Role of epitope density in the induction of immunity and tolerance with thymus-independent antigens. IV. Selective tolerance and IgE response by DNP-levan conjugates.

Tolerance induction of IgE antibody-forming cells by dinitrophenylated levan (DNP-LE) conjugates was investigated in CBA mice. Antibody production was measured by passive coutaneous anaphylaxis on rat skin. Specific antibody neutralization was obtained with conjugates of different substitution degrees. Specific inhibition of IgE antibody production was obtained only with highly substituted conjugates. However, the epitope density required for inducing tolerance to antibody synthesis of the IgE class was lower than for other classes of immunoglobulins. The tolerance induced by DNP-LE was shown to affect only the anti-DNP B cells of IgE class without T cell participation. These results demonstrate that B cell precursors of all Ig classes are susceptible to tolerance induction and not to triggering by T-independent antigens.     

Molecular Basis of B-Cell Activation

The present experiments were performed in an attempt to investigate the nature of the surface receptor on B lymphocytes responsible for triggering of these cells. B-cell mitogenicity of unsubstituted dextrans, quantitated by activation of DNA and antibody synthesis was detected only if the ligand had a MVC higher than 7 × 10⁴. Above this threshold mitogenicity increased linearly with the log of the MW Substitution of the polymeric structure with lipid residues did not result in increased mitogenicity of the conjugate. However, sulphate substitution of the sugar units greatly enhanced the ability of the conjugates to activate DNA synthesis and. to a much smaller extent, antibody synthesis. Mitogenicity of sulphate derivatives was independent of their MW. Another polyanionic derivative (carboxymethyl) did not show increased mitogenicity. whereas a low-MW compound very similar to dextran sulphate (pentosan sulphate) was highly active. The activation induced by dextrans was immunologically nonspecific and caused induction of polyclonal antibody synthesis. The activated cells presumably belong to a subset of B cells at a rather premature stage of differentiation. These findings suggest that the mitogenic signal is delivered to the cells by single sites at the membrane. These sites appear to have the capacity to interact with polysaccharide structures or releated conformations The polymeric structure of the active ligands is not a necessary requirement for mitogenicity and seems to have the accessory function of providing multipoint binding to low-affinity receptors.     

Mitogenic activities of synthetic lipid A analogs and suppression of mitogenicity of lipid A.

The effect of synthetic lipid A analogs on murine spleen cells was studied. The preparations represented D-glucosamine and D-glucosaminyl-beta 1,6-D-glucosamine disaccharide derivatives substituted in different combinations by ester- and amide-bound fatty acids and by phosphate groups. Significant mitogenic activity was demonstrated with a number of synthetic disaccharide preparations; however, their potency was lower than that of lipid A. The synthetic preparations were not mitogenic for spleen cells from C3H/HeJ mice. Furthermore, the mitogenicity of the synthetic preparations was abolished after binding with polymyxin B. The results indicate that for expression of mitogenicity, a phosphate group at position 1 of the reducing glucosamine and amide-bound acyloxyacyl residues are important factors. Some of the synthetic preparations containing the diglucosamine backbone and expressing relatively low mitogenicity suppressed B-cell mitogenicity of lipid A. Although these preparations were lytic for erythrocytes, they did not affect the viability of the splenic lymphocytes. Suppression was seen when the synthetic preparations were added simultaneously with or after the lipid A mitogen, but optimal suppression was expressed when the preparations were added to the system 3 h before lipid A. Washing of the cells before the addition of lipid A did not affect the results. The suppression was not due to the induction of suppressor cells by the synthetic preparations. The disaccharide preparations did not inhibit T-cell mitogenicity of concanavalin A. In contrast to the disaccharide preparations, the monosaccharide preparations suppressed mitogenicity of both lipid A and concanavalin A, probably because of their direct toxicity for lymphocytes.     

Tobacco constituents are mitogenic for arterial smooth-muscle cells.

