Serum sC5b-9 Research Articles

MRNA-LNPs induce immune activation and cytokine release in human whole blood assays across diverse health conditions.

RNA medicines have become a promising platform for therapeutic use in recent years. Understanding the immunomodulatory effects of novel mRNA-LNPs is crucial for future therapeutic development. An in vitro whole blood assay was developed to assess the impact of mRNA-LNPs on immune cell function, cytokine release, and complement activation. mRNA-LNPs significantly increased CD69 expression on T cells and natural killer (NK) cells, and CD80/CD86 on myeloid subsets, in a dose-dependent fashion. Furthermore, mRNA-LNPs elicited a robust release of pro-inflammatory cytokines, including TNF-α, IL-1β, MCP-1, IL-6, and IP-10, indicating a potent immune response. Notably, mRNA-LNPs stimulate early cytokine production prior to triggering immune cell activation, suggesting a temporal and biological relationship. Moreover, mRNA-LNPs induce complement activation via the alternative pathway, as evidenced by increased serum sC5b-9, C3a, and Bb which can amplify the inflammatory response and potentially impact safety. In vitro effects of mRNA-LNPs in whole blood of healthy human donors were compared to those from disease cohorts including systemic lupus erythematosus (SLE), type 2 diabetes mellitus (T2DM), and cancer donors. The differences in mRNA-LNPs effects on samples from healthy and diseased populations may impact therapeutic efficacy or toxicity, indicating a need for tailoring LNPs for specific target populations.

Predictive value of soluble CD59 for poor 28-day neurological prognosis and all-cause mortality in patients after cardiopulmonary resuscitation: a prospective observatory study

BackgroundsCD59, as a soluble form of CD59, is observed in multiple types of body fluids and correlated with the cell damage after ischemia/reperfusion injury. This study aims to observe the dynamic changes of serum sCD59 in patients after restoration of spontaneous circulation (ROSC) and explore the association of serum sCD59 with neurological prognosis and all-cause mortality in patients after ROSC.MethodsA total of 68 patients after ROSC were prospectively recruited and divided into survivors (n = 23) and non-survivors (n = 45) groups on the basis of 28-day survival. Twenty healthy volunteers were enrolled as controls. Serum sCD59 and other serum complement components, including sC5b-9, C5a, C3a, C3b, C1q, MBL, Bb, and pro-inflammatory mediators tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), neurological damage biomarkers neuron-specific enolase (NSE) and soluble protein 100β (S100β) were measured by enzyme linked immunosorbent assay on day 1, 3, and 7 after ROSC. Neurologic outcome was assessed using cerebral performance category scores, with poor neurologic outcome defined as 3–5 points.ResultsIn the first week after ROSC, serum levels of sCD59, sC5b-9, C5a, C3a, C3b, C1q, MBL, Bb, TNF-α, IL-6, NSE and S100β were significantly elevated in patients after ROSC compared to healthy volunteers, with a significant elevation in the non-survivors compared to survivors except serum C1q and MBL. Serum sCD59 levels were positively correlated with serum sC5b-9, TNF-α, IL-6, NSE, S100β, SOFA score and APACHE II score. Moreover, serum sCD59 on day 1, 3, and 7 after ROSC could be used for predicting poor 28-day neurological prognosis and all-cause mortality. Serum sCD59 on day 3 had highest AUCs for predicting poor 28-day neurological prognosis [0.862 (95% CI 0.678–0.960)] and 28-day all-cause mortality [0.891 (95% CI 0.769–0.962)]. In multivariate logistic regression analysis, the serum level of sCD59D1 was independently associated with poor 28-day neurological prognosis and all-cause mortality.ConclusionsThe elevated serum level of sCD59 was positively correlated with disease severity after ROSC. Moreover, serum sCD59 could have good predictive values for the poor 28-day neurological prognosis and all-cause mortality in patients after ROSC.

Soluble terminal complement activation fragment sC5b-9: a new serum biomarker for traumatic brain injury?

