Wolfgang Jungraithmayr

Ex vivo reconditioning of marginal donor lungs injured by acid aspiration

BACKGROUND Injured lungs due to gastric acid aspiration may be rejected for transplantation because of the possibility of early graft dysfunction. We hypothesized that diluted surfactant administration during ex vivo perfusion would recondition the lungs injured by acid aspiration and permit their use as suitable grafts for transplantation. METHODS Using a pig model, lung injury was induced with 5-ml/kg administration of a betaine-HCl/pepsin mixture via a flexible bronchoscope. After injury, animals were randomly assigned to three study groups (n = 6/group): saline lavage during ex vivo perfusion (control); surfactant lavage ex vivo (SL-Exvivo); and surfactant lavage before harvest (SL-Pre); and a normal group (n = 4), with no lung injury. Cold storage time was 3 hours. A volume of 10 ml/kg (4 mg/ml, 40 mg/kg) surfactant (Curosurf) was used for lavage. Bronchoalveolar lavage (BAL) was performed before and after injury and at the end of the experiment. Protein and neutrophil percentage in BAL were assessed. Hemodynamic and aerodynamic parameters were measured every 30 minutes during a 2-hour observation period. RESULTS An approximately 50% decrease in Pao(2) was observed in all animals after injury. Ex vivo surfactant lavage resulted in lower pulmonary vascular resistance, lower oxygenation index and higher Pao(2)/Fio(2) ratio compared with the control group (p = 0.001, p = 0.0001 and p = 0.0001, respectively, according to analysis of variance for repeated measures). Wet-to-dry weight ratio was lower in the SL-Exvivo group compared with the control group (p = 0.015). BAL neutrophil percent at the end of the experiment differed significantly between control and all other groups (p < 0.05). CONCLUSION Diluted surfactant lavage during ex vivo perfusion improves graft function of lungs injured by gastric acid aspiration.

A mouse model of orthotopic, single-lung transplantation

OBJECTIVES Progress in studying acute and chronic pulmonary allograft rejection has been hampered by the lack of feasible experimental animal transplantation models. Contemporary approaches are limited by anatomic applicability (heterotopic tracheal implantation) and lack of genetic variability (rat model). To utilize the breadth of available genetic modifications in a physiologic setup, we optimized and validated a procedure of orthotopically transplanted, perfused, and ventilated single pulmonary transplantation in mice. METHODS C57BL/6 mice served as recipient, with Balb/c as donor. At time of harvest, explanted lungs were perfused with Perfadex, and the heart-lung block excised. Under 30 to 40x magnification, vessels and bronchus were cuffed. Following left thoracotomy in the recipient, hilar structures were incised and cuff-anastomosed with the corresponding donor parts. Allogeneic and syngeneic transplantations (n = 12/group) were performed with a follow-up period of 5 days and up to 90 days, respectively. RESULTS The success rate of lung transplantation in mice was 87.5% (21/24). Mean cold ischemia time was 32.3 +/- 3.7 minutes, and warm ischemia time was 30.8 +/- 9.5 minutes. Deaths were due to bleeding during dissection of the hilus and/or caused by thrombosis postoperatively. Allogeneic grafts were rejected by day 5; syngeneic grafts were slightly congested but mainly unchanged up to day 90 posttransplantation. CONCLUSIONS Unilateral lung transplantation in mice can be performed in a standardized and controlled fashion with low mortality, comparable to the rat. Employing transgenic and knockout mice strains, this procedure holds great promise to advance the understanding of immunologic pathways in acute and chronic rejection in a physiologic model of pulmonary transplantation.

CD26/DPP-4 inhibition recruits regenerative stem cells via stromal cell-derived factor-1 and beneficially influences ischaemia-reperfusion injury in mouse lung transplantation

