Comparison of the effects of e-cigarette vapor with cigarette smoke on lung function and inflammation in mice.

Electronic cigarettes (e-cigs) are advertised as a less harmful nicotine delivery system or as a new smoking cessation tool. We aimed to assess the in vivo effects of e-cig vapor in the lung and to compare them to those of cigarette smoke (CS). We exposed C57BL/6 mice for either 3 days or 4 wk to ambient air, CS, or e-cig vapor containing 1) propylene glycol/vegetable glycerol (PG:VG-Sol; 1:1), 2) PG:VG with nicotine (G:VG-N), or 3) PG:VG with nicotine and flavor (PG:VG-N+F) and determined oxidative stress, inflammation, and pulmonary mechanics. E-cig vapors, especially PG:VG-N+F, increased bronchoalveolar lavage fluid (BALF) cellularity, Muc5ac production, as well as BALF and lung oxidative stress markers at least comparably and in many cases more than CS. BALF protein content at both time points studied was only elevated in the PG:VG-N+F group. After 3 days, PG:VG-Sol altered tissue elasticity, static compliance, and airway resistance, whereas after 4 wk CS was the only treatment adversely affecting these parameters. Airway hyperresponsiveness in response to methacholine was increased similarly in the CS and PG:VG-N+F groups. Our findings suggest that exposure to e-cig vapor can trigger inflammatory responses and adversely affect respiratory system mechanics. In many cases, the added flavor in e-cigs exacerbated the detrimental effects of e-cig vapor. We conclude that both e-cig vaping and conventional cigarette smoking negatively impact lung biology.

Highly Selective Endothelin-1 Receptor A Inhibition Prevents Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Mice.

BACKGROUND: Pulmonary fibrosis is a chronic disease, which progressively leads to respiratory failure and ultimately death. Endothelin-1 (ET-1), a vasoconstrictor secreted by endothelial cells, promotes vasoconstriction by activation of its receptors A and B. OBJECTIVES: We addressed the role of highly selective ET-1 receptor A (ETA) inhibition in the pathogenesis of experimental pulmonary fibrosis by bleomycin (BLM). METHODS: BLM sulfate (2 U/mL) or saline was intratracheally administered to C57/Bl6 mice (4 groups; n = 5-11/group). Pretreatment with the highly selective ETA receptor inhibitor sitaxentan (15 mg/kg/day) was started 1 day prior to BLM injection and continued for the duration of the experiment. Lung mechanics were assessed prior to sacrifice at days 7, 14, and 21 after BLM, followed by procurement of bronchoalveolar lavage fluid (BALF), blood, and lung tissue samples. RESULTS: Time-dependent effects of BLM exposure included decreased static compliance and increased lung elastance, airspace inflammation and microvascular permeability, histological acute lung injury and fibrosis, and lung collagen deposition. Pretreatment with highly selective ETA receptor inhibitor had no adverse effect on control mice but improved lung mechanics and lung injury score in addition to decreasing BALF pleocytosis, protein content, and collagen deposition in BLM-treated mice. Mortality from BLM reached 40% and occurred primarily during the inflammatory stage of the model but was abrogated by sitaxentan pretreatment. CONCLUSIONS: We conclude that in our BLM-induced pulmonary fibrosis model, prophylactic highly selective ETA inhibition improves survival, preserves lung function, attenuates lung injury, and reduces collagen deposition.

Guanylyl cyclase activation reverses resistive breathing-induced lung injury and inflammation.

