Mechanism of chlorogenic acid in alveolar macrophage polarization in Klebsiella pneumoniae-induced pneumonia.

Chlorogenic acid (CA) has been discovered to regulate macrophage polarization in pneumonia. This study aims to analyze the functional mechanism of CA in alveolar macrophage (AM) polarization and provide a theoretical basis for treatment of Klebsiella pneumoniae (Kp)-induced pneumonia. Mice were infected with Kp, and treated with CA and silent information regulator 1 (SIRT1) inhibitor (Selisistat). Mouse survival rate was recorded and bacterial burden was detected. AM polarization and pathologic change of lung tissues were evaluated. Expressions of SIRT1 and HMGB1 and cytokine levels were detected. MH-S cells were infected with Kp to establish the pneumonia cell model, followed by transfection of si-SIRT1 and HMGB1 overexpression vector. The HMGB1 expression in the nucleus and cytoplasm was detected. HMGB1 subcellular localization and HMGB1 acetylation level were detected. Kp led to high death rates, SIRT down-regulation and increases in inflammatory factor level and bacterial burden, and promoted M1 polarization. CA treatment improved mouse survival rate and promoted M2 polarization and SIRT1 expression. SIRT1 decreased HMGB1 acetylation level to inhibit nuclear to the cytoplasm translocation. Silencing SIRT1 or HMGB1 overexpression reversed the effect of CA on Kp-induced pneumonia. Overall, CA activated SIRT1 to inhibit HMGB1 acetylation level and nuclear translocation, thereby promoting M2 polarization in AMs and alleviating Kp-induced pneumonia.

Complement component 3 prevents imiquimod-induced psoriatic skin inflammation by inhibiting apoptosis in mice.

Complement component 3 (C3), a pivotal molecule in the complement system, is an essential immune mediator in various diseases, including psoriasis. However, the mechanistic role of C3 in psoriasis pathology and development remains elusive. Here, we showed that C3 deficiency dramatically augmented imiquimod-induced psoriasis-like skin inflammation, characterized by greater epidermal hyperplasia, inflammatory cell infiltration, and inflammatory gene expression than those in wild-type counterparts. In addition, C3 deficiency promoted imiquimod-induced skin cell apoptosis and supported greater proportions of IFN-γ+ T cells in the inflamed tissues. Accordingly, C3 supplement in the C3 deficient mice reduced skin inflammation and cells apoptosis. Moreover, blocking apoptosis with Z-VAD-FMK, a broad caspase inhibitor, markedly attenuated imiquimod-induced psoriasis-like skin inflammation and IFN-γ+ T cell responses in C3-deficient mice. Collectively, our results suggest that C3 prevents imiquimod-induced psoriasis-like skin inflammation by inhibiting apoptosis.

Ag nanoparticles generated using bio-reduction and -coating cause microbial killing without cell lysis.

Cost-effective "green" methods of producing Ag nanoparticles (NPs) are being examined because of the potential of these NPs as antimicrobials. Ag NPs were generated from Ag ions using extracellular metabolites from a soil-borne Pythium species. The NPs were variable in size, but had one dimension less than 50 nm and were biocoated; aggregation and coating changed with acetone precipitation. They had dose-dependent lethal effects on a soil pseudomonad, Pseudomonas chlororaphis O6, and were about 30-fold more effective than Ag(+) ions. A role of reactive oxygen species in cell death was demonstrated by use of fluorescent dyes responsive to superoxide anion and peroxide accumulation. Also mutants of the pseudomonad, defective in enzymes that protect against oxidative stress, were more sensitive than the wild type strain; mutant sensitivity differed between exposure to Ag NPs and Ag(+) ions demonstrating a nano-effect. Imaging of bacterial cells treated with the biocoated Ag NPs revealed no cell lysis, but there were changes in surface properties and cell height. These findings support that biocoating the NPs results in limited Ag release and yet they retained potent antimicrobial activity.

Thyroid function is associated with non-alcoholic fatty liver disease in chronic hepatitis B-infected subjects.

