[Study on the dendritic cell subsets in peripheral blood and its relationship with the expressions of Gata-3 and T-bet in lymphocytes of patients with immune thrombocytopenia].

OBJECTIVE: To explore the relationship between the dendritic cell (DC) subsets and abnormal expression of transcription factors Gata-3 and T-bet in patients with immune thrombocytopenia (ITP). METHODS: The plasmacytoid DC (pDC) and myeloid DC (mDC) of 33 ITP (16 untreated, 17 remitted) patients and 12 healthy controls were analyzed by flow cytometry (FCM) . The expressions of Gata-3 mRNA and T-bet mRNA in peripheral blood mononuclear cell (PBMNC) were detected by reverse transcription-polymerase chain reaction (RT-PCR) .The levels of interleukin-4 (IL-4) and interferon-gamma (IFN-γ) were measured by FCM in 33 ITP patients and 12 healthy controls. RESULTS: The percentage of pDC in PBMNC was 0.49% ± 0.18% in untreated and it was higher than that in remitted ITP patients (0.27% ± 0.17%) and in controls (0.32% ± 0.13%) (both P < 0.05). The percentage of mDC in PBMNC was 0.23% ± 0.17% in untreated, which was lower than that in remitted ITP patients (0.33% ± 0.18)% and in controls (0.31% ± 0.11%), but no statistic difference in mDC expression existed among 3 groups (P > 0.05). pDC/mDC ratios was (3.15 ± 2.01) in untreated ITP patients and it was higher than that in remitted ITP patients (0.81 ± 0.32) and in controls (1.07 ± 0.44) (both P < 0.05). The relative mRNA expression levels of Gata-3 were 2775 ± 489, 1357 ± 307 and 652 ± 165 respectively. And the expression of Gata-3mRNA in untreated group was higher than that in remission group or healthy controls (both P < 0.05). The relative mRNA expression levels of T-bet were 782 ± 394, 583 ± 176 and 576 ± 120. No statistic difference in T-bet expression existed among 3 groups (P > 0.05). Gata-3mRNA/T-bet mRNA ratio was (4.13 ± 1.69 ) in untreated group and it was higher than that of remission group (2.45 ± 0.69) or controls (1.15 ± 0.27) (both P < 0.05). The level of IL-4 in the untreated group was 9.14% ± 4.34% and it was higher than that of remission group (4.78% ± 1.69%) or controls (4.86% ± 1.41%). The level of IFN-γ in the untreated group was lower than that of controls (P < 0.05). Significant positive correlations existed between Gata-3 and pDC/mDC ratio (r = 0.585, P < 0.01). Significant positive correlations existed between Gata-3 and IL-4 ( r = 0.463, P < 0.05). CONCLUSION: The mechanism of ITP may be due to a disorder of DC subsets and a high expression of Gata-3.

[Inhibitory effects of tacrolimus on effector T cells from patients with severe aplastic anemia].

OBJECTIVE: To explore the inhibitory effects of tacrolimus (FK506) on effector T cells in vitro and examine the relationship between effector T cells and clinical features in patients with severe aplastic anemia (SAA) to elucidate its immune mechanism. METHODS: The CD8(+) HLA-DR(+) cells, sorted by immunomagnetic separation from bone marrow mononuclear cells (BMMNC) of 16 SAA patients, were cultured in different concentrations of interleukin-2 (IL-2) alone or with FK506 for 72 hours. The proliferation effect was measured with methyl thiazolyl tetrazolium (MTT) method. The T lymphocytes were sorted from the SAA patients by lymphocyte separation medium and cultured alone or with IL-2 or with FK506 or FK506 plus cyclosporin A (CsA) for 18 hours. The expression of tumor necrosis factor-ß (TNF-ß) in CD8(+) HLA-DR(+) T cells was analyzed by flow cytometry. The relationship between the expression of TNF-ß and the clinical data, including percentages of reticulocyte and lymphocytes in peripheral blood cell count and ratio of CD4(+) T cells and CD8(+)T cells, was also analyzed. RESULTS: At the concentration of IL-2 greater than or equal to 20 U/ml, the cell proliferation (A values, 0.538 ± 0.142) were significantly higher than that in the blank culture hole (0.505 ± 0.153) (P < 0.05). The A values significantly decreased (0.386 ± 0.124) after the addition of FK506 (P < 0.05). Compared with control group, the expression of TNF-ß was significantly higher in IL-2 group (73.36% ± 16.73% vs 66.61% ± 16.20%, P < 0.05), significantly lower in FK506 and FK506 plus CsA groups (P < 0.05). No significant differences existed between the FK506 and FK506 plus CsA groups (47.78% ± 20.09% and 42.23% ± 21.35%, P > 0.05). The expression of TNF-ß in SAA was negatively correlated with the percentage of reticulocyte and the ratio of CD4(+) T cell and CD8(+) T cell, positively correlated with the percentage of lymphocyte in peripheral blood count (r = -0.86, -0.90, 0.77, all P < 0.05). CONCLUSIONS: IL-2 can enhance the proliferation and expression of TNF-ß of CD8(+)HLA-DR(+)T cells from SAA patients. Such an effect is inhibited by FK506. And FK506 and FK506 plus CsA have similar effects.

