Adolescent ; Adult ; Aged ; Burns ; drug therapy ; physiopathology ; Epidermal Growth Factor ; therapeutic use ; Humans ; Middle Aged ; Recombinant Proteins ; therapeutic use ; Wound Healing ; drug effects ; Young Adult

Objective To explore the mechanisms underlying the mediating effect of regulatory B (Breg)cells in the pathogenesis of pemphigus.Methods Peripheral blood specimens were collected from 48 patients with pemphigus at different degrees of severity and 20 healthy controls.Flow cytometry was conducted to analyze the frequency of Breg(CD19 +CD24hiCD38hi)cells in peripheral CD19+ B cells,enzyme linked immunosorbent assay(ELISA)to determine the titer and subtypes of anti-desmoglein(Dsg)1 and 3 antibodies in sera from these subjects.The relationship of anti-Dsg 1 and 3 antibodies with the frequency of Breg cells was assessed by Spearman's rank correlation test.Results Significant differences were observed in the proportion of Breg cells in CD19+ B cells between patients with pemphigus and healthy controls(11.54％ ± 0.97％ vs.8.19％ ± 0.85％,P =0.04),as well as between patients with acute mild pemphigus and those with moderate and severe pemphigus(5.17％ ± 2.14％ vs.11.38％ ± 5.30％ and 11.17％ ± 5.31％,P =0.042,0.046,respectively).The frequency of Breg cells was positively correlated with the absorbance value for anti-Dsg1 antibodies of IgG4 subclass and IgG4/IgG1 ratio(r =0.527,0.565,respectively,both P ＜ 0.01).Conclusions Breg cells are mainly associated with the production of antibodies in and disease severity of pemphigus,while the actual mechanism of action remains unknown.

Objective To investigate the role of nentrophils in the pathogenesis of psoriasis vulgaris. Methods Neutrophils were isolated from venous blood samples of 25 patients with psoriasis vulgaris (including 13 cases of active psoriasis and 12 cases of inactive psoriasis) as well as 25 normal human con-trols, and cultured. Then, these neutrophils were grouped and treated with lipopolysaccharide (LPS, 100 g/L),CpG-A (50 mg/L), CpG-B (50 mg/L), and RPMI 1640 culture medium, respectively, for 24 hours followed by the collection of culture supematants. Human keratinocytes (HaCaT) were cultured in the presence of su-pematants of treated or untreated nentrophils for 72 hours followed by the detection of cell proliferation with MTT assay. To determine the role of proinflammatory factors, SOD/CAT and monoclonal antibody to IL-8 and TNF-alpha of 400 u/mL were used to pretreat HaCaT cells 1 hour prior to the stimulation with super-natants of neutrophils. Results Compared with culture medium, the supematant of unstimulated neutrophils from normal controls or patients with inactive psoriasis had no significant effect on the proliferation of HaCaT cells (P ＞ 0.05), but that from patients with active psoriasis markedly promoted the proliferation of HaCaT cells (t = 2.41, P < 0.05). ARe, stimulation by LPS, CpG-A and CpG-B, the supematant of active patient-derived neutrophils significantly promoted the proliferation of HaCaT cells compared with that of normal control-derived nentrophils (t = 3.11, 2.89, 2.29, respectively, all P < 0.05). In comparison with tmstimulated neutrophils, the supematant from LPS- and CpG-A stimulated nentrophiles significantly accelerated the pro-liferation of HaCaT cells. Furthermore, the proliferation of HaCaT cells induced by the supematants of LPS-,CpG-A-, CpG-B-stimulated neutrophils from psoriatic patients was statistically suppressed by the pretreat-ment with the monoclonal antibody to IL-8, TNF-alpha and SOD/CAT (all P < 0.05). Conclusions In patients with psoriasis vulgaris, there is an abnormal secretion of IL-8, TNF-alpha and superoxide by neutrophils in peripheral blood, and these proinflammatory factors could promote the proliferation of HaCaT cells.

