Dendritic cells under allergic condition enhance the activation of pruritogen-responsive neurons via inducing itch receptors in a co-culture study.

BACKGROUND: Itch sensitization has been reported in patients with chronic allergic skin diseases and observed in a mouse model of allergic contact dermatitis (ACD). There is evidence suggesting that neuroimmune interactions may contribute to itch sensitization, as an increase in dendritic cells (DCs) within ganglia has been observed during allergic conditions. However, how DCs interact with sensory neurons in ganglia during allergic conditions is still not known. This study aims to investigate the role of DCs in dorsal root ganglion (DRG) under ACD conditions, specifically focusing on itch sensitization within the DRG. The tolylene-2,4-diisocyanate (TDI) mouse model for ACD and the co-culture model of DCs and DRG neurons was employed in this study. RESULTS: We successfully induced ACD by TDI, as evidenced by the development of edema, elevated total serum IgE levels, and an observed itch reaction in TDI-sensitized mice. Calcium imaging and RT-qPCR analysis revealed that TDI-sensitized mice exhibited signs of peripheral sensitization, including a higher percentage of neurons responding to pruritogens and increased activation and expression of itch receptors in excised DRG of TDI-sensitized mice. Immunofluorescence and flow cytometric analysis displayed an increase of MHCII+ cells, which serves as a marker for DCs, within DRG during ACD. The co-culture study revealed that when DRG neurons were cultured with DCs, there was an increase in the number of neurons responsive to pruritogens and activation of itch receptors such as TRPA1, TRPV1, H1R, and TRPV4. In addition, the immunofluorescence and RT-qPCR study confirmed an upregulation of TRPV4. CONCLUSIONS: Our findings indicate that there is an increase of MHCII+ cells and itch peripheral sensitization in DRG under TDI-induced ACD condition. It has been found that MHCII+ cells in DRG might contribute to the itch peripheral sensitization by activating itch receptors, as shown through co-culture studies between DRG neurons and DCs. Further studies are required to identify the specific mediator(s) responsible for peripheral sensitization induced by activated DCs.

Prevalence of MRSA in canine and feline clinical samples from one-third of veterinary practices in Germany from 2019-2021.

BACKGROUND: MRSA is a major contributor to AMR-related deaths. The WHO's global action plan emphasizes a One Health approach, acknowledging the connection between humans and their companion animals. It is agreed on that comprehensive AMR surveillance is needed. OBJECTIVES: This study provides a large-scale overview of MRSA occurrence in cats and dogs in Germany, serving as a foundation for continuous surveillance. METHODS: The study analysed all results of canine and feline bacterial diagnostic samples from a large laboratory, encompassing samples received from veterinary practices between January 2019 and December 2021. MRSA prevalence between host species, sample types and geographical distribution were compared. Additionally, data were contrasted with human MRSA surveillance data from Germany. RESULTS: Samples originated from 3491 German veterinary practices, representing 33.1% of practices and clinics nationally. Bacterial examination results from 175 171 samples were analysed, identifying S. aureus in 5526 of these samples (3.2% isolation rate). S. aureus in clinical samples was more prevalent in cats (5.6%) than dogs (2.0%). Methicillin resistance was found in 17.8% of S. aureus samples and was higher in dogs (20.4%, 95%CI 18.9-22.0) than cats (15.6%, 95%CI 14.3-17.0). The highest MRSA prevalence was found in canine wound samples (32%), compared to skin/soft tissue, respiratory tract and other (<23% respectively). CONCLUSION: The study reveals a 17.8% MRSA prevalence, which is higher than the human outpatient MRSA prevalence (5.4%). Restriction and regulation of veterinary antibiotic use should be validated with AMR surveillance. Our study shows that this is feasible in companion animals with significant coverage.

Sphingosine 1-Phosphate as Essential Signaling Molecule in Inflammatory Skin Diseases.

