Bennett G. Smith

Immune interferon induction by T-cell mitogens involves different T-cell subpopulations

Abstract Recent studies of mitogen-induced DNA synthesis in cells from various lymphoid tissues indicate that certain mitogens may selectively activate specific T-cell subpopulations. Staphylococcal enterotoxin A (SEA) optimally stimulates thymocytes and spleen cells (presumed T1 cells) and phytohemagglutinin P (PHA) optimally stimulates lymph node and spleen cells (presumed T2 cells); concanavalin A (Con A) stimulates cells from all these sources well. To determine further the specificity of mitogens for T-cell subpopulations, immune interferon (IIF) production was studied in spleen cells from mice treated in vivo with cortisone acetate (CA), preferentially a T1-cell inactivator, and antithymocyte serum (ATS), preferentially a T2-cell inactivator. The effect of administering gallic acid (3,4,5 trihydroxy-benzoic acid) (GA) in vivo was also studied, since in vitro studies showed GA to be capable of blocking IIF production. Results indicate that SEA induces IIF primarily in T1 cells, PHA induces IIF primarily in T2 cells, and Con A induces IIF in both T1 and T2 cells. Furthermore, GA was shown to mimic the ability of CA to inactivate T1 cells selectively in vivo . The data indicate that more than one type of T-cell subpopulation is capable of producing IIF.

Use of an in vitro Antibody-Producing System for Recognizing Potentially Immunosuppressive Compounds

An in vitro primary antibody-forming system was used to detect immunosuppressive compounds, some of which were food additives and/or metabolites of food additives. The system used was found to be specific with regard to chemical inhibitors and advantageous because of the cellular interactions required for an antibody response.

Inhibitory effect of an anti-oxidant, butylated hydroxyanisole, on the primary in vitro immune response.

Summary Butylated hydroxyanisole (BHA), an anti-oxidant food additive, inhibited the primary in vitro antibody (PFC) response of C57B1/6 spleen cells to both a thymus-dependent antigen (SRBC) and a thymus-independent antigen (E. coli 0127:B8); the PFC response of athymic nude spleen cells to E. coli 0127:B8 was inhibited to the same degree. BHA inhibited both B- and T-lymphocyte function. BHA needed to be present only for the first 4 hr of culture to exert its inhibitory effect on the PFC response. DNA synthesis in C57B1/6, athymic nude, and T-cell mito-gen-induced C57B1/6 spleen cell cultures was inhibited by BHA; however, short-term exposure of cultures to BHA at certain PFC inhibitory concentrations resulted in stimulation of DNA synthesis. The mechanism of the inhibitory effect of BHA on the in vitro PFC response is unknown, but may possibly involve activation of regulatory cell activity. Certainly, the data warrant further study of the effect of this ubiquitous food additive on the immune response.

Suppression of macrophage-dependent T-lymphocyte function(s) by gallic acid, a food additive metabolite.

Summary At concentrations as low as 5 × 10−6 M, gallic acid (GA), a metabolite of the food additives propyl gallate and tannic acid, suppressed the anti-sheep erythrocyte (SRBC) plaque-forming cell (PFC) response of C57B1/6 mouse spleen cells when added to cultures as late as 48 hr after antigen addition. GA-induced suppression was reversed by 5 × 10-5 M 2-mercaptoeth-anol (2ME) added at the same time as, or up to 48 hr after, antigen and was reversed less efficiently by adherent-cell supernatant (ACS). GA also suppressed mitogen-in-duced DNA synthesis of C57B1/6 T lymphocytes, and this suppression was reversed by 2ME. GA had no effect on the response of athymic nude mouse spleen cells to the thymus-independent antigen Escherichia coli 0127:B8, and failed to suppress lipo-polysaccharide (LPS)-induced B-lympho-cyte DNA synthesis. The data suggest that GA selectively suppresses a macrophage (MØ)-dependent T-lymphocyte function(s).

Inhibitory effect of synthetic polyribonucleotides on the primary in vitro immune response.

Summary The synthetic double-stranded polyribonucleotides, poly (rA):poly (rU) and poly (rl):poly (rC), were shown to be potent inhibitors of the in vitro plaque-forming cell (PFC) response to a thymus-dependent (SRBC) and thymus-independent (E. coli 0127 LPS) antigen in mouse C57BL/6 spleen cell cultures. The same polynucleotides had no effect on the PFC response of nude (athymic) mouse spleen cells to E. coli 0127 LPS, suggesting that functional T lymphocytes are necessary for the inhibitory effect. Enhancement effects were modest and inconsistent in the cultures. Poly (rA) and poly (rU) were ineffective as inhibitors. The data indirectly suggest that the inhibition may be due to the early production of interferon by functional T lymphocytes.

