Tumor neoantigens and tumor immunotherapies.

As a high-risk group of patients with cancer, the elderly exhibit limited efficacy with traditional treatments. Immunotherapy emerges as a promising adjunctive therapeutic approach that holds potential in addressing the needs of geriatric patients with cancer. Neoantigens, a unique class of tumor-specific antigens generated by non-synonymous mutations, are garnering increasing attention as targets for immunotherapy in clinical applications. Newly developed technologies, such as second-generation gene sequencing and mass spectrometry, have provided powerful technical support for the identification and prediction of neoantigens. At present, neoantigen-based immunotherapy has been extensively applied in clinical trials and has demonstrated both safety and efficacy, marking the beginning of a new era for cancer immunotherapy. This article reviews the conception, classification, inducers, and screening process of tumor neoantigens, as well as the application prospects and combination therapy strategies of neoantigen-based cancer immunotherapy.

Surface charge regulation of imidazolium salts/starch/carboxymethyl cellulose ternary polymer blend films for controlling antimicrobial performance and hydrophobicity.

Starch (SR)/carboxymethyl cellulose (CMC) based antimicrobial films have been widely applied in packaging field. As a high-effect antimicrobial agent, the surface charge of imidazolium salt plays an important effect on antimicrobial performances of starch/carboxymethyl cellulose. Here in, the surface charge of dodecyl imidazolium bromide salt was regulated via thiol-ene reaction. Furthermore, antibacterial films were prepared by mixing imidazolium salts with SR/CMC via solution casting method. Under the optimized ratio of CMC to SR, the antibacterial activity for as-prepared ternary polymer blend films was enhanced with the increasing of surface charge of imidazolium salt. The sample of ADSC-01 film with highest surface charge showed best antibacterial properties for E. coli and S. aureus with the inhibition zone of 3.20 cm and 3.00 cm, respectively. In addition, hydrophobic property exhibited similar positive correlation with the surface charge. Therefore, this work provides a new route to regulate the antibacterial activity of bio-based ternary polymer blend films in the packaging.

Investigation of macrophage polarization in herniated nucleus pulposus of patients with lumbar intervertebral disc herniation.

Macrophage infiltration and polarization during lumbar intervertebral disc herniation (LDH) have attracted increased attention but their role remains unclear. To explore macrophage polarization in herniated nucleus pulposus (NP) tissue of patients with LDH and investigate the association between cell frequency and different clinical characteristics or symptoms, we conducted a retrospective study by analyzing NP tissue samples from 79 patients. Clinical features and symptoms, using the visual analog scale (VAS) and Oswestry disability index (ODI), were collected. The macrophage markers CD68, CCR7, CD163, and CD206; pro-inflammatory cytokine TNF-α; and anti-inflammatory factor IL-4 were analyzed by immunohistochemistry. The frequency of polarized macrophages and positivity rate of pro- and anti-inflammatory cytokines showed significant differences in some of clinical characteristics. Specifically, higher CCR7+ and TNF-α + proportions were identified in the high-intensity zone (HIZ) and the type of extrusion and sequestration NP tissue than in non-HIZ and protrude NP tissue. Higher CD206+ and IL-4+ proportion were detected in Modic changes. However, no differences in gender, age, smoking status, Pfirrmann grade, analgesic use, leg pain duration, and segments were found between groups. CD68+ , CCR7+ , and CD206+ cell proportions, and TNF-α and IL-4 showed positive associations with VAS scores preoperation. Associations between ODI and the macrophages markers were weak/insignificant. Our results indicated that macrophage polarization or macrophage-like cells contribute to LDH pathological features. Macrophage populations displaying significant associations with VAS score reflected continuous M1/M2 transition contributing to pain during LDH. These findings may contribute to enhanced/personalized pharmacological interventions for patients with LDH considering pain heterogeneity.

