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Effects of palmitoylethanolamide on immunologically induced histamine, PGD2 and TNFα release from canine skin mast cells.

Palmitoylethanolamide (PEA) is an endocannabinoid-like compound and the parent molecule of the aliamide family, a group of fatty acid amides able to act through the down-regulation of mast cell degranulation. PEA has been proven to exert both analgesic and anti-inflammatory activity, and recent studies have shown its ability in reducing clinical symptoms of inflammatory skin diseases, both in humans and in animals. Although its pharmacological efficacy is well known, the mechanism of action of this family of compounds is still unclear. To better understand the cellular effects of aliamides in dogs, canine mast cells freshly isolated from skin biopsies were incubated with IgE-rich serum and were challenged with anti-canine IgE. Histamine, prostaglandin D(2) (PGD(2)) and tumour necrosis factor-alpha (TNFalpha) release was measured in the presence and absence of increasing concentrations of PEA, ranging from 10(-8)M to 10(-5)M. Histamine, PGD(2) and TNFalpha release, immunologically induced by canine anti-IgE, were significantly inhibited in the presence of PEA. The maximum inhibitory effect on histamine release was observed at 3x10(-6)M PEA concentration achieving an inhibition of 54.3+/-5.2%. PGD(2) release was significantly inhibited at 10(-5)M and 10(-6)M PEA concentrations with 25.5+/-10.2% and 14.6+/-5.6% of inhibition, respectively. Finally, PEA inhibited TNFalpha release to 29.2+/-2.0% and 22.1+/-7.2%, at concentrations of 10(-5)M and 3x10(-6)M, respectively. The results obtained in the present study showed the ability of the aliamide PEA to down-modulate skin mast cell activation. Therefore, our findings suggest that the beneficial effect of PEA, observed in inflammation and pain clinical studies, could be due, at least in part, to its ability to inhibit the release of both preformed and newly synthesised mast cell mediators.

In vitro inhibitory effect of rupatadine on histamine and TNF-α release from dispersed canine skin mast cells and the human mast cell line HMC-1

Abstract.Objective and design: To examine the inhibitory potential of rupatadine, a new H1-antihistamine and anti-PAF agent, on histamine and TNF-α release. Comparison with an H1-antihistamine (loratadine) and a PAF-antagonist (SR-27417A).¶Material: Dispersed canine skin mast cells were used to assess the effect of the drugs tested on FcεRI-dependent and -independent histamine release; the human HMC-1 cell line was used to study TNF-α release.¶Treatment and methods: Before stimulation mast cell populations were treated with increasing concentrations of rupatadine, loratadine and SR-27417A. Histamine and TNF-α release were measured following 15-30 min and 3 h activation, respectively.¶Results: The IC50 for rupatadine in A23187, concanavalin A and anti-IgE induced histamine release was 0.7 ± 0.4 μM, 3.2 ± 0.7μM and 1.5 ± 0.4 μM, respectively whereas for loratadine the IC50 was 2.1 ± 0.9 μM, 4.0 ± 1.3 M and 1.7 ± 0.5 μM. SR-27417A exhibited no inhibitory effect. Rupatadine, loratadine and SR-27417A inhibited TNF-α release with IC50 2.0 ± 0.9 μM, 2.1 ± 1.1 M and 4.3 ± 0.6 μM, respectively.¶Conclusions: Rupatadine and loratadine showed similar inhibitory effect on histamine and TNF-α release, whereas SR-27417A only exhibited inhibitory effect against TNF-α.

Evaluation of storage mite contamination of commercial dry dog food.

Storage mites may be considered important allergens in dogs with atopic dermatitis. High sensitization rates to Tyrophagus, Acarus, and Lepidoglyphus species have been reported in atopic dogs, and dry pet food has been suggested as a potential source of storage mite exposure. The aim of the present study was to evaluate commercial dry dog food for contamination with storage mites, and how storage time and conditions could influence the risk of contamination. Ten different premium commercial dry dog foods formulated for skin disorders were selected. Food bags were opened and stored for 6 weeks under two different environmental conditions. At different time points, samples from each bag were collected and analysed by microscopy, guanine test, storage mite-specific traps, and a modified flotation technique. On opening, two storage mites identified as Acarus siro were isolated from one of the 10 bags by flotation technique, indicating that storage mites can be present in packaged dry dog food bags. After 5 weeks of storage under environmental conditions optimal for mite growth (23.2 +/- 2.1 degrees C and 71 +/- 5.6% of relative humidity), mites were detected by microscopic observation in nine of the 10 diets. When mites were identified by the flotation technique, Tyrophagus spp. were found to be the most common contaminating species. These results show that dry dog food can be a suitable substrate for storage mite reproduction, and that environmental and storage conditions may influence food contamination and mite development.

