Modification of delayed-type hypersensitivity reactions by muramyldipeptide in guinea pigs

Abstract N -Acetylmuramyl- l -alanyl- d -isoglutamine (muramyldipeptide, MDP) modulated delayed-type hypersensitivity (DTH) reactions and induced severe inflammatory lesions in guinea pigs. The animals immunized with heat-killed Mycobacterium tuberculosis were challenged with the purified protein derivative (PPD) at the flanks and the corneas to prepare DTH reactions at 2 weeks after the immunization, thereafter 24 h the animals received subcutaneous injections of MDP at the flanks of the opposite side. At the skin with the DTH reaction, increase of swelling and redness accompanied with hemorrhage and necrosis were observed. As corneal reactions in the animals that had received MDP, increase of cornea thickness, opaque and grayish-white and the projection of eyes accompanied with severe iritis were observed. Modification of the skin reaction occurred from 2 h after the MDP injection, rapidly increased to the maximum level around 10 h, maintained the level until 24 h, then slowly decreased. The polymorphonuclear leukocyte infiltration was observed from 15 min after the MDP injection, and tumor necrosis factor alpha, interleukin (IL)-1, and IL-6 levels in the serum and skin lesions increased after the MDP injection. Synthetic muramyltripeptide ( N -acetylmuramyl- l -alanyl- d -isoglutaminyl- l -lysine) also provoked definite skin reactions, while the larger peptidoglycan fragments and various inflammatory agents including cytokines so far examined were inactive in this respect. Cortisone and heparin inhibited definitely and slightly the reaction, respectively. A comparison was made with the modified DTH reaction and the necrotic reactions which we reported previously.     

Endotoxin Induces Severe Inflammatory Reactions with Necrosis at Sites Primed with Delayed-Type Hypersensitivity Reactions in Guinea Pigs

Guinea pigs immunized with Freund's complete adjuvant received challenge injection of the purified protein derivative of Mycobacterium tuberculosis in the flanks and the corneas to prepare delayed-type hypersensitivity (DTH) reactions. The animals were injected subcutaneously with lipopolysaccharide (LPS) or a synthetic lipid A (LA-15-PP). At the skin site primed with DTH reaction, increased swelling and hemorrhagic reaction followed by a definite necrotic reaction occurred. Severe corneal reactions were also observed in the animals. These findings indicate that bacterial endotoxin modulates DTH reactions and induces severe inflammatory reactions.     

Immunoadjuvant activities of synthetic 6-O-acyl-N-acetylmuramyl-L-alanyl-D-isoglutamine with special reference to the effect of its administration with liposomes

Addition of a lauroyl, stearoyl or docosanoyl group to the primary hydroxy group at the C-6 position of N-acetylmuramyl-L-alanyl-D-isoglutamine gave lipophilic derivatives that had definite adjuvancies in induction of delayed-type hypersensitivity and enhancement of antibody production against a test protein antigen, ovalbumin, when administered to guinea pigs as liposomes, that is without mineral oil. When administered as mineral oil-in-water emulsion, including Ribitype emulsions, rather than as water-in-mineral oil emulsions, N-acetylmuramyl-L-alanyl-D-isoglutamine and its 6-O-acyl derivatives showed only weak immunoadjuvancies.     

Delayed hypersensitivity and granulomatous response after immunization with protein antigens associated with a mycobacterial glycolipid and oil droplets.

A myocardial glycolipid (P3) mixed with protein antigens in oil-in-water emulsion induced lasting delayed hypersensitivity (DH) and granulomatous inflammation after intradermal injection into guinea pigs. This did not occur when P3 and bovine serum albumin (BSA) were given in Freund's incomplete adjuvant. The oil-in-water emulsions consisted of microscopic oil droplets suspended in aqueous medium. By separating oil and aqueous phases from BSA + P3 emulsion it was shown that antigen retained with oil droplets led to DH and granuloma formation. The association of antigen with oil droplets was P3 dependent and was quantitated with 125I-labeled BSA. The same phenomenon occurred with 125I-labeled rabbit gamma-globulin (RGG) + P3 emulsion. Fluorescein-conjugated RGG was observed in a particulate state within or on oil droplets in emulsion containing P3. These physical characteristics of antigen + P3 emulsion appeared to be important for immunogenicity.     

