Katsushi Kurosu

The role of EBUS-TBNA for the diagnosis of sarcoidosis – comparisons with other bronchoscopic diagnostic modalities

BACKGROUND The diagnosis of sarcoidosis requires both compatible clinical features and pathologic findings as a means to exclude other differential diagnoses. The utility of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for diagnosis of sarcoidosis has been reported, although its indication remains unclear for cases of suspicious sarcoidosis. To clarify the role of EBUS-TBNA for the diagnosis of sarcoidosis, we compared three diagnostic modalities: EBUS-TBNA, transbronchial lung biopsy (TBLB) and bronchoalveolar lavage fluid analysis (BAL). METHODS Thirty-eight patients with suspicious sarcoidosis who had enlarged hilar and/or mediastinal lymph nodes on chest CT were retrospectively reviewed. Patients with malignancies or prior established diagnosis of sarcoidosis were excluded. BAL was initially performed followed by TBLB and finally EBUS-TBNA at the same setting. Microbacterial examinations were also performed from all samples. RESULTS Pathological findings compatible with sarcoidosis were obtained in 32 patients. The remaining 6 patients were diagnosed as one case each of chronic eosinophilic pneumonia, atypical mycobacterial infection and tuberculosis, and the remaining three were pathologically indefinite cases. Clinically, 35 patients were diagnosed with sarcoidosis. The diagnostic accuracy of sarcoidosis was significantly better by EBUS-TBNA (91.4%, p<0.001) compared to the other two modalities. According to chest roentgenogram classifications, there were 31 stage I patients and 4 stage II patients. For stage I patients, EBUS-TBNA was significantly better (90.3%, p<0.001), but each modality showed 100% accuracy for stage II patients. CONCLUSION It is recommended that EBUS-TBNA is added to the conventional diagnostic modalities for patients with suspicious stage I sarcoidosis on chest roentgenogram.

VEGF-R blockade causes endothelial cell apoptosis, expansion of surviving CD34+ precursor cells and transdifferentiation to smooth muscle-like and neuronal-like cells

Severe pulmonary hypertension (PH) is characterized by complex precapillary arteriolar lesions, which contain phenotypically altered smooth muscle (SM) and endothelial cells (EC). We have demonstrated that VEGF receptor blockade by SU5416 {3‐[(2,4‐dimethylpyrrol‐5‐yl)methylidenyl]‐indolin 2‐one} in combination with chronic hypoxia causes severe angioproliferative PH associated with arterial occlusion in rats. We postulate that endothelial‐mesenchymal transdifferentiation can take place in the occlusive lesions and that endothelium‐derived mesenchymal cells can further differentiate toward a SM phenotype. To examine this hypothesis, we incubated human pulmonary microvascular endothelial cells (HPMVEC) with SU5416 and analyzed these cells utilizing quanti‐tative‐PCR, immunofluorescent staining and flow cytometry analysis. In vitro studies in HPMVEC demonstrated that SU5416 suppressed PGI2S gene expression while potently inducing COX‐2, VEGF, and TGF‐β1 expression;and caused transdifferentiation of mature vascular endothelial cells (defined by Dil‐ac‐LDL, Lectin and Factor VIII) to SM‐like (as defined by expression of α‐SM actin) “transitional” cells, coexpressing both endothelial and SM markers. SU5416 expanded the number of CD34 and/or c‐kit positive cells and caused transdifferentiation of CD34 positive cells but not negative cells. In conclusion, our data show that SU5416 generated a selection pressure that killed some EC and expanded progenitor‐like cells to transdiffer‐entiate to SM‐like and neuronal‐like cells.—Sakao, S., Taraseviciene‐Stewart, L., Cool, C. D., Tada, Y., Kasahara, Y., Kurosu, K., Tanabe, N., Takiguchi, Y., Tatsumi, K., Kuriyama, T., and Voelkel, N. F. VEGF‐R blockade causes endothelial cell apoptosis, expansion of surviving CD34+ precursor cells and transdifferentiation to smooth muscle‐like and neuronal‐like cells. FASEB J. 21, 3640–3652 (2007)

Identification of Annexin 1 as a Novel Autoantigen in Acute Exacerbation of Idiopathic Pulmonary Fibrosis

