Questionnaire for diagnosing asthma-COPD overlap in COPD: Development of ACO screening questionnaire (ACO-Q).

BACKGROUND: The considerable prevalence and worse outcomes of asthma-COPD overlap (ACO) in COPD have been reported, and optimal introduction of ICS is essential for ACO. However, diagnostic criteria for ACO consist of multiple laboratory tests, which is challenging during this COVID-19 era. The purpose of this study was to create a simple questionnaire to diagnose ACO in patients with COPD. METHODS: Among 100 COPD patients, 53 were diagnosed with ACO based on the Japanese Respiratory Society Guidelines for ACO. Firstly, 10 candidate questionnaire items were generated and further selected by a logistic regression model. An integer-based scoring system was generated based on the scaled estimates of items. RESULTS: Five items, namely a history of asthma, wheezing, dyspnea at rest, nocturnal awakening, and weather- or season-dependent symptoms, contributed significantly to the diagnosis of ACO in COPD. History of asthma was related to FeNO >35 ppb. Two points were assigned to history of asthma and 1 point to other items in the ACO screening questionnaire (ACO-Q), and the area under the receiver operating characteristic curve was 0.883 (95% CI: 0.806-0.933). The best cutoff point was 1 point, and the positive predictive value was 100% at a cutoff of 3 points or higher. The result was reproducible in the validation cohort of 53 patients with COPD. CONCLUSIONS: A simple questionnaire, ACO-Q, was developed. Patients with scores ≥3 could be reasonably recommended to be treated as ACO, and additional laboratory testing would be recommended for patients with 1 and 2 points.

Pulmonary Apical Opacities on Thin-Section Computed Tomography: Relationship to Primary Spontaneous Pneumothorax in Young Male Patients and Corresponding Histopathologic Findings.

OBJECTIVE: The purpose of this study was to test the hypothesis that apical opacities on computed tomography (CT) are related to occurrence of primary spontaneous pneumothorax (PSP) in young male patients. METHODS: We compared the frequency of apical opacities on thin-section CT between 70 male patients with PSP (PSP group) and 74 male patients without a history of PSP (non-PSP group). We also evaluated histopathologic findings of 39 specimens from 37 surgical cases in the PSP group. RESULTS: Apical opacities were significantly more frequent in the PSP group than in the non-PSP group (right side, P = 0.01; left side, P = 0.005). Histopathologically, subpleural band-like alveolar collapse was seen in 35 specimens (89.7%), which was always accompanied by fibroelastosis and fibroblastic foci. CONCLUSIONS: Apical opacities on CT were significantly associated with PSP in young male patients. These apical opacities histopathologically correspond to fibrotic pleural thickening with subpleural alveolar collapse.

Matrix metalloproteinase-9 activates TGF-ß and stimulates fibroblast contraction of collagen gels.

Matrix metalloproteinase-9 (MMP-9) is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in three-dimensional (3D) collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 small-interfering RNA (siRNA) were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without transforming growth factor (TGF)-ß1 for 5 days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA-treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a nonspecific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9-deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9-deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9-deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-ß1 and reduced several TGF-ß1-driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Because TGF-ß1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-ß1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3D collagen gels mediated by fibroblasts.

Differential knockdown of TGF-ß ligands in a three-dimensional co-culture tumor- stromal interaction model of lung cancer.

BACKGROUND: Transforming growth factor (TGF)-ß plays a pivotal role in cancer progression through regulating cancer cell proliferation, invasion, and remodeling of the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are the predominant type of stromal cell, in which TGF-ß signaling is activated. Among the strategies for TGF-ß signaling inhibition, RNA interference (RNAi) targeting of TGF-ß ligands is emerging as a promising tool. Although preclinical studies support the efficacy of this therapeutic strategy, its effect on the tumor microenvironment in vivo remains unknown. In addition, differential effects due to knockdown of various TGF-ß ligand isoforms have not been examined. Therefore, an experimental model that recapitulates tumor-stromal interaction is required for validation of therapeutic agents. METHODS: We have previously established a three-dimensional co-culture model of lung cancer, and demonstrated the functional role of co-cultured fibroblasts in enhancing cancer cell invasion and differentiation. Here, we employed this model to examine how knockdown of TGF-ß ligands affects the behavior of different cell types. We developed lentivirus vectors carrying artificial microRNAs against human TGF-ß1 and TGF-ß2, and tested their effects in lung cancer cells and fibroblasts. RESULTS: Lentiviral vectors potently and selectively suppressed the expression of TGF-ß ligands, and showed anti-proliferative effects on these cells. Furthermore, knockdown of TGF-ß ligands attenuated fibroblast-mediated collagen gel contraction, and diminished lung cancer cell invasion in three-dimensional co-culture. We also observed differential effects by targeting different TGF-ß isoforms in lung cancer cells and fibroblasts. CONCLUSIONS: Our findings support the notion that RNAi-mediated targeting of TGF-ß ligands may be beneficial for lung cancer treatment via its action on both cancer and stromal cells. This study further demonstrates the usefulness of this three-dimensional co-culture model to examine the effect of therapeutic agents on tumor-stromal interaction.

