Application of modified Matutes score containing CD200 in diagnosis of chronic lymphocytic leukemia / 中南大学学报(医学版)

OBJECTIVES@#Immunophenotyping technique is a powerful tool for the diagnosis and differential diagnosis of chronic lymphocytic leukemia (CLL) and other B-cell chronic lymphoproliferative diseases (B-CLPD). CD200 is strongly expressed in CLL. This study aims to analyze the clinical value of modified Matutes score (MMS) containing CD200 in the diagnosis of CLL.@*METHODS@#We retrospectively analyzed 103 B-CLPD patients diagnosed from January 2020 to July 2021, including 64 CLL patients, 11 follicular lymphoma (FL) patients, 14 mantle cell lymphoma (MCL) patients, 6 marginal zone lymphoma (MZL) patients, 1 hairy cell leukemia (HCL) patient, and 7 lymphoplasmic lymphoma/Waldenstrom macroglobulinemia (LPL/WM) patients. The expression of CD markers between the CLL group and the non-CLL group was compared, and the sensitivity, specificity, and clinical consistency of MMS and Royal Marsden Hospital (RMH) immunophenotyping score system were analyzed.@*RESULTS@#There were significant differences in the expressions of CD5, CD23, FMC7, CD22, CD79b, CD200, and sIg between the CLL group and the non-CLL group (χ2 values were 37.42, 54.98, 30.71, 11.67, 55.26, 68.48, and 17.88, respectively, all P<0.01). When the RMH immunophenotyping score≥4, the sensitivity was 79.7%, and the specificity was 100%. When the MMS≥3, the sensitivity was 95.3%, and the specificity was 100%. The Kappa coefficient of RMH immunophenotyping system was 0.677, and the Kappa coefficient of MMS system was 0.860.@*CONCLUSIONS@#The MMS system containing CD200 has better sensitivity and same specificity compared with RMH immunophenotyping system, and MMS system may be more useful in the diagnosis of CLL.

Risk factors and survival analysis for central nervous system complications after allogeneic hematopoietic stem cell transplantation / 中南大学学报(医学版)

OBJECTIVES@#To investigate the risk factors as well as their impact on patients' survival of central nervous system (CNS) complications following allogeneic hematopoietic stem cell transplantation (HSCT).@*METHODS@#All relevant clinical data from a total of 323 patients, who underwent allogeneic HSCT in Xiangya Hospital of Central South University from September 2016 to September 2019, were retrospectively reviewed in this study. The complications' occurrence time, common symptoms and some other clinical data of the patients who developed CNS complications were analyzed descriptively. The risk factors for CNS complications following allogeneic HSCT were analyzed through univariate and multivariate analysis. And the survival analysis was conducted as well.@*RESULTS@#Among the 323 patients who underwent allogeneic HSCT, 32 patients developed CNS complications. These complications occurred in these patients at a median of 32 (range from -1 to 584) d after transplantation. Common symptoms were disturbance of consciousness (78.1%), convulsion (59.4%), and headache (12.5%). Univariate analysis showed that there were significant differences in neutrophil engraftment, platelet (PLT) engraftment, serum cytomegalovirus (CMV) DNA positive, combined with acute graft-versus-host disease (aGVHD), donor selection (@*CONCLUSIONS@#The delay or the failure of PLT engraftment and combined with aGVHD are the risk factors for CNS complications. The facts indicate that we should prevent CNS complications when patients who underwent allogeneic HSCT with the delay or the failure of PLT engraftment or aGVHD. Compared with non-CNS complication group, patients who developed CNS complications usually have poor prognosis.

Regulation of DNA demethylation of STAT3 promoter in CD4+ T cells from aGVHD patients by HMGB1/GADD45A / 中南大学学报(医学版)

To study the molecular mechanism for DNA hypomethylation of STAT3 promoter in CD4+ T cells from acute graft-versus-host disease (aGVHD) patients.
 Methods: We collected CD4+ T cells from peripheral blood of 42 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) from HLA-identical sibling donors. GADD45A expression level in CD4+ T cells was measured by real-time PCR and Western blot. The binding level between HMGB1 and GADD45A in CD4+ T cells was analyzed by co-immunoprecipitation, while the binding levels of HMGB1/GADD45A with STAT3 promoter were detected by chromatin immunoprecipitation-quantitative real-time PCR (ChIP-qPCR). After overexpression of HMGB1 and knockdown of GADD45A in normal CD4+ T cells, STAT3 expression and DNA methylation were measured by Western blot and bisulfite sequencing PCR, respectively.
 Results: GADD45A expression was significantly up-regulated in patients with aGVHD compared with that in the patients without aGVHD. More HMGB1-GADD45A complexes were found in CD4+ T cells from patients with aGVHD compared with that in patients without aGVHD. The bindings of HMGB1/GADD45A with STAT3 promoter were significantly increased, and the binding levels of HMGB1/GADD45A were negatively correlated with STAT3 promoter DNA methylation. The expression of STAT3 was significantly reduced and the DNA methylation of STAT3 promoter was significantly increased in CD4+ T cells with overexpression of HMGB1 and knockdown of GADD45A compared with CD4+ T cells only with overexpression of HMGB1.
 Conclusion: The increased expression of HMGB1/GADD45A plays an importent role in STAT3 promoter DNA hypomethylation, thereby promoting STAT3 expression in CD4+ T cells from aGVHD patients.

