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Tobacco smoking causes a variety of smoking-related diseases, death, and economic damage. Despite targeted anti-smoking campaigns, tobacco-related deaths are expected to increase in Japan. We investigated the current state of non-cancerous lung diseases such as idiopathic interstitial pneumonias (IIPs), chronic obstructive pulmonary disease (COPD), and combined pulmonary fibrosis and emphysema (CPFE), which are known to be highly related to tobacco smoking.

The interaction of programmed cell death ligand 2 (PD-L2) with programmed cell death 1 is implicated in tumor immune escape. The regulation of PD-L2 expression in tumor cells has remained unclear, however. We here examined intrinsic and extrinsic regulation of PD-L2 expression in NSCLC.

Interstitial lung disease (ILD) is a risk factor for lung cancer development and is frequently observed in patients with lung cancer. Individuals with ILD have been excluded from most prospective clinical trials of lung cancer therapies because of the risk of ILD acute exacerbation. Thus, the optimal anticancer drug treatment for such patients has yet to be established. Tyrosine kinase inhibitors are avoided for the treatment of advanced non–small cell lung cancer (NSCLC) with ILD because of the concern of acute exacerbation, and information on the effects of immune-checkpoint inhibitors is limited in these patients. Only three prospective single-arm studies of cytotoxic chemotherapies for advanced lung cancer with ILD have been reported. Based on the results of these studies and those of retrospective analyses, carboplatin and either paclitaxel or nab-paclitaxel are often selected in daily clinical practice for patients with NSCLC and ILD, whereas platinum plus etoposide is selected for those with small cell lung cancer and ILD. Although the antitumor activity of first-line platinum-based chemotherapy appears similar in advanced lung cancer patients with and without ILD, its impact on overall survival of the former patients is limited. The risks and benefits of chemotherapy must therefore be carefully explained before treatment initiation, and careful follow-up is necessary for such patients, especially those with the usual interstitial pneumonia pattern, a risk factor for chemotherapy-related exacerbation. Prospective clinical studies with large patient populations are still required to establish the appropriate treatments for advanced lung cancer with ILD.

T helper 17 (Th17) cell development is programmed by the orphan nuclear receptor RORγt, but the underlying mechanism is not well understood. Nuclear receptor-mediated transcriptional activation depends on coactivators. Here, we show that steroid receptor coactivator-3 (SRC-3) critically regulates Th17 cell differentiation. Reduced incidence of experimental autoimmune encephalitis (EAE) associated with decreased Th17 cell generation in vivowas observed in mice with SRC-3 deletion specifically in T cells. In vitro, SRC-3 deficiency did not affect TGF-β/IL-6-induced Th17 cell generation but severely impaired pathogenic Th17 differentiation induced by IL-1/IL-6/IL-23. Microarray analysis revealed that SRC-3 not only regulates IL-17A but also IL-1R1 expression. SRC-3 bound to Il17aand Il1r1loci in a RORγt-dependent manner and was required for recruitment of the p300 acetyltransferase. Thus, SRC-3 is critical for RORγt-dependent gene expression in Th17 cell-driven autoimmune diseases.

Mechanisms underlying differentiation of embryonic hematopoietic stem/progenitor cells (HSPCs) remain unclear. In mouse, intra-aortic clusters (IACs) form in the aorta-gonad-mesonephros region and acquire HSPC potential after 9.5 days postcoitum (dpc). In this study we demonstrate that Twist1 is highly expressed in c-Kit+CD31+CD34+IACs, which are equivalent to embryonic HSPCs, compared with adult HSPCs. Progenitor activities of colony-forming unit (CFU) of granulocytes and macrophages, CFU of macrophages, burst-forming unit of erythroid, and B lymphopoiesis were impaired in IACs of Twist1−/−relative to wild-type embryos. Microarray analysis and real-time polymerase chain reaction showed downregulated expression of Myband Gata2transcripts in Twist1−/−IACs. Chromatin immunoprecipitation and promoter binding assays indicated that Twist1 directly binds the Myband Gata2promoters in 10.5-dpc IACs. We conclude that Twist1 is a novel transcriptional regulator of HSPC differentiation through direct binding to promoter regions of key regulators of the process.

Mechanisms underlying differentiation of embryonic hematopoietic stem/progenitor cells (HSPCs) remain unclear. In mouse, intra-aortic clusters (IACs) form in the aorta-gonad-mesonephros region and acquire HSPC potential after 9.5 days postcoitum (dpc). In this study we demonstrate that Twist1 is highly expressed in c-Kit+CD31+CD34+ IACs, which are equivalent to embryonic HSPCs, compared with adult HSPCs. Progenitor activities of colony-forming unit (CFU) of granulocytes and macrophages, CFU of macrophages, burst-forming unit of erythroid, and B lymphopoiesis were impaired in IACs of Twist1-/- relative to wild-type embryos. Microarray analysis and real-time polymerase chain reaction showed downregulated expression of Myb and Gata2 transcripts in Twist1-/- IACs. Chromatin immunoprecipitation and promoter binding assays indicated that Twist1 directly binds the Myb and Gata2 promoters in 10.5-dpc IACs. We conclude that Twist1 is a novel transcriptional regulator of HSPC differentiation through direct binding to promoter regions of key regulators of the process.

