We examined the effects of lung volume on the bronchoconstriction induced by inhaled aerosolized methacholine (MCh) in seven normal subjects. We constructed dose-response curves to MCh, using measurements of inspiratory pulmonary resistance (RL) during tidal breathing at functional residual capacity (FRC) and after a change in end-expiratory lung volume (EEV) to either FRC -0.5 liter (n = 5) or FRC +0.5 liter (n = 2). Aerosols of MCh were generated using a nebulizer with an output of 0.12 ml/min and administered for 2 min in progressively doubling concentrations from 1 to 256 mg/ml. After MCh, RL rose from a base-line value of 2.1 +/- 0.3 cmH2O. 1-1 X s (mean +/- SE; n = 7) to a maximum of 13.9 +/- 1.8. In five of the seven subjects a plateau response to MCh was obtained at FRC. There was no correlation between the concentration of MCh required to double RL and the maximum value of RL. The dose-response relationship to MCh was markedly altered by changing lung volume. The bronchoconstrictor response was enhanced at FRC - 0.5 liter; RL reached a maximum of 39.0 +/- 4.0 cmH2O X 1-1 X s. Conversely, at FRC + 0.5 liter the maximum value of RL was reduced in both subjects from 8.2 and 16.6 to 6.0 and 7.7 cmH2O X 1-1 X s, respectively. We conclude that lung volume is a major determinant of the bronchoconstrictor response to MCh in normal subjects. We suggest that changes in lung volume act to alter the forces of interdependence between airways and parenchyma that oppose airway smooth muscle contraction.

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Human peripheral blood lymphocytes (HPBLs) are one of the most sensitive cells to ionizing radiation (IR) in the human body, and IR-induced DNA damage and functional impairment of HPBLs are the adverse consequences of IR accidents and major side effects of radiotherapy. Phosphorylated H2AX (γH2AX) is a sensitive marker for DNA double-strand breaks, but the role and regulation of the pan-nuclear γH2AX response in HPBLs after IR remain unclear. We herein demonstrated that the pan-nuclear γH2AX signals were increased in a time- and dose-dependent manner, colocalized with >94% of TUNEL apoptotic staining, and displayed a typical apoptotic pattern in resting HPBLs after low LET X-ray IR. In addition, the X-irradiation-induced pan-nuclear p-ATM and p-DNA-PKcs responses also occurred in resting HPBLs, and were colocalized with 92–95% of TUNEL staining and 97–98% of the pan-nuclear γH2AX signals, respectively, with a maximum at 6 h post irradiation, but disappeared at 24 h post irradiation. Moreover, ATM/DNA-PKcs inhibitor KU55933, p53 inhibitor PFT-μ and pan-caspase inhibitor ZVAD-fmk significantly decreased X-irradiation-induced pan-nuclear γH2AX signals and TUNEL staining, protected HPBLs from apoptosis, but decreased the proliferative response to mitogen in X-irradiated HPBLs. Notably, whereas both KU55933 and PFT-μ increased the IR-induced chromosome breaks and mis-repair events through inhibiting the formation of p-ATM, p-DNA-PKcs and γH2AX foci in X-irradiated HPBLs, the ZVAD-fmk did not increase the IR-induced chromosomal instability. Taken together, our data indicate that pan-nuclear γH2AX response represents an apoptotic signal that is triggered by the transient pan-nuclear ATM and DNA-PKcs activation, and mediated by p53 and pan-caspases in X-irradiated HPBLs, and that caspase inhibitors are better than ATM/DNA-PKcs inhibitors and p53 inhibitors to block pan-nuclear γH2AX response/apoptosis and protect HPBLs from IR.

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Triple-negative breast cancer (TNBC) is one of the most difficult breast cancers to treat because there is no targeted treatment, and conventional cytotoxic chemotherapy followed by adjuvant radiation therapy is the standard of care for patients with TNBC. We herein reported that ionizing radiation (IR) induced Wnt3a, LRP6 and β-catenin expression and consequently activated Wnt/β-catenin signaling in TNBC MDA-MB-231, MDA-MB-468 and Hs578T cells. Moreover, depletion of β-catenin by shRNA sensitized TNBC cells to IR, whereas treatment of Wnt3a protein or overexpression of β-catenin resulted in radioresistance of TNBC cells. Niclosamide, a potent inhibitor of Wnt/β-catenin signaling, not only inhibited constitutive Wnt/β-catenin signaling, but also blocked IR-induced Wnt/β-catenin signaling in TNBC cells. In addition, niclosamide sensitized TNBC cells to IR, prevented Wnt3a-induced radioresistance, and overcame β-catenin-induced radioresistance in TNBC cells. Importantly, animals treated with the combination of niclosamide and γ-ray local tumor irradiation had significant inhibition of MDA-MB-231 tumor growth compared with treated with local tumor irradiation alone. These findings indicate that Wnt/β-catenin signaling pathway plays an important role in the development of radioresistance of TNBC cells, and that niclosamide had significant radiosensitizing effects by inhibiting Wnt/β-catenin signaling in TNBC cells. Our study also provides rationale for further preclinical and clinical evaluation of niclosamide in TNBC management.

