Induction of a memory-like CD4+ T-cell phenotype by airway smooth muscle cells.

In asthma, CD4+ T-cell interaction with airway smooth muscle (ASM) may enhance its contractile properties and promote its proliferation. However, less is known about the effects of this interaction on T cells. To explore the consequences of interaction of CD4+ T cells with ASM we placed the cells in co-culture and analyzed the phenotypic and functional changes in the T cells. Effector status as well as cytokine expression was assessed by flow cytometry. An increase in CD45RA-CD45RO+ memory T cells was observed after co-culture; however, these cells were not more responsive to CD3/28 restimulation. A reduction in mitochondrial coupling and an increase in the production of mitochondrial reactive oxygen species by CD4+ T cells post-restimulation suggested altered mitochondrial metabolism after co-culture. RNA sequencing analysis of the T cells revealed characteristic downregulation of effector T-cell-associated genes, but a lack of upregulation of memory T-cell-associated genes. The results of this study demonstrate that ASM cells can induce a phenotypic shift in CD4+ T cells into memory-like T cells but with reduced capacity for activation.

[Current status of breast cancer undetected by mammography in population-based breast cancer screening and associated factors].

Objective　This study aimed to promote the application of mammography (MMG) screening without performing a clinical breast examination (CBE). It examined population-based screening data, including history taking findings, to elucidate the status of breast cancer cases, detected solely by CBE, and to reveal the factors associated with breast cancer. Through this, it explored alternative methods for evaluating breast cancer cases, undetected by MMG, when CBE is omitted.Methods　The linked anonymized data from women, who underwent breast cancer screening in 2014, 2016, or 2017, were prepared. The data were obtained from the Nishinomiya City database. Breast cancer, undetected by MMG, were defined as breast cancer cases diagnosed by close examination based on CBE only (MMG findings were category 2 or lower, with no abnormalities). To assess the quality of breast cancer screening, process indices were calculated for the overall population, and for patients indicated for close examination based on CBE but not MMG. The association of breast cancer with each factor was statistically analyzed (χ2 test, etc.).Results　In total, 13,504 women underwent breast cancer screening. Close examination was required in 1,247 women (9.2%). Breast cancer was diagnosed in 44 women (3.5%), including four, who had breast cancer undetected by MMG. All of the process indices satisfied the acceptable values. Three of the four women with breast cancer, undetected by MMG, noticed a lump. Breast cancer was significantly associated with "subjective symptoms". The presence of a "lump" and "nipple discharge" were significantly more common in breast cancer patients.Conclusion　Three of the four breast cancer cases, undetected by MMG screening, had a subjective symptom (lump). There was a significant association between subjective symptoms (lump and nipple discharge) and breast cancer. To avoid missing breast cancer, undetected by MMG alone, these symptoms should be especially evaluated in women to compensate for the omission of a CBE. This can be achieved by encouraging women experiencing symptoms to seek consult, performing a thorough history-taking and observation, engaging in multidisciplinary collaboration (communication from medical staff to physicians), and promoting breast awareness.

Naftopidil reduced the proliferation of lung fibroblasts and bleomycin-induced lung fibrosis in mice.

Naftopidil, an α-1 adrenoceptor antagonist with few adverse effects, is prescribed for prostate hyperplasia. Naftopidil inhibits prostate fibroblast proliferation; however, its effects on lung fibroblasts and fibrosis remain largely unknown. Two normal and one idiopathic pulmonary fibrosis human lung fibroblast lines were cultured with various naftopidil concentrations with or without phenoxybenzamine, an irreversible α-1 adrenoceptor inhibitor. We examined the incorporation of 5-bromo-2'-deoxyuridine into DNA and lactic acid dehydrogenase release by enzyme-linked immunosorbent assay, cell cycle analysis by flow cytometry, scratch wound-healing assay, and mRNA expressions of type IV collagen and α-smooth muscle actin by polymerase chain reaction. Effects of naftopidil on bleomycin-induced lung fibrosis in mice were evaluated using histology, micro-computed tomography, and surfactant protein-D levels in serum. Naftopidil, dose-dependently but independently of phenoxybenzamine, inhibited 5-bromo-2'-deoxyuridine incorporation in lung fibroblasts. Naftopidil induced G1 cell cycle arrest, but lactic acid dehydrogenase release and migration ability of lung fibroblasts were unaffected. Naftopidil decreased mRNA expressions of type IV collagen and α-smooth muscle actin in one normal lung fibroblast line. Histological and micro-computed tomography examination revealed that naftopidil attenuated lung fibrosis and decreased serum surfactant protein-D levels in bleomycin-induced lung fibrosis in mice. In conclusion, naftopidil may have therapeutic effects on lung fibrosis.

