Molecular interactions between a diphenyl scaffold and PED/PEA15: Implications for type II diabetes therapeutics targeting PED/PEA15 - Phospholipase D1 interaction.

In a recent study, we have identified BPH03 as a promising scaffold for the development of compounds aimed at modulating the interaction between PED/PEA15 (Phosphoprotein Enriched in Diabetes/Phosphoprotein Enriched in Astrocytes 15) and PLD1 (phospholipase D1), with potential applications in type II diabetes therapy. PED/PEA15 is known to be overexpressed in certain forms of diabetes, where it binds to PLD1, thereby reducing insulin-stimulated glucose transport. The inhibition of this interaction reestablishes basal glucose transport, indicating PED as a potential target of ligands capable to recover glucose tolerance and insulin sensitivity. In this study, we employ computational methods to provide a detailed description of BPH03 interaction with PED, evidencing the presence of a hidden druggable pocket within its PLD1 binding surface. We also elucidate the conformational changes that occur during PED interaction with BPH03. Moreover, we report new NMR data supporting the in-silico findings and indicating that BPH03 disrupts the PED/PLD1 interface displacing PLD1 from its interaction with PED. Our study represents a significant advancement toward the development of potential therapeutics for the treatment of type II diabetes.

Characterization of Complexes between Imidacloprid and ß-Cyclodextrin: Evaluation of the Toxic Activity in Algae and Rotifers.

The development of new formulations can be driven by the knowledge of host-guest complexes using cyclodextrins which have the ability to include guest molecules within their hydrophobic cavities, improving the physicochemical properties of the guest. To rationally explore new pesticide formulations, the effects of cyclodextrins on the properties of such guest molecules need to be explored. Imidacloprid is a neonicotinoid systemic insecticide used worldwide. In this study, the inclusion complexes of Imidacloprid (IMI) with ß-cyclodextrin (ß-CD) were prepared in the solid state by co-precipitation and the physical mixing method, with a stoichiometry of 1:1 and 1:2 molar ratios. The obtained products, Imidacloprid:ß-cyclodextrin inclusion complex (IMI:ß-CD), were characterized in the solid state by Fourier transform-infrared (FT-IR) spectroscopy and X-ray powder diffractometry (XRD). In solution, the 1:1 stoichiometry for the inclusion complexes was established by the Job plot method, and the binding constant of IMI:ß-CD was determined by UV-vis titration. The toxicity was determined in producers and primary consumers of the freshwater trophic chain, the green alga Raphidocelis subcapitata and the rotifer Brachionus calyciflorus, respectively. The results indicated that Imidacloprid forms inclusion complexes with CDs showing improved physicochemical properties compared to free Imidacloprid. The formation of the inclusion complex reduced the chronic toxicity in rotifers when IMI concentrations were close to those of environmental concern (tenths/hundredths of micromoles/L). Therefore, CD inclusion complexes could provide important advantages to be considered for the future industrial production of new formulations.

Palmitoyl-glucosamine co-micronized with curcumin for maintenance of meloxicam-induced pain relief in dogs with osteoarthritis pain.

