Terminal Complement Complex Levels Research Articles

Increased plasma level of terminal complement complex in AMD patients: potential functional consequences for RPE cells.

Polymorphisms in complement genes are risk-associated for age-related macular degeneration (AMD). Functional analysis revealed a common deficiency to control the alternative complement pathway by risk-associated gene polymorphisms. Thus, we investigated the levels of terminal complement complex (TCC) in the plasma of wet AMD patients with defined genotypes and the impact of the complement activation of their plasma on second-messenger signaling, gene expression, and cytokine/chemokine secretion in retinal pigment epithelium (RPE) cells. Collection of plasma from patients with wet AMD (n = 87: 62% female and 38% male; median age 77 years) and controls (n = 86: 39% female and 61% male; median age 58 years), grouped for risk factor smoking and genetic risk alleles CFH 402HH and ARMS2 rs3750846, determination of TCC levels in the plasma, in vitro analysis on RPE function during exposure to patients' or control plasma as a complement source. Genotyping, measurement of TCC concentrations, ARPE-19 cell culture, Ca2+ imaging, gene expression by qPCR, secretion by multiplex bead analysis of cell culture supernatants. TCC concentration in plasma, intracellular free Ca2+, relative mRNA levels, cytokine secretion. TCC levels in the plasma of AMD patients were five times higher than in non-AMD controls but did not differ in plasma from carriers of the two risk alleles. Complement-evoked Ca2+ elevations in RPE cells differed between patients and controls with a significant correlation between TCC levels and peak amplitudes. Comparing the Ca2+ signals, only between the plasma of smokers and non-smokers, as well as heterozygous (CFH 402YH) and CFH 402HH patients, revealed differences in the late phase. Pre-stimulation with complement patients' plasma led to sensitization for complement reactions by RPE cells. Gene expression for surface molecules protective against TCC and pro-inflammatory cytokines increased after exposure to patients' plasma. Patients' plasma stimulated the secretion of pro-inflammatory cytokines in the RPE. TCC levels were higher in AMD patients but did not depend on genetic risk factors. The Ca2+ responses to patients' plasma as second-messenger represent a shift of RPE cells to a pro-inflammatory phenotype and protection against TCC. We conclude a substantial role of high TCC plasma levels in AMD pathology.

Common complement factor H polymorphisms are linked with periodontitis in elderly patients.

In our recent genome-wide association study, we found that genetic polymorphisms in the complement factor H (CFH) gene and S100A gene region are strongly associated with serum matrix metalloproteinase 8 (MMP-8) concentration and the release of MMP-8 from neutrophils. As MMP-8 is centrally involved in the pathogenesis of periodontitis, we aimed to evaluate the presence of genetic polymorphisms of S100A8/A9/A12, MMP8, and CFH in periodontitis. In addition, we studied whether polymorphisms of these genes affect the concentrations of S100A8, S100A12, MMP-8, or complement activation marker in saliva. We genotyped four single-nucleotide polymorphisms (SNPs, rs1560833 in S100A8/A9/A12, rs11225395 in MMP8, rs800292 in CFH, and rs1061170 in CFH) and measured salivary concentrations of S100A8, S100A12, MMP-8, and terminal complement complex (TCC) in the Parogene cohort (n = 508). The cohort was composed of patients with an indication to coronary angiography and all underwent a clinical and radiographic oral examination. CFH polymorphisms rs800292 and rs1061170 were associated with periodontal parameters. None of the polymorphisms showed association with salivary proteins. However, salivary concentrations of S100A8, S100A12, MMP-8, and TCC were strongly associated with the number of periodontal pockets and alveolar bone loss. Interestingly, genetic variants of CFH, MMP8, and S100A8/A9/A12 gene regions did not affect salivary levels of measured proteins. However, saliva levels of S100A8, S100A12, MMP-8, and TCC, and CFH polymorphisms were associated with clinical and radiographic signs of periodontitis. Our study further supports the observations that any dysregulation of complement may increase the risk of inflammatory disorders, such as periodontitis.

Antirheumatic therapy is associated with reduced complement activation in rheumatoid arthritis.

