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PubMed1998

Structural properties of group B streptococcal type III polysaccharide conjugate vaccines that influence immunogenicity and efficacy.

In this study, we tested the hypothesis that the immunogenicity and protective efficacy of polysaccharide-protein conjugate vaccines are influenced by three variables: (i) molecular size of the conjugate, (ii) molecular size of the polysaccharide used for conjugation, and (iii) extent of polysaccharide-to-protein cross-linking. Type III group B Streptococcus capsular polysaccharide was linked by reductive amination at multiple sites to tetanus toxoid to create a polysaccharide-protein conjugate (III-TT). A single lot of III-TT was fractionated into small, medium, and large Mr pools. Whereas all three conferred protection in a maternal immunization-neonatal challenge model in mice, the smallest Mr conjugate evoked less polysaccharide-specific immunoglobulin G (IgG) than the two larger Mr conjugates. To test whether the molecular size of the polysaccharide used for conjugation also affected the immunogenicity of the conjugate, vaccines were synthesized using capsular polysaccharides with Mrs of 38,000, 105,000, and 349,000. Polysaccharide-specific IgG responses in mice increased with the Mr of the polysaccharides, and protective efficacy was lower for the smallest polysaccharide conjugate compared to the other two vaccines. Immunogenicity testing of a series of vaccines prepared with different degrees of polysaccharide-to-protein cross-linking demonstrated higher polysaccharide-specific antibody responses as the extent of cross-linking increased. However, opsonic activity was greatest in mouse antiserum raised to a moderately cross-linked conjugate, suggesting that some antibodies evoked by highly cross-linked conjugates were directed to a nonprotective epitope. We conclude that conjugate size, polysaccharide size, and degree of polysaccharide-protein cross-linking influence the immunogenicity and protective efficacy of III-TT conjugate vaccines.

Animals↗
PubMed1983

Effect of growth conditions on production of rhamnose-containing cell wall and capsular polysaccharides by strains of Lactobacillus casei subsp. rhamnosus.

Strains of Lactobacillus casei subsp. rhamnosus possessing two cell wall polysaccharides, a hexosamine-containing H-polysaccharide and a rhamnose-containing R-polysaccharide, were examined for the effect of growth conditions on the production of these two components. In strain NCTC 6375, R- and H-polysaccharides accounted for an estimated 44 and 20%, respectively, of the cell wall for organisms grown in batch culture with glucose as the carbohydrate source. Growth on fructose-containing media reduced the amount of R-polysaccharide by approximately 50% without affecting the amount of H-polysaccharide. Subculture of fructose-grown organisms in glucose restored the original proportions of the two polysaccharides. Galactose- and sucrose-grown cells behaved similarly to glucose-grown cells with respect to polysaccharide production, whereas growth in rhamnose or ribose showed values close to those for fructose-grown cells. Continuous culture of strain NCTC 6375 for more than 100 generations showed a gradual and irreversible reduction of the R-polysaccharide to less than 5% of the cell wall and an increase of the H-polysaccharide to 40% of the cell wall. Other type culture strains of L. casei subsp. rhamnosus, NCIB 7473 and ATCC 7469, behaved similarly in batch and continuous culture. In contrast, strains of L. casei subsp. rhamnosus isolated at the Institute of Dental Research showed phenotypic stability with respect to the relative proportions of R- and H-polysaccharides in both batch and continuous culture. Changes in polysaccharide composition of type culture strains were also mirrored in changes in the immunogenicity of the two components and resistance to the rate of enzymic lysis of whole organisms. For L. casei subsp. rhamnosus strain NCTC 10302 the R-polysaccharide is present entirely as capsular material. The amount of R-polysaccharide produced was also markedly dependent on the carbohydrate component of the medium in batch culture and both dilution rate and nature of the limiting carbohydrate in continuous culture, varying over a 10-fold range, whereas the cell wall H-polysaccharide remained constant.

Cell Wall↗
PubMed1988

Chemically modified capsular polysaccharides as vaccines.

