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Im Folgenden wird die Planung und Organisation eines Kochworkshops beschrieben, der von Studierenden der FH für Gesundheitsberufe, Studiengang Diätologie durchgeführt wurde. Der Workshop wurde im Rahmen der Lehrveranstaltung Projektmanagement mit und für die Schülerinnen und Schüler der 4. Klasse einer privaten Volks- und Mittelschule konzipiert.

Living organisms use a large repertoire of anabolic and catabolic reactions to maintain their physiological body functions, many of which include oxidation and reduction of substrates. The scientific field of redox biology tries to understand how redox homeostasis is regulated and maintained and which mechanisms are derailed in diverse pathological developments of diseases, where oxidative or reductive stress is an issue. The term “oxidative stress” is defined as an imbalance between the generation of oxidants and the local antioxidative defense. Key mediators of oxidative stress are reactive species derived from oxygen, nitrogen, and sulfur that are signal factors at physiological concentrations but can damage cellular macromolecules when they accumulate. However, therapeutical targeting of oxidative stress in disease has proven more difficult than previously expected. Major reasons for this are the very delicate cellular redox systems that differ in the subcellular compartments with regard to their concentrations and depending on the physiological or pathological status of cells and organelles (i.e., circadian rhythm, cell cycle, metabolic need, disease stadium). As reactive species are used as signaling molecules, non-targeted broad-spectrum antioxidants in many cases will fail their therapeutic aim. Precision medicine is called to remedy the situation.

Ventilator-induced lung injury (VILI) is a major contributor to morbidity and mortality in critically ill patients. Mechanical damage to the lungs is potentially aggravated by the activation of the renin-angiotensin system (RAS). This article describes RAS activation profiles in VILI and discusses the effects of angiotensin (Ang) 1-7 supplementation or angiotensin-converting enzyme (ACE) inhibition with captopril as protective strategies.Animal study.University research laboratory.C57BL/6 mice.Anesthetized mice ( n = 12-18 per group) were mechanically ventilated with low tidal volume (LV T , 6 mL/kg), high tidal volume (HV T , 15 mL/kg), or very high tidal volume (VHV T , 30 mL/kg) for 4 hours, or killed after 3 minutes (sham). Additional VHV T groups received infusions of 60 μg/kg/hr Ang 1-7 or a single dose of 100 mg/kg captopril.VILI was characterized by increased bronchoalveolar lavage fluid levels of interleukin (IL)-6, keratinocyte-derived cytokine, and macrophage inflammatory protein-2 (MIP2). The Ang metabolites in plasma measured with liquid chromatography tandem mass spectrometry showed a strong activation of the classical (Ang I, Ang II) and alternative RAS (Ang 1-7, Ang 1-5), with highest concentrations found in the HV T group. Although the lung-tissue ACE messenger RNA expression was unchanged, its protein expression showed a dose-dependent increase under mechanical ventilation. The ACE2 messenger RNA expression decreased in all ventilated groups, whereas ACE2 protein levels remained unchanged. Both captopril and Ang 1-7 led to markedly increased Ang 1-7 plasma levels, decreased Ang II levels, and ACE activity (Ang II/Ang I ratio), and effectively prevented VILI.VILI is accompanied by a strong activation of the RAS. Based on circulating Ang metabolite levels and tissue expression of RAS enzymes, classical ACE-dependent and alternative RAS cascades were activated in the HV T group, whereas classical RAS activation prevailed with VHV T ventilation. Ang 1-7 or captopril protected from VILI primarily by modifying the systemic RAS profile.

Acute respiratory distress syndrome (ARDS) is a major concern in critical care medicine with a high mortality of over 30%. Injury to the lungs is caused not only by underlying pathological conditions such as pneumonia, sepsis, or trauma, but also by ventilator-induced lung injury (VILI) resulting from high positive pressure levels and a high inspiratory oxygen fraction. Apart from mechanical factors that stress the lungs with a specific physical power and cause volutrauma and barotrauma, it is increasingly recognized that lung injury is further aggravated by biological mediators. The COVID-19 pandemic has led to increased interest in the role of the renin-angiotensin system (RAS) in the context of ARDS, as the RAS enzyme angiotensin-converting enzyme 2 serves as the primary cell entry receptor for severe acute respiratory syndrome (SARS) coronavirus (CoV)-2. Even before this pandemic, studies have documented the involvement of the RAS in VILI and its dysregulation in clinical ARDS. In recent years, analytical tools for RAS investigation have made major advances based on the optimized precision and detail of mass spectrometry. Given that many clinical trials with pharmacological interventions in ARDS were negative, RAS-modifying drugs may represent an interesting starting point for novel therapeutic approaches. Results from animal models have highlighted the potential of RAS-modifying drugs to prevent VILI or treat ARDS. While these drugs have beneficial pulmonary effects, the best targets and application forms for intervention still have to be determined to avoid negative effects on the circulation in clinical settings.

