Objectives To detect the abnormalities of CD34+ cells differentiation and bone marrow cell cycle in myelodysplastic syndrome (MDS). Methods Fifty newly diagnosed MDS ( 17 in low risk and 33 in high risk), 8 acute myeloid leukemia preceded by MDS (MDS-AML) and 25 normal controls were enrolled into this study. Their CD34+ CD38+, CD34+CD38- bone marrow cells and bone marrow cell cycle were measured with flow cytometry. Results The mean percentages of CD34+ cells in bone marrow karyocyte of high risk [ (2.29 ±2.17)% ] and MDS-AML groups [ ( 18.69 ± 17.47)% ] were significantly higher than that of control group [ ( 0.36 ± 0.49 )%, P ＜ 0.05 ]. The mean percentages of CD34+CD38+ cells were significantly lower in low risk, high risk and MDS-AML groups [ ( 86.09 ± 7.79 )%, ( 81.68 ± 11.82)% and (82.88 ±2.60)%, respectively] than that in control group [ (92.21 ±3.85)%, P＜0.05], thus the percentages of CD34+CD38- cells were significantly higher in either MDS (low risk and high risk) or MDS-AML groups [ (13.91 ±7.79)%, (18.32 ±11.82)% or (17.13 ±2.60)%, respectively] than that in control group [ (7.79 ± 3.85 )%, P ＜ 0.05 ]. The percentages of CD+34 CD-38 cells of MDS cases correlated directly with their International Prognostic Scoring System (IPSS) (r =0.493, P =0.001 ) and WHO Adapted Prognostic Scoring System (WPSS) ( r = 0.586, P = 0.000 ) scores. The percentages of bone marrow mononuclea cells (BMMNCs) in G0/G1 phase of in low risk, high risk and MDS-AML groups [ (94.52 ±4.32)%, (96.07 ± 3.88 )% and (94.65 ± 4.55 )%, respectively ] were significantly higher than that in control group[ (88.94 ±7.30)%, P ＜0.01 ], thus the percentages of BMMNCs in S and G2/M phase were significantly lower in either MDS (low risk and high risk) or MDS-AML groups than that in control group (P＜0.05). MDS patients with low percentages of CD34+CD38- cells presented higher therapeutic efficacy than those with high percentages of CD34+CD38- cells, while without significant differences ( P ＞ 0.05 ) .Conclusions There are abnormalities of differentiation of CD34+ bone marrow cells and high proportion of G0/G1 cells which indicates a G1 phase arrest in MDS that might be involved in the pathogenesis of MDS. So the examination of CD34+ bone marrow cells and cell cycle might be helpful for MDS diagnosis and assessment of prognosis and therapeutic effects.

To investigate the role of CD4+, CD25+, and CD127low regulatory T cells (Tregs) in multiple myeloma (MM). Methods:Levels of CD4+T cells and Tregs, as well as expression of CTLA-4 and apoptosis-related proteins, such as CD95, bcl-2, and Caspase3 of Tregs in peripheral blood of 30 patients with newly diagnosed cases, 27 patients under of complete remission (CR) from multiple myeloma patients, and 25 healthy adults were analyzed by flow cytometry. Results:The percentage of CD4+T cells in the untreated group was significantly lower than that of the control group (P<0.05). The percentage of Tregs in CD4+T cells in the untreated group was significantly higher than that of the CR group and control group (P<0.05), which in ISSⅢpatients of the untreated group was significantly higher than that in I/II(P<0.05). No significant difference of CD95 expression in Tregs was observed among the three groups. The expression of CTLA-4 in Tregs from the untreated group was significantly higher than that of the CR group (P<0.05) and control group (P<0.01), and so was in CR group than this in controls (P<0.05). The expression of bcl-2 in Tregs in the untreated group was significantly higher than that of the CR group (P<0.05) and control group (P<0.01), and so was in CR group than this in controls(P<0.05). The expression of Caspase3 in Tregs from the untreated group and CR group were all significantly lower than that of the control group (P<0.05). The percentage of Tregs in CD4+T cells in the untreated group was positively correlated with the proportion of bone marrow plasma cells (P<0.05). The percentage of Tregs in CD4+T cells from 15 MM patients who received bortezamib and dexamethasone (VD) chemotherapy was negatively correlated to the ratio of plasma cell reduction after the first VD chemotherapy (r=0.735, P<0.01). Conclusion:The level of Tregs in the peripheral blood of MM patients was positively correlated with tumor burden and progression of disease, but was negatively correlated with curative effect. The increased level of Tregs was associated with their strengthened anti-apoptosis function.

