In this review article, we focus on activation of the soluble components of the innate immune system triggered by nonbiological compounds and stress variances in activation due to the difference in size between nanoparticles (NPs) and larger particles or bulk material of the same chemical and physical composition. We then discuss the impact of the so-called protein corona which is formed on the surface of NPs when they come in contact with blood or other body fluids. For example, NPs which bind inert proteins, proteins which are prone to activate the contact system (e.g., factor XII), which may lead to clotting and fibrin formation or the complement system (e.g., IgG or C3), which may result in inflammation and vascular damage. Furthermore, we describe a whole blood model which we have developed to monitor activation and interaction between different components of innate immunity: blood protein cascade systems, platelets, leukocytes, cytokine generation, which are induced by NPs. Finally, we describe our own studies on innate immunity system activation induced by three fundamentally different species of NPs (two types of engineered NPs and diesel NPs) as demonstrator of the utility of an initial determination of the composition of the protein corona formed on NPs exposed to ethylenediaminetetraacetic acid (EDTA) plasma and subsequent analysis in our whole blood model. [GRAPHICS] .

C3 glomerulopathy (C3G) is a severe kidney disease, which is caused by defective regulation of the alternative complement pathway. Disease pathogenesis is heterogeneous and is caused by both autoimmune and genetic factors. Here we characterized IgG autoantibodies derived from 33 patients with autoimmune C3 glomerulopathy. Serum antibodies from all 33 patients as well as purified IgGs bound to the in vitro assembled C3-convertase. Noteworthy, two groups of antibodies were identified: group 1 with strong (12 patients) and group 2 with weak binding C3-convertase autoantibodies (22 patients). C3Nef, as evaluated in a standard C3Nef assay, was identified in serum from 19 patients, which included patients from group 1 as well as group 2. The C3-convertase binding profile was independent of C3Nef. Group 1 antibodies, but not the group 2 antibodies stabilized the C3-convertase, and protected the enzyme from dissociation by Factor H. Also, only group 1 antibodies induced C3a release. However, both group 1 and group 2 autoantibodies bound to the C5-convertase and induced C5a generation, which was inhibited by monoclonal anti-C5 antibody Eculizumab in vitro. In summary, group 1 antibodies are composed of C3Nef and C5Nef antibodies and likely over-activate the complement system, as seen in hemolytic assays. Group 2 antibodies show predominantly C5Nef like activities and stabilize the C5 but not the C3-convertase. Altogether, these different profiles not only reveal a heterogeneity of the autoimmune forms of C3G (MPGN), they also show that in diagnosis of C3G not all autoimmune forms are identified and thus more vigorous autoantibody testing should be performed.

Objectives: C1q is a valuable biomarker of disease activity in systemic lupus erythematosus (SLE). The "gold standard" assay, rocket immunoelectrophoresis (RIE), is time-consuming, and thus a shift to soluble immune precipitation techniques such as nephelometry has occurred. However, quantification of C1q with these techniques has been questioned as a result of the antibody binding properties of C1q. In the present work, we have compared results using various techniques (RIE, nephelometry, and ELISA) and have developed and validated a new magnetic bead-based sandwich immunoassay (MBSI). Methods: C1q was quantified by nephelometry and the new sandwich immunoassay in 45 serum samples analyzed using RIE. C1q was also assessed in plasma using RIE and sandwich immunoassay in samples from SLE patients with nephritis (n = 69), SLE patients without nephritis (n = 310) as classified by BILAG score, and matched controls (n = 322). In addition, cerebrospinal fluid (CSF) samples from 31 patients, previously analyzed with ELISA, were also analyzed with the MBSI to test the behavior of this new assay in the lower detection range. Results: We found a strong correlation between the new MBSI, RIE, and ELISA, but not with nephelometry. The MBSI demonstrated lower levels of C1q in SLE patients than in matched controls (p < 0.0001), and patients with nephritis had lower levels than patients without nephritis (p < 0.01). Similarily, RIE showed significant differences between the patient groups (p < 0.0001). An association was also found between the levels of C1q and the SLE disease activity index (SLEDAI). Furthermore, there was good correlation between the values obtained by MBSI and ELISA, in both serum (r = 0.960) and CSF (r = 0.786), underscoring the ability of both techniques to measure low concentrations of C1q with high accuracy. Conclusion: The sandwich immunoassay correlated well with RIE, but soluble immune precipitation techniques, such as nephelometry, did not appear suitable alternatives, since C1q itself, and possibly anti-C1q antibodies, interfered with the measurements. The new sandwich immunoassay is therefore a good replacement for RIE in monitoring SLE disease activity.

