The Characteristics of T Lymphocyte Reconstitution after Haploid Hematopoietic Stem Cell Transplantation in SAA and Its Relationship with aGVHD / 中国实验血液学杂志

OBJECTIVE@#To investigate the recovery characteristics of T cell subsets in patients with severe aplastic anemia (SAA) who received haploid hematopoietic stem cell transplantation(HSCT) and its relationship with acute graft-versus-host disease(aGVHD).@*METHODS@#The clinical data of 29 SAA patients who received haploid hematopoietic stem cell transplantation in the department of hematology, Shanxi Bethune Hospital from June 2018 to January 2022 were retrospectively analyzed. The absolute counts of CD3+T, CD4+T, CD8+T lymphocytes and the ratio of CD4+T/CD8+T lymphocytes in all patients before transplantation, 14, 21, 30, 60, 90 and 120 days after transplantation were analyzed. The proportion of T lymphocytes was compared in the non-aGVHD group, the grade Ⅰ-Ⅱ aGVHD group and the grade III-IV aGVHD group.@*RESULTS@#The counts of all T cells in 27 patients were far below the normal level at 14 and 21 days after transplantation, but there was obvious heterogeneity. There was a certain relationship between T cell immune reconstitution and conditioning regimen, age, and immunosuppressive treatment before transplantation. CD3+T cells showed a steady upward trend at 30, 60, 90, and 120 days after transplantation, and returned to the normal levels at 120 days after transplantation; faster recovery of CD4+T cells was closely related to aGVHD, which was at 30, 60, 90, 120 days after transplantation showed a slow upward trend, and which was still far below the normal level of 120 days after transplantation. CD8+T cell counts began to recover at 14 and 21 days after transplantation, and the recovery was earlier than the CD4+T cells, and its recovery speed was rapid 30 and 60 days after transptantation, which showed an upward trend and exceeded the normal levels 90 days after transplantation. Since CD8+ T cells reconstituted quickly, while the CD4+ T cells reconstitution was slowly, which made the long-term CD4+T/CD8+T cell ratio after transplantation was inverted . Compared with the non-aGVHD group, the absolute counts of CD3+T, CD4+T, and CD8+T cells in the aGVHD group were significantly higher than those in the non-aGVHD group at each time period after transplantation. In the aGVHD group, grade Ⅲ-Ⅳ aGVHD occurred more frequently in the early post-transplantation period (within 14-21 days), the grade Ⅰ-Ⅱ aGVHD group mostly occurred within 30-90 days after transplantation, and CD3+T, CD4+T, CD8+T cell counts in the grade Ⅲ-Ⅳ aGVHD group were significantly higher than those in the grade Ⅰ-Ⅱ aGVHD group; and the greater the proportion of CD4+T, the more severe the degree of aGVHD.@*CONCLUSION@#The speed of T cell immune reconstitution after SAA haploid transplantation is different, which is related to the conditioning regimen, age, and immunosuppressive therapy before transplantation. The rapid recovery of CD4+ T cells is closely related to the occurrence of aGVHD.

Clinical Characteristics and Nomogram Model of Nosocomial Infection in Patients with Newly Diagnosed Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To explore the clinical characteristics of nosocomial infection in newly diagnosed multiple myeloma(NDMM) patients, and establish a predictive nomogram model.@*METHODS@#The clinical data of 164 patients with MM who were treated in Shanxi Bethune Hospital from January 2017 to December 2021 were retrospectively analyzed. The clinical characteristics of infection were analyzed. Infections were grouped as microbiologically defined infections and clinically defined infections. Univariate and multivariate regression models were used to analyze the risk factors of infection. A nomogram was established.@*RESULTS@#164 patients with NDMM were included in this study, and 122 patients (74.4%) were infected. The incidence of clinically defined infection was the highest (89 cases, 73.0%), followed by microbial infection (33 cases, 27.0%). Among 122 cases of infection, 89 cases (73.0%) had CTCAE grade 3 or above. The most common site of infection was lower respiratory in 52 cases (39.4%), upper respiratory tract in 45 cases (34.1%), and urinary system in 13 cases (9.8%). Bacteria(73.1%) were the main pathogens of infection. Univariate analysis showed that ECOG ≥2, ISS stage Ⅲ, C-reactive protein ≥10 mg/L, serum Creatinine ≥177 μmol/L had higher correlation with nosocomial infection in patients with NDMM. Multivariate regression analysis showed that C-reactive protein ≥10 mg/L (P＜0.001), ECOG ≥2 (P=0.011) and ISS stage Ⅲ （P=0.024） were independent risk factors for infection in patients with NDMM. The nomogram model established based on this has good accuracy and discrimination. The C-index of the nomogram was 0.779（95%CI: 0.682-0.875）. Median follow-up time was 17.5 months, the median OS of the two groups was not reached (P=0.285).@*CONCLUSION@#Patients with NDMM are prone to bacterial infection during hospitalization. C-reactive protein ≥10 mg/L, ECOG ≥2 and ISS stage Ⅲ are the risk factors of nosocomial infection in NDMM patients. The nomogram prediction model established based on this has great prediction value.

