The inflammatory response induced by Aspergillus fumigatus conidia is dependent on complement activation - Insight from a whole blood model.

INTRODUCTION: We aimed to elucidate the inflammatory response of Aspergillus fumigatus conidia in a whole-blood model of innate immune activation and to compare it with the well-characterized inflammatory reaction to E. coli. METHODS: Employing a human lepirudin whole-blood model, we analyzed complement and leukocyte activation by measuring the sC5b-9 complex and assessing CD11b expression. A 27-multiplex system was used for quantification of cytokines. Selective cell removal from whole blood and inhibition of C3, C5, and CD14 were also applied. RESULTS: Our findings demonstrated a marked elevation in sC5b-9 and CD11b post-A. fumigatus incubation. Thirteen cytokines (TNF, IL-1ß, IL-1ra, IL-4, IL-6, IL-8, IL-17, IFNγ, MCP-1, MIP-1α, MIP-1ß, FGF-basic, and G-CSF) showed increased levels. A generally lower level of cytokine release and CD11b expression was observed with A. fumigatus conidia than with E. coli. Notably, monocytes were instrumental in releasing all cytokines except MCP-1. IL-1ra was found to be both monocyte and granulocyte-dependent. Pre-inhibiting with C3 and CD14 inhibitors resulted in decreased release patterns for six cytokines (TNF, IL-1ß, IL-6, IL-8, MIP-1α and MIP-1ß), with minimal effects by C5-inhibition. CONCLUSION: A. fumigatus conidia induced complement activation comparable to E. coli, whereas CD11b expression and cytokine release were lower, underscoring distinct inflammatory responses between these pathogens. Complement C3 inhibition attenuated cytokine release indicating a C3-level role of complement in A. fumigatus immunity.

Trauma-associated extracellular histones mediate inflammation via a MYD88-IRAK1-ERK signaling axis and induce lytic cell death in human adipocytes.

Despite advances in the treatment and care of severe physical injuries, trauma remains one of the main reasons for disability-adjusted life years worldwide. Trauma patients often suffer from disturbances in energy utilization and metabolic dysfunction, including hyperglycemia and increased insulin resistance. White adipose tissue plays an essential role in the regulation of energy homeostasis and is frequently implicated in traumatic injury due to its ubiquitous body distribution but remains poorly studied. Initial triggers of the trauma response are mainly damage-associated molecular patterns (DAMPs) such as histones. We hypothesized that DAMP-induced adipose tissue inflammation contributes to metabolic dysfunction in trauma patients. Therefore, we investigated whether histone release during traumatic injury affects adipose tissue. Making use of a murine polytrauma model with hemorrhagic shock, we found increased serum levels of histones accompanied by an inflammatory response in white adipose tissue. In vitro, extracellular histones induced an inflammatory response in human adipocytes. On the molecular level, this inflammatory response was mediated via a MYD88-IRAK1-ERK signaling axis as demonstrated by pharmacological and genetic inhibition. Histones also induced lytic cell death executed independently of caspases and RIPK1 activity. Importantly, we detected increased histone levels in the bloodstream of patients after polytrauma. Such patients might benefit from a therapy consisting of activated protein C and the FDA-approved ERK inhibitor trametinib, as this combination effectively prevented histone-mediated effects on both, inflammatory gene activation and cell death in adipocytes. Preventing adipose tissue inflammation and adipocyte death in patients with polytrauma could help minimize posttraumatic metabolic dysfunction.

Transdiagnostic Associations between Anger Hostility and Chemokine Interferon-gamma Inducible Protein 10.

Objective: Many psychiatric disorders are linked to low grade systemic inflammation as measured by systemic cytokine levels. Exploration of cytokines and immune activity and their role in psychiatric symptoms may inform pathobiology and treatment opportunities. The aim of this study is to explore if there are associations between cytokines and psychiatric symptom clusters. Comparison between patients regularly using and those not using psychotropic medication is also conducted. Methods: This was a cross sectional naturalistic study with 132 participants from a general open inpatient psychiatric ward at the Nordland Hospital Trust, Norway. Serum levels of 28 different cytokines were assessed. Psychiatric symptoms the last week were assessed by a self-rating scale (Symptom check list, SCL-90-R) and grouped in defined clusters. Multiple linear regression model was used for statistical analyses of associations between levels of cytokines and symptoms, adjusting for possible confounding factors. Results: We found a positive association (p = 0.009) between the chemokine interferon-gamma inducible protein 10 (CXCL 10; IP-10) and the anger hostility cluster. No associations were found between the other symptom clusters and cytokines. IP-10 and the anger hostility cluster were positively associated (p = 0.002) in the subgroup of patients using psychotropic medication, not in the subgroup not using psychotropic medication. Conclusion: Our analyses revealed a significant positive association between the symptom cluster anger hostility in SCL-90-R and the chemokine IP-10 in the subgroup of patients using psychotropic medications.

