The donor organ shortage in clinical pulmonary transplantation could be further improved by the use of pulmonary lobes of living related donors. In this project, a porcine model of left lung transplantation will be used as a model of lung lobe transplantation to evaluate methods of improved graft preservation of living related lung donors. This is of importance because the standard technique of antegrade perfusion through the trunk of the pulmonary artery for preservation in cadaver donors cannot be used for living lung lobe donation. Thus alternative techniques taking into account prolonged warm ischemia during dissection of the lobe have to be developed. Using our established porcine lung transplantation model, pulmonary function will be monitored for a 7 hour period after LTx. Monitoring will include analysis of respiratory and hemodynamic data, analysis of surfactant function from bronchoalveolar lavages as well as tissue myeloperoxidase assays, in order to quantify tissue damage. Multiple techniques using both Celsior and low-potassium-dextrane preservation solutions, the two most commonly used solutions, will be evaluated. In conclusion, living related donor lung transplantation has not only the general potential to increase the donor organ pool, but the technique could potentially also improve outcome due to the use of healthier donor lungs (as compared with cadaver donation) providing, however, that the issue of preservation is resolved.

Bronchiolitis obliterans syndrome (BOS) remains to be a critical factor, determining long term survival and quality of life following lung transplantation. While the diagnosis of BOS is based on clinical findings, the underlying pathology is characterized by chronic inflammatory processes, accompanied by altered tissue repair. Recently, it has been demonstrated that, in addition to granulocytes and macrophages, lymphocytes are significantly involved in this process, also. However, there is still lack of knowledge regarding the mechanisms that direct the activation of T- and B-lymphocytes and their consequent migration to the transplanted lung. In the previous funding period, we proposed to establish a mouse tracheal transplantation model to study the role of specific factors, including donor age, ischemia time and specific chemokines and their receptors, in order to analyze their role in leukocyte infiltration and the development of BOS.
Resulting data demonstrate, that ischemia induces specific changes in the tissue similar to those obtained after allogeneic transplantation. Moreover, the experimental data strongly suggests, that while ischemia itself leads to downstream changes, it renders the graft susceptible to additional processes, which are independent of but triggered by an anti-allogeneic response of the recipient. Surprisingly and in contrast to data of the ISHLT and retrospective clinical studies that a donor age of above 30 may have a significant impact on the long term outcome after lung transplantation, we could not detect any difference between grafts from young and old donors in our experimental mouse model.
Finally, we analyzed whether molecules involved in T-cell homing affect the development of BOS. Unexpectedly, an effect, diametrically opposed to the initial hypothesis, was observed: Compared to controls, allogeneic (CCR7+/+) wild type tracheae transplanted into CCR7-/- recipients showed a massive infiltration of leukocytes, accompanied by a significant obstruction of the airway lumen. This may suggest, that the defect in CCR7-/- recipients prevent leukocytes specifically from emigrating from wild type (CCR7+/+) tissue.
Based on the above results, we now propose to analyze further, which factors are triggered by the ischemic insult and which specific types of lymphocytes are triggered to migrate into syngeneic ischemic grafts by the anti-allo response. Furthermore, we will elucidate the factors involved in the non-destructive, infiltrative and obliterative process in CCR7-/- recipients. Special emphasis will be placed on the role of T-cells and their interaction with chemokines/receptors in the development of BOS.
Finally, we will use adoptive transfer experiments to analyze a possible role of regulatory T-cells influence the process of graft preservation/destruction and the development of BOS.