Terminal complement pathway activation after traumatic brain injury (TBI) leads to formation of the membrane attack complex (MAC/C5b-9) which induces neuronal cell death and host-mediated secondary brain injury. Serum levels of soluble MAC (sC5b-9) have not been previously determined in patients with isolated TBI. A prospective observational cohort study was performed during a 5-year time-period on adult patients with isolated TBI admitted to an academic level I trauma center in the United States. Controls consisted of patients with femur shaft fractures with or without TBI to mitigate the effect of systemic complement activation by peripheral trauma. Healthy volunteers served as internal controls. The sC5b-9 serum concentrations were measured on the day of admission by enzyme-linked immunosorbent assay (ELISA) and compared between the study cohorts. Univariate analysis was performed to determine independent predictive variables of major complications during hospital admission. Serum sC5b-9 levels were significantly elevated in patients with isolated TBI (n = 42), compared to patients with isolated femoral shaft fractures (n = 36) or combined TBI and femoral shaft fractures (n = 30; p < 0.05). There was no significant difference in serum sC5b-9 levels between the femur group and the combined injury group, compared to the healthy volunteers (n = 21). Univariate analysis revealed serum sC5b-9 levels as an independent predictor of major postinjury complications after isolated TBI (p < 0.01). The soluble terminal complement complex sC5b-9 represents a potential novel serum biomarker specific for isolated head injuries, since peripheral trauma did not appear to affect the serum sC5b-9 levels.

Activation of serum complement in Posner-Schlossman syndrome patients

Background Activation of serum complement system is involved in the pathological process of uveitis and open angle glaucoma.Pathogenesis and pathological characteristics of Posner-Schlossman syndrome (PSS) are similar to uveitis and open angle glaucoma.However, etiology of PSS remains unelucidated.The activation complement in PSS patients’ serum is rarely reported. Objective The aim of this study was to investigate the activation of serum complement in PSS patients for PSS pathogenesis. Methods A prospective case-controlled study was designed.The peripheral blood simples of 79 PSS patients were collected from Shenzhen Eye Hospital during December 2013 to December 2015, and the peripheral blood simples were obtained from 83 unrelated healthy blood donors as healthy control group.Immuno-scatter turbidmetry was adopted to detect the common activated components in complement pathway in each group including complement C3 (a vital intersection molecule in the three pathways), C4 (the vital molecule both the complement classical and lectin pathways), split products C3a, soluble membrane attack complex (sC5b-9), C1q (complement classical pathway), L-ficolin (complement lectin pathway), complement factor Bb (complement alternative pathway), IgG, IgA and IgM.The correlation between serum C3a content and sC5b-9 content in PSS group was analyzed.The serum contents of fabric binding protein 2 (FCN2) (a marker of serum classical pathway), factor Bb (a marker of complement alternative pathway), C3a (the common activation products of three complement activation pathways), and sC5b-9 were assayed by ELISA.This research protocal was approved by Shenzhen Eye Hospital and written informed consent was obtained from each PSS patient prior to any medical examination. Results Compared with normal control group, the serum levels of C3, C4, C3a, sC5b-9, C1q, FCN2, IgG, IgA and IgM were significantly higher in PSS group (Z=-4.743, -2.913, -1.985, -2.620, -2.062, -2.500, -7.010, -6.327, -3.652, all at P<0.05). The serum complement factor Bb level was 13.87 (9.24, 32.00)μg/ml in PSS group, which was significantly lower than 20.51 (12.90, 33.50)μg/ml in normal control group (Z=-2.515, P=0.012). Serum C3a content was positively correlated with the serum sC5b-9 content in PSS group (rs=0.832, P<0.001). Conclusions The serum complement system is activated in PSS patients.Complement alternative pathway, classical pathway and lectin pathway might all be involved in the activative process of complement system. Key words: Glaucoma, open-angle; Syndrome; Iridocyclitis; Complement activation/ physiology; Complement pathway, activation /physiology

