Multiplex suspension array for human anti-carbohydrate antibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.

Evaluation of multimeric tyrosine-O-sulfate as a cytoprotectant in an in vivo model of acute myocardial infarction in pigs

Intracoronary administration of glycosaminoglycan analogs, including the complement inhibitor dextran sulfate, attenuates myocardial ischemia/reperfusion injury (I/R injury). However, dextran sulfate has a distinct anticoagulatory effect, possibly limiting its use in specific situations in vivo. We therefore developed multimeric tyrosine sulfate (sTyr-PAA), a novel, minimally anticoagulatory, fully synthetic non-carbohydrate-containing polyacrylamide conjugate, for in vivo testing in an acute closed-chest porcine model of acute myocardial infarction.

Normal human serum contains high levels of anti-Gal alpha 1-4GlcNAc antibodies

BACKGROUND: Natural xenoreactive antibodies (Abs) directed against the Bdi-epitope (Gal alpha 1-3Gal beta) on the cells of non-primate mammals take part in hyperacute rejection of xenotransplanted organs. We found that some Abs, which were one-step affinity purified on Bdi-Sepharose, cross-reacted with the disaccharide Gal alpha 1-4GlcNAc beta. The epitope Gal alpha 1-4GlcNAc has not been identified on mammals or bacterial polysaccharides yet. METHODS: To isolate the antibodies of the corresponding specificity the disaccharide was immobilized on Sepharose and antibodies were affinity purified from pooled serum of blood group O individuals. RESULTS: These one-step purified Abs cross-reacted with Bdi, but after a prior absorption step on Bdi-Sepharose no cross-reactivity with Bdi was observed any longer. Surprisingly, the quantity of anti-Gal alpha 1-4GlcNAc isolated from the same serum pool, 4-7 microg/ml, was equal to that of anti-Bdi or more. Independently of ABO blood groups all the tested healthy donors had anti-Gal alpha 1-4GlcNAc Abs at a similar level. Monospecific anti-Gal alpha 1-4GlcNAc Abs were not cytotoxic towards porcine cells. CONCLUSIONS: 1. The actual concentration of monospecific, xenoreactive Gal alpha 1-3Gal beta Abs in blood may be considerably lower than the value referred to in the literature for 'anti-alpha Gal' or 'anti-Galili' antibodies. 2. Anti-Gal alpha 1-4GlcNAc Abs seem not to be important for xenotransplantation.

Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood

BACKGROUND: Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting. METHODS: Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads. RESULTS: Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS. CONCLUSIONS: This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ''endothelial protective'' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.

Locally targeted cytoprotection with dextran sulfate attenuates experimental porcine myocardial ischaemia/reperfusion injury

AIMS: Intravascular inflammatory events during ischaemia/reperfusion injury following coronary angioplasty alter and denudate the endothelium of its natural anticoagulant heparan sulfate proteoglycan (HSPG) layer, contributing to myocardial tissue damage. We propose that locally targeted cytoprotection of ischaemic myocardium with the glycosaminoglycan analogue dextran sulfate (DXS, MW 5000) may protect damaged tissue from reperfusion injury by functional restoration of HSPG. METHODS AND RESULTS: In a closed chest porcine model of acute myocardial ischaemia/reperfusion injury (60 min ischaemia, 120 min reperfusion), DXS was administered intracoronarily into the area at risk 5 min prior to reperfusion. Despite similar areas at risk in both groups (39+/-8% and 42+/-9% of left ventricular mass), DXS significantly decreased myocardial infarct size from 61+/-12% of the area at risk for vehicle controls to 39+/-14%. Cardioprotection correlated with reduced cardiac enzyme release creatine kinase (CK-MB, troponin-I). DXS abrogated myocardial complement deposition and substantially decreased vascular expression of pro-coagulant tissue factor in ischaemic myocardium. DXS binding, detected using fluorescein-labelled agent, localized to ischaemically damaged blood vessels/myocardium and correlated with reduced vascular staining of HSPG. CONCLUSION: The significant cardioprotection obtained through targeted cytoprotection of ischaemic tissue prior to reperfusion in this model of acute myocardial infarction suggests a possible role for the local modulation of vascular inflammation by glycosaminoglycan analogues as a novel therapy to reduce reperfusion injury.

