A Remote Secondary Binding Pocket Promotes Heteromultivalent Targeting of DC-SIGN.

Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN's carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general.

Cancer cell death using metabolic glycan labelling techniques.

Herein we describe a method for inducing cancer cell death, which relies on the use of a H2O2-responsive glycan metabolic precursor in conjunction with antibody-dependent cellular cytotoxicity (ADCC) or photodynamic therapy (PDT).

Cloning and Partial Characterization of an Endo-α-(1→6)-d-Mannanase Gene from Bacillus circulans.

Mycobacteria produce two major lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), whose broad array of biological activities are tightly related to the fine details of their structure. However, the heterogeneity of these molecules in terms of internal and terminal covalent modifications and complex internal branching patterns represent significant obstacles to their structural characterization. Previously, an endo-α-(1→6)-D-mannanase from Bacillus circulans proved useful in cleaving the mannan backbone of LM and LAM, allowing the reducing end of these molecules to be identified as Manp-(1→6) [Manp-(1→2)]-Ino. Although first reported 45 years ago, no easily accessible form of this enzyme was available to the research community, a fact that may in part be explained by a lack of knowledge of its complete gene sequence. Here, we report on the successful cloning of the complete endo-α-(1→6)-D-mannanase gene from Bacillus circulans TN-31, herein referred to as emn. We further report on the successful production and purification of the glycosyl hydrolase domain of this enzyme and its use to gain further insight into its substrate specificity using synthetic mannoside acceptors as well as LM and phosphatidyl-myo-inositol mannoside precursors purified from mycobacteria.

Multivalent glycoligands with lectin/enzyme dual specificity: self-deliverable glycosidase regulators.

Multivalent mannosides with inherent macrophage recognition abilities, built on ß-cyclodextrin, RAFT cyclopeptide or peptide dendrimer cores, trigger selective inhibition of lysosomal ß-glucocerebrosidase or α-mannosidase depending on valency and topology, offering new opportunities in multitargeted drug design.

Flavonol Biosynthesis Genes and Their Use in Engineering the Plant Antidiabetic Metabolite Montbretin A.

The plant metabolite montbretin A (MbA) and its precursor mini-MbA are potential new drugs for treating type 2 diabetes. These complex acylated flavonol glycosides only occur in small amounts in the corms of the ornamental plant montbretia (Crocosmia × crocosmiiflora). Our goal is to metabolically engineer Nicotiana benthamiana using montbretia genes to achieve increased production of mini-MbA and MbA. Two montbretia UDP-dependent glycosyltransferases (UGTs), CcUGT1 and CcUGT2, catalyze the formation of the first two pathway-specific intermediates in MbA biosynthesis, myricetin 3-O-rhamnoside and myricetin 3-O-glucosyl rhamnoside. In previous work, expression of these UGTs in N. benthamiana resulted in small amounts of kaempferol glycosides but not myricetin glycosides, suggesting that myricetin was limiting. Here, we investigated montbretia genes and enzymes of flavonol biosynthesis to enhance myricetin formation in N. benthamiana We characterized two flavanone hydroxylases, a flavonol synthase, a flavonoid 3'-hydroxylase (F3'H), and a flavonoid 3'5'-hydroxylase (F3'5'H). Montbretia flavonol synthase converted dihydromyricetin into myricetin. Unexpectedly, montbretia F3'5'H shared higher sequence relatedness with F3'Hs in the CYP75B subfamily of cytochromes P450 than with those with known F3'5'H activity. Transient expression of combinations of montbretia flavonol biosynthesis genes and a montbretia MYB transcription factor in N. benthamiana resulted in availability of myricetin for MbA biosynthesis. Transient coexpression of montbretia flavonol biosynthesis genes combined with CcUGT1 and CcUGT2 in N. benthamiana resulted in 2 mg g-1 fresh weight of the MbA pathway-specific compound myricetin 3-O-glucosyl rhamnoside. Additional expression of the montbretia acyltransferase CcAT1 led to detectable levels of mini-MbA in N. benthamiana.

Mannose-Decorated Multicomponent Supramolecular Polymers Trigger Effective Uptake into Antigen-Presenting Cells.

