Parathyroid hormone-related protein (PTHRP) is responsible for production of bone metastasis, but not visceral metastasis, by human small cell lung cancer SBC-5 cells in natural killer cell-depleted SCID mice.

We previously established an osteolytic bone metastasis model with multiorgan dissemination in natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice using human small cell lung cancer cells (SBC-5), which highly express the parathyroid hormone-related protein (PTHrP). In our present study, we evaluated the role of PTHrP on bone metastasis by SBC-5 cells using anti-PTHrP neutralizing antibody (Ab). Anti-PTHrP Ab did not affect the proliferation or cytokine production of SBC-5 cells in vitro. Repeated intravenous injection with anti-PTHrP Ab inhibited the formation of bone metastasis in a dose-dependent manner, while the same treatment had no significant effect on the metastasis to visceral organs (lung, liver, kidney and lymph node). In addition, treatment with anti-PTHrP Ab improved the elevated serum calcium level, associated with inhibition of osteolytic bone metastasis, suggesting that anti-PTHrP Ab inhibited bone metastasis via suppression of bone resorption probably by neutralizing PTHrP. These findings suggest that PTHrP is essential for bone metastasis, but not visceral metastasis, by small cell lung cancer SBC-5 cells.


Long-term Follow-up of Pulmonary Function in Bronchial Asthma Patients Treated with Pranlukast

Clinical studies have shown that pranlukast, a selective cysteinyl leukotriene antagonist, is effective for bronchial asthma. In the present paper, we retrospectively analyzed long-term asthma control by pranlukast treatment in patients treated with inhaled corticosteroids. We analyzed medical records and asthma diaries of 21 patients (9 males, 12 females) (52.1 +/- 3.5 years of age) with bronchial asthma who experienced increase of more than 10 L/min in peak expiratory flow in the first 4 weeks of treatment with pranlukast (450 mg/day) and were subsequently treated with pranlukast for more than 1 year. They all received inhaled corticosteroids (400-1600 microg/day of beclomethasone dipropionate or equivalent). We examined clinical control in terms of time course of self-monitored peak expiratory flow. During the analyzed period, the dose of inhaled corticosteroids was tapered in 4 patients, constant in 15 patients and increased in 2 patients. In 19 patients treated with unchanged or tapered dose of inhaled corticosteroids, improvement in the increase of mean PEF at 4-week treatment was maintained for 1 year. No difference in the add-on effect of pranlukast was observed in patients treated with less than 800 microg and more than or equal to 800 microg of inhaled corticosteroids. Four patients underwent reduction of inhaled corticosteroids in the analyzed period and PEF was well-maintained and even increased by pranlukast treatment. In 11 patients in whom data for 3 years were available, the improvement in PEF persisted for 3 years. Although the present investigation is a retrospective analysis, these data may suggest that pranlukast has no tachyphylaxis and its effect continues for more than 1 year.


Stimulation of human bronchial epithelial cells by IgE-dependent histamine-releasing factor

An IgE-dependent histamine-releasing factor (HRF p23; also known as translationally controlled tumor protein or p23) stimulates the release of histamine, IL-4, and IL-13 from a subpopulation of highly allergic donor basophils. It has also been shown to act as a chemoattractant for eosinophils. To elucidate novel functions of HRF p23 in airway inflammation, we examined the effects of human recombinant HRF p23 (hrHRF) on bronchial epithelium and found that hrHRF stimulated the secretions of IL-8 and granulocyte/macrophage colony-stimulating factor by both primary cultures of human bronchial epithelial cells and BEAS-2B cells. In response to hrHRF, these cells induced IL-8 mRNA expression within 4 h. H2O2, but not IL-1 beta or tumor necrosis factor-alpha, stimulated secretion of HRF p23 by BEAS-2B cells, suggesting that oxidative stress may trigger the release of HRF p23 from bronchial epithelial cells. Bronchoalveolar lavage (BAL) from healthy volunteers contained only trivial or undetectable amounts of HRF p23. Significantly higher amounts of HRF p23 were recovered from BAL fluid taken from asthmatic patients, and the amounts of HRF p23 were further elevated in patients with idiopathic eosinophilic pneumonia. Our results demonstrate for the first time that HRF p23 can stimulate nonimmune epithelium. HRF p23 derived from bronchial epithelial cells may regulate complex cytokine networks in eosinophil-dependent inflammation of the human airway.

American Journal of Physiology. Lung Cellular and Molecular Physiology


Increased macrophage inflammatory protein-1α and -1β in BAL fluid of bronchiolitis obliterans organizing pneumonia

CC chemokines are mainly chemotactic for monocytes and lymphocytes. The aim of this study was to evaluate the involvement of the CC chemokines, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, in the pathogenesis of bronchiolitis obliterans organizing pneumonia (BOOP). The concentrations of MIP-1alpha and MIP-1beta in BAL fluid (BALF) obtained from patients with BOOP (n = 13) and control patients (CP, n= 18) were measured by enzyme-linked immunosorbent assay. MIP-1alpha in BALF was significantly higher in patients with BOOP (mean +/- SD; 123.8 +/- 98.0 pg/mL) than in CP (62.5 +/- 46.1 pg/mL). Significantly higher MIP-1beta was also detected in patients with BOOP (51.6 +/- 72.5 pg/mL) than in CP (6.4 +/- 3.7 pg/mL). The concentration of MIP-1alpha significantly correlated with the percentage of lymphocytes in BALF, and the concentration of MIP-1beta significantly correlated with the numbers of lymphocytes, neutrophils and eosinophils in BALF. Both MIP-1alpha and MIP-1beta in BALF were decreased after corticosteroid therapy and this was accompanied by decreased lymphocytes in BALF. This study suggests that MIP-1alpha and MIP-1beta may play important roles in the recruitment of immuno-inflammatory cells into the lungs, and may contribute to the pathogenesis of BOOP.


Combined therapy with a new bisphosphonate, minodronate (YM529), and chemotherapy for multiple organ metastases of small cell lung cancer cells in severe combined immunodeficient mice.

Lung cancer in the advanced stage frequently metastasizes to multiple organs, including the liver, lungs, lymph nodes, and bone. Bisphosphonates have been widely used to treat osteolytic bone metastasis in the past years; however, many studies have implicated that a single use of bisphosphonates could not prolong the survival of patients. In the present study, using a multiple-organ metastasis model of human lung cancer cells, we examined the effect of combined therapy with a new bisphosphonate (YM529) and etoposide (VP-16). Human small cell lung cancer (SBC-5) cells i.v. inoculated into natural killer cell-depleted severe combined immunodeficient mice metastasized to multiple organs, including the lungs, liver, kidneys, lymph nodes, and bone. SBC-5-bearing mice were treated with YM529 and/or VP-16 and sacrificed 5 weeks after tumor cell inoculation. Bone metastasis was assessed by X-ray photographs, and visceral metastasis was evaluated macroscopically. The number of osteoclasts in the bone lesions was examined by tartrate-resistant acid phosphatase staining. Monotherapy with YM529 suppressed the production of bone metastases, but not visceral metastasis. Histological analyses revealed that the number of osteoclasts in bone lesions was lower in YM526-treated mice, compared with control mice. VP-16 inhibited both bone metastasis and visceral (lung and liver) metastasis. However, neither YM529 alone nor VP-16 alone significantly prolonged the survival of SBC-5-bearing mice. Combined use of YM529 and VP-16 further inhibited the production of bone metastasis and significantly prolonged survival. Combined therapy with bisphosphonate and chemotherapy may be useful for small cell lung cancer patients with multiple organ metastases including bone metastasis.


Add-on Effects of Suplatast Tosilate in Bronchial Asthma Patients Treated With Inhaled Corticosteroids

Th2 cytokines play an important role in the pathogenesis of asthma. In the present study, we investigated the effect of suplatast tosilate, a selective Th2 cytokine inhibitor, on asthma control, in terms of subjective symptoms and pulmonary function in patients treated with inhaled corticosteroids. Thirty-eight patients with bronchial asthma being treated with inhaled corticosteroids were given suplatast tosilate (100 mg three times daily) for 12 weeks, in a multicenter setting. During the study period, other medications were continued. Morning and evening peak expiratory flow, asthma symptoms, blood eosinophil count and serum IgE levels were monitored. Suplatast tosilate treatment was associated with a significant improvement in mean morning peak expiratory flow (from 295 L/min to 348 L/min, P < 0.01) and evening peak expiratory flow (from 313 L/min to 357 L/min, P < 0.01). The mean daily variation in peak expiratory flow was significantly reduced (from 11.6% to 7.3%, P < 0.01) by suplatast tosilate treatment. The greatest improvement in peak expiratory flow was observed in patients whose blood eosinophil counts were decreased by suplatast tosilate treatment. Treatment with suplatast tosilate improved pulmonary function in patients with bronchial asthma. Our results suggest the therapeutic effects observed may occur through suppression of eosinophilic inflammation.


To determine the significance of CD13/aminopeptidase N in collagen vascular diseases (CVD), we examined its activity and expression in sera and disease sites of patients with CVD. Significantly higher aminopeptidase activity was detected in bronchoalveolar lavage fluid from patients with interstitial lung diseases due to rheumatoid arthritis (RA), polymyositis/dermatomyositis (PM/DM), systemic sclerosis (SSc), and Sjögren's syndrome than from control subjects. Increased aminopeptidase activity and increased expression of CD13/aminopeptidase N protein were found in alveolar macrophages from CVD patients with interstitial lung diseases. Significantly higher aminopeptidase activity was detected in pleural effusions from patients with systemic lupus erythematosus (SLE) than in transudate effusions. The mean aminopeptidase activity in synovial fluids from RA patients was significantly higher than from patients with osteoarthritis. The mean value of serum aminopeptidase activity was significantly higher in patients with SLE, RA, SSc, and PM/DM than in normal subjects. This study suggests that the activity of CD13/aminopeptidase N, locally produced in the disease site, is a useful marker for CVD and that CD13/aminopeptidase N may have an important role in the pathogenesis of CVD.


Adenoviral interleukin-12 gene transduction into human bronchial epithelial cells:up-regulation of pro-inflammatory cytokines and its prevention by corticosteroids

One of the potential effects of IL-12 is to restore Th1/Th2 balance. Therefore, we investigated the possibility of developing a system for local delivery of IL-12 into the airways by examining protein expression in a human bronchial epithelial cell line (BEAS-2B) after adenoviral IL-12 gene transduction. The effects of dexamethasone on the gene-modified cells were also examined. Adenoviral vectors AxCAegfp and Ax1CIhp40ip35 were used to transduce enhanced green fluorescence protein and IL-12 genes, respectively, into BEAS-2B cells. Wild-type and IL-12 gene-transduced BEAS-2B cells were then incubated with or without dexamethasone, and concentrations of IL-12, IFN-gamma, IL-6, IL-8, granulocyte macrophage-colony stimulating factor and chemokines (TARC and RANTES) in the supernatant were measured by ELISA. IL-12 receptor expression was analysed by flow cytometry and RT-PCR. The efficiency of transgene expression in BEAS-2B cells at a multiplicity of infection of 30 was approximately 80%. Gene-modified BEAS-2B cells produced biologically active IL-12, regardless of dexamethasone treatment. While IL-12 gene transduction led to increased production of IL-6 and IL-8 by BEAS-2B cells, expressions of these proteins were suppressed by dexamethasone. Addition of exogenous IL-12 failed to augment BEAS-2B cell IL-6 and IL-8 production, and IL-12 receptor expression by BEAS-2B cells was not detected. Our findings suggest that adenoviral IL-12 gene transduction may be effective in inducing IL-12 expression in the airways, and could be a potential approach in the management of bronchial asthma.


