OBJECTIVE: To establish venetoclax-resistant acute myeloid leukemia (AML) cell lines, assess the sensitivity of venetoclax-resistant cell lines to the BCL-2 protein family, and investigate their resistance mechanisms. METHODS: CCK-8 method was used to screen AML cell lines (MV4-11, MOLM13, OCI-AML2) that were relatively sensitive to venetoclax. Low concentrations of venetoclax continuously induced drug-resistance development in the cell lines. Changes in cell viability and apoptosis rate before and after resistance development were measured using the CCK-8 method and flow cytometry. BH3 profiling assay was performed to anayze the transform of mitochondrion-dependent apoptosis pathway as well as the sensitivity of resistant cell lines to BCL-2 family proteins and small molecule inhibitors. Real-time fluorescence quantitative PCR (RT-qPCR) was utilized to examine changes in the expression levels of BCL-2 protein family members in both venetoclax-resistant cell lines and multidrug-resistant patients. RESULTS: Venetoclax-resistant cell lines of MV4-11, MOLM13, and OCI-AML2 were successfully established, with IC₅₀ values exceeding 10-fold. Under the same concentration of venetoclax, the apoptosis rate of resistant cells decreased significantly (P < 0.05). BH3 profiling assay revealed that the drug-resistant cell lines showed increased sensitivity to many pro-apoptotic proteins (such as BIM,BID and NOXA). RT-qPCR showed significantly upregulated MCL1 and downregulated NOXA1 were detected in drug-resistant cell lines. Expression changes in MCL1 and NOXA1 in venetoclax-resistant patients were consistent with our established drug-resistant cell line results. CONCLUSION The venetoclax-resistant AML cell lines were successfully established through continuous induction with low concentrations of venetoclax. The venetoclax resistance resulted in alterations in the mitochondrial apoptosis pathway of the cells and an increased sensitivity of cells to pro-apoptotic proteins BIM, BID, and NOXA, which may be associated with the upregulation of MCL1 expression and downregulation of NOXA1 expression in the drug-resistant cells.
Humans ; Sulfonamides/pharmacology* ; Drug Resistance, Neoplasm ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology* ; Leukemia, Myeloid, Acute/pathology* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Cell Line, Tumor ; Apoptosis ; Antineoplastic Agents/pharmacology*

OBJECTIVE: To establish the in vivo traceable acute myeloid leukemia mice model with Luciferase-Expressing KG1a Cells. METHODS: KG1a cells with stable luciferase gene expression (called as KG1a-Luc cells) were constructed by lentivirus transfection, then sifted out by puromycin. Eighteen male NOD-SCID-IL2rg RESULTS: KG1a cells expressing luciferase stably were successfully obtained. The tumor luminescence wildly spread at day 17 captured by in vivo imaging. The KG1a-Luc tumor cells could be detected in the peripheral blood of the mice, with the average percentage of (16.27±6.66)%. The morphology and pathology result showed that KG1a-Luc cells infiltrate was detected in bone marrow, spleens and livers. The survival time of the KG1a-Luc mice was notably shorter as compared with those in the control group, the median survival time was 30.5 days (95%CI: 0.008-0.260). CONCLUSION The acute myeloid leukemia NOD-SCID-IL2rg
Animals ; Disease Models, Animal ; Interleukin Receptor Common gamma Subunit ; Leukemia, Myeloid, Acute ; Luciferases/genetics* ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID

