ObjectiveTo investigate the awareness and clinical practice of guidelines for the prevention and treatment of chronic hepatitis B （2022 edition） among clinicians. MethodsFrom July 19 to December 31， 2024， a self-designed electronic questionnaire was distributed via the WeChat mini program to collect related data from 1 588 clinicians nationwide， including their awareness and practice based on 18 questions regarding testing and referral， diagnosis and treatment， and follow-up. ResultsAmong all respondents， only 350 clinicians correctly understood all the updated key points of antiviral indications and treatment for special populations in the 2022 edition of guidelines for the prevention and treatment of chronic hepatitis B， with an overall awareness rate of 22.0%. Only 20% ‍—‍ ‍40% of the patients with positive HBV DNA and an age of >30 years receive antiviral therapy， while 80% ‍—‍ ‍100% of the patients with positive HBV DNA and a family history of hepatitis B cirrhosis or hepatocellular carcinoma receive antiviral therapy. The median follow-up rates at 1 year， 3 years， and 5 years were 67.5% 57.5% and 47.5%，respectively， showing a trend of gradual reduction， which might be associated with the influencing factors such as insufficient time for follow-up management by clinicians， insufficient awareness of the disease among patients， and poor adherence to follow-up. ConclusionThere is a gap between the awareness and practice of guidelines for the prevention and treatment of chronic hepatitis B （2022 edition） among clinicians. It is recommended to further strengthen training and focus on the whole process of “detection， diagnosis， treatment， and management” for patients with chronic hepatitis B in healthcare institutions， in order to promote the implementation of the guidelines.

Objective:To establish a noninvasive prediction model for liver fibrosis in chronic hepatitis B (CHB) virus infection patients with alanine aminotransferase (ALT) less than upper limit of normal level.Methods:A total of 183 CHB patients with ALT <40 U/L admitted in the First Affiliated of Wenzhou Medical University from January 2014 to September 2017 were enrolled. There were 149 cases of non-marked liver fibrosis (F0-F2) and 34 cases of marked liver fibrosis (F3-F6) according to the Ishak scoring system. The clinical data, liver stiffness measurement (LSM), liver ultrasound imaging, serum biochemical features and hepatitis B virus related indexes of patients were retrospectively analyzed. The independent predictors of liver fibrosis were screened and a prediction model was constructed based on the results of multivariate logistic regression analysis. The receiver operating characteristic (ROC) curve was applied to evaluate the established model and other indicators in predicting liver fibrosis.Results:Multivariate logistic regression analysis showed that LSM ( OR=1.327, 95% CI 1.149-1.531), liver ultrasound scores ( OR=6.610, 95% CI 2.704-16.156) were independent predictors of liver fibrosis. The area under ROC curve(AUC)of the established model (LU) in predicting liver fibrosis was 0.873(95% CI 0.799-0.947), which was significantly greater than that of the ultrasound score, LSM, FIB-4, APRI and GPR (AUC=0.790, 0.804, 0.654, 0.673 and 0.770; Z=3.394, 1.982, 2.077, 3.168 and 2.165, all P<0.05 or <0.01). With the cut-off value of -1.787, the sensitivity and specificity of LU model were 85.3% and 77.2%, respectively. Conclusion:The established model (LU) can effectively predict the liver fibrosis in CHB patients with ALT less than upper limit of normal.

Objective:To investigate the prenatal genetic testing for suspected Beckwith-Wiedemann syndrome (BWS) to improve its prenatal diagnosis rate.Methods:This study reported a pregnant woman, who had a pregnant history of termination due to the same reason at 18 weeks, with fetal acromphalus and unusually thickened placenta indicated by ultrasound examination at 13 weeks of gestation. After chorionic villus sampling, single nucleotide polymorphism (SNP) array was used to analyze copy number variations in the whole genome, and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was also performed to detect the methylation and copy number variations in H19 and KCNQ1 genes on chromosome 11p15. Peripheral blood samples were collected from the couple for chromosome G-banding karyotype analysis and SNP array. Results:The SNP array indicated a 176 kb heterozygous deletion in the 11p15.5 region. MS-MLPA revealed a loss of methylation at imprinting control region 2 and a 50% reduction of copy numbers of KCNQ1 (L02903) gene. No abnormality was found in the parents in the SNP array and G-banding karyotype analysis. The fetus was prenatally diagnosed with BWS. Conclusions:When intrauterine abnormalities, such as acromphalus and abnormal thickening of the placenta, are found by ultrasound during early pregnancy, prenatal genetic tests related to BWS, including MS-MLPA and SNP array, are suggested to avoid a missed diagnosis of BWS.

