Background:Complement activation is increasingly recognized as a key driver in various critical diseases and a potential therapeutic target. However, conventional enzyme-linked immunosorbent assays (ELISAs) for complement protein detection are laboratory-bound and unsuitable for rapid clinical decision-making in the emergency and intensive care unit setting. We evaluated the feasibility of a point-of-care (POC) multiplex micro-ELISA for complement component C3, activation marker C3d and the soluble terminal complement complex (TCC) in a heterogeneous intensive care unit (ICU) population. Method:Thirty-eight critically ill and three cardiopulmonary bypass patients were prospectively enrolled. Fifteen age- and sex-matched healthy volunteers served as controls. Plasma samples collected at ICU admission and days 2-4 were analyzed using a novel POC multiplex micro-ELISA platform and compared with established in-house and commercial ELISAs using Spearman rank correlation and receiver operating characteristic (ROC) analysis. Additional complement activation markers (C3bc, C3bBbP, C1s/C1-INH, MASP-1/C1-INH and C4d) were assessed to explore specific pathway involvement. Results:TCC levels were significantly elevated in patients (median 3575, IQR 2048-6504 mAU/ml) compared with controls (median 1286, IQR 1032-1903 mAU/ml; p < 0.0001) at both sampling time points. The POC assay demonstrated strong rank correlation with the in-house TCC ELISA (r = 0.83, p < 0.001) and the commercial Hycult TCC ELISA (r = 0.88, p < 0.001). The POC TCC assay accurately discriminated patient samples above and below the ELISA-defined threshold (AUC 0.97, p < 0.001). In contrast, C3 and C3d showed limited patient-control discriminatory ability and poor POC-ELISA agreement. C3bc and C3bBbP were elevated in patients, suggesting alternative pathway amplification. C1s/C1-INH and C4d were not significantly elevated, whereas MASP-1-C1-INH complexes were decreased in patients compared to healthy controls. Conclusion:Point-of-care TCC quantification is feasible, correlates well with established ELISAs and accurately identifies elevated TCC levels in critically ill patients. Although formal method-comparison analyses were not possible to establish, TCC emerged as the most robust marker of complement activation in this ICU cohort using the evaluated POC prototype. POC testing may facilitate timely identification of complement activation in acute care settings and support integration of complement-directed diagnostics and therapeutics into clinical workflows.

Left ventricular assist device (LVAD) implantation is a life-saving therapy for end-stage heart failure but may compromise immune integrity. Mechanical shear stress and surface-induced innate immune activation can trigger bleeding and thromboembolic complications. While thrombotic mechanisms are well characterized, the associated inflammatory response remains poorly studied. We investigated thromboinflammation in patients with terminal heart failure (n = 8) implanted with the HeartWare ventricular assist device. Blood samples were collected before and immediately after implantation, daily for five days, and three months post-implantation. Ten age- and sex-matched healthy individuals served as controls. Samples were analyzed for a broad panel of thromboinflammatory and cell damage biomarkers. Twenty-eight of 43 biomarkers were significantly elevated (p < 0.05) at patient baseline compared with controls, indicating a pre-existing low-grade inflammatory state prior to LVAD implantation. Complement activation products increased markedly immediately after implantation—C3bc, C3bBbP, and the terminal complement complex C5b-9 rose 2.8-, 8.9-, and 6.6-fold, respectively, compared with baseline (p < 0.0001), but returned toward baseline within 24 h. A similar transient increase was observed for TNF, IL-6, IL-8, IL-10, IFN-γ, MMP-8, MMP-9, tissue factor, and prothrombin fragment 1.2 (p < 0.05). LVAD implantation with cardiopulmonary bypass induces a strong but transient immune response, including robust complement activation. Targeting upstream complement pathways may help attenuate downstream thromboinflammatory processes during the acute post-implantation period.