Lung transplantation (LTx) provides a new therapeutic option for patients with end stage lung disease. Nevertheless, chronic graft dysfunction still represents a major obstacle. Bronchiolitis obliterans syndrome (BOS) is the resulting clinical entity, defined by a significant reduction of maximal mid-expiratory flow. Up to now, no reliable diagnostic markers for early diagnosis of BOS have been available. Therefore, the present project aims to identify diagnostic and prognostic markers by analysis of gene and protein expression profiles of cells obtained by brush aspirates and by bronchial lavage (BALF) from patients who have undergone bilateral lung transplantation. Employing microarray analysis, differentially expressed genes, e.g. HBB, CD99, HBA2, HBE1, ALDH3B2, HNRPH1, H1F0, PALMD and PSAP, were found comparing bronchoepithelial cells from LTx patients with controls. BALF and brush aspirates were taken at defined times after transplantation. Gene expression profiles of these consecutive samples revealed 17 differentially expressed genes in samples with BOS when compared to those without BOS. Of these 17 genes, GZMA, PRF1, SFTPB, MELK, RCSD1 and COL3A1 were upregulated in samples from LTx patients with BOS. Moreover, a highly reproducible method for proteome analysis from BALF was established. Important steps are for example the application of paramagnetic beads with a C1 or C8 surface to separate peptides and proteins with a hydrophobic functionality. HBB, which was found to be overexpressed by gene expression analysis, was also identified by proteome analysis.
Four major aims have to be targeted for the next funding period: 1) The results have to be evaluated on a large cohort of LTx patients who do and do not develop BOS after lung transplantation to identify new differentially expressed genes and proteins. 2) It has to be investigated whether the differentially expressed genes and proteins can be used as candidate biomarkers for the prediction of BOS. 3) The data obtained by global gene and protein expression analyses have to be validated using other methods like real-time PCR and immunohistochemistry. 4) Bioinformatic analyses will help to clarify whether deregulated genes and proteins belong to signal transduction pathways that may play a role in the pathogenesis of BOS.
Thus, global expression analyses of bronchial epithelial cells is a new approach to identify new diagnostic markers and to gain further insight into the pathogenesis of inflammatory processes or into further studies of bronchiolitis obliterans syndrome.

Living donor organ transplantation offers several unique advantages in the setting of tolerance induction. These comprise the availability of donor material such as leukocytes well-ahead of the procedure and the possibility of planning the time of transplantation in order to enable conditioning protocols of the recipient. We wish to perform experiments evaluating protocols to induce peripheral T cell regulation of alloantigens in a modified preclinical allogeneic porcine lung transplantation system modelling living-related donation. T cell regulation has the potential to revolutionize the field of transplantation, but its usefulness in large animals and humans remains to be proven. It is likely that partially MHC-matched donors and recipients in living-related lung transplantation will be especially suitable for the application of T cell regulation. Successful results from these experiments are planned to be transferred to our clinical program in due course in the future.

Type II alveolar epithelial cells (AT2 cells) have important functions, including the production of surfactant and regeneration of lost type I alveolar epithelial cells (AT1 cells). Therefore, the ability of in vitro production of human AT2 cells would offer a variety of new therapeutic options in treating pulmonary injuries and diseases, including genetic disorders and ischemia-reperfusion-injury after lung transplantation. We have now established highly efficient formation of mesendoderm and differentiation of AT2 cells from murine ES cells (mESCs) under serum-free conditions as well as robust differentiation of mesendoderm from rhesus monkey ES-cells (RESCs). Moreover lentiviral vectors mediating highly AT2 cell specific GFP and dsRed expression have been constructed and characterized in detail.
In our established mouse ES cell system, we now aim (1) to identify key factors of AT2 differentiation, (2) to proof functionality of mouse ES-cell derived type II alveolar epithelial cells in a murine pulmonary cell transplantation model and (3) to establish generation of AT2 cells from rhesus monkey ES-cell derived mesendodermal progenitors. The project represents a logical continuation of our recent work on differentiation of AT2 cells from mESCs and represents an important step towards cell-based restoration of destructed lung epithelium or lost surfactant production.

