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Nilsson Ekdahl, KristinaORCID iD iconorcid.org/0000-0001-7888-1571
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Publications (10 of 269) Show all publications
Lindelöf, L., Dahlqvist, S. R., Lundtoft, C., Nilsson Ekdahl, K., Nilsson, B., Gunnarsson, I., . . . Eriksson, O. (2023). 62 Acquired ficolin-3 deficiency in patients with Systemic Lupus Erythematosus. Immunobiology, 228(5), 152515-152515, Article ID 62.
Open this publication in new window or tab >>62 Acquired ficolin-3 deficiency in patients with Systemic Lupus Erythematosus
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2023 (English)In: Immunobiology, ISSN 0171-2985, E-ISSN 1878-3279, Vol. 228, no 5, p. 152515-152515, article id 62Article in journal, Meeting abstract (Refereed) Published
Abstract [en]

Background: Ficolin-3 is the main initiator of the lectin pathway in humans. Case reports of ficolin-3 deficient patients have suggested that ficolin-3 deficiency may be enriched in patients with Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease where complement plays an important role. Therefore, this study aimed to investigate the activity levels of ficolin-3 and to identify potential ficolin-3 deficient individuals in two Swedish SLE cohorts.

Methods: Serum or plasma samples from SLE patients (n=810) and matched controls (n=566) were collected from the Karolinska Institute (KI) and Umeå University Hospital. The ficolin-3 activity levels were measured by an in-house developed functional ELISA with a pooled normal human serum sample as a reference. Serial samples were analyzed for ficolin-3 deficient patients when available. Sequencing data were analyzed for FCN3 frame-shift mutation +1637delC (rs532781899) and other potential loss-of-function (LoF) variants.

Results: This screening revealed that the level of ficolin-3 activity varies largely in patients with SLE. The activity levels also show that SLE patients seem to generally have elevated ficolin-3 activity compared to the control group (p<0.0001). Out of 810 patients with SLE, four patients were determined to be ficolin-3 deficient. For two of these patients, the ficolin-3 activity was at normal levels at the time of diagnosis and thereafter depleted over time, indicating an acquired deficiency. For deficient patients, no or very low ficolin-3 protein levels and no lectin pathway-dependent complement activation could be detected. Autoantibodies against ficolin-3 were not detectable. No patients were homozygous for the +1637delC frameshift mutation, whereas in total 10 patients were determined to be heterozygous carriers. These heterozygous patients displayed lower levels of ficolin-3 activity but did not include the deficient patients. Additional possible LoF variants were analyzed but none were enriched in either patients or controls.

Conclusions: Contrary to the classical pathway of the complement system we show that genetic ficolin-3 deficiency is not a risk factor for SLE. Instead, acquired ficolin-3 deficiency was observed in a subgroup of SLE patients, possibly due to a potent activation of the lectin pathway that depleted ficolin-3 plasma levels in these individuals.

Place, publisher, year, edition, pages
Elsevier, 2023
National Category
Immunology in the medical area
Research subject
Biomedical Sciences, Immunology
Identifiers
urn:nbn:se:lnu:diva-126339 (URN)10.1016/j.imbio.2023.152515 (DOI)001057448500053 ()
Available from: 2024-01-10 Created: 2024-01-10 Last updated: 2024-01-10Bibliographically approved
Kokelj, S., Ostling, J., Fromell, K., Vanfleteren, L. E. G., Olsson, H. K., Nilsson Ekdahl, K., . . . Olin, A.-C. (2023). Activation of the Complement and Coagulation Systems in the Small Airways in Asthma. Respiration, 102(8), 621-631
Open this publication in new window or tab >>Activation of the Complement and Coagulation Systems in the Small Airways in Asthma
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2023 (English)In: Respiration, ISSN 0025-7931, E-ISSN 1423-0356, Vol. 102, no 8, p. 621-631Article in journal (Refereed) Published
Abstract [en]

Background: Several studies have shown the importance of the complement and coagulation systems in the pathogenesis of asthma. Objectives: We explored whether we could detect differentially abundant complement and coagulation proteins in the samples obtained from the small airway lining fluid by collection of exhaled particles in patients with asthma and whether these proteins are associated with small airway dysfunction and asthma control. Method: Exhaled particles were obtained from 20 subjects with asthma and 10 healthy controls (HC) with the PExA method and analysed with the SOMAscan proteomics platform. Lung function was assessed by nitrogen multiple breath washout test and spirometry. Results: 53 proteins associated with the complement and coagulation systems were included in the analysis. Nine of those proteins were differentially abundant in subjects with asthma as compared to HC, and C3 was significantly higher in inadequately controlled asthma as compared to well-controlled asthma. Several proteins were associated with physiological tests assessing small airways. Conclusions: The study highlights the role of the local activation of the complement and coagulation systems in the small airway lining fluid in asthma and their association with both asthma control and small airway dysfunction. The findings highlight the potential of complement factors as biomarkers to identify different sub-groups among patients with asthma that could potentially benefit from a therapeutic approach targeting the complement system.

