Carbohydrate-binding Receptors Research Articles

Quality by design (QbD) approach-based development of optimized nanocarrier to achieve quality target product profile (QTPP)-targeted lymphatic delivery

Background. Insulin, commonly used for diabetes treatment, needs better ways to improve its effectiveness and safety due to its challenges with poor permeability and stability. Various system has been developed for oral peptide delivery. The non-targeted system can prevent gastric and enzymatic degradation of peptides but cannot increase the bulk transport of peptides across the membrane. However, the non-selectivity is the limitation of the existing system. Numerous carbohydrate-binding receptors overexpressed on intestinal macrophage cells (M-cells) of gut-associated lymphoid tissue. It is the most desirable site for receptor-mediated endocytosis and lymphatic drug delivery of peptides. Objective. The prime objective of the study was to fabricate mannose ligand conjugated nanoparticles (MNPs) employing a quality-by-design approach to address permeability challenges after oral administration. Herein, the study’s secondary objective of this study is to identify the influencing factor for producing quality products. Considering this objective, the Lymphatic uptake of NPs was selected as a quality target product profile (QTPP), and a systematic study was conducted to identify the critical formulation attributes (CFAs) and critical process parameters (CPP) influencing critical quality attributes (CQAs). Mannosylated Chitosan concentrations (MCs) and TPP concentrations were identified as CFAs, and stirring speed was identified as CPP. Methods. MNPs were prepared by the inotropic gelation method and filled into the enteric-coated capsule to protect from acidic environments. The effect of CFAs and CPP on responses like particle size (X) and entrapment (Y) was observed by Box-Behnken design (BBD). ANOVA statistically evaluated the result to confirm a significant level (p < 0.05). The optimal conditions of NPs were obtained by constructing an overlay plot and determining the desirability value. HPLC and zeta-seizer analysis characterized the lyophilized NPs. Cell-line studies were performed to confirm the safety and M-cell targeting of NPs to enhance Insulin oral bioavailability. Results. The morphology of NPs was revealed by SEM. The developed NPs showed a nearly oval shape with the average size, surface potential, and % drug entrapment were 245.52 ± 3.37 nm, 22.12 ± 2.13 mV, and 76.15 ± 1.3%, respectively. MTT assay result exhibited that MNPs safe and Confocal imaging inference that NPs selectively uptake by the M-cell. Conclusion. BBD experimental design enables the effective formulation of optimized NPs. The statistical analysis estimated a clear assessment of the significance of the process and formulation variable. Cell line study confirms that NPs are safe and effectively uptake by the cell.

A protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors.

Sensing, transport, and utilization of glucose is pivotal to the maintenance of energy homeostasis in animals. Although transporters involved in mobilizing glucose across different cellular compartments are fairly well known, the receptors that bind glucose to mediate its effects independently of glucose metabolism remain largely unrecognized. Establishing precise and reproducible methods to identify glucose receptors in the brain or other peripheral organs will pave the way for comprehending the role of glucose signaling pathways in maintaining, regulating, and reprogramming cellular metabolic needs. Identification of such potential glucose receptors will also likely lead to development of effective therapeutics for treatment of diabetes and related metabolic disorders. Commercially available biotin or radiolabeled glucose conjugates have low molecular weight; therefore, they do not provide enough sensitivity and density to isolate glucose receptors. Here, we describe a protocol to isolate, identify, and verify glucose-binding receptor/s using high molecular weight glucose (or other carbohydrate) conjugates. We have produced 30 kDa glucose- (or other carbohydrate-) biotin-polyacrylamide (PAA) conjugates with mole fractions of 80:5:15% respectively. These conjugates are used with biotin-streptavidin biochemistry, In-cell ELISA, and surface plasmon resonance (SPR) methods to isolate, identify, and verify glucose- or carbohydrate-binding receptors. We first demonstrate how streptavidin-coated magnetic beads are employed to immobilize glucose-biotin-PAA conjugates. Then, these beads are used to enrich and isolate glucose-binding proteins from tissue homogenates or from single-cell suspensions. The enriched or isolated proteins are subjected to mass spectrometry/proteomics to reveal the identity of top candidate proteins as potential glucose receptors. We then describe how the In-cell ELISA method is used to verify the interaction of glucose with its potential receptor through stable expression of the receptor in-vitro. We further demonstrate how a highly sensitive SPR method can be used to measure the binding kinetics of glucose with its receptor. In summary, we describe a protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors using magnetic beads, In-cell ELISA, and SPR. This protocol will form the future basis of studying glucose or carbohydrate receptor signaling pathways in health and in disease.