Objective:To study the pathological changes of the spleen in patients with COVID-19 and to analyze the relationship between the weakened immune system and splenic lesions.Methods:Postmortem needle autopsies from the spleen were carried out on 10 patients who died from COVID-19 in Wuhan. Routine hematoxylin and eosin (HE) staining was used to observe the pathological changes. The changes of lymphocytes were studied further with immunohistochemistry.RT-PCR was used to detect 2019-nCoV RNA in the spleen. In addition,the Epstein-Barr virus (EBV) was detected by in situ hybridization, and coronavirus particles were detected by transmission electron microscopy in 2 cases.Results:There were 7 males and 3 females, with an average age of 68.3 years.Of the 10 cases, 4 had cancer history and another 4 had other underlying diseases respectively.Cough, fever, malaise and dyspnea were the main clinical symptoms.The time from onset to death was 15-45 days.Ten cases patients had normal or slight increase in peripheral blood leukocyte count in the early stage of the disease, 6 cases had significant increase before death. Five patients′ peripheral blood lymphocyte count decreased in the early stage of the disease, and 10 patients′ peripheral blood lymphocyte count decreased significantly before the disease progressed or died. Seven cases were treated with corticosteroid (methylprednisolone ≤40?mg/d, not more than 5 days). Histopathological examination showed that the cell composition of the spleen decreased, white pulp atrophied at different levels, meanwhile lymphoid follicles decreased or absent;in addition, the ratio of red pulp to white pulp increased with varying degrees. In 7 cases, more neutrophil infiltration was found, and in 5 cases, scattered plasma cell infiltration was found. Macrophage proliferation and hemophagocytic phenomena in a few cells were found in a case. Meanwhile, necrosis and lymphocyte apoptosis were detected in 2 cases, small artery thrombosis and spleen infarction in 1 case, and fungal infection in 1 case. The results of immunohistochemistry showed that the T and B lymphocyte components of the spleen in all cases decreased in varying degrees. CD20 + B cells were found to accumulate in the lymphoid sheath around the splenic artery in 8 cases. However, CD20 and CD21 immunostaining in 2 cases showed that the number of white pulp was almost normal, and splenic nodules were atrophic. CD3 +, CD4 + and CD8 +T cells were decreased. In 9 cases,CD68 + macrophages were no significant changes in the distribution and quantity. While more CD68 + cells were found in the medullary sinuses of 1 case (related to fungal infection). Few CD56 + cells were found. EBV was negative by in situ hybridization. RT-PCR was used to detect the nucleic acid of 2019-nCoV. One of 10 cases was positive, 39 years old,who was the youngest patient in this group, and the other 9 cases were negative. Coronavirus particles were found in the cytoplasm of macrophage under electron microscope in 2 cases. Conclusions:The death of COVID-19 occurs mainly in the elderly, and some cases have no underlying diseases. Spleen may be one of the organs directly attacked by the virus in some patients who died from COVID-19. T and B lymphocyte in the spleen decrease in varying degrees, lymphoid follicles are atrophied, decreased or absent, and the number of NK cells do not change significantly. And the pathological changes of the spleen are not related to the use of low dose corticosteroid, which may be related to the direct attack of virus and the attack of immune system on its own tissues.

OBJECTIVE: To investigate the expression and significance of B and T lymphocyte weakening factor (BTLA) in patients with chronic myelomonocytic leukemia (CMML). METHODS: Real-time PCR was used to detect the expression of BTLA and its ligand HVEM mRNA in 11 patients with chronic myelomonocytic leukemia and 11 normal donors. Flow cytometry was used to detect expression of BTLA and its HVEM on the cell surface of peripheral blood T lymphocytes and γδ T cells. RESULTS: The median values of BTLA and its ligand HVEM mRNA expression in peripheral blood of patients with CMML were 0.009% and 559.4%, respectively, which were significantly lower than those of normal controls (0.053% and 1031%)(P<0.001). The expression level of BTLA and HVEM on cell surface of peripheral lymphocytes was not significantly different from that in normal controls (P=0.3031 and 0.2576), however, the proportion of peripheral blood T lymphocytes in patients with CMML (median: 37.73%) was significantly lower than that in controls (median 69.23%)(P=0.0005). The expression of BTLA on the surface of γδ T cells in peripheral blood of patients with CMML (median: 23.26%) was significantly lower than that of the controls (median: 52.64%) (P<0.05), and there was no significant abnormality in HVEM expression (P=0.2791). CONCLUSION The expression of BTLA and its ligand HVEM, the proportion of T lymphocytes and the expression of BTLA on the surface of γδ T cells in patients with CMML are reduced. The effects of these abnormalities on T cell function and prognosis and efficacy of patients need to be further observed.

