CF mice We tested whether Peptamen R alters ceramide levels in the lungs of CF mice and fed our Cftr deficient mice, which express a residual activity of Cftr (Cftr MHH strain) or express Cftr in the intestinum as a transgen (Cftr KO strain), with Peptamen R. The data (Supplementary Figure 1a) demonstrated that Peptamen R reduced ceramide levels in lungs of Cftr MHH mice by 92% compared to untreated Cftr MHH mice and in lungs of Cftr KO mice by 86%. Compared to untreated wild type mice, Peptamen R treated Cftr MHH mice indeed revealed reduced ceramide levels. To address the mechanisms that mediate the reduction of ceramide upon Peptamen R feeding, we determined the concentration of cholesterol in the lungs of animals fed with Peptamen R. Cholesterol has been previously shown to critically influence the activity of the Asm and high levels of cholesterol significantly reduce the activity of the Asm 1. Our data demonstrated a 3 fold increase of cholesterol in the lung upon Peptamen R feeding (Supplementary Figure 1b). Simultaneous treatment of CF mice with simvastatin (40 mg/kg/day i.p.), a known blocker of cholesterol biosynthesis 2 prevented the cholesterol accumulation (Supplementary Figure 1b) as well as the reduction of ceramide in the lungs of these animals (Supplementary Figure 1a). Consistent with its effect on ceramide, Peptamen R diet resulted in 60% inhibition of the activity of the Asm in the lung, an effect that was abrogated by treatment with simvastatin (Supplementary Figure 1c). These in vivo data are supported by in vitro studies that show a dose dependent inhibition of the Asm in isolated respiratory epithelial cells incubated with increasing doses of cholesterol (Supplementary Figure 1d).

4 - 4 - Supplementary Fig. 1. High cholesterol levels inhibit acid sphingomyelinase (Asm) activity and normalize ceramide levels in CF mice. (a,b) Feeding of Cftr MHH or Cftr KO mice for 3 weeks with Peptamen R ad libitum reduces ceramide (a) and increases cholesterol (b) concentrations in the lung, effects that are blocked by simvastatin (Sim) treatment. Data represent means ± SD from each 5 independent experiments. (a) *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA; (b) *P < 0.04; Mann Whitney test. (c) Feeding of mice with Peptamen R for 14 d reduced Asm activity in mouse lungs, which was prevented by treatment with simvastatin. (d) Similarly, Asm activity was reduced in isolated epithelial cells, incubated with increasing doses of cholesterol. Displayed are means ± SD of 5 independent experiments each. Significant differences to untreated controls are indicated by asterisks (**P < 0.01, ***P < 0.002; exact Mann Whitney test). References: 1. Bhuvaneswaran, C., Venkatesan, S. & Mitropoulos K.A. Lysosomal accumulation of

5 - 5 - cholesterol and sphingomyelin: evidence for inhibition of acid sphingomyelinase. Eur. J. Cell Biol. 73, (1985). 2. Mol, M.J., Erkelens, D.W., Leuven J.A., Schouten J.A. & Stalenhoef, A.F. Effects of synvinolin (MK 733) on plasma lipids in familial hypercholesterolaemia. Lancet 25, (1986).

