Objective To study the effect of radiofrequency ablation (RFA) with sublethal temperature on the production of liver cancer stem cells (LCSCs) and on the expression of LCSCs-related transcriptional factors.Methods Mouse hepl-6 hepatoma cell line and clinical samples of patients with hepatocellular carcinoma (HCC) were used to test the expressions of LCSCs-related markers and transcriptional factors.Results Different temperatures were used to stimulate Hep1-6 cells,and it was proved that the temperature of 45℃ was a sublethal temperature that could not induce cell death.Flow cytometry testing showed that treatment with 45℃ could obviously increase CD13+,CD44+,CD90 and CD133+ Hep1-6 cells,suggesting that treatment with 45℃ could increase the production of above mentioned types of LCSCs in hep1-6 cells.Real-time quantitative polymerase chain reaction (RT-qPCR) assay indicated that the temperature of 45℃could cause significant increase in CD13,CD90 and CD133 mRNA.In all 5 HCC patients,CD13 mRNA in the recurrent HCC lesions was remarkably increased,CD133 mRNA was increased in 4 patients with recurrent HCC,and CD90 mRNA was increased in only one patient with recurrent HCC.Flow cytometry testing revealed that CD13+ LCSCs were strikingly increased in 4 recurrent HCC patients,while CD133+LCSC was increased in only one patient,suggesting that more close correlation existed between the increase of CD13+ LCSCs and the temperature of 45℃.RT-qPCR assay showed that in 4 recurrent HCC patients with increased CD13+ LCSC,the Sox2 and Stat2 among 13 LCSCs-related transcriptional factors were obviously increased.Flow cytometry testing showed that 45℃ treatment also increased the expression of Sox2 and Stat1 mRNA in Hep1-6 cells.Finally,Sox2 and Stat1 could be knockdown by siRNAs,indicating that both Sox2 and Stat1 transcriptional factors were involved in 45℃-induced production of CD13+ LCSCs in Hep1-6 cells.Conclusion In RFA therapy,the use of sublethal temperature of 45℃ can increase CD13+LCSCs,which is related to the promotion of Sox2 and Stat1 expression.The results of this study can be used for reference in the research of liver cancer recurrence.

Objective To study the prevalence and genotypes of rotavirus (RV) among children,< 5 years old hospitalized with viral diarrhea in Tianjin. Methods Stool specimens were collected from hospitalized diarrhea children in Tianjin children's hospital between May 2008 and April 2009. Detection of rotavirus was employed by Colloidal Gold Device. The detected positives were inoculated to MA-104 cells. The total RNA of virus was extracted after CPE which was caused by rotavirus were observed, The VP7 serotypes were determined by using RT-PCR to amplify the VP7 gene and sequencing the RT-PCR products.The clinical data for each patient were also collected. Results Among 837 specimens, the RV antigen positive rate was 26. 3% (220/837). Among all the children with rotavirus diarrhea, 90. 5% (199/220)were < 2 years old. The prevalence of rotavirus diarrhea in children peaked during Oct. 2008 through Apr.2009. Of the 208 rotavirus positive specimens, 95 were successfully identified by RT-PCR Thirty-five positive strains of RV were sequenced, and the sequencing results showed that 32 positive strains were belonged to rotavirus G1 type, 2 positive strains were belonged to rotavirus G3 type and 1 positive strain were belonged to rotavirus C9 type. Conclusion RV was the dominant etiological agent for infantile diarrhea infection in Tianjin, and the predominant serotype was G1.

