Kazue Fujita

Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis

BackgroundBone marrow-derived fibrocytes reportedly play important roles in the pathogenesis of idiopathic pulmonary fibrosis. Pirfenidone is an anti-fibrotic agent; however, its effects on fibrocytes have not been investigated. The aim of this study was to investigate whether pirfenidone inhibits fibrocyte pool size in the lungs of bleomycin-treated mice.MethodsBleomycin (100 mg/kg) was infused with osmotic pumps into C57BL/6 mice, and pirfenidone (300 mg/kg/day) was orally administered daily for 2 wk. The lungs were removed, and single-cell suspensions were subjected to fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes, which were defined as CD45 and collagen-I double-positive cells. Immunohistochemistry was performed on the lung specimens to quantify fibrocytes. Chemokines in the lung digests were measured with enzyme-linked immunosorbent assay. The effect of pirfenidone on alveolar macrophages was evaluated with bronchoalveolar lavage (BAL). In a therapeutic setting, pirfenidone administration was initiated 10 days after bleomycin treatment. For chemotaxis assay, lung fibrocytes were isolated with immunomagnetic selection (CD45-positive mesenchymal cells) after culture and allowed to migrate toward chemokines in the presence or absence of pirfenidone. Moreover, the effect of pirfenidone on the expression of chemokine receptors on fibrocytes was evaluated.ResultsPirfenidone significantly ameliorated bleomycin-induced pulmonary fibrosis as assessed with quantitative histology and collagen measurement. Fibrocyte pool size in bleomycin-treated mice lungs was attenuated from 26.5% to 13.7% by pirfenidone on FACS analysis. This outcome was also observed in a therapeutic setting. Immunohistochemistry revealed that fibrocytes were significantly decreased by pirfenidone administration compared with those in bleomycin-treated mice (P = 0.0097). Increased chemokine (CC motif) ligand-2 (CCL2) and CCL12 production in bleomycin-treated mouse lungs was significantly attenuated by pirfenidone (P = 0.0003 and P < 0.0001, respectively). Pirfenidone also attenuated macrophage counts stimulated by bleomycin in BAL fluid. Fibrocyte migration toward CCL2 and chemokine (CC motif) receptor-2 expression on fibrocytes was significantly inhibited by pirfenidone in vitro.ConclusionsPirfenidone attenuated the fibrocyte pool size in bleomycin-treated mouse lungs via attenuation of CCL2 and CCL12 production in vivo, and fibrocyte migration was inhibited by pirfenidone in vitro. Fibrocyte inhibition is considered a mechanism of anti-fibrotic action of pirfenidone.

Sequential changes of Legionella antigens and bacterial load in the lungs and urines of a mouse model of pneumonia.

Legionella pneumophila is an important cause of community- and hospital-acquired pneumonia. In spite of the introduction of the urinary antigen detection method, Legionella pneumonia may be still underdiagnosed. We performed kinetic and quantitative analysis of diagnostic markers, such as bacterial loads, DNA assays, and antigen titers, in a 28-day time course murine model of L. pneumophila pneumonia. L. pneumophila replicated approximately 100-fold in the lungs of A/J mice in the first 48 h, and then became undetectable on day 14. Unexpectedly, pathogens other than L. pneumophila were consistently recovered from the lungs and livers at the acute phases, although those numbers were far below Legionella loads in the lungs. The peaks of specific antigen titer were observed on 48 h in the lungs, bronchoalveolar lavage (BAL) fluids, and urines and sustained positive even at 28 days after the infection. Especially, the lung homogenates and BAL fluids demonstrated 16 to 64 times higher levels of antigen titer than the urines by the end of observation. Legionella-specific DNA in the lungs was detected by polymerase chain reaction and loop-mediated isothermal amplification methods until 7 and 14 days after the infection, respectively. The inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, interleukin 6, and MIP-2, exhibited a peak on the acute phase, whereas the maximal production of high mobility group box 1 in the serum was observed on day 7. These results characterized the kinetic nature of diagnostic markers in L. pneumophila pneumonia. The present data suggested prolonged and compartmentalized deposition of antigen in the lungs, which may have an impact on the diagnosis of L. pneumophila pneumonia, especially in missed cases even after recovery from disease.

