Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.

Escalated complement activation during hospitalization is associated with higher risk of 60-day mortality in SARS-CoV-2-infected patients.

BACKGROUND: The 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. METHODS: Serial 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. FINDINGS: During 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. CONCLUSION: Hospitalized 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.

Thrombin Differentially Modulates the Acute Inflammatory Response to Escherichia coli and Staphylococcus aureus in Human Whole Blood.

Thrombin plays a central role in thromboinflammatory responses, but its activity is blocked in the common ex vivo human whole blood models, making an ex vivo study of thrombin effects on thromboinflammatory responses unfeasible. In this study, we exploited the anticoagulant peptide Gly-Pro-Arg-Pro (GPRP) that blocks fibrin polymerization to study the effects of thrombin on acute inflammation in response to Escherichia coli and Staphylococcus aureus Human blood was anticoagulated with either GPRP or the thrombin inhibitor lepirudin and incubated with either E. coli or S. aureus for up to 4 h at 37°C. In GPRP-anticoagulated blood, there were spontaneous elevations in thrombin levels and platelet activation, which further increased in the presence of bacteria. Complement activation and the expression of activation markers on monocytes and granulocytes increased to the same extent in both blood models in response to bacteria. Most cytokines were not elevated in response to thrombin alone, but thrombin presence substantially and heterogeneously modulated several cytokines that increased in response to bacterial incubations. Bacterial-induced releases of IL-8, MIP-1α, and MIP-1ß were potentiated in the thrombin-active GPRP model, whereas the levels of IP-10, TNF, IL-6, and IL-1ß were elevated in the thrombin-inactive lepirudin model. Complement C5-blockade, combined with CD14 inhibition, reduced the overall cytokine release significantly, both in thrombin-active and thrombin-inactive models. Our data support that thrombin itself marginally induces leukocyte-dependent cytokine release in this isolated human whole blood but is a significant modulator of bacteria-induced inflammation by a differential effect on cytokine patterns.

Storage of Transfusion Platelet Concentrates Is Associated with Complement Activation and Reduced Ability of Platelets to Respond to Protease-Activated Receptor-1 and Thromboxane A2 Receptor.

Platelet activation and the complement system are mutually dependent. Here, we investigated the effects of storage time on complement activation and platelet function in routinely produced platelet concentrates. The platelet concentrates (n = 10) were stored at 22 °C for seven days and assessed daily for complement and platelet activation markers. Additionally, platelet function was analyzed in terms of their responsiveness to protease-activated receptor-1 (PAR-1) and thromboxane A2 receptor (TXA2R) activation and their capacity to adhere to collagen. Complement activation increased over the storage period for all analyzed markers, including the C1rs/C1-INH complex (fold change (FC) = 1.9; p < 0.001), MASP-1/C1-INH complex (FC = 2.0; p < 0.001), C4c (FC = 1.8, p < 0.001), C3bc (FC = 4.0; p < 0.01), and soluble C5b-9 (FC = 1.7, p < 0.001). Furthermore, the levels of soluble platelet activation markers increased in the concentrates over the seven-day period, including neutrophil-activating peptide-2 (FC = 2.5; p < 0.0001), transforming growth factor beta 1 (FC = 1.9; p < 0.001) and platelet factor 4 (FC = 2.1; p < 0.0001). The ability of platelets to respond to activation, as measured by surface expression of CD62P and CD63, decreased by 19% and 24% (p < 0.05) for PAR-1 and 69-72% (p < 0.05) for TXA2R activation, respectively, on Day 7 compared to Day 1. The extent of platelet binding to collagen was not significantly impaired during storage. In conclusion, we demonstrated that complement activation increased during the storage of platelets, and this correlated with increased platelet activation and a reduced ability of the platelets to respond to, primarily, TXA2R activation.

Cytokines in hepatitis C-infected patients with or without opioid maintenance therapy.