Capsular polysaccharides have assumed an important role as vaccines against disease caused by bacteria in humans. The concept of using pure definable polysaccharides devoid of their accompanying complex bacterial mass is technically elegant and is obviously capable of extension into other areas of immunoprophylaxis. However, problems have been identified which will need to be solved in order that the concept may be more widely adopted. Focusing on the meningococcal polysaccharides, possible solutions to two of these important problems, namely, the poor immunogenicity of the A and C polysaccharides in infants, and the poor immunogenicity of the B polysaccharide in all humans, are proposed. These solutions involve the use of a new generation of artificial synthetic antigens for modulating the immune response. For instance, conjugation of the A and C polysaccharides to tetanus toxoid converted them to T-cell dependent antigens in mice, thus making these conjugates potential infant vaccine candidates. Although a similar conjugation of the B polysaccharide failed to substantially enhance its immunogenicity in mice, this could be achieved by further chemical manipulation of the basic structure of the B polysaccharide. N-propionylation of the B polysaccharide, followed by its conjugation to tetanus toxoid, yielded an antigen, which when injected in mice, induced in them high titers of cross-reactive B polysaccharide-specific IgG antibodies. The chemical modification of polysaccharides requires an understanding of the interrelation between their structures and immunospecificities, and the structural elucidation of polysaccharides and the resultant monitoring of their structural modifications, can be conveniently accomplished using a wide range of NMR spectroscopic techniques. The capsular polysaccharides of many of the bacteria which cause meningitis in humans contain sialic acid and have extensive structural homology with human tissue. As a result of this homology the immunospecificities of these polysaccharides are complex, being based on unconventional conformational determinants.

Antibodies, Bacterial↗
PubMed2005

Immuno-stimulating effect of the endo-polysaccharide produced by submerged culture of Inonotus obliquus.

Inonotus obliquus BELYU1102 was selected from 12 different strains of Inonotus as a producer of immuno-stimulating polysaccharide. After a batch fermentation of I. obliquus BELYU1102 was carried out in a 300 l pilot vessel, endo-polysaccharide and exo-polysaccharide were both obtained. The proliferation activity of endo-polysaccharide for splenic cells was much higher than the activity of exo-polysaccharide. The active endo-polysaccharide was produced primarily during the late stationary phase. Enhanced proliferation and polyclonal IgM antibody production were observed in B cells by purified water-soluble endo-polysaccharide. Nitrite production and expression of IL-1beta, IL-6, TNF-alpha, and iNOS in macrophages were also enhanced. However, the endo-polysaccharide did not affect the proliferation of T cells, the IL-2 expression of Th1 cells, or the IL-4 expression of Th2 cells. The endo-polysaccharide showed activities similar to lipopolysaccharide (LPS) for B cells and macrophages, but there was a large difference between the two polysaccharides because cellular activations induced by endo-polysaccharide were not affected by polymyxin B, a specific inhibitor of LPS. The endo-polysaccharide appeared to have other cellular binding sites with TLR-4 and did not show a direct toxicity against tumor cells. However, indirect anti-cancer effects via immuno-stimulation were observed. The mycelial endo-polysaccharide of I. obliquus is a candidate for use as an immune response modifier. Submerged mycelial cultures are advantageous for industrial production of polysaccharides.

Adjuvants, Immunologic↗
PubMed1985

Immunochemical analysis of the types Ia and Ib group B streptococcal polysaccharides.

The types Ia and Ib group B streptococcal type-specific polysaccharides have remarkable immunologic differences despite a great deal of structural similarity. Although these two complex polysaccharides differ only by a single glycosidic linkage, they are antigenically distinct. Furthermore, terminal sialic acid residues appear to be critical to the immunodeterminant on the type Ia polysaccharide, whereas the antigenicity of the type Ib polysaccharide does not show this dependence on sialic acid. In the current investigation we defined better the immunodeterminant of these polysaccharides. With homologous rabbit antiserum, the type Ia native and core polysaccharides demonstrated partial serologic identity, whereas the type Ib native and core polysaccharides demonstrated complete serologic identity. Surprisingly, the type I degalactosylated polysaccharide, degraded structure, was capable of reacting with a population of antibodies present in type Ia antiserum similar to the complete type Ia native polysaccharide, although demonstrating a reduced level of immunodeterminant expression. Unlike the reactions of the type Ia polysaccharides with homologous rabbit antiserum, the Ib native and core polysaccharides were able to react with identical populations of antibodies in type Ib-specific antiserum. A minor population of antibodies was demonstrated in the type Ib antiserum, which was reactive with the degalactosylated polysaccharide. That a population of antibodies reactive toward the degalactosylated polysaccharide is present in both type Ia and type Ib antisera suggests that the Iabc cross-reacting determinant is due to the presence of serum antibodies reactive with this trisaccharide repeating unit, which is shared by both the type Ia and the type Ib native and core polysaccharides.

