C5b-9 Levels Research Articles

Functional Activity of the Complement System in Deceased Donors in Relation to Kidney Allograft Outcome

Complement activation is considered one of the mediators of renal ischemia-reperfusion injury. Elevated levels of C5b-9, C3a, and C5a are detected in sera of deceased kidney donors. The goal of the study was to characterize the functional activity of complement pathways in donor sera and to assess their influence on transplant outcome. Materials and methodsSixty-four deceased kidney donors (age 45 ± 16 years; 28 female, 36 male) and 27 healthy controls (age 42 ± 12 years; 14 female, 13 male) were enrolled in the study. The results of transplantation for the respective 122 kidney recipients were included in the analysis. The functional activities of classical (CP), lectin (LP), and alternative (AP) pathways were measured using Wielisa-kit (reference normal level = 100%). In most cases, decreased functional activity reflects the activation status of the pathway. ResultsThe median (interquartile range) functional activities of the pathways in donor sera were CP 118 (89–150)%, LP 80 (20–127)%, and AP 74 (50–89)%, and did not differ from the control values CP 110 (102–115)%, LP 81 (26–106)%, AP 76 (61–88)%. The frequency of pathway activation observed in controls was CP 0%, LP 11%, and AP 0%. Deceased donors did not differ in activation of classical (11%) and lectin (13%) pathways, but presented a higher rate of alternative pathway activation (19%, P = .03). No significant influence of any pathway functional activity or its activation was proved to influence the transplant outcome. ConclusionComplement activation via alternative pathway was observed in diseased donor sera. No predictive potential of donor complement functional activity on the transplant outcome could be proved.

Pathogenic role of inflammatory response during Shiga toxin-associated hemolytic uremic syndrome (HUS).

Hemolytic uremic syndrome (HUS) is defined as a triad of noninmune microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. The most frequent presentation is secondary to Shiga toxin (Stx)-producing Escherichia coli (STEC) infections, which is termed postdiarrheal, epidemiologic or Stx-HUS, considering that Stx is the necessary etiological factor. After ingestion, STEC colonize the intestine and produce Stx, which translocates across the intestinal epithelium. Once Stx enters the bloodstream, it interacts with renal endothelial and epithelial cells, and leukocytes. This review summarizes the current evidence about the involvement of inflammatory components as central pathogenic factors that could determine outcome of STEC infections. Intestinal inflammation may favor epithelial leakage and subsequent passage of Stx to the systemic circulation. Vascular damage triggered by Stx promotes not only release of thrombin and increased fibrin concentration but also production of cytokines and chemokines by endothelial cells. Recent evidence from animal models and patients strongly indicate that several immune cells types may participate in HUS physiopathology: neutrophils, through release of proteases and reactive oxygen species (ROS); monocytes/macrophages through secretion of cytokines and chemokines. In addition, high levels of Bb factor and soluble C5b-9 (sC5b-9) in plasma as well as complement factors adhered to platelet-leukocyte complexes, microparticles and microvesicles, suggest activation of the alternative pathway of complement. Thus, acute immune response secondary to STEC infection, the Stx stimulatory effect on different immune cells, and inflammatory stimulus secondary to endothelial damage all together converge to define a strong inflammatory status that worsens Stx toxicity and disease.

306: Preeclampsia with severe features, defining a subset of women with complement-associated disease

Preeclampsia is heterogeneous. Detection of a universal biomarker may be unrealistic due to distinct pathways of disease. Complement activation, which predisposes to kidney injury, hemolysis and thrombocytopenia, is increased in preeclampsia. We hypothesize that urinary complement biomarker C5b-9, the membrane attack complex, may define a subset of cases with severe, complement-associated disease. Project COPA (COmplement and Preeclampsia in the Americas), an IRB approved, multi-center observational study, enrolled subjects prospectively from 6 centers and 3 cities in Colombia (Bogotá, Cartagena and Medellín (Nov ’15-Jul ’16). Subjects were enrolled in blocks by gestational age (< or > 34 weeks) and diagnosis (ACOG criteria): 1. healthy; 2. chronic hypertension (CHTN); 3. gestational hypertension (GHTN); 4. preeclampsia (PE) and; 5. PE with severe features (PE-SF). COPA was powered for PE-SF (target, n=100). Clinical data and urine samples were collected by trained coordinators. C5b-9 was measured by enzyme linked immunosorbent assays (Human C5b-9 ELISA, BD Biosciences). For this study, we utilized receiver operating characteristic (ROC) curve analysis, chi-square test and multivariable logistic regression. 352 subjects were enrolled [healthy (n=59), CHTN (n=42), GHTN (n=92), PE (n=58), PE-SF (n=101)]. Using ROC analysis, we found that urinary C5b-9 has significant diagnostic utility for PE-SF (Figure 1), with area under the curve = 0.74 (0.67-0.80). Urinary C5b-9 level ≥22 ng/ml was the optimal cut-point for PE-SF with 96.4% specificity, positive likelihood ratio 11.3. The odds of PE-SF in those with urinary C5b-9 ≥22 ng/ml was significantly increased, and the association remained strong after adjustment for confounding factors, including total urine protein (Table 1). We found that 40% (41/101) of subjects with PE-SF had urinary C5b-9 levels ≥22 ng/ml, compared to 12% (7/58) of those with PE alone (p<0.001) and 0% (0/134) of those with CHTN or GHTN (p<0.001). Our data provide strong evidence that a subset of women with preeclampsia with severe features have complement-associated disease, characterized by increased urinary excretion of C5b-9. We propose that women with complement-associated preeclampsia may benefit from targeted complement blockade. Clinical studies are warranted.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

