ObjectiveTo assess the efficacy of herpes virus entry mediator (HVEM) gene modifled dendritic cells (DCs) in protecting against myosin induced myocarditis,and to investigate the involving mechanism.MethodsWe treated experimental autoimmune myocarditis (EAM) mice with myosin-pulsed DCs which were transfected with HVEM-expressing adenovirus (Ad-HVEM) or control vectors,then evaluated myocarditis,plasm cTn [ and autoantibody by histopathology,fluoroimmunoassay,and ELISA,respectively.ResultsWe found that DCs transfected with Ad-HVEM (DC-Ad-HVEM) could protect against EAM.Further study showed DC-Ad-HVEM could produce regulatory cytokine IL-10,and IL-10 promoted the production of a key regulatory T cell subset which is important in peripheral tolerance.The T cells mediated protection against EAM.ConclusionThis study suggest that myosin-DC-Ad-HVEM cell gene therapy is a safe and effective way for inhibiting the development of EAM,and the signal net mediated by HVEM plays different roles in different cells.

Objective To assess the sequence variations in preS/S regions of occult hepatitis B virus (OHB) and their relationship to severe chronic hepatic injury. MethodsWe collected samples from HBsAg negative patients, and evaluated their HBV-DNA by nest-PCR. HBV-DNA positive samples were used for analysis of preS/S region by PCR sequencing. Results Sixty-nine cases with HBV-DNA were identified in 468 cases without HBsAg. The positive percents were 16％, 8.7％, 36.4％, 18.3％ and 0％in group of only HBcAb positive, only HBeAb positive, only HBeAg positive, both HBcAb and HBsAb positive and all indexes negative, respectively. The level of HBV-DNA of OHB was significant lower than that in HBsAg positive patients. Compared with HBsAg positive controls, preS/S deletion, M1I and Q2K in preS2 region, Q129N/R/P,G185R and S210R in S region were more common in OHB. Moreover, M1I and Q2K in preS2 region, G185R and S210R in S region in OHB with severe chronic hepatic injury were more common that those in OHB without severe chronic hepatic injury. Compared with HBsAg positive patients with severe chronic hepatic injury, the level of HBV-DNA was lower, while the frequency of M1I and Q2K mutation in preS2 region, G185R and S210R in S region were more common in OHB patients with severe chronic hepatic injury. ConclusionThe virological factors were different between OHB and HBsAg positive patients. The M1I and Q2K in preS2 region, G185R and S210R in S region might be useful for prognosis evaluation of OHB patients.

Objective:To evaluate the effect of lung recruitment maneuvers combined with individualized positive end-expiratory pressure(PEEP) on the degree of postoperative atelectasis in elderly patients undergoing laparoscopic surgery.Methods:One hundred and forty-three elderly patients, aged ≥65 yr, with body mass index of 18.5-30.0 kg/m 2, scheduled for elective laparoscopic surgery, were assigned to either individualized PEEP combined with recruitment maneuvers (group Ⅱ) or fixed PEEP (group Ⅰ) using a random number table method. PEEP was maintained at 6 cmH 2O starting from the beginning of procedure until the end of the procedure in group I. Individualized PEEP titration was performed after induction of anesthesia in group Ⅱ. The primary outcome measure was the 12-zone lung ultrasound score at 15 min after tracheal extubation. Other outcome measures were the occurrence of postoperative pulmonary complications within 7 days after surgery, Quality of Recovery-15 scale score on 3rd day after surgery, rate of unplanned admission to intensive care units, length of hospital stay, incidence of intraoperative hypoxemia, usage rate of intraoperative vasoactive drugs, and incidence of postoperative hypotension. Results:Compared with group Ⅰ, the lung ultrasound score, driving pressure and postoperative pulmonary complications were significantly decreased, the dynamic lung compliance was increased ( P<0.05 or 0.01), and no significant changes were found in the other parameters in group Ⅱ ( P>0.05). Conclusions:Individualized PEEP combined with recruitment maneuvers can reduce the degree of postoperative atelectasis in elderly patients undergoing laparoscopic surgery.

