ObjectiveTo investigate the protective effect of transfected microRNA-146a (miR-146a) on mice with sepsis-induced acute lung injury (ALI) in vivo.Methods Twenty-four healthy male BALB/C mice were randomly divided into sham group, sepsis group, transfection group and transfection control group, eachn = 6. Mice in transfection group were given miR-146a agomir loaded by in vivo-jetPEITM via airway before reproduction of model, and mice in transfection control group were given negative control loaded by in vivo-jetPEITM only via airway. The septic model was reproduced by cecal ligation and puncture (CLP) 12 hours after transfection , and the mice in the sham group underwent laparotomy and closure only without ligation and puncture of the cecum. The mice of each group were sacrificed at 24 hours post-operation. The expression of miR-146a in lung tissue was determined by real time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the quantity of tumor necrosis factor-α (TNF-α) in the bronchial alveolar lavage fluid (BALF) was determined with enzyme-linked immunosorbent assay (ELISA). The wet/dry ratio of lung (W/D) was determined. The pathohistological changes in the lung were observed and scored. Results The expression of miR-146a showed a significant increase in sepsis group, transfection group and transfection control group, which were (3.56±0.43), (27.64±3.46) and (3.72±0.54) folds of that in sham group, respectively (P< 0.05 orP< 0.01). The miR-146a expression in transfection group was significantly increased compared with sepsis group and transfection control group (bothP< 0.01), but no statistical difference in the expression was found between sepsis group and transfection control group (P> 0.05). Compared with the sham group, higher level of TNF-αin the BALF was found in the sepsis group, transfection group and transfection control group (ng/L: 511.65±43.47, 305.74±34.76, 492.27±42.21 vs. 50.72±7.23, allP< 0.01). The level of TNF-α in transfection group was significantly lower than that in sepsis group and transfection control group (bothP< 0.01). Compared with the sham group, the W/D ratio of lung in sepsis group, transfection group and transfection control group showed a significant increase (6.11±0.32, 5.02±0.29, 6.05±0.43 vs. 4.18±0.10, allP< 0.01). The W/D ratio of lung in transfection group was significantly lower than that of sepsis group and transfection control group (bothP< 0.01). The lung injury score of transfection group was significantly lower than that of sepsis group and transfection control group (6.12±0.75 vs. 10.53±1.52, 9.73±1.08, bothP< 0.01).Conclusions miR-146a agomir loaded by in vivo-jetPEITM instillation into airway was able to increase the expression of miR-146a in the lung tissue of septic mice. Up-regulation of miR-146a inhibit the release of the inflammatory cytokine TNF-α stimulated by sepsis, and alleviate inflammatory reaction and lung tissue injury in mice with sepsis-induced ALI.

Objective To screen and identify the predicted epitopes of synthesized HLA-A*0201restricted CTL derived from HPVll E7 antigen.Methods Five HPVll E7 CTL epitope peptides and terramers consisting of HLA-A*0201 were selected by way of computer and synthesized by Sanquin company,including HPVllE7 7-15(TLKDIVLDL),15-23(LQPPDPVGL),47-55(PLTQHYQIL),81-89(DLLLGTLNI)and 82-90(LLLGTLNIV).These peptides binding to human peripheral blood-derived DCs were tested for their ability to activate T cells isolated from peripheral blood lymphocytes of HLA-A*0201 healthy individuals.the number of specific tetramer+CD8+T cells by flow cytometry,the level of the section of IFN-γ by ELISA,and the ability of the CTL to kill the target cells were observed.Results The immature DCs could be fully activated by all the five HPV11 E7 peptides.Peptide-loaded mature DCs were able to stimulate the epitope-specific T cells responses in vitro.An increased frequency(P<0.05)of T ceils specific for the E7 7-15 epitope compared to other epitopes of HPV11E7.The epitope-specific CTL of E7 7-15 induced by the activated DCs specifically killed HPV11E7 expressing 293 cell line,and in a ratio of 50:1,the specific cytolytic activity was the strongest than the others(P<0.05).Conclusion DCs loaded with HPV11 E7 7-15(TLKDIVLDL)peptide can induce highly effective and specific ectogenic processed epitopespecific CTL responses in vitro.This peptide may be the candidate for development of CTL based vaccine in the treatment of HPV infeetions.

