[Expression and Significance of PD-1 and ICOS in Patients with Primary Immune Thrombocytopenia].

OBJECTIVE: To investigate the expression of programmed death receptor-1 (PD-1) and inducible costimulator (ICOS) on the surface of CD8+ T cells in peripheral blood of patients with primary immune thrombocytopenia (ITP), and explore the roles of PD-1 and ICOS in the occurrence and development of ITP. METHODS: A total of 28 ITP patients treated in Tianjin Medical University General Hospital from September to December 2020 were selected, including 13 patients with newly diagnosed ITP, 15 patients with chronic ITP, and 22 healthy volunteers were recruited as control group. Flow cytometry was used to detect the expression levels of PD-1 and ICOS, and evaluate their correlation with clinical indicators. RESULTS: The percentage of CD8 + T cells in ITP patients of chronic group was higher than that of the newly diagnosed group and the control group (P<0.05). The expression level of PD-1 on CD8+ T cells in ITP patients of newly diagnosed group and chronic group were significantly lower than that of the control group (P<0.05), while the expression level of ICOS were significantly higher (P<0.05). In ITP patients, PD-1 was negatively correlated with platelet count (r=-0.4942, P<0.01), but positively with ICOS (r=0.4342). PD-1 and ICOS were both negatively correlated with lymphocyte count (rPD-1=-0.4374; rICOS=-0.4492). CONCLUSION: In ITP patients, the unbalanced expression of PD-1 and ICOS may interfere with the immune homeostasis of the body, which can be used as a therapeutic target for ITP patients.

The Risk of Clonal Evolution of Granulocyte Colony-Stimulating Factor for Acquired Aplastic Anemia: A Systematic Review and Meta-Analysis.

OBJECTIVES: This meta-analysis aimed to evaluate the risk of clonal evolution of granulocyte colony-stimulating factor (G-CSF) in acquired aplastic anemia (AA), and whether the use of G-CSF increases the occurrence of secondary malignant neoplasms, mainly myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) or paroxysmal nocturnal hemoglobinuria (PNH). METHODS: Data were gathered from randomized controlled trials (RCTs) to evaluate the effect of G-CSF versus no G-CSF at the risk of developing the clonal complications of acquired AA. Electronic searches in PubMed, Embase, and the Cochrane Library were performed to identify studies up to 1 January 2017. Only RCTs performed on patients who were randomly assigned to receive G-CSF or not to receive G-CSF were included. RESULTS: Four relevant trials that met the inclusion criteria were identified. In a pooled analysis, the G-CSF groups of AA patients were not associated with a statistically significant higher occurrence of secondary malignant neoplasm, mainly MDS and AML (relative risk [RR] 0.86; 95% confidence interval [CI] 0.34-2.19; 4 trials). No significant heterogeneity was found (p = 0.67, I2 = 0%). There was no statistically significant higher occurrence of PNH in the G-CSF groups with AA (RR 1.17; 95% CI 0.51-2.71; 4 trials) and no significant heterogeneity was found (p = 0.42, I2 = 0%). CONCLUSIONS: G-CSF for patients with AA is not associated with a higher occurrence of secondary malignant neoplasm, mainly MDS/AML, or PNH.

[Influence of Costimulatory Signaling Molecules on Immun Functons of B Lymphocytes in Patients with Immune Thrombocytopenia].

