Hyaluronidase splice variants are associated with histology and outcome in adenocarcinoma and squamous cell carcinoma of the lung

Heterogeneity of hyaluronidase (HYAL) expression has been identified in tumors and shows promise as an indicator of disease progression. The expression profile of alternatively spliced forms of HYAL was evaluated in tumors and normal lung tissue from 69 resected tumors of patients with adenocarcinomas and squamous cell carcinomas. HYAL1-wild-type (wt) and variants 1 to 5, HYAL2-wt, and HYAL3-wt, and variants 1 to 3 were identified by polymerase chain reaction and direct sequencing. Different proportions of the 3 HYAL-wt and variants were expressed in tumor and normal lung tissues. HYAL1-wt was associated with a poorer prognosis and HYAL3-vl with a better prognosis. HYAL splice variants are associated with histology and outcome, suggesting that strategies aimed at modulating their levels may be effective for lung cancer treatment. (C) 2012 Elsevier Inc. All rights reserved.

Comparison of Celsior and Perfadex lung preservation solutions in rat lungs subjected to 6 and 12 hours of ischemia using an ex-vivo lung perfusion system

OBJECTIVE: This study evaluated the performance of lungs that were preserved with different solutions (Celsior, Perfadex or saline) in an ex vivo rat lung perfusion system. METHODS: Sixty Wistar rats were anesthetized, anticoagulated and randomized into three groups (n = 20). The rats were subjected to antegrade perfusion via the pulmonary artery with Perfadex, Celsior, or saline, followed by 6 or 12 hours of ischemia (4 degrees C, n = 10 in each group). Respiratory mechanics, gas exchange and hemodynamics were measured at 10-minute intervals during the reperfusion of heart-lung blocks in an ex vivo system (IL2-Isolated Perfused Rat or Guinea Pig Lung System, Harvard Apparatus, Holliston, Massachusetts, USA; Hugo Sachs Elektronik, Germany) for 60 minutes. The lungs were prepared for histopathology and evaluated for edema following reperfusion. Group comparisons were performed using ANOVA and the Kruskal-Wallis test with a 5% level of significance. RESULTS: Gas exchange was not significantly different between lungs perfused with either Perfadex or Celsior at the same ischemic times, but it was very low in lungs that were preserved with saline. Airway resistance was greater in the lungs that were preserved for 12 hours. Celsior lungs that were preserved for 6 and 12 hours exhibited lower airway resistance (p = 0.01) compared to Perfadex lungs. Pulmonary artery pressure was not different between the groups, and no significant differences in histopathology and apoptosis were observed between the groups. CONCLUSIONS: Lungs that were preserved with Celsior or Perfadex exhibited similar gas exchange and histopathological findings. Airway resistance was slightly lower in the Celsior-preserved lungs compared with the Perfadex-preserved lungs.

Impaired lung function in individuals chronically exposed to biomass combustion

Background: The use of biomass for cooking and heating is considered an important factor associated with respiratory diseases. However, few studies evaluate the amount of particulate matter less than 2.5 mu in diameter (PM2.5), symptoms and lung function in the same population. Objectives: To evaluate the respiratory effects of biomass combustion and compare the results with those of individuals from the same community in Brazil using liquefied petroleum gas (Gas). Methods: 1402 individuals in 260 residences were divided into three groups according to exposure (Gas, Indoor-Biomass, Outside-Biomass). Respiratory symptoms were assessed using questionnaires. Reflectance of paper filters was used to assess particulate matter exposure. In 48 residences the amount of PM2.5 was also quantified. Pulmonary function tests were performed in 120 individuals. Results: Reflectance index correlated directly with PM2.5 (r=0.92) and was used to estimate exposure (ePM2.5). There was a significant increase in ePM2.5 in Indoor-Biomass and Outside-Biomass, compared to Gas. There was a significantly increased odds ratio (OR) for cough, wheezing and dyspnea in adults exposed to Indoor-Biomass (OR=2.93, 2.33, 2.59, respectively) and Outside-Biomass (OR=1.78, 1.78, 1.80, respectively) compared to Gas. Pulmonary function tests revealed both Non-Smoker-Biomass and Smoker-Gas individuals to have decreased %predicted-forced expiratory volume in the first second (FEV1) and FEV1/forced vital capacity (FVC) as compared to Non-Smoker-Gas. Pulmonary function tests data was inversely correlated with duration and ePM2.5. The prevalence of airway obstruction was 20% in both Non-Smoker-Biomass and Smoker-Gas subjects. Conclusion: Chronic exposure to biomass combustion is associated with increased prevalence of respiratory symptoms, reduced lung function and development of chronic obstructive pulmonary disease. These effects are associated with the duration and magnitude of exposure and are exacerbated by tobacco smoke. (C) 2011 Elsevier Inc. All rights reserved.

