In vitro studies of poison oak immunity. II. Effect of urushiol analogues on the human in vitro response.

Studies were performed to ascertain the effect of urushiol analogues on the in vitro lymphocyte blastogenesis elicited by urushiol in peripheral blood lymphocytes taken from individuals sensitized to poison oak or ivy. Urushiol is a mixture of alkylcatechols composed of a catechol ring coupled to mono-, di-, or tri-unsaturated C-15 or C-17 carbon side chains. Each of these two moieties, catechol ring and side chain, was tested for its role in eliciting reactivity. Analogues tested represented the catechol ring (3-methylcatechol), the mono- or di-unsaturated side chain (oleic or linoleic acid), and the saturated side chain coupled to a catechol ring (pentadecylcatechol), a blocked catechol ring (heptadecylveratrole), or a resorcinol (pentadecylresorcinol). Urushiol with a blocked catechol ring (urushiol dimethyl ether) was also included. Of these, only pentadecylcatechol evoked reactivity in sensitized lymphocytes, and this reactivity was only a fraction of that evoked by urushiol. This suggested that the system has some requirement for the side chain, and that the catechol ring is critical for reactivity. This was further investigated by testing the ability of some of these analogues to inhibit urushiol-specific blastogenesis. No inhibition was noted with compounds bearing the saturated side chain with modified ring structures (pentadecylresorcinol and heptadecylveratrole). However, both 3-methylcatechol and pentadecylcatechol (at equimolar concentrations) blocked reactivity. The results of our experiments suggested that although both the side chain and the catechol ring are required for reactivity, the latter is most critical. Unsaturation in the side chain is important for maximal reactivity because the saturated catechols were only partially as active as the urushiol oil. There may be a greater dose requirement for the catechol ring than for the side chain.

In vitro studies of poison oak immunity. I. In vitro reaction of human lymphocytes to urushiol.

Poison oak, ivy, and sumac dermatitis is a T-cell-mediated reaction against urushiol, the oil found in the leaf of the plants. This hapten is extremely lipophilic and concentrates in cell membranes. A blastogenesis assay employing peripheral blood lymphocytes obtained from humans sensitized to urushiol is described. The reactivity appears 1--3 wk after exposure and persists from 6 wk to 2 mon. The dose-response range is narrow, with inhibition occurring at higher antigen concentrations. Urushiol introduced into the in vitro culture on autologous lymphocytes, erythrocytes and heterologous erythrocytes produces equal results as measured by the optimal urushiol dose, the intensity of reaction, and the frequency of positive reactors. This suggests that the urushiol is passed from introducer to some other presenter cell. Although the blastogenically reactive cell is a T cell, there is also a requirement for an accessory cell, found in the non-T-cell population, for reactivity. Evidence is presented that this cell is a macrophage.

Processing of urushiol (poison ivy) hapten by both endogenous and exogenous pathways for presentation to T cells in vitro.

The antigen processing requirements for urushiol, the immunogen of poison ivy (Toxicodendron radicans), were tested by presentation of urushiol to cultured human urushiol-responsive T cells. Urushiol was added to antigen-presenting cells (APC) either before or after fixation with paraformaldehyde. Three distinct routes of antigen processing were detected. CD8+ and CD4+ T cells, which were dependent upon processing, proliferated if urushiol was added to APC before fixation, but did not proliferate when urushiol was added to APC after fixation. Processing of urushiol for presentation to CD8+ T cells was inhibited by azide, monensin, and brefeldin A. This suggests that urushiol was processed by the endogenous pathway. In contrast, presentation of urushiol to CD4+ T cells was inhibited by monensin but not by brefeldin A. This was compatible with antigen processing by the endosomal (exogenous) pathway. Finally, certain CD8+ T cells recognized urushiol in the absence of processing. These cells proliferated in response to APC incubated with urushiol after fixation. Classification of contact allergens by antigen processing pathway may predict the relative roles of CD4+ and CD8+ cells in the immunopathogensis of allergic contact dermatitis.

Immunologic studies of poisonous Anacardiaceae: I. Production of tolerance and desensitization to poison Ivy and oak urushiols using esterified urushiol derivatives in guinea pigs.

