Noninvasive Diagnostic Technique for Nonalcoholic Fatty Liver Disease Based on Features of Tongue Images / 中国结合医学杂志

OBJECTIVE@#To investigate a new noninvasive diagnostic model for nonalcoholic fatty liver disease (NAFLD) based on features of tongue images.@*METHODS@#Healthy controls and volunteers confirmed to have NAFLD by liver ultrasound were recruited from China-Japan Friendship Hospital between September 2018 and May 2019, then the anthropometric indexes and sampled tongue images were measured. The tongue images were labeled by features, based on a brief protocol, without knowing any other clinical data, after a series of corrections and data cleaning. The algorithm was trained on images using labels and several anthropometric indexes for inputs, utilizing machine learning technology. Finally, a logistic regression algorithm and a decision tree model were constructed as 2 diagnostic models for NAFLD.@*RESULTS@#A total of 720 subjects were enrolled in this study, including 432 patients with NAFLD and 288 healthy volunteers. Of them, 482 were randomly allocated into the training set and 238 into the validation set. The diagnostic model based on logistic regression exhibited excellent performance: in validation set, it achieved an accuracy of 86.98%, sensitivity of 91.43%, and specificity of 80.61%; with an area under the curve (AUC) of 0.93 [95% confidence interval (CI) 0.68-0.98]. The decision tree model achieved an accuracy of 81.09%, sensitivity of 91.43%, and specificity of 66.33%; with an AUC of 0.89 (95% CI 0.66-0.92) in validation set.@*CONCLUSIONS@#The features of tongue images were associated with NAFLD. Both the 2 diagnostic models, which would be convenient, noninvasive, lightweight, rapid, and inexpensive technical references for early screening, can accurately distinguish NAFLD and are worth further study.

Effect of a novel phosphodiesterase type 5 inhibitor, CPD1, on carbon tetrachloride-induced liver fibrosis in mice / 中国药理学通报

Aim To investigate the effects of CPD1, a novel phosphodiesterase 5 inhibitor, on liver pathological phenotype and hepatic stellate cells (HSCs) activation in hepatic fibrosis model mice caused by carbon tetrachloride ( CCl

Clinical Significance of Minimal Residual Disease in Pediatric Patients with TCF3/PBX1+ B-cell Acute Lymphoblastic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the consistency of flow cytometry (FCM) method and polymerase chain reaction (PCR) technique in the detection of minimal residual disease (MRD) at different treatment stages in pediatric patients with TCF3/PBX1+ B-cell acute lymphoblastic leukemia (B-ALL) and the correlations between the detection results and prognosis.@*METHODS@#The clinical data of 64 newly diagnosed pediatric patients with TCF3/PBX1+ B-ALL admitted to the Department of Pediatrics of Peking University People's Hospital from January 2005 to December 2017 were retrospectively analyzed. FCM and PCR methods were used to monitor the MRD level in bone marrow samples from 64 children during the same period of treatment on d33 and d90 respectively, and the detection results were analyzed.@*RESULTS@#There were 37 males and 27 females in the 64 patients, with a median age of 8 years(range 0.8 to 16 years). The complete remission (CR) rate after the first cycle of induction chemotherapy was 98.4% (62/63), with overall CR rate of 100%. 12 patients experienced recurrence, with a median recurrence time of 16.9 (5.3-46.3) months. The median follow-up time of the 64 patients was 77.2 (1.0-184.8) months , and the 5-year overall survival (OS) rate and event-free survival (EFS) rate were 82.8%±4.7% and 75.0%±5.4%, respectively. On d90, the concordance rate of the MRD results from the two methods was 98.4%, and the related kappa value was 0.792 (P < 0.001), which were significantly higher than those on d33. After induction chemotherapy (d33), the 5-year EFS rate of MRD-FCM- group (79.3%±5.3%) was significantly better than that of MRD-FCM+ group (40.0%±21.9%) (P =0.028), there were no significant differences in the 5-year OS rate and EFS rate between MRD-PCR+ group and MRD-PCR- group, and the 5-year EFS rate of MRD-FCM-/PCR- group (85.4%±5.5%) was significantly better than that of MRD-FCM+/PCR+ group (40.0 %±21.9%) (P =0.026).@*CONCLUSION@#In children with TCF3/PBX1+ B-ALL, the MRD results detected by FCM and PCR methods show good consistency, especially in consolidation therapy period (d90). The MRD level at the end of induction therapy (d33) is an important factor affecting the long-term prognosis, especially the MRD results detected by FCM method, which is significantly associated with prognosis.

