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1.
Xenotransplantation ; 30(5): e12820, 2023.
Article in English | MEDLINE | ID: mdl-37735958

ABSTRACT

Xenotransplantation is a promising approach to reduce organ shortage, while genetic modification of donor pigs has significantly decreased the immunogenic burden of xenotransplants, organ rejection is still a hurdle. Genetically modified pig organs are used in xenotransplantation research, and the first clinical pig-to-human heart transplantation was performed in 2022. However, the impact of genetic modification has not been investigated on a cellular level yet. Endothelial cells (EC) and their sugar-rich surface known as the glycocalyx are the first barrier encountering the recipient's immune system, making them a target for rejection. We have previously shown that wild type venous but not arterial EC were protected against heparan sulfate (HS) shedding after activation with human serum or human tumor necrosis factor alpha (TNF𝛼). Using a 2D microfluidic system we investigated the glycocalyx dynamics of genetically modified porcine arterial and venous EC (Gal𝛼1,3 Gal knock-out, transgenic for human CD46 and thrombomodulin, GTKO/hCD46/hTM) after activation with human serum or human TNF𝛼. Interestingly, we observed that GTKO/hCD46/hTM arterial cells, additionally to venous cells, do not shed HS. Unscathed HS on GTKO/hCD46/hTM EC correlated with reduced complement deposition, suggesting that protection against complement activation contributes to maintaining an intact glycocalyx layer on arterial EC. This protection was lost on GTKO/hCD46/hTM cells after simultaneous perfusion with human serum and human TNF𝛼. HS shedding on arterial cells and increased complement deposition on both arterial and venous cells was observed. These findings suggest that GTKO/hCD46/hTM EC revert to a proinflammatory phenotype in an inflammatory xenotransplantation setting, potentially favoring transplant rejection.


Subject(s)
Endothelial Cells , Glycocalyx , Animals , Humans , Swine , Transplantation, Heterologous , Animals, Genetically Modified , Complement System Proteins
2.
Sci Rep ; 13(1): 4483, 2023 03 18.
Article in English | MEDLINE | ID: mdl-36934164

ABSTRACT

Endothelial dysfunction is an early event of vascular injury defined by a proinflammatory and procoagulant endothelial cell (EC) phenotype. Although endothelial glycocalyx disruption is associated with vascular damage, how various inflammatory stimuli affect the glycocalyx and whether arterial and venous cells respond differently is unknown. Using a 3D round-channel microfluidic system we investigated the endothelial glycocalyx, particularly heparan sulfate (HS), on porcine arterial and venous ECs. Heparan sulfate (HS)/glycocalyx expression was observed already under static conditions on venous ECs while it was flow-dependent on arterial cells. Furthermore, analysis of HS/glycocalyx response after stimulation with inflammatory cues revealed that venous, but not arterial ECs, are resistant to HS shedding. This finding was observed also on isolated porcine vessels. Persistence of HS on venous ECs prevented complement deposition and clot formation after stimulation with tumor necrosis factor α or lipopolysaccharide, whereas after xenogeneic activation no glycocalyx-mediated protection was observed. Contrarily, HS shedding on arterial cells, even without an inflammatory insult, was sufficient to induce a proinflammatory and procoagulant phenotype. Our data indicate that the dimorphic response of arterial and venous ECs is partially due to distinct HS/glycocalyx dynamics suggesting that arterial and venous thrombo-inflammatory disorders require targeted therapies.


Subject(s)
Endothelial Cells , Heparitin Sulfate , Animals , Swine , Endothelial Cells/metabolism , Heparitin Sulfate/metabolism , Glycocalyx/metabolism , Arteries/metabolism , Veins/metabolism
3.
Blood ; 134(22): 1941-1950, 2019 11 28.
Article in English | MEDLINE | ID: mdl-31537530

ABSTRACT

Immune system failure in primary antibody deficiencies (PADs) has been linked to recurrent infections, autoimmunity, and cancer, yet clinical judgment is often based on the reactivity to a restricted panel of antigens. Previously, we demonstrated that the human repertoire of carbohydrate-specific immunoglobulin G (IgG) exhibits modular organization related to glycan epitope structure. The current study compares the glycan-specific IgG repertoires between different PAD entities. Distinct repertoire profiles with extensive qualitative glycan-recognition defects were observed, which are characterized by the common loss of Galα and GalNAc reactivity and disease-specific recognition of microbial antigens, self-antigens, and tumor-associated carbohydrate antigens. Antibody repertoire analysis may provide a useful tool to elucidate the degree and the clinical implications of immune system failure in individual patients.


