Multimeric bivalent immunogens from recombinant tetanus toxin H<Subscript>C</Subscript> fragment,synthetic hexasaccharides,and a glycopeptide adjuvant

Using recombinant tetanus toxin H_C fragment (rTT-H_C) as carrier, we prepared multimeric bivalent immunogens featuring the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Ogawa, in combination with either the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Inaba, or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant. The conjugation reaction was effected by squaric acid chemistry and monitored in virtually real time by SELDI-TOF MS. In this way, we could prepare well-defined immunogens with predictable carbohydrate–carrier ratio, whose molecular mass and the amount of each saccharide attached could be independently determined. The ability to prepare such neoglycoconjugates opens unprecedented possibilities for preparation of conjugate vaccines for bacterial diseases from synthetic carbohydrates.     

Monoclonal antibodies to antigens abnormally expressed in breast cancer

We report the production, screening, and characterization of ten murine monoclonal antibodies directed at antigens that are expressed abnormally in human breast tumors. Immunoperoxidase staining of frozen and fixed tissues shows the antigens to be present at low levels on the luminal membrane of normal breast cells and at high levels in the cytoplasm and surface membrane of breast tumor cells. The ten antibodies appear to recognize six different epitopes on the basis of their quantitative differences in reactivity against four antigen preparations, as measured by ELISA. Immunoblots show that eight of the ten antibodies recognize a 300,000 MW molecule from breast tumor preparations; six of these antibodies also react with a second molecule from the same tumor preparations of 280,000 MW. Seven antibodies react with an antigen from milk fat globule membrane of 330,000 MW. It therefore appears that the two molecules from tumor tissue and the one molecule from normal tissue share common epitopes. Selected antibodies were tested for reactivity against 25 primary breast tumors and 14 pairs of primary and metastatic breast tumors. Three antibodies have broad reactivity and stain more than 80% of primary tumors; the three other antibodies identify subsets of those tumors. Results of staining pairs of primary and metastatic lesions show that metastases continue to express antigens of the primary lesion in a high percentage of cells.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-β- -galactopyranoside and methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-O-β- -galactopyranosyl-(1→6)-β- -galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α- -galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β- -galactopyranoside (4) gave a fully acetylated (1→6)-β- -galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α- -galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β- -galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β- -galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

The molecular basis of the requirement for antigen processing of pigeon cytochrome c prior to T cell activation

Antigen-induced activation of T lymphocytes that co-recognize Ia molecules has been shown to require an antigen-processing step by the presenting cell before T cell stimulation can occur. In this report, we demonstrate that antigen presentation of pigeon cytochrome c to an E kappa beta:E kappa alpha-restricted T cell hybridoma, 2C2, is inhibited by pretreatment of the antigen-presenting cells (APC) either with chloroquine or with fixation by paraformaldehyde. The chloroquine effect was partially reversible after 22 hr; the paraformaldehyde effect was not. In contrast, these treatments had little or no effect on the presentation of the carboxy-terminal cyanogen bromide cleavage fragment of pigeon cytochrome c, residues 81 to 104. There was at least a 50-fold greater potency of the fragment, as compared to that of the intact molecule, when paraformaldehyde-fixed APC were used. In addition, the fixed cells did not present synthetic fragments of the cytochrome c that were nonstimulatory when presented by unfixed cells. This observation showed that the loss of potency, demonstrated previously for analogs of pigeon cytochrome c with single amino acid substitutions at positions such as 99, was not a consequence of an alteration in the rate of antigen-processing. This result is consistent with our earlier hypothesis that these residues are contact amino acids with the antigen-specific T cell receptor or the Ia molecule. The major goal of these experiments was to define the molecular transition that occurred as a result of antigen processing. To achieve this end, we tested a variety of pigeon cytochrome c molecules and fragments for their ability to be presented by paraformaldehyde-fixed APC. Apocytochrome c, the denatured form of the molecule with the heme removed, could not be presented by the fixed cells, nor could the fragment 60-104, derived by acid cleavage of the tryptophan at position 59. Both molecules stimulated an IL 2 response from the T cell hybridoma when unfixed APC were utilized, demonstrating that the conditions used to prepare these two molecules did not destroy their antigenic determinant. In contrast, carboxy-terminal fragments, both native and synthetic, ranging in size from 16 to 39 amino acids, were capable of stimulating in the presence of paraformaldehyde-fixed APC. In particular, the partial-digest cyanogen bromide fragment, residues 66 to 104, was only twofold less potent than the pigeon fragment 81-104.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemotype pattern differentiation of Thymus pulegioides on different substrates

