Minimally invasive diagnostic methods for canine disorders

Abstract

This disclosure relates to a method for detecting or measuring the content of one or more selected biomarkers in a canine skin sample, comprising the steps of: a) applying an adhesive tape to an exposed skin target area of ​​a dog, thereby collecting an epidermal sample adhered to the adhesive tape; and b) detecting the presence and / or measuring the content of one or more selected biomarkers in the epidermal sample obtained in step a). This further relates to a method for determining the presence of a skin disorder condition in the skin of a dog, a method for diagnosing atopic dermatitis conditions in the skin of a dog, a method for determining the beneficial effects of a drug and / or a selected dietary regimen in a dog suffering from atopic dermatitis, and a kit of components for detecting and / or measuring canine biomarkers in a canine epidermal sample.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from European Patent Application No. 23306455.9, filed on August 31, 2023, the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present disclosure relates to the field of diagnosis of inflammatory and non-inflammatory disorders of canine skin, including allergic disorders such as canine atopic dermatitis. BACKGROUND ART

[0003] Diagnosis of a disease or disorder in dogs is conventionally performed through skin biopsy.

[0004] Skin biopsy is an important diagnostic tool used by veterinarians to gather information on a wide variety of skin disorders and diseases in dogs. Skin biopsy involves removing a small sample of skin tissue for examination. The information obtained from a biopsy can help identify the cause of a skin problem and guide appropriate treatment.

[0005] In fact, skin biopsy is inevitably traumatic and is generally performed when clinical signs are already detectable. Therefore, invasive methods are generally not performed at the early stages of a suspected disorder or disease. As a result, besides being traumatic for the animal, skin biopsy has limited use in large-scale clinical trials and longitudinal studies.

[0006] Minimally invasive methods are desirable to enable more dynamic and repeated skin sampling over time, including to study associations between treatment, environment, and disease relapse. Furthermore, it is desirable to reduce canine discomfort, potential infection, and scarring caused by biopsy, including skin biopsy.

[0007] Furthermore, minimally invasive methods are desirable because they allow for the detection of the risk of developing a disorder or disease before it actually occurs. Alternatively, minimally invasive methods can be used to assess the actual occurrence of a disease or disorder in the early stages after its onset, particularly before clinical symptoms appear.

[0008] In humans, several minimally invasive diagnostic methods have been disclosed in the art, including collecting skin samples by tape stripping.

[0009] Therefore, sampling human skin material by tape stripping is well known in the art, particularly for the purpose of studying skin physiological parameters in subjects suffering from skin diseases such as atopic dermatitis. Human skin samples can be collected and studied in terms of their protein content (Clausen et al., 2020, Nature Research. 10: 21895), their nucleic acid content such as mRNA content (Dyjack et al., 29, J Allergy Clin Immunol, Vol. 141 (4): 1298-1309), or their microRNA content (Morlang et al., 2021, Vet Dermatol, Vol. 32: 331-e92).

[0010] In this field, the provision of minimally invasive methods for qualitatively and / or quantitatively analyzing proteins in canine skin samples remains technically necessary, particularly for the diagnosis of canine conditions, especially non-inflammatory or inflammatory skin diseases, such as canine atopic dermatitis. [Overview of the project]

[0011] This text relates to a method for detecting or measuring the content of one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in a canine skin sample, comprising the steps of: a) attaching an adhesive tape to an exposed skin target area of ​​a dog and thereby collecting an epidermal sample that adheres to the adhesive tape; and b) detecting the presence of one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in the epidermal sample obtained in step a), and / or measuring their content.

[0012] In certain embodiments, in step b), one or more selected proteins and / or nucleic acid canine biomarkers may indicate the immune status of the canine. In other embodiments, in step b), one or more selected proteins and / or nucleic acid canine biomarkers may be canine biomarkers indicating the inflammatory response of the canine. In other embodiments, in step b), one or more selected proteins and / or nucleic acid canine biomarkers may be canine biomarkers indicating the canine Th2 immune response. In particular, one or more selected proteins and / or nucleic acid canine biomarkers may be selected from proteins present in the epidermis. In particular, one or more selected proteins present in the epidermis are: a) interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33); b) tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ); and / or c) thymic stromal lymphoproteins Ethin (TSLP); and / or d) selected from one or more proteins selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha 1 (IL13RA1), interleukin-13 receptor subunit alpha 2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof. In certain embodiments, the detection of the presence of the protein biomarker selected in step b) and / or the measurement of its content may be carried out by immunological methods. In particular, the selected one or more nucleic acid biomarkers may be selected from mRNA and miRNA. In particular, the detection of the presence of the mRNA or miRNA biomarker selected in step b) and / or the measurement of its content may be carried out by nucleic acid amplification methods.

[0013] A further objective of this text is a method for determining the presence of a skin disorder in the skin of a dog, comprising the steps of: a) applying an adhesive tape to an exposed target area of ​​the dog's skin and thereby collecting an epidermal sample that adheres to the adhesive tape; b) detecting statistically significant different levels of one or more proteins and / or one or more nucleic acid canine biomarkers in the epidermal sample; and c) determining the presence of a skin disorder if, in step b), one or more statistically significant different levels of the biomarkers are measured. In particular, the skin disorder may be an inflammatory disorder and is preferentially selected from canine atopic dermatitis, pyoderma, contact dermatitis, folliculitis, acne, seborrheic dermatitis, panniculitis, cellulitis, licking granuloma (stomatitis, histiocytoma, eosinophilic granuloma complex), nasal dermatitis (Collie nose), interdigital cysts and furunculosis, ichthyosis, demodectic mange (red scabies), ringworm (dermatophytosis), and vasculitis. In particular, the skin disorder may be a non-inflammatory disorder and is preferred from canine atopic dermatitis, colordilution alopecia, pattern alopecia, congenital hypotrichosis, hyperkeratosis, seborrheic lymphadenitis, follicular dysplasia, sinusoidal ichthyosis X-linked, and seasonal follicular dysplasia. Priority is given to selecting from nasal plane hyperkeratosis, hereditary palmoplantar keratosis, primary seborrhea, vitamin A-responsive dermatopathy, cutaneous mastocytosis, hypothyroidism-related skin lesions, and canine epidermolysis bullosa.

[0014] The text further relates to a method for diagnosing atopic dermatitis in the skin of dogs, comprising the steps of: a) applying adhesive tape to an exposed skin target area of ​​a dog and thereby collecting an epidermal sample adhered to the adhesive tape; b) detecting statistically significant different levels of one or more proteins and / or nucleic acid canine biomarkers in the epidermal sample; c) determining the presence of atopic dermatitis if statistically significant different levels of one or more such proteins and / or nucleic acid canine biomarkers are measured in step b); and d) selecting one or more proteins and / or nucleic acid canine biomarkers selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha 1 (IL13RA1), interleukin-13 receptor subunit alpha 2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0015] In particular, the protein and / or nucleic acid canine biomarkers may be selected from the group consisting of a) interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33); b) tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ); c) thymic stromal lymphopoietin (TSLP); and d) tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha 1 (IL13RA1), interleukin-13 receptor subunit alpha 2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0016] Furthermore, this text relates to a method for determining the beneficial effects of a drug and / or selected dietary regimen in dogs suffering from atopic dermatitis, comprising the steps of: a) applying adhesive tape to an exposed skin target area of ​​the dog and thereby collecting an epidermal sample that adheres to the adhesive tape; b) measuring the level of one or more selected proteins and / or nucleic acid canine biomarkers in the epidermal sample collected in step b); c) treating the dog with the drug and / or selected dietary regimen; d) applying adhesive tape to an exposed skin target area of ​​the dog and thereby collecting an epidermal sample that adheres to the adhesive tape; e) detecting and / or measuring the level of one or more selected proteins and / or nucleic acid canine biomarkers in the epidermal sample collected in step d); and f) determining the beneficial effects of the drug and / or dietary therapy if an increase and / or decrease in the level of one or more biomarkers is measured in step e) compared with the level of the same selected biomarkers measured in step b), etc. In particular, steps a) (and d)) include a1) (and d1) applying adhesive tape to an exposed target area of ​​the dog's skin about 1 to 50 times, preferably about 10 to 30 times, to remove the surface layer of the skin, most preferably the stratum corneum, and a2) (and d2) applying an adhesive tape different from the adhesive tape of step a1) to the exposed target area of ​​the dog's skin, thereby collecting an epidermal sample that adheres to the adhesive tape. In particular, in each of steps a) and d), the adhesive tape may be applied under constant pressure.

[0017] Furthermore, the protein or nucleic acid canine biomarker may be selected from a) interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33); b) tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ); c) thymic stromal lymphopoietin (TSLP); and d) tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha 1 (IL13RA1), interleukin-13 receptor subunit alpha 2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0018] Furthermore, this text relates to a kit of components for detecting and / or measuring protein and / or nucleic acid canine biomarkers in canine epidermal samples, comprising: a) one or more adhesive tapes for peeling skin; b) reagents for collecting epidermal samples adhered to the surface of the adhesive tapes; c) reagents for detecting and / or quantifying one or more protein biomarkers; and / or d) reagents for detecting and / or quantifying one or more nucleic acids, particularly for detecting and / or quantifying one or more mRNAs or miRNAs; and e) optionally, recommendations for using the kit of components.

