Moritaka Suga

Quantitative Analysis of Mycobacterial and Propionibacterial DNA in Lymph Nodes of Japanese and European Patients with Sarcoidosis

ABSTRACT The cause(s) of sarcoidosis is unknown. Mycobacterium spp. are suspected in Europe and Propionibacterium spp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes of Propionibacterium acnes, Propionibacterium granulosum, Mycobacterium tuberculosis, Mycobacterium avium subsp. paratuberculosis, and Escherichia coli (as the control) were counted by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was found in no sarcoid sample. M. tuberculosis was found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum were far more than those of M. tuberculosis. P. acnes or P. granulosum was found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in such samples were less than those in sarcoid samples. Propionibacterium spp. are more likely than Mycobacteria spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.

Interstitial lung disease in Japanese patients with lung cancer: a cohort and nested case-control study.

RATIONALE Interstitial lung disease (ILD) occurs in Japanese patients with non-small cell lung cancer (NSCLC) receiving gefitinib. OBJECTIVES To elucidate risk factors for ILD in Japanese patients with NSCLC during treatment with gefitinib or chemotherapy. METHODS In a prospective epidemiologic cohort, 3,166 Japanese patients with advanced/recurrent NSCLC were followed for 12 weeks on 250 mg gefitinib (n = 1,872 treatment periods) or chemotherapy (n = 2,551). Patients who developed acute ILD (n = 122) and randomly selected control subjects (n = 574) entered a case-control study. Adjusted incidence rate ratios were estimated from case-control data by odds ratios (ORs) with 95% confidence intervals (CIs) using logistic regression. Crude (observed) incidence rates and risks were calculated from cohort data. MEASUREMENTS AND MAIN RESULTS The observed (unadjusted) incidence rate over 12 weeks was 2.8 (95% CI, 2.3-3.3) per 1,000 person-weeks, 4.5 (3.5-5.4) for gefitinib versus 1.7 (1.2-2.2) for chemotherapy; the corresponding observed naive cumulative incidence rates at the end of 12-week follow-up were 4.0% (3.0-5.1%) and 2.1% (1.5-2.9%), respectively. Adjusted for imbalances in risk factors between treatments, the overall OR for gefitinib versus chemotherapy was 3.2 (1.9-5.4), elevated chiefly during the first 4 weeks (3.8 [1.9-7.7]). Other ILD risk factors in both groups included the following: older age, poor World Health Organization performance status, smoking, recent NSCLC diagnosis, reduced normal lung on computed tomography scan, preexisting chronic ILD, concurrent cardiac disease. ILD-related deaths in patients with ILD were 31.6% (gefitinib) versus 27.9% (chemotherapy); adjusted OR, 1.05 (95% CI, 0.3-3.2). CONCLUSIONS ILD was relatively common in these Japanese patients with NSCLC during therapy with gefitinib or chemotherapy, being higher in the older, smoking patient with preexisting ILD or poor performance status. The risk of developing ILD was higher with gefitinib than chemotherapy, mainly in the first 4 weeks.

CD44 cleavage induced by a membrane-associated metalloprotease plays a critical role in tumor cell migration

CD44 is a cell surface receptor for hyaluronate, a component of the extracellular matrix (ECM). Although CD44 has been implicated in tumor invasion and metastasis, the molecular mechanisms remain to be elucidated. Here we find that CD44 expressed in cancer cells is cleaved at the membrane-proximal region of the ectodomain and the membrane-bound cleavage product can be detected using an antibody against the cytoplasmic domain of CD44. Furthermore, we report that CD44 cleavage is mediated by a membrane-associated metalloprotease expressed in cancer cells. A tissue inhibitor of metalloproteases-1 (TIMP-1), as well as metalloprotease inhibitors, inhibit CD44 cleavage in the cell-free assay. Contrary, serine protease inhibitors enhance CD44 cleavage, and the enhancement can be prevented by pretreatment with a metalloprotease inhibitor. Thus, CD44 cleavage is regulated by an intricate balance between some proteases and their inhibitors. Interestingly, treatment with the metalloprotease blocker 1,10-phenanthroline, which strongly prevent the CD44 cleavage, suppressed RERF-LC-OK lung cancer cell migration on a hyaluronate substrate, but not on several other substrates. These results suggest that CD44 cleavage plays a critical role in an efficient cell-detachment from a hyaluronate substrate during the cell migration and consequently promotes CD44-mediated cancer cell migration. Our present data indicate that CD44, not only ECM per se, is one of the targets of pericellular proteolysis involved in tumor invasion and metastasis.

