A survey of ficolin-3 activity in Systemic Lupus Erythematosus reveals a link to hematological disease manifestations and autoantibody profile.

The complement system plays a central role in the pathogenesis of Systemic Lupus Erythematosus (SLE), but most studies have focused on the classical pathway. Ficolin-3 is the main initiator of the lectin pathway of complement in humans, but its role in systemic autoimmune disease has not been conclusively determined. Here, we combined biochemical and genetic approaches to assess the contribution of ficolin-3 to SLE risk and disease manifestations. Ficolin-3 activity was measured by a functional assay in serum or plasma samples from Swedish SLE patients (n = 786) and controls matched for age and sex (n = 566). Genetic variants in an extended 300 kb genomic region spanning the FCN3 locus were analyzed for their association with ficolin-3 activity and SLE manifestations in a Swedish multicenter cohort (n = 985). Patients with ficolin-3 activity in the highest tertile showed a strong enrichment in an SLE cluster defined by anti-Sm/DNA/nucleosome antibodies (OR 3.0, p < 0.001) and had increased rates of hematological disease (OR 1.4, p = 0.078) and lymphopenia (OR = 1.6, p = 0.039). Genetic variants associated with low ficolin-3 activity mapped to an extended haplotype in high linkage disequilibrium upstream of the FCN3 gene. Patients carrying the lead genetic variant associated with low ficolin-3 activity had a lower frequency of hematological disease (OR 0.67, p = 0.018) and lymphopenia (OR 0.63, p = 0.031) and fewer autoantibodies (p = 0.0019). Loss-of-function variants in the FCN3 gene were not associated with SLE, but four (0.5 %) SLE patients developed acquired ficolin-3 deficiency where ficolin-3 activity in serum was depleted following diagnosis of SLE. Taken together, our results provide genetic and biochemical evidence that implicate the lectin pathway in hematological SLE manifestations. We also identify lectin pathway activation through ficolin-3 as a factor that contributes to the autoantibody response in SLE.

Activation of the Complement and Coagulation Systems in the Small Airways in Asthma.

BACKGROUND: Several studies have shown the importance of the complement and coagulation systems in the pathogenesis of asthma. OBJECTIVES: We explored whether we could detect differentially abundant complement and coagulation proteins in the samples obtained from the small airway lining fluid by collection of exhaled particles in patients with asthma and whether these proteins are associated with small airway dysfunction and asthma control. METHOD: Exhaled particles were obtained from 20 subjects with asthma and 10 healthy controls (HC) with the PExA method and analysed with the SOMAscan proteomics platform. Lung function was assessed by nitrogen multiple breath washout test and spirometry. RESULTS: 53 proteins associated with the complement and coagulation systems were included in the analysis. Nine of those proteins were differentially abundant in subjects with asthma as compared to HC, and C3 was significantly higher in inadequately controlled asthma as compared to well-controlled asthma. Several proteins were associated with physiological tests assessing small airways. CONCLUSIONS: The study highlights the role of the local activation of the complement and coagulation systems in the small airway lining fluid in asthma and their association with both asthma control and small airway dysfunction. The findings highlight the potential of complement factors as biomarkers to identify different sub-groups among patients with asthma that could potentially benefit from a therapeutic approach targeting the complement system.

Properdin binding to complement activating surfaces depends on initial C3b deposition.

Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenous molecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions.

Complement C5a Functions as a Master Switch for the pH Balance in Neutrophils Exerting Fundamental Immunometabolic Effects.

During sepsis, excessive activation of the complement system with generation of the anaphylatoxin C5a results in profound disturbances in crucial neutrophil functions. Moreover, because neutrophil activity is highly dependent on intracellular pH (pHi), we propose a direct mechanistic link between complement activation and neutrophil pHi In this article, we demonstrate that in vitro exposure of human neutrophils to C5a significantly increased pHi by selective activation of the sodium/hydrogen exchanger. Upstream signaling of C5a-mediated intracellular alkalinization was dependent on C5aR1, intracellular calcium, protein kinase C, and calmodulin, and downstream signaling regulated the release of antibacterial myeloperoxidase and lactoferrin. Notably, the pH shift caused by C5a increased the glucose uptake and activated glycolytic flux in neutrophils, resulting in a significant release of lactate. Furthermore, C5a induced acidification of the extracellular micromilieu. In experimental murine sepsis, pHi of blood neutrophils was analogously alkalinized, which could be normalized by C5aR1 inhibition. In the clinical setting of sepsis, neutrophils from patients with septic shock likewise exhibited a significantly increased pHi These data suggest a novel role for the anaphylatoxin C5a as a master switch of the delicate pHi balance in neutrophils resulting in profound inflammatory and metabolic changes that contribute to hyperlactatemia during sepsis.

