Disrupting RhoA activity by blocking Arhgef3 expression mitigates microglia-induced neuroinflammation post spinal cord contusion.

Excess inflammatory microglia activation deteriorates the pathological degree of spinal cord injury (SCI). We here employed microglia samples in vitro and murine model in vivo to trace the role of inhibition of Arhgef3 in inflammatory response post SCI. From the specimen analysis of lipopolysaccharide (LPS)-induced inflammatory microglia, we found that Arhgef3 expression was positively relative to microglia activation. In vitro, LPS caused the microglia inflammatory activation and induced upregulation of the Arhgef3 expression. Interestingly, presence of Arhgef3 could activate RhoA through promoting Rho GTPases, but silencing of Arhgef3 decreased RhoA activation and inhibited the microglia inflammation. Moreover, disruption of Arhgef3 inhibited the GTP-RhoA, resulted in a suppression of proinflammatory cytokines, and alleviated the LPS-elicited inflammatory genes expression. Moreover, artificially decreasing Arhgef3 expression remarkedly reduced ROS generation after LPS treatment. In vivo of a mouse mechanical contusion-induced SCI model, inhibition of Arhgef3 reduced the ratio of GTP-RhoA/Total-RhoA, and prevented SCI via mitigating the microglial inflammatory phenotype and decreased secondary neurological injury. Besides, inhibition of Arhgef3 prevented alleviated the degree of demyelination but did not affect neuronal regeneration. Meaningfully, absence of Arhgef3 improved mouse locomotor recovery post SCI. Taken together, Arhgef3 involves the microglial activation and inflammatory response following neural injury, and targeted disrupting of which may indicate a promising therapeutic direction in preventing SCI.

[Association of interleukin 28B polymorphisms with response to pegylatedinterferon plus ribavirin therapy for chronic hepatitis C].

OBJECTIVE: To investigate the characteristics of gene polymorphisms at rs12979860 of interleukin 28B (IL28B) and explore the relationships between the genetic polymorphisms and the antiviral therapy efficiency for chronic hepatitis C patients in the Sichuan region of China. METHODS: Data from 56 patients treated for 48 weeks with PEG-IFN alpha-2a plus weight-based Ribavirin (RBV), which were followed for 24 weeks after the end of treatment, were analyzed. And the IL28B rs12979860 genotype was detected by polymerase chain reaction-restriction techniques (PCR) and sequencing analysis. RESULTS: Two genotypes, CC (76.8%) and CT (23.2%), were identified in the study. There are no significant correlation in sex, age, body weight, routes of infection, baseline ALT value, baseline viral load and hepatitis C viral (HCV) genotype between the patients with CC genotype and CT genotype (P>0.05). PEG-IFN alpha-2a plus RBV showed a conspicuous therapeutic effect in patients of the Sichuan region of China, and the rate of sustained virological response (SVR) was 82.1% (46/56). The higher rates of SVR were observed in patients with IL28B genotype CC than genotype CT (90.7% versus 53.8%, P=0.009). Statistically higher proportion of SVR wasn't observed in patients with lower baseline viral load (< or =6 x 10(5) IU/mL) [88.2% versus 79.5% in patients with higher baseline viral load (> or = 6 x 10(5) IU/mL), P=0.684] and statistically lower proportion of SVR wasn't observed in patients infected with HCV genotype 1 (76.9% versus 92.9% in patients infected with HCV genotype non-1, P = 0.363). The higher rates of SVR were observed in patients with IL28B genotype CC than patients with genotype CT in the group of higher baseline viral load (> or = 6 x 10(5) IU/mL) (87.5% versus 42.9%, P=0.033) and in HCV genotype 1 infected patients (89.7% versus 50.0%, P=0.025). CONCLUSION: CC genotype was accounted for the majority at rs12979860 in patients of the Sichuan region of China. The higher rates of SVR were observed in IL28B genotype CC than genotype CT. Compared to HCV viral genotype and baseline viral load, IL28B genotype may predict the treatment effect of greater value.

[Lamivudine-resistant analysis and management for chronic hepatitis B patients with initial lamivudine therapy].

