N-glycolylneuraminic acid in red meat and processed meat is a health concern: A review on the formation, health risk, and reduction.

One of the most consistent epidemiological associations between diet and human disease risk is the impact of consuming red meat and processed meat products. In recent years, the health concerns surrounding red meat and processed meat have gained worldwide attention. The fact that humans have lost the ability to synthesize N-glycolylneuraminic acid (Neu5Gc) makes red meat and processed meat products the most important source of exogenous Neu5Gc for humans. As our research of Neu5Gc has increased, it has been discovered that Neu5Gc in red meat and processed meat is a key factor in many major diseases. Given the objective evidence of the harmful risk caused by Neu5Gc in red meat and processed meat to human health, there is a need for heightened attention in the field of food. This updated review has several Neu5Gc aspects given including biosynthetic pathway of Neu5Gc and its accumulation in the human body, the distribution of Neu5Gc in food, the methods for detecting Neu5Gc, and most importantly, a systematic review of the existing methods for reducing the content of Neu5Gc in red meat and processed meat. It also provides some insights into the current status and future directions in this area.

Influenza D virus utilizes both 9-O-acetylated N-acetylneuraminic and 9-O-acetylated N-glycolylneuraminic acids as functional entry receptors.

Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other hosts. We have previously demonstrated that IDV binds both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac). Bovines produce both Neu5,9Ac2 and Neu5Gc9Ac, while humans are genetically unable to synthesize Neu5Gc9Ac. 9-O-Acetylation of sialic acids is catalyzed by CASD1 via a covalent acetyl-enzyme intermediate. To characterize the role of Neu5,9Ac2 and Neu5Gc9Ac in IDV infection and determine which form of 9-O-acetylated sialic acids drives IDV entry, we took advantage of a CASD1 knockout (KO) MDCK cell line and carried out feeding experiments using synthetic 9-O-acetyl sialic acids in combination with the single-round and multi-round IDV infection assays. The data from our studies show that (i) CASD1 KO cells are resistant to IDV infection and lack of IDV binding to the cell surface is responsible for the failure of IDV replication; (ii) feeding CASD1 KO cells with Neu5,9Ac2 or Neu5Gc9Ac resulted in a dose-dependent rescue of IDV infectivity; and (iii) diverse IDVs replicated robustly in CASD1 KO cells fed with either Neu5,9Ac2 or Neu5Gc9Ac at a level similar to that in wild-type cells with a functional CASD1. These data demonstrate that IDV can utilize Neu5,9Ac2- or non-human Neu5Gc9Ac-containing glycan receptor for infection. Our findings provide evidence that IDV has acquired the ability to infect and transmit among agricultural animals that are enriched in Neu5Gc9Ac, in addition to posing a zoonotic risk to humans expressing only Neu5,9Ac2.IMPORTANCEInfluenza D virus (IDV) has emerged as a multiple-species-infecting pathogen with bovines as a primary reservoir. Little is known about the functional receptor that drives IDV entry and promotes its cross-species spillover potential among different hosts. Here, we demonstrated that IDV binds exclusively to 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and non-human 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac) and utilizes both for entry and infection. This ability in effective engagement of both 9-O-acetylated sialic acids as functional receptors for infection provides an evolutionary advantage to IDV for expanding its host range. This finding also indicates that IDV has the potential to emerge in humans because Neu5,9Ac2 is ubiquitously expressed in human tissues, including lung. Thus, results of our study highlight a need for continued surveillance of IDV in humans, as well as for further investigation of its biology and cross-species transmission mechanism.

Detection of N-glycolyl-neuraminic acid-containing glycolipids in human skin.