CISH is a negative regulator of IL-13-induced CCL26 production in lung fibroblasts.

BACKGROUND: Bronchial asthma is a chronic airway disease characterized by eosinophilic airway inflammation. Lung fibroblasts activated by IL-13 serve as important sources of chemokines, such as eotaxins, contributing to persistent eosinophilic inflammation. Src-homology 2-containing protein (CISH), belonging to the suppressor of cytokine signaling (SOCS) family, acts as a negative regulator of cytokine induction. The aim of this study was to elucidate the role of CISH in the production of eosinophil chemotactic chemokines in human lung fibroblasts. METHODS: Normal human lung fibroblasts were stimulated by IL-13, and global gene expression profile was assessed by cDNA microarray. Expression changes and downstream of IL-13 signaling were evaluated by quantitative RT-PCR, ELISA or western blotting. Loss- and gain-of-function analyses of CISH were performed by small interfering RNA and vector overexpression, respectively. RESULTS: Ingenuity pathway analysis revealed that IL-13 induced chemokine signaling, including the eotaxin family, while significantly suppressing IFN-α/ß signaling. Among eight SOCS family members, CISH was most strongly induced by IL-13 via phosphorylation of signal transducer and activator of transcription 6 (STAT6). Loss- and gain-of-function studies demonstrated that CISH negatively regulated the expression of CCL26. CONCLUSIONS: These findings suggest that CISH plays a key role in the eosinophilic inflammation associated with bronchial asthma by regulating IL-13-induced CCL26 production. Augmentation of CISH function could be a novel approach for treating eosinophilic inflammation in severe asthma.

Mechanism of Periostin Production in Human Bronchial Smooth Muscle Cells.

BACKGROUND: Asthma is a chronic airway inflammatory disease characterized by airway remodeling, in which the bronchial smooth muscle (BSM) cells play an important role. Periostin, a biomarker that reflects Th2-driven inflammatory diseases such as asthma, may play an important role in the asthmatic airway. Although periostin is mainly produced in airway epithelial cells and fibroblasts after interleukin (IL)-13 stimulation, whether BSM cells produce periostin remains unclear. Therefore, we investigated periostin production in BSM cells and the mechanisms involved. METHODS: Human BSM cells were cultured, and the effect of IL-13 stimulation on periostin production was evaluated using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). We evaluated the phosphorylation of signal transducer and activator of transcription factor 6 (STAT6), extracellular signal-regulated kinase (ERK)1/2, and Akt after IL-13 stimulation. Furthermore, using ELISA, we evaluated the influence of several phosphorylation inhibitors on periostin production. RESULTS: Periostin mRNA expression increased in a dose- and time-dependent manner after IL-13 stimulation; periostin production was induced 24 and 48 h after stimulation. IL-13 stimulation induced the phosphorylation of STAT6, ERK1/2, and Akt. IL-13-induced periostin production was attenuated by inhibiting STAT6 phosphorylation and strongly suppressed by inhibiting mitogen-activated protein kinase kinase 1/2 phosphorylation or phosphatidylinositol 3-kinase (PI3K) phosphorylation. CONCLUSIONS: BSM cells produced periostin after IL-13 stimulation, via the JAK/STAT6, ERK1/2, and PI3K/Akt pathways. Understanding the mechanism of periostin production in BSM cells may help to clarify asthma pathogenesis.

Lysophosphatidylcholine Acyltransferase 1 Deficiency Promotes Pulmonary Emphysema via Apoptosis of Alveolar Epithelial Cells.