[Telomere length and gene expression of shelterin in CD3(+) T cell of severe aplastic anemia patients].

OBJECTIVE: To explore the changes in telomere length and gene expression of complex shelterin (composed of 6 core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1) in severe aplastic anemia (SAA). METHODS: Bone marrow samples were obtained from 20 SAA patients and 10 normal controls. CD3(+)T cells were sorted by immunomagnetic separation. Telomere length was tested by Southern blot and the gene expressions of TRF1, TRF2, POT1, TIN2, TPP1 and RAP1 were detected by reverse transcription-PCR(RT-PCR). RESULTS: Telomeres of CD3(+)T cells were found significantly shorter in SAA untreated ((4.4 ± 1.1) kb, n = 9) and recovering groups((5.8 ± 1.0) kb, n = 11) than control group ((9.2 ± 3.3) kb, P < 0.05). Telomere length of CD3(+)T cells shortened with TH/S decreasing (r = 0.564, P = 0.029). The mRNA expression of POT1 decreased in untreated SAA patients (0.16(0.02-0.29)) and over-expressed in recovering patients (1.17(0.82-1.86), P < 0.05). The mRNA expression of RAP1 was significantly higher in untreated patients (4.14 (1.93-6.92)) than that in recovering group (0.87 (0.30-1.73) ) and controls (0.62 (0.45-4.07) , both P < 0.05). CONCLUSION: Changes in telomere length and shelterin gene expression occur in CD3(+)T cells of SAA patients and may be correlated with disease severity.

[The long-term efficacy and safety of rituximab combined with cyclophosphamide in treatment of seven patients with refractory and recurrent autoimmune hemolytic anemia].

OBJECTIVE: To assess the efficacy and safety of monoclonal antibody rituximab combined with cyclophosphamide (CTX) in the treatment of refractory and recurrent autoimmune hemolytic anemia. METHODS: Seven cases with refractory and recurrent autoimmune hemolytic anemia (including 1 case of Evans syndrome) were recruited during January, 2007 to December, 2010. Treatment regimens were as follows: rituximab: 375 mg/m², 1 time/week, 2-6 courses; CTX:1 g, 1/10 d, 2-7 courses; combined with intravenous immunoglobulin (IVIG) 5 g, 1 time/week, given 1 day after rituximab administration. The efficacy and safety of this regimen were assessed during follow-up. RESULTS: All the patients showed good responses (7/7). Six patients achieved complete remission (6/7) and one achieved partial remission (1/7). Average follow-up time for the patients was 27 months. All patients remained in remission during the 12-month follow-up visits. Two patients showed elevated indirect bilirubin and increased reticulocyte counts within 24 months. One patient achieved complete remission after additional rituximab therapy, and another patient remained partial remission after cyclosporine therapy. At the time of 36-month follow-up visit, the patient relapsed and was retreated with 3 courses of rituximab combined with CTX and eventually achieved partial remission. All patients tolerated the treatment well with few mild side effects. CONCLUSIONS: Rituximab combined with CTX is effective and relatively safe in patients with refractory and recurrent autoimmune hemolytic anemia. Additional treatment to relapse patients about 12 - 24 months after drug withdrawal continues to be effective.

[TET2 and DLK1 gene expression and their clinical significance in bone marrow CD(3)(+) T cells of patients with myelodysplastic syndrome].

OBJECTIVE: To investigate the expression of TET2 and DLK1 mRNA in bone marrow CD(3)(+) T cells of patients with myelodysplastic syndrome (MDS) and their clinical significance and to explore the potential mechanism of abnormal cell-mediated immunity. METHODS: CD(3)(+) T cells were sorted by magnetic activated cell-sorting system. The expressions of TET2 and DLK1 mRNA in bone marrow CD(3)(+) T cells from 26 MDS patients and 16 healthy controls were detected by fluorescence quantitative PCR. RESULTS: The expression of TET2 mRNA in CD(3)(+) T cells was down-regulated in the MDS patients by (0.16 ± 0.15) fold compared with the controls (P < 0.05). The expression of TET2 mRNA in CD(3)(+) T cells of MDS patients was positively correlated with serum complement C(3) (r = 0.404, P < 0.05). The expression of DLK1 mRNA in CD(3)(+) T cells was up-regulated in the MDS patients by (1.61 ± 0.88) folds compared with the controls (P < 0.05). Grouped by the chromosomes, the patients with chromosome abnormalities presented significantly higher DLK1 mRNA level than those with normal chromosomes [(1.45 ± 0.44) folds, P < 0.05]. The expression of DLK1 mRNA in CD(3)(+) T cells of MDS patients was positively correlated with the proportion of bone marrow blasts (r = 0.343, P < 0.05). CONCLUSIONS: The mRNA expression of TET2 in CD(3)(+) T cells of MDS patients was decreased while the mRNA expression of DLK1 was increased, which might decline the immune surveillance function. The findings would be useful for exploring the mechanism of immune tolerance.

[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.

[Research of regulative factors on CD8(+)HLA-DR(+) effector T cells in severe aplastic anemia].

