Studies on hypersensitivity. II. Delayed hypersensitivity to denatured proteins in guinea pigs

The capacity of denatured proteins to provoke anaphylactic sensitivity and delayed sensitivity in guinea pigs has been investigated. It has been found that heat-denatured proteins are as effective as native proteins in provoking delayed hypersensitivity to either material, in spite of the fact that such denaturation greatly modifies their antigenicity from the point of view of antibody production. Pure delayed hypersensitivity, moreover, occurs regularly in response to extremely minute amounts of a good antigen, whether native or denatured.

It is further shown that immunological cross-reactivity can be demonstrated regularly by means of the delayed hypersensitivity reaction in guinea pigs, at a time when no cross-reacting antibodies of conventional type can be found.

The significance of these observations in regard to the relationship between delayed hypersensitivity and antibody production is discussed.

     

Delayed hypersensitivity to hapten-skin protein conjugates in guinea pigs sensitized to benzo(a)pyrene

Guinea pigs were sensitized to 3,4-benzo(a)pyrene, by epicutaneous application or by footbad injection in Freund's complete adjuvant. Conjugates of benzo(a)pyrene and guinea pig skin protein formed by ultraviolet radiation could elicit cutaneous delayed hypersensitivity and could inhibit the migration of macrophages obtained from guinea pigs sensitized to the carcinogen. Extracts of benzo(a)pyrene-treated guinea pig skin and conjugates formed in vitro with benzo(a)pyrene isocyanate were unable to consistently elicit delayed hypersensitivity reactions in vivo or in vitro. The results indicate a high degree of hapten-carrier specificity to contact sensitivity to benzo(a)pyrene.     

Studies on sperm antigenicity. I. Delayed hypersensitivity to spermatozoa

Delayed hypersensitivity to human and guinea pig sperm was demonstrated in guinea pigs of the Rockefeller strain by immunization with H₃₇Ra adjuvant. The reaction in vivo was demonstrated by skin testing the animals and in vitro by the capillary method. It was found that the sensitivity is not only directed towards the sensitizing antigen, but also shows cross-reactivity. Thus, peritoneal exudate cells derived from guinea pigs sensitized to human sperm were inhibited by guinea pig sperm. This cross-reactivity revealed the possibility of a tissue specific antigen. In addition, supernatants obtained after incubation of the sensitized lymph node cells with the specific antigen were found to be spermatotoxic.     

Antibody-mediated and delayed-type hypersensitivity reactions to Brucella skin test antigens in guinea pigs.

Cutaneous hypersensitivity responses to brucella antigens of different composition were studied in guinea pigs sensitized by infection with smooth brucella or immunization with killed rough brucella in adjuvant. These animals had circulating antibodies to smooth lipopolysaccharide or protein antigens, respectively. Intradermal skin tests, active cutaneous anaphylaxis, passive cutaneous anaphylaxis, and immunodiffusion tests were performed. Delayed-type hypersensitivity reactions uncomplicated by accompanying antibody-mediated reactions were seen only in infected guinea pigs with protein antigen that was entirely free of lipopolysaccharide. In the adjuvant-immunized animals, the protein antigen evoked overlapping antibody-mediated and delayed-type reactions. Lipopolysaccharide and polysaccharide preparations contained varying amounts of protein components. In infected animals, reactions of these antigens were clearly antibody mediated, but participation of delayed-type hypersensitivity could not be excluded. In adjuvant-immunized animals, the antibody-mediated reaction to the lipopolysaccharide preparation was caused by its protein component.     

Delayed hypersensitivity and acquired cellular resistance in guinea pigs infected with Listeria monocytogenes.

Randomly bred pigs of both sexes were injected intracardially with one-half of a 50% lethal dose of Listeria monocytogenes. When infected animals were skin tested with 30 mug of a water-soluble extract of sonically disrupted Listeria, both males and females had uniformly detectable levels of delayed hypersensitivity (DH) 4 days after infection. In males, cutaneous hypersensitivity to Listeria antigens reached a peak on day 5 or 6 of infection, and high levels of DH persisted through the 7th week. In females, DH reached a peak on day 6 or 7, remained at this level through the 4th week, and then dropped sharply. Cutaneous reactivity was usually higher for males than for females, and differences between the sexes were statistically significant 5, 6, and 7 weeks after infection. Low levels of DH were still present 41 weeks (females) or 46 weeks (males) after infection. Assays to determine the number of viable Listeria present in spleen homogenates indicated that bacterial multiplication occurred only during the first 24 hours of infection. The number of Listeria declined steadily thereafter, and by day 13 no bacteria could be recovered from the spleens of infected animals. Spleen assays indicated that Listeria-infected animals of both sexes were resistant to a small challenge dose of Listeria given 48 hours, 7 days, or 2 weeks after the primary infection. Resistance to re-infection was absent in females challenged at 41 weeks and in males challenged at 46 weeks.     