Comparative morphofunctional study of dispersed mature canine cutaneous mast cells and BR cells, a poorly differentiated mast cell line from a dog subcutaneous mastocytoma

The dog mastocytoma BR cell line provides us with a permanent source of canine mast cells, allowing a characterization of secretory mediators that exert important effects in canine allergic and nonallergic diseases and in physiological processes. We studied the ultrastructural characteristics and histamine releasing activity after immunological and non-immunological stimuli of the dog mastocytoma BR cell line, and compared the cell line to normal skin mast cells enzymatically isolated from healthy dogs. The histamine content of BR cells was 0.04 +/- 0.002 pg/cell, approximately 100-fold less than that found in canine skin mast cells. Non-immunologic stimuli induced similar concentration-dependent histamine release from skin mast cells and BR cells: 29.3 +/- 0.9% vs. 12.7 +/- 0.7% (calcium ionophore A23187), 23.3 +/- 0.7% vs. 18.8 +/- 0.7% (substance P) and 12.5 +/- 0.3% vs. 12.1 +/- 0.9% (compound 48/80), respectively. Immunologic stimulation, however, was only effective on canine skin mast cells, causing 30.9 +/- 1.7%, 27.7 +/- 0.6% and 12.2 +/- 0.9% histamine release in response to anti-canine IgE, concanavalin A, and antigen Asc S 1, respectively. The absence of functional IgE receptors in BR cells was confirmed by the lack of response to anti-IgE and antigen Asc S 1 following passive sensitization with dog atopic serum and dog antigen sensitized serum. We conclude that BR cells are able to release histamine after non-immunologic stimulation in a similar manner to canine skin mast cells, but that there are morphological and functional differences possibly due to different states of maturity or differentiation. For this reason the study of the highly homogeneous BR cells could offer insights into dog mast cell biology in contexts where freshly isolated cells cannot be used because of low purity and recovery.

Inhibitory effects of rupatadine on mast cell histamine release and skin wheal development induced by Ascaris suum in hypersensitive dogs

The present studies were performed to compare the cutaneous antiallergic effects of rupatadine, a new potent dual antagonist of histamine and platelet‐activating factor (PAF), with those of loratadine (an H1‐antihistamine) and SR‐27417A (a PAF antagonist). Two experimental models were used: an in vivo skin challenge by Ascaris suum extract administered intradermically in conscious hypersensitive dogs and an in vitro assay of Asc S 1 antigen‐induced histamine release from isolated canine skin mast cells. In antigen‐induced skin inflammation, both rupatadine and loratadine administered orally inhibited wheal formation in a dose‐dependent manner at the studied doses. At the 0.1 mg/kg dose, maximum inhibition values for rupatadine and loratadine were similar, but the effect of rupatadine lasted 24 h, whereas loratadines effect disappeared after 8 h. At the 1 mg/kg dose, the two compounds behaved similarly, with maximum effects of about 65% (4 h after treatment) and activity lasting more than 24 h in both cases. At the 10 mg/kg dose, rupatadine and loratadine exhibited maximum effects of 84 and 64% in wheal inhibition 2 and 4 h after drug administration, respectively. SR‐27417A did not inhibit ascaris‐induced wheal at the doses studied (1 and 10 mg/kg p.o.). In isolated canine skin mast cells, rupatadine and loratadine inhibited antigen‐induced histamine release in a concentration‐dependent manner. Rupatadine was more potent than loratadine at each concentration studied (100 nM–30 μM). Maximum inhibitory effects were 83 and 67% for rupatadine and loratadine, respectively, after a 30 μM concentration. Rupatadine, with an IC50 value of 5.3 μM, was about fourfold more potent than loratadine, with an IC50 value of 19 μM. SR‐27417A exhibited no effect in our experimental model. Moreover, neither loratadine or rupatadine showed cytotoxic or prodegranulating effects at any concentration, whereas SR‐27417A was cytotoxic at the highest concentration (30 μM). Thus, rupatadine is effective in controlling inflammatory reactions in dog skin and the effect may be partly due to its modulation of mast cell degranulation, but not to its PAF‐antagonist properties. Drug Dev. Res. 44:49–55, 1998.

