Impact of the Graphene Production Methods Sonication and Microfluidization on In Vitro and In Vivo Toxicity, Macrophage Response, and Complement Activation.

Graphene, a material composed of a two-dimensional lattice of carbon atoms, has due to its many unique properties a wide array of potential applications in the biomedical field. One of the most common production methods is exfoliation through sonication, which is simple but has low yields. Another approach, using microfluidization, has shown promise through its scalability for commercial production. Regardless of their production method, materials made for biomedical applications need to be tested for biocompatibility. Here, we investigated the differences in toxicity, macrophage response, and complement activation of similar-sized graphene flakes produced through sonication and microfluidization, using in vitro cell assays and in vivo assays on zebrafish larvae. In vitro toxicity testing showed that sonicated graphene had a high toxicity, with an EC50 of 100 µg mL-1 for endothelial cells and 60 µg mL-1 for carcinoma cells. In contrast, microfluidized graphene did not reach EC50 at any of the tested concentrations. The potency to activate the complement system in whole blood was 10-fold higher for sonicated than for microfluidized graphene. In zebrafish larvae, graphene of either production method was found to mainly agglomerate in the caudal hematopoietic tissue; however, no acute toxic effects were found. Sonicated graphene led to an increase in macrophage count and a macrophage migration to the ventral tail area, while microfluidized graphene led to a transient reduction in macrophage count and fewer cells in the ventral trail area. The observed reduction in macrophages and change in macrophage distribution following exposure to microfluidized graphene was less pronounced compared to sonicated graphene and contributed to masking of the fluorescent signal rather than cytotoxic effects. Summarized, we observed higher toxicity, macrophage response, and complement activation with graphene produced through sonication, which could be due to oxygen-containing functional groups introduced to the edge of the carbon lattice by this production method. These findings indicate that microfluidization produces graphene more suitable for biomedical applications.

Damage Control Orthopaedics Induced Less Trauma-Induced Coagulopathy than Early Total Care in a Porcine Polytrauma Model.

INTRODUCTION: Coagulopathic disorders (CDs) complicate treatment in polytraumatised patients. Against this background, operative strategies for fracture management are controversial in this cohort. This study therefore investigated the effects of two established operative concepts, early total care (ETC) and damage control orthopaedics (DCO), on CD in a large-animal polytrauma (PT) model. METHODS: Twenty-two animals (Sus scrofa domesticus) sustained PT involving blunt-chest trauma, liver laceration, bilateral femur fracture, and pressure-controlled haemorrhagic shock. After resuscitation, animals were allocated to ETC (n = 8), DCO (n = 8), or served as a non-traumatised control group (CG, n = 6). Animals were ventilated and monitored under ICU standards for 72 h. Blood samples were collected at baseline and post-trauma after 1.5, 2.5, 24, 48, and 72 h. Plasminogen activator inhibitor-1 (PAI-1) and thrombin-antithrombin (TAT) complex concentrations were determined by ELISA. RESULTS: Compared to the CG, ETC and DCO subjects had significantly increased plasma concentrations of PAI-1 after 2.5 h (CG vs. ETC: p = 0.0050, CG vs. DCO: p = 0.0016). Furthermore, the ETC group showed significantly increased plasma PAI-1 concentrations after 24 h compared to the CG and DCO groups (CG vs. ETC: p = 0.0002, DCO vs. ETC: p = 0.0004). During the later clinical course, concentrations of TAT were significantly increased in the ETC group compared to the CG and DCO group after 72 h (CG vs. ETC: p = 0.0290, DCO vs. ETC: p = 0.0322). CONCLUSION: PT is strongly associated with CD in the early post-traumatic course. In comparison to DCO, ETC appeared to be negatively associated with CD. Future studies must investigate this impact, especially in those patients admitted with trauma-induced coagulopathy, to improve outcomes.

Femoral nailing associated with bone marrow emboli in pigs induced a specific increase in blood IL-6 and broad inflammatory responses in the heart and lungs.