Animals↗
PubMed1964

METABOLISM OF INTRACELLULAR POLYSACCHARIDE BY STREPTOCOCCUS MITIS AND ITS RELATION TO INDUCIBLE ENZYME FORMATION.

Gibbons, R. J. (Forsyth Dental Center, Boston Mass.). Metabolism of intracellular polysaccharide by Streptococcus mitis and its relation to inducible enzyme formation. J. Bacteriol. 87:1512-1520. 1964.-The synthesis and catabolism of an intracellular iodine staining polysaccharide produced from glucose by Streptococcus mitis was investigated. Approximately 15% of the total glucose metabolized by buffered suspensions of S. mitis was assimilated. Over 90% of the assimilated glucose was converted into a polysaccharide of the glycogen-amylopectin type. Use of uniformly labeled C(14)-glucose provided a convenient method for determining polysaccharide accumulation in this organism. Glucose assimilation occurred at a rate of over 80 mug of glucose per hr per 100 mug of starting dry cell weight. Prolonged assimilation produced cells containing over 50% polysaccharide on a dry weight basis. Accumulated polysaccharide was catabolized at the same rate when the organism was suspended in buffer, sugar-free broth, or sugar-free broth containing thiomethyl galactoside. Metabolic intermediates produced from polysaccharide catabolism did not markedly repress inducible enzyme synthesis. The last glucose molecules incorporated into polysaccharide were among the first molecules to be removed during catabolism. Catabolism of polysaccharide provides S. mitis with energy in a utilizable form, for cells containing polysaccharide increased in beta-galactosidase activity when induced with thiomethyl galactoside in the absence of an exogenous energy source. Cells devoid of polysaccharide, and a polysaccharide-negative variant of S. mitis did not increase in beta-galactosidase activity when induced in a similar manner. It appears that the intracellular polysaccharide is the sole substrate for the endogenous metabolism of S. mitis.

Carbohydrate Metabolism↗
PubMed1987

Local response in rat to liposome-associated Streptococcus mutans polysaccharide-protein conjugate.

The effect of gastric intubation with soluble or liposome-associated Streptococcus mutans serovar polysaccharide, 74-kDa saliva receptor (74K SR protein) or polysaccharide-74K SR protein conjugate on the locally induced salivary IgA response and memory in rats was investigated. Animals immunized on four successive days with soluble antigens showed a weak salivary anti-74K SR protein or anti-polysaccharide IgA response. Rats primed and boosted by a single injection of liposome-associated 74K SR protein or polysaccharide-74K SR protein conjugate developed a salivary anti-74K SR protein IgA and IgG primary and secondary response. A primary anti-polysaccharide response was only observed in saliva of animals immunized with either high concentration of liposome-associated polysaccharide or liposome-associated polysaccharide-74K SR protein conjugate. However, a secondary local anti-polysaccharide IgA response was detected in animals boosted with liposome-polysaccharide-74K SR protein conjugate. No such anamnestic response was seen when high dose of liposome-associated polysaccharide was used to boost the animals. Furthermore, the salivary anti-polysaccharide IgA response paralleled the anti-74K SR protein IgA response. These studies showed that intragastric immunization of rats with liposome-associated polysaccharide-74K SR protein conjugate produced a local anti-polysaccharide IgA memory.

Animals↗
PubMed1994

Structural characteristics of polysaccharides that induce protection against intra-abdominal abscess formation.

