Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis

Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis

Accepted Manuscript Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis Anna Serrano-Mollar, PhD, Gemma ...

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Accepted Manuscript Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis Anna Serrano-Mollar, PhD, Gemma Gay-Jordi, PhD, Raquel Guillamat-Prats, PhD, Daniel Closa, PhD, Fernanda Hernandez-Gonzalez, MD, Pedro Marin, PhD, Felip Burgos, PhD, Jaume Martorell, PhD, Marcelo Sánchez, MD, Pedro Arguis, MD, Dolors Soy, PhD, José M. Bayas, PhD, José Ramirez, PhD, Teresa D. Tetley, PhD, Laureano Molins, PhD, Jordi Puig de la Bellacasa, PhD, Camino RodríguezVillar, PhD, Irene Rovira, PhD, Juan José Fiblà, MD, Antoni Xaubet, PhD, for the Pneumocyte Study Group PII:

S0012-3692(16)45721-1

DOI:

10.1016/j.chest.2016.03.021

Reference:

CHEST 385

To appear in:

CHEST

Received Date: 17 November 2015 Revised Date:

1 February 2016

Accepted Date: 1 March 2016

Please cite this article as: Serrano-Mollar A, Gay-Jordi G, Guillamat-Prats R, Closa D, HernandezGonzalez F, Marin P, Burgos F, Martorell J, Sánchez M, Arguis P, Soy D, Bayas JM, Ramirez J, Tetley TD, Molins L, Puig de la Bellacasa J, Rodríguez-Villar C, Rovira I, Fiblà JJ, Xaubet A, for the Pneumocyte Study Group, Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis, CHEST (2016), doi: 10.1016/j.chest.2016.03.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis

Anna Serrano-Mollar, PhD

*, Gemma Gay-Jordi, PhD

, Pedro Marin, PhD 3, Felip Burgos, PhD

2,15

, Jaume Martorell, PhD 4,

Marcelo Sánchez, MD 5, Pedro Arguis, MD 5, Dolors Soy, PhD Bayas, PhD 7, José Ramirez, PhD

8,14, 15

16

, Jordi Puig de la Bellacasa, PhD

Rodríguez-Villar; PhD

6,15,16

, José M

, Teresa D. Tetley, PhD 9, Laureano

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10,

, Raquel

, Daniel Closa, PhD 1, Fernanda Hernandez-Gonzalez,

1, 2, 15

Molins, PhD

1,15

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1,15

Guillamat-Prats, PhD MD

1,15

12

11,16

, Camino

, Irene Rovira, PhD13, Juan José Fiblà, MD

14

, Antoni

CORRESPONDENCE Anna Serrano-Mollar, PhD.

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Xaubet, PhD 2,15,16 *, for the Pneumocyte Study Group#

Department of Experimental Pathology, IIBB-CSIC

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Rosselló, 161, 7a, 08036 Barcelona, Spain. E-mail: [email protected]

1 Departamento de Patología Experimental. Instituto de Investigaciones

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Biomédicas de Barcelona IIBB- CSIC. Barcelona.

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2 Servicio de Neumología. Hospital Clínic. Barcelona. 3 Área de Criopreservación. Servicio de Hemoterapia y Hemostasia. Hospital Clínic. Barcelona.

4 Servicio de Inmunología. Hospital Clínic. Barcelona. 5 Departamento de Radiología. Hospital Clínic. Barcelona. 6 Servicio de Farmacia. Hospital Clínic. Barcelona. 7 Servicio de Medicina Preventiva y Epidemiología. Hospital Clínic. Barcelona 8 8 Servicio de Anatomía Patológica. Hospital Clínic. Barcelona.

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ACCEPTED MANUSCRIPT 9 Section of Pharmacology and Toxicology, National Heart and Lung Institute, Imperial College. London. 10 Servicio de Cirugía Torácica. Hospital Clínic. Barcelona.

12 Unidad de Donación. Hospital Clínic. Barcelona.

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11 Servicio de Microbiología. Hospital Clínic. Barcelona.

13 Servicio de Anestesiología y Reanimación. Hospital Clínic. Barcelona.

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14 Servicio de Cirugía Torácica. Hospital Universitario Sagrat Cor. Barcelona 15 Centro de Investigaciones Biomédicas en Red de Enfermedades

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Respiratorias (CIBERES). 16 Universidad de Barcelona. Barcelona.

*Drs Anna Serrano-Mollar and Antoni Xaubet contributed equally to this article as first author and also as senior author. Drs Anna Serrano-Mollar and Antoni

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Xaubet share both authorships.

#Members of the Pneumocyte Study group are listed in the e-appendix 1.

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Running head: Alveolar Type II Cell Transplantation in IPF

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Key words: Idiopathic pulmonary fibrosis, alveolar type II cells, cell therapy.

Word count: 4015

Conflict of interest statements: The author A. Serrano-Mollar, D.Closa and O.Bulbena (from the Pneumocyte Study Group) have a patent for the Use of type II pneumocytes in the treatment of pulmonary diseases associated with pulmonary fibrosis. Granted European patent EP1961423, based on Spanish priority patent application ES200502939.

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All the other authors declare that they have no competing financial interests.

