The CD26/DPP4-inhibitor vildagliptin suppresses lung cancer growth via macrophage-mediated NK cell activity.

CD26/dipeptidyl peptidase 4 (DPP4) is a transmembrane protein which is expressed by various malignant cells. We found that the expression of CD26/DPP4 was significantly higher in lung adenocarcinoma samples in our own patient cohort compared to normal lung tissue. We therefore hypothesize that the inhibition of CD26/DPP4 can potentially suppress lung cancer growth. The CD26/DPP4 inhibitor vildagliptin was employed on Lewis Lung Carcinoma (LLC) cell line and a human lung adenocarcinoma (H460) cell line. Two weeks after subcutaneous injection of tumor cells into C57BL/6 and CD1/nude mice, the size of LLC and H460 tumors was significantly reduced by vildagliptin. Immunohistochemically, the number of macrophages (F4/80+) and NK cells (NKp46+) was significantly increased in vildagliptin-treated tumor samples. Mechanistically, we found in vitro that lung cancer cell lines expressed increased levels of surfactant protein upon vildagliptin treatment thereby promoting the pro-inflammatory activity of macrophages. By the depletion of macrophages with clodronate and by using NK cell deficient (IL-15-/-) mice, tumors reversed to the size of controls, suggesting that indeed macrophages and NK cells were responsible for the observed tumor-suppressing effect upon vildagliptin treatment. FACS analysis showed tumor-infiltrating NK cells to express tumor necrosis-related apoptosis-inducing ligand (TRAIL) which induced the intra-cellular stress marker γH2AX. Accordingly, we found upregulated γH2AX in vildagliptin-treated tumors and TRAIL-treated cell lines. Moreover, the effect of vildagliptin-mediated enhanced NK cell cytotoxicity could be reversed by antagonizing the TRAIL receptor. Our data provide evidence that the CD26/DPP4-inhibitor vildagliptin reduces lung cancer growth. We could demonstrate that this effect is exerted by surfactant-activated macrophages and NK cells that act against the tumor via TRAIL-mediated cytotoxicity.

A strategy to analyse activity-based profiling of tyrosine kinase substrates in OCT-embedded lung cancer tissue.

The use of optimal cutting temperature (OCT) medium has served to improve the long-term preservation of surgical tissue specimens. Unfortunately, the presence of polymers in OCT has been found to generate signal interference in proteomic-based techniques. Indeed the presence of OCT medium in tissue lysates precludes the analysis of activity based proteomic profiles obtained from lung adenocarcinoma (LuAdCa) resection specimens. In order to probe this question further tissue lysates were prepared from 47 lung non-neoplastic and tumour, node, metastasis (TNM) stage 1 LuAdCa resection specimens embedded with or without OCT, and data of activity based multiplex profiles of protein tyrosine kinase peptide substrates were obtained. We found that changes in overall phosphorylation level coincided with the use of OCT and subsequently developed an OCT per peptide median correcting strategy by performing median centering on the values of each peptide. Application of this post-analytical strategy not only can identify changes in kinase activity but can also assist in identifying novel targets for therapeutic intervention against LuAdCa.

Ex vivo treatment with inhaled N-acetylcysteine in porcine lung transplantation.

BACKGROUND: We have shown the beneficial effects of N-acetylcysteine (NAC) on posttransplant lung function, when both donor and recipient were pretreated intravenously. However, systemic treatment of multiorgan donors may not be clinically relevant. Thus, we hypothesized that ex vivo treatment of donors with nebulized NAC would be adequate to prevent from ischemia-reperfusion injury after lung transplantation. METHODS: Lungs were retrieved from domestic pigs and stored at 4°C for 24 h followed by 2 h of ex vivo lung perfusion (EVLP) to administer 50 mg/kg of NAC via nebulization in the NAC group (n = 6). The control group received nebulized saline (n = 5). Left lungs were transplanted and isolated at 1 h of reperfusion by occluding the right main bronchus and pulmonary artery, followed by 5 h of observation. Physiological data during EVLP and after reperfusion were recorded. Inflammatory response, markers of oxidative stress, and microscopic lung injury were analyzed. RESULTS: There was a trend toward better oxygenation throughout reperfusion period in the treatment group, which was accompanied by inhibited inflammatory response related to reduction in myeloperoxidase activity during EVLP and nuclear factor-κB activation at the end of reperfusion. CONCLUSIONS: Ex vivo treatment of donor lungs with inhaled NAC reduced inflammatory response via its antioxidant activity in experimental porcine lung transplantation.

