Stretch induces a growth factor in alveolar cells via protein kinase.

Positive-pressure mechanical ventilation can injure the lung, causing edema and alveolar inflammation in a complication termed ventilator-induced lung injury (VILI). Cytokines such as interleukin-8 (IL-8) reportedly are important in this inflammatory response. On the other hand, hepatocyte growth factor (HGF) promotes regeneration of the lung, and delays pulmonary fibrosis. We postulated that cyclic stretch upregulates production and release of both of mediators. Human alveolar epithelial cells (A549) cultured on a silicoelastic membrane were tested for mRNA expression and release of IL-8 and HGF after cyclic stretch in vitro. Stretch induced mRNA expression and release of these mediators. The signaling pathway from cyclic stretch to release of IL-8 and HGF appeared to involve protein kinase C in the signal transduction pathway.

Double-stranded rna dependence of nitric oxide synthase 2 expression in human bronchial epithelial cell lines BET-1A and BEAS-2B.

The human airway epithelium expresses abundant nitric oxide synthase 2 (NOS2) in vivo. Although NOS2 is easily induced by cytokines in primary cultured human airway epithelial cells and lung adenocarcinoma cell line A549, the human bronchial epithelial cell lines BEAS-2B and BET-1A do not express NOS2 in response to cytokines. Mechanisms regulating NOS2 expression in human respiratory epithelial cells are complex, but we have recently shown that NOS2 expression in primary human airway epithelial cells occurs in response to double-stranded RNA (dsRNA) through activation of signaling proteins including nuclear factor (NF)-kappaB and interferon (IFN) regulatory factor (IRF)-1. In this context, we hypothesized that BEAS-2B and BET-1A cells may express NOS2 in response to dsRNA. Here, we show that although cytokines (IFN-gamma, tumor necrosis factor-alpha and interleukin-1beta) do not induce NOS2 expression in BEAS-2B or BET-1A cells, addition of dsRNA to this cytokine mix enables BEAS-2B cells to express NOS2. IFN-gamma and dsRNA induction of NOS2 in BET-1A cells occurs in a serum concentration-dependent manner, with a minimum of 3 d of serum treatment necessary for BET-1A cells to acquire the potential to induce NOS2. Importantly, dsRNA strongly activates NF-kappaB and IRF-1 in BEAS-2B cells, transcription factors essential for NOS2 gene expression in other cell lines. On the basis of these results, dsRNA-activated signaling pathways are clearly important for NOS2 expression in human respiratory epithelial cells. With conditions for NOS2 expression characterized, these cell lines are a convenient in vitro system to investigate the mechanisms regulating NOS2 expression in human respiratory epithelial cells.

Nitric oxide synthase 2 through an autocrine loop via respiratory epithelial cell-derived mediator.

Respiratory epithelium expresses nitric oxide synthase 2 (NOS2) continuously in vivo; however, mechanisms responsible for its expression are only partially understood. We definitively identify an autocrine mechanism of induction and maintenance of NOS2 in human airway epithelial cells through the synthesis and secretion of a soluble mediator. Short exposure of human airway cells to interferon (IFN)-gamma leads to prolonged NOS2 expression. Transfer of the overlying culture medium (conditioned medium) induces NOS2 expression in other airway epithelial cells, suggesting the presence of an intermediary substance regulating NOS2 expression in an autocrine loop. Characterization of the soluble mediator reveals that it is stable and transferable in conditioned medium for up to 7 days. However, soluble mediator does not induce NOS2 mRNA in human alveolar macrophages, indicating that the response to soluble mediator is unique to human respiratory epithelium. Soluble mediator is heat labile but is not inactivated by acid treatment, unlike IFN-gamma itself. Importantly, IFN regulatory factor-1, which is critical for murine NOS2 expression, is expressed and activated by soluble mediator through the signal transducer and activator of transcription-1-dependent pathway. Based on these findings, we propose novel regulatory mechanisms for NOS2 expression in human airway epithelium.

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase.

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.

A dedicated Z-stent for acquired saber-sheath tracheobronchomalacia.

