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PubMed1993

Molecular size analysis of capsular polysaccharide preparations from Streptococcus pneumoniae.

Purified capsular polysaccharide preparations from Streptococcus pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were analyzed by high performance size exclusion chromatography (HPSEC) with multi-angle laser light scattering (MALLS), specific viscosity (SV), and refractive index (RI) detection to determine the molecular size and molar mass of each of the pneumococcal (Pn) polysaccharides. The Mw's of the polysaccharides ranged from a low of 606 kg/mol for Pn4 to a high of 1145 kg/mol for Pn9V, and the z-average radii of gyration ranged from 59 nm for Pn14 to 72 nm for Pn18C. Estimations of molar mass of the highly anionic polysaccharides (all but Pn14) by the universal calibration approach were unsuccessful, resulting in a 27-53% overestimate of the Mw's though application of Mark-Houwink-Sakurada coefficients calculated from the HPSEC-MALLS/SV/RI data resulted in estimates of Mw that were in agreement with the MALLS estimates for all but the Pn4 preparation. These results emphasize the need for direct measurement of both molecular size and intrinsic viscosity distributions for definitive characterization of the molar mass, hydrodynamic volume, rigidity, and drainage of complex biological polymers such as the pneumococcal polysaccharides.

Carbohydrate Conformation↗
PubMed1993

Klebsiella K43 capsular polysaccharide: primary structure and depolymerisation by a viral-borne endoglycanase.

The capsular polysaccharide of Klebsiella K43 has been studied by glycose analysis, methylation analysis, and NMR spectroscopy, and by bacteriophage depolymerisation of the native polysaccharide. Additional evidence for the structure of the repeating unit came from base-catalysed degradation of the methylated polysaccharide, and from NMR spectroscopic analysis of the lithium-degraded polysaccharide and of the oligosaccharide-alditol derived from the repeating unit oligosaccharide obtained from a bacteriophage degradation. The polysaccharide was shown to have the repeating unit: [formula: see text]

Bacteriophages↗
PubMed1994

Analysis by the reductive-cleavage method of a polysaccharide containing 2-acetamido-2,6-dideoxy-D- and -L-galactopyranosyl residues.

Modifications of the reductive-cleavage method for the analysis of 2-acetamido sugars were tested using a polysaccharide containing alpha-linked 2-acetamido-2,6-dideoxy-L-galactopyranosyl (Fuc pNAc) residues, beta-linked D-Fuc pNAc residues and 2-linked D-glucopyranosyl residues. Reductive cleavage of the fully methylated O-antigenic polysaccharide of Pseudomonas aeruginosa ATCC 33358 in the presence of triethylsilane and trimethylsilyl trifluoromethanesulfonate, followed by quenching with methanol and subsequent acetylation, unexpectedly resulted in nearly complete cleavage of all glycosidic linkages to yield 2-O-acetyl-1,5-anhydro-3,4,6-tri-O-methyl-D-glucitol (1a) and methyl 3-O-acetyl-2,6-dideoxy-4-O-methyl-2-(N-methylacetamido)-beta-D- and -L- galactopyranosides (3a and 4a) as the major products. When the reductive-cleavage reaction was quenched with (S)-2-butanol, the major FucNAc derivatives were the diastereomeric (S)-2-butyl glycosides 15a and 17a, confirming the presence of enantiomeric FucNAc residues in the repeating unit of the polysaccharide. However, compounds 15a and 17a were not detected in equimolar proportions, presumably as a consequence of diastereoselectivity in the reaction of the chiral alcohol with the respective intermediate oxazolinium ions. Reductive cleavage of the fully methylated polysaccharide in the presence of a mixture of trimethylsilyl methanesulfonate and boron trifluoride etherate, followed by quenching with methanol, resulted in incomplete cleavage, giving rise to three disaccharide derivatives whose sequences overlap that of the trisaccharide repeating unit in the polysaccharide. The lack of selectivity for cleavage at beta-D-FucNAc residues suggests that the alpha-L-FucNAc residues underwent anomerization prior to transglycosidation.

Benzoates↗
PubMed1992

Human anti-pneumococcal polysaccharide antibodies are secreted by the CD5- B cell lineage.

