Objective Three kinds of different internal promoters were inserted into the lentiviral vector to drive the expression of CD19-specifc chimeric antigen receptor (CAR)-redirected T lymphocytes with the green fluorescent protein(GFP) and the GFP expression efficiency of transduced cells driven by lentivirus were compared.Methods Three GFP reporter lentivirus vectors carrying different promoters and anti-CD19-CAR,including CMV,EF1α,and CMV-EF1α were selected.Three different vectors' names abbreviate to pHAGE-CMV-EFlα-EGFP-CD19,pHAGE-CMV-EGFP-CD19,pHAGE-EF1α-EGFP-CD19.Human embroic kideny 293T cells were cotransfected with the three plasmids by calcium phosphate DNA precipation.And the expression of GFP was observed under fluorescent microscope after transfecting 293T cells,and virus supernatant was collected after 72 h and centrifuged.Nucleic acid copy number in RNA and the expression of EGFP and CD3 zeta were identified through fluorescence quantitative PCR,flow cytometry and western blotting,respectively.The titers of the lentiviral vectors were determinde by scoring GFP expression following swerial dilutions of the viral supernatant on 293T cells.T lymphocytes was infected with lentivims produced from these reporter vectors.Then,fluorescence microscope,wesyern blotting were used to detect the GFP expression strength.Results Results of fluorescence microscope maintained that different internal promotors driving the expressing effect is different.293T as targeted cell,the transfected 293T cells were found containing strong expression of GFP.The amount of the 293T cells expressing GFP driven by CMV-EF1α promoter was the largest,while the EF1α promoter was the smallest.Flow cytometry results show that the rate of transduction is 72.4％,20.6％ and 14.5％,respectively.T lymphocytes as targeted cell,Western blot showed that CMV-EF1α was the largest among all promoters,while the CMV was the smallest.Conclusion In the study of gene expression mediated by lenuvirus,proper cell lines and promoters should be selected to obtain high efficiency.

0.05).The death rate of cases with pneumonia combined with ascites was higher than that of cases with ascites only(?2=4.894,P=0.027) and cases without ascites and infections(?2=9.260,P=0.002).Unfavorable prognosis was found in cases with Enterococcus faecium isolated before OLT.CONCLUSIONS Severe lung infection before OLT is one of the main reasons of death.It is important to grasp characteristics of infection,evaluate risk fully,control infections and screen cases strictly before OLT to improve survival rate.

Objective To analyze the clinical characteristics of children with cyclic vomiting syndrome (CVS), summarize the clinical experience, and improve the awareness,diagnosis and treatment level of CVS.Methods The clinical data and results of long-term follow-up of the children with CVS were collected and analyzed from 1995 to 2009 in our department.Results Forty-six children were enrolled in the study, including 22 boys and 24 girls.Mean onset age was 4.5 years(ranged from 1 to 11 years)and the mean age at final diagnosis was 8.5 years (ranged from 4.5 to 14.5 years).Sixty-six and seventy-four percent of patients had family history of migraine and motional sickness respectively,whereas 83% patients had triggers.The clinical manifestation of CVS was severe episodic vomiting.The episodes had a rapid onset and sudden ending,persisted for several hours to days,and were separated by symptom-free intervals.The incidental symptoms were pallor, lethargy,intractable nausea,abdominal pain,headache,photophobia and dizzy.Twenty-nine patients diagnosed were followed up for 5 years at average.Five patients were treated with valproate,4 with cyproheptadine, 5 with cyproheptadine and valproate,5 with amitriptyline,cyprohep tadine and valproate,and 3 with cyproheptadine and flunarizine.All treated patients recovered mean 10 months(1.5 months to 2 years) years later and displayed the reduced number of episodes or the severity of episodes except 3 patients.Twenty-eight percent (8/29) of patients progressed to migraine headaches.Conclusion CVS is a relatively common disease in children and awareness of the condition should be increased.CVS should be considered when patients had recurrent vomitting and were completely healthy between the two episodes after excluding other pathological conditions.

