Dalestones A and B, two anti-inflammatory cyclopentenones from Daldinia eschscholzii with an edited strong promoter for the global regulator LaeA-like gene.

Replacement of the native promoter of theglobal regulator LaeA-like gene of Daldinia eschscholzii by a strong gpdA promoter led to the generation of two novel cyclopentenone metabolites, named dalestones A and B, whose structures were assigned by a combination of spectroscopic analysis, modified Mosher's reaction, and electronic circular dichroism (ECD). Dalestones A and B inhibit the gene expression of TNF-α and IL-6 in LPS-induced RAW264.7 macrophages.

Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells.

BACKGROUND: Treatments that target cancer stem cells play an important role in the controlling and eliminating of tumor initiation as well as in development, progression, and chemotherapy/radiotherapy resistance. In our previous study, we cultured and harvested human laryngeal cancer stem cells (CSCs) and applied microRNA biochips to screen differentially expressed miRNAs that were related to radiation tolerance in irradiated human laryngeal CSCs. According to the predicted genes and pathways of differential miRNAs target, down-regulated expression of hsa-miR-138-2-3p under radiation was thought to play a key role in enhancing the radio-sensitivity in human laryngeal squamous cancer stem cells. METHOD: To investigate the radiational enhancement of hsa-miR-138-2-3p, we transfected hsa-miR-138-2-3p mimics that were synthesized based on the sequences of hsa-miR-138-2-3p in vitrointo human laryngeal CSCs (Hep-2, M2e, and TU212 cell lines) to make hsa-miR-138-2-3p overexpressed, and the tumorous specialities of CSCs, like cell proliferation, invasion, apoptosis, cell cycle arrest, and DNA damage were evaluated by CCK-8 assay, clone formation assay, invasion assay, flow cytometry, and comet assay. Furthermore, we explored the signal transduction pathways that regulated the cancer stem cell initiation, development, invasion, apoptosis and cell cycle arrest, which were controlled by hsa-miR-138-2-3p. RESULT: Overexpressed hsa-miR-138-2-3p played a key role in many anti-cancer biological processes in human laryngeal CSCs: (1) it decreased laryngeal CSCs proliferation and invasion in response to radiotherapy; (2) it increased the proportion of early and late apoptosis in laryngeal CSCs after radiation, raised G1 phase arrest in laryngeal CSCs after radiation, and decreased the proportion of S stage cells of cell cycle that were related to radio-resistance in laryngeal CSCs; (3) it down-regulated the expression of ß-catenin in Wnt signal pathway that was related to the tolerance of laryngeal CSCs to radiotherapy; (4) it down-regulated the expression of YAP1 in Hippo signal pathway that regulated cell proliferation, invasion and apoptosis; (5) it up-regulated the expression of p38 and JNK1 in MAPK signal pathway that was concerned to radio-sensitivity. CONCLUSION: In the present study, it was found that hsa-miR-138-2-3p regulated the Wnt/ß-catenin pathways, the Hippo/YAP1 pathways, and the MAPK/p38/JNK1 pathways that were involved in cell proliferation, invasion, apoptosis, cell cycle arrest, radio-resistance and radio-sensitivity in laryngeal CSCs. These results will be useful for a better understanding of the cell biology of hsa-miR-138-2-3p in laryngeal CSCs, and for serving hsa-miR-138-2-3p as a promising biomarker and as a target for diagnosis and for novel anti-cancer therapies for laryngeal cancers.

Anti-inflammatory and protective properties of daphnetin in endotoxin-induced lung injury.