Chronic obstructive pulmonary disease (COPD) is primarily caused by inhalation of cigarette smoke and is the third leading cause of death worldwide. Pulmonary surfactant, a complex of phospholipids and proteins, plays an essential role in respiration by reducing the surface tension in the alveoli. Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is an enzyme that catalyzes the biosynthesis of surfactant lipids and is expressed in type 2 alveolar epithelial cells. Its dysfunction is suggested to be involved in various lung diseases; however, the relationship between LPCAT1 and COPD remains unclear. To investigate the role of LPCAT1 in the pathology of COPD, we analyzed an elastase-induced emphysema model using Lpcat1 knockout (KO) mice. In Lpcat1 KO mice, elastase-induced emphysema was significantly exacerbated with increased apoptotic cells, which was not ameliorated by supplementation with dipalmitoylphosphatidylcholine, which is a major component of the surfactant synthesized by LPCAT1. We subsequently evaluated the effects of cigarette smoking on primary human type 2 alveolar epithelial cells (hAEC2s) and found that cigarette smoke extract (CSE) downregulated the expression of Lpcat1. Furthermore, RNA sequencing analysis revealed that the apoptosis pathway was significantly enriched in CSE-treated primary hAEC2s. Finally, we downregulated the expression of Lpcat1 using small interfering RNA, which resulted in enhanced CSE-induced apoptosis in A549 cells. Taken together, cigarette smoke-induced downregulation of LPCAT1 can promote the exacerbation of pulmonary emphysema by increasing the susceptibility of alveolar epithelial cells to apoptosis, thereby suggesting that Lpcat1 is a novel therapeutic target for irreversible emphysema.

ASCL1 promotes tumor progression through cell-autonomous signaling and immune modulation in a subset of lung adenocarcinoma.

The master regulator of neuroendocrine differentiation, achaete-scute complex homolog 1 (ASCL1) defines a subgroup of lung adenocarcinoma. However, the mechanistic role of ASCL1 in lung tumorigenesis and its relation to the immune microenvironment is principally unknown. Here, the immune landscape of ASCL1-positive lung adenocarcinomas was characterized by immunohistochemistry. Furthermore, ASCL1 was transduced in mouse lung adenocarcinoma cell lines and comparative RNA-sequencing and secretome analyses were performed. The effects of ASCL1 on tumorigenesis were explored in an orthotopic syngeneic transplantation model. ASCL1-positive lung adenocarcinomas revealed lower infiltration of CD8+, CD4+, CD20+, and FOXP3+ lymphocytes and CD163+ macrophages indicating an immune desert phenotype. Ectopic ASCL1 upregulated cyclin transcript levels, stimulated cell proliferation, and enhanced tumor growth in mice. ASCL1 suppressed secretion of chemokines, including CCL20, CXCL2, CXCL10, and CXCL16, indicating effects on immune cell trafficking. In accordance with lower lymphocytes infiltration, ASCL1-positive lung adenocarcinomas demonstrated lower abundance of CXCR3-and CCR6-expressing cells. In conclusion, ASCL1 mediates its tumor-promoting effect not only through cell-autonomous signaling but also by modulating chemokine production and immune responses. These findings suggest that ASCL1-positive tumors represent a clinically relevant lung cancer entity.

Utility of bronchoscopy in the definitive diagnosis of patients with haematological malignancies presenting with radiological abnormalities.

INTRODUCTION: Patients with haematological malignancies usually have a plethora of respiratory complications. Bronchoscopy is one of the most important procedures used to diagnose respiratory complications. Despite enormous benefit, patients should be carefully selected for bronchoscopy as the process is invasive; however, there are only few reports evaluating the contributing factors of bronchoscopy that result in the definitive diagnosis of respiratory complications in these patients. OBJECTIVE: This study aimed to elucidate and identify the contributing factors of bronchoscopy for definitive diagnosis in patients with haematological malignancies. METHODS: We retrospectively analysed 275 patients with haematological malignancies who later showed respiratory complications, requiring consultation with pulmonologists. We found that 62 patients underwent bronchoscopy. Our data analysis focused on this particular subset of patients to identify the factors crucial for definitive diagnosis via bronchoscopy. RESULTS: Bronchoscopy provided definitive diagnosis for 25 patients (diagnostic yield = 40.3%). We determined that nodular shadow was associated with high diagnostic yields by multivariate logistic regression [odds ratio (OR): 6.6 (2.1-23)]. Furthermore, in several bronchoscopic procedures, biopsy also contributed to definitive diagnosis of patients with nodular shadow [OR: 17 (1.5-180)]. Life-threatening complications were not observed due to bronchoscopy in our study. CONCLUSIONS: Our study demonstrated that patients with haematological malignancies who showed lung nodular shadows are more likely to be definitively diagnosed by bronchoscopy, whereas transbronchial biopsy may also be beneficial for these patients.