The bronchiolitis obliterans syndrome (BOS) represents a major obstacle to long-term survival after lung transplantation (LTx). BOS has to be considered as the main cause for a 5-year-survival after LTx not exceeding 50% (ISHLT). Recently we could demonstrate for the first time that recipient cells of bone marrow origin contribute to the obliterating intrabronchial mesenchymal proliferation. In this proposal, we aim to investigate the mechanisms which govern the recruitment of recipient derived fibroblast precursor cells and activate them to proliferation and matrix production. By combining laser microdissection with quantitative real-time PCR on the one hand and fluorescent double labeling with confocal laser microscopy on the other, we aim to identify the cellular sources of fibrogenic cytokines (e.g. SDF-1, SLC, TGF-ß, CRC7L, CXCL12/CXCR4L). The activation status of myofibroblasts will be examined by the analysis of collagen and matrix proteinase gene expression. Furthermore, the mRNA profile of activated myofibroblasts will be determined. This profile and other fibroblast specific mRNA-species will be used to detect fibroblast precursors in the mononuclear blood cell fraction of LTx-patients and their number will be related to functional and clinical outcome data. With the help of an established micro RNA array resident and activated pulmonary fibroblasts will be investigated for the expression of these regulatory molecules. Because BOS is accompanied by remodelling of the vasculature, vascular endothelial cells will be analyzed for their donor- or recipient derivation as has been done for fibroblasts. We expect that with the help of these studies mechanisms of recruitment and activation of mesenchymal cells in remodelling fibrogenic processes within lung transplants undergoing chronic injury will be delineated. Finally, we aim to identify potential target molecules, which govern the deteriorating processes in BOS, and to develop diagnostic instruments useful in monitoring patients after lung transplantation.

Living donor lung transplantation could alleviate the donor organ shortage, could provide improved HLA-matching and could enable pretreatment protocols aimed at the deliberate induction of transplantation tolerance. The aim of the herein described project is to evaluate psychological, physiologic and immunologic parameters in young patients undergoing living-related lung transplantation in a newly implemented clinical program. Control patients will be age-matched recipients of cadaver lungs. Psychological parameters to be evaluated longitudinally will comprise perception of health-related quality of life, signs and symptoms of anxiety and depression, and interpersonal and familial coping strategies. Physiologic parameters include routine lung function tests (VCmax, FEV1), analyses of exhaled nitric oxide, blood gas analyses (arterial and/or capillary), chest radiographs, bronchoscopies with bronchoalveolar lavages and ergospirometry tests. Immunologic parameters will be evaluated in an attempt to characterize T cell-mediated mechanisms of alloantigen-regulation. This includes FACS-assays and slide based cytometry for phenotype analyses of leukocyte population, and mixed lymphocyte reaction assays for functional analyses of T cell regulation. We hypothesize that living donor lung transplantation will lead to improvements of multiple of the aforementioned parameters as compared to control recipients of cadaver lungs. In conclusion, the implementation of a living donor lung transplantation program will make this procedure available in the Eurotransplant zone for the first time, opening exciting prospects for the scientific monitoring of the program described in this project.

The extent of the lung transplantation (LTx) program at Hannover Medical School allows the development of a worldwide unique data and material base of human and animal tissues that can be extremely useful for a multitude of scientific and clinical questions. During the first funding period, BAL, pulmonary brush aspirates and blood samples were already collected from LTx patients for use in project 3. With regard to the growing demand for collection of such materials including lung biopsies from lung transplanted patients and large animal recipients, a Central Service Unit for obtaining, processing, storage, and documentation of such materials will be established. The resulting data base of the Central Service Unit will be integrated into the already existing clinical data base. In general, all procedures, which have been previously applied in project 3, like bronchial-alveolar lavage, bronchial brushing, as well as collection of lung tissue samples will be accessible for the clinical, as well as for the experimental projects.
In addition, a surveillance biopsy program will be established in all patients in the first 12 months after lung transplantation. In preliminary studies with indication bronchoscopies, we could demonstrate that a biopsy program is feasible, safe, and important for clinical decisions. The specimen obtained in the surveillance biopsy program are essential for the projects no. 3 and 9, but will be available for all other projects with requirement of human biopsy material.
The establishment of this Central Service Unit will be of major importance for sustainability for the clinical research group during and beyond the granted period.