To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.

We report in this paper that glycophorin C (GPC) is the receptor for PfEBP-2 (baebl, EBA-140), the newly identified erythrocyte binding ligand of Plasmodium falciparum. PfEBP-2 is a member of the Duffy binding-like erythrocyte binding protein (DBL-EBP) family. Although several reports have been published characterizing PfEBP-2, the identity of its erythrocytic receptor was still unknown. Using a combination of enzymatically treated red blood cells (RBCs) and rare, variant RBCs lacking different surface proteins, we have shown that PfEBP-2 does not bind to cells lacking GPC. Additionally, we found that PfEBP-2 binds differentially to variants of GPC lacking exon 2 or exon 3, and determined that the binding domain on GPC is potentially restricted to amino acid residues 14 through 22 within exon 2. Thus PfEBP-2 is involved in a sialic acid-dependent pathway of invasion, which does not involve glycophorin A or glycophorin B and represents a novel route of entry into the RBCs.

The addition of sialic acid to T cell surface glycoproteins influences essential T cell functions such as selection in the thymus and homing in the peripheral circulation. Sialylation of glycoproteins can be regulated by expression of specific sialyltransferases that transfer sialic acid in a specific linkage to defined saccharide acceptor substrates and by expression of particular glycoproteins bearing saccharide acceptors preferentially recognized by different sialyltransferases. Addition of alpha2,6-linked sialic acid to the Galbeta1,4GlcNAc sequence, the preferred ligand for galectin-1, inhibits recognition of this saccharide ligand by galectin-1. SAalpha2,6Gal sequences, created by the ST6Gal I enzyme, are present on medullary thymocytes resistant to galectin-1-induced death but not on galectin-1-susceptible cortical thymocytes. To determine whether addition of alpha2,6-linked sialic acid to lactosamine sequences on T cell glycoproteins inhibits galectin-1 death, we expressed the ST6Gal I enzyme in a galectin-1-sensitive murine T cell line. ST6Gal I expression reduced galectin-1 binding to the cells and reduced susceptibility of the cells to galectin-1-induced cell death. Because the ST6Gal I preferentially utilizes N-glycans as acceptor substrates, we determined that N-glycans are essential for galectin-1-induced T cell death. Expression of the ST6Gal I specifically resulted in increased sialylation of N-glycans on CD45, a receptor tyrosine phosphatase that is a T cell receptor for galectin-1. ST6Gal I expression abrogated the reduction in CD45 tyrosine phosphatase activity that results from galectin-1 binding. Sialylation of CD45 by the ST6Gal I also prevented galectin-1-induced clustering of CD45 on the T cell surface, an initial step in galectin-1 cell death. Thus, regulation of glycoprotein sialylation may control susceptibility to cell death at specific points during T cell development and peripheral activation.

Interleukin (IL)-13 is a newly described cytokine expressed by activated lymphocytes. We examined the effects of the murine recombinant cytokine on the phenotype and activation status of elicited peritoneal macrophages (M phi), concentrating on activities which are known to be modulated by interferon-gamma and IL-4. IL-13 markedly suppressed nitric oxide release and to a lesser extent secretion of the pro-inflammatory cytokine tumor necrosis factor-alpha. However, antimicrobial capacity was not completely jeopardized as the respiratory burst was unaffected, and indeed the enhanced expression of M phi mannose receptor and major histocompatibility class II, and regulation of sialoadhesin, the M phi sialic acid-specific receptor involved in hemopoietic and lymphoid interactions, suggest that these cells are not simply deactivated, but primed for an active role in immune and inflammatory responses. These activities closely mimic those of IL-4, but mediation of the effects by IL-4 was discounted by the use of a neutralizing monoclonal antibody. Thus, IL-13, like IL-4, is a cytokine which has complex effects on M phi behavior, inducing activities characteristic of both activation and deactivation.