BACKGROUND: Osteoarthritis (OA) pain is the number one cause of chronic pain in dogs. Multimodal treatment, including combining safe and effective nutritional interventions with non-steroidal anti-inflammatory drugs (NSAIDs), is currently considered one of the most appropriate choices for managing OA pain. Palmitoyl-glucosamine is a feed material belonging to the ALIAmide family, whose parent molecule is the prohomeostatic lipid amide N-palmitoyl-ethanolamine. Curcumin is a promising plant antioxidant. The present study aimed at investigating whether 18-week dietary integration with palmitoyl-glucosamine co-micronized with curcumin was able to maintain pain relief in dogs with OA-associated chronic pain receiving meloxicam (1.5 mg/ml oral suspension) on a tapering regimen (progressive 25% decrease of the original 0.1 mg/kg/day dose, on a biweekly basis) during the first 8 weeks of treatment. Pain was assessed both by the owners and veterinary surgeons, with the first using both subjective evaluation and validated metrology instruments-i.e., Helsinki Chronic Pain Index (HCPI) and Canine Brief Pain Inventory (CBPI)-while the second rating the severity of lameness and pain on palpation on two previously used 5-point scales. RESULTS: A total of fifty-eight dogs with OA chronic pain entered the uncontrolled study. Pain on HCPI was considered severe at baseline (range 18-39). Based on owner's assessment, 90% of dogs who responded to meloxicam at the full-dose regimen could reduce meloxicam up to 25% of the original dose without experiencing pain worsening. Moreover, 75% of dogs was assessed as having no pain increase ten weeks after meloxicam withdrawal. A statistically significant decrease of pain severity as scored by HCPI (P < 0.0001) was observed two and ten weeks after meloxicam withdrawal compared to study entry (17.0 ± 1.05 and 15.1 ± 1.02, respectively, vs 29.0 ± 0.74; mean ± SEM). After meloxicam withdrawal, no statistically significant change in the CBPI scores was recorded. Pain on palpation and lameness significantly changed to less severe distributions along the study period (P < 0.0001). CONCLUSION: The findings appear to suggest that dietary integration with palmitoyl-glucosamine co-micronized with curcumin was able to maintain meloxicam-induced pain relief in dogs with severe OA chronic pain.

Psychometric Testing and Validation of the Italian Version of the Helsinki Chronic Pain Index (I-HCPI) in Dogs with Pain Related to Osteoarthritis.

Pain assessment is of paramount importance for properly managing dogs with osteoarthritis (OA) pain. The aim of the present study was to develop and psychometrically validate the Italian version of the Helsinki Chronic Pain Index (I-HCPI). Owners of OA painful (n = 87) and healthy dogs (n = 40) were administered the I-HCPI once or twice after an eight-week meloxicam treatment. Sixty-nine owners of healthy and OA dogs also completed the Italian version of the Canine Brief Pain Inventory (I-CBPI). Pain on palpation on a 0-4 scale was assessed on all recruited dogs. Construct validity was tested both with hypothesis testing and principal component analysis, confirming the I-HCPI accurately measured chronic pain. Good convergent and criterion validity were shown through correlations with I-CBPI subscores and distribution among pain on palpation scores (p < 0.0001). The significant difference between the pre- and post-treatment I-HCPI scores (p < 0.0001) and Cohen's effect size (2.27) indicated excellent responsiveness. The I-HCPI was shown to be reliable through communalities (range 0.47-0.90) and Cronbach α (≥0.95). Discriminative ability and cut-off point, as tested through Receiver Operating Characteristic analysis, showed excellent diagnostic accuracy with a threshold value of 11 (specificity 0.98 and sensitivity 0.94). The I-HCPI was confirmed to be a valid, sensitive, reliable, and accurate tool to discriminate between dogs with and without pain.

Screening a Molecular Fragment Library to Modulate the PED/PEA15-Phospholipase D1 Interaction in Cellular Lysate Environments.

The overexpression of PED/PEA15, the phosphoprotein enriched in diabetes/phosphoprotein enriched in the astrocytes 15 protein (here referred simply to as PED), observed in some forms of type II diabetes, reduces the transport of insulin-stimulated glucose by binding to the phospholipase D1 (PLD1). The inhibition of the PED/PLD1 interaction was shown to restore basal glucose transport, indicating PED as a pharmacological target for the development of drugs capable of improving insulin sensitivity and glucose tolerance. We here report the identification and selection of PED ligands by means of NMR screening of a library of small organic molecules, NMR characterization of the PED/PLD1 interaction in lysates of cells expressing PLD1, and modulation of such interactions using BPH03, the best selected ligand. Overall, we complement the available literature data by providing detailed information on the structural determinants of the PED/PLD1 interaction in a cellular lysate environment and indicate BPH03 as a precious scaffold for the development of novel compounds that are able to modulate such interactions with possible therapeutic applications in type II diabetes.