ABSTRACTAccumulation of profibrotic myofibroblasts is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF) pathogenesis. TGFB (transforming growth factor β) is one of the major profibrotic cytokines for myofibroblast differentiation and NOX4 (NADPH oxidase 4) has an essential role in TGFB-mediated cell signaling. Azithromycin (AZM), a second-generation antibacterial macrolide, has a pleiotropic effect on cellular processes including proteostasis. Hence, we hypothesized that AZM may regulate NOX4 levels by modulating proteostasis machineries, resulting in inhibition of TGFB-associated lung fibrosis development. Human lung fibroblasts (LF) were used to evaluate TGFB-induced myofibroblast differentiation. With respect to NOX4 regulation via proteostasis, assays for macroautophagy/autophagy, the unfolded protein response (UPR), and proteasome activity were performed. The potential anti-fibrotic property of AZM was examined by using bleomycin (BLM)-induced lung fibrosis mouse models. TGFB-induced NOX4 and myofibroblast differentiation were clearly inhibited by AZM treatment in LF. AZM-mediated NOX4 reduction was restored by treatment with MG132, a proteasome inhibitor. AZM inhibited autophagy and enhanced the UPR. Autophagy inhibition by AZM was linked to ubiquitination of NOX4 via increased protein levels of STUB1 (STIP1 homology and U-box containing protein 1), an E3 ubiquitin ligase. An increased UPR by AZM was associated with enhanced proteasome activity. AZM suppressed lung fibrosis development induced by BLM with concomitantly reduced NOX4 protein levels and enhanced proteasome activation. These results suggest that AZM suppresses NOX4 by promoting proteasomal degradation, resulting in inhibition of TGFB-induced myofibroblast differentiation and lung fibrosis development. AZM may be a candidate for the treatment of the fibrotic lung disease IPF.

Alectinib has shown marked efficacy and safety in patients with anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangement–positive NSCLC and a good performance status (PS). It has remained unclear whether alectinib might also be beneficial for such patients with a poor PS.

Despite the success of cancer immunotherapies such as programmed cell death–1 (PD-1) and PD-1 ligand 1 (PD-L1) inhibitors, patients often develop resistance. New combination therapies with PD-1/PD-L1 inhibitors are needed to overcome this issue. Bezafibrate, a ligand of peroxisome proliferator–activated receptor–γ coactivator 1α/peroxisome proliferator–activated receptor complexes, has shown a synergistic antitumor effect with PD-1 blockade in mice that is mediated by activation of mitochondria in T cells. We have therefore now performed a phase 1 trial (UMIN000017854) of bezafibrate with nivolumab in previously treated patients with advanced non–small cell lung cancer. The primary end point was the percentage of patients who experience dose-limiting toxicity, and this combination regimen was found to be well tolerated. Preplanned comprehensive analysis of plasma metabolites and gene expression in peripheral cytotoxic T cells indicated that bezafibrate promoted T cell function through up-regulation of mitochondrial metabolism including fatty acid oxidation and may thereby have prolonged the duration of response. This combination strategy targeting T cell metabolism thus has the potential to maintain antitumor activity of immune checkpoint inhibitors and warrants further validation.

Emphysema is thought to be a risk factor for lung cancer in smokers, with emphysematous bullae (EBs), which are believed to have the potential to give rise to lung cancer. The clinical characteristics of patients with lung cancer with EBs have remained incompletely defined, however.

Elucidating the prevalence of asthma and chronic obstructive pulmonary disease (COPD) is important for designing a public health strategy. Recent studies have discriminated a phenotype of COPD with variable airflow limitation (COPD-VAL) associated with asthma–COPD overlap syndrome. Its prevalence remains uncertain. The age and occupational distributions in the town of Hisayama and in Japan are nearly identical. Each disease׳s prevalence was estimated for the town׳s residents.

Chronic obstructive pulmonary disease (COPD) is highly prevalent worldwide. COPD is a treatable disease and it is important to identify COPD subjects, highlighting the need for an efficient screening measure. Although the COPD screening questionnaire (COPD Population Screener, COPD-PS) was developed as a screening tool, its validity is not clear in population-based studies. This study determines the validity of the COPD-PS in the general Japanese population.

