Relationship of Peripheral Blood IL-37 Expression with T Lymphocytes Subsets and NK Cells in Patients with Primary Immune Thrombocytopenia / 中国实验血液学杂志

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-18RαCD4 T cells and Tim-3NK 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-18RαCD4T 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 / 中国实验血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>When adult ITP disease is remitted, the number of MDSC rises and correlates with the therapeutic response and PGE2 level in the bone marrow.</p>

Single Nucleotide Polymorphism of Mitochondrial DNA D-LOOP Region in Peripheral Blood Lymphocytes of Immuno-related Pancytopenia Patients / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the single nucleotide polymorphism(SNP) of mitochondrial DNA (mtDNA) D-LOOP region in peripheral blood lymphocytes of immuno-related pancytopenia (IRP) patients and its correlation with immune parameters.</p><p><b>METHODS</b>The D-LOOP region in mitochondrial DNA of lymphocytes in peripheral blood mononuclear cells from 43 patients with untreated IRP was detected by polymerase chain reaction(PCR). The PCR products were sequenced by the pros and cons direct sequencing methods. The sequencing results were compared with the revised Cambridge reference sequence (rCRS) and the Polymorphic Sites of Human Mitochondrial Genome Database.</p><p><b>RESULTS</b>Among total of 110 variant positions of D-LOOP region in 43 patients, 62 was SNP sites and 48 was mutation sites, of which 14 were the new mutation sites not yet registered in the database, 516 base variations were observed at 110 positions, the most common variations were base substitutions, among them T/C and A/G was 184/410 and 113/410 respectively. In the 110 variant positions, the high frequency variation sites were 73 and 263 for 43/43,311 for 32/43,310 and 16 224 for 27/43,16 519 for 25/43, 489 and 16 362 for 24/43. By the analysis of mitochondrial DNA D-LOOP polymorphism and related clinical immunology indicators of the patient's lymphocytes, it was found that D-loop in adult patients (age≥ 18 years old) significantly correlated with CD15 IgM, GLYCoACells IgM, CD34CellsIgG, CD34Cells IgM correlation.</p><p><b>CONCLUSION</b>The high frequency of polymorphism exists in mitochondrial DNA D-loop region of lymphocytes in IRPA patients, and was significantly correlates with the autoantibodies in bone marrow mononuclear cells in adult patients, which may be associated with the IRP occurrence.</p>

Quantity and function of T follicular helper cells in the bone marrow of patients with immune thrombocytopenia / 中国实验血液学杂志

This study was purposed to detect the quantity and function of bone marrow (BM) T follicular helper (Tfh) cells of patients with immune thrombocytopenia, and to explore the role of Tfh cells in the pathogenesis of ITP. Twenty-one newly diagnosed ITP patients, twenty ITP patients in recovery stage and eighteen normal controls were enrolled in this study. The percentages of Tfh cells, Tfh-related molecules ICOS, CD40L, IL-21 in BM were detected by flow cytometry (FCM), and the mRNA expression of BCL-6 in BMMNC was determined by semi-quantitive RT-PCR. Correlation of Tfh cell level with the disease severity of ITP patients was analysed. The results showed that the ratio of CD4(+)CXCR5(+)/CD4(+) cells in newly diagnosed ITP patients [(5.532 ± 2.599)%] was significantly higher than that in ITP patients with recovery stage [(4.064 ± 2.026)%] and controls [(4.048 ± 1.413)%] (P < 0.05). The ratio of CD4(+)CXCR5(+)ICOS(+)/CD4(+) CXCR5(+) cells in newly diagnosed ITP patients [(14.586 ± 8.561)%] was higher than that in recovery stage ITP patients [(12.884 ± 10.161)%] and controls [(7.487 ± 5.176)%]. The differences be-tween newly diagnosed ITP patients and controls were statistically significant (P < 0.05). The ratio of CD4(+)CXCR5(+) CD40L(+)/CD4(+) CXCR5(+) cells in newly diagnosed ITP patients [(15.309 ± 10.756)%] and in ITP patients with recovery stage [(18.242 ± 12.243)%] were significantly higher than that in controls [(8.618 ± 5.719) %] (P < 0.05). The ratio of intracytoplasm CD4(+) CXCR5(+) IL-21(+)/CD4(+)CXCR5(+) cells in newly diagnosed ITP patients [(58.560 ± 26.285)%] and in ITP patients with recovery stage [(57.035 ± 30.936)%] were significantly higher than that in controls [(36.289 ± 24.868)%] (P < 0.05). The relative expression levels of BCL-6 mRNA in BMMNC of three groups were (1.407 ± 0.264), (1.149 ± 0.217) and (0.846 ± 0.157), respectively. The differences between 3 groups were significant(P < 0.05). It is concluded that the quantity and function of Tfh cells in ITP patients increase, which may play an important role in the pathogenesis of ITP.