The expression of chemokine receptor CXCR3 : relevance to disease activity of rheumatoid arthritis


Molecular mechanisms of angiogenesis in non-small cell lung cancer, and therapeutics targeting related molecules.

Angiogenesis, neovascularization from pre-existing vasculature, is necessary to supply oxygen and nutrition for tumor growth in both primary and distant organs. It consists of sprouting and non-sprouting (the enlargement, splitting, and fusion of pre-existing vessels) processes, and both can occur concurrently. Growth of solid tumors, including non-small cell lung cancer (NSCLC), is usually dependent on angiogenesis, which is regulated by complex mechanisms involving various angiogenesis-related molecules. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), one of the most potent angiogenic molecules, regulates both angiogenesis and vascular permeability, and hence promotes tumor progression and development of malignant pleural effusions in NSCLC. Signals via epidermal growth factor receptor (EGFR) promote not only the tumor cell cycle, but also the process of angiogenesis. Therefore, these molecules are potential targets for anti-tumor vasculature therapy. Many agents targeting tumor vasculature have been developed, and several compounds have shown anti-tumor potential in preclinical studies. Their efficacy against NSCLC is currently being evaluated in clinical trials.


Increased binding and chemotactic capacities of PDGF-BB on fibroblasts in radiation pneumonitis

Although pulmonary fibrosis is a frequent and serious consequence of radiotherapy for thoracic malignant diseases such as lung cancer, the pathogenesis of this radiation-induced lung disorder remains unclear. To clarify the mechanisms underlying radiation pneumonitis and pulmonary fibrosis, we investigated the expression of platelet-derived growth factor receptor (PDGFR) on fibroblasts obtained from irradiated rat lungs and on control fibroblasts. Whole lungs of male Wistar rats were irradiated with a single dose of 15 Gy, and lung fibroblasts were isolated at 4 weeks after the irradiation. The chemotactic response of irradiated lung fibroblasts to PDGF-BB was significantly higher than that of control lung fibroblasts, whereas there was no significant difference between irradiated lung fibroblasts and control lung fibroblasts in the response to PDGF-AA. Receptor binding assay showed more specific binding sites for PDGF-BB on irradiated lung fibroblasts than on control lung fibroblasts, and the displacement of (125)I-labeled PDGF binding to fibroblasts by unlabeled PDGF showed that (125)I-labeled PDGF-BB was displaced by PDGF-BB but not by PDGF-AA. These results suggest that the increased binding sites for PDGF-BB on irradiated lung fibroblasts correspond mainly to PDGFRB. Scatchard analysis of the saturation data demonstrated an approximately twofold increase both in the number of PDGF-BB binding sites and in the binding affinity in irradiated lung fibroblasts compared to that in control lung fibroblasts. Those results suggest that the increased chemotactic response of irradiated lung fibroblasts to PDGF-BB is related to the overexpression of PDGFRB, which may have an important role in the pathogenesis of radiation-induced pneumonitis and pulmonary fibrosis.


Genome-Wide Analysis of Organ-Preferential Metastasis of Human Small Cell Lung Cancer in Mice

Although a number of molecules have been implicated in the process of cancer metastasis, the organ-selective nature of cancer cells is still poorly understood. To investigate this issue, we established a metastasis model in mice with multiple organ dissemination by i.v. injection of human small cell lung cancer (SBC-5) cells. We analyzed gene-expression profiles of 25 metastatic lesions from four organs (lung, liver, kidney, and bone) using a cDNA microarray representing 23,040 genes and extracted 435 genes that seemed to reflect the organ specificity of the metastatic cells and the cross-talk between cancer cells and microenvironment. Furthermore, we discovered 105 genes that might be involved in the incipient stage of secondary-tumor formation by comparing the gene-expression profiles of metastatic lesions classified according to size (<1 or >2 mm) as either "micrometastases" or "macrometastases." This genome-wide analysis should contribute to a greater understanding of molecular aspects of the metastatic process in different microenvironments, and provide indicators for new strategies to predict and prevent metastasis.


Differential effects of IL-12 on the generation of alloreactive CTL mediated by murine and human dendritic cells: a critical role for nitric oxide

We examined the mechanisms involved in interleukin (IL)-12-mediated suppression of cellular immunity in mice using allogeneic mixed leukocyte reaction (MLR) stimulated by dendritic cells (DCs) in vitro and compared the effect of IL-12 on MLR in mice and humans. Although IL-12 stimulated human MLR, the addition of IL-12 or interferon-gamma (IFN-gamma) resulted in a dose-dependent suppression of MLR in mice. The treatment with N(G)-monomethyl-L-arginine (L-NMMA) completely abrogated IL-12- and IFN-gamma-mediated suppression of MLR in mice. Furthermore, IL-12 enhanced the alloreactive cytolytic T lymphocyte (CTL) induction in human MLR, whereas the addition of L-NMMA was required to generate alloreactive CTLs in the presence of IL-12 in mice. Nitric oxide (NO) was detected only in mouse MLR. Murine DCs could produce NO, but neither human CD34(+) cell- nor monocyte-derived DCs produced a detectable amount of NO. These results suggest that NO produced by DCs might play an important role in IL-12-mediated immune suppression in mice but not in humans.


A report of two bronchioloalveolar carcinoma cases which were rapidly improved by treatment with the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 ("Iressa").

Bronchioloalveolar carcinoma (BAC), a form of pulmonary adenocarcinoma, presents unique clinical features, such as endobronchial spread and bronchorrhea in advanced stages. The prognosis for BAC patients in advanced stages is poor, as is the case for patients with other non-small-cell lung cancer (NSCLC) types, because of low susceptibility to conventional chemotherapy. Recently, an orally active, selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI), ZD1839 ("Iressa"), has been investigated in phase II clinical studies (IDEAL 1 and IDEAL 2) as monotherapy against chemotherapy-refractory NSCLC, and provided clinically significant antitumor activity. In this study, we examined the therapeutic efficiency of ZD1839 in chemotherapy-refractory BAC patients with bronchorrhea. Two female BAC patients with bronchorrhea were treated once daily with ZD1839 (250 mg/day). In both cases, serous sputum production was dramatically reduced within 3 days of starting the treatment, and hypoxia and radiographic signs of bilateral lung consolidation were visibly improved within 7 days. Following more than 8 months of treatment, no evidence of recurrence or severe adverse events has been observed. These results suggest that this selective EGFR-TKI, ZD1839, may be a powerful agent for treatment of chemotherapy-refractory BAC patients with bronchorrhea.


A third-generation matrix metalloproteinase (MMP) inhibitor (ONO-4817) combined with docetaxel suppresses progression of lung micrometastasis of MMP-expressing tumor cells in nude mice.

The lung is the common target organ of hematogenous metastasis that restricts the prognosis of cancer patients. MMPs play a pivotal role in metastasis by promoting tumor invasion and angiogenesis; therefore, a large number of MMPIs have been developed. Our purpose was to determine the therapeutic efficacy of a selective-spectrum MMPI, ONO-4817 (inhibits MMP-2 and MMP-9 but not MMP-1), against established lung micrometastasis in combination with a cytotoxic anticancer drug, DOC, in a nude mouse model. Human non-small cell lung cancer PC14PE6 (adenocarcinoma) or H226 (squamous cell carcinoma) cells, expressing MMP-2, MMP-9 and/or MMP-1, were injected i.v. into nude mice on day 0. Mice received a single injection of DOC on day 7 (after establishment of micrometastasis) and/or ONO-4817 mixed with food from day 7 to the end of experiments. Monotherapy with ONO-4817 or DOC inhibited formation of lung metastasis by PC14PE6 and H226 cells. In addition, combined use of ONO-4817 with DOC significantly suppressed the tumor burden of H226 and PC14PE6 cells in the lung and prolonged the survival of PC14PE6-bearing mice compared to ONO-4817 or DOC alone. These therapies did not affect the body weight or food intake of tumor-bearing mice. FIZ revealed that lung lesions, but not nontumor parenchyma of the lung, expressed gelatinolytic activity and that treatment with ONO-4817 abrogated the gelatinolytic activity in lung lesions. These results suggest that the combined use of MMPIs with cytotoxic anticancer drugs may be helpful in the control of established lung micrometastasis by tumor cells expressing MMPs.


Soluble Fas in malignant pleural effusion and its expression in lung cancer cells

Soluble Fas (sFas) has the ability to block Fas-mediated apoptosis, suggesting that sFas at tumor sites might inhibit tumor cell-killing by immune effector cells. We examined the sFas level in pleural effusion associated with lung cancer. The level of sFas in malignant pleural effusion was significantly higher than those in transudate and tuberculous pleural effusion. There was no significant difference in the sFas concentration among various histological types of lung cancer. The cytotoxicity mediated by anti-Fas agonistic antibody against Jurkat cells was inhibited by the addition of malignant pleural effusion, being inversely correlated with the sFas concentration. When Fas expression was examined using flow cytometry, eight of ten (80%) lung cancer cell lines expressed cell surface Fas. On the other hand, sFas protein and mRNA were detected in six of ten (60%) lung cancer cell lines, but there was no correlation between Fas and sFas expression. Furthermore, although the expressions of Fas and sFas were clearly detected in tumor cells derived from malignant effusion, the sFas expression was down-regulated in an in vitro culture. These results suggest that sFas in malignant pleural effusion is at least in part produced by lung cancer cells, and might play a role in local immunosuppression by tumor cells.


Multifunctional interleukin-1beta promotes metastasis of human lung cancer cells in SCID mice via enhanced expression of adhesion-, invasion- and angiogenesis-related molecules.

We examined whether interleukin-1 (IL-1), a multifunctional proinflammatory cytokine, progresses or regresses metastasis of lung cancer. Exogenous IL-1beta enhanced expression of various cytokines (IL-6, IL-8, and vascular endothelial growth factor (VEGF)) and intracellular adhesion molecule-1 (ICAM-1) by A549, PC14, RERF-LC-AI, and SBC-3 cells expressing IL-1 receptors. A549 cells transduced with human IL-1beta-gene with the growth-hormone signaling-peptide sequence (A549/IL-1beta) secreted a large amount of IL-1beta protein. Overexpression of IL-1beta resulted in augmentation of expression of the cytokines, ICAM-1, and matrix metalloproteinase-2 (MMP-2). A549/IL-1beta cells intravenously inoculated into severe combined immunodeficiency (SCID) mice distributed to the lung more efficiently and developed lung metastasis much more rapidly than did control A549 cells. Treatment of SCID mice with anti-IL-1beta antibody inhibited formation of lung metastasis by A549/IL-1beta cells. Moreover, A549/IL-1beta cells inoculated in the subcutis grew more rapidly, without necrosis, than did control A549 cells, which produced smaller tumors with central necrosis, suggesting involvement of angiogenesis in addition to enhanced binding in the high metastatic potential of A549/IL-1beta cells. Histological analyses showed that more host-cell infiltration, fewer apoptotic cells, more vascularization, and higher MMP activity were observed in tumors derived from A549/IL-1beta cells, compared with tumors derived from control A549 cells. These findings suggest that IL-1beta facilitates metastasis of lung cancer via promoting multiple events, including adhesion, invasion and angiogenesis.


