Author Correction: 'Stealth' corporate innovation: an emerging threat for therapeutic drug development.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Application of the C3 inhibitor compstatin in a human whole blood model designed for complement research - 20 years of experience and future perspectives.

The complex molecular and cellular biological systems that maintain host homeostasis undergo continuous crosstalk. Complement, a component of innate immunity, is one such system. Initially regarded as a system to protect the host from infection, complement has more recently been shown to have numerous other functions, including involvement in embryonic development, tissue modeling, and repair. Furthermore, the complement system plays a major role in the pathophysiology of many diseases. Through interactions with other plasma cascades, including hemostasis, complement activation leads to the broad host-protective response known as thromboinflammation. Most complement research has been limited to reductionistic models of purified components and cells and their interactions in vitro. However, to study the pathophysiology of complement-driven diseases, including the interaction between the complement system and other inflammatory systems, holistic models demonstrating only minimal interference with complement activity are needed. Here we describe two such models; whole blood anticoagulated with either the thrombin inhibitor lepirudin or the fibrin polymerization peptide blocker GPRP, both of which retain complement activity and preserve the ability of complement to be mutually reactive with other inflammatory systems. For instance, to examine the relative roles of C3 and C5 in complement activation, it is possible to compare the effects of the C3 inhibitor compstatin effects to those of inhibitors of C5 and C5aR1. We also discuss how complement is activated by both pathogen-associated molecular patterns, inducing infectious inflammation caused by organisms such as Gram-negative and Gram-positive bacteria, and by sterile damage-associated molecular patterns, including cholesterol crystals and artificial materials used in clinical medicine. When C3 is inhibited, it is important to determine the mechanism by which inflammation is attenuated, i.e., whether the attenuation derives directly from C3 activation products or via downstream activation of C5, since the mechanism involved may determine the appropriate choice of inhibitor under various conditions. With some exceptions, most inflammatory responses are dependent on C5 and C5aR1; one exception is venous air embolism, in which air bubbles enter the blood circulation and trigger a mainly C3-dependent thromboembolism, with the formation of an active C3 convertase, without a corresponding C5 activation. Under such conditions, an inhibitor of C3 is needed to attenuate the inflammation. Our holistic blood models will be useful for further studies of the inhibition of any complement target, not just C3 or C5. The focus here will be on targeting the critical complement component, activation product, or receptor that is important for the pathophysiology in a variety of disease conditions.

Synthetic Oligodeoxynucleotide CpG Motifs Activate Human Complement through Their Backbone Structure and Induce Complement-Dependent Cytokine Release.

Bacterial and mitochondrial DNA, sharing an evolutionary origin, act as danger-associated molecular patterns in infectious and sterile inflammation. They both contain immunomodulatory CpG motifs. Interactions between CpG motifs and the complement system are sparsely described, and mechanisms of complement activation by CpG remain unclear. Lepirudin-anticoagulated human whole blood and plasma were incubated with increasing concentrations of three classes of synthetic CpGs: CpG-A, -B, and -C oligodeoxynucleotides and their GpC sequence controls. Complement activation products were analyzed by immunoassays. Cytokine levels were determined via 27-plex beads-based immunoassay, and CpG interactions with individual complement proteins were evaluated using magnetic beads coated with CpG-B. In whole blood and plasma, CpG-B and CpG-C (p < 0.05 for both), but not CpG-A (p > 0.8 for all), led to time- and dose-dependent increase of soluble C5b-9, the alternative complement convertase C3bBbP, and the C3 cleavage product C3bc. GpC-A, -B, and -C changed soluble fluid-phase C5b-9, C3bBbP, and C3bc to the same extent as CpG-A, -B, and -C, indicating a DNA backbone-dependent effect. Dose-dependent CpG-B binding was found to C1q (r = 0.83; p = 0.006) and factor H (r = 0.93; p < 0.001). The stimulatory complement effect was partly preserved in C2-deficient plasma and completely preserved in MASP-2-deficient serum. CpG-B increased levels of IL-1ß, IL-2, IL-6, IL-8, MCP-1, and TNF in whole blood, which were completely abolished by inhibition of C5 and C5aR1 (p < 0.05 for all). In conclusion, synthetic analogs of bacterial and mitochondrial DNA activate the complement system via the DNA backbone. We suggest that CpG-B interacts directly with classical and alternative pathway components, resulting in complement-C5aR1-dependent cytokine release.