OBJECTIVE: Both chronic hepatitis C virus (HCV) infection and opioids cause altered blood levels of cytokines. Previous studies have investigated levels of selected groups of cytokines in patients on opioid maintenance treatment. Little is known about the levels of multiple cytokines in patients with chronic HCV infection on opioid maintenance treatment. Our aim was to investigate the cytokine profile in patients with active HCV infection with and without opioid maintenance treatment. METHODS: We conducted a cross-sectional study in an out-patients population included upon referral for antiviral hepatitis C infection treatment. The level of 27 cytokines was measured in serum using multiplex technology. Patients were interviewed using a modified version of the European addiction severity index. Data pertaining to weight, height, current medication, smoking habits, allergies, previous medical history and ongoing withdrawal symptoms were collected. Non-parametric testing was used to investigate differences in levels of cytokines between the two groups. A 3-model hierarchical regression analysis was used to analyse associations between cytokines and confounding variables. RESULTS: Out of 120 included patients, 53 were on opioid maintenance treatment. Median duration of opioid treatment was 68.4 months. There were no demographical differences between the two groups other than age. IL-1ß was lower and eotaxin-1 higher in the group on opioid maintenance treatment than in the non-opioid group. No other inter-group differences in the remaining cytokine levels were found. CONCLUSION: In HCV infection patients, the impact of chronic opioid administration on peripheral circulating cytokine level is minimal.

Plasma Inflammatory Biomarkers Are Associated With Poststroke Cognitive Impairment: The Nor-COAST Study.

BACKGROUND: Inflammation is proposed to be involved in the pathogenesis of poststroke cognitive impairment. The aim of this study was to investigate associations between concentrations of systemic inflammatory biomarkers after ischemic stroke and poststroke cognitive impairment. METHODS: The Nor-COAST study (Norwegian Cognitive Impairment After Stroke) is a prospective observational multicenter cohort study, including patients hospitalized with acute stroke between 2015 and 2017. Inflammatory biomarkers, including the TCC (terminal C5b-9 complement complex) and 20 cytokines, were analyzed in plasma, collected at baseline, 3-, and 18 months poststroke, using ELISA and a multiplex assay. Global cognitive outcome was assessed with the Montreal Cognitive Assessment (MoCA) scale. We investigated the associations between plasma inflammatory biomarkers at baseline and MoCA score at 3-, 18-, and 36-month follow-ups; the associations between inflammatory biomarkers at 3 months and MoCA score at 18- and 36-month follow-ups; and the association between these biomarkers at 18 months and MoCA score at 36-month follow-up. We used mixed linear regression adjusted for age and sex. RESULTS: We included 455 survivors of ischemic stroke. Higher concentrations of 7 baseline biomarkers were significantly associated with lower MoCA score at 36 months; TCC, IL (interleukin)-6, and MIP (macrophage inflammatory protein)-1α were associated with MoCA at 3, 18, and 36 months (P<0.01). No biomarker at 3 months was significantly associated with MoCA score at either 18 or 36 months, whereas higher concentrations of 3 biomarkers at 18 months were associated with lower MoCA score at 36 months (P<0.01). TCC at baseline and IL-6 and MIP-1α measured both at baseline and 18 months were particularly strongly associated with MoCA (P<0.01). CONCLUSIONS: Higher concentrations of plasma inflammatory biomarkers were associated with lower MoCA scores up to 36 months poststroke. This was most pronounced for inflammatory biomarkers measured in the acute phase following stroke. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT02650531.

Complement factors B, D, C3bBbP and risk of future venous thromboembolism.

The complement system appears to be involved in the pathogenesis of venous thromboembolism (VTE). We investigated the association of complement factors (CF) B, D, and the alternative pathway convertase, C3bBbP, measured at inclusion, with the risk of future VTE in a nested case-control study; 380 VTE patients and 804 age- and sex-matched controls derived from the Tromsø study. Odds ratios (ORs) with 95% confidence intervals (95% CI) for VTE across tertiles of CF concentrations were estimated using logistic regression. There was no association between CFB or CFD and risk of future VTE. Higher levels of C3bBbP gave an increased risk of provoked VTE; subjects in Q4 had a 1.68-fold higher OR compared with Q1 in the age-, sex- and BMI-adjusted model (OR 1.68; 95% CI 1.08-2.64). There was no increased risk of future VTE in individuals with higher levels of complement factors B or D of the alternative pathway. Increased levels of the alternative pathway activation product, C3bBbP, showed an association with future risk of provoked VTE.

Increased thromboinflammatory load in hereditary angioedema.

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 (ß-thromboglobulin (ß-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)-1ß, 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), ß-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.

Elevated plasma concentration of complement factor C5 is associated with risk of future venous thromboembolism.

