Effects of palmitoylethanolamide on the cutaneous allergic inflammatory response in Ascaris hypersensitive Beagle dogs

Palmitoylethanolamide (PEA) is an endogenous lipid mediator with anti-inflammatory and anti-hyperalgesic properties. The main objective of the present study was to evaluate the effects of PEA on the cutaneous allergic inflammatory reaction induced by different immunological and non-immunological stimuli in hypersensitive dogs. Six spontaneously Ascaris hypersensitive Beagle dogs were challenged with intradermal injections of Ascaris suum extract, substance P and anti-canine IgE, before and after a single oral administration of PEA at doses of 3, 10 and 30 mg/kg. A significant reduction in wheal area induced by both antigen and anti-canine IgE challenge was observed after PEA administration. No significant differences were observed between the two higher doses studied, suggesting that the 10 mg/kg dose had exerted the maximum inhibitory effect. When blood levels of PEA were compared with the effects at different times, an evident correlation was obtained. However, the anti-inflammatory effects of PEA were more long-lasting than their plasma concentrations. The intradermal injection of substance P did not reveal any skin reaction (wheal or erythema formation) at any of the concentrations tested. In conclusion, PEA might constitute a new therapeutic strategy for the treatment of allergic inflammatory skin diseases in companion animals.     

Effects of cyclosporin A and cilomilast on activated canine, murine and human keratinocytes

The calcineurin inhibitor cyclosporin A and the phosphodiesterase 4 inhibitor cilomilast exhibit potent immunomodulatory properties which make them interesting therapeutics for the treatment of skin disorders like canine and human atopic dermatitis. Cyclosporin A and phosphodiesterase 4 inhibitors have already demonstrated clinical efficacy in the therapy of canine and human atopic dermatitis. Their direct impact on keratinocytes, especially canine keratinocytes, is less obvious. Thus, an investigation was carried out to ascertain whether cyclosporin A and cilomilast modulate keratinocyte proliferation and secretion of proinflammatory mediators. Cyclosporin A inhibited canine and murine keratinocyte proliferation, whereas cilomilast had no affect. Cyclosporin A and cilomilast reduced the lipopolysaccharide-induced prostaglandin E2 synthesis in canine and murine keratinocytes. Both immunomodulators also inhibited the production of the CXC chemokine KC and CCL2 in the murine keratinocyte cell line MSC-P5. The two immunomodulators also significantly reduced the interferon-gamma-induced production of interferon-gamma-inducible protein 10 in human keratinocytes (HaCaT cells). Thus, cyclosporin A and cilomilast directly modulate keratinocyte functions which might contribute to the anti-inflammatory and immunomodulatory action of these compounds in the treatment of allergic skin diseases.     

Skin mast cell histamine release following stem cell factor and high-affinity immunoglobulin E receptor cross-linking in dogs with atopic dermatitis

Stem cell factor (SCF) influences mast cell activation and inflammatory mediator release, and is elevated in tissues undergoing allergic inflammation. Wheal formation in response to the injection of SCF or anti-immunoglobulin (Ig)E antibody injection was compared between normal (n = 10) and nonlesional atopic (n = 10) canine skin. In situ SCF secretion was compared between lesional and nonlesional skin using immunohistochemistry. Histamine release by skin cell suspensions after stimulation with SCF, concanavalin A (ConA) or rabbit anticanine IgE antibodies was compared between normal and atopic dogs. All dogs exhibited strong responses to intradermal SCF injection at 10 and 50 ng mL(-1). Atopic dogs had significantly (P = 0.002) larger wheal responses to anti-IgE than normal dogs; but there was no difference in numbers of skin mast cells bearing IgE as detected by immunohistochemistry. Only atopic dogs exhibited interstitial deposition of SCF in both lesional and nonlesional skin specimens. Median histamine release stimulated by SCF in the absence of IgE from lesional skin cells was higher in atopic than normal dogs (P = 0.04). These experiments suggest that dermal SCF secretion could potentiate histamine release following IgE receptor cross-linking and thus, could be one of the explanations for the inherent mast cell hyperexcitability observed in canine atopic dermatitis.     

