Contact sensitivity to topical antimicrobials

A predictive study comparing the sensitizing potentials of some topical antimicrobials, using a modified Beuhler's technique, showed that over-the-counter (OTC) antimicrobials were more sensitizing than prescribed topical antibiotics. Among OTC antimicrobials, proflavine was the most potent sensitizer (4/10 guinea pigs); parachlorometaxylenol, benzalkonium chloride and propamidine isethionate moderate sensitizers (2/10 guinea pigs); iodine a weak sensitizer (1/10 guinea pigs); and chlorhexidine and cetrimide very weak sensitizers (0/10 guinea pigs). Among prescribed topical antibiotics, neomycin was a moderate sensitizer (2/10 guinea pigs); gentamycin and chloramphenicol weak sensitizers (1/10 guinea pigs); kanamycin, clioquinol, polymyxin B, bacitracin, tetracycline, sodium fusidate and fusidic acid very weak sensitizers (0/10 guinea pigs). There was good correlation between sensitizing potentials in animal studies and clinical experience of contact allergy to these topical antimicrobials.     

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.     

Induction of delayed reaction to dinitrophenylated lymphocytes in guinea pigs with contact sensitivity to DNCB.

The incidence of dinitrophenylated cells in guinea pig lymphocytes incubated with 0-30 mM concentrations of DNBSO3Na in phosphate-buffered saline was examined by an immunofluorescence method using fluorescence-labelled anti-DNP antibody. Under our experimental conditions, the incidence was roughly proportional to the concentration used. Using DNP-lymphocytes as an antigen for skin testing, a marked delayed reaction was induced in guinea pigs sensitized by painting with DNCB and intradermal injection of Freund's complete adjuvant. The significance of these findings is discussed.     

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.     

Antigen-specific IgG1-mediated epidermal cell injury: a component of contact hypersensitivity reactions in guinea pigs, measurable in vitro in full-thickness skin explants.

Guinea pigs were sensitized by the topical application of either dinitrochlorobenzene (DNCB) or oxazolone on days 1, 2, 3, and 10. Seventeen days after the first treatment with the sensitizer, full-thickness 1.0-cm2 explants of untreated areas of skin were topically exposed in vitro to these contactants. Compared to the response of skin from control guinea pigs, skin from specifically sensitized animals showed a dose-related increase in the number of epidermal cells containing vacuoles. A specific increase in epidermal microblistering paralleled the increase in epidermal vacuolization. In addition, skin explants from sensitized animals (exposed to the contactant) showed a specific decrease in the incorporation of [14C]leucine. Full-thickness skin explants from unsensitized guinea pigs were sensitized in vitro by the intradermal injection of serum IgG1 fraction from oxazolone-sensitized guinea pigs. In such passively sensitized explants, the specific contactant produced an increase in the number of epidermal vacuoles, an increase in the amount of microblistering, and a decrease in the number of mast cells detectable by Giemsa staining. To elicit this specific response, the concentration of the specific contactant had to be mildly injurious, as well as antigenic. This requirement for nonspecific injury could be met by topically exposing skin explants to a nonspecific irritant followed by a sub-threshold concentration of the specific contactant. In contrast to vacuole formation and blistering, contactant-specific degranulation of mast cells (measured by the decrease in their number) did not require irritant levels of the contactant. These studies show that several components of contact sensitivity reactions can be reproduced in vitro by the passive transfer of sera containing antigen-specific immunoglobulins. Banks of such sera might, therefore, be useful in identifying (in human populations) many pre-existing sensitivities to chemical compounds.     

Cross sensitivity between aminoside antibiotics (author's transl)

The great number of topical preparation containing neomycin and their frequent use for a long period induce a lot of contact dermatitis. It is interesting to detect the importance of cross sensitivity between aminosides among the patients whose dermatitis was caused by neomycin. 15 neomycin eczematous patients were investigated. The research about cross-sensitivity concerned 6 antibiotics: streptomycin, framycetin, ribostamycin, gentamycin, kanamycin, tobramycin. Patch-tests (20 p. 100 in petrolatum) were used in order to show this cross-sensitivity. The results of these tests are nearly the same as in the other studies, as for 5 of the antibiotics; but it is the first time, to our knowledge, that a cross-sensitivity between ribostamycin and neomycin is observed. The cross-sensitivity between these two aminosides can be explained by their chemical structure. There are four rings in neomycin: the main components are neamin and neobiosamin. Only three rings are found in ribostamycin: ribostamycin is constitued of neomycin lacking neosamin of neobiosamin. Thus the structure of the 3 rings of ribostamycin is identical with the structure of 3 out of 4 rings of neomycin. The study about cross-sensitivity between aminosides suggests two conclusions: 1. The use of local antibiotics must be prescribed only when is it really necessary. 2. It seems wise to avoid all systemic aminosid antibiotics for patients who are sensitized by neomycin ointment.     