Histamine induces human lung fibroblast-mediated collagen gel contraction via histamine H1 receptor.

BACKGROUND: Airway remodeling is implicated in irreversible airflow limitation of refractory asthma, which includes increased smooth muscle mass and subepithelial fibrosis. Activated fibroblasts acquire contractile phenotype to participate in tissue contraction and structural alteration of extracellular matrices. Histamine is a potent mediator of allergic inflammation, substantially involved in asthmatic pathophysiology. OBJECTIVE: We hypothesized that histamine might play a role in airway remodeling, and investigated its effect on fibroblast-mediated collagen gel contraction. METHODS: Fibroblast-mediated collagen gel contraction was studied. Histamine's regulation of collagen gel contraction was characterized by using specific histamine-receptor antagonists, an IP3 receptor antagonist and a PKC inhibitor. RESULTS: Histamine induced contraction of collagen gels embedded with human lung fibroblasts, in a time-dependent manner, and at the concentration more than 10(-6) M, both in four primary cultured adult lung fibroblasts and three fetal lung fibroblast cell lines. This effect was attenuated by H1 receptor antagonist, whereas those for H2 to H4 receptors failed to show an inhibitory effect. Furthermore, IP3 receptor-mediated Ca(2+) mobilization was implicated in histamine's action on collagen gel contraction. CONCLUSIONS: Our results suggest that histamine is involved in airway remodeling through its action on lung fibroblasts, and antihistamine drugs, especially H1 receptor antagonists, might be potentially beneficial for a subset of asthmatic patients.

Tumor necrosis factor superfamily member LIGHT induces epithelial-mesenchymal transition in A549 human alveolar epithelial cells.

Fibrosis is an abnormal response to organ injury, characterized by accumulation of activated fibroblasts at the sites of injury. Fibroblasts arise from several sources, including resident fibroblasts and circulating fibrocytes that infiltrate organ tissue. Recently, epithelial-mesenchymal transition (EMT) has been recognized as a source of mesenchymal cells. EMT is induced by various growth factors, such as transforming growth factor (TGF)-ß1, and enhanced by inflammatory cytokines. Recently the tumor necrosis factor superfamily member LIGHT has been implicated in the pathogenesis of inflammatory disease and airway remodeling in severe asthma. We hypothesized that LIGHT might contribute to the pathogenesis of airway fibrosis via enhancement of EMT. Therefore, we investigated LIGHT's ability to induce EMT. A549 cells were stimulated with LIGHT, TGF-ß1 or both for 48h. To estimate EMT, we evaluated the expression of epithelial and mesenchymal markers using immunocytochemistry, Western blotting and quantitative RT-PCR. Signaling pathways for EMT were characterized by Western analysis to detect phosphorylation of Erk1/2 and smad2. LIGHT enhanced TGF-ß1-induced EMT both morphologically, by suppressing E-cadherin and enhancing vimentin, and functionally, by enhancing cell contractility. Additionally, LIGHT induced EMT without TGF-ß1. Evaluation of the mechanism showed that LIGHT did not induce TGF-ß1 production or affect the smad-snai1 pathway. Inhibition of Erk1/2 phosphorylation reduced LIGHT-induced EMT, indicating the Erk1/2 pathway to be a key pathway in LIGHT-induced EMT. In summary, LIGHT enhanced TGF-ß1-induced EMT but also induced EMT via the Erk1/2 pathway by itself, without TGF-ß1 signaling. LIGHT may contribute to the pathogenesis of airway fibrosis through enhancement of EMT.