The role of complement in the pathogenesis of venous thromboembolism (VTE) is unclear. We wanted to investigate (1) whether plasma complement component C5 (C5) levels are influenced by genetic variants or chronic inflammation and (2) the association between plasma C5 and risk of future VTE in a nested case-control study of 415 patients with VTE and 848 age- and sex-matched controls derived from the Tromsø Study. Plasma C5 levels were measured at inclusion. Odds ratios (ORs) with 95% confidence intervals (95% CIs) for provoked and unprovoked VTE across tertiles of C5 concentrations were estimated by logistic regression. Adjustment for C-reactive protein (CRP) served as a proxy for general inflammation. Whole-exome sequencing and protein quantitative trait loci analyses were performed to assess genetic influence on C5 concentrations. There was no association between genome-wide or C5-related gene variants and C5 levels. The association between plasma C5 levels and VTE risk displayed a threshold effect, where subjects with C5 levels above the lowest tertile had increased risk of VTE. Subjects in tertile 3 (highest C5 levels) had an age- and sex-adjusted OR of 1.45 (95% CI, 1.07-1.96) compared with tertile 1 (lowest). These statistics were more pronounced for unprovoked VTE (OR, 1.70; 95% CI, 1.11-2.60). Adjustments for body mass index and CRP had minor impact on risk estimates. The OR increased substantially with shorter time between blood sampling and VTE event. In conclusion, plasma C5 was associated with risk of future VTE. C5 levels were not genetically regulated and were only slightly influenced by chronic inflammation.

Dual inhibition of complement C5 and CD14 attenuates inflammation in a cord blood model.

BACKGROUND: Escherichia coli and Group B streptococci (GBS) are the main causes of neonatal early-onset sepsis (EOS). Despite antibiotic therapy, EOS is associated with high morbidity and mortality. Dual inhibition of complement C5 and the Toll-like receptor co-factor CD14 has in animal studies been a promising novel therapy for sepsis. METHODS: Whole blood was collected from the umbilical cord after caesarean section (n = 30). Blood was anti-coagulated with lepirudin. C5 inhibitor (eculizumab) and anti-CD14 was added 8 min prior to, or 15 and 30 min after adding E. coli or GBS. Total bacterial incubation time was 120 min (n = 16) and 240 min (n = 14). Cytokines and the terminal complement complex (TCC) were measured using multiplex technology and ELISA. RESULTS: Dual inhibition significantly attenuated TCC formation by 25-79% when adding inhibitors with up to 30 min delay in both E. coli- and GBS-induced inflammation. TNF, IL-6 and IL-8 plasma concentration were significantly reduced by 28-87% in E. coli-induced inflammation when adding inhibitors with up to 30 min delay. The dual inhibition did not significantly reduce TNF, IL-6 and IL-8 plasma concentration in GBS-induced inflammation. CONCLUSION: Dual inhibition of C5 and CD14 holds promise as a potential future treatment for severe neonatal EOS. IMPACT: Neonatal sepsis can cause severe host inflammation with high morbidity and mortality, but there are still no effective adjunctive immunologic interventions available. Adding CD14 and complement C5 inhibitors up to 30 min after incubation of E. coli or Group B streptococci in a human umbilical cord blood model significantly reduced complement activation and cytokine release. Dual inhibition of C5 and CD14 is a potential future therapy to modulate systemic inflammation in severe cases of neonatal sepsis.

High Levels of Complement Activating Enzyme MASP-2 Are Associated With the Risk of Future Incident Venous Thromboembolism.