[0019] The following figures are included to illustrate specific aspects of the Disclosure and should not be considered exclusive embodiments. The subject matter of the Disclosure can be substantially modified, altered, combined, and made equivalent in form and function without departing from the scope of the Disclosure. [Brief explanation of the drawing]

[0020] [Figure 1]Comparison of Elisa magnification changes (log2 scale) and response states for TNF in dogs 5 and 6. Dog IDs are indicated within dots. [Figure 2] Comparison of Elisa magnification changes (log2 scale) and response states for dogs 5 and 6 for IL13. Dog IDs are indicated within dots. [Figure 3] Comparison of TNF transcriptome expression levels and Elisa expression levels in dogs 5 and 6. The line is a linear regression line. Dog IDs are indicated within the dots. [Figure 4] Comparison of transcriptome and Elisa expression levels for IL13 in dogs 5 and 6. The line is a linear regression line. Dog IDs are indicated within the dots. [Figure 5] Comparison of transcriptome magnification changes (log2 scale) and response states for dogs 5 and 6 for TNFRSF4 (ENSCAFG00845022948). Dog IDs are indicated within the dots. [Figure 6] Comparison of transcriptome magnification changes (log2 scale) and response states for dogs 5 and 6 for FA2H (ENSCAFG00845025633). Dog IDs are indicated within the dots. [Figure 7] Comparison of transcriptome magnification changes (log2 scale) and response states for dogs 5 and 6 for IL1B (ENSCAFG00845011661). Dog IDs are indicated within dots. [Figure 8] Comparison of transcriptome magnification changes (log2 scale) and response states for dogs 5 and 6 for IL13RA1 (ENSCAFG00845019238). Dog IDs are indicated within dots. [Figure 9] Comparison of transcriptome magnification changes (log2 scale) and response states for dogs 5 and 6 for IL13RA2 (ENSCAFG00845016176). Dog IDs are indicated within the dots. [Figure 10]Comparison of transcriptome fold changes (log2 scale) and response status in dogs 5 and 6 for IL22 (ENSCAFG00845013728). The dog IDs are shown within the dots. [Figure 11] Comparison of transcriptome fold changes (log2 scale) and response status in dogs 5 and 6 for IL4R (ENSCAFG00845004286). The dog IDs are shown within the dots. MODES FOR CARRYING OUT THE INVENTION

[0021] Definitions Terms used herein generally have their ordinary meanings in the art, within the context of the present disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance in describing the compositions and methods of the present disclosure and how to make and use them.

[0022] Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0023] Units, prefixes, and symbols are given in the form accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range. Headings provided herein are not intended to limit the various aspects of the present disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0024] All publications and other references mentioned herein are incorporated by reference in their entirety.

[0025] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural subjects unless otherwise explicitly indicated by the context. For example, a reference to "a compound" may include a mixture of at least two compounds.

[0026] Where used in this disclosure, the terms “approximately” or “about” mean an acceptable margin of error for a particular value as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limits of the measuring system. Generally, the term “approximately” may qualify a number that is, for example, above or below a variance of ±10 percent (higher or lower) from the stated value. In some embodiments, the term indicates a deviation of ±15%, ±10%, ±5%, ±4%, ±3%, ±2%, or ±1% from the stated number.

[0027] Where used in this disclosure, the terms “comprise,” “comprising,” “include,” and “including,” or any other variation thereof, are intended to encompass non-exclusive inclusion, so that a food composition, process, or method containing a list of elements or components does not consist solely of those elements or components, but may include other elements or components not expressly enumerated or specific to such food composition, process, or method. The term “consist of” indicates the inclusion of the described elements, excluding any additional elements. The term “essentially consisting of” indicates the inclusion of the described elements, and, where applicable, other elements that do not substantially affect the fundamental and novel features of this disclosure. Different embodiments of this disclosure using the term “consist of” or its equivalents are understood to encompass embodiments in which the term is substituted with “consist of” or “essentially consisting of.”

[0028] In the detailed description herein, references to “embodiments,” “certain embodiments,” “one embodiment,” “in some embodiments,” or any other variations thereof indicate that the embodiments described may include certain features, components, structures, or characteristics, but not all embodiments may necessarily include those particular features, components, structures, or characteristics. Furthermore, such phrases do not necessarily refer to the same embodiments. Moreover, if certain features, components, structures, or characteristics are described in relation to a particular embodiment, it is proposed that making such features, components, structures, or characteristics work in relation to other embodiments, whether expressly described or not, is within the knowledge of those skilled in the art. By reading this specification, methods for carrying out the disclosure in alternative embodiments will be apparent to those skilled in the art in the relevant field.

[0029] Where used in this disclosure, the term “Canidae” encompasses animals including pet animals selected from recognized breeds (some of which are further subdivided), such as Afhan Hound, Airedale, Akita Inu, Alaskan Malamute, Basset Hound, Beagle, Belgian Shepherd, Bloodhound, Border Collie, Border Terrier, Borzoi, Boxer, Bulldog, French Bulldog, Bullmastiff, Cairn Terrier, Chihuahua, Chow Chow, Cocker Spaniel, Collie, Corgi, Dachshund, Dalmatian, Doberman, Shar Pei, English Setter, Fox Terrier, German Shepherd, Golden Retriever, Great Dane, Greyhound, Brussels Griffon, Irish Setter, Irish Wolfhound, King Charles Spaniel, Labrador Retriever, Lhasa Apso, Mastiff, Newfoundland. Examples include the Dorland, Old English Sheepdog, Papillon, Pekingese, Pointer, Pomeranian, Poodle, Pug, Rottweiler, Saint Bernard, Saluki, Samoyed, Schnauzer, Scottish Terrier, Shetland Sheepdog, Shih Tzu, Siberian Husky, Skye Terrier, Vizsla, Rhodesian Ridgeback, Staffordshire Bull Terrier, Jack Terrier, Springer Spaniel, West Highland White Terrier, Whip Companion, Yorkshire Terrier, and Bichon Frise.

[0030] As used herein, “skin” means the outermost protective covering of mammals, particularly dogs, and includes three main layers: the ectoderm (epidermis), the mesoderm (dermis), and the hypoderm (hypoderm). The epidermis consists of the following five layers, from top to bottom (in contact with the dermis): (i) stratum corneum, (ii) stratum lucidum, (iii) stratum granulosum, (iv) stratum spinosum, and (v) stratum basale.

[0031] As used herein, “tape stripping” means a minimally invasive method of collecting biological material (in particular proteins and nucleic acids (e.g., mRNA and miRNA)) using a skin substrate, in particular canine skin substrate, with an adhesive placed on the skin substrate. According to this disclosure, tape stripping is used to collect biological material (in particular proteins and nucleic acids, e.g., mRNA and miRNA) from the surface of a dog's skin by bringing a suitable adhesive-containing substrate into contact with the surface of the dog's skin, thereby causing the biological material to adhere to the adhesive.

[0032] As used herein, the terms “skin disorder,” “skin disease,” or “dermatitis” refer to a broad range of conditions affecting the appearance, function, and health of the skin. These disorders may manifest as a variety of changes in the texture, color, structure, and overall condition of the skin. Skin disorders may be caused by genetic factors, infections, immune system reactions, environmental factors, allergies, hormonal imbalances, and other underlying causes. These may affect the outermost layer of the skin (epidermis), the deeper layers (dermis), or both.

[0033] As used herein, the term “inflammatory disease” means any disease / disorder associated with inflammation, including, but not limited to, those related to allergic (or atopic) disorders, inflammatory skin disorders, inflammatory gastrointestinal disorders, and respiratory and tracheal disorders, as selected from these. For example, the term may encompass one or more of the following conditions and syndromes: atopic disease, feline atopic syndrome (FAS), feline atopic dermatological syndrome (FASS), feline asthma, and feline food allergy (FFA). The term may further include allergic dermatitis, allergic enteritis, and asthma.

[0034] As used herein, the term “non-inflammatory disease” means a medical condition or disorder that does not primarily involve inflammation as a major component of its pathology.

[0035] Non-inflammatory diseases often have different underlying causes and mechanisms compared to inflammatory conditions. They can arise from genetic factors, metabolic imbalances, structural abnormalities, hormonal disorders, or other non-immune sources. Non-inflammatory diseases can affect various organs and systems of the body and can cause a wide range of symptoms and clinical manifestations.

[0036] As used herein, the terms “atopy” or “atopic disease” mean, but are not limited to, the commonly recognized definitions. The essential features of atopic dermatitis are defined as a predisposition to allergic diseases affecting the skin. Thus, the term encompasses any clinical dermatitis hypersensitivity associated with a genetic predisposition to produce immunoglobulin E (IgE) antibodies in response to environmental proteins such as pollen, house dust mites, and food allergens.

[0037] As used herein, “atopic dermatitis” (AD) refers to an inflammatory skin disease characterized by severe pruritus (e.g., severe itching) and dry, eczematous lesions with scaling. The term “atopic dermatitis” includes, but is not limited to, AD caused by or associated with epidermal barrier dysfunction, allergies (e.g., allergies to certain foods, pollen, mold, mites, animals, etc.), radiation exposure, and / or asthma. This disclosure encompasses methods for determining the occurrence or risk of occurrence of moderate to severe or severe AD. As used herein, “moderate to severe AD” is characterized by widespread skin lesions with severe itching, often complicated by persistent bacterial, viral, or fungal infections. Moderate to severe AD also includes chronic AD in dogs. Often, chronic lesions include thickening of skin plaques, lichenification, and fibrous papules. Furthermore, dogs with moderate to severe Alzheimer's disease (AD) generally have more than 20% or 10% of their skin affected, with lesions also observed in the eyes, paws, and body wrinkles. Moderate to severe AD can also be present in dogs receiving treatment with topical corticosteroids. In some cases, dogs can be said to have moderate to severe AD if they are resistant or refractory to treatment with either topical corticosteroids or calcineurin inhibitors.

