[Analysis of the prognosis and survival of patients with acute-on-chronic liver failure].

Objective: To explore the influencing factors and the impact of artificial liver treatment on the prognosis and survival of patients with acute-on-chronic liver failure (ACLF). Methods: Clinical data from 201 cases with ACLF from January 2016 to December 2019 was retrospectively analyzed. The survival rate was calculated by the Kaplan-Meier method, the log-rank test of univariate analysis, and the multivariate analysis of the stepwise Cox regression forward method. Results: The median survival time of patients was 6 months, and the survival rates at 6, 9, and 12 months were 51.2%, 38.3%, and 29.9%, respectively. In univariate analysis, age, presence or absence of hypertension and upper gastrointestinal bleeding, treatment method, model for end-stage liver disease (MELD) score, and cholinesterase were associated with prognosis (P < 0.05). Multivariate regression analysis results showed that MELD score was the main factor affecting the 1-year prognosis of ACLF patients (P = 0.002). Artificial liver treatment was beneficial for the 1-year prognosis of ACLF patients aged < 50 years or with a MELD score of ≥20 (P < 0.05 ). The relative risk ratio (RR) of mortality was 2.55 times higher in patients with advanced age (≥50 years old) than that of younger patients (P < 0.001). Regression analysis was performed using age as a stratification factor, and upper gastrointestinal bleeding was related to the prognosis of younger patients, while choline esterase was related to the prognosis of advanced age. Regression analysis after stratified MELD score showed that age and hypertension were related to the prognosis of patients with MELD score < 20, and treatment method and age were related to the prognosis of patients with MELD score≥20. Conclusion: Artificial liver treatment is beneficial for the 1-year prognosis of ACLF patients. Age, MELD score, hypertension, and upper gastrointestinal bleeding are independent risk factors affecting the prognosis of ACLF patients.

[Short-term prognostic predictive value of the neutrophil/lymphocyte ratio combined with prognostic nutritional index in hepatitis B virus-related acute-on-chronic liver failure].

Objective: To explore the prognostic predictive value of neutrophil/lymphocyte ratio (NLR) combined with prognostic nutritional index (PNI) in patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). Methods: Clinical data from 149 HBV-ACLF patients admitted to the infectious diseases Department of the General Hospital of Ningxia Medical University were retrospectively analyzed. Demographic data of the enrolled patients and the initial clinical-related data after admission were collected. Patients were divided into survival (93 cases) and death groups (56 cases) according to their prognostic condition 90 days after discharge. Demographic and clinical differences were compared between the two groups data. Receiver operating characteristic (ROC) curves were plotted to determine the optimal cutoff values for NLR and PNI in predicting the 90-day mortality rate of HBV-ACLF patients. The COX regression model was used to conduct univariate and multivariate analyses to investigate the correlation between NLR and PNI and the prognosis of HBV-ACLF patients. Kaplan-Meier survival analysis was used to explore the effects of NLR and PNI on the survival of HBV-ACLF patients. Results: The death group NLR was higher than that of the survival group, while the PNI was lower than that of the survival group, with a statistically significant difference. The area under the receiver operating characteristic curve (0.842, 95% CI: 0.779-0.906) showed patients with adverse prognosis assessed by NLR combined with PNI had a superior prognosis than that of the Model for End-Stage Liver Disease (MELD) and its combined serum sodium (MELD-Na) and Child-Turcotte-Pugh (CTP) scores. COX regression analysis showed that NLR≥3.03 and MELD score were independent risk factors affecting the prognosis of HBV-ACLF patients. PNI > 36.13 was a protective factor for evaluating the prognosis of HBV-ACLF patients. Conclusion: NLR combined with PNI can enhance the prognostic predictive value of HBV-ACLF.

Evaluation of highly sensitive diagnostic tools for the detection of P. falciparum in pregnant women attending antenatal care visits in Colombia.