Duration of inhibition of immediate skin test reactivity by hydroxyzine hydrochloride in dogs

The duration of hydroxyzine-mediated suppression of the immediate hypersensitivity reaction in the skin of dogs was assessed by intradermal administration of various dilutions of histamine phosphate and of aqueous flea antigen in 18 dogs known to be allergic to fleas. Wheal diameters and scores were used to evaluate the strength of the resulting reactions. In most dogs, significant (P less than 0.05) inhibition lasted from 3 to 5 days after withdrawal from treatment. Some dogs took up to 9 days to equal or exceed their pretreatment wheal diameters and scores.     

Inhibition of histamine release from dispersed canine skin mast cells by cyclosporin A, rolipram and salbutamol, but not by dexamethasone or sodium cromoglycate

Despite the important role that canine skin mast cells play in IgE-mediated allergic inflammation, clinically useful compounds for modulating mediator release from these cells or for suppressing cell response are lacking in the dog. The ability of five compounds to inhibit histamine release induced by non immunological (calcium ionophore A23187 and substance P) and IgE-dependent (concanavalin A) stimuli were compared. Sodium cromoglycate, a mast cell stabilizer, and dexamethasone, a glucocorticoid, failed to inhibit histamine release from isolated skin mast cells following any kind of stimulation. Salbutamol, a β-adrenergic agonist, exhibited inhibitory activity (46.0%) only after concanavalin A activation. In contrast, rolipram, a selective phosphodiesterase IV inhibitor and cyclosporin A, an immunosuppressor, showed potent anti allergic actions, inhibiting both IgE-dependent and -independent stimuli. Rolipram inhibited 42.8%, 44.7% and 19.2% of the mediator release induced by ionophore A23187, substance P and concanavalin A, respectively. Similarly cyclosporin A induced 85.9%, 14.9% and 67.3% inhibition after ionophore A23187, substance P and concanavalin A stimulation, respectively. These results suggest that rolipram and cyclosporin A merit to be clinically tested as agents for the treatment of chronic allergic diseases in the dog.     

Effects of a 1 per cent hydrocortisone conditioner on the prevention of immediate and late-phase reactions in canine skin

Ten laboratory beagles were used to determine whether a 1 per cent hydrocortisone conditioner applied topically for three consecutive days would inhibit IgE-mediated immediate and late-phase reactions induced in their skin. The trial was blinded, controlled with the product's vehicle and designed with a crossover. It consisted of three phases: one period without treatment (control phase) and two periods of treatment with either the active ingredient or the vehicle. Immediate and late-phase reactions were induced by the intradermal injection of rabbit anti-canine IgE polyclonal antibodies. Twenty minutes after the intradermal challenge, the diameter of the wheal, but not the erythematous flares, were significantly reduced after the application of the active product. In contrast, IgE-mediated cutaneous late-phase reactions, evaluated by measurements of dermal thickness and eosinophil counts six hours after challenge and the numbers of dermal CD3-positive T lymphocytes after 24 hours, were not reduced by its application.     

Stem cell factor enhances IgE-mediated histamine and TNF-alpha release from dispersed canine cutaneous mast cells