Objective: To investigate the effects of artesunate treatment on chronic graft-versus-host disease (cGVHD). Methods: Recipient BALB/c mice received 8 × 10(6) bone marrow cells with 8×10(6) spleen cells from B10D2 mice. Artesunate solubilized in acetone was injected intraperitoneally every day at the dose of 1 mg/kg at Day 28 after BMT. The clinical scores, survival and histopathological damage were analyzed. The frequency of Th17 and Tregs in PB and spleens from the mice were evaluated by flow cytometry. In addition, CD4(+) T cells from the spleens of mice were cultured in vitro, then stimulated with artesunate, the frequency of Th17 and Tregs in these splenocytes were evaluated by flow cytometry. Results: Artesunate administration diminished clinical and histopathological damage, and improved the survival of cGVHD mice[(46.57±7.83)% vs (55.71±6.99)%, χ(2)=5.457, P=0.020]; Artesunate contributed to Tregs development [(4.45±0.04)% vs (8.40±0.23)%, t=15.679, P<0.001; (6.62±0.24)% vs (10.48±0.48)%, t=6.587, P=0.003] while decreased Th17 cells [(1.51±0.18)% vs (0.58±0.19)%, t=7.233, P<0.001; (1.48±0.38)% vs (0.71±0.18)%, t=3.653, P=0.011] expressions in both PB and spleens, and decreased the Th17/Treg ratio (0.34±0.05 vs 0.09±0.03, t=7.621, P=0.002; 0.19±0.03 vs 0.06±0.02, t=6.993, P=0.002). Moreover, artesunate suppressed the Th17 cells expressions [(0.82±0.37) % vs (3.39±1.22) %, t=4.044, P=0.007] and contributed to Tregs development [(34.63±1.29) % vs (14.28±1.69) %, t=19.119, P<0.001], and also decreased the Th17/Treg ratio (0.24±0.09 vs 0.02±0.01, t=4.780, P=0.003) in vitro. Conclusions: Artesunate suppressed the Th17 cells expressions and contributed to Tregs development, which provided new sights into the development of a novel drug for cGVHD, e.g., artemisinin.
Animals ; Artesunate ; Graft vs Host Disease ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes, Regulatory ; Th17 Cells

Objective: To monitor the WT1 mRNA level and its dynamic changes in patients with myelodysplastic syndromes (MDS) after hypomethylating agents (HMA) , as well as to assess the significance of WT1 mRNA levels and its dynamic changes in evaluating the efficacy of HMA and distinguishing the disease status of heterogeneous patients with stable disease (SD) . Methods: Bone marrow or peripheral blood samples of 56 patients with MDS who underwent hypomethylating agents (≥4 cycles) from November 2009 to March 2018 were tested by real-time quantitative polymerase chain reaction (PCR) to detect the expression of WT1 mRNA, and to observe the correlation between the dynamic changes of WT1 mRNA expression and clinical efficacy and prognosis of patients. Results: WT1 mRNA expression levels of MDS patients decreased significantly after 3 cycles of hypomethylating agent treatment. Besides, the WT1 mRNA expression levels of patients increased significantly after diseases progression. According to the dynamic changes of WT1 mRNA expression levels during SD, 45 cases could be further divided into increased group and non-increased group. In those SD patients with increased WT1 mRNA expression level, the ratio of suffering disease progression or transformation to AML was 95.65% (22/23) , whereas the ratio turned to be 9.09% (2/22) for the non-increased group (χ(2)=33.852, P<0.001) . Compared with those SD patients reporting no increase in WT1 mRNA expression level, the overall survival[17 (95%CI 11-23) months vs not reached, P<0.001] and progression-free survival [13 (95%CI 8-18) months vs not reached, P<0.001] of those SD patients reporting increase in WT1 mRNA expression level were significantly shorter. Conclusion: WT1 mRNA expression level is a useful indicator to assess the efficacy of hypomethylating agents in MDS patients. Especially in patients with SD, detection of the changes in WT1 mRNA expression level is able to predict disease progression and help to make clinical decision.
Bone Marrow ; Humans ; Myelodysplastic Syndromes/genetics* ; Prognosis ; RNA, Messenger ; WT1 Proteins/genetics*

