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The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa , is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa . Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of c bpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions. The Pseudomonas aeruginosa lytic polysaccharide monooxygenase CbpD, prevalent in clinical isolates, has been proposed to act as a virulence factor. Here, the authors combine structural work, in silico simulations, enzymatic activity and in vitro and in vivo experiments to further delineate the role of CbpD and show that its deletion renders P. aeruginosa unable to establish a lethal systemic infection, leading to enhanced bacterial clearance in a mouse model of infection.

In SARS-CoV-2 infection there is an urgent need to identify patients that will progress to severe COVID-19 and may benefit from targeted treatment. In this study we analyzed plasma cytokines in COVID-19 patients and investigated their association with respiratory failure (RF) and treatment in Intensive Care Unit (ICU). Hospitalized patients (n = 34) with confirmed COVID-19 were recruited into a prospective cohort study. Clinical data and blood samples were collected at inclusion and after 2–5 and 7–10 days. RF was defined as PaO2/FiO2 ratio (P/F) < 40 kPa. Plasma cytokines were analyzed by a Human Cytokine 27-plex assay. COVID-19 patients with RF and/or treated in ICU showed overall increased systemic cytokine levels. Plasma IL-6, IL-8, G-CSF, MCP-1, MIP-1α levels were negatively correlated with P/F, whereas combinations of IL-6, IP-10, IL-1ra and MCP-1 showed the best association with RF in ROC analysis (AUC 0.79–0.80, p  < 0.05). During hospitalization the decline was most significant for IP-10 ( p  < 0.001). Elevated levels of pro-inflammatory cytokines were present in patients with severe COVID-19. IL-6 and MCP-1 were inversely correlated with P/F with the largest AUC in ROC analyses and should be further explored as biomarkers to identify patients at risk for severe RF and as targets for improved treatment strategies.

Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining regions that harbour cysteine-rich knob domains. To produce high-affinity peptides, we previously isolated autonomous 3–6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 Å from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors, and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential. Antibodies are proteins produced by the immune system that can selectively bind to other molecules and modify their behaviour. Cows are highly equipped at fighting-off disease-causing microbes due to the unique shape of some of their antibodies. Unlike other jawed vertebrates, cows’ antibodies contain an ultra-long loop region that contains a ‘knob domain’ which sticks out from the rest of the antibody. Recent research has shown that when detached, the knob domain behaves like an antibody fragment, and can independently bind to a range of different proteins. Antibody fragments are commonly developed in the laboratory to target proteins associated with certain diseases, such as arthritis and cancer. But it was unclear whether the knob domains from cows’ antibodies could also have therapeutic potential. To investigate this, Macpherson et al. studied how knob domains attach to complement C5, a protein in the inflammatory pathway which is a drug target for various diseases, including severe COVID-19. The experiments identified various knob domains that bind to complement C5 and inhibits its activity by altering its structure or movement. Further tests studying the structure of these interactions, led to the discovery of a common mechanism by which inhibitors can modify the behaviour of this inflammatory protein. Complement C5 is involved in numerous molecular pathways in the immune system, which means many of the drugs developed to inhibit its activity can also leave patients vulnerable to infection. However, one of the knob domains identified by Macpherson et al. was found to reduce the activity of complement C5 in some pathways, whilst leaving other pathways intact. This could potentially reduce the risk of bacterial infections which sometimes arise following treatment with these types of inhibitors. These findings highlight a new approach for developing drug inhibitors for complement C5. Furthermore, the ability of knob domains to bind to multiple sites of complement C5 suggests that this fragment could be used to target proteins associated with other diseases.

Ficolins are a family of pattern recognition molecules that are capable of activating the lectin pathway of complement. A limited number of reports have demonstrated a protective role of ficolins in animal models of infection. In addition, an immune modulatory role of ficolins has been suggested. Yet, the contribution of ficolins to inflammatory disease processes remains elusive. To address this, we investigated ficolin deficient mice during a lipopolysaccharide (LPS)-induced model of systemic inflammation. Although murine serum ficolin was shown to bind LPS in vitro , there was no difference between wildtype and ficolin deficient mice in morbidity and mortality by LPS-induced inflammation. Moreover, there was no difference between wildtype and ficolin deficient mice in the inflammatory cytokine profiles after LPS challenge. These findings were substantiated by microarray analysis revealing an unaltered spleen transcriptome profile in ficolin deficient mice compared to wildtype mice. Collectively, results from this study demonstrate that ficolins are not involved in host response to LPS-induced systemic inflammation.

