Objective:To construct a novel dual-specific antibody targeting human CD123 (CD123 DuAb) and study its effects in acute myeloid leukemia (AML) .Methods:Based on the variable region of the CD123 monoclonal antibody independently developed at our institution, the CD123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein. Through a series of in vitro experiments, its activation and proliferation effect on T cells, as well as the effect of promoting T-cell killing of AML cells, were verified.Results:① A novel CD123 DuAb plasmid targeting CD123 was successfully constructed and expressed in the Expi-CHO eukaryotic system. ②The CD123 DuAb could bind both CD3 on T cells and CD123 on CD123 + tumor cells. ③When T cells were co-cultured with MV4-11 cells with addition of the CD123 DuAb at a concentration of 1 nmol/L, the positive expression rates of CD69 and CD25 on T cells were 68.0% and 44.3%, respectively, which were significantly higher than those of the control group ( P<0.05). ④Co-culture with CD123 DuAb at 1 nmol/L promoted T-cell proliferation, and the absolute T-cell count increased from 5×10 5/ml to 3.2×10 6/ml on day 9, and CFSE fluorescence intensity decreased significantly. ⑤ With the increase in CD123 DuAb concentration in the culture system, T-cell exhaustion and apoptosis increased. When the CD123 DuAb was added at a concentration of 1 nmol/L to the culture system, the proportion of CD8 + PD-1 + LAG-3 + T cells was 10.90%, and the proportion of propidium iodide (PI) - Annexin Ⅴ + T cells and PI + Annexin Ⅴ + T cells was 18.27% and 11.43%, respectively, which were significantly higher than those in the control group ( P<0.05). ⑥ The CD123 DuAb significantly activated T cells, and the activation intensity was positively correlated with its concentration. The expression rate of CD107a on T cells reached 16.05% with 1 nmol/L CD123 DuAb, which was significantly higher than that of the control group ( P<0.05). ⑦The CD123 DuAb promoted cytokine secretion by T cells at a concentration of 1 nmol/L, and the concentration of IFN-γ and TNF-α in the supernatant of the co-culture system reached 193.8 pg/ml and 169.8 pg/ml, respectively, which was significantly higher than that of the control group ( P<0.05). ⑧When CD123 DuAb was added at a concentration of 1 nmol/L to the co-culture system of T cells and CD123 + tumor cells, the killing intensity of T cells significantly increased, and the residual rates of CD123 + MV4-11 cells, CD123 + Molm13 cells, and CD123 + THP-1 cells were 7.4%, 6.7%, and 14.6% on day 3, respectively, which were significantly lower than those in the control group ( P<0.05) . Conclusion:In this study, a novel CD123 DuAb was constructed and expressed. In vitro experiments verified that the DuAb binds to CD123 + tumor cells and T cells simultaneously, promotes T-cell activation and proliferation, and facilitates their anti-leukemia effect, which provides a basis for further clinical research.