Characterization of human lung cancer-associated fibroblasts in three-dimensional in vitro co-culture model.

Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher α-smooth muscle actin (α-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.

A phase II study of S-1 and cisplatin with concurrent thoracic radiotherapy followed by durvalumab for unresectable, locally advanced non-small-cell lung cancer in Japan (SAMURAI study): primary analysis.

Background: The standard of care for unresectable, locally advanced non-small-cell lung cancer (LA-NSCLC) is chemoradiotherapy (CRT) followed by durvalumab, based on the PACIFIC study. Although multiple Japanese phase II studies have shown high efficacy and tolerability of CRT with cisplatin plus S-1 (SP), no prospective study using durvalumab after SP-based CRT has been reported. Objectives: We conducted a multicenter phase II study of this approach, the interim analysis of which showed a high transition rate to durvalumab consolidation therapy. Here, we report the primary analysis results. Design: In treatment-naïve LA-NSCLC, cisplatin (60 mg/m2, day 1) and S-1 (80-120 mg/body, days 1-14) were administered with two 4-week cycles with concurrent thoracic radiotherapy (60 Gy) followed by durvalumab (10 mg/kg) every 2 weeks for up to 1 year. Methods: The primary endpoint was 1-year progression-free survival (PFS). The expected 1-year PFS and its lower limit of the 80% confidence interval (CI) were set as 63% and 47%, respectively, based on the results of TORG1018 study. Results: In all, 59 patients were enrolled, with 51 (86.4%) proceeding to durvalumab. The objective response rate throughout the study was 72.9% (95% CI: 59.7-83.6%). After median follow-up of 21.9 months, neither median PFS nor OS was reached. The 1-year PFS was 72.5% (80% CI: 64.2-79.2%, 95% CI: 59.1-82.2%), while the 1-year overall survival was 91.5% (95% CI: 80.8-96.4%). No grade 5 adverse events were observed throughout the study. The most common adverse event during the consolidation phase was pneumonitis (any grade, 78.4%; grade ⩾3, 2.0%). Eventually, 52.5% of patients completed 1-year durvalumab consolidation therapy from CRT initiation. Conclusion: This study of durvalumab after SP-based CRT met its primary endpoint and found a 1-year PFS of 73% from CRT initiation. This study provides the first prospective data on the prognosis and tolerability of durvalumab consolidation from the initiation of CRT. Trial registration: Japan Registry of Clinical Trials, jRCTs031190127, registered 1 November, 2019, https://jrct.niph.go.jp/latest-detail/jRCTs031190127.

Prospective analysis of factors precluding the initiation of durvalumab from an interim analysis of a phase II trial of S-1 and cisplatin with concurrent thoracic radiotherapy followed by durvalumab for unresectable, locally advanced non-small cell lung cancer in Japan (SAMURAI study).

Background: The standard of care for unresectable, locally advanced non-small cell lung cancer (LA-NSCLC) is chemoradiotherapy (CRT) followed by durvalumab, based on the PACIFIC trial. Disease progression and pneumonitis were reported as the main reasons to preclude the initiation of durvalumab in multiple retrospective studies. However, the transition rate and the reasons for failure to proceed to consolidation therapy with durvalumab after CRT were not evaluated prospectively. Although phase II studies in Japan have shown high efficacy and tolerability of CRT with cisplatin + S-1 (SP), no prospective study using durvalumab after SP-based CRT has yet been reported. We therefore conducted a phase II study to verify the efficacy and safety of durvalumab following SP-based CRT. In this interim analysis, we report the transition rate and the reasons for its failure. Methods: In treatment-naïve LA-NSCLC, cisplatin (60 mg/m2, day 1) and S-1 (80-120 mg/body, days 1-14) were administered with two 4-week cycles with concurrent thoracic radiotherapy (60 Gy) followed by durvalumab every 2 weeks for up to 12 months. The primary endpoint was 12 month progression-free survival rate. Results: Fifty-nine patients were enrolled, of whom 86.4% (51/59) proceeded to durvalumab. All of them initiated durvalumab within 42 days after CRT [median 18 days (range: 3-38)], including 27.5% (14/51) in <14 days. Common reasons for failure to proceed to durvalumab were disease progression (2/59, 3.4%) and adverse events (6/59, 10.2%). Among the latter cases, four resumed treatment and proceeded to durvalumab within 42 days on off-protocol. The objective response rate and the disease control rate were 62.7% and 93.2%, respectively. The incidences of ⩾grade 3 pneumonitis, febrile neutropenia, and esophagitis were 0%, 8.5%, and 3.4%, respectively. Conclusion: Regarding durvalumab after CRT, this interim analysis of the SAMURAI study clarified the high transition rate, early introduction, and reasons for failure to proceed to consolidation therapy, which were not determined in the PACIFIC trial. Trial registration: Japan Registry of Clinical Trials, jRCTs031190127, registered 1 November, 2019, https://jrct.niph.go.jp/latest-detail/jRCTs031190127.