BackgroundThe complement system plays an important role in pathophysiology of cardiovascular disease (CVD), and might be involved in accelerated atherogenesis in rheumatoid arthritis (RA). The role of complement activation in response to treatment, and in development of premature CVD in RA, is limited. Therefore, we examined the effects of methotrexate (MTX) and tumor necrosis factor inhibitors (TNFi) on complement activation using soluble terminal complement complex (TCC) levels in RA; and assessed associations between TCC and inflammatory and cardiovascular biomarkers.MethodsWe assessed 64 RA patients starting with MTX monotherapy (n = 34) or TNFi with or without MTX co-medication (TNFi±MTX, n = 30). ELISA was used to measure TCC in EDTA plasma. The patients were examined at baseline, after 6 weeks and 6 months of treatment.ResultsMedian TCC was 1.10 CAU/mL, and 57 (89%) patients had TCC above the estimated upper reference limit (<0.70). Compared to baseline, TCC levels were significantly lower at 6-week visit (0.85 CAU/mL, p<0.0001), without significant differences between the two treatment regimens. Notably, sustained reduction in TCC was only achieved after 6 months on TNFi±MTX (0.80 CAU/mL, p = 0.006). Reductions in TCC after treatment were related to decreased C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and interleukin 6, and increased levels of total, high and low-density lipoprotein cholesterol. Similarly, baseline TCC was significantly related to baseline CRP, ESR and interleukin 6. Patients with endothelial dysfunction had higher baseline TCC than those without (median 1.4 versus 1.0 CAU/mL, p = 0.023).ConclusionsPatients with active RA had elevated TCC, indicating increased complement activation. TCC decreased with antirheumatic treatment already after 6 weeks. However, only treatment with TNFi±MTX led to sustained reduction in TCC during the 6-month follow-up period. RA patients with endothelial dysfunction had higher baseline TCC compared to those without, possibly reflecting involvement of complement in the atherosclerotic process in RA.

Complement Activation Profiles in Patients with Juvenile Idiopathic Arthritis

Objectives: Besides T- and B-cells also the innate immunity is playing an effective role in the pathogenesis of Juvenile idiopathic arthritis (JIA) reported in literature. The complement system (CS) is activated by the three different ways classical pathway (CP), lectin pathway (LP) and alternative pathway (AP). This is a controlled prospective observational single center study focused on the three pathways of CS and its effector, the terminal complement complex (TCC), associated with disease activity in all clinical JIA subgroups. Methods: Peripheral blood (PB) samples (n=159) of 60 JIA patients (partially also longitudinal visits) were analyzed for specific complement pathway activation (COMPL300 ELISA), complement factor H (CFH)-autoantibodies (CFHAb ELISA), and the soluble TCC (sC5b-9 ELISA) in serum (S) and EDTA plasma (P). TCC was also analyzed in synovial fluid (SF) of JIA patients (n=9) controlled with samples of healthy adults (n=12) obtained by arthroscopy (primarily gonarthrosis). As healthy controls (HC) adults (n=100) and children (n=18) without inflammatory diseases were tested. The JADAS10 defined the acute phase of the disease. Results: We compared the elevated serum TCC levels of JIA patients in the acute phase of the disease (13.32 IQR 8.09-19.48; p&lt;0,001) with the controls' levels (7.78 IQR 4.93-10.31). Subgroup analyses showed statistically significant (s.s.) elevation in PArf+, OAper, and OAext. COMPL300 analyses revealed that JIA patients with acute disease had lower CP (82% IQR 37-97% vs 105% IQR 98-115%; p&lt;0.001), LP (31% IQR 2-86% vs 80% IQR 20-101%; p&lt;0.001), and AP (25% IQR 2-88% vs 85% IQR 70-98%; p&lt;0.001) capacity compared to the HC average due to chronic activation in all three pathways. Both TCC and COMPL300 analyses during acute phase and remission showed a clear tendency, especially in PArf+ but only s.s. in the individual course. Remarkable sJIA patients neither in TCC nor in COMPL300 analyses showed complement activation. No evidence of CFH-autoantibodies was found in our study group. The TCC levels in SF showed a median of 2.48 IQR 1.71-2.76 AU/ml in JIA patients and surprisingly high levels in HC with 6.7 IQR 3.9-9.24 AU/ml. Conclusion: Specific subgroups of JIA patients (extOA, PARf+, ERA) showed increased CS activation in TCC in PB in the acute course of the disease. Furthermore, the decreased CS capacity, especially in the CP and AP, confirms the consumption of complement components as an additional contributor to the disease pathogenesis and acute course. Due to the CS activation throughout all disease activity states, we could not determine s.s. between the acute and remission phases in our cohort. However, COMPL300 in combination with TCC seems to be a helpful acute disease biomarker in the individual course. The pharmacological modulation of CS components might be a valid alternative approach in treatmentresistant patients, although further investigations are needed.