Reconditioning of an injured lung graft with intrabronchial surfactant instillation in an ex vivo lung perfusion system followed by transplantation.

BACKGROUND: We tested whether an injured lung graft from category-3 donation after cardiac death donor could be reconditioned with an ex vivo lung perfusion (EVLP) system by intrabronchial diluted surfactant lavage before transplantation. METHODS: In a pig model, cardiac arrest was induced by deconnecting from the ventilator. Left lung injury was done by intrabronchial instillation of 1 mL/kg pepsin + HCl. After retrieval, the heart-lung block was stored at 4°C for 2 h. In the treated group, transplantation was performed after reconditioning with intrabronchial diluted surfactant lavage in EVLP system. RESULTS: During EVLP, surfactant group showed better oxygenation and lower pulmonary vascular resistance. After transplantation, better oxygenation, lower mean pulmonary artery pressure, and lower lung edema were observed in surfactant group. Lower blood IL-1 beta and IL-6 cytokine levels were measured in the surfactant group. In bronchoalveolar lavage, the percentage of neutrophils, IL-1 beta and IL-6 cytokine levels, amount of protein, and neutrophil infiltration in the lung tissue at the end of the experiment were significantly lower in the surfactant group. CONCLUSIONS: Our data demonstrate the feasibility of reconditioning and transplantation of an acutely damaged lung graft due to aspiration from a category-3 DCD donor. Implementation of an EVLP system is an efficacious tool to recondition and assess a questionable graft before transplantation.

Perioperative diclofenac application during video-assisted thoracic surgery pleurodesis modulates early inflammatory and fibrinolytic processes in an experimental model.

BACKGROUND/PURPOSE: It has been substantiated that the quality of pleurodesis is reduced when non-steroidal anti-inflammatory drugs (NSAIDs) are used perioperatively. The effects of NSAID administration on the early inflammatory and fibrinolytic processes after mechanical pleurodesis were investigated in an established pig model. METHODS: Left-sided mechanical pleural abrasion was performed on 24 pigs assigned to either an NSAID or a control group. Pleural fluid and blood samples were analysed over a 24-hour period. Histological evaluation of neutrophil influx at the site of pleural abrasion was performed. RESULTS: The volume of pleural effusion was significantly decreased in the diclofenac group at 10 and 24 h, and the protein content was significantly lower. The diclofenac group at 24 h had a diminished total number of white blood cells and a reduced content of transforming growth factor-ß. Moreover, the diclofenac group had a reduced percentage of neutrophils at 6 h. Significantly increased levels of D-dimers and tissue plasminogen activator were measured at 6 h and of interleukin-10 at 24 h. Neutrophils at the site of pleural abrasion were significantly reduced. CONCLUSIONS: Systemic application of diclofenac led to a local enhancement of fibrinolysis and attenuation of pro-inflammatory and fibrotic processes necessary for adhesion formation in our model.

Hemoadsorption in Organ Preservation and Transplantation: A Narrative Review.