Promising cell therapies using mesenchymal stem cells (MSCs) is proposed for stroke patients. Therefore, we aimed to efficiently accumulate human MSC (hMSC) to damaged brain area to improve the therapeutic effect using poly(ethylene glycol) (PEG)-conjugated phospholipid (PEG-lipid) carrying an oligopeptide as a ligand, specific for E-selectin which is upregulated on activated endothelial cells under hypoxia-like stroke. Here we synthesized E-selectin-binding oligopeptide (ES-bp) conjugated with PEG spacer having different molecular weights from 1 to 40 kDa. We found that ES-bp can be immobilized onto the hMSC surface through PEG-lipid without influence on cell growth and differentiation into adipocytes and osteocytes, respectively. It is also possible to control the immobilization of ES-bp on hMSC surface (<10(8) ES-bp per cell). Immobilized ES-bp can be continuously immobilized at the outside of cell membrane when PEG-lipids with PEG 5 and 40 kDa were used. In addition, the modified hMSC can specifically attach onto E-selectin-immobilized surface as a model surface of activated endothelium in human blood, indicating the sufficient number of immobilized ES-bp onto hMSC. Thus, this technique is one of the candidates for hMSC accumulation to cerebral infarction area.

Trauma-induced hemorrhagic shock (HS) plays a decisive role in the development of immune, coagulation, and organ dysfunction often resulting in a poor clinical outcome. Imbalanced complement activation is intricately associated with the molecular danger response and organ damage after HS. Thus, inhibition of the central complement component C3 as turnstile of both inflammation and coagulation is hypothesized as a rational strategy to improve the clinical course afterHS. Applying intensive care conditions, anaesthetized, monitored, and protectively ventilated nonhuman primates (NHP; cynomolgusmonkeys) received a pressure-controlled severe HS (60min at mean arterial pressure 30 mmHg) with subsequent volume resuscitation. Thirty minutes after HS, animals were randomly treated with either an analog of the C3 inhibitor compstatin (i.e., Cp40) in saline (n =4) or with saline alone (n =4). The observation period lasted 300 min after induction of HS. We observed improved kidney function in compstatin Cp40-treated animals after HS as determined by improved urine output, reduced damage markers and a tendency of less histopathological signs of acute kidney injury. Sham-treated animals revealed classical signs ofmucosal edema, especially in the ileum and colon reflected by worsened microscopic intestinal injury scores. In contrast, Cp40-treated HS animals exhibited only minor signs of organ edema and significantly less intestinal damage. Furthermore, early systemic inflammation and coagulation dysfunction were both ameliorated by Cp40. The data suggest that therapeutic inhibition of C3 is capable to significantly improve immune, coagulation, and organ function and to preserve organ-barrier integrity early after traumatic HS. C3-targeted complement inhibition may therefore reflect a promising therapeutic strategy in fighting fatal consequences of HS.

Exposure to traumatic or infectious insults results in a rapid activation of the complement cascade as major fluid defense system of innate immunity. The complement system acts as a master alarm system during the molecular danger response after trauma and significantly contributes to the clearance of DAMPs and PAMPs. However, depending on the origin and extent of the damaged macro- and micro-milieu, the complement system can also be either excessively activated or inhibited. In both cases, this can lead to a maladaptive immune response and subsequent multiple cellular and organ dysfunction. The arsenal of complement-specific drugs offers promising strategies for various critical conditions after trauma, hemorrhagic shock, sepsis, and multiple organ failure. The imbalanced immune response needs to be detected in a rational and real-time manner before the translational therapeutic potential of these drugs can be fully utilized. Overall, the temporal-spatial complement response after tissue trauma and during sepsis remains somewhat enigmatic and demands a clinical triad: reliable tissue damage assessment, complement activation monitoring, and potent complement targeting to highly specific rebalance the fluid phase innate immune response.

