Clinical characteristics and risk factors of patients with eosinophilic fasciitis associated with pleural effusion.

To investigate the risk factors of eosinophilic fasciitis (EF) associated with pleural effusion (PE). A retrospective analysis was performed on 22 patients with EF diagnosed by skin biopsy in our hospital, and they were divided into EF-PE and EF according to chest computed tomography examination. The clinical characteristics, clinical manifestations, comorbidities and laboratory test indicators of the two groups were collected and compared, and the risk factors for occurring PE in patients with EF were determined by multivariate logistic regression analysis. Among 22 patients with EF, 8 had PE. The age, course of disease, incidence of fever, weight loss, cough and shortness of breath, pulmonary infection, hypothyroidism, hydronephrosis and kidney stone, swelling rate of small vascular endothelial cells, consolidation shadows, C-reactive protein and thyroid stimulating hormone in EF-PE group were higher than those in EF group, while free triiodothyronine and thyroxine were lower than those in EF group. Age, fever, shortness of breath, C-reactive protein, ESR, thyroid stimulating hormone, pulmonary infection, hypothyroidism, hydronephrosis, kidney stones, swollen small vascular endothelial cells and chest CT consolidation shadows were identified as risk factors for happening PE in patients with EF, while free triiodothyronine and free thyroxine were identified as protective factors against PE in patients with EF. The incidence of EF-PE was 36.36% in this study. Advanced age, high C-reactive protein, ESR, thyroid stimulating hormone, incidence of fever, shortness of breath, pulmonary infection, hydronephrosis, kidney stones, swollen small vascular endothelial cells, chest CT consolidation shadows, and low free triiodothyronine and thyroxine suggest that patients with EF are significantly at increased risk of PE.

The Pharmacokinetics/Pharmacodynamics and Neurotoxicity of Tigecycline Intraventricular Injection for the Treatment of Extensively Drug-Resistant Acinetobacter baumannii Intracranial Infection.

Objective: This study aimed to provide feasible suggestions for intraventricular injection of tigecycline to treat intractable Acinetobacter baumannii intracranial infections by studying its pharmacokinetics/pharmacodynamics and neurotoxicity. Methods: A simple and reliable two-dimensional high-performance liquid chromatography (2D-HPLC) method was used to determine tigecycline concentration. The pharmacokinetics (PK) of tigecycline in cerebrospinal fluid (CSF) was investigated by performing therapeutic drug monitoring (TDM). The pharmacodynamics (PD) of tigecycline was evaluated by its minimum inhibitory concentration (MIC) against XDR A. baumannii. CCK8 assay was used to evaluate the cytotoxicity of different concentrations of tigecycline effect on PC12 cells, and apoptosis assay was analyzed by flow cytometry. Results: Tigecycline retention time in 2D-HPLC was 7.636 min. The lower limit of quantitation (LLOQ) was 0.1mg/L, which met the requirements of concentration determination for TDM. The MIC50 and MIC90 values of tigecycline for A. baumannii were 2 and 4 mg/L, respectively. After a dose of 5mg tigecycline, Cmax in CSF was 37.894 mg/L which was high above the MIC values. The t 1/2 of tigecycline was estimated to be 2.73 hours. Tigecycline significantly decreased cell viability as assessed and induced apoptosis of the PC12 cell. The IC50 value of PC12 cells treated with tigecycline was about 51.35 mg/L. Conclusion: Intraventricular injection of tigecycline is a promising method for treating XDR A. baumannii intracranial infection. Since a high concentration of tigecycline in CSF may have potential neurotoxicity, and the t 1/2 was short, giving small doses of less than 5 mg at least twice a day may be safer and more effective. Intraventricular injection of tigecycline must be selected cautiously and best carried out under TDM.

Loss of MBD2 ameliorates LPS-induced alveolar epithelial cell apoptosis and ALI in mice via modulating intracellular zinc homeostasis.