Initial Psychometric Testing and Validation of the Italian Version of the Canine Brief Pain Inventory in Dogs With Pain Related to Osteoarthritis.

The Canine Brief Pain Inventory (CBPI) is an owner-administered questionnaire, originally developed and validated in English, used to assess canine chronic pain in terms of severity and interference with daily life activities. The aim of the present study was to perform a preliminary validation of an Italian version of the CBPI. Translation was performed and the resulting questionnaire was administered to 45 native Italian speaking owners of dogs suffering from chronic pain due to radiographically confirmed osteoarthritis. Psychometric properties of the Italian CBPI including construct validity, convergent validity and reliability were evaluated. Construct validity was assessed by factor analysis and confirmed a two-factor model (i.e., pain severity and interference factors). The respective scores, that is, the pain severity score (PSS) and pain interference score (PIS), exhibited a substantial negative correlation with overall quality of life score. Pain severity and interference items showed a mean inter-item correlation of 0.90 and 0.80, respectively. For each question, communality ranged from 0.84 to 0.97, highlighting strong internal consistency and suggesting that PSS and PIS can be calculated by averaging the items contained within each factor. Cronbach's α was 0.97 and 0.96 for PSS and PIS, respectively. The present findings confirmed the main psychometric properties of the Italian version of the CBPI, providing clinicians and researchers with a useful metrology instrument to evaluate the severity of chronic pain and its interference with daily life activities in dogs with osteoarthritis owned by Italian speaking people. Further properties of the questionnaire need to be evaluated in future research and larger studies are warranted.

Effect of dietary supplementation with ultramicronized palmitoylethanolamide in maintaining remission in cats with nonflea hypersensitivity dermatitis: a double-blind, multicentre, randomized, placebo-controlled study.

BACKGROUND: Feline nonflea hypersensitivity dermatitis (NFHD) is a frequent cause of over-grooming, scratching and skin lesions. Multimodal therapy often is necessary. HYPOTHESIS/OBJECTIVES: To investigate the efficacy of ultramicronized palmitoylethanolamide (PEA-um) in maintaining methylprednisolone-induced remission in NFHD cats. ANIMALS: Fifty-seven NFHD cats with nonseasonal pruritus were enrolled originally, of which 25 completed all study requirements to be eligible for analysis. METHODS AND MATERIALS: Cats were randomly assigned to PEA-um (15 mg/kg per os, once daily; n = 29) or placebo (n = 28) while receiving a 28 day tapering methylprednisolone course. Cats responding favourably to methylprednisolone were then administered only PEA-um (n = 21) or placebo (n = 23) for another eight weeks, followed by a four week long treatment-free period. Cats were maintained in the study until relapse or study end, whichever came first. Primary outcome was time to relapse. Secondary outcomes were pruritus Visual Analog Scale (pVAS), SCORing Feline Allergic Dermatitis scale (SCORFAD) and owner Global Assessment Score (GAS). RESULTS: Mean relapse time was 40.5 days (±7.8 SE) in PEA-um treated cats (n = 13) and 22.2 days (±3.7 SE) for placebo (n = 12; P = 0.04). On Day 28, the severity of pruritus was lower in the PEA-um treated cats compared to placebo (P = 0.03). Mean worsening of pruritus at the final study day was lower in the PEA-um group compared to placebo (P = 0.04), whereas SCORFAD was not different between groups. Mean owner GAS at the final study day was better in the PEA-um than the placebo-treated group (P = 0.05). CONCLUSION AND CLINICAL IMPORTANCE: Ultramicronized palmitoylethanolamide could represent an effective and safe option to delay relapse in NFHD cats.

Expression of functional TRPV1 receptor in primary culture of canine keratinocytes.

