The effects of the Smad3- knockout on the hematopoiesis of mouse were investigated in this work. Five pairs of wild type and Smad3- null mice were studied. White blood cell(WBC), red blood cell(RBC) and platelet (PLT) counting of peripheral blood cells were performed with blood obtained from tails. And white blood cells were classified by their morphology. Bone marrow nucleated cells (BMNCs) were counted and classified. The CFU-GM, BFU-E, CFU-GEMM yields were measured in each pair of mice. CFU-S yield of each mouse was measured by injecting bone marrow cells into lethally irradiated 8-10 weeks old wild type female mice. And the pathomorphism of their bone marrows, spleens and livers were observed. As a result, WBC and PLT of Smad3- null mice were significantly higher than those in wild type mice. Smad3- null mice had much more proportion of granulocytes in classification. There wasn't any difference in RBC counting and BFU-E measurement. The yield of CFU-GM increased, while the yields of CFU-GEMM and CFU-S markedly reduced. Bone marrows are actively proliferative, with granulocytosis. The granulocyte/erythrocyte ratio increased. There were no obviously alterative in spleen and liver. Thus Smad3- knockout results in a decreased number of stem and progenitor cells. Moreover hematopoietic differentiation is abnormal with a tendency to forming more granulocytes and platelets. The effect of Smad3 on hematopoiesis is correlative to that of TGF-beta.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Erythrocytes ; cytology ; metabolism ; Erythroid Precursor Cells ; cytology ; metabolism ; Female ; Granulocyte-Macrophage Progenitor Cells ; cytology ; metabolism ; Granulocytes ; cytology ; metabolism ; Hematopoiesis ; genetics ; Mice ; Mice, Knockout ; Myeloid Progenitor Cells ; cytology ; metabolism ; Smad3 Protein ; genetics

Flow cytometry was used to identify and characterize monoclonal antibodies (mAbs) that react with rabbit leukocyte differentiation molecules (LDM). Screening sets of mAbs, developed against LDM in other species, for reactivity with rabbit LDM yielded 11 mAbs that recognize conserved epitopes on rabbit LDM orthologues and multiple mAbs that recognize epitopes expressed on the major histocompatibility class I or class II molecules. Screening of mAbs submitted to the Animal Homologues Section of the Eighth Human Leukocyte Differentiation Workshop yielded 7 additional mAbs. Screening of mAbs generated from mice immunized with leukocytes from rabbit thymus or spleen or concanavalin A activated peripheral blood and/or spleen lymphocytes has yielded 42 mAbs that recognize species restricted epitopes expressed on one or more lineages of leukocytes. Screening of the anti-rabbit mAbs against leukocytes from other species yielded one additional mAb. The studies show that screening of existing sets of mAbs for reactivity with rabbit LDM will not be productive and that a direct approach will be needed to develop mAbs for research in rabbits. The flow cytometric approach we developed to screen for mAbs of interest offers a way for individual laboratories to identify and characterize mAbs to LDM in rabbits and other species. A web-based program we developed provides a source of information that will facilitate analysis. It contains a searchable data base on known CD molecules and a data base on mAbs, known to react with LDM in one or more species of artiodactyla, equidae, carnivora, and or lagomorpha.
Animals ; Antibodies, Monoclonal/*immunology ; Antigens, Differentiation/*metabolism ; B-Lymphocytes/cytology/metabolism ; Basophils/cytology/metabolism ; Epitopes/genetics/metabolism ; *Flow Cytometry ; Gene Expression Regulation ; Granulocytes/cytology/metabolism ; Leukocytes/immunology/*metabolism ; Mice ; Monocytes/cytology/metabolism ; Rabbits ; T-Lymphocytes/cytology/metabolism

OBJECTIVE: To investigate the diagnostic significance of basophil activation test (BAT) in anaphylaxis to non-ionic contrast media through testing the content of CD63, mast cell-carboxypeptidase A3 (MC-CPA3), and terminal complement complex SC5b-9 of the individuals by testing their levels in the normal immune group and the anaphylaxis groups to β-lactam drugs and non -ionic contrast media. METHODS: The CD63 expression of basophilic granulocyte in blood was detected by flow cytometry. The levels of MC-CPA3 in blood serum and SC5b-9 in blood plasma were detected by ELISA. RESULTS: The CD63 expression of basophilic granulocyte in blood, the levels of MC-CPA3 and SC5b-9 of anaphylaxis to non-ionic contrast media and β-lactam drugs were significantly higher than that in normal immune group (P < 0.05). CONCLUSION There is activation of basophilic granulocytes, mast cells and complement system in anaphylaxis to non-ionic contrast media. BAT can be used to diagnose the anaphylaxis to non-ionic contrast media.
Anaphylaxis/diagnosis* ; Basophils/cytology* ; Carboxypeptidases A/metabolism* ; Complement Membrane Attack Complex/metabolism* ; Contrast Media ; Flow Cytometry ; Granulocytes/cytology* ; Humans ; Mast Cells/cytology* ; Tetraspanin 30/metabolism*