Place, publisher, year, edition, pages
S. Karger, 2023
Keywords
Asthma control, Asthma, Complement system, Coagulation, Small airways, Exhaled particles
National Category
Immunology in the medical area
Research subject
Biomedical Sciences, Immunology
Identifiers
urn:nbn:se:lnu:diva-123593 (URN)10.1159/000531374 (DOI)001026727600001 ()37423212 (PubMedID)2-s2.0-85165921610 (Scopus ID)
Available from: 2023-08-10 Created: 2023-08-10 Last updated: 2023-11-07Bibliographically approved
Martin, M., Nilsson, S. C. C., Eikrem, D., Fromell, K., Scavenius, C., Vogt, L. M. M., . . . Blom, A. M. M. (2023). Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis. Frontiers in Immunology, 14, Article ID 1203506.
Open this publication in new window or tab >>Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis
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2023 (English)In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 14, article id 1203506Article in journal (Refereed) Published
Abstract [en]

BackgroundDysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. MethodsCitrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. ResultsC1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. ConclusionCitrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2023
Keywords
citrullination, C1-inhibitor, complement system, PAD, rheumatoid arthritis, synovial fluid, ACPA
National Category
Rheumatology and Autoimmunity Immunology in the medical area
Research subject
Biomedical Sciences, Immunology
Identifiers
urn:nbn:se:lnu:diva-123557 (URN)10.3389/fimmu.2023.1203506 (DOI)001020025000001 ()37426666 (PubMedID)2-s2.0-85164147618 (Scopus ID)
Available from: 2023-08-09 Created: 2023-08-09 Last updated: 2024-01-17Bibliographically approved
Mannes, M., Halbgebauer, R., Wohlgemuth, L., Messerer, D. A., Savukoski, S., Schultze, A., . . . Huber-Lang, M. (2023). Combined Heterozygous Genetic Variations in Complement C2 and C8B: An Explanation for Multidimensional Immune Imbalance?. Journal of Innate Immunity, 15(1), 412-427
Open this publication in new window or tab >>Combined Heterozygous Genetic Variations in Complement C2 and C8B: An Explanation for Multidimensional Immune Imbalance?
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2023 (English)In: Journal of Innate Immunity, ISSN 1662-811X, E-ISSN 1662-8128, Vol. 15, no 1, p. 412-427Article in journal (Refereed) Published
Abstract [en]

The complement system plays a crucial role in host defense, homeostasis, and tissue regeneration and bridges the innate and the adaptive immune systems. Although the genetic variants in complement C2 (c.839_849+17del; p.(Met280Asnfs*5)) and C8B (c.1625C>T; p.(Thr542Ile)) are known individually, here, we report on a patient carrying their combination in a heterozygous form. The patient presented with a reduced general condition and suffers from a wide variety of autoimmune diseases. While no autoimmune disease-specific autoantibodies could be detected, genetic analysis revealed abnormalities in the two complement genes C2 and C8B. Therefore, we performed a comprehensive investigation of the innate immune system on a cellular and humoral level to define the functional consequences. We found slightly impaired functionality of neutrophils and monocytes regarding phagocytosis and reactive oxygen species generation and a diminished expression of the C5aR1. An extensive complement analysis revealed a declined activation potential for the alternative and classical pathway. Reconstitution with purified C2 and C8 into patient serum failed to normalize the dysfunction, whereas the addition of C3 improved the hemolytic activity. In clinical transfer, in vitro supplementation of the patient's plasma with FFP as a complement source could fully restore full complement functionality. This study describes for the first time a combined heterozygous genetic variation in complement C2 and C8B which, however, cannot fully explain the overall dysfunctions and calls for further complement deficiency research and corresponding therapies.

