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

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*

Mast cells, which are major effector cells in allergic reactions, are found in the perivascular spaces of most tissues and contain pro-inflammatory and vasoactive mediators. These mediators are released after IgE receptor cross-linking induced by allergens or other stimuli, including anaphylatoxins (C3a and C5a), aggregated IgG, certain drugs, venoms, and physical stimuli (pressure and temperature changes), as well as cytokines and neuropeptides. The excess release of these mediators can cause variable allergic symptoms and signs, such as bronchospasm, itching, flushing, nausea, vomiting, diarrhea, abdominal pain, vascular instability, and anaphylaxis. Furthermore, mast cell disorders may involve either excessive proliferation of mast cells or abnormal mast cell reactivity. Mast cell disorders can be broadly divided into 3 types: primary, secondary, and idiopathic. All of these disorders present with signs and symptoms of mast cell activation and differ in severity and involvement of various organ systems. The best characterized primary disorder is mastocytosis. Systemic and cutaneous forms of the disease are well described. Secondary disorders include typical allergic diseases and some types of urticarial diseases. In this article, the biochemical characteristics of mast cells and the role of mast cells in allergic inflammation, as well as the classification, diagnosis, and management of mast cell-related disorders, will be reviewed.
Abdominal Pain ; Allergens ; Allergy and Immunology ; Anaphylatoxins ; Anaphylaxis ; Bronchial Spasm ; Classification ; Cytokines ; Diagnosis ; Diarrhea ; Flushing ; Hypersensitivity ; Immunoglobulin E ; Immunoglobulin G ; Inflammation* ; Mast Cells* ; Mastocytosis ; Nausea ; Neuropeptides ; Pruritus ; Venoms ; Vomiting

BACKGROUND: Several recent studies have reported that generation of inflammatory cytokines and activation of complements may be associated with febrile nonhemolytic transfusion reactions (FNHTR). However, few data are available for whole blood, which is still commonly utilized for massive transfusion and for autologous transfusion. METHODS: A total of 15 whole blood units from healthy adult donors was collected and stored at 4degrees C for 35 days. During the storage time, samples for analyses of cytokines including interleukin-1alpha (IL-1alpha), IL-2, IL-6, IL-8, and tumor necrosis factor alpha (TNFalpha) and anaphylatoxins such as C3a and C5a were obtained on day 0, 1, 3, 5, 7, 14, 28, and 35. Cytokines were measured by enzyme-linked immunosorbent assay and anaphylatoxins by radioimmunoassay. RESULTS: IL-1alpha (<0.5 pg/mL), IL-2 (<7 pg/mL), and TNFalpha (<4.4 pg/mL) were not detectable. IL-6 was measured in 4 units with low level (1.1-4.0 pg/mL) and IL-8 showed slightly higher level (10.5 pg/mL) on day 35. Anaphylatoxins (C3a and C5a) were detectable at the level of 1350.0 ng/mL on day 21 and of 14.6 ng/mL on day 14, respectively, which were significantly increased levels compared with those on day 0. The levels of C3a and C5a reached 2513.3 ng/mL and 18.4 ng/mL on day 35, respectively. CONCLUSIONS: It is not likely that cytokines generated during storage of whole blood under normal blood banking condition could explain FNHTR. However, anaphylatoxins are elevated in whole blood after 2 weeks of storage, which might be due to complement activation by the plastic surface of blood bag.
Adult ; Anaphylatoxins* ; Blood Banks* ; Blood Group Incompatibility ; Complement Activation ; Complement System Proteins ; Cytokines* ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-1alpha ; Interleukin-2 ; Interleukin-6 ; Interleukin-8 ; Plastics ; Radioimmunoassay ; Tissue Donors ; Tumor Necrosis Factor-alpha

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

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

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

OBJECTIVETo investigate the effect of Liuwei Wuling tablets on the cytoplasmic translocation and release of high-mobility group box-1 (HMGB1) in hepatocytes in mice with acute live injury induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS).

METHODSA Balb/c mouse model of acute liver injury was established by intraperitoneal injection of D-GalN (400 mg/kg) and LPS (5 ug/kg). A total of 24 healthy mice were randomly and equally divided into acute liver injury control group and Liuwei Wuling tablet treatment group. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in both groups at each time point within one week. Liver tissues were collected at 36 hours to perform pathological examination. The serum levels of HMGB1, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), complement 3a (C3a), and complement 5a (C5a) were measured. Immunohistochemistry was used to determine the expression and cytoplasmic translocation of HMGB1 in hepatocytes.

