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.
CD52 Antigen ; Cell Line, Tumor ; Humans ; Immunotherapy, Adoptive/methods* ; Lentivirus/genetics* ; Leukemia ; Receptors, Antigen, T-Cell ; Receptors, Chimeric Antigen/genetics*

OBJECTIVES: To investigate the clinical treatment outcomes and the changes of the outcomes over time in extremely preterm twins in Guangdong Province, China. METHODS: A retrospective analysis was performed for 269 pairs of extremely preterm twins with a gestational age of <28 weeks who were admitted to the department of neonatology in 26 grade A tertiary hospitals in Guangdong Province from January 2008 to December 2017. According to the admission time, they were divided into two groups: 2008-2012 and 2013-2017. Besides, each pair of twins was divided into the heavier infant and the lighter infant subgroups according to birth weight. The perinatal data of mothers and hospitalization data of neonates were collected. The survival rate of twins and the incidence rate of complications were compared between the 2008-2012 and 2013-2017 groups. RESULTS: Compared with the 2008-2012 group, the 2013-2017 group (both the heavier infant and lighter infant subgroups) had lower incidence rates of severe asphyxia and smaller head circumference at birth (P<0.05). The mortality rates of both of the twins, the heavier infant of the twins, and the lighter infant of the twins were lower in the 2013-2017 group compared with the 2008-2012 group (P<0.05). Compared with the 2008-2012 group, the 2013-2017 group (both the heavier infant and lighter infant subgroups) had lower incidence rates of pulmonary hemorrhage, patent ductus arteriosus (PDA), periventricular-intraventricular hemorrhage (P-IVH), and neonatal respiratory distress syndrome (NRDS) and a higher incidence rate of bronchopulmonary dysplasia (P<0.05). CONCLUSIONS There is a significant increase in the survival rate over time in extremely preterm twins with a gestational age of <28 weeks in the 26 grade A tertiary hospitals in Guangdong Province. The incidences of severe asphyxia, pulmonary hemorrhage, PDA, P-IVH, and NRDS decrease in both the heavier and lighter infants of the twins, but the incidence of bronchopulmonary dysplasia increases. With the improvement of diagnosis and treatment, the multidisciplinary collaboration between different fields of fetal medicine including prenatal diagnosis, obstetrics, and neonatology is needed in the future to jointly develop management strategies for twin pregnancy.
Bronchopulmonary Dysplasia/epidemiology* ; Female ; Gestational Age ; Humans ; Infant ; Infant, Extremely Premature ; Infant, Newborn ; Pregnancy ; Respiratory Distress Syndrome, Newborn/epidemiology* ; Retrospective Studies ; Treatment Outcome

Objective To explore the possibility of adipose-derived stem cells (ADSCs) to differentiate into fibroblasts, and to provide an effective way for the effective solution of skin tissue engineering seed cells. Methods Primary ADSCs were obtained from inguinal fat of ten healthy adult SD rats,weighing 280-320 g,and cultured in vitro and purified. When primary ADSCs expansion to the 3rd passages,the following experiments were performed alkaline phosphatase test on the 16th day after osteogenesis induction,staining of alizarin red mineralized nodules on day 23 after osteogenesis induction, oil red O staining on day 12 after adipogenic induction; Flow cytometry detection of cell surface markers; Addition of conditioned medium to induce differentiation into fibroblasts,Photograph the changes of cell morphology on the 2nd, 4th, 6th, and 8th days after induction,MTT to detect cell viability at various time points;Scanning electron microscopy on day 6 and day 8 after induction;Immunocytochemical staining on day 8 after induction,detect the expression of vimentin, the main marker of fibroblasts. Results Primary ADSCs grew in long spindles, showed strong positive expression of alkaline phosphatase (ALP) after osteogenesis induction,and alizarin red staining showed red mineralized nodules;Aggregation of intracellular red-stained lipid droplets after adipogenic induction were found;Flow cytometry showed positive expression of mesenchymal stem cell-related marker CD90,and hematopoietic stem cell marker CD45 was negative. Morphology of ADSCs started to change on day 2 after induction into fibroblasts. On the 4th day after induction, the cells were in the shape of water droplets or short rods. On the 6th day after induction, the cells were protruded polygonal or triangular. Cells crowded and covered the bottom of the bottle on day 8 after induction,becoming slender fibrous. MTT test showed that the cell viability was significantly lower on the second day after induction than in the control group. There were no significant differences in cell viability on the 4th, 6th, and 8th days after induction compared with the control group. Scanning electron microscopy showed that the cells were triangular on the 6th day after induction, and the surface had more cilia. On the 8th day after induction, the cells were slender and fibrous, with small protrusions, and the surface cilia were dense. Vimentin was positively expressed in most cells on the 8th day after induction. Conclusion ADSCs can have the morphological characteristics of fibroblasts after induced differentiation in vitro; that can express fibroblasts marker protein.