Comparative study of histamine release from skin mast cells dispersed from atopic, ascaris-sensitive and healthy dogs

Atopic dermatitis results from the interaction between allergen and allergen-specific IgE bound to the mast cell surface receptors. This process triggers mast cell degranulation and accounts at least for early phase reaction. Furthermore, there is increasing in vitro and in vivo evidence that IgE has the ability to induce overexpression of the Fc epsilonRI receptor on the mast cell plasma membrane. In order to study the potential effect of an increase in serum IgE on mast cell activity, the histamine releasability of mature mast cells isolated from the skin of atopic, ascaris-sensitive and healthy dogs was analyzed. No histamine release was detected upon the immunological stimulation of cells that were not previously sensitized with atopic or ascaris-sensitive dog serum. However, when passively sensitized, skin mast cells were challenged with either Asc SI antigen or anti-IgE, the mast cell histamine release increased in a stimulus concentration-dependent manner. The amount of histamine released was significantly higher in response to anti-IgE than in response to Asc SI antigen. However. the difference in the percentage of mast cell histamine release between atopic (26.3+/-2.8%) and non-atopic (30.9+/-1.7%) dogs was not statistically significant, similar to what occurred when ascaris-sensitive (12.8+/-1.6%) and non-sensitive (13.2+/-1.7%) dogs were compared. Although these results could suggest that there is either little or no increase in the density of IgE receptors in atopic or ascaris-hypersensitive dogs versus controls, we strongly consider either the possibility that the digestion procedure might affect cell behaviour in vitro or that an underlying increase of receptors poorly affects the release of granule-stored mediators but influences mast cell activity in a different manner.

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.

Development and characterization of a canine skin equivalent

Abstract:  The development of a complex cellular model, which incorporates the basic cell components of the dog skin, would be a useful tool to investigate the biology and pathology of canine skin and also to replace animal testing partially. The aim of the present study was to develop and characterize a canine skin equivalent. Epidermal keratinocytes and dermal fibroblasts were freshly isolated from skin biopsies from healthy dogs. Fibroblasts were embedded into a bio‐matrix from collagen type I matrix protein; this built the scaffold where the keratinocytes were seeded, at air exposed conditions. At 3, 7, 15 and 21 days of culture in special growth media, skin equivalents were analysed by histological, immunohistochemical and electron microscopical techniques. At 15 days, keratinocytes underwent differentiation to a multilayer epidermis with stratum basal, stratum spinosum, stratum granulosum and stratum corneum. Expression of epidermal cytokeratins in keratinocytes was detected by immunhistochemistry, and followed the same pattern than in the normal canine epidermis. Fibroblasts from the skin equivalent expressed vimentin as dermal fibroblasts do. A basement membrane (BM) was observed underneath the epidermis; ultrastructurally, it was similar to the normal canine BM and collagen IV and laminin 5 were detected immunohistochemically as major components of this structure. Skin equivalents developed from canine cutaneous cells presented a similar morphological structure than healthy canine skin. Moreover, the immunohistochemical analysis revealed the expression of the major markers of the epidermis (keratins), dermis (vimentin) and BM (collagen type IV, laminin 5).

Stem cell factor enhances IgE-mediated histamine and TNF-α 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.

Bovine Colostrum Increases Proliferation of Canine Skin Fibroblasts

Bovine colostrum (BC) is a milk secreted during the first few days after calving and is a rich source of bioactive components such as immunoglobulins, insulin-like growth factor (I, II) (IGF I-II), transforming growth factor-b (TGFb), platelet-derived growth factor, epidermal growth factor, tumor necrosis factor, basic fibroblast growth factor, vasoendothelial growth factor, and telomerase (1–3). The importance of colostrum for the health of young animals has been known for a long time (1–4), but other systemic effects in adult humans fed BC orally have also been demonstrated, such as increased salivary IgA and plasma IGF-I (5), reduced symptoms of upper respiratory tract infection in humans (6), and changes in body composition of human athletes (7). Specific growth factors have been studied in different cultured cells, such as cementoblasts (8), chondrocytes (9), fibroblasts (10), and myoblasts (11) to evaluate improved regenerative therapies for periodontal tissues, osteoarthritis, and wound healing. The aim of this study was to evaluate the effects of a skim freeze-dried BC source (Colexan, Colostrum Technologies GmbH) rich in bioactive components (immunoglobulins, natural growth factors, and hormones), as an in vitro stimulator of canine skin fibroblast proliferation and activity. This in vitro model could be used as a model for wound repair efficiency and periodontal tissue repair for topical applications. MATERIALS AND METHODS