The performance of scientifically and ethically valid prospective clinical trials is the only means by which to obtain reliable clinical evidence that can improve clinical practice and thus the outcome of patients with lung cancer. The efficacy of treatment for advanced lung cancer remains limited; many cooperative study groups for lung cancer have been established in Japan since 1990s, and they have completed several landmark investigator-initiated clinical trials. This review highlights eight active Japanese cooperative study groups for lung cancer and summarizes their achievements made through clinical trials. In addition to their benefits, the existence of multiple study groups for a single disease such as lung cancer presents several challenges including the provision of infrastructure to ensure the scientific integrity of trial results, the unnecessary duplication of effort and the wasting of limited resources, and the accrual and completion of large-scale phase III trials in the shortest possible time. Collaboration among Japanese cooperative groups has recently increased in order to overcome these challenges. Although institutional barriers to the performance of such large intergroup trials remain, further harmonization and collaboration among cooperative groups will be vital in allowing Japanese investigators to make further important contributions for the development of new lung cancer therapies.

A 59-year-old woman with epidermal growth factor receptor gene (EGFR) mutation–positive advanced lung adenocarcinoma was treated with afatinib after a diagnosis of chronic myelomonocytic leukemia (CMML). Twenty-one weeks later, she developed agranulocytosis, and CMML subsequently progressed to blast crisis. After complete remission of CMML, gefitinib was initiated; however, agranulocytosis recurred. This is the first reported case of both EGFR mutation–positive advanced non-small cell lung cancer with CMML, and of CMML blast crisis. Physicians should be aware of such risks and monitor EGFR-TKI–treated patients with myeloid neoplasms accordingly.

Background:Severe hypocalcemia sometimes develops during denosumab treatment for bone metastases from cancer and is, therefore, an important issue. However, limited information is available on the risk factors for hypocalcemia and the appropriate interval for monitoring serum calcium concentration. Objective:The present study aimed to identify the risk factors for grade ≥2 hypocalcemia and to investigate the time course of serum calcium concentrations in patients receiving denosumab for bone metastases from cancer. Method:The medical records of 66 cancer patients treated with denosumab between April 2012 and August 2013 were retrospectively reviewed. Result:Of the 66 enrolled patients, 11, 5, and 1 developed grade 1, 2, and 3 hypocalcemia, respectively. All 4 patients with a baseline estimated glomerular filtration rate (eGFR) of <30 mL/min developed hypocalcemia. Hypocalcemia occurred in only 20%, 24%, and 15% of patients with an eGFR of 30 to 59, 60 to 89, and ≥90 mL/min, respectively. Multivariate logistic regression analysis revealed that lower eGFR values (odds ratio, 1.72 per 10 mL/min decrease, P= 0.02) were significantly associated with grade ≥2 hypocalcemia. In 11 patients who developed hypocalcemia during the first treatment course, the mean calcium concentrations decreased from 9.8 mg/dL at baseline to 8.4 mg/dL during the first week and reached a nadir of 8.1 mg/dL during the second week. Conclusion:Our results support more frequent monitoring of serum calcium concentrations at baseline and during the first 2 weeks of treatment in patients receiving denosumab, especially those with an eGFR <30 mL/min.

Nursing and healthcare-associated pneumonia (NHCAP) is a relatively new condition that was recently defined by the Japanese Respiratory Society. Previous reports and guidelines have not thoroughly investigated the adverse prognostic factors and validity of the selection criteria for NHCAP. The purpose of this research was to clarify the adverse prognostic factors of NHCAP and investigate the validity of the selection criteria with respect to patient deaths.

BLT1 is a high-affinity receptor for leukotriene B4 (LTB4) that is a potent lipid chemoattractant for myeloid leukocytes. The role of LTB4/BLT1 axis in tumor immunology, including cytokine-based tumor vaccine, however, remains unknown. We here demonstrated that BLT1-deficient mice rejected subcutaneous tumor challenge of GM-CSF gene-transduced WEHI3B (WGM) leukemia cells (KO/WGM) and elicited robust antitumor responses against second tumor challenge with WEHI3B cells. During GM-CSF–induced tumor regression, the defective LTB4/BLT1 signaling significantly reduced tumor-infiltrating myeloid-derived suppressor cells, increased the maturation status of dendritic cells in tumor tissues, enhanced their CD4+ T-cell stimulation capacity and migration rate of dendritic cells that had phagocytosed tumor-associated antigens into tumor-draining lymph nodes, suggesting a positive impact on GM-CSF–sensitized innate immunity. Furthermore, KO/WGM mice displayed activated adaptive immunity by attenuating regulatory CD4+ T subsets and increasing numbers of Th17 and memory CD44hiCD4+ T subsets, both of which elicited superior antitumor effects as evidenced by adoptive cell transfer. In vivo depletion assays also revealed that CD4+ T cells were the main effectors of the persistent antitumor immunity. Our data collectively underscore a negative role of LTB4/BLT1 signaling in effective generation and maintenance of GM-CSF–induced antitumor memory CD4+ T cells.

