Effect of an anti-inflammatory pomegranate otic treatment on the clinical evolution and microbiota profile of dogs with otitis externa.

BACKGROUND: Canine otitis externa (OE) is a common disease characterised by inflammation of the epithelial tissue of the external ear canal. Secondary infections are frequent, and Malassezia pachydermatis and Staphylococcus pseudintermedius are routinely isolated and treated with antifungal and antibiotic compounds. HYPOTHESIS/OBJECTIVES: To analyse the otitis ear microbiome before and after a treatment with prednisolone plus pomegranate or antimicrobial drugs ANIMALS: 15 dogs with nonpurulent OE. METHODS AND MATERIALS: A 30 day, double-blinded, multicentre, randomized and controlled parallel-group (1:1) trial was conducted in 15 dogs with nonpurulent OE, following two different topical treatments (prednisolone plus pomegranate versus prednisolone plus antibiotic and antifungal drugs). On days (D)0, D15 and D30, serum and skin otic samples were collected, and clinical examination and microbiome analysis (bacteria and fungi) were performed. Results were compared with validated otitis clinical scores to assess the effectiveness of both treatments. RESULTS: Nine bacterial and four fungal families were detected during the three time-points tested. An increase in fungal diversity (Shannon index) and composition was the most significant change observed after both treatments. At treatment D15 and D30, the reduction in clinical signs was statistically significant in both treatment groups (P ≤ 0.05). Prednisolone plus pomegranate cleanser treatment was able to control the clinical signs of otitis as well as the bacterial and fungal overgrowth. CONCLUSIONS AND CLINICAL IMPORTANCE: Mild otitis cases associated with microbial overgrowth may be managed with topical antiseptic and anti-inflammatory agents without the need for antibiotic and/or antifungal compounds.

Specific Dermatophagoides farinae extract for canine immunotherapy.

BACKGROUND: Canine atopic dermatitis (cAD) is a pruritic allergic skin disease most often caused by Dermatophagoides farinae. Differences in the sensitization profile to D. farinae have been reported between people and dogs. However, allergic dogs traditionally have been treated with extracts intended for human immunotherapy. HYPOTHESIS/OBJECTIVES: To develop a specific allergen immunotherapy for veterinary practice enriched in canine major allergens and to demonstrate its in vitro efficacy. ANIMALS: Twenty privately owned dogs, clinically diagnosed with cAD, and three healthy dogs. METHODS AND MATERIALS: A veterinary D. farinae allergen extract was manufactured and characterized compared to D. farinae extract used for human immunotherapy. The protein profile was analysed by SDS-PAGE and size exclusion chromatography and Der f 15 and Der f 18 allergens quantified by mass spectrometry. The allergenic profile was studied by immunoblot and the biological potency by enzyme-linked immunosorbent assay-inhibition assays. The extract's capacity to induce cytokine production [interleukin (IL)-10, interferon (IFN)-Ɣ] by peripheral blood mononuclear cells also was evaluated. RESULTS: The veterinary extract showed a higher content of high molecular weight proteins, preferentially recognized by atopic dog sera. The fold-increases in Der f 15 and Der f 18 with respect to the human extract were 2.07 ± 0.32 and 1.63 ± 0.15, respectively. The veterinary extract showed higher biological potency (0.062 versus 0.132 µg required for 50% inhibition of dogs sera) compared to the human extract and induced significantly higher levels of IL-10 (1,780 pg/mL) and IFN-Ɣ (50.4 pg/mL) with respect to the negative control. CONCLUSIONS AND CLINICAL IMPORTANCE: A veterinary D. farinae extract with a higher content of dog major allergens was developed and in vitro efficacy demonstrated by immunological parameters.

Allergen-specific immunotherapy in dogs with atopic dermatitis: is owner compliance the main success-limiting factor?