Offsetting Low-Affinity Carbohydrate Binding with Covalency to Engage Sugar-Specific Proteins for Tumor-Immune Proximity Induction.

Carbohydrate-binding receptors are often used by the innate immune system to potentiate inflammation, target endocytosis/destruction, and adaptive immunity (e.g., CD206, DC-SIGN, MBL, and anticarbohydrate antibodies). To access this class of receptors for cancer immunotherapy, a growing repertoire of bifunctional proximity-inducing therapeutics use high-avidity multivalent carbohydrate binding domains to offset the intrinsically low affinity associated with monomeric carbohydrate-protein binding interactions (Kd ≈ 10-3-10-6 M). For applications aimed at recruiting anticarbohydrate antibodies to tumor cells, large synthetic scaffolds are used that contain both a tumor-binding domain (TBD) and a multivalent antibody-binding domain (ABD) comprising multiple l-rhamnose monosaccharides. This allows for stable bridging between tumor cells and antibodies, which activates tumoricidal immune function. Problematically, such multivalent macromolecules can face limitations including synthetic and/or structural complexity and the potential for off-target immune engagement. We envisioned that small bifunctional "proximity-inducing" molecules containing a low-affinity monovalent ABD could efficiently engage carbohydrate-binding receptors for tumor-immune proximity by coupling weak binding with covalent engagement. Typical covalent drugs and electrophilic chimeras use high-affinity ligands to promote the fast covalent engagement of target proteins (i.e., large kinact/KI), driven by a favorably small KI for binding. We hypothesized the much less favorable KI associated with carbohydrate-protein binding interactions can be offset by a favorably large kinact for the covalent labeling step. In the current study, we test this hypothesis in the context of a model system that uses rhamnose-specific antibodies to induce tumor-immune proximity and tumoricidal function. We discovered that synthetic chimeric molecules capable of preorganizing an optimal electrophile (i.e., SuFEx vs activated ester) for protein engagement can rapidly covalently engage natural sources of antirhamnose antibody using only a single low-affinity rhamnose monosaccharide ABD. Strikingly, we observe chimeric molecules lacking an electrophile, which can only noncovalently bind the antibody, completely lack tumoricidal function. This is in stark contrast to previous work targeting small molecule hapten and peptide-specific antibodies. Our findings underscore the utility of covalency as a strategy to engage low-affinity carbohydrate-specific proteins for tumor-immune proximity induction.

Mannose in vaccine delivery.

Adjuvants and vaccine delivery systems are used widely to improve the efficacy of vaccines. Their primary roles are to protect antigen from degradation and allow its delivery and uptake by antigen presenting cells (APCs). Carbohydrates, including various structures/forms of mannose, have been broadly utilized to target carbohydrate binding receptors on APCs. This review summarizes basic functions of the immune system, focusing on the role of mannose receptors in antigen recognition by APCs. The most popular strategies to produce mannosylated vaccines via conjugation and formulation are presented. The efficacy of mannosylated vaccines is discussed in detail, taking into consideration factors, such as valency and number of mannose in mannose ligands, mannose density, length of spacers, special arrangement of mannose ligands, and routes of administration of mannosylated vaccines. The advantages and disadvantages of mannosylation strategy and future directions in the development of mannosylated vaccines are also debated.

Carbohydrates: Binding Sites and Potential Drug Targets for Neural-Affecting Pathogens.

A number of viruses that have caused wide spread concern e.g. Ebola, Zika, and SARS-CoV2 (severe acute respiratory syndrome coronavirus 2 also known as COVID 19) have at various times, become newsworthy as a result of being newly discovered, mutations enabling them to more efficiently infect humans or modern modes of transportation moving them to areas with naive, susceptible populations. As more is learned about the mechanisms whereby these pathogens enter human cells it has become increasingly evident that carbohydrates expressed on the surface of either target cells or the pathogens themselves are essential. Variability in carbohydrate structures as well as the presence of carbohydrate binding receptors (lectins) provides a plethora of potential binding interactions by which infection of cells can occur. Identification of specific lipid- or protein-associated carbohydrates essential for infection provides support for research being done to develop carbohydrate related inhibitors of those interactions. This chapter (1) discusses scenarios for how carbohydrates affect the ability of specific infectious agents to interact with neural cells, (2) gives examples of problems that may result from development of antibodies to carbohydrate antigens found on pathogens that are similar to epitopes expressed on mammalian cells, and (3) provides examples of approaches either in use or under consideration for translational uses of this information.