Breast Cancer Resistance Protein Impacts Clinical Outcome in Platinum-Based Chemotherapy for Advanced Non-Small Cell Lung Cancer

Purpose: The purpose of this study was to investigate the relationship between the level of expression of ATP-binding cassette (ABC) transporter proteins, and response to chemotherapy and prognosis in advanced non-small cell lung cancer (NSCLC). Experimental Design: Expression of ABC transporter proteins, including P-glycoprotein, multidrug resistance protein (MRP) 1, MRP2, MRP3, and breast cancer resistance protein (BCRP), was examined immunohistochemically in 72 formalin-fixed tumor samples from untreated stage IIIB or IV NSCLC patients. All of the patients received platinum-based chemotherapy. Response to chemotherapy, progression-free survival (PFS), and overall survival were compared in relation to expression of each of the ABC transporter proteins and clinicopathological factors. Results: Expression of P-glycoprotein, MRP1, and MRP3 was not significantly associated with response to chemotherapy or survival. MRP2 expression was associated with overall survival (P = 0.002) but not with response to chemotherapy and PFS. By contrast, the response rate to chemotherapy of patients with BCRP-negative tumors was 44%, as opposed to 24% in patients with BCRP-positive tumors. Response rate was lower in BCRP-positive tumors, although this difference was not statistically significant (P = 0.08). BCRP-positive patients had also shorter PFS (P = 0.0003) and overall survival (P = 0.004) than BCRP-negative patients. Multivariate analysis confirmed BCRP status as an independent variable related to PFS (P = 0.001). Conclusions: Positive immunostaining for BCRP appears to be a predictor of survival in patients with advanced NSCLC. These findings indicate that BCRP may serve as a molecular target for reducing drug resistance to chemotherapy in advanced NSCLC patients.

Activation of Human Matrix Metalloproteinases by Various Bacterial Proteinases

Matrix metalloproteinases (MMPs) are zinc-containing proteinases that participate in tissue remodeling under physiological and pathological conditions. To test the involvement of bacterial proteinases in tissue injury during bacterial infections, we investigated the activation potential of various bacterial proteinases against precursors of MMPs (proMMPs) purified from human neutrophils (proMMP-8 and −9) and from human fibrosarcoma cells (proMMP-1). Each proMMP was subjected to treatment with a series of bacterial proteinases at molar ratios of 0.01-0.1 (bacterial proteinase to proMMP), and activities of MMPs generated were determined. Among six different bacterial proteinases, thermolysin family enzymes (family M4) such as Pseudomonas aeruginosa elastase, Vibrio cholerae proteinase, and thermolysin strongly activated all three proMMPs via limited proteolysis to generate active forms of the MMPs. N-terminal sequence analysis of the active MMPs revealed that cleavage occurred at the Val82-Leu83 and Thr90-Phe91 bonds of proMMP-1 and proMMP-9, respectively, which are located near the N terminus of the catalytic domain of MMPs. In contrast, Serratia 56-kDa proteinase and Pseudomonas alkaline proteinase, both of which are classified as members of the serralysin subfamily of zinc metalloproteinases (family M10), and Serratia 73-kDa thiol proteinase did not evidence proteolytic processing or activation of proMMP-1, −8, and −9 under these experimental conditions. These results indicate that bacterial proteinases may play an important role in tissue destruction and disintegration of extracellular matrix at the site of infections.

Prognostic value of circulating KL-6 in idiopathic pulmonary fibrosis

Objective:  Circulating levels of KL‐6, a high MW glycoprotein (MUC1 mucin), are elevated in a majority of patients with a number of interstitial lung diseases, including idiopathic pulmonary fibrosis (IPF). However, KL‐6 levels vary from patient to patient. The aim of the present study was to determine whether the serum KL‐6 level at the time of diagnosis predicts prognosis in IPF.

Expression of constitutively activated EGFRvIII in non-small cell lung cancer.

The epidermal growth factor receptor (EGFR) variant type III (variously called EGFRvlll, de2–7 EGFR or ΔGFR) has an in‐frame deletion of the extracellular domain and is found in numerous types of human tumors. Since EGFRvlll has been reported to be tumorspecific and has oncogenic potential, it is being investigated as a potential therapeutic target. Because the cell‐specific expression of EGFRvlll in lung has not been well documented, we examined the expression of EGFRvlll in 76 non‐small cell lung cancers (NSCLCs) and 10 non‐neoplastic lung tissues by immunohistochemistry using a new monoclonal antibody specific for this variant receptor. We found a higher incidence (30 of 76, 39%) of enhanced EGFRvlll expression in NSCLC than previously described. Interestingly, the presence of EGFRvlll was also observed in several normal tissue components of lung (e.g., normal bronchial epithelium). Given the high prevalence of EGFRvlll in NSCLC, a newly developed phospho‐specific (activated) EGFR antibody was employed for immunohistochemical analysis that permitted visualization of activated EGFR and/or EGFRvlll in tumors. This study presents evidence, for the first time, that EGFRvlll expressed in human tumors is phosphorylated and hence activated. Our results suggest that the sustained activation of EGFRvlll is implicated in the pathogenesis of NSCLC and thus EGFRvlll is a potential therapeutic target in this challenging disease. (Cancer Sci 2003; 94: 50–56)

Free Radicals in Viral Pathogenesis: Molecular Mechanisms Involving Superoxide and NO