Bacteroides fragilis is the anaerobe most commonly isolated from clinical cases of intra-abdominal sepsis. In a rodent model of this disease process, intraperitoneal injection of the capsular polysaccharide complex (CPC) from B. fragilis provokes abscess formation, while subcutaneous administration of this complex confers protection against B. fragilis-induced intra-abdominal abscesses. The CPC consists of two discrete polysaccharides, polysaccharides A and B (PS A and PS B), each possessing oppositely charged structural groups critical to the ability of these carbohydrates to induce the formation of abscesses. Other bacterial polysaccharides that possess oppositely charged groups (such as the group antigen or capsular polysaccharide from Streptococcus pneumoniae type 1 strains) also exhibited potent abscess-inducing capabilities. We report here that positively and negatively charged groups on polysaccharides are also essential for inducing protection against abscess formation. Vaccination of rats with B. fragilis PS A, PS B, or the S. pneumoniae type 1 capsule protected against intra-abdominal abscesses subsequent to intraperitoneal challenge with each of these polysaccharides. Chemical conversion of the free amino or carboxyl groups on PS A to uncharged N-acetyl or hydroxymethyl groups, respectively, abrogated the ability of this polymer to confer protection against polysaccharide-mediated abscess formation. Adoptive transfer of splenic T cells from polysaccharide-vaccinated rats to naive animals demonstrated that T cells mediated this protective activity. T cells transferred from animals vaccinated with a polysaccharide repeating unit (Salmonella typhi Vi antigen) that normally contains one carboxyl group but was chemically converted to a polymer that possesses both free amino and carboxyl groups (accomplished by de-N-acetylating the Vi antigen) protected naive T-cell recipients against polysaccharide-induced abscesses. These results demonstrate that a distinct structural motif associated with the B. fragilis polysaccharides is necessary for induction of protective immunity against abscess formation associated with intra-abdominal sepsis. However, protection is not antigen specific in a traditional sense. Rather, the protective ability of these structurally dissimilar polysaccharides is conferred by, and perhaps specific for, a motif of oppositely charged groups.

Abdominal Abscess↗
PubMed1986

Purification and characterization of complement activating-acidic polysaccharide from the root of Lithospermum euchromum Royle.

An extraordinary potent anti-complementary substance was isolated from the root of Lithospermum euchromum Royle (Japanese name: Nan-shikon) which activates the complement system in vitro, and the active principle was shown to be acidic polysaccharide (LR-polysaccharide IIa). LR-polysaccharide IIa was purified by chromatographies on DEAE-Sepharose, Sephadex G-100, concanavalin A-Sepharose, Ricinus communis agglutinin conjugated Sepharose, Sepharose CL-6B and Sepharose CL-2B. LR-polysaccharide IIa was found to be composed of rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose in the molar ratios of 2.0:2.5:3.4:2.8:5.6:9.6:14.4. The polysaccharide also contained 15% of galacturonic acid and 3.8% of protein. The methylation analysis of the polysaccharide showed that rhamnose, arabinofuranose, xylose, glucose and galactose are present as a part of the nonreducing terminal residues. The main chain and side chains are composed of ----3Glc1----,----3Gal1----,----6Man1----,----4Gal1, [corrected] and the branching points consist of (formula; see erratum) These results indicated that LR-polysaccharide IIa has a highly complicated structure. A marked consumption of C4 was observed after the incubation of the serum with LR-polysaccharide IIa in the presence of the Ca++ ion. The anti-complementary activity of LR-polysaccharide IIa was reduced partially in the absence of the Ca++ ion. After the incubation of the serum with LR-polysaccharide IIa in the absence of Ca++ ion, a cleavage of C3 in the serum was found to have occurred through immunoelectrophoresis as well as from the consumption of the complement when rabbit erythrocytes were used in the assay system. These results indicate that the mode of complement activation by LR-polysaccharide IIa is via both the alternative and classical pathways. Complement titer also decreased in guinea pigs upon i.p. injection of LR-polysaccharide IIa.

Animals↗
PubMed1976

Isolation and characterization of a Ca2+ -binding polysaccharide associated with coccoliths of Emiliania huxleyi (Lohmann) Kamptner.