Pentraxin 3 accelerates lung injury in high tidal volume ventilation in mice

Mechanical ventilation is the major cause of iatrogenic lung damage in intensive care units. Although inflammation is known to be involved in ventilator-induced lung injury (VILI), several aspects of this process are still unknown. Pentraxin 3 (PTX3) is an acute phase protein with important regulatory functions in inflammation which has been found elevated in patients with acute respiratory distress syndrome. This study aimed at investigating the direct effect of PTX3 production in the pathogenesis of VILI. Genetically modified mice deficient and that over express murine Ptx3 gene were subjected to high tidal volume ventilation (V-T = 45 mL/kg, PEEPzero). Morphological changes and time required for 50% increase in respiratory system elastance were evaluated. Gene expression profile in the lungs was also investigated in earlier times in Ptx3-overexpressing mice. Ptx3 knockout and wild-type mice developed same lung injury degree in similar times (156 +/- 42 min and 148 +/- 41 min, respectively: p = 0.8173). However, Ptx3 overexpression led to a faster development of VILI in Ptx3-overexpressing mice (77 +/- 29 min vs 118 +/- 41 min, p = 0.0225) which also displayed a faster kinetics of Il1b expression and elevated Ptx3, Cxcl1 and Ccl2 transcripts levels in comparison with wild-type mice assessed by quantitative real-time polymerase chain reaction. Ptx3 deficiency did not impacted the time for VILI induced by high tidal volume ventilation but Ptx3-overexpression increased inflammatory response and reflected in a faster VILI development. (C) 2012 Elsevier Ltd. All rights reserved.

Sepsis-Induced Acute Lung Injury (ALI) Is Milder in Diabetic Rats and Correlates with Impaired NFkB Activation

Acute lung injury (ALI) develops in response to a direct insult to the lung or secondarily to a systemic inflammatory response, such as sepsis. There is clinical evidence that the incidence and severity of ALI induced by direct insult are lower in diabetics. In the present study we investigated whether the same occurs in ALI secondarily to sepsis and the molecular mechanisms involved. Diabetes was induced in male Wistar rats by alloxan and sepsis by caecal ligation and puncture surgery (CLP). Six hours later, the lungs were examined for oedema and cell infiltration in bronchoalveolar lavage. Alveolar macrophages (AMs) were cultured in vitro for analysis of I kappa B and p65 subunit of NF kappa B phosphorylation and MyD88 and SOCS-1 mRNA. Diabetic rats were more susceptible to sepsis than non-diabetics. In non-diabetic rats, the lung presented oedema, leukocyte infiltration and increased COX2 expression. In diabetic rats these inflammatory events were significantly less intense. To understand why diabetic rats despite being more susceptible to sepsis develop milder ALI, we examined the NF kappa B activation in AMs of animals with sepsis. Whereas in non-diabetic rats the phosphorylation of I kappa B and p65 subunit occurred after 6 h of sepsis induction, this did not occur in diabetics. Moreover, in AMs from diabetic rats the expression of MyD88 mRNA was lower and that of SOCS-1 mRNA was increased compared with AMs from non-diabetic rats. These results show that ALI secondary to sepsis is milder in diabetic rats and this correlates with impaired activation of NF kappa B, increased SOCS-1 and decreased MyD88 mRNA.

Different morphology, stage and treatment affect immune cell infiltration and long-term outcome in patients with non-small-cell lung carcinoma

Aims: Development of effective immune-based therapies for patients with non-small-cell lung carcinoma (NSCLC) depends on an accurate characterization of complex interactions that occur between immune cells and the tumour environment. Methods and results: Innate and adaptive immune responses were evaluated in relation to prognosis in 65 patients with surgically excised NSCLC. Immunohistochemistry and morphometry were used to determine the abundance and distribution of immune cells. We found low numbers of immune cells and levels of cytokines in the tumour environment when compared with surrounding parenchyma. Smoking was associated inversely with the adaptive immune response and directly with innate immunity. We observed a prominent adaptive immune response in squamous cell carcinomas (SCC) but greater innate immune responses in adenocarcinomas and large cell carcinomas. Cox model analysis showed a low risk of death for smoking <41 packs/year, N-0 tambour stage, squamous carcinoma, CD4(+) > 16.81% and macrophages/monocytes >4.5%. Collectively, the data indicate that in NSCLC there is not a substantive local immune cell infiltrate within the tumour. Conclusion: Although immune cell infiltration is limited in NSCLC it appears to have an impact on prognosis and this may be of relevance for new immunotherapeutic approaches.