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ACCEPTED MANUSCRIPT ABSTRACT Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited response to currently available therapies. Alveolar type II

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cells (ATII) act as progenitor cells in the adult lung, contributing to alveolar repair during pulmonary injury. However, in IPF ATII die and are replaced by fibroblasts and myofibroblasts. In previous preclinical studies, we demonstrated

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that ATII intratracheal-transplantation was able to reduce pulmonary fibrosis. The main objective of this study was to investigate the safety and tolerability of

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ATII intratracheal-transplantation in IPF patients.

Methods: We enrolled 16 patients with moderate and progressive IPF who underwent ATII intratracheal-transplantation by fiber-optic bronchoscopy. We evaluated the safety and tolerability of ATII transplantation by the emergent adverse

side-effects

within

12-months.

Moreover,

pulmonary

function,

assessed.

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respiratory symptoms and disease extent during 12-months of follow-up were

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Results: No significant adverse events were associated with the ATII intratracheal-transplantation. After 12-months of follow-up, there was no

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deterioration in pulmonary function, respiratory symptoms and disease extent. Conclusion: Our results support the hypothesis that ATII intratrachealtransplantation is safe and well tolerated in IPF patients. This study opens the door to design a clinical trial to elucidate the potential beneficial effects of the ATII cell therapy in IPF.

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ACCEPTED MANUSCRIPT ABBREVIATION LIST ATI: alveolar type I cells ATII: alveolar type II cells

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BDI: baseline dyspnea index DLco: single-breath diffusing capacity for carbon monoxide FVC: forced vital capacity

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HRCT: high-resolution computed tomography IPF: idiopathic pulmonary fibrosis

SD: standard deviation

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6MWT: 6-minute walk test

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SaO2: arterial oxygen saturation

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ACCEPTED MANUSCRIPT INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and severe disease with no known cause and a limited response to currently available therapies.

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IPF is the most common form of idiopathic interstitial pneumonia and its prevalence has been increasing in recent years. Most patients show a progressive decline in pulmonary function, which quickly leads to respiratory

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failure and death. The poor prognosis, combined with the scarcity of treatment options, provides a strong rationale for development of novel therapeutic

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alternatives for this disease (1).

Damage to alveolar epithelium is believed to be an important early pathogenic event in IPF. Epithelial cell damage and death result in gaps in epithelial basement membranes. Migration of fibroblasts into alveolar space via these gaps leads to intra-alveolar fibrosis. Under normal conditions, proliferation of

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alveolar type II cells (ATII) and their subsequent differentiation into alveolar type I cells (ATI) contribute to alveolar repair. However, in IPF, both ATII and ATI die

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and are replaced by fibroblasts and myofibroblasts (2-4). The loss of ATII compromises the reparative mechanism and is thought to play a significant role

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in the development and progression of pulmonary fibrosis. Based on this evidence, we hypothesized that administration of ATII directly to alveoli could restore the ability to repair the epithelium, thus attenuating or even reversing the progression of IPF. Indeed, our group has previously demonstrated, in an experimental model of bleomycin-induced lung fibrosis in rats, that ATII intratracheal-transplantation clearly reduces the fibrogenic process (5-7). In the present work, we initiated a clinical study to evaluate the safety and tolerability of ATII intratracheal-transplantation in humans, as well as to obtain a

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ACCEPTED MANUSCRIPT preliminary assessment of this cell therapy in patients with moderate and

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progressive IPF.

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ACCEPTED MANUSCRIPT METHODS Legal regulation ATII were obtained from deceased organ donors. Cells were isolated and not subject to any in vitro procedure; therefore, ATII intratracheal-transplantation

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procedure was considered within the law of organ transplantation and not as a drug. The study was conducted in compliance with current Good Clinical and Laboratory Practice standards and in accordance with the principles set forth

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under the Declaration of Helsinki. The study was approved by the Ethics

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Committee of Hospital Clínic (Barcelona) (approval nº: 2007/3571, 2007/3818), Organización Nacional de Trasplantes (ONT, Spain), and Organització Catalana de Trasplantaments (OCATT, Catalonia, Spain) (approval nº: P-9343, P-9531). Families or legal surrogates provided consent for organ donation which included authorization for research purposes. Also, all study patients

Study design

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provided written informed consent.

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Given the positive results obtained with ATII intratracheal-transplantation in our

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previous preclinical studies, we initiated a study focused on assessing the safety and tolerability of ATII intratracheal-transplantation in patients with moderate and progressive IPF. We enrolled 16 patients who had been diagnosed with IPF (Table 1). Each patient received four cell intratrachealtransplantations. Additionally, in order to avoid cell rejection reactions, all patients followed a vaccination program before cell instillation and were treated with low doses of immunosuppressive drugs and antibiotics. After intratracheal-

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ACCEPTED MANUSCRIPT transplantations the enrolled patients were monitored for a year (see flow diagram, Figure 1).

