Treatment of pulmonary fibrosis with siRNA against a collagen-specific chaperone HSP47 in vitamin A-coupled liposomes.

BACKGROUND: Pulmonary fibrosis is a life-threatening pathological state of progressive interstitial lung diseases, such as idiopathic pulmonary fibrosis. Myofibroblasts are known to play a critical role in the pathogenesis of pulmonary fibrosis. This study aimed to evaluate the inhibitory effect of a small interfering RNA (siRNA) on a collagen-specific chaperone heat shock protein 47 (HSP47). The siRNA was preferentially delivered to myofibroblasts in a bleomycin (BLM)-induced pulmonary fibrosis rat model using siRNA against HSP47, encapsulated in a vitamin A-coupled liposome (VA-lip-siRNA HSP47). METHODS AND RESULTS: Male Sprague-Dawley rats were treated with an intratracheal injection of BLM or phosphate buffered saline followed by an intravenous injection of VA-lip-siRNA HSP47 three times per week under preventive administration schedules from day 1 to day 21 and therapeutic administration schedules from day 15 to day 35. The expression of HSP47 after the treatment was assessed by immunoblotting. The specific delivery of VA-lip-siRNA HSP47 conjugated with 6'-carboxyfluoresce into myofibroblasts was examined by immunofluorescence staining. The effect of VA-lip-siRNA HSP47 on fibrosis was analyzed by morphological and biochemical methods. Preferential delivery of VA-lip-siRNA HSP47 to myofibroblasts in fibrotic areas in BLM-treated rats was verified by immunofluorescence staining. Treatment of VA-lip-siRNA HSP47 clearly suppressed HSP47 expression and induced apoptosis of myofibroblasts in the lung of BLM-treated rats. Hydroxyproline levels and inflammatory cytokines in the lungs, and the number of inflammatory cells in the bronchial alveolar lavage of BLM-treated rats were significantly suppressed by the treatment. Morphological assessment showed that VA-lip-siRNA HSP47 also significantly improved the morphological pulmonary fibrosis of BLM-treated rats in both preventive and therapeutic schedules. CONCLUSIONS: These results suggest that VA-lip-siRNA HSP47 improves pulmonary fibrosis in not only preventive, but also therapeutic schedules, and thus, this drug delivery system should provide a novel therapy for refractory pulmonary fibrosis.

Serum surfactant protein D predicts the outcome of patients with idiopathic pulmonary fibrosis treated with pirfenidone.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal pulmonary disease with poor prognosis. Pirfenidone, the first antifibrotic drug, suppresses the decline in forced vital capacity (FVC) and improves prognosis in some, but not all, patients with IPF; therefore, an indicator for identifying improved outcomes in pirfenidone therapy is desirable. This study aims to clarify whether baseline parameters can be predictors of disease progression and prognosis in patients with IPF treated with pirfenidone. METHODS: We retrospectively investigated patients with IPF who started treatment with pirfenidone between December 2008 and November 2014 at the Sapporo Medical University Hospital. Patients treated with pirfenidone for ≥6 months were enrolled in this study and were observed until November 2015. We investigated the association of clinical characteristics, pulmonary function test results, and blood examination results at the start of pirfenidone with the outcome of patients. RESULTS: Sixty patients were included in this study. In multivariate logistic regression analysis, % predicted FVC and serum surfactant protein (SP)-D levels were predictors of a ≥10% decline in FVC in the initial 12 months. In the Cox proportional hazards model, these two factors predicted progression-free survival. Pack-years, % predicted diffusing capacity for carbon monoxide, and SP-D levels predicted overall survival. CONCLUSIONS: The serum SP-D level was a predictor of disease progression and prognosis in patients with IPF treated with pirfenidone. In addition, this analysis describes the relative usefulness of other clinical parameters at baseline in estimating the prognosis of patients with IPF who are candidates for pirfenidone therapy.

Distinct compartmentalization of SP-A and SP-D in the vasculature and lungs of patients with idiopathic pulmonary fibrosis.

