Elena Korchagina

Synthesis of polymeric neoglycoconjugates based on N-substituted polyacrylamides

Several types of polymeric glycoconjugates,N-substituted polyacrylamides, have been synthesized by the reaction of activated polymers with ω-aminoalkylglycosides: (i) (carbohydrate-spacer)n-polyacrylamide, ‘pseudopolysaccharides’; (ii) (carbohydrate-spacer)n-phosphatidylethanolaminem-polyacrylamide, neoglycolipids, derivatives of phosphatidylethanolamine; (iii) (carbohydrate-spacer)n-biotinm-polyacrylamide, biotinylated probes; (iv) (carbohydrate-spacer)n-polyacrylamide-(macroporous glass), affinity sorbents based on macroporous glass, covalently coated with polyacrylamide. An almost quantitative yield in the conjugation reaction makes it possible to insert in the conjugate a predetermined quantity of the ligand(s).Pseudopolysaccharides proved to be a suitable form of antigen for activation of polystyrene and poly(vinyl chloride) plates (ELISA) and nitrocellulose membranes (dot blot), being advantageous over traditional neoglycoproteins. Polyvalent glycolipids insert well in biological membranes: their physical properties, particularly solubility, can be changed in a desired direction. Biotinylated derivatives were used as probes for detection and analysis of lectins.

In vivo immunoadsorption of antipig antibodies in baboons using a specific Gal(alpha)1-3Gal column.

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.

Detection, immunoabsorption, and inhibition of cytotoxic activity of anti‐αGal antibodies using newly developed substances with synthetic Gal α1–3Gal disaccharide epitopes

Abstract: The presence of naturally occurring anti‐Galα1–3Gal (anti‐αGal) antibodies in human serum is believed to be a major factor in the hyperacute rejection of discordant organ xenografts such as the pig‐to‐human combination. Galα1–3Gal epitopes are expressed on pig tissues and the binding of anti‐αGal leads to endothelial cell activation and complement‐mediated, hyperacute graft rejection. One possible method to overcome this problem is to absorb anti‐αGal antibodies from the plasma of the xenograft recipient using a suitable immunoabsorbent or to interfere with their binding to tissues and thus prevent their cytotoxic activity by the intravenous injection of soluble antigen. We describe here the use of new synthetic antigens containing the Galα1–3Gal disaccharide (Bdi) epitope. Soluble conjugates of the Bdi with polyacrylamide (PAA‐Bdi) were used as coating antigens for an anti‐αGal ELISA as well as for in vitro inhibition of the cytotoxicity of anti‐αGal. An immunoabsorbent consisting of PAA‐Bdi coupled to macroporous glass (Sorbent Bdi) was tested for absorption of anti‐αGal from human serum.

Synthetic glycolipid modification of red blood cell membranes

BACKGROUND: Glycolipids have a natural ability to insert into red cell (RBC) membranes. Based on this concept the serology of RBCs modified with synthetic analogs of blood group glycolipids (KODE technology) was developed, which entails making synthetic glycolipid constructs engineered to have specific performance criteria. Such synthetic constructs can be made to express a potentially unlimited range of carbohydrate blood group determinants.

Dextran sulfate acts as an endothelial cell protectant and inhibits human complement and natural killer cell-mediated cytotoxicity against porcine cells.

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-&agr; 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.”

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.

Microcalorimetric indications for ligand binding as a function of the protein for galactoside-specific plant and avian lectins

The process cascade leading to the final accommodation of the carbohydrate ligand in the lectins 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.

Structural Basis for Substrate Specificity of Mammalian Neuraminidases

The removal of sialic acid (Sia) residues from glycoconjugates in vertebrates is mediated by a family of neuraminidases (sialidases) consisting of Neu1, Neu2, Neu3 and Neu4 enzymes. The enzymes play distinct physiological roles, but their ability to discriminate between the types of linkages connecting Sia and adjacent residues and between the identity and arrangement of the underlying sugars has never been systematically studied. Here we analyzed the specificity of neuraminidases by studying the kinetics of hydrolysis of BODIPY-labeled substrates containing common mammalian sialylated oligosaccharides: 3′Sia-LacNAc, 3′SiaLac, SiaLex, SiaLea, SiaLec, 6′SiaLac, and 6′SiaLacNAc. We found significant differences in substrate specificity of the enzymes towards the substrates containing α2,6-linked Sia, which were readily cleaved by Neu3 and Neu1 but not by Neu4 and Neu2. The presence of a branching 2-Fuc inhibited Neu2 and Neu4, but had almost no effect on Neu1 or Neu3. The nature of the sugar residue at the reducing end, either glucose (Glc) or N-acetyl-D-glucosamine (GlcNAc) had only a minor effect on all neuraminidases, whereas core structure (1,3 or 1,4 bond between D-galactose (Gal) and GlcNAc) was found to be important for Neu4 strongly preferring β3 (core 1) to β4 (core 2) isomer. Neu3 and Neu4 were in general more active than Neu1 and Neu2, likely due to their preference for hydrophobic substrates. Neu2 and Neu3 were examined by molecular dynamics to identify favorable substrate orientations in the binding sites and interpret the differences in their specificities. Finally, using knockout mouse models, we confirmed that the substrate specificities observed in vitro were recapitulated in enzymes found in mouse brain tissues. Our data for the first time provide evidence for the characteristic substrate preferences of neuraminidases and their ability to discriminate between distinct sialoside targets.

Fluorescein and radiolabeled Function-Spacer-Lipid constructs allow for simple in vitro and in vivo bioimaging of enveloped virions

Tools that can aid in vitro and in vivo imaging and also noninvasively determine half-life and biodistribution are required to advance clinical developments. A Function-Spacer-Lipid construct (FSL) incorporating fluorescein (FSL-FLRO4) was used to label vesicular stomatitis virus (VSV), measles virus MV-NIS (MV) and influenza virus (H1N1). The ability of FSL constructs to label these virions was established directly by FACScan of FSL-FLRO4 labeled VSV and MV, and indirectly following labeled H1N1 and MV binding to a cells. FSL-FLRO4 labeling of H1N1 was shown to maintain higher infectivity of the virus when compared with direct fluorescein virus labeling. A novel tyrosine (125)I radioiodinated FSL construct was synthesized (FSL-(125)I) from FSL-tyrosine. This was used to label VSV (VSV-FSL-(125)I), which was infused into the peritoneal cavity of laboratory mice. Bioscanning showed VSV-FSL-(125)I to localize in the liver, spleen and bloodstream in contrast to the free labels FSL-(125)I or (125)I, which localized predominantly in the liver and thyroid respectively. This is a proof-of-principle novel and rapid method for modifying virions and demonstrates the potential of FSL constructs to improve in vivo imaging of virions and noninvasively observe in vivo biodistribution.

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

Abstract: 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.