Invariant natural killer T cells stimulated with cholesteryl glycosides modulate immune responses in allergy and delayed-type hypersensitivity.

Invariant NKT cells were stimulated with cholesteryl O-acyl α-glycosides in the context of CD1d. The activated NKT cells have potential to sustain the homeostasis in the body exposed to excess in either Th1- or Th2-immunity.

Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage.

GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors.

Total synthesis of legionaminic acid as basis for serological studies.

Legionaminic acid is a nine-carbon diamino monosaccharide that is found coating the surface of various bacterial human pathogens. Its unique structure makes it a valuable biological probe, but access via isolation is difficult and no practical synthesis has been reported. We describe a stereoselective synthesis that yields a legionaminic acid building block as well as linker-equipped conjugation-ready legionaminic acid starting from cheap d-threonine. To set the desired amino and hydroxyl group pattern of the target, we designed a concise sequence of stereoselective reactions. The key transformations rely on chelation-controlled organometallic additions and a Petasis multicomponent reaction. The legionaminic acid was synthesized in a form that enables attachment to surfaces. Glycan microarray containing legionaminic acid revealed that human antibodies bind the synthetic glycoside. The synthetic bacterial monosaccharide is a valuable probe to detect an immune response to bacterial pathogens such as Legionella pneumophila, the causative agent of Legionnaire's disease.

Anti-Allergic Activities of Smilax glabra Rhizome Extracts and Its Isolated Compounds.

BACKGROUND: The rhizomes of Smilax glabra (SG) has long been used in Traditional Chinese and Thai herbal medicine to treat a variety of infectious diseases and immunological disorders. OBJECTIVE: To investigate the in vitro anti-allergic activities of crude extracts andpure isolated flavonoid compounds from SG by determination of inhibitory effect on antigen-induced release of ß-hexosaminidasefrom RBL-2H3 cells. MATERIAL AND METHOD: The in vitro inhibitory effects ofcrude aqueous and organic extracts on ß-hexosaminidase release in RBL-2H3 cells were evaluated as an in vitro indication ofpossible anti-allergic activity. Bioassay-guided fractionation of extracts was used to isolate flavonoid compounds from the ethanolic extracts. RESULTS: The 95% and 50% ethanolic extracts of SG showed remarkably high anti-allergic activity, with IC50 values of 5.74 ± 2.44 and 23.54 ± 4.75 µg/ml, much higher activity than that for Ketotifen (IC50 58.90 µM). The water extract had negligible activity (IC50 > 100 µg/ml). The two isolated flavonols, Engeletin and Astilbin, showed weak anti-allergic activity, IC50 values 97.46 ± 2.04 and >100 µg/ml, respectively. CONCLUSION: The 95% and 50% ethanolic extracts of SG showed strong anti-allergic activity, but two flavonol constituents did not show any significant anti-allergic activity. These findings suggest that a combination of effects of various phytochemicals in crude extracts used in traditional medicine, are responsible for the purported anti-allergic activity of SG herbal preparations. The plethora of constituents in crude extracts, as yet unidentified, are likely to be acting synergistically to account for the strong observed anti-allergic in vitro activity.

Tubular immunostimulating complex based on cucumarioside A2-2 and monogalactosyldiacylglycerol from marine macrophytes.

BACKGROUND: There is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity. RESULTS: The cucumarioside A2-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times. CONCLUSIONS: We propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.

Investigating the elusive mechanism of glycosaminoglycan biosynthesis.

Glycosaminoglycan (GAG) biosynthesis requires numerous biosynthetic enzymes and activated sulfate and sugar donors. Although the sequence of biosynthetic events is resolved using reconstituted systems, little is known about the emergence of cell-specific GAG chains (heparan sulfate, chondroitin sulfate, and dermatan sulfate) with distinct sulfation patterns. We have utilized a library of click-xylosides that have various aglycones to decipher the mechanism of GAG biosynthesis in a cellular system. Earlier studies have shown that both the concentration of the primers and the structure of the aglycone moieties can affect the composition of the newly synthesized GAG chains. However, it is largely unknown whether structural features of aglycone affect the extent of sulfation, sulfation pattern, disaccharide composition, and chain length of GAG chains. In this study, we show that aglycones can switch not only the type of GAG chains, but also their fine structures. Our findings provide suggestive evidence for the presence of GAGOSOMES that have different combinations of enzymes and their isoforms regulating the synthesis of cell-specific combinatorial structures. We surmise that click-xylosides are differentially recognized by the GAGOSOMES to generate distinct GAG structures as observed in this study. These novel click-xylosides offer new avenues to profile the cell-specific GAG chains, elucidate the mechanism of GAG biosynthesis, and to decipher the biological actions of GAG chains in model organisms.