A modular route to prepare functional self-assembling dendritic peptide amphiphiles decorated with mannosides, to effectively target antigen-presenting cells, such as macrophages, is reported. The monomeric building blocks were equipped with tetra(ethylene glycol)s (TEGs) or labeled with a Cy3 fluorescent probe. Experiments on the uptake of the multifunctional supramolecular particles into murine macrophages (Mφs) were monitored by confocal microscopy and fluorescence-activated cell sorting. Mannose-decorated supramolecular polymers trigger a significantly higher cellular uptake and distribution, relative to TEG carrying bare polymers. No cytotoxicity or negative impact on cytokine production of the treated Mφs was observed, which emphasized their biocompatibility. The modular nature of the multicomponent supramolecular polymer coassembly protocol is a promising platform to develop fully synthetic multifunctional vaccines, for example, in cancer immunotherapy.

Phenolic Compounds, Volatiles and Antioxidant Capacity of White Myrtle Berry Liqueurs.

The aim of this research was to evaluate the antioxidant capacity and physical-chemical characteristics of commercial white myrtle berry (Myrtus communis L. var. leucocarpa DC) liqueur (WMBL). The total phenolic (TP) content was measured spectrophotometrically, applying a modified Folin-Ciocalteu's method, and phenolic compounds were identified by high-performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry, and quantified by HPLC coupled with ultraviolet/visible detection. The antioxidant capacities were evaluated by FRAP, CUPRAC, DPPH•, and ABTS•+ assays. The volatiles were assessed by gas chromatography and mass spectrometry (GC-MS/FID) after headspace solid-phase microextraction (HS-SPME) and liquid-liquid extraction (LLE). WMBL showed lower TP levels (636.3 ± 39.2 mg GAE/L) than in purple myrtle berry liqueur (PMBL). Nevertheless, WMBL exhibited better antioxidant capacities, potentially due to high concentrations of gallic acid (294.2 ± 14.2 mg/L) and its derivatives (58.3 ± 2.1 mg/L). Other phenolic compounds detected by HPLC-DAD and LC-MS/MS were flavonols like myricetin and its derivatives (myricetin-3-O-galactoside and myricetin-3-O-rhamnoside) with concentrations similar to those found in PMBL. GC-MS/FID analysis revealed 44 compounds (terpenes, higher aliphatic compounds and shikimic acid pathway derivatives). 1,8-Cineole was the most abundant terpene in the liqueur (26.5% (HS-SPME) and 9.6% (LLE)).

Tri- and tetravalent mannoclusters cross-link and aggregate BC2L-A lectin from Burkholderia cenocepacia.

The opportunistic Gram-negative bacterium Burkholderia cenocepacia causes lethal infections in cystic fibrosis patients. Multivalent mannoside derivatives were prepared as potential inhibitors of lectin BC2L-A, one of the virulence factors deployed by B. cenocepacia in the infection process. An (α1→2)-thio-linked mannobioside mimic bearing an azide functionalized aglycon was conjugated to different multivalent scaffolds such as propargylated calix[4]arenes, methyl gallate and pentaerythritol by azide-alkyne 1,3-dipolar cycloaddition. The interaction between the glycoclusters and the mannose binding BC2L-A lectin from B. cenocepacia was examined by isothermal microcalorimetry, surface plasmon resonance, inhibition of yeast agglutination and analytical ultracentrifugation.

The Antiadhesive Strategy in Crohn's Disease: Orally Active Mannosides to Decolonize Pathogenic Escherichia coli from the Gut.

Blocking the adherence of bacteria to cells is an attractive complementary approach to current antibiotic treatments, which are faced with increasing resistance. This strategy has been particularly studied in the context of urinary tract infections (UTIs), in which the adhesion of pathogenic Escherichia coli strains to uroepithelial cells is prevented by blocking the FimH adhesin expressed at the tips of bacteria organelles called fimbriae. Recently, we extended the antiadhesive concept, showing that potent FimH antagonists can block the attachment of adherent-invasive E. coli (AIEC) colonizing the intestinal mucosa of patients with Crohn's disease (CD). In this work, we designed a small library of analogues of heptyl mannoside (HM), a previously identified nanomolar FimH inhibitor, but one that displays poor antiadhesive effects in vivo. The anomeric oxygen atom was replaced by a sulfur or a methylene group to prevent hydrolysis by intestinal glycosidases, and chemical groups were attached at the end of the alkyl tail. Importantly, a lead compound was shown to reduce AIEC levels in the feces and in the colonic and ileal mucosa after oral administration (10 mg kg(-1) ) in a transgenic mouse model of CD. The compound showed a low bioavailability, preferable in this instance, thus suggesting the possibility of setting up an innovative antiadhesive therapy, based on the water-soluble and non-cytotoxic FimH antagonists developed here, for the CD subpopulation in which AIEC plays a key role.

