Pulmonary immune responses to 2009 pandemic influenza A (H1N1) virus in mice.

BACKGROUND: Well-characterized mice models will afford a cheaper, easy-handling opportunity for a more comprehensive understanding of 2009 influenza A (H1N1) virus's pathogenesis potential. We aimed to provide a robust description of pulmonary immune responses in the mice infected by the virus. METHODS: BALB/c mice were inoculated intranasally with A/Beijing/501/2009(H1N1) (BJ501) and A/PR/8/34(H1N1) (PR8) viruses and compared for survival rate, viral replication, and kinetics of pulmonary immune responses. RESULTS: BJ501 virus replicated less efficiently in the lungs than PR8, and both caused lethal illness in the mice. The transient increases of pulmonary TNF-α 2 days post infection for BJ501 and of INF-γ and IL-10 at 6 days post infection for PR8 were observed. IL-2+ and IL-4+ secreting cells showed significant increase 12 days post infection, while IFN-γ+, IgG+ and IgA+ secreting cells increased 6 days post infection. The different patterns of pulmonary immunological parameters between two viruses were at most seen in IL-6, IL-17 secretion and IgG1/IgG2a ratio. CONCLUSIONS: The BALB/c mouse is evaluated as a good pathogenic model for studying BJ501 2009 H1N1 virus. The work provided some basic and detailed data, which might be referred when further evaluating innate and adapted pulmonary immune responses and local viral load in mice.

Enriched environment ameliorates fear memory impairments induced by sleep deprivation via inhibiting PIEZO1/calpain/autophagy signaling pathway in the basal forebrain.

AIMS: To verify the hypothesis that an enriched environment (EE) alleviates sleep deprivation-induced fear memory impairment by modulating the basal forebrain (BF) PIEZO1/calpain/autophagy pathway. METHODS: Eight-week-old male mice were housed in a closed, isolated environment (CE) or an EE, before 6-h total sleep deprivation. Changes in fear memory after sleep deprivation were observed using an inhibitory avoidance test. Alterations in BF PIEZO1/calpain/autophagy signaling were detected. The PIEZO1 agonist Yoda1 or inhibitor GsMTx4, the calpain inhibitor PD151746, and the autophagy inducer rapamycin or inhibitor 3-MA were injected into the bilateral BF to investigate the pathways involved in the memory-maintaining role of EE in sleep-deprived mice. RESULTS: Mice housed in EE performed better than CE mice in short- and long-term fear memory tests after sleep deprivation. Sleep deprivation resulted in increased PIEZO1 expression, full-length tropomyosin receptor kinase B (TrkB-FL) degradation, and autophagy, as reflected by increased LC3 II/I ratio, enhanced p62 degradation, increased TFEB expression and nuclear translocation, and decreased TFEB phosphorylation. These molecular changes were partially reversed by EE treatment. Microinjection of Yoda1 or rapamycin into the bilateral basal forebrain induced excessive autophagy and eliminated the cognition-protective effects of EE. Bilateral basal forebrain microinjection of GsMTx4, PD151746, or 3-MA mimicked the cognitive protective and autophagy inhibitory effects of EE in sleep-deprived mice. CONCLUSIONS: EE combats sleep deprivation-induced fear memory impairments by inhibiting the BF PIEZO1/calpain/autophagy pathway.

A three serum miRNA panel as diagnostic biomarkers of radiotherapy-related metastasis in non-small cell lung cancer.