BACKGROUND: Surfactant proteins SP-A and SP-D are useful biomarkers in diagnosis, monitoring, and prognosis of idiopathic pulmonary fibrosis (IPF). Despite their high structural homology, their serum concentrations often vary in IPF patients. This retrospective study aimed to investigate distinct compartmentalization of SP-A and SP-D in the vasculature and lungs by bronchoalveolar lavage fluid (BALF)/serum analysis, hydrophilicity and immunohistochemistry. METHODS: We included 36 IPF patients, 18 sarcoidosis (SAR) patients and 20 healthy subjects. Low-speed centrifugal supernatants of BALF (Sup-1) were obtained from each subject. Sera were also collected from each patient. Furthermore, we separated Sup-1 of IPF patients into hydrophilic supernatant (Sup-2) and hydrophobic precipitate (Ppt) by high-speed centrifugation. We measured SP-A and SP-D levels of each sample with the sandwich ELISA technique. We analyzed the change of the BALF/serum level ratios of the two proteins in IPF patients and their hydrophilicity in BALF. The distribution in the IPF lungs was also examined by immunohistochemical staining. RESULTS: In BALF, SP-A levels were comparable between the groups; however, SP-D levels were significantly lower in IPF patients than in others. Although IPF reduced the BALF/serum level ratios of the two proteins, the change in concentration of SP-D was more evident than SP-A. This suggests a higher disease impact for SP-D. Regarding hydrophilicity, although more than half of the SP-D remained in hydrophilic fractions (Sup-2), almost all of the SP-A sedimented in the Ppt with phospholipids. Hydrophilicity suggests that SP-D migrates into the blood more easily than SP-A in IPF lungs. Immunohistochemistry revealed that SP-A was confined to thick mucus-filling alveolar space, whereas SP-D was often intravascular. This data also suggests that SP-D easily leaks into the bloodstream, whereas SP-A remains bound to surfactant lipids in the alveolar space. CONCLUSIONS: The current study investigated distinct compartmentalization of SP-A and SP-D in the vasculature and lungs. Our results suggest that serum levels of SP-D could reflect pathological changes of the IPF lungs more incisively than those of SP-A.

Development of an enzyme-linked immunosorbent assay for measurement of rat pulmonary surfactant protein D using monoclonal antibodies.

Surfactant protein D (SP-D) has been used as a biomarker of lung inflammation. In rat, several types of enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies have been reported. The purpose of this study was the development of a sensitive ELISA for rat SP-D using monoclonal antibodies. The authors developed a sandwich ELISA using monoclonal antibodies that were obtained by immunizing with purified rat SP-D. The ELISA was evaluated by performance tests. Furthermore, concentrations of serum SP-D were measured in normal control and bleomycin-treated rats. The working range of ELISA was between 0.47 and 30 ng/mL. Different concentrations of added SP-D were recovered, between 94.1% and 102.8%. Serum SP-D levels of bleomycin-treated rats were significantly higher than those of normal rats. In conclusion, this newly developed ELISA for rat SP-D using monoclonal antibodies is applicable for research on the mechanism and therapy of lung injury.

Circulating surfactant protein A (SP-A), a marker of lung injury, is associated with insulin resistance.

OBJECTIVES: Impaired lung function and inflammation have both attracted interest as potentially novel risk factors for glucose intolerance, insulin resistance, and type 2 diabetes. We hypothesized that circulating levels of surfactant protein (SP)-A, which reflects interstitial lung injury, could be associated with altered glucose tolerance and insulin resistance. RESEARCH DESIGN AND METHODS: Circulating SP-A concentration and metabolic variables (including insulin sensitivity by minimal model method, n = 89) were measured in 164 nonsmoking men. RESULTS: Circulating SP-A concentration was significantly higher among patients with glucose intolerance and type 2 diabetes than in subjects with normal glucose tolerance, even after adjustment for BMI, age, and smoking status (ex/never). The most significant differences were found in overweight and obese subjects with altered glucose tolerance (n = 59) who showed significantly increased serum SP-A concentrations (by a mean of 24%) compared with obese subjects with normal glucose tolerance (n = 58) (log SP-A 1.54 +/- 0.13 vs. 1.44 +/- 0.13; P < 0.0001). Insulin sensitivity (P = 0.003) contributed independently to 22% of SP-A variance among all subjects. In subjects with altered glucose tolerance, insulin sensitivity (P = 0.01) and fasting triglycerides (P = 0.02) contributed to 37% of SP-A variance. Controlling for serum creatinine or C-reactive protein in these models did not significantly change the results. CONCLUSIONS: Lung-derived SP-A protein was associated with altered glucose tolerance and insulin resistance in 164 nonsmoking men.

