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The recruitment of leukocytes from blood is one of the most important cellular processes in response to tissue damage and inflammation. This multi-step process includes rolling leukocytes and their adhesion to endothelial cells (EC), culminating in crossing the EC barrier to reach the inflamed tissue. Galectin-8 and galectin-9 expressed on the immune system cells are part of this process and can induce cell adhesion via binding to oligolactosamine glycans. Similarly, these galectins have an order of magnitude higher affinity towards glycans of the ABH blood group system, widely represented on ECs. However, the roles of gal-8 and gal-9 as mediators of adhesion to endothelial ABH antigens are practically unknown. In this work, we investigated whether H antigen-gal-9-mediated adhesion occurred between Jurkat cells (of lymphocytic origin and known to have gal-9) and EA.hy 926 cells (immortalized endothelial cells and known to have blood group H antigen). Baseline experiments showed that Jurkat cells adhered to EA.hy 926 cells; however when these EA.hy 926 cells were defucosylated (despite the unmasking of lactosamine chains), adherence was abolished. Restoration of fucosylation by insertion of synthetic glycolipids in the form of H (type 2) trisaccharide Fucα1-2Galβ1-4GlcNAc restored adhesion. The degree of lymphocyte adhesion to native and the "H-restored" (glycolipid-loaded) EA.hy 926 cells was comparable. If this gal-9/H (type 2) interaction is similar to processes that occur in vivo, this suggests that only the short (trisaccharide) H glycan on ECs is required.

Modification of the cell surface with synthetic glycolipids opens up a wide range of possibilities for studying the function of glycolipids. Synthetic glycolipids called Function-Spacer-Lipids (FSL; where F is a glycan or label, S is a spacer, and L is dioleoylphosphatidyl ethanolamine) easily and controllably modify the membrane of a living cells. This current study investigates the dynamics and mechanism of the FSL insertion and release/loss. FSL insert into the cell membrane (~1 million molecules per cell) within tens of minutes, almost regardless of the nature of the cells (including the thickness of their glycocalyx) and the size of the FSL glycan. FSLs do not accumulate uniformly, but instead form patches >300 nm in size either entrapped in the glycocalyx, or integrated in the plane of the plasma membrane, but always outside the cell rafts. The natural release (loss) of FSL from the modified cell was two orders of magnitude slower than attachment/insertion and occurred mainly in the form of released microvesicles with a size of 140 ± 5 nm. The accumulation of FSL as patches in the cell membrane is similar to the coalescence of natural glycosphingolipids and supports (along with their long residence time in the membrane) the use of FSL as probes for the study of glycosphingolipid-protein interactions., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Agglutination of red blood cells (RBCs) remains the only practical method for routine use for ABH typing in clinical practice. However, exact mechanistic details of agglutination are not yet thoroughly studied. In this research, RBCs of blood group O were converted to blood group A through two approaches: by chemical ligation of the cells' glycocalyx with synthetic blood group A tetrasaccharide, and by insertion of synthetic glycolipid carrying the same A antigen into the cells' membranes. The O→A ligated RBCs and natural A RBCs showed comparable agglutination characteristics with antibodies. As expected, RBCs with inserted glycolipid showed lower agglutination scores. This approach could help cell biologists in site-specific and cell-friendly modification of glycocalyx by other ligands., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Gram scale synthesis of A (type 2) and B (type 2) tetrasaccharides in the form of 3-aminopropyl glycosides is proposed starting from 3-O-benzoyl-1,6-anhydro-N-acetylglucosamine. Its galactosylation followed by re-protection gave lactosamine derivative with single free 2'-OH group in total yield 75%. Standard fucosylation and next run of re-protection in total yield 88% gave a trisaccharide Fuc-Gal-anhydroGlcNAc with single free 3'-OH group. Its standard α-galactosylation gave protected B (type 2) tetrasaccharide. For synthesis of correspondent A tetrasaccharide seven different 2-azido-2-deoxygalactosyl (GalN 3 ) donors were tested: 6-O-acetyl-3,4-O-isopropylidene-GalN 3 thioglycoside was shown to provide the best yield (89%) and stereoselectivity (α/β = 24:1). Further 1,6-anhydro cycle opening, spacer-arming and complete deprotection resulted in the target 3-aminopropyl glycosides of A (type 2) and B (type 2) tetrasaccharides, yields 87 and 85% correspondingly., (Copyright © 2017. Published by Elsevier Ltd.)