Angiogenesis in the progression of lung cancer.


Effect of human airway trypsin-like protease on intracellular free Ca2+ concentration in human bronchial epithelial cells

It has been shown that human airway trypsin-like protease (HAT) is localized in human bronchial epithelial cells (HBEC), and trypsin activates protease-activated receptor-2 (PAR-2). Activation of PAR-2 activates G-protein followed by an increase of intracellular free Ca2+, [Ca2+]in. This study was undertaken to clarify whether HAT can activate PAR-2 in HBEC or not. RT-PCR showed that HAT mRNA is expressed in HBEC, and PAR-2 mRNA is the most strongly expressed of the known PARs in HBEC. Both PAR-2 agonist peptide (PAR-2 AP) and HAT increased [Ca2+]in in HBEC in a biphasic fashion; a prompt, sharp increase (peak I) and a sustained low plateau (peak II). PAR-2 AP over 100-200 microM and HAT over 200-300 mU/ml (0.08-0.12 microM) induced both peak I and II, and PAR-2 AP below 100 microM and HAT below 200 mU/ml induced only peak II. Both PAR-2 AP-induced and HAT-induced peak I were induced by Ca2+ mobilization from intracellular stores, because they appeared even in Ca2+-free medium. Both PAR-2 AP-induced and HAT-induced peak II were induced by an influx of extracellular Ca2+, because they were abolished in Ca2+-free medium. The Ca2+ response to HAT was desensitized by exposure of HBEC to PAR-2 AP. These results indicate that HBEC have a functional PAR-2, and HAT regulates cellular functions of HBEC via activation of PAR-2.

The Journal of Medical Investigation : JMI


Inhaled steroid therapy and hospitalization for bronchial asthma:trend in Tokushima University Hospital

With the recognition that airway inflammation is present even in patients with mild bronchial asthma, therapy with inhaled corticosteroids is now indicated in various stages of patients. In the present article, we retrospectively examined the prescriptions for inhaled corticosteroids and other drugs for the treatment of outpatients with bronchial asthma at Tokushima University Hospital. We also analyzed asthma control in these patients, in terms of the incidence of emergency consultations and hospitalizations due to asthma exacerbations. To analyze the recent trend, the patients observed from 1998 to 2000 (recent years) were included, and for control purpose, those in 1990 and 1991 (earlier years) were also included. The percentage of patients treated with inhaled corticosteroids remarkably increased in recent years (mean; 81.3%) compared to earlier years (mean; 23.5%). In contrast, the usage of oral corticosteroids, oral xanthine derivatives, beta-adrenergic receptor agonists and anti-allergic agents tended to decrease in the 10 years period. After the introduction in 1995, considerable patients up to 25% have been treated with anti-leukotrienes. Emergency consultations decreased in recent years (mean; 0.18/patient/year) compared to earlier years (mean; 0.79/patient/year). Emergency hospitalizations also decreased in recent years (mean; 0.043/patient/year) compared to earlier years (mean; 0.23/patient/ year). In the present study, spread of inhaled corticosteroid therapy and decline in incidence of emergency consultation and hospitalization were simultaneously observed at Tokushima University Hospital, and the former has, at least in part, a contribution to the latter.


三尖弁感染症心内膜炎および化膿性脊椎炎に併発した敗血症性肺塞栓症の1例(症例報告)

The journal of the Japanese Respiratory Society


Phosphorylation, but not overexpression, of epidermal growth factor receptor is associated with poor prognosis of non-small cell lung cancer patients

Epidermal growth factor receptor (EGFR) is commonly overexpressed in non-small cell lung cancer (NSCLC) and its tyrosine kinase phosphorylation is thought to be an ideal target in the treatment of patients with NSCLC. In the present study, we examined surgically obtained specimens from a series of 36 NSCLC patients for expression of EGFR, phosphorylated EGFR (p-EGFR), and HER2 by immunohistochemistry, and also examined the correlation with clinical characteristics. The positive rate of EGFR, p-EGFR, and HER2 was 97.2%, 44.4%, and 88.6%, respectively, and the overexpression rate was 80.6%, 0.0%, and 27.8%, respectively. EGFR overexpression and phosphorylation were seen at almost the same rate in each histological type of squamous and nonsquamous cell carcinoma (squamous vs. nonsquamous; 78.6% vs. 81.8% for EGFR, 35.7% vs. 50.0% for p-EGFR), while HER2 overexpression was seen less frequently in squamous cell carcinoma than in nonsquamous cell carcinoma (0.0% vs. 45.5%, P = 0.003). Univariate analysis revealed that EGFR overexpression was related to good performance status (P = 0.038) but not related to EGFR phosphorylation. EGFR phosphorylation was correlated to short time to progression (TTP) (P = 0.002) and poor prognosis (P = 0.002), although EGFR overexpression, HER2 overexpression, or EGFR-HER2 coexpression were not correlated to TTP or survival. Bivariate analysis showed EGFR phosphorylation was related to short TTP and poor prognosis both in early and advanced stages. Multivariate analyses confirmed that clinical stage, performance status, and p-EGFR expression were independently associated with increasing risk of short TTP and poor prognosis. These results suggest that phosphorylation, but not overexpression, of EGFR may be an important predictor for clinical outcome of NSCLCs.


A novel bisphosphonate minodronate (YM529) specifically inhibits osteolytic bone metastasis produced by human small-cell lung cancer cells in NK-cell depleted SCID mice.

In the present study, we examined the effects of a newly developed bisphosphonate, minodronate (YM529), on osteolytic bone metastasis caused by lung cancer. Human small-cell lung cancer (SBC-5) cells, injected intravenously into natural killer cell-depleted SCID mice, produced radiologically detectable bone metastasis by day 18 and macroscopically visible visceral metastases (lung, liver, kidney, systemic lymph node) by day 35. Prophylactic treatment with YM529 on day 1 significantly inhibited the formation of osteolytic bone metastasis evaluated on X-ray photographs in a dose-dependent manner. In addition, treatment with YM529 after establishment of bone metastasis (on day 21) also inhibited bone metastasis, although the treatment was more effective when started earlier. Single administration was as effective as repeated treatment, suggesting a sustained inhibitory effect of YM529 on bone metastasis. YM529 reduced the number of osteoclasts in the bone metastatic lesions in vivo, but had no effect on the proliferation or cytokine production of SBC-5 cells in vitro. These results suggest that YM529 is a potent inhibitor of bone metastasis of human lung cancer, probably by suppressing osteoclastic bone resorption. In contrast, treatment with YM529 had no effect on visceral metastasis, even if started on day 1, and did not prolong the survival of the mice. Therefore, development of a combined modality is necessary for prolonging the survival of small-cell lung cancer patients with multiple-organ metastasis.


Organ Heterogeneity of Host-derived Matrix Metalloproteinase Expression and Its Involvement in Multiple-Organ Metastasis by Lung Cancer Cell Lines

Cancer metastasis is tightly regulated by the interaction of tumor cells and host organ microenvironments. Matrix metalloproteinases (MMPs), produced by both tumor cells and host stromal cells, play a central role in tumor invasion and angiogenesis. We determined whether metastatic potential of lung cancer to multiple organs is dependent solely on the expression of MMPs by tumor cells, using two metastasis models of human lung cancer cell lines expressing various levels of MMPs and a MMP inhibitor (ONO-4817). In the lung metastasis model, tumor cells (PC14, PC14PE6, H226, A549) inoculated i.v. into nude or SCID mice metastasized only in the lung. In the multiple-organ metastasis model, tumor cells (RERF-LC-AI, SBC-3/DOX, H69/VP, which express low levels of MMPs) inoculated i.v. into natural killer cell-depleted SCID mice metastasized into the liver, kidneys, and systemic lymph nodes. Film in situ zymography analysis revealed that the nontumor parenchyma of the lung had no gelatinolytic activity, whereas gelatinolytic activity of the liver and kidney was high and low, respectively. In the lung metastasis model, gelatinolytic activity of lung nodules directly correlated with the in vitro expression of MMP-2 and MMP-9 by tumor cells. Inhibition of MMP activity by ONO-4817 suppressed lung metastasis by the cell lines that expressed MMPs, but not those that did not express MMP, via the inhibition of MMP activity of lung tumors. In the multiple-organ metastasis model, liver parenchyma, but not liver nodules, showed gelatinolytic activity. The MMP inhibition reduced metastasis to the liver, but not to the kidney or lymph nodes, via inhibition of MMP activity of liver parenchyma. These findings suggest that MMP expression varies among the host organ microenvironments and that stromal MMPs may promote metastasis of lung cancer. Therefore, antimetastatic effects based on MMP inhibition may be dependent on MMPs derived not only from tumor cells but also from organ-specific microenvironments.


CD13/aminopeptidase N Induced Lymphocyte Involvement in Inflamed Joints of Rheumatoid Arthritis

We previously showed that CD13/aminopeptidase N (EC 3.4.11.2) induces chemotactic migration of T lymphocytes by its enzymatic activity. In this study, we examined the role of CD13/aminopeptidase N in lymphocyte involvement in rheumatoid arthritis (RA). Synovial fluids were obtained from 27 RA patients and 6 osteoarthritis (OA) patients. Synovial tissue specimens were obtained from 3 RA patients and 3 OA patients. Protease activity of aminopeptidase in synovial fluids and synovial fibroblasts was assayed fluorometrically using the specific substrate. Expression of CD13/aminopeptidase N in synovial fibroblasts was determined by flow cytometry analyses, Western blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR). The mean value of aminopeptidase activity in synovial fluid samples from RA patients was significantly higher than that in samples from OA patients. Increased enzymatic activity of aminopeptidase was detected on synovial fibroblasts from RA patients compared with those from OA patients. Flow cytometry showed that the expression of CD13/aminopeptidase N on synovial fibroblasts from RA patients was higher than the expression on synovial fibroblasts from OA patients, and Western blots and RT-PCR showed that synovial fibroblasts from RA patients contained a greater amount of CD13/aminopeptidase N. The activity of CD13/aminopeptidase N correlated significantly with lymphocyte counts in synovial fluids from RA patients. Synovial fluids from RA patients in which high aminopeptidase activity was detected contained considerable chemotactic activity for lymphocytes, and bestatin, a specific inhibitor of aminopeptidases, partially inhibited the chemotactic activity. CD13/aminopeptidase N may participate in the mechanism of lymphocyte involvement in inflamed joints of RA patients as a lymphocyte chemoattractant.


