Comparative characteristics of the diagnostic properties of staphylococcal allergens in an experiment

The diagnostic value of five staphylococcal allergens prepared from a single S. aureus strain by different methods and in different institutions has been tested on the experimental models of delayed, immediate and mixed (immediate and delayed) hypersensitivity in guinea pigs. The advantages of the allergens prepared in Kazan (USSR) for the detection of delayed hypersensitivity and the ultrasonicated allergen, as well as the allergen made in Czechoslovakia, for the detection of immediate hypersensitivity have been noted.     

Delayed hypersensitivity to house dust-storage mites Thyrophagus putrescentiae in experimental allergic dermatoses]

House dust mites and storage mites proved to be one of the main allergens causing hypersensitivity in atopic dermatitis in allergic patients. The authors reproduced experimental allergic dermatosis on the model of delayed type hypersensitivity in guinea pigs, caused by sensitization to the allergen from the mite's bodies Tyrophagus putrescentiae--species having wide distribution in the country. The results characteristic for T-cell type hypersensitivity have been obtained: delayed positive skin tests after 24 hours, typical histomorphological picture (strong allergic alteration, epidermis desquamation, vasculitis, dermis T-cell infiltration and spongiosis--Waksman's syndrome). It is concluded that side by side with humoral IgE-mediated reaction there is strong delayed T-cell hypersensitivity to the storage mites Tyrophagus putrescentiae.     

Follicular contact dermatitis due to coloured permanent-pressed sheets

A delayed hypersensitivity type of allergic contact dermatitis was observed following exposure to certain brands of 50% cotton, 50% polyester coloured permanent-pressed sheets produced by a particular manufacturer. The dermatitis presented as an extremely pruritic follicular eczema of the body and vesicular edema of the ears and face. Patch testing excluded formalin as the allergen but suggested permanent-pressing chemicals as a possibility. Several washings of the sheets did not prevent the development of the dermatitis. The removal of sheets did not immediately result in improvement: the condition could persist for up to eight weeks after their discontinuance.     

Genetically engineered and synthetic allergen derivatives: candidates for vaccination against type I allergy.

Type I allergy, a hypersensitivity disease affecting almost 20% of the population worldwide, is based on the IgE recognition of otherwise harmless antigens (i.e., allergens). Allergen-induced crosslink of effector cell-bound IgE antibodies leads to the release of biological mediators and thus to immediate disease symptoms (allergic rhinitis, conjunctivitis and asthma). Specific immunotherapy, the only causative treatment of Type I allergy, is based on the administration of increasing doses of allergens to allergic patients in order to yield allergen-specific non-responsiveness. Major disadvantages are 1. that current forms of allergen immunotherapy are performed with allergens difficult to standardize which cannot be matched to the patients reactivity profile and 2. that the administration of active allergen preparations can cause anaphylactic side effects. Through the application of molecular biological techniques many relevant environmental allergens have been produced as active recombinant proteins which allow component-resolved allergy diagnosis and thus represent the basis for patient-tailored forms of immunotherapy. Here we review molecular strategies which have been recently applied to generate genetically engineered and synthetic hypoallergenic allergen derivatives for patient-tailored and safe vaccination against Type I allergy.     

Editorial comment

A single injection of Cyclophosphamide (CY) in a dose of 300 mg of CY/kg of mice resulted in enhanced delayed hypersensitivity (DH) when administered between at least 7 days prior to and 15 days after intracutaneous (ic) immunization of sheep red blood cells in Freund's complete adjuvant. The maximal enhancement occurred when CY was applied 8 hr before the antigen. Using the latter interval, the effect of varying the dose of CY before ic or intraperitoneal (ip) injections of antigen was studied. Combined with ic immunization, increasing doses of CY resulted in increasing DH. The ip route of immunization needed CY in amounts of at least 100 mg/kg to augment DH to a detectable level. The enhancing effect of lower doses of CY was more pronounced when the interval between immunizing and eliciting injections was reduced. Administration of 300 or 200 mg of CY/kg before ip immunization suppressed the antibody formation, when measured S and 7 days later. A dose of 100 mg of CY/kg caused a suppression of antibody formation on Day 5, but an enhancement on Day 7. With this dose, a maximal enhancement of DH was found on both days. The results suggest that CY interferes with more than one regulatory mechanism of the immune response.     

