Delayed hypersensitivity against staphylococcal antigens in guinea pigs (author's transl)]

Delayed type hypersensitivity against staphylococcal antigens could be induced in guinea pigs by injecting the animals with a staphylococcal homogenate in Freund's adjuvant in all four foot pads and the nuchal skin. Maximal skin reactivity, tested intracutaneously, was observed 21 days after sensitization. Highest stimulation of lymph node cells and peripheral blood lymphocytes was also obtained 21 days after sensitization. When comparing cell walls, cell materials (nonsoluble material obtained after the separation of the cell wall fraction) and soluble fraction (obtained after sedimentation of all nonsoluble material, the strongest skin test reactions occurred after testing intracutaneously with the cell wall fraction.     

Skin test and lymphocyte stimulation in delayed hypersensitivity against staphylococcal antigens

Summary Staphylococcal homogenate was fractionated into cell walls (CW), cell membranes (CM) (insoluble part left after removal of the cell wall fraction) and a soluble fraction. The capacity of these fractions to evoke delayed skin reactions and to stimulate lymph node and peripheral blood lymphocytes from guinea pigs sensitized with staphylococcal homogenate in Freund's complete adjuvant was tested 21 days after sensitization. Highest skin reactivity was observed with the cell wall fraction. In the lymphocyte stimulation test similar results were obtained with all three fractions. With peripheral blood lymphocytes higher stimulation indices were observed than with lymph node lymphocytes.These investigations were performed under the supervision of the retired director Prof. Dr. H. Storck     

Inhibitory effect of 8-methoxysporalen plus UVA (PUVA) on the systemic induction of contact sensitivity to dinitrochlorbenzene (DNCB) in guinea pigs

Summary To investigate the influence of 8-MOP and UVA on the induction of cell-mediated immunity, guinea pigs, sensitized with a single injection of dinitrofluorbenzene (DNFB) in Freund's complete adjuvant (FCA) in all footpads and the nuchal skin, were treated with 8-MOP, UVA, or 8-MOP+ UVA on days 0, 2, and 5 and tested with dinitrochlorbenzene (DNCB) on day 14 after sensitization. Two control groups, exposed in a covered condition to the PUVA 4000 lamp, to observe the heat effect, showed a slightly enhanced contact sensitivity in comparison to the only sensitized control group. No altered reactivity was observed after irradiation with longwave UV light alone, whereas a statistically significant enhanced resp. reduced contact sensitivity was obtained after the treatment with 8-MOP alone and the combined action of 8-MOP +UVA, respectively.Dedicated to Prof. Dr. P. Laugier, Chairman of the Department of Dermatology, University of Geneva, Switzerland, on the occasion of his 70th anniversary     

Studies of new short-period method for delayed contact hypersensitivity assay in the guinea pig

A new method for delayed contact hypersensitivity assay of chemical compounds in guinea pigs, a short-period method (14 days) with a high detection sensitivity, has been developed. The new method was as follows; a combination of a Freund's complete adjuvant (FCA, undiluted) intradermal injection and a 24–h occusive patch on a guinea pig was performed 2x at an interval of 4 days and challenged by non-occlusive topical application II days after the first sensitization (with benzyl alcohol during test development). Acanthosis and spongiosis in the epidermis and mononuclear cell infiltration into the dermis were observed histopathologically at the skin reaction site. This newly developed method (adjuvant and 24–h occusive patch 2 test: AP2 test) could equally and/or better detect the allergenicities of 6 other chemical compounds (bromostyrol, citronellal, benzyl salicyfate. p -aminobenzoic acid ethyl ester, phenylenediamine and formaldehyde) as compared with the cumulative contact enhancement test (CCET) and the guinea pig maximization test (GPMT).     

Route of contact sensitization and in vitro lymphocyte transformation

A guinea pig skin extract conjugate with dinitrofluorobenzene elicited significant in vitro transformation of cultured lymphnode lymphocytes from 19 of 27 guinea pigs sensitized by footpad injection of dinitrochlorobenzene in Freund's complete adjuvant, as compared to only 1 of 26 guinea pigs topically sensitized to dinitrochlorobenzene. Topically sensitized guinea pigs appear to be more appropriate models for contact allergy in man than guinea pigs sensitized by other methods. Other sensitization procedures are likely to produce more heterogeneous forms of sensitization, with features of contact allergy, tuberculin-type allergy, antibody-mediated hypersensitivity and cutaneous basophile hypersensitivity.     

