Anti-pig antibodies in swine veterinarian serum: Implications for clinical xenotransplantation.

Recent clinical xenotransplantation and human decedent studies demonstrate that clinical hyperacute rejection of genetically engineered porcine organs can be reliably avoided but that antibody mediated rejection (AMR) continues to limit graft survival. We previously identified porcine glycans and proteins which are immunogenic after cardiac xenotransplantation in non-human primates, but the clinical immune response to antigens present in glycan depleted triple knockout (TKO) donor pigs is poorly understood. In this study we use fluorescence barcoded human embryonic kidney cells (HEK) and HEK cell lines expressing porcine glycans (Gal and SDa) or proteins (tetraspanin-29 [CD9], membrane cofactor protein [CD46], protectin, membrane attack complex inhibition factor [CD59], endothelial cell protein C receptor, and Annexin A2) to screen antibody reactivity in human serum from 160 swine veterinarians, a serum source with potential occupational immune challenge from porcine tissues and pathogens. High levels of anti-Gal IgM were present in all samples and lower levels of anti-SDa IgM were present in 41% of samples. IgM binding to porcine proteins, primarily CD9 and CD46, previously identified as immunogenic in pig to non-human primate cardiac xenograft recipients, was detected in 28 of the 160 swine veterinarian samples. These results suggest that barcoded HEK cell lines expressing porcine protein antigens can be useful for screening human patient serum. A comprehensive analysis of sera from clinical xenotransplant recipients to define a panel of commonly immunogenic porcine antigens will likely be necessary to establish an array of porcine non-Gal antigens for effective monitoring of patient immune responses and allow earlier therapies to reverse AMR.

CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan.

Invasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only known pathogen recognition receptor on NK cells triggering potent antifungal activity against Aspergillus fumigatus. However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, and ger mutants with altered cell wall composition, we herein found that CD56 interacts with the A. fumigatus cell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK-cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identified A. fumigatus GAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells.

High-mannose glycans from Schistosoma mansoni eggs are important for priming of Th2 responses via Dectin-2 and prostaglandin E2.

The parasitic helminth Schistosoma mansoni is a potent inducer of type 2 immune responses by stimulating dendritic cells (DCs) to prime T helper 2 (Th2) responses. We previously found that S. mansoni soluble egg antigens (SEA) promote the synthesis of Prostaglandin E2 (PGE2) by DCs through ERK-dependent signaling via Dectin-1 and Dectin-2 that subsequently induces OX40L expression, licensing them for Th2 priming, yet the ligands present in SEA involved in driving this response and whether specific targeting of PGE2 synthesis by DCs could affect Th2 polarization are unknown. We here show that the ability of SEA to bind Dectin-2 and drive ERK phosphorylation, PGE2 synthesis, OX40L expression, and Th2 polarization is impaired upon cleavage of high-mannose glycans by Endoglycosidase H treatment. This identifies high-mannose glycans present on glycoproteins in SEA as important drivers of this signaling axis. Moreover, we find that OX40L expression and Th2 induction are abrogated when microsomal prostaglandin E synthase-1 (mPGES) is selectively inhibited, but not when a general COX-1/2 inhibitor is used. This shows that the de novo synthesis of PGE2 is vital for the Th2 priming function of SEA-stimulated DCs as well as points to the potential existence of other COX-dependent lipid mediators that antagonize PGE2-driven Th2 polarization. Lastly, specific PGE2 inhibition following immunization with S. mansoni eggs dampened the egg-specific Th cell response. In summary, our findings provide new insights in the molecular mechanisms underpinning Th2 induction by S. mansoni and identify druggable targets for potential control of helminth driven-Th2 responses.

Methods of Cryptococcal Polysaccharide Analysis Using ELISA.