Expression and clinical significance of miR-21 in diffuse large B cell lymphoma / 中国实验血液学杂志

This study was aimed to investigate the expression of microRNA-21 and its correlation with PTEN in diffuse large B cell lymphoma (DLBCL) paraffin-embedded tissues, and evaluate its potential relevance with clinical characteristics. The expression levels of miR-21 in 26 primary DLBCL and 10 normal lymph node tissue specimens were examined by real-time polymerase chain reaction. The expression of PTEN was detected by immunohistochemical staining. The results indicated that the expression of miR-21 was significantly higher in tumor tissues [6.586(1.10,38.22)] than that in normal tissues [0.791 (0.35,2.87)] (P < 0.05). Among 26 patients with DLBCL the expression of PTEN protein was positive in 6 patients (23%), and was negative in 20 patients (77%). In patients with DLBCL, the expression level of miR-21 was negatively correlated with the level of PTEN protein. The high expression of miR-21 was positively correlated with the level of serum LDH. The expression level of miR-21 in patients with Ann Arbor III-IV stage was obviously higher than that of patients with Ann Arbor I-II stage, but did not correlate with the subtype of patients in clinic (P > 0.05). It is concluded that the expression of miR-21 is high in DLBCL and its overexpression may be related with poor prognosis of DLCBL. These findings suggest that PTEN is possibly one of the targets of miR-21 in DLBCL.

Preliminary study on the quantity and function of T follicular helper cells in the cytopenic patients with positive BMMNC-Coombs test / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the quantity and function of bone marrow (BM) T follicular helper (Tfh) cells of the cytopenia patients with positive bone marrow mononuclear cells (BMMNC)- Coombs test (also known as immuno-related pancytopenia, IRP), and explore the role of Tfh cells in the pathogenesis of IRP.</p><p><b>METHODS</b>Forty- three untreated IRP patients, 47 recovered IRP patients and 25 healthy donors were enrolled in this study. The percentages of Tfh cells, Tfh-related molecules ICOS, CD40L, IL-21 and Bcl-6 in BM were investigated by flow cytometry and semiquantitive RT-PCR.</p><p><b>RESULTS</b>The ratio of CD4⁺CXCR5⁺/CD4⁺ cells of untreated IRP patients [(28.79 ± 19.70)%] was significantly higher than that of recovered IRP patients [(21.15 ± 12.81)% ] and normal controls ([ 13.42 ± 6.72)% ](P<0.05). The ratio of CD4⁺CXCR5⁺ICOS⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.05 ± 4.71)% ] was significantly higher than that of recovered IRP patients [(2.96 ± 2.89)% ] and normal controls [(2.99 ± 2.23)% ] (P<0.05). The ratio of CD4⁺CXCR5⁺CD40L⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.87 ± 4.14)%] and recovered IRP patients [(6.52±5.47)%] were significantly higher than that of normal controls [(2.93 ± 2.92)%] (P<0.05). The ratio of intracytoplasmic CD4⁺CXCR5⁺IL-21⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(8.20 ± 7.41)% ] and recovered IRP patients [(6.30 ± 6.03)% ] were significantly higher than that of normal controls [(3.43 ± 3.40)%] (P<0.05). The relative expressions of Bcl-6 mRNA in BMMNC were 0.625 ± 0.248, 0.485 ± 0.253, 0.306 ± 0.210 in three groups, respectively. The differences between untreated IRP patients, recovered IRP patients and normal controls were significant (P<0.05).</p><p><b>CONCLUSION</b>There exists increased quantity and hyperfunction of Tfh cells in the IRP patients, they may play important role in the pathogenesis of IRP. Tfh cells and their related effector molecules could be a potential therapeutic target for the disease.</p>

The mechanisms underlying bone marrow damage by iron overload in pancytopenic patients with positive BMMNC-Coombs test / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the mechanisms underlying bone marrow damage by iron overload in pancytopenic patients with positive BMMNC-Coombs test (IRP).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Expression of microRNA-223 in lymphocytic leukemia cells and its action mechanism / 中国实验血液学杂志