[0038] In this specification, “marker,” “markers,” “biomarker,” or “biomarkers” refers to proteins or nucleic acids (e.g., mRNA or miRNA) expressed in cells or tissues that are useful for assessing the physiological state of canids, particularly dogs, including non-inflammatory and inflammatory states, and encompass the prediction or diagnosis of disorders or diseases such as inflammatory responses. Protein markers or biomarkers in the context of this disclosure include, for example, cytokines, chemokines, growth factors, other proteins and peptides. Nucleic acid markers or biomarkers in the context of this disclosure include, for example, mRNA encoding cytokines, mRNA encoding chemokines, mRNA encoding growth factors, and mRNA encoding other proteins. Examples of nucleic acid markers or biomarkers include, but are not limited to, FLG, LOR, IVL, CCL11, CCL13, CCL17, CCL18, CCL22, CCL18, CCL26, CCL27, CCL28, CXCL1, CXCL9, CXCL10, CXCL20, IL36R, IL18, IL6, IL7, IL15, IL17Rb, IL23, S100A12, S100A8, MMP12, MAN1A1, TNFRSF4, and FCER1A. According to this disclosure, nucleic acid biomarkers include mRNA and miRNA.

[0039] As used herein, the terms “mRNA” or “messenger RNA” refer to any polynucleotide that encodes at least one peptide or polypeptide of interest and can be translated to produce the encoded peptide polypeptide of interest in vitro, in vivo, in situ, or ex vivo. mRNA can be isolated from tissues or cells by a variety of methods. For example, total RNA extraction can be performed on cell-containing samples such as canine epithelial samples, and the resulting extracted total RNA can be purified (e.g., on a column containing oligo-dT beads) to obtain extracted mRNA.

[0040] As used herein, the terms “microRNA” or “miRNA” refer to an RNA interfering molecule (RNAi) that is approximately 21–23 nucleotides (nt) in length. miRNAs can range in length from 18–26 nucleotides. Typically, miRNAs are single-stranded. However, in some embodiments, miRNAs may be at least partially double-stranded. In certain embodiments, miRNAs may include an RNA double-strand (referred to herein as a “double-stranded region”) and may further include one or two single-stranded overhangs. In some embodiments, the RNAi agent includes a double-stranded region ranging in length from 15–29 bp and may further include one or two single-stranded overhangs. miRNAs may be formed from two RNA molecules that hybridize together, or alternatively, from a single RNA molecule containing a self-hybridizing portion. Generally, the free 5' end of a miRNA molecule has a phosphate group, and the free 3' end has a hydroxyl group. The double-stranded portion of a miRNA typically contains one or more bulges consisting of one or more unpaired nucleotides, but this is not always the case. The single-stranded portion of a miRNA contains a region that hybridizes with the target RNA. In certain embodiments of the present invention, the single-stranded portion of the miRNA is not exactly complementary to the region of the target RNA, meaning that the miRNA hybridizes to the target RNA with one or more mismatches. In other embodiments of the present invention, the single-stranded portion of the miRNA is exactly complementary to the region of the target RNA, meaning that the miRNA hybridizes to the target RNA without any mismatches. Typically, miRNAs are thought to mediate inhibition of gene expression by inhibiting the translation of the target transcript. However, in some embodiments, miRNAs may mediate inhibition of gene expression by causing degradation of the target transcript.

[0041] As used herein, “statistically significant different levels,” “different levels,” or “different expression” of a protein or nucleic acid may include any increase or decrease in the tested dog compared to a reference level of the protein or nucleic acid in a healthy dog, more specifically, in dogs without inflammatory disorders or diseases, and even more specifically, in dogs without skin inflammatory disorders or diseases, such as dogs that are not affected by atopic dermatitis. In one embodiment, a statistically significant different level means an increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more. In another embodiment, statistically significant levels mean levels of reduction of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. As used herein, the terms “food composition,” “wet food composition,” “complete nutrition wet food composition,” or “diet” encompass all foods, diets, nutritional supplements, or materials that may contain proteins, fiber, carbohydrates, and / or fats that can be used in the body of an organism to sustain growth, repair, and vital processes and to provide energy. Carbohydrates may include NFE (nitrogen-free extracts). Food compositions may also contain auxiliary substances or additives, such as flavorings, minerals, and vitamins. Food compositions according to this disclosure may consist of complete nutrition food compositions. In some particular embodiments, wet food compositions according to this disclosure may be complete nutrition food compositions.

[0042] Where used in this disclosure, “complete nutrition” means that the composition of this disclosure provides complete and balanced nutritional requirements for a companion animal such as a cat or a dog. Specifically, a complete nutrition food composition may contain all known necessary nutrients for the intended recipient of the food composition in appropriate amounts and proportions, for example, based on the recommendations of recognized supervisory authorities in the field of animal nutrition. Thus, a nutritionally adequate diet is one that can be fed as the sole food source and sustain life without additional food (except water). A complete nutrition food composition may further include carriers, diluents, and / or excipients. Depending on the intended use, the carriers, diluents, or excipients may be selected to be suitable for companion animal use, specifically for companion animals such as cats and dogs. Furthermore, the complete nutrition food composition may not cause significant changes in the companion animal, i.e., body weight and / or body condition score (BCS). Exemplary examples of food compositions described herein may include, but are not limited to, proteins, fats, ash, fiber (TDF), carbohydrates, starch, calcium, phosphorus, sodium, chloride, sodium tripolyphosphate, potassium, magnesium, iron, water, copper, manganese, zinc, selenium, flavorings, vitamin A, vitamin D3, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin B7, vitamin B9, choline chloride, arachidonic acid, W3 fatty acids, or W6 fatty acids.

[0043] explanation This disclosure provides a minimally invasive method for detecting and / or quantifying proteins or nucleic acids in canine skin samples, particularly for the purpose of determining the likelihood of developing or detecting skin disorders or diseases, including inflammatory skin disorders such as atopic dermatitis.

[0044] Unexpectedly, the applicant has found that reproducible qualitative and / or quantitative analysis of proteins and nucleic acids contained in canine skin samples can be performed by using a method in which canine skin samples are collected by applying an adhesive support to a skin target area, thereby allowing the skin material to migrate to the surface of the adhesive support, and then the migrated skin material can be analyzed for the presence and / or content of proteins and / or nucleic acids contained therein. The method of collecting skin material by applying an adhesive support to a skin target area is known in the art and may also be called "tape stripping".

[0045] To the applicant's knowledge, the technique of tape stripping for the purpose of analyzing the protein and / or nucleic acid content of canine skin samples, as well as the protein and / or nucleic acid biomarker content of canine skin samples, is neither disclosed nor suggested in the art.

[0046] This disclosure relates to a method for detecting or measuring the content of one or more selected protein biomarkers and / or one or more selected nucleic acid biomarkers in a canine skin sample, a) A step of applying adhesive tape to an exposed skin target area of ​​a dog and thereby collecting epidermal samples that adhere to the adhesive tape, and The present invention relates to a method comprising the steps of: b) detecting the presence of one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in the epidermal sample obtained in step a), and / or measuring their content.

[0047] This disclosure relates to a method for detecting or measuring the content of one or more selected protein biomarkers in a canine skin sample, a) A step of applying adhesive tape to an exposed skin target area of ​​a dog and thereby collecting epidermal samples that adhere to the adhesive tape, and The present invention relates to a method comprising the steps of (b) detecting the presence of one or more selected protein biomarkers in the epidermal sample obtained in step (a) and / or measuring their content.

[0048] This disclosure relates to a method for detecting or measuring the content of one or more selected nucleic acid biomarkers in a canine skin sample, a) A step of applying adhesive tape to an exposed skin target area of ​​a dog and thereby collecting epidermal samples that adhere to the adhesive tape, and The present invention relates to a method comprising the steps of (b) detecting the presence of one or more nucleic acid biomarkers in the epidermal sample obtained in step (a) and / or measuring their content.

[0049] This disclosure relates to a method for detecting or measuring the content of one or more selected protein biomarkers and / or one or more selected nucleic acid biomarkers in a canine skin sample, a) A step of providing an adhesive tape on which a sample of dog skin that has been previously attached to the adhesive tape has been collected, and b) The method comprises the step of detecting the presence of one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in an epidermal sample and / or measuring their content.

[0050] This disclosure relates to a method for detecting or measuring the content of one or more selected protein biomarkers in a canine skin sample, a) A step of providing an adhesive tape on which a sample of dog skin that has been previously attached to the adhesive tape has been collected, and b) The method comprises the step of detecting the presence of one or more selected protein biomarkers in an epidermal sample and / or measuring their content.

[0051] This disclosure relates to a method for detecting or measuring the content of one or more selected nucleic acid biomarkers in a canine skin sample, a) A step of providing an adhesive tape on which a sample of dog skin that has been previously attached to the adhesive tape has been collected, and b) The method comprises the step of detecting the presence of one or more selected nucleic acid biomarkers in an epidermal sample and / or measuring their content.

[0052] According to the method described above, the nucleic acid biomarker most preferably consists of an mRNA or miRNA biomarker.

[0053] By definition, the protein biomarkers and nucleic acid biomarkers used herein are canine biomarkers.

[0054] In some embodiments of this method, one or more selected protein biomarkers indicate the physiological state of the tested dog, encompassing the physiological state of the skin of the tested dog. Therefore, according to these most preferred embodiments, the one or more selected protein biomarkers may consist of one or more protein biomarkers indicating the physiological state of the tested dog, particularly biomarkers indicating the physiological state of the skin of the tested dog.

[0055] In some embodiments of this method, one or more selected nucleic acid biomarkers (e.g., mRNA or miRNA) indicate the physiological state of the tested dog, encompassing the physiological state of the skin of the tested dog. Therefore, according to these most preferred embodiments, the selected one or more nucleic acid biomarkers consist of one or more nucleic acid biomarkers indicating the physiological state of the tested dog, in particular biomarkers indicating the physiological state of the skin of the tested dog.

[0056] The examples described herein demonstrate the validation of such a canine skin sampling method in sensitized canine animals that induce an inflammatory response, more precisely an inflammatory response related to an inflammatory immune response, and in particular mimic the development of sensitization in canine atopic dermatitis.