Delayed type hypersensitivity in guinea pigs infected subcutaneously with Naegleria fowleri Carter

Summary A soluble fraction, derived from Naegleria fowleri trophozoites disrupted by freeze-thawing, was tested for antigenic properties. Intradermal injections of this preparation were administered to guinea pigs previously infected subcutaneously with viable N. fowleri. Delayed hypersensitivity to the antigen and loss of weight, the diagnostic symptom of visceral naegleriasis, were observed in the surviving animals. Fifty percent of the guinea pigs, however, did not lose weight and had a reduced reaction to the antigen. The apparent differences in the immunocompetence of guinea pigs inoculated subcutaneously and intranasally with N. fowleri are compared.This report is a part of a thesis presented by Peter Diffley in partial fulfillment of the requirements designated by the Department of Zoology, University of Montana, for the M.A. degree.     

A high-molecular-weight trypsinlike protease in the skin sites of delayed hypersensitivity in guinea pigs.

A trypsinlike protease was extracted from the delayed hypersensitivity skin sites in guinea pigs. Extractable amounts of the enzyme were chronologically paralleled with the gross appearance of the inflammation, and the maximum activity from the inflamed sites at 24-36 hours was about 20 times stronger than that from normal skin, suggesting a potential role in the pathogenesis of the delayed hypersensitivity reaction. The enzyme, which suitably hydrolyzed t-butyloxycarbonyl-phenylalanyl-seryl-arginine 4-methylcoumaryl-7-amide, was partially purified by isoelectric focusing or by gel filtration. The enzyme demonstrated a single peak of activity on the former column with an apparent isoelectric point of 4.2, and in the latter it showed an apparent molecular weight of 600,000 (600K-protease). When incubated with 3H-diisopropylfluorophosphate, the enzyme lost all amidolytic activity and yielded a single band of radioactivity in polyacrylamide disk gel electrophoresis in the presence of sodium dodecyl sulfate, and a single peak of radioactivity in gel filtration, both having an apparent molecular weight of 31,000-33,000 (31K-protease). That the 600K-protease might be a complex with alpha 2-macroglobulin was ruled out. The 31K-protease was separated from the 600K-protease by gel filtration in the presence of 6 M guanidine hydrochloride, and was renatured to an active form. An apparent isoelectric point of the 31K-protease observed was 9.4, suggesting that the 600K-protease may be a complex of 31K-protease with an acidic carrier molecule(s). Both proteases, 31K- and 600K-protease, had identical substrate specificity, a pH profile of amidolytic activity, and susceptibility to exogenous protease inhibitors. However, when sensitivities to intrinsic protease inhibitors in guinea pig plasma, two kinds of trypsin inhibitor, and alpha 2-macroglobulin were compared, the 600K-protease was at least 100 times more resistant than the 31K-protease. It was supposed that one of the pathophysiologically significant functions of the complex formation might be to maintain the enzyme activity longer in vivo.     

Nasal mucosal hypersensitivity in guinea pigs intermittently exposed to cold.

To develop an animal model for experimental nasal hypersensitivity and hyperreactivity, guinea pigs were subjected to intermittent exposure to cold temperature (intermittent cold stress, SART stress) for 5 consecutive days. In SART-stressed guinea pigs, nasal mucosal hypersensitivity to histamine evoking sneeze response and nasal hypersecretion in response to methacholine were observed. The hypersensitivity remained for further 7 days after being released from SART stress. On the other hand, such nasal mucosal hypersensitivity was not caused by a continuous cold stress alone, suggesting that intermittent exposure to cold may be of importance for the appearance of nasal mucosal hypersensitivity. In passively sensitized SART-stressed guinea pigs, the quantity of nasal secretion induced by an allergen was significantly increased compared with that of a group of normal animals. The expression of muscarinic acetylcholine receptor (m-ACh.R) became higher in SART-stressed guinea pigs. Thus, hypersensitivity and hyperreactivity in this system were found to be associated with an increase in density of m-ACh.R. SART-stressed guinea pigs will serve as an animal model for hypersensitivity in nasal mucosa, which would be useful for the study of nasal allergy.     