Seven lipophilic constructs containing Lewis (Le a , Le b , Le y ) or chimeric Lewis/ABH (ALe b , BLe b , ALe y , BLe y ) glycans were obtained starting from corresponding oligosaccharides in form of 3-aminopropyl glycosides. ALe b and BLe b pentasaccharides were synthesized via [3 + 1] blockwise approach. The constructs (neoglycolipids, or FSLs) were inserted in erythrocyte membrane, and obtained "kodecytes" were used to map the immunochemical specificity of historical and contemporary monoclonal and polyclonal blood group system Lewis reagents., (Copyright © 2016. Published by Elsevier Ltd.)

Herein we report the synthesis of 3-aminopropyl glycosides of A (type 2) and B (type 2) tetrasaccharides via [3 + 1] block scheme. Peracetylated trichloroacetimidates of A and B trisaccharides were used as glycosyl donors. The well-known low reactivity of 4-OH group of N-acetyl-d-glucosamine forced us to test four glucosamine derivatives (3-Bz-1,6-anhydro-GlcNAc and 3-trifluoroacetamidopropyl β-glycosides of 3-Ac-6-Bn-GlcNAc, 3-Ac-6-Bn-GlcN3, and 3-Ac-6-Bn-GlcNAc2) to select the best glycosyl acceptor for the synthesis of type 2 tetrasaccharides. The desired tetrasacchrides were not isolated, when 3-trifluoroacetamidopropyl glycosyde of 3-Ac-6-Bn-GlcNAcβ was glycosylated. Glycosylation of 3-Bz-1,6-anhydro-GlcNAc derivative resulted in α-glycoside as a major product. High stereospecificity was achieved only in the synthesis of B (type 2) tetrasaccharide, when 3-trifluoroacetamidopropyl 3-Ac-6-Bn-GlcNAc2β was applied as the glycosyl acceptor (β/α 5:1), whereas glycosylation with trichloroacetimidate of A trisaccharide was not stereospecific (β/α 1.3:1). Glycosylation of 3-trifluoroacetamidopropyl glycoside of 3-Ac-6-Bn-GlcN3β with trichloroacetimidates of A and B trisaccharides provided the same stereochemical yield (β/α 1.5:1)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Function-spacer-lipid (FSL) constructs are amphiphilic molecules that are able to disperse in water and then self-assemble into cell membranes or onto solid surfaces. Modification of a biological or non-biological surface is very easy and achieved by simple contact of the surface with an appropriately buffered solution containing one or more FSLs. When the functional head group of the FSL is a glycan, glycan modified surfaces can be rapidly formed. Once cells, viruses, or solid surfaces are FSL modified with either simple or complex glycans, they can be used in vitro and/or in vivo to measure interactions with cells, viruses, antibodies, and lectins. FSLs have already been used in a variety of techniques including antibody specificity mapping, antibody/toxin neutralization, diagnostic assays, immune system manipulation, and animal modeling of transfusion reactions. FSLs offer the easiest and fastest method available to achieve a glycan-modified surface.

A major aspect of carbohydrate-dependent galectin functionality is their cross-linking capacity. Using a cell surface as biorelevant platform for galectin binding and a panel of 40 glycans as sensor part of a fluorescent polyacrylamide neoglycopolymer for profiling galectin reactivity, properties of related proteins can be comparatively analyzed. The group of the chicken galectins (CGs) is an especially suited system toward this end due to its relatively small size, compared with mammalian galectins. The experiments reveal particularly strong reactivity toward N-acetyllactosamine repeats for all tested CGs and shared reactivity of CG-1A and CG-2 to histo-blood group ABH determinants. In cross-species comparison, CG-1B's properties closely resembled those of human galectin-1, as was the case for the galectin-2 (but not galectin-3) ortholog pair. Although binding-site architectures are rather similar, reactivity patterns can well differ., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Direct labeling of virus particles is a powerful tool for the visualization of virus-cell interaction events. However, this technique involves the chemical modification of viral proteins that affects viral biological properties. Here we describe an alternative approach of influenza virus labeling that utilizes Function-Spacer-Lipid (FSL) constructs that can be gently inserted into the virus membrane. We assessed whether labeling with fluorescent (fluo-Ad-DOPE) or biotin-labeled (biot-CMG2-DOPE) probes has any deleterious effect on influenza virus hemagglutinin (HA) receptor specificity, neuraminidase (NA) activity, or replicative ability in vitro. Our data clearly show that neither construct significantly affected influenza virus infectivity or viral affinity to sialyl receptors. Neither construct influenced the NA activities of the influenza viruses tested, except the A/Puerto Rico/8/34 (H1N1) strain. Our data indicate that lipid labeling provides a powerful tool to analyze influenza virus infection in vitro.