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Enrolled patients Enrolled patients were diagnosed with IPF during three years before ATII intratracheal-transplantation, according to ATS/ERS/JRS/ALAT guidelines (1). A

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histological diagnosis of definite usual interstitial pneumonia was obtained by surgical lung biopsy in 13 of these 16 patients (81.25%). Inclusion criteria

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followed those used by different IPF clinical trials (8): a) aged < 75 years; and b) single-breath diffusing capacity for carbon monoxide (DLco) > 35% predicted and forced vital capacity (FVC) > 50% predicted. Moreover, patients were included only if disease was in a progressive phase; this new criterion was added in order to establish any changes induced by cell transplantation.

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Progressive disease was defined as an increase in the degree of dyspnea, increase in the extent of disease in high-resolution computed tomography

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(HRCT), and/or evidence of deterioration in lung function: change of FVC ≥ 10% and/or change of DLco ≥ 15% of predicted values during the last year. These

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parameters were selected because they are proven independent predictors of increased short-term mortality (9, 10). Patients were excluded if they had IPF complications (respiratory failure, pulmonary hypertension, pulmonary infection, lung

cancer,

pulmonary

thromboembolism,

pneumothorax,

or

acute

exacerbation), presence of emphysema in HRCT, and/or any other severe concomitant illness that could reduce life expectancy. Before being included in this study, four patients were treated with prednisone, azathioprine and N-acetylcysteine, three with prednisone and N-acetylcysteine,

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ACCEPTED MANUSCRIPT two with bosentan, one with prednisone, four with N-acetylcysteine and two had not received any treatment. During the study period, concomitant treatment with

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any medication used for IPF was not allowed.

Screening visit

In order to set the baseline status of the patients a screening visit was

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performed 30 days before the first cell transplantation. This included taking a detailed history, blood analysis (hematological and biochemical determinations)

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pulmonary function tests, arterial blood gases, 6-minutes walking test (6MWT), HRCT, assessing the degree of cough and dyspnea and a physical examination.

Vaccination program, immunosuppressive regimen and antimicrobial

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prophylaxis

As it is not known which vaccines are indicated in patients undergoing ATII

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instillation, we adopted the general criteria used for patients undergoing solid organ transplantation (11). The following serological tests were carried out

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before vaccination: anti-HBc, anti-HBs (titers), HBsAg, HAV (IgG) and measles, rubella,

mumps

and

varicella-zoster

antibodies.

Immunizations

were

administered according to the results obtained one month before ATII transplantation. Donors and recipients were typed for ABO blood group and HLA antigens. However, to prevent cell rejection reactions, patients were treated with immunosuppressive and prophylaxis drugs based on a combination of tacrolimus, mycophenolate mofetil, steroids, trimethoprim-sulfamethoxazole in combination folinic acid, valganciclovir and mouthwashes of liquid-oral

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ACCEPTED MANUSCRIPT nystatin. This treatment was started two hours after the first cell instillation. See e-Appendix 2 and 3.

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Donor selection and cell isolation Lungs were obtained from deceased organ donors aged < 80 years and from donors with traumatic lungs which were useful to isolate ATII cells but were not

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suitable for whole lung transplantation. A history of smoking and the presence of chest trauma did not prevent the use of donor lungs for cell isolation.

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Selection criteria included clear chest X-ray, normal gas exchange (PaO2 (arterial partial oxygen pressure) > 300 mmHg on FiO2 (fraction of inspired oxygen) = 1.0, PEEP (positive end expiratory pressure) = 5 cm H2O, no evidence of aspiration or sepsis, absence of microorganisms on bronchial aspirate samples, no history of primary pulmonary disease or active pulmonary

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infection, and ABO blood group compatibility. Standard criteria for donor exclusion were applied to minimize the risk of donor-derived infection or cancer.

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To treat the 16 patients, we used 18 lungs. ATII were isolated, cryopreserved and instilled. ATII purity was established by

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presence of intracellular alkaline-phosphatase, lamellar bodies and microvilli at electron microscope level and by flow cytometry (Figure 2). Purity measured by positive staining with alkaline-phosphatase was 78.92 ± 4.64 (n=18) at the end of isolation procedure and 77.05 ± 5.32 (n=18) after thawing. Purity measured by flow cytometry after thawing was (80.97 ± 4.25, n=9). Additionally, as macrophages are the main contaminant cell in this procedure, a CD32 staining was performed to evaluate their presence by flow cytometry and it was found to

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ACCEPTED MANUSCRIPT be 10.82 ± 4.83 % (n=9). Viability of ATII before cryopreservation was (99.54 ± 1.25, n=18) and (84.38 ± 5.99, n=18) after thawing. ATII purity was similar to that obtained in previous preclinical studies in which was demonstrated the

Alveolar type II cell transplantation

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specificity of ATII to halt pulmonary fibrosis (5, 6). e-Appendix 4.

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ATII intratracheal-transplantation was performed via fiber-optic bronchoscopy (Pentax EB 1570K, Tokyo, Japan). Thawed ATII were resuspended in 10 ml

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aliquots of sterile saline solution, and instilled into the lung through the biopsy channel of the fiber-optic bronchoscope. Cells were instilled in the lower lobes, middle lobe and lingula, since these are the areas most affected by IPF. Four cell instillations were performed for each patient: two in the right lung, and two in the left. Based on the half-life of ATII, the time period selected between cell

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instillations was 15 days. The total number of cells used was 1000 to 1200 x 106. The amount of cells instilled was determined via extrapolation of the

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number of cells that was previously used, in terms of lung size, in the animal

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transplantation model (5).