Activity of a New Vascular Targeting Agent, ZD6126, in Pulmonary Metastases by Human Lung Adenocarcinoma in Nude Mice

ZD6126 (ANG453) is a novel vascular targeting agent that selectively disrupts the cytoskeleton of endothelial cells in tumor. In mouse s.c. xenograft models, ZD6126 was found to induce selective occlusion of tumor blood vessels, cessation of tumor blood flow, and death of tumor cells because of the starvation of oxygen and nutrition. Here, we investigated whether ZD6126 inhibited the metastatic formation of human non-small cell lung cancer cells. PC14PE6 (adenocarcinoma) and H226 (squamous cell carcinoma) cells were injected into the tail vein of nude mice, and lung metastases were estimated. ZD6126 treatment involved either a single dose on 24 h before killing or daily doses from day 14 until the end of the experiment. Single treatment with i.p. injection of 200 mg/kg ZD6126 caused bleeding and necrotic changes in the tumor by 24 h. Histological analysis revealed that apoptotic tumor cells were markedly increased in the ZD6126-treated group. Moreover, ZD6126 induced the apoptosis of CD31-positive vascular endothelial cells in tumors but not in the normal lung parenchyma. When mice were treated daily with 100 mg/kg ZD6126 from day 14 until the end of the experiment, the lung weight was significantly less in the ZD6126-treated group than that of the control group, despite no difference in the number of metastatic nodules. These data suggest that ZD6126 could demonstrate its antitumor activity against both already established and early phase of lung cancer metastasis by causing the selective apoptosis of tumor endothelial cells and destruction of the tumor vasculature.


Chemokines in synovial inflammation in rheumatoid arthritis: basic and clinical aspects

Abstract Rheumatoid arthritis (RA) is a chronic, multisystem autoimmune disease characterized by persistent synovitis. Since chemotactic cytokines (chemokines) may play critical roles in the recruitment of leukocytes in RA, analyses of chemokines and their receptors should provide insight into events in synovial inflammation in RA. The production of chemokines is regulated by cytokines such as tumor necrosis factor (TNF)-α produced in the inflamed joint, suggesting that the efficacy of anti-TNF-α therapy is mediated at least partly by the reduction of chemokine production. Chemokines have a role in joint inflammation not only by inducing leukocyte chemotaxis, but also by activating immune cells and angiogenesis. The pathogenesis of RA has been shown to be mediated by Th1-type T cells, because Th1-related chemokine receptors are preferentially expressed on cells in synovial fluid and synovial tissue. Accordingly, antichemokine therapy may be important as a possible new approach to therapeutic intervention in RA.


Age-related changes in control of blood pressure and heart rate during sleep in the rat

The aim of this study was to determine age-related changes in the control of mean arterial pressure (MAP) and heart rate (HR) during sleep, and its relationship to the baroreflex in aging. MAP, HR, body temperature (TP), spontaneous activity (ACT), and sleeping/waking duration were monitored for 24 hours in groups of young (10-12 wk old) and old (23-24 mo old) rats. The sleep laboratory at the University of Tokushima. Subjects were 8 young (10-12 wk old) and 7 old (23-24 mo old) Wistar rats. Reflex control of HR was evaluated by examining various pressure responses to an intravenous bolus injection of phenylephrine and sodium nitroprusside. MAP and TP were recorded by a radiotelemetry system. HR was detected from the AP signal. ACT was counted by a photo-sensor system. In the case of old rats, the sensitivity of baroreflex control of HR was significantly depressed, and the spontaneous increase of MAP and HR during REM sleep and the MAP drop at the end of REM sleep were significantly enhanced. The old rats showed no large deterioration of the circadian profiles of MAP, HR, TP, and the amount of sleep. The baroreflex dysfunction is considered to appear in an early stage of the aging process, and to affect the control of MAP and HR during sleep.


Antiangiogenic Effect by SU5416 Is Partly Attributable to Inhibition of Flt-1 Receptor Signaling

Interaction between vascular endothelial growth factor (VEGF) and its cognate receptors, KDR/Flk-1 and Flt-1, of vascular endothelial cells is expected to induce an angiogenesis "switch" in tumors and other angiogenesis-associated diseases. SU5416, a selective inhibitor of the KDR/Flk-1 tyrosine kinase, is known to be a potent inhibitor of tumor angiogenesis. In this study, we first observed that SU5416 inhibited Flt-1 tyrosine kinase activity at similar doses, in addition to inhibiting KDR/Flk-1 tyrosine kinase activity in response to VEGF. SU5416 inhibited cell migration of human vascular endothelial cells expressing both Flt-1 and KDR in response to VEGF and also inhibited the cell migration in response to placenta growth factor (PIGF), a specific ligand for Flt-1. Chemotaxis of monocytes expressing only Flt-1 was also inhibited by SU5416 in a dose-dependent manner. Moreover, SU5416 was found to inhibit tyrosine kinase of Flt-1 in response to PIGF in vitro. And angiogenesis induced by PIGF in a Matrigel plug assay was inhibited by administration of SU5416. The antiangiogenic effects by this VEGF receptor-targeting compound appeared to be mediated through interference not only with KDR/Flk-1 but also with Flt-1. Cell migration of vascular endothelial cells and monocytic cells through Flt-1, thus, might play a key role in VEGF-induced tumor angiogenesis in concert with KDR/Flk-1.


IL-12-induced production of IL-10 and interferon-γ by mononuclear cells in lung cancer-associated malignant pleural effusions

Interleukin (IL)-12 enhances natural killer (NK) activity and induces interferon gamma (IFN-gamma) production. Recently, it was shown that IL-12 induces IL-10 production by human T cells and NK cells, as a negative feedback for IL-12-induced immune responses. In the present study, in order to investigate the functions of host immune cells existing in contact with cancer cells, we examined the effect of IL-12 on the induction of non-major histocompatibility complex (MHC)-restricted killer activity and of IFN-gamma and IL-10 production by pleural and peripheral blood mononuclear cells (MNC), isolated from 40 lung cancer patients and 20 control subjects. IL-12 induced significant killer activity in pleural MNC from lung cancer patients, as well as those in peripheral blood, against a small cell lung cancer cell line (SBC-3). In lung cancer patients, pleural MNC incubated with IL-12 produced more IFN-gamma than blood MNC. In addition, when stimulated with both IL-12 and IL-2, pleural MNC produced more IL-10 than blood MNC. This is the first study reporting that MNC from pleural effusions of patients with lung cancer can produce both type 1 (IFN-gamma) and type 2 (IL-10) cytokines following exposure to IL-2 and IL-12. These observations suggest that control of IL-10 production at the microenvironment level may be important for the efficacy of human lung cancer immunotherapy with IL-12.


Interleukin(IL)-12 gene transduction and its functional expression into human bronchial epithelial cells (BEAS-2B) by adenovirus vector

Interleukin (IL)-12 is known as a cytokine that augments the Th1 type response. Especially in allergic diseases such as a bronchial asthma, IL-12 induced restoration of the balance of the Th1/Th2 type immune response is an attractive strategy. In this study, the functional properties of the human bronchial epithelial cell line (BEAS-2B) transduced by an adenoviral vector encoding the human IL-12 gene were examined. Adenovirus vectors, AxCAegfp and Ax1CIhp40ip35 were transduced into BEAS-2B cells. Wild and gene-transduced BEAS-2B cells were incubated and the concentrations of IL-12 and IFN-gamma produced by co-cultured lymphocytes in the supernatant were measured using ELISA. The expressions of surface adhesion molecules, such as CD54 and CD106 were analyzed using flow cytometry. The efficiency of transgene expression of BEAS-2B cells was in a multiplicity of infection (MOI)-dependent manner and at an MOI of 30, the efficiency was approximately 80%. The gene-modified BEAS-2B cells produced biologically active IL-12 in dose- and time-dependent manners. IL-12 gene transduction did not significantly affect the expression of adhesion molecules (CD 54, CD106 and HLA-A,B,C) by BEAS-2B cells. These results suggest that the IL-12 gene may be successfully transduced into human bronchial epithelial cells by adenoviral vector to express IL-12 activity in vivo.

The Journal of Medical Investigation : JMI


Genetic modification of dendritic cells and its application for cancer immunotherapy

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs). DCs pulsed with peptides of tumor-associated antigens (TAA) and tumor lysate have been used in cancer immunotherapy. An early clinical study demonstrated the safety of the use of DCs, but the clinical response was not sufficient. The gene-modification of DCs with TAA and soluble factor genes such as cytokine and chemokine genes has been examined to enhance the antigen-presenting capacity of DCs. Viral vectors including retroviruses and adenoviruses have been reported to be useful to obtain a sufficient transduction efficiency into DCs. TAA gene-transduced DCs could have several advantages compared with TAA peptide-pulsed DCs as follows: 1) The use of TAA gene-modified DCs are not restricted by MHC haplotypes. 2) The gene transduction with TAA genes is likely to present the unknown TAA peptides on DCs. 3) The gene-modified DCs show the prolonged presentation of TAA peptides. The transduction of DCs with cytokine genes including IL-12 and GM-CSF have also been reported to argument the antitumor effects of DCs. Although the results in the experimental systems were promising, the clinical application of gene-modified DCs includes several problems such as the standardization of methods of manipulation and gene-transduction of DCs. Approaches to solve them require further studies.


Role of CD13/Aminopeptidase N in Rat Lymphocytic Alveolitis Caused by Thoracic Irradiation

CD13/aminopeptidase N is a cell surface glycoprotein that is widely distributed in a variety of mammalian cells. It was recently shown to have chemotactic activity for T lymphocytes. This study examined the role of CD13/aminopeptidase N in lymphocytic alveolitis in radiation-induced lung injury caused by a single-dose thoracic irradiation (15 Gy) in rats. Significantly increased aminopeptidase activity was detected in bronchoalveolar lavage fluid obtained from irradiated rats at 4 weeks after irradiation compared to the activity in unirradiated rats. Significantly higher aminopeptidase activity was detected on alveolar macrophages from irradiated rats at 2 and 4 weeks than on those from unirradiated rats. Western blot analysis showed an increased expression of CD13/aminopeptidase N protein in alveolar macrophages from irradiated rats at 4 weeks. Chemotactic activity for normal rat lymphocytes was detected in bronchoalveolar lavage fluid from irradiated rats at 4 weeks, and approximately 60% of the activity was inhibited by pretreatment of bronchoalveolar lavage fluid with bestatin, a specific aminopeptidase inhibitor. This study suggests that CD13/aminopeptidase N may play an important role as a lymphocyte chemoattractant in lymphocyte-mediated alveolitis in experimental radiation-induced lung injury.


The Significance of CD13/Aminopeptidase N in Interstitial Lung Diseases

CD13/aminopeptidase N (E.C.3.4.11.2) is an ectoenzyme located in the outer cell membrane in a variety of cells. Since CD13/aminopeptidase N was shown to induce in vitro chemotactic migration of human lymphocytes, we examined here the significance of CD13/aminopeptidase N in pulmonary sarcoidosis and radiation pneumonitis caused by a single-dose thoracic irradiation (15 Gy) in a rat model. The activity of aminopeptidase in bronchoalveolar lavage fluid (BALF) was significantly higher in sarcoidosis patients than in normal volunteers (NV) and control patients (CP). CD13/aminopeptidase N protein was detectable in alveolar macrophages (AN) from sarcoidosis patients at higher levels than in those from NV. Higher chemotactic activity for lymphocytes was detected in the BALF from sarcoidosis patients that in that from NV, and the activity was significantly decreased by the treatment with bestatin, an specific inhibitor for aminopeptidase N. Significantly increased CD13/aminopeptidase N activity and expression were also detected in BALF and AM obtained from irradiated rats at 4 weeks after irradiation compared with the activity in unirradiated rats. Chemotactic activity for normak rat lymphocytes was detected in BALF from irradiated rats at 4 weeks, and approximately 60% of the activity was inhibited by pretreatment of BALF with bestatin. This study suggests that CD13/aminopeptidase N may play an important role as a lymphocyte chemoattractant in lymphocyte-mediated alveolitis in interstitial lung diseases.