Blood group A tetrasaccharides of different types have the same terminal trisaccharide fragment that allows using a block scheme in their synthesis. 3-Aminopropyl glycosides of tetrasaccharides GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ (A type 1), GalNAcα1-3(Fucα1-2)Galβ1-3GalNAcα (A type 3), and GalNAcα1-3(Fucα1-2)Galβ1-3GalNAcβ (A type 4) were synthesised using acetylated Galα1-3(Fucα1-2)Gal trichloroacetimidate as a glycosyl donor at the key stage., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objectives: 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., Methods: Following balloon occlusion of the left anterior descending artery just after the first diagonal branch (60-minute ischemia), sTyr-PAA (approx. 10 mg/kg bodyweight, fraction with strongest complement-inhibitory and minimal anticoagulatory properties, n = 11) or phosphate-buffered saline (controls, n = 9) was administered intracoronarily into ischemic myocardium prior to 120 min of reperfusion., Results: sTyr-PAA significantly reduced infarct size (from 61.0 ± 12.0% of the ischemic area at risk to 39.4 ± 17.0%), plasma creatine kinase, local complement deposition and tissue factor upregulation, without affecting systemic coagulation. Protection was associated with significantly reduced myocardial neutrophil extravasation and translated into a significant improvement of ejection fraction and left ventricular enddiastolic pressure., Conclusions: sTyr-PAA protected significantly against myocardial I/R injury without substantially affecting systemic coagulation. Local intravascular sTyr-PAA administration may prove advantageous in situations where bleeding complications are likely or are to be avoided at all costs., (Copyright © 2012 S. Karger AG, Basel.)

The network of adhesion/growth-regulatory galectins in chicken (chicken galectin, CG) has only one tandem-repeat-type protein, CG8. Using a cell-based assay and probing galectin reactivity with a panel of fluorescent neoglycoconjugates (glycoprobes), its glycan-binding profile was determined. For internal validation, human galectin-8 (HG8) was tested. In comparison to HG8, CG8 showed a rather similar specificity: both galectins displayed high affinity to blood group ABH antigens as well as to 3'-sialylated and 3'-sulfated lactosamine chains. The most remarkable difference was found to be an ability of HG8 (but not CG8) to bind the disaccharide Galβ1-3GlcNAc (Le(c)) as well as branched and linear oligolactosamines. The glycan-binding profile was shown to be influenced by glycocalix of the cell, where the galectin is anchored. Particularly, glycosidase treatment of galectin-loaded cells led to the change of the profile. Thus, we suppose the involvement of cis-glycans in the interaction of cell-anchored galectins with external glycoconjugates.

Removal of Anti-A/B antibodies prior to ABO-incompatible transplantation can prevent hyperacute organ rejection. We are developing a specific antibody filter (SAF) device to selectively remove ABO blood group antibodies from the whole blood by utilizing immunoaffinity adsorption. The device consists of ultrafiltration hollow fiber membranes with synthetic antigens specific to bind blood group antibodies immobilized on the inner lumenal walls of the fibers. The aim of this study was to evaluate the effect of antigen molecular weight and surface activation process to increase the antibody binding capacity of the fiber membrane surface. A new higher molecular weight antigen Atri-pNSA-1000 compared with Atri-pNPA-30 (A-trisaccharide (Atri) conjugated to activated polymers of Mol. wt. 1000 kDa and 30 kDa, respectively) was employed to improve accessibility of the antigen to bind antibodies. Also, a cyanogen bromide (CNBr) based surface activation method mediated by TEA in neutral pH medium was used to enhance the number of active sites for antigen binding compared to a strong basic medium of NaOH. Using a CNBr/TEA activation method and by immobilizing Atri-pNSA-1000 antigen, an antibody binding capacity (∼0.01 monoclonal anti-A IgM nmol/cm(2)) was achieved on the fiber surface. This binding capacity was sufficient to reduce monoclonal antibody titer from 1:128 to final titer below 1:4 with a surface area to volume ratio that is similar to commercial dialysis device (∼1.1 m(2) surface area for an average body blood volume of 5 L)., (© 2010 Wiley Periodicals, Inc.)

Specific immunoadsorption of blood group antibodies by synthetic antigens immobilized on support matrices in the peri-transplantation period provides a promising solution to hyperacute rejection risk following ABO-incompatible transplantation. In this study, we investigated binding interactions between anti-A antibodies and synthetic blood group A trisaccharide conjugated with polyacrylamide of different molecular weights (30 and 1000 kDa). The glycopolymers were equipped with biotin tags and deposited on streptavidin-coated sensor chips. The affinity and kinetics of anti-A antibodies binding to glycoconjugates were studied using surface plasmon resonance (SPR). The high molecular weight conjugate (Atri-PAA(1000)-biotin) enhanced antibody binding capacity by two to three fold compared with the low molecular weight conjugate (Atri-PAA(30)-biotin), whereas varying the carbohydrate content in Atri-PAA(1000)-biotin (20 mol % or 50 mol %) did not affect antibody binding capacity of the glycoconjugate. The obtained results suggest that immunoadsorption devices, especially hollow fiber-based antibody filters which are limited in available surface area for antigen immobilization, may greatly benefit from the new synthetic high molecular weight polyacrylamide glycoconjugates.