Tobacco glycoprotein (TGP) purified from flue-cured tobacco leaves, tar-derived material (TAR), the water soluble, nondialyzable, delipidized extract of cigarette smoke condensate, rutin-bovine serum albumin conjugates, quercetin, and chlorogenic acid are mitogenic for bovine aortic smooth-muscle cells, but not adventitial fibroblasts. The mitogenicity appears to depend on polyphenol epitopes on carrier molecules. Ellagic acid, another plant polyphenol, inhibited arterial smooth-muscle proliferation. These results suggest that a number of ubiquitous, plant-derived substances may influence smooth-muscle cell proliferation in the arterial wall.     

Mitogenic effect of Bartonella bacilliformis on human vascular endothelial cells and involvement of GroEL

Bartonellae are bacterial pathogens for a wide variety of mammals. In humans, bartonellosis can result in angioproliferative lesions that are potentially life threatening to the patient, including bacillary angiomatosis, bacillary peliosis, and verruga peruana. The results of this study show that Bartonella bacilliformis, the agent of Oroya fever and verruga peruana, produces a proteinaceous mitogen for human vascular endothelial cells (HUVECs) that acts in a dose-dependent fashion in vitro with maximal activity at >or=72 h of exposure and results in a 6- to 20-fold increase in cell numbers relative to controls. The mitogen increases bromodeoxyuridine (BrdU) incorporation into HUVECs by almost twofold relative to controls. The mitogen is sensitive to heat and trypsin but is not affected by the lipopolysaccharide inhibitor polymyxin B. The mitogen does not affect caspase 3 activity in HUVECs undergoing serum starvation-induced apoptosis. The Bartonella mitogen was found in bacterial culture supernatants, the soluble cell lysate fraction, and, to a lesser degree, in insoluble cell fractions of the bacterium. In contrast, soluble cell lysate fractions from closely related B. henselae, although possessing significant mitogenicity for HUVECs, resulted in only about a twofold increase in cell numbers. Biochemical and immunological analyses identified GroEL as a participant in the observed HUVEC mitogenicity. A B. bacilliformis strain containing the intact groES-groEL operon on a multicopy plasmid was generated and used to demonstrate a correlation between HUVEC mitogenicity and GroEL levels in the lysate (r(2) = 0.85). Antiserum to GroEL significantly inhibited mitogenicity of the lysate. Data also show that GroEL is located in the soluble and insoluble fractions (including inner and outer membranes) of the cell and is actively secreted by B. bacilliformis.     

Ganglioside and monosaccharide inhibition of nonspecific lymphocyte mitogenicity by group A streptococcal pyrogenic exotoxins.

Group A streptococcal pyrogenic exotoxins (SPE) types A, B, and C are potent nonspecific lymphocyte mitogens. The mitogenicity of these exotoxins was inhibited by gangliosides and sialic acid, whereas concanavalin A was unaffected. The capacity of both concanavalin A and SPE-A to stimulate lymphocytes was suppressed by alpha-methyl-D-mannopyranoside. Galactose reduced the activity of SPE-C. The sugars, glucose, N-acetylglucosamine, alpha-methyl-D-glucopyranoside, and fucose, did not affect SPE mitogenicity.     

Relationship between biological activities of polymers. I. Immunogenicity, C3 activation, mitogenicity for B cells and adjuvant properties.

The effect of thirteen compounds on C3 activation, mitogenicity for B lymphocytes and on the immune response was tested. Only two of the compounds tested, polyinosinic acid and a high molecular weight polyethylenesulphate induced C3 conversion, while eleven out of the thirteen compounds (negative charged immunogenic or non-immunogenic polymers including the three C3 active compounds) stimulated thymidine incorporation in B lymphocytes. The non-charged (T-independent) immunogens dextran and polyvinylpyrrolidone neither activated C3 nor BP lymphocytes. With the exception of single-stranded polyribonucleic acids, the compounds stimulating DNA synthetis in B cells enhanced the response of the mice to a suboptimal dose of sheep red blood cells. In conclusion C3 activation, intrinsic (T-independent) immunogenicity and B-cell stimulation are not correlated properties. However, B-cell mitogenicity and adjuvanticity seem to be related.     