Preparation of CD52-targeted chimeric antigen receptor-modified T cells and their anti-leukemia effects / 中华血液学杂志

Objective: To construct chimeric antigen receptor (CAR) T cells targeting CD52 (CD52 CAR-T) and validate the effect of CD52 CAR-T cells on CD52-positive leukemia. Methods: A second-generation CD52-targeting CAR bearing 4-1BB costimulatory domain was ligated into a lentiviral vector through molecular cloning. Lentivirus was prepared and packaged by 293 T cells with a four-plasmid system. Fluorescein was used to label cell surface antigens to evaluate the phenotype of CD52 CAR-T cells after infection. Flow cytometry and ELISA were used to evaluate the specific cytotoxicity of CD52 CAR-T cells to CD52-positive cell lines in vitro. Results: ①A pCDH-CD52scFv-CD8α-4-1BB-CD3ζ-GFP expressing plasmid was successfully constructed and used to transduce T cells expressing a novel CD52-targeting CAR. ②On day 6, CD52-positive T cells were almost killed by CD52-targeted CAR-T post lentivirus transduction [CD52 CAR-T (4.48 ± 4.99) %, vs Vector-T (56.58±19.8) %, P=0.011]. ③T cells transduced with the CAR targeting CD52 showed low levels of apoptosis and could be expanded long-term ex vivo. ④The CD52 CAR could promote T cell differentiation into central and effector memory T cells, whereas the proportion of T cells with a CD45RA(+) effector memory phenotype were reduced. ⑤CD52 CAR-T cells could specifically kill CD52-positive HuT78-19t cells but had no killing effect on CD52-negative MOLT4-19t cells. For CD52 CAR-T cells, the percentage of residual of HuT78-19t cells was (2.66±1.60) % at an the E:T ratio of 1∶1 for 24 h, while (56.66±5.74) % of MOLT4-19t cells survived (P<0.001) . ⑥The results of a degranulation experiment confirmed that HuT78-19t cells significantly activated CD52 CAR-T cells but not MOLT4-19t cells[ (57.34±11.25) % vs (13.06± 4.23) %, P<0.001]. ⑦CD52 CAR-T cells released more cytokines when co-cultured with HuT78-19t cells than that of vector-T cells [IFN-γ: (3706±226) pg/ml, P<0.001; TNF-α: (1732±560) pg/ml, P<0.01]. Conclusions: We successfully prepared CD52 CAR-T cells with anti-leukemia effects, which might provide the foundation for further immunotherapy.

Preparation of CD33 targeted bispecific- and trispecific-T cell engagers and their cytotoxicity on leukemia cells / 中华血液学杂志

Objective: To investigate the effect of CD33-targeted bi-specific and tri-specific T-cell engagers on T-cell proliferation and explore their cytotoxicity on leukemia cells. Methods: The CD33-targeted bi-specific T-cell engager (CD33-BiTE) and tri-specific T-cell engager (CD33-TriTE) expression vectors were successfully constructed and expressed through a eukaryotic cell expression system. CD33-BiTE and CD33-TriTE were purified by affinity chromatography. The effects of CD33-BiTE and CD33-TriTE on T cells were analyzed through in vitro experiments. Results: ① CD33-BiTE and CD33-TriTE were successfully constructed and purified and could compete with flow cytometry antibodies for binding to the target cells. ② After 12 days of co-culture with CD33-BiTE and CD33-TriTE, the number of human T cells were expanded to 33.89±19.46 and 81.56±23.62 folds, respectively. CD33-TriTE induced a stronger proliferation of T cells than CD33-BiTE (P<0.05) . ③ Both CD33-BiTE and CD33-TriTE induced specific dose-dependent cytotoxicity on CD33(+) leukemia cells. ④ Compared to CD33-TriTE, leukemia cells were prone to express PD-L1 when co-cultured with T cells and CD33-BiTE. CD33-TriTE induced powerful cytotoxicity on leukemia cells with high PD-L1 expression. Conclusion: CD33-BiTE and CD33-TriTE expression vectors were constructed, and fusion proteins were expressed in eukaryotic cells. Our results support the proliferative and activating effects of BiTE and TriTE on T cells. Compared to that of CD33-BiTE, CD33-TriTE induced a stronger proliferative effect on T cells and a more powerful cytotoxicity on leukemia cells with high PD-L1 expression.

Functional investigation of chimeric antigen receptor T cells targeting LMP1 antigen / 中华血液学杂志