One of the standard assays for the fungal pathogen Cryptococcus neoformans is the glucuronoxylomannan (GXM) ELISA. This assay utilizes monoclonal antibodies targeted against the critical virulence factor, the polysaccharide (PS) capsule. GXM ELISA is one of the most used assays in the field used for diagnosis of cryptococcal infection, quantification of PS content, and determination of binding specificity for antibodies. Here we present three variations of the GXM ELISA used by our group-indirect, capture, and competition ELISAs. We have also provided some history, perspective, and notes on these methods, which we hope will help the reader choose, and implement, the best assay for their research.While it has long been referred to as the GXM ELISA, we also suggest a name update to better reflect our updated understanding of the polysaccharide antigens targeted by this assay. The Cryptococcal PS ELISA is a more accurate description of this set of methodologies and the antigens they measure. Finally, we discuss the limitations of this assay and put forth future plans for expanding the antigens assayed by ELISA.

Semisynthetic Glycoconjugate Vaccine Candidates against Cryptococcus neoformans.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, which poses a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semisynthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semisynthetic glycoconjugate vaccines contain an identical synthetic decasaccharide (M2 motif) antigen. This antigen is present in serotype A strains, which constitute 95% of the clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity toward M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced weakly opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). These findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. This antigen could serve as a component in a multivalent GXM motif vaccine.

Macrophage- and CD4+ T cell-derived SIV differ in glycosylation, infectivity and neutralization sensitivity.

The human immunodeficiency virus (HIV) envelope protein (Env) mediates viral entry into host cells and is the primary target for the humoral immune response. Env is extensively glycosylated, and these glycans shield underlying epitopes from neutralizing antibodies. The glycosylation of Env is influenced by the type of host cell in which the virus is produced. Thus, HIV is distinctly glycosylated by CD4+ T cells, the major target cells, and macrophages. However, the specific differences in glycosylation between viruses produced in these cell types have not been explored at the molecular level. Moreover, it remains unclear whether the production of HIV in CD4+ T cells or macrophages affects the efficiency of viral spread and resistance to neutralization. To address these questions, we employed the simian immunodeficiency virus (SIV) model. Glycan analysis implied higher relative levels of oligomannose-type N-glycans in SIV from CD4+ T cells (T-SIV) compared to SIV from macrophages (M-SIV), and the complex-type N-glycans profiles seem to differ between the two viruses. Notably, M-SIV demonstrated greater infectivity than T-SIV, even when accounting for Env incorporation, suggesting that host cell-dependent factors influence infectivity. Further, M-SIV was more efficiently disseminated by HIV binding cellular lectins. We also evaluated the influence of cell type-dependent differences on SIV's vulnerability to carbohydrate binding agents (CBAs) and neutralizing antibodies. T-SIV demonstrated greater susceptibility to mannose-specific CBAs, possibly due to its elevated expression of oligomannose-type N-glycans. In contrast, M-SIV exhibited higher susceptibility to neutralizing sera in comparison to T-SIV. These findings underscore the importance of host cell-dependent attributes of SIV, such as glycosylation, in shaping both infectivity and the potential effectiveness of intervention strategies.

Human T-cell activation with Toxoplasma gondii antigens loaded in maltodextrin nanoparticles.

Toxoplasmosis is the most prevalent parasitic zoonosis worldwide, causing ocular and neurological diseases. No vaccine has been approved for human use. We evaluated the response of peripheral blood mononuclear cells (PBMCs) to a novel construct of Toxoplasma gondii total antigen in maltodextrin nanoparticles (NP/TE) in individuals with varying infectious statuses (uninfected, chronic asymptomatic, or ocular toxoplasmosis). We analyzed the concentration of IFN-γ after NP/TE ex vivo stimulation using ELISA and the immunophenotypes of CD4+ and CD8+ cell populations using flow cytometry. In addition, serotyping of individuals with toxoplasmosis was performed by ELISA using GRA6-derived polypeptides. Low doses of NP/TE stimulation (0.9 µg NP/0.3 µg TE) achieved IFN-γ-specific production in previously exposed human PBMCs without significant differences in the infecting serotype. Increased IFN-γ expression in CD4+ effector memory cell subsets was found in patients with ocular toxoplasmosis with NP/TE but not with TE alone. This is the first study to show how T-cell subsets respond to ex vivo stimulation with a vaccine candidate for human toxoplasmosis, providing crucial insights for future clinical trials.

Impaired Response to Polysaccharide Vaccine in Selective IgE Deficiency.