OBJECTIVETo analyze the effect of dexamethason (Dex) on blast composition in patients with myelodysplastic syndrome (MDS) and investigate its significance in diagnosis of MDS.

METHODSThe flow cytometry (FCM) was used to detect the blast rate and the expression of its antigens in 30 cases of MDS (10 cases were treated with Dex as DX group and 20 cases were treated without Dex as control group).

RESULTSThe difference of the CD34(+) cell number detected by FCM was not statistically significant between DX group and control group (P > 0.05); The rate of BM B cell precursors (BCP CD34(+)/CD19(+)/CD10(+) cells) increased in DX group significantly, and BM CD117(+) cells in CD34(+) cells was decreased significantly as compared with control group (P < 0.001). The expression of antigens between granulocyte and monocyte was not significantly different (P > 0.05).

CONCLUSIONThe dexamethasone can increase the rate of BCP significantly and decreased the rate of BM CD117(+) cells in CD34(+) cells significantly. There is significant influence on the blast composition in MDS patients after dexamethasone treatment and without significant influence on the other phenotypcs.

Antigens, CD34 ; metabolism ; Dexamethasone ; therapeutic use ; Flow Cytometry ; Granulocytes ; cytology ; Humans ; Monocytes ; cytology ; Myelodysplastic Syndromes ; drug therapy ; Precursor Cells, B-Lymphoid ; cytology ; Proto-Oncogene Proteins c-kit ; metabolism

BACKGROUND: Final diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) may take years demanding a quick diagnosis measure. We used the facts that PNH cells are damaged in acid, and reagents for measuring reticulocytes in Coulter DxH800 (Beckman Coulter, USA) are weakly acidic and hypotonic, to create a new PNH screening marker. METHODS: We analyzed 979 complete blood counts (CBC) data from 963 patients including 57 data from 44 PNH patients. Standard criteria for PNH assay for population selection were followed: flow cytometry for CD55 and CD59 on red blood cells (RBCs) to a detection level of 1%; and fluorescent aerolysin, CD24 and CD15 in granulocytes to 0.1%. Twenty-four PNH minor clone-positive samples (minor-PNH+) were taken, in which the clone population was <5% of RBCs and/or granulocytes. Excluding PNH and minor-PNH+ patients, the population was divided into anemia, malignancy, infection, and normal groups. Parameters exhibiting a distinct demarcation between PNH and non-PNH groups were identified, and each parameter cutoff value was sought that includes the maximum [minimum] number of PNH [non-PNH] patients. RESULTS: Cutoff values for 5 selected CBC parameters (MRV, RDWR, MSCV, MN-AL2-NRET, and IRF) were determined. Positive rates were: PNH (86.0%), minor-PNH+ (33.3%), others (5.0%), anemia (13.4%), malignancy (5.3%), infection (3.7%), normal (0.0%); within anemia group, aplastic anemia (40.0%), immune hemolytic anemia (11.1%), iron deficiency anemia (1.6%). Sensitivity (86.0%), specificity (95.0%), PPV (52.1%), and NPV (99.1%) were achieved in PNH screening. CONCLUSION: A new PNH screening marker is proposed with 95% specificity and 86% sensitivity. The flag identifies PNH patients, reducing time to final diagnosis by flow cytometry.
Antigens, CD15/metabolism ; Antigens, CD24/metabolism ; Antigens, CD55/metabolism ; Antigens, CD59/metabolism ; Biomarkers/metabolism ; Blood Cell Count ; Erythrocytes/cytology/metabolism ; Flow Cytometry ; Granulocytes/cytology/metabolism ; Hemoglobinuria, Paroxysmal/*diagnosis/metabolism ; Humans ; Sensitivity and Specificity