BACKGROUND: In low transmission settings early diagnosis is the main strategy to reduce adverse outcomes of malaria in pregnancy; however, microscopy and rapid diagnostic tests (RDTs) are inadequate for detecting low-density infections. We studied the performance of the highly sensitive-RDT (hsRDT) and the loop mediated isothermal DNA amplification (LAMP) for the detection of P. falciparum in pregnant women. METHODS: A cross-sectional study was conducted in two malaria-endemic municipalities in Colombia. We screened pregnant women in the context of an antenatal care program in health facilities and evaluated five tests (microscopy, conventional RDT, hsRDT, LAMP and nested polymerase chain reaction-PCR) for the detection of P. falciparum in peripheral blood, using a quantitative reverse transcription PCR (qRT-PCR) as the reference standard. Diagnostic performance of hsRDT and LAMP were compared with routine testing. RESULTS: The prevalence of P. falciparum was 4.5% by qRT-PCR, half of those infections were subpatent. The sensitivity of the hsRDT (64.1%) was slightly better compared to microscopy and cRDT (59 and 53.8% respectively). LAMP had the highest sensitivity (89.7%) for detecting P. falciparum and the ability to detect very low-density infections (minimum parasite density detected 0.08 p/µL). CONCLUSIONS: There is an underestimation of Plasmodium spp. infections by tests routinely used in pregnant women attending antenatal care visits. LAMP methodology can be successfully implemented at local hospitals in malaria-endemic areas. The relevance of detecting and treating this sub-patent P. falciparum infections in pregnant women should be evaluated. TRIAL REGISTRATION: ClinicalTrials.gov, Identifier: NCT03172221 , Date of registration: May 29, 2017.

[Deleted in lymphocytic leukemia 1 promoted proliferation and apoptosis of nephroblastoma cells through regulating miR-513a-5p and RANBP2 pathway].

Objective: To study the regulatory effects and mechanisms of deleted in lymphocytic leukemia 1 (DLEU1), microRNA-513a-5p (miR-513a-5p), and RAN binding protein 2 (RANBP2) in nephroblastoma. Methods: The GHINK-1 cells were transfected with pcDNA (pcDNA group), pcDNA-DLEU1 (pcDNA-DLEU1 group), miR-NC (miR-NC group), miR-513a-5p mimics (miR-513a-5p group), pcDNA-RANBP2 (pcDNA-RANBP2 group), pcDNA-DLEU1 and miR-NC (pcDNA-DLEU1+ miR-NC group), pcDNA-DLEU1 and miR-513a-5p mimics (pcDNA-DLEU1+ miR-513a-5p group), miR-513a-5p mimics and pcDNA (miR-513a-5p+ pcDNA group), miR-513a-5p mimics and pcDNA-RANBP2 (miR-513a-5p + pcDNA-RANBP2 group). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expressions of DLEU1, miR-513a-5p, RANBP2 in nephroblastoma tissues, normal adjacent tissues, normal kidney cell HK2, and hemangioblastoma cell GHINK-1. Western blot was used to detect the expressions of proliferating cell nuclear antigen (PCNA), B cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 related X (Bax). Cell counting kit 8 (CCK-8) was used to detect the cell survival rate. Flow cytometry was used to detect the apoptosis rate. Dual luciferase report test was used to detect the luciferase activity of cells. Results: The expression levels of DLEU1, miR-513a-5p and RANBP2 in adjacent tissues were 1.02±0.08, 1.01±0.06, 1.00±0.05, respectively, significantly lower than 5.16±0.24, 0.23±0.02, 1.67±0.09 in nephroblasts tumor tissues (P<0.05). Their expression levels in HK2 cells were 1.00±0.06, 1.00±0.08, 1.02±0.09, respectively, significantly lower than 3.15±0.21, 0.18±0.01, 1.54±0.10 in GHINK-1 cells (P<0.05). Overexpression of DLEU1 significantly reduced the apoptosis rate (7.35±0.41 vs 12.35±1.12, P<0.05). Overexpression of RANBP2 significantly reduced the apoptosis rate (8.89±0.48 vs 12.64±1.12, P<0.05). Compared with the miR-NC group (1.01±0.06, 0.99±0.06), the luciferase activity of DLEU1-WT (0.43±0.04) and RANBP2-WT (0.61±0.07) in miR-513a-5p group were significantly reduced (P<0.05). Compared with anti-miR-NC group (0.99±0.07, 0.98±0.05), the luciferase activity of DLEU1-WT (1.34±0.11) and RANBP2-WT (1.39 ±0.13) in anti-miR-513a-5p group was significantly increased (P<0.05). Simultaneous overexpression of pcDNA-DLEU1 and miR-513a-5p in GHINK-1 cells significantly reduced the apoptosis rate (11.34±1.03 vs 8.51±0.69, P<0.05). Simultaneous overexpression of miR-513a-5p and RANBP2 in GHINK-1 cells significantly reduced the apoptosis rate (9.96±0.72 vs 15.94±1.00, P<0.05). Conclusions: The long-chain non-coding RNA (lncRNA) DLEU1 can promote the proliferation and inhibit the apoptosis of nephroblastoma cells. The mechanism is related to the targeted regulation of miR-513a-5p and RANBP2 function, which will provide theoretical support for the nephroblastoma treatment.

