[Synchronization isolation method for multiple types of cells from mouse liver].

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.

[Effects of hepatitis B virus on Th17, Treg and Th17/Treg ratio in different alanine aminetransferase stages].

OBJECTIVE: To evaluate the effects of hepatitis B virus (HBV) on helper T lymphocytes 17 (Th17), regulatory T lymphocyte (Treg) and Th17/Treg ratio in chronic hepatitis B patients in different alanine aminetransferase (ALT) stages. METHODS: In the study, 336 chronic hepatitis B patients in the first hospital of Lanzhou University were analyzed. The hepatitis B antigen antibody parameters were measured by chemiluminescence immunoassay analyzer, the liver function parameters were measured by automatic biochemical analyzer, the HBV loads were measured by quantitative PCR, Th17, Treg and Th17/Treg ratios were detected by flow cytometry. Among them, 111 cases (ALT < 40 U/L) of ALT were normal hepatitis B, 108 cases of chronic hepatitis B with ALT above normal upper limit and < 2 times higher (40 U/L≤ALT < 80 U/L), and 117 cases of chronic hepatitis B with ALT above 2 times normal upper limit (80 U/L≤ALT). According to the viral load, they were divided into low replication group with HBV DNA < 4.0 lg copies/mL, medium replication group with 4.0 lg copies/mL≤HBV DNA < 6.0 lg copies/mL and high replication group with HBV DNA ≥ 6.0 lg copies / mL. Dunnett T3 variance analysis were used to analyze the effects of HBV on Th17, Treg and Th17/Treg ratio in the chronic hepatitis B patients in different ALT stages. The changes of virological and immunological indexes before and after treatment were observed for 24 weeks of antiviral therapy in the hepatitis B patients with ALT≥double upper limit of normal group. RESULTS: In the ALT normal group, different virus load HBV had minor effects on Th17, Treg and Th17/Treg ratio. In the ALT≥2 times upper limit of normal group, with the virus load increased, Th17 (3.18%±0.79% in low replication group, 3.78%±0.92% in medium replication group and 4.57%±1.15% in high replication group), Treg cells (5.52%±1.58% in low replication group, 5.89%±1.84% in medium replication group and 6.37%±2.35% in high replication group) and their ratio Th17/Treg (0.57±0.25 in low replication group, 0.65±0.29 in medium replication group and 0.73±0.36 in high replication group) were significantly increased (P < 0.05). After entecavir treatment 24 weeks, the patient' s HBV-DNA decreased significantly, Th17 (3.89%±1.02% vs. 2.06%±0.46%), Treg (6.02%±2.03% vs. 5.06%±1.25%), Th17/Treg ratio (0.65±0.28 vs. 0.41±0.14) decreased significantly (P < 0.05). CONCLUSION: Investigation on the effects of HBV on Th17 and Treg cells and their ratios in different ALT states can clarify the effects of HBV on the body from the immunological perspective and can further understand the ALT grouping for antiviral treatment theoretical significance, which is helpful for clinical treatment.

[A phase II, single-arm, open-label, multicenter clinical study to evaluate the efficacy and safety of sofosbuvir combined with ribavirin in patients with genotype 2 chronic hepatitis C virus infection].

Objective: To evaluate the efficacy and safety of sofosbuvir (Nanjing Zhengda Tianqing Pharmaceutical Co., Ltd.) combined with ribavirin in patients with genotype 2 chronic hepatitis C virus infection. Methods: Treatment-naïve or treatment experienced genotype 2 chronic hepatitis C patients from sixteen research centers of China were screened. All subjects received once-daily dose of sofosbuvir (400 mg) combined with ribavirin (body weight < 75 kg, 1 000 mg/day, 400 mg in the morning and 600 mg in the evening; body weight > 75 kg, 1 200 mg/d, 600 mg in the morning and 600 mg in the evening) for 12 weeks. Patients were followed-up for a period of 12 weeks after discontinuation of treatment. Continuous variables were expressed as mean ± standard deviation. The proportion of subjects with virologic response at different follow-up time points and 95% confidence intervals were estimated by maximum likelihood ratio and Clopper-Pearson interval. Results: 132 cases with genotype 2 chronic hepatitis C virus infection from sixteen research centers of China were included, 12 cases of whom were associated with cirrhosis, and the remaining 120 cases were not associated with cirrhosis. One hundred and thirty-one cases completed the study, and one patient lost to follow-up at week 4 after the end of treatment. The sustained virological response rate was 96.2% (95% confidence interval: 92.37% - 99.16%) after 12 weeks of drug withdrawal. Virological relapse occurred in four cases. Of the 132 subjects enrolled in the study, 119 (90.2%) reported 617 adverse events during treatment, of which 359 (76.5%) were TEAE related to sofosbuvir and/or ribavirin. There were nine TEAEs of grade 3 and above, and six cases (4.5%) of them had six severe adverse events. Only one serious adverse event was associated with sofosbuvir and ribavirin (unstable angina pectoris). There were no adverse events leading to drug discontinuation or death. Conclusion: Sofosbuvir combined with ribavirin has a high SVR rate in the treatment of genotype 2 chronic hepatitis C virus infection, and most of the adverse events occurred were mild with acceptable safety profile.

Quantification of colony-forming units for M. tuberculosis complex using gyrB-based real-time PCR assay.