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

Frequent downregulation of the transcription factor Foxa2 in lung cancer through epigenetic silencing

Purpose: We sought to determine the mechanisms of downregulation of the airway transcription factor Foxa2 in lung cancer and the expression status of Foxa2 in non-small-cell lung cancer (NSCLC). Methods: A series of 25 lung cancer cell lines were evaluated for Foxa2 protein expression, FOXA2 mRNA levels, FOXA2 mutations, FOXA2 copy number changes and for evidence of FOXA2 promoter hypermethylation. In addition, 32 NSCLCs were sequenced for FOXA2 mutations and 173 primary NSCLC tumors evaluated for Foxa2 expression using an immunohistochemical assay. Results: Out of the 25 cell lines, 13 (52%) had undetectable FOXA2 mRNA. The expression of FOXA2 mRNA and Foxa2 protein were congruent in 19/22 cells (p = 0.001). FOXA2 mutations were not identified in primary NSCLCs and were infrequent in cell lines. Focal or broad chromosomal deletions involving FOXA2 were not present. The promoter region of FOXA2 had evidence of hypermethylation, with an inverse correlation between FOXA2 mRNA expression and presence of CpG dinucleotide methylation (p < 0.0001). In primary NSCLC tumor specimens, there was a high frequency of either absence (42/173, 24.2%) or no/low expression (96/173,55.4%) of Foxa2. In 130 patients with stage I NSCLC there was a trend towards decreased survival in tumors with no/low expression of Foxa2 (HR of 1.6, 95%CI 0.9-3.1; p = 0.122). Conclusions: Loss of expression of Foxa2 is frequent in lung cancer cell lines and NSCLCs. The main mechanism of downregulation of Foxa2 is epigenetic silencing through promoter hypermethylation. Further elucidation of the involvement of Foxa2 and other airway transcription factors in the pathogenesis of lung cancer may identify novel therapeutic targets. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Histologic and functional evaluation of lungs reconditioned by ex vivo lung perfusion

BACKGROUND: Only about 15% of donor lungs are considered suitable for transplantation (LTx). Ex vivo lung perfusion (EVLP) has been developed as a method to reassess and repair damaged lungs. We report our experience with EVLP in non-acceptable donor lungs and evaluate its ability to recondition these lungs. METHODS: We studied lungs from 16 brain-dead donors rejected for LTx. After harvesting, the lungs were stored at 4 degrees C for 10 hours and subjected to normothermic EVLP with Steen Solution (Vitro life, Goteborg, Sweden) for 60 minutes. For functional evaluation, the following variables were assessed: partial pressure of arterial oxygen (Pao(2)), pulmonary vascular resistance (PVR), and lung compliance (LC). For histologic assessment, lung biopsy was done before harvest and after EVLP. Tissue samples were examined under light microscopy. To detect and quantify apoptosis, terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling assay was used. RESULTS: Thirteen lima donors were refused for having impaired lung function. The mean Pao(2) obtained in the organ donor at the referring hospital was 193.7 mm Hg and rose to 489 mm Hg after EVLP. During EVLP, the mean PVR was 652.5 dynes/sec/cm(5) and the mean LC was 48 ml/cm H2O. There was no significant difference between the mean Lung Injury Score before harvest and after EVLP. There was a trend toward a reduction in the median number of apoptotic cells after EVLP. CONCLUSIONS: EVLP improved lung function (oxygenation capacity) of organs considered unsuitable for transplantation. Lung tissue structure did not deteriorate even after 1 hour of normothermic perfusion. J Heart Lung Transplant 2012;31:305-9 (C) 2012 International Society for Heart and Lung Transplantation. All rights reserved.

In vivo hydroquinone exposure impairs MCP-1 secretion and monocyte recruitment into the inflamed lung