OBJECTIVE: To study the factors influencing the long-term usage of lamivudine (LAM) in chronic hepatitis B (CHB) patients and the management after drug-resistance. METHODS: 383 cases of naive CHB patients in our outpatient clinic were treated with lamivudine (100 mg/d) and followed up for at least over 1 year from 2001 to 2010. 129 cases encountered lamivudine-resistance and were then divided into two groups: patients in group A were switched to adefovir dipivoxil (ADV) alternative treatment and those patients in group B were added with ADV for continuous treatment. Efficacy assessment factors included serum HBV markers, HBV DNA, ALT, AFP and other biochemical indicators. RESULTS: Among the 383 cases, patients with HBV DNA negative conversion (PCR below test line) at 3 months, 6 months, 1 year, 2 years, 3 years and > 3 years after initial treatment were respectively 156 cases (40.7%), 213 cases (55.6%), 228 cases (59.5% ), 217cases (56.7%), 214 cases (55.9%) and 213 cases (55.6%). HBeAg/HBeAb seroconversion occurred in 62 cases (22.6%). 12 cases were found with primary LAM resistance, 129 cases with HBV breakthrough and rebound, the cumulative resistance rate was 36.8% and the cumulative rebound rate was 34.8%. High baseline viral load, baseline ALT levels < 2 ULN, Lower virologic response rate at week 24 were associated with a higher rebound rate (chi2 is 35.716, 8.728, 43.534, respectively, all with P < 0.01).Viral breakthrough and rebound occurred in 112 patients (86.8%) within 1 year and a half, 123 patients (95.3%) occurred at the end of 2 years and no patient with viral breakthrough and rebound after 5 years. For the patients with viral rebound in group A and group B, the rates of HBV DNA loss were 22.7% (15/66) and 58.7% (37/63) respectively, and the viral response rates were 59.1% (39/66) and 87.3% (52/63) respectively, with chi2 values equaled 17.364 and 12.975 respectively (P < 0.01). CONCLUSION: For the chronic hepatitis B patients on initial treatment with lamivudine, the viral rebound occurred mainly within 2 years. LAM combined with ADV is more effective than ADV alone for lamivudine-resistant patients.

[Association between the influential factors and the effectiveness of pegylated interferon alpha-2a plus ribavirin as a combination treatment for chronic hepatitis C patients].

To evaluate the efficacy and to investigate the association between the length of the treatment period and the cumulative dose of pegylated interferon alpha-2a (PegIFN alpha-2a) plus ribavirin (RBV) and the effectiveness of antiviral therapy. We analyzed data from 117 patients treated for 48 weeks with PEG-IFN alpha-2a (135mug or 180mug/week) plus weight-based RBV (800 mg/d for patients is less than or equal to 65 kg, 1000 mg/d for patients 65-75 kg and 1200 mg/d for patients is more than or equal to 75 kg) under care at West China Hospital. HCV RNA was assessed at baseline, Week 4, 12 and 24, the end of treatment (EOT) and after 24 weeks follow-up (sustained virological response; SVR) with a test range of 1.0*10(3) to 5.0*10(7) IU/ml. Patients were stratified by age, gender, weight, route of transmission, duration of infection, baseline HCV RNA level and PegIFN alpha-2a or RBV dosage. HCV genotype was assessed in 29 patients (genotype 1b, 21; genotype 2a, 7; genotype 1b/2a, 1). Rapid virological response (RVR; HCV RNA negative at week 4), complete early virological response (cEVR; HCV RNA negative at week 12), EOT response, and SVR were achieved in 88 (75.2%), 110 (94%), 114 (97.4%) and 96 (82.1%) patients, respectively. Younger age, lower weight and shorter speculated infection years were associated with higher SVR rates (91.4% vs 72.9%, x2=6.796, P value is less than 0.05; 85% vs 50%, x2=5.433, P value is less than 0.05; 96.7% vs 77%, x2=5.852, P value is less than 0.05). SVR significantly increased with treatment length (38.5%, 66.7%, and 88.8% for is less than or equal to 29 weeks, 29-38 weeks, and is more than or equal to 38 weeks, respectively). SVR significantly increased with total cumulative treatment doses (38.5%, 66.7% and 88.8% for is less than or equal to 60%, 60%-80% and is more than or equal to 80% of PegIFN dose respectively; 33.3%, 85.3% and 96.8% for is less than or equal to 60%, 60%-80% and is more than or equal to 80% in RBV dose respectively) in all patients. Less than 80% of standard dose of RBV was not sufficient even if given enough PegIFN (is more than or equal to 80% cumulative treatment dose) in patients who achieved RVR. Chinese patients treated with peginterferon alpha-2a plus ribavirin have high rates of SVR. It is important to complete the target length of treatment and to continue the target dosage to achieve SVR.

[A double-blind, randomized, lamivudine-controlled clinical trial of DAIDING (adefovir dipivoxil) for lamivudine-resistant patients with chronic hepatitis B].

