Comparison of lung preservation solutions in human lungs using an ex vivo lung perfusion experimental model

OBJECTIVE: Experimental studies on lung preservation have always been performed using animal models. We present ex vivo lung perfusion as a new model for the study of lung preservation. Using human lungs instead of animal models may bring the results of experimental studies closer to what could be expected in clinical practice. METHOD: Brain-dead donors whose lungs had been declined by transplantation teams were used. The cases were randomized into two groups. In Group 1, Perfadex (R) was used for pulmonary preservation, and in Group 2, LPDnac, a solution manufactured in Brazil, was used. An ex vivo lung perfusion system was used, and the lungs were ventilated and perfused after 10 hours of cold ischemia. The extent of ischemic-reperfusion injury was measured using functional and histological parameters. RESULTS: After reperfusion, the mean oxygenation capacity was 405.3 mmHg in Group 1 and 406.0 mmHg in Group 2 (p=0.98). The mean pulmonary vascular resistance values were 697.6 and 378.3 dyn.s.cm(-5), respectively (p=0.035). The mean pulmonary compliance was 46.8 cm H2O in Group 1 and 49.3 ml/cm H2O in Group 2 (p=0.816). The mean wet/dry weight ratios were 2.06 and 2.02, respectively (p=0.87). The mean Lung Injury Scores for the biopsy performed after reperfusion were 4.37 and 4.37 in Groups 1 and 2, respectively (p=1.0), and the apoptotic cell counts were 118.75/mm(2) and 137.50/mm(2), respectively (p=0.71). CONCLUSION: The locally produced preservation solution proved to be as good as Perfadex (R). The clinical use of LPDnac may reduce costs in our centers. Therefore, it is important to develop new models to study lung preservation.

Regular and moderate exercise before experimental sepsis reduces the risk of lung and distal organ injury

de Araujo CC, Silva JD, Samary CS, Guimaraes IH, Marques PS, Oliveira GP, do Carmo LGRR, Goldenberg RC, Bakker-Abreu I, Diaz BL, Rocha NN, Capelozzi VL, Pelosi P, Rocco PRM. Regular and moderate exercise before experimental sepsis reduces the risk of lung and distal organ injury. J Appl Physiol 112: 1206-1214, 2012. First published January 19, 2012; doi:10.1152/japplphysiol.01061.2011.-Physical activity modulates inflammation and immune response in both normal and pathologic conditions. We investigated whether regular and moderate exercise before the induction of experimental sepsis reduces the risk of lung and distal organ injury and survival. One hundred twenty-four BALB/c mice were randomly assigned to two groups: sedentary (S) and trained (T). Animals in T group ran on a motorized treadmill, at moderate intensity, 5% grade, 30 min/day, 3 times a week for 8 wk. Cardiac adaptation to exercise was evaluated using echocardiography. Systolic volume and left ventricular mass were increased in T compared with S group. Both T and S groups were further randomized either to sepsis induced by cecal ligation and puncture surgery (CLP) or sham operation (control). After 24 h, lung mechanics and histology, the degree of cell apoptosis in lung, heart, kidney, liver, and small intestine villi, and interleukin (IL)-6, KC (IL-8 murine functional homolog), IL-1 beta, IL-10, and number of cells in bronchoalveolar lavage (BALF) and peritoneal lavage (PLF) fluids as well as plasma were measured. In CLP, T compared with S groups showed: 1) improvement in survival; 2) reduced lung static elastance, alveolar collapse, collagen and elastic fiber content, number of neutrophils in BALF, PLF, and plasma, as well as lung and distal organ cell apoptosis; and 3) increased IL-10 in BALF and plasma, with reduced IL-6, KC, and IL-1 beta in PLF. In conclusion, regular and moderate exercise before the induction of sepsis reduced the risk of lung and distal organ damage, thus increasing survival.