Investigation and Analysis of the Current Situation of Quality Management in Drug Clinical Trial Institutions / 医药导报

Objective To understand the current situation of quality management and implementation of risk-based quality management in clinical trial institutions.Methods In the form of questionnaires,Questionnaire Star was used to create the questionnaire,which was sent to clinical trial organisations in all provinces and cities to fill in through emails and institutional WeChat groups.Results The questionnaire results from 123 clinical trial institutions from 25 provinces were included for analysis.The quality management status was as follows:104(84.6%)institutions of the main quality management mode were the internal quality control by GCP department.On average,each institution had 3、4 full-time staff for quality management,with an average of 1 full-time staff to manage 16.8 projects and 10.9 specialties.In the last three years,65(52.8%)institutions conducted training for researchers 2-3 times/year.Ninety-five(77.2%)institutions had established a clinical trial funding allocation system.Forty-nine(39.2%)institutions had established GCP informatization management system and 44(35.2%)institutions had established risk-based quality management model.In the case of coordination between the clinical trial institution and the sponsor,47(41.2%)institutions did not not aware of the sponsor's/CRO's quality management plan in their collaboration with the sponsor.Conclusion The surveyed institutions have weaknesses,such as insufficient dedicated personnel for quality management,low utilization rate of GCP information systems.The collaborative capacity between clinical trial institutions and sponsors needs to be improved,and the implementation rate of risk-based quality management is also low.

Structural optimization and antibacterial property of alkylimidazole salt/carboxymethyl cellulose/starch composite films.

As a green solvent, alkylimidazolium salt has attracted much attention due to high antibacterial activity and excellent biocompatibility. In this work, 1-allyl-3-alkylimidazolium bromide ionic liquids (AIMB) were synthesized in a closed system. Using carboxymethyl cellulose (CMC) and starch (SR) as carriers, 1-allyl-3-dodecylimidazolium bromide ionic liquid (AIMC12)/CMC/SR (AIMCS) films were prepared via solution casting method. The AIMC12 exhibited the highest antibacterial activity. Under the optimized ratio of CMC to SR, the AIMCS-1-1 film showed effective antibacterial properties for E. coli and S. aureus with the inhibition zone of 3.50 cm and 3.02 cm, respectively. In addition, the tensile strength and elongation at break of AIMCS-1-1 reached to 4.5 MPa and 111.6 %, and its Young's modulus was 1.4 GPa. Therefore, as-prepared AIMB will be expected to replace traditional antibacterial agents in antibacterial applications, and as-prepared AIMCS films as the green packaging materials have potential application in antibacterial packaging.

Oligomerization of the HBV/HDV functional receptor NTCP expressed in Sf9 insect cell.

BACKGROUND: Sodium taurocholate co-transportering polypeptide (NTCP, SLC10A1) is a vital bile acid transporter and the functional receptor of hepatitis B and D virus. The oligomerization of NTCP is important for the structural study of its interaction with HBV preS1 peptide. METHODS: Recombinant NTCPs were expressed in Sf9 host cell using baculoviruses. Function of recombinant NTCP was verified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method. A quantitative fluorescence resonance energy transfer (FRET) method was established to analyze the interaction between NTCP wild type (WT) and mutants. Co-immunoprecipitation (Co-IP) was used to test the interaction between NTCP variants. RESULTS: Sub-cellular location of recombinant NTCPs varies with the modification of NTCP. Bands of monomer, dimer and oligomers were shown in gel analysis of NTCP. Significant FRET was observed between cyan florescence protein (CFP) tagged NTCP and yellow florescence protein (YFP) tagged NTCP. FRET efficiency between CFP- and YFP-NTCP S267F mutants was lower than WT. Co-IP results showed that S267F interacts with WT NTCP when co-expressed in cell. CONCLUSION: Dimer is the predominant form of NTCP expressed in Sf9 when solubilized with detergent. FRET and Co-IP analysis support that NTCP forms oligomers in Sf9 cell. GENERAL SIGNIFICANCE: Our results showed that NTCP formed oligomers in Sf9 cell. Meanwhile the FRET analysis of NTCP variants further elucidated the molecular mechanism of NTCP oligomerization.

Mutations of epidermal growth factor receptor gene in non-small cell lung cancer patients and the correlation with clinical characteristics and therapeutic effects / 肿瘤研究与临床