The development of contact sensitivity to poison ivy urushiol in Hartley guinea pigs was inhibited by i.v. injection of the diacetate esters of poison ivy and oak urushiols into guinea pigs 2 weeks prior to attempted sensitization with homologous antigen. Immune tolerance to urushiols of poison ivy and oak developed in 80% or more of the treated animals and persisted for the duration of the study, 8 weeks. The tolerance was immunologically specific for urushiols since the tolerant animals were sensitizable to the unrelated sensitizer 2, 4-dinitrochlorobenzene. Guinea pigs already sensitive to urushiol were also desensitized or hyposensitizied by i.v. injection of urushiol acetates in successively increasing doses. After receiving the equivalent of 16 mg of poison ivy and oak urushiols in the acetate form over a period of 12 weeks, 54% of a group of guinea pigs were desensitized to poison ivy. all of the remaining 46% of the guinea pigs still sensitive to poison ivy were substantially hyposensitized (no longer responded to 1.5 or 0.80 microgram test doses of PDC). A control group of guinea pigs was not hyposensitized by injection of vehicle, and remained highly sensitive throughout the 15 week study. The majority of treated animals (less than 80%) were also hyposensitized to poison sumac and cashew nut shell liquid allergens.

Allergic contact dermatitis to tulips: an example of enantiospecificity.

Allergic contact dermatitis (ACD), an immunological reaction of the skin resulting from contact with reactive compounds occurring in plants was shown to the enantiospecific (animals sensitized to a compound do not react to its nonsuperimposable mirror image). Thus, when guinea pigs were experimentally sensitized to (+)-tulipalin B (a compound present in tulip bulbs) they did not react to its enantiomer, (-)-tulipalin B. This was also true for (+)- and (-)-beta-hydroxy-gamma-methyl-alpha-methylene-gamma-butyrolactones.

Oral and parenteral vaccination of mice with protein-ergotamine conjugates and evaluation of protection against fescue toxicosis.

Acremonium coenophialum produces ergopeptide alkaloids in tall fescue (Festuca arundinacea Schreb.). These ergot alkaloids decrease serum alkaline phosphatase (ALP) activity, serum cholesterol and prolactin concentrations, as well as average daily gains (ADG) in cattle. The objective of this study was to evaluate the protection of anti-ergotamine antibodies induced by either oral or parenteral vaccination with protein-ergotamine conjugates or passive vaccination with anti-ergovaline, monoclonal antibodies in a murine model of fescue toxicosis. Ergotamine (EG) was conjugated to bovine serum albumin (BSA) and cholera toxin subunit B (CTB) by the Mannich reaction. Mice were blocked based on weight and randomly allocated into five groups of 10 mice each. Treatment groups were as follows: (1) group vaccinated intraperitoneally (ip) with a BSA-EG conjugate and fed an endophyte-infected (EI) fescue diet (BSA-EG group); (2) group orally vaccinated with a CTB-EG conjugate mixed with free cholera toxin (CT) and fed an EI fescue diet (CTB-EG group); (3) nonvaccinated group fed an EI fescue diet (EI group); (4) group passively vaccinated with anti-ergovaline, monoclonal antibodies and fed an EI fescue diet (MoAB group); and (5) nonvaccinated group fed an endophyte-free (EF) fescue diet (EF group). The EI diet contained 1.5 ppm of Ergovaline (EV), whereas no EV was detected in the EF diet.Respective diets were similar upon nutritional analysis. Unvaccinated mice in the EI group exhibited features of fescue toxicosis as indicated by decreased serum ALP activity and cholesterol, and decreased weight gain as compared to mice in the EF group. Antibodies against EG and EV were present in sera of mice in the BSA-EG and MoAB groups, respectively. Mice orally vaccinated with the CTB-EG conjugate developed secretory IgA (sIgA) antibodies and short-lived, systemic IgG responses against EG. Weight gains were increased in the BSA-EG and CTB-EG groups and tended to be increased in the MoAB group vs. the unvaccinated EI group. Serum ALP activity was decreased in the BSA-EG and MoAB groups as compared to the EF group. Serum ALP activity was further decreased in the BSA-EG vaccinated group as compared to the EI group. Cholesterol concentrations were decreased in the EI, BSA-EG and MoAB groups as compared to the EF group. Prolactin concentrations were similar in all groups.

Evaluation of physiologic indices in mice vaccinated with protein-ergotamine conjugates and fed an endophyte-infected fescue diet.

