Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac).We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described

The association between palliative care team consultation and hospital costs for patients with advanced cancer: An observational study in 12 Dutch hospitals

Background: Early palliative care team consultation has been shown to reduce costs of hospital care. The objective of this study was to investigate the association between palliative care team (PCT) consultation and the content and costs of hospital care in patients with advanced cancer. Material and Methods: A prospective, observational study was conducted in 12 Dutch hospitals.

Siglec-F is a novel intestinal M cell marker

Intestinal microfold (M) cells are epithelial cells primarily present on Peyer's patches (PPs) in the small intestine. The ability of M cells to shuttle antigens into the PP for appropriate immune responses makes M cells a target for next-generation oral vaccine delivery. In this regard, discovery of M cell specific receptors are of great interest, which could act as molecular tags for targeted delivery of cargo to M cells. Here, using a monoclonal antibody we generated to the Sialic acid-binding immunoglobulin-like lectin F (Siglec-F), we show that Siglec-F is expressed on mouse M cells in the small intestine. Immunohistochemical analysis of the PP tissue sections shows that Siglec-F is expressed on the surface of the M cell membrane exposed to the intestinal lumen. Anti-Siglec-F antibody injected into the mouse small intestine bound to M-cells, demonstrating the potential to target M cells via Siglec-F

Siglec-5 and Siglec-14 are polymorphic paired receptors that modulate neutrophil and amnion signaling responses to group B Streptococcus

Group B Streptococcus (GBS) causes invasive infections in human newborns. We recently showed that the GBS beta-protein attenuates innate immune responses by binding to sialic acid-binding immunoglobulin-like lectin 5 (Siglec-5), an inhibitory receptor on phagocytes. Interestingly, neutrophils and monocytes also express Siglec-14, which has a ligand-binding domain almost identical to Siglec-5 but signals via an activating motif, raising the possibility that these are paired Siglec receptors that balance immune responses to pathogens. Here we show that beta-protein-expressing GBS binds to both Siglec-5 and Siglec-14 on neutrophils and that the latter engagement counteracts pathogen-induced host immune suppression by activating p38 mitogen-activated protein kinase (MAPK) and AKT signaling pathways. Siglec-14 is absent from some humans because of a SIGLEC14-null polymorphism, and homozygous SIGLEC14-null neutrophils are more susceptible to GBS immune subversion. Finally, we report an unexpected human-specific expression of Siglec-5 and Siglec-14 on amniotic epithelium, the site of initial contact of invading GBS with the fetus. GBS amnion immune activation was likewise influenced by the SIGLEC14-null polymorphism. We provide initial evidence that the polymorphism could influence the risk of prematurity among human fetuses of mothers colonized with GBS. This first functionally proven example of a paired receptor system in the Siglec family has multiple implications for regulation of host immunity

The pattern of influenza virus attachment varies among wild bird species

The ability to attach to host cells is one of the main determinants of the host range of influenza A viruses. By using virus histochemistry, we investigate the pattern of virus attachment of both a human and an avian influenza virus in colon and trachea sections from 12 wild bird species. We show that significant variations exist, even between closely related avian species, which suggests that the ability of wild birds to serve as hosts for influenza viruses strongly varies among species. These results will prove valuable to assess the possibilities of interspecies transmission of influenza viruses in natural environments and better understand the ecology of influenza

B cell antigen receptor signal strength and peripheral B cell development are regulated by a 9-O-acetyl sialic acid esterase

We show that the enzymatic acetylation and deacetylation of a cell surface carbohydrate controls B cell development, signaling, and immunological tolerance. Mice with a mutation in sialate:O-acetyl esterase, an enzyme that specifically removes acetyl moieties from the 9-OH position of α2–6-linked sialic acid, exhibit enhanced B cell receptor (BCR) activation, defects in peripheral B cell development, and spontaneously develop antichromatin autoantibodies and glomerular immune complex deposits. The 9-O-acetylation state of sialic acid regulates the function of CD22, a Siglec that functions in vivo as an inhibitor of BCR signaling. These results describe a novel catalytic regulator of B cell signaling and underscore the crucial role of inhibitory signaling in the maintenance of immunological tolerance in the B lineage

The Infection of Chicken Tracheal Epithelial Cells with a H6N1 Avian Influenza Virus

Sialic acids (SAs) linked to galactose (Gal) in α2,3- and α2,6-configurations are the receptors for avian and human influenza viruses, respectively. We demonstrate that chicken tracheal ciliated cells express α2,3-linked SA, while goblet cells mainly express α2,6-linked SA. In addition, the plant lectin MAL-II, but not MAA/MAL-I, is bound to the surface of goblet cells, suggesting that SA2,3-linked oligosaccharides with Galβ1–3GalNAc subterminal residues are specifically present on the goblet cells. Moreover, both α2,3- and α2,6-linked SAs are detected on single tracheal basal cells. At a low multiplicity of infection (MOI) avian influenza virus H6N1 is exclusively detected in the ciliated cells, suggesting that the ciliated cell is the major target cell of the H6N1 virus. At a MOI of 1, ciliated, goblet and basal cells are all permissive to the AIV infection. This result clearly elucidates the receptor distribution for the avian influenza virus among chicken tracheal epithelial cells and illustrates a primary cell model for evaluating the cell tropisms of respiratory viruses in poultry

Chemical characterization of oligosaccharides in the milk of six species of New and Old world monkeys

Human and great ape milks contain a diverse array of milk oligosaccharides, but little is known about the milk oligosaccharides of other primates, and how they differ among taxa. Neutral and acidic oligosaccharides were isolated from the milk of three species of Old World or catarrhine monkeys (Cercopithecidae: rhesus macaque (Macaca mulatta), toque macaque (Macaca sinica) and Hamadryas baboon (Papio hamadryas)) and three of New World or platyrrhine monkeys (Cebidae: tufted capuchin (Cebus apella) and Bolivian squirrel monkey (Saimiri boliviensis); Atelidae: mantled howler (Alouatta palliata)). The milks of these species contained 6–8% total sugar, most of which was lactose: the estimated ratio of oligosaccharides to lactose in Old World monkeys (1:4 to 1:6) was greater than in New World monkeys (1:12 to 1:23). The chemical structures of the oligosaccharides were determined mainly by 1H-NMR spectroscopy. Oligosaccharides containing the type II unit (Gal(β1-4)GlcNAc) were found in the milk of the rhesus macaque, toque macaque, Hamadryas baboon and tufted capuchin, but oligosaccharides containing the type I unit (Gal(β1-3)GlcNAc), which have been found in human and many great ape milks, were absent from the milk of all species studied. Oligosaccharides containing Lewis x (Gal(β1-4)[Fuc(α1-3)]GlcNAc) and 3-fucosyl lactose (3-FL, Gal(β1-4)[Fuc(α1-3)]Glc) were found in the milk of the three cercopithecid monkey species, while 2-fucosyl lactose (5'-FL, Fuc(α1-2)Gal(β1-4)Glc) was absent from all species studied. All of these milks contained acidic oligosaccharides that had N-acetylneuraminic acid as part of their structures, but did not contain oligosaccharides that had N-glycolylneuraminic acid, in contrast to the milk or colostrum of great apes which contain both types of acidic oligosaccharides. Two GalNAc-containing oligosaccharides, lactose 3′-O-sulfate and lacto-N-novopentaose I (Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc) were found only in the milk of rhesus macaque, hamadryas baboon and tufted capuchin, respectively. Further research is needed to determine the extent to which the milk oligosaccharide patterns observed among these taxa represent wider phylogenetic trends among primates and how much variation occurs among individuals or species