Cytokine adsorption can resolve different complications characteristic of transplantation medicine, such as cytokine storm activation and blood ABO and immune incompatibilities. Cytokine adsorption is also performed for the treatment of various life-threatening conditions, such as endotoxic septic shock, acute respiratory distress syndrome, and cardiogenic shock, all potentially leading to adverse clinical outcomes during transplantation. After surgery, dysmetabolism and stress response limit successful graft survival and can lead to primary or secondary graft dysfunction. In this clinical context, and given that a major problem in transplant medicine is that the demand for organs far exceeds the supply, a technological innovation such as a hemoadsorption system could greatly contribute to increasing the number of usable organ donors. The objectives of this review are to describe the specific advantages and disadvantages of the application of cytokine adsorption in the context of transplantation and examine, before and/or after organ transplantation, the benefits of the addition of a cytokine adsorption therapy protocol.

Effect of ß-Nicotinamide Adenine Dinucleotide on Acute Allograft Rejection After Rat Lung Transplantation.

Acute rejection is still a major limitation for a successful outcome in lung transplantation. Since ß-nicotinamide adenine dinucleotide (NAD+) has been shown to have various immunomodulatory properties on the innate and adaptive immune system, we evaluate here a potential protective effect of NAD+ against acute lung rejection. Methods: Rat single-lung transplantation was performed in 2 groups (n = 8 per group), using Brown-Norway donors and major histocompatibility complex-mismatched Lewis recipients. Recipients of the NAD+ group received 1000 mg/kg NAD+ intraperitoneally before transplantation and daily thereafter until euthanasia, whereas the control group received saline solution. At autopsy on day 5, blood samples were analyzed and the lung allograft was assessed by bronchioalveolar lavage, histology, and immunochemistry. Results: The NAD+ group maintained an intact compliant lung tissue, a strong trend of lower acute cellular rejection (A3 versus A3-A4) and significantly less lymphocytic bronchiolitis (B0-B2R versus B1R-Bx). In addition, a trend of fewer alveolar CD68+ macrophages and significantly fewer interstitial CD163+ macrophages was observed. Bronchoalveolar lavage in the NAD+ group showed significantly fewer proinflammatory cytokines interleukin (IL)-6, IL-13, TNFα, and a protective IL-6/IL-10-ratio. In blood samples, we observed significantly fewer neutrophils, and proinflammatory GRO/KC in the NAD+ group. Conclusions: NAD+ might be a promising substance in prevention of acute allograft rejection in lung transplantation.

Extracorporeal cytokine adsorption reduces systemic cytokine storm and improves graft function in lung transplantation.

Objectives: Ischemia-reperfusion injury often coincides with a cytokine storm, which can result in primary graft dysfunction following lung transplantation. Our previous research has demonstrated allograft improvement by cytokine adsorption during ex vivo lung perfusion. The aim of this study was to investigate the effect of in vivo extracorporeal cytokine adsorption in a large animal model. Materials and Methods: Pig left lung transplantation was performed following 24 hours of cold ischemic storage. Observation period after transplantation was 24 hours. In the treatment group (n = 6), extracorporeal CytoSorb adsorption was started 30 minutes before reperfusion and continued for 6 hours. A control group (n = 3) did not receive adsorber treatment. Results: During adsorption, we consistently noticed a significant decrease in plasma proinflammatory interleukin (IL)-2, trends of less proinflammatory, tumor necrosis factor- α, IL-1α, and granulocyte-macrophage colony-stimulating factor as well as significantly reduced systemic neutrophils. In addition, a significantly lower peak airway pressure was detected during the 6 hours of adsorption. After 24 hours of observation, when evaluating the left lung allograft independently, we observed significantly improved CO2 removal, partial pressure of oxygen/inspired oxygen fraction ratio, and less acidosis in the treatment group. At autopsy, bronchoalveolar lavage results exhibited significantly lower recruitment of cells and less pro-inflammatory IL-1α, IL-1ß, IL-6, and IL-8 in the treatment group. Histologically, the treatment group had a strong trend, indicating less neutrophil invasion into the alveolar space. Conclusions: Based on our findings, cytokine adsorption during and after reperfusion is a viable approach to reducing posttransplant inflammation following lung transplantation. CytoSorb may increase the acceptance of extended criteria donor lungs, which are more susceptible to ischemia-reperfusion injury.

