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INTRODUCTION: The role of inflammation and immunity in COPD treatment is increasingly being recognized. The relationship between anti-inflammation/immunoregulation and emphysema in COPD lungs remains to be elucidated. The aim of this study was to investigate the effects of azithromycin (Azm) on the development of emphysema in smoking-induced COPD in rats. METHODS: Sprague-Dawley rats (n = 50) were randomly assigned to normal, COPD, saline-treated, Azm-treated, and levofloxacin-treated (Lev) groups. The effects of treatment were assessed by measuring the levels of vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay and measuring the numbers of neutrophil and macrophage in bronchoalveolar lavage fluid, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) protein expression by western blotting. Lung function measurements and histopathological evaluations (mean linear intercept and destructive index) were performed. RESULTS: FEV0.3/FVC and peak expiratory flow were lower in the COPD group than in the normal group. Mean linear intercept and destructive index were lower in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. The numbers of neutrophil and macrophage in bronchoalveolar lavage fluid were lower in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. As confirmed by western blotting, the levels of VEGF in lung homogenates were higher in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. VEGFR2 protein expression was higher in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. CONCLUSIONS: Azm attenuates pulmonary emphysema by partly reversing the decrease in the numbers of inflammatory cells (neutrophil and macrophage) and VEGF secretion and VEGFR2 protein expression in smoking-induced COPD in rats.

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OBJECTIVE: To study the immunoregulatory effect of hepatitis B virus (HBV) e antigen (HBeAg) on peripheral blood monocytes (PBMCs). METHODS: PBMCs were isolated from patients with chronic hepatitis B (CHB; both HBeAg- and HBeAg+) and healthy controls, and cultured with recombinant HBeAg. The HBeAg-induced changes in expression of PD-1/PD-L1 were measured by flow cytometry of the cells and in secreted cytokines were measured by enzyme-linked immunosorbent assay of the supernatants. Comparisons between two groups were made by the independent-samples t-test; the relationship between PD-1/B7-H1 level and HBV DNA copy number was evaluated by Spearman's correlation analysis. RESULTS: Exposure to HBeAg led to a significant decrease in CD3+CD4+ T lymphocyte-specific expression of IFNa for both the CHB patients' and healthy controls' samples (t = 2.382 and -4.190 respectively, P less than 0.01). For the HBeAg- CHB patients' and healthy controls' samples, the HBeAg exposure led to increased levels of secreted cytokines IL-6, IL-10 and TNFa (t = 2.504, 3.583 and 4.324, P less than 0.01 and t = 3.542, 6.246 and 5.273, P less than 0.01 respectively) and of CD14+ PBMC-specific expression of PD-L1 (t = 4.815 and 3.454, P less than 0.05 respectively). Compared to the HBeAg-negative CHB patients' and healthy controls' samples, the HBeAg+ CHB patients' samples had significantly lower CD3+CD4+ T cell-specific expression of IFNa (t = -3.177 and -4.541, P less than 0.01 respectively), but significantly higher levels of secreted IL-4 (t = 3.382 and 4.393, P less than 0.01 respectively), of CD3+ T cells-specific expression of PD-1/PD-L1 (t = 4.755, 2.942 and 4.518, 4.595, P less than 0.01 respectively), and of CD14+ T cells-specific expression of PD-L1 (t = 5.092 and 5.473, P less than 0.01 respectively). The CD3+ T cells-specific expression of PD-L1 was significantly higher in the samples from HBeAg- CHB patients than from the healthy controls (t = 3.214, P less than 0.01). CONCLUSION: HBeAg was able to down-regulate the production of Th1-type cytokines (IFNgamma), and up-regulate the secretion of Th2-type cytokines (IL-6, IL-10) and the expression of PD-1/PD-L1on monocytes. These changes are conducive to the formation of immune tolerance to HBV. Therefore, HBeAg may play an important role in immune tolerance to chronic HBV infection.

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AIM: To study the magnitude and correlation of humoral and cellular immune responses to hepatitis surface antigen(HBsAg), preS1+S2 antigen, and core antigen(HBcAg) in the blood donors. METHODS: Enzyme linked immunosorbent assay (ELISA) was employed to scan the humoral immune responses to HBsAg, preS1+S2 antigen, and HBcAg. Enzyme linked immunospot assay (ELISPOT) was used to check the antigen specific IFN-γ secreting cells stimulated by HBsAg, preS1+S2 antigen or HBcAg in peripheral blood mononuclear cell from blood donors. RESULTS: The prevalence of humoral immune response to preS1+S2 antigen was as high as 85.7% (48/56), compared with the lower response to HBcAg (30.4%, 17/56). In the cellular immune response, there was no difference between preS1+S2 antigen and HBcAg, both of which were stronger than the response stimulated by HBsAg. PreS1+S2 antigen or HBsAg specific humoral immune and cellular immune responses were highly correlated, which could not be observed in the HBcAg specific humoral immune and cellular immune responses(P>0.05). CONCLUSION: Either humoral or cellular immune responses to HBsAg, preS1+S2 antigen and HBcAg could be detected in the blood donors. PreS1+S2 antigen immune responses, however, were most powerful, which may suggest that PreS1+S2 antigen may be a candidate for the further HBV vaccine.