Background Primary graft dysfunction (PGD) after lung transplantation (LTx) carries significant morbidity and mortality in the early post-operative period and is associated with the development of chronic lung allograft dysfunction. AP301, an activator of epithelial sodium channel–mediated Na + uptake represents a new concept for prevention and treatment of pulmonary edema and has shown promising results in the pre-clinical setting. This pilot study investigated the clinical effect of inhaled AP301 on patients with development of PGD > 1 according to International Society of Heart and Lung Transplantation criteria after primary LTx in a high-volume center and was conducted as a randomized, placebo-controlled, single-center pilot-study including 20 patients. All consecutive patients fulfilling inclusion criteria were screened for PGD at arrival on the intensive care unit (ICU) after LTx. After randomization, inhaled AP301 or placebo was administered by nebulizer twice daily for 7 days or until extubation. Otherwise, patients were treated according to routine clinical protocol. Partial pressure of arterial oxygen (Pa o 2 )/fraction of inspired oxygen (F io 2 ) values were obtained until extubation and assessed as a primary outcome parameter. Patients were monitored for 30 days within the study protocol. Results From July 2013 to August 2014, 20 patients were randomized 1:1 to AP301 (Group 1) or placebo (Group 2). Both groups were comparable with regard to sex (40% women/60% men vs 50% women/50% men), mean age (55 ± 13 vs 54 ± 6 years), comorbidities, and diagnosis leading to LTx. The Pa o 2 /F io 2 ratio at the time of inclusion was comparable in both groups, with a mean 235.65 ± 90.78 vs 214.2 ± 95.84 ( p = 0.405), and there was no significant difference in the extravascular lung water index (13.88 ± 5.28 vs 16 ± 6.29 ml/kg, p = 0.476). The primary end point was mean Pa o 2 /F io 2 ratio values between baseline and Day 3. In the AP301 group, only 1 patient was ventilated at Day 4 and no patients were ventilated after Day 4. In the placebo group, 5 patients were ventilated on Day 4 and 2 patients on Days 5, 6, and 7. The mean increase in the Pa o 2 /F io 2 ratio was significantly higher in Group 1 patients, and the mean between baseline and at 72 hours was 365.6 ± 90.4 in Group 1 vs 335.2 ± 42.3 in Group 2 ( p = 0.049). The duration of intubation was shorter in Group 1 than in Group 2 patients (2 ± 0.82 vs 3.7 ± 1.95 days; p = 0.02). ICU stay was 7.5 ± 3.13 days in Group 1 vs 10.8 ± 8.65 days in group 2 ( p = 0.57). Survival at 30 days was 100%. No severe adverse events were recorded. Conclusions This study was designed as a proof-of-concept pilot study. Although it was not powered to achieve statistical significances, the study demonstrated relevant clinical effects of inhaled AP301 on patients with PGD after primary LTx. The improved gas exchange led to a significantly shorter duration of mechanical ventilation and a trend towards a shorter ICU stay. Further investigation of AP301 for treatment of PGD in larger studies is warranted. Trial Registration The trial is registered at https://www.clinicaltrialsregister.eu/ctr-search/trial/2013-000716-21/AT .

Background High-permeability pulmonary edema is a hallmark of acute respiratory distress syndrome (ARDS) and is frequently accompanied by impaired alveolar fluid clearance (AFC). AP301 enhances AFC by activating epithelial sodium channels (ENaCs) on alveolar epithelial cells, and we investigated its effect on extravascular lung water index (EVLWI) in mechanically ventilated patients with ARDS. Methods Forty adult mechanically ventilated patients with ARDS were included in a randomized, double-blind, placebo-controlled trial for proof of concept. Patients were treated with inhaled AP301 (n = 20) or placebo (0.9% NaCl; n = 20) twice daily for 7 days. EVLWI was measured by thermodilution (PiCCO®), and treatment groups were compared using the nonparametric Mann–Whitney U test. Results AP301 inhalation was well tolerated. No differences in mean baseline-adjusted change in EVLWI from screening to day 7 were found between the AP301 and placebo group (p = 0.196). There was no difference in the PaO2/FiO2 ratio, ventilation pressures, Murray lung injury score, or 28-day mortality between the treatment groups. An exploratory subgroup analysis according to severity of illness showed reductions in EVLWI (p = 0.04) and ventilation pressures (p

