Effects of clonidine on the dermal inflammatory cell response of experimental toxic and allergic contact reactions and intradermal hypersensitivity

In previous studies, the alpha 2-adrenoceptor agonist clonidine has been shown to suppress the wheal and flare reaction in guinea pigs sensitized to ovalbumin. This phenomenon has been further studied with special reference to effects on the dermal inflammatory cell infiltrate and mast cells. Clonidine lessens the degranulation of mast cells seen in control untreated immediate hypersensitivity reactions. Less neutrophils and eosinophils arrive to the treated reactions. Basophils and mononuclear cells (chiefly lymphocytes) which characterize the late phase of the wheal and flare reaction were not influenced by clonidine. Clonidine had a possible minimal effect on allergic contact (delayed hypersensitivity) reactions. The toxic contact reaction to croton oil (nonspecific cutaneous inflammation) was not affected.     

Transdermal clonidine application: Long-term results in essential hypertension

Summary Skin patches of a clonidine transdermal therapeutic system (clonidine-TTS) with a constant release rate of either 0.1 or 0.2 mg clonidine/24 h continuously over 7 days were used in 32 essential hypertensives. These self-adhesive drug delivery systems (3.5 cm²), which were affixed to the upper outer arm, were changed by the patients at weekly intervals.During a mean observation period of 7 months (range 1–19 months) transdermal clonidine reduced the blood pressure from 162±15/107±5 mmHg to normal values (diastolic 95 mmHg) in 63% of our patients. However, chronic use of clonidine-TTS was accompanied by a high frequency of contact dermatitis (type IV allergy) in nearly half of our patients (n=15, 47%). In 11 of these 15 patients transdermal clonidine administration had to be stopped because of intolerable local skin reactions (pruritus, erythema, vesiculation, and/or infiltration). Subsequent patch testing with all components of clonidine-TTS was performed in eight cases. Whereas in seven cases an allergic contact dermatitis to clonidine was found, only one patient showed an allergy to another component of clonidine-TTS (polyisobutylene).We conclude that this strikingly high incidence of local allergic skin reactions limits the use of clonidine-TTS in essential hypertension.     

Development of a concomitant nickel and chromium sensitization model in the guinea pig.

Although nickel allergy is the most frequent contact hypersensitivity in man, reports on successful nickel sensitization in experimental animals are scarce. Chromium hypersensitivity, on the other hand, is readily induced in guinea pigs. In this study we set out to obtain reproducible nickel sensitization in guinea pigs, in order to establish an animal model for immunospecific tolerance and desensitization studies in which two non-cross-reacting metal allergens, chromium and nickel, could be studied simultaneously. Strong and reproducible sensitization to nickel was achieved by injecting low amounts of Freund's complete adjuvant and nickel sulfate in a split-adjuvant procedure. Strong erythematous reactions were observed as early as 14 days after sensitization and could be elicited both by intradermal and open epicutaneous challenges. Optimal evaluation was with nickel sulfate administered epicutaneously in 40% dimethyl sulfoxide to enhance skin penetration. Hypersensitivity could be transferred with lymphocytes and not with serum. Sensitization procedures for nickel and chromium then could successfully be combined in a double sensitization procedure. With four different guinea pig strains no genetic restriction was observed for the induction of nickel or chromium sensitivity. However, for both metals a clear sex and age dependence was observed: female guinea pigs reached a higher degree of sensitization than males, whereas sensitization in young animals was relatively weak.     

土贝母皂苷抑制对苯二胺诱发豚鼠接触性皮炎效应的研究

目的:研究土贝母皂苷(Tubeimosides,TBMSs,简称皂苷)抑制对苯二胺诱发的豚鼠接触性皮炎的效应。方法:从土贝母块茎中提取分离出有效部位土贝母皂苷。实验所用皂苷的主要成分是土贝母苷甲和苷乙(简称苷甲、苷乙),以及微量土贝母苷丙(简称苷丙),其标志性成分苷甲的含量逸90%。采用异体和同体两种对照的局部涂皮封闭实验(Buehler test),研究皂苷对对苯二胺(p-phenylenediamine,PPDA)诱发豚鼠接触性皮炎的效应的影响。结果:异体和同体对照实验结果表明,皂苷提前或与PPDA 混合应用,均可剂量依赖性地改善,甚至完全抑制PPDA 诱发豚鼠接触性皮炎。一分子的土贝母皂苷能抑制或完全逆转6 到12 分子对苯二胺诱发的豚鼠接触性皮炎。结论:土贝母皂苷有显著抑制对苯二胺诱发豚鼠接触性皮炎的活性。     