Objective: This study aimed to create a type of CAR-T cells that targets LMP1 antigen and study its immunotherapeutic effect on LMP1-positive hematological malignancies. Methods: To generate LMP1 CAR-T cells, a plasmid expressing LMP1 CAR was created using molecular cloning technology, and T cells were infected with LMP1 CAR lentivirus. The effects of LMP1 CAR-T cells on specific cytotoxicity against LMP1-positive tumor cell lines infected with the EB virus had been confirmed. Results: ① LMP1 protein expressing on EB virus-positive lymphoma cells surface was verified. ② The LMP1 CAR-expressing plasmid was created, and LMP1 CAR-T cells were obtained by infecting T cells with a lentivirus packaging system, with an infection efficiency of more than 80% . ③LMP1 CAR-T cells have a 4∶1 effect-to-target ratio in killing LMP1-positive lymphoma cells. The killing effect of LMP1 CAR-T cells on Raji cells was enhanced after 48 h of coculture, but there was no significant killing effect on Ramos, which are LMP1-negative lymphoma cells. ④After coculture with LMP1-positive lymphoma cells at a ratio of 1∶1 for 5 h, the degranulation effect was enhanced. The proportion of CD107a(+) T cells in the LMP1 CAR-T cell treatment group was significantly higher than that in the vector-T cell group [ (13.25±2.94) % vs (1.55±0.05) % , t=3.972, P=0.017]. ⑤After coculture with LMP1-positive lymphoma cells, the proportion of CD69(+) and CD25(+) T cells in the LMP1 CAR-T cell group was significantly higher than that in vector-T cell group [ (7.40±0.41) % vs (3.48±0.47) % , t=6.268, P=0.003; (73.00±4.73) % vs (57.67±2.60) % , t=2.842, P=0.047]. ⑥After coculture with LMP1-positive lymphoma cells, cytokine secretion in the LMP1 CAR-T cell group was higher than that in the vector-T cell group [interferon-gamma: (703±73) ng/L vs (422±87) ng/L, t=2.478, P=0.068; tumor necrosis factor-alpha: (215±35) ng/L vs (125±2) ng/L, t=2.536, P=0.064]. Conclusion: In this study, we found that the LMP1 protein is only found on the surface of the EBV-positive tumor cell. Simultaneously, we created an LMP1 CAR-expressing plasmid and obtained LMP1 CAR-T cells by infecting T cells with a lentivirus packaging system. Furthermore, we demonstrated that LMP1 CAR-T cells could specifically kill LMP1-positive tumor cells in vitro. The degranulation and activation effects of LMP1 CAR-T cells were enhanced after coculture with LMP1-positive tumor cells, indicating a potential clinical application.

Clinical features and prognosis of childhood B-lineage acute lymphoblastic leukemia expressing the PRAME gene / 中国当代儿科杂志

OBJECTIVES@#To study the clinical and prognostic significance of the preferentially expressed antigen of melanoma (PRAME) gene in the absence of specific fusion gene expression in children with B-lineage acute lymphoblastic leukemia (B-ALL).@*METHODS@#A total of 167 children newly diagnosed with B-ALL were enrolled, among whom 70 were positive for the PRAME gene and 97 were negative. None of the children were positive for MLL-r, BCR/ABL, E2A/PBX1, or ETV6/RUNX1. The PRAME positive and negative groups were analyzed in terms of clinical features, prognosis, and related prognostic factors.@*RESULTS@#Compared with the PRAME negative group, the PRAME positive group had a significantly higher proportion of children with the liver extending >6 cm below the costal margin (P<0.05). There was a significant reduction in the PRAME copy number after induction chemotherapy (P<0.05). In the minimal residual disease (MRD) positive group after induction chemotherapy, the PRAME copy number was not correlated with the MRD level (P>0.05). In the MRD negative group, there was also no correlation between them (P>0.05). The PRAME positive group had a significantly higher 4-year event-free survival rate than the PRAME negative group (87.5%±4.6% vs 73.5%±4.6%, P<0.05), while there was no significant difference between the two groups in the 4-year overall survival rate (88.0%±4.4% vs 85.3%±3.8%, P>0.05). The Cox proportional-hazards regression model analysis showed that positive PRAME expression was a protective factor for event-free survival rate in children with B-ALL (P<0.05).@*CONCLUSIONS@#Although the PRAME gene cannot be monitored as MRD, overexpression of PRAME suggests a good prognosis in B-ALL.

The Impact of Induction Treatment Response on the Prognosis of Pediatric Core Binding Factor-Acute Myeloid Leukemia Patients / 中国实验血液学杂志

OBJECTIVE@#To explore the impact of induction treatment response on the prognosis of pediatric core binding factor-acute myeloid leukemia (CBF-AML).@*METHODS@#The result of induce reaction and survival data of 157 pediatric CBF-AML patients in our hospital from September 2008 to December 2018 were retrospectively analyzed．The survival rate of the patients with different degrees of morphological remission after induction chemotherapy was comparative analyzed.@*RESULTS@#Among the 157 children with CBF-AML, 113 (72.4%) patients achieved morphologic leukemia-free state (MLFS) after the first course of induction chemotherapy, 153 (98.1%) patients achieved MLFS after the second course of induction chemotherapy. The 5-year event-free survival (EFS) rate and 5-year overall survival (OS) rate of patients with non-remission (NR) status after the first course of induction of chemotherapy was significantly lower than the patients achieved MLFS and the patients achieved partial remission (PR). The 5-year EFS rate and 5-year OS rate of the patients with PR status after the second course of induction chemotherapy were lower than the patients achieved MLFS, but the difference was not statistically significant. Multivariable analyze showed that NR after the first course of induction chemotherapy and myeloid sarcoma were the independent risk factors affecting EFS of the patients. There were six patients with NR status after the first course of induction chemotherapy, in which all of them harbored t(8;21), three of them with sex chromosome deletion, two of them with myeloid sarcoma.@*CONCLUSION@#NR status after the first course of induction chemotherapy was the independent risk factor affecting EFS and OS of CBF-AML patients, it can be taken as an indicator for higher risk stratification. PR status after the first course of induction chemotherapy may not be used as a diagnostic criterion for primary drug resistance.

