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Protein-carbohydrate binding depends on multivalent ligand display that is even more important for low affinity carbohydrate-carbohydrate interactions. Detection and analysis of these low affinity multivalent binding events are technically challenging. We describe the synthesis of dual-fluorescent sugar-capped silicon nanoparticles that proved to be an attractive tool for the analysis of low affinity interactions. These ultrasmall NPs with sizes of around 4 nm can be used for NMR quantification of coupled sugars. The silicon nanoparticles are employed to measure the interaction between the cancer-associated glycosphingolipids GM3 and Gg3 and the associated kD value by surface plasmon resonance experiments. Cell binding studies, to investigate the biological relevance of these carbohydrate-carbohydrate interactions, also benefit from these fluorescent sugar-capped nanoparticles.

The antimicrobial peptide LL-37 is generated upon proteolytic cleavage of cathelicidin and limits invading pathogens by directly targeting microbial membranes as well as stimulating innate immune cell function. However, some microbes evade LL-37-mediated defense. Notably, group A Streptococcus (GAS) strains belonging to the hypervirulent M1T1 serogroup are more resistant to human LL-37 than other GAS serogroups. We show that the GAS surface-associated M1 protein sequesters and neutralizes LL-37 antimicrobial activity through its N-terminal domain. M1 protein also binds the cathelicidin precursor hCAP-18, preventing its proteolytic maturation into antimicrobial forms. Exogenous M1 protein rescues M1-deficient GAS from killing by neutrophils and within neutrophil extracellular traps and neutralizes LL-37 chemotactic properties. M1 also binds murine cathelicidin, and its virulence contribution in a murine model of necrotizing skin infection is largely driven by its ability to neutralize this host defense peptide. Thus, cathelicidin resistance is essential for the pathogenesis of hyperinvasive M1T1 GAS., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The coiled-coil folding motif represents an ideal scaffold for the defined presentation of ligands due to the possibility of positioning them at specific distances along the axis. We created a coiled-coil glycopeptide library to characterize the distances between the carbohydrate-binding sites of the asialoglycoprotein receptors (ASGPR) on hepatocytes. The components of the glycopeptide library vary for the number of displayed ligands (galactose), their position on the peptide sequence, and the space between peptide backbone and carbohydrate. We determined the binding of the glycopeptides to the hepatocytes, and we established the optimal distance and orientation of the galactose moieties for interaction with the ASGPR using flow cytometry. We confirmed that the binding occurs through endocytosis mediated by ASGPR via inhibition studies with cytochalasin D; fluorescence microscopy studies display the uptake of the carrier peptides inside the cell. Thus, this study demonstrates that the coiled-coil motif can be used as reliable scaffold for the rational presentation of ligands.

Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is an abundant cell wall glycolipid and major virulence factor of Mycobacterium tuberculosis. Its synthetic analog trehalose-6,6-dibehenate (TDB) is a new adjuvant currently in phase I clinical trials. In rodents, the C-type lectin receptors Mincle and Mcl bind TDB/TDM and activate macrophages and dendritic cells (DC) through the Syk-Card9 pathway. However, it is unknown whether these glycolipids activate human innate immune cells through the same mechanism. We performed in vitro analysis of TDB/TDM-stimulated primary human monocytes, macrophages, and DC; determined C-type lectin receptor expression; and tested the contribution of SYK, MINCLE, and MCL by small interfering RNA knockdown and genetic complementation. We observed a robust chemokine and cytokine release in response to TDB or TDM. MCSF-driven macrophages secreted higher levels of IL-8, IL-6, CCL3, CCL4, and CCL2 after stimulation with TDM, whereas DC responded more strongly to TDB and GM-CSF-driven macrophages were equally responsive to TDB and TDM. SYK kinase and the adaptor protein CARD9 were essential for glycolipid-induced IL-8 production. mRNA expression of MINCLE and MCL was high in monocytes and macrophages, with MINCLE and MCL proteins localized intracellularly under resting conditions. Small interfering RNA-mediated MINCLE or MCL knockdown caused on average reduced TDB- or TDM-induced IL-8 production. Conversely, retroviral expression in murine Mincle-deficient DC revealed that human MINCLE, but not MCL, was sufficient to confer responsiveness to TDB/TDM. Our study demonstrates that SYK-CARD9 signaling plays a key role in TDB/TDM-induced activation of innate immune cells in man as in mouse, likely by engagement of MINCLE., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Near-infrared (NIR) fluorescent dyes are gaining increased attention due to their potential to serve as molecular probes for in vivo imaging. Here, we demonstrate that oligoglycerol dendrons effectively enhance the fluorescence properties of an NIR dye by increasing the solubility in water and the prevention of aggregate formation. First- and second-generation oligoglycerol dendrons were conjugated to an NIR dye via a dipolar-cycloaddition (click) reaction. The two new dye conjugates exhibited enhanced NIR fluorescent emission and considerably higher fluorescent quantum yields than the dye alone. The high photostability measured for one of the oligoglycerol-linked dyes, in comparison to commonly used fluorogenic dyes such as Cy5 and Cy7, was validated using fluorescence microscopy of macrophages.

Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3(-/-) mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD., (© 2015 The Authors.)

Carbohydrates can be found on the cell surface of nearly every cell ranging from bacteria to fungi right up to mammalian cells. Carbohydrates and their interactions with carbohydrate-binding proteins play crucial roles in multiple biological processes including immunity, homeostasis, cellular communication, cell migration, and the regulation of serum glycoprotein levels. In the last decades, the interest in exploiting the biological activity of glycans as vaccine components has considerably increased. On the one hand, carbohydrates display epitopes to generate protective antibodies against pathogen-derived cell wall structures and on the other hand, glycans have the potential to stimulate the immune system; thus they can act as potent vaccine adjuvants.An effective vaccine consists of two major components, the vaccine antigen and an adjuvant. The vaccine antigen is an original or modified part of the pathogen that causes the disease. The immune response triggered by vaccination should induce antigen-specific plasma cells secreting protective antibodies as well as the development of memory T and B cells. Carbohydrate structures on pathogens represent an important class of antigens that can activate B cells to produce protective anti-carbohydrate antibodies in adults. A major breakthrough in vaccine development was the design of conjugate vaccines that evoke protective antibody responses against encapsulated bacteria strains such as Haemophilus influenzae, Streptococcus pneumoniae, or Neisseria meningitidis in adults, but also in young children. The first part of this chapter focuses on immune responses triggered by carbohydrate-based vaccines. The second part of the chapter discusses the immunological mechanisms of carbohydrate-based adjuvants to increase the immunogenicity of vaccines.

Vaccination is one of the key developments in the fight against infectious diseases. It is based on the principle that immunization with pathogen-derived antigens provides protection from the respective infection by inducing an antigen-specific immune response. The discovery by Avery and Heidelberger in the 1920s that capsular polysaccharides (CPS) from Streptococcus pneumoniae are immunoreactive was the starting point of the development of carbohydrate-based vaccines. CPS-specific neutralizing antibodies were found to mediate protection against S. pneumoniae infection. Since the majority of bacterial pathogens carry a dense array of polysaccharides on their surface, the carbohydrate-based vaccine approach was applied to a variety of bacterial strains. The first CPS-based vaccines against S. pneumoniae were licensed in the 1940s. The increasing emergence of antibiotic-resistant bacterial strains since the 1960s boosted the development of carbohydrate-based vaccines and led to the approval of CPS-based vaccines against Neisseria meningitidis, Haemophilus influenzae type b (Hib), and Salmonella typhi. Meanwhile, it was observed that CPS generally do not elicit protective antibody responses in children below the age of 2 years who are at the greatest risk of infection. As a consequence, studies refocused on the conjugation of oligosaccharides to proteins in order to increase vaccine immunogenicity which led to the introduction of the first glycoconjugate vaccine against Hib in 1987. Due to the success of the first glycoconjugate vaccines, higher valent formulations were developed against numerous bacterial infections to achieve broad serotype coverage. Current research also focuses on the development of carbohydrate-based vaccines against other pathogens such as viruses, fungi, protozoan parasites, or helminths.