Shiga toxin 2a binds antithrombin and heparin, but does not directly activate platelets.

Escherichia coli-induced hemolytic uremic syndrome (eHUS) is a life-threatening complication of infection with Shiga toxin (Stx), in particular Stx2a-producing Escherichia coli. Enhanced coagulation activation with formation of microthrombi seems to be a key event in development of eHUS. Platelet activation has been postulated as a possible, but controversially debated mechanism. The present study investigated the effect of Stx2a on plasmatic coagulation and platelets. Binding studies were initially performed with ELISA and co-immunoprecipitation and supported by quartz crystal microbalance with dissipation monitoring (QCM-D). Antithrombin (AT) activity was measured using the automated BCS XP® system. ROTEM® was used for functional coagulation testing. Platelet binding and activation was studied with FACS and light-transmission aggregometry. We found binding of Stx2a to AT, an important inhibitor of blood coagulation, but only a mild albeit significant reduction of AT activity against FXa in the presence of Stx2a. QCM-D analysis also showed binding of Stx2a to heparin and an impaired binding of AT to Stx2a-bound heparin. ROTEM® using Stx2a-treated platelet-poor plasma revealed a significant, but only moderate shortening of clotting time. Neither binding nor activation of platelets by Stx2a could be demonstrated. In summary, data of this study suggest that Stx2a binds to AT, but does not induce major effects on plasmatic coagulation. In addition, no interaction with platelets occurred. The well-known non-beneficial administration of heparin in eHUS patients could be explained by the interaction of Stx2a with heparin.

Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?

We have recently reported that therapeutic mesenchymal stromal cells (MSCs) have low engraftment and trigger the instant blood mediated inflammatory reaction (IBMIR) after systemic delivery to patients, resulting in compromised cell function. In order to optimize the product, we compared the immunomodulatory, blood regulatory, and therapeutic properties of freeze-thawed and freshly harvested cells. We found that freeze-thawed MSCs, as opposed to cells harvested from continuous cultures, have impaired immunomodulatory and blood regulatory properties. Freeze-thawed MSCs demonstrated reduced responsiveness to proinflammatory stimuli, an impaired production of anti-inflammatory mediators, increased triggering of the IBMIR, and a strong activation of the complement cascade compared to fresh cells. This resulted in twice the efficiency in lysis of thawed MSCs after 1 hour of serum exposure. We found a 50% and 80% reduction in viable cells with freshly detached as opposed to thawed in vitro cells, indicating a small benefit for fresh cells. In evaluation of clinical response, we report a trend that fresh cells, and cells of low passage, demonstrate improved clinical outcome. Patients treated with freshly harvested cells in low passage had a 100% response rate, twice the response rate of 50% observed in a comparable group of patients treated with freeze-thawed cells at higher passage. We conclude that cryobanked MSCs have reduced immunomodulatory and blood regulatory properties directly after thawing, resulting in faster complement-mediated elimination after blood exposure. These changes seem to be paired by differences in therapeutic efficacy in treatment of immune ailments after hematopoietic stem cell transplantation.