Objective:To investigate the clinical characteristics of non-small cell lung cancer (NSCLC) patients with different epidermal growth factor receptor (EGFR) gene mutations and the comparison of therapeutic effects.Methods:The clinical data of 324 patients with NSCLC admitted to the 904th Hospital of the Joint Service Support Force of PLA from April 2018 to June 2020 were retrospectively analyzed. Gene sequencing method was used to detect EGFR gene and mutations of exons 19 and 21. NSCLC patients with EGFR gene mutations were divided into group A (mutation of exon 19 of EGFR gene) and group B (mutation of exon 21 of EGFR gene). Both groups were treated with gefitinib combined with TP (paclitaxel + cisplatin) regimen for 3 months. The clinical features, efficacy and adverse reactions of the two groups were compared.Results:Among 234 NSCLC patients, 107 cases (45.73%) had EGFR gene mutations. Among them, there were 49 cases in group A (including delE746-A750 mutation in 32 cases, delL747-P753insS 3 mutation in 8 cases, delL747-A750 1 mutation in 6 cases, delL747-T751 1 mutation in 3 cases), and there were 58 cases in group B (all L858R mutations), and no double mutations in exons 19 and 21 were found in both groups. There were no significant differences in gender, TNM staging, pathological type, smoking history, age, degree of differentiation, tumor location, tumor diameter, and lymph node metastasis in the two groups (all P > 0.05). The difference in the clinical control rates of group A and group B was not statistically significant [91.8% (45/49) vs. 89.7% (52/58), χ2=0.15, P = 0.699]. The incidence of grade Ⅲ-Ⅳ adverse reactions in the two groups during treatment had no statistically significant differences (all P > 0.05). Conclusions:EGFR mutation rate in NSCLC patients is relatively high, most of which are EGFR exons 19 and 21 mutations. Gefitinib combined with TP regimen in the treatment of EGFR exons 19 and 21 mutations in NSCLC patients has good curative effects and high safety.

Efficacy and safety of fruquintinib in treatment of elderly patients with advanced metastatic colorectal cancer who failed chemotherapy / 肿瘤研究与临床

Objective:To investigate the clinical efficacy and safety of fruquintinib in elderly patients with advanced metastatic colorectal cancer who failed chemotherapy.Methods:Ninety-nine elderly patients with advanced metastatic colorectal cancer who failed chemotherapy in No. 904 Hospital of Joint Logistics Support Force from September 2018 to July 2020 were selected. All patients were given furquintinib capsules, 1 time/d, 5 mg/time, and 28 days was 1 cycle. All patients were treated continuously for 2 cycles and the effect was observed. The patient's recent anti-tumor efficacy was counted. The serum levels of carbohydrate antigen 125 (CA125), carcinoembryonic antigen (CEA) and carbohydrate antigen 199 (CA199) in patients before and after treatment were compared. The safety of the medication during the patient's treatment was recorded, and the Kaplan-Meier method was used for survival analysis.Results:A total of 99 elderly patients with advanced metastatic colorectal cancer who failed chemotherapy were treated for 2 cycles, with an objective response rate (ORR) of 22.22% (22/99) and a clinical control rate (CCR) of 75.76% (75/99). The serum levels of CA125, CA199 and CEA after treatment were lower than those before treatment (all P<0.05). The drug adverse reactions in 99 patients during the treatment were mostly grade Ⅰ-Ⅱ, and grade Ⅲ-Ⅳ were rare. The most common gradeⅠ-Ⅱ adverse reactions were hypertension (45.45%, 45/99), hand-foot syndrome (40.40%, 40/99), and elevated aspartate transferase (36.36%, 36/99). Followed up for 12 months, 5 cases were lost to follow-up, the follow-up rate was 94.95%, the median progression-free survival time of the remaining 94 patients was 5.62 months (95% CI 3.57-8.75 months), and the median overall survival time was 8.41 months (95% CI 4.85-11.14 months). Conclusions:Fruquintinib has good efficacy in the treatment of elderly patients with advanced metastatic colorectal cancer who failed chemotherapy. It can reduce the levels of tumor markers, the survival status of patients is good, and the adverse reactions are controllable.

Effect of hot spring bathing therapy on human serum immunoglobulins and complements / 上海预防医学