Objective To analyze the levels of cytokines (IL-2,IFN-γ,IL-6,IL-10) associated with Th1 and Th2 cells in HLA-DQ8 transgenic mice model of ocular experimental autoimmune myasthenia gravis (oEAMG) induced by recombinant H-AChR γ subunit immunization.Methods DQ8 mice were immunized with 20 μg of AChR γ subunit,20 μg of crude E. coli extract (E. coli group),or complete Freund's adjuvant (CFA) only (CFA group). All mice were immunized on days 0,30,and 60. Mice were euthanized 28 days after the third immunization,and draining lymph node cells (LNC) and spleen lymphocytes were cultured in vitro. The supernatant was collected to observe the interleukin(IL)-2,interferon(IFN)-γ,IL-6,IL-10 production by ELISA.Results LNCs and spleen lymphocytes of H-AChR γ subunit-immunized mice exhibited significantly enhanced IFN-γ (F=76.332,P<0.001;F=34.865,P<0.001) and IL-2 (F=42.835,P<0.001;F=38.030,P<0.001),which associated with Th1 cells,as compared to E. coli group and CFA group. There were no significant differences in IL-6 (F=1.325,P=0.284;F=1.935,P=0.166) and IL-10 (F=0.908,P=0.417;F=1.189,P=0.322) levels,which secreted by Th2 cells,among these three groups.Conclusion Th1 cytokines play key roles in the pathogenesis of oEAMG,while the mechanism of Th2 cytokines for oEAMG remains unclear.
Animals ; Cytokines ; Escherichia coli ; HLA-DQ Antigens ; Interferon-gamma ; Mice ; Mice, Transgenic ; Receptors, Cholinergic ; Th1 Cells ; Th2 Cells

Ocular myasthenia gravis(OMG)is an autoimmune disease caused by neuromuscular junction transmission disorders and manifested mainly as fluctuating blepharoptosis and diplopia,with the extraocular muscles as the main involveed sites.While the pathogenesis of OMG remains unclear,some antibodies,complements,and cytokines may be the contributing factors.The diagnosis and treatment of OMG have been defined in recent years.This article reviews the pathogenesis,diagnosis,and treatment of OMG.
Antibodies ; Complement System Proteins ; Cytokines ; Humans ; Myasthenia Gravis ; diagnosis ; pathology ; therapy ; Oculomotor Muscles ; pathology

Retinopathy of prematurity(ROP)is a pathological neovascularization with fibrotic changes in the fundus of premature infants.It is a major cause of preventable blindness in children in both developing and developed countries.Treatment of ROP has long been a hot research topic in ophthalmology and pediatrics.With a clearer knowledge of the pathogenesis of ROP,more basic and clinical studies have been carried out.The anti-vascular endothelial growth factor therapy and surgical treatment have become mature strategies,and a variety of therapeutic drugs including insulin-like growth factor-1,transforming growth factor-β,polyunsaturated fatty acids,and β-adrenergic receptor blockers have been developed.This article reviews the recent advances in ROP.
Child ; Humans ; Infant, Newborn ; Infant, Premature ; Neovascularization, Pathologic ; therapy ; Retinopathy of Prematurity ; drug therapy ; surgery ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors

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.
Animals ; Cytokines ; Humans ; Immunotherapy, Adoptive ; Mice ; Multiple Myeloma/therapy* ; Receptors, Antigen, T-Cell ; Receptors, Chimeric Antigen ; T-Lymphocytes

To study the characteristics of adsorption and desorption of Scutellaria baicalensis pieces. On the basic thermodynamic theory, thestatic method was adopted to obtain S. baicalensis pieces' isothermal adsorption and desorption data at 25, 35, 45 ℃, with the water activity between 0.10 and 0.85. Eight moisture models were selected to fit the data and then evaluated to determine the thermodynamic properties of S. baicalensis pieces. The results show that, among the eight adsorption models, Peleg fit the best, in which absolutely-safe and the relatively-safe moisture contents of S. baicalensis pieces were around 9.22% and 13.51% respectively; the net equivalent heat adsorption and desorption and the differential entropy of S. baicalensis pieces were closely related to moisture content, and decrease within crease of water content; when drying the pieces, 12.0% of moisture content can be taken as the drying end; both of adsorption and desorption processes were driven by enthalpy and non-spontaneous. The study on S. baicalensis pieces' isothermal adsorption and desorption rules can help advance studies on adsorption and desorption rules of Chinese herbal pieces, and play a good guiding role in optimizing storage conditions and drying process of Chinese herbal pieces.

Objective To analyze the contact failure of monolithic lithium disilicate CAD/CAM crowns by experiment and numerical simulation, and explore the influences of adhesives aging in water on load-bearing capacity of the crowns. Methods The specimens of sectioned monolithic lithium disilicate crowns were designed and manufactured, and evenly divided into two groups and stored in the air and in the distilled water for 30 days, respectively. The specimens were then subjected to monotonic contact loads to compare and analyze their load-bearing capacity. The fractured surfaces and adhesive interfaces of the specimens were observed by scanning electronic microscope. Meanwhile, the stress distribution on the crowns was calculated by numerical simulation to analyze the adhesives aging influence on load-bearing capacity of the crowns. Results The fracture loads on crowns stored in the air and in the water were (561.51 ± 65.66) N and (398.09 ± 90.20) N, respectively, indicating a significant difference. The tensile stress increased considerably at lower surface of the ceramic crown due to the reduction of adhesive strength at the interface of ceramic crown and substrate, which could increase the propensity of contact failure. Conclusions The adhesives aging in water reduces the bonding strength, and accordingly changes the tensile stress distributions, which can lower the load bearing capacity of the lithium disilicate crowns. The research finding provides references for the design and manufacturing of all-ceramic CAD/CAM restored crowns in clinic.

The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3. Seventy-six percent of full amino acid sequence of the fusion protein FP3 was measured by LC-ESI-Q-TOF with the remaining 24% completed by LC-ESI-Trap. As LC-MS and tandem mass spectrometry are rapid, sensitive, accurate to measure the protein amino acid sequence, they are important approach to structure analysis and identification of recombinant protein.
Amino Acid Sequence ; Chromatography, High Pressure Liquid ; Molecular Sequence Data ; Peptide Mapping ; Recombinant Fusion Proteins ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Tandem Mass Spectrometry ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; chemistry