Streptomyces oryzae sp. nov., an endophytic actinomycete isolated from stems of rice plant.

An actinomycete strain S16-07(T), isolated from surface-sterilized stems of rice plant (Oryza sativa L.), was characterized using a polyphasic approach. Phylogenetic analysis of 16S rRNA gene sequences indicated affiliation of the strain belonged to the genus Streptomyces. The highest levels of sequence similarity were found with Streptomyces smyrnaeus SM3501(T) (97.7% similarity), S. abikoensis NBRC 13860(T) (97.6% similarity) and S. thermocarboxydovorans NBRC 16324(T) (97.5% similarity). The cell wall of strain S16-07(T) contained LL-diaminopimelic acid. The predominant menaquinones were MK-9(H6) and MK-9(H8). Phospholipids detected were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, hydroxy-phosphatidylethanolamine, hydroxy-phosphatidylmonomethylethanolamine and phosphatidylinositol mannosides. The major cellular fatty acids were ai-C(15:0), i-C(16:0) and ai-C(17:0). The G+C content of strain S16-07(T) was 70.4 mol%. On the basis of the phylogeny of the isolate and its differences from the most closely related species, the isolate S16-07(T) represents a novel species for which the name S. oryzae sp. nov. is proposed. The type strain is S16-07(T) (=BCC 60400(T)=NBRC 109761(T)).

Enhanced bioavailability and safety of curcumagalactomannosides as a dietary ingredient.

In spite of the various bioavailable formulations of curcumin for pharma and dietary supplement applications, food grade formulations suitable as a dietary ingredient, and capable of providing significant levels of plasma curcumin, are limited. The present contribution describes the safety and oral bioavailability of a novel water soluble formulation of curcumin, curcumagalactomannosides (CGM), when used as a dietary ingredient in selected food items. CGM was prepared using a food grade hydrocolloid (galactomannans) derived from the kitchen spice fenugreek (Trigonella foenum graccum), without using any synthetic excipients. The safety of the formulation was assessed through acute and subchronic toxicity studies on Wistar rats and genotoxicity studies. The efficacy of CGM as a bioavailable dietary ingredient was assessed by successfully preparing various food items and by measuring the post-blood plasma curcumin levels at various time intervals after the consumption of food items. High performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) and electrospray ionization tandem mass spectrometer (ESI-MS/MS) was employed for the quantification of plasma curcuminoids. It was observed that CGM is safe and suitable for further development and clinical studies, with a no observable adverse effect level (NOAEL) up to 2.0 g kg⁻¹ per day b.wt. CGM was found to offer seven to ten times higher bioavailability of curcumin in humans, when incorporated into various food/beverage items at 100 mg CGM per serving size, as compared to the standard unformulated curcumin.

Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms.

Mycobacteria develop strategies to evade the host immune system. Among them, mycobacterial LAM or PIMs inhibit the expression of pro-inflammatory cytokines by activated macrophages. Here, using synthetic PIM analogues, we analyzed the mode of action of PIM anti-inflammatory effects. Synthetic PIM(1) isomer and PIM(2) mimetic potently inhibit TNF and IL-12 p40 expression induced by TLR2 or TLR4 pathways, but not by TLR9, in murine macrophages. We show inhibition of LPS binding to TLR4/MD2/CD14 expressing HEK cells by PIM(1) and PIM(2) analogues. More specifically, the binding of LPS to CD14 was inhibited by PIM(1) and PIM(2) analogues. CD14 was dispensable for PIM(1) and PIM(2) analogues functional inhibition of TLR2 agonists induced TNF, as shown in CD14-deficient macrophages. The use of rough-LPS, that stimulates TLR4 pathway independently of CD14, allowed to discriminate between CD14-dependent and CD14-independent anti-inflammatory effects of PIMs on LPS-induced macrophage responses. PIM(1) and PIM(2) analogues inhibited LPS-induced TNF release by a CD14-dependent pathway, while IL-12 p40 inhibition was CD14-independent, suggesting that PIMs have multifold inhibitory effects on the TLR4 signalling pathway.