Therapeutic Targeting of the Complement System: From Rare Diseases to Pandemics.

The complement system was discovered at the end of the 19th century as a heat-labile plasma component that "complemented" the antibodies in killing microbes, hence the name "complement." Complement is also part of the innate immune system, protecting the host by recognition of pathogen-associated molecular patterns. However, complement is multifunctional far beyond infectious defense. It contributes to organ development, such as sculpting neuron synapses, promoting tissue regeneration and repair, and rapidly engaging and synergizing with a number of processes, including hemostasis leading to thromboinflammation. Complement is a double-edged sword. Although it usually protects the host, it may cause tissue damage when dysregulated or overactivated, such as in the systemic inflammatory reaction seen in trauma and sepsis and severe coronavirus disease 2019 (COVID-19). Damage-associated molecular patterns generated during ischemia-reperfusion injuries (myocardial infarction, stroke, and transplant dysfunction) and in chronic neurologic and rheumatic disease activate complement, thereby increasing damaging inflammation. Despite the long list of diseases with potential for ameliorating complement modulation, only a few rare diseases are approved for clinical treatment targeting complement. Those currently being efficiently treated include paroxysmal nocturnal hemoglobinuria, atypical hemolytic-uremic syndrome, myasthenia gravis, and neuromyelitis optica spectrum disorders. Rare diseases, unfortunately, preclude robust clinical trials. The increasing evidence for complement as a pathogenetic driver in many more common diseases suggests an opportunity for future complement therapy, which, however, requires robust clinical trials; one ongoing example is COVID-19 disease. The current review aims to discuss complement in disease pathogenesis and discuss future pharmacological strategies to treat these diseases with complement-targeted therapies. SIGNIFICANCE STATEMENT: The complement system is the host's defense friend by protecting it from invading pathogens, promoting tissue repair, and maintaining homeostasis. Complement is a double-edged sword, since when dysregulated or overactivated it becomes the host's enemy, leading to tissue damage, organ failure, and, in worst case, death. A number of acute and chronic diseases are candidates for pharmacological treatment to avoid complement-dependent damage, ranging from the well established treatment for rare diseases to possible future treatment of large patient groups like the pandemic coronavirus disease 2019.

Systemic complement activation is associated with respiratory failure in COVID-19 hospitalized patients.

Respiratory failure in the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is hypothesized to be driven by an overreacting innate immune response, where the complement system is a key player. In this prospective cohort study of 39 hospitalized coronavirus disease COVID-19 patients, we describe systemic complement activation and its association with development of respiratory failure. Clinical data and biological samples were obtained at admission, days 3 to 5, and days 7 to 10. Respiratory failure was defined as PO2/FiO2 ratio of ≤40 kPa. Complement activation products covering the classical/lectin (C4d), alternative (C3bBbP) and common pathway (C3bc, C5a, and sC5b-9), the lectin pathway recognition molecule MBL, and antibody serology were analyzed by enzyme-immunoassays; viral load by PCR. Controls comprised healthy blood donors. Consistently increased systemic complement activation was observed in the majority of COVID-19 patients during hospital stay. At admission, sC5b-9 and C4d were significantly higher in patients with than without respiratory failure (P = 0.008 and P = 0.034). Logistic regression showed increasing odds of respiratory failure with sC5b-9 (odds ratio 31.9, 95% CI 1.4 to 746, P = 0.03) and need for oxygen therapy with C4d (11.7, 1.1 to 130, P = 0.045). Admission sC5b-9 and C4d correlated significantly to ferritin (r = 0.64, P < 0.001; r = 0.69, P < 0.001). C4d, sC5b-9, and C5a correlated with antiviral antibodies, but not with viral load. Systemic complement activation is associated with respiratory failure in COVID-19 patients and provides a rationale for investigating complement inhibitors in future clinical trials.