Effect of AB serum on human terminal erythroid differentiation ex vivo / 中南大学学报(医学版)

To demonstrate the effect of AB serum on terminal erythroid differentiation ex vivo.
 Methods: After separation of CD34+ cells from cord blood, the cells were cultured and divided into a control group and an experimental group. The effects of AB serum were examined by the expressions of different markers (GPA, Band3 and α4-integrin) for erythroblast differentiation and enucleation by flow cytometry. 
 Results: The CD34+ cells were successfully differentiated to enucleated red blood cells. There were evident differences among the expressions of GPA, Band3 and α4-integrin between the 2 groups. The percentage of GPA positive cells in the experimental group was bigger than that in the control group in every time point. The expression of Band3 in the experimental group was higher than that in the control group. The expression of α4-integrin in the experimental group was lower than that in the control group. In addition, the enucleation rate in the experimental group was higher than that in the control group.
 Conclusion: AB serum can promote the cell differentiation and enucleation during terminal erythroid differentiation in vitro.

In vitro selection of single strand deoxyribonucleic acid aptamers binding to cells from patients with acute myeloblastic leukemia / 中南大学学报(医学版)

OBJECTIVE@#To screen aptamers binding CD33+/CD34- cells from patients with acute myeloblastic leukemia M2 subtype (AML-M2).@*METHODS@#CD33+/CD34- cells from patients with AML-M2 were taken as targeted cells, CD33+/ CD34- cells from normal people were taken as anti-selecting cells, and aptamers in the single strand deoxyribonucleic acid (ssDNA) library were then selected repeatedly by cell-systematic evolution of ligands by exponential enrichment (C-SELEX) technology, and amplified by polymerase chain reaction (PCR) to generate sub-ssDNA library. During the experiment, PCR amplification with fluorescently labeled primer and flow cytometry were performed to analyze the aptamers'enrichment of sub-library, and the final round product of the sub-ssDNA library was cloned. After the sequencing, the primary and secondary structures of the aptamers were analyzed.@*RESULTS@#Electrophoresis indicated that the product of PCR amplification for each round subssDNA library was able to see a clear DNA band in the agarose gel. After 13 rounds of screening, the fluorescence intensity of the sub-ssDNA library binding the cells ranged from 2.14% to 51.12%, reaching a steady state at the 13th round. A total of 30 clones were selected and sequenced, 22 of which contained 1 of the 4 conserved sequences of AAGTA, TATCT, AGATG and AAATT in their primary structure, but the remained eight aptamers contained none of the conserved sequence. Secondary structure analysis indicated that four stem-loops and loop simulation convex structures existed in the aptamers.@*CONCLUSION@#C-SELEX technology can be used to screen the aptamers binding primary cells from patients with leukemia. The aptamers selected from the CD33+/CD34- cells from the patients of AML-M2 subtype might be used for the diagnosis and treatment for leukemia.

Effect of extracts from Radix Trichosanthis on the expression of HBsAg and HBeAg in HepG2.2.15 cells / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of extracts with water and alcohol from Radix Trichosanthis on the cell survival and the expression of hepatitis B surface antigen (HBsAg) and hepatitis B e-antigen (HBeAg) in HepG2.2.15 cell supernatant.@*METHODS@#The cell survival rate of HepG2.2.15 cells was detected by MTT assay. The HBsAg and HBeAg in HepG 2.2.15 cell supernatant were evaluated by enzyme linked immunosorbent assay.@*RESULTS@#The water and alcohol soluble extracts from Radix Trichosanthis significantly inhibited the levels of HBsAg and HBeAg in HepG2.2.15 cells in a time-and-concentration-dependent manner. However, the therapeutic index of extracts with water from Radix Trichosanthis was better than that in the alcohol group.@*CONCLUSION@#The activity of water-soluble extract from Radix Trichosanthis is stronger on anti-hepatitis B virus than that of the alcohol-soluble extract.