Recombinant human erythropoietin produced in transfected Chinese hamster ovary cells is glycosylated much the same way as the erythropoietin present in human urine. To determine the role of carbohydrates in the stability of recombinant human erythropoietin in vivo, [125I]-labeled recombinant erythropoietin was intravenously infused into rats. The erythropoietin was slowly cleared from the blood with a half-life of approximately two hours. Asialoerythropoietin, which was produced by treatment of recombinant human erythropoietin with sialidase, was found to be cleared rapidly from circulation within ten minutes. These data suggest that the galactose binding protein of hepatic cells is involved in the clearance of asialoerythropoietin. Erythropoietin also contains N-glycans with a few N-acetyllactosamine repeats, which can be enriched by tomato lectin affinity chromatography. The lectin-bound fraction was cleared to a larger extent than was the unfractionated erythropoietin, while the component that did not bind the lectin was found to be stable in the circulation. Authentic N-acetyllactosamine repeats (polylactosaminoglycans) prepared from erythrocytes were similarly rapidly cleared from the circulation to the liver, and this clearance was inhibitable with asialo-alpha 1-acid glycoprotein. These results suggest that (a) the sialic acid of the recombinant erythropoietin is necessary for this glycoprotein hormone to circulate stably and (b) glycoproteins with more than three lactosaminyl repeat units may be cleared by the galactose binding protein of hepatocytes.

Plasmodium falciparum malaria parasites with different capabilities of invading sialic acid-deficient erythrocytes were identified. Thai-2 parasites cultured in Tn erythrocytes invaded neuraminidase-treated and Tn erythrocytes twice as efficiently as Thai-2 parasites cultured in normal erythrocytes and seven to ten times more efficiently than a cloned line of Camp parasites cultured in normal erythrocytes. All three parasite lines required sialic acid for optimal invasion, but Thai-2 parasites cultured in Tn erythrocytes invaded neuraminidase-treated erythrocytes with 45% efficiency whereas Camp parasites invaded neuraminidase-treated erythrocytes with less than 10% efficiency. P falciparum malaria parasites probably possess two receptors: one that binds to a sialic acid-dependent ligand and another that binds to a sialic acid-independent ligand. Parasites may differ in the quantity or affinity of their receptors for the sialic acid-independent ligand.

Here we report the isolation of influenza virus A/turkey/Minnesota/833/80 (H4N2) with a mutation at the catalytic residue of the neuraminidase (NA) active site, rendering it resistant to the novel NA inhibitor 4-guanidino-Neu5Ac2en (GG167). The resistance of the mutant stems from replacement of one of three invariant arginines (Arg 292->>Lys) that are conserved among all viral and bacterial NAs and participate in the conformational change of sialic acid moiety necessary for substrate catalysis. The Lys292 mutant was selected in vitro after 15 passages at increasing concentrations of GG167 (from 0.1 to 1,000 microM), conditions that earlier gave rise to GG167-resistant mutants with a substitution at the framework residue Glu119. Both types of mutants showed similar degrees of resistance in plaque reduction assays, but the Lys292 mutant was more sensitive to the inhibitor in NA inhibition tests than were mutants bearing a substitution at framework residue 119 (Asp, Ala, or Gly). Cross-resistance to other NA inhibitors (4-amino-Neu5Ac2en and Neu5Ac2en) varied among mutants resistant to GG167, being lowest for Lys292 and highest for Asp119. All GG167-resistant mutants demonstrated markedly reduced NA activity, only 3 to 50% of the parental level, depending on the particular amino acid substitution. The catalytic mutant (Lys292) showed a significant change in pH optimum of NA activity, from 5.9 to 5.3. All of the mutant NAs were less stable than the parental enzyme at low pH. Despite their impaired NA activity, the GG167-resistant mutants grew as well as parental virus in Madin-Darby canine kidney cells or in embryonated chicken eggs. However, the infectivity in mice was 500-fold lower for Lys292 than for the parental virus. These findings demonstrate that amino acid substitution in the NA active site at the catalytic or framework residues, followed by multiple passages in vitro, in the presence of increasing concentrations of the NA inhibitor GG167, generates GG167-resistant viruses with reduced NA activity and decreased infectivity in animals.

Maturation of the vesicular stomatitis virus (VSV) glycoprotein (G) to the cell surface is blocked at the nonpermissive temperature in cells infected with temperature-sensitive mutants in the structural gene encoding for G. We show here that these mutants fall into two discrete classes with respect to the stage of post-translational processing at which the block occurs. In all cases the mutant glycoproteins are inserted normally into the endoplasmic reticulum membrane, receive the two-high-mannose oligosaccharides, and apparently lose the NH2-terminal signal sequence of 16 amino acids. In cells infected with one class of mutants, no further processing of the glycoprotein occurs, and we conclude that the mutant protein is blocked at a pre-Golgi stage. In cells infected with ts L511(V), however, addition of the terminal sugars galactose and sialic acid occurs normally. Thus the maturation of G proceeds through several Golgi functions but is blocked before its appearance on the cell surface. The oligosaccharide chain of ts L511(V) G, accumulated at either the permissive (where surface maturation occurs) or the nonpermissive temperature, lacks one saccharide residue, probably fucose. In addition, no fatty acid residues are added to the ts L511(V) G protein at the nonpermissive temperature, although addition does occur under permissive conditions.