BACKGROUND: Experimental studies have shown that the complement activating enzyme MASP-2 (mannose-binding lectin associated serine protease 2) exhibits a thrombin-like activity and that inhibition of MASP-2 protects against thrombosis. In this study, we investigated whether plasma MASP-2 levels were associated with risk of future venous thromboembolism (VTE) and whether genetic variants linked to MASP-2 levels were associated with VTE risk. METHODS: We conducted a population-based nested case-control study involving 410 VTE patients and 842 age- and sex-matched controls derived from the Norwegian Tromsø Study. Logistic regression was used to estimate odds ratios (ORs) of VTE across MASP-2 quartiles. Whole-exome sequencing and protein quantitative trait loci analyses were performed to assess genetic variants associated with MASP-2 levels. A 2-sample Mendelian randomization study, also including data from the INVENT consortium (International Network of Venous Thrombosis), was performed to assess causality. RESULTS: Subjects with plasma MASP-2 in the highest quartile had a 48% higher OR of VTE (OR, 1.48 [95% CI, 1.06-2.06]) and 83% higher OR of deep vein thrombosis (OR, 1.83 [95% CI, 1.23-2.73]) compared with those with MASP-2 levels in the lowest quartile. The protein quantitative trait loci analysis revealed that 3 previously described gene variants, rs12711521 (minor allele frequency, 0.153), rs72550870 (minor allele frequency, 0.045; missense variants in the MASP2 gene), and rs2275527 (minor allele frequency, 0.220; exon variant in the adjacent MTOR gene) explained 39% of the variation of MASP-2 plasma concentration. The OR of VTE per 1 SD increase in genetically predicted MASP-2 was 1.03 ([95% CI, 1.01-1.05] P=0.0011). CONCLUSIONS: Our findings suggest that high plasma MASP-2 levels are causally associated with risk of future VTE.

Air Bubbles Activate Complement and Trigger Hemostasis and C3-Dependent Cytokine Release Ex Vivo in Human Whole Blood.

Venous air embolism, which may complicate medical and surgical procedures, activates complement and triggers thromboinflammation. In lepirudin-anticoagulated human whole blood, we examined the effect of air bubbles on complement and its role in thromboinflammation. Whole blood from 16 donors was incubated with air bubbles without or with inhibitors of C3, C5, C5aR1, or CD14. Complement activation, hemostasis, and cytokine release were measured using ELISA and quantitative PCR. Compared with no air, incubating blood with air bubbles increased, on average, C3a 6.5-fold, C3bc 6-fold, C3bBbP 3.7-fold, C5a 4.6-fold, terminal complement complex sC5b9 3.6-fold, prothrombin fragments 1+2 (PTF1+2) 25-fold, tissue factor mRNA (TF-mRNA) 26-fold, microparticle tissue factor 6.1-fold, ß-thromboglobulin 26-fold (all p < 0.05), and 25 cytokines 11-fold (range, 1.5-78-fold; all p < 0.0001). C3 inhibition attenuated complement and reduced PTF1+2 2-fold, TF-mRNA 5.4-fold, microparticle tissue factor 2-fold, and the 25 cytokines 2.7-fold (range, 1.4-4.9-fold; all p < 0.05). C5 inhibition reduced PTF1+2 2-fold and TF-mRNA 12-fold (all p < 0.05). C5 or CD14 inhibition alone reduced three cytokines, including IL-1ß (p = 0.02 and p = 0.03). Combined C3 and CD14 inhibition reduced all cytokines 3.9-fold (range, 1.3-9.5-fold; p < 0.003) and was most pronounced for IL-1ß (3.2- versus 6.4-fold), IL-6 (2.5- versus 9.3-fold), IL-8 (4.9- versus 8.6-fold), and IFN-γ (5- versus 9.5-fold). Antifoam activated complement and was avoided. PTF1+2 was generated in whole blood but not in plasma. In summary, air bubbles activated complement and triggered a C3-driven thromboinflammation. C3 inhibition reduced all mediators, whereas C5 inhibition reduced only TF-mRNA. Combined C5 and CD14 inhibition reduced IL-1ß release. These data have implications for future mechanistic studies and possible pharmacological interventions in patients with air embolism.

A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity.

