Objective To prepare specific mouse monoclonal antibodies against Homo sapiens dynein,axonemal, heavy chain 2 (DNAH2). Methods Firstly, recombinant plasmid encoding His tagged immunogen, targeting N-terminal sequence of DNAH2 protein (1-300 aa), in E. coli was constructed. IPTG was used to induce the expression of His-immunogen, which was then purified and immunized in BALB/c mice. Hybridoma cells were obtained through the fusion between myeloma cells and splenocytes isolated from BALB/c mice. Finally, ELISA and Western blot assays were performed to screen the positive hybridoma. Results IPTG was used efficiently to induce the expression of DNAH2 immunogen in E. coli. DNAH2 protein bands were detected in screened positive hybridoma. Conclusion Mouse monoclonal anti-DNAH2 antibody is prepared successfully.

Objective:To investigate the effects of rituximab on lymphocytes and immunoglobulin in the treatment of focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD).Methods:The subjects were FSGS and MCD patients admitted to Ruijin Hospital affiliated to Shanghai Jiaotong University on July 1, 2014 and July 1, 2019. All the enrolled patients were confirmed by clinical examination and renal biopsy, and received rituximab treatment (4 infusions of 375 mg/m 2 with the interval of 7-14 d). The levels of immunoglobulin IgA, IgG, IgM, and lymphocytes of CD19 +, CD20 +, CD3 +, CD3 +CD4 +, CD3 +CD8 + and natural killer cells (CD56 +CD16 +) were compared between baseline and the third month, the sixth month, the ninth month and the twelfth month after treatment. Results:Ninety-six patients with FSGS or MCD were enrolled in this study. The midian age was 28 years old (14-77 years old). The ratio of men to woman was 1.8∶1. There were 65 cases of MCD and 31 cases of FSGS. After rituximab treatment, the 24 h-proteinuria was significantly lower than that before treatment, and the serum albumin level was increased (both P<0.05). After rituximab treatment of 3 months, 6 months, 9 months and 12 months, CD19 + and CD20 + lymphocyte counts were significantly decreased (all P<0.01), and gradually recovered after 6 months. Compared with baseline, at 3, 6, 9, 12 months after rituximab treatment, the level of blood IgG was significantly increased ( P=0.004,<0.001,<0.001,<0.001, respectively), and the level of blood IgM was significantly decreased ( P<0.001, =0.008, =0.005,<0.001, respectively) but the median level still within the normal range (400-3 450 mg/L). The level of blood IgA was not significantly changed (all P<0.05). T lymphocytes (CD3 +, CD3 +CD4 + and CD3 +CD8 +) and natural killer cells (CD56 +CD16 +) showed no significant difference from baseline (all P>0.05). Conclusions:Rituximab can effectively eliminate CD19 + and CD20 + lymphocytes, and has little influence on peripheral blood lymphocyte count and immunoglobulin level except CD19 + and CD20 + lymphocytes. The standard administration of rituximab is safe for patients with FSGS and MCD.

Objective To evaluate the relationship between plasma cystatin C concentration (PcyC) and coronary artery diseases (CAD). Method A total of 126 subjects with CAD evidenced by coronary angiography admitted from April 2007 to March 2009 were divided into three groups: stable angina pectoris (SAPs, n = 34),unstable angina pectoris (UAPs, n = 56) and acute myocardial infarction (AMIs, n = 36), according to the diag-nostic criteria of CAD set by WHO. Another 34 subjects without CAD were taken as controls. There were no statis-tical differences in demographics among four groups. Serum lipids profile, uric acid (UA), PcyC and high-sensi-tive C-reactive protein (hs-CRP) were determined. And in the meantime, all patients were followed up for six months and adverse cardiovascular events were recorded. Comparisons were made between groups with a number of independent-sample t -tests. Data were processed with analysis of variance to test the differences in means among four groups, and the means were compared with chi-square test. Statistical significance was established at a P val-ue of less than 0.05. Results Cystatin C levels were significantly higher in UAPs than that in SAPs and in controls (P < 0.05), but were much lower than that in AMIs (P < 0.05). And much higher concentration of hs-CRP was found in UAPs (P < 0.05) and in AMIs (P < 0.01). Cystatin C was positively and significantly corre-lated with age, hs-CRP, WBC, creatinine and UA (r > 0, P < 0.05), whereas a significantly negative correla-tion with high-density lipoprotein cholesterol was found (r = - 0.227, P < 0.05). These coefficients were obvi-ously high for creatinine (r = + 0. 612), and WBC (r = + 0.459). During the period of six-month follow-up, 26 patients with adverse cardiovascular events were found, and had significantly higher cystatin C levels than 22 con-trols at admission (P < 0.01). Conclusions Cystatin C plays a pivotal role in the course of CAD, and the PcyC is a strong predictor for the risk of cardiovascular events.