OBJECTIVE: To investigate the effects of exosomes from human umbilical cord mesenchymal stem cells on the development of Treg and TH17 cells. METHODS: Exosomes from the serum-free-culture supernatants of hUC-MSC were harvested by ultracentrifugation. The electron microscopy, nanoparticle tracking analysis and western blot were used to identify the hUC-MSC-exosomes, such as the morphology, the paticle chameter, and the protein content. The PBMC stimulated with anti-CD3/CD28 were incubated with the exosomes for five days, and then the percentage changes of Treg and TH17 cells were analyzed by using flow cytometry. RESULTS: The hUC MSC-derived exosomes were saucer-like in morphology the averge diameter was approximately 142 nm. They were identified as positive for CD9 and CD63. Flow cytometry showed that the proportion of CD4CD25Foxp3 Treg cells in the PBMC were significantly higher, but the proportion of CD4IL17A T cells in the hUC-MSC-exosome group was obviously lower than that in the group without the hUC-MSC-exosom (control group) (P<0.05). CONCLUSION The hUC-MSC-exosomes have an immunomodulatory effect on T cells in vitro by increasing the ratio of Treg and reducing the ratio of TH17 cells, expecting the hUC-MSC-exosom as a novel cell-free target for immunotherapy.
Exosomes ; Humans ; Leukocytes, Mononuclear ; Mesenchymal Stem Cells ; T-Lymphocytes, Regulatory ; Th17 Cells ; Umbilical Cord

Objective: To investigate the influence of different feeding ways on the incidence rate of complication in patients with congenital cleft lip and their family satisfaction degree. Methods: A retrospective analysis were conducted from 90 cases underwent surgery of congenital cleft lip with clinical data, were randomly assigned to the control group and the observation group, 45 cases in each. The control group routinely take spoon feeding way after surgery, whereas patients in the observation group does not change the feeding way in the past, continuely taking the nipple feeding or breastfeeding. Wound infection rate, wound rupture rate, crying in the process of feeding and parents' satisfaction were compared between the two groups. Results: The wound infection rate and wound rate of the observation group were 2.2% and 0 respectively, the control group were 4.4% and 0 respectively. There was no significant difference between the 2 groups in the 2 index (P > 0.05). In the observation group, the crying children were less than the control group, In the observation group, the degree of coordination, parental satisfaction and acceptance of feeding were 84.4%, 88.9%, 93.3%, respectively, the control group were 55.6%, 60.0%, 66.7%. The observation group were better than the control group. The differences between groups was statistically significant (χ2coordination degree = 8.94, χ2satisfaction degree = 9.87, χ2recognition degree = 10.00, P < 0.05). Conclusion Children under the age of 1 with congenital grade Ⅰ and Ⅱ cleft lip do not change the way of feeding after surgery , who continue to take the bottle or breastfeeding can better meet the needs of children fed liquid diet, crying reduce, get parents' recogniton and reduce the burden on parents than the control group conventionally fed with a spoon.

OBJECTIVETo understand the predictive value of early monitoring BCR-ABL transcripts in patients with chronic myeloid leukemia (CML) after treatment with tyrosine kinase inhibitor (TKI), and to provides the information for early assessment of prognosis and treatment options.

METHODSBCR-ABL transcripts of 53 CML patients before and after TKI treatment were detected by using real-time quantitative RT-PCR. The relationship between BCR-ABL transcripts level after TKI treatment for 3 months and the later molecular response, progression and mutation was analyzed.