The interest for the endovanilloid system and for transient receptor potential vanilloid 1 (TRPV1) is continuously increasing, due to their involvement in inflammation, nociception and pruritus. Even if TRPV1 enrolment was highlighted in both physiological and pathological conditions, some aspects remain unclear, mostly in veterinary medicine. This study aimed to verify the expression and functionality of TRPV1 in canine keratinocytes to investigate in vitro the role of TRPV1 in these cells that are involved in different cutaneous pathologies. Keratinocytes primary cultures were isolated from bioptical samples and cultivated. Binding assay (using 3 [H]-resiniferatoxin), displacement assay (in the presence of 1.2 nM 3 [H]-resiniferatoxin) and functional assays (in the presence of 1 µCi/45 Ca2+ ) with vanilloid agonists and antagonists, specifically addressed to TRPV1 receptor, were performed. Binding assay demonstrated the presence of measurable concentrations of TRPV1 (Bmax  = 1,240 ± 120 fmol/mg protein; Kd  = 0.01 ± 0.004 nM). Displacement assay highlighted the highest affinity for resiniferatoxin (RTX) and 5-iodo-resiniferatoxin (5-I-RTX), among agonists and antagonists, respectively. The same compounds results as the most potent in the functional assays. This study demonstrated the identification and the characterization of TRPV1 receptor in primary canine keratinocytes cultures. The results are promising for a clinical use, but further in vivo investigations are required.

Sobre la adherencia al tratamiento: Análisis de un caso clínico a la luz del modelo transteórico / PATIENT'S ADHESION TO TREATMENT: ANALYSIS OF A CLINICAL CASE REFERRED TO A TRANSTHEORETICAL MODEL

Antecedentes: Determinadas patologías crónicas, asociadas con hábitos perniciosos y con las que convive el enfermo durante muchos años afectando cada vez más su calidad de vida y eventualmente amenazando su vida misma, requieren abordajes terapéuticos interdisciplinarios que modifiquen la resistencia a su corrección. Objetivo: demostrar la utilidad del modelo transteórico. Diseño: observacional-descriptivo de un caso clìnico. Lugar de aplicación: Hospital Universitario de atención terciaria. Método: consta de cinco etapas: Precontemplación, Contemplación, Preparación para la acción, Acción y Mantenimiento del cambio logrado (Método Transteórico de Prochaska y Di Clemente). Resultado: Se logró la adhesión del paciente a conductas e indicaciones rechazadas en un principio. Logrando mejorar su control metabólico, y de otros factores de riesgo. Conclusiones:el modelo terapéutico aplicado a este paciente con una enfermedad de evolución crónica sin conciencia de la misma, resultó eficaz

Background: Certain chronic diseases associated with harmful habits with which some patients live for many years that affect both their quality of life and eventually threaten life itself, require multidisciplinary therapeutic approaches that change the patient´s resistance to habits being corrected. Objective: To show the benefits of the transtheoretical model. Design: Observation of case report Setting: tertiary care at University Hospital Method: The method consists of five stages: Precontemplation, Contemplation, Preparation for action, Action, and Maintenance of the achieved change (The Transtheoretical Model by Prochaska & Di Clemente) Result: The patient´s adhesion to behaviour and medical advice which had initially been rejected was achieved. The insulin dose was reduced obtaining adequate glycaemic control, there was an improvement in the lipid profile, and adhesion to the nutritional treatment strengthened. This led to the beginning of body weight loss and the treatment of an ulcer on the sole of the patient´s foot. Conclusions: The therapeutic model applied to this patient with a chronic disease without awareness was effective

The validation of pharmacogenetics for the identification of Fabry patients to be treated with migalastat.

