Methods and compositions for identification of diagnostically and therapeutically relevant proteins

Abstract

The present disclosure generally relates to compositions, methods, and kits for the identification of diagnostically and therapeutically relevant proteins. In some instances, the identification of the diagnostically and therapeutically relevant proteins can be used to aid in treatment of a subject in need thereof.

Description

[0001] METHODS AND COMPOSITIONS FOR IDENTIFICATION OF DIAGNOSTICALLY AND THERAPEUTICALLY RELEVANT PROTEINS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 579,364, filed August 29, 2023; and U.S. Provisional Patent Application No. 63 / 594,532, filed October 31, 2023, the disclosures of each of which are hereby incorporated herein by reference their entireties.

[0004] BACKGROUND

[0005] While common inflammatory skin conditions such as atopic dermatitis (eczema) and psoriasis can often be diagnosed clinically, there are certain cases in which this distinction is challenging. In such cases, making the correct diagnosis is often the key to successful therapy, particularly in the current era of highly effective targeted biologic and small molecule therapeutics. Further within each diagnosis of atopic dermatitis and psoriasis, there are different immunologic disease endotypes in which the underlying immunologic drivers are different from other endotypes. Choosing the wrong medication can lead to suboptimal clinical responses or even worsening of the underlying condition.

[0006] For instance, fungal infections of the skin are very common and account for over 4 million medical visits in the U.S. yearly. However, other inflammatory skin conditions are many times misdiagnosed as fungal infections, or vice versa. Oftentimes, patients with inflammatory skin conditions are initially treated with antifungal s, which could represent an inappropriate treatment for the patient’s skin condition. As an additional concern, skin lymphomas may often mimic psoriasis and eczema. If patients are treated with biologies for psoriasis and eczema, the underlying lymphoma may worsen.

[0007] Currently, though it is possible to culture skin samples for fungus, it is challenging to culture fungus in the microbiology lab. Also, culture-based fungal tests often takes weeks to come back, and as such this type of test is not generally useful for treatment decisions in a clinically reasonable timeframe. Another option is to scrape a portion of scale from a patient, place it on a slide, stain it with potassium hydroxide (KOH), and then look under the microscope for signs of fungus. While this test is inexpensive, the test is highly user dependent and without significant training, the test is challenging to perform reliably. Additionally, the test also requires that practices using the test to maintain a license to do so, and the cost of the license can be prohibitive in some instances.

[0008] At present, there is no available tool for personalized medicine based on non-invasive immunologic profiling of skin in dermatology. As such, there is a need in the field for the diagnosis and selection of therapy for individuals with inflammatory skin disease using non- invasive immunologic profiling of skin in dermatology.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0011] FIG. 1 presents a schematic representation of a process workflow for collecting and analyzing a sample comprising one or more analytes in accordance with the present disclosure.

[0012] FIG. 2 presents a schematic representation of the contents of a test kit for identifying diagnostically and therapeutically relevant proteins in accordance with the present disclosure.

[0013] FIG. 3 presents a schematic representation of an indicator card for analyzing a sample in accordance with the present disclosure.

[0014] FIG. 4 presents a graphical representation of IL-13 levels in various different skin samples. NL - control sample; AD - atopic dermatitis; Ps - Psoriasis.

[0015] FIG. 5 presents a graphical representation of sample analysis performed with samples collected from patients with atopic dermatitis and patients with psoriasis.

[0016] FIG. 6 presents a graphical representation of interferon gamma and interferon gamma receptor 2 protein levels in non-responders (black dots) and responders (white dots).

[0017] FIG. 7 presents a flow diagram of patient diagnosis with a focus on unmet needs concerning the diagnosis of skin conditions such as psoriasis, eczema, and fungal infections.

[0018] FIG. 8 presents a graphical representation of the IL17A:IL13 ratio of samples derived from various different subjects (control, atopic dermatitis, psoriasis).

[0019] FIG. 9 presents a graphical representation of the immunologic heterogeneity within each diagnosis of the subjects of FIG. 8. FIG. 10 presents a flow diagram highlighting the challenges of misdiagnosing new psoriasis and eczema cases.

[0020] FIG. 11 presents a flow diagram presenting potential cost savings that could be obtained by using the methods and compositions described herein.

[0021] FIG. 12 presents a comparative table presenting the advantages of the methods and compositions described herein (referred to as “Dermcyf ’) as compared to other available methods of sample analysis.

[0022] FIG. 13A-FIG. 13F present images and schematics related to a histologic and clinical overview of noninvasive, detergent based epidermal sampling (NIDBES). FIG. 13 A presents a representative image of H&E stain showing sampled and non-sampled epidermis (arrow denotes transition point, 40X). FIG. 13B presents a representative image of H&E stain (arrow denotes transition point, 200X). FIG. 13C presents a representative image of PAS stain (arrow denotes transition point, 200X). FIG. 13D presents representative images of a psoriasis lesion before and after NIDBES. FIG. 13E presents representative images of the clinical course of healthy control skin after NIDBES. FIG. 13F presents a schematic of an overview of an approach utilizing NIDBES and downstream proteomics. H&E, hematoxylin & eosin; PAS, periodic acid-Schiff.

[0023] FIG. 14 presents data and results related to proof-of-concept detection of recombinant IL- 13. Measured levels of IL-13, IL-17A, and IFN-y in ex-vivo mammoplasty skin following superficial dermal injection of 1 mL of 1000 pg / mL solution of recombinant IL-13 incubated for 1 hour at 37°C. Epidermis was collected via NIDBES and proteins were measured using a human cytokine / chemokine protein array 71-plex panel. IL-17A and IFN-y serve as controls. IL, interleukin; IFN, interferon.

[0024] FIG. 15A-FIG. 15F present data and results related to a comparison of the proteomes of psoriasis and eczema. FIG. 15A presents a volcano plot of differentially expressed proteins between psoriasis and eczema measured using Explore 3072. FIG. 15B presents a volcano plot of differential upstream regulator proteins between psoriasis and eczema as determined via pathway analysis. FIG. 15C presents a volcano plot of differentially expressed proteins between psoriasis and eczema as measured using Target 48. FIG. 15D presents a scatterplot of psoriasis and eczema samples on PCI and PC2 after PC A using the top 10 DEPs in each diagnosis. FIG. 15E presents the composition of clusters identified via gaussian mixtures modeling performed on PCI and PC2. FIG. 15F presents a biplot derived from PCA using the top 10 DEPs in psoriasis and eczema. LogFC, log base 2 fold-change; -Log(FDR), negative log base 10 of false discovery rate; PC, principal component; PCA, principal component analysis; DEP, differentially expressed protein.

[0025] FIG. 16A-FIG. 16B present data and results related to cytokine concentration correlation between Explore 3072 and Target 48. Correlation plots of IL-13 (FIG. 16A) and IL-17A (FIG. 16B) as measured using Explore 3072 and Target 48 are presented. IL, interleukin.

[0026] FIG. 17A-FIG. 17B present data and results related to Bland-Altman analysis of Explore 3072 and Target 48. Bland-Altman plots of IL-13 (FIG. 17A) and IL-17A (FIG. 17B) as measured using Explore 3072 and Target 48 are presented. IL, interleukin.

[0027] FIG. 18 presents data and results related to principle component analysis and gaussian mixtures-based clustering of psoriasis and eczema samples. Visualization of gaussian mixtures- based clusters of psoriasis and eczema samples on PCI and PC2 after PCA using the top 10 DEPs in each diagnosis are presented. PC, principal component; PCA, principal component analysis; DEP, differentially expressed protein.

[0028] FIG. 19A-FIG. 19B present data and results related to additional principal component analysis of psoriasis and eczema samples. FIG. 19A presents a scatterplot of psoriasis and eczema samples on PCI and PC2 after PCA using all measured proteins. FIG. 19B presents a scatterplot of psoriasis and eczema samples on PCI and PC2 after PCA using only cytokines and chemokines. PC, principal component; PCA, principal component analysis.

[0029] FIG. 20 presents data and results related to UMAP analysis of psoriasis and eczema samples. UMAP projection of psoriasis and eczema samples using the top 10 DEPs in each diagnosis are presented. DEP, differentially expressed protein; UMAP, uniform manifold approximation and projection.

[0030] FIG. 21 presents data and results related to NOS2 and CCL17-based separation of psoriasis and eczema which identifies low NOS2 psoriasis and high NOS2 eczema subtypes. A scatterplot of eczema and psoriasis samples as a function of NOS2 and CCL17 expression is presented.

[0031] FIG. 22 presents data and results related to logistic regression classification of psoriasis and eczema. A scatterplot of psoriasis and eczema samples on PC 1 and PC2 after PCA using PI3, IL-12B, CCL17, CCL2, CXCL13 is presented. Samples are colored by their logistic regression classification, which agreed with the clinical diagnosis in all cases. PC, principal component; PCA, principal component analysis.

[0032] FIG. 23A-FIG. 23E present data and results related to evaluation of immunologic heterogeneity in psoriasis samples. FIG. 23A presents representative clinical and H&E histological images from low NOS2 psoriasis patients (40X), FIG. 23B presents a volcano plot of differentially expressed proteins between low NOS2 and high NOS2 psoriasis patients. FIG. 23C presents a scatterplot of LR classification of low NOS2 and high NOS2 psoriasis on PCI and PC2 after PCA using NOS2 and CCL17, FIG. 23D presents a heatmap of all measured cytokines after hierarchical unsupervised clustering. FIG. 23E presents a heatmap of all measured chemokines after hierarchical unsupervised clustering. LogFC, log base 2 fold-change; - LogFDR, negative log base 10 of false discovery rate; LR, logistic regression; PC, principal component; PCA, principal component analysis; GT, guttate.

[0033] FIG. 24A-FIG. 24B present data and results related to differences in treatment relevant cytokines and select chemokines between NOS2 low and high psoriasis. FIG. 24A presents boxplots of IL-17A, IL-17F, IL-12B, and IL-36A. FIG. 24B presents boxplots of IFN-y, CCL17, CCL18, and CCL22. * P<0.05, ** P<0.01, *** P<0.001, N.S. not significant. Cont, Control; lowNOS2, low NOS2 psoriasis; PsO, psoriasis.

[0034] FIG. 25A-FIG. 25D present data and results related to evaluation of additional cytokine and chemokine heterogeneity in psoriasis samples. FIG. 25A presents boxplots of IL-17C, IL36G, IL-12, and IL6. FIG. 25B presents boxplots of IL-4, IL-13, IL-22, IL-31. FIG. 25C presents boxplots of IL-1B, IL-14, IL-16, and IL-18. FIG. 25D presents boxplots of IL-19, IL-20, IL-25, and IL-32. * P<0.05, ** P<0.01, *** P<0.001, N.S. not significant. Cont, Control; lowNOS2, low NOS2 psoriasis; PsO, psoriasis.

[0035] FIG. 26A-FIG. 26D present data and results related to evaluation of immunologic heterogeneity in eczema samples and correlation with response to therapy. FIG. 26A presents a heatmap of all measured cytokines after hierarchical unsupervised clustering (responder samples highlighted in blue, non-responders in red). FIG. 26B presents a scatterplot of all eczema samples on PCI and PC2 after PCA using all measured proteins. FIG. 26C presents a scatterplot of LR classification of responders vs non-responders to IL- 13 blockade on PCI and PC2 after PCA using CXCL9, CXCL14, IFN-y, and IL-9. FIG. 26D presents a heatmap of CXCL9, CXCL14, IFN-y, and IL-9 in responders and non-responders after hierarchical unsupervised clustering. PC, principal component; GT, guttate.

[0036] FIG. 27A-FIG. 27B present clinical images of representative non-responders (FIG. 27A) and responders (FIG. 27B) to IL- 13 blockade.

[0037] DETAILED DESCRIPTION

[0038] Diagnosis of various different diseases and disorders often relies on clinical and histopathologic morphology without consideration of immune signals. Routine clinical methods including histopathology can be unable to distinguish among disease endotypes. Selection of therapy, thus, can occur by trial and error without consideration of immune signals and disease endotypes.