Ischemia/reperfusion injury leads to activation of graft endothelial cells (EC), boosting antigraft immunity and impeding tolerance induction. We hypothesized that the complement inhibitor and EC-protectant dextran sulfate (DXS, MW 5000) facilitates long-term graft survival induced by non-depleting anti-CD4 mAb (RIB 5/2). Hearts from DA donor rats were heterotopically transplanted into Lewis recipients treated with RIB 5/2 (20 mg/kg, days-1,0,1,2,3; i.p.) with or without DXS (grafts perfused with 25 mg, recipients treated i.v. with 25 mg/kg on days 1,3 and 12.5 mg/kg on days 5,7,9,11,13,15). Cold graft ischemia time was 20 min or 12 h. Median survival time (MST) was comparable between RIB 5/2 and RIB 5/2+DXS-treated recipients in the 20-min group with >175-day graft survival. In the 12-h group RIB 5/2 only led to chronic rejection (MST = 49.5 days) with elevated alloantibody response, whereas RIB 5/2+DXS induced long-term survival (MST >100 days, p < 0.05) with upregulation of genes related to transplantation tolerance. Analysis of the 12-h group treated with RIB 5/2+DXS at 1-day posttransplantation revealed reduced EC activation, complement deposition and inflammatory cell infiltration. In summary, DXS attenuates I/R-induced acute graft injury and facilitates long-term survival in this clinically relevant transplant model.

Removal of blood group anti-A and anti-B antibodies can prevent hyperacute organ rejection in ABO-incompatible transplantation. We are developing an extracorporeal-specific antibody filter (SAF) as an immunoadsorption device for direct removal of ABO blood group antibodies from whole blood, without the need for plasma separation and plasma exchange. A hollow fiber-based small scale SAF (mini-SAF) device was fabricated and synthetic A antigen, Atrisaccharide (Atri) conjugated to activated polyacrylic acid, was immobilized on the fiber lumen surface. Monoclonal antibody anti-A IgM were specifically removed up to 70% of initial antibodies using mini-SAF device. The monoclonal anti-A capture experiments on mini-SAF indicated that antibody removal relative to the initial concentration is independent of inlet concentration in the beginning; however, as the surface starts saturating with bound antibodies, removal becomes dependent on inlet concentration. No significant effect of flow rate on removal rate was observed. The radial diffusion and axial convection-based mathematical model developed for unsteady state antibody removal was in good agreement with the experimental data and showed that the antibody removal rate can be maximized by increasing the antibody-binding capacity of the SAF., (Copyright 2007 Wiley Periodicals, Inc.)

Removal of blood group antibodies against the donor organ prior to ABO-incompatible transplantation can prevent episodes of hyperacute rejection. We are developing a specific antibody filter (SAF) device consisting of immobilized synthetic Atrisaccharide antigens conjugated to polyacrylamide (Atri-PAA) to selectively remove anti-A antibodies directly from whole blood. In this study, we evaluated eight anti-A IgM monoclonal antibodies (mAbs) using Enzyme-Linked Immunosorbent Assay (ELISA) to determine their specificity for binding to Atri-PAA. Five of the eight mAbs met our criteria for specificity by binding to Atri-PAA with at least five times greater affinity compared to the negative controls. These selected mAbs will be studied for their binding characteristics to Atri-PAA which will aid in the development of the SAF. The study of kinetics of antibody removal and quantification of antibody removal will be used in our mathematical model to maximize the antibody removal rate and binding capacity of the SAF.

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.

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.

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.

