Choice of immunoassay to evaluate porcine cytokine levels

Ueland, Nora Lamark; Ludvigsen, Judith K.; Hellerud, Bernt Christian; Mollnes, Tom Eirik; Skjeflo, Espen Waage

doi:10.1016/j.vetimm.2020.110129

Cited by 4 publications

(5 citation statements)

References 7 publications

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“…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%

“…Despite many similarities between humans and pigs, human immunoassays may not reliably cross-react and work in pigs. In this study, we limited complement readouts to C3a and TCC, and we used only cytokine assays shown to work in pigs as detailed in ( 43 ). Many commercial immunological assays, including complement and cytokine assays, are marketed as working in pigs.…”

Section: Discussionmentioning

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%

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

et al. 2022

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“…Terminal complement complex (TCC) and C3a were measured using in-house ELISA, as described in detail previously [ 22 , 23 ]. Tumor necrosis factor (TNF), IL-1b, IL-6, IL-8, and IL-10 were measured using commercially available ELISA and multiplex assays as described previously described [ 24 ].…”

Section: Methodsmentioning

confidence: 99%

H2S-Enriched Flush out Does Not Increase Donor Organ Quality in a Porcine Kidney Perfusion Model

et al. 2023

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Kidney extraction time has a detrimental effect on post-transplantation outcome. This study aims to improve the flush-out and potentially decrease ischemic injury by the addition of hydrogen sulphide (H2S) to the flush medium. Porcine kidneys (n = 22) were extracted during organ recovery surgery. Pigs underwent brain death induction or a sham operation, resulting in four groups: donation after brain death (DBD) control, DBD H2S, non-DBD control, and non-DBD H2S. Directly after the abdominal flush, kidneys were extracted and flushed with or without H2S and stored for 13 h via static cold storage (SCS) +/− H2S before reperfusion on normothermic machine perfusion. Pro-inflammatory cytokines IL-1b and IL-8 were significantly lower in H2S treated DBD kidneys during NMP (p = 0.03). The non-DBD kidneys show superiority in renal function (creatinine clearance and FENa) compared to the DBD control group (p = 0.03 and p = 0.004). No differences were seen in perfusion parameters, injury markers and histological appearance. We found an overall trend of better renal function in the non-DBD kidneys compared to the DBD kidneys. The addition of H2S during the flush out and SCS resulted in a reduction in pro-inflammatory cytokines without affecting renal function or injury markers.

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“…The immunoassays for these analyses were chosen based on previous assessment of the different commercially available kits ( 33 ). Porcine ELISAs were used to quantify the interleukin 6 (IL-6) (lower detection limit: 9.4 pg/ml) and tumor necrosis factor (TNF) (lower detection limit: 11.7 pg/ml) (R&D Systems, MN); plasminogen activator inhibitor-1 (PAI-1) (lower detection limit: 3.1 ng/ml) (Nordic BioSite AB, Taby, Sweden) and neutrophil gelatinase-associated lipocalin (NGAL) (lower detection limit: 4.0 pg/ml) (Abcam, Cambridge, UK).…”

Section: Methodsmentioning

confidence: 99%

Simultaneous C5 and CD14 inhibition limits inflammation and organ dysfunction in pig polytrauma

et al. 2022

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Dysfunctional complement activation and Toll-like receptor signaling immediately after trauma are associated with development of trauma-induced coagulopathy and multiple organ dysfunction syndrome. We assessed the efficacy of the combined inhibition therapy of complement factor C5 and the TLR co-receptor CD14 on thrombo-inflammation and organ damage in an exploratory 72-h polytrauma porcine model, conducted under standard surgical and intensive care management procedures. Twelve male pigs were subjected to polytrauma, followed by resuscitation (ATLS® guidelines) and operation of the femur fracture (intramedullary nailing technique). The pigs were allocated to combined C5 and CD14 inhibition therapy group (n=4) and control group (n=8). The therapy group received intravenously C5 inhibitor (RA101295) and anti-CD14 antibody (rMil2) 30 min post-trauma. Controls received saline. Combined C5 and CD14 inhibition reduced the blood levels of the terminal complement complex (TCC) by 70% (p=0.004), CRP by 28% (p=0.004), and IL-6 by 52% (p=0.048). The inhibition therapy prevented the platelet consumption by 18% and TAT formation by 77% (p=0.008). Moreover, the norepinephrine requirements in the treated group were reduced by 88%. The inhibition therapy limited the organ damage, thereby reducing the blood lipase values by 50% (p=0.028), LDH by 30% (p=0.004), AST by 33%, and NGAL by 30%. Immunofluorescent analysis of the lung tissue revealed C5b-9 deposition on blood vessels in five from the untreated, and in none of the treated animals. In kidney and liver, the C5b-9 deposition was similarly detected mainly the untreated as compared to the treated animals. Combined C5 and CD14 inhibition limited the inflammatory response, the organ damage, and reduced the catecholamine requirements after experimental polytrauma and might be a promising therapeutic approach.

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Choice of immunoassay to evaluate porcine cytokine levels

Cited by 4 publications

References 7 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

H2S-Enriched Flush out Does Not Increase Donor Organ Quality in a Porcine Kidney Perfusion Model

Simultaneous C5 and CD14 inhibition limits inflammation and organ dysfunction in pig polytrauma

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