Non-human Sialic Acid Research Articles

Profiling temporal changes in bovine milk lactoferrin glycosylation using lectin microarrays

The bovine milk glycoprotein bovine lactoferrin (bLF) has a variety of biological activities related to its constituent glycans. However, little is known about bLF’s oligosaccharide structural changes over the course of lactation. BLF was isolated at 13 time points during the first three months of lactation from three individual cows and glycosylation changes were profiled by lectin microarrays. Substantial profile differences between early and late lactation were observed and accompanying monosaccharide analysis revealed that the occurrence of the non-human sialic acid, N-glycolylneuraminic acid, was greater during early stage milk production. Overall, the data suggested that more diverse complex-type oligosaccharide structures were present on bLF during early lactation with an abundance of oligomannose type glycans in later lactation. The differences in the glycoprofiles of bLF from colostrum to mature milk suggest that these may have different functionality in vivo.

Corrigendum: involvement of a non-human sialic Acid in human cancer.

Antibodies 14F7 and the corresponding anti-idiotype 1E10 (racotumomab, misspelled as racotumumab) were mistakenly conflated in this review. 14F7 reacts with (Neu5Gc)GM3, was recently humanized (1), but has not yet been tested in clinical trials. Racotumomab induced a human anti-(Neu5Gc)GM3 immune response, which correlated with longer median survival in non-small cell lung cancer (2). The first clinical trial result using racotumomab was actually published in 2002 (3). A phase III trial testing racotumomab in advanced non-small cell lung cancer began in 2011, and is currently recruiting (NCT01460472). These studies do not consider dietary Neu5Gc intake and incorporation as a variable that could affect (Neu5Gc)GM3 expression by human cancers.

Inverse hormesis of cancer growth mediated by narrow ranges of tumor-directed antibodies

Compelling evidence for naturally occurring immunosurveillance against malignancies informs and justifies some current approaches toward cancer immunotherapy. However, some types of immune reactions have also been shown to facilitate tumor progression. For example, our previous studies showed that although experimental tumor growth is enhanced by low levels of circulating antibodies directed against the nonhuman sialic acid N-glycolyl-neuraminic acid (Neu5Gc), which accumulates in human tumors, growth could be inhibited by anti-Neu5Gc antibodies from a different source, in a different model. However, it remains generally unclear whether the immune responses that mediate cancer immunosurveillance vs. those responsible for inflammatory facilitation are qualitatively and/or quantitatively distinct. Here, we address this question using multiple murine tumor growth models in which polyclonal antibodies against tumor antigens, such as Neu5Gc, can alter tumor progression. We found that although growth was stimulated at low antibody doses, it was inhibited by high doses, over a linear and remarkably narrow range, defining an immune response curve (IRC; i.e., inverse hormesis). Moreover, modulation of immune responses against the tumor by altering antibody avidity or by enhancing innate immunity shifted the IRC in the appropriate direction. Thus, the dualistic role of immunosurveillance vs. inflammation in modulating tumor progression can be quantitatively distinguished in multiple model systems, and can occur over a remarkably narrow range. Similar findings were made in a human tumor xenograft model using a narrow range of doses of a monoclonal antibody currently in clinical use. These findings may have implications for the etiology, prevention, and treatment of cancer.

Uniquely human changes in expression and binding specificity of Siglec‐11: implications for human brain evolution (787.2)

We are exploring the role of Siglec‐11, a member of the sialic acid‐recognizing immunoglobulin‐like lectin family, in the modulation of microglial functions in the human brain. Human SIGLEC11 underwent two tandem and likely simultaneous gene conversions by the adjacent pseudogene SIGLEC16P (Wang et al., Mol. Biol. Evol., 29:2073‐2086, 2012). These genes conversions preserved an intact open reading frame, but changed 5' untranslated sequences, likely altering transcription of SIGLEC11. Using specific antibodies, we confirmed the resulting uniquely human expression of Siglec‐11 in brain microglia, and next wished to define its ligands. Surprisingly, we did not observe robust binding of human Siglec‐11 to any sialic acid‐containing glycan in multiple assay platforms. In contrast chimpanzee Siglec‐11 strongly recognized several glycans terminating with sialic acid, with a preference for the non‐human sialic acid N‐glycolylneuraminic acid (Neu5Gc). Accordingly, chimp Siglec‐11 bound to cells proportionally to their Neu5Gc content, but human Siglec‐11 exhibited minimal binding to the same cells. We also found that human Siglec‐11 binds to a microglial cell line independently of sialic acid, and are characterizing the nature of this ligand. Alterations in the expression pattern and ligand‐binding specificity of human Siglec‐11 may have evolved to generate novel, human‐specific functions in the brain.Grant Funding Source: Supported by P01 HL057345