Sphingolipids are crucial molecules of the mammalian epidermis. The formation of skin-specific ceramides contributes to the formation of lipid lamellae, which are important for the protection of the epidermis from excessive water loss and protect the skin from the invasion of pathogens and the penetration of xenobiotics. In addition to being structural constituents of the epidermal layer, sphingolipids are also key signaling molecules that participate in the regulation of epidermal cells and the immune cells of the skin. While the importance of ceramides with regard to the proliferation and differentiation of skin cells has been known for a long time, it has emerged in recent years that the sphingolipid sphingosine 1-phosphate (S1P) is also involved in processes such as the proliferation and differentiation of keratinocytes. In addition, the immunomodulatory role of this sphingolipid species is becoming increasingly apparent. This is significant as S1P mediates a variety of its actions via G-protein coupled receptors. It is, therefore, not surprising that dysregulation in the signaling pathways of S1P is involved in the pathophysiological conditions of skin diseases. In the present review, the importance of S1P in skin cells, as well as the immune cells of the skin, is elaborated. In particular, the role of the molecule in inflammatory skin diseases will be discussed. This is important because interfering with S1P signaling pathways may represent an innovative option for the treatment of inflammatory skin diseases.

Early inflammatory events of mastitis-a pilot study with the isolated perfused bovine udder.

BACKGROUND: Bovine mastitis is an important health and cost factor in the milk industry. To elucidate whether isolated perfused bovine udders can be used to study early inflammatory events of mastitis, 1 mg of lipopolysaccharide (LPS) was instilled into quarters of 10 isolated perfused bovine udders. Three hours and 6 h after LPS instillation, tissue samples were taken from the gland cistern and base of the udder, subsequently stored in RNAlater and processed for the determination of inflammation-dependent gene regulation by real-time RT-qPCR. Gene expression analysis was performed using delta-delta Ct method. To translate mRNA results to protein, IL-1ß and IL-6 were determined in tissue homogenate by ELISA. RESULTS: The instillation of 1 mg LPS lead to an increased expression of pro-inflammatory cytokines and chemokines like TNF-α, CCL20, CXCL8 as well as of IL-1 ß, IL-6 and IL-10, lingual antimicrobial peptide (LAP) and S100A9. However, the degree of elevation differed slightly between gland cistern and udder base and markedly between 3 and 6 h after instillation, with a distinct increase in mediator expression after 6 h. IL-1ß protein increased in a time-dependent manner, whereas IL-6 was unchanged within 6 h of LPS instillation. CONCLUSION: Compared to in vivo studies with instillation of LPS into udders of living cows, a similar inflammation-dependent gene regulation profile can be mimicked in the isolated perfused bovine udder, indicating a supplementation of animal experiments.

Surface distribution of pyrethroids following topical application to veterinary species: Implications for lateral transport.

Pyrethroids like permethrin have been used as topical formulations for their ectoparasiticidal effects since the 1970s. There are numerous efficacy studies in dogs and livestock animals that indicate a fast spread of pyrethroids after topical administration onto rather confined areas of the skin. Some studies correlate the efficacy against ticks, fleas or lice with concentrations of pyrethroids in hair and, less frequently, stratum corneum samples. It is often stated that lateral transport is responsible for the distribution of the pyrethroids over the body surface. With this review, we attempt to demonstrate evidence for lateral transport of pyrethroids after topical administration in dogs, cattle and sheep and to present data gaps that should be addressed in follow-up studies.

Histamine 2 Receptor Agonism and Histamine 4 Receptor Antagonism Ameliorate Inflammation in a Model of Psoriasis.

Psoriasis is a chronic inflammatory skin disorder characterized by hyperproliferative keratinocytes and immune cell infiltration into the skin, often accompanied by itch. Histamine, acting via histamine 1-4 receptors, is known to modulate immune responses in the skin and to induce itch. The aim of this study was to test the role of histamine 2 receptors and histamine 4 receptors in the imiquimod-induced psoriasis-like skin inflammation model. BALB/c mice were treated intraperitoneally with amthamine (histamine 2 receptor agonist), JNJ-39758979 (histamine 4 receptor antagonist), a combination of both, or vehicle twice daily in a preventive manner. Imiquimod was applied once daily onto the back skin for 10 consecutive days. Stimulation of histamine 2 receptors and blockade of histamine 4 receptors ameliorated imiquimod-induced skin inflammation. The combination of amthamine and JNJ-39758979 reduced skin inflammation even more pronounced, diminished epidermal hyperproliferation, and inhibited spontaneous scratching behaviour. A combination of histamine 2 receptor agonist and histamine 4 receptor antagonists could represent a new strategy for the treatment of psoriasis.