Staphylococcal infection in guinea pigs sensitized with a commercial staphylococcal allergen

Guinea pigs, previously injected with commercial staphylococcal allergen to induce delayed hypersensitivity, were infected by the intramuscular injection of S. aureus in a nonlethal dose. For control, the animals receiving only S. aureus were used. The dynamic study of the degree of septicemia and some lymphocytic characteristics in the animals was made. The study revealed that delayed hypersensitivity did not aggravate the course of the main disease; on the contrary, it rendered protection against the subsequent infection. Increased resistance to infection was manifested by a decrease in the degree of septicemia, determined from the decreased number of colony-forming units of S. aureus in the splenic tissue as assessed by inoculation into agar, as well as from a higher level of the activation of lymphocytes as assessed by rosette formation.     

Effect of detergent and glycerin on the efficacy of nasal immunotherapy with allergens and on the level of the lymphocyte subpopulation in the organs of local immunity

Experimental mixed allergy to staphylococcal antigens in guinea pigs was treated by the intranasal administration of a staphylococcal allergen with a surfactant or glycerin added. The treatment was found to produce a hyposensitizing effect with respect to immediate and delayed hypersensitivity. The addition of glycerine enhanced this effect. At the same time the level of T-lymphocytes in the lungs and the lymph nodes of the respiratory tract returned to normal. Detergent used at a concentration of 2% abolished the hyposensitizing effect of the allergen, stimulated T-lymphocytes in the lymph nodes of the respiratory tract and the lungs; the number of T-suppressors decreased.     

Induction of in vivo hyporesponsiveness to contact allergens by hapten-modified Ia+ keratinocytes.

Because our previous in vitro studies of hapten-modified Ia+ keratinocytes (KC) indicated that these cells induced anergy in Ag-specific Th1 cells, we assayed such cells for their ability to induce unresponsiveness in an in vivo animal model system of delayed type hypersensitivity (allergic contact dermatitis). Naive animals that were treated with i.p. injections of FITC-modified Ia+ cultured Langerhans cells (cLC) developed allergic contact dermatitis to subsequent hapten challenge; whereas, animals treated with similar doses of FITC-Ia+ KC failed to become sensitized to epicutaneous application of FITC, as evidenced by absent ear swelling responses to a FITC challenge. Those animals that were first treated with intraperitoneal injections of hapten modified Ia+ KC could not be sensitized when they were subsequently exposed to sensitizing doses of FITC; whereas a similar first exposure to FITC-cultured Langerhans cells did not interfere with epicutaneous sensitization. This hyporesponsiveness to sensitization was hapten specific, as FITC-Ia+ KC-treated animals were hyporesponsive only to FITC but not to the irrelevant hapten, TNCB. Additionally, Ia- KC failed to induce unresponsiveness. Additional studies indicate that the hyporesponsiveness was not passively transferrable with splenocytes and was not related to the I-J MHC locus. In contrast to our in vitro studies, the unresponsiveness induced by hapten-modified Ia+ KC in vivo was transient in nature. These data indicate that hapten-modified Ia+ KC function in vivo as nonstimulatory accessory cells, by generating down-regulatory signals that can interfere with the induction of contact hypersensitivity.     