Abstract The importance of free radical molecular species in the pathogenesis of various viral diseases has been increasingly recognized in recent years. Oxygen radicals such as superoxide (O2-) and hydroxyl radical (OH) have been implicated as possible pathogenic molecules in viral disease pathogenesis. Much attention has been given to another simple inorganic radical [nitric oxide (NO)] in the hosts defense mechanism and pathogenesis of virus infection. The NO synthesis pathway, in particular, the inducible isoform of NO synthase (INOS), is expressed in different viral diseases via induction of proinflammatory cytokines such as interferon-γ. iNOS produces an excessive amount of NO for a long time compared with other constitutive isoforms of NOS (i.e., neuronal NOS and endothelial NOS). Recent studies indicate that NO and O2 - are produced in excess during the hosts defense responses against various intruding microbes. Reactive nitrogen oxide species such as peroxynitrite (ONOO-) and NOx (NO2, and N2O3) are produced in biological systems through the reaction of NO with either O2 - or O2. Among these reactive nitrogen species, ONOO-and its biological actions are of considerable interest in that ONOO- causes oxidation and nitration of amino acid residues of proteins and guanine of DNA, lipid peroxidation, and DNA cleavage. Because the ONOO- is formed via a diffusion-limited fast reaction of NO and O2 -, it may be a dominant nitrogen oxide species during the hosts defense reactions, when both NO and O2 - are produced in excess. Thus, understanding the role of NO and oxygen radical generation in virus infections will provide insight into not only viral pathogenesis but also the host-pathogen interaction in microbial infections at a molecular level.

Chronic hypersensitivity pneumonitis in Japan: A nationwide epidemiologic survey☆☆☆★

BACKGROUND Pulmonary fibrosis inevitably develops in patients with chronic hypersensitivity pneumonitis (HP). OBJECTIVE We conducted a nationwide epidemiologic study in Japan to evaluate the frequency and clinical characteristics of chronic HP. METHODS This report is on 36 cases of chronic HP, including 10 patients with summer-type HP, 5 patients with home-related HP, 7 patients with bird fanciers lung, 5 patients with isocyanate-induced HP, 4 patients with farmers lung, and 5 patients with other types of chronic HP. Chronic HP was further subgrouped into 2 types: one type of patients were first seen with chronic disease (9 patients), and the other type became chronic with fibrosis after repeated acute episodes (27 patients). RESULTS The upper lung field was frequently involved in chronic HP (17%). Ground-glass opacities were observed in 57% and air space consolidation in 30% of the patients. Honeycombing was apparent in 37%. Twenty-six of 28 patients had antibodies to the presumptive antigens. Five of 8 patients with chronic HP were positive for antigen-induced lymphocyte proliferation. In 2 cases patients did not have detectable antibodies to causative antigens, although antigen-induced lymphocyte proliferation was detectable. The ratio of CD4 to CD8 in BAL lymphocytes was lowest in isocyanate-induced HP (mean 0.22) and tended to be high in farmers lung and bird fanciers lung. Granulomas were observed in 39% and Masson bodies in 42% of specimens on histologic examination. Administration of prednisolone was effective in 58% of patients. CONCLUSIONS The insidious form of chronic HP has probably been misdiagnosed as idiopathic pulmonary fibrosis when a good history was not taken and immunologic (especially antigen-induced lymphocyte proliferation) and BAL testing were not counted.

Regulated CD44 Cleavage under the Control of Protein Kinase C, Calcium Influx, and the Rho Family of Small G Proteins

CD44 is a cell surface receptor for several extracellular matrix components and is implicated in tumor cell invasion and metastasis. Our previous studies have shown that CD44 expressed in cancer cells is proteolytically cleaved at the extracellular domain through membrane-associated metalloproteases and that CD44 cleavage plays a critical role in CD44-mediated tumor cell migration (Okamoto, I., Kawano, Y., Tsuiki, H., Sasaki, J., Nakao, M., Matsumoto, M., Suga, M., Ando, M., Nakajima, M., and Saya, H. (1999)Oncogene 18, 1435–1446). In the present study, we first demonstrate rapid degradation of the membrane-tethered CD44 cleavage product through intracellular proteolytic pathways, and it occurs only after CD44 extracellular cleavage. To address the mechanisms regulating CD44 cleavage at the extracellular domain, we show that 12-O-tetradecanoylphorbol 13-acetate (TPA) and the calcium ionophore ionomycin rapidly enhance metalloprotease-mediated CD44 cleavage in U251MG cells via protein kinase C-dependent and -independent pathways, respectively, suggesting the existence of multiple distinct pathways for regulation of CD44 cleavage. Concomitant with TPA-induced CD44 cleavage, TPA treatment induces redistribution of CD44 and ERM proteins (ezrin, radixin, and moesin) to newly generated membrane ruffling areas. Treatment with lysophosphatidic acid, which is known to activate the Rho-dependent pathway, inhibits TPA-induced CD44 redistribution and CD44 cleavage. Furthermore, overexpression of Rac dominant active mutants results in the redistribution of CD44 to the Rac-induced ruffling areas and the enhancement of CD44 cleavage. These results suggest that the Rho family proteins play a role in regulation of CD44 distribution and cleavage.