OBJECTIVE: To explore the regulative factors on CD8(+)HLA-DR(+) T cells in the patients with severe aplastic anemia (SAA) and examine the roles of these cells in the immunopathogenesis of SAA. METHODS: CD8(+)HLA-DR(+) T cells were sorted from bone marrow mononuclear cells of 13 SAA patients from July 2011 to March 2012 by magnetic activated cell sorting system and were divided into 3 groups: interleukin 2 (IL-2) group (0, 0.1, 1, 10, 100 and 1000 U/ml), cyclosporine A (CsA) group (addition of 400 ng/ml CsA in each IL-2-containing well),receptor antagonist group (addition of IL-2 receptor antagonist 8 µg/ml in each IL-2-containing well). Then cell proliferation rate was evaluated by MTT assay after a 72-hour culturing. Bone marrow mononuclear cells of the SAA patients were divided into CsA group, IL-2 group and control group and cultured for 18 hours and another 4 hours following the dosing of phorbol ester. The expression of tumor necrosis factor ß (TNF-ß) in CD8(+)HLA-DR(+) T cells was analyzed by flow cytometry. RESULTS: The cell proliferations of IL-2 wells at the concentrations of 10, 100 and 1000 U/L (0.36 ± 0.12, 0.41 ± 0.12, 0.46 ± 0.14) were significantly higher than those of the control wells (0.23 ± 0.11), CsA group (0.18 ± 0.05, 0.19 ± 0.00, 0.20 ± 0.04) and receptor antagonist group (0.18 ± 0.05, 0.17 ± 0.04, 0.18 ± 0.03, all P < 0.05). No statistic difference existed between CsA and receptor antagonist groups (P > 0.05). The expressions of TNF-ß of CD8(+)HLA-DR(+)T cells of the IL-2 group were higher than those of the control group (64% ± 25% vs 46% ± 22%) whereas the CsA group (27% ± 20%) were lower than those of the control group (both P < 0.05). CONCLUSIONS: IL-2 can significantly stimulate the proliferation of CD8(+)HLA-DR(+) T cells and accelerate the in vitro secretion of TNF-ß in SAA patients. The proliferation may be inhibited by CsA and receptor antagonist. And the expression of TNF-ß is suppressed significantly by CsA.

[Expression of lymphokines in CD8(+)HLA-DR(+) T lymphocytes of patients with severe aplastic anemia].

OBJECTIVE: To examine the expression of lymphokines damaging hematopoietic cells by CD8(+)HLA-DR(+) effector T cells in peripheral blood (PB) of the patients with severe aplastic anemia (SAA) and explore further the heterogeneous immunopathogenesis of SAA. METHODS: The CD8(+)HLA-DR(+) cells were sorted by immunomagnetic separation from the PB of 24 untreated SAA patients and 23 normal controls. The mRNA expressions of perforin, granzyme B, FasL and tumor necrosis factor ß (TNF-ß) of sorted cells were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The mRNA levels of perforin, granzyme B of CD8(+)HLA-DR(+)T cells in untreated group were higher than those of the controls (0.66 ± 0.25, 0.56 ± 0.26 vs 0.53 ± 0.14, 0.40 ± 0.13, P = 0.042, 0.012). The mRNA level of FasL in CD8(+)HLA-DR(+) T cells of untreated SAA patients was higher than that of the controls (0.77 ± 0.24 vs 0.61 ± 0.16, P = 0.011). The mRNA of TNF-ß in CD8(+)HLA-DR(+) T cells of untreated SAA patients was also higher than that of the controls (0.58 ± 0.16 vs 0.46 ± 0.15, P = 0.011). CONCLUSIONS: CD8(+)HLA-DR(+) T cells may damage hematopoiesis through the actions of perforin, granzyme B, TNF-ß and FasL. And it thus contributes to the immunopathogenesis of SAA.

[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.

[Percentages and functions of natural killer cell subsets in peripheral blood of patients with severe aplastic anemia].