Safety assessments, outcome measures and follow-up To asses for possible adverse effects triggered by cell administration, different protocols were performed throughout all the study period. During the first 24 h after each cell instillation, blood analysis including hematological and biochemical determinations, arterial blood gases, monitoring of vital signs, (temperature, oxygen saturation, respiratory and heart rate), electrocardiogram and chest X-ray were performed. Additionally, changes of

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ACCEPTED MANUSCRIPT cough and dyspnea and costal pain were also evaluated by asking patients about any alteration. Before each ATII instillation a fiber-optic bronchoscopy with bronchial aspirate

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and/or bronchoalveolar lavage was undertaken to exclude the presence of pulmonary infection. However, this procedure was also scheduled in case of clinical manifestations suggestive of respiratory infection, and/or deterioration of

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lung function during follow-up.

The follow-up visits were programmed two, six and twelve months after the first

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ATII instillation and were complemented with a monthly visit and unscheduled visits, if necessary, for up to 12-months. Main outcome measures included pulmonary function tests, arterial blood gases and 6MWT, all of them according to the guidelines of ATS/ERS/JRS/ALAT (1). Dyspnea was assessed using Baseline Dyspnea Index and Transition Dyspnea Index, and cough was

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assessed by Leicester Cough Questionnaire. Additionally, extent of disease was evaluated by HRCT scoring. The extent of parenchymal abnormalities was

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evaluated as qualitative and semi-quantitative analysis. To perform the semiquantitative analysis, we followed the method previously described by our group

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(12). Changes in any of these parameters were evaluated and compared from baseline. Severe side effects were defined as those requiring hospitalization. For more details regarding methodology see e-Appendixes 5-7. Further, changes in all of these measurements were also used to obtain preliminary data of whether ATII cells were able to modify IPF progression. Flaherty et al and du Bois et al (9, 10) have described that IPF can be considered stable when changes from baseline are <15% for DLco and <5% for FVC, a stabilization in the 6-minute walk distance, stabilization and/or

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ACCEPTED MANUSCRIPT improvement in the HRCT and in the cough and dyspnea degree. Thus, we followed these criteria to classify the disease between stable and progressive

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during the 12-months of follow-up.

Statistical analysis

Quantitative variables are expressed as median and range (minimum–

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maximum) or mean and standard deviation (SD). Data in graphs are represented as mean with individual data points. Data were compared using

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Two-tailed Mann-Whitney exact test and no correction for multiplicity was done. Statistical significance was set at p < 0.05. Analysis was performed using SAS version 9.2 software (SAS Institute Inc., Cary, NC, USA).

RESULTS

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Cell transplantation did not induce any severe adverse effects and all patients were discharged 24 hours after every cell instillation, given that they were

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afebrile with no signs of infection, with normal blood analysis and hemodynamically stable. Measures of blood gases did not present any

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significant changes respect to baseline determinations (see e-Table 1). Cough and dyspnea did not increase after 24 h of cell instillation (e-Table 1) and any patients had hemoptysis and neither did costal pain. Moreover, none of the patients showed signs of cardiac dysfunction (e-Table 1). Microbiological analysis of bronchial aspirates and bronchoalveolar lavage, undertaken before ATII instillations and during follow-up, were negative in all patients. The only mild side-effect was observed in one patient who presented a transitory alveolar infiltrate in the lower left lung and hypoxemia after the second cell instillation.

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ACCEPTED MANUSCRIPT This patient presented an increase in dyspnea and cough (e-Table 1, patient 6) with no manifestations of respiratory infection, no fever, neither purulent sputum and performed hemocultures were negative. This patient was treated with 1 g of

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prednisone as a single dose, alveolar infiltrate and dyspnea disappeared after 24 hours and the patient was discharged from hospital. During follow-up, two patients showed a mild bronchial infection that was reflected by an increase of

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cough and presence of sputum, these infections were resolved in both patients after azithromycin antibiotic treatment during 3 days. Additionally, no serious

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side-effects were reported during the entire study period.

Immunosuppressive and antibiotic therapies were also well tolerated, with no serious adverse effects. However some mild side-effects attributed to immunosuppressive and antibiotic therapies were observed: all patients had mild transitory cramps in the legs, one patient developed a hyperglycemia, and

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another one presented a cutaneous rash. None of the patients had any detectable anti-HLA antibodies before or after first

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and second cell instillations. After third cell instillation, however, four patients developed antibodies against both HLA-I and HLA-II, and one only against HLA-

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II. Antibodies became spontaneously negative in two of these patients two months after the last cell instillation. Regarding FVC, DLco, distance walked and oxygen saturation (SaO2) at the beginning and at the end of 6MWT, no statistically significant differences were observed between baseline and 12-months of follow-up (Table 2). Further, no changes in disease extent evaluated by HRCT and no additional structural alterations were observed (Table 2). Finally, a decrease in severity of cough and dyspnea, in the majority of transplanted patients, was also noted (e-Table