Efficacy study of a topical treatment with a plant extract with antibiofilm activities using an in vivo model of canine superficial pyoderma.

BACKGROUND: Canine pyoderma is a common skin infection caused predominantly by staphylococcal bacteria. Because of increasing rates of antimicrobial resistance in bacterial isolates, there is an urgent need for alternative or supplementary treatment options. W16P576, a Water Extract of Complex Mix of Edible Plants (WECMEP), has shown in vitro activity against a variety of bacteria, including Staphylococcus pseudintermedius. A canine model of pyoderma was developed which allows in vivo testing of antimicrobial agents in a controlled environment. OBJECTIVE: To evaluate the antibacterial efficacy of topical application of W16P576 in a model of canine pyoderma. ANIMALS: Nine laboratory housed beagle dogs. METHODS AND MATERIALS: In an evaluator-blinded cross-over study with an eight week washout period, dogs were treated topically twice daily with W16P576 WECMEP or its vehicle, starting three days before bacterial challenge. On the day of challenge, each dog was treated with two concentrations of a clinical S. pseudintermedius strain on opposite sides of the body. Topical treatment was continued for 11 days and lesions of pyoderma were evaluated and scored for 14 days. RESULTS: All dogs developed lesions consistent with bacterial pyoderma. Lesion scores were generally higher on the side inoculated with a higher concentration of bacteria. Treatment with W16P576 significantly reduced lesion development and hastened resolution of lesions, compared to placebo. CONCLUSION: Topical application of W16P576 markedly reduced lesion development in this proof of principle study. Clinical trials are warranted to estimate benefits for dogs with naturally occurring pyoderma under field conditions.

Neuromedin B Induces Acute Itch in Mice via the Activation of Peripheral Sensory Neurons.

Neuromedin B is expressed in nociceptive and itch-sensitive dorsal root ganglia neurons, but its peripheral pruritogenic potential is not well described. The potential of neuromedin B as a pruritogen and pro-inflammatory peptide in the skin was tested in vivo in an acute model in mice and monkeys as well as an allergic dermatitis model in mice. To identify the underlying mechanisms in vitro real time PCR analysis for neuromedin B and its receptor expression in murine mast cells and dorsal root ganglia as well as functional calcium imaging in the ganglia was applied. Neuromedin B induces itch when injected intradermally, and the peripheral signal is likely transmitted through the activation of dorsal root ganglia. Thus, neuromedin B could be an interesting new therapeutic target for peripheral processing of itch at the level of sensory neurons.

Exon skipping of FcεRIß eliminates expression of the high-affinity IgE receptor in mast cells with therapeutic potential for allergy.

Allergic diseases are driven by activation of mast cells and release of mediators in response to IgE-directed antigens. However, there are no drugs currently available that can specifically down-regulate mast cell function in vivo when chronically administered. Here, we describe an innovative approach for targeting mast cells in vitro and in vivo using antisense oligonucleotide-mediated exon skipping of the ß-subunit of the high-affinity IgE receptor (FcεRIß) to eliminate surface high-affinity IgE receptor (FcεRI) expression and function, rendering mast cells unresponsive to IgE-mediated activation. As FcεRIß expression is restricted to mast cells and basophils, this approach would selectively target these cell types. Given the success of exon skipping in clinical trials to treat genetic diseases such as Duchenne muscular dystrophy, we propose that exon skipping of FcεRIß is a potential approach for mast cell-specific treatment of allergic diseases.

Diphenhydramine pharmacokinetics after oral and intravenous administration of diphenhydramine and oral administration of dimenhydrinate to healthy dogs, and pharmacodynamic effect on histamine-induced wheal formation: a pilot study.