[0039] In dermatology, the use of targeted injectable biologic agents that inhibit the activity of specific cytokines that are key to the pathophysiology of inflammatory skin diseases are becoming more prevalent. In some instances, there are multiple different agents that target distinct cytokines based on each diagnosis. Within each diagnosis of eczema and psoriasis, distinct immunologic endotypes, driven by distinct cytokines, exist. At the present time, decisions on which agent to use are made based on comorbidities and often insurance approval. Importantly, there are no currently available personalized medicine approaches that allow physicians to select an agent for a patient based on the immune signature in the patient’s skin. Such an approach would improve upon the current trial -and-error based methods and likely optimize outcomes, increase efficiency, and save healthcare costs.

[0040] Prior attempts to evaluate immunologic heterogeneity in skin diseases, such as psoriasis and eczema, have largely relied on mRNA sequencing from skin biopsies, tape strips, and / or skin scrapings. These approaches have several limitations. Tape stripping, for example, can be time consuming and mostly samples the cornified layer and not the underlying epidermis. Furthermore, tape stripping for sample collection may dilute signal concentrated in the basal and spinous layers of the epidermis. Curettage is likely highly user-dependent and may not ensure consistent sampling depth. Further, mRNA is unstable and requires specialized collection approaches. Analysis of mRNA with RNA-sequencing is labor intensive, requires specialized equipment, expertise, and data normalization, and may miss changes occurring at the post- transcriptional level, which is important for many diagnostically and therapeutically relevant cytokines including TNF, IL-36, and IL-1B.

[0041] Enrollment of patients in clinical trials can be improved by inclusion of biomarkers that predict whether a given patient could potentially respond to a particular intervention. For example, in atopic dermatitis, when an IL-22 blocking medication was used in clinical trials, it was determined retrospectively that those expressing low levels of IL-22 did not respond to the drug, while those expressing high levels did (Brunner P.M., et al., J Allergy Clin Immunol. 2019; 143(1): 142-154.). Knowing this fact regarding low expression levels of IL-22 ahead of time could have led to a more efficient and more rational enrollment of patients for the clinical trial. As more targeted agents are evaluated in dermatology, patient stratification methods hold great promise. Moreover, such an approach could provide valuable information to help differentiate characteristics between responders and non-responders for clinical trial participants. As the generation and use of targeted therapies in medicine increases, the importance of predictive biomarkers should likewise increase.

[0042] As such, in one aspect, the present disclosure generally relates to a method for preparing a sample from a subject, the method comprising: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; and d. analyzing the sample, wherein the analysis comprises a proximity extension assay. Moreover, in one aspect, the present disclosure generally relates to a kit for generating a sample from a subject, the kit comprising: a. a composition comprising: i. a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. a collection device; c. an indicator card; and d. instructions for use. Furthermore, in one aspect, the present disclosure generally relates to a method of detecting an immune-mediated disease, a fungal infection, or a skin lymphoma in a subject, wherein the method comprises: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; d. analyzing the sample to determine a level of at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma- related molecule; and comparing the level of the at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma- related molecule with a predetermined reference level for at least one immune-mediated disease- related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and wherein if the level of the at least one immune-mediated disease-related molecule is above the respective reference level, the subject is determined to have an immune-mediated disease; wherein if the level of the at least one fungal infection-related molecule is above the respective reference level, the subject is determined to have a fungal infection; wherein if the level of the at least one skin lymphoma-related molecule is above the respective reference level, the subject is determined to have skin lymphoma. In one aspect, the present disclosure also generally relates to a method of preparing and analyzing a sample representative of a fungal infection, or a skin lymphoma in a subject, wherein the method comprises: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; d. analyzing the sample to determine a level of at least one immune-mediated disease-related molecule, at least one fungal infection- related molecule, and / or at least one skin lymphoma-related molecule; and e. comparing the level of the at least one immune-mediated disease-related molecule, at least one fungal infection- related molecule, and / or at least one skin lymphoma-related molecule with a predetermined reference level for at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and wherein if the level of the at least one immune-mediated disease-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of an immune-mediated disease; wherein if the level of the at least one fungal infection-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of a fungal infection; wherein if the level of the at least one skin lymphoma-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of a skin lymphoma. Moreover, in one aspect, the present disclosure generally relates to a method for preparing a sample from a subject, the method comprising: a. collecting a sample of a region of interest from the subject, wherein the sample comprises skin cells; b. contacting the sample with a composition comprising: i. a 3- (alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; and c. analyzing the sample, wherein the analysis comprises a proximity extension assay. In some aspects, the sample is collected by any means known in the art, such as a biopsy, e.g., hole punch biopsy. Such a method has the advantages of being able to be applied to skin samples collected by various different means, such as by traditional biopsy techniques, e.g., hole punch biopsy. Such a method is minimally invasive and provides the sample analysis advantages as discussed herein.

[0043] In light of the above-discussed challenges in the field, the present disclosure generally describes compositions, methods, and kits developed for measuring biomarkers (including treatment relevant cytokines) obtained easily and painlessly from a patient, such as obtaining a skin sample where the skin is impacted by a rash. The present approach facilitates characterization of rashes that are challenging to classify, providing diagnostic clarity and aiding in the selection of effective therapy. The instant compositions, methods, and kits are designed to make sample collection and characterization rapid, straightforward, painless, and non-scarring. Using the compositions, methods, and kits described herein, collection requires minimal training or other skill, unlike more invasive diagnostic procedures such as skin biopsies, and with rapid turnaround time, in some use instances, point-of-care results. Unlike a skin biopsy, the compositions, methods, and kits provided herein are relatively painless when implemented and require no local anesthesia. Moreover, the collection site of the patient heals without scarring and / or significant pigmentary alteration. Furthermore, the protein level data obtained using such compositions, methods, and kits as described herein also advantageous as proteins can drive the underlying disease process.

[0044] In some aspects, the instant approach utilizes a noninvasive sampling technique as described, for instance, by U.S. patents 9,909,098 and 9,814,422, each of which are incorporated by reference in their entireties herein. The approach described in these documents employs a mixture of two detergents to solubilize skin. Tn some instances, such an approach can be combined with commercially available downstream analysis for protein quantification, such as, for instance, OLINK®. However, the approaches described by U.S. Patent Nos. 9,909,098 and 9,814,422, which use the detergent combined with another downstream analytic approach to quantify proteins, gave results that appear inaccurate and only evaluated the approach in mouse skin as opposed to human skin.

[0045] For example, in U.S. Patent No. 9,909,098 (‘098 patent) analysis of protein collected from a mouse model of atopic dermatitis using a 62-plex cytokine array was performed and showed that IL-13 was not statistically significantly upregulated compared to healthy control skin (see ‘098 patent, FIG. 9, left panel entitled “Interleukins”). IL-13 is a hallmark of atopic dermatitis and there are multiple approved agents targeting IL- 13 in atopic dermatitis. This would have given a potential user no indication that this approach was likely to work.

[0046] Moreover, a second example where the same approach was used to analyze proteins in a mouse model of psoriasis is as follows. Hallmarks of psoriasis include upregulation of IL-17 and IL- 12, against which there are multiple approved psoriasis medications. Referring again to U.S. Patent No. 9,909,098, when comparing protein levels to normal affected skin, a significant upregulation of IL- 17 and IL- 12 was not found. Instead, upregulation of IL-4 was found (see ‘098 patent, FIG. 10, left panel entitled “Interleukins”). It is noted that IL-4 upregulation is instead a hallmark of atopic dermatitis. Again, such a result would give a potential user no indication that this approach would work for immune profiling of inflammatory skin disease.

[0047] As compared to U.S. patents 9,909,098 and 9,814,422, the compositions, methods, and kits of the present disclosure provide a number of advantages, such as optimization of collection probe, optimization of collection technique in human subjects (so that no scarring occurs), and processing and storing of the tissue.

[0048] For instance, the ‘098 patent studied mouse skin models (psoriasis and atopic dermatitis models). See US patent 9,909,098. The approach of the ‘098 patent was never tested in a human patient, including a human patient with these disorders. Furthermore, the immune profiles determined in the mouse models were not as would be predicted; that is, the cytokine levels seen were not typically the ones associated with each disorder in human skin, and for some cytokines was opposite what would have been expected. This is particularly notable because detergents, including those used to collect the samples, are pro-inflammatory because they wound the skin (Masutani, Y., et al., Int Arch Allergy Immunol. 2022; 183(10): 1040-1049.; Yoshimura, T., et al., J Invest Dermatol. 2023:S0022-202X(23)02080-8). Therefore, it was surprising and unexpected, particularly in the setting of the prior mouse data, that the compositions, methods, and kits described herein could give immunologically accurate downstream information in human skin. Without wishing to be bound by theory, it is believed that the accuracy relies on proper downstream analysis of the material as multiple other downstream approaches (including in our hands) were unable to accurately measure relevant cytokines (see Example 2 below).

[0049] Moreover, the detergent formulations and compositions discussed herein differ significantly from those of US patent 9,814,422. For instance, in some aspects, the instant detergent formulations and compositions differ from those of the ‘422 patent at least in the following ways: 1) addition of particular protease inhibitor(s), as, for instance, while it is noted the ‘422 patent does discuss use of a protease inhibitor, but it was found that all protease inhibitors do not work equally and certain protease inhibitor(s) perform optimally, and 2) addition of a phosphatase inhibitor, as it was found that a phosphatase inhibitor can promote preservation of activation status of proteins.

[0050] In regard to the protease inhibitor, not all protease inhibitors performed optimally. For example an exemplary protease inhibitor selected for use with the methods and compositions described herein does not interfere with downstream protein concentration quantification; whereas another protease inhibitor did interfere.

[0051] Furthermore, the compositions, methods, and kits described herein can be used to identify cytokines that are targeted by approved therapeutic agents in dermatology to potentially personalize treatment. Specifically, based on the cytokine profile in an individual patient, a therapy that targets cytokines that are most abundant may be the most effective. Medications that target cytokines expressed at low or undetectable levels would not be advisable. As such, the compositions, methods, and kits described herein could improve outcomes, be efficient, and be cost effective by, for instance, saving wasted healthcare cost on obtaining approval and paying for a medication that may not work well when there is another medication that would work better for an individual patient.

[0052] Moreover, the compositions, methods, and kits described herein could be applied in a similar way to inflammatory conditions in other organs. For example, the compositions, methods, and kits described herein could be applied to other organs as well including but not limited to other parts of the GI tract (mucous membranes, esophagus, stomach, small intestine), eyes, nasal sinuses, bronchopulmonary tissue, genital mucosa, and many others. These organs are all affected by diseases that require cytokine blockade in some instances for treatment. Moreover, using novel machine learning and artificial intelligence approaches, the output from this approach can be combined with methods currently used to select therapies (other medical conditions, for example) to help drive and simplify optimal therapeutic selection.

[0053] Furthermore, the present disclosure describes simple and straightforward approaches for readily identifying the types of fungus (dermatophytes) organisms that most commonly cause skin infections and create confusion for non-dermatologic providers. These conditions are frequently misdiagnosed and the wrong treatment may be initiated, which can sometimes lead to worsening of the rash. In some instances, by using the approaches described herein, DNA from samples can be obtained and collected, which could then be analyzed using PCR to identify pathogenic fungal organisms. This approach is technically simple and easy to interpret, allowing patients with fungal infections to be treated with antifungals and patients with rashes that mimic fungal infections to be treated appropriately with other means. This approach could also be applied to detection of other infections by bacteria, viruses, arthropods, and other organisms. Additionally, the ability to detect DNA could facilitate evaluation of clonal T cell populations in the skin, enabling appropriate diagnosis and treatment of skin lymphomas. Additionally, the compositions, methods, and kits described herein could be applied to non-dermatologic diseases such as inflammatory bowel disease, Crohn’s disease, and ulcerative colitis, and has wide- reaching potential applications in dermatology, such as, for instance, in the treatment of inflammatory diseases, infectious diseases, and skin cancer. Moreover, the approaches described herein could be applied to any organ or tissue where inflammation is treated with cytokine blockade.