Inspiratory resistive breathing (RB), encountered in obstructive lung diseases, induces lung injury. The soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is down-regulated in chronic and acute animal models of RB, such as asthma, chronic obstructive pulmonary disease, and in endotoxin-induced acute lung injury. Our objectives were to: (1) characterize the effects of increased concurrent inspiratory and expiratory resistance in mice via tracheal banding; and (2) investigate the contribution of the sGC/cGMP pathway in RB-induced lung injury. Anesthetized C57BL/6 mice underwent RB achieved by restricting tracheal surface area to 50% (tracheal banding). RB for 24 hours resulted in increased bronchoalveolar lavage fluid cellularity and protein content, marked leukocyte infiltration in the lungs, and perturbed respiratory mechanics (increased tissue resistance and elasticity, shifted static pressure-volume curve right and downwards, decreased static compliance), consistent with the presence of acute lung injury. RB down-regulated sGC expression in the lung. All manifestations of lung injury caused by RB were exacerbated by the administration of the sGC inhibitor, 1H-[1,2,4]oxodiazolo[4,3-]quinoxalin-l-one, or when RB was performed using sGCα1 knockout mice. Conversely, restoration of sGC signaling by prior administration of the sGC activator BAY 58-2667 (Bayer, Leverkusen, Germany) prevented RB-induced lung injury. Strikingly, direct pharmacological activation of sGC with BAY 58-2667 24 hours after RB reversed, within 6 hours, the established lung injury. These findings raise the possibility that pharmacological targeting of the sGC-cGMP axis could be used to ameliorate lung dysfunction in obstructive lung diseases.

Inspiratory resistive breathing induces MMP-9 and MMP-12 expression in the lung.

Inspiratory resistive breathing (IRB) is characterized by large negative intrathoracic pressures and was shown to induce pulmonary inflammation in previously healthy rats. Matrix metalloproteinases (MMP)-9 and -12 are induced by inflammation and mechanical stress in the lung. We hypothesized that IRB induces MMP-9 and -12 in the lung. Anesthetized, tracheostomized rats breathed spontaneously through a two-way valve, connected to an inspiratory resistance, with the tidal inspiratory tracheal pressure set at 50% of the maximum. Quietly breathing animals served as controls. After 3 and 6 h of IRB, respiratory mechanics were measured, bronchoalveolar lavage (BAL) was performed, lung injury score was estimated, and lung MMP-9 was estimated by zymography and ELISA. MMP-9 and MMP-12 immunohistochemistry was performed. Isolated normal alveolar macrophages were incubated with BAL from rats that underwent IRB. After 18 h, MMP-9 and -12 levels were measured in supernatants, and immunocytochemistry was performed. Macrophages were treated with IL-1ß, IL-6, or TNF-α, and MMP-9 in supernatants was measured. After 6 h of IRB, leukocytes in BAL increased, and IL-1ß and IL-6 levels were elevated. Elasticity and injury score were increased after 3 and 6 h of IRB. Lung MMP-9 levels increased after 6 h of IRB. MMP-9 and MMP-12 were detected in alveolar macrophages and epithelial (bronchial/alveolar) cells after 3 and 6 h of IRB. MMP-9 and MMP-12 were found in supernatants after treatment with 6 h of IRB BAL. Cytosolic immunostaining was detected after treatment with 3 and 6 h of IRB BAL. All cytokines induced MMP-9 in culture supernatants. In conclusion, IRB induces MMP-9 and -12 in the lung of previously healthy rats.

Evaluation of the prognostic significance of HER family mRNA expression in high-risk early breast cancer: a Hellenic Cooperative Oncology Group (HeCOG) validation study.

BACKGROUND: The aim of the study was to evaluate the prognostic ability of the transcriptional profiling of the HER family genes in early breast cancer, as a validation analysis of another previously published HeCOG study. METHODS: RNA was extracted from 663 formalin-fixed paraffin-embedded (FFPE) tumor tissue samples of high-risk early breast cancer patients enrolled in the randomized HE10/00 trial. Relative mRNA expression of all four HER family members was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: In compliance with our previous study, the overall agreement between qRT-PCR and IHC/FISH for HER2 status determination was good (69%). Likewise, the overall concordance between qRT-PCR and IHC for EGFR status was high (81%). In line with our previously reported data, we demonstrated a positive association between HER2 and HER3 mRNA expression. Similarly, mRNA expression of HER3 and HER4 was positively associated with each other and negatively associated with EGFR. Regarding relationships with clinico-pathological parameters, our findings are also in agreement with our previous results. Generally, increased EGFR and HER2 mRNA expression was related to unfavorable, whereas high HER3 and HER4 mRNA expression was associated with favorable clinico-pathological parameters. In univariate analysis, no significant association between EGFR, HER2 and HER3 mRNA expression and overall survival (OS) or disease-free survival (DFS) was demonstrated. However, high EGFR protein expression was associated with significantly shorter OS (log-rank, p = 0.015). In compliance with our previously published data, increased HER4 mRNA expression had a significantly favorable prognostic value in terms of OS (p = 0.044) and DFS (p = 0.047). In multivariate analysis, among all HER receptors, only EGFR protein expression was found to affect OS (Wald's p = 0.028) and DFS (p = 0.015) independently. Concerning the combined expression of all four HER family receptors, the combination of high EGFR, high HER2, low HER3 and low HER4 mRNA expression was associated with a trend for shorter OS (log-rank, p = 0.065) and significantly worse DFS (p = 0.033), compared with all other co-expression profiles. CONCLUSIONS: These data indicate that qRT-PCR may represent a valid alternative method for evaluating the expression of HER family members in FFPE breast carcinoma tissue samples. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12609001036202.

