Author Correction: 'Stealth' corporate innovation: an emerging threat for therapeutic drug development.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Therapeutic regulation of complement activation in extracorporeal circuits and intravascular treatments with special reference to the alternative pathway amplification loop.

A number of clinical treatment modalities involve contact between blood and biomaterials: these include extracorporeal circuits such as hemodialysis, cardiopulmonary bypass, plasmapheresis, and intravascular treatments. Common side effects arising from these treatments are caused by activation of the cascade systems of the blood. Many of these side effects are mediated via the complement system, including thromboinflammatory reactions and rejection of implants. Depending on the composition of the materials, complement activation is triggered via all the activation pathways but is by far mostly driven by the alternative pathway amplification loop. On biomaterial surfaces the alternative pathway amplification is totally unregulated and leads under optimal conditions to deposition of complement fragments, mostly C3b, on the surface leading to a total masking of the underlying surface. In this review, we discuss the mechanism of the complement activation, clinical consequences of the activation, and potential strategies for therapeutic regulation of the activation, using hemodialysis as demonstrator.

Complement C3 activation in the ICU: Disease and therapy as Bonnie and Clyde.

Patients in the intensive care unit (ICU) often straddle the divide between life and death. Understanding the complex underlying pathomechanisms relevant to such situations may help intensivists select broadly acting treatment options that can improve the outcome for these patients. As one of the most important defense mechanisms of the innate immune system, the complement system plays a crucial role in a diverse spectrum of diseases that can necessitate ICU admission. Among others, myocardial infarction, acute lung injury/acute respiratory distress syndrome (ARDS), organ failure, and sepsis are characterized by an inadequate complement response, which can potentially be addressed via promising intervention options. Often, ICU monitoring and existing treatment options rely on massive intervention strategies to maintain the function of vital organs, and these approaches can further contribute to an unbalanced complement response. Artificial surfaces of extracorporeal organ support devices, transfusion of blood products, and the application of anticoagulants can all trigger or amplify undesired complement activation. It is, therefore, worth pursuing the evaluation of complement inhibition strategies in the setting of ICU treatment. Recently, clinical studies in COVID-19-related ARDS have shown promising effects of central inhibition at the level of C3 and paved the way for prospective investigation of this approach. In this review, we highlight the fundamental and often neglected role of complement in the ICU, with a special focus on targeted complement inhibition. We will also consider complement substitution therapies to temporarily counteract a disease/treatment-related complement consumption.

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.

Impact of spontaneous liposome modification with phospholipid polymer-lipid conjugates on protein interactions.

Liposome surface coating has been studied to avoid the immunological responses caused by the complement system, and alternative materials to poly(ethylene glycol) (PEG) have been explored recently since the production of anti-PEG IgM antibodies has been found in humans. We previously reported a liposome coating with poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC))-conjugated lipids (PMPC-lipids) and demonstrated its protective effect on blood protein interactions. Here, we attempted to modify the liposome surface by exogenously adding PMPC-lipids, which were spontaneously incorporated into the outer membrane via hydrophobic interactions. The polymerization degree of the PMPC segment was regulated from 10 to 100. The incorporated ratio of PMPC-lipid increased with a decrease in the degree of PMPC polymerization. Due to surface modification with PMPC-lipids, increase in the length of the PMPC-chain increased the size of the liposomes. The modified liposomes were kept stable for 14 d in terms of their size, polydispersity, and surface properties, where approximately 70% of PMPC-lipids were incorporated into the liposome surface. We demonstrated that liposome surface modification with PMPC-lipids can inhibit protein adsorption when exposed to serum, regardless of the degree of polymerization of PMPC. In addition, the PMPC-lipid modified surface was not recognized by the anti-PEG IgM antibody, whereas PEG-lipid was recognized by the antibody. Thus, we successfully fabricated an inert liposome surface via spontaneous modification with PMPC-lipids, where only the outer bilayer surface was modified. This technique can be available for full loading of water-soluble active pharmaceutical ingredient inside the modified liposome.

Induction of Spontaneous Liposome Adsorption by Exogenous Surface Modification with Cell-Penetrating Peptide-Conjugated Lipids.