Effect of methylation inhibitor on demethylation pattern of the PD-1 gene in promoter region and PD-1 expression in human T lymphocyte cell line / 中南大学学报(医学版)

OBJECTIVE@#To observe the demethylation effect of demethylation inhibitor 5-azacytidine (5-Zac) on programmed death receptor 1 (PD-1) in Molt-4 cells (T lymphocyte cell line) and to investigate the relationship between DNA demethylation and expression of PD-1.@*METHODS@#Molt-4 cells were cultured in the medium containing different concentrations of 5-Zac(0, 5, 10 μmol/L) for 72 h. According to the concentrations of 5-Zac, the Molt-4 cells were divided into a 0 μmol/L 5-Zac group, a 5 μmol/L 5-Zac group, and a 10 μmol/L 5-Zac group. The expression of PD-1 in Molt-4 cells was detected by flow cytometry and the apoptosis rate was calculated. The mRNA transcription level of PD-1 was detected by real-time polymerase chain reaction; Molt-4 cell DNA in all groups were treated by sodium bisulfite. The PD-1 promoter fragment was amplified by PCR, the amplification fragments were transformed into E. coli., the positive clones were selected for equencing, and the methylation status of the fragments of PD-1 promoter was examined. RESULTS Seventy-two hours after the 5-Zac treatment, the expression rate of PD-1 in the Molt-4 cells in the 0 μmol/L 5-Zac group, the 5 μmol/L 5-Zac group, and the 10 μmol/L 5-Zac group was (1.13 ± 0.01)%, (18.96 ± 1.87)%, and (63.09 ± 6.25)% respectively, in a low concentration-dependent way. The PD-1 mRNA expression level was increased significantly with the 5-Zac treatment. Cells apoptosis showed that:compared with the 0 μmol/L 5-Zac group, the apoptosid rate in the 5 μmol/L 5-Zac group and 10 μmol/L 5-Zac group was signficantly increased, which was (1.9 ± 0.06)%, (8.89 ± 1.36)%, and (24.50 ± 3.68)% in the 0 μmol/L 5-Zac group, the 5 μmol/L 5-Zac group, and the 10 μmol/L 5-Zac mol/L group respectively. The bisulfite genomic sequencing showed that the demethylation probability of CpG points on -601 bp and -553 bp was significantly increased in the 5-Zac treated cells compared with those untreated.@*CONCLUSION@#5-Zac can result in the increase of PD-1 expression in the human lymphoid cell series Molt-4 in vitro, and the apoptosis rate increases, which is related to PD-1 gene promoter demethylation.

5-azacytidine induces PD-1 gene promoter demethylation and PD-1 protein expression in human lymphoid cell series Molt-4 cells / 中华微生物学和免疫学杂志

Objective To investigate the demethylation and changes in gene expression of programmed death receptor-1 ( PD-1) caused by methylation inhibitor 5- azacytidine (5-Zac) in lymphocyte series Molt-4 cells and its mechanism. Methods Molt-4 cells were cultured in different concentrations of 5-Zac(0, 5, 10 Umol/L)for 72 h, ratio of cell expressing PD-1 and apoptosis rate were detected by FCM, transcription of PD-1 gene mRNA was detected by RT-PCR. Molt-4 cell DNA of all groups were disposed by sodium bisulfite, PD-1 gene promoter fragment binded with transcription factor Brn-2 was amplified by PCR,these amplification fragments were transformed into E. coli. Positive clones were selected by sequencing,methylation status of the fragments binded with transcription factor Brn-2 was examined. Results S-Zac could increase the PD-1 expression of Molt-4 cells. PD-1 expression rate in 0 μmol/L 5-Zac( 1. 13%±0.01% ) treated cells was found more lower than that in both 5 μmol/L and 10 μmol/L 5-Zac treated cells (18. 96% ±1. 87% , 63. 09% ± 6. 25% , P < 0. 05 ) , and they showed concentration-dependent (P <0.01). Cells apoptosis rate and PD-1 mRNA expression were also observed increased significantly with 5-Zac treating. Demethylation probability of CG points showed significant difference between transcription factor Brn-2 binding site and other four locations (P < 0.05 ). Conclusion 5 -Zac inhibits cell grouth in human lymphoid cell series Molt-4 by inducing PD-1 gene expression and promoter demethylation. PD-1 gene promoter binding transcription factor Brn-2 fragment CG point demethylation may be one of the important mechanisms in 5-Zac treated Molt-4 cells.