OBJECTIVE: It has been proposed that the contact between blood and dialysis membrane during hemodialysis therapy induces harmful reactions to patients. Membrane biocompatibility is the characteristic that makes less adverse reaction. We tried to compare the biocompatibility between Cuprophane and Polysulfone membranes. MRTHODS: Nine chronic renal failure patients who have undergone maintenance hemrodialysis therapy with Hemophan membrane three times per week were included in this study. Cuprophane membranes were used in the first week: Hemophan membranes in the second week; Polysulfone membranes in the third week. Each membrane was used three times a week. On the day of third dialysis with the Cuprophane membrane(first week) and Polysulfone membrane(third week), serial blood sampling was obtained from the afferent line at hemodialysis initiation, 15 minutes, 30 minutes, 60 minutes, 120 minutes and 30 minutes to measure serum complement activity(C3a, C5a), blood polymorphonuclear leukocyte and platelet count, and arterial oxygen pressure which have been regarded as biocompatibility parameters. The parameters measured during the first week(Cuprophane) and the third week(Polysulfone) were compared to evaluate the difference in biocompatibility of both membranes. RESULTS: 1) C3a desArg In both groups, the level of C3a desArg increased significantly from basal level and the Cuprophane group showed significantly higher level of C3a desArg than that of Polysulfone group. 2) C5a desArg : In both groups, the level of C5a desArg did not increased sigpificantly from basal level. Only at 30 minute after hemodialysis, Cuprophane group showed significantly higher level of C5a desArg than that of Polysulfone group(p=0.037). 3) Polymorphonuclear leukocyte : In both groups, the polymorphonuclear leukocyte counts decreased significantly at 15 minutes and 30 minutes from basal level. The polymorphonuclear leukocyte count was lower in Cuprophane group than that of Polysulfone group at 15 minute after hemodialysis(p=0.001). 4) Platelet: In Cuprophane group, the platelet count was decreased significantly at 15 minute(p=0.004) but there were no difference in platelet counts between the two groups. 5) Arterial oxygen pressure Both group showed no consistent pattern of variation of oxygen pressure and there was no significant difference between the two groups. CONCLUSION: The biocompatibility of Polysulfone membrane was superior to the Cuprophane membrane with respect to the increased activation of complement C3a. decrease of polymorphonuclear leukocyte and decrease of platelet count.
Blood Platelets ; Complement C3a ; Complement System Proteins ; Dialysis ; Humans ; Kidney Failure, Chronic ; Membranes* ; Neutrophils ; Oxygen ; Platelet Count ; Renal Dialysis

OBJECTIVETo investigate the clinical significance of anaphylatoxin C3a in induced sputum in patients with asthma.

METHODSThe patients with acute exacerbation of asthma treated at our department between September, 2006 and February, 2007 were included in the study. The demographic data, medical history, levels of lung function and C3a levels in induced sputum were assessed.

RESULTSA total of 33 patients were included in the study. The level of C3a in induced sputum was significantly higher in patients with acute exacerbation of asthma (2.24 ng/ml, range 1.68-5.58 ng/ml) than that in patients with asthma remission (0.7 ng/ml, range 0.24-2.31 ng/ml, P<0.05). Sputum C3a levels in the remission patients were significantly higher than those in the healthy controls (0.12 ng/ml, range 0.07-0.39 ng/ml, P<0.05). The levels of C3a in patients with severe exacerbation (4.69 ng/ml, range 2.69-6.59 ng/ml) were significantly higher than those in patients with mild exacerbation (0.25 ng/ml, range 0.09-0.40 ng/ml) and moderate exacerbation (2.21 ng/ml, range 1.16-3.41 ng/ml) (P<0.01), and were significantly higher in patients with moderate exacerbation than in those in mild exacerbation (P<0.01). The level of C3a in induced sputum was positively correlated with the number of total cell count (r=0.718, P<0.05), eosinophils (r=0.495, P<0.05) and macrophages (r=0.600, P<0.05) in patients with acute exacerbation of asthma.

CONCLUSIONInduced sputum C3a level can serve as an important clinical biomarker for clinical asthma management.