Elevated plasma sTIM-3 levels in patients with severe COVID-19.

BACKGROUND: The pathogenesis of coronavirus disease 2019 (COVID-19) is still incompletely understood, but it seems to involve immune activation and immune dysregulation. OBJECTIVE: We examined the parameters of activation of different leukocyte subsets in COVID-19-infected patients in relation to disease severity. METHODS: We analyzed plasma levels of myeloperoxidase (a marker of neutrophil activation), soluble (s) CD25 (sCD25) and soluble T-cell immunoglobulin mucin domain-3 (sTIM-3) (markers of T-cell activation and exhaustion), and sCD14 and sCD163 (markers of monocyte/macrophage activation) in 39 COVID-19-infected patients at hospital admission and 2 additional times during the first 10 days in relation to their need for intensive care unit (ICU) treatment. RESULTS: Our major findings were as follows: (1) severe clinical outcome (ICU treatment) was associated with high plasma levels of sTIM-3 and myeloperoxidase, suggesting activated and potentially exhausted T cells and activated neutrophils, respectively; (2) in contrast, sCD14 and sCD163 showed no association with need for ICU treatment; and (3) levels of sCD25, sTIM-3, and myeloperoxidase were inversely correlated with degree of respiratory failure, as assessed by the ratio of Pao2 to fraction of inspired oxygen, and were positively correlated with the cardiac marker N-terminal pro-B-type natriuretic peptide. CONCLUSION: Our findings suggest that neutrophil activation and, in particular, activated T cells may play an important role in the pathogenesis of COVID-19 infection, suggesting that T-cell-targeted treatment options and downregulation of neutrophil activation could be of importance in this disorder.

Cholesterol Crystals Induce Coagulation Activation through Complement-Dependent Expression of Monocytic Tissue Factor.

Cholesterol crystals (CC) are strong activators of complement and could potentially be involved in thromboinflammation through complement-coagulation cross-talk. To explore the coagulation-inducing potential of CC, we performed studies in lepirudin-based human whole blood and plasma models. In addition, immunohistological examinations of brain thrombi and vulnerable plaque material from patients with advanced carotid atherosclerosis were performed using polarization filter reflected light microscopy to identify CC. In whole blood, CC exposure induced a time- and concentration-dependent generation of prothrombin fragment 1+2 (PTF1.2), tissue factor (TF) mRNA synthesis, and monocyte TF expression. Blocking Abs against TF abolished CC-mediated coagulation, thus indicating involvement of the TF-dependent pathway. Blockade of FXII by corn trypsin inhibitor had a significant inhibitory effect on CC-induced PTF1.2 in platelet-free plasma, although the overall activation potential was low. CC exposure did not induce platelet aggregation, TF microparticle induction, or TF on granulocytes or eosinophils. Inhibition of complement C3 by CP40 (compstatin), C5 by eculizumab, or C5aR1 by PMX53 blocked CC-induced PTF1.2 by 90% and reduced TF+ monocytes from 18-20 to 1-2%. The physiologic relevance was supported by birefringent CC structures adjacent to monocytes (CD14), TF, and activated complement iC3b and C5b-9 in a human brain thrombus. Furthermore, monocyte influx and TF induction in close proximity to CC-rich regions with activated complement were found in a vulnerable plaque. In conclusion, CC could be active, releasable contributors to thrombosis by inducing monocyte TF secondary to complement C5aR1 signaling.

Open chest and pericardium facilitate transpulmonary passage of venous air emboli.