Objective To analyze changes in the percentages of circulating follicular helper T (Tfh) cells and Tfh subsets in patients with systemic lupus erythematosus (SLE) for better understanding their relationships with SLE , and to investigate effects of steroids on circulating Tfh cells .Methods Pe-ripheral blood mononuclear cells (PBMCs) from 27 patients with SLE (including 10 inactive patients and 17 active patients ) and 21 sex-and age-matched healthy donors were analyzed by flow cytometry to detect the percentage of CD4+CD45RA-CXCR5+Tfh-like cells.Disease activity and the concentration of anti-double-stranded DNA ( anti-dsDNA) antibody were evaluated by SLEDAI score ( SLE disease activity index ) and ELISA, respectively.PBMCs from healthy donors were treated with or without prednisone to evaluate its effects on circulating Tfh cells .Twelve patients with SLE were treated with high-dose steriods ( 200-500 mg/d, 2-3 d) and the percentages of circulating Tfh cells and Tfh subsets in them were analyzed before and after treatment .Results No significant difference in the percentage of circulating Tfh cells was ob-served between patients with SLE and healthy donors (P>0.05), but the percentage of Tfh17 cells in pa-tients with SLE was significantly higher than that in healthy donors (P<0.05).Compared with patients with inactive SLE and healthy donors , patients with active SLE had a lower percentage of Tfh 1 cells (P<0.05).Moreover, the percentage of Tfh1 cells was negatively correlated with SLEDAI score (r=-0.44, P<0.05). The percentage of Tfh2 cells in anti-dsDNA antibody-positive group was significantly higher than that in anti-dsDNA antibody-negative group (P<0.05).In vitro treatment of PBMCs from healthy donors with predni-sone could significantly down-regulate the percentage of circulating Tfh (P<0.01), Tfh1 (P<0.05) and Tfh2 cells (P<0.01), and up-regulate the percentage of Tfh17 cells (P<0.01).In vivo treatment of pa-tients with SLE with steriods could significantly down-regulate the percentage of circulating Tfh (P<0.01), Tfh1 (P<0.05) and Tfh2 cells (P<0.01) and up-regulate the percentage of Tfh17 cells (P<0.01).Con-clusion Abnormal distribution of Tfh subsets is correlated with SLE disease activity and anti -dsDNA anti-body .Steroids in the treatment of SLE could affect the percentage of circulating Tfh cells and the distribution of Tfh subsets .

Objective To investigate the phenotypic and functional properties of in vitro-induced He-lios+regulatory T cells(Helios+iTreg) and to analyze the differences between Helios+iTreg and Helios+thymic-derived natural Treg (Helios+nTreg). Methods CD4+CD25- effector T cells (CD4+CD25- Teff) that were separated from healthy subjects were cultured with anti-CD3 and anti-CD28 antibodies and stimulated with IL-2 and TGF-β to induce the generation of Helios+iTreg. Flow cytometry and real-time PCR were respectively used to analyze the differences in phenotype and CpG methylation in Treg-specific demethylated region (TSDR) be-tween Helios+iTreg and Helios+nTreg derived from the same donor. Results CD45RA was highly expressed on Helios+iTreg, but not on Helios+nTreg. Additionally, Helios+iTreg expressed significantly higher levels of CD127,ICOS and PD-1,but lower levels of CXCR3,CCR4 and CD25 than Helios+nTreg did. IFN-γ and IL-2 that expressed by Helios+iTreg were more than those by Helios+nTreg. It was also found that the level of CpG methylation in TSDR was much higher in Helios+iTreg than in Helios+nTreg. Conclusion Both nTreg and iTreg expressed Helios,but the phenotypic and functional properties of the two Treg subsets were different. It was suggested that Helios+iTreg and Helios+nTreg might play different roles in the immune system.

Objective:To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion.Methods:Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay.Results:The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin ( r2: 0.438, 0.695, and 0.736, respectively, all P＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P=0.034). Traf4 -/- cells had a weakened proliferative capacity than traf4+/+ cells and formed tumors with smaller size ( P＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4 -/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4+/+ cells [(62.3±10.3)%, P=0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4 -/- cells were lower than those of the traf4+/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions:TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