Induced sensitization to nickel in guinea pigs immunized with mycobacteria by injection of purified protein derivative with nickel

Nickel has been reported to be one of the most common causes of allergic contact dermatitis. Despite the fact that nickel is a frequent sensitizer in humans, establishing animal models for nickel allergy has met with considerable difficulties. In clinical cases, allergic contact hypersensitivity to nickel develops much more readily in inflamed skin than normal skin. In this study, we tried to induce nickel sensitization when inflammation has been evoked in guinea pigs immunized with mycobacteria followed by co-administration of a mycobacterial component with nickel. We first examined the delayed-type hypersensitivity (DTH) reaction of mycobacterial components such as the cell wall, cell membrane, 70S ribosomal fraction, cytoplasm, tuberculin purified protein derivative (PPD), RNA and DNA from Mycobacterium bovis BCG in guinea pigs immunized with live M. bovis BCG or heat killed M. tuberculosis. When PPD was used, the hypersensitivity reaction was strongest. Next, we tested whether PPD with nickel could induce nickel sensitivity in guinea pigs immunized with mycobacteria. Strong sensitization to nickel was achieved by injecting PPD with nickel. However, if too large an amount of PPD or nickel salts was used, sensitization to nickel decreased. In this way, sensitization of nickel developed much more easily in guinea pigs immunized with mycobacteria by injection of an appropriate amount of nickel at the inflammation site induced by a suitable amount of PPD.     

Allergenicity of Mycobacterial Ribosomal and Ribonucleic Acid Preparations in Mice and Guinea Pigs

Guinea pigs were injected subcutaneously with mycobacterial ribosomal fraction incorporated in Freund's incomplete adjuvant and tested 6 and 12 weeks later by the intradermal injection of 0.5 μg (25 TU) of Purified Protein Derivative. No evidence of delayed-type hypersensitivity could be detected in these animals, although large necrotic reactions were obtained in guinea pigs sensitized with living, attenuated mycobacterial cells. Mice also were vaccinated by the intraperitoneal injection of mycobacterial ribosomal fraction or ribonucleic acid (RNA) and tested for sensitivity to tuberculin at various subsequent times. No evidence of true tuberculin hypersensitivity could be detected at any time, although what appeared to be small Arthus type reactions were seen in mice given the largest vaccinating doses. Attempts to recall tuberculin sensitivity in vaccinated mice by the intravenous injection, 4 weeks after vaccination of living cells, of either the virulent or attenuated mycobacterial strains were unsuccessful. Instead, when the virulent cells were injected, a suppression of footpad reactivity was noted in animals made sensitive to tuberculin by the previous intraperitoneal injection of viable attenuated mycobacterial cells. Both guinea pigs and mice, vaccinated as described above, were also skin tested or footpad tested, respectively, with 2 μg of the ribosomal fraction or RNA used for vaccination. No evidence of true tuberculin hypersensitivity could be obtained; instead, in guinea pig skin very small dermonecrotic areas were noted, and in mice swelling and redness of the footpad occurred to an equal extent in both vaccinated and nonvaccinated mice. The possible role of tuberculin hypersensitivity in acquired immunity to tuberculosis is discussed, and the conclusion is reached that its part, if any, is minor.     

Further study of the problem of specific cellular receptors in delayed hypersensitivity]

The work presents the materials obtained as a result of the further study of specific T lymphocyte receptors with the use of so-called receptor antisera, i. e. antisera against the lymphoid cells of mice sensitized with one of the two antigens (Macobacterium tuberculosis or bovine gamma globulin); thus these differed from control antisera against the lymphoid cells of intact mice. These mouse antisera reacted with the lymphoid cells of guinea pigs in experiments of delayed-type hypersensitivity transfer. The cells of sensitized guinea pigs lost their ability to transfer hypersensitivity if, prior to their injection into the recipient guinea pigs, these cells were treated with the above-mentioned mouse antisera, i. e. antisera against the lymphoid cells of mice had a blocking effect on the lymphoid cells of guinea pigs. The blocking action of the antisera proved to be specific: antisera against the lymphoid cells of mice sensitized to bovine gamma gloublin blocked the cells of guinea pigs, also sensitized to bovine gamma globulins, but did not block the cells sensitized to Mycobacterium tuberculosis. The control antisera, taken in the same doses as the factor antisera, did not show a blocking effect on the specific activity of lymphoid cells.     

