Benjamin Prescott

Separation of immunomodulatory effects of mannan from Candida albicans into stimulatory and suppressive components

Mannan extracted from Candida albicans was studied for its immunomodulatory activity on in vivo antibody responses to type III pneumococcal polysaccharide (SSS-III), a helper-T-cell-independent antigen, and to sheep erythrocytes (SRBC), a helper-T-cell-dependent antigen. In some studies, the antibody response to SSS-III was converted to a helper-T-cell-dependent response by attaching it to a carrier (horse erythrocytes, HRBC); this complex then was used to immunize mice primed with a subimmunogenic dose of HRBC. Mannan enhanced the antibody response to both SSS-III and SRBC when administered at the same time or 1 or 2 days after immunogen. However, when both mannan and SSS-III were coated onto HRBC for immunization, either enhancement or suppression was noted; the effect depended upon the amount of mannan used. Larger amounts stimulated, whereas smaller amounts suppressed, the antibody response to SSS-III. The enhancing and suppressive components of mannan could be separated by molecular size or charge by chromatography on Sepharose 4B or on DEAE-Sephadex A-50 columns, indicating that mannan extracts contain individual components having opposing immunomodulatory properties. These components can be separated on the basis of molecular size and charge.

IMMUNOLOGICAL PARALYSIS TO TYPE III PNEUMOCOCCAL POLYSACCHARIDE AS ASSESSED BY AN IMMUNO‐PLAQUE PROCEDURE

Two mechanisms have been proposed to explain the development of immunological paralysis or tolerance to pneumococcal polysaccharides. These deal with whether paralysis is the result of the neutralization of antibody by persistent undegraded antigen (the “treadmill” hypothesis) ,l or whether a suppression of antibody synthesis, i.e., a central failure of the immune mechanism, is involved.z These proposed mechanisms are not necessarily mutually exclusive and persuasive evidence has been advanced to support both points of view.l> 3-7 However, in order to evaluate fully the significance of either process in the development of paralysis, more precise quantitative information is required concerning the antibody response to pneumococcal polysaccharides at the cellular level. Recently, we developed an adaptation of the technique of localized hemolysisin-gel that permits one to determine not only the number of antibody-forming cells produced in response to pneumococcal polysaccharides but also the rate at which antibody is synthesized and released by such cells.x,9 By means of this method we have been able to show that while the neutralization of antibody by excess antigen may be a factor in the development of paralysis to large doses of pneumococcal polysaccharides, a central failure of the immune mechanism clearly plays a significant, if not a dominant, role.

Antiviral activity of a fraction of abalone juice.

Conclusion The present experiments indicate that the antibacterial and antiviral activities of untreated abalone juice are readily separable by ion exchange chromatography.

Strain differences in the ability of antithymocyte serum (ATS) to enhance the antibody response of inbred mice to type III pneumococcal polysaccharide

Abstract Treatment with antithymocyte serum (ATS), prepared in burros or rabbits, significantly enhanced the antibody response to Type III pneumococcal polysaccharide (SSS-III) in 5 inbred and 2 hybrid strains of mice. The degree of enhancement attained depended upon the dose of ATS employed. Strains of mice differed with respect to the amount of enhancement produced following treatment with a given dose of the same preparation of ATS.

The role of antigen in the activation of regulatory T cells by immune B cells

The transfer of B cells from mice immunized with Type III pneumococcal polysaccharide (SSS-III) results in the activation of suppressor and amplifier T cells that control the magnitude of the antibody response in recipient mice, immunized subsequently with SSS-III. Prior treatment of transferred B cells with an excess of enzyme (polysaccharide depolymerase) capable of hydrolyzing SSS-III, does not alter the capacity of these cells to activate regulatory T cells. These findings indicate that the activation of regulatory T cells by immune B cells is not mediated by residual antigen on the surface of transferred cells.

Electron microscopy of solubilized Acholeplasma laidlawii membrane proteins reaggregated with Mycoplasma pneumoniae glycolipids

Abstract 1. 1. The purified glycolipid haptens of Mycoplasma pneumoniae were reaggregated with Acholeplasma laidlawii membrane proteins. The process consisted of the solubilization of lipid-depleted A. laidlawii membranes and M. pneumoniae glycolipids in 20 mM sodium dodecyl sulfate, and dialysis of the solution separately or in mixtures against 20 mM Mg2+. 2. 2. The reaggregated material collected by centrifugation of the dialyzed solution of lipid-depleted A. laidlawii membrane proteins consisted of amorphous clumps, while the reaggregated M. pneumoniae glycolipids consisted of “myelin-like” globules and sheets composed of lamellae with a mean center-to-center distance of 37 A. The reaggregated material of a mixture of lipid-depleted A. laidlawii membrane proteins and M. pneumoniae glycolipids contained, in addition to the amorphous clumps representing reaggregated proteins and the “myelin-like” structures representing reaggregated glycolipids, also long membranous sheets having a triple-layered structure with a mean center-to-center distance of the dense lines of 54 A. The appearance and dimensions are closely similar to those of the original or reagregated A. laidlawii membranes. 3. 3. It is suggested that these membrane-like structures are formed by the association of A. laidlawii membrane protein and M. pneumoniae glycolipids, and that these “hybrid” structures are responsible for the increased antigenicity of the reaggregated glycolipids.