The detrimental effects of intestinal injury mediated by inflammation are limited in cardiac arrest patients: A prospective cohort study.

Background: Ischaemic intestines could be a driver of critical illness through an inflammatory response. We have previously published reports on a biomarker for intestinal injury, plasma Intestinal Fatty Acid Binding Protein (IFABP), and inflammatory biomarkers after out-of-hospital cardiac arrest (OHCA). In this post-hoc study we explored the potential indirect effects of intestinal injury mediated through the inflammatory response on organ dysfunction and mortality. Methods: We measured IFABP and twenty-one inflammatory biomarkers in 50 patients at admission to intensive care unit after OHCA. First, we stratified patients on median IFABP and compared biomarkers between "low" and "high" IFABP. Second, by causal mediation analysis, we assessed effects of IFABP through the two most important inflammatory biomarkers, interleukin (IL)-6 and terminal complement complex (TCC), on day two circulatory variables, Sequential Organ Failure Assessment (SOFA)-score, and 30-day mortality. Results: Cytokines and complement activation were higher in the high IFABP group. In mediation analysis, patients on the 75th percentile of IFABP, compared to the 25th percentile, had 53% (95% CI, 33-74; p < 0.001) higher risk of dying, where 13 (95% CI, 3-23; p = 0.01) percentage points were mediated through an indirect effect of IL-6. Similarly, the indirect effect of IFABP through IL-6 on SOFA-score was significant, but smaller than potential other effects. Effects through IL-6 on circulatory variables, and all effects through TCC, were not statistically significant and/or small. Conclusion: Effects of intestinal injury mediated through inflammation on organ dysfunction and mortality were limited. Small, but significant, effects through IL-6 were noted.Trial registration: ClinicalTrials.gov: NCT02648061.

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.

Femoral Nailing in a Porcine Model Causes Bone Marrow Emboli in the Lungs and Systemic Emboli in the Heart and Brain.

Background: Shaft fractures of the femur are commonly treated with intramedullary nailing, which can release bone marrow emboli into the bloodstream. Emboli can travel to the lungs, impairing gas exchange and causing inflammation. Occasionally, emboli traverse from the pulmonary to the systemic circulation, hindering perfusion and resulting in injuries such as heart and brain infarctions, known as fat embolism syndrome. We studied the extent of systemic bone marrow embolization in a pig model. Methods: Twelve anesthetized pigs underwent bilateral intramedullary nailing of the femur, while 3 animals served as sham controls. Monitoring included transesophageal echocardiography (TEE), pulse oximetry, electrocardiography, arterial blood pressure measurement, and blood gas and troponin-I analysis. After surgery, animals were monitored for 240 minutes before euthanasia. Post mortem, the heart, lungs, and brain were biopsied. Results: Bone marrow emboli were found in the heart and lungs of all 12 of the pigs that underwent intramedullary nailing and in the brains of 11 of them. No emboli were found in the sham group. The pigs subjected to intramedullary nailing exhibited significant hypoxia (PaO2/FiO2 ratio, 410 mm Hg [95% confidence interval (CI), 310 to 510) compared with the sham group (594 mm Hg [95% CI, 528 to 660]). The nailing group exhibited ST-segment alterations consistent with myocardial ischemia and a significant increase in the troponin-I level compared with the sham group (1,580 ng/L [95% CI, 0 to 3,456] versus 241 ng/L [95% CI, 0 to 625] at the 240-minute time point; p = 0.005). TEE detected emboli in the right ventricular outflow tract, but not systemically, in the nailing group. Conclusions: Bilateral intramedullary nailing caused bone marrow emboli in the lungs and systemic emboli in the heart and brain in this pig model. The observed clinical manifestations were consistent with coronary and pulmonary emboli. TEE detected pulmonary but not systemic embolization. Clinical Relevance: Femoral intramedullary nailing in humans is likely to result in embolization as described in our pig model. Focused monitoring is necessary for detection of fat embolism syndrome. Absence of visual emboli in the left ventricle on TEE does not exclude the occurrence of systemic bone marrow emboli.

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.

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.

A novel selective leukocyte depletion human whole blood model reveals the specific roles of monocytes and granulocytes in the cytokine response to Escherichia coli.