Pathological study of Streptococcus faecalis antigen-induced arthritis in New Zealand white rabbits

The pathological examination of the rabbit knee joint with antigen-induced arthritis produced by heat-killed Streptococcus faecalis (Str. faec.) as antigen was carried out. Macroscopically, there were findings of acute inflammation about five hours after the injection. Histopathologically, very remarkable acute exudative inflammation was seen 48 hours later. This supported the picture of Arthus reaction. The Arthus reaction disappeared with time, and this supported the view of delayed-type hypersensitivity three weeks later. After that, an obvious chronic inflammation was admitted in 10 weeks. This resembled the histopathological feature of rheumatoid arthritis (RA) when these findings are summarized. It was suggested that arthritis produced by Str. faec. progresses from an acute condition to a one with time. As mentioned above, it thought that Arthus reaction of both immediate hypersensitivity and delayed-type hypersensitivity are necessary in the occurrence of this arthritis.     

CLA reduces antigen-induced histamine and PGE(2) release from sensitized guinea pig tracheae

Conjugated linoleic acid (CLA) has been shown to enhance immune reactions such as lymphocyte blastogenesis and delayed-type hypersensitivity. We investigated the role of CLA in type I (immediate) hypersensitivity, using a guinea pig tracheal superfusion model for measuring antigen-induced airway smooth muscle contraction and inflammatory mediator release. Female Hartley guinea pigs were fed a diet supplemented with 0.25 g corn oil or linoleic acid/100 g of diet (control) or 0.25 g CLA/100 g of diet for at least 1 wk before and during active sensitization to ovalbumin antigen. Tracheae from sensitized guinea pigs were suspended in air-filled water-jacketed (37 degrees C) tissue chambers in a superfusion apparatus. Tracheae were superfused with buffer containing antigen, and tissue contraction was recorded. Superfusate was collected at 90-s intervals for evaluation of histamine and PGE(2) release. CLA did not affect antigen-induced tracheal contractions when expressed as gram contraction per gram tissue. CLA significantly reduced antigen-induced histamine and PGE(2) release. CLA appears to decrease release of some inflammatory mediators during type I hypersensitivity reactions.     

Evolutionary Divergence in the Structure of Myelin Basic Protein: Comparison of Chondrichthye Basic Proteins with Those from Higher Vertebrates

Abstract: A basic protein has been purified from the CNS myelin of the gummy shark (Mustelus antarticus). Electroblotting was used to examine the capacity of rabbit antisera raised against this electrophoretically pure protein to recognize myelin basic protein from higher vertebrates. The antisera bound to two shark proteins including the original polypeptide antigen and to chicken, bovine, and human myelin basic proteins. Thus, the shark protein appeared to possess antigenic determinants that have been retained through evolutionary divergence of these proteins. Whereas bovine basic protein caused experimental allergic encephalomyelitis in guinea pigs, animals that received injections of the shark protein showed neither clinical nor histological signs of this disease. However, tests for delayed-type hypersensitivity and for Arthus reaction following injection with the shark protein revealed a T-cell-mediated response to this antigen and substantial cross-reactivity with higher vertebrate basic proteins. Analysis of the amino acid composition of the shark protein, and comparison of its tryptic peptide map with that of the bovine protein, revealed substantial changes in the amino acid sequence. Although the shark protein has some antigenic determinants in common with the proteins from higher vertebrates, it appears that much of the structure differs.     

Suppression and reversal of allergic encephalomyelitis in guinea pigs with a non-encephalitogenic analogue of the tryptophan region of the myelin basic protein.

The administration of synthetic peptide S42 leads to suppression and reversal of experimental allergic encephalomyelitis (EAE) induced in guinea pigs by myelin basic protein. Peptide S42 contains a linear sequence of 21 amino acid residues, H-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Gly-OH, made up of four repeating unit sequences of H-Phe-Ser-Trp-Gln-Lys-OH in addition to a C-terminal glycine. Injected at relatively high doses, peptide S42 is non-encephalitogenic. It induces delayed-type hypersensitivity which is not followed by EAE, and elicits delayed-type hypersensitivity responses in peptide S42, encephalitogenic trytophan peptide, or BP-challenged animals for either of the three antigens. The repeating unit sequence of peptide S42 is analogous to the encephalitogenic tryptophan region of the BP molecules . The sequence homology is responsible for cellular recognition of this antigen by the skin test assay and suggests in vivo interaction between peptide S42 and EAE-inducing cells leading to suppression and reversal of disease.     