Asthma ; physiopathology ; Biomarkers ; chemistry ; Case-Control Studies ; Complement C3a ; chemistry ; Eosinophils ; Humans ; Leukocyte Count ; Macrophages ; Sputum ; chemistry

BACKGROUND: Complementary receptors have been suggested to play causative roles in the neuroinflammatory process of Alzheimer's disease (AD). The genetic expressions of the C3a receptor (C3aR), C5a receptor (C5aR) and the protein expressions of the C3aR and C5aR were examined in the human neuroblastoma cell line, SK-N-SH, after the administration of amyloid peptide (A1-42). METHODS: SK-N-SH cells were incubated overnight with a single dose of 20 M of aggregated A (A1-42). An inhibition study was done with actinomycin D (ActD, 2.5 M) or with the administration of cycloheximide (CHX, 2.5 M) to the cell suspension. Messenger RNA expressions of C3aR and C5aR were detected by RT-PCR. The intensity of bands from 6% polyacrylamide electrophoretic gel was analyzed by a bioimage analyzer. The protein production of C3aR and C5aR in the A-treated cells was also measured by flow cytometry. NFB activation after treatment of A in the cells was detected by an electrophoretic mobility-shift assay. RESULTS: A1-42 increased the expression of C3aR and C5aR. ActD inhibited the expression of both anaphylatoxin receptors but CHX only suppressed C5aR mRNA expression. Activated NFB was demonstrated in the A-stimulated cells. CONCLUSIONS: C3aR and C5aR were constitutively expressed in the human neuroblastoma SK-N-SH cell. Expression of these anaphylatoxin receptors was upregulated after A1-42 stimulation, which as a result, may contribute to the complement-mediated neuroinflammation of AD.
Alzheimer Disease ; Amyloid ; Amyloid beta-Peptides ; Anaphylatoxins ; Cell Line* ; Complement C3a* ; Complement System Proteins* ; Cycloheximide ; Dactinomycin ; Flow Cytometry ; Humans* ; Neuroblastoma* ; RNA, Messenger

OBJECTIVETo determine the levels of complement components C3a and C5a in the kidneys of trichloroethylene (TCE)-sensitized BALB/c mice, and to investigate the role of complement components in TCE-induced renal injury among BALB/c mice.

METHODSSixty-two female BALB/c mice were randomly divided into blank control group, vehicle control group, and TCE sensitization group. The mice in TCE sensitization group were sensitized by one intracutaneous injection and one abdominal smear of TCE. At 24 h, 48 h, 72 h, and 7 d after the second sensitization, mice were sacrificed, and the blood and kidneys were collected. An automatic biochemical analyzer was used in the determination of serum blood urea nitrogen (BUN) and creatinine (Cr). The levels of C3a and C5a in the kidneys were determined by immunohistochemistry.

RESULTSThe sensitization rate of TCE sensitization group was 42.0%. Kidney coefficient and serum levels of BUN and Cr were significantly increased in the TCE sensitization group as compared with the vehicle control group at 48 h and 72 h after sensitization (P < 0.05). The kidney coefficients of the TCE sensitization group at 48 h and 72 h were significantly higher than those of the control groups (P < 0.05). In comparison with the vehicle control group, however, no significant change was found in kidney coefficient, serum BUN, or serum Cr at 7 d after TCE sensitization (P > 0.05). Levels of C3a and C5a at 48 h (3.80±0.84 and 4.00±1.00, respectively) and 72 h (4.40 ± 1.14 and 4.40 ± 1.14, respectively) after sensitization were all significantly higher than those of the vehicle control group (P < 0.05), but no significant difference was found in level of C3a (1.80±0.45) or C5a (2.00 ± 0.71) at 7 d (P > 0.05).

CONCLUSIONTCE sensitization can induce renal injury in mice. Levels of complement components C3a and C5a are elevated in the kidneys of sensitized mice, indicating that C3a and C5a may be involved in the renal injury induced by TCE sensitization.

Animals ; Blood Urea Nitrogen ; Complement C3a ; metabolism ; Complement C5a ; metabolism ; Creatinine ; blood ; Female ; Kidney ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Trichloroethylene ; toxicity

BACKGROUND: Cardiopulmonary bypass induces an acute systemic inflammatory response mediated by complement activation and cytokine release. This response is likely to cause capillary leak syndrome and organ dysfunction in infants. Removing harmful cytokines and complement anaphylatoxins after cardiopulmonary bypass may attenuate this response. This study was conducted to see if the modified ultrafiltration and postoperative peritoneal dialysis can reduce plasma inflammatory mediators in pediatric cardiac surgery. MATERIAL AND METHOD: 30 infants (age 1.1 to 12.6 months) who underwent closures of ventricular septal defect using cardiopulmonary bypass (CPB) were enrolled in this study. These patients were divided into three groups; 10 patients selected randomly underwent modified ultrafiltration (Group U), 10 with small body weights (C3a and interleukin-6 (IL-6) were measured by radioimmunoassay and enzyme- linked immunosorbent assay respectively. RESULT: There was no differences in CPB time, aortic cross-clamping time, and lowest temperature during CPB. The effluents of peritoneal dialysis contained significant amount of C3a and IL-6, but there was no definitive decrease of serum concentration of C3a and IL-6. The effluents of modified ultrafiltration had some amount of C3a and negligible IL-6, and there was no decrease of serum concentration of these factors. CONCLUSION: The effluents of peritoneal dialysis contained significant amount of proinflammatory cytokine, IL-6 and complement, C3a. However this study failed to elucidate the decrease in serum levels of these factors. The modified ultrafiltration also was not able to reduce the serum levels of C3a or IL-6 in our study as well.
Anaphylatoxins ; Body Weight ; Capillary Leak Syndrome ; Cardiopulmonary Bypass ; Complement Activation ; Complement C3a ; Complement System Proteins ; Cytokines ; Heart Septal Defects, Ventricular ; Humans ; Infant ; Interleukin-6 ; Peritoneal Dialysis* ; Plasma ; Radioimmunoassay ; Thoracic Surgery* ; Ultrafiltration*