OBJECTIVES: To assess effects of vaccination against fescue toxicosis on weight gain, serum prolactin and cholesterol concentrations, and alkaline phosphatase (ALP) activity in mice fed an endophyte-infected (EI) or endophyte-free (EF) fescue diet. ANIMALS: 50 six-week-old male BALB/c mice. PROCEDURE: Mice were randomly allocated to the following 5 groups: 1, vaccinated intraperitoneally with a bovine serum albumin-ergotamine (EG) conjugate and fed an EI fescue diet; 2, orally vaccinated with cholera toxin (CT) subunit B-EG conjugate mixed with free CT and fed an EI fescue diet; 3, not vaccinated and fed an EI fescue diet; 4, passively vaccinated with monoclonal antibodies specific for ergovaline (EV) and fed an EI fescue diet; and 5, not vaccinated and fed an EF fescue diet. RESULTS: Antibodies against EG and EV were in serum of mice of groups 1 and 4, respectively. Secretory IgA and IgG coproantibodies against EG were induced in mice of group 2. Weight increased in groups 1 and 2 and tended to be increased in group 4 versus group 3. Prolactin concentration was similar in all groups; cholesterol concentration was decreased in groups 1, 3, and 4, compared with group 5. Compared with that in group 5, serum ALP activity decreased in groups 1 and 4 and was further decreased in group 1, compared with that in groups 2 and 3; it was negatively correlated with anti-EG titer. CONCLUSIONS AND CLINICAL RELEVANCE: Induction of anti-EG antibodies and administration of EV monoclonal antibodies tended to increase short-term weight gain in this murine model of fescue toxicosis. However, systemic IgG antibodies against EG or EV antibodies were not protective against decreases in serum ALP activity and cholesterol concentrations. Clinical significance of decreased ALP activity associated with vaccination is unknown, but represents a worsening of a response often associated with fescue toxicosis in cattle.

Detection and characterization of bovine rumen microorganismsresistant to sodium fluoroacetate

Background: Poisoning of animals due to toxic plants is found in Brazil and other countries. One of the known toxic plantsin Brazil, with the active ingredient sodium fluoroacetate (SF), is Palicourea marcgravii. Dehalogenases that inactivatethe fluor-carbon bonds are enzymes found in microorganisms and may prevent intoxication. This study evaluated the occurrence of rumen microorganisms naturally resistant to SF.Materials, Methods & Results: Two samples of rumen fluid of cattle from the Experimental Farm of Federal University ofMato Grosso fed with Brachiaria sp. were obtained via fistula in flasks. An aliquot of 2 mL was placed in a microtube andcentrifuged at 9000 g for 1 min. Then, the sample was inoculated into 2 tubes, one containing 100 µL of clarified rumenfluid in 2 mL of modified liquid culture medium (0.1% ammonium sulfate, 0.1% potassium phosphate monobasic, 0.05%sodium phosphate dibasic, 0.01% magnesium sulfate, 0.01% yeast extract, pH 7.0) and 0.4% of SF and the other samplecontaining 2 mL of liquid culture medium and 100 µL of clarified rumen fluid. The 2 samples were incubated at 40°C for24 h. Dilutions were performed under the same conditions every 24 h until the attainment of microorganisms resistant toSF, and the finaldilution containing 50 µL of each sample was plated in the middle containing SF (0.4%) and incubated at40°C for 24 h for the isolation of bacteria. The bacterial colonies resistant to SF were identified by morphological methods, stained, and subjected to DNA extraction sequencing using the universal primers 27f and 1492r (16S rDNA) for theidentification of the bacterial genus using Blast DNA identity analysis. These bacteria were cultured with and without SF(0.4%), and the presence of fluoride ions was detected by an ion-selective electrode (fluoride) during incubation for 0, 30,60, 90, and 120 min. Two resistant microorganisms were isolated, one was a Gram-positive coccus and the other was aGram-positive rod...

Detection and characterization of bovine rumen microorganismsresistant to sodium fluoroacetate

Background: Poisoning of animals due to toxic plants is found in Brazil and other countries. One of the known toxic plantsin Brazil, with the active ingredient sodium fluoroacetate (SF), is Palicourea marcgravii. Dehalogenases that inactivatethe fluor-carbon bonds are enzymes found in microorganisms and may prevent intoxication. This study evaluated the occurrence of rumen microorganisms naturally resistant to SF.Materials, Methods & Results: Two samples of rumen fluid of cattle from the Experimental Farm of Federal University ofMato Grosso fed with Brachiaria sp. were obtained via fistula in flasks. An aliquot of 2 mL was placed in a microtube andcentrifuged at 9000 g for 1 min. Then, the sample was inoculated into 2 tubes, one containing 100 µL of clarified rumenfluid in 2 mL of modified liquid culture medium (0.1% ammonium sulfate, 0.1% potassium phosphate monobasic, 0.05%sodium phosphate dibasic, 0.01% magnesium sulfate, 0.01% yeast extract, pH 7.0) and 0.4% of SF and the other samplecontaining 2 mL of liquid culture medium and 100 µL of clarified rumen fluid. The 2 samples were incubated at 40°C for24 h. Dilutions were performed under the same conditions every 24 h until the attainment of microorganisms resistant toSF, and the finaldilution containing 50 µL of each sample was plated in the middle containing SF (0.4%) and incubated at40°C for 24 h for the isolation of bacteria. The bacterial colonies resistant to SF were identified by morphological methods, stained, and subjected to DNA extraction sequencing using the universal primers 27f and 1492r (16S rDNA) for theidentification of the bacterial genus using Blast DNA identity analysis. These bacteria were cultured with and without SF(0.4%), and the presence of fluoride ions was detected by an ion-selective electrode (fluoride) during incubation for 0, 30,60, 90, and 120 min. Two resistant microorganisms were isolated, one was a Gram-positive coccus and the other was aGram-positive rod...(AU)