BACKGROUND: Histamine type-1 (H1) receptor antagonists such as diphenhydramine are frequently used for treatment of pruritus in dogs, yet therapeutic efficacy for allergic disorders is reported to be highly variable. Dimenhydrinate is a salt of diphenhydramine and 8-chlorotheophylline, and has been reported to produce superior oral absorption of diphenhydramine. HYPOTHESIS/OBJECTIVE: To determine the pharmacokinetic and pharmacodynamic properties of diphenhydramine in dogs after intravenous (1 mg/kg) and oral (5 mg/kg) administration, and when given orally as dimenhydrinate at a dose of 10 mg/kg (≈5 mg/kg diphenhydramine). ANIMALS: Each drug was administered to six healthy, fasted mixed-breed dogs in a research facility, using a cross-over design. METHODS AND MATERIALS: Blood samples were collected for pharmacokinetic analysis of diphenhydramine and chlorotheophylline at defined intervals. Pharmacodynamic response was measured by histamine-mediated cutaneous wheal formation. RESULTS: There was great variability in the data and one dog was an extreme outlier. The mean systemic availabilities of diphenhydramine were 7.8% and 22.0% after oral administration of diphenhydramine and dimenhydrinate, respectively, whereas the mean maximum concentrations were 36 (± 20) and 124 (± 46) ng/mL. The terminal elimination half-lives of diphenhydramine and dimenhydrinate were 5.0 (± 7.1) and 11.6 (± 17.7) h, respectively. Plasma diphenhydramine concentrations did not correlate with the percentage reduction in histamine-induced wheal formation. Theophylline reached plasma concentrations considered to be therapeutic for dogs. CONCLUSION: Oral absorption of diphenhydramine was approximately three times greater with a longer half-life when it was administered as the combination product dimenhydrinate.

Diluted sodium hypochlorite (bleach) in dogs: antiseptic efficacy, local tolerability and in vitro effect on skin barrier function and inflammation.

BACKGROUND: Diluted sodium hypochlorite represents an inexpensive and widely available topical antiseptic, but there are no tolerability and efficacy data in veterinary dermatology. OBJECTIVES: To determine the in vivo antibacterial effect and tolerability of topical diluted bleach application and to assess its in vitro effect on skin barrier lipids and anti-inflammatory properties on keratinocytes. METHODS: Topical hypochlorite at 0.05% and tap water were applied to both sides of the thorax of four healthy dogs. The anti-inflammatory effect on canine keratinocytes was determined by real-time polymerase chain reaction; skin barrier integrity was assessed by evaluating stratum corneum lipid changes in canine stratified epidermal constructs. RESULTS: The cell viability of primary keratinocytes treated with water and diluted hypochlorite at 0.005 and 0.01%, reduced the percentage of viable cells by 10%. The exposure of primary keratinocytes to 0.005% diluted hypochlorite significantly reduced the induction of inflammatory genes chemokine ligand-2 (CCL2; P = 0.015) and thymus and activation-regulated chemokine (TARC/CCL17, P = 0.032). There were no changes in skin lipid ceramide and nonceramide fractions in stratified epidermal constructs cultured for 17 days with 0.05% hypochlorite. Topical hypochlorite at 0.05% and tap water were well-tolerated without signs of skin irritation. Although a marked reduction in bacterial counts was seen within 20 min of diluted bleach application compared to the tap water control, this was only marginally significant (P = 0.06). CONCLUSIONS AND CLINICAL IMPORTANCE: The results indicate that a topical diluted bleach solution, at either 0.05 or 0.005% hypochlorite concentrations, is a well-tolerated antiseptic that also exhibits anti-inflammatory properties.

The selective glucocorticoid receptor agonist mapracorat displays a favourable safety-efficacy ratio for the topical treatment of inflammatory skin diseases in dogs.

BACKGROUND: Mapracorat is a nonsteroidal Selective Glucocorticoid Receptor Agonist (SEGRA) that is presumed to have a better therapeutic index compared to classical glucocorticoids. OBJECTIVES: To compare the efficacy and safety of mapracorat with classical glucocorticoids used for the treatment of allergic skin diseases in dogs. ANIMALS: Six laboratory beagles. METHODS: The effect of mapracorat on lipopolysaccharide-induced TNFα secretion from canine peripheral blood derived mononuclear cells (PBMC) was tested. In vivo, mapracorat was compared to triamcinolone acetonide using a skin inflammation model. Skin fold thickness was determined after daily administration of mapracorat and triamcinolone acetonide over 14 days. RESULTS: Mapracorat concentration dependently inhibited TNFα secretion from activated canine PBMC with a half maximal inhibitory concentration (IC50 ) value of approximately 0.2 nmol/L. Intradermal injection of compound 48/80 (50 µg in 50 µL saline) resulted in a clear wheal and flare reaction over the 60 min observation period. Topical pre-treatment with mapracorat (0.1%) and triamcinolone acetonide (0.015%) led to significant reduction in the wheal and flare responses compared to vehicle (acetone) treated areas. However, once daily topical administration of triamcinolone acetonide significantly reduced skin fold thickness from day 8 to 14, whereas no such reduction was observed for mapracorat. CONCLUSION: These results demonstrate that mapracorat has comparable anti-inflammatory efficacy to classical steroidal glucocorticoids under these experimental settings and maintenance of skin fold thickness indicates a better safety profile compared to triamcinolone acetonide at equipotent concentrations. This profile further suggests that SEGRAs show promise in the management of inflammatory and pruritic skin diseases in dogs.