Glucopyranosyl lipid adjuvant enhances immune response to Ebola virus-like particle vaccine in mice.

The identification of adjuvants that promote lasting antigen-specific immunity and augment vaccine efficacy are integral to the development of new protein-based vaccines. The Ebola virus-like particle (VLP) vaccine expressing Ebola virus glycoprotein (GP) and matrix protein (VP40) was used in this study to evaluate the ability of TLR4 agonist glucopyranosyl lipid adjuvant (GLA) formulated in a stable emulsion (SE) to enhance immunogenicity and promote durable protection against mouse-adapted Ebola virus (ma-EBOV). Antibody responses and Ebola-specific T cell responses were evaluated post vaccination. Survival analysis after lethal ma-EBOV challenge was performed 4 weeks and 22 weeks following final vaccination. GLA-SE enhanced EBOV-specific immunity and resulted in long-term protection against challenge with ma-EBOV infection in a mouse model. Specifically, GLA-SE elicited Th1-skewed antibodies and promoted the generation of EBOV GP-specific polyfunctional T cells. These results provide further support for the utility of TLR4 activating GLA-SE-adjuvanted vaccines.

Structural characterization and immunosuppressive activity of a new pregnane glycoside from Epigynum cochinchinensis.

Phytochemical investigation on the ethyl acetate fraction of the leaves of Epigynum cochinchinensis led to the isolation of a new C21 pregnane glycoside, epigycoside B (1), together with three known analogues. Their structures were elucidated on the basis of extensive spectroscopic techniques, including UV, MS, and NMR experiments, as well as the chemical methods. Compound 1 displayed in vitro immunosuppressive activity against concanavalin A (Con A)/Lipopolysaccharides (LPS)-stimulated proliferation of mice splenocyte. The activity was significant as compared with control group at 50 µM concentration.

Immunochemical detection of flavonoid glycosides: development, specificity, and application of novel monoclonal antibodies.

Flavonoid-rich diets are expected to decrease the risk of cardiovascular diseases. The localization and target sites of flavonoids underlying the protective mechanism in vivo have not been fully investigated because the methods for detection of flavonoids have been limited to chemical analysis such as high-performance liquid chromatography. To further understand the actions of flavonoids in vivo, we developed a novel methodology that immunochemically evaluates flavonoids using specific antibodies. Quercetin-3-glucuronide (Q3GA), a major metabolite in human plasma, was coupled with keyhole limpet hemocyanin. Alternatively, the sugar moiety of quercetin-3-glucoside (Q3G) was succinylated and then coupled with a carrier protein. Using these two immunogens, we finally obtained two monoclonal antibodies, mAb14A2 and mAb11G6, from the immunogen using Q3GA and Q3G, respectively. Competitive enzyme-linked immunosorbent assay showed the unique difference in the specificity between the two similar antibodies: mAb14A2 recognized several quercetin-3-glycosides including Q3G and rutin but mAb11G6 was highly specific to the Q3G structure. The macrophage-derived foam cells in human atherosclerotic lesions were significantly stained with mAb14A2 but scarcely with mAb11G6. These results showed that the anti-flavonoid glycoside antibodies are useful tools for evaluating their localization in tissues and that the specificities strongly depend on the immunogen design for synthesizing the hapten-protein conjugates.

Development of an FHbp-CTB holotoxin-like chimera and the elicitation of bactericidal antibodies against serogroup B Neisseria meningitidis.

The Neisseria meningitidis factor H binding protein (FHbp) is an important virulence factor and vaccine antigen contained in both USA licensed serogroup B meningococcal vaccines. Recent studies in human factor H (hFH) transgenic mice suggest that hFH-FHbp interactions lower FHbp-elicited immunogenicity. To provide tools with which to characterize and potentially improve FHbp immunogenicity, we developed an FHbp-cholera holotoxin-like chimera vaccine expression system in Escherichia coli that utilizes cholera toxin B (CTB) as both a scaffold and adjuvant for FHbp. We developed FHbp-CTB chimeras using a wild-type (WT) FHbp and a low hFH-binding FHbp mutant R41S. Both chimeras bound to GM1 ganglioside and were recognized by the FHbp-specific monoclonal antibody JAR4. The R41S mutant had greatly reduced hFH binding compared to the WT FHbp-CTB chimera. WT and R41S FHbp-CTB chimeric antigens were compared to equimolar amounts of FHbp admixed with CTB or FHbp alone in mouse immunogenicity studies. The chimeras were significantly more immunogenic than FHbp alone or mixed with CTB, and elicited bactericidal antibodies against a panel of MenB isolates. This study demonstrates a unique and simple method for studying FHbp immunogenicity. The chimeric approach may facilitate studies of other protein-based antigens targeting pathogenic Neisseria and lay groundwork for the development of new protein based vaccines against meningococcal and gonococcal disease.