Apoptosis of alveolar epithelial cells is a critical initial link in the pathogenesis of acute lung injury (ALI), recent studies have revealed that Methyl-CpG binding domain protein 2 (MBD2) was involved in the execution of apoptosis, yet its role in ALI remained unclear. In the present study, we aim to explore the role and mechanism of MBD2 in the pathogenesis of ALI. We have found that MBD2 expression, in parallel to apoptosis, increased in alveolar epithelial cells of mice treated with LPS, knockout of MBD2 reduced apoptosis and protected mice from LPS-induced ALI. In MLE-12 cells, a cell line of murine alveolar epithelial cells, LPS induced MBD2 expression and apoptosis in a dose- and time-dependent manner. Knockdown of MBD2 with shRNA alleviated, while overexpression of MBD2 increased LPS-induced apoptosis. Mechanistically, intracellular zinc level decreased when MLE-12 cells were treated with LPS. MBD2 knockdown restored intracellular zinc level after LPS treatment, and MBD2 overexpression further aggravated LPS-induced intracellular zinc loss. Metal transcription factor 1 (MTF1) is a critical transcription factor in charge of intracellular zinc efflux. LPS treatment induced MTF1 expression both in vivo and in vitro. Inhibition of MTF1 reduced LPS-induced apoptosis in MLE-12 cells. MBD2 could bind to the promoter region of MTF1 and promote MTF1 expression. Collectively, these data indicated that loss of MBD2-ameliorated LPS-induced alveolar epithelial cell apoptosis and ALI in mice via modulating intracellular zinc homeostasis by upregulating MTF1.

Increased Proportion of Dual-Positive Th2-Th17 Cells Promotes a More Severe Subtype of Asthma.

Asthma is a heterogeneous disease, and abnormal activation of T cells is the driving link of asthma's pathophysiological changes. Dual-positive Th2-Th17 cells, as newly discovered T-helper cells, have the functions of Th2 and Th17 cells and can coproduce Th2 and Th17 cytokines. Previous studies have shown that dual-positive Th2-Th17 cells increase the chances of asthma and correlate with asthma severity. However, the exact role of dual-positive Th2-Th17 cells in asthma is not known. Since there is no mature differentiation method for dual-positive Th2-Th17 cells, the present study aimed to clarify the strict differentiation conditions and reveal how dual-positive Th2-Th17 cells regulate asthma phenotypes. In this study, we confirmed that IL-1ß, IL-6, anti-IFN-γ, and IL-21 promoted biphenotypic cell differentiation. Moreover, more proportion of dual-positive Th2-Th17 cells can be obtained by conditioned differentiation of mouse CD4+ T cells after classical allergic asthma modeling. Before asthma modeling, adoptive dual-positive Th2-Th17 cells promoted T cells to differentiate into the same biphenotype cells and exacerbated the severity of asthma. Together, our results clarify the differentiation conditions of dual-positive Th2-Th17 cells and further confirm that it stimulates asthma T cells to differentiate into the same biphenotype cells, leading to exacerbation of asthma.

MicroRNA-16 inhibits the proliferation and metastasis of human lung cancer cells by modulating the expression of YAP1.

PURPOSE: Accounting for significant human morbidity and mortality across the globe, lung cancer is the most prevalent type of cancer as far as incidence and mortality is concerned. MicroRNAs (miRs) have shown an amazing potential to act as therapeutic agents for the management of several human diseases. This study investigated the function of miR-16 in lung cancer. METHODS: The normal lung cancer cell line MRC3 and lung cancer cell lines SK-MES-1, A549, MS-53 and SK-LU-1 were used in the present study. The qRT-PCR was used for expression profiling of miR-16 and yes associated protein 1 (YAP1). WST-1 assay was used to monitor the proliferation rate. Flow cytometry was used for cell cycle analysis. Apoptosis was examined by DAPI and annexin V/propidium iodide (PI) staining. TargetScan analysis was performed to identify the potential target of miR-16 and western blot analysis was done to estimate the protein expression. RESULTS: The gene expression analysis showed miR-16 to be suppressed in lung cancer tissues and cell lines. Overexpression of miR-16 inhibited the growth and metastasis of the DMS-53 lung cancer cells via induction of the apoptotic cell death. Bioinformatic approaches revealed miR-16 exerts its effects by targeting YAP1. YAPI expression was found elevated in lung cancer tissues and its silencing halted the growth of the DMS-53 lung cancer cells. Nonetheless, YAP1 overexpression could reverse the growth inhibitory effects of miR-16. CONCLUSION: Taken together, miR-16 may serve as novel therapeutic target for the treatment of lung cancer.

