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 cbpD 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 alternative complement pathway revisited Harboe, Morten; Mollnes, Tom Eirik
Journal of Cellular and Molecular Medicine,
August 2008, Letnik:
12, Številka:
4
Journal Article
Recenzirano
Odprti dostop
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Introduction
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Methods to study and modify the alternative pathway
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Inhibition by monoclonal antibodies
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Knockout models
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Influence of AP amplification on the effect of specific activation ...of the other initial pathways
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Direct activation versus amplification
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Mechanisms of amplification
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Properdin
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Control protein in the AP amplification loop
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Recognition molecule in AP
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Influence of AP in deficiency states
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Factor H deficiency and variants
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C2 bypass in CP and LP
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Properdin deficiency
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Complement in sepsis
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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.
The liver is both an immunologically complex and a privileged organ. The innate immune system is a central player, in which the complement system emerges as a pivotal part of liver homeostasis, ...immune responses, and crosstalk with other effector systems in both innate and adaptive immunity. The liver produces the majority of the complement proteins and is the home of important immune cells such as Kupffer cells. Liver immune responses are delicately tuned between tolerance to many antigens flowing in from the alimentary tract, a tolerance that likely makes the liver less prone to rejection than other solid organ transplants, and reaction to local injury, systemic inflammation, and regeneration. Notably, complement is a double‐edged sword as activation is detrimental by inducing inflammatory tissue damage in, for example, ischemia–reperfusion injury and transplant rejection yet is beneficial for liver tissue regeneration. Therapeutic complement inhibition is rapidly developing for routine clinical treatment of several diseases. In the liver, targeted inhibition of damaged tissue may be a rational and promising approach to avoid further tissue destruction and simultaneously preserve beneficial effects of complement in areas of proliferation. Here, we argue that complement is a key system to manipulate in the liver in several clinical settings, including liver injury and regeneration after major surgery and preservation of the organ during transplantation.
Polyethylene glycol (PEG)-coated nanopharmaceuticals can cause mild to severe hypersensitivity reactions (HSRs), which can occasionally be life threatening or even lethal. The phenomenon represents ...an unsolved immune barrier to the use of these drugs, yet its mechanism is poorly understood. This study showed that a single i.v. injection in pigs of a low dose of PEGylated liposomes (Doxebo) induced a massive rise of anti-PEG IgM in blood, peaking at days 7–9 and declining over 6 weeks. Bolus injections of PEG-liposomes during seroconversion resulted in anaphylactoid shock (pseudo-anaphylaxis) within 2–3 min, although similar treatments of naı̈ve animals led to only mild hemodynamic disturbance. Parallel measurement of pulmonary arterial pressure (PAP) and sC5b-9 in blood, taken as measures of HSR and complement activation, respectively, showed a concordant rise of the two variables within 3 min and a decline within 15 min, suggesting a causal relationship between complement activation and pulmonary hypertension. We also observed a rapid decline of anti-PEG IgM in the blood within minutes, increased binding of PEGylated liposomes to IgM + B cells in the spleen of immunized animals compared to control, and increased C3 conversion by PEGylated liposomes in the serum of immunized pigs. These observations taken together suggest rapid binding of anti-PEG IgM to PEGylated liposomes, leading to complement activation via the classical pathway, entailing anaphylactoid shock and accelerated blood clearance of liposome–IgM complexes. These data suggest that complement activation plays a causal role in severe HSRs to PEGylated nanomedicines and that pigs can be used as a hazard identification model to assess the risk of HSRs in preclinical safety studies.
The host is protected by pattern recognition systems, including complement and TLRs, which are closely cross‐talking. If improperly activated, these systems might induce tissue damage and disease. ...Inhibition of single downstream proinflammatory cytokines, such as TNF, IL‐1β, and IL‐6, have failed in clinical sepsis trials, which might not be unexpected, given the substantial amounts of mediators involved in the pathogenesis of this condition. Instead, we have put forward a hypothesis of inhibition at the recognition phase by “dual blockade” of bottleneck molecules of complement and TLRs. By acting upstream and broadly, the dual blockade could be beneficial in conditions with improper or uncontrolled innate immune activation threatening the host. Key bottleneck molecules in these systems that could be targets for inhibition are the central complement molecules C3 and C5 and the important CD14 molecule, which is a coreceptor for several TLRs, including TLR4 and TLR2. This review summarizes current knowledge of inhibition of complement and TLRs alone and in combination, in both sterile and nonsterile inflammatory processes, where activation of these systems is of crucial importance for tissue damage and disease. Thus, dual blockade might provide a general, broad‐acting therapeutic regimen against a number of diseases where innate immunity is improperly activated.
Review of how targeting key upstream molecules at the recognition phase of innate immunity exert anti‐inflammatory effects; a potential therapeutic regimen for inflammatory diseases.
The complement system is involved in pathogenesis of cardiovascular disease, and might play a role in accelerated atherogenesis in spondylarthropathies (SpA). Hence, we examined complement activation ...in SpA, and its relationship to antirheumatic treatment, inflammatory and cardiovascular markers.
