Quantification of Porcine Complement Activation Fragment C3a by a Neoepitope-Based Enzyme-Linked Immunosorbent Assay

Nilsson, Per H.; Pettersen, Kristin; Oppermann, Martin; Skjeflo, Espen Waage; Fure, Hilde; Christiansen, Dorte; Mollnes, Tom Eirik

doi:10.1007/978-1-0716-1016-9_5

Cited by 5 publications

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“…Many commercial immunological assays, including complement and cytokine assays, are marketed as working in pigs. Over the years, we have thoroughly tested most available complement assays, comparing them to the reliable C3a and TCC assays described above and in (22)(23)(24); disappointingly, in such comparisons, most assays failed to detect complement activation reliably. Likewise, we have recently studied porcine-specific cytokine assays, finding that many of these are also unreliable (43).…”

Section: Discussionmentioning

confidence: 99%

“…This contrasts the complement activation otherwise seen. For example, in a porcine study of polymicrobial sepsis ( 35 ), both C3a and TCC increased substantially, with C3a appearing first followed by TCC ( 22 ).…”

Section: Discussionmentioning

confidence: 99%

“…In our study, we measured C3a using an ELISA assay with a porcine-specific antibody ( 22 ) and TCC using a human assay shown to work well in swine ( 23 , 24 ), as no reliable porcine C5a assay is commercially available. This is a reasonable approach, as C5a and TCC (sC5b-9) are released in equimolar concentration in plasma.…”

Section: Discussionmentioning

confidence: 99%

“…We measured complement C3a using ELISA with porcine-specific C3a monoclonal antibodies as previously described in detail ( 22 ). The antibody binds to a neoepitope exposed when C3a is cleaved off C3, and the assay only detects free C3a in the fluid phase.…”

Section: Methodsmentioning

confidence: 99%

“…The TNF mRNA expression did not differ between the groups. In line with the mRNA findings, the median lung tissue cytokines measured as protein in pigs receiving air infusion compared to sham animals were: IL-1b 302 pg/mL (IQR 190-437) versus 107 pg/ mL (IQR 66-120), IL-6 644 pg/mL (IQR 358-1094) versus 25 pg/ mL (IQR 23-30), IL-8 203 pg/mL (IQR 81-377) versus 21 pg/mL (IQR [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35], and TNF 113 pg/mL (IQR 96-147) versus 16 pg/mL (IQR 13-22) (all p<0.001) (Figure 4C). Thus, TNF increased as measured by protein but not by mRNA.…”

Section: Cytokines In Plasma and Lung Tissuementioning

confidence: 99%

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Venous Air Embolism Activates Complement C3 Without Corresponding C5 Activation and Trigger Thromboinflammation in Pigs

et al. 2022

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IntroductionAir embolism may complicate invasive medical procedures. Bubbles trigger complement C3-mediated cytokine release, coagulation, and platelet activation in vitro in human whole blood. Since these findings have not been verified in vivo, we aimed to examine the effects of air embolism in pigs on thromboinflammation.MethodsForty-five landrace pigs, average 17 kg (range 8.5-30), underwent intravenous air infusion for 300 or 360 minutes (n=29) or served as sham (n=14). Fourteen pigs were excluded due to e.g. infections or persistent foramen ovale. Blood was analyzed for white blood cells (WBC), complement activation (C3a and terminal C5b-9 complement complex [TCC]), cytokines, and hemostatic parameters including thrombin-antithrombin (TAT) using immunoassays and rotational thromboelastometry (ROTEM). Lung tissue was analyzed for complement and cytokines using qPCR and immunoassays. Results are presented as medians with interquartile range.ResultsIn 24 pigs receiving air infusion, WBC increased from 17×109/L (10-24) to 28 (16-42) (p<0.001). C3a increased from 21 ng/mL (15-46) to 67 (39-84) (p<0.001), whereas TCC increased only modestly (p=0.02). TAT increased from 35 µg/mL (28-42) to 51 (38-89) (p=0.002). ROTEM changed during first 120 minutes: Clotting time decreased from 613 seconds (531-677) to 538 (399-620) (p=0.006), clot formation time decreased from 161 seconds (122-195) to 124 (83-162) (p=0.02) and α-angle increased from 62 degrees (57-68) to 68 (62-74) (p=0.02). In lungs from pigs receiving air compared to sham animals, C3a was 34 ng/mL (14-50) versus 4.1 (2.4-5.7) (p<0.001), whereas TCC was 0.3 CAU/mL (0.2-0.3) versus 0.2 (0.1-0.2) (p=0.02). Lung cytokines in pigs receiving air compared to sham animals were: IL-1β 302 pg/mL (190-437) versus 107 (66-120), IL-6 644 pg/mL (358-1094) versus 25 (23-30), IL-8 203 pg/mL (81-377) versus 21 (20-35), and TNF 113 pg/mL (96-147) versus 16 (13-22) (all p<0.001). Cytokine mRNA in lung tissue from pigs receiving air compared to sham animals increased 12-fold for IL-1β, 121-fold for IL-6, and 17-fold for IL-8 (all p<0.001).ConclusionVenous air embolism in pigs activated C3 without a corresponding C5 activation and triggered thromboinflammation, consistent with a C3-dependent mechanism. C3-inhibition might represent a therapeutic approach to attenuate this response.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Cytokines In Plasma and Lung Tissuementioning

confidence: 99%

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Venous Air Embolism Activates Complement C3 Without Corresponding C5 Activation and Trigger Thromboinflammation in Pigs