Increase in the Complement Activation Product C4d and the Terminal Complement Complex sC5b-9 Is Associated with Disease Severity and a Fatal Outcome in Necrotizing Soft-Tissue Infection

The hyperinflammatory burden is immense in necrotizing soft-tissue infection (NSTI). The complement system is a key during the innate immune response and may be a promising target to reduce the inflammatory response, potentially improving the clinical outcome. However, complement activation and its association to disease severity and survival remain unknown in NSTI. Therefore, we prospectively enrolled patients with NSTI and sampled blood at admission and once daily for the following 3 days. Plasma C4c, C4d, C3bc, and C3dg and the terminal complement complex (TCC) were evaluated using ELISA techniques. In total, 242 patients were included with a median age of 62 years, with a 60% male predominance. All-cause 30-day mortality was 17% (95% confidence interval [CI] 13–23) with a follow-up of >98%. C4c and C3dg were negatively correlated with Simplified Acute Physiology Score II (Rho −0.22, p < 0.001 and Rho −0.17, p = 0.01). Patients with septic shock (n = 114, 47%) had higher levels of baseline TCC than those in non-shock patients (18 vs. 14, p < 0.001). TCC correlated with the Sequential Organ Failure Assessment (SOFA) score (Rho 0.19, p = 0.004). In multivariate Cox regression analysis (adjusted for age, sex, comorbidity, and SOFA score), high baseline C4d (>20 ng/mL) and the combination of high C4d and TCC (>31 arbitrary units/mL) were associated with increased 30-day mortality (hazard ratio [HR] 3.26, 95% CI 1.56–6.81 and HR 5.12, 95% CI 2.15–12.23, respectively). High levels of both C4d and TCC demonstrated a negative predictive value of 0.87. In conclusion, we found that in patients with NSTI, complement activation correlated with the severity of the disease. High baseline C4d and combination of high C4d and TCC are associated with increased 30-day mortality. Low baseline C4d or TCC indicates a higher probability of survival.

Complement activation is associated with neutrophil extracellular traps and all-cause mortality in ST-elevation myocardial infarction

Abstract Background The complement system and neutrophil extracellular traps (NETs) are both parts of the innate immune system, and have been implicated in the ischemia-reperfusion injury in patients with ST-elevation myocardial infarction (STEMI). There is experimental evidence of reciprocal activation between the complement system and NETs. Any such link in patients with STEMI has not been investigated. Purpose To investigate a potential association between complement activation and clinical outcomes after STEMI, and assess any interplay between complement activation and NETs in this situation. Methods Patients with ST-elevation myocardial infarction were included at a median of 18 hours after percutaneous coronary intervention (n=864). The terminal complement complex (TCC) was measured by ELISA as a marker of complement activation. As markers of NETs were myeloperoxidase-deoxynucleic acid (MPO-DNA) and citrullinated histone 3 (CitH3) measured by ELISAs, while double stranded DNA (dsDNA) was measured by a nucleic acid stain. Patients were followed for a median of 4.6 years. The primary endpoint was a composite of new myocardial infarction, unscheduled revascularization, stroke, hospitalization for heart failure and death, whichever occurred first. All-cause mortality was also recorded. Results The composite endpoint occurred in 184 (21.3%) patients, while 70 (8.1%) died during follow-up. When dichotomizing at median TCC, the group with above-median TCC levels did not have an increased risk of reaching the composite endpoint (hazard ratio (HR): 1.069, 95% CI: [0.801, 1.428], p=0.651). However, this group exhibited an increased risk of all-cause mortality (HR: 1.650, 95% CI: [1.020, 2.671], p=0.041). This risk persisted when adjusting for age, sex, hypertension and LDL-cholesterol (HR: 1.673, 95% CI: [1.014, 2.761], p=0.044), but the significance was lost when adjusting for NT-proBNP (HR: 1.492, 95% CI: [0.885, 2.515], p=0.133). TCC was correlated to dsDNA (r=0.127, p&amp;lt;0.001) and CitH3 (r=0.102, p=0.003), but not MPO-DNA. The group with both TCC and dsDNA in the highest quartile exhibited a significantly higher incidence of all-cause mortality than the remaining population (17.6% vs, 7.2%, p=0.002). When examining the predictive value of TCC and dsDNA on all-cause mortality in ROC curve analysis, the area under the curve (AUC) for TCC was 0.549 (95% CI: [0.472, 0.625]), while the AUC for dsDNA was 0.653 (95% CI: [0.584, 0.722]). When combining TCC and dsDNA the predictive value was marginally higher than for TCC alone (AUC: 0.649, 95% CI: [0.579, 0.720]) Conclusion In this STEMI population, complement activation measured by TCC was not associated with the primary composite endpoint, but was associated with increased risk of death. TCC was weakly correlated with markers of NETs. Despite a high mortality rate in patients with high levels of TCC and dsDNA, combining these variables did not increase the prognostic value compared to TCC alone. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Stein Erik Hagen's Foundation for Clinical Heart Research Survival according to cox regression

Complement Activation in Association with Markers of Neutrophil Extracellular Traps and Acute Myocardial Infarction in Stable Coronary Artery Disease.

