| 1. |
- Amara, Umme, et al.
(författare)
-
Molecular Intercommunication between the Complement and Coagulation Systems
- 2010
-
Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 185:9, s. 5628-5636
-
Tidskriftsartikel (refereegranskat)abstract
- The complement system as well as the coagulation system has fundamental clinical implications in the context of life-threatening tissue injury and inflammation. Associations between both cascades have been proposed, but the precise molecular mechanisms remain unknown. The current study reports multiple links for various factors of the coagulation and fibrinolysis cascades with the central complement components C3 and C5 in vitro and ex vivo. Thrombin, human coagulation factors (F) XIa, Xa, and IXa, and plasmin were all found to effectively cleave C3 and C5. Mass spectrometric analyses identified the cleavage products as C3a and C5a, displaying identical molecular weights as the native anaphylatoxins C3a and C5a. Cleavage products also exhibited robust chemoattraction of human mast cells and neutrophils, respectively. Enzymatic activity for C3 cleavage by the investigated clotting and fibrinolysis factors is defined in the following order: FXa > plasmin > thrombin > FIXa > FXIa > control. Furthermore, FXa-induced cleavage of C3 was significantly suppressed in the presence of the selective FXa inhibitors fondaparinux and enoxaparin in a concentration-dependent manner. Addition of FXa to human serum or plasma activated complement ex vivo, represented by the generation of C3a, C5a, and the terminal complement complex, and decreased complement hemolytic serum activity that defines exact serum concentration that results in complement-mediated lysis of 50% of sensitized sheep erythrocytes. Furthermore, in plasma from patients with multiple injuries (n = 12), a very early appearance and correlation of coagulation (thrombin-antithrombin complexes) and the complement activation product C5a was found. The present data suggest that coagulation/fibrinolysis proteases may act as natural C3 and C5 convertases, generating biologically active anaphylatoxins, linking both cascades via multiple direct interactions in terms of a complex serine protease system. The Journal of Immunology, 2010, 185: 5628-5636.
|
|
| 2. |
- Danielsson, Angelika, 1981-, et al.
(författare)
-
An ex vivo loop system models the toxicity and efficacy of PEGylated and unmodified adenovirus serotype 5 in whole human blood
- 2010
-
Ingår i: Gene Therapy. - : Nature Publishing Group. - 0969-7128 .- 1476-5462. ; 17:6, s. 752-762
-
Tidskriftsartikel (refereegranskat)abstract
- Polyethylene glycol coating (PEGylation) of adenovirus serotype 5 (Ad5) has been shown to effectively reduce immunogenicity and increase circulation time of intravenously administered virus in mouse models. Herein, we monitored clot formation, complement activation, cytokine release and blood cell association upon addition of uncoated or PEGylated Ad5 to human whole blood. We used a novel blood loop model where human blood from healthy donors was mixed with virus and incubated in heparin-coated PVC tubing while rotating at 37°C for up to 8 hours. Production of the complement components C3a and C5a and the cytokines IL-8, RANTES and MCP-1 was significantly lower with 20K-PEGylated Ad5 than with uncoated Ad5. PEGylation prevented clotting and reduced Ad5 binding to blood cells in blood with low ability to neutralize Ad5. The effect was particularly pronounced in monocytes, granulocytes, B-cells and T-cells, but could also be observed in erythrocytes and platelets. In conclusion, PEGylation of Ad5 can reduce the immune response mounted in human blood, although the protective effects are rather modest in contrast to published mouse data. Our findings underline the importance of developing reliable models and we propose the use of human whole blood models in pre-clinical screening of gene therapy vectors.
|
|
| 3. |
|
|
| 4. |
- Engberg, Anna E., et al.