OBJECTIVETo investigate the role of sulfated tyrosine in regulating the activity of tyrosylprotein sulfotransferases (TPST) 1 and TPST2.

METHODSConstructs of TPST 1 and TPST2 were amplified by polymerase chain reaction (PCR), then fused into immunoglobulin G1 Fc region. All the variants in which sulfated tyrosines were mutated to phenylalanine were made by the PCR-based Quick Change method and confirmed by sequencing the entire reading frame. Small hairpin RNA (shRNA) constructs-targeting nucleotides 259-275 of TPST1 and nucleotides 73-94 of TPST2 were generated and subcloned into pBluescript. Human embryonic kidney (HEK) 293T cells were transfected with these plasmids. One day later, cells were split: one part was labeled with 35S-cysteine and methionine or 35S-Na2SO3 overnight, the second part was used for 125I labeled binding experiment, and the third part was retained for binding and flow cytometry.

RESULTSTyrosines at position 326 of TPST1 and position 325 of TPST2 were sulfated posttranslationally. Tyrosine sulfation of TPSTs was effectively inhibited by sulfation inhibitors, including specific shRNAs and non-specific NaCIO3. shRNAs reduced the sulfation of C3a receptor and C5a receptor, and partially blocked the binding of these two receptors to their respective ligands.

CONCLUSIONSThe activities of TPSTs were regulated by tyrosine sulfation. Inhibition of sulfotyrosine decreases the binding ability of C3a receptor and C5a receptor to their respective ligands.

Cell Line ; Complement C3a ; metabolism ; Complement C5a ; metabolism ; Humans ; Protein Binding ; Protein Processing, Post-Translational ; Receptor, Anaphylatoxin C5a ; metabolism ; Receptors, Complement ; metabolism ; Sulfotransferases ; genetics ; metabolism ; Transfection ; Tyrosine ; analogs & derivatives ; metabolism

Perilipin and adipophilin, two significant lipid droplet (LD)-specific proteins, participate in storing fat or ectopic lipid deposition and fat mobilization in many types of mammalian cells. Acylation stimulating protein (ASP) is a novel adipocyte-derived hormone known for a major determinant for triglyceride synthesis (TGS) and lipid metabolism. The present study was aimed to investigate: (1) whether ASP, rather than insulin, is a powerful potentiator which could physiologically and directly influence TGS during 3T3-L1 preadipocyte differentiation; (2) whether ASP exposure at indicated time points during 3T3-L1 preadipocyte differentiation could influence the gene/protein expression of adipophilin and perilipin. 3T3-L1 preadipocytes were differentiated by traditional hormone cocktail and divided into control, ASP and insulin groups according to the treatment of ASP (1 mmol/L) or insulin (100 nmol/L). ASP-stimulated and insulin-stimulated TGS rate at indicated time points (0 d, 3 d, 6 d, 9 d) were assayed by measuring the incorporation of [(3)H]-oleic acid into TG, and the corresponding glucose transport was assayed by [(3)H]-2-DG uptake. The effects of ASP or insulin on gene/protein expression of adipophilin and perilipin at indicated time points were evaluated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The results obtained were as follows: (1) on the 3rd and 6th day of differentiation, ASP dramatically enhanced TGS rate compared with control group (P<0.05, P<0.01); There was no significant difference in TGS rate between insulin group and control group; (2) on the 6th and 9th day of differentiation, both ASP and insulin promoted glucose uptake (P<0.05, P<0.01), and the promoting effect in ASP group was greater than that in insulin group; (3) ASP elevated adipophilin gene and protein expression at the very early stage of differentiation (P<0.05, P<0.001) and had no significant effect from the 4th day of differentiation. Perilipin gene and protein expression increased throughout preadipocyte differentiation and its expression was up-regulated following ASP stimulation from the 3rd day of differentiation (P<0.05, P<0.001) to the end of differentiation (P<0.05); (4) Insulin did not affect gene and protein variation pattern of adipophilin and perilipin. Taken together, this study provides evidence that ASP-evoked changes in gene and protein expression of adipophilin and perilipin correlate with ASP-stimulated TGS acceleration, and adipophilin and perilipin are involved in the molecular mechanism of ASP-induced adipogenesis and LD formation.
3T3-L1 Cells ; Adipocytes ; cytology ; Animals ; Carrier Proteins ; metabolism ; Cell Differentiation ; Complement C3a ; pharmacology ; Gene Expression ; Insulin ; pharmacology ; Membrane Proteins ; metabolism ; Mice ; Perilipin-1 ; Perilipin-2 ; Phosphoproteins ; metabolism