Detection of hypersensitivity toLegionella pneumophila in guinea pigs by skin test

Cross-reacting antigens of three serogroups ofLegionella pneumophila differed serologically and immunologically from the serogroup-specific antigens. Intradermal injection of cross-reacting antigens into sensitized guinea pigs evoked skin hypersensitivity. Animals were sensitized either by injection of inactivatedL. pneumophila in adjuvant or by infection with live organisms. Skin reactions were measurable about 2–4 h after injection and continued to increase in intensity for the first 24 h, followed by a gradual decline over the next 48 h. Histological examination of skin reactions taken from test sites at 48 h revealed infiltration of mononuclear cells in and about the small subcutaneous blood vessels and throughout the dermis, compatible with a delayed-type reaction. The overall appearance and time course of the reaction resembled a combination of immediate and delayed types of hypersensitivity. Each cross-reacting antigen of the three serogroups evoked skin reactions in animals which had been sensitized to any of those serogroups, but was not reactive in nonsensitized animals. These observations indicate the possibility of detecting present or past infection ofL. pneumophila by skin tests.     

Properties of TAS-1D3, a Tuberculin-Active Substance from BCG,in Regard to Delayed Hypersensitivity

The properties of TAS-1D3, a tuberculin-active substance purified from the cell extract of Mycobacterium bovis BCG, were studied in vivo and in vitro. In the delayed hypersensitivity skin reaction, TAS-1D3 showed far more potent activity than tuberculin purified protein derivative (PPD) in guinea pigs sensitized with BCG vaccine. This was consistently observed from 6 to 24 weeks after sensitization. The histological findings of the skin reaction to TAS-1D3 were similar to those of the reaction to PPD. Moreover, TAS-1D3 induced well both thymidine incorporation and the production of migration inhibitory factor (MIF) by the spleen cells from guinea pigs sensitized with BCG vaccine. In contrast, TAS-1D3 showed weaker activity than PPD in guinea pigs sensitized with either heat-killed M. tuberculosis Aoyama B or heat-killed M. tuberculosis H37Ra, and it weakly stimulated the spleen cells from animals sensitized with M. tuberculosis Aoyama B to incorporate thymidine and to produce MIF.     

Pathological observations in experimental candida infection of sensitized guinea pigs

Guinea pigs immunized intramuscularly with heat-killed or viable Candida albicans were infected intracutaneously with C. albicans. Animals with negative delayed hypersensitivity against C. albicans antigen showed similar lesions with non-immunized controls. Delayed hypersensitivity-positive guinea pigs, which were detected in the animals immunized with heat-killed C. albicans in CFA and IFA, demonstrated a delay of the resolution of the inflammatory tissue reaction and, in the animals immunized with C. albicans in CFA, developed a granuloma.These results suggest that both humoral and cell-mediated immunities do not play a significant role for protection against candidiasis and at a late stage of infection, cell-mediated immunity may play a secondary role of the enhancement of resistance to candida infection associated with granuloma formation.     

Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern.

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitro sensitized T lymphocytes of guinea pigs immunized with living BCG. The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.     

Tuberculin Activity of Mycobacterial Fractions Obtained by Chromatography

Commercial purified protein derivatives (PPD), old tuberculin (OT), the bacillary extract, and the culture filtrate of Mycobacterium tuberculosis H37Ra were submitted to Sephadex G-25 and diethylaminoethyl (DEAE)-cellulose chromatography. The ability of the fractions obtained to elicit delayed dermal hypersensitivity in M. tuberculosis H37Ra-infected guinea pigs was studied. Skin tests with Sephadex fractions in M. tuberculosis H37Ra-infected guinea pigs showed that the tuberculin activity was localized in the first fraction. All other Sephadex fractions were nonessential and nonspecifically irritating. Fractions from chromatography of Sephadex G-25 fraction 1 on DEAE-cellulose columns showed that all but the first were able to elicit delayed hypersensitivity reactions. There was a variability in the capacity to elicit the tuberculin reaction according to the fraction injected and the stage of tuberculous infection in guinea pigs. Compared to the others, the seven lots of commercial PPD were variable in composition and content. They contained both essential and nonessential materials for the tuberculin reaction. Sephadex fraction 1 would appear to be a better tuberculin as it excludes nonessential nonspecifically irritating elements and contains the complement able to elicit the tuberculin reaction. Its methodological simplicity would be economically advantageous.     