Complement activation and neutrophil extracellular traps (NETs) have both been suggested to drive atherosclerotic plaque progression. Although experimental studies suggest interplay between these two innate immunity components, the relevance in patients with coronary artery disease (CAD) is unclear. The aim of this study was to assess associations between complement activation and NETs in patients with stable CAD and examine the role of complement activation on clinical outcome. Blood samples from a cohort of patients with angiographically verified stable CAD (n = 1001) were analyzed by ELISA for the terminal complement complex (TCC) and by relative quantification for gene expression of the C5a receptor 1 (C5aR1) as markers of complement activation. As markers of NETs, dsDNA was analyzed by fluorescent nucleic acid stain and myeloperoxidase-DNA (MPO-DNA) by ELISA. Clinical outcome was defined as unstable angina, nonhemorrhagic stroke, acute myocardial infarction (MI), or death (n = 106, whereof 36 MI). Levels of TCC and C5aR1 were not significantly correlated to dsDNA (TCC: r = −0.045, p = 0.153; C5aR1: r = −0.060, p = 0.434) or MPO-DNA (TCC: r = 0.026, p = 0.414; C5aR1: r = 0.123, p = 0.107). When dividing TCC and C5aR1 levels into quartiles (Q), levels of MPO-DNA differed significantly across quartiles (TCC: p = 0.008, C5aR1: 0.049), while dsDNA did not (TCC: p = 0.181, C5aR1: p = 0.771). Patients with TCC levels in Q4 had significantly higher levels of MPO-DNA than Q1-3 (p = 0.019), and C5aR1 levels in Q3-4 had significantly higher levels of MPO-DNA than Q1-2 (p = 0.046). TCC levels did not differ between patients experiencing a clinical endpoint or not, but high levels were associated with increased risk of acute MI (OR. 1.97, 95% CI: 0.99-3.90, p = 0.053) during two-year follow up, also when adjusted for relevant covariates. In conclusion, TCC and C5aR1 were moderately associated with the NET marker MPO-DNA, and TCC levels were related to the risk of future MI in this cohort of patients with stable CAD.

Increased Complement Factor B and Bb Levels Are Associated with Mortality in Patients with Severe Aortic Stenosis

Inflammation is involved in initiation and progression of aortic stenosis (AS). However, the role of the complement system, a crucial component of innate immunity in AS, is unclear. We hypothesized that circulating levels of complement factor B (FB), an important component of the alternative pathway, are upregulated and could predict outcome in patients with severe symptomatic AS. Therefore, plasma levels of FB, Bb, and terminal complement complex were analyzed in three cohorts of patients with severe symptomatic AS and mild-to-moderate or severe asymptomatic AS (population 1, n = 123; population 2, n = 436; population 3, n = 61) and in healthy controls by enzyme immunoassays. Compared with controls, symptomatic AS patients had significantly elevated levels of FB (2.9- and 2.8-fold increase in population 1 and 2, respectively). FB levels in symptomatic and asymptomatic AS patients were comparable (population 2 and 3), and in asymptomatic patients FB correlated inversely with valve area. FB levels in population 1 and 2 correlated with terminal complement complex levels and measures of systemic inflammation (i.e., CRP), cardiac function (i.e., NT-proBNP), and cardiac necrosis (i.e., Troponin T). High FB levels were significantly associated with mortality also after adjusting for clinical and biochemical covariates (hazard ratio 1.37; p = 0.028, population 2). Plasma levels of the Bb fragment showed a similar pattern in relation to mortality. We concluded that elevated levels of FB and Bb are associated with adverse outcome in patients with symptomatic AS. Increased levels of FB in asymptomatic patients suggest the involvement of FB from the early phase of the disease.

Cytokine responses, microbial aetiology and short-term outcome in community-acquired pneumonia.