Inhibition of HMGCoA reductase by simvastatin protects mice from injurious mechanical ventilation.

BACKGROUND: Mortality from severe acute respiratory distress syndrome exceeds 40% and there is no available pharmacologic treatment. Mechanical ventilation contributes to lung dysfunction and mortality by causing ventilator-induced lung injury. We explored the utility of simvastatin in a mouse model of severe ventilator-induced lung injury. METHODS: Male C57BL6 mice (n = 7/group) were pretreated with simvastatin or saline and received protective (8 mL/kg) or injurious (25 mL/kg) ventilation for four hours. Three doses of simvastatin (20 mg/kg) or saline were injected intraperitoneally on days -2, -1 and 0 of the experiment. Lung mechanics, (respiratory system elastance, tissue damping and airway resistance), were evaluated by forced oscillation technique, while respiratory system compliance was measured with quasi-static pressure-volume curves. A pathologist blinded to treatment allocation scored hematoxylin-eosin-stained lung sections for the presence of lung injury. Pulmonary endothelial dysfunction was ascertained by bronchoalveolar lavage protein content and lung tissue expression of endothelial junctional protein Vascular Endothelial cadherin by immunoblotting. To assess the inflammatory response in the lung, we determined bronchoalveolar lavage fluid total cell content and neutrophil fraction by microscopy and staining in addition to Matrix-Metalloprotease-9 by ELISA. For the systemic response, we obtained plasma levels of Tumor Necrosis Factor-α, Interleukin-6 and Matrix-Metalloprotease-9 by ELISA. Statistical hypothesis testing was undertaken using one-way analysis of variance and Tukey's post hoc tests. RESULTS: Ventilation with high tidal volume (HVt) resulted in significantly increased lung elastance by 3-fold and decreased lung compliance by 45% compared to low tidal volume (LVt) but simvastatin abrogated lung mechanical alterations of HVt. Histologic lung injury score increased four-fold by HVt but not in simvastatin-pretreated mice. Lavage pleocytosis and neutrophilia were induced by HVt but were significantly attenuated by simvastatin. Microvascular protein permeability increase 20-fold by injurious ventilation but only 4-fold with simvastatin. There was a 3-fold increase in plasma Tumor Necrosis Factor-α, a 7-fold increase in plasma Interleukin-6 and a 20-fold increase in lavage fluid Matrix-Metalloprotease-9 by HVt but simvastatin reduced these levels to control. Lung tissue vascular endothelial cadherin expression was significantly reduced by injurious ventilation but remained preserved by simvastatin. CONCLUSION: High-dose simvastatin prevents experimental hyperinflation lung injury by angioprotective and anti-inflammatory effects.

Nationwide Real-World Data of Microsatellite Instability and/or Mismatch Repair Deficiency in Cancer: Prevalence and Testing Patterns.