Inhibition of B-Cell Function by Concanavalin-A-Induced Suppressor Cells

The in vitro immune response of normal spleen cells to the thymus-independent antigen dinitrophenylated levan (DNP-LE) was found to be inhibited by suppressor cells induced previously by concanavalin A (Con A) in vitro. At various suppressor to target cell ratios, the level of suppression observed was comparable to that seen in anti-sheep erythrocyte (SRBC) responses. Consequently, it is suggested that B cells constitute a direct target for Con-A-induced suppressor cell activity.     

A comparison of specificity and biological activity of polyclonal and monoclonal antibodies raised against Salmonella minnesota R595 lipopolysaccharide

Murine monoclonal antibodies (MAbs) and immune rabbit serum were raised against the rough mutant of Salmonella minnesota strain R595. These antibodies were tested for their ability to inhibit LPS-induced B-cell mitogenicity and neutralise LPS toxicity in chick embryos. Immune rabbit serum inhibited both mitogenicity and LPS lethality. None of the MAbs or a cocktail of antibodies were able to neutralise LPS lethality in chick embryos. However, they were able to inhibit mitogenicity by varying degrees.     

Novel hyperactive mitogen to endothelial cells: human decidual NKG5

PROBLEM: The purpose of this study was the isolation and characterization of decidual extract proteins that exhibit mitogenicity on endothelial cells. METHOD OF STUDY: A partially purified extract (F1 fraction) was obtained from human decidua of the first trimester of pregnancy. F1 was separated by heparin-sepharose column and showed significant mitogenicity on bovine brain capillary endothelial (BBCE) cells in vitro, using methylene blue stain nuclear assay. Sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed four fractions with MW less than 30 KDa. RESULTS: Mitogenic fraction, E11-12 (eluted at 1.5 M NaCl) was identified as basic fibroblast growth factor (bFGF). Vascular endothelial growth factor (VEGF) and acidic (a)FGF were not identified as one of the mitogenic fractions. However, fractions E5-6, and E7-8 induced statistically significant mitogenicity at concentration of 1 pg/mL, which is 10(3) times lower than bFGF. Sequencing of E5-6 fraction identified NKG5 protein, a putative secreted polypeptide derived from human natural killer (NK) cells and activated T cells of unknown function. CONCLUSION: This work demonstrates that NKG5 stimulates mitogenicity of endothelial cells and may be involved in angiogenesis.     

Mitogenicity of a lipid-deficient lipoprotein from a mutant Escherichia coli strain.

From the mutant bacterial strain Escherichia coli JE5511 lpp lpm, muropeptide-containing and muropeptide-free lipoproteins were prepared. By gas chromatography and by infrared spectroscopy we showed that the products were deficient in the two ester-bound N-terminal fatty acids, but still carried the amide-linked fatty acid. Mutant lipoproteins were tested for mitogenicity in lipopolysaccharide nonresponder C3H/HeJ mice by incorporation of [3H]thymidine and [3H]uridine and by hemolytic plaque assays for immunoglobulin-secreting plasma cells. Our results showed that the mutant lipoproteins still exhibited marked mitogenicity toward mouse B-lymphocytes, although the activity of the products was reduced in comparison to the wild-type lipoprotein. Thus, the presence of one fatty acid in the N-terminal part of lipoprotein is sufficient to bring about mitogenicity.     

Use of rabbit Fab'-peroxidase conjugates prepared by the maleimide method for detecting plant viruses by ELISA

In contrast to antibodies conjugated to enzyme with glutaraldehyde or by the periodate method, monomeric Fab' fragments conjugated to enzyme by means of a maleimide compound are not adversely affected by the conjugation procedure. We used such Fab'-enzyme conjugates prepared with antibody to tobacco mosaic virus (TMV), to TMV coat protein and to rabbit IgG for the detection of different tobamoviruses by direct and indirect double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Compared to conjugates prepared by other methods, the sensitivity of TMV detection with Fab'-enzyme conjugates by direct DAS-ELISA was markedly increased. However, because of their monomeric nature, these FAb'-enzyme conjugates did not cross-react with serologically related tobamoviruses. Anti-globulin Fab'-enzyme conjugate was found to be the most efficient anti-globulin conjugate for detecting TMV by indirect DAS-ELISA. Because of their high sensitivity and serotype specificity, FAb'-enzyme conjugates are useful for detecting low amounts of contaminating viruses present in crude viral preparations.     

