Evaluation of contact allergy to chemicals using laser Doppler flowmetry (LDF) technique

Skin blood flow in allergic contact reactions and cross-sensitivity were evaluated using laser Doppler flowmetry (LDF) to study the dose-response relationships in phases of induction and challenge in guinea pigs. Guinea pigs were sensitized with different doses of 1-chloro-2,4-dinitrobenzene (DNCB) and challenged with different doses of DNCB and 2,4-dinitrobenzene sulfonic sodium salt (DNBS). The skin reactions were evaluated by LDF and visual reading score. The results indicated that there were dose-response relationships between the doses of DNCB and LDF measurements in both phases of induction and challenge, that there was a cross-reaction between DNCB and DNBS, and that the reactions at 24 h were greater than that at 48 h after removal of the patches. LDF may discriminate between positive patch test reactions and negative or doubtful reactions, but not between weak positive and strong positive reactions. This is because vascular dilatation and increase of flow already reaches a maximum in weak reactions. The more advanced phases are dominated by oedema formation. This is simply the nature of the inflammatory reaction, rather than a methodological error. The important point is that LDF can separate positive reactions from negative/uncertain reactions. The results indicated that LDF, as a non-invasive technique, may objectively and quantitatively evaluate the dose-response relationships of contact sensitivity of sensitizers.     

Tierexperimentelle Studien zum Kontaktekzem

Zusammenfassung In Sensibilisierungsstudien mit 2,4-Dinitrochlorbenzol (DNCB) wird an 197 Meerschweinchen die Morpho-Histo-Genese der mit stets gleichen atoxischen Allergen-Dosen ausgelösten Epicutantest-Reaktionen systematisch analysiert. Insgesamt wurden 253 Test-Excisionen ausgewertet.Die Testapplikation verursacht bei epi- und intracutan sensibilisiertee Meerschweinchen epidermo-cutane, bei intravenös und intraperitoneal sensibiliserten Tieren dagegen cutane Reaktionen. Werden Versuchstiere mit cutanen Reaktionen anschließend mit DNCB epicutan nachbehandelt, so entstehen sekundär epidermocutane Epicutantest-Reaktionen.

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Summary Several groups of guinea pigs were sensitized by means of epi- and intracutaneous as well as by intravenous and intraperitoneal treatment with 2,4-dinitrochlorobenzene. From 197 animals 253 samples of epicutaneous test reactions produced in every case by the same patch test method, were excised and histologically analysed.The test areas from epi- and intracutaneously sensitized guinea pigs showed regularly an epidermo-cutaneous type of reaction. In animals with intravenous and intraperitoneal sensitization on the other hand appeared a cutaneous type of reaction. Guinea pigs with such cutaneous reactions got secondary, after additional epicutaneous treatment with 2,4-dinitrochlorobenzene, also characteristic epidermocutaneous test reactions.

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Mit Unterstützung des Senators für Wirtschaft, Berlin.     

Effect of vehicle on elicitation of DNCB contact allergy in the guinea pig

22 DNCB sensitized guinea pigs were challenged with varying amounts dissolved in alcohol, acetone and olive oil. DNCB applied in alcohol resulted in almost 100% positive reactions; the test scores correlated to dose. When similar amounts were applied in alcohol and acetone, the former produced a significantly higher degree of positivity. The importance of defining allergen concentration, volume or weight of test substance applied and test are size, when comparing test results in guinea pigs and humans, is emphasized.     

The mechanisms of histological incontinence of pigment: light and electron microscopic studies of lesions and patch test sites in female facial melanosis and DNCB reactions in guinea pigs

Histological incontinence of pigment (HIP) was studied using light and electron microscopy in a pigmented lesion from a female facial melanosis (FFM) patient and in another lesion produced by isoeugenol when the patient was patch tested. The following sequence of events is thought to underlie HIP. Phagocytes invade the epidermis and phagocytize melanosomes either in keratinocytes or in cytoid bodies, which are degenerated keratinocytes. These phagocytes then return to the dermis through gaps in the basal lamina. In brown guinea pigs sensitized to DNCB, 0.02% DNCB in acetone was applied repeatedly to the same area of the abdomen once per week for six weeks. HIP was observed in 28% of the animals following the fifth application of DNCB. In contrast to the HIP process observed in the patient, phagocytes in brown guinea pigs phagocytized free melanosomes. In irritant reactions to DNCB in brown guinea pigs, only concentrations which produced epidermal necrosis induced HIP.     