BACKGROUND: Transpulmonary passage of air emboli can lead to fatal brain- and myocardial infarctions. We studied whether pigs with open chest and pericardium had a greater transpulmonary passage of venous air emboli than pigs with closed thorax. METHODS: We allocated pigs with verified closed foramen ovale to venous air infusion with either open chest with sternotomy and opening of the pleura and pericardium (n = 8) or closed thorax (n = 16). All pigs received a five-hour intravenous infusion of ambient air, starting at 4-6 mL/kg/h and increased by 2 mL/kg/h each hour. We assessed transpulmonary air passage by transesophageal M-mode echocardiography and present the results as median with inter-quartile range (IQR). RESULTS: Transpulmonary air passage occurred in all pigs with open chest and pericardium and in nine pigs with closed thorax (56%). Compared to pigs with closed thorax, pigs with open chest and pericardium had a shorter to air passage (10 minutes (5-16) vs. 120 minutes (44-212), P < .0001), a smaller volume of infused air at the time of transpulmonary passage (12 mL (10-23) vs.170 mL (107-494), P < .0001), shorter time to death (122 minutes (48-185) vs 263 minutes (248-300, P = .0005) and a smaller volume of infused air at the time of death (264 mL (53-466) vs 727 mL (564-968), P = .001). In pigs with open chest and, infused air and time to death correlated strongly (r = 0.95, P = .001). CONCLUSION: Open chest and pericardium facilitated the transpulmonary passage of intravenously infused air in pigs.

Comparison of cytokine changes in three different lipoprotein apheresis systems in an ex vivo whole blood model.

INTRODUCTION: Even if proprotein convertase subtilisin/kexin type 9 inhibitors have replaced lipoprotein apheresis in many patients, lipoprotein apheresis still is an important option in homozygous familial hypercholesterolemia, progressive atherosclerosis or when removal of lipoprotein(a) is indicated. Additional possible favorable effects beyond lipid lowering could include changes in the concentration of cytokines and improvement of hemorheology. METHODS: We evaluated how whole blood adsorption, dextran sulfate plasma adsorption, and double filtration plasmapheresis lipoprotein apheresis systems affected cytokine concentrations, using a human whole blood ex vivo model differentiating the effect of the lipoprotein apheresis and plasma separation columns and describing temporal changes. RESULTS: Compared to the control bag, the whole blood adsorption system reduced Interferon-γ (IFN-γ), IL-8, IL-1ra, eotaxin, tumor necrosis factor (TNF), monocyte chemoattractant protein 1 (MCP-1), platelet derived growth factor (PDGF)-BB, regulated on activation T cell expressed and secreted (RANTES), macrophage inflammatory protein-1ß (MIP-1ß), and IP-10 (P < .05). The dextran sulfate plasma adsorption system reduced IFN-γ, IL-8, IL-1ra, eotaxin, TNF, MCP-1, PDGF-BB, MIP-1ß, and IP-10 (P < .05). Vascular endothelial growth factor (VEGF) and granulocyte macrophage colony stimulating factor (GM-CSF) were increased in the whole blood and dextran sulfate plasma adsorption systems (P < .05). The double filtration plasmapheresis system reduced IFN-γ, IL-1ra, TNF, MIP-1ß, and IP-10 (P < .05), while MCP-1,VEGF, GM-CSF, and RANTES were increased (P < .05). The plasma separation column increased concentration of RANTES, and was a barrier to reduction of eotaxin. Temporal patterns of concentration change indicated first pass increase of PDGF-BB and first pass reduction of IP-10. CONCLUSION: There were marked differences in how the three systems affected total and temporal cytokine concentration changes in this in vitro model, as well as compared to former in vivo studies.

Biphasic Release of the Alarmin High Mobility Group Box 1 Protein Early After Trauma Predicts Poor Clinical Outcome.