The lepirudin-based human whole blood model is a well-established ex vivo system to characterize inflammatory responses. However, the contribution of individual cell populations to cytokine release has not been investigated. Thus, we modified the model by selectively removing leukocyte subpopulations to elucidate their contribution to the inflammatory response. Lepirudin-anticoagulated whole blood was depleted from monocytes or granulocytes using StraightFrom Whole Blood MicroBeads. Reconstituted blood was incubated with Escherichia coli (108/mL) for 2 hours at 37 °C. CD11b, CD62P, and CD63 were detected by flow cytometry. Complement (C3bc, sC5b-9) and platelet activation (platelet factor 4, NAP-2) were measured by enzyme-linked immunosorbent assay. Cytokines were quantified by multiplex assay. A significant (P < 0.05) specific depletion of the monocyte (mean = 86%; 95% confidence interval = 71%-92%) and granulocyte (mean = 97%; 95% confidence interval = 96%-98%) population was obtained. Background activation induced by the depletion protocol was negligible for complement (C3bc and sC5b-9), leukocytes (CD11b), and platelets (NAP-2). Upon Escherichia coli incubation, release of 10 of the 24 cytokines was solely dependent on monocytes (interleukin [IL]-1ß, IL-2, IL-4, IL-5, IL-17A, interferon-γ, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein-1α, and fibroblast growth factor-basic), whereas 8 were dependent on both monocytes and granulocytes (IL-1ra, IL-6, IL-8, IL-9, IL-10, macrophage inflammatory protein-1ß, tumor necrosis factor, and eotaxin). Six cytokines were not monocyte or granulocyte dependent, of which platelet-derived growth factor and RANTES were mainly platelet dependent. We document an effective model for selective depletion of leukocyte subpopulations from whole blood, without causing background activation, allowing in-depth cellular characterization. The results are in accordance with monocytes playing a major role in cytokine release and expand our knowledge of the significant role of granulocytes in the response to E. coli.

Longitudinal Associations Between Persistent Post-Concussion Symptoms and Blood Biomarkers of Inflammation and CNS-Injury After Mild Traumatic Brain Injury.

The aim of our study was to investigate the biological underpinnings of persistent post-concussion symptoms (PPCS) at 3 months following mild traumatic brain injury (mTBI). Patients (n = 192, age 16-60 years) with mTBI, defined as Glasgow Coma Scale (GCS) score between 13 and 15, loss of consciousness (LOC) <30 min, and post-traumatic amnesia (PTA) <24 h were included. Blood samples were collected at admission (within 72 h), 2 weeks, and 3 months. Concentrations of blood biomarkers associated with central nervous system (CNS) damage (glial fibrillary acidic protein [GFAP], neurofilament light [NFL], and tau) and inflammation (interferon gamma [IFNγ], interleukin [IL]-8, eotaxin, macrophage inflammatory protein-1-beta [MIP]-1ß, monocyte chemoattractant protein [MCP]-1, interferon-gamma-inducible protein [IP]-10, IL-17A, IL-9, tumor necrosis factor [TNF], basic fibroblast growth factor [FGF]-basic platelet-derived growth factor [PDGF], and IL-1 receptor antagonist [IL-1ra]) were obtained. Demographic and injury-related factors investigated were age, sex, GCS score, LOC, PTA duration, traumatic intracranial finding on magnetic resonance imaging (MRI; within 72 h), and extracranial injuries. Delta values, that is, time-point differences in biomarker concentrations between 2 weeks minus admission and 3 months minus admission, were also calculated. PPCS was assessed with the British Columbia Post-Concussion Symptom Inventory (BC-PSI). In single variable analyses, longer PTA duration and a higher proportion of intracranial findings on MRI were found in the PPCS group, but no single biomarker differentiated those with PPCS from those without. In multi-variable models, female sex, longer PTA duration, MRI findings, and lower GCS scores were associated with increased risk of PPCS. Inflammation markers, but not GFAP, NFL, or tau, were associated with PPCS. At admission, higher concentrations of IL-8 and IL-9 and lower concentrations of TNF, IL-17a, and MCP-1 were associated with greater likelihood of PPCS; at 2 weeks, higher IL-8 and lower IFNγ were associated with PPCS; at 3 months, higher PDGF was associated with PPCS. Higher delta values of PDGF, IL-17A, and FGF-basic at 2 weeks compared with admission, MCP-1 at 3 months compared with admission, and TNF at 2 weeks and 3 months compared with admission were associated with greater likelihood of PPCS. Higher IL-9 delta values at both time-point comparisons were negatively associated with PPCS. Discriminability of individual CNS-injury and inflammation biomarkers for PPCS was around chance level, whereas the optimal combination of biomarkers yielded areas under the curve (AUCs) between 0.62 and 0.73. We demonstrate a role of biological factors on PPCS, including both positive and negative effects of inflammation biomarkers that differed based on sampling time-point after mTBI. PPCS was associated more with acute inflammatory processes, rather than ongoing inflammation or CNS-injury biomarkers. However, the modest discriminative ability of the models suggests other factors are more important in the development of PPCS.