[0057] The examples herein demonstrate that in these sensitized canine animals, both protein and nucleic acid biomarkers indicating a Th2-type immunological response are increased. More precisely, the quantification of proteins contained in skin samples collected by tape stripping from dogs is shown herein, and particularly in sensitized sites, a significant increase in the content of interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-33 (IL-33), and tumor necrosis factor alpha (TNF-α) can be determined, where skin inflammation is artificially induced by sensitization to an allergen compared to control skin sites of the same animals that are not sensitized. The presence of IL-4 and IL-13, as well as the absence of an increase in gamma interferon (IFN-γ), actually indicates a Th2-led rather than Th1-led inflammatory response, and its presence and levels are shown to be measured in canine skin samples in this study.

[0058] As used herein, the name of a biomarker, for example, interleukin-4 (IL-4), is intended to mean either a protein biomarker, for example, the IL-4 protein, or a protein-coding nucleic acid, for example, mRNA encoding IL-4.

[0059] Regarding other nucleic acid biomarkers, particularly miRNA biomarkers for canine atopic dermatitis, these can be selected as non-limiting examples from among miR-203 and miR-483. These miRNAs are described in particular by Koury et al. (2019, PlosOne, Vol. 14 (6): e0128670).

[0060] Furthermore, as confirmed by differential mRNA expression results, increased skin content is associated with increased expression levels of the corresponding gene encoding the protein biomarker.

[0061] The applicant's findings are more surprising regarding the substantial differences between canine and human skin, particularly its thinness, as lower thickness reduces the likelihood that nucleic acids and proteins will be properly retained in an undegraded form within canine skin. Furthermore, there is no evidence in this art that cells attached to hair follicles in canine skin, including those attached to the hair follicle sheath and appendages, have similar cell division and / or chemokinetic synthesis, and therefore nuclear expression, to keratinocytes in human skin. Moreover, given the high density of hair on canine skin, the amount of skin collected per surface is much smaller, and therefore the likelihood that concentrations of cytokines and / or chemokines and nucleic acids are detectable on canine skin, and especially on adhesive tape, is proportionally lower.

[0062] In step a), the selected target area is first gently clipped and shaved to expose the epidermis in the skin target area to which the adhesive tape is applied. The selected area is gently clipped or shaved to avoid wounds, cuts, abrasions, or erosion in the clipped or shaved selected skin target area.

[0063] In certain embodiments, the selected target region may be a lesional area of ​​the skin. In particular, the selected target region may be an area of ​​skin damaged by a skin disorder or disease, especially by an inflammatory skin disorder such as atopic dermatitis.

[0064] In another specific embodiment, the selected target area is not a lesion area of ​​the skin. This may be particularly the case when the method described herein is used as a preventive measure.

[0065] In step a), the exposed skin target area may preferably be a skin area from which the stratum corneum has been previously removed in order to expose the underlying skin layer, particularly the underlying granular skin layer, after shearing or shaving. This means that, according to the embodiments described above, the adhesive tape is applied in step a) on the surface of the granular layer. In some embodiments, the removal of the stratum corneum is carried out by gently and continuously applying the adhesive tape over the skin area until the underlying skin layer, particularly the underlying granular layer, is exposed, thereby obtaining the desired skin target area. Exemplarily, exposing the skin target area may be carried out by continuously applying the adhesive tape over the selected skin area until the underlying skin layer, most preferably the underlying granular layer, is exposed, or by continuously applying the adhesive tape over the selected skin area. Exemplarily, in embodiments in which the stratum corneum is removed using adhesive tape, the adhesive tape is typically applied over the selected skin target area 5 to 30 times, for example 3 to 50 times. The same adhesive tape can be applied a desired number of times, and / or multiple adhesive tapes can be used.

[0066] In step a), the epidermal sample collected is a canine skin material sample belonging to the epidermis that does not contain or substantially contains material derived from the stratum corneum. Most preferably, the majority of the epidermal sample is derived from the granular layer of the epidermis.

[0067] Step a) can be carried out using known skin adhesive tapes, such as non-polar and flexible adhesive tapes, such as flexible tapes using rubber-based adhesives. For example, skin adhesive tapes can be selected from D-Squame® (polyacrylate ester adhesive; CuDerm; Dallas, Texas, Duraper), Sebutape® (acrylic polymer film; CuDerm; Dallas, Texas), Tegerm®, Duct tape (333 Duct tape, Nashua tape products), Scotch® tape (3M Scotch 810, St. Paul, Minn.), Gold® (The Sellotape Company; Eindhoven, Netherlands), and Sentega® (polypropylene tape; Sentega Eiketten BV, Utrecht, Netherlands). The adhesive tape can be selected from any of the commonly used pressure-sensitive adhesives or adhesives that solidify rapidly with skin contents (such as cinaoacylates). The adhesive tape may be on a flexible or solid backing to facilitate sampling.

[0068] In step a), the same strip of tape can be repeatedly applied to the target skin area and then removed. In other exemplary embodiments, multiple fresh pieces of adhesive tape are applied sequentially to the same target skin area. The individual tape strips used to sample the skin target area can then be combined into a single extraction vessel for further processing, such as protein extraction or nucleic acid extraction, most preferably mRNA and miRNA extraction.

[0069] In some embodiments, the collected epidermal sample adhered to the adhesive tape can be at least partially removed from the tape, thereby providing the epidermal sample for further use in step b) of the method.

[0070] In these embodiments, step a) may include a1) applying an adhesive tape to an exposed skin target area of ​​a dog to collect an epidermal sample that adheres to the adhesive tape, and a2) removing the collected epidermal sample that adheres to the adhesive tape so that an epidermal sample is provided for analysis in step b).

[0071] In step a2), the epidermal sample can be removed according to methods well known in the art. Step a2) can generally be carried out by extraction of the collected material, such as by performing extraction using an appropriate extraction buffer.

[0072] In step a2), the protein can be extracted from the epidermal sample adhered to an adhesive tape bay by any known method, such as by using a known buffer of water and ethanol, for further analysis in step b).

[0073] In step a2), for nucleic acids, particularly mRNA and / or miRNA, these RNAs can be extracted from the adhesive tape through a procedure that allows for the direct qualitative and quantitative evaluation of nucleic acid biomarkers, i.e., mRNA and miRNA biomarkers. Commercially available kits can be used, such as the "RNeasy" RNA extraction kit and the "Sensiscript Reverse Transcriptase" kit, PCR primers, and probes (including all buffers and enzymes necessary for amplification of the target nucleic acid biomarker and subsequent fluorescence detection of specific cDNA, TaqMan® Pre-Developed Assay Reagents and TaqMan® Universal Master Mix) (Applied Biosystems, Foster City, CA, USA), which are commercially available from Quiagen Company (Valebcia, CA, USA).

[0074] In step b), the detection or quantification of the selected one or more protein biomarkers can be carried out according to any method known in the art. In some embodiments, the detection and / or quantification of the selected one or more protein biomarkers can be carried out by an immunological method, such as the ELISA method disclosed in the Examples, by using an antibody against the protein to be detected and / or quantified.

[0075] In step b), the detection or quantification of the presence of the selected one or more nucleic acid biomarkers, particularly mRNA or miRNA, can be carried out according to any method known in the art. Typically, the detection and / or quantification of the selected one or more nucleic acid biomarkers can be carried out by, if necessary, reverse transcription of mRNA or miRNA, followed by amplification of the target nucleic acid, preferably the target mRNA or miRNA, and then detection and / or quantification of the amplified nucleic acid by using a well-designed nucleic acid probe, for example, a labeled nucleic acid probe, as shown in the examples herein.

[0076] Amplification of nucleic acid molecules can be carried out in vitro by biochemical processes known to those skilled in the art. The amplifier may be any compound or system that functions to achieve the synthesis of a primer extension product, including an enzyme. It will be recognized that by utilizing various amplification methodologies, the copy number of target nucleic acids can be increased in nucleic acid skin samples obtained using the methods provided herein, both before and after detection. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Taq polymerase, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, T4 or T7 RNA polymerase, polymerase mutein, reverse transcriptase, ligase, other enzymes, and thermostable enzymes (i.e., enzymes that perform primer extension after being subjected to a temperature sufficiently elevated to cause denaturation, or enzymes that produce aRNA from a DNA template using an RNA polymerase promoter, i.e., linear amplification of aRNA). One method of in vitro amplification that may be used in accordance with this disclosure is described in U.S. Patents No. 4,683,202 and No. 4,683,195.

[0077] In some embodiments of step a) of the method, the first adhesive tape is applied to a plurality of "first adhesive tapes" used in step a) to detect and / or quantify one or more selected protein biomarkers, and the second adhesive tape is applied to a plurality of second adhesive tapes used in step a) to detect and / or quantify one or more selected nucleic acid biomarkers, for example, one or more selected mRNAs or miRNAs, regardless of the order in which the first and second adhesive tapes are applied.

[0078] In some embodiments, the selected one or more proteins and / or nucleic acid canine biomarkers indicate the immune status of the canine.

[0079] Protein and / or nucleic acid biomarkers indicative of a canine immune status may include (i) protein and / or nucleic acid biomarkers indicating inflammatory responses, (ii) protein and / or nucleic acid biomarkers indicating skin barrier changes, (iii) protein and / or nucleic acid biomarkers indicating Th2 immune responses, (iv) protein and / or nucleic acid biomarkers indicating Th1 immune responses, and (v) protein and / or nucleic acid biomarkers indicating pruritus.

[0080] In some embodiments, one or more selected proteins and / or nucleic acid canine biomarkers indicate an inflammatory response in dogs.

[0081] Canine biomarkers indicating an inflammatory response may be selected from the following protein or nucleic acid biomarkers, as non-limiting examples: C-reactive protein (CRP), tumor necrosis factor (TNF), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), myeloperoxidase (MPO), matrix metalloproteinase (MMP), adiponectin, monocyte chemotactic protein 1 (MCP-1), and CD40 ligand.

[0082] An inflammatory response is determined when the content of at least one, preferably two or more, of the inflammatory biomarkers identified above in the epidermal sample is increased.

