Biomimetics ; Blood Cells ; metabolism ; CD55 Antigens ; blood ; CD59 Antigens ; blood ; Cell Membrane ; Humans ; Male ; Submarine Medicine

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

OBJECTIVEDifferent from primary membranous nephropathy, hepatitis B virus associated membranous nephropathy (HBV-MN) shows lower deposits of membrane attack complex (C5b-9) in glomerular subepithelium. The causes of relatively low complement activation in this disease remain unclear. The aim of this study was to investigate the influence of hepatitis B x protein (HBx) on the expression of CD59 and Crry in mouse podocytes.

METHODCultured mouse podocytes were divided into adenovirus vector hepatitis B virus X gene (Ad-HBx) transfected group (Ad-X group), blank podocytes group (B group) and adenovirus vector transfected group (Ad group). CD59 and Crry mRNA expression were assayed by semiquantitative RT-PCR. CD59 and Crry expression were tested by flow cytometry. The effect of HBx on complement activation was evaluated with MTT method. And then, the effects of P38MAPK, PI-3K and ERK1/2 pathway inhibitors (SB203580, LY294002, U0126) and DMSO on CD59 and Crry expression were respectively detected by flow cytometry.

RESULTProteins CD59 and Crry expression rates (%) in group B, Ad group and Ad-X group were 17.71 ± 3.81, 18.29 ± 3.36 and 45.7 ± 9.01; 18 ± 2.31, 21.78 ± 2.01 and 47.45 ± 9.95, respectively. Compared with group B, CD59 and Crry expression in group Ad was not significantly different (P values for both > 0.05), but CD59 and Crry protein expression in Ad-X group was significantly higher than that in groups B and Ad (P values for both < 0.005); CD59 and Crry gene expression in group Ad was not significantly different from that in group B (P values for both > 0.05). However, CD59 and Crry gene expression of Ad-X group was significantly higher than that in groups B and Ad (P values for both < 0.05). Flow cytometry detected CD59 protein expression rates (%) were 17.35 ± 1.24, 46.19 ± 9.77, 43.03 ± 6.83 and 40.04 ± 6.39 and Crry protein expression rates (%) were 18.14 ± 3.56, 31.95 ± 1.68, 31.95 ± 1.69 and 37.14 ± 3.92 after SB203580, LY294002, U0126 and DMSO were added to Ad-X group respectively. P38 pathway inhibition resulted in significantly lower CD59 and Crry expression than Ad-X group (P values for all < 0.005), but PI-3K, ERK1/2 pathway inhibitors and DMSO had no significant effect on the expression of CD59 and Crry (P values for all > 0.05). The inhibition rates of cell lysis were significantly higher in Ad-X group than in groups B and Ad at each serum dilution point (P values for all < 0.05), while groups B and Ad had no significant difference in cell viability.

CONCLUSIONHBx can up-regulate CD59 and Crry expression in podocytes through activating P38 pathway, resulting in decreased complement activation, which may facilitate latent HBV infection in podocytes and play a role in development of hepatitis B virus associated glomerulonephritis (HBV-GN).

Animals ; CD59 Antigens ; metabolism ; Cells, Cultured ; Mice ; Podocytes ; immunology ; metabolism ; Receptors, Complement ; metabolism ; Trans-Activators ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

CD59 is a glycosyl-phosphatidyl inositol-anchored protein with the capacity to block the formation of membrane-attack complex, and protect the cells from complement-mediated cytolysis. The study was aimed to investigate whether CD59 is deficient in acute promyelocytic leukemia (APL) blast cells. Expression of CD59 on APL blast cells was analysed by flow cytometry. Expression of CD59 on NB4 cells was determined by flow cytometry before and after treating with all trans retinoic acid (ATRA). Pig-A gene coding region was sequenced. The results showed that the deficiency of CD59 expression in 12 out of 19 APL samples was found, its incidence was significantly higher than that in other acute myeloid leukemia (AML) samples (deficiency of CD59 expression in 14 of 40 non-APL AML samples, p=0.042). The expression of CD59 became normal after the patients achieved complete remission (CR), which indicated that the deficient of CD59 expression was only found in APL blast cells, but also found in APL cell line NB4 cells. The expression of CD59 was not changed after NB4 cells were induced to differentiate by ATRA. Sequencing pig-A gene coding region of NB4 cells and one APL patient with deficiency of CD59 displayed that the mutation of pig-A gene was not observed, therefore the deficiency of CD59 expression in APL cells did not result from mutation of pig-A gene. It is concluded that the deficiency of CD59 expression exists in APL blast cells more probably.
Adolescent ; Adult ; CD59 Antigens ; genetics ; metabolism ; Female ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; metabolism ; Male ; Membrane Proteins ; metabolism ; Middle Aged ; Tretinoin ; pharmacology ; Tumor Cells, Cultured ; Young Adult

OBJECTIVETo estimate whether the expression of glycosylphosphatidylinositol (GPI)-anchored protein in B lymphocytes could be an indicator for detecting paroxysmal nocturnal hemoglobinuria (PNH).