Performance Verification of SeqType® P52 Human Ancestry Identification SNP Detection Kit / 法医学杂志

Objective To evaluate the discrimination efficiency of the SeqType® P52 Human Ancestry Identification SNP Detection Kit based on a high-throughput sequencing platform in five Chinese ethnic groups. Methods Using the SeqType® P52 Human Ancestry Identification SNP Detection Kit based on a high-throughput sequencing platform, a total of 350 samples from Han, Tibetan, Mongolian, Uygur, and Yi populations in China were detected and population cluster analysis was performed. Results The effective sequencing depth of a single site in a single sample was ≥720×, and the average report rate was 96%. The mean values of allele frequency differences between the Tibetan, Mongolian, Uygur, Yi and Han population were 0.20, 0.05, 0.24 and 0.11, respectively. Using Structure 2.3.4 software under K=5 mode, independent ancestral component in Han, Tibetan and Uygur could be detected, which was consistent with the result observed from the principal component analysis （PCA）. For the Yi population, two thirds of them had relatively independent ancestral component close to the Tibetan population and one third were similar to the Uygur population. The Mongolian population had similar ancestral origin component with Han population. Conclusion The composite detection system with 52 screened ancestry-informative SNP sites has been established in this study, which can effectively analyze the composition and individual genetic components of populations from Han, Tibetan and Uygur. The ability to discriminate among Han, Mongolian and Yi needs to be further improved. The SeqType® P52 Human Ancestry Identification SNP Detection Kit can be used to infer the origin of an individual's ancestors in some forensic DNA cases.

Free anterolateral thigh flap with superficial temporal vessels as recipient vessels for reconstruction of defect after maxillectomy in malignant tumor / 中华耳鼻咽喉头颈外科杂志

Objective: To study the effects of superficial temporal artery and vein as recipient vessels for the free anterolateral thigh flap on the appearance and functions after maxillectomy. Methods: Clinical data of 21 patients with malignant maxillary tumors in Department of Oral and Maxillofacial Surgery, the Second Xiangya Hospital of Central South University from January 2014 to November 2019, who were treated by free anterolateral thigh flap with temporal superficial vessels as the recipient vessels were analyzed retrospectively. There were 18 males and 3 females, with the age ranging from 29 to 73 years old, including 19 cases of squamous carcinoma, 1 case of adenoid cystic carcinoma and 1 case of osteosarcoma. Of those 7 patients underwent primary surgery, 14 patients received resurgery, and 6 patients had a history of postoperative radiotherapy and chemotherapy. Among 14 patients with resurgery, 13 had recurrent ipsilateral second site tumor and 1 had recurrent tumor, and all of them received the maxillectomy and reconstructive surgery with the free anterolateral thigh flap. Patients were evaluated with water swallow test and speech intelligibility score in 1, 3 and 6 months after operation. The data were statistically analyzed with SPSS 22.0 statistical software. Water swallow test results before and after operation were compared using the Wilcoxon rank sum test. The mean speech intelligibility scores before and after operation were compared by the paired t test. Results: Patients were followed up for 10-60 months. All free flaps survived after operation. No diplopia occurred. Breathing, swallowing and speaking functions were normal. No movement disorders caused by the donor of thigh flap. Water swallow test showed no phenomenon of water flowing into the nasal cavity or oral and nasal leakage with level Ⅰ for 4 cases, level Ⅱ for 13 cases, level Ⅲ for 3 cases and level Ⅳ for 1 case. The mean speech intelligibility scores before surgery and 1, 3 and 6 months after surgery were 4.31±0.13, 1.46±0.21, 2.15±0.45 and 2.87±0.76 respectively. There was statistically significant difference in the mean speech intelligibility scores between 1 and 6 months after surgery (F=78.456, P<0.05). Conclusion: It is safe and reliable to use the superficial temporal vessels as recipient vessels for free anterolateral thigh flap in the reconstruction of defect after maxillectomy in malignant tumors, with good outcomes of functions and a satisfactory restoration of outward appearance.