The sensitization potential of D & C Yellow No. 11 in guinea pigs

The certified drug and cosmetic dye substance known as D & C Yellow No. 11 [2-(2-quinolyl)-1,3 indandione] was tested for its dermal sensitization potential in guinea pigs. An induction regimen consisting of a single footpad injection of Complete Freund's Adjuvant diluted with antigen, and an intradermal injection of antigen, produced delayed-type hypersensitivity reactions. A high sensitization frequency was observed with 50 μg D & C Yellow No. 11 and the positive control 2,4-dinitrochlorobenzene (DNCB). The reaction severities of both DNCB and 50 μg D & C Yellow No. 11 were comparable, A dose-response profile was obtained with D & C Yellow No. 11 employing several concentrations (50, 250 and 500 μg/ml). Histologic examination of selected skin sites demonstrated a celluar inflammatory response which was consistent with delayed-type hypersensitivity.     

Prevention of nickel-induced allergic contact reactions with pentoxifylline

In this study, we investigated the effect of pentoxifylline, an inhibitor of TNF-α, on the contact sensitivity response induced by nickel. For induction, open epicutaneous sensitization by NiSO₄. 6H₂O (25% aq.) solution was applied on the backs of 38 albino guinea pigs 5 days a week for 4 weeks. NaCl (0.9%) solution was applied epicutaneously to 10 albino guinea pigs as a control group. 19 were sensitized by nickel and developed positive patch test reactions. Patch tests were repeated after 10 of the sensitized pigs were given pentoxifylline 20 mg/kg/day orally. At the end of this study, only 2 positive patch test reactions were observed in the pentoxifylline-treated group, while 7/9 of the untreated guinea pigs developed positive reactions. These results suggest that pentoxifylline inhibits the contact sensitivity response induced by nickel only during drug administration.     

Topical application of cyclosporine on guinea pig allergic contact dermatitis

Topical cyclosporine applied to the test site substantially inhibited the elicitation reaction of contact sensitivity in the guinea pigs previously sensitized with 2,4-dinitrochlorobenzene (DNCB). This suppressive effect of the drug was short lived and reversible. Cyclosporine was not effective when given six hours or later after antigenic challenge to the test site. Cyclosporine had no effect on the toxic contact reaction in normal animals either to croton oil or to DNCB in high concentration. Cyclosporine applied topically to the challenge site also resulted in a reduction of retest and flare-up reactions of contact sensitivity to DNCB, but did not affect the production of generalized rash in the same animals. These results indicate that in the future local topical application of cyclosporine may make treatment of human cutaneous immune-mediated disorders a possibility without serious side effects.     

A new animal model for contact dermatitis: the hairless guinea pig.

Allergic and irritant contact reactions were evaluated in the recently identified hairless guinea pig, Crl:IAF(HA)BR, a mutant from the Hartley strain. The cutaneous changes were observed macro- and microscopically. The irritant contact dermatitis was induced by croton oil, 2,4-dinitrochlorobenzene (DNCB), or anthralin. Both hairless and hairy guinea pigs developed similar reactions to these chemicals. The density of the epidermal Langerhans cells (LC) of hairless guinea pigs was significantly higher than that in the hairy strain. Allergic contact sensitization was easily induced with DNCB. Photoallergic contact sensitization was also induced with tetrachlorosalicylanilide (TCSA) but not with tribromosalicylanilide (TBS). However, by administration of cyclophosphamide before sensitization, positive photocontact responses were seen with TBS. These results indicate that hairless guinea pigs can be used as animal models for investigation of immunologic and nonimmunologic contact reactions.     

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.     

Subcutaneous fat necrosis of the newborn

I. A group of twenty-one Caucasian volunteers was patch tested without preliminary sensitization with 3, 10, 30, 50 and 90 mug 2,4-dinitrochlorobenzene (DNCB)/cm2. Of this group seven persons were read after 24 h; with one exception all showed a toxic erythematous reaction to 30 mug and higher amounts of DNCB/cm2. The other fourteen volunteers were read after 48 h. All except one reacted positively to 30 mug and higher amounts of DNCB/cm2. Histologically no changes could be noted after application of 3 and 10 mug DNCB/cm2; they started to appear in patch tests with 30 mug DNCB/cm2 or more. II. A second group of twenty-one Caucasian volunteers was sensitized with 2000 mug DNCB on a surface area of 3-14 cm2. Biopsies were performed after 1 h, 5 h, 1 day and then daily for 17 days. The histological changes occurring in the sensitization site during the primary irritant reaction of the first days were compared with the changes occurring during the flare-up reaction. No essential qualitative differences could be noted. III. Four Caucasian volunteers were sensitized with 2000 mug DNCB on 3-14 cm2. Fourteen days later challenge patch test doses of 3, 10, 30, 50 and 90 mug DNCB/cm2 were applied. Biopsies of the challenge sites were performed after 48 h. In contrast to the results in group I, histological changes could already be noted after challenge patch tests with 3 mug DNCB/cm2. The nature ofthe histological changes in primary irritant and allergic reactions to DNCB appeared to be identical and consisted of spongiosis, epidermal degeneration and lymphocytic infiltration around vessles and epidermal appendages and penetrating into the epidermal layers. It is concluded that, in order to avoid errors through misinterpretation, challenge patch tests with DNCB must be performed with low amounts, e.g. 3 or 10 mug/cm2.     