[A preliminary study of serum marker alpha-enolase in the diagnosis of hepatocellular carcinoma].

Objective: To investigate the diagnostic value of serum α-enolase (ENO1) in the primary hepatocellular carcinoma. Methods: From May 2012 to March 2017, 163 cases with liver diseases who met the inclusion and exclusion criteria were admitted to the Infectious Diseases Department of the General Hospital of Ningxia Medical University. Among them, 28 cases were of chronic hepatitis B (CHB), 31 cases with liver cirrhosis (LC), 104 cases with hepatocellular carcinoma (HCC), and 18 healthy volunteers (NC). Patient data and serum samples were collected and liver disease related indicators were measured to detect ENO1 levels with enzyme-linked immunosorbent assay (ELISA). The measured indicators were expressed in median. Mann-Whitney U nonparametric test was used to analyze the differences between the data. A Spearman's correlation analysis was used for bivariate correlation analysis. The sensitivity and specificity of ENO1 and alpha-fetoprotein in the diagnosis of liver cancer were analyzed by ROC curve. Results: Serum level of ENO1 in CHB group, LC group and HCC group was significantly higher than normal group. Serum level of ENO1 in HCC group was higher than CHB group (P = 0.001) and LC group (P < 0.01). Area under the curve (AUC) for serum ENO1 and alpha-fetoprotein were 0.782 (cut-off value 75.96, P = 0.000 1) and 0.800 (cut-off value 27.02, P = 0.000 1), respectively. There was a positive correlation between ENO1 and AFP (P = 0.001). The combined detection had significantly improved the detection efficiency (AUC = 0.835). Serum ENO1 was statistically significant (P < 0.05) in HCC tumor size (AUC = 0.663), tumor metastasis (AUC = 0.681), TNM stage (AUC = 0.710, stage I vs. II), and Edmondson grade (AUC = 0.685) (P < 0.05) and the elevated levels of ENO1 had significantly reduced (P < 0.05) the survival time. Conclusion: ENO1 can be a new candidate marker for the diagnosis of early stage HCC and its progression.

[Role of enolase 1 in hepatocellular carcinoma and possible mechanism].

Objective: To investigate the role of enolase 1 (ENO1) in hepatocellular carcinoma (HCC) and possible mechanism. Methods: Real-time PCR and Western blot were used to measure the expression of ENO1 in HCC tissue, adjacent tissue, hepatoma cells, and normal hepatocytes. The siRNA interference technique was used for ENO1 knockout in HepG2 cells, and then CCK-8, colony formation assay, and transwell assay were used to measure the proliferation, migration, and invasion abilities of HepG2 cells. Real-time PCR and Western blot were used to measure the expression of proteins and genes involved in the activation of the Notch signaling pathway. The two-independent-samples t test and a one-way analysis of variance were used for comparison. Results: HCC tissue and HepG2 cells had significantly higher expression of ENO1 than adjacent tissue and normal hepatocytes (P < 0.05). There were significant reductions in the proliferation, migration, and invasion abilities of HepG2 cells after siRNA interference (P < 0.05). There were also significant reductions in the expression of N1ICD, snail, slug, HEY1, HES1, and HES5 (P < 0.05). Conclusion: ENO1 may promote the development of HCC, possibly by participating in the regulation of the Notch signaling pathway.

[Role of glucose-6-phosphate dehydrogenase in hepatitis B virus replication and its possible mechanism of action].

OBJECTIVE: To investigate the role of glucose-6-phosphate dehydrogenase (G6PD) in hepatitis B virus (HBV) replication and its possible mechanism of action. METHODS: Tissue microarray, quantitative real-time PCR, and Western blot were performed to analyze the differences in G6PD expression levels in the HBV-positive and HBV-negative liver tissues, HepG2.2.15 cells, and HepG2 cells. The siRNA transfection technique was used to knock down G6PD gene in HepG2.2.15 cells for 48 hours. Chemiluminescence was used for HBsAg and HBeAg quantification in supernatant, and quantitative real-time PCR was used to measure HBV DNA, type I interferon (IFN), and downstream IFN-stimulated genes. The t-test was used for comparison between groups. RESULTS: G6PD expression was significantly upregulated in the HBV-positive liver tissues and cells compared with HBV-negative liver tissues and cells, and the stain intensity and immunohistochemical scores were 89.69±54.92 and 31.90±18.62, respectively (P < 0.05). After G6PD expression in HepG2.2.15 cells was interfered by siRNA, the quantitative levels of HBV DNA, HBsAg, and HBeAg in supernatant were reduced significantly, and the mRNA expression levels of IFNα1, IFNß1, and five downstream IFN-stimulated genes (OAS1, ISG15, OAS3, EIF2α, and PKR) increased significantly (all P < 0.05). CONCLUSION: G6PD plays a vital role in HBV replication, and its mechanism of action in regulating HBV replication may be related to type I IFN signaling pathway.