Introduction: Bone marrow embolization may complicate orthopedic surgery, potentially causing fat embolism syndrome. The inflammatory potential of bone marrow emboli is unclear. We aimed to investigate the inflammatory response to femoral intramedullary nailing, specifically the systemic inflammatory effects in plasma, and local tissue responses. Additionally, the plasma response was compared to that following intravenous injection of autologous bone marrow. Methods: Twelve pigs underwent femoral nailing (previously shown to have fat emboli in lung and heart), four received intravenous bone marrow, and four served as sham controls. Blood samples were collected hourly and tissue samples postmortem. Additionally, we incubated bone marrow and blood, separately and in combination, from six pigs in vitro. Complement activation was detected by C3a and the terminal C5b-9 complement complex (TCC), and the cytokines TNF, IL-1ß, IL-6 and IL-10 as well as the thrombin-antithrombin complexes (TAT) were all measured using enzyme-immunoassays. Results: After nailing, plasma IL-6 rose 21-fold, compared to a 4-fold rise in sham (p=0.0004). No plasma differences in the rest of the inflammatory markers were noted across groups. However, nailing yielded 2-3-times higher C3a, TCC, TNF, IL-1ß and IL-10 in lung tissue compared to sham (p<0.0001-0.03). Similarly, heart tissue exhibited 2-times higher TCC and IL-1ß compared to sham (p<0.0001-0.03). Intravenous bone marrow yielded 8-times higher TAT than sham at 30 minutes (p<0.0001). In vitro, incubation of bone marrow for four hours resulted in 95-times higher IL-6 compared to whole blood (p=0.03). Discussion: A selective increase in plasma IL-6 was observed following femoral nailing, whereas lung and heart tissues revealed a broad local inflammatory response not reflected systemically. In vitro experiments may imply bone marrow to be the primary IL-6 source.

The function of the complement system remains fully intact throughout the course of allogeneic stem cell transplantation.

Introduction: Hematopoietic stem cell transplantation (HSCT) is associated with immune complications and endothelial dysfunction due to intricate donor-recipient interactions, conditioning regimens, and inflammatory responses. Methods: This study investigated the role of the complement system during HSCT and its interaction with the cytokine network. Seventeen acute myeloid leukemia patients undergoing HSCT were monitored, including blood sampling from the start of the conditioning regimen until four weeks post-transplant. Clinical follow-up was 200 days. Results: Total complement functional activity was measured by WIELISA and the degree of complement activation by ELISA measurement of sC5b-9. Cytokine release was measured using a 27-multiplex immuno-assay. At all time-points during HSCT complement functional activity remained comparable to healthy controls. Complement activation was continuously stable except for two patients demonstrating increased activation, consistent with severe endotheliopathy and infections. In vitro experiments with post-HSCT whole blood challenged with Escherichia coli, revealed a hyperinflammatory cytokine response with increased TNF, IL-1ß, IL-6 and IL-8 formation. Complement C3 inhibition markedly reduced the cytokine response induced by Staphylococcus aureus, Aspergillus fumigatus, and cholesterol crystals. Discussion: In conclusion, HSCT patients generally retained a fully functional complement system, whereas activation occurred in patients with severe complications. The complement-cytokine interaction indicates the potential for new complement-targeting therapeutic strategies in HSCT.

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.

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.

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.

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.

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.

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.

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.

Inflammatory Markers and Radiotherapy Response in Patients With Painful Bone Metastases.

CONTEXT: Inflammation is proposed to influence tumor response in radiotherapy (RT). Clinical studies to investigate the relationship between inflammatory markers and RT response is warranted to understand the variable RT efficacy in patients with painful bone metastases. OBJECTIVES: To evaluate the association between inflammatory markers and analgesic response to RT in patients with painful bone metastases. METHODS: Adult patients from 7 European study sites undergoing RT for painful bone metastases were included in this prospective and longitudinal analysis. The association between RT response and 17 inflammatory markers at baseline, as well as the association between RT response and the changes observed in inflammatory markers between baseline and three and eight weeks after RT, was analyzed with univariate regression analyses. Baseline analyses were adjusted for potential clinical predictors of RT response. RESULTS: None of the inflammatory markers were significantly associated with an upcoming RT response in the analysis of 448 patients with complete baseline data. In patients available for follow-up, the three-week change in TNF (P 0.017), IL-8 (P 0.028), IP-10 (P 0.032), eotaxin (P 0.043), G-CSF (P 0.033) and MCP-1 (P 0.002) were positively associated with RT response, while the three-week change in CRP (P 0.006) was negatively associated. CONCLUSION: Results from this study show an association between RT response and change in pro-inflammatory mediators and indicate that inflammation may be important to achieve an analgesic RT response in patients with painful bone metastases. None of the investigated inflammatory markers were found to be pre-treatment predictors of RT response.

