Anna Puigdemont

Canine mast cell tumors express stem cell factor receptor

c-kit protooncogene encodes a type III transmembrane receptor kinase, the stem cell factor receptor, or KIT. The ligand of the KIT. stem cell factor, is a cytokine that stimulates mast cell growth and differentiation. We have studied immunohistochemically KIT expression in 23 canine mast cell tumors (MCTs), 10 histiocytomas, 5 malignant melanomas, and in 2 cell lines derived from mast cells (HMC-1, human and C2, canine). As expected, KIT was detected both in the human mast cell leukemia cell line (HMC- ) and in the canine mastocytoma cell line C2. In normal canine skin, KIT expression was confined to mast cells. All canine MCTs expressed KIT, although the intensity of the staining reaction varied considerably among the 23 neoplasms. Grade III tumors showed the highest expression of KIT, whereas grade I tumors showed the lowest expression of KIT. Two patterns of KIT expression were detected in mast cells. In normal canine mast cells and in some neoplastic mast cells, KIT appeared mainly on the cell membrane. However, in many canine MCTs, KIT is accumulated in the cytoplasm, usually near the cell nucleus. The meaning of these two patterns is not clear. Expression of KIT could not be detected immunohistochemically in any of the other neoplasias investigated. According to our results, it can be concluded that most, if not all, canine MCT express KIT. Furthermore, there is an inverse correlation between the degree of differentiation and the expression of KIT. Moreover, according to our results, KIT can be used as a reliable immunohistochemical marker for canine mast cells and undifferentiated mast cell tumors.

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.

The anti‐inflammatory mediator palmitoylethanolamide enhances the levels of 2‐arachidonoyl‐glycerol and potentiates its actions at TRPV1 cation channels

Palmitoylethanolamide (PEA) is an endogenous congener of anandamide and potentiates its actions at cannabinoid CB1 and CB2 receptors, and at transient receptor potential vanilloid type‐1 (TRPV1) channels. The other endocannabinoid, 2‐arachidonoylglycerol (2‐AG), was recently suggested to act as a TRPV1 channel agonist. We investigated if PEA enhanced levels of 2‐AG in vitro or in vivo and 2‐AG activity at TRPV1 channels.

Skin mast cell releasability in dogs with atopic dermatitis.

Isolated dermal mast cells from atopic dogs are a valuable tool for the analysis of their functional properties in atopic dermatitis. We have characterized the histamine secretory pattern of mast cells enzymatically dispersed from the skin of dogs naturally suffering from this condition. The total histamine content found per isolated skin mast cell was higher in the allergic dogs than in nonatopic (control) animals (8.7 pg/mast cell versus 5.2 pg/mast cell). This phenomenon together with the well known higher concentration of skin mast cell number in atopic dermatitis lesions might account for the observed increase in local histamine concentration (15.0 μg/g versus 9.0 μg/g). Atopic dog-derived mast cells were highly reactive to both non-immunological (ionophore A23187) and an immunological-like (concanavalin A) stimulus. Furthermore, histamine net release induced by concanavalin A (1 mg/ml) stimulation was clearly enhanced in the atopic dogs (33.3% net release versus 15.4% in controls). These results have not been described in dermal mast cells dispersed from the skin of individuals with atopic dermatitis and clearly support the hypothesis that mast cells play a major role in causing and possibly modulating atopic dermatitis, through enhanced sensitivity or releasability. however, whether these two phenomena are primary abnormalities of atopic dermatitis, or only secondary changes, remains undetermined.

Rapid headspace gas chromatographic method for the determination of liquid/gas partition coefficients

A rapid, efficient and low-cost headspace technique useful for the determination of liquid/gas partition coefficients of gases and volatile substances of low and intermediate solubility is described. The equilibration step is carried out at constant pressure using glass syringes, with a ratio of liquid/gas phase volumes of ca. 1:3; after 30 min at the desired temperature, the headspace is recovered by transfer into another syringe and analyzed by gas chromatography. A study of the partition coefficients in water at 37 degrees C of 27 volatile compounds demonstrated that the method is fully applicable for all gases, with exception of those with a partition coefficient higher than 300.

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.

The role of mast cells in atopy: what can we learn from canine models? A thorough review of the biology of mast cells in canine and human systems

Mast cell research has largely focused on the role of these cells in the early phase of allergic reactions. However, their involvement may well extend beyond this stage, and even reach across nonallergic conditions. Mast cells from different sources have helped advance our knowledge of their biology. Although in vitro and in vivo research in this area has mainly focused on humans, such studies are limited by the extent to which cells from certain human tissues and/or human patients can be collected or studied. While rodents also provide valuable models with which to further our understanding of the behaviour of mast cells and their contribution to allergy, reported differences between human and murine mast cells, and, in some instances, the limitations of in vivo rodent models of mast cell‐mediated allergic conditions, preclude their use. In this review, we introduce a relatively unknown mast cell population, that of the dog. Canine mast cells display many phenotypic and functional similarities with their human counterparts, and dogs develop spontaneous and induced allergic diseases that share clinical and pathophysiological features with the human condition. Therefore, the use of canine cells can shed light on the general role of mast cells, particularly in relation to allergic diseases given the potential of in vivo dog models within this field. Here we provide a detailed review of the data reported from in vitro and in vivo studies of canine mast cells, and compare them with results obtained in human systems. We also highlight direct evidence of the mast cell contribution to canine atopy. We conclude that the dog offers useful in vitro and in vivo models in which to investigate mast cell behaviour, and that its use should be considered when undertaking studies aimed either at elucidating the role of mast cells in health and disease, or at prescreening novel therapies prior to entry into man.

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.