Objective To assess the correlation between the subclasses of antibodies against desmoglein (Dsg) 1 and Dsg3 and disease activity in patients with pemphigus. Methods Sera were collected from 47 patients with pemphigus, and ELISA was performed to determine the titers and subclasses of antibodies against Dsg1 and Dsg3. The correlation of antibody titers and subclasses with disease activity was assessed.Results Clinical phenotype was associated with antibody profiles in these patients with pemphigus. Of 17 patients with mucocutaneous involvement, 14 (82.4%) had both anti-Dsg3 and anti-Dsg1 antibodies; of 16 patients with cutaneous involvement, 15 (93.7%) had anti-Dsg1 antibody, only 1 (6.3%) developed anti-Dsg3 antibody; of 6 patients with mucosal involvement, all ( 100% ) had only anti-Dsg3 antibody. The serum levels of antibodies against Dsg1 and Dsg3 were increased, but not in parallel with the disease severity in these patients.Moreover, the subclasses of antibodies were correlated with disease severity. IgG4 subclass antibodies against Dsg1 and Dsg3 predominated in patients with pemphigus at active stage, whereas IgG1 subclass in those at remission stage. The serum levels (expressed as absorbance value) of IgG4 and IgG1 subclass antibodies were 1.92 ± 1.21 and 0.60 ± 0.61 respectively, with the ratio of IgG4 to IgG1 more than 1, in patients with antiDsg1 antibodies at active stage, 0.03 ± 0.02 and 0.22 ± 0.11 respectively with the ratio of IgG4 to IgG1 less than 1, in those at remission stage, 2.35 ± 2.17 and 1.84 ± 1.16 respectively with the ratio of IgG4 to IgG1 more than 1 in patients with anti-Dsg3 antibodies at active stage, 0.15 ± 0.16 and 1.05 ± 0.77 respectively with the ratio of IgG4 to IgG1 less than 1 in those at remission stage. Conclusions The subclasses of pemphigus-specific antibodies are closely correlated with the disease severity in patients with pemphigus. The detection of subclasses and titers of anti-Dsg1 and anti-Dsg3 antibodies may aid in the diagnosis of and monitoring of disease severity in pemphigus.

Objective To investigate the role of IL-13 in the patients with acute and chronic urticaria. Methods In 22 patients with acute urticaria, 20 patients with chronic urticaria and 19 normal controls, the levels of IL-13, IL-4 and IFN-? of peripheral T lymphocytes were measured by flow cytometry. The serum concentrations of IL-13 and total IgE were tested by ELISA. Results The results of flow cytometry showed that the level of IL-13 of the patients with acute urticaria was significantly higher than that of the normal controls (P

Objective To study the prognostic value of procalcitonin(PCT) and C?reactive protein ( CRP ) in critically ill patients with ventilator?associated pneumonia ( VAP )?Methods A single?center prospective observational study was conducted?A total of 67 cases patients with VAP admitted into intensive care unit(ICU) from November 2013 to October 2015 were enrolled and grouped as survivors(43 cases) and non?survivors(24 cases)?Blood samples for PCT and CRP were collected on the day of the pneumonia diagnosis,and the 4th and 8th day after the diagnosis?Results There were 24 cases(35?8 %) died among the 28 days after the pneumonia diagnosis?There was no significant difference between the survivor and non?survivor groups in terms of PCT on the day of the pneumonia diagnosis( P>0?05) ,or CRP on the day of the pneumonia diagnosis, and the 4th and 8th day after the diagnosis ( P>0?05)?But the PCT values on the 4th and 8th day were significantly higher in the non?survivor group than the survivor group(4 d:0?4(0?3,1?1) μg/L vs?4?7(2?3, 10?8) μg/L,P<0?05;8 d:0?2(0?1,1?7) μg/L vs?3?9(3?2,14?8) μg/L,P<0?05)?PCT levels decreased significantly from the day of the pneumonia diagnosis(0?7(0?4,4?2) μg/L) to the 8th day after the diagnosis (0?2(0?1,1?7) μg/L,P<0?05) in the survivor group?The PCT level above 1 μg/L on the 4th day after the diagnosis was the strongest predictor of mortality,with an odds ratio of 23?Conclusion PCT is found to be a more important prognostic marker compared to CRP in terms of predicting mortality in critically ill patients with VAP?The PCT level on the 4th day after the diagnosis is the strongest predictor of mortality in VAP.