Inhibition of delayed hypersensitivity reactions by a new agent, cis-1-methyl-4-isohexylcyclohexane carboxylic acid (IG-10)

A newly synthesized compound, cis-1-methyl-4-isohexylcyclohexane carboxylic acid (IG-10), has been reported as an inhibitor of delayed hypersensitivity reaction. In the present paper, the mechanisms regarding the inhibitory action of IG-10 was investigated on delayed hypersensitivity reactions. p-Phenylenediamine-induced contact dermatitis in guinea pigs was significantly inhibited when the drug was given in a dose of 100 mg/kg p.o. at various times after challenge with the antigen. IG-10 inhibited both contact dermatitis and monocytes or neutrophils infiltrations induced by picryl chloride in mice. A skin reaction, similar to that seen in the case of delayed hypersensitivity reaction, was induced by an intradermal injection of phytohemagglutinin-P (PHA-P) stimulated lymphocytes in guinea pigs. This reaction was a useful method for assessing the effect of drugs on the release of lymphokines, particularly skin reactive factor (SRF). IG-10 in a concentration of 10(-5) g/ml inhibited the release of SRF as well as the release of migration inhibitory factor (MIF) from guinea pig lymphocytes stimulated by PHA-P. The reduction of delta 4-3-ketone of aldosterone and/or hydrocortisone by rat liver homogenates was not affected with IG-10, unlike that seen in the case of glycyrrhizin.     

Production and isolation of guinea pig IgE antibody

Immunoglobulins of the IgE and IgG classes have been causally associated with hypersensitivity reactions in man and in numerous animal species including mice, rats and guinea pigs. The use of the guinea pig as an animal model for both pulmonary and dermal hypersensitivity reactions, and the recent recognition of the importance of IgE antibodies in both early- and late-onset hypersensitivity responses, has heightened interest in production, separation, and isolation of this immunoglobulin class from the guinea pig. IgE antibodies were produced by treatment of strain 13 guinea pigs with cyclophosphamide followed by injection with S. aureus enterotoxin. Serum was obtained and the globulin fraction isolated by addition of caprylic acid then ammonium sulfate. Immunoglobulins were separated into classes using fast protein liquid chromatography (FPLC) employing a Mono Q column and a linear gradient of 0.01-0.3 M Na,K phosphate buffer, pH 7.5 (buffer B). IgG eluted in two major peaks. IgG2 was not retained on the column and emerged with the starting buffer; IgG1 was eluted with 15-20% buffer B. IgE, detected as heat labile homocytotropic antibody, was found in the fraction eluting with 30-35% buffer B. The elution profile of the guinea pig immunoglobulins was predicted from the pattern obtained with immunoglobulin classes from other species. This chromatographic procedure enabled rapid isolation of immunoglobulin classes from guinea pig sera and effectively separated IgG1 from IgE, the two classes associated with hypersensitivity reactions.     

Nasal mucosal hypersensitivity in guinea pigs intermittently exposed to cold.

To develop an animal model for experimental nasal hypersensitivity and hyperreactivity, guinea pigs were subjected to intermittent exposure to cold temperature (intermittent cold stress, SART stress) for 5 consecutive days. In SART-stressed guinea pigs, nasal mucosal hypersensitivity to histamine evoking sneeze response and nasal hypersecretion in response to methacholine were observed. The hypersensitivity remained for further 7 days after being released from SART stress. On the other hand, such nasal mucosal hypersensitivity was not caused by a continuous cold stress alone, suggesting that intermittent exposure to cold may be of importance for the appearance of nasal mucosal hypersensitivity. In passively sensitized SART-stressed guinea pigs, the quantity of nasal secretion induced by an allergen was significantly increased compared with that of a group of normal animals. The expression of muscarinic acetylcholine receptor (m-ACh.R) became higher in SART-stressed guinea pigs. Thus, hypersensitivity and hyperreactivity in this system were found to be associated with an increase in density of m-ACh.R. SART-stressed guinea pigs will serve as an animal model for hypersensitivity in nasal mucosa, which would be useful for the study of nasal allergy.     

