Minimally invasive method of diagnosing a canine condition

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 adhesive tape to a target area of ​​exposed skin of the dog, thereby collecting an epidermal sample adhered to the adhesive tape, and b) detecting the presence and / or measuring the content of the one or more selected biomarkers in the epidermal sample obtained at step a). This disclosure also relates to methods for determining the presence of a skin condition in canine skin, methods for diagnosing atopic dermatitis in canine skin, methods for determining the beneficial effects of pharmaceutical preparations and / or selected dietary regimens in dogs suffering from atopic dermatitis, and component kits for detecting and / or measuring canine biomarkers in canine epidermal samples.

Description

[0001] Cross-reference to related applications This application claims priority to European Patent Application No. 23306455.9, filed on August 31, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates to the diagnosis of inflammatory and non-inflammatory skin conditions in dogs, including allergic conditions such as canine atopic dermatitis. Background Technology

[0003] Diagnosis of diseases or conditions in dogs is usually performed through skin biopsy.

[0004] Skin biopsy is an important diagnostic tool used by veterinarians to obtain information about a variety of skin conditions and diseases in dogs. A skin biopsy involves taking a small sample of skin tissue for examination. The information obtained from the biopsy can help determine the underlying cause of the skin problem and guide appropriate treatment.

[0005] In practice, skin biopsies are inherently invasive and are typically performed only after clinical signs have been detected. Therefore, invasive methods are generally not used in suspected cases or early stages of disease. Consequently, in addition to causing trauma to animals, the use of skin biopsies in large-scale clinical trials and longitudinal studies is limited.

[0006] Minimally invasive methods are desirable for enabling more dynamic and repeatable skin sampling over time, including sampling related to treatment, environment, and disease outbreaks. Furthermore, reducing discomfort, potential infection, and scarring in dogs due to biopsies (including skin biopsies) is desirable.

[0007] Furthermore, minimally invasive methods are anticipated because they allow for the detection of the risk of a symptom or disease before it occurs. Alternatively, minimally invasive methods may allow for the assessment of the actual occurrence of a symptom or disease early in the course of its development, particularly before any clinical signs appear.

[0008] In the field of human medicine, existing technologies disclose a number of minimally invasive diagnostic methods, including those that involve collecting skin samples by peeling off adhesive tape.

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

[0010] There is still a need in the art for minimally invasive methods for qualitative and / or quantitative analysis of proteins in canine skin samples, particularly for canine diagnosis, especially for the diagnosis of canine skin conditions or diseases (whether non-inflammatory or inflammatory, such as canine atopic dermatitis). Summary of the Invention

[0011] This article 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) applying adhesive tape to a target area of ​​exposed skin of a dog to collect an epidermal sample adhered to the adhesive tape, and b) detecting the presence and / or measuring the content of the one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in the epidermal sample obtained at step a).

[0012] In a particular embodiment, at step b), one or more selected protein and / or nucleic acid canine biomarkers may indicate the canine's immune status. In another embodiment, at step b), one or more selected protein and / or nucleic acid canine biomarkers may be canine biomarkers indicating a canine inflammatory response. In yet another embodiment, at step b), one or more selected protein and / or nucleic acid canine biomarkers may be canine biomarkers indicating a canine Th2 immune response. Specifically, one or more selected protein and / or nucleic acid canine biomarkers may be selected from proteins present in the epidermis. Specifically, one or more selected proteins present in the epidermis may be selected from: a) interleukin-1β (IL-1... (a) 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 α (TNF-α) ) and gamma interferon (IFN-γ) (c) and / or (d) thymic stromal lymphopoietin (TSLP); and / or (e) 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 α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof. In a particular embodiment, the detection of the presence and / or measurement of the content of the selected protein biomarker at step b) can be performed by immunological methods. In particular, one or more selected nucleic acid biomarkers can be selected from mRNA and miRNA. In particular, the detection of the presence and / or measurement of the content of the selected mRNA or miRNA biomarker at step b) can be performed by nucleic acid amplification methods.

[0013] Another objective of this document relates to a method for determining the presence of a skin condition in canine skin, comprising the following steps: a) applying adhesive tape to an exposed target area of ​​the dog, thereby collecting an epidermal sample adhered to the adhesive tape; b) detecting statistically significant differences in one or more protein and / or one or more nucleic acid canine biomarkers in the epidermal sample; and c) determining the presence of a skin condition when a statistically significant difference in one or more of the biomarkers is measured at step b). Specifically, the skin condition may be an inflammatory condition, preferably selected from canine atopic dermatitis, pyoderma, contact dermatitis, folliculitis, acne, seborrheic dermatitis, panniculitis, cellulitis, licking granuloma (acromial licking dermatitis, histiocytoma, eosinophilic granuloma complex), nasal photodermatitis (Collie nose), interdigital cysts and furuncles, ichthyosis, demodicosis (demodectic mange), tinea (dermatomycosis), and vasculitis. Specifically, the skin condition may be a non-inflammatory condition, preferably selected from alopecia areata, color dilution alopecia, patterned alopecia, congenital sparse hair, hyperkeratosis, sebaceous gland inflammation, follicular dysplasia, dermoid sinus, X-linked ichthyosis, periodic follicular dysplasia, nasal plane hyperkeratosis, hereditary paw pad hyperkeratosis, primary seborrhea, vitamin A-reactive dermatitis, cutaneous mastocytosis, hypothyroidism-related dermatitis, and canine bullous epidermolysis.

[0014] This article also relates to a method for diagnosing atopic dermatitis in canine skin, comprising the following steps: a) applying adhesive tape to a target area of ​​exposed skin of a dog, thereby collecting an epidermal sample adhered to the adhesive tape; b) detecting statistically significant differences in one or more canine biomarkers of proteins and / or nucleic acids in the epidermal sample; c) determining the presence of atopic dermatitis when a statistically significant difference in one or more of the canine biomarkers of proteins and / or nucleic acids is measured at step b); and d) one or more canine biomarkers of proteins and / or nucleic acids selected from the group consisting of: tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0015] Specifically, the canine biomarkers for the proteins and / or nucleic acids may be selected from: a) interleukin-1β (IL-1 (a) 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 α (TNF-α) ) and gamma interferon (IFN-γ) (c) thymic stromal lymphopoietin (TSLP), and (d) one or more canine biomarkers of proteins and / or nucleic acids selected from the group consisting of: tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0016] Furthermore, this article relates to a method for determining the beneficial effects of a pharmaceutical preparation and / or a selected dietary regimen in dogs suffering from atopic dermatitis, comprising the following steps: a) applying adhesive tape to a target area of ​​exposed skin of the dog, thereby collecting epidermal samples adhered to the adhesive tape; b) measuring the levels of one or more selected protein and / or nucleic acid canine biomarkers in the epidermal samples collected at step b); c) treating the dog with the pharmaceutical preparation and / or the selected dietary regimen; d) applying adhesive tape to a target area of ​​exposed skin of the dog, thereby collecting epidermal samples adhered to the adhesive tape; e) detecting and / or measuring the levels of one or more selected protein and / or nucleic acid canine biomarkers in the epidermal samples collected at step d); f) determining that the pharmaceutical preparation and / or the dietary regimen has a beneficial effect when the level of one or more of the biomarkers measured at step e) is higher and / or lower than the level of the same selected biomarker measured at step b). Specifically, steps a) (and d) may include: a1) (and d1) applying adhesive tape about 1 to about 50 times, preferably about 10 to about 30 times, to the exposed target area of ​​the dog's skin 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 used in step a1) to the exposed target area of ​​the dog's skin, thereby collecting an epidermal sample adhered to the adhesive tape. Specifically, in each of steps a) and d), the adhesive tape may be applied under constant pressure.

[0017] In addition, the protein or nucleic acid canine biomarker may be selected from: a) interleukin-1β (IL-1 (a) 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 α (TNF-α) ) and gamma interferon (IFN-γ) c) thymic stromal lymphopoietin (TSLP); and d) one or more canine biomarkers of proteins or nucleic acids selected from the group consisting of: tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0018] Furthermore, this article relates to a component kit for detecting and / or measuring canine biomarkers of proteins and / or nucleic acids in canine epidermal samples, comprising: a) one or more adhesive tapes for peeling skin off, b) reagents for collecting epidermal samples adhered to the surface of the 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, and e) optional suggestions for using the component kit. Attached Figure Description

[0019] The following figures are included to illustrate certain aspects of this disclosure and should not be considered as exclusive embodiments. The disclosed subject matter can be modified, altered, combined, and equivalent in form and function without departing from the scope of this disclosure.

