Objective:To clarify the effect of ubiquitination hepatitis B virus core antigen (Ub-HBcAg) on dendritic cells (DC) autophagy, and to explore the mechanism of autophagy in enhancing DC antigen presentation and inducing hepatitis B virus-specific cytotoxic T lymphocyte (CTL) responses.Methods:Ub-HBcAg lentiviral vector (LV-Ub-HBcAg), lentiviral vector-hepatitis B virus core antigen (LV-HBcAg) and no-load plasmid LV (LV) were constructed and packaged. DC2.4 cells were divided into LV-Ub-HBcAg group, LV-HBcAg group and LV group. The blank control group (NC group) was also set. The protein expression of autophagy-related protein P62, microtubule associated protein 1 light chain 3 beta (LC3B), autophagy related 5(ATG5) and Beclin-1 were detected by Western blotting. The expressions of co-stimulatory molecules such as CD86, CD80 and major histocompatibility complex (MHC)-Ⅱ were detected by flow cytometry. Cell counting kit-8 (CCK-8) method was used to detect T lymphocytes proliferation. The non-radioactive lactic acid dehydrogenase (LDH) release method was applied to detect the killing ability of CTL. Statistical analysis was conducted by independent sample t test. Results:The relative protein expressions of LC3B-Ⅱ/LC3B-Ⅰ, Beclin-1 and ATG5 in NC group were 0.445±0.076, 0.522±0.026 and 0.761±0.038, respectively, which were all lower than those in LV-Ub-HBcAg group (0.926±0.021, 0.919±0.016 and 1.451±0.028, respectively). The relative protein expression of P62 in the NC group was higher than that in LV-Ub-HBcAg group ((1.875±0.016) vs (0.647±0.121)). The differences were all statistically significant ( t=6.102, 9.842, 17.490 and 10.590, respectively, all P<0.01). The expressions of CD86 (75.51%), CD80 (83.35%), MHC-Ⅱ (66.66%) in the LV-Ub-HBcAg group were high, and those in the NC group were 8.03%, 7.49%, 0.04%, respectively. The specific CTL killing rate ((65.310±2.091)%) of the LV-Ub-HBcAg group was significantly higher than both NC group ((14.400±0.497)%) and LV-HBcAg group ((54.870±1.443)%), and the differences were both statistically significant ( t=23.690 and 4.111, respectively, both P<0.05). Conclusion:Ub-HBcAg promotes the DC autophagy, up-regulates the expressions of costimulatory molecules on cell surface of DC to induce the maturation and activation, and then stimulates T lymphocyte to induce a stronger specific CTL response under the effort of ubiquitination.

Objective To observe the changes of calcitonin gene-related peptide(CGRP) -immunoreactive positive nerve fibers innervation in bone tissue of femoral heads during the pathological process of early steroid-induced avascular necrosis of the femoral head (SANFH), and explore its significance.Methpathological group of SANFH was induced.Immunohistochemical technique was used and the changes of CGRP-immunoreactive positive nerve fibers innervation in weight bearing area of the femoral heads during the pathological process of early SANFH were observed.Result The number of CGRP-immunoreactive nerves increased first and then decreased ( Peaked at 6 weeks, 10.28 ± 0.66 ), but it was more than that ofnormal control group.There was significant difference between two groups ( P ＜ 0.01 ).Conclusion There were changes in the distribution of CGRP-immunoreactive positive nerves fiber during the process of bone repair after SANFH.CGRP-immunoreactive positive nerves fiber might take part in the process of bone repair in SANFH.