Objective:To construct a novel chimeric antigen receptor T (CAR-T) cell targeting CD138 and to investigate its cytotoxicity against myeloma cells.Methods:The hybridoma strain that can stably secrete the CD138 monoclonal antibody (mAb) was prepared and obtained through monoclonal antibody screening technology. The hybridoma strain cells were intraperitoneally injected into mice to produce ascites containing monoclonal antibodies, which were then collected and purified to obtain pure CD138 mAb. Further examinations were performed to assess the biological characteristics of CD138 mAb. The variable region sequence of this antibody was amplified through reverse transcription polymerase chain reaction and was used as the antigen recognition domain of CD138 CAR, which was subsequently expressed on the surface of T cells by lentiviral infection. Flow cytometry was employed to assess the phenotype of CD138 CAR-T cells. In vitro cytotoxicity and degranulation assays were performed to evaluate their antitumor effects.Results:① We successfully prepared anti-human CD138 antibody hybridoma cell lines and screened a hybridoma cell strain, 5G2, which could persistently and stably secrete the anti-CD138 antibody. ② The purified CD138 (5G2) mAb can especially recognize CD138 + cells with a binding affinity constant (K D) of 6.011×10 -9 mol/L and showed no significant binding activity with CD138 - cells. ③The variable region sequence of the CD138 (5G2) antibody was obtained using molecular cloning technology, and CD138 (5G2) CAR was successfully constructed and expressed on T cells through lentivirus infection and, concurrently, demonstrated effective binding to recombinant human CD138 protein.④ The proliferation of T cells transduced with the CD138 (5G2) CAR was highly efficient. The phenotype analysis revealed that CD138 (5G2) CAR-T cells exhibited a greater tendency to differentiate into central memory T cells and memory stem T cells, with a reduced proportion of terminally differentiated effector memory subsets. ⑤CD138 (5G2) CAR-T cells demonstrated specific cytotoxicity against CD138 + myeloma cell line H929, whereas CD138 - cell line K562 remained unaffected. The percentage of residual H929 cells was (12.92±8.02) % after co-culturing with CD138 (5G2) CAR-T cells, while (54.25±15.79) % was left in the Vector-T group (E∶T=1∶2; P<0.001). ⑥Results of degranulation assays demonstrated a significant activation of CD138 (5G2) CAR-T cells after co-culture with the H929 cell line, whereas no significant activation was observed in Vector-T cells [ (25.78±3.35) % vs (6.13±1.30) %, P<0.001]. ⑦After co-culturing with CD138 + cells, CD138 (5G2) CAR-T cells exhibited a significant increase in cytokine secretion compared to the Vector-T group [interleukin-2: (1 697.52±599.05) pg/ml vs (5.07±1.17) pg/ml, P<0.001; interferon-γ: (3 312.20±486.38) pg/ml vs (9.28±1.46) pg/ml, P<0.001; and tumor necrosis factor-α: (1 837.43±640.49) pg/ml vs (8.75±1.65) pg/ml, P<0.001]. However, no significant difference was observed in cytokine secretion levels between the two groups after co-culturing with CD138 - cells. Conclusion:This study successfully prepared a novel monoclonal antibody against CD138, and CAR-T cells constructed with the antigen recognition domain derived from this 5G2 mAb demonstrated effective antitumor activity against myeloma cells. This can be used as a new option for the detection of the CD138 antigen and proposes a novel strategy for multiple myeloma immunotherapy.

OBJECTIVE: To assess the practical and health economical values of non-invasive prenatal test (NIPT) in Changsha Municipal Public Welfare Program. METHODS: A retrospective analysis was carried out on 149 165 women undergoing NIPT test from April 9, 2018 to December 31, 2019. For pregnant women with high risks, invasive prenatal diagnosis and follow-up of pregnancy outcome were conducted. The cost-benefit of NIPT for Down syndrome was analyzed. RESULTS: NIPT was carried out for 149 165 pregnant women and succeeded in 148 749 cases (99.72%), for which outcome were available in 148 538 (99.86%). 90% of pregnant women from the region accepted the screening with NIPT. 415 (0.27%) were diagnosed as high risk. Among these, 381 (91.81%) accepted amniocentesis, which led to the diagnosis of 212 cases of trisomy 21 (PPV=85.14%), 41 cases with trisomy 18 (PPV=48.81%) and 10 cases with trisomy 13 (PPV=20.83%). The sensitivity and specificity of NIPT for trisomy 21, trisomy 18 and trisomy 13 were (97.70%, 99.98%), (97.62%, 9.97%) and (100%, 99.97%), respectively. In addition, 213 and 30 cases were diagnosed with sex chromosomal aneuploidies (PPV=46.2%) and other autosomal anomalies (PPV=16.57%), respectively. For Down syndrome screening, the cost and benefit of the project was 120.79 million yuan and 1,056.95 million yuan, respectively. The cost-benefit ratio was 1: 8.75, and safety index was 0.0035. CONCLUSION NIPT is a highly accurate screening test for trisomy 21, which was followed by trisomy 18 and sex chromosomal aneuploidies, while it was less accurate for other autosomal aneuploidies. The application of NIPT screening has a high health economical value.
Aneuploidy ; Cost-Benefit Analysis ; Female ; Humans ; Noninvasive Prenatal Testing ; Pregnancy ; Retrospective Studies ; Trisomy 18 Syndrome/genetics*