Dexmedetomidine attenuates the toxicity of ß­amyloid on neurons and astrocytes by increasing BDNF production under the regulation of HDAC2 and HDAC5.

Cytotoxicity of ß-Amyloid (Aß) is a major contributor to the pathogenesis of Alzheimer's disease. Dexmedetomidine (Dex) has been revealed to have multiple neuroprotective actions as a clinical anesthetic agent. The aim of the present study was to investigate the protection of Dex against Aß in neurons and astrocytes, and the possible protective mechanisms. Primary neurons and astrocytes were isolated respectively from the hippocampus and cerebral cortex of neonatal Sprague Dawley rats. The neurons and astrocytes were incubated with Aß in the presence or absence of Dex, which was followed by evaluation of the cell viability and apoptosis. Reverse transcription­quantitative polymerase chain reaction, western blotting and ELISA assays were performed to assess the levels of specific genes or proteins. The results revealed that Aß decreased the viabilities of neurons and astrocytes in a dose­dependent manner, and elevated the rate of apoptosis. However, Dex attenuated the detrimental effects of Aß. Aß caused deacetylation of histone H3 by promoting the accumulation of histone deacetylase (HDAC)­2 and HDAC5 in the cell nucleus, resulting in the reduced production of brain­derived neurotrophic factor (BDNF). However, Dex reversed the Aß­induced deacetylation of histone H3 and thus, increased BDNF production. Using a HDAC inhibitor or recombinant BDNF protein also protected the neurons and astrocytes against Aß cytotoxicity. These results suggested that the protective effect of Dex against Aß is particularly relevant to BDNF. Thus, the present study provides a foundation for the further study of Dex protection against Aß in animal models and pre­clinical researches.

Bronchial epithelial cells of young and old mice directly regulate the differentiation of Th2 and Th17.

To determine whether or not house dust mite (HDM) and HDM+lipopolysaccharide (LPS) exposure causes a difference in T-cell subsets from young and old mice. The bronchial epithelial cells (BECs) from young and old mice were divided into three groups (PBS (control), HDM, and HDM+LPS). CD4+ naive T cells from the spleen and lymph nodes were collected after 24 h of co-culture with BECs. The number of Th2 and Th17 cells was elevated in the HDM and HDM+LPS groups compared with the control group; these responses were exacerbated when exposed to HDM+LPS. The number of HDM- and HDM+LPS-specific Th2/Th17 cells in young mice was higher than old mice; however, the Th2:Th17 cell ratio was greater in young mice, whereas the Th17:Th2 cell ratio was greater in old mice. The expression of GATA-3 and RORc was increased in the HDM+LPS and HDM groups compared with the PBS group and exhibited most in HDM+LPS group. The expression of HDM+LPS-specific GATA-3 in young mice was higher, while the expression of HDM+LPS-specific RORc in old mice was higher. Murine BECs directly regulated CD4+ naive T-cell differentiation under allergen exposure.

MBD2-mediated Th17 differentiation in severe asthma is associated with impaired SOCS3 expression.

T helper 17 (Th17) cells has proven to be crucial in the pathogenesis of severe asthma. Although it is known that Suppressor of cytokine signaling 3 (SOCS3) is involved in differentiation of Th17 cells but, how it affects severe asthma is uncertain. Since previous studies indicated that Methtyl-CpG binding domain protein 2 (MBD2) null mice was deficient in Th17 cell differentiation, the aim of the present study was to understand how MBD2 interacts with SOCS3 to regulate Th17 cell differentiation in severe asthma. Here, we show that SOCS3 expression was significantly decreased in Th17-mediated severe asthmatic mice, accompanied by elevated STAT3 phosphorylation and RORγt expression. Knock-down of SOCS3 promoted the differentiation of naïve T cells into Th17 cells through STAT3/RORγt pathway. Meanwhile, MBD2 was overexpressed in Th17-mediated severe asthmatic mice. Intervention of MBD2 expression lead to a negative change of SOCS3 expression, whereas the differentiation of Th17 cells showed positive change. In addition, MBD2 knockout (MBD2-KO) mice displayed increased SOCS3 expression and decreased Th17 differentiation after severe asthma modeling. Taken together, our results suggest that MBD2 might facilitate Th17 cell differentiation via down-regulating SOCS3 expression in severe asthma. These findings uncover new roles for SOCS3 and MBD2, and provide a potential target for treatment of severe asthma.