Objective To study the changes of immune function of endotoxin tolerant dendritic cell (ETDC) and to observe its effect on sepsis in mouse model.Methods ETDC were prepared by pretreated bone marrow dendritic cells derived from BALB/c mice with lipopolysaccharide stimulation.The cells were collected and the expressions of surface markers including major histocompatibility complex ( MHC)Ⅱ, CD86 and CD11c were detected by flow cytometry.The proliferation rate of T lymphocytes was evaluated by cell counting kit-8 and the concentrations of cytokines in the supernatant were detected by enzyme linked immuno sorbent assay.Afterwards, 36 mice were randomly assigned into 4 groups.The blank control group did not receive any treatment, the sham-operated group underwent simple incision suture, the sepsis group and ETDC reinfusion group underwent cecal ligation and puncture to establish sepsis.Before sepsis model establishment, 0.9% sodium chloride solution or suspension of ETDC and 0.9%sodium chloride were reinfused by tail vein.The serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), tumor necrosis factor (TNF)-α, interleukin(IL)-6 and IL-10, and the proportion of help T cell ( Th) 17/regulatory T cell ( Treg) in spleen of each group were detected.The pathological manifestations of liver, kidney and ileum in each group were observed.T test and χ2 test were used for comparisons between groups.Results The results of flow cytometry showed that MHCⅡ, CD86 and CD11c of ETDC were 70.4%, 43.1%, and 73.1%, respectively, which were significantly lower than those of mature dendritic cell (mDC) (96.1%, 89.5%, and 84.6%, respectively) (χ2 ＝56.47, 83.78, and 23.29, respectively, all P＜0.01).The concentrations of IL-10, TNF-αand IL-6 in the supernatant of ETDC were (978.04 ±56.70), (980.34 ±111.96) and (12 743.03 ±865.81) ng/L, respectively, and those of mDC were (741.35 ±99.23), (1 703.11 ±117.00) and (19 052.28 ±1 145.84) ng/L, respectively.The differences were all statistically significant (t＝5.073, 10.93, and 10.76, respectively, all P＜0.01).The proliferation rates of T lymphocytes co-cultured with ETDC in 1∶5 and 1∶10 ratio group were (676.95 ±85.99)%and (514.00 ±106.39)%, respectively, which were lower than those of the mDC group (956.25 ±127.12)%and (772.07 ±214.08)%, respectively.The pathological injuries of liver, kidney and ileum in ETDC treatment group were significantly lighter than those in sepsis group.Serum ALT and AST levels in the ETDC reinfusion group were (299.71 ±36.91) and (690.39 ±154.92) U/L, respectively, and TNF-α, IL-6 and IL-10 were (0.067 ±0.005), (0.428 ±0.051) and (0.058 ±0.005) ng/L, respectively.Serum ALT and AST levels in the sepsis group were (620.67 ±69.27) and (1 430.17 ±134.05) U/L, respectively, and TNF-α, IL-6 and IL-10 were (0.085 ±0.007), (0.774 ±0.088) and (0.036 ±0.005) ng/L, respectively.The differences were all statistically significant (t＝11.60, 10.96, 5.991, 8.657, and 8.04, respectively, all P ＜0.01).The proportion of Treg/Th17 in the ETDC reinfusion group was 23.4%, and that in the sepsis group was 60.8%(χ2 ＝28.69, P＜0.01).Conclusion The reinfusion of ETDC has a protective effect on sepsis in mouse model, which may play a negative immune regulatory role by regulating the differentiation of T cells.

