Oligonucleotide-targeting periostin ameliorates pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with a median survival of 3-4 years after diagnosis. It is the most frequent form of a group of interstitial pneumonias of unknown etiology. Current available therapies prevent deterioration of lung function but no therapy has shown to improve survival. Periostin is a matricellular protein of the fasciclin 1 family. There is increased deposition of periostin in lung fibrotic tissues. Here we evaluated whether small interfering RNA or antisense oligonucleotide against periostin inhibits lung fibrosis by direct administration into the lung by intranasal route. Pulmonary fibrosis was induced with bleomycin and RNA therapeutics was administered during both acute and chronic phases of the disease. The levels of periostin and transforming growth factor-ß1 in airway fluid and lung tissue, the deposition of collagen in lung tissue and the lung fibrosis score were significantly reduced in mice treated with siRNA and antisense against periostin compared to control mice. These findings suggest that direct administration of siRNA or antisense oligonucleotides against periostin into the lungs is a promising alternative therapeutic approach for the management of pulmonary fibrosis.

Circulating fibrocytes correlate with the asthma control test score

BACKGROUND: Bronchial asthma is characterised by airway inflammation and remodelling with a decline of lung function. Fibrocytes are bone marrow-derived mesenchymal progenitor cells that play important roles in the pathogenesis of airway remodelling. Several clinical parameters are currently being used in routine clinical practice to assess outcome of therapy in asthma including frequency of rescue with short-acting β2-agonist and the asthma control test. In this study, we hypothesised that asthma control test is associated with circulating levels of fibrocytes in bronchial asthma. METHODS: There were 20 patients with asthma and seven healthy controls. The number of CD45+Collagen I+circulating fibrocytes was assessed in the peripheral blood by flow cytometry. RESULTS: The number of circulating fibrocytes was significantly increased in asthma patients with moderate and severe disease compared to controls, and it was inversely correlated with % forced expiratory volume in one second and % forced vital capacity (%FVC). The frequency of inhalation of short-acting β2 agonist and the asthma control test score was significantly and inversely correlated with the number of circulating fibrocytes. CONCLUSION: The results of this study showed that the number of circulating fibrocytes is inversely correlated with clinical asthma control parameters, further supporting the relevance of measuring circulating fibrocytes as a marker of clinical control in bronchial asthma

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Protein S is protective in pulmonary fibrosis.

UNLABELLED: Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis. SUMMARY: Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar epithelial cells in vitro. Conclusions These observations suggest clinical relevance and a protective role of protein S in pulmonary fibrosis.

Circulating fibrocytes correlate with the asthma control test score.

BACKGROUND: Bronchial asthma is characterised by airway inflammation and remodelling with a decline of lung function. Fibrocytes are bone marrow-derived mesenchymal progenitor cells that play important roles in the pathogenesis of airway remodelling. Several clinical parameters are currently being used in routine clinical practice to assess outcome of therapy in asthma including frequency of rescue with short-acting ß2-agonist and the asthma control test. In this study, we hypothesised that asthma control test is associated with circulating levels of fibrocytes in bronchial asthma. METHODS: There were 20 patients with asthma and seven healthy controls. The number of CD45(+)Collagen I(+) circulating fibrocytes was assessed in the peripheral blood by flow cytometry. RESULTS: The number of circulating fibrocytes was significantly increased in asthma patients with moderate and severe disease compared to controls, and it was inversely correlated with % forced expiratory volume in one second and % forced vital capacity (%FVC). The frequency of inhalation of short-acting ß2 agonist and the asthma control test score was significantly and inversely correlated with the number of circulating fibrocytes. CONCLUSION: The results of this study showed that the number of circulating fibrocytes is inversely correlated with clinical asthma control parameters, further supporting the relevance of measuring circulating fibrocytes as a marker of clinical control in bronchial asthma.

Dose-dependent differential effects of thrombin in allergic bronchial asthma.