Dual-targeting daunorubicin liposomes improve the therapeutic efficacy of brain glioma in animals.

Chemotherapy for brain glioma has been of limited value due to the inability of transport of drug across the blood-brain barrier (BBB) and poor penetration of drug into the tumor. For overcoming these hurdles, the dual-targeting daunorubicin liposomes were developed by conjugating with p-aminophenyl-alpha-D-manno-pyranoside (MAN) and transferrin (TF) for transporting drug across the BBB and then targeting brain glioma. The dual-targeting effects were evaluated on the BBB model in vitro, C6 glioma cells in vitro, avascular C6 glioma tumor spheroids in vitro, and C6 glioma-bearing rats in vivo, respectively. After applying dual-targeting daunorubicin liposomes, the transport ratio across the BBB model was significantly increased up to 24.9%. The most significant uptake by C6 glioma was evidenced by flow cytometry and confocal microscope. The C6 glioma spheroid volume ratio was significantly lowered to 54.7%. The inhibitory rate to C6 glioma cells after crossing the BBB was significantly enhanced up to 64.0%. The median survival time of tumor bearing rats after administering dual-targeting daunorubicin liposomes (22 days) was significantly longer than that after giving free daunorubicin (17 days, P=0.001) or other controls. In conclusion, the dual-targeting daunorubicin liposomes are able to improve the therapeutic efficacy of brain glioma in vitro and in animals.

A long-wavelength fluorescent substrate for continuous fluorometric determination of alpha-mannosidase activity: resorufin alpha-D-mannopyranoside.

A simple and reliable continuous assay for measurement of alpha-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin alpha-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK(a) of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi alpha-mannosidases. The assay performed using recombinant Drosophila Golgi alpha-mannosidase (dGMII) has been shown to give the kinetic parameters K(m) of 200 microM and V(max) of 11 nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known alpha-mannosidase inhibitor swainsonine are also presented, demonstrating a potential for use of the assay as a simple method for high-throughput screening of inhibitors potentially useful in cancer treatment.

Intervening with urinary tract infections using anti-adhesives based on the crystal structure of the FimH-oligomannose-3 complex.

BACKGROUND: Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and alpha3beta1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that alpha-D-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl alpha-D-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl alpha-D-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl alpha-D-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Man alpha1,3Man beta1,4GlcNAc beta1,4GlcNAc in an extended binding site. The interactions along the alpha1,3 glycosidic bond and the first beta1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl alpha-D-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group. CONCLUSIONS/SIGNIFICANCE: The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection.

Affinity and kinetics of sialyl Lewis-X and core-2 based oligosaccharides binding to L- and P-selectin.

Soluble oligosaccharide mimetics of natural selectin ligands act as competitive inhibitors of leukocyte adhesion in models of inflammation. We quantified the binding of simple oligosaccharides based on sialyl Lewis-X (sLe(X)) and complex molecules with the core-2 structure to L- and P-selectin, under both static and fluid flow conditions. Isolated human neutrophils were employed to mimic the physiological valency of selectins and selectin ligands. Surface plasmon resonance studies quantified binding kinetics. We observed the following: (i) The functional group at the anomeric position of carbohydrates plays an important role during selectin recognition, since sLe(X) and sialyl Lewis-a (sLe(a)) were approximately 5-7-fold poorer inhibitors of L-selectin mediated cell adhesion compared to their methyl glycosides. (ii) Despite their homology to physiological glycans, the putative carbohydrate epitopes of GlyCAM-1 and PSGL-1 bound selectins with low affinity comparable to that of sLe(X)-selectin interactions. Thus, besides the carbohydrate portion, the protein core of GlyCAM-1 or the presentation of carbohydrates in clusters on this glycoprotein may contribute to selectin recognition. (iii) A compound Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,6(GalNAcbeta1,3)GalNAcalpha-OMe was identified which blocked L- and P-selectin binding at 30-100-fold lower doses than sLe(X). (iv) Surface plasmon resonance experiments determined that an sLe(X) analogue (TBC1269) competitively inhibited, via steric/allosteric mechanisms, the binding of two anti-P-selectin function blocking antibodies that recognized different epitopes of P-selectin. (v) TBC1269 bound P-selectin via both calcium-dependent and -independent mechanisms, with K(D) of approximately 111.4 microM. The measured on- and off-rates were high (k(off) > 3 s(-)(1), k(on) > 27,000 M(-)(1) s(-)(1)). Similar binding kinetics are expected for sLe(X)-selectin interactions. Taken together, our study provides new insight into the kinetics and mechanisms of carbohydrate interaction with selectins.

