RESUMEN

Seven novel saponins were isolated from a bark extract of Quillaja saponaria Molina. the compounds were characterized, using mainly NMR spectroscopy, mass spectrometry and chemical methods, as quillaic acid substituted at C-3 with oligosaccharides consisting of various compositions of D-glucuronic acid D-galactose, D-xylose, and L-rhamnose and at C-28 with complex oligosaccharide structures consisting of various compositions of D-xylose, L-rhamnose, D-apiose and a branched 4-O-acetyl-D-fucose residue.

Asunto(s)

RESUMEN

A fraction of saponins from Quillaja saponaria Molina, QH-B, was fractionated by consecutive separations on three different reverse-phase HPLC systems. Eight compounds were isolated and the structures of these were elucidated mainly by sugar analysis and NMR spectroscopy. The structures consisted of a quillaic acid substituted with two different trisaccharides at C-3, beta-D-Galp-(1-->2)-[alpha-L-Rhap-(1-->3)]-beta-D-GlcpA and beta-D-Galp-(1-->2)-[beta-D-Xylp-(1-->3)]-beta-D-GlcpA, and a tetra- or pentasaccharide at C-28, beta-D-Xylp-(1-->4)-[beta-D-Glcp-(1-->3)]-alpha-L-Rhap-(1--> 2)-beta-D-Fucp and beta-D-Apif-(1-->3)-beta-D-Xylp-(1-->4)-[beta-D-Glcp-(1-->3) ]-alpha-L- Rhap-(1-->2)-beta-D-Fucp. These compounds were further substituted with an acyl group either at O-3 or O-4 of the fucose residue, which is the sugar linked to C-28 of the quillaic acid.

Asunto(s)

RESUMEN

Three new saponins were isolated from a commercial bark extract of Quillaja saponaria Molina. These compounds were also obtained as degradation products from larger saponins in this extract when treated with strong alkali. The compounds were characterized, using mainly NMR spectroscopy, mass spectrometry and chemical methods, as quillaic acid 3-O-¿beta-D-galactopyranosyl-(1-->2)-beta-D-glucopyranosiduronic acid¿, 3-O-¿alpha-L-rhamnopyranosyl-(1-->3)-[beta-D-galactopyranosyl-(1-->2)] -beta-D-glucopyranosiduronic acid¿ and 3-O-¿beta-D-xylopyranosyl-(1-->3)-[beta-D-galactopyranosyl -(1-->2)]-beta-D-glucopyranosiduronic acid¿, respectively.

Asunto(s)

RESUMEN

The iscom is an efficient antigen-presenting system for various antigens inducing both MHC class I and class II restricted immune responses. Protective immunity has been evoked against a variety of infectious agents. The saponin adjuvant Quil A, which was originally used to form iscoms, is composed of a mixture of structurally similar triterpenoids from Quillaja saponaria Molina having different biological activities. A purified, toxic Quillaja triterpenoid fraction with strong adjuvant activity, designated QH-B, was used to study whether modification of the carbohydrate moiety with sodium periodate would alter the toxicity without harming adjuvant activity and cholesterol-binding capacity. Most sugars, and in particular Api, Gal and Xyl, were modified by periodate treatment with only minor changes of the molecular weights indicating no loss of sugar residues. The adjuvant activity of QH-B was reduced in a dose-related manner, and at a concentration of 25 mM sodium periodate a significant reduction in toxicity was observed. The differences in both toxicity and adjuvant activity of the periodate-treated QH-B could be derived from alterations in the structure of the sugars Gal and Xyl, while modification of Api may influence adjuvant activity but not toxicity in vivo. The cholesterol-binding capacity, a prerequisite for iscom formation, was not affected by periodate oxidation at the doses tested. However, the use of modified QH-B as described in the present study for iscom-matrix formation resulted in "saponin-lipid complexes" which, to a various degree or totally, deviated from the characteristic iscom morphology.

Asunto(s)

RESUMEN

In the iscom, multiple copies of antigen are attached by hydrophobic interaction to a matrix which is built up by Quillaja triterpenoid saponins and lipids. Thus, the iscom presents antigen in multimeric form in a small particle with a built-in adjuvant resulting in a highly immunogenic antigen formulation. We have designed a chloroform-methanol-water extraction procedure to isolate the triterpenoid saponins and lipids incorporated into iscom-matrix and iscoms. The triterpenoids in the triterpenoid phase were quantitated using orcinol sulfuric acid detecting their carbohydrate chains and by HPLC. The cholesterol and phosphatidylcholine in the lipid phase were quantitated by HPLC and a commercial colorimetric method for the cholesterol. The quantitative methods showed an almost total separation and recovery of triterpenoids and lipids in their respective phases, while protein was detected in all phases after extraction. The protein content was determined by the method of Lowry and by amino acid analysis. Amino acid analysis was shown to be the reliable method of the two to quantitate proteins in iscoms. In conclusion, simple, reproducible and efficient procedures have been designed to isolate and quantitate the triterpenoids and lipids added for preparation of iscom-matrix and iscoms. The procedures described should also be useful to adequately define constituents in prospective vaccines.