Zinc is an essential element required for the cell metabolism, including gene transcription, signal transduction, immunity, and apoptosis. The pathophysiological role of zinc in asthma, however, is not entirely clear. Mast cells have been implicated in atopic asthma, and zinc deprivation has been reported to reduce mast cell activation. Here, we investigate the effects of a zinc chelator, N,N,N',N'-tetrakis (2-pyridyl-methyl) ethylenediamine (TPEN), on asthmatic responses in mouse models of ovalbumin (OVA)-induced airway hyperresponsiveness and allergic airway inflammation.

Experiments using cancer cell lines have revealed that CHFR methylation correlates with sensitivity to microtubule inhibitors. However, this marker may not benefit actual clinical cases because it is difficult to detect CHFR methylation without surgically resected samples. Thus, a more easily accessible marker that correlates with sensitivity to microtubule inhibitors might be useful in NSCLC, especially in advanced cases. In this study, we show that EGFR gene status and smoking are predict the efficacy of treatment with microtubule inhibitors in NSCLC. Chemosensitivity to paclitaxel and six other chemotherapeutic agents was evaluated using the succinate dehydrogenase inhibition (SDI) method in 69 NSCLC cases, consisting of 48 adenocarcinomas, 20 squamous cell carcinomas and 1 large cell carcinoma. Next, we evaluated the relationships between CHFR or EGFR status and clinicopathologic data. Methylation-specific PCR (MSP) and direct DNA sequencing were performed to detect aberrant methylation of CHFR and EGFR mutations, respectively. CHFR gene promoter methylation and EGFR gene mutation were observed in 11 cases (15.9%) and 7 cases (10.1%), respectively. The SDI method revealed that CHFR gene methylation was significantly related to high sensitivity to paclitaxel (p

Doxycycline is a semi-synthetic tetracycline, which was invented and clinically developed in the early 1960s. Doxycycline works by inhibiting protein synthesis and it is also bacteriostatic. Doxycycline is highly effective against all of the common pathogens that cause upper respiratory tract infections. Doxycycline is particularLY effective for the treatment of atypical pneumonia due to Mycoplasma, Chlamydia and Legionella. Recently, doxycycline has been reported to have a biological function apart from its antimicrobial function. Doxycycline is known to inhibit the release of reactive oxygen species, while also inducing apoptosis, decreasing neutrophil chemotaxis and inhibiting matrix metalloproteinases. Regarding animal models, doxycycline is able to attenuate lung inflammation caused by several agents. Recently, several clinical trials using doxycycline have also been reported. In this review, we provide a comprehensive, yet concise analysis of the two different functions of doxycycline, while particularly focusing on respiratory diseases.

Purpose:Amrubicin is a novel 9-aminoanthracycline. This multicenter phase II study was conducted to evaluate the efficacy and safety of amrubicin in patients with non-small-cell lung cancer (NSCLC).Purpose:Amrubicin is a novel 9-aminoanthracycline. This multicenter phase II study was conducted to evaluate the efficacy and safety of amrubicin in patients with non-small-cell lung cancer (NSCLC).

ABSTRACTThe administration of doxycycline prior to bleomycin in mice attenuated pulmonary fibrosis. Bronchoalveolar neutrophil influx and gelatinase activity, but not caseinolytic activity, were attenuated by doxycycline. Established fibrosis was not affected by doxycycline. Thus, doxycycline might be useful for slowing down pulmonary fibrosis by biological activity other than antibacterial activity.

Microsomal epoxide hydrolase 1 (EPHX1) plays an important role in both the activation and detoxification of tobacco-derived carcinogens. Polymorphisms at exons 3 and 4 of the EPHX1gene have been reported to be associated with variations in EPHX1 activity. The aim of this study is to review and summarize the available molecular epidemiologic studies of lung cancer and EPHX1.

The aim of this study is to summarize the available molecular epidemiologic studies of lung cancer and metabolic genes, such as NAD(P)H quinone reductase 1 (NQO1) and myeloperoxidase (MPO). NQO1 plays a dual role in the detoxification and activation of procarcinogens whereas MPO has Phase I activity by converting lipophilic carcinogens into hydrophilic forms. Variant genotypes of both NQO1 Pro187 Ser and MPO G-463A polymorphisms may be related to low enzyme activity. The Pro/Ser and Ser/Ser genotypes combined of NQO1 was significantly associated with decreased risk of lung cancer in Japanese [random effects odds ratio (OR) = 0.70, 95% confidence interval (CI) = 0.56—0.88] among whom the variant allele is common. The variant genotype of MPO was associated with decreased risk of lung cancer among Caucasians (random effects OR = 0.70, 95% CI = 0.47--1.04). Gene-environment interactions in both polymorphisms may be hampered by inaccurate categorization of tobacco exposure. Evidence on gene-gene interactions is extremely limited. As lung cancer is a multifactorial disease, an improved understanding of such interactions may help identify individuals at risk for developing lung cancer. Such a study should include larger sample size and other polymorphisms in the metabolism of tobacco-derived carcinogens and address interactions with smoking status. The effects of polymorphisms are best represented by their haplotypes. In future studies on lung cancer, the development of haplotype-based approaches will facilitate the evaluation of haplotypic effects, either for selected polymorphisms physically close to each other or for multiple genes within the same drug-metabolism pathway.