[0083] In some embodiments, one or more selected protein and / or nucleic acid canine biomarkers may be canine biomarkers indicating a canine Th2 immune response.

[0084] Canine biomarkers exhibiting a Th2 response can be selected from protein or nucleic acid biomarkers of interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-17 (IL-17), interleukin-25 (IL-25), and interleukin-33 (IL-33), and in particular from protein or nucleic acid biomarkers of interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13).

[0085] A Th2 response can be determined if the content of the epidermal sample increases in at least one, particularly two or more of the Th2 biomarkers specified above. Alternatively, a Th2 response can be determined if the content of the epidermal sample increases in at least one Th2 biomarker and decreases in at least one other biomarker. In some embodiments, one or more selected protein and / or nucleic acid canine biomarkers are canine biomarkers that indicate a canine Th1 immune response.

[0086] Canine biomarkers that indicate a Th1 response can be selected from the following protein or nucleic acid biomarkers: gamma interferon (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), interleukin-1 beta (IL-1β), interleukin-18 (IL-18), interleukin-27 (IL-27), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), and T-bet (T-box transcription factor), particularly IFN-γ and IL-2.

[0087] A Th1 response can be determined when the content of at least one, particularly two or more, of the Th1 biomarkers identified above increases in the epidermal sample. Alternatively, a Th1 response can be determined when the content of at least one Th1 biomarker increases in the epidermal sample, and the content of at least one other biomarker decreases.

[0088] In some embodiments, one or more selected protein and / or nucleic acid canine biomarkers may be canine biomarkers indicating canine pruritus.

[0089] Canine biomarkers indicating pruritus can be selected from the following protein or nucleic acid biomarkers: gamma interferon (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), interleukin-33 (IL-33), C-reactive protein (CRP), matrix metalloproteinase, substance P, eosinophil cationic protein (ECP), and thymic interstitial lymphoetin (TSLP), IFN-γ, IL-2, IL-31, and TSLP, with IL-2 being particularly noteworthy.

[0090] Pruritus can be determined when the content of at least one, preferably two or more, of the pruritus biomarkers identified above increases in the epidermal sample.

[0091] In some embodiments, one or more selected proteins and / or nucleic acid canine biomarkers may indicate atopic dermatitis in dogs.

[0092] The Disclosure further relates to a method for determining the occurrence or likelihood of atopic dermatitis in dogs, comprising the steps of: a) providing an epidermal sample previously collected from an adhesive tape to which a sample is attached; and b) measuring the content of two or more selected protein biomarkers and / or two or more nucleic acid biomarkers in the epidermal sample obtained in step a), wherein the biomarkers are interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin- The present invention relates to a method for determining the presence or likelihood of atopic dermatitis in a dog, comprising the steps of: a) selecting from IL-25, interleukin-31, interleukin-33, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ); and c) determining the presence or likelihood of atopic dermatitis in a dog if the content of the selected biomarker measured in step b) is increased compared to the content of the same biomarker in a dog unaffected by skin inflammation, and most preferably compared to the content of the same biomarker in a dog not suffering from atopic dermatitis.

[0093] In some embodiments, the selected one or more proteins and / or nucleic acid canine biomarkers can be selected from the following proteins or nucleic acid biomarkers, i.e., proteins present in the epidermis.

[0094] In some embodiments, the proteins present in the epidermis are: a) interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33); b) tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ); c) thymic stromal lymphoproteins Ethin (TSLP); and / or d) at least one protein selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha-1 (IL13RA1), interleukin-13 receptor subunit alpha-2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0095] In some embodiments, the proteins present in the epidermis can be selected from interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33). In some embodiments, the proteins present in the epidermis include one or more proteins selected from the group consisting of interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), interleukin-33 (IL-33), and combinations thereof. In some embodiments, the proteins present in the epidermis include interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33). In some embodiments, the proteins present in the epidermis further include at least one protein selected from the group consisting of IL-1-1β, IL-13, TNF-α, IFN-γ, TSLP, and combinations thereof. In some embodiments, the proteins present in the epidermis further include at least one protein selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha 1 (IL13RA1), interleukin-13 receptor subunit alpha 2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0096] The present disclosure further relates to a method for determining the presence of a skin disorder in the skin of a dog, comprising the steps of: a) applying an adhesive tape to an exposed skin target area of ​​the dog and thereby collecting an epidermal sample that adheres to the adhesive tape; b) detecting statistically significant different levels of one or more proteins and / or one or more nucleic acid biomarkers in the epidermal sample; and c) determining the presence of a skin disorder if, in step b), one or more statistically significant different levels of the biomarkers are measured.

[0097] As used herein, “statistically significant levels of protein” typically refers to the amount of a particular protein in a biological sample (here within skin tissue, more precisely within canine epidermis lacking the stratum corneum) that deviates significantly from what is normal or expected for a particular individual or population of dogs. This abnormality may indicate a variety of potential conditions, diseases, or physiological changes.

[0098] In some embodiments of the above method, the protein and / or nucleic acid biomarkers are a) interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33); b) tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ); c) thymic stromal lymphopoietic enzymes The biomarkers are selected from tin (TSLP), and d) at least one protein and / or nucleic acid biomarker selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha-1 (IL13RA1), interleukin-13 receptor subunit alpha-2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0099] In some embodiments, the protein and / or nucleic acid biomarker can be selected from interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33). In some embodiments, the protein and / or nucleic acid biomarker includes one or more proteins and / or nucleic acid biomarkers selected from the group consisting of interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), interleukin-33 (IL-33), and combinations thereof. In some embodiments, the protein and / or nucleic acid biomarker includes interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33). In some embodiments, the protein and / or nucleic acid biomarker further includes at least one protein and / or nucleic acid biomarker selected from the group consisting of IL-1-1β, IL-13, TNF-α, IFN-γ, TSLP, and combinations thereof. In some embodiments, the protein and / or nucleic acid biomarker further comprises at least one protein and / or nucleic acid biomarker selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha 1 (IL13RA1), interleukin-13 receptor subunit alpha 2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0100] In some embodiments of the above method, the skin disorder is an inflammatory disorder, and is preferentially selected from canine atopic dermatitis, pyoderma, contact dermatitis, folliculitis, acne, seborrheic dermatitis, panniculitis, cellulitis, licking granuloma (stomatitis, histiocytoma, eosinophilic granuloma complex), nasal dermatitis (Collie nose), interdigital cysts and furunculosis, ichthyosis, demodectic mange (red scabies), ringworm (dermatophytosis), and vasculitis.

[0101] In some embodiments of the above method, the skin disorder is a non-inflammatory disorder and is preferentially selected from alopecia areata, colordilution alopecia, pattern alopecia, congenital hypotrichosis, hyperkeratosis, seborrheic lymphadenitis, follicular dysplasia, sinusoidal ichthyosis, X-linked ichthyosis, seasonal follicular dysplasia, nasal plane hyperkeratosis, hereditary palmoplantar keratosis, primary seborrhea, vitamin A-responsive dermatopathy, cutaneous mastocytosis, hypothyroidism-related skin lesions, and canine epidermolysis bullosa.

[0102] This disclosure further relates to a method for diagnosing atopic dermatitis in the skin of dogs, comprising: a) applying an adhesive tape to an exposed skin target area of ​​a dog and thereby collecting an epidermal sample that adheres to the adhesive tape; and b) detecting statistically significant different levels of one or more selected proteins and / or nucleic acid canine biomarkers in the epidermal sample, in particular, interleukin-1 beta (IL-1β), interleukin-4 (IL-4), inter The present invention relates to a method comprising the steps of: a) selecting from leukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), interleukin-33 (IL-33), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ); and c) determining the presence of an atopic dermatitis condition if, in step b), one or more statistically significant different levels are measured among the protein and / or nucleic acid canine biomarkers.

[0103] In some embodiments of the above method, the protein and / or nucleic acid canine biomarker is a) interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33); b) tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ); c) thymic stromal lymphopoietin ( TSLP); and d) can be selected from at least one protein and / or nucleic acid canine biomarker selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha-1 (IL13RA1), interleukin-13 receptor subunit alpha-2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0104] In some embodiments, the protein and / or nucleic acid canine biomarker may be selected from interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33). In some embodiments, the protein and / or nucleic acid canine biomarker includes one or more proteins and / or nucleic acid canine biomarkers selected from the group consisting of interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), interleukin-33 (IL-33), and combinations thereof. In some embodiments, the protein and / or nucleic acid canine biomarker comprises interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33). In some embodiments, the protein and / or nucleic acid canine biomarker further comprises at least one protein and / or nucleic acid canine biomarker selected from the group consisting of IL-1-1β, IL-13, TNF-α, IFN-γ, TSLP, and combinations thereof. In some embodiments, the protein and / or nucleic acid canine biomarker further comprises at least one protein and / or nucleic acid canine biomarker selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha 1 (IL13RA1), interleukin-13 receptor subunit alpha 2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0105] The Disclosure also relates to a method for determining the beneficial effects of a drug and / or selected dietary regimen in dogs suffering from atopic dermatitis, comprising: a) applying an adhesive tape to an exposed skin target area of ​​the dog and thereby collecting an epidermal sample that adheres to the adhesive tape; b) measuring the level of one or more selected proteins and / or nucleic acid canine biomarkers in the epidermal sample collected in step b); c) treating the dog with the drug and / or selected dietary regimen; d) applying an adhesive tape to an exposed skin target area of ​​the dog and thereby collecting an epidermal sample that adheres to the adhesive tape; e) detecting and / or measuring the level of one or more selected proteins and / or nucleic acid canine biomarkers in the epidermal sample collected in step d); and f) determining the beneficial effects of the drug and / or dietary therapy if an increase and / or decrease in the level of one or more biomarkers is measured in step e) compared with the level of the same selected biomarkers measured in step b); In some embodiments, steps a) (and d)) of the above method may include a1) (and d1) applying adhesive tape to an exposed target area of ​​the dog's skin about 1 to about 50 times, preferably about 10 to about 30 times, to remove the surface layer of the skin, most preferably the stratum corneum, and a2) (and d2) applying an adhesive tape different from the adhesive tape of step a1) to an exposed target area of ​​the dog's skin, thereby collecting an epidermal sample that adheres to the adhesive tape.

