Søren E. Pischke

Combined Inhibition of Complement (C5) and CD14 Markedly Attenuates Inflammation, Thrombogenicity, and Hemodynamic Changes in Porcine Sepsis

Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli–induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin–antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1β, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic changes.

Double Blockade of CD14 and Complement C5 Abolishes the Cytokine Storm and Improves Morbidity and Survival in Polymicrobial Sepsis in Mice

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

Inflammatory response after laparoscopic versus open resection of colorectal liver metastases: Data from the oslo-comet trial

Abstract Laparoscopic and open liver resection have not been compared in randomized trials. The aim of the current study was to compare the inflammatory response after laparoscopic and open resection of colorectal liver metastases (CLM) in a randomized controlled trial. This was a predefined exploratory substudy within the Oslo CoMet-study. Forty-five patients with CLM were randomized to laparoscopic (n = 23) or open (n = 22) resection. Ethylenediaminetetraacetic acid-plasma samples were collected preoperatively and at defined time points during and after surgery and snap frozen at −80 oC. A total of 25 markers were examined using luminex and enzyme-linked immunosorbent assay techniques: high-mobility box group 1(HMGB-1), cell-free DNA (cfDNA), cytokines, and terminal C5b-9 complement complex complement activation. Eight inflammatory markers increased significantly from baseline: HMGB-1, cfDNA, interleukin (IL)-6, C-reactive protein, macrophage inflammatory protein -1&bgr;, monocyte chemotactic protein -1, IL-10, and terminal C5b-9 complement complex. Peak levels were reached at the end of or shortly after surgery. Five markers, HMGB-1, cfDNA, IL-6, C-reactive protein, and macrophage inflammatory protein -1&bgr;, showed significantly higher levels in the open surgery group compared with the laparoscopic surgery group. Laparoscopic resection of CLM reduced the inflammatory response compared with open resection. The lower level of HMGB-1 is interesting because of the known association with oncogenesis.

Systemic CD14 Inhibition Attenuates Organ Inflammation in Porcine Escherichia coli Sepsis

ABSTRACT Sepsis is an infection-induced systemic inflammatory response syndrome. Upstream recognition molecules, like CD14, play key roles in the pathogenesis. The aim of the present study was to investigate the effect of systemic CD14 inhibition on local inflammatory responses in organs from septic pigs. Pigs (n = 34) receiving Escherichia coli-bacteria or E. coli-lipopolysaccharide (LPS) were treated with an anti-CD14 monoclonal antibody or an isotype-matched control. Lungs, liver, spleen, and kidneys were examined for bacteria and inflammatory biomarkers. E. coli and LPS were found in large amounts in the lungs compared to the liver, spleen, and kidneys. Notably, the bacterial load did not predict the respective organ inflammatory response. There was a marked variation in biomarker induction in the organs and in the effect of anti-CD14. Generally, the spleen produced the most cytokines per weight unit, whereas the liver contributed the most to the total load. All cytokines were significantly inhibited in the spleen. Interleukin-6 (IL-6) was significantly inhibited in all organs, IL-1β and IP-10 were significantly inhibited in liver, spleen, and kidneys, and tumor necrosis factor, IL-8, and PAI-1 were inhibited only in the spleen. ICAM-1 and VCAM-1 was significantly inhibited in the kidneys. Systemic CD14-inhibition efficiently, though organ dependent, attenuated local inflammatory responses. Detailed knowledge on how the different organs respond to systemic inflammation in vivo, beyond the information gained by blood examination, is important for our understanding of the nature of systemic inflammation and is required for future mediator-directed therapy in sepsis. Inhibition of CD14 seems to be a good candidate for such treatment.

Perioperative detection of myocardial ischaemia/reperfusion with a novel tissue CO2 monitoring technology

OBJECTIVES Detection of perioperative myocardial ischaemia in cardiac surgery remains challenging, as current clinical bedside monitoring is insufficient in making proper diagnoses in real-time. Cellular metabolism gets altered during ischaemia and tissue PCO2 is produced in the course of buffering anaerobic lactic acidosis. Myocardial tissue PCO2 has been suggested as a parameter of ischaemia, but PCO2 measurement devices for routine clinical usage are lacking. Study aims were to (i) evaluate the diagnostic potential of PCO2 in early detection of localized myocardial metabolic changes, (ii) compare PCO2 obtained by novel conductometric PCO2 sensors (IscAlert) with fibre-optical sensors (Neurotrend), and (iii) investigate the relationship between myocardial PCO2, PO2 and parameters of energy consumption during regional myocardial ischaemia/reperfusion. METHODS In nine pigs, IscAlert sensors, Neurotrend sensors and microdialysis catheters were placed in the myocardium in the supply region of the left anterior descending (LAD) or circumflex (CX) coronary artery. LAD was occluded for 1, 3, 5 and 15 min, with 30 min of reperfusion between occlusion intervals. PCO2, PO2 and pH were measured continuously, microdialysis samples were obtained intermittently. The generation rate of CO2 (time-derivative of PCO2, TDPCO2) was calculated. RESULTS Myocardial ischaemia was confirmed by PO2 and pH decline, accompanied by lactate and lactate/pyruvate ratio increase. PCO2 measured by IscAlert increased significantly (P<0.01) during all occlusions and the increase was related to duration of ischaemia. PCO2 normalized during reperfusion. No significant changes were observed in CX region, indicating high regional sensitivity and specificity. Similar results were found with fibre-optically measured PCO2 and maximum PCO2 values during each interval correlated well with PCO2 values measured by IscAlert (R=0.93±0.05, P<0.001). Maximum TDPCO2 depicted beginning of anoxia and diminishing metabolism during anaerobic conditions. CONCLUSIONS IscAlert sensors enable reliable and continuous detection of myocardial ischaemia by measuring myocardial PCO2. A combination of PCO2 and TDPCO2 seems promising in revealing information about substrate supply and cellular homeostasis during ischaemic events.