Objective To investigate the effect of hot spring baths on human immune function by analyzing the changes of immunoglobulins and complements in serum of residents after hot spring baths in order to provide a theoretical reference for the therapeutic effect of hot spring bathing. Methods After physical examination, 421 volunteers from five hot spring areas with three types of hot springs(temperature type hot springs, metasilicic acid type hot springs, and warm mineral spring type hot springs)in Guizhou Province were selected as the subjects. Under the guidance of professionals, the volunteers took a hot spring bath with the whole body immersed for four weeks, once a day, 5 times a week and 40-50 minutes each time. Finally, 311 volunteers completed the standard bath required by this study. The transmission immunoturbidimetric method was used to determine the content of immunoglobulins reflecting mucosal anti-infective immunity(IgA), anti-pathogenic microorganisms(IgG), recent infections(IgM)and the level of important immune effect factors(C3, C4)in the serum. Paired T test was used to compare the changes of serum immunoglobulin and complement before and after the hot spring bath therapy. Results Before the hot spring baths, the content of serum IgG, IgA, IgM, and complements C3 and C4 was(12.169±2.358)g/L, (2.285±0.891)g/L, (1.430±0.660)g/L, (1.224±0.186)g/L, and(0.257±0.073)g/L, respectively. After the hot spring baths, the content of serum IgG, IgA, IgM, and complements C3 and C4 was(12.769±2.984)g/L, (2.397±0.909)g/L, (1.497±0.715)g/L, (1.242±0.169)g/L, and(0.266±0.074)g/L, respectively.Comparison of results of different types of hot springs showed that warm mineral type of hot springs and metasilicic acid type of hot springs could significantly increase the serum levels of main immunoglobulins IgG and IgA(P < 0.05), while water temperature type of hot springs could increase the serum IgA content of the population(P < 0.05), but the effect on IgG was not significant(P > 0.05). Compared with before the bath intervention, the level of complement C4 in the serum increased in the population after the intervention of metasilicic acid type of hot springs and water temperature type of hot springs(P < 0.05). Conclusion Hot spring bathing can enhance the body's humoral immune function. Given that IgG is the most important anti-pathogenic microorganism antibody in body fluids, the result suggests that metasilicic acid hot spring and warm mineral hot spring are better than pure water temperature hot spring in terms of improving the body's humoral immune function.

Homosexual behavior and related factors among married men who have sex with men in Mianyang city, Sichuan province / 中华流行病学杂志

Objective@#To understand the homosexual behavior and related factors among married MSM in Mianyang city.@*Methods@#Between January and October in 2017, a snowball sampling method was adopted to carry out cross-sectional survey through questionnaires plus HIV testing among those MSM in Mianyang city. Logistic regression model was used to analyze homosexual behaviors and related factors among married MSM under study. Statistical analysis was used by EpiData 3.1 and SPSS 19.0 software.@*Results@#A total of 234 MSM participated in this survey. The overall rate of homosexual behavior in these married MSM appeared as 94.9% (222/234). Rate of having anal sex behavior was 94.4% (221/234) in the past 6 months, with rate of condom use as 57.9% (128/221). HIV positive rate was 8.1% (18/222). As for the motives for homosexual behavior after marriage, 87.8% (195/222) were driven by feelings of love, 12.2% (27/222) due to 'releasing pressure’. Proportion of male sex partners would include occasional sex partners (62.2%, 138/222), stable male sex partners (26.1%, 58/222) and stable boyfriends (11.7%, 26/222). Factors from logistic regression analysis showed that homosexual behaviors were related to the factors including education level of senior high school or above vs. education level of junior middle school or below (OR=3.65, 95%CI: 1.33-9.98); local residency over one year vs. the ones having local residency less than one year (OR=23.28, 95%CI:1.67-324.89); having 10 or more friends in the MSM community vs. having below 10 friends in MSM community (OR=4.15, 95%CI: 1.28-13.43); without sex pleasure with spouse vs. having sex pleasure with spouse (OR=3.25, 95%CI: 1.22-8.62); having 2 or more anal sex partners in the past 6 months vs. having less than 2 anal sex partners in the past 6 months (OR=0.28,95%CI: 0.09-0.81).@*Conclusions@#The rate of homosexual behavior and HIV positive rate were high among MSM in Mianyang city. Homosexual behaviors after marriage were influenced by multiple factors among MSM. The motives of homosexual behavior after marriage were driven by feelings of love, the related factors were education level of senior high school or above, local residency over one year, having 10 or more friends in the MSM community and without sex pleasure with spouse. As for the motives of these behaviors was caused by releasing pressure, the related factors was having more than 2 anal sex partners.

Designed inhibitor for nuclear localization signal of polo-like kinase 1 induces mitotic arrest.