Effects of overinflation on procollagen type III expression in experimental acute lung injury

Abstract Introduction In acute lung injury (ALI), elevation of procollagen type III (PC III) occurs early and has an adverse impact on outcome. We examined whether different high-inflation strategies of mechanical ventilation (MV) in oleic acid (OA) ALI alter regional expression of PC III. Methods We designed an experimental, randomized, and controlled protocol in which rats were allocated to two control groups (no injury, recruited [alveolar recruitment maneuver after tracheotomy without MV; n = 4 rats] and control [n = 5 rats]) or four injured groups (one exposed to OA only [n = 10 rats] and three OA-injured and ventilated). The three OA-injured groups were ventilated for 1 hour according to the following strategies: LVHP-S (low volume-high positive end-expiratory pressure [PEEP], supine; n = 10 rats, tidal volume [VT] = 8 ml/kg, PEEP = 12 cm H2O), HVLP-S (high volume-low PEEP, supine; n = 10 rats, VT = 20 ml/kg, PEEP = 5 cm H2O), and HVLP-P (high volume-low PEEP, prone; n = 10 rats). Northern blot analysis for PC III and interleukin-1-beta (IL-1β) and polymorphonuclear infiltration index (PMI) counting were performed in nondependent and dependent regions. Regional differences between groups were assessed by two-way analysis of variance after logarithmic transformation and post hoc tests. Results A significant interaction for group and region effects was observed for PC III (p = 0.012) with higher expression in the nondependent region for HVLP-S and LVHP-S, intermediate for OA and HVLP-P, and lower for control (group effect, p < 0.00001, partial η2 = 0.767; region effect, p = 0.0007, partial η2 = 0.091). We found high expression of IL-1β (group effect, p < 0.00001, partial η2 = 0.944) in the OA, HVLP-S, and HVLP-P groups without regional differences (p = 0.16). PMI behaved similarly (group effect, p < 0.00001, partial η2 = 0.832). Conclusion PC III expression is higher in nondependent regions and in ventilatory strategies that caused overdistension. This response was partially attenuated by prone positioning.

Impact of CD39 and purinergic signalling on the growth and metastasis of colorectal cancer

Despite improvements in prevention and management of colorectal cancer (CRC), uncontrolled tumor growth with metastatic spread to distant organs remains an important clinical concern. Genetic deletion of CD39, the dominant vascular and immune cell ectonucleotidase, has been shown to delay tumor growth and blunt angiogenesis in mouse models of melanoma, lung and colonic malignancy. Here, we tested the influence of CD39 on CRC tumor progression and metastasis by investigating orthotopic transplanted and metastatic cancer models in wild-type BALB/c, human CD39 transgenic and CD39 deficient mice. We also investigated CD39 and P2 receptor expression patterns in human CRC biopsies. Murine CD39 was expressed by endothelium, stromal and mononuclear cells infiltrating the experimental MC-26 tumors. In the primary CRC model, volumes of tumors in the subserosa of the colon and/or rectum did not differ amongst the treatment groups at day 10, albeit these tumors rarely metastasized to the liver. In the dissemination model, MC-26 cell line-derived hepatic metastases grew significantly faster in CD39 over-expressing transgenics, when compared to CD39 deficient mice. Murine P2Y2 was significantly elevated at both mRNA and protein levels, within the larger liver metastases obtained from CD39 transgenic mice where changes in P2X7 levels were also noted. In clinical samples, lower levels of CD39 mRNA in malignant CRC tissues appeared associated with longer duration of survival and could be linked to less invasive tumors. The modulatory effects of CD39 on tumor dissemination and differential levels of CD39, P2Y2 and P2X7 expression in tumors suggest involvement of purinergic signalling in these processes. Our studies also suggest potential roles for purinergic-based therapies in clinical CRC.

Early occurrence of lung adenocarcinoma and breast cancer after radiotherapy of a chest wall sarcoma in a patient with a de novo germline mutation in TP53

We report a 26-year-old female patient who was diagnosed within 4 years with chest sarcoma, lung adenocarcinoma, and breast cancer. While her family history was unremarkable, DNA sequencing of TP53 revealed a germline de novo non-sense mutation in exon 6 p.Arg213X. One year later, she further developed a contralateral ductal carcinoma in situ, and 18 months later a jaw osteosarcoma. This case illustrates the therapeutic pitfalls in the care of a young cancer patient with TP53 de novo germline mutations and the complications related to her first-line therapy. Suggestion is made to use the less stringent Chompret criteria for germline TP53 mutation screening. Our observation underlines the possibly negative effect of radiotherapy in generating second tumors in patients with a TP53 mutation. We also present a review of six previously reported cases, comparing their cancer phenotypes with those generally produced by TP53 mutations.