Objective:To study the influence of G protein-coupled estrogen receptor 1 (GPER1) specific agonist G1 and antagonist G2 in epilepsy susceptibility of temporal lobe epileptic rats.Methods:Sixty rats were randomly divided into control group, G1 treatment group and G15 treatment group ( n=20). Rats in the latter two groups were intraperitoneally injected with GPER1 agonist G1 (10 μg) or antagonist G15 (40 μg) for a consecutive 12 d. Lithium chloride pilocarpine epilepsy models were prepared in the 3 groups. The behavior manifestations of these rats were observed within 1 h of intraperitoneal injection of pilocarpine; Racine grading was used to evaluate the severity of epileptic seizures every 5 min; the latency of epileptic seizures (Racine grading IV) and epileptic seizure grading at different time points in the 3 groups were compared. The EEG monitoring was performed to these rats, and EEG data were recorded from 10 min before pilocarpine injection to 2 h after pilocarpine injection; EEG time-frequency was analyzed by Fast-Fourier transform (FFT); distribution of brain electrical energy and changes of θ and α wave energy during 20 min of epileptic status were compared among the 3 groups. Results:(1) As compared with that in the control group and G1 treatment group, the latency of epileptic seizures in the G15 treatment group was significantly shortened ( P<0.05); 15 and 20 min after pilocarpine injection, the epileptic seizure grading of rats in G1 treatment group was statistically lower than that in control group ( P<0.05); 15-35 min after pilocarpine injection, the epileptic seizure grading of rats in G15 treatment group was significantly higher than that in control group ( P<0.05). (2) As compared with those in the control group, rats in the G1 treatment group had smaller brain wave amplitude, while the rats in the G15 treatment group had earlier seizure time, larger brain wave amplitude and higher frequency. There were no obvious changes in the amount of brain electrical energy between the G1 treatment group and control group; while the amount of brain electrical energy in the G15 treatment group 2 h after pilocarpine injection was higher than that in the control group. As compared with those in the control group and G1 treatment group, the θ and α wave energy values of rats in the G15 treatment group were significantly increased within 20 min of epileptic status ( P<0.05). Conclusion:Activation level of GPER1 might be associated with susceptibility to epileptic seizures, and specific inhibition of GPER1 activation can enhance the susceptibility to epilepsy and increase the energy values of specific frequency bands during epilepsy.

Objective To study the role and mechanism of orexin A in cell viability of Alzheimer's disease (AD) cell model PC12. Methods PC12 cells were treated with 0, 10, 20, 30, 40 and 50 μmol/L Aβ25-35 for 24 h, and then, cell viability was measured by MTT to confirm which concentration was the suitable one to establish the AD cell models. (1) AD cell models were treated with 0, 0.01, 0.1, 1 and 2μmol/L orexin A for 24 h, and then, 30μmol/L Aβ25-35 was added for 24 h; MTT assay was used to determine the cell viability to conform the suitable concentration of orexin A. (2) Inverted phase contrast microscope was employed to observe the morphology changes of PC12 cells from the control group, 30 μmol/L Aβ25-35 treatment group, and 0.01 μmol/L orexin A+30 μmol/L Aβ25-35 treatment group. (3) The PC12 cells were given pretreatment of orexin A receptor inhibitor SB408124 for 2 h, and cell viability was detected. Results (1) Aβ25-35 at concentration 30μmol/L was the suitable one to establish the AD cell models;after being pretreated with different concentrations of orexin A, the cell viability showed significant differences (F=27.120, P=0.000), and 0.01μmol/L orexin A was the suitable concentration. (2) Some of the cells from the 30μmol/L Aβ25-35 treatment group had breaking-off of protuberance and damaged soma;cells from 0.01μmol/L orexin A+30μmol/L Aβ25-35 treatment group had breaking-off of protuberance, and the degree of damaged soma was eased as compared with that in the 30μmol/L Aβ25-35 treatment group. (3) If the cell viability of the control group was 100％, cell viability of orexin A receptor inhibitor group was 109.10％±0.36％, which was significantly decreased as compared with that in the 0.01 μmol/L orexin A pretreated group (117.24％±2.72％, P<0.05). Conclusion Orexin A can improve the cell viability via combination of its specific receptor; orexin A and its specific receptor may be new targets for prevention and cure of AD.