Determination of microsatellite instability (MSI)/mismatch repair (MMR) status in cancer has several clinical implications. Our aim was to integrate MSI/MMR status from patients tested in Greece to assess the prevalence of MSI-high (MSI-H)/deficient MMR (dMMR) per tumor type, testing patterns over time and concordance between MSI and MMR status. We retrospectively recorded MSI/MMR testing data of patients with diverse tumor types performed in pathology and molecular diagnostics laboratories across Greece. Overall, 18 of 22 pathology and/or molecular diagnostics laboratories accepted our invitation to participate. In the 18 laboratories located across the country, 7916 tumor samples were evaluated for MSI/MMR status. MSI/MMR testing significantly increased in patients with colorectal cancer (CRC) and other tumor types overtime (p < 0.05). The highest prevalence was reported in endometrial cancer (47 of 225 patients, 20.9%). MSI-H/dMMR was observed in most tumor types, even in low proportions. Among 904 tumors assessed both for MSI and MMR status, 21 had discordant results (overall discordance rate, 2.3%). We reported MSI-H/dMMR prevalence rates in patients with diverse cancers, while demonstrating increasing referral patterns from medical oncologists in the country overtime. The anticipated high rate of concordance between MSI and MMR status in paired analysis was confirmed.

Synergistic Effects of Resistive Breathing on Endotoxin-Induced Lung Injury in Mice.

Introduction: Resistive breathing (RB) is characterized by forceful contractions of the inspiratory muscles, mainly the diaphragm, resulting in large negative intrathoracic pressure and mechanical stress imposed on the lung. We have shown that RB induces lung injury in healthy animals. Whether RB exerts additional injurious effects when added to pulmonary or extrapulmonary lung injury is unknown. Our aim was to study the synergistic effect of RB on lipopolysaccharide (LPS)-induced lung injury. Methods: C57BL/6 mice inhaled an LPS aerosol (10mg/3mL) or received an intraperitoneal injection of LPS (10 mg/kg). Mice were then anaesthetized, the trachea was surgically exposed, and a nylon band of a specified length was sutured around the trachea, to provoke a reduction of the surface area at 50%. RB through tracheal banding was applied for 24 hours. Respiratory system mechanics were measured, BAL was performed, and lung sections were evaluated for histological features of lung injury. Results: LPS inhalation increased BAL cellularity, mainly neutrophils (p < 0.001 to ctr), total protein and IL-6 in BAL (p < 0.001 and p < 0.001, respectively) and increased the lung injury score (p = 0.001). Lung mechanics were not altered. Adding RB to inhaled LPS further increased BAL cellularity (p < 0.001 to LPS inh.), total protein (p = 0.016), lung injury score (p = 0.001) and increased TNFa levels in BAL (p = 0.011). Intraperitoneal LPS increased BAL cellularity, mainly macrophages (p < 0.001 to ctr.), total protein levels (p = 0.017), decreased static compliance (p = 0.004) and increased lung injury score (p < 0.001). Adding RB further increased histological features of lung injury (p = 0.022 to LPS ip). Conclusion: Resistive breathing exerts synergistic injurious effects when combined with inhalational LPS-induced lung injury, while the additive effect on extrapulmonary lung injury is less prominent.

Prognostic significance of phosphorylated STAT-1 expression in premenopausal and postmenopausal patients with invasive breast cancer.

AIMS: STAT-1 is the first member of the family of signal transducers and activators of transcription (STATs). In breast cancer experimental models, an apoptotic and antiproliferative effect has been demonstrated. Our aim was to study the role of phosphorylated STAT-1 (pSTAT-1) in invasive breast carcinoma and its prognostic significance in premenopausal and postmenopausal patients. METHODS AND RESULTS: Immunohistochemistry was performed in 165 patients in order to detect the expression of pSTAT-1 and its correlation with oestrogen receptor (ER), progesterone receptor (PR), caspase-3, and pAkt. pSTAT-1 was immunodetected in the cytoplasm of the malignant cells (11.6%). In premenopausal patients, cytoplasmic pSTAT-1 was positively correlated with stage (P = 0.014), ER (P = 0.008), caspase-3 (P = 0.029), and pAkt (P = 0.045). Univariate analysis showed that cytoplasmic pSTAT-1 was associated with poor overall survival (P = 0.042) and the phenotype of pSTAT-1/ER or PR coexpression with shorter disease-free survival (P = 0.012). In contrast, in postmenopausal patients, no association with clinicopathological parameters and survival was observed, except for the relationship of pSTAT-1/ER or PR coexpression with longer disease-free survival (P = 0.034). CONCLUSIONS: This is the first study examining the role of pSTAT-1 in premenopausal and postmenopausal women with invasive breast cancer. Our results suggest that pSTAT-1 is related to tumour progression in premenopausal patients through the advanced stage and worse survival.