OBJECTIVE

To determine whether serum concentrations of the cytokines tumour necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6), which regulate C reactive protein, are associated with cardiovascular risk factors and prevalent coronary heart disease.

METHODS

A population based cross sectional study.

METHODS

198 men aged 50 to 69 years were part of a random population sample drawn from south London. Serum cytokine and C reactive protein concentrations were determined by enzyme linked immunosorbent assay. The presence of coronary heart disease was determined by Rose angina questionnaire and Minnesota coded electrocardiogram.

RESULTS

Serum TNF alpha concentrations were positively related to body mass index and Helicobacter pylori infection, but inversely related to alcohol consumption. IL-6 concentrations were positively associated with smoking, symptoms of chronic bronchitis, age, and father having a manual occupation. TNF alpha was associated with increased IL-6 and triglycerides, and reduced high density lipoprotein cholesterol. IL-6 was associated with raised fibrinogen, sialic acid, and triglycerides. ECG abnormalities were independently associated with increases in IL-6 and TNF alpha, each by approximately 50% (P < 0.05 for TNF alpha, P < 0.1 for IL-6). The corresponding increases in men with an abnormal ECG or symptomatic coronary heart disease were 28% for TNF alpha and 36% for IL-6 (P = 0.14 for TNF alpha and P < 0.05 for IL-6).

CONCLUSIONS

This study confirms that many of the phenomena with which C reactive protein is associated, are also associated with serum levels of cytokine, which may be the mechanism.

Human influenza viruses are more efficiently isolated by inoculating patient samples into the amniotic rather than the allantoic cavity of embryonated chicken eggs. This type of cultivation selects virus variants with mutations around the hemagglutinin (HA) receptor binding site. To understand the molecular basis of these phenomena, we investigated the abundances of sialic acid (SA) linked to galactose (Gal) by the alpha-2,3 linkage (SA alpha2,3Gal) and SA alpha2,6Gal in egg amniotic and allantoic cells and in Madin-Darby canine kidney (MDCK) cells. Using SA-Gal linkage-specific lectins (Maackia amurensis agglutinin specific for SA alpha2,6Gal and Sambucus nigra agglutinin specific for SA alpha2,3Gal), we found SA alpha2,3Gal in both allantoic and amniotic cells and SA alpha2,6Gal in only the amniotic cells. MDCK cells contained both linkages. To investigate how this difference in abundances of SA alpha2,3Gal and SA alpha2,6Gal in allantoic and amniotic cells affects the appearance of host cell variants in eggs, we determined the receptor specificities and HA amino acid sequences of two different patient viruses which were isolated and passaged in the amnion or in the allantois and which were compared with MDCK cell-grown viruses. We found that the viruses maintained high SA alpha2,6Gal specificities when grown in MDCK cells or following up to two amniotic passages; however, further passages in either the amnion or allantois resulted in the acquisition of, or a complete shift to, SA alpha2,3Gal specificity, depending on the virus strain. This change in receptor specificity was accompanied by the appearance of variants in the population with Leu-to-Gln mutations at position 226 in their HA. These findings suggest that lack of SA alpha2,6Gal linkages in the allantois of chicken eggs is a selective pressure for the appearance of host cell variants with altered receptor specificities and amino acid changes at position 226.

Influenza is a highly infectious disease characterized by recurrent annual epidemics and unpredictable major worldwide pandemics. Rapid spread of the highly pathogenic avian H5N1 strain and escalating human infections by the virus have set off the alarm for a global pandemic. To provide an urgently needed alternative treatment modality for influenza, we have generated a recombinant fusion protein composed of a sialidase catalytic domain derived from Actinomyces viscosus fused with a cell surface-anchoring sequence. The sialidase fusion protein is to be applied topically as an inhalant to remove the influenza viral receptors, sialic acids, from the airway epithelium. We demonstrate that a sialidase fusion construct, DAS181, effectively cleaves sialic acid receptors used by both human and avian influenza viruses. The treatment provides long-lasting effect and is nontoxic to the cells. DAS181 demonstrated potent antiviral and cell protective efficacies against a panel of laboratory strains and clinical isolates of IFV A and IFV B, with virus replication inhibition 50% effective concentrations in the range of 0.04 to 0.9 nM. Mouse and ferret studies confirmed significant in vivo efficacy of the sialidase fusion in both prophylactic and treatment modes.