Involvement of a Non-Human Sialic Acid in Human Cancer

Sialic acids are common monosaccharides that are widely expressed as outer terminal units on all vertebrate cell surfaces, and play fundamental roles in cell–cell and cell–microenvironment interactions. The predominant sialic acids on most mammalian cells are N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Neu5Gc is notable for its deficiency in humans due to a species-specific and species-universal inactivating deletion in the CMAH gene encoding the hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc. However, Neu5Gc is metabolically incorporated into human tissues from dietary sources (particularly red meat), and detected at even higher levels in some human cancers. Early life exposure to Neu5Gc-containing foods in the presence of certain commensal bacteria that incorporate dietary Neu5Gc into lipooligosaccharides can lead to generation of antibodies that are also cross-reactive against Neu5Gc-containing glycans in human tissues (“xeno-autoantigens”). Such anti-Neu5Gc “xeno-autoantibodies” are found in all humans, although ranging widely in levels among individuals, and displaying diverse and variable specificities for the underlying glycan. Experimental evidence in a human-like Neu5Gc-deficient Cmah−/−mouse model shows that inflammation due to “xenosialitis” caused by this antigen–antibody interaction can promote tumor progression, suggesting a likely mechanism for the well-known epidemiological link between red meat consumption and carcinoma risk. In this review, we discuss the history of this field, mechanisms of Neu5Gc incorporation into tissues, the origin and specificities of human anti-Neu5Gc antibodies, their use as possible cancer biomarkers, implications of xenosialitis in cancer initiation and progression, and current and future approaches toward immunotherapy that could take advantage of this unusual human-specific phenomenon.

Long-Term IgG Response to Porcine Neu5Gc Antigens without Transmission of PERV in Burn Patients Treated with Porcine Skin Xenografts

Acellular materials of xenogenic origin are used worldwide as xenografts, and phase I trials of viable pig pancreatic islets are currently being performed. However, limited information is available on transmission of porcine endogenous retrovirus (PERV) after xenotransplantation and on the long-term immune response of recipients to xenoantigens. We analyzed the blood of burn patients who had received living pig-skin dressings for up to 8 wk for the presence of PERV as well as for the level and nature of their long term (maximum, 34 y) immune response against pig Ags. Although no evidence of PERV genomic material or anti-PERV Ab response was found, we observed a moderate increase in anti-αGal Abs and a high and sustained anti-non-αGal IgG response in those patients. Abs against the nonhuman sialic acid Neu5Gc constituted the anti-non-αGal response with the recognition pattern on a sialoglycan array differing from that of burn patients treated without pig skin. These data suggest that anti-Neu5Gc Abs represent a barrier for long-term acceptance of porcine xenografts. Because anti-Neu5Gc Abs can promote chronic inflammation, the long-term safety of living and acellular pig tissue implants in recipients warrants further evaluation.

Canine and feline parvoviruses preferentially recognize the non-human cell surface sialic acid N-glycolylneuraminic acid

Feline panleukopenia virus (FPV) is a pathogen whose canine-adapted form (canine parvovirus (CPV)) emerged in 1978. These viruses infect by binding host transferrin receptor type-1 (TfR), but also hemagglutinate erythrocytes. We show that hemagglutination involves selective recognition of the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) but not N-acetylneuraminic acid (Neu5Ac), which differs by only one oxygen atom from Neu5Gc. Overexpression of α2-6 sialyltransferase did not change binding, indicating that both α2-3 and α2-6 linkages are recognized. However, Neu5Gc expression on target cells did not enhance CPV or FPV infection in vitro. Thus, the conserved Neu5Gc-binding preference of these viruses likely plays a role in the natural history of the virus in vivo. Further studies must clarify relationships between virus infection and host Neu5Gc expression. As a first step, we show that transcripts of CMAH (which generates Neu5Gc from Neu5Ac) are at very low levels in Western dog breed cells.

