Onset of Inflammation With Ischemia: Implications for Donor Lung Preservation and Transplant Survival.

Lungs stored ahead of transplant surgery experience ischemia. Pulmonary ischemia differs from ischemia in the systemic organs in that stop of blood flow in the lung leads to loss of shear alone because the lung parenchyma does not rely on blood flow for its cellular oxygen requirements. Our earlier studies on the ischemia-induced mechanosignaling cascade showed that the pulmonary endothelium responds to stop of flow by production of reactive oxygen species (ROS). We hypothesized that ROS produced in this way led to induction of proinflammatory mediators. In this study, we used lungs or cells subjected to various periods of storage and evaluated the induction of several proinflammatory mediators. Isolated murine, porcine and human lungs in situ showed increased expression of cellular adhesion molecules; the damage-associated molecular pattern protein high-mobility group box 1 and the corresponding pattern recognition receptor, called the receptor for advanced glycation end products; and induction stabilization and translocation of hypoxia-inducible factor 1α and its downstream effector VEGFA, all of which are participants in inflammation. We concluded that signaling with lung preservation drives expression of inflammatory mediators that potentially predispose the donor lung to an inflammatory response after transplant.

The putative role of mast cells in lung transplantation.

Mast cells (MCs) were primarily recognized as effector cells of allergy. These cells are acting predominantly at the interface between the host and the external environment, such as skin, gastrointestinal and the respiratory tract. Only recently, MCs have gained increased recognition as cells of functional plasticity with immune-regulatory properties that influence both the innate and the adaptive immune response in inflammatory disorders, cancer and transplantation. Through the secretion of both proinflammatory and antiinflammatory mediators, MCs can either ameliorate or deteriorate the course and outcome in lung transplantation. Recent research from other models recognized the immune-protective activity of MCs including its role as an important source of IL-10 and TGF-ß for the modulation of alloreactive T cell responses or assistance in Treg activity. This paper summarizes the current understanding of MCs in lung transplantation and discusses MC-mediated immune-mechanisms by which the outcome of the engrafted organ is modulated.

The technique of orthotopic mouse lung transplantation as a movie-improved learning by visualization.

The introduction of the mouse model of orthotopic single-lung transplantation has significantly advanced research in experimental lung transplantation. Technically, this model is demanding and presents major challenges to the researcher. In order to facilitate and accelerate the successful acquisition of this technique, we provide here a narrated movie in which the full transplantation procedure is comprehensively illustrated, featuring all key operative steps, but also highlighting potential pitfalls.

CD154 blockade abrogates allospecific responses and enhances CD4(+) regulatory T-cells in mouse orthotopic lung transplant.

Acute cellular rejection (ACR) is a common and important clinical complication following lung transplantation. While there is a clinical need for the development of novel therapies to prevent ACR, the regulation of allospecific effector T-cells in this process remains incompletely understood. Using the MHC-mismatched mouse orthotopic lung transplant model, we investigated the short-term role of anti-CD154 mAb therapy alone on allograft pathology and alloimmune T-cell effector responses. Untreated C57BL/6 recipients of BALB/c left lung allografts had high-grade rejection and diminished CD4(+) : CD8(+) graft ratios, marked by predominantly CD8(+) >CD4(+) IFN-γ(+) allospecific effector responses at day 10, compared to isograft controls. Anti-CD154 mAb therapy strikingly abrogated both CD8(+) and CD4(+) alloeffector responses and significantly increased lung allograft CD4(+) : CD8(+) ratios. Examination of graft CD4(+) T-cells revealed significantly increased frequencies of CD4(+) CD25(+) Foxp3(+) regulatory T-cells in the lung allografts of anti-CD154-treated mice and was associated with significant attenuation of ACR compared to untreated controls. Together, these data show that CD154/CD40 costimulation blockade alone is sufficient to abrogate allospecific effector T-cell responses and significantly shifts the lung allograft toward an environment predominated by CD4(+) T regulatory cells in association with an attenuation of ACR.