Objective To investigate the expression of Toll-like receptors(TLRs) in condyloma acuminatum(CA) lesions and their possible roles in the pathogenesis of CA. Methods The expressions of TLR1-10 mRNA level in the lesions of CA and in the cervix scrape cells from the patients with human papillomavirus(HPV) negative chronic cervicitis were detected by real-time quantitative fluorescent PCR. HPV typing was detected by HPV GenoArray test kit. Results Low-risk HPV type 6 and type 11 were the most prevalent types in the forty CA cases with positive rate of 77.5% and 55% respectively. 55% CA patients were found infected with more than two types of HPV. 35% CA patients were concurrently infected with high-risk HPV. The expressions of TLR3, 7, 8 mRNA were higher than other TLRs and the expression of TLR9 mRNA was lower than others in the lesions of CA. No significant differences of the TLR1-10 mRNA levels were found between HPV6 and HPV11 positive CA lesions, so did it between low-risk and high-risk HPV concurrent infected CA lesions. The expressions of TLR1-3, TLR5-8, TLR10 mRNA, especially TLR2, TLR7 and TLR8 in the lesions of CA were significantly higher than that in cervix scrape cells of HPV negative chronic cervicitis. There were no significant differences of TLR4 and TLR9 mRNA levels between the two groups. Conclusion There were higher expressions of some TLRs (3, 7, 8) and lower expression of TLR9 in the lesions of CA. Compared with HPV negative chronic cervicitis, the expressions of TLR1-3, TLR5-8, TLR10 mRNA in the lesions of CA were up-regulated. The expression profile of TLRs in different type of HPV infected CA lesions had no significant differences. Our results suggested that the expression profile of TLRs in CA may be associated with the HPV infection. Whether it was associated with the immune escape mechanism and persistent infection of HPV need further demonstration.

Objective To investigate the expression and clinical significance of intergrin linked kinase (ILK) in pancreatic carcinoma.Methods ILK protein was detected by immunohistochemistry and Western blotting in 60 cases of pancreatic carcinoma and 32 cases of normal pancreatic tissue,and the relationship with the clinicopathological characteristics were analyzed.Results Immunohistochemistry showed ILK was expressed in cytoplasm and membrane of pancreatic carcinoma cells and the positive rate was 65% (39/60),which was significantly higher than 18.75% (6/32) of normal pancreatic tissue(P ＜0.05 ).Western blotting showed the expression of ILK in pancreatic carcinoma tissue was 303933±195116,which was significantly higher than 144613±30074 of normal pancreatic tissue(P＜0.05 ).In pancreatic carcinoma,the expression of ILK was correlated with clinical stage and lymph metastasis(P＜0.05 ),but not correlated with tumor cell differentiation(P＞0.05 ).Conclusions ILK protein was highly expressed in pancreatic carcinoma tissue and it was correlated with the degree of malignancy.

Objective To observe the levels of Foxp3+ CD+ CD25+ regulatory T cells in the peripheral blood of condyloma acuminatum (CA) patients and investigate their roles in the pathogenesis of CA. Methods The peripheral blood was collected from 30 CA patients (including 15 with relapsing and 15 with first onset) and 20 healthy controls. Peripheral blood mononuclear ceils (PBMC) were isolated and stained with anti-human CD4-PE-Cy5 and anti-human CD25-fluorescein isothiocyanate (FITC) monoclonal antibodies on cell membrane, followed by intraeellular staining with anti-human Foxp3-PE. The percentage of Foxp3+ CD4+-CD25+ regulatory T cells was detected by three-color flow cytometry. Comparison between groups was done by ANOVA test. Results The percentages of Foxp3+ CD4+ CD25+ regulatory T cells among total CD4 + T cells in CA patients and relapsing CA group were (3.4 ± 1.0) % and (4.7 ±+ 1.2) %, respectively, which were both significantly higher than that in healthy control group [(1.2±0. 5)%, P<0.01]. Furthermore, that in first onset CA group was (2. 1 ± 1.0) %, which was higher than that in healthy control group, but without statistical significance; but that in relapsing CA group was significantly higher than that in first onset group (P<0.05). Conclusions The number of Foxp3+ CD4+ CD25+ regulatory T cells increases in the peripheral blood of CA patients. The disorder of cellular immunity may be involved in the immunopathogenesis of CA.