The tracheobronchial lumen has a continuous horseshoe arch morphology. We formed Z-stents accordingly to support the weakened cartilagenous portions. With this type of stent we treated a patient with acquired saber-sheath type tracheobronchomalacia (TBM), Rayl's type II, Johnson's grade III, whose condition was aggravated even under positive end expiratory pressure (PEEP) therapy. The patient improved gradually. No immediate complication was observed. Bronchofiberscopic examination revealed that the tracheobronchial arcade was closely strut-braced and showed no expiratory collapse. Six months later, when the patient was intubated due to asthmatic attacks, tissue ingrowth through the stent was found and removed. There was no recurrence of TBM. The patient died 2 years later of pneumoconiosis.

Interferon gamma and interleukin 4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators.

Human respiratory epithelium expresses inducible nitric oxide synthase (iNOS) continuously in vivo, however mechanisms responsible for maintenance of expression are not known. We show that IFNgamma is sufficient for induction of iNOS in primary human airway epithelial cells (HAEC) in vitro, and IL-4 potentiates IFNgamma-induced iNOS expression in HAEC through stabilization of iNOS mRNA. IFNgamma/IL-4- induced iNOS expression in HAEC was delayed in onset and prolonged with expression up to 1 wk. Removal of overlying culture media resulted in loss of expression, while transfer of conditioned media induced iNOS mRNA in other HAEC. IFNgamma and IL-4 stimulation activated STAT1 and STAT6 in HAEC, but conditioned media transfer to HAEC produced even higher levels of STAT1 activation than achieved by direct addition of cytokines. Although cytokine induction of iNOS was dependent on new protein synthesis, conditioned media induction of iNOS in HAEC was not. Further, removal of overlying culture media from cells at different times after cytokine stimulation demonstrated that mediator synthesis and/or secretion important for induction and maintenance of iNOS occurs early after cytokine stimulation. In conclusion, a combination of IFNgamma/ IL-4, which occurs naturally in the lung epithelial lining fluid, leads to maintenance of iNOS expression in human airway epithelium through production of soluble mediators and stabilization of mRNA.

1 alpha, 25-dihydroxyvitamin D3 and all-trans-retinoic acid inhibit the growth of a lung cancer cell line.

The secosteroid 1 alpha, 25-dihydroxyvitamin D3 (calcitriol) and retinoic acid are the major biologically active metabolites of vitamins D and A, respectively. Their antitumor activity has been observed in several cancer cells in vitro apart from lung cancer cells. The purpose of this study was to examine the possible effects of the agents on lung cancer cell lines. The responses of five lung cancer cell lines to calcitriol or all-transretinoic acid (RA) were assessed by a colorimetric MTT assay. Calcitriol inhibited growth in one of the tested cell lines, i.e. EBC-1 squamous cell carcinoma, dose dependently. RA also exhibited the same effect in EBC-1 cells. However neither agent affected the growth of other lung cancer cell lines. Subsequently we examined the mRNA expression of vitamin D receptor (VDR) and retinoic acid receptor (RAR alpha) in these lung cancer cells by quantitative RT-PCR. EBC-1 cells expressed high levels of mRNA for both VDR and RAR alpha, while other cell lines expressed much lower mRNA levels for the receptors. These data suggest that the growth inhibitory effects of the vitamins are associated with mRNA expression for VDR and RAR alpha.

[A case of chronic hypersensitivity pneumonitis due to long term exposure to toluene diisocyanate].

A 55-year-old paint sprayer, who had been working with paint containing toluence diisocyanate since age 20, was admitted to our hospital with a complaint of exertional dyspnea. Physical examination revealed clubbed fingers, and fine crackles were audible in both lower lung fields. The thoracic CT film showed diffuse linear and ringed shadows in both lung fields. Open lung biopsy disclosed alveolitis and fibrosis as well as infiltration of mononuclear cells, but no Masson bodies or granulomas. Toluene diisocyanate-specific antibody was positive. Based on these results, we diagnosed chronic hypersensitivity pneumonitis due to the chemical. To our knowledge, there has been no previously reported case of chronic hypersensitivity pneumonitis due to isocyanate.