Experimental nickel sensitization in the guinea pig: comparison of different protocols

3 different sensitization protocols were compared for inducing delayed-type nickel contact hypersensitivity in guinea pigs. Open epicutaneous sensitization (OE) induced nickel allergy in 11/22 (50%) guinea pigs. When intradermal injections of Freund's complete adjuvant into the nickel-painted skin was added to the same protocol. 4/13 (31 %) became sensitized. The guinea pig maximization protocol induced nickel allergy in only 7/31 (23%) of the animals. Compared with the 2 other methods, animals sensitized with open epicutaneous applications reacted more rapidly (maximum at 6 h) and strongly (2+ reaction in 12/22 of animals) in previous patch lest sites upon systemic (i p.) nickel challenge. Open epicutaneous sensitization of guinea pigs should he a useful model for studying cellular and immunological mechanisms in nickel contact sensitivity.     

Persistent light reaction: induction in the guinea pig

Persistent light reactions similar to those in humans were observed in the study of photoallergenicity of chemicals in guinea pigs. The animals photoinduced with chemicals reacted to long-wavelength ultraviolet (UVA) radiation in the absence of test materials at the challenge stage. The sensitivity of the animals to UVA persisted for more than 1 year. The minimum erythema dose to UVB of animals in the treated group was less than that in the control group. Our investigations indicate that the main factors influencing the elicitation of persistent light reaction were the amount of Freund's complete adjuvant (FCA) used to enhance the allergic response in animals, and the UVA dose at the induction stage. Based on these findings, we have developed a method to make animals persistent light reactors with high frequency. This method consists of 1 intradermal injection of 1.2 ml emulsified FCA, 5 irradiations with 20.4 J/cm2 of UVA, and topical applications of 5% p-aminoethylbenzoate (benzocaine) at the induction stage. We also found that photosensitivity could be induced using FCA and UVA without photosensitizers.     

Nickel-sulphate-induced contact dermatitis in the guinea pig maximization test: a dose-response study

Nickel sulphate is a sensitizer in guinea pigs, but the frequency of sensitization varies from study to study. The dose-response relationship for NiSO4.6H2O was evaluated in the guinea pig maximization test in this study. 6 intradermal (0.01%-3.0% aq.) and 6 topical (0.25%-10.0% pet.) concentrations were chosen for induction and NiSO4.6H2O 1% pet. was used for challenge, based on the absence of skin irritation in a pilot study. Blind reading was performed. A logistic dose-response model was applied to the challenge results. At 48 h, a linear relationship was obtained between the intradermal induction dose (but not topical dose) and the response, resulting in a maximum sensitization rate of 40% after 3% i.d. The reactivity disappeared at re-challenge 1 week later. Following a booster closed patch on day 35, using NiSO4 10% pet., the animals were challenged with NiSO4 2% pet. and statistical analyses of 72-h readings revealed a non-linear dose-response relationship, giving a maximum response frequency of 40% after initial induction with NiSO4 3% i.d. and 2% topical.     

Studies on contact hypersensitivity in the guinea pig The cumulative contact enhancement test

A method to determine the quantitative induction and challenge of the allergenicity of externally applied toiletories and cosmetics, including their components, is described.
The experiment used oil-soluble cinnamic aldehyde and water-soluble formalin as allergens, and guinea pigs as the experimental animals. A high sensitization method resulted, carried out as follows. A 24-h closed patch is attached to the skin every other day over a period of 2 weeks (a total of 4 applications). Freund's complete adjuvant is administered intradermally just before the 3rd application of the patch. The challenge step is performed by directly applying the test material.
This method was compared with other allergenicity evaluation methods. As a result, this method was found to be in no way inferior in sensitization performance to the other methods. The method was used cm perfume mixtures and tested for its evaluation effectiveness. It proved to be satisfactory.     

Induction of contact sensitivity to a broad variety of allergens with haptenized macrophages

Using guinea pigs an analysis could be made of various aspects of contact sensitivity (CS) induced by subcutaneous injection of syngeneic haptenized macrophages (oil-induced peritoneal exudate cells, PEC) as compared to epicutaneous sensitization. Very little PEC-bound hapten (dinitrochlorobenzene, or oxazolone) is needed for optimum sensitization. Nevertheless, both sensitization methods induce a state of CS that may last for over 6 months, give rise to hapten-specific antibodies with a similar isotype distribution, and show susceptibility to cyclophosphamide pretreatment. In addition, time courses and microscopic appearance of skin test reactions after either way of sensitization are identical. CS to a broad variety of physicochemically different antigens, including nickel, penicillin, and acrylates, is readily induced by syngeneic PEC, haptenized following a standardized procedure. As Freund's complete adjuvant is known to cause serious side effects like ulceration and long-lasting granuloma formation, immunization with haptenized PEC should now be considered as a clean and effective alternative in experimental CS studies.     