Humans lack the enzyme that produces the sialic acid N-glycolyl neuraminic acid (Neu5Gc), but several lines of evidence have shown that Neu5Gc can be taken up by mammalian food sources and replace the common human sialic acid N-acetyl neuraminic acid (Neu5Ac) in glycans. Cancer tissue has been shown to have increased the presence of Neu5Gc and Neu5Gc-containing glycolipids such as the ganglioside GM3, which have been proposed as tumor-specific antigens for antibody treatment. Here, we show that a previously described antibody against Neu5Gc-GM3 is binding to Neu5GC-containing gangliosides and is strongly staining different cancer tissues. However, we also found a strong intracellular staining of keratinocytes of healthy skin. We confirmed this staining on freshly isolated keratinocytes by flow cytometry and detected Neu5Gc by mass spectrometry. This finding implicates that non-human Neu5Gc can be incorporated into gangliosides in human skin, and this should be taken into consideration when targeting Neu5Gc-containing gangliosides for cancer immunotherapy.

Heterophile carbohydrate antigen N-glycolylneuraminic acid as a potential biomarker in patients with hepatocellular carcinoma.

BACKGROUND AND OBJECTIVES: Hepatocellular carcinoma (HCC) has a high recurrence rate even after radical hepatectomy. More optimal biomarkers may help improve recurrence and prognosis. METHODS: We investigated whether the oncological properties of N-glycolylneuraminic acid (NeuGc) can participate in the prognosis of HCC. We evaluated the NeuGc antigen (Ag) expression in the HCC tissues and measured the preoperative anti-NeuGc IgG antibodies (Abs) in the sera of the patients with HCC. We compared the clinical characteristics and survival rate in the hepatectomized patients (initial; n = 66, recurrent; n = 34) with and without the NeuGc Ag or Abs. RESULTS: Multivariate analyses showed positive expression of NeuGc Ag in HCC tissues (Odds ratio; initial = 6.3, recurrent = 14.0) and higher titers of preoperative anti-NeuGc Ab (Odds ratio; initial = 4.9; recurrent = 3.8), which could be the predictive factors related to early recurrence. Both the NeuGc Ag-positive and Ab-positive groups in the initial hepatectomized patients exhibited significantly shorter recurrent free survival compared to those in the negative groups. CONCLUSIONS: Our findings suggested that anti-NeuGc Ab titers and NeuGc Ag expression in the HCC tissues can be used as the predictive factors for the postoperative recurrence and prognosis of HCC.

The generation of 5-N-glycolylneuraminic acid as a consequence of high levels of reactive oxygen species.

The presence of N-glycolylneuraminic acid (Neu5Gc), a non-human sialic acid in cancer patients, is currently attributed to the consumption of red meat. Excess dietary red meat has been considered a risk factor causing chronic inflammation and for the development of cancers. However, it remains unknown whether Neu5Gc can be generated via a chemical reaction rather than via a metabolic pathway in the presence of high levels of reactive oxygen species (ROS) found in the inflammatory and tumor environments. In this study, the conversion of N-acetylneuraminic acid (Neu5Ac) to Neu5Gc has been assessed in vitro under conditions mimicking the hydroxyl radical-rich humoral environment found in inflammatory and cancerous tissues. As a result, Neu5Gc has been detected via liquid chromatography-multiple reaction monitoring mass spectrometry. Furthermore, this conversion has also been found to take place in serum biomatrix containing ROS and in cancer cell cultures with induced ROS production.

Implication of N-glycolylneuraminic acid in regulation of cell adhesiveness of C2C12 myoblast cells during differentiation into myotube cells.