Brain death (BD) induces a systemic inflammation impairing donor organ quality. Complement and Toll-like receptors (TLRs), with the key co-receptor CD14 molecule, are key innate recognition immune systems. We hypothesized that dual inhibition of complement (C5) and TLRs (CD14) will prevent BD-mediated innate immune inflammation. BD was induced in mice either untreated, treated with a C5 inhibitor, a CD14 inhibitor, or both. Blood and kidneys were collected after three hours. Cytokines were analyzed using enzyme-immuno assays and qPCR. In plasma, a substantial increase in interleukin-6 (IL-6), KC (IL-8 analogue), IL-12, monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein MIP-1α, MIP-1β, eotaxin, RANTES and G-CSF (median 90-fold increase) were observed in BD animals compared to sham (all p < 0.01). In kidneys, BD substantially induced IL-6, KC, TNF, MCP-1, P-Selectin, and VCAM-1 (all p < 0.01). In plasma, C5 and CD14 inhibition, either single or in combination, virtually abolished all cytokines in the BD animals (> 90% for six cytokines and 70–90% for three) (all p < 0.01). In kidneys, the effect of inhibition was similar (> 90% for IL-6 and KC and 60–80% for TNF and MCP-1 (all p < 0.01). Single and combined inhibition of C5 and CD14 efficiently prevented BD-induced systemic inflammation and reduced local kidney inflammation in a mouse model.

BackgroundThe complement system, an upstream recognition system of innate immunity, is activated upon SARS-CoV-2 infection. To gain a deeper understanding of the extent and duration of this activation, we investigated complement activation profiles during the acute phase of COVID-19, its persistence post-recovery and dynamic changes in relation to disease severity. MethodsSerial blood samples were obtained from two cohorts of hospitalized COVID-19 patients (n = 457). Systemic complement activation products reflecting classical/lectin (C4d), alternative (C3bBbP), common (C3bc) and terminal pathway (TCC and C5a) were measured during hospitalization (admission, days 3-5 and days 7-10), at 3 months and after 1 year. Levels of activation and temporal profiles during hospitalization were related to disease severity defined as respiratory failure (PO2/FiO2 ratio <26.6 kPa) and/or admission to intensive care unit, 60-day total mortality and pulmonary pathology after 3 months. FindingsDuring hospitalization, TCC, C4d, C3bc, C3bBbP and C5a were significantly elevated compared to healthy controls. Severely ill patients had significantly higher levels of TCC and C4d (p < 0.001), compared to patients with moderate COVID-19. Escalated levels of TCC and C4d during hospitalization were associated with a higher risk of 60-day mortality (p < 0.001), and C4d levels were additionally associated with chest CT changes at 3 months (p < 0.001). At 3 months and 1 year, we observed consistently elevated levels of most complement activation products compared to controls. ConclusionHospitalized COVID-19 patients display prominent and long-lasting systemic complement activation. Optimal targeting of the system may be achieved through enhanced risk stratification and closer monitoring of in-hospital changes of complement activation products.

