iTRAQ-Based Quantitative Proteomic Analysis of Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a common disease in the southern provinces of China with a poor prognosis. To better understand the pathogenesis of NPC and identify proteins involved in NPC carcinogenesis, we applied iTRAQ coupled with two-dimensional LC-MS/MS to compare the proteome profiles of NPC tissues and the adjacent non-tumor tissues. We identified 54 proteins with differential expression in NPC and the adjacent non-tumor tissues. The differentially expressed proteins were further determined by RT-PCR and Western blot analysis. In addition, the up-regulation of HSPB1, NPM1 and NCL were determined by immunohistochemistry using tissue microarray. Functionally, we found that siRNA mediated knockdown of NPM1 inhibited the migration and invasion of human NPC CNE1 cell line. In summary, this is the first study on proteome analysis of NPC tissues using an iTRAQ method, and we identified many new differentially expressed proteins which are potential targets for the diagnosis and therapy of NPC.

[Comparison of the efficacy of 48 week-Entecavir therapy with that of Adefovir therapy for chronic hepatitis B patients].

OBJECTIVE: To compare the efficacy of 48 week-Entecavir therapy with that of Adefovir therapy for chronic hepatitis B patients. METHODS: In this open-label study we randomly assigned 125 CHB patients to receive 0.5 mg of entecavir (n = 56) or 10mg of adefovir (n = 69) once daily for 48 weeks. RESULTS: HBV DNA, ALT and HBeAg were quantified at baseline and at 0, 24, 48 weeks. At week 24 and 48, more patients in entecavir group than in adefovir group achieved undetectable serum HBV DNA level (68% vs 35%, 84% vs 49%, P < 0.05). The percentage of patients with normal ALT level in the two groups at week 48 was similar (100% vs 94%, P > 0.05). Among the HBeAg positive patients, more patients in entecavir group than in adefovir group had HBeAg loss at week 24 and 48 (23% vs 7%, 44% vs 15%, P < 0.05). The ratio of HBeAg seroconversion was similar in the two groups at week 24 (18% vs 7%, P > 0.05), but more patients in entecavir group than in adefovir group achieved HBeAg seroconversion at week 48 (33% vs 12%, P < 0.05). The retreated patients in the entecavir group had a higher chance to achieve undetectable serum HBV DNA level (79% vs 34%, P < 0.05), HBeAg loss (42% vs 17%, P > 0.05), and seroconversion (26% vs 17%, P > 0.05), than these in the adefovir group. The safety profiles and adverse event profiles were similar in the two groups. CONCLUSIONS: Compared to adefovir, entecavir is more potent to suppress HBV replication.

[Analysis of spontaneous decline of HBV DNA in chronic hepatitis B patients in 12 weeks].

OBJECTIVE: To analyze the spontaneous decline of HBV DNA in chronic hepatitis B patients in 12 weeks. METHODS: Chronic hepatitis B patients not receiving antiviral treatment from 2003 to 2005 were divided into two groups according to the baseline value of ALT and TBil. Spontaneous decline of HBV DNA were retrospected, and the influence of the baseline value of ALT and TBil on spontaneous decline of HBV DNA was analyzed. RESULTS: Total of 213 chronic hepatitis B patients (male 174, female 39, aged from 18 to 65) were recruited in this study, including 124 mild and moderate type of hepatitis B, 89 severe type of hepatitis B, and 19 patients (8.92%) were lost at the end of the 12th week. The mean baseline value of HBV DNA of all the patients was (6.66+/-1.03) log10 copies/ml, at 12 week the mean value of HBV DNA of all the patients was (5.98+/-1.53) log10 copies/ml (compared to baseline P<0.01), the decline value of HBV DNA was (0.68+/-1.46) log10 copies/ml. The mean baseline value of HBV DNA of patients with the severe type of hepatitis B was lower than that with the mild or moderate type of hepatitis B patients [(6.45+/-0.99) log10 copies/ml and (6.81+/-1.04) log10 copies/ml respectively] (P<0.05). However, there was no significant difference in the mean and the declined value of HBV DNA between these two groups at the 12th week (P<0.05). At the 12th week, the baseline values of ALT and TBil were higher in patients with HBV DNA3 log10 copies/ml (P>0.05); And there were no significant difference in the baseline values of ALT and TBil between patients with the declined value of HBV DNA>or=2 log10 copies/ml and patients with declined value of HBV DNA less than 2 log10 copies/ml. At the 12th week, the mean and the declined value of HBV DNA were similar between the patients with ALT5xULN at baseline. The mean baseline value of HBV DNA of patients in the group of patients whose baseline value of ALT5xULN while TBil>5xULN, ALT5xULN, ALT>5xULN while TBil0.05 respectively). CONCLUSIONS: There is spontaneous decline of HBV DNA in patients with chronic hepatitis B in 12 weeks, but the level of liver injury is not correlated with the level of spontaneous decline of HBV DNA in chronic hepatitis B patients in 12 weeks.