Delayed hypersensitivity responses in mice and guinea pigs to Mycobacterium leprae, Mycobacterium vaccae, and Mycobacterium nonchromogenicum cytoplasmic proteins.

Antigenic relationships between Mycobacterium vaccae, M. nonchromogenicum, and M. leprae were examined in mice and guinea pigs injected with M. vaccae or M. nonchromogenicum suspensions. The growth of both organisms in outbred ICR and four inbred mouse strains was followed up to 30 days. M. nonchromogenicum persisted in the livers and spleens of the inbred mice substantially better than did the M. vaccae population in the same mouse strains. A translucent colony variant of M. vaccae isolated from the opossum survived in vivo better than the opaque colony isolated from opossums and cattle. Persistence of M. vaccae and M. nonchromogenicum was not markedly increased in T-cell-depleted (nude) mice. Normal mice infected with increasing numbers of M. vaccae did not develop delayed-type hypersensitivity to the homologous M. vaccae cytoplasmic protein antigen. When heat-killed M. vaccae were incorporated into Freund adjuvant, both mice and guinea pigs developed delayed hypersensitivity to cytoplasmic antigens prepared from M. vaccae, M. nonchromogenicum and M. vaccae vaccines cross-sensitized guinea pigs to the M. leprae cytoplasmic antigens.     

Delayed hypersensitivity to myxoviruses and myxovirus vaccines in the guinea pig

Influenza, mumps and measles viruses were examined for their ability to induce in guinea pigs homologous and cross-reactive delayed hypersensitivity. A majority of the animals skin tested with homologous and a portion of the animals skin tested with heterologous viruses and vaccines developed positive reactions. Findings with the heterologous preparations suggest that the observed cross-reactive hypersensitivity might be due to shared antigens of viral or substrate origin in the influenza, mumps and measles preparations. The present findings in guinea pigs suggest that the adverse effects, attributed in whole or in part to induced hypersensitivity, observed in man following injection of killed influenza, mumps and measles vaccines could be due to cross-reactive delayed hypersensitivity resulting from the use of these preparations.     

Relationship between intracellular calcium and airway reactivity in guinea pigs.

The present study was carried out to examine the relationship between intracellular free calcium ion concentrations and its regulatory enzymes, sodium potassium adenosine triphosphatase (Na(+),K(+)-ATPase) and calcium adenosine triphosphatase (Ca(2+)-ATPase), with airway reactivity to inhaled histamine in guinea pigs. Forty-nine guinea pigs were included in this study. Of these, 34 animals responded to histamine bronchoprovocation challenge in vivo with a greater than 35% fall in specific airways conductance and were labeled as "reactive," and the remaining 15 were "nonreactive." The dose of histamine producing a 35% fall in specific airways conductance was labeled as ED(35) SGaw. The animals were then sacrificed, and the following biochemical measurements were carried out: intracellular free calcium ion concentrations [Ca(2+)](i) in leukocytes and isolated tracheal smooth muscle cells, activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase in tracheal homogenate, and plasma levels of lysophosphatidylcholine (LPC). Reactive guinea pigs showed significantly higher [Ca(2+)](i) and Na(+),K(+)-ATPase and Ca(2+)-ATPase activities. Airway reactivity (ED(35) SGaw) had significant negative correlation with [Ca(2+)](i), with activities of each of the ATPases and with plasma lysophosphatidylcholine. It is concluded that the level of [Ca(2+)](i) is an important determinant of airway reactivity. Intracellular calcium levels modulate airway response to histamine with higher levels being associated with greater reactivity.     

Circulatory basophilia in guinea pigs with delayed-type hypersensitivity

Circulatory basophilia could be induced in inbred guinea pigs systematically immunized with ovalbumin and consequently provoked repeatedly with dissolved ovalbumin applied onto the mucosa of the nares or the outer eye. The degree of the increase in circulatory basophil granulocytes depended on the adjuvant used and was significantly more pronounced after immunization with Freund's complete adjuvant than with alhydrogel (A1(OH)3). The degree of basophilia was also dependent on the animal strain, but different in two strains selected for high-asthma trait.