(författare)
-
Blood protein-polymer adsorption : Implications for understanding complement-mediated hemoincompatibility
- 2011
-
Ingår i: Journal of Biomedical Materials Research - Part A. - : Wiley. - 1549-3296. ; 97A:1, s. 74-84
-
Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to create polymeric materials with known properties to study the preconditions for complement activation. Initially, 22 polymers were screened for complement activating capacity. Based on these results, six polymers (P1-P6) were characterized regarding physico-chemical parameters, for example, composition, surface area, pore size, and protein adsorption from human EDTA-plasma. P2, P4, and reference particles of polystyrene and polyvinyl chloride, were hydrophobic, bound low levels of protein and were poor complement activators. Their accessible surface was limited to protein adsorption in that they had pore diameters smaller than most plasma proteins. P1 and P3 were negatively charged and adsorbed IgG and C1q. A 10-fold difference in complement activation was attributed to the fact that P3 but not P1 bound high amounts of C1-inhibitor. The hydrophobic P5 and P6 were low complement activators. They selectively bound apolipoproteins AI and AIV (and vitronectin), which probably limited the binding of complement activators to the surface. We demonstrate the usefulness of the modus operandi to use a high-throughput procedure to synthesize a great number of novel substances, assay their physico-chemical properties with the aim to study the relationship between the initial protein coat on a surface and subsequent biological events. Data obtained from the six polymers characterized here, suggest that a complement-resistant surface should be hydrophobic, uncharged, and have a small available surface, accomplished by nanostructured topography. Additional attenuation of complement can be achieved by selective enrichment of inert proteins and inhibitors.
|
|
| 5. |
- Hallström, Teresia, et al.
(författare)
-
Immune Evasion of Moraxella catarrhalis Involves Ubiquitous Surface Protein A-Dependent C3d Binding.
- 2011
-
Ingår i: Journal of immunology. - : The American Association of Immunologists. - 1550-6606 .- 0022-1767. ; 186, s. 3120-3129
-
Tidskriftsartikel (refereegranskat)abstract
- The complement system plays an important role in eliminating invading pathogens. Activation of complement results in C3b deposition (opsonization), phagocytosis, anaphylatoxin (C3a, C5a) release, and consequently cell lysis. Moraxella catarrhalis is a human respiratory pathogen commonly found in children with otitis media and in adults with chronic obstructive pulmonary disease. The species has evolved multiple complement evasion strategies, which among others involves the ubiquitous surface protein (Usp) family consisting of UspA1, A2, and A2 hybrid. In the present study, we found that the ability of M. catarrhalis to bind C3 correlated with UspA expression and that C3 binding contributed to serum resistance in a large number of clinical isolates. Recombinantly expressed UspA1 and A2 inhibit both the alternative and classical pathways, C3b deposition, and C3a generation when bound to the C3 molecule. We also revealed that the M. catarrhalis UspA-binding domain on C3b was located to C3d and that the major bacterial C3d-binding domains were within UspA1(299-452) and UspA2(165-318). The interaction with C3 was not species specific since UspA-expressing M. catarrhalis also bound mouse C3 that resulted in inhibition of the alternative pathway of mouse complement. Taken together, the binding of C3 to UspAs is an efficient strategy of Moraxella to block the activation of complement and to inhibit C3a-mediated inflammation.
|
|
| 6. |
- Hamad, Osama A., 1978-, et al.