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ACCEPTED MANUSCRIPT 2, 3). Altogether, these data indicate that ATII transplantation was safe and well tolerated. Individualized evaluation of patients revealed that none of them showed disease

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progression in the first two months after the first cell instillation. However, after this time, in 13 of the 16 enrolled patients (81.25%) IPF remained stable while in the other 3 patients the disease progressed (Figure 3). In these 13 patients, the

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decline from baseline to 12-months of follow-up in DLco was -0.46 ± 6.1 % and for FVC was -0.69 ± 5.8%. Moreover, the walked distance in 6MWT tended to

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increase (55.6 ± 70.9 m). Changes in desaturation at the beginning and at the end of 6MWT between baseline and after 12-months of follow-up were -0.2 ± 0.8% and 0.1 ± 2.9% respectively. Additionally, changes in the degree of cough were 19.73 ±1.29 and in dyspnea were 3.18 ± 2.56. Finally, HRCT images did not show any structural change from baseline to 12 months of follow-up (Table

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2, e-Tables 2 and 3, Figure 4).

Conversely, in the remaining 3 patients, the decline from baseline to 12-months

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of follow-up in DLco was -10.3 ± 6.6 % and for FVC was -15.3 ± 4.6 %. Walked distance in 6MWT was also decreased (-116.3 ± 56.1 m) and changes in

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desaturation at the beginning and at the end of the 6MWT were -3.0 ± 1.0% and -13.3 ± 2.3% respectively. In addition, these patients also showed an increase in the degree of cough (13.67 ± 6.35) and dyspnea (-7.67 ± 2.31) (e-Table 3 and 4). Differences between these 3 patients and the other 13 were found in baseline measures, concretely in DLco. The 13 patients that remained stable had a DLco above 40%, while the other 3 patients with progressive disease had lower values for this parameter.

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ACCEPTED MANUSCRIPT DISCUSSION Cell therapies represent a promising therapeutic strategy for IPF. To the best of our knowledge, this is the first study which investigates intratracheal epithelial

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cell transplantation in IPF patients. Our results showed that ATII intratrachealtransplantation is safe, well tolerated and not associated with significant adverse effects.

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This study shows that ATII intratracheal-transplantation has a satisfactory safety profile in all the enrolled patients. In this sense, it is important to remark that

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after 24 h of each cell instillations all patients were released from the hospital as none of them showed severe or clinically significant side-effects. Moreover, no patients showed any signs of pulmonary infection. The only mild side-effect was observed in one patient (patient 6) who presented an alveolar infiltrate 2 h after the second cell transplantation. This incident was resolved

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after 24 h and was not observed in any other cell administration. In this case, the patient was discharged from the hospital after 24 h as usual. This event

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could be attributed to the fiber-optic bronchoscopy procedure rather than a rejection episode as this patient showed no anti-HLA before ATII transplantation

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or during the 12-months of follow-up. Additionally, results indicate that there was no deterioration of lung function and/or disease progression during followup, directly related to cell administration. All in all, these data suggest that epithelial cell therapy in IPF is feasible and reliable. A matter of concern could be the immune response triggered against instilled cells. A relationship between outcome and alloantibody status has been reported in different heterologous cell therapies (13, 14). However, in this study, no clinical, functional or radiological findings suggestive of rejection of instilled

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ACCEPTED MANUSCRIPT cells were observed. It is important to remark that none of the patients produced antibodies that can be detected by cytotoxicity. Although five patients (31.25 %) developed anti-HLA antibodies detected by more sensitive Solid Phase

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techniques, at the end of follow-up these antibodies become negative in two of the patients. In no case a relationship between the presence of these antibodies and disease evolution was found. In fact, the rate of sensitization due to ATII

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intratracheal-transplantation is similar to that observed in a blood transfusion (15). In our case they react with a reduced number of specificities, no one of the

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recipients enters in to the rage of Highly Sensitized. The low level of sensitization observed in our patients probably would not difficult a future organ transplantation more than a blood transfusion.

Although immunosuppressive and prophylactic treatment was well tolerated, some mild side-effects characteristics of these drugs were observed. The most

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common side-effect associated to tacrolimus is the presence of cramps in the lower limbs (16). All study patients reported this effect, however, after dose

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adjustment cramps disappeared in all of them. Therefore, this seems to be related to tracrolimus. Another side-effect linked to these drugs was the

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cutaneous rash reported by one single patient. This effect was associated to trimethoprim-sulfamethoxazole as is one of the typical side-effects of this drug. In this case, the rash disappeared spontaneously after some days with no modification the dose. The last side-effect related to immunosuppressive treatment was a hyperglycemia observed only in one patient. Hyperglycemia is a common side-effect in patients treated with steroids, decreasing the dose was enough to reverse it.

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ACCEPTED MANUSCRIPT After 12-months of follow-up, we have observed that disease seems to remain stable in 13 of the 16 patients. The threshold values currently used to define significant decline in lung function parameters are a reduction in baseline values

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of 15% for DLco and 10% for FVC (9, 10), although some reports suggest that disease should only be regarded stable if the FVC has declined by < 5% (17, 18). In our study, changes observed in these 13 patients were less than 5% for

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FVC and less than 15% for DLco.