Comparison of the roles of house dust mite allergens, ovalbumin and lipopolysaccharides in the sensitization of mice to establish a model of severe neutrophilic asthma.

Neutrophilic asthma (NA) is associated with a severe disease course and poor response to corticosteroids. The present study aimed to compare the effects of various concentrations of house dust mite (HDM) allergens, ovalbumin (OVA), the major egg allergen, and lipopolysaccharide (LPS) in combination on the onset of severe NA. Female C57BL/6 mice were grouped according to a random number table and intranasally sensitized with HDM/LPS/OVA extracts on days 0, 1 and 2 of the study. In group 1, mice received 50 µg HDM + 50 µg OVA + 15 µg LPS, mice in group 2 received 50 µg HDM + 100 µg OVA + 15 µg LPS, mice in group 3 received 100 µg HDM + 50 µg OVA + 15 µg LPS and those in group 4 received 100 µg HDM + 100 µg OVA + 15 µg LPS, while mice in the control group received saline only. The mice were then challenged by OVA solution with atomized excitation on days 14, 15, 18, 19 and 20 for 30 min each. Ethology, airway hyperresponsiveness (AHR), immune cell distributions in bronchoalveolar lavage fluid (BALF), and specific cytokines interleukin 17A (IL-17A) and IL-4 in serum were assessed. Histological examination of inflammation by hematoxylin and eosin staining and immunohistochemical assessment of neutrophils (NEU), eosinophils (EOS), IL-17A and IL-4 were also performed. Compared with the control group, the HDM/OVA/LPS-sensitized groups 1-4 had markedly increased BALF cells, serum interleukin IL-17A and IL-4, inflammatory cell infiltration, EOS as well as IL-17A and IL-4 by immunohistochemical staining (all P<0.05). Among the four HDM/OVA/LPS-sensitized groups, mice of group 4 had higher AHR, a significantly higher total cell number, NEU and EOS in BALF as well as significantly higher NEU and NEU/EOS ratios according to immunohistochemical staining when compared to groups 1-3 (P<0.05 for all). In conclusion, sensitization with 100 µg HDM + 100 µg OVA + 15 µg LPS successfully established a severe asthma model with a predominantly neutrophilic inflammatory phenotype.

MBD2 Regulates Th17 Cell Differentiation and Experimental Severe Asthma by Affecting IRF4 Expression.

Th17 cells and IL-17 participate in airway neutrophil infiltration characteristics in the pathogenesis of severe asthma. Methyl-CpG binding domain protein 2 (MBD2) expression increased in CD4+ T cells in peripheral blood samples of asthma patients. However, little is known about that epigenetic regulation of MBD2 in both immunological pathogenesis of experimental severe asthma and CD4+ T cell differentiation. Here, we established a neutrophil-predominant severe asthma model, which was characterized by airway hyperresponsiveness (AHR), BALF neutrophil granulocyte (NEU) increase, higher NEU and IL-17 protein levels, and more Th17 cell differentiation. In the model, MBD2 and IRF4 protein expression increased in the lung and spleen cells. Under overexpression or silencing of the MBD2 and IRF4 gene, the differentiation of Th17 cells and IL-17 secretion showed positive changes. IRF4 protein expression showed a positive change with overexpression or silencing of the MBD2 gene, whereas there was no significant difference in the expression of MBD2 under overexpression or silencing of the IRF4 gene. These data provide novel insights into epigenetic regulation of severe asthma.

Parecoxib Protects Mouse Cortical Neurons Against OGD/R Induced Neurotoxicity by Up-Regulating Bcl-2.