C-occolithophoridae, a group of mostly unicellular algae, possess a cell wall containing calcified plates, called coccoliths. The coccoliths from the species Emilania huxleyi (Lohmann) Kamptner contain a water-soluble acid polysaccharide. In this paper we describe the isolation and some characteristic properties of the polysaccharide, in particular its Ca2+ -binding capacity. A large-scale cultivation of the Coccolithophoridae was worked out and a new procedure for isolating coccoliths was developed. The polysaccharide obtained from the coccoliths contained two types of monobasic acid groups in a total amount of 1.8 mumol/mg polysaccharide. One type consisted of weakly acid groups which were identified as uronic acids. The nature of the stronger acid groups remains to be established. The ratio between the respective groups was 1:0.8. Studies with 45Ca2+ demonstrated that the isolated polysaccharide is capable of binding Ca2+. Equilibrium dialysis revealed that the maximum amount of Ca2+ which can be bound in 0.92 +/- 0.05 mumol/mg polysaccharide. Flow-rate dialysis experiments strongly suggested the presence of two classes of Ca2+ -binding sites differing in affinity for Ca2+. High-affinity sites (dissociation constant Kd for Ca2+ :2.2 +/- 1.0 X 10(-5) M) were found to be present in amounts (0.38 +/- 0.04 mumol/mg polysaccharide) approximately equivalent to the strongly acid monovalent groups mentioned above (0.8 mumol/mg polysaccharide). Low-affinity sites (Kd for Ca2+: -11 +/- 39 X 10(-5) M) were estimated at 0.74 +/- 0.11 mumol/mg polysaccharide. Although this figure could be determined less accurately, it is suggested that the uronic acids (1.0 mumol/mg polysaccharide) are identical to the low-affinity sites. Preferential binding of Ca2+ occurred in a 100-fold excess of Na+ and Mg2+ as was shown by gel filtration. A 100-fold excess of Sr2+ inhibited Ca2+ binding to a great extent while no Ca2+ was bound in the presence of an equimolar amount of La3+. The dissociation constants of the high-affinity sites for Na+, Mg2+, Sr2+ and La3+ (in the presence of Ca2+) were determined with the flow-rate dialysis technique. They confirm the order of binding preference found with gel filtration. A polysaccharide with similar properties could be isolated from subfossil coccoliths of E. hyxleyi (about 1000 years old). The possible role of the polysaccharide as a heterogeneous matrix in coccolith formation is discussed.

Binding Sites↗
PubMed1989

Isolation of a coaggregation-inhibiting cell wall polysaccharide from Streptococcus sanguis H1.

Coaggregation between Streptococcus sanguis H1 and Capnocytophaga ochracea ATCC 33596 cells is mediated by a carbohydrate receptor on the former and an adhesin on the latter. Two methods were used to release the carbohydrate receptor from the gram-positive streptococcus, autoclaving and mutanolysin treatment. The polysaccharide released from the streptococcal cell wall by either treatment was purified by ion-exchange chromatography; this polysaccharide inhibited coaggregation when preincubated with the gram-negative capnocytophaga partner. After hydrolysis of the polysaccharide by hydrofluoric acid (HF), the major oligosaccharide of the polysaccharide was purified by high-performance liquid chromatography. By analysis of the HF hydrolysis of the polysaccharide and the purified oligosaccharide, this major oligosaccharide appeared to be the repeating unit of the polysaccharide, with minor components resulting from internal hydrolysis of the major oligosaccharide. Gas chromatography results showed that the oligomer was a hexasaccharide, consisting of rhamnose, galactose, and glucose, in the ratio of 2:3:1, respectively. By weight, the purified hexasaccharide was a fourfold-more-potent inhibitor of coaggregation than the native polysaccharide. Resistance to hydrolysis by sulfuric acid alone and susceptibility to hydrolysis by HF suggested that oligosaccharide chains of the polysaccharide are linked by phosphodiester bonds. Studies with a coaggregation-defective mutant of S. sanguis H1 revealed that the cell walls of the mutant contained neither the polysaccharide nor the hexasaccharide repeating unit. The purification of both a polysaccharide and its constituent hexasaccharide repeating unit, which both inhibited coaggregation, and the absence of this polysaccharide or hexasaccharide on a coaggregation-defective mutant strongly suggest that the hexasaccharide derived from the polysaccharide functions as the receptor for the adhesin from C. ochracea ATCC 33596.

Adhesins, Bacterial↗
PubMed1981

Immunochemical relations between pneumococcal group 19 and Klebsiella capsular polysaccharides.

Pneumococcal type 19F polysaccharide, contained in the current U.S. licensed pneumococcal vaccine, is a poor immunogen in young children. Capsular polysaccharides of Klebsiella, reported to be cross-reactive with the pneumococcal type 19F, were isolated. Immunochemical relations between pneumococcal group 19 and Klebsiella capsular polysaccharides were studied by immunodiffusion with hyperimmune typing antisera, immune passive hemolysis, and plaque-forming cell response in mice following injection of polysaccharides. Klebsiella K2, K9, K32, K47, and K48 polysaccharides were reported to cross-react with horse pneumococcal type 19 antiserum by direct precipitation. However, these Klebsiella polysaccharides did not react in immunodiffusion with horse or with rabbit pneumococcal 19F and 19A typing antisera. Furthermore, pneumococcal type 19F and 19A polysaccharides did not precipitate with rabbit K2, K9, K32, K47, and K49 typing antisera. The cross-reactivity of the Klebsiella and pneumococcal polysaccharides was studied by 2 other methods. Rabbit pneumococcal 19F antiserum induced by low passive hemolysis of sheep erythrocytes coated with Klebsiella polysaccharides. In contrast, Klebsiella typing antisera exhibited higher hemolytic activity with erythrocytes coated with type 19F polysaccharides. The cross-reactivity of K2 and pneumococcal 19F polysaccharides was also exhibited by the direct IgM plaque-forming cell response. The cross-reactivity, determined by precipitin reactions using animal antisera, may not predict the cross-immunogenicity of purified polysaccharides in humans.