Objective:To prepare a novel tri-specific T cell engager (19TriTE) targeting CD19 antigen, and to investigate its immunotherapeutic effect on CD19-positive hematological malignancies.Methods:19TriTE was constructed by molecular cloning technology and successfully expressed through the eukaryotic expressing system. The effects of 19TriTE on the proliferation and activation of T cells, as well as the specific cytotoxicity against CD19 positive tumor cell lines were verified.Results:①19TriTE expressing plasmid was constructed and successfully expressed through the eukaryotic expressing system. ②19TriTE can specifically bind to T cells and Nalm6 cells, with equilibrium dissociation constants of 19.21 nmol/L and 11.67 nmol/L, respectively. ③The expression rates of CD69 positive T cells and CD25 positive T cells were 35.4% and 49.8% respectively, when 2 nmol/L 19TriTE were added in the co-culture system, which were significantly higher than those in the control group. ④19TriTE can significantly promote the proliferation of T cells. The absolute count of T cells expanded from the initial one million to 74 million with an 74 fold increase at the concentration of 1 nmol/L on day 12. ⑤19TriTE can significantly mediate T cells killing of CD19 positive target cells in a dose-dependent manner. At the concentration of 10 nmol/L, the target cells lysis reached 50%. ⑥Degranulation experiment verified that 19TriTE can activate T cells in the presence of CD19 positive target cells, and the activation of T cells positively correlated with the dose of 19TriTE. ⑦When 19TriTE fusion protein co-cultured with T cells and target cells overexpression RFP and luciferase genes respectively, 19TriTE can notably mediate T cells killing of CD19 positive target cells through fluorescent microscope or bioluminescence imaging technology.Conclusion:In this study, we successfully constructed and expressed 19TriTE fusion protein and verified that it can effectively activate T cells and promote their proliferation in vitro. At the same time, it can bind to CD19 positive target cells and T cells, as well as enhance T cells anti-leukemia effect in vitro, providing the foundation for further clinical research.

Objective:To discuss whether platelet distribution width (PDW) can effectively predict the prognosis of neuroblastoma (NB).Methods:The clinical data of 67 NB patients in the First Affiliated Hospital of Zhengzhou University between January 2014 and January 2018 were retrospectively analyzed.They were divided into low PDW group and high PDW group according to the PDW level, and the differences in clinical indicators between the 2 groups were compared.The prognostic effects of PDW were assessed by using the Kaplan- Meier method and Cox regression model. Results:Among the 67 patients, 41 cases were male, 26 cases were female, with the ratio of male to female being 1.58∶1.00, and the average age was 44 months (2-156 months). Five cases were in stage Ⅰ, 1 case in stage Ⅱ, 15 cases in stage Ⅲ and 46 cases in stage Ⅳ.At the first time of diagnosis, there were 14 cases with age ≤ 18 months, 53 cases with age > 18 months, 47 cases with neuron specific enolase (NSE) level ≥ 100 μg/L, 20 cases with NSE level<100 μg/L.The median follow-up time was 20.4 months.At the end of follow-up, 35 cases died and 32 cases survived.There was no statistical difference in age, gender, primary site of tumor, tumor stage and mean platelet volume between the low PDW group and the high PDW group (all P>0.05). The proportion of high-risk patients, the level of NSE, bone marrow metastasis rate, MYCN gene amplification rate and the red blood cell distribution width in the high PDW group were significantly higher than those in the low PDW group, but the high PDW group had a lower level of thrombocytocrit than the low PDW group, and the differences were statistically significant(all P<0.05). Survival analysis revealed that the 2-year overall survival of the low PDW group was significantly higher than that of the high PDW group (69.8% vs.25.3%, χ2=15.761, P<0.05). Univariate analysis showed that NSE ( HR=6.606, 95% CI: 2.018-21.620), MYCN gene ( HR=1.977, 95% CI: 0.794-4.919), tumor risk stratification ( HR=5.926, 95% CI: 1.416-24.794), PDW ( HR=4.036, 95% CI: 1.957-8.322), and red blood cell distribution width ( HR=1.120, 95% CI: 1.005-1.249) were the adverse factors affecting the overall survival, and thrombocytocrit was a protective factor for the prognosis of NB.Multivariate analysis indicated that PDW was an independent risk factor of NB ( HR=2.524, 95% CI: 1.017-6.264, P=0.046). Conclusions:There is a good consistency between the increase of PDW and the known prognostic risk factors, elevated tumor markers and bone marrow metastasis.Increased PDW is associated with poor prognosis in NB patients, and PDW is an independent risk factor for the poor prognosis of NB.