Differential proliferative responses of cultured Schwann cells to axolemma and myelin-enriched fractions. II. Morphological studies

Summary Axolemma-enriched and myelin-enriched fractions were prepared from bovine CNS white matter and conjugated to fluorescein isothiocyanate (FITC). Both unlabelled and FITC-labelled axolemma and myelin were mitogenic for cultured rat Schwann cells. Treatment of Schwann cells with the FITC-labelled mitogens for up to 24 h resulted in two distinct morphological appearances. FITC-myelin-treated cells were filled with numerous round, fluorescent-labelled intracellular vesicles, while FITC-axolemma-treated cells appeared to be coated with a patchy, ill-defined fluorescence, primarily concentrated around the cell body but extending onto the cell processes. These observations were corroborated under phase microscopy. Electron microscopy revealed multiple, membrane-bound, membrane-containing phagosomes within myelin-treated cells and to a far lesser extent in axolemma-treated cells. The effect on the expression of the myelin-mediated and axolemma-mediated mitogenic signal when Schwann cells were treated with the lysosomal inhibitors, ammonium chloride and chloroquine, was evaluated. The mitogenicity of myelin was reduced 70–80% by these agents whereas the mitogenicity of axolemma was not significantly altered under these conditions. These results suggest that axolemma and myelin stimulate the proliferation of cultured Schwann cells by different mechanisms. Myelin requires endocytosis and lysosomal processing for expression of its mitogenic signal; in contrast, the mitogenicity of axolemma may be transduced at the Schwann cell surface.     

Comparison of the Mitogenic Activities of Streptococcal Protein-G and Staphylococcal Protein-A on Human Mononuclear Cells

In the present work we report data from experiments comparing the proliferative stimuli demonstrated by Streptococcal Protein-G and Staphylococcal Protein A on human peripheral blood mononuclear cells. Protein-G and Protein-A are presented to the cells in solution as well as linked to plastic or Sepharose beads, or incorporated within the cell wall of whole bacteria. The cellular response is measured by incorporation of 3H-thymidine in 72 hr cultures.

The soluble and the immobilized forms of Protein-A, but not those of Protein-G, displayed high mitogenicity. Possible explanations for the absence of Protein-G induced mitogenicity are discussed.     

Comparison of the Mitogenic Activities of Streptococcal Protein-G and Staphylococcal Protein-A on Human Mononuclear Cells

In the present work we report data from experiments comparing the proliferative stimuli demonstrated by Streptococcal Protein-G and Staphylococcal Protein A on human peripheral blood mononuclear cells. Protein-G and Protein-A are presented to the cells in solution as well as linked to plastic or Sepharose beads, or incorporated within the cell wall of whole bacteria. The cellular response is measured by incorporation of 3H-thymidine in 72 hr cultures.

The soluble and the immobilized forms of Protein-A, but not those of Protein-G, displayed high mitogenicity. Possible explanations for the absence of Protein-G induced mitogenicity are discussed.     

Are mitogenic sera necessary for the generation of human plaque-forming cells?

Human plaque-forming cells (PFC) have been quantitated following pokeweed mitogen stimulation by a protein A plaque technique using human erythrocytes. The PFC are generated in cultures supplemented with non-mitogenic human AB serum of foetal bovine serum (FBS) of varying degrees of inherent mitogenicity. In the latter case, no correlation exists between the mitogenicity of the serum supplement used in these cultures and the PFC response obtained. This fact, in addition to the demonstrated usefulness of the AB sera in facilitating the generation of PFC makes it unlikely that serum mitogenicity is a requirement for in vitro human immunoglobulin synthesis and secretion.     