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.

Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, and its synthetic analog Trehalose-6,6-dibehenate (TDB) bind to the C-type lectin receptors macrophage-inducible C-type lectin (Mincle) and Mcl to activate macrophages. Genetically, the transcriptional response to TDB/TDM has been defined to require FcRγ-Syk-Card9 signaling. However, TDB/TDM-triggered kinase activation has not been studied well, and it is largely unknown which transcriptional regulators bring about inflammatory gene expression. In this article, we report that TDB/TDM caused only weak Syk-phosphorylation in resting macrophages, consistent with low basal Mincle expression. However, LPS-priming caused MYD88-dependent upregulation of Mincle, resulting in enhanced TDB/TDM-induced kinase activation and more rapid inflammatory gene expression. TLR-induced Mincle expression partially circumvented the requirement for Mcl in the response to TDB/TDM. To dissect transcriptional responses to TDB/TDM, we mined microarray data and identified early growth response (Egr) family transcription factors as direct Mincle target genes, whereas upregulation of Cebpb and Hif1a required new protein synthesis. Macrophages and dendritic cells lacking C/EBPβ showed nearly complete abrogation of TDB/TDM responsiveness, but also failed to upregulate Mincle. Retroviral rescue of Mincle expression in Cebpb-deficient cells restored induction of Egr1, but not of G-CSF. This pattern of C/EBPβ dependence was also observed after stimulation with the Dectin-1 ligand Curdlan. Inducible expression of hypoxia-inducible factor 1α (HIF1α) also required C/EBPβ. In turn, HIF1α was not required for Mincle expression, kinase activation, and Egr1 or Csf3 expression, but critically contributed to NO production. Taken together, we identify C/EBPβ as central hub in Mincle expression and inflammatory gene induction, whereas HIF1α controls Nos2 expression. C/EBPβ also connects TLR signals to cord factor responsiveness through MYD88-dependent upregulation of Mincle., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract. Though its exact etiology is still unclear, it is proposed that an imbalance in the intestinal homeostasis leads to a disturbed interaction between commensal microbiota and the mucosal immune system. Previous studies have shown that both innate and adaptive immunity are involved in an overwhelming colon inflammation, and thus contribute to the pathogenesis of IBD. In innate immunity, several pattern recognition receptors such as Toll-like receptors, NOD-like receptors or C-type lectin receptors (CLRs) are involved in IBD pathogenesis. Myeloid CLRs are mainly expressed by antigen-presenting cells and bind to glycan structures present on self or foreign antigens. The Macrophage-restricted C-type lectin (MCL) and the Dendritic cell immunoreceptor (DCIR) are two poorly characterized members of the CLR family. In this study, we investigated the role of MCL and DCIR in the pathogenesis of murine colitis. Both CLRs bound to intestinal microbiota to a different extent. They modulated the production of pro-inflammatory cytokines by antigen-presenting cells upon stimulation with heat-killed microbiota and impacted subsequent T cell responses. To analyze whether MCL and DCIR contribute to the pathogenesis of IBD, the dextran sulfate sodium (DSS) murine colitis model was employed. MCL-/- as well as DCIR-/- mice exhibited only a slightly increased severity of disease compared to wild-type mice indicating a limited role for MCL and DCIR in the regulation of intestinal immunity.

Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes, gacA through gacL. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAc side-chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum, and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis, and a deeper understanding of this unique polysaccharide has implications for vaccine development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Myeloid C-type lectin receptors (CLRs) expressed by antigen-presenting cells are pattern-recognition receptors involved in the recognition of pathogens as well as of self-antigens. The interaction of carbohydrate ligands with a CLR can trigger immune responses. Although several CLR ligands are known, there is limited insight into CLR targeting by carbohydrate ligands. The weak affinity of lectin-carbohydrate interactions often renders multivalent carbohydrate presentation necessary. Here, we have analyzed the impact of multivalent presentation of the trisaccharide Lewis X (Le(X) ) epitope on its interaction with the CLR macrophage galactose-type lectin-1 (MGL-1). Glycan arrays, including N-glycan structures with terminal Le(X) , were prepared by enzymatic extension of immobilized synthetic core structures with two recombinant glycosyltransferases. Incubation of arrays with an MGL-1-hFc fusion protein showed up to tenfold increased binding to multiantennary N-glycans displaying Le(X) structures, compared to monovalent Le(X) trisaccharide. Multivalent presentation of Le(X) on the model antigen ovalbumin (OVA) led to increased cytokine production in a dendritic cell /T cell coculture system. Furthermore, immunization of mice with Le(X) -OVA conjugates modulated cytokine production and the humoral response, compared to OVA alone. This study provides insights into how multivalent carbohydrate-lectin interactions can be exploited to modulate immune responses., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cerebral malaria (CM) is the most severe complication of malaria. The murine Plasmodium berghei ANKA (PbA) infection model has helped to identify crucial players in the pathogenesis of CM. However, the role of pattern recognition receptors in innate immunity to CM induction is still poorly understood. C-type lectin receptors (CLRs) represent a family of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-Ags often in a Ca(2+)-dependent manner. In this study, we investigated the role of the CLR dendritic cell immunoreceptor (DCIR) in the genesis of CM. Using the murine PbA infection, we show in this article that DCIR is essential for the development of CM. Although PbA infection led to 80% CM in wild-type C57BL/6 mice, DCIR-deficient mice were highly protected with only 15% CM development. In accordance with the reduced CM incidence in DCIR(-/-) mice, CD8(+) T cell sequestration was markedly reduced in brains of PbA-infected DCIR(-/-) mice, which was accompanied by reduced brain inflammation. Reduced T cell sequestration in the brain was caused by decreased TNF-α levels in sera, as well as a modulated activation of CD4(+) and CD8(+) T cells in spleen of PbA-infected DCIR(-/-) mice. This study indicates that DCIR is critically involved in CM induction, thus highlighting the importance of this CLR in innate immunity during malaria infection.