No Expression of Non-Human Sialic Acid in Human Embryonic Stem Cell Lines Cultured and Derived in Xeno-Free Culture Condition.

No Expression of Non-Human Sialic Acid in Human Embryonic Stem Cell Lines Cultured and Derived in Xeno-Free Culture Condition.

Successful Prediction of Substrate-binding Pocket in SLC17 Transporter Sialin

Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.

LC-MS analysis of polyclonal human anti-Neu5Gc xeno-autoantibodies immunoglobulin G Subclass and partial sequence using multistep intravenous immunoglobulin affinity purification and multienzymatic digestion.

Human polyclonal IgG antibodies directly against the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc) are potential biomarkers and mechanistic contributors to cancer and other diseases associated with chronic inflammation. Using a sialoglycan microarray, we screened the binding pattern of such antibodies (anti-Neu5Gc IgG) in several samples of clinically approved human IVIG (IgG). These results were used to select an appropriate sample for a multistep affinity purification of the xeno-autoantibody fraction. The sample was then analyzed via our multienzyme digestion procedure followed by nano liquid chromatography (nanoLC) coupled to linear ion trap-Fourier transform mass spectrometry (LTQ-FTMS). We used characteristic and unique peptide sequences to determine the IgG subclass distribution and thus provided direct evidence that all four IgG subclasses can be generated during a xeno-autoantibody immune response to carbohydrate Neu5Gc-antigens. Furthermore, we obtained a significant amount of sequence coverage of both the constant and variable regions. The approach described here, therefore, provides a way to characterize these clinically significant antibodies, helping to understand their origins and significance.

Nontypeable Haemophilus influenzae: the role of N-acetyl-5-neuraminic acid in biology

Nontypeable Haemophilus influenzae (NTHi) is an exclusive human pathogen, which has evolved a number of unique mechanisms to survive within the human environment. An important part of this is the ability of the organism to take up and incorporate sialic acid into its surface structures. This protects the organism against host adaptive and innate immune factor as well as serving as a mechanism for sustaining itself within biofilms. Recent evidence suggests that this also may be the source of the evolution of human antibodies to non-human sialic acid structures, which can lead to inflammation in the host. In very rare instances, evolution of antibodies to sialylated lipooligosaccharide (LOS) mimics of human antigens can result in autoimmune disease.

Development of a Human Extracellular Matrix for Applications Related with Stem Cells and Tissue Engineering