Alveolar macrophages (AMs) are important cells in the resolution of the inflammatory process and they come into direct contact with inhaled pollutants. Hydroquinone (HQ) is an environmental pollutant and a component of cigarette smoke that causes immunosuppressive effects. In the present work, we showed that mice exposed to low levels of aerosolized HQ (25 ppm; 1 h/day/5 days) presented impaired mononuclear cell migration to the lipopolysaccharide (LPS)-inflamed lung. This may have been due to reduced monocyte chemoattractant protein-1 (MCP-1) secretion into bronchoalveolar lavage fluid (BALF), and it was not related to alterations to mononuclear cell mobilization into the blood or adhesion molecules expression on mononuclear cell membranes. Corroborating the actions of HQ on MCP-1 secretion, reduced MCP-1 concentrations were also found in the supernatant of ex vivo AM and tracheal tissue collected from HQ-exposed mice. A direct action of HQ on MCP-1 secretion, resulting from impaired gene synthesis, was verified by in vitro incubation of naive AMs or tracheal tissue with HQ. The role of reduced levels of MCP-1 in the BALF on monocyte migration was analysed in the human monocytic lineage THP-1 in in vitro chemotaxis assays, which showed that the reduced concentrations of MCP-1 found in the BALF or cell supernatants from HQ-exposed mice impaired cell migration. Considering the fact that MCP-1 presents a broad spectrum of actions on pathophysiological conditions and that resident mononuclear cells are involved in lung tissue homeostasis and in immune host defence, the mechanism of HQ toxicity presented herein might be relevant to the genesis of infectious lung diseases in smokers and in inhabitants of polluted areas. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Effects of Positive End-expiratory Pressure Titration and Recruitment Maneuver on Lung Inflammation and Hyperinflation in Experimental Acid Aspiration-induced Lung Injury

Background: In acute lung injury positive end-expiratory pressure (PEEP) and recruitment maneuver are proposed to optimize arterial oxygenation. The aim of the study was to evaluate the impact of such a strategy on lung histological inflammation and hyperinflation in pigs with acid aspiration-induced lung injury. Methods: Forty-seven pigs were randomly allocated in seven groups: (1) controls spontaneously breathing; (2) without lung injury, PEEP 5 cm H2O; (3) without lung injury, PEEP titration; (4) without lung injury, PEEP titration + recruitment maneuver; (5) with lung injury, PEEP 5 cm H2O; (6) with lung injury, PEEP titration; and (7) with lung injury, PEEP titration + recruitment maneuver. Acute lung injury was induced by intratracheal instillation of hydrochloric acid. PEEP titration was performed by incremental and decremental PEEP from 5 to 20 cm H2O for optimizing arterial oxygenation. Three recruitment maneuvers (pressure of 40 cm H2O maintained for 20 s) were applied to the assigned groups at each PEEP level. Proportion of lung inflammation, hemorrhage, edema, and alveolar wall disruption were recorded on each histological field. Mean alveolar area was measured in the aerated lung regions. Results: Acid aspiration increased mean alveolar area and produced alveolar wall disruption, lung edema, alveolar hemorrhage, and lung inflammation. PEEP titration significantly improved arterial oxygenation but simultaneously increased lung inflammation in juxta-diaphragmatic lung regions. Recruitment maneuver during PEEP titration did not induce additional increase in lung inflammation and alveolar hyperinflation. Conclusion: In a porcine model of acid aspiration-induced lung injury, PEEP titration aimed at optimizing arterial oxygenation, substantially increased lung inflammation. Recruitment maneuvers further improved arterial oxygenation without additional effects on inflammation and hyperinflation.

Overall Survival Improvement in Patients with Lung Cancer and Bone Metastases Treated with Denosumab Versus Zoledronic Acid Subgroup Analysis from a Randomized Phase 3 Study

Introduction: Denosumab, a fully human anti-RANKL monoclonal antibody, reduces the incidence of skeletal-related events in patients with bone metastases from solid tumors. We present survival data for the subset of patients with lung cancer, participating in the phase 3 trial of denosumab versus zoledronic acid (ZA) in the treatment of bone metastases from solid tumors (except breast or prostate) or multiple myeloma. Methods: Patients were randomized 1:1 to receive monthly subcutaneous denosumab 120 mg or intravenous ZA 4 mg. An exploratory analysis, using Kaplan-Meier estimates and proportional hazards models, was performed for overall survival among patients with non-small-cell lung cancer (NSCLC) and SCLC. Results: Denosumab was associated with improved median overall survival versus ZA in 811 patients with any lung cancer (8.9 versus 7.7 months; hazard ratio [HR] 0.80) and in 702 patients with NSCLC (9.5 versus 8.0 months; HR 0.78) (p = 0.01, each comparison). Further analysis of NSCLC by histological type showed a median survival of 8.6 months for denosumab versus 6.4 months for ZA in patients with squamous cell carcinoma (HR 0.68; p = 0.035). Incidence of overall adverse events was balanced between treatment groups; serious adverse events occurred in 66.0% of denosumab-treated patients and 72.9% of ZA-treated patients. Cumulative incidence of osteonecrosis of the jaw was similar between groups (0.7% denosumab versus 0.8% ZA). Hypocalcemia rates were 8.6% with denosumab and 3.8% with ZA. Conclusion: In this exploratory analysis, denosumab was associated with improved overall survival compared with ZA, in patients with metastatic lung cancer.