Objective:To analyze the predictive value of von Willebrand factor (vWF) for venous thromboembolism (VTE) of patients in intensive care unit (ICU) by using propensity score matching (PSM).Methods:Patients admitted to ICU of the Second Affiliated Hospital of Kunming Medical University from December 2020 to June 2022 who stayed in ICU for ≥72 hours and underwent daily bedside vascular ultrasound screening were included. Baseline data such as age, gender, primary disease, and chronic comorbidities were collected. Coagulation indexes before admission to ICU and 24 hours and 48 hours after ICU admission were collected, including prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), international normalized ratio (INR), fibrinogen (Fib), fibrin monomer (FM), vWF, D-dimer, antithrombin Ⅲ (ATⅢ), etc. Patients were divided into VTE group and non-VTE group according to whether they had VTE or not [diagnosis of VTE: patients underwent daily ultrasound screening of bedside blood vessels (both upper and lower limbs, visceral veins), and those suspected of having thrombosis were confirmed by ultrasonographer or pulmonary angiography]. Using PSM analysis method, the VTE group was used as the benchmark to conduct 1 : 1 matching of age, whether there was malignant tumor, whether there was infection, whether there was diabetes, and coagulation indicators before admission to ICU. Finally, the cases with balanced covariates between the two groups were obtained. The risk factors of VTE were analyzed by multivariate Logistic regression analysis. Receiver operator characteristic curve (ROC curve) was drawn to evaluate the predictive value of vWF in the occurrence of VTE in critically ill patients.Results:A total of 120 patients were enrolled, of which 18 (15.0%) were diagnosed with VTE within 72 hours after admission to ICU, and 102 (85.0%) were not found to have thrombus in ICU. Before PSM, there were significant differences in age, gender, proportion of malignant tumor and infection, and coagulation indexes between VTE group and non-VTE group. After PSM, 14 pairs were successfully matched, and the unbalanced covariables between the two groups reached equilibrium. Multivariate Logistic regression analysis showed that vWF was an independent risk factor for VTE at 48 hours after ICU admission in critically ill patients [odds ratio ( OR) = 1.165, 95% confidence interval (95% CI) was 1.000-1.025, P = 0.004]. ROC curve analysis showed that the area under the ROC curve (AUC) of vWF at 48 hours after ICU admission for predicting VTE was 0.782, 95% CI was 0.618-0.945, P = 0.007. When the optimal cut-off value was 312.12%, the sensitivity was 67.7% and the specificity was 93.0%. Conclusion:Dynamic monitoring of vWF is helpful to predict the occurrence of VTE in ICU patients, and vWF at 48 hours after ICU admission has certain value in predicting the occurrence of VTE.

Objective To analyze the clinicopathological characteristics of renal lesions in type 2 diabetic patients and to differentiate diabetic nephropathy (DN) from non-diabetic renal diseases (NDRD).Methods Type 2 diabetic patients who received renal biopsy in Ruijin Hospital from January 2011 to December 2015 were recruited in this study. Clinical history, laboratory results and pathological data were retrospectively collected. According to the pathological findings, the patients were divided into 3 groups: DN, NDRD, DN+NDRD. Logistic model was applied to explore the independent clinical predictive factors in differentiating DN from NDRD. Results A total of 207 type 2 diabetic patients received renal biopsy, accounting for 6.82% of all biopsy population. Fifty-one patients were diagnosed with DN, 142 with NDRD and 14 with both DN and NDRD. In NDRD, membranous nephropathy(MN) (34.5%) was the most common finding, followed by IgA nephropathy(19.7%).By contrast, NDRD patients manifested a shorter diabetic course, a higher baseline hemoglobin level, a lower baseline serum creatinine, a higher prevalence of hematuria, a lower prevalence of hypertension and diabetic retinopathy, a better control of blood glucose, better compliance of monitoring blood glucose and less family history of diabetes. In multivariate logistic model, diabetic family history(OR=4.68, P=0.04) and long history of diabetes(OR=1.01, P=0.02) were risk factors of DN. Conclusion There is a high prevalence of NDRD in diabetic patients with renal lesions. Family history of diabetes and duration of diabetes are independent predictors of DN.