Construction of a new anti-CD123 chimeric antigen receptor T cells and effect of anti-acute myeloid leukemia / 中华血液学杂志

Objective: To construct a new CD123- specific chimeric antigen receptor in order to provide a foundation for immunotherapy of CD123 positive leukemia. Methods: A hybridoma strain (6E11) capable of stably secreting CD123 antibody was obtained by a monoclonal screening technique, and the hybridoma cells were expanded and injected intraperitoneally to the pretreated Balb/c mice. Ascites was collected and purified to obtain the monoclonal antibody (mAb) . The affinity and specificity of 6E11 mAb were measured. The variable regions of the heavy and light chains of the 6E11 mAb were cloned by RT-PCR from the 6E11 mouse hybridoma. We generated a new CD123 specific chimeric antigen receptor with a scFv fragment derived from 6E11 antibody, designated as 6E11 CAR. T cells were transduced with lentiviral supernatant from 293T cells transfected with 6E11 CAR plasmid to generate 6E11 CAR-T cells. The specific cytotoxicity of 6E11 CAR-T against CD123(+) acute myeloid leukemia (AML) cell lines and primary AML cells in vitro were evaluated by co-culture experiments, degranulation experiments and cytokine releasing assay. Results: ① A hybridoma cell line 6E11 stably secreting anti-human CD123 antibody was developed and its variable region sequences were obtained. ② The 6E11 mAb has high affinity for CD123 protein (Kd value: 2.1 nmol/L) . The 6E11 mAb specifically recognizes CD123(+) cell line THP-1 cells and does not respond to CD123(-) cell line Jurkat cells. ③ 6E11 CAR-T cells were successfully generated with a CAR expression rate higher than 60%. ④ 6E11 CAR-T cells could specifically kill CD123(+) MV4-11 cell line but had no killing effect on the CD123(-) K562 cell line. Compared with vector-T cells, 6E11 CAR-T cells have higher killing rate to MV4-11 cells[ (98.60±1.20) %vs (20.28±6.74) %, P<0.001]. ⑤ MV4-11 cells activated 6E11 CAR-T cells significantly but not Vector-T cells[ (26.33±3.30) %vs (1.17±0.06) %, P<0.001]. ⑥ 6E11 CAR-T cells released more cytokines than vector-T cells when co-cultured with MV4-11[IL-2: (92.90±1.51) pg/ml vs (6.05±3.41) pg/ml, P<0.001; TNF-α: (1 407.20±91.95) pg/ml vs (7.86±0.85) pg/ml, P<0.001; IFN-γ: (5 614.60±170.17) pg/ml vs (8.42±2.70) pg/ml, P<0.001]. The IFN-γ, IL-2 and TNF-α in the 6E11 CAR-T group were similar to those in the Vector-T group when co-cultured with K562. ⑦ 6E11 CAR-T cells could be activated by bone marrow mononuclear cells (BMMNC) derived from CD123(+) AML patients and effectively kill these BMMNC cells from CD123(+) AML patients. Conclusion: 6E11 hybridoma cell line can stably secrete highly specific monoclonal antibodies against human CD123, which can be used to detect the expression of human CD123. It can also be used to target human CD123 protein in tumor immunotherapy. CD123 CAR-T cells with 6E11 Ig variable region sequence have specific anti-leukemic activity in vitro, which may provide a new option for further clinical research of AML.

Clinical characteristics and prognostic analysis of pediatric pro-B cell acute lymphoblastic leukemia / 中国当代儿科杂志

OBJECTIVE@#To explore the clinical-biological characteristics and prognosis of pediatric pro-B cell acute lymphoblastic leukemia (pro-B-ALL).@*METHODS@#A total of 64 patients aged less than 18 years old with pro-BALL were enrolled. Clinical characteristics, therapeutic effect and prognostic factors were retrospectively analyzed.@*RESULTS@#Pro-B-ALL occurred in 6.23% (64/1 028) of pediatric ALL. Among the 64 patients, 35 were male and 29 were female. The median age was 7.0 years (range 0.4-16.0 years) at diagnosis, of which 39% and 6% were ≥ 10 years old and < 1 year old respectively. The median WBC count was 25.5×10@*CONCLUSIONS@#Pediatric pro-B ALL is a heterogeneous disease with clinical and biological diversity. Biological characteristics, such as immunological markers, genetic alterations, and MRD at 3 months after chemotherapy may be important factors for the long-term prognosis.

Influence of dasatinib treatment on body height in children with acute myeloid leukemia / 中国当代儿科杂志

