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Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y.pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y.pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y.pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.

Many pathogenic bacteria secrete AB

Dietary habits have been associated with alterations of the human gut resident microorganisms contributing to obesity, diabetes, and cancer1. In Western diets, red meat is one of the frequently eaten foods2, but long-term consumption has been associated with increased risk of disease3,4. Red meat is enriched in N-glycolylneuraminic acid (Neu5Gc) that cannot be synthesized by humans5. However, consumption can cause Neu5Gc incorporation into cell surface glycans6, especially in carcinomas4,7. As a consequence, an inflammatory response is triggered when Neu5Gc-containing glycans encounter circulating anti-Neu5Gc antibodies8,9. Although bacteria can utilize free sialic-acids as a nutrient source10-12, it is currently unknown if gut microorganisms contribute to releasing Neu5Gc from food. We found that a Neu5Gc-rich diet induces changes in the gut microbiota, with Bacteroidales and Clostridiales responding for the most. Genome assembling of mouse and human shotgun metagenomic sequencing identified bacterial sialidases with previously unobserved substrate preference for Neu5Gc-containing glycans. X-ray crystallography revealed key amino-acids potentially contributing to substrate preference. Additionally, we verified that mouse and human sialidases were able to release Neu5Gc from red meat. The release of Neu5Gc from red meat using bacterial sialidases could reduce the risk of inflammatory diseases associated with red meat consumption, including colorectal cancer4 and atherosclerosis13.

Hypertriglyceridemia results from accumulation of triglyceride (TG)-rich lipoproteins (TRLs) in the circulation and is associated with increased CVD risk. ApoC-III is an apolipoprotein on TRLs and a prominent negative regulator of TG catabolism. We recently established that in vivo apoC-III predominantly inhibits LDL receptor-mediated and LDL receptor-related protein 1-mediated hepatic TRL clearance and that apoC-III-enriched TRLs are preferentially cleared by syndecan-1 (SDC1). In this study, we determined the impact of apoE, a common ligand for all three receptors, on apoC-III metabolism using apoC-III antisense oligonucleotide (ASO) treatment in mice lacking apoE and functional SDC1 (Apoe -/- Ndst1 f/f Alb-Cre +). ApoC-III ASO treatment significantly reduced plasma TG levels in Apoe -/- Ndst1 f/f Alb-Cre + mice without reducing hepatic VLDL production or improving hepatic TRL clearance. Further analysis revealed that apoC-III ASO treatment lowered plasma TGs in Apoe -/- Ndst1 f/f Alb-Cre + mice, which was associated with increased LPL activity in white adipose tissue in the fed state. Finally, clinical data confirmed that ASO-mediated lowering of APOC-III via volanesorsen can reduce plasma TG levels independent of the APOE isoform genotype. Our data indicate that apoE determines the metabolic impact of apoC-III as we establish that apoE is essential to mediate inhibition of TRL clearance by apoC-III and that, in the absence of functional apoE, apoC-III inhibits tissue LPL activity.

A well known, epidemiologically reproducible risk factor for human carcinomas is the long-term consumption of "red meat" of mammalian origin. Although multiple theories have attempted to explain this human-specific association, none have been conclusively proven. We used an improved method to survey common foods for free and glycosidically bound forms of the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc), showing that it is highly and selectively enriched in red meat. The bound form of Neu5Gc is bioavailable, undergoing metabolic incorporation into human tissues, despite being a foreign antigen. Interactions of this antigen with circulating anti-Neu5Gc antibodies could potentially incite inflammation. Indeed, when human-like Neu5Gc-deficient mice were fed bioavailable Neu5Gc and challenged with anti-Neu5Gc antibodies, they developed evidence of systemic inflammation. Such mice are already prone to develop occasional tumors of the liver, an organ that can incorporate dietary Neu5Gc. Neu5Gc-deficient mice immunized against Neu5Gc and fed bioavailable Neu5Gc developed a much higher incidence of hepatocellular carcinomas, with evidence of Neu5Gc accumulation. Taken together, our data provide an unusual mechanistic explanation for the epidemiological association between red meat consumption and carcinoma risk. This mechanism might also contribute to other chronic inflammatory processes epidemiologically associated with red meat consumption.