329: Excess complement activation is associated with adverse outcomes in women with hypertensive disorders of pregnancy

Complement activation occurs in normal pregnancy, but excess activation is associated with preeclampsia. Terminal complement activation generates C5b-9, the lytic membrane attack complex, which mediates organ damage. We hypothesize that activation of C5b-9 is increased in women with hypertensive disorders of pregnancy and adverse outcomes We assessed urine and plasma C5b-9 levels in hypertensive subjects from project COPA (COmplement and Preeclampsia in the Americas), an IRB approved, multi-center observational study, which enrolled subjects from 6 centers and 3 cities in Colombia (Bogotá, Cartagena and Medellín; Nov 15-Jul 16). Hypertensive subjects enrolled in blocks by gestational age ( ≤ or ≥ 34 weeks) and diagnosis (ACOG criteria): 1. chronic hypertension (CHTN); 2. gestational hypertension (GHTN); 3. preeclampsia (PE) and; 4. PE with severe features (PE-SF). COPA was powered for PE-SF (n=100). Clinical data, urine and plasma were collected by trained coordinators, with C5b-9 measured by enzyme linked immunosorbent assays (Human C5b-9 ELISA, BD Biosciences). Maternal and neonatal outcomes were assessed individually and as composite outcomes. Data were analyzed by Chi-square, t-test and logistic regression 293 subjects were evaluated [CHTN (n=42), GHTN (n=92), PE (n=58), PE-SF (n=101)]. Adverse maternal and neonatal outcomes, by plasma C5b-9 quartiles 1-4 (pC5b9, Q1-4), are shown in Table 1. Composite maternal outcomes were increased with low pC5b9 (Q1, ≤1443 ng/ml), particularly for those ≥ 34wks (OR 2.93, 95% CI 1.0-8.6, p=0.05). For neonates, preterm birth (PTB) was increased with lower pC5b9 levels (PTB, 2870 ± 1904 vs. term, 3572 ± 2262 ng/ml, p=0.006). Adverse outcomes, by urine C5b-9 (uC5b9) quartiles, are shown in Table 2. They were more common with high uC5b9 levels (Q4, ≥8.49 ng/ml), predominantly due to increased kidney injury (OR 3.0, 95%CI 1.1-8.4, p=0.036) and PTB (OR 2.0, 95% CI 1.2-3.5, p=0.01) We describe a novel pattern of complement activation (low plasma / high urine C5b-9), which associates with adverse maternal and neonatal outcomes in women with hypertensive disorders of pregnancy. We postulate that excess complement activation results in kidney injury and depletion of complement factors in plasma, with resultant pregnancy complications.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Dynamic changes of complement level in patients with acute coronary syndrome and its relationships with myocardial injury

