C3a Concentrations Research Articles

Platelets promote CRC by activating the C5a/C5aR1 axis via PSGL-1/JNK/STAT1 signaling in tumor-associated macrophages.

Rationale: Platelets can influence the progression and prognosis of colorectal cancer (CRC) through multiple mechanisms, including crosstalk with tumor-associated macrophages (TAMs). However, the mechanisms underlying the crosstalk between platelets and TAMs remain unclear. The present study aimed to investigate the role of intratumoral platelets in regulating the function of TAMs and to identify the underlying mechanisms. Methods: The interaction of platelets with macrophages was assessed in the presence or absence of the indicated compounds in vivo. An azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC mouse model was used to investigate the role of platelets in controlling CRC development. Multiplexed immunofluorescence staining, fluorescence-activated cell sorting (FACS), and RNA sequence analysis were used to examine the changes in TAMs. TAMs and bone marrow-derived macrophages (BMDMs) were treated with the indicated compounds or siRNA against specific targets, and the expression levels of signal transducer and activator of transcription 1 (STAT1), c-Jun N-terminal kinase (JNK), and P-selectin glycoprotein ligand-1 (PSGL-1) were measured by Western blotting. The mRNA expression levels of complement 5 (C5), complement 5a receptor 1 (C5ar1), Arginase 1 (Arg1) and Il10 were measured by real-time RT-PCR, and the complement 5a (C5a) concentration was measured by ELISA. The dual-luciferase reporter assay and ChIP assay were performed to examine the potential regulatory mechanisms of platelet induction of C5 transcription in TAMs. Results: In our study, we found that an increase in platelets exacerbated CRC development, while inhibiting platelet adhesion attenuated tumor growth. Platelets signal TAMs through P-selectin (CD62P) binding to PSGL-1 expressed on TAMs and activating the JNK/STAT1 pathway to induce the transcription of C5 and the release of C5a, shifting TAMs toward a protumor phenotype. Inhibiting the C5a/C5aR1 axis or PSGL-1 significantly reduced CRC growth. Conclusions: An increase in intratumoral platelets promoted CRC growth and metastasis by CD62P binding to PSGL-1 expressed on TAMs, leading to JNK/STAT1 signaling activation, which promoted C5 transcription and activated the C5a/C5aR1 axis in TAMs. Our study examined the mechanism of the crosstalk between platelets and TAMs to exacerbate CRC development and proposed a potential therapeutic strategy for CRC patients.

Plasma complement C3 and C3a are increased in major depressive disorder independent of childhood trauma

BackgroundDysregulated complement system is linked to pathophysiology of major depressive disorder (MDD). Childhood trauma has been associated with an increased incidence of adult depression via a putative mechanism of immune activation. This study aimed to measure and compare peripheral levels of complement C3, C3a, C1q and C-reactive protein (CRP) in MDD patients and healthy controls and explore the relationship between these molecule levels and childhood trauma history in the participants.MethodsThe participants were 49 medication-free MDD patients and 45 healthy controls. All participants were asked to finish the Childhood Trauma Questionnaire, followed by blood sampling for measurement of plasma complement C3, C3a, C1q and CRP by means of enzyme-linked immunosorbent assay.ResultsPeripheral plasma concentration of C3 and C3a in medication-free MDD group was significantly higher than that in the healthy controls; whereas the concentration of plasma C1q and CRP in depressed patients was comparable to that in healthy controls. All these inflammatory factors were not associated to childhood trauma experience in patients with MDD.ConclusionOur data suggest that complement C3 and C3a may be implicated in the pathophysiology of MDD, although traumatic childhood experiences were not associated with the circulating levels of complement C3, C3a, C1q and CRP.

Complement and endothelial cell activation in COVID-19 patients compared to controls with suspected SARS-CoV-2 infection: A prospective cohort study.