OBJECTIVE: To investigate the efficacy and safety of adefovir dipivoxil (ADV, DAIDING) for Chinese chronic hepatitis B patients with lamivudine (LAM) resistance. METHODS: This study was a multicenter, double-blind clinical trial. 209 chronic hepatitis B patients with LAM resistance were randomly put in an ADV, DAIDING or a LAM group. After 24 and 48-weeks of treatment, serum HBV DNA levels were measured by quantitative PCR and liver function tests; HBV serology and safety assessments were also conducted. RESULTS: The mean reduction of HBV DNA from baseline at 24 and 48 weeks was significantly greater in the ADV group compared with that in the LAM group (2.40 log10 vs 0.94 log10, P < 0.01; 2.71 log10 vs 1.07 log10, P < 0.01). In the ADV group, the virological response and ALT normalization at 24 and 48 weeks were significantly higher than those in the LAM group. There was no significant difference between the two groups in the portion of HBeAg reduction, HBeAg seroconversion and incidence of adverse events. There was no severe adverse event related to the investigational product, DAIDING, in this trial. CONCLUSION: DAIDING (ADV) is effective and safe for the treatment of chronic hepatitis B patients with LAM resistance.

[A study of the dosage and efficacy of entecavir for treating hepatitis B virus].

OBJECTIVE: To evaluate the antiviral activity and safety of entecavir in patients with chronic HBV infection as a preliminarily step in selecting 0.1 mg or 0.5 mg as a better dosage for a further large scale clinical trial. METHODS: This was a randomized, double-blinded, placebo-controlled and dose-ranging trial of entecavir usage in 212 patients with chronic HBV infection. The patients were randomly assigned to 3 groups: 0.1 mg entecavir (69), 0.5 mg entecavir (72) and, placebo (71) groups and treated for 28 days. The patients were then followed for 56 days without treatment. RESULTS: The proportion of subjects who achieved the primary endpoint at day 28, with their HBV DNA level decreased >2 log or undetectable, was significantly greater in the entecavir 0.1 mg and 0.5 mg dose groups compared with the placebo group (P < 0.01 for both comparisons). The mean change from baseline in HBV DNA levels at day 28 was greater for entecavir 0.1mg and 0.5 mg groups compared with the placebo group (both P < 0.01). The mean change from baseline in HBV DNA levels at day 28 for entecavir 0.5 mg group was greater than that of the entecavir 0.1 mg group (P < 0.01). During the 56-day post-dosing follow-up phase, the entecavir 0.5 mg group was associated with greater and more sustained suppression of viral replication than the entecavir 0.1 mg group (P < 0.01). There were no clinically meaningful differences in the incidence of any adverse events between the entecavir dosing and the placebo groups. CONCLUSION: Entecavir at both 0.1 mg and 0.5 mg doses demonstrated superior antiviral activity compared with a placebo. Since the entecavir 0.5 mg dose appears to have greater antiviral activity than the 0.1 mg dose and with a comparable safety and tolerability profile, the 0.5 mg entecavir dose could be used in further trials.

[Influence of wen-yang herbs on hemodynamics in liver fibrotic rats].

OBJECTIVES: To observe the influence of wen-yang herbs on the hemodynamics in liver fibrotic rats. METHODS: Wistar rats with liver fibrosis, induced by carbon tetrachloride and alcohol, were randomly divided into a treatment group and a control group. The treatment group was administered wen-yang herbs and the control group saline. At the end of the experiment, the hemodynamic markers of the liver and the mesentery, the liver function and hydroxyproline content of liver tissues between the two groups were compared. Blood volume of the livers and hydroxyproline content of liver tissues were also determined. RESULTS: Blood volume of the liver and mesentery (P < 0.01) and blood flow velocity of small vein of mesentery (P < 0.05) of the treatment group were distinctly higher than the control group. The hydroxyproline content (P < 0.01) of the treatment group was remarkably reduced and liver function was improved. CONCLUSION: Wen-yang herbs can activate microcirculation of the liver and mesentery, decrease the deposit of collagen in the liver and improve liver function.

Tissue microarray for high-throughput analysis of gene expression profiles in hepatocellular carcinoma.