Metformin attenuates ventilator-induced lung injury.

INTRODUCTION: Diabetic patients may develop acute lung injury less often than non-diabetics; a fact that could be partially ascribed to the usage of antidiabetic drugs, including metformin. Metformin exhibits pleiotropic properties which make it potentially beneficial against lung injury. We hypothesized that pretreatment with metformin preserves alveolar capillary permeability and, thus, prevents ventilator-induced lung injury. METHODS: Twenty-four rabbits were randomly assigned to pretreatment with metformin (250 mg/Kg body weight/day per os) or no medication for two days. Explanted lungs were perfused at constant flow rate (300 mL/min) and ventilated with injurious (peak airway pressure 23 cmH2O, tidal volume ≈17 mL/Kg) or protective (peak airway pressure 11 cmH2O, tidal volume ≈7 mL/Kg) settings for 1 hour. Alveolar capillary permeability was assessed by ultrafiltration coefficient, total protein concentration in bronchoalveolar lavage fluid (BALF) and angiotensin-converting enzyme (ACE) activity in BALF. RESULTS: High-pressure ventilation of the ex-vivo lung preparation resulted in increased microvascular permeability, edema formation and microhemorrhage compared to protective ventilation. Compared to no medication, pretreatment with metformin was associated with a 2.9-fold reduction in ultrafiltration coefficient, a 2.5-fold reduction in pulmonary edema formation, lower protein concentration in BALF, lower ACE activity in BALF, and fewer histological lesions upon challenge of the lung preparation with injurious ventilation. In contrast, no differences regarding pulmonary artery pressure and BALF total cell number were noted. Administration of metformin did not impact on outcomes of lungs subjected to protective ventilation. CONCLUSIONS: Pretreatment with metformin preserves alveolar capillary permeability and, thus, decreases the severity of ventilator-induced lung injury in this model.

mRNA expression of specific HER ligands and their association with clinical outcome in patients with metastatic breast cancer treated with trastuzumab.

Prognostic and predictive biomarkers are being studied for the diagnosis and treatment of breast cancer. The present study retrospectively assessed the mRNA expression of HER family receptor ligands and of other potential prognostic biomarkers and their association with time to progression (TTP), survival and clinicopathological characteristics in patients with metastatic breast cancer (MBC) treated with trastuzumab. A total of 145 tumour tissue samples were analysed. mRNA expression analysis of the transcripts of interest was performed and the association of these markers with selected clinicopathological parameters was examined. HER2 status was centrally re-evaluated. Only 67.6% of patients were truly HER2-positive according to the central HER2 re-evaluation. Heparin binding epidermal growth factor (EGF)-like growth factor, transforming growth factor ß1 (TGFB1) and thyroid hormone receptor α (THRA) mRNA expression was higher in HER2-positive patients (P=0.026, P<0.001 and P<0.001). Insulin-like growth factor binding protein 4 was correlated with retinoic acid receptor α, TGFB1 and THRA (rho=0.45, rho=0.60 and rho=0.45). In HER2-positive patients, high neuregulin 1 and high betacellulin were unfavourable factors for TTP [hazard ratio (HR) = 1.78, P=0.040 and HR=2.00, P=0.043, respectively]. In patients with de novo MBC, high EGF expression was associated with a non-significant prolongation of TTP (HR=0.52, P=0.080) and significantly longer survival (HR=0.40, P=0.020). The present study examined clinical and biological implications of specific genes and it was concluded that their expression has an impact on the outcome of trastuzumab-treated patients with MBC.

Androgen Receptor and PIM1 Expression in Tumor Tissue of Patients With Triple-negative Breast Cancer.