Delayed hypersensitivity to hapten-skin protein conjugates in guinea pigs sensitized to benzo(a)pyrene

Guinea pigs were sensitized to 3,4-benzo(a)pyrene, by epicutaneous application or by footbad injection in Freund's complete adjuvant. Conjugates of benzo(a)pyrene and guinea pig skin protein formed by ultraviolet radiation could elicit cutaneous delayed hypersensitivity and could inhibit the migration of macrophages obtained from guinea pigs sensitized to the carcinogen. Extracts of benzo(a)pyrene-treated guinea pig skin and conjugates formed in vitro with benzo(a)pyrene isocyanate were unable to consistently elicit delayed hypersensitivity reactions in vivo or in vitro. The results indicate a high degree of hapten-carrier specificity to contact sensitivity to benzo(a)pyrene.     

Immunological cross-reactivity between the collagen-like fragment of human Clq and human type I collagen

In guinea pigs immunized with human acid-soluble type I collagen in the native form immunological cross-reactivity with the collagen-like fragment of human Clq, CLF,4 was observed. At the cellular level, this fragment elicited a clear-cut delayed-type hypersensitivity reaction in 11 out of 20 collagen-immunized guinea pigs as demonstrated in skin tests after injection of 10–100 μg CLF. These 11 animals, and only these, also showed positive skin reactions when challenged with low doses of native collagen (2.0 μg). At the humoral level, only one animal had anticollagen antibodies that cross-reacted with CLF both in passive hemagglutination and solid-phase radioimmunoassay. Depending on the detection method used, only five (seven) animals showing cell-mediated cross-reactivity with CLF were also found to be anticollagen-antibody-positive.     

Effects of AHR-5333, a new potential antiallergy compound, in in vivo models of immediate hypersensitivity

AHR-5333 [1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1-piperidinyl] propoxy]-3-methoxyphenyl]ethanone] possessed potent, long-acting activity in rat and guinea pig in vivo models of immediate hypersensitivity: AHR-5333 was more potent than azatadine (2 X), ketotifen (approximately 3 X), oxatomide (approximately 5 X), albuterol (6 X) and aminophylline (approximately 50 X) in a passive, foot anaphylaxis model in rats and more potent than diphenhydramine (approximately 237 X), oxatomide (approximately 5.6 X) and theophylline (approximately 295 X) in guinea pigs challenged with aerosolized antigen. A long duration of action was noted after oral dosing of guinea pigs (24 h PD50, 0.78 mg/kg). Administration by aerosol (1%) to sensitized, spontaneously breathing, conscious guinea pigs protected against antigen-induced anaphylactic collapse; this protection persisted through 8 h. When administered prior to antigen challenge, AHR-5333 (10 mg/kg, p.o.) effectively inhibited ascaris antigen-induced skin hypersensitivity reactions in both dogs and cynomolgus monkeys.     

Delayed hypersensitivity to myxoviruses and myxovirus vaccines in the guinea pig

Influenza, mumps and measles viruses were examined for their ability to induce in guinea pigs homologous and cross-reactive delayed hypersensitivity. A majority of the animals skin tested with homologous and a portion of the animals skin tested with heterologous viruses and vaccines developed positive reactions. Findings with the heterologous preparations suggest that the observed cross-reactive hypersensitivity might be due to shared antigens of viral or substrate origin in the influenza, mumps and measles preparations. The present findings in guinea pigs suggest that the adverse effects, attributed in whole or in part to induced hypersensitivity, observed in man following injection of killed influenza, mumps and measles vaccines could be due to cross-reactive delayed hypersensitivity resulting from the use of these preparations.     