A transition of sialic acid (Sia) species on GM3 ganglioside from N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc) takes place in mouse C2C12 myoblast cells during their differentiation into myotube cells. However, the meaning of this Sia transition remains unclear. This study thus aims to gain a functional insight into this phenomenon. The following lines of evidence show that the increased de novo synthesis of Neu5Gc residues in differentiating myoblast cells promotes adhesiveness of the cells, which is beneficial for promotion of differentiation. First, the Sia transition occurred even in the C2C12 cells cultured in serum-free medium, indicating that it happens through de novo synthesis of Neu5Gc. Second, GM3(Neu5Gc) was localized in myoblast cells, but not in myotube cells, and related to expression of the CMP-Neu5Ac hydroxylase (CMAH) gene. Notably, expression of CMAH precedes myotube formation not only in differentiating C2C12 cells, but also in mouse developing embryos. Since the myoblast cells were attached on the dish surface more strongly than the myotube cells, expression of GM3(Neu5Gc) may be related to the surface attachment of the myoblast cells. Third, exogenous Neu5Gc, but not Neu5Ac, promoted differentiation of C2C12 cells, thus increasing the number of cells committed to fuse with each other. Fourth, the CMAH-transfected C2C12 cells were attached on the gelatin-coated surface much more rapidly than the mock-cells, suggesting that the expression of CMAH promotes cell adhesiveness through the expression of Neu5Gc.

N-glycolylneuraminic acid serum biomarker levels are elevated in breast cancer patients at all stages of disease.

BACKGROUND: Normal human tissues do not express glycans terminating with the sialic acid N-glycolylneuraminic acid (Neu5Gc), yet Neu5Gc-containing glycans have been consistently found in human tumor tissues, cells and secretions and have been proposed as a cancer biomarker. We engineered a Neu5Gc-specific lectin called SubB2M, and previously reported elevated Neu5Gc biomarkers in serum from ovarian cancer patients using a Surface Plasmon Resonance (SPR)-based assay. Here we report an optimized SubB2M SPR-based assay and use this new assay to analyse sera from breast cancer patients for Neu5Gc levels. METHODS: To enhance specificity of our SPR-based assay, we included a non-sialic acid binding version of SubB, SubBA12, to control for any non-specific binding to SubB2M, which improved discrimination of cancer-free controls from early-stage ovarian cancer. We analysed 96 serum samples from breast cancer patients at all stages of disease compared to 22 cancer-free controls using our optimized SubB2M-A12-SPR assay. We also analysed a collection of serum samples collected at 6 monthly intervals from breast cancer patients at high risk for disease recurrence or spread. RESULTS: Analysis of sera from breast cancer cases revealed significantly elevated levels of Neu5Gc biomarkers at all stages of breast cancer. We show that Neu5Gc serum biomarker levels can discriminate breast cancer patients from cancer-free individuals with 98.96% sensitivity and 100% specificity. Analysis of serum collected prospectively, post-diagnosis, from breast cancer patients at high risk for disease recurrence showed a trend for a decrease in Neu5Gc levels immediately following treatment for those in remission. CONCLUSIONS: Neu5Gc serum biomarkers are a promising new tool for early detection and disease monitoring for breast cancer that may complement current imaging- and biopsy-based approaches.

N-Glycolylneuraminic Acid Binding of Avian and Equine H7 Influenza A Viruses.

Influenza A viruses (IAV) initiate infection by binding to glycans with terminal sialic acids on the cell surface. Hosts of IAV variably express two major forms of sialic acid, N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). NeuGc is produced in most mammals, including horses and pigs, but is absent in humans, ferrets, and birds. The only known naturally occurring IAV that exclusively bind NeuGc are extinct highly pathogenic equine H7N7 viruses. We determined the crystal structure of a representative equine H7 hemagglutinin (HA) in complex with NeuGc and observed high similarity in the receptor-binding domain with an avian H7 HA. To determine the molecular basis for NeuAc and NeuGc specificity, we performed systematic mutational analyses, based on the structural insights, on two distant avian H7 HAs and an H15 HA. We found that the A135E mutation is key for binding α2,3-linked NeuGc but does not abolish NeuAc binding. The additional mutations S128T, I130V, T189A, and K193R converted the specificity from NeuAc to NeuGc. We investigated the residues at positions 128, 130, 135, 189, and 193 in a phylogenetic analysis of avian and equine H7 HAs. This analysis revealed a clear distinction between equine and avian residues. The highest variability was observed at key position 135, of which only the equine glutamic acid led to NeuGc binding. These results demonstrate that genetically distinct H7 and H15 HAs can be switched from NeuAc to NeuGc binding and vice versa after the introduction of several mutations, providing insights into the adaptation of H7 viruses to NeuGc receptors. IMPORTANCE Influenza A viruses cause millions of cases of severe illness and deaths annually. To initiate infection and replicate, the virus first needs to bind to a structure on the cell surface, like a key fitting in a lock. For influenza A viruses, these "keys" (receptors) on the cell surface are chains of sugar molecules (glycans). The terminal sugar on these glycans is often either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc). Most influenza A viruses bind NeuAc, but a small minority bind NeuGc. NeuGc is present in species like horses, pigs, and mice but not in humans, ferrets, and birds. Here, we investigated the molecular determinants of NeuGc specificity and the origin of viruses that bind NeuGc.