Dual inhibition of complement and Toll-like receptors as a novel approach to treat inflammatory diseases—C3 or C5 emerge together with CD14 as promising targets

The host is protected by pattern recognition systems, including complement and TLRs, which are closely cross‐talking. If improperly activated, these systems might induce tissue damage and disease. Inhibition of single downstream proinflammatory cytokines, such as TNF, IL‐1β, and IL‐6, have failed in clinical sepsis trials, which might not be unexpected, given the substantial amounts of mediators involved in the pathogenesis of this condition. Instead, we have put forward a hypothesis of inhibition at the recognition phase by “dual blockade” of bottleneck molecules of complement and TLRs. By acting upstream and broadly, the dual blockade could be beneficial in conditions with improper or uncontrolled innate immune activation threatening the host. Key bottleneck molecules in these systems that could be targets for inhibition are the central complement molecules C3 and C5 and the important CD14 molecule, which is a coreceptor for several TLRs, including TLR4 and TLR2. This review summarizes current knowledge of inhibition of complement and TLRs alone and in combination, in both sterile and nonsterile inflammatory processes, where activation of these systems is of crucial importance for tissue damage and disease. Thus, dual blockade might provide a general, broad‐acting therapeutic regimen against a number of diseases where innate immunity is improperly activated.

Organ inflammation in porcine Escherichia coli sepsis is markedly attenuated by combined inhibition of C5 and CD14.

Sepsis is an infection-induced systemic inflammatory syndrome, potentially causing organ failure. We previously showed attenuating effects on inflammation, thrombogenicity and haemodynamics by inhibiting the Toll-like receptor co-factor CD14 and complement factor C5 in a porcine Escherichia coli-induced sepsis model. The present study explored the effect on organ inflammation in these pigs. Tissue samples were examined from the combined treatment group (n = 8), the positive (n = 8) and negative (n = 6) control groups after 4h of sepsis. Inflammatory biomarkers were measured using ELISA, multiplex and qPCR analysis. Combined inhibition of C5 and CD14 markedly attenuated IL-1β by 31-66% (P < 0.05) and IL-6 by 54-96% (P < 0.01) in liver, kidney, lung and spleen; IL-8 by 65-100% in kidney, lung, spleen, and heart (P < 0.05) and MCP-1 by 46-69% in liver, kidney, spleen and heart (P < 0.05). Combined inhibition significantly attenuated tissue factor mRNA upregulation in spleen (P < 0.05) and IP-10 mRNA upregulation in four out of five organs. Finally, C5aR mRNA downregulation was prevented in heart and kidney (P < 0.05). Combined inhibition of C5 and CD14 thus markedly attenuated inflammatory responses in all organs examined. The anti-inflammatory effects observed in lung and heart may explain the delayed haemodynamic disturbances observed in septic pigs receiving combined inhibition of C5 and CD14.

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

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

Acute heart failure following myocardial infarction: complement activation correlates with the severity of heart failure in patients developing cardiogenic shock: Complement activation in severe heart failure

Heart failure (HF) is an impending complication to myocardial infarction. We hypothesized that the degree of complement activation reflects severity of HF following acute myocardial infarction.

A neglected organ in multiple organ failure - 'skin in the game'?

In a recent issue of this journal, Drs. Koskela et al. investigated immunological changes in the skin in sepsis patients. They obtained fluid from non-injured skin by applying vacuum and collected the fluid of the resulting blisters. This can remind one of the images from the recent summer Olympic games where a number of athletes had undergone the alternative medicine treatment of ‘cupping’. Although samples were collected in this study from healthy looking skin, index cytokines for sepsis severity were increased in septic patients in comparison to healthy controls. Interestingly, concentration of many cytokines was higher in skin blister fluid than in blood from sepsis patients. Sepsis is defined in the new sepsis guidelines as ‘life-threatening organ dysfunction caused by a dysregulated host response to infection’ and diagnosed by an increase of two points or more in the now renamed Sepsis-related Organ Failure Assessment (SOFA) score. Thus, it is acknowledged that sepsis affects the whole organism and not only compartments. However, the SOFA score does not include the biggest organ in the body, which is the only organ that has always direct contact with the environment, the skin. Skin has a finely balanced homeostasis to ensure protection of the body and allow growth of protective commensal bacteria, which is each individual’s microbiome. Thus, skin is a large and vulnerable organ. Extensive skin injury poses an immediate threat to the patient and a considerable challenge to the intensivist. It is widely recognized that there are high mortality rates associated with toxic epidermal necrolysis and extensive burn injury. At the same time, almost every intensive care patient shows sign of disturbed skin homeostasis, resulting in high-risk for pressure ulcers and prolonged wound healing. In the affected skin, the microbiome is changed, that is, pathogenic bacteria like S. aureus dominate and increase the risk for systemic infections leading to sepsis and poor outcome. Disturbed skin homeostasis during septic shock may result from ischaemia/reperfusion injury caused by dysfunction of the microcirculation in addition to blood flow diversion from the periphery to central organs. Ischaemia/reperfusion injury is known to cause activation of the innate immune system with subsequent cytokine production resulting in increased skin vulnerability. Additionally, stressors like catecholamines, acetylcholine and glucocorticoids, all produced in vast amounts during sepsis, have been shown to affect the dermal microbiome, resulting in impaired