A VersaTile-driven platform for rapid hit-to-lead development of engineered lysins.

Health care authorities are calling for new antibacterial therapies to cope with the global emergence of antibiotic-resistant bacteria. Bacteriophage-encoded lysins are a unique class of antibacterials with promising (pre)clinical progress. Custom engineering of lysins allows for the creation of variants against potentially any bacterial pathogen. We here present a high-throughput hit-to-lead development platform for engineered lysins. The platform is driven by VersaTile, a new DNA assembly method for the rapid construction of combinatorial libraries of engineered lysins. We constructed approximately 10,000 lysin variants. Using an iterative screening procedure, we identified a lead variant with high antibacterial activity against Acinetobacter baumannii in human serum and an ex vivo pig burn wound model. This generic platform could offer new opportunities to populate the preclinical pipeline with engineered lysins for diverse (therapeutic) applications.

Neutrophil activation identifies patients with active polyarticular gout.

BACKGROUND: Gout is the most prevalent inflammatory arthritis in developed countries. A gout flare is mediated by phagocytosis of monosodium urate crystals by macrophages and neutrophils leading to subsequent activation of neutrophils contributing to synovitis, local joint destruction, and systemic inflammation. We hypothesize that biomarkers from activated neutrophils reflect gout disease activity. The objective of this study therefore was to investigate the clinical utility of neutrophil-derived biomarkers in gout disease activity. METHODS: Plasma samples from 75 gout patients participating in the "Reade gout cohort Amsterdam" were compared with 30 healthy controls (HC). Levels of neutrophil extracellular traps (NETs) and neutrophil activation markers (calprotectin and peroxidase activity) were analyzed by ELISA and fluorimetry, compared to healthy controls, and related to markers of inflammation and disease activity. RESULTS: Levels of NETs, as well as neutrophil activation markers, were increased in gout patients compared to HC (p < 0.01). No associations were found between markers of cell death (cell-free DNA and NETs) and disease activity. Cell-free levels of genomic DNA were elevated among gout patients compared to HC (p < 0.05) and related to the number of gout attacks in the last year (ß = 0.35, p < 0.01). Peroxidase activity correlated with disease activity (RAPID score: ß = 0.49, p < 0.01, MHAQ: ß = 0.66, p < 0.01) and inflammation markers (CRP: ß = 0.25, p = 0.04, and ESR: ß = 0.57, p < 0.001). Involvement of ankle or wrist resulted in significant higher peroxidase levels compared to mono-articular disease (ß = 0.34, p < 0.01), indicating that peroxidase activity is a marker of poly-articular gout. Calprotectin (S100A8/A9) correlated with the inflammation marker CRP (ß = 0.23, p = 0.05) and morning stiffness, especially in patients with chronic poly-articular gout (ß = 0.71, p < 0.01). CONCLUSIONS: Neutrophil activation markers are associated with characteristics of active, polyarticular gout. Furthermore, NETs are present in the peripheral blood of gout patients. However, NETs do not associate with markers of disease activity or inflammation. Future research should point out if peroxidase and calprotectin could be used in clinical practice as biomarkers for monitoring gout disease activity.

Pro-inflammatory S100 proteins are associated with glomerulonephritis and anti-dsDNA antibodies in systemic lupus erythematosus.

Objectives Systemic lupus erythematosus (SLE) is associated with elevated levels of S100A8/A9, pro-inflammatory proteins mainly secreted by activated polymorphonuclear neutrophils (PMNs). The underlying mechanisms for increased S100A8/A9 levels and their relation to the clinical phenotype have not been carefully investigated. We assessed S100A8/A9 and S100A12 levels in SLE patient sera in relation to disease activity, clinical phenotype, presence of anti-dsDNA antibodies and ability to promote phagocytosis of necrotic cells (NCs) by PMNs. Methods Serum levels of S100A8/A9 and S100A12 were measured by ELISA in paired samples of 100 SLE patients at time points of higher and lower disease activity. Serum-mediated phagocytosis of NCs by PMNs was analysed by flow cytometry. Clinical data were recorded at time points of blood sampling. Results Serum levels of S100A8/A9 and S100A12 were increased in SLE patients with high disease activity compared to paired samples at low disease activity ( p = 0.01 and p = 0.008, respectively). Elevated levels of S100A8/A9 were particularly seen in patients with anti-dsDNA antibodies ( p = 0.01) and glomerulonephritis before treatment ( p = 0.02). Immunosuppressive therapy was associated with a reduction of S100A8/A9 serum levels ( p = 0.002). The ability of serum to support phagocytosis of NCs by PMNs was related to increased S100A8/A9 levels ( p = 0.01). Conclusions Elevated serum levels of S100A8/A9 may be used to monitor disease activity and response to treatment in SLE patients, especially in patients with glomerulonephritis. S100A12 may be a marker of disease activity in SLE. Increased S100A8/A9 levels may reflect immune-pathological processes involving phagocytosis of immune complexes by PMNs.

The lectin complement pathway serine proteases (MASPs) represent a possible crossroad between the coagulation and complement systems in thromboinflammation.