Ex Vivo Lung Perfusion with ß-Nicotinamide Adenine Dinucleotide (NAD+) Improves Ischemic Lung Function.

Ischemia-reperfusion injury compromises short- and long-term outcomes after lung transplantation. The scarce existing data on NAD+ suggest effects on hypoxia-induced vasoconstriction, on reactive oxygen species and on tampering inflammation. We exposed rat lungs to 14 h of cold ischemic storage and perfused them in a rat ex vivo lung perfusion (EVLP) system for 4 h. A control group (n = 6) was compared to groups receiving 100 µM (n = 6) or 200 µM NAD+ (n = 6) in the preservation solution and groups receiving 200 µM (n = 4) or 2000 µM (n = 6) NAD+ every 30 min in the perfusate, starting at 1 h of EVLP. Compared to the control, significant effects were only achieved in the 2000 µM NAD+ group. During the 4 h of EVLP, we monitored higher vascular flow, lower mean pulmonary arterial pressure and increased oxygenation capacity. Tissue inflammation estimated with the myeloperoxidase assay was lower in the 2000 µM NAD+ group. We observed higher levels of anti-inflammatory IL-10, higher anti-inflammatory IL-6/IL-10 ratios and lower levels of pro-inflammatory IL-12 and IL-18 as well as a trend of more anti-inflammatory IFNy in the 2000 µM NAD+ perfusate. In the bronchoalveolar lavage, the pro-inflammatory levels of IL-1α and IL-1ß were lower in the 2000 µM NAD+ group. NAD+ administered during EVLP is a promising agent with both anti-inflammatory properties and the ability to improve ischemic lung function.

A Comprehensive Review on the Surgical Aspect of Lung Transplant Models in Mice and Rats.

Lung transplantation improves the outcome and quality of life of patients with end-stage pulmonary disease. However, the procedure is still hampered by the lack of suitable donors, the complexity of the surgery, and the risk of developing chronic lung allograft dysfunction. Over the past decades, translational experiments in animal models have led to a better understanding of physiology and immunopathology following the lung transplant procedure. Small animal models (e.g., rats and mice) are mostly used in experiments regarding immunology and pathobiology and are preferred over large animal models due to the ethical aspects, the cost-benefit balance, and the high throughput possibility. In this comprehensive review, we summarize the reported surgical techniques for lung transplantation in rodent models and the management of perioperative complications. Furthermore, we propose a guide to help identify the appropriate species for a given experiment and discuss recent experimental findings in small animal lung transplant models.

Surfactant alterations following donation after cardiac death donor lungs.

The use of lungs from donation after cardiac death (DCD) donors is one of the strategies to increase the donor pool. The aim of this study was to assess the surfactant alterations in DCD donor lungs. Pigs were sacrificed and left untouched for 1 (DCD1), 2 (DCD2) and 3 (DCD3) h. Lungs were then topically cooled with saline for 1, 2 or 3 h to reach a total ischemic time of 4 h. Heart-beating donors (HBD) served as control group. Bronchoalveolar lavage (BAL) samples were assessed for protein levels and surfactant function. Left lungs were prepared for ex-vivo evaluation. Pulmonary vascular resistance (PVR), oxygenation, airway pressure (AWP) and wet-to-dry weight ratio were significantly different between HBD and DCD3 groups (P < 0.05). BAL protein levels were statistically higher in DCD3 compared with HBD group (P < 0.05). Surface tension and surface tension measured at minimal bubble diameter (adsorption) were lower in HBD compared with DCD groups (P < 0.05). Adsorption was also lower in DCD1 compared with DCD2 (P < 0.05). Adsorption and surface tension were correlated with oxygenation and AWP (P < 0.05). This study has shown that lung function deteriorates with increasing warm ischemic time intervals. BAL protein, surface tension, adsorption, peak AWP and PVR increase significantly after 2 h of warm ischemia together with a significant reduction of the ratio PaO(2)/FiO(2).