Seminal Plasma Glycoproteins as Potential Ligands of Lectins Engaged in Immunity Regulation.

Environmental pollution, chronic stress, and unhealthy lifestyle are factors that negatively affect reproductive potential. Currently, 15–20% of couples in industrialized countries face the problem of infertility. This growing health and social problem prompts researchers to explore the regulatory mechanisms that may be important for successful fertilization. In recent years, more attention has been paid to male infertility factors, including the impact of seminal plasma components on regulation of the female immune response to allogenic sperm, embryo and fetal antigens. Directing this response to the tolerogenic pathway is crucial to achieve a healthy pregnancy. According to the fetoembryonic defense hypothesis, the regulatory mechanism may be associated with the interaction of lectins and immunomodulatory glycoepitopes. Such interactions may involve lectins of dendritic cells and macrophages, recruited to the cervical region immediately after intercourse. Carbohydrate binding receptors include C type lectins, such as DC-SIGN and MGL, as well as galectins and siglecs among others. In this article we discuss the expression of the possible lectin ligands, highly fucosylated and high mannose structures, which may be recognized by DC-SIGN, glycans of varying degrees of sialylation, which may differ in their interaction with siglecs, as well as T and Tn antigens in O-glycans.

Circulating platelet count and glycans.

This review highlights recent advancements in understanding the regulation of platelet numbers, focusing on mechanisms by which carbohydrates (glycans) link platelet removal with platelet production in the bone marrow in health and disease. This review is focused on the role of carbohydrates, specifically sialic acid moieties, as a central mediator of platelet clearance. We discuss recently identified novel mechanisms of carbohydrate-mediated platelet removal and carbohydrate-binding receptors that mediate platelet removal. The platelet production rate by megakaryocytes and removal kinetics controls the circulating platelet count. Alterations in either process can lead to thrombocytopenia (low platelet count) or thrombocytosis (high platelet count) are associated with the risk of bleeding or overt thrombus formation and serious complications. Thus, regulation of a steady-state platelet count is vital in preventing adverse events. There are few mechanisms delineated that shed light on carbohydrates' role in the complex and massive platelet removal process. This review focuses on carbohydrate-related mechanisms that contribute to the control of platelet numbers.

The Role of Lectin Receptors and Their Ligands in Controlling Allergic Inflammation.

More than fifty c-type lectin receptors (CLR) are known and have been identified so far. Moreover, we know the group of galectins and sialic acid-binding immunoglobulin-type lectins that also belong to the carbohydrate-binding receptors of the immune system. Thus, the lectin receptors form the largest receptor family among the pathogen recognition receptors. Similar to the toll-like receptors (TLRs), the CLR do not only recognize foreign but also endogenous molecules. In contrast to TLRs, which have a predominantly activating effect on the immune system, lectin receptors also mediate inhibitory signals. They play an important role in innate and adaptive immunity for the induction, regulation and shaping of the immune response. The hygiene hypothesis links enhanced infection to protection from allergic disease. Yet, the microbial substances that are responsible for mediating this allergy-protective activity still have to be identified. Microbes contain both ligands binding to TLRs and carbohydrates that are recognized by CLR and other lectin receptors. In the current literature, the CLR are often recognized as the ‘bad guys’ in allergic inflammation, because some glycoepitopes of allergens have been shown to bind to CLR, facilitating their uptake and presentation. On the other hand, there are many reports revealing that sugar moieties are involved in immune regulation. In this review, we will summarize what is known about the role of carbohydrate interaction with c-type lectins and other sugar-recognizing receptors in anti-inflammation, with a special focus on the regulation of the allergic immune response.

The C-type Lectin Receptor CLEC12A Recognizes Plasmodial Hemozoin and Contributes to Cerebral Malaria Development

SummaryMalaria represents a major cause of death from infectious disease. Hemozoin is a Plasmodium-derived product that contributes to progression of cerebral malaria. However, there is a gap of knowledge regarding how hemozoin is recognized by innate immunity. Myeloid C-type lectin receptors (CLRs) encompass a family of carbohydrate-binding receptors that act as pattern recognition receptors in innate immunity. In the present study, we identify the CLR CLEC12A as a receptor for hemozoin. Dendritic cell-T cell co-culture assays indicate that the CLEC12A/hemozoin interaction enhances CD8+ T cell cross-priming. Using the Plasmodium berghei Antwerpen-Kasapa (ANKA) mouse model of experimental cerebral malaria (ECM), we find that CLEC12A deficiency protects mice from ECM, illustrated by reduced ECM incidence and ameliorated clinical symptoms. In conclusion, we identify CLEC12A as an innate sensor of plasmodial hemozoin.