A model of chronic lung allograft rejection in the rat.

Bronchiolitis obliterans, the pathological hallmark of chronic pulmonary rejection, severely impacts long-term survival following lung transplantation. However, experimental reproduction of this pathophysiological phenomenon has not been achieved with contemporary in vivo models. Here, a model of chronic rejection is described, with sensitised recipients receiving unilateral orthotopic rat lung transplants. Lewis rats, sensitised with skin from brown Norway rats 7 days before receiving left lung transplants from donors that were Lewis x brown Norway F(1) hybrids, were analysed during day 21-84. The development of chronic rejection was modulated by a treatment with rapamycin and cyclosporin, and characterised histologically, immunohistochemically and by reverse transcriptase PCR. Characteristic histopathological changes leading to chronic rejection were induced over time by an initial treatment with cyclosporin in the presence of continuous rapamycin application. At day 84, fibrotic lesions replaced the respiratory epithelium within small bronchioles, with strong expression of smooth muscle alpha-actin and upregulation of mRNA for T-helper cell type-1 cytokines, smooth muscle alpha-actin, transforming growth factor-beta and CC chemokine ligand 5, but decreased forkhead box protein P3 gene expression. A reproducible and clinically relevant experimental set-up for progressive chronic rejection in rat pulmonary allografts is described. This model will permit better understanding of the pathological changes of small airways during the development of bronchiolitis obliterans, and may serve as an in vivo set-up for testing the efficacy of novel therapeutic interventions.

Ischemia/reperfusion injury: The role of CD26/dipeptidyl-peptidase-IV-inhibition in lung transplantation.

UNLABELLED: CD26/Dipeptidyl peptidase (DPP) IV is an integral membrane protein of lymphocytes that modulates the activities of chemokines, interleukins, and neuropeptides. We investigated the effect of enzymatic DPP IV inhibition on ischemia/reperfusion injury after extended ischemia prior to transplantation. MATERIALS AND METHODS: We used a syngeneic rat (Lewis) orthotopic left lung transplantation model. In the control group (group I), donor lungs were flushed and preserved in Perfadex for 18 hours at 4 degrees C, then transplanted and reperfused for 2 hours. Group II donor lungs were perfused with and stored in Perfadex +25mol/L AB192 (bis(4-acetamidophenyl) 1-(S)-prolylpyrrolidine-2(R,S)-phosphonate), a small molecular weight DPP IV inhibitor. After 2-hour reperfusion, we measured blood gas, peak airway pressure, and thiobarbituric acid reactive substances. RESULTS: Grafts from group II versus group I showed a significantly increased oxygenation capacity (II: 298.4 +/- 87.6 mm Hg vs 120.9 +/- 48.0, P < .01), lower peak airway pressure (11.8 +/- 0.9 mm Hg vs 16.0 +/- 1.4, P < .01), and less lipid peroxidation (9.3 +/- 2.0 micromol/L vs 13.8 +/- 1.8, P < .01). CONCLUSION: Inhibition of intragraft DPP IV enzymatic activity significantly reduced ischemia/reperfusion-associated pulmonary injury, allowing for successful transplantation after 18 hours of ischemia.

Decrease of Airway Allergies After Lung Transplantation Is Associated With Reduced Basophils and Eosinophils.