BackgroundThromboinflammation may influence disease outcome in COVID-19. We aimed to evaluate complement and endothelial cell activation in patients with confirmed COVID-19 compared to controls with clinically suspected but excluded SARS-CoV-2 infection.MethodsIn a prospective, observational, single-center study, patients presenting with clinically suspected COVID-19 were recruited in the emergency department. Blood samples on presentation were obtained for analysis of C5a, sC5b-9, E-selectin, Galectin-3, ICAM-1 and VCAM-1.Results153 cases and 166 controls (suffering mainly from non-SARS-CoV-2 respiratory viral infections, non-infectious inflammatory conditions and bacterial pneumonia) were included. Hospital admission occurred in 62% and 45% of cases and controls, respectively. C5a and VCAM-1 concentrations were significantly elevated and E-selectin concentrations decreased in COVID-19 out- and inpatients compared to the respective controls. However, relative differences in outpatients vs. inpatients in most biomarkers were comparable between cases and controls. Elevated concentrations of C5a, Galectin-3, ICAM-1 and VCAM-1 on presentation were associated with the composite outcome of ICU- admission or 30-day mortality in COVID-19 and controls, yet more pronounced in COVID-19. C5a and sC5b-9 concentrations were significantly higher in COVID-19 males vs. females, which was not observed in the control group.ConclusionsOur data indicate an activation of the complement cascade and endothelium in COVID-19 beyond a nonspecific inflammatory trigger as observed in controls (i.e., “over”-activation).

Impact of Dialysis Clinical Operating Conditions on Human Serum Protein-Mediated Inflammatory Biomarkers Released in Patients Using Polyarylethersulfone Membranes

Hemodialysis (HD) is a life-sustaining treatment of crucial importance in managing end-stage renal disease (ESRD). However, this membrane-based therapy is associated with acute side-effects due to bioincompatibility issues and limitations on the removal of uremic toxins. The present study assessed the influence of hydrodynamic conditions applied during HD treatment on protein-mediated inflammatory and thrombotic responses. The membrane modules considered are commonly used in Canadian hospitals and are comprised of a polymer blend of polyarylether sulfone-polyvinylpyrrolidone (PAES). The membranes morphology and hydrophilicity were assessed using SEM, AFM, BET, and zeta potential. An in vitro study evaluated the adsorptive behavior of fibrinogen (FB) to the membrane under different flow conditions. Lower rates of 200 mL/min promoted slower and significant FB adsorption, leading to more severe inflammatory and thrombotic responses. Hydrodynamic conditions also affected the concentration of all inflammatory biomarkers. Lower flow rates triggered more complement activation as well as coagulation, clotting, and inflammatory responses compared to higher flow rates. At the end of the dialysis session, patients treated with a Qb of 200 mL/min presented a significant increase in the concentration of C5a (232%), properdin (114%), serpin (545%), IL-1α (50%), IL-6 (450%), and vWF (212%). IL-1β and TNF-α concentrations declined by 12.5 and 35.5%, respectively. Male patients experienced more severe inflammatory responses than female patients at the operating conditions considered. Comparing the pre- and post-dialysis levels of female and male patients, female patients experienced significantly higher levels of IL-6 and properdin, while male patients presented higher levels of C5a, IL-1α, and IL-6. The results of this study will help clinical doctors evaluate the impact of HD operating conditions on blood activations before prescribing treatment and inform expectations for outcomes in female and male patients.

The Role of Complement Split-products as Biomarkers for Acute Antibody-mediated Rejection of Kidney Allografts.

Background.Acute antibody-mediated rejection (AMR) is mediated by the activation of the classical complement system in addition to noncomplement-dependent inflammatory pathways. Complement fixation by donor-specific antibodies leads to cleavage of the complement proteins C4, C3, and C5 to produce multiple complement split-products (CSP) and the end-effector membrane attack complex, C5b-9. In this study, we investigate CSP as potential biomarkers for AMR.Methods.In an Institutional Review Board–approved, prospective, controlled study, CSP levels were measured in blood and urine samples from consecutive kidney transplant recipients with biopsy-proven AMR (n = 10), acute cellular rejection (ACR) (n = 5), or no rejection (n = 5). After obtaining informed consent, samples were collected at the time of biopsy (day 0) and days 15 (end of rejection treatment) and 30 postbiopsy for AMR and ACR patients. ELISA was used to measure C5a, C4d, and soluble C5b-9 concentrations in blood and urine, in addition to factor Bb (Bb) concentration in blood only. Kidney transplant histopathology was evaluated using the Banff 2013 classification. Rejection treatment and follow-up were performed per standard of care.Results.Blood and urine CSP levels adjusted to urine creatinine were not elevated in AMR compared to no rejection and ACR arms. There was significant variability in CSP concentration within each of the study groups.Conclusion.Our study does not support the utility of CSP as surrogate biomarkers of AMR; however, it is limited by the small sample size and larger studies may be warranted.

Complement 3a Mediates CCN2/CTGF in Human Retinal Pigment Epithelial Cells.