Objectives Plasminogen activator inhibitor 1 is the primary regulator of urokinase plasminogen activator and tissue plasminogen activator. Plasminogen activator inhibitor 1 is essential in the control of the thrombotic/fibrinolytic balance and is a marker of endothelial cell injury. Idiopathic dilated cardiomyopathy is reportedly associated with endothelial cell dysfunction. Whether endothelial cell damage plays a role in patients with dilated cardiomyopathy after cardiac transplantation remains unknown. Methods In this study explanted hearts of cardiac transplant recipients with ischemic cardiomyopathy and dilated cardiomyopathy, as well as control myocardial tissue, were investigated for expression of urokinase plasminogen activator, tissue plasminogen activator, urokinase plasminogen activator receptor, and plasminogen activator inhibitor 1 and 2. Furthermore, plasminogen activator inhibitor 1 expression was examined in endomyocardial biopsy specimens and sera of patients with ischemic cardiomyopathy and those with dilated cardiomyopathy during the first posttransplantation year. The effect of the patient's serum on endothelial cells was assessed in vitro to examine the role of circulating endothelial cell damage–related factors. Results Plasminogen activator inhibitor 1 expression was upregulated in ischemic cardiomyopathy and dilated cardiomyopathy myocardial tissue versus that seen in control tissue. After transplantation, plasminogen activator inhibitor 1 expression returned to control levels in patients with ischemic cardiomyopathy. In patients with dilated cardiomyopathy, plasminogen activator inhibitor 1 expression increased at 24 weeks after transplantation in both biopsy specimens and sera versus that seen in control tissue. Sera of patients with dilated cardiomyopathy, but not that of patients with ischemic cardiomyopathy, inhibited vascular endothelial growth factor A–induced proliferation of endothelial cells, although downstream target gene activation of early growth response factor 1 and NGFI-A binding protein 2 was not affected. Conclusions These data suggest for the first time that the endothelial cell damage–related process recurs in patients with dilated cardiomyopathy after transplantation, which, independently of vascular endothelial growth factor, is associated with increased plasminogen activator inhibitor 1 expression, and that this pathology might play a role in allograft remodeling in patients with dilated cardiomyopathy.

Cardiac allograft rejection is currently diagnosed from endomyocardial biopsies (EMB) that are invasive and impractical to repeat. A serological marker could facilitate rejection monitoring and minimize EMB-associated risks. We investigated the relation of serum matrix metalloprotease (MMP)-1 and vascular endothelial growth factor (VEGF)-A concentrations to cardiac allograft rejection, using 1176 EMBs and serum samples obtained from 208 recipients. Acute cellular rejection was diagnosed in 186 EMBs. Mean week 1 and week 2 serum MMP-1 concentrations predicted rejection (p = 0.001, AUC = 0.80). At the optimal cut-off level of ≥7.5 ng/mL, MMP-1 predicted rejection with 82% sensitivity and 72% specificity. Initial serum MMP-1

Background: PGD following LuTX carries significant morbidity and mortality. AP301, an activator of ENaC-mediated Na(+) uptake represents a new concept for treatment of hyper-permeability oedema with promising preclinical results. Aims: This study aims to investigate the effect of AP301 on patients with PGD≥1 at ICU arrival after LuTX. Methods: Prospective randomised, placebo-controlled pilot-study. All patients fulfilling inclusion criteria are screened for PGD at ICU arrival. After randomization AP301 or placebo is administered via nebulizer twice daily for 7 days or until extubation. Otherwise patients are treated according to clinical routine protocol and followed for 30 days within the study. Results: From 7/2013-8/2014 20 patients were randomised 1:1 to AP301 (group 1) or placebo (group 2). Demographics were comparable in both groups as well as mean PaO2/FiO2 ratio at time of inclusion (236±91 vs 214±96). Patients in group 1 had a significantly shorter duration of intubation with 2±0,8 vs 3,7±2 days (p=0,02). There was a trend towards a shorter ICU stay (7,5±3,1 vs 10,8±8,7 days) and increased PaO2/FiO2 ratio in group 1 at 24h (313±126 vs 240±90), 48h (486±59 vs 460±33), 72h (504±40 vs 427±78) and 96h postoperative (549±0 vs 370±96). Thereafter no patient in group 1 was ventilated. 30-day survival was 100%. Conclusion: This pilot study showed impressive clinical effects of AP301 on patients with PGD after primary LuTX leading to a significantly shorter intubation time and a trend towards improved gas exchange and shorter ICU stay. Further investigation of AP301 for PGD treatment is warranted.

Objective Azithromycin has become a standard of care in therapy of bronchiolitis obliterans following lung transplantation. Matrix metalloprotease-9 broncho-alveolar lavage levels increase in airway neutrophilia and bronchiolitis obliterans. Interleukin-17 may play a role in lung allograft rejection, and interleukin-12 is downregulated in bronchiolitis obliterans. Whether these mechanisms can be targeted by azithromycin remains unclear. Methods Bronchiolitis obliterans was induced by transplantation of Fischer F344 rat left lungs to Wistar Kyoto rats. Allografts with azithromycin therapy from day 1 to 28 or 56 and mono- or combination therapy with the broad-spectrum matrix metalloprotease inhibitor tanomastat from day 1 to 56 were compared to control allografts and isografts. Graft histology was assessed, and tissue cytokine expression studied using Western blotting and immunofluorescence. Results The chronic airway rejection score in the azithromycin group did not change between 4 and 8 weeks after transplantation, whereas it significantly worsened in control allografts ( P = .041). Azithromycin+tanomastat prevented complete allograft fibrosis, which occurred in 40% of control allografts. Azithromycin reduced interleukin-17 expression ( P = .049) and the number of IL-17 + /CD8 + lymphocytes at 4 weeks, and active matrix metalloprotease-9 at 8 weeks ( P = .017), and increased interleukin-12 expression ( P = .025) at 8 weeks following transplantation versus control allografts. Conclusions The expression of interleukin-17 and matrix metalloprotease-9 in bronchiolitis obliterans may be attenuated by azithromycin, and the decrease in interleukin-12 expression was prevented by azithromycin. Combination of azithromycin with a matrix metalloprotease inhibitor is worth studying further because it prevented complete allograft fibrosis in this study.

