Study on construction of c-Met specific CAR-T cells and its killing effect on non-small cell lung carcinoma / 中华肿瘤杂志

Objective: To produce chimeric antigen receptor T cells (CAR-T) targeting human hepatocyte growth factor/c-Met (HGF/c-Met) protein and detect its cytotoxicity against non-small cell lung cancer (NSCLC) cells H1975 in vitro. Methods: The whole gene sequence of c-Met CAR containing c-Met single-chain fragment variable was synthesized and linked to lentiviral vector plasmid, plasmid electrophoresis was used to detect the correctness of target gene. HEK293 cells were transfected with plasmid and the concentrated solution of the virus particles was collected. c-Met CAR lentivirus was transfected into T cells to obtain second-generation c-Met CAR-T and the expression of CAR sequences was verified by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, and the positive rate and cell subtypes of c-Met CAR-T cells were detected by flow cytometry. The positive expression of c-Met protein in NSCLC cell line H1975 was verified by flow cytometry, and the negative expression of c-Met protein in ovarian cancer cell line A2780 was selected as the control. The cytotoxicity of c-Met CAR-T to H1975 was detected by lactate dehydrogenase (LDH) cytotoxicity assay at 1∶1, 5∶1, 10∶1 and 20∶1 of effector: target cell ratio (E∶T). Enzyme-linked immunosorbent assay (ELISA) was used to detect the release of cytokines such as TNF-α, IL-2 and IFN-γ from c-Met CAR-T co-cultured with H1975. Results: The size of band was consistent with that of designed c-Met CAR, suggesting that the c-Met CAR plasmid was successfully constructed. The results of gene sequencing were consistent with the original design sequence and lentivirus was successfully constructed. CAR molecules expression in T cells infected with lentivirus was detected by western blot and RT-qPCR, which showed c-Met CAR-T were successfully constructed. Flow cytometry results showed that the infection efficiency of c-Met CAR in T cells was over 38.4%, and the proportion of CD8(+) T cells was increased after lentivirus infection. The NSCLC cell line H1975 highly expressed c-Met while ovarian cancer cell line A2780 negatively expressed c-Met. LDH cytotoxicity assay indicated that the killing efficiency was positively correlated with the E∶T, and higher than that of control group, and the killing rate reached 51.12% when the E∶T was 20∶1. ELISA results showed that c-Met CAR-T cells released more IL-2, TNF-α and IFN-γ in target cell stimulation, but there was no statistical difference between c-Met CAR-T and T cells in the non-target group. Conclusions: Human NSCLC cell H1975 expresses high level of c-Met which can be used as a target for immunotherapy. CAR-T cells targeting c-Met have been successfully produced and have high killing effect on c-Met positive NSCLC cells in vitro.

Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro / 中西医结合学报

OBJECTIVE@#Qili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model.@*METHODS@#The main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia-reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.@*RESULTS@#Apoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX.@*CONCLUSION@#QLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.

Advances in dendritic cell-based vaccine delivery / 药学学报

Dendritic cells (DCs) are the most powerful and professional antigen-presenting cells (APCs) known at present. They play vital roles in the initiation and regulation of immune responses in body. Therefore, DC-based vaccine delivery system has gradually become a hotspot of basic scientific research and clinical treatment. DCs can be loaded with whole-cell antigens, nucleic acids, peptides, proteins (such as neoantigens) and nanoparticles to induce specific cellular immune responses and humoral immune responses after antigen processing, presentation and targeting delivery in vivo for the prevention and treatment of various diseases including cancers and microorganism infections. Vaccine-based on this technique is called dendritic cell (DC) vaccines. Great process in DC vaccines has been achieved in recent years. Therefore, we reviewed the characteristics of DC, types of DC vaccines and their clinical research progress in this paper.

Progress on Anticancer Effects of PD-1/PD-L1 Checkpoint Inhibitors / 中国药学杂志

This artical explained the biological action mechanism of PD-1/PD-L1 monoclonal antibodies through reactivating the immune response of T cells to tumor cells immune response, analyzed the detection reports on the targets, introduced the clinical manifestations and indications research results, and looked into their development potential in the field of anticancer therapy. It's important to rationally use PD-1/PD-L1 monoclonal antibodies to decrease immune-mediated adverse events as well as maximizing the therapeutic effect. Furthermore, we should not only see the accomplishment in anticancer therapy, but also gain insight into its development direction in the field of anticancer research based on the therapeutic idea of inducing autoimmune system to kill tumor cells.