This study was aimed to investigate the expression level and mechanism of microRNA-223 and LMO2 in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) cells and the mechanism. MicroRNA-223 mimics was transfected to increase the expression of MicroRNA-223 in the lymphocytes sorted by ficoll separation from the bone marrow mononuclear cells (BMMNC) of ALL and CLL patients. MicroRNA-223 inhibitor was transfected to decrease the expression of the MicroRNA-223 in the lymphocytes of normal controls. Then the expression of the MicroRNA-223 and LMO2 in transfected lymphocytes before and after cultivating for 72 hours were detected by RT-PCR, the apoptosis and cell cycle of these cells were measured by flow cytometery. The results indicated that before the transfection, the expression of MicroRNA-223 in ALL and CLL cells was (433.11 ± 144.88), which was significantly lower than that in the normal lymphocyte (949.59 ± 267.39); the expression of LMO2 was (807.10 ± 238.41), which was significantly higher than that in the normal lymphocytes (455.32 ± 176.83) (P < 0.05); after the transfection, the expression of MicroRNA-223 was (571.86 ± 142.00) in ALL and CLL cells, which was significantly higher than that before transfection (P < 0.05), but the expression of LMO2 was significantly lower than that before transfection (651.97 ± 230.12) (P < 0.05); in the normal control the expression of MicroRNA-223 obviously decreased (646.32 ± 172.93) (P < 0.05), the expression of LMO2 was significantly increased (541.27 ± 158.86.2) (P < 0.05). After transfection, the cell cycle G1/G2 phase and apoptosis changed in ALL and CLL cells. Before transfection the cell ratio in cell cycle G1/G2 phase was (94.75 ± 3.15)%, the cell ratio in S phase was (5.14 ± 3.12)%; after transfection the cell ratio in cell cycle G1/G2 phase was (97.03 ± 2.08)% and obviously increased (P < 0.05), the cell ratio in S phase was (2.97 ± 2.08)% and significantly decreased (P < 0.05). Before transfection the apoptosis rate was (54.47 ± 8.72)%, and obviously was higher than that after transfection (60.48 ± 8.81)%. And in the normal control, the cell ratio in G1/G2 phase was significantly higher than that after transfection [(96.73 ± 2.26)%, (94.55 ± 2.77)%, P < 0.05)], and the cell ratio in S phase was significantly increased [(3.25 ± 2.26)%, (5.45 ± 2.77)% (P < 0.05)]. The apoptotic rate in the ALL and CLL patients was significantly higher than that after the transfection [(54.47 ± 8.72)% vs (60.48 ± 8.81)%, respectively (P < 0.05)]. The apoptotic rate in the normal control was significantly lower than that after the transfection [(59.02 ± 10.20)%, (51.96 ± 10.20)%, respectively (P < 0.05)]. It is concluded that the expression of MicroRNA-223 decreases, and the expression of LMO2 increases in lymphocytic leukemia cells which leads to the lymphocytes over-proliferation and abnormal apoptosis, thus may be one of pathogenesis in lymphocytic leukemia.

Expression and clinical significance of Notch1 on the membrane of bone marrow CD38(+)CD138(+) plasma cells in the patients with multiple myeloma / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of Notch1 on the membrane of bone marrow CD38(+)CD138(+) plasma cells in the patients with multiple myeloma (MM), and explore the importance of Notch signaling pathway in the formation and progression of MM.</p><p><b>METHODS</b>Thirty three MM patients and 15 healthy controls were enrolled in this study. The expression of Notch1 on the membrane of bone marrow CD38(+)CD138(+) and CD38(+)CD138(-) plasma cells were analyzed by flow cytometry. The clinical data of MM patients were also analyzed.</p><p><b>RESULTS</b>The ratio of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients was (60.21 ± 25.06)% which was significantly higher than those of CD38(+)CD138(-) plasma cells of MM patients (39.84 ± 18.94)% (P = 0.000) and controls (38.34 ± 19.39)% (P = 0.004). There was no statistical difference between the two latter groups (P > 0.05). The expression of Notch1 on CD38(+)CD138(+)plasma cells from 24 newly diagnosed MM patients was correlated to the level of malignant plasma cells in there bone marrow (r = 0.914, P = 0.000), serum level of lactate dehydrogenase (LDH) (r = 0.754, P = 0.007), and β(2)-MG(r = 0.716, P = 0.013). The ratio of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients who had renal dysfunction was correlated to their abnormal serum creatinine levels. The expression of Notch1 on CD38(+)CD138(+) plasma cells from 17 MM patients who received VD (bortezamib and dexamethasone) chemotherapy was correlated to the ratio of plasma cell reduction after the first VD chemotherapy (r = 0.842, P = 0.000).</p><p><b>CONCLUSION</b>The expression of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients was significantly higher than those of CD38(+)CD138(-) plasma cells of MM patients and controls. Notch1 overexpressed plasma cells were sensitive to the early VD therapy, and correlated to the progression and long term outcome of MM.</p>