PURPOSE: Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene. Migalastat, a pharmacological chaperone, binds to specific mutant forms of α-galactosidase A to restore lysosomal activity. METHODS: A pharmacogenetic assay was used to identify the α-galactosidase A mutant forms amenable to migalastat. Six hundred Fabry disease-causing mutations were expressed in HEK-293 (HEK) cells; increases in α-galactosidase A activity were measured by a good laboratory practice (GLP)-validated assay (GLP HEK/Migalastat Amenability Assay). The predictive value of the assay was assessed based on pharmacodynamic responses to migalastat in phase II and III clinical studies. RESULTS: Comparison of the GLP HEK assay results in in vivo white blood cell α-galactosidase A responses to migalastat in male patients showed high sensitivity, specificity, and positive and negative predictive values (≥0.875). GLP HEK assay results were also predictive of decreases in kidney globotriaosylceramide in males and plasma globotriaosylsphingosine in males and females. The clinical study subset of amenable mutations (n = 51) was representative of all 268 amenable mutations identified by the GLP HEK assay. CONCLUSION: The GLP HEK assay is a clinically validated method of identifying male and female Fabry patients for treatment with migalastat.Genet Med 19 4, 430-438.

Establishment of a 2-week canine skin organ culture model and its pharmacological modulation by epidermal growth factor and dexamethasone.

Although canine skin models are already available as either monocellular or organotypic cultures, they only partly recapitulate normal skin morphological features and function. The objective of this study was to establish a canine serum-free skin organ culture model and verify whether dexamethasone could rescue epidermal growth factor-induced changes. The study of morphological changes as a response to pharmacological substances may indeed help to investigate skin physiology and pathology. Normal skin was obtained from five client-owned dogs subjected to surgical procedures unrelated to dermatological conditions. Two experimental designs were performed: (i) two-week viability of the skin culture; (ii) dexamethasone (DMS) inhibition of epidermal growth factor (EGF)-induced effects. Serum-free submerged organ cultures were established in Williams' E medium supplemented with penicillin-streptomycin, insulin, hydrocortisone and l-glutamine. General morphological features of skin anatomical structures were well maintained up to day 14, scattered pyknotic nuclei were visible in the epidermis from day 7. Normal keratinocyte differentiation was confirmed by cytokeratin (K) 10, K14 and loricrin immunostaining. Epidermal thickness did not decrease throughout the study. A decrease in keratinocyte proliferation was observed at day 7 and 14. Treatment with EGF induced both keratinocyte proliferation and thickening of the epidermis; both responses were counteracted by DMS. Treatment with EGF increased the length of epithelial tongues at the edge of the skin explants; this effect was further enhanced by DMS supplementation. Our findings demonstrate the potential use of a full-thickness canine skin organ culture model for the study of skin physiology and pharmacological response to exogenous compounds, especially in the field of re-epithelialisation and keratinization disorders.

Reply to: "Palmitoylethanolamide: problems regarding micronization, ultra-micronization and additives" Inflammopharmacology DOI:10.1007/s10787-014-0202-3.

This is a reply to a recently published Commentary: "Palmitoylethanolamide: problems regarding micronization, ultra-micronization and additives" Inflammopharmacology DOI: 10.1007/s10787-014-0202-3 , written in relation to our review article: Skaper SD, Facci L, Fusco M, della Valle MF, Zusso M, Costa B, Giusti P (2014) "Palmitoylethanolamide, a naturally occurring disease-modifying agent in neuropathic pain" Inflammopharmacology 22:79-94 DOI: 10.1007/s10787-013-0191-7 . We believe that the Commentary by Kriek contains a number of erroneous statements and misinterpretations of the published scientific/medical literature which our reply shall elaborate on. Further, the writer of the Commentary has a direct connection to a company, JP Russell Science Ltd that sells palmitoylethanolamide. The take-home message of our review remains as originally stated: "Collectively, the findings presented here propose that palmitoylethanolamide merits further consideration as a disease-modifying agent for controlling inflammatory responses and related chronic and neuropathic pain".

Increased levels of palmitoylethanolamide and other bioactive lipid mediators and enhanced local mast cell proliferation in canine atopic dermatitis.