Place, publisher, year, edition, pages
S. Karger, 2023
Keywords
Genetic complement variants, Complement imbalance, Complement reconstitution
National Category
Immunology in the medical area
Research subject
Biomedical Sciences, Immunology
Identifiers
urn:nbn:se:lnu:diva-120768 (URN)10.1159/000528607 (DOI)000960514200001 ()36858027 (PubMedID)2-s2.0-85151848436 (Scopus ID)
Available from: 2023-05-17 Created: 2023-05-17 Last updated: 2023-05-31Bibliographically approved
Nilsson, B., Eriksson, O., Fromell, K., Persson, B. & Nilsson Ekdahl, K. (2023). How COVID-19 and other pathological conditions and medical treatments activate our intravascular innate immune system. Frontiers in Immunology, 13, Article ID 1030627.
Open this publication in new window or tab >>How COVID-19 and other pathological conditions and medical treatments activate our intravascular innate immune system
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2023 (English)In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 13, article id 1030627Article in journal (Refereed) Published
Abstract [en]

COVID-19 has been shown to have a multifaceted impact on the immune system. In a recently published article in Front Immunol, we show that the intravascular innate immune system (IIIS) is strongly activated in severe COVID-19 with ARDS and appears to be one of the causes leading to severe COVID-19. In this article, we describe the IIIS and its physiological function, but also the strong pro-inflammatory effects that are observed in COVID-19 and in various other pathological conditions and treatments such as during ischemia reperfusion injury and in treatments where biomaterials come in direct contact with blood in, e.g., extracorporeal and intravasal treatments. In the present article, we describe how the IIIS, a complex network of plasma proteins and blood cells, constitute the acute innate immune response of the blood and discuss the effects that the IIIS induces in pathological disorders and treatments in modern medicine.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2023
Keywords
leukocytes, platelets, blood cascade systems, intravascular innate immune system, cross talk
National Category
Immunology in the medical area
Research subject
Biomedical Sciences, Immunology
Identifiers
urn:nbn:se:lnu:diva-119797 (URN)10.3389/fimmu.2022.1030627 (DOI)000934791500001 ()36820001 (PubMedID)2-s2.0-85148512719 (Scopus ID)
Available from: 2023-03-16 Created: 2023-03-16 Last updated: 2024-01-17Bibliographically approved
Spiegelburg, D. T., Mannes, M., Schultze, A., Scheibenberger, F., Mueller, F., Klitzing, A., . . . Braun, C. K. (2023). Impact of surface coating and systemic anticoagulants on hemostasis and inflammation in a human whole blood model. PLOS ONE, 18(1), Article ID e0280069.
Open this publication in new window or tab >>Impact of surface coating and systemic anticoagulants on hemostasis and inflammation in a human whole blood model
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2023 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 18, no 1, article id e0280069Article in journal (Refereed) Published
Abstract [en]

BackgroundSurface compatibility with blood is critical both for scientific investigations on hemostasis and clinical applications. Regarding in vitro and ex vivo investigations, minimal alteration in physiological hemostasis is of particular importance to draw reliable conclusions on the human coagulation system. At the same time, artificial coagulation activation must be avoided, which is relevant for the patient, for example to prevent stent graft occlusion. The aim was to evaluate the advantages and disadvantages of antithrombotic and antifouling surface coatings in the context of their suitability for ex vivo incubation and the study of coagulation properties. MethodsWe investigated the impact of different protocols for surface coating of synthetic material and different anticoagulants on hemostasis and platelet activation in ex vivo human whole blood.Blood samples from healthy donors were incubated in coated microtubes on a rotating wheel at 37 degrees C. Two protocols for surface coating were analyzed for hemostatic parameters and metabolic status, a heparin-based coating (CHC, Corline Heparin Conjugate) without further anticoagulation and a passivating coating (MPC, 2-methacryloyloxethyl phosphorylcholine) with added anticoagulants (enoxaparin, ENOX; or fondaparinux, FPX). Employing the MPC-based coating, the anticoagulants enoxaparin and fondaparinux were compared regarding their differential effects on plasmatic coagulation by thrombelastometry and on platelet activation by flowcytometry and platelet function assays. ResultsUsing the CHC coating, significant coagulation cascade activation was observed, whereas parameters remained mostly unchanged with MPC-based protocols. Extended incubation caused significantly elevated levels of the soluble membrane attack complex. Neither ENOX nor FPX caused a relevant impairment of platelet function or activation capacity and thrombelastometric parameters remained unchanged with both protocols. For translational purposes, we additionally modeled endotoxemia with the MPC-based protocols by incubating with lipopolysaccharide plus/minus thrombin. While coagulation parameters remained unchanged, elevated Interleukin 8 and Matrix Metalloproteinase 9 demonstrated preserved immune cell responsiveness. ConclusionsThe MPC-based protocols demonstrated better hemocompatibility compared to CHC, and ENOX and FPX proved useful for additional anticoagulation. Furthermore, this simple-to-use whole blood model may be useful for experimental analyses of the early coagulatory and immunological response without decalcification.