Is cardiac tissue more susceptible than lung to oxidative effects induced by chronic nasotropic instillation of residual oil fly ash (ROFA)?

The current study aimed to determine the role of oxidants in cardiac and pulmonary toxicities induced by chronic exposure to ROFA. Eighty Wistar rats were divided into four groups: G1 (10 mu L Saline), G2 (ROFA 50 mu g/10 mu L), G3 (ROFA 250 mu g/10 mu L) and G4 (ROFA 500 mu g/10 mu L). Rats received ROFA by nasotropic instillation for 90 days. After that, they were euthanized and bronchoalveolar lavage (BAL) was performed for total count of leukocytes, protein and lactate dehydrogenase (LDH) determinations. Lungs and heart were removed to measure lipid peroxidation (MDA), catalase (CAT) and superoxide dismutase (SOD) activity. BAL presented an increase in leukocytes count in G4 in comparison to the Saline group (p = 0.019). In lung, MDA level was not modified by ROFA, while CAT was higher in G4 when compared to all other groups (p = 0.013). In heart, G4 presented an increase in MDA (p = 0.016) and CAT (p = 0.027) levels in comparison to G1. The present study demonstrated cardiopulmonary oxidative changes after a chronic ROFA exposure. More specifically, the heart tissue seems to be more susceptible to oxidative effects of long-term exposure to ROFA than the lung.

Effects of different tidal volumes in pulmonary and extrapulmonary lung injury with or without intraabdominal hypertension

We hypothesized that: (1) intraabdominal hypertension increases pulmonary inflammatory and fibrogenic responses in acute lung injury (ALI); (2) in the presence of intraabdominal hypertension, higher tidal volume reduces lung damage in extrapulmonary ALI, but not in pulmonary ALI. Wistar rats were randomly allocated to receive Escherichia coli lipopolysaccharide intratracheally (pulmonary ALI) or intraperitoneally (extrapulmonary ALI). After 24 h, animals were randomized into subgroups without or with intraabdominal hypertension (15 mmHg) and ventilated with positive end expiratory pressure = 5 cmH(2)O and tidal volume of 6 or 10 ml/kg during 1 h. Lung and chest wall mechanics, arterial blood gases, lung and distal organ histology, and interleukin (IL)-1 beta, IL-6, caspase-3 and type III procollagen (PCIII) mRNA expressions in lung tissue were analyzed. With intraabdominal hypertension, (1) chest-wall static elastance increased, and PCIII, IL-1 beta, IL-6, and caspase-3 expressions were more pronounced than in animals with normal intraabdominal pressure in both ALI groups; (2) in extrapulmonary ALI, higher tidal volume was associated with decreased atelectasis, and lower IL-6 and caspase-3 expressions; (3) in pulmonary ALI, higher tidal volume led to higher IL-6 expression; and (4) in pulmonary ALI, liver, kidney, and villi cell apoptosis was increased, but not affected by tidal volume. Intraabdominal hypertension increased inflammation and fibrogenesis in the lung independent of ALI etiology. In extrapulmonary ALI associated with intraabdominal hypertension, higher tidal volume improved lung morphometry with lower inflammation in lung tissue. Conversely, in pulmonary ALI associated with intraabdominal hypertension, higher tidal volume increased IL-6 expression.

Cigarette smoke dissociates inflammation and lung remodeling in OVA-sensitized and challenged mice

We evaluated the effects of cigarette smoke (CS) on lung inflammation and remodeling in a model of ovalbumin (OVA)-sensitized and OVA-challenged mice. Male BALB/c mice were divided into 4 groups: non-sensitized and air-exposed (control); non-sensitized and exposed to cigarette smoke (CS), sensitized and air-exposed (OVA) (50 mu g + OVA 1% 3 times/week for 3 weeks) and sensitized and cigarette smoke exposed mice (OVA + CS). IgE levels were not affected by CS exposure. The increases in total bronchoalveolar fluid cells in the OVA group were attenuated by co-exposure to CS, as were the changes in IL-4, IL-5, and eotaxin levels as well as tissue elastance (p < 0.05). In contrast, only the OVA + CS group showed a significant increase in the protein expression of IFN-gamma, VEGF, GM-CSF and collagen fiber content (p < 0.05). In our study, exposure to cigarette smoke in OVA-challenged mice resulted in an attenuation of pulmonary inflammation but led to an increase in pulmonary remodeling and resulted in the dissociation of airway inflammation from lung remodeling. (C) 2012 Elsevier B.V. All rights reserved.