[0054] Additionally, the present compositions, methods, and kits are designed such that high- throughput proteomics to solubilized skin samples can be applied so as to generate proteomic profiles, as prior approaches in the field have been limited to mRNA and / or smaller groups of proteins. Prior approaches do not describe the use of proximity extension assay-based high throughput proteomics on skin samples, as is described herein. For instance, other methods, such as multiplex array assays, have not provided adequate sensitivity or specificity to be particularly useful. In many instances, protein level data can be advantageous as proteins directly generally drive the underlying disease process, whereas mRNA levels do not necessarily correlate with protein expression.

[0055] Furthermore, the compositions, methods, and kits described herein are designed to be used with both supervised and unsupervised machine learning approaches (including but not limited to naive bayes, logistic regression, quadratic / linear discriminant analysis, support vector machines, k-nearest neighbors, random forests, gradient boosting, deep leaming / neural networks, principal component analysis, k-means clustering, and gaussian mixtures) being applied to the generated proteomic data to identify combinations of proteins that are specific to certain skin diseases, and protein combinations that predict response to a particular therapy. Additionally, biomarker profiles generated using the approaches described herein, which will include hundreds of human samples across dozens of inflammatory skin diseases, will add additional novelty.

[0056] As such, the compositions, methods, and kits described herein represent a simple and straightforward test to aid clinicians in determining whether a skin rash is fungal or not, ultimately helping them to determine the best treatment for patients. Beyond fungal infections, other infectious diseases may be readily diagnosed using this approach. The ability to evaluate for a clonal T cell population using this approach has the potential to inform these challenging cases and ensure an accurate diagnosis as rapidly as possible

[0057] DEFINITIONS

[0058] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0059] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and nucleic acid hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0060] Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0061] That the disclosure may be more readily understood, select terms are defined below.

[0062] As used herein, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. By way of example, “an element” means one element or more than one element. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.”

[0063] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0064] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by any degree of suppression, remission, or eradication of a disease state.

[0065] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0066] As used herein, “region of interest” refers to an area of the subject’s body, in various embodiments, the skin, that is suspected to be affected by a disease. In various embodiments, the region of interest presents signs of inflammation. In various embodiments, the region of interest presents signs of thickening, redness, itchiness, ulceration, flaking and / or cracking.

[0067] As used herein, the term “immune-mediated disease-related molecule” refers to a molecule that may be detected by the methods of the invention and is indicative that the region of interest is affected by an auto-immune disease. In various embodiments, the immune-mediated disease-related molecule is a cytokine.

[0068] As used herein, the term “fungal disease-related molecule” refers to a molecule that may be detected by the methods of the invention and is indicative that the region of interest is affected by a fungal disease. In various embodiments, the fungal-disease related molecule may be a fragment of the genome of a pathogenic fungus or a fungal protein, such as elements of the fungal wall or other structures of the fungal organism.

[0069] As used herein, the term “skin lymphoma-related molecule” refers to a molecule that may be detected by the methods of the invention and is indicative that the region of interest is affected by skin lymphoma. In various embodiments, the skin lymphoma-related molecule is a marker for a clonal T cell population.

[0070] As used herein, the term “psoriasis” refers to a long-lasting, noncontagious immune- mediated disease characterized by plaques of abnormal skin displaying signs such as redness and scale.

[0071] As used herein, the term “eczema” refers to a group of conditions that cause the skin to become red, itchy and inflamed. In various embodiments, eczema is atopic eczema (atopic dermatitis).

[0072] As used herein, the term “skin lymphoma” refers to a group of cancers of the lymphocytes which affect the skin. In various embodiments, the skin lymphoma is a cutaneous T-cell lymphoma. In various embodiments, the skin lymphoma is a cutaneous B-cell lymphoma

[0073] A “reference level” of a biomarker means a level of the biomarker that is indicative of the absence of a particular disease state or phenotype. When the level of a biomarker in a subject is above the reference level of the biomarker it is indicative of the presence of a particular disease state or phenotype. When the level of a biomarker in a subject is within the reference level of the biomarker it is indicative of a lack of a particular disease state or phenotype.

[0074] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 and so forth, as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0075] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0076] The term “subject” is intended to include living organisms that may be subjected to treatment for a given disease, e.g., mammals. A “subject” or “patient,” as used herein, can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as simian and non- human primate mammals. Preferably, the subject is human.

[0077] As used herein, the terms “effective amount” and “therapeutically effective amount” are used interchangeably and refer to the amount required to reduce or improve at least one symptom or change in a clinical marker of a disease, e.g., a respiratory disorder, relative to an untreated patient, and / or the amount required to promote the protection of endothelial cell integrity, e.g., lung endothelial cell integrity. The effective amount of the treatment, e.g., VEGF-D, used for therapeutic treatment of the disease, e.g., respiratory disorder, varies depending upon the manner of the specific disorder, condition or disease, extent of the disorder, condition or disease, and administration of the cells, as well as the age, body weight, and general health of the subject. The effective amount is capable of achieving a particular desired biological result and / or provides a therapeutic or prophylactic benefit.

[0078] An “analyte” as used herein means any biomolecule (e.g., polypeptide, nucleic acid, lipid, and the like), drug (e.g., therapeutic drugs, drugs-of-abuse, metabolites, and the like), small molecule (e.g., natural moisturizing factors, nicotine, and the like, with the understanding that small molecules can also be drugs), warfare agent, environmental contaminant (e.g., pesticides, etc.), microbe (e.g., bacterium, virus, fungus, yeast, and the like) and the like that is present in or on the tissue and can be extracted from the tissue of interest (e.g., skin, a mucosal membrane, and the like) and detected, analyzed, and / or quantified.

[0079] The term “liquefaction” is used to describe the process by which tissue and / or tissue constituents are converted to a liquid state through exposure to sufficient energy and / or a liquefaction promoting medium, and can involve conversion of at least a portion of a tissue structure of interest to a liquid form. A tissue sample that has been subjected to liquefaction as sometimes referred to herein as a “liquefied” sample.

[0080] A “biomolecule” as used herein means any molecule or ion which has a biological origin or function. Non-limiting examples of biomolecules include proteins (e.g., disease biomarkers such as cancer biomarkers, antibodies: IgE, IgG, IgA, IgD, or IgM, and the like), peptides, lipids (e.g., cholesterol, ceramides, or fatty acids), nucleic acids (RNA and DNA), small molecules (e g., glucose, urea, creatine), small molecule drugs or metabolites thereof, microbes, inorganic molecules, elements, or ions (e.g., iron, Ca2+, K+, Na+, and the like). In some embodiments, the biomolecule is other than glucose and / or is other than a cancer marker.

[0081] As used herein, the term “sample” generally refers to any material or substance that may contain an analyte, such as an analyte of interest. In some aspects, the sample comprises more than one analyte, e.g., more than one analyte of interest.

[0082] “Energy” as used herein means any appropriate energy that can be applied to tissue to accomplish the objective of the methods disclosed herein (e.g., liquefying tissue). Exemplary types of energy include mechanical energy (e.g., abrasion, shear, vacuum, pressure, suction), ultrasound, optical (e.g., laser), thermal, and electrical energy.

[0083] As used herein, the term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a polypeptide naturally present in a living animal is not “isolated,” but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0084] An ’’isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0085] The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA methods.

[0086] The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[0087] As used herein, the term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T.”

[0088] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, “nucleic acid” and “polynucleotide” as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides” and which comprise one or more “nucleotide sequence(s)”. The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences (i.e., “nucleotide sequences”) which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0089] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0090] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0091] METHODS

[0092] In one aspect, the present disclosure generally relates to a method for preparing a sample from a subject, the method comprising: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; and d. analyzing the sample, wherein the analysis optionally comprises a proximity extension assay. Tn some aspects, the composition comprises both the protease inhibitor and the phosphatase inhibitor. In some aspects, the protease inhibitor and phosphatase inhibitor can be any of those described herein.

[0093] In some aspects, the methods described herein enable diagnosis and immunologic profiling of a rash without a skin biopsy. In some aspects, the method comprises sampling an area of about 0.5 mm2to about 5 mm2. In some aspects, the method comprises sampling an area of about 0.5 mm2 to about 5 mm2, about 0.75 mm2to about 4 mm2, or about 1 mm2to about 3 mm2. In some aspects, the method comprises sampling an area of about 1 mm2to about 3 mm2. In some aspects, the methods described herein do not leave a scar on the subject from which a sample is derived. In some aspects, the method comprises use of samples of full-thickness epidermis.

[0094] In some aspects, step b. of the method further comprises applying energy to the region of interest using the collection device. In some aspects, the energy is applied in the form of ultrasound, mechanical, optical, thermal, or electrical energy. In some aspects, the mechanical energy is applied by an abrasive material. In some aspects, the thermal energy is applied in the form of radio frequency energy. In some aspects, the optical energy is applied in the form of a laser. In some aspects, steps a. and b. of the method promote solubilization of the region of interest.

[0095] Sample and Analytes

[0096] In some aspects, a sample is obtained from a subject. In some aspects, the sample is derived from a region of interest. In some aspects, the sample comprises at least one analyte. In some aspects, the at least one analyte comprises at least one protein. In some aspects, the at least one protein comprises at least one cytokine. In some aspects, the analyte comprises any one or more of IL-1 A, IL-1B, IL-1R1, IL-1R2, IL-1RAP, IL-1RL1, IL-1RL2, IL-1RN, IL-2, IL-2RA, IL-2RB, IL-2RG, IL-3, IL-3RA, IL-4, IL-4R, IL-5, IL-5RA, IL-6, IL-6R, IL-6ST, IL-7, IL-7R, IL-9, IL-10, IL-10RA, IL-10RB, IL-11, IL-12, IL-12A, IL-12B, IL12-RB 1, IL-12RB2, IL-13, IL-13RA1, IL-13RA2, IL-15, IL-15RA, IL-16, IL-17A, IL-17C, IL-17D, IL-17F, IL17-RA, IL17-RB, IL-18, IL-18-BP, IL-18R1, IL-18RAP, IL-19, IL-20, IL-20RA, IL-20RB, IL-21R, IL- 22, IL-22RA1, IL-24, IL-25, IL 31, IL-31RA, IL-32, IL-33, IL-34, IL-36A, IL-36G, GM-CSF, R ANTES, MCP-4, CCL2, CTACK / CCL27, IFN-g, TNFa, CD23, CD-40, Eotaxin-2, TARC, IL- 1, IL-6, CXCL8 (formerly IL-8), CXCL11, CCL11, CXCL13, CCL13, CXCL16, CCL17, CCL20, CCL26, G-CSF, NOS2, PI3, TMPRSS11D, BLMH, IL1R2, IFNGR1, IFNGR2, CCL14, CCL15, CCL16, CCL18, CCL19, CCL2, CCL21, CCL22, CCL23, CCL24, CCL25, CCL27, CCL28, CCL3, CCL4, CCL5, CCL7, CCL8, CXCL1, CXCL10, CXCL12, CXCL14, CXCL17, CXCL3, CXCL5, CXCL6, CXCL8, CXCL9, IRF4. In some aspects, the analyte comprises any one or more of IL- 1 (IL- la and -P), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12, IL-12A, IL-12B, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17A, IL-17C, IL-17F, IL-18, IL- 31, IL-33, IFN-a, IFN-P, IFN-y, TNF-a, and TNF-p. In some aspects, the analyte comprises any one or more of IL-13, IL-23, IL-17, interferon (IFN)-gamma, or combinations thereof. In some aspects, the analyte comprises a combination of IL-13, IL-23, IL- 17, and interferon (IFN)- gamma. In some aspects, the analyte comprises a combination of at least two of IFN-gamma, IL- 22, and IL-13. In some aspects, the analyte comprises a combination of at least two of IFN- gamma, IL-22, IL-13, IL-17A, and IL-31. In some aspects, the analyte comprises any one or more of IL-17A, IL-17F, IFN-y, IL- 12, IL- 12p40, CXCL9, CXCL10, CXCL11, IL-4, CCL2, CCL13, CCL17, CCL18, CCL19, and CCL22, and combinations thereof. In some aspects, the analyte comprises any one or more of NOS2, NT5C3A, GBP1, PI3, TMPRSS1 ID, and combinations thereof. In some aspects, the analyte comprises any one or more of MMP7, MMP10, ITGAX, EGF, and combinations thereof. In some aspects, the analyte comprises any one or more of IL-17A, IL-23, IFN- y, IL-12, IL-15, STAT1, TNF, NF-KP, IL-36, and combinations thereof. In some aspects, the analyte comprises any one or more of IL-13, IL-10, CCL2, and combinations thereof. In some aspects, the analyte comprises any one or more of PI3, IL-12B, CCL17, CCL2, CXCL13, and combinations thereof. In some aspects, the analyte comprises NOS2, CCL17, or a combination thereof. In some aspects, the analyte comprises any one or more of IL-17A, IL-17F, IL- 12, IL-12p40, IL-17C, IL-36 A, CXCL1, CXCL8, CCL20, and combinations thereof. In some aspects, the analyte comprises any one or more of CXCL9, CXCL14, IFN- y, IL-9, and combinations thereof. In some aspects, the analyte comprises one or more of IL-4, CCL17, CCL22, and combinations thereof. In some aspects, the analyte comprises IL-17A, CCL17, or a combination thereof.