A novel C3 mutation causing increased formation of the C3 convertase in familial atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome has been associated with dysregulation of the alternative complement pathway. In this study, a novel heterozygous C3 mutation was identified in a factor B-binding region in exon 41, V1636A (4973 T > C). The mutation was found in three family members affected with late-onset atypical hemolytic uremic syndrome and symptoms of glomerulonephritis. All three patients exhibited increased complement activation detected by decreased C3 levels and glomerular C3 deposits. Platelets from two of the patients had C3 and C9 deposits on the cell surface. Patient sera exhibited more C3 cleavage and higher levels of C3a. The C3 mutation resulted in increased C3 binding to factor B and increased net formation of the C3 convertase, even after decay induced by decay-accelerating factor and factor H, as assayed by surface plasmon resonance. Patient sera incubated with washed human platelets induced more C3 and C9 deposition on the cell surface in comparison with normal sera. More C3a was released into serum over time when washed platelets were exposed to patient sera. Results regarding C3 and C9 deposition on washed platelets were confirmed using purified patient C3 in C3-depleted serum. The results indicated enhanced convertase formation leading to increased complement activation on cell surfaces. Previously described C3 mutations showed loss of function with regard to C3 binding to complement regulators. To our knowledge, this study presents the first known C3 mutation inducing increased formation of the C3 convertase, thus explaining enhanced activation of the alternative pathway of complement.

Are therapeutic human mesenchymal stromal cells compatible with human blood?

Multipotent mesenchymal stromal cells (MSCs) are tested in numerous clinical trials. Questions have been raised concerning fate and function of these therapeutic cells after systemic infusion. We therefore asked whether culture-expanded human MSCs elicit an innate immune attack, termed instant blood-mediated inflammatory reaction (IBMIR), which has previously been shown to compromise the survival and function of systemically infused islet cells and hepatocytes. We found that MSCs expressed hemostatic regulators similar to those produced by endothelial cells but displayed higher amounts of prothrombotic tissue/stromal factors on their surface, which triggered the IBMIR after blood exposure, as characterized by formation of blood activation markers. This process was dependent on the cell dose, the choice of MSC donor, and particularly the cell-passage number. Short-term expanded MSCs triggered only weak blood responses in vitro, whereas extended culture and coculture with activated lymphocytes increased their prothrombotic properties. After systemic infusion to patients, we found increased formation of blood activation markers, but no formation of hyperfibrinolysis marker D-dimer or acute-phase reactants with the currently applied dose of 1.0-3.0 × 10(6) cells per kilogram. Culture-expanded MSCs trigger the IBMIR in vitro and in vivo. Induction of IBMIR is dose-dependent and increases after prolonged ex vivo expansion. Currently applied doses of low-passage clinical-grade MSCs elicit only minor systemic effects, but higher cell doses and particularly higher passage cells should be handled with care. This deleterious reaction can compromise the survival, engraftment, and function of these therapeutic cells.

Protection of nonself surfaces from complement attack by factor H-binding peptides: implications for therapeutic medicine.

Exposure of nonself surfaces such as those of biomaterials or transplanted cells and organs to host blood frequently triggers innate immune responses, thereby affecting both their functionality and tolerability. Activation of the alternative pathway of complement plays a decisive role in this unfavorable reaction. Whereas previous studies demonstrated that immobilization of physiological regulators of complement activation (RCA) can attenuate this foreign body-induced activation, simple and efficient approaches for coating artificial surfaces with intact RCA are still missing. The conjugation of small molecular entities that capture RCA with high affinity is an intriguing alternative, as this creates a surface with autoregulatory activity upon exposure to blood. We therefore screened two variable cysteine-constrained phage-displayed peptide libraries for factor H-binding peptides. We discovered three peptide classes that differed with respect to their main target binding areas. Peptides binding to the broad middle region of factor H (domains 5-18) were of particular interest, as they do not interfere with either regulatory or binding activities. One peptide in this group (5C6) was further characterized and showed high factor H-capturing activity while retaining its functional integrity. Most importantly, when 5C6 was coated to a model polystyrene surface and exposed to human lepirudin-anticoagulated plasma, the bound peptide captured factor H and substantially inhibited complement activation by the alternative pathway. Our study therefore provides a promising and novel approach to produce therapeutic materials with enhanced biocompatibility.

Impact of surface coating and systemic anticoagulants on hemostasis and inflammation in a human whole blood model.