[0020] Figure 1 Comparison of TNF fold change (log2 scale) and response status in dogs 5 and 6. Dog ID is indicated within the data points.

[0021] Figure 2 Comparison of IL13 fold change (log2 scale) and response status for dogs 5 and 6. Dog ID is indicated within the data points.

[0022] Figure 3 Comparison of TNF transcriptome and ELISA expression values ​​in dogs 5 and 6. The straight line in the figure is the linear regression line. The canine ID is indicated within the data points.

[0023] Figure 4 Comparison of IL13 transcriptomic and ELISA expression values ​​in dogs 5 and 6. The straight line in the figure is a linear regression line. The dog ID is indicated within the data points.

[0024] Figure 5 Comparison of transcriptomic fold change (log2 scale) and response status of TNFRSF4 (ENSCAFG00845022948) in dogs 5 and 6. Dog ID is indicated within the data points.

[0025] Figure 6 Comparison of transcriptome fold change (log2 scale) and response status for FA2H (ENSCAFG00845025633) in dogs 5 and 6. Dog ID is indicated within the data points.

[0026] Figure 7Comparison of transcriptomic fold change (log2 scale) and response status of IL1B (ENSCAFG00845011661) in dogs 5 and 6. Dog ID is indicated within the data points.

[0027] Figure 8 Comparison of transcriptomic fold change (log2 scale) and response status of IL13RA1 (ENSCAFG00845019238) in dogs 5 and 6. Dog ID is indicated within the data points.

[0028] Figure 9 Comparison of transcriptomic fold change (log2 scale) and response status of IL13RA2 (ENSCAFG00845016176) in dogs 5 and 6. Dog ID is indicated within the data points.

[0029] Figure 10 Comparison of transcriptomic fold change (log2 scale) and response status of IL22 (ENSCAFG00845013728) in dogs 5 and 6. Dog ID is indicated within the data points.

[0030] Figure 11 Comparison of transcriptomic fold change (log2 scale) and response status of IL4R (ENSCAFG00845004286) in dogs 5 and 6. Dog ID is indicated within the data points. Detailed Implementation

[0031] definition Within the context of this disclosure and in the specific context in which each term is used, the terms used herein generally have their common meaning in the art. Certain terms are discussed below or elsewhere in this specification to provide additional guidance in describing the compositions and methods of this disclosure and how they are prepared and used.

[0032] Exemplary methods and materials are described below, but methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this disclosure. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms.

[0033] Units, prefixes, and symbols are represented in a form acceptable in the International System of Units (SI). Numerical ranges include the numerical endpoints of the defined range. The headings provided herein are not intended to limit any aspect of this disclosure. Therefore, the terms defined below are defined more fully by reference to the entire contents of this specification.

[0034] All publications and other references mentioned in this article are included in their entirety by way of citation.

[0035] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used in the specification and appended claims include plural indicators. Thus, for example, a reference to “a compound” may include a mixture of at least two compounds.

[0036] As used herein, the terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by one of ordinary skill in the art, depending in part on how the value was measured or determined. Right now Limitations of the measurement system. Typically, the term "about" modifies values ​​higher or lower than the specified value by, for example, a variance of more than or less than 10% (higher or lower). In some implementations, the term indicates a deviation from the indicated value of ±15%, ±10%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0037] As used herein, the terms “comprising,” “including,” “containing,” “having,” or any other variation thereof are intended to cover non-exclusive inclusion, such that a food composition, process, or method that comprises a list of elements or ingredients includes not only those elements or ingredients but may also include other elements or ingredients not expressly listed or inherent to such food composition, process, or method. The term “consisting of” means including the specified elements but excluding any additional elements. The term “substantially consisting of” means including the specified elements as well as possible other elements, wherein the other elements do not materially affect the basic and novel characteristics of this disclosure. It should be understood that different embodiments of this disclosure using the term “comprising / including” or equivalent forms cover embodiments in which such term is replaced by “consisting of” or “substantially consisting of”.

[0038] In the detailed description herein, references to "implementation," "an implementation," "one implementation," "some implementations," or any other variations thereof indicate that the described implementation may include a particular feature, component, structure, or characteristic, but not every implementation necessarily includes that particular feature, component, structure, or characteristic. Furthermore, such phrases do not necessarily refer to the same implementation. Moreover, when a particular feature, component, structure, or characteristic is described in conjunction with an implementation, whether explicitly stated or not, it is considered that implementation in conjunction with other implementations is within the knowledge of those skilled in the art. After reading this specification, those skilled in the art will understand how to implement this disclosure in alternative implementations.

[0039] As used herein, the term "dog" can include animals, including companion animals selected from recognized breeds (some of which are further subdivided), which may include: Afghan Hound, Elkie, Akita, Alaskan Malamute, Basset Hound, Beagle, Belgian Shepherd, Bloodhound, Border Collie, Border Terrier, Borzoi, Boxer, Bulldog, French Bulldog, Bulldog, Bullmastiff, Cairn Terrier, Chihuahua, Chow Chow, Cocker Spaniel, Collie, Corgi, Dachshund, Dalmatian, Doberman Pinscher, Shar Pei, English Setter, Fox Terrier, German Shepherd, Golden Retriever Retrievers, Great Danes, Greyhounds, Brussels Griffons, Irish Setters, Irish Wolfhounds, Cavalier King Charles Spaniels, Labrador Retrievers, Lhasa Apso Dogs, Mastiffs, Newfoundlands, Old English Sheepdogs, Papillons, Pekingese, Pointers, Pomeranians, Poodles, Pugs, Rottweilers, Saint Bernards, Salukis, Samoyeds, Schnauzers, Scottish Terriers, Shetland Sheepdogs, Shih Tzus, Siberian Huskies, Skye Terriers, Vizsla, Rhodesian Terriers, Staffordshire Terriers, Jack Terriers, Springer Spaniels, West Highland White Terriers, Whippets, Yorkshire Terriers, Bichon Frises, etc.

[0040] As used in this article, "skin" refers to the outermost protective covering of mammals, especially dogs, and mainly consists of three layers: the outer epidermis, the middle dermis, and the bottom subcutaneous layer. subcutaneous tissue The epidermis itself consists of five layers, from top (exposed to the environment) to bottom (in contact with the dermis): (i) stratum corneum, (ii) stratum lucidum, (iii) stratum granulosum, (iv) stratum spinosum and (v) stratum basale.

[0041] As used in this article, "tape peeling" refers to the collection of biological materials, especially proteins and nucleic acids. For example Minimally invasive methods for collecting biological materials, particularly proteins and nucleic acids (mRNA and miRNA), involving the use of an adhesive placed on a skin base, particularly in dogs. According to this disclosure, tape peeling is used to collect biological materials, particularly proteins and nucleic acids (mRNA and miRNA), from the surface of canine skin. For example (mRNA and miRNA), by contacting the adaptable adhesive substrate with the surface of the dog's skin, the biomaterials adhere to the adhesive.

[0042] As used herein, the terms "dermatitis" or "skin disease" or "dermatopathy" refer to a variety of conditions that affect the appearance, function, and health of the skin. These conditions can manifest as various changes in skin texture, color, structure, and overall condition. Dermatitis can be caused by genetic factors, infections, immune system responses, environmental factors, allergies, hormonal imbalances, and other underlying causes. They may affect the outermost layer of the skin (epidermis), deeper layers (dermis), or both.

[0043] As used herein, the term "inflammatory disease" means any disease / condition associated with inflammation, and without limitation includes those associated with a selection of allergic (or atopic) conditions, inflammatory skin conditions, inflammatory gastrointestinal conditions, and respiratory conditions. For example, the term may cover one or more of the following conditions and syndromes: atopic diseases, feline atopic syndrome (FAS), feline atopic skin syndrome (FASS), feline asthma, and feline food allergy (FFA). The term also covers atopic dermatitis, allergic enteritis, and asthma.

[0044] As used in this article, the term “non-inflammatory disease” refers to a medical condition or symptom that does not primarily involve inflammation as a major component of its pathology.

[0045] Compared to inflammatory conditions, non-inflammatory diseases often have different underlying causes and mechanisms. They can be caused by genetic factors, metabolic imbalances, structural abnormalities, hormonal disorders, or other non-immune-related sources. Non-inflammatory diseases can affect multiple organs and systems in the body and can lead to a wide range of symptoms and clinical manifestations.