Analysis of WRKY transcription factor family based on full-length transcriptome sequencing in Polygonatum cyrtonema / 中国中药杂志

WRKY transcription factor family plays an important role in plant growth and development, secondary metabolite synthesis, and biotic and abiotic stress responses. The present study performed full-length transcriptome sequencing of Polygonatum cyrtonema by virtue of the PacBio SMRT high-throughput platform, identified the WRKY family by bioinformatics methods, and analyzed the physicochemical properties, subcellular localization, phylogeny, and conserved motifs. The results showed that 30.69 Gb nucleotide bases and 89 564 transcripts were obtained after redundancy removal. These transcripts had a mean length of 2 060 bp and an N50 value of 3 156 bp. Based on the full-length transcriptome sequencing data, 64 candidate proteins were selected from the WRKY transcription factor family, with the protein size of 92-1 027 aa, the relative molecular mass of 10 377.85-115 779.48 kDa, and the isoelectric point of 4.49-9.84. These WRKY family members were mostly located in the nucleus and belonged to the hydrophobic proteins. According to the phylogenetic analysis of WRKY family in P. cyrtonema and Arabidopsis thaliana, all WRKY family members were clustered into seven subfamilies and WRKY proteins from P. cyrtonema were distributed in different numbers in these seven subgroups. Expression pattern analysis confirmed that 40 WRKY family members had distinct expression patterns in the rhizomes of 1-and 3-year-old P. cyrtonema. Except for PcWRKY39, the expression of 39 WRKY family members was down-regulated in 3-year-old samples. In conclusion, this study provides abundant reference data for genetic research on P. cyrtonema and lays a foundation for the in-depth investigation of the biological functions of the WRKY family.

The Prognostic Value of Early Relapse after Autologous Hematopoietic Stem Cell Transplantation in Newly Diagnosed Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To assess the impact of early relapse (ER) after autologous hematopoietic stem cell transplan-tation (AHSCT) on overall survival (OS) for multiple myeloma (MM) patients.@*METHODS@#Clinical data of 37 patients with MM undergoing AHSCT in department of hematology of Shanxi Bethune Hospital from January 2012 to December 2017 were retrospectively analyzed. The effect of ER on OS of patients was analyzed. The effects of international staging system (ISS) staging, cytogenetics, pre-transplant efficacy, minimal residual disease, and age on OS of the patients were also analyzed respectively.@*RESULTS@#Among the 37 patients, 13 cases (35.1%) had ER, and 24 cases (64.9%) had non-ER. 3 patients with ER had extramedullary disease, but none with non-ER showed extramedullary disease. More than or equal to very good partial rate (VGPR) in patients with ER and without ER were 3 cases (23.1%) and 15 cases (62.5%), respectively, and the curative effect of the former was significantly lower than that of the latter (P<0.05). The median follow-up time was 31 (12-96) months, and median OS time was 93 months in all the patients. The median survival time of patients with ER was 17 months, and the median progression free survival was 7 months, both were significantly shorter than 93 months and 38 months of patients with non-ER (P<0.05). Univariate analysis showed that the OS was affected by ER, cytogenetic abnormalities (FISH), and ≥VGPR before transplantation. Multivariate analysis showed that ER was an independent prognostic factor.@*CONCLUSION@#The prognosis of patients with ER after AHSCT in newly diagnosed MM is poor. ER is an independent prognostic factor of survival.

Effect of Serum Free Light Chain Ratio and Normalization Ratio after Treatment on Diagnosis and Prognosis of Patients with Newly Diagnosed Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To evaluate the value of serum free light chain (sFLC) κ/λ ratio (sFLCR) on the diagnosis and prognosis of patients with newly diagnosed multiple myeloma(MM), and explore the effect of sFLCR normalization on the prognosis of patients after 4 courses of induction therapy.@*METHODS@#The clinical data of 43 newly diagnosed MM patients from January 2014 to January 2019 were analyzed retrospectively. Immunoturbidimetry was used to detect the expression levels of sFLC κ and λ. According to the ratio of involved and uninvolved sFLC, using 100 as a boundary, the MM patients were divided into the high ratio group (sFLCR≥100 or ≤0.01) and the low ratio group (0.010.05).@*CONCLUSION@#Patients in the high ratio group at the initial diagnosis have worse renal function, later stage of disease, lower deep remission rate, earlier disease progression, shorter survival time, and worse clinical prognosis.