Background Complement 3 (C3) is the crucial component of the complement cascade when retina was exposed to external stimulus. Cellular communication network 2/connective tissue growth factor (CCN2/CTGF) is important in response of retinal stress and a fulcrum for angiogenesis and fibrosis scar formation. Our study aims to explore the interaction between C3 and CCN2/CTGF via bioinformatics analyses and in vitro cell experiments. Methods The GSE dataset was selected to analyse the chemokine expression in human retinal pigment epithelium (ARPE-19) cells under stimulus. Then, RPE cells were further transfected with or without C3 siRNA, followed by C3a (0.1 μM or 0.3 μM) for 24, 48, and 72 hours. Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to measure CCN2/CTGF mRNA and protein levels. Results The GSE36331 revealed C3 expression was significantly elevated in RPE under stimulus. Compared with negative control, CCN2/CTGF mRNA was increased with all types of C3a treatments, whereas a significant increase of protein level was only observed with high concentration of 0.3 μM C3a for a prolonged 72-hour time. Compared with nontransfected cells, significant reductions of CCN2/CTGF mRNA were observed in the C3 siRNA transfected cells with 0.3 μM C3a for 24, 48, and 72 hours, and a significant reduction of CCN2/CTGF protein was observed with 0.3 μM C3a for 48 hours. Conclusions C3 was elevated in RPE under environmental stimulus and long-term exposure to specified concentration of C3a increased CCN2/CTGF expression in RPE, which could be partially reversed by C3 siRNA.

Assaying Macrophage Chemotaxis Using Fluid‐Walled Microfluidics

AbstractWhile many tools exist to study immune‐cell chemotaxis in vitro, current methods often lack desirable features. Using fluid‐walled microfluidics, circuits are built around primary murine macrophages deposited in pre‐defined patterns on Petri dishes or microplates. Concentration gradients of complement component 5a (C5a) are established in flow‐free or flowing environments, cell migration imaged, and cell directionality and velocity correlated to calculated local C5a concentrations. In flow‐free circuits built around patterned macrophages, only cells nearest the C5a source migrate regardless of local attractant concentration. Conversely, in flowing circuits free from intercellular signaling and attractant degradation, only cells distant from the source migrate. In both systems, cells respond to lower C5a concentrations than previously reported (≈0.1 pM). Finally, macrophages follow instantly‐shifted gradients better than slowly‐shifting ones, suggesting that migration depends on both spatial and temporal responses to concentration.

Cold storage alters the immune characteristics of platelets and potentiates bacterial-induced aggregation.

Cold-stored platelets are currently under clinical evaluation and have been approved for limited clinical use in the United States. Most studies have focused on the haemostatic functionality of cold-stored platelets; however, limited information is available examining changes to their immune function. Two buffy-coat-derived platelet components were combined and split into two treatment arms: room temperature (RT)-stored (20-24°C) or refrigerated (cold-stored, 2-6°C). The concentration of select soluble factors was measured in the supernatant using commercial ELISA kits. The abundance of surface receptors associated with immunological function was assessed by flow cytometry. Platelet aggregation was assessed in response to Escherichia coli and Staphylococcus aureus, in the presence and absence of RGDS (blocks active conformation of integrin α2 β3 ). Cold-stored platelet components contained a lower supernatant concentration of C3a, RANTES and PF4. The abundance of surface-bound P-selectin and integrin α2 β3 in the activated conformation increased during cold storage. In comparison, the abundance of CD86, CD44, ICAM-2, CD40, TLR1, TLR2, TLR4, TLR3, TLR7 and TLR9 was lower on the surface membrane of cold-stored platelets compared to RT-stored components. Cold-stored platelets exhibited an increased responsiveness to E. coli- and S. aureus-induced aggregation compared to RT-stored platelets. Inhibition of the active conformation of integrin α2 β3 using RGDS reduced the potentiation of bacterial-induced aggregation in cold-stored platelets. Our data highlight that cold storage changes the in vitro immune characteristics of platelets, including their sensitivity to bacterial-induced aggregation. Changes in these immune characteristics may have clinical implications post transfusion.

Age-Related Maculopathy Susceptibility 2 and Complement Factor H Polymorphism and Intraocular Complement Activation in Neovascular Age-Related Macular Degeneration.

