Margriet Jansze

Penicillin and clindamycin differentially inhibit the production of pyrogenic exotoxins A and B by group A streptococci

Streptococcal pyrogenic exotoxins A (SPE-A) and B (SPE-B) have been implicated in the pathogenesis of serious group A streptococcal infections including streptococcal toxic shock-syndrome. Current antibiotics used for the treatment of these infections are penicillin and clindamycin. The effects of sub- and suprainhibitory concentrations of penicillin and clindamycin were evaluated in 14 isolates of Streptococcus pyogenes that were fully susceptible to both antibiotics. Clindamycin was superior to penicillin in reducing the production of SPE-A and SPE-B by invasive and non-invasive Dutch group A streptococcal isolates in vitro.

CD16 on human γδ T lymphocytes: Expression, function, and specificity for mouse IgG isotypes☆

Abstract We examined the expression, the signal transduction capacity and mouse IgG-isotype specificity of CD16 on human γδ T cells. CD16 is expressed by the majority of γδ T cells in peripheral blood and by part of the γδ T cell clones. The amount of CD16 expressed on γδ T cell clones varied considerably with passaging of the cells, but was always significantly less than on freshly isolated γδ T cells. Like CD16 on CD3−CD16+ natural killer (NK) cells, CD16 on γδ T cells can act as an activation site triggering cytotoxic activity. CD16+ γδ T cell clones exerted antibody-dependent cellular cytotoxicity (ADCC) which could be blocked by anti-CD16 mAb. ADCC activity of γδ T cell clones was also inhibited by anti-CD3 mAb, suggesting a functional linkage between the CD16 and CD3 activation pathways. MAb directed against CD16 induced lysis of FcγR+ target cells by CD16+ γδ T cell clones. The mouse IgG-isotype specificity of CD16 on γδ T cells was analyzed using isotype switch variants of a murine anti-glycophorin A mAb in EA rosette assays, and was found to be identical to that of CD16 on CD3−CD16+ NK cells, i.e., highest affinity for mIgG2a, intermediate affinity for mIgG2b, and undetectable binding of mIgG1-sensitized erythrocytes. CD16 was partly modulated from the cell surface of both γδ T cells and NK cells after rosette formation with mIgG2a-sensitized erythrocytes, indicating that the rosette formation was indeed mediated via the CD16 molecule.

Combinations of two synthetic adjuvants: Synergistic effects of a surfactant and a polyanion on the humoral immune response

Synergistic effects of two synthetic adjuvants, dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) on the humoral response to sheep red blood cells (SRBC) were investigated. Mice received intraperitoneal (ip) injections of adjuvant and antigen simultaneously. The number of plaque-forming cells (PFC) in the spleen were determined 5 days later and circulating anti-SRBC antibodies were measured till 16 weeks after immunization. Although combinations of DDA and DXS were very effective in enhancing the PFC response to both moderate (2 X 10(7] and low (2 X 10(6] doses of SRBC, synergy between the adjuvants was only observed at the low dose of SRBC. Optimal augmentation of the primary response to the low antigen dose was evoked by the combination of the highest dose tested of either adjuvant (1 mumol DDA and 1 nmol DXS) resulting in a 560-fold increase of the number of PFC in the spleen as compared to controls. Even combinations of relatively small amounts of both adjuvants were very effective in augmenting the response to SRBC. Mice receiving half the amounts of both adjuvants with 2 X 10(6) SRBC displayed increased numbers of PFC in the spleen at Day 5 as well as increased titers of total anti-SRBC antibodies at Week 1 and Week 2 and 2-mercaptoethanol-resistant antibodies from Week 4 till Week 16 as compared to the calculated sum of responses in mice which received either DDA (0.05 mumol per mouse) or DXS (0.05 nmol per mouse). The mechanism behind the synergy between these adjuvants is discussed and the possibility of discerning adjuvants on their modes of action is suggested.