The rare NOR erythrocytes, which are agglutinated by most human sera, contain unique glycosphingolipids (globoside elongation products) terminating with the sequence Galalpha1-4GalNAcbeta1-3Gal- recognized by common natural human antibodies. Anti-NOR antibodies were isolated from several human sera by affinity procedures, and their specificity was tested by inhibition of antibody binding to NOR-tri-polyacrylamide (PAA) conjugate (ELISA) by the synthetic oligosaccharides, Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), Galalpha1-4GalNAc (NOR-di), Galalpha1-4Galbeta1-3Galbeta1-4Glc ((Gal)3Glc), and Galalpha1-4Gal (P1-di). Two major types of subspecificity of anti-NOR antibodies were found. Type 1 antibodies were found to react strongly with (Gal)3Glc and NOR-tri and weakly with P1-di and NOR-di, which indicated specificity for the trisaccharide epitope Galalpha1-4Gal/GalNAcbeta1-3Gal. Type 2 antibodies were specific to Galalpha1-4GalNAc, because they were inhibited most strongly by NOR-tri and NOR-di and were not (or very weakly) inhibited by (Gal)3Glc and P1-di. Monoclonal anti-NOR antibodies were obtained by immunizing mice with NOR-tri-human serum albumin (HSA) conjugate and were found to have type 2 specificity. All anti-NOR antibodies reacted specifically with NOR glycolipids on thin-layer plates. The cross-reactivity of type 1 anti-NOR antibodies with Galalpha1-4Gal drew attention to a possible antigenic relationship between NOR and blood group P system glycolipids. The latter glycolipids include Pk (Galalpha1-4Galbeta1-4Glc-Cer) present in all normal erythrocytes and P1 (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) present only in P1 erythrocytes. Sera of some P2 (P1-negative) persons contain natural anti-P1 antibodies. This prompted us to test the specificity of anti-P1 antibodies. Natural human anti-P1 isolated from serum of P2 individual and mouse monoclonal anti-P1 were best inhibited by Galalpha1-4Galbeta1-4GlcNAc (P1-tri) and did not react with NOR-tri and NOR-di. Monoclonal anti-P1 bound to Pk and P1 glycolipids and not to NOR glycolipids. These results indicated an entirely different specificity of anti-NOR and anti-P1 antibodies. Human serum samples differed in the content of anti-alpha-galactosyl antibodies, including both types of anti-NOR. In the sera of some individuals, type 1 or type 2 anti-NOR antibodies dominated, and other samples contained mixtures of both types of anti-NOR. The biological significance of these new abundant anti-alpha-galactosyl antibodies still awaits elucidation.

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., (Copyright Blackwell Munksgaard, 2004)

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.

Endogenous lectins induce effects on cell growth by binding to antennae of natural glycoconjugates. These complex carbohydrates often present more than one potential lectin-binding site in a single chain. Using the growth-regulatory interaction of the pentasaccharide of ganglioside GM(1) with homodimeric galectin-1 on neuroblastoma cell surfaces as a model, we present a suitable strategy for addressing this issue. The approach combines NMR spectroscopic and computational methods and does not require isotope-labeled glycans. It involves conformational analysis of the two building blocks of the GM(1) glycan, i.e., the disaccharide Galbeta1-3GalNAc and the trisaccharide Neu5Acalpha2-3Galbeta1-4Glc. Their bound-state conformations were determined by transferred nuclear Overhauser enhancement spectroscopy. Next, measurements on the lectin-pentasaccharide complex revealed differential conformer selection regarding the sialylgalactose linkage in the tri- versus pentasaccharide (Phi and Psi value of -70 degrees and 15 degrees vs 70 degrees and 15 degrees, respectively). To proceed in the structural analysis, the characteristic experimentally detected spatial vicinity of a galactose unit and Trp68 in the galectin's binding site offered a means, exploiting saturation transfer from protein to carbohydrate protons. Indeed, we detected two signals unambiguously assigned to the terminal Gal and the GalNAc residues. Computational docking and interaction energy analyses of the entire set of ligands supported and added to experimental results. The finding that the ganglioside's carbohydrate chain is subject to differential conformer selection at the sialylgalactose linkage by galectin-1 and GM(1)-binding cholera toxin (Phi and Psi values of -172 degrees and -26 degrees, respectively) is relevant for toxin-directed drug design. In principle, our methodology can be applied in studies aimed at blocking galectin functionality in malignancy and beyond glycosciences.

Background: The innate immune system, including complement and natural killer (NK) cells, plays a critical role in activation and damage of endothelial cells (ECs) during xenograft rejection. The semisynthetic proteoglycan analog dextran sulfate (DXS, molecular weight 5,000) is known to inhibit the complement and coagulation cascades. We hypothesized that DXS may act as an "EC-protectant" preventing complement and NK lysis by functionally replacing heparan sulfate proteoglycans that are shed from the EC surface on activation of the endothelium., Methods: Binding of DXS to ECs, deposition of human complement, cytotoxicity, and heparan sulfate expression after exposure to normal human serum were analyzed by flow cytometry. The efficacy of DXS to protect ECs from xenogeneic NK cell-mediated cytotoxicity was tested in standard 51Cr-release assays., Results: DXS dose-dependently inhibited all three pathways of complement activation. Binding of DXS to porcine cells increased on treatment with human serum or heparinase I and correlated positively with the inhibition of human complement deposition. This cytoprotective effect of DXS was still present when the challenge with normal human serum was performed up to 48 hr after DXS treatment of the cells. DXS incubation of porcine ECs with and without prior tumor necrosis factor-alpha stimulation reduced xenogeneic cytotoxicity mediated by human NK cells by 47.3% and 25.3%, respectively., Conclusions: DXS binds to porcine cells and protects them from complement- and NK cell-mediated injury in vitro. It might therefore be used as a novel therapeutic strategy to prevent xenograft rejection and has potential for clinical application as an "EC protectant."