[Short-term results of telbivudine versus entecavir treatments in HBeAg-positive chronic hepatitis B patients in China].

OBJECTIVE: To evaluate the efficacy and safety of telbivudine (LDT) versus entecavir treatments in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients. METHODS: Eighty HBeAg-positive compensated CHB patients with HBV DNA more than 6 log10 copies/ml and serum ALT 2 x ULN were divided into two groups: a telbivudine treatment group, and a entecavir treatment group. HBV DNA, ALT and HBeAg were surveyed at baseline and at 12 and 24 weeks. The efficacy and safety of the two nucleoside analogues were assessed at 12 and 24 weeks. RESULTS: Undetectable serum HBV DNA levels of the telbivudine group (50% and 80%) were similar to those of the entecavir B group (50% and 70%) according to the polymerase-chain-reaction assay at week 12 and 24. There were no significant differences in the normalization of alanine aminotransferase levels between the two groups at week 12 and 24 (52.5% vs 60.0%, 77.5% vs 75.0%). The mean reductions in serum HBV DNA from the baseline levels at week 12 and 24 were similar between the two groups [5.27 vs.5.36, 6.49 vs.6.18 log (on a base-10 scale) copies per milliliter]. More patients in the telbivudine group had HBeAg seroconversion at week 12 than those in the entecavir group (20.0% vs 5.0%, P = 0.043); however, there was no significant difference between the two groups at week 24 (27.5% vs 17.5%). No adverse reactions were found in either group. CONCLUSION: There was no significant difference in HBV DNA undetectable rates and the ALT normalization rates between the two groups in a short-term therapy (24 weeks), but the telbivudine group had a higher rate in HBeAg seroconversion than that in the entecavir group at week 12.

Therapeutic effect of autologous dendritic cell vaccine on patients with chronic hepatitis B: a clinical study.

AIM: To investigate the therapeutic effect of autologous HBsAg-loaded dendritic cells (DCs) on patients with chronic hepatitis B. METHODS: Monocytes were isolated from fresh peripheral blood of 19 chronic HBV-infected patients by Ficoll-Hypaque density gradient centrifugation and cultured by plastic-adherence methods. DCs were induced and proliferated in the culture medium with recombinant human granulocyte-macrophage-colony-stimulating factor (rhGM-CSF) and human interleukin-4 (rhIL-4). DCs pulsed with HBsAg for twelve hours were injected into patients subcutaneously twice at intervals of two weeks. Two patients received 100 mg oral lamivudine daily for 12 mo at the same time. HBV-DNA and viral markers in sera of patients were tested every two months. RESULTS: By the end of 2003, 11 of 19 (57.9%) patients had a clinical response to DC-treatment. HBeAg of 10 (52.6%) patients became negative, and the copies of HBV-DNA decreased 10(1.77+/-2.39) on average (t = 3.13, P<0.01). Two cases co-treated with DCs and lamivudine had a complete clinical response. There were no significant differences in the efficient rate between the cases with ALT level lower than 2XULN and those with ALT level higher than 2XULN before treatment (chi(2) = 0.0026). CONCLUSION: Autologous DC-vaccine induced in vitro can effectively suppress HBV replication, reduce the virus load in sera, eliminate HBeAg and promote HBeAg/anti-HBe transformation. Not only the patients with high serum ALT levels but also those with normal ALT levels can respond to DC vaccine treatment, and the treatment combining DCs with lamivudine can eliminate viruses more effectively.

[A multi-center clinical study of N-acetylcysteine on chronic hepatitis B].

OBJECTIVES: To evaluate the effectiveness and safety of N-acetylcysteine (NAC) in treating chronic hepatitis B patients. METHODS: 144 patients with chronic hepatitis B (total bilirubin, TBil>170 mmol/L) from several centers were chosen for a randomized and double blind clinical trial. The patients were divided into a NAC group and a placebo group and all of them were treated with an injection containing the same standardized therapeutic drugs. A daily dose of 8 microgram NAC was added to the injection of the NAC group. The trial lasted 45 days. Hepatic function and other biochemistry parameters were checked at the experimental day 0 and days 15, 30, 45. RESULTS: Each group consisted of 72 patients of similar demology and disease characteristics. During the trial, 28 cases of the 144 patients dropped out. In the NAC group, at day 0 and day 30, the TBil were 401.7 vs. 149.2 and 160.1+/-160.6. In the placebo group, the TBil on the corresponding days were 384.1+/-134.0 and 216.3+/-199.9. Its decrease in the NAC group was 62% and 42% in the placebo group. At day 0 and day 45 of treatment, the effective PTa increase rate was 72% in the NAC group and 54% in the placebo group. The total effective rate (TBil + PTa) was 90% in the NAC group and 69% in the placebo group. The parameters of the two groups showed a remarkable difference. The rate of side effects was 14% in the NAC and 5% in the placebo groups. CONCLUSION: NAC can decrease the level of serum TBil, increase the PTa and reduce the time of hospitalization. NAC showed no serious adverse effects during the period of our treatment. We find that NCA is effective and secure in treating chronic hepatitis B patients.

