Non-glycosidic compounds can stimulate both human and mouse iNKT cells.

Invariant natural killer T (iNKT) cells recognize CD1d/glycolipid complexes and upon activation with synthetic agonists display immunostimulatory properties. We have previously described that the non-glycosidic CD1d-binding lipid, threitolceramide (ThrCer) activates murine and human iNKT cells. Here, we show that incorporating the headgroup of ThrCer into a conformationally more restricted 6- or 7-membered ring results in significantly more potent non-glycosidic analogs. In particular, ThrCer 6 was found to promote strong anti-tumor responses and to induce a more prolonged stimulation of iNKT cells than does the canonical α-galactosylceramide (α-GalCer), achieving an enhanced T-cell response at lower concentrations compared with α-GalCer both in vitro, using human iNKT-cell lines and in vivo, using C57BL/6 mice. Collectively, these studies describe novel non-glycosidic ThrCer-based analogs that have improved potency in iNKT-cell activation compared with that of α-GalCer, and are clinically relevant iNKT-cell agonists.

Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells.

Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than alpha-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/alpha-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.

Anaphylaxis to excipient mannitol: evidence for an immunoglobulin E-mediated mechanism.

BACKGROUND: Anaphylaxis to mannitol present naturally in pomegranate and cultivated mushroom in a sensitized subject has been described recently, and an IgE-mediated mechanism to this sugar alcohol has been proposed. The same subject also experienced severe allergic reactions to a chewable pharmaceutical (cisapride drug). OBJECTIVE: The purpose of the study was to identify allergenic component in the pharmaceutical preparation, and also, to understand the mechanism of immediate hypersensitivity to mannitol. METHODS: Methodology involved skin prick tests (SPTs), high-performance liquid chromatographic (HPLC) analysis of pharmaceutical preparations, separation of mannitol by Ca++-ion-moderated cation-exchange chromatography, preparation of alditol-protein conjugates by reductive amination, SPT using the conjugates, hapten affinity purification of the allergic serum on D-mannitol-keyhole limpet haemocyanin (KLH)-Sepharose CL-6B, and detection of serum mannitol-specific IgE by ELISA. RESULTS: Component testing by SPT, and HPLC analysis of various pharmaceuticals indicated that the excipient mannitol is the causative allergen. Mannitol separated from Cisapid MPS showed allergenic activity by SPT. Among the several conjugates tested by SPT, D-mannitol-bovine serum albumin and D-mannitol-KLH showed positive weal/flare reaction, demonstrating the presence of cell-bound mannitol-specific IgE in vivo. Negative results with D-glucitol, D-galactitol, meso-erythritol, and L-mannitol protein conjugates clearly showed that the mannitol-specific human IgE is very specific to the D-isomer of mannitol. ELISA using the hapten affinity-purified allergic serum was positive, demonstrating the presence of mannitol-specific serum IgE in the allergic subject. CONCLUSION: Mannitol, which is widely used as a food and drug additive (excipient), can rarely cause IgE-mediated anaphylaxis. This study is the first one to demonstrate the presence of mannitol-specific human IgE in a sensitized allergic subject to validate an IgE-mediated hypersensitivity mechanism for mannitol.

[Antigenicity study on lactitol (NS-4)].

Lactitol hydrate (lactitol) was tested for its antigenicity in guinea pigs and mice. The following results were obtained. 1. No active systemic anaphylaxis reactions were found in guinea pigs sensitized subcutaneously with lactitol alone or in combination with Freund's complete adjuvant (FCA). 2. No 24 hr heterologous passive cutaneous anaphylaxis reactions were elicited in rats by sera from mice sensitized intraperitoneally with lactitol alone or in combination with 3% aluminum hydroxide gel. 3. No passive hemagglutination reactions were elicited by sera from mice sensitized subcutaneously with lactitol in combination with FCA. From these results, it is concluded that lactitol has no antigenicity under the present experimental conditions.

An evaluation of serodiagnostic tests in patients with candidemia: beta-glucan, mannan, candida antigen by Cand-Tec and D-arabinitol.

The serodiagnostic tests, beta-glucan, mannan, candida antigen by Cand-Tec, and D-arabinitol were evaluated in 10 patients with candidemia, 14 patients with suspected fungemia, and 10 healthy persons. By blood culture or lysis centrifugation, C. albicans was isolated from 5 patients, C. parapsilosis from 4, and C. tropicalis from 1 patient; no organisms were isolated from the 14 patients with suspected fungemia or the 10 healthy subjects. Beta-glucan was measured by the difference between two chromogenic limulus tests (Endotoxin test-D and Endospecy), which was more than 60 pg/ml in 7 of 9 (78%) candidemic patients and 1 of 12 (8%) patients with suspected fungemia. Mannan was positive in 6 of 10 (60%) candidemic patients and 1 of 13 (8%) patients with suspected fungemia. Both antigens were very sensitive and highly specific for candidemia. However, the Cand-Tec assay was less specific, because titers of more than 4 were observed in 5 of 14 (34%) patients with suspected fungemia. D-Arabinitol was the least sensitive, because a D-arabinitol/creatinine ratio greater than 2.0 mumol/mg was observed in only 2 of 7 (29%) candidemic patients. The titers of serodiagnostic tests decreased after successful treatment with an anti-fungal agent. Our results show that the combined use of the assays in necessary for accurate serological diagnosis of candidemia.