(författare)
-
Complement component C3 binds to activated normal platelets without preceding proteolytic activation and promotes binding to complement receptor 1
- 2010
-
Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 184:5, s. 2686-2692
-
Tidskriftsartikel (refereegranskat)abstract
- It has been reported that complement is activated on the surface of activated platelets, despite the presence of multiple regulators of complement activation. To reinvestigate the mechanisms by which activated platelets bind to complement components, the presence of complement proteins on the surfaces of nonactivated and thrombin receptor-activating peptide-activated platelets was analyzed by flow cytometry and Western blot analyses. C1q, C4, C3, and C9 were found to bind to thrombin receptor-activating peptide-activated platelets in lepirudin-anticoagulated platelet-rich plasma (PRP) and whole blood. However, inhibiting complement activation at the C1q or C3 level did not block the binding of C3 to activated platelets. Diluting PRP and chelating divalent cations also had no effect, further indicating that the deposition of complement components was independent of complement activation. Furthermore, washed, activated platelets bound added C1q and C3 to the same extent as platelets in PRP. The use of mAbs against different forms of C3 demonstrated that the bound C3 consisted of C3(H(2)O). Furthermore, exogenously added soluble complement receptor 1 was shown to bind to this form of platelet-bound C3. These observations indicate that there is no complement activation on the surface of platelets under physiological conditions. This situation is in direct contrast to a number of pathological conditions in which regulators of complement activation are lacking and thrombocytopenia and thrombotic disease are the ultimate result. However, the generation of C3(H(2)O) represents nonproteolytic activation of C3 and after factor I cleavage may act as a ligand for receptor binding.
|
|
| 7. |
- Hamad, Osama A., 1978-, et al.
(författare)
-
Contribution of chondroitin sulfate A to the binding of complement proteins to activated platelets
- 2010
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:9, s. e12889-
-
Tidskriftsartikel (refereegranskat)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.
|
|
| 8. |
- Klapper, Yvonne, et al.
(författare)
-
Mediation of a non-proteolytic activation of complement component C3 by phospholipid vesicles
- 2014
-
Ingår i: Biomaterials. - : Elsevier BV. - 0142-9612 .- 1878-5905. ; 35:11, s. 3688-3696
-
Tidskriftsartikel (refereegranskat)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.
|
|
| 9. |
- Moll, Guido, et al.
(författare)
-
Do Cryopreserved Mesenchymal Stromal Cells Display Impaired Immunomodulatory and Therapeutic Properties?
- 2014
-
Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 32:9, s. 2430-2442
-
Tidskriftsartikel (refereegranskat)abstract
- We have recently reported that therapeutic mesenchymal stromal cells (MSCs) have low engraftment and trigger the instant blood mediated inflammatory reaction (IBMIR) after systemic delivery to patients, resulting in compromised cell function. In order to optimize the product, we compared the immunomodulatory, blood regulatory, and therapeutic properties of freeze-thawed and freshly harvested cells. We found that freeze-thawed MSCs, as opposed to cells harvested from continuous cultures, have impaired immunomodulatory and blood regulatory properties. Freeze-thawed MSCs demonstrated reduced responsiveness to proinflammatory stimuli, an impaired production of anti-inflammatory mediators, increased triggering of the IBMIR, and a strong activation of the complement cascade compared to fresh cells. This resulted in twice the efficiency in lysis of thawed MSCs after 1 hour of serum exposure. We found a 50% and 80% reduction in viable cells with freshly detached as opposed to thawed in vitro cells, indicating a small benefit for fresh cells. In evaluation of clinical response, we report a trend that fresh cells, and cells of low passage, demonstrate improved clinical outcome. Patients treated with freshly harvested cells in low passage had a 100% response rate, twice the response rate of 50% observed in a comparable group of patients treated with freeze-thawed cells at higher passage. We conclude that cryobanked MSCs have reduced immunomodulatory and blood regulatory properties directly after thawing, resulting in faster complement-mediated elimination after blood exposure. These changes seem to be paired by differences in therapeutic efficacy in treatment of immune ailments after hematopoietic stem cell transplantation.
|
|
| 10. |
- Moll, Guido, et al.
(författare)
-
Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses (Open Access)
- 2011
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:7, s. e21703-
-
Tidskriftsartikel (refereegranskat)abstract
- Infusion of human third-party mesenchymal stromal cells (MSCs) appears to be a promising therapy for acute graft-versus-host disease (aGvHD). To date, little is known about how MSCs interact with the body's innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46) and DAF (CD55), but were protected from complement lysis via expression of protectin (CD59). Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells.
|
|