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, alpha2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to alpha2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with alpha2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.

The proper glycosylation of erythropoietin is essential for its function in vivo. Human erythropoietins were isolated from Chinese hamster ovary cells transfected with a human erythropoietin cDNA and from human urine. Carbohydrate chains attached to these proteins were isolated and fractionated by anion-exchange high performance liquid chromatography (HPLC) and HPLC employing a Lichrosorb-NH2 column. The structures of fractionated saccharides were analyzed by fast atom bombardment-mass spectrometry and methylation analysis before and after treatment with specific exoglycosidases. Both erythropoietins were found to contain one O-linked oligosaccharide/mol of the proteins, and its major component was elucidated to be NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH (where NeuNAc represents N-acetylneuraminic acid) in both proteins. The N-linked saccharides of recombinant erythropoietin were found to consist of biantennary (1.4% of the total saccharides), triantennary (10%), triantennary with one N-acetyllactosaminyl repeat (3.5%), tetraantennary (31.8%), and tetraantennary with one (32.1%), two (16.5%), or three (4.7%) N-acetyllactosaminyl repeats. All of these saccharides are sialylated by 2----3-linkages. Tetraantennary with or without polylactosaminyl units are mainly present as disialosyl or trisialosyl forms, and these structures exhibit the following unique features. alpha 2----3-Linked sialic acid and N-acetyllactosaminyl repeats are selectively present in the side chains attached to C-6 and C-2 of 2,6-substituted alpha-mannose and C-4 of 2,4-substituted alpha-mannose. We have also shown that the carbohydrate moiety of urinary erythropoietin is indistinguishable from recombinant erythropoietin except for a slight difference in sialylation, providing the evidence that recombinant erythropoietin is valuable for biological as well as clinical use.

Adherence of Helicobacter pylori to inflamed gastric mucosa is dependent on the sialic acid-binding adhesin (SabA) and cognate sialylated/fucosylated glycans on the host cell surface. By in situ hybridization, H. pylori bacteria were observed in close association with erythrocytes in capillaries and post-capillary venules of the lamina propria of gastric mucosa in both infected humans and Rhesus monkeys. In vivo adherence of H. pylori to erythrocytes may require molecular mechanisms similar to the sialic acid-dependent in vitro agglutination of erythrocytes (i.e., sialic acid-dependent hemagglutination). In this context, the SabA adhesin was identified as the sialic acid-dependent hemagglutinin based on sialidase-sensitive hemagglutination, binding assays with sialylated glycoconjugates, and analysis of a series of isogenic sabA deletion mutants. The topographic presentation of binding sites for SabA on the erythrocyte membrane was mapped to gangliosides with extended core chains. However, receptor mapping revealed that the NeuAcalpha2-3Gal-disaccharide constitutes the minimal sialylated binding epitope required for SabA binding. Furthermore, clinical isolates demonstrated polymorphism in sialyl binding and complementation analysis of sabA mutants demonstrated that polymorphism in sialyl binding is an inherent property of the SabA protein itself. Gastric inflammation is associated with periodic changes in the composition of mucosal sialylation patterns. We suggest that dynamic adaptation in sialyl-binding properties during persistent infection specializes H. pylori both for individual variation in mucosal glycosylation and tropism for local areas of inflamed and/or dysplastic tissue.

Sialic acid (Sia) is a family of acidic nine-carbon sugars that occupies the nonreducing terminus of glycan chains. Diversity of Sia is achieved by variation in the linkage to the underlying sugar and modification of the Sia molecule. Here we identified Sia-dependent epitope specificity for GL7, a rat monoclonal antibody, to probe germinal centers upon T cell-dependent immunity. GL7 recognizes sialylated glycan(s), the alpha2,6-linked N-acetylneuraminic acid (Neu5Ac) on a lactosamine glycan chain(s), in both Sia modification- and Sia linkage-dependent manners. In mouse germinal center B cells, the expression of the GL7 epitope was upregulated due to the in situ repression of CMP-Neu5Ac hydroxylase (Cmah), the enzyme responsible for Sia modification of Neu5Ac to Neu5Gc. Such Cmah repression caused activation-dependent dynamic reduction of CD22 ligand expression without losing alpha2,6-linked sialylation in germinal centers. The in vivo function of Cmah was analyzed using gene-disrupted mice. Phenotypic analyses showed that Neu5Gc glycan functions as a negative regulator for B-cell activation in assays of T-cell-independent immunization response and splenic B-cell proliferation. Thus, Neu5Gc is required for optimal negative regulation, and the reaction is specifically suppressed in activated B cells, i.e., germinal center B cells.

