| 1. |
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| 2. |
- Girardi, Benedetta, et al.
(author)
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Selective Monovalent Galectin-8 Ligands Based on 3-Lactoylgalactoside
- 2022
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In: ChemMedChem. - : Wiley. - 1860-7179 .- 1860-7187. ; 17:3
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Journal article (peer-reviewed)abstract
- Galectin-8 has gained attention as a potential new pharmacological target for the treatment of various diseases, including cancer, inflammation, and disorders associated with bone mass reduction. To that end, new molecular probes are needed in order to better understand its role and its functions. Herein we aimed to improve the affinity and target selectivity of a recently published galectin-8 ligand, 3-O-[1-carboxyethyl]-β-d-galactopyranoside, by introducing modifications at positions 1 and 3 of the galactose. Affinity data measured by fluorescence polarization show that the most potent compound reached a KD of 12 μM. Furthermore, reasonable selectivity versus other galectins was achieved, making the highlighted compound a promising lead for the development of new selective and potent ligands for galectin-8 as molecular probes to examine the protein's role in cell-based and in vivo studies.
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| 3. |
- Hamad, Osama A., et al.
(author)
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Contact activation of C3 enables tethering between activated platelets and polymorphonuclear leukocytes via CD11b/CD18
- 2015
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In: Thrombosis and Haemostasis. - 0340-6245 .- 2567-689X. ; 114:6, s. 1207-1217
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Journal article (peer-reviewed)abstract
- Complement component C3 has a potential role in thrombotic pathologies. It is transformed, without proteolytic cleavage, into C3(H2O) upon binding to the surface of activated platelets. We hypothesise that C3(H2O) bound to activated platelets and to platelet-derived microparticles (PMPs) contributes to platelet-PMN complex (PPC) formation and to the binding of PMPs to PMNs. PAR-1 activation of platelets in human whole blood from normal individuals induced the formation of CD16(+)/CD42a(+) PPC. The complement inhibitor compstatin and a C5a receptor antagonist inhibited PPC formation by 50 %, while monoclonal antibodies to C3(H2O) or anti-CD11b inhibited PPC formation by 75-100 %. Using plasma protein-depleted blood and blood from a C3-deficient patient, we corroborated the dependence on C3, obtaining similar results after reconstitution with purified C3. By analogy with platelets, PMPs isolated from human serum were found to expose C3(H2O) and bind to PMNs. This interaction was also blocked by the anti-C3(H2O) and anti-CD11b monoclonal antibodies, indicating that C3(H2O) and CD11b are involved in tethering PMPs to PMNs. We confirmed the direct interaction between C3(H2O) and CD11b by quartz crystal microbalance analysis using purified native C3 and recombinant CD11b/CD18 and by flow cytometry using PMP and recombinant CD11b. Transfectants expressing CD11b/CD18 were also shown to specifically adhere to surface-bound C3(H2O). We have identified contact-activated C3(H2O) as a novel ligand for CD11b/CD18 that mediates PPC formation and the binding of PMPs to PMNs. Given the various roles of C3 in thrombotic reactions, this finding is likely to have important pathophysiological implications.
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| 4. |
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| 5. |
- Hamad, Osama A., 1978-, et al.
(author)
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Contribution of chondroitin sulfate A to the binding of complement proteins to activated platelets
- 2010
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:9, s. e12889-
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Journal article (peer-reviewed)abstract
- Exposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established. The aim of the present study was to investigate to what extent CS-A contributes to the binding of C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets. Human serum was passed over Sepharose conjugated with CS-A, and bound proteins were identified by Western blotting, and mass spectrometric analysis. C1q was identified as the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were shown to bind also to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets. In conclusion, this study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases.
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| 6. |
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| 7. |
- Klapper, Yvonne, et al.