[0106] In some embodiments of the above method, the adhesive tape is applied under constant pressure in each of steps a) and d).

[0107] According to some embodiments of the above method, the protein and / or nucleic acid canine biomarker is a) interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33); b) tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ); c) thymic stromal lymphopoietin ( TSLP); and d) can be selected from at least one protein and / or nucleic acid canine biomarker selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha-1 (IL13RA1), interleukin-13 receptor subunit alpha-2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0108] In some embodiments, the protein and / or nucleic acid canine biomarker may be selected from interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33). In some embodiments, the protein and / or nucleic acid canine biomarker includes one or more proteins and / or nucleic acid canine biomarkers selected from the group consisting of interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), interleukin-33 (IL-33), and combinations thereof. In some embodiments, the protein and / or nucleic acid canine biomarker comprises interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33). In some embodiments, the protein and / or nucleic acid canine biomarker further comprises at least one protein and / or nucleic acid canine biomarker selected from the group consisting of IL-1-1β, IL-13, TNF-α, IFN-γ, TSLP, and combinations thereof. In some embodiments, the protein and / or nucleic acid canine biomarker further comprises at least one protein and / or nucleic acid canine biomarker selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha 1 (IL13RA1), interleukin-13 receptor subunit alpha 2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0109] In some embodiments of the above method for determining the beneficial effects of a pharmaceutical and / or selected dietary regimen in dogs suffering from atopic dermatitis, step c) comprises subjecting the dog to treatment with the pharmaceutical. The pharmaceutical may be selected from antihistamines, corticosteroids, cyclosporine, oclacitinib, lokibetsumab, and immunotherapies, among others, from graded exposure to small amounts of allergens.

[0110] In some other embodiments of the above method for determining the beneficial effects of a pharmacokinetic and / or selected dietary regimen in dogs suffering from atopic dermatitis, the dietary regimen may be selected from among diets containing hydrolyzed protein, grain-free diets, omega-3 supplement diets, antioxidant-rich diets, pantothenate supplement diets, choline supplement diets, nicotinamide supplement diets, histidine supplement diets, zinc supplement diets, copper supplement diets, flavonoid supplement diets, terpenoid supplement diets, and inositol supplement diets.

[0111] In all methods of this disclosure, the detection of the presence of the protein biomarker selected in step b) and / or the measurement of its content can be carried out by immunological methods.

[0112] In all the methods of this disclosure, the selected nucleic acid biomarker can be chosen from mRNA and miRNA.

[0113] In all methods of this disclosure, the detection of the presence of the mRNA or miRNA biomarker selected in step b) and / or the measurement of its content can be carried out by a nucleic acid amplification method.

[0114] This disclosure further relates to a kit of components for detecting and / or measuring protein and / or nucleic acid biomarkers in canine epidermal samples, including one or more adhesive tapes for tapering skin according to this specification.

[0115] The Disclosure further relates to a kit of components for detecting and / or measuring protein and / or nucleic acid canine biomarkers in canine epidermal samples, comprising: a) one or more adhesive tapes for peeling skin with tape according to the specification of the Disclosure; b) reagents for collecting epidermal samples adhered to the surface of the adhesive tapes; c) reagents for detecting and / or quantifying one or more protein biomarkers; and / or d) reagents for detecting and / or quantifying one or more nucleic acids, particularly for detecting and / or quantifying one or more mRNAs or miRNAs; and e) optionally, a recommendation for using the kit of components.

[0116] In embodiments in which the kit includes reagents for detecting and / or quantifying one or more protein biomarkers, the kit may include one or more biomarker-specific antibodies and optionally include means for detecting the binding of one or more biomarker-specific antibodies to their respective target proteins in an epidermal sample initially adhered to the adhesive tape.

[0117] In embodiments of the kit, which includes reagents for detecting and / or quantifying one or more nucleic acid biomarkers, most preferably one or more mRNA or miRNA biomarkers, the kit includes, as reagents, an RNA extraction reagent, a reverse transcriptase, a DNA polymerase, and optionally primers and probes for mRNA or miRNA biomarkers whose abnormal expression correlates with a skin disease or pathological skin condition. Furthermore, the kit may include primers and probes for control genes, such as housekeeping genes. Primers and probes for control genes can be used, for example, in normalization calculations. The kit may also include instructions for performing tape stripping and for analyzing gene expression using normalization calculations. In some embodiments of the kit, the kit may include probes that bind to a given nucleic acid biomarker, most preferably a given mRNA or miRNA biomarker. In further embodiments, the kit may include a microarray containing amplification products of selected nucleic acid biomarkers and one or more nucleic acid probes that can specifically hybridize, most preferably with the mRNA or miRNA biomarkers according to this disclosure. In some embodiments, many reagents may be provided in the kit of the present invention, and only some of them should be used together in a particular reaction or procedure. For example, multiple primers may be provided, and only two of them may be required for a particular application.

[0118] This disclosure is further illustrated by the following embodiments, but is not limited in any way to them. [Examples]

[0119] The Disclosure may be further illustrated by the following embodiments of its particular form, but it should be understood that these embodiments are included for illustrative purposes only and are not intended to limit the scope of the Disclosure.

[0120] Example 1: Determination of the occurrence of atopic dermatitis in dogs A. Materials and Methods A.1. Dog Protocol Eight healthy beagle dogs were epidermally sensitized to Dermatophagoides farinae extract twice a week for 49 days after tape stripping. The control site received only the vehicle. Adhesive tape strips D-Squame® (DS 100, France, Monaderm) were subjected to a standardized pressure (225 g.cm²) using a DSquame® pressure gauge (DS500, France, Monaderm). -2 It was applied to the skin.

[0121] A.2. ELISA assay The commercially available ELISA kits used in the assay are listed in Table 1 at the end of this specification.

[0122] These assays employ quantitative sandwich enzyme immunoassay techniques. Monoclonal antibodies specific to particular cytokines are pre-coated onto microplates. Standards, controls, and samples are pipetted into wells, and any present cytokines are bound with immobilized antibodies. After washing away any unbound substances, biotinylated mouse monoclonal antibodies specific to the cytokine being tested are added to the wells. After washing to remove unbound antibodies, streptavidin bound to HRP is added to the wells. Subsequent washing removes unbound streptavidin-HRP, and the substrate solution is added to the wells. The enzymatic reaction yields a blue product that turns yellow when stop solution is added. The intensity of the measured color is proportional to the amount of cytokine bound in the first step. Sample values ​​are then read from the standard curve.

[0123] A.3. Transcriptome assay Total RNA was extracted using promoters, and these samples were subjected to deep sequencing (strand protocol, 2 × 150 bp paired ends). This specification reports the quality control, pretreatment, and statistical analysis performed on this dataset.

[0124] RNA samples were quantified using a Qubit4.0 Fluorometer (Life Technologies, Carlsbad, California), and RNA integrity was confirmed using an RNA Kit on an Agilent 5600 Fragment Analyzer (Agilent Technologies, Palo Alto, California).

[0125] RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina, following the manufacturer's instructions (New England Biolabs, Ipsowitch, Massachusetts, USA). Briefly, mRNA was first enriched with oligo-d(T) beads. The enriched mRNA was fragmented at 94°C for 15 minutes. Subsequently, primary and secondary cDNA were synthesized. The cDNA fragments were repaired at the ends, adenylated at the 3' end, ligated to the cDNA fragments with a universal adapter, and then indexed and enriched by PCR in limited cycles. The sequencing libraries were validated using a DNA kit on an Agilent 5600 Fragment Analyzer (Agilent Technologies, Palo Alto, California) and quantified using a Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, California).

[0126] The sequencing library was multiplexed and clustered on a flow cell. After clustering, the flow cell was loaded into an Illumina NovaSeq 6000 instrument according to the manufacturer's instructions. Samples were sequenced using a 2×150 paired-end (PE) configuration. Image analysis and base recall were performed using NovaSeq control software v1.6 on the NovaSeq instrument. Raw sequence data (.bcl files) generated from Illumina NovaSeq were converted to fastq files and multiplexed using the Illumina bcl2fastq program version 2.20. One mismatch was permitted for index sequence identification.

[0127] A.4. Correlation between ELISA and transcriptome assay results The goal was to test the effect of antigen sensitization on canine skin and compare ELISA data with transcriptomics data.

[0128] Elisa's data was generated as disclosed in §A.2 above.

[0129] The transcriptome data was generated as disclosed in §A.3 above.

[0130] Nine cytokines (IFNG (=IFN-γ), IL-10, IL-13, IL-1B, IL-25, IL-33, IL-4, TNF, and TSLP) were measured. Their names were used to identify the corresponding ENSEMBL IDs listed in Table 2a below.

[0131] [Table 1]

[0132] ENEMBL v105 does not contain the IL-25 gene for the reference canine assembly used for transcriptome analysis. A search for canine IL-25 in NCBI returned an entry corresponding to another ENSEMBL gene with the symbol CMTM5. Therefore, this ENEMBL ID was used for IL-25.

[0133] In the original filtering procedure, transcripts encoding the cytokines IFNG, IL-25 / CM5, IL-33, and IL-4 were considered to have low expression and were filtered from the transcriptomics dataset. These were then recovered for this analysis.

[0134] A.5. Correlation between genes or proteins and clinical responses in dogs, and / or given cytokines. The objective was to test the effect of antigen-induced sensitization on canine skin and to compare clinical responses with transcriptome data.