Objective To observe the effects of cytoplasmic transduction peptide (CTP)-HBcAg18-27-Tapasin induced murine bone marrow-derived dendritic cell (DC) maturation on T lymphocyte proliferation in vitro,Methods Bone marrow derived DC isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleutin (IL)-4 for 5 days followed by lipopolysaccharide added to induce DC maturation.10 μg/L CTP-HBcAg18-27-Tapasin,50 μg/L CTP-HBcAg18-27-Tapasin,10 μg/L CTP-HBcAg18-27 or RPMI-1640 were added into culture medium to induce DC maturation.DC phenotypes were analyzed by flow cytometry.The level of IL-12p70 in the supernatant was detected by enzyme linked immunosorbent assay.The proliferation of.T lymphocytes was performed by using cell counting kit-8 and intracellular cytokine of proliferative T cells were analyzed by flow cytometry.The means among groups were compared using one-way ANOVA and those between two groups were compared by least significant difference test.Results DC were cultured and induced successfully.The molecules on DC surface,such as CD80,CD86 and major histocompatibility antigen-Ⅰ were upregulated by CTP-HBcAg18-27-Tapasin.IL-12p70 level induced by 50 μg/L CTP-HBcAg18-27-Tapasin was (61.12±10.25) pg/mL,which was higher than those induced by 10 μg/L CTP-HBcAg18-27-Tapasin (50.43±10.42) pg/mL,10μg/L CTP-HBcAg18-27 (40.17±8.54) pg/mL and medium control (30.51±8.03) pg/mL (F=15.85,P=0.030 and 0.037).The proliferation of T lymphocytes induced by CTP- HBcAg18-27 -Tapasin was higher than control groups.The amounts of cytotoxic T lymphocyte (CTL) induced by 50 μg/L CTP-HBcAg18-27-Tapasin [(2.05±0.41) ％] and 10 μg/L CTP-HBcAg18-27-Tapasin [(1.06 ±0.10 )％] were both significantly higher than the 10 μg/L CTP-HBcAg18-27 group [(0.45±0.11)％] and medium group [(0.09±0.02)％,F=60.22,P=0.003].Conclusions CTP HBcAg18- 27 Tapasin could promote the differentiation and maturation of DC,and enhance the ability of DC stimulating T lymphocytes proliferation and increase CTL expression effectively.

AIM: To Screen and identify human single-chain variable fragment (ScFv) specific to hepatitis B virus core protein and determine its gene sequence. METHODS: The recombinant phages were panned by HBcAg coated in a 96-pore plate and 48 clones were identified specific to HBc after three rounds of panning. The specificity of ScFv from the positive clone was determined by ELISA. Then, the soluble ScFv was expressed in E.coli. HB2151 and secreted in the supernatant. Subsequently, SDS-PAGE and dot blot were performed to identify the ScFv in the supernatant and cell lysate. The gene of ScFv specific to hepatitis B virus core protein was sequenced. RESULTS: The ScFv screened from phage antibodies has a specific combination character with hepatitis B virus core antigen. Soluble ScFv was confirmed to express in E.coli. HB2151 and secrete in the supernatant. The sequence of ScFv gene conformed to that of heavy chain and kappa chain of human immunoglubulin. CONCLUSION: Human ScFv specific to hepatitis B virus core protein has been identified by means of the phage display technology, and its gene sequence has been determined.

Objective: To study the influencing factors of fracture nonunion after intramedullary nailing for subtrochanteric fracture and construct a risk assessment model. Methods: A retrospective analysis was performed on 251 patients with intramedullary nail fractures of the femoral subtrochanteric fracture from February 2006 to January 2018. According to the different treatment time, the 251 patients included in this study were divided into the modeling group and the verification group. In the modeling group, postoperative fracture nonunion rate, general data, fracture related factors, surgical reduction related factors, mechanical and biological factors were calculated, and the influencing factors of fracture nonunion were screened by univariate analysis. Indicators with statistical differences in univariate analysis were analyzed using Logistic regression model for multivariate analysis to build the risk assessment model. The influencing factors were re-evaluated through the verification group, and the differentiation and calibration of the model were evaluated. Results: Fracture nonunion occurred in 34 of 149 patients in the modeling group. Among the 13 potential influencing factors, univariate analysis and logistic regression analysis showed that postoperative hip varus, intramedullary nail fixation failure and complete open reduction were the risk factors of fracture nonunion. Postoperative reduction of medial cortex was a protective factor for fracture nonunion, and a regression equation was established. Based on the logistic regression model, the Nomogram diagram was drawn. In the verification group, fracture nonunion occurred in 24 of 149 patients. The area under the ROC curve was AUC=0.883>0.7, indicating that there was a moderate differentiation to evaluate the occurrence of fracture nonunion after operation. The goodness of fit test: the H-L test (χ²=2.921, P=0.712) showed that the model had a good calibration. Conclusion The risk factors of fracture nonunion were hip varus, failure of intramedullary nail fixation and complete open reduction after intramedullary nailing of subtrochanteric fracture, and postoperative reduction of medial cortex was the protective factor. The risk assessment model has moderate differentiation and good calibration, which can provide reference for the risk assessment of fracture nonunion after subtrochanteric fracture operation.