To study relationships between myocardial injury and the levels of serum complement C3, C4 and C5b-9 in patients with acute coronary syndrome (ACS). A retrospectively analysis was conducted. 170 ACS patients [including 110 cases of ST-segment elevation myocardial infarction (STEMI) and 60 cases of non-ST-segment elevation acute coronary syndrome (NSTE-ACS)] with ischemic chest pain or chest discomfort onset within the prior 12 hours admitted to the cardiology department of Tianjin Union Medicine Center from January 2014 to July 2016 were enrolled. Thirty-six healthy cases were enrolled as control during the same time. The levels of serum complement C3, C4 and C5b-9 on 1, 3 and 7 days after admission and myocardial function indicators were analyzed. Major adverse cardiovascular events (MACE) and readmission rate were analyzed after 1 year follow-up. The correlation between serum complement levels and myocardial function indicators was analyzed by Pearson correlation analysis. (1) The levels of serum C3, C4 and C5b-9 on the first day in NSTE-ACS group and STEMI group were significantly higher than control group [C3 (g/L): 1.04±0.33, 1.26±0.35 vs. 0.39±0.21, C4 (g/L): 0.31±0.14, 0.33±0.10 vs. 0.19±0.07, C5b-9 (g/L): 575.46±197.26, 659.26±160.77 vs. 501.40±141.51, all P < 0.05]. There were no changes of serum C3, C4 in NSTE-ACS group, but C5b-9 decreased after a peak (g/L: 700.63±218.42) at 3 days. Serum complements in STEMI group reached peak on the third day [C3 (g/L): 1.37±0.33, C4 (g/L): 0.42±0.12, C5b-9 (g/L): 754.72±136.22]. The levels of serum C4 and C5b-9 in STEMI group were higher than NSTE-ACS group on the third and seventh day. (2) The levels of troponin T (TnT), creatine kinase-MB (CK-MB), solution intercellular adhesion molecule-1 (sICAM-1), global registry of acute coronary events (GRACE) scores and percutaneous coronary intervention (PCI) numbers in STEMI group were significantly higher than those in the NSTE-ACS group, which were as opposite as left ventricular ejection fraction (LVEF). However, there were no significant differences in levels of serum N-terminal pro-brain nitric peptide (NT-proBNP), Fibrinogen (Fib), readmission rate and incidence of MACE between STEMI and NSTE-ACS groups. (3) According to GRACE, patients with ACS were divided into low risk group (≤ 108 scores, 26 cases), intermediate risk group (109-140 scores, 61 cases) and highest group (> 140 scores, 83 cases). TnT and sICAM-1 in intermediate risk group were significantly increased as compared with low risk group. Levels of TnT, sICAM-1, C3, C4 and C5b-9 in the highest group were significantly higher than the low and intermediate risk groups, however the lowest LVEF was found in the highest group. (4) It was shown by Pearson correlation analyses that levels of serum C3, C4, C5b-9 were positively correlated with TnT (r value was 0.481, 0.367, 0.292, respectively, all P < 0.01), sICAM-1 (r value was 0.298, 0.249, 0.365, respectively, all P < 0.01), but negatively correlated with LVEF (r value was -0.384, -0.260, -0.200, respectively, all P < 0.01). In addition sICAM-1 positively correlated with TnT (r = 0.536, P = 0.000), but negatively correlated with LVEF (r = -0.341, P = 0.001). Serum complements activation was found in the acute phase of ACS patients. Serum complement C3, C4 and C5b-9 are involved in the process of myocardial injury, and may reflect severity of myocardial injury and cardiac dysfunction.

Accuracy of complement activation product levels to detect infected pleural effusion in rats.

Pleural empyema is a well-known complication of pneumonia. If treatment is delayed, empyema may increase morbidity and mortality in affected patients. Therefore, the identification of empyema biomarkers in parapneumonic pleural effusion is desirable. Previous research has suggested complement activation products as candidate empyema markers. To compare the levels of complement activation products C3a, C5a, and C5b9 in pleural effusion induced by Staphylococcus aureus (SA), Streptococcus pneumoniae (SP), or turpentine (control). Thirty-nine male Wistar rats (mean weight 414 g; 290-546 g) were allocated as follows: 17 animals in the SA group, 12 in the SP group, and 10 in the control group. Bacteria or turpentine were injected into the pleural space. After 12 hr, intrapleural fluid was collected using ultrasound-guided thoracentesis. Levels of complement activation products were determined using ELISA kits. Two SA and one SP animals died before 12 hr. Mean levels were as follows: C3a: 1066.82 µg/ml (937.29-1196.35 µg/ml) in SA, 1188.28 µg/ml (1095.65-1280.92 µg/ml) in SP, and 679.13 µg/ml (601.29-756.98 µg/ml) in controls (P < 0.001); C5a: 55.727 ng/ml (41.22-70.23 ng/ml) in SA, 520.107 ng/ml (278.92-761.3 ng/ml) in SP, and 5.268 ng/ml (1.68-8.85 ng/ml) in controls (P < 0.001); C5b9: 15.02 ng/ml (13.1-16.94 ng/ml) in SA, 16.63 ng/ml (14.37-18.9 ng/ml) in SP, and 14.05 ng/ml (9.8-18.29 ng/ml) in controls (P = 0.692). ROC analysis revealed an area under the curve of 0.987 (95% CI: 0.953-1) for C3a; 1 (1-1) for C5a; and 0.757 for C5b9 (0.523-0.990). In the present rat model, complement activation fragments C3a and C5a accurately detected infected pleural effusion. Pediatr Pulmonol. 2017;52:757-762. © 2017 Wiley Periodicals, Inc.

Estudio de las variables asociadas a la activación local del complemento en la nefropatía IgA idiopática

Objectives1. To identify the variables that are associated with urinary levels of properdin, MBL, C4d, and C5b-9 in patients with idiopathic IgA nephropathy. 2. To analyse whether urinary levels of MBL and/or C4d are useful for identifying the presence of mesangial deposits of C4d/MBL. Patients and methodA total of 96 patients with IgA nephropathy were studied. Demographic, clinical and biochemical variables were recorded at the time of diagnosis. Renal lesions were quantified using the Oxford classification. Immunohistochemical staining for MBL, MASP-2, properdin, C4d, and C5b-9 was performed in kidney biopsies, and in urine, the levels of properdin, MBL, C4d and C5b-9 were determined. ResultsIn multivariate analysis, the independent predictors of C4d and MBL levels in urine were the mesangial deposits of each protein and, to a lesser extent, the urinary protein excretion. The independent predictors of urinary levels of C5b-9 were MBL properdin and proteinuria. Urinary excretion of C4d had a sensitivity of 90% (95% CI: 58,7 to 99) and a specificity of 73% (95% CI: 54-87) for detecting mesangial C4d deposits, and the level of MBL had a sensitivity of 83.9% (95% CI: 62-95) and a specificity of 81.6% (95% CI: 65-92) for identifying mesangial deposits of MBL. ConclusionThe main predictor of urinary concentration of C4d and MBL was the presence of their respective mesangial deposits. Urine MBL may contribute to complement activation in the tubular luz through the lectin pathway. Urinary levels of MBL and C4d could be sensitive and specific biomarkers for the identification of patients with mesangial deposits of MBL and C4d.