The Dysfunction of Platelets in Paroxysmal Nocturnal Hemoglobinuria

Introduction Thrombosis is one of the most dangerous complications in PNH, which can cause high mortality. However, its underlying mechanism remains unclear. To explore the mechanism of thrombosis in PNH, the function of platelets and complements were investigated in our study.Material and Methods Serum level of terminal complement complex (sC5b-9) was detected by ELISA. The quantities of C5b-9,CD61 and CD62p on the membrane of platelets were detected by flow cytometry. Clinical tests were collected, including D-Dimer and platelet aggregation rates induced by adenosine diphosphate (ADP) and arachidonic acid (ARA).Results The serum sC5b-9 level was significantly lower in the PNH/PNH-AA group than that in the control group (p<0.01). The deposition of C5b-9 on CD59- platelets (17.53%±6.27%) was higher than those on CD59+ platelets in PNH/PNH-AA patients(11.33%±5.03%) and normal controls(10.88%±3.58%) (p<0.01). D-dimer was significantly higher in PNH/PNH-AA patients (485ng/dL) than that in normal controls (311ng/dL)(p<0.05),especially in patients with LDH>1000U/L(789ng/dL). CD61 and CD62p expression on CD59+ platelets in PNH patients (76.87%,39.99%) were significantly lower than those in normal controls(98.41%,64.21%)(p<0.05,p<0.01).CD62p expression on CD59- platelets (40.07%) was significantly lower than normal controls(p<0.01). Platelet aggregation stimulated by agonists ADP (37.54±24.25)% and ARA (24.60%) were lower in the PNH/PNH-AA group than that in the control group(59.20±23.30)%, (14.54%) (p < 0.05). Interestingly,CD61 expression on CD59+ platelets in PNH patients was higher in the patients with higher type II PNH clone.Conclusions The function of platelets was inversely inhibited,especially CD59+ platelets even if hypercoagulation continuously exists in PNH/PNH-AA. DisclosuresNo relevant conflicts of interest to declare.

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。

Functional assessment of rat complement pathway activities and quantification of soluble C5b-9 in an experimental model of renal ischemia/reperfusion injury

There is a growing interest in the monitoring of complement activation, not only in clinical settings but also in experimental models. However, for rodents only a limited number of tools are available to assess complement activity and activation. Here we describe three ELISAs for the measurement of rat classical (CP), MB-lectin (LP) and alternative (AP) pathway activities in serum and plasma. Moreover, we optimised a soluble C5b-9 (sC5b-9) ELISA for the detection of low level complement activation in rat. We determined the conditions for correct sample handling and showed that the assays had low inter- and intra-assay variation. We applied these assays to monitor complement activation in an experimental rat model of renal ischemia/reperfusion injury. We did not observe major complement consumption following reperfusion in CP or LP, and only minor AP consumption at 24h post reperfusion. However, MBL depletion prior to ischemia/reperfusion using a monoclonal antibody, transiently and specifically inhibited 75% of LP activity and ameliorated the AP consumption at 24h. To further assess complement activation during renal IRI, we monitored serum sC5b-9 and found that it was only significantly increased 72h post-reperfusion, but not when rats were pre-treated with anti-MBL or after sham surgery. In conclusion the described assays enable sensitive, reproducible and comprehensive assessment of complement activation in experimental rat models.

Hypersensitivity reaction studies of a polyethoxylated castor oil-free, liposome-based alternative paclitaxel formulation

The commercial drug paclitaxel (Taxol) may introduce hypersensitivity reactions associated with the polyethoxylated castor oil-ethanol solvent. To overcome these problems, we developed a polyethoxylated castor oil-free, liposome-based alternative paclitaxel formulation, known as Lipusu. In this study, we performed in vitro and in vivo experiments to compare the safety profiles of Lipusu and Taxol, with special regard to hypersensitivity reactions. First, Swiss mice were used to determine the lethal dosages, and then to evaluate hypersensitivity reactions, followed by histopathological examination and enzyme-linked immunosorbent assays (ELISAs) of serum SC5b-9 and lung histamine. Additionally, healthy human serum was used to analyze in vitro complement activation. Finally, an MTT assay was used to determine the in vitro anti-proliferation activity. Our data clearly showed that Lipusu displayed a much higher safety margin and did not induce hypersensitivity or hypersensitivity-related lung lesions, which may be associated with the fact that Lipusu did not activate complement or increase histamine release in vivo. Moreover, Lipusu did not promote complement activation in healthy human serum in vitro, and demonstrated anti-proliferative activity against human cancer cells, similar to that of Taxol. Therefore, the improved formulation of paclitaxel, which exhibited a much better safety profile and comparable cytotoxic activity to Taxol, may bring a number of benefits to cancer patients.