OBJECTIVETo investigate the effect of protein transduction domain-hepatitis B virus core antigen (CTP-HBcAg18-27)-Tapasin fusion protein-induced specific cytotoxic T lymphocyte (CTL) response on hepatitis B virus (HBV) replication in HBV transgenic mice.

METHODSTwenty HBV-transgenic mice were randomly divided into two groups for a 3-week course of once weekly subcutaneous immunizations with either CTP-HBcAg18-27-Tapasin fusion protein or CTP-HBcAg18-27. Mice administered isotonic saline served as blank controls. Expressions of cytokines in splenocytes were analyzed by flow cytometry. Serum levels of hepatitis B surface antigen (HBsAg) and HBV DNA were determined by microparticle enzyme immunoassay and real-time fluorescent PCR assay, respectively. Expression of HBsAg in hepatic tissues was detected by immunohistochemistry.

RESULTSImmunization with 100 mug of CTP-HBcAg18-27-Tapasin fusion protein led to a significant increase in proportions of CTLs in spleen (2.70%+/-0.20% vs. 50 mug of CTP-HBcAg18-27-Tapasin: 1.66%+/-0.53%, 50 mug of CTP-HBcAg18-27: 1.26%+/-0.56%, and blank controls: 0.75%+/-0.71%; F = 741.45, P = 0.000) and up-regulation of inflammatory cells in hepatic tissue. In addition, both immunizations of CTP-HBcAg18-27-Tapasin led to significant decreases in serum HBsAg and HBV DNA levels compared to those in the CTP-HBcAg18-27 group.

CONCLUSIONHBV-related modification of the expression of the molecular chaperone Tapasin may affect its interaction with intracellular antigen peptides, thereby leading to increases the number of specific CTLs in the spleen, decreases in serum HBsAg and HBV DNA levels, and down-regulation of HBsAg expression in hepatic tissue. These results obtained in HBV-transgenic mice suggest that the CTP-HBcAg18-27-Tapasin fusion protein has anti-HBV activity.

Animals ; DNA, Viral ; blood ; Female ; Hepatitis B ; immunology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; physiology ; Male ; Membrane Transport Proteins ; genetics ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Virus Replication

Bladder cancer is a kind of urothelial cancer with high incidence and heterogeneity. From superficial bladder tumors to muscular invasive malignant tumors, due to their high-frequency recurrence and metastatic characteristics, they are inherently refractory. Although a comprehensive treatment with surgery as the main focus while chemotherapy, radiotherapy, and immunotherapy as a supplement has been formed, the limitations of chemotherapy regimens, the unpopularity of radiotherapy, and the low efficiency of immunotherapy, make clinical decision-making still difficult. Recently, a number of targeted therapies have emerged in bladder cancer and have shown good responses. Transient receptor potential (TRP) channel are novel therapeutic targets and current research hotspots in bladder cancer. This review discusses the anti-tumoral molecular mechanism of TRP channel in bladder cancer, its feasibility as a potential therapeutic target, and the prospects of drug combination to sensitize platinum-based chemotherapy.

Objective:To investigate the effects of antibiotic treatment and antibiotics combined with surgery treatment on the prognosis of patients with infective endocarditis (IE).Methods:The clinical data and prognosis of all patients diagnosed as IE discharged from Shanghai Jiao Tong University Affiliated Sixth People′s Hospital from June 2011 to May 2021 were collected. There were 240 IE patients, divided into antibiotic treatment group and the antibiotics combined with surgery group according to the treatment methods. The clinical characteristics and prognosis of the IE patients were compared between the two groups, so as to investigate the timing of surgery for IE patients and to analyze the effects of the two treatment methods on the prognosis of IE patients.Statistical analysis methods including Wilcoxon rank sum test, chi-square test, Kaplan-Meier survival analysis and Cox regression analysis were used when appropriate.Results:Of the 240 patients with IE, 63 cases were only treated with antibiotics and 177 cases were treated with antibiotics combined with surgery. After propensity score matching (PSM), one-year mortality rate of the IE patients in the antibiotics combined with surgery group was 11.1%(4/36), which was significantly lower than that in the antibiotic treatment group (33.3%(12/36), χ2=5.14, P=0.023). The median values of left ventricular ejection fraction (LVEF), left ventricular end diastolic diameter (LVEDD) and left ventricular fractional shortening (LVFS) in the antibiotics combined with surgery group were 59%, 47 mm and 31%, respectively, which were significantly lower than those before surgery (63%, 54 mm and 34%, respectively, Z=6.19, 9.36 and 6.11, respectively, all P<0.001). The most common surgical indication was moderate to severe heart failure, and there was no significant difference between the early operation group and the late operation group (both P>0.050). The one-year cumulative survival rate of antibiotics combined with surgery group was 94.9%, which was significantly higher than that in the antibiotic treatment group (83.2%, χ2=7.38, P=0.007). Heart failure and Pitt bacteremia scores≥4 were the independent risk factors for one-year all-cause death of the IE patients (hazard ratio ( HR)=5.668 and 19.392, respectively, both P<0.050). Hospital days and antibiotics combined with surgery were independent related factors for reducing the risks of one-year all-cause death ( HR=0.931 and 0.299, respectively, both P<0.050). Pitt bacteremia scores≥4 had the greatest impact on one-year prognosis of the IE patients. Conclusions:Surgery could significantly improve cardiac function and one-year prognosis of the IE patients. IE patients with heart failure and Pitt bacteremia score≥4 should be actively treated.