Structure of an allergenic pentasaccharitol, Gp-1 beta-b6, isolated from a sea squirt antigen, Gi-rep, as a minimum structural unit responsible for its allergenicity.

An allergenic pentasaccharitol, Gp-1 beta-b6, was isolated as a minimum structural unit responsible for the allergic reaction in skin of patients with sea squirt allergy from a saccharitol fraction, Gp-1 beta-b, that had been liberated by beta-elimination from a glycopeptide in a Pronase digest of a sea squirt antigen, Gi-rep. Methylation/GC-MS and FAB-MS analyses indicated the sugar sequence of Gp-1 beta-b6 to be GalNAcl----2Fucl----(GalNAc1----) 3,4GlcNAc1----3GalNAc-ol. To analyze the structure in more detail, Gp-1 beta-b6 was labeled with p-aminobenzoic acid ethyl ester (ABEE), i.e., the reducing terminal 3-O-substituted GalNAc-ol of the saccharitol was oxidized to 2-O-substituted L-ThrNAc with equimolar periodate, and the resultant aldehyde was labeled with ABEE by reductive amination. The ABEE-labeled Gp-1 beta-b6 was subjected to sequential exoglycosidase digestion with beta-N-acetylhexosaminidase, alpha-N-acetylgalactosaminidase, and alpha-fucosidase, and the digests were chromatographed on an HPLC column of TSK gel Amide 80. From the results of the HPLC, methylation/GC-MS, and FAB-MS analyses of the digests of the labeled substrate, the structure of Gp-1 beta-b6 was determined to be GalNAc alpha 1----2Fuc alpha 1----3(GalNAc beta 1----4)GlcNAc beta 1----3GalNAc-ol. Enzymatic elimination of either the non-reducing terminal beta-GalNAc or the non-reducing terminal alpha-GalNAc led to inactivation of the allergenic pentasaccharitol. Accordingly, it is possible that the allergenic saccharitol contains two disaccharide units as the allergy-specific epitopes, one GalNAc alpha 1----2Fuc alpha 1---- and the other GlcNAc beta 1----4GLcNAc beta 1----.

A B72.3 second-generation-monoclonal antibody (CC49) defines the mucin-carried carbohydrate epitope Gal beta(1-3) [NeuAc alpha(2-6)]GalNAc.

Mucus glycoproteins from bovine seminal plasma were demonstrated to express the MAbs CC49- and B72.3-defined epitopes on TAG 72 antigen. Inhibition studies with reductively cleaved mucin O-glycans from bovine seminal plasma and submaxillary glands revealed that CC49 binds specifically to a core-type sialyl-oligosaccharide alditol (fraction 2c), which could be defined with regard to its primary structure by FAB- and EI-mass spectrometry combined with methylation analysis: (formula; see text) Structurally related alditols NeuAc alpha(2-6)-GalNAc-ol, Gal beta(1-3)GalNAc-ol, NeuAc alpha(2-3)-Gal beta(1-3) [NeuAc alpha(2-6)] GalNAc-ol, GlcNAc beta(1-3) [NeuAc alpha(2-6)] GalNAc-ol or NeuAc alpha(2-3) Gal beta(1-3)GalNAc-ol were not inhibitory.

Primary structures and Lewis blood-group-dependent expression of major sialylated saccharides from mucus glycoproteins of human seminal plasma.

The primary structures of four major sialylated saccharide alditols derived from mucus glycoproteins of human seminal plasma were established by fast-atom-bombardment mass spectrometry and methylation analysis. Anomeric configurations of the glycosidic bonds were determined by exoglycosidase digestion and CrO3 oxidation. (Formula: see text) Two minor components represent isomers of the major saccharide in A6, which are probably characterized by terminal sequences NeuAc(2----3)Gal(1----4)[Fuc(1----3)]GlcNAc(1---- and Fuc(1----2)Gal(1----4)[NeuAc(2----6)]GlcNAc(1----, respectively. Based on quantitative data from high-pressure liquid chromatography and on structural information, the biosynthetic pathways for neutral and sialylated saccharides related to the Lewis system were proposed. Expression of saccharides A6, A7 and A8 and their asialo counterparts, which are characterized by antigenic determinants H, Lex and Ley, respectively, is qualitatively and quantitatively dependent on the Lewis blood type of the respective donor and correlates with Ca 19-9 activity of its seminal plasma.

Serological investigations on ribitol-containing lipopolysaccharides from Proteus mirabilis.

Lipopolysaccharides containing or noncontaining ribitol derived from several Proteus mirabilis strains were studied using passive hemagglutination, hemagglutination-inhibition and semi-quantitative precipitin tests. The results indicate that ribitol plays a role in the serological specificity of the respective lipopolysaccharides.