Polo-like kinase 1 (Plk1), a member of polo-like kinase family, regulates multiple essential steps of the cell cycle progression. Plk1 is overexpressed in multiple cancer cell lines and considered to be a prime anticancer target. Plk1 accumulates in the nucleus during S and G2 phases by its bipartite nuclear localization signal (NLS) sequence, which is crucial for Plk1 regulation during normal cell cycle progression. Here, through combined computational and experimental studies, we identified compound D110, which inhibits Plk1 kinase activity with an IC50 of 85 nm and blocks the nuclear localization of Plk1 during S and G2 phases. D110-treated cancer cells were arrested at mitosis with monopolar spindle, indicating the inhibition of the Plk1 kinase activity in cell. As D110 interacts with both the ATP site and the NLS in Plk1, it demonstrates good selectivity toward Plk2 and Plk3. The strategy of simultaneously inhibiting kinase activity and its subcellular translocations offers a novel approach for selective kinase inhibitor design.

Assessing placental corticotrophin-releasing hormone disruption by hexestrol in a cell model.

Studies have shown that corticotrophin-releasing hormone (CRH) and relaxin are associated with early delivery. Our lab previously has shown the mycotoxin zeranol increases placental CRH expression. The mycotoxin is used in the farming industry to promote cattle growth, and some synthetic hormones are also used for the same purposes. In order to complete the picture of these growth promoting agents, we attempted to examine the synthetic hormones on the placental gene expression in the current study. Among the tested compounds, hexestrol induced the CRH mRNA and protein expression at 100nM in JEG-3 cells. As signal transduction pathways have been described in the transcriptional control previously, the activations of several protein kinases were determined. P38, PKCß and JNK were activated upon hexestrol treatment. Since the P38-inhibitor SB203580 prevented hexestrol from inducing CRH in a subsequent experiment, P38 was likely involved in the transcriptional regulation. Electrophoretic mobility shift assay revealed an increase in the CRE binding activity in CRH promoter. This study showed that hexestrol exposure might be a concern for pregnant women.

Identification of acylthiourea derivatives as potent Plk1 PBD inhibitors.

Thiourea derivatives have drawn much attention for their latent capacities of biological activities. In this study, we designed acylthiourea compounds as polo-like kinase 1 (Plk1) polo-box domain (PBD) inhibitors. A series of acylthiourea derivatives without pan assay interference structure (PAINS) were synthesized. Four compounds with halogen substituents exhibited binding affinities to Plk1 PBD in low micromole range. The most potent compound (3v) showed selectivity over other subtypes of Plk PBDs and inhibited the kinase activity of full-length Plk1.

Discovery of Novel Polo-Like Kinase 1 Polo-Box Domain Inhibitors to Induce Mitotic Arrest in Tumor Cells.

Polo-like kinase 1(Plk1) is vital for cell mitosis and has been identified as anticancer target. Its polo-box domain (PBD) mediates substrate binding, blocking of which may offer selective Plk1 inhibition compared to kinase domain inhibitors. Although several PBD inhibitors were reported, most of them suffer from low cell activity. Here, we report the discovery of novel inhibitors to induce mitotic arrest in HeLa cells by virtual screening with Plk1 PBD and cellular activity testing. Of the 81 compounds tested in the cell assay, 10 molecules with diverse chemical scaffolds are potent to induce mitotic arrest of HeLa at low micromolar concentrations. The best compound induces mitotic arrest of HeLa cells with an EC50 of 4.4 µM. The cellular active inhibitors showed binding to Plk1 PBD and compete with PBD substrate in microscale thermophoresis analysis.

Discovery of Non-ATP-Competitive Inhibitors of Polo-like Kinase 1.

Polo-like kinase 1 (Plk1) is an evolutionarily conserved serine/threonine kinase, and its N-terminal kinase domain (KD) controls cell signaling through phosphorylation. Inhibitors of Plk1 are potential anticancer drugs. Most known Plk1 KD inhibitors are ATP-competitive compounds, which may suffer from low selectivity. In this study we discovered novel non-ATP-competitive Plk1 KD inhibitors by virtual screening and experimental studies. Potential binding sites in Plk1 KD were identified by using the protein binding site detection program Cavity. The identified site was subjected to molecular-docking-based virtual screening. The activities of top-ranking compounds were evaluated by in vitro enzyme assay with full-length Plk1 and direct binding assay with Plk1 KD. Several compounds showed inhibitory activity, and the most potent was found to be 3-((2-oxo-2-(thiophen-2-yl)ethyl)thio)-6-(pyridin-3-ylmethyl)-1,2,4-triazin-5(4H)-one (compound 4) with an IC50 value of 13.1 ± 1.7 µm. Our work provides new insight into the design of kinase inhibitors that target non-ATP binding sites.