(author)
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Mediation of a non-proteolytic activation of complement component C3 by phospholipid vesicles
- 2014
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In: Biomaterials. - : Elsevier BV. - 0142-9612 .- 1878-5905. ; 35:11, s. 3688-3696
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Journal article (peer-reviewed)abstract
- Liposomes are becoming increasingly important as drug delivery systems, to target a drug to specific cells and tissues and thereby protecting the recipient from toxic effects of the contained drug. Liposome preparations have been described to activate complement. In this study, we have investigated complement activation triggered by neutral dimyristoyl-phosphocholine (DMPC) liposomes in human plasma and whole-blood systems. Incubation in plasma led to the generation of complement activation products (C3a and sC5b-9). Unexpectedly, investigations of surface-bound C3 revealed contact activated, conformationally changed C3 molecules on the liposomes. These changes were characterized by Western blotting with C3 monoclonal antibodies, and by incubating liposomes with purified native C3 and factors I and H. Quartz crystal microbalance analysis confirmed binding of C3 to planar DMPC surfaces. In addition, we demonstrated that DMPC liposomes bound to or were phagocytized by granulocytes in a complement-dependent manner, as evidenced by the use of complement inhibitors. In summary, we have shown that C3 is activated both by convertase-dependent cleavage, preferentially in the fluid phase, by mechanisms which are not well elucidated, and also by contact activation into C3(H2O) on the DMPC surface. In particular, this contact activation has implications for the therapeutic regulation of complement activation during liposome treatment. (C) 2013 Elsevier Ltd. All rights reserved.
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| 8. |
- Lambris, John D., et al.
(author)
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Preface
- 2015
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In: IMMUNE RESPONSES TO BIOSURFACES. - 9783319186030 - 9783319186023 ; , s. V-VI
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Conference paper (peer-reviewed)
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| 9. |
- Mastellos, Dimitrios C., et al.
(author)
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Compstatin : a C3-targeted complement inhibitor reaching its prime for bedside intervention
- 2015
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In: European Journal of Clinical Investigation. - : Wiley. - 0014-2972 .- 1365-2362. ; 45:4, s. 423-440
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Research review (peer-reviewed)abstract
- There is a growing awareness that complement plays an integral role in human physiology and disease, transcending its traditional perception as an accessory system for pathogen clearance and opsonic cell killing. As the list of pathologies linked to dysregulated complement activation grows longer, it has become clear that targeted modulation of this innate immune system opens new windows of therapeutic opportunity for anti-inflammatory drug design. Indeed, the introduction of the first complement-targeting drugs has reignited a vibrant interest in the clinical translation of complement-based inhibitors. Compstatin was discovered as a cyclic peptide that inhibits complement activation by binding C3 and interfering with convertase formation and C3 cleavage. As the convergence point of all activation pathways and a molecular hub for crosstalk with multiple pathogenic pathways, C3 represents an attractive target for therapeutic modulation of the complement cascade. A multidisciplinary drug optimization effort encompassing rational wet' and in silico synthetic approaches and an array of biophysical, structural and analytical tools has culminated in an impressive structure-function refinement of compstatin, yielding a series of analogues that show promise for a wide spectrum of clinical applications. These new derivatives have improved inhibitory potency and pharmacokinetic profiles and show efficacy in clinically relevant primate models of disease. This review provides an up-to-date survey of the drug design effort placed on the compstatin family of C3 inhibitors, highlighting the most promising drug candidates. It also discusses translational challenges in complement drug discovery and peptide drug development and reviews concerns related to systemic C3 interception.
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| 10. |
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| 11. |
- Nilsson, Per H., et al.
(author)
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Autoregulation of thromboinflammation on biomaterial surfaces by a multicomponent therapeutic coating
- 2013
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In: Biomaterials. - : Elsevier BV. - 0142-9612 .- 1878-5905. ; 34:4, s. 985-994
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Journal article (peer-reviewed)abstract
- Activation of the thrombotic and complement systems is the main recognition and effector mechanisms in the multiple adverse biological responses triggered when biomaterials or therapeutic cells come into blood contact. We have created a surface which is auto-protective to human innate immunity by combining three fundamentally different strategies, all developed by us previously, which have been shown to induce substantial, but incomplete hemocompatibility when used separately. In summary, we have conjugated a factor H-binding peptide; and an ADP-degrading enzyme; using a PEG linker on both material and cellular surfaces. When exposed to human whole blood, factor H was specifically recruited to the modified surfaces and inhibited complement attack. In addition, activation of platelets and coagulation was efficiently attenuated, by degrading ADP. Thus, by inhibiting thromboinflammation using a multicomponent approach, we have created a hybrid surface with the potential to greatly reduce incompatibility reactions involving biomaterials and transplantation.