Immunomodulatory effect of Polypodium leucotomos (Anapsos) in child palatine tonsil model.

BACKGROUND: Recurrent tonsillitis might reduce the immunological capability of fighting against the infection of tonsil tissue. Polypodium leucotomos (Anapsos) immunomodulating effect has been subject of research in the last years. The aim of this research is to test the in vitro immunomodulating capacity of Anapsos in a child palatine tonsil explants model. METHODS: Palatine tonsils explants of children undergoing amigdalectomy were stimulated with mononuclear cells obtained from their own blood by density gradient centrifugation. Some were then treated with Anapsos while others rest untreated. Cytokines were measured by ELISA, immune cells activation was measured by flow cytometry and activation of immunoglobulins was appreciated by indirect immunofluorescence in tonsils tissue. RESULTS: Anapsos activates Natural Killers cells. It increases IL-2 and IFN-γ levels by the activation of Th2 lymphocytes, and IL-10, by the Th1 lymphocytes. Anapsos also increases immunoglobulins IgM, IgD and IgG4 by B-lymphocyte activation in tonsils tissue. CONCLUSION: Anapsos has an immunomodulating effect, both in humoral and cellular responses, which might benefit children suffering of recurrent tonsillitis as it could enhance their immune system. This effect might reduce the number of episodes suffered and therefore the number of children undergoing surgery.

Possible role of human natural anti-Gal antibodies in the natural antitumor defense system.

Expression of Gal alpha 1-3Gal cell surface residues has been correlated with the metastatic potential of murine tumor cells. We report that Gal alpha 1-3Gal residues are expressed at the cell surface of malignant human cancer cells, including four cell lines and 50% of the malignant breast specimens obtained by aspiration biopsy. In contrast, all benign breast biopsies and normal cells were Gal alpha 1-3Gal negative. Affinity-purified anti-alpha-galactosyl IgG (anti-Gal) antibody, which specifically recognizes Gal alpha 1-3Gal residues, significantly inhibited cell attachment in two in vitro assays thought to indicate tumor cell extravasation of the circulatory system during the metastatic process: attachment to perfused human umbilical vein endothelium, and attachment to isolated laminin. Since anti-Gal antibody is a natural component of all human sera, we propose that it may be part of the natural antitumor defense system in humans.

Modulatory potency of the beta-galactoside-specific lectin from mistletoe extract (Iscador) on the host defense system in vivo in rabbits and patients.

Proprietary extract of mistletoe (Iscador) that has federal approval for clinical application can exhibit immunomodulatory capacity. However, the nature of this responsible substance has still remained elusive. To validate the hypothesis that specific lectin-carbohydrate interactions at least participate in eliciting immunomodulation, the modulatory efficiency of the major beta-galactoside-specific mistletoe lectin (ML I) from the clinically applied extract on selected immunological parameters was monitored "in vivo" in rabbits. Injections of nontoxic doses of the purified lectin or even only of its carbohydrate-binding subunit (0.25-1.0 ng/kg) into rabbits yielded significant increases in natural killer cytotoxicity, frequency of large granular lymphocytes, and phagocytic activity of granulocytes. In the clinically relevant situation, changes in these parameters were also determined in cancer patients after extract (Iscador) injection s.c. as well as i.v., emphasizing the potential relevance of the lectin. Comparative analyses of the changes in the selected parameters following injection of extract with normal lectin content as well as of extract, selectively depleted of lectin, into healthy volunteers corroborated this inference. Lectin depletion by affinity chromatography was highly specific and did not affect any other substance in the extract. Remarkably, contamination by endotoxin has been rigorously excluded in each applied specimen. These results encourage detailed elucidation of lectin action on various parts of the tumor defense system "in vitro" with the long range goal of achieving progress in the treatment of cancer through immunological strategies, exploring selective mediatory lectin-carbohydrate interactions.

Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (k(off )< 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

Ricin-Holotoxin-Based Vaccines: Induction of Potent Ricin-Neutralizing Antibodies.

Ricin is one of the most potent and lethal toxins known to which there is no available antidote. Currently, the most promising therapy is based on neutralizing antibodies elicited by active vaccination or given passively. Here, detailed protocols are provided for the production of two ricin holotoxin-based vaccines: monomerized subunit-based vaccine, and a formaldehyde-based ricin toxoid vaccine. Both vaccines were found to be stable with no toxic activity reversion even after long-term storage while eliciting high anti-ricin antibody titers possessing a potent neutralizing activity. The use of these vaccines is highly suitable for both the production of sera that can be used in passive protection experiments and immunization aimed to isolate potent anti-ricin monoclonal antibodies.