Influence of spacer length on interaction of mannosylated liposomes with human phagocytic cells.

PURPOSE: To improve target specificity and uptake of liposomes by macrophages, one can improve high-affinity receptor binding to mannose determinants with their 175-kDa mannose receptor (MR), which is mainly influenced by the length and flexibility of the spacer between the carbohydrate head group and liposome surface. Liposomes containing alkylmannosides with hydrophilic spacers 0 to 8 ethyleneoxy units (EO) long (Man0...Man8) were used to investigate systematically the effects of spacer length on liposome-cell interactions. METHODS: Concanavalin A (ConA)-induced liposome aggregation was studied by turbidity measurement and cell uptake using PMA-induced HL-60 cells or native human macrophages by determining 6-CF after cell lysis or NBD-fluorescence with flow cytometry. Detection of MR in native cell populations was carried out by an antibody assay using flow cytometry; MR-representing cells were selected analytically. RESULTS: Liposomes containing mannosides with more than one EO spacer length were specifically aggregated by ConA, indicating accessibility of the carbohydrate ligands of these derivatives. Increase in EO spacer units of incorporated mannosides (two or more EO) led to suppression of cellular uptake of mannosylated liposomes by phagocytes lacking MR (HL60, U937). The extent of suppression increased with spacer length. Liposome uptake by native macrophages expressing MR was, on the contrary, improved, particularly by Man6 and Man8. CONCLUSIONS: Uptake of liposomes modified with Man6 or Man8 by native cells was enhanced but did not reach an optimum. Thus, Man6, Man8, and mannosides with even longer spacer arms are of potential use in receptor-mediated targeting.

Analysis of binding of mannosides in relation to Langerin (CD207) in Langerhans cells of normal and transformed epithelia.

Tandem-repeat C-type lectins (pattern-recognition receptors) with specificity for mannosides are intimately involved in antigen recognition, uptake, routing and presentation in macrophages and dendritic cells. In Langerhans cells, Langerin (CD207), a type-II transmembrane protein with a single C-type carbohydrate recognition domain attached to a heptad repeat in the neck region, which is likely to establish oligomers with an alpha-coiled-coil stalk, has been implicated in endocytosis and the formation of Birbeck granules. The structure of Langerin harbours essential motifs for Ca2+-binding and sugar accommodation. Lectin activity has previously been inferred by diminished antibody binding to cells in the presence of the glycan ligand mannan. In view of the complexity of the C-type lectin/lectin-like network, it is unclear what role Langerin plays for Langerhans cells in binding mannosides. In order to reveal in frozen tissue sections to what extent mannose-binding activity co-localizes with Langerin, we have used a synthetic marker, i.e. a neoglycoprotein carrying mannose maxiclusters, as a histochemical ligand, and computer-assisted fluorescence monitoring in a double-labelling procedure. Mannoside-binding capacity was detected in normal epithelial cells. Double labelling ensured the unambiguous assessment of the binding of the neoglycoprotein in Langerhans cells. Light-microscopically, its localization profile resembled the pattern of immunohistochemical detection of Langerin. This result has implications for suggesting rigorous controls in histochemical analysis of this cell type, because binding of kit reagents, i.e. mannose-rich glycoproteins horseradish peroxidase or avidin, to Langerin (or a spatially closely associated lectin) could yield false-positive signals. To show that recognition of carbohydrate ligands in dendritic cells is not restricted to mannose clusters, we have also documented binding of carrier-immobilized histo-blood group A trisaccharide, a ligand of galectin-3, which was not affected by the presence of a blocking antibody to Langerin. Remarkably, access to the carbohydrate recognition domain of Langerin appeared to be impaired in proliferatively active environments (malignancies, hair follicles), indicating presence of an endogenous ligand with high affinity to saturate the C-type lectin under these conditions.