C-type lectin receptors (CLRs) represent a large receptor family including collectins, selectins, lymphocyte lectins, and proteoglycans. CLRs share a structurally homologous carbohydrate-recognition domain (CRD) and often bind carbohydrates in a Ca²⁺-dependent manner. In innate immunity, CLRs serve as pattern recognition receptors (PRRs) and bind to the glycan structures of pathogens and also to self-antigens. In nature, the low affinity of CLR/carbohydrate interactions is overcome by multivalent ligand presentation at the surface of cells or pathogens. Thus, multivalency is a promising strategy for targeting CLR-expressing cells and, indeed, carbohydrate-based targeting approaches have been employed for a number of CLRs, including asialoglycoprotein receptor (ASGPR) in the liver, or DC-SIGN expressed by dendritic cells. Since CLR engagement not only mediates endocytosis but also influences intracellular signaling pathways, CLR targeting may allow for cell-specific drug delivery and also the modulation of cellular functions. Glyconanoparticles, glycodendrimers, and glycoliposomes were successfully used as tools for CLR-specific targeting. This review will discuss different approaches for multivalent CLR ligand presentation and aims to highlight how CLR targeting has been employed for cell specific drug delivery. Major emphasis is directed towards targeting of CLRs expressed by antigen-presenting cells to modulate immune responses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bacterial or viral infection of the airway plays a critical role in the pathogenesis and exacerbation of chronic obstructive pulmonary disease (COPD) which is expected to be the 3rd leading cause of death by 2020. The induction of inflammatory responses in immune cells as well as airway epithelial cells is observed in the disease process. There is thus a pressing need for the development of new therapeutics. Keratan sulfate (KS) is the major glycosaminoglycans (GAGs) of airway secretions, and is synthesized by epithelial cells on the airway surface. Here we report that a KS disaccharide, [SO3(-)-6]Galβ1-4[SO3(-)-6]GlcNAc, designated as L4, suppressed the production of Interleukin-8 (IL-8) stimulated by flagellin, a Toll-like receptor (TLR) 5 agonist, in normal human bronchial epithelial (NHBE) cells. Such suppressions were not observed by other L4 analogues, N-acetyllactosamine or chondroitin-6-sulfate disaccharide. Moreover, treatment of NHBE cells with L4 inhibited the flagellin-stimulated phosphorylation of epidermal growth factor receptor (EGFR), the down stream signaling pathway of TLRs in NHBE cells. These results suggest that L4 specifically blocks the interaction of flagellin with TLR5 and subsequently suppresses IL-8 production in NHBE cells. Taken together, L4 represents a potential molecule for prevention and treatment of airway inflammatory responses to bacteria infections, which play a critical role in exacerbation of COPD., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Bacteria will gain an advantage if they are able to metabolize nutrients that are inaccessible for other bacteria. To demonstrate this principle, we developed a simple model system, which mimics how bacteria exploit natural carbon sources. A masked glucose precursor that is activated by β-galactosidase was used as a carbon source for bacterial growth in a glucose-deficient medium. No bacterial growth was observed in the presence of control substances in which β-galactosidase mediated cleavage did not lead to glucose release. This study represents a proof-of-principle example in which a bacterium can grow in a nutrient-free medium by inducible, enzyme-mediated nutrient release from a precursor.

The glycoproteins hemagglutinin (HA) and neuraminidase are the major determinants of host range and tissue tropism of the influenza virus. HA is the most abundant protein in the virus particle membrane and represents the basis of most influenza vaccines. It has been reported that influenza virus HA N-glycosylation markedly depends on the host cell line used for virus production. However, little is known about how differential glycosylation affects immunogenicity of the viral proteins. This is of importance for virus propagation in chicken eggs as well as for innovative influenza vaccine production in mammalian cell lines. In this study, we investigated the impact of the differential N-glycosylation patterns of two influenza A virus PR/8/34 (H1N1) variants on immunogenicity. Madin-Darby canine kidney cell-derived and Vero cell-derived glycovariants were analyzed for immunogenicity in a TCR-HA transgenic mouse model. Next-generation pyrosequencing validated the congruence of the potential HA N-glycosylation sites as well as the presence of the HA peptide recognized by the TCR-HA transgenic T cells. We show that differential HA N-glycosylation markedly affected T cell activation and cytokine production in vitro and moderately influenced IL-2 production in vivo. Cocultivation assays indicated that the difference in immunogenicity was mediated by CD11c(+) dendritic cells. Native virus deglycosylation by endo- and exoglycosidases dramatically reduced cytokine production by splenocytes in vitro and markedly decreased HA-specific Ab production in vivo. In conclusion, this study indicates a crucial importance of HA N-glycosylation for immunogenicity. Our findings have implications for cell line-based influenza vaccine design.