BACKGROUND: Surface compatibility with blood is critical both for scientific investigations on hemostasis and clinical applications. Regarding in vitro and ex vivo investigations, minimal alteration in physiological hemostasis is of particular importance to draw reliable conclusions on the human coagulation system. At the same time, artificial coagulation activation must be avoided, which is relevant for the patient, for example to prevent stent graft occlusion. The aim was to evaluate the advantages and disadvantages of antithrombotic and antifouling surface coatings in the context of their suitability for ex vivo incubation and the study of coagulation properties. METHODS: We investigated the impact of different protocols for surface coating of synthetic material and different anticoagulants on hemostasis and platelet activation in ex vivo human whole blood. Blood samples from healthy donors were incubated in coated microtubes on a rotating wheel at 37°C. Two protocols for surface coating were analyzed for hemostatic parameters and metabolic status, a heparin-based coating (CHC, Corline Heparin Conjugate) without further anticoagulation and a passivating coating (MPC, 2-methacryloyloxethyl phosphorylcholine) with added anticoagulants (enoxaparin, ENOX; or fondaparinux, FPX). Employing the MPC-based coating, the anticoagulants enoxaparin and fondaparinux were compared regarding their differential effects on plasmatic coagulation by thrombelastometry and on platelet activation by flowcytometry and platelet function assays. RESULTS: Using the CHC coating, significant coagulation cascade activation was observed, whereas parameters remained mostly unchanged with MPC-based protocols. Extended incubation caused significantly elevated levels of the soluble membrane attack complex. Neither ENOX nor FPX caused a relevant impairment of platelet function or activation capacity and thrombelastometric parameters remained unchanged with both protocols. For translational purposes, we additionally modeled endotoxemia with the MPC-based protocols by incubating with lipopolysaccharide plus/minus thrombin. While coagulation parameters remained unchanged, elevated Interleukin 8 and Matrix Metalloproteinase 9 demonstrated preserved immune cell responsiveness. CONCLUSIONS: The MPC-based protocols demonstrated better hemocompatibility compared to CHC, and ENOX and FPX proved useful for additional anticoagulation. Furthermore, this simple-to-use whole blood model may be useful for experimental analyses of the early coagulatory and immunological response without decalcification.

Combined Heterozygous Genetic Variations in Complement C2 and C8B: An Explanation for Multidimensional Immune Imbalance?

The complement system plays a crucial role in host defense, homeostasis, and tissue regeneration and bridges the innate and the adaptive immune systems. Although the genetic variants in complement C2 (c.839_849+17del; p.(Met280Asnfs*5)) and C8B (c.1625C>T; p.(Thr542Ile)) are known individually, here, we report on a patient carrying their combination in a heterozygous form. The patient presented with a reduced general condition and suffers from a wide variety of autoimmune diseases. While no autoimmune disease-specific autoantibodies could be detected, genetic analysis revealed abnormalities in the two complement genes C2 and C8B. Therefore, we performed a comprehensive investigation of the innate immune system on a cellular and humoral level to define the functional consequences. We found slightly impaired functionality of neutrophils and monocytes regarding phagocytosis and reactive oxygen species generation and a diminished expression of the C5aR1. An extensive complement analysis revealed a declined activation potential for the alternative and classical pathway. Reconstitution with purified C2 and C8 into patient serum failed to normalize the dysfunction, whereas the addition of C3 improved the hemolytic activity. In clinical transfer, in vitro supplementation of the patient's plasma with FFP as a complement source could fully restore full complement functionality. This study describes for the first time a combined heterozygous genetic variation in complement C2 and C8B which, however, cannot fully explain the overall dysfunctions and calls for further complement deficiency research and corresponding therapies.

Modulation of Neutrophil Activity by Soluble Complement Cleavage Products-An In-Depth Analysis.

The cellular and fluid phase-innate immune responses of many diseases predominantly involve activated neutrophil granulocytes and complement factors. However, a comparative systematic analysis of the early impact of key soluble complement cleavage products, including anaphylatoxins, on neutrophil granulocyte function is lacking. Neutrophil activity was monitored by flow cytometry regarding cellular (electro-)physiology, cellular activity, and changes in the surface expression of activation markers. The study revealed no major effects induced by C3a or C4a on neutrophil functions. By contrast, exposure to C5a or C5a des-Arg stimulated neutrophil activity as reflected in changes in membrane potential, intracellular pH, glucose uptake, and cellular size. Similarly, C5a and C5a des-Arg but no other monitored complement cleavage product enhanced phagocytosis and reactive oxygen species generation. C5a and C5a des-Arg also altered the neutrophil surface expression of several complement receptors and neutrophil activation markers, including C5aR1, CD62L, CD10, and CD11b, among others. In addition, a detailed characterization of the C5a-induced effects was performed with a time resolution of seconds. The multiparametric response of neutrophils was further analyzed by a principal component analysis, revealing CD11b, CD10, and CD16 to be key surrogates of the C5a-induced effects. Overall, we provide a comprehensive insight into the very early interactions of neutrophil granulocytes with activated complement split products and the resulting neutrophil activity. The results provide a basis for a better and, importantly, time-resolved and multiparametric understanding of neutrophil-related (patho-)physiologies.