BACKGROUND

A major determinant of influenza infection is the presence of virus receptors on susceptible host cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid alpha2,3-galactose (SAalpha2,3-Gal) linked receptors, whereas human strains bind to sialic acid alpha2,6-galactose (SAalpha2,6-Gal) linked receptors. To date, there has been no detailed account published on the distribution of SA receptors in the pig, a model host that is susceptible to avian and human influenza subtypes, thus with potential for virus reassortment. We examined the relative expression and spatial distribution of SAalpha2,3-GalG(1-3)GalNAc and SAalpha2,6-Gal receptors in the major organs from normal post-weaned pigs by binding with lectins Maackia amurensis agglutinins (MAA II) and Sambucus nigra agglutinin (SNA) respectively.

RESULTS

Both SAalpha2,3-Gal and SAalpha2,6-Gal receptors were extensively detected in the major porcine organs examined (trachea, lung, liver, kidney, spleen, heart, skeletal muscle, cerebrum, small intestine and colon). Furthermore, distribution of both SA receptors in the pig respiratory tract closely resembled the published data of the human tract. Similar expression patterns of SA receptors between pig and human in other major organs were found, with exception of the intestinal tract. Unlike the limited reports on the scarcity of influenza receptors in human intestines, we found increasing presence of SAalpha2,3-Gal and SAalpha2,6-Gal receptors from duodenum to colon in the pig.

CONCLUSIONS

The extensive presence of SAalpha2,3-Gal and SAalpha2,6-Gal receptors in the major organs examined suggests that each major organ may be permissive to influenza virus entry or infection. The high similarity of SA expression patterns between pig and human, in particular in the respiratory tract, suggests that pigs are not more likely to be potential hosts for virus reassortment than humans. Our finding of relative abundance of SA receptors in the pig intestines highlights a need for clarification on the presence of SA receptors in the human intestinal tract.

The human JC polyomavirus (JCV) causes a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML), in immunocompromised individuals. Current treatment options for PML are inadequate. Sialylated oligosaccharides and the serotonin receptor are known to be necessary for JCV entry, but the molecular interactions underlying JCV attachment remain unknown. Using glycan array screening and viral infectivity assays, we identify a linear sialylated pentasaccharide with the sequence NeuNAc-α2,6-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc (LSTc) present on host glycoproteins and glycolipids as a specific JCV recognition motif. The crystal structure of the JCV capsid protein VP1 was solved alone and in complex with LSTc. It reveals extensive interactions with the terminal sialic acid of the LSTc motif and specific recognition of an extended conformation of LSTc. Mutations in the JCV oligosaccharide-binding sites abolish cell attachment, viral spread, and infectivity, further validating the importance of this interaction. Our findings provide a powerful platform for the development of antiviral compounds.

The effect of meningococcal cell-associated sialic acid on activation of the human alternative complement pathway was examined by using a quantitative fluorescence immunoassay to assess alternative pathway-mediated C3 binding to a group B strain of Neisseria meningitidis from which graded amounts of sialic acid had been removed with neuraminidase. Using human serum absorbed with strain B16B6 (B:2a:L2,3) and chelated with 10 mM MgCl2 and 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we found an increase in the amount of C3 bound by enzymatically desialylated B16B6 organisms over the amount bound by fully sialylated organisms. This increase was proportional to the amount of sialic acid cleaved from the bacteria. Enhanced C3 binding was accompanied by an increase in factor B deposition. A sialic acid-deficient mutant of strain B16B6, designated 2T4-1, bound C3 via the alternative pathway at a level equivalent to that bound by wild-type meningococci from which 88% of the sialic acid had been removed. Strain B16B6 was resistant to the alternative pathway-mediated bactericidal activity of both absorbed and hypogammaglobulinemic human sera, whereas noncapsular variant 2T4-1 was sensitive to these sera. The addition of purified immune immunoglobulin M (IgM) and IgG significantly increased the alternative pathway-mediated killing of strain B16B6 organisms. IgM mediated increased bactericidal activity without an increase in C3 or factor B deposition. In contrast, the IgG-mediated killing was associated with increased binding of C3 and factor B to the organisms. Absorption studies showed that the IgM bound to the sialic acid capsule, whereas the IgG bound to noncapsular surface antigens. We conclude from these results that the group B meningococcal sialic acid capsule inhibits activation of the alternative pathway in the nonimmune host and that both IgM and IgG, although specific for different surface antigens, are capable of augmenting the alternative pathway-mediated killing of group B meningococci.