Objective To evaluate the diagnostic value of immunohistochemistry (IHC) for the identification and localization of Treponema pallidum (TP).Methods Rabbit anti-human TP polyclonal antibody labeled IHC was used to detect 20 paraffin-embedded biopsy samples from lesions of 14 patients with syphilis or suspected syphilis in Sir Run Run Shaw Hospital of Zhejiang University from January 2004 to May 2012.Results TP was detected in 80％ of all the 20 samples by IHC assay,including 83.3％ (5/6) in patients with primary syphilis,100.0％ (10/10) in patients with secondary syphilis,and 25.0％ (1/4) in patients with tertiary syphilis,with a positive diagnostic accuracy of 100.0％.TP was mainly present in lower part of epidermis or perivascular,characterized by an endotheliotropic and epitheliotropic patterns or in the tissue of granulomatous inflammation.Besides,the density of TP was associated with types of lesions.There were more TP in the lesions of syphilis chancre,syphilis proctitis and condyloma latum,and fewer TP in the lesions of squamous erythema,greyish-black plaque,ulcer of chest wall from tertiary syphilis,and least in syphilitic lymphadenitis.There were no correlations between the quantity of TP and the rapid plasma regain (RPR) test titer (P＞0.05).Conclusions IHC for TP is of both high sensitivity and specificity for the diagnosis of syphilis,suggesting that TP-IHC is helpful for the diagnosis of syphilis,especially for the diagnosis of early suspected syphilis with negative serological results,systemic damage of syphilis,or syphilis in untypical locations and unusual lesions.It can serve as an alternative method for the diagnosis of syphilis.

Objective To study the treatment effect and the mechanism of high volume hemofiltration(HVHF)on acute lung injury(ALl)induced by endotoxin in dogs.Methods Sixteen healthy hybrid male dogs were injected LPS(650?g/kg)via central vein within 30 minutes.After model establishment,all animals were divided into two groups randomly( n=8).One group received the treatment of HVHF,while another group received routine treatment.PH,PaO_2,PaCO_2 in arterial blood were recorded at O h after LPS model establishment and 4h after HVHF.Contents of TNF-?,IL-6,and IL- 10 in plasma were measured by radioirnmunity,mRNA expression of TNF-?,IL-6,and IL-10 in lung tissue homogenate were measured by RT-PCR and NF-?B activity by flow cytometer.Results After injection of LPS,PaO_2 and PaO_2/FiO_2 began to decrease,and PaO_2/FiO_2 value was