Abstract B63: Bittersweet symphony: How tumor-associated glycan structures orchestrate immune evasion

Abstract The tumor microenvironment is immune suppressive, allowing tumor cells to escape from immune attack. By relieving this immune suppression, immune cells might be able to effectively eliminate the tumor. Therefore, a detailed understanding on how tumor cells induce an immunosuppressive tumor microenvironment is required. Glycosylation is the most abundant post-translational modification of proteins. It is highly diverse on a cellular level and strongly affected by oncogenesis. Tumor cells generally display an aberrant glycosylation profile and this is now well accepted as a new hallmark of cancer. Overexpression of glycan structures containing sialic acids (Sia) is frequently observed on tumors. Glycan structures can be recognized by lectin receptors expressed on immune cells, whereby sialic acids specifically bind to the Siglec receptor family. The majority of Siglec receptors has an immunoreceptor tyrosine-based inhibition motif and therefore, sialylated glycan structures are known to suppress ongoing immune responses. Hence, we hypothesize that tumor cells overexpress sialylated glycan structures in order to communicate with the immune system and to dampen immune attack. We have previously demonstrated that a genetically engineered Sialow glycovariant of the mouse melanoma B16 model displayed delayed growth in vivo compared to wild-type B16 Siahigh tumors (1). Moreover, higher frequencies of IFNγ-producing effector T-cells were detected in B16 Sialow tumors. This was paralleled by a 50% reduction in FoxP3+CD4+ regulatory T-cell frequencies in Sialow tumors (1), together indicating that tumor-associated sialic acids indeed play, indeed, a crucial role in suppressing the anti-tumor immune response. To check whether the inhibitory effect of sialic acids on the effector T cell response is tumor type specific, we investigated the role of sialic acids in a mouse colorectal cancer model (MC38). With the use of Crispr-Cas9 we successfully knocked out the N-acylneuraminate cytidylyltransferase (CMAS) gene, which is responsible for the sialylation pathway and thus the generation of sialylated glycans on the cell surface. In contrast to the B16 Sialow tumors, the Sianeg MC38 CMAS KO tumors displayed an increased tumor growth in vivo compared to the Siahigh MC38 Mock tumors. Moreover, less viable leukocytes could be recovered from the Sianeg MC38 CMAS KO tumors compared to the Siahigh MC38 Mock tumors. With the use of mass spectrometry, we analyzed the complete glycosylation profile of our Sianeg MC38 CMAS KO and Siahigh MC38 Mock cells and observed, in addition to the absence of sialylation, an increase in LacNAc (Galβ1-4GlcNAc) structures on the Sianeg MC38 CMAS KO cells. This glycan structure is a ligand for carbohydrate-binding Galectin receptors. Galectins are well known for their potential to induce leukocyte apoptosis after binding specific glycoproteins on the leukocyte surface. Using qPCR we observed higher mRNA levels of Galectin-1 in our MC38 CMAS KO cells. The enhanced production of Galectin-1 and the elevated expression of LacNAc together suggest that MC38 CMAS KO cells are able to recruit Galectin-1, thereby facilitating the enhanced immune suppression in these tumors. Currently, we are confirming the role of Galectins in our MC38 CMAS KO tumors. In conclusion, tumor cells alter their glycosylation profile and based on our data, we propose that tumor cells use these glycan structures as a mechanism to influence antitumor immunity and that this phenomenon might be tumor-type specific. Their immune inhibitory nature and their tumor-specific expression identifies tumor-associated glycans as potent candidates for future immune checkpoint blockade therapy. Reference: 1. Perdicchio M. et al. Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells. Oncotarget 2016;7(8):8771-8. Citation Format: Lenneke A.M. Cornelissen, Athanasios Blanas, Joost C. Van der Horst, Laura Kruijssen, Yvette Van Kooyk, Sandra J. Van Vliet. Bittersweet symphony: How tumor-associated glycan structures orchestrate immune evasion [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B63.