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Published data on the association between N-acetyltransferase 1 (NAT1) gene polymorphisms and colorectal carcinoma (CRC) susceptivity are inconclusive. To derive a more precise estimation of the association, we conducted this meta-analysis. Data were collected from electronic databases: PubMed, EMBASE, with the last report up to May 2010. The odds ratio (OR) and corresponding 95% confidence intervals (CIs) were used to assess the strength of the association. A total of 20 individual studies including 8,219 cases and 11,498 controls based on the search criteria were involved. Meta-analysis was performed for slow versus rapid acetylation genotypes of NAT1. We found no association between NAT1 polymorphisms and CRC in overall population (OR = 0.96, 95% CI = 0.88-1.05 P = 0.05 for heterogeneity) without significant publication bias present. In subgroup analyses, similar results were found in different ethnicities, source of controls, genotyping methods and adjustment. Current meta-analysis suggests that lack of association between the NAT1 polymorphisms and individual risk to CRC.

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AIM: To expand HBV special interferon-γ secreting human T lymphocytes in vitro and sustain the ability to recognize special T cell epitodes of HBcAg. METHODS: After stimulated by peptide 22 hours, feeded with autologous peripheral blood mononuclear cells ( PBMCs) deproliferated by mitomycin C and low concentration of IL-2 to expand in vitro for other 4 weeks. Then cells were collected and tested by flow cells assay every week. The ability of these cells to recognize special epitodes was tested by enzyme linked immunospot assay in which autologous lymphoblastoid cell lines (LCLs) produced from PBMC was transfected by epstein-barr virus pulsed with peptides and used as target cell. RESULTS: HBV special interferon-secreting human T lymphocytes were selected and expanded more than 1000 times in vitro. The ability of the T cells to recognize special epitodes is as same as unexpanded cells and the proportion of CD4 and CD8 positive cells likely remained native state. CONCLUSION: HBV special interferon-γ secreting T lymphocytes are effectively expanded in vitro and keep the ability to recognize stringently special T cell epotides.

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This report aims to investigate the dynamical changes of HBcAg18-27 epitope specific cytotoxic T lymphocytes(CTL), alanine aminotransferase (ALT), HBV DNA and HBsAg in peripheral blood of acute hepatitis B patients, and to explore the roles of HBcAg18-27-specific CTLs in virus clearance and liver injury. Acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients were divided into two groups according to results of HLA-A0201. Patients with positive HLA-A0201 were classified into HBcAg-specific CTL group and those with negative HLA-A0201 were referred as control group. The specific CTLs were stained with HLA-A0201 limited HBcAg18-27 epitope MHC-Pentamer and the frequencies of CTLs, T, B, NK and NKT cells were detected by flow cytometry (FCM). The serum ALT, HBV DNA and HBsAg were examined using speed analysis, quantitative PCR and abbott chemiluminescent technology. The frequencies of HBcAg18-27-specific CTLs in AHB patients were higher in the early three weeks as compared to the late three weeks. The apex time of HBV-specific CTL frequencies lagged behind those of HBV DNA, HBsAg and ALT. The loss of HBsAg in patients with high frequencies of HBV-specific CTL was earlier than that in patients with low frequencies (t = 2.018, P value is less than 0.05). In the second week the peak frequencies of CD3+CD8+ cells overlapped with that of HBcAg18-27-specific CTLs and with a positive correlation between (r = 0.420, P value is less than 0.05). During the early stages of AHB, the frequencies of NK and NKT cells were found significantly lower than that of control group and CHB group and the levels were back to normal after recovery. Moreover, a negative correlation existed between the frequencies of NK cells and the dynamic changes of HBcAg18-27-specific CTLs (r = -0.435, P value is less than 0.01) in AHB group. The frequencies of HBcAg18-27-specific CTLs were significantly higher as compared to CHB group in the first three weeks (z = -3.258, -4.04, and -3.259, P value is less than 0.01). The early loss of HBsAg was closely related to the high frequencies of HBcAg18-27 specific CTLs in AHB patients. HBcAg-specific CTL frequencies in peripheral blood could be used to predict clinical outcome after HBV infection. The frequencies of CD8+ T cells can reflect the changes of frequencies of HBcAg-specific CTL during acute HBV infection.