•  Introduction •  Methods to study and modify the alternative pathway ‐  Inhibition by monoclonal antibodies ‐  Knockout models •  Influence of AP amplification on the effect of specific activation of the other initial pathways ‐  Direct activation versus amplification ‐  Mechanisms of amplification •  Properdin ‐  Control protein in the AP amplification loop ‐  Recognition molecule in AP •  Influence of AP in deficiency states ‐  Factor H deficiency and variants ‐  C2 bypass in CP and LP ‐  Properdin deficiency •  Complement in sepsis •  Concluding remarks Alternative pathway amplification plays a major role for the final effect of initial specific activation of the classical and lectin complement pathways, but the quantitative role of the amplification is insufficiently investigated. In experimental models of human diseases in which a direct activation of alternative pathway has been assumed, this interpretation needs revision placing a greater role on alternative amplification. We recently documented that the alternative amplification contributed to 80–90% of C5 activation when the initial activation was highly specific for the classical pathway. The recent identification of properdin as a recognition factor directly initiating alternative pathway activation, like C1q in the classical and mannose‐binding lectin in the lectin pathway, initiates a renewed interest in the reaction mechanisms of complement. Complement and Toll‐like receptors, including the CD14 molecule, are two main upstream recognition systems of innate immunity, contributing to the inflammatory reaction in a number of conditions including ischaemia‐reperfusion injury and sepsis. These systems act as ‘double‐edged swords’, being protective against microbial invasion, but harmful to the host when activated improperly or uncontrolled. Combined inhibition of complement and Toll‐like receptors/CD14 should be explored as a treatment regimen to reduce the overwhelming damaging inflammatory response during sepsis. The alternative pathway should be particularly considered in this regard, due to its uncontrolled amplification in sepsis. The alternative pathway should be regarded as a dual system, namely a recognition pathway principally similar to the classical and lectin pathways, and an amplification mechanism, well known, but quantitatively probably more important than generally recognized.

Intravenously injected nanopharmaceuticals, including PEGylated nanoparticles, induce adverse cardiopulmonary reactions in sensitive human subjects, and these reactions are highly reproducible in pigs. Although the underlying mechanisms are poorly understood, roles for both the complement system and reactive macrophages have been implicated. Here, we show the dominance and importance of robust pulmonary intravascular macrophage clearance of nanoparticles in mediating adverse cardiopulmonary distress in pigs irrespective of complement activation. Specifically, we show that delaying particle recognition by macrophages within the first few minutes of injection overcomes adverse reactions in pigs using two independent approaches. First, we changed the particle geometry from a spherical shape (which triggers cardiopulmonary distress) to either rod- or disk-shape morphology. Second, we physically adhered spheres to the surface of erythrocytes. These strategies, which are distinct from commonly leveraged stealth engineering approaches such as nanoparticle surface functionalization with poly(ethylene glycol) and/or immunological modulators, prevent robust macrophage recognition, resulting in the reduction or mitigation of adverse cardiopulmonary distress associated with nanopharmaceutical administration. The transitional link from robust clearance of intravenously injected nanoparticles by strategically placed macrophages in systemic circulation to adverse haemodynamic reactions can be overcome by changing nanoparticle geometry or by prior nanoparticle attachment to erythrocytes

The overuse of compression ultrasound procedures on patients with suspected deep vein thrombosis compromise the cost-effectiveness of deep vein thrombosis management and emphasize room for improvement of the current diagnostic algorithm. As the complement system and hemostasis are comprehensively intertwined, we aimed to investigate complement activation products as diagnostic tests for acute deep vein thrombosis alone or together with D-dimer. We performed a cross-sectional study using consecutive sampling of outpatients referred to the emergency department with suspected first-time deep vein thrombosis of the lower limbs, to investigate the diagnostic utility of the index tests C3bc and C5b-C9 terminal complex (TCC) with compression ultrasound as reference standard. For comparison of receiver operating characteristics, analyses were also performed for D-dimer and C-reactive protein in addition to analyses for the index tests on a D-dimer positive patient subgroup. Of the 366 included patients, 103 had deep vein thrombosis. The calculated effect sizes of differences between groups (Cohen's d) with 95% confidence intervals (95%CI) were 0.25 (95%CI 0.03-0.48) for C3bc, 0.33 (95%CI 0.09-0.57) for C5b-C9 terminal complex (TCC), 0.88 (95%CI 0.61-1.15) for C-reactive protein, and 1.64 (95%CI 1.37-1.91) for D-dimer. The areas under the curves derived from comparison receiver operating characteristics analyses with corresponding 95%CIs for C3bc, C5b-C9 terminal complex (TCC), C-reactive protein and D-dimer were 0.56 (95%CI 0.50-0.63), 0.64 (95%CI 0.58-0.69), 0.73 (95%CI 0.67-0.79) and 0.92 (95%CI 0.89-0.95), respectively. The plasma levels of the complement activation products C3bc and C5b-C9 terminal complex (TCC) were elevated in patients with acute deep vein thrombosis but displayed low diagnostic performance for deep vein thrombosis alone or together with D-dimer.