The Journal of the Japan Society for Respiratory Endoscopy


Effect generation of dendritic cells from alveolar and pleural macrophages as well as blood monocytes with lung cancer

In this study, we investigated the generation of dendritic cells (DCs) from blood monocytes and mature macrophages from untreated primary lung cancer patients. Blood monocytes were separated by adherence from blood mononuclear cells (MNC) from ten lung cancer patients and ten control subjects, and cultured for 7 days in medium with granulocyte/macrophage colony-stimulating factor (GM-CSF) plus interleukin (IL-) 4. In all cases examined, DCs with typical characteristics were obtained even in lung cancer patients after 7 days culture with these cytokines, and there was no significant difference in phenotype and stimulatory activity in allogeneic lymphocyte proliferation between DCs derived from monocytes from lung cancer patients and those from control subjects. Next, we examined whether alveolar and pleural macrophages in malignant pleural effusion separated by magnetic beads could differentiate to immunostimulatory DCs. Conventional culture conditions with GM-CSF and IL-4 did not induce efficient numbers of DCs from mature macrophages, whereas the addition of tumor necrosis factor-alpha (TNF-alpha) to GM-CSF and IL-4 effectively contributed to generate DCs. These findings suggest that both mature macrophages and blood monocytes from lung cancer patients could differentiate to DCs, and might be a useful source of DCs for immunotherapy.


Increased CCR4 expression in active systemic lupus erythematosus

CC chemokine receptor (CCR)4 is selectively expressed on Th2-type T cells and has been shown to be responsible for Th2-dominant immune responses. In this study, we analyzed the expression of CCR4 in active systemic lupus erythematosus (SLE) patients by FACS analysis using anti-human CCR4 monoclonal antibody and determined the clinical relevance in this disease. Higher expression of CCR4 was found on peripheral blood CD4+ T lymphocytes of active SLE patients than was found with healthy controls and inactive SLE patients. The CCR4 expression significantly correlated with the SLE disease activity index (SLEDAI) scores. The expression was dramatically decreased after the corticosteroid therapy in parallel with a serum level of double-stranded DNA antibody and SLEDAI scores. Moreover, we found that serum levels of IL-10 were increased in active SLE patients and significantly correlated with the CCR4 expression. This study suggests that Th2 immune response is predominant in the active state of SLE, and CCR4 may have relevance in regard to the disease course in SLE patients.


Immunological circumvention of multiple organ metastases of multidrug resistant human small cell lung cancer cells by mouse-human chimeric anti-ganglioside GM2 antibody KM966.

serum against SBC-3/DOX cells to a similar extent compared with parental SBC-3 cells. Pretreatment of human effector cells with various cytokines induced further enhancement of the KM966-dependent ADCC against SBC-3/DOX cells. Intravenous injection of SBC-3 or SBC-3/DOX cells into natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice developed metastases in multiple organs (liver, kidneys and lymph nodes). Interestingly, SBC-3/DOX cells produced metastases more rapidly than SBC-3 cells, suggesting more aggressive phenotype of SBC-3/DOX cells than their parental cells in vivo. Systemic treatment with KM966, given on days 2 and 7, drastically inhibited the formation of multiple-organ metastases produced by both SBC-3 and SBC-3/DOX cells, indicating that KM966 can eradicate metastasis by SCLC cells irrespective of MDR phenotype. These findings suggest that the mouse-human chimeric KM966 targets the GM2 antigen, and might be useful for the immunological circumvention of multiple-organ metastases of refractory SCLC.


Human monocyte-derived and CD83(+) blood dendritic cells enhance NK cell-mediated cytotoxicity.

Dendritic cells (DC) are known to be the most potent APC and to stimulate antigen-specific T cell responses. Recently it was reported that murine DC were also capable of modulating the innate immunity by stimulating NK cells through cell-to-cell contact. In the present study, we examined whether human DC could affect NK activity. Both monocyte-derived and CD83(+) blood DC were tested. The addition of DC to cultures of CD56(+) cells resulted in the significant dose-dependent enhancement of the killing activity against various NK-sensitive targets. The resultant activity was comparable to that induced by optimal concentrations of various cytokines, including IL-2, IL-12, IL-15 and IFN-gamma. Interestingly, DC enhanced the cytotoxicity of CD3(-)CD56(+) NK cells, but not that of CD3(+)CD56(+) T cells. Experiments using transwells clearly demonstrated that the enhancement of NK activity by DC was mediated by soluble factors produced by DC. The culture supernatants of DC also stimulated NK activity. The treatment of both DC and their supernatants with anti-human IL-12 or IL-18 antibodies did not block the enhancement of NK cell-mediated cytolysis by DC, indicating that other factor(s) produced by DC were responsible for the enhancement of NK activity. These results suggest that human myeloid DC can modulate innate immunity by enhancing NK activity.


Protease-induced leukocyte chemotaxis and activation: roles in host defense and inflammation

The migration of leukocytes such as neutrophils, monocytes and lymphocytes into inflamed lesions is one of the critical events of inflammation. Although the traditional function of neutrophil-derived antimicrobial proteases is to ingest and kill bacteria, some neutrophil serine proteases have been shown to induce leukocyte migration and activation. Mast cell-derived chymase also has the chemotactic activity for leukocytes. During the acute phase of inflammatory and allergic diseases, the predominantly migrated cells are neutrophils and mast cells, respectively, and in the subsequent chronic phase, monocytes and lymphocytes are mainly migrated. The chemotactic activity for monocytes and lymphocytes of neutrophil-derived serine proteases and mast cell-derived chymase may have a role in switching acute inflammation to chronic inflammation and delayed-type hypersensitivity. Recently, aminopeptidase N and endothelin were shown to induce chemotactic migration of leukocytes. Thus, protease-induced leukocyte chemotaxis and activation may play an important role in immunologic events of inflammatory and allergic diseases.

The Journal of Medical Investigation : JMI


Autoantibodies to IL-1 alpha in sera from rapidly progressive idiopathic pulmonary fibrosis

To clarify the clinical significance of autoantibodies to interleukin-1 alpha (IL-1 alpha autoantibodies) in rapidly progressive idiopathic pulmonary fibrosis (IPF), we measured the level of IL-1 alpha autoantibodies in serum of 11 patients on the first hospital day, when patients were admitted due to severe symptoms, and on the 21st hospital day. IL-1 alpha autoantibodies in serum were measured using radioimmunoassay, and the limitation of this assay for IL-1 alpha autoantibodies was 5 ng/ml. These antibodies were detected in 5 of 11 patients on the first hospital day. On the 21st hospital day, these antibodies were detected in all patients, and its level was increased compared with that on the first hospital day. IL-1 alpha autoantibodies that appeared in patients corresponded to that of IgG. The half life of exogenous autoantibodies was investigated following administration of autoantibody rich plasma obtained from healthy blood donors to 6 control patients (CP) and 6 progressive IPF patients. These autoantibody levels in their serum were less than 5 ng/ml before administration. Serum was obtained at the indicated time after administration of IL-1 alpha autoantibodies and the level of these autoantibodies in serum was measured, then the half life was calculated. Half life of exogenous IL-1 alpha autoantibodies in progressive IPF patients was significantly shorter than that in CP (71.3 +/- 31.8 hr vs 352.0 +/- 98.3 hr, p < 0.01). These findings suggested that IL-1 alpha autoantibodies were generated in response to the inflammatory process of rapidly progressive IPF and may act as a regulatory factor for IL-1 alpha.


A novel bioactive 31-amino acid endothelin-1 is a potent chemotactic peptide for human neutrophils and monocytes

Endothelin (ET)-1(1-31) is a novel 31-amino acid-length peptide derived from big ET-1 by chymase or other chymotrypsin-type proteases and is a major ET derivative in human neutrophils. In this study, we revealed that ET-1(1-31), but not big ET, exhibited chemotactic activities toward human neutrophils and monocytes as an inflammatory mediator, although the effects were less potent than those of formyl-methionyl-leucyl-phenylalanine or interleukin-8. However, the chemotactic effects of ET-1(1-31) were much greater than those of the 21-amino acid ET-1, ET-1(1-21). Checkerboard analyses revealed that the effects are chemotactic rather than chemokinetic. The effects of ET-1(1-31) are not mediated by interleukin-8 or monocyte chemoattractant protein-1. The chemotactic effects and an increase in intracellular-free Ca(2)(+) caused by ET-1(1-31) were significantly inhibited by BQ123, an ET(A) receptor antagonist, but not by BQ788, an ET(B) receptor antagonist, suggesting that ET-1(1-31) mediates chemotaxis through an ET(A) or ET(A)-like receptor.


Transduction of KAI1/CD82 cDNA promotes hematogenous spread of human lung-cancer cells in natural killer cell-depleted SCID mice

KAI1, which is identical to CD82, was initially identified as a metastasis-suppressor gene for human prostate cancer, and its expression is reported to be a favorable prognostic factor for operable human lung cancer. In this study, we examined the functional role of KAI1/CD82 in the late phase of metastatic spread of human lung-cancer cells. For this, KAI1/CD82 cDNA was introduced into KAI1/CD82 low-expressing human lung-cancer cell lines, SBC-3 and PC-14, and then the metastatic potential of the transformants was analyzed by i.v. inoculation of KAI1/CD82-transduced cells, SBC-3/KAI1 and PC-14/KAI1, into NK cell-depleted SCID mice. Contrary to our expectations, KAI1/CD82 gene transfer promoted multiorgan metastasis of i.v.-inoculated human lung-cancer cells, while s.c. tumor growth was unaffected. Cancer cells from metastatic tumors of NK cell-depleted SCID mice injected i.v. with SBC-3/KAI1 expressed appreciable cell-surface KAI1/CD82, and cells not expressing KAI1/CD82 (revertants) were not detected in the tumors. Our findings indicate that under conditions where the host's natural cytotoxicity is suppressed, KAI1/CD82 may enhance the formation of tumors by circulating lung-cancer cells at metastatic sites.