Restoration of immunoregulation in splenic lymphocyte populations of mice fed reduced dietary protein

Abstract Moderate, acute reduction of dietary protein in young mice often leads to increases in humoral immune responses. To test the hypothesis that reduction of dietary protein affects T lymphocyte regulation of the humoral immune response, in vitro humoral immune responses to sheep erythrocytes (SRBC) and bromelain-treated mouse erythrocytes (BrMRBC), were examined in BDF 1 and BALB/c mice fed diets low in protein (4% or 6% casein) compared with well-fed (20% casein) controls. In addition, the immunoregulatory splenic T lymphocyte subset profile was evaluated in mice fed the 4% and 20% casein diets. Groups of female BDF1 and BALB/c mice 5 weeks of age were fed one of the three diets for 5 weeks. Mice fed the 4% casein diet exhibited less body weight and splenic lymphocyte number than mice from the other two dietary groups. BDF 1 but not BALB/c mice fed the 4% casein diet exhibited significantly increased in vitro and in vivo immune responses to optimal or high doses of SRBC. On the other hand, both BDF 1 and BALB/c mice fed the 4% casein diet exhibited a significantly higher immune response to BrMRBC. The increased response of 4% casein-fed BDF 1 mice was related to an inability to generate adequate specific immunoregulatory T cells involved in suppressing the response. Co-culturing with Lyt1 + or Lyt2 + T cells from well-fed mice regulated downwardly the enhanced autoimmune, anti-BrMRBC response of mice fed the 4% casein diet. Decreased suppressor T lymphocyte activity in BDF 1 mice, but not BALB/c mice fed the 4% casein diet was confirmed by the significant depression of the Lyt2 + suppressor T cell number in the spleen evaluated by direct immunofluorescence using monoclonal antibodies which identify T lymphocyte subsets. These results indicate that moderate, acute reduction of dietary protein in young mice affects the regulatory functions of T cells but not the differentiation of B cells to antibody-forming plasma cells. This effect seems to be dependent upon the strain of mouse used.

Tartrazine: Quantitative passive hemagglutination studies on a food-borne allergen of small molecular weight☆

Abstract The quantitative hemagglutination migration procedure was adapted for the measurement of the food-borne allergen, tartrazine (FD&C Yellow No. 5). Tartrazine and sulfanilic acid are structurally and haptenically related, so the migration system was developed with azo sulfanilate coupled to sheep red blood cells and with antisera to azo sulfanilate. Tartrazine was a more effective inhibitor of the system than was sulfanilic acid. The system was most appropriately represented by the empirical mathematical model Log 10 Y = β 0 + β 1 X + ϵ , where Y = mm cell migration, X = serum concentration, β 0 and β 1 = regression coefficients, and ϵ = the residual error.

Haptenic relationships of p-azobenzenesulfonate and some structurally-related food dyes.

Abstract Tartrazine, an FD&C approved azo dye, has been implicated in food and drug-borne allergic reactions. The dye was coupled to bovine serum albumin (BSA) by a water-soluble carbodiimide and by bis-diazotized benzidine. Both conjugates, as immunogens in rabbits, elicited the production of antibodies specific for tartrazine. The antibodies cross-reacted with other FD&C dyes, the extent of the reaction depending on the structural relationship of these dyes to tartrazine. Tartrazine is structurally and haptenically related to p -azobenzenesulfonate. Antibodies to tartrazine, however, are serologically unique. The tartrazine-BSA conjugates should be useful in skin-test studies for tartrazine allergies and in immunoassays for the dye.

Serological Specificity of Types A and B Botulinal Toxins and Antitoxins

Summary Rabbit, National Communicable Disease Center (NCDC) equine, and International Standard antitoxins to types A and B botulinal toxins were compared by passive hemagglutination (HA), hemagglutination inhibition (HI), and gel-diffusion. The HA reactions in general were quite specific, although cross-reactions were observed. The extent of cross-reaction was greater with B antitoxins and A botulinal toxoid-sensitized red blood cells. The different antitoxins behaved similarly in HI and gel-diffusions, though the reactions of the NCDC and International antitoxins were weaker than those of rabbit antitoxins in gel-diffusions. Hemagglutination inhibition and gel-diffusion reactions with toxic cultures suggested that the in vitro specificity of the A toxin-antitoxin did not involve neutralizing antibodies. Hemagglutination inhibitions and gel-diffusions with B toxins did not resolve the question of the involvement of neutralizing antibodies in in vitro specificity, since fairly toxic cultures inhibited HA and formed bands believed to be associated with type specificity, whereas a virtually non-toxic B culture neither inhibited HA nor formed a line of identity with the type specific systems in gel-diffusion.

Tartrazine: Solid-Phase Radioimmunoassay Studies of an Azo Dye Implicated in Allergic Reactions (Azo Dyes and Allergy)

Summary A solid-phase radioimmunoassay procedure was adapted for the haptenic study of tartrazine, an azo dye implicated in various forms of allergy. Further, the haptenic relationship of tartrazine and aspirin was investigated, since sensitivity of individuals to the two substances is often clinically associated. The specificity of antibody to tartrazine was directed strongly toward a pyrazolone intermediate of the molecule, 1-(4-sulfophenyl)-3-carboxy-5-hydroxy-pyrazole. Aspirin did not cross-react with anti-tartrazine, suggesting that the clinical association of aspirin and tartrazine sensitivity in patients is a nonimmunological phenomenon.