BACKGROUND: While the efficacy of allergen-specific immunotherapy (ASIT) to treat canine atopic dermatitis has been well established, it remains unclear why not all dogs show the same response to treatment. The goal of the study was to determine the relationship between duration of ASIT and two measurements of success: disease severity and concomitant medication sparing effect. METHODS: Data were retrospectively compiled for 145 dogs with atopic dermatitis treated with ASIT. As a measure of treatment compliance, cases were stratified into dogs treated for less than 12 months or for at least 12 months. Treatment efficacy, defined as a reduction in disease severity score (scale 0-10), was compared between both groups, and correlations between treatment success and several related factors were examined. RESULTS: ASIT treatment duration was strongly correlated with treatment efficacy. Animals treated for less than 12 months showed lower efficacy rates (22 per cent) than those treated for at least 12 months (65 per cent). Further, in animals treated for at least 12 months, concomitant medications were reduced more (87 per cent) than in animals treated for less than 12 months (39 per cent). CONCLUSION: A lack of owner compliance emerged as the main factor explaining the reduced effectiveness of ASIT. To improve treatment adherence, veterinarians and owners need to be better informed about ASIT mechanisms of action before starting treatment.

"In vitro" capacitation and further progesterone-induced acrosome exocytosis are linked to specific changes in the expression and location of threonine phosphorylation of boar spermatozoa.

The aim of this study was to determine if the achievement of the "in vitro" capacitation (IVC) status and subsequent progesterone-induced "in vitro" acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono- and bi-dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono-dimensional Western blot in non-capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi-dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non-capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm.

Topical polyhydroxy acid treatment for autosomal recessive congenital ichthyosis in the golden retriever: a prospective pilot study.

BACKGROUND: Autosomal recessive congenital ichthyosis (ARCI) in golden retrievers is due to a PNPLA1 gene mutation, which plays a role in epidermal lipid organization and metabolism. Topical therapies are used to reduce scaling; however, there are few published efficacy studies. OBJECTIVES: To examine the efficacy of topical treatment based on gluconolactone, a polyhydroxy acid with known beneficial effects on stratum corneum structure. ANIMALS: Sixteen golden retriever dogs with clinical signs of ARCI and PCR-confirmed PNPLA1 gene mutation. METHODS: This was a prospective, multicentre, noncontrolled study. Dogs were treated with a shampoo and lotion containing gluconolactone and other hydroxyl acids. Treatments were administered initially twice weekly for two weeks, then once weekly for two weeks and finally once monthly. Examinations were performed prior to and at 14 and 30 days of treatment to assess scaling, presence of other skin lesions and pruritus. In two dogs, pre- and 30 day post-treatment, skin biopsies were obtained. RESULTS: The extent and size of the scales were reduced by 60% and 75% after 14 and 30 days of treatment, respectively (P < 0.001). In 20% of the dogs, scaling was no longer observed after the first 30 days of treatment. No other skin lesions or pruritus were observed in any dog. Post-treatment biopsies showed normalization of the stratum corneum morphology and reduced hyperpigmentation. CONCLUSION AND CLINICAL IMPORTANCE: The frequent use of a shampoo and lotion containing gluconolactone may be an effective measure to improve skin scaling in golden retrievers with ARCI.

Effects of sphingolipid extracts on the morphological structure and lipid profile in an in vitro model of canine skin.

Ceramides (CER) are essential sphingolipids of the stratum corneum (SC) that play an important role in maintaining cutaneous barrier function. Skin barrier defects occur in both human beings and dogs affected with atopic dermatitis, and have been associated with decreased CER concentrations and morphological alterations in the SC. The aim of the present study was to investigate the changes induced by three different sphingolipid extracts (SPE-1, SPE-2 and SPE-3) on the morphological structure and lipid composition of canine skin, using an in vitro model, whereby keratinocytes were seeded onto fibroblast-embedded collagen type I matrix at the air-liquid interface. Cell cultures were supplemented with SPE-1, SPE-2, SPE-3 or vehicle (control) for 14 days. The relative concentrations of lipids were determined by ultra-performance liquid chromatography coupled to mass spectrometry. The ultrastructural morphology of samples was examined by transmission electron microscopy. SPE-1 induced significant elevation in total CERs, CER[NS], CER[NDS], CER[NP], CER[AS], CER[AP], CER[EOS] and CER[EOP] subclasses, whereas SPE-2 induced a significant elevation in total CER, CER[AP] and CER[EOS] compared with control conditions. Ultrastructural analysis revealed an increase in lamellar-lipid structures in the SC of SPE-1-treated samples. The findings demonstrated that SPE-1 stimulates production of CERs, as shown by changes in lipid composition and ultrastructural morphology. Thus, SPE-1 contributes to the formation of a well-organised SC and represents a potential therapeutic target for improving skin barrier function in atopic dermatitis.