Stimulatory effect of 1 alpha,25-dihydroxyvitamin D3 on mouse alveolar macrophage tumor necrosis factor-alpha production in vitro: involvement of protein kinase C and Ca2+/calmodulin-dependent kinase.

1 alpha,25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3, calcitriol] has been shown to modulate the immune function of peripheral monocytes and peritoneal macrophages. However, its effect on alveolar macrophage (AM) cytokine secretion has not been reported. We therefore investigated the influence of calcitriol on tumor necrosis factor (TNF-alpha) production by murine AMs and attempted to elucidate changes in the signal transduction system involved in such effects. Calcitriol significantly enhanced TNF-alpha secretion by AM stimulated with either lipopolysaccharide (LPS; 10 micrograms/ml; p < 0.005) or phorbol 12-myristate 13-acetate (PMA; 100 ng/ml; p < 0.05) at low doses (between 10(-11) and 10(-9) M). However the protein kinase C (PKC) inhibitor, H7 (10 microM), and the Ca2+/calmodulin inhibitor, W7 (25 microM), reversed such calcitriol effects. Calcitriol increased the total PKC activity of AMs. These findings indicate that calcitriol enhances both LPS- and PMA-stimulated TNF-alpha secretion through PKC- or Ca2+/calmodulin-dependent pathways.

[Influence of age on mouse pulmonary alveolar macrophage clonal growth].

Although monocyte influx has been suggested as the primary source of pulmonary alveolar macrophages (AM), increasing evidence from recent studies has indicated that AM may be sustained through a self-renewal mechanism. We evaluated the age-related changes of the clonal growth (colony formation) of AM in mice (C57BL/6N mice and senescence accelerated mice). The colony forming unit (CFU) of AM of 24 month old C57BL/6N mice was lower than that of AM of 4-month-old mice (p < 0.05). In SAMP6 (senescence accelerated mice), CFU of AM was decreased with aging (p < 0.05). In SAMR1 (controls for SAMP6), CFU of AM was decreased with aging (p < 0.001). In SAMR1, CFU of bone marrow (BM) adherent cells of 12-month-old mice was similar to that of 4-month-old mice. In SAMP6, CFU of BM adherent cells of 12-month-old mice was larger than that of 4-month-old mice (P < 0.005). It was concluded that the CFU of AM declined with aging, but the CFU of the BM adherent cells did not. The decline of the AM CFU may be partly responsible for the defect of the immune response of the alveolar space in the elderly.

Effect of aging on plasma 1,5-anhydroglucitol levels in humans and rats.

Since normal reference values change with age in some clinical parameters, we measured the plasma levels of 1,5-anhydroglucitol (AG), a new marker of glycemic control in diabetes mellitus, in healthy subjects and in rats. Our results showed a significantly negative correlation of the marker with age in humans and that the plasma AG levels of older rats were markedly lower than those of younger counterparts. This remarkable reduction of AG in the older rat group can be partially explained by our finding that aged animals excreted AG more rapidly in the urine than younger ones, besides a decrease in food intake. We therefore suggest that normal clinical reference values for plasma AG levels should be modified according to age.

Pharmacokinetic re-evaluation and phase I study of high dose epirubicin in advanced non-small cell lung cancer.

We performed a phase I trial to evaluate the toxicity and the maximum tolerated dose of high dose epirubicin on a three-consecutive-day schedule on Japanese patients with advanced non-small cell lung cancer. Fourteen patients were entered in the study. At least three patients were assigned to each different dose level. Epirubicin was given intravenously daily for three day by bolus injection. The dose was started at 60 mg/m2/course and escalated by 30 mg/m2/course. Granulocytopenia was found to be the dose limiting toxicity with a maximum tolerated dose of 150 mg/m2/course. Thrombocytopenia and non-hematological toxicities were mild and well tolerated. The maximum tolerated dose was lower than that in Europe and Canada. Partial responses were observed in two out of five patients on 150 mg/m2/course. The recommended phase II dose for high dose epirubicin was demonstrated to be 120 mg/m2/course. A further dose-escalating study of epirubicin in conjunction with the administration of granulocyte colony stimulating factor is scheduled for the determination of its antitumor activity in non-small cell lung cancer.