Objective To investigate the pathogenic significance of antigenic epitopes and their relevant antibodies in pemphigus vulgaris (PV) by neonatal mouse model. Methods The extracellular domain 1-2 (EC1-2) fusion protein was expressed and purified by glutathione affinity chromatography on the basis of construction of recombinant EC1-2 vector, and then the New Zealand white rabbits were immunized to obtain the specific antisera. The IgG fraction was transferred into the neonatal mice passively after it was purified from the antisera. After 15-18 hours of injection, the abdomen skin and the sera of the mice were examined by light microscopy, electron microscopy, direct immunofluorescence and indirect immunofluorescence. Results In the evaluation of the study group of mice, the intraepithelial vesicle formation was observed. Electron microscopy showed that intercellular spaces were widened, desmosome split and disappeared. In immunofluorescence, the fluorescence-labeled IgG deposied between the acantholytic cells. In the control group of mice there were no pathogenic changes observed, except very weak fluorescence between intercellular spaces. Conclusion The PV mouse model established shows that the EC1-2 epitope in PVA antigen and its relevant antibodies were pathogenic, and can be used as a tool in studying the pathogenesis of PV.

Objective To determine the molecular characteristics of predominant Salmonella typhi and Salmonella paratyphi A strains prevalent in Hangzhou area from 2002 to 2008.Methods Pulse field gel electrophoresis (PFGE),multi-locus variable-number tandem repeat analysis (MLVA) and multi-locus sequence typing (MLST) were applied for typing as well as analysis of the molecular characteristics of 31 S.typhi isolates and 404 S.paratyphi A isolates from Hangzhou area during 2002 to 2008.Results The 404 S.paratyphi A isolates could be divided into six PFGE types (P1-P6).99.0％ of the S.paratyphi A isolates (400/404) belonged to the same one clone family (P1 and P2 types),in which P1 strains occupied 93.3％ (373/400) of the isolates.The 31 S.typhi isolates displayed a high diversity,which could be classified into 14 PFGE types,28 MLVA types with 90.3％ resolving power and 3 MLST types.The S.typhi strains prevalent in Hangzhou area were similar to those in Southeast Asia but different from those in Europe.The variable number tandem repeat (VNTR) sites with high polymorphism,TR1,TR2 and Sal02,could be used to the markers for diagnosis of S.typhi isolates in the area.The MLST types of 31 S.typhi isolates included all the three types currently found in the world but the ST2 type of S.typhi strains was predominant (23/31,74.2％).Conclusion The paratyphoid A prevalence in Hangzhou area in the recent years is caused by infection of the same clone family of S.paratyphi A whereas the S.typhi strains prevalent in the area display a high diversity.