OBJECTIVE: To analyze the percentage and functional changes of natural killer (NK) cell subsets in peripheral blood of severe aplastic anemia (SAA) patients before and after immunosuppressive therapy (IST) so as to evaluate the relationships between these changes and hematopoietic functions and explore the role of NK cells in the pathogenesis of SAA. METHODS: By flow cytometry, the percentages of NK cells (CD3(-)CD56(+)CD16(+)) and its subsets [CD3(-)CD56(bright)CD16(-)(CD56(bright)), CD3(-)CD56(dim) CD16(+)(CD56(dim)), CD3(-)CD56(-)CD16(+)] in peripheral blood lymphocytes were detected in 12 untreated patients, 30 recovered patients and 13 normal controls respectively from April 2010 to December 2010 in our hospital. NK cells activating receptors (NKG2D and NKp46), perforin and granzyme-ß of patients and normal controls were also detected. The correlation between these changes and hematopoietic functions, including the percentages of neutrophil granulocyte (ANC%), lymphocyte and reticulocyte absolute value in peripheral blood, and hyperplasia degree, percentage of granulocytes, erythrocytes, lymphocytes and megakaryocytes absolute value in bone marrow were evaluated. RESULTS: (1) The percentages of NK cells (10.30% ± 6.08%) and CD56 bright cells (0.11%) in untreated patients were significantly lower than those of recovered patients (16.47% ± 8.29%, 0.68%, both P < 0.05) or normal controls (19.45% ± 6.88%, 0.53%, both P < 0.05). The percentage of CD56(dim) cells in untreated patients was significantly lower than that of normal controls (9.62% ± 6.04% vs 18.21% ± 7.16%, P < 0.05). The percentage of CD3(-)CD56(-)CD16(+) cells was significantly higher in recovered patients than that of untreated patients or normal controls (0.79% vs 0.37%, 0.41%, both P < 0.05). (2) The expression of NKp46 and perforin of NK cells in untreated (88.23%, 64.97% ± 21.61%) and recovered patients (82.97%, 66.14% ± 20.73%) were significantly higher than those of healthy controls (40.99%, 42.11% ± 27.25%, all P < 0.05). (3) The percentage of NK CD56(bright) and CD3(-)CD56(-)CD16(+) cells of patients was positively correlated with ANC% (r = 0.423, 0.609, 0.468 respectively, all P < 0.05) and the percentage of granulocytes in bone marrow (r = 0.357, 0.517, 0.434 respectively, all P < 0.05). The percentages of NK, CD56(bright), CD56(dim) and CD3(-)CD56(-)CD16(+) cells were positively correlated with the hyperplasic degree of bone marrow (r = 0.455, 0.412, 0.404, 0.451 respectively, all P < 0.05), but they were negatively correlated with the percentage of lymphocytes in bone marrow (r = -0.522, -0.435, -0.411, -0.547 respectively, all P < 0.05). The expression of NKG2D, NKp46, perforin and granzyme-ß of NK cells had no correlation with hematopoiesis (all P > 0.05). CONCLUSION: The lowered percentage of NK CD56(bright), CD56(dim) cells and a higher expression of perforin may cause the over-function of T lymphocytes and thus lead to hematopoietic failure in SAA.

[Count and function of CD8(+)CXCR3(+) regulatory T cells in peripheral blood of patients with autoimmune hemolytic anemia].

OBJECTIVE: To explore the effects of CD8(+)CXCR3(+)T cells on autoimmune hemolytic anemia (AIHA). METHODS: Twenty-two AIHA patients, including 11 untreated and 11 recovered ones, and 23 normal controls were recruited from July 2010 to November 2010. The percentage of CD8(+)CXCR3(+)/CD8(+)T cells in peripheral blood and the expression of interleukin-10 (IL-10) in CD8(+)CXCR3(+)T cells were detected by flow cytometry. Their correlations with the count of CD3(+)CD4(+)cells and the percentage of CD5(+)CD19(+) in CD19(+) B cells were analyzed. The expression level of CXCR3 mRNA in PBMC was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The percentage of CD8(+)CXCR3(+)/CD8(+) of untreated AIHA patients was (39.80 ± 19.96)%. And it was lower than that of recovered patients [(58.76 ± 14.22)%, P < 0.05] and normal controls [(59.66 ± 12.62)%, P < 0.01]. The percentage of IL-10(+) T cells in CD8(+)CXCR3(+)T cells of untreated patients was (22.98 ± 14.96)% and it was lower than that of normal controls [(38.15 ± 17.03)%, P < 0.05]. The expression level of CXCR3 mRNA for untreated AIHA patients was (0.51 ± 0.19) and it was lower than that of normal controls (1.67 ± 1.17, P < 0.01). The percentage of CD8(+)CXCR3(+)/CD8(+)T cells had a negative correlation with the count of CD3(+)CD4(+) cells and the percentage of CD5(+)CD19(+)/CD19(+) B cells (r = -0.571, -0.583, both P < 0.05). So did the percentage of IL-10(+) T cells in CD8(+)CXCR3(+)T cells (r = -0.524, -0.523, both P < 0.05). CONCLUSION: The decreased count of CD8(+)CXCR3(+)T cells and the lowered level of IL-10 may disturb the immune tolerance and lead to the occurrence of AIHA.

[Differentiation and expression of membrane hemopoietic cytokine receptors on CD34+ bone marrow cells in patients with myelodysplastic syndromes].