PURPOSE: Immunoglobulin E deficiency (IgED) (defined as IgE < 2 IU/mL) is enriched in patients with primary antibody deficiency (PAD). We hypothesized that selective IgED (sIgED) is a more sensitive predictor of the development of PAD than declining IgG, as IgE production typically requires two class switch recombination (CSR) events in contrast to IgG. Thus, the inability of patients with sIgED to mount an appropriate antibody response to a T-cell independent antigen or evidence of aberrant induction of ɛ germ line (ɛGL) or IgE heavy chain (IgEHC) transcripts in vitro would support the concept that sIgED is a biomarker for emerging PAD. METHODS: We compared pre- and post-polysaccharide vaccination titers in healthy patients with sIgED without a history of recurrent infections or autoimmunity (n = 20) and in healthy controls (HCs) (n = 17). Subsequently, we assessed in vitro induction of εGL and IgEHC transcripts in patients with sIgED and HC (n = 6) in response to IL-4 + CD40L stimulation. RESULTS: Thirty percent of patients with sIgED did not have a robust vaccine response compared to 0% of HCs (p = 0.017). Individuals with sIgED with an abnormal vaccine response demonstrated persistent germline mRNA expression in their B-cells at day 5, with lower levels of IgEHC, compared to both HCs and sIgED participants with a normal vaccine response. CONCLUSION: Patients with sIgED are more likely to have abnormal antibody responses to a T cell-independent antigen and may have dysregulated CSR machinery. Following individuals with sIgED longitudinally may be beneficial in the early identification of PAD.

Novel method to specifically determine the structures of non-N297 glycans in IgGs.

Monoclonal antibodies comprise a major class of biologic therapeutics and are also extensively studied in immunology. Given the importance of glycans on antibodies, fluorescent labeling of enzymatically released glycans and their LC/MS analysis is routinely used for in-depth characterization of antibody glycosylation. In this technical note, we propose a method for facile characterization of glycans in the variable region of antibodies using sequential enzymatic digests with Endoglycosidase-S2 and RapidTM Peptide-N-Glycosidase-F followed by labeling with a fluorescent dye carrying an NHS-carbamate moiety. The results and proposed mechanism also suggest that the choice of glycosidases along with the labeling chemistry is critical for accurate glycan analysis for a desired application.

Variations within the Glycan Shield of SARS-CoV-2 Impact Viral Spike Dynamics.

The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity, whether arisen naturally or through vaccination. Understanding the structure of the viral spike assists in determining the impact of mutations on the antigenic surface. One class of mutation impacts glycosylation attachment sites, which have the capacity to influence the antigenic structure beyond the immediate site of attachment. Here, we compare the site-specific glycosylation of recombinant viral spike mimetics of B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), B.1.1.529 (Omicron). The P.1 strain exhibits two additional N-linked glycan sites compared to the other variants analyzed and we investigate the impact of these glycans by molecular dynamics. The acquired N188 site is shown to exhibit very limited glycan maturation, consistent with limited enzyme accessibility. Structural modeling and molecular dynamics reveal that N188 is located within a cavity by the receptor binding domain, which influences the dynamics of these attachment domains. These observations suggest a mechanism whereby mutations affecting viral glycosylation sites have a structural impact across the protein surface.

Modification of Hinge/Transmembrane and Signal Transduction Domains Improves the Expression and Signaling Threshold of GXMR-CAR Specific to Cryptococcus spp.