Objective To study the pathogenic prevalence and genotypes of astrovirus among children under 5 years old hospitalized with diarrhea in Tianjin. Methods A total 837 stool specimens were collected from children with diarrhea hospitalized in Tianjin children's hospital from May 2008 to April 2009. Astrovirus antigens were detected using ELISA and the postive specimens were inoculated in CaCo-2cells. After the CPE caused by virus were observed, the total RNA of virus was extracted, then the genomc fragments of the strains were amplified by using RT-PCR and confirmed by sequencing of the RT-PCR products. Detection of rotavirus was employed by Colloidal Gold Device. Results Astrovirus antigen was found positive in 3.0% of the patients. The coinfection rate of astrovirus and rotavirus was 0. 7% (6/837).Ninety-six persent of children with astrovirus diarrhea were younger than 2 years of age, Forty-eight persent of children with astrovirus diarrhea were younger than 6 months. The astrovirus infections occurred mainly between August 2008 and April 2009. Of the 21 astrovirus positive specimens, 11 cases were successfully identified by RT-PCR and they were all serotype 1. Conclusion Astrovirus is a major cause of nonbacterical diarrhea between 2008 and 2009 in Tianjin, and the predominant serotype is type 1.

Objective:To investigate the risk factors for short-term mortality and long-term survival after liver transplantation in patients with hepatitis B related acute-on-chronic liver failure.Methods:Forty patients with hepatitis B related acute-on-chronic liver failure performing liver transplantation were prospectively collected from August 2018 to July 2021 in Beijing YouAn Hospital of Capital Medical University. The mean age was (44.5±8.79) years, there were 36 males and 4 females. The basic data, including liver and kidney function, blood routine, coagulation function, lactic acid, infection indexes as well as MELD score, MELD-Na score, CLIF-C ACLFs score, CLIF OFs score, CLIF grade within 48 hours before liver transplantation were counted. The post-LT mortality within 90 days and long-term survival were observed for these patients who were divided into survival group ( n=34) and death group ( n=6) according to the survival in 3 months after liver transplantation. The measurement data conforming to the normal distribution were expressed by mean ± standard deviation ( ± s), and the comparison was performed by t-test between groups; The skewness data were expressed by M ( Q1, Q3), and the rank sum test was used for inter-group comparison. The counting data were tested by Chi square test or Fisher exact probability method. The risk factors of short-term mortality and long-term survival were analyzed through univariate and multivariate analysis as well as survival analysis. The sensitivity, specificity and cut off value were calculated by ROC curve. The patients were divided into ≥ 48.5 scores group ( n=10) and < 48.5 scores group ( n=30) by CLIF-C ACLFs score 48.5 as cut-off value. Kaplan Meier was used for survival analysis and comparison. Results:The total bilirubin (TBIL), creatinine (CR), platelet count, international normalized ratio (INR), lactic acid and neutrophil/lymphocyte ratio (NLR) within 48 hours before liver transplantation were 24.30 (13.45, 33.95) mg/dL, 0.68 (0.53, 1.11) mg/dL and 56 (39, 82)×10 9/L, 3.12(2.33, 4.46), 2.14(1.59, 4.14) mmol/L, 4.06(2.12, 9.13) for all forty patients, respectively. The mean MELD, MELD Na, CLIF OFs, CLIF-C ACLFs and AARC scores within 48 hours before transplantation were (32.1±6.3), (33.2±5.3), (11.2±2.6), (43.8±8.8) and (10.6±2.4) scores, respectively. 65% of patients were complicated with hepatic encephalopathy, 17 patients with controllable systemic infection, 10 patients with renal function injury, 2 patients with variceal bleeding and 6 patients underwent ventilator-assisted ventilation (endotracheal intubation). All 40 patients underwent emergency orthotopic liver transplantation. The incidence of postoperative complications was 47.5%, the most common was post-LT infection (27.5%), followed by renal insufficiency (17.5%). There were significant differences in neutrophil lymphocyte ratio (NLR), lactic acid, MELD score, MELD Na score, CLIF-C ACLFs score, CLIF OFs score, CLIF grades, hepatic encephalopathy, infection and renal injury between survival group and death group ( P<0.05). Univariate logistic regression analysis showed that NLR, MELD Na score, CLIF-C ACLFs score and CLIF OFs score were the risk factors for short-term mortality after liver transplantation ( P<0.05). Multivariate logistic regression analysis showed that CLIF-C ACLFs score was an independent risk factor for 3-month mortality after liver transplantation. ROC curve showed that the area under the curve of CLIF-C ACLFs score was 0.895 (95% CI: 0.779-1.000, P=0.002), and the diagnostic sensitivity and specificity were the highest, 83.3% and 85.3% respectively when cut off value was 48.5. Meanwhile, there was significant difference in long-term survival between the patients with CLIF-C ACLFs score ≥ 48.5 and < 48.5 ( P=0.001). Conclusions:NLR, MELD Na score, CLIF-C ACLFs score and CLIF OFs score within 48 hours before liver transplantation are the risk factors for short-term mortality after liver transplantation, however CLIF-C ACLFs score is an independent risk factor for three-months mortality and long-term survival in patients with hepatitis B related acute-on-chronic liver failure after liver transplantation.