OBJECTIVES The CD26 antigen is a transmembrane glycoprotein that is constitutively expressed on activated lymphocytes and in pulmonary parenchyma. This molecule is also identified as dipeptidyl peptidase-4 (DPP-4) that cleaves a host of biologically active peptides. Here, we aimed to identify an important substrate of CD26/DPP-4-stromal cell-derived factor-1 (SDF-1/CXCL12)-as a key modulator for stem-cell homing together with its receptor CXCR4 in response to ischaemic injury of the lung. METHODS Orthotopic single lung transplantation (Tx) was performed between syngeneic C57BL/6 mice. Inhibition of CD26/DPP-4 activity in recipients was achieved using vildagliptin (10 mg/kg, every 12 h) subcutaneously, and 6 h ischaemia time was applied prior to implantation. Forty-eight hours after Tx, lung histology, SDF-1 levels (enzyme-linked immunosorbent assay) in lung, spleen and plasma, and expression of the SDF-1 receptor CXCR4 in blood and lung were assessed. Homing of regenerative progenitor cells to the transplanted lung was evaluated using fluorescent-activated cell sorting. RESULTS Compared with untreated lung transplanted mice, systemic DPP-4 inhibition of Tx recipients resulted in an increase in protein concentration of SDF-1 in plasma, spleen and lung. Concordantly, the frequency of cells bearing the SDF-1 receptor CXCR4 rose significantly in the circulation and also in the lungs of DPP-4-inhibited recipients. We found co-expression of CXCR4/CD34 in the grafts of animals treated with vildagliptin, and the stem-cell markers Flt-3 and c-kit were present on a significantly increased number of cells. The morphology of grafts from DPP-4 inhibitor-treated recipients revealed less alveolar oedema when compared with untreated recipients. CONCLUSIONS Targeting the SDF-1-CXCR4 axis through CD26/DPP-4 inhibition increased the intragraft number of progenitor cells contributing to the recovery from ischaemia-reperfusion lung injury. Stabilization of endogenous SDF-1 is achievable and may be a promising strategy to intensify sequestration of regenerative stem cells and thus emerges as a novel therapeutic concept.

Dipeptidyl peptidase 4 as a therapeutic target in ischemia/reperfusion injury.

Dipeptidyl peptidase 4 (DPP4, DPPIV, CD26, EC 3.4.14.5) was discovered more than four decades ago as a serine protease that cleaves off N-terminal dipeptides from peptide substrates. The development of potent DPP4 inhibitors during the past two decades has led to the identification of DPP4 as a target in the treatment of type 2 diabetes. The favorable effect of DPP4 inhibitors is based on prevention of the in vivo inactivation of the incretin hormone, glucagon-like peptide-1 (GLP-1) by DPP4. Apart from GLP-1, a number of other biologically active peptides are truncated by DPP4. For these peptides, the physiological relevance of their truncation has yet to be fully elucidated. Within the last 10years, DPP4 inhibitors have been employed in several animal models of lung and heart disease, in which injury was induced by an ischemic insult followed by subsequent reperfusion. In this review, we present a state-of-the-art of the ischemia/reperfusion injury (IRI)-related pharmacological actions of DPP4 substrates, including GLP-1, stromal cell-derived factor-1 alpha and vasoactive intestinal peptide. Furthermore, we discuss the large body of experimental work that now provides compelling evidence for the advantageous impact of DPP4 targeting in IRI. However, possible risks as well as underlying mechanisms are yet to be elucidated before translating these promising treatment strategies into clinical practice.

Method comparison of dipeptidyl peptidase IV activity assays and their application in biological samples containing reversible inhibitors.

BACKGROUND Dipeptidyl peptidase IV (DPPIV, DPP4) is a serine protease that releases N-terminal dipeptides. It is a validated drug target for type 2 diabetes and DPPIV inhibitors are currently evaluated for other therapeutic applications. Various assays are used for DPPIV activity measurements in biological samples. Highly sensitive methods are needed to measure also very low activities in inhibited samples. METHODS Here, the three most extensively used substrates to quantify DPPIV activity are compared using in-house methods. A luminescent kit was also included. In addition, one of the in-house fluorometric assays was elaborated for use in biological samples containing reversible DPPIV inhibitors to estimate residual DPPIV activity which is usually underestimated due to sample dilution. RESULTS The in-house methods showed a good precision, linearity and specificity. Both fluorometric substrates had a 10-fold higher sensitivity compared to the colorimetric assay. The luminescent kit was found to be the most sensitive. CONCLUSIONS All three in-house methods can be used to measure DPPIV activity in non-inhibited biological samples. The more sensitive fluorometric assays are recommended when sample volumes are limited or when using inhibited samples. The elaborated fluorometric method can be used to estimate the residual in vivo DPPIV activity in inhibitor treated subjects.

Inhibition of CD26/DPP IV attenuates ischemia/reperfusion injury in orthotopic mouse lung transplants: the pivotal role of vasoactive intestinal peptide.