From PSARA, a prospective observational study, we examined 51 SpA patients (31 psoriatic arthritis (PsA), and 20 ankylosing spondylitis (AS)), starting tumor necrosis factor (TNF) inhibitor alone (n = 25), combined with methotrexate (MTX) (n = 10), or MTX monotherapy (n = 16). Complement activation was determined by the soluble terminal complement complex (sC5b-9), inflammation by erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), and endothelial function by finger plethysmography (Endopat) at baseline, after 6 weeks and 6 months of treatment.
SpA patients had sC5b-9 levels at (PsA) or above (AS) the upper limit of the estimated reference range. Median sC5b-9 levels decreased significantly from baseline to 6 weeks, with no significant difference between the AS and PsA group. Notably, a significant reduction in sC5b-9 was observed after administration of TNF inhibitor ± MTX, whereas no significant changes were observed in patients treated with MTX alone. Between 6 weeks and 6 months, sC5b-9 remained stable across all subgroups. Reduction in sC5b-9 was independently related to decreased ESR and CRP, and to increased high density cholesterol and total cholesterol. Reduction in sC5b-9 from baseline to 6 weeks was associated with improved EF in age and gender adjusted analyses.
TNF-inhibition, but not MTX monotherapy, led to rapid and sustained reduction of complement activation in SpA. Thus, the observed decrease in cardiovascular morbidity in patients treated with TNF-inhibitors might be partly due to its beneficial effect on complement.
Clinical Trials (NCT00902005), retrospectively registered on the 14th of May 2009.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gelatin based adhesives have been used in the last decades in different biomedical applications due to the excellent biocompatibility, easy processability, transparency, non-toxicity, and reasonable ...mechanical properties to mimic the extracellular matrix (ECM). Gelatin adhesives can be easily tuned to gain different viscoelastic and mechanical properties that facilitate its ocular application. We herein grafted glycidyl methacrylate on the gelatin backbone with a simple chemical modification of the precursor, utilizing epoxide ring-opening reactions and visible light-crosslinking. This chemical modification allows the obtaining of an elastic protein-based hydrogel (GELGYM) with excellent biomimetic properties, approaching those of the native tissue. GELGYM can be modulated to be stretched up to 4 times its initial length and withstand high tensile stresses up to 1.95 MPa with compressive strains as high as 80% compared to Gelatin-methacryloyl (GeIMA), the most studied derivative of gelatin used as a bioadhesive. GELGYM is also highly biocompatible and supports cellular adhesion, proliferation, and migration in both 2 and 3-dimensional cell-cultures. These characteristics along with its super adhesion to biological tissues such as cornea, aorta, heart, muscle, kidney, liver, and spleen suggest widespread applications of this hydrogel in many biomedical areas such as transplantation, tissue adhesive, wound dressing, bioprinting, and drug and cell delivery.
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•Gelatin based adhesives have been used in the last decades in different biomedical applications.•Gelatin adhesives can be easily tuned to gain different viscoelastic and mechanical properties that facilitate its ocular application.•We herein grafted glycidyl methacrylate on the gelatin backbone with a simple chemical modification of the precursor.•Epoxide ring-opening reactions and visible light-crosslinking allows the obtaining of an elastic gelatin-based hydrogel (GELGYM).•GELGYM shows excellent biomimetic properties, approaching those of the native tissue.
The complement system plays an important role in pathophysiology of cardiovascular disease (CVD), and might be involved in accelerated atherogenesis in rheumatoid arthritis (RA). The role of ...complement activation in response to treatment, and in development of premature CVD in RA, is limited. Therefore, we examined the effects of methotrexate (MTX) and tumor necrosis factor inhibitors (TNFi) on complement activation using soluble terminal complement complex (TCC) levels in RA; and assessed associations between TCC and inflammatory and cardiovascular biomarkers.
We assessed 64 RA patients starting with MTX monotherapy (n = 34) or TNFi with or without MTX co-medication (TNFi±MTX, n = 30). ELISA was used to measure TCC in EDTA plasma. The patients were examined at baseline, after 6 weeks and 6 months of treatment.
Median TCC was 1.10 CAU/mL, and 57 (89%) patients had TCC above the estimated upper reference limit (<0.70). Compared to baseline, TCC levels were significantly lower at 6-week visit (0.85 CAU/mL, p<0.0001), without significant differences between the two treatment regimens. Notably, sustained reduction in TCC was only achieved after 6 months on TNFi±MTX (0.80 CAU/mL, p = 0.006). Reductions in TCC after treatment were related to decreased C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and interleukin 6, and increased levels of total, high and low-density lipoprotein cholesterol. Similarly, baseline TCC was significantly related to baseline CRP, ESR and interleukin 6. Patients with endothelial dysfunction had higher baseline TCC than those without (median 1.4 versus 1.0 CAU/mL, p = 0.023).
Patients with active RA had elevated TCC, indicating increased complement activation. TCC decreased with antirheumatic treatment already after 6 weeks. However, only treatment with TNFi±MTX led to sustained reduction in TCC during the 6-month follow-up period. RA patients with endothelial dysfunction had higher baseline TCC compared to those without, possibly reflecting involvement of complement in the atherosclerotic process in RA.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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