Consistent with the hypothesis that pulmonary epithelial apoptosis is the key to the acute exacerbation of idiopathic pulmonary fibrosis (IPF), we conducted serological identification of Ags by recombinant expression cloning (SEREX) analysis using type II alveolar cell carcinoma (A549) cell lines to identify disease-related Abs. In a survey of Abs to the recombinant autoantigens identified by SEREX analysis, five Abs were identified as novel candidates for the acute exacerbation of IPF. Abs to annexin 1 were detected in 47 and 53% of the sera and bronchoalveolar lavage materials from patients with acute exacerbation of IPF. Some identical TCR Vβ genes were identified in sequential materials obtained at 1–3 mo in all 10 acute exacerbation IPF cases, suggesting that some infiltrating CD4-positive T cells sharing limited epitopes expand by Ag-driven stimulation during disease extension. The CDR3 region of these identical TCR Vβ genes showed high homology with the N-terminal portion of annexin 1, including in the HLA-DR ligand epitopes predicted by TEPITOPE analysis. By Western blotting analysis and observation of the CD4-positive T cell responses in bronchoalveolar lavage samples, the N-terminal portion of annexin 1 was cleaved and found to induce marked proliferative responses of CD4-positive T cells in three patients. Our study demonstrates that annexin 1 is an autoantigen that raises both Ab production and T cell response in patients with acute exacerbation of IPF, and that the N-terminal portion of annexin 1 plays some role in the pathogenesis of acute exacerbation in IPF patients.

Feasibility of combination chemotherapy with cisplatin and etoposide for haemodialysis patients with lung cancer.

Cancer chemotherapy for haemodialysis patients has never been established. To elucidate the feasibility of cisplatin-based combination chemotherapy for haemodialysis patients with lung cancer, a dose escalation study was conducted. Five haemodialysis patients with lung cancer were treated with cisplatin and etoposide. A starting dose of 40 mg m−2 of cisplatin on day 1 and 50 mg m−2 of etoposide on days 1, 3 and 5 were administered as the first course for the first patient. Membrane haemodialysis was regularly performed three times a week and soon after the completion of therapy. By monitoring toxicity and pharmacokinetics data, the dose was escalated course by course and patient by patient. Dose escalation was completed for the first two patients resulting in full-dose chemotherapy consisting of 80 mg m−2 of cisplatin on day 1 and 100 mg m−2 of etoposide on days 1, 3 and 5. Multiple courses of the full-dose chemotherapy were administered to the other three patients. Toxicity was manageable and tolerable for all. Pharmacokinetics data were comparable to those from patients with normal renal function, except for potential long-lasting higher levels of free platinum in the renal insufficiency group. In conclusion, this standard-dose combination chemotherapy was feasible even for haemodialysis patients.

Clinical phenotypes of COPD: results of a Japanese epidemiological survey.

Objective:  The Global Initiative for Obstructive Lung Disease characterizes COPD as airflow limitation caused by parenchymal destruction and/or small airway disease. This report characterizes the clinical features of these two phenotypes of COPD in Japan.

Antiproliferative action of metformin in human lung cancer cell lines

The oral antidiabetic agent metformin has anticancer properties, probably via adenosine monophosphate-activated protein kinase activation. In the present study, growth inhibition was assessed by a clonogenic and by a cell survival assay, apoptosis induction was assessed by Hoechst staining and caspase activities and cell cycle alteration after exposure to metformin, and the interaction of metformin with cisplatin in vitro were elucidated in four human lung cancer cell lines representing squamous, adeno-, large cell and small cell carcinoma. Clonogenicity and cell proliferation were inhibited by metformin in all the cell lines examined. This inhibitory effect was not specific to cancer cells because it was also observed in a non-transformed human mesothelial cell line and in mouse fibroblast cell lines. Inhibition of clonogenicity was observed only when the cells were exposed to metformin for a long period, (10 days) and the surviving fraction, obtained after inhibiting proliferation by increasing the dose, reached a plateau at approximately 0.1-0.3, indicating the cytostatic characteristics of metformin. Metformin induced significant apoptosis only in the small cell carcinoma cell line. A tendency of cell cycle accumulation at the G0/G1 phase was observed in all four cell lines. Cisplatin, in a dose-dependent manner, severely antagonized the growth inhibitory effect of metformin, and even reversed the effect in three cell lines but not in the adenocarcinoma cell line. The present data obtained using various histological types of human lung cancer cell lines in vitro illustrate the cytostatic nature of metformin and its cytoprotective properties against cisplatin.