Cumulative irritancy in the guinea Pig from low grade irritant vehicles and the angry skin syndrome

A 4-week open cumulative irritancy test in guinea pigs discriminated between two low grade irritant vehicles, nonionic base (anhydrous) and hydrophilic ointment. The procedure might be useful as a predictive test for low grade irritants. The angry skin syndrome was established in the guinea pigs by one Freunds complete adjuvant intradermal injection on day 0, or by daily open applications of 10% sodium lauryl sulfate in petrolatum to the neck area of the animals.     

Allergic contact dermatitis to methylenelactones use of lymphocyte transformation test

Summary Seventeen guinea pigs were sensitized to alantolactone, a natural sesquiterpene lactone known for its sensitizing properties, using intradermal injections in Freund's complete adjuvant. Guinea pig skin protein extracts (SPE) were used to make conjugates with alantolactone and an isomer, isoalantolactone. Lymphocyte blastogenesis was observed with SPE-alantolactone conjugates, as well as with guinea pig albumin-lactone conjugates. In six cases, stimulation was noted with unconjugated haptens. These results do not show a high degree of hapten-carrier specificity in contact sensitivity to alantolactone, induced by unconjugated hapten injections. Cross-sensitivity with SPE-isoalantolactone conjugate was also observed.     

Contact hypersensitivity reaction to ovalbumin in newborn guinea pigs from maternally sensitized animals.

An animal study was conducted to elucidate the role of ovalbumin (OA) in the development of eczematous lesions in intrauterine sensitized newborns. Four groups of pregnant guinea pigs were used: group A, immunized by oral administration of 1% OA in drinking water until parturition; group B, immunized by intradermal injection of OA with Freund's complete adjuvant; group C, immunized by both methods; and group D (control), not immunized. The newborn guinea pigs of each group were patch tested with 10% OA in white petrolatum. Positive reactions were seen in the newborns of groups B and C, but not in those in groups A and D. By enzyme-linked immunosorbent assay and passive cutaneous anaphylaxis, a high titre of OA-specific IgG was detected in the group B and C newborns. The number of positive patch test reactions decreased concomitantly with the decline of specific IgG. Histologically, eczematous changes were observed in the positive reaction sites. Many OA antigen-bearing Langerhans cells were found by the immuno-double labelling technique. Immuno-electron microscopic findings revealed the presence of OA antigens as well as IgG molecules on the cytoplasmic membranes of Langerhans cells. Our studies demonstrated that maternal sensitization with OA can induce an eczematous reaction in the newborns to OA patch testing under the presence of high levels of OA-specific IgG in the serum. From these findings it is suggested that IgG plays an essential role in the development of contact hypersensitivity reaction to OA.     

INDUCTION OF PSORIASIFORM CHANGES IN GUINEA PIG SKIN BY PROPRANOLOL

Background. The ability of β-adrenergic blocking agents to induce psoriasis as an adverse effect prompted us to use such an agent to induce psoriasis in guinea pigs. Methods. Thirty female albino guinea pigs were divided into four groups. Group 1 received propranolol, 0.1 mg/day, dissolved in 2 mL of normal saline, orally by gavage for 30 days. Group 2 was given the same treatment, but in addition intradermal injections of propranolol with Freund's complete adjuvant, injected at weekly intervals. Group 3 (five animals) received 2 mL saline, and group 4 additional injections of adjuvant without propranolol. Groups 3 and 4 served as normal controls. Results. All animals of group 2 (which received propranolol orally and in addition intradermal injections of adjuvant) developed psoriasiform epidermal hyperplasia with acanthosis. Parakeratosis, papillomatosis, and formation of microabscesses, all characteristic signs of psoriasis, have not been seen in any of the skin samples of this group. Skin samples from group 1 animals receiving propranolol orally showed normal epidermis and dermis. They showed exactly the same histologic picture as the control groups 3 and 4. Conclusions. Beta-blockers given orally for 30 days do not cause any significant skin changes in guinea pigs. When given with a weekly intradermal injection of Freund's complete adjuvant, they cause psoriasiform epidermal hyperplasia. Although the overall histologic appearance of the skin of group 2 resembled psoriasis, it lacked important histologic features characteristic of this disease. It seems, therefore, that the model, per se, does not fulfill the initial expectations as an experimental model for psoriasis; however, this model has potential in the study of adverse drug reactions. Perhaps by introducing modifications to the experimental protocol, we may succeed also in developing a better model for experimental psoriasis.     