Bronchiolitis obliterans syndrome (BOS), pathognomonic for chronic graft-versus-host disease (cGVHD) of the lung, is a progressive and often fatal complication after allogeneic hematopoietic cell transplantation (HCT). Biomarkers for the prediction and diagnosis of BOS are urgently needed to improve patients' prognosis. We prospectively evaluated B-cell subpopulations and B-cell activating factor (BAFF) in 136 patients (46 BOS, 41 no cGVHD, 49 cutaneous cGVHD) to define novel biomarkers for early diagnosis of National Institutes of Health-defined BOS diagnosed a median of 11 mo after HCT. Patients with newly diagnosed BOS had significantly higher percentages of CD19(+)CD21(low) B cells (25.5 versus 6.6%, P.0001), BAFF (7.3 versus 3.5 ng/mL, P = .02), and BAFF/CD19(+) ratio (0.18 versus 0.02 ng/10(3) CD19(+) B cells, P 5 .007) compared with patients without cGVHD. The area under the receiver operating curve for CD19(+)CD21(low) B cells was 0.97 (95% confidence interval, 0.94-0.99) and a cutoff point9% was optimal for diagnosing BOS in patients with first drop of pulmonary function tests with a sensitivity of 96% and a negative predictive value of 94%. Thus, elevated levels of CD19(+)CD21(low) B cells are a potential novel biomarker for HCT patients at risk for developing BOS at an early stage and could allow improvement of patient outcome.

Chronic graft-versus-host disease (GVHD) remains a serious complication after allogeneic hematopoietic stem cell transplantation (HCT). In 2005 the National Institutes of Health (NIH) established new criteria for chronic GVHD based on retrospective data and expert recommendations. We prospectively evaluated the incidence of NIH-defined chronic GVHD and its prognostic impact in 178 consecutive patients. The cumulative incidence of chronic GVHD at 3 years was 64, 48 and 16% for chronic classic GVHD and overlap syndrome. Prior acute GVHD and myeloablative conditioning were significantly associated with increased risk of chronic GVHD. Three-year survival (overall survival (OS)) for late-acute GVHD, chronic classic and overlap chronic GVHD when assigned on day 100 were 69, 83 and 73%. OS was significantly worse for patients with platelet counts below 100 g/l at onset of chronic GVHD (35% versus 86%, P

Background. Endomyocardial biopsy (EMB) remains the gold standard for acute cellular rejection (ACR) diagnosis in cardiac transplantation yet is subject to interobserver variability. A method that could avoid discordant EMB analysis would be desireable. The apoptosis rate in EMB correlates with ACR severity. Apollon inhibits apoptosis, and RNF41 catalyzes its degradation. Whether tissue Apollon/RNF41 could diagnose ACR is not known. This study addressed this issue. Methods. Apollon/RNF41 messenger RNA (mRNA) was measured by real time reverse-transcriptase polymerase chain reaction and apoptosis was quantified with TUNEL assays in EMBs of 268 transplant recipients. EMBs were obtained at 1, 2, 3, 4, 7, 12, 24, and 52 posttransplant weeks. Results. At all time points posttransplant, Apollon mRNA decreased significantly in EMBs with ACR grades 2R/3R combined (P≤0.0010) compared with 0/1R combined, although RNF41 mRNA significantly increased in EMBs with ACR grade 1R (P

Background— Primary graft dysfunction (PGD) is a life-threatening complication in cardiac transplantation. A sensitive, specific, and easily measurable predictor in donors could facilitate PGD prevention. Methods and Results— SMARCAL1 is a matrix-associated regulator of chromatin with helicase and ATPase activities, and its serum concentrations were significantly increased in a targeted protein array in donors whose grafts developed PGD. Therefore, this study analyzed SMARCAL1 serum concentrations by ELISA in 336 heart donors before and after aortic cross-clamping (ACC) and in recipients at 10, 30, and 60 minutes reperfusion. Demographic and hemodynamic parameters of donors and recipients as well as transplant procedure characteristics were documented. PGD (n=68) was defined as ventricular dilation and hypocontractility associated with systolic blood pressure 20 mm Hg, and decreased mixed venous oxygen saturation necessitating mechanical circulatory support. SMARCAL1 serum protein concentration was significantly increased only before and after ACC in donors ( P P Conclusions— Donor serum SMARCAL1 may serve as a specific, sensitive, and noninvasive predictive marker in the assessment of cardiac graft quality.