BACKGROUND/AIM: Effective targeted therapies for triple-negative breast cancer (TNBC) are limited. In a subset of TNBC, androgen receptor (AR) plays an important role, while the human proviral integration site for Moloney murine leukemia virus-1 (PIM1) overexpression is also implicated. PIM1 kinases phosphorylate AR, thus regulating its transcriptional activity, regardless of the presence or not of androgens. We evaluated the expression of AR and PIM1 and their prognostic significance in TNBC. MATERIALS AND METHODS: AR and PIM1 transcripts were quantified by quantitative reverse transcription polymerase chain reaction in formalin-fixed paraffin-embedded tumor from 141 patients with TNBC. RESULTS: AR was expressed in 38.3%, PIM1 in 10.6%, while co-expression of AR and PIM1 was detected in 7/141 cases (5.0%). No prognostic significance of AR or PIM1 was reached for overall or disease-free survival. CONCLUSION: Co-expression of AR and PIM1 exists in only in a small percentage of patients with TNBC. The implications of this finding in the therapeutic management of patients with TNBC should be investigated in larger patient cohorts.

Pretreatment with atorvastatin attenuates lung injury caused by high-stretch mechanical ventilation in an isolated rabbit lung model.

OBJECTIVE: We hypothesized that pretreatment with atorvastatin improves alveolar capillary permeability and hemodynamics and, thus, confers protection against lung injury caused by high-stretch mechanical ventilation. METHODS: Twenty-four isolated sets of normal rabbit lungs were utilized. Treated animals received atorvastatin (20 mg/kg body weight/day by mouth) for 3 days before surgery. Lungs were perfused constantly (300 mL/min) and ventilated for 1 hr with pressure-control ventilation at either 23 (high pressure; resulting in tidal volume approximately 22 mL/kg) or 11 (low pressure; tidal volume approximately 10 mL/kg) cm H2O peak inspiratory pressure and positive end-expiratory pressure of 3 cm H2O. Four groups were examined: high pressure-no statin, high pressure-statin pretreatment, low pressure-no statin, and low pressure-statin pretreatment. RESULTS: The high-pressure-no statin group sustained more damage than the low-pressure groups. In high-pressure groups, lungs of statin-pretreated vs. no statin-pretreated animals sustained a significantly lower increase in ultrafiltration coefficient (an accurate marker of alveolar capillary permeability; high-pressure-statin pretreatment vs. high-pressure-no statin, -0.013 +/- 0.017 g/min/mm Hg/100g vs. 1.723 +/- 0.495 g/min/mm Hg/100g; p < .001), lower weight gain (i.e., less edema formation; 4.62 +/- 1.50 grams vs. 17.75 +/- 4.71 grams; p = .005), improved hemodynamics (i.e., lower increase in mean pulmonary artery pressure; 0.56 +/- 0.51 mm Hg vs. 5.62 +/- 1.52 mm Hg; p = .04), lower protein concentration in bronchoalveolar lavage fluid (p < .001), and fewer histologic lesions (p = .013). Apoptosis of lung parenchyma cells was not different (p = .97). There was no difference between low-pressure-statin pretreatment and low-pressure-no statin groups regarding these outcomes. CONCLUSION: In this model, atorvastatin improves alveolar capillary permeability and hemodynamics and, thus, attenuates lung injury caused by high-stretch mechanical ventilation.

P38 expression in colorectal adenomas: relationships to cell proliferation, stem phenotype and AKT pathway proteins.

BACKGROUND: The P38-protein is known to be expressed in colorectal adenomas (CRA). Expression in low- and high-grade tubular adenomas is decreased when compared to adenocarcinomas and increased with regard to normal mucosa. We aimed to study P38 expression in human CRAs and the relationships to cell proliferation Ki67-protein, stem-phenotype CD133-protein and, to mTOR-protein (AKT pathway). METHODS: The immunohistochemical expression of P38 was evaluated in CRAs on tissue microarrays. Data were analyzed with the Kendall-rank-correlation test. RESULTS: Nuclear P38 correlated to low-grade dysplasia (Kendall P<0.01/tau=-0.254) and to decreased adenoma size (P<0.01/tau=-0.267). Nuclear P38 also correlated to cytoplasmic or membrane mTOR (P<0.01/tau=-0.223 and P<0.01/tau=-0.340) and to cytoplasmic CD133 (P<0.01/0.293). An inverse relationship was observed to Ki67 (P<0.00/ tau=-0.110). CONCLUSIONS: Our results suggest an interference of P38 with initial steps of colorectal adenomagenesis. The correlation to mTOR suggests a biological crosstalk between the MAPK- and AKT-signaling-pathways in colorectal adenomagenesis at P38 level.