Glycomics and glycoproteomics have become indispensible tools in the study of glycoconjugates. Mass spectrometry based methods are standardly used to study the proteome and/or glycome and these approaches are capable of providing both, qualitative and quantitative information using top down techniques. The human immune system marks a particular area of interest for glycomics and glycoproteomics research since a large number of key proteins in innate and adaptive immunity are glycoproteins. In numerous examples, the crucial influence of glycosylation on critical steps such as receptor interaction and binding has been demonstrated. In this review, we focus on different glycomics and glycoproteomics approaches and their application for studying protein glycosylation in the immune system., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A series of well-defined polymer-drug conjugates were prepared in order to modify the physical properties of a known cytotoxic drug, 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11). Reversible addition-fragmentation chain transfer (RAFT) polymerisation was used to covalently and site-specifically append a defined N-(2-hydroxypropyl)methacrylamide (HPMA) polymer to SN-38 using a graft-from process. These poly-HPMA-SN-38 conjugates displayed excellent aqueous solubility and stability, whilst retaining the cytotoxic activity of the parent SN-38. In vitro co-culture assays containing both cancer and noncancer cell lines demonstrated the specificity of RAFT-derived poly-HPMA-SN-38 conjugates for cancerous cells. The concept of post-optimisation modification of small-molecule drugs through a graft-from polymer conjugation method is introduced., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new method for the spatially defined alignment of carbohydrates on a duplex DNA scaffold is presented. The use of an N-hydroxysuccinimide (NHS)-ester phosphoramidite along with carbohydrates containing an alkylamine linker allows for on-column labeling during solid-phase oligonucleotide synthesis. This modification method during solid-phase synthesis only requires the use of minimal amounts of complex carbohydrates. The covalently attached carbohydrates are presented in the major groove of the B-form duplex DNA as potential substrates for murine type II C-type lectin receptors mMGL1 and mMGL2. CD spectroscopy and thermal melting revealed only minimal disturbance of the overall helical structure. Surface plasmon resonance and cellular uptake studies with bone-marrow-derived dendritic cells were used to assess the capability of these carbohydrate-modified duplexes to bind to mMGL receptors., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Carbohydrates are integral to biological signaling networks and cell-cell interactions, yet the detection of discrete carbohydrate-lectin interactions remains difficult since binding is generally weak. A strategy to overcome this problem is to create multivalent sensors, where the avidity rather than the affinity of the interaction is important. Here we describe the development of a series of multivalent sensors that self-assemble via hydrophobic supramolecular interactions. The multivalent sensors are comprised of a fluorescent ruthenium(II) core surrounded by a heptamannosylated β-cyclodextrin scaffold. Two additional series of complexes were synthesized as proof-of-principle for supramolecular self-assembly, the fluorescent core alone and the core plus β-cyclodextrin. Spectroscopic analyses confirmed that the three mannosylated sensors displayed 14, 28, and 42 sugar units, respectively. Each complex adopted original and unique spatial arrangements. The sensors were used to investigate the influence of carbohydrate spatial arrangement and clustering on the mechanistic and qualitative properties of lectin binding. Simple visualization of binding between a fluorescent, multivalent mannose complex and the Escherichia coli strain ORN178 that possesses mannose-specific receptor sites illustrates the potential for these complexes as biosensors.

Targeting glycan-binding receptors is an attractive strategy for cell-specific drug and gene delivery. The C-type lectin asialoglycoprotein receptor (ASGPR) is particularly suitable for liver-specific delivery due to its exclusive expression by parenchymal hepatocytes. In this study, we designed and developed an efficient synthesis of carbohydrate-functionalized β-cyclodextrins (βCDs) and liposomes for hepatocyte-specific delivery. For targeting of ASGPR, rhodamine B-loaded βCDs were functionalized with glycodendrimers. Liposomes were equipped with synthetic glycolipids containing a terminal D-GalNAc residue to mediate binding to ASGPR. Uptake studies in the human hepatocellular carcinoma cell line HepG2 demonstrated that βCDs and liposomes displaying terminal D-Gal/D-GalNAc residues were preferentially endocytosed. In contrast, uptake of βCDs and liposomes with terminal d-Man or D-GlcNAc residues was markedly reduced. The d-Gal/d-GalNAc-functionalized βCDs and liposomes presented here enable hepatocyte-specific targeting. Gal-functionalized βCDs are efficient molecular carriers to deliver doxorubicin in vitro into hepatocytes and induce apoptosis.