[0097] In some aspects, the region of interest comprises a fungal infection, a viral infection, a bacterial infection, and / or an arthropod infestation. In some aspects, the region of interest comprises an immune-mediated disease. In some aspects, the immune-mediated disease is psoriasis or eczema. In some aspects, the region of interest comprises a skin lymphoma.

[0098] In some aspects, the subject is a mammal. In some aspects, the mammal is a human. In some aspects, the subject can include livestock and / or pets, such as ovine, bovine, porcine, canine, feline and murine mammals, and further includes simian and non-human primate mammals.

[0099] In some aspects, the sample comprises a tissue sample. In some aspects, the tissue is mucosal membrane tissue, skin tissue, breast tissue, prostate tissue, eye tissue, vagina tissue, bladder tissue, nail tissue, hair tissue, colon tissue, testicle tissue, intestine tissue, lung tissue, brain tissue, pancreas tissue, liver tissue, heart tissue, bone tissue, or aorta wall tissue. In some aspects, the tissue sample comprises an epidermal tissue sample. In some aspects, the sample comprises a tissue that is skin, which can be skin of the face, arms, hands, legs, back, or any other location. In some aspects, the area of the subject from which the sample is derived is from about 1 mm2to about 3 mm2.

[0100] Sample Analysis

[0101] In some aspects, a sample, such as a sample generated by any of the method described herein, is analyzed. In some aspects, the sample analysis comprises quantifying one or more analytes of the sample. In some aspects, the analysis comprises generating a cytokine profile of the subject. In some aspects, the analysis comprises generating a biomarker profile of the subject. In some aspects, the analysis comprises machine learning and / or artificial intelligence-based approaches. In some aspects, the machine learning comprises supervised and / or unsupervised machine learning approaches. In some aspects, the supervised and / or unsupervised machine learning approaches are naive bayes, logistic regression, quadratic / linear discriminant analysis, support vector machines, k-nearest neighbors, random forests, gradient boosting, deep learning / neural networks, principal component analysis, k-means clustering, and / or gaussian mixtures.

[0102] In some aspects, the analysis is performed on from about 1 to about 1,000 different individual samples. In some aspects, the different individual samples represent at least two different diseases. In some aspects, the analysis comprises analysis of T cell populations of the sample. Tn some aspects, the analysis comprises nucleic acid sequencing. In some aspects, the analysis is used to select a therapy to administer to the subject.

[0103] In some aspects, the analysis comprises a proximity extension assay. In general, proximity extension assays are immunohistochemical tools that utilize two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridization of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein-protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualize single protein molecules or complexes with high spatial accuracy. In some instances, a proximity extension assay features use of proximity extension assay (PEA) probes, wherein one of the PEA probes consists of a double stranded- oligonucleotide attached to the antibody at its 3 '-end, with a nine nucleotide or so 3 '-overhang at its 5'-end. This overhang is complementary to the 3'-end of the oligonucleotide bound to the other antibody partner. Following incubation of the proximity probes with a sample containing antigen hybridization by the probes, the overhanging 3 '-end could hybridize to the 5'- oligonucleotide and, following the addition of a DNA polymerase, the free 3'-OH was extended in the 5'— 3 ' direction towards the attachment site of the 5 ’-oligonucleotide. This generated a full- length amplicon and hybridization site for the upstream primer and thus allows for the amplification and detection of the target antigen by PCR. This arrangement has now been replaced by a modification, where each of the two single-stranded oligonucleotides contain a complementary site for pair-wise annealing with the other oligonucleotide, allowing extension by a DNA polymerase. In some aspects, the DNA polymerase is a DNA polymerase with intact 3'- 5' exonuclease activity.

[0104] In some aspects, analysis, such as protein quantification and subsequent analysis, is aided using OLINK®-based methods. Such OLINK®-based methods are described, for example, in U.S. Patent Nos. 9,677,131; 9,777,315; 10,731,206; and 9,902,993, each of which is incorporated in their entireties herein.

[0105] Treatments

[0106] In some aspects, the method further comprises administering at least one therapeutic composition to the region of interest. In some aspects, the therapeutic composition is a composition for treating a fungal infection. In some aspects, the therapeutic composition is a composition for treating a skin disease. In some aspects, the therapeutic composition comprises an immunotherapeutic. In some aspects, the therapeutic composition comprises a biologic. In some aspects, the therapeutic composition comprises an small molecule, such as an oral small molecule. In some aspects, the therapeutic composition comprises a Janus kinase (JAK) inhibitor. In some aspects, the therapeutic composition is a therapeutic composition for treating a disease that has been identified using any of the methods and / or compositions described herein.

[0107] Collection Devices

[0108] In some aspects, the collection device comprises an abrasive device. In some aspects, the collection device comprises an abrasive surface. In some aspects, the collection device comprises a chamber for dispensing the composition. In some aspects, the collection device comprises a cotton swab or swab with microfiber tip.

[0109] In some aspects, the collection device comprises an indicator card. In some aspects, the indicator card is used to analyze a sample. In some aspects, the indicator card is used to detect one or more analytes comprised by a sample. In some aspects, the card is used to detect one or more proteins. In some aspects, the one or more proteins comprise one or more cytokines. In some aspects, the cytokines are interleukin IL-13, IL-17, IL-23, interferon (IFN)-gamma, NOS2, CCL17 or combinations thereof. In some aspects, the indicator card comprises one or more immobilized antibodies. In some aspects, the one or more immobilized antibodies bind to a cytokine. In some aspects, the one or more antibodies are each attached to an enzyme. In some aspects, the collection device comprises a component capable of exerting an abrasive force. In some aspects, the abrasive force can be generated by setting an abrasive surface in motion, with the abrasive surface held in contact with a tissue of interest. For this purpose a mechanized device that bears the abrasive surface and is capable of setting the surface into motion can be introduced into a chamber containing a composition as described herein. Such a chamber, when placed in contact with the tissue, can expose the tissue to the mechanized abrasion device such that the abrasive surface of the device can be held in contact with the tissue. Setting the abrasive surface in motion (e.g., by rotating, or a similar periodic oscillatory movement) can dislodge the tissue constituents and collect them directly into the composition. Non-limiting examples of abrasive surface can be a disc made of abrasive material (e.g., fabric, polymer, abrasive crystals (e.g., quartz, metal, silica, silicon carbide, dust and derivatives of aluminum (such as AIO2), diamond dust, and the like), etc.); a disc bearing sanding paper with its abrasive side designed to face the tissue of interest; and a brushing device bearing bristles which form the means for abrading tissues. In some aspects, the pressure applied by the abrasive surface onto the tissue may be varied in the range of 1-2000 N / m2. Higher pressures may also be applied to the tissue, but such an application would require careful control of the applicator device. A preferred range of pressure is about 100 to about 1000 N / m2. In some aspects, the duration of contact time between the tissue and the surface may range from 1 second to 60 minutes; however, application time may be more depending on the tissue type, the amount of tissue constituent, and the area of tissue to be sampled.

[0110] Methods of Use

[0111] In some aspects, the methods disclosed herein can be used for a broad range of tissue evaluations, including assessment of the presence or absence of an analyte(s) of interest to facilitate diagnosis of a condition of interest. In some aspects, the methods find use where, for example, the patient presents with clinical signs and symptoms suggestive of one or more conditions, where the methods disclosed herein can facilitate making the correct diagnosis among the entities included in the differential diagnosis.

[0112] In some aspects, the methods described herein are used for a broad range of sample evaluations. For instance, in some aspects, energy-assisted tissue liquefaction can provide a quantitative evaluation and profile of normal tissue. Comparison of the normal tissue profile with a profile of tissue under investigation can facilitate diagnosis of changes in tissue microenvironment (e.g. up / down-regulation of several proteins, such as cytokines) which can indicate various diseased conditions. In some aspect, the methods described herein can also be used as a tool for monitoring tissue recovery and evaluating therapeutic efficacy of various treatments (as in monitoring of therapy, which can be combined with modification of therapy as desired or needed).

[0113] In some aspects, the methods described herein can be used to evaluate the therapeutic effect of various treatments, including bioavailability of therapeutics in samples of interest. The analyte in the liquefied skin sample derived from a region of interest can be quantified to indicate how much of the analyte is present in the sample. The quantitative presence or absence of a certain analyte or composition of analytes present in a sample under investigation, when compared to the quantitative presence or absence of the same analytes in a reference sample, can indicate whether or not the dosed therapeutic agent is staying in specific skin long enough to achieve its desired effect. The reference sample is usually skin that is in a healthy state.

[0114] In some aspects, the methods described herein can be used for detecting or diagnosing pathogenic microbes (e.g., fungi). Current methodologies for microbial diagnostics in tissues, such as replica plating, swabbing, and washing, are unattractive due to large variability and low dispersion of extracts, which leads to decreased sensitivity and high protocol -dependency. Various tests can be performed upon the liquefied tissue sample to isolate and identify the microbial analytes present in the tissue.

[0115] Methods of Discriminating Diseases

[0116] In some instance, the methods described herein can be used to discriminate between various different disease types in a subject, such that a subject’s condition can be accurately diagnosed and subsequently treated. For instance, in one aspect, the present disclosure generally relates to a method of detecting an immune-mediated disease, a fungal infection, or a skin lymphoma in a subject, wherein the method comprises: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; d. analyzing the sample to determine a level of at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma- related molecule; and comparing the level of the at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma- related molecule with a predetermined reference level for at least one immune-mediated disease- related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and wherein if the level of the at least one immune-mediated disease-related molecule is above the respective reference level, the subject is determined to have an immune-mediated disease; wherein if the level of the at least one fungal infection-related molecule is above the respective reference level, the subject is determined to have a fungal infection; wherein if the level of the at least one skin lymphoma-related molecule is above the respective reference level, the subject is determined to have skin lymphoma.

[0117] In some aspects, the composition comprises any of the compositions described herein. In some aspects, the collection device comprises any of the collection devices described herein. In some aspect, the analysis comprises any form of analysis described herein. In some aspects, the analysis comprises a proximity extension assay. In some aspects, the immune-mediated disease is psoriasis or eczema. In some aspects, the analysis comprises generating a cytokine profde of the subject. In some aspects, the at least one immune-mediated disease-related molecule, at least one fungal infection -related molecule, and / or at least one skin lymphoma-related molecule comprises at least one protein. In some aspects, the method comprises at least one cytokine. In some aspects, the at least one cytokine is a combination of IL-13, IL-17, IL -23, NOS2, CCL17 and / or interferon (IFN)-gamma. In some aspects, the analysis is used to select a therapy to administer to the subject. In some aspects, the subject is administered a therapy to treat the immune-mediated disorder, fungal infection, or skin lymphoma.