STAT5 phosphorylation in CD34(+)CD38(-)CD123(+) bone marrow cells of the patients with myelodysplastic syndrome / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expressions of STAT5 phosphorylation in CD34(+)CD38(-)CD123(+) bone marrow cells of the patients with myelodysplastic syndromes (MDS), and then evaluate the level of activation of STAT5 associated with cell proliferation in MDS clone cells.</p><p><b>METHODS</b>The bone marrow mononuclear cells (BMMNC) were extracted from 36 MDS patients and 14 normal controls. The mean fluorescence intensities (MFI) of phosphorylated STAT5(P-STAT5) in CD34(+)CD38(-)CD123(+) and CD34(+)CD38(-)CD123(-)cells, with or without the stimulation of 10 U/ml EPO, were examined by flow cytometry (FCM).</p><p><b>RESULTS</b>Without stimulation, the P-STAT5 MFI in CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients was 113.71 ± 67.22/173.05 ± 102.78, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (58.84 ± 27.51/68.99 ± 50.42, P < 0.01, P < 0.05) and the normal controls CD34(+)CD38(-)CD123(-) cells (63.06 ± 21.06, P < 0.05), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal control CD34(+)CD38(-)CD123(-) cells; With the EPO stimulation, the P-STAT5 MFI in CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients was 144.04 ± 58.11/239.45 ± 152.05, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (68.41 ± 25, 10/64.21 ± 23.43, P < 0.01) and the normal controls CD34(+)CD38(-)CD123(-) cells (75.21 ± 27.02, P < 0.01), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal control CD34(+)CD38(-)CD123(-) cells; The P-STAT5 MFI in the CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients with or without EPO stimulation were 21.80/28.86, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (7.42/5.50, P < 0.01, P < 0.05) and the normal controls CD34(+)CD38(-)CD123(-) cells (6.39, P < 0.05), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal controls CD34(+)CD38(-)CD123(-) cells; There was no significant difference of P-STAT5 MFI with or without EPO stimulation and the increased P-STAT5 MFI between the CD34(+)CD38(-)CD123(+) cells of low and high risk MDS.</p><p><b>CONCLUSION</b>STAT5 associated with cell proliferation was activated in CD34(+)CD38(-)CD123(+) bone marrow cells in MDS, which had more significant reactions to EPO than CD34(+)CD38(-)CD123(-) cells, indicating that CD34(+)CD38(-)CD123(+) bone marrow cells might be the real malignant MDS clone cells in MDS.</p>

Expressions of miR-21, miR-155 and miR-210 in plasma of patients with lymphoma and its clinical significance / 中国实验血液学杂志

This study was purposed to investigate the expressions of miR-21, miR-155 and miR-210 in plasma of patients with lymphoma, and explore their role played in diagnosis, evaluation of chemotherapy effect and prognosis of lymphoma. The expressions of miR-21, miR-155 and miR-210 were assayed by RT-PCR in plasma of 54 cases of lymphoma, 10 cases of lymphonode inflammation and 27 cases of normal controls. The results indicated that the expressions of miR-21, miR-155 and miR-210 in plasma of lymphoma patients were higher than those of control group and lymphonode inflammation group (P < 0.05). The expressions of miR-21 and miR-210 in plasma of control group and lymphonode inflammation group had no significant differences (P > 0.05). The expression of miR-21 in plasma of lymphoma patient group significantly correlated with their serum LDH level. The expressions of miR-21 and miR-210 in plasma of previously untreated lymphoma patient group were higher than those of the patients treated for 6 or more courses (P < 0.05). The diagnostic accuracy of miR-21, miR-155 and miR-210 used for lymphoma patients was 56, 65, 48 respectively, and reached to 83 when combined three of them. It is concluded that the expressions of miR-21, miR-155 and miR-210 in plasma of lymphoma patients were significantly higher. Detection of these 3 miRNA in plasma of patients can contribute to the clinical diagnosis, treatment and prognosis evaluation of lymphoma.

Expression of CD80 and CD86 on dendritic cells of patients with immune related pancytopenia and its clinical significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the function of dendritic cells (DC) of patients with immune related pancytopenia (IRP) and explore the role of DC in IRP.</p><p><b>METHODS</b>The expression of CD80 and CD86 on myeloid DC (mDC, Lin-HLA-DR(+) CD11c(+) cells) and plasmacytoid DC (pDC, Lin-HLA-DR(+) CD123(+) cells) of 65 IRP (37 untreated and 28 remitted) patients and 17 healthy controls were analyzed by flow cytometry.</p><p><b>RESULTS</b>The expression of CD86 on pDC was (82.47 ± 13.17)% in untreated group and (60.08 ± 14.29)% in remission group, which were significantly higher than that of controls (47.95 ± 18.59)% (P < 0.05), while the expression in untreated group was higher than that of remission group (P < 0.05). The expression of CD80 on pDC was (6.31 ± 4.49)% in untreated group, which was significantly higher than that of remitted patients (3.09 ± 2.93)% and controls (2.33 ± 2.25)% (P < 0.05). The expression of CD86 on mDC was (97.06 ± 4.82)% in untreated group and (91.35 ± 12.20)% in control group, while the expression in untreated group was higher than that of control group (P < 0.05). The expression of CD80 on mDC was (6.20 ± 5.44)% in untreated group and (3.97 ± 3.24)% in remission group, which were significantly higher than that of controls (1.86 ± 1.73)% (P < 0.05). The expression of CD86 on pDC was negatively correlated to Th1/Th2 (r = -0.733, P < 0.05), it was positively correlated to the antibody on membrane of BMMNC (r = 0.283, P < 0.05) and the quantity of CD5(+)B cells (r = 0.436, P < 0.05), while it was negatively correlated to the level of hemoglobin, platelets and white blood cells (r = -0.539, P < 0.05; r = -0.519, P < 0.05; r = -0.567, P < 0.05, respectively). The expression of CD80 on pDC was negatively correlated to the level of hemoglobin and platelets (r = -0.431, P < 0.05; r = -0.464, P < 0.05).</p><p><b>CONCLUSION</b>The function of pDC in PB of IRP were strengthened, which was relevant to the immunopathogenesis of IRP.</p>