BACKGROUND: Apart from its role in the coagulation system, thrombin plays an important role in the inflammatory response through its protease-activated receptors (PARs). However, the role of thrombin in the immune response is not clear. OBJECTIVE: To evaluate whether thrombin has a modulatory role in allergic bronchial asthma. METHODS: Bronchial asthma was induced in mice by intraperitoneal sensitization and inhalation challenge with ovalbumin. Thrombin or its inhibitors were administered by inhalation before each allergen challenge. RESULTS: Mice with low but sustained coagulation activation had reduced allergic inflammation, and allergic asthma was inhibited by low doses of thrombin but worsened by high doses. Allergic asthma was worsened by antithrombin, argatroban, hirudin, and anti-thrombomodulin antibody. Mice with a higher level of an inhibitor of both thrombin and activated protein C had worse disease. Heterozygous PAR-1 mice had less allergic inflammation, but PAR-1 agonist worsened it. Allergic bronchial inflammation was worsened in mice that received adoptive transfer of PAR-1 agonist-treated Th2 cells as compared with controls. Low levels of thrombin suppressed the maturation and secretion of cytokines in dendritic cells, but high levels enhanced this. CONCLUSIONS: The effects of thrombin on allergic asthma are dose-dependent, with detrimental effects at high doses and protective effects at low doses. These data demonstrate that thrombin modulates the outcome in allergic bronchial asthma.

Pulmonary hypertension is ameliorated in mice deficient in thrombin-activatable fibrinolysis inhibitor.

BACKGROUND: The fibrinolytic system has been implicated in the pathogenesis of pulmonary hypertension (PH). Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis and therefore its absence would be expected to increase fibrinolysis and ameliorate PH. OBJECTIVE: The objective of the present study was to evaluate the effect of TAFI deficiency on pulmonary hypertension in the mouse. METHODS AND RESULTS: PH was induced in C57/Bl6 wild-type (WT) or TAFI-deficient (KO) mice by weekly subcutaneous treatment with 600 mg kg(-1) monocrotaline (MCT) for 8 weeks. PH was inferred from right heart hypertrophy measured using the ratio of right ventricle-to-left ventricle-plus-septum weight [RV/(LV+S)]. Pulmonary vascular remodeling was analyzed by morphometry. TAFI-deficient MCT-treated and wild-type MCT-treated mice suffered similar weight loss. TAFI-deficient MCT-treated mice had reduced levels of total protein and tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta) and monocyte chemoattractant protein-1 (MCP-1) in bronchial alveolar lavage compared with wild-type MCT-treated mice. The ratio of RV to (LV+S) weight was significantly higher in WT/MCT than in KO/MCT mice. The pulmonary artery wall area and vascular stenosis were both greater in MCT-treated WT mice compared with MCT-treated TAFI-deficient mice. CONCLUSIONS: TAFI-deficient MCT-treated mice had less pulmonary hypertension, vascular remodeling and reduced levels of cytokines compared with MCT-treated WT animals, possibly as a result of reduced coagulation activation.

Direct effects of protein S in ameliorating acute lung injury.

OBJECTIVE: Protein S may exert an anticoagulant activity by enhancing the anticoagulant activity of activated protein C and/or by directly inhibiting the prothrombinase complex. Protein S itself may also directly regulate inflammatory responses and apoptosis. The role of protein S in acute lung injury (ALI) was unknown. This study evaluated the effect of protein S on ALI in the mouse. METHODS: Animal ALI was induced in C57/BL6 mice by intratracheal instillation of lipopolysaccharide (LPS). Mice were treated with protein S or saline by intraperitoneal injection 1 h before LPS instillation. RESULTS: Activated protein or protein S alone and combined activated protein C + protein S therapy decreased inflammatory markers and cytokines in mice with acute lung injury. In LPS-treated mice compared with controls ALI was induced as shown by significantly increased levels of total protein, tumor necrosis factor-alpha, interleukin-6 and monocyte chemoattractant protein-1 in the bronchoalveolar lavage fluid. Mice with ALI treated with protein S had significantly decreased concentrations of tumor necrosis factor-alpha and interleukin-6 in the lung compared with untreated animals. Thrombin-antithrombin III, a marker of the activity of the coagulation cascade, was unchanged. Protein S inhibited the expression of cytokines in vitro and increased activation of the Axl tyrosine kinase pathway in A549 epithelial cells. CONCLUSION: Protein S protects against LPS-induced ALI, possibly by directly inhibiting the local expression of inflammatory cytokines without affecting coagulation.

A prospective comparison of nursing home-acquired pneumonia with hospital-acquired pneumonia in non-intubated elderly.