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Patients with advanced hepatocellular carcinoma (HCC) have shown to benefit from tamoxifen treatment. The mechanisms of tamoxifen effects in HCC, however, are not yet clearly understood. The PI3K/Akt/mTOR signal pathway is involved in cell proliferation, tumorigenesis, and apoptosis. Over-expression of survivin has played an important role in leading to antiapoptosis. The current study investigated changes in mTOR pathway and survivin expression in hepatocarcinoma cell line HepG2 treated with tamoxifen. We detected apoptosis of hepatocarcinoma cells by flow cytometry assay. Survivin transcription level and p70S6k was demonstrated by PCR, dual-luciferase reporter assay and western blot analysis respectively. Our results are showed as follows: tamoxifen leads to apoptosis of the cells, and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6 kinase. Twenty micromoles tamoxifen treatment significantly depresses transcription of survivin mRNA. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduce survivin protein level but did not affect survivin transcription, which indicated that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells. Our results suggest that tamoxifen down-regulate survivin expression in HepG2 cells is mediated by transcriptional and posttranscriptional level via PI3K/Akt/mTOR pathway to induce apoptosis.

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OBJECTIVE: The aim of this study was to investigate the changes in mTOR activity and survivin expression in liver cancer cell line HepG2 cells treated with tamoxifen. METHODS: Survivin transcription level and p70S6K was demonstrated by PCR, dual-luciferase reporter assay and Western blot analysis, respectively, and the apoptosis in the HepG2 cells was detected by flow cytometry. RESULTS: Tamoxifen leads to apoptosis of the cells and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6K. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduced the survivin protein level but not affected the survivin transcription, indicating that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells. CONCLUSIONS: Our results suggest that tamoxifen down-regulates survivin expression in HepG2 cells and it is mediated by transcriptional and post-transcriptional level via PI3K/Akt/mTOR pathway to induce apoptosis.

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OBJECTIVE: To develop a method for long-term culture of human B cells from peripheral blood mononuclear cells (PBMCs) by means of human soluble CD40 ligand (sCD40L), and to investigate the proliferation and antigen-presenting function of the CD40-activated B cells. METHOD: Peripheral blood sample of 30 ml was collected from a healthy person. PBMCs were isolated and cultured in the presence of sCD40L. Flow cytometry was used to examine the proliferation, DNA cycle, and cell surface markers: CD86, CD80, major histocompatibility complex (MHC) class II, and MHC Class I of the B cells. The cells cultured for 45 days were divided into 2 groups: Group 1 incubated with the fluorescein isothiocyanate (FITC)-conjugated hepatitis-B core antigen (HBcAg-FITC) and Group 2 used as control group. Eighteen hours later cytometry and fluorescence microscopy were used to detect the peptide pulsing. RESULTS: The B cells could be cultured up to 50 days in the sCD40L culture system. The ratio of B cells in the PBMCs was 8.21% at the beginning, and increased to 70.67% by day 47, and the B cell absolute count was 6.56 x 10(5) at the beginning and increased to 8.61 x 10(6) at day 50, about 10 times higher. sCD40L promoted a strong up-regulation of cell surface markers. The expression rates of CD80, CD86, MHC-II, and MHC-I of the sCD40L-activated B cells were 83.97%, 84.73%, 99.59%, and 98.70% respectively. Flow cytometry showed that 98.10% of the B cells co-incubated with HBcAg-FITC were loaded with HBcAg. Fluorescence microscopy showed that the HBcAg was in the cytoplasm of the B cells. CONCLUSION: A human sCD40L culture system has been developed with the ability to culture human peripheral blood B cells, thus realizing the long-term proliferation and activation of human peripheral blood B cells that function as antigen-presenting cells.

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We report here novel array of gene cassettes found in single variable region of class 1 integron disseminated in Pseudomonas aeruginosa isolated from a teaching hospital in Nanjing, Jiangsu Province, China. 29 of 47 (61%) P. aeruginosa strains were confirmed haboured class 1 integron, and all the positive strains have the same variable region confirmed by PCR and RFLP methods. The variable region contained an unreported order of four gene cassettes aac(6')-II-aadA13-cmlA8-oxa-10. Of those, cmlA8 gene was a variant of cmlA5 encoding non-enzymatic protein which putatively confer resistance to chloramphenicol. Susceptibility testing revealed multidrug-resistant mechanisms were involved in the class 1 integron positive clinical isolates. And the class 1 integron located on an about 15 kb transferable plasmid was certified by conjugation experiment and plasmid DNA analysis. The macro restriction profile indicated those clinical strains were clonally related.