Place, publisher, year, edition, pages
Public Library of Science (PLoS), 2023
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-123714 (URN)10.1371/journal.pone.0280069 (DOI)000945466300001 ()36634087 (PubMedID)2-s2.0-85146176032 (Scopus ID)
Available from: 2023-08-15 Created: 2023-08-15 Last updated: 2023-09-07Bibliographically approved
Adler, A., Fritsch, M., Fromell, K., Leneweit, G., Nilsson Ekdahl, K., Nilsson, B. & Teramura, Y. (2023). Regulation of the innate immune system by fragmented heparin-conjugated lipids on lipid bilayered membranes in vitro. Journal of materials chemistry. B, 11(46), 11121-11134
Open this publication in new window or tab >>Regulation of the innate immune system by fragmented heparin-conjugated lipids on lipid bilayered membranes in vitro
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2023 (English)In: Journal of materials chemistry. B, ISSN 2050-750X, E-ISSN 2050-7518, Vol. 11, no 46, p. 11121-11134Article in journal (Refereed) Published
Abstract [en]

Surface modification with heparin is a powerful biomaterial coating strategy that protects against innate immunity activation since heparin is a part of the proteoglycan heparan sulfate on cell surfaces in the body. We studied the heparinization of cellular and material surfaces via lipid conjugation to a heparin-binding peptide. In the present study, we synthesized fragmented heparin (fHep)-conjugated phospholipids and studied their regulation of the innate immune system on a lipid bilayered surface using liposomes. Liposomes have versatile applications, such as drug-delivery systems, due to their ability to carry a wide range of molecules. Owing to their morphological similarity to cell membranes, they can also be used to mimic a simple cell-membrane to study protein-lipid interactions. We investigated the interaction of complement-regulators, factor H and C4b-binding protein (C4BP), as well as the coagulation inhibitor antithrombin (AT), with fHep-lipids on the liposomal surface. Herein, we studied the ability of fHep-lipids to recruit factor H, C4BP, and AT using a quartz crystal microbalance with dissipation monitoring. With dynamic light scattering, we demonstrated that liposomes could be modified with fHep-lipids and were stable up to 60 days at 4 degree celsius. Using a capillary western blot-based method (Wes), we showed that fHep-liposomes could recruit factor H in a model system using purified proteins and assist in the degradation of the active complement protein C3b to iC3b. Furthermore, we found that fHep-liposomes could recruit factor H and AT from human plasma. Therefore, the use of fHep-lipids could be a potential coating for liposomes and cell surfaces to regulate the immune system on the lipid surface.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2023
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-125917 (URN)10.1039/d3tb01721d (DOI)001103685900001 ()37953734 (PubMedID)2-s2.0-85176929830 (Scopus ID)
Available from: 2023-12-07 Created: 2023-12-07 Last updated: 2024-01-18Bibliographically approved
Huang, T., Sato, Y., Kuramochi, A., Ohba, Y., Sano, M., Miyagishi, M., . . . Teramura, Y. (2023). Surface modulation of extracellular vesicles with cell-penetrating peptide-conjugated lipids for improvement of intracellular delivery to endothelial cells. Regenerative Therapy, 22, 90-98
Open this publication in new window or tab >>Surface modulation of extracellular vesicles with cell-penetrating peptide-conjugated lipids for improvement of intracellular delivery to endothelial cells
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2023 (English)In: Regenerative Therapy, ISSN 2352-3204, Vol. 22, p. 90-98Article in journal (Refereed) Published
Abstract [en]

Exosomes (diameter 30-200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG -lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exo-some surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluores-cence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.(c) 2022, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

Place, publisher, year, edition, pages
Elsevier, 2023
Keywords
Extracellular vesicles, Exosome, Cell-penetrating peptide (CPP), Tat peptide, Poly(ethylene glycol)-conjugated lipid (PEG-Lipid)
National Category
Biochemistry and Molecular Biology Biomaterials Science
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-123543 (URN)10.1016/j.reth.2022.12.007 (DOI)001010895000001 ()36712957 (PubMedID)2-s2.0-85146352336 (Scopus ID)
Available from: 2023-08-09 Created: 2023-08-09 Last updated: 2023-09-07Bibliographically approved
Nilsson Ekdahl, K., Fromell, K., Mannes, M., Grinnemo, K.-H., Huber-Lang, M., Teramura, Y. & Nilsson, B. (2023). Therapeutic regulation of complement activation in extracorporeal circuits and intravascular treatments with special reference to the alternative pathway amplification loop. Immunological Reviews, 313(1), 91-103
Open this publication in new window or tab >>Therapeutic regulation of complement activation in extracorporeal circuits and intravascular treatments with special reference to the alternative pathway amplification loop
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2023 (English)In: Immunological Reviews, ISSN 0105-2896, E-ISSN 1600-065X, Vol. 313, no 1, p. 91-103Article in journal (Refereed) Published
Abstract [en]