The T cell activation Ag CD26/dipeptidylpeptidase IV (DPP IV) combines co-stimulatory and enzymatic properties. Catalytically, it functions as an exopeptidase, modulating biological activity of key chemokines and peptides. Here we investigated the effect of organ-specific inhibition of DPP IV catalytic activity on ischemia/reperfusion injury after extended ischemia in the mouse model of orthotopic single lung transplantation. C57BL/6 mice were syngeneically, transplanted, grafts were perfused and stored in Perfadex with (treated) or without (control) a DPP IV enzymatic activity inhibitor (AB192). Transplantation was performed after 18h cold ischemia time; following 2-h reperfusion, grafts were analyzed for oxygenation, thiobarbituric acid-reactive substances, histomorphology, and immunohistochemistry was performed for leukocyte Ag 6, myeloperoxidase, hemoxygenase 1, vasoactive intestinal protein (VIP), and real-time PCR for VIP. Treatment with the DPP IV inhibitor AB192 resulted in significant improvement of gas exchange, less lipid oxidation, preservation of parenchymal ultrastructure, reduced neutrophil infiltration, reduced myeloperoxidase expression, increased hemoxygenase 1 expression, pronounced expression of VIP in alveolar macrophages and increased mRNA expression of VIP. Inhibition of intragraft DPP IV catalytic activity with AB192 strikingly ameliorates ischemia/reperfusion injury after extended ischemia. Furthermore, preservation of endogenous intragraft VIP levels correlate with maintaining lung function and structural integrity.

Persistent Benefit From Lung Volume Reduction Surgery in Patients With Homogeneous Emphysema

BACKGROUND The purpose of this study was to evaluate whether favorable short-term results achieved by lung volume reduction surgery in selected patients with homogeneous emphysema would persist for longer periods. Their symptoms, lung function, and survival for several years were analyzed in comparison to patients with heterogeneous emphysema. METHODS Two hundred fifty consecutive patients (105 women), mean (+/- standard deviation) age 64 +/- 8.4 years, with advanced emphysema underwent bilateral thoracoscopic lung volume reduction surgery. Forced expiratory volume in 1 second was 28% +/- 8% of predicted, 6-minute walking distance was 245 +/- 118 m, and Medical Research Council dyspnea score was 3.5 +/- 0.7. In 138 patients (55%) computed tomography revealed homogeneous emphysema (including 82 intermediate type) distribution and in 112 patients (45%) heterogeneous emphysema. Baseline characteristics were otherwise similar in the two groups that were prospectively observed for several years. RESULTS Thirty-day mortality was 2.4%. Both groups revealed significant improvements 3 months after lung volume reduction surgery: in homogeneous emphysema, predicted forced expiratory volume in 1 second was 38% +/- 14% (35% improvement), 6-minute walk distance was 324 +/- 87 m, and dyspnea score was 1.8 +/- 0.9 (p < 0.05 all outcomes). Corresponding results in heterogeneous emphysema were 44% +/- 15% (61% improvement), 382 +/- 95 m, and 1.3 +/- 0.9 points (p < 0.05 versus baseline; not significant versus homogeneous). Median time until predicted forced expiratory volume in 1 second and 6-minute walk distance had returned to baseline was 36 months in both groups. One-year survival was similar in both groups. At 5 years, median survival without lung transplantation was 64% in the homogeneous and 73% in the heterogeneous group (Cox proportional hazard, 0.81; 95% confidence interval, 0.66 to 0.98; p = 0.03). CONCLUSIONS In selected patients with homogeneous pulmonary emphysema, lung volume reduction surgery can be successfully performed with low perioperative mortality. Significant improvements in dyspnea, lung function, and exercise capacity are maintained for several years.

Chronic rejection pathology after orthotopic lung transplantation in mice: the development of a murine BOS model and its drawbacks.

Almost all animal models for chronic rejection (CR) after lung transplantation (LTx) fail to resemble the human situation. It was our attempt to develop a representative model of CR in mice. Orthotopic LTx was performed in allografts receiving daily immunosuppression with steroids and cyclosporine. Controls included isografts and mice only undergoing thoracotomy (SHAM). Allografts were sacrificed 2, 4, 6, 8, 10 or 12 weeks after LTx. Pulmonary function was measured repeatedly in the 12w allografts, isografts and SHAM mice. Histologically, all allografts demonstrated acute rejection (AR) around the blood vessels and airways two weeks after LTx. This decreased to 50–75% up to 10 weeks and was absent after 12 weeks. Obliterative bronchiolitis (OB) lesions were observed in 25–50% of the mice from 4–12 weeks. Isografts and lungs of SHAM mice were normal after 12 weeks. Pulmonary function measurements showed a decline in FEV0.1, TLC and compliance in the allografts postoperatively (2 weeks) with a slow recovery over time. After this initial decline, lung function of allografts increased more than in isografts and SHAM mice indicating that pulmonary function measurement is not a good tool to diagnose CR in a mouse. We conclude that a true model for CR, with clear OB lesions in about one third of the animals, but without a decline in lung function, is possible. This model is an important step forward in the development of an ideal model for CR which will open new perspectives in unraveling CR pathogenesis and exploring new treatment options.