6 - 6 - Supplementary note 2 Methods Mouse strains. We bred and housed Cftr tm1unc -Tg (FABPCFTR) (abbreviated Cftr KO ) Jaw mice (Jackson Laboratories) in the vivarium of the University of Duisburg-Essen, Germany. These mice, which are on a mixed background consisting of C57BL/6, FVB/N and 129, are genetically deficient for the mouse equivalent to human CFTR (Cftr), but express human CFTR in the gut under control of a fatty acid binding protein (FABP) promoter, which prevents acute intestinal obstruction 1,2. C3He mice were used as controls. Additionally, B6.129P2(CF/3)-Cftr TgH(neoim)Hgu (abbreviated Cftr MHH ) congenic mice were used 3. The inbred CF strain CF/3-Cftr TgH(neoim)Hgu was established by brother-sister mating from the original Cftr TgH(neoim)Hgu mutant mouse that produces low levels of Cftr 4-6. Then, the congenic Cftr MHH strain was generated by backcrossing the targeted mutation into the B6 inbred background by speed congenic production. Syngenic B6 mice were used as controls. Cftr MHH and control mice were housed both in the Central Laboratory Animal Facility of the Medizinische Hochschule Hannover and the University of Duisburg-Essen, Germany. Furthermore, 12 week old C57BL/6 congenic B6.129P2-Cftr tm1unc mice bearing the S489X mutation in Cftr (Cftr S489X ) were used; Cftr +/+ mice were used as wild type littermate controls. Cftr S489X mice were housed and maintained in the vivarium at Case Western Reserve University, Cleveland, USA 7. Finally, Cftr KO mice were bred with Smpd1 -/- mice that are on a C57BL/6 background. To obtain Cftr KO /Smpd1 +/- mice, the heterozygous offspring was backcrossed to Cftr KO mice for at least 5 generations. The different laboratories, housing the mice in isolator cages, provided a pathogen-free environment. The mice were routinely investigated for microbial infections by bacterial culturing and serology and remained negative during the study period. The hygienic status was repeatedly tested by a panel of common mouse pathogens according to the FELASA recommendations of

7 - 7 - Bacterial lung infection. Mice were challenged with the P. aeruginosa strain 762, obtained from a patient with urosepsis, strain 769, from a patient with pneumonia or the laboratory strain ATCC 14115, obtained from ATCC. Bacteria were grown for 14 h to 14.5 h on fresh (maximally 2 weeks old) Trypticase Soy Agar (TSA) plates (Becton Dickinson, # ). Bacteria were transferred into 40 ml 37 o C pre-warmed Trypticase Soy Broth (TSB) (Becton Dickinson, # ) in Erlenmeyer flasks at an optical density (OD) (550 nm) of 0.225/ml (equals to 5.5 x 10 8 colony forming units (CFU)/ml) and the bacteria were incubated for 60 min at 37 o C with shaking at 125 rpm. The quality and source of the agar plates and the TSB, the shaking speed and the incubation time are critical to obtain reproducible results. Bacteria were centrifuged at 2,800 rpm (1600 x g) and the cell pellet re-suspended in 10 ml prewarmed RPMI-1640, supplemented with 10 mm HEPES, ph 7.3. The bacterial suspension was washed twice and finally re-suspended at 10 8 CFU/20 µl in pre-warmed HEPES/Saline (H/S) consisting of 132 mm NaCl, 20 mm HEPES, ph 7.4, 5 mm KCl, 1 mm CaCl 2, 0.7 mm MgCl 2, and 0.8 mm MgSO 4. Mice were then infected within the next 10 min. We employed a 30-gauge needle of a 1 ml insulin syringe covered with a tightly fitting, smooth plastic tube to avoid injuries in the nose. The mice were anesthesized for sec in ether (in a covered two liter beaker filled with a few paper towels to get a saturated ether athmosphere), the needle was immediately inserted for 2 mm into the nose and the bacteria were carefully injected. Between the 1st and the last mouse infected, we allowed a time interval of maximally 10 min, to exclude a change in viable counts of the bacteria. Usually, we infected not more than 10 mice/experiment. Of note, we never infected more than one mouse per group and the numbers given in the text indicate the number of independent experiments with one mouse each. P. aeruginosa cell numbers were determined in mouse lungs 15, 30, 60 or 120 min after challenge. Lungs were removed, homogenised and lysed for 10 min in 5 mg/ml saponin to release intracellular bacteria. Dilutions of the homogenates were cultured on TSB plates in