The recent progress in stem cell biology has created new approaches for their study as well as their application to the treatment of human diseases [1–3]. The success of stem-cell based technologies in the clinical setting [4, 5] has emphasized the need to improve the standards of quality for all phases of cell therapy, particularly the development of culture methods that circumvent products of animal origin since these might provoke infections or immune rejection following transplantation in patients. Indeed, since Martin et al. (2005) [6] demonstrated that hESC cultured with animal or serum products retained non-human sialic acid which was immunogenic when transplanted in humans, the establishment of animal-free conditions to support the maintenance and differentiation of human stem cells has been a major goal of the field of regenerative medicine [7]. Thus, chemically-defined culture systems that are devoid of non-human substances will greatly facilitate the use of stem cells in regenerative strategies. The concept of a niche is crucial for the organization of stem cells. A niche is consider as a subset of tissue cells and extracellular substrates (matrix and soluble factors) that can support stem cells and control their self-renewal in vivo [8]. Extracellular matrices help to structure niches spatially and modulate the concentration of adhesive and signalling molecules locally. The ECM is a molecular complex that contains collagens and other glycoproteins, hyaluronic acid, proteoglycans, glycosaminoglycans (GAGs), and elastins. Additionally, the ECM harbours growth factors or cytokines to protect against degradation [9]. ECM components are responsible for adhesion during the majority of cell interactions and are implicated in the maintenance of embryonic induction during development as well as stem cell differentiation in vitro [10]. Thus, local changes in ECM can dramatically modulate the proliferation and migration of stem cells and may participate in the specification of lineages. hESC have provided invaluable tools for gaining insight into the developmental origins of human tissues. However, to realize the full biological and clinical potential of hESC, certain problems related with the routine culture of these cells must be solved. Mouse embryonic fibroblasts (MEFs) and murine derivatives such as Matrigel are widely used in the maintenance and differentiation of hESC. Recently, considerable effort has been dedicated to the elimination of animal-derived reagents from the culture of hESC and in parallel, to the control of cell growth parameters by avoiding human feeder cells. For example, in 2006 Ludwig et al. reported the use of conditioned media and high doses of FGF to maintain the undifferentiated state in hESC cultured on plastic, but some abnormalities were detected at passage 20 under these conditions [11, 12]. The use of human feeders complicates the growth and molecular analysis of both pluripotency and differentiation since experimental data may reflect the combined effects of hESC and feeder cells in the culture. Given these considerations, the use of an ECM of human or synthetic origin would provide many advantages. Indeed, there have been attempts [13, 14] to produce such a tool but the results have not been satisfactory because the products were unable to maintain hESC in the undifferentiated state over time. Matrix proteins have been used as coating for in vitro cultures of human stem cells but they have usually been applied as undefined protein mixtures [11] of animal origin [15] with undefined media [13], serum [16] or a synthetic mixture [17, 18]. However, most of these human biological reagents are expensive to manufacture and thus, are cost-prohibitive for many laboratories. Decellularization procedures have been used traditionally to isolate ECM from cells in culture, tissues or organs [19]. The goal of decellularization protocols is to efficiently remove cellular and nuclear material while minimizing any adverse effect on the composition, biological activity and mechanical and structural integrity of the remaining ECM [20, 21]. Decellularized human scaffolds have facilitated the remodelling of various tissues in both animal models and humans [4, 22]. However, any biochemical procedure employed to remove cells may also alter the native three-dimensional architecture of the ECM and thus, a balance must be achieved between chemical and physical treatments during the decellularization process [4, 5, 23]. Given the potential importance of hESC in translational research and regenerative medicine, the aim of the present study was to develop a simple, efficient protocol for the production of a human ECM that is both safe and economical. Here we report that hypotonic lysis of human foreskin fibroblasts (HFF) generates a human ECM that retains protein components which are essential for attachment and cell-cell interaction. This hffECM was capable of maintaining the pluripotency of hESC and supporting their differentiation when used with the appropriate medium. Therefore, our results reveal hffECM as a novel tool which may facilitate the clinical application of hESC-based technologies.

Human Xeno-Autoantibodies against a Non-Human Sialic Acid Serve as Novel Serum Biomarkers and Immunotherapeutics in Cancer

Human carcinomas can metabolically incorporate and present the dietary non-human sialic acid Neu5Gc, which differs from the human sialic acid N-acetylneuraminic acid (Neu5Ac) by 1 oxygen atom. Tumor-associated Neu5Gc can interact with low levels of circulating anti-Neu5Gc antibodies, thereby facilitating tumor progression via chronic inflammation in a human-like Neu5Gc-deficient mouse model. Here we show that human anti-Neu5Gc antibodies can be affinity-purified in substantial amounts from clinically approved intravenous IgG (IVIG) and used at higher concentrations to suppress growth of the same Neu5Gc-expressing tumors. Hypothesizing that this polyclonal spectrum of human anti-Neu5Gc antibodies also includes potential cancer biomarkers, we then characterize them in cancer and noncancer patients' sera, using a novel sialoglycan microarray presenting multiple Neu5Gc-glycans and control Neu5Ac-glycans. Antibodies against Neu5Gcα2-6GalNAcα1-O-Ser/Thr (GcSTn) were found to be more prominent in patients with carcinomas than with other diseases. This unusual epitope arises from dietary Neu5Gc incorporation into the carcinoma marker Sialyl-Tn, and is the first example of such a novel mechanism for biomarker generation. Finally, human serum or purified antibodies rich in anti-GcSTn-reactivity kill GcSTn-expressing human tumors via complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity. Such xeno-autoantibodies and xeno-autoantigens have potential for novel diagnostics, prognostics, and therapeutics in human carcinomas.