Certain pathogenic bacteria are known to modulate the innate immune response by decorating themselves with sialic acids, which can engage the myelomonocytic lineage inhibitory receptor Siglec-9, thereby evading immunosurveillance. We hypothesized that the well-known up-regulation of sialoglycoconjugates by tumors might similarly modulate interactions with innate immune cells. Supporting this hypothesis, Siglec-9-expressing myelomonocytic cells found in human tumor samples were accompanied by a strong up-regulation of Siglec-9 ligands. Blockade of Siglec-9 enhanced neutrophil activity against tumor cells in vitro. To investigate the function of inhibitory myelomonocytic Siglecs in vivo we studied mouse Siglec-E, the murine functional equivalent of Siglec-9. Siglec-E-deficient mice showed increased in vivo killing of tumor cells, and this effect was reversed by transgenic Siglec-9 expression in myelomonocytic cells. Siglec-E-deficient mice also showed enhanced immunosurveillance of autologous tumors. However, once tumors were established, they grew faster in Siglec-E-deficient mice. In keeping with this, Siglec-E-deficient macrophages showed a propensity toward a tumor-promoting M2 polarization, indicating a secondary role of CD33-related Siglecs in limiting cancer-promoting inflammation and tumor growth. Thus, we define a previously unidentified impact of inhibitory myelomonocytic Siglecs in cancer biology, with distinct roles that reflect the dual function of myelomonocytic cells in cancer progression. In keeping with this, a human polymorphism that reduced Siglec-9 binding to carcinomas was associated with improved early survival in non-small-cell lung cancer patients, which suggests that Siglec-9 might be therapeutically targeted within the right time frame and stage of disease.

Humans can incorporate the xenoglycan N-glycolylneuraminic acid (Neu5Gc) from the diet into reproductive tissues and secretions. Most humans also have circulating antibodies specific for this dietary xenoglycan. The potential for inflammation induced by incorporated Neu5Gc and circulating anti-Neu5Gc antibodies, termed xenosialitis, has been discussed as a factor influencing several human diseases. Potential effects of xenosialitis on human fertility remain unknown. Here, we investigate possible adverse effects of the presence of Neu5Gc on sperm or endometrium combined with anti-Neu5Gc antibodies in semen or uterine secretions in a mouse model. We use Cmah(-/-) mice, humanized for Neu5Gc deficiency. We find that the viability, migration, and capacitation of sperm with incorporated Neu5Gc are negatively affected when these are exposed to anti-Neu5Gc antibodies. In addition, we find that after copulation, activated uterine neutrophils and macrophages show increased phagocytosis of sperm in the presence of anti-Neu5Gc antibodies via the complement receptor 3 (C3R) and Fcγ I/II/III (Fc receptor). Furthermore, Neu5Gc in endometrial cells combined with the presence of anti-Neu5Gc antibodies alters the receptivity and decidualization of endometrial explants. These studies provide mechanistic insights on how Neu5Gc on sperm and/or endometrium combined with anti-Neu5Gc antibodies in semen and uterine fluid might contribute to unexplained human infertility.

Background: Mammalian sperm lose sialic acids during capacitation through unknown mechanisms. Results: Sialidases Neu1 and Neu3 are present on sperm. Their activity is required for capacitation and zona pellucida binding. Conclusion: Sperm sialidases modulate sperm surface sialic acids en route to fertilization. Significance: Understanding the mechanism of deciduous sialylation in sperm provides novel insights into sperm function and glycan-mediated fertility., Sialic acids (Sias) mediate many biological functions, including molecular recognition during development, immune response, and fertilization. A Sia-rich glycocalyx coats the surface of sperm, allowing them to survive as allogeneic cells in the female reproductive tract despite female immunity. During capacitation, sperm lose a fraction of their Sias. We quantified shed Sia monosaccharides released from capacitated sperm and measured sperm sialidase activity. We report the presence of two sialidases (neuraminidases Neu1 and Neu3) on mammalian sperm. These are themselves shed from sperm during capacitation. Inhibiting sialidase activity interferes with sperm binding to the zona pellucida of the ovum. A survey of human sperm samples for the presence of sialidases NEU1 and NEU3 identified a lack of one or both sialidases in sperm of some male idiopathic infertility cases. The results contribute new insights into the dynamic remodeling of the sperm glycocalyx prior to fertilization.

Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1 , an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1 HEP mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1 HEP mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1 HEP mice. FX remained essential for Ad5 transduction in vivo in Ext1 HEP mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo . This study reopens the question of how adenovirus enters cells in vivo . IMPORTANCE Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo . The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptor in vivo and emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cells in vivo and for optimization of Ad5 vectors as therapeutic agents.