Development of allergic contact dermatitis requires activation of both tumor necrosis factor-receptors

We investigated the role of the TNF receptors, type I (p55TNFR) and type II (p75TNFR), in a mouse model of contact hypersensitivity, i.e., a model of a delayed type hypersensitivity (DTH) allergic reaction. Mice deficient for p55TNFR or p75TNFR were used to investigate the functions of these receptors in development of the DTH reaction. We show that both TNF receptors have a strong influence on the overall outcome of the DTH reaction, with the two TNF receptors exerting distinct functions. Dendritic cells of mice lacking p55TNFR had a defect in allergen uptake but showed normal migration into regional lymph nodes. In contrast, dendritic cells of p75TNFR-deficient mice showed diminished migration into regional lymph nodes after allergen contact, whereas the allergen uptake was independent of the p75TNFR. Thus, both TNF receptors are required for the development of a complete DTH reaction.     

Identification and characterisation of an aspartyl protease inhibitor homologue as a major allergen of Trichostrongylus colubriformis

Allergens were identified from the gastrointestinal nematode of sheep, Trichostrongylus colubriformis, by probing Western blots of infective larvae (third stage) somatic antigen with IgE purified from the serum of sheep grazed on worm contaminated pasture. A 31 kDa allergen was frequently recognised by sera from immune sheep, particularly those deriving from a line that has been genetically selected over 23 years for parasite resistance. Using a proteomic approach, the 31 kDa allergen was identified as an aspartyl protease inhibitor homologue. The entire coding sequence of T. colubriformis aspartyl protease inhibitor (Tco-api-1) was obtained and the mature protein expressed in Escherichia coli. Anti-Tco-API-1 antibodies revealed that a commonly observed 21 kDa T. colubriformis allergen species is a truncated form of Tco-API-1. Specific IgE responses to T. colubriformis aspartyl protease inhibitor were significantly correlated with the degree of resistance to nematode infection as measured by faecal egg count in sheep. Surprisingly, IgE responses to Tco-API-1 were not correlated with breech soiling (dag score), which is thought to be caused, in part, by allergic hypersensitivity to worms. Therefore, a specific IgE response to this allergen may be a suitable marker for identifying lambs at an early age that will develop strong immunity to gastrointestinal nematodes.     

Immunomorphologic features of experimental allergic encephalomyelitis induced by the administration of tryptophan peptide

Three patterns of EAE with different morbidity and mortality rates were induced in the guinea pigs inoculated with various doses of tryptophane peptide (TP) and complete Freund's adjuvant. TP-sensitized animals manifested the delayed type hypersensitivity (DTH) reactions to TP and circulating anti-TP and -BPF antibodies were not found, polypeptide fraction of myelin basic protein (BPF while a A correlation was revealed between the DTH-reactions and EAE development. Intracutaneous TP and BPF injections at the early period before the EAE onset resulted in reduction of morbidity rate from 90 to 50 per cent. For the first time with the help of modified Marchi method demyelination has been shown to be highly marked in CNS tissue process in CNS can be caused by cell-mediated immune of animals given TP. It is supposed that the demyelinating reaction to encephalitogenic fragment of BP molecule. The data indicate a possibility of EAE inhibition by means of TP and BPF injections in saline solution.     

Effects of pretreatment of mice with BCG cell walls in saline on subsequent vaccination with BCG oil droplet vaccine

Multiple intravenous injections of bacillus Calmette-Guérin cell walls (BCG CW) suspended in Tween-saline into mice produced an unresponsive state to a subsequent vaccination with BCG-CW-oil droplet mixture. This was manifest by loss of delayed hypersensitivity to PPD as measured by footpad swelling, a diminished granulomatous response in pulmonary tissue, and almost complete abrogation of protection to an aerosol challenge with virulent Mycobacterium tuberculosis, strain H37Rv. The unresponsive state disappeared 20 days after stopping pretreatment. There was evidence of immunity early after H37Rv challenge, but by 4 wk it had markedly diminished and by 6 wk there was no difference between nonvaccinated animals and the pretreated vaccinated animals. Unresponsiveness was specific since pretreated mice were capable of developing delayed type reaction to immunization with modified sheep red blood cells in the usual manner. Relationships among delayed hypersensitivity, granulomatous inflammation, and protection against aerosol infection with virulent M. tuberculosis are discussed in the light of these findings.     