Multimeric tyrosine sulfate acts as an endothelial cell protectant and prevents complement activation in xenotransplantation models

BACKGROUND: Activation of endothelial cells (EC) in xenotransplantation is mostly induced through binding of antibodies (Ab) and activation of the complement system. Activated EC lose their heparan sulfate proteoglycan (HSPG) layer and exhibit a procoagulant and pro-inflammatory cell surface. We have recently shown that the semi-synthetic proteoglycan analog dextran sulfate (DXS, MW 5000) blocks activation of the complement cascade and acts as an EC-protectant both in vitro and in vivo. However, DXS is a strong anticoagulant and systemic use of this substance in a clinical setting might therefore be compromised. It was the aim of this study to investigate a novel, fully synthetic EC-protectant with reduced inhibition of the coagulation system. METHOD: By screening with standard complement (CH50) and coagulation assays (activated partial thromboplastin time, aPTT), a conjugate of tyrosine sulfate to a polymer-backbone (sTyr-PAA) was identified as a candidate EC-protectant. The pathway-specificity of complement inhibition by sTyr-PAA was tested in hemolytic assays. To further characterize the substance, the effects of sTyr-PAA and DXS on complement deposition on pig cells were compared by flow cytometry and cytotoxicity assays. Using fluorescein-labeled sTyr-PAA (sTyr-PAA-Fluo), the binding of sTyr-PAA to cell surfaces was also investigated. RESULTS: Of all tested compounds, sTyr-PAA was the most effective substance in inhibiting all three pathways of complement activation. Its capacity to inhibit the coagulation cascade was significantly reduced as compared with DXS. sTyr-PAA also dose-dependently inhibited deposition of human complement on pig cells and this inhibition correlated with the binding of sTyr-PAA to the cells. Moreover, we were able to demonstrate that sTyr-PAA binds preferentially and dose-dependently to damaged EC. CONCLUSIONS: We could show that sTyr-PAA acts as an EC-protectant by binding to the cells and protecting them from complement-mediated damage. It has less effect on the coagulation system than DXS and may therefore have potential for in vivo application.

Endothelial cell protection by dextran sulfate: a novel strategy to prevent acute vascular rejection in xenotransplantation

We showed recently that low molecular weight dextran sulfate (DXS) acts as an endothelial cell (EC) protectant and prevents human complement- and NK cell-mediated cytotoxicity towards porcine cells in vitro. We therefore hypothesized that DXS, combined with cyclosporine A (CyA), could prevent acute vascular rejection (AVR) in the hamster-to-rat cardiac xenotransplantation model. Untreated, CyA-only, and DXS-only treated rats rejected their grafts within 4-5 days. Of the hearts grafted into rats receiving DXS in combination with CyA, 28% survived more than 30 days. Deposition of anti-hamster antibodies and complement was detected in long-term surviving grafts. Combined with the expression of hemoxygenase 1 (HO-1) on graft EC, these results indicate that accommodation had occurred. Complement activity was normal in rat sera after DXS injection, and while systemic inhibition of the coagulation cascade was observed 1 h after DXS injection, it was absent after 24 h. Moreover, using a fluorescein-labeled DXS (DXS-Fluo) injected 1 day after surgery, we observed a specific binding of DXS-Fluo to the xenograft endothelium. In conclusion, we show here that DXS + CyA induces long-term xenograft survival and we provide evidence that DXS might act as a local EC protectant also in vivo.

Design of the blood group AB glycotope

Although the nature of the blood groups A and B has been comprehensively studied for a long time, it is still unclear as to what exactly is the epitope that is recognized by antibodies having AB specificity, i.e. monoclonal and polyclonal antibodies which are capable of interacting equally well with the antigens GalNAcalpha 1-3(Fucalpha 1-2)Gal (A trisaccharide) and Galalpha 1-3(Fucalpha 1-2)Gal (B trisaccharide), but do not react with their common fragment Fucalpha 1-2Gal. We have supposed that besides Fucalpha 1-2Gal, A and B antigens have one more shared epitope. The trisaccharides A and B are practically identical from the conformational point of view, the only difference being situated at position 2 of Galalpha residue, i.e. trisaccharide A has a NHAc group, whereas trisaccharide B has a hydroxyl group (see formulas). We have hypothesized that the AB-epitope should be situated in the part of the molecule that is opposite to the NHAc group of GalNAc residue. In order to test this hypothesis we have synthesized a polymeric conjugate in such a way that de-N-acetylated A-trisaccharide is attached to a polymer via the nitrogen in position C-2 of the galactosamine residue. In this conjugate the supposed AB-epitope should be maximally accessible for antibodies from the solution, whereas the discrimination site of antigens A and B by the antibodies should be maximally hidden due to the close proximity of the polymer. Interaction with several anti-AB monoclonal antibodies revealed that a part of them really interacted with the synthetic AB-glycotope, thus confirming our hypothesis. Moreover, similar antibodies were revealed in the blood of healthy blood group 0 donors. Analysis of spatial models was performed in addition to identify the hydroxyl groups of Fuc, Galalpha, and Galbeta residues, which are particularly involved in the composition of the AB-glycotope.