Pig-to-human xenotransplantation might be an option to overcome the increasing shortage of human donor organs. However, naturally occurring antibodies in human blood against the Galalpha1-->3Gal antigen on pig endothelial cells lead to hyperacute or, if prevented, acute or delayed vascular rejection of the pig graft. The purpose of this study was therefore to evaluate synthetic oligosaccharides with terminal Galalpha1-->3Gal to inhibit antigen-binding and cytotoxicity of anti-alphaGal antibodies against pig cells. Different oligosaccharides were synthesized chemically and by a combined chemico-enzymatic approach. These included monomeric di-, tri-, and pentasaccharides, a polyacrylamide-conjugate (PAA-Bdi), as well as di-, tetra-, and octamers of Galalpha1-->3Gal. All were tested for inhibitory activity by anti-alphaGal ELISA and complement-dependent cytotoxicity tests. PAA-Bdi was the best inhibitor of binding as well as cytotoxicity of anti-alphaGal antibodies. Monomeric oligosaccharides efficiently prevented binding of anti-alphaGal IgG, but less well that of anti-alphaGal IgM, with tri- and pentasaccharides showing a better efficacy than the disaccharide. The two trisaccharides Galalpha1-->3Galbeta1-->4GlcNAc and Galalpha1-->3Galbeta1-->3GlcNAc were equally effective. Oligomers of Galalpha1-->3Gal were more effective than monomers in blocking the binding of anti-alphaGal IgG. However, they could not block IgM binding, nor could they match the efficacy of PAA-Bdi. We conclude that oligosaccharides with terminal Galalpha1-->3Gal, most effectively as PAA-conjugates, can prevent binding and cytotoxicity of human anti-alphaGal in vitro. The PAA-Bdi conjugate might be most suited for use as a Sepharose-bound immunoabsorption material.

The process cascade leading to the final accommodation of the carbohydrate ligand in the lectin's binding site comprises enthalpic and entropic contributions of the binding partners and solvent molecules. With emphasis on lactose, N-acetyllactosamine, and thiodigalactoside as potent inhibitors of binding of galactoside-specific lectins, the question was addressed to what extent these parameters are affected as a function of the protein. The microcalorimetric study of carbohydrate association to the galectin from chicken liver (CG-16) and the agglutinin from Viscum album (VAA) revealed enthalpy-entropy compensation with evident protein type-dependent changes for N-acetyllactosamine. Reduction of the entropic penalty by differential flexibility of loops or side chains and/or solvation properties of the protein will have to be reckoned with to assign a molecular cause to protein type-dependent changes in thermodynamic parameters for lectins sharing the same monosaccharide specificity.

To study conformational parameters of ligands before and after complex formation with the galactoside-binding agglutinin of Viscum album L. (VAA) in solution, combined computer-assisted random walk molecular mechanics (RAMM) calculations extended by conformational clustering analysis (CCA), molecular dynamics (MD) simulations as well as two-dimensional rotating-frame nuclear Overhauser effect (ROE) and two-dimensional nuclear Overhauser effect (NOE) spectroscopy NMR experiments were employed. Derivatives of the naturally occurring disaccharides Galbeta1-3GlcNAcbeta1-R and Galbeta1-3GalNAcbeta1-R as well as of a synthetic high-affinity binding partner, i.e. the disaccharide Galbeta1-2Galbeta1-R', were chosen as ligands in this study. The disaccharides displayed inherent flexibility in the valley of the global minimum between phi/psi combinations of (40 degrees/60 degrees) and (40 degrees/-60 degrees). Calculations of the de-N-acetylated sugars revealed that presence of this group did not markedly influence the distribution of low-energy conformers in the phi, psi, epsilon plot. Occupation of side minima at phi/psi (180 degrees/0 degrees) or (0 degrees/180 degrees) is either unlikely or low according to the results of MD simulations and RAMM calculations extended by CCA. Notably, these side minima define conformations which are not stable during a MD simulation. Transitions to other minima occur already a few picoseconds after the start of the simulation. NMR experiments of the free-state ligand confirmed the validity of the data sets obtained by the calculations. Following the description of the conformational space in the free-state NMR experiments were performed for these disaccharides complexed with VAA. They yielded two interresidual contacts for Galbeta1-3GlcNAcbeta1-R and Galbeta1-2Galbeta1-R'. The ligand conformations in the complex did not deviate markedly from those of a minimum conformation in the free state. One- and two-dimensional transferred nuclear Overhauser enhancement (TRNOE) experiments at different mixing times excluded the influence of spin-diffusion effects. When the NOE build-up curves in the three studied cases were compared, the residual mobility of the penultimate carbohydrate unit of Galbeta1-3GalNAcbeta1-R was observed to be higher than that of the respective hexopyranose unit of the other two bound ligands. Due to the availability of the conformational parameters of Galbeta1-2Galbeta1-R' in association with a galectin, namely the beta-galactoside-binding protein from chicken liver, it is remarkable to note that this ligand displays different conformations in the binding sites of either the plant or the animal lectin. They correspond to local energy-minimum conformations in the phi,psi, epsilon plot and substantiate differential conformer selection by these two lectins with identical nominal monosaccharide specificity.