8 - 8 - duplicates. Bacterial counts, determined after 18 h, were normalized for lung weight. Additionally, the survival of infected mice was determined for 7 d after challenge. Control experiments reveal that 10 mg/l amitriptyline did not affect growth of the bacteria. Streptococcus pneumoniae strain R6 was grown on sheep blood tryptic soy agar for 12 h, removed from the plate and grown in TSB for 1 h. The mice were intranasally infected with 10 7 CFU and the number of bacteria in the lungs was determined 3 h after infection as above. Drug administration to mouse lungs and pulmonary lavage. We administered 1 ml of recombinant human DNase (rhdnase) (Roche) (25 µg/ml) to the mice using a Pariboy SX nebulizer apparatus and a LL-Nebulizer mask (Pari). The mask was clipped at the sides to cover only the nose and manually pressed on the mouse nose for 20 min. Similarly, we let mice inhale 1 ml of a solution of bafilomycin (1 µm), nigericin (2.5 µm), NH 4 Cl (25 mm), chloroquine (10 µm) and n-oleoylethanolamine (100 µm) in 0.9% NaCl. Optimal drug concentrations were determined in pilot experiments to achieve maximum DNA digestion in the bronchi, or sufficient alkalinisation of vesicles in lung cells and to cause minimal adverse effects such as increased breathing frequency. To obtain mouse lung macrophages, mice were killed and the trachea canuled with a plastic catheter (at least 5 mm inner diameter) connected to a syringe using surgical thread. The thorax was carefully opened, lungs flushed six times with 1 ml of a 0.9% NaCl solution each and the lavage fluid carefully collected by slow suction. Uptake of sphingosine/sphingosine-1-phosphate. We removed the trachea from wild type-, Cftr KO and Cftr MHH mice and opened it after suspending it in RPMI 1640, supplemented with 10 mm HEPES and 10% fetal calf serum (FCS). Then we incubated the trachea with 5 µci [ 3 H]sphingosine (20 Ci/mmol, American Radiolabelled Chemicals) and 2 µm sphingosine or 5 µci [ 14 C]sphingosine-1-phosphate (25 mci/mmol, American Radiolabelled Chemicals) and

9 - 9-2 µm sphingosine-1-phosphate for 45 min at 37 o C as described 9. We washed the trachea in PBS and determined the uptake of the radioactivity by liquid scintillation counting. Asm activity. We homogenized mouse lungs in 250 mm sodium acetate buffer, ph 5.0, supplemented with 1.3 mm EDTA and 1% NP40 employing a dounce homogenisator followed by 3 rounds of sonication (Ultrasonic Processor tip sonicator). An aliquot of the samples was diluted to 0.1% NP40 and incubated with 0.05 µci per sample [ 14 C]sphingomyelin (52 mci/mmol; MP Biomedicals). Prior to addition we dried the substrate, then re-suspended it in 250 mm sodium acetate buffer (ph 4.5, 5.0 or 5.9), supplemented with 1.3 mm EDTA, and 0.1% NP40 and bath sonicated the samples for 10 min. The samples were incubated for 30 min at 37 o C. The enzymatic reaction was terminated by extraction in 4 volumes of CHCl 3 :CH 3 OH (2:1, v/v), the samples were centrifuged and an aliquot of the upper aqueous phase was scintillation counted to determine the release of [ 14 C]phosphorylcholine from [ 14 C]sphingomyelin. Acid ceramidase activity. We lysed mouse lung cells in 200 mm citrate/phosphate buffer (ph 4.5, 5.0 or 5.9), supplemented with 300 mm NaCl, and 1% Triton X-100, on ice for 15 min. After dilution to 0.1% Triton X-100 we added the substrate [ 14 ]C 16 -ceramide (0.1 µci/sample, 55 mci/mmol, ARC). Prior to addition, the substrate was dried, re-suspended in 150 mm NaCl, supplemented with 0.05% Triton X-100, and sonicated to get micelles. The enzyme reaction was performed for 30 min at 37 o C. We terminated the reaction by addition of 20 volumes ethanol, centrifuged the samples, dried the supernatants, re-suspended in CHCl 3 :CH 3 OH (1:1, v/v) and separated lipids on TLC employing CHCl 3 :CH 3 OH:28% ammonium hydroxide (90:20:0.5, v/v/v). The TLC plates were exposed to X-ray films, the substrate and the product were identified by co-migration with standards, removed from the plate and liquid scintillation counted. To determine the reverse Ac activity, the enzyme