Clinical features of 26 patients with H7N9 avian influenza and characteris-tics of viral gene / 中国感染控制杂志

Objective To evaluate the clinical,epidemiological,and viral molecular biology features of 26 patients infected with H7N9 avian influenza A virus. Methods Clinical and epidemiological data of 26 patients with con-firmed avian influenza A (H7N9)infection in 2013 and 2014 were collected,virus isolated from human and poultry were identified and typed through sequencing.Results Of 26 patients,fever and cough were the most common symptoms,all patients had pneumonia;20 patients (76. 92% )developed acute respiratory distress syndrome (ARDS);25 patients (96.15% )had leucopenia or normal leukocytes at the initial diagnosis;treatment with antivi-ral drugs was initiated in 25 patients at a median of 10 days after the onset of illness;10 patients (38.46% )died. Gene sequencing indicated Gln226Leu and Gly186Val substitutions in human virus H7 gene and the PB2 Asp701Asn mutation. Conclusion Acute respiratory system damage is the main clinical manifestation of avian influenza (H7N9)virus infection in humans,live poultry exposure is an important risk factor for H7N9 infection in humans, adaptive mutation occurred at partial site of avian virus gene,which can be more easily be spread from birds to hu-man and cause serious diseases,it is necessary to strengthen the pathogen monitoring.

Construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct the expression vector pLCK-CD69-IRES-EGFP that contains mouse cell surface activation protein CD69 and enhanced green fluorescent protein(EGFP),and to generate CD69 transgenic mice based on this vector.</p><p><b>METHODS</b>First, RNA was extracted from mouse lung tissue and cDNA was synthesized via reverse transcription. PCR primer was designed through the PubMed searching, then mouse CD69 DNA fragment was amplified with PCR. Second, this DNA fragment was subcloned to the pInsulater-LCK-IRES-EGFP plasmid and constructed the transgenic vector after the verification of nucleotide sequence. Third, the expression vector was then transfected into 293 T cells and its expression in 293 T cells was observed under fluorescence microscope. Last, microinjection was performed to transfer the expression vector pLCK-CD69-IRES-EGFP into fertilized eggs, which were implanted into pseudo-pregnant recipient mice. After birth the tail samples of the pups were obtained for the purpose of genotyping to determine the transgenic founders. Fluorescence microscope and flow cytometer were used to measure the expression of CD69 on cells.</p><p><b>RESULTS</b>The construction of the expression vector pLCK-CD69-IRES-EGFP was verified by enzyme digestion and DNA sequencing. The transfected 293 T cell showed expression of the protein under fluorescence microscope. Identification of PCR for the tail tissue of the pups confirmed the present of CD69 transgene and resting lymphocytes demonstrated the expression of CD69.</p><p><b>CONCLUSION</b>The construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice have been successfully processed, which lays a foundation of the solid pattern studies in inflammatory diseases.</p>

Analysis on evaluation tool for literature quality in clinical study / 中国针灸

The tools used for the literature quality evaluation are introduced. The common evaluation tools that are publicly and extensively used for the evaluation of clinical trial literature quality in the world are analyzed, including Jadad scale, Consolidated Standards of Reporting Trials (CONSORT) statement and Grades of Recommendations Assessment, Development and Evaluation (GRADE) system and the others. Additionally, the present development, updates and applications of these tools are involved in analysis.

Effects of trichloroethylene toxicity on normal human liver cells and hepatocytes with CYP2E1 gene overexpression / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effects of trichloroethylene (TCE) toxicity on the normal human liver cells (L02 cells) and hepatocytes with CYP2E1 gene overexpression which was constructed through molecular cloning technology in our laboratory, then to explore the roles of CYP2E1 gene in TCE toxicity.</p><p><b>METHODS</b>L02 cells and hepatocytes with CYP2E1 overexpression were treated with various doses of TCE (0,0.25, 0.5, 1.0, 2.0, 4.0 mmol/L) for 12h, the expression of apoptosis genes (Bcl-2, Caspase-3, Caspase-8, Caspase-9) and oncogenes (c-fos, c-myc, k-ras, p53) were determined by real-time fluorescent PCR.</p><p><b>RESULTS</b>Bcl-2 mRNA expression levels increased significantly in normal liver cells and CYP2E1-overexpressing cells after TCE treatment, Bcl-2 levels were 20%∼50%higher in CYP2E1-overexpressing cells than in L02 liver cells at doses of 0.25∼2.0 mmol/L TCE. Caspase-3, Caspase-8 and caspase-9 mRNA expression increased by 30%∼600% in CYP2E1-overexpressing cells at doses of 0.5∼4.0 mmol/L TCE when compared with L02 cells (P < 0.01). Additionally, c-fos, k-ras and c-myc mRNA expression levels were 25%∼120% higher in CYP2E1-overexpressing cells than in L02 cells (P < 0.01), p53 mRNA expression levels were lower 10%∼50% in CYP2E1-overexpressing cells than in L02 cells (P < 0.05 or P < 0.01).</p><p><b>CONCLUSIONS</b>There were significant differences for apoptosis gene and oncogene expression levels between normal liver cells and CYP2E1-overexpressing cells after they were treated with TCE, these findings indicated that CYP2E1 might play an important role in TCE metabolism in vivo.</p>