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

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

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.

Antirheumatic therapy is associated with reduced complement activation in rheumatoid arthritis.

BACKGROUND: The complement system plays an important role in pathophysiology of cardiovascular disease (CVD), and might be involved in accelerated atherogenesis in rheumatoid arthritis (RA). The role of complement activation in response to treatment, and in development of premature CVD in RA, is limited. Therefore, we examined the effects of methotrexate (MTX) and tumor necrosis factor inhibitors (TNFi) on complement activation using soluble terminal complement complex (TCC) levels in RA; and assessed associations between TCC and inflammatory and cardiovascular biomarkers. METHODS: We assessed 64 RA patients starting with MTX monotherapy (n = 34) or TNFi with or without MTX co-medication (TNFi±MTX, n = 30). ELISA was used to measure TCC in EDTA plasma. The patients were examined at baseline, after 6 weeks and 6 months of treatment. RESULTS: Median TCC was 1.10 CAU/mL, and 57 (89%) patients had TCC above the estimated upper reference limit (<0.70). Compared to baseline, TCC levels were significantly lower at 6-week visit (0.85 CAU/mL, p<0.0001), without significant differences between the two treatment regimens. Notably, sustained reduction in TCC was only achieved after 6 months on TNFi±MTX (0.80 CAU/mL, p = 0.006). Reductions in TCC after treatment were related to decreased C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and interleukin 6, and increased levels of total, high and low-density lipoprotein cholesterol. Similarly, baseline TCC was significantly related to baseline CRP, ESR and interleukin 6. Patients with endothelial dysfunction had higher baseline TCC than those without (median 1.4 versus 1.0 CAU/mL, p = 0.023). CONCLUSIONS: Patients with active RA had elevated TCC, indicating increased complement activation. TCC decreased with antirheumatic treatment already after 6 weeks. However, only treatment with TNFi±MTX led to sustained reduction in TCC during the 6-month follow-up period. RA patients with endothelial dysfunction had higher baseline TCC compared to those without, possibly reflecting involvement of complement in the atherosclerotic process in RA.

Vitamin C, Hydrocortisone, and the Combination Thereof Significantly Inhibited Two of Nine Inflammatory Markers Induced by Escherichia Coli But Not by Staphylococcus Aureus - When Incubated in Human Whole Blood.

ABSTRACT: Vitamin C combined with hydrocortisone is increasingly being used to treat septic patients, even though this treatment regimen is based on questionable evidence. When used, a marked effect on key players of innate immunity would be expected, as sepsis is featured by a dysregulated immune response.Here, we explored the effect of vitamin C and hydrocortisone alone and combined, in an ex vivo human whole-blood model of Escherichia coli- or Staphylococcus aureus-induced inflammation. Inflammatory markers for activation of complement (terminal C5b-9 complement complex [TCC]), granulocytes (myeloperoxidase), platelets (ß-thromboglobulin), cytokines (tumor necrosis factor [TNF], IL-1ß, IL6, and IL-8), and leukocytes (CD11b and oxidative burst) were quantified, by enzyme-linked immunosorbent assay, multiplex technology, and flow cytometry.In E. coli- and S. aureus-stimulated whole blood, a broad dose-titration of vitamin C and hydrocortisone alone did not lead to dose-response effects for the central innate immune mediators TCC and IL-6. Hence, the clinically relevant doses were used further. Compared to the untreated control sample, two of the nine biomarkers induced by E. coli were reduced by hydrocortisone and/or vitamin C. TNF was reduced by hydrocortisone alone (19%, P = 0.01) and by the combination (31%, P = 0.01). The oxidative burst of monocytes and granulocytes was reduced for both drugs alone and their combination, (ranging 8-19%, P < 0.05). Using S. aureus, neither of the drugs, alone nor in combination, had any effects on the nine biomarkers.In conclusion, despite the limitation of the ex vivo model, the effect of vitamin C and hydrocortisone on bacteria-induced inflammatory response in human whole blood is limited and following the clinical data.