Idiopathic dilated cardiomyopathy (DCM) is associated with reduced myocardial vascular density and increased collagen deposition with subsequent myocardial fibrosis and heart failure. The urokinase plasminogen activator (uPA)/plasminogen activator inhibitor (PAI) system plays an important role in extracellular matrix homeostasis as well as in preserving the integrity of the vascular endothelial cell (EC) intimal layer and EC regeneration, proliferation and motility. However, the involvement of the uPA/PAI system in DCM and graft remodeling following cardiac transplantation remains unclear. Gene expression of uPA, the uPA receptor uPAR and PAI-1 was examined in left ventricular (LV) biopsies of explanted hearts in 24 ischemic cardiomyopathy (ICM) and 60 DCM patients and 12 controls by real time RT-PCR and Western blotting. Vascular endothelial growth factor (VEGF) and uPA/PAI interplay was examined in stimulated DCM and ICM myocardium in vitro. Expression of uPA and PAI-1 mRNA was evaluated in endomyocardial biopsies (EMB) at 1, 2, 3, 4, 7, 12, 24 and 52 weeks post-transplant. The mRNA levels of uPA and uPAR were unchanged in both ICM and DCM patients compared to controls. PAI-1 mRNA and protein were up-regulated in DCM compared to ICM and controls (pin vitro did not affect uPA, uPAR or PAI-1 expression. Following transplantation, PAI-1 expression returned to baseline control levels in EMB from both patient groups, although PAI-1 expression increased at 24 and 52 weeks post-transplant in DCM patients (p

Knowledge on interplay between the cardiac molecular response to transplantation-induced stress and primary graft dysfunction (PGD) is limited. A cDNA array identified HIF-1, EGR-1, NAB-2, VEGF-A and uPA as mediators of cardiac tissue response to transplantation-induced stress. mRNA expression of these molecules was measured in left ventricular biopsies from 200 donors before and after aortic cross-clamping and at 10-, 30- and 60-min reperfusion by real-time RT-PCR. HIF-1alpha expression at two time points was significantly associated with PGD, as shown by univariate analysis, receiver operating characteristic curve and multivariate logistic regression. At a cut-off level of 200 arbitrary units, HIF-1alpha after aortic cross-clamping in donors (78% sensitivity, 83% specificity) and at 10-min reperfusion (85% sensitivity, 83% specificity) identified PGD. HIF-1alpha demonstrates the potential to be a predictive marker for PGD; however, as multiple factors were tested at different time points, prospective evaluation is clearly necessary to confirm this observation.

Primary graft dysfunction (PGD) is a severe complication in lung transplantation. Therapeutic strategies are limited and there exist no predictive markers for PGD. To investigate whether vascular endothelial growth factor (VEGF) that regulates vascular permeability could predict PGD, pretransplant VEGF serum concentrations were measured in 150 lung transplant patients and 12 controls by ELISA. PGD was scored from 0 to 3 using chest radiographs and PaO(2)/FiO(2) ratios according to the International Society for Heart and Lung Transplantation guidelines. The mean graft ischemia time was 5 h 47 min and the donors' PaO(2)/FiO(2) ratios were300. PGD grades 0-3 occurred in 23%, 44%, 21%, and 11% of patients, respectively. Pre-operative VEGF serum concentrations were significantly higher in PGD grade 3 (p0.0001) versus grade 0-2 and controls. VEGF concentrations significantly predicted PGD grade 3 versus 0-2 in logistic regression analysis (p0.0001) and receiver operating analysis (AUC = 0.778). At a cut-off level ofor =650 pg/mL VEGF had 86% sensitivity and 62% specificity to identify PGD grade 3 versus 0-2. Pre-operative VEGF serum concentrations could identify lung transplant recipients with high PGD risk.

Objective: Multiple studies haveshown that matrixmetalloproteases(MMPs) areinvolvedinthepathologicreactionsoccurringasa consequence ofcardiactransplantation,includingischemia—reperfusioninjuryandallograftrejection.ThisstudysoughttodeterminethetemporalprofileofMMP serumlevelsfollowingcardiactransplantation.Methods:Endomyocardialbiopsiesandserumsampleswereobtainedfrom66recipientsat1,2,3,4, 7, 12, 24, and 52 weeks post-transplant during the routine follow-up protocol, and MMP-1, MMP-8, MMP-9, and tissue inhibitor of metalloproteases (TIMP)-1serumconcentrationsweremeasuredbyenzyme-linkedimmunosorbentassay(ELISA).ImmunosuppressioncomprisedcyclosporineA(CyA; n = 46) or tacrolimus (TAC; n = 20) with mycophenolate mofetil and steroids. Results: Increased MMP-8, MMP-9, and TIMP-1 serum levels were observedduringthefirst2weeksfollowingtransplantationcomparedtothelatertimepoints.MMP-1wasincreasedat2and3weekspost-transplant compared to all later time points. No correlation of MMP or TIMP serum concentrations with infection episodes was observed. Conclusions: Early increaseinMMPandTIMPserumlevelsfollowingcardiactransplantationindicatesinvolvementofthesemoleculesinthereactionofthetransplantto ischemia—reperfusionorearlyimmunologicadaptationprocessesofthehost.FurtherinvestigationoftherelationshipbetweenMMPandTIMPserum levels and clinical conditions following transplantation including allograft rejection and hemodynamic graft function is necessary. # 2007 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