Thrombin activation of C5 connects thrombosis to inflammation. Complement research in whole blood ex vivo necessitates anticoagulation, which potentially interferes with the inflammatory modulation by thrombin. We challenged the concept of thrombin as an activator of native C5 by analyzing complement activation and C5 cleavage in human whole blood anticoagulated with Gly-Pro-Arg-Pro (GPRP), a peptide targeting fibrin polymerization downstream of thrombin, allowing complete endogenous thrombin generation. GPRP dose-dependently inhibited coagulation but allowed for platelet activation in accordance with thrombin generation. Spontaneous and bacterial-induced complement activation by Escherichia coli and Staphylococcus aureus, analyzed at the level of C3 and C5, were similar in blood anticoagulated with GPRP and the thrombin inhibitor lepirudin. In the GPRP model, endogenous thrombin, even at supra-physiologic concentrations, did not cleave native C5, despite efficiently cleaving commercially sourced purified C5 protein, both in buffer and when added to C5-deficient serum. In normal serum, only exogenously added, commercially sourced C5 was cleaved, whereas the native plasma C5 remained intact. Crucially, affinity-purified C5, eluted under mild conditions using an MgCl2 solution, was not cleaved by thrombin. Acidification of plasma to pH ≤ 6.8 by hydrochloric or lactic acid induced a C5 antigenic change, nonreversible by pH neutralization, that permitted cleavage by thrombin. Circular dichroism on purified C5 confirmed the structural change during acidification. Thus, we propose that pH-induced conformational change allows thrombin-mediated cleavage of C5 and that, contrary to previous reports, thrombin does not cleave plasma C5 in its native form, suggesting that thrombin cleavage of C5 may be restricted to certain pathophysiological conditions.

Inflammation in the early phase after kidney transplantation is associated with increased long-term all-cause mortality.

In the general population, low-grade inflammation has been established as a risk factor for all-cause mortality. We hypothesized that an inflammatory milieu beyond the time of recovery from the surgical trauma could be associated with increased long-term mortality in kidney transplant recipients (KTRs). This cohort study included 1044 KTRs. Median follow-up time post-engraftment was 10.3 years. Inflammation was assessed 10 weeks after transplantation by different composite inflammation scores based on 21 biomarkers. We constructed an overall inflammation score and five pathway-specific inflammation scores (fibrogenesis, vascular inflammation, metabolic inflammation, growth/angiogenesis, leukocyte activation). Mortality was assessed with Cox regression models adjusted for traditional risk factors. A total of 312 (29.9%) patients died during the follow-up period. The hazard ratio (HR) for death was 4.71 (95% CI: 2.85-7.81, p < .001) for patients in the highest quartile of the overall inflammation score and HRs 2.35-2.54 (95% CI: 1.40-3.96, 1.52-4.22, p = .001) for patients in the intermediate groups. The results were persistent when the score was analyzed as a continuous variable (HR 1.046, 95% CI: 1.033-1.056, p < .001). All pathway-specific analyses showed the same pattern with HRs ranging from 1.19 to 2.70. In conclusion, we found a strong and consistent association between low-grade systemic inflammation 10 weeks after kidney transplantation and long-term mortality.

A Complement C3-Specific Nanobody for Modulation of the Alternative Cascade Identifies the C-Terminal Domain of C3b as Functional in C5 Convertase Activity.

The complement system is an intricate cascade of the innate immune system and plays a key role in microbial defense, inflammation, organ development, and tissue regeneration. There is increasing interest in developing complement regulatory and inhibitory agents to treat complement dysfunction. In this study, we describe the nanobody hC3Nb3, which is specific for the C-terminal C345c domain of human and mouse complement component C3/C3b/C3c and potently inhibits C3 cleavage by the alternative pathway. A high-resolution structure of the hC3Nb3-C345c complex explains how the nanobody blocks proconvertase assembly. Surprisingly, although the nanobody does not affect classical pathway-mediated C3 cleavage, hC3Nb3 inhibits classical pathway-driven hemolysis, suggesting that the C-terminal domain of C3b has an important function in classical pathway C5 convertase activity. The hC3Nb3 nanobody binds C3 with low nanomolar affinity in an SDS-resistant complex, and the nanobody is demonstrated to be a powerful reagent for C3 detection in immunohistochemistry and flow cytometry. Overall, the hC3Nb3 nanobody represents a potent inhibitor of both the alternative pathway and the terminal pathway, with possible applications in complement research, diagnostics, and therapeutics.

Association between brain-derived neurotropic factor (BDNF), high-sensitivity C-reactive protein (hs-CRP) and psychiatric symptoms in medicated and unmedicated patients.