Dietary Neu5Ac Intervention Protects Against Atherosclerosis Associated With Human-Like Neu5Gc Loss-Brief Report.

Objective: Species-specific pseudogenization of the CMAH gene during human evolution eliminated common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) biosynthesis from its precursor N-acetylneuraminic acid (Neu5Ac). With metabolic nonhuman Neu5Gc incorporation into endothelia from red meat, the major dietary source, anti-Neu5Gc antibodies appeared. Human-like Ldlr-/-Cmah-/- mice on a high-fat diet supplemented with a Neu5Gc-enriched mucin, to mimic human red meat consumption, suffered increased atherosclerosis if human-like anti-Neu5Gc antibodies were elicited. Approach and Results: We now ask whether interventional Neu5Ac feeding attenuates metabolically incorporated Neu5Gc-mediated inflammatory acceleration of atherogenesis in this Cmah-/-Ldlr-/- model system. Switching to a Neu5Gc-free high-fat diet or adding a 5-fold excess of Collocalia mucoid-derived Neu5Ac in high-fat diet protects against accelerated atherosclerosis. Switching completely from a Neu5Gc-rich to a Neu5Ac-rich diet further reduces severity. Remarkably, feeding Neu5Ac-enriched high-fat diet alone has a substantial intrinsic protective effect against atherosclerosis in Ldlr-/- mice even in the absence of dietary Neu5Gc but only in the human-like Cmah-null background. Conclusions: Interventional Neu5Ac feeding can mitigate or prevent the red meat/Neu5Gc-mediated increased risk for atherosclerosis, and has an intrinsic protective effect, even in the absence of Neu5Gc feeding. These findings suggest that similar interventions should be tried in humans and that Neu5Ac-enriched diets alone should also be investigated further.

Dietary spinach reshapes the gut microbiome in an Apc-mutant genetic background: mechanistic insights from integrated multi-omics.

Complex interrelationships govern the dynamic interactions between gut microbes, the host, and exogenous drivers of disease outcome. A multi-omics approach to cancer prevention by spinach (SPI) was pursued for the first time in the polyposis in rat colon (Pirc) model. SPI fed for 26 weeks (10% w/w, freeze-dried in the diet) exhibited significant antitumor efficacy and, in the Apc-mutant genetic background, ß-catenin remained highly overexpressed in adenomatous polyps. However, in both wild type and Apc-mutant rats, increased gut microbiome diversity after SPI consumption coincided with reversal of taxonomic composition. Metagenomic prediction implicated linoleate and butanoate metabolism, tricarboxylic acid cycle, and pathways in cancer, which was supported by transcriptomic and metabolomic analyses. Thus, tumor suppression by SPI involved marked reshaping of the gut microbiome along with changes in host RNA-miRNA networks. When colon polyps were compared with matched normal-looking tissues via metabolomics, anticancer outcomes were linked to SPI-derived linoleate bioactives with known anti-inflammatory/ proapoptotic mechanisms, as well as N-aceto-2-hydroxybutanoate, consistent with altered butanoate metabolism stemming from increased α-diversity of the gut microbiome. In colon tumors from SPI-fed rats, L-glutamate and N-acetylneuraminate also were reduced, implicating altered mitochondrial energetics and cell surface glycans involved in oncogenic signaling networks and immune evasion. In conclusion, a multi-omics approach to cancer prevention by SPI provided mechanistic support for linoleate and butanoate metabolism, as well as tumor-associated changes in L-glutamate and N-acetylneuraminate. Additional factors, such as the fiber content, also warrant further investigation with a view to delaying colectomy and drug intervention in at-risk patients.