[0046] As used herein, the terms “atopic” or “atopic disease” are intended to refer to the generally accepted definition without limitation. The essential characteristic of atopic disease is defined as a predisposition to allergic diseases affecting the skin. Thus, the term encompasses any clinical dermatitis-related hypersensitivity reaction associated with a genetic predisposition manifested as the production of immunoglobulin E (IgE) antibodies in response to environmental proteins such as pollen, house dust mites, and food allergens.

[0047] As used herein, “atopic dermatitis” (AD) refers to an inflammatory skin condition characterized by intense itching (e.g., severe itching) and scaly, dry, eczematous lesions. 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, dust mites, animals, etc.), radiation exposure, and / or asthma. This disclosure covers methods for determining the occurrence or risk of moderate to severe AD. As used herein, “moderate to severe AD” is characterized by intensely itchy, widely distributed skin lesions, often secondary to persistent bacterial, viral, or fungal infections. Moderate to severe AD also includes chronic AD in dogs. In many cases, chronic lesions include hypertrophic plaques, lichenification, and fibrous papules. Dogs with moderate to severe AD typically also have more than 20% of their body skin affected, or an additional 10% of the skin area affected in addition to the eyes, feet, and body folds. Moderate to severe AD can also be seen in dogs receiving topical corticosteroid treatment. In some cases, a dog may be classified as having moderate to severe AD when it is resistant or refractory to topical corticosteroids or calcineurin inhibitors.

[0048] The terms "marker," "biomarker," or "biomarker" in this document refer to proteins or nucleic acids expressed in cells or tissues. For example Protein biomarkers (mRNAs or miRNAs) can be used to assess the physiological state of dogs, including non-inflammatory, inflammatory, or immune states, particularly in dogs, and can be used to predict or diagnose conditions or diseases, such as inflammatory responses. In the context of this disclosure, protein biomarkers or biomarkers encompass, for example, cytokines, chemokines, growth factors, other proteins, and peptides. In the context of this disclosure, nucleic acid biomarkers or biomarkers encompass, for example, mRNAs encoding cytokines, mRNAs encoding chemokines, mRNAs encoding growth factors, and mRNAs encoding other proteins. Examples of nucleic acid biomarkers 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 encompass both mRNA and miRNA.

[0049] As used herein, the term "mRNA" or "messenger RNA" refers to... in vitro , in vivo , In situ or In vitro mRNA is any polynucleotide that encodes at least one target peptide or polypeptide and can be translated to produce the encoded target peptide or polypeptide. mRNA can be isolated from tissues or cells using various methods. For example, total RNA can be extracted from cell-containing samples (such as canine epidermal samples), and the resulting extracted total RNA can be purified. For example, (On a column containing oligo-dT microspheres) to obtain extracted mRNA.

[0050] As used herein, the term "microRNA" or "miRNA" refers to an RNA interference (RNAi) agent with a length of about 21-23 nucleotides (nt). The length of miRNA can range from 18 to 26 nucleotides. Typically, miRNA is single-stranded. However, in some embodiments, miRNA may be at least partially double-stranded. In some embodiments, miRNA may contain an RNA double-stranded region (referred to herein as the "double-stranded region") and optionally also contain one or two single-stranded overhanging ends. In some embodiments, the RNAi agent contains a double-stranded region ranging in length from 15 to 29 bp and optionally also contains one or two single-stranded overhanging ends. miRNA may be formed from two RNA molecules hybridized together, or alternatively from a single RNA molecule including a self-hybridized portion. Typically, the free 5′ end of the miRNA molecule has a phosphate group, and the free 3′ end has a hydroxyl group. The double-stranded portion of the miRNA typically, but does not necessarily, contain one or more protrusions consisting of one or more unpaired nucleotides. One strand of the miRNA includes the portion that hybridizes with the target RNA. In some embodiments of the invention, one strand of the miRNA is not precisely complementary to a region of the target RNA, meaning that the miRNA hybridizes with the target RNA with one or more mismatches. In other embodiments of the invention, one strand of the miRNA is precisely complementary to a region of the target RNA, meaning that the miRNA hybridizes with the target RNA with zero mismatches. Generally, miRNAs are thought to mediate the repression of gene expression by inhibiting the translation of the target transcript. However, in some embodiments, miRNAs may mediate the repression of gene expression by inducing the degradation of the target transcript.

[0051] As used in this article, protein or nucleic acid ( For exampleThe term "statistically significant level" (mRNA or miRNA), "level of difference," or "differential expression" can include any elevated or decreased level in the tested dogs compared to a reference level of the protein or nucleic acid in healthy dogs, more particularly dogs without inflammatory conditions or diseases, and even more particularly dogs without dermatitis or diseases such as atopic dermatitis. In one embodiment, a statistically significant 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 higher. In another embodiment, a statistically significant level means a decrease 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%, or at least 100%. As used herein, the terms "food composition," "wet food composition," "complete wet food composition," or "formulated diet" encompass all foods, feeds, food supplements, or materials that may contain protein, fiber, carbohydrates, and / or fats, and are intended for use in an organism to maintain growth, repair, and life processes and to provide energy. Carbohydrates include NFE (nitrogen-free extract). Food compositions may also contain supplements or additives, such as flavoring agents, minerals, and vitamins. Food compositions according to this disclosure may consist of complete food compositions. In some specific embodiments, wet food compositions according to this disclosure may be complete wet food compositions.

[0052] As used herein, “complete nutrition” means that the compositions of this disclosure provide companion animals (such as cats or dogs) with a complete and balanced nutritional requirement. Specifically, a complete nutrition food composition may contain all known nutrients required by the intended recipient of the food composition, in appropriate amounts and proportions based on recommendations from, for example, recognized authorities in the field of animal nutrition. Thus, a nutritionally adequate diet is one that the companion animal can use as its sole food source and can sustain life without additional food (other than water). The complete nutrition food composition may also contain carriers, diluents, and / or excipients. Depending on the intended use, carriers, diluents, and / or excipients suitable for use in companion animals, particularly those such as cats and dogs, may be selected. Furthermore, the complete nutrition food composition may not cause significant changes in the companion animal, i.e., it will not cause significant changes in body weight and / or body condition score (BCS). Illustratively, the food composition described herein may include, but is not limited to, protein, fat, ash, fiber (TDF), carbohydrates, starch, calcium, phosphorus, sodium, chloride, sodium tripolyphosphate, potassium, magnesium, iron, water, copper, manganese, zinc, selenium, flavoring agents, vitamin A, vitamin D3, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin B7, vitamin B9, choline chloride, arachidonic acid, W3 fatty acid, or W6 fatty acid.

[0053] describe 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 or occurrence of a condition or disease, including skin conditions or diseases, especially inflammatory skin conditions such as atopic dermatitis.

[0054] Surprisingly, the applicant has discovered that reproducible qualitative and / or quantitative analysis of proteins and nucleic acids contained in canine skin samples can be performed using a method in which canine skin samples are collected by applying an adhesive carrier to a skin target area, thereby transferring skin material to the surface of the adhesive carrier, and then the presence and / or content of proteins and / or nucleic acids contained in the transferred skin material can be analyzed. In the art, methods for collecting skin material by applying an adhesive carrier to a skin target area are available. itself It is known and can also be called "tape peeling".

[0055] To the applicant's knowledge, the prior art has never disclosed or revealed the use of the tape peeling technique for analyzing the protein and / or nucleic acid content of canine skin samples, nor has it involved the analysis of protein and / or nucleic acid biomarker content in canine skin samples.

[0056] This disclosure relates to a method for detecting or measuring the levels of one or more selected protein biomarkers and / or one or more selected nucleic acid biomarkers in a canine skin sample, comprising the following steps: a) Apply adhesive tape to the exposed target area of ​​the dog, and collect epidermal samples adhering to the adhesive tape, and b) Detect the presence and / or measure the content of one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in the epidermal sample obtained in step a).

[0057] This disclosure relates to a method for detecting or measuring the content of one or more selected protein biomarkers in a canine skin sample, comprising the following steps: a) Apply adhesive tape to the exposed target area of ​​the dog, and collect epidermal samples adhering to the adhesive tape, and b) Detect the presence and / or measure the content of one or more selected protein biomarkers in the epidermal sample obtained in step a).

[0058] 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, comprising the following steps: a) Apply adhesive tape to the exposed target area of ​​the dog, and collect epidermal samples adhering to the adhesive tape, and b) Detect the presence and / or measure the content of one or more nucleic acid biomarkers in the epidermal sample obtained in step a).