In these 13 patients, we also found a significant increase in the walked distance

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in 6MWT and a stabilization of SaO2 at the end of 6MWT. Moreover, these patients reported an improvement on health-related quality of life after ATII transplantation, mainly due to a favorable effect on the degree of cough and dyspnea. Finally, none of these patients showed increases in the extent of disease evaluated by HRCT. All together, these results indicate that ATII

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transplantation might exert a favorable effect in the stabilization of IPF. It is noteworthy the difference between these 13 patients and the other 3 in

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which disease progressed. The only parameter that showed differences was DLco at the beginning of the study, being below 40% in these 3 patients, while

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in the other 13 patients DLco was above this level. Although the low number of patients could have limited the ability to detect relevant differences, this results might suggest that the values of DLco at the beginning of cell treatment may establish a threshold below which ATII intratracheal-transplantation is no longer able to have any effect. An additional aspect to consider is that the majority of enrolled patients received IPF treatment before cell therapy (14 patients from the 16 total). They were treated with N-acetylcisteine associated or not to prednisone and azathioprine,

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ACCEPTED MANUSCRIPT or bosentan. In this context, it is important to point out that several clinical trials have demonstrated that these drugs do not halt disease progression (19). In this study all enrolled patients had a progressive disease, thus, previous

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pharmacological treatments probably did not have a positive effect in IPF. Taking into account that ATII express HLA-I and HLA-II molecules, ATII transplantation should be administrated together with immunosuppressive drugs

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and prophylaxis. Thus, the observed stabilization of IPF in these 13 patients could be due to some of these drugs. It has been reported that the addition of

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co-trimoxazole to standard treatment for fibrotic idiopathic interstitial pneumonia resulted in an improved quality of life, however it has no effect on lung function (20). Our study patients were treated with co-trimoxazole for 48 weeks, which could have influenced the improvement in quality of life observed. Our patients were also treated with prednisone and mycophenolate mofetil that could be

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related to the disease stabilization. Nonetheless, it have been reported that steroids and mycophenolate mofetil do not have any proven efficacy in IPF (21,

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22). Finally, there are no data for tacrolimus and IPF treatment, therefore, the effect of this drug on IPF evolution cannot be predicted. Although it seems

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unlikely that none of the drugs individually is related to observed effects, combination of all them can provide unexpected synergies. Currently, there are no clinical trials in IPF with the same combination of immunosuppressive and prophylactic drugs that we have administered. However, Elicker et al. demonstrated that, in IPF patients who underwent unilateral lung transplantation, combination of prednisone, mycophenolate mofetil and tacrolimus did not prevent fibrosis progression in the native lung

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ACCEPTED MANUSCRIPT (23).Therefore, more studies are needed to identify which is the ultimately responsible of the stabilization of disease in ATII cell therapy. After procedure, patients have been monitored for a year. Although the results

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could be considered encouraging, further studies are needed to determine longterm effects of ATII transplantation and establish the mechanisms involved in this cell therapy. However, we can speculate on some explanations for the

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apparently favorable effects of ATII administration. Possible mechanism included the well-known role played by ATII as ATI progenitors in response to

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injury (24, 25). Thus, instilled ATII may proliferate, leading to an increased number of alveolar cells for resolving disrupted alveolar surfaces. Although there are no data on the fate of ATII instilled in patients, in our previous experimental studies, we effectively observed engraftment and proliferation of these cells (5, 6).

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On the other hand, ATII has a number of physiological functions, including synthesis, secretion and turnover of pulmonary surfactant (24, 26, 27). Thus,

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the effects of this cell therapy could also be related with restoring the pulmonary surfactant pool, helping to open up collapsed but still functional alveoli and

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thereby enhancing breathing capacity. In this line, in an animal model of bleomycin-induced pulmonary fibrosis, we have recently demonstrated that ATII transplantation was able to restore synthesis and release of pulmonary surfactant proteins (6). This could help to understand the transitory effect observed in the 3 patients who only showed stabilization during the first two months of cell treatment. Certainly, this study has limitations and the most obvious is the lack of a control group. To apply the instillation of vehicle and immunosuppressive and

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ACCEPTED MANUSCRIPT prophylactic drugs to IPF patients was ethically unacceptable by our ethical committee. Another limitation, in this case to interpret the mechanisms underlying procedure, is the impossibility to track administered cells. In previous

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experimental studies we demonstrated that, after instillation, ATII were engrafted in pulmonary parenchyma and acquired characteristics of ATI (5, 6). Such confirmation could not be addressed in this study without lung biopsies

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and therefore we could only speculate on the mechanisms post-instillation.

In conclusion, this study indicates that ATII intratracheal-transplantation is safe

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and well tolerated in IPF patients. This study opens the door to design a clinical

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trial to elucidate the potential beneficial effects of the ATII cell therapy.

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ACKNOWLEDGMENTS

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Guarantor statement: Anna Serrano-Mollar and Antoni Xaubet

Authors’ Contribution:

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Anna Serrano-Mollar: She contributed to the design, implementation and coordination of the study. She also participated in the development of protocols for cell extraction and cryopreservation, in the cell extraction as well as in the monitoring of patients. She participated in data collection, and in their analysis and interpretation. She also wrote the manuscript. Gemma Gay-Jordi: She contributed to cell extraction. She participated in data collection, and in their analysis and interpretation. She also participated in the writing of the manuscript.