BackgroundThe inflammatory response to community‐acquired pneumonia (CAP) is orchestrated through activation of cytokine networks and the complement system. We examined the association of multiple cytokines and the terminal complement complex (TCC) with microbial aetiology, disease severity and short‐term outcome.Materials and methodsPlasma levels of 27 cytokines and TCC were analysed in blood samples obtained at hospital admission, clinical stabilization and 6‐week follow‐up from 247 hospitalized adults with CAP. Fourteen mediators were included in final analyses. Adverse short‐term outcome was defined as intensive care unit (ICU) admission and 30‐day mortality.ResultsCytokine and TCC levels were dynamic in the clinical course of CAP, with highest levels seen at admission for most mediators. Admission levels of cytokines and TCC did not differ between groups of microbial aetiology. High admission levels of IL‐6 (odds ratio [OR] 1.47, 95% confidence interval [CI] 1.18‐1.84, P = .001), IL‐8 (OR 1.79, 95% CI 1.26‐2.55, P = .001) and MIP‐1β (OR 2.28, 95% CI 1.36‐3.81, P = .002) were associated with a CURB‐65 severity score of ≥3, while IL‐6 (OR 1.37, 95% CI 1.07‐1.74, P = .011) and MIP‐1β (OR 1.86, 95% CI 1.03‐3.36, P = .040) were associated with a high risk of an adverse short‐term outcome.ConclusionsIn this CAP cohort, admission levels of IL‐6, IL‐8 and MIP‐1β were associated with disease severity and/or adverse short‐term outcome. Still, for most mediators, only nonsignificant variations in inflammatory responses were observed for groups of microbial aetiology, disease severity and short‐term outcome.

Complement factors C4 and C3 are down regulated in response to short term overfeeding in healthy young men

Insulin resistance is associated with high circulating level of complement factor C3. Animal studies suggest that improper complement activation mediates high-fat-diet-induced insulin resistance. Individuals born with low birth weight (LBW) are at increased risk of developing insulin resistance. We hypothesized that high-fat overfeeding (HFO) increase circulating C3 and induce complement activation in a birth weight differential manner. Twenty LBW and 26 normal birth weight (NBW) young men were studied using a randomised crossover design. Insulin resistance was measured after a control-diet and after 5-days HFO by a hyperinsulinemic-euglycemic-clamp. Circulating C4, C3, ficolins, mannose-binding-lectin, complement activation products C3bc, terminal complement complex (TCC) and complement activation capacity were determined using turbidimetry and ELISA. HFO induced peripheral insulin resistance in LBW individuals only, while both groups had the same degree of hepatic insulin resistance after HFO. Viewing all individuals circulating levels of C4, C3, C3bc, TCC and complement activation capacity decreased paradoxically along the development of insulin resistance after HFO (P = 0.0015, P < 0.0001, P = 0.01, P < 0.0001, P = 0.0002, P < 0.0001, P = 0.0006). Birth weight did not influence these results. This might reflect a hitherto unrecognized down-regulatory mechanism of the complement system. More human studies are needed to understand the underlying physiology and the potential consequences of these findings.

Complement factors C4 and C3 are down regulated in response to short term overfeeding in healthy young men

Insulin resistance is associated with high circulating level of complement factor C3. Animal studies suggest that improper complement activation mediates high-fat-diet-induced insulin resistance. Individuals born with low birth weight (LBW) are at increased risk of developing insulin resistance. We hypothesized that high-fat overfeeding (HFO) increase circulating C3 and induce complement activation in a birth weight differential manner. Twenty LBW and 26 normal birth weight (NBW) young men were studied using a randomised crossover design. Insulin resistance was measured after a control-diet and after 5-days HFO by a hyperinsulinemic-euglycemic-clamp. Circulating C4, C3, ficolins, mannose-binding-lectin, complement activation products C3bc, terminal complement complex (TCC) and complement activation capacity were determined using turbidimetry and ELISA. HFO induced peripheral insulin resistance in LBW individuals only, while both groups had the same degree of hepatic insulin resistance after HFO. Viewing all individuals circulating levels of C4, C3, C3bc, TCC and complement activation capacity decreased paradoxically along the development of insulin resistance after HFO (P = 0.0015, P < 0.0001, P = 0.01, P < 0.0001, P = 0.0002, P < 0.0001, P = 0.0006). Birth weight did not influence these results. This might reflect a hitherto unrecognized down-regulatory mechanism of the complement system. More human studies are needed to understand the underlying physiology and the potential consequences of these findings.

Complement activation in leprosy: a retrospective study shows elevated circulating terminal complement complex in reactional leprosy.