METHODSFlow cytometry and two-color McAbs were used to detect CD59 expression in B lymphocytes and granulocytes of 92 clinical samples. The feasibility of the indicators for diagnosis of PNH was assessed.

RESULTSB lymphocytes from healthy individuals mainly showed a single population of cells strongly positive for CD59, as the percentage of CD59 expression was (96.3 +/- 1.2)%. In PNH patients, B lymphocytes showed decreased expression of CD59, as well as negative cells and partly-positive cells were observed, as the percentage of CD59 expression was 10.5%-92.3%. In 26 of 28 non-PNH anemia patients, CD59 expression in B lymphocyte was normal, and in the other 2 patients, CD59 expression were decreased (< 95%), but no negative cell peak was observed. The precision CV of CD59 in B lymphocyte assay is less than 4.4%, stained samples could be kept stable for 24 h, the CD59 in B lymphocyte assay was 100% in accordance with routine assay (CD59 in granulocyte assay). The sensitivity of the indicators (CD59 in B lymphocytes and granulocytes) was 100%, the specificity was 97.4%, misdiagnosis rate was 2.6%, the rate of failing to diagnosis was 0%, the rate of positive forecast was 87.5%, the rate of negative forecast was 100%, the accuracy was 97.8%.

CONCLUSIONSThe test of CD59 expression in B lymphocytes by flow cytometry was simple, accurate and reproduceable. It could be a good marker for diagnosis of PNH because of high sensitivity and specificity.

B-Lymphocytes ; metabolism ; Biomarkers ; CD59 Antigens ; blood ; Female ; Flow Cytometry ; Glycosylphosphatidylinositols ; biosynthesis ; Granulocytes ; metabolism ; Hemoglobinuria, Paroxysmal ; diagnosis ; Humans ; Male ; Membrane Proteins ; metabolism ; Sensitivity and Specificity

The remarkable change of phenomenon induced by stress increase energy metabolism that can induce many reactive oxygen species (ROS) production. ROS can peroxidize cellular macromolecules including lipid and protein. The object of this study was to investigate whether stress may induce cellular damage by producing ROS and whether vitamin E, as a strong lipid-soluble antioxidant, can protect cells against reactive oxygen species produced by noise and immobilization stress in SD rats. The stress group increased 5-hydroxyindole aceti acid (5-HIAA) , one of the stress hormone, in brain tissue and free fatty acid in plasma. Vitamin I treatment had no effect on 5-HIAA but free fatty acid contents decreased with a fortified vitamin I diet. Furthermore, the body weight of vitamin I-treated rats increased more than that of the stress group. Lipid peroxidation and protein degradation as an index of oxidative damage in brain tissue decreased with the use of the fortified vitamin I diet supplement. The results suggest that vitamin I supplements have a protective effect against noise and immobilization stress-induced oxidative damage in brain tissue.
Animals ; Antigens, CD59* ; Body Weight ; Brain* ; Diet ; Energy Metabolism ; Hydroxyindoleacetic Acid ; Immobilization* ; Lipid Peroxidation ; Noise ; Plasma ; Proteolysis ; Rats* ; Reactive Oxygen Species ; Vitamin E* ; Vitamins*

OBJECTIVETo study the expressions of CD55 and CD59 in patients with hyperlipidemia and the effects of atorvastatin on it, and to identify the possible influential factors.

METHODSWe selected 67 patients with hyperlipidemia, and 24 healthy people matched in terms of age, sex and body weight as control. The expressions of CD55 and CD59 on white blood cells were detected by flow cytometry, and their relationships to blood lipids, complement activation indexes (C(5a), sC(5b-9)), inflammatory factors (high sensitivity C-reactive protein (hsCRP), TNF-alpha, IL-6 were analyzed. 24 patients with hyperlipidemia were treated with atorvastatin for 8-12 weeks and the expressions of CD55 and CD59 were measured before and after atorvastatin therapy.

RESULTSThe mean fluorescence intensity (MFI) of CD55 lymphocytes and monocytes were decreased in patients with hyperlipidemia compared with control (2.07 +/- 0.28 vs 2.29 +/- 0.44 and 3.45 +/- 1.02 vs 4.33 +/- 2.32, P < 0.01 and P < 0.05, respectively). CD55 positive lymphocyte MFI was negatively correlated with waist circumference, waist-hip ratio, hsCRP and C(5a). C(5a) was negatively correlated with the MFIs of CD55 positive lymphocytes, monocytes, granulocytes, and positively with TG and diastolic blood pressure. After atorvastatin therapy, the MFIs of CD59 positive lymphocytes, monocytes and granulocytes increased (4.34 +/- 1.16 vs 3.69 +/- 0.76, 4.52 +/- 1.36 vs 3.91 +/- 0.89, 5.67 +/- 1.72 vs 4.56 +/- 1.03, P < 0.05, < 0.05 and < 0.01 respectively), which were not correlated with changes of blood lipids.