To determine whether human antibody responses to T cell-independent pneumococcal polysaccharide antigens are derived from CD5+ or CD5- B cells, we utilized an ELISPOT assay to detect individual anti-polysaccharide antibody-secreting cells. Human anti-type IV pneumococcal polysaccharide antibody-secreting cells were found in the CD5- B cell subpopulation. An EBV transformed anti-pneumococcal antibody-secreting B cell line was also CD5-. The ontogeny of CD5 expressing B cells correlated with the age at which polysaccharide responsiveness is acquired (generally around age 2 years in humans). The CD5- B cell subset represents only 25-30% of the B cells in young children, but this fraction increases throughout childhood to a plateau of 70-80% of the B cells in adults. These results support the hypothesis that the developmental change in responsiveness to T cell-independent polysaccharide antigens in humans is associated with maturation of the CD5- B cell subset.

Adolescent↗
PubMed1984

Effect of thyrotropin-releasing hormone and its metabolites on the secretion of sulfated polysaccharides by foot integument of a pond snail.

The effect of thyrotropin-releasing hormone (TRH), TRH metabolites, and a TRH analog on the secretion of 35S-labeled sulfated polysaccharides from in vitro incubated foot integument of the pond snail Lymnaea stagnalis palustris was assessed. Whereas TRH significantly inhibited the secretion of 35S-labeled polysaccharides, its metabolite deamido TRH significantly stimulated polysaccharide secretion. Histidyl-proline-diketopiperazine did not alter the secretion of 35S-labeled sulfated polysaccharides. Foot integument exhibited considerable TRH-peptidase activity in vitro. The principal proline-containing TRH metabolites were deamido-TRH and proline. The deamido-TRH content of the foot, mantle, and hemolymph was determined by radioimmunoassay. All tissues contained deamido-TRH. These findings suggest that the secretory activity of skin mucus glands of L. stagnalis palustris is the result of the stimulatory action of deamido-TRH and the inhibitory action of TRH. 3Methyl-2histidyl-TRH, TRH, vasotocin, and bombesin did not alter the polysaccharide secretion by in vitro incubated foot integument, indicating that the changes in mucus secretion after TRH and deamido-TRH administration are specific and that the gastropod "TRH receptors" may differ from the mammalian brain and anterior pituitary gland TRH receptors.

Animals↗
PubMed1994

Application of capillary ion electrophoresis and ion chromatography for the determination of O-acetate groups in bacterial polysaccharides.

Many bacterial polysaccharides possess O-linked acetate groups as constituents of their repeating units which often can serve as immunological determinants. It is therefore important to develop analytical methods for process monitoring as well as product characterization when such O-acetylated polysaccharides are used as components of vaccines. This is the case in a polysaccharide conjugate vaccine under development for treatment of diseases caused by Streptococcus pneumoniae. An ion chromatographic (IC) method utilizing suppressed conductivity detection (SCD) was developed to quantitatively measure O-acetate groups in the capsular polysaccharides from S. pneumoniae types 18C and 9V following hydrolytic release of O-acetate from the polysaccharide backbones using 2 mM sodium hydroxide. IC was carried out using an OmniPac PAX-500 column and 0.98 mM NaOH in 2% methanol as the mobile phase. Capillary ion electrophoresis (CIE) with indirect photometric detection was evaluated as an alternative method. The CIE method utilized a 72 cm x 75 microns I.D. fused-silica capillary and an electrolyte composed of 5 mM potassium hydrogenphthalate, 0.5 mM tetradecyltrimethylammonium bromide, and 2 mM sodium tetraborate, pH 5.88. A comparison of CIE and IC-SCD in terms of reproducibility, accuracy, linearity, and sensitivity will be presented.

Acetates↗
PubMed1990

A monoclonal antibody that recognizes 2,3-, 2,6-, and 4,6-disulphate ester ring substitution in pyranose-containing polysaccharides. Its production, characterization and application for the quantitation of pentosan polysulphate, dextran sulphate, glycosaminoglycan polysulphate and chondroitin sulphate E.