Novel centrifugal method for simple and highly efficient adenovirus-mediated green fluorescence protein gene transduction into human monocyte-derived dendritic cells

Dendritic cells (DC) are professional antigen-presenting cells in the immune system. Gene transduction of DC with tumor-associated antigen (TAA) or other genes that enhance the immune reaction has been considered theoretically useful for DC-based immunotherapy. However, gene transduction of DC generated from human peripheral blood monocytes has been difficult due to its low efficiency, even when adenoviral vector was used at high multiplicity of infection (MOI). In the present study, we examined the effect of centrifugal force to enhance efficiency of adenovirus-mediated gene transduction into human monocyte-derived DC at various rotor speeds at various temperatures for various times. We judged the transduction efficiency using enhanced green fluorescence protein (EGFP)-expressing adenoviral vector, and the best condition for centrifugal transduction was determined as 2000 x g at 37 degrees C for 2 h at an MOI of 10 or greater. At an MOI of 50 without centrifugation, the gene transduction efficiency was about 66% and mean fluorescence intensity (MFI) of EGFP expression was about 150 (at 37 degrees C for 2 h). With centrifugal transduction (2000 x g at an MOI of 50 at 37 degrees C for 2 h), 86% or more DC were gene-modified, and especially, MFI of EGFP expression was highly enhanced (MFI: about 3100 or greater). Centrifugally gene-transduced DC were not damaged and were thoroughly functional as measured by mixed lymphocyte reaction (MLR). The centrifugal method was also applicable to human monocytes and K562 cells. The centrifugal transduction method with adenoviral vector might be helpful for the generation of gene-modified DC.


A new quinoline derivative MS-209 reverse multidrug resistance and inhibits multiorgan metastases by P-glycoprotein-expressing human small cell lung cancer cells

Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3 / ADM and H69 / VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3 / ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro, compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3 / ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3 / ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3 / ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3 / ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3 / ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3 / ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3 / ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.


Huang, L., Ogushi, F., Tani, K., Ogawa, H., Kawano, T., Endo, T., Izumi, K., Ueno, J., Nishitani, H. and Sone, S. Thrombin Promotes Fibroblast Proliferation during the Early Stages of Experimental Radiation Pneumonitis. Radiat. Res. 156, 45-52 (2001). To clarify the role of thrombin in the pathogenesis of radiation-induced pneumonitis, we measured the thrombin activity and fibroblast growth-inducing activity in bronchoalveolar lavage fluid obtained from the irradiated lungs of rats at 1, 2, 4, 8 and 18 weeks after irradiation. Thrombin activity was not detected in the bronchoalveolar lavage fluid from unirradiated rats, but the bronchoalveolar lavage fluid from irradiated rats showed significantly increased thrombin activity which reached a maximum at 4 weeks after treatment. Higher fibroblast growth-inducing activity was detected in the bronchoalveolar lavage fluid from irradiated rats at 4 and 18 weeks than in fluid from unirradiated rats. Bronchoalveolar lavage fluid from irradiated rats that were pretreated with the thrombin inhibitors antithrombin III and argatroban showed significantly inhibited fibroblast growth-inducing activity and thrombin activity at 4 weeks. However, these thrombin inhibitors did not inhibit fibroblast growth-inducing activity in bronchoalveolar lavage fluid from irradiated rats at 18 weeks. Purified rat thrombin similarly induced proliferation of fibroblasts derived from irradiated and unirradiated rats. These findings suggest that thrombin may play an important role as a fibroblast growth-inducing factor during the early stages of radiation pneumonitis.


Humanized anti-ganglioside GM2 antibody is effective to induce antibody-dependent cell-mediated cytotoxicity in mononuclear cells from lung cancer patirnts

Ganglioside GM2 is one of the major gangliosides expressed on the cell surface of human tumors including lung cancer. We have previously reported that a mouse-human chimeric monoclonal antibody (mAb), KM966, against GM2 promotes the lysis of lung cancer cells by human blood mononuclear cells (MNC) of healthy donors. In this study, we examined antibody-dependent cell-mediated cytotoxicity (ADCC) of MNC, using KM966 mAb and its humanized counterpart, KM8969, in 16 lung cancer patients and 18 control patients. The ADCC activity was assessed by 4-h (51)Cr release from GM2 positive SBC-3 small cell lung cancer cells. MNC from lung cancer patients exhibited similar ADCC activity to those from control patients when KM966 and KM8969 were used as mAb. Moreover, effective ADCC activity was observed even in MNC from advanced lung cancer patients. These observations suggest the potential activity of humanized anti-GM2 mAb (KM8969), as well as chimeric KM966, in biological therapy for lung cancer patients.


Enhanced efficiency by centrifugal manipulation of adenovirus-mediated interleukin 12 gene transduction into human monocyte-derived dendritic cells.

Transduction of dendritic cells (DCs) with genes encoding tumor-associated antigen or with other genes that enhance immune reaction has been theorized to be potentially useful for enhancing the efficiency of DC-based immunotherapy. However, gene transduction of DCs generated from human peripheral blood monocytes has been of limited use because of the low efficiency. Here, we report that the efficiency of in vitro adenovirus-mediated gene transduction into human monocyte-derived DCs can be dramatically enhanced by centrifugation. The best conditions for centrifugal gene transduction were determined to be as follows: 2000 x g at 37 degrees C for 2 hr at a multiplicity of infection (MOI) of 10 or greater. By this centrifugal method, approximately 88 and 70% of DCs were gene transducible at an MOI of 50 and 10, respectively. Functional analysis showed that DCs transduced with human interleukin 12 (IL-12)-expressing adenoviral vector under the optimal conditions of centrifugation stably produced IL-12 protein at high levels (8.1 ng/10(6) cells/48 hr). IL-12 gene-modified DCs (DC/IL-12) displayed a more mature phenotype than nontransduced DCs, as judged by decreased expression of CD1a and increased expression of CD83, B7.1 (CD80), B7.2 (CD86), and MHC class I and II molecules. DC/IL-12 showed a high phagocytic ability similar to nontransduced DCs and were significantly superior to control DCs in the stimulation of autologous and allogeneic T lymphocyte responses. The centrifugal transduction method with adenoviral vector might be useful for efficient generation of gene-modified DCs because it is very simple, highly efficient, reproducible, and not cytopathic. IL-12 gene-modified human DCs may be therapeutically useful as a good adjuvant in DC-based immunotherapy.


Symposium on molecular pathogenesis of respiratory diseases and its clinical implication. 4. Molecular pathogenesis of lung cancer and its molecular targeted therapy


CD40 and IFN-γ dependent T cell activation by human bronchial epithelial cells

We examined whether freshly isolated human bronchial cells (HBEC) and bronchial epithelial cell line/BEAS-2B cells expressed surface molecules required for APC function. These cells expressed CD40 and ICAM-1, but not B7-1, B7-2 or HLA-DR molecules. Treatment of these cells with IFN-gamma resulted in enhanced expression of CD40 and ICAM-1 as well as induction of HLA-DR expression. Th2 cytokines such as IL-4 and IL-5, proinflammatory cytokine of GM-CSF and nonspecific activator endotoxin had no effect on these phenotypic expressions. Functional examinations showed that allogeneic lymphocytes purified from peripheral blood strongly proliferated in response to BEAS-2B cells cultured with IFN-gamma, but only weakly compared with those without IFN-gamma. When allogeneic lymphocytes were purified to CD4+ cells, the proliferative response against BEAS-2B cells was abolished. Blockade of CD40-CD40L interaction by anti-CD40 antibody also inhibited the proliferation of lymphocytes to BEAS-2B cells, although this treatment showed a minimum effect on the response to allogeneic MNC. Thus, bronchial epithelial cells have the ability to present allogeneic antigens to T cells in both CD40- and IFN-gamma-dependent manners under the presence of third party cells that transduce co-stimulatory signals.

The Journal of Medical Investigation : JMI


Nuclear Factor-kB-dependent Expression of Metastasis Suppressor KAI1/CD82 Gene in Lung Cancer Cell Lines Expressing Mutant p531

KAI1/CD82 has been shown to be a metastasis suppressor for several human cancers, and a recent study revealed that wild-type tumor suppressor p53 can directly activate KAI1/CD82 gene expression. However, the response of KAI1/CD82 expression in cancer cells to exogenous stimulants has not been investigated. The present study examined whether tumor necrosis factor (TNF), which mediates many of the cellular responses associated with inflammatory reactions or cancer progression, can affect the KAI1/CD82 expression in lung cancer cells and, if so, whether nuclear factor (NF)-kappaB, a key molecule in TNF-mediated gene expression, is involved in the mechanism of KAI1/CD82 induction. Our results demonstrated that expression of KAI1/CD82 in PC-14 cells expressing mutant p53 could be augmented by TNF-alpha, and that transfer of the gene for a specific inhibitor of NF-kappaB, IkappaB alphaSR (mutant IkappaB alpha; NF-kappaB super-repressor), into PC-14 cells could inhibit this augmentation. The amount of NF-kappaB in the nucleus of PC-14/IkappaB alphaSR cells correlated well with KAI1/CD82 mRNA and protein expression. In addition, IkappaB alphaSR gene transfer inhibited the spontaneous expression of KAI1/CD82 protein in KAI1/CD82-high-expressing RERF-LC-OK cells, which contain a mutant-type p53. These observations indicate that NF-kappaB activation may play a role in the regulation of KAI1/CD82 expression in lung cancer cells independently of wild-type p53, and suggest that KAI1/CD82 expression may be regulated by interaction with the host microenvironment.


INTERLEUKIN (IL-)15 HAS LESS ACTIVITY THAN IL-2 TO PROMOTE TYPE 2 CYTOKINE PREDOMINANCE IN TUMOUR-ASSOCIATED MONONUCLEAR CELLS FROM LUNG CANCER PATIENTS


嚢胞状変化を呈した肺サルコイドーシスの1例

嚢胞状変化を呈した肺サルコイドーシスの1例

The journal of the Japanese Respiratory Society


Natural killer cell-dependent suppression of systemic spread of human lung adenocarcinoma cells by monocyte chemoattractant protein-1 gene transfection in severe combined immunodeficient mice.


Down-regulation by a new anti-inflammatory compound, FR167653, of differentiation and maturation of human monocytes and bone marrow CD34(+) cells to dendritic cells

International Journal of Immunopharmacology


Expression of a TGF-β1 inducible gene, TSC-36, causes growth inhibition in human lung cancer cell lines


Chymase is a potent chemoattractant for human monocytes and neutrophils

Chymase is a major chymotrypsin-like serine protease expressed in the secretory granules of mast cells in many mammalian species. In this study, we revealed the chemotactic activity of chymase for human mononuclear cells and neutrophils with a 48-well microchemotaxis chamber technique. Human chymase showed the potent chemotactic activity for monocytes and neutrophils dose-dependently in a concentration range from 0.1 to 10 microg/mL, corresponding to about 4-400 microM. The activity was as potent as that of N-formyl-methionyl-leucyl-phenylalanine. Chymase also stimulated cell migration of lymphocytes and purified T cells, but checkerboard analysis revealed that the effect was chemokinetic rather than chemotactic. Inhibition of chymase activities with chymase inhibitors, such as antileukoprotease and Bowman-Birk soybean trypsin inhibitor, significantly inhibited the chemotactic activity of chymase, suggesting that the proteolytic activity of chymase participates in the chemotactic activity. Our results suggest that mast cell chymase acts as a chemoattractant, and may play a role in the accumulation of inflammatory cells in development of the chronic inflammatory responses of allergic and nonallergic diseases.