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| 12. |
- Primikyri, Alexandra, et al.
(author)
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Method development and validation for the quantitation of the complement inhibitor Cp40 in human and cynomolgus monkey plasma by UPLC-ESI-MS
- 2017
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In: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 1041, s. 19-26
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Journal article (peer-reviewed)abstract
- Cp40 is a 14-amino acid cyclic analog of the peptidic complement inhibitor compstatin that binds with sub-nanomolar affinity to complement component C3 and has already shown promise in various models of complement-related diseases. The preclinical and clinical development of this compound requires a robust, accurate, and sensitive method for quantitatively monitoring Cp40 in biological samples. In this study, we describe the development and validation of an ultra-high performance liquid chromatography electrospray mass spectrometry method for the quantitation of Cp40 in human and non-human primate (NHP) plasma. Isotope-labeled Cp40 was used as an internal standard, allowing for the accurate and absolute quantitation of Cp40. Labeled and non-labeled Cp40 were extracted from plasma using reversed phase-solid phase extraction, with recovery rates exceeding 80%, indicating minor matrix effects. The triply charged states of Cp40 and isotope-labeled Cp40 were detected at m/z 596.60 and 600.34, respectively, via a Q-TOF mass spectrometer and were used for quantitation. The method was linear in the range of 0.18-3.58 mu g/mL (r(2) >= 0.99), with precision values below 0.71% in NHP and 0.77% in human plasma. The accuracy of the method ranged from -2.17% to 17.99% in NHP and from -0.26% to 15.75% in 'human plasma. The method was successfully applied to the quantitation of Cp40 in cynomolgus monkey plasma after an initial intravenous bolus of 2 mg/kg followed by repetitive subcutaneous administration at 1 mg/kg. The high reproducibility, accuracy, and robustness of the method developed here render it suitable for drug monitoring of Cp40, and potentially other compstatin analogs, in both human and NHP plasma samples during pharmacokinetic and pharmacodynamic studies.
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| 13. |
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| 14. |
- Sfyroera, Georgia, et al.
(author)
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Rare loss-of-function mutation in complement component C3 provides insight into molecular and pathophysiological determinants of complement activity
- 2015
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In: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 194:7, s. 3305-3316
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Journal article (peer-reviewed)abstract
- The plasma protein C3 is a central element in the activation and effector functions of the complement system. A hereditary dysfunction of C3 that prevents complement activation via the alternative pathway (AP) was described previously in a Swedish family, but its genetic cause and molecular consequences have remained elusive. In this study, we provide these missing links by pinpointing the dysfunction to a point mutation in the beta-chain of C3 (c.1180T > C; p.Met(373)Thr). In the patient's plasma, AP activity was completely abolished and could only be reconstituted with the addition of normal C3. The M373T mutation was localized to the macroglobulin domain 4 of C3, which contains a binding site for the complement inhibitor compstatin and is considered critical for the interaction of C3 with the AP C3 convertase. Structural analyses suggested that the mutation disturbs the integrity of macroglobulin domain 4 and induces conformational changes that propagate into adjacent regions. Indeed, C3 M373T showed an altered binding pattern for compstatin and surface-bound C3b, and the presence of Thr(373) in either the C3 substrate or convertase-affiliated C3b impaired C3 activation and opsonization. In contrast to known gain-of-function mutations in C3, patients affected by this loss-of-function mutation did not develop familial disease, but rather showed diverse and mostly episodic symptoms. Our study therefore reveals the molecular mechanism of a relevant loss-of-function mutation in C3 and provides insight into the function of the C3 convertase, the differential involvement of C3 activity in clinical conditions, and some potential implications of therapeutic complement inhibition.
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| 15. |
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| 16. |
- van Griensven, Martijn, et al.
(author)
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PROTECTIVE EFFECTS OF THE COMPLEMENT INHIBITOR COMPSTATIN CP40 IN HEMORRHAGIC SHOCK
- 2019
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In: Shock. - Alphen aan den Rijn : LIPPINCOTT WILLIAMS & WILKINS. - 1073-2322 .- 1540-0514. ; 51:1, s. 78-87
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Journal article (peer-reviewed)abstract
- 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.
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| 17. |
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