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 x 109/L, positive fungus culture, and bacteria infection were related with the response to itraconazole significantly, while patient's age, application of other antibiotics,positive C 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.

Objective To investigate the expression of interleukin-3 receptor alpha(CD123)on bone marrow cells in acute myelocytic leukemia(AML)and its clinical significances.Methods By means of Fluorescence-activated cell sorer(FACS)and semi-quantity reverse transcripition polymerase chain reaction(RT-PCR),the expression of IL-3R?(CD123+)protein on CD34+CD38-cells and mRNA in BMMNCs of 62 AML patients of Tianjin Medical University General Hospital from March 2008 to January 2009 and 12 normal controls were detected respectively;Then the correlation between IL-3R? and the clinical stages of AML were analyzed.Results CD34+CD38-CD123+/CD34+CD38-and IL-3R? mRNA in BMMNC of 33 deno-vo or relapsed AML patients were higher than those of control group(P

Objective To investigate the expression of granulocyte colony-stimulating factor receptor(G-CSFR,CD114)in acute myelocytic leukemia(AML)bone marrow CD34+cells and evaluate G-CSF's secutiy of applying to the patients of AML after chemotherapy.Methods From March 2008 to January 2009,62 AML patients[33 deno-vo or relapsed AML patients,29 AML patients in complete remission(CR)]and 16 normal controls in the General Hospital,Tianjin Medical University were detected for the expression of G-CSFR in CD34+cells by Fluorescence-activated cell sorer(FCM)and Semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR).Results The ratio of CD114+CD34+/CD34+ in the deno-vo or relapsed group,CR group and the control group were(11.69?2.91)%,(31.84?8.62)%,(32.87?8.44)% respectively(P0.05),G-CSFR mRNA expression in BMNNCs of the deno-vo or relapsed group,CR group and the control group were(30.52?6.21)%,(85.13?21.25)%,(91.57?18.64)% respectively(P0.05).13 AML patients were followed up.The ratio of CD114+CD34+/CD34+ before treatment and in CR were(12.58?2.00)% and (30.13?7.09)% respectively.The ratio before treatment was lower than that in CR(P

Objective To study the effect of iron overload on the bone marrow hematopoietic function on immuo-related pancytopenia(IRP)patients by hematopoietic progenitor cell(HPC)culture of bone marrow(BM).Methods BM liquid 4～5 mL was taken from 46 IRP patients of General Hospital Tianjin Medical University from July 2009 to February 2010 to detect colony-forming-unit of erythrocyte,blast-forming-unit of erythrocyte and colony-forming-unit of granulocyte-monocyteby HPC culture of BM.And according to the serum ferritin(SF)level,these patients were classified into 2 groups to compare HPC proliferation,blood cell counts,BM proliferation,blood transfusion and treatment effects.Results The mean values of 3 different colonies of IRP patients with high SF level [(43.33?17.74),(1.50?2.2),(11.06?5.83)/105BMMNC] were significantly lower than those of the patients with normal SF level [(77.43?40.64),(9.57?7.99),(21.25?11.41)/105BMMNC](P