Glucose and fructose have sugar-specific effects in both liver and skeletal muscle in vivo: a role for liver fructokinase.

We examined glucose and fructose effects on serine phosphorylation levels of a range of proteins in rat liver and muscle cells. For this, healthy adult rats were subjected to either oral glucose or fructose loads. A mini-array system was utilized to determine serine phosphorylation levels of liver and skeletal muscle proteins. A glucose oral load of 125 mg/100 g body weight (G 1/2) did not induce changes in phosphorylated serines of the proteins studied. Loading with 250 mg/100 g body weight of fructose (Fr), which induced similar glycemia levels as G 1/2, significantly increased serine phosphorylation of liver cyclin D3, PI3 kinase/p85, ERK-2, PTP2 and clusterin. The G 1/2 increased serine levels of the skeletal muscle proteins cyclin H, Cdk2, IRAK, total PKC, PTP1B, c-Raf 1, Ras and the ß-subunit of the insulin receptor. The Fr induced a significant increase only in muscle serine phosphorylation of PI3 kinase/p85. The incubation of isolated rat hepatocytes with 10 mM glucose for 5 min significantly increased serine phosphorylation of 31 proteins. In contrast, incubation with 10 mM fructose produced less intense effects. Incubation with 10 mM glucose plus 75 µM fructose counteracted the effects of the incubation with glucose alone, except those on Raf-1 and Ras. Less marked effects were detected in cultured muscle cells incubated with 10 mM glucose or 10 mM glucose plus 75 µM fructose. Our results suggest that glucose and fructose act as specific functional modulators through a general mechanism that involves liver-generated signals, like micromolar fructosemia, which would inform peripheral tissues of the presence of either glucose- or fructose-derived metabolites.

Oligomycin A-induced inhibition of mitochondrial ATP-synthase activity suppresses boar sperm motility and in vitro capacitation achievement without modifying overall sperm energy levels.

Incubation of boar spermatozoa in a capacitation medium with oligomycin A, a specific inhibitor of the F0 component of the mitochondrial ATP synthase, induced an immediate and almost complete immobilisation of cells. Oligomycin A also inhibited the ability of spermatozoa to achieve feasible in vitro capacitation (IVC), as measured through IVC-compatible changes in motility patterns, tyrosine phosphorylation levels of the acrosomal p32 protein, membrane fluidity and the ability of spermatozoa to achieve subsequent, progesterone-induced in vitro acrosome exocytosis (IVAE). Both inhibitory effects were caused without changes in the rhythm of O2 consumption, intracellular ATP levels or mitochondrial membrane potential (MMP). IVAE was accompanied by a fast and intense peak in O2 consumption and ATP levels in control spermatozoa. Oligomycin A also inhibited progesterone-induced IVAE as well as the concomitant peaks of O2 consumption and ATP levels. The effect of oligomycin on IVAE was also accompanied by concomitant alterations in the IVAE-induced changes on intracellular Ca(2+) levels and MMP. Our results suggest that the oligomycin A-sensitive mitochondrial ATP-synthase activity is instrumental in the achievement of an adequate boar sperm motion pattern, IVC and IVAE. However, this effect seems not to be linked to changes in the overall maintenance of adequate energy levels in stages other than IVAE.

Development and characterization of an equine skin-equivalent model.

BACKGROUND: There is increasing interest in the biological and pathological study of equine skin owing to the high prevalence of cutaneous diseases in horses. However, knowledge of equine skin cell biology and cultures is limited by the low number of in vitro studies in the literature. HYPOTHESIS/OBJECTIVES: The objective of the study was to develop and characterize an in vitro equine skin equivalent. METHODS: Cultures of pure equine keratinocytes and dermal fibroblasts were obtained by enzymatic digestion of skin biopsies. Fibroblasts were embedded into type I collagen matrices to obtain dermal scaffolds, the surface of which was seeded with keratinocytes. The three-dimensional cultures were exposed to the air-liquid interface to enable epidermal stratification. RESULTS: After 14 days in air-exposed conditions, histological analysis showed that keratinocytes underwent differentiation into a multilayered epidermis. Immunohistochemical studies revealed the expression of epidermal cytokeratin in keratinocytes, whereas vimentin was expressed in dermal fibroblasts, as expected in equine skin. Immunostaining of Ki67 showed proliferative keratinocytes in the stratum basale. A continuous basement membrane at the dermo-epidermal junction was also detected immunohistochemically through the expression of its major components (type IV collagen and laminin 5). Ultrastructural analysis by electron microscopy showed desmosomes located among keratinocytes in all layers and hemidesmosomes among the basal keratinocytes and lamina densa. CONCLUSIONS AND CLINICAL IMPORTANCE: This study reports, for the first time, the development of an in vitro equine skin-equivalent model that resembles equine skin morphologically, immunohistochemically and ultrastructurally.