BACKGROUND: Despite the precise pathogenesis of atopic dermatitis (AD) is unknown, an immune dysregulation that causes Th2-predominant inflammation and an intrinsic defect in skin barrier function are currently the two major hypotheses, according to the so-called outside-inside-outside model. Mast cells (MCs) are involved in AD both by releasing Th2 polarizing cytokines and generating pruritus symptoms through release of histamine and tryptase. A link between MCs and skin barrier defects was recently uncovered, with histamine being found to profoundly contribute to the skin barrier defects.Palmitoylethanolamide and related lipid mediators are endogenous bioactive compounds, considered to play a protective homeostatic role in many tissues: evidence collected so far shows that the anti-inflammatory effect of palmitoylethanolamide depends on the down-modulation of MC degranulation.Based on this background, the purpose of the present study was twofold: (a) to determine if the endogenous levels of palmitoylethanolamide and other bioactive lipid mediators are changed in the skin of AD dogs compared to healthy animals; (b) to examine if MC number is increased in the skin of AD dogs and, if so, whether it depends on MC in-situ proliferation. RESULTS: The amount of lipid extract expressed as percent of biopsy tissue weight was significantly reduced in AD skin while the levels of all analyzed bioactive lipid mediators were significantly elevated, with palmitoylethanolamide showing the highest increase.In dogs with AD, the number of MCs was significantly increased in both the subepidermal and the perifollicular compartments and their granule content was significantly decreased in the latter. Also, in situ proliferation of MCs was documented. CONCLUSIONS: The levels of palmitoylethanolamide and other bioactive lipid mediators were shown to increase in AD skin compared to healthy samples, leading to the hypothesis that they may be part of the body's innate mechanisms to maintain cellular homeostasis when faced with AD-related inflammation. In particular, the increase may be considered a temptative response to down-regulating the observed elevation in the number, functionality and proliferative state of MCs in the skin of AD dogs. Further studies are warranted to confirm the hypothesis.

Palmitoylethanolamide, a naturally occurring disease-modifying agent in neuropathic pain.

Persistent pain affects nearly half of all people seeking medical care in the US alone, and accounts for at least $80 billion worth of lost productivity each year. Among all types of chronic pain, neuropathic pain stands out: this is pain resulting from damage or disease of the somatosensory nervous system, and remains largely untreatable. With few available treatment options, neuropathic pain represents an area of significant and growing unmet medical need. Current treatment of peripheral neuropathic pain involves several drug classes, including opioids, gabapentinoids, antidepressants, antiepileptic drugs, local anesthetics and capsaicin. Even so, less than half of patients achieve partial relief. This review discusses a novel approach to neuropathic pain management, based on knowledge of: the role of glia and mast cells in pain and neuroinflammation; the body's innate mechanisms to maintain cellular homeostasis when faced with external stressors provoking, for example, inflammation. The discovery that palmitoylethanolamide, a member of the N-acylethanolamine family which is produced from the lipid bilayer on-demand, is capable of exerting anti-allodynic and anti-hyperalgesic effects by down-modulating both microglial and mast cell activity has led to the application of this fatty acid amide in several clinical studies of neuropathic pain, with beneficial outcome and no indication of adverse effects at pharmacological doses. Collectively, the findings presented here propose that palmitoylethanolamide merits further consideration as a disease-modifying agent for controlling inflammatory responses and related chronic and neuropathic pain.

Inhibitory effect of topical adelmidrol on antigen-induced skin wheal and mast cell behavior in a canine model of allergic dermatitis.