Role of SIRT1 in C5b-9 mediated oligodendrocyte cell cycle activation

Abstract SIRT1 is a NAD-dependent histone deacetylase involved in the regulation of transcription, apoptosis, metabolism and differentiation. We have previously demonstrated that sublytic levels of complement C5b-9 terminal complex increased the survival of oligodendrocytes (OLGs) and induced their dedifferentiation. In this study we investigated the role of SIRT1 in OLGs differentiation and the effect of sublytic levels of C5b-9 on SIRT1 expression. We also investigated the downstream effects of SIRT1 by measuring histone H3 Lysine 9 trimethylation (H3K9me3) and expression of cyclin D1. OLG progenitor cells purified from the brain of rat pups were differentiated in vitro and stimulated with sublytic C5b-9 or C5b6 for 3, 6 and 18 h. The level of SIRT1 mRNA was measured using real-time PCR and SIRT1, cyclin D1 and H3K9me3 protein expression were measured using western blotting. We found a decreased expression of SIRT1 mRNA and protein, a decreased level of H3K9me3 and an increased expression of cyclin D1 during OLG differentiation. Stimulation of OLGs with sublytic C5b-9 for 3h resulted in a significant decrease in SIRT1 mRNA and protein levels while stimulation with C5b6 had no effect. SIRT1 protein level in OLGs after 8h of exposure to C5b-9 were significantly lower than in C5b6 treated cells. H3K9me3 levels also decreased significantly after stimulation with C5b-9 as compared with unstimulated or C5b6-treated OLGs. Cyclin D1 expression increased after stimulation with C5b-9, indicating cell cycle activation. Our data show that C5b-9 stimulation of OLGs reduces SIRT1 expression, contributing to cell cycle activation by decreasing repressive trimethylation of histone H3 lysine 9.

Proteomic Profiling Analysis Reveals a Link between Experimental Autoimmune Uveitis and Complement Activation in Rats

Uveitis is an autoimmune disease that usually damages the vision function, leading to poor visual quality in patients. As an autoimmune ocular inflammatory disease, the pathogenesis of uveitis is associated with abnormal expression of some proteins and aberrant regulation of multiple signalling pathways. Nevertheless, the detailed mechanism remains unclear. In this study, we induced an experimental autoimmune uveitis (EAU) model in rats. We determined the levels of C3a and membrane attack complex C5b-9 (soluble C5b-9, sC5b-9) in both plasma and aqueous humour, identified the differentially expressed proteins in plasma by liquid chromatography-tandem mass spectrometry and employed bioinformatics algorithms to analyse differentially expressed proteins in EAU rat plasma. The results demonstrate that there were 168 differentially expressed plasma proteins in EAU rats versus control subjects. The levels of sC5b-9 and C3a were elevated in the plasmas and aqueous humours of EAU rats. Gene ontology enrichment analysis showed that the differentially expressed proteins in EAU rat plasma were mainly involved in metabolic and immune processes. Kyoto encyclopedia of genes and genomes (KEGG) pathway annotation, database for annotation, visualization and integrated discovery (DAVID) and protein-protein interaction analyses revealed that the differentially expressed proteins in EAU rat plasmas were closely associated with complement and coagulation cascades, metabolic pathways, NF-kappa B, PI3K-Akt, Toll-like receptors and autophagy. Overall, the differentially expressed proteins in EAU rat plasmas are mainly involved in the complement and coagulation cascades. The pathogenesis of uveitis closely correlates with complement activation.

Study of the variables associated with local complement activation in IgA nephropathy