Diabetic Retinopathy Is Associated with Decreased Tyrosine Nitrosylation of Vitreous Interleukins IL-1α, IL-1ß, and IL-7.

OBJECTIVE: To simultaneously evaluate tyrosine nitrosylation and phosphorylation levels of vitreous interleukins of patients with diabetic retinopathy, in which abnormal tyrosine phosphorylation has been previously described. RESEARCH DESIGN AND METHODS: Specific immunoprecipitation of interleukins IL-1α, IL-1ß, IL-2 and IL-7 was carried out in samples obtained during vitrectomy performed for proliferative diabetic retinopathy in patients (n=12) and for macular hole in controls (n=12). Tyrosine nitrosylation and phosphorylation levels of the immunoprecipitated interleukins were analysed by Western blot with the respective specific antibodies and correlated. The results were also correlated with the total amount of immunoprecipitated interleukin protein. The mean phosphorylation/nitrosylation ratios of these proteins in vitreous humour of both the control group and diabetic patients were determined. RESULTS: Diabetes was associated with decreased tyrosine nitrosylation of IL-1α, IL-1ß and IL-7 and an increased tyrosine phosphorylation/nitrosylation ratio with respect to controls in IL-1α (1.58±0.22 vs. 2.74±0.39, respectively; p<0.05) and IL-7 (2.15±0.01 vs. 3.26±0.57, respectively; p<0.05). No significant changes were observed in nitrotyrosine or in the tyrosine phosphorylation/nitrosylation ratio of IL-2. CONCLUSIONS: Proliferative diabetic retinopathy is associated with concomitant and simultaneous changes in both tyrosine phosphorylation and tyrosine nitrosylation status of specific pro-inflammatory interleukins present in the vitreous fluid such as IL-1α, IL-1ß and IL-7. These changes could be related to the increase in pro-inflammatory activity detected in diabetes-induced retinopathy.

Tyrosine phosphorylation of vitreous inflammatory and angiogenic peptides and proteins in diabetic retinopathy.

PURPOSE: To evaluate the degree of phosphorylation of vitreous proteins in patients with type 2 diabetes mellitus and diabetic retinopathy compared with a group of control subjects without diabetes and of similar age and sex. METHODS: In samples obtained after vitrectomy for diabetic retinopathy in patients and for macular hole in control subjects, immunoblot techniques were applied to a mini-array system for quantification of a wide range of chemokines and vasoactive peptides and proteins. Antiphosphotyrosine antibody was used for tyrosine phosphorylation evaluation and results were expressed as the percentage of variation compared with that in control subjects. RESULTS: Samples from eight patients with type 2 diabetes and from eight control subjects were analyzed. The total quantity of proteins analyzed was similar in both patients and control subjects. Tyrosine phosphorylation was very significantly decreased (<20%, P < 0.05) in diabetic patients with respect to the control group in growth-related oncogene, human cytokine I-309, interleukin-13, monocyte colony-stimulating factor, macrophage-derived chemokine, stem cell factor, transforming growth factor-beta1, angiogenin, and oncostatin M. A significant decrease in phosphorylation (between 20% and 40%, P < 0.05) was observed in epithelial neutrophil-activating peptide 78; granulocyte colony-stimulating factor; granulocyte-monocyte-stimulating colony factor; IL-5, -6, -7, -8, -10, and -12p40p70; monokine induced by interferon-gamma; macrophage inflammatory protein 1-gamma; and normal T expressed and secreted cytokine (RANTES) in comparison with that in the control subjects. The greatest decrease in phosphorylation status was found in IL-1-alpha and -1beta. CONCLUSIONS: Diabetic retinopathy is associated with a decrease in tyrosine phosphorylation of many vitreous proteins which may indicate an alteration in protein functionality or action even before significant quantitative variations.