BACKGROUND:Growth factor is the key effect molecule that plays a role in platelet-rich plasma in clinical treatment.There are differences in the concentration of growth factor after different activators activate platelet-rich plasma,which is an important factor affecting clinical efficacy. OBJECTIVE:To analyze the influence of different activators on the mass concentration of growth factors in platelet-rich plasma. METHODS:Totally 12 healthy volunteers were recruited to collect EDTA-K2 anticoagulant venous blood.Secondary centrifugation was used to prepare platelet-rich plasma.The difference in mass concentrations of growth factors was compared between venous blood and platelet-rich plasma.The platelet-rich plasma was mixed with four activators(normal saline,thrombin,calcium gluconate,calcium gluconate+thrombin)according to the volume ratio of 10:1,and incubated in a constant temperature water bath at 37 °C for 30 minutes.After centrifugation,the supernatant was extracted and the mass concentration of growth factor was detected.The bacterial growth in supernatant was measured by blood agar plate.Pearson correlation was used to analyze the correlation between different activators and the mass concentration of growth factor in platelet-rich plasma,and the correlation between the value of thrombocytometer and the mass concentration of growth factors in platelet-rich plasma. RESULTS AND CONCLUSION:(1)The mass concentrations of platelet-derived growth factor-BB,platelet-derived growth factor-AB,vascular endothelial growth factor,and epidermal growth factor in platelet-rich plasma were 8.7,22.2,2.3,and 2.8 times of those in venous blood,respectively(P<0.05).(2)Compared with normal saline group,the mass concentrations of platelet-derived growth factor BB,platelet-derived growth factor AB,vascular endothelial growth factor,and epidermal growth factor were increased in the thrombin group,calcium gluconate group,and calcium gluconate+thrombin group(P<0.05).The mass concentration of platelet-derived growth factor BB in the thrombin group and calcium gluconate group was higher than that in the calcium gluconate+thrombin group(P<0.05),and the mass concentration of platelet-derived growth factor AB in the thrombin group was higher than that in the calcium gluconate group and calcium gluconate+thrombin group(P<0.05).Epidermal growth factor mass concentration in the thrombin group was lower than that in the calcium gluconate group and calcium gluconate+thrombin group(P<0.05).(3)The results of blood agar plate test showed no bacterial growth in the supernatant of the four groups.(4)Pearson correlation analysis showed that the mass concentration of platelet-derived growth factor BB in platelet-rich plasma was strongly positively correlated with thrombin(r=0.683,P<0.05),and the mass concentration of vascular endothelial growth factor was strongly positively correlated with thrombin,calcium gluconate,calcium gluconate+thrombin stimulant(r=0.730,0.789,0.686,P<0.05).There was no correlation between the value of thrombocytometer and the mass concentration of four kinds of growth factors(P>0.05).(5)The results suggest that different activators have an impact on the concentration of growth factors in platelet-rich plasma.It is suggested to choose different activators to improve clinical efficacy according to different growth factor mass concentrations and treatment needs.

Based on his extensive clinical experience and the team's mechanistic research, Professor XU Nenggui has proposed the academic concept that "the governor vessel governs the brain and treats cerebral viscus diseases", and established a novel acupuncture approach for encephalopathy treatment centered on the integrated theory of "governor vessel-brain-mind", and developed a staged acupuncture protocol of "governor vessel regulating spirit" for ischemic stroke. This article introduces the academic features of this method in treating post-stroke dysphagia from four aspects: theoretical framework, treatment principles and point selection, mechanistic research, and clinical case studies. In clinical application, the method emphasizes syndrome differentiation based on meridians, harmonization between the conception and governor vessels; precise acupoint selection to treat both form and spirit; stage-specific differentiation with targeted needling. Furthermore, Professor XU integrates basic research with clinical practice, focusing on the neurobiological mechanisms underlying the efficacy of acupuncture in treating post-stroke dysphagia.
Acupuncture Therapy/methods* ; Humans ; Deglutition Disorders/psychology* ; Stroke/complications* ; Meridians ; Acupuncture Points

BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

Objective This study aimed to explore the differences in global and local brain network topological properties between deficient pattern(DP)and excess pattern(EP)of mild vascular cognitive impairment caused by subcortical small vessel disease based on graph theory network.Methods Patients were recruited prospectively and were classified with DP and EP subtype.The global small-world topological attributes and local nodes were calculated for the comparison of DP,EP,and healthy controls(CN)using the GRETNA platform.Results The three groups all had small-world attributes,but only the patients in EP had a significantly lower small world attribute δ in the range of 0.05-0.26 than the control group(P<0.05).The node efficiency and node strength indicators of multiple brain region were able to significantly distinguish the DP group from the EP group.However,there was no positive brain region in the node efficiency of the DP patients(P>0.05),and only a few brain regions showed increased node strength efficiency(P<0.05).Conclusion The results indicate that the syndrome of DP and EP have significantly different neuroimaging phenotypes,providing a basis for further research of biological classification based on Chinese Medicine syndromes.