10 reaction was performed with 200 µm [ 14 C] lauric acid (50 mci/mmol) and sphingosine (50 nmol/reaction) at ph 4.5, 5.0 and 5.9. Ceramide was identified by TLC and quantified as above. Ceramide measurements. Mouse lungs were removed, transferred into 1 ml CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v) and homogenized 30-times in a Dounce homogenisator and three times for 20 sec each with a tip sonicator. After addition of 200 µl H 2 O, the samples were centrifuged for 5 min at 14,000 rpm. Serum samples were directly added to 1 ml CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v). The lower phase was collected, dried and subjected to alkaline hydrolysis of diacylglycerol in 0.1 N methanolic KOH at 37 o C for 60 min. The samples were re-extracted, the lower phase dried and re-suspended in 20 µl of a detergent solution (7.5% (w/v) n-octylglucopyranoside, 5 mm cardiolipin in 1 mm diethylene-triaminepentaacetic acid). After sonication (10 min), the samples were added to 70 µl of a reaction mixture, containing 10 µl diacylglycerol kinase (GE Healthcare Europe), 0.1 M imidazole/hcl, ph 6.6, 0.2 mm diethylenetriamine-pentaacetic acid, ph 6.6, 70 mm NaCl, 17 mm MgCl 2, 1.4 mm EGTA, 2 mm DTT, 1 µm ATP and 10 µci [ 32 P]γATP. The kinase reaction was performed for 30 min at room temperature and terminated by addition of 1 ml CHCl 3 :CH 3 OH:1N HCl (100:100:1, v/v/v), 170 µl buffered saline solution (135 mm NaCl, 1.5 mm CaCl 2, 0.5 mm MgCl 2, 5.6 mm glucose, 10 mm HEPES), ph 7.2, and 30 µl of a 100 mm EDTA solution. The samples were vortexed and the phases separated. The lower phase was collected, dried, dissolved in 20 µl CHCl 3 :CH 3 OH (1:1, v/v) and separated on Silica G60 TLC plates employing CHCl 3 :CH 3 OH:CH 3 COOH (65:15:5, v/v/v). The TLC-plates were exposed to x-ray films and ceramide spots were identified by co-migration with a C 16 - ceramide standard. Incorporation of [ 32 P] into ceramide was quantified by liquid scintillation counting after removal of the spots from the plates. Ceramide amounts were determined by

11 comparison with a standard curve using C 16 -ceramide as substrate. We performed six independent experiments with each one mouse per group. Cholesterol measurements. Cholesterol was determined using the ECCH-100 kit (BioAssay Systems), following the protocol of the vendor. Briefly, cholesterol esters are converted to cholesterol, cholesterol is then metabolized to cholest-4-ene-3-one and NAD reduced to NADH by cholesterol dehydrogenase. Absorbance at 340 nm was determined to measure cholesterol concentrations. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) of lung sections. We fixed lungs in 4% buffered paraformaldehyde (PFA), ph 7.3, for 36 h and embedded them in paraffin. We de-paraffinized, re-hydrated and treated the samples with 0.1 M citrate buffer, ph 6.0, at 350 W for 5 min in a microwave, washed them in PBS, blocked them in 0.1 M Tris/HCl buffer, ph 7.5, supplemented with 3% bovine serum albumin and 20% FCS and incubated them with fluorescein-coupled dutp and TUNEL enzyme (Roche Diagnostics) in the presence of terminal deoxynucleotidyl transferase. Then, we washed the samples again with PBS, incubated them for 10 min at 70 o C to remove unspecific binding and labeled them with an antibody to FITC, coupled to alkaline phosphatase, in PBS. The signal was then visualized by a colorimetric reaction, the sections were mounted in moviol and analyzed by light microscopy. Ceramide staining. To visualize ceramide in epithelial cells, we isolated large bronchi from the indicated mice, fixed them in 2% PFA, ph 7.3, in PBS for 30 min and scratched ciliated epithelial cells from the tissue. The cells were blocked in PBS, supplemented with 5% FCS, and stained with monoclonal antibodies to ceramide (1:100 dilution, clone 15B4 from Alexis or clone MAS 0010 from Glycobiotech), followed by incubation with a secondary Cy3-