Prevention of primary graft dysfunction in lung transplantation by N-acetylcysteine after prolonged cold ischemia

BACKGROUND N-Acetylcysteine (NAC), a thiol-containing compound that has been used as an anti-oxidant, may also lead to an increased glutathione synthesis. This study assessed the protective effect of NAC on primary graft dysfunction after lung transplantation. METHODS Porcine single left-lung transplantation was performed in 2 experimental groups after 24 hours of cold storage. Donor and recipient animals were treated with intravenous injection of 150 mg/kg NAC 60 minutes before harvest and reperfusion, followed by 12.5 mg/kg/hour continuous perfusion during the 8-hour observation period (NAC). Control animals did not receive any treatment. Hemodynamic and respiratory parameters were recorded throughout the observation period. Bronchoalveolar lavage (BAL) nitrite, neutrophil elastase (NE), protein accumulation, interleukin (IL)-8, nuclear factor-κB (p50 sub-unit), and reduced glutathione (GSH) in lung tissue and red blood were measured. RESULTS During the observation period, the mean pulmonary artery pressure, oxygenation, airway pressure, and static lung compliance were significantly better in NAC animals compared with controls (p < 0.05). Extravascular lung water index was higher at points during the reperfusion in the control group. BAL protein, nitrite, NE, and IL-8 cytokine levels at the end of the experiment were significantly higher in the controls than in the NAC group (p < 0.05). Lung tissue reduced GSH levels were significantly higher in the NAC group than in the control group. Red blood cell GSH levels were always higher in the NAC group during the reperfusion period. Reverse transcription polymerase chain reaction for IL-8 messenger RNA was significantly higher in controls during the reperfusion period than in the NAC group (p = 0.001). The amount of lung tissue nuclear NF-κB (p50 sub-unit) was significantly higher in controls than in NAC pigs (p = 0.03). CONCLUSION In this model, donor and recipient treatment with NAC effectively protected the lung from primary graft dysfunction after prolonged cold ischemia.

Primary graft dysfunction in lung transplantation: the role of CD26/dipeptidylpeptidase IV and vasoactive intestinal peptide.

Background. Enzymatic activity inhibition of CD26/dipeptidylpeptidase IV (CD26/DPP IV) attenuated short-term post-Tx (transplantation) ischemia-reperfusion injury after 18-hr-ischemia. Here, we investigated the effect of intragraft CD26/DPP IV catalytic inhibition on primary graft dysfunction during 7 day post-Tx, following extended ischemia. Methods. A syngeneic rat (LEW [Lewis abstract]) orthotopic lung Tx model was used, grafts exposed to 18 hr cold ischemia before Tx. Controls were flushed and preserved in Perfadex, and harvested after 1 day (CON1) or 7 day (CON7) post-Tx. Investigational groups IN1, IN3, and IN7 grafts were perfused with and stored in Perfadex + inhibitor (AB192) and harvested at 1, 3, and 7 days post-Tx, respectively. Blood gas analysis, peak airway pressure (PAwP), wet/dry weight ratio, myeloperoxidase thiobarbituric acid reactive substances (TBARS), and staining for vasoactive intestinal peptide (VIP) were analyzed. Results. IN1 versus CON1 showed preserved histology, increased pO2 (P<0.01), lowered PAwP (P<0.01), less edema (P<0.05) and decreased TBARS (P<0.05). Survival was better for IN7 versus CON7 (P<0.01). The course of AB192-perfused grafts from 1 to 7 days displayed improved values for pO2 (P<0.01), PAwP (P<0.01), edema (P<0.05), TBARS (P<0.05), and myeloperoxidase (P<0.05). Compared with controls, VIP was preserved during 18 hr ischemia in alveolar macrophages (P=0.0001) and respiratory epithelial cells (P=0.001). Conclusions. Perfusion with an inhibitor of CD26/DPP IV enzymatic activity significantly reduced the incidence and severity of pulmonary primary graft dysfunction and enabled recovery after extended ischemia. This is the first report that CD26/DPPIV inhibitor treatment increases local pulmonary VIP levels, which correlate with preserved ventilatory function and pulmonary structural integrity.