Objective: To investigate the change of autophagy level of bone marrow nucleated red blood cell (RBC) in patients with myelodysplastic syndromes (MDS) . Methods: Fifty-four MDS patients and thirty-three controls were enrolled in this study. The mitophagy were observed by transmission electron microscopy (TEM) . The level of autophagy-associated protein LC3B in GlycoA⁺ nucleated RBC was measured by flow cytometry. The expressions of ULK1 and mTOR mRNA in GlycoA⁺ nucleated RBC were measured by real-time PCR. The expression of the mitochondrial outer membrane protein TOM20 in GlycoA⁺ nucleated RBC was detected by Western blot. Results: Autophagosomes or autolysosomes were scarcely observed by TEM in MDS patients. The expression of LC3B in GlycoA⁺ nucleated RBC in high-risk MDS patients (0.22±0.12) was significantly lower than that in normal controls (0.43±0.22, P<0.001) , and lower than that in low-risk MDS patients (0.40±0.16, P=0.001) . The expression of AMPK [0.26 (0.60) ] in GlycoA⁺ nucleated RBC in high-risk MDS patients was significantly lower than that in controls [1.00 (2.07) , P<0.017) . The expression of ULK1 mRNA in GlycoA⁺ nucleated RBC in high-risk MDS patients [0.27 (3.31) ] was significantly lower than that in controls [1.07 (4.41) , P<0.017]. The level of mTOR mRNA in GlycoA⁺ nucleated RBC in high-risk MDS patients [1.82 (3.74) ] was significantly higher than that in controls [1.26 (1.38) , P<0.017]. The level of LC3B in GlycoA⁺ nucleated RBC was negatively correlated with the HGB (r=0.529, P=0.009) in high-risk MDS patients. The expression of mitochondrial outer membrane protein TOM20 in high-risk MDS patients was 9.42±4.42. Conclusion Autophagy is impaired in nucleated RBC of MDS patients.

Objective: To explore characteristic and function of peripheral blood mononuclear cells (PBMNC) -induced macrophages in patients with myelodysplastic syndrome (MDS) to couple with its progression. Methods: A total of 24 MDS patients (11 low-risk patients and 13 high-risk group patients) referred to Department of Hematology of Tianjin Medical University General Hospital and normal controls were enrolled from September 2014 to December 2015. PBMNC was stimulated with GM-CSF to transform to macrophages. The morphology of macrophages was observed by microscope. The quantity of macrophages, CD206 and SIRPα on surface of macrophages were detected by flow cytometry. The phagocytic function of macrophages was analyzed by fluorescence microscopy and flow cytometry. Results: The morphology of macrophages from MDS patients was abnormal. The percentage of transformed macrophages was (5.17±3.47) % in patients with MDS, which was lower than that in controls significantly[ (66.18±13.43) %, t=3.529, P=0.001]. The expression of CD206 on macrophages from MDS patients was significantly lower than that of controls[ (9.73±2.59) % vs (51.15±10.82) %, t=4.551, P<0.001]. The SIRPα level of macrophages from MDS patients was significantly lower than that of controls [ (0.51±0.09) % vs (0.77±0.06) %, t=2.102, P=0.043]. The phagocytic index and the percentage of phagocytic of macrophages from MDS patients were significantly lower than those of macrophages from normal controls[0.45±0.08 vs 0.92±0.07, t=-6.253, P=0.008; (23.69±3.22) % vs (42.75±2.13) %, t=-6.982, P=0.006 respectively]by flow cytometry. The phagocytic index of MDS patients was significantly lower than that of controls (0.24±0.04 vs 0.48±0.96, t=3.464, P=0.001) by fluorescence microscopy. Conclusion The quantity, recognization receptors and phagocytosis of PBMNC-induced macrophages decreased in MDS patients.

OBJECTIVETo explore the expression levels of terminal complement complex (C5b-9) and CD62p on platelets and the soluble C5b-9 (sC5b-9) level in serum in patients with PNH or PNH-aplastic anemia (AA).

METHODSSerum levels of sC5b-9, complement C3 and C4 were detected by using ELISA in 25 patients with PNH/PNH-AA. The quantities of C5b-9 and CD62p on the membrane of platelets were detected by flow cytometry.

