[Effect of Eltrombopag on Response to Immunosuppressive Therapy in Patients with Transfusion-Dependent Non-Severe Aplastic Anemia].

OBJECTIVE: To compare the efficacy of eltrombopag combined with cyclosporine A (CsA) and CsA alone in patients with transfusion-dependent non-severe aplastic anemia (TD-NSAA). METHODS: The clinical data of 76 patients with treatment-naive TD-NSAA in Ningde Municipal Hospital of Ningde Normal University and Affiliated Hospital of Nantong University from December 2017 to June 2021 were retrospectively analyzed. Among them, 45 cases were treated with eltrombopag combined with CsA, and 31 patients with compatible baseline characters were treated with CsA alone. The efficacy of patients between the two groups was compared, and the factors affecting the curative effects were also analyzed. RESULTS: There were significant differences in hematological response (HR) and complete response(CR) rates between the two groups at 3, 6, 12 months, and follow-up endpoint of treatment (P<0.05). With the prolongation of eltrombopag treatment time, the curative effect increased gradually, and the patients achieved more CR and HR rates by the end of the follow-up period. Simultaneously, with the increase in the maximum stable dose of eltrombopag, the HR rate increased gradually. The megakaryocyte count in eltrombopag group was higher than that in control at 6 and 12 months (P<0.05). Compared with the control group, the median time of platelet transfusion independence in eltrombopag group was more shorter (P=0.018), and the median platelets transfusion volume was lower (P=0.009). At 3, 6, 12 months after eltrombopag, the change of platelet in eltrombopag group was higher than that in the control group (P<0.05). Analysis of related factors affecting the efficacy showed that sex, age, iron overload, platelet count before treatment had no effect on the efficacy, and the median maximum stable dosage and the administration period for eltrombopag were related to the curative effect. The patients of eltrombopag group experienced adverse events of varying degrees, but the reactions were mild and mostly tolerated. CONCLUSION: Eltrombopag can effectively improve the hematopoietic response and promote platelet recovery for TD-NSAA patients with relatively more residual hematopoietic cells, and it is safe and well tolerated.

[Effects of the ITGA2B Nonsense Mutation (c.2659C > T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann's Thrombasthenia Generated with CRISPR/Cas9 Technology].

OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbß3 on the surface of platelet membrane. METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbß3 on the surface of platelet. RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbß3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). CONCLUSION: A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.

Long non-coding RNA HAGLROS promotes the development of diffuse large B-cell lymphoma via suppressing miR-100.

BACKGROUND: Long non-coding RNAs (lncRNAs), a vital component of functional regulators, are involved in various human cancers development, including diffuse large B-cell lymphoma (DLBCL). In particular, lncRNA HAGLROS has been reported to be associated with several types of cancer in humans. Nevertheless, the role of HAGLROS in DLBCL has yet to be described. METHODS: The HAGLROS expression patterns and its relationship with clinicopathological features and survival were investigated in DLBCL patients. CCK-8 and transwell assays were used to analyze the cell proliferation, migration, and invasion capacities. AGO2-RIP, dual-luciferase assay, RT-qPCR, and rescue experiments were fulfilled to measure the physical interaction between HAGLROS and miR-100. Xenograft assay was conducted to test tumor growth ability. RESULTS: HAGLROS was upregulated in DLBCL tissues and cells, and closely associated with advanced clinicopathological features. Upregulation of HAGLROS resulted in poor survival outcomes in DLBCL patients. In addition, HAGLROS knockdown inhibited the proliferation, migration, and invasion of DLBCL cells in vitro. Further experiments revealed that HAGLROS negatively regulated the expression of miR-100 in DLBCL, and the expression of miR-100 and HAGLROS showed an inverse correlation in DLBCL tissues. HAGLROS functioned as a competing endogenous RNA for miR-100 in DLBCL cells, and miR-100 overexpression abolished the oncogenic effects of HAGLROS upregulation on DLBCL progression. Besides, in-vivo assays revealed that HAGLROS knockdown suppressed tumor growth in nude mice. CONCLUSION: HAGLROS overexpression contributes to DLBCL development and poor prognosis via targeting miR-100, which could be a potential prognostic biomarker and therapeutic target for DLBCL patients.

Atorvastatin improves bone marrow endothelial progenitor cell function from patients with immune-related hemocytopenia.