OBJECTIVE@#To study the influence of dasatinib treatment on body height in children with acute myeloid leukemia (AML).@*METHODS@#A retrospective analysis was performed for the clinical data of 86 AML children aged <17 years. According to the treatment regimen, these children were divided into a conventional chemotherapy group and a dasatinib chemotherapy group. The 57 children in the conventional chemotherapy group were given conventional chemotherapy drugs without tyrosine kinase inhibitor, and the 29 children in the dasatinib chemotherapy group were given conventional chemotherapy drugs and dasatinib. The two groups were compared in terms of height standard deviation score (HtSDS) at the beginning of treatment and after treatment, as well as the change in HtSDS after 1 and 2 years of treatment.@*RESULTS@#There was no significant difference in HtSDS between the conventional and dasatinib chemotherapy groups before treatment. Within the first two years of treatment, the dasatinib chemotherapy group had a similar change trend of HtSDS as the conventional chemotherapy group. Four children in the dasatinib chemotherapy group reached the final adult height during follow-up, which was significantly lower than the target height (P=0.044). In the conventional chemotherapy group, there was no significant difference between final adult height and target height. In the dasatinib chemotherapy group, the children in adolescence had a significant change in HtSDS after treatment (P=0.032).@*CONCLUSIONS@#Dasatinib treatment may affect the final height of children with AML, and the use of dasatinib after the beginning of adolescence may lead to growth disorder, but dasatinib treatment has little effect on body height in the short-term treatment.

Oxidative Damage of Bone Marrow Stromal Cells Caused by Chemotherapy Drugs / 中国实验血液学杂志

OBJECTIVE@#To explore the oxidative damage of OP9 cells induced by daunorubicin (DNR) treatment.@*METHODS@#The TMRM probe was used to detect mitochondrial membrane potential by flow cytometry; the reactive oxygen species (ROS) was determined by flow cytometry DCFDA probe; the real-time PCR was used to detect the molecular expression of antioxidant enzyme，glutathione peroxidase (GPX) in OP9 cells; the expression of γ-H2AX was determined by flow cytometry.@*RESULTS@#Compared with normal OP9 cells, the positive rate of TMRM in DNR-treated OP9 cells decreased by 56.7% (P＜0.05); the positive rate of DCFDA in DNR-treated OP9 cells increased by 3.52 times (P＜0.01). Compared with normal OP9 cells, DNR-treated OP9 cells showed a decrease in the expression of GPX4 by 44.22% (P＜0.001); the expression of GPX7 decreased by 65.7% (P＜0.001); the expression of GPX8 decreased by 24.7% (P＜0.001); the positive rate of γ-H2AX in DNR-treated OP9 cells increased (P＜0.05).@*CONCLUSION@#After DNR treatment, mitochondrial membrane potential of OP9 cells decreases; the level of reactive oxygen species increases; the expression of glutathione peroxidase (GPX) molecules decreases significantly; genomic instability increases obviously; the oxidative damage of cells increased.

Effect of Chemotherapeutic Drug-Induced Damage of Bone Marrow Stroma Cells on Normal Hematopoietic Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of damage of bone marrow stroma cells induced by chemotherapeutic drug on the function of normal hematopoitic cells.@*METHODS@#Senescence cells were detected by flow cytometry after SA-β-gal staining; real-time PCR was used to detect the expression of a serial molecules in bone marrow stromal cell line OP9 cells; the expression of γ-H2AX was determined by flow cytometry after histone γ-H2AX staining; the colony forming ability of hematopoietic cells was tested by colony formation assay.@*RESULTS@#The percentage of senescence cells in OP9 cells after DNR treatment was 2.24 times as much as that in untreated OP9 cells (P<0.05). Compared with normal OP9 cells, the expression levels of IL-6 and TNF-alpha in DNR-treated OP9 cells increased by 2.73 times (P<0.01) and 0.56 times (P<0.01), and the expression levels of N-cadherin, alpha smooth muscle actin (alpha-SMA), angiopoietin1 (Angpt1) and osteopontin (OPN) decreased by 69.54%（P<0.01），63.90%（P<0.01），87.41%（P<0.01）and 42.78%（P<0.01）respectively. After the co-culture with DNR-treated OP9 cells, the colony formation of normal hematopoietic cells decreased by 47.10% than that co-cultured with untreated OP9 cells (P< 0.05), meanwhile, the percentage of γ-H2AX+ cells in normal hematopoietic cells increased by 2.19 times (P<0.05).@*CONCLUSION@#After treatment with DNR, the senescence cell number of OP9 cells sgnificantly increases; the expression of TNF-α and IL-6 is up-regulated, while the expression of α-SMA, Angpt-1 and OPN is down-regulated as compared with normal OP9 cells. In addition, after co-culture of DNR-treated OP9 cells with normal hematopoietic cells, the colony formation ability of hematopoietic cells decreases and the genome instability of hematopoietic cells increases as compared with normal hematopoietic cells.