Nonspecific hypersensitivity: false-positive responses with the use of Freund's complete adjuvant

While conducting a guinea pig sensitization protocol, using the maximization test, it was discovered, at challenge, that the test animals were more responsive to the vehicle (acetone) than they were to the proprietary lest material. During rechallenge, conducted to clarify the specific immune status of the test animals, it was determined that they were also hyperreactive to an alternate vehicle (diethyl phthalate), to which they were naïve. This bizarre set of data is presented and it is suggested that this type of response is the prototype for the presence of false-positive responses experienced by loxicologists using this test. The test conditions imposed on the immune system by the maximization test that could result in these anomalous results are discussed. These data suggest that investigators need cautiously to interpret data that are produced by the injection of Freund's complete adjuvant (FCA).     

MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) IN GUINEA PIGS AND HUMANS WITH DELAYED HYPERSENSITIVITY

When lymph node cells from PPD-sensitive guinea pigs were incubated with PPD for 20–24 hours, they produced a soluble mediator which inhibited the migration of normal guinea pig peritoneal exudate cells (GP-PEC) out of capillary tubes. Also, human lymphocytes stimulated by antigen produced a soluble mediator which inhibited migration of normal GP-PEC and peripheral leukocytes. Guinea pig migration inhibitory factor (MIF) was eluted in the 34,000–67,000 molecular weight (MW) region of cultured supernatants. Human MIF was harvested from the 25,000 MW region of cultured supernatants from human lymphocytes fractionated on Sephadex G-100. Intradermal injection of the MIF into normal guinea pigs provoked reactions characterized by erythema and induration. The histologic response was found to be similar to that induced by antigen injection into sensitized animals. MIF from antigen stimulated lymphocytes may not have species specificity between guinea pig and human, although their molecular weights are different. No correlation was found between delayed skin reactivity induced by antigen in vivo and MIF production in vitro, and it is suggested that a substance inhibitory to MIF activity may be released in the lymphocyte culture.     

Sensitizing potential of 12di(meth)acrylates in the guinea pig *

The aim of this study was to investigate the sensitizing potential of 12di(meth)acrylates, when tested in the Guinea Pig Maximization Test (GPMT) and Freund's Complete Adjuvant Test (FCAT). In these tests the same (molar) concentration was used for intradermal induction. The results (see table, below) show that dimethacrylates are moderate to strong sensitizers in the guinea pig. In the group of diacrylates, no sensitization to diethylene glycol diacrylate was observed. 1,2-ethanediol diacrylate and 1,6-hexanediol diacrylate sensitized a number of animals. The challenge reaction pattern of the FCAT with 1,4-butanediol diacrylate, neopentane diol diacrylate and 1,5-pentanediol diacrylate differed from that commonly observed with sensitizers. Positive reactions were seen only to the maximum non-irritant concentration at 24 h in the challenge at day 21. At 48 h and in the second challenge at day 35, the reactivity had decreased or disappeared. This different reaction pattern makes it difficult to classify these monomers with certainty as sensitizers. In most of the FCATs, a decrease in reactivity in successive challenges was found. Possible explanations for this decrease and for the different challenge reaction patterns of some diacrylates are discussed. The skin irritant capacity of di(meth)acrylates was estimated after one open application. Diacrylates are very strong irritants on guinea pig skin. Dimethacrylates are weak irritants.     

Experimentally induced malignant lymphoma due to chronic antigen stimulation

Animal experiments were started in 1988 with the aim of inducing malignant lymphomas. 52 guinea pigs were exposed to potassium dichromate using the TINA test (plus 39 control animals). Following the sensitization procedure, the test animals and 15 control guinea pigs were exposed daily (5 × a week) epicutaneously to the antigen up to the present time. Currently, 30 exposed and 17 control animals are still in the experiment. In 3 out of 17 post-mortem examinations, histologically evident lymphomas were found. In the control animals, no tumor has been seen. The experiments support lymphoma cassation by chronic antigen stimulation. The experiments are continuing.     

Studies on the retest reaction in contact sensitivity to DNCB

DNCB-sensitized guinea pigs demonstrated an accelerated reactivity on retest of DNCB at the site of prior contact reaction, though presenting normal contact sensitivity at the virgin site. The retest reaction reached maximal at 9 h and waned at 24 h after antigenic challenge. Massive accumulation of eosinophils in either the epidermis or dermis was its distinguishing histologic feature. The reaction was induced at the site of delayed skin reaction to DNP-GPE in the animals sensitized with DNCB or DNP-GPE. A retest reaction in delayed sensitivity to DNP-GPE was also elicited at the site of contact reaction to DNCB in the animals. The significance of these findings is discussed.     