Serum microRNAs (miRNAs) have been implicated as noninvasive biomarkers for lung cancer diagnosis. However, there are no sensitive and specific biomarkers for the detection of radiotherapy-related non-small cell lung cancer (NSCLC) metastasis. The present study aimed to investigate the role of three serum miRNAs, namely miRNA (miR)-130a, miR-25 and miR-191*, in diagnosing NSCLC, and their biological functions in radiation-mediated development of metastatic properties in A549 cells. To determine this, serum samples were collected from 84 patients with NSCLC and 42 age- and sex-matched healthy controls. Differential expression of serum miRNAs was analyzed by quantitative PCR. Significant associations between miRNA expression and overall survival of patients with NSCLC were identified using the Cox proportional regression model. A receiver operating characteristic curve was generated to evaluate diagnostic accuracy. The functions of miR-130a, miR-25 and miR-191* in lung cancer cells were studied by transfecting A549 cells with miRNA mimics and inhibitors. The results of the present study demonstrated that the expression levels of miR-130a, miR-25 and miR-191* in the serum of patients with NSCLC were increased compared with those in healthy controls, and these increases were associated with advanced age (≥60 years), radiotherapy, histological type (squamous carcinoma), low survival rate and low median survival time. Additionally, irradiation induced the upregulation of miR-130a, miR-25 and miR-191* expression in A549 cells in vitro and in a xenograft mouse model. Irradiation also promoted the invasiveness of A549 cells in vitro and metastasis in vivo. In conclusion, miR-130a, miR-25 and miR-191* may be potential biomarkers for the diagnosis of patients with NSCLC and may serve oncogenic roles in radiation-mediated metastasis of NSCLC.

PMN apoptosis and its relationship with the lung injury after chest impact trauma.

BACKGROUND: Polymorphonuclear neutrophil (PMN), one of the most important inflammatory cells, functions throughout the initiation, progression and resolution of inflammation. This study aimed at investigating the relationship between PMN apoptosis and the lung injury after chest impact trauma. METHODS: PMNs were purified from rabbits subjected to the chest impact trauma and their apoptosis, necrosis, survival and respiratory burst were detected by flow cytometry. Meanwhile, lactate dehydrogenase and (LDH) [Ca2+]i were measured. RESULTS: The delayed apoptosis of PMNs in bronchoalveolar lavage fluid was observed from 2 hours to 12 hours after trauma, and viable cells increased. Respiratory burst of PMNs in bronchoalveolar lavage fluid was increased significantly from 2 hours with the peak at 8 hours. Meanwhile, lactate dehydrogenase in bronchoalveolar lavage fluid was higher than that in control (P < 0.05) from 4 hours to 24 hours, and intracellular free Ca2+ in PMN was increased temporarily. CONCLUSIONS: Retention of PMN in tissues and the abnormality in apoptotic pathway inevitably generate persistent activation of PMN and excessive release of toxic substances, resulting in tissue injury. The temporary increase of intracellular free Ca2+ may be responsible for the delayed apoptosis of PMN.

Design and screening of antisense oligodeoxynucleotides against PAI-1 mRNA in endothelial cells in vitro.

AIM: To design and screen antisense oligodeoxynucleotides (ASODNs), which inhibit type-1 plasminogen activator inhibitor (PAI-1) expression in human umbilical vein endothelial cells (HUVEC) in vitro. METHODS: Twenty seven ASODNs against different sites of PAI-1 mRNA were designed and transfected to HUVEC by lipofectin in vitro. The effects of ASODNs on PAI-1 antigen, PAI-1 activity and PAI-1 mRNA expression were detected by ELISA, amidolytical assay and RT-PCR, respectively. RESULTS: Transforming growth factor beta1 (TGF-beta1)-treated HUVEC increased the expression of PAI-1 compared with the normal HUVEC. Five among twenty seven designed ASODNs were effective in inhibiting the increase in PAI-1 antigen and PAI-1 activity in a dose-dependent manner after 48-h transfection. In particular, ASODN 14 (AO14) exhibited the best inhibitory effect. The control sequences of AO14, including sense, scramble, and mismatch sequences, did not significantly inhibit PAI-1 activity. It was revealed that the inhibitory efficacy of AO14 was in a sequence-specific manner. RT-PCR showed that ASODN 1, 7, 8, 14, and 15 decreased PAI-1 mRNA expression induced by TGF-beta1 and AO14 showed the best inhibitory effect. CONCLUSION: ASODN 1, 7, 8, 14, and 15, among twenty seven designed ASODNs against PAI-1 mRNA, significantly decreased PAI-1 antigen and PAI-1 activity induced by TGF-beta1 in a dose-dependent manner in HUVEC in vitro. AO14 showed the best inhibitory effect on PAI-1 expression in a sequence-specific manner. The results of RT-PCR indicated that inhibitory effects of ASODNs on PAI-1 biosynthesis occurred at the mRNA level. Four among five effective target sites of ASODNs located at the translation initiation site or within the translation area of PAI-1 mRNA, suggesting that these sites may be promising sites for the design of effective ASODNs.