BACKGROUND: It is inappropriate to select insertion sequence (IS) 6110 as the amplification target for the quantitative analysis of Mycobacterium tuberculosis complex (MTC). OBJECTIVE: To develop a novel Taqman real-time polymerase chain reaction (RT-PCR) for the quantitative analysis of MTC by amplifying a single-copy PCR target. DESIGN: Analytic sensitivity and specificity, repeatability and reproducibility, and standard curve were estimated by analysing 18 reference strains and 100 clinical isolates. Diagnostic sensitivity and specificity, positive predictive value (PPV) and negative predictive value (NPV) were evaluated by detecting 50 clinical specimens. Quantitative accuracies of commercial and in-house Taqman RT-PCR were also compared. RESULTS: Analytic sensitivity of this method was 30 colony-forming units/ml and analytic specificity was 100%. In comparison with commercial Taqman RT-PCR (reference), diagnostic sensitivity, diagnostic specificity, PPV and NPV of the novel Taqman RT-PCR were respectively 85.7%, 94.4%, 85.7% and 94.4%. There was no significant difference between the two methods (χ(2) = 0.25, P > 0.05) and there was strong agreement between them (κ = 0.802, P < 0.05). In-house Taqman RT-PCR was more accurate than commercial assay. CONCLUSIONS: The novel RT-PCR is sensitive and specific for the detection of MTC and it is also accurate for quantitative analysis of MTC.

Nicotinamide mononucleotide adenylyltransferase promotes hypoxic survival by activating the mitochondrial unfolded protein response.

Gain-of-function mutations in the mouse nicotinamide mononucleotide adenylyltransferase type 1 (Nmnat1) produce two remarkable phenotypes: protection against traumatic axonal degeneration and reduced hypoxic brain injury. Despite intensive efforts, the mechanism of Nmnat1 cytoprotection remains elusive. To develop a new model to define this mechanism, we heterologously expressed a mouse Nmnat1 non-nuclear-localized gain-of-function mutant gene (m-nonN-Nmnat1) in the nematode Caenorhabditis elegans and show that it provides protection from both hypoxia-induced animal death and taxol-induced axonal pathology. Additionally, we find that m-nonN-Nmnat1 significantly lengthens C. elegans lifespan. Using the hypoxia-protective phenotype in C. elegans, we performed a candidate screen for genetic suppressors of m-nonN-Nmnat1 cytoprotection. Loss of function in two genes, haf-1 and dve-1, encoding mitochondrial unfolded protein response (mitoUPR) factors were identified as suppressors. M-nonN-Nmnat1 induced a transcriptional reporter of the mitoUPR gene hsp-6 and provided protection from the mitochondrial proteostasis toxin ethidium bromide. M-nonN-Nmnat1 was also protective against axonal degeneration in C. elegans induced by the chemotherapy drug taxol. Taxol markedly reduced basal expression of a mitoUPR reporter; the expression was restored by m-nonN-Nmnat1. Taken together, these data implicate the mitoUPR as a mechanism whereby Nmnat1 protects from hypoxic and axonal injury.

[Effects and clinical significance of virus load on red blood cell parameters in different stage of hepatitis B].

OBJECTIVE: To evaluate the effect of the viral load on the red blood cell parameters in chronic hepatitis B patients and its clinical significance. METHODS: In the study, 373 chronic hepatitis B patients were recruited, including 123 alanine transaminase (ALT) normal patients (ALT<40 U/L),128 ALT greater than or equal to the upper limit of normal, and less than 2 times higher than the upper limit of normal patients(40 U/L ≤ALT<80 U/L), and 122 ALT greater than or equal to 2 times higher than the upper limit of normal patients (ALT≥80 U/L). The blood routine parameters were measured by automatic blood cell counter. The liver function parameters were measured by automatic biochemical analyzer, the hepatitis B virus loads were measured by quantitative PCR analyzer and the results were analyzed by covariance analysis. RESULTS: In the ALT normal chronic hepatitis B patients group, the viral load had minor effects on the red blood cell parameters.But in the ALT abnormal chronic hepatitis B patients group, the viral load had a significant effect on the red blood cell parameters, and the effect was most manifest in the ALT≥ double upper limit of normal group. The specific performance was that with the viral load increasing, the red blood cell [low copies group (4.10±0.67)×10(12)/L,medium copies group (3.92±0.69)×10(12)/L,high copies group (3.54±0.90) ×10(12)/L], the hemoglobin[low copies group (129.66±21.12 ) g/L, medium copies group (126.23±23.38) g/L, high copies group (112.98±27.77) g/L], the hematocrit (low copies group 37.66±5.68, medium copies group 37.03±6.03, high copies group 33.34±8.15) decreased(P=0.006,0.007,0.010),the mean corpuscular volume [low copies group (92.17±6.53) fL, medium copies group (94.85±7.95) fL, high copies group (101.63±11.33) fL], the mean corpuscular hemoglobin [low copies group (31.70±2.22) pg, medium copies group (33.11±3.62) pg, high copies group (34.65±3.13) pg], the mean corpuscular hemoglobin concentration [low copies group (344.28±17.17) g/L, medium copies group (351.33±16.90) g/L, high copies group (358.12±15.67) g/L], and the red blood cell distribution width-standard deviation [low copies group (52.49±9.04) fL, medium copies group (56.96±7.19) fL, high copies group (61.23 ±7.23) fL] increased(P=0.000,0.000,0.002,0.000). CONCLUSION: Observing the effect of the viral load on the red blood cell parameters in chronic hepatitis B patients can reflect the effect of hepatitis B virus on the immune response and liver function in the different pathological stages, providing theoretical support for the clinical antiviral treatment.