[0059] This disclosure relates to a method for detecting or measuring the levels of one or more selected protein biomarkers and / or one or more selected nucleic acid biomarkers in a canine skin sample, comprising the following steps: a) Provide adhesive tape on which a sample of dog skin has been pre-collected and adhered to. b) Detect the presence and / or measure the content of one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in the epidermal sample.

[0060] This disclosure relates to a method for detecting or measuring the content of one or more selected protein biomarkers in a canine skin sample, comprising the following steps: a) Provide adhesive tape on which a sample of dog skin has been pre-collected and adhered to. b) Detect the presence and / or measure the content of one or more selected protein biomarkers in the epidermal sample.

[0061] 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, comprising the following steps: a) Provide adhesive tape on which a sample of dog skin has been pre-collected and adhered to. b) Detect the presence and / or measure the content of one or more selected nucleic acid biomarkers in the epidermal sample.

[0062] According to the above method, the nucleic acid biomarker is preferably composed of mRNA or miRNA biomarkers.

[0063] By definition, the protein biomarkers and nucleic acid biomarkers in this article are canine biomarkers.

[0064] In some embodiments of this method, one or more selected protein biomarkers indicate the physiological state of the test dog, encompassing the physiological state of the test dog's skin. 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 test dog, particularly biomarkers indicating the physiological state of the test dog's skin.

[0065] In some implementations of this method, one or more selected nucleic acid biomarkers ( For example, mRNA or miRNA This indicates the physiological state of the test dog, encompassing the physiological state of the test dog's skin. Therefore, according to these preferred embodiments, the one or more selected nucleic acid biomarkers consist of one or more nucleic acid biomarkers indicating the physiological state of the test dog, particularly biomarkers indicating the physiological state of the test dog's skin.

[0066] The examples in this paper demonstrate an illustrative validation of this canine skin sampling method in sensitized canines that induce an inflammatory response, or more precisely, an inflammatory response associated with an inflammatory immune response, thereby specifically mimicking the sensitization phenomenon in canine atopic dermatitis.

[0067] In the examples presented in this paper, both protein and nucleic acid biomarkers indicating a Th2-type immune response were increased in these sensitized canines. More specifically, this paper demonstrates that quantifying proteins in skin samples collected by tape peeling from dogs (whose skin inflammation was artificially induced through sensitization to an allergen) allows for the determination of interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-25 (IL-25), interleukin-33 (IL-33), and tumor necrosis factor-α (TNF-α) levels compared to control skin sites from the same unsensitized animal. The levels of [unclear - possibly IL-4 and IL-13] were significantly elevated, especially at sensitization sites. The combination of biomarkers (especially IL-4 and IL-13) was increased, and interferon-gamma (IFN-γ) was absent. The increase in ) actually indicates a Th2-driven rather than Th1-driven inflammatory response, the presence of which and its level are shown in this paper as determined in canine skin samples.

[0068] When used in this article, the name of the biomarker, For example Interleukin-4 (IL-4) is a protein biomarker. For example IL-4 protein, or the nucleic acid encoding the protein, For example mRNA encoding IL-4.

[0069] Regarding other nucleic acid biomarkers, particularly miRNA biomarkers for canine atopic dermatitis, these may be selected from miR-203 and miR-483 as non-limiting examples. These miRNAs are specifically described by Koury et al. (2019, PlosOne, Vol. 14(6): e0128670).

[0070] It also showed elevated expression levels of the corresponding genes encoding the protein biomarkers for increased skin content, as confirmed by the results of differential mRNA expression.

[0071] The applicant's findings are particularly surprising because canine skin differs significantly from human skin, especially in its thinner thickness. This thinner thickness reduces the likelihood that nucleic acids and proteins are properly preserved in undegraded forms within canine skin. Furthermore, there is no evidence in the prior art that the cells of hair follicles, their follicular sheaths, and follicular appendages attached to canine skin possess the same cell kinetics and / or chemokine synthesis capacity as keratinocytes in human skin, and therefore have the same nucleic acid expression. Moreover, given the higher hair density and significantly less skin per unit collection surface on canine skin, the probability of detectable concentrations of cytokines and / or chemokines and nucleic acids on canine skin, especially on adhesive tape, is also much lower.

[0072] In step a), the selected target area is first finely trimmed or shaved to expose the epidermis of the skin target area, and adhesive tape is applied to it. The selected area is gently trimmed or shaved to avoid wounds, cuts, abrasions or erosions in the trimmed or shaved selected skin target area.

[0073] In a particular implementation, the selected target area may be a lesion area of ​​the skin. In particular, the selected target area may be a skin area with lesions caused by skin conditions or diseases, especially inflammatory skin conditions such as atopic dermatitis.

[0074] In another specific implementation, the selected target area is not a lesion area of ​​the skin. This is especially likely to occur when the methods described herein are used as a preventative measure.

[0075] At step a), the exposed target skin area is preferably a skin area in which the stratum corneum has been removed prior to trimming or shaving to expose the underlying skin layer, particularly the underlying granular skin layer. This means that, according to the embodiment, the adhesive tape is applied to the surface of the granular layer at step a). In some embodiments, the removal of the stratum corneum can be achieved by gently and continuously applying the adhesive tape to the skin area until the skin layer beneath the stratum corneum is exposed, particularly until the underlying granular skin layer is exposed, thereby obtaining the desired target skin area. Illustratively, the target skin area can be exposed by applying the adhesive tape repeatedly to the selected skin area, or by continuously applying the adhesive tape to the selected skin area until the skin layer beneath the stratum corneum is exposed, most preferably until the underlying granular layer is exposed. Illustratively, in embodiments where the stratum corneum is removed by using adhesive tape, the adhesive tape is typically applied to the selected target skin area 3 to 50 times, such as 5 to 30 times. The same adhesive tape can be applied the desired number of times and / or multiple adhesive tapes can be used.

[0076] Therefore, in step a), the collected epidermal sample is a canine skin material sample belonging to the epidermal layer, which contains little or no material derived from the stratum corneum. Most preferably, the majority of the epidermal sample originates from the granular epidermal layer.

[0077] Step a) can be performed using known skin adhesive tapes, such as non-polar, flexible adhesive tapes, like flexible tapes with a rubber adhesive. For example, skin adhesive tapes selected from the following can be used: D-Squame® (polyacrylate adhesive; CuDerm, Dallas Tex.), Durapor, Sebutape™ (acrylic polymer film; CuDerm, Dallas, Tex.), Tegaderm™, Duct tape (333 Duct tape, Nashua tape products), Scotch® tape (3M Scotch 810, St. Paul, Minn.), Diamond™ (The Sellotape Company, Eindhoven, Netherlands), and Sentega™ (polypropylene tape; Sentega Eiketten BV, Utrecht, Netherlands). The adhesive tape can be selected from commonly used pressure-sensitive adhesives or those that cure rapidly on skin contents (such as cyanoacrylates). The adhesive tape can be placed on a flexible or solid backing to facilitate sampling.

[0078] At step a), the same adhesive tape may be repeatedly applied to and removed from the target skin area. In other illustrative embodiments, multiple new adhesive tapes are sequentially applied to the same target skin area. The individual tapes used for sampling the skin target area can then be combined into an extraction container for further processing, such as protein extraction or nucleic acid extraction, most preferably for mRNA and miRNA extraction.

[0079] In some embodiments, the collected epidermal samples adhered to the adhesive tape can be at least partially removed from the adhesive tape, thereby providing an epidermal sample for further use in step b) of the method.

[0080] In these implementations, step a) may include the following steps: a1) applying adhesive tape to a target area of ​​the dog’s exposed skin to collect an epidermal sample adhered to the adhesive tape, and a2) removing the collected epidermal sample adhered to the adhesive tape to provide an epidermal sample for analysis at step b).

[0081] In step a2), the epidermal sample may be removed according to methods well known in the art. Step a2) can typically be performed by extracting the collected material, such as by extraction with a suitable extraction buffer.

[0082] In step a2), the protein can be extracted from the epidermal sample adhered to the adhesive tape by any known method, such as by using a known water and ethanol buffer for further analysis at step b).