Objective To determine kinetics of TNF-α and miR-146a (microRNA-146a)expressions in lipopolysaccharide (LPS)-induced NR8383 alveolar macrophages (AM) at different intervals and their relationships in order to explore regulatory effect and mechanism of miR-146a on alveolar macrophages inflammatory responses.Methods NR8383 alveolar macrophages were seeded in a 6-well plate,and stimulated with 1 μg/ml of LPS for 0 h,3 h,6 h and 12 h separately after 90 min.Cells were harvested and supernatant were collected 0 h,3 h,6 h and 12 h after incubation.The expressions of miR146a and TNF-α mRNA in cells were detected by real-time qPCR and the levels of TNF-α protein in the supematant of cells were assayed by enzyme-linked immunosorbent assay ( ELISA ).Pearson correlation analysis was used to analyze the correlation between miR-146a and TNF-α mRNA.Results ①The level of TNF-α protein in the supernatant of cell was significantly increased 3 h after LPS challenge ( 359.80 ±57.54) pg/ml (P ＜0.01 ),and peaked 12 h later (729.22 ±50.40) pg/ml (P＜0.01 ) ; ②the expression of TNF-α mRNA peaked 3 h after LPS challenge (67.48 ±24.52) fold,P ＜0.01 ),and then decreased gradually; ③the expression of miR-146a mRNA increased continuously until 6 h or 12 h after LPS challenge 6 h:(5.33 ±0.81) fold,12 h:(8.21 ±1.19) fold,(P＜0.01),and it showed an upward tendency;④ the expression of miR-146a mRNA was negatively correlated with TNF-α mRNA ( r =-0.895,P ＜0.01).Conclusions The miR-146a mRNA showed a negative correlation with TNF-α mRNA present in lipopolysaccharide-stimulated alveolar macrophages,suggesting miR-146a mRNA involved in regulating the inflammatory response of alveolar macrophages.

OBJECTIVETo develop a RP-HPLC method for simultaneous determination of liquiritin, naringin, hesperidin and glycyrrhizic acid in extraction of Wendan formula.

METHODDIKMA Diamonsil(2)-C18 column (4.6 mm x 250 mm, 5 microm) was used at 25 degrees C with the mobile phase of acetonitrile-0.1% phosphatic acid in a gradient manner. The flow rate was set at 1.0 mL min(-1). The detection wavelength was 237, 283 nm.

RESULTThe linear responses ranged from 0.0199-0.1191 microg for liquiritin (r = 0.9997, n = 6), 0.1800-1.0800 microg for naringin (r = 0.9997, n = 5), 0.1455-0.8730 microg for hesperidin (r = 0.9998, n = 6), 0.0393-0.2355 microg for monoammonium glycyrrhizinate (r = 0.9997, n = 6), respectively. The average recoveries were 97.7% with RSD 1.5% for liquiritin, 97.7% with RSD 2.0% for naringin, 97.1% with RSD 2.0% for hesperidin and 98.5% with RSD 1.9% for glycyrrhizic acid, respectively.

CONCLUSIONThe method is quick, simple and repeatable for simultaneous determination of liquiritin, naringin, hesperidin and glycyrrhizic acid in extraction of Wendan formula.

Chromatography, High Pressure Liquid ; methods ; Chromatography, Reverse-Phase ; methods ; Drugs, Chinese Herbal ; analysis ; isolation & purification ; Flavanones ; analysis ; isolation & purification ; Glucosides ; analysis ; isolation & purification ; Glycyrrhizic Acid ; analysis ; isolation & purification ; Hesperidin ; analysis ; isolation & purification

OBJECTIVETo investigate the effect of triptolide on the differentiation of human Th17 cells.

METHODHuman peripheral blood mononuclear cells, purified CD4+ T cells and CD4+CD45RA- memory T cells were treated with various concentrations of triptolide in vitro. Cell proliferation was determined by MTT assay. Flow cytometry was used to analyze the intracellular expression of IL-17 and IFN-gamma. Cytokine production of IL-17 and IFN-gamma was measured by ELISA.

RESULTCell proliferation, intracellular expression of IL-17 and IL-17 secretion were inhibited by triptolide in a dose-dependent manner. IFN-gamma expression and production were also inhibited by triptolide.

CONCLUSIONTriptolide inhibits the differentiation of human Th17 cell. The observation may indicate at least one of the mechanisms of the immunosuppressive and anti-inflammatory effects of triptolide.

Anti-Inflammatory Agents, Non-Steroidal ; CD4-Positive T-Lymphocytes ; drug effects ; metabolism ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Diterpenes ; pharmacology ; Dose-Response Relationship, Drug ; Epoxy Compounds ; pharmacology ; Humans ; Interferon-gamma ; drug effects ; metabolism ; Interleukin-17 ; metabolism ; secretion ; Phenanthrenes ; pharmacology ; Th17 Cells ; cytology ; drug effects ; Tripterygium ; chemistry