C-Type Lectin Receptor (CLR)–Fc Fusion Proteins As Tools to Screen for Novel CLR/Bacteria Interactions: An Exemplary Study on Preselected Campylobacter jejuni Isolates

C-type lectin receptors (CLRs) are carbohydrate-binding receptors that recognize their ligands often in a Ca2+-dependent manner. Upon ligand binding, myeloid CLRs in innate immunity trigger or inhibit a variety of signaling pathways, thus initiating or modulating effector functions such as cytokine production, phagocytosis, and antigen presentation. CLRs bind to various pathogens, including viruses, fungi, parasites, and bacteria. The bacterium Campylobacter jejuni (C. jejuni) is a very frequent Gram-negative zoonotic pathogen of humans, causing severe intestinal symptoms. Interestingly, C. jejuni expresses several glycosylated surface structures, for example, the capsular polysaccharide (CPS), lipooligosaccharide (LOS), and envelope proteins. This “Methods” paper describes applications of CLR–Fc fusion proteins to screen for yet unknown CLR/bacteria interactions using C. jejuni as an example. ELISA-based detection of CLR/bacteria interactions allows a first prescreening that is further confirmed by flow cytometry-based binding analysis and visualized using confocal microscopy. By applying these methods, we identified Dectin-1 as a novel CLR recognizing two selected C. jejuni isolates with different LOS and CPS genotypes. In conclusion, the here-described applications of CLR–Fc fusion proteins represent useful methods to screen for and identify novel CLR/bacteria interactions.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Macrophage exosomes as natural nanocarriers for protein delivery to inflamed brain.

Recent work has stimulated interest in the use of exosomes as nanocarriers for delivery of small drugs, RNAs, and proteins to the central nervous system (CNS). To overcome the blood-brain barrier (BBB), exosomes were modified with brain homing peptides that target brain endothelium but likely to increase immune response. Here for the first time we demonstrate that there is no need for such modification to penetrate the BBB in mammals. The naïve macrophage (Mϕ) exosomes can utilize, 1) on the one hand, the integrin lymphocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), and, 2) on the other hand, the carbohydrate-binding C-type lectin receptors, to interact with brain microvessel endothelial cells comprising the BBB. Notably, upregulation of ICAM-1, a common process in inflammation, promotes Mϕ exosomes uptake in the BBB cells. We further demonstrate invivo that naïve Mϕ exosomes, after intravenous (IV) administration, cross the BBB and deliver a cargo protein, the brain derived neurotrophic factor (BDNF), to the brain. This delivery is enhanced in the presence of brain inflammation, a condition often present in CNS diseases. Taken together, the findings are of interest to basic science and possible use of Mϕ-derived exosomes as nanocarriers for brain delivery of therapeutic proteins to treat CNS diseases.

Identification and Characterization of Carbohydrate-Based Adjuvants.

Modern vaccines such as recombinant proteins or nucleic acids are usually of pure origin, enhancing their tolerability and overall safety. However, this purity often renders them less immunogenic, creating the need for potent adjuvants. Carbohydrates are promising candidates to fulfill this role as they enable direct targeting of dendritic cells and modulation of adaptive immunity. C-type lectin receptors (CLRs) comprise a major group of carbohydrate binding receptors. As they are predominantly expressed by cells of innate immunity, CLR targeting can enhance or dampen early stages of cytokine secretion and antigen presentation, thus modulate the activation and differentiation of T cells. Here, we provide a protocol for the identification of novel CLR ligands by glycan array using recombinant CLR-Fc chimeras followed by the covalent conjugation of carbohydrate CLR ligands to the model antigen ovalbumin (OVA). The resulting glycoconjugates are subsequently used to evaluate T cell activation in vitro and immunomodulation in vivo.

Synthesis and evaluation of glyco-coated liposomes as drug carriers for active targeting in drug delivery systems

Novel sugar-conjugated cholesterols, β-Gal-, α-Man-, β-Man-, α-Fuc-, and β-Man-6P-S-β-Ala-Chol, were synthesized and incorporated into liposomes. In vitro experiments using the glyco-coated liposomes showed that the glyco-coated liposomes are efficiently taken up by cells expressing carbohydrate-binding receptors selectively. Glyco-coated liposomes are promising candidates for drug delivery vehicles.

Roles of carbohydrates in the interaction of pathogens with neural cells.