Application of bone marrow indirect Coombs test and its clinical significance in diagnosis of immuno-related pancytopenia / 中国实验血液学杂志

This study was purposed to applicate the bone marrow indirect Coombs test and investigate its clinical significancies in diagnosis of immuno-related pancytopenia (IRP). 30 patients with pancytopenia including 22 cases of IRP and 8 cases of idiopathic cytopenia of undetermined significance (ICUS), and 15 patients with iron-deficiency anemia as controls were enrolled in this study. After incubation of the bone marrow supernatant of IRP patients and bone marrow nucleated cell (BMNC) of controls was used as experiment group, while the incubation of BMNC and bone marrow supernatant of controls was used as control group. After incubation for 45 min, the positive rate of membrane antibodies in bone marrow hematopoietic cells (CD15(+), GlyCoA(+) and CD34(+)cells) was detected by flow cytometry, and correlation analysis of positive rate with clinical data of patients were analyzed. The results showed that among 30 patients with pancytopenia (16 positive and 14 negative for bone marrow direct Coombs test) 16 cases showed positive for bone marrow indirect Coombs test, with positive rate 53.33. In the experiment group, the median positive rate of CD15(+)IgM was 0.34, which was significantly higher than that in control group (0.20, P < 0.05); the median positive rates of CD34(+) IgG and IgM were 0.64 and 0.21 respectively, which were significantly higher than those in control group (0.00, P < 0.05) and (0.00, P < 0.05); the positive rates of GlyCoA(+)IgG and IgM were (0.83 ± 0.75) and (2.12 ± 1.98) respectively, which were significantly higher than those in control group [(0.47 ± 0.43), P < 0.05, (0.68 ± 0.64), P < 0.01]; the positive rates of CD15(+) IgG and IgM were positively correlated with the ratio of CD5(+)B cells. The positive rates of GlyCoA(+) IgG and IgM negatively correlated with the Hb level, percentage of reticulocytes, the ratio of bone marrow erythroid lineage and DC1/DC2 positively correlated with the ratio of CD5(+)B cells and indirect bilirubin level. It is concluded that antibodies (IgG or IgM) aiming at the bone marrow hematopoietic cells exist in the supernatant of some IRP and ICUS patients, and may act on the membrane protein of the normal BMNC. These antibodies correlate with the prognosis of IRP.

Quantity and subtypes of dendritic cells in patients with immune related pancytopenia and their clinical significance / 中国实验血液学杂志

This study was aimed to investigate the quantity and subtypes of dendritic cells (DC) in patients with immune related pancytopenia (IRP) and to explore the role of DC in pathogenesis of IRP. The quantity of plasmacytoid dendritic cells (pDC, Lin(-)HLA-DR(+) CD123(+) cells) and myeloid dendritic cells (mDC, Lin(-)HLA-DR(+) CD11c(+)cells) in peripheral blood of 65 patients with IRP (37 new diagnosed and 28 remitted) and 17 healthy controls were analyzed by flow cytometry. The results indicated that the ratio of pDC in peripheral blood mononuclear cells (PBMNC) was (0.91 ± 064)% in new diagnosed group, which was significantly higher than that in remission group (0.39 ± 0.11)% and control group (0.29 ± 0.13)% (P < 0.01), while this ratio of pDC in remission group was higher than that in control group (P < 0.05). The ratio of mDC in PBMNC was (0.21 ± 0.20)% in new diagnosed group and (0.34 ± 0.21)% in remission group respectively, there was no statistical difference as compared with control group (0.29 ± 0.09)% (P > 0.05). The ratio of pDC to mDC in new diagnosed group was 6.75 ± 7.11, which was significantly higher than that in remission group (1.55 ± 0.93) and control group (1.07 ± 0.43, P < 0.01), there was no statistical difference between the ratio of remission group and control group (P > 0.05). The ratio of pDC in PBMNC of IRP group negatively correlated to ratio of Th1/Th2 (r = -0.347, P < 0.05), and positively correlated to the ratio of auto-antibody on membrane of BMMNC (r = 0.606, P < 0.05) and to the quantity of CD5(+)B cells (r = 0.709, P < 0.05), while it negatively correlated to the levels of hemoglobin (r = -0.381, P < 0.01) and platelets (r = -0.343, P < 0.01). The ratio of mDC in PBMNC positively correlated to the ratio of Th1/Th2 (r = 0.595, P < 0.05) and the level of hemoglobin (r = 0.292, P < 0.05). The ratio of pDC/mDC negatively correlated to ratio of Th1/Th2 (r = -0.395, P < 0.05), it positively correlated to the level of antibody on membrane of BMMNC (r = 0.421, P < 0.05) and the quantity of CD5(+)B cells (r = 0.423, P < 0.05), while it negatively correlated to the levels of hemoglobin (r = -0.304, P < 0.05) and platelets (r = -0.287, P < 0.05). It is concluded that the quantity of pDC in peripheral blood of IRP patients increases, which may be related to the immunopathogenesis of IRP.