OBJECTIVES: The causal role of the prototype alarmin high mobility group box 1 protein in systemic inflammation and remote organ injury after trauma and shock is established in animal models but not in humans. Our aim was therefore to determine high mobility group box 1 protein concentration kinetics with high time resolution during the first hours after trauma in individual patients and investigate the association with outcome. DESIGN: Prospective single-center observational study. SETTING: University hospital Level I trauma center. PATIENTS: Convenience recruitment of 136 trauma patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Total plasma high mobility group box 1 protein levels were analyzed with enzyme-linked immunosorbent assay in repeated samples. Relationships between predefined predictor variables and outcome were examined in multivariable linear regression models. Ventilator-free days was used as primary outcome measure. Two distinct high mobility group box 1 protein release phases were identified. An initial exponential decay phase with half-life 26 minutes was not correlated with outcome. In contrast, a second high mobility group box 1 protein wave peaking 3-6 hours after trauma in the most severely injured and physiologically deranged patients was consistently the most important predictor of outcome in our multivariable models, rendering all other predictor variables insignificant except for smaller contributions from age and sex, and of admission base excess for maximal creatinine concentration. CONCLUSIONS: High mobility group box 1 protein was released in two consecutive phases. Only the second high mobility group box 1 protein wave was a significant predictor of outcome. Patients with a high high mobility group box 1 protein concentration between 3 and 6 hours after trauma might hypothetically benefit from high mobility group box 1 protein-specific antagonist therapy.

Cyclodextrin Reduces Cholesterol Crystal-Induced Inflammation by Modulating Complement Activation.

Cholesterol crystals (CC) are abundant in atherosclerotic plaques and promote inflammatory responses via the complement system and inflammasome activation. Cyclic oligosaccharide 2-hydroxypropyl-ß-cyclodextrin (BCD) is a compound that solubilizes lipophilic substances. Recently we have shown that BCD has an anti-inflammatory effect on CC via suppression of the inflammasome and liver X receptor activation. The putative effects of BCD on CC-induced complement activation remain unknown. In this study, we found that BCD bound to CC and reduced deposition of Igs, pattern recognition molecules, and complement factors on CC in human plasma. Furthermore, BCD decreased complement activation as measured by terminal complement complex and lowered the expression of complement receptors on monocytes in whole blood in response to CC exposure. In line with this, BCD also reduced reactive oxygen species formation caused by CC in whole blood. Furthermore, BCD attenuated the CC-induced proinflammatory cytokine responses (e.g., IL-1α, MIP-1α, TNF, IL-6, and IL-8) as well as regulated a range of CC-induced genes in human PBMC. BCD also regulated complement-related genes in human carotid plaques treated ex vivo. Formation of terminal complement complex on other complement-activating structures such as monosodium urate crystals and zymosan was not affected by BCD. These data demonstrate that BCD inhibits CC-induced inflammatory responses, which may be explained by BCD-mediated attenuation of complement activation. Thus, these findings support the potential for using BCD in treatment of atherosclerosis.

Cytokine responses, microbial aetiology and short-term outcome in community-acquired pneumonia.

BACKGROUND: The inflammatory response to community-acquired pneumonia (CAP) is orchestrated through activation of cytokine networks and the complement system. We examined the association of multiple cytokines and the terminal complement complex (TCC) with microbial aetiology, disease severity and short-term outcome. MATERIALS AND METHODS: Plasma levels of 27 cytokines and TCC were analysed in blood samples obtained at hospital admission, clinical stabilization and 6-week follow-up from 247 hospitalized adults with CAP. Fourteen mediators were included in final analyses. Adverse short-term outcome was defined as intensive care unit (ICU) admission and 30-day mortality. RESULTS: Cytokine and TCC levels were dynamic in the clinical course of CAP, with highest levels seen at admission for most mediators. Admission levels of cytokines and TCC did not differ between groups of microbial aetiology. High admission levels of IL-6 (odds ratio [OR] 1.47, 95% confidence interval [CI] 1.18-1.84, P = .001), IL-8 (OR 1.79, 95% CI 1.26-2.55, P = .001) and MIP-1ß (OR 2.28, 95% CI 1.36-3.81, P = .002) were associated with a CURB-65 severity score of ≥3, while IL-6 (OR 1.37, 95% CI 1.07-1.74, P = .011) and MIP-1ß (OR 1.86, 95% CI 1.03-3.36, P = .040) were associated with a high risk of an adverse short-term outcome. CONCLUSIONS: In this CAP cohort, admission levels of IL-6, IL-8 and MIP-1ß were associated with disease severity and/or adverse short-term outcome. Still, for most mediators, only nonsignificant variations in inflammatory responses were observed for groups of microbial aetiology, disease severity and short-term outcome.