AIM: To study the expression profiles of HBsAg, HBcAg, p21WAF1/CIP1 (p21), Rb genes in hepatocellular carcinoma (HCC) and to investigate their roles in the hepatocar-cinogenesis. METHODS: HCC tissue microarray containing 120-min tissues of 40 HCC cases was constructed. HBsAg, HBcAg, p21 and Rb proteins were immunohistochemically stained by streptavidin-peroxidase conjugated method (S-P). The expression loss of these genes in cancerous, para-cancerous tissues and adjacent normal liver tissues of 40 HCCs were comparatively examined. RESULTS: The positive rate of HBsAg expression in cancerous tissues of 40 HCCs was 7.5%, which was lower than that in para-cancerous and adjacent normal liver tissues (chi2 =12.774, P < 0.01; chi2 = 18.442, P < 0.01). The positive rate of HBcAg expression in cancerous tissues of 40 HCCs was 20.0%, which was also lower than that in para-cancerous and adjacent normal liver tissues (chi2 = 9.482, P < 0.01; chi2 = 14.645, P < 0.01). p21 protein deletion rate in cancerous tissues of 40 HCCs was 27.5%, which was higher than that in para-cancerous and adjacent normal liver tissues (chi2 = 7.439, P < 0.01; chi2 = 11.174, P < 0.01). p21 protein deletion correlated remarkably with the pathological grade of HCC (chi2 = 0.072, P < 0.05). Rb protein deletion rate in cancerous tissues of 40 HCCs was 42.5%, which was also higher than that in para-cancerous and adjacent normal liver tissues (chi2 = 10.551, P < 0.01; chi2 = 18.353, P < 0.01). Rb protein deletion rate did not correlate remarkably with tumor size or pathological grade of HCC (chi2 = 0.014, P > 0.05; chi2 = 0.017, P > 0.05). CONCLUSION: Expression deletion of HBsAg, HBcAg, p21 and Rb proteins in HCCs may play important roles in the carcinogenesis of HCC. Tissue microarray is an effective high-throughput technique platform for cancer research.

[Studies on the nucleotides sequences of extended-spectrum-beta-lactamases encoding genes of Escherichia coli and Klebsiella pneumoniae and the related molecular evolution].

OBJECTIVE: To identify TEM-type and SHV-type ESBLs encoding genes of ESBLs-producing Klebsiella pneumoniae and Escherichia coli isolated from clinical species in West China Hospital of Sichuan University and study the molecular evolution of the ESBLs. METHODS: The nucleotide sequences of TEM-type and SHV-type ESBLs encoding genes amplified by PCR were detected by automatic sequencer, and the subtypes of the encoding genes were determined by Blastx searching. The molecular evolution of ESBLs was studied by means of bioinformatics. RESULTS: In this study, the subtypes of ESBLs were SHV-2 and TEM-19, the distribution of silent mutation in ten bla(SHV-2) was identical, and that of two bla(TEM-19) was the same; the distribution of silent mutation of bla(TEM-19) was the same as that of bla(TEM-1). The distribution of silent mutation of bla(SHV-2) observed here was different from that observed in other countries. CONCLUSION: SHV-2 was the main ESBLs in this study. The bla(SHV-2) and bla(TEM-19) in this study originated from the same transferable variants respectively. The prevalent SHV-2 in different countries resulted from convergent evolution. It seems possible that the bla(TEM-19) identified by this study might originate directly from the transferable bla(TEM-1) identified in this study.

[Pathological study of the therapeutic effect of wen-yang herbs on experimental liver cirrhosis].

OBJECTIVE: To observe the pathological effect of wen-yang herbs on experimental hepatic fibrosis. METHODS: Thirty-two male Wistar rats were used in this study comprising four groups. To start with, 24 rats of three groups were given subcutaneous injection of CCl4 and drinking 10% alcohol so as to make the model of hepatic fibrosis. After the establishment of the pathologic model, the rats were divided into the model group, the pathological control group and the therapeutic group by randomization. The rats of the therapeutic group were given the herbal remedies via gastrogavage, q.d. x 30. The rats of the pathological control group were given normal saline via gastrogavage, q.d. x 30. Then liver tissue Hydroxyproline (Hyp) content was examined in these 3 groups and the normal group. Quantitative marks were done according to a modified semiquantitative scoring system (SSS). The results of SSS marks and Hyp contents were analysed using Pearson's coefficient of correlation. RESULTS: The Hyp content and SSS marks of the therapeutic group decreased remarkably as compared with those of the control group (P<0.01), and the SSS marks had a strong positive correlation with Hyp content (r=0.804). CONCLUSION: Wen-yang herbs can mitigate the rats' hepatic fibrosis and promote a recovery of their experimental illness.

[Effects of Radix Salviae Miltiorrhizae on the PMN-EC adhesion in vitro at the early stage of endotoxemia].