The How, Why and When of Immunological Testing

Immunotoxic effects include direct immunotoxicity, hypersensitivity and autoimmunity. Each effect requires a specific strategy to identify and assess the risks involved. At the moment, 2- or 3-tier protocols are considered the best modalities to detect an unexpectedly immunosuppressive compound and presumably an immunoenhancing compound as well. Such protocols are best conducted as 21–90 day rat studies. Contact sensitisation and anaphylaxis models in guinea pigs or mice are also useful tools to predict hypersensitivity. Finally, only a few systemic autoimmune reactions may be predicted in mice or rats using the popliteal lymph node assay. Thanks to improved standardisation and interlaboratory validation, an increased risk for infections and lymphomas (immunosuppression), contact dermatitis, anaphylaxis (macromolecular compounds only) and systemic autoimmune reactions (e.g. lupus) can reasonably be predicted. As few guidelines are available concerning the immunotoxicity assessment of novel chemicals, it is proposed that tier-1 and contact sensitisation should be performed routinely whatever the compound whereas additional testing may be required on a case-by-case approach, taking into account the modalities of exposure (e.g. dose and duration), the exposed population and the nature of the compound (e.g. pharmaceuticals).Originally presented at ECCP 93.     

Histological study of mast cells in the actively sensitized guinea pig lung and after challenge: effect of a corticosteroid

Although mast cells are sometimes considered to play a minor role in hypersensitivity reactions, we used a histological method to show the modifications in the guinea pig lung mast cell population in the course of such reactions. We sensitized guinea pigs with ovalbumin and studied the effect of the challenge with and without corticosteroid treatment. We observed that the mast cell count was not modified after sensitization but was decreased after challenge. Twenty-four hours after challenge, the number of mast cells returned to the control value, indicating a renewal of the mast cell pool. A second challenge, 1 week after the first, did not provoke the same mast cell degranulation, suggesting a non-responsiveness to aerosol antigen. Betamethasone dipropionate treatment protected mast cells against challenge: in treated guinea pigs, mast cell degranulation was prevented, and we did not observe any change in mast cell count after challenge. The present study was useful to show an effect of corticosteroids on mast cell degranulation in immediate hypersensitivity reactions in vivo.     

Human and guinea pig immune responses to Legionella pneumophila protein antigens OmpS and Hsp60.

We studied the immune responses of guinea pigs and humans to two Legionella pneumophila antigens. Guinea pigs surviving a lethal intraperitoneal challenge dose of virulent L. pneumophila exhibited strong cutaneous delayed-type hypersensitivity (DTH) reactions to purified OmpS (28-kDa major outer membrane protein) and Hsp60 (heat shock protein or common antigen), while weak DTH reactions were noted for extracellular protease (major secretory protein [MSP] [ProA]) and no reaction was observed with an ovalbumin (OA) control. Lymphocyte proliferation responses (LPRs) were measured for peripheral blood and spleen lymphocytes from guinea pigs surviving sublethal and lethal challenge doses of L. pneumophila. Lymphocytes from uninfected animals showed no proliferation to Hsp60 or OmpS, while lymphocytes from sublethally and lethally challenged animals exhibited strong proliferative responses to Hsp60 and OmpS. Guinea pigs vaccinated with purified OmpS exhibited low antibody titers and strong DTH and LPRs to OmpS, whereas lymphocytes from animals vaccinated with Hsp60 exhibited weak DTH responses and high antibody titers to Hsp60. All guinea pigs immunized with OmpS survived experimental challenge with L. pneumophila (two of two in a pilot study and seven of seven in trial 2) versus zero of seven OA-immunized controls (P = 0.006 by Fisher's exact test). In three vaccine trials in which animals were vaccinated with Hsp60, only 1 guinea pig of 15 survived lethal challenge. Peripheral blood lymphocytes (PBLs) from humans with legionellosis showed stronger LPRs to OmpS than PBLs from humans with no history of legionellosis (P = 0.0002 by Mann-Whitney test). PBLs of humans surviving legionellosis exhibited a lower but highly significant proliferative response to Hsp60 (P < 0.0001 compared with controls by Mann-Whitney test). These studies indicate that OmpS and Hsp60 are important antigens associated with the development of protective cellular immunity. However, as determined in vaccine trial studies in the guinea pig model for legionellosis, the species-specific antigen OmpS proved much more effective than the genus-common Hsp60 antigen.     

