Biomedical subjects

I van de Rijn

Publications and source records attributed to I van de Rijn.

At least 37 records · Page 2Linked to original sources

PubMed1991

Evaluation of the immune response in protection against experimental Streptococcus defectivus endocarditis.

Immune protection from endocarditis caused by Streptococcus defectivus was examined by using a rabbit model. Previously we had demonstrated that immunization with nutritionally variant streptococci (NVS, now referred to as Streptococcus, species defectivus and adjacens) protected rabbits against endocarditis when they were challenged with the homologous strain. However, when high-titer immune globulin was transferred to nonimmunized rabbits, no protection was achieved. In the present study, immunosuppressive treatments were given to previously immunized rabbits, and alterations in the level of protection were determined by using the rabbit endocarditis model. Control-immunized rabbits as well as immunized rabbits receiving cyclosporin A or methylprednisolone treatments were protected from S. defectivus endocarditis at levels between 50% and 67%. Rabbits in each of these groups cleared S. defectivus organisms from the circulation by 3 hours after infection. Nitrogen mustard-treated rabbits (immunized or nonimmunized), however, were unable to clear S. defectivus organisms as efficiently (almost 100 times as many organisms in the blood when compared with other groups) by 3 hours, and all were susceptible to endocarditis. These data suggest that circulating phagocytes such as monocytes and granulocytes function to a certain extent in protection against S. defectivus endocarditis. Moreover, when neutrophils were transfused into granulocytopenic and monocytopenic rabbits, efficient clearance was prolonged, indicating that polymorphonuclear leukocytes were involved in the later (greater than 1 hour after infection) phase of protection.

Animals↗

PubMed1988

Nutritionally variant streptococcal serotype I antigen. Characterization as a lipid-substituted poly(ribitol phosphate).

Initial characterization of the nutritionally variant streptococcal serotype I amphipathic polymer indicated an estimated m.w. of 360,000 for the aggregated form of the molecule, whereas the dissociated form had an estimated m.w. of 38,000 based on dextran m.w. standards. In addition, SDS-PAGE indicated an amphiphile with a stepladder appearance made up of several components with m.w. larger than the major electrophoresis components of LPS or lipoteichoic acid. Chemically, the serotype I amphiphile appeared to be a lipid-substituted poly(ribitol phosphate) with galactose and alanine substitution on the ribitol phosphate backbone. This report represents the first characterization of a bacterial amphiphile with a chemical composition and structure as proposed here. As is the case with other bacterial amphiphiles, the nutritionally variant streptococcal serotype I amphiphile was found both intra- and extracellularly. Finally, immunofluorescence studies demonstrated that the amphiphile was expressed on the cell surface.

Alanine↗

PubMed1988

Analysis of cross-protection between serotypes and passively transferred immune globulin in experimental nutritionally variant streptococcal endocarditis.

Nutritionally variant streptococci (NVS), which account for 5 to 10% of all cases of streptococcal endocarditis, were recently subdivided into three serotypes. In the past, using a rabbit endocarditis model, I demonstrated that by immunization of rabbits with NVS and by challenge with a strain from the homologous serotype a level of 90 to 100% protection was elicited. In the present study, the level of cross-protection between strains from different serotypes was measured. No cross-protection was demonstrated between serotype I and II or III strains. However, significant cross-protection was observed when serotype II and III strains were analyzed in the model. Since high levels of immunoglobulin G were demonstrated against the surface of the NVS after immunization, passive transfer experiments were initiated. Even at comparable levels of surface immunoglobulin G, none of the rabbits given immune globulin were protected against challenge with a dose of live NVS that equaled 20 times the 50% infective dose. Therefore, it appears that components from the immune system, in addition to humoral components, are required for active protection against NVS endocarditis.

Animals↗

PubMed1988

Purification of serotype I antigen from nutritionally variant streptococci.

The nutritionally variant streptococci (NVS) are a fastidious group of bacteria first recognized in the early 1960s in the blood cultures of patients with subacute bacterial endocarditis. Since that time, the NVS have been implicated in 5 to 6% of all cases of human bacterial endocarditis. The NVS possess membrane-associated amphipathic molecules different from those described for other streptococci. Unitl recently, chemical characterization of these new amphipathic polymers was hampered by unsuccessful attempts at isolating large quantities of these molecules in a form free from other bacterial components. Presently, stationary-phase-culture supernatants provide an optimum source of crude material for amphiphile purification procedures. Hydrophobic-interaction chromatography in conjunction with immunoaffinity chromatography yields an NVS serotype I amphiphile preparation free of contaminants, as determined by immunoelectrophoretic and chemical analyses. Tandem crossed immunoelectrophoresis of the purified extracellular NVS amphiphile demonstrated that it is immunologically similar to the intracellular amphiphile. Finally, this amphiphile serves as the NVS serotype I antigen.