Biomedical Differences Between Human and Nonhuman Hominids: Potential Roles for Uniquely Human Aspects of Sialic Acid Biology

Although humans are genetically very similar to the evolutionarily related nonhuman hominids (chimpanzees, bonobos, gorillas, and orangutans), comparative studies suggest a surprising number of uniquely human differences in the incidence and/or severity of biomedical conditions. Some differences are due to anatomical changes that occurred during human evolution. However, many cannot be explained either by these changes or by known environmental factors. Because chimpanzees were long considered models for human disease, it is important to be aware of these differences, which appear to have been deemphasized relative to similarities. We focus on the pathophysiology and pathobiology of biomedical conditions that appear unique to humans, including several speculative possibilities that require further study. We pay particular attention to the possible contributions of uniquely human changes in the biology of cell-surface sialic acids and the proteins that recognize them. We also discuss the metabolic incorporation of a diet-derived nonhuman sialic acid, which generates a novel xeno-autoantigen reaction, and chronic inflammation known as xenosialitis.

Cytofluorimetric evaluation of N-glycolylated GM3 ganglioside expression on murine leukocytes

Gangliosides are considered relevant components of lipid rafts at the plasma membrane. Antigen encounter, immunological synapse assembly and signal transduction modify lipid raft composition and distribution on immune system cells. On the contrary of other gangliosides, differential expression of the N-glycolylated variant of GM3 (NGcGM3) on murine leukocytes has received limited attention. In particular, whether cell activation modulates the expression of NGcGM3 on lymphoid and myeloid cells is still unexplored. Availability of the NGcGM3 specific 14F7 MAb allows us to characterize by cytofluorimetric assays the presence of this molecule on resting and activated immune system cells. On T cells, preferential expression of NGcGM3 was detected on CD4 + single positive thymocytes, peripheral CD4 + lymphocytes and natural occurring regulatory T cells. In comparison with peritoneal B1 cells, reduced expression of NGcGM3 was observed in peritoneal B2 and splenic B cell subpopulations. Of note, activation of CD4 + and NK 1.1 + cells abrogated NGcGM3 expression while LPS-maturated DC increased the ganglioside level at the plasma membrane. Modifications on the presence of NGcGM3 mediated by cell activation did not influence the expression of the N-acetylated variant of GM3 (NAcGM3). In addition to extend previous descriptions of NGcGM3 expression on immunity cell subpopulations, this work highlights the opposite effect of cellular activation over NGcGM3 levels on lymphoid and myeloid cellular series. Obtained results complement the evaluation of a tumor-specific, non-human sialic acid containing ganglioside that has been considered an attractive target for cancer immunotherapy.

Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins

Recombinant glycoprotein therapeutics produced in nonhuman mammalian cell lines and/or with animal serum are often modified with the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc; refs. 1,2). This documented contamination has generally been ignored in drug development because healthy individuals were not thought to react to Neu5Gc (ref. 2). However, recent findings indicate that all humans have Neu5Gc-specific antibodies, sometimes at high levels. Working with two monoclonal antibodies in clinical use, we demonstrate the presence of covalently bound Neu5Gc in cetuximab (Erbitux) but not panitumumab (Vectibix). Anti-Neu5Gc antibodies from healthy humans interact with cetuximab in a Neu5Gc-specific manner and generate immune complexes in vitro. Mice with a human-like defect in Neu5Gc synthesis generate antibodies to Neu5Gc after injection with cetuximab, and circulating anti-Neu5Gc antibodies can promote drug clearance. Finally, we show that the Neu5Gc content of cultured human and nonhuman cell lines and their secreted glycoproteins can be reduced by adding a human sialic acid to the culture medium. Our findings may be relevant to improving the half-life, efficacy and immunogenicity of glycoprotein therapeutics.

Novel mechanism for the generation of human xeno-autoantibodies against the nonhuman sialic acid N-glycolylneuraminic acid

The nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc) is metabolically incorporated into human tissues from certain mammalian-derived foods, and this occurs in the face of an anti-Neu5Gc “xeno-autoantibody” response. Given evidence that this process contributes to chronic inflammation in some diseases, it is important to understand when and how these antibodies are generated in humans. We show here that human anti-Neu5Gc antibodies appear during infancy and correlate with weaning and exposure to dietary Neu5Gc. However, dietary Neu5Gc alone cannot elicit anti-Neu5Gc antibodies in mice with a humanlike Neu5Gc deficiency. Other postnatally appearing anti-carbohydrate antibodies are likely induced by bacteria expressing these epitopes; however, no microbe is known to synthesize Neu5Gc. Here, we show that trace exogenous Neu5Gc can be incorporated into cell surface lipooligosaccharides (LOS) of nontypeable Haemophilus influenzae (NTHi), a human-specific commensal/pathogen. Indeed, infant anti-Neu5Gc antibodies appear coincident with antibodies against NTHi. Furthermore, NTHi that express Neu5Gc-containing LOS induce anti-Neu5Gc antibodies in Neu5Gc-deficient mice, without added adjuvant. Finally, Neu5Gc from baby food is taken up and expressed by NTHi. As the flora residing in the nasopharynx of infants can be in contact with ingested food, we propose a novel model for how NTHi and dietary Neu5Gc cooperate to generate anti-Neu5Gc antibodies in humans.