C1 inhibitor (C1Inh) is a serine protease inhibitor involved in the kallikrein-kinin system, the complement system, the coagulation system, and the fibrinolytic system. In addition to the plasma leakage observed in hereditary angioedema (HAE), C1Inh deficiency may also affect these systems, which are important for thrombosis and inflammation. The aim of this study was to investigate the thromboinflammatory load in C1Inh deficiency. We measured 27 cytokines including interleukins, chemokines, interferons, growth factors, and regulators using multiplex technology. Complement activation (C4d, C3bc, and sC5b-C9/TCC), haemostatic markers (beta-thromboglobulin (beta-TG), thrombin-antithrombin complexes (TAT), prothrombin fragment 1 + 2 (F1 + 2), active plasminogen activator inhibitor-1 (PAI-1), and the neutrophil activation marker myeloperoxidase (MPO) were measured by enzyme immunoassays. Plasma and serum samples were collected from 20 patients with HAE type 1 or 2 in clinical remission and compared with 20 healthy age- and sex-matched controls. Compared to healthy controls, HAE patients had significantly higher levels of tumour necrosis factor (TNF), interleukin (IL)-1 beta, IL-2, IL-4, IL-6, IL-7, IL-9, IL-12, and IL-17A, chemokine ligand (CXCL) 8, chemokine ligand (CCL) 3, CCL4, IL-1 receptor antagonist (IL-1RA), granulocyte-macrophage colony-stimulating factor (GM-CSF), fibroblast growth factor (FGF) 2 and platelet-derived growth factor (PDGF)-BB. HAE patients also had higher levels of TAT and F1 + 2. Although granulocyte colony-stimulating factor (G-CSF), beta-TG and PAI-1 were higher in HAE patients, the differences did not reach statistical significance after correction for multiple testing. In conclusion, C1Inh deficiency is associated with an increased baseline thromboinflammatory load. These findings may reflect that HAE patients are in a subclinical attack state outside of clinically apparent oedema attacks. C1 inhibitor deficiency leads to dysregulation of the contact and kallikrein-kinin systems. This results in an increased inflammatory load in hereditary angioedema.

IntroductionHematopoietic stem cell transplantation (HSCT) is associated with immune complications and endothelial dysfunction due to intricate donor-recipient interactions, conditioning regimens, and inflammatory responses.MethodsThis study investigated the role of the complement system during HSCT and its interaction with the cytokine network. Seventeen acute myeloid leukemia patients undergoing HSCT were monitored, including blood sampling from the start of the conditioning regimen until four weeks post-transplant. Clinical follow-up was 200 days.ResultsTotal complement functional activity was measured by WIELISA and the degree of complement activation by ELISA measurement of sC5b-9. Cytokine release was measured using a 27-multiplex immuno-assay. At all time-points during HSCT complement functional activity remained comparable to healthy controls. Complement activation was continuously stable except for two patients demonstrating increased activation, consistent with severe endotheliopathy and infections. In vitro experiments with post-HSCT whole blood challenged with Escherichia coli, revealed a hyperinflammatory cytokine response with increased TNF, IL-1β, IL-6 and IL-8 formation. Complement C3 inhibition markedly reduced the cytokine response induced by Staphylococcus aureus, Aspergillus fumigatus, and cholesterol crystals.DiscussionIn conclusion, HSCT patients generally retained a fully functional complement system, whereas activation occurred in patients with severe complications. The complement-cytokine interaction indicates the potential for new complement-targeting therapeutic strategies in HSCT.

Organs obtained from brain dead donors can have suboptimal outcomes. Activation of the innate immune system and translocation of intestinal bacteria could be causative. Thirty two pigs were assigned to control, brain death (BD), BD + luminal intestinal polyethylene glycol (PEG), and BD + luminal intestinal University of Wisconsin solution (UW) groups. Animals were observed for 360 min after BD before organ retrieval. 2,000 mL luminal intestinal preservation solution was instilled into the duodenum at the start of organ procurement. Repeated measurements of plasma C3a, Terminal Complement Complex (TCC), IL-8, TNF, and lipopolysaccharide binding protein were analysed by immunoassays. C3a was significantly higher in the BD groups compared to controls at 480 min after brain death. TCC was significantly higher in BD and BD + UW, but not BD + PEG, compared to controls at 480 min. TNF was significantly higher in the BD group compared to all other groups at 480 min. LPS binding protein increased following BD in all groups except BD + PEG, which at 480 min was significantly lower compared with all other groups. Brain death induced innate immune system activation was decreased by luminal preservation using PEG during organ procurement, possibly due to reduced bacterial translocation.