Chimeric antigen receptors (CARs) redirect T cells to recognize a specific target. CAR components play a pivotal role in antigen specificity, structure stability, expression on cell surface, and induction of cellular activation, which together determine the success of CAR T-cell therapy. CAR products targeting B-cell lymphoma encouraged the development of new CAR applications beyond cancer. For example, our group developed a CAR to specifically target glucuronoxylomannan (GXM) in the capsule of Cryptococcus species, called GXMR-CAR or GXMR-IgG4-28ζ. Cryptococcus are fungi that cause the life-threatening disease cryptococcosis, and GXMR-IgG4-28ζ redirected T cells to target yeast and titan cell forms of Cryptococcus spp. Here, we replaced the IgG4-hinge and CD28-transmembrane domains from GXMR-CAR with a CD8α molecule as the hinge/transmembrane and used CD28 or 4-1BB molecules as co-stimulatory domains, creating GXMR-8-28ζ and GXMR-8-BBζ, respectively. Jurkat cells expressing GXMR-CAR containing CD8α as the hinge/transmembrane improved the CAR expression and induced a tonic signaling. GXMR-8-28ζ and GXMR-8-BBζ induced high levels of IL-2 and up-regulation of CD69 expression in the presence of reference strains of C. neoformans and C. gattii. Moreover, GXMR-8-28ζ and GXMR-8-BBζ showed increased strength in response to incubation with clinical isolates of Cryptococcuss spp., and 4-1BB co-stimulatory domain triggered a more pronounced cellular activation. Dasatinib, a tyrosine kinase inhibitor, attenuated the GXMR-CAR signaling cascade's engagement in the presence or absence of its ligand. This study optimized novel second-generation GXMR-CARs containing the CD8-hinge/transmembrane domain that improved CAR expression, antigen recognition, and signal strength in T-cell activation.

The weaker-binding Fc γ receptor IIIa F158 allotype retains sensitivity to N-glycan composition and exhibits a destabilized antibody-binding interface.

Antibodies engage Fc γ receptors (FcγRs) to elicit healing cellular immune responses following binding to a target antigen. Fc γ receptor IIIa/CD16a triggers natural killer cells to destroy target tissues with cytotoxic proteins and enhances phagocytosis mediated by macrophages. Multiple variables affect CD16a antibody-binding strength and the resulting immune response, including a genetic polymorphism. The predominant CD16a F158 allotype binds antibodies with less affinity than the less common V158 allotype. This polymorphism likewise affects cellular immune responses and clinical efficacy of antibodies relying on CD16a engagement, though it remains unclear how V/F158 affects CD16a structure. Another relevant variable shown to affect affinity is composition of the CD16a asparagine-linked (N)-glycans. It is currently not known how N-glycan composition affects CD16a F158 affinity. Here, we determined N-glycan composition affects the V158 and F158 allotypes similarly, and N-glycan composition does not explain differences in V158 and F158 binding affinity. Our analysis of binding kinetics indicated the N162 glycan slows the binding event, and shortening the N-glycans or removing the N162 glycan increased the speed of binding. F158 displayed a slower binding rate than V158. Surprisingly, we found N-glycan composition had a smaller effect on the dissociation rate. We also identified conformational heterogeneity of CD16a F158 backbone amide and N162 glycan resonances using NMR spectroscopy. Residues exhibiting chemical shift perturbations between V158 and F158 mapped to the antibody-binding interface. These data support a model for CD16a F158 with increased interface conformational heterogeneity, reducing the population of binding-competent forms available and decreasing affinity.

N-Linked Glycans Shape Skin Immune Responses during Arthritis and Myositis after Intradermal Infection with Ross River Virus.

Arthritogenic alphaviruses are mosquito-borne arboviruses that include several re-emerging human pathogens, including the chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), and o'nyong-nyong (ONNV) virus. Arboviruses are transmitted via a mosquito bite to the skin. Herein, we describe intradermal RRV infection in a mouse model that replicates the arthritis and myositis seen in humans with Ross River virus disease (RRVD). We show that skin infection with RRV results in the recruitment of inflammatory monocytes and neutrophils, which together with dendritic cells migrate to draining lymph nodes (LN) of the skin. Neutrophils and monocytes are productively infected and traffic virus from the skin to LN. We show that viral envelope N-linked glycosylation is a key determinant of skin immune responses and disease severity. RRV grown in mammalian cells elicited robust early antiviral responses in the skin, while RRV grown in mosquito cells stimulated poorer early antiviral responses. We used glycan mass spectrometry to characterize the glycan profile of mosquito and mammalian cell-derived RRV, showing deglycosylation of the RRV E2 glycoprotein is associated with curtailed skin immune responses and reduced disease following intradermal infection. Altogether, our findings demonstrate skin infection with an arthritogenic alphavirus leads to musculoskeletal disease and envelope glycoprotein glycosylation shapes disease outcome. IMPORTANCE Arthritogenic alphaviruses are transmitted via mosquito bites through the skin, potentially causing debilitating diseases. Our understanding of how viral infection starts in the skin and how virus systemically disseminates to cause disease remains limited. Intradermal arbovirus infection described herein results in musculoskeletal pathology, which is dependent on viral envelope N-linked glycosylation. As such, intradermal infection route provides new insights into how arboviruses cause disease and could be extended to future investigations of skin immune responses following infection with other re-emerging arboviruses.

