Damage to the valves caused by decellularization technique is much less than the damage caused by the recipient's immune response. In vitro removal of viable cells in (cryopreserved) homografts may decrease graft failure. Seeding with autologous or major histocompatibility complex-matched donor endothelial cells will be necessary to diminish damage induced by an absent blood-tissue barrier.
BackgroundAnti-Citrullinated Protein Antibodies (ACPAs) are specific for Rheumatoid Arthritis (RA) and have been implicated in disease pathogenesis. The fragment antigen-binding domain of ACPA was recently shown to be extensively glycosylated. It is known that glycans play a key role in controlling innate and adaptive immunity, however to date there is limited understanding on the mode of action of glycans in RA. We hypothesise that the glycans on ACPA interact with glycan binding receptors and thus modulate immune responses in RA. Therefore, our aim is to elucidate the glycan effect of ACPA and other glycans on immune cells of RA patients to increase our understanding of RA pathogenesis.Materials and methodsA whole blood flow assay is used to study glycan interactions with leukocytes. Leukocytes were isolated from blood using Ficoll density centrifugation and lysis of erythrocytes. Cells were incubated for 4 hours with 4 µg/ml glycan at 4°C. Used biotinylated glycans include: sialic acid, Lewis-x, mannose, lactosamine, galactosamine and as a negative control glucitol. Glycan binding and identification of immune cell subsets was assessed with flow cytometry using a whole blood flow antibody panel.ResultsA whole blood flow assay of four healthy donors showed consistent high binding of mannose to B-cells. Interestingly, no binding of mannose was observed to other immune cells indicating that mannose binds specifically to B cells. At this concentration there was no binding of other glycans to leukocytes. When there is a difference in glycan binding between healthy and RA patients CyTOF3-Helios mass cytometer will be used to have a more in-depth analyse of the interacting leukocyte cell subsets.ConclusionThis study examines the glycan-binding capacity of leukocytes in healthy donors and RA patients via the whole blood flow assay. Our preliminary data indicates specific binding of mannose to B cells. This is an important finding because B cells play a key role in the pathogenesis of RA, as they produce ACPA and are very efficient in antigen presentation. Further studies on glycan binding to other key immune cells in RA may aid in elucidating their role in the pathogenesis of RA.
Career situation of first and presenting authorStudent for a master or a PhDIntroductionIn rheumatoid arthritis (RA) activated neutrophils produce neutrophil extracellular traps (NETs), which provide a source of autoantigens that drives the autoimmune process.1 To identify novel immune processes that dampened neutrophil activity, we investigated a family of inhibitory glycan-binding receptors (Siglecs) that bind a specific type of glycan; called sialic acids.2 We hypothesize that sialic acid-mediated triggering of siglecs on neutrophils, which express siglec-5, –9 and −14, will reduce their activation. In this study we focused on dampening the activity of neutrophils and thereby NET formation.MethodsPolymorphonuclear cells (PMNs) were isolated from healthy donors. Neutrophil binding of sialic acid-containing glycoconjugates was assessed by flow cytometry. Neutralizing antibodies for siglec-5/14 and −9 were used to block the interaction with the sialic acid glycoconjugates. For functional assays a branched synthetic molecule containing sialic acids (sialic acid dendrimer) was used. PMNs were rested for 1 hour at 37°C followed by stimulation of sialic acids dendrimers for 30 min. Subsequently, IgA coated beads were added for 30 min to activate the neutrophils. NETosis was quantified via Sytoxgreen and visualised via microscopy, and phagocytosis was measured by flow cytometry.ResultsBinding of sialic acid glycoconjugates was observed on neutrophils. Neutralizing siglec-5/14 and −9 receptor almost completely abolished sialic acid glycoconjugate binding to neutrophils. Neutrophils activated with IgA beads released NETs, as confirmed via microscopy. Triggering neutrophils with sialic acid dendrimer reduced this process of NETosis. The capacity to engulf IgA beads was not affected by sialic acid dendrimer stimulation.ConclusionsNeutrophils stimulated with sialic acid dendrimers show reduced activation. Patients with RA might benefit from treatment with sialic acid to dampen neutrophil-mediated autoimmune response.ReferencesWright HL, Moots RJ, Edwards SW. The multifactorial role of neutrophils in rheumatoid arthritis. Nat Rev Rheumatol 2014;10(10):593–601.Macauley MS, Crocker PR, Paulson JC. Siglec-mediated regulation of immune cell function in disease. Nat Rev Immunol 2014;14(10):653–66.Disclosure of InterestNone declared.
Career situation of first and presenting authorStudent for a master or a PhD.IntroductionIn rheumatoid arthritis the imbalance between osteoblasts and osteoclasts leads to an increased bone resorption, resulting in extensive bone destruction. Osteoclasts are multinucleated cells formed after fusion of macrophages. Macrophages are known to acquire an anti-inflammatory phenotype upon engagement of the carbohydrate sialic acid1, however whether triggering by sialic acids affects osteoclastogenesis is unknown. Therefore, we investigated the effect of sialic acids on osteoclastogenesis and their expression of the sialic acid binding receptors, called Siglecs.MethodsHuman monocytes were plated on either plastic, glass or bone slices and stimulated with M-CSF and RANK-L to generate osteoclasts. We designed a novel sorting methodology to separate the osteoclasts and the residual macrophages in our osteoclast-derived cultures. Purified osteoclasts were reanalyzed by microscopy and assessed for gene expression of osteoclast-specific genes by RT-PCR. Flow cytometry was used to obtain a full expression profile of the different siglec receptors in the osteoclasts, macrophages and monocytes precursor cells. Stimulation of pre-osteoclasts with sialic acids was done at day 7 and 10 of the culture. Supernatants were assessed for IL-6 and IL-10 production.ResultsOur novel osteoclast purification method resulted in pure osteoclasts expressing the classical genetic osteoclast markers such as cathepsin K and TRAP, while being negative for CD14. During the transition to osteoclasts, expression of siglec-1, −5/14 and −7 was lost, while siglec-6, –9 and −15 expression was maintained or gained. Osteoclasts are known for their effectively bone resorption, however after adding sialic acids to the pre-osteoclasts, both osteoclast formation and bone resorption were reduced, while an increase of IL-6 and IL-10 cytokines was observed.ConclusionsPre-osteoclasts stimulated with sialic acids display reduced osteoclast formation and bone resorption. Based on the siglec profiling we hypothesize that sialic acids engage siglec-9 on osteoclasts. Macrophages stimulated via siglec-9 produce IL-10 cytokine,1 similarly as we observed in our stimulation experiments. Currently, we are addressing the role of siglec-9 in osteoclastogenesis.ReferenceAndo M, et al. Siglec-9 enhances IL-10 production in macrophages via tyrosine-based motifs. Biochem Biophys Res Commun 2008;369(3):878–83.Disclosure of InterestNone declared.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.