Effect of Eltrombopag on Response to Immunosuppressive Therapy in Patients with Transfusion-Dependent Non-Severe Aplastic Anemia / 中国实验血液学杂志

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 / 中国实验血液学杂志

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 αIIbβ3 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 αIIbβ3 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 αIIbβ3 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.

Rapamycin affect the apoptosis of splenic CD4+CD25+ regulatory T cells of mouse severe aplastic anemia model / 中华血液学杂志

Objective: To explore the effects and possible mechanism of rapamycin (RAPA) on apoptosis of CD4+CD25+ Tregs from the mouse severe aplastic anemia (SAA) model. Methods: The BALB/c female SAA model mice were induced by interferon-gamma in combination with busulphan. The SAA model mice were intraperitoneal injection with RAPA at daily dose of 0.5 mg/kg for 5 days (the RAPA-treated group, n=15) in the SAA group (n=15) and the un-treated group (n=15) were control. Bone marrow hematopoiesis changes were observed by the patho-morphological examination of femurs. The mononuclear cells of the peripheral blood and spleen were subjected to assess the intracellular Foxp3 expression in CD4+CD25+ Tregs by flow cytometry (FCM). In addition, after being pured by immunomagnetic beads, the splenic CD4+CD25+ Tregs was subjected to assess apoptosis by FCM and the Akt and Stat3 phosphorylation by using of western blot. Results: The patho-morphological examination of femurs showed normal marrow cell proliferation in un-treated group and hypocellularity in both SAA group and RAPA-treat group, with an increase in the number of fat cells. The bone marrow hematopoietic tissue ratio in RAPA-treat group was higher than SAA group [(9.75±1.83)% vs (7.00±2.00)%, Δx=2.15% (95%CI 0.15%-5.35%), P=0.037]. In the SAA group, FCM analysis showed down-expression of Foxp3 in CD4+CD25+ Tregs compared with the un-treated group. However, after treatment with RAPA, the expression of Foxp3 in CD4+CD25+ Tregs was increased (P<0.017). Compared with the un-treated group, increased CD4+CD25+ Tregs apoptosis [(19.84±1.39)% vs (29.85±2.72)%] with increased Akt phosphorylation accompanied by increased Stat3 phosphorylation was found in SAA group (P<0.05, respectively). On the contrary, RAPA-treated group exhibited CD4+ CD25+ Tregs with a reduction in apoptosis rate [(22.39±3.71)%], Akt phosphorylation and Stat3 phosphorylation compared with the SAA group (P<0.05, respectively). Conclusion: These results indicate that RAPA may increase the expression of Foxp3 by down-regulation the levels of Akt and Stat3 phosphorylation and reduce apoptosis in splenic CD4+CD25+ Tregs from the mice model of SAA.

Apoptosis and its mechanisms of spleen CD4⁺ CD25⁺ regulatory T cells in severe aplastic anemia mouse model / 中华血液学杂志

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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-β expression was measured by Western blot.</p><p><b>RESULTS</b>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-β 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).</p><p><b>CONCLUSION</b>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-β could play a role in increased apoptosis of Treg cells. Our data may provide a new treatment strategy in AA.</p>

The changes of regulatory T cells and Th17 cells in a novel mouse severe aplastic anemia model / 中华血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>

A novel severe aplastic anemia mouse model induced by IFN-γ plus busulphan / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish a novel severe aplastic anemia (SAA) mouse model by interferon-γ (IFN-γ) plus busulphan.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>IFN-γ plus busulphan can establish a SAA mouse model in a relatively short period, which is more resemble with human SAA.</p>

The Effects of IFN-γ on AKT activated 32D Cells and its Mechanisms / 中华血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>(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).</p><p><b>CONCLUSIONS</b>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.</p>