Correspondence

Correspondence

Serum SC5b-9 (terminal complement complex) level, a sensitive indicator of disease activity in patients with Henoch-Schönlein purpura.

The concentration of the terminal complement complex (TCC), SC5b-9, was determined by enzyme immunoassay using 95 serum samples from 30 patients with Henoch-Schönlein purpura (HSP), 27 with other forms of inflammatory skin disease and 20 normal healthy donors. Twenty-five patients with HSP showed significantly increased TCC concentration in the active phase of the disease, during which newly formed urticarial or purpuric macules/papules could be seen. Skin biopsy specimens of skin lesions from patients with elevated TCC levels in nearly all cases contained the membrane attack complex of complement and consisted of C5b, C6, C7, C8, C9 without S protein on the vessel walls. Systemic and local activation of complement may thus possibly occur in HSP. Three patients with various manifestations of the disease were followed over a period of several years during which the active and inactive phases were scanned. TCC elevation in all cases was correlated with exacerbation of the disease. In contrast, C3, C4 and CH50 levels either remained normal or increased and thus were not reliable indicators of disease activity. Measurement of TCC should thus prove quite useful for monitoring the activity of HSP in patients in whom there is complement activation and also serve to facilitate clarification of the functions of complement in the pathogenesis of the disease.

Serum SC5B-9 levels as a sensitive indicator of Henoch-Schonlein purpura activity

Serum SC5B-9 levels as a sensitive indicator of Henoch-Schonlein purpura activity

Serum complement activation in central nervous system disease in sjögren's syndrome

Central nervous system disease and vasculitis are extraglandular manifestations of Sjögren's syndrome. In our experience, central nervous system disease develops in approximately 70 percent of patients with Sjögren's syndrome and biopsy documented peripheral vasculitis. In order to further investigate the pathogenesis of central nervous system disease and its relationship to peripheral vasculitis in Sjögren's syndrome, we examined sera of patients with Sjögren's syndrome with and without focal central nervous system involvement for evidence of terminal complement pathway activation. Patients were classified as having active focal central nervous system involvement only when they had focal neurologic deficits on physical examination, plus at least one abnormal neurodiagnostic test result. Two thirds of these patients also had cognitive or psychiatric dysfunction. Patients were classified as having peripheral vasculitis if they had clinical and histopathologic documentation of vascular inflammation. Serum SC5b-9 was measured by a sensitive enzyme-linked immunoabsorbent assay. Total hemolytic complement assay, measurement of serum C3 and C4 by radial immunodiffusion, and determination of immune complexes were performed. Fluid-phase terminal complement complexes (SC5b-9) were detected in the sera of 25 of 30 (83 percent) patients with focal central nervous system involvement, but in only seven of 21 (33 percent) patients with Sjögren's syndrome without focal central nervous system disease (p = 0.00084 by Yates' chi-square analysis). Four of these seven patients without focal central nervous system disease, but who had serum SC5b-9, had psychiatric or cognitive dysfunction. SC5b-9 was also detected in sera from 14 of 15 (93 percent) patients with active biopsy-documented peripheral vasculitis in contrast to 18 of 36 (50 percent) patients without clinical evidence of peripheral vasculitis (p = 0.0094). Serum SC5b-9 was a more sensitive indicator of complement activation than circulating immune complex or complement assays. These findings suggest that terminal complement activation may participate in the pathophysiology of both central nervous system and peripheral vasculitis in Sjögren's syndrome. Serum SC5b-9 appears to be a useful diagnostic indicator of vascular inflammation in Sjögren's syndrome and appears to identify those patients at risk for central nervous system complications.