Psychiatric disorders and chronic hepatitis virus C infection are known to alter blood cytokines levels. However, little is known about the association between cytokines and psychiatric symptoms in patients with chronic hepatitis C virus infection. This study aimed at exploring this association. Moreover, since nearly half of the patients receive opioid maintenance treatment, we also investigated if long-term opioid treatment had any impact on these associations. We conducted a cross-sectional study on 120 outpatients referred for antiviral hepatitis C treatment. Serum level of 27 cytokines was measured using multiplex technology, and psychiatric symptom clusters were assessed using the Symptoms Check-List-90-R. Data on confounding factors including age, gender, weight, height, current medication and smoking habits were collected. Multiple linear regression analysis was performed to examine associations, adjusting for confounding factors. After adjusting for the most commonly known confounding factors, IP-10 and GM-CSF were negatively associated with depression, and GM-CSF was negatively associated with phobic anxiety. Subgroup analyses revealed that these associations were present only in patients receiving opioid maintenance treatment, as demonstrated by repeated regression analysis. In patients with chronic hepatitis C viral infection, only IP-10 and GM-CSF were negatively associated with self-reported psychiatric symptom clusters. These associations were observed exclusively in patients receiving opioid maintenance treatment. Our study contributes to others investigations pointing to a possible immune dampening caused by long-term opioid treatment.

[...]complement plays key roles in (i) preventing the spread of infection to other cells and tissues, (ii) participating in the clearance of damaged cells and tissues, and (iii) preventing the development of chronic inflammation and/or cancer. FH bound to monocyte cell surface complement receptor (CR3) in the absence of deposited C3b and increased ApoE binding to monocytes and THP-1 cells, possibly through simultaneous interaction with sialic acids. Complement biomarkers are especially useful to follow the activity of diseases if they are compared with baseline values under physiological conditions.Gaya da Costa et al.have investigated the serum concentrations of 19 complement components and the activity of the three complement pathways in a healthy Caucasian population of 120 individuals (50% women/men, 20–69 years old). Substitution therapies with plasma-derived MBL (pdMBL) proved to be safe with restoration of MBL levels but not of a functional LP due to inactivation of pre-activated MBL-MASPs complexes present in pdMBL.Keizer et al.demonstrated that recombinant MBL (rMBL) can associate with non-complexed circulating forms of proenzyme MASPs and restore LP activity of MBL-deficient serum, providing a rationale for new clinical rMBL substitution studies.

Following extensive trauma surgery,Teuben et al.recorded a shift in composition of the bone marrow (BM) neutrophil pool, which was associated with relative circulatory neutropenia in a porcine polytrauma model. [...]the authors reported that the CXCR4high-neutrophil subset became overrepresented, possibly reflecting remigration of aged neutrophils into the BM. Whereas MIF caused organ injury and/or dysfunction and hypotension in rats, treatment with ISO-1 attenuated organ injury and dysfunction, and reduced the activation of NF-kB and NLRP3 pathways in the rat kidney and liver. [...]the authors pointed out that MIF inhibitors may be used as a potential therapeutic approach for MODS after trauma and/or hemorrhage. By hypergraph analysis and principal component analysis of 20 proteins sampled from the heart, gut, lung, liver, spleen, kidney and systemic circulation, the authors found that IL-17A was present persistently in all tissues at all sampled time points (except for its absence in the plasma at 0.5 h) in the wild type strain compared to TLR4-null (TLR4 -/-) animals. [...]this Research Topic presents the latest scientific insights on immunological and cellular reactions among different organs after severe trauma or during the further clinical course of critically ill patients.