Objective To investigate the therapeutic effects and mechanisms of interleukin-10 (IL-10) gene-modified dendritic cells (DC-IL-10) in mice with liver fibrosis. Methods DC-IL-10 was constructed in vitro, the phenotype and function of which were evaluated by flow cytometry. BALB/c mice were treated with intraperitoneal injection of carbon tetrachloride(CCl4)to establish liver fibrotic model. DC-IL-10 was administrated via tail vein. Animals were divided into 4 groups including normal dendritic cell (DC) control , liver fibrosis only, negative lentiviral transfection DC (DC-mock) and DC-IL-10. Liver function, cytokine secretion, T lymphocyte differentiation and liver histomorphology were tested. Real-time PCR and western blot were used to analyze the effect of DC-IL-10 on Wnt/β-catenin signaling pathway and its role in liver fibrosis. Results When compared with DC control and DC-mock, the expression of DC-IL-10 surface stimulating molecules (major histocompatibity complex-Ⅱ, CD80, CD86) were significantly decreased (F=14.708, 22.503, 12.595, respectively, all P<0.05), and DC-IL-10 significantly inhibited T lymphocyte proliferation (F=50.295, P<0.05). When compared with liver fibrosis group, serum alanine aminotransferase and aspartate transaminase were decreased in DC-IL-10 treated group (all P<0.05), other parameters including inflammatory factors (tumor necrosis factor α, IL-6, IL-1β) reduced (all P<0.05), the proportion of regulatory T cells (Treg) increased (F=6.742, P<0.05), pathological damage improved, the expression of Wnt3a, α-SMA and β-catenin mRNA and protein significantly reduced in DC-IL-10 treatment group(all P<0.001). Conclusions DC-IL-10 induces elevation of Treg for immune tolerance, as well as inhibition of inflammatory response, block of Wnt/β-catenin signaling pathway, which translates into improvement of liver fibrosis.

Objective To investigate the impact of suppressor of cytokine signaling 1 (SOCS1) overexpression on dendritic cells (DC) functions and its therapeutic effect on acute liver failure (ALF) in mice.Methods Bone marrow derived dendritic cells (BMDC) from C57BL/6 mice were transfected with lentivirus encoding SOCS1 and negative control lentivirus at a MOI=50, and labeled as DC-SOCS1and DC-VNG, respectively after 96 hours of successful transduction.Then DCs were stimulated with lipopolysaccharides(LPS)1 mg/L and collected for flow cytometry analysis of surface costimulatory molecules, allogeneic mixed lymphocyte reaction (MLR) and western blot test of Janus kinase (JAK)/signaling transducers and activators of transcription (STAT) pathway.Afterwards, 90 mice were randomly assigned into 4 groups including 12 in normal control group, 26 in ALF group, 26 in treatment groups with DC-SOCS1 and 26 with the treatment of DC-VNG.All were received tail vein injection with normal saline, modified DC-VNG and DC-SOCS1 suspended in normal saline, respectively.Twelve hours after injection, LPS (10 μg/kg)/D-GaIN (600 mg/kg) were injected intraperitoneally to induce ALF model.The mortality, serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), liver pathology and proportion of splenic regulatory T cells of each group were observed.Means in different groups were compared with one-way ANOVA analysis.Categorical variables were analyzed with x2 test.Variables were examined with normality test and homogeneity of variance with LSD test.Results The results of mixed lymphocyte reaction (MLR) revealed that T cell proliferation ratio in DC-SOCS1 group with mixture ratio of 100∶1 were (25.87±0.38)%, which was lower than that of mixture ratio of 10∶1 in the mDC group ([84.29±3.25]%) with statistical significance (x2=49.821, P<0.01);interleukin (IL)-10 concentration was higher than that in mDC group with mixture ratio of 10∶1 with statistical significance (F=20.112, P<0.05);IL-6 concentration was also lower with statistical significance (F=47.718, P<0.05).Compared to imDC, expression of JAK2 (t=0.525,0.523 and 0.489, respectively, all P<0.01), signal transduction factors and activation of transcription factors-1 (STAT1) (t=0.442,0.400 and 0.402, respectively, all P<0.01) and SOCS1 (t=0.322,0.363 and 1.090, respectively, all P<0.01) of mDC, DC-VNG and DC-SOCS1 after LPS stimulation increased significantly.Furthermore, the expressions of phosphorylated STAT1 (p-STAT1) and phosphorylated JAK2 (p-JAK2) of DC-SOCS1 were much lower than those of the mDC, with statistically significant difference (t=-3.840 and 0.254, respectively, both P<0.01).Pathological analysis revealed that there existed moderate hepatic cells necrosis and less immune cell infiltration in DC-SOCS1 group accompanied with higher regulatory T lymphocytes proportion than those in ALF group and DC-VNG group.Survival rate of ALF with DC-SOCS1 treatment group was significantly higher than that of ALF group with statistical difference (x2=12.87, P<0.05).Conclusions DC-SOCS1could sustain an immature state and exhibit as regulatory DC through negative regulation of JAK2/STAT1 pathway with overexpression of SOCS1.Infusion of DC-SOCS1 could ameliorate ALF by inhibiting aggressive inflammation response with increased proportion of regulatory T cells in mice, which shows good therapeutic effect for ALF mice.