A number of clinical treatment modalities involve contact between blood and biomaterials: these include extracorporeal circuits such as hemodialysis, cardiopulmonary bypass, plasmapheresis, and intravascular treatments. Common side effects arising from these treatments are caused by activation of the cascade systems of the blood. Many of these side effects are mediated via the complement system, including thromboinflammatory reactions and rejection of implants. Depending on the composition of the materials, complement activation is triggered via all the activation pathways but is by far mostly driven by the alternative pathway amplification loop. On biomaterial surfaces the alternative pathway amplification is totally unregulated and leads under optimal conditions to deposition of complement fragments, mostly C3b, on the surface leading to a total masking of the underlying surface. In this review, we discuss the mechanism of the complement activation, clinical consequences of the activation, and potential strategies for therapeutic regulation of the activation, using hemodialysis as demonstrator.

Place, publisher, year, edition, pages
John Wiley & Sons, 2023
Keywords
alternative pathway, complement regulation, extracorporeal treatment, hemodialysis, thromboinflammation
National Category
Immunology
Research subject
Biomedical Sciences, Immunology
Identifiers
urn:nbn:se:lnu:diva-117469 (URN)10.1111/imr.13148 (DOI)000869553100001 ()36258635 (PubMedID)2-s2.0-85140016281 (Scopus ID)
Available from: 2022-11-11 Created: 2022-11-11 Last updated: 2023-02-21Bibliographically approved
Adler, A., Manivel, V. A., Fromell, K., Teramura, Y., Nilsson Ekdahl, K. & Nilsson, B. (2022). A Robust Method to Store Complement C3 With Superior Ability to Maintain the Native Structure and Function of the Protein. Frontiers in Immunology, 13, Article ID 891994.
Open this publication in new window or tab >>A Robust Method to Store Complement C3 With Superior Ability to Maintain the Native Structure and Function of the Protein
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2022 (English)In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 13, article id 891994Article in journal (Refereed) Published
Abstract [en]

Complement components have a reputation to be very labile. One of the reasons for this is the spontaneous hydrolysis of the internal thioester that is found in both C3 and C4 (but not in C5). Despite the fact that approximate to 20,000 papers have been published on human C3 there is still no reliable method to store the protein without generating C3(H2O), a fact that may have affected studies of the conformation and function of C3, including recent studies on intracellular C3(H2O). The aim of this work was to define the conditions for storage of native C3 and to introduce a robust method that makes C3 almost resistant to the generation of C3(H2O). Here, we precipitated native C3 at the isoelectric point in low ionic strength buffer before freezing the protein at -80 degrees C. The formation of C3(H2O) was determined using cation exchange chromatography and the hemolytic activity of the different C3 preparations was determined using a hemolytic assay for the classical pathway. We show that freezing native C3 in the precipitated form is the best method to avoid loss of function and generation of C3(H2O). By contrast, the most efficient way to consistently generate C3(H2O) was to incubate native C3 in a buffer at pH 11.0. We conclude that we have defined the optimal storage conditions for storing and maintaining the function of native C3 without generating C3(H2O) and also the conditions for consistently generating C3(H2O).

Place, publisher, year, edition, pages
Frontiers Media S.A., 2022
Keywords
C3, C3(H2O), thioester, storage, freezing
National Category
Immunology
Research subject
Biomedical Sciences, Immunology
Identifiers
urn:nbn:se:lnu:diva-114110 (URN)10.3389/fimmu.2022.891994 (DOI)000797921100001 ()35592325 (PubMedID)2-s2.0-85130368668 (Scopus ID)
Available from: 2022-06-16 Created: 2022-06-16 Last updated: 2024-01-17Bibliographically approved
Projects
MECHANISTIC STUDIES OF THE INTRAVASCULAR INNATE IMMUNE SYSTEM (IIIS) IN COVID19 AND ARDS OF OTHER ORIGIN [2021-02252_VR]; Uppsala University
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-7888-1571

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