Macrophage infiltration correlates with tumor stage and angiogenesis in human malignant melanoma: Possible involvement of TNFα and IL-1α

We examined whether macrophage infiltration is associated with angiogenesis in cutaneous melanoma. The numbers of macrophages and microvessels increased significantly with increasing depth of tumor and with tumor angiogenesis. Macrophage infiltration thus appeared to provide a useful diagnostic marker for the progression of cutaneous melanoma. We further examined whether human melanoma cells produce angiogenic factors in response to macrophage-derived cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 alpha (IL-1alpha). Treatment of melanoma cells with TNFalpha and IL-1alpha in vitro enhanced the production of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and of basic fibroblast growth factor (bFGF) to a lesser degree, in human melanoma cells. Lipopolysaccharide (LPS)-activated human monocytes enhanced production of IL-8, VEGF, TNF alpha, as well as IL-1alpha, but not bFGF. Co-culture of human monocytes and human melanoma cells was also found to significantly enhance production of IL-8 and VEGF in the absence and presence of LPS, compared with either monocytes or melanoma cells alone. The production of IL-8 and VEGF from co-cultured melanoma cells and LPS-activated monocytes was blocked when anti-TNF-alpha antibody or anti-IL-1alpha antibody was co-administrated. This is direct evidence that production of the potent angiogenic factors IL-8 and VEGF from melanoma cells is up-regulated through TNFalpha and/or IL-1alpha secreted by activated monocytes/macrophages, influencing both tumor growth and angiogenesis in melanomas.


Augmentation by interleukin-18 of MHC-nonrestricted killer activity of human peripheral blood mononuclear cells in response to interleukin-12

Interleukin (IL)-18 is a novel cytokine with pleiotropic functions. In the present study, we examined the induction of the killer activity of peripheral blood mononuclear cells (MNC) against lung cancer cell lines upon treatment with IL-18 in combination with IL-12. Cytotoxic activity was measured by standard (51)Cr release assay. IL-18 (100 ng/ml) was found to significantly augment IL-12-induced killer activity in a MHC-nonrestricted manner against allogeneic NK-resistant Daudi cells and lung cancer cell lines: SBC-3, RERF-LC-AI and A549. IL-18 could augment IL-12-induced killer activity both at the optimal as well as suboptimal doses of the latter. However, IL-18 was found to have little effect on the killer activity of MNC induced by optimal or suboptimal dose of IL-2 or IL-15. Treatment of MNC with IL-18 in combination with IL-12 for a period of more than 4 days was observed to optimally induce the killer activity. As for induction of IFN-gamma production by MNC, IL-18 augmented that induced by IL-2 and IL-15, as well as that induced by IL-12. These results show the potential of IL-18 in combination with IL-12 for clinical application in treatment of cancer.


Bone metastasis model with multi-organ dissemination of human small cell lung cancer (SBC-5) cells in nutural killer-cell depleted SCID mice

Lung cancer is commonly associated with multiorgan metastasis, and bone is a frequent metastatic site for lung cancer. Nevertheless, no bone metastasis model of lung cancer with multiorgan dissemination is available, which could provide opportunity to study the molecular pathogenesis. We examined the abilities of eight human lung cancer cell lines injected intravenously into natural killer (NK) cell-depleted SCID mice to generate metastatic nodules in bone and multiple organs, and explored the correlation of the parathyroid hormone-related protein (PTHrP) with the bone metastasis. Although all the small-cell carcinoma cell lines (SBC-5, SBC-3, SBC-3/ADM, H69, H69/VP) formed metastatic nodules in multiple organs (liver, kidney, and lymph nodes), only SBC-5 cells reproducibly developed bone metastases. Squamous cell carcinoma (RERF-LC-AI) cells metastasized mainly into the liver and kidneys, whereas adenocarcinoma (PC-14, A549) mainly produced colonies in the lungs. As assessed by X-ray photography, the osteolytic bone metastases produced by SBC-5 cells were detected as early as on day 28, and all recipient mice developed bone metastasis by day 35. The expression of PTHrP in eight cell lines was directly correlated with the formation of bone metastasis. No correlation was observed between the formation of bone metastasis and the expression of other metastasis-related cytokines (IL-1, IL-6, IL-8, IL-10, IL-11, TNF-alpha, VEGF, M-CSF). Consistent with the formation of bone metastasis by SBC-5 cells, the levels of PTHrP and calcium in the mouse serum were increased in a time-dependent manner, suggesting that PTHrP produced by human lung cancer may play a crucial role in the formation of bone metastasis and hypercalcemia. These findings indicate that a bone metastasis model of SBC-5 cells may be useful for clarifying the molecular aspects of the metastatic processes in different organ microenvironments and the development of therapeutic modalities for lung cancer patients with bone metastases.


Demethylation by 5-aza-2'-deoxycytidine (5-azadC) of p16INK4A gene results in downregulation of vascular endothelial growth factor expression in human lung cancer cell lines

Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor progression via angiogenesis. Recently, gene transduction of wild-type p16INK4A, tumor suppressor gene, has been shown to result in downregulation of VEGF expression in p16INK4A-deleted glioma cells. Because expression of p16INK4A is regulated by methylation of the p16INK4A gene, we examined whether demethylation of the p16INK4A gene by 5-aza-2'-deoxycytidine (5-azadC) could cause the protein expression of VEGF as well as of p16INK4A in human lung cancer cells. For this, five different lung cancer cell lines with or without loss of p16 activity were used. H841 and Ma-10 cells had the methylated p16INK4A gene without expression of p16INK4A protein, whereas Ma-1 and H209 cells had the unmethylated p16INK4A gene with constitutive expression of p16INK4A protein. Neither the p16INK4A gene nor p16INK4A protein was detected in A549 cells. Treatment with 5-azadC caused demethylation of the p16INK4A gene with reexpression of p16INK4A protein in H841 and Ma-10 (methylated p16INK4A gene dominant) cell, but not in other cell lines such as Ma-1, H209 (unmethylated p16INK4A gene dominant), or A549 (p16INK4A gene deleted). In a parallel experiment, 5-azadC inhibited production of VEGF protein by H841 and Ma-10 cells, especially in the later hypermethylated cells, but not Ma-1, H209, or A549 cells. RT-PCR analysis showed that Ma-10 cells expressed VEGF isoforms 121, 165, and 189, all of which were inhibited by 5-azadC. These findings indicate that the methylation status of the p16INK4A gene plays an important role in the regulation of angiogenesis associated with progression of lung cancer, through regulation of VEGF expression.


Interleukin-10 inhibits the production of inflammatory cytokines by antigen-stimulated mononuclear cells from asthmatic patients


Human interferon-gamma enhances expression of ganglioside GM2 on human lung cancer cells and their susceptibility for antiganglioside GM2 monoclonal antibody-dependent cellular cytotoxicity.

Interferons are known to modulate several cellular functions by the induction of various proteins. In this study, we demonstrated that human interferon-gamma (HuIFN-gamma) enhanced the expression of ganglioside GM2 (GM2), which is a kind of tumor-associated antigen substantially expressed in human lung cancer and that human lung cancer cells expressing GM2 became more susceptible to anti-GM2 monoclonal antibody (mAb)-dependent tumor cell killing mediated by human effector cells after HuIFN-gamma treatment. GM2 expression on human lung cancer cells treated with or without HuIFN-gamma was measured by flow cytometry. The antibody-dependent cellular cytotoxicity (ADCC) activity was assessed by 4-h 51Cr release assay. HuIFN-gamma enhanced GM2 expression on human small-cell lung cancer (SCLC), SBC-3, and human non-small-cell lung cancer (NSCLC), A549 cells in a dose-dependent manner. The optimal concentration of HulIFN-gamma was 1,000 U/ml. The effect of HulFN-gamma reached maximum after 4 days of culture. HulFN-gamma did not have any effect to enhance the expression of other gangliosides in SBC-3 cells. No other cytokines used in this study modulated GM2 expression in SBC-3 cells. Anti-GM2 mAb-dependent ADCC activities induced by lymphocytes and monocytes were more potent against IFN-gamma-treated SBC-3 and A549 cells than nontreated cells. Taken together, HulFN-gamma combined with anti-GM2 mAb may be useful for immunotherapy against GM2-positive human lung cancer.


Apoptosis induction of human lung cancer cell line in multicellular heterospheroids with humanized antiganglioside GM2 monoclonal antibody.

The chimeric antiganglioside GM2 monoclonal antibody (MAb) KM966, which showed high effector functions such as complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), potently suppressed growth and metastases of GM2-positive human cancer cells inoculated into mice. To further improve the therapeutic efficacy of the anti-GM2 MAb in humans, we constructed a humanized anti-GM2 MAb, KM8969. The humanized KM8969 was more efficient in supporting ADCC against GM2-positive human cancer cell lines than the chimeric KM966, whereas complement-dependent cytotoxicity was slightly reduced in the humanized KM8969. In addition, the humanized KM8969 was shown to exert potent ADCC mediated by both lymphocytes and monocytes. To investigate the effect of the humanized KM8969 on the biological function of GM2 in the condition physiologically mimicking formation and growth of cancer masses, the heterospheroids composed of normal human dermal fibroblasts and GM2-positive human lung cancer cells were developed. Interestingly, the humanized KM8969 gave rise to growth inhibition of heterospheroids without dependence of the effector functions. Morphological and immunocytochemical analysis suggested that the inhibitory effect was due to the apoptosis of GM2-positive cancer cells in the heterospheroids. The result indicates that GM2 captured by the antibody on the cell surface loses its physiological function that plays a critical role in maintaining the three-dimensional growth of cancer cells in contact with its own cells or other type of cells in a microenvironment. The humanized KM8969, which can destroy the cancer cells via blocking functional GM2 on the cell surface as well as the effector functions, would have extraordinary potential in human cancer therapy.


A case of resectable lung adenocarcinoma associated with sarcoidosis

A 71-year-old woman with uveitis was referred to our hospital for further examination of the possible underlying diseases. In roentgenological examination with plain X-ray and CT scan, hilar and mediastinal lymphadenopathy and a mass shadow in the right upper lung field was observed, whereas fibrotic changes were not obvious in both lung fields. Transbronchial lung biopsy with fiberoptic bronchoscope revealed granulomatous interstitial pneumonia. CD4-positive lymphocytes were increased in bronchoalveolar lavage. The patient was diagnosed as having sarcoidosis. Subsequently, right upper lobectomy was performed, and Stage I lung adenocarcinoma was diagnosed. The patient is under follow up without medication and the disease has been stable for two years. A relationship between epithelioid granulomatosis and malignant diseases is discussed and a review of the literature is given. Since it is still controversial as to the incidence of malignant diseases in sarcoidosis patients, it is important to accumulate data on these associations.