Local Postoperative Graft Inflammation in Pancreas Transplant Patients With Early Graft Thrombosis.

Background: Graft thrombosis is the main cause of early graft loss following pancreas transplantation, and is more frequent in pancreas transplant alone (PTA) compared with simultaneous pancreas-kidney (SPK) recipients. Ischemia-reperfusion injury during transplantation triggers a local thromboinflammatory response. We aimed to evaluate local graft inflammation and its potential association with early graft thrombosis. Methods: In this observational study, we monitored 67 pancreas-transplanted patients using microdialysis catheters placed on the pancreatic surface during the first postoperative week. We analyzed 6 cytokines, interleukin-1 receptor antagonist (IL-1ra), IL-6, IL-8, interferon gamma-induced protein 10 (IP-10), macrophage inflammatory protein 1ß (MIP-1ß), IL-10, and the complement activation product complement activation product 5a (C5a) in microdialysis fluid. We compared the dynamic courses between patients with pancreas graft thrombosis and patients without early complications (event-free) and between PTA and SPK recipients. Levels of the local inflammatory markers, and plasma markers C-reactive protein, pancreas amylase, and lipase were evaluated on the day of thrombosis diagnosis compared with the first week in event-free patients. Results: IL-10 and C5a were not detectable. Patients with no early complications (n = 34) demonstrated high IL-1ra, IL-6, IL-8, IP-10, and MIP-1ß concentrations immediately after surgery, which decreased to steady low levels during the first 2 postoperative days (PODs). Patients with early graft thrombosis (n = 17) demonstrated elevated IL-6 (P = 0.003) concentrations from POD 1 and elevated IL-8 (P = 0.027) concentrations from POD 2 and throughout the first postoperative week compared with patients without complications. IL-6 (P < 0.001) and IL-8 (P = 0.003) were higher on the day of thrombosis diagnosis compared with patients without early complications. No differences between PTA (n = 35) and SPK (n = 32) recipients were detected. Conclusions: Local pancreas graft inflammation was increased in patients experiencing graft thrombosis, with elevated postoperative IL-6 and IL-8 concentrations, but did not differ between PTA and SPK recipients. Investigating the relationship between the local cytokine response and the formation of graft thrombosis warrants further research.

Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody.

INTRODUCTION: The complement system anaphylatoxin C5a is a critical player in inflammation. By binding to complement C5a receptor 1 (C5aR1/CD88), C5a regulates many cellular functions, mainly as a potent pro-inflammatory inducer. We describe the generation and selection of a potent antagonistic C5aR1 mouse monoclonal antibody (mAb). METHODS: Initial C5aR1 hybridoma clone selection was performed with a cell-binding study in human whole blood. In-house C5aR1 mAb assessment for C5aR1 inhibition was done via the iLite® C5a assay. C5aR1 mAb specificity was investigated on C5aR1his- and C5aR2his-expressing Flp-In™-CHO cells. Physiological C5aR1 inhibition was assessed via a C5a-driven calcium flux assay and stimulation assay based on isolated polymorphonuclear leukocytes (PMNs) and a whole blood model stimulated with Escherichia coli. RESULTS: The supernatant of hybridoma clones targeting the N-terminal section of C5aR1 displayed efficient binding to C5aR1 in whole blood, which was confirmed for purified mAbs. The C5aR1 mAb 18-41-6 was selected following the assay of in-house C5aR1 mAbs via the iLite® C5a assay. The mAb 18-41-6 was specific for C5aR1. Full-size and/or F(ab')2 preparations of mAb 18-41-6 were found to efficiently abrogate C5a-induced calcium flux in neutrophils and to significantly reduce the upregulation of the activation markers CD11b (neutrophils, monocytes) and CD66b (neutrophils). CONCLUSION: Our results demonstrate that mAb 18-41-6 is a valuable tool for investigating the C5a-C5aR1 axis and a potential therapeutic candidate for inflammatory disease treatment.

Ocrelizumab and ofatumumab, but not rituximab, trigger complement induction in vitro.