[0135] The transcriptome data was generated as disclosed in §A.3 above.

[0136] The correlation between 19 proteins (CCL17, CCL20, CCL22, CCL5, CD2, CD3E, ELOVL3, FA2H, FCER1A, ICOS, IL13, IL17RB, IL1B, IL1RL2, IL22, IL36B, IVL, RORA, TNFRSF4) and clinical responses was evaluated. Their names were used to identify the corresponding ENSEMBL IDs listed in Table 2b below.

[0137] [Table 2]

[0138] A first analysis is provided, taking into account Spearman's correlation coefficients for both transcriptomics data and clinical responses.

[0139] Next, for each ELISA protein, heatmaps were created for the 100 genes with the highest Pearson correlation. ELISA signal data were initially converted using the formula "log2(1+signal)" (IL1B was excluded because its signal was flat). Only genes with at least a twofold change in transcriptome amplitude were considered.

[0140] The same process was applied to the transcriptome signals of CCL17, CCL5, and CCL20.

[0141] B. Results B.1. Correlation between ELISA and transcriptome assay results The results regarding the presence and content of each selected protein biomarker and gene biomarker are summarized in Table 3, localized at the end of the explanation.

[0142] Table 3 also shows the correlation between (i) the detection and quantification of selected protein biomarkers and (ii) the detection and quantification of selected mRNA biomarkers. Therefore, Table 3 compares the values ​​observed by ELISA with those observed by transcriptome analysis for each cytokine (transcriptome values ​​are raw count values ​​normalized by a sample size factor estimated by SESeq2).

[0143] In Table 3, each line corresponds to the dog that was tested.

[0144] In Table 3, each column corresponds to a selected biomarker, which is indicated at the bottom of the column.

[0145] Table 3 is detailed below, following the format for reading from left to right.

[0146] Findings regarding the visual skin lesion response to sensitization are clearly stated in the second column of Table 3 for each sensitized dog.

[0147] In each table cell containing the results, (i) The upper left of the cell shows the progression of the level of the protein specified in the column. (ii) The symbol on the right in the upper right part of the cell indicates the change in the level of mRNA encoding the protein specified in the column: "-" corresponds to no change, "+" to a slight increase in protein / mRNA level, and "++" to a significant increase in protein / mRNA level. (iii) The symbol in the lower center of each cell indicates whether there is a correlation between the protein level and the corresponding mRNA level with respect to the lesion (check mark symbol), whether there is no correlation between the protein level and the corresponding mRNA level with respect to the lesion (cross symbol), or whether there is a greater or lesser correlation between the protein level and the corresponding mRNA level with respect to the lesion (almost equal symbol).

[0148] On day 49, these tape strips were used for ELISA and transcriptome analysis to test for inflammatory cytokines and mRNA in the epidermis. ELISA showed a significant increase in the concentrations of interleukins IL-4, IL-13, IL-33, IL-25, and TNF-α at the sensitized site on day 49 compared to the control site (Friedman test, p<0.05). IL-4, IL-13, IL-33, IL-25, TNF-α, IFN-γ, and TSLP concentrations were significantly increased after sensitization compared to D0 (p<0.05). IL-1β and IL-10 were not detected. mRNAs with differential expression were selected based on changes in gene expression levels (cutoff factor change ≥2 and statistical significance p ≤0.05). On day 49, 210 differentially regulated genes were identified between the sensitized and control sites. The main pathways modified by these gene dysregulations are cell adhesion, immune response, and cell migration, which perfectly match the overall immune response generated by our sensitization procedure.

[0149] B2: Deeper results obtained with two dogs A deeper analysis was conducted to integrate the results. The results obtained from two dogs representing the tested population are presented below.

[0150] B2.1: Correlation between ELISA and clinical signs The results obtained demonstrate a good correlation between ELISA and clinical signs, thus demonstrating that the genes analyzed correlate well with the canine response. Figures 1 and 2 show the results obtained for TNF (Figure 1) and IL13 (Figure 2) from two different dogs, Dog 5 and Dog 6, respectively, demonstrating that ELISA correlates with clinical response (Dog 5 showed a very good response, and Dog 6 showed a moderate response).

[0151] B2.2: Correlation between ELISA (proteins) and their own genes The results demonstrate a good correlation between ELISA and mRNA for the relevant proteins / cytokines in the same two dogs, 5 and 6. Figures 3 and 4 show the results obtained for TNF (Figure 3) and IL13 (Figure 4) using the same dogs.

[0152] B2.3: Correlation between other Th2 or Th1 genes and canine responses. The results obtained demonstrate a good correlation between other Th2 or Th1 genes and the canine response. Figures 5–11 show these results for the genes TNFRSF4 (Figure 5), FA2H (Figure 6), IL1B (Figure 7), IL13RA1 (Figure 8), IL13RA2 (Figure 9), IL22 (Figure 10), and IL4R (Figure 11).

[0153] In conclusion, DSquame® is considered a suitable method for measuring stratum corneum cytokine concentrations and mRNA mapping. mRNA analysis showed correlation with the Th2 response generated by the sensitization procedure, despite known individual variability.

[0154] [Table 3]

[0155] [Table 4]

[0156] literature 1. Clausen et al.,2020,Nature Research. 10 : 21895 2. Dyjack et al., 29, J Allergy Clin Immunol, Vol. 141(4): 1298-1309 3. Morlang et al., 2021, Vet Dermatol, Vol. 32 : 331 - e92 4. Koury et al., 2019, PlosOne, Vol. 14 (6) : e0128670

[0157] While the subject matter and its merits have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of the subject matter of this disclosure. Furthermore, the scope of this application is not intended to be limited to specific embodiments of the processes, machines, products, material compositions, means, methods, and steps described in the specification. As will be readily apparent to those skilled in the art from the subject matter, processes, machines, products, material compositions, means, methods, or steps of this disclosure, existing or future developments that perform substantially the same functions or achieve substantially the same results as the corresponding embodiments described herein can be utilized in accordance with the subject matter of this disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, products, material compositions, means, methods, or steps. Various patents, patent applications, publications, product descriptions, protocols, and sequence acceptance numbers are referenced throughout this application, and their inventions are incorporated herein by reference in their entirety for all purposes.

[0158] Possible Embodiments Examples of possible embodiments intended by the present invention are as follows:

[0159] 1. A method for detecting or measuring the content of one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in a canine skin sample, a) A step of applying adhesive tape to the exposed skin target area of ​​the dog and thereby collecting epidermal samples that adhere to the adhesive tape, and b) A method comprising the steps of detecting the presence of one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in the epidermal sample obtained in step a), and / or measuring their content.

[0160] 2. The method according to Embodiment 1, wherein in step b), the selected canine biomarker of one or more proteins and / or nucleic acids indicates the immune status of the canine.

[0161] 3. The method according to Embodiment 1, wherein in step b), the selected canine biomarker of one or more proteins and / or nucleic acids is a canine biomarker indicating an inflammatory response in dogs.

[0162] 4. The method according to Embodiment 1, wherein in step b), the selected canine biomarker of one or more proteins and / or nucleic acids is a canine biomarker that exhibits a canine Th2 immune response.

[0163] 5. The method according to Embodiment 1, wherein the selected one or more proteins and / or nucleic acid canine biomarkers are selected from among the proteins present in the epidermis.

[0164] 6. The selected one or more proteins present in the epidermis, a) Interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33), b) Tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), c) Thymus interstitial lymphopoietin (TSLP), and / or d) The method according to Embodiment 5, wherein one or more proteins are selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha 1 (IL13RA1), interleukin-13 receptor subunit alpha 2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0165] 7. The method according to any one of Embodiments 1 to 6, wherein the presence of the protein biomarker selected in step b) and / or the content thereof is detected by an immunological method.

[0166] 8. The method according to any one of Embodiments 1 to 7, wherein the selected one or more nucleic acid biomarkers are selected from mRNA and miRNA.

[0167] 9. The method according to Embodiment 8, wherein the presence of the mRNA or miRNA biomarker selected in step b) and / or the content is measured by a nucleic acid amplification method.

[0168] 10. A method for determining the presence of a skin disorder in the skin of a dog, a) A step of applying adhesive tape to an exposed skin target area of ​​the dog and thereby collecting an epidermal sample that adheres to the adhesive tape, b) A step of detecting statistically significant different levels of one or more proteins and / or one or more nucleic acid canine biomarkers in the epidermal sample. c) A method comprising the step of determining the presence of a skin disorder if, in step b), one or more of the biomarkers are measured at statistically significantly different levels.

[0169] 11. The method according to Embodiment 11, wherein the skin disorder is an inflammatory disorder, and is preferentially selected from canine atopic dermatitis, pyoderma, contact dermatitis, folliculitis, acne, seborrheic dermatitis, panniculitis, cellulitis, licking granuloma (stomatitis, histiocytoma, eosinophilic granuloma complex), nasal dermatitis (Collie nose), interdigital cysts and furunculosis, ichthyosis, demodectic mange (red scabies), ringworm (dermatophytosis), and vasculitis.

[0170] 12. The method according to Embodiment 10, wherein the skin disorder is a non-inflammatory disorder, and is preferentially selected from alopecia areata, colordilution alopecia, pattern alopecia, congenital hypotrichosis, hyperkeratosis, seborrheic lymphadenitis, follicular dysplasia, sinusoidal dysplasia, X-linked ichthyosis, seasonal follicular dysplasia, nasal plane hyperkeratosis, hereditary palmoplantar keratosis, primary seborrhea, vitamin A-responsive dermatopathy, cutaneous mastocytosis, hypothyroidism-related skin lesions, and canine epidermolysis bullosa.

[0171] 13. A method for diagnosing atopic dermatitis in the skin of dogs, a) A step of applying adhesive tape to an exposed skin target area of ​​the dog and thereby collecting an epidermal sample that adheres to the adhesive tape, b) A step of detecting statistically significant different levels of one or more proteins and / or nucleic acid canine biomarkers in the epidermal sample. c) A method comprising determining the presence of an atopic dermatitis condition if, in step b), one or more statistically significant levels of the protein and / or nucleic acid canine biomarkers are measured.