BACKGROUND: Immune-related hemocytopenia (IRH) is a type of autoimmune disease that targets bone marrow hematopoietic cells. This study investigated the influence of atorvastatin on the functional aspects of bone marrow endothelial progenitor cells (BM EPCs) in IRH patients. METHODS: BM EPCs were isolated from 15 patients with IRH and 20 normal controls. The isolated BM EPCs were characterized by flow cytometry. Cell counting kit-8, flow cytometry, and Transwell migration assays were used to determine the proliferation, apoptosis, and migration of BM EPCs, respectively. Protein levels were determined by western blot assay. RESULTS: The BM EPCs isolated from IRH patients showed reduced proliferation, increased apoptosis, and attenuated migratory ability compared to those from normal controls. Western blot analysis showed that the protein level of p-p38 was significantly increased, while that of Phosphorylated protein kinase B (p-AKT) was significantly decreased in the BM EPCs from IRH patients, compared to BM EPCs from healthy subjects. Cell proliferation and migration were significantly enhanced by atorvastatin, recombinant human thrombopoietin, and SB20358 compared to the untreated BM EPCs from IRH patients. Atorvastatin, Recombinant human thrombopoietin (TPO), and SB20358 treatment significantly suppressed the protein levels of p-p38 protein, but increased those of p-AKT in BM EPCS from IRH patients. CONCLUSIONS: In summary, atorvastatin increases the number and function of BM EPCs in IRH patients by regulating the p38 and AKT signaling pathways.

Low-dose bortezomib and dexamethasone as primary therapy in elderly patients with WaldenstrÓ§m macroglobulinemia.

OBJECTIVE: This retrospective study was designed to determine the efficacy and safety of low-dose bortezomib and dexamethasone (lBD) in elderly Chinese patients with WaldenstrÓ§m macroglobulinemia (WM). METHODS: Ten patients with WM aged over 60 years received first-line treatment with lBD. RESULTS: The median age was 70 years (range, 61-77 years). The overall response rate was 80%, including 1 patient who achieved a complete response, 1 patient with very good partial response, and 6 patients with a partial response. Median time to response was 1.8 months after treatment with lBD. Six (60%) patients achieved a partial response, including 2 (20%) patients who had a more than 75% reduction in serum immunoglobulin M levels. A rapid reduction in paraprotein was observed in three patients who received plasmapheresis. After a median follow-up period of 36 months, all patients were still alive and six had no disease progression. The estimated median time to progression was 39 months (range, 15-60 months). The most common adverse events were anemia, thrombocytopenia, neuropathy, and neutropenia. Peripheral neuropathy was the most common non-hematological toxicity in six (60%) patients, but did not result in the discontinuation of bortezomib. CONCLUSIONS: Our findings show that lBD is an effective and tolerable treatment regimen for elderly patients with WM.

Establishment and characterization of a rare atypical chronic myeloid leukemia cell line NT-1.

Human leukemia cell lines are of great value in leukemia research. In this study, we established and described the biological characteristics of a rare atypical chronic myeloid (aCML) leukemia cell line (NT-1). Mononuclear cells were isolated from the bone marrow of a patient with atypical chronic myeloid leukemia (Ph(-)/bcr(-)/abl(-)), and were passaged by liquid culture. Cells were maintained without any cytokines for over 1 year, and named NT-1. This cell line was extensively characterized using morphological assays, flow cytometry, cytogenetic analysis, clonogenic culture, quantitative fluorescent PCR, short tandem repeating sequence PCR (STR-PCR) and array-CGH. Its tumorigenic capacity was also examined in nude mice. The NT-1 cell line had morphological features of chronic myeloid leukemia and major myeloid markers (CD13, CD33, CD11b). Additionally, NT-1 expressed progenitor cells and natural killer cell-related antigens such as CD34, CD117, CD56. Cytogenetic analysis initially demonstrated two abnormalities: 47, xx, +8 and 47, xx, +8 accompanied by t(5;12)(q31;p13) translocation. The one-year passage process did not alter the karyotype. NT-1 cells maintained the same morphology, immunophenotyping and cytogenetic features as primary leukemia cells, which was strongly supported by STR-PCR results. Neither Epstein-Barr virus nor mycoplasma was detected in the NT-1 line. In addition, NT-1 cells showed high tumorigenic capacity in nude mice. NT-1 is a new atypical chronic myeloid leukemia cell line with the +8 and t(5,12) translocation, and exhibits high tumorigenicity in nude mice. This new cell line provides a useful tool for the study of leukemogenesis.

[Apoptosis and its mechanisms of spleen CD4⁺ CD25⁺ regulatory T cells in severe aplastic anemia mouse model].

OBJECTIVE: To explore the apoptosis and its mechanisms of spleen CD4⁺ CD25⁺ regulatory T (Treg) cells in severe aplastic anemia (SAA) mouse model induced by interferon (IFN-γ) in combination with busulphan (BU). METHODS: The BALB/c female mice SAA model was induced by intraperitoneal injection with IFN-γ and intragastric administration with BU (combined group, n=16), with BU group (n=16), IFN-γ group (n=16) and normal group (n=16) as controls. Spleen Treg cells were purified by using of immunomagnetic beads. Apoptosis was detected by flow cytometry. AKt and TGF-ß expression was measured by Western blot. RESULTS: Apoptosis of spleen Treg cells in combined group [(33.21±0.65)%] was significantly higher than that in BU group [(21.58±0.64)%], IFN-γ group [(17.62±1.05)%], and control[(27.38±1.89)%] (P<0.05). A significantly lower expression of AKt and TGF-ß protein was also seen in combined group (0.30±0.05 and 0.17±0.05), as compared to the other three groups (P<0.05). CONCLUSION: Excessive apoptosis of Treg cells was found in SAA mouse model, which may be a cause of Treg cells decrease in patients with AA. The down-regulated expression of AKt and TGF-ß could play a role in increased apoptosis of Treg cells. Our data may provide a new treatment strategy in AA.