T helper 2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, play a critical role in allergic asthma. These cytokines transmit signals through the Janus kinase/signal transducer and activator of transcription (STAT) and the Ras–extracellular signal-regulated kinase (ERK) signaling pathways. Although the suppressor of cytokine signaling (SOCS) family proteins have been shown to regulate the STAT pathway, the mechanism regulating the ERK pathway has not been clarified. The Sprouty-related Ena/VASP homology 1–domain-containing protein (Spred)-1 has recently been identified as a negative regulator of growth factor–mediated, Ras-dependent ERK activation. Here, using Spred-1–deficient mice, we demonstrated that Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness, without affecting helper T cell differentiation. Biochemical assays indicate that Spred-1 suppresses IL-5–dependent cell proliferation and ERK activation. These data indicate that Spred-1 negatively controls eosinophil numbers and functions by modulating IL-5 signaling in allergic asthma.

Apoptosis plays a major role in homeostasis as well as proliferation and differentiation. The important role of epithelial and endothelial cell apoptosis in lung injury and repair has been demonstrated. Modulating signaling molecules of apoptosis may be effective treatment against lung injury.

We describe the rare case of a Japanese male with multifocal lupus vulgaris that preceded asymptomatic pulmonary tuberculosis and adult T‐cell leukemia/lymphoma (ATL). He visited our hospital with multiple reddish plaques and erythema of 4–12 months duration. A skin biopsy revealed non‐caseating epithelioid granulomas. Mycobacterium tuberculosiswas detected by polymerase chain reaction (PCR)‐hybridization from a skin biopsy specimen and was also isolated from a culture of the skin biopsy sample. The result of chest roentogenography was compatible with pulmonary tuberculosis. In addition, the diagnosis of ATL was based upon the presence of atypical lymphocytes with convoluted nuclei in his peripheral blood and a positive anti‐ATL antibody reaction. Cases of cutaneous tuberculosis presenting with unusual clinical features may be on the increase, accompanying the spread of tuberculosis in immunosuppressed patients, including those with ATL and acquired immunodeficiency syndrome (AIDS).

Pulmonary fibrosis is a common response to injuries to the lung. Although there are various initiating factors or causes, the terminal stages are characterized by proliferation and progressive accumulation of connective tissue replacing normal functional parenchyma. The pathogenesis of pulmonary fibrosis includes endothelial and epithelial cell injury, production of inflammatory cells and their mediators, and fibroblast activation. Conventional therapy consisting of glucocorticoids or cytotoxic drugs is usually ineffective in preventing progression of the disease. Apoptosis plays a major role in homeostasis as well as proliferation and differentiation. Failure to clear unwanted cells by apoptosis will prolong the inflammation because of the release of their toxic contents. In contrast, excessive apoptosis may cause diseases. DNA damage and apoptosis in lung epithelial and endothelial cells have been reported in acute lung injury and diffuse alveolar damage, as well as pulmonary fibrosis. Protecting epithelial and endothelial cells from injury may be effective and physiological treatment.

Bleomycin is a radiomimetic antitumor agent with unique genotoxic properties. 1-nitropyrene is an environmental mutagen and carcinogen that undergoes both oxidative and reductive metabolism. In the present study, hepatocellular carcinomas were induced in male A/J mice by the intraperitoneal injection of bleomycin (120 mg/kg) followed by the intraperitoneal administration of 1-nitropyrene (total dose: 1,575 mg/kg). In order to understand the mechanism by which these two compounds induce hepatocellular carcinomas, the incidence and spectrum of mutations in the K-ras proto-oncogene in these hepatocellular carcinomas were analyzed. The hepatocellular carcinomas were induced by the administration of bleomycin and 1-nitropyrene were evaluated for point mutations in exon 1 and exon 2 of the K-ras gene by the polymerase chain reaction and a sequencing analysis. No mutation was found in the hotspots regions of the K-ras gene codon 12, 13, or 61. However, the codon 64 of the K-ras gene mutation was identified in 10 of 10 (100%) hepatocellular carcinomas. All mutations showed the same pattern, which was TAC-CAC transition. Codon 64 of the K-ras gene mutation may thus play an important role in the induction of hepatocellular carcinomas by bleomycin in the existence of 1-nitropyrene. As far as we know, this is the first report of a codon 64 mutation in the K-ras gene in a chemically induced tumor. Teratogenesis Carcinog. Mutagen. Suppl 1:161–170, 2003. © 2003 Wiley-Liss, Inc.