Efficacy and prognostic factors of abiraterone combined with prednisone treating metastatic castration-resistant prostate cancer patients / 上海交通大学学报（医学版）

Objective· To assess the efficacy of abiraterone acetate (AA) plus prednisone treating metastatic castration-resistant prostate cancer (mCRPC) patients and analyze the prognostic factors for this treatment. Methods · The medical history of 112 patients with mCRPC treated in Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine, including 70 patients in the chemotherapy-na?ve setting and 42 in the post-chemotherapy setting, were retrospectively reviewed. Coprimary end points were prostate specific antigen progression-free survival (PSA PFS), radiographic PFS (rPFS) and overall survival (OS). Univariable and multivariable Cox analyses were performed to determine prognostic factors that were associated with PSA PFS, rPFS and OS. Results · At a median follow-up of 20.2 months, 59 (52.7%) patients had died. The median PSA PFS, rPFS and OS were 8.9 (7.8~10.0) months, 9.7 (9.0~10.4) months, and 22.2 (20.3~24.1) months, respectively. In multivariate analysis, previous chemotherapy, neutrophil lymphocyte ratio(≥3 vs<3),serum lactate dehydrogenase level(≥196 U/L vs<196 U/L)and ECOG PS(≤?1 vs 2)were independent predictors for PSA PFS and rPFS,and previous chemotherapy,ECOG PS(≤?1 vs 2)remained significant predictors for OS. Conclusion·These results further support the favourable profile of AA plus prednisone in patients with mCRPC in China.Previous chemotherapy,ECOG PS(≤?1 vs 2)remained significant predictors for OS.

Combined screening report of hearing screening and deafness susceptibility genes screening for newborns / 实用医学杂志

Objective To investigate the clinic signification of newborn hearing screening combined with deafness susceptibility genes screening. Methods 1 440 newborns(3 ~ 5 days after birth) were screened for 8 hot spot hearing loss associated mutations from GJB2, mt12S rRNA and SLC26A4. At the same time, all infants received hearing screening. Those who failed to pass two-step test were referred to further audiological assessment. Results The carrier rate of commonmutations was 1.46% for GJB2 c.235delC, 0.35% for GJB2 c.299-300delAT, 0.42% for mt12S rRNA c.1555A > G, 0.42% for SLC26A4 c.IVS7-2A > G and 0.14% for SLC26A4 c.2168A > G. The total carrier rate was 2.78%. 10 infants were diagnosed as hearing loss in the hearing screening and follow-up audiology assessment (6.94‰) and 5 were diagnosed as severe hearing loss (3.47‰). 32 hearing loss associated mutation carriers passed the hearing screening. Conclusions Genetic screening of newborn hearing screening can be helpful to find out neonates with late-onset and progressive hearing impairment, which were significant for early intervention, regular follow-up and reduction of deafness.

Spectrum of gene deletion in 471 children with α-thalassemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the distribution of common α-thalassemia gene deletion in children.</p><p><b>METHODS</b>Blood cell analysis was performed on children who visited the clinic of the Foshan Women and Children's Hospital. Blood samples (2 mL, EDTA anticoagulant) was collected from children with MCV<82 fl for analysis of α-thalassemia gene using the GAP-PCR method.</p><p><b>RESULTS</b>MCV<82 fl was found in 1341 children. Of the 1341 children, 471 (35.1%) were diagnosed with α-thalassemia. The prevalence of α-thalassemia increased with increasing age. --SEA was a major type of α-thalassemia gene deletion (75.3%), followed by -a3.7 (17.0%) and -a4.2 (7.7%) in the 471 patients. The top three genotypes were --SEA/aa (73.2%), aa/-a3.7 (12.5%) and --SEA/-a3.7 (5.5%).</p><p><b>CONCLUSIONS</b>Genetic testing is necessary for the diagnosis of α-thalassemia in children with MCV<82 fl. --SEA is a common type of α-thalassemia gene deletion, and -SEA/aa is a common gene type of α-thalassemia in the subjects of this study.</p>