BACKGROUND: Adelmidrol is a semisynthetic derivative of azelaic acid and analogue of the anti-inflammatory compound palmitoylethanolamide (PEA). Based upon its physicochemical properties, adelmidrol is suitable for topical application. The main objective of the present study was to evaluate the efficacy of a topical adelmidrol emulsion on early and late inflammatory responses in hypersensitive dogs. Repeated intradermal injections of Ascaris suum extract were performed in both lateral thoracic areas of six conscious hypersensitive Beagle dogs, topically treated during 8 consecutive days. Adelmidrol (2%) was applied to one side and vehicle to the other. 24 hours after the last antigen challenge, two biopsies (adelmidrol- and vehicle-treated side) were obtained for each dog at the antigen injection site. RESULTS: A significant reduction in the antigen-induced wheal areas was observed on the 4th and 7th day of adelmidrol treatment. Moreover, cutaneous mast cell numbers were significantly decreased in biopsies obtained after 8 consecutive days of topical adelmidrol treatment. CONCLUSIONS: The results obtained in the present study show that topical treatment with adelmidrol might represent a new therapeutic tool in controlling the early and late allergic inflammatory skin responses in companion animals.

Cannabinoid receptor type 1 and 2 expression in the skin of healthy dogs and dogs with atopic dermatitis.

OBJECTIVE: To determine the distribution of cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2) in skin (including hair follicles and sweat and sebaceous glands) of clinically normal dogs and dogs with atopic dermatitis (AD) and to compare results with those for positive control samples for CB1 (hippocampus) and CB2 (lymph nodes). SAMPLE: Skin samples from 5 healthy dogs and 5 dogs with AD and popliteal lymph node and hippocampus samples from 5 cadavers of dogs. PROCEDURES: CB1 and CB2 were immunohistochemically localized in formalin-fixed, paraffin-embedded sections of tissue samples. RESULTS: In skin samples of healthy dogs, CB1 and CB2 immunoreactivity was detected in various types of cells in the epidermis and in cells in the dermis, including perivascular cells with mast cell morphology, fibroblasts, and endothelial cells. In skin samples of dogs with AD, CB1 and CB2 immunoreactivity was stronger than it was in skin samples of healthy dogs. In positive control tissue samples, CB1 immunoreactivity was detected in all areas of the hippocampus, and CB2 immunoreactivity was detected in B-cell zones of lymphoid follicles. CONCLUSIONS AND CLINICAL RELEVANCE: The endocannabinoid system and cannabimimetic compounds protect against effects of allergic inflammatory disorders in various species of mammals. Results of the present study contributed to knowledge of the endocannabinoid system and indicated this system may be a target for treatment of immune-mediated and inflammatory disorders such as allergic skin diseases in dogs.

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.

A pharmacogenetic approach to identify mutant forms of α-galactosidase A that respond to a pharmacological chaperone for Fabry disease.

Fabry disease is caused by mutations in the gene (GLA) that encodes α-galactosidase A (α-Gal A). The iminosugar AT1001 (GR181413A, migalastat hydrochloride, 1-deoxygalactonojirimycin) is a pharmacological chaperone that selectively binds and stabilizes α-Gal A, increasing total cellular levels and activity for some mutant forms (defined as "responsive"). In this study, we developed a cell-based assay in cultured HEK-293 cells to identify mutant forms of α-Gal A that are responsive to AT1001. Concentration-dependent increases in α-Gal A activity in response to AT1001 were shown for 49 (60%) of 81 mutant forms. The responses of α-Gal A mutant forms were generally consistent with the responses observed in male Fabry patient-derived lymphoblasts. Importantly, the HEK-293 cell responses of 19 α-Gal A mutant forms to a clinically achievable concentration of AT1001 (10 µM) were generally consistent with observed increases in α-Gal A activity in peripheral blood mononuclear cells from male Fabry patients orally administered AT1001 during Phase 2 clinical studies. This indicates that the cell-based responses can identify mutant forms of α-Gal A that are likely to respond to AT1001 in vivo. Thus, the HEK-293 cell-based assay may be a useful aid in the identification of Fabry patients with AT1001-responsive mutant forms.

Classification of subcellular location by comparative proteomic analysis of native and density-shifted lysosomes.

One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for identification of lysosomal proteins and underscore the necessity of balance sheet analysis for localization studies.