Objectives1. To identify the variables that are associated with urinary levels of properdin, MBL, C4d, and C5b-9 in patients with idiopathic IgA nephropathy. 2. To analyse whether urinary levels of MBL and/or C4d are useful for identifying the presence of mesangial deposits of C4d/MBL. Patients and methodA total of 96 patients with IgA nephropathy were studied. Demographic, clinical and biochemical variables were recorded at the time of diagnosis. Renal lesions were quantified using the Oxford classification. Immunohistochemical staining for MBL, MASP-2, properdin, C4d, and C5b-9 was performed in kidney biopsies, and in urine, the levels of properdin, MBL, C4d and C5b-9 were determined. ResultsIn multivariate analysis, the independent predictors of C4d and MBL levels in urine were the mesangial deposits of each protein and, to a lesser extent, the urinary protein excretion. The independent predictors of urinary levels of C5b-9 were MBL properdin and proteinuria. Urinary excretion of C4d had a sensitivity of 90% (95% CI: 58.7–99) and a specificity of 73% (95% CI: 54–87) for detecting mesangial C4d deposits, and the level of MBL had a sensitivity of 83.9% (95% CI: 62–95) and a specificity of 81.6% (95% CI: 65–92) for identifying mesangial deposits of MBL. ConclusionThe main predictor of urinary concentration of C4d and MBL was the presence of their respective mesangial deposits. Urine MBL may contribute to complement activation in the tubular luz through the lectin pathway. Urinary levels of MBL and C4d could be sensitive and specific biomarkers for the identification of patients with mesangial deposits of MBL and C4d.

Treatment of B-cell disorder improves renal outcome of patients with monoclonal gammopathy–associated C3 glomerulopathy

AbstractThe high frequency of monoclonal gammopathy in adult patients with C3 glomerulopathy (C3G) emphasizes the role of monoclonal immunoglobulin (MIg) in the occurrence of renal disease and raises the issue of the therapeutic management. The aim of the study was to evaluate the effect of chemotherapy in a large cohort of patients with MIg-associated C3G. Fifty adult patients with MIg and biopsy-proven C3G were extracted from the French national database of C3G. We retrospectively compared renal outcomes in patients who either received or did not receive chemotherapy targeting the underlying B-cell clone. At diagnosis, renal disease was severe, with nephrotic-range proteinuria in 20/46 (43%) patients and chronic kidney disease stage 3 or above in 42/49 (86%) patients. Monoclonal gammopathy was of IgG type in 47 (94%) patients. Hematological diagnosis was monoclonal gammopathy of renal significance in 30 (60%), multiple myeloma in 17 (34%), and chronic lymphocytic leukemia in 3 (6%) patients. Complement studies showed low C3 level in 22/50 (43%) and elevated soluble C5b-9 level in 27/34 (79%) patients. Twenty-nine patients received chemotherapy (including bortezomib in 22), whereas 8 and 13 patients received various immunosuppressive drugs or symptomatic measures alone, respectively. Patients who achieved hematological response after chemotherapy had higher renal response rates (P = .0001) and median renal survival (hazard ratio, 0.22; 95% confidence interval, 0.05-0.92; P = .009) than those receiving conservative/immunosuppressive therapy. In conclusion, our results suggest that chemotherapy adapted to the B-cell clone may constitute an efficient strategy for C3G in the setting of MIg, as rapid achievement of hematological response appears to result in improved renal survival.

Patients with hypertension-associated thrombotic microangiopathy may present with complement abnormalities

Thrombotic microangiopathy (TMA) is a pattern of endothelial damage that can be found in association with diverse clinical conditions such as malignant hypertension. Although the pathophysiological mechanisms differ, accumulating evidence links complement dysregulation to various TMA syndromes and in particular the atypical hemolytic uremic syndrome. Here, we evaluated the role of complement in nine consecutive patients with biopsy-proven renal TMA attributed to severe hypertension. Profound hematologic symptoms of TMA were uncommon. In six out of nine patients, we found mutations C3 in three, CFI in one, CD46 in one, and/or CFH in two patients either with or without the risk CFH-H3 haplotype in four patients. Elevated levels of the soluble C5b-9 and renal deposits of C3c and C5b-9 along the vasculature and/or glomerular capillary wall, confirmed complement activation invivo. In contrast to patients without genetic defects, patients with complement defects invariably progressed to end-stage renal disease, and disease recurrence after kidney transplantation seems common. Thus, a subset of patients with hypertension-associated TMA falls within the spectrum of complement-mediated TMA, the prognosis of which is poor. Hence, testing for genetic complement abnormalities is warranted in patients with severe hypertension and TMA on renal biopsy to adopt suitable treatment options and prophylactic measures.

Anti-Factor B and Anti-C3b Autoantibodies in C3 Glomerulopathy and Ig-Associated Membranoproliferative GN.

In C3 glomerulopathy (C3G), the alternative pathway of complement is frequently overactivated by autoantibodies that stabilize the C3 convertase C3bBb. Anti-C3b and anti-factor B (anti-FB) IgG have been reported in three patients with C3G. We screened a cohort of 141 patients with C3G and Ig-associated membranoproliferative GN (Ig-MPGN) for anti-FB and anti-C3b autoantibodies using ELISA. We identified seven patients with anti-FB IgG, three patients with anti-C3b IgG, and five patients with anti-FB and anti-C3b IgG. Of these 15 patients, ten were diagnosed with Ig-MPGN. Among those patients with available data, 92% had a nephrotic syndrome, 64% had AKI, and 67% had a documented infection. Patients negative for anti-C3b and anti-FB IgG had much lower rates of infection (17 [25%] patients with C3G and one [10%] patient with Ig-MPGN). After 48 months, four of 15 (26%) positive patients had developed ESRD or died. All 15 patients had high plasma Bb levels, six (40%) patients had low levels of C3, and nine (60%) patients had high levels of soluble C5b9. In vitro, IgG purified from patients with anti-FB Abs selectively enhanced C3 convertase activity; IgG from patients with anti-C3b/anti-FB Abs enhanced C3 and C5 cleavage. IgG from patients with anti-C3b Abs stabilized C3bBb and perturbed C3b binding to complement receptor 1 but did not perturb binding to factor H. In conclusion, the prevalence of anti-C3b/anti-FB Abs and alternative pathway activation is similar in Ig-MPGN and C3G, suggesting similar pathogenic mechanisms. Identification of the underlying defect in Ig-MPGN could lead to improved treatment.

