Methods for detecting food-specific immune responses

Abstract

Methods for diagnosing and treating a subject with active or inactive eosinophilic esophagitis (EoE) are disclosed. The methods may include detecting whether the level of one or more immunoglobulin antibodies is elevated in an esophageal secretion sample obtained from the subject, diagnosing the subject with active EoE when the level of the one or more immunoglobulin antibodies in the sample is elevated above a predetermined cutoff value, diagnosing the subject with inactive EoE when the level of the one or more immunoglobulin antibodies in the sample is below the predetermined cutoff value, and treating the subject diagnosed with active EoE.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 912,505, filed October 8, 2019. The contents of this previously filed application are hereby incorporated by reference in their entirety. [Background technology]

[0002] Food antigens are involved in the induction and maintenance of eosinophilic esophagitis. Dietary elimination to identify triggers is invasive, laborious, and expensive. Alternatives are needed that can reduce costs and improve patients' quality of life during this process. Summary of the Invention

[0003] Disclosed herein is a method for detecting binding of one or more food-specific immunoglobulin (Ig) antibodies to one or more food antigens in a sample, the method comprising contacting a sample obtained from a subject with one or more food antigens, and detecting binding of the one or more food-specific Ig antibodies to the one or more food antigens.

[0004] Disclosed herein is a method for detecting a food-specific immune response in a sample, the method comprising: a) contacting the sample with one or more food antigens; b) determining the presence or level of one or more immunoglobulin (Ig) antibodies bound to the one or more food antigens; and c) comparing the level of the one or more Ig antibodies in the sample bound to the one or more food antigens of b) with the level of the one or more Ig antibodies bound to the one or more food antigens in a reference sample, thereby detecting a food-specific immune response in the sample when the level of the one or more Ig antibodies bound to the one or more food antigens in the sample is higher than the level of the one or more Ig antibodies in the reference sample.

[0005] Disclosed herein is a method for identifying food allergies in a subject suffering from eosinophilic esophagitis (EoE), the method comprising: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more food antigens in a sample; and b) comparing the level of the one or more Ig antibodies bound to the one or more food antigens in the sample with the level of the one or more Ig antibodies bound to the one or more food antigens in a reference sample, wherein the level of the one or more Ig antibodies bound to the one or more food antigens in the sample is higher than the level of the one or more Ig antibodies bound to the one or more food antigens in the reference sample, identifying the food allergy in the subject.

[0006] Disclosed herein is a method of treating a subject with active or inactive eosinophilic esophagitis (EoE) in a subject suffering from EoE, the method comprising: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more food antigens in a sample; b) comparing the level of one or more Ig antibodies bound to the one or more food antigens in the sample with the level of one or more Ig antibodies bound to the one or more food antigens in a reference sample; and c) treating the subject.

[0007] Disclosed herein is a method of treating a subject with active eosinophilic esophagitis (EoE) in a subject suffering from EoE, the method comprising: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more food antigens in a sample; b) comparing the level of one or more Ig antibodies bound to the one or more food antigens in the sample with the level of one or more Ig antibodies bound to the one or more food antigens in a reference sample; and c) treating the subject.

[0008] Disclosed herein are methods for diagnosing and treating active or inactive Eosinophilic Esophagitis (EoE) in a subject suffering from EoE, the methods comprising: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more food antigens in a sample; b) comparing the level of the one or more Ig antibodies bound to the one or more food antigens in the sample with the level of the one or more Ig antibodies bound to the one or more food antigens in a reference sample; and c) detecting whether the level of the one or more Ig antibodies bound to the one or more food antigens in the sample is greater than or equal to the level of the one or more food antigens in the reference sample. and diagnosing the subject with active EoE when the level of one or more Ig antibodies bound to the one or more food antigens in the sample is higher than the level of one or more Ig antibodies bound to the one or more food antigens in the reference sample levels, and diagnosing the subject with inactive EoE when the level of one or more Ig antibodies bound to the one or more food antigens in the sample is lower than the level of one or more Ig antibodies bound to the one or more food antigens in the reference sample levels; and d) treating the subject diagnosed with active EoE, wherein the level of one or more Ig antibodies bound to the one or more food antigens in the sample is higher than the level of one or more Ig antibodies bound to the one or more food antigens in the reference sample.

[0009] Disclosed herein is a method for diagnosing and treating eosinophilic esophagitis in a subject in need thereof, the method comprising: (i) measuring a gene expression level of at least one gene in an esophageal biopsy sample from a patient, wherein the at least one gene is selected from the group consisting of eosinophil peroxidase, periostin, eotaxin-3, stem cell factor, or CCL26, KITLG, and POSTN, and measuring the gene expression level is performed by a method comprising DNA microarray analysis, polymerase chain reaction analysis, or both; ii) comparing the gene expression level of the at least one gene to its expression level in an esophageal biopsy sample from a normal individual, defined as zero eosinophils per high power field and no basal lamina swelling, and diagnosing eosinophilic esophagitis in the subject if the expression level of the at least one gene is increased by more than 10-fold compared to its expression level in the esophageal biopsy sample from the normal individual; and (iv) treating eosinophilic esophagitis in the subject diagnosed according to step (iii) with one or more therapies selected from anti-inflammatory therapy, allergen removal, cytokine inhibitors, immunosuppressants, and complement inhibitors.

[0010] Disclosed herein are methods for diagnosing Eosinophilic Esophagitis (EoE) by detecting one or more biomarkers selected from the group consisting of eosinophil peroxidase, periostin, eotaxin-3, stem cell factor, or CCL26, KITLG, and POSTN.