Simultaneous stimulation with TGF-ß1 and TNF-α induces epithelial mesenchymal transition in bronchial epithelial cells.

BACKGROUND: Airway remodeling is an important feature of chronic airway disease, but the mechanisms involved remain unclear. Recently, epithelial mesenchymal transition (EMT) was reported to be associated with tissue fibrosis. TGF-ß1, which is a potent inducer of EMT, is thought to be related to the pathogenesis of airway remodeling. We investigated whether TGF-ß1 and/or TNF-α induce EMT in bronchial epithelial cells. METHODS: Cultured BEAS-2B cells and primary normal human bronchial epithelial cells (NHBE) were treated with TGF-ß1 and/or TNF-α. Morphological changes and the expression of EMT-related markers were evaluated by immunocytochemical staining. Expressions of EMT-related markers, extracellular matrix (ECM) components (collagen type I and versican), and TGF-ß receptors I, II, and III were analyzed by quantitative RT-PCR. Migration was evaluated using the Boyden chamber technique. RESULTS: The TGF-ß1-induced EMT in BEAS-2B cells was demonstrated on the basis of morphological changes and the downregulation of E-cadherin. Costimulation with TNF-α enhanced the TGF-ß1-induced morphological changes and increased vimentin expression. Treatment with TGF-ß1 increased the expression of collagen type I and versican. EMT induced with TGF-ß1 plus TNF-α promoted cell migration. Stimulation of NHBE with TGF-ß1 led to EMT. CONCLUSION: TGF-ß1 induced EMT in BEAS-2B cells, and costimulation with TNF-α enhanced the EMT. As a result of the EMT process, BEAS-2B cells acquired functions of mesenchymal cells. In addition, TGF-ß1 treatment induced EMT in NHBE as shown by changes in EMT-related markers. Bronchial epithelial cells might contribute to airway remodeling through EMT.

A phase II study of S-1 and cisplatin with concurrent thoracic radiotherapy followed by durvalumab for unresectable, locally advanced non-small-cell lung cancer in Japan (SAMURAI study).

BACKGROUND: Based on the results of the PACIFIC study, chemoradiotherapy followed by 1-year consolidation therapy with durvalumab was established as the standard of care for unresectable, locally advanced non-small-cell lung cancer (LA-NSCLC). However, some topics not foreseen in that design can be explored, including progression-free survival (PFS) and overall survival (OS) after the start of chemoradiotherapy, the proportion of patients who proceeded to consolidation therapy with durvalumab, and the optimal chemotherapeutic regimens. In Japan, the combination regimen of S-1 + cisplatin (SP), for which the results of multiple clinical studies have suggested a good balance of efficacy and tolerability, is frequently selected in clinical settings. However, the efficacy and safety of consolidation therapy with durvalumab following this SP regimen have not been evaluated. We therefore planned a multicenter, prospective, single-arm, phase II study. METHODS: In treatment-naïve LA-NSCLC, two cycles of combination chemotherapy with S-1 (80-120 mg/body, Days 1-14) + cisplatin (60 mg/m2, Day 1) will be administered at an interval of 4 weeks, with concurrent thoracic radiotherapy (60 Gy). Responders will then receive durvalumab every 2 weeks for up to 1 year. The primary endpoint is 1-year PFS rate. DISCUSSION: Compared with the conventional standard regimen in Japan, the SP regimen is expected to be associated with lower incidences of pneumonitis, esophagitis, and febrile neutropenia, which complicate the initiation of consolidation therapy with durvalumab, and have higher antitumor efficacy during chemoradiotherapy. Therefore, SP-based chemoradiotherapy is expected to be successfully followed by consolidation therapy with durvalumab in more patients, resulting in prolonged PFS and OS. Toxicity and efficacy results of the SP regimen in this study will also provide information important to the future establishment of the concurrent combination of chemoradiotherapy and durvalumab. TRIAL REGISTRATION: Japan Registry of Clinical Trials, jRCTs031190127, registered 1 November 2019, https://jrct.niph.go.jp/latest-detail/jRCTs031190127.