Background:Previous studies have reported a synergistic effect between irinotecan and cisplatin. We have conducted a phase I trial combining these agents to find the optimal dose of irinotecan in combination with a fixed dose of cisplatin. Methods:Patients with advanced solid tumors, aged ≤75 years, performance status ≤2, and adequate organ function were enrolled in this study. They were treated at 4-week intervals with irinotecan plus 20 mg/m2 cisplatin on days 1, 8, and 15. The starting dose of irinotecan of 40 mg/m2 was escalated in 10 mg/m2 increments until a maximum dose of 90 mg/m2 was reached. Results:The recommended dose for phase II studies is 90 mg/m2 of irinotecan and 20 mg/m2 of cisplatin on days 1, 8, and 15. Overall response to the chemotherapy was 35% (95% confidential interval, 19.2–54.6%). Conclusion:This combination seems to be active against lung cancer with acceptable toxicity. A phase II study is now ongoing.

The effect of genetic polymorphism of DNA repair enzyme on the DNA adduct levels was evaluated in this study. We explored the relationship between polymorphism in the nucleotide excision repair enzyme XPD and DNA adduct levels in lymphocytes. Lymphocyte DNA adducts were measured by a 32

Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase) uses PPias an energy donor and requires free Mg2+for enzyme activity and stability. To determine the catalytic domain, we analyzed charged residues (Asp253, Lys261, Glu263, Asp279, Asp283, Asp287, Asp723, Asp727, and Asp731) in the putative PPi-binding site and two conserved acidic regions of mung bean V-PPase by site-directed mutagenesis and heterologous expression in yeast. Amino acid substitution of the residues with alanine and conservative residues resulted in a marked decrease in PPihydrolysis activity and a complete loss of H+transport activity. The conformational change of V-PPase induced by the binding of the substrate was reflected in the susceptibility to trypsin. Wild-type V-PPase was completely digested by trypsin but not in the presence of Mg-PPi, while two V-PPase mutants, K261A and E263A, became sensitive to trypsin even in the presence of the substrate. These results suggest that the second acidic region is also implicated in the substrate hydrolysis and that at least two residues, Lys261and Glu263, are essential for the substrate-binding function. From the observation that the conservative mutants K261R and E263D showed partial activity of PPihydrolysis but no proton pump activity, we estimated that two residues, Lys261and Glu263, might be related to the energy conversion from PPihydrolysis to H+transport. The importance of two residues, Asp253and Glu263, in the Mg2+-binding function was also suggested from the trypsin susceptibility in the presence of Mg2+. Furthermore, it was found that the two acidic regions include essential common motifs shared among the P-type ATPases.

CTLs recognize 8- to 10-mer peptides on MHC class I molecules. Recent studies have shown that human CTLs kill autologous tumor cells in an HLA-restricted and peptide-specific manner, and that artificial pep- tides can stimulate tumor-specific CTLs both in vitroand in vivo. Accordingly, several human clinical trials using such peptides are ongoing worldwide. In such methods, the amount of peptide-MHC complexes that remain on the cell surface of APCs after peptide administration is crucial, because CTL activation depends on the number of ligated TCRs and co-stimulation. However, it remains uncertain how many peptide-MHC complexes are reconstituted and remain on live cells after peptide administration. We herein examined the binding affinities of five HLA-A∗0201 restricted peptides—four TAAs and one HIV antigen—to HLA-A∗0201 molecules and their decay rates on a live B cell line using tandem mass spectrometry. Our experiments showed that nearly 105peptide-MHC complexes per cell could be reconstituted on a cell surface by pulsing a high dose of peptide even if the binding affinities were intermediate or low. However, the decay rates observed for these pep- tide-MHC complexes on a B cell line were faster than previously estimated.