ObjectiveWe investigated the relationship between plasma miRNAs levels and inflammatory characteristics in asthmatic patients.MethodsEligible adults with untreated asthma (n = 35) underwent a clinical assessment, sputum induction, and assessment of pulmonary function test and Asthma Control Test (ACT) scores. Asthma phenotypes were defined using the sputum cell count. miR-199a-5p expression was measured using quantitative real-time polymerase chain reaction (qPCR). Lipopolysaccharide (LPS) stimulation was used to detect miR-199a-5p secretion from peripheral blood-derived neutrophil, lymphocyte, macrophage and BEAS-2B cells. The correlation of miR-199a-5p expression with clinical parameters was analyzed using multiple linear regression analysis. In silico analysis predicted the target genes and signaling pathway of miR-199a-5p. Transfection of miR-199a-5p mimics in human airway smooth muscle cells (HASMCs) was performed in vitro.ResultsThe miRNA-199a-5p levels in plasma and sputum increased significantly in patients with neutrophilic asthma compared to healthy subjects (ps = 0.014 and 0.006, respectively). Expression of miR-199a-5p in the plasma of asthmatic patients positively correlated with sputum miR-199a-5p expression (r = 0.511, p = 0.021). The miR-199a-5p level was only elevated with LPS stimulation in neutrophils but not macrophages, lymphocytes, or epithelial cells from healthy controls (p < 0.01). miR-199a-5p expression increased in response to LPS (p = 0.005) and LPS combined with IL-4 (p = 0.003), but not IL-4 alone. However, peripheral neutrophils from eosinophilic asthma patients did not respond to LPS with increased miR-199a-5p expression (n = 5, p > 0.05) in contrast to the significant response from neutrophilic patients (n = 4, p < 0.0001). miR-199a-5p negatively correlated with FEV1, FVC and PEF (r = -0.377, p = 0.026; r = -0.419, p = 0.012; and r = -0.392, p = 0.024, respectively). Multivariate correlation analysis confirmed that the plasma miR-199a-5p levels negatively correlated with FEV1 in patients with asthma (Adjusted R2 = 0.164, p = 0.015). In silico analysis suggested that the WNT signaling pathway participates in miR-199a-5p mediation of smooth muscle cell hypertrophy. In vitro experiment, miR-199a-5p mimics inhibited the protein expressions of WNT2 and WNT4, decreased the c-myc expression and dramatically increased the Sm-MHC expression in HASMCs.ConclusionPlasma miR-199a-5p was increased in neutrophilic asthma and negatively correlated with pulmonary function, which suggests that miR-199a-5p actively contributes to disease pathogenesis by modulating the inflammatory process and transferring the signal from inflammatory cells to structure cells.

The biodosimetric information is critical for evaluating the human health hazards caused by radon and its progeny. Here, we demonstrated that the formation of phosphorylated histone variant H2AX (γ-H2AX), p53-binding protein 1 (53BP1) and phosphorylated KRAB-associated protein 1 (pKAP-1) foci and their linear tracks in human peripheral blood lymphocytes (HPBLs) in vitro exposed to radon and its progeny were dependent on the cumulative absorbed dose of radon exposure but was unrelated to the concentration of radon. Among them, γ-H2AX foci and its linear tracks were the most sensitive indicators with the lowest estimable cumulative absorbed dose of 1.74 mGy from their linear dose-response curves and sustained for 12 h after termination of radon exposure. In addition, three types of foci showed an overdispersed non-Poisson distribution in HPBLs. The ratios of pKAP-1/γ-H2AX foci co-localization, 53BP1/γ-H2AX foci co-localization and 53BP1/pKAP-1 foci co-localization were significantly increased in HPBLs exposed to radon while they were unrelated to the cumulative dose of radon exposure, suggesting that γ-H2AX, pKAP-1 and 53BP1 play an important role in the repair of heterochromatic double-strand breaks. Altogether, our findings provide an experimental basis for estimating the biological dose of internal α-particle irradiation from radon and its progeny exposure in humans.