Mycobacterium leprae infection gives rise to the immunologically and histopathologically classified spectrum of leprosy. At present, several tools for the stratification of patients are based on acquired immunity markers. However, the role of innate immunity, particularly the complement system, is largely unexplored. The present retrospective study was undertaken to explore whether the systemic levels of complement activation components and regulators can stratify leprosy patients, particularly in reference to the reactional state of the disease. Serum samples from two cohorts were analysed. The cohort from Bangladesh included multi-bacillary (MB) patients with (n = 12) or without (n = 46) reaction (R) at intake and endemic controls (n = 20). The cohort from Ethiopia included pauci-bacillary (PB) (n = 7) and MB (n = 23) patients without reaction and MB (n = 15) patients with reaction. The results showed that the activation products terminal complement complex (TCC) (P≤0·01), C4d (P≤0·05) and iC3b (P≤0·05) were specifically elevated in Bangladeshi patients with reaction at intake compared to endemic controls. In addition, levels of the regulator clusterin (P≤0·001 without R; P<0·05 with R) were also elevated in MB patients, irrespective of a reaction. Similar analysis of the Ethiopian cohort confirmed that, irrespective of a reaction, serum TCC levels were increased significantly in patients with reactions compared to patients without reactions (P≤0·05). Our findings suggests that serum TCC levels may prove to be a valuable tool in diagnosing patients at risk of developing reactions.

Assessment of Proteins Associated With Complement Activation and Inflammation in Maculae of Human Donors Homozygous Risk at Chromosome 1 CFH-to-F13B.

To determine the effects of chromosome 1 genotype and cigarette smoking on levels of complement activation and inflammation in the human macula. Donor macular tissue was stratified into three groups by diplotype at the AMD-associated CFH-to-F13B locus: homozygous "risk" (n = 9, 56-78 years), homozygous neutral (n = 2, 64-79 years), and homozygous "protective" (n = 6, 61-78 years) diplotype. Importantly, all donors were homozygous nonrisk at the ARMS2/HTRA1 locus, so that purely chromosome 1-directed pathways were examined. Immunohistochemistry was performed by using 14 antibodies, mostly against markers of complement and inflammation, followed by confocal microscopy and immunofluorescence quantification (all masked to donor status). Donors homozygous risk at CFH-to-F13B exhibited significantly higher levels of terminal complement complex (TCC) in macular Bruch's membrane (BM; P = 0.03), choriocapillaris (CC; P = 0.04), and choriocapillaris intercapillary septa (CC IS; P = 0.03), compared to homozygous protected donors. Smoking was associated with increased TCC in BM (P = 0.05), CC IS (P = 0.03), and choroidal stroma (CS; P = 0.01), and with substantially elevated C-reactive protein (CRP) levels in RPE (P = 0.04), BM (P = 0.01), CC (P = 0.05), and CS (P = 0.05). Smoking was associated with higher levels of oxidative stress in macular RPE (P = 0.04) and CS (P = 0.01). Genetic risk at the CFH-to-F13B locus was associated with higher levels of complement activation at the human macular RPE-choroid interface, as was cigarette smoking. Levels of CRP were substantially elevated in risk donors with smoking history. Examination of human macular tissue from donors with "pure" diplotypes allows assessment of AMD-associated pathways driven solely by CFH-to-F13B. These findings have important implications for identifying chromosome 1-directed pathways and therapeutic targets.

Study on C5b-9 deposited on the membrane of platelets and its dysfunction in patients with paroxysmal nocturnal hemoglobinuria

目的通过检测阵发性睡眠性血红蛋白尿症（PNH）伴或不伴再生障碍性贫血（AA）患者血小板膜补体复合物（C5b-9）、血小板活化分子（CD62p）表达及血清可溶性C5b-9(sC5b-9）水平探究PNH血栓形成的病理机制。方法用ELISA方法检测25例PNH/PNH-AA患者血清sC5b-9、补体C3和C4水平，并以30名健康志愿者作为正常对照；采用流式细胞术检测PNH/PNH-AA患者与正常人血小板PNH克隆数（CD59−CD61+/CD61+）、血小板膜C5b-9沉积率（C5b-9+CD61+/CD61+）以及血小板活化标志分子CD62p表达率（CD62p+CD61+/CD61+），并进行相关性分析。结果①PNH/PNH-AA组患者血清sC5b-9水平为390.27（265.73~676.87）µg/L，显著低于正常对照组的540.39（344.20~1 576.78）µg/L（P<0.01）。②PNH/PNH-AA组患者血小板PNH克隆数[50.58（23.29~81.60）%]显著高于正常对照组[23.57（15.58~29.02）%]（P<0.01）；PNH/PNH-AA组患者PNH克隆血小板膜C5b-9沉积率[（17.53± 6.27）%]与患者正常血小板[（11.33±5.03）%]及正常对照组血小板[（10.88±3.58）%]相比均显著增高（P<0.01）。③PNH/PNH-AA组患者PNH克隆血小板CD62p表达率[（61.98±11.71）%]与患者正常血小板[（43.76±11.30）%]及正常对照组血小板[（38.23±8.07）%]相比均显著升高（P<0.01）；PNH/PNH-AA组患者正常血小板膜CD62p表达率比正常对照组血小板显著增高（P<0.05）。④血小板膜C5b-9沉积率与CD62p表达率呈显著正相关（r=0.449，P=0.002）。结论PNH/PNH-AA患者血小板锚连蛋白（CD59）缺失导致补体复合物C5b-9沉积于异常血小板膜并使其激活，这一过程可能参与PNH血栓的形成。