[0083] In step a2), nucleic acids, especially mRNA and / or miRNA, can allow the expression of nucleic acid biomarkers ( Right now The procedure for direct qualitative and quantitative evaluation of mRNA and miRNA biomarkers is performed from adhesive tape. Commercial kits such as the "RNeasy" RNA extraction kit and the "Sensiscript reverse transcriptase" kit sold by Quiagen (Valebcia, CA, USA), along with PCR primers and probes, can be used. Pre-developed assay reagents and Universal premixed solutions, which include all buffers and enzymes necessary for amplifying target nucleic acid biomarkers and subsequent fluorescence detection of specific cDNAs (Applied Biosystems, Foster City, CA, USA).

[0084] At step b), the detection of the presence or quantification of the one or more selected protein biomarkers can be performed according to any method known in the art. In some embodiments, the detection and / or quantification of the one or more selected protein biomarkers can be performed by an immunological method (such as the ELISA method disclosed in the examples) using antibodies against the protein to be detected and / or quantified.

[0085] At step b), the detection or quantification of the presence of one or more selected nucleic acid biomarkers, particularly mRNA or miRNA, can be performed according to any method known in the art. Typically, the detection and / or quantification of one or more selected nucleic acid biomarkers can be performed by the following steps: after reverse transcription of the mRNA or miRNA, if necessary, amplification of the target nucleic acid, preferably the target mRNA or miRNA, and then, as illustrated in the examples herein, detection and / or quantification of the amplified nucleic acid, for example, by using a suitably designed nucleic acid probe (which covers labeled nucleic acid probes).

[0086] The amplification of nucleic acid molecules can In vitro This is performed using biochemical methods known to those skilled in the art. The amplifying agent can be any compound or system, including enzymes, that will function to complete the synthesis of the primer extension product. It should be recognized that various amplification methodologies can be used to increase the copy number of the target nucleic acid in the nucleic acid skin sample obtained using the methods provided herein, both before and after detection. Suitable enzymes for this purpose include, for example, *Escherichia coli* (…). E. coli DNA polymerase I, Taq polymerase, E. coliKlenow fragment of DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, T4 or T7 RNA polymerases, polymerase mutants, reverse transcriptases, ligases, and other enzymes, including thermostable enzymes (i.e., those that extend primers after being subjected to sufficiently high temperatures to induce denaturation, or those that prepare aRNA from a DNA template using an RNA polymerase promoter, i.e., linearly amplified aRNA). One in vitro amplification method that may be used according to this disclosure is polymerase chain reaction (PCR) as described in U.S. Patent Nos. 4,683,202 and 4,683,195.

[0087] In some embodiments of step a) of the method, a first adhesive tape or multiple "first adhesive tapes" are applied for detection and / or quantification of one or more selected protein biomarkers at step a), and a second adhesive tape or multiple "second adhesive tapes" are applied for detection and / or quantification of one or more selected nucleic acid biomarkers (such as one or more selected mRNAs or miRNAs), regardless of the order in which the first adhesive tape and the second adhesive tape are applied.

[0088] In some implementations, the one or more selected proteins and / or nucleic acids canine biomarkers indicate the immune status of dogs.

[0089] Canine biomarkers of proteins and / or nucleic acids that indicate the immune status of dogs may include: (i) those indicating inflammatory responses, (ii) those indicating changes in the skin barrier, (iii) those indicating Th2 immune responses, (iv) those indicating Th1 immune responses, and (v) those indicating pruritus.

[0090] In some implementations, the one or more selected protein and / or nucleic acid canine biomarkers indicate inflammatory responses in dogs.

[0091] As a non-limiting example, canine biomarkers indicating inflammatory responses may be selected from the following protein or nucleic acid biomarkers: C-reactive protein (CRP), tumor necrosis factor (TNF), interleukin-1β (IL-1). Interleukin-6 (IL-6), interleukin-10 (IL-10), myeloperoxidase (MPO), matrix metalloproteinase (MMP), adiponectin, monocyte chemoattractant protein 1 (MCP-1), and CD40 ligand.

[0092] An inflammatory response can be identified when the level of at least one, preferably two or more, of the inflammatory biomarkers specified above is elevated in an epidermal sample.

[0093] In some implementations, one or more selected protein and / or nucleic acid canine biomarkers may be canine biomarkers indicating a canine Th2 immune response.

[0094] Canine biomarkers indicating Th2 response can be selected from the following protein or nucleic acid biomarkers: 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), especially interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13).

[0095] A Th2 response can be determined when the level of at least one, particularly two or more, of the Th2 biomarkers specified above is elevated in an epidermal sample. Alternatively, a Th2 response can be determined when the level of at least one Th2 biomarker is elevated and the level of at least one other biomarker is decreased in an epidermal sample. In some embodiments, one or more selected protein and / or nucleic acid canine biomarkers are canine biomarkers indicating a canine Th1 immune response.

[0096] Canine biomarkers indicating Th1 response can be selected from the following protein or nucleic acid biomarkers: interferon-gamma (IFN-γ) Interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) Interleukin-12 (IL-12), interleukin-1β (IL-1) Interleukin-18 (IL-18), interleukin-27 (IL-27), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), and T-bet (T-box transcription factor), especially IFN-γ. And IL-2.

[0097] A Th1 response can be determined when the level of at least one, and particularly two or more, of the Th1 biomarkers specified above is elevated in an epidermal sample. Alternatively, a Th1 response can be determined when the level of at least one Th1 biomarker is elevated and the level of at least one other biomarker is decreased in an epidermal sample.

[0098] In some implementations, one or more selected protein and / or nucleic acid canine biomarkers may be used as canine biomarkers to indicate itching in dogs.

[0099] Canine biomarkers indicating itching can be selected from the following protein or nucleic acid biomarkers: interferon-gamma (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 metalloproteinases, substance P, eosinophil cationic protein (ECP), and thymic stromal lymphopoietin (TSLP), IFN-γ IL-2, IL-31 and TSLP, especially IL-2.

[0100] Itching can be determined when the level of at least one, preferably two or more, of the pruritus biomarkers specified above is elevated in an epidermal sample.

[0101] In some implementations, one or more selected protein and / or nucleic acid canine biomarkers can indicate canine atopic dermatitis.

[0102] This disclosure also relates to determining the occurrence or likelihood of atopic dermatitis in dogs, comprising the following steps: a) providing an epidermal sample previously collected from an adhesive tape to which the sample is adhered, b) measuring the levels of two or more selected protein biomarkers and / or two or more nucleic acid biomarkers in the epidermal sample obtained at step a), wherein the biomarkers are selected from interleukin-1β (IL-1). Interleukin-4 (IL-4), Interleukin-10 (IL-10), Interleukin-13 (IL-13), Interleukin-25 (IL-25), Interleukin-31 (IL-31), Interleukin-33 (IL-33), Tumor Necrosis Factor α (TNF-α) ) and gamma interferon (IFN-γ) (c) and determine the occurrence or likelihood of canine atopic dermatitis in the dogs, wherein, most preferably, the content of the selected biomarker measured at step b) is increased compared to the content of the same biomarker in dogs not affected by skin inflammation.

[0103] In some implementations, one or more selected protein and / or nucleic acid canine biomarkers may be selected from proteins or nucleic acid biomarkers present in the epidermis.

[0104] In some implementations, the proteins present in the epidermis may be selected from: a) interleukin-1β (IL-1 (a) 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 α (TNF-α) ) and gamma interferon (IFN-γ) (c) thymic stromal lymphopoietin (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 α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0105] In some embodiments, the proteins present in the epidermis 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 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-1-1. IL-13, TNF-α IFN- TSLP and combinations thereof. In some embodiments, the proteins present in the epidermis also 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 α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0106] This disclosure also relates to a method for determining the presence of a skin condition in the skin of a dog, comprising the steps of: a) applying adhesive tape to a target area of ​​exposed skin of a dog to collect an epidermal sample adhered to the adhesive tape; b) detecting statistically significant differences in one or more protein and / or one or more nucleic acid biomarkers in the epidermal sample; and c) determining the presence of a skin condition when a statistically significant difference in one or more of the biomarkers is measured at step b).

[0107] As used in this article, "statistically significant protein levels" typically refers to the amount of a specific protein in a biological sample (here, in skin tissue, more precisely in the akeratinized canine epidermis) that deviates significantly from the amount considered normal or expected for a particular individual dog or canine population. Such abnormalities can indicate a variety of underlying symptoms, diseases, or physiological changes.