[0118] Moreover, in one aspect, the present disclosure also generally relates to a method of preparing and analyzing a sample representative of a fungal infection, or a skin lymphoma in a subject, wherein the method comprises: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; d. analyzing the sample to determine a level of at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and e. comparing the level of the at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule with a predetermined reference level for at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and wherein if the level of the at least one immune-mediated disease-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of an immune-mediated disease; wherein if the level of the at least one fungal infection-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of a fungal infection; wherein if the level of the at least one skin lymphoma-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of a skin lymphoma. In some aspects, the composition comprises any of the compositions described herein. In some aspects, the collection device comprises any of the collection devices described herein. In some aspect, the analysis comprises any form of analysis described herein. In some aspects, the analysis comprises a proximity extension assay.

[0119] COMPOSITIONS

[0120] As discussed above, in one aspect, the present disclosure generally relates to a composition comprising a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; a protease inhibitor; and a phosphatase inhibitor.

[0121] In some aspects, the zwitterionic surfactant and / or the nonionic surfactant is present in the composition at a concentration of from about 0.1% to about 2.0% w / v. In some aspects, the zwitterionic surfactant and / or the nonionic surfactant is present in the composition at a concentration of about 0.5% w / v.

[0122] In some aspects, the protease inhibitor is a protease inhibitor cocktail, such as a commercially available protease inhibitor cocktail. In some aspects, the protease inhibitor is the complete™ mini protease inhibitor cocktail (Roche). In some aspects, the protease inhibitor is selected from the group consisting of AEBSF-HC1, aprotinin, bestatin, E-64, EDTA, leupeptin, pepstatin A, and combinations thereof.

[0123] In some aspects, the phosphatase inhibitor is a phosphatase inhibitor cocktail, such as a commercially available phosphatase inhibitor cocktail. In some aspects, the phosphatase inhibitor cocktail is PhosSTOP™ (Roche).

[0124] In some aspects, the pH of the composition is from about 5 to about 11, about 6 to about 10, or about 7 to about 9. In some aspects, the pH of the composition is from about 7 to about 9.

[0125] In some aspects, the zwitterionic surfactant and the nonionic surfactant are not simultaneously and respectively 3 -(decyl dimethyl ammonia) propane sulfonate and polyoxyethylene (4) lauryl ether. In some aspects, the composition comprises a buffer solution. In some aspects, the composition comprises one or more of: phosphate-buffered saline, Tris-buffered saline, Trishydrochloride, and ethylene diamine tetraacetic acid. In some aspects, the composition comprises phosphate buffered saline. In some aspects, the pH of the composition in the buffer solution is about 8.8.

[0126] In some aspects, wherein the composition comprises a total (w / v) concentration of the zwitterionic surfactant of one or more of: between about 0.01% and about 10%; between about 0.01% and about 5%; between about 0.1% and about 2%; between about 0.1% and about 0.5%; and about 1%. In some aspects, wherein the composition comprises a total (w / v) concentration of the nonionic surfactant of one or more of: between about 0.01% and about 10%; between about 0.01% and about 5%; between about 0.1% and about 2%; between about 0.1% and about 0.5%; and about 1%. In some aspects, wherein the composition comprises a total (w / v) concentration of the zwitterionic and nonionic surfactants of one or more of: between about 0.01% and about 10%; between about 0.01% and about 5%; between about 0.1% and about 2%; between about 0.1% and about 0.5%; and about 1%. In some aspects, the composition comprises a concentration ratio of the zwitterionic surfactant to the nonionic surfactant of between about 1 :5 and about 5: 1, between about 1 :4 and about 4: 1; between about 1 :3 and about 3: 1; between about 2:5 and about 5:2, between about 2:4 and about 4:2, or about between about 2:3 and about 3:2. In some aspects, the composition comprises a concentration ratio of the zwitterionic surfactant to the nonionic surfactant of between about 1 :3 and about 3: 1. In some aspects, the composition comprises a concentration ratio of the zwitterionic surfactant to the nonionic surfactant of between about 2:3 and about 3:2.

[0127] In some aspects, the composition comprises an anionic surfactant. In some aspects, the composition comprises an alkyl sulfate surfactant or a salt thereof. In some aspects, the composition comprises a total (w / v) concentration of the anionic surfactant of one or more of: between about 0.01% and about 10%; between about 0.01% and about 5%; between about 0.1% and about 2%; between about 0.1% and about 0.5%; and about 1%.

[0128] In some aspects, the compositions described herein are used to probe protein functional states and related skin cell signaling pathways. Skin cell signaling pathways are in some instances stress-induced, and can change over minutes to hours. Phosphorylation is a highly labile post-translational modification that regulates many aspects of protein function. The ability to probe these functional states in the epidermis necessitates a fast and efficient method to solubilize and isolate phosphoproteins. Thus, in some aspects, a method is provided for recovering signaling proteins from skin cells, the method comprising: contacting the skin cells with a composition as described herein to provide solubilized signaling proteins; and subjecting the solubilized signaling proteins to analysis.

[0129] KITS

[0130] Furthermore, the present disclosure also encompasses kits for practicing the current methods. As such, in one aspects, the present disclosure generally relates to a kit for generating a sample from a subject, the kit comprising: a. a composition comprising a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; a protease inhibitor; and a phosphatase inhibitor, a protease inhibitor, and a phosphatase inhibitor; b. a collection device; c. an indicator card; and d. instructions for use. In some aspects, the composition comprises a composition as described herein, e.g., a liquefication medium. In some aspects, the collection device comprises a collection device as described herein.

[0131] The composition may be provided in ready -to-use form, or may be provided as components (e.g., in separate containers) that can be mixed together to formulate the composition suitable for the use to which the kit is to be applied. The various components of the kit may be present in separate containers, or certain compatible components may be precombined into a single container, as desired. The various components may also be provided treated so as to be sterile prior to use, and maintained in sterile packaging.

[0132] In some aspects, the kit comprises a kit as depicted in the schematic of FIG. 2. For instance, the kit (200) comprises a test probe (201), e g., an abrasive device, e.g., a device comprising an abrasive surface, a solution (202) comprising reagents needed for the sample collection, such as a solution comprising any one or more of the compositions described herein, and an indicator card (203), such as any of the indicator cards described herein.

[0133] In addition to the above-mentioned components, in some aspects, the kits typically include instructions for using the components of the kit to practice the methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In some aspects, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium. In some aspects, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this aspect is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0134] EXAMPLES

[0135] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0136] Example 1: Collection and Analysis of a Sample Comprising Analytes

[0137] The present example describes a process workflow for the collection and analysis of a sample comprising one or more analytes.

[0138] Referring now to the process 100 of FIG. 1, a desired amount, generally between 10 pL and 100 pL volume, of a composition comprising from about 0.1% to about 2.0% w / v, e.g., 0.5% w / v, of a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; from about 0.1% to about 2.0% w / v, e.g., about 0.5%, of a polyethylene glycol alkyl ether nonionic surfactant; a protease inhibitor, which in some instances can be cOmplete™ protease inhibitor cocktail (Roche); and a phosphatase inhibitor, which in some instances can be PhosSTOP™ (Roche), is used to contact tissue from which a sample is to be derived (101). After contacting the tissue with the composition, a collection device, such as a cotton swab, microfiber swab, or a device otherwise comprising an abrasive surface, is used to contact the tissue and composition (102). Energy is applied in the form of rotating the swab or device at such a speed and with such a force so as to promote liquefication of a tissue sample and subsequently to collect the liquified sample on the swab or device (103). After sample collection, the swab or device comprising the same is transferred to a container comprising a medium containing reagents for subsequent analysis (304). After transfer of the sample to the medium containing reagents for subsequent analysis, an indicator card is used to contact the sample (305). After contacting the sample, the analysis assay is performed on the card. Subsequently, protein quantification is performed.

[0139] Protein quantification and downstream data analysis can proceed using, for instance, proximity extension assays and analytic algorithms. For instance, each target biomarker of a sample is addressed by a matched pair of antibodies, coupled to unique, partially complementary oligonucleotides, and measured by quantitative real-time PCR. Such an approach promotes a high level of sample multiplexing.

[0140] In some instances, protein quantification and analysis is aided using OLINK®-based methods. Such OLINK®-based methods are described, for example, in U.S. Patent Nos. 9,677,131; 9,777,315; 10,731,206; and 9,902,993, each of which is incorporated in their entireties herein.

[0141] Furthermore, in some instances, sample analysis is performed using an indicator card (FIG. 3). An indicator card can be a lateral flow assay to help detect the presence and / or amounts of a desired analyte or analytes comprised by a sample. In some instances, the indicator card is placed into the sample. In some instances, the indicator card is part of a collection device. In general, the indicator card contains immobilized antibodies to the analytes, e g., cytokines of interest, that become attached to an enzyme. Once the indicator card is exposed to sample, the sample, with the analytes of interest, e.g., cytokines of interest attached to the antibody-enzyme combination, the sample flows up the card, and the enzyme causes the test lines for each of the analytes of interest to change color. The indicator card also has a control line that changes color with the enzyme to indicate that the test worked and was functional.

[0142] Example 2: Comparative Sample Analysis

[0143] In the present example, other means for sample analysis were analyzed for comparison to the methods and compositions described herein. In particular, other downstream approaches for analysis of relevant proteins / cytokines were tested, such as, for example, by using a bead-based multiplexed approach. It was found that this bead-based approach resulted in data that were not useful for measuring treatment-relevant proteins. For instance, IL-13 is upregulated in atopic dermatitis, but not in psoriasis. Using the bead-based approach, it was found that there was no difference in IL-13 levels between atopic dermatitis and psoriasis (Ps) (FIG. 4; note that NL represents normal skin controls).

[0144] Example 3: Sample Analysis

[0145] The present example describes sample analysis performed using the methods and compositions described herein. It was discovered that protein biomarkers collected using the described approach could be used for distinction between atopic dermatitis and psoriasis, and further to subset patients into molecular endotypes with treatment relevance. For example, when analyzing samples collected from patients with atopic dermatitis and psoriasis, it was discovered that the biomarkers NOS2 and CCL17 were useful and were able to classify patients having typical psoriasis (PsO), atopic dermatitis-like psoriasis (depicted as low NOS2), and patients with atopic dermatitis (FIG. 5).

[0146] In another example, it was found that certain protein biomarkers could distinguish patients that responded to a particular therapeutic intervention. Analysis was performed related to atopic dermatitis (AD) patients treated with dupilumab, which is an FDA approved medication for atopic dermatitis that inhibits IL-4 and IL-13, but not other cytokines, such as interferon gamma. It was shown that patients with atopic dermatitis treated with dupilumab did not respond to therapy and that this could be predicted based alone on interferon gamma and interferon gamma receptor 2 protein levels (FIG. 6). Referring now to FIG. 6, black dots represent nonresponders and white dots represent responders, and the red and blue regions represent a trained gradient boosting classifier that could discriminate dupilumab responders from non-responders based on protein values alone.

[0147] Example 4: Sample Analysis

[0148] The present example describes sample analysis performed using the methods and compositions described herein. For instance, use of the methods and compositions described herein resulted in successful classification of psoriasis as compared to atopic dermatitis diagnosis based on a cytokine profile alone (FIG. 8). Moreover, the methods and compositions as described herein were able to detect immune heterogeneity within each diagnosis (FIG. 9). Example 5: Non-invasive epidermal proteome-based diagnosis and molecular subclassification of psoriasis and eczema

[0149] Materials and Methods

[0150] Non-invasive epidermal protein biomarker sampling

[0151] A surfactant was used, herein referred to as BN, consisting of an equimolar ratio (0.5% w / v each) of Brij-30 and N-decyl-N,N-dimethyl-3-ammonio-l- propanesulfonate, that, when coupled to dermabrasion, enables solubilization of the epidermis without breaching of the dermis or scarring. Skin was swabbed using a microfiber cosmetic applicator and approximately 40 pL of surfactant. Swabbing was performed by rubbing the surfactant-coated applicator tip in a back- and-forth motion for 45 seconds, applying light pressure, over a 2mm-by-lmm rectangular area on lesional psoriasis or eczema skin. The tip was then inserted into an Eppendorf tube containing 130 pL of phosphate buffered saline and a protease inhibitor cocktail (Sigma- Aldrich #11836153001), let sit for 1 minute, and then rubbed against the interior of the tube to dislodge any attached material. Samples were then immediately placed on wet ice for transport and later were frozen at -80 °C.