Complement Alternative Pathway׳s Activation in Patients With Lupus Nephritis

ObjectiveThe aim of this study was to detect the spectrum of complement activation pathways in circulation and to assess their correlations with clinical and pathologic features in a large lupus nephritis cohort from China. Materials and MethodsPlasma levels of C1q, mannose-binding lectin, C4d, Bb, C3, C3a, C5a and soluble C5b-9 were detected by enzyme-linked immunosorbent assay in 222 patients with active biopsy-proven lupus nephritis, 34 patients with lupus nephritis at remission, 82 patients with active systemic lupus erythematosus without renal involvement and 39 normal controls. The correlations between levels of complement components and clinicopathological features of these patients were further analyzed. ResultsPlasma levels of C1q and C3 significantly decreased, and the levels of Bb, C3a, C5a and soluble C5b-9 were significantly elevated in patients with active lupus nephritis compared with those in remission, active systemic lupus erythematosus without renal involvement group and normal controls. In the lupus nephritis group, soluble C5b-9 levels were inversely correlated with C1q and C4d levels (r = −0.412, P < 0.001 and r = −0.221, P = 0.002, respectively), but more strongly correlated with the level of Bb (r = 0.546, P < 0.001). C3b, Bb and C5b-9 could colocalize on glomeruli in lupus nephritis. Plasma Bb level was significantly correlated with some renal disease activity indices and was a risk factor for renal outcomes (hazard ratio = 1.745; 95% CI: 1.106-2.754; P = 0.017) in the lupus nephritis group. ConclusionsOur findings suggested that the activation of the complement alternative pathway might play a more important role in the pathogenesis of lupus nephritis, and factor Bb might be a useful marker for evaluating renal disease activity and outcomes.

Neuroprotective effects of intravenous immunoglobulin are mediated through inhibition of complement activation and apoptosis in a rat model of sepsis

BackgroundIntravenous (IV) immunoglobulin (Ig) treatment is known to alleviate behavioral deficits and increase survival in the experimentally induced model of sepsis. To delineate the mechanisms by which IVIg treatment prevents neuronal dysfunction, an array of immunological and apoptosis markers was investigated.MethodsSepsis was induced by cecal ligation perforation (CLP) in rats. The animals were divided into five groups: sham, control, CLP + saline, CLP + immunoglobulin G (IgG) (250 mg/kg, iv), and CLP + immunoglobulins enriched with immunoglobulin M (IgGAM) (250 mg/kg, iv). Blood and brain samples were taken in two sets of experiments to see the early (24 h) and late (10 days) effects of treatment. Total complement activity, complement 3 (C3), and soluble complement C5b-9 levels were measured in the sera of rats using ELISA-based methods. Cerebral complement, complement receptor, NF-κB, Bax, and Bcl-2 expressions were analyzed by western blot and/or RT-PCR methods. Immune cell infiltration and gliosis were examined by immunohistochemistry using CD3, CD4, CD8, CD11b, CD19, and glial fibrillary acidic protein antibodies. Apoptotic neuronal death was investigated by TUNEL staining.ResultsIVIgG and IgGAM administration significantly reduced systemic complement activity and cerebral C5a and C5a receptor expression. Likewise, both treatment methods reduced proapoptotic NF-κB and Bax expressions in the brain. IVIgG and IgGAM treatment induced considerable amelioration in glial cell proliferation and neuronal apoptosis which were increased in non-treated septic rats.ConclusionsWe suggest that IVIgG and IgGAM administration ameliorates neuronal dysfunction and behavioral deficits by reducing apoptotic cell death and glial cell proliferation. In both treatment methods, these beneficial effects might be mediated through reduction of anaphylatoxic C5a activity and subsequent inhibition of inflammation and apoptosis pathways.