There are no prospective comparison of the etiology and clinical outcome between hospital-acquired pneumonia (HAP) and nursing home-acquired pneumonia (NHAP) in non-intubated elderly. This study prospectively evaluated the etiology of HAP and NHAP in non-intubated elderly. A prospective cohort study was carried out in a rural region of Japan where the population over 65 years of age represents 30% of the population. A total of 108 patients were enrolled. There were 33 patients with HAP and 75 with NHAP. Etiologic diagnosis was established in 78.8% of HAP and in 72% of NHAP patients. The most frequent pathogens were Chlamydophila pneumoniae followed by Streptococcus pneumoniae, Staphylococcus aureus and Influenza virus. The frequency of Streptococcus pneumoniae and Influenza virus was significantly higher, whereas the frequency of Staphylococcus aureus and Enterobacteriaceae was significantly lower in NHAP compared to HAP. Performance and nutritional status were significantly worse in patients with HAP than in those with NHAP. Hospital mortality was significantly lower in patients with NHAP compared to those with HAP. This study demonstrated that C. pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus and Influenza virus are frequent causative agents of pneumonia in non-intubated elderly and that the responsible pathogens and clinical outcome differ between NHAP and HAP.

Exposure to hardly soluble indium compounds in ITO production and recycling plants is a new risk for interstitial lung damage.

OBJECTIVES: To identify the effects of indium on the lung and to assess exposure-effect and exposure-response relations between indium exposure and effects on the lungs. METHODS: Ninety three male indium exposed and 93 male non-exposed workers from four ITO manufacturing or ITO recycling plants were analysed in a cross-sectional study. Indium in serum (In-S) was determined as a biological exposure index. Geometric means (GSD) of In-S were 8.25 ng/ml (4.55) in the exposed workers and 0.25 (2.64) in the non-exposed workers. The maximum concentration of In-S was 116.9 ng/ml. A questionnaire for respiratory symptoms and job histories, spirometry, high-resolution computerised tomography (HRCT) of the chest, serum KL-6, serum SP-A, serum SP-D and serum CRP were measured as the effect indices. RESULTS: Spirometry, subjective symptoms and the prevalence of interstitial or emphysematous changes on lung HRCT showed no differences between exposed and non-exposed workers. Geometric means (GSD) of KL-6, SP-D and SP-A in the exposed workers were 495.4 U/ml (2.26), 85.2 ng/ml (2.02) and 39.6 ng/ml (1.57), and were significantly higher than those in the non-exposed workers. The prevalence (%) of the exposed and non-exposed workers exceeding the reference values were also significantly higher in KL-6 (41.9 vs 2.2), SP-D (39.8 vs 7.5), and SP-A (43.0 vs 24.7). Very sharp exposure-effect and exposure-response relations were discovered between In-S and KL-6 and between In-S and SP-D when the exposed workers were classified into seven groups by In-S. CONCLUSIONS: The study outcomes with regard to the basis of serum immunochemistry biomarkers and HRCT indicate that exposure to hardly soluble indium compound dust may represent a risk for interstitial lung damage.

Protective role of thrombin activatable fibrinolysis inhibitor in obstructive nephropathy-associated tubulointerstitial fibrosis.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) has been reported to affect wound healing and fibrotic processes, but its role in renal tubulointerstitial fibrosis remains unknown. OBJECTIVE: To study its potential role, we compared TAFI-deficient and wild-type mice for the degree of renal fibrosis caused by unilateral ureteral obstruction (UUO). METHODS: The grade of tubulointerstitial fibrosis, the activity of plasmin, MMP-2 and MMP-9 were evaluated on days 4 and 9 after UUO. RESULTS: The renal content of hydroxyproline and the activity of plasmin, MMP-2 and MMP-9 were significantly increased in kidneys with UUO from TAFI-deficient mice compared with those from wild-type mice. These differences disappeared when animals with UUO from both groups were treated with the plasmin inhibitor tranexamic acid. The renal concentrations of fibrogenic cytokines were also significantly elevated in kidneys with UUO from TAFI-deficient mice compared with those from wild-type mice. CONCLUSION: The results of this study suggest that increased renal activity of plasmin in TAFI-deficient mice causes increased renal interstitial fibrosis in obstructive nephropathy.

Inverse correlation between activated protein C generation and carotid atherosclerosis in Type 2 diabetic patients.