Combined therapy with anti-p-glycoprotein antibody and macrophage colony-stimulating factor gene transduction for multiorgan metastases of multidrug-resistant human small cell lung cancer in nk cell-depleted scid mice

Our aim was to determine the antimetastatic potential of anti-P-glycoprotein (P-gp) antibodies (Abs) against multidrug-resistant (MDR) human small cell lung cancer (SCLC) cells expressing P-gp. Human SCLC cells H69 (P-gp negative) and its etoposide-resistant variant H69/YP (P-gp positive) were used. H69 and H69/VP cells injected i.v. metastasized to the liver, kidneys and systemic lymph nodes of NK cell-depleted severe combined immunodeficient (SCID) mice. H69/VP cells, but not H69 cells, were resistant to treatments with vindesine. Treatment with mouse-human chimeric anti-P-gp Ab (MH162) and its mouse counterpart (MRK-16) reduced metastasis of H69/VP cells in various organs and prolonged the survival of tumor-bearing mice, although they were less effective if injected at late times (after 28 days). Treatment with another mouse anti-Pgp Ab, MRK-17, was effective only against liver metastasis. MH162 and MRK-16 efficiently induced Ab-dependent cellular cytotoxicity (ADCC) by peritoneal macrophages against H69/VP cells in vitro, but MRK-17 was less effective, in accordance with their in vivo antimetastatic potential. Gene transfection of macrophage colony-stimulating factor (M-CSF) into H69/VP cells to augment macrophage-mediated ADCC resulted in inhibition of metastasis to the liver and lymph nodes, but not kidneys. Combined treatment with a low dose of MRK-16 completely cured metastasis of M-CSF transfectant, but not of the mock transfectant. Our findings suggest that while anti-P-gp Abs had antimetastatic potential against SCLC cells expressing P-gp, combined treatment with M-CSF gene transduction to augment the therapeutic efficacy of anti-P-gp Abs may be beneficial for eradicating metastatic MDR SCLC in humans.


Cytolysis of human dendritic cells by autologous lymphokine-activated killer cells: participation of both T cells and NK cells in the killing.

Dendritic cells (DC) play a key role in the initiation of immune response by stimulating the naive T cells. The fate of DC after the initiation of immune response is not clearly understood. Although there are few reports implicating natural killer (NK) cells in the elimination of DC, killing of DC by LAK cells, and specifically by T cells, has not been studied. In this study, we observed that DC, generated from monocytes, in vitro in the presence of granulocyte-macrophage colony-stimulating factor, interleukin-4 (IL-4), and tumor necrosis factor alpha were susceptible to cytolysis by lymphokine-activated killer (LAK) cells induced in the presence of IL-2 and IL-15 but not IL-12 alone. However, LAK cells induced by a combination of IL-12 and suboptimal dose of IL-2 were cytotoxic to DC. When purified lymphocytes were activated with IL-2, the CD8+/CD57- fraction (T-LAK), but not the CD8-/CD57+ fraction (NK-LAK) was cytotoxic to autologous DC. However, when unseparated peripheral blood mononuclear cells were used to generate LAK cells, both T-LAK and NK-LAK fractions showed equal cytotoxicity against autologous DC. Monoclonal antibodies against CD54, CD11a, and CD18 significantly inhibited the cytolysis, indicating that the killing involves the engagement of CD54 with its ligands.


Monocyte chemoattractant protein-1 gene modification of multidrug-resistant human lung cancer enhances antimetastatic effect of therapy with anti-P-glycoprotein antibody in SCID mice

Distant metastases and multidrug resistance are critical problems in the therapy of human small cell lung cancer (SCLC). In this study, we investigated whether transduction of the monocyte chemoattractant protein-1 (MCP-1) gene into multidrug-resistant (MDR) human lung cancer cells affected the formation of metastases or their inhibition by the anti-P-glycoprotein (P-gp) monoclonal antibody (MAb) MRK16. MDR human SCLC (H69/VP) cells were transduced with the human MCP-1 gene inserted into the expression vector BCMGSNeo. MCP-1 gene transduction had no effect on drug sensitivity, the expression of surface antigens or the in vitro proliferation of H69/VP cells. Using the metastatic model of NK cell-depleted SCID mice, H69/VP cells transduced with the MCP-1 gene were inoculated intravenously (i.v.) and formed metastatic colonies in the liver, kidneys and lymph nodes, similar to those formed by parent or mock-transduced cells. However, systemic treatment of the mice with MRK16 reduced the metastases of H69/VP cells in the liver, kidneys and lymph nodes, and was significantly more effective in inhibiting the metastases of MCP-1 producing H69/VP than those of mock-transduced cells. MCP-1 gene transduction significantly prolonged the survival of tumor-bearing mice treated with MRK16. Our findings suggest that local production of MCP-1 in the tumor site increases the anti-P-gp antibody-dependent cell-mediated cytotoxicity, and the MCP-1 gene-induced modification of MDR human SCLC cells thereby enhances the antimetastatic effect of therapy with anti-P-gp antibody. Thus, the accumulation of effector cells in the tumor site is a very important factor in the therapy using the anti-P-gp antibody.


Therapeutic Efficacy of a New Topoisomerase I and II Inhibitor TAS-103, Against Both P-Glycoprotein Expressing and Non-expressing Drug-Resistant Human Small-Cell Lung Cancer

We examined the effect of a novel topoisomerase I and II (topo I and II) inhibitor, TAS-103, on P-glycoprotein (P-gp)-expressing and -nonexpressing drug-resistant human small-cell lung cancer (SCLC) cells in vitro and in vivo. We observed that TAS-103 was effective in inhibiting in vitro proliferation of human SCLC (SBC-3 and H69) cells and their drug-resistant variants SBC-3/ADM or SBC-3/CDDP and H-69/VP, respectively. SBC-3/ADM and H-69/VP expressed high P-gp, whereas SBC-3/CDDP did not. TAS-103 also effectively reduced the tumor growth (more than 50% inhibition) of the parental as well as MDR SCLC cells grown SC in nude mice. Adriamycin (ADM) and cisplatin (CDDP), on the other hand, were effective only against the parental cells, while these drugs failed to inhibit the respective drug-resistant variants in vitro or in vivo. TAS-103 was observed to induce apoptosis dose dependently in the parental as well as drug-resistant SCLC cells as analyzed after 48 h of in vitro treatment, suggesting that the stabilization of cleavable topo I- or II-DNA complexes by topo I and II inhibitors like TAS-103 is followed by apoptosis of the cells. Overall, our study suggests that TAS-103 may have clinical application against drug-resistant human SCLC.


Heterogeneity of multiorgan metastases of human lung cancer cells genetically engineered to produce cytokines and reversal using chimeric monoclonal antibodies in natural killer cell-depleted severe combined immunodeficient mice

Lung cancer is a major cause of cancer deaths, most of which can be attributed to distant multiorgan metastases. To examine the cellular and molecular mechanisms of lung cancer metastasis to distant organs, we have established novel models of human lung cancer (small cell and non-small cell lung cancer) metastasis in natural killer cell-depleted severe combined immunodeficient (SCID) mice. We investigated whether local production of the cytokines responsible for regulation of macrophage function at tumor growth sites affects the pattern of lung cancer metastasis in distant organs. Several lung cancer cell lines were genetically engineered to produce human macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein-1 (MCP-1), and their metastatic potentials were assessed. Interestingly, M-CSF gene transduction had an antimetastatic effect for the liver and lymph nodes, but not the kidneys. In contrast, MCP-1 gene-modified lung cancer cells and their parent cells had identical metastatic potentials. These findings indicate a possible role for cytokines and suggest that lung cancer has metastatic heterogeneity. Examining ways of controlling human lung cancer metastases, we investigated the antimetastatic effect of chimeric monoclonal antibodies (MAbs) against P-glycoprotein and ganglioside GM2 (MH162 and KM966, respectively). Both MAbs, when given on days 2 and 7, inhibited the development of distant metastases of lung cancer in a dose-dependent fashion. Combined use of anti-P-glycoprotein MAb with M-CSF or MCP-1 gene transduction caused complete inhibition of metastasis of H69/VP cells. The antimetastatic effect of these MAbs in vivo was mainly due to an antibody-dependent cell-mediated cytotoxicity reaction mediated by mouse macrophages. These findings suggest that the mouse-human chimeric MAb in combination with cytokine gene transduction may be useful for the eradication of lung cancer metastases in humans.


Therapeutic efficacy of mouse-human chimeric anti-ganglioside GM2 monoclonal antibody against multiple organ micrometastases of human lung cancer in NK cell-depleted SCID mice

The development of distant metastases to multiple organs is a critical problem in the treatment of human lung cancer. In this study, we evaluated the therapeutic efficacy of a mouse-human chimeric anti-ganglioside GM2 (GM2) monoclonal antibody (MAb), KM966 against metastasis formation of GM2-positive human lung cancer cells inoculated intravenously (i.v.) into natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice. GM2-positive human small cell lung cancer (SCLC), SBC-3 cells (1 x 10(6)), injected through a tail vein into NK cell-depleted SCID mice, formed large number of metastatic colonies in the liver, kidneys and lymph nodes by 42 days after inoculation (day 42). KM966, but not control MAb, given on days 2 and 7, almost completely inhibited metastasis formation of SBC-3 cells in the liver, kidneys and lymph nodes in a dose-dependent fashion. Moreover, treatment with KM966 at advanced stages of metastasis (even from day 28) significantly suppressed multiple organ metastases of SBC-3 cells. The anti-metastatic effect of KM966 in vivo was mainly due to an antibody-dependent cell-mediated cytotoxicity (ADCC) reaction mediated by macrophages of the SCID mice. Our findings suggest that the mouse-human chimeric anti-GM2 MAb, KM966 may be useful for eradicating multiple organ micrometastases of lung cancer in humans.


Circumvention of multidrug resistance by a quinoline derivative, MS-209, in multidrug-resistant human small-cell lung cancer cells and its synergistic interaction with cyclosporin A or verapamil

To develop a clinically useful approach to circumvent P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MDR human small-cell lung cancer (SCLC), we examined the ability of a novel quinoline compound, MS-209, to reverse MDR by inhibition of P-gp function in combination with other MDR-reversing drugs using a cytotoxicity assay. We established MDR human SCLC cells by culture in medium with gradually increasing concentrations of adriamycin (ADM). Compared with the parental human SCLC cells, SBC-3, the MDR variant SBC-3 cells obtained (SBC-3/ADM) were highly resistant to various chemotherapeutic agents due to P-gp expression. MS-209 reversed the resistance to ADM and vincristine (VCR) of SBC-3/ADM and H69/VP cells in a dose-dependent manner. Moreover, MS-209 in combination with cyclosporin A (CsA) or verapamil (VER) synergistically enhanced the antitumor effects of ADM and VCR on SBC-3/ADM cells. MS-209 restored ADM incorporation and this effect was enhanced by CsA and VER, suggesting that these synergistic effects were due to competitive inhibition of P-gp function. MS-209 in combination with CsA or VER might increase the efficacy of these chemotherapeutic agents against MDR human SCLC cells.


An autopsy case of cytokeratin 7-positive minute adenocarcinoma of the lung with systemic metastases

We describe a 60-year-old woman with leg pain. Although metastatic bone tumor and atypical cells mimicking signet-ring cells in the bone marrow picture were observed, systemic survey revealed no primary lesion. The patient died two months after admission from systemic progress of the disease. Autopsy revealed a small focus of adenocarcinoma within the right upper lobe of the lung and systemic metastases without any particular changes in the gastrointestinal tract. The tumor cells of the lung were diffusely positive for cytokeratin 7, whereas cytokeratin 20 immunoreactivity was weak and focal, and that supported the lung origin of the present tumor. Moreover, the tumor cells in the bone marrow showed a similar pattern in immunoreactivity. These findings suggest that cytokeratin 7 and cytokeratin 20 immunoreactivity is helpful for the premortem diagnosis of the metastatic tumor of unknown origin.