Hyperventilation-induced bronchoconstriction in guinea pigs

The purpose of this study was to investigate the pulmonary effects of hyperventilation in anesthetized, mechanically ventilated guinea pigs. Airway resistance (Raw), dynamic lung compliance (CDyn), blood pressure (BP), heart rate (HR), arterial blood gases (PaO2, PaCO2), pH and arterial plasma HCO3- were measured before and after a 10-min period of hyperventilation produced by increasing the respiratory rate from 60 to 120 breaths/min while maintaining tidal volume at 4 ml. There was a significant increase in Raw and decrease in CDyn lasting up to 20 min after hyperventilation was stopped with no change in BP and HR. PaO2 was reduced from 109 +/- 3 mm Hg before to 53 +/- 7 mm Hg at 5 min after hyperventilation. The Raw and CDyn changes were prevented and reversed with the bronchodilators salbutamol and aminophylline indicating that reversible bronchospasms are induced in guinea pigs following a period of hyperventilation. Additional studies demonstrated that the pulmonary mechanical responses to hyperventilation were not changed by vagotomy, ventilation with high CO2 or by pretreatment with chlorpheniramine, methysergide, atropine, indomethacin, FPL 55712 or calcium-influx blockers. These results indicate that neither vagal reflexes, airway hypocapnia, receptors of histamine, serotonin, acetylcholine nor the products of arachidonic acid metabolism were involved in hyperventilation-induced bronchospasm in guinea pigs.     

Inhibition of the platelet activating factor mediated component of guinea pig anaphylaxis by receptor antagonists

The role of platelet activating factor (PAF) and its inhibition by specific receptor antagonists was studied in hypersensitivity reactions in guinea pig lung parenchymal strips and in guinea pigs in vivo. Immunological challenge of isolated lung parenchymal strips with ovalbumin resulted in a contractile response of 184 +/- 16 mg (n = 38). Pretreatment of the strips with a combination of the antihistamine diphenhydramine, the dual lipoxygenase and cyclooxygenase product synthesis inhibitor BW-755C, and the PAF receptor antagonist kadsurenone totally inhibited the increase in tension. The bronchoconstrictor effects induced by immunological challenge were also totally inhibited when the leukotriene receptor antagonist FPL-55,712 was used to replace BW-755C or another PAF receptor antagonist, alprazolam was used to replace kadsurenone. Ovalbumin challenge in sensitized guinea pigs in vivo resulted in airway constriction, circulatory failure and an abrupt fall in continuously measured platelet count by 75 +/- 12%, followed by death of all animals within 5-8 min. Pretreatment with diphenhydramine, BW-755C and kadsurenone (or alprazolam) improved survival to 64% (i.e., 7 of 11 animals) compared to a survival rate of 0% (i.e., 0 of 8 vehicle treated controls). Despite the improved survival, the severe thrombocytopenia was unaltered with the combined therapy. PAF, histamine and peptide leukotrienes appear to act in concert in mediating the anaphylaxis-induced airway constriction and circulatory failure in guinea pigs, but are not apparently responsible for the accompanying thrombocytopenia.     

Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis.