[0152] High throughput proteomic analysis

[0153] All samples were analyzed using the Olink Explore 3072 Panel (Olink Proteomics, Uppsala, Sweden), which provides relative quantification of 3072 proteins related to inflammation, oncology, neurology, and cardiometabolics. This assay utilizes a proximity extension assay (PEA)-based technique coupled with NGS readouts. A subset of samples was also analyzed using the Olink Target 48 Cytokine Panel, which provides absolute quantification of 45 cytokine and chemokine proteins using a PEA-based technique coupled with real-time qPCR.

[0154] Patient enrollment

[0155] Patients were enrolled from a single outpatient dermatology clinic at our tertiary referral center. Criteria for enrollment included age >18 years and clinical and / or histologic diagnosis of psoriasis or eczema by a board-certified dermatologist. At least one non-invasive lesional skin sample was obtained per patient, along with demographic information, treatment information, and clinical photographs.

[0156] Statistics

[0157] Protein quantification data was analyzed using Python v3.8.8 and R v3.6.1. Differentially expressed protein values were computed using the limma v3.42.2 package in R. Machine learning analyses were performed using sci-kit learn vl .1.3 and UMAP was performed using umap-leam v0.5.3 in python. PCA was performed using sci-kit learn vl.1.3 and FactoMineR v2.4 in python and R, respectively. Comparisons between two groups were performed using two- tailed t-tests, and comparisons between multiple groups were performed using ANOVA.

[0158] Results

[0159] Development and evaluation of NIDBES for downstream proteomics

[0160] The ability of BN surfactant to solubilize human epidermis ex vivo when applied and rubbed against the skin with a small applicator was first evaluated. Six different applicators were evaluated and a microfiber-tipped cosmetic swab was selected due to its superior ability to rapidly solubilize full -thickness epidermis in a small area, easily transfer the specimen to an aqueous solution with minimal volume loss, yield a maximal amount of protein, and result in suitable material for downstream proteomic analysis.

[0161] Using this approach on ex vivo skin with the microfiber cosmetic swab, it was sought to determine the optimal collection time to reach, but not significantly breach, the dermoepidermal junction (an important consideration that suggests that the approach should be minimally painful and not leave a scar). Post-collection histologic analysis of skin was utilized to evaluate the tissue. It was found that a 45 second swab duration resulted in optimal, full thickness epidermal sampling while generally leaving the dermoepidermal junction intact (FIG. 13A-FIG. 13B). Periodic acid-Schiff (PAS) staining further demonstrated that the basement membrane remained largely intact (FIG. 13C). It was found that NIDBES could be used to collect and transfer epidermal material from a 2x1 mm swab area to phosphate buffered saline (PBS) supplemented with protease inhibitors, and to detect recombinant IL- 13 using the human cytokine / chemokine protein array 71-plex panel (Eve Technologies) after the IL-13 was injected into the superficial dermis and allowed to diffuse for 1 hour at 37°C (FIG. 14).

[0162] Based on ex vivo experiments, BN surfactant, the microfiber cosmetic swab, a 45 second collection duration, and a 2x1 mm collection area with subsequent transfer to PBS supplemented with protease inhibitors were used. In order to evaluate the utility of NIDBES in patients with eczema and psoriasis, 76 patients seen in an outpatient dermatology clinic who underwent NIDBES using the developed parameters after informed consent were enrolled. Patients had a diagnosis of psoriasis (n=43 patients) or eczema (n=27 patients) as determined by one of two board-certified dermatologists using clinical examination and occasionally histopathology when it was deemed clinically necessary to determine the diagnosis. A subset of the cases included non-classical morphologies, allowing for evaluation of the utility of the approach in such a setting as well. Healthy control patients (n=6) were also enrolled, and normal skin was sampled.

[0163] Clinical severity was assessed using body surface area (BSA) as well as Investigator’s Global Assessment (IGA) score and Validated Investor Global Assessment for Atopic Dermatitis (vIGA-AD) score for psoriasis and eczema, respectively. BSA was not significantly different between psoriasis and eczema patients (P=0.60), while IGA was higher in the psoriasis group than vIGA-AD in eczema (P=0.0032). Additional patient information is available in Table 1 below.

[0164] TABLE 1 - PATIENT INFORMATION

[0165] IGA, Investigator’s Global Assessment; vIGA-AD, Validated Investor Global Assessment for

[0166] Atopic Dermatitis; BSA, body surface area. It was found that NIDBES was very well tolerated. Patients reported either no or, at most, minimal discomfort during sample collection. The swabbing procedure resulted in a visible depress! on / erosi on after the collection was complete (FIG. 13D). It was found that the sampled skin rapidly re-epithelized, and at 3 -month follow-up there was no significant scarring or pigmentary alteration (FIG. 13E), even in patients with darker skin tones (data not shown).

[0167] NIDBES enables detection of differentially expressed immunologically-relevant protein in psoriasis and eczema

[0168] Samples collected using NIDBES were stored at -80°C until analysis. These samples were subsequently subjected to affinity -based proteomic analysis using a proximity extension assay (PEA)-based approach (Olink Explore 3072). Olink was selected due to its high specificity, requiring binding of two distinct antibodies to each target for signal detection. The Explore 3072 panel provides quantitative measurement of >3000 proteins, including numerous cytokines and chemokines (FIG. 13F).

[0169] First, it was asked if the approach was able to detect immunologic differences characteristic of psoriasis and eczema as a whole. Individual protein concentrations were not normalized to the median protein concentration within each sample in order to preserve relative protein concentrations between samples and the observation that similar results were obtained with or without normalization. Log base 2-fold change was computed for each measured protein and was compared between psoriasis and eczema patients; adjusted p-values were calculated to evaluate significance. Many of the most significantly differentially expressed proteins (DEPs) were related to Th mediated immunity, including Type 3 (IL-17A and IL-17F) and Type 1 (IFN- y, IL-12, IL- 12p40, CXCL9, CXCL10, and CXCL11) polarized responses in psoriasis, and a Type 2 (IL-4, CCL2, CCL13, CCL17, CCL18, CCL19, and CCL22) polarized response in eczema. Several innate immune mediators were also upregulated in psoriasis including IL-36A and IL17-C. Previously described psoriasis biomarkers were also upregulated in psoriasis including NO S2, NT5C3A, GBP1, PI3 and TMPRSS11D, while eczema demonstrated significant upregulation of previously described biomarkers including MMP7, MMP10, ITGAX, and EGF, among others (FIG. 15 A).

[0170] Upstream regulator analysis was then performed on the DEPs in order to infer key signaling molecules leading to the observed DEP patterns. It was found that in the psoriasis cases, some of the most significant predicted upstream regulators were IL-17A and IL-23 (Type 3), IFN- y, IL-12, IL-15, and STAT1 (Type 1), TNF, NF-KP, and IL-36. In eczema, they were IL- 13 (Th2), IL- 10, and CCL2 (FIG. 15B). IRF4, which can drive dendritic cells to polarize T cells towards Th2, was also upregulated in eczema. These patterns were conserved independent of the individual NTDBES sample collector (data not shown).

[0171] It was surprising that while IL- 13 was a highly significant upstream regulator in eczema, its differential expression between psoriasis and eczema was minimal. Given that IL- 13 is an important mediator of and key treatment target in eczema, it was postulated that a technical limitation in IL-13 detection could have been responsible for its low differential expression in the Olink Explore 3072 panel. To investigate this, a subset of samples was analyzed (eczema = 14, psoriasis = 20, control = 2) using the Olink Target 48 Cytokine Panel, which utilizes real-time quantitative polymerase chain reaction (qPCR) readout of antibody barcodes for absolute protein quantification of a smaller subset of 45 cytokine / chemokine proteins, rather than a next generation sequencing (NGS)-based readout for relative quantification of 3072 proteins as does the Explore assay.

[0172] Using the Target 48 Cytokine Panel, it was found that IL-13 was significantly upregulated in eczema (mean ± standard deviation; 3.5±5.3 pg / pL) compared to psoriasis (0.16±0.17, P<0.01) and controls (0.12±0.01). Other targets identified using the Explore 3072 panel showed expected differences between psoriasis and eczema (FIG. 15C). Additionally, while other canonical cytokines such as IL-17A were generally well correlated between the Explore 3072 and Target 48 assays (Pearson correlation coefficient (PCC) = 0.93), IL- 13 was less well correlated (PCC = 0.74) (FIG. 16A-FIG. 16B). Bland-Altman analysis of IL-13, which evaluates the difference in measured values between the two assays as a function of the mean of both values, demonstrated that the bias changed directionality for high versus low IL- 13 values, which was not the case for other cytokines such as IL-17A. Thus, the less accurate detection of IL- 13 in the Explore 3072 panel may reflect difficulty in detecting IL- 13 in cases with relatively lower concentrations of IL-13 (FIG. 17A-FIG. 17B).

[0173] Distinct protein signatures differentiate psoriasis from eczema

[0174] It was then sought to determine whether psoriasis and eczema patients could be accurately discriminated using relative protein abundances measured with this approach. Principal component analysis (PCA) was performed using the top 10 DEPs in both psoriasis and eczema (20 DEPs total), which resulted in relatively distinct clustering of eczema and psoriasis with minimal admixing (FIG. 15D). To quantify the extent of overlap, unsupervised Gaussian mixture modeling was performed on principal components (PC) 1 and 2, which optimally identified two clusters. Cluster 1 contained 88.6% of psoriasis samples (39 / 44) while cluster 2 contained 96.8% of eczema samples (30 / 31) (FIG. 15E, FIG. 18).

[0175] It was then investigated which protein biomarkers were most representative of each PC, and it was found that NOS2 was most positively correlated with PCI while CCL17 and CCL19 were most positively correlated with PC2 (FIG. 15F). Similar analyses were also performed using all measured proteins as well as all cytokines / chemokines (FIG. 19A-FIG. 19B). Considerable overlap was found using all measured proteins, highlighting similarities in the overall proteome between the two diseases; in contrast, when only cytokines / chemokines were used, the result was comparable to that of the top 20 DEPs in accordance with the distinct immunology of the two conditions.

[0176] As an alternative form of dimensionality reduction, uniform manifold approximation and projection (UMAP) was also performed using the top 10 DEPs for each condition. This resulted in two spatially distinct clusters, one containing the majority of psoriasis (38 / 44, 86.4%) and the other containing the majority of eczema samples (30 / 31, 96.8%) (FIG. 20). Notably, all overlapped samples in the PCA analysis were also identified as overlapped in the UMAP analysis, suggesting that unique underlying proteomic changes might account for the clustering patterns observed in these samples, as opposed to a limitation in accuracy.

[0177] Given the close association of NOS2 and CCL17 with PCI and PC2 from the PCA analysis, it was inquired whether these two biomarkers alone would enable discrimination between psoriasis and eczema. Indeed, it was found that this analysis yielded similar results to the PCA analysis; one majority psoriasis cluster (38 / 39, 97%) and one majority eczema cluster (30 / 36, 83%) emerged. A single eczema sample with elevated NOS2 and 6 psoriasis samples with low NOS2 overlapped between the clusters (FIG. 21). These were the same overlapping samples identified in the PCA and UMAP analyses, indicating that low NOS2 expression was a hallmark of the psoriasis subset that proteomically clustered with eczema, and high NOS2 expression characterized the eczema sample that clustered with psoriasis. Lastly, a logistic regression (LR) machine learning classifier was trained and tested to identify a set of protein biomarkers that enabled optimal classification of psoriasis and eczema samples. All measured protein values were first standardized by removing the mean and scaling to unit variance, and then recursive feature elimination (RFE) was used to select a subset of proteins that resulted in the best accuracy. The LR classifier identified a minimum set of 5 proteins (PI3, IL-12B, CCL17, CCL2, CXCL13) that led to 100% accurate discrimination between psoriasis and eczema (FIG. 22). Interestingly, when the NOS2-low psoriasis and NOS2- high eczema patients were removed from the analysis, an LR classifier could be trained using only NOS2 and CCL17 that enabled 100% accurate diagnosis of psoriasis and eczema. Taken together, these findings implied to us that these “outlier” cases are biologically unique with non- canonical immunology as opposed to an error in the clustering approach.