Effect of the anticoagulant, storage time and temperature of blood samples on the concentrations of 27 multiplex assayed cytokines - Consequences for defining reference values in healthy humans.

Cytokines are potentially useful biomarkers of sepsis and other inflammatory conditions. Many cytokines can be released by leukocytes and platelets after sampling. The sampling and processing techniques are consequently critically important to measure the in vivo levels. We therefore examined the effects of four different anticoagulants, EDTA, citrate, lepirudin, heparin compared to serum, on the levels of 27 different cytokines. The effects of storage temperature, freezing and thawing on the plasma cytokines were examined. Cytokines were analysed using a multiplex immunoassay. The cytokine levels in serum were significantly higher compared with plasma, consistent with release of cytokines in vitro during coagulation. In general, the lowest values for all cytokines were found in EDTA samples, stored on crushed ice, centrifuged within 4h and thereafter stored at -80°C. MCP-1 and MIP-1ß levels were highest in heparin plasma and storage of blood for up to 4h at room temperature significantly increased the interleukin (IL)-2, IL-6, IL-8, IFN-γ and GM-CSF levels in EDTA plasma, indicating post-sampling release. In contrast, the IP-10 levels were unaffected by sample storage at both temperatures. Our results indicate that the cytokines were more stable in plasma than in whole blood after sampling. Thus, cytokines should be analysed in EDTA plasma samples stored on ice and centrifuged within 4h. Based on these data, the reference ranges of 27 cytokines in EDTA plasma in 162 healthy human donors were calculated.

TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-ß Production via a TAK1-IKKß-IRF5 Signaling Pathway.

Staphylococcus aureus may cause serious infections and is one of the most lethal and common causes of sepsis. TLR2 has been described as the main pattern recognition receptor that senses S. aureus and elicits production of proinflammatory cytokines via MyD88 -: NF-κB signaling. S. aureus can also induce the production of IFN-ß, a cytokine that requires IFN regulatory factors (IRFs) for its transcription, but the signaling mechanism for IFN-ß induction by S. aureus are unclear. Surprisingly, we demonstrate that activation of TLR2 by lipoproteins does not contribute to IFN-ß production but instead can suppress the induction of IFN-ß in human primary monocytes and monocyte-derived macrophages. The production of IFN-ß was induced by TLR8-mediated sensing of S. aureus RNA, which triggered IRF5 nuclear accumulation, and this could be antagonized by concomitant TLR2 signaling. The TLR8-mediated activation of IRF5 was dependent on TAK1 and IκB kinase (IKK)ß, which thus reveals a physiological role of the recently described IRF5-activating function of IKKß. TLR8 -: IRF5 signaling was necessary for induction of IFN-ß and IL-12 by S. aureus, and it also contributed to the induction of TNF. In conclusion, our study demonstrates a physiological role of TLR8 in the sensing of entire S. aureus in human primary phagocytes, including the induction of IFN-ß and IL-12 production via a TAK1 -: IKKß -: IRF5 pathway that can be inhibited by TLR2 signaling.

Reconstituted High-Density Lipoprotein Attenuates Cholesterol Crystal-Induced Inflammatory Responses by Reducing Complement Activation.