OBJECTIVE: To explore the effect of costimulatory signaling molecules (CD80, CD86) expression on the quantity and function of B lymphocytes in peripheral blood (PB) of the patients with immune thrombocytopenia (ITP). METHODS: A total of 55 ITP patients (30 cases were newly diagnosed and 25 cases were in remission), 25 healthy volunteers as controls were enrolled in this study. The levels of CD19(+)CD5(+), CD19(+)CD80(+), CD19(+)CD86(+), CD41a(+)IgG, CD41a(+)IgM and IgG, IgM in CD19(+)B cells were measured by flow cytometry (FCM). The correlation of CD19(+)B cells with clinical parameters of ITP patients was analyzed. RESULTS: The level of B1 (CD19(+)CD5(+)) of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P<0.05). The level of CD19(+)CD80(+) of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P<0.05). And the expression of IgG and IgM in CD19(+)B cells of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P<0.05). The levels of IgG and IgM in remitted ITP patients after treatment were significantly lower than that before treatment (P<0.05). The level differences of IgG and IgM before and after treatment in refractory ITP patients were not statistically significant (P>0.05). The expression of CD19(+)CD80(+) in newly diagnosed ITP patients positively correlated with the level of Th1 and Th1/Th2 (r=0.502, r=0.471, P<0.05). The expression of CD19(+)CD80(+) of newly diagnosed ITP patients positively correlated with the level of IgG and IgM in CD19(+)B cells (r=0.552, r=0.467, P<0.05), and negatively correlated with PB platelet count (r= -0.424, P<0.05). The levels of IgG and IgM in CD19(+)B cells of newly diagnosed ITP patients negati- vely correlated with PB platelet count (r=-0.658, r=-0.526, P<0.05). CONCLUSION: The enhacement of costimulatory signaling pathway of CD19(+)B cells in ITP patients results in the abnormal activation of B lymphocytes, thereby mediates the dysfunction of immune system and involves in the pathogenesis of ITP.

Expression and function of hematopoiesis-stimulating factor receptors on the GPI- and GPI+ hematopoietic stem cells of patients with paroxysmal nocturnal hemoglobinuria/aplastic anemia syndrome.

Paroxysmal nocturnal hemoglobinuria/aplastic anemia (PNH/AA) syndrome presents a markedly increased population of cells deficient in glycophosphatidylinositol (GPI- cells) and signs of bone marrow failure, which requires treatment with hematopoiesis-stimulating factors, such as granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). However, little is known about the effects of these stimulating factors on GPI- cells. In order to explore the effects of stimulating factors in PNH/AA, G-CSF receptor (CD114) and SCF receptor (CD117) expression levels on GPI+ and GPI- hematopoietic stem cells (HSCs) were measured by flow cytometry (FCM). The mean fluorescence intensity (MFI) values of signal transducer and activator of transcription 5 (STAT5) and phosphorylated (P)-STAT5 were measured in GPI+ and GPI- HSCs by FCM following stimulation with G-CSF or SCF in vitro. The expression levels of CD114 and CD117 on GPI- HSCs were significantly lower (P<0.01) than those on GPI+ HSCs in PNH/AA patients and normal controls. The MFI values of STAT5 in the GPI- and GPI+ HSCs of PNH/AA patients and normal controls were not significantly different. However, the MFI values of P-STAT5 in the GPI- HSCs of PNH/AA patients were significantly lower than those in the GPI+ HSCs of PNH/AA patients and normal controls prior to and following stimulation with G-CSF or SCF (P<0.01). The GPI- HSCs of PNH/AA patients responded poorly to stimulation by hematopoiesis-stimulating factors, which indicates that these factors can be used safely in patients with PNH/AA.

[The mechanisms underlying bone marrow damage by iron overload in pancytopenic patients with positive BMMNC-Coombs test].