Patient Derived Colonoids as Drug Testing Platforms-Critical Importance of Oxygen Concentration.

Treatment of inflammatory bowel disease (IBD) is challenging, with a series of available drugs each helping only a fraction of patients. Patients may face time-consuming drug trials while the disease is active, thus there is an unmet need for biomarkers and assays to predict drug effect. It is well known that the intestinal epithelium is an important factor in disease pathogenesis, exhibiting physical, biochemical and immunologic driven barrier dysfunctions. One promising test system to study effects of existing or emerging IBD treatments targeting intestinal epithelial cells (IECs) is intestinal organoids ("mini-guts"). However, the fact that healthy intestinal epithelium is in a physiologically hypoxic state has largely been neglected, and studies with intestinal organoids are mainly performed at oxygen concentration of 20%. We hypothesized that lowering the incubator oxygen level from 20% to 2% would recapitulate better the in vivo physiological environment of colonic epithelial cells and enhance the translational value of intestinal organoids as a drug testing platform. In the present study we examine the effects of the key IBD cytokines and drug targets TNF/IL17 on human colonic organoids (colonoids) under atmospheric (20%) or reduced (2%) O2. We show that colonoids derived from both healthy controls and IBD-patients are viable and responsive to IBD-relevant cytokines at 2% oxygen. Because chemokine release is one of the important immunoregulatory traits of the epithelium that may be fine-tuned by IBD-drugs, we also examined chemokine expression and release at different oxygen concentrations. We show that chemokine responses to TNF/IL17 in organoids display similarities to inflamed epithelium in IBD-patients. However, inflammation-associated genes induced by TNF/IL17 were attenuated at low oxygen concentration. We detected substantial oxygen-dependent differences in gene expression in untreated as well as TNF/IL17 treated colonoids in all donors. Further, for some of the IBD-relevant cytokines differences between colonoids from healthy controls and IBD patients were more pronounced in 2% O2 than 20% O2. Our results strongly indicate that an oxygen concentration similar to the in vivo epithelial cell environment is of essence in experimental pharmacology.

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.

Innate Immune Invisible Ultrasmall Gold Nanoparticles-Framework for Synthesis and Evaluation.

Nanomedicine is seen as a potential central player in the delivery of personalized medicine. Biocompatibility issues of nanoparticles have largely been resolved over the past decade. Despite their tremendous progress, less than 1% of applied nanosystems can hit their intended target location, such as a solid tumor, and this remains an obstacle to their full ability and potential with a high translational value. Therefore, achieving immune-tolerable, blood-compatible, and biofriendly nanoparticles remains an unmet need. The translational success of nanoformulations from bench to bedside involves a thorough assessment of their design, compatibility beyond cytotoxicity such as immune toxicity, blood compatibility, and immune-mediated destruction/rejection/clearance profile. Here, we report a one-pot process-engineered synthesis of ultrasmall gold nanoparticles (uGNPs) suitable for better body and renal clearance delivery of their payloads. We have obtained uGNP sizes of as low as 3 nm and have engineered the synthesis to allow them to be accurately sized (almost nanometer by nanometer). The synthesized uGNPs are biocompatible and can easily be functionalized to carry drugs, peptides, antibodies, and other therapeutic molecules. We have performed in vitro cell viability assays, immunotoxicity assays, inflammatory cytokine analysis, a complement activation study, and blood coagulation studies with the uGNPs to confirm their safety. These can help to set up a long-term safety-benefit framework of experimentation to reveal whether any designed nanoparticles are immune-tolerable and can be used as payload carriers for next-generation vaccines, chemotherapeutic drugs, and theranostic agents with better body clearance ability and deep tissue penetration.