Leukocyte differentiation antigens (LDAs) play important roles in the immune system, by serving as surface markers and participating in multiple biological activities, such as recognizing pathogens, mediating membrane signals, interacting with other cells or systems, and regulating cell differentiation and activation. Data mining is a powerful tool used to identify novel LDAs from whole genome. LRRC25 (leucine rich repeat-containing 25) was predicted to have a role in the function of myeloid cells by a large-scale "omics" data analysis. Further experimental validation showed that LRRC25 is highly expressed in primary myeloid cells, such as granulocytes and monocytes, and lowly/intermediately expressed in B cells, but not in T cells and almost all NK cells. It was down-regulated in multiple acute myeloid leukemia (AML) cell lines and bone marrow cells of AML patients and up-regulated after all-trans retinoic acid (ATRA)-mediated granulocytic differentiation in AML cell lines and acute promyelocytic leukemia (APL; AML-M3, FAB classification) cells. Localization analysis showed that LRRC25 is a type I transmembrane molecule. Although ectopic LRRC25 did not promote spontaneous differentiation of NB4 cells, knockdown of LRRC25 by siRNA or shRNA and knockout of LRRC25 by the CRISPR-Cas9 system attenuated ATRA-induced terminal granulocytic differentiation, and restoration of LRRC25 in knockout cells could rescue ATRA-induced granulocytic differentiation. Therefore, LRRC25, a potential leukocyte differentiation antigen, is a key regulator of ATRA-induced granulocytic differentiation.
Antigens, Differentiation ; immunology ; metabolism ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Granulocytes ; cytology ; drug effects ; immunology ; metabolism ; Humans ; Leukocytes ; cytology ; drug effects ; immunology ; metabolism ; Membrane Proteins ; antagonists & inhibitors ; immunology ; metabolism ; RNA, Small Interfering ; pharmacology ; Tretinoin ; pharmacology

5' nucleotides (5'NT), a purine degradative enzyme, is capable of hydrolyzing nucleotide and acting as a phosphotransferase simultaneously. It has critical role in maintaining nucleotide metabolism balance. The present study was aimed to investigate the expression of 5'NT in bone marrow granulocytes (BMGs) from patients with acute myeloid leukemia (AML) and healthy donors comparatively. The BMGs were isolated from bone marrow of 33 patients with AML and 6 healthy donors by using lymphocyte isolating solution. The reactivity of 5'NT was detected by electron microscope and cytochemistry of cytidine monophosphate (CMP). The positive BMG ratio and their index were calculated on the base of ultrastructural observation semiquantitatively. The results indicated that electron microscopy revealed plasma membrane reacting pattern of CMP. Most BMGs from normal donors were CMP negative or exhibited lower active degree. All cases of M(0), M(1), M(2) and t (8; 21) showed high positive percentages and high indexes of BMGs, but no statistic differences between them. APL of t (15; 17) shared lower percentages and indexes than other subtypes. There was no significant difference between APL and normal donors statistically. In conclusions, the results suggested the expression of 5'NT may be associated with BMG differentiation in AML, and APL of t (15; 17) may be a highly differentiated leukemia subtype.
5'-Nucleotidase ; metabolism ; ultrastructure ; Adolescent ; Adult ; Aged ; Bone Marrow Cells ; cytology ; enzymology ; Child ; Female ; Granulocytes ; enzymology ; Humans ; Leukemia, Myeloid, Acute ; classification ; enzymology ; Male ; Middle Aged ; Young Adult

Human 8-oxo-G-DNA glycosylase 1 (hOGG1) is a DNA glycosylase to cleave 8-oxo-7,8-dihydroguanine (8-oxo-G), a mutagenic DNA adduct formed by oxidant stresses. Here, we examined hOGG1 protein expression and repair activity to nick a DNA strand at the site of 8-oxo-G during differentiation of hematopoietic cells using HL-60 cells. Overall expression of hOGG1 protein was increased during granulocytic differentiation of HL-60 cells induced by DMSO and monocytic differentiation by vitamine D3. Greater level of hOGG1 protein was expressed in DMSO-treated cells. However, change in the DNA nicking activity was not in parallel with the change in hOGG1 protein expression, especially in PMA-treated cells. In PMA- treated cells, the level of hOGG1 protein was lowered, even though the DNA nicking activity was elevated, in a manner similar to the changes in serum- deprived HL-60 cells. These results indicate that hOGG1 expression change during differentiation of hematopoietic stem cells for adaptation to new environments. And the DNA cleaving activity may require additional factor(s) other than expressed hOGG1 protein, especially in apoptotic cell death.
Blotting, Western ; *Cell Differentiation ; Culture Media, Serum-Free/pharmacology ; DNA Glycosylases/*metabolism ; Enzyme Activation ; *Gene Expression Regulation, Enzymologic/drug effects ; Granulocytes/cytology/drug effects/metabolism ; HL-60 Cells ; Human ; Monocytes/cytology/drug effects/metabolism

OBJECTIVETo explore the high risk factors of thrombosis in paroxysmal nocturnal hemoglobinuria (PNH). It has been reported that in Chinese patients with venous thrombosis, the mutation frequency in PROC c.574_576 del (rs199469469), PROC c.565C>T (rs146922325) and THBD c.-151G>T (rs1698852) was higher than that of normal controls, indicating its importance in thrombophilia pathogenesis.