Short-term administration of a high oxygen concentration is not injurious in an ex-vivo rabbit model of ventilator-induced lung injury.

BACKGROUND: Mechanical ventilation and administration of a high oxygen concentration are simultaneously used in the management of respiratory failure. We conducted this study to evaluate the effect of a high inspired oxygen concentration on ventilator-induced lung injury. METHODS: Forty sets of isolated/perfused rabbit lungs were randomized for 60 min of pressure-control ventilation at a plateau inspiratory pressure of 25 or 15 cm H(2)O and positive end-expiratory pressure of 3 cm H(2)O while receiving 100% or 21% O(2). The temperature, pH, and partial pressure of CO(2) in the perfusate were maintained the same in all groups (n = 10 for each group). The outcome measures used to assess lung injury included: the change in weight gain and ultrafiltration coefficient, the frequency of vascular failure, the histological lesions and the concentration of tumor necrosis factor-alpha and malondialdehyde in the bronchoalveolar lavage fluid. RESULTS: The two groups ventilated at the higher inspiratory pressure/tidal volume experienced greater weight gain and increases in the ultrafiltration coefficient, more frequently suffered vascular failure, and presented higher composite scores of histological damage than the two groups ventilated at the lower inspiratory pressure/tidal volume. Hyperoxia was not found to further increase any of the monitored markers of lung injury. No difference was noticed among the four experimental groups in the alveolar lavage fluid levels of tumor necrosis factor-alpha or malondialdehyde. CONCLUSIONS: These findings suggest that short-term administration of a high oxygen concentration is not a major determinant of ventilator-induced lung injury in this experimental model.

Orotracheal treprostinil administration attenuates bleomycin-induced lung injury, vascular remodeling, and fibrosis in mice.

Pulmonary fibrosis is a progressive disease characterized by disruption of lung architecture and deregulation of the pulmonary function. Prostacyclin, a metabolite of arachidonic acid, is a potential disease mediator since it exerts anti-inflammatory and anti-fibrotic actions. We investigated the effect of treprostinil, a prostacyclin analogue, in bleomycin-induced experimental pulmonary fibrosis. Bleomycin sulfate or saline was administrated intratracheally to mice (n = 9-10/group) at day 0. Orotracheal aspiration of treprostinil or vehicle was administered daily and started 24 h prior to bleomycin challenge. Evaluation of lung pathology was performed in tissue samples and bronchoalveolar lavage fluid collected 7, 14 and 21 days after bleomycin exposure. Lung injury was achieved due to bleomycin exposure at all time points as indicated by impaired lung mechanics, pathologic lung architecture (from day 14), and cellular and protein accumulation in the alveolar space accompanied by a minor decrease in lung tissue VE-cadherin at day 14. Treprostinil preserved lung mechanics, and reduced lung inflammation, fibrosis, and vascular remodeling (day 21); reduced cellularity and protein content of bronchoalveolar lavage fluid were additionally observed with no significant effect on VE-cadherin expression. Bleomycin-induced collagen deposition was attenuated by treprostinil from day 14, while treprostinil involvement in regulating inflammatory processes appears mediated by NF-κB signaling. Overall, prophylactic administration of treprostinil, a stable prostacyclin analogue, maintained lung function, and prevented bleomycin-induced lung injury, and fibrosis, as well as vascular remodeling, a hallmark of pulmonary hypertension. This suggests potential therapeutic efficacy of treprostinil in pulmonary fibrosis and possibly in pulmonary hypertension related to chronic lung diseases.

Expression of the epidermal growth factor receptor (EGFR) and the phosphorylated EGFR in invasive breast carcinomas.