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Raquel Guillamat-Prats: She contributed to cell extraction. She participated in data collection, and in their analysis and interpretation. Daniel Closa: Hi contributed to the design of the study. He also contributed to writing of the manuscript.

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Fernanda Hernandez-Gonzalez: She contributed to the alveolar type II cell transplantation, and data collection and interpretation.

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Pedro Marin: He contributed to the development of the cryopreservation and microbiological protocol for the cell viability. Felip Burgos: He performed the pulmonary function test. He also participated in data interpretation and in the writing of the manuscript. Jaume Martorell: He performed the immunological tests, and also participated in data interpretation and in the writing of the manuscript. Marcelo Sánchez: He performed the high-resolution computed tomography and its quantification. He also participated in data interpretation and in the writing of the manuscript. Pedro Arguis: He performed the high-resolution computed tomography and its quantification. He also participated in data interpretation and in the writing of the manuscript.

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ACCEPTED MANUSCRIPT Dolors Soy: She contributed to the setting up of the post-transplantation treatment (immunosuppressive antimicrobial prophylaxis). José M Bayas: He contributed to the setting up of the vaccination program. He also participated in the writing of the manuscript.

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José Ramirez: He participated in the diagnosis of patients. Teresa D. Tetley: She contributed to the development of protocols for cell extraction and cryopreservation. Laureano Molins: He participated in the diagnosis of the patients.

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Jordi Puig de la Bellacasa: He participated in the microbiological studies of the cells. He also participated in the writing of the manuscript.

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Camino Rodríguez-Villar: She participated in the establishment of the donor selection criteria. She contributed to the extraction of donor lungs and to the testing of their viability. Irene Rovira: She contributed to the implantation of the cells. She is the anesthetist. She also participated in the writing of the manuscript. Juan José Fiblà: He participated in the diagnosis of the patients.

Funding

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Antoni Xaubet: He contributed to the design, implementation and coordination of the study. He performed the alveolar type II cell transplantation. He participated in data collection, and in their analysis and interpretation. He also wrote the manuscript.

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This work was supported by grants from Fundación Genoma España, Consejo Superior de Investigaciones Científicas (CSIC), Ministerio de Sanidad Política Social e Igualdad (TRA-020 and EC-10-89), Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (PI12/01122 and PI13/00282). “Cofinanciado por el Fondo Europeo de Desarrollo Regional (FEDER). Unión Europea. Una manera de hacer Europa” and by Fundació la Marató de TV3 (MTV3 122410). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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ACCEPTED MANUSCRIPT REFERENCES 1. Raghu G, Collard HR, Egan JJ, et al. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence – based guidelines for

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diagnosis and management. Am J Respir Crit Care Med 2011;183:788824.

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3. Phan SH. Biology of fibroblasts and myofibroblasts. Proc Am Thorac Soc

4. Wynes MW, Edelman BL, Kostyk AG, et al. Increased cell surface Fas expression is necessary and sufficient to sensitize lung fibroblasts to Fas ligation-induced apoptosis: implications for fibroblast accumulation in

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idiopathic pulmonary fibrosis. J Immunol 2011;187:527-37. 5. Serrano-Mollar A, Nacher M, Gay-Jordi G, et al. Intratracheal-

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transplantation of alveolar type II cells reverses bleomycin-induced lung fibrosis. Am J Respir Crit Care Med 2007;176:1261-1268.

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6. Guillamat-Prats R, Gay-Jordi G, Xaubet A, et al. Alveolar type II cell transplantation restores pulmonary surfactant protein levels in lung fibrosis. J Heart Lung Transplant. 2014;33:758-65.

7. Serrano-Mollar A, Closa D, Bulbena O. Use of type II pneumocytes in the treatment of pulmonary diseases associated with pulmonary fibrosis. Granted European patent EP1961423, based on Spanish priority patent application ES200502939.

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ACCEPTED MANUSCRIPT 8. Noble PW, Albera C, Bradford WZ, et al. Pirfenidone in patients with idiopathic pulmonary fibrosis (CAPACITY): two randomised trials. Lancet. 2011;377:1760-1769.

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9. Flaherty KR, Andrei AC, Murray S, et al. Idiopathic pulmonary fibrosis: prognostic value of changes in physiology and six minute hallwalk. Am J Respir Crit Care Med 2006;174:803-809.

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10. du Bois RM, Weycker D, Albera C, et al. Forced vital capacity in patients with idiopathic pulmonary fibrosis: test properties and minimal clinically

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important difference. Am J Respir Crit Care Med 2011;184:1382-1389. 11. Danzinger-Isakov L, Kumar D. AST Infectious Diseases Community of Practice. Guidelines for vaccination of solid organ transplant candidates and recipients. Am J Transplant 2009; Suppl 4: S258-262. 12. Xaubet A, Agustí C, Luburich P, et al. Pulmonary function tests and CT

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2006;355:1318-1330. 15. Karpinski M, Pochinco D, Dembinski I, et al. Leukocyte reduction of red blood cell transfusions does not decrease allosensitization rates in

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ACCEPTED MANUSCRIPT potential kidney transplant candidates. J Am Soc Nephrol. 2004;15:81824. 16. http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_(accessed

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_Product_Information/human/000712/WC500022234.pdf May 2015).