[Objective] To describe a case of a rare,novel mutation causing recurrent chorioamniotic membrane separation in a Chinese family with combined next-generation sequencing (NGS) and Sanger sequencing.[Methods] For the affected fetus,potential mutation were detected by the conbinedcombined next-generation sequencing (NGS) and Sanger sequencing.And the prenatal diagnosis were identified by Sanger sequencing.[Results] A frameshifting mutation c.1389_1390delAG (inherited from mother),and a missense mutationc.1006 G ＞ C (inherited from mother) have been identified in the affected fetus (the second pregnancy).The prenatal diagnosis of the third fetus turns out to be a carrier,the mutation was inherited from father.[Conclusions] We describe a novel mutation in gene ZMPSTE24,which was considered with mandibuloacral dysplasia with type B,and that may be the cousecoursecausing of recurrent chorioamniotic membrane separation.This rare mutation constitutes an additional heterogeneous defect causing chorioamniotic membrane separation.And the conbinedcombined next-generation sequencing (NGS) and Sanger sequencing allows high resolution characterization of novel mutions that are not readily detected by present methods.

Objective To analyze the fetus chromosome abnormalities in women with advanced maternal age under the universal two-child policy. Methods A total of 10607 women underwent prenatal diagnosis proce-dures from the year of 2015 to 2016,among which 3569 cases were with advanced maternal age. Cytogenetic karyotyping was conducted with chromosomal microarray analysis(CMA)in 854 cases. The ration of chromosome abnormalities in the two population groups(aged from 35~39 and over 40)were counted. The type of abnormali-ties were also analyzed. Results The ration of women with advanced maternal age in 10607 cases underwent pre-natal diagnosis between 2015 and 2016 was 33.6%. The respective rations were 27.2%and 37.9%. The detection rates of chromosome abnormalities were 7.9% and 10.8%,with significantly difference. The significant differences were also found in the detection rate of chromosome aneuploidies ,but no differences in other chromosome abnor- malities. CMA was benefit to detect the micro chromosome abnormalities. Conclusions In 2016,the number of prenatal diagnosis procedures increased under the two-child policy. The detection rate of chromosome abnormalities also significantly increased. The incidence of chromosome abnormalities was higher with the growing age of preg-nant women. Genetic counseling must be presented and prenatal diagnosis should be promoted in women with ad-vanced age.