Analysis of the glyco-code in pancreatic ductal adenocarcinoma identifies glycan-mediated immune regulatory circuits.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a 5-year survival rate of only 9%. Despite the fact that changes in glycosylation patterns during tumour progression have been reported, no systematic approach has been conducted to evaluate its potential for patient stratification. By analysing publicly available transcriptomic data of patient samples and cell lines, we identified here two specific glycan profiles in PDAC that correlated with progression, clinical outcome and epithelial to mesenchymal transition (EMT) status. These different glycan profiles, confirmed by glycomics, can be distinguished by the expression of O-glycan fucosylated structures, present only in epithelial cells and regulated by the expression of GALNT3. Moreover, these fucosylated glycans can serve as ligands for DC-SIGN positive tumour-associated macrophages, modulating their activation and inducing the production of IL-10. Our results show mechanisms by which the glyco-code contributes to the tolerogenic microenvironment in PDAC.

CAR-Ts: new perspectives in cancer therapy.

Chimeric antigen receptor (CAR)-T-cell therapy is a promising anticancer treatment that exploits the host's immune system to fight cancer. CAR-T cell therapy relies on immune cells being modified to express an artificial receptor targeting cancer-specific markers, and infused into the patients where they will recognize and eliminate the tumour. Although CAR-T cell therapy has produced encouraging outcomes in patients with haematologic malignancies, solid tumours remain challenging to treat, mainly due to the lack of cancer-specific molecular targets and the hostile, often immunosuppressive, tumour microenvironment. CAR-T cell therapy also depends on the quality of the injected product, which is closely connected to CAR design. Here, we explain the technology of CAR-Ts, focusing on the composition of CARs, their application, and limitations in cancer therapy, as well as on the current strategies to overcome the challenges encountered. We also address potential future targets to overcome the flaws of CAR-T cell technology in the treatment of cancer, emphasizing glycan antigens, the aberrant forms of which attain high tumour-specific expression, as promising targets for CAR-T cell therapy.

Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drives rapid and potent immunogenicity capable of single-dose protection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may target epitopes that reduce durability or increase the potential for escape from vaccine-induced immunity. Using synthetic vaccinology, we have developed rationally immune-focused SARS-CoV-2 Spike-based vaccines. Glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and in vitro screening, we have incorporated glycans into the receptor-binding domain (RBD) and assessed antigenic profiles. We demonstrate that glycan-coated RBD immunogens elicit stronger neutralizing antibodies and have engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicits potent neutralizing antibodies in guinea pigs, hamsters, and multiple mouse models, including human ACE2 and human antibody repertoire transgenics. RBD nanoparticles induce high levels of cross-neutralizing antibodies against variants of concern with durable titers beyond 6 months. Single, low-dose immunization protects against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of potent and durable coronavirus vaccines.

Sulfation Pattern Dependent Iron(III) Mediated Interleukin-8 Glycan Binding.

Cytokines such as interleukin-8 activate the immune system during infection and interact with sulfated glycosaminoglycans with specific sulfation patterns. In some cases, these interactions are mediated by metal ion binding which can be used to tune surface-based glycan-protein interactions. We evaluated the effect of both hyaluronan sulfation degree and Fe3+ on interleukin-8 binding by electrochemical impedance spectroscopy and surface characterizations. Our results show that sulfation degree and metal ion interactions have a synergistic effect in tuning the electrochemical response of the glycated surfaces to the cytokine.

Nonhuman glycans can regulate anti-factor VIII antibody formation in mice.