[The changes of regulatory T cells and Th17 cells in a novel mouse severe aplastic anemia model].

OBJECTIVE: To explore the changes of regulatory T (Treg) cells and Th17 cells in a novel mouse severe aplastic anemia (SAA) model induced by interferon-γ (IFN-γ) combined with busulphan (BU), and to demonstrate the rationality of the model in immunology level. METHODS: The BALB/c female mice SAA model was constructed by intraperitoneal injection with IFN-γ and intragastric administration with BU (combined group), with BU group, IFN-γ group and normal group as controls. After collecting the mononuclear cells in the peripheral blood (PB) and spleen of mice in each group, the percentage of CD4(+)CD25(+)FOXP3(+) Treg cells and Th17 cells in the mononuclear cells were detected by flow cytometry(FCM), and to analyze the changes. RESULTS: The percentage of the Treg cells in PB and spleen was (3.19 ± 0.76)% and (4.77 ± 1.05)% respectively in combined group, being significantly lower than in other three groups (all P < 0.01). The percentage of the Th17 cells in PB and spleen was (2.07 ± 0.12)% and (3.18 ± 0.46)% respectively in combined group, being significantly higher than that in other three groups (P < 0.05 and 0.01, respectively). CONCLUSIONS: Lower Treg cells and higher Th17 cells was found in the novel mouse SAA model induced by IFN-γ combined with BU, which demonstrates that this SAA model may be more close to the human immune-mediated marrow failure.

[A novel severe aplastic anemia mouse model induced by IFN-γ plus busulphan].

OBJECTIVE: To establish a novel severe aplastic anemia (SAA) mouse model by interferon-γ (IFN-γ) plus busulphan. METHODS: Thirty clean-class BALB/c female mice were intraperitoneally injected with IFN-γ and intragastrically administrated with busulphan (group I), meanwhile busulphan alone group (n = 30, group II) and normal control group (n = 30, group III). Multi-parameters were compared among the three groups. RESULTS: In group I at day 10 after treatment, the incidence of SAA was 100% and mortality 20% respectively; the WBC, HGB, PLT, absolute reticulocyte count (Ret) and tibial nucleated cell count (TNCC) were (0.8 ± 0.3) × 10(9)/L, (45 ± 20) g/L, (10 ± 8) × 10(9)/L, (15.2 ± 10.2) × 10(9)/L, (12 ± 7) × 10(6)/tibia, respectively, which were significantly different from the other two groups (all P < 0.05). The bone marrow smears and patho-histological examinations showed marked reductions of marrow cell proliferation, and increases of the percentages of non-hematopoietic cells and cellular adipose. The depression was severe and irreversible. In group II, the blood cells count, TNCC and marrow proliferation recovered gradually with erythroid hyperplasia and hematopoietic dysplasia. CONCLUSIONS: IFN-γ plus busulphan can establish a SAA mouse model in a relatively short period, which is more resemble with human SAA.

[The Effects of IFN-γ on AKT activated 32D Cells and its Mechanisms].

OBJECTIVE: To investigate the effects of activated AKT on murine myeloid precursor cells (32D cells), and the effects of IFN-γ on 32D cells and its mechanisms. METHODS: Plasmid transduction was used to enhance the expression of AKT on 32D cells. After the transfected cells treated with IFN-γ for 24 hours, proliferation rate was tested by WST-1, apoptosis by flow cytometry, expression of phosphorylated Erk1/2, Stat3 and phosphorylated Stat3 was determined by Western blot. RESULTS: (1) IFN-γ at low concentration (100 U/ml) enhanced the growth and proliferation of 32D cells, while at high concentration (1000 U/ml) suppressed them. (2) Compared with control groups, low concentration IFN-γ increased (1124 ± 13) Stat3 phosphorylation in 32D-cell, while it high concentration IFN-γ decreased (601 ± 13). 32D cells transfected with activated Akt grew rapidly (0.287 ± 0.010) and had a low apoptotic rate [(9.57 ± 0.17)% (P < 0.05)]. (3) The expression of p-Erk1/2 in transfected 32D-cell was significantly reduced (P < 0.05). (4) Apoptosis rate of IFN-γ treated group was significantly decreased in transfected 32D cells (P < 0.05). CONCLUSIONS: IFN-γ has dual effects on 32D cells, namely, at low concentration enhanced the growth and proliferation of 32D cells, while at high concentration suppressed them. Its mechanisims is possibly through Stat3 pathway. Activated Akt can significantly promote the growth and proliferation of 32D cell and significantly inhibit apoptosis and IFN-γ can regulate cell proliferation and apoptosis through AKT. AKT activation can inhibit the Erk signal pathway, which may be affected by inhibition the modificaton of Raf1.