Objective: Vascular endothelial growth factor (VEGF) is the prime regulator of angiogenesis and vascular permeability and its serum levels increase in cystic fibrosis (CF). The mechanisms of VEGF overproduction and its impact on CF lung pathology and pulmonary vascular permeability during lung transplantation are not fully understood. Methods: The expression of VEGF, its receptors, hypoxia inducible factor (HIF)-1a, β, angiopoietins, and endothelial cell marker CD31 were studied in lung biopsies of CF and COPD patients and controls, using real time reverse transcription (RT)-PCR and Western blotting. DNA binding activity of HIF-1 to VEGF-A promoter was assessed by electrophoretic mobility shift assay (EMSA) and wet-to-dry lung weight ratios as well as microvascular density (MVD) were determined. Serum VEGF-A concentrations in enzyme-linked immunosorbent assay (ELISA) and wet-to-dry weight ratios of donor lungs were monitored during transplantation in CF and COPD patients. Primary graft dysfunction (PGD) was diagnosed and graded according to the guidelines of the International Society for Heart and Lung Transplantation. Results: VEGF-A 165 and Flt-1 mRNA expression (P < 0.05), VEGF-A (P < 0.05), and HIF-1α (P < 0.05) protein levels, DNA binding activity of HIF-1 to VEGF promoter (P < 0.001) and extravascular lung water content (P < 0.05) were increased in CF lungs versus controls, whereas MVD was unchanged. Before and during lung transplantation, VEGF-A serum concentrations were higher in CF versus COPD patients (P < 0.05) and 60 min following reperfusion donor lungs transplanted to CF patients had higher tissue water contents than in COPD patients (P < 0.05). PGD grade 3 occurred more frequently in CF (22.7%) versus COPD patients (4%). PGD grade 3 patients had significantly higher VEGF serum concentrations versus PGD grade 0-2 patients (P < 0.001). Conclusions: These data indicate that upregulated VEGF-A levels are most likely induced by enhanced HIF-1 binding to VEGF-A promoter, possibly contributing to elevated serum VEGF-A levels in CF. Furthermore, CF patients undergoing lung transplantation are possibly more susceptible to PGD because of increased VEGF-A expression that mediates increased lung graft vascular permeability.

Collagen overproduction characteristic for dilated cardiomyopathy (DCM) is coregulated by endothelin (ET)-1, transforming growth factor (TGF)-beta1, basic fibroblast growth factor (bFGF) and matrix metalloproteases (MMPs). Whether these molecules affect grafts transplanted to heart failure patients is unknown. In 67 idiopathic DCM patients, 31 patients with ischemic cardiomyopathy (ICM) and 16 controls, the myocardial bFGF, TGF-beta1, pro-collagen (PrCol) type 1 (PrCol1-alpha1, -alpha2) and MMP expressions were examined using real-time RT-PCR or Western blotting. mRNA expression was measured in grafts for 1 year. TGF-beta1/bFGF stimulation or gene silencing was used to examine their effect on collagen synthesis in cardiac tissue cultures. TGF-beta1 and PrCol1 were upregulated in DCM only, while bFGF was upregulated in both groups versus controls. TGF-beta1 downregulated MMP-1 and upregulated collagen 1, whereas bFGF upregulated MMP-13 in DCM tissue. Post-transplant PrCol1-alpha1, -alpha2 and ET-1 mRNA increased over time in grafts of DCM patients only, while other factors returned to control baseline levels in DCM and ICM. These data indicate that cardiac transplantation corrects the dysregulated TGF/bFGF/MMP-1/MMP-13, but not the excess collagen and ET-1 synthesis in cardiac grafts transplanted to DCM patients. ET-1 might be a major pathologic trigger for graft fibrosis in DCM.