Uncontrolled inflammatory responses cause tissue injury and severe immunopathology. Pharmacological interference of intracellular pro-inflammatory signaling may confer a therapeutic benefit under these conditions. Daphnetin, a natural coumarin derivative, has been used to treat inflammatory diseases including bronchitis. However, the protective effect of daphnetin in inflammatory airway disorders has yet to be determined, and the molecular basis for its anti-inflammatory properties is unknown. This paper shows that daphnetin treatment conferred substantial protection from endotoxin-induced acute lung injury (ALI), in parallel with reductions in the production of inflammatory mediators, symptoms of airway response, and infiltration of inflammatory cells. Further studies indicate that activation of macrophage and human alveolar epithelial cells in response to lipopolysaccharide (LPS) was remarkably suppressed by daphnetin, which was related to the down-regulation of NF-κB-dependent signaling events. Importantly, this study demonstrates that TNF-α-induced protein 3 (TNFAIP3), also known as A20, was significantly induced by daphnetin, which appeared to be largely responsible for the down-regulation of NF-κB activity through modulation of nondegradative TRAF6 ubiquitination. Accordingly, the deletion of TNFAIP3 in primary macrophages reversed daphnetin-elicited inhibition of immune response, and the beneficial effect of daphnetin in the pathogenesis of ALI was, partially at least, abrogated by TNFAIP3 knockdown. These findings demonstrate the anti-inflammatory and protective functions of daphnetin in endotoxin-induced lung inflammation and injury and also reveal the key mechanism underlying its action in vitro as well as in vivo.

Efficacy and significance of various scores for pneumonia severity in the management of patients with community-acquired pneumonia in China.

BACKGROUND: Community-acquired pneumonia (CAP) remains one of the leading causes of death from infectious diseases around the world. Most severe CAP patients are admitted to the intensive care unit (ICU), and receive intense treatment. The present study aimed to evaluate the role of the pneumonia severity index (PSI), CURB-65, and sepsis score in the management of hospitalized CAP patients and explore the effect of ICU treatment on prognosis of severe cases. METHODS: A total of 675 CAP patients hospitalized in the Second Affiliated Hospital of Zhejiang University School of Medicine were retrospectively investigated. The ability of different pneumonia severity scores to predict mortality was compared for effectiveness, while the risk factors associated with 30-day mortality rates and hospital length of stay (LOS) were evaluated. The effect of ICU treatment on the outcomes of severe CAP patients was also investigated. RESULTS: All three scoring systems revealed that the mortality associated with the low-risk or intermediate-risk group was significantly lower than with the high-risk group. As the risk level increased, the frequency of ICU admission rose in tandem and LOS in the hospital was prolonged. The areas under the receiver operating characteristic curve in the prediction of mortality were 0.94, 0.91 and 0.89 for the PSI, CURB-65 and sepsis score, respectively. Compared with the corresponding control groups, the mortality was markedly increased in patients with a history of smoking, prior admission to ICU, respiratory failure, or co-morbidity of heart disease. The differences were also identified in LOS between control groups and patients with ICU treatment, heart, or cerebrovascular disease. Logistic regression analysis showed that age over 65 years, a history of smoking, and respiratory failure were closely related to mortality in the overall CAP cohort, whereas age, ICU admission, respiratory failure, and LOS at home between disease attack and hospital admission were identified as independent risk factors for mortality in the high-risk CAP sub-group. The 30-day mortality of patients who underwent ICU treatment on admission was also higher than for non-ICU treatment, but much lower than for those patients who took ICU treatment subsequent to the failure of non-ICU treatment. CONCLUSIONS: Each severity score system, CURB-65, sepsis severity score and especially PSI, was capable of effectively predicting CAP mortality. Delayed ICU admission was related to higher mortality rates in severe CAP patients.

[Effects of Sema3A derived from tumor cells on functions of dendritic cells].