Antigens, Bacterial↗

PubMed1986

Solubilization of hyaluronic acid synthetic activity from streptococci and its activation with phospholipids.

To date all hyaluronic acid synthetic systems have been of a particulate nature, and attempts at solubilization have been unsuccessful. This has hampered attempts to elucidate the mechanism by which hyaluronic acid is produced. In this paper we demonstrate that the hyaluronic acid synthetic activity from group C streptococcal membranes was solubilized using 2% digitonin and that the activity was optimized by reconstitution with cardiolipin at an optimum phospholipid/protein ratio (microgram/microgram) of 5:1. Furthermore, chromatography of the solubilized synthetase demonstrated that it eluted after the void volume of a Sepharose CL-6B column. CHAPSO, octyl glucopyranoside, sodium cholate, Triton X-100, and zwittergent 314 either inhibited or failed to solubilize the synthetic activity. Phospholipids other than cardiolipin also reconstituted the activity from the digitonin extract, particularly phosphatidylethanolamine and phosphatidylserine. In our system, the specific activity of hyaluronic acid synthetase was increased up to 63 times that of the system of the intact membrane. Furthermore, the total activity of the reconstituted system was 4.9 times greater than that of intact membranes. The soluble enzyme system showed similarities to the membrane-bound synthetase in the kinetics of production of trichloroacetic acid-soluble and -insoluble hyaluronic acid, and the hyaluronic acid produced was of comparable molecular weight.

Carbon Radioisotopes↗

PubMed1986

Enzyme-linked immunosorbent assay for the detection of antibodies to nutritionally variant streptococci in patients with endocarditis.

The viridans streptococci are responsible for 50%-55% of microbial endocarditis. Among these varied species, the nutritionally variant streptococci (NVS) have recently been associated with culture-negative endocarditis and are responsible for 5%-10% of all streptococcal endocarditis. The isolation and identification of these bacteria has been hampered by the extremely fastidious nature of their growth requirements as well as by their slow growth rate. In addition, their antibiotic sensitivity varies greatly, a characteristic leading to a higher rate of morbidity and mortality than is found in patients with non-NVS endocarditis. For these reasons sera from patients with NVS endocarditis were examined for antibodies to the NVS serotype I antigen by using an enzyme-linked immunosorbent assay. Seventy-four percent of patients with NVS endocarditis had elevated titers to this antigen. None of the sera from controls and only 6.7% of the sera from patients with non-NVS streptococcal endocarditis showed increased titers. These results indicate that antibody to the NVS serotype I antigen can serve as a marker for NVS endocarditis.

Antibodies, Bacterial↗

PubMed1985

Quantitative analysis of cell walls of nutritionally variant streptococci grown under various growth conditions.

Strains of nutritionally variant streptococci are usually isolated from patients with subacute bacterial endocarditis. Only recently have these strains been subdivided into three serotypes; however, no group-specific antigen has been described. To understand the immunochemical basis for the serology of these microorganisms as well as set the groundwork for adherence studies, quantitative analysis of the cell walls of nutritionally variant streptococci was undertaken. The bacteria were grown in semisynthetic medium or pyridoxal-supplemented Todd-Hewitt broth and harvested during the exponential or stationary phase. Cell walls were isolated and analyzed for amino sugars, sugars, polyalcohols, amino acids, and phosphorus by gas chromatography, high-pressure liquid chromatography, or colorimetric assays. The peptidoglycans of the cell walls of the prototype strains from the three serotypes were representative of other streptococcal cell walls, including the presence of alanine as the possible cross-bridge. The composition of the peptidoglycan was similar for all three strains and included a decreased concentration of peptidoglycan in their cell walls during the stationary phase. Glucosamine, glucose, galactose, ribitol, and a small amount of rhamnose were found in each of the cell wall polysaccharides. Galactosamine was only found in serotype II and III cell walls and might be responsible for the previously described cross-reaction between these strains. The concentration of the other sugars and amino sugars varied in each of the cell wall preparations, depending on the growth conditions. Finally, all three strains expressed both ribitol and phosphorus in their cell walls, characteristic of the presence of a ribitol teichoic acid. Therefore the cell wall composition of the nutritionally variant streptococci varies depending on the growth conditions, and their composition appears similar to that of strains of Streptococcus mitis.