PurposeTo investigate the association of risk alleles in complement factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) with complement activation products in the aqueous humor in eyes with neovascular age-related macular degeneration (nAMD) including polypoidal choroidal vasculopathy (PCV), retinal angiomatous proliferation (RAP), and pachychoroid neovasculopathy (PNV).DesignProspective, comparative, observational study.ParticipantsTreatment-naïve patients with nAMD and cataract patients as controls.MethodsThe study included 236 eyes of 236 patients with nAMD and 49 control eyes of 49 patients. Aqueous humor samples were collected from 67 eyes with drusen-associated nAMD, 72 eyes with PCV, 26 eyes with RAP, and 71 eyes with PNV before intravitreal anti-VEGF injection and cataract surgery in the 49 control eyes. Clinical samples were measured for complement component 3a (C3a), C4a, and C5a using a bead-based immunoassay. Genotyping of the ARMS2 A69S (rs10490924), CFH I62V (rs800292), and CFH Y402H (rs1061170) was performed using TaqMan genotyping.Main Outcome MeasuresThe levels of complement activation products (C3a, C4a, and C5a) in the aqueous humor in each genotype of ARMS2 and CFH.ResultsThe C3a level in the aqueous humor was significantly elevated (P = 0.006) in patients with nAMD and the ARMS2 A69S risk allele, whereas the levels of the complement activation products were not associated with CFH I62V and Y402H genotypes. Among the control eyes, no significant differences were seen in any complement activation products for all genetic polymorphisms. The levels of the complement activation products in the aqueous humor of eyes with the nAMD subtypes for each genetic polymorphism did not show significant differences.ConclusionsThe C3a concentration in the aqueous humor was significantly higher in Japanese nAMD patients with the ARMS2 A69S risk allele, whereas it was not elevated in the patients with CFH I62V. Age-related maculopathy susceptibility 2 A69S polymorphism is strongly associated with local complement activation in nAMD patients.

Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

BackgroundThrombotic events cannot be completely prevented by antithrombotics, implicating a therapeutic gap due to inflammation, a not yet sufficiently addressed mechanism. Neutrophil extracellular traps (NETs) are an essential interface between inflammation and thrombosis, but exactly how the NETotic process is initiated and maintained during arterial thrombosis remains incompletely understood.Methods and resultsWe found that the plasma concentrations of C5a were higher in patients with ST-elevation myocardial infarction (STEMI) than in patients with angina and higher in mice with left common carotid artery (LCCA) thrombosis induced by FeCl3 than in control mice. We observed that the thrombus area and weight were decreased and that NET formation in the thrombi was reduced in the group treated with the selective C5aR1 receptor inhibitor PMX53 compared with the NaCl group. In vitro, NETosis was observed when C5a was added to neutrophil cultures, and this effect was reversed by PMX53. In addition, our data showed that C5a increased the production of mitochondrial reactive oxygen species (ROS) and that the promotion of NET formation by C5a was mitochondrial ROS (Mito-ROS) dependent. Furthermore, we found that C5a induced the production of Mito-ROS by inhibiting mitochondrial STAT3 activity.ConclusionsBy inhibiting mitochondrial STAT3 to elicit Mito-ROS generation, C5a triggers the generation of NETs to promote the development of arterial thrombosis. Hence, our study identifies complement C5a as a potential new target for the treatment and prevention of thrombosis.

Abstract 96: CompLement C5 Antibodies For Decreasing Brain Injury After Aneurysmal Subarachnoid Hemorrhage (CLASH): A Randomized Controlled Phase II Clinical Trial

Introduction: The inflammatory response after aneurysmal subarachnoid hemorrhage (aSAH) has been associated with early brain injury, delayed cerebral ischemia, and poor functional outcome. In experimental SAH studies, complement C5 antibodies administered shortly after SAH reduced brain injury with approximately 40%. We investigated the pharmacodynamic efficacy and safety of eculizumab (a complement C5 antibody) in aSAH patients. Methods: We conducted a randomized, controlled, open-label, phase II clinical trial with blinded outcome assessment in the Netherlands. Patients were randomized (1:1) to receive eculizumab plus standard care or to standard care alone. Eculizumab (1200 mg) was administered intravenously < 12 h, on day 3 and on day 7 after ictus. Patients in the intervention group received prophylactic antibiotics for 4 weeks and prophylactic antifungal therapy if the patient had a central line or an external ventricular shunt and a positive fungal or yeast culture. The primary outcome was C5a concentration in the cerebrospinal fluid (CSF) on day 3 after ictus. Secondary outcomes included the occurrence of adverse events, eculizumab concentration and inflammatory parameters in the blood and CSF, cerebral infarction on magnetic resonance imaging, and clinical and cognitive outcomes. We aimed to evaluate 26 patients with CSF assessments, 13 in the intervention group and 13 in the comparator group. Results: A total of 31 patients have been included, of which 26 with CSF samples. Conclusions: Final results will be presented at the conference. Trial registration: Netherlands Trial Register: NTR6752. European Clinical Trials Database (EudraCT): 2017-004307-51.