Normothermic machine perfusion (NMP) is a clinical strategy to reduce renal ischemia-reperfusion injury (IRI). Optimal NMP should restore metabolism and minimize IRI induced inflammatory responses. Microdialysis was used to evaluate renal metabolism. This study aimed to assess the effect of complement inhibition on NMP induced inflammatory responses. Twenty-two pig kidneys underwent 18 h of static cold storage (SCS) followed by 4 h of NMP using a closed-circuit system. Kidneys were randomized to receive a C5-inhibitor or placebo during SCS and NMP. Perfusion resulted in rapidly stabilized renal flow, low renal resistance, and urine production. During SCS, tissue microdialysate levels of glucose and pyruvate decreased significantly, whereas glycerol increased (p < 0.001). In the first hour of NMP, glucose and pyruvate increased while glycerol decreased (p < 0.001). After 4 h, all metabolites had returned to baseline. Inflammatory markers C3a, soluble C5b-9, TNF, IL-6, IL-1β, IL-8, and IL-10 increased significantly during NMP in perfusate and kidney tissue. C5-inhibition significantly decreased perfusate and urine soluble C5b-9 (p < 0.001; p = 0.002, respectively), and tissue IL-1β (p = 0.049), but did not alter other inflammatory markers. Microdialysis can accurately monitor the effect of NMP on renal metabolism. Closed-circuit NMP induces inflammation, which appeared partly complement-mediated. Targeting additional immune inhibitors should be the next step.

Despite extensive treatment with surgery and chemotherapy many patients with peritoneal metastases from colorectal cancer experience intraperitoneal disease relapse. The α-emitting 224radium-labelled microparticle radionuclide therapeutic Radspherin® is being explored as a novel treatment option for these patients. Radspherin® is specially designed to give local radiation to the surface of the peritoneal cavity and potentially kill remaining attached micrometastases as well as free-floating cancer cells, thus preventing future relapse. The effect of Radspherin® on the immune system is not known. Systemic and local inflammatory responses were analyzed in plasma, intraperitoneal fluid and urine collected prospectively as part of a phase 1 dose-escalation study of intraperitoneal instillation of the α-emitting therapeutic radiopharmaceutical Radspherin®, at baseline and the first 7 postoperative days from nine patients undergoing cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. All patients additionally received intraperitoneal instillation of Radspherin® on postoperative day 2. Complement activation products C3bc and the terminal complement complex were analyzed using enzyme-linked immunosorbent assay. Cytokines (n = 27), including interleukins, chemokines, interferons and growth factors, were analyzed using multiplex technique. The time course and magnitude of the postoperative cytokine response after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy displayed a modest systemic response in plasma, in contrast to a substantial local intraperitoneal response. After administration of Radspherin®, a significant increase (P < 0.05) in TNF and MIP-1β was observed in both plasma and peritoneal fluid, whereas IL-9 increased only in plasma and IFNγ and IL1-RA only in peritoneal fluid. Only minor changes were seen for the majority of the inflammatory markers after Radspherin® administration. Our study showed a predominately local rather than systemic inflammatory response to cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. Radspherin® had overall modest impact on the inflammation.