Ischemic stroke remains a significant problem that is the major cause of death and disability worldwide. Parecoxib is clinically used for short-term management of postoperative pain. Administration of parecoxib in rats has been reported to protect against the cerebral ischemia/reperfusion. However, the neuroprotective mechanism of parecoxib is still largely unknown. In this study, we found parecoxib could protect against neurotoxicity induced by 4 h oxygen-glucose deprivation (OGD) plus reoxgenation for 20 h, a widely used in vitro model of ischemia/reperfusion. In addition, we characterized the molecular mechanism of parecoxib's neuroprotection. We found parecoxib was able to activate CREB, and subsequently maintained the expression of Bcl-2, which is an important mitochondria-associated protein. Inhibition of endogenous Bcl-2 expression by transfection of Bcl-2-shRNA significantly attenuated the neuroprotective effects of parecoxib treatment. Furthermore, ATP production assay and mitochondrial membrane potential (ΔΨm) assay suggested that parecoxib exerted neuroprotective effect against OGD/R by maintaining the function of mitochondria. These data suggested that parecoxib treatment is a potential therapeutic approach for protecting against ischemia/reperfusion injury.

Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars differing in salt tolerance.

A sand culture experiment was conducted to answer the question whether or not exogenous KNO(3) can alleviate adverse effects of salt stress in winter wheat by monitoring plant growth, K(+)/Na(+) accumulation and the activity of some antioxidant enzymes. Seeds of two wheat cultivars (CVs), DK961 (salt-tolerant) and JN17 (salt-sensitive), were planted in sandboxes and controls germinated and raised with Hoagland nutrient solution (6 mM KNO(3), no NaCl). Experimental seeds were exposed to seven modified Hoagland solutions containing increased levels of KNO(3) (11, 16, 21 mM) or 100 mM NaCl in combination with the four KNO(3) concentrations (6, 11, 16 and 21 mM). Plants were harvested 30 d after imbibition, with controls approximately 22 cm in height. Both CVs showed significant reduction in plant height, root length and dry weight of shoots and roots under KNO(3) or NaCl stress. However, the combination of increased KNO(3) and NaCl alleviated symptoms of the individual salt stresses by improving growth of shoots and roots, reducing electrolyte leakage, malondialdehyde and soluble sugar contents and enhancing the activities of antioxidant enzymes. The salt-tolerant cultivar accumulated more K(+) in both shoots and roots compared with the higher Na(+) accumulation typical for the salt-sensitive cultivar. Soluble sugar content and activities of antioxidant enzymes were found to be more stable in the salt-tolerant cultivar. Our findings suggest that the optimal K(+)/Na(+) ratio of the nutrient solution should be 16:100 for both the salt-tolerant and the salt-sensitive cultivar under the experimental conditions used, and that the alleviation of NaCl stress symptoms through simultaneously applied elevated KNO(3) was more effective in the salt-tolerant than in the salt-sensitive cultivar.

[Effect of losartan on the expressions of TGF-beta1, p-Smad2/3, and Smad7 in the remnant renal tissues of 5/6 nephrectomized rats].

OBJECTIVE: To investigate the mechanism of losartan treating glomerulosclerosis and to observe the effect of losartan on the expressions of TGF-beta1, p-Smad2/3, and Smad7 in the renal tissues of 5/6 nephrectomized rats. METHODS: Male Wistar rats were randomly divided into a sham-operated group, a 5/6 nephrectomized model group, and a losartan treated group. The rats in the model group and the losartan treated group were performed 5/6 nephrectomy by the method with 2 procedures. Twelve weeks after of the operation, all rats were killed. The 24-hour urinary protein, serum creatinine, and urea nitrogen were detected. Pathological changes of the renal tissues were observed by HE and Masson staining, and the expressions of TGF-beta1, p-Smad2/3, and Smad7 were detected by immunohistochemical staining. RESULTS: The 24-hour urinary protein, serum creatinine, urea nitrogen, and the relative area of collagen in the renal tissues of the rats in the model group significantly increased (P<0.01), and losartan could reduce these indexes. The expressions of TGF-beta1 and p-Smad2/3 were just at a low level in the renal tissues of the rats in the sham-operated group, and were strongly positive in the model group; but losartan could decrease the expressions of TGF-beta1 and p-Smad2/3 (P<0.01). The expression of Smad7 in the model group was fewer than that in the sham-operated group (P<0.01), but losartan could improve the expression of Smad7 (P<0.01). CONCLUSION: Losartan may implement its anti-glomerulosclerosis by affecting TGF-beta1, p-Smad2/3, and Smad7 of TGF-beta/Smads pathway of the renal tissues of 5/6 nephrectomized rats.