Animals↗
PubMed1994

Comparison of peptidoglycan-polysaccharide and lipopolysaccharide stimulation of Kupffer cells to produce tumor necrosis factor and interleukin-1.

Endotoxin (lipopolysaccharide) is a cell wall polymer from gram-negative bacteria that stimulates Kupffer cell release of cytokines such as tumor necrosis factor-alpha and interleukin-1. Another bacterial cell wall polymer in both gram-negative and gram-positive organisms is peptidoglycan-polysaccharide. Lipopolysaccharide and peptidoglycan-polysaccharide exist together in the intestinal lumen and can cross the intestinal mucosa, enter the portal vein and activate Kupffer cells. The purpose of this study was to compare the effects of lipopolysaccharide stimulation and peptidoglycan-polysaccharide stimulation of Kupffer cells on release of tumor necrosis factor-alpha and interleukin-1. Both bacterial polymers caused maximum tumor necrosis factor-alpha release from Kupffer cells after incubation for 4 to 8 hr. Maximum tumor necrosis factor-alpha release induced by 400 ng/ml lipopolysaccharide was 704 +/- 258 pg/ml, compared with 329 +/- 91 pg/ml tumor necrosis factor-alpha after 100 micrograms/ml peptidoglycan-polysaccharide (p < 0.001). Polymyxin B blocked lipopolysaccharide stimulation of tumor necrosis factor-alpha by 95% +/- 5% but blocked peptidoglycan-polysaccharide-stimulated tumor necrosis factor-alpha by 30% +/- 14% (p < 0.001). Repeat incubation of Kupffer cells with lipopolysaccharide after prior lipopolysaccharide incubation induced low tumor necrosis factor-alpha release (tolerance). Repeat incubation with peptidoglycan-polysaccharide induced no tolerance to tumor necrosis factor-alpha release. Incubation of lipopolysaccharide plus peptidoglycan-polysaccharide released less tumor necrosis factor-alpha than did each polymer used alone, but this inhibition was prevented by indomethacin. Dibutyryl cyclic AMP, prostaglandin E1, prostaglandin E2 and the adenosine A2-receptor agonist N-ethylcarboxyamideadenosine inhibited lipopolysaccharide-stimulated tumor necrosis factor-alpha release by 83%, 97%, 90% and 94%, respectively, but inhibited peptidoglycan-polysaccharide-stimulated tumor necrosis factor-alpha release by 52%, 60%, 45% and 51%, respectively (p < 0.001 for each). This indicates that intracellular signaling pathways differ for lipopolysaccharide-stimulated and peptidoglycan-polysaccharide-stimulated tumor necrosis factor-alpha release. After incubation for 8 and 24 hr, 100 micrograms/ml peptidoglycan-polysaccharide had induced significantly more interleukin-1 release from cultured Kupffer cells than had 400 ng/ml lipopolysaccharide (p < 0.001). Lipopolysaccharide induced tolerance to interleukin-1 release after repeat incubation, but peptidoglycan-polysaccharide caused no tolerance. These studies show that peptidoglycan-polysaccharide, a ubiquitous bacterial cell wall polymer, shares several proinflammatory properties with lipopolysaccharide but that there are differences that may have pathophysiological significance.

Animals↗
PubMed1998

Induction of immunologic memory by conjugated vs plain meningococcal C polysaccharide vaccine in toddlers: a randomized controlled trial.