OBJECTIVE: To investigate the mechanisms underlying bone marrow damage by iron overload in pancytopenic patients with positive BMMNC-Coombs test (IRP). METHODS: Twenty-one iron overloading, 26 non-iron overloading IRP patients and 10 normal controls were enrolled in this study. The expressions of ROS, Bcl-2, Caspase-3 and apoptosis of BMMNC were analyzed by flow cytometry (FCM). Antioxidants were added to iron overloading IRP BMMNC, and then the changes of indices above were detected by FCM. The number and apoptosis of T lymphocytes of IRP patients were also detected. RESULTS: ROS and apoptosis of BMMNC, myelocytes, erythrocytes and stem cells of iron overloading IRP patients were significantly higher than that of non-iron overloading IRP ones and normal controls (P < 0.05). The expressions of Bcl-2 on BMMNC, erythrocytes and stem cells of iron overloading IRP patients were significantly lower than those of non-iron overloading IRP ones (P < 0.05). The levels of Caspase-3 on myelocytes, erythrocytes and stem cells of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP ones and normal controls (P < 0.05). After treatment with antioxidants, the expressions of ROS, Caspase-3 and apoptosis of iron overloading IRP BMMNC significantly decreased, but opposite for Bcl-2. The percentages of CD4(+) lymphocytes [ ( 40.86 ± 8.74）%] and CD4(+)/CD8(+) (1.44 ± 0.36) in PB of iron overloading IRP patients were significantly higher than that of non-iron overloading IRP ones [(35.96 ± 7.03)% and 1.14 ± 0.37] and normal controls [(28.00 ± 6.73)% and 0.79 ± 0.21], respectively (P < 0.05), as opposite for CD8(+) lymphocytes (P < 0.05). The apoptosis of CD8(+) lymphocytes [(27.35 ± 10.76)%] and the ratio of CD8(+) apoptosis/CD4(+) apoptosis (2.51 ± 0.81) in BM of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP ones [(15.47 ± 8.99)%] and normal controls (1.39 ± 0.47), respectively (P < 0.05). The apoptosis of erythrocytes and stem cells coated with auto-antibodies in BM of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP and normal controls. CONCLUSION: Mechanisms underlying bone marrow damage by iron overload might be through the follows: ①The increased ROS induced by excessive iron deposition affected the expressions of Caspase-3 and Bcl-2, which caused more BMMNC apoptosis; ②The abnormal number and ratio of T lymphocytes caused by iron overload aggravated the abnormality of immunity of IRP; ③Iron overload may increase the damage to erythrocytes and stem cells coated with auto-antibodies.

[Expression of phosphorylated STAT5 in bone marrow hematopoietic stem cells of patients with paroxysmal nocturnal hemoglobinuria before and after in vitro G-CSF or SCF stimulation].

OBJECTIVE: To study the expressions of phosphorylated STAT5 (P-STAT5) in CD34(+)CD59(-) and CD34(+)CD59(+) bone marrow cells of the patients with paroxysmal nocturnal hemoglobinuria (PNH) before and after in vitro G-CSF or SCF stimulation, then evaluate the functions of G-CSF and SCF receptors in PNH clone cells. METHODS: Bone marrow mononuclear cells (BMMNC) of 26 PNH patients and 14 normal controls were stimulated in vitro with G-CSF (100 ng/ml) or SCF (100 ng/ml) for 10 min. Before and after these stimulations, the mean fluorescence intensity (MFI) of P-STAT5 in CD34(+)CD59(+) BMMNC and CD34(+)CD59(-) BMMNC were measured by flow cytometry. RESULTS: (1) The P-STAT5 MFI was (24 ± 18) in unstimulated CD34(+)CD59(-) cells. And it was significantly lower than that in unstimulated CD34(+)CD59(+) cells of PNH patients (64 ± 49) and normal controls (61 ± 33) (both P < 0.01). No statistic difference existed between the latter two. (2) The P-STAT5 MFI was (36 ± 35) in G-CSF stimulated CD34(+)CD59(-) cells of PNH patients. And it was significantly lower than that in G-CSF stimulated CD34(+)CD59(+) cells of PNH patients (124 ± 84) and normal controls (116 ± 59) (both P < 0.01). There was no statistic difference between the latter two. (3) The P-STAT5 MFI was (34 ± 27) in SCF stimulated CD34(+)CD59(-) cells of PNH patients. And it was significantly lower than that in SCF stimulated CD34(+)CD59(+) cells of PNH patients (124 ± 97) and normal controls (128 ± 62) (both P < 0.01). And no statistic difference existed between the latter two. (4) The increased P-STAT5 MFI in G-CSF stimulated CD34(+)CD59(-) cells of PNH patients was significantly lower than that in G-CSF stimulated CD34(+)CD59(+) cells of PNH patients and normal controls (both P < 0.01). No statistic difference existed between the latter two. The increase of P-STAT5 MFI in SCF stimulated CD34(+)CD59(-) cells of PNH patients was significantly lower than that in SCF stimulated CD34(+)CD59(+) cells of PNH patients and normal controls (both P < 0.01). No statistic difference existed between the latter two. CONCLUSION: There is a lower expression of P-STAT5 expressed in G-CSF or SCF stimulated PNH clone cells compared to that in normal clone cells.