OBJECTIVETo evaluate the hemocompatibility of glutaraldehyde (GA)-tanned bovine pericardium additionally treated by sodium bisulfite (SOB) solution.

METHODSThe hemocompatibility of GA-tanned bovine pericardium treated by SOB solution is evaluated by using dynamic clotting time test, blood platelet adhension test, D-dimeride determination, and complement activation test. The GA-tanned bovine pericardium was used as control.

RESULTSThe curve of absorbance-clotting time of two kinds of bovine pericardium was similar in dynamic clotting time test. There was no significant difference between SOB-treated and control groups in blood platelet adhension test. The D-dimeride contents of all bioprostheses were at normal level, and the D-dimeride content of GA-tanned bovine pericardium treated by SOB solution was significantly lower than that of control group (P < 0.05). In complement activation test, the level of complement C3a in SOB-treated group was significantly lower than that in control group (P < 0.05).

CONCLUSIONGA-tanned bovine pericardium treated by SOB solution meets the demands of cardiac interstitial implanted materials in hemocompatibility.

Animals ; Biocompatible Materials ; Bioprosthesis ; Blood Coagulation ; Cattle ; Complement C3a ; analysis ; Fibrin Fibrinogen Degradation Products ; metabolism ; Glutaral ; pharmacology ; Materials Testing ; Pericardium ; drug effects ; metabolism ; Platelet Adhesiveness ; Sulfites ; pharmacology

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

BACKGROUNDTubulointerstitial renal fibrosis is the common end point of progressive kidney diseases, and tubular epithelial-myofibroblast transdifferentiation (TEMT) plays a key role in the progress of tubulointerstitial renal fibrosis. Anaphylatoxin C3a and C5a are identified as novel profibrotic factors in renal disease and as potential new therapeutic targets. The aim of this study was to investigate whether C3a, C5a can regulate TEMT by transforming growth factor-β1 (TGF-β)/connective tissue growth factor (CTGF) signaling pathway and the effects of C3a and C5a receptor antagonists (C3aRA and C5aRA) on C3a- and C5a-induced TEMT.

METHODSHK-2 cells were divided into C3a and C5a groups which were subdivided into four subgroups: control group, 10 ng/ml TGF-β1 group, 50 nmol/L C3a group, 50 nmol/L C3a plus 1 µmol/L C3aRA group; control group, 10 ng/ml TGF-β1 group, 50 nmol/L C5a group, 50 nmol/L C5a plus 2.5 µmol/L C5aRA group. TGF-β1 receptor antagonist (TGF-β1RA) 10 µg/ml was used to investigate the mechanism of C3a- and C5a-induced TEMT. Electron microscopy was used to observe the morphological changes. Immunocytochemistry staining, real-time PCR and Western blotting were used to detect the expressions of a smooth muscle actin (α-SMA), E-cadherin, Col-I, C3a receptor (C3aR), C5aR, CTGF and TGF-β1.

RESULTSHK-2 cells cultured with C3a and C5a for 72 hours exhibited strong staining of α-SMA, lost the positive staining of E-cadherin, and showed a slightly spindle-like shape and loss of microvilli on the cell surface. The expressions of α-SMA, E-cadherin, Col-I, C3aR, C5aR, TGF-β1 and CTGF in C3a- and C5a-treated groups were higher than normal control group (P < 0.05). C3aRA and C5aRA inhibited the expressions of α-SMA, Col-I, C3aR, C5aR, and up-regulated the expression of E-cadherin (P < 0.05). TGF-β1 and CTGF mRNA expressions induced by C3a and C5a were partly blocked by TGF-β1RA (P < 0.05).

CONCLUSIONC3a and C5a can induce TEMT via the up-regulations of C3aR and C5aR mRNA and the activation of TGF-β1/CTGF signaling pathway in vitro.

Blotting, Western ; Cadherins ; genetics ; Cell Line ; Cell Transdifferentiation ; drug effects ; Complement C3a ; pharmacology ; Complement C5a ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; ultrastructure ; Humans ; Immunohistochemistry ; Microscopy, Electron, Scanning ; Myofibroblasts ; cytology ; drug effects ; ultrastructure ; Real-Time Polymerase Chain Reaction