RESULTS①In PNH/PNH-AA group, the serum sC5b-9 level [390.27(265.73-676.87) μg/L] was lower than that in control group [540.39(344.20-1 576.78) μg/L] (P<0.01). ②The platelet PNH clone (CD59⁻CD61⁺/CD61⁺) size [50.58(23.29-81.60)%] was bigger in the PNH/PNH-AA group than that [23.57(15.58-29.02)%] in control group (P<0.01). The percentages of C5b-9 deposition (C5b-9⁺CD61⁺/CD61⁺) were higher on the PNH clone platelets (CD59⁻CD61⁺) in the PNH/PNH-AA group [(17.53 ± 6.27)%] than those on the normal platelets (CD59⁺CD61⁺) in PNH patients 11.33±5.03）%］ and control [(10.88±3.58)%] group (P<0.01). ③ The expression of CD62p (CD62p⁺CD61⁺/CD61⁺) on PNH clone platelets in PNH patients [(61.98 ± 11.71)%] was higher than that on the normal platelets in PNH patients [(43.76±11.30)%] and control group [(38.23±18.07)%] (P<0.01). In addition, the expression of CD62p on normal platelets was higher in PNH patients than control (P<0.05). ④The deposition of C5b-9 positively correlated with the expression of CD62p on the platelets (r=0.559, P=0.002).

CONCLUSIONDeficiency of CD59 antigen on platelets in PNH patients may lead to the deposition of C5b-9 on its membrane and its dysfunction, which may contribute to thrombosis events in PNH.

Anemia, Aplastic ; Blood Platelets ; Clone Cells ; Complement Membrane Attack Complex ; Flow Cytometry ; Hemoglobinuria, Paroxysmal ; Humans ; P-Selectin ; Thrombosis

OBJECTIVETo investigate the change of autophagy level of bone marrow mononuclear cells（BMMNCs）in patients with myelodysplastic syndromes（MDS）.

METHODSThirty- eight patients with MDS and 26 megaloblastic anemia patients were enrolled in this study. The autophagic vacuoles were observed by transmission electron microscopy （TEM） and the quantity of autophagic vacuoles was detected by monodansylcadaverine （MDC） staining. The LC3 protein positive cells were counted by immunofluorescence assays. The expression of Beclin 1, LC3A, mTOR mRNA were measured by real time PCR. The expression of Beclin 1 proteins were detected by Western blotting.

RESULTSThe autophgic vacuoles of double membrane that surrounds lysosomes appeared in MDS patients. The percentage of MDC positive cells was significantly higher in MDS patients［（9.75±2.63）%］than that of controls［（2.90± 0.89）%, P＜0.05）. The percentage of LC3 protein cells was also increased in MDS patients（6.13±1.03）% vs（1.5±0.58）%, P＜0.05）. The expression of Beclin 1 and LC3A mRNA in low-risk and intermediate-1 MDS were higher compared with controls （3.61 ± 3.02 vs 1.55 ± 1.03 and 6.56 ± 3.97 vs 1.21 ± 0.95 respectively, both P＜0.05）. The expression of mTOR mRNA was down- regulated in low- risk and intermediate-1 MDS compared with controls（0.39±0.37 vs 1.50±1.03, P＜0.05）. There were no significant difference in expression of Beclin 1, LC3 and mTOR mRNA among intermediate-2 and high-risk MDS and controls. Beclin 1 protein expression was higher in low- risk and intermediate- 1 MDS patients（1.257 ± 0.197）than that of controls（0.528±0.086）and inermediate-2 and high-risk MDS patients（0.622±0.118）.

CONCLUSIONThe autophagy levels were increased in low- risk and intermediate- 1 MDS, while not enhanced in intermediate-2 MDS. Autophagy might be considered as a cell protective mechanism in MDS. The relatively defective autophagy in intermediate- 2 and high- risk MDS might contribute to disease's progression.

Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; Beclin-1 ; Bone Marrow Cells ; cytology ; Humans ; Membrane Proteins ; metabolism ; Microscopy, Electron, Transmission ; Microtubule-Associated Proteins ; metabolism ; Myelodysplastic Syndromes ; pathology ; TOR Serine-Threonine Kinases ; metabolism ; Vacuoles ; ultrastructure