Structures of complement component C3 provide insights into the function and evolution of immunity.

The mammalian complement system is a phylogenetically ancient cascade system that has a major role in innate and adaptive immunity. Activation of component C3 (1,641 residues) is central to the three complement pathways and results in inflammation and elimination of self and non-self targets. Here we present crystal structures of native C3 and its final major proteolytic fragment C3c. The structures reveal thirteen domains, nine of which were unpredicted, and suggest that the proteins of the alpha2-macroglobulin family evolved from a core of eight homologous domains. A double mechanism prevents hydrolysis of the thioester group, essential for covalent attachment of activated C3 to target surfaces. Marked conformational changes in the alpha-chain, including movement of a critical interaction site through a ring formed by the domains of the beta-chain, indicate an unprecedented, conformation-dependent mechanism of activation, regulation and biological function of C3.

The Complex Relationship between C4b-Binding Protein, Warfarin, and Antiphospholipid Antibodies.

BACKGROUND: Low levels of total C4b-binding protein (C4BPt), a circulating inhibitor of the classical/lectin complement pathways, were observed in patients with antiphospholipid antibodies (aPLs) and during warfarin treatment. OBJECTIVES: To investigate the associations between aPL and C4BPt in patients with persistently positive (++) aPL, with/without clinical manifestations and systemic lupus erythematosus (SLE), and in controls. Furthermore, we explored the impact of anticoagulation on C4BPt and in relation to complement activation. METHODS: In a cross-sectional design we investigated defined subgroups: primary (p) antiphospholipid syndrome (APS, N = 67), aPL++ individuals without clinical manifestations (aPL carriers, N = 15), SLE-aPL++ (N = 118, among them, secondary [s] APS, N = 56), aPL negative (-) SLE (SLE-aPL-, N = 291), and 322 controls. Clinical characteristics, including treatment, were tabulated. C4BPt was determined with a magnetic bead method. Complement proteins (C1q, C2, C3, C4, C3a, C3dg, sC5b-9, factor I [FI]) were measured. A mediation analysis was performed to decompose the total effect of aPL++ on C4BPt into the direct and indirect effects of aPL++ through warfarin. RESULTS: Overall, C4BPt is 20% decreased in aPL++ patients, regardless of SLE, APS, clinical manifestations, and aPL profile. C4BPt levels associate positively with complement proteins C1q, C2, C3, and C4, and negatively with complement activation product C3dg. In the SLE group, warfarin treatment contributes to approximately half of the C4BPt reduction (9%) CONCLUSION: Both aPLs and warfarin are associated with C4BPt reduction. Complement activation in aPL++ patients may partly be explained by impaired inhibition through depressed C4BPt levels. Further studies are needed to understand the clinical implications.

Interdialytic weight gain of less than 2.5% seems to limit cardiac damage during hemodialysis.

AIMS: To investigate if a single low-flux HD induces a rise in cardiac biomarkers and if a change in clinical approach may limit such mechanism. MATERIAL AND METHODS: A total of 20 chronic HD patients each underwent three different study-dialyses. Dialyzers (low-flux polysulfone, 1.8 sqm) had been stored either dry or wet (Wet) and the blood level in the venous chamber kept low or high. Laboratory results were measured at baseline, 30 and 180 min, adjusted for the effect of fluid shift. Ultrasound measured microemboli signals (MES) within the return line. RESULTS: Hemodialysis raised cardiac biomarkers (p < 0.001): Pentraxin 3 (PTX) at 30 min (by 22%) and at 180 min PTX (53%), Pro-BNP (15%), and TnT (5%), similarly for all three HD modes. Baseline values of Pro-BNP correlated with TnT (rho = 0.38, p = 0.004) and PTX (rho = 0.52, p < 0.001). The changes from pre- to 180 min of HD (delta-) were related to baseline values (Pro-BNP: rho = 0.91, p < 0.001; TnT: rho = 0.41, p = 0.001; PTX: rho = 0.29, p = 0.027). Delta Pro-BNP (rho = 0.67, p < 0.001) and TnT (rho = 0.38, p = 0.004) correlated with inter-dialytic-weight-gain (IDWG). Biomarkers behaved similarly between the HD modes. The least negative impact was with an IDWG ⩽ 2.5%. Multiple regression analyses of the Wet-High mode does not exclude a relation between increased exposure of MES and factors such as release of Pro-BNP. CONCLUSION: Hemodialysis, independent of type of dialyzer storage, was associated with raised cardiac biomarkers, more profoundly in patients with higher pre-dialysis values and IDWG. A limitation in IDWG to <2.5% and prolonged ultrafiltration time may limit cardiac strain during HD, especially in patients with cardiovascular risk.