The anti-C5a antibody vilobelimab efficiently inhibits C5a in patients with severe COVID-19.

Recently, we reported the phase II portion of the adaptive phase II/III PANAMO trial exploring potential benefit and safety of selectively blocking C5a with the monoclonal antibody vilobelimab (IFX‐1) in patients with severe coronavirus disease 2019 (COVID‐19). The potent anaphylatoxin C5a attracts neutrophils and monocytes to the infection site, causes tissue damage by oxidative radical formation and enzyme releases, and leads to activation of the coagulation system. Results demonstrated that C5a inhibition with vilobelimab was safe and secondary outcomes appeared in favor of vilobelimab. We now report the pharmacokinetic/pharmacodynamic (PK/PD) analysis of the phase II study. Between March 31 and April 24, 2020, 30 patients with severe COVID‐19 pneumonia confirmed by real‐time polymerase chain reaction were randomly assigned 1:1 to receive vilobelimab plus best supportive care or best supportive care only. Samples for measurement of vilobelimab, C3a and C5a blood concentrations were taken. Vilobelimab predose (trough) drug concentrations in plasma ranged from 84,846 to 248,592 ng/ml (571 to 1674 nM) with a geometric mean of 151,702 ng/ml (1022 nM) on day 2 and from 80,060 to 200,746 ng/ml (539 to 1352 nM) with a geometric mean of 139,503 ng/ml (939 nM) on day 8. After the first vilobelimab infusion, C5a concentrations were suppressed in the vilobelimab group (median 39.70 ng/ml 4.8 nM, IQR 33.20–45.55) as compared to the control group (median 158.53 ng/ml 19.1 nM, IQR 60.03–200.89, p = 0.0006). The suppression was maintained on day 8 (p = 0.001). The current PK/PD analysis shows that vilobelimab efficiently inhibits C5a in patients with severe COVID‐19.

Complement Activation Dramatically Accelerates Blood Plasma Fouling On Antifouling Poly(2-hydroxyethyl methacrylate) Brush Surfaces.

Non-specific protein adsorption (fouling) triggers a number of deleterious events in the application of biomaterials. Antifouling polymer brushes successfully suppress fouling, however for some coatings an extremely high variability of fouling for different donors remains unexplained. The authors report that in the case of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) this variability is due to the complement system activation that causes massive acceleration in the fouling kinetics of blood plasma. Using plasma from various donors, the fouling kinetics on poly(HEMA) is analyzed and correlated with proteins identified in the deposits on the surface and with the biochemical compositions of the plasma. The presence of complement components in fouling deposits and concentrations of C3a in different plasmas indicate that the alternative complement pathway plays a significant role in the fouling on poly(HEMA) through the "tick-over" mechanism of spontaneous C3 activation. The generated C3b binds to the poly(HEMA) surface and amplifies complement activation locally. Heat-inactivated plasma prevents accelerated fouling kinetics, confirming the central role of complement activation. The results highlight the need to take into account the variability between individuals when assessing interactions between biomaterials and blood plasma, as well as the importance of the mechanistic insight that can be gained from protein identification.

Association between peripheral blood WBCs C3aR mRNA level and plasma C3a, C3aR, IL-1β concentrations and acute exacerbation of chronic obstructive pulmonary disease