A method is described for the preparation of a monoclonal antibody (MAb) which binds specifically to polysaccharides which contain 2,3-, 2,6- and 4,6-disulphate ester pyranose ring substitution. Such molecules include the semisynthetic heparinoids, pentosan polysulphate (PPS), dextran sulphate (DS) and glycosaminoglycan polysulphates (GAGPS), as well as the naturally occurring polysulphated polysaccharides, chondroitin sulphate E, and the 2,6-disulphated galactoses of carrageenans. The antibody (MAb 5-B-10) did not significantly cross-react with other sulphated polysaccharides such as heparin, heparan sulphate, the chondroitin sulphates, A, B, C or D, or keratan sulphate. No cross-reactivity was found with non-sulphated polysaccharides or polyanions including hyaluronic acid, xylan, or DNA. MAb 5-B-10 was characterized as IgM and kappa light chains, and was to develop an amplified enzyme-linked immunosorbent inhibition assay (ELISIA) to detect and quantitate some of the polysulphated polysaccharides in biological fluids. Using this assay, the lower limits of detection of these compounds in serum were in the order of 50 ng/ml; however 50% inhibition was obtained between 200-500 ng/ml. The intra- and inter-assay coefficients of variation for PPS were 4.2 +/- 2.8 and 16.7 +/- 13.8% respectively. The MAb 5-B-10 and the ELISIA were used to determine the levels of PPS in plasma of three healthy non-fasted volunteers for up to 120 min post-intravenous infusion (1.0 mg/kg). The results obtained compared favourably with kinetic data reported by others using a competitive binding assay method for this drug.

Animals↗
PubMed1993

Defective antipneumococcal polysaccharide antibody response in children with recurrent respiratory tract infections.

BACKGROUND: Recurrent pyogenic infections are known to occur in patients with an impaired response to polysaccharide antigens. We investigated the occurrence of deficient responses to pneumococcal capsular polysaccharides in patients with recurrent respiratory tract and recurrent systemic infections. METHODS: Forty-five patients, 1.7 to 17.1 years of age, were immunized with 23-valent pneumococcal polysaccharide vaccine. Antibody levels to seven pneumococcal serotypes (3, 4, 6A, 9N, 14, 19F, 23F) were determined by ELISA before and after immunization. In addition, patients received a booster immunization with diphtheria toxoid, tetanus toxoid, and poliomyelitis virus vaccine. RESULTS: Thirty-five patients had normal serum immunoglobulin levels. Five of these patients (14%) had low antipneumococcal preimmunization antibody levels and failed to respond to pneumococcal vaccination, whereas the response to booster immunization with protein antigens was appropriate. Three patients were younger than 3 years old, and one had a family history of IgG2 deficiency. Low IgG developed in a fifth patient during follow-up. Ten patients had a humoral immunodeficiency. Seven of these patients failed to respond to pneumococcal vaccination. CONCLUSIONS: We conclude that a defective immune response to polysaccharide antigens in patients requires long-term follow-up to distinguish transient maturational delay from a persistent selective impaired response to polysaccharide antigens, which on occasion may precede the development of humoral immunodeficiency disease.

Adult↗
PubMed1994

A simple and nondestructive method for the separation of polysaccharides from beta-glucosidase produced extracellularly by Aspergillus niger.

An apparatus based on electrophoresis has been devised that removes noncovalently bound polysaccharides from extracellular proteins of Aspergillus niger with concomitant partial beta-glucosidase purification and concentration. The apparatus consists of a series of three chambers separated by polyacrylamide gels. Dialyzed and concentrated crude extract of Aspergillus niger containing beta-glucosidase was poured into the middle chamber, while smaller anodic and cathodic chambers contained buffer. When electric current was applied, negatively charged protein-polysaccharide complexes moved toward the anode. Most of the negatively charged proteins, including beta-glucosidase, crossed the gel barrier into the anodic compartment, while neutral polysaccharides were either trapped in the gels or remained in the middle chamber. In this way, 125 ml of dialyzed and concentrated crude extract of Aspergillus niger was processed. Therefore, after 24 h of electrophoresis, 68% of the proteins and 90% of the beta-glucosidase activity, but only negligible amounts of polysaccharide, were transferred to the anodic chamber. The removal of high-molecular-weight polysaccharide from beta-glucosidase had a detrimental effect on the stability of the enzyme.

Aspergillus niger↗
PubMed1989

Structural similarity of the type-specific group B streptococcal polysaccharides and the carbohydrate units of tissue glycoproteins: evaluation of possible cross-reactivity.

Type-specific capsular polysaccharides of group B streptococci show striking structural similarity with the terminal sugar sequences of tissue glycoconjugates. The polysaccharides have been put forward as vaccines against neonatal meningitis. A potential source of hazard in immunization of pregnant mothers may be the presence of the cross-reactive components in adult or fetal tissues. A radioactive ligand binding assay was used to test human immune sera to type Ia, II and III group B streptococcal polysaccharides for binding to tissue-derived glycopeptides showing structural similarities with the streptococcal polysaccharides. Of the 13 glycopeptides of human and rat tissues studied, representing a wide selection of structures known to occur in glycoproteins, only two showed some reactivity with the antisera. The reactivity with human small intestinal glycopeptides could be explained by the presence of natural blood group A antibodies, and was not related to the streptococcal group B antibodies. The basis of the reactivity of a high-molecular-weight glycopeptide from rat kidney with some of the sera was unknown, but was unrelated to the vaccination and clearly could not be inhibited with the streptococcal polysaccharides. Thus, no immunological cross-reactions of the tissue glycopeptides studied could be demonstrated with the group B streptococcal antisera.