N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus.

The first step in influenza virus infection is the binding of hemagglutinin to sialic acid-containing glycans present on the cell surface. Over 50 different sialic acid modifications are known, of which N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two main species. Animal models with α2,6 linked Neu5Ac in the upper respiratory tract, similar to humans, are preferred to enable and mimic infection with unadapted human influenza A viruses. Animal models that are currently most often used to study human influenza are mice and ferrets. Additionally, guinea pigs, cotton rats, Syrian hamsters, tree shrews, domestic swine, and non-human primates (macaques and marmosets) are discussed. The presence of NeuGc and the distribution of sialic acid linkages in the most commonly used models is summarized and experimentally determined. We also evaluated the role of Neu5Gc in infection using Neu5Gc binding viruses and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)-/- knockout mice, which lack Neu5Gc and concluded that Neu5Gc is unlikely to be a decoy receptor. This article provides a base for choosing an appropriate animal model. Although mice are one of the most favored models, they are hardly naturally susceptible to infection with human influenza viruses, possibly because they express mainly α2,3 linked sialic acids with both Neu5Ac and Neu5Gc modifications. We suggest using ferrets, which resemble humans closely in the sialic acid content, both in the linkages and the lack of Neu5Gc, lung organization, susceptibility, and disease pathogenesis.

The non-human glycan, N-glycolylneuraminic acid (Neu5Gc), is not expressed in all organs and skeletal muscles of nine animal species.

Red meat-derived sialic acid (Sia), N-glycolylneuraminic acid (Neu5Gc), promotes the risk of carcinoma and inflammation. Expression in skeletal muscle and organs across animal species remains unknown. We measured Neu5Gc in skeletal muscle and organ tissues from nine species using UHPLC and found that: (1) neu5Gc concentration in skeletal muscle was highest in goats (166 ± 48.7 µg/g protein), followed by cattle, pig, sheep, horse, cat and deer: >75% was conjugated. No Neu5Gc was detected in kangaroo and dog muscles; (2) total Neu5Gc in organ meats was generally about 2-54% higher than in muscle. Surprisingly Neu5Gc was absent in seven organs of female deer; (3) nine commercial ovine meat cuts contained similar Neu5Gc levels. Thus, red meat Neu5Gc concentration is tissue and species-specific and absent in muscle and organ tissue of some species. Our study provides guidelines for animal meat preferences for consumers and sheds light on the functionality of Neu5Gc.

High-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues are unique to porcine islets.

OBJECTIVES: Islet transplantation is an emerging treatment option for type 1 diabetes but its application is limited by the shortage of human pancreas donors. Characterization of the N- and O-glycan surface antigens that vary between human and genetically engineered porcine islet donors could shed light on targets of antibody mediated rejection. METHODS: N- and O-glycans were isolated from human and adult porcine islets and analyzed using matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS/MS). RESULTS: A total of 57 porcine and 34 human N-glycans and 21 porcine and 14 human O-glycans were detected from cultured islets. Twenty-eight of which were detected only from porcine islets, which include novel xenoantigens such as high-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues. Porcine islets have terminal N-glycolylneuraminic acid (NeuGc) residue in bi-antennary N-glycans and sialyl-Tn O-glycans. No galactose-α-1,3-galactose (α-Gal) or Sda epitope were detected on any of the islets. CONCLUSIONS: These results provide important insights into the potential antigenic differences of N- and O-glycan profiles between human and porcine islets. Glycan differences may identify novel gene targets for genetic engineering to generate superior porcine islet donors.