Stem cell factor (SCF), the c-kit receptor ligand, plays a critical role in mast cell (MC) development and differentiation. In addition, SCF has recently been found to both modulate and induce MC activation. To investigate the effect of SCF on canine cutaneous MC function, we have characterized the ability of SCF to modulate the release by mature canine MC of preformed (histamine) and newly generated (TNF-alpha) mediators. Mature MC were isolated from skin and cultured in the absence or presence of exogenous SCF (6 ng/ml) for up to 5 days and then challenged with anti-IgE (1 microg/ml) alone for 30 min or with a combination of SCF (50 ng/ml) and anti-IgE. SCF alone failed to trigger either histamine or TNF-alpha release at any time. However, we observed that SCF used as a co-stimulus significantly potentiated histamine and TNF-alpha release in canine MC activated through Fc epsilon RI regardless of whether or not SCF was added to the medium during culturing. Thus, the mean histamine release (%) and TNF-alpha production (pg/ml) were found to be significantly higher if cells were maintained in culture in SCF-supplemented medium compared with cells cultured in the absence of exogenous SCF. We also observed that MC responsiveness to immunological stimulation increased with culture time, the percentage of histamine released being higher in cells cultured for at least 3 days when compared to freshly isolated MC. Taken together these findings suggest that canine skin MC releasability can be enhanced independently either through prolonged incubation with SCF and/or through anti-IgE and SCF co-stimulation.     

A histamine release assay to identify sensitization to Culicoides allergens in horses with skin hypersensitivity

Skin hypersensitivity is an allergic disease induced in horses by allergens of Culicoides midges. The condition is typically diagnosed by clinical signs and in some horses in combination with allergy testing such as intradermal skin testing or serological allergen-specific IgE determination. Here, we describe an alternative method for allergy testing: a histamine release assay (HRA) that combines the functional aspects of skin testing with the convenience of submitting a blood sample. The assay is based on the principle that crosslinking of allergen-specific IgE bound via high-affinity IgE receptors to the surfaces of mast cells and basophils induces the release of inflammatory mediators. One of these mediators is histamine. The histamine was then detected by a colorimetric enzyme-linked immunosorbent assay. The histamine assay was used to test 33 horses with skin hypersensitivity and 20 clinically healthy control animals for histamine release from their peripheral blood basophils after stimulation with Culicoides allergen extract or monoclonal anti-IgE antibody. An increased histamine release was observed in the horses with skin hypersensitivity compared to the control group after allergen-specific stimulation with Culicoides extract (p=0.023). In contrast, stimulation with anti-IgE induced similar amounts of released histamine in both groups (p=0.46). For further evaluation of the HRA, we prepared a receiver operating-characteristic (ROC) curve and performed a likelihood-ratio analysis for assay interpretation. Our results suggested that the assay is a valuable diagnostic tool to identify sensitization to Culicoides allergens in horses. Because some of the clinically healthy horses also showed sensitization to Culicoides extract, the assay cannot be used to distinguish allergic from non-allergic animals. The observation that sensitization is sometimes detectable in non-affected animals suggested that clinically healthy horses use immune mechanisms to control the reaction to Culicoides allergens that are different or absent in allergic horses.     

Comparative studies of experimental oral inoculation of pigs with Ascaris suum eggs or third stage larvae

This experimental study was designed to compare the acquired resistance in pigs to Ascaris suum eggs following 4-weekly oral immunizations with either 200 A. suum infective eggs or 50 A. suum third stage larvae (L3). The two immunized groups (n = 7) together with an unimmunized control group (n = 7) of pigs were challenged orally with 50 infective A. suum eggs per kilogram bodyweight on day 19 after the last immunization. Seven days post-challenge the group immunized with eggs showed signs of resistance as evidenced by reduced lung larval counts compared with the challenge control group. Such significant resistance was not observed in the L3-immunized group. However, a markedly increased inflammatory liver reaction and white spot formation was demonstrated in the L3-immunized pigs after challenge compared with both control animals and egg-immunized pigs. On the day of challenge only the egg-immunized pigs mounted an anti-Ascaris antibody response both in serum and in lung lavage fluid. Ascaris-antigen induced increased histamine release from peripheral leucocytes following both immunization and challenge could only be demonstrated in the egg-immunized pigs. On day 7 post-challenge local IgA-anti-Ascaris antibodies were further demonstrated in bile of the egg-immunized group and in the small intestine of both immunized groups. In conclusion, oral A. suum egg immunization of pigs induced a significant reduction in lung larval counts upon challenge. In contrast, oral L3 immunization seemed to prime the pigs as observed by the presence of stunted lung larval growth and increased liver reaction post-challenge with A. suum eggs.     