Effect of low doses of internal irradiation resulting from the administration of 75Se- and 35S-labeled compounds on the cellular immune response

The administration to rats of radioactive preparations. 75Se- selenomethionine, 35S-methionine or sodium 75Se-selenite in the amounts creating the absorbed dose in the body of 0.5 Gy suppressed the delayed hypersensitivity reaction as was registered 1-12 months after the injection. The delayed hypersensitivity was tested in vitro by the method of inhibimacrophage spreding inhibition. The degree of suppression of the cellular immune response depended upon the type of the compound administered, of which the radionuclide was a component, and upon characteristics of radiation spectrum of the radionuclide.     

Immunomodulatory effects of ultra-low-dose interleukin-2 in cancer patients: a phase-IB study

High-dose interleukin-(IL-2) has been broadly studied in tumour therapy, yet it may be inhibitory to T-cell-dependent immunity. Therefore immune and tumour responses mediated by low-dose IL-2 were studied systematically with respect to the feedback organisation of immune responses. IL-2 was administered once daily at three dose levels: 0.18, 0.9, 4.5 MIU/m² according to three different schedules requiring subcutaneous (s.c.) injection once weekly (four doses, stratum I), thrice weekly every other day (nine doses, stratum II), or five times weekly every other week (ten doses, stratum III). A total of 46 patients with advanced cancer were randomly assigned to one of the nine treatment groups. Systemic effects were induced at doses as low as 0.18 MIU/m² IL-2 s.c. as demonstrated from measurable IL-2 serum levels, induction of circulating IL-6, a transient lymphopenia, and stimulation of delayed-type hypersensitivity (DTH) responses of the skin. Analysis of the different IL-2 schedules demonstrated (a) prolonged effects of once-weekly injections on DTH responses, lymphocyte and eosinophil counts, and (b) maximum increase of eosinophil counts and preferential expansion of activated NK cells with repeated injections every 48 h or 72 h (stratum II), while sequential treatment according to stratum III was found to be more potent in increasing the number of activated T cells. A tumour response was observed in 1/15 patients with renal cell carcinoma who experienced more than 50% tumour regression for 8 months; 12 patients had stable disease for 4 months (median). These data demonstrate prolonged immunological effects of ultra-low doses of s. c. IL-2 despite its short half-life. Furthermore, scheduling of IL-2 was found to affect immune responsiveness specifically as demonstrated by the differential effects on natural killer and T cell populations.Supported by Bundesministerium für Forschung und Technologie, Förderkennzeichen 01GA8901/3 to R. M.     

Desensitization of delayed-type hypersensitivity in mice: suppressive environment

The systemic injection of high doses of antigen into a preimmunized animal results in transient unresponsiveness of cell-mediated immune responses. This phenomenon is known as desensitization. Serum interleukin 2 (IL-2) activity was found transiently in desensitized mice at 3 h after the antigen challenge. These mice could not reveal antigen nonspecific delayed-type hypersensitivity (DTH) 1 d after the challenge. Specific suppression of DTH was observed at later stages. Sera from 3 h desensitized mice showed suppressive effects on DTH in preo immunized mice. Administration of recombinant IL-2 into preimmunized mice led to the failure of development of DTH to antigens. These observations suggest that IL-2 plays an important role in the suppressive environment.     