Translational Animal Models of Atopic Dermatitis for Preclinical Studies.

There is a medical need to develop new treatments for patients suffering from atopic dermatitis (AD). To improve the discovery and testing of novel treatments, relevant animal models for AD are needed. Generally, these animal models mimic different aspects of the pathophysiology of human AD, such as skin barrier defects and Th2 immune bias with additional Th1 and Th22, and in some populations Th17, activation. However, the pathomechanistic characterization and pharmacological validation of these animal models are generally incomplete. In this paper, we review animal models of AD in the context of preclinical use and their possible translation to the human disease. Most of these models use mice, but we will also critically evaluate dog models of AD, as increasing information on disease mechanism show their likely relevance for the human disease.

Topically Administered Janus-Kinase Inhibitors Tofacitinib and Oclacitinib Display Impressive Antipruritic and Anti-Inflammatory Responses in a Model of Allergic Dermatitis.

The prevalence of allergic skin disorders has increased rapidly, and development of therapeutic agents to alleviate the symptoms are still needed. In this study, we orally or topically administered the Janus kinase (JAK) inhibitors, tofacitinib and oclacitinib, in a mouse model of dermatitis, and compared the efficacy to reduce the itch and inflammatory response. In vitro effects of JAK inhibitors on bone marrow-derived dendritic cells (BMDCs) were analyzed. For the allergic dermatitis model, female BALB/c mice were sensitized and challenged with toluene-2,4-diisocyanate (TDI). Each JAK inhibitor was orally or topically applied 30 minutes before and 4 hours after TDI challenge. After scratching bouts and ear thickness were measured, cytokines were determined in challenged skin and the cells of the draining lymph node were analyzed by means of flow cytometry. In vitro, both JAK inhibitors significantly inhibited cytokine production, migration, and maturation of BMDCs. Mice treated orally with JAK inhibitors showed a significant decrease in scratching behavior; however, ear thickness was not significantly reduced. In contrast, both scratching behavior and ear thickness in the topical treatment group were significantly reduced compared with the vehicle treatment group. However, cytokine production was differentially regulated by the JAK inhibitors, with some cytokines being significantly decreased and some being significantly increased. In conclusion, oral treatment with JAK inhibitors reduced itch behavior dramatically but had only little effect on the inflammatory response, whereas topical treatment improved both itch and inflammatory response. Although the JAK-inhibitory profile differs between both JAK inhibitors in vitro as well as in vivo, the effects have been comparable.

Atopic itch in dogs: pharmacology and modeling.

Itch is the most common clinical problem seen in dogs with skin diseases. Although an etiological classification of canine pruritus does not yet exist, most causes would likely fall into the IFSI class I (dermatological) itch. One of the most common causes of canine itch is that associated with atopic dermatitis, and there is randomized controlled trial grade evidence of the efficacy of several antipruritic interventions. At this time, the mainstay of treatment of canine atopic itch relies principally on the use of topical and/or oral glucocorticoids and oral cyclosporine. Type 1 receptor antihistamines are notorious in their inconsistency in reducing pruritus in atopic dogs. A new Janus kinase (JAK)-1 inhibitor has recently been approved for treatment of allergic itch in dogs, and its onset of efficacy is remarkably fast. Modeling itch in dogs can be achieved by allergen sensitization (fleas, house dust mites), and challenges that elicit pruritic manifestation can be used for mechanistic studies as well as for testing of novel anti-itch modalities.