Immunomodulating properties of two synthetic adjuvants: Dependence upon type of antigen, dose, and time of administration

The effects of two synthetic adjuvants on the antibody response to sheep red blood cells (SRBC) as a thymus dependent (TD) antigen and to dinitrophenyl59-Ficoll as a thymus-independent (TI-1) antigen were investigated in mice. Both dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) augmented the humoral response to SRBC but not to dinitrophenyl59-Ficoll if injected simultaneously with antigen. Dose-response curves of both antigen and adjuvant revealed that DXS compared to DDA is a more effective adjuvant for the induction of a humoral response to SRBC. Intraperitoneal injection of DDA or DXS evoked a sequence of distinct immune responsive states in mice, measured by the capacity to develop an anti-SRBC response. A short immune-potentiating period (less than 6 hr) is followed by a suppressive, second immune-potentiating state. The immune suppressive state lasted for a period of about 8 days and was restricted to TD-antigens. Suppression could be totally overridden by injection of DDA or DXS simultaneously with antigen, suggesting that the suppressive state was reversible. The kinetics of the observed alteration of the immune response by DDA and DXS were very similar. It is concluded that differences in the modulation of the immune response by DDA and DXS are limited to the initial state. Long-term effects like the induction of a succession of distinct immune responsive states, are more or less similar for both adjuvants. Possible mechanisms by which these immunomodulators interfere with the immune system are discussed.

Measurement of the humoral immune response against Streptococcus pneumoniae type 3 capsular polysaccharide and oligosaccharide containing antigens by ELISA and ELISPOT techniques

A sensitive ELISA has been developed to study immune responses in mice against Streptococcus pneumoniae type 3 capsular polysaccharide (S3PS) and hexasaccharide (HS)-protein conjugates derived therefrom. An advantage of the described system is that the same microtiter plates can be used for both ELISA and ELISPOT tests with a standardized washing procedure and diluent composition. S3PS induced predominantly IgM antibodies and minute amounts of IgG as measured by ELISA in serum. This was accompanied by large numbers (greater than 14000) of IgM spot-forming cells in the spleen. A shift towards IgG production was achieved by addition of lipid A. HS-protein conjugates induced predominantly IgG antibodies after booster immunization(s). Furthermore these conjugates induced large numbers (greater than 40000) of IgG spot-forming cells (SFC) in the spleen. ELISA and ELISPOT assays on microtiter plates are both reliable and highly reproducible assays for the evaluation of immune responses to S. pneumoniae antigens.

Measurement of the humoral immune response against streptococcus pneumoniae type 14-derived antigens by an ELISA and ELISPOT assay based on biotin-avidin technology

A Streptococcus pneumoniae type 14-specific ELISA and ELISPOT assay have been developed based on the use of biotinylated type 14 capsular polysaccharide (S14PS-biotin). A major advantage of this application over other methods is the use of 10-100-fold less antigen than that reported in the literature for other similar assays. Moreover, the prepared biotinylated polysaccharides are very stable and it is possible to use the same procedures for other pneumococcal polysaccharide antigens (e.g., S6BPS) with no major changes necessary in the ELISA and ELISPOT protocols. Furthermore, a simple thin layer chromatography method has been developed as a method for quality control of the biotinylated polysaccharide. Immunization with the thymus-independent antigen S14PS resulted in the induction of IgM spot-forming cells (SFC) and antibodies while S14PS-protein conjugates induced a thymus-dependent response. The immune response to the conjugates was enhanced by the addition of the adjuvant Quil A resulting in high levels of both IgG SFC and antibodies at day 14 after immunization. The developed assays are reliable and reproducible tools for studying the humoral immune response against Streptococcus pneumoniae type 14 capsular polysaccharide derived antigens.

Effect of in vivo administration of different adjuvants on the in vitro candidacidal activity of mouse peritoneal cells

The candidacidal activity (CA) of peritoneal cells (PC) in vitro was used as a measure of nonspecific microbicidal activity of phagocytes after intraperitoneal injection of mice with different adjuvants. Dilutions of PC were incubated with constant numbers of C. parapsilosis in a 96-well culture plate. The PC number causing 50% reduction of yeast colonies formed after 48 hr at 37 degrees C was called 1 CA50 unit. CA was expressed in CA50 units per 10(6) PC. Optimal reduction of the number of viable candida cells in vitro was established within 1.5 hr while 50% reduction was reached after 0.5 hr. In this test CA was, within limits, independent of the number of viable candida cells added per well (22 to 152 yeast cells), of the concentration of fetal calf serum (1-20%) and of the presence of heat-labile serum components. The CA of PC of individual mice was measured 6, 24, and 96 hr after injection of an adjuvant. In most instances optimal CA was observed 6 hr after administration of adjuvant and varied from 3.7 (methylamine) to 50 (Corynebacterium parvum strain 4982) units. With respect to the titer and duration of CA, the adjuvants were arranged in the following order of increasing efficacy: methylamine, heparin, polyol L 121, suramin, dextran sulfate, polyol L 101, dimethyldioctadecylammonium bromide, Liquoid, heat-killed Listeria monocytogenes, formalin-killed C. parvum strain 10387, and strain 4982. The CA induced by the latter strain persisted at least till 96 hr after injection. The induction of CA was accompanied by recruitment of polymorphonuclear cells. The contribution of distinct phagocytic effector cells to CA and the correlation between modulation of the specific and nonspecific immunity are discussed.