CONTEXT: Meningococcal polysaccharide vaccines are not used routinely in infants and toddlers, the groups at highest risk of invasive disease, because of poor immunologic responses to the Neisseria meningitidis serogroup C polysaccharide in these age groups. Meningococcal C conjugate vaccines offer the prospect of circumventing this problem. OBJECTIVE: To assess the immunogenicity and the induction of immunologic memory in toddlers by meningococcal C conjugate vaccine. DESIGN: A multicenter, randomized, observer-blinded controlled trial. SETTING: Urban and suburban family medicine or pediatric practices. PARTICIPANTS: Two hundred eleven healthy toddlers aged 15 to 23 months. INTERVENTION: Two injections at 2 months apart of meningococcal C conjugate (group 1, n = 69), plain meningococcal polysaccharide (group 2, n = 72), or hepatitis B virus vaccine (group 3, n = 70). All toddlers received a follow-up dose of plain meningococcal polysaccharide vaccine 12 months later. MAIN OUTCOME MEASURES: IgG meningococcal C anticapsular antibody concentrations determined by enzyme-linked immunosorbent assay and complement-mediated bactericidal antibody. RESULTS: In group 1, the magnitude of the IgG response to meningococcal C conjugate vaccine was more than 4-fold higher after dose 1 and more than 10-fold higher after dose 2 compared with meningococcal polysaccharide vaccine (group 2) (P<.001). Higher titers persisted in the meningococcal C conjugate group for at least 12 months (P<.001). Group 1, primed with meningococcal C conjugate, had 25-fold higher IgG responses to the meningococcal polysaccharide 1-year booster dose than the controls who had received hepatitis B virus vaccine initially and were given meningococcal polysaccharide vaccine 1 year later for the first time (P<.001). In contrast, group 2, primed with meningococcal polysaccharide, had a 2-fold lower response to the 1-year booster meningococcal polysaccharide dose than the hepatitis B virus control group (P = .006). Serum bactericidal responses paralleled the enzyme-linked immunosorbent assay responses. CONCLUSIONS: Immunization of toddlers with meningococcal C conjugate vaccine induces high titers of anticapsular and bactericidal antibody. Furthermore, this vaccine induces immunologic memory to meningococcal C polysaccharide. In contrast, meningococcal polysaccharide vaccine is less immunogenic than the conjugate vaccine and also induces a hyporesponsive state that persists for at least 12 months.

Antibodies, Bacterial↗
PubMed2004

Base hydrolysis of phosphodiester bonds in pneumococcal polysaccharides.

A comprehensive study of the base hydrolysis of all phosphodiester bond-containing capsular polysaccharides of the 23-valent pneumococcal vaccine is described here. Capsular polysaccharides from serotypes 6B, 10A, 17F, 19A, 19F, and 20 contain a phosphodiester bond that connects the repeating units in these polysaccharides (also referred to as backbone phosphodiester bonds), and polysaccharides from serotypes 11A, 15B, 18C, and 23F contain a phosphodiester bond that links a side chain to their repeating units. Molecular weight measurements of the polysaccharides, using high performance size exclusion chromatography with tandem multiangle laser light scattering and refractive index detection, was used to evaluate the kinetics of hydrolysis. The measurement of molecular weight provides a high degree of sensitivity in the case of small extents of reaction, thus allowing reliable measurements of the kinetics over short times. Pseudo-first-order rate constants for these polysaccharides were estimated using a simple model that accounts for the polydispersity of the starting sample. It was found that the relative order of backbone phosphodiester bond instability due to base hydrolysis was 19A > 10A > 19F > 6B > 17F, 20. Degradation of side-chain phosphodiester bonds was not observed, although the high degree of sensitivity in measurements is lost in this case, due to the low contribution of the side chains to the total polysaccharide molecular weight. In comparison with literature data on pneumococcal polysaccharide 6A, 19A was found to be the more labile, and hence appears to be the most labile pneumococcal polysaccharide studied to date. The rate of hydrolysis increased at higher pH and in the presence of divalent cation, but the extent was lower than expected based on similar data on RNA. Finally, the differences in the phosphodiester bond stabilities were analyzed by considering stereochemical factors in these polysaccharides. These results also provide a framework for evaluation of molecular integrity of phosphodiester-bond-containing polysaccharides in different solution conditions.

Carbohydrate Conformation↗
PubMed1986

Modification of lymphocyte migration by sulfated polysaccharides.