OBJECTIVE: To detect the abnormalities of differentiation and expression of membrane hemopoietic cytokine receptors on CD34+ bone marrow cells in patients with myelodysplastic syndromes (MDS). METHODS: Forty-five newly diagnosed MDS cases from July 2008 to March 2010 in our hospital and 30 normal controls were enrolled. There were 17 low-risk and 28 high-risk patients. The CD34+CD38+ and CD34+CD38- bone marrow cells and the expressions of stem cell factor receptor (SCF-R), erythropoietin receptor (EpoR), granulocyte colony-stimulating factor receptors (G-CSFR) and thrombopoietin receptor (TpoR) on those cells were measured by flow cytometry. RESULTS: The mean percentage of CD34+ in karyocyte of MDS cases in high-risk patients [0.53% (0.10%-1.68%)] was significantly higher than that of control group [0.13% (0.08%-0.32%), P<0.01]. The mean percentages of CD34+CD38+ cells were significantly lower in low and high-risk groups (86.3%±8.5% and 82.6%±11.1%) than those in control group (92.3%±3.4%). And the percentage of CD34+CD38- cells was significantly higher in either low-risk or high-risk group (13.7%±8.5% and 17.4%±11.0%) than that in control group (7.7%±3.4%, both P<0.05). In control group, the mean percentage of antigen expression of EpoR was significantly lower in CD34+CD38+ cells than that in CD34+CD38- cells (18.7%±18.3% vs 63.6%±20.0%, P<0.01). The expressions of SCF-R, G-CSFR and TpoR were not significantly different between two cell populations. The expressions of EpoR on CD34+CD38+ cells of low and high-risk MDS groups [9.0% (1.4%-12.7%), 5.2% (1.1%-14.1%)] were significantly lower than those of control group [9.6% (5.1%-30.1%), both P<0.05]. The expressions of G-CSFR on CD34+CD38+ cells of low and high-risk MDS groups (29.8%±19.1%, 28.7%±21.1%) were significantly lower than those of control group (44.4%±23.4%, both P<0.05). The quantities of EpoR on CD34+CD38- cells of low and high-risk MDS groups (42.2%±21.9%, 25.7%±15.6%) were significantly lower than those of control group (63.6%±20.0%, both P<0.01). The expressions of TpoR on CD34+CD38- cells of low and high-risk MDS groups (5.4%±4.7%, 4.1%±4.0%) were significantly lower than those of control group (10.1%±8.3%, both P<0.05). The incidence of cytopenia with low expression rates of hemopoietic cytokine receptors on CD34+ cells was higher than that of MDS with high expression rates. CONCLUSION: The abnormalities of differentiation and membrane hemopoietic cytokine receptors expression of CD34+ bone marrow cells in MDS are associated with MDS cytopenia and may be useful for the diagnosis of MDS.

[STAT5 phosphorylation levels of erythropoietin and thrombopoietin receptors in CD34(+)CD59(-) and CD34(+)CD59(+) bone marrow cells of patients with paroxysmal nocturnal hemoglobinuria].

OBJECTIVE: To study the STAT5 phosphorylation levels of erythropoietin receptor (EPOR) and thrombopoietin receptor (TPOR) in CD34(+)CD59(-) and CD34(+)CD59(+) bone marrow cells of the patients with paroxysmal nocturnal hemoglobinuria (PNH). METHODS: The bone marrow mononuclear cells (BMMNC) were extracted from 23 PNH patients treated at our department from April 2010 to February 2011 and 11 normal controls. The mean fluorescence intensity (MFI) of phosphorylated STAT5 (P-STAT5) in CD34(+)CD59(+) cells and CD34(+)CD59(-) cells with or without the stimulation of 10 U/ml EPO and 50 U/ml TPO were examined by flow cytometry. RESULTS: (1) Without stimulation, the P-STAT5 MFI in CD34(+)CD59(-) cells of PNH patients was significantly lower than that of CD34(+)CD59(+) cells (31 ± 15 vs 74 ± 47, P < 0.01). And it was 59 ± 23 in normal control CD34(+)CD59(+) cells (P < 0.05). No statistic difference existed between the CD34(+)CD59(+) cells of PNH patients and the normal control CD34(+)CD59(+) cells. (2) Under the stimulations of EPO and TPO, the P-STAT5 MFI was significantly lower in CD34(+)CD59(-) cells of PNH patients than that of CD34(+)CD59(+) cells (49 ± 24 and 51 ± 41 vs 120 ± 82 and 124 ± 87, both P < 0.01). For the normal control CD34(+)CD59(+) cells, they were 79 ± 47 and 98 ± 53 respectively (P < 0.05). No statistic difference existed between the CD34(+)CD59(+) cells of PNH patients and the normal control CD34(+)CD59(+) cells. P-STAT5 MFI was elevated after the stimulations of EPO and TPO. The increments of CD34(+)CD59(+) cells in PNH patients were significantly higher than those of CD34(+)CD59(-) cells (49 ± 11 and 54 ± 43 vs 17 ± 4 and 16 ± 6, both P < 0.01). CONCLUSION: Under the in vitro stimulations of EPO and TPO, the STAT5 phosphorylation levels of EPO and TPO receptors in normally cloned hematopoietic stem cells in PNH patients are obviously superior to those in abnormally cloned counterparts.

[Expression of bone marrow macrophages antigen activation and its clinical significance in pancytopenia patients with positive bone marrow mononuclear cells-Coombs test.].