SEROLOGICAL AND IMMUNOGENIC ACTIVITIES OF DIFFERENT FRACTIONS OF MYCOPLASMA PNEUMONIAE

Several serological tests have been recently developed for the study of M . pneumoniae. The test methods involve complement-fixation, indirect-hemagglutination, gel-diffusion and growth-inhibition (tetrazolium reduction-inhibition) . The aim of this study was to determine the nature of various antigens of M . pneumoniae which react in these different serological procedures. In addition, we were interested in defining the nature of the antigens of this organism which stimulate antibodies measurable by the different serological techniques. Such information would have relevance to our basic understanding of this important respiratory tract pathogen and to the future preparation of purified vaccines containing only those antigens which stimulate protective antibodies. It should be emphasized that our major interest was in recovering serologically active fractions and not in describing the chemical composition of the organism. M . pneumoniae, strain FH, grown in broth medium was fractionated by chemical procedures and by chromatography on Sephadex G 2 5 and GlOO and on thin-layer silica gel. After sonication at lOKC for one hour, the sediment of the M . pneumoniae suspension was fractionated by chemical methods while the supernatant fluid was fractionated by Sephadex G25 chromatography. All fractions were lyophilized and tested for serological and immunogenic activity as 1 % solutions or suspensions in phosphate buffered saline (PBS) . Complement-fixation (CFT) ,l indirect-hemagglutination (IHA) ,2 growth-inhibition (tetrazolium reduction-inhibition [TRI]3) and gel-diffusion tests4 were performed as described previously. In addition, various fractions of M . pneumoniae were tested for their capacity to block the effect of indirect-hemagglutinating and growth-inhibiting antibody. A convalescent human serum and a hyperimmune rabbit serum were used to measure the serologic activity of the various fractions of M . pneumoniae. These sera contained high titers of specific antibodies and lacked both antibodies against growth medium constituents and antibodies against other human mycoplasma species, except for a low titer of complement-king and indirect-hemagglutinating antibodies for M . salivarium in the rabbit antiserum. The rabbit serum was used at a dilution at which heterotypic reactivity could not be demonstrated. To study the immunogenicity of different fractions of M . pneumoniae, rabbits and guinea pigs were inoculated twice intradermally with a mixture of fraction and adjuvant. The method used for chemical fractionation of the sediment of the sonicated organism suspension is shown in TABLE 1. This procedure yielded one lipid fraction, three protein fractions and four polysaccharide fractions. The supernatant fluid from the sonicated organism suspension yielded four fractions following chromatography on Sephadex G-25 (TABLE 2 ) . The first SG-25 fraction was rechromatographed on Sephadex GlOO and one major frac-

Lack of involvement of auto-anti-idiotypic antibody in the regulation of oscillations and tolerance in the antibody response to levan

Abstract Bacterial levan (BL) induces a cyclic (oscillatory) antibody response in both euthymic and athymic BALB/c mice. Significant numbers of plaque-forming cells (PFC) making auto-anti-idiotypic antibody directed against a major cross-reactive idiotype expressed on antibodies specific for BL were detected in euthymic—but not athymic—mice. This suggests that auto-anti-idiotypic antibody, the formation of which requires the participation of mature T cells, does not play a decisive role in generating the cyclic patterns observed. However, such antibody might still influence the proportion of PFC making antibody of complementary idiotype. No relationship between the suppressive property of auto-anti-idiotypic antibody and either the induction or maintenance of immunological tolerance to BL was evident.

Chemical Studies of Paolin II, an Antiviral Substance from Oysters

Summary An antiviral thermostable substance (paolin II) is isolated from oysters by extraction with acetic acid followed by fractional precipitation with ethanol. The substance shows one peak on ultracentrifugation, has a sedimentation constant of 1.6-1.8 and a molecular weight of approximately 10,000. The material yields 16 amino acids on hydrolysis with a composition of 8.6% nitrogen and 23.4% carbohydrate, indicating the mucoprotein nature of the substance. The authors are indebted to A.M. Young, Division of Biologies Standards, Laboratory of Biophysics and Biochemistry, for his technical assistance with the sedimentation studies.

Lectin-induced modulation of the antibody response to type III pneumococcal polysaccharide

Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG1 antibody response was noted after the administration of Con A whereas profound enhancement of IgG2a antibody response was noted after PHA was given.