Classical complement pathway activation in the nasal tissue of patients with chronic rhinosinusitis

Complement plays a major role in inflammatory diseases, but its involvement and mechanisms of activation in patients with chronic rhinosinusitis (CRS) are not known. After earlier studies discovering autoantibodies in patients with CRS, we sought to investigate the nature, extent, and location of complement activation in nasal tissue of patients with CRS. Specifically, we were interested in whether antibody-mediated activation through the classical pathway was a major mechanism for complement activation in patients with CRS. Nasal tissue was obtained from patients with CRS and control subjects. Tissue homogenates were analyzed for complement activation products (ELISA-C5b-9, C4d, activated C1, and C5a) and major complement-fixing antibodies (Luminex). Tissue sections were stained for C5b-9, C4d, and laminin. Antibodies were purified with protein A/G columns from nasal polyps (NP), matching patient serum, and control serum and assayed for basement membrane binding by means of ELISA. C5b-9 levels were significantly increased in NP tissue compared with uncinate tissue (UT) of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and those with chronic rhinosinusitis without nasal polyps (CRSsNP; P<.01). Similarly, C4d levels were increased in NPs compared with UT of patients with CRSwNP, patients with CRSsNP, and control subjects (P<.05). Activated C1 levels were also increased in NP tissue compared with UT of patients with CRSsNP and control subjects (P<.05) and correlated with levels of C5a (P<.01), local immunoglobulins (especially IgM, P<.0001), and anti-double-stranded DNA IgG (P<.05). Immunofluorescence showed that C5b-9 and C4d deposition occurred linearly along the epithelial basement membrane. NP tissue extracts had significantly more anti-basement membrane antibodies than sera from patients with CRSwNP and control subjects (P<.0001). Levels of C5b-9, C4d, and activated C1 were significantly increased locally in NP tissue. C5b-9 and C4d were almost universally deposited linearly along the basement membrane of NP tissue. Furthermore, activated C1 levels were best correlated with local immunoglobulin and C5a levels. Together, these data suggest that the classical pathway plays a major role in complement activation in patients with CRS.

Antiphospholipid Syndrome (APS) Patient Plasmas Consistently Activate Complement: A Novel Approach to Detecting a Pathogenic Mechanism for APS

INTRODUCTION: The antiphospholipid syndrome (APS) has remained an enigmatic condition for over 3 decades. Pathogenic mechanism(s) have not been established, and the disorder has therefore continued to be defined by non-mechanistic assays such as the lupus anticoagulant and antiphospholipid immunoassays. Although evidence from animal models has indicated a role for complement activation in the disease process, it is has been remarkable that activation markers are not consistently detected in human APS plasmas. We hypothesized that this might be due to rapid clearance of activation products and that we might be better able to detect a role for complement in APS through a 2 stage system - i.e. by first allowing APS immune complexes to form on phospholipid vesicles and then measuring whether those vesicles might activate complement in normal serum.METHODS: Complement activation markers - specifically C5a and the C5b-9 membrane attack complex - were measured in plasmas of 5 groups of plasmas (n= 10 for each group): normal healthy controls, APS with clinical histories for thrombosis (8 with venous thromboembolism (VTE), 1 venous stroke, and 1 arterial stroke), cancer patients without thrombosis, systemic lupus erythematosus (SLE) without APS, and VTE without APS. These were compared to measurements of complement activation induced by phospholipid vesicles that were pre-incubated with the same plasmas. Additional experiments were then done to: 1) characterize whether the effects could be reproduced with purified APS vs control IgGs and β2-glycoprotein I (β2GPI), 2) define the complement activation pathways involved with factor B and C2 deficient sera and with serum in the presence of EGTA and MgCl2, and 3) determine whether the activation could be inhibited by the monoclonal anti-C5 antibody, eculizimab.RESULTS: When the plasmas themselves were assayed, we found no significant differences in the plasmas levels of C5a or C5b-9 between APS and controls or among the 5 groups (Fig 1 A, B). In contrast, preincubation of phospholipid vesicles with plasmas resulted in significant elevations of both C5a (Fig 1C) and C5b-9 (Fig 1D) in APS compared to controls. Also, C5a levels in cancer, SLE and VTE were significantly lower than in APS, but above the controls. C5b-9 levels were elevated in APS and the other disease groups compared to controls, with the APS levels that were significantly higher than only the cancer group. The results were confirmed using purified IgGs and β2GPI in place of the whole plasmas. In addition, we found that APS-treated vesicles triggered complement activation via both the classical/lectin pathway and the alternative pathway. Eculizimab completely abrogated the complement activation.CONCLUSIONS: To our knowledge, this is the first demonstration that APS patient plasmas clearly and consistently activate complement. This was achieved through the novel approach of first incubating the plasmas with phospholipid vesicles which serve as platforms for the assembly of APS plasma-derived protein complexes that, in turn, activate complement. In addition, these findings may provide the basis for a mechanistic biomarker for disease activity in APS, by detecting complement activation at the site of injury.* P<0.05 vs Controls. # P<0.05 vs APS [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The profile of adsorbed plasma and serum proteins on methacrylic acid copolymer beads: Effect on complement activation.