Sialic acids expression in newborn rat lungs: implications for pulmonary developmental biology.

Mammalian lung development proceeds during the postnatal period and continues throughout life. Intricate tubular systems of airways and vessels lined by epithelial cells are developed during this process. All cells, and particularly epithelial cells, carry an array of glycans on their surfaces. N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic (Neu5Gc) acids, two most frequently-occurring sialic acid residues, are essential determinants during development and in the homeostasis of cells and organisms. However, systematic data about the presence of cell surface sialic acids in the postnatal lung and their content is still scarce. In the present study, we addressed the histochemical localization of Neu5Ac > Neu5Gc in 0-day-old rat lungs. Furthermore, both residues were separated, identified and quantified in lung membranes isolated from 0-day-old rat lungs using high-performance liquid chromatography (HPLC) methodologies. Finally, we compared these results with those previously reported by us for adult rat lungs. The Neu5Ac > Neu5Gc residues were located on the surface of ciliated and non-ciliated cells and the median values for both residues in the purified lung membranes of newborn rats were 5.365 and 1.935 µg/mg prot., respectively. Comparing these results with those reported for the adults, it was possible to observe a significant difference between the levels of Neu5Ac and Neu5Gc (p < 0.001). A more substantial change was found for the case of Neu5Ac. The preponderance of Neu5Ac and its expressive increase during the postnatal development points towards a more prominent role of this residue. Bearing in mind that sialic acids are negatively charged molecules, the high content of Neu5Ac could contribute to the formation of an anion "shield" and have a role in pulmonary development and physiology.

Gram-Negative Bacterial Endotoxin LPS Induces NeuGc Loss through Ets1-Dependent Downregulation of Intestine-Specific pcmah Transcript in Porcine Intestinal Cells.

N-glycolylneuraminic acid (NeuGc), a non-human sialic acid derivative synthesized by cytidine-5'-monophospho-N-acetylneuraminic acid hydroxylase (CMAH), plays a crucial role in mediating infections by certain pathogens. Although it has been postulated that NeuGc biosynthesis and CMAH expression are downregulated during microbial infection, the underlying mechanisms remain unclear. The present study showed that exposure to lipopolysaccharide (LPS), a Gram-negative bacterial endotoxin, leads to loss of NeuGc biosynthesis in pig small intestinal I2I-2I cells. This LPS-induced NeuGc loss was accompanied by decreased CMAH transcript levels, especially intestine-specific 5'pcmah-1. Furthermore, LPS suppressed the activity of the Pi promoter responsible for 5'pcmah-1 by inhibiting DNA binding of Est1. These findings provide insight into the regulatory mechanisms of Neu5Gc biosynthesis during pathogenic infectious events, which may represent a host defense mechanism that protects the self against pathogenic bacterial infections even in non-sanitary environments.

Osmolality as a lever to modulate the N-glycolylneuraminicacid (Neu5Gc) level of a recombinant glycoprotein produced in Chinese hamster ovary cells.

Glycoproteins could be highly sialylated, and controlling the sialic acid levels for some therapeutic proteins is critical to ensure product consistency and efficacy. N-acetylneuraminic acid (Neu5Ac, or NANA) and N-glycolylneuraminic acid (Neu5Gc, or NGNA) are the two most common forms of sialic acids produced in mammalian cells. As Neu5Gc is not produced in humans and can elicit immune responses, minimizing Neu5Gc formation is important in controlling this quality attribute for complex glycoproteins. In this study, a sialylated glycoprotein was used as the model molecule to study the effect of culture osmolality on Neu5Gc. A 14-day fed-batch process with osmolality maintained at physiological levels produced high levels of Neu5Gc. Increase of culture osmolality reduced the Neu5Gc level up to 70-80%, and the effect was proportional to the osmolality level. Through evaluating different osmolality conditions (300-450 mOsm/kg) under low or high pCO2 , we demonstrated that osmolality could be an effective process lever to modulate the Neu5Gc level. Potential mechanism of osmolality impact on Neu5Gc is discussed and is hypothesized to be cytosol NADH availability related. Compared with cell line engineering efforts, this simple process lever provides the opportunity to readily modulate the Neu5Gc level in a cell culture environment.