Recombinant factor VIII (FVIII) products represent a life-saving intervention for patients with hemophilia A. However, patients can develop antibodies against FVIII that prevent its function and directly increase morbidity and mortality. The development of anti-FVIII antibodies varies depending on the type of recombinant product used, with previous studies suggesting that second-generation baby hamster kidney (BHK)-derived FVIII products display greater immunogenicity than do third-generation Chinese hamster ovary (CHO)-derived FVIII products. However, the underlying mechanisms responsible for these differences remain incompletely understood. Our results demonstrate that BHK cells express higher levels of the nonhuman carbohydrate α1-3 galactose (αGal) than do CHO cells, suggesting that αGal incorporation onto FVIII may result in anti-αGal antibody recognition that could positively influence the development of anti-FVIII antibodies. Consistent with this, BHK-derived FVIII exhibits increased levels of αGal, which corresponds to increased reactivity with anti-αGal antibodies. Infusion of BHK-derived, but not CHO-derived, FVIII into αGal-knockout mice, which spontaneously generate anti-αGal antibodies, results in significantly higher anti-FVIII antibody formation, suggesting that the increased levels of αGal on BHK-derived FVIII can influence immunogenicity. These results suggest that posttranslational modifications of recombinant FVIII products with nonhuman carbohydrates may influence the development of anti-FVIII antibodies.

Isolation and immune activity of a new acidic Cordyceps militaris exopolysaccharide.

A new type of acidic exopolysaccharide (AESP-II) was extracted and separated from the fermentation broth of Cordyceps militaris (C. militaris), which was further purified to elucidate its structural characteristics and immunological activity. AESP-II was confirmed to be an acidic pyranose with a molecular weight of 61.52 kDa, which consisted of mannose, glucuronic acid, rhamnose, galactose acid, N-acetyl-galactosamine, glucose, galactose and arabinose with a molar ratio of 1.07: 5.38: 1: 3.14: 2.23: 15: 6.09: and 4.04. Animal experiment results verified that AESP-II can significantly promote the proliferation of spleen T and B lymphocytes in mice with immune injury caused by cyclophosphamide (CTX). In particular, the promotion of B lymphocytes presented a dose-effect relationship. In addition, the levels of the cytokines IL-2, IL-4, and IFN-γ, which are mainly secreted by T lymphocytes, and immunoglobulin IgG, IgM and IgA, which are mainly secreted by B lymphocytes, were increased after AESP-II treatment. The above results suggest that fluid immunity is involved in the immunomodulatory function of AESP-II. Simultaneously, AESP-II was detected significantly to promote the phosphorylation expression of p38 kinase (p38), extracellular regulated protein kinases (ERK) and c-Jun N-terminal kinase (JNK) by Western blot, further suggesting that the activation of the MAPK signaling pathway mediates the immunoregulatory function of AESP-II.

Prospects and Challenges of the Study of Anti-Glycan Antibodies and Microbiota for the Monitoring of Gastrointestinal Cancer.

Over the past decades, a large amount of data has been accumulated in various subfields of glycobiology. However, much clinically relevant data and many tools are still not widely used in medicine. Synthetic glycoconjugates with the known structure of glycans are an accurate tool for the study of glycan-binding proteins. We used polyacrylamide glycoconjugates (PGs) including PGs with tumour-associated glycans (TAGs) in immunoassays to assess the prognostic potential of the serum level of anti-glycan antibodies (AG Abs) in gastrointestinal cancer patients and found an association of AG Abs with survival. The specificity of affinity-isolated AG Abs was investigated using synthetic and natural glycoconjugates. AG Abs showed mainly a low specificity to tumour-associated and tumour-derived mucins; therefore, the protective role of the examined circulating AG Abs against cancer remains a challenge. In this review, our findings are analysed and discussed in the context of the contribution of bacteria to the AG Abs stimulus and cancer progression. Examples of the influence of pathogenic bacteria colonising tumours on cancer progression and patient survival through mechanisms of interaction with tumours and dysregulated immune response are considered. The possibilities and problems of the integrative study of AG Abs and the microbiome using high-performance technologies are discussed.