Carbohydrates and glycoconjugates are involved in a number of biological processes including host-pathogen interactions, cell communication, proliferation and differentiation as well as the initiation of immune responses. However, access to synthetic oligosaccharide structures has been limited for long time since no standardized protocols for the synthesis of complex oligosaccharides from monomeric building block existed. Recent advances in glycochemistry and glycobiology have helped elucidate biological functions of carbohydrates. Novel methods such as automated synthesis of vaccine candidates and immunologically relevant carbohydrates have contributed to this success as well as high-throughput methods such as glycan arrays. This review illustrates the impact of carbohydrate synthesis on the design of efficient carbohydrate-based antimicrobial and anticancer vaccines. Furthermore, this review focuses on how immunological research can benefit from these novel tools provided by glycochemistry.

Access to synthetic carbohydrates is an urgent need for the development of carbohydrate-based drugs, vaccines, adjuvants as well as novel drug delivery systems. Besides traditional synthesis in solution, synthetic carbohydrates have been generated by chemoenzymatic methods as well as automated solid-phase synthesis. Synthetic oligosaccharides have proven to be useful for identifying ligands of carbohydrate-binding proteins such as C-type lectins and siglecs using glycan arrays. Furthermore, glyconanoparticles and glycodendrimers have been used for specific targeting of lectins of the immune system such as selectins, DC-SIGN, and CD22. This review focuses on how diverse carbohydrate structures can be synthetically derived and highlights the benefit of synthetic carbohydrates for glycobiology., (Copyright 2010 Elsevier Ltd. All rights reserved.)

PEGylated quantum dots (QDs) capped with d-mannose, d-galactose, and d-galactosamine have been synthesized. The stable, high quantum yield fluorescence of QDs was exploited to study specific carbohydrate-protein interactions in vitro and in vivo.

The emergence of multidrug-resistant tuberculosis (TB) and problems with the BCG tuberculosis vaccine to protect humans against TB have prompted investigations into alternative approaches to combat this disease by exploring novel bacterial drug targets and vaccines. Phosphatidylinositol mannosides (PIMs) are biologically important glycoconjugates and represent common essential precursors of more complex mycobacterial cell wall glycolipids including lipomannan (LM), lipoarabinomannan (LAM), and mannan capped lipoarabinomannan (ManLAM). Synthetic PIMs constitute important biochemical tools to elucidate the biosynthesis of this class of molecules, to reveal PIM interactions with host cells, and to investigate the function of PIMs as potential antigens and/or adjuvants for vaccine development. Here, we report the efficient synthesis of all PIMs including phosphatidylinositol (PI) and phosphatidylinositol mono- to hexa-mannoside (PIM1 to PIM6). Robust synthetic protocols were developed for utilizing bicyclic and tricyclic orthoesters as well as mannosyl phosphates as glycosylating agents. Each synthetic PIM was equipped with a thiol-linker for immobilization on surfaces and carrier proteins for biological and immunological studies. The synthetic PIMs were immobilized on microarray slides to elucidate differences in binding to the dendritic cell specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) receptor. Synthetic PIMs served as immune stimulators during immunization experiments in C57BL/6 mice when coupled to the model antigen keyhole-limpet hemocyanin (KLH).