OBJECTIVE: To evaluate the effects of Radix Salviae Miltiorrhizae (RSM) on the PMN-EC adhesion in vitro at the early stage of endotoxemia, and to probe into the mechanism there in involved. METHODS: The rabbit endotoxemia model was made, and one group was treated with RSM injection (2 ml/kg) instantly and 24 h after the modeling of endotoxemia. PMN-EC adhesion rate, adhesion molecule (CD11a/CD18 and CD11b/CD18) and TNF-alpha level were measured instantly, 2 h, 4 h, 8 h, 16 h, 24 h and 48 h after endotoxemia modeling. RESULTS: It was found that after the modeling of endotoxemia, the levels of CD11a/CD18, CD11b/CD18 and PMN-EC adhesion rate instantly increased in the endotoxemia group and RSM group, they went up to the peak at 4 h in the endotoxemia group, and at 2 h, 4 h, 8 h, 16 h, 24 h and 48 h, they were markedly lower in the RSM group than in the endotoxemia group (P < 0.05). The TNF-alpha level in the endotoxemia group began to increase 2 h after the modeling and reached to the peak at 8 h, with its level still higher than normal at 48 h, while the RSM group had lower level of TNF-alpha at all time-points(P < 0.05). CONCLUSION: The data from this animal experiment indicate that Radix Salviae Miltiorrhizae can decrease PMN-EC adhesion rate and the levels of CD11a/CD18, CD11b/CD18 and TNF-alpha in case of endotoxemia so as to improve the microcirculation.

Pathomorphological study on location and distribution of Kupffer cells in hepatocellular carcinoma.

AIM: To clarify the location and distribution of Kupffer cells in hepatocellular carcinoma (HCC), and to investigate their role in hepatocarcinogenesis. METHODS: Kupffer cells were immunohistochemically stained by streptavadin-peroxidase conjugated method (S-P). The numbers of Kupffer cells in cancerous, para-cancerous and adjacent normal liver tissues of 48 HCCs were comparatively examined. RESULTS: The mean number of Kupffer cells in cancerous, para-cancerous and adjacent normal liver tissues was 12.7+/-6.8, 18.1+/-8.2 and 18.9+/-7.9 respectively. The number of Kuppfer cells in cancerous tissues was significantly lower than that in para-cancerous tissues (t=2.423, P<0.05) and adjacent normal liver tissues (t=2.521, P<0.05). As tumor size increased, the number of Kupffer cells in cancerous tissues significantly decreased (F=4.61, P<0.05). Moreover, there was also a significant difference in the number of Kupffer cells among well-differentiated, moderately-differentiated and poorly-differentiated cases(F=4.49, P<0.05). CONCLUSION: This study suggests that decrease of Kupffer cells in HCCs may play an important role in the carcinogenesis of HCC, the number of Kupffer cells in HCC is closely related to the size and differentiation grade of the tumor.

DNA-based vaccination induces humoral and cellular immune responses against hepatitis B virus surface antigen in mice without activation of C-myc.

AIM:To develop a safe and effective DNA vaccine for inducing humoral and cellular immunological responses against hepatitis B virus surface antigen (HBsAg).METHODS:BALB/c mice were inoculated with NV-HB/s, a recombinant plasmid that had been inserted S gene of hepatitis B virus genome and could express HBsAg in eukaryotes. HBsAg expression was measured by ABC immunohis tochemical assay, generation of anti-HBs by ELISA and cytotoxic T lymphocyte (CTL), by MTT method, existence of vaccine DNA by Southern blot hybridization and activation of oncogene C-myc by in situ hybridization.RESULTS:With NV-HB/s vaccination by intramuscular injection, anti-HBs was initially positive 2 weeks after inoculation while all mice tested were HBsAg positive in the muscles.The titers and seroconversion rate of anti-HBs were steadily increasing as time went on and were dose dependent. All the mice inoculated with 100&mgr;g NV-HB/s were anti-HBs positive one month after inoculation, the titer was 1 1024 or more. The humoral immune response was similar induced by either intramuscular or intradermal injection. CTL activities were much stronger (45.26%) in NV-HB/s DNA immunized mice as compared with those (only 6%) in plasma-derived HBsAg vaccine immunized mice. Two months after inoculation, all muscle samples were positive by Southernblot hybridization for NV-HB/s DNA detection, but decreased to 25% and all were undetectable by in situ hybridiza-tion after 6 months.No oncogene C-myc activation was found in the muscle of inoculation site.CONCLUSION:NV-HB/s could generate humoral and cellular immunolo-gical responses against HBsAg that had been safely expressed in situ by NV-HB/s vaccination.