Nrf2 is closely related to allergic airway inflammatory responses induced by low-dose diesel exhaust particles in mice.

We have recently reported that disruption of nuclear erythroid 2 P45-related factor 2 (Nrf2) enhances susceptibility to airway inflammatory responses induced by low-dose diesel exhaust particles (DEP) in mice. C57BL/6 Nrf2 knockout (Nrf2(-/-)) mice and wild-type (Nrf2(+/+)) mice were further exposed to low-dose DEP for 7h/day, 5 days/week, for a maximum of 8 weeks. After exposure to DEP for 5 weeks, allergic airway inflammation was generated in the mice by intraperitoneal sensitization with OVA followed by intranasal challenge. Nrf2(-/-) mice exposed to relatively low-dose DEP showed significantly increased percentage changes relative to the OVA alone group in terms of airway hyperresponsiveness (AHR) and inflammatory cells, levels of IL-5 and thymus and activation regulated chemokine (TARC) in bronchoalveolar lavage (BAL) fluid than did Nrf2(+/+) mice. Lung tissues of Nrf2(-/-) mice after DEP exposure showed inflammatory cell infiltrates, and increased PAS staining-positive mucus cell hyperplasia. In contrast, the percentage changes relative to the OVA group in the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in whole blood was higher in Nrf2(+/+) mice than in Nrf2(-/-) mice. By using Nrf2(-/-) mice, it was shown for the first time that relatively low-dose DEP exposure induces oxidant stress, and that host anti-oxidant responses play a key role in the development of DEP-induced exacerbation of allergic airway inflammation.

Histamine stimulates human lung fibroblast migration.

Histamine is a potent mediator in allergic inflammatory processes and is released by basophils and mast cells. The aim of this study was to investigate the effect of histamine on in vitro migration of human fetal lung fibroblasts (HFL-1) to human plasma fibronectin (HFn), a chemoattractant. Using the blindwell chamber technique, histamine alone had no chemotactic activity. However, histamine augmented HFn-induced HFL-1 migration at concentrations ranging between 0 and 10(-7) M (290.6 +/- 20.8%) (P < 0.05). The concentration-response was bell-shaped. The effect of histamine increased with time. The stimulatory effect of histamine on HFL-1 migration was inhibited by an H4 receptor antagonist, JNJ7777120 (10(-5) M). Histamine's effect was also inhibited by pertussis toxin (50 ng/ml), showing that the effect was mediated by the H4 receptor. This study demonstrated that histamine has the potential to stimulate human lung fibroblast migration, and thus may contribute to regulation of wound healing and the development of fibrotic disorders of the lung.

Tumor necrosis factor-alpha enhances both epithelial-mesenchymal transition and cell contraction induced in A549 human alveolar epithelial cells by transforming growth factor-beta1.

Recently, epithelial-mesenchymal transition (EMT) has been reported to contribute to tissue fibrosis through enhanced transforming growth factor (TGF)-beta1 signaling. Tumor necrosis factor (TNF)-alpha has also been implicated in tissue fibrosis. Therefore, the authors investigated whether TNF-alpha affected TGF-beta1-induced EMT. Cultured alveolar epithelial cells (A549 cells) were stimulated with TGF-beta1 (5 ng/mL), with/without TNF-alpha (10 ng/mL). TGF-beta1 induced EMT of A549 cells, with loss of E-cadherin and acquisition of vimentin. Combination of TNF-alpha with TGF-beta1 enhanced EMT, causing morphological changes, while quantitative polymerase chain reaction (PCR) showed suppression of E-cadherin mRNA and expression of vimentin mRNA. In addition, the gel contraction method revealed that cells that had undergone EMT acquired cell contractility, which is a feature of mesenchymal cells. Stimulation with TGF-beta1 induced cell contraction, as did TNF-alpha. Moreover, costimulation with TGF-beta1 and TNF-alpha enhanced the cell contraction. Although IFN-gamma suppressed spontaneous cell contraction, it did not suppress cell contraction, which was induced by TGF-beta1. In conclusion, TNF-alpha enhances not only EMT but also cell contraction induced by TGF-beta1. EMT might contribute to tissue fibrosis through induction of cell contraction.