The role of sulfated polysaccharides in lymphocyte migration has been analyzed in vivo using lymphocytes labeled with an intracellular DNA-binding fluorochrome Hoechst 33342. The influence of a panel of sulfated polysaccharides on entry (by injecting the sulfated polysaccharide prior to the labeled cells) and displacement from lymphoid organs (by injecting the sulfated polysaccharide after the labeled cells have localized) indicated that different sulfated polysaccharides have selective effects on entry and displacement, and furthermore positioning of subpopulations within organs. Additional experiments suggested that receptors for sulfated polysaccharides on high endothelial venules may interact with complementary structures on lymphocytes. The data supporting this conclusion were: (a) the normal localization behavior of lymphocytes preincubated with sulfated polysaccharides; (b) an inverse relationship between the expression of lymphocyte surface receptors for sulfated polysaccharides and the ability of the lymphocytes to enter lymphoid organs and (c) the selective binding of sulfated polysaccharide-coupled fluoresceinated beads to high endothelial venules. In this case only the beads coupled with the sulfated polysaccharides that inhibited entry bound to the high endothelial venules. These findings are discussed in terms of a fundamental cellular recognition system utilizing sulfated polysaccharides.

Animals↗
PubMed1996

The stability of extracellular beta-glucosidase from Aspergillus niger is significantly enhanced by non-covalently attached polysaccharides.

The removal of noncovalently bound polysaccharide coating from the extracellular enzymes of Aspergillus niger, by the technique of compartmental electrophoresis, had a very dramatic effect on the stability of beta-glucosidase. The polysaccharide-beta-glucosidase complex was extremely resistant to proteinases and far more stable against urea and temperature as compared with polysaccharide-free beta-glucosidase. The beta-glucosidase-polysaccharide complex was 18-, 36-, 40- and 82-fold more stable against chymotrypsin, 3 mol/L urea, total thermal denaturation and irreversible thermal denaturation, respectively, as compared with polysaccharide-free beta-glucosidase. The activation energy of polysaccharide-complexed beta-glucosidase (55 kJ/mol) was lower than polysaccharide-free enzyme (61 kJ/mol), indicating a slight activation of the enzyme by the polysaccharide. No significant difference could be detected in the specificity constant (V/K(m)) for A-nitrophenyl beta-D-glucopyranoside between polysaccharide-free and polysaccharide-complexed beta-glucosidase. We suggest that the function of these polysaccharides secreted by fungi including A. niger might be to protect the extracellular enzymes from proteolytic degradation, hence increasing their life span.

Aspergillus niger↗
PubMed2002

Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides.

The number of mushrooms on Earth is estimated at 140,000, yet maybe only 10% (approximately 14,000 named species) are known. Mushrooms comprise a vast and yet largely untapped source of powerful new pharmaceutical products. In particular, and most importantly for modern medicine, they represent an unlimited source of polysaccharides with antitumor and immunostimulating properties. Many, if not all, Basidiomycetes mushrooms contain biologically active polysaccharides in fruit bodies, cultured mycelium, culture broth. Data on mushroom polysaccharides have been collected from 651 species and 7 infraspecific taxa from 182 genera of higher Hetero- and Homobasidiomycetes. These polysaccharides are of different chemical composition, with most belonging to the group of beta-glucans; these have beta-(1-->3) linkages in the main chain of the glucan and additional beta-(1-->6) branch points that are needed for their antitumor action. High molecular weight glucans appear to be more effective than those of low molecular weight. Chemical modification is often carried out to improve the antitumor activity of polysaccharides and their clinical qualities (mostly water solubility). The main procedures used for chemical improvement are: Smith degradation (oxydo-reducto-hydrolysis), formolysis, and carboxymethylation. Most of the clinical evidence for antitumor activity comes from the commercial polysaccharides lentinan, PSK (krestin), and schizophyllan, but polysaccharides of some other promising medicinal mushroom species also show good results. Their activity is especially beneficial in clinics when used in conjunction with chemotherapy. Mushroom polysaccharides prevent oncogenesis, show direct antitumor activity against various allogeneic and syngeneic tumors, and prevent tumor metastasis. Polysaccharides from mushrooms do not attack cancer cells directly, but produce their antitumor effects by activating different immune responses in the host. The antitumor action of polysaccharides requires an intact T-cell component; their activity is mediated through a thymus-dependent immune mechanism. Practical application is dependent not only on biological properties, but also on biotechnological availability. The present review analyzes the pecularities of polysaccharides derived from fruiting bodies and cultured mycelium (the two main methods of biotechnological production today) in selected examples of medicinal mushrooms.

Adjuvants, Immunologic↗