Subnormothermic ex vivo lung perfusion attenuates ischemia reperfusion injury from donation after circulatory death donors.

Use of normothermic ex vivo lung perfusion (EVLP) was adopted in clinical practice to assess the quality of marginal donor lungs. Subnormothermic perfusion temperatures are in use among other solid organs to improve biochemical, clinical and immunological parameters. In a rat EVLP model of donation after circulatory death (DCD) lung donors, we tested the effect of four subnormothermic EVLP temperatures that could further improve organ preservation. Warm ischemic time was of 2 hours. EVLP time was of 4 hours. Lung physiological data were recorded and metabolic parameters were assessed. Lung oxygenation at 21°C and 24°C were significantly improved whereas pulmonary vascular resistance and edema formation at 21°C EVLP were significantly worsened when compared to 37°C EVLP. The perfusate concentrations of potassium ions and lactate exiting the lungs with 28°C EVLP were significantly lower whereas sodium and chlorine ions with 32°C EVLP were significantly higher when compared to 37°C EVLP. Also compared to 37°C EVLP, the pro-inflammatory chemokines MIP2, MIP-1α, GRO-α, the cytokine IL-6 were significantly lower with 21°C, 24°C and 28°C EVLP, the IL-18 was significantly lower but only with 21°C EVLP and IL-1ß was significantly lower at 21°C and 24°C EVLP. Compared to the 37°C EVLP, the lung tissue ATP content after 21°C, 24°C and 28°C EVLP were significantly higher, the carbonylated protein content after 28°C EVLP was significantly lower and we measured significantly higher myeloperoxidase activities in lung tissues with 21°C, 24°C and 32°C. The 28°C EVLP demonstrated acceptable physiological variables, significantly higher lung tissue ATP content and decreased tissue carbonylated proteins with reduced release of pro-inflammatory cytokines. In conclusion, the 28°C EVLP is a non inferior setting in comparison to the clinically approved 37°C EVLP and significantly improve biochemical, clinical and immunological parameters and may reduce I/R injuries of DCD lung donors.

Subnormothermic Ex Vivo Lung Perfusion Temperature Improves Graft Preservation in Lung Transplantation.

Normothermic machine perfusion is clinically used to assess the quality of marginal donor lungs. Although subnormothermic temperatures have proven beneficial for other solid organ transplants, subnormothermia-related benefits of ex vivo lung perfusion (EVLP) still need to be investigated. Material and Methods: In a rat model, we evaluated the effects of 28 °C temperature on 4-h EVLPs with subsequent left lung transplantation. The recipients were observed for 2 h postoperatively. Lung physiology data were recorded and metabolic parameters were assessed. Results: During the 4-h subnormothermic EVLP, the lung oxygenation was significantly higher (p < 0.001), pulmonary vascular resistance (PVR) lower and dynamic compliance (Cdyn) higher when compared to the 37 °C EVLP. During an end-of-EVLP stress test, we recorded significantly higher flow (p < 0.05), lower PVR (p < 0.05) and higher Cdyn (p < 0.01) in the 28 °C group when compared to the 37 °C group. After the left lung transplantation, Cdyn and oxygenation improved in the 28 °C group, which were comparable to the 37 °C group. Chemokines RANTES, MIP-3α, MIP-1α MCP-1 GRO/KC and pro-inflammatory mediators GM-CSF, G-CSF and TNFα were significantly lower after the 28 °C EVLP and remained low in the plasma of the recipient rats after transplantation. The lungs of the 28 °C group showed significantly lowered myeloperoxidase activity and lowered levels of TNFα and IL-1ß. Conclusions: Compared to the normothermic perfusion, the 28 °C EVLP improved Cdyn and PVR and reduced both the release of pro-inflammatory cytokines and myeloperoxidase activity in lung tissue. These observations were also observed after the left lung transplantation in the subnormothermic group. The 28 °C EVLP significantly improved biochemical, physiological and inflammatory parameters in lung donors.