C-reactive protein modulates human lung fibroblast migration.

C-reactive protein (CRP) has been classically used as a marker of inflammation. The aim of this study was to investigate the effect of CRP on migration of human fetal lung fibroblasts (HFL-1) to human plasma fibronectin (HFn). Using the blindwell chamber technique, CRP inhibited HFL-1 migration in a dose-dependent fashion (at 1 microg/mL, inhibition: 32.5% +/- 7.1%; P < .05). Western blot analysis showed that CRP inhibited the p38 mitogen-activated protein kinase (MAPK) activity in the presence of HFn. Moreover, the MAPK inhibitors SB202190 (25 microM) and SB203580 (25 microM) inhibited HFn-induced cell migration, suggesting an important role of p38 MAPK in HFn-induced migration. Taken together, these results suggest that the inhibitory effect of CRP is mediated by blocking MAPK. In summary, this study demonstrates that CRP directly modulates human lung fibroblasts migration. Thus, CRP may contribute to regulation of wound healing and may be endogenous antifibrotic factor acting on lung fibrosis.

The effects of oxidative stress induced by prolonged low-dose diesel exhaust particle exposure on the generation of allergic airway inflammation differ between BALB/c and C57BL/6 mice.

We have recently reported that airway inflammatory responses to the oxidative stress induced by prolonged low-dose diesel exhaust particle (DEP) exposure differ markedly between BALB/c and C57BL/6 mice. In the present study, the effects of genetic differences in the response to prolonged low-dose DEP exposure on the generation of ovalbumin-induced allergic airway inflammation were further explored using the same mouse strains. In BALB/c mice, eosinophils and mucous goblet cells in histopathological pulmonary specimens increased significantly after DEP exposure, and were more marked than in C57BL/6 mice. Interleukin (IL)-5 and IL-13 levels in bronchoalveolar lavage (BAL) fluid were increased significantly by DEP exposure only in BALB/c mice. The DEP-induced increases in peribronchial eosinophils and mucous goblet cells in the lung tissues, and of IL-5 and IL-13 in the BAL fluid, were significantly attenuated by the antioxidant N-acetylcysteine. Thus, the effects of prolonged low-dose DEP exposure on the generation of allergic airway inflammation differed markedly between the mouse strains. These differences may be caused by different antioxidant responses to the oxidative stress induced by DEP exposure. Our results contribute more information to the search for genetic susceptibility factors in the response to DEP, and may thus assist in the discovery of new biomarkers for DEP-related disease.

Disruption of Nrf2 enhances susceptibility to airway inflammatory responses induced by low-dose diesel exhaust particles in mice.

To test our hypothesis that diesel exhaust particle (DEP)-induced oxidative stress and host antioxidant responses play a key role in the development of DEP-induced airway inflammatory diseases, C57BL/6 nuclear erythroid 2 P45-related factor 2 (Nrf2) knockout (Nrf2(-/-)) and wild-type mice were exposed to low-dose DEP for 7 h/day, 5 days/week, for 8 weeks. Nrf2(-/-) mice exposed to low-dose DEP showed significantly increased airway hyperresponsiveness and counts of lymphocytes and eosinophils, together with increased concentrations of IL-12 and IL-13, and thymus and activation-regulated chemokine (TARC), in BAL fluid than wild-type mice. In contrast, expression of antioxidant enzyme genes was significantly higher in wild-type mice than in Nrf2(-/-) mice. We have first demonstrated that disruption of Nrf2 enhances susceptibility to airway inflammatory responses induced by inhalation of low-dose DEP in mice. These results strongly suggest that DEP-induced oxidative stress and host antioxidant responses play some role in the development of DEP-induced airway inflammation.