OBJECTIVE: To explore the expression of antigen activated of macrophages (MPhi) of bone marrow and its clinical significance in pancytopenia patients with positive bone marrow mononuclear cells (BMMNC)-Coombs test (immunorelated pancytopenia, IRP). METHODS: Sixty-one IRP patients, 10 severe aplastic anemia (SAA) patients and 13 healthy controls were enrolled in this study. The categories of auto-antibodies (IgG, IgM) on BMMNC (CD(34)(+)/CD(15)(+)/GlycoA(+) hematocytes), the quantity (CD(68)(+)/CD(45)(+))% and expression of antigen activated (CD(69)) of MPhi (CD(68)(+)CD(69)(+)/CD(+)(68))% in bone marrow of all cases and controls were measured by fluorescence activated cell sorting (FACS). RESULTS: The quantity and expression ratio of activated antigen of bone marrow (BM) MPhi in IRP patients [(0.57 +/- 0.30)% and (40.30 +/- 18.49)%] were respectively significantly higher than those in SAA [(0.46 +/- 0.08)% and (32.44 +/- 19.37)%] and healthy controls [(0.44 +/- 0.69)% and (29.71 +/- 11.67)%] (both P < 0.05). The quantity presented high-positive correlation with the expression ratio of activated antigen of BM MPhi (r = 0.89, P < 0.01). Patients with IRP were classified into two subgroups according to the quantity of MPhi: Group A (MPhi >/= 0.5%, 34 cases) and Group B (MPhi < 0.5%, 27 cases). Thirty-two cases (94.12%) were with auto antibody (IgG) in Group A, while only 2 (7.41%) with auto antibody (IgG) in Group B. There was significant difference in expression ratio of activated antigen of BM MPhi between Group A (49.19 +/- 16.63)% and Group B (29.11 +/- 14.30)% (P < 0.05), but no difference was found between Group B and the control group (P > 0.05). Total curative rates at 3 and 6 month (47.06% and 79.41%) of Group A were better than those of Group B (22.22% and 51.85%). Thirty-four IRP patients with autoantibody (IgG)(+) were divided into two subgroups according to the quantity of MPhi: high level group (>/= 0.75%, 9 cases) and low level group (< 0.75%, 25 cases), 24 cases (96%) in MPhi low level group were found auto-antibody (IgG) on one hemotopoietic cell lineage, 1 on two lineages, while 8 (88.89%) in MPhi high level group were detected auto-antibody (IgG) on two cell lineages, and 1 on three cell lineages. Expression ratio of activated antigen (56.12 +/- 15.11)% was much higher in MPhi high level group than that in MPhi low level group (44.58 +/- 18.16)% (P < 0.05). The count of red blood cell concentration of hemoglobin and platelet in peripheral blood in MPhi high level group were respectively lower than those in MPhi low level group, while the percentage of Ret, the level of total bilirubin and indirect bilirubin, the ratio of erythroid of sternal bone marrow in MPhi high level group were higher than those in MPhi low level group. CONCLUSION: The expression of activated antigen of BM MPhi was enhanced in IRP especially with auto-antibody (IgG), which might be involved in damage process of hemotopoietic cell.

[An analysis of efficacy and related factors of itraconazole in the treatment of invasive fungal infection in hematological diseases].

OBJECTIVE: To investigate the effects and related factors of itraconazole in the treatment of invasive fungal infection (IFI) in the patients with blood diseases (BD). METHODS: A total of 156 BD patients with IFI treated with itraconazole in General Hospital, Tianjin Medical University from 2005 to 2008, were retrospectively analyzed. RESULTS: Of these patients, 92 were with underlying malignant BD, and 64 with non-malignant BD; 77 possible IFI, and others proven IFI. A total of 94 (63.5%) patients were responded to itraconazole successfully, while 54 (36.5%) failed. The underlying malignant BD, post-chemotherapy, neutrophil count less than 0.5×10(9)/L, positive fungus culture, and bacteria infection were related with the response to itraconazole significantly, while patient's age, application of other antibiotics, positive G test, IFI localization, haemoglobin level and platelet counts were not. Five patients was changed other anti-IFI therapy because of side effects, including gastrointestinal ill (3 cases with nausea or vomiting) and tachycardia (2 cases). CONCLUSIONS: Itraconazole was effective and safe in the treatment of IFI in the patients with BD. Underlying malignant BD, agranulocytosis, bacteria infection, and delayed anti-IFI therapy might reduce itraconazole therapeutic effects.

[Expression of differentiation antigens on bone marrow myeloid cells of the patients with myelodysplastic syndromes and its clinical significances].

OBJECTIVE: To detect the abnormal differentiation of bone marrow myeloid cells in myelodysplastic syndromes (MDS) and its correlation with the prognosis of MDS patients. METHODS: Quantitative assessment of CD11b, CD13, CD16 and HLA-DR expression on the membrane of bone marrow granulocytes, and CD71 and glycophorin A on erythroblasts of 12 MDS patients in low-risk, 22 in high-risk and 31 normal controls was conducted with flow cytometry. The correlation between the abnormality and the prognosis of MDS cases were analyzed. RESULTS: The granulocytic differentiation was analyzed with the combinations of CD13/CD11b, CD13/CD16 and CD11b/CD16. The "right hook", "sickle" and "retroflex 7" shape expressions were found in normal controls while there were various changes in MDS groups. The ratios of CD11b-/CD11b+ (0.39 +/- 0.34) and CD16-/CD16+ (1.33 +/- 0.77) were significantly higher in high-risk MDS group than those of control group (0.07 +/- 0.05 and 0.39 +/- 0.31 respectively) (P < 0.05). The MFI (mean fluorescence index) of SSC (side scatter) in the granulocyte gate of MDS groups was lower while MFI of CD13 was higher. The mean percentages of CD11b-HLA-DR+ 3.88% +/- 3.07%, CD11b-HLA-DR- 16.23% +/- 15.59%, CD16-HLA-DR- 41.12% +/- 24.53%, CD11b+CD16- 33.53% +/- 17.26% and CD13+CD16- 44.51% +/- 21.99% granulocytes of high-risk MDS group were significantly higher than those of low-risk and control groups (P < 0.05). The erythroid cell lineage differentiation was analyzed with CD71/glycophorin A combination. Double positive expression was found in all controls, but asynchronous expression of CD71/glycophorin A was found in some MDS cases. The mean percentage of double positive cells in CD45- and glycophorin A+ cell population was significantly lower in low-risk and high-risk MDS groups. The abnormal numbers and patterns of the antigen expression of MDS cases per case correlated directly with their IPSS (international prognostic scoring system) (r = 0.690, P = 0.000) and WPSS (WHO adapted prognostic scoring system) (r = 0.651, P = 0.000) scores. CONCLUSION: There is an abnormal expression of differentiation antigens on bone marrow myeloid cells of MDS patients. And the severity is correlated with the prognosis. The abnormal differentiation of myeloid cells is probably involved in the pathogenesis of MDS. So the examination of these antigenic expressions with flow cytometry might be helpful for diagnosis and prognosis of MDS.