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Complement Is Activated During Normothermic Machine Perfusion of Porcine and Human Discarded Kidneys

et al. 2022

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BackgroundThe gap between demand and supply of kidneys for transplantation necessitates the use of kidneys from extended criteria donors. Transplantation of these donor kidneys is associated with inferior results, reflected by an increased risk of delayed graft function. Inferior results might be explained by the higher immunogenicity of extended criteria donor kidneys. Normothermic machine perfusion (NMP) could be used as a platform to assess the quality and function of donor kidneys. In addition, it could be useful to evaluate and possibly alter the immunological response of donor kidneys. In this study, we first evaluated whether complement was activated during NMP of porcine and human discarded kidneys. Second, we examined the relationship between complement activation and pro-inflammatory cytokines during NMP. Third, we assessed the effect of complement activation on renal function and injury during NMP of porcine kidneys. Lastly, we examined local complement C3d deposition in human renal biopsies after NMP.MethodsNMP with a blood-based perfusion was performed with both porcine and discarded human kidneys for 4 and 6 h, respectively. Perfusate samples were taken every hour to assess complement activation, pro-inflammatory cytokines and renal function. Biopsies were taken to assess histological injury and complement deposition.ResultsComplement activation products C3a, C3d, and soluble C5b-9 (sC5b-9) were found in perfusate samples taken during NMP of both porcine and human kidneys. In addition, complement perfusate levels positively correlated with the cytokine perfusate levels of IL-6, IL-8, and TNF during NMP of porcine kidneys. Porcine kidneys with high sC5b-9 perfusate levels had significantly lower creatinine clearance after 4 h of NMP. In line with these findings, high complement perfusate levels were seen during NMP of human discarded kidneys. In addition, kidneys retrieved from brain-dead donors had significantly higher complement perfusate levels during NMP than kidneys retrieved from donors after circulatory death.ConclusionNormothermic kidney machine perfusion induces complement activation in porcine and human kidneys, which is associated with the release of pro-inflammatory cytokines and in porcine kidneys with lower creatinine clearance. Complement inhibition during NMP might be a promising strategy to reduce renal graft injury and improve graft function prior to transplantation.

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A Novel Porcine Model of Ischemia-Reperfusion Injury After Cross-Clamping the Thoracic Aorta Revealed Substantial Cardiopulmonary, Thromboinflammatory and Biochemical Changes Without Effect of C1-Inhibitor Treatment

et al. 2022

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Ischemic injury worsens upon return of blood and innate immunity including the complement system play a central role in ischemia-reperfusion injury (IRI) as in thoracic aortic surgery. Complement component1 inhibitor (C1-INH) has been shown to reduce IRI and is a broad-acting plasma cascade inhibitor. We established a new porcine model of IRI by cross-clamping the thoracic aorta and evaluated the global changes occurring in organ function, systemic inflammatory response and organ damage with or without treatment with C1-INH-concentrate. Twenty-four piglets (8.8-11.1 kg) underwent 45 minutes clamping of the thoracic aorta at the Th8 level. Upfront 12 piglets received human saline and 12 received C1-INH (250 IU/kg) intravenously. Three sham animals received thoracic opening without clamping. Reperfusion lasted 5 hours. We studied ten cardiorespiratory markers, three hematologic markers, eleven inflammatory markers, and twelve organ damage markers over the whole experimental period. Postmortem tissue homogenates from seven organs were examined for inflammatory markers and analysed by two-way repeated-measures ANOVA, area under the curve or unpaired t-tests. By excluding sham and combining treated and untreated animals, the markers reflected a uniform, broad and severe organ dysfunction. The mean and range fold change from before cross-clamp onset to maximum change for the different groups of markers were: cardiorespiratory 1.4 (0.2-3.7), hematologic 1.9 (1.2-2.7), plasma inflammatory 19.5 (1.4-176) and plasma organ damage 2.9 (1.1-8.6). Treatment with C1-INH had only a marginal effect on the IRI-induced changes, reaching statistical significance only for the plasma complement activation product TCC (p=0.0083) and IL-4 (p=0.022) and INF-α (p=0.016) in the colon tissue. In conclusion, the present novel model of porcine global IRI is forceful with regards to central markers and could generally be applicable for pathophysiological studies. C1-INH treatment had no significant effect, but the model allows for future testing of other drugs attenuating IRI globally.

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Quantification of Porcine Complement Activation Fragment C3a by a Neoepitope-Based Enzyme-Linked Immunosorbent Assay

Cited by 5 publications

References 5 publications

Venous Air Embolism Activates Complement C3 Without Corresponding C5 Activation and Trigger Thromboinflammation in Pigs

Venous Air Embolism Activates Complement C3 Without Corresponding C5 Activation and Trigger Thromboinflammation in Pigs

Complement Is Activated During Normothermic Machine Perfusion of Porcine and Human Discarded Kidneys

A Novel Porcine Model of Ischemia-Reperfusion Injury After Cross-Clamping the Thoracic Aorta Revealed Substantial Cardiopulmonary, Thromboinflammatory and Biochemical Changes Without Effect of C1-Inhibitor Treatment

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