Chronic inflammation of the arterial wall is a key element in the development of atherosclerosis, and cholesterol crystals (CC) that accumulate in plaques are associated with initiation and progression of the disease. We recently revealed a link between the complement system and CC-induced inflammasome caspase-1 activation, showing that the complement system is a key trigger in CC-induced inflammation. HDL exhibits cardioprotective and anti-inflammatory properties thought to explain its inverse correlation to cardiovascular risk. In this study, we sought to determine the effect of reconstituted HDL (rHDL) on CC-induced inflammation in a human whole blood model. rHDL bound to CC and inhibited the CC-induced complement activation as measured by soluble terminal C5b-9 formation and C3c deposition on the CC surface. rHDL attenuated the amount of CC-induced complement receptor 3 (CD11b/CD18) expression on monocytes and granulocytes, as well as reactive oxygen species generation. Moreover, addition of CC to whole blood resulted in release of proinflammatory cytokines that were inhibited by rHDL. Our results support and extend the notion that CC are potent triggers of inflammation, and that rHDL may have a beneficial role in controlling the CC-induced inflammatory responses by inhibiting complement deposition on the crystals.

Plasma Levels of Marine n-3 Fatty Acids Are Inversely Correlated With Proinflammatory Markers sTNFR1 and IL-6 in Renal Transplant Recipients.

OBJECTIVE: Marine n-3 polyunsaturated fatty acids (PUFAs) exert potential anti-inflammatory effects and might improve long-term outcomes after renal transplantation. We assessed associations between plasma phospholipid levels of marine n-3 PUFAs and plasma inflammatory biomarkers 10 weeks after renal transplantation. DESIGN: Cross-sectional single-center study. SUBJECTS: A study population of 861 renal transplant recipients transplanted at Oslo University Hospital between 2007 and 2011. METHODS AND MAIN OUTCOME MEASURE: Plasma phospholipid fatty acids were determined by gas chromatography. Marine n-3 PUFA levels were defined as the sum of eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid levels in weight percentage of total plasma phospholipid fatty acids. Plasma inflammatory biomarkers were measured by enzyme immunoassays. We used multivariable linear regression analysis to assess associations between levels of marine n-3 PUFAs and inflammatory biomarkers in plasma. RESULTS: Plasma marine n-3 PUFA levels were inversely associated with plasma levels of proinflammatory biomarkers soluble tumor necrosis factor receptor 1 (standardized regression coefficient -0.11, P < .001) and interleukin-6 (standardized regression coefficient -0.09, P = .01). In contrast, there was no association between plasma levels of marine n-3 PUFAs and the anti-inflammatory mediator interleukin-10. CONCLUSIONS: In this renal transplant cohort, inverse associations between plasma levels of marine n-3 PUFAs and markers of inflammation were demonstrated.

Combined Inhibition of Complement and CD14 Attenuates Bacteria-Induced Inflammation in Human Whole Blood More Efficiently Than Antagonizing the Toll-like Receptor 4-MD2 Complex.

BACKGROUND: Single inhibition of the Toll-like receptor 4 (TLR4)-MD2 complex failed in treatment of sepsis. CD14 is a coreceptor for several TLRs, including TLR4 and TLR2. The aim of this study was to investigate the effect of single TLR4-MD2 inhibition by using eritoran, compared with the effect of CD14 inhibition alone and combined with the C3 complement inhibitor compstatin (Cp40), on the bacteria-induced inflammatory response in human whole blood. METHODS: Cytokines were measured by multiplex technology, and leukocyte activation markers CD11b and CD35 were measured by flow cytometry. RESULTS: Lipopolysaccharide (LPS)-induced inflammatory markers were efficiently abolished by both anti-CD14 and eritoran. Anti-CD14 was significantly more effective than eritoran in inhibiting LPS-binding to HEK-293E cells transfected with CD14 and Escherichia coli-induced upregulation of monocyte activation markers (P < .01). Combining Cp40 with anti-CD14 was significantly more effective than combining Cp40 with eritoran in reducing E. coli-induced interleukin 6 (P < .05) and monocyte activation markers induced by both E. coli (P < .001) and Staphylococcus aureus (P < .01). Combining CP40 with anti-CD14 was more efficient than eritoran alone for 18 of 20 bacteria-induced inflammatory responses (mean P < .0001). CONCLUSIONS: Whole bacteria-induced inflammation was inhibited more efficiently by anti-CD14 than by eritoran, particularly when combined with complement inhibition. Combined CD14 and complement inhibition may prove a promising treatment strategy for bacterial sepsis.