Subject(s)
Autoantigens/immunology , Carbohydrates/immunology , Epitopes/immunology , Immunoglobulin G/immunology , Primary Immunodeficiency Diseases/immunology , Female , Humans , Male
4.
PLoS One ; 14(4): e0214674, 2019.
Article in English | MEDLINE | ID: mdl-30943234

ABSTRACT

PURPOSE: Various profibrotic and proinflammatory cytokines have been found upregulated in uncomplicated primary retinal detachment (pRD), but without providing a uniform picture. Here, we compare the cyto- and chemokine profiles in pRD with and without proliferative vitreoretinopathy (PVR) in an attempt to unravel relevant differences not in single cytokines, but in the cytokine profiles at diagnosis. METHODS: Undiluted vitreous fluid (VF) was obtained at the beginning of surgery from 174 eyes with pRD without relevant PVR (maximally grade B; group 1; n = 81) and with moderate or advanced PVR requiring a gas tamponade (group 2; n = 49) or silicon oil filling (group 3; n = 44). VF of eyes undergoing macular hole (MH) surgery served as controls (group 4; n = 26). Forty-three cytokines were quantified in parallel using a multiplex cytokine analysis system (Bioplex). For all comparisons we applied Holm's correction to control for multiple comparisons. RESULTS: 44.9% of group 2 eyes presented grade C1 and 55.1% C2-C3, whereas 86.4% of group 3 eyes exhibited a PVR grade of C2-D. CCL19 was the only cytokine that displayed higher concentrations in the vitreous of eyes with PVR C1 compared to lower PVR grades. Eyes with PVR C2-D showed higher levels of CCL27, CXCL6, IL4, IL16, CXCL10, CCL8, CCL22, MIG/CXCL9, CCL15, CCL19, CCL 23 and CXCL12 compared to controls. Interestingly, no difference of cytokine levels was detected between C1 and C2-D PVR. CONCLUSIONS: CCL19 may represent a potential biomarker for early PVR progression that holds promise for future diagnostic and therapeutic applications.


Subject(s)
Cytokines/metabolism , Retinal Detachment/metabolism , Vitreoretinopathy, Proliferative/metabolism , Aged , Biomarkers/metabolism , Female , Humans , Male , Middle Aged , Retinal Detachment/complications , Vitreoretinopathy, Proliferative/complications , Vitreoretinopathy, Proliferative/diagnosis
5.
Sci Rep ; 8(1): 5898, 2018 04 12.
Article in English | MEDLINE | ID: mdl-29651108

ABSTRACT

In the context of xenotransplantation, in ischemia/reperfusion injury as well as in cardiovascular research, the study of the fascinating interplay between endothelial cells (EC) and the plasma cascade systems often requires in vitro models. Blood vessels are hardly reproducible with standard flat-bed culture systems and flow-plate assays are limited in their low surface-to-volume ratio which impedes the study of the anticoagulant properties of the endothelial cells. According to the 3R regulations (reduce, replace and refine animal experimentation) we developed a closed circuit microfluidic in vitro system in which endothelial cells are cultured in 3D round section microchannels and subjected to physiological, pulsatile flow. In this study, a 3D monolayer of porcine aortic EC was perfused with human serum to mimic a xenotransplantation setting. Complement as well as EC activation was assessed in the presence or absence of complement inhibitors showing the versatility of the model for drug testing. Complement activation products as well as E-selectin expression were detected and visualized in situ by high resolution confocal microscopy. Furthermore, porcine pro-inflammatory cytokines as well as soluble complement components in the recirculating fluid phase were detected after human serum perfusion providing a better overview of the artificial vascular environment.


Subject(s)
Cell Culture Techniques , Complement System Proteins/genetics , Endothelial Cells/immunology , Lab-On-A-Chip Devices , Animals , Aorta/immunology , Aorta/ultrastructure , Complement Activation/drug effects , Complement Inactivating Agents/pharmacology , Complement System Proteins/immunology , Cytokines/genetics , Cytokines/immunology , Dextran Sulfate/pharmacology , E-Selectin/genetics , E-Selectin/immunology , Endothelial Cells/drug effects , Endothelial Cells/ultrastructure , Gene Expression , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/immunology , Microscopy, Confocal , Models, Biological , Pulsatile Flow , Rheology , Swine , Transplantation, Heterologous
6.
J Vis Exp ; (133)2018 03 12.
Article in English | MEDLINE | ID: mdl-29578502

ABSTRACT

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. °°°This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.