RESULTSAt 6, 12, and 24 hours, the Liuwei Wuling tablet treatment group had significantly lower serum levels of ALT than the control group (225.33±181.64 U/L vs 471.17±174.72 U/L, t = 3.38, P < 0.01; 1509.53±182.51 U/L vs 7149.52±734.25 U/L, t = 25.82, P < 0.01; 162.89±86.51 U/L vs 1318.16±557.71 U/L, t = 7.09, P < 0.01), as well as significantly lower serum levels of AST than the control group (179.22±94.57 U/L vs 561.91±209.6 U/L, t = 5.76, P < 0.01; 590.92±190.92 U/L vs 2266.48±705.64 U/L, t = 7.94, P < 0.01; 231.24±87.7 U/L vs 444.32±117.01 U/L, t = 5.05, P < 0.01). The treatment group had significantly lower levels of HMGB1 than the control group at 6 and 12 hours (54.21±11.89 ng/ml vs 72.07±13.65 ng/ml, t = 3.41, P < 0.01; 49.23±5.97 ng/ml vs 68.71±13.07 ng/ml, t = 4.70, P < 0.01). The treatment group had significantly lower levels of TNF-α, IL-1β, and IL-6 than the control group at 12 hours (163.62±9.12 pg/ml vs 237.09±51.47 pg/ml, t = 4.86, P < 0.01; 15.66±13.13 pg/ml vs 37.43±18.68 pg/ml, t = 3.30, P < 0.01; 7.10±3.06 pg/ml vs 21.42±8.23 pg/ml, t = 5.65, P < 0.01). The treatment group had significantly lower levels of C3a and C5a than the control group at 12 hours (2.52±1.27 pg/ml vs 9.83±2.96 ng/ml, t = 7.86, P < 0.01; 2.16±1.03 ng/ml vs 7.23±1.55 ng/ml, t = 9.67, P < 0.01). Compared with the control group, the treatment group had significantly reduced liver inflammation and necrosis, and a significantly lower cytoplasmic translocation rate of HMGB1 in hepatocytes (38.76%±7.37% vs 8.15%±2.11%, P < 0.01).

CONCLUSIONLiuwei Wuling tablets can reduce the cytoplasmic translocation of HMGB1 in hepatocytes and relieve liver inflammation in mice with acute liver injury.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Complement C3a ; analysis ; Complement C5a ; analysis ; Cytoplasm ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Galactosamine ; HMGB1 Protein ; metabolism ; Hepatocytes ; drug effects ; Interleukin-1beta ; blood ; Interleukin-6 ; blood ; Lipopolysaccharides ; Liver Failure, Acute ; drug therapy ; Mice ; Mice, Inbred BALB C ; Protein Transport ; Tablets ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo observe serum C4a and platelet aggregation rates changes in acute myocardial infarction (AMI) patients before and after percutaneous coronary intervention (PCI) and association with the development of no-reflow phenomenon.

METHODSFrom June 2006 to August 2009, 119 AMI patients underwent PCI (28 cases of no-reflow group, 91 cases of reflow group) and 30 subjects with suspected coronary heart diseases and normal coronary angiography results (control group) were enrolled in this study. C4a and platelet aggregation rate were measured at 30 minutes before PCI, immediately after PCI, 30 minutes, 1 hour, 2 hour, and 6 months post PCI in AMI patients and at before coronary angiography in control subjects.

RESULTSThe levels of serum C4a at 30 minutes prior to PCI in control, no-reflow, and reflow groups were similar (P > 0.05). Platelet aggregation rate at 30 minutes prior to PCI was significantly higher in no-reflow group and reflow group than in control group (all P < 0.05). Serum C4a and platelet aggregation rates were significantly higher in no-reflow group at immediate, 30 minutes and 1 hour after PCI than at 30 minutes prior to PCI, two hours and 6 months after PCI (all P < 0.05), and were significantly higher than in reflow group at immediate, 30 minutes and 1 hour after PCI (all P < 0.05). Serum C4a and platelet aggregation rates were similar at different time points in reflow group (all P > 0.05). The levels of C4a in no-reflow group at immediate, 30 minutes and 1 hour after PCI were positively correlated with platelet aggregation rates (r = 0.91, 0.79, 0.60, respectively, all P < 0.01).

CONCLUSIONThe transient increase on levels of C4a and platelet aggregation rate early post PCI are verified in no-reflow patients with AMI undergoing PCI.

Aged ; Angioplasty, Balloon, Coronary ; Case-Control Studies ; Complement C4a ; metabolism ; Humans ; Middle Aged ; Myocardial Infarction ; blood ; No-Reflow Phenomenon ; Platelet Aggregation ; Postoperative Period

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