Canine epidermal lipid sampling by skin scrub revealed variations between different body sites and normal and atopic dogs.

BACKGROUND: Previously, we evaluated a minimally invasive epidermal lipid sampling method called skin scrub, which achieved reproducible and comparable results to skin scraping. The present study aimed at investigating regional variations in canine epidermal lipid composition using the skin scrub technique and its suitability for collecting skin lipids in dogs suffering from certain skin diseases. Eight different body sites (5 highly and 3 lowly predisposed for atopic lesions) were sampled by skin scrub in 8 control dogs with normal skin. Additionally, lesional and non-lesional skin was sampled from 12 atopic dogs and 4 dogs with other skin diseases by skin scrub. Lipid fractions were separated by high performance thin layer chromatography and analysed densitometrically. RESULTS: No significant differences in total lipid content were found among the body sites tested in the control dogs. However, the pinna, lip and caudal back contained significantly lower concentrations of ceramides, whereas the palmar metacarpus and the axillary region contained significantly higher amounts of ceramides and cholesterol than most other body sites. The amount of total lipids and ceramides including all ceramide classes were significantly lower in both lesional and non-lesional skin of atopic dogs compared to normal skin, with the reduction being more pronounced in lesional skin. The sampling by skin scrub was relatively painless and caused only slight erythema at the sampled areas but no oedema. Histological examinations of skin biopsies at 2 skin scrubbed areas revealed a potential lipid extraction from the transition zone between stratum corneum and granulosum. CONCLUSIONS: The present study revealed regional variations in the epidermal lipid and ceramide composition in dogs without skin abnormalities but no connection between lipid composition and predilection sites for canine atopic dermatitis lesions. The skin scrub technique proved to be a practicable sampling method for canine epidermal lipids, revealed satisfying results regarding alterations of skin lipid composition in canine atopic dermatitis and might be suitable for epidermal lipid investigations of further canine skin diseases. Although the ceramide composition should be unaffected by the deeper lipid sampling of skin scrub compared to other sampling methods, further studies are required to determine methodological differences.

Histamine induces proliferation in keratinocytes from patients with atopic dermatitis through the histamine 4 receptor.

BACKGROUND: Epidermal hyperproliferation resulting in acanthosis is an important clinical observation in patients with atopic dermatitis, and its underlying mechanisms are not completely understood. OBJECTIVE: Because increased levels of histamine are present in lesional skin, we investigated the effect of histamine, especially with regard to histamine 4 receptor (H4R) activation, on the proliferation of human and murine keratinocytes. METHODS: The expression of H4R on human and murine keratinocytes was detected by using real-time PCR. Keratinocyte proliferation was evaluated by using different in vitro cell proliferation assays, scratch assays, and measurement of the epidermal thickness of murine skin. RESULTS: We detected H4R mRNA on foreskin keratinocytes and on outer root sheath keratinocytes; H4R mRNA was more abundant in keratinocytes from patients with atopic dermatitis compared with those from nonatopic donors. Stimulation of foreskin keratinocytes, atopic dermatitis outer root sheath keratinocytes, and H4R-transfected HaCaT cells with histamine and H4R agonist resulted in an increase in proliferation, which was blocked with the H4R-specific antagonist JNJ7777120. Abdominal epidermis of H4R-deficient mice was significantly thinner, and the in vitro proliferation of keratinocytes derived from H4R-deficient mice was lower compared with that seen in control mice. Interestingly, we only detected H4R expression on murine keratinocytes after stimulation with LPS and peptidoglycan. CONCLUSION: H4R is highly expressed on keratinocytes from patients with atopic dermatitis, and its stimulation induces keratinocyte proliferation. This might represent a mechanism that contributes to the epidermal hyperplasia observed in patients with atopic dermatitis.

PCR detects bands consistent with the expression of receptors associated with pruritus in canine dorsal root ganglia.