GM3 synthase gene is a novel biomarker for histological classification and drug sensitivity against epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.

Expression of gangliosides and alterations in their composition have been observed during cell proliferation and differentiation and in certain cell cycle phases, brain development and cancer malignancy. To investigate the characteristics of GM3 synthase, SAT-I mRNA and ganglioside GM3 expression levels in lung cancer, we examined the expression levels of SAT-I mRNA as well as GM3 in 40 tumor tissues surgically removed from non-small cell lung cancer patients. Adenocarcinoma tissues expressed SAT-I mRNA levels that were significantly higher than those of squamous and other carcinomas (P < 0.0001). Moreover, the SAT-I mRNA levels were high in the bronchioalveolar carcinoma subtype and low in the solid and mucin subtypes of adenocarcinomas (P = 0.049, 0.049 and 0.013, respectively). To clarify the relationship between SAT-I mRNA and epidermal growth factor receptor (EGFR)-tyrosine kinase (TK) inhibitor sensitivity, we carried out drug sensitivity tests for the EGFR-TK inhibitors gefitinib and AG1478 using eight adenocarcinoma cell lines expressing no EGFR mutations. The IC(50) values for gefitinib and AG1478 decreased dramatically with increasing SAT-I mRNA levels (R(2) = 0.81 and 0.59, respectively), representing a wide range of drug sensitivities among adenocarcinoma cell lines. To explore a possible mechanism of how GM3 could enhance the sensitivity to EGFR-TK inhibitors, the SAT-I gene was introduced stably into a GM3-negative clone of murine 3LL lung cancer cells to produce GM3-reconstituted clones. We found an increase in EGFR protein levels and gefitinib sensitivity in GM3-reconstituted cells, suggesting the involvement of GM3 in the turnover of EGFR protein. Therefore, it is highly expected that, by measuring the expression levels of SAT-I mRNA in lung biopsy samples from non-small cell lung cancer patients, enhanced pathological identification and individualized chemotherapeutic strategies can be established for the appropriate use of EGFR-TK inhibitors.

Elevated serum surfactant protein A and D in pulmonary alveolar microlithiasis.

Pulmonary alveolar microlithiasis (PAM) is a rare disease characterized by widespread localization of calcispherites in the alveolar spaces. The authors report two cases of PAM, with markedly elevated sera concentrations of surfactant protein-A and surfactant protein-D, which showed a tendency to increase as the disease progressed. Therefore, surfactant protein-A and surfactant protein-D may function as serum markers to monitor the disease activity and progression of PAM.

Monitoring markers of disease activity for interstitial lung diseases with serum surfactant proteins A and D.

OBJECTIVES: Surfactant protein (SP) A and D are specific serum markers for interstitial lung diseases including idiopathic pulmonary fibrosis (IPF). The authors evaluated the critical roles of these markers on the prognoses of patients with IPF and the mechanisms of their elevation in sera. METHODOLOGY: The authors evaluated the relationship between prognosis and the serum markers in 82 IPF patients. The protein content and mRNA expression of the markers were evaluated using rats with interstitial pneumonia induced by bleomycin administration. RESULTS: Higher levels of serum SP-D at the time of the initial visit to the Sapporo Medical University Hospital were associated with poorer prognoses, while SP-A showed no significant affect on survival. Causes of the elevation in sera were due to the acceleration of, not only production in the lungs, leakage into the circulation. The elevation was associated with alveolitis but not fibrosis. CONCLUSIONS: SP-D is a good predictor of the prognosis in patients with IPF.

Surfactant protein gene expressions for detection of lung carcinoma cells in peripheral blood.