Carbohydrates↗

PubMed1985

Role of culture conditions and immunization in experimental nutritionally variant streptococcal endocarditis.

The nutritionally variant streptococci (NVS) are usually isolated from patients with NVS endocarditis and recently have been serotyped into three groups. In the past, studies on microbial endocarditis have not analyzed the effect of the growth medium or growth phase on the bacteria used to induce the disease in the experimental rabbit model. Therefore, in this study various bacterial growth parameters were examined, including growth in semisynthetic or complex medium to the exponential or stationary phase of growth. The 50% infective dose ranged from 3.7 X 10(5) to 8.5 X 10(6) CFU for representative strains from each of the three serotypes grown under these conditions, indicating that there was no significant difference. The role of immunization was also examined in this model using organisms grown to the exponential phase in semisynthetic medium. Rabbits were immunized with heat-killed whole cells, high titres of specific antibody were produced as demonstrated by enzyme-linked immunosorbent assay, and then the rabbits were challenged with 20- to 100-fold 50% infective dose of the homologous strain. A total of 90 to 100% of the rabbits were protected from the disease process, as shown by the absence of the organisms from the heart valve 3 days after the challenge. Rabbits immunized with the amphiphile that replaces lipoteichoic acid in these organisms were not protected from challenge, demonstrating that another surface component is responsible for adherence or colonization or both. Finally NVS were incubated with radioiodinated fibronectin, fibrinogen, or laminin to determine whether these molecules aided in the adherence of the organisms to the heart valve. Only minor amounts of these components were bound to NVS as compared with controls. Therefore, NVS bind directly to the damaged heart valve or through an unknown mechanism.

Animals↗

PubMed1984

Characterization of a pH-dependent chromophore from nutritionally variant streptococci.

Strains of nutritionally variant streptococci and Streptococcus mitis produce a chromophore when they are heated at acid pH. No other strains of streptococci elaborated this chromophore. Furthermore, the nutritionally variant streptococci produced approximately twice the amount of chromophore as the S. mitis strains. The chromophore was localized in the cell wall of these streptococcal strains and appeared to be resistant to trypsin treatment. Hydrolysis apparently is required because elevated temperatures at pH 2 are necessary for demonstration of the chromophore. The chromophore has a maximal absorbance at 504 nm with a pKa of 3.6. The chromophore absorbance spectrum showed an isosbestic point at 400 nm. This is the first example of a pH-dependent chromophore to be found in streptococci and serves as a positive characteristic for the description of nutritionally variant streptococci as well as S. mitis.

Chromogenic Compounds↗

PubMed1984

Immunological studies of post-streptococcal sequelae. Evidence for presence of streptococcal antigens in circulating immune complexes.

Since elevated levels of circulating complexes have been noted to occur in the sera of patients with post-streptococcal sequelae, the possibility that these complexes contained streptococcal antigens within the complex was investigated. Sera from these patients were precipitated with polyethylene glycol to extract a fraction rich in these complexes, which was then injected into rabbits. The rabbit sera were then reacted with both cellular and extracellular fractions obtained from streptococcal strains associated with either acute post-streptococcal nephritis (APSGN) or acute rheumatic fever (ARF) by using immunoelectrophoresis and ELISA techniques. The data demonstrate that both ARF and APSGN complexes contain streptococcal antigens. However, APSGN complexes react uniquely to certain extracellular antigens present in those strains associated with nephritis, while ARF complexes react specifically to certain streptococcal extracellular antigens excreted by strains associated with rheumatic fever. Neither of the two groups of complexes appear to contain streptococcal antigens related to any cellular antigens derived from the group A streptococcus. Additionally, a rabbit serum immunized with streptococcal extracellular products reacted directly with complexes isolated from nephritis patients. Removal of the gamma globulin by absorption with an anti-human Fc serum resulted in the concomitant loss of reactivity with the anti-streptococcal serum, strongly suggesting an intimate association of the streptococcal antigen with these complexes. The presence of streptococcal antigens within the circulating immune complex of patients with APSGN coupled with their specific presence in those strains associated with post-streptococcal glomerulonephritis argues strongly for a causal role of these antigens in the disease process.