Objective: To investigate the genetic characteristics of Lamivudine-resistant mutation patterns and HBV S gene mutants in patients with chronic hepatitis disease of different disease progression. Methods: Blood samples of LAM-resistant patients with chronic hepatitis disease were collected. HBV RT gene nucleotide sequences were obtained, and then differences in drug-resistant mutation patterns, drug susceptibility and HBV S gene mutants characteristics between the two groups were analyzed. Results: Forty-seven chronic hepatitis B (CHB) patients and 16 HBV-related liver cirrhosis (LC)/HBV-related hepatocellular carcinoma (HCC) patients were included in this study. M204I single point mutation and L180M+ M204I/V were the most common pattern during patients with chronic hepatitis disease (35/63, 55.56%). The numbers of resistant to three nucleos(t)ide analogues in LC/HCC group was higher than CHB group’s (62.50% vs 34.04%, P=0.046). In HBV S gene, more immune associated HBsAg-escape mutations were detected in LC/HCC group than that in CHB group (62.50% vs 31.91%, P=0.031). I126T/V and G145A (for LCC/HCC group, 60%), I126S/T and S117T (for CHB group, 46.67%) were showed as the most common form for HBsAg escape mutations in the two groups. The two groups both detected RT mutations concomitantly with stop codon mutations in S gene (rtA181T/sW172* and rtM204I/sW196*). Conclusions Different characteristics in Lamivudine-resistant mutations and associated HBV S gene mutants were found in patients with chronic hepatitis disease of different disease progression, and LC/HCC patients exhibit more multi-drug resistant variants and immune associated HBsAg-escape mutants than CHB patients.

Objective:To investigate the safety and efficacy of combining programmed death-1 (PD-1) with tyrosine kinase inhibitors (TKIs) in patients with advanced hepatocellular carcinoma (HCC) before liver transplantation(LT).Methods:The data of six males with a mean ± s. d. age of (57.5±4.3) years who were treated with PD-1 inhibitors combined with TKIs for advanced HCC before LT at Beijing You'an Hospital, Capital Medical University and the First Medical Center of Chinese PLA General Hospital were retrospectively analysed. The tumor stagings, the use of PD-1 inhibitors and TKIs with their discontinuation in pre-LT/post-LT liver function recovery durations, incidences of complication. The tumor recurrence and disease-free survival rates were determined on follow-up of these patients at outpatients clinics.Results:For the 6 patients included in this study, four patients were classified by the Barcelona Clinic Liver Cancer Staging (BCLC) as C and the China Liver Cancer Staging (CNLC) as Ⅲa, and two patients were classified by the BCLC staging as B and the CNLC asⅡb. The mean cycle of PD-1 inhibitor used was 5.5 (1-20), and the mean duration of PD-1 inhibitor discontinuation was 19.5 (12-45) days pre-LT. All patients who were treated with PD-1 inhibitors combined with TKIs reached the liver transplantation standard, and all successfully underwent orthotopic liver transplantation. The liver function recovered well without any serious complications post-LT. All the patients survived without developing any acute rejection or other complications. The follow-up time ranged from 8.2 to 27.3 months, with a median of 11.9 months. No patients had died, and 2 patients developed tumor recurrence. The median (range) tumor-free survival time was 10.9 (2.9-27.3) months.Conclusion:Patients with advanced HCC could benefit from combined PD-1 inhibitors with TKIs therapy pre-LT. There were no increased incidences of acute rejection and other complications post-LT.