Perfluorocarbon-Based Oxygen Carriers and Subnormothermic Lung Machine Perfusion Decrease Production of Pro-Inflammatory Mediators.

The quality of marginal donor lungs is clinically assessed with normothermic machine perfusion. Although subnormothermic temperature and perfluorocarbon-based oxygen carriers (PFCOC) have proven favourable for other organ transplants, their beneficial use for ex vivo lung perfusion (EVLP) still requires further investigation. In a rat model, we evaluated on a 4 h EVLP time the effects of PFCOC with either 28 °C or 37 °C perfusion temperatures. During EVLP at 28 °C with PFCOC, we recorded significantly lower lung pulmonary vascular resistance (PVR), higher dynamic compliance (Cdyn), significantly lower potassium and lactate levels, higher lung tissue ATP content, and significantly lower myeloperoxidase tissue activity when compared to the 37 °C EVLP with PFCOC. In the subnormothermic EVLP with or without PFCOC, the pro-inflammatory mediator TNFα, the cytokines IL-6 and IL-7, the chemokines MIP-3α, MIP-1α, MCP-1, GRO/KC as well as GM-CSF, G-CSF and the anti-inflammatory cytokines IL-4 and IL-10 were significantly lower. The 28 °C EVLP improved both Cdyn and PVR and decreased pro-inflammatory cytokines and pCO2 levels compared to the 37 °C EVLP. In addition, the 28 °C EVLP with PFCOC produced a significantly lower level of myeloperoxidase activity in lung tissue. Subnormothermic EVLP with PFCOC significantly improves lung donor physiology and ameliorates lung tissue biochemical and inflammatory parameters.

Ex Vivo Lung Perfusion with K(ATP) Channel Modulators Antagonize Ischemia Reperfusion Injury.

Ex vivo lung perfusion (EVLP) has been implemented to increase the number of donor lungs available for transplantation. The use of K(ATP) channel modulators during EVLP experiments may protect against lung ischemia-reperfusion injury and may inhibit the formation of reactive oxygen species. In a rat model of donation after circulatory death with 2 h warm ischemic time, we evaluated rat lungs for a 4-hour time in EVLP containing either mitochondrial-specific or plasma membrane and/or sarcolemmal-specific forms of K(ATP) channel modulators. Lung physiological data were recorded, and metabolic parameters were assessed. When compared to the control group, in the EVLP performed with diazoxide or 5-hydroxydecanoic acid (5-HD) we recorded significantly lower pulmonary vascular resistance and only in the diazoxide group recorded significant lung weight loss. In the perfusate of the 5-HD group, interleukin-1ß and interleukin-1α were significantly lower when compared to the control group. Perfusate levels of calcium ions were significantly higher in both 5-HD and cromakalim groups, whereas the levels of calcium, potassium, chlorine and lactate were reduced in the diazoxide group, although not significantly when compared to the control. The use of a diazoxide mitochondrial-specific K(ATP) channel opener during EVLP improved lung physiological and metabolic parameters and reduced edema.

Perfusate adsorption during ex vivo lung perfusion improves early post-transplant lung function.