Effect of CD8+ effector T cells on the hematopoiesis pathway damage in the patients with severe aplastic anemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the quantity and the pathway to damage hematopoietic cells of CD8+CD25+ and CD8+ HLA-DR+ effector T cells in peripheral blood (PB) of severe aplastic anemia(SAA) patients and explore the immunopathogenesis of SAA.</p><p><b>METHODS</b>The quantity of CD8+ CD25+ and CD8+ HLA-DR+ cells in PB and the expressions of perforin, granzyme B, tumor necrosis factor-beta (TNF-beta) and FasL in 29 SAA (14 untreated and 15 recovered) patients and 12 normal controls were analyzed by flow cytometry.</p><p><b>RESULTS</b>The fraction of CD8+ CD25+ T cells in CD8+ T cells was (3.67 +/- 2.58)% in untreated SAA patients, (5.19 +/- 4. 29)% in recovered patients and (4.84 +/- 2.31)% in normal controls, and that of CD8+ CD25+ T cells in CD3+ cells in the three groups was (2.25 +/- 1.35)%, (2.98 +/- 1.35)% and (2.11 +/- 1.88)%, respectively. They had no statistic difference among the 3 groups (P >0.05). The fraction of CD8+ HLA-DR+ T cells in CD8+ T cells was (39.30 +/- 8.13)% in untreated patients, which was significantly higher than that in recovered patients [(20.65 +/- 5.38)%] and controls [(18.34 +/- 6.68)%] (P<0.001), while there was no statistic difference between the latter two groups (P>0.05). CD8+ HLA-DR+ T cells in CD3+ cells was (27.81 +/- 7.10)% in untreated group, which was significantly higher than that of recovered group [(12.02 +/- 3.03)%] and controls [(8.50 +/-2.33)%] (P<0.01). And that in recovered group was higher than that in control group (P<0.05). The expressions of perforin, granzyme B, TNF-beta and FasL of CD8+ HLA-DR+ T cells in untreated group were 8.51%, 96.08%, 72.11% and 94.25% respectively, which were higher than those in recovered group (1.78%, 85.20%, 34.38% and 51.20%) and controls (1.86%, 82.09% ,17.92% and 32.91%). There was no statistic difference between recovered patients and controls (P>0.05).</p><p><b>CONCLUSION</b>There were elevated quantity of CD8+ HLA-DR+ T cells and high expressions of perforin, granzyme B, TNF-beta and FasL in SAA, which might contribute to the bone marrow failure.</p>

EPOR and TPOR expressions on CD34+ CD59- and CD34+ CD59+ bone marrow cells from patients with paroxysmal nocturnal hemoglobinuria / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expressions of erythropoietin receptor (EPOR) and thrombopoietin receptor (TPOR) on CD34+ CD59- and CD34+ CD59+ bone marrow (BM) cells from patients with paroxysmal nocturnal hemoglobinuria (PNH).</p><p><b>METHODS</b>(1) The expressions of EPOR and TPOR on CD34+ CD59- and CD34+ CD59- BM cells from 26 PNH patients and 16 normal controls were examined by flow cytometry (FCM). (2) The mRNA expression of the EPOR and the TPOR in BM mononuclear cells (BMMNC) from 25 PNH patients and 13 normal controls were examined by RT-PCR.</p><p><b>RESULTS</b>(1) The percentage of EPOR positive cells in PNH CD34+ CD59+ BMMNC [(30.67 +/- 18.30)%] was significantly higher than that in PNH CD34+ CD59- BMMNC [(8.05 +/- 3.51)%] (P < 0.01) and than that in control CD34+ CD59+ BMMNC [(8.24 +/- 6.51)%] (P < 0.01), but there was no obvious difference between the CD34+ CD59-BMMNC in PNH and CD34+ CD59+ BMMNC in control. (2) The percentage of TPOR positive cells in PNH CD34+ CD59+ BMMNC [(28.15 +/- 17.75)%] was significantly higher than that in PNH CD34+ CD59-BMMNC [(15.65 +/- 14.45)%] (P < 0.05) and than that in control CD34+ CD59+ BMMNC [(10.77 +/- .39)%] (P < 0.01), but there was no obvious difference between the CD34+ CD59- BMMNC in PNH and CD34+ CD59+ BMMNC in control. (3) There was no statistic difference in EPOR mRNA and TPOR mRNA expressions in BMMNCs between PNH patients group [(0.41 +/- 0.37) and (0.32 +/- 0.19), respectively] and control group [(0.47 +/- 0.33) and (0.40 +/- 0.29), respectively].</p><p><b>CONCLUSION</b>The expression of EPOR and TPOR of PNH patients on BM CD34+ CD59+ cells are significantly higher than those on BM CD34+ CD59- cells. The difference may be due to abnormal transcription of both receptor coding genes.</p>