Bronchiolitis obliterans (BO) is a survival-limiting factor in lung transplantation. There are no common BO markers in use. Since BO is associated with extracellular matrix remodeling, we asked whether matrix metalloproteases (MMPs) and their tissue inhibitors (TIMPs) could serve as BO markers. In 72 lung transplant patients (34 BO syndrome (BOS) 0, 15 BOS 0-p, and 23 BOS 1) serum and broncho-alveolar lavage (BAL) MMP and TIMP levels were examined by ELISA. The BAL cell counts were additionally analyzed. The serum MMP-2, MMP-8, MMP-9 and TIMP-2 levels were not different in all groups. In contrast, the BAL MMP-8, -9 and TIMP-1 levels were significantly elevated in BOS 0-p (p = 0.003; p = 0.007; p = 0.0003, respectively) and BOS 1 (p = 0.003; p = 0.001; p = 0.0004, respectively) as compared to BOS 0 patients. The BAL MMP-8, -9 and TIMP-1 levels were significant predictors of BOS 0-p (p = 0.01; p = 0.01; p = 0.01, respectively) and BOS-1 (p = 0.007; p = 0.01; p = 0.006, respectively) in receiver operating characteristic analysis. Except for BAL macrophages that were significantly decreased in BOS 0-p versus BOS 0 patients; other cell counts were not different between the groups. BAL MMP-8, -9 and TIMP-1 might be useful markers to detect BO in lung transplant patients.

Transplantation-induced hypoxia results in enhanced vascular permeability and tissue vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) overexpression in donor lung grafts. Promoter studies have uncovered a hypoxia-inducible factor (HIF)-1 binding site (HBS) in 5'-flanking region of VEGF gene that regulates the hypoxia-induced expression of VEGF; and ET-1 potently stimulates VEGF-A production. We hypothesized that HIF-1 regulates VEGF-mediated vascular permeability in lung grafts.We studied the mRNA and protein expression of HIF-1 and its protein-binding capacity to the HBS of the VEGF gene in biopsies of preserved donor and control lungs, using real-time reverse transcription-polymerase chain reaction, Western blotting, and electrophoretic mobility shift assay. Wet-to-dry lung weight ratio was measured in donor and control lungs.While HIF-1 alpha mRNA expression was unchanged, HIF-1 beta was downregulated (p0.05) in donor versus control lungs. Protein expression of both, HIF-1 alpha and -beta was significantly upregulated in donor lung grafts. HIF-1 binding to the HBS of the VEGF promoter as well as tissue fluid content were increased in donor lung biopsies versus controls (p0.05).These data indicate that upregulated HIF-1 DNA binding activity to the HBS of VEGF-A most likely contributes to elevated VEGF levels in preserved lung grafts. Unchanged HIF-1 alpha mRNA expression did not affect HIF-1 alpha protein levels. Endothelin-1 increases HIF-1 alpha accumulation and activates HIF-1 transcription complex in vitro. Therefore, ET-1-mediated increased HIF-1 alpha protein stability most likely leads to transcriptional activation of VEGF during lung graft preservation. Targeting HIF might be of benefit to counteract edema formation in preserved lung grafts.

Abstract 900 Introduction: Bronchiolitis obliterans syndrome (BOS) characterized by the new development of fixed airflow obstruction after allogeneic hematopoietic cell transplantation (HCT) is an increasingly recognized manifestation of chronic graft-versus-host disease (cGVHD) affecting up to 30% of patients. BOS is associated with increased non-relapse mortality and poor patient outcome after HCT. Its cause is currently unknown but allorecognition of lung antigens is the suspected etiology since BOS has been observed in states of alloimmunity such as cGVHD and after lung transplantation. Currently, the diagnosis of BOS is based on radiologic findings in high resolution expiratory chest CTs (HR-CT) and pulmonary function tests (PFTs). For early diagnosis of BOS biomarkers would be highly desirable allowing efficient treatment of early disease before progression to irreversibility is observed. B-cells are of importance in cGVHD and excess of B-cell activation factor (BAFF), increased CD19+CD21- immature transitional B-cells and dysgammaglobulinemia characterize cGVHD. Objective: We investigated circulating B-cell subpopulations in cGVHD patients to define novel cellular biomarkers for diagnosis of BOS and scoring its severity. Patients and methods: One hundred and thirty-five patients (74 males, 61 females) with a median age of 46 (range, 20–65) years, given myeloablative (n=76) or reduced-intensity (n=59) conditioning for related (n=108) or unrelated donor (n=27) HCT were prospectively evaluated for presence of cGVHD from day 100 after HCT and thereafter serially every 3 months for 2 years. At the same time points PFTs, multicolor flow cytometric analyses of peripheral blood (PB) cells stained for CD19, CD21, CD27, CD10, CD38, IgM, IgD, CD3, CD4, CD8, and CD56, measurements of serum immunoglobulin levels and BAFF were performed. BOS and its severity were defined according to the NIH consensus criteria. All patients with BOS except 2 had HR-CTs of the lung, bronchoalveolar lavage and transbronchial biopsies performed. Results: After a mean follow-up of 250 (range, 100–640) days after HCT 35 of 135 (26%) patients developed BOS. Other manifestations of cGVHD included skin in 72%, eyes in 58%, oral mucosa in 50%, and liver in 65% of patients. Severity of BOS consisted of NIH score I (FEV1=60-79%) in 14 (40%), NIH score II (FEV1=40-59%) in 16 (46%), and NIH score III (FEV Conclusion: Disturbance of B-cell homeostasis is more pronounced in cGVHD of the lung compared to other main organ manifestations. B-cell subpopulation numbers and excess of BAFF can serve as novel cellular biomarkers for BOS. Disclosures: No relevant conflicts of interest to declare.