ABO Blood-Group System↗
PubMed1992

Intranasal immunization with bacterial polysaccharide containing liposomes enhances antigen-specific pulmonary secretory antibody response.

Enhancement of bacterial antigen-specific secretory IgA (sIgA) titres in the lungs may enhance resistance to infections, such as pneumonia, occurring at this mucosal surface. To examine this issue, we intranasally administered liposomes containing bacterial polysaccharide antigens from Aerobacter levanicum, Pseudomonas aeruginosa and Streptococcus pneumoniae. In each case, increased titres of bacterial polysaccharide-specific sIgA could be achieved in the lungs following intranasal immunization with antigen encapsulated in liposomes. In comparison with oral immunization, which required high doses of polysaccharide antigen even when coadministered with adjuvant, intranasal administration of liposomes containing polysaccharide antigens achieved a similar pulmonary sIgA response with approximately 1/30 the amount of antigen necessary with oral immunization. In the case of P. aeruginosa, the magnitude of the sIgA response following intranasal immunization was sufficient to significantly reduce mortality from pneumonia produced by this organism. These results demonstrate that liposome-based mucosal immunization strategies can induce increased bacterial polysaccharide antigen-specific sIgA titres in the lung, and reduce susceptibility to pneumonia.

Administration, Intranasal↗
PubMed1995

Adjuvant activity of QS-21 for experimental E. coli 018 polysaccharide vaccines.

Three types of experimental vaccines containing O-side-chain polysaccharide from the enterotoxigenic strain Escherichia coli 018 were evaluated. The immunogenicity of free O-polysaccharide (PS), a polysaccharide-diphtheria toxoid conjugate (PS-conj), and detoxified lipopolysaccharide (dLPS) was tested in female ICR mice, either alone or in combination with QS-21, a purified saponin adjuvant derived from the bark of the tree Quillaja saponaria Molina. Both the number of individual mice responding and the titres of O-polysaccharide specific antibodies in pools of sera were increased by the addition of QS-21. The immune response to both O-specific polysaccharide and carrier was primarily IgM and IgG1. The addition of QS-21 not only increased the level of IgG1, but also had a significant adjuvant effect on antigen-specific IgG2a, IgG2b and IgG3.

Adjuvants, Immunologic↗
PubMed1996

Immunization with a pneumococcal capsular polysaccharide vaccine during pregnancy.

The feasibility of preventing invasive pneumococcal infections during the first few months of life by immunization during pregnancy has been investigated. One hundred and fifty Gambian women were immunized with either a 23-valent pneumococcal polysaccharide vaccine or a meningococcal polysaccharide vaccine during the last trimester of pregnancy. Pregnant women showed a good antibody response to five of the six pneumococcal polysaccharides tested (types 1, 3, 5, 6, 14 and 19) but not to type 6 polysaccharide. Mean cord blood/maternal blood IgG antibody ratios varied from 24% (type 1) to 49% (type 3) and differed substantially between individual mother/infant pairs. Pneumococcal antibody levels were higher at birth in infants of women immunized with pneumococcal polysaccharide vaccine than in control infants. However, these antibodies disappeared rapidly during the first few months of life and it is uncertain how much clinical protection against pneumococcal infection maternal immunization would have provided.

Antibodies, Bacterial↗
PubMed1978

Polysaccharides of crithidia fasciculata. Identification and partial characterization of a cell surface constituent.

A carbohydrate-containing fraction was extracted from the trypanosomatid Crithidia fasciculata by a phenol-water procedure. Ion-exchange chromatography separated this fraction into three components: a polysaccharide which was not retained on the column; RNA which eluted upon addition of salt; and, another polysaccharide which eluted upon addition of detergent. The unretained fraction was shown to be composed solely of D-mannose. The mannan, which was heterodisperse on Sephadex G-100, had an average molecular weight of approx. 14 000 as based on analysis of reducing groups. The detergent-eluted material yielded arabinose and galactose upon acid hydrolysis. The arabinogalactan was excluded from Sephadex G-100 and Sephacryl S-200 molecular sieve columns, suggesting a molecular weight greater than or equal to 200 000. Cell fractionation studies showed the bulk of extractable polysaccharide was associated with a particulate fraction. Further determination of the cellular localization of the polysaccharide was accomplished by employing a specific antiserum prepared from rabbits immunized with the polysaccharide extract. The cell surface localization of the arabinogalactan was demonstrated by cell agglutination studies as well as immunocytochemical techniques using fluorescein and ferritin conjugated antibodies.