The overexpression of lectins by malignant cells was applied for in vitro targeting of liposomes equipped with a saccharide vector and loaded in the lipid phase with a lipid derivative of anticancer agent sarcolysine. The lectin specificity of human leukemia HL-60 and human lung adenocarcinoma ACL cells was revealed by tests with fluorescein-labeled sugar probes. With the help of fluorescent lipid dye it was shown that active saccharide ligands increased the level of the vectored liposome binding to malignant cells by 50-80% as compared to liposomes without vector or with inactive one. The degree of liposome/cell membrane fusion was monitored fluorometrically and was shown to be complete and independent of the vectors. The targeted drug-loaded liposomes had the cytotoxic activity 2-4 times higher as compared to the vector-free ones.

Purpose: Targeted label or drug delivery requires access to convenient carrier systems and methods for efficient ligand conjugation. The main purpose of this study is to design an iodinatable synthetic polymer, whose application in vivo in tumor-bearing mice is tested with several related carbohydrate ligands, namely ABH and Lewis blood group epitopes., Methods: Tyramine and aminopropyl derivatives of the synthetic oligosaccharides were attached to poly(4-nitrophenylacrylate). Following iodination, the biodistribution of the sugar-free and the substituted polymers was determined in tumor-bearing mice. Flow cytofluorimetric analysis assessed tumor cell binding of further ligand types to human tumor cells in vitro., Results: Quantitative ligand incorporation was achieved under mild conditions. Whereas the ligand-free poly[N-(2-hydroxyethyl)acrylamide] (MW 30 kDa) showed preferential accumulation in kidney, neoglycopolymers were found in substantial amounts in liver, kidney or spleen. The nature of the carbohydrate structure quantitatively influenced the distribution pattern. Tumor cell binding of blood group determinants and three further ligand types revealed non-uniform intensity in labeling and percentage of positive cells even in comparison between lines with identical histogenetic origin., Conclusions: Carbohydrate-exposing poly[N-(2-hydroxyethyl)acrylamide] polymers with tyramine as an iodine acceptor distribute in mice with a profile which is quantitatively influenced by small structural variations of the ligand part. Further refinement of the ligand structure may increase the level of selectivity for organ and tumor accumulation.

We have tested 88 monoclonal antibodies with proposed specificities against ABH- and related antigens in an ELISA with synthetic oligosaccharide conjugates as coating substances. All mabs were tested for binding towards A-, B-, and H type I trisaccharides, the B disaccharide, and the proposed anti-A and -AB were also tested on A tetrasaccharide type I. Of the 50 mabs with proposed A- and/or B-specificity, 28 showed a specific reaction in our ELISA. Cross-reactivity with other ABH-antigens was observed for 7 of these 50 anti-A/B mabs and 15 of them did not react in the ELISA. Only 2 of the 17 mabs submitted as anti-H bound to H trisaccharide type I, one of them showed a polyspecific reactivity pattern, and the remaining 14 did not react with our type I antigen. Of the mabs with other than ABH-specificities only one, supposedly anti-P1, showed cross-reactivity with one of our coating antigens, in this case the B trisaccharide.