Polymer beads made of 45% methacrylic acid co methyl methacrylate (MAA beads) promote vascular regenerative responses in contrast to control materials without methacrylic acid (here polymethyl methacrylate beads, PMMA). Invitro and invivo studies suggest that MAA copolymers induce differences in macrophage phenotype and polarization and inflammatory responses, presumably due to protein adsorption differences between the beads. To explore differences in protein adsorption in an unbiased manner, we used high resolution shotgun mass spectrometry to identify and compare proteins that adsorb from human plasma or serum onto MAA and PMMA beads. From plasma, MAA beads adsorbed many complement proteins, such as C1q, C4-related proteins and the complement inhibitor factor H, while PMMA adsorbed proteins, such as albumin, C3 and apolipoproteins. Because of the differences in complement protein adsorption, follow-up studies focused on using ELISA to assess complement activation. When incubated in serum, MAA beads generated significantly lower levels of soluble C5b9 and C3a/C3adesarg in comparison to PMMA beads, indicating a decrease in complement activation with MAA beads. The differences in adsorbed protein on the two materials likely alter subsequent cell-material interactions that ultimately result in different host responses and local vascularization.

The dysfunction of platelets in paroxysmal nocturnal hemoglobinuria

IntroductionThrombosis is a dangerous complication of paroxysmal nocturnal hemoglobinuria (PNH) and has a high mortality rate. However, the mechanism underlying the development of thrombosis in PNH remains unclear. To explore this, platelet function and serum complement activity were investigated in 14 patients with classical PNH, 11 with PNH aplastic anemia (AA) and 30 healthy controls. Material and methodsSerum concentrations of the terminal complement complex (sC5b-9) were determined by enzyme-linked immunofluorescence assay (ELISA), and the levels of C5b-9, CD61 and CD62p on platelet membranes were determined by flow cytometry. Clinical parameters were assessed, including D-dimer and platelet aggregation induced by adenosine diphosphate (ADP) and arachidonic acid (ARA). ResultsSerum sC5b-9 concentrations were significantly lower in the PNH/PNH-AA than in the control group (P<0.01). C5b-9 deposition was significantly higher on CD59− platelets than on CD59+ platelets in PNH/PNH-AA patients and healthy controls (P<0.01 for each). D-dimer concentration was significantly higher in PNH/PNH-AA patients – especially those with lactate dehydrogenase (LDH) concentrations>1000U/L – than in controls (P<0.05). CD61 (P<0.05) expression was lower on CD59+ platelets in PNH than in controls and CD5− platelets in PNH. Expression of CD62p (P<0.01) was lower on CD59− and CD59+ platelets (P<0.01) in PNH cases than in controls. Platelet aggregation stimulated by the agonists ADP and ARA in the PNH/PNH-AA patients was significantly lower than that in controls (P<0.05). ConclusionsThe adhesion and aggregation of platelets, especially of CD59+ platelets, were compensatively decreased in PNH/PNH-AA patients without active thrombosis.

Peripheral trafficking of bone marrow-derived stem cells in patients with different types of gastric neoplasms: Significance of complement and interleukins.

49 Background: Increasing evidence suggests that gastric malignancy may be a stem cell disorder. Methods: Using FACS analysis supported by ImageStream technique verification, various populations of bone marrow-derived stem cells (BMSCs), such as Lin-CD45-CD133+CXCR4+ very small embryonic/epiblast-like stem cells (VSELs), CD45-STRO-1+CD105+ mesenchymal stem cells (MSCs), Lin-CD45+CD133+ hematopoietic stem/progenitor cells (HSPCs), and CD34 +/ KDR +/ CD31 +/ CD45- endothelial progenitor cells (EPCs) were enumerated and sorted from peripheral blood samples of patients with gastric cancer (n = 23) and other gastric tumors (GISTs, NENs, lymphomas; n = 15) and control patients (n = 15). RT-qPCR analysis for the expression of cancerous, embryonic, and tissue trans-differentiation markers (Oct-4, Nanog, TFF-1, TFF-2, Lgr-5, BMI-1) in circulating VSELs was employed. The plasma levels of stromal-derived factor-1 (SDF-1), complement-derived anaphylatoxins/molecules (C3a, C5a, C5b-9/MAC), and interleukins (IL-6, IL-8, IL-17 and IL-23) were measured. Results: Significantly lower numbers of circulating HSPCs, and intensified peripheral trafficking of both VSELs and MSCs was observed in patients with gastric cancer, but not in those with other types of gastric tumors. In gastric cancer patients, the circulating VSELs presented higher gene expression of Oct-4, Nanog, Lgr-5, BMI-1, and TFF-1/2. The abnormal balance in the peripheral trafficking of BMSCs in patients with gastric cancer was not associated with the clinical stage of the disease or with the systemic levels of stromal-derived factor-1 (SDF-1), but correlated with the levels of C5b-9/MAC, IL-6, IL-8 and IL-23. Conclusions: Abnormal peripheral trafficking of BMSCs occurs in patients with gastric cancer, but not in those with other types of gastric neoplasms. Selected interleukins and complement cascade-derived molecules, but not SDF-1, seem to be significant players associated with this phenomenon (POIG.01.01.02-00-109/09 and IP2014003273).