Low biological activity of Helicobacter pylori lipopolysaccharide.

Lipopolysaccharide from the gastroduodenal pathogen Helicobacter pylori was tested for its ability to induce mitogenicity in mouse spleen cells, pyrogenicity in rabbits, and toxic lethality in galactosamine-sensitized mice. Compared with those for enterobacterial lipopolysaccharide, mitogenicity and pyrogenicity were a thousand-fold lower and lethal toxicity was 500-fold lower. We suggest that the phosphorylation pattern and acylation in lipid A are responsible for the low biological activity.     

Hypersensitivity reactions to acetylsalicylic acid. I. Detection of antibodies in human sera using acetylsalicylic acid attached to proteins through the carboxyl group

Aspirin–protein conjugates (aspirin coupled to rabbit anti-human `O' antibody and to anti-bacteriophage-Fab') were prepared by a method which links aspirin through its carboxyl group. The protein conjugates were used to raise specific aspirin antibodies in guinea-pigs. Such antibodies helped to characterize the other preparations which were subsequently used to test suspected hypersensitive sera for aspirin antibodies. Although an antibody of aspirin specificity was detected it could not be related to the clinical hypersensitivity state.     

Comparison of Techniques and Immunoreagents Used for Indirect Immunofluorescence and Immunoperoxidase Identification of Enteroviruses

Peroxidase-conjugated antibodies were found to be as sensitive as those conjugated to fluorescein-isothiocyanate (FITC) for identification of selected enterovirus types by the indirect technique. Peroxidase conjugates, however, were found to give fewer nonspecific reactions. The reasons for the higher specificity of the immunoperoxidase technique appear to be related to the relative size, charge, and uniformity of the preparations. Monomers of peroxidase-conjugated globulin are larger than those of FITC conjugates, but the latter readily form aggregates. This was shown by chromatography on Sepharose 6B columns: various FITC-conjugated globulins eluted before those conjugated to peroxidase and before (125)I-labeled immunoglobulin G. The net charge of the conjugates was determined by adsorption to ion-exchange columns. FITC-labeled globulins had a negative net charge, eluting at pH 5.1 from diethylamino-ethyl-Sephadex A-50 columns. Peroxidase conjugates were not retained by either cationic or anionic exchangers at pH values ranging from 4.0 to 10.5. Further, the fluorescein/protein ratios of FITC conjugates from different commercial sources and of those prepared in the laboratory were found to be variable; higher fluorescein/protein ratios (>2:1) give a higher degree of nonspecific reactions, whereas the peroxidase/protein ratio does not appear to affect specificity. These characteristics of peroxidase conjugates make the immunoperoxidase technique easier to standardize and more reliable for enterovirus identification than the immunofluorescence technique.     

Immunological specificity of chloramine-T-induced IgE antibodies in serum from a sensitized worker

A procedure for the preparation of chloramine-T (CT) conjugates used to assay IgE antibodies was developed using response surface methodology and serum from a subject occupationally exposed to the substance. The conjugates, synthesized by reacting CT with human serum albumin (HSA) and other protein carriers, were used as antigens in a radio-allergosorbent test (RAST). Human serum albumin was found to be a suitable carrier, although other protein carriers also gave specific IgE-binding of a similar extent. The CT-HSA conjugates used in the RAST were characterized by high performance liquid chromatography, electrophoresis, immunodiffusion and ammonium sulphate precipitation. However, no strong correlation was seen between the ability of the conjugates to bind IgE and their physical or immuno-chemical properties. The hapten and carrier specificity of CT-induced IgE antibodies in the subject's serum were studied by direct RAST and RAST inhibition. No existence of new antigenic determinants related to the carrier could be demonstrated. Although HSA as a carrier was altered immunochemically by CT, the IgE antibodies were found to be specific to hapten only. Chloramine-T-specific IgG antibodies could not be demonstrated in the subject's serum.