Effect of Low-Dose Triptolide and Sorafenib Alone and Their Combination on AML Cell Line MV411 and the Pathway of STAT5 / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of inhibiting proliferation and inducing apoptosis of low-dose triptolide and sorafenib alone or in combination on FLT3-ITD acute myeloid leukemia cell line MV4-11 and STAT5 pathway.@*METHODS@#The MV4-11 cells were treated with low dose triptolide(IC) and sorafenib(IC) alone or in combination for 48 hours. The cell proliferation and inhibition were detected by using CCK-8 kit, the cell apoptosis was detected by flow cytometry, the expression of FLT3,STAT5 in mRNA and protein levels was detected by RT-PCR and Western blot respectively.@*RESULTS@#The treatment of MV4-11 cells with low dose triptolide and sorafenib alone and in combination for 48 hours could inhibit cell proliferation and induce cell apoptosis, moreover the inhibitory rate and apoptotic rate of MV4-11 cells in drug-combination group both were higher than those in single drug group. The mRNA expression and protein expression of FLT3,STAT5 signaling pathway in drug combination group were significantly lower than those in single drug group.@*CONCLUSION@#Low-dose triptolide combined with sorafenib can synergistically inhibit the proliferation and induce the apoptosis of MV4-11 cells, which may be related with the inhibition of FLT3 and STAT5 pathway.

Isoliquiritigenin relaxes the cerebral basilar artery by enhancing BKCa current in spontaneously hypertensive rat: role of sGC/cGMP / 生理学报

The purpose of the present study is to investigate the effect of isoliquiritigenin (ISL) on the cerebral basilar artery in spontaneously hypertensive rats (SHR). The change of SHR systolic pressure was measured by tail artery pressure measurement instrument before and after ISL intervention. After perfusion with 1 × 10(-5) mol/L phenylephrine (PE), 1 × 10(-5) mol/L PE + 1 × 10(-4) mol/L ISL and 1 × 10(-5) mol/L PE, the diameter of the cerebral basilar artery separated from SHR was measured by pressure myograph. The current of large-conductance calcium-activated potassium (BKCa) channel of SHR single vascular smooth muscle cell (VSMC) was recorded by whole-cell patch-clamp technique and the cGMP levels of basilar artery was evaluated by ELISA. The results showed that 1) after intervention with ISL for 14 days, the systolic pressure of SHR was decreased from (218.3 ± 1.6) mmHg to (119.2 ± 1.9) mmHg (P < 0.01), but there was no difference in systolic pressure between ISL-treated SHR and Wistar-Kyoto (WKY) rat; 2) 1 × 10(-4) mol/L ISL relaxed the SHR cerebral basilar artery (P < 0.01); 3) ISL significantly increased the outward current density of VSMC from SHR cerebral basilar artery (P < 0.01, n = 6), and the effect could be reversed by 1 × 10(-3) mol/L TEA (a BKCa channel inhibitor), but 3 × 10(-4) mol/L 4-AP (a Kv channel inhibitor) had no effect on the enhanced current density induced by ISL in VSMC; 4) 1 × 10(-5) mol/L Methylene blue (a sGC inhibitor) significantly inhibited the ISL-enhanced current density in VSMC (P < 0.05, n = 6); 5) ISL significantly increased the cGMP level of SHR basilar artery (P < 0.05, n = 6). The results suggest that the role of the ISL in relaxing the SHR cerebral basilar artery may be related to its effect in enhancing BKCa current by increasing the levels of cGMP in the VSMC.

Effects of visfatin on NG2 cells following cerebral ischemia/reperfusion injury in rats / 第二军医大学学报

Objective To investigate the effect of visfatin on the NG2 cell number in the brain of normal rats and rats with cerebral ischemia/reperfusion (I/R) injury. Methods Transient focal cerebral I/R model was established by middle cerebral artery occlusion (MCAO) and a monofilament suture via intraluminal approach in rats. Thirty adult male Wistar-Kyoto rats were randomly divided in to four groups, namely, a sham operation group (Sham), a cerebral I/R model group(MCAO), Sham+FK866(a specific inhibitor of visfatin) group, and cerebral I/R+FK866 group. Animats nn the latter two groups were intragastsically given FK866 (1 mg/[kg • d]) for a consecutive of 14 days; Sham operation or MCAO was pefformed at the 7th day of FK866 treatment. The NG2 cells were observed by immunofluorescence staining in animal brains at the 7th day after surgery. Results There was no signficant difference in NG2 cell numbeis between the Sham group and Sham + FK866 group. Cerebral I/R model group had significantly less NG2 cells in the core region of infarction(P<0. 01) and significantly more in the penumbra area (P<0. 01) compared with the Sham group, with no significant change in the contralateral side. The NG2 cell numbers were not significantly different between the cerebral I/ R model group and cerebral I/R + FK866 group in the above mentioned areas. Conclusion Inhibition of visfatin shows no noticeable effect on NG2 cell number in the brains of normal and cerebral I/R rats, indicating that the neuroprotective effect of visfatin is not assoiiated with NG2 cells.

Construction of fusion gene vaccine of WT1 multi-epitope fused with stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and its expression and immunogenicity / 中国实验血液学杂志

This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.