[Expression and Significance of IL-9 and IL-6 in Patients with BCR-ABL- MPN].

OBJECTIVE: To investigate the expression of IL-9 and IL-6 in patients with BCR-ABL- bone marrow proli- ferative tumor (MPN), and to explore its role in the occurrence and development of MPN. METHODS: A total of 71 newly diagnosis MPN patients treated in Tianjin Medical University General Hospital from 2018 to 2019 were selected, including 32 patients with polycythemia vera (PV) and 22 patients with primary thrombocytosis (ET), and 17 patients with primary myelofibrosis (PMF). Then 58 patients who retestine after treatment were selected as therapy group，and 20 healthy volunteers were recruited as control group. ELISA was used to detect the expression level of IL-6 and IL-9 in bone marrow supernatant, and the relative expression level of IL-6 and IL-9 mRNA in BMMNC was detected by real-time PCR. The proportion of Th9 cells in peripheral blood were detected by flow cytometry (FCM). The expression level of IL-6 mRNA and IL-9 mRNA of BMMNC and clinical indicators were analyzed, and the correlation between JAK2 gene mutation load and IL-9 level was further analyzed. RESULT: The level of IL-6 in bone marrow supernatant and the expression of IL-6 mRNA in BMMNC were higher in the newly diagnosed group as compared with those in the treated group and the control group (P<0.001). The expression level of IL-9 in bone marrow supernatant and the expression of IL-9 mRNA in BMMNC were lower in the newly diagnosed group as compared with those in the treated group and the control group (P<0.05). The proportion of Th9 cells in peripheral blood was lower in the newly diagnosed group as compared with that in the treated group and the control group (P<0.001). The level of IL-6 in bone marrow supernatant and the expression of IL-6 mRNA in BMMNC in JAK2+ group were higher than those in JAK2- group (P<0.05). The expression level of IL-9 in bone marrow supernatant and the expression of IL-9 mRNA in BMMNC were lower in JAK2+ group as compared with those in JAK2- group (P<0.05). The expression of IL-6 and IL-9 in the patient group showed correlation with the number of lymphocytes (IL-6: r=-0.49, P<0.01; IL-9: r=0.53, P<0.001), and also related with Hb in PV patients (IL-6: r= 0.87, P<0.001; IL-9: r=-0.54, P<0.01), and platelets in ET patients (IL-6: r=0.64, P<0.05; IL-9: r=-0.46, P<0.05). CONCLUSION: The increased expression of IL-6 in MPN and hyperfunction may promote the progression of BCR-ABL- MPN disease. The expression of IL-9 in MPN decreases, and it negatively correlates with the mutation load of JAK2 gene, which may be related with the decrease of tumor environmental antitumor immune effect.

[In vitro induction of allo-T lymphocytes proliferation by myeloid dendritic cells in patients with severe aplastic anemia.].

OBJECTIVE: To investigate the function of myeloid dendritic cells (mDCs) from severe aplastic anemia (SAA) patients in stimulating allogeneic T lymphocytes proliferation in vitro and then explore the immunopathogenesis of SAA. METHODS: Twenty-five SAA patients (15 untreated and 10 recovered after immunosuppressive therapy) and 12 normal controls were enrolled in this study. Their mature mDCs were induced from their bone marrow monocytes with recombined human interleukin-4 (rhIL-4), recombined human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombined human tumor necrosis factor (rhTNF) in vitro. Then mDCs were co-cultured with allogeneic lymphocytes (mixture lymphocyte reaction, MLR) at a ratio of 1:100 or 1:50. The growth rate of lymphocyte was measured with methyl thiazolyl tetrazolium (MTT) colorimetry. The concentrations of interleukin (IL)-12 and interferon gamma (IFNgamma) in MLR supernatant were measured with ELISA. The correlation between the growth rate and the concentration of IL-12 or IFNgamma was analyzed. RESULTS: When mDCs and lymphocytes were co-cultured at the ratio of 1:100, the growth rates of lymphocytes stimulated with mDCs from untreated, recovered SAA patients and controls were (219.8 +/- 94.0)%, (159.1 +/- 66.0)% and (160.1 +/- 91.9)% respectively. The concentrations of IL-12 in MLR supernatant were (8.2 +/- 3.6) ng/L, (6.5 +/- 2.8) ng/L and (6.1 +/- 2.6) ng/L and the concentrations of IFNgamma were (21.8 +/- 8.7) ng/L, (25.5 +/- 9.1) ng/L and (22.6 +/- 7.8) ng/L respectively. All of them had no statistical differences among the three groups (P > 0.05). When mDCs and lymphocytes were co-cultured at the ratio of 1:50, the growth rate of lymphocytes stimulated with mDCs from untreated patients was (322.1 +/- 171.1)%, which was higher than that of recovered patients [(180.9 +/- 79.1)% and controls (192.3 +/- 91.9)%]. The concentrations of IL-12 in MLR supernatant in the three groups were (12.6 +/- 4.4) ng/L, (9.4 +/- 3.3) ng/L and (8.5 +/- 3.7) ng/L, and the concentrations of IFNgamma were (32.3 +/- 9.2) ng/L, (27.4 +/- 6.5) ng/L and (24.4 +/- 7.4) ng/L. Both of the values in untreated cases were higher than those of the recovered cases or controls (P < 0.05), but there were no statistical difference between the recovered and control groups (P > 0.05). The concentration of IL-12 in MLR supernatant correlated positively with the growth rate of lymphocyte (r = 0.529, P < 0.01) and so did the concentration of IFNgamma (r = 0.381, P < 0.05). CONCLUSION: The function of mDCs to stimulate T lymphocytes proliferation in SAA was enhanced; it might play an important role in the immunopathogenesis of SAA.