INTRODUCTION: Epidermal growth factor receptor (EGFR) is involved in regulating cell growth in breast carcinomas. Its activated form, phosphorylated EGFR (pEGFR), is correlated with poor prognosis in lung cancer, but it has not yet been fully investigated in breast cancer. The aim of this study was to investigate the expressions of EGFR and pEGFR and their correlation with overall and disease-free survival, clinicopathological parameters and biological markers of invasion and angiogenesis (phosphorylated Akt [pAkt], urokinase plasminogen activator receptor [uPAR], matrix metalloproteinase [MMP]-14, vascular endothelial growth factor receptor [VEGFR]-1/Flt-1). METHODS: A three-step immunohistochemical method was applied to paraffin-embedded sections from 154 patients with invasive breast carcinoma in order to detect expressions of the proteins EGFR, pEGFR, oestrogen receptor, progesterone receptor, c-erbB-2, pAkt, VEGFR-1/Flt-1, MMP-14 and uPAR. The results were evaluated statistically using the chi2 test. Overall and disease-free survival distribution curves were assessed using the Kaplan-Meier test and log-rank statistics, followed by Cox proportional hazards regression model. RESULTS: EGFR and pEGFR proteins were immunodetected in the membrane of the malignant cells (11.3% and 35.7%, respectively). EGFR expression was positively correlated with nuclear grade (P = 0.001) and negatively correlated with the hormonal receptor oestrogen receptor (P = 0.005). pEGFR was positively related to the Akt pathway (P = 0.008) and appeared to participate in invasion and metastasis (uPAR, P = 0.049; MMP-14, P = 0.025; VEGFR-1/Flt-1, P = 0.016). Univariate analysis showed that the EGFR/pEGFR phenotype was associated with poor overall survival (P = 0.019), a finding further supported by multivariate analysis (P = 0.013). CONCLUSION: These data provide evidence that pEGFR expression is related to angiogenesis (via VEGFR-1/Flt-1, MMP-14 and pAkt pathways) and invasiveness (via uPAR, MMP-14 and pAkt pathways) and that the EGFR/pEGFR phenotype is associated with poor patient survival in invasive breast cancer.

Tiotropium bromide exerts anti-inflammatory effects during resistive breathing, an experimental model of severe airway obstruction.

INTRODUCTION: Resistive breathing (RB), a hallmark of obstructive airway diseases, is characterized by strenuous contractions of the inspiratory muscles that impose increased mechanical stress on the lung. RB is shown to induce pulmonary inflammation in previous healthy animals. Tiotropium bromide, an anticholinergic bronchodilator, is also shown to exert anti-inflammatory effects. The effect of tiotropium on RB-induced pulmonary inflammation is unknown. METHODS: Adult rats were anesthetized, tracheostomized and breathed spontaneously through a two-way non-rebreathing valve. Resistances were connected to the inspiratory and/or expiratory port, to produce inspiratory resistive breathing (IRB) of 40% or 50% Pi/Pi,max (40% and 50% IRB), expiratory resistive breathing (ERB) of 60% Pe/Pe,max (60% ERB) or combined resistive breathing (CRB) of both 40% Pi/Pi,max and 60% Pe/Pe,max (40%/60% CRB). Tiotropium aerosol was inhaled prior to RB. After 6 h of RB, mechanical parameters of the respiratory system were measured and bronchoalveolar lavage (BAL) was performed. IL-1ß and IL-6 protein levels were measured in lung tissue. Lung injury was estimated histologically. RESULTS: In all, 40% and 50% IRB increased macrophage and neutrophil counts in BAL and raised IL-1ß and IL-6 lung levels, tissue elasticity, BAL total protein levels and lung injury score. Tiotropium attenuated BAL neutrophil number, IL-1ß, IL-6 levels and lung injury score increase at both 40% and 50% IRB. The increase in macrophage count and protein in BAL was only reversed at 40% IRB, while tissue elasticity was not affected. In all, 60% ERB raised BAL neutrophil count and total protein and reduced macrophage count. IL-1ß and IL-6 levels and lung injury score were increased. Tiotropium attenuated these alterations, except for the decrease in macrophage count and the increase in total protein level. In all, 40%/60% CRB increased macrophage and neutrophil count in BAL, IL-1ß and IL-6 levels, tissue elasticity, total protein in BAL and histological injury score. Tiotropium attenuated the aforementioned alterations. CONCLUSION: Tiotropium inhalation attenuates RB-induced pulmonary inflammation.