The use of amphiphilic molecules such as poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) enables incorporation into liposome surfaces by exogenous addition as a result of the self-assembly with lipids. This technique can be applicable for manipulation of both liposomes and cells. In this study, we aimed to characterize Tat peptide (YGRKKRRQRRR)-conjugated PEG-lipids when used to exogenously surface modify liposomes (size: ca. 100 nm). We earlier reported that cells, which were surface modified with Tat peptides conjugated to PEG-lipids could attach spontaneously to material surfaces without any chemical modification. Here, we synthesized different types of Tat-PEG-lipids by combining PEG of different molecular weights (5 and 40 kDa) with different lipids with three acyl chains (myristoyl, palmitoyl, and stearoyl, respectively) and then studied the spontaneous adsorption of modified liposomes onto a substrate surface induced by the different Tat-PEG-lipids. The amount of adsorbed liposomes strongly depended on the number of incorporated Tat-PEG-lipid moieties: a decrease in both the PEG and the acyl chain lengths led to adsorption of higher amounts of liposomes. Furthermore, when a collagenase-cleavable amino acid sequence was inserted between the Tat sequence and the PEG segment, adsorbed liposomes could be harvested from the substrate by collagenase treatment with no difference in desorption efficiency between the different Tat-PEG-lipids. Thus, Tat-PEG-lipid can be a suitable tool for the manipulation of liposomes and cells.

Pluronic Micelle-Mediated Tissue Factor Silencing Enhances Hemocompatibility, Stemness, Differentiation Potential, and Paracrine Signaling of Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) evoke great excitement for treating different human diseases due to their ability to home inflamed tissues, suppress inflammation, and promote tissue regeneration. Despite great promises, clinical trial results are disappointing as allotransplantation of MSCs trigger thrombotic activity and are damaged by the complement system, compromising their survival and function. To overcome this, a new strategy is presented by the silencing of tissue factor (TF), a transmembrane protein that mediates procoagulant activity. Novel Pluronic-based micelles are designed with the pendant pyridyl disulfide group, which are used to conjugate TF-targeting siRNA by the thiol-exchange reaction. This nanocarrier design effectively delivered the payload to MSCs resulting in ∼72% TF knockdown (KD) without significant cytotoxicity. Hematological evaluation of MSCs and TF-KD MSCs in an ex vivo human whole blood model revealed a significant reduction in an instant-blood-mediated-inflammatory reaction as evidenced by reduced platelet aggregation (93% of free platelets in the TF-KD group, compared to 22% in untreated bone marrow-derived MSCs) and thrombin-antithrombin complex formation. Effective TF silencing induced higher MSC differentiation in osteogenic and adipogenic media and showed stronger paracrine suppression of proinflammatory cytokines in macrophages and higher stimulation in the presence of endotoxins. Thus, TF silencing can produce functional cells with higher fidelity, efficacy, and functions.

Complement C3 vs C5 inhibition in severe COVID-19: Early clinical findings reveal differential biological efficacy.

Growing clinical evidence has implicated complement as a pivotal driver of COVID-19 immunopathology. Deregulated complement activation may fuel cytokine-driven hyper-inflammation, thrombotic microangiopathy and NET-driven immunothrombosis, thereby leading to multi-organ failure. Complement therapeutics have gained traction as candidate drugs for countering the detrimental consequences of SARS-CoV-2 infection. Whether blockade of terminal complement effectors (C5, C5a, or C5aR1) may elicit similar outcomes to upstream intervention at the level of C3 remains debated. Here we compare the efficacy of the C5-targeting monoclonal antibody eculizumab with that of the compstatin-based C3-targeted drug candidate AMY-101 in small independent cohorts of severe COVID-19 patients. Our exploratory study indicates that therapeutic complement inhibition abrogates COVID-19 hyper-inflammation. Both C3 and C5 inhibitors elicit a robust anti-inflammatory response, reflected by a steep decline in C-reactive protein and IL-6 levels, marked lung function improvement, and resolution of SARS-CoV-2-associated acute respiratory distress syndrome (ARDS). C3 inhibition afforded broader therapeutic control in COVID-19 patients by attenuating both C3a and sC5b-9 generation and preventing FB consumption. This broader inhibitory profile was associated with a more robust decline of neutrophil counts, attenuated neutrophil extracellular trap (NET) release, faster serum LDH decline, and more prominent lymphocyte recovery. These early clinical results offer important insights into the differential mechanistic basis and underlying biology of C3 and C5 inhibition in COVID-19 and point to a broader pathogenic involvement of C3-mediated pathways in thromboinflammation. They also support the evaluation of these complement-targeting agents as COVID-19 therapeutics in large prospective trials.

Complement deposition, C4d, on platelets is associated with vascular events in systemic lupus erythematosus.