OBJECTIVE: To investigate the effects of tumor cell-derived Sema3A on the immunological functions of murine dendritic cells (DCs). METHODS: Lung adenocarcinoma A549 cells were transfected with small interference RNA, Si-Sema and Si-mut, and the interference efficiency was determined by real-time PCR and Western-blot. The concentrated supernatants from cultured tumor cells, Si-Sema and Si-mut-infected tumor cells were subjected to DCs respectively. The immunophenotypes of DCs were analyzed by flow cytometry, the production of IL-12P70 and the ability of DCs to stimulate DO11. 10 T cells secreting IFN-gamma and IL-2 were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Knockdown with Si-Sema3A significantly decreased the secretion of Sema3A by A549 cells in comparison with the Si-mut cells. DCs exposed to supernatants from Si-Sema cells showed elevated levels of MHC, CD40 and CD80, more production of IL-12P70, and enhanced capability of activating antigen-specific T cells, as evidenced by the remarkably increased levels of IFN-gamma and IL-2. CONCLUSION: A549 cells secrete Sema3A to inhibit the maturation and functions of DCs, which might be associated with the unidentified mechanism of immune evasion by tumor cells.

[Adjuvant effect of co-stimulatory molecule CD137L on cellular responses to HBsAg DNA vaccination in mice].

OBJECTIVE: To investigate the adjuvant effect of co-stimulatory molecule CD137L on cellular responses to HBsAg DNA vaccination in mice. METHODS: Eukaryotic expression vector containing the full length of mouse CD137L cDNA sequence (pcD137L) was transfected into NIH3T3 cells, and then the expression of CD137L mRNA and protein in the transfected cells were detected by RT-PCR, flow cytometry and immunofluorescence method, respectively. The BALB/c mice were co-immunized with pcD137L and HBsAg DNA vaccine (pcDS) by intramuscular injection. HBsAg-specific activity of splenic cytotoxic T lymphocyte (CTL) in the immunized mice was measured by LDH release assay. The splenic memory CD8+ T cells, and intracellular IFN-gamma and IL-4 of splenic lymphocytes and CD8+ T cells after immunization were detected by flow cytometry. RESULTS: The NIH3T3 cells transfected with pcD137L efficiently expressed mouse CD137L mRNA and protein. HBsAg-specific CTL responses induced by the pcDS plus pcD137L group were much stronger than those induced by pcDS alone at a week after immunization (P<0.05). Compared to mice immunized with pcDS alone, CD44high and CD127(IL-7R) were all significantly up-regulated in memory CD8+ T cells from the mice immunized with pcDS combined CD137L both at a week and 12 weeks after immunization (P<0.05 and P<0.01). The pcDS plus CD137L group also elicited higher levels of IFN-gamma secreted by CD8+ T cells and splenic lymphocytes than pcDS alone at a week, 12 and 13 weeks after immunization, respectively (all P<0.01). CONCLUSION: DNA, viral/immunol; Co-stimulatory molecule CD137L can enhance the Tc1 (type I) cell-mediated immunity, HBsAg-specific CTL and memory responses induced by HBsAg DNA vaccine, and may be an efficient adjuvant in priming HBV-specific T cell response.

[Construction and expression of recombinant adenovirus containing human thymic stromal lymphopoietin gene].

AIM: To construct and express recombinant adenovirus bearing human thymic stromal lymphopoietin (TSLP) gene. METHODS: TSLP gene amplified from human fetal lung cells was first cloned into eukaryotic expression vector pcDNA3.1, and then subcloned into shuttle vector pShuttle. The resultant plasmid was subsequently cotransformed into E. coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1. The recombinant adenovirus plasmid containing TSLP was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. The TSLP gene of the recombinant virus was detected by PCR, and its expression was analyzed by Western blot. RESULTS: Recombinant adenovirus vector bearing human TSLP gene was constructed by homologous recombination in E.coli, and recombinant adenovirus was obtained by transfecting HEK293 cells with this infectious DNA. PCR test indicated that TSLP gene was successfully integrated into the adenoviral genome, and the titer of the recombinant Ad reached 1 x 10(11) pfu/L. Meanwhile, expression of TSLP in the infected Hela cells was confirmed by Western blot. CONCLUSION: The construction of recombinant adenovirus bearing human TSLP gene and its expression mediated by this adenovirus pave a foundation for the study on the biological function of this novel cytokine.