Immunomodulating Properties of Substances to Be Used in Combination with Liposomes

Liposomes haptenated with tripeptide-enlarged dinitrophenyl (DNP) are known to act as thymus-independent antigens which induce a strong IgM response and only limited amounts of circulating IgG. When haptenated liposomes are used in vaccination studies, it is of practical importance to improve the immunogenicity of these complexes. Therefore, an evaluation was made of the potency of various substances to modulate the immune response in such a way that the total antibody production is increased, including a relative great increase of 2-mercaptoethanol (2-ME)-resistant antibodies and immunological memory is induced. The following substances were used: glycophorin A (GP-A), sialogangliosides (monosialo-, disialo- and trisialoganglioside), 6-0-stearoyl-MDP (MDP-SA) and lipid A (lip A). Lip A incorporated into liposomes was the only substance inducing considerable increases of both total and 2-ME-resistant haemagglutination (HA) titre after immunization. Depending on the dose tested, the sialic-acid-containing protein GP-A had a small and varying influence on the serum antibody response. Sialogangliosides transiently decreased in a dose-dependent manner the total antibody titre in serum. In contrast to lip A, the lipophilic bacterial adjuvant MDP-SA did not influence HA titres significantly. The number of plaque-forming cells (PFC) in the spleen was enhanced considerably after both primary and secondary immunization with liposomes containing lip A. The other substances tested induced only minor differences of the number of PFC. To some extent, lip A induced immunological memory. In conclusion, it can be stated that of the agents tested, only lip A is a potent and consistent stimulator of the humoral immune response to liposomes haptenated with DNP groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Route-Dependent Immunomodulation: Local Stimulation by a Surfactant and Systemic Stimulation by a Polyanion

Immunomodulatory activity of the two synthetic adjuvants dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) in relation to route and time of injection was investigated in mice. Humoral responses to sheep red blood cells (SRBC) were measured as the number of direct anti-SRBC plaque-forming cells (PFC) in the spleen 5 days after immunization. Both adjuvants stimulated the anti-SRBC response if adjuvant and antigen were injected simultaneously via the same route (either intraperitoneally or intravenously). Administration of adjuvant and antigen via different routes (intraperitoneally or intravenously, respectively or vice versa) resulted in enhanced humoral responses after DXS, but not after DDA. Intraperitoneal immunization of mice which were injected intraperitoneally with either adjuvant 4 days earlier resulted in diminished humoral responses. Immune responses in pretreated mice were not suppressed when the antigen was injected intravenously instead of intraperitoneally. In conclusion, DDA and DXS differ in immunostimulating properties as DDA enhanced only a response to antigen injected via the same route whereas DXS induced a systemic state of increased immunoresponsiveness. The immunosuppressive state induced by intraperitoneal injection of either adjuvant prior to immunization is restricted to the peritoneal compartment. Mechanisms underlying differences between both adjuvants and aspects of systemic immunopotentiation are discussed.

Multivalent binding of toxin A from Clostridium difficile to carbohydrate receptors

A specific monoclonal antibody against toxin A from Clostridium difficile was generated that did not show thermolabile binding. Nonspecific murine monoclonal antibodies bound toxin A at 4 degrees C, but less effectively at 37 degrees C. Nonspecific human monoclonal antibodies did not bind to toxin A at 4 degrees C. Cytotoxic properties of purified toxin A were not inhibited by Bandeiraea simplicifolia lectin. This points to a carbohydrate moiety on the cell surface and a multivalent nonspecific carbohydrate binding ligand on toxin A.