BCL-2/IgH and IgH gene rearrangements in bone marrow mononuclear cells of patients with non-Hodgkin's lymphoma / 中国实验血液学杂志

This study was purposed to investigate the BCL-2/IgH gene rearrangement in major break point region (MBR) and IgH gene rearrangements of patients with non-Hodgkin's lymphoma (NHL), and explore their significance for improving early diagnosis and accurately evaluating chemotherapy effect. DNA for BCL-2/IgH and IgH gene assays was extracted from bone marrow mononuclear cells in 70 cases of lymphoma (60 cases of B-NHL and 10 cases of T-NHL), 7 cases of lymph node inflammatory and 20 healthy controls. The BCL-2/IgH, IgH gene rearrangements were assayed by polymerase chain reaction (PCR), the assayed results were compared with results of pathological biopsy; the factors related with occurrence of these 2 kinds of gene rearrangement were analyzed and the dynamic changes of BCL-2/IgH and IgH gene rearrangements after chemotherapy were compared, the chemotherapy effect was evaluated. The results indicated that (1) BCL-2/IgH gene rearrangement in bone marrow mononuclear cells was observed in 10 cases out of 30 DLBCL cases (33.3%), and was more frequent than that in 30 other B-NHL cases (6.7%), 10 T-NHL cases (0%), 7 lymph nodes inflammatory cases (0%) and 20 healthy controls (5%) (p < 0.05). (2) the quantity of rearranged BCL-2/IgH gene of 8 DLBCL cases reduced from 0.59 to 0.16 (p < 0.05) after 2 courses of R-CHOP chemotherapy and completely disappeared after 6 courses of R-CHOP chemotherapy. (3) 81.8% patients with BCL-2/IgH gene rearrangement showed high serum LDH level, while it was observed in 28.6% patients without this gene rearrangement (p < 0.05). Lymphoma staging, systemic symptoms, β(2)-MG level, bone marrow involvement, infiltration of liver and spleen were not significantly correlated with BCL-2/IgH gene rearrangement. (4) IgH gene rearrangement was found in 9 cases out of 20 DLBCL patients (all newly diagnosed patients) (45%), IgH rearrangement was observed in 14 cases out of 30 other B-NHL (all newly diagnosed or relapsed patients, except patients with DLBCL) (46.7%) and there was no statistical difference between these 2 groups, however IgH rearrangement all were not observed in 20 healthy persons, 10 T-NHL cases and 7 lymph nodes inflammatory cases. (5) the quantity of rearranged IgH gene in 7 DLBCL cases was reduced from 0.42 to 0.13 after one course of R-CHOP chemotherapy (p < 0.05) and completely disappeared after 2 courses of R-CHOP chemotherapy. (6) 90% patients with IgH gene rearrangement had high serum LDH level, while it was found in 30% patients without this gene rearrangement (p < 0.05). Lymphoma staging, systemic symptoms, β(2)-MG levels, bone marrow involvement, infiltrations liver and spleen all were not significantly correlated with IgH gene rearrangement. It is concluded that the BCL-2/IgH and IgH gene rearrangements may be used as specific indicators in early diagnosis and accurate evaluation of therapy efficacy in B-NHL, these 2 kind of rearrangement correlate with LDH level. The BCL-2/IgH gene rearrangement is more specific for in DLBCL.

Perforin gene mutations in patients with acquired severe aplastic anemia / 中国实验血液学杂志

This study was aimed to explore whether the perforin gene 1 (PRF1) mutation is the basis of genetic susceptibility to pathogenesis of acquired severe aplastic anemia (SAA). DNA exon2 and exon3 of PRF1 gene in peripheral blood mononuclear cells in 31 SAA patients and 15 normal controls were amplified by PCR; the sequencing was performed by using ABI pRISM 373OXL sequencer; the mutation loci were sought through checking sequences with GenBank-reported sequences; after the mutation sequences were found, those were cloned into M13 phage vector, then the corresponding sequences of gained 2 chromosomes were sequenced respectively to determine the distribution of different mutations on chromosomes. The results showed that (1) one homozygous mutation (822 C > T, synonymous mutation) and one heterozygous mutation (907 G > A, methionine 303 valine) were found in PRF1 coding region of 2 SAA patients. These mutations were not detected in normal controls. (2) 1 SNP (rs885822) in the coding region was detected in SAA patients and controls, and the heterozygosity rate between the 2 groups was different (p < 0.05). It is concluded that perforin gene mutation may be one risk factor in the aberrant proliferation and activation of cytotoxic T cells in pathogenesis of a part of patients with aplastic anemia.