Targeting BCMA in multiple myeloma using chimeric antigen receptor-engineered T cells / 中华血液学杂志

Objective: To construct the BCMA-CAR using the B-cell maturation antigen (BCMA) specific ligand APRIL as antigen binding region and to validate the effect of BCMA-CAR modified T cells (BCMA-CAR-T) on myeloma cells. Methods: The BCMA-CAR was constructed using the BCMA specific ligand APRIL as antigen binding domain and 4-1BB as the costimulatory domain. The specific cytotoxicity against BCMA(+) myeloma cell lines and primary multiple myeloma (MM) cells in vitro were evaluated. In addition, BCMA(+) myeloma xenograft mouse model was established to assess the anti-tumor effect of BCMA-CAR-T cell therapy in vivo. Results: BCMA-CAR-T cells could specifically kill BCMA(+) myeloma cell lines (For BCMA-CAR-T cells, BCMA(+) cells are almost undetectable in the E∶T ratio of 1∶4) and MM patients' bone marrow mononuclear cells (the proportion of residual cells in BCMA-CAR-T and vector-T groups was 16.0% vs 66.85%, P=0.003) with significant degranulation (CAR-T and vector-T cells cocultured with MM1.S, H929 and U266 had degranulation levels of 33.30% vs 5.62%, 16.97% vs 2.95% and 25.87% vs 2.97%, respectively, P<0.001) and cytokines release (P<0.01) in vitro. In a human BCMA(+) myeloma xenograft mouse model, BCMA-CAR-T cells could significantly prolong the survival of mice (The median survival time of mice treated with BCMA-CAR-T and vector-T cells was 87.5 days and 67.5 days, respectively, P<0.001) . Conclusion: The ligand-based BCMA-CAR-T cells could be a promising strategy for BCMA(+) multiple myeloma treatment.

Chimeric antigen receptors T cells for treatment of 48 relapsed or refractory acute lymphoblastic leukemia children: long term follow-up outcomes / 中华血液学杂志

Objective: To evaluate the safety and efficacy of chimeric antigen receptors T cells (CAR-T) in childhood acute B lymphoblastic leukemia (B-ALL) to probe the prognosis-related factors. Methods: Forty-eight children, 29 boys and 19 girls, aged 3-17years old (median age was 8 years old) , with recurrent or refractory CD19 positive B-ALL, were treated by the CD19 specific CAR-T cells. A total of 48 cases received 61 infusions. Flow cytometry or real-time quantitative polymerase chain reaction method were used to monitor micro residual disease (MRD) . The follow-up period was from 16 to 1 259 days with the median follow-up of 406 days. SPSS software was used to statistical analysis. Results: No adverse reaction was observed during 61 infusions. The most common adverse reaction after CAR-T cell infusions was cytokine-release syndrome (CRS) . Only 2 cases experienced level 3 CRS performance, including continuous high fever, convulsions, delirium, serous cavity effusion, and decreasing of blood pressure. Tocilizumab was given to release CRS performance. No treatment-related death occurred. Thirty-seven patients showed response during 7 to 28 days after infusions. The early response rate was 77.1%, with MRD before infusion less than 5% group higher than the MRD more than 5% group (87.1% vs 58.8%, χ2=4.968, P=0.036) . For the 37 patients who showed response to CAR-T cell infusions, univariate analysis identified that age, disease status at the time of treatment, MRD before infusion affected 2-year OS rate (P<0.05) . Multivariate prognostic analysis for EFS disclosed that the MRD before infusion more than 5% (RR=3.433, 95% CI 1.333-8.844, P=0.011) and not bridge to HSCT (RR=4.996, 95% CI 1.852-13.474, P=0.001) were the independent risk factors. Conclusion: The fourth generation CAR-T cells directed against CD19 could effectively and safely treat relapsed and refractory B-ALL, which implicated that CAR-T therapy as a novel therapeutic approach could be useful for patients with relapsed or refractory B-ALL who have failed all other treatment options. Reducing MRD as far as possible by effective pretreatment chemotherapy was in favor of increasing the response rate. Bridging HSCT after CAR-T cell treatment might be a better therapeutic strategy for the patient with refractory or molecular relapsed B-ALL.

Study of clinical outcome and prognosis in pediatric core binding factor-acute myeloid leukemia / 中华血液学杂志

Objective: To analyze the clinical outcome and the prognostic factor in pediatric patients with core binding factor-acute myeloid leukemia (CBF-AML). Methods: A total of 121 newly diagnosed pediatric CBF-AML patients enrolled from Aug. 2005 to Sep. 2017 were retrospectively reviewed. Cumulative incidence of relapse (CIR), event-free survival (EFS) and overall survival (OS) rates were estimated by Kaplan-Meier method and prognostic factors were evaluated by Cox regression with SPSS. Results: Of the 121 patients, 120 patients were assessed for bone marrow remission after induction chemotherapy. 100 cases (83.3%) achieved complete remission (CR) after the first course of chemotherapy. 119 cases (99.2%) achieved CR after the second course of chemotherapy. Of the 121 patients, 13 patients (10.7%) had recurrence with the median interval of recurrence as 13.8 months (3.7 to 58.8 months). 17 patients (14.0%) died. The CIR, EFS and OS at 3 years were 12.7%, 77.5% and 82.8%, respectively. The factors including age at diagnosis, sex, initial WBC count, presence of extramedullary leukemia, C-KIT expression, additional chromosomal abnormalities, and CR after the first course of chemotherapy were analyzed by multivariate regression analysis of Cox. Multivariate analysis identified that additional chromosomal abnormalities was the only independent risk factor affecting OS (HR=4.289, 95%CI 1.070-17.183, P=0.040). Conclusions: Pediatric CBF-AML was a unique setting of prognostic subtypes. Chemotherapy produced good responses. Additional chromosomal abnormalities was the only independent risk factor for OS in pediatric CBF-AML.