Complement activation, placental malaria infection, and birth weight in areas characterized by unstable malaria transmission in central Sudan.

BackgroundThe pathogenesis of malaria during pregnancy is not completely understood. There are few published data on complement activation and malaria during pregnancy. This study aimed to investigate complement activation and malaria during pregnancy, and their association with hemoglobin and birth weight.MethodsA cross-sectional study was conducted at Medani, Sudan. Soluble terminal complement complex (TCC) levels were measured using ELISA in maternal and cord blood samples from 126 parturient women.ResultsThere were no Plasmodium falciparum-positive blood films from maternal peripheral blood, the placenta, or cord blood samples. Three (2.4%) and 22 (17.5%) of the placentas showed chronic and previous infection with histopathological examination, respectively, while 101 (80.2%) of them had no malaria infection. The mean [SD] of the maternal (22.4 [6.1] vs. 26.5 [3.5] ng/ml, P < 0.001) and cord blood (24.5 [4.5] vs. 26.8 [4.4] ng/ml, P = 0.024) TCC levels were significantly lower in cases of placental malaria infection (n = 25) than in those without placental malaria infection (n = 101). Linear regression showed that placental malaria infection was significantly associated with birth weight (−0.353 g, P = 0.013), but there were no associations between maternal and cord TCC levels and maternal hemoglobin, or between TCC levels and birth weight.ConclusionMaternal and cord blood TCC levels are lower in women with placental malaria infection than in those without placental malaria infection.Virtual SlideThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9600054761463915

Blood levels of histone-complexed DNA fragments are associated with coagulopathy, inflammation and endothelial damage early after trauma

Background:Tissue injury increases blood levels of extracellular histones and nucleic acids, and these may influence hemostasis, promote inflammation and damage the endothelium. Trauma-induced coagulopathy (TIC) may result from an endogenous response to the injury that involves the neurohumoral, inflammatory and hemostatic systems.Aims:To study the contribution of extracellular nucleic constituents to TIC, inflammation and endothelial damage.Setting and Design:Prospective observational study.Materials and Methods:We investigated histone-complexed DNA fragments (hcDNA) along with biomarkers of coagulopathy, inflammation and endothelial damage in plasma from 80 trauma patients admitted directly to the Trauma Centre from the scene of the accident. Blood was sampled a median of 68 min (IQR 48-88) post injury. Trauma patients with hcDNA levels >median or ≤median were compared.Results:Trauma patients with high plasma hcDNA had higher Injury Severity Score (ISS) and level of sympathoadrenal activation (higher adrenaline and noradrenaline) and a higher proportion of prolonged activated partial thromboplastin time (APTT) and higher D-dimer, tissue-type plasminogen activator (tPA), Annexin V and soluble CD40 ligand (sCD40L) concurrent with lower plasminogen activator inhibitor (PAI)-1) and prothrombin fragment (PF) 1 + 2 (all P < 0.05), all indicative of impaired thrombin generation, hyperfibrinolysis and platelet activation. Furthermore, patients with high hcDNA had enhanced inflammation and endothelial damage evidenced by higher plasma levels of terminal complement complex (sC5b-9), IL-6, syndecan-1, thrombomodulin and tissue factor pathway inhibitor (all P < 0.05).Conclusions:Excessive release of extracellular histones and nucleic acids seems to contribute to the hypocoagulability, inflammation and endothelial damage observed early after trauma.