BACKGROUND: Allergies are hypersensitive reactions of the immune system on antigen exposure similar to immune reactions after transplantation (Tx). Their activity can change after Tx. The lung as a transplantable organ is challenged two-fold, by antigens from the blood and the air environment. Herein we analyzed if airway allergies change after lung Tx. METHODS: We systematically reviewed patients' airway allergies before and after lung Tx between 1992 and 2014. The course of lymphocytes, thrombocytes, and leukocytes, among them neutrophils, eosinophils, and basophils, was analyzed in patients in whom airway allergies have changed and in whom they did not change. RESULTS: From 362 lung transplanted patients, 44 patients had suffered from allergies before Tx (12.2%). In 20 of these patients (45.5%), airway allergies disappeared completely within 1 year after lung Tx and were persistently absent thereafter. In these patients, basophils and eosinophils decreased significantly (P < .0012); in contrast, cells did not decrease in patients whose allergies did not disappear. Leukocytes overall, and in particular, neutrophils, decreased significantly in patients whose allergy disappeared (P < .014, P < .012, respectively). CONCLUSIONS: Airway allergies disappeared in almost half of cases after lung Tx. Along with this reduction, basophils and eosinophils decreased as potentially responsible cells for this phenomenon. These findings may stimulate intensified research on basophils and eosinophils as major drivers of airway allergies.

The role of recipient derived interleukin-17A in a murine orthotopic lung transplant model of restrictive chronic lung allograft dysfunction.

The single most important cause of late mortality after lung transplantation is chronic lung allograft dysfunction (CLAD). However, the pathological development of CLAD was not as simple as previously presumed and subclassification phenotypes, bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD), have been introduced. We want to re-investigate how CLAD manifests in the murine orthotopic lung transplant model and investigate the role of interleukin 17A (IL-17A) within this model. Orthotopic LTx was performed in CB57BL/6, IL-17 WT and IL-17 KO mice. In a first experiment, CB57BL/6 mice receiving an isograft (CB57BL/6) or allograft (BALB/C) were compared. In a second experiment IL-17 WT and IL-17 KO mice (both CB57BL/6 background) received an allograft (BALB/C). Mice received daily immunosuppression with steroids and cyclosporine and were sacrificed 10weeks after transplantation for histopathological analysis by an experienced lung pathologist. After murine orthotopic lung transplantation, the allograft histopathologically presented features of human rCLAD (i.e. overt inflammation, pleural/parenchymal fibrosis and obliterative bronchiolitis). In the IL-17A KO group, less inflammation in the bronchovascular axis (p=0.03) was observed and a non-significant trend towards less bronchovascular fibrosis, pleural/septal inflammation and fibrosis, and parenchymal inflammation and fibrosis when compared to WT mice. The major mismatch orthotopic lung transplant model resembles features of human rCLAD. IL-17A mediated immunity is involved in the inflammatory component, but had little influence on the degree of fibrosis. Further mechanistic and therapeutic studies in this mouse model are needed to fully understand the mechanisms in rCLAD.

[Completion pneumonectomy. Indications and results]. / Sekundäre Pneumonektomien. Indikationen und Langzeitergebnisse.

Completion pneumonectomy (CP) is widely known to be associated with high morbidity and lethality. However, in certain instances, it offers the only chance for cure. The results of the following CPs (N=86) were investigated: progressive or recurrent benign disease (N=6, group I), recurrence of a malignant tumor (N=41, group II), and complication after lung resection (N=39, group III). Right completion pneumonectomy was carried out in 48 cases and left completion pneumonectomy in 38. The overall 30-day lethality of CP was 20.2%, 0% in group I, 10% n group II, and 33.3% n group III. This lethality was significantly higher on the right side (29.8%) than on the left (7.7%; P=0.014). Differentiation between emergency and urgent indications resulted in 30-day lethalities of 54% and 23%, respectively. This difference is significant (P=0.002). The 30-day lethality for patients with anastomotic or stump insufficiency was 41% (P=0.002). Five-year survival was 26% in the group of patients with malignant disease and 32% in those with complications after lung resection. The results show: the lethality of CP remains high, especially after complications from operating in emergency conditions. However, considering the long-term survival, CP is certainly justified.

[Radiofrequency ablation of malignant lung tumours. Judicious approach?]. / Radiofrequenzablation maligner Lungentumore. Eine sinnvolle Methode?