Multiglycosidorum tripterygii versus Tacrolimus for rat tracheal allografts.

OBJECTIVE: Several other immunosuppressive agents still need to be found for rejection as alternatives to Tacrolimus in lung transplantation. We tried to elucidate the treatment effect of Multiglycosidorum tripterygii on tracheal allografts in comparison to that of Tacrolimus. METHODS: Treatment effect of agents on tracheal allografts, undergoing incomplete immunosuppression for 12 weeks after transplantation, was investigated using a heterotopic rat tracheal transplantation model. Treatments with Tacrolimus (1.0 or 1.5mg/kg per day), Multiglycosidorum tripterygii (150 or 225mg/kg per day) and a combination of Tacrolimus (1.0mg/kg per day) and Multiglycosidorum tripterygii (150mg/kg per day) were applied as a therapy for allografts. Four weeks after administering this therapy, the effect of each treatment was investigated by the morphologic assessment of transplants. RESULTS: Treatment group with high doses of Multiglycosidorum tripterygii demonstrated a significantly better graft patency and lower cartilage dislocation than that without any treatment and tended to show better morphological findings than the other treatment groups, in addition to being safe. Some of allografts with high doses of Tacrolimus or Multiglycosidorum tripterygii therapy had a viable epithelium and viable tracheal glands in part, whereas the allografts with other treatments showed almost a completely denuded epithelium. High doses of Multiglycosidorum tripterygii therapy demonstrated less infiltration of mononuclear cells into the allografts, whereas other therapies showed a higher infiltration of such cells. CONCLUSIONS: We conclude that high doses of Multiglycosidorum tripterygii may be a useful alternative to Tacrolimus as an immunosuppressant for rat tracheal allografts.

Human IgE binding to the glycosidic moiety of bovine kappa-casein.

IgE ability for recognizing milk proteins was assayed in the serum of an adult atopic patient who outgrew cow milk allergy in early childhood. A number of protein species included in casein from bovine milk were detected by human IgE in immunoblotting experiments. Comparing these results with those obtained from an analysis using antibody preparations specifically directed toward the different casein fractions, IgE-reactive bands were identified as isoforms of kappa-casein. IgE-reactive protein was not present in neither bovine cheese, regardless of cheese-making technology and time ripening, nor milk from any other dairy animal, such as ewe, goat, and water buffalo. Chemical deglycosylation of protein bands immobilized onto nitrocellulose proved that the glycosidic moiety of bovine kappa-casein was principally involved in IgE recognition.

Probing the combining site of monoclonal IgA J539 using deoxyfluoro- and other galactosides as ligands.

IgA J539 is a monoclonal anti beta-(1----6)-D-galactopyranan. Measurement of its affinity with a number of synthetic galactosyl oligosaccharides of that linkage type, in which one or more hydroxyl groups have been replaced by a fluorine atom, confirm the assignment of the relative binding strength of the subsites in the protein for the individual galactosyl residues of the antigen.

Allergic contact dermatitis to Alstroemeria.

Two female florists developed dermatitis of the fingertips. Patch testing revealed allergic contact dermatitis to the flower, Alstroemeria, used in floral arrangements. They had positive patch tests to portions of Alstroemeria, and to tuliposide A, the allergen in this plant. Vinyl gloves were not helpful since tuliposide A readily penetrates through these gloves. Nitrile gloves may be protective since they prevented positive patch test to tuliposide A.

The preparation of carbohydrate-protein conjugates: cyanuric trichloride coupling of 2-aminoethyl glycosides, and mixed-anhydride coupling of 8-carboxyoctyl glycosides to bovine serum albumin.

Preparation of the following glycosides is described: 2-aminoethyl beta-D-glycosides of (A) 2-acetamido-3,4,6-trio-O-acetyl-2-deoxy-D-glucopyranose, (B) 2-acetamido-4-O-(2-acetamido-3,4,6-trio-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-3,6-di-O-acetyl-2-deoxy-beta-D-glucopyranose (N,N'-diacetylchitobiose pentaacetate), (C) 4-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-2,3,6-trio-O-acetyl-beta-D-glucopyranose (cellobiose heptaacetate); 8-carboxyoctyl glycosides of (D) cellobiose, and (E) N,N'-diacetylchitobiose. Conjugates were prepared from (A), (B), and (C) by coupling to bovine serum albumin by cyanuric trichloride and subsequent deacetylation; (D) and (E) were coupled to bovine serum albumin by the mixed-anhydride reaction. Conjugates (A) and (B) were insoluble; conjugates (C), (D), and (E) functioned as artificial antigens and gave rise to precipitating antibodies in rabbits. Specificities of the antisera were determined by inhibition studies.