A monosialoganglioside antigen of gastrointestinal adenocarcinomas defined by murine monoclonal antibody was demonstrated by immunoperoxidase (IP) assay in fixed paraffin-embedded tumors in 59% of colonic adenocarcinomas, 86% of pancreatic adenocarcinomas, and 89% of all gastric adenocarcinomas. In all patients with detectable levels of antigen in circulation, the resected tumors also expressed the antigen in IP assay. Six of eight individuals with no detectable levels of antigen in their serum samples expressed the antigen in the tumor tissue. Removal of the sialic acid residue of the antigen abolished the IP reaction. The successful use of the IP assay on fixed tissue to demonstrate the specific sites of gastrointestinal cancer antigen localization in human tumors and normal tissues provides an important tool for the study of developing neoplasia.

The application of 13-C nuclear magnetic resonance to the analysis of some sialic acid-containing meningococcal polysaccharide antigens is described. Complete assignments of the spectra of both the native serogroup B and the de-O-acetylated serogroup C polysaccharides have been made. These assignments were based on the corresponding data for some related monomers (sialic acid and its alpha-and beta-methylglycosides) and on supportive chemical evidence. The data indicate that the serogroup B polysaccharide is a 2 yields 8-alpha-linked homopolymer of sialic acid, identical in structure with colominic acid from Escherichia coli, whereas the de-O-acetylated serogroup C polysaccharide is a 2 yield 9-alpha-linked homopolymer. The native serogroup C polysaccharide is O-acetylated (1.16 mol of O-acetyl per sialic acid residue), all the O-acetyl substituents being located only at C-7 and C-8 of the sialic acid residues, and in addition contains unacetylated residues (24%). The polysaccharide contains di-O-acetylated residues (O-acetyl on C-7 and C-8), and at least one of the possible monoacetylated residues at C-7 or C-8.

Streptococcus sanguis is the most common oral bacterium causing infective endocarditis and its ability to adhere to platelets, leading to their activation and aggregation, is thought to be an important virulent factor. Previous work has shown that S. sanguis can bind directly to platelet glycoprotein (GP) Ib but the nature of the adhesin was unknown. Here, we have shown that a high molecular weight glycoprotein of S. sanguis mediates adhesion to glycocalacin. The bacterial glycoprotein was purified from cell extracts by chromatography on GPIb- and wheatgerm agglutinin affinity matrices and its interaction with GPIb was shown to be sialic acid-dependent. We designated the glycoprotein serine-rich protein A (SrpA). An insertional inactivation mutant lacking the SrpA of S. sanguis showed significantly reduced binding to glycocalacin, reduced adherence to platelets and a prolonged lag time to platelet aggregation. In addition, under flow conditions, platelets rolled and subsequently adhered on films of wild-type S. sanguis cells at low shear (50/s) but did not bind to films of the SrpA mutant. Platelets did not bind to wild-type bacterial cells at high shear (1500/s). These findings help to understand the mechanisms by which the organism might colonize platelet-fibrin vegetations.

Studies on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of the molecule and its serum half-life and in vivo biological activity, but an inverse relationship with its receptor-binding affinity. These observations led to the hypothesis that increasing the carbohydrate content, beyond that found naturally, would lead to a molecule with enhanced biological activity. Hyperglycosylated recombinant human EPO (rHuEPO) analogues were developed to test this hypothesis. Darbepoetin alfa (novel erythropoiesis stimulating protein, NESP, ARANESP, Amgen Inc, Thousand Oaks, CA), which was engineered to contain 5 N-linked carbohydrate chains (two more than rHuEPO), has been evaluated in preclinical animal studies. Due to its increased sialic acid-containing carbohydrate content, NESP is biochemically distinct from rHuEPO, having an increased molecular weight and greater negative charge. Compared with rHuEPO, it has an approximate 3-fold longer serum half-life, greater in vivo potency, and can be administered less frequently to obtain the same biological response. NESP is currently being evaluated in human clinical trials for treatment of anaemia and reduction in its incidence.

Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.