We have studied the capacity of a selected fraction of Mycobacterium tuberculosis extracellular proteins (EP) released into broth culture by mid-logarithmic-growth-phase organisms to induce cell-mediated immune responses and protective immunity in a guinea pig model of pulmonary tuberculosis. Guinea pigs infected with M. tuberculosis by aerosol but not uninfected control guinea pigs exhibit strong cell-mediated immune responses to EP, manifest by dose-dependent cutaneous delayed-type hypersensitivity and splenic lymphocyte proliferation. Guinea pigs immunized subcutaneously with EP but not sham-immunized control guinea pigs also develop strong cell-mediated immune responses to EP, manifest by dose-dependent cutaneous delayed-type hypersensitivity and splenic lymphocyte proliferation. EP is nonlethal and nontoxic to guinea pigs upon subcutaneous immunization. Guinea pigs immunized with EP and then challenged with aerosolized M. tuberculosis exhibit protective immunity. In five independent experiments, EP-immunized guinea pigs were consistently protected against clinical illness, including weight loss. Compared with EP-immunized guinea pigs, sham-immunized control guinea pigs lost 12.9 +/- 2.0% (mean +/- SE) of their total weight. EP-immunized guinea pigs also had a 10-fold reduction in viable M. tuberculosis bacilli in their lungs and spleens (P = 0.004 and 0.001, respectively) compared with sham-immunized control animals. In the two experiments in which some guinea pigs died after aerosol challenge, EP-immunized animals were protected from death. Whereas all 12 (100%) EP-immunized guinea pigs survived challenge with aerosolized M. tuberculosis, only 6 of 12 (50%) sham-immunized control guinea pigs survived challenge (P = 0.007, Fisher exact test). This study demonstrates that actively growing M. tuberculosis cells release immunoprotective molecules extracellularly, that a subunit vaccine against tuberculosis is feasible, and that extracellular molecules of M. tuberculosis are potential candidates for a subunit vaccine.     

Experimental model for dermal granulomatous hypersensitivity in Q fever.

Q fever has been associated with granulomatous changes in clinical biopsy material obtained from liver and bone marrow. Local reactions to skin testing have been described in previously sensitized humans, but histological studies of such reactions have not been reported. We note that delayed hypersensitivity reactions to whole-cell phase I Q fever vaccine in immunized guinea pigs have a time course of development of induration characteristic of granulomatous hypersensitivity. Histological examination of such skin reactions on day 9 after testing revealed epithelioid cell infiltration and the presence of large numbers of multinucleated giant cells. Prominent in the sections were fragments of disintegrating polymorphonuclear leukocytes having the appearance of leukocytoclasis. Electron microscopic studies confirmed the presence of epithelioid changes in cells of the mononuclear phagocyte series, as well as extensive collagen deposition. This animal system affords a readily reproducible model of dermal granulomatous hypersensitivity and an opportunity to analyze the immunological basis of this reaction.     

Comparison of clinical and experimental data from an animal model of pulmonary immunologic sensitivity.

Isocyanates are highly reactive chemicals capable of causing a multitude of toxicologic effects including respiratory irritation, dermal irritation, contact sensitivity, and pulmonary hypersensitivity. In order to probe the mechanism(s) underlying these reactions, an animal model has been developed. The guinea pig model reproduces both the respiratory and immunologic effects of isocyanates that have been observed clinically. In experimental animals and in humans, isocyanates induce immunologic reactions with specific antibody formation, including IgE. This finding allows development of diagnostic reagents to assess isocyanate sensitivity. Further characterization of immunologic components in the model is expected to increase understanding of the mechanisms of this immunotoxic disease and develop strategies for treatment and prevention.     

A comparison of the effects of hydrocortisone on peritoneal inflammatory exudates, the tuberculin reaction and contact sensitivity-to DNFB in the guinea pig

Hydrocortisone acetate, injected subcutaneously in five doses in a depot producing vehicle, causes prolonged suppression of the ability of guinea pigs to produce a chronic inflammatory reaction in the peritoneal cavity and also of the tuberculin reaction. This suppression of the tuberculin reaction persists for at least 65 days after the last dose of hydrocortisone. Suppression of both peritoneal exudate and the tuberculin reaction was associated with a preferential depletion of macrophages at the reaction site. A similar regime had no effect on the ability of guinea pigs to manifest contact sensitivity reactions to 2,4-dinitrofluorobenzene although there was a similar drop in macrophages in the lesion. It appears that the effect of hydrocortisone on delayed hypersensitivity reactions is not directly related to the functional contribution of macrophages in the inflammatory reactions, and may be related to the site of the inflammatory reaction in the skin.