BACKGROUND: Radiofrequency ablation (RFA) is a minimally invasive technique and well established in the treatment of malignant hepatic tumours. This method could also find application in patients with malignant lung tumours who, for functional reasons, have to be excluded from standard surgery. Until now, however, very little data have been available on the application of RFA in malignant pulmonary tumours. PATIENTS AND METHODS: From November 2001 to January 2004, eleven malignant lesions of the lung were treated with RFA. The indication for RFA resulted from an inadequate pulmonary reserve and additional severe risk factors. RESULTS: Eleven lesions were treated in ten patients with RFA. The malignancies were primary non-small cell bronchial carcinomas (n=9) as well as metastases of non-small cell carcinomas (n=2). Early complications of RFA were pneumothorax, hemorrhagic intrapleural effusion, bronchopleural fistula and pericarditis. Two weeks after RFA, pneumonia appeared as a late complication. No patient's death was related to the RFA procedure. After a mean follow-up of 8.5 months five patients died. Five patients are still alive, two of whom exhibit no tumour recurrence. CONCLUSION: RFA in patients with lung tumours is possible from a technical viewpoint. It is possibly a therapeutic alternative for patients with localized tumours that are inoperable. However, in this series, the morbidity of the procedure -- taking the degree of invasiveness into account -- is high, and the oncological results are unsatisfactory, possibly due to a small cohort of patients.

Completion pneumonectomy for lung metastases.

OBJECTIVE: Completion pneumonectomy (CP) for malignant disease is generally accepted but controversial for lung metastases. The data available show a high perioperative morbidity and mortality with a poor long-term prognosis. We analysed the postoperative outcome and long-term results of our patients undergoing CP. PATIENTS AND METHODS: Between January 1986 and May 2003, nine patients underwent completion pneumonectomy for lung metastases. This represents 10% (9/86) of all CPs performed and 1.7% (9/525) of all pneumonectomies. RESULTS: One to three metastasectomies in the form of wedge resection (16), segment resection (5) and lobectomies (3) were performed prior to CP. The mean time interval between the operation of the primary tumour and the first metastasectomy was 38 months, the first and second metastasectomy 12 months, the second and third metastasectomy 14 months, and the third metastasectomy and CP 25 months. Six patients had an extended completion pneumonectomy. Operative morbidity and mortality was 0%. One patient is still alive and recurrence-free 9 months after CP. Two patients have recurrent pulmonary contralateral metastases under chemotherapy and six patients died of metastatic disease. Actual survival is 33%, recurrence-free survival (RFS) is 11%. The 3-year survival is 34%. CONCLUSION: Since there was no morbidity and mortality in our series, CP for lung metastases seems to be justified but the long-term survival is limited by the occurrence of contralateral or extrapulmonary metastatic disease. Multiple resections of metastases have a positive influence on survival, but the last step of resection in the form of CP does not seem to improve long-term survival.

An experimental approach toward chronic pulmonary allograft rejection: Orthotopic lung versus heterotopic tracheal segment transplantation in rats.

BACKGROUND: Despite the impact of chronic rejection (CR) on long-term outcomes, clinically relevant experimental models are sparse, often including a design of subcutaneous implantation of tracheal segments. However, this latter site lacks anatomic correlation, adequate perfusion, and ventilatory function. In this study, we compared the spatial and sequential course of CR in models of orthotopic single lung transplantation (LT) versus heterotopically implanted tracheal segments in rats. METHODS: We performed 30 orthotopic left single LTs from Fisher 344 (F344) to Wistar Kyoto (WKY) rats for comparison with the outcomes of 3 tracheal segments implanted subcutaneously in every recipient. As a control group, 3 syngeneic tracheal segments were implanted into 12 WKY rats. For histopathologic examinations, tracheal segments and pulmonary allografts were harvested between days 1 and 112 and between weeks 4 and 18, respectively. RESULTS: Allogeneic tracheal segments showed rapid fragmentation of the respiratory epithelium, with complete luminal occlusion by week 4, whereas the lumen in isografts remained unobstructed. In contrast, bronchioles from orthotopically transplanted lungs did not show epithelial changes before week 14. However, marked lymphocytic sequestration into bronchioles occurred by week 8 with sequential destruction of all layers of the small airways, with loss of respiratory epithelium by week 16. CONCLUSIONS: Based on the different histomorphologic dynamics of CR, direct comparison between those 2 models is limited. When investigating CR in future studies, initial findings based on tracheal implantation experiments should be expanded in the site of orthotopic pulmonary transplantation.