Comparison of adjuvants with Leishmania antigens in a guinea pig model to induce delayed-type hypersensitivity responses.

BACKGROUND AND PURPOSE: Guinea pigs have been a traditional model for studies of delayed-type hypersensitivity. They are the natural host of Leishmania enriettii and have been experimentally infected with other species of Leishmania. They have been used as a skin-test model to screen potential antigens for use in diagnostic tests for Leishmania. Use of complete Freund's adjuvant (CFA), along with whole promastigote Leishmania antigen, was necessary to sensitize guinea pigs to invoke a sufficient cell-mediated immune response. However, use of CFA has come under scrutiny by Animal Care and Use Committees due to the pathologic changes associated with its use. METHODS: Thirty-two specific-pathogen-free male Hartley guinea pigs were inoculated with Leishmania antigens alone or mixed with one of three adjuvants (CFA, TiterMax, and liposomes), and were skin tested 2 weeks later. RESULTS: For the Leishmania antigens tested, guinea pigs that received liposomes as an adjuvant had skin-test responses comparable to those of guinea pigs that received CFA. TiterMax was also tested, but cellular responses at antigen test sites were poor. CONCLUSIONS: Liposomes can be used in this model as a safe, effective adjuvant.     

Production of a Mycobacterium avium ssp. paratuberculosis purified protein derivative (PPD) and evaluation of potency in guinea pigs.

A Mycobacterium avium ssp. paratuberculosis purified protein derivative (PPD) was produced and the biologic activity evaluated in sensitized guinea pigs. The PPD when adjusted to a protein concentration of 1mg/ml induced a delayed-type hypersensitivity response comparable to USDA Johnin OT 133-8707.     

Role of mediators of cellular hypersensitivity in the stimulation of the antibody formation to unrelated antigen

The effect of a concurrent delayed hypersensitivity reaction on the antibody response to sheep red cells was assessed by a plaque assay. Guinea pigs with delayed hypersensitivity to tuberculin purified protein derivative (PPD) or egg albumin showed an increased antibody response to sheep red cells when the cells were injected intravenously at the same time as PPD or egg albumin. This effect was transferred to normal guinea pigs by serum from guinea pigs with delayed hypersensitivity to PPD or egg albumin taken 24 hr after injecting the corresponding antigen. Supernatants containing migratory inhibitory factor were prepared by incubating lymphocytes from sensitized rabbits with antigen. These supernatants were injected with sheep red cells and gave rise to an enhanced plaque response. Similar results were obtained with supernatants from normal rabbit thymus cells. The role of mediators of delayed hypersensitivity in enhancing antibody formation and in T cell/B cell cooperation is discussed.     

Study of Delayed Hypersensitivity to Myxoviruses Induced by Vaccines

The delayed hypersensitivity response of guinea pigs to Bacillus Calmette-Gúerin (BCG) and myxovirus vaccines was investigated by use of the techniques of skin testing and inhibition of macrophage migration. A serum antibody response to the injected vaccines was readily demonstrable. Parainfluenza type 2 virus consistently failed to induce a delayed hypersensitivity state in 15 animals, even with the use of a virus adjuvant emulsion. Respiratory syncytial virus occupied an intermediate position in that positive delayed type skin tests of an erythematous nature were elicited following inoculation, but only two of seven guinea pigs yielded a positive migration inhibition test. In mumps-inoculated animals, skin testing gave rise to erythematous delayed skin reactions which varied from 0 to 20 mm in size. Migration inhibition could be demonstrated in 7 of 21 animals. In almost all guinea pigs inoculated with BCG, large, indurated, erythematous skin reactions were elicited, and inhibition of macrophage migration was readily demonstrated. The degree of skin reactivity was positively correlated with inhibition of macrophage migration. If the skin reaction to a specific antigen exceeded 9 mm of erythema, that antigen also inhibited macrophage migration. Skin testing proved to be the most sensitive indicator of viral hypersensitivity. Migration inhibition was demonstrated only when a greater than 8-mm skin reaction was evoked. This cellular hypersensitivity appeared to be a qualitative phenomenon, the expression of which could be heightened by the use of adjuvant. The applicability and sensitivity of the migration inhibition technique is considered relative to its use for in vitro monitoring of effects of viral vaccine inoculations.