In vitro studies in nickel allergy: diagnostic value of a dual parameter analysis

A comparison was made between the diagnostic value of assaying nickel-induced lymphocyte proliferation (lymphocyte transformation test, LTT) and migration inhibition factor (MIF) production in nickel contact sensitivity. Although lymphocyte proliferation was significantly increased in the group of patients with skin test reactivity to nickel, positive LTT were also frequently found in skin test-negative subjects: in 63% of subjects with and in 30% of subjects without a history of metal allergy. This would limit the value of the LTT as an in vitro correlate of skin test reactivity. However, in certain patients positive lymphocyte transformation may reveal nickel sensitization at a time of undetectable skin reactivity. Data obtained with the macrophage migration inhibition test (MMIT) showed a good correlation with nickel patch test reactions. Accurate determination of MIF became feasible by using cells from the human monocytoid cell line U937 as target cells in a microdroplet agarose assay. Using this MMIT, positive reactions occurred in 13% of the healthy controls and false-negative reactions were found in 26% of patients with positive skin test reactivity to nickel. As LTT and MMIT data appeared to be only weakly correlated in the individuals tested, a dual parameter analysis was performed. An excellent correlation [p = 1.8 (10(-8]] was found between skin test and in vitro reactivity for individuals with matching in vitro results (60% of all individuals tested). In those individuals with discordant in vitro data, skin testing will remain indispensable for diagnosing nickel allergy.     

Optimization of an in vitro lymphocyte blastogenesis assay for predictive assessment of immunologic responsiveness to contact sensitizers

Current methods for the predictive and diagnostic assessment of contact sensitization rely on the visual scoring of skin reactions. Predictive animal tests, generally using guinea pigs, require a relatively large number of animals to produce a sufficient database for interpreting skin reaction scores. In vitro assays have the potential of being more quantitative than skin testing and, if so, would require fewer animals. However, although in vitro assays are commonly used to study the cellular immune response to strong contact sensitizers, there has been little effort to validate them for predictive assessment purposes. We have optimized an in vitro lymphocyte blastogenesis assay for detecting the response of mouse lymphocytes to strong contact sensitizers with the eventual objective of applying this assay to moderate and weak sensitizers as well. Lymph node lymphocytes from mice sensitized to the strong contact allergens, dinitrochlorobenzene (DNCB), dinitrofluorobenzene (DNFB), or trinitrochlorobenzene (TNCB), responded [greater than or equal to 12,000 counts per minute (CPM) above background] when cultured with water soluble chemical analogues, di- or trinitrobenzene sulfonic acid (DNBS or TNBS). However, the strong sensitizer, oxazolone (OXAZ), has no water soluble analogue and lymphocytes from mice sensitized to OXAZ responded poorly in vitro (less than 2000 CPM) to an ethanol-solubilized OXAZ preparation in spite of very strong in vivo sensitization (ear swelling assay). To increase the assay sensitivity, for OXAZ, we modified the antigen presentation conditions by using 1) solubilized antigen-modified adherent spleen cells, 2) dendritic cells from the draining lymph nodes of antigen painted mice, and 3) antigen-modified Langerhans cell-enriched cultured epidermal cells (EC). These approaches increased OXAZ-directed responses to greater than 7000, greater than 20,000, and greater than 100,000 CPM, respectively, under culture conditions optimized for cell density, responder: stimulator cell ratio, culture duration, and responder cell type. Our results represent a first attempt to directly modify cultured epidermal cells with OXAZ and use these cells to stimulate OXAZ-directed blastogenesis in microtiter plate cultures. This optimized assay is now under evaluation for predictive assessment of contact sensitizers relevant to occupational and consumer exposures.     

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.     

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.     

Immunological studies in psoriasis. The quantitative evaluation of cell-mediated immunity in patients with psoriasis by experimental sensitization to 2,4-dinitrochlorobenzene.

Quantitative techniques of sensitization to 2,4-dinitrochlorobenzene (DNCB) was used to determine in psoriasis the intensity and frequency of allergic reactions to DNCB following primary challenge with 2,000 microgram allergen and secondary challenge with decreasing doses of DNCB. 56 patients with psoriasis and 23 healthy volunteers were examined. Frequency of positive reactions to DNCB was similar in both groups, since all normal controls were sensitized, whereas only 8 of 56 psoriasis cases failed to develop delayed hypersensitivity to DNCB. However, the intensity of acquired contact allergy was significantly diminished in psoriasis in comparison with controls. The patients with stationary skin lesions resembled the normal population in the intensity of reaction to DNCB. Decreased intensity of DNCB sensitization seemed to be related to the activity of the disease, but not correlated with the extent of the lesions. A relationship was found between reduced reactivity to DNCB and decrease in E rosette-forming lymphocytes. The data suggest that the impaired function of T lymphocytes in active psoriasis could be responsible for both, defective recognition of contact antigens, such as DNCB, and the alteration of secondary response to DNCB.     