Background: Organs obtained from brain dead (BD) donors often have worse outcomes. Activation of the complement system and translocation of intestinal bacteria could be causative. We aimed to examine activation of the complement system following BD and evaluate the systemic and local effect of adding luminal intestinal preservation to classical vascular preservation. Methods and material: BD was induced in 30 pigs (four groups: control (n=7), BD alone (n=8), BD + luminal intestinal polyethylene glycol (PEG, n=7) and BD + luminal intestinal University of Wisconsin solution (UW, n=8) using a previously validated method and all animals were observed for 6 hours before organ retrieval. In the PEG and UW groups, 2000 ml of the selected solution was instilled into the duodenum during the organ procurement surgery. Repeated measurements of C3a, Terminal Complement Complex (TCC), IL-8 and TNF were performed in plasma at baseline, BD, 30, 60, 120, 240 and 360 minutes after BD, and following the intestinal intervention (480). Plasma lipopolysaccharide binding protein (LPS-BP) was measured at baseline, BD, and 480 minutes after BD. All were normalised to albumin concentration. Biopsies were taken from jejunum and ileum at time of removal and following 8, 14 and 24 hours of static cold storage (SCS) using UW. Preliminary analysis has been performed at 24 hours of SCS for the BD groups, full histology will be available soon. Results: All animals were kept circulatory- and respiratory stable until organ procurement. At 480 minutes, C3a was significantly higher in BD, BD+PEG, and BD+UW groups compared to control group (all p<0.05) (fig. 1A). TCC was significantly higher in the combined BD group compared to control at 360 minutes, at 480 minutes, the BD and BD+UW groups were significantly higher compared to the control group (all p<0.05) (fig. 1B). IL-8 and TNF were significantly higher in the BD group compared to all other groups at 480 minutes (p=0,003 and p=0.001) (fig. 1C and D). LPS-BP increased following induction of BD in all groups except BD+PEG, which at 480 minutes were significantly lower (p=0.002) (fig. 1E and F) compared with all other groups. Preliminary biopsies from the Jejunum after 24 hours of SCS show a reduced median Chiu/Park score in the BD+PEG (2.5) and BD+UW (2.0) groups compared to the BD group (fig. 2). Conclusion: The complement system is activated following BD independently of intestinal and luminal preservation and may lead to inflammation. Luminal intestinal preservation during organ procurement led to lower Chiu/Park scores, and reduced cytokine and LPS-BP expression, which may be due to reduced bacterial translocation occurring during surgery independent of BD. Luminal PEG intervention may be combined with early innate immune system inhibition in BD donors to prevent systemic inflammation, which hampers organ function.

The lepirudin-based human whole blood model is a well-established ex vivo system to characterize inflammatory responses. However, the contribution of individual cell populations to cytokine release has not been investigated. Thus, we modified the model by selectively removing leukocyte subpopulations to elucidate their contribution to the inflammatory response. Lepirudin-anticoagulated whole blood was depleted from monocytes or granulocytes using StraightFrom Whole Blood MicroBeads. Reconstituted blood was incubated with Escherichia coli (10(8)/mL) for 2 hours at 37 degrees C. CD11b, CD62P, and CD63 were detected by flow cytometry. Complement (C3bc, sC5b-9) and platelet activation (platelet factor 4, NAP-2) were measured by enzyme-linked immunosorbent assay. Cytokines were quantified by multiplex assay. A significant (P < 0.05) specific depletion of the monocyte (mean = 86%; 95% confidence interval = 71%-92%) and granulocyte (mean = 97%; 95% confidence interval = 96%-98%) population was obtained. Background activation induced by the depletion protocol was negligible for complement (C3bc and sC5b-9), leukocytes (CD11b), and platelets (NAP-2). Upon Escherichia coli incubation, release of 10 of the 24 cytokines was solely dependent on monocytes (interleukin [IL]-1 beta, IL-2, IL-4, IL-5, IL-17A, interferon-gamma, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein-1 alpha, and fibroblast growth factor-basic), whereas 8 were dependent on both monocytes and granulocytes (IL-1ra, IL-6, IL-8, IL-9, IL-10, macrophage inflammatory protein-1 beta, tumor necrosis factor, and eotaxin). Six cytokines were not monocyte or granulocyte dependent, of which platelet-derived growth factor and RANTES were mainly platelet dependent. We document an effective model for selective depletion of leukocyte subpopulations from whole blood, without causing background activation, allowing in-depth cellular characterization. The results are in accordance with monocytes playing a major role in cytokine release and expand our knowledge of the significant role of granulocytes in the response to E. coli.