BackgroundThe relationship between C3a-C3aR, IL-1β, and the acute exacerbation of chronic obstructive pulmonary disease is still unclear. This study aims to explore the expression levels of C3aR in peripheral blood WBCs and the concentrations of C3a, C3aR, and IL-1β in plasma in healthy controls and patients with chronic obstructive pulmonary disease (COPD). MethodsWBCs C3aR level in the peripheral blood, the concentrations of C3a, C3aR, and IL-1β in plasma were measured in 60 patients with acute exacerbation of COPD (AECOPD), 30 patients with stable COPD (SCOPD), and 30 healthy controls. The baseline characteristics and clinical data collected from enrolled patients, including age, gender, laboratory indicators, and lung function. We analyzed the correlation between C3a, C3aR, IL-1β, and lung function indicators (forced expiratory volume in the first second as a percentage of predicted value, FEV1%pred) in the AECOPD group. ResultsThe white blood cell count (WBC), neutrophil/lymphocyte ratio (NLR), and C-reactive protein (CRP) of patients in COPD were higher than in healthy controls (P < 0.05). The peripheral blood WBCs C3aR mRNA and plasma C3a, C3aR, and IL-1β in AECOPD were higher than in SCOPD and healthy controls (P < 0.05). The peripheral blood WBCs C3aR mRNA and plasma C3aR, and IL-1β in AECOPD combined with respiratory failure were higher than in the non-respiratory failure group (P < 0.05). The peripheral blood WBCs C3aR mRNA and plasma C3a, C3aR, and IL-1β in AECOPD with high-risk were higher than in the low-risk group (P < 0.05). The peripheral blood WBCs C3aR mRNA and plasma C3a, C3aR, and IL-1β in AECOPD were negatively correlated with FEV1pred%. The peripheral blood WBCs C3aR mRNA, the plasma C3a and C3aR in AECOPD were positively correlated with IL-1β. ConclusionThe peripheral blood WBCs C3aR mRNA and plasma C3a, C3aR, and IL-1β in COPD patients were significantly related to the risk of disease deterioration. The C3a-C3aR axis may be involved in airway inflammation in patients with COPD.

Complement Activation Fragments in Cervicovaginal Fluid Are Associated with Intra-Amniotic Infection/Inflammation and Spontaneous Preterm Birth in Women with Preterm Premature Rupture of Membranes.

We sought to determine whether the levels of complement and other inflammatory and angiogenic mediators in cervicovaginal fluid (CVF) are independently associated with intra-amniotic infection and/or inflammation (IAI) and imminent spontaneous preterm birth (SPTB, £48 hours of sampling) in women with preterm premature rupture of membranes (PPROM). This was a retrospective study consisting of 85 singleton pregnant women with PPROM at 200/7 to 336/7 weeks. Amniotic fluid (AF) obtained via amniocentesis was cultured and assayed for interleukin-6. CVF samples collected at the time of amniocentesis were assayed for complement C3a, C4a, and C5a, HSP70 (heat shock protein 70), M-CSF (macrophage colony-stimulating factor), M-CSF-R (macrophage colony-stimulating factor-receptor), S100 A8, S100 A9, thrombospondin-2, VEGF (vascular endothelial growth factor-receptor), and VEGFR-1 (vascular endothelial growth factor-receptor 1) by enzyme-linked immunosorbent assay. Multivariate logistic regression analyses revealed that elevated CVF concentrations of complement C3a, 4a, and 5a were significantly associated with an increased risk of IAI and imminent SPTB, whereas those of M-CSF were associated with IAI, but not imminent SPTB (p = 0.063), after adjustment for baseline covariates (e.g., gestational age at sampling). However, univariate, and multivariate analyses showed that the CVF concentrations of angiogenic (thrombospondin-2, VEGF, and VEGFR-1) and inflammatory (HSP70, M-CSF-R, S100 A8, and S100 A9) proteins were not associated with either IAI or imminent SPTB. In women with PPROM, elevated CVF concentrations of complement C3a, C4a, and C5a are independently related to an increased risk of IAI and imminent SPTB. These findings suggest that complement activation in CVF is significantly involved in mechanisms underlying preterm birth and in the host response to IAI in the context of PPROM. · Elevated CVF levels of C3a, 4a and 5a are associated with IAI and SPTB.. · CVF C3a, 4a and 5a have better predictability for SPTB, compared to AF WBC.. · Elevated CVF levels of M-CSF were associated with IAI, but not SPTB..

Activation of the Complement System in Patients with Cancer Cachexia.