The major role of anti-alphaGal antibodies in the hyperacute rejection of pig organs by humans and baboons has been clearly demonstrated. Spacered alpha-galactose disaccharide (Gal(alpha1)-3Gal) hapten was produced by chemical synthesis and covalently attached to a flexible, hydrophilic polymer (PAA), which in turn was covalently coupled to macroporous glass beads, forming an immunoadsorbent that is mechanically and chemically stable and can be sterilized. The extracorporeal immunoadsorption (EIA) of anti-alphaGal antibodies using this column has been investigated in vivo in 3 baboons. In Baboon 1 (which had hyperacutely rejected a pig heart transplant 4 months previously, was not splenectomized, and did not receive any pharmacologic immunosuppression) the levels of anti-alphaGal antibody and antipig IgM and IgG, as well as serum cytotoxicity, fell significantly after each of 3 EIAs but were not eliminated. Serum cytotoxicity, antipig immunoglobulin and anti-alphaGal antibody rose steeply within 24 hr of the final EIA, suggesting that the return of cytotoxicity was associated with anti-alphaGa1 antibody. In Baboons 2 and 3 (which were immunologically naive and splenectomized, and received triple drug immunosuppressive therapy) serum cytotoxicity was totally eliminated and anti-alphaGal antibody and antipig IgM and IgG levels were greatly reduced by courses of EIA. In Baboon 2, cytotoxicity and all antibody levels remained negligible for approximately one week after the final (fourth) daily EIA. In Baboon 3, cytotoxicity and antibody levels were maintained low by intermittent EIA (over a period of 13 days) for almost 3 weeks, although antipig IgM began to rebound 4 days after the final EIA. We conclude that, in an immunosuppressed, splenectomized baboon, repeated EIA using a specific alphaGal disaccharide column will reduce antipig and anti-alphaGal antibody levels and serum cytotoxicity significantly for several days. This reduction in cytotoxicity will almost certainly be sufficient to delay the hyperacute rejection of a transplanted pig organ, but further studies are required to investigate whether it will be sufficient to allow accommodation to develop.

The binding of a carbohydrate to a lectin may affect the conformation of the ligand. To address this question for the galectin from chicken liver, the conformation of Gal beta 1-2Gal beta 1-R was analyzed in the free and in the galectin-bound state with 2D-ROESY- and 1D- as well as 2D-transferred NOE-experiments. A computer-assisted analysis of spatial parameters of the ligand by molecular dynamics (MD) and random walk molecular mechanics (RAMM) calculations, taking different dielectric constraints from epsilon = 1 to epsilon = 80 and various force fields into account, were instrumental to define the energetic minima of the free state. NMR-derived interresidual distance constraints enabled a conformational mapping. The two overlapping interresidual distance constraints obtained from transferred-NOE experiments of the galectin-ligand complex clearly support the notion that the conformation of the disaccharide in the bound state is at least very close to its global energy minimum state in solution.

Synthetic ABO immunoabsorbents (known as Synsorbs) were in use for several years to specifically eliminate ABO antibodies from the patient's circulation before ABO-incompatible organ or bone marrow transplantation. Because Synsorbs are no longer available, we have developed new ABO immunoabsorbents. These substances, termed BioSorbents A and B, respectively, consist of synthetic A or B trisaccharides covalently coupled to macroporous glass beads via polyacrylamide. Here we report the evaluation of BioSorbents in regard to efficacy, specificity, and biocompatibility. Using a closed-circuit in vitro system, representing a 1:10-1:20 scale as compared with the immunoabsorption procedure with an adult patient, blood group O plasma was run through columns filled with ethylene oxide-sterilized BioSorbent. Hemagglutination was reduced by 4 titer steps after absorption, and anti-A and/or anti-B IgM/G/A, as measured by ABO ELISA, dropped by 85% or more, while no nonspecific absorption of immunoglobulins occurred. No significant changes could be observed for complement (C3, C4, and total hemolytic complement of the classical pathway) or for coagulation parameters (fibrinogen, prothrombin time, activated partial thromboplastin time). As monitored by immunoblotting, neither factor XII nor high molecular weight kininogen was cleaved. In addition, a monocyte phagocytosis inhibition test provided evidence that no significant aggregation of IgG had occurred during absorption. We conclude that BioSorbents A and B are efficient, specific, and biocompatible with human plasma.

Carrier-immobilised carbohydrates were used to monitor the presence of specific carbohydrate-binding sites in tissue sections. Sarcolectin, an interferon-alpha/beta antagonist and growth regulator, had been shown to bind the lymphokine macrophage migration inhibitory factor (MIF). The lectin is thus a MIF-specific probe. Biotinylated sarcolectin, neoglycoproteins with lactose or N-acetylglucosamine residues, and polyacrylamide-attached trisaccharides, that represent the ABH histo-blood group antigens, were applied to sections of 187 primary lung carcinomas. The panel of cases consisted of 57 epidermoid carcinomas, 55 adenocarcinomas, 43 large cell anaplastic carcinomas and 32 small cell anaplastic carcinomas. 47 cases with intrapulmonary metastatic tumours were also included. Expression of binding sites of both sarcolectin and trisaccharides of histo-blood group antigens A and H correlated with patient survival in lung cancer. In view of the widely performed analysis of the presence of histo-blood group antigens, concomitant profiling of binding sites for these sugar components is suggested to be of potential benefit.