OBJECTIVE: Improvement in ex vivo lung perfusion protocols could increase the number of donors available for transplantation and protect the lungs from primary graft dysfunction. We hypothesize that perfusate adsorption during ex vivo lung perfusion reconditions the allograft to ischemia-reperfusion injury after lung transplantation. METHODS: Donor pig lungs were preserved for 24 hours at 4°C, followed by 6 hours of ex vivo lung perfusion according to the Toronto protocol. The perfusate was additionally adsorbed through a CytoSorb adsorber (CytoSorbents, Berlin, Germany) in the treatment group, whereas control lungs were perfused according to the standard protocol (n = 5, each). Ex vivo lung perfusion physiology and biochemistry were monitored. Upon completion of ex vivo lung perfusion, a left single lung transplantation was performed. Oxygenation function and lung mechanics were assessed during a 4-hour reperfusion period. The inflammatory response was determined during ex vivo lung perfusion and reperfusion. RESULTS: The cytokine concentrations in the perfusate were markedly lower with the adsorber, resulting in improved ex vivo lung perfusion physiology and biochemistry during the 6-hour perfusion period. Post-transplant dynamic lung compliance was markedly better during the 4-hour reperfusion period in the treatment group. Isolated allograft oxygenation function and dynamic compliance continued to be superior in the adsorber group at the end of reperfusion, accompanied by a markedly decreased local inflammatory response. CONCLUSIONS: Implementation of an additional cytokine adsorber has refined the standard ex vivo lung perfusion protocol. Furthermore, cytokine removal during ex vivo lung perfusion improved immediate post-transplant graft function together with a less intense inflammatory response to reperfusion in pigs. Further studies are warranted to understand the beneficial effects of perfusate adsorption during ex vivo lung perfusion in the clinical setting.

Functional, Metabolic and Morphologic Results of Ex Vivo Donor Lung Perfusion with a Perfluorocarbon-Based Oxygen Carrier Nanoemulsion in a Large Animal Transplantation Model.

BACKGROUND: Ex vivo lung perfusion (EVLP) is a technology that allows the re-evaluation of questionable donor lung before implantation and it has the potential to repair injured donor lungs that are otherwise unsuitable for transplantation. We hypothesized that perfluorocarbon-based oxygen carrier, a novel reconditioning strategy instilled during EVLP would improve graft function. METHODS: We utilized perfluorocarbon-based oxygen carrier (PFCOC) during EVLP to recondition and improve lung graft function in a pig model of EVLP and lung transplantation. Lungs were retrieved and stored for 24 h at 4 °C. EVLP was done for 6 h with or without PFCOC. In the transplantation groups, left lung transplantation was done after EVLP with or without PFCOC. Allograft function was assessed by means of pulmonary gas exchange, lung mechanics and vascular pressures, histology and transmission electron microscopy (TEM). RESULTS: In the EVLP only groups, physiological and biochemical markers during the 6-h perfusion period were comparable. However, perfusate lactate potassium levels were lower and ATP levels were higher in the PFCOC group. Radiologic assessment revealed significantly more lung infiltrates in the controls than in the PFCOC group (p = 0.04). In transplantation groups, perfusate glucose consumption was higher in the control group. Lactate levels were significantly lower in the PFCOC group (p = 0.02). Perfusate flavin mononucleotide (FMN) was significantly higher in the controls (p = 0.008). Post-transplant gas exchange was significantly better during the 4-h reperfusion period in the PFCOC group (p = 0.01). Plasma IL-8 and IL-12 levels were significantly lower in the PFCOC group (p = 0.01, p = 0.03, respectively). ATP lung tissue levels at the end of the transplantation were higher and myeloperoxidase (MPO) levels in lung tissue were lower in the PFCOC group compared to the control group. In the PFCOC group, TEM showed better tissue preservation and cellular viability. CONCLUSION: PFCOC application is safe during EVLP in lungs preserved 24 h at 4 °C. Although this strategy did not significantly affect the EVLP physiology, metabolic markers of the donor quality such as lactate production, glucose consumption, neutrophil infiltration and preservation of mitochondrial function were better in the PFCOC group. Following transplantation, PFCOC resulted in better graft function and TEM showed better tissue preservation, cellular viability and improved gas transport.

Identification and functional characterization of the promoter of the mouse sodium-activated sodium channel Na(x) gene (Scn7a).