BACKGROUND AND AIMS: Associations between thyroid function and non-alcoholic fatty liver disease (NAFLD) are unknown in chronic hepatitis B (CHB)-infected patients. Thus, the aim of the study was to investigate the prevalence of thyroid dysfunction and its relationship with NAFLD in CHB. METHODS: Consecutive naive CHB infected patients that had undergone liver biopsy and serum thyroid function tests between January 2007 and December 2011 were retrospective analyzed. NAFLD was diagnosed as at least 5% biopsy-proven hepatic steatosis without significant alcohol consumption. RESULTS: A total of 1154 non-alcoholics with CHB were included, 270 (23.39%) patients were found to have NAFLD, most of them (88.5%) with mild steatosis. The prevalence of hyperthyroidism and hypothyroidism (including subclinical and overt) was 1.56% and 1.64%, respectively, both with similar rates in patients with and without NAFLD (1.85% vs 1.47%, 1.48% vs 1.69%, respectively, both P > 0.05). The serum thyroid-stimulating hormone (TSH) level in NAFLD patients was significantly higher than that in patients without NAFLD (2.22 ± 2.13 vs 1.61 ± 1.20 mIU/L, P < 0.05). After adjustment for age and gender, the elevated TSH level was associated with increased odds of having steatosis (odds ratio1.54, 95% confidence interval 1.049-2.271) instead of viral factors and hepatic inflammation and fibrosis. CONCLUSIONS: Thyroid dysfunction is not common in CHB-infected patients, and the prevalence of hypothyroidism in CHB individuals with or without NAFLD is similar. However, increased serum TSH concentration at the normal range is a significant predictor of hepatic steatosis in patients with CHB.

Extracellular production of a novel endo-ß-agarase AgaA from Pseudomonas vesicularis MA103 that cleaves agarose into neoagarotetraose and neoagarohexaose.

The gene agaA, of the isolated marine bacterium Pseudomonas vesicularis MA103, comprised 2958-bp nucleotides encoding a putative agarase AgaA of 985 amino acids, which was predicted to contain a signal peptide of 29 amino acids in the N-terminus, a catalytic domain of glycoside hydrolase 16 (GH16) family, a bacterial immunoglobulin group 2 (Big 2), and three carbohydrate binding modules 6 (CBM 6). The gene agaA was cloned and overexpressed in Escherichia coli, and the optimum temperatures for AgaA overexpression were 16, 20 and 24 °C. The agaA was cloned without its signal peptide for cytosolic production overexpression, whereas it was cloned with the heterologous signal peptide PelB and its endogenous signal peptide for periplasmic and extracellular productions, respectively. Extracellular and periplasmic rAgaA showed greater activity than that of cytosolic rAgaA, indicating that membrane translocation of AgaA may encourage proper protein folding. Time-course hydrolysis of agarose by rAgaA was accomplished and the products were analyzed using thin layer chromatography and matrix-assisted laser desorption inoization-time of flight mass spectrometry, indicating that AgaA from P. vesicularis was an endo-type ß-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products.

Effect of lactose monolaurate on pathogenic and nonpathogenic bacteria.

The antimicrobial activities of sucrose monolaurate and a novel ester, lactose monolaurate (LML), were tested. Gram-positive bacteria were more susceptible than Gram-negative bacteria to both esters. The minimal bactericidal concentrations of LML were 5 to 9.5 mM for Listeria monocytogenes isolates and 0.2 to 2 mM for Mycobacterium isolates.

[Anticancer therapeutic effect of SEA-linked and membrane-bound HSP70-expressed intestine-carcinoma vaccine].

OBJECTIVE: To develop a novel dual-modified vaccine, the superantigen-linked intestine-carcinoma cells expressing membrane-bound heat shock protein 70 (HSP70), and further examine its anticancer therapeutic effect. METHODS: The pre-established intestine carcinoma CT26 line expressing membrane-bound heat shock protein 70 (HSP70) was amplified and incubated with superantigen fusion protein, staphylococcal enterotoxin A (SEA) fused with transmembrane sequence (SEA-TM), thereby the dual-modified vaccine was prepared after inactivation. The anticancer efficacy of the vaccine was examined. RESULTS: The laser confocal microscopy and flow cytometry showed that there co-existed much HSP70 and SEA on the vaccine membrane surface. Both of the single-modified vaccines, the SEA-linked vaccine and membrane-bound-HSP70-expressing one, displayed marked tumor suppression, a prolonged survival period, augmented lymphocyte proliferation and higher NK and CTL activity in the vaccinated mice when compared with its counterpart. Furthermore, the dually modified vaccine induced lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest tumor rejection in the vaccinated mice. The survival period of the mice was further prolonged. CONCLUSION: A new vaccine, SEA-linked and membrane-bound-HSP70-expressing intestine-carcinoma cells can induce more potent anticancer immunity and produce better therapeutic efficacy.

Receptor-mediated gene delivery using polyethylenimine (PEI) coupled with polypeptides targeting FGF receptors on cells surface.

OBJECTIVE: To construct a novel kind of nonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability. METHODS: The synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo. RESULTS: The polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides. CONCLUSION: The synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.

[Construction of recombinant GST-RCAS1 fusion gene and its expression in E. Coli].