[0172] 14. The protein and / or nucleic acid canine biomarkers are a) Interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33), b) Tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), c) Thymus interstitial lymphopoietin (TSLP), and d) The method according to any one of Embodiments 10 to 13, selected from one or more proteins and / or nucleic acid canine biomarkers selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha 1 (IL13RA1), interleukin-13 receptor subunit alpha 2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0173] 15. A method for determining the beneficial effects of a drug and / or a selected dietary regimen in dogs suffering from atopic dermatitis, a) A step of applying adhesive tape to an exposed skin target area of ​​the dog and thereby collecting an epidermal sample that adheres to the adhesive tape, b) A step of measuring the level of one or more selected proteins and / or nucleic acid canine biomarkers in the epidermal sample collected in step b), c) The step of treating the dog with a drug and / or a selected dietary regimen, d) A step in which an adhesive tape is applied to an exposed skin target area of ​​the dog, thereby collecting an epidermal sample that adheres to the adhesive tape, e) A step of detecting and / or measuring the level of one or more selected proteins and / or nucleic acid canine biomarkers in the epidermal sample collected in step d), A method comprising the step of determining the beneficial effects of the drug and / or the dietary therapy if an increase and / or decrease in the level of one or more biomarkers is measured in step e) compared with the level of the same selected biomarker measured in step b).

[0174] 16. Steps a) and d) are a1) (and d1) Applying adhesive tape to the exposed target area of ​​the dog's skin about 1 to about 50 times, preferably about 10 to about 30 times, to remove the surface layer of the skin, most preferably the stratum corneum, and The method according to Embodiment 15, comprising the steps of a2) (and d2) applying an adhesive tape different from the adhesive tape of step a1) to an exposed skin target area of ​​the dog, thereby collecting an epidermal sample that adheres to the adhesive tape.

[0175] 17. The method according to Embodiment 15 or 16, wherein in each of steps a) and d), the adhesive tape is applied at a constant pressure.

[0176] 18. The protein or nucleic acid canine biomarker is a) Interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33), b) Tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), and c) Thymus interstitial lymphopoietin (TSLP), and d) The method according to any one of Embodiments 15 to 17, wherein one or more proteins or nucleic acid canine biomarkers are selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha-1 (IL13RA1), interleukin-13 receptor subunit alpha-2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0177] 19. The method according to any one of Embodiments 1 to 18, wherein the selected one or more protein biomarkers and / or one or more nucleic acid biomarkers are selected from interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33).

[0178] 20. A kit of components for detecting and / or measuring protein and / or nucleic acid canine biomarkers in canine epidermal samples, a) One or more adhesive tapes for peeling off skin, b) Reagents for collecting epidermal samples adhered to the surface of adhesive tape, c) Reagents for detecting and / or quantifying one or more protein biomarkers, and / or d) Reagents for detecting and / or quantifying one or more nucleic acids, particularly for detecting and / or quantifying one or more mRNAs or miRNAs. e) Kits, including recommendations for using a parts kit in some cases.

Claims

1. A method for detecting or measuring the content of one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in a canine skin sample, a) A step of applying adhesive tape to an exposed skin target area of ​​the dog and thereby collecting epidermal samples that adhere to the adhesive tape, and b) A method comprising the steps of detecting the presence of one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in the epidermal sample obtained in step a), and / or measuring their content.

2. The method according to claim 1, wherein in step b), the selected canine biomarker of one or more proteins and / or nucleic acids indicates the immune status of the canine.

3. The method according to claim 1, wherein in step b), the selected canine biomarker of one or more proteins and / or nucleic acids is a canine biomarker indicating an inflammatory response in dogs.

4. The method according to claim 1, wherein in step b), the selected canine biomarker of one or more proteins and / or nucleic acids is a canine biomarker that exhibits a canine Th2 immune response.

5. The method according to claim 1, wherein the selected one or more proteins and / or nucleic acid canine biomarkers are selected from among the proteins present in the epidermis.

6. The selected one or more proteins present in the epidermis, a) Interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33), b) Tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), c) Thymic interstitial lymphopoietin (TSLP), and / or d) The method according to claim 5, wherein one or more proteins are selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha-1 (IL13RA1), interleukin-13 receptor subunit alpha-2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

7. The method according to any one of claims 1 to 6, wherein the detection of the presence of the selected protein biomarker in step b) and / or the measurement of its content is carried out by an immunological method.

8. The method according to any one of claims 1 to 7, wherein the selected one or more nucleic acid biomarkers are selected from mRNA and miRNA.

9. The method according to claim 8, wherein the detection of the presence of the mRNA or miRNA biomarker selected in step b) and / or the measurement of its content is carried out by a nucleic acid amplification method.

10. A method for determining the presence of a skin disorder in the skin of a dog, a) A step of applying adhesive tape to an exposed skin target area of ​​the dog and thereby collecting an epidermal sample that adheres to the adhesive tape, b) A step of detecting statistically significant different levels of one or more proteins and / or one or more nucleic acid canine biomarkers in the epidermal sample. c) A method comprising the step of determining the presence of a skin disorder if, in step b), one or more of the biomarkers are measured at statistically significantly different levels.

11. The method according to claim 11, wherein the skin disorder is an inflammatory disorder, and is preferentially selected from canine atopic dermatitis, pyoderma, contact dermatitis, folliculitis, acne, seborrheic dermatitis, panniculitis, cellulitis, licking granuloma (stomatitis, histiocytoma, eosinophilic granuloma complex), nasal dermatitis (Collie nose), interdigital cysts and furunculosis, ichthyosis, demodectic mange (red scabies), ringworm (dermatophytosis), and vasculitis.

12. The aforementioned skin disorders are non-inflammatory disorders, including alopecia areata, color dilution alopecia, pattern alopecia, congenital hypotrichosis, hyperkeratosis, seborrheic lymphadenitis, follicular dysplasia, sinusoidal dysplasia, X-linked ichthyosis, and seasonal follicular dysplasia. The method according to claim 10, which is preferentially selected from nasal plane hyperkeratosis, hereditary palmoplantar keratosis, primary seborrhea, vitamin A-responsive dermatopathy, cutaneous mastocytosis, hypothyroidism-related skin lesions, and canine epidermolysis bullosa.

13. A method for diagnosing atopic dermatitis in the skin of dogs, a) A step of applying adhesive tape to an exposed skin target area of ​​the dog and thereby collecting an epidermal sample that adheres to the adhesive tape, b) A step of detecting statistically significant different levels of one or more proteins and / or nucleic acid canine biomarkers in the epidermal sample. c) A method comprising determining the presence of an atopic dermatitis condition if, in step b), one or more statistically significant levels of the protein and / or nucleic acid canine biomarkers are measured.

14. The aforementioned protein and / or nucleic acid canine biomarkers a) Interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33), b) Tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), c) Thymic interstitial lymphopoietin (TSLP), and d) The method according to any one of claims 10 to 13, wherein one or more proteins and / or nucleic acid canine biomarkers selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha-1 (IL13RA1), interleukin-13 receptor subunit alpha-2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

15. A method for determining the beneficial effects of a drug and / or a selected dietary regimen in dogs suffering from atopic dermatitis, a) A step of applying adhesive tape to an exposed skin target area of ​​the dog and thereby collecting an epidermal sample that adheres to the adhesive tape, b) A step of measuring the level of one or more selected proteins and / or nucleic acid canine biomarkers in the epidermal sample collected in step b), c) The step of treating the dog with a drug and / or a selected dietary regimen. d) A step in which an adhesive tape is applied to an exposed skin target area of ​​the dog, thereby collecting an epidermal sample that adheres to the adhesive tape. e) A step of detecting and / or measuring the level of one or more selected proteins and / or nucleic acid canine biomarkers in the epidermal sample collected in step d), A method comprising the step of determining the beneficial effects of the drug and / or the dietary therapy when an increase and / or decrease in the level of one or more biomarkers is measured in step e) compared with the level of the same selected biomarker measured in step b).

16. Steps a) and d) are a1) (and d1) Applying adhesive tape to the exposed target area of ​​the dog's skin about 1 to about 50 times, preferably about 10 to about 30 times, to remove the surface layer of the skin, most preferably the stratum corneum, and a2) (and d2) the method of claim 15, comprising the step of applying an adhesive tape different from the adhesive tape of step a1) to an exposed skin target area of ​​the dog, thereby collecting an epidermal sample that adheres to the adhesive tape.

17. The method according to claim 15 or 16, wherein in each of steps a) and d), the adhesive tape is applied at a constant pressure.

18. The aforementioned protein or nucleic acid canine biomarker a) Interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33), b) Tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), and c) Thymic interstitial lymphopoietin (TSLP), and d) The method according to any one of claims 15 to 17, wherein one or more proteins or nucleic acid canine biomarkers selected from the group consisting of tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit alpha-1 (IL13RA1), interleukin-13 receptor subunit alpha-2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

19. The method according to any one of claims 1 to 18, wherein the selected one or more protein biomarkers and / or one or more nucleic acid biomarkers are selected from interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-25 (IL-25), interleukin-31 (IL-31), and interleukin-33 (IL-33).

20. A kit of components for detecting and / or measuring protein and / or nucleic acid canine biomarkers in canine epidermal samples, a) One or more adhesive tapes for peeling off skin, b) Reagents for collecting epidermal samples adhered to the surface of adhesive tape, c) Reagents for detecting and / or quantifying one or more protein biomarkers, and / or d) Reagents for detecting and / or quantifying one or more nucleic acids, particularly for detecting and / or quantifying one or more mRNAs or miRNAs. e) Kits, including recommendations for using a parts kit in some cases.

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