[0108] In some embodiments of the above method, the protein and / or nucleic acid biomarkers are selected from: a) interleukin-1β (IL-1 (a) 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 α (TNF-α) ) and gamma interferon (IFN-γ) (c) thymic stromal lymphopoietin (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 α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0109] In some embodiments, the protein and / or nucleic acid biomarkers 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 biomarkers comprise one or more protein 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 biomarkers comprise 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 biomarkers further include at least one protein and / or nucleic acid biomarker selected from the group consisting of: IL-1-1 IL-13, TNF-α IFN- TSLP and combinations thereof. In some embodiments, the protein and / or nucleic acid biomarkers further include 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 α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0110] In some embodiments of the above methods, the skin condition is an inflammatory condition, preferably selected from canine atopic dermatitis, pyoderma, contact dermatitis, folliculitis, acne, seborrheic dermatitis, panniculitis, cellulitis, licking granuloma (acral licking dermatitis, histiocytoma, eosinophilic granuloma complex), nasal photodermatitis (Collie nose), interdigital cysts and furuncles, ichthyosis, demodicosis (demodectic mange), tinea (dermatomycosis), and vasculitis.

[0111] In some embodiments of the above methods, the skin condition is a non-inflammatory condition, preferably selected from alopecia areata, color dilution alopecia, patterned alopecia, congenital sparse hair, hyperkeratosis, sebaceous gland inflammation, follicular dysplasia, dermoid sinus, X-linked ichthyosis, periodic follicular dysplasia, nasal plane hyperkeratosis, hereditary paw pad hyperkeratosis, primary seborrhea, vitamin A-reactive dermatitis, cutaneous mastocytosis, hypothyroidism-associated dermatitis, and canine epidermolysis bullosa.

[0112] This disclosure also relates to a method for diagnosing atopic dermatitis in the skin of dogs, comprising the steps of: a) applying adhesive tape to a target area of ​​exposed skin of a dog, thereby collecting an epidermal sample adhered to the adhesive tape; b) detecting statistically significant differences in one or more selected protein and / or nucleic acid canine biomarkers in the epidermal sample, particularly wherein the selected protein and / or nucleic acid biomarkers are selected from interleukin-1β (IL-1). Interleukin-4 (IL-4), Interleukin-10 (IL-10), Interleukin-13 (IL-13), Interleukin-25 (IL-25), Interleukin-31 (IL-31), Interleukin-33 (IL-33), Tumor Necrosis Factor α (TNF-α) ) and gamma interferon (IFN-γ) (b) and (c) when a statistically significant difference in one or more of the canine biomarkers of the protein and / or nucleic acid is measured at step (b), an atopic dermatitis state is determined.

[0113] In some embodiments of the above method, the canine protein and / or nucleic acid biomarker may be selected from: a) interleukin-1β (IL-1 (a) 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 α (TNF-α) ) and gamma interferon (IFN-γ) c) thymic stromal lymphopoietin (TSLP); and d) at least one canine biomarker of protein and / or nucleic acid selected from the group consisting of: tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0114] In some embodiments, the protein and / or nucleic acid canine biomarkers 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 biomarkers comprise one or more protein 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 biomarkers 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 protein and / or nucleic acid canine biomarkers further include at least one protein and / or nucleic acid canine biomarker selected from the group consisting of: IL-1-1 IL-13, TNF-α IFN- TSLP and combinations thereof. In some embodiments, the canine protein and / or nucleic acid biomarkers further include at least one canine 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 α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0115] This disclosure also relates to a method for determining the beneficial effects of a pharmaceutical formulation and / or a selected dietary regimen in dogs suffering from atopic dermatitis, comprising the steps of: a) applying adhesive tape to a target area of ​​exposed skin of the dog, thereby collecting an epidermal sample adhered to the adhesive tape; b) measuring the levels of one or more selected protein and / or nucleic acid canine biomarkers in the epidermal sample collected at step b); c) treating the dog with the pharmaceutical formulation and / or the selected dietary regimen; d) applying adhesive tape to a target area of ​​exposed skin of the dog, thereby collecting an epidermal sample adhered to the adhesive tape; e) detecting and / or measuring the levels of one or more selected protein and / or nucleic acid canine biomarkers in the epidermal sample collected at step d); and f) determining that the pharmaceutical formulation and / or the dietary regimen has a beneficial effect when the level of one or more of the biomarkers measured at step e) is higher and / or lower than the level of the same selected biomarker measured at step b).

[0116] In some embodiments, the above method steps a) (and d) may include: a1) (and d1) applying adhesive tape about 1 to about 50 times, preferably about 10 to about 30 times, to the exposed target area of ​​the dog's skin 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 used in step a1) to the exposed target area of ​​the dog's skin to collect epidermal samples adhered to the adhesive tape.

[0117] In some embodiments of the above method, in each of steps a) and d), adhesive tape is applied under constant pressure.

[0118] According to some implementation schemes of the above method, the canine protein and / or nucleic acid biomarkers may be selected from: a) interleukin-1β (IL-1 (a) 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 α (TNF-α) ) and gamma interferon (IFN-γ) c) thymic stromal lymphopoietin (TSLP); and d) at least one canine biomarker of protein and / or nucleic acid selected from the group consisting of: tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0119] In some embodiments, the protein and / or nucleic acid canine biomarkers 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 biomarkers comprise one or more protein 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 biomarkers 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 protein and / or nucleic acid canine biomarkers further include at least one protein and / or nucleic acid canine biomarker selected from the group consisting of: IL-1-1 IL-13, TNF-α IFN- TSLP and combinations thereof. In some embodiments, the canine protein and / or nucleic acid biomarkers further include at least one canine 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 α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

[0120] In some embodiments of the methods described above for determining the beneficial effects of a pharmaceutical preparation and / or a selected dietary regimen in dogs suffering from atopic dermatitis, step c) comprises treating the dog with the pharmaceutical preparation. The pharmaceutical preparation may be selected from antihistamines, corticosteroids, cyclosporine, olaratinib, logivisumab, and immunotherapy through gradual exposure to a small amount of allergen.

[0121] In some other embodiments of the methods described above for determining the beneficial effects of pharmaceutical preparations and / or selected dietary regimens in dogs with atopic dermatitis, the dietary regimen may be selected from formula foods containing hydrolyzed protein, grain-free foods, omega-3 supplemented formula foods, antioxidant-rich formula foods, pantothenic acid supplemented formula foods, choline supplemented formula foods, nicotinamide supplemented formula foods, histidine supplemented formula foods, zinc supplemented formula foods, copper supplemented formula foods, flavonoid supplemented formula foods, terpene supplemented formula foods, and inositol supplemented formula foods.

[0122] In all methods of this disclosure, the detection of the presence of the selected protein biomarker and / or the measurement of its content at step b) can be performed by immunological methods.

[0123] In all methods disclosed herein, one or more selected nucleic acid biomarkers may be selected from mRNA and miRNA.

[0124] In all methods of this disclosure, the detection of the presence and / or measurement of the content of the selected mRNA or miRNA biomarker at step b) can be performed by nucleic acid amplification methods.

[0125] This disclosure also relates to a component kit for detecting and / or measuring protein and / or nucleic acid biomarkers in canine skin samples, comprising one or more adhesive tapes for peeling skin according to this specification.

[0126] The present invention also relates to a component kit for detecting and / or measuring canine biomarkers of proteins and / or nucleic acids in canine epidermal samples, comprising: a) one or more adhesive tapes for peeling skin according to the present specification, b) reagents for collecting epidermal samples adhered to the surface of the 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) optionally, a suggestion for using the component kit.

[0127] In embodiments where the combo kit includes reagents for detecting and / or quantifying one or more protein biomarkers, the combo kit may include one or more biomarker-directed antibodies, and optionally means for detecting the binding of one or more biomarker-directed antibodies to their respective target proteins in an epidermal sample initially adhered to the adhesive tape.

[0128] In embodiments where the combo kit includes reagents for detecting and / or quantifying one or more nucleic acid biomarkers, most preferably for detecting and / or quantifying one or more mRNA or miRNA biomarkers, the combo kit may include RNA extraction reagents, reverse transcriptase, DNA polymerase, and optional primers and probes for the mRNA or miRNA biomarker, the abnormal expression of which is associated with skin disease or pathological skin conditions. Additionally, the combo kit may include primers and probes for a control gene (such as a housekeeping gene). Primers and probes for the control gene can be used, for example, for normalization calculations. The combo kit may also include instructions for performing tape peeling and analyzing gene expression using normalization calculations. In some embodiments of the combo kit, it may include probes that bind to a given nucleic acid biomarker, most preferably a portion of a given mRNA or miRNA biomarker. In yet another embodiment, the combo kit may include a microarray containing one or more nucleic acid probes that can specifically hybridize with the amplification product of a selected nucleic acid biomarker, most preferably with the mRNA or miRNA biomarker according to this disclosure. In some embodiments, multiple reagents may be provided in the combination kit of the present invention, of which only some are intended to be used together in a particular reaction or procedure. For example, multiple primers may be provided, of which only two are required for a particular application.