Platelets play an essential role in maintaining homeostasis in the circulatory system after an injury by forming a platelet thrombus, but they also occupy a central node in the intravascular innate immune system. This concept is supported by their extensive interactions with immune cells and the cascade systems of the blood. In this review we discuss the close relationship between platelets and the complement system and the role of these interactions during thromboinflammation. Platelets are protected from complement-mediated damage by soluble and membrane-expressed complement regulators, but they bind several complement components on their surfaces and trigger complement activation in the fluid phase. Furthermore, localized complement activation may enhance the procoagulant responses of platelets through the generation of procoagulant microparticles by insertion of sublytic amounts of C5b9 into the platelet membrane. We also highlight the role of post-translational protein modifications in regulating the complement system and the critical role of platelets in driving these reactions. In particular, modification of disulfide bonds by thiol isomerases and protein phosphorylation by extracellular kinases have emerged as important mechanisms to fine-tune complement activity in the platelet microenvironment. Lastly, we describe disorders with perturbed complement activation where part of the clinical presentation includes uncontrolled platelet activation that results in thrombocytopenia, and illustrate how complement-targeting drugs are alleviating the prothrombotic phenotype in these patients. Based on these clinical observations, we discuss the role of limited complement activation in enhancing platelet activation and consider how these drugs may provide opportunities for further dissecting the complex interactions between complement and platelets.

Background: Previous studies and own clinical observations of patients with systemic lupus erythematosus (SLE) suggest that SLE harbors distinct immunophenotypes. This heterogeneity might result in differences in response to treatment in different subgroups and obstruct clinical trials. Our aim was to understand how SLE subgroups may differ regarding underlying pathophysiology and characteristic biomarkers.

Methods: In a cross-sectional study, including 378 well-characterized SLE patients and 316 individually matched population controls, we defined subgroups based on the patients’ autoantibody profile at inclusion. We selected a core of an antiphospholipid syndrome-like SLE (aPL+ group; positive in the lupus anticoagulant (LA) test and negative for all three of SSA (Ro52 and Ro60) and SSB antibodies) and a Sjögren’s syndrome-like SLE (SSA/SSB+ group; positive for all three of SSA (Ro52 and Ro60) and SSB antibodies but negative in the LA test). We applied affinity-based proteomics, targeting 281 proteins, together with well-established clinical biomarkers and complementary immunoassays to explore the difference between the two predefined SLE subgroups.

Results: The aPL+ group comprised 66 and the SSA/SSB+ group 63 patients. The protein with the highest prediction power (receiver operating characteristic (ROC) area under the curve = 0.89) for separating the aPL+ and SSA/SSB+ SLE subgroups was integrin beta-1 (ITGB1), with higher levels present in the SSA/SSB+ subgroup. Proteins with the lowest p values comparing the two SLE subgroups were ITGB1, SLC13A3, and CERS5. These three proteins, rheumatoid factor, and immunoglobulin G (IgG) were all increased in the SSA/SSB+ subgroup. This subgroup was also characterized by a possible activation of the interferon system as measured by high KRT7, TYK2, and ETV7 in plasma. In the aPL+ subgroup, complement activation was more pronounced together with several biomarkers associated with systemic inflammation (fibrinogen, α-1 antitrypsin, neutrophils, and triglycerides).

Conclusions: Our observations indicate underlying pathogenic differences between the SSA/SSB+ and the aPL+ SLE subgroups, suggesting that the SSA/SSB+ subgroup may benefit from IFN-blocking therapies while the aPL+ subgroup is more likely to have an effect from drugs targeting the complement system. Stratifying SLE patients based on an autoantibody profile could be a way forward to understand underlying pathophysiology and to improve selection of patients for clinical trials of targeted treatments.

Artificial surfaces that come into contact with blood induce an immediate activation of the cascade systems of the blood, leading to a thrombotic and/or inflammatory response that can eventually cause damage to the biomaterial or the patient, or to both. Heparin coating has been used to improve hemocompatibility, and another approach is 2-methacryloyloxyethyl phosphorylcholine (MPC)-based polymer coatings. Here, the aim is to evaluate the hemocompatibility of MPC polymer coating by studying the interactions with coagulation and complement systems using human blood in vitro model and pig in vivo model. The stability of the coatings is investigated in vitro and MPC polymer-coated catheters are tested in vivo by insertion into the external jugular vein of pigs to monitor the catheters' antithrombotic properties. There is no significant activation of platelets or of the coagulation and complement systems in the MPC polymer-coated one, which was superior in hemocompatibility to non-coated matrix surfaces. The protective effect of the MPC polymer coat does not decline after incubation in human plasma for up to 2 weeks. With MPC polymer-coated catheters, it is possible to easily draw blood from pig for 4 days in contrast to the case for non-coated catheters, in which substantial clotting is seen.