Preischemic iloprost application for improvement of graft preservation: which route is superior in experimental pig lung transplantation: inhaled or intravenous?

BACKGROUND: Optimal allograft protection is essential in lung transplantation to reduce postoperative organ dysfunction. Although intravenous prostanoids are routinely used to ameliorate reperfusion injury, the latest evidence suggests a similar efficacy of inhaled prostacyclin. Therefore, we compared donor lung-pretreatment using inhaled lioprost (Ventavis) with the commonly used intravenous technique. METHODS: Five pig lungs were each preserved with Perfadex and stored for 27 hours without (group 1) or with (group-2, 100 prior aerosolized of iloprost were (group 3) or iloprost (IV). Following left lung transplantation, hemodynamics, Po(2)/F(i)o(2), compliance, and wet-to-dry ratio were monitored for 6 hours and compared to sham controls using ANOVA analysis with repeated measures. RESULTS: The mortality was 100% in group 3. All other animals survived (P < .001). Dynamic compliance and PVR were superior in the endobronchially pretreated iloprost group as compared with untreated organs (P < .05), whereas oxygenation was comparable overall W/D-ratio revealed significantly lower lung water in group 2 (P = .027) compared with group 3. CONCLUSION: Preischemic alveolar deposition of iloprost is superior to IV pretreatment as reflected by significantly improved allograft function. This strategy offers technique to optimize pulmonary preservation.

Lung growth after experimental pulmonary transplantation

Replacement of the heart and both lungs or single lung transplantation has been performed in a few cases of terminal (cardio) pulmonary disease in childhood. It remains unclear whether pulmonary allografts will meet the demands of a growing organism. Six domestic pigs (mean body weight, 24 kg) underwent left lung transplantation from donors of equal weight. Immunosuppression consisted of cyclosporine, azathioprine, and corticosteroids. After the pigs doubled their body weight, growth of the lung was assessed by bronchography and pulmonary angiography. In transplant animals it took 11 weeks (normal animals, 6 weeks) for their weight to double. At that time, the bronchial tree showed similar growth when compared with nontransplant animals of equal weight. The diameter of the left lower lobe bronchus (9.2 +/- 0.4 mm) was significantly greater than that of animals of 24 kg body weight (7.5 +/- 0.3 mm; p less than 0.01) but comparable to that of normal pigs of similar weight (9.0 +/- 0.5 mm). The same applied for length of the left lower lobe bronchus (transplants, 95 +/- 6.7 mm; controls 24 kg, 67 +/- 2 mm [p less than 0.01]; controls 48 kg, 93 +/- 3 mm). Similar growth tendencies were observed in the pulmonary vascular tree. The diameter of the left lower lobe artery was 9.4 +/- 98 mm in 48 kg transplant pigs, compared with 9.7 +/- 1.2 mm in 24 kg control pigs and 8.5 +/- 0.8 mm in 48 kg control pigs. In one case of recurrent severe pulmonary rejection, the lung did not grow. We conclude from this study that growth is retarded by immunosuppression.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth of lung allografts after experimental transplantation

Heart and lung transplantation has been performed in cases of end-stage cardiopulmonary disease in infants. Nevertheless, it still remains unclear whether lung allografts adjust to a growing organism. In 6 young domestic pigs unilateral left lung allotransplantation was performed. Immunosuppression consisted of a triple drug therapy including cyclosporine, azathioprine, and corticosteroids. Lung growth was studied by using bronchography, pulmonary angiography, and lung histology. After 11 weeks the transplanted animals had doubled their body weight from 24 kg to 48 kg. Non-transplanted animals in contrast doubled their weight within only 6 weeks. The growth retardation was attributed to the immunosuppressive therapy. The bronchial tree and pulmonary vasculature of lung allografts showed a similar growth potential to non-transplanted lungs in animals of equivalent body weight. In one case of recurrent severe rejection of the lung no growth was observed. Therefore it was concluded that lung allografts grow adequately according to the development of the recipient organism. Lung transplantation in children does not seem to be restricted by a limited growth potential of the graft.