Chemo-enzymatic synthesis of the carbohydrate antigen N-glycolylneuraminic acid from glucose

N-Glycolylneuraminic acid (Neu5Gc) is a non-human sialic acid, which may play a significant role in human pathologies, such as cancer and vascular disease. Further studies into the role of Neu5Gc in human disease are hindered by limited sources of this carbohydrate. Using a chemo-enzymatic approach, Neu5Gc was accessed in six steps from glucose. The synthesis allows access to gram-scale quantities quickly and economically and produces Neu5Gc in superior quality to commercial sources. Finally, we demonstrate that the synthesized Neu5Gc can be incorporated into the cell glycocalyx of human cells, which do not naturally synthesize this sugar. The synthesis produces Neu5Gc suitable for in vitro or in vivo use.

Identification of α-Gal and non-Gal Epitopes in Pig Corneal Endothelial Cells and Keratocytes by Using Mass Spectrometry

Purpose: To investigate the expression of α-Gal or unidentified non-Gal antigens in pig corneal endothelial cells and keratocytes, we performed the qualitative and quantitative analysis by using mass spectrometry.Methods: The N-glycans from common adult pig corneal endothelial cells and keratocytes cultured in vitro were directly analyzed by using mass spectrometric approaches. In addition, immunochemical staining was added to confirm the non-Gal antigen expression in pig corneal cells.Results: Totally, 34 of the sialylated N-glycans from pig corneal endothelial cells and 27 from pig keratocytes were identified and observed to contain nonhuman sialic acid, NeuGc as well as NeuAc. In addition, we were able to detect 25 of α-galactosylated N-glycan structures (22.2% of total) from the pig corneal endothelial cells and 18 of that (17.5% of total) from the pig keratocytes by using mass spectrometric approaches. On immunofluorescent staining, the expression of sialylated glycans was also observed.Conclusions: As well as α-Gal epitopes, several promising non-Gal antigens were widely expressed on both pig corneal endothelial cells and keratocytes. The detailed structural information of the α-Gal and non-Gal epitopes would be a tremendous value to develop a new strategy for the successful corneal xenotransplantation in future.

A dietary non-human sialic acid may facilitate hemolytic-uremic syndrome

Hemolytic-uremic syndrome (HUS) is a systemic disease characterized by microvascular endothelial damage, mainly in the gastrointestinal tract and the kidneys. A major cause of HUS is Shiga toxigenic Escherichia coli (STEC) infection. In addition to Shiga toxin, additional STEC virulence factors may contribute to HUS. One is the newly discovered subtilase cytotoxin (SubAB), which is highly toxic to eukaryotic cells, and when injected intraperitoneally into mice causes pathology resembling that associated with human HUS. Recent data show that SubAB exhibits a strong preference for glycans terminating in alpha2-3-linked N-glycolylneuraminic acid (Neu5Gc), a sialic acid that humans are unable to synthesize, because we genetically lack the necessary enzyme. However, Neu5Gc can still be found on human cells due to metabolic incorporation from the diet. Dietary incorporation happens to be highest in human endothelium and to a lesser extent in the intestinal epithelium, the two affected cell types in STEC-induced HUS. Mammalian-derived foods such as red meat and dairy products appear to be the primary source of dietary Neu5Gc. Ironically, these are also common sources of STEC contamination. Taken together, these findings suggest a 'two-hit' process in the pathogenesis of human SubAB-induced disease. First, humans eat Neu5Gc-rich food, leading to incorporation of Neu5Gc on the surfaces of endothelial and intestinal cells. Second, when exposed to a SubAB-producing STEC strain, the toxin produced would be able to bind to the intestinal epithelial cells, perhaps causing acute gastrointestinal symptoms, and eventually damaging endothelial cells in other organs like the kidney, thereby causing HUS.