Although the effects of polychlorinated biphenyls (PCBs) on human lung carcinogenesis are suggested from the massive PCBs poisoning that occurred in Japan designated “Yusho,” the detailed molecular mechanism are unknown. 1 nitropyrene (1-NP), an ubiquitous and abundant environmental pollutant, is known to be detected in lung tissues derived from patients with lung cancer in Japan, and its relation to lung carcinogenesis is also suggested. We investigated the effects of PCBs (Kanechlor-400) on 1-NP-induced lung tumorigenesis in A/J mice. PCBs were administered intraperitoneally followed by ip injection of 1-NP. The lung lesions were examined 18 weeks after the final treatment. In the control group, no neoplastic lesions were induced in the lung. In the PCB group, preneoplastic lesions such as hyperplasia and adenoma were induced in 2/10 (20%) mice. In 1-NP group and in PCB + 1-NP group, lung lesions including adenocarcinoma were induced in 16/20 (80%) and 13/13 (100%) mice, respectively. Both the number and the size of tumors in PCB + 1-NP group were significantly greater than those in 1-NP group. K-ras gene mutation, CAA to CGA in codon 61 or GGT to GAT in codon 12, was found in either 1-NP group or PCB + 1-NP group but not in the PCB group. There was no difference in the pattern of K-ras mutation associated with the pretreatment with PCBs. These results suggest that PCBs promote 1-NP-induced lung tumorigenesis and may support, at least in part, the mechanism of the high incidence of lung cancer in patients with Yusho. Teratogenesis Carcinog. Mutagen. 21:395–403, 2001. © 2001 Wiley-Liss, Inc.

AbstractBackground:Bronchial artery embolization (BAE) is a well-accepted and widely used treatment modality for the management of massive and recurrent hemoptysis. However, few reports have previously investigated the long-term results. Objectives:To investigate the prognosis of patients with hemoptysis who had undergone BAE. Methods:Twenty-two patients with hemoptysis underwent BAE. The underlying diseases included bronchiectasis in 9, aspergillosis in 3, chronic bronchitis in 2, idiopathic bronchial bleeding in 4, and other diseases in 4. The follow-up period ranged from 25 to 88 months (median 47 months). Results:After the initial BAE, 11 of 22 (50%) patients had re-bleeding (5 patients with hemoptysis and 6 patients with minor hemosputa). Among them, 1 patient suffered from recurrent massive hemoptysis and died from airway obstruction within 1 month after BAE. In addition, 10 of these 11 (90.9%) patients experienced recurrent airway bleeding within 3 years after BAE. Recurrent cases of hemoptysis were seen in 6 of 22 patients (27.3%) within 3 years and no case recurred later than 3 years after BAE. A recurrence of hemoptysis was frequently seen in patients with either bronchiectasis or pulmonary-bronchial artery (P-B) shunt. Although BAE is an effective treatment for the immediate control of hemoptysis, 5 of the patients experienced recurrent bleeding in the long-term follow-up. Conclusions:It is important to follow-up such patients until 3 years after initial BAE, especially when either ectatic changes of the bronchi on a CT scan or a P-B shunt on angiographic findings are detected.Copyright © 2000 S. Karger AG, Basel

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones.

Because aryl hydrocarbon hydroxylase (AHH) is considered to be responsible for the activation of benzo(a)pyrene and other polyaromatic hydrocarbons in cigarette smoke to carcinogens, it is important to examine CYP1A1 (AHH) activity in the determination of susceptibility to lung cancer. We investigated AHH activity in peripheral mitogen-treated lymphocytes in 108 lung cancer patients and 95 healthy control individuals. Non-induced AHH activity was detectable in all the samples. AHH inducibility (3-methyIcholanthrene-inducednon-induced AHH activity) showed a very wide interindividual variation as well as non-induced AHH activity. No significant associations were found between adjusted AHH activity and histologic type of tumor among lung cancer patients. Adjusted AHH inducibility of genotype C geometric mean and 95 confidence interval (CI); 15.56 and 11.69-20.71 in MspI polymorphism was significantly higher than those of the other two genotypes (P0.0001), while no significant difference was observed between genotypes A (4.76 and 3.82-5.93) and B (5.60 and 4.57-6.86). On the other hand, non-induced AHH activity of genotype ValVal (0.121 and 0.082-0.178 pmolmin106cells) in isoleucinevaline (He-Val) polymorphism was significantly higher than those of genotypes IleIle (0.042 and 0.034-0.052 pmolmin106cells) and IleVal (0.040 and O.O3O-O.O53 pmolmin106 cells) (P<0.0001). Even after controlling for age, cigarettes smoked per day and season of the year, high AHH inducibility (7.0 < versus 0 < ≤ 3.0 OR and 95 CI, 12.4 and 2.88-53.4) was an independent risk factor for lung cancer. The data indicate that high AHH inducibility may strongly associate With the Susceptibility to lung Carcinogenesis.

Abstract: Plant growth results from the division, enlargement and specialization of cells. The two processes of the enlargement and the differentiation of cells are not spatially separated in plant tissue. We focus our attention here on the enlargement and elongation of cells. In most cases, growing plant cells contain a large central vacuole. The acid growth theory is based on the space-filling function of the large vacuole. The active transport systems in the vacuolar membrane are essential for maintenance of high osmotic pressure and for the expansion of the vacuole. The secondary active transport systems of the vacuole for sugars and ions are driven by the proton-motive force which is generated by the vacuolar H+-ATPase and H+-translocating inorganic pyrophosphatase. In this review, the relationship between cell elongation and these enzymes of the vacuolar membrane is emphasized.