BACKGROUND: Various mediators are involved in the induction of itch, i.e. pruritus; however, the in vivo pharmacology of pruritus seems to be different in distinct species, and little is known about receptors that are involved in the induction of itch in dogs. The species differences in the mediation of pruritus might be explained by species differences in receptor expression in the sensory nerves, including the dorsal root ganglia (DRG). HYPOTHESIS/OBJECTIVES: The aim of the study was to analyse the expression of receptors for various mediators of pruritus in canine DRG. METHODS: Dorsal root ganglia of 14 dogs, which were euthanized for reasons not related to this study, were analysed. Multiple DRG per dog were dissected and, after homogenization of the DRG tissues, total RNA was isolated, reverse transcribed to complementary DNA and amplified with custom-synthesized primers. RESULTS: The following receptors were found in canine DRG: transient receptor potential cation channel subfamily V member 1, tachykinin receptor 1, Toll-like receptor 7, endothelin receptor type A, opioid receptors µ1 and κ1, histamine H1 -H4 receptors and the interleukin-31 receptor complex. CONCLUSIONS AND CLINICAL IMPORTANCE: PCR analysis detected bands consistent with the expression of receptors associated with pruritus in canine DRG.

LEKTI domain 6 displays anti-inflammatory action in vitro and in a murine atopic dermatitis model.

BACKGROUND: Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a serine protease inhibitor consisting of multiple domains. A loss of function mutation is described in Netherton patients that show severe symptoms of atopic lesions and itch. OBJECTIVES: LEKTI domain 6 (LD6) has shown strong serine protease-inhibitory action in in vitro assays and thus it was tested in vitro and in vivo for potential anti-inflammatory action in models of atopic skin disease. METHODS: Human skin equivalents were treated with LD6 and an inflammatory reaction was challenged by kallikrein-related endopeptidase 5 (KLK5). Furthermore, LD6 was tested on dorsal root ganglia cells stimulated with KLK5, SLIGRL and histamine by calcium imaging. The effect of topically administered LD6 (0.4-0.8%) in lipoderm was compared to a topical formulation of betamethasone-diproprionate (0.1%) in a therapeutic setting on atopic dermatitis-like lesions in NC/Nga mice sensitized to house dust mite antigen. Endpoints were clinical scoring of the mice as well as determination of scratching behaviour. RESULTS: KLK5 induced an upregulation of CXCL-8, CCL20 and IL-6 in skin equivalents. This upregulation was reduced by pre-incubation with LD6. KLK5 as well as histamine induced calcium influx in a population of neurons. LD6 significantly reduced the calcium response to both stimuli. When administered onto lesional skin of NC/Nga mice, both LD6 and betamethasone-dipropionate significantly reduced the inflammatory reaction. The effect on itch behaviour was less pronounced. CONCLUSION: Topical administration of LD6 might be a new therapeutic option for treatment of lesional atopic skin.

Topical Delivery of Tofacitinib in Dermatology: The Promise of a Novel Therapeutic Class Using Biodegradable Dendritic Polyglycerol Sulfates.

Inflammatory skin diseases, such as psoriasis, atopic dermatitis, and alopecia areata, occur when the regulatory tolerance of the innate immune system is disrupted, resulting in the activation of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) inflammatory signaling pathway by interleukin 6 (IL-6) and other key inflammatory cytokines. JAK inhibitors, such as tofacitinib, bind to these enzymes which are coupled to receptors on cell surfaces and block the transcription of inflammatory cytokine-induced genes. The first topical applications are being marketed, yet insufficient effects regarding indications, such as alopecia areata, suggest that improved delivery technologies could help increase the efficacy. In this study, we used sulfated dendritic polyglycerol with caprolactone segments integrated in its backbone (dPGS-PCL), with a molecular weight of 54 kDa, as a degradable carrier to load and solubilize the hydrophobic drug tofacitinib (TFB). TFB loaded in dPGS-PCL (dPGS-PCL@TFB), at a 11 w/w% loading capacity in aqueous solution, showed in an ex-vivo human skin model better penetration than free TFB in a 30:70 (v/v) ethanol/water mixture. We also investigated the anti-inflammatory efficacy of dPGS-PCL@TFB (0.5 w/w%), dPGS-PCL, and free TFB in the water/ethanol mixture by measuring their effects on IL-6 and IL-8 release, and STAT3 and STAT5 activation in ex vivo skin models of simulated inflamed human skin. Our results suggest that dPGS-PCL@TFB reduces the activation of STAT3 and STAT5 by increasing the penetration of the tofacitinib. However, no statistically significant differences with respect to the inhibition of IL-6 and IL-8 were observed in this short incubation time.