UNLABELLED: ESSENTIALS: The lectin pathway's MASP-1/2 activates coagulation factors but the trigger of the activation is unknown. MASP-1/2 activation was assessed by quantifying complexes between MASPs and antithrombin/C1-inhibitor. Activated platelets and fibrin were demonstrated to activate MASP-1 and MASP-2 both in vitro and in vivo. These findings may represent a crossroad between the complement and the coagulation systems. BACKGROUND: The activated forms of the complement lectin pathway (LP) proteases MASP-1 and MASP-2 are able to cleave the coagulation factors prothrombin, fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor in vitro. In vivo studies also show that MASP-1 is involved in thrombogenesis. OBJECTIVES: To clarify the not yet identified mechanisms involved in triggering activation of the LP during thrombotic reactions. METHODS: Novel sandwich-ELISAs for detection of complexes between MASP-1 or MASP-2 and the serpins C1 inhibitor (C1-INH) or antithrombin (AT), were used to specifically detect and quantify the activated forms of MASP-1 and MASP-2. RESULTS: Activated platelets were shown by flow cytometry to bind Ficolin-1, -2 and -3 but not MBL, which was associated with activation of MASP-1 and MASP-2. We also demonstrated that fibrin and the plasmin-generated fibrin fragment DD in plasma, bind and activate MASP-1 and MASP-2. As demonstrated by the ELISA and SDS-PAGE/Western blotting, the fibrin-associated activation was reflected in a specific inactivation by AT during clotting without the assistance of heparin. In all other cases the MASPs were, as previously reported, inactivated by C1-INH. In systemic lupus erythematosus patients with thrombotic disease and in polytrauma patients, the levels of activated MASP-1 and MASP-2 in complex with both AT and C1-INH were associated with markers of thrombotic disease and contact/coagulation system activation. CONCLUSIONS: MASP-1 and MASP-2 are activated during blood clotting. This activation is triggered by activated platelets and by the generation of fibrin during thrombotic reactions in vitro and in vivo, and may represent a novel activation/amplification mechanism in thromboinflammation.

Increased serum type I interferon activity in early systemic sclerosis patients is associated with antibodies against Sjögren's syndrome antigens and nuclear ribonucleoprotein antigens.

OBJECTIVE: To study serum type I interferon (IFN) activity in patients with early systemic sclerosis (SSc). METHOD: Serum type I IFN activity was measured in 33 consecutive patients with SSc and a disease duration of < 2 years and in 13 healthy individuals by calculating a type I IFN score according to the induction of six IFN-α regulated genes in a reporter cell line. RESULTS: Twenty-seven per cent of the SSc patients had an increased type I IFN score compared to none of the healthy individuals (p < 0.05). The clinical SSc phenotype associated with high serum type I IFN activity did not differ from patients with low serum type I IFN activity regarding the presence of skin or lung fibrosis, pulmonary hypertension, or digital complications. Patients with high serum type I IFN activity were younger (p < 0.01) and had a lower frequency of cardiac involvement (p = 0.053), lower leucocyte count (p < 0.001), higher immunoglobulin (Ig)G levels (p < 0.05), and a higher amount of antibodies against extractable nuclear antigens (p < 0.01) than patients with low serum type I IFN activity. The presence of antibodies against topoisomerase I, Sjögren's syndrome antigen, and nuclear ribonucleoprotein antigens was associated with higher type I IFN activity (p < 0.05 for all comparisons). CONCLUSIONS: Our study indicates that increased serum type I IFN activity in early SSc patients is associated with an antibody and laboratory profile that may reflect a subclinical overlap of SSc with other type I IFN-driven connective tissue diseases (CTDs).

Increased C1q, C4 and C3 deposition on platelets in patients with systemic lupus erythematosus--a possible link to venous thrombosis?

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have an increased risk of developing vascular diseases (VD) such as myocardial infarction, stroke and venous thrombosis, which can only partly be explained by traditional risk factors. The role of platelets in this process has not been extensively studied. Platelet activation supports complement binding to the platelet surface, and increased C4d has been seen on platelets in SLE patients as well as in non-rheumatic patients with stroke. In this study we investigated in vivo platelet deposition of the classical complement pathway components C1q, C4d and C3d in relation to VD in SLE patients. Furthermore, the ability of serum to support in vitro complement deposition on fixed heterologous platelets was analyzed. METHODS: Blood from 69 SLE patients and age- and sex-matched healthy individuals was collected in sodium-citrate tubes and platelets isolated by centrifugation. Complement deposition on platelets was detected by flow cytometry. RESULTS: We could demonstrate that SLE patients had increased C1q, C3d and C4d deposition on platelets as compared to healthy controls (p < 0.0001). SLE patients with a history of venous thrombosis had increased complement deposition on platelets as compared to SLE patients without this manifestation (p < 0.05). In vitro studies demonstrated that serum from patients with lupus anticoagulant, venous thrombosis or antiphospholipid antibody syndrome supported increased platelet C4d deposition in vitro as compared to SLE patients without these manifestations (p < 0.05). Our data support the hypothesis that platelet activation and the subsequent complement deposition on platelets are central in the development of venous thrombosis in SLE. CONCLUSIONS: Altogether we suggest that complement deposition on platelets could reflect important pathogenetic events related to the development of venous thrombosis in SLE and might be used as a marker for venous thrombosis in SLE.