Jan Holgersson

Characteristics of protein–carbohydrate interactions as a basis for developing novel carbohydrate-based antirejection therapies

The relative shortage of human organs for transplantation is today the major barrier to a broader use of transplantation as a means of treating patients with end‐stage organ failure. This barrier could be partly overcome by an increased use of blood group ABO‐incompatible live donors, and such trials are currently underway at several transplant centres. If xenotransplantation can be used clinically in the future, the human organ shortage will, in principle, be eradicated. In both these cases, carbohydrate antigens and the corresponding anti‐carbohydrate antibodies are the major primary immunological barriers to overcome. Refined carbohydrate‐based therapeutics may permit an increased number of ABO‐incompatible transplantations to be carried out, and may remove the initial barriers to clinical xenotransplantation. Here, we will discuss the chemical characteristics of protein–carbohydrate interactions and outline carbohydrate‐based antirejection therapies as used today in experimental as well as in clinical settings. Novel mucin‐based adsorbers of natural anti‐carbohydrate antibodies will also be described.

A comparison of fetal and adult porcine islets with regard to Gal alpha(1,3)Gal expression and the role of human immunoglobulins and complementin islet cell cytotoxicity.

BACKGROUND It is still debated whether fetal or adult porcine islets should be the preferred choice for future clinical islet xenotransplantation. Each type of islet preparation has advantages and disadvantages compared with the other. Here we present a direct comparison between fetal and adult porcine islets with regard to Gal alpha(1,3)Gal expression, immunoglobulin and complement binding, and cytotoxicity after exposure to fresh human serum. METHOD Islet single cell suspensions were prepared from adult and fetal islets by trypsin digestion. Fluorescein isothiocyanate-conjugated Bandeiraea simplicifolia isolectin B4 (BS-IB4) and affinity-purified chicken anti-Gal alpha(1,3)Gal antibody was used to detect Gal alpha(1,3)Gal expression. Immunoglobulin and complement binding to the islet cells and cytotoxicity for islet cells was compared after incubation with fresh and heat-inactivated human sera and with an immune serum from a diabetic patient who received a fetal porcine islet transplant. Furthermore, two pools of human AB sera were depleted of porcine endothelial cell cytotoxic human anti-Gal alpha(1,3)Gal antibodies by absorption and were used to analyze the effect of Gal alpha(1,3)Gal antibody removal on islet cell cytotoxicity. RESULTS Fetal islet cells readily bound both BS-IB4 and the chicken anti-Gal alpha(1,3)Gal antibody. None of 10 adult porcine islet preparations were stained by BS-IB4. In comparison, IgY anti-Gal Ab binding was detected in two of eight adult islet isolations, whereas the other six preparations showed marginal/no binding. After incubation of fetal islet cells with fresh human serum, C3c binding was strongly positive and IgM binding variable, with occasional binding of IgG and no detectable binding of IgA. Adult islet cells were also strongly positive for C3c but did not bind detectable amounts of IgM, IgG, or IgA. Immune sera from a patient who had received fetal porcine islets showed the presence of induced antibodies that bound to fetal islet cells and to porcine peripheral blood lymphocytes, whereas binding to adult islet cells was barely detectable. Fresh human sera showed a high and similar level of complement-mediated lytic activity for both adult islet cells (78+/-22%) and fetal islet cells (75+/-16%). Cytotoxicity for fetal islet cells and peripheral blood lymphocytes was significantly reduced when the corresponding sera were depleted of anti-Gal antibodies before use (P=0.002 and P=0.003, respectively). In contrast, no difference in cytotoxicity for adult islet cells was detected when anti-Gal-depleted human sera were used. CONCLUSION Gal alpha(1,3)Gal expression is occasionally detectable on adult porcine islet cells, but not as readily and at a lower level, compared with fetal islet cells. Thus, as porcine fetal islets mature to adult islets, the expression of the Gal alpha(1,3)Gal epitope gradually diminishes. Consequently, cytotoxic anti-Gal alpha(1,3)Gal antibodies in human serum play an important role in the lysis of fetal but not adult porcine islet cells.

Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells.

Helicobacter pylori colonizes the mucus niche of the gastric mucosa and is a risk factor for gastritis, ulcers and cancer. The main components of the mucus layer are heavily glycosylated mucins, to which H. pylori can adhere. Mucin glycosylation differs between individuals and changes during disease. Here we have examined the H. pylori response to purified mucins from a range of tumor and normal human gastric tissue samples. Our results demonstrate that mucins from different individuals differ in how they modulate both proliferation and gene expression of H. pylori. The mucin effect on proliferation varied significantly between samples, and ranged from stimulatory to inhibitory, depending on the type of mucins and the ability of the mucins to bind to H. pylori. Tumor-derived mucins and mucins from the surface mucosa had potential to stimulate proliferation, while gland-derived mucins tended to inhibit proliferation and mucins from healthy uninfected individuals showed little effect. Artificial glycoconjugates containing H. pylori ligands also modulated H. pylori proliferation, albeit to a lesser degree than human mucins. Expression of genes important for the pathogenicity of H. pylori (babA, sabA, cagA, flaA and ureA) appeared co-regulated in response to mucins. The addition of mucins to co-cultures of H. pylori and gastric epithelial cells protected the viability of the cells and modulated the cytokine production in a manner that differed between individuals, was partially dependent of adhesion of H. pylori to the gastric cells, but also revealed that other mucin factors in addition to adhesion are important for H. pylori-induced host signaling. The combined data reveal host-specific effects on proliferation, gene expression and virulence of H. pylori due to the gastric mucin environment, demonstrating a dynamic interplay between the bacterium and its host.

Glycolipids of human large intestine: difference in glycolipid expression related to anatomical localization, epithelial/non-epithelial tissue and the ABO, Le and Se phenotypes of the donors

Human large intestine specimens were obtained during elective surgery from donors of known blood group ABO, Lewis and secretor phenotypes. The intestinal epithelial cells were isolated from the non-epithelial tissue in one case and in another case mucosa tissue was obtained by scraping. Total non-acid glycolipid and ganglioside fractions were isolated from the tissue specimens, analyzed by thin-layer chromatography and detected by chemical reagents and autoradiography after staining the plate with various blood group monoclonal antibodies and bacterial toxins. The amount of non-acid glycolipids present in the large intestine epithelial cells was 3.9 micrograms/mg of cell protein and in the non-epithelial tissue 0.39 mg/g dry tissue weight. The epithelial cells contained monoglycosylceramides and blood group Lea pentaglycosylceramides as major compounds together with small amounts of diglycosylceramides. In addition, trace amounts of tri- and tetra-glycosylceramides together with more complex glycolipids were present. The non-epithelial tissue contained mono-, di-, tri- and tetra-glycosylceramides as major non-acid components. Blood group ABH glycolipids were present in trace amounts in the non-epithelial part of the large intestine. Lea pentaglycosylceramide was the major blood group glycolipid present in all Le-positive individuals independent of the secretor status. Leb glycolipids were present in trace amounts in secretor individuals but completely lacking in non-secretors. Trace amounts of X antigens were found in all individuals, while Y antigens were only present in secretor individuals. The Lea, Leb, X and Y glycolipids were located in the epithelial cells. The gangliosides were present mainly in the non-epithelial tissue (65-350 nmol of sialic acid/g dry weight) and only trace amounts (less than 0.014 nmol/mg of cell protein) were found in the epithelial cells. The major gangliosides of the non-epithelial tissue were identified as GM3, GM1, GD3, GD1b, GT1b and GQ1b. In addition, several minor gangliosides were also present. Binding of cholera toxin to the thin-layer plate revealed trace amounts of the GM1 ganglioside in the epithelial cell ganglioside fraction.

The Extracellular Adherence Protein from Staphylococcus aureus Inhibits Neutrophil Binding to Endothelial Cells

ABSTRACT Extracellular adherence protein (Eap) from Staphylococcus aureus inhibits the adherence of neutrophils to nonstimulated and tumor necrosis factor alpha-stimulated endothelial cells in both static adhesion assays and flow adhesion assays. Consequently, Eap also impaired their transendothelial migration. During an S. aureus infection, Eap may thus serve to reduce inflammation by inhibiting neutrophil adhesion and extravasation.