Hyposensitization to urushiol among Japanese lacquer craftsmen: results of patch tests on students learning the art of lacquerware.

8 subjects learning the art of lacquerware were patch tested to urushiol before and after contact with lacquer, in order to document whether hyposensitization to urushiol occurred among Japanese lacquer craftsmen. Simultaneously, we performed patch tests on 2 urushiol-sensitized controls who had no contact with lacquer during the investigation. Lacquer is made from the sap of the Japanese lacquer tree and raw lacquer is composed of 60-65% urushiol and its oligomer. 5 of the 8 subjects showed positive reactions to urushiol 1 month after their first contact. They became negative or less positive after prolonged (9 or 10 months) exposure to lacquer. As reactions to urushiol decreased, dermatitis became less severe. Controls showed consistently high reactions. However, 1 subject showed persistently strong reactions to urushiol. Unlike the other 7 subjects, he was previously sensitized to urushiol before the first contact with lacquer. The remaining 2 subjects showed no reaction throughout our investigation. These results strongly suggest that hyposensitization to urushiol does occur among Japanese lacquer craftsmen.

Immunoreactive leukotrienes in nettle plants (Urtica urens).

In order to clarify the mechanisms of urtication after contact with stinging plants, nettle (Urtica urens) hair and whole-plant extracts were examined for the presence of leukotriene (LT) B4 and LTC4 by reverse phase high-pressure liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) and for in vitro neutrophil chemotactic activity and histamine contents. Both hair and plant extracts contained high levels of LTB4 and LTC4 by RIA as well as histamine. The presence of LTB4 was supported by RP-HPLC elution profiles and by in vitro chemotaxis. Nettle hairs therefore resemble insect venoms and cutaneous mast cells with regard to their spectrum of mediators.

Screening for IgE mediated allergy among people working in the Marseilles harbour.

Screening for IgE mediated allergy by RASTs to professional (castor bean, green coffee, peanut, soy protein, wheat, rice), and non professional (pollens, mites, cat, Alternaria tenuis) air borne antigens among 36 people working in the Marseilles harbour has showed rather unexpected findings: only one case of IgE positivity to Dermatophagoides pteronyssinus (class I) and one case of IgE positivity to castor bean seed (Ricinus communis) (class IV). IgG4 specific antibodies against castor bean and green coffee were also measured by an ELISA technique, with eleven cases of positivity to castor bean and only one case to green coffee being recorded. Several explanations can be put forward for the low incidence of IgE responses to the commonest airborne antigens and to the professional antigens (castor bean being the only offender), and for the rather high incidence of specific IgG4 antibodies to castor bean. Most likely, the low incidence of latent atopy is the result of a natural selection among the workers who gave up their job if experiencing of discomfort. As far as the elevated IgG4 antibody levels to castor bean are concerned, these are probably natural blocking antibodies.

V beta-specific deletion of mature thymocytes induced by the plant superantigen Urtica dioica agglutinin.

Urtica dioica agglutinin (UDA), a plant protein, is a superantigen activating in a MHC class II-restricted manner the V beta 8. 3-bearing T-cells (Galelli and Truffa-Bachi, J. Immunol. 151, 1821, 1993). Administration of UDA to adult mice provokes the clonal expansion of the responding cells which is followed by the deletion of the major fraction of the UDA-sensitive cells, whereas the remaining cells become anergic (Galelli et al., J. Immunol. 154, 2600, 1995). We have analyzed the effect of UDA on thymocytes. Injection of UDA resulted in a rapid, but transient, deletion of a large fraction of the V beta 8.3-bearing mature T-cells. In contrast to other exogenous superantigens, this deletion was not preceded by the clonal expansion of the UDA-responding thymocytes. Moreover, the V beta 8.3-bearing mature T-cells escaping the deletion were not anergic to an in vitro UDA restimulation. UDA and the other superantigens also differ as the general, V beta-unrestricted, thymic atrophy induced by classical superantigens was not observed with UDA.