The effects of 2-bromopropane on viability and testosterone production ability of rat Leydig cells in primary culture.

Epidemiological surveys and animal experiments have shown that 2-bromopropane induces oligozoospermia in exposed workers and inhibits spermatogensis in laboratory animals. However, the mechanism by which 2-bromopropane exerts its effects is unknown. To this end, we examined the formation of testosterone by the Leydig cells and their survival of these cells in the presence of different concentrations of 2-bromopropane in vitro. Leydig cells were isolated following vascular perfusion, enzymatic dissociation and Percoll gradient centrifugation techniques. The cells were cultured in culture dishes. After 8 h, different cultures were exposed to 2-bromopropane at concentrations of 0.01 mmol/L, 0.10 mmol/L and 1.00 mmol/L. In order to stimulate Leydig cells to secrete testosterone, human chorionic gonadotropin (hCG) was also added. Cell viability was determined using the trypan blue dye exclusion test and cell numbers were counted by hemocytometer. Testosterone secretion was detected by radioimmunoassay. The cell viability decreased after exposure to 2-bromopropane in a dose-dependent way, but no morphological change was observed. The cell number decreased in the 2-bromopropane-treated cultures. The secretion of testosterone did not manifest defectable changes in the culture treated with 0.10 mmol/L and 0.01 mmol/L of 2-bromopropane; however, it decreased significantly (P < 0.02) in the presence of 1.00 mmol/L. Therefore, our results strongly suggest that 2-bromopropane may exert its cytotoxic effects on Leydig cells in vitro. We speculate that the decrease in the numbers of Leydig cells caused by 2-bromopropane was mediated by a feedback mechanism resulting from a lower testosterone concentration.

[Determination of the progesterone antagonist lilopristone in serum by RP-HPLC].

To 1.0 ml of serum containing lilopristone were added RU486 solution (internal standard, IS) and 1 ml of 1.0 mol.L-1 NaOH. The mixture was extracted with diethyl ether for 2 times. After extraction, the combined organic phase was evaporated to dryness and the residue was dissolved in the mobile phase and washed with petroleum ether. After centrifugation, 20 microliters of the lower layer was subjected to HPLC. A muBondapak-C18 (10 microns) column (30 cm x 3.9 mm) was used and the column temperature was kept at 50 degrees C. The flow rate of mobile phase (methanol-dichloromethane--0.01 mol.L-1 phosphate buffer, pH 4.0, 67:5:28 v/v) was 1.1 ml.min-1 and UV detection was performed at 302 nm. The retention times of lilopristone and IS were 6.85 and 9.07 min respectively and the detection limit was 10 ng.ml-1 (S/N > or = 4) serum. The extraction recoveries of lilopristone and IS were over 85%. The relative standard deviations were 2.21 to 4.23%. This method has been applied to study the pharmacokinetic of lilopristone in rats.

Anaphylactoid reactions induced by fluorocarbon blood substitute.

The potential mechanism of fluorocarbon emulsion (FCE)-induced anaphylactoid reactions characterized by transient flush and respiratory disturbance in a few patients were elucidated by detecting the immunological response and the blood vessel vasoactive amine content. The passive cutaneous anaphylaxis (PCA) test in rats immunized with FCE was negative. The precipitating FCE reaction antibody (FRA) in sera of rabbits at 20 min and 24 h after i.v. infusion of FCE 10 ml/kg by reversible single radioactive immunodiffusion (RSRI) was positive, the densities of FRA were negatively related to the dilution of FCE (24 h, r = -0.9998). Histamine and 5-hydroxytryptamine (5-HT) content in peripheral blood of rabbits using fluorochromatography were decreased by 79% and 92% respectively at 10 min after i.v. infusion of FCE 10 ml/kg. Platelet counts fell to 67%, WBC to 47% and the ratio of polymorphonuclear cells and lymphocytes (P/L) was depressed by as much as half of that before infusion. Small arterioles of lungs were filled with macrophages. These results indicated that the mechanisms of FCE-induced anaphylactoid reactions did not involve IgE-dependent mediator release (histamine and 5-HT), but was associated with FCE-FRA complex formation and macrophage-stasis in pulmonary vessels.