The effects of exogenous and endogenous granulocyte colony-stimulating factor (G-CSF) on invasion by cancer cells were studied, using lung cancer cell lines that produce G-CSF (NCI-H157) and lines that do not (PC-9 and NCI-H23). The invasive capacity of NCI-H157 cells was 26- to 27-fold higher than that of PC-9 and NCI-H23 cells. The invasiveness of PC-9 cells was stimulated by exogenous G-CSF, while that of NCI-H157 cells was not. Antibodies against G-CSF blocked the stimulation of PC-9 cell invasiveness by exogenous G-CSF. Anti G-CSF antibodies also inhibited invasion by NCI-H157 cells in the absence of exogenous G-CSF. These results suggest that endogenous and exogenous G-CSF both stimulate invasion by lung cancer cells.

Abstract Group C adenovirus is latent in human tissues and can malignantly transform cells. The purpose of this study was to investigate the association between this virus and lung cancer. We investigated latent adenoviral infection using the nested polymerase chain reaction and in situ hybridization in transbronchial biopsy specimens from patients with small-cell lung cancer and non-small-cell lung cancer. The polymerase chain reaction was performed on DNA extracts with two sets of primers directed at a 261-base-pair target sequence of the E1A region of the adenoviral genome. In situ hybridization was performed on histological sections using DNA representing the entire adenovirus type 5 genome. E1A target DNA was present in 11 (31%) of 35 cases of small-cell lung cancer but in none of the 40 cases of non-small-cell lung cancer (P&lt;0.01). Of the 11 cases found positive by PCR, 8 were positive for adenovirus DNA by in situ hybridization. Adenovirus was prominent in tumor cells in 5 of the 8 cases, and in normal epithelial cells in the 3 remaining cases. Adenovirus DNA was not detected by in situ hybridization in specimens in which E1A DNA was not detected by the polymerase chain reaction. Small-cell lung cancer has mutations or deletions in the p53 and retinoblastoma genes more frequently than are found in non-small-cell lung cancer. Therefore, we speculate that adenovirus infection might participate in the pathogenesis of SCLC by producing mutation in these genes, rather than by inhibiting the function of these proteins.

Abstract: Purpose: A phase II trial combining cisplatin, carboplatin and etoposide was conducted in previously untreated patients with stage IIIB and IV small-cell lung cancer, in an attempt to increase response rates and prolong survival. Methods: Previously untreated patients with small-cell lung cancer, with measurable disease, aged ≤ 72 years, performance status ≤ 2, and adequate hematologic, hepatic and renal function were enrolled in the study. They were treated with 80 mg/m2 cisplatin on day 1, 100 mg/m2 carboplatin on days 2, 3 and 8, and 50 mg/m2 etoposide on days 1, 2, 3 and 8. Results: A total of 46 patients (20 with stage IIIB and 26 with stage IV disease) were enrolled in the study. A total of 186 courses of chemotherapy were given, and the dose was reduced in 27 courses (15%). The chemotherapy was repeated for four or more courses in 30 patients. There were 10 complete responses and 32 partial responses, for a total response rate of 91% (95% confidence interval, 79% to 98%). The median survival time and 2-year survival rates were 18 months and 22% for stage IIIB disease, and 14 months and 15% for stage IV disease. Major side effects were hematologic: leukopenia, anemia, and thrombocytopenia of grade 3 or more occurred in 48%, 46%, and 43% of patients, respectively. Conclusions: The three-drug regimen of cisplatin, carboplatin and etoposide is feasible and active against small-cell lung cancer.

In seven patients with nonmalignant disease, we bronchoscopically observed various types of bronchial arterial lesions that may have caused hemorrhage. Five of the seven showed a bulging lesion, and the other two demonstrated an intrabronchial mass. We also examined these seven patients using selective bronchial arteriography. Herein we report our comparative study of the bronchoscopic findings and the bronchial arteriograms of these bronchial arterial lesions. The bulge observed in bronchoscopy corresponded either to an aneurysm or to a hypervascular area in the bronchial arteriogram. The mass lesions corresponded to a hypervascular area or a focal dilatation in the bronchial arteriogram. The intrabronchial lesions observed bronchoscopically either disappeared or were significantly diminished by bronchial arterial embolization for management of the hemorrhage. A histologic examination in two patients who underwent surgery revealed vascular lesions corresponding to the intrabronchial lesions in bronchoscopy. The results of this comparative study have important application in the bronchoscopic examination of bronchial arterial lesions in patients with hemoptysis.