Combined inhibition of complement and CD14 efficiently attenuated the inflammatory response induced by Staphylococcus aureus in a human whole blood model.

The complement and TLR systems are activated in sepsis, contributing to an unfavorable inflammatory "storm." Combined inhibition of these systems has been documented to efficiently attenuate the inflammatory responses induced by Gram-negative bacteria. In this study, we hypothesized that the combined inhibition would attenuate the inflammatory responses induced by Gram-positive bacteria. Staphylococcus aureus bacteria (strains Cowan and Wood), as well as S. aureus cell wall lipoteichoic acid (LTA), were incubated in thrombin-inhibited human whole blood. Complement was inhibited at the level of C3 and C5, and the TLRs by inhibiting CD14 and TLR2. Thirty-four inflammatory markers were measured by multiplex technology and flow cytometry. Thirteen markers increased significantly in response to Cowan and Wood, and 12 in response to LTA. Combined inhibition with the C3 inhibitor compstatin and the anti-CD14 Ab 18D11 significantly reduced 92 (Cowan, LTA) and 85% (Wood) of these markers. Compstatin alone significantly reduced 54 (Cowan), 38 (Wood), and 83% (LTA), whereas anti-CD14 alone significantly reduced 23, 15, and 67%, respectively. Further experiments showed that the effects of complement inhibition were mainly due to inhibition of C5a interaction with the C5a receptor. The effects on inhibiting CD14 and TLR2 were similar. The combined regimen was more efficient toward the bacterial effects than either complement or anti-CD14 inhibition alone. Complement was responsible for activation of and phagocytosis by both granulocytes and monocytes. Disrupting upstream recognition by inhibiting complement and CD14 efficiently attenuated S. aureus-induced inflammation and might be a promising treatment in both Gram-negative and Gram-positive sepsis.

Double blockade of CD14 and complement C5 abolishes the cytokine storm and improves morbidity and survival in polymicrobial sepsis in mice.

Sepsis and septic shock, caused by an excessive systemic host-inflammatory response, are associated with high morbidity and mortality. The complement system and TLRs provide important pattern recognition receptors initiating the cytokine storm by extensive cross-talk. We hypothesized that double blockade of complement C5 and the TLR coreceptor CD14 could improve survival of experimental polymicrobial sepsis. Mice undergoing cecal ligation and puncture (CLP)-induced sepsis were treated with neutralizing anti-CD14 Ab biG 53, complement C5 inhibitor coversin (Ornithodoros moubata C inhibitor), or a combination thereof. The inflammatory study (24-h observation) revealed statistically significant increases in 22 of 24 measured plasma biomarkers in the untreated CLP group, comprising 14 pro- and anti-inflammatory cytokines and 8 chemokines, growth factors, and granulocyte activation markers. Single CD14 or C5 blockade significantly inhibited 20 and 19 of the 22 biomarkers, respectively. Combined CD14 and C5 inhibition significantly reduced all 22 biomarkers (mean reduction 85%; range 54-95%) compared with the untreated CLP group. Double blockade was more potent than single treatment and was required to significantly inhibit IL-6 and CXCL1. Combined inhibition significantly reduced morbidity (motility and eyelid movement) and mortality measured over 10 d. In the positive control CLP group, median survival was 36 h (range 24-48 h). Combined treatment increased median survival to 96 h (range 24-240 h) (p = 0.001), whereas survival in the single-treatment groups was not significantly increased (median and range for anti-CD14 and anti-C5 treatment were 36 h [24-48 h] and 48 h [24-96 h]). Combined with standard intervention therapy, specific blockade of CD14 and C5 might represent a promising new therapeutic strategy for treatment of polymicrobial sepsis.