Persistent activity of doramectin and ivermectin against Ascaris suum in experimentally infected pigs.

A study was conducted to investigate the persistent nematocidal activity of two avermectins against experimentally-induced infections of Ascaris suum in swine. Seventy-two nematode-free cross-bred pigs of similar bodyweight were randomly allotted to nine treatment groups of eight pigs each. Eight of the groups were treated with injectable solutions containing 300 microg of doramectin/kg (IM) or 300 microg of ivermectin/kg (SC) either 0 (same day), 7, 14, or 21 days prior to an oral challenge of 50000 embryonated A. suum eggs. The ninth group (control) was challenged in parallel without any avermectin treatment. At 41 or 42 days after challenge, pigs were euthanatized and adult and larval stages of A. suum were collected from the gastrointestinal tract of each pig and counted. Both avermectins significantly (P < 0.0002) reduced nematode counts when given on the day of challenge (0 days prior), and the efficacy was 100% and 97.5% for doramectin and ivermectin, respectively. Doramectin given 7 days prior to challenge significantly (P < 0.0001) reduced nematode counts, and the efficacy was 98.4%. For all other avermectin-treatment groups, nematode counts were not significantly reduced compared to those in control pigs. These data indicated that anthelmintic activity of ivermectin against A. suum persisted for less than 7 days and the activity of doramectin persisted for more than 7, but less than 14 days.     

Influence of vitamin E on mast cell mediator release

Abstract We investigated the influence of vitamin E on mediator activity and release in a canine mastocytoma cell line (C2) as a model for canine atopic dermatitis. Cells were incubated without and with vitamin E (100 µ m ) for 24 h. The histamine and prostaglandin D₂ (PGD₂) release as well as the chymase and tryptase activity were measured. To stimulate the PGD₂ and histamine release, cells were incubated with the wasp venom peptide mastoparan (50 µ m ) for 30 or 45 min. Nonstimulated as well as mastoparan-stimulated histamine and PGD₂ release was reduced significantly in vitamin E-treated cells. The activity of chymase tended to decrease, but the tryptase activity of C2 cells was not influenced by vitamin E. These results indicate that vitamin E decreased the production and release of inflammatory mediators in C2 cells, suggesting that vitamin E might have a possible beneficial effect in inflammatory diseases.     

Characterization of isolated porcine intestinal mucosal mast cells following infection with Ascaris suum

Porcine intestinal mucosal mast cells (IMMC) were isolated from intestinal tissues of swine by enzymatic digestion and density gradient separation. Helminth-free swine and swine exposed to the nematode parasite, Ascaris suum, were used as a source of intestinal tissue. Up to 40% of the isolated intestinal cells stained metachromatically with toluidine blue pH 3.0, indicating the presence of IMMC. The histamine content of this cell population ranged from 2.9-8.9 pg per toluidine blue-positive IMMC, regardless of the animal source. Enrichment procedures that increased the proportion of toluidine blue-positive IMMC from the isolated intestinal cell population correlated with an increase in the amount of histamine detected in the cell population, indicating that toluidine blue-positive IMMC were the major source of histamine in this heterogeneous cell population. However, only cells isolated from the intestines of parasite-exposed swine released histamine in vitro after mixing with antigens derived from A. suum. Cells from the intestines of both helminth-free and parasite-exposed swine did not release histamine after mixing with a non-parasite hapten-protein molecule DNP-human serum albumin, but did release greater than 90% of their total histamine after lysis with Triton X-100 or with the Ca2+ ionophore (A23187). The stimulus for acquired responsiveness of IMMC to A. suum antigens in vitro was parasitic infection in vivo because helminth-free swine maintained in confinement on concrete yielded IMMC that specifically released histamine in the presence of parasite antigens only after 3 weeks of daily experimental inoculations with A. suum eggs. IMMC isolated from the entire length of the small intestines of infected pigs were responsive to antigens in vitro, but the relative number of IMMC isolated and their level of histamine release decreased from the anterior to the posterior end. IMMC isolated from infected swine were also stimulated to release histamine in vitro by viable second stage larvae of A. suum and by treatment with anti-swine immunoglobulin. Responsiveness to both parasite antigens and anti-immunoglobulin were totally eliminated, however, by a brief treatment of the cells with acidic buffer, suggesting that an acid-dissociable cell-bound antibody molecule was responsible for specific antigen-induced histamine release by IMMC.     