12 labelled antibody against mouse IgM. We analysed the cells on a Leica fluorescence microscope DMIRE2 with an exposure time of 150 ms. All microscopy studies were done at 1000x magnification if not otherwise noted. We collected human epithelial cells by nasal brushings using cotton tips and washed the cells carefully five times in H/S buffer, fixed them in 4% PFA for 15 min and stained them as described above. The fluorescence signal was quantified in areas of 0.5 x 0.5 cm of the fluorescence pictures using the QFluoro software (Leica). We analysed nasal epithelial cells on a DMIRE 2 fluorescence microscope (Leica) with an exposure time of 140 ms. We stained ceramide in mouse lung sections after fixation, embedding in paraffin, dewaxing and re-hydration by incubating the slides in PBS, supplemented with 0.01% Tween 20 for 15 min. The slides were washed in PBS and the tissue was incubated with monoclonal antibodies to ceramide (1:100 dilution, clone MAS 0010) for 45 min at room temperature, followed by incubation with a Cy3-labelled antibody against mouse IgM (1:1000) for 45 min at room temperature. The sections were mounted in moviol and analyzed by fluorescence microscopy as above or by confocal microscopy. Confocal microscopy studies were done with the same microscopy settings on paraffin sections of 16 week old wild type and Cftr MHH mice. FACS studies were performed on crude vesicle preparations from lungs of the indicated mice. Vesicles were stained with Cy3 coupled ceramide antibodies. Analysis was performed on a BD Calibur with standard settings. We stained ceramide in human lung sections using 5 µm cryostat thin sections, prepared from lung tissues of CF and normal individuals. We fixed the sections on glass slides with acetone, washed them with PBS-Tween (0.1%), and pre-incubated them with normal goat serum, 1:10 diluted in PBS-Tween (0.1%). The sections were incubated with monoclonal antibodies to ceramide (Axxora), 1:20 diluted in PBS, at room temperature for 2 h. Then, we washed the sections with PBS-Tween (0.1%) and incubated them with Cy2-labeled goat

13 antibodies to mouse IgM for 1 h at room temperature, followed by three wash steps and an incubation with 2 µg/ml of 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI; Roche Mannheim) in PBS-Tween (0.1%) for 5 min at room temperature. We washed the sections again and embedded them with fluorescence mounting medium. Lung tissues were analysed with a Zeiss Axioplan microscope at an exposure time of 800 ms. We analysed a similar number of airways each in healthy and CF tissues. For ceramide staining by transmission electron microscopy, we studied 16 week old wild type and Cftr MHH mice. We fixed mouse lung tissue (1 mm 3 cubes) with 4% PFA, dehydrated it in a graded ethanol series and embedded it in LR-White (Plano). We performed immunogold double staining on ultrathin sections of 70 nm. After blocking for 1 h, we incubated the sections with monoclonal antibodies to ceramide and cathepsin D specific antibodies at 4 C overnight, followed by four wash steps with PBS-Tween and an incubation with secondary antibodies, i.e. anti-mouse IgM conjugated with 25 nm gold and anti-goat IgG conjugated with 10 nm gold, each diluted 1:25 for 1 h. We washed the samples four times with TBS-Tween and finally with H 2 O, and post-stained them with uranyl acetate. We investigated the sections with an EM 910 microscope (Zeiss). Image documentations were done with Kodak 4489 macro EM film. Corrections of background illumination and contrast enhancement were done in ImageJ (Wayne Rusband, NIH). DNA staining of lung sections and adhesion of P. aeruginosa to DNA. Re-hydrated sections from mouse lungs were incubated for 5 min in 4 µg/ml ethidium bromide, washed in PBS, mounted in moviol and analyzed by fluorescence microscopy. To determine P. aeruginosa adhesion to and growth on DNA, we pre-incubated A549 respiratory cells with 10 µg denatured salmon sperm DNA or medium for 30 min at 37 C. Then we incubated cells with a suspension of 1x 10 7 CFU/ml of P. aeruginosa for 2 h at 37 C. We removed the