Interleukin 8 Elicits Rapid Physiological Changes in Neutrophils That Are Altered by Inflammatory Conditions.

A sufficient response of neutrophil granulocytes stimulated by interleukin (IL)-8 is vital during systemic inflammation, for example, in sepsis or severe trauma. Moreover, IL-8 is clinically used as biomarker of inflammatory processes. However, the effects of IL-8 on cellular key regulators of neutrophil properties such as the intracellular pH (pHi) in dependence of ion transport proteins and during inflammation remain to be elucidated. Therefore, we investigated in detail the fundamental changes in pHi, cellular shape, and chemotactic activity elicited by IL-8. Using flow cytometric methods, we determined that the IL-8-induced cellular activity was largely dependent on specific ion channels and transporters, such as the sodium-proton exchanger 1 (NHE1) and non-NHE1-dependent sodium flux. Exposing neutrophils in vitro to a proinflammatory micromilieu with N-formyl-Met-Leu-Phe, LPS, or IL-8 resulted in a diminished response regarding the increase in cellular size and pH. The detailed kinetics of the reduced reactivity of the neutrophil granulocytes could be illustrated in a near-real-time flow cytometric measurement. Last, the LPS-mediated impairment of the IL-8-induced response in neutrophils was confirmed in a translational, animal-free human whole blood model. Overall, we provide novel mechanistic insights for the interaction of IL-8 with neutrophil granulocytes and report in detail about its alteration during systemic inflammation.

Poly(2-aminoethyl methacrylate)-based polyampholyte brush surface with carboxylic groups to improve blood compatibility.

Zwitterionic material-based polymer brush significantly prevents protein adsorption and cell adhesion, which leads to the blood compatibility. However, zwitterionic polymer itself is difficult to be modified further, for the blood compatibility since the charged balance is impaired after the modification. In this research, chemically modifiable mixed charge polymer brush is designed, without impairing its characteristics. Condensed mixed charge polymer brush will work like zwitterionic material because neighbouring opposite charge is reported to be important in the zwitterionic material. Cationic polymer brush with primary amine group, which is based on 2-aminoethyl methacrylate (AEMA), was prepared and modified by succinic anhydride to obtain carboxylic group induced poly(AEMA). The ratio of primary amine group and carboxylic group was optimized to obtain the polyampholyte brush. The blood compatibility was evaluated by measuring coagulation/complement activation, protein adsorption and cell adhesion induced by the polymer. Our designed cationic-based polyampholyte brush prevented coagulation/complement activation comparable to poly(2-methacryloyloxyethyl phosphorylcholine) brush, based on intra-monomer interaction, because condensed mix charge works like zwitterion.

Animal-Free Human Whole Blood Sepsis Model to Study Changes in Innate Immunity.

Studying innate immunity in humans is crucial for understanding its role in the pathophysiology of systemic inflammation, particularly in the complex setting of sepsis. Therefore, we standardized a step-by-step process from the venipuncture to the transfer in a human model system, while closely monitoring the inflammatory response for up to three hours. We designed an animal-free, human whole blood sepsis model using a commercially available, simple to use, tubing system. First, we analyzed routine clinical parameters, including cell count and blood gas analysis. Second, we demonstrated that extracellular activation markers (e.g., CD11b and CD62l) as well as intracellular metabolic (intracellular pH) and functional (generation of radical oxygen species) features remained stable after incubation in the whole blood model. Third, we mimicked systemic inflammation during early sepsis by exposure of whole blood to pathogen-associated molecular patterns. Stimulation with lipopolysaccharide revealed the capability of the model system to evoke a sepsis-like inflammatory phenotype of innate immunity. In summary, the presented model serves as a convenient, economic, and reliable platform to study innate immunity in human whole blood, which may yield clinically important insights.