OBJECTIVE: Complement components, including C4d, can be found on activated platelets, a process associated with vascular disease in SLE. We investigated whether platelet C4d (PC4d) adds additional value to traditional and known lupus-associated risk factors when identifying SLE patients with vascular disease. METHODS: This cross-sectional study included 308 well-characterized SLE patients and 308 matched general population controls. PC4d deposition was analysed using flow cytometry. Values >95% of controls were considered as PC4d positive (+). aPL were determined by Luminex, and the LA test was performed by DRVVT. History of vascular disease (composite and as separate outcomes) was defined at inclusion. RESULTS: SLE patients had increased PC4d deposition as compared with population controls (50 vs 5%, P < 0.0001). PC4d+ positively associated with any vascular events, and separately with venous and cerebrovascular events, and also with all investigated aPL profiles. The association for any vascular event remained statistically significant after adjustment for traditional and SLE-associated risk factors (odds ratio: 2.3, 95% CI: 1.3, 4.3, P = 0.008). Compared with patients negative for both PC4d and LA, patients with double positivity were more likely to have vascular disease (odds ratio: 12.3, 95% CI: 5.4, 29.3; attributable proportion due to interaction 0.8, 95% CI: 0.4, 1.1). CONCLUSION: PC4d+ is associated with vascular events in SLE, independently of traditional and SLE-associated risk factors. Concurrent presence of PC4d and LA seem to interact to further increase the odds for vascular events. Prospective studies should examine whether the aPL/PC4d combination can improve prediction of vascular events in SLE and/or APS.

Potential of Cell Surface Engineering with Biocompatible Polymers for Biomedical Applications.

The regulation of the cellular surface with biomaterials can contribute to the progress of biomedical applications. In particular, the cell surface is exposed to immunological surveillance and reactions in transplantation therapy, and modulation of cell surface properties might improve transplantation outcomes. The transplantation of therapeutic cells, tissue, and organs is an effective and fundamental treatment and has contributed to saving lives and improving quality of life. Because of shortages, donor cells, tissues, and organs are carefully transplanted with the goal of retaining activity and viability. However, some issues remain to be resolved in terms of reducing side effects, improving graft survival, managing innate and adaptive immune responses, and improving transplant storage and procedures. Given that the transplantation process involves multiple steps and is technically complicated, an engineering approach together with medical approaches to resolving these issues could enhance success. In particular, cell surface engineering with biocompatible polymers looks promising for improving transplantation therapy and has potential for other biomedical applications. Here we review the significance of polymer-based surface modification of cells and organs for biomedical applications, focusing on the following three topics: Cell protection: cellular protection through local immune regulation using cell surface modification with biocompatible polymers. This protection could extend to preventing attack by the host immune system, freeing recipients from taking immunosuppressive drugs, and avoiding a second transplantation. Cell attachment: cell manipulation, which is an important technique for delivery of therapeutic cells and their alignment for recellularization of decellularized tissues and organs in regenerative therapy. Cell fusion: fusion of different cells, which can lead to the formation of new functional cells that could be useful for generating, e.g., immunologically competent or metabolically active cells.

Complement inhibition in biomaterial- and biosurface-induced thromboinflammation.

Therapeutic medicine today includes a vast number of procedures involving the use of biomaterials, transplantation of therapeutic cells or cell clusters, as well as of solid organs. These treatment modalities are obviously of great benefit to the patient, but also present a great challenge to the innate immune system, since they involve direct exposure of non-biological materials, cells of non-hematological origin as well as endothelial cells, damaged by ischemia-perfusion in solid organs to proteins and cells in the blood. The result of such an exposure may be an inappropriate activation of the complement and contact/kallikrein systems, which produce mediators capable of triggering the platelets and PMNs and monocytes, which can ultimately result in thrombotic and inflammatory (i.e., a thrombo-inflammatory) response to the treatment modality. In this concept review, we give an overview of the mechanisms of recognition within the innate immunity system, with the aim to identify suitable points for intervention. Finally, we discuss emerging and promising techniques for surface modification of biomaterials and cells with specific inhibitors in order to diminish thromboinflammation and improve clinical outcome.

Obstacles to intergenerational communication in caregivers' narratives regarding young people's sexual and reproductive health and lifestyle in rural South Africa.