Recent developments in canine atopic dermatitis: a review

Atopic dermatitis (AD) is a genetically predisposed inflammatory and pruritic allergic skin disease with characteristic clinical features. New results on the pathogenesis and therapeutic aspects are discussed in this review. IgE-mediated hypersensitivity may be involved in the largest subset of atopic patients, yet there is another subset for which such involvement cannot be documented. Alterations in epidermal barrier function, priming of cutaneous antigen-presenting cells with IgE, intrinsic keratinocyte defects, and development of autoimmunity are also factors that contribute to the primary disease. Polymorphisms in regions of the genome that are of key importance to the inflammatory response contribute to the patient's clinical picture. Secondary infections, especially with Staphylococcus and yeast organisms, strongly modify or augment the inflammatory response, which changes over time. After the treatment of secondary infections and skin inflammation the avoidance of causal allergens would prevent relapse. Another causative therapy is the variously effective allergen-specific immunotherapy. The newest treatments for canine AD (cyclosporin A and tacrolimus) are highly effective at suppressing the allergic response and comparable to treatment with glucocorticoids. Canine AD presents a substantial diagnostic and therapeutic challenge over a patient's lifetime, and no single treatment is universally effective.     

Abnormal cutaneous response to mitogens and a contact allergen in dogs with atopic dermatitis

Antigen specific and nonspecific T-lymphocyte activity was evaluated in normal dogs and in dogs with atopic dermatitis by measuring the increase in skin thickness after application of the contact allergen dinitrochlorobenzene and after intradermal injection of the mitogens phytohemagglutinin and concanavalin A. The atopic dogs had a significantly reduced response to the contact allergen (P less than or equal to 0.001) but a significantly increased response to the mitogens (P less than or equal to 0.001). The atopic and normal dogs responded similarly to intradermally injected histamine. The response of dogs with non-atopic skin conditions to the cutaneous mitogen test was like that of normal dogs. Pre-existing dermatitis does not apparently influence cutaneous response to mitogens in dogs. The cutaneous response of atopics during treatment with corticosteroids is not different from normal controls. These results suggest a role for altered cell-mediated immunity in the pathogenesis of canine atopy and that the cutaneous mitogen test may have value as a rapid screening test for the disease.     

Modulation of canine lymphocyte blastogenesis via histamine

The effect of histamine on in vitro T cell blastogenic responses of canine peripheral blood lymphocytes to phytohemagglutinin-P (PHA-P) was investigated. A dose dependent inhibition of blastogenesis was observed; an effect which could be blocked by cimetidine, a type II histamine receptor antagonist, but not by diphenhydramine, a type I receptor antagonist, suggesting that histamine's inhibitory effect is mediated through a type II histamine receptor. The inhibitory effect of histamine on blastogenesis was also reversible by indomethacin, a prostaglandin synthetase inhibitor, implicating prostaglandin involvement in histamine suppression. Histamine release at sites of inflammation may result in down regulation of local immune responses by activation of specific immunoregulatory cells. This could permit the escape of certain neoplasia from local immunosurveillance mechanisms. Cimetidine may block activation of histamine responsive regulatory cells bearing type II receptors, which may help explain the beneficial effect cimetidine therapy has on regression of certain human tumors (i.e., malignant melanomas).     