T-cadherin loss promotes experimental metastasis of squamous cell carcinoma

T-cadherin is gaining recognition as a determinant for the development of incipient invasive squamous cell carcinoma (SCC). However, effects of T-cadherin expression on the metastatic potential of SCC have not been studied. Here, using a murine model of experimental metastasis following tail vein injection of A431 SCC cells we report that loss of T-cadherin increased both the incidence and rate of appearance of lung metastases. T-cadherin-silenced SCC metastases were highly disordered with evidence of single cell dissemination away from main foci whereas SCC metastases overexpressing T-cadherin developed as compact, tightly organised sheets. SCC cell adhesion to vascular endothelial cells (EC) in culture was increased for T-cadherin-silenced SCC and decreased for T-cadherin-overexpressing SCC. Confocal microscopy showed that T-cadherin-silenced SCC adherent on EC display an elongated morphology with long thin extensions and a high degree of intercalation within the EC monolayer, whereas SCC overexpressing T-cadherin formed poorly-spread multicellular aggregates that remain on the outer surface of the EC monolayer. T-cadherin-deficient SCC or human keratinocyte cells exhibited increased transendothelial migration in vitro which could be attenuated in the presence of EGFR inhibitor gefitinib. Our data suggest that loss of T-cadherin can increase metastatic potential and aggressiveness of SCC, possibly due to facilitating arrest and extravasation through the vascular wall and/or more efficient establishment of metastases in the new microenvironment.

Investigating the Effects of Silencing EphA2 in Metastatic Breast Cancer Cells

EphA2, also known as ECK (epithelial cell kinase), is a transmembrane receptor tyrosine kinase that is commonly over-expressed in cancers such as those of the prostate, colon, lung, and breast. For breast cancers, EphA2 overexpression is most prominent in the ER-negative subtype, and is associated with a higher rate of lung metastasis. Studies conducted to demonstrate the role of EphA2 in a non-cancerous environment have shown that it is very important in developmental processes, but not in normal adult tissues. These results make EphA2 a prospective therapeutic target since new therapies are needed for the more aggressive ER-negative breast cancers. A panel of breast cancer cell lines was screened for expression of EphA2 by immunoblotting. Several of the overexpressing cell lines, including BT549, MDA-MB-231, and HCC 1954 were selected for experiments utilizing siRNA for transient knockdown and shRNA for stable knockdown. Targeted knockdown of EphA2 was measured using RT-PCR and immunoblotting techniques. Here, the functions of EphA2 in the process of metastasis have been elucidated using in vitro assays that indicate cancer cell metastatic potential and in vivo studies that reveal the effect of EphA2 on mammary fat pad tumor growth, vessel formation, and the effect of using EphA2-targeting siRNA on pre-established mammary fat pad tumors. A decrease in EphA2 expression both in vitro and in vivo correlated with reduced migration and experimental metastasis of breast cancer cells. Current work is being done to investigate the mechanism behind EphA2’s participation in some of these processes. These studies are important because they have contributed to understanding the role that EphA2 plays in the progression of breast cancers to a metastatic state.

Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

INTRODUCTION: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. METHODS: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. RESULTS: Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. CONCLUSION: We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.

LY2109761, a novel transforming growth factor beta receptor type I and type II dual inhibitor, as a therapeutic approach to suppressing pancreatic cancer metastasis.

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.

The Role of Cancer-Associated Fibroblasts in Lung Tumorigenesis

The extracellular milieu is rich in growth factors that drive tumor progression,but the mechanisms that govern tumor cell sensitivity to those ligands have notbeen fully defined. In this study, we address this question in mice that developmetastatic lung adenocarcinomas through the suppression of the microRNA-200 (miR-200) family. Cancer-associated fibroblasts (CAF) enhance tumorgrowth and invasion by secreting VEGF-A that binds to VEGFR1, a processrequired for tumor growth and metastasis in mice and correlated with a poorprognosis in lung adenocarcinoma patients. In this study, we discovered thatmiR-200 blocked CAF-induced tumor cell invasion by directly targetingVEGFR1 in tumor cells. In the context of previous studies, our findings suggestthat the miR-200 family is a point of convergence for diverse biologic processesthat regulate tumor cell proliferation, invasion, and metastasis; its target genesixdrive epithelial-to-mesenchymal transition (ZEB1 and ZEB2) and promotesensitivity to a potent tumor growth factor emanating from the microenvironment(VEGFR1). Clinical trials should focus not only on the role of VEGFR1 inangiogenesis but also on the expression and activation of VEGFR1 in tumorcells by stromal sources of VEGF-A in the tumor microenvironment as a targetfor metastasis prevention.