Antibodies, Bacterial↗

PubMed1984

Immunochemical study of nutritionally variant streptococci.

Nutritionally variant streptococci (NVS) have been characterized by their growth as satellite colonies around colonies of staphylococci or several other gram-positive or gram-negative bacterial strains. The majority of the NVS strains were isolated from patients with subacute bacterial endocarditis. Organisms identified as NVS were subdivided into three serotypes by rocket-line electrophoresis and hemagglutination inhibition assays. Ninety-nine of 103 strains expressed one or more of the three serotype antigens; however, a group antigen was not demonstrated in the various extracts of these streptococci. Surface protein studies confirmed the NVS differentiation into serotypes. Serotype I organisms expressed surface protein(s) specific for the serotype, whereas the serotype II and III NVS demonstrated common protein(s) on their surface. Furthermore, SDS extraction released a greater amount of radioiodinatable surface protein from serotypes I and III bacteria than serotype II. Finally, there was no correlation between the serotype or the disease of the patients from which the NVS strains were isolated.

Animals↗

PubMed1983

Streptococcal hyaluronic acid: proposed mechanisms of degradation and loss of synthesis during stationary phase.

Streptococcal hyaluronic acid was found to distribute into two discrete sizes. Cellular hyaluronic acid from strain D181 had an average molecular weight of 10 X 10(6), whereas the average molecular weight of extracellular hyaluronic acid from the same strain was 2 X 10(6). Cellular streptococcal hyaluronic acid was purified to homogeneity. Proteases were unable to cleave the purified cellular polymer, indicating that a peptide was not involved in cross-linking five extracellular hyaluronate polymers to form a cell-bound complex. Lipids apparently are not part of the cellular hyaluronic acid because phosphorus and glycerol were not detected by radioisotopic techniques, and denaturing conditions did not change the size of the polymer. Membranes obtained from various strains of group A and C streptococci cleaved the cellular form of the hyaluronate polymer demonstrating the presence of a membrane-bound hyaluronidase-like activity. By contrast, this activity was not found in the extracellular products of the strains studied. Furthermore, membranes derived from streptococci at the stationary phase of growth no longer had the capacity to synthesize hyaluronic acid. The loss of this property appeared to be due to changes in the structure of the membrane.

Cell Membrane↗

PubMed1983

Immunological studies of post-streptococcal sequelae: serological studies with an extracellular protein associated with nephritogenic streptococci.

Using the Ouchterlony double diffusion and the crossed-immunoelectrophoresis techniques the reactivity to a purified extracellular product of nephritogenic group A streptococci (NASP) was examined with both acute and convalescent sera obtained from patients with documented post-streptococcal glomerulonephritis and patients with documented acute rheumatic fever. The streptococcal antigen utilized in these studies was first purified on SDS gels and then eluted from the gel, resulting in a single protein band on SDS electrophoresis. Double diffusion studies revealed that only nephritis patients reacted to this extracellular product associated with nephritogenic strains, whereas rheumatic fever sera produced no line of precipitation. An assay of serial bleedings from nephritis patients suggested that the antibody reactive to the NASP was in higher titre in the acute phase of the disease and decreased with convalescence. In confirmation of these findings, crossed-immunoelectrophoresis experiments were conducted with a battery of sera from acute nephritic and non-nephritic patients against the NASP antigen. A striking increase was detected in the reactivity of nephritis patients (96%) compared to non-nephritis sera (15-20%). Comparison between acute and convalescent sera using this technique confirmed the finding of decreasing antibody titre with resolution of disease. These findings of a specific humoral response in patients with acute post-streptococcal nephritis to the NASP of nephritogenic strains further implicates an aetiological function to this protein.

Adolescent↗

PubMed1982

Changes in T-lymphocyte subsets during acute rheumatic fever.

Relative proportions and numbers of helper [OKT4(+)] and suppressor [OKT8(+)] peripheral blood lymphocytes were examined in 32 young patients during the acute phase of rheumatic fever. No significant decrease in percentage T cells during acute rheumatic fever was noted in comparison to normal children controls from the same population. A decrease in absolute numbers of OKT4(+) T cells was also noted in acute rheumatic subjects not receiving corticosteroids (P less than 0.05). In addition, a significant decrease was documented in both proportions and total numbers of OKT8(+) putative suppressor cells during acute rheumatic attacks. C-reactive protein binding to T-lymphocyte subsets showed no preferential reactivity in vivo for suppressor or helper T cells. Antigen-reactive T lymphocytes identified with the T29 mouse hybridoma reagent showed similar proportions and numbers in rheumatic children as were noted in controls. The present data indicate profound alterations in both helper- and suppressor-cell types in the peripheral blood profile of children with acute rheumatic fever.