A novel Candida albicans skin test antigen: efficacy and safety in man

Yeast phase Candida albicans (ATCC No. 10231) was grown in a nonantigenic medium, harvested and lyophilized. Ammonium sulfate fractions of an aqueous extract of the lyophilized cells were evaluated and the fraction yielding the highest specific delayed cutaneous reactivity in sensitized guinea-pigs was used to prepare a C. albicans skin test antigen (CASTA). The safety of the antigen was evaluated by measuring immediate and delayed (0.25, 6, 24, 48 and 72 h) cutaneous reactions in atopic and nonatopic human subjects. The outcome of three repetitive monthly Mantoux skin tests with 0.01-1 microgram antigen doses was used to test for booster effects in 14 subjects and to estimate a safe initial test antigen dose. The utility of a single skin test as a measure of cell-mediated immunity was evaluated in 40 healthy subjects. Reactor rates (greater than or equal to 2 mm, 48 h) of 40% and 85% were detected, respectively, with doses of 0.0316 and 1 microgram. Using a skin test reaction diameter greater than or equal to 5 mm at 48 h, the reactor rate was 50% for the 1-microgram dose. The only adverse reaction (45 mm, 0.25 h) was detected with the 1-microgram dose in an atopic subject who also exhibited exquisite scratch test reaginic hypersensitivity to C. albicans allergen. The prevalence of other adverse reactions to this antigen compared favorably with that to other antigens used for recall antigen testing. These studies suggest the 1-microgram CASTA dose can be used for effective, safe recall antigen skin tests.     

Delayed hypersensitivity in mast-cell-deficient mice

The ability of mast cell-deficient Wf/Wf and W/Wv mice to produced delayed hypersensitivity responses was examined. The W/Wv mice did not have detectable mast cells and could not produce IgE-mediated passive cutaneous anaphylaxis. Mice of both genotypes produced large delayed hypersensitivity responses to the contact sensitizers oxazolone and picryl chloride. The responses were indistinguishable from responses of control mice when challenged with optimal or suboptimal doses of antigen. Delayed hypersensitivity could be transferred into Wf/Wf mice by an antigen-specific T cell line, and the proliferative responses in the lymph nodes of these mice after, painting with sensitizer, were normal.     

Lethal infection with BCG in thymectomized mice]

Investigations carried out on CBA mice demonstrated that adult mice to which mycobacteria BCG were injected intravenously 12 months after thymectomy (in a dose of 2 mg for a period of 2 1/2 months) died of disseminated BCG infection against the background of depression of hypersensitivity of delayed type. The usual vaccine process developed in the sham-operated animals.     

Hypersensitivity of the human gonadotrophs to the repeated administration of small doses of LH-RH.

The functional capacity of the gonadotrophs was assessed by repeated stimulation with small doses of LH-RH (5 microgram intravenously at 2-hour intervals for 3 injections) in normal women during the early and late follicular phases of the menstrual cycle. The results were compared to those obtained when a single dose (100 microgram) of the neurohormone was administered. During the early follicular phase, the release of LH and FSH remained about equal after the 3 successive injections of the small and after the large dose of LH-RH. During the late follicular phase, the release of LH and fsh increased progressively after the repeated administration of the 5 microgram of the neurohormone while the large dose induced a more pronounced and a more sustained pituitary response. This hypersensitivity of the gonadotrophs is observed when the E2 concentrations are higher than in the early follicular phase of the menstrual cycle.     

Elicitation of Delayed Type Hypersensitivity in Chicks after In Ovo Sensitization with Different Molecular Forms of the Same Hapten

Lymphocyte sensitization, which participates in delayed type hypersensitivity (DTH) in chick embryos, was studied. The in ovo injection of dinitrophenylated keyhole limpet hemocyanine (DNP-KLH) or DNP-dextran (DNP-D) led to delayed onset of the hapten-specific reaction as shown by allergic contact dermatitis (ACD) after hatching. The extent of the ACD response was not directly dependent on the antigen dosage or the number of injections given for sensitizing. The magnitude of the ACD response was higher in chicks sensitized with DNP-D than in those given DNP-KLH. These findings suggest the presence of embryonic lymphocytes which can be sensitized by in ovo antigenic stimulation at the later stage of embryogenesis and may make possible the differentiation of functional lymphocytes. Antigen stimulation with higher doses may be inadequate for the in ovo sensitization of embryonic lymphocytes. The ACD response elicited by DNFB in chicks primed with either DNP-KLH or DNP-D was thought to be T-cell dependent, from the kinetics of the ACD peak within a period of 24 to 48 hr. Furthermore the conditions for in ovo sensitization of embryonic lymphocytes by DNP-D seem to be different from those for sensitization by DNP-KLH.