METHODS142 patients with PNH diagnosed between 2009 and 2015 were enrolled in the study. Clinical data were analyzed and thrombophilia risk factors, such as the level of protein C, protein S, antithrombin III, APC resistance, blood fat, phospholipid antibody, were evaluated. Samples from patients and 100 normal controls were detected for the mutations of PROC c.574_576 del (rs199469469), PROC c.565C>T (rs146922325) and THBD c.-151G>T (rs1698852) by Sanger sequence.

RESULTSOf the 142 PNH patients, 21 (14.8%) patients had at least 1 episode of thrombotic event. Only 2 patients had arterial thrombosis and 19 patients had venous thrombosis. The median age of patients with thrombosis was 35 years old, similar to those without episode (40 years old, P=0.687). The ratios of males and females were 1.33 in thrombosis group and 1.57 in non-thrombosis group (P=0.728) , respectively. Patients with thrombosis had the same disease pattern compared with those without episode. Although there was no difference in the level of hemoglobin, WBC and PLT count, and LDH level between patients with thrombosis and those without episode, patients with thrombosis showed higher RBC, higher percentage of CD59(-) granulocytes and RBC, and Flaer(-) granulocytes compared with those without episode. The routine thrombophilia screening tests did not show any difference either between PNH patients and normal controls, or between patients with or without thrombosis. There were two mutations in rs199469469 and rs16984852 sites in patients with PNH, but the mutated patients did not have any thrombosis. Mutation rs146922325 was found in PNH patients. The mutation rate was similar between PNH patients and normal controls, thrombotic PNH and non-thrombotic PNH (P>0.05).

CONCLUSIONSCompared with non-thrombotic patients, PNH thrombotic patients have bigger PNH clone and higher RBC count. There are no differences among the routine thrombophilia factors and the three known venous eligible genes either between PNH patients and normal controls or between thrombotic and non-thrombotic PNH patients.

Adult ; Antithrombin III ; metabolism ; Case-Control Studies ; Clone Cells ; cytology ; Female ; Granulocytes ; cytology ; Hemoglobinuria, Paroxysmal ; genetics ; physiopathology ; Humans ; Leukocyte Count ; Male ; Protein C ; metabolism ; Protein S ; metabolism ; Risk Factors ; Thrombosis ; genetics ; physiopathology

The study was purposed to investigate the expression and function of non-muscle myosin heavy chain-IIA (NMMHC-IIA) in Fechtner syndrome in order to explore the pathologic changes of kindy disease and the mechanism of granulocyte inclusion body formation. NMMHC-IIA levels in granulocytes were analyzed by Western-blot, the expressions of NMMHC-IIA, IIB in HEK-293 cells were detected by RT-PCR and were analyzed by co-immunoprecipitation. The results indicated that the IIA/beta-actin ratio for Fechtner syndrome granulocytes was (0.35 +/- 0.12), and obviously decreased as compared with that of normal control (0.87 +/- 0.18) (p < 0.01). The IIA and IIB expressed higher in HEK-293 cells. The interaction of IIA and IIB was confirmed by co-immunoprecipitation in HEK-293 cells. It is concluded that dominant-negative effect of NMMHC-IIA is involved in the formation of inclusion bodies. IIA and IIB show obvious interaction, IIB partly compensates the IIA defect derived from MYH9 mutations, and may delay or prevent the development of clinically relevant abnormalities.
Blood Platelet Disorders ; genetics ; metabolism ; pathology ; Cell Line ; Granulocytes ; pathology ; Humans ; Inclusion Bodies ; pathology ; Kidney ; cytology ; embryology ; metabolism ; Mutation ; Nonmuscle Myosin Type IIA ; genetics ; metabolism ; physiology ; Nonmuscle Myosin Type IIB ; genetics ; metabolism ; physiology ; Syndrome ; Thrombocytopenia ; genetics ; metabolism ; pathology