[Relationship of Peripheral Blood IL-37 Expression with T Lymphocytes Subsets and NK Cells in Patients with Primary Immune Thrombocytopenia].

OBJECTIVE: To study the correlation of IL-37 with T lymphocytes subsets and NK cells in ITP patients, and to explore its possible mechanisms involved in the pathogenesis of ITP. METHODS: Forty-five patients with newly diagnosed ITP(newly diagnosed group), 32 patients of complete remission (remission group) and 22 healthy persons(control group) were selected. The serum level of IL-37 in 3 groups was determined by enzyme linked immunosorbent assay (ELISA). The mRNA expression of IL-37, IL-17 and IL-18 in peripheral blood mononuclear cells（PBMNC） in 3 groups was measured by real-time fluorescence quantitative polymerase chain reaction (PCR). The number of IL-18Rα+CD4+ T cells and Tim-3+NK cells in the peripheral blood in 3 groups was detected by flow cytometry (FCM). RESULTS: The serum level of IL-37 in the peripheral blood of ITP patients in the newly diagnosed group was significantly higher than that in the control group and the remission group(P＜0.01) . The expression level of IL-37 in PBMNC of the ITP patients in newly diagnosed group was higher than that in the control group and the remission group(P＜0. 05). The expression level of IL-17 and IL-18 in PBMNC of the ITP patients in newly diagnosed group was higher than that in the control group and the remission group(P＜0. 01); the expression of IL-18Rα in CD4+ T cells in newly diagnosed group was significantly higher than that in both the control and the remission group(P＜0.01).The expression of Tim-3 in NK cells in ITP patients was significantly lower than that in the control group (P＜0. 01). In ITP patients, the serum IL-37 level and IL-18Rα+CD4+T cells ratio both negatively correlated with Plt count (r=-0.58, r=-0.48) moreo-ver the serum IL-37 level also negatively correlated with amount of CD4+ T cells and NK cells (r=-0.29, r=-0.28), but positively correlated with amount of CD8+ T cells (r=0.329). CONCLUSION: The IL-37 and its receptors may play an immunoregulatory role in CD4+ T cells and NK cells, the IL-37 may be a therapeutic target for ITP patients.

[Number and Function of Myeloid-Derived Suppressor Cells in Patients with Adult Primary Immune Thrombocytopenia].

OBJECTIVE: To analyze the number of myeloid-derived suppressor cells(MDSC) and the level of prostaglandin E2(PGE2) in the bone marrow of adult ITP patients, and to explore their possible mechanisms involved in the pathogenesis of this disease. METHODS: Twenty-five patients of newly diagnosed ITP, 25 patients of complete remission group and 15 patients of control group were selected. The number of MDSC in the bone marrow between 3 groups was detect by flow cytometry (FCM). The serum level of prostaglandin E2 (PGE2) in 3 groups was determined by enzyme linked immunosorbent assay (ELISA). The relative expression of IFN-γ mRNA in bone marrow mononuclear cells was measured by real time fluorescence quantitative polymerase chain reaction (RT-qPCR) in each groups. RESULTS: The number of MDSC in the complete remission group was significantly higher than that in the control group (P<0.05); the number of MDSC in the newly diagnosed group was higher than that in the control group; the number of MDSC in the complete remission group was higher than that in the newly diagnosed group. The serum level of PGE2 in bone marrow of ITP patients in the newly diagnosed group was higher than that of the control group（P<0.05）. The serum level of PGE2 in the bone marrow of ITP patients of the complete remission group was higher than that of the control group (P<0.05）. The level of PGE2 in bone marrow serum of ITP patients of the newly diagnosed group was lower than that in the complete remission group(P<0.05）. The relative expression level of IFN-gamma in bone marrow mononuclear cells of the ITP patients in newly diagnosed group was higher than that in the control group and the complete remission group（P<0.001）. The relative quantification (RQ) of IFN-γ in bone marrow mononuclear cells was 2.60 between the newly diagnosed group and the complete remission group. CONCLUSION: When adult ITP disease is remitted, the number of MDSC rises and correlates with the therapeutic response and PGE2 level in the bone marrow.