Objective:To evaluate the predictive role of ETV6-RUNX1 fusion gene in protocol CCLG-ALL-2008 as well as identify the prognostic factors that influence the outcome of ALL with ETV6-RUNX1 fusion gene.Methods:One hundred and seventy-eight patients newly diagnosed with pediatric acute lymphoblastic leukemia with ETV6-RUNX1 rearrangement from April 2008 to April 2015 were enrolled in CCLG-ALL-2008. The follow up period ended in July 2018; we performed retrospective analyses of their data to determine the efficacy of the regimen and the prognostic factors.Results:The median age of the study population (178 pediatric patients) , including 100 boys and 78 girls was 4 (1-13) y, and the median white blood cell count at diagnosis was 9.46 (1.25-239.83) ×10 9/L. Three patients died, and 1 was lost to follow up by the end of the first induction chemotherapy, resulting in an induced remission rate of 97.8% (174/178) . The cumulative incidence of relapse was 15.9% with a median follow up of 73.5 mon. Total 83.3% of the relapse cases were those of isolated bone marrow relapse, while 79.2% of the cases were those of late relapse. The median interval time between relapse and first complete remission was 35.5 mon (range, 1-62 months) . One of the 5 patients with early recurrence and 7 of the 19 with late recurrence cases survived. The 5-year-OS and 5-year-EFS of ETV6-RUNX1 positive children was (89.4±2.4) % and (82.1±6.9) %, respectively. The estimated 10-year-OS and 10-year-EFS of ETV6-RUNX1 positive children was (88.6±2.5) % and (77.3±4.0) %, respectively. The Kaplan-Meier method and Log-rank test were used to estimate and compare the survival. Univariate statistical analysis showed that poor prognostic factors that influenced survival included central nervous system state 2 at diagnosis, poor prednisone response, high risk, gene positivity after induction chemotherapy, as well as MRD positivity and gene positivity at the 12 th week. In the multivariate analysis, only the central nervous system state 2 at diagnosis and MRD positivity at the 12 th week were associated with the outcome. Conclusion:ETV6-RUNX1-positive ALL is a subgroup with a favorable prognosis as per the CCLG-ALL-2008 protocol. Patients with ETV6-RUNX1 should be given more intensive therapy, including hematopoietic stem cell transplantation when they are CNS2 at diagnosis or have high level of MRD at the 12 th week after treatment.

Objective:To analyze the clinical features, bone marrow features, and gene mutations of children with familial platelet disorder with predisposition to myeloid leukemia (FPD/AML) caused by a RUNX1 germline mutation as well as their family members.Methods:The clinical data and gene mutations of a child with FPD/AML hospitalized in the Pediatric Blood Disease Center, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, and some family members were extracted and analyzed. The literature was searched using "RUNX1 germline mutation" and "FPD/AML" as keywords in the Chinese databases; also PubMed was reviewed until September 2020.Results:A male patient aged 5 with dermatorrhagia was admitted due to thrombocytopenia for more than 3 years. The laboratory tests revealed a peripheral blood routine (WBC 6.38×10 9/L, HGB 113 g/L, PLT 54×10 9/L, NEUT 4.03×10 9/L, and MPV 9.1 fl) . Bone marrow smear revealed dysplasia of megakaryocytes. The immunohistochemistry for CD42b and CD41 highlighted small mononuclear megakaryocytes. Second generation sequencing revealed RUNX1 (exon3:c.520delC: p.R174Efs*10, NM_001001890) frameshift mutations, and its germline mutation was verified via genetic detection of oral epithelial cells. Five members of the family had blood diseases and successively died. The child's mother and maternal grandfather were sequenced for the second generation, and RUNX1 frameshift mutation was detected in the same locus as the child. However, the clinical features among them were different. A total of 37 English literatures were retrieved, and more than 70 FPD/AML families were reported. No relevant Chinese literature was retrieved. Conclusion:Runx1 germline mutations cause FPD/AML with a high risk of progression to myeloid malignancy. Family members carrying the same mutations may exhibit different clinical features and severity.

Objective:To analyze the relationship between FAB morphological classification and World Health Organization (WHO) 2016 classification in children with acute erythroid leukemia(AEL), and to summarize the clinical features and prognosis.Methods:Clinical data of de nova childhood AEL patients from January 1, 2002 to December 31, 2019, in Pediatric Blood Disease Center, Institute of Hematology & Blood Disease Hospital were retrospectively analyzed.All of them were re-evaluated according to the WHO 2016 classification.Results:(1) A total of 20 patients were diagnosed as AEL by FAB classification.According to the criteria of WHO 2016, they were re-diagnosed as myelodysplastic syndromes (MDS)- refractory anemia with excess of blasts (11 cases), acute myeloid leukemia with MDS-related changes (3 cases), acute monocytic leukemia (1 case), and pure red leukemia (PEL, 5 cases). (2) Pathological hematopoiesis was frequently detected in bone marrow smears.Auer bodies were seen occasionally in some blasts.The most common antigen expressing were CD 117, CD 13, CD 33, CD 34, CD7, and CD 38.Karyotype analysis was performed in 18 cases successfully, involving 6 cases with abnormal karyotypes, including + 8, -7, 22p+ , t (3; 5: ? ), + 3q-, 15q-, and del (9)(q13). (3) Thirteen cases were treated by chemotherapy, and the one-course complete remission rate was 38.5%.By July 1, 2020, only 2 cases were alive without disease.The overall survival was 49 months and 11 months, respectively. Conclusions:Childhood AEL is susceptible to pathological hematopoiesis, poor response to early chemotherapy and poor prognosis.After re-evaluation according to WHO 2016 classification, most of them were diagnosed as MDS-related.Therefore, adjusting the suitable induction regimen with allogeneic hematopoietic stem cell transplantation may improve the prognosis.