Asunto(s)

RESUMEN

It is well established that ISCOMs function efficiently as an antigen-presenting system and protective immunity has been evoked against a variety of infectious agents. The built-in saponin adjuvant from Quillaja saponaria Molina is responsible for the strong immunoenhancing activity displayed by the ISCOM. However, to allow the use of ISCOMs in human vaccines it is necessary to determine the immunological properties and toxicity of chemically defined Quillaja components. Thus, the present study was carried out in a mouse model to determine the adjuvant activity and toxicity of "free", isolated Quillaja components, as well as formulated into particles, i.e. ISCOM matrix. The purified Quillaja components and the ISCOM matrix formulations were examined for their adjuvant activity in a model system consisting of purified influenza virus antigen and Quillaja saponins. It was demonstrated that a Quillaja component, designated QH-C, either as a "free" component or in an ISCOM matrix, has a strong adjuvant activity, but little or no toxicity in the doses tested. In addition, QH-C in the form of ISCOM matrix does not induce any local reactions at the site of injection. Thus, ISCOMs containing the QH-C component, devoid of toxicity, but with strong adjuvant activity, can prove to be useful in adjuvant formulations for human use.

Asunto(s)

Asunto(s)

RESUMEN

The cellular regulation of diphtheria toxin cell surface receptors was studied. Treatment of Vero cells with cycloheximide reduced their diphtheria toxin binding capacity, while cells treated with actinomycin D did not lose their ability to bind diphtheria toxin. A non-toxic analogue of diphtheria toxin, CRM 197, produced a dose-related depletion of cell surface diphtheria toxin binding capacity that was reversible upon washing the cells. Vero cells depleted of toxin receptors by CRM 197 did not restore their ability to bind diphtheria toxin in the presence of cycloheximide. Phospholipase C treatment of Vero cells reduced their diphtheria toxin binding capacity in a dose-dependent manner. The loss of diphtheria toxin binding capacity was recovered within 2 hr after removal of the enzyme. Protein synthesis inhibition blocked this recovery while actinomycin D partially inhibited it. Receptors prebound with toxin were resistant to phospholipase C treatment, suggesting that the action of the enzyme was directly on the receptor. Inhibition of glycosylation with tunicamycin did not prevent reappearance of toxin receptors after CRM 197 or phospholipase C treatment. These data establish the requirement of a continuous protein synthesis for the maintenance of diphtheria toxin cell surface receptors and also suggest that these receptors do not recycle after binding ligand. A hypothesis is put forward that the diphtheria toxin receptor might be a lipid-linked cell surface protein.

Asunto(s)

RESUMEN

Mice were immunized with a cell line (Vero) that possesses a high number of membrane receptors for diphtheria toxin. Spleen cells from these mice were fused with SP2/0-Ag14 cells and two cell lines (1A2 and 2D2) isolated by screening for the ability of their secreted antibodies to inhibit binding of radiolabeled diphtheria toxin to Vero cells. These antibodies protected Vero cells from the inhibition of protein synthesis mediated by diphtheria toxin. The antibodies were purified, iodinated, and their binding characteristics investigated. At 4 degrees C, the association of 1A2 and 2D2 with Vero cells was saturable (KD approximately 10(-8) M) and indicated about 10(6) binding sites/cell. Diphtheria toxin did not inhibit the binding of either radiolabeled antibody. Monoclonal antibody 1A2 completely inhibited 125I-2D2 binding and vice versa. Trypsin or phospholipase C treatment of Vero cells had no effect on the ability of the monoclonal antibodies to bind to the cells. These findings suggest that: (1) the two monoclonal antibodies recognize the same or closely related epitopes and (2) the antibodies bind a domain distinct from the toxin binding site or to a subcomponent of the diphtheria toxin receptor that is present at many other cell surface sites. These antibodies offer a powerful tool to study the structure, processing and mode of action of diphtheria toxin receptors.

Asunto(s)

RESUMEN

The gene for Escherichia coli heat-stable enterotoxin type II (STII) was fused to the genes for protein A from Staphylococcus aureus and beta-galactosidase in two different expression systems. Antibodies raised in rabbits against the protein A-STII fusion protein recognized the beta-galactosidase-STII fusion protein. The latter fusion protein was used as the immobilized antigen in an enzyme-linked immunosorbent assay (ELISA) for detection of STII. The correlation between the results of the ELISA and the intestinal loop test in piglets was 95%, suggesting that the ELISA can be used to reliably detect STII.

Asunto(s)

Asunto(s)

RESUMEN

A number of different assay methods has been developed for the detection of the heat-labile enterotoxin (LT) of ETEC strains isolated from humans and animals. In the present study 40 Escherichia coli strains were subject of a comparative study of the staphylococcal coagglutination (Coa-LT) test, the GM1-ELISA and the Y-1 adrenal cell test. All but two of 20 "LT-ST" or "LT-only" producing ETEC strains gave identical results in both the Coa-LT test and the bioassay. None of the 14 "ST only" producing ETEC strains gave false positive results. It is concluded that the Coa-LT test is a simple and rapid assay method for the routine diagnosis of LT producing ETEC strains.