Coronary computed tomographic angiography using low-dose prospectively electrocardiographic triggered high-pitch spiral acquisition by dual-source computed tomography: image quality and radiation dose / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate image quality (IQ) and radiation exposure of coronary computed tomographic angiography (CTA) with prospectively electrocardiographic (ECG) triggered high-pitch spiral acquisition using dual source CT.</p><p><b>METHODS</b>Totally 75 consecutive patients with a stable heart rate (HR) ≤65 bpm underwent coronary CTA. patients were divided into two groups according to their HR (group A HR≤60 bpm, group B HR >60 bpm to≤65 bpm) . A dual-source CT scanner was used (0.6mm collimation, 0.28s rotation time, 80~100 kV, 370 mAs/rot) . Data acquisition was prospectively ECG-triggered at 60% of the R-R interval with a pitch of 3.4. Images were reconstructed with 75ms temporal resolution, 0.75mm slice thickness and 0.5mm increment. IQ was evaluated using a four-point scale (1=excellent, 4=unevaluable) .</p><p><b>RESULTS</b>The mean HR and scan time of all patients was (57.2 ± 4.8) bpm and (0.42 ± 0.02) s. Of 1103 coronary artery segments, 934 (84.7%) had an IQ score of 1, 135 (12.2%) score of 2, 18 (1.6%) score of 3,and 16 (1.5%) were rated as unevaluable. There was no significant difference between the two groups in IQ [mean score (1.19 ± 0.52 vs. 1.22 ± 0.55;Z=-1.107,P=0.268) . The rate of evaluable segments showed no significant difference between the two groups (98.5% vs. 98.6%;X2=0.000,P=1.000) . Mean dose-length product of all patients was (67.2 ± 30.4) mGy × cm, mean effective dose was (0.94 ± 0.43) mSv.</p><p><b>CONCLUSION</b>In patients with a stable HR of 65 bpm or less, prospectively ECG-triggered high-pitch spiral CT acquisition provides high IQ at low radiation dose.</p>

Association of killer cell immunoglobulin-like receptor gene polymorphisms with seronegative spondylarthropathies / 中华风湿病学杂志

Objective To investigate whether the killer cell immunoglobulin like receptor (KIR) gene polymorphisms are associated with seronegative spondylarthropathies.Methods All 197 HLA B27 positive patients with seronegative spondyloarthro pathies (SpA) and 83 randomly ethnically matched healthy controls were enrolled to detect the KIR genotype using PCR SSP in Shanghai area.Results The KIR3DL1 gene frequency was significantly lower in the patient group (0 763) than in the control group (1 00) (RR=0 76, P =0 003).Meanwhile,the gene frequencies of two pseudogenes (KIR2DP1,KIR3DP1) were significantly higher in SpA group than in healthy control group (RR=1 1, P =0 004).Conclusion There may be a negative association between pathogenesis of SpA and KIR3DL1 gene.The function of KIR3DL1 molecule should be investigated deeply.

Influences of two different HLA-B antigens on receptors expression of NK cells from peripheral blood lymphocytes / 中国免疫学杂志

Objective:To investigate influences of two different HLA-B antigens expressed on K562 cells on receptors expression of NK cells from peripheral blood lymphocytes.Methods:Studied the alteration of the percentage of CD16+CD56+ cells and the percentage of KIR3DL1+ cells before and after PBMC interaction with K562 cells for 24 hours,and also compared the percentage of CD16+CD56+ cells and the percentage of KIR3DL1+ cells after PBMC interaction with two different kind of K562 cells transfected with HLA-B39 and HLA-B51 respectively.Results:After PBMCs were incubated with K562 cells for 24 hours,the percentage of CD16+CD56+ cells and the percentage of KIR3DL1+ cells were both increased.However,after PBMCs were incubated with K562-HLA-B51 cells for 24 hours,the percentage of KIR3DL1+ cells and the percentage of CD16+CD56+ cells were both decreased in comparison with that interaction with K562-HLA-B39 cells.Conclusion:CD16 up-regulation was associated with an up-regulation of inhibitory receptors(KIR3DL1).The interaction between HLA-Bw4 and KIR3DL1 would down-regulate the expression of KIR3DL1.In addition,KIR3DL1 down-regulation was associated with down-regulation of activating receptors(CD16).

Association of MICA gene exon 2～4 polymorphism with seronegative spondylarthropathies / 中华风湿病学杂志

Objective To investigate whether MICA gene exon 2,3 and 4 polymorphism is associated with seronegative spondylarthropathies (SpA) or not in Chinese Han population.Methods All 199 B27 positive patients with SpA and 183 randomly ethnically matched healthy controls,and 12 B27 positive controls were enrolled to detect the MICA genotype from its exons 2,3 and 4 by using PCR SSOP method in Shanghai area.Results The MICA007 allele frequency was significantly more in the patient group (18 0%) than in the randomly healthy control group (6 6%) (RR=3 04, P =0 000 045).However,the MICA007 allele frequency was not significantly higher in the B27 positive patient group than in the B27 positive control group.Conclusion The MICA007 allele itself may not be the real disease susceptibility gene involved in the development of ankylosing spondylitis.The increased frequency of MICA007 allele is supposed to be due to a strong linkage disequilibrium between MICA and HLA B genes.