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of mortality for patients with idiopathic pulmonary fibrosis. Am J Respir

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18. Best AC, Meng J, Lynch AM, et al. Idiopathic pulmonary fibrosis: physiologic test, quantitative CT indexes, and CT visual scores as predictors of mortality. Radiology 2008;246:935-940. 19. Spagnolo P, Wells AU, Collard HR. Pharmacological treatment of

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ACCEPTED MANUSCRIPT 24. Fehrenbach H. Alveolar epithelial type II cell: defender of the alveolus revisited. Respir Res 2001;2:33–46. 25. Zoz DF, Lawson WE, Blackwell TS. Idiopathic pulmonary fibrosis: a

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27. Burkhardt A. Alveolitis and collapse in the pathogenesis of pulmonary

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fibrosis. Am Rev Respir Dis. 1989:140:513-524.

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ACCEPTED MANUSCRIPT Tables: Table 1. Demographics and clinical characteristics of study patients (n=16) at baseline. Median

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(minimum-maximum) (n=16) Age (years)

61.5 (51 - 73)

Gender (male/ female)

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13/3

Smoking history (n) Once smoked (former)

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Time since diagnosis (months) Score of Basal Dyspnea Index (score 0-12) *

21 (6 - 36 )

9 (4 - 12)

Score of Leicester questionnaire (score 3-21) *

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FVC (liters)

18.9 (5.50 - 21)

FVC (% of predicted value)

DLco

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FEV1/FVC%

3 (1.7 - 5.6) 67 (50 - 105) 83.5 (61 - 90.1) 13.2 (8.6 - 19)

(ml/min/mmHg)

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DLco (% of predicted value) AaPO2 at rest (mmHg)

48.5 (35 - 59) 19.4 (7.6 - 39.7)

6-minute walk test Arterial oxygen saturation, initial (%)

95.5 (92 - 98)

Arterial oxygen saturation, final (%)

88 (73 - 95)

Distance (meters)

552.5 (342 - 690)

HRCT scan score (%) Overall disease extent

26.5 (10 – 40)

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ACCEPTED MANUSCRIPT *A higher score indicates better function. FVC denotes forced vital capacity, FEV1/FVC % of forced expiratory volume in 1 second to FVC, DLco singlebreath diffusing capacity for carbon monoxide, AaPO2 alveolar-arterial oxygen

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partial pressure gradient and HRCT high-resolution computed tomography.

Table 2. Changes from diagnosis to 12-months of follow-up in pulmonary

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function testing, 6-minute walk test, SaO2 at the beginning and at the end of the 6-minute walk test and the HRCT, in all the study patients (n=16).

n =16

p

Median (minimum-maximum)

FVC (%)

67 (50 – 105)

63.5 (32 – 105)

0.947

DLco (%)

48.5 (35 – 59)

47 (21 - 63)

0.782

552.5 (342 – 690)

574 (364 – 738)

0.752

95.5 (92 – 98)

95 (97 – 88)

0.159

88 (73 – 95)

88 (64 – 94)

0.676

26.5 (10 – 40)

26.5 (16 – 42)

0.966

6MWT Distance (m)

6MWT

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SaO2% initial

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Median (minimum-maximum)

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n = 16

12-months

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Baseline

6MWT

SaO2% final

HRCT scan score (%)

Overall disease extent

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and HRCT high-resolution computed tomography

Figure legends

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Figure 1. Clinical study flow diagram. DLco denotes single-breath diffusing capacity for carbon monoxide, FVC forced vital capacity, 6MWT 6-minute walk

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test and HRCT high-resolution computed tomography.

Figure 2. Alveolar type II cells. A) Cytospin preparation showing alkaline phosphatase-positive alveolar type II cells (in red color). Magnification x 200. B and C) Transmission electron micrographs of alveolar type II cells (arrows)

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showing lamellar bodies. D) Detail of alveolar type II cell. E) Detail of the lamellar bodies. F) Representative histogram of flow cytometry analysis of

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CD32 in isolated cells. Cells were immunostained with PE-labelled anti CD32 antibody to evaluate the macrophage population in the isolated cells. H)

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Representative histogram of flow cytometry analysis of p180 lamellar body in isolated cells. Cells were immunostained with PE-labelled anti p180 lamellar body antibody to evaluate ATII in the isolated cells.

Figure 3. Changes from diagnosis-baseline to 12-months in all the study patients, data in graph represent individual data points and mean (black squares). Solid line: patients who remained stables n=13, dotted line: patients with deterioration of the disease n=3. A) FVC (%), B) DLco (%) C) 6MWT

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test and HRCT high-resolution computed tomography.

Figure 4. High-resolution computed tomography (HRCT). HRCT images

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representative from two different patients of the 13 patients that remained stable (A and B) at baseline and after 12-months of follow-up. No changes were

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observed in the disease extent between baseline and 12-months of follow-up.

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