14 supernatant, fixed the cells with 4% formaldehyde, and stained P. aeruginosa with a Cy3- coupled antibody to P. aeruginosa. We visualized eukaryotic cells with DAPI. Determination of neutrophil and macrophage numbers and submucosal glands in mouse lung tissues. We prepared cryostat thin sections (5 µm) from lung tissue of uninfected Cftr KO, Cftr S489X mice, wild type or Cftr -/- /Smpd1 +/- mice. We fixed sections on glass slides with acetone, washed them with PBS-Tween (0.2%) and pre-incubated them with normal goat serum, 1:10 diluted in PBS-Tween (0.2%). Then, we incubated the sections with a 1:50 dilution of monoclonal rat antibodies to mouse neutrophils (clone 7/4) or to macrophages (CD68) (Acris) in PBS-Triton (0.5%) overnight at 4 C. After washing with PBS-Tween, (0.1%) we incubated the sections with Cy2-labeled goat antibodies against mouse IgM for 1 h at room temperature, followed by three washing steps with PBS-Tween (0.1%) and an incubation with 2 µg/ml of DAPI (Roche) in PBS-Tween (0.1%) at room temperature. The sections were washed and embedded with mounting medium. For quantitative determination of cell numbers, we examined five sections per mouse lung. From every section, we took 6 to 10 digitalized images (magnification: 200x; area: 850 x 650 µm) and analysed the sections with an Axioplan microscope (Zeiss) using Axiovision (Zeiss). We determined submucosal glands in mouse lungs by staining with the periodic acid Schiff (PAS) reagent. References 1. Snouwaert, J.N. et al. An animal model for cystic fibrosis made by gene targeting. Science 257, (1992). 2. Zhou, L. et al. Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR. Science 266, (1994). 3. Charizopoulou, N. et al. Instability of the insertional mutation in CftrTgH(neoim)Hgu

15 cystic fibrosis mouse model. BMC Genet. 5, 6 (2004). 4. Charizopoulou N, et al. Spontaneous rescue from cystic fibrosis in a mouse model. BMC Genet. 7, 18 (2006). 5. Dorin, J.R. et al. Cystic fibrosis in the mouse by targeted insertional mutagenesis. Nature 359, (1992). 6. Dorin JR, et al. Long-term survival of the exon 10 insertional cystic fibrosis mutant mouse is a consequence of low level residual wild type Cftr gene expression. Mammalian Genome 5, (1994). 7. van Heeckeren, A.M., Schluchter, M.D., Xue, W. & Davis, P.B. Response to acute lung infection with mucoid Pseudomonas aeruginosa in cystic fibrosis mice. Am. J. Respir. Crit. Care Med. 173, (2006). 8. Nicklas, W. et al. Recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units. Lab. Anim. 36, (2002). 9. Boujaoude, L.C. et al. Cystic fibrosis transmembrane regulator regulates uptake of sphingoid base phosphates and lysophosphatidic acid: modulation of cellular activity of sphingosine 1-phosphate. J. Biol. Chem. 276, (2001).

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