Acute Disease↗

PubMed1982

Identification of bacterial antigens in circulating immune complexes of infective endocarditis.

The presence of circulating immune complexes (IC) in patients with infective endocarditis has been well documented but the contributions of host and bacterial components to these IC have not been defined. To study this question, IC were isolated from serum of a patient with Streptococcus faecalis endocarditis by differential polyethylene glycol precipitation and competitive binding to staphylococcal protein A. A rabbit antiserum raised against the purified IC had reactivity by crossed immunoelectrophoresis primarily with an antigen derived from the cytoplasm of the infective organism. The antigen was a protein with a 12,000-dalton molecular mass. In situ radiolabeling of the IC bound to the protein A demonstrated a component of the same molecular mass as the bacterial antigen recognized by the antiserum. The patient serum had multiple antibody specificities reactive with bacterial antigens, including the antigen recognized by the rabbit anti-IC antiserum. These techniques for isolation and characterization of circulating IC may have value in the study of IC diseases in which the inciting antigens are not known.

Animals↗

PubMed1981

Effects of penicillin on group A streptococci: loss of viability appears to precede stimulation of release of lipoteichoic acid.

The rate of killing of a group A streptococcal species by penicillin was compared with the release of lipoteichoic acid (LTA) and its deacylated derivative, dLTA. Although there was no stimulation of release from stationary-phase cells in the presence of penicillin, there was dramatic release of LTA and dLTA from exponential-phase cells after the addition of penicillin. Although decreases in viability were observed within 15 min after addition of penicillin, culture mass and LTA content did not appear to be affected until after 30 min. Stimulation of release of LTA and dLTA appeared to take place after 15 but before 30 min after addition of penicillin. These observations are interpreted to indicate that the stimulation of release of LTA and dLTA in response to penicillin is secondary to the killing event.

Bacteriolysis↗

PubMed1981

Immunochemical analysis of intact M protein secreted from cell wall-less streptococci.

M protein is a major virulence factor of group A streptococci, which provides these organisms with protection against phagocytosis in the absence of specific antibody. To gain insight into the nature of the native M-protein molecule, type 12 M protein was isolated and purified from the extracellular supernatants of a group A streptococcal L form and stabilized protoplasts. The intact purified M protein from both sources had a molecular weight of 58,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is in contrast to the 32,0000-dalton molecule isolated from the parent type 12 organism by using a nonionic detergent. The purified secretory M protein removed opsonic antibodies from type 12 rabbit immune serum, as demonstrated by a bactericidal assay. Therefore, it appears that either previous nondestructive methods of M-protein isolation have not removed intact M protein from cell walls or part of the molecule is fragmented during its association with cell walls.

Animals↗

PubMed1981

Nutritionally variant streptococci from patients with endocarditis: growth parameters in a semisynthetic medium and demonstration of a chromophore.

Nutritionally variant streptococci have been characterized in the past by their growth as satellite colonies and by their nutrient requirements of cysteine or vitamin B6 for growth in complex media. To further understand the growth characteristics of these strains, we studied fresh isolates from patients with endocarditis by using chemically defined medium enriched with 2% Todd-Hewitt dialysate. Under anaerobic conditions, growth yields of the strains in this medium were comparable to those obtained from a complex medium supplemented with vitamin B6, whereas under aerobic conditions, most of the strains had higher growth yields in the semisynthetic medium. Furthermore, the requirement for cysteine and vitamin B6 in the semisynthetic medium was no greater than that of other Streptococcus species. Electron microscopic studies demonstrated normal cell wall structures in organisms grown in the semisynthetic medium as compared with abnormal and irregular cell wall thickening in organisms grown in supplemented complex medium. Finally, these strains appeared to contain a common component when grown in the semisynthetic medium as demonstrated by the appearance of a chromophore after boiling the bacteria at pH 2. Therefore, the demonstration of a medium which permits adequate growth with a normal ultrastructure of nutritionally variant streptococci will permit the further study of this group of important streptococci.

Culture Media↗