Simple SummaryPatients with cancer often suffer from severe weight loss as a result of the wasting of skeletal muscle and fat tissue. This has a strong negative impact on the patient’s prognosis and quality of life. Inflammation is thought to contribute to weight loss in cancer. To enable the future targeting of inflammation in patients with cancer who experience weight loss, we set out to characterize an important pro-inflammatory component of the immune system: the complement system. The blood levels of several elements of the complement system were analyzed in patients with and without weight loss and inflammation. We found that complement factors were activated specifically in patients with both weight loss and inflammation. Since complement inhibitory drugs are already on the market, these findings may open new opportunities for treating inflammation-mediated weight loss in patients with cancer.Systemic inflammation is thought to underlie many of the metabolic manifestations of cachexia in cancer patients. The complement system is an important component of innate immunity that has been shown to contribute to metabolic inflammation. We hypothesized that systemic inflammation in patients with cancer cachexia was associated with complement activation. Systemic C3a levels were higher in cachectic patients with inflammation (n = 23, C-reactive protein (CRP) ≥ 10 mg/L) as compared to patients without inflammation (n = 26, CRP < 10 mg/L) or without cachexia (n = 13) (medians 102.4 (IQR 89.4–158.0) vs. 81.4 (IQR 47.9–124.0) vs. 61.6 (IQR 46.8–86.8) ng/mL, respectively, p = 0.0186). Accordingly, terminal complement complex (TCC) concentrations gradually increased in these patient groups (medians 2298 (IQR 2022–3058) vs. 1939 (IQR 1725–2311) vs. 1805 (IQR 1552–2569) mAU/mL, respectively, p = 0.0511). C3a and TCC concentrations were strongly correlated (rs = 0.468, p = 0.0005). Although concentrations of C1q and mannose-binding lectin did not differ between groups, C1q levels were correlated with both C3a and TCC concentrations (rs = 0.394, p = 0.0042 and rs = 0.300, p = 0.0188, respectively). In conclusion, systemic inflammation in patients with cancer cachexia is associated with the activation of key effector complement factors. The correlations between C1q and C3a/TCC suggest that the classical complement pathway could play a role in complement activation in patients with pancreatic cancer.

Efficacy and Safety of Vilobelimab (IFX-1), a Novel Monoclonal Anti-C5a Antibody, in Patients With Early Severe Sepsis or Septic Shock-A Randomized, Placebo-Controlled, Double-Blind, Multicenter, Phase IIa Trial (SCIENS Study).

This phase II clinical trial was conducted to study safety, tolerability, pharmacokinetics, and pharmacodynamics of vilobelimab, a recombinant monoclonal antibody against C5a, in patients with severe sepsis or septic shock. Multicenter, randomized, and placebo-controlled study. Eleven multidisciplinary ICUs across Germany. Adult patients with severe sepsis or septic shock and with early onset of infection-associated organ dysfunction. Patients were randomly assigned in a ratio of 2:1 to three subsequent dosing cohorts for IV vilobelimab or placebo receiving either 2 × 2 mg/kg (0 and 12 hr), 2 × 4 mg/kg (0 and 24 hr), and 3 × 4 mg/kg (0, 24, and 72 hr). Co-primary endpoints were pharmacodynamics (assessed by C5a concentrations), pharmacokinetics (assessed by vilobelimab concentrations), and safety of vilobelimab. Preliminary efficacy was evaluated by secondary objectives. Seventy-two patients were randomized (16 patients for each vilobelimab dosing cohort and eight patients for each placebo dosing cohort). Vilobelimab application was associated with dosing dependent decrease in C5a compared with baseline (p < 0.001). Duration of C5a decrease increased with more frequent dosing. Membrane attack complex lysis capacity measured by 50% hemolytic complement was not affected. Vilobelimab was well tolerated with similar safety findings in all dose cohorts. No vilobelimab-specific adverse events emerged. For vilobelimab-treated patients, investigators attributed less treatment-emergent adverse events as related compared with placebo. Dosing cohorts 2 and 3 had the highest ICU-free and ventilator-free days. There was no difference in mortality, vasopressor-free days, or renal replacement therapy-free days between the groups. Administration of vilobelimab in patients with severe sepsis and septic shock selectively neutralizes C5a in a dose-dependent manner without blocking formation of the membrane attack complex and without resulting in detected safety issues. The data warrant further investigation of C5a inhibition in sepsis.

Role of uromodulin and complement activation in the progression of kidney disease.