AIMS: Activated protein C (APC) is a key regulator of the clotting system and immune responses. We studied the relationship between the degree of atherosclerosis as measured by the intima-media thickness (IMT) of carotid artery and APC generation in Type 2 diabetic patients. METHODS: Eighty-seven Type 2 diabetic patients and 35 control subjects participated. APC generation was assessed by the plasma APC-protein C inhibitor complex (APC-PCI) levels and the mean IMT of carotid artery was measured by ultrasonography. The plasma levels of the thrombin-anti-thromobin complex (TAT) and platelet-derived growth factor (PDGF) were measured by enzyme-linked immunoassays. RESULTS: Plasma TAT levels were significantly higher in diabetic patients [2.03 (1.12, 2.56) ng/ml, median (25th, 75th percentile)] compared with control subjects [0.85 (0.55, 2.08) ng/ml, P < 0.01]. Plasma APC-PCI levels were significantly lower in diabetic patients [0.93 (0.74, 1.22) ng/ml], than in control subjects [1.66 (1.25, 2.36) ng/ml, P < 0.001]. The mean IMT was significantly increased in diabetic patients (0.881 +/- 0.242 mm; mean +/- sd) compared with control subjects (0.669 +/- 0.140 mm; P < 0.01). Univariate analysis showed a significant and inverse correlation between plasma APC-PCI levels and mean IMT (r = -0.32, P < 0.005), and multivariate regression analysis confirmed the independent correlation (P < 0.05). Moreover, plasma APC-PCI levels significantly and inversely correlated with plasma PDGF levels in diabetic patients (r = -0.30, P < 0.01). CONCLUSIONS: These results suggest that decreased APC generation is associated with vascular atherosclerotic changes in Type 2 diabetic patients.

Interstitial pulmonary disorders in indium-processing workers.

The production of indium-tin oxide has increased, owing to the increased manufacture of liquid-crystal panels. It has been reported that interstitial pneumonia occurred in two indium-processing workers; therefore, the present study aimed to evaluate whether interstitial pulmonary disorders were prevalent among indium workers. The study was carried out in 108 male workers in the indium plant where the two interstitial pneumonia patients mentioned above were employed, and included high-resolution computed tomography (HRCT) of the lungs, pulmonary function tests and analysis of serum sialylated carbohydrate antigen KL-6 and the serum indium concentration. Significant interstitial changes were observed in 23 indium workers on HRCT and serum KL-6 was abnormally high (>500 U x mL(-1)) in 40 workers. Workers with serum indium concentrations in the highest quartile had significantly longer exposure periods, greater HRCT changes, lower diffusing capacity of the lung for carbon monoxide and higher KL-6 levels compared with those in the lowest quartile. The serum indium concentration was positively correlated with the KL-6 level and with the degree of HRCT changes. In conclusion, the results of the present study indicated that serum KL-6 and high-resolution computed tomography abnormalities were prevalent among indium workers and that these abnormalities increased with the indium burden, suggesting that inhaled indium could be a potential cause of occupational lung disease.

Protective role of protein C inhibitor in monocrotaline-induced pulmonary hypertension.

BACKGROUND: Protein C inhibitor (PCI) plays a role in multiple biological processes including fertilization, coagulation, fibrinolysis and kinin systems. OBJECTIVES: We hypothesized that PCI participates in the pathogenesis of pulmonary hypertension. To demonstrate this, we compared the development of pulmonary hypertension in mice overexpressing PCI in the lung with wild-type (WT) mice. Pulmonary hypertension was induced by s.c. injection of 600 mg kg-1 of monocrotaline weekly for 8 weeks. RESULTS: Right ventricular arterial pressure was significantly increased in monocrotaline-treated WT mice compared with that in monocrotaline-treated transgenic mice. Bronchoalveolar lavage fluid (BALF) levels of thrombin-antithrombin complex, monocyte chemoattractant protein-1 and platelet-derived growth factor, and the plasma level of tumor necrosis factor-alpha were significantly increased in monocrotaline-treated WT mice as compared with monocrotaline-treated PCI transgenic mice. Increased level of PCI-thrombin complex was detected in BALF from monocrotaline-treated PCI transgenic mice as compared with saline-treated PCI transgenic mice. CONCLUSIONS: This study showed that increased expression of PCI in the lung is protective against monocrotaline-induced pulmonary hypertension, suggesting a potential beneficial effect of PCI for the therapy of this disease.