Complement activation in primiparous women from a malaria endemic area is associated with reduced birthweight

The hallmark of placental malaria (PM) due to Plasmodium falciparum infection is the accumulation of mature-stage parasites, monocytes and macrophages in the maternal vascular bed of the placenta. The mechanisms leading to morbidity and mortality in PM are incompletely understood. However, an inflammatory response in the placenta has been related to both severe anemia in the mother and low birthweight (<2500 g) in the newborn. In this study we analyzed whether complement activation as a mediator of inflammation could contribute to poor pregnancy outcome in PM. The concentrations of the soluble terminal complement complex (TCC) were measured as an indicator of complement activation in placental, cord and peripheral blood samples from 146 women from a malaria endemic area. Placental and cord plasma samples of primiparous women, a group vulnerable to PM, showed significantly higher levels of TCC than multiparous women. Additionally, in women with malaria history during pregnancy or placental infection by P. falciparum at delivery, the TCC levels in the corresponding placental and cord plasma samples were significantly higher than in the malaria negative group. In multiple regression analysis parity was shown to be the main determinant of TCC levels. Placental plasma samples corresponding to babies weighing less than 2700 g had significantly higher levels of TCC than babies carrying more weight. In conclusion, both primiparity and P. falciparum infection were related to a local increase of complement activation in the placentas. Association between reduced birthweight and higher levels of TCC in placental blood suggests a role for complement activation in influencing the pregnancy outcome in malaria exposed women.

Local and systemic activation of the whole complement cascade in human leukocytoclastic cutaneous vasculitis; C3d,g and terminal complement complex as sensitive markers.

We have studied complement activation both in plasma samples and in lesional skin from patients with leukocytoclastic cutaneous vasculitis (LCV). Enzyme immunoassay (EIA) quantification of the complement activation markers, C3d,g and the terminal complement complex (TCC) in plasma, showed that their levels were significantly increased in 66% and 55% of the patients, respectively (n = 29) compared with healthy controls, whereas the standard measurements of C3, factor B, C1q, C4 and C2 were generally within normal range. Elevations of C3d,g and TCC levels in plasma were significantly correlated. Importantly, a significant correlation was found between the severity of the vasculitis and both C3d,g and TCC plasma levels. Immunofluorescence studies of skin biopsy specimens demonstrated simultaneous presence of perivascular dermal deposits of C3d,g and TCC in lesional skin from 96% and 80% respectively of the patients (n = 25). There was a significant correlation between the intensity of the deposits of both markers. Clusterin, a TCC inhibitory protein, was always found at the same sites of perivascular TCC deposits. Immunofluorescence studies at the epidermal basement membrane zone (BMZ) revealed in each case deposits of C3d,g which were accompanied by TCC deposits in 52% of the biopsy specimens. These data demonstrate that there is a local and systemic activation of the whole complement cascade in human LCV. The presence of both C3d,g and clusterin-associated TCC perivascular deposits suggests an intervention of a regulatory mechanism of local complement activation in LCV. Finally, measurement of plasma C3d,g and TCC appears to be a sensitive indicator of systemic complement activation and disease severity in LCV.

Terminal complement complex in septic shock with capillary leakage

The aim of this study was to evaluate the value of terminal complement complex (C5b-9) plasma levels as a marker for complement activation in septic shock with concomitant capillary leak syndrome. In a prospective animal study 10 fasted, anaesthetized, mechanically ventilated and multi-catheterized pigs (20.6 +/- 1.3 kg) were investigated over a period of 8 h. Sepsis was induced by faecal peritonitis (1 g kg(-1) body weight faeces, n = 5) and compared to controls (n = 5). The animals received 6% hydroxyethyl starch 200/0.5 to maintain a central venous pressure of 12 mmHg. To quantify capillary leak syndrome, albumin escape rate was measured using 99mTc-labelled human serum albumin. Plasma levels of terminal complement complex were measured in a double antibody immunoassay (neoepitope-specific MoAb aE 11 as catching antibody). Immunohistological studies of renal specimens were performed to detect terminal complement complex deposition. Albumen escape rate increased in septic animals (+ 52%) compared to controls (+ 3%, P < 0.05). Plasma levels of terminal complement complex decreased during the study period in both groups. In septic animals this finding was accompanied by a significant deposition of terminal complement complex in renal specimens (P < 0.05). We found an activation of the complement system proven by marked deposition of terminal complement complex in renal specimen, while its plasma levels decreased during the study period in septic and control animals. These results suggest that in septic shock with capillary leak syndrome plasma level of terminal complement complex may not be a reliable marker of complement activation.

Rapid activation of the complement system by cuprophane depends on complement component C4

Rapid activation of the complement system by cuprophane depends on complement component C4