Objective This study aimed to investigate the effect of splenic CD11clow CD45RBhigh dendritic cell (DC)derived from endotoxin tolerance (ET)mice on the expression of zinc finger protein A20 in acute liver failure (ALF)and to clarify the possible mechanism.Methods ET mice were modeled. CD11clow CD45RBhigh DC were isolated from spleen by magnetic activated cell sorting (MACS).One hundred and twenty-six healthy male BALB/c mice were randomly divided into four groups:control group (group A,n=6),ALF group (group B,n =40),normal CD11clow CD45RBhigh DC-treated group (group C,n=40),ET-CD11clow CD45RBhigh DC-treated group (group D,n=40).Mice in group B,C and D were injected with D-galactosamine (D-GalN)600 mg/kg and lipopolysaccharides (LPS)10 μg/mouse.Mice in group A were given the same volume of normal saline (NS).Half an hour after the D-GalN/LPS injection,mice in group C were treated with splenic CD11clow CD45RBhigh DC derived from normal mice (1 ×10 6/mouse,0.2 mL/mouse).Mice in group D were treated with splenic CD11clow CD45RBhigh DC derived from ET mice (1 × 10 6/mouse,0.2 mL/mouse).Mice in group A and B were given the same volume of 0.9% NaCl solution (0.2 mL/mouse).Alanine aminotransferase (ALT)and aspartate aminotransferase (AST)levels were measured at each time point.Liver histopathological changes were confirmed by hematoxglin and eosin methods.Expressions of tumor necrosis factor-α (TNF-α),nuclear factor-kappa B (NF-κB),and zinc finger protein A20 were measured by reverse transcriptase polymerase chain reaction(RT-PCR)and Western blot.One-way analysis of variance was used to compare means between groups.Normal distribution and homogeneity of variance were tested.LSD test was conducted in patients accorded with homogeneity of variance.Results ALT and AST levels increased 2 h after modeling in group B and peaked at 24 h,which were significantly higher than groups A (t = 31 .00, 11 .52,both P <0.05).ALT and AST levels also increased after 2 h after modeling and peaked at 24 h in group C and group D,which were both significantly higher than group B (t =14.60,26.43,both P <0.05).The mRNA levels and protein expressions of TNF-αand NF-κB in group B increased gradually and peaked at 12 h after D-GalN/LPS injection.Compared to that of group A,the differences were both statistically significant (t = 427.58,122.42,179.35 ,165 .98,all P < 0.05 ).The mRNA level and protein expression of zinc finger protein A20 in group B decreased gradually and reached the minimum at 12 h after D-GalN/LPS injection,which was statistically different compared to group A (t = 90.80, 160.43,both P <0.05).On the contrary,the levels of zinc finger protein A20 in group C and D increased gradually and peaked at 12 h after D-GalN/LPS injection.The expression level of zinc finger protein A20 in group D was significantly higher than group C (t = 11 .21 ,24.80,both P < 0.05 ).Conclusion Treatment of splenic CD11clow CD45RBhigh DC derived from ET mice contributes to liver protection against D-GalN/LPS-induced ALF.

Objective To isolate and culture splenic CD11clow CD45RBhigh dendritic cells (DC) derived from endotoxin tolerance (ET) mice and investigate its biological characterization.Methods Mice weighed 20 to 25 gram were completely randomized into two groups including ET group and control group with 6 each.ET mice were modeled by intraperitoneal injection of low-dose lipopolysaccharide (LPS) for several days (pretreated with LPS 0.1 μg/mouse for 5 d).Mice in control group were given the same volume of normal saline (NS).CD11clowCD45RBhighDC were isolated from spleen by magnetic activated cell sorting (MACS).The immunological phenotypes were detected by flow cytometry.The suppressive capacity of CD11clow CD45RBhigh DC was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in allogenic mixed T cells reaction.The expressions of interleukin (IL)-10 and IL-12 produced by CD11clow CD45RBhigh DC were measured by enzyme-linked immunosorbent assay (ELISA).Statistical significance was analyzed through one-way analysis of variance (ANOVA).The homogeneity of variances was detected by Levene test.If variances were homogeneous,the least significant difference (LSD) test was used.If not,Dunnett T3 test was applied.Results The consistence of CD1 1 clow CD45RBhigh DC in control group was 30 ％,reaching the amount of (5.30±0.12) × 105/mouse ;In ET group,the percentage of CD11clow CD45RBhighDC achieved 80 ％ and the production was (1.20 ± 0.13) × 106/mouse the difference was statistically significant (t=3.23,P＜0.01).The cellar morphology in two groups showed no obvious difference.Compared to expression levels of all cell phenotypes (histocompatibility complex-Ⅱ,CD40 and CD80) in normal mice,the cell surface expression levels of CD11clowCD45RBhigh DC in ET mice were much lower.The difference in two groups was statistically significant.Splenic CD11clowCD45RBhighDC derived from ET mice with cell concentration of 1∶ 10,1∶50and 1∶100 had more obvious prohibitory effects on allogenic T cells (t1∶0 =1.36,P1∶10 ＜0.01,t1∶50 =2.49,P1∶50 ＜0.01,1∶100 =1.88,Pm00 ＜0.01).Secretion of IL-10 produced by CD11clowCD45RBhighDC of ET mice was significantly increased (t1∶0=13.63,P1∶10 ＜0.01,t1∶50 =13.45,P1∶50 ＜0.01,t1∶00 =9.31,P1∶00 ＜0.01),but the expression of IL-12 was lower (t1∶0 =2.62,P1∶0 =0.02,1∶∶50 =2.74,P1∶0=0.02,t1∶100 =2.99,P1∶100 =0.01).Conclusion Splenic CD11clow CD45RBhigh DC from ET mice have weaker ability of antigen presenting and allogeneic lymphocytes proliferation stimulating than those from normal mice.