BACKGROUND: Statistics from South Africa show the world's highest HIV prevalence with an estimated seven million people living with the virus. Several studies have pointed to communication about sexuality between parents/caregivers and children as a protective factor. However, communication between generations has been described as problematic, especially due to discomfort in discussing sexual matters. The aim of this study was to explore how caregivers in a poor, rural part of South Africa talked about young people in general, their sexuality, and their lifestyle practices. A particular interest was directed towards central discourses in the caregivers' narratives and how these discourses were of importance for the caregivers to function as conversation partners for young people. METHODS: In this qualitative study convenience sampling was used to select and invite participants. Information was collected from nine one-on-one interviews conducted with caregivers from rural areas within South Africa. The interview guide included nine main questions and optional probing questions. Each interview took place in an uninterrupted setting of choice associated with the caregivers' home environment. The interviews were transcribed and analyzed using discourse analysis. RESULTS: Interview narratives were characterized by three central discourses - demoralized youths in a changing society, prevailing risks and modernity and a generation gap. The youths were discursively constructed as a problematic group relating to specific prevailing risks such as early pregnancies, modern technologies, STI/HIV and contraceptives. The interview narratives illustrated that caregivers tried to impose their views of a respectable lifestyle in young people. At the same time caregivers expressed a morality of despair mirroring a generation gap which counteracted their ability to communicate with their children and grandchildren. CONCLUSIONS: The findings add to the body of earlier research illustrating that rural South African caregivers and their children/grandchildren hold different moral standards. The interview material reflected a 'clash' between generations relating to their differing perceptions of a desirable lifestyle. To overcome the generational gap, we recommend further research about how a well-founded national and community collaboration linked to school-based programs can support family participation in order to empower adults in their communication with young people.

Dangerous liaisons: complement, coagulation, and kallikrein/kinin cross-talk act as a linchpin in the events leading to thromboinflammation.

Innate immunity is fundamental to our defense against microorganisms. Physiologically, the intravascular innate immune system acts as a purging system that identifies and removes foreign substances leading to thromboinflammatory responses, tissue remodeling, and repair. It is also a key contributor to the adverse effects observed in many diseases and therapies involving biomaterials and therapeutic cells/organs. The intravascular innate immune system consists of the cascade systems of the blood (the complement, contact, coagulation, and fibrinolytic systems), the blood cells (polymorphonuclear cells, monocytes, platelets), and the endothelial cell lining of the vessels. Activation of the intravascular innate immune system in vivo leads to thromboinflammation that can be activated by several of the system's pathways and that initiates repair after tissue damage and leads to adverse reactions in several disorders and treatment modalities. In this review, we summarize the current knowledge in the field and discuss the obstacles that exist in order to study the cross-talk between the components of the intravascular innate immune system. These include the use of purified in vitro systems, animal models and various types of anticoagulants. In order to avoid some of these obstacles we have developed specialized human whole blood models that allow investigation of the cross-talk between the various cascade systems and the blood cells. We in particular stress that platelets are involved in these interactions and that the lectin pathway of the complement system is an emerging part of innate immunity that interacts with the contact/coagulation system. Understanding the resulting thromboinflammation will allow development of new therapeutic modalities.

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.

A human whole-blood model to study the activation of innate immunity system triggered by nanoparticles as a demonstrator for toxicity.

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.

Safety profile after prolonged C3 inhibition.

The central component of the complement cascade, C3, is involved in various biological functions, including opsonization of foreign bodies, clearance of waste material, activation of immune cells, and triggering of pathways controlling development. Given its broad role in immune responses, particularly in phagocytosis and the clearance of microbes, a deficiency in complement C3 in humans is often associated with multiple bacterial infections. Interestingly, an increased susceptibility to infections appears to occur mainly in the first two years of life and then wanes throughout adulthood. In view of the well-established connection between C3 deficiency and infections, therapeutic inhibition of complement at the level of C3 is often considered with caution or disregarded. We therefore set out to investigate the immune and biochemical profile of non-human primates under prolonged treatment with the C3 inhibitor compstatin (Cp40 analog). Cynomolgus monkeys were dosed subcutaneously with Cp40, resulting in systemic inhibition of C3, for 1 week, 2 weeks, or 3 months. Plasma concentrations of both C3 and Cp40 were measured periodically and complete saturation of plasma C3 was confirmed. No differences in hematological, biochemical, or immunological parameters were identified in the blood or tissues of animals treated with Cp40 when compared to those injected with vehicle alone. Further, skin wounds showed no signs of infection in those treated with Cp40. In fact, Cp40 treatment was associated with a trend toward accelerated wound healing when compared with the control group. In addition, a biodistribution study in a rhesus monkey indicated that the distribution of Cp40 in the body is associated with the presence of C3, concentrating in organs that accumulate blood and produce C3. Overall, our data suggest that systemic C3 inhibition in healthy adult non-human primates is not associated with a weakened immune system or susceptibility to infections.

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.