RESULTSThe median values of BCR-ABL transcripts in peripheral blood samples from 30 newly diagnosed patients were 43.99%, which was used as a baseline of BCR-ABL transcripts for molecular response evaluation. Of 53 patients, 31 (58.49%) had a BCR-ABL mRNA ≤ 4.40% (reduced more than 1 log) and 22 (41.51%) greater than 4.40% (reduced to less than 1 log) after 3 months of TKI treatment. The former 31 patients had a significantly higher 18-months cumulative incidence of major molecular response (MMR) (90.32% vs 18.18%, P=0.000) and 3-year cumulative incidence of complete molecular response (CMR) (48.39% vs 0, P=0.000) compared with the latter 22 patients. The lower BCR-ABL level was, the earlier MMR reached. The proportion of patients with a mutation in group of BCR-ABL mRNA>4.40% was significantly higher than that of BCR-ABL mRNA ≤ 4.40% (22.73% vs 0, P=0.021). The incidence of progression increased in group of BCR-ABL mRNA>4.40%, but the difference was not statistically significant (P=0.052).

CONCLUSIONIt is important for the prognosis evaluation of the patients to monitor the level of BCR-ABL transcripts at 3 months after TKI treatment, which might help to early optimization of treatment and to improve curative effect of CML patients.

Adult ; Aged ; Female ; Fusion Proteins, bcr-abl ; blood ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; diagnosis ; drug therapy ; Male ; Middle Aged ; Predictive Value of Tests ; Prognosis ; Protein Kinase Inhibitors ; therapeutic use ; RNA, Messenger ; genetics ; Treatment Outcome ; Young Adult

In vitro amplified human leukocyte antigen (HLA)-haploidentical donor immune cell infusion (HDICI) is not commonly used in children. Therefore, our study sought to evaluate its safety for treating childhood malignancies. Between September 2011 and September 2012, 12 patients with childhood malignancies underwent HDICI in Sun Yat-sen University Cancer Center. The median patient age was 5.1 years (range, 1.7-8.4 years). Of the 12 patients, 9 had high-risk neuroblastoma (NB) [7 showed complete response (CR), 1 showed partial response (PR), and 1 had progressive disease (PD) after multi-modal therapies], and 3 had Epstein-Barr virus (EBV)-positive lymphoproliferative disease (EBV-LPD). The 12 patients underwent a total of 92 HDICIs at a mean dose of 1.6×10(8) immune cells/kg body weight: 71 infusions with natural killer (NK) cells, 8 with cytokine-induced killer (CIK) cells, and 13 with cascade primed immune cells (CAPRIs); 83 infusions with immune cells from the mothers, whereas 9 with cells from the fathers. Twenty cases (21.7%) of fever, including 6 cases (6.5%) accompanied with chills and 1 (1.1%) with febrile convulsion, occurred during infusions and were alleviated after symptomatic treatments. Five cases (5.4%) of mild emotion changes were reported. No other adverse events occurred during and after the completion of HDIDIs. Neither acute nor chronic graft versus host disease (GVHD) was observed following HDICIs. After a median of 5.0 months (range, 1.0-11.5 months) of follow-up, the 2 NB patients with PR and PD developed PD during HDICIs. Of the other 7 NB patients in CR, 2 relapsed in the sixth month of HDICIs, and 5 maintained CR with disease-free survival (DFS) ranging from 4.5 to 11.5 months (median, 7.2 months). One EBV-LPD patient achieved PR, whereas 2 had stable disease (SD). Our results show that HDICI is a safe immunotherapy for childhood malignancies, thus warranting further studies.
Child ; Child, Preschool ; Cytokine-Induced Killer Cells ; immunology ; Epstein-Barr Virus Infections ; therapy ; Female ; Follow-Up Studies ; Graft vs Host Disease ; etiology ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Immunotherapy, Adoptive ; Infant ; Killer Cells, Natural ; immunology ; Lymphoproliferative Disorders ; therapy ; virology ; Male ; Neuroblastoma ; therapy ; Transplantation, Homologous ; Treatment Outcome