[0129] This disclosure is further illustrated by the examples below, but is not limited to those examples in any way.

[0130] Example This disclosure may be further illustrated by the following examples of its particular embodiments, but it should be understood that these examples are included for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0131] Example 1: Determining the occurrence of atopic dermatitis in dogs A. Materials and Methods A.1. Canine Program Eight healthy beagles were stripped of tape and subjected to treatment of dust mites on their skin twice a week for 49 days. Dermatophagoides farinae The extract was sensitized. Control sites received only carrier treatment. Standardized pressure (225 g / cm³) was applied using a DSquame® pressure gauge (DS500, Monaderm, France). -2 Apply adhesive tape strips of D-Squame® (DS 100, Monaderm, France) to the skin. A.2. ELISA assay The commercial ELISA kits used in the assay are listed in Table 1 at the end of the instruction manual.

[0132] These assays employed a quantitative sandwich enzyme immunoassay technique. Monoclonal antibodies specific to a particular cytokine were pre-coated onto microplates. Standards, controls, and samples were pipetted into the wells, and any present cytokines were bound by the immobilized antibodies. After washing away any unbound material, biotinylated mouse monoclonal antibodies specific to the tested cytokine were added to the wells. After washing to remove any unbound antibodies, streptavidin conjugated with HRP was added to the wells. Subsequent washing to remove any unbound streptavidin-HRP was followed by adding the substrate solution to the wells. The enzymatic reaction produced a blue product, which turned yellow upon addition of a stop solution. The measured color intensity was proportional to the amount of cytokine bound in the initial step. Sample values ​​were then read from a standard curve.

[0133] A.3. Transcriptome assay Total RNA was extracted via promoter extraction, and these samples were subjected to deep sequencing (strand-specific library construction, 2x150bp paired-end sequencing). This document reports the quality control, preprocessing, and statistical analysis performed on this dataset.

[0134] RNA samples were quantified using a Qubit 4.0 fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using an RNA kit on an Agilent 5600 fragment analyzer (Agilent Technologies, Palo Alto, CA, USA).

[0135] RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Preparation Kit for Illumina (New England Biolabs, Ipswich, MA, USA) following the manufacturer's instructions. In short, mRNA was initially enriched using Oligod(T) microspheres. The enriched mRNA was fragmented at 94°C for 15 min. First- and second-strand cDNAs were then synthesized. The cDNA fragments underwent end repair and 3' adenylation, and universal adapters were ligated into the cDNA fragments. Indexing and library enrichment were then performed using finite-cycle PCR. The sequencing libraries were validated using a DNA kit on an Agilent 5600 fragment analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantification was performed using a Qubit 4.0 fluorometer (Invitrogen, Carlsbad, CA).

[0136] Sequencing libraries were multiplexed and clustered on a flow cell. After clustering, the flow cell was loaded onto the Illumina NovaSeq 6000 instrument according to the manufacturer's instructions. Samples were sequenced using a 2x150 paired-end sequencing (PE) configuration. Image analysis and base identification were performed on the NovaSeq instrument using NovaSeq control software v1.6. The raw sequence data (.bcl files) generated by Illumina NovaSeq were converted to fastq files and demultiplexed using Illuminabcl2fastq program version 2.20. One mismatch was allowed for index sequence identification.

[0137] A.4. Correlation between ELISA and transcriptome assay results The aim was to study the sensitizing effect of antigens on canine skin and to compare ELISA data with transcriptomics data.

[0138] As disclosed in §A.2 above, generate Elisa data.

[0139] As disclosed in §A.3 above, transcriptomics data are generated.

[0140] Nine cytokines (IFN-γ, IFN-γ, IFN-γ) were measured. (IL-10, IL-13, IL-1B, IL-25, IL-33, IL-4; TNF and TSLP). Their names are used to identify the corresponding ENSEMBL IDs, listed in Table 2a below. Table 2a: List of protein content / gene expression comparisons For the reference canine assembly version used in transcriptome analysis, the IL-25 gene was not included in ENEMBL v105. A query for canine IL-25 in NCBI returned an entry for another ENEMBL gene named CMTM5. Therefore, this ENEMBL id was used for IL-25.

[0141] Using the original filtering procedure, transcripts encoding the cytokines IFNG, IL-25 / CM5, IL-33, and IL-4 were considered to be of low expression and filtered out from the transcriptome dataset. They were recovered for this analysis.

[0142] A.5. Correlation between genes or proteins and canine clinical responses and / or predefined cytokines The aim was to study the sensitizing effect of antigens on canine skin and to compare clinical responses with transcriptomic data.

[0143] As disclosed in §A.3 above, transcriptomics data are generated.

[0144] The association between 19 proteins (CCL17, CCL20, CCL22, CCL5, CD2, CD3E, ELOVL3, FA2H, FCER1A, ICOS, IL13, IL17RB, IL1B, IL1RL2, IL22, IL36B, IVL, RORA, and TNFRSF4) and clinical response was evaluated. Their names were used to identify the corresponding ENSEMBL IDs and are listed in Table 2b below. Table 2b: List of compared protein content / gene expression A preliminary analysis was provided, taking into account the Spearman correlation coefficients between each of the transcriptomic data and the clinical response.

[0145] Then, for each ELISA protein, heatmaps were created for the 100 genes with the highest Pearson correlation. ELISA signal data were first transformed using the formula “log2(1+signal value)” (IL1B was removed due to its average signal value). Only genes with at least a 2-fold transcriptome variation were considered.

[0146] The above process was applied to transcriptomic signals from CCL17, CCL5, and CCL20.

[0147] B. Result B.1. Correlation between ELISA and transcriptome assay results The results of the presence and content of each of the selected protein biomarkers and gene biomarkers are summarized in Table 3 at the end of the instruction manual.

[0148] 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, for each cytokine, Table 3 compares the values ​​observed by ELISA with those observed by transcriptomics (transcriptomics values ​​are obtained by normalizing the original counts with the sample size factor estimated by SESeq2).

[0149] In Table 3, each row corresponds to one dog being tested.

[0150] In Table 3, each column corresponds to a selected biomarker, which is indicated at the bottom of the column.

[0151] Table 3 is explained in detail below based on the readings from left to right.

[0152] For each of the sensitized dogs, comments on the sensitized visual skin lesions are indicated in the second column of Table 3.

[0153] In each table cell containing the results, (i) The upper left of the cell indicates the dynamic changes in the protein levels specified in that column. (ii) The upper right corner of the cell indicates the dynamic changes in the mRNA level of the protein specified in that column, where "-" corresponds to no change, "+" corresponds to a small increase in protein / mRNA level, and "++" corresponds to a large increase in protein / mRNA level; and (iii) The symbols centered at the bottom of each cell indicate: for lesions, there is a correlation between protein levels and corresponding mRNA levels (check mark); for lesions, there is no correlation between protein levels and corresponding mRNA levels (cross mark); or for lesions, there is a certain degree of correlation between protein levels and corresponding mRNA levels (approximately equal sign).

[0154] On day 49, these adhesive tape strips were subjected to ELISA and transcriptomic analysis to investigate inflammatory cytokines and mRNAs in the epidermis. ELISA showed that on day 49, compared with the control site, the concentrations of interleukins IL-4, IL-13, IL-33, IL-25, and TNF-α were significantly increased in the sensitized site (Friedman test, p<0.05). Compared with D0, the concentrations of IL-4, IL-13, IL-33, IL-25, TNF-α, IFN-α, and TSLP were significantly increased after sensitization (p<0.05). IL-1β and IL-10 were not detected. Differentially expressed mRNAs were selected by changes in gene expression levels (screening criteria: fold 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 altered by these gene dysregulations are cell adhesion, immune response, and cell migration, which perfectly match the overall immune response generated during the sensitization process.

[0155] B2: Further results obtained from the two dogs To solidify the findings, further analysis has been conducted. The results obtained from two dogs representing the test group are presented below.