Cerebral Arterial Air Embolism after Diagnostic Flexible Fiberoptic Bronchoscopy: A Case Report and Review of the Literature.

Cerebral arterial air embolism (CAAE) is an extremely rare complication of diagnostic flexible fiberoptic bronchoscopy, reported to occur once about every 103978 examinations. In all the eight cases of CAAE reported previously, the patients had undergone transbronchial lung biopsy (TBLB) or transbronchial needle aspiration (TBNA) prior to the onset of CAAE. Herein, we describe the case of a 77-year-old patient with double primary lung cancer who developed CAAE after bronchial curette cytology, which is considered to be less invasive than TBLB or TBNA. The patient was treated with supplemental oxygen, but paresis of the left upper arm and left spatial neglect remained. This is the first report of CAAE occurring after bronchial curettage during diagnostic flexible fiberoptic bronchoscopy.

TAZ contributes to pulmonary fibrosis by activating profibrotic functions of lung fibroblasts.

Transcriptional coactivator with PDZ-binding motif (TAZ) regulates a variety of biological processes. Nuclear translocation and activation of TAZ are regulated by multiple mechanisms, including actin cytoskeleton and mechanical forces. TAZ is involved in lung alveolarization during lung development and Taz-heterozygous mice are resistant to bleomycin-induced lung fibrosis. In this study, we explored the roles of TAZ in the pathogenesis of idiopathic pulmonary fibrosis (IPF) through histological analyses of human lung tissues and cell culture experiments. TAZ was highly expressed in the fibroblastic foci of lungs from patients with IPF. TAZ controlled myofibroblast marker expression, proliferation, migration, and matrix contraction in cultured lung fibroblasts. Importantly, actin stress fibers and nuclear accumulation of TAZ were more evident when cultured on a stiff matrix, suggesting a feedback mechanism to accelerate fibrotic responses. Gene expression profiling revealed TAZ-mediated regulation of connective tissue growth factor (CTGF) and type I collagen. Clinical relevance of TAZ-regulated gene signature was further assessed using publicly available transcriptome data. These findings suggest that TAZ is involved in the pathogenesis of IPF through multifaceted effects on lung fibroblasts.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition.

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor ß, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor α. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of Pulmonary Artery Aneurysms Due to Behçet's Disease and Resolution after Treatment.

We herein describe a patient with Behçet's disease in whom we followed the development and resolution of pulmonary artery aneurysms. He presented with intermittent hemoptysis, pulmonary thromboembolism was initially diagnosed, and anticoagulant therapy was started. Over the next several months, the expansion of pulmonary arteries was noted. Five months after his initial admission, he was readmitted for massive hemoptysis, and further examinations revealed that he had Behçet's disease. Corticosteroids and intravenous cyclophosphamide were started. Over the next five months, the pulmonary artery aneurysms and thrombosis resolved. The development of pulmonary artery aneurysms led to the diagnosis of Behçet's disease, and they resolved after immunosuppressive therapy.

Usefulness of low-dose methotrexate monotherapy for treating sarcoidosis.

OBJECTIVE: Methotrexate (MTX) is a cytotoxic agent that is commonly employed as an alternative to corticosteroids to treat sarcoidosis, although the proper use and efficacy of MTX as a single agent remain unclear. METHODS: The clinical records of patients newly diagnosed with sarcoidosis who were admitted to our institution between 2000 and 2009 were reviewed. Among these patients, 26 received 7.5 mg of MTX per week as a single agent, and the independent effects of MTX were analyzed. RESULTS: Six of the 26 patients (23%) exhibited an improvement of sarcoidosis-related lesions. The skin lesions demonstrated a relatively higher response rate (37%) than the pulmonary lesions (9%). Ten of the 26 patients (39%) experienced adverse effects, mostly mild hepatotoxicity. No severe adverse effects, including irreversible hepatotoxicity, were observed. CONCLUSION: Although the efficacy of low-dose MTX monotherapy for sarcoidosis in this study was not high (23%), some patients exhibited definite improvements, and the drug proved to be safe, suggesting its possible benefits as a single agent for treating sarcoidosis.