Objective To investigate the long-term safety and effectiveness of withdrawal of hepatitis B immuneglobulin (HBIG) and/or nucleos(t)ide analogues (NAs) to prevent hepatitis B virus (HBV) reinfection in liver transplant recipients with hepatitis B-related diseases after successful vaccination. Methods Baseline data of 76 liver transplant recipients undergoing hepatitis B immune reconstitution after receiving hepatitis B vaccines were retrospectively analyzed. The vaccination and response, the follow-up results of respondents with HBIG and/or NAs withdrawal, and the reinfection of HBV after withdrawal of HBIG and/or NAs were analyzed. Results The time interval from liver transplantation to hepatitis B vaccination was 26 (20, 40) months. The time interval from vaccination to response was 15 (8,27) months. Initially, 76 recipients withdrew HBIG, and 36 recipients withdrew HBIG and NAs. During the follow-up, 12 of 76 recipients who withdrew HBIG resumed use of HBIG, and 16 of 36 recipients who withdrew HBIG and NAs resumed use of NAs. The withdrawal time of HBIG and NAs was 135 (98,150) and 133 (34,149) months, respectively. Sixteen respondents did not receive booster, and 36 respondents received boosters on a regular basis. The time interval between the first booster and HBIG withdrawal was 44 (11,87) months. No significant differences were observed in baseline data between the respondents with and without boosters (all P>0.05). During the follow-up, 9 recipients were lost to follow-up, 5 were re-infected with HBV, 3 died, and 1 recipient developed graft loss and underwent secondary liver transplantation. Among 5 recipients re-infected with HBV, 4 cases had virus mutation. Significant differences were found between re-infected and uninfected patients regarding withdrawal of NAs and hepatitis B e antigen (HBeAg) positive before transplantation (both P<0.05). Conclusions Long-term withdrawal of HBIG is feasible and safe for recipients with successful hepatitis B immune reconstitution after liver transplantation for hepatitis B-related diseases. Nevertheless, whether antiviral drugs can be simultaneously withdrawn remains to be validated.

OBJECTIVETo study resistance evolution pathway of HIV-1 CRF_BC under drug selection pressure, and compare with B subtype.

METHODSBased on the reverse transcriptase region of CRF_ 97BC HIV-1 from 588 treatment-naive and 274 treatment patients, selection pressure based method was used to select resistance-associated mutations, and Bayesian network was used to construct the resistance evolutionary pathway under antiretroviral therapy. Meanwhile, it was constructed that the resistance evolutionary pathway for B subtype with the same regimens using the data from HIV resistance database, and made a comparison with CRF_07BC.

RESULTSThe major resistance mutations for CRF_07BC were identified including K103N, Q197K, V179D and Y188L. While for B subtype, the major resistance mutations include M184V, K103N,Y181C, T69N,G190A, K238T,Y188H and P225H. Much difference was observed between these two classes. However, the classical TMA1 (41L, 210W and 215Y) and TMA2 (67N, 70R and 219E/Q) pathways exist in both pathways. As different from B subtype, the predicted major drug resistance mutations for CRF_07BC did not contain TAM-related mutations, and nucleoside reverse transcriptase inhibitor-related mutations and non-nucleoside reverse transcriptase inhibitor-related mutations were mutually depending on each other.

CONCLUSIONHIV-1 CRF_07BC showed distinctive resistance evolutionary pathway, the mutations K103N,Q197K,V179D and Y188L were the major resistance mutations, and different resistance evolutionary pathways were observed between HIV-1 CRF_07BC and B subtype.

Anti-HIV Agents ; pharmacology ; Bayes Theorem ; Drug Resistance, Viral ; genetics ; Evolution, Molecular ; HIV-1 ; drug effects ; enzymology ; genetics ; Humans ; Mutation ; RNA-Directed DNA Polymerase ; genetics