The Inhibitor of Apoptosis Birc6 Is a Sensitive and Specific Marker of Cardiac Allograft Rejection S. Aharinejad, O. Andrukhova, R. Schaefer, K. Krenn, C. May, A. Zuckermann, D. Zimpfer, M. Grimm, Dept. of Cardiothoracic Surgery, Medical University of Vienna, Vienna, Austria; Lab. for Cardiovasc. Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria; Dept. of Internal Medicine II, Medical University of Vienna, Vienna, Austria

was more effective than TA (p 0.05). Although TO and TA treatments both markedly suppressed the cellular (IFNspots: TA and TO vs UA p 0.001; IL-4 spots: TA p 0.579 and TO p 0.001 vs UA) and humoral response (IgM: TA p 0.004 and TO p 0.001 vs UA; IgG: TA p 0.241 and TO p 0.011 vs UA), TO was far more effective. Due to the insufficient systemic cellular immunosuppression, discontinuation of TA treatment resulted in a far stronger (3.5-fold) graft infiltration on POD 8 compared to TO (p 0.001). TO animals showed reduced weight gain and, after 60 days, BUN was significantly elevated (p 0.001). Cholesterol and triglycerides were significantly higher with TO than with TA (p 0.001 and p 0.041, respectively). Interestingly, after 60 days, luminal obliteration was similarly inhibited with TO (15 3%) and TA (13 4%) vs UA (44 7%, p 0.001) and the airway epithelium was preserved (TO: 96 7%, TA 97 6% vs UA 67 26%). Conclusions: Tacrolimus aerosol inhalation provides adequate trachea tissue concentrations, reduces systemic side effects and effectively protects the airway graft from early cellular rejection as well as from chronic obliterative airway disease. However, systemic immunosuppression is inferior to oral treatment and short treatment discontinuation results in rapid graft damage by activated lymphocytes.

Purpose: Antibody mediated Rejection (AMR) is associated with poor outcome. We hypothesized that specific gene expression profiles may be present in patients with AMR. Methods and Materials: Based on the Columbia University cohort from the CARGO study, we analyzed PBMC gene microarrays from patients meeting criteria for AMR by using SAM, Gene ontology analysis and hierarchical clustering. A FDR 5-10% was considered significant. Results: Five patients fulfilled criteria for AMR. We found dramatic changes in the expression profiles and coherent enrichment of gene ontology categories. Represented genes included BCL11A (leukemia and B-Cell lymphomas), BNIP3L (suppression of cell proliferation), AGPTA1, HLA-DRA, CD1D and CD74 (MHC-related); FADD (apoptosis), TNFRSF25 and TNFRSF10 (T cell development); GZMB (target cell apoptosis),IGHG-1, 4 (immunoglobulin chains), IGLL1 (cellular proliferation and differentiation), IL1R2 (inhibits the activity of its ligands), IL-4 (antagonize IL-1), IL7R (differentiation and activation of T cells), IRF4 (B-cell proliferation and differentiation, T-Cell differentiation and induction of IL-10 and IL-2), ITK (T-cell kinase containing SH2 and SH3), OSM (regulates IL-6, G-CSF and GM-CSF in endothelial cells), PTPRC (regulates Tand B-cell antigen receptor signaling in hematopoietic cells) and TNFAIP6 (induced in vascular smooth muscle). The figure show high correlation in patients with AMR (right). Conclusions: Patients suffering AMR have specific gene expression profiles involving several B-cell but also T-Cell-related genes. These results should encourage the design of future studies aimed to dissect the cross-talk between the different known forms of rejection.

single, 3 heart-lung) survived more than 3 months following transplant. Only 1 of 194 patients (0.05%) developed PTLD. The patient was EBV seropositive prior to transplant, but CMV negative with a CMV positive donor. She had complete resolution of PTLD with Rituximab, and upon autopsy, there was no evidence of recurrent PTLD. 122 of 194 patients (63%) were documented to be EBV seropositive prior to transplantation, while 20 (10%) were EBV seronegative. Repeat EBV serologies were available for 6 of 20 patients who were previously negative. 5 of the 6 became EBV seropositive following transplantation with no evidence of PTLD. Of the 113 living patients, 82 (72.6%) remain on valgancyclovir, 3 (2.7%) are on acyclovir, and 28 (24.8%) are no longer on antiviral therapy. Conclusions: The incidence of PTLD in patients undergoing lung transplantation at UCSF is only 0.05%, with 0% mortality secondary to PTLD, which is much lower than previously reported. Antiviral therapy may decrease the incidence of PTLD either by direct inhibitory effects on EBV or by indirectly preventing CMV disease. The implications of this study are limited by its small sample size and retrospective design, but further investigation is indicated to determine whether antiviral prophylaxis decreases the incidence of PTLD.