Delayed-type skin reaction to 2,4-dinitrophenylated epidermal cells in guinea pigs with contact sensitivity to 2,4-dinitrochlorobenzene

Summary Contact sensitivity (CS) induced by hapten has been thought to be analogous to delayed-type hypersensitivity, such as the Mantoux reaction, because of outstanding similarities between the two phenomena. It can be suggested that animals with CS respond also to intradermal injection of the conjugate of hapten and protein as well as to epicutaneous application of hapten. However, evidence against this has been reported. In the present experiments, delayed-type skin reaction (DSR) was successfully obtained in JY1 strain guinea pigs sensitized by painting the skin with 2,4-dinitrochlorobenzene using in vitro dinitrophenylated epidermal cell suspension (DNP-EC) as antigen for a delayed intradermal test. The experiment using anti-Ia alloantiserum and complement showed that the elicitation of DSR is due to the presence of Ia-positive cells (presumably Langerhans cells) among DNP-ECs. The delayed intradermal test with the conjugates such as haptenated ECs in the animals with CS is considered to be an experimentally useful way of analysing the antigen in the sensitivity.     

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).     

Possibility of passive transfer of cold urticaria to guinea pigs

Serum from one out of six patients with idiopathic acquired cold urticaria produced a short lived edematous reaction in the pinna of guinea pigs 24 hours after intradermal injection of the patient's serum and subsequent cold challenge. No relationship was observed between the transfer activity and IgE titer or atopic status.     

Absence of specific histologic changes in guinea pig skin treated with bullous pemphigoid antibodies

Previous studies have reported that intradermal injections of bullous pemphigoid antibodies into guinea pigs can reproduce the histologic and immunohistologic features of bullous pemphigoid lesions. In this study we examined this model to determine its reproducibility and suitability for testing other types of anti-BMZ antibodies. Twenty guinea pigs were injected intradermally with 0.1, 0.3, or 0.5 ml of either bullous pemphigoid serum or IgG fraction containing high-titer complement-binding anti-BMZ antibodies or an equivalent volume of normal human serum or IgG fraction as control. Sites were biopsied at intervals after injection and were examined by routine histology and direct immunofluorescence. The results showed (a) no difference in the incidence of dermal epidermal separation or type of inflammation in experimental and control sites; (b) no evidence of an eosinophil-rich inflammatory reaction typical of bullous pemphigoid; (c) an absence of linear BMZ deposits of IgG and complement in the majority of sites injected with bullous pemphigoid antibodies; and (d) no correlation between dermal-epidermal separation and deposition of immune reactants at the BMZ. These results suggest the histologic changes seen in guinea pigs that are administered intradermal injections of bullous pemphigoid antibodies are nonspecific and that the model is not suitable for testing the pathogenicity of anti-BMZ antibodies in sera or IgG fractions.     

Epidermal cell proliferation following an active arthus reaction in the guinea pig

Active Arthus reactions were provoked by injections of 100 micrograms horseradish peroxidase (HRP), 10 micrograms HRP and 100 micrograms bovine serum albumin (BSA) into the skin of sensitized guinea pigs. Labeling indices (LI) of epidermal basal cells were measured 1, 4, 8, 24, 48 and 72 h later by the in vivo 3H-thymidine labeling technique, and compared with those obtained with injections of antigens into the skin of non-sensitized guinea pigs. From 1-8 h after the induction of an active Arthus reaction, the LI of epidermal basal cells of the skin injected with 100 micrograms HRP decreased to a remarkably low value. On the other hand, those obtained with the reaction against 10 micrograms HRP were significantly high. At 24 h after the reaction, LI were as high as those obtained in non-sensitized guinea pigs with control intradermal injections, though the former persisted high until 48 h after the injection. In addition, decreased LI of the epidermal basal cells were observed in the skin 4 h after intradermal injections of immune complexes. It was suggested that DNA synthetic activity of the epidermis increases in a mild active Arthus reaction, while the activity may be suppressed in a severe active Arthus reaction up to 8 h after provocation.     