The clinical and adverse effects of the therapeutic monoclonal antibodies (mAb) ocrelizumab, ofatumumab and rituximab in multiple sclerosis (MS) are presently subject to extensive study. While the two former are approved for MS, the older and less costly rituximab is used off label, and adverse effect profiles are important in their evaluation. The three mAbs all induce B cell depletion, with complement-dependent cytotoxicity (CDC) as one of several mechanisms of action. Complement activation is also postulated to underlie adverse reactions related to infusion/injection. Such administration-related reactions are associated with all three mAbs, but comparisons have so far been indirect, resting on incidence reports from separate clinical trials. The objective of this study was to perform head-to-head comparison of complement activation by ofatumumab, ocrelizumab and rituximab. In vitro experiments were performed in whole blood from healthy donors. The complement-activating potential of the three mAbs was analyzed after 30 min of exposure to 0.3 mg/mL or 0.9 mg/mL of each drug, and compared with those of the well-known TNF inhibitory mAbs adalimumab and infliximab, the latter with recognized potential for infusion reactions. Ofatumumab, ocrelizumab, and infliximab, but not rituximab and adalimumab, triggered statistically significant complement activation measured as increased levels of terminal C5b-9 complement complex (TCC), a sensitive marker of such activation. While results demand careful interpretation, they provide an indication of distinct complement-inducing potential among anti-CD20 mAbs currently used to treat MS.

Effect of Intraperitoneal 224Radium-Labelled Microparticles on Compartmentalized Inflammation After Cytoreductive Surgery and Hypertherm Intraperitoneal Chemotherapy.

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

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.

A role for the terminal C5-C9 complement pathway in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by damage to the alveolar epithelium, leading to fibrosis and excessive accumulation of extracellular matrix in the interstitium of the lung. In the present study we performed high-resolution proteomic profiling of bronchoalveolar lavage (BAL) from IPF patients and controls, and found that the complement pathway was highly upregulated in IPF. The proteins C5, C6, C7, C8, and C9, all of which are part of the complement end product, TCC, were all upregulated. We also found that TCC levels were increased in plasma among IPF patients compared to controls, after adjustment for age, sex and BMI [mean (SD) 0.62 (0.24) vs. 0.33 (0.10), p = 0.031]. These findings suggest a role for the complement system in the pathogenesis of IPF.

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.

Thromboinflammatory response is increased in pancreas transplant alone versus simultaneous pancreas-kidney transplantation and early pancreas graft thrombosis is associated with complement activation.

Background: Pancreas transplant alone (PTA) recipients are more affected by pancreas graft thrombosis, and graft loss compared to simultaneous pancreas-kidney (SPK) recipients. The pathophysiology is unknown, but an increased immune response has been suggested in the PTA recipients. In this observational study, we compared perioperative thromboinflammation between PTA (n=32) and SPK (n=35) recipients, and between PTA recipients with (n=14) versus without (n=18) early graft thrombosis. Methods: We measured C-reactive protein (CRP), plasma markers of activated coagulation and complement, and cytokines preoperatively and daily during the first postoperative week. Results: Preoperatively, coagulation and complement activation markers were comparable between PTA and SPK recipients, while cytokine concentrations were higher in SPK recipients (TNF, IL-8, IP-10, MCP-1, MIP-1α; all p<0.05). On the first postoperative day, PTA recipients had higher coagulation activation, measured as thrombin-antithrombin complex (TAT), than SPK recipients (p=0.008). In the first postoperative week, PTA recipients showed higher relative cytokine release (IL-6, IL-8, G-CSF, IP-10, MCP-1, and MIP-1α; all p<0.05) while SPK recipients showed higher absolute cytokine concentrations (TNF, IL-1ra, IL-8, MIP-1α, and IL-4; all p<0.05). PTA and SPK recipients showed similar terminal complement complex (TCC, sC5b-9) activation. On the first postoperative day, TCC (OR 1.2 [95% CI 1.0-1.5] for 0.1 CAU/ml increase, p=0.02) and CRP (OR 1.2 [95% CI 1.0-1.3] for 10 mg/L increase, p=0.04) were associated with an increased risk of early graft thrombosis. TCC was specific for graft thrombosis, while CRP increased with several complications. PTA recipients with compared to those without graft thrombosis had higher TCC pre- (p=0.04) and postoperatively (p=0.03). Conclusion: The relative increase in postoperative thromboinflammatory response was more pronounced in PTA recipients. Complement activation was associated with an increased risk of graft thrombosis. This study indicates that innate immune activation rather than elevated levels may affect early postoperative pancreas graft thrombosis. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT01957696, identifier NCT01957696.

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.