[0156] B2.1: Correlation between ELISA and clinical signs The results showed a good correlation between ELISA and clinical signs, thus demonstrating a good correlation between the genes to be analyzed and the canine response. Figure 1 and Figure 2 This indicates the TNF (tumor) obtained from two different dogs (dog 5 and dog 6). Figure 1 ) and IL13 ( Figure 2 The results showed that ELISA was associated with clinical response (dog 5 was a highly effective responder, and dog 6 was a moderate responder).

[0157] B2.2: Correlation between ELISA (protein) and its own genes The results obtained indicate that for the two dogs, No. 5 and No. 6, there is a good correlation between the ELISA results and the mRNA of the relevant proteins / cytokines. Figure 3 and Figure 4 This indicates the TNF ( ) obtained from the above-mentioned dogs. Figure 3 ) and IL13 ( Figure 4 ) results B2.3: The correlation between other Th2 or Th1 genes and canine responses.

[0158] The results indicate that there is a strong correlation between other Th2 or Th1 genes and canine responses. Figures 5 to 11 The gene TNFRSF4 ( Figure 5 ), FA2H ( Figure 6 ), IL1B ( Figure 7 ), IL13RA1 ( Figure 8 ), IL13RA2 ( Figure 9 ), IL22 ( Figure 10 ) and IL4R ( Figure 11 These results.

[0159] In short, DSquame® appears to be a measure of... stratum corneum Appropriate methods for analyzing cytokine concentrations and mRNA profiling. Despite known individual variability, mRNA analysis has shown a correlation with the Th2 response generated during the sensitization procedure. Table 1: ELISA Kit Reference Information Bibliography Clausen et al., 2020, Nature Research. 10 : 21895 Dyjack et al., 29, J Allergy Clin Immunol, Vol. 141 (4) : 1298-1309 Morlang et al., 2021, Vet Dermatol, Vol. 32 : 331 – e92 Koury et al., 2019, PlosOne, Vol. 14 (6) : e0128670 * * * Although the subject matter and advantages of this disclosure have been described in detail, it should be understood that various changes, substitutions, and alterations may be made herein without departing from the spirit and scope of this disclosure. Furthermore, the scope of this application is not intended to be limited to specific embodiments of the processes, machines, articles, and substances, compositions, apparatuses, methods, and steps described in the specification. Those skilled in the art will readily understand from the disclosure of the subject matter that existing or future-developed processes, machines, articles, substances, compositions, means, methods, or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized. Therefore, the appended claims are intended to include such processes, machines, articles, substances, compositions, apparatuses, methods, or steps within their scope. Various patents, patent applications, publications, product specifications, schemes, and serial number references are incorporated herein by reference in their entirety for all purposes.

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, comprising the following steps: a) Apply adhesive tape to the exposed skin target area of ​​the dog, thereby collecting epidermal samples adhering to the adhesive tape, and b) Detect the presence and / or measure the content of one or more selected protein biomarkers and / or one or more nucleic acid biomarkers in the epidermal sample obtained in step a).

2. The method of claim 1, wherein at step b), the one or more selected protein and / or nucleic acid canine biomarkers indicate the canine immune status.

3. The method of claim 1, wherein at step b), the one or more selected protein and / or nucleic acid canine biomarkers are canine biomarkers indicating inflammatory responses in dogs.

4. The method of claim 1, wherein at step b), the one or more selected protein and / or nucleic acid canine biomarkers are canine biomarkers indicating a canine Th2 immune response.

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

6. The method of claim 5, wherein the one or more selected proteins present in the epidermis are selected from: a) Interleukin-1β (IL-1) 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 α (TNF-α) ) and gamma interferon (IFN-γ) ); c) Thymic stromal lymphopoietin (TSLP); and / or d) 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 α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

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

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

9. The method of claim 8, wherein the detection of the presence and / or measurement of the content of the selected mRNA or miRNA biomarker at step b) is performed by a nucleic acid amplification method.

10. A method for determining the presence of a skin condition in a dog's skin, comprising the following steps: a) Apply adhesive tape to the exposed target area of ​​the dog, thereby collecting epidermal samples adhered to the adhesive tape. b) Detect the statistically significant differences in one or more protein and / or one or more nucleic acid canine biomarkers in the epidermal sample. c) When a statistically significant difference in one or more of the biomarkers is measured at step b), a skin condition is determined to be present.

11. The method of claim 11, wherein the skin condition is an inflammatory condition, preferably selected from canine atopic dermatitis, pyoderma, contact dermatitis, folliculitis, acne, seborrheic dermatitis, panniculitis, cellulitis, licking granuloma (acromial licking dermatitis, histiocytoma, eosinophilic granuloma complex), nasal photodermatitis (Collie nose), interdigital cysts and furuncles, ichthyosis, demodicosis (demodectic mange), tinea (dermatomycosis), and vasculitis.

12. The method of claim 10, wherein the skin condition is a non-inflammatory condition, preferably selected from alopecia areata, color dilution alopecia, patterned alopecia, congenital sparse hair, hyperkeratosis, sebaceous gland inflammation, follicular dysplasia, dermoid sinus, X-linked ichthyosis, periodic follicular dysplasia, nasal plane hyperkeratosis, hereditary paw pad hyperkeratosis, primary seborrhea, vitamin A-reactive dermatitis, cutaneous mastocytosis, hypothyroidism-associated dermatitis, and canine epidermolysis bullosa.

13. A method for diagnosing atopic dermatitis in the skin of dogs, comprising the following steps: a) Apply adhesive tape to the exposed skin target area of ​​the dog, thereby collecting epidermal samples adhered to the adhesive tape. b) Detect the statistically significant differences in one or more canine biomarkers of proteins and / or nucleic acids in the epidermal sample. c) When a statistically significant difference in one or more of the canine biomarkers of the protein and / or nucleic acid is measured at step b), an atopic dermatitis state is determined.

14. The method of any one of claims 10 to 13, wherein the canine protein and / or nucleic acid biomarkers are selected from: a) Interleukin-1β (IL-1) 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 α (TNF-α) ) and gamma interferon (IFN-γ) ); c) Thymic stromal lymphopoietin (TSLP); and d) One or more canine biomarkers of proteins and / or nucleic acids selected from the group consisting of: tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

15. A method for determining the beneficial effects of a pharmaceutical preparation and / or a selected dietary regimen in dogs suffering from atopic dermatitis, comprising the following steps: a) Apply adhesive tape to the exposed skin target area of ​​the dog, thereby collecting epidermal samples adhered to the adhesive tape. b) Measure the levels of one or more selected protein and / or nucleic acid canine biomarkers in the epidermal sample collected at step b). c) Treat the dogs with medication and / or with a selected diet regimen. d) Apply adhesive tape to the exposed skin target area of ​​the dog, thereby collecting epidermal samples adhering to the adhesive tape. e) Detect and / or measure the levels of one or more selected canine protein and / or nucleic acid canine biomarkers in the epidermal sample collected in step d). f) When the level of one or more of the biomarkers measured at step e) is higher and / or lower than the level of the same selected biomarker measured at step b), the pharmaceutical preparation and / or the dietary regimen are determined to have a beneficial effect.

16. The method of claim 15, wherein steps a) and d) comprise: a1) (and d1) apply 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) Apply an adhesive tape different from the adhesive tape of step a1) to the exposed skin target area of ​​the dog, thereby collecting epidermal samples adhered to the adhesive tape.

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

18. The method of any one of claims 15 to 17, wherein the canine protein or nucleic acid biomarker is selected from: a) Interleukin-1β (IL-1) 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 α (TNF-α) ) and gamma interferon (IFN-γ) );and c) Thymic stromal lymphopoietin (TSLP); and d) One or more canine biomarkers of proteins or nucleic acids selected from the group consisting of: tumor necrosis factor superfamily member 4 (TNFRSF4), fatty acid 2-hydroxylase (FA2H), interleukin-13 receptor subunit α1 (IL13RA1), interleukin-13 receptor subunit α2 (IL13RA2), interleukin-22 (IL-22), interleukin-4 receptor (IL-4R), and combinations thereof.

19. The method of any one of claims 1 to 18, wherein the one or more selected 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 component kit for detecting and / or measuring canine biomarkers of proteins and / or nucleic acids in canine epidermal samples, comprising: a) One or more adhesive tapes used for peeling skin off. b) Reagents for collecting epidermal samples adhering to the surface of adhesive tape. c) Reagents for the detection and / or quantification of one or more protein biomarkers, and / or d) Reagents for the detection and / or quantification of one or more nucleic acids, particularly for the detection and / or quantification of one or more mRNAs or miRNAs. e) Optional, use the recommended component kit.

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