Objective To investigate the correlation between the frequency of myeloid-derived suppressor cells (MDSC) and the frequency of regulatory T cells (Treg) in the peripheral blood in patients with chronic hepatitis B (CHB) and its clinical significance.Methods A total of 45 CHB patients including 23 mild-to-moderate CHB patients,22 severe CHB patients,and 15 healthy controls were enrolled.The frequencies of MDSC and Treg in the peripheral blood were studied using flow cytometry and its correlation with clinical data was analyzed by Sepearman correlation analysis.Results The median frequency of MDSC in CHB patients was 0.414％,which was significantly higher than that in healthy controls 0.226％ (Z=-2.356,P=0.018 9).The frequency of MDSC in CHB patients was negatively correlated with the level of alanine transaminase (ALT) and aspartate transaminase (AST) (r=-0.480,-0.478; both P＜0.01),but had no relations with hepatitis B virus (HBV) viral load (r=-0.049,P=0.75).An increase frequency of MDSC was observed in CHB patients with an ALT of 5 × upper limits of normal (ULN) or less or AST of 3 × ULN or less.The frequency of MDSC in CHB patients was positively correlated with that of Treg (r =0.345,P =0.02).Conclusions The activation and proliferation of MDSC may facilitate and maintain HBV persistent infection.The change of the frequency of MDSC is in line with that of Treg,indicating that immunosuppressive functions of MDSC may be related with the development of Treg in CHB.

Objective To investigate HBV genotypes and gene mutations in chrenic hepatitis B (CHB) patients with liver failure after lamivudine withdrawal. Methods Twenty four patients with relapsing CHB after lamivudine withdrawal were divided into liver failure group ( n = 12 ) and chronic hepatitis group ( n = 12 ). HBV DNA from these patients was amplified by PCR. The PCR products were cloned into PGEM-T vector and HBV DNA sequences were analyzed. Results In liver failure group, there were 6 sequences detected, in which 3 were of genotype B and 3 were of genotype C. In chronic hepatitis group, there were 9 sequences detected, in which 2 were of genotype B and 7 were of genotype C. Compared with the wild type HBV sequences, there were multiples mutations in S, P, C, X regions. Gene mutations in high conservative sequences of BCP and P regions were detected in liver failure patients after lamivudine withdrawal. Conclusions In HBV patients with liver failure after lamivudine withdrawal, half of them were of genotype B and the others were of genotype C. Some mutations in high conservative sequences of BCP and P regions may be related to the liver failure in these patients.

Objective To study the expression of signaling lymphocytic activation molecule (SLAM)CD150 in peripheral blood mononuclear cells(PBMCs)isolated from adult non-responders to recombined yeast gene hepatitis B vaccine.Methods A total of 202 cases were recruited.All these subjects had been immunized with recombined yeast gene hepatitis B vaccine for more than one standard scheme in two years(from Sep 2007 to Dec 2009)and remained negative for hepatitis B markers(HBsAg,anti-HBs,HBeAg,anti-HBe and anti-HBc).After recruitment,all 202 subjects received another standard scheme(0,1 and 6 month)revaccination.The blood samples were collected 7 months later after the first injection of revaccination to detect anti-HBs titer.The PBMCs were isolated from 18 adult non-responders(anti-HBs titer<10 mIU/mL)and 18 adult responders(antiHBs titer≥100 mIU/mL).CD150 expression on cell surface was analyzed by flow cytometry.SAS package was used for t test and spearman rank correlation analysis.Results After rHBsAg stimulation,the percentage of PBMCs expressed CD150 was significantly higher in non-responders (39.20%±10.66%)than responders(23.73%±12.41%)(t=2.1947,P<0.05).The same trend was also observed in rHBsAg stimulated C133+CD4+T cells,but the difference was not statistically significant(49.64%±11.94%vs 37.73%±11.02%)(t=1. 7175,P>0.05).After phytohaemagglutinin (PHA)stimulation,the percentage of CD150-positive PBMCs was also significantly higher in non-responders (39.21%±7.37%)than responders(23.18%±12.68%)(t=2.2835,P<0.05).CD150 expressions in both PBMCs and CD3+CD4+T cells were negatively correlated with anti-HBs titer after rHBsAg stimulation (r=-0.726,P<0.05).Conclusion Activation of CD150 may contribute to the non-response to hepatitis B vaccine.