BACKGROUND: There is evidence that brain-derived neurotropic factor (BDNF) plays a protective role in the brain. Peripheral levels of BDNF correlate with its concentration in the brain. Previous studies have revealed lower serum BDNF levels in patients with mental illnesses. In most studies serum BDNF correlates negatively with psychiatric disorders and disease severity. Most studies in this field are on psychiatric diagnosis and personality traits. The aim of our study is to explore associations between general psychiatric symptoms, independent of diagnostic groups, and serum BDNF as well as the inflammatory biomarker high-sensitive CRP (hs-CRP). Comparison between the group regularly using psychotropic medication and those not using psychotropic medication is conducted. METHODS: The study is a cross sectional study with 132 participants from a general open inpatient psychiatric ward at the Nordland Hospital Trust, Bodoe, Norway. Participants were assessed on serum levels of BDNF and hs-CRP. Psychiatric symptoms were assessed by a self-rating scale (Symptom check list, SCL-90- R). Multiple linear regression model was used for statistical analyses of associations between levels of BDNF, hs-CRP and symptoms. RESULTS: We found a positive association (p < 0.05), for most SCL-90 symptom clusters with BDNF in the psychotropic medication-free group. No associations were found in the group of patients using psychotropic medication, except one, the paranoid ideation cluster (p 0.022). No associations were found between hs-CRP and symptom clusters. CONCLUSION: We found no relation between symptom clusters and the inflammatory biomarker hs-CRP. Serum BDNF levels were positively associated with intensity of psychiatric symptoms in the group of patients not using psychotropic medication. Our findings are in conflict with several previous studies reporting increased hs-CRP as well as decreased rather than increased BDNF in mental suffering. Patients on psychotropic medication may not require the same upregulation because the medication is modulating the underlying biological pathology.

Rapid systemic surge of IL-33 after severe human trauma: a prospective observational study.

BACKGROUND: Alarmins are considered proximal mediators of the immune response after tissue injury. Understanding their biology could pave the way for development of new therapeutic targets and biomarkers in human disease, including multiple trauma. In this study we explored high-resolution concentration kinetics of the alarmin interleukin-33 (IL-33) early after human trauma. METHODS: Plasma samples were serially collected from 136 trauma patients immediately after hospital admission, 2, 4, 6, and 8 h thereafter, and every morning in the ICU. Levels of IL-33 and its decoy receptor sST2 were measured by immunoassays. RESULTS: We observed a rapid and transient surge of IL-33 in a subset of critically injured patients. These patients had more widespread tissue injuries and a greater degree of early coagulopathy. IL-33 half-life (t1/2) was 1.4 h (95% CI 1.2-1.6). sST2 displayed a distinctly different pattern with low initial levels but massive increase at later time points. CONCLUSIONS: We describe for the first time early high-resolution IL-33 concentration kinetics in individual patients after trauma and correlate systemic IL-33 release to clinical data. These findings provide insight into a potentially important axis of danger signaling in humans.

Increased complement activation 3 to 6 h after trauma is a predictor of prolonged mechanical ventilation and multiple organ dysfunction syndrome: a prospective observational study.

BACKGROUND: Complement activation is a central mechanism in systemic inflammation and remote organ dysfunction following major trauma. Data on temporal changes of complement activation early after injury is largely missing. We aimed to describe in detail the kinetics of complement activation in individual trauma patients from admission to 10 days after injury, and the association with trauma characteristics and outcome. METHODS: In a prospective cohort of 136 trauma patients, plasma samples obtained with high time resolution (admission, 2, 4, 6, 8 h, and thereafter daily) were assessed for terminal complement complex (TCC). We studied individual TCC concentration curves and calculated a summary measure to obtain the accumulated TCC response 3 to 6 h after injury (TCC-AUC3-6). Correlation analyses and multivariable linear regression analyses were used to explore associations between individual patients' admission TCC, TCC-AUC3-6, daily TCC during the intensive care unit stay, trauma characteristics, and predefined outcome measures. RESULTS: TCC concentration curves showed great variability in temporal shapes between individuals. However, the highest values were generally seen within the first 6 h after injury, before they subsided and remained elevated throughout the intensive care unit stay. Both admission TCC and TCC-AUC3-6 correlated positively with New Injury Severity Score (Spearman's rho, p-value 0.31, 0.0003 and 0.21, 0.02) and negatively with admission Base Excess (- 0.21, 0.02 and - 0.30, 0.001). Multivariable analyses confirmed that deranged physiology was an important predictor of complement activation. For patients without major head injury, admission TCC and TCC-AUC3-6 were negatively associated with ventilator-free days. TCC-AUC3-6 outperformed admission TCC as a predictor of Sequential Organ Failure Assessment score at day 0 and 4. CONCLUSIONS: Complement activation 3 to 6 h after injury was a better predictor of prolonged mechanical ventilation and multiple organ dysfunction syndrome than admission TCC. Our data suggest that the greatest surge of complement activation is found within the first 6 h after injury, and we argue that this time period should be in focus in the design of future experimental studies and clinical trials using complement inhibitors.