Hemoglobin Kansas as a Rare Cause of Cyanosis: A Case Report and Review of the Literature.

Hemoglobin (Hb) Kansas is an inherited Hb variant with a low oxygen affinity that is associated with low oxygen saturation on pulse oximetry (SpO2). It leads to asymptomatic cyanosis. Patients with Hb Kansas do not require any specific treatment and the prognosis is good. In patients with unexplained cyanosis, we should thus consider Hb variants, including Hb Kansas and avoid unnecessary investigations and managements. We herein report the case of 65-year-old woman with Hb Kansas and review five other cases (three lineages) that have been reported in Japan.

Serum Reactive Oxygen Metabolite Levels Predict Severe Exacerbations of Asthma.

BACKGROUND AND PURPOSE: Bronchial asthma (BA) is a chronic airway disease characterized by airway hyperresponsiveness and remodeling, which are intimately linked to chronic airway inflammation. Reactive oxygen species (ROS) such as hydrogen peroxide are generated by inflammatory cells that are involved in the pathogenesis of BA. However, the role of ROS in the management of BA patients is not yet clear. We attempted to determine the role of ROS as a biomarker in the clinical setting of BA. SUBJECTS AND METHODS: We enrolled patients with BA from 2013 through 2015 and studied the degrees of asthma control, anti-asthma treatment, pulmonary function test results, fractional exhaled nitric oxide (FeNO), serum reactive oxygen metabolite (ROM) levels, and serum levels of interleukin (IL)-6 and IL-8. RESULTS: We recruited 110 patients with BA. Serum ROM levels correlated with white blood cell (WBC) count (rs = 0.273, p = 0.004), neutrophil count (rs = 0.235, p = 0.014), CRP (rs = 0.403, p < 0.001), and IL-6 (rs = 0.339, p < 0.001). Serum ROM levels and IL-8 and CRP levels negatively correlated with %FEV1 (rs = -0.240, p = 0.012, rs = -0.362, p < 0.001, rs = -0.197, p = 0.039, respectively). Serum ROM levels were significantly higher in patients who experienced severe exacerbation within 3 months than in patients who did not (339 [302-381] vs. 376 [352-414] CARR U, p < 0.025). Receiver-operating characteristics analysis showed that ROM levels correlated significantly with the occurrence of severe exacerbation (area under the curve: 0.699, 95% CI: 0.597-0.801, p = 0.025). CONCLUSIONS: Serum levels of ROM were significantly associated with the degrees of airway obstruction, WBC counts, neutrophil counts, IL-6, and severe exacerbations. This biomarker may be useful in predicting severe exacerbations of BA.

TGF-beta1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels.

Apoptosis of fibroblasts may be key for the removal of cells following repair processes. Contraction of three-dimensional collagen gels is a model of wound healing and remodeling. Here two potent inducers of contraction, TGF-beta1 and fetal calf serum (FCS) were evaluated for their effect on fibroblast apoptosis in contracting collagen gels. Human fetal lung fibroblasts were cultured in floating type I collagen gels, exposed to TGF-beta1 or FCS, and allowed to contract for 5 days. Apoptosis was evaluated using TUNEL and confirmed by DNA content profiling. Both TGF-beta1 and serum significantly augmented collagen gel contraction. TGF-beta1 also increased apoptosis assessed by TUNEL positivity and DNA content analysis. In contrast, serum did not affect apoptosis. TGF-beta1 induction of apoptosis was associated with augmented expression of Bax, a pro-apoptotic member of the Bax/Bcl-2 family, inhibition of Bcl-2, an anti-apoptotic member of the same family, and inhibition of both cIAP-1 and XIAP, two inhibitors of the caspase cascade. Serum was associated with an increase in cIAP-1 and Bcl-2, anti-apoptotic proteins. Interestingly, serum was also associated with an apparent increase in Bax, a pro-apoptotic protein. Blockade of Smad3 with either siRNA or by using murine fibroblasts deficient in Smad3 resulted in a lack of TGF-beta induction of augmented contraction and apoptosis. Contraction induced by different factors, therefore, may be differentially associated with apoptosis, which may be related to the persistence or resolution of the fibroblasts that accumulate following injury.