Objective:To analyze the influence of CD19 isoforms to the efficacy of CD19/CD3 Bispecific T-cell Engager (BiTE) antibody, and explore the resistance mechanism of BiTE immunotherapy.Methods:Semi-quantitative RT-PCR (qRT-PCR) was used to detect the expression of CD19 mRNA isoforms before and after BiTE treatment in a patient with CD19 + B cell acute lymphoblastic leukemia (ALL) . CD19 isoforms were analyzed by Sanger sequencing. Flow cytometry and transcriptome sequencing were performed to analyze the expression of cell lineage specific molecules before and after BiTE treatment. Results:The expression of CD19 isoform with exon 2 deletion was identified at diagnosis. After relapsed and treatment of BiTE antibody, the patient did not achieve remission and CD19 antigen on leukemic cells turned negative detected by flow cytometry after BiTE treatment. However the expression ratio of CD19 isoform with exon 2 deletion was not increased. Flow cytometry phenotype and transcriptome sequencing confirmed that no linage switching developed, which suggested the expression of CD19 isoform caused by exon alternative splicing and lineage switching was not related to CD19 epitope loss in this patient. This patient achieved complete remission by sequential administration of self-developed CD22 CAR-T and CD19 CAR-T after disease progression.Conclusion:Targeting or combining an alternative antigen specific CAR-T may be a promising treatment option after losing CD19 expression in relapsed ALL.

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.

Objective To summarize the cLinicaL features of cerebraL venous sinus thrombosis (CVST) in 6 chiLdren, and to improve the understanding of CVST in chiLdren. Methods The risk factors, cLinicaL presentations, Laboratory findings, imaging manifestations, treatments and outcomes of 6 chiLdren (3 maLes, 3 femaLes) with CVST admitted to the Department of Pediatrics, First AffiLiated HospitaL of Zhengzhou University from January 2012 to December 2017 were anaLyzed retrospectiveLy. ResuLts The risk factors of disease were found in 5 cases, incLuding 3 cases of infection, 1 case of L?asparaginase and dexamethasone chemotherapy and 1 case of oraL prednisone aLone. No definite risk factor was found in 1 case. The cLinicaL presentations were headache in aLL cases, vomiting in 3 cases, convuLsion in 2 cases, hemipLegia, photophobia, phonophobia and Limitation of eyebaLL abduction in 1 case respectiveLy. Magnetic resonance imaging (MRI) and magnetic resonance venography (MRV) showed 2 cases of intracraniaL hemorrhage, 2 cases of cerebraL parenchymaL infarction and 2 cases of abnormaL signaL in venous sinus. Thrombus Located in superior sagittaL sinus in 4 cases, transverse sinus in 4 cases, sigmoid sinus in 3 cases and straight sinus in 1 case. After anticoaguLation treatment, headache and vomiting were aLLeviated, and veins were recanaLized in 6 cases. Two cases were diagnosed with eyebaLL abduction Limitation and hemipLegia but did not improve in the short term after treatment. ConcLusions Infection is the main risk factor of CVST in chiLdren and headache is often the cause of medicaL consuLtation. Brain MRI and MRV are heLpfuL in diagnosis and timeLy treatment can improve prognosis.

Objective To compare the quality of Lonicerae flos and Lonicerae japonicae flos by determine the main active ingredients. Methods According to methods and standards of determination in Chinese Pharmacopoeia (2015 Edition), the content of chlorogenic acid , galuteolin , macranthoidin B and asperosaponin B in 50 batches of samples of Lonicerae japonicae flos and .Lonicerae flos were determined. Results For Lonicerae japonicae flos (Lonicera japonica Thunb.), the contents of galuteolin was higher, and chlorogenic acid was lower, less or no contain saponins. For the main species of Lonicerae flos (Lonicera macranthoides Hand.-Mazz. and Lonicera hypoglauca Miq.), the contents of chlorogenic acid and the sum of saponins were higher, less or no galuteolin. Conclusions The main active ingredients of Lonicerae japonicae flos and Lonicerae flos were different. The contents of saponins in some samples of Lonicerae japonicae flos were higher, so the test of saponin should be considered when its raw material for injection.