Aging is a multifactorial process that includes the lifelong accumulation of molecular damage, leading to age-related frailty, disability and disease, and eventually death. In this study, we report evidence of a significant correlation between the number of genes encoding the immunomodulatory CD33-related sialic acid-binding immunoglobulin-like receptors (CD33rSiglecs) and maximum lifespan in mammals. In keeping with this, we show that mice lacking Siglec-E, the main member of the CD33rSiglec family, exhibit reduced survival. Removal of Siglec-E causes the development of exaggerated signs of aging at the molecular, structural, and cognitive level. We found that accelerated aging was related both to an unbalanced ROS metabolism, and to a secondary impairment in detoxification of reactive molecules, ultimately leading to increased damage to cellular DNA, proteins, and lipids. Taken together, our data suggest that CD33rSiglecs co-evolved in mammals to achieve a better management of oxidative stress during inflammation, which in turn reduces molecular damage and extends lifespan.

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.

Humans lack the common mammalian cell surface molecule N -glycolylneuraminic acid (Neu5Gc) due to a CMAH gene inactivation, which occurred approximately three million years ago. Modern humans produce antibodies specific for Neu5Gc. We hypothesized that anti-Neu5Gc antibodies could enter the female reproductive tract and target Neu5Gc-positive sperm or fetal tissues, reducing reproductive compatibility. Indeed, female mice with a human-like Cmah (−/−) mutation and immunized to express anti-Neu5Gc antibodies show lower fertility with Neu5Gc-positive males, due to prezygotic incompatibilities. Human anti-Neu5Gc antibodies are also capable of targeting paternally derived antigens and mediate cytotoxicity against Neu5Gc-bearing chimpanzee sperm in vitro. Models of populations polymorphic for such antigens show that reproductive incompatibility by female immunity can drive loss-of-function alleles to fixation from moderate initial frequencies. Initially, the loss of a cell-surface antigen can occur due to drift in isolated populations or when natural selection favors the loss of a receptor exploited by pathogens, subsequently the same loss-of-function allele can come under sexual selection because it avoids being targeted by the female immune system. Thus, we provide evidence of a link between sexual selection and immune function: Antigenicity in females can select against foreign paternal antigens on sperm and rapidly fix loss-of-function alleles. Similar circumstances existed when the CMAH null allele was polymorphic in ancestral hominins, just before the divergence of Homo from australopithecines.

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. Cancer Res; 71(9); 3352–63. ©2011 AACR.

Wingless and iNT-1 (WNT) pathway members are critical for pancreatic development and exocrine tissue formation. Recently, much attention has focused on delineating the roles of beta-catenin in pancreatic organogenesis. However, little is known about the involvement of beta-catenin in the endocrine or exocrine function of the mature pancreas. We report for the first time the impact of beta-catenin deletion in the pancreatic beta cells.We targeted the deletion of the beta-catenin gene in pancreatic beta cells by crossing a floxed beta-catenin mouse strain with a RIP-Cre mouse strain.Surprisingly, the majority of the mutant mice died shortly after birth and had deregulated glucose and insulin levels. The newborn mutant pancreases demonstrated increased insulin content, reflecting a defect in insulin release confirmed in vitro. Moreover, there was a reduction in total endocrine tissue at birth, while cellularity in islets was greater, suggesting that lack of beta-catenin affects beta cell size. Some newborns survived beta-catenin deletion and showed a milder phenotype during adulthood.The deletion of beta-catenin in the maturing beta cells negatively impacts on islet morphology and function. This work reveals that lack of beta-catenin in early life is related to severe deregulation of glucose homeostasis.

Although lymphocyte infiltration and islet destruction are hallmarks of diabetes, the mechanisms of beta-cell destruction are not fully understood. One issue that remains unresolved is whether cytokines play a direct role in beta-cell death. We investigated whether beta-cell cytokine signaling contributes to autoimmune type 1 diabetes. We demonstrated that NOD mice harboring beta-cells expressing the suppressor of cytokine signaling-1 (SOCS-1), an inhibitor of Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, have a markedly reduced incidence of diabetes. Similar to their non-transgenic (Tg) littermates, SOCS-1-Tg mice develop insulitis and their splenocytes transfer disease to NODscid recipients. Disease protection correlates with suppression of cytokine-induced STAT-1 phosphorylation in SOCS-1-expressing beta-cells and with a reduced sensitivity of these cells to destruction by diabetogenic cells in vivo. Interestingly, lymphocytes recruited to the pancreas of SOCS-1-Tg mice transferred diabetes to NODscid recipients with a reduced efficiency, suggesting that the pancreatic environment in SOCS-1-Tg mice does not support the maintenance of functionally differentiated T-cells. These results suggest that cytokines contribute to the development of type 1 diabetes by acting directly on the target beta-cell. Importantly, given that the SOCS-1-expressing mouse maintain normal blood glucose levels throughout life, this study also showed that SOCS-1 expression by beta-cells can represent a promising strategy to prevent type 1 diabetes.