Detection of double mutant X antigen/antibody in sera of patients with hepatitis B virus infection and its implication / 中华传染病杂志

Objective To explore the correlation between serum hepatitis B virus (HBV) X antigen/antibody (HBxAg-wild/HBxAb-wild,and HBxAg-mutant/HBxAb-mutant) and the disease progression in patients with chronic HBV infection.Methods A direct enzyme immunosorbent asssay (ELISA) was performed to detect HBxAb using recombinant antigen,and a double antibody sandwich ELISA assay to detect HBxAg using monoclonal antibody and specific rabbit polyclonal antibody.HBxAg-wild/HBxAb-wild and HBxAg-mutant/HBxAb-mutant were tested in sera from cases at different stages of chronic HBV infection.A chi-square test was employed to examine statistical significance.Results The positive rates of HBxAg-wild and HBxAg-mutant in the chronic asymptomatic HBV carriers,chronic hepatitis,hepatitis B-related cirrhosis and liver cancer were 6.2％ (2/32),10.7％ (3/28),28.6％ (6/21),43.6％ (17/39) and 3.1％ (1/32),10.7％ (3/28),33.3％ (7/21),48.7％ (19/39),respectively.The positive rates of HBxAb-wild and HBxAb-mutant in the above mentioned groups were 6.2％ (2/32),21.4％ (6/28),38.1％ (8/21),53.8％ (21/39)and 6.2％ (2/32),25.0％ (7/28),42.9％ (9/21),61.5％ (24/39) respectively.The positive rates of HBxAg-wild and HBxAg-mutant were not significantly different among the above groups (x2 =0.871,0.780,0.565 and 0.317,respectively; all P＞0.05) ; The positive rates of HBxAb-wild and HBxAb-mutant were also similar among all the groups (x2 =0.780,0.709,0.580 and 0.210,respectively; all P＞0.05).The positive rates of HBxAg-wild,HBxAb-wild,HBxAg-mutant,HBxAb-mutant in patients with low viral loads (HBV DNA＜1 × 104 copy/mL) were 36.5％ (23/63),44.4％ (28/63),42.9％ (27/63) and 54.0％ (34/63),respectively,those in patients with high viral loads (HBVDNA≥1×104 copy/mL) were 8.8％ (5/57),15.8％ (9/57),5.3％ (3/57) and 14.0％ (8/57),respectively.The positive rates of HBxAg and HBxAb were significantly higher in cases with low viral loads than those with high viral loads (x2 =12.869,11.522,22.556 and 20.976,respectively; all P＜0.05).The positive rates of HBxAg-wild,HBxAb-wild,HBxAg-mutant,HBxAb-mutant in the HBeAg positive group were 21.7％ (18/83),30.1％ (25/83),22.9％ (19/83) and 32.5％ (27/83),respectively,while those in the HBeAg negative group were 27.0％ (10/37),32.4％ (12/37),29.7％ (11/37) and 40.5％ (15/37),respectively.No significant difference of HBxAg/HBxAb positive rates between HBeAg positive group and HBeAg negative group was noticed (x2 =0.408,0.064,0.638 and 0.722,respectively; all P＞0.05).Conclusions The antigenicity and specificity of HBV X protein remains similar after the occurrence of A1762T/G1764A double mutant in X gene.It is also found that the positive rates of HBxAg and HBxAb increase with disease progression.HBxAg/HBxAb might be promoting factors for tumorigenesis in chronic HBV infection.HBxAg and HBxAb might have negative influence on HBV replication.