Objective To detect the mRNA and protein expressions of desmoglein 1 (DSG1) and DSG3 in different types of keratinocytes (KCs).Methods Two keratinocyte cell lines HaCaT and A431,as well as primary keratinocytes from human abdomenal skin served as the object of this study.Direct immunofluorescence assay was performed to observe and quantify the expressions of DSG1 and DSG3,and quantitative PCR (qPCR) to determine the mRNA expressions of DSG1 and DSG3,in these cells.Results Both DSG1 and DSG3 were expressed in all the three types of keratinocytes,and the fluorescence intensity of DSG1 and DSG3 in HaCaT cells was higher than that in primary keratinocytes but lower than that in A431 cells.Similarly,all the keratinocytes expressed DSG1 and DSG3 mRNA,with the relative expression levels of DSG1 and DSG3 mRNA in primary keratinocytes being 291.7％ and 237.4％ of those in HaCaT cells respectively (both P ＜ 0.01),and those in A431 cells being 0.1％ and 18.8％ of those in HaCaT cells respectively (both P ＜ 0.05).Conclusions HaCaT cells,A431 cells and primary keratinocytes all can be used for the study of DSG1 and DSG3,of which,A431 cells show the strongest expressions of DSG1 and DSG3,and primary keratinocytes display the highest expressions of DSG1 and DSG3 mRNAs.

OBJECTIVE: To investigate the protective effect of protein kinase C (PKC) inhibitor rottlerin on rat renal vascular endothelial injury induced by lipopolysaccharide (LPS). METHODS: Rat renal microvascular endothelial cells cultured for 3-6 generations were divided into three groups according to random number table: blank control group in which cells were not challenged, LPS group in which cells were only stimulated by LPS 10 mg/L for 24 hours, and PKC inhibitor group in which cells were treated with PKC inhibitor rottlerin 2 μmol/L 30 minutes before LPS stimulation. The levels of tumor necrosis factor-α (TNF-α) and interleukins (IL-1β, IL-8) were determined by enzyme-linked immunosorbent assay (ELISA). Monolayer permeability was determined by Transwell assay. The expressions of PKC, RhoA and vascular endothelial-cadherin (VE-cadherin) were detected by Western Blot. The morphological characteristic and distribution of F-actin was measured by laser confocal fluorescence microscope. RESULTS: Compared with blank control group, the levels of inflammatory cytokines at 24 hours after 10 mg/L LPS stimulation were significantly increased in LPS group [TNF-α (ng/L): 397.3±25.4 vs. 46.8±8.9, IL-1β (ng/L): 76.7±11.2 vs. 12.6±3.2, IL-8 (ng/L): 574.5±31.4 vs. 73.2±9.6, all P < 0.05], the permeability of endothelial cells was significantly increased (A value: 1.32±0.03 vs. 0.36±0.02, P < 0.05), while the expressions of PKC and RhoA were significantly up-regulated (PKC/β-actin: 0.88±0.02 vs. 0.61±0.03, RhoA/β-actin: 0.96±0.01 vs. 0.49±0.03, both P < 0.05), VE-cadherin expression was significantly down-regulated (VE-cadherin/β-actin: 0.51±0.01 vs. 0.72±0.04, P < 0.05), and the F-actin distribution disorder had obvious stress fiber formation. Compared with LPS group, the levels of inflammatory cytokines were significantly lowered in PKC inhibitor group [TNF-α (ng/L): 127.4±14.6 vs. 397.3±25.4, IL-1β(ng/L): 43.2±7.8 vs. 76.7±11.2, IL-8 (ng/L): 212.7±18.2 vs. 574.5±31.4, all P < 0.05], the permeability of endothelial cells was significantly decreased (A value: 0.81±0.02 vs. 1.32±0.03, P < 0.05), the expressions of PKC and RhoA were significantly down-regulated (PKC/β-actin: 0.44±0.03 vs. 0.88±0.02, RhoA/β-actin: 0.63±0.05 vs. 0.96±0.01, both P < 0.05), the VE-cadherin expression was significantly up-regulated (VE-cadherin/β-actin: 0.69±0.03 vs. 0.51±0.01, P < 0.05), and the F-actin remodeling and stress fiber formation were significantly reduced. CONCLUSIONS PKC inhibitor could significantly attenuate the damage of vascular endothelial barrier induced by LPS, and plays an important role in endothelial cell barrier.
Acute Kidney Injury/prevention & control* ; Animals ; Endothelium, Vascular/drug effects* ; Interleukin-1beta ; Lipopolysaccharides/toxicity* ; Protein Kinase C/antagonists & inhibitors* ; Protein Kinase Inhibitors/pharmacology* ; Random Allocation ; Rats