Subject(s)
Blood Specimen Collection/methods , Myocardial Infarction/blood , Myocardial Infarction/pathology , Myocardial Reperfusion Injury/blood , Myocardial Reperfusion Injury/pathology , Specimen Handling/methods , Animals , Disease Models, Animal , Myocardial Infarction/diagnosis , Myocardial Reperfusion Injury/diagnosis , Swine
7.
Invest Ophthalmol Vis Sci ; 57(14): 6320-6326, 2016 11 01.
Article in English | MEDLINE | ID: mdl-27893098

ABSTRACT

Purpose: Cytokines play an important role in cell signaling in inflammatory and repair processes, also within the posterior segment of the eye. These molecules are thus implicated in the pathophysiology of several vitreoretinal diseases. In the present study, we compared vitreal cytokine profiles in patients with idiopathic epiretinal membranes (ERMs) and idiopathic full-thickness macular holes (MHs) without epiretinal membranes. Methods: Native vitreal humor was collected during elective pars plana vitrectomy for the treatment of macular pathologies (group 1: ERM; group 2: MH) from patients without any other ocular or systemic disease. The concentrations of 43 chemokines and cytokines were measured in parallel by multiplex beads analysis. Intergroup comparisons were conducted using the Mann-Whitney U test and Bonferroni's correction, at a level of significance of P < 0.0012. Results: Vitreal samples from 31 patients with ERMs (group 1) and from 30 with MHs (group 2) were analyzed. For 12 of the tested cytokines (GM-CSF, MCP-1, MIF, CCL15, CCL20, CCL17, CX3CL1, CXCL10, CXCL16, and TGF-ß-1, -2, and -3), no intergroup differences were revealed; for the other 31, the concentrations were higher in the ERM than in the MH group (P < 0.0012 in each case). Conclusions: The vitreal levels of 72% of the tested cytokines were higher in ERM than in MH. This indicates that even in the absence of clinical markers, activation of inflammatory and profibrotic mechanisms is implicated in the progression of ERMs. Although frequently used as such in the past, eyes with ERMs should be considered with caution as a healthy control group.


Subject(s)
Cytokines/metabolism , Epiretinal Membrane/metabolism , Retinal Perforations/metabolism , Vitreous Body/metabolism , Aged , Biomarkers/metabolism , Epiretinal Membrane/diagnosis , Epiretinal Membrane/surgery , Female , Follow-Up Studies , Humans , Male , Prospective Studies , Retinal Perforations/diagnosis , Tomography, Optical Coherence , Visual Acuity , Vitrectomy , Vitreous Body/diagnostic imaging
8.
J Cell Physiol ; 229(8): 1106-17, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24402969

ABSTRACT

Many studies in the field of cell-based cartilage repair have focused on identifying markers associated with the differentiation status of human articular chondrocytes (HAC) that could predict their chondrogenic potency. A previous study from our group showed a correlation between the expression of S100 protein in HAC and their chondrogenic potential. The aims of the current study were to clarify which S100 proteins are associated with HAC differentiation status and to provide an S100-based assay for measuring HAC chondrogenic potential. The expression patterns of S100A1 and S100B were investigated in cartilage and in HAC cultured under conditions promoting dedifferentiation (monolayer culture) or redifferentiation (pellet culture or BMP4 treatment in monolayer culture), using characterized antibodies specifically recognizing S100A1 and S100B, by immunohistochemistry, immunocytochemistry, Western blot, and gene expression analysis. S100A1 and S100B were expressed homogeneously in all cartilage zones, and decreased during dedifferentiation. S100A1, but not S100B, was re-expressed in pellets and co-localized with collagen II. Gene expression analysis revealed concomitant modulation of S100A1, S100B, collagen type II, and aggrecan: down-regulation during monolayer culture and up-regulation upon BMP4 treatment. These results strongly support an association of S100A1, and to a lesser extent S100B, with the HAC differentiated phenotype. To facilitate their potential application, we established an S100A1/B-based flow cytometry assay for accurate assessment of HAC differentiation status. We propose S100A1 and S100B expression as a marker to develop potency assays for cartilage regeneration cell therapies, and as a redifferentiation readout in monolayer cultures aiming to investigate stimuli for chondrogenic induction.


Subject(s)
Chondrocytes/cytology , Chondrocytes/metabolism , Gene Expression Regulation/physiology , S100 Calcium Binding Protein beta Subunit/metabolism , S100 Proteins/metabolism , Antibodies , Cell Differentiation , Cells, Cultured , Humans , S100 Calcium Binding Protein beta Subunit/genetics , S100 Proteins/genetics
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