Thirty-Eight-Negative Kinase 1 Is a Mediator of Acute Kidney Injury in Experimental and Clinical Traumatic Hemorrhagic Shock.

Trauma represents a major socioeconomic burden worldwide. After a severe injury, hemorrhagic shock (HS) as a frequent concomitant aspect is a central driver of systemic inflammation and organ damage. The kidney is often strongly affected by traumatic-HS, and acute kidney injury (AKI) poses the patient at great risk for adverse outcome. Recently, thirty-eight-negative kinase 1 (TNK1) was proposed to play a detrimental role in organ damage after trauma/HS. Therefore, we aimed to assess the role of TNK1 in HS-induced kidney injury in a murine and a post hoc analysis of a non-human primate model of HS comparable to the clinical situation. Mice and non-human primates underwent resuscitated HS at 30 mmHg for 60 min. 5 h after the induction of shock, animals were assessed for systemic inflammation and TNK1 expression in the kidney. In vitro, murine distal convoluted tubule cells were stimulated with inflammatory mediators to gain mechanistic insights into the role of TNK1 in kidney dysfunction. In a translational approach, we investigated blood drawn from either healthy volunteers or severely injured patients at different time points after trauma (from arrival at the emergency room and at fixed time intervals until 10 days post injury; identifier: NCT02682550, https://clinicaltrials.gov/ct2/show/NCT02682550). A pronounced inflammatory response, as seen by increased IL-6 plasma levels as well as early signs of AKI, were observed in mice, non-human primates, and humans after trauma/HS. TNK1 was found in the plasma early after trauma-HS in trauma patients. Renal TNK1 expression was significantly increased in mice and non-human primates after HS, and these effects with concomitant induction of apoptosis were blocked by therapeutic inhibition of complement C3 activation in non-human primates. Mechanistically, in vitro data suggested that IL-6 rather than C3 cleavage products induced upregulation of TNK1 and impaired barrier function in renal epithelial cells. In conclusion, these data indicate that C3 inhibition in vivo may inhibit an excessive inflammatory response and mediator release, thereby indirectly neutralizing TNK1 as a potent driver of organ damage. In future studies, we will address the therapeutic potential of direct TNK1 inhibition in the context of severe tissue trauma with different degrees of additional HS.

One-lung ventilation during thoracoabdominal esophagectomy elicits complement activation.

BACKGROUND: One-lung ventilation (OLV) during thoracoabdominal esophagectomy may induce an inflammatory response that can contribute to the induction and propagation of frequently occurring postoperative respiratory distress. Markers of such a response might be detected in the pulmonary as well as in the systemic circulation. Inflammation and tissue damage may be key pathogenetic pathways and we hypothesized that 1-lung ventilation may induce an inflammatory cascade reflected by markers for such a response. MATERIALS AND METHODS: Thirty patients with esophageal cancer were randomized to OLV (n = 16) or 2-lung ventilation (TLV; n = 14) during the thoracic part of the operation. Compounds involved in inflammation and coagulation were measured perioperatively and during the 1st, 2nd, 3rd, and 10th postoperative d. RESULTS: During the perioperative phase, the proinflammatory cytokine interleukin-6 and thrombin, measured as thrombin-antithrombin complexes, started to increase. Thrombin, which can induce complement activation, peaked at the end of surgery and interleukin-6 at the 1st to 2nd postoperative d, but there were no differences between the OLV and TLV groups. C3a and terminal complement complex (TCC) started to increase on the 2nd postoperative d and continued to do so for the rest of the study period. The increase of TCC was significantly higher in the OLV group compared to the TLV group, whereas C3a attained similar levels in the 2 groups. CONCLUSIONS: OLV is associated with an augmented inflammatory response as reflected by the activation of the TCC. This may induce pulmonary tissue damage and recruitment of inflammatory cells.