Removal of xenoreactive human anti-pig antibodies by absorption on recombinant mucin-containing glycoproteins carrying the Gal alpha1,3Gal epitope.

BACKGROUND The hyperacute rejection caused by preformed natural antibodies in the recipient species reacting with donor species endothelial antigens is one of the major obstacles preventing routine use of clinical xenotransplantation. Based on the known structure and biosynthetic pathway of the major porcine xenoantigen, Gal alpha1,3Gal, we developed a novel strategy aimed at specific removal of human, natural anti-pig antibodies. METHODS Cotransfection of COS cells with expression plasmids encoding a secreted mucin/immunoglobulin chimera and the porcine alpha1,3-galactosyltransferase facilitated simple immunoaffinity purification of a highly Gal alpha1,3Gal-substituted mucin/immunoglobulin fusion protein from transfected cell supernatants. RESULTS Cotransfection of COS cells resulted in a mucin/Ig concentration in the supernatants ranging from 150 to 200 ng/ml. Approximately 300 ng of mucin/Ig chimeras absorbed onto 50 microl of packed anti-mouse IgG agarose beads could completely remove cytotoxic human anti-pig antibodies from 1 ml of human AB serum, as estimated in porcine endothelial cell cytotoxicity assays. Purified human IgG, IgM, and IgA all bound porcine endothelium, but only IgG and IgM were cytotoxic in the presence of rabbit complement. When the cytotoxicity of human IgG at 8 mg/ml and IgM at 1 mg/ml was completely removed by absorption on the mucin/Ig chimera, the binding to porcine endothelium was only partly reduced. Antibodies mediating antibody-dependent cellular cytotoxicity against porcine endothelium were also absorbed, indicating the importance of Gal alpha1,3Gal epitopes for this effect. CONCLUSIONS We describe the construction and production of a new and effective Gal alpha1,3Gal-substituted, mucin domain-containing absorber that can be used in a pretransplant extracorporeal immunoabsorption setting to remove anti-pig antibodies involved in antibody-dependent, complement- and cell-mediated cytotoxicity of pig endothelial cells.

Porcine endothelium supports transendothelial migration of human leukocyte subpopulations: anti-porcine vascular cell adhesion molecule antibodies as species-specific blockers of transendothelial monocyte and natural killer cell migration.

BACKGROUND In cases where hyperacute rejection has been prevented, pig to primate organ transplantation results in a delayed rejection mediated by graft-infiltrating leukocytes. The migration of human leukocytes across porcine endothelium is poorly characterized, but may offer targets for species-specific antirejection therapy. METHODS Transwell tissue culture inserts with endothelial cells growing on polycarbonate filters were used to characterize the migration of peripheral blood monocuclear cells and purified leukocyte subpopulations across pig and human endothelial cells and cell lines. Endothelial cell morphology was evaluated by scanning and transmission electron microscopy, and the contribution of different adhesion receptor pairs to transendothelial migration was evaluated by antibody blocking experiments. RESULTS There were no evident quantitative or qualitative differences in the capacity of human and porcine endothelium to support transendothelial migration of human leukocytes [T, B, and natural killer (NK) cells, monocytes, and neutrophils]. Monocytes and large granular CD3+ lymphocytes migrated most efficiently across the endothelium. Antiporcine vascular cell adhesion molecule-1 antibodies blocked transendothelial migration of human monocytes and NK cells across tumor necrosis factor-alpha stimulated pig endothelium by at least 60%. Anti-CD18 antibodies had no effect on the migration of human NK cells across pig endothelium, whereas they partly blocked migration of NK cells across human endothelium and migration of monocytes across porcine endothelium. Interleukin-2 stimulated, but not unstimulated, T and NK cells were cytotoxic to porcine endothelium. CONCLUSIONS Porcine endothelium supports transendothelial migration of human leukocyte subpopulations as efficiently as human endothelium. Incompatibilities in some adhesion receptor pairs may be compensated for by other adhesion receptor pairs, as exemplified by human NK cells whose migration across human, but not pig, endothelium was blocked by anti-CD18 antibodies. Antiporcine vascular cell adhesion molecule-1 antibodies may be used as species-specific blockers of transendothelial NK cell and monocyte migration, and as such may prove to be useful inhibitors of cellular organ xenograft rejection.