Objective:To analyze the mutations of globin genes among patients suspected for thalassemia from the Shanghai area.Methods:A total of 4 644 patients diagnosed at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine between June 2016 and December 2019 were selected as the study subjects. The patients were tested for common mutations associated with thalassemia gene by Gap-PCR and reverse dot blotting (RDB). Patients were suspected to harbor rare mutations based on the inconsistency between hematological phenotypes and results of common mutation detection, and were further analyzed by Gap-PCR and Sanger sequencing.Results:Among the 4 644 patients, 2 194 (47.24%) were found to carry common thalassemia mutations, among which 701 (15.09%) were α-thalassemia, 1 448 (31.18%) were β-thalassemia, and 45 (0.97%) were both α- and β-thalassemia. Forty six samples were found to harbor rare mutations, which included 17 α-globin gene and 29 β-globin gene mutations. CD77(CCC>ACC) ( HBA2: c. 232C>A) of the α-globin gene, NG_000007.3: g. 70567_71015del449, codon 102(-A) ( HBB: c. 308_308delA) and IVS-Ⅱ-636 (A>G) ( HBB: c. 316-215A>G) of the β-globin gene were previously unreported new types of globin gene mutations. Conclusion:Among the 4 644 patients, the detection rate for common thalassemia mutations was 47.24%, whilst 46 samples were detected with rare gene mutations. The type of gene mutation types were diverse in the Shanghai area. The study has provided more accurate results for genetic diagnosis and counseling.

Objective:To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion.Methods:Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay.Results:The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin ( r2: 0.438, 0.695, and 0.736, respectively, all P＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P=0.034). Traf4 -/- cells had a weakened proliferative capacity than traf4+/+ cells and formed tumors with smaller size ( P＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4 -/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4+/+ cells [(62.3±10.3)%, P=0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4 -/- cells were lower than those of the traf4+/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions:TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

Objective To evaluate the role of combined detections of routine blood test,serum iron and hemoglobin electrophoresis in screening thalassemia in non-high incidence area.Methods Peripheral blood and serum samples of 1 000 outpatients from the department of hematology and the department of gynecology and obstetrics were obtained.Common mutations of thalassemia were detected by using GAP-PCR and reverse dot blotting,and Sanger sequencing was performed to discover rare mutations of ct-and β-thalassemia.Routine blood test,serum iron and hemoglobin electrophoresis were also performed for every patient.Results Among 1 000 samples,225 (22.5％) are detected as α-thalassemia,403 (40.3％) β-thalassemia and 15 (1.5％) composite thalassemia.Among 225 α-thalassemia patients,28 were silent,138 were intermedia,and 59 were HbH disease.Of 403 β-thalassemia,390 were carriers,7 were double heterozygote,and 6 were homozygote.In all samples,there were 357 patients detected with no common mutations,38 patients had higher result values for both MCV and MCH and none detected with thalassemia gene.There were 48 patients who had higher serum iron but normal or lower MCV,42 of them (87.5％) had thalassemia gene.Furthermore,38 patients showed abnormal hemoglobin electrophoresis,35 of them were HbH disease,while the other 3 were HbF-related thalassemia.Five patients showed abnormal hemoglobin electrophoresis,lower MCV and MCH,as well as higher serum iron,had no frequent mutation but rare ones.Conclusion Patients with higher MCV and MCH can mostly be excluded to have thalassemia,while higher serum iron represents thalassemia possibility and can provide a preliminary indication of thalassemia type,and last but not least abnormal hemoglobin electrophoresis indicates the disease.It is recommended to further carry out sequencing of rare mutations for those who had abnormal results in the combined screening,and detected with no frequent mutation.Combination of these three examinations can improve the detection efficiency of patients with thalassemia.

Objective:To evaluate the predictive value of preoperative frailty combined with nutritional status for prolonged postoperative ileus (PPOI) in the patients with gynecological malignancies.Methods:Patients undergoing elective surgery for gynecological malignancies in the Affiliated Hospital of Jiangnan University from April 2022 to February 2023 were selected. The Frail scale was used to evaluate the frailty within 24 h of admission, and the nutritional status was evaluated by the Controlling Nutritional Status score. The general characteristics of patients and occurrence of PPOI were recorded, and the risk factors for PPOI were analyzed by multivariate logistic regression. The ability of frailty, nutritional status and their combination to predict PPOI was assessed by the receiver operating characteristic curve.Results:Two hundred and fourteen patients were finally included, 52 cases developed of PPOI, and 98 cases were frail patients. Preoperative frailty combined with moderate to severe malnutrition was an independent risk factor for PPOI in the patients with gynecological malignancies ( P<0.05), and the area under the curve in predicting the occurrence of PPOI was 0.796 (95% confidence interval 0.736-0.857) in the patients with gynecological malignancies. Conclusions:Preoperative frailty combined with moderate to severe malnutrition has a higher accuracy in predicting PPOI in the patients with gynecological malignancies.