Specific Detection of Neu5Gc in Animal Tissues by Immunohistochemistry.

One of the major obstacles in xenotransplantation is tissue expression of the non-human mammalian carbohydrate N-glycolylneuraminic acid (Neu5Gc). This 9-carbon backbone acidic sugar is the hydroxylated form of N-acetylneuraminic acid (Neu5Ac), and both constitute the two most common sialic acid types in mammals. Loss of CMP-Neu5Ac hydroxylase encoding gene in humans dictates the immunogenic nature of Neu5Gc-containing xenografts. Here we describe an immunohistochemistry method for the detection of Neu5Gc in mammalian-derived tissues using affinity-purified chicken anti-Neu5Gc IgY. Specificity is further demonstrated by competitive inhibition with free Neu5Gc or Neu5Gc-glycoproteins, but not with Neu5Ac or Neu5Ac-glycoproteins. This method can be used to evaluate potential Neu5Gc-immunogenicity of xenografts.

Influence of sulfonated and diet-derived human milk oligosaccharides on the infant microbiome and immune markers.

Human milk oligosaccharides (HMOs) promote the development of the neonatal intestinal, immune, and nervous systems and has recently received considerable attention. Here we investigated how the maternal diet affects HMO biosynthesis and how any diet-induced HMO alterations influence the infant gut microbiome and immunity. Using capillary electrophoresis and MS-based analyses, we extracted and measured HMOs from breast milk samples and then correlated their levels with results from validated 24-h diet recall surveys and breast milk fatty acids. We found that fruit intake and unsaturated fatty acids in breast milk were positively correlated with an increased absolute abundance of numerous HMOs, including 16 sulfonated HMOs we identified here in humans for the first time. The diet-derived monosaccharide 5-N-glycolyl-neuraminic acid (Neu5Gc) was unambiguously detected in all samples. To gain insights into the potential impact of Neu5Gc on the infant microbiome, we used a constrained ordination approach and identified correlations between Neu5Gc levels and Bacteroides spp. in infant stool. However, Neu5Gc was not associated with marked changes in infant immune markers, in contrast with sulfonated HMOs, whose expression correlated with suppression of two major Th2 cytokines, IL-10 and IL-13. The findings of our work highlight the importance of maternal diet for HMO biosynthesis and provide as yet unexplored targets for future studies investigating interactions between HMOs and the intestinal microbiome and immunity in infants.

Effects of sialic acid linkage on antibody-fragment crystallizable receptor binding and antibody dependent cytotoxicity depend on levels of fucosylation/bisecting.

Aim: Fragment crystallizable (Fc) glycosylation of immunoglobulin G-type monoclonal antibodies applied to therapeutic applications is regarded a critical quality attribute and can influence bioactivity, pharmacokinetics and/or immunogenicity/safety. Investigating the impact of certain Fc N-glycans is therefore of importance to assess its criticality for a therapeutic product. This has been done for N-glycan types like fucosylation, galactosylation or sialylation. There were contradictory results reported for functionality especially with regard to sialylation. Material & methods: We elucidated the effect of terminal sialic acid residues on Fcγ receptor binding and antibody dependent cytotoxicity activity of two immunoglobulin G1 antibodies with different levels of fucosylation/bi-secting. Conclusion: We found the impact to be specific to the sialylation linkage type, in other words, α2,3- versus α2,6-linked sialic acid attached to the terminal galactose residues.