BACKGROUND: Inflow of tumor cells to circulation is an essential step for metastasis of primary tumors. To know its state may contribute to therapeutic strategy. However, methodology to detect lung carcinoma cells floating in peripheral blood has not been established. Pulmonary surfactant protein (SP)-A, SP-C and Clara cells-10 kd protein (CC10) are specific to the lung and often expressed in primary lung carcinomas. We evaluated the worth of these gene expressions for the detection of carcinoma cells in peripheral blood. METHODS: The expressions in 5 ml of venous blood were tested by RT-PCR. Ninety-nine patients with non-small cell lung carcinoma (NSCLC) and 17 with small cell lung carcinoma (SCLC) were compared to 13 with secondary lung tumor, 48 with non-malignant respiratory diseases and 19 healthy volunteers. RESULTS: The mRNA expressions of SP-A and SP-C were completely specific to NSCLC when compared to SCLC and secondary lung tumors. All of the healthy volunteers and patients with non-malignant respiratory diseases showed negative for these mRNA expressions, except for one sample. The positive rate of SP-A, SP-C and CC10 mRNA in patients with NSCLC was 33.3%, 14.1%, 3.3%, respectively. The rates of SP-A and SP-C mRNA were higher than that (11.1%) in CEA mRNA. The increased positive rate of mRNA of SP-A and SP-C was significantly dependent on the clinical stage and the existence of distant metastasis. CONCLUSION: These results demonstrate that the detection of mRNA of SP-A and SP-C would give clinicians valuable information suggesting the presence of blood-floating carcinoma cells as a step toward metastasis.

Acute lung injury as an adverse event of gefitinib.

Gefitinib, a selective epidermal growth factor receptor tyrosine kinase inhibitor, is an effective treatment for patients with non-small cell lung cancer (NSCLC). Some investigators have recently reported several patients complicated by acute lung injury after the initiation of gefitinib administration. In this report, we investigated the efficacy and adverse events during treatment with gefitinib. The subjects of this study were all of the 110 patients with NSCLC who were treated in our hospital and its eight branch hospitals. Patients received gefitinib at a dose of 250 mg once daily. The response rate was 30%. The frequently reported adverse events were skin disorders, gastrointestinal disturbances, liver dysfunction and acute lung injury. Five of the 12 patients who were considered to have suffered acute lung injury died of progressive respiratory failure. Of the nine patients who had pulmonary fibrosis before use of gefitinib, five developed acute lung injury during the treatment. Sera from three of the 12 patients were evaluated and all three showed increases of surfactant protein (SP)-A, SP-D and KL-6. We conclude that gefitinib was clinically useful. However, several patients suffered acute lung injury which could have been caused by gefitinib. A detection system including SP-A, SP-D and KL-6 as prime candidates as markers should be established as promptly as possible. Clinicians should be aware that treatment of NSCLC with gefitinib involves the risk of acute lung injury and therefore careful consideration should be given before deciding whether or not gefitinib is indicated for treatment. Further study is necessary to elucidate the mechanism of acute lung injury by gefitinib.

[Two younger male patients with rapidly progressing pulmonary pleomorphic carcinoma].

We report 2 cases of pulmonary pleomorphic carcinoma. The patient in case 1 was a 44-year-old man who was admitted to our hospital complaining of dry cough and dyspnea on exertion. Chest radiography and CT showed a huge tumor with left pleural effusion. Percutaneous biopsy suggested malignancy. As distant metastasis was not found and as cytological examination of pleural effusion gave a result of class II, we attempted to perform left pneumonectomy. However, we performed partial resection of the tumor because it had invaded the aorta, pulmonary artery, pericardium and pleura. The pathological diagnosis was pleomorphic carcinoma, p-T4 N2 M0. After the operation, we performed systemic chemotherapy, including cisplatin and irinotecanm with little effect (PD). The patient died of progression of the tumor. Case 2 was a 34-year-old man who was admitted to our hospital complaining of fever and general malaise. Chest radiography and CT showed a tumor in the left upper lobe. As no distant metastasis was found, we tried left upper lobectomy. However, we performed left upper segmentectomy and lymph node dissection because of invasion of the aorta and the chest wall, and because of hypersegmentation of the left upper lobe. Pathological diagnosis was pleomorphic carcinoma, p-T4 N0 M0. After the operation, the mediastinum was subjected to radiation therapy. However, a metastatic tumor was found in the pelvis after this radiation was given. We performed systemic chemotherapy with substances including cisplatin, gemcitabine and vinorelbine, but with little effect (PD). The patient died of peritonitis and pleuritis due to the recurrence and progression of the tumor. Both cases had rapidly growing neoplasms showing little sensitivity to chemotherapy or radiotherapy. Pulmonary pleomorphic carcinoma is suggested to be type of lung cancer with a poor prognosis when the tumor is not resected in the early stages.

