Delayed Hypersensitivity, Macrophage Migration, and Antibodies in Guinea-Pigs Immunized with Diphtheria Toxoid

Guinea-pigs were immunized with diphtheria toxoid (DT) in Freund's complete adjuvant (FCA) and boosted three times with DT in saline. Boosting increased anti-DT passive haemagglutinins, haemolysins, and cytophilic antibody on the surface of peritoneal exudate cells, but skin reactions to DT became negative and peritoneal cell migration was no longer inhibited by DT. When normal peritoneal cells were incubated in the sera of boosted animals, their migration was not inhibited by antigen, although they had cytophilic antibody on their surface as shown by rosette formation.     

The Effect of an Unrelated Delayed-Type Hypersensitivity Reaction on the Antibody Response to Diphtheria Toxoid

Four Lf fluid diphtheria toxoid were injected intracutaneously, together with tuberculin PPD, into guinea-pigs sensitized to tuberculin by a water-in-oil emulsion of killed tubercle bacilli 4–5 months previously. Antibody production, judged by antibody levels at 4 weeks, was two to four times greater than that in control guinea-pigs. Such an increase in antitoxin would correspond to a ten- to twelve-fold increase in the effective dose of toxoid, which is much greater than any likely non-specific retention at the injection site. It is suggested that the effect of a local tuberculin reaction in enhancing the immunogenic capacity of diphtheria toxoid is due to an increase in the number of immunologically competent cells to which the toxoid has access.     

Antibodies Produced by Rabbits Immunized with Visna Virus

Immunization of New Zealand White rabbits with purified visna virus elicited antibody activity demonstrated by passive hemagglutination (PHA), complement fixation (CF), and indirect immunofluorescent tests. The antibody activities of hyperimmune sera and Sephadex G-200 fractions of the sera were studied. It was found that the PHA test was 10 to 100 times more sensitive than the CF test in detecting visna antibodies in rabbits. It was also found that the immunoglobulin M fractions from Sephadex G-200 filtration displayed greater PHA activity than did the immunoglobulin G fractions. Although neutralizing antibody was demonstrated in the serum of the natural host (sheep), our attempts to demonstrate neutralizing antibody in the sera from hyperimmunized rabbits (non-natural host) so far have failed.     

Delayed Hypersensitivity Reactions Provoked by Ribosomes from Acid-Fast Bacilli I. Ribosomal Isolation, Characterization, Delayed Hypersensitivity, and Specificity

Ribosomes and ribosomal subunits of Mycobacterium bovis (strain BCG) and M. smegmatis have been isolated and employed as skin test antigens in guinea pigs sensitized with homologous or heterologous organisms. Ribosomes and ribosomal subunits were found to be potent antigens for skin test purposes, and the 30S subunits were found to be more specific and active than the 50S subunits.     

The Macrophage Disappearance Reaction (MDR) as an in Vivo Test of Delayed Hypersensitivity in Mice

The macrophage disappearance reaction (MDR), as an in vivo analogue of in vitro tests for delayed hypersensitivity, was utilized in mice immunized with M. tuberculosis or mouse thyroid extract (MTE). The optimal schedule for the induction of macrophages in peritoneal exudates and the optimal concentration of antigen for the MDR were determined. Macrophage disappearance occurred 4 hr after immunized mice received an intraperitoneal injection of 200 μg of soluble antigen. The MDR was found to be antigen specific. Intraperitoneal injection of an unrelated antigen or tissue culture medium alone did not cause macrophage disappearance; however, the re-injection of the antigen used for immunization caused a 70-80% reduction of macrophages. Macrophage disappearance was greater in mice immunized with M. tuberculosis than in mice immunized with MTE. Comparison of the MDR with the footpad test in these 2 groups showed that mice immunized with M. tuberculosis developed a higher degree of delayed hypersensitivity than the group immunized with MTE. These results demonstrate that the MDR represents a specific and quantitative method for detecting the delayed type of cellular immune response in mice.     