CONCLUSIONSThe expression of CD55 is down-regulated in hyperlipidemia, which might be influenced by obesity, abdominal distribution of adipose tissue and inflammatory status of hyperlipidemia, but not by blood lipids. The expression of CD55 is related with complement activation; The expression of CD59 is up-regulated after atorvastatin treatment independently of blood lipids.

Aged ; Atorvastatin Calcium ; CD55 Antigens ; metabolism ; CD59 Antigens ; metabolism ; Case-Control Studies ; Complement Activation ; Gene Expression Regulation ; Heptanoic Acids ; therapeutic use ; Humans ; Hyperlipidemias ; drug therapy ; immunology ; metabolism ; Hypolipidemic Agents ; therapeutic use ; Male ; Middle Aged ; Pyrroles ; therapeutic use

OBJECTIVETo construct a recombinant human CD59 gene containing intercellular adhesion molecule-2 promoter for high level endothelial-specific expression in xenotransplantation.

METHODSICAM-2 promotor fragment and CD59-intron 1 fragment were produced by PCR from the human blood genome, and then clone these fragments into a pcDNA3-CD59 eukaryotic expression vector which was followed by digestion with the specific restricted endonuclease (for example: EcoRI, Hind III). The ICAM-2 promoter and CD59-intron 1 fragments were identified by PCR, and sequencing. The recombinant was then transfected into pig aorta endothelial cells with Lipofection, and the expression was measured by flow cytometer.

RESULTSProducts of the sequences measured were in accord with the frames of the gene bank. The expression of the protein of this recombinant was positive.

CONCLUSIONThe CD59 recombinant gene is constructed successfully, providing a basis for transgenic research.

Animals ; Antigens, CD ; genetics ; CD59 Antigens ; biosynthesis ; genetics ; Cell Adhesion Molecules ; genetics ; Cloning, Molecular ; Endothelium, Vascular ; cytology ; metabolism ; Eukaryotic Cells ; metabolism ; Gene Expression Regulation ; Humans ; Promoter Regions, Genetic ; Recombinant Proteins ; biosynthesis ; genetics ; Swine ; Transfection ; Transplantation, Heterologous

OBJECTIVETo 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).

METHODS(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.

RESULTS(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].

CONCLUSIONThe 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.

Adult ; Bone Marrow Cells ; metabolism ; CD59 Antigens ; metabolism ; Case-Control Studies ; Cells, Cultured ; Female ; Flow Cytometry ; Hemoglobinuria, Paroxysmal ; metabolism ; Humans ; Male ; Middle Aged ; Receptors, Erythropoietin ; metabolism ; Receptors, Thrombopoietin ; metabolism ; Young Adult

OBJECTIVETo assess complement-mediated myocardial injury on isolated guinea pig working hearts and cardioprotective effects of CD59.

METHODSUsing a modified Langendorff apparatus, isolated guinea-pig working hearts were perfused with a modified Krebs Henseleit buffer containing 3% heat-inactivated human plasma and zymosan (IPZ) (control) (n = 10), 3% normal human plasma and zymosan (NPZ) (n = 10), or 3% normal human plasma and zymosan and 1.5 microg/ml CD59 (NPZC) (n = 10), respectively. Epicardial electrocardiogram (ECG), cardiac output (CO), coronary arterial flow (CF), maximum left ventricular developed pressure (LVP(max)), maximum left ventricular developed pressure increase rate (+ dp/dt(max)), maximum left ventricular developed pressure decrease rate (- dp/dt(max)) and heart rate (HR) were recorded at 0, 15, 30, 45 and 60 min of treatment. After the experiment, immunohistochemical examination was performed to detect the presence of C3a or C5b-9 in the myocardium of the isolated hearts.

RESULTSCompared the IPZ group, hearts treated with NPZ showed a slight depression on ST segments of epicardial ECG at 15 min, a significant elevation between 30 min to 60 min, a decrease in CF, CO, LVP(max), + dp/dt(max) and - dp/dt(max), and an increase in HR at 15 min. The observed alterations in CF, CO, LVP(max), + dp/dt(max) and - dp/dt(max) remained decreased, while the HR remained increased until the end of the protocol. The all above parameters of hearts treated with NPZC were similar to the control group (IPZ) at any given time. Immunohistochemical examination showed positive signals of C3a and C5b-9 in the myocardium of hearts treated with NPZ. C3a was positive in NPZC, and C3a and C5b-9 were negative in IPZ.

CONCLUSIONSActivated human complements directly damage isolated guinea pig working hearts, and CD59 offers a significant protection against the injuries.

Animals ; CD59 Antigens ; pharmacology ; Complement C3a ; antagonists & inhibitors ; metabolism ; Complement Inactivator Proteins ; pharmacology ; Electrocardiography ; Guinea Pigs ; Heart ; drug effects ; physiology ; Immunohistochemistry ; In Vitro Techniques ; Male ; Myocardium ; metabolism ; pathology ; Time Factors