Mutagenicity of various organic fractions of diesel exhaust particles.

Treated with various organic fractions of Diesel Exhaust Particles (DEP), the Ames test withSalmonella typhimurium strains TA98 and TA100, and the mice micronucleus test were employed to study the mutagenic activity in the bacterial reverse mutation system, with and without a mammalian S(9) activation component, and the clastogenic activity in mice polychromatic erythrocyte (PCE) stem cells. Extracted ultrasonically with dichloromethane then using the acid and base separated reaction and column chromatography, DEP were divided into five organic fractions. They are the organic acid fraction (Fl), the organic base fraction (F2), the aliphatic hydrocarbon fraction (F3), the aromatic hydrocarbon fraction (F4) and the polar fraction (F5). Results showed that an increase in the counted numbers of histidine revertants on theSalmonella TA100 and TA98 was observed with or without (S(9) mix), but these activities were more pronounced in the TA98 strains especially in the absence of the S(9) mix. These results suggest that the organic fractions of DEP contain mainly compounds with direct frame-shift mutaganicity. Positive results were also obtained from mice micronucleus assay. The frequency of mice bone marrow micronucleated polychromatic erythrocytes (PCE) was increased using this assay and it showed a definite dose-response relationship. The results suggest that various organic fractions could affect spindle fiber function or formation in mammalian cells. Compared with the results of different organic fraction, the effects of the F2, F4 and F5 were found to be stronger than those of other fractions. Based on the findings obtaind in the Ames and micronucleus tests, DEPs have genotoxic effects in both of the test systems.

[Scavenging effect of EDTA-fluorocarbon microspheres on 210lead].

EDTA-fluorocarbon microspheres (EDTAFM), calcium disodium ethylene diaminetetraacetate (CaNa2EDTA), calcium- or zinc-diethylene triamine pentaacetate (Ca- or Zn-DTPA) were investigated for their ability to treat experimental lead intoxication in mice. The 48 ICR mice were divided into six groups. Group I = no treatment; The other groups were injected with single ip doses of 210Pb (10 mg Pb2+ +555 kBq/kg). After 24 h they were injected in the tail vein with the chelating agents (20 mg/kg) or an equal volume of 10% glucose (10 mg/kg). Each mouse was housed in one metabolic cage, and urine was collected daily for 3 d. After 3 d, the mice were sacrificed for comparison of lead distribution within the liver, kidney, femur and the entire carcass as measured by 0.047 Mev gamma emission from 210Pb. The results reveal that injection of EDTA-FM to lead poisoned mice pretreated with 210Pb was more effective than Zn- or Ca-DTPA and CaNa2EDTA in reducing the lead induced inhibition in the activity of blood ALAD, and that it increased the excretion of 210Pb into the urine. The hepatic, renal and femur 210Pb contents after treatment with EDTAFM were much more decreased than Zn- or Ca-DTPA and CaNa2-EDTA. The order of effectiveness was EDTAFM greater than Zn-DTPA greater than Ca-DTPA greater than CaNa2-EDTA.

[Expression of cDNA of human chorionic gonadotropin beta-subunit (beta-hCG) cDNA in insect cells and effect of expressed product on mouse lymphocytes in vitro].

Expression vector pVL 1393-hCG beta containing beta-hCG cDNA has been constructed using an unfused protein nuclear polyhedrosis virus (AcNPV) expression vector. The insect cells (Sf 9) were cotransfected by the expression vector and nuclear polyhedrosis virus genomic DNA, and recombinant virus AcNPV-hCG beta was screened out, beta-hCG cDNA was expressed in insect cells infected by recombinant virus and recombinant beta-hCG (r beta-hCG) was secreted into medium. The purity of r beta-hCG, purified by immuno-affinity chromatography, was about 90% and the molecular weight of r beta-hCG was 22,500 Da. Like hCG, r beta-hCG suppressed significantly proliferation of induced lymphocytes, as well as production of IL-2 to some extent, on a parallel with suppression of lymphoproliferation.