Activation of T lymphocytes through TCR signalling takes place within the context of numerous other cell surface proteins. To prevent unnecessary activation of T cells the immune system has developed an intricate balance between positive and negative costimulatory signals. Positive costimulatory signals determine whether antigen recognition by T cells leads to full activation or to anergy/death. In contrast, the expression of negative costimulatory molecules by T cells such as cytotoxic T lymphocyte antigen 4 (CTLA-4) mediates the regulation of an immune response and thus plays a pivotal role in the maintenance of peripheral tolerance. The new members of the CD28 family members, programmed death-1 (PD-1) and B and T lymphocyte attenuator (BTLA) are expressed more broadly on various immune cells and are also induced upon inflammation. There is increasing evidence that negative costimulators are critically involved in the regulation of immune responses against parasitic infections. The production of pro-inflammatory cytokines is often required to control parasites but the same cytokines contribute to immunopathology. Recent studies indicate that these negative costimulators are not redundant but fulfil specific functions in different tissues. This might represent a means to provoke a fine-tuning of the immune response and to define tissue specific limits of inflammation that ensure proper physiological function of the respective organ. Thus, negative costimulators represent possible drug targets since their blockade leads to an increased immune response whereas their ligation dampens T cell activation and thereby might prevent immunopathology. This review examines the role of negative costimulators during parasitic infections, and we also discuss their therapeutic potential.

Plasmodium falciparum GPI contributes to malaria pathology by inducing cytokine release. It has been shown to be recognized through TLR2 and to a lesser extent TLR4 in vitro. However, previous findings on the role of TLRs in parasite clearance or pathology in vivo are conflicting. Thus, we analyzed the impact of TLR-signalling on protection using the P. yoelii infection model. Deficiency of single TLRs as well as triple TLR2/4/9-deficiency had no impact on parasitaemia. In contrast, mice deficient for the adaptor protein MyD88 were more susceptible to P. yoelii infection in that they exhibited an increased parasitaemia in the early phase of the infection and a higher lethality. This phenotype was caused mainly by impaired IL-18 signalling since parasitaemia in IL-18-deficient mice was also increased at early time points during P. yoelii infection compared to wild-type control mice. However, no lethality was observed in IL-18-deficient mice. Since parasitaemia in IL-1R-deficient mice was also slightly increased during P. yoelii infection, impaired IL-1R signalling contributed to the increased susceptibility of MyD88-deficient mice to a lesser extent. These findings correlated with a reduced IFN-gamma production in MyD88- and IL-18-deficient mice, but not in TLR2/4/9-deficient mice. We conclude that mainly IL-18/MyD88-dependent signalling but not TLR2/4/9-signalling is important for early parasite control in our model.

The contribution of the Toll-like receptor (TLR) cascade to the pathogenesis of cerebral malaria (CM) is controversially discussed. TLR2 and TLR9 were reported to be involved in the induction of CM in a study while recently TLR signaling was shown to be dispensable for the development of CM. Using Plasmodium berghei ANKA (PbA) infection of mice as a model of CM, we demonstrate here that the induction of CM is independent of TLR2, 4 and 9. Using triple TLR2/4/9-deficient mice, we exclude synergistic effects between the single TLRs that have been previously implicated with malaria pathology. In conclusion, this study shows that the activation of the innate immune response and the development of CM is not dependent on the engagement of TLR2/4/9.

B and T lymphocyte attenuator (BTLA; CD272) is a coinhibitory receptor that is predominantly expressed on T and B cells and dampens T cell activation. In this study, we analyzed the function of BTLA during infection with Plasmodium berghei ANKA. Infection of C57BL/6 mice with this strain leads to sequestration of leukocytes in brain capillaries that is associated with a pathology resembling cerebral malaria in humans. During the course of infection, we found an induction of BTLA in several organs, which was either due to up-regulation of BTLA expression on T cells in the spleen or due to infiltration of BTLA-expressing T cells into the brain. In the brain, we observed a marked induction of BTLA and its ligand herpesvirus entry mediator during cerebral malaria, which was accompanied by an accumulation of predominantly CD8+ T cells, but also CD4+ T cells. Application of an agonistic anti-BTLA mAb caused a significantly reduced incidence of cerebral malaria compared with control mice. Treatment with this Ab also led to a decreased number of T cells that were sequestered in the brain of P. berghei ANKA-infected mice. Our findings indicate that BTLA-herpesvirus entry mediator interactions are functionally involved in T cell regulation during P. berghei ANKA infection of mice and that BTLA is a potential target for therapeutic interventions in severe malaria.