A rare case of pulmonary bronchogenic cyst associated with bronchial atresia in the same lobe.

A rare case of 49-year-old woman having pulmonary bronchogenic cyst associated with bronchial atresia in the same lobe was presented. The diagnosis was confirmed by aortography and operation specimen. Three-dimensional reconstructed images of computed tomography clearly demonstrated the defference between mucoid impaction of bronchial trees in the left S9 not communicating with hilar bronchus and the cyst in the left S10 oppressing surrounding vessels.

[A case of sarcoidosis with primary pulmonary cavitation].

A 30-year-old woman presented with multiple nodular shadows which enclosed a cavity on a chest radiograph. Chest computed tomographic (CT) images showed mediastinal lymphadenopathy, and multiple nodular opacities enclosing a cavity. Histopathological findings of biopsy specimens from the lung and mediastinal lymph nodes revealed noncaseating epithelioid cell granulomas without any evidence of Mycobacterium or fungal growth. The lesion in the lung included granulomatous vasculitis. Even without corticosteroid or any other therapy, the lung lesions resolved and the cavity disappeared. We report a case of sarcoidosis with primary acute cavitation.

Pulmonary lymphangioleiomyomatosis; unusual radiological manifestation of multiple large nodules.

A 44-year-old woman was admitted to our hospital in August 1999 for multiple large nodules detected on chest roentgenogram in an annual health check. Chest CT scans showed bilateral large nodules (>10 mm in diameter) with irregular margins and multiple thin walled cystic lesions. From these radiologic examinations, we suspected pulmonary Langerhans cell histiocytosis. Histological examination of the biopsy specimen by video-assisted thoracoscopy revealed a marked proliferation of the spindle cells, which were immunologically positive for alpha-smooth muscle actin and HMB-45, in the cyst walls and lung parenchyma. The large nodules consisted of proliferation of the smooth muscle cells surrounded by a dense layer of hemosiderinladen macrophages. During the two years subsequent to these 1999 examinations, the opacities have gradually diminished and the patient was found to have pulmonary lymphangioleiomyomatosis. This case exhibited rare radiologic manifestations of multiple large nodules mimicking Langerhans cell histiocytosis.

Assessment of differentiation in adenocarcinoma cells from pleural effusion by peripheral airway cell markers and their diagnostic values.

Ultrastructural studies have shown that Clara cell-type is a more common type of adenocarcinoma than alveolar type II cell-type, and that both types may provide better prognosis than other types, indicating an importance of differentiation toward peripheral airway cells. Pulmonary surfactant protein (SP)-A is a specific marker for both alveolar type II cells and Clara cells in peripheral lung tissues, while SP-C and Clara cell 10 kD protein (CC10) may be particularly and highly specific to alveolar type II cells and Clara cells, respectively. The aims of this study were to assess the differentiation of adenocarcinoma cells in pleural effusions by evaluating the expression of these cell markers and to evaluate their values as diagnostic tools for judging the cause of pleural effusion. We examined pleural effusions from 52 patients; 20 with primary lung adenocarcinomas, 6 with small cell lung carcinomas, 11 with metastatic malignant tumors and 15 with non-neoplastic diseases. The cell pellets from effusions were subjected to immunocytochemical staining for SP-A, proSP-C, a precursor of SP-C, and CC10. By this immunocytochemical study for SP-A and proSP-C, 10 (50%) and 6 (30%) of 20 adenocarcinomas, respectively, showed a positive immunoreactivity in their effusion cells, while none of them expressed CC10. Alveolar type II cells therefore may be the main progenitor cells of some adenocarcinomas. In pleural effusions from patients with primary lung adenocarcinomas, reverse transcriptase-polymerase chain reaction (RT-PCR) for SP-A mRNA showed a sensitivity of 83%, while, in all remaining patients, these assays were negative. In conclusion, we demonstrated that lung adenocarcinomas, which are partially differentiated toward alveolar type II cells, are not as rare as previously thought, and that both the RT-PCR and immunocytochemical analyses for SP-A and pro-SP-C could be worthy indicators of differential diagnosis.