Objective To evaluate the effects of nucleoside/nucleotide analogue treatment on immunoglobulin and complement in patients with chronic hepatitis B (CHB).MethodsA total of 157 CHB patients were recruited and divided into CHB group,liver cirrhosis (LC) group and severe hepatitis B (SHB) group.There were 50 patients who received oral antiviral treatment (lamivudine 100 mg/d,or entecavir 0.5 mg/d,or telbivudine 600 mg/d).Serum levels of complement 3 and 4 (C3,C4),C-reaction protein (CRP),hemolytic complement (CH50),immunoglobulin G,M,A (IgG,IgM,IgA),hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were detected by enzyme-linked immunosorbent assay (ELISA) or immunoturbidimetry.Hepatitis B virus (HBV) DNA was quantified by real-time polymerase chain reaction (RT-PCR) before and 1,2,3 and 4 weeks after nucleoside antiviral therapy.Comparison of means was done by t test and Mann-Whitney test.The correlation was analyzed by Pearson correlation coefficient test.ResultsSerum IgA and IgM levels of SHB and LC patients were significantly higher than those of CHB patients (P＜0.01).Levels of C3,C4,CH50 and CRP were significantly different among three groups.Levels of C3,IgM,IgG and HBV DNA in HBeAg positive patients were significantly different from those in HBeAg negative patients.There was a statistically significant difference of IgA,IgM,C3 and CH50 levels between patients with high HBV DNA level and low HBV DNA level in HBeAg-positive patients.While in the HBeAg-negative patients,only the IgA level was significantly different with HBV DNA levels.After anti-viral treatment,immunoglobulin and HBV DNA levels were all decreased in three groups,while the serum complement level was increased compared to baseline,and the differences became significant at week 4 of treatment. HBV DNA level was negatively correlated with C3 (r=-0.78,P=0.021) and HBeAg titer was positively correlated with C3 (r=0.87,P=0.015).ConclusionsThe immunoglobulin,CRP,C3,C4,and C H50 could reflect the inflammatory activity in liver.The changes of C3 level can predict the efficacy of antiviral therapy.

Objective To study the relationship between programmed death-1 (PD-1)/programmed death-1 ligand (PD-L1) expressions and serum hepatitis B virus (HBV) DNA levels in chronic hepatitis B (CHB) patients. Methods A total of 137 CHB patients and 10 healthy controls were enrolled in the study. The peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples. HBV-specific cytotoxic T lymphocyte (CTL) was expanded in vitro in 64 human leucocyte antigen (HLA)-A2 positive patients. Flow cytometry was used to detect HLA-A2 type,expressions of PD-1/PD-L1 on PBMCs and PD-1 on HBV specific CTL. Interferon gamma (IFN-γ)was measured by commercial enzyme-linked immunosorbent assay (ELISA) kits. PD-1/PD-L1expressions on PBMCs, HBV-specific CTL and IFN-γ level in PBMC culture medium were compared among patients with different baseline HBV DNA levels. Ten hepatitis B e antigen (HBeAg) positive patients were treated with telbivudine for 24 weeks. The above mentioned parameters were determined and compared before and after the antiviral treatment. Independent-samples t test were used to compare means between two groups and one-way A NOVA were used to compare means among multigroups. We used the pearson corretation test to assess corretation significance. Results The PD-1 and PD-L1 expressions on PBMCs in patients with baseline HBV DNA＜3 lg copy/mL, 3-6 lg copy/mL and ＞6 lg copy/mL were all significant higher than those in healthy control group, but no statistical differences were found. PD-1 expressions on HBV-specific CTL in the three CHB patient groups were (69.3±11.2)%, (76.5±9. 1)% and (78.0±11.7)%, respectively. However, PD-1 expression on HBV-specific CTL was higher, while the frequency of HBV-specific CTL cells was lower in HBV DNA ＞6 lg copy/mL group compared to HBV DNA＜3 lg copy/mL group. The above parameters, including expressions of PD-1 and PD-L1, the frequency of HBV-specific CTL and its PD-1 expression were not significantly different between HBeAg-positive group and HBeAg-negative group. Compared with baseline, PD-1 and PD-L1 expression decreased obviously accompanying with increase of HBV-specific CTL cells frequency and IFN-γ level after 12 weeks and 24 weeks of telbivudine treatment. Conclusions PD-1 expression on HBV-specific CTL correlates with serum HBV DNA level, but not HBeAg status in CHB patients. Suppression of HBV replication can reduce PD-1/PD-L1 expressions and partially restore HBV specific CTL function.