Numerous pathogens that can affect neural function utilize oligosaccharide-protein interactions as a first step in the infection process. The variability in carbohydrate structures as well as the presence of carbohydrate binding receptors on the surface of cells provides a plethora of potential binding sites for viruses, bacteria, and bacterial toxins. This chapter discusses scenarios for how carbohydrates may affect the ability of infectious agents to interact with neural cells, provides examples of problems that may result from development of antibodies to carbohydrate antigens found on pathogens that are similar to epitopes expressed on mammalian cells, and presents approaches either in use or under consideration for translational uses of this information.

Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.

Sugar/gadolinium-loaded gold nanoparticles for labelling and imaging cells by magnetic resonance imaging

Targeted magnetic resonance imaging (MRI) probes for selective cell labelling and tracking are highly desired. We here present biocompatible sugar-coated paramagnetic Gd-based gold nanoparticles (Gd-GNPs) and test them as MRI T1 reporters in different cellular lines at a high magnetic field (11.7 T). With an average number of 20 Gd atoms per nanoparticle, Gd-GNPs show relaxivity values r1 ranging from 7 to 18 mM-1 s-1 at 1.41 T. The multivalent presentation of carbohydrates on the Gd-GNPs enhances the avidity of sugars for carbohydrate-binding receptors at the cell surface and increases the local concentration of the probes. A large reduction in longitudinal relaxation times T1 is achieved with both fixed cells and live cells. Differences in cellular labelling are obtained by changing the type of sugar on the gold surface, indicating that simple monosaccharides and disaccharides are able to modulate the cellular uptake. These results stress the benefits of using sugars to produce nanoparticles for cellular labelling. To the best of our knowledge this is the first report on labelling and imaging cells with Gd-based gold nanoparticles which use simple sugars as receptor reporters.

Microglial activatory (immunoreceptor tyrosine‐based activation motif)‐ and inhibitory (immunoreceptor tyrosine‐based inhibition motif)‐signaling receptors for recognition of the neuronal glycocalyx

Microglia sense intact or lesioned cells of the central nervous system (CNS) and respond accordingly. To fulfill this task, microglia express a whole set of recognition receptors. Fc receptors and DAP12 (TYROBP)-associated receptors such as microglial triggering receptor expressed on myeloid cells-2 (TREM2) and the complement receptor-3 (CR3, CD11b/CD18) trigger the immunoreceptor tyrosine-based activation motif (ITAM)-signaling cascade, resulting in microglial activation, migration, and phagocytosis. Those receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif (ITIM)-signaling receptors, such as sialic acid-binding immunoglobulin superfamily lectins (Siglecs). Siglecs recognize the sialic acid cap of healthy neurons thus leading to an ITIM signaling that turns down microglial immune responses and phagocytosis. In contrast, desialylated neuronal processes are phagocytosed by microglial CR3 signaling via an adaptor protein containing an ITAM. Thus, the aberrant terminal glycosylation of neuronal surface glycoproteins and glycolipids could serve as a flag for microglia, which display a multitude of diverse carbohydrate-binding receptors that monitor the neuronal physical condition and respond via their ITIM- or ITAM-signaling cascade accordingly.

Targeting carbohydrates: a novel paradigm for fungal control

The tendentious decrease in global arable land and the fast increment in world population will inevitably lead to a critical point. It is therefore imperative to protect crops from pathogen attack. Their deleterious impact on human health is not better, with several invasive infections reaching mortality rates up to 80%. The door is definitely open for biological control, based on molecular interactions between host and pathogen. The remarkable coding capacity of oligosaccharides, far superior to those of nucleotides and amino acids, justifies the emergence of the sugar code as the third life code. The word exoglycome is proposed to represent the oligosaccharide side chains presented at the outer surface of cell membranes, thus excluding the cell wall carbohydrates. The exoglycome plays a fundamental role in cell-cell recognition, in distinction between self from non-self, in the warfare between host and pathogen before infection is established, and in certain diseases such as cancer. Unlike pathogen cell wall carbohydrates, the exoglycome has been neglected as a target for developing effective ways to control disease. Pathogens often induce changes in their cell wall and/or exoglycome to elude and/or circumvent host defences. Identification of such host-induced alterations may prove extremely important for both treatment and diagnosis. Reading the sugar-encoded messages is mainly played by a class of carbohydrate-binding receptors called lectins. The available evidence strongly suggests that a large number of lectins remain to be discovered. Their role in defence should not be underestimated. A high-throughput procedure capable of single-step isolation of novel lections from previously unknown biological samples has recently been developed.