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OBJECTIVES: In order to investigate the relationship among the toll-like receptor 2 (TLR2), hepatitis B e antigen and HBV DNA, the expression levels of TLR2 on peripheral blood monocytes of chronic hepatitis B (CHB) patients as well as on their monocytes stimulated by ligand of TLR2 (Pam3CSK4) and HBeAg were analyzed. METHODS: Sixty-eight adults with CHB were enrolled, including 37 HBeAg-positive patients, 17 HBeAg-negative and HBV DNA negative patients, and 14 HBeAg-negative and HBV DNA positive patients. Sixteen healthy volunteers were also studied as controls. TLR2 expression levels on their peripheral blood monocytes stimulated with Pam3CSK4 or not stimulated were analyzed by FACS Caliber. The relationship of the expression levels of TLR2, HBeAg and HBV DNA were also analyzed. The level of TLR2 on peripheral blood monocytes of healthy volunteers and HBeAg-negative CHB patients stimulated by HBeAg was examined for six hours. RESULTS: The TLR2 expression levels on CD14+ cells were significantly reduced in HBeAg-positive patients (47.57%+/-21.40 %) compared to both healthy volunteers (76.51%+/-7.46%) and HBeAg-negative patients (HBV DNA positive group 73.2%+/-14.2%, HBV DNA negative group 75.2%+/-11.3%); but there was no difference between those of the HBeAg-negative patients and the healthy volunteers. Expression levels of TLR2 on monocytes stimulated by TLR2 ligand in HBeAg-positive patients were obviously increased, and reached the basic levels of the healthy volunteers and the HBeAg-negative patients. The expression levels of TLR2 on monocytes stimulated by HBeAg of the healthy volunteers and the HBeAg-negative patients were markedly reduced. CONCLUSIONS: In the presence of HBeAg, HBV down-regulates the expressions of TLR2 on CD14+ cells from peripheral blood, and there is no correlation between HBV-DNA and TLR2. Pam3CSK4 can boost the TLR2 expression in HBeAg-positive patients. The proposed interaction between HBV and TLR2 may provide an important clue to explain the reasons of the establishment of persistent HBV infection.

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OBJECTIVE: To determine the expression level changes of survivin, a inhibitor of apoptosis protein, followed by activation of insulin receptors in human hepatocellular carcinoma HepG2 cell line, and to investigate the signalling pathway involved in the regulation. METHODS: Human hepatocellular carcinoma HepG2 cells were treated with insulin alone or pre-treated with LY294002, a specific inhibitor of PI3K signalling pathway, to determine whether blocking PI3K signaling can attenuate the up-regulation of survivin expression. Real time RT-PCR and Western blot analysis were used to measure survivin mRNA and protein changes before and after treatment, respectively. RESULTS: Without serum supplement, HepG2 cells expressed a small amount of survivin. Insulin induced survivin expression in a dose- and time-dependent fashion. Survivin expression was blocked if cells were pre-treated with LY294002 prior to insulin stimulation. CONCLUSION: Insulin induces survivin expression via PI3K signalling pathway, suggesting that to interfere the key gene in this signalling pathway may block survivin expression, therefore, promoting apoptosis in hepatocellular carcinoma cells.

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AIM: To investigate the immunogenicity of a novel DNA vaccine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes (CTLs) of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS: HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1:97,200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector:target ratio of 20:1 in mice that received pSW3891/HBc DNA vaccine. CONCLUSION: pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.

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OBJECTIVES: To observe immunogenicity of new DNA vaccine encoding for hepatitis B virus core antigen (HBcAg). METHODS: A new DNA vaccine (pSW3891/HBc) encoding for hepatitis B virus core antigen was constructed using plasmid pSW3891 which can be used in human. Control and experiment groups of Balb/c mice were immunized with pSW3891 or pSW3891/HBc by gene gun. Anti-HBc in sera of mice was tested by ELISA (enzyme linked immune sorbent assay). Specific cytotoxicity of cytotoxic T lymphocyte (CTL) of mice was detected by LDH release assay. RESULTS: pSW3891/HBc can express in 293T cell line effectively. Mice immunized with pSW3891/HBc showed strong anti-HBc response and specific high cytotoxicity of CTL. CONCLUSION: pSW3891/HBc induced significantly humoral and cellular immune responses in Balb/c mice.

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