Lymphocyte Response to Staphylococcal Enterotoxin B in Patients with Annular Erythema Associated with Sjögren Syndrome

Lymphocyte response to staphylococcal enterotoxin B (SEB) was analysed in 11 cases with Sjögren syndrome (SjS) who developed annular erythema during the course. Increased lymphocyte response against SEB was observed on day 5 in those patients who developed annular erythema at the time of examination. A similar reaction pattern was observed when Con A and PWM, but not protein A, were used as the mitogen. No significant lymphocyte response was observed in the patients in the inactive stage without annular erythema or in normal controls. Furthermore, peripheral blood lymphocytes express LFA-1 after SEB stimulation. These results suggest that lymphocytes from SjS patients react to various types of antigenic or mitogenic stimulation and that they express cell adhesion molecules, especially in patients with active annular erythema.     

Contact sensitivity and bioavailability of chlorocresol

Chlorocresol sensitization from 5 topical preparations was determined in guinea pigs using the cumulative contact enhancement test. Chlorocresol 5% in olive oil/acetone (4/1), and 5% in aqueous suspension stabilized with carbomer 941 were more sensitizing (55% and 60% of the animals positive, respectively) than chlorocresol 5% in propylene glycol with or without carbomer 941 (20% positive). The sensitization from a saturated aqueous chlorocresol solution (about 0.38% w/v) was comparable to that of a 5% propylene glycol solution containing 13 times more chlorocresol. The fraction of the applied dose (from each preparation) that remained in the bandage material and the patch test skin site was determined by combined gas chromatography-mass spectrometry using an isotopic dilution technique. From 0.2% to 1.6% of the applied doses remained at the patch test skin sites as free chlorocresol. 75% of the chlorocresol in aqueous suspension permeated the skin in contrast to 34% and 35% of the chlorocresol in olive oil/acetone (4/1) and propylene glycol, respectively. In spite of the same amount of chlorocresol absorption from the 2 latter preparations, they showed a significant difference in sensitizing capacity. No simple relationship between the sensitization rates and the calculated bioavailability was observed with the preparations tested.     

Experimental characteristics of the immune response of lymphoid organs to fungal and staphylococcal antigens

Increased proliferative and functional activities of the lymphoid organs and a high mitotic activity of the lymphocyte blast forms were recorded by autohistoradiography (3H thymidine and 35S-labeled sulfate) in 60 guinea pigs in acute experiments (a challenge with staphylococcal or fungal antigen). Degeneration of the lymphoid tissue and substitution of the lymphoid tissue with fatty tissue were observed in a chronic experiment. Discoordination of the immune response consisted in depression of the T-component, with the B-component retaining high activity; these shifts were the most manifest after challenge with fungal antigen.     

Attempts to induce sensitization in guinea pigs with nickel complexes.

A technique for the induction of sensitization in pigs with a nickel-alanine conjugate has been reported by other investigators. Similar results were observed in our experiments in mini-pigs. However, using the identical technique, we were unable to induce sensitization in guinea pigs with NiSO4, with nickel-alanine or other nickel-amino acid complexes, or with a complex of nickel with soluble guinea pig skin extract. These results indicate that nickel-amino acid complexes and nickel-guinea pig skin complex were not antigenic in guinea pigs with this technique. Hypotheses for the apparent failure to induce sensitization are proposed.     

Experimental infection of inbred guinea pigs with Treponema pallidum: development of lesions and formation of antibodies.

Inbred strain 2 and strain 13 guinea pigs were infected intradermally in the hind legs with different numbers of the virulent Nichols strain of Treponema pallidum. About 91% of the animals developed clearly visible lesions after being injected with 5 X 10(6) to 10 X 10(6) treponemes. T pallidum organisms could be isolated from skin lesions at various stages after infection. Infected animals were monitored for the production of specific treponemal and non-specific cardiolipin antibodies by the fluorescent treponemal antibody (FTA) and microhaemagglutination (MHA-TP) tests and the rapid plasma reagin (RPR) card test. Low levels of specific antibodies could be detected by both FTA and MHA-TP tests from three to four weeks after infection. Maximum titres of treponemal antibody generally occurred after week 6 and persisted for several more months. These peak titres ranged from 1/40 to 1/80 in the FTA test and 1/160 to 1/320 in the MHA-TP test. During the same period infected guinea pigs, unlike rabbits with syphilis, did not produce detectable quantities of antibodies against cardiolipin.