Despite extensive treatment with surgery and chemotherapy many patients with peritoneal metastases from colorectal cancer experience intraperitoneal disease relapse. The α-emitting 224 radium-labelled microparticle radionuclide therapeutic Radspherin® is being explored as a novel treatment option for these patients. Radspherin® is specially designed to give local radiation to the surface of the peritoneal cavity and potentially kill remaining attached micrometastases as well as free-floating cancer cells, thus preventing future relapse. The effect of Radspherin® on the immune system is not known. Systemic and local inflammatory responses were analyzed in plasma, intraperitoneal fluid and urine collected prospectively as part of a phase 1 dose-escalation study of intraperitoneal instillation of the α-emitting therapeutic radiopharmaceutical Radspherin®, at baseline and the first 7 postoperative days from nine patients undergoing cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. All patients additionally received intraperitoneal instillation of Radspherin® on postoperative day 2. Complement activation products C3bc and the terminal complement complex were analyzed using enzyme-linked immunosorbent assay. Cytokines ( n = 27), including interleukins, chemokines, interferons and growth factors, were analyzed using multiplex technique. The time course and magnitude of the postoperative cytokine response after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy displayed a modest systemic response in plasma, in contrast to a substantial local intraperitoneal response. After administration of Radspherin®, a significant increase ( P < 0.05) in TNF and MIP-1β was observed in both plasma and peritoneal fluid, whereas IL-9 increased only in plasma and IFNγ and IL1-RA only in peritoneal fluid. Only minor changes were seen for the majority of the inflammatory markers after Radspherin® administration. Our study showed a predominately local rather than systemic inflammatory response to cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. Radspherin® had overall modest impact on the inflammation. Keywords inflammation , colorectal cancer , peritoneal metastasis , cytoreductive surgery , hyperthermic intraperitoneal chemotherapy HIPEC , α-emitting radionuclide therapy

The innate immune system is rapidly activated during myocardial infarction and blockade of extracellular complement system reduces infarct size. Intracellular complement, however, appears to be closely linked to metabolic pathways and its role in ischemia-reperfusion injury is unknown and may be different from complement activation in the circulation. The purpose of the present study was to investigate the role of intracellular complement in isolated, retrogradely buffer-perfused hearts and cardiac cells from adult male wild type mice (WT) and from adult male mice with knockout of complement component 3 (C3KO). Main findings: (i) Intracellular C3 protein was expressed in isolated cardiomyocytes and in whole hearts, (ii) after ischemia-reperfusion injury, C3KO hearts had larger infarct size (32 ± 9% in C3KO vs. 22 ± 7% in WT; p=0.008) and impaired post-ischemic relaxation compared to WT hearts, (iii) C3KO cardiomyocytes had lower basal oxidative respiration compared to WT cardiomyocytes, (iv) blocking mTOR decreased Akt phosphorylation in WT, but not in C3KO cardiomyocytes, (v) after ischemia, WT hearts had higher levels of ATP, but lower levels of both reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) compared to C3KO hearts. Conclusion: intracellular C3 protected the heart against ischemia-reperfusion injury, possibly due to its role in metabolic pathways important for energy production and cell survival.

ObjectiveDysregulation of the complement system has been described in patients with heart failure (HF). However, data on the alternative pathway are scarce and it is unknown if levels of factor B (FB) and the C3 convertase C3bBbP are elevated in these patients. We hypothesized that plasma levels of FB and C3bBbP would be associated with disease severity and survival in patients with HF.MethodsWe analyzed plasma levels of FB, C3bBbP, and terminal C5b-9 complement complex (TCC) in 343 HF patients and 27 healthy controls.ResultsCompared with controls, patients with HF had elevated levels of circulating FB (1.6-fold, p < 0.001) and C3bBbP (1.3-fold, p < 0.001). In contrast, TCC, reflecting the terminal pathway, was not significantly increased (p = 0.15 vs controls). FB was associated with NT-proBNP, troponin, eGFR, and i.e., C-reactive protein. FB, C3bBbP and TCC were not associated with mortality in HF during a mean follow up of 4.3 years.ConclusionOur findings suggest that in patients with HF, the alternative pathway is activated. However, this is not accompanied by activation of the terminal pathway.