The morphology of chromium allergic skin reactions at electron microscopic resolution: studies in man and guinea pig.

Previous studies on clinical patch test reactions have been expanded to short-term studies in humans hypersensitive to chromium. Preliminary results are discussed in relation to experiments in normal and sensitized guinea pigs.     

Modified short-term guinea pig sensitization tests for detecting contact allergens as an alternative to the conventional test

The conventional adjuvant and patch test (APT) method of guinea pig sensitization testing was modified in 2 ways, s-APT and s-APT(2), in order to shorten the test period. These short-term test methods consist of 72-h closed application of test material with intradermal injection of emulsified Freund's complete adjuvant (E-FCA) for 1st induction, 48-h closed application of test material with (s-APT) or without (s-APT(2)) intradermal injection of E-FCA on the 7th day for 2nd induction, and open application on the 14th day for challenge. They were compared with conventional APT by using 8 allergenic chemicals (formaldehyde, nickel sulfate, cobalt sulfate, ethyl-p-aminobenzoate (benzocaine), isoeugenol, 2-mercaptobenzothiazole, 2,4-dinitrochlorobenzene (DNCB) and 1-phenylazo-2-naphthol (Sudan I)). The short-term methods gave similar results to those of conventional APT in terms of mean response, sensitization rate and sensitization potency (challenge concentration that induces a mean response equal to 1.0). Thus, our short-term methods, which are capable of evaluating skin sensitization within 17 days, are sufficiently sensitive to detect potentially hazardous contact allergens.     

The loss of contact sensitization in man

Little is yet known about the duration of contact sensitivity, but frequent exposure of a target to allergen seems to reduce skin reactivity. The aim of this study was to study the persistence of a specific contact sensitivity in 66 patients with alopecia areata, previously sensitized to DNCB (31 patients) and SADBE (35 patients) between 1978-1985. Patch tests were performed with 0.020 ml of different concentrations of DNCB or SADBE in acetone (0.05%, 0.10%, 0.20%, 1%). The results were read in a standardized manner. Of 66 patients, 47 (71%) had positive reactions and 19 (29%) negative. 8 of the 19 negative patients had been treated with DNCB, 11 with SADBE. Approximately 1/3 of the patients previously sensitized had lost their original sensitivity, and this did not seem to be time-dependent. This phenomenon seemed to be clinically correlated because the majority of the patients were from the "low responders" group. We think that acquired unresponsiveness to topical antigen in man is a possible phenomenon, but that it occurs more rarely than in mice and guinea pigs.     

Comparison of methods for the macroscopic assessment of epicutaneous allergic contact reactions in guinea pigs

40 guinea pigs were sensitised with a 50% solution of 2,4-dinitro-1-chlorobenzene (DNCB) and challenged 14 days later with DNCB 0.05%. Four parameters were determined to evaluate the challenge reaction after 24, 48, 72 and 96 h: (a) intensity of erythema, (b) reaction area (product of the largest diameters of the reaction in vertical alignment), (c) increase in skinfold thickness and (d) reaction volume (product of the reaction area and the increase in skinfold thickness). The test reactions were read blind by 2 independent observers, yielding small but significant differences in all methods except determination of the reaction area. Further statistical analysis revealed a linear correlation between the intensity of erythema and the other 3 parameters determined, as well as between the reaction area and the reaction volume. In contrast, the increase in skinfold thickness did not correlate linearly either with the reaction area or the reaction volume. When the results of the 24- and 48-h readings were compared, the characteristic crescendo reaction of contact allergy was demonstrable by all methods except the determination of the reaction area. After the 48-h reading, a continuous decrease of reactions was found with all methods. It is concluded that the determination of the reaction area and consequently of the reaction volume are not suitable for exact measurement of epicutaneous allergic contact reactions in guinea pigs. The most precise results will be obtained by measuring the increase in skinfold thickness, whereas the determination of the intensity of erythema, which is easier to perform, may be sufficient for many purposes.(ABSTRACT TRUNCATED AT 250 WORDS)     

AN IN VIVO AND IN VITRO STUDY OF THE CYCLOPHOSPHAMIDE-INDUCED ENHANCEMENT OF CONTACT SENSITIVITY TO 2,4-DINITRO-1-CHLOROBENZENE (DNCB)

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