[0178] Analysis of psoriasis samples reveals immunologic heterogeneity and distinct NOS2 high and low subtypes

[0179] Next, it was sought to better understand differences between the low NOS2 (n=6) and high NOS2 (n=38) psoriasis samples. Clinically, there was no significant difference in BSA involvement between the NOS2-low and NOS2-high cohorts (P=0.57) although the IGA was significantly lower in the NOS2-low cohort (P=0.026) (FIG. 23 A). Histologically, those cases that were biopsied from the NOS2-low demonstrated histologic features of psoriasis (regular acanthosis, confluent parakeratosis, diminution of the granular cell layer, and dilated, tortuous vessels in the dermal papillae) but also showed superimposed changes of eczema-like histology, including focal spongiosis and serum, supporting an overlap phenotype (FIG. 23 A).

[0180] DEPs between the two groups were then examined. It was found that in addition to NOS2, many of the most significant DEPs in the NOS2-high group related to classic psoriasis immunology and included IL-17A and IL-17F (Thl7), IL-12 and IL-12p40 (Thl), IL-17C, IL- 36A, CXCL1, CXCL8, and CCL20 (FIG. 23B). Interestingly, several proteins classically associated with eczema, including IL-4, CCL17, and CCL22, were differentially upregulated in the low NOS2 group, again supporting the notion of overlapping immunologic features with eczema.

[0181] To identify a minimum subset of immunologic proteins that could distinguish the NOS2- high and NOS2-low cohorts, an LR classifier was trained and tested on all cytokines and chemokines. Protein values were standardized, and RFE was used to select a subset of proteins that resulted in the best accuracy. This process identified IL-17A and CCL17 as a minimum set of proteins that enabled prediction of low NOS2 versus high N0S2 cases with 100% accuracy by the LR classifier (FIG. 23C). IL-17A was downregulated and CCL17 was upregulated in the NOS2-low cases. Together, these findings explained the initial clustering of the NOS2-low psoriasis cases more closely to eczema compared other psoriasis cases (FIG. 15D).

[0182] To further explore immunologic heterogeneity within psoriasis, heatmaps were created using hierarchical unsupervised clustering of all cytokines and chemokines (FIG. 23D-FIG. 23E). The low NOS2 psoriasis cases were generally found to have cytokine / chemokine profiles that were more similar to healthy controls than were the profiles of high NOS2 psoriasis cases. Interestingly, 3 out of 4 psoriasis samples with the highest mean cytokine and chemokine protein values were obtained from patients with new onset guttate psoriasis secondary to group A strep infection and were the only such patients in this study (A200, A201, and A207). Treatment relevant cytokines, such as IL17-A, IL-17F, IL-12B, IFN-y, and IL-36A, also tended to be lower in the low NOS2 cases compared to high NOS2 cases; however, these differences were only significant in IL17-A, IL- 17F, and IL-12B (FIG. 24A-FIG. 24B / Figure 4). Further, significant intra-patient variability in cytokine / chemokine profiles within each group was apparent, particularly for IL-17F and IFN-y. Given that each of these cytokines is targeted by distinct therapies, these differences may have implications for treatment response. Such variability was present in other cytokines as well (FIG. 25A-FIG. 25D).

[0183] Non-canonical eczema immunology is associated with non-response to IL-4 / IL-13 blockade

[0184] Many cases of eczema are driven predominantly by IL- 13 and are effectively treated with IL-4Ra blockade or IL- 13 blockade. Other cases may have more cytokine driver diversity and may be better suited for treatment with a JAK inhibitor or other approaches. Presently, there is no clinically relevant way to distinguish between the two groups other than therapeutic trial and error. To explore protein heterogeneity in eczema and its implications for therapeutic response, several unsupervised and supervised machine learning approaches were used to analyze the dataset.

[0185] First, unsupervised hierarchical clustering was performed using all ILs (FIG. 26A). This analysis identified two overarching distinct clusters; cluster 1 contained samples that were relatively more inflamed with elevated levels of multiple cytokines, while cluster 2 samples tended to be relatively less inflamed. The samples in cluster 1 were A34, A96, A105, Al 14, A126 / 127 (both from the same patient), A204, and A220. Of these, patients A105, A126 / 127, and A204 had moderate-to-severe disease and were initiated on dupilumab. Interestingly, each of these patients failed to respond or worsened on dupilumab as measured by BSA involvement and vIGA-AD score after approximately 3 months. One patient (A126 / 127) was subsequently initiated on a JAK1 inhibitor with improvement. Also of note, patient Al 14 was treated with the IL- 13 blocker tralokinumab with minimal response and was also subsequently successfully treated with a JAK1 inhibitor. The remainder of samples in Cluster 1 did not receive systemic therapy. These results suggested that certain patterns of cytokine expression, such as those identified in cluster 1, correlate with response to therapy.

[0186] Next, PCA was performed using all measured proteins (FIG. 26B). This analysis identified a central cluster with several outlier cases. The main cluster contained all patients who were responders to dupilumab (A76 and A89 / 116). Patients who were proteomic outliers included A34, A96, A105, A126 / 127, A204, and A220, all of which were contained in cluster 1 in the prior analysis, which suggested that outlier overall protein profiles also correlated with nonresponse to therapy.

[0187] After utilizing the unsupervised learning approaches above, it was then sought to use supervised learning approaches to determine whether a limited set of immune protein biomarkers could accurately predict non-response to IL- 13 blockade. In the entire dataset, 3 samples from 2 patients were identified who responded to IL- 13 blockade and 10 samples from 7 patients were identified who did not after 3-month follow-up. Notably, all of the non-responders had biopsies performed. The predominant histologic finding in all cases was spongiotic dermatitis without evidence of cutaneous T-cell lymphoma, interface dermatitis, or other processes. There was no significant difference in BSA or IGA between responders and non-responders (FIG. 27A-FIG. 27B).

[0188] An LR classifier was then trained and tested on all cytokines and chemokines; protein values were standardized, and RFE was used to select a subset of proteins that resulted in the best accuracy. It was found that 4 proteins, CXCL9, CXCL14, IFN-g, and IL-9, enabled 100% accurate classification of responders and non-responders (FIG. 26C). Heatmaps of these proteins are given in FIG. 26D. IFN-g (and downstream target CXCL9) reflect activated Type 1 immunity, which is inhibited by JAK inhibition but not TL-4 / IL-13 blockade. IL-9 is a common gamma chain cytokine relying on JAK-STAT signaling to exert its effect and similarly is inhibited by JAK inhibition but not dupilumab.

[0189] Enumerated Embodiments

[0190] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0191] Embodiment 1: A method for preparing a sample from a subject, the method comprising: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3 -(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; and d. analyzing the sample, wherein the analysis comprises a proximity extension assay.

[0192] Embodiment 2: The method of embodiment 1, wherein the composition comprises both the protease inhibitor and the phosphatase inhibitor.

[0193] Embodiment 3: The method of embodiment 1 or embodiment 2, wherein the protease inhibitor is cOmplete™ protease inhibitor cocktail.

[0194] Embodiment 4: The method of any one of embodiments 1-3, wherein the phosphatase inhibitor is PhosSTOP™.

[0195] Embodiment 5: The method of any one of embodiments 1-4, wherein the sample comprises at least one analyte.

[0196] Embodiment 6: The method of embodiment 5, wherein the at least one analyte comprises at least one cytokine.

[0197] Embodiment 7: The method of embodiment 5 or embodiment 6, wherein the at least one analyte is IL-13, IL-23, IL-17, NOS2, CCL17, interferon (IFN)-gamma, or any combination thereof.

[0198] Embodiment 8: The method of embodiment 5 or embodiment 6, wherein the at least one analyte is IL-17A, IL-17F, IFN-y, IL-12, IL- 12p40, CXCL9, CXCL10, CXCL11, IL-4, CCL2, CCL13, CCL17, CCL18, CCL19, CCL22, or any combination thereof. Embodiment 9: The method of embodiment 5 or embodiment 6, wherein the at least one analyte is NOS2, NT5C3A, GBP1, PI3, TMPRSS1 ID, or any combination thereof.

[0199] Embodiment 10: The method of embodiment 5 or embodiment 6, wherein the at least one analyte is MMP7, MMP10, ITGAX, EGF, or any combination thereof.

[0200] Embodiment 11 : The method of embodiment 5 or embodiment 6, wherein the at least one analyte is IL-17A, IL-23, IFN- y, IL-12, IL-15, STAT1, TNF, NF-K , IL-36, or any combination thereof.

[0201] Embodiment 12: The method of embodiment 5 or embodiment 6, wherein the at least one analyte is IL- 13, IL- 10, CCL2, or any combination thereof.

[0202] Embodiment 13: The method of embodiment 5 or embodiment 6, wherein the at least one analyte is PI3, IL-12B, CCL17, CCL2, CXCL13, or any combination thereof

[0203] Embodiment 14: The method of embodiment 5 or embodiment 6, wherein the at least one analyte is NOS2, CCL17, or a combination thereof.

[0204] Embodiment 15: The method of embodiment 5 or embodiment 6, wherein the at least one analyte is IL-17A, IL-17F, IL-12, IL-12p40, IL-17C, IL-36A, CXCL1, CXCL8, CCL20, or any combination thereof.

[0205] Embodiment 16: The method of embodiment 5 or embodiment 6, wherein the at least one analyte is CXCL9, CXCL14, IFN- y, IL-9, or any combination thereof.

[0206] Embodiment 17: The method of embodiment 5 or embodiment 6, wherein the at least one analyte is IL-4, CCL17, CCL22, or any combination thereof.

[0207] Embodiment 18: The method of embodiment 5 or embodiment 6, wherein the at least one analyte is IL-17A, CCL17, or any combination thereof.

[0208] Embodiment 19: The method of any one of embodiments 1-18, wherein the analysis comprises generating a cytokine profde of the subject.

[0209] Embodiment 20: The method of any one of embodiments 1-19, wherein the analysis comprises machine learning and / or artificial intelligence-based approaches.

[0210] Embodiment 21 : The method of any one of embodiments 1-20, wherein the analysis is performed on from about 1 to about 1,000 different individual samples.

[0211] Embodiment 22: The method of embodiment 21, wherein the different individual samples represent at least two different diseases. Embodiment 23: The method of any one of embodiments 1-22, wherein the analysis is used to select a therapy to administer to the subject.

[0212] Embodiment 24: The method of any one of embodiments 1-23, wherein the region of interest comprises a fungal infection, a bacterial infection, a viral infection, and / or an arthropod infestation.

[0213] Embodiment 25: The method any one of embodiments 1-24, wherein the region of interest comprises an immune-mediated disease.

[0214] Embodiment 26: The method of embodiment 25, wherein the immune-mediated disease is psoriasis or eczema.

[0215] Embodiment 27: The method of any one of embodiments 1-26, wherein the region of interest comprises a skin lymphoma.

[0216] Embodiment 28: The method of any one of embodiments 1-27, wherein the area of the subject from which the sample is derived is from about 1 mm2to about 3 mm2.

[0217] Embodiment 29: The method of any one of embodiments 1-28, wherein the collection device comprises an indicator card.

[0218] Embodiment 30: The method of embodiment 29, wherein the card is used to detect one or more proteins.

[0219] Embodiment 31 : The method of embodiment 29 or embodiment 30, wherein the indicator card comprises one or more immobilized antibodies.

[0220] Embodiment 32: The method of embodiment 31, wherein the one or more immobilized antibodies bind to a cytokine.

[0221] Embodiment 33: A kit for generating a sample from a subject, the kit comprising: a. a composition comprising: i. a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. a collection device; c. an indicator card; and d. instructions for use.

[0222] Embodiment 34: A method of detecting an immune-mediated disease, a fungal infection, or a skin lymphoma in a subject, wherein the method comprises: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; d. analyzing the sample to determine a level of at least one immune-mediated disease- related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and comparing the level of the at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule with a predetermined reference level for at least one immune- mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and wherein if the level of the at least one immune- mediated disease-related molecule is above the respective reference level, the subject is determined to have an immune-mediated disease; wherein if the level of the at least one fungal infection-related molecule is above the respective reference level, the subject is determined to have a fungal infection; wherein if the level of the at least one skin lymphoma-related molecule is above the respective reference level, the subject is determined to have skin lymphoma.