Uromodulin (UMOD) is a glycoprotein that is selectively expressed on the epithelial cells of the thick ascending limb of Henle's loop and the early distal renal tubule. The present study aimed to investigate whether UMOD was associated with complement activation in patients with renal diseases. In addition, its biological function was examined in vitro. The expression levels of UMOD and complement components, including C1q, C3, C4 and C3a, and membrane attack complex (MAC) in the plasma of patients with IgA nephropathy (IgAN; n=58) and lupus nephritis (LN; n=36) were detected using ELISA, which was used to determine the association between UMOD expression and complement components. In addition, a simulated hypoxia-reoxygenation (H/R) model was used to stimulate UMOD expression in mouse inner medullary collecting duct cells. Additionally, the association between UMOD expression and complement components C1q and C3d at the cellular level was identified using western blotting and immunofluorescence, respectively. It was revealed that the plasma UMOD concentration was significantly decreased in patients with IgAN and LN compared with in healthy controls, and the levels of C3a and MAC were significantly increased in the plasma of patients with IgAN and LN. Furthermore, the plasma levels of C1q, C3 and C4 in patients with LN, but not in patients with IgAN, were significantly decreased compared with in healthy controls. The plasma levels of UMOD were negatively correlated with the plasma C3a and MAC concentrations. However, the plasma levels of UMOD were significantly and positively correlated with the plasma C1q concentration, but not with that of C3 and C4. It was identified that UMOD expression started to increase after 1 h of simulated H/R, and continued to increase at 6 and 12 h. In addition, cells with lower UMOD expression had higher C3d expression in vitro. Collectively, the present results suggested that UMOD was associated with severe complement activation and may be involved in complement-mediated immune protection by inhibiting complement activation in renal disease.

Cryopreservation alters the immune characteristics of platelets.

Cryopreserved platelets are under clinical evaluation as they offer improvements in shelf-life and potentially hemostatic effectiveness. However, the effect of cryopreservation on characteristics related to the immune function of platelets has not been examined. Buffy coat derived platelets were cryopreserved at -80°C using 5%-6% dimethylsulfoxide (DMSO, n=8). Paired testing was conducted pre-freeze (PF), post-thaw (PT0), and after 24 h of post-thaw storage at room temperature (PT24). The concentration of biological response modifiers (BRMs) in the supernatant was measured using commercial ELISAs and surface receptor abundance was assessed by flow cytometry. Cryopreservation resulted in increased RANTES, PF4, and C3a but decreased IL-1β, OX40L, IL-13, IL-27, CD40L, and C5a concentrations in the supernatant, compared to PF samples. C4a, endocan, and HMGB1 concentrations were similar between the PF and PT0 groups. The abundance of surface-expressed P-selectin, siglec-7, TLR3, TLR7, and TLR9 was increased PT0; while CD40, CLEC2, ICAM-2, and MHC-I were decreased, compared to PF. The surface abundance of CD40L, B7-2, DC-SIGN, HCAM, TLR1, TLR2, TLR4, and TLR6 was unchanged by cryopreservation. Following 24 h of post-thaw storage, all immune associated receptors and TLRs increased to levels higher than observed on PF and PT0 platelets. Cryopreservation alters the immune phenotype of platelets. Understanding the clinical implications of the observed changes in BRM release and receptor abundance are essential, as they may influence the likelihood of adverse events.

Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

BackgroundDuring the process of deep decay, when decay approaches the pulp, an immune response is triggered inside the pulp, which activates the complement cascade. The effect of complement component 5a (C5a) on the differentiation of dental pulp mesenchymal stem cells (DPSCs) is related to dentin reparation. The aim of the present study was to stimulate DPSCs with different concentrations of C5a and evaluate the differentiation of odontoblasts using dentin sialoprotein (DSP).MethodsDPSCs were divided into the following six groups: (i) Control; (ii) DPSCs treated with 50 ng/ml C5a; (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a. Flow cytometry and multilineage differentiation potential were used to identify DPSCs. Mineralization induction, Real-time PCR and Western blot were conducted to evaluate the differentiation of odontoblast in the 6 groups.ResultDPSCs can express mesenchymal stem cell markers, including CD105, CD90, CD73 and, a less common marker, mesenchymal stromal cell antigen-1. In addition, DPSCs can differentiate into adipocytes, neurocytes, chondrocytes and odontoblasts. All six groups formed mineralized nodules after 28 days of culture. Reverse transcription-quantitative PCR and western blotting indicated that the high concentration C5a groups expressed higher DSP levels and promoted DPSC differentiation, whereas the low concentration C5a groups displayed an inhibitory effect.ConclusionIn this study, the increasing concentration of C5a, which accompanies the immune process in the dental pulp, has demonstrated an enhancing effect on odontoblast differentiation at higher C5a concentrations in vitro.