Clinical analysis of 102 blood disease patients with gram positive cocci infection treated with Linezolid / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the efficacy and safety of linezolid for the treatment of gram positive coccus infections in hematological disease patients.</p><p><b>METHODS</b>One hundred and two hematological disease patients with suspected or proven gram positive coccus bacteria infection were enrolled in this study. Linezolid was given at a dosage of 600 mg, iv, q12h. The mean treatment period was (10.82 ± 5.12) days (1 to 51 days) with 74.5% over 7 d and 51.0% over 10 d.</p><p><b>RESULTS</b>Among 102 patients, 57 were male, 45 female aged 11 to 81 years, with a mean of (45.26 ± 19.15) years. Ninety four cases were nosocomial infection (92.2%) and 8 community infection (7.8%); There were pneumonia in 80 (78.4%), septicemia in 11 (10.8%), and infection of other organsin 11 (10.8%); Forty five cases were proven gram positive coccus bacteria infection, and 57 were suspected infection; Fifty one bacteria strains were isolated from cultivated samples of proven patients, in which 22 were staphylococcus aureus with 19 methicillin resistant 13 hemolytic streptococcus, 9 staphylococcus epidermidis with 7 methicillin resistant 6 enterococcus faecom, and 1 enterococcus hirae. Seven cases were mixed with one kind gram negative bacillus infection, 4 mixed with two kinds of gram negative bacillus infection, and 12 mixed with fungal infection; Total clinical response rates by ITT (intention to treatment) analysis was 69.6%, in which 40 (39.2%) were curative and 31 (30.4%) obviously effective; PP (per-protocol) analysis was 70.9%, in which 39 (41.9%) were curative and 27 (29.0%) obviously effective. Bacteria clearance rate was 70.6%, and in this group the clinical effective rate was 88.9%; Adverse effect rate was 2.9%, being transient thrombocytopenia and increased transaminase.</p><p><b>CONCLUSION</b>Linezolid is a safe and effective antibiotic used in hematological disease patients complicated with infections of gram positive coccus.</p>

Study on quantity and function of bone marrow macrophages in patients with BMMNC-Coombs Test(+) pancytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the quantity and function of bone marrow (BM) macrophages in patients with BMMNC-Coombs Test(+) pancytopenia (BCTP).</p><p><b>METHODS</b>Sixty-one patients with BCTP, 10 with severe aplastic anemia (SAA) and 13 normal controls were enrolled in this study. The quantity of BM macrophages was measured by FACS and their function was evaluated by phagocytosis test.</p><p><b>RESULTS</b>The number of macrophages, phagocytosis ratio and index of cock's red blood cells (CRBC) in BCTP patients were (0.57 +/- 0.30)%, (37.56 +/- 15.20)%, and 0.75 +/- 0.34, respectively, being significantly higher than those in SAA group \[0.46 +/- 0.08)%, (28.26 +/- 10.46)%, and in 0.59 +/- 0.39\] and in normal control \[0.44 +/- 0.69)% (25.63 +/- 14.75)%, and 0.55 +/- 0.16\] (P < 0.05). The BCTP patients were classified into two subgroups according to the quantity of macrophages: Group A (M(Phi) > or = 0.5%, 34 cases) and Group B (M(Phi) < 0.5%, 27 cases). There were 32 cases (94.12%) with BMMNC-IgG(+) in Group A and only 2 cases (7.41%) in Group B. There were significant differences in phagocytosis ratio and index of macrophages between Group A \[46.62 +/- 13.38)% and 0.91 +/- 0.36\] and Group B \[(28.67 +/- 12.59)% and 0.61 +/- 0.30\] (P < 0.05), while no statistical differences between group B and other two control groups (P > 0.05). Thirty-four BMMNC-IgG(+) patients were further divided into two subgroups: High level (HL) group \[> or = 0.75%, 9 cases (26.47%)\] and Low level (LL) group \[< 0.75%, 25 cases (73.53%)\]. Only one lineage of BMMNC-IgG could be detected in LL group. Among 9 patients in HL group, 8(23.53%) had two lineages of BMMNC-IgG and 1(2.94%) had three lineages. Phagocytosis ratio and index of macrophages were significantly higher in HL group \[(60.22 +/- 12.51)% and 1.23 +/- 0.23\] than in LL group \[(43.32 +/- 9.24)% and 0.84 +/- 0.24\] (P < 0.05). The level of peripheral blood(PB) RBC, HGB and PLT in HL group were significantly lower than in LL group (P < 0.05), while the percentage of Ret, the level of TBIL and the ratio of erythroid of sternal BM in HL group were significantly higher than in LL group (P < 0.05).</p><p><b>CONCLUSION</b>More quantity and stronger function of macrophages are observed in BCTP patients with BMMNC-IgG(+). One of the mechanism of hematocytopenia might be that macrophages activated by IgG autoantibodies phagocytose hematopoietic cells in BM. Macrophages do not involve in damage process of BM in BCTP with IgM or cold autoantibodies.</p>