Na(x) is a sodium channel, thought to be a descendant of the voltage-gated sodium channel family. Nevertheless, Na(x) is not activated by voltage but rather by augmentation of extracellular sodium over 150 mM. In the brain, it is localized to the circumventricular organs, important regions for salt and water homeostasis in mammals, where it operates as a sodium-level sensor of body fluid. Na(x) channel is expressed in lung, uterus, and heart, and it is also found in trigeminal and dorsal root ganglia and in nonmyelinating Schwann cells, where its physiological role remains unclarified. Here we identified the promoter and transcription start sites of Na(x) sodium channel in dorsal root ganglia neurons from mouse. We report a characterization of the basal TATA-less promoter and the sequence requirements for promoter activity in Neuro 2A neuroblastoma cells and in dorsal root ganglia neurons, where basal promoter activity seems to require NGFI-C and Ebox DNA elements. Finally, we provide evidence that a repression mechanism that inhibits Na(x) expression may be present in certain tissues. These findings provide the basis with which to understand tissue-specific regulation of Na(x) sodium channel gene (Scn7a) expression.

Comparative immunohistochemistry of L19 and F16 in non-small cell lung cancer and mesothelioma: two human antibodies investigated in clinical trials in patients with cancer.

The antibody-mediated targeted delivery of therapeutics to tumor sites is an attractive avenue for combating cancer while sparing normal tissues. Indeed, five derivatives of the human monoclonal antibodies L19 and F16, specific to splice isoforms of fibronectin and tenascin-C, are currently being investigated in clinical trials in patients with malignancies. Until now, a comparative immunohistochemical analysis of these antibodies, which recognize components of the modified extracellular matrix, was missing. Here, we report that the majority of NSCLC and mesothelioma specimens are stained with both antibodies in the stroma, while non-tumoral lung and mesothelium samples rarely exhibit reactivity with either L19 or F16. In our analysis, the anti-tenascin F16 antibody was found to generally exhibit a stronger staining of desmoplastic stroma surrounding tumor. This superior performance was found to be particularly striking in the case of low-grade non-small cell lung cancer.

Local recurrence model of malignant pleural mesothelioma for investigation of intrapleural treatment.

OBJECTIVE: Local recurrence remains a major problem in the treatment of malignant pleural mesothelioma. The aim of the underlying study was to establish a standardised local recurrence model in rats which enables to study different intrapleural therapies. MATERIALS AND METHODS: Fifty microlitre containing 1 x 10(6) cells of a syngeneic rat malignant mesothelioma cell line (II-45), established from mesothelioma in Fischer 344 rats exposed to asbestos, were inoculated subpleurally via a left-sided thoracotomy. Tumour size was assessed 6 days later and the tumour nodule completely resected. Evaluation of recurrence at the resection site was performed after 10 days (n=6) and 6 days (n=6). The recurrent nodule was histopathologically confirmed. In a second experiment, this new recurrence model was evaluated for the effect of intrapleural therapy with different agents: 4 ml of cisplatin-solution (100mg(2)/kg BW), cisplatin combined with the fibrin-based sealant Vivostat, 4 ml taurolidine 2%, repeated injection of 1 microg of the chemokine CCL-19 at the tumour site and 4 ml povidone-iodine in a dilution 1:10. In a control group, the chest cavity was filled with 4 ml 0.9% NaCl. The primary endpoint was the extent of tumour recurrence. RESULTS: Six days after inoculation, all animals presented a standardised tumour nodule at the injection site of a mean diameter of 5.1 (+/-0.8)mm. Evaluation of the recurrence after 10 days showed a relapse directly at the resection site, but additional tumour nodules on the ipsi- and contralateral chest wall were found and histologically confirmed. The animals that were sacrificed 6 days after resection of the tumour nodule showed a recurrence only at the resection site with no macroscopic or microscopic evidence of other tumour. Resection of the tumour nodule combined with intrapleural application of the different agents lead to clear reduction of recurrence. The strongest effect was observed after intrapleural application of cisplatin-Vivostat with significant decrease of the longest, widest and thickest diameter of the recurrence. CONCLUSIONS: With this new recurrence model for investigation of malignant pleural mesothelioma in rats, we were able to investigate new intrapleural therapies after pneumonectomy. The intrapleural application of cisplatin-Vivostat significantly reduced the extent of local recurrence.