Characterization of myofibroblasts in chronic thromboembolic pulmonary hypertension

BACKGROUND It has been generally accepted that chronic thromboembolic pulmonary hypertension (CTEPH) results from pulmonary embolism arising from deep vein thrombosis. An unresolved question regarding the etiology of CTEPH is why pulmonary thromboemboli are stable and resistant to effective anticoagulation. Recently non-resolving pulmonary thromboemboli in CTEPH have been shown to include myofibroblasts. This study investigates the cellular characteristics of myofibroblasts included in the organized thrombotic tissues of CTEPH. METHODS Organized thrombotic tissues of patients with CTEPH were obtained following pulmonary endarterectomy. We isolated cells from endarterectomized tissue from patients with CTEPH and identified them as endothelial-like cells and myofibroblast-like cells. RESULTS Myofibroblast-like cells were characterized as hyperproliferative, anchorage-independent, invasive and serum-independent. CONCLUSIONS Here we report the presence of active myofibroblast-like cells in endarterectomized tissue of CTEPH. We suggest that the formation of myofibroblasts with a high growth potential in the organized thrombotic tissues may be an important event in the pathobiology of this disease.

Endothelial-like cells in chronic thromboembolic pulmonary hypertension: crosstalk with myofibroblast-like cells

BackgroundChronic thromboembolic pulmonary hypertension (CTEPH) is characterized by intravascular thrombus formation in the pulmonary arteries.Recently, it has been shown that a myofibroblast cell phenotype was predominant within endarterectomized tissues from CTEPH patients. Indeed, our recent study demonstrated the existence of not only myofibroblast-like cells (MFLCs), but also endothelial-like cells (ELCs). Under in vitro conditions, a few transitional cells (co-expressing both endothelial- and SM-cell markers) were observed in the ELC population. We hypothesized that MFLCs in the microenvironment created by the unresolved clot may promote the endothelial-mesenchymal transition and/or induce endothelial cell (EC) dysfunction.MethodsWe isolated cells from these tissues and identified them as MFLCs and ELCs. In order to test whether the MFLCs provide the microenvironment which causes EC alterations, ECs were incubated in serum-free medium conditioned by MFLCs, or were grown in co-culture with the MFLCs.ResultsOur experiments demonstrated that MFLCs promoted the commercially available ECs to transit to other mesenchymal phenotypes and/or induced EC dysfunction through inactivation of autophagy, disruption of the mitochondrial reticulum, alteration of the SOD-2 localization, and decreased ROS production. Indeed, ELCs included a few transitional cells, lost the ability to form autophagosomes, and had defective mitochondrial structure/function. Moreover, rapamycin reversed the phenotypic alterations and the gene expression changes in ECs co-cultured with MFLCs, thus suggesting that this agent had beneficial therapeutic effects on ECs in CTEPH tissues.ConclusionsIt is possible that the microenvironment created by the stabilized clot stimulates MFLCs to induce EC alterations.

Chronic obstructive pulmonary disease and interstitial lung disease in patients with lung cancer

Background and objective:  Although lung cancer is frequently accompanied by COPD and interstitial lung disease (ILD), the precise coincidence of these diseases with lung cancer is not well understood. The objectives of this study were to determine the prevalence of abnormal CT and spirometric findings suggestive of COPD or ILD in a population of patients with untreated lung cancer, and to estimate the lung cancer risk in this population.

Tiotropium Bromide Attenuates Respiratory Syncytial Virus Replication in Epithelial Cells

Background: Respiratory syncytial virus (RSV) infection could be related to airway inflammation as well as exacerbation of chronic obstructive pulmonary disease (COPD). Tiotropium bromide decreases the frequency of exacerbation in patients with COPD; however, the mechanisms of tiotropium bromide to reduce the chances of exacerbation have not been defined. One potential mechanism could be that tiotropium bromide protects against RSV infection in epithelial cells. Objective: To examine whether tiotropium bromide affects RSV replication in HEp-2 cells. Methods: The supernatant titer of RSV was calculated by methylcellulose plaque assay after RSV innoculation. Intracellular RSV and ICAM-1 mRNA were measured by PCR. Syncytium formation was observed by light microscopy. Intracellular RSV fusion protein and RhoA protein were detected by Western blot analysis. Furthermore, RhoA activity, ICAM-1 expression and inflammatory cytokines in cultured supernatant were measured by binding assay, immunofluorescence staining and ELISA, respectively. Results: Tiotropium bromide decreased the supernatant titer of RSV, and it inhibited syncytium formation, RhoA activation and ICAM-1 expression. Moreover, it suppressed the production of IL-6 and IL-8 after RSV infection. Conclusions:The antiviral effects of tiotropium bromide regarding RSV replication are partly due to inhibition of RhoA activity and ICAM-1 expression. Tiotropium bromide decreases RSV replication and may modulate airway inflammation by reducing the production of inflammatory cytokines.