Construction of mammalian cell expression vector of human leukocyte antigen-C gene and expression in JAR cells / 中国免疫学杂志

Objective:To construct mammalian cell expression vector of human leukocyte antigen C gene and express it in JAR cell and study its functions.Methods:Total cell RNA was extracted from peripheral blood mononuclear cells and the cDNA was amplified by RT PCR;the cDNA fragments were directly inserted into Pbluescript KS(+/-) and the recombinant PBS HLA C was identified by restriction endonuclease digestion and sequencing;then the plasmid of pcDNA HLA C was transfected into JAR cells.Lactate Dehydrogenase(LDH) release assay was employed to detect the cytotoxicities of NK cells proliferated in vitro against HLA C transfectants JAR cells.Results:The recombinant mammalian cell expression vectors of pcDNA3 HLA C was constructed,the sequences of the insert was identical to the published sequences encoding HLA Cw *0602 gene.Several transfected JAR clones were obtained,which expressed HLA Cw *0602 in high level and constitutively.The specific lysis was detected for NK cells stimulated by IL 2 against JAR cells and HLA C transfectants JAR cells.But both cells were resistant to freshly isolated PBL cells.Conclusion:The recombinant mammalian cell expression vectors of pcDNA3 HLA Cw *0602 was successfully constructed,JAR cells resistance to NK lysis could involve an HLA C independent mechanism.

Study on association of PIH with HLA-G allele / 中国免疫学杂志

Objective:To investigate the mechanism of association between Pregnancy induced Hypertension and HLA G.Methods:The distribution and frequencies of HLA G alleles were analyzed in 54 PIH patients and 146 random healthy controls with nonradioactive digoxigenin labeled PCR SSO method.Results:The results showed that the same nine HLA G alleles were detected and the G*01011 was the commonest allele with the frequency of 31.38% and 37.33% respectively in both PIH and controls groups,the next was G*01013,25.00% and 20.21% separately.Statistic analysis showed that the frequencies of HLA G alleles were similar to in the two groups and there was no significant difference between PIH patients and controls.Conclusion:These data suggest that there is no significant detectable relation between the HLA G polymorphism and PIH.

Study on association of HLA-E with recurrent spontaneous abortion / 中国免疫学杂志

Objective:To explore the mechanism of association between recurrent spontaneous abortion(RSA) and the polymorphism of HLA E,had detected the frequencies of five HLA E alleles in 53 women with RSA and 156 random Shanghai Chinese controls.Methods:PCR sequence specific oligonucleotide (PCR SSO) typing was carried to detect polymorphism in exon2 and exon3 of HLA E gene.Results:HLA E *0101 is the most common allele in both RSA patients and controls(50% and 43.09% respectively).HLA E *01031 and E *01032 were detected with allele freqencies of 23.79% and 33.12% in controls,20.75% and 29.25% in RSA.No HLA E *0102 and E *0104 alleles could be detected in both groups.Conclusion:Maternal HLA E polymorphism does not associated with pathogenesis of RSA.

Relationship between human leukocyte antigen-DRB1 allele gene polymorphism and intrahepatic cholestasis of pregnancy / 中华妇产科杂志

Objective To study the relationship between human leukocyte antigen-DRB1 (HLA-DRB1) allele genes polymorphism and intrahepatic cholestasis of pregnancy (ICP). Methods Forty-two patients with ICP were tested for HLA-DRB1 allele genes polymorphism with the polymerase chain reaction technique and sequence specific oligonucleotide (PCR-SSO) probes hybridization, 56 normal pregnant women as control group were also tested. In addition, the phenotype frequencies of HLA-DRB1 alleles were compared with it′s clinical character in patients with ICP. Results The higher frequencies were observed for alleles DR9, DR12 and DR4 in both groups. DR6 alleles were detected in 14 cases out of 42 patients. Patients with ICP had a significantly higher frequency of the allele DR6 when compared to control group (16.7% vs 3.6%), with a relative risk (RR) as 6.5 (P

The influence of tapasin on HLA-E expression on cell surface / 中国免疫学杂志

Objective:To explore the influence of chaperone tapasin on HLA E expression on cell surface.Methods:The infection method was used to transfer the recombinant retrovirus expression vector which was constructed with HLA E molecules on target cells was detected with FACS technique.Results:Exogenous HLA E expression on T2 cell surface was detected with 74.13% and no HLA E expression was detected on control cells and that was transfected with empty vector.Conclusion:It is possible that the expression of HLA E molecules on cell surface involves in TAP independent mechanism.