[0223] Embodiment 35: The method of embodiment 34, wherein the analysis comprises a proximity extension assay.

[0224] Embodiment 36: The method of embodiment 34 or embodiment 35, wherein the immune- mediated disease is psoriasis or eczema.

[0225] Embodiment 37: The method of any one of embodiments 34-36, wherein the analysis comprises generating a cytokine profde of the subject.

[0226] Embodiment 38: The method of any one of embodiments 34-37, wherein the at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule comprises at least one protein.

[0227] Embodiment 39: The method of embodiment 38, wherein the method comprises at least one cytokine.

[0228] Embodiment 40: The method of embodiment 38 or embodiment 39, wherein the at least one protein is IL-13, IL-17, IL-23, CCL17, NOS2, interferon (IFN)-gamma, or any combination thereof.

[0229] Embodiment 41 : The method of embodiment 38 or embodiment 39, wherein the at least one protein is IL-17A, IL-17F, IFN-y, IL- 12, IL- 12p40, CXCL9, CXCL10, CXCL11, IL-4, CCL2, CCL13, CCL17, CCL18, CCL19, CCL22, or any combination thereof. Embodiment 42: The method of embodiment 38 or embodiment 39, wherein the at least one protein is NOS2, NT5C3A, GBP1, PI3, TMPRSS11D, or any combination thereof.

[0230] Embodiment 43: The method of embodiment 38 or embodiment 39, wherein the at least one protein is MMP7, MMP10, ITGAX, EGF, or any combination thereof.

[0231] Embodiment 44: The method of embodiment 38 or embodiment 39, wherein the at least one protein is IL-17A, IL-23, IFN- y, IL-12, IL-15, STAT1, TNF, NF-KP, IL-36, or any combination thereof.

[0232] Embodiment 45: The method of embodiment 38 or embodiment 39, wherein the at least one protein is PI3, IL-12B, CCL17, CCL2, CXCL13, or any combination thereof.

[0233] Embodiment 46: The method of embodiment 38 or embodiment 39, wherein the at least one protein is NOS2, CCL17, or a combination thereof.

[0234] Embodiment 47: The method of embodiment 38 or embodiment 39, wherein the at least one protein is IL-17A, IL-17F, IL- 12, IL-12p40, IL-17C, IL-36A, CXCL1, CXCL8, CCL20, or any combination thereof.

[0235] Embodiment 48: The method of embodiment 38 or embodiment 39, wherein the at least one protein is CXCL9, CXCL14, IFN- y, IL-9, or any combination thereof.

[0236] Embodiment 49: The method of embodiment 38 or embodiment 39, wherein the at least one protein is IL-4, CCL17, CCL22, or any combination thereof.

[0237] Embodiment 50: The method of embodiment 38 or embodiment 39, wherein the at least one protein is IL-17A, CCL17, or any combination thereof.

[0238] Embodiment 51 : The method of any one of embodiments 34-50, wherein the analysis is used to select a therapy to administer to the subject.

[0239] Embodiment 52: The method of any one of embodiments 34-51, wherein the subject is administered a therapy to treat the immune-mediated disorder, fungal infection, or skin lymphoma.

[0240] Embodiment 53 : a method of preparing and analyzing a sample representative of a fungal infection, or a skin lymphoma in a subject, wherein the method comprises: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; d. analyzing the sample to determine a level of at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and e. comparing the level of the at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule with a predetermined reference level for at least one immune- mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and wherein if the level of the at least one immune- mediated disease-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of an immune-mediated disease; wherein if the level of the at least one fungal infection-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of a fungal infection; wherein if the level of the at least one skin lymphoma-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of a skin lymphoma.

[0241] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

[0242] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0243] In sum, while this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS1. A method for preparing a sample from a subject, the method comprising: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3 -(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; and d. analyzing the sample, wherein the analysis comprises a proximity extension assay.

2. The method of claim 1, wherein the composition comprises both the protease inhibitor and the phosphatase inhibitor.

3. The method of claim 1 or claim 2, wherein the protease inhibitor is cOmplete™ protease inhibitor cocktail.

4. The method of any one of claims 1-3, wherein the phosphatase inhibitor PhosSTOP™.

5. The method of any one of claims 1-4, wherein the sample comprises at least one analyte.

6. The method of claim 5, wherein the at least one analyte comprises at least one cytokine.

7. The method of claim 5 or claim 6, wherein the at least one analyte is IL-13, IL -23, IL-17, interferon (IFN)-gamma, or any combination thereof.

8. The method of claim 5 or claim 6, wherein the at least one analyte is IL-17A, IL-17F, IFN-y, IL-12, IL- 12p40, CXCL9, CXCL10, CXCL11, IL-4, CCL2, CCL13, CCL17, CCL18, CCL19, CCL22, or any combination thereof.

9. The method of claim 5 or claim 6, wherein the at least one analyte is NOS2, NT5C3A, GBP1, PI3, TMPRSS1 ID, or any combination thereof.

10. The method of claim 5 or claim 6, wherein the at least one analyte is MMP7, MMP10, ITGAX, EGF, or any combination thereof.

11. The method of claim 5 or claim 6, wherein the at least one analyte is IL-17A, IL-23, IFN- y, IL- 12, IL- 15, STAT1, TNF, NF-KP, IL-36, or any combination thereof.

12. The method of claim 5 or claim 6, wherein the at least one analyte is IL-13, IL-10, CCL2, or any combination thereof.

13. The method of claim 5 or claim 6, wherein the at least one analyte is PI3, IL-12B, CCL17, CCL2, CXCL13, or any combination thereof.

14. The method of claim 5 or claim 6, wherein the at least one analyte is NOS2, CCL17, or a combination thereof.

15. The method of claim 5 or claim 6, wherein the at least one analyte is IL-17A, IL-17F, IL- 12, IL-12p40, IL-17C, IL-36A, CXCL1, CXCL8, CCL20, or any combination thereof.

16. The method of claim 5 or claim 6, wherein the at least one analyte is CXCL9, CXCL14, IFN- y, IL-9, or any combination thereof.

17. The method of claim 5 or claim 6, wherein the at least one analyte is IL-4, CCL17, CCL22, or any combination thereof.

18. The method of claim 5 or claim 6, wherein the at least one analyte is IL-17A, CCL17, or any combination thereof.

19. The method of any one of claims 1-18, wherein the analysis comprises generating a cytokine profde of the subject.

20. The method of any one of claims 1-19, wherein the analysis comprises machine learning and / or artificial intelligence-based approaches.21 . The method of any one of claims 1-20, wherein the analysis is performed on from about 1 to about 1,000 different individual samples.

22. The method of claim 21, wherein the different individual samples represent at least two different diseases.

23. The method of any one of claims 1-22, wherein the analysis is used to select a therapy to administer to the subject.

24. The method of any one of claims 1-23, wherein the region of interest comprises a fungal infection, a bacterial infection, a viral infection, and / or an arthropod infestation.

25. The method any one of claims 1-24, wherein the region of interest comprises an immune- mediated disease.

26. The method of claim 25, wherein the immune-mediated disease is psoriasis or eczema.

27. The method of any one of claims 1-26, wherein the region of interest comprises a skin lymphoma.

28. The method of any one of claims 1-27, wherein the area of the subject from which the sample is derived is from about 1 mm2to about 3 mm2.

29. The method of any one of claims 1-28, wherein the collection device comprises an indicator card.

30. The method of claim 29, wherein the card is used to detect one or more proteins.

31. The method of claim 29 or claim 30, wherein the indicator card comprises one or more immobilized antibodies.

32. The method of claim 31, wherein the one or more immobilized antibodies bind to a cytokine.

33. A kit for generating a sample from a subject, the kit comprising: a. a composition comprising:i. a 3-(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. a collection device; c. an indicator card; and d. instructions for use.

34. A method of detecting an immune-mediated disease, a fungal infection, or a skin lymphoma in a subject, wherein the method comprises: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3 -(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor; b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; d. analyzing the sample to determine a level of at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and e. comparing the level of the at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule with a predetermined reference level for at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; andwherein if the level of the at least one immune-mediated disease-related molecule is above the respective reference level, the subject is determined to have an immune- mediated disease; wherein if the level of the at least one fungal infection-related molecule is above the respective reference level, the subject is determined to have a fungal infection; wherein if the level of the at least one skin lymphoma-related molecule is above the respective reference level, the subject is determined to have skin lymphoma.

35. The method of claim 34, wherein the analysis comprises a proximity extension assay.

36. The method of claim 34 or claim 35, wherein the immune-mediated disease is psoriasis or eczema.

37. The method of any one of claims 34-36, wherein the analysis comprises generating a cytokine profile of the subject.

38. The method of any one of claims 34-37, wherein the at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule comprises at least one protein.

39. The method of claim 38, wherein the at least one protein comprises at least one cytokine.

40. The method of claim 38 or 39, wherein the at least one protein is IL-13, IL-17, IL-23, CCL17, NOS2, interferon (IFN)-gamma, or any combination thereof.

41. The method of claim 38 or claim 39, wherein the at least one protein is IL-17A, IL-17F, IFN-Y, IL-12, IL- 12p40, CXCL9, CXCL10, CXCL11, IL-4, CCL2, CCL13, CCL17, CCL18, CCL19, CCL22, or any combination thereof.

42. The method of claim 38 or claim 39, wherein the at least one protein is NOS2, NT5C3A, GBP1, PI3, TMPRSS1 ID, or any combination thereof.

43. The method of claim 38 or claim 39, wherein the at least one protein is MMP7, MMP10, ITGAX, EGF, or any combination thereof.

44. The method of claim 38 or claim 39, wherein the at least one protein is IL-17A, IL-23, IFN- y, IL-12, IL-15, STAT1, TNF, NF-K|3, IL-36, or any combination thereof.

45. The method of claim 38 or claim 39, wherein the at least one protein is PI3, IL-12B, CCL17, CCL2, CXCL13, or any combination thereof.

46. The method of claim 38 or claim 39, wherein the at least one protein is NOS2, CCL17, or a combination thereof.

47. The method of claim 38 or claim 39, wherein the at least one protein is IL-17A, IL-17F, IL-12, IL-12p40, IL-17C, IL-36A, CXCL1, CXCL8, CCL20, or any combination thereof.

48. The method of claim 38 or claim 39, wherein the at least one protein is CXCL9, CXCL14, IFN- y, IL-9, or any combination thereof.

49. The method of claim 38 or claim 39, wherein the at least one protein is IL-4, CCL17, CCL22, or any combination thereof.

50. The method of claim 38 or claim 39, wherein the at least one protein is IL-17A, CCL17, or any combination thereof.

51. The method of any one of claims 34-50, wherein the analysis is used to select a therapy to administer to the subject.

52. The method of any one of claims 34-51, wherein the subject is administered a therapy to treat the immune-mediated disorder, fungal infection, or skin lymphoma.

53. A method of preparing and analyzing a sample representative of a fungal infection, or a skin lymphoma in a subject, wherein the method comprises: a. contacting skin cells of a region of interest of the subject with a composition comprising: i. a 3 -(alkyl dimethyl ammonia) propane sulfonate zwitterionic surfactant; a polyethylene glycol alkyl ether nonionic surfactant; and ii. a protease inhibitor and / or a phosphatase inhibitor;b. contacting the region of interest with a collection device; c. collecting a sample of the region of interest using the collection device; and d. analyzing the sample to determine a level of at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and e. comparing the level of the at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule with a predetermined reference level for at least one immune-mediated disease-related molecule, at least one fungal infection-related molecule, and / or at least one skin lymphoma-related molecule; and wherein if the level of the at least one immune-mediated disease-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of an immune-mediated disease; wherein if the level of the at least one fungal infection-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of a fungal infection; wherein if the level of the at least one skin lymphoma-related molecule is above the respective reference level, the sample derived from the subject is determined to be representative of a skin lymphoma.

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