Increased T-bet to GATA-3 ratio during acute allograft rejection in the rat lung.

Acute allograft rejection (AR) remains a major problem in solid organ transplantation. The pivotal mechanism hinges on alloantigen recognition by recipient T helper (T(h)) cells that differentiate into T(h)1 and T(h)2. This study investigated the association of mRNA levels of the transcription factors T-box expressed in T cells and GATA-binding protein 3 with the development of T(h)1/T(h)2-directed immune responses. We investigated the expression of T-bet and GATA-3 mRNA levels and the protein levels of their marker cytokines interleukin (IL)-2 and IL-4 in orthotopically transplanted rat lungs during AR. We observed a nonsignificant increase in T-bet expression following allografting at days 3 and 5 but there was a significant reduction in GATA-3 expression on day 5 compared with controls. The ratio of T-bet to GATA-3 expression showed a trend to increase at day 3 following allografting reaching significance at 5 days. These changes were associated with a significant increase in the expression of IL-2 over IL-4 on days 3 and 5. This study suggests that T(h)1 responses play a major role during AR in the rat lung, and that this differentiation can be monitored by measuring mRNA levels of T-bet and GATA-3.

Significance of total vascular exclusion for hepatic cryotherapy: an experimental study.

BACKGROUND: In the liver, efficacy of cryosurgical ablation of tumors located near the retrohepatic vena cava is impaired by the heat-sink effect. This could be overcome by total vascular exclusion (TVE) of the liver. In this study, the effect of TVE on cryosurgical ablation of liver tissue close to the retrohepatic vena cava was investigated with regard to the extent of the cryolesion and complications arising from necrosis of the caval wall. METHODS: Of a total of 28 pigs, 14 underwent cryotherapy with TVE compared to 14 without TVE, both involving the vena cava. 7 animals in each group were subjected to one freeze cycle and 7 in each group to two freeze cycles. Temperatures in the cryolesion were monitored and cryolesions were documented sonographically. Laboratory parameters were determined pre- and postoperatively. Follow-up was 14 days. Morphology, extent of the cryolesion, damage to the vena cava and complications were assessed after autopsy. RESULTS: With TVE, freezing rates were increased and cryolesions were significantly larger than without TVE. Transmural necroses of the vena cava with complete necrosis of the intima occurred significantly more frequently after TVE. Macro- and microscopically, the damage to the caval wall was considerably more marked after cryotherapy under TVE but in all cases the continuity of the vessel wall remained intact. There were no ruptures, thrombosis, or strictures of the vena cava. CONCLUSIONS: The combination of cryotherapy and TVE increases the effectiveness of cryoablation in the liver involving the retrohepatic vena cava without any severe vascular complications occurring in the pig.

Hepatic cryotherapy involving the vena cava. Experimental study in a pig liver model.

Liver tumors located near the retrohepatic vena cava are often considered nonresectable. For these patients cryoablation could be a therapeutic option. In this study the safety and efficacy of hepatic cryosurgery involving the retrohepatic vena cava were investigated. Cryolesions involving the vena cava were created in 26 pigs. Follow-up was 24 h and 14 days. The extent of the cryolesion, damage to the vena cava and complications were assessed after autopsy. The cyronecrosis extended into the wall of the vena cava in 81% of the animals. All animals had an uneventful recovery without any complications such as ruptures of the vessel, thrombosis or pulmonary embolism. Microscopically elastic and collagenous fibers of the cava wall remained intact. The continuity of the vessel wall was conserved. In conclusion, the safety and efficacy of cryosurgical treatment involving the retrohepatic vena cava were shown in a pig model.