Nintedanib modulates surfactant protein-D expression in A549 human lung epithelial cells via the c-Jun N-terminal kinase-activator protein-1 pathway.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a high mortality rate. Signalling pathways activated by several tyrosine kinase receptors are known to be involved in lung fibrosis, and this knowledge has led to the development of the triple tyrosine kinase inhibitor nintedanib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), for the treatment of IPF. Pulmonary surfactant protein D (SP-D), an important biomarker of IPF, reportedly attenuates bleomycin-induced pulmonary fibrosis in mice. In this study, we investigated whether nintedanib modulates SP-D expression in human lung epithelial (A549) cells using quantitative real-time reverse transcriptase polymerase chain reaction and western blotting. To investigate the mechanisms underlying the effects of nintedanib, we evaluated the phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream target c-Jun. The effect of the JNK inhibitor SP600125 on c-Jun phosphorylation was also tested. Activation of activator protein-1 (AP-1) was examined using an enzyme-linked immunosorbent assay-based test, and cell proliferation assays were performed to estimate the effect of nintedanib on cell proliferation. Furthermore, we treated mice with nintedanib to examine its in vivo effect on SP-D levels in lungs. These experiments showed that nintedanib up-regulated SP-D messenger RNA expression in a dose-dependent manner at concentrations up to 5 μM, with significant SP-D induction observed at concentrations of 3 μM and 5 μM, in comparison with that observed in vehicle controls. Nintedanib stimulated a rapid increase in phosphorylated JNK in A549 cells within 30 min of treatment and stimulated c-Jun phosphorylation, which was inhibited by the JNK inhibitor SP600125. Additionally, nintedanib was found to activate AP-1. A549 cell proliferation was not affected by nintedanib at any of the tested concentrations. Moreover, blocking FGFR, PDGFR, and VEGFR function did not affect nintedanib-induced SP-D expression, suggesting that nintedanib mediates its effects through a mechanism that is distinct from its known role as a tyrosine kinase inhibitor. Nintedanib is also reported to inhibit Src kinase although pre-treatment of cells with a Src kinase inhibitor had no effect on nintedanib-induced SP-D expression. Increased expression of SFTPD mRNA and SP-D protein in the lungs of nintedanib-treated mice was also observed. In this work, we demonstrated that nintedanib up-regulated SP-D expression in A549 cells via the JNK-AP-1 pathway and did not affect cell proliferation. This is the first report describing SP-D induction by nintedanib.

Fatal Pneumonia Associated with Temozolomide Therapy in Patients with Malignant Glioma

This report presents the cases of three patients with fatal pneumonia that was highly suspected to be Pneumocystis pneumonia (PCP) based on serological diagnosis. Their chest radiographs showed bilateral pneumonia and each had presented with severe respiratory failure requiring mechanical ventilation when they arrived at the hospital. Although bronchoscopical sampling could not be performed, their chest computed tomography imaging and a marked elevation of serum KL-6 and β-D-glucan levels were characteristic of Pneumocystis pneumonia. All three were found to have been treated with temozolomide after surgery for malignant glioma. Temozolomide can cause Pneumocystis pneumonia. The three patients did not receive prophylactic medication against Pneumocystis pneumonia during treatment with temozolomide, and their histories suggested that all had delayed seeking treatment. It may be difficult to diagnose Pneumocystis pneumonia because the symptoms are not specific for Pneumocystis pneumonia and they tend to be similar to those of common respiratory infectious diseases. Therefore, patients who receive temozolomide therapy have the potential to develop fatal pneumonia and should be carefully observed. The patients should also be adequately informed about Pneumocystis pneumonia, and prophylaxis against Pneumocystis pneumonia should be considered proactively before treatment with temozolomide is initiated.

Prognostic Factors in the Acute Exacerbation of Idiopathic Pulmonary Fibrosis: A Retrospective Single-center Study

Objectives Acute exacerbation of idiopathic pulmonary fibrosis (IPF-AE) has been recognized as a fatal pulmonary disorder, but the exact prognostic factors are unknown. The aim of the present study was to analyze the clinical characteristics of patients with IPF-AE and identify the prognostic factors. Methods The medical records of 59 cases of IPF-AE were retrospectively reviewed. Clinical data, laboratory data, radiographic findings, treatment, and time from the onset of symptoms to the initiation of corticosteroid pulse therapy, i.e. symptom duration, and outcome were analyzed. Results The IPF Stage, Gender-Age-Physiology (GAP) Index, symptom duration, and the high-resolution computed tomography (HRCT) score were significantly related to the prognosis in the univariate analysis. In the multivariate analysis, the symptom duration remained a significant prognostic factor (hazard ratio of 1-day increase, 1.11; 95% confidence interval, 1.01-1.15; p=0.0427). The area under the receiver operating characteristics curve of symptom duration was statistically significant for survivors versus non-survivors (area under the curve, 0.73; p=0.012). The survival period was significantly shorter in the late-treatment groups (≥5 days; n=30) than in the early-treatment groups (<5 days; n=29; log-rank test; p<0.0001). Conclusion The time interval between the onset of symptoms and the initiation of corticosteroid pulse therapy may be an independent prognostic factor in patients with IPF-AE.

A case of cutaneous metastatic lung cancer difficult to distinguish from malignant nodular hidradenoma

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