[One year and half treatment with lamivudine in active cirrhosis resulting from chronic hepatitis B].

OBJECTIVE: To study the therapy effect of long term lamivudine treatment on active cirrhosis following chronic hepatitis B, and explore the methods for abnormalities resulting from lamivudine withdrawing. METHODS: 58 patients received lamivudine 100 mg orally everyday for 18 months. The changes were observed and wrote down, including clinical symptoms and signs, aminotransferase, virology indexes, and the abnormalities after lamivudine withdrawing, then further to find out plans for the latter. RESULTS: (1) After lamivudine treatment, there were 35 patients whose situation stabilized, life quality improved, child-pugh score declined, and liver function turned better. (2) The level of HBV DNA decreased at least 10(3) copies/ml. HBeAg of 33.3% patients (13/39) became negative. (3) Among the 10 patients who stopped lamivudine of their own accord, and came again after 3 - 6 months because of hepatitis B recurring, two were treated with interferon for one month, then turning to liver-protecting methods for deteriorating, the other eight only received liver-protecting and immune-regulating treatment, whose liver function improved. CONCLUSIONS: Long term treatment with lamivudine for active cirrhosis following chronic hepatitis B can improve liver function and life quality, prevent exacerbation. And it is not advisable to use interferon for hepatitis B relapsing after lamivudine withdrawing.

[Clinical research on the treatment effect of autologous dendritic cell vaccine on the patients with chronic hepatitis B].

OBJECTIVE: To investigate the treatment effect of autologous HBsAg-loaded dendritic cells (DCs) on patients with chronic hepatitis B (CHB). METHODS: Monocytes were isolated from fresh peripheral blood of 19 CHB patients by Ficoll-Hypaque density gradient centrifugating and cultured with plastic -adherence method. DCs were induced and proliferated from the monocytes with granulocyte-macrophage clony stimulating factor (GM-CSF) and interleukin-4 (IL-4) for seven days. After being incubated with HBsAg for two hours, DCs were injected to patients subcutaneously twice at the interval of two weeks. HBV DNA level, alanine aminotransferase (ALT) level, and HBV markers in the serum of patients were tested every two months. RESULTS: 11 of the 19 (57.9%) patients responded to DC-treatment clinically. The rates of HBeAg clearance and HBeAg/anti-HBe seroconversion were 52.6% (10/19) and 26.3% (5/19) respectively, and the copies of HBV DNA decreased by 10(1.77 2.39) (t = 3.13, P < 0.01). Two patients who were treated in combination with lamivudine had complete clinical response. There was no difference in the trial effect between the DC treatment and the other two antiviral methods, and in the efficient rate between the patients whose ALT levels were high before treatment and those whose ALT levels were normal. CONCLUSION: The autologous HBsAg-loaded DCs can effectively suppress HBV replication, reduce virus load in serum, eliminate HBeAg and promote HBeAg/ anti-HBe seroconversion. The patients whose ALT levels are high or normal can response clinically to DCs treatment. DCs in combination with lamivudine can eliminate virus more effectively.

A new method to isolate and culture rat kupffer cells.

BACKGROUND: Previous methods for Kupffer cells (KCs) isolation require sophisticated skills and tedious procedures. Few studies have attempted to explore the self-renewal capacity of KCs in vitro. Therefore, the aim of this study was to establish a simple method for rat KCs isolation and further investigate the mitotic potential of KCs in vitro. METHODS: KCs were obtained by performing one-step perfusion, enzymatic tissue treatment, differential centrifugation and selective adherence. The proliferation ability of cultured KCs was determined by MTT assay and Propidium Iodide FACS analysis. Phagocytic assay and ED-1, ED-2 immunofluorescence were used to identify cell phenotype. After stimulation with LPS, the expression of surface antigens (MHCII, CD40, CD80, and CD86) and the production of cytokines (NF-κB, TNF-α, IL-6 and IL-10) were measured for cell function identification. RESULTS: KCs were isolated with certain numbers and reasonable purities. The KCs were able to survive until at least passage 5 (P5), and at P3 showed equally strong phagocytic activity as primary KCs (P0). After stimulation with LPS, the change in the expression of surface antigens and the production of cytokines for P3 cells was similar to that for P0 cells. CONCLUSIONS: Our study provides a simple and efficient method for KCs isolation, and reveals that self-renewing KCs have the same phagocytic activity and functions as primary KCs.