Relationships between soil chemistry and population chemotype structure of Thymus pulegioides have been studied. The analysis of correlations suggest that an increased carbonate content in soil decreases the chemotype diversity of a population (as calculated by use of the Shannon index): the proportion of linalool chemotype plants rises and that of the phenol chemotype plants declines. In addition, the chemotype diversity decreases with increasing frequency of linalool chemotype, and increases with increase of carvacrol chemotype.     

Human Ia molecules carrying DC1 or BR4X7 determinants are not homologous to murine I-E molecules

An anti-I-E^k alloantiserum was shown to react with purified human Ia preparation. All Ia preparations tested were actively bound irrespective of their HLA-DR phenotypes. However, from a quantitative point of view, DR7 molecules were significantly less reactive. No reaction was observed with isolated Ia subunits. Only molecules carrying DR determinants, but not those carrying either DC1 or BR4X7 determinants, were bound.     

Glycosylation changes in different developmental stages of Trichinella

The in situ identification of carbohydrate structures in Trichinella spiralis intestinal larvae, adults and L1 muscular larvae was carried out by lectin histochemistry, with emphasis on the O-linked glycans. The absence of reactivity with two lectins-TML and MAL indicated that Trichinella spiralis does not synthesize sialic acid. Reactivity with HPA, VVL-B4, PNA and UEA-I staining suggested that T. spiralis synthesizes and expresses on its cuticle O-linked glycans analogous to Tn-antigen (GalNAc-α-Ser/Thr), T-antigen (Gal-β1,3-GalNAc-α-Ser/Thr) and also structures analogous to A-blood group antigens (GalNAc-α1,3-Gal-β1,3(4)-(Fuc-α1,2-)-R). Expression of the saccharidic moieties is stage-specific. Blood group-A and T-antigen structures were identified on the cuticle of the intestinal and muscular larvae. The Tn-antigen structure was missing in the intestinal larvae. Appropriate ligands for WGA were not identified in the adult individuals. The obtained results may contribute to a better understanding of the glycobiology of this parasitic nematode in relation to occupation of its intracellular niche. The presence of saccharidic structures analogous to some of those expressed on the intestinal epithelial cells may serve as a protective shield on the surface of the parasite.     

The incorporation of macromolecules into the germ cells of male mice via electroporation and dimethyl sulfoxide

Electroporation (incorporation of macromolecules into the living cells by means of electric pulses) provides inclusion of plasmid 14C-DNA into immature cells of spermatogenic epithelium. The highest level of foreign DNA incorporation into spermatocytes and spermatids has been induced by 8kV electric pulses applied 3 times with 20 sec intervals. Meanwhile, mature sperms are found to be exclusively resistant to exogenous DNA irrespective of the voltage level, the number of pulses and Ca++ uptake (contents). Incubation of mature sperms for two hours in the medium with Ca++ (10 mM) and dimethylsulfoxide--(DMSO, 33%) provides highly reliable incorporation of plasmid 14C-DNA into sperm heads. The sperm cells with foreign DNA incorporated by means of Ca++ and DMSO treatment still remain alive and mobile. The possibilities of mature sperms loaded with foreign DNA for the creation of transgenic mammals are discussed.