Animals↗
PubMed1989

Identification of polysaccharides in pharmaceuticals by capillary gas chromatography.

A sensitive method for the identification of polysaccharides in pharmaceuticals is described. Polysaccharides are isolated by gel filtration and subsequently hydrolysed. The monomeric carbohydrates obtained are transformed into oxime-trimethylsilyl derivatives and analysed by capillary gas chromatography. Profiles of 13 different natural or semi-synthetic polysaccharides are discussed. The profiles of the hydrolysis products can be used to identify the polysaccharides mentioned above. Possible interferences by other polymers are given. The method can be used to identify most polysaccharides used as pharmaceutical adjuvants.

Alcohols↗
PubMed1987

Etiologic diagnosis of pneumonia by antigen detection: crossreactions between pneumococcal C-polysaccharide and oral microorganisms.

Crossreactions between bacteria occurring more or less frequently in the respiratory tract were investigated using an enzyme-linked immunosorbent assay (ELISA) developed for the detection of pneumococcal C-polysaccharide. A collection of 218 strains was investigated: 30 Streptococcus pneumoniae, 120 alpha-streptococci, and 68 strains representing other species. Strong crossreactions were observed with 36% of the alpha-streptococci and with two of 11 Staphylococcus aureus strains. The collection of alpha-streptococci consisted of 90 fresh clinical isolates and 30 stock strains. Almost all crossreactions of alpha-streptococci were found among the clinical isolates. Among the stock strains only one of four Streptococcus mitis strains was positive. Pneumococcal C-polysaccharide and phosphorylcholine inhibited the reactions in ELISA with monoclonal antibodies against pneumococcal C-polysaccharide, as well as with a polyclonal antiserum against pneumococcal C-polysaccharide. We suggest that the cross reactions between alpha-streptococci and pneumococci depend on the presence of phosphorylcholine as a common antigenic determinant. The crossreaction in the ELISA with some Staphylococcus aureus strains may be explained by the presence of protein A binding to the Fc portion of the antibodies. When the 10 alpha-streptococci that showed the strongest crossreactions and ten pneumococci representing different types were tested in different concentrations the absorbance values were lower for most alpha-streptococci compared with the pneumococci. This explains that false positive results with alpha-streptococci do not seem to constitute a practical problem in this ELISA developed for detection of pneumococcal C-polysaccharide in samples from patients with pneumonia.

Antigens, Bacterial↗
PubMedDate not supplied

Isolation of type 5 capsular polysaccharide from Staphylococcus aureus.

Clinical surveys have indicated the predominance of type 5 and type 8 capsular polysaccharides among clinical isolates of Staphylococcus aureus. The type 5 capsular polysaccharide was extracted from three clinical isolates of S. aureus grown on solid media, and purified by ion exchange chromatography and gel filtration. Chemical analysis showed that the type 5 capsular polysaccharide is composed of N-acetylfucosamine and N-acetylhexosaminuronic acid. Immunodiffusion and 13C nuclear magnetic resonance experiments showed that the type 5 capsular polysaccharide is both immunologically and chemically distinct from the type 8 capsular polysaccharide and the teichoic acid of S. aureus.

Bacterial Capsules↗
PubMed2005

Development of a high-resolution magic-angle spinning nuclear magnetic resonance identity assay of the capsular polysaccharide from Haemophilus influenzae type b present in cetavlon precipitate.

We describe the use of high-resolution magic-angle spinning nuclear magnetic resonance to control the identity of the capsular polysaccharide from Haemophilus influenzae type b (Hib) present in the cetavlon precipitate. This step is one of the earliest in the purification of this polysaccharide, which is further used in the production of Hib polysaccharide-protein conjugate vaccine. The effects of sample procedure and magnetic field strength have been investigated. Since this assay is rapid and simple, it may represent a useful technique for characterization of polysaccharides present in complex and insoluble matrices. Moreover, it allows a rapid evaluation of the structure of the produced polysaccharides very early on during the production process and is as such an essential analytical tool before starting the purification process.

Bacterial Capsules↗