Background and purpose:The pro-oncogene Bmi-1 is a member of the polycomb-group family, can regulation of the proliferation and self-renewal of normal and tumor stem cells. In recent years, Bmi-1 has been found that it is overexpressed in varieties of human malignant tumors. The study aimed to observe the effects of Bmi-1-siRNA on the growth capacity of lung cancer cell line A549 in vivo and in vivo, and explore its mechanism. Methods:The most effective one as a target sequence was chosen from four Bmi-1 siRNA sequences which were designed by our lab, and one random sequence was chosen as a negative control. In short, the chemically synthesized siRNA and control sequences were connected to a retrovirus expressing vector, pSUPERretro-Neo plasmid, and then transfected into A549 cells. The stably transfected cells were cultured and passed. The level of mRNA and protein of Bmi-1 in A549 cells were assessed by RT-PCR and Western blot respectively. The proliferations of A549 cells in vivo was analyzed with MTT, trypan blue exclusion and plate colony forming methods. Flow cytometry was used for cell cycle analysis. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of A549 cells. The expressions of cyclin D, p21/27, p-AKT and PTEN were analyzed by Western blot. Results:Compared to A549-ctr and A549-wt cells, Bmi-1 mRNA and protein levels all signiifcantly reduced in A549-Bmi-1-siRNA cells. Bmi-1-siRNA inhibited the growth, colony formation in vitro and tumorigenesis in vitro of A549 cells, and the interference cells cell cycle arrested in G1 phase. In A549-Bmi-1-siRNA cells, p-AKT and cyclinD1 expression were down-regulated while p21/p27 and PTEN were up-regulated. Conclusion:Silencing Bmi-1 gene inhibits the proliferation of A549 cells through G1 phase arrest, which involves the downregulation of cyclin D/p-AKT and upregulation of p21/p27/PTEN.

Background and purpose:The human oncogene B-cell-speciifc moloney murine leukemia virus integration site 1 (Bmi-1) is an important member of the polycomb group family, and it regulates cell proliferation and senescence via INK4a/ARF locus. This study investigated the effects of Bmi-1-siRNA on the proliferation of lung adenocarcinoma cell line SPC-A1 cells with INK4a/ARF locus and clarify the mechanism of Bmi-1-mediated effect on proliferation of lung adenocarcinoma cells. Methods:In this study, we chose the most efifcient siRNA chain the pGeneshl-2-Bmi-1 sense-1 and inserted into a pSUPER-retro-neo retroviral vector. The packaged si-Bmi-1 pSUPERret-ro-neo retroviral vector was stably transfected into lung adenocarcinoma SPC-A1 cell line. The stably transfected cells were cultured and passed. After transfection, the levels of Bmi-1 mRNA and protein expression of SPC-A1 cells were analyzed by RT-PCR and Western blot respectively. Trypan blue, MTT and plate colony forming assay were performed to observe the proliferation capibility of SPC-A1 cells and evaluate the cloning forming ability in vitro. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of SPC-A1 cells. Cell cycle distribu-tion was analyzed by lfow cytometry (FCM) in SPC-A1 cells. The expression levels of proliferation proteins including p16INK4a, p53, Cyclin D1, PTEN, Akt and Ser473p-Akt were analyzed by Western blot. Results:The mRNA and protein expression levels of Bmi-1 were signiifcantly reduced in SPC-A1-Bmi-1-siRNA cells transfected with pSUPER-retro-neo retroviral vector. Knockdown of Bmi-1 could inhibit the growth, colony formation in vitro and tumorigenesis in vivo of SPC-A1 cells (P<0.01). The transfected SPC-A1 cells were arrested in G1 phase [(64.6±1.2)%, P<0.05]. Compared with two control groups, p16INK4a, p53 and Akt were not affected (P>0.05), while Cyclin D1 and Ser473p-Akt were downregulated (P<0.01) and PTEN was up-regulated (P<0.01) in the SPC-A1-Bmi-1-siRNA cells. SPC-A1-Bmi-1-siRNA cells were treated with various concentrations of PTEN inhibitor to determine expression levels of PTEN, Bmi-1 and Ser473p-Akt protein. Ablation of PTEN rescued Bmi-1 and Ser473p-Akt expression in SPC-A1-Bmi-1-siRNA cells. Conclusion:Knockdown of Bmi-1 gene can arrest the proliferation of SPC-A1 cells through G0/G1 phase arrest by inhibiting Cyclin D1 expression indirectly, which may be not associated with p16INK4a signaling pathway.