Blood group type glycosphingolipids of human kidneys. Structural characterization of extended globo-series compounds.

Blood group type glycosphingolipids present in kidneys of blood group A and B human individuals have been isolated and structurally characterized by mass spectrometry, proton NMR spectroscopy, degradation studies and by their reactivity with various monoclonal antibodies andEscherichia coli bacteria. The two major complex glycolipids present in the blood group A and B kidneys were globopentaosylceramide (IV3Galβ-Gb4Cer) and the X pentaglycosylceramide (III3Fucα-nLc4Cer). The major blood group A glycolipid in the blood group A kidneys was based on the type 4 chain (globo-series). There were also small amounts of the type 2 chain and trace amounts of the type 1 and type 3 chain based A glycolipids. In addition, the blood group H type 4 chain structure was present together with Lea and Leb compounds. In the blood group B kidneys, the major B glycolipids were monofucosylated hexa- and octaglycosylceramides, where the former were based on the type 2 carbohydrate chain. The blood group B type 4 chain heptaglycosylceramide was found to be a minor component making up only about 1% of the total blood group B structures.

Porcine endothelium activated by anti-??-GAL antibody binding mediates increased human neutrophil adhesion under flow

Background. Neutrophils participate in acute vascular rejection (AVR) of organ xenografts. Induced antibodies (Abs), including anti-Gal&agr;1,3Gal (&agr;-Gal) Abs, have been suggested to cause AVR. We investigated the adhesion of naive human neutrophils to porcine aortic endothelial cells (PAECs) stimulated with anti-&agr;-Gal Abs under conditions of flow. In addition, the ability of human neutrophils to adhere to human and porcine endothelium under static and flow conditions was evaluated. Methods and Results. In a flow-adhesion assay, a significant increase in adhesion of human neutrophils to PAECs, but not to human aortic endothelial cells (HAECs), was detected 6 hours after anti-&agr;-Gal Ab-binding. After Ab stimulation, PAECs expressed CD62E and increased levels of CD106, indicating an activated endothelial cell (EC) phenotype. In a migration assay, supernatants from Ab-stimulated PAECs induced migration of human neutrophils, which was partially blocked by anti-porcine (p) interleukin (IL)-8 Abs and an antagonist to platelet-activating factor (PAF). In static and flow-adhesion assays, no difference in adhesion of human neutrophils to unstimulated or tumor necrosis factor (TNF)-&agr;-stimulated HAECs and PAECs could be detected. Conclusions. Our data suggest that anti-&agr;-Gal Abs play an important role in the initiation of AVR by mediating adhesion and recruitment of neutrophils within an organ xenograft. In contrast with previous investigations, our data argues against a differential recognition of PAECs and HAECs by human neutrophils. Thus, to prevent AVR and accomplish long-term xenograft survival, it will be important to remove anti-&agr;-Gal Abs before and after pig-to-human transplantation.

Glycosylation of extracellular superoxide dismutase studied by high-performance liquid chromatography and mass spectrometry

Extracellular superoxide dismutase, EC-SOD, the main superoxide dismutase in biological fluids, is known from its lectin binding to be a glycoprotein. We have characterized the glycosylation of recombinant EC-SOD. A tryptic digest of the protein contained only one glycosylated peptide. This peptide was specifically bound to lectins and stained by periodic acid-Schiff stain. Although appearing very large on size-exclusion chromatography, it was shown to be glycosylated at only one site, asparagine-89, by specific cleavage with glycanases followed by mass spectrometry of the resulting peptide. Based on the binding properties of the peptide to concanavalin A and lentil lectin and the elution profile of N-glycanase-treated glycopeptide on ion-exchange chromatography, the carbohydrate appears to be the complex biantennary type with a core fucose.