Asunto(s)

RESUMEN

A competitive enzyme-linked immunosorbent assay (ELISA) was compared with the conventional suckling mouse assay for the identification of heat-stable enterotoxin (STa) in samples from piglets suffering from diarrhea. A total of 110 Escherichia coli isolates, 22 primary cultures, and 26 fecal samples from piglets up to 8 weeks of age with diarrhea were compared in parallel by both assays. Of the 110 isolates tested, all gave consistent results by the ELISA and the suckling mouse assay; 60 strains were negative and 50 strains produced STa by both tests. Identical results were obtained when 22 primary agar cultures were screened for STa production by both methods; 6 were found to produce STa, while 16 did not. When 26 fecal samples were tested for the presence of STa, 10 were negative and 12 were positive by both assays. One of the remaining four samples gave questionable positive results by both the suckling mouse assay and the ELISA, but E. coli isolated from this sample gave positive results by both tests. The remaining three samples were negative by the suckling mouse assay, but gave questionable positive results by the ELISA. E. coli isolates from these three samples were always negative by both assays. The ELISA used in this study provides a reliable and convenient method for diagnosing STa-producing enterotoxigenic E. coli of porcine origin.

Asunto(s)

RESUMEN

Colonies of Escherichia coli from blood agar plates were suspended and lysed in saline containing polymyxin B and a detergent (Triton X-100). The lysates were assayed for heat-labile enterotoxin (LT) by a coagglutination test (coa-test). The coa-test reagent consisted of Formalin-treated and heat-killed cells of Staphylococcus aureus, strain Cowan 1, sensitized with a high-titer rabbit anti-LT serum. Purified LT was detected in the coa-test at the nanogram level (2 to 5 ng), whereas larger amounts of cholera toxin (50 ng) were required to give a positive test. The coa-test was compared with the CHO cell test for detection of LT among E. coli strains isolated from human and animal stool cultures. The results of the coa-test and the CHO cell test correlated with 63 of 67 strains of human origin. Six of nine animal strains, defined as LT positive by the CHO cell test, gave positive results in the coa-test. We conclude that the coa-test is probably accurate and sensitive enough to be used in routine diagnosis of LT-producing E. coli strains isolated from human stool cultures. No special laboratory equipment is required, which makes the coa-test suited for diagnosis of enterotoxigenic E. coli in small hospital laboratories and in developing countries.

Asunto(s)

Asunto(s)

RESUMEN

Protein A positive Staphylococcus aureus cells, strain Cowan 1, were coated with a high titer anti-LT serum produced in rabbits. Purified E. coli heat labile enterotoxin (LT) was detected in a slide coagglutination (coa-LT) test with anti-LT coated staphylococci at the nanogram level (0.1-1 ng) while cholera toxin (CT) was detected only at much higher concentrations (10-50 ng). This LT immunoassay was evaluated in different modifications on enterotoxigenic E. coli (ETEC) isolated from humans and animal stool cultures. Colonies from primary stool cultures on blood agar were suspended and lysed in saline containing polymyxin B and a detergent (Triton X100), centrifuged and assayed for LT. The coa-LT and CHO-cell tests regularly detected LT among human LT producing ETEC, while ETEC strains from piglets and calves usually gave only weak reactions in the standard coa-LT test. We conclude that this coa-LT test is accurate and sensitive enough to be used in routine diagnosis of LT producing ETEC strains in stools from human patients. This test requires no special laboratory equipment and is well suited for rapid diagnosis of ETEC in small hospital laboratories and in developing countries. Heat stable enterotoxin (ST) giving a positive suckling mouse test, has been purified by hydrophobic interaction chromatography (HIC) on Octyl Sepharose. Purified ST was covalently linked to different proteins and such carrier complexes used for immunization of rabbits. The ST antibody titers obtained by various ST-carriers were compared in a ST coagglutination test (ST-coa) and in a conventional ELISA microtiter assay. The results and detection limits for ST in polymyxin-detergent cell lysates in human ETEC strains producing LT-ST or ST only will be presented.

Asunto(s)

RESUMEN

A heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli has been purified to homogeneity by hydrophobic interaction, molecular-sieve and high performance liquid chromatography with a recovery of 48%. The toxin is composed of 10 different amino acids with a total of 18 amino acid residues, one-third of which are half-cystine. Purified enterotoxin contains no carbohydrate and is biologically active in the suckling mouse test in 2.1-ng quantities. The molecule was heat-stable (80 degrees C, 20 min.) at pH 7 and pH 2 but lost biological activity at pH 12. Biological activity was lost when treated with the reducing agent dithiothreitol, suggesting that the presence of disulfide bridges is required for biological activity.

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)