Increased Local Inflammatory Response to MOC31PE Immunotoxin After Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy.

BACKGROUND: Despite extensive cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC), most patients with resectable peritoneal metastases from colorectal cancer experience disease relapse. MOC31PE immunotoxin is being explored as a novel treatment option for these patients. MOC31PE targets the cancer-associated epithelial cell adhesion molecule, and kills cancer cells by distinct mechanisms, simultaneously causing immune activation by induction of immunogenic cell death (ICD). METHODS: Systemic and local cytokine responses were analyzed in serum and intraperitoneal fluid samples collected the first three postoperative days from clinically comparable patients undergoing CRS-HIPEC with (n = 12) or without (n = 26) intraperitoneal instillation of MOC31PE. A broad panel of 27 pro- and antiinflammatory interleukins, chemokines, interferons, and growth factors was analyzed using multiplex technology. RESULTS: The time course and magnitude of the systemic and local postoperative cytokine response after CRS-HIPEC were highly compartmentalized, with modest systemic responses contrasting substantial intraperitoneal responses. Administration of MOC31PE resulted in changes that were broader and of higher magnitude compared with CRS-HIPEC alone. Significantly increased levels of innate proinflammatory cytokines, such as interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF) as well as an interesting time response curve for the strong T-cell stimulator interferon (IFN)-γ and its associated chemokine interferon gamma-induced protein/chemokine (C-X-C motif) ligand 10 (IP-10) were detected, all associated with ICD. CONCLUSIONS: Our study revealed a predominately local rather than systemic inflammatory response to CRS-HIPEC, which was strongly enhanced by MOC31PE treatment. The MOC31PE-induced intraperitoneal inflammatory reaction could contribute to improve remnant cancer cell killing, but the mechanisms remain to be elucidated in future studies.

Investigating novel biomarkers of immune activation and modulation in the context of sedentary behaviour: a multicentre prospective ischemic stroke cohort study.

BACKGROUND: Sedentary behaviour is associated with disease, but the molecular mechanisms are not understood. Valid biomarkers with predictive and explanatory properties are required. Therefore, we have investigated traditional and novel biomarkers of inflammation and immune modulation and their association to objectively measured sedentary behaviour in an ischemic stroke population. METHODS: Patients admitted to hospital with acute ischemic stroke were included in the multicentre Norwegian Cognitive Impairment After Stroke (Nor-COAST) study (n = 815). For this sub-study (n = 257), sedentary behaviour was registered 3 months after stroke using position transition data from the body-worn sensor, ActivPal®. Blood samples were analysed for high sensitive C-reactive protein (hsCRP), the cytokines interleukin-6 (IL-6) and 10 (IL-10), neopterin, tryptophan (Trp), kynurenine (kyn), kynurenic acid (KA), and three B6 vitamers, pyridoxal 5'-phosphate (PLP), pyridoxal (PL), and pyridoxic acid (PA). The kynurenine/tryptophan ratio (KTR) and the pyridoxic acid ratio index (PAr = PA: PL + PLP) were calculated. RESULTS: Of the 815 patients included in the main study, 700 attended the three-month follow-up, and 257 fulfilled the inclusion criteria for this study. Sedentary time was significantly associated with levels of hsCRP, IL-6, neopterin, PAr-index, and KA adjusted for age, sex, waist circumference, and creatinine. In a fully adjusted model including all the significant biomarkers except hsCRP (because of missing values), sedentary time was independently positively associated with the PAr-index and negatively with KA. We did not find an association between sedentary behaviour, IL-10, and KTR. CONCLUSIONS: The PAr-index is known to capture several modes of inflammation and has previously shown predictive abilities for future stroke. This novel result indicates that the PAr-index could be a useful biomarker in future studies on sedentary behaviour and disease progression. KA is an important modulator of inflammation, and this finding opens new and exciting pathways to understand the hazards of sedentary behaviour. TRIAL REGISTRATION: The study was registered at Clinicaltrials.gov ( NCT02650531 ). First posted 08/01/2016.