Effectiveness analysis of intensive management of dyslipidemia in community based on family doctor contracting service / 中华健康管理学杂志

Objective@#To analysis of the effect of strengthening management of dyslipidemia in community based on family doctor contracting service.@*Methods@#In December 2014, 1267 cases patients with dyslipidemia aged over 18 years were selected from three community health service centers in Hangzhou, including 645 in intervention group (311 males and 334 females) and 622 in control group (307 males and 315 females). In the intervention group, the management of dyslipidemia in community was strengthened by graded follow-up and personalized diagnosis and treatment based on the contracted services of family doctors, while the control group adopted the general management of dyslipidemia in the community. After 12 months of intervention, the changes of lifestyle (smoking, drinking, body mass index, waist circumference), regularly taking lipid-regulating drugs, blood lipid, blood pressure, blood sugar levels and their control rates were compared by χ2 test or t test before and after intervention between the intervention group and the control group. Non-conditional logistic regression analysis was used to analyze the influencing factors of blood lipid attainment.@*Results@#Before intervention, there were no significant differences in gender, age, cardiovascular risk stratification, the levels of lipid and other metabolic indicators, lipid compliance rate between intervention group and control group (P> 0.05). After intervention, the intervention group improved in drinking, overweight,obesity, abdominal obesity, and the rate of regularly taking lipid-regulating drugs increased,compared with the control group, the difference was statistically significant (χ2=5.923,4.765,8.587,5.341, 5.654; all P<0.05). The levels of total cholesterol, triglyceride, low density lipoprotein cholesterol, body mass index, waist circumference, systolic blood pressure, diastolic blood pressure, fasting blood glucose, glycosylated hemoglobinin the intervention group were lower than those in the control group, the differences were statistically significant (t=-4.987,-3.207, -6.280, -3.339, -2.466, -4.052, -5.012, -2.865, -2.450; all P<0.05), while the HDL-C level in the intervention group was higher than that in the control group (t=2.294; P<0.05). The control rate of blood lipids, the control rate of blood pressure, and the combined control rates of blood lipid, blood pressure and blood sugar in the intervention group were higher than those in the control group, the differences were all significant (χ2=31.262,4.818,17.245; all P<0.05). Unconditional logistic regression analysis showed that family doctor contracted services (OR=1.961, 95%CI: 1.485-2.589), gender (OR=0.662, 95%CI: 0.471-0.930), smoking (OR=0.498, 95%CI: 0.332-0.745), obesity (OR=0.570, 95%CI: 0.359-0.904), hypertension (OR=0.353, 95%CI: 0.259-0.480), diabetes mellitus (OR=0.340, 95%CI: 0.239-0.483) was the influencing factor of blood lipid reaching the target (all P<0.05).@*Conclusion@#Intensive management of dyslipidemia in community based on family doctor's contracting service is helpful to improve the management effect of dyslipidemia.

Exploration of Mechanism for Meisoindigo-Inducing K562 Cell Apoptosis by Using Quantitative Proteomic Analysis / 中国实验血液学杂志

OBJECTIVE@#To screen the differentially expressed proteins at the early stage of K562 cells treated with meisoindigo by using tandem mass tags (TMT)-based proteomics technology, and to explore the mechanism for meisoindigo-inducing apoptosis.@*METHODS@#The half inhibitory concentration (IC) of mesoindigo on K562 cells was determined by CCK8. The flow cytometry was used to assay the apoptosis of K562 cells treated by meisoindigo or DMSO. Total proteins were extracted from the cells treated with 0.2% DMSO (control) or 20 μmol/L meisoindigo (Test) for 2 hours. Then, the TMT-labeling HPLC-MS/MS was used to identify and quantify the peptides and their abundance, all the tests were repeated for 3 times. The Mascot software was used to identify the proteins; the GO annotations, enrichment and cluster analysis were used to analyze the differentially expressed proteins.@*RESULTS@#Meisoindigo-induced K562 cell apoptosis in a dose-dependent manner (r=0.98), 5 544 proteins were identified, 4792 of which were quantified. The protein with expression difference>1.5-folds in Test group accoanted for 8, out of which the expression of 4 proteins were up-regulated and 4 were down-regulated. The differentially expressed proteins mainly associated with reactive oxygen species (ROS).@*CONCLUSION@#Several proteins including DDIT4 were found to have dramatic changes in the early stage of K562 cells treated with meisoindigo by using quantitative proteomics technology. The ROS metabolic process may play important roles in meisoindigo-inducing apoptosis of K562 cells.