Anti-inflammatory effect of activated protein C in gastric epithelial cells.

It has been previously demonstrated that activated protein C (APC) plays an important role in the inhibition of inflammation in the gastric mucosa from patients with Helicobacter pylori infection. However, the role of gastric epithelial cells in the anti-inflammatory activity of APC remains unknown. In the present study, we evaluated the anti-inflammatory activity of APC and the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in gastric epithelial cells. The gastric epithelial cell lines, MKN-1 and AGS, and gastric biopsy samples from patients with and without H. pylori infection were used in the experiments. Polymerase chain reaction showed that gastric epithelial cell lines express EPCR and TM. Flow cytometry analysis also showed EPCR expression in both cells. H. pylori infection significantly increased EPCR expression compared with non-infected cells. Similar results were observed in vivo when samples from patients with and without H. pylori infection were analyzed for EPCR protein expression. Significant TM activity was found on AGS and MKN-1 cells stimulated with LPS from Escherichia coli and VacA antigen. APC was able to significantly inhibit the secretion of MCP-1 and IL-1beta induced by H. pylori homogenate in AGS cells. APC also remarkably suppressed the mRNA expression and secretion of MCP-1 from AGS cells infected with H. pylori. These results demonstrated the expression of components of the protein C pathway on gastric epithelial cells and that APC may play a critical role in the protection against gastric mucosal inflammation.

[Recurrent lung cancer with interstitial pneumonia treated with percutaneous radiofrequency ablation].

We performed computed tomography (CT)-guided percutaneous radiofrequency ablation (RFA) for postoperative recurrent pulmonary metastases developed in a 77-year-old man with interstitial pneumonia. He had received left upper segmentectomy with ND 2a nodal dissection. RFA was safely performed for pulmonary metastases in right S6 and left S6. There was no evidence to suggest any deterioration on interstitial pneumonia, including KL 6 and CT findings. Autopsy revealed residual cancer cells in peripheral lesion in 1 of 2 tumors treated by RFA. Although RFA is palliative, it is a promising treatment for local control of pulmonary malignancy in high-risk patients.

Expression and cytoprotective effect of protease-activated receptor-1 in gastric epithelial cells.

Thrombin is a serine protease involved in many physiological functions and its receptor. the protease-activated receptor-1 (PAR-1), has a wide tissue distribution. We hypothesized that PAR-1 is expressed in gastric epithelial cells and that thrombin can modulate defence mechanisms through PAR-1. The rat gastric epithelial cell line (RMG1) and gastric biopsy specimens from gastritis patients were used in the study. Reverse transcriptase polymerase chain reaction analysis showed that the thrombin receptors PAR-1, PAR3 and PAR-4 are expressed by RGM1 gastric epithelial cell line. Immunohistochemical and electron microspcopic studies also showed PAR-1 expression in human gastric epithelial cells. Thrombin stimulated the secretion of mucin and prostaglandin E2 (PGE2) formation in RGM1 cells in a dose-dependent manner. PAR-1 agonist also stimulated PGE2 formation. In addition, thrombin significantly increases the expression of the PGE2 receptors EP2-R and EP4-R in RGM1 cells. In conclusion, the results of the present study showed for the first time that gastric epithelial cells express thrombin receptors and that these receptors may play a protective role in the gastric mucosa.

Activation of protein C pathway in the airways.

The protein C (PC) pathway plays important roles in the regulation of the coagulation system and inflammatory response. This study evaluated the degree of PC activation in the airway of patients with bronchial asthma (BA), and the expression and regulation of PC and its receptor in airway epithelial cell lines. Thirteen BA patients and 8 healthy volunteers were enrolled in the study. BEAS-2B and A549 epithelial cell lines were used in experimental assays. Expression of anticoagulant factors was evaluated by RT-PCR and Western blotting. The activated protein C (APC)/thrombin (1.65 +/- 0.35 vs 3.34 +/- 0.59) and APC/PC (8.30 +/- 2.26 vs 24.41 +/- 9.88) ratios were significantly decreased and the concentrations of soluble thrombomodulin (TM) were significantly increased in induced sputum from BA patients compared with healthy subjects. Airway epithelial cells express PC, its receptor, and TM. PC antigen prepared from epithelial cells was significantly activated in the presence of thrombin. Thrombin increased the expression of PC antigen from lung epithelial cells. However, tumor necrosis factor-alpha, eotaxin, and RANTES (regulated on activation, normal T-cell expressed and secreted) decreased the expression of PC and its receptor in bronchial epithelial cells. Overall, these results showed for the first time that reduced activation of PC pathway occurs in the airway of BA patients and that TM, PC, and its receptor, are expressed by human airway epithelial cells. The expression of these PC pathway components was found to be downregulated by inflammatory cytokines. The decrease in PC activation may contribute to exacerbation of the inflammatory response in the airway of asthmatic patients.

Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins.

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-less enhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.

Intratracheal administration of activated protein C inhibits bleomycin-induced lung fibrosis in the mouse.

It is well recognized that activation of the coagulation system plays an important role in bleomycin (BLM)-induced lung injury and fibrosis. The protein C (PC) pathway is an important regulator of the coagulation system. In this study, we evaluated the bronchoalveolar lavage fluid (BALF) concentration of activated PC (APC) and the therapeutic effect of the intratracheal administration of APC on BLM-induced lung fibrosis in mice. APC levels in BALF were significantly lower in BLM-treated animals than in the saline-treated group. Fibrotic changes were progressive in mice treated with BLM and intratracheal instillation of vehicle (BLM/Veh) after 14 and 21 d of BLM infusion. Compared with the BLM/Veh group, histologic findings on Days 14 and 21 in mice treated with BLM and intratracheal instillation of APC (BLM/APC) showed less fibrotic lesions in the subpleural and central areas of the lung. The mean Aschcroft's fibrosis score in the BLM/Veh group was significantly (p < 0.05) higher than in the BLM/APC group. The lung hydroxyproline content on Day 21 was significantly higher (p < 0.05) in the BLM/Veh group (1.78 +/- 0.07 micromol/lung weight) than in the BLM/APC (1.30 +/- 0.06 micromol/lung weight) group. The ratio of plasminogen activator activity to thrombin level in BALF was significantly increased in the BLM/APC group compared with the BLM/ Veh group on Day 21. The expression of tumor necrosis factor-alpha and interleukin-1beta was significantly decreased in the lungs of the BLM/APC group compared with the BLM/Veh group on Day 14 after BLM infusion. These results showed that intratracheal APC administration inhibits the development of lung fibrosis in BLM-induced lung injury, giving further support to the important role that the PC pathway plays in the mechanism of lung fibrosis.

Long-term inhalation of high-dose nitric oxide increases intraalveolar activation of coagulation system in mice.

Inhalation of nitric oxide (NO) is useful for the treatment of patients with pulmonary hypertension. However, the potential toxicity of inhaled NO is still unclear. Coagulation activation plays an important role in lung injury. We assessed the effect of low- and high-dose inhaled NO on the coagulation system in the intraalveolar space of mice. The animals were assigned to five groups (n = 6): [RA] group, mice exposed to fresh air alone; [RA+2 ppm NO] group, fresh air and 2 ppm NO; [RA+40 ppm NO] group, fresh air and 40 ppm NO; [RA+2 ppm NO+O(2)] group, fresh air, 2 ppm NO and O(2); and [RA+40 ppm NO+O(2)] group, fresh air, 40 ppm NO and O(2). Each group was treated for 3 wk. Lung specimens of [RA+40 ppm NO] and [RA+40 ppm NO+O(2)] groups showed significant nitrotyrosine immunoreactivity. BALF concentrations of total protein, thrombin and soluble tissue factor were significantly increased in mice of [RA+40 ppm NO] and [RA+40 ppm NO+O(2)] groups compared with [RA] group. However, BALF concentrations of total protein, thrombin, and soluble tissue factor were not significantly increased in mice of [RA+2 ppm NO] and [RA+2 ppm NO+O(2)] groups compared with [RA] group. Lung tissue factor mRNA expression was higher in the high-dose NO group than in the low-dose NO group. NO donor increased significantly tissue factor activity on alveolar epithelial cells. This study has shown for the first time that long-term inhalation of high, but not low, concentration of NO may activate the clotting system by increasing the lung expression of tissue factor.