[Effects of aerosol oxitropium bromide and fenoterol on maximal exercise capacity in chronic obstructive pulmonary disease and their correlation with air flow during exercise and with parameters of maximal exercise].

To examine the effects of bronchodilators on maximal exercise capacity and their correlation with airflow during exercise in patients with chronic obstructive pulmonary disease (COPD), we conducted a double-blind, randomized comparison between inhaled fenoterol (beta 2-agonist) and oxitropium bromide (anticholinergic agent) in 8 patients with stable COPD (mean age 73 years, mean FEV1 1.1 L, mean FEV1% 50%). Only oxitropium bromide resulted in statistically significant improvement in FEV1 40 min after inhalation. On maximal exercise, fenoterol did not affect oxygen uptake (VO2 max), minute ventilation (VEmax), respiratory frequency (Rfmax), ventilatory efficacy (VEmax/VO2 max), peak expiratory flow during exercise (PEFmax), heart rate (HRmax) and dyspnea (Borg Scale Slope). After oxitropium bromide, dyspnea during exercise and HRmax decreased significantly, but PEFmax and other parameters did not change significantly compared with control. There was no correlation between changes in dyspnea during exercise and changes in FEV1 and PEFmax after oxitropium bromide inhalation. We conclude that inhaled oxitropium bromide, an anticholinergic agent, reduces dyspnea during exercise in patients with COPD. This favorable effect was not due to change of airflow limitation during exercise, and other factors can thus influence reduction of dyspnea during exercise in these patients.

[A case of pulmonary embolism probably induced by long term use of oral contraceptive].

We present a 53-year-old female with pulmonary embolism (PE), who had been taking oral contraceptive for 13 years. She was admitted to our hospital with upper abdominal pain and was found to be in shock. The diagnosis of PE was made from chest X-ray examination, electrocardiogram and pulmonary angiography. Pulmonary hypertension was observed on right heart catheterization, and an anticoagulant was administered. Multiple defects of the right pulmonary artery were detected on lung perfusion scan, and there were no significant findings on leg phlebogram. Home oxygen therapy was effective for the treatment of pulmonary hypertension and chronic hypoxemia which still persisted after her recovery from the acute stage. We are concerned that wide spread use of oral contraceptives will increase the incidence of PE in the near future in this country. We conclude that contraceptive users should be warned of their higher risk of PE, and that they should visit a clinic for examination.

[A case of Wegener's granulomatosis complicated by hypopituitarism].

A 71-year-old male complaining of chest pain was admitted to our hospital. A single cavitary mass shadow was observed on chest X-ray films. Urinalysis revealed microscopic hematuria. CT examination demonstrated a tumorous shadow in the maxillary sinus. The diagnosis of Wegener's granulomatosis was histologically established by biopsy specimens from the nasal mucosa which showed necrotizing vasculitis and granuloma with fibrinoid degeneration. He was treated with combination therapy of prednisolone and cyclophosphamide. The abnormal shadows on chest X-ray and in the maxillary sinus on CT improved rapidly, but the patient developed progressive weight loss and complained of cold intolerance, weakness and dysphagia. Serum T3, T4 and TSH were found to be reduced. Anterior pituitary function tests showed reduction of TSH, GH and ACTH responses, which was probably due to irreversible vasculitis.

Idiopathic acute eosinophilic pneumonia.

A previously healthy young man presented with acute respiratory distress, high fever and bilateral ground-glass appearance on chest radiograph. Bronchoalveolar lavage analysis demonstrated significant eosinophilia (72%) with no evidence of infection. The transbronchial lung biopsy showed that the walls of bronchioli and alveolar septa were markedly infiltrated with eosinophils. The patient rapidly improved with corticosteroid therapy. This case exemplifies the recently described idiopathic acute eosinophilic pneumonia. Similar cases published in the Japanese literature were reviewed and discussed.