The Macrophage Disappearance Reaction (MDR) as an in Vivo Test of Delayed Hypersensitivity in Mice

The macrophage disappearance reaction (MDR), as an in vivo analogue of in vitro tests for delayed hypersensitivity, was utilized in mice immunized with M. tuberculosis or mouse thyroid extract (MTE). The optimal schedule for the induction of macrophages in peritoneal exudates and the optimal concentration of antigen for the MDR were determined. Macrophage disappearance occurred 4 hr after immunized mice received an intraperitoneal injection of 200 μg of soluble antigen. The MDR was found to be antigen specific. Intraperitoneal injection of an unrelated antigen or tissue culture medium alone did not cause macrophage disappearance; however, the re-injection of the antigen used for immunization caused a 70-80% reduction of macrophages. Macrophage disappearance was greater in mice immunized with M. tuberculosis than in mice immunized with MTE. Comparison of the MDR with the footpad test in these 2 groups showed that mice immunized with M. tuberculosis developed a higher degree of delayed hypersensitivity than the group immunized with MTE. These results demonstrate that the MDR represents a specific and quantitative method for detecting the delayed type of cellular immune response in mice.     

Effect of Encephalitogenic Protein, PPD, and Tetanus Toxoid on Leukocyte Migration in Agarose

The reactivity to three antigens: bovine encephalitogenic protein (EP), PPD, and tetanus toxoid, was studied with blood leukocytes from healthy humans using Clausen's (5) leukocyte migration in agarose technique. There is an obvious correlation between the reactivity to EP (all concentrations studied) and to low concentrations of PPD; and between the reactivity to low concentrations of EP and low concentrations of tetanus toxoid. After vaccination with tetanus vaccine, a marked increase in reactivity to the toxoid sometimes occurred: at the same time, a marked reactivity to EP appeared. Various explanations are discussed: a true immunological cross-reactivity, that the correlations are due to a variability in individual response with respect to lymphokine production, and that BCG and tetanus vaccinations produce an adjuvant effect increasing a pre-existing low reactivity to EP.     

Eosinophils in Delayed Hypersensitivity Skin Reaction Sites

Guinea pigs immunized with egg albumin or an azobenzene arsanate conjugate of acetyl-tyrosine (ABA-tyr) in complete or incomplete Freund's adjuvant can develop eosinophil-containing delayed hypersensitivity skin reactions when challenged with specific antigen. Retest, achieved by the re-injection of antigen at the site of a prior skin test, leads to an accelerated reaction which may contain increased numbers of eosinophils. ABA-tyr which induces delayed hypersensitivity without antibody formation, dissociates the two components of the retest reaction; that antigen leads to an accelerated reaction, but one with only small numbers of eosinophils.     

Leucocyte Migration Inhibition Test and Tuberculin Hypersensitivity

Tuberculin hypersensitivity was evaluated in 71 Mantoux-positive and 58 Mantoux-negative persons by the leucocyte migration inhibition test. The capillary lube technique discriminated these two groups, bill variability of results and overlapping of the groups were considerable. The shorter the incubation period, the better discrimination between the groups. Only after short incubation (2 h) was the correlation between the intensities of Mantoux reactions and migration inhibitions evident. The observed variability in migration areas, similar in the Mantoux-positives and controls, was the result of technical causes and variations in cells' ability to migrate. Further, in Mantoux-positive persons the migration progression (per min) increased during the first 2 h, but slowed down later on, more in controls than in chamber with antigen. Smaller migration areas in controls (early intervals) tended to produce lower migration indices than the larger ones. Finally, it was possible, using the standard score c, to calculate whether each individual result differed significantly from controls. By this procedure the intensity of cell-mediated immunity in individual patients could be estimated.     

Leucocyte Migration Test and Hypersensitivity to Glafenin

The leucocyte migration test (LMT) was performed on 20 patients with an intolerance to glafenin--a non-narcotic analgesic drug. LMT was found to be positive in 50% of the subjects with intolerance, a highly significant percentage as compared with the control groups. HSA-glafenin was found to be the most appropriate method for presenting the antigen, but glafenin and its hydroxylated metabolites were only found to induce a migration inhibition in the subjects intolerant to glafenin.     