Objective To observe the metabolic changes of subcortical structures in children with intractable epilepsy using 18 F-FDG PET/CT,and to investigate the mechanism of subcortical structure involvement in epileptic seizures and its clinical significance.Methods Features of 18F-FDG PET/CT imaging in 611 intractable epilepsy children were analyzed.The metabolic changes of cortex and subcortical structures (basal ganglia,thalamus and cerebellum) were observed.The children were divided into three groups (young,middle and older groups) according to age,also mild group and severe group according to the number of involved lobar,respectively.The incidence of metabolic abnormalities in subcortical structures of different groups were analyzed.Results Among 611 children,unilateral cortical metabolic abnormality was found in 525,and bilateral cortical metabolic abnormalities were found in 86 children.The involvement of subcortical structures was detected in 190 children,including basal ganglia (n=64),thalamus (n=113) and cerebellum (n=105).The incidence of metabolic abnormality in subcortical structures under different age groups was not statistically different (all P＞ 0.05),while the incidence of metabolic abnormality in subcortical structures of severe group was significantly higher than that of mild group (all P＜0.001).Conclusion 18 F-FDG PET/CT might be able to detect the metabolic abnormalities of subcortical structures,therefore indicating the involvement of cerebral cortex.

Objective: To observe the expression levels of PD-1/PD-L1 costimulatory molecules and explore the clinical significance in patients with chronic lymphocytic leukemia (CLL) . Methods: The expression of PD-1/PD-L1 in peripheral blood CD8⁺ T cells, CD4⁺T cells, CD19⁺B, and dendrites cells (DC) was detected by flow cytometry in 57 CLL patients and 20 healthy controls. The correlations of PD-1/PD-L1 expression with disease stage, CD38 expression, ZAP-70 expression, chromosome karyotype abnormality and β₂-MG expression were analyzed. Results: ①Compared with control, CLL patients, including 39 males and 18 females with the median age of (63.7±10.7) years, had no statistically significant difference in age and gender (P>0.05) . CLL patients had the higher PD-1/PD-L1 expression than healthy controls (P<0.05) . ②In Rai staging, the later the stage, the higher expression of PD-1/PD-L1 (P<0.05) . ③PD-1 expression in CD8⁺CD38⁺ group (11 cases) was higher than that in CD8⁺CD38^- group (46 cases) (P=0.004) , and CD8⁺ poor prognosis chromosome group (14 cases) also had significant higher PD-1 expression than CD8⁺good prognosis chromosome group (28 cases) (P=0.004) . ④The expression of PD-L1 was higher in CD38⁺group, ZAP-70⁺group, and poor prognosis group, as compared to that in CD38^-group (P=0.002) , in ZAP-70^-group (P<0.001) , in good prognosis group (P=0.023) . There was no correlation between the expression of PD-1/PD-L1 and β₂-MG (P>0.05) . Conclusion This data reveals that PD-1/PD-L1 was highly expressed in CLL patients. Its expression levels were correlated with Rai stage, CD38, ZAP-70, chromosome karyotype, but not with β₂-MG. PD-1/PD-L1 may be a prognostic factor in patients with CLL.