The suitability of medetomidine sedation for intradermal skin testing in dogs

The objective of this study was to determine the suitability of medetomidine sedation for facilitating intradermal skin testing in dogs. Quality of sedation and immobilization, and effects of sedation on responses to intradermally injected histamine were evaluated. Ten clinically normal dogs were injected intradermally before and after medetomidine sedation (10 μg kg^?1 intravenously) with diminishing concentrations of histamine (100–10^?5μg mL^?1) and a negative control. Mean wheal responses at injection sites were compared before and during sedation, and no significant suppression of responses occurred during sedation. Medetomidine produced sedation that notably increased the ease of performing multiple intradermal injections in all dogs and sedative effects were rapidly reversed by the antagonist atipamezole. It was concluded that medetomidine may be an excellent sedative for facilitating intradermal skin testing in dogs provided further studies similarly reveal no inhibition of responses to intradermally injected allergens in atopic dogs.     

Evaluation of cell-surface IgE receptors on the canine mastocytoma cell line C2 maintained in continuous culture

OBJECTIVE: To assess expression and function of cell-surface IgE receptors on the canine mastocytoma cell line C2 maintained in continuous culture. SAMPLE POPULATION: C2 cells maintained in medium lacking IgE for up to 10 passages before being stored at -80 C. PROCEDURE: Cells were thawed, cultured in medium without IgE for 1 to 3 passages, sensitized for 7 days with IgE-rich serum from dogs naturally sensitized to Ascaris suum, and stimulated with antigen Asc S1 from A suum, goat polyclonal anti-canine IgE, or calcium ionophore and phorbol myristate acetate (PMA). Percentage of intracellular beta-hexosaminidase released and concentration of tumor necrosis factor-alpha (TNF-alpha) synthesized after stimulation were determined. Expression of cell-surface IgE receptors was assessed by use of a flow cytometry. RESULTS: Immunologic stimulation (antigen or anti-IgE) failed to induce release or synthesis of detectable amounts of beta-hexosaminidase or TNF-alpha. In contrast, nonimmunologic stimulation (calcium ionophore and PMA) led to release of beta-hexosaminidase (mean +/- SEM maximum release, 23.95+/-1.96%) and synthesis of TNF-alpha (maximum concentration, 34.34+/-2.34 pg/10(6) cells). As revealed by use of flow cytometry, C2 cells expressed surface IgE receptors that bound canine IgE in vitro. CONCLUSIONS: Continuous culture of the canine mastocytoma cell line C2 in medium without exogenous IgE or cytokines and other growth factors resulted in cell-surface expression of nonfunctional IgE receptors. However, C2 cells maintained in continuous culture may still be a useful tool for the evaluation of mast cell responses to nonimmunologic stimulation and IgE receptor differentiation and maturity.     

Determination of threshold concentrations of allergens and evaluation of two different histamine concentrations in canine intradermal testing

The purpose of this study was to determine the optimal histamine concentration and allergen threshold concentrations for canine intradermal testing. Thirty healthy dogs were tested using two different concentrations of histamine and four different concentrations of each allergen. The optimal histamine concentration was determined to be 1:10 000 w/v. The threshold concentration was at least 1750 PNU/mL for all tested grasses, weeds, trees, moulds and insects, except for fleas which was as least 1:500 w/v. For Dermatophagoides pteronyssinus, the optimal threshold concentration was 250 PNU/mL, whereas for Dermatophagoides farinae and Tyrophagus putrescentiae, it was 100 PNU/mL. Threshold concentration for all epidermals except human dander was at least 1250 PNU/mL. The optimal threshold concentration for human dander was 300 PNU/mL. Our results suggest that the currently used 1:100 000 w/v concentration of histamine and the 1000 PNU/mL concentration for most grasses, weeds, trees, moulds, epidermals and insects may not be appropriate for canine intradermal testing.