The Loss of Macrophages from Peritoneal Exudates following the Injection of Antigens into Guinea-Pigs with Delayed-Type Hypersensitivity

Exudates were induced in the peritoneal cavities of guinea-pigs by the injection of glycogen. The cell content of these exudates was examined after 4 days in normal and hypersensitive animals. In animals uninjected with antigens, the exudates contained a high proportion of macrophages, together with lymphocytes and polymorphs. In BCG-vaccinated animals, with delayed-type hypersensitivity to tuberculin, subcutaneous, intravenous or intraperitoneal injection of tuberculin resulted in a profound fall in the macrophage content of the exudates. This effect was apparent within an hour of intraperitoneal injection and occurred with very small doses of tuberculin. No such effect occurred after the intraperitoneal injection of tuberculin into guinea-pigs with Arthus hypersensitivity to tuberculin. Ovalbumin injected intraperitoneally into guinea-pigs with mixed delayed-type and Arthus hypersensitivity to ovalbumin also resulted in a marked fall in the macrophage content of peritoneal exudates, but had no effect on the peritoneal macrophages of animals with pure Arthus hypersensitivity. Bacterial endotoxin injected intraperitoneally caused a similar fall in the macrophage content of exudates of both normal and BCG-vaccinated animals. It is concluded that this loss of macrophages from peritoneal exudates after the injection of antigen is the consequence of an immunological reaction which is a manifestation of a state of delayed-type hypersensitivity.     

Localization of the Antigen and Antibodies in the Placenta of Immunized Sheep

ABSTRACT: Placentomata of sheep immunized with human serum albumin (HSA) were examined. Both HSA and immunoglobulins were found in the maternal part and maternofetal border of the placenta using FITC labelled antisera on paraffin sections. Radiolabelled HSA was also detected in the fetal blood. The ultrastructure of placentomata revealed immunopathological process.     

In Vitro Correlates of Delayed Hypersensitivity in Man: Ambiguity of Polymorphonuclear Neutrophils as Indicator Cells in Leukocyte Migration Test

Delayed cutaneous hypersensitivity (DCH) of 12 normal adult subjects to purified protein derivative (PPD) of Mycobacterium tuberculosis, streptococcal streptokinase-streptodornase (SK-SD), and Candida albicans Dermatophytin O (DO) was assayed in vivo by skin testing and compared with such in vitro correlates of cellular immunity as lymphocyte transformation (LT) and inhibition of leukocyte migration (ILM) from microcapillary tubes or in agarose gel. LT was shown to be the best in vitro correlate of specific lymphocyte sensitization with all antigens. In the ILM assays, PPD showed good correlation with in vivo DCH and in vitro LT; SK-SD showed partial correlation; DO showed no correlation, not being active in any of the ILM tests. Cell distribution and morphology of stained migration patterns, ILM tests performed on separated populations of lymphocytes and polymorphonuclear leukocytes (PMN), as well as the ability of test antigens to stimulate PMN cells to reduce nitroblue-tetrazolium dye, indicated that in ILM tests mononuclear cells were not inhibited in their migration, whereas migration of PMN cells appeared to depend on their direct reaction with the test antigens.     

The Fate of [131I]Labelled Antigens in the Skin of Normal Guinea-Pigs and those with Delayed-Type Hypersensitivity

A comparison was made of the retention of three antigens PPD (protein purified derivative of tuberculin), HSA (human serum albumin) and HGG (human γ globulin) labelled with ¹³¹I in the skin of normal guinea-pigs. It was found that PPD was eliminated more rapidly than HSA or HGG in the first 24 hours after skin test. In contrast PPD was found to be more readily adsorbed to collagen and cellular tissues in vitro.

The retention of these antigens at 24 hours in normal and sensitized guinea-pigs was also compared. It was found that PPD was retained in the tuberculin lesion as compared with normal skin, whereas HSA was eliminated more rapidly from a delayed-type hypersensitivity lesion to HSA when compared with normal skin. It was concluded that retention of PPD was probably not specific to delayed-type hypersensitivity but was related to the difference in character and severity of the inflammatory process, which itself was related to the chemical nature of the antigen involved.