Induction of formaldehyde contact sensitivity: dose response relationship in the guinea pig maximization test

The sensitizing potential of aqueous formaldehyde was evaluated with the guinea pig maximization test (GPMT) in two laboratories (Copenhagen and Stockholm) using different guinea pig strains. Six intradermal (0.01%-3%), and 6 topical (0.5%-20%) concentrations were used for induction, and formaldehyde 1% and 0.1% was used for challenge. The incidence of contact sensitivity depended on the intradermal, but not on the topical induction dose. Statistical analyses showed a non-monotonous (non-linear) dose response relationship. The estimated maximal sensitization rate in Copenhagen was 80% after intradermal induction with 0.65% formaldehyde; in Stockholm it was 84% after induction with 0.34%. The data from the two laboratories could be described by parallel displaced dose response curves suggesting that the guinea pig strain used in Stockholm was significantly more susceptible to formaldehyde than the strain used in Copenhagen. The EC50 (formaldehyde concentration at which 50% of the guinea pigs were sensitized) at the 72 h scoring and a 1% challenge concentration, was 0.061% in Copenhagen and 0.024% in Stockholm.     

A modified technique of guinea pig testing to identify delayed hypersensitivity allergens

A modified guinea pig testing technique was developed For the detection of weak allergens and allergenicity of materials unsuitable for testing by intradermal injection. This test involved the use of Freund's complete adjuvant to stimulate the immune system of the animal, and external application instead of intradermal injection of the test compound in the induction stage. The allergenicity of Sudan III, Brilliant Lake Red R and Sudan I was tested by this procedure.
In the dose-effect study of Sudan I, the dose dependency of a positive reaction of the induction and challenge concentrations was recognised.
The test was compared with three other guinea pig sensitization tests. The results obtained with this test correlated well with those obtained with the guinea pig maximization test.     

Influence of PUVA and UVB radiation on delayed hypersensitivity in the guinea pig

Exposure of guinea pigs to UVA (320--400 nm) radiation following administration of 8-methoxypsoralen by gavage (referred to by the acronym, PUVA) or exposure to UVB (290--320 nm) radiation, produced suppression of the cutaneous delayed hypersensitivity reaction at the site of exposure to radiation and at distant nonexposed sites. In these experiments, the animals were immunized by injection of dinitrophenyl-bovine gamma-globulin (DNP-BGG) in complete Freund's adjuvant and delayed hypersensitivity responses were provoked by intradermal injections of DNP-BGG, DNP and BGG on the flanks. Exposure to erythemogenic doses of either PUVA or UVB radiation for 7 days prior to immunization and for the 7 days between immunization and challenge (total period of radiation: 14 days) produced inhibiton of responses to each of the test substances. In addition, treatment with erythemogenic doses of PUVA either for 7 days prior to immunization or during the interval between immunization and challenge with DNP-BGG, inhibited the delayed hypersensitivity responses at the site of irradiation and at a nonexposed site. These findings suggest that in vivo exposure to nonionizing radiation leads to both local and systemic alteration of certain immune responses.     

Comparative studies of the sensitization potential of morpholine, 2-mercaptobenzothiazole and 2 of their derivatives in guinea pigs

The aim of this study was to determine the sensitization potential of morpholine (M), 4,4'-dithiodimorpholine (DTDM), morpholinyl-mercaptobenzothiazole (MMBT) and 2-mercaptobenzothiazole (MBT) in guinea pigs. 5% and 10% DTDM, MMBT and MBT produced irritation reactions. M up to 10% failed to irritate. Sensitization tests showed that all the guinea pigs treated with DTDM and MMBT were sensitized. Cross-sensitization tests showed that 60% of the DTDM-sensitized animals reacted to challenge with MMBT; 30% of the animals sensitized to DTDM reacted to challenge with M; 80% of the MMBT-sensitized animals reacted to challenge with MBT and 10% to challenge with M; 42% of the MBT-sensitized animals reacted to MMBT. The rank order of sensitization potential in guinea pigs observed from this study is DTDM, MMBT and then MBT. It appears that the disulfide bond, sulfur linkage and the sulfhydryl group may each play a role in the sensitizing capacity of these compounds.     

Stereospecificity of allergic contact dermatitis (ACD) to enantiomers

Summary Guinea pigs were sensitized to polygodial 1, a natural sesquiterpene dialdehyde, using intradermal injections in Freund's complete adjuvant.Cross-reactions with a (±)-mixture of warburganal and (-)-warburganal 2 (a natural hydroxylated derivative of polygodial) showed a high specificity of the allergic response since the (-)-pseudoenantiomer (having the same configuration as the primary sensitizer) gave a stronger skin reaction than the racemic mixture.