OBJECTIVE: To construct the recombinant plasmid of RCAS1, to express and purify its fusion protein GST-RCAS1, and to investigate its biological function. METHODS: RCAS1 encoding gene was amplified by RT-PCR from total RNA extract of MCF-7 cells and was ligated with expression plasmid vector pGEX-2T by T4 DNA ligase after digested by the restricted endonucleases BamH I and EcoR I. Then the ligated products were inserted into competence JM109 E. Coli and the positive recombinants were identified by restriction endonuclease digestion assay and DNA sequencing. The GST-RCAS1 fusion protein expression was induced by IPTG in BL21 E. Coli and was purified with GST column and identified by SDS-PAGE and Western blotting with anti-GST monoclonal antibody, anti-RCAS1 (N-18) and anti-RCAS1 (C-20) polyclonal antibody. The apoptosis of activated T cells induced by GST-RCAS1 fusion protein was detected by flow cytometry with Annexin V and propidium iodide (PI) staining. RESULT: A 642 bp product was cloned by RT-PCR and the recombinant plasmid was constructed successfully. The GST-RCAS1 fusion protein was recognized by GST monoclonal antibody and RCAS1(N-18 and C-20) polyclonal antibody. FACS analysis showed that GST-RCAS1 fusion protein induced apoptosis in activated T cells. CONCLUSION: The recombinant plasmid of RCAS1 has been successfully constructed and the GST-RCAS1 fusion protein expressed and purified. The apoptosis inducing effect of GST-RCAS1 fusion protein on activated T cells is demonstrated.

[Antitumor therapeutic effect induced by intestine-carcinoma cells vaccine expressing membrane-bound heat shock protein 70].

OBJECTIVE: To develop a new vaccine expressing membrane-bound heat shock protein 70 (mbHSP70) and further study its antitumor therapeutic effect. METHOD: The pre- established vector expressing mbHSP70 was transfected into CT26 cells of colorectal cancer. After the CT26 cells were incubated with 900 microg/ml G418, the sub-clones resistant to G418 were harvested and the HSP70 positive clones were selected by limiting dilution. The clones were amplified and inactivated, thereby the vaccine expressing mbHSP70 was prepared. Lymphocyte proliferation stimulated by the vaccines, NK and CTL activity was observed. The antitumor efficacy of vaccine was observed in BALB/c mice model with colorectal cancer. RESULTS: The laser confocal microscopy and flow cytometry showed that there existed much HSP70 on the vaccine membrane surface. The HSP70 gene-modified vaccine displayed augmented lymphocyte proliferation and higher NK and CTL activity in vitro,and marked tumor suppression and prolonged survival time of the vaccinated micein vivo, when compared with its counterpart. Furthermore, mbHSP70-expression vaccine elicited lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest antitumor effect, and prolonged survival time of the vaccinated mice. CONCLUSION: A new vaccine expressing mbHSP70 has more potent antitumor immunity and better therapeutic efficacy than HSP70 gene-modified vaccine did.

[Study on lysosomes degradation of ricin A chain].

OBJECTIVE: To study lysosomes involvement in the degradation of ricin A chain. METHODS: A lysosome-targeted singal KFERQ was added to the C terminus of rRTA by DNA recombinant technology. A pKK223.3 expression system in E. coli was used to produce recombinant ricine A chain (rRTA) and rRTA-KFERQ. Recombinant proteins were purified by affinity chromatography using Blue-Sepharose 6B. The cytotoxicity of recombinant proteins was measured by the MTT method. RESULTS: Recombinant RTA-KFERQ was 49.87%, 54.18% and 88.68% less cytotoxic than RTA itself on the three cell lines HEPG2, Hela and A549, respectively. CONCLUSION: Lysosomes can degrade, but not completely inactivate RTA in different cells, suggesting cells may have other degradation pathways for RTA.

[Gene transfer by novel non-viral vector polyethylenimine].

OBJECTIVE: To investigate the influencing factors of polyethylenimine (PEI) in gene transfer in vitro. METHODS: Cytotoxic effects of PEI on in vitro cultured NIH 3T3 cells were quantified by MTT assay. The interaction between PEI and DNA at different charge ratios was analyzed by agarose gel electrophoresis retardation assay. The expression of gene transfer was monitored in Cos-7 cells using pEGFP and pSV beta plasmids as the reporter gene systems. Influences of chloroquine, albumin, serum, salt ion strength, and Mg(2+) ion and other factors on PEI/DNA transfer efficiency were evaluated. RESULT: The survival rate of NIH3T3 cells at 6 mg/L of PEI was 64.2% and at 7 mg/L of PEI was 54.4%. Gel electrophoresis retardation assays showed that PEI completely retarded DNA migration at 3.0 PEI nitrogen per DNA phosphate. Chloroquine enhanced the transfection efficiency of PEI. Albumin and serum in the culture medium decreased the transfection efficiency. HBS(HEPES buffered solution) or 150 mmol/L NaCl as the dilution solution of PEI/DNA was superior over 278 mmol/L glucose solution in the transfection efficiency. Mg(2+) in the dilution solution decreased the transfer efficiency of PEI/DNA. CONCLUSION: PEI is efficient gene transfer agent of eukaryotes in vitro, and can be possibly used in vivo.