Objective:To assess the effect of low-dose esketamine on postoperative cognitive function in elderly patients undergoing non-cardiac surgery.Methods:One hundred and twenty-four patients, aged 65-80 yr, regardless of gender, with a body mass index of 18-35 kg/m 2, of American Society of Anesthesiologists Physical Status classification Ⅰ or Ⅱ, were assigned to either esketamine group (group E, n=64) or control group (group C, n=60) using a random number table method. Group E received intravenous esketamine at a dose of 0.5 mg/kg, while group C received an equal volume of normal saline intravenously. Anesthesia was induced with intravenous fentanyl, propofol and rocuronium and maintained using combined intravenous-inhalational anesthesia in both groups. Patient-controlled analgesia was carried out postoperatively. Cognitive function tests including a simple mental state examination, auditory word learning test, tracking connection test A and B, number symbol replacement test, Boston naming test and complex graph test were performed at 1 day before surgery and 30 days after surgery, and postoperative cognitive dysfunction was determined using Z-score method. Delirium was assessed using Confusion Assessment Method from 1 to 7 days after operation. The operative hypotension, postoperative delayed emergence, nausea and vomiting, and hallucinations were recorded. The recovery time of spontaneous breathing, eye opening to verbal command and extubation time were recorded. Results:Compared with group C, the incidence of cognitive dysfunction at 30 days after surgery and intraoperative hypotension was significantly decreased ( P<0.05), and no significant change was found in the recovery time of spontaneous breathing, eye opening to verbal command, extubation time, incidence of postoperative delayed emergence, delirium, nausea and vomiting, and hallucinations in group E ( P>0.05). Conclusions:Low-dose esketamine can improve postoperative cognitive function in elderly patients undergoing non-cardiac surgery.

BACKGROUND:Adjustable piezoelectric effect can promote tissue regeneration and repair.Piezoelectric materials are widely used in weight-bearing tissue engineering. OBJECTIVE:To prepare a piezoelectric film material that can promote bone regeneration,and to explore its structural characterization,electrical output performance,biocompatibility,and effect of electrical output on osteogenic differentiation of rabbit bone marrow mesenchymal stem cells. METHODS:Using poly-3-hydroxybutyrateco/4-hydroxybutyrate(P34HB)as raw material,barium calcium stannate titanate powder(Ba0.94Ca0.06Sn0.08Ti0.92O3,BCST)was added according to mass ratios of 0%,5%,10%,15%,and 20%.Dichloromethane was added to solve P34HB,and the thickness of 150-200 μm BCST/P34HB piezoelectric film was prepared by vacuum drying method.After polarization in the oil bath,the surface morphology,crystal phase composition,piezoelectric coefficient and open circuit voltage were tested.The effect of BCST/P34HB electrical output at 110 Hz and 0.25 N force on the proliferation and osteogenic differentiation of rabbit bone marrow mesenchymal stem cells was tested. RESULTS AND CONCLUSION:(1)Scanning electron microscopy,X-ray diffraction,water contact angle,piezoelectric coefficient and electrical output performance tests showed that when the mass ratio of BCST increased to 20%,the BCST/P34HB piezoelectric film had good piezoelectric properties(d33=5.9 pC/N)and electrical output performance(180 mV),which was closer to the suitable range of 500 mV for electrical stimulation.(2)Live and dead staining showed that on the first day of co-culture,15%group and 20%group showed less red fluorescence.On the 5th day of culture,the number of green fluorescence in each group was significantly higher than that on the first day,and the red fluorescence was not observed in the 10%,15%and 20%groups,and only a small amount of red fluorescence was observed in the 0%and 5%groups.(3)On the 1st,3rd and 5th days of co-culture with rabbit bone marrow mesenchymal stem cells,Almar blue staining exhibited that the number of cells in each group showed an increasing trend with the increase of time.On the 5th day of culture,the number of cells in the 20%group was significantly more than that in the 0%group(P<0.05).(4)On day 10 of osteogenic induction,alkaline phosphatase staining results showed that the positive rate of the 20%group was significantly higher than that of the 0%group(P=0.000 1).On day 21,alizarin red staining and quantitative analysis of calcium nodules showed a similar trend to alkaline phosphatase staining.Compared with the 0%group,the 15%group and 20%group showed significant differences(P<0.01,P<0.000 1).(5)The results showed that 20%BCST/P34HB films had good piezoelectric properties,electrical output properties,biocompatibility and the ability of promoting osteogenic differentiation of bone marrow mesenchymal stem cells.

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 understand the molecular characteristics of the mutant strain of polymerase basic protein 2 (PB2) gene of influenza A (H1N1pdm) in Guangdong province, and to explore its specific molecular sites, so as to provide scientific basis for the prevention and control of influenza virus.Methods:Throat swab samples were collected from 2 cases infected with PB2 gene variant strains for virus isolation, and 23 influenza virus strains were selected from Guangdong province for sequencing analysis. The reference sequences and vaccine strain sequences provided by GISAID were used to perform evolutionary analysis on hemagglutinin (HA) and PB2 genes. Virus strain antigen analysis and neuraminidase (NA) inhibition test were carried out. PB2 protein model was constructed and polymerase activity was analyzed.Results:H399N amino acid mutation occurred in the HA gene of PB2-D701N and PB2-A271S variant strains, both of which belonged to the branch of 6B.1A.5a.2a. They belonged to the same big branch and different small branches as the vaccine strain A/Victoria/4897/2022, and they are all vaccine-like strains. In the three-dimensional structure, the mutations of PB2-D701N and PB2-A271S change charge and hydrophobicity.Conclusions:PB2-D701 and A271 were highly conserved, and PB2 mutant strains were not the dominant strains. The PB2 mutant had high antigenicity with the vaccine. The PB2 mutant strain is sensitive to NA inhibitors. The three-dimensional model predicted that PB2-D701N mutation could enhance virulence and affect transmissibility of influenza virus, while PB2-A271S mutation could affect polymerase activity and polymerase complex synthesis of influenza virus.