Determination of UDP-N-acetylglucosamine: beta-D-mannoside-1,4-N-acetylglucosaminyltransferase-III in patients sera with chronic hepatitis and liver cirrhosis using a monoclonal antibody.

The glycoprotein UDP-N-acetylglucosamine: beta-D-mannoside-1,4-N-acetylglucosaminyltransferase-III (GnT-III) catalyzes the addition of N-acetylglucosamine via a beta-1, 4-linkage to the beta-linked mannose of the trimannosyl core of N-linked glycans. It has been reported that the expression of GnT-III increases in many oncogenically transformed cells and human hepatocellular carcinoma (HCC) tissues, and GnT-III enzyme activity in serum can be used for the detection and monitoring of primary hepatomas and hepatocellular carcinomas. A solid-phase enzyme-linked immunosorbent sandwich assay in which a polyclonal antibody (PAb) to aglycosylrecombinant GnT-III (AGR-GnT-III) and a monoclonal antibody (mAb) are employed as a capture protein and probe protein, respectively, is described. The sensitivity of the PAb-mAb sandwich assay, as determined by the dose-response effect for AGR-GnT-III, was 10 ng/ml. This assay was specific for GnT-III and did not detect beta-1, 6-N-acetylglucosaminyltrasferase-V (GnT-V). AGR-GnT-III concentrations in 377 serum specimens were determined by the PAb-mAb sandwich assay and the results were analyzed based on the disease category, using 1.99 microg/mL (AGR-GnT-III) as a cut-off value. The AGR-GnT-III level of 61 normal serum samples was 0.57 +/- 0.71 microg/ml (mean +/- SD). The results revealed an elevation in serum AGR-GnT-III levels in 60 of 86 patients (3.03 +/- 2.04 microg/ml) with liver cirrhosis (LC) and 86 of 91 patients (2.73 +/- 0.59 microg/ml) with chronic hepatitis (CH). By contrast, 3 of 61 normal subjects, 9 of 34 patients (1.02 +/- 1.03 microg/ml) with acute hepatitis and 8 of 38 patients (1.79 +/- 0.56 microg/ml) with a variety of non-hepatic diseases exhibited a slight increase above the cut-off value. These results indicate that serum AGR-GnT-III levels are elevated predominantly in LC or CH cases. Serum AGR-GnT-III concentration, as measured by the developed PAb-mAb sandwich assay, may be a useful differential marker as a diagnostic aid for CH and/or LC and warrants further investigations with expanded serum panels.

Uptake of dimannoside clusters and oligomannosides by human dendritic cells.

Knowing that human blood monocyte-derived dendritic cells express cell-surface mannose-specific lectins, we prepared various mannoses containing glycoconjugates with the aim of developing highly specific synthetic carriers of oligonucleotides and genes. Conjugates were prepared from oligosaccharides obtained by hydrazinolysis of Saccharomyces cerevisiae invertase glycopeptides. The reducing saccharides were converted into glycosynthons, i.e., into glyco-amino acids. Fluorescein derivatives were obtained by coupling the free carboxyl group of oligosaccharyl-pyroglutamate to the alpha-amino group of epsilon-fluoresceinyl-thiocarbamyl lysine methyl ester. It has been shown by others that glycosylated linear oligolysines containing up to six alpha-D-mannopyranosylphenylthiocarbamyl units have a high affinity for the human mannose receptor. In order to obtain fully biodegradable clusters and to improve both the specificity and the selectivity, disaccharides transformed into glycosynthons were coupled to pentalysine carriers (Lys5-Ala-Cys-NH2). Glycosylated pentalysyl cysteine conjugates were made fluorescent upon substitution of the cysteine thiol group with fluorescein iodoacetamide. As shown by flow cytofluorimetry, both the dimannoside clusters and yeast oligomannosides were very efficiently taken up by DC, conversely lactoside clusters were not.