[0011] Disclosed herein is a method for detecting the cause of symptoms of eosinophilic esophagitis (EoE), which symptoms of EoE are caused by an immune response to an antigen in a patient diagnosed with or suffering from EOE, the method comprising obtaining or obtaining an esophageal mucosal sample from a subject, where the esophageal mucosal sample obtained from the subject is derived from a site of an immune response, and where the esophageal mucosal sample contains one or more immunoglobulin (Ig) antibodies, contacting the sample with one or more antigens, and detecting binding of the one or more specific Ig antibodies to the one or more antigens. [Brief description of the drawings]

[0012] [Figure 1] Immunoglobulin A response values ​​for gluten, casein, soy, and egg are shown for the cohorts: EoE (n=43), resolved EoE (n=13), and controls (n=12). Resolved patients were not significantly different from controls in any pairwise tests. Active EoE differed from resolved patients in terms of antibody response levels, with significantly increased antibody values ​​to gluten, casein, and egg compared to the other two groups. IgA=immunoglobulin A. [Diagram 2] Immunoglobulin G4 response values ​​to gluten, casein, soy, and egg cohorts (active EoE, resolved EoE, and controls) are shown. Active EoE showed significantly higher Immunoglobulin G4 antibody levels to the four food antigens compared to resolved EoE and controls. IgG4 = Immunoglobulin G4. [Diagram 3] Esophageal eosinophil infiltration and endoscopic scoring before and after introduction of an elimination diet in 17 patients are shown. [Figure 4A] Figure 4 shows Immunoglobulin A and Immunoglobulin G4 antibodies in esophageal secretions of EoE patients, compared with trigger and non-trigger foods. Figure 4A shows the Immunoglobulin A response to trigger and non-trigger foods. IgA = Immunoglobulin A. IgG4 = Immunoglobulin G4 [Figure 4B]Figure 4B shows Immunoglobulin A and Immunoglobulin G4 antibodies in esophageal secretions of EoE patients, comparing trigger foods with non-trigger foods. Figure 4B shows a heat map depicting the Immunoglobulin A response in each individual to trigger foods compared to non-trigger foods. Red represents the higher response levels detected. Most trigger foods produced response values ​​in the higher range. IgA = Immunoglobulin A. IgG4 = Immunoglobulin G4 [Figure 4C] Figure 4 shows Immunoglobulin A and Immunoglobulin G4 antibodies in esophageal secretions of EoE patients, compared with trigger and non-trigger foods. Figure 4C shows the Immunoglobulin G4 response to trigger and non-trigger foods. IgA = Immunoglobulin A. IgG4 = Immunoglobulin G4. [Figure 4D] Figure 4 shows Immunoglobulin A and Immunoglobulin G4 antibodies in esophageal secretions of EoE patients, compared with trigger and non-trigger foods. Figure 4D shows a heat map of Immunoglobulin G4 responses to trigger and non-trigger foods in each individual. IgA = Immunoglobulin A. IgG4 = Immunoglobulin G4. [Diagram 5] Immunoglobulin A and Immunoglobulin G4 antibody levels against gluten and casein as trigger and non-trigger foods are shown. Casein Immunoglobulin A responses are significantly elevated in those with a dairy trigger as opposed to those with no dairy trigger. Immunoglobulin G4 response values ​​were not significantly different between those with and without a known dairy trigger. However, this was likely due to one large outlier in the non-trigger group. Wheat response values ​​for Immunoglobulin A were significantly elevated in those whose EoE trigger was wheat. Immunoglobulin G4 response values ​​again did not reliably distinguish between those in whom wheat was a trigger food or not in this limited cohort. IgA=Immunoglobulin A. IgG4=Immunoglobulin G4. [Figure 6A]Uniform sampling across the area of ​​esophageal eosinophilia shows similar affinities for specific foods and that sampling is easily reproducible. This indicates that sampling is uniform across the affected area and results are similar in two different scrapings. Figure 6A shows food-specific antibody (FSA)-IgA diffuse panesophageal sampling - peak count 132 eosinophils / HPF and panesophagitis (i.e., involvement of the entire esophagus with inflammation). [Figure 6B] Uniform sampling across the area of ​​esophageal eosinophilia shows similar affinities for specific foods and that sampling is easily reproducible. This indicates that sampling is uniform across the affected area and results are similar in two different scrapings. Figure 6B shows FSA-IgG4 pan-sampling of the esophagus at 95 eosinophils and total disease. [Figure 6C] Uniform sampling across the area of ​​esophageal eosinophilia shows similar affinities for specific foods and that sampling is easily reproducible. This indicates that sampling is uniform across the affected area and results are similar in two different scrapings. Figure 6C shows FSA-IgA pan-esophageal sampling of the esophagus with 95 eosinophils and total disease. [Figure 6D] Uniform sampling across the area of ​​esophageal eosinophilia shows similar affinities for specific foods and that sampling is easily reproducible. This indicates that sampling is uniform across the affected area and results are similar in two different scrapings. Figure 6D shows FSA-IgA pan-sampling of the esophagus, 25-30 eosinophils, and the entire area. [Figure 7A]Results are shown for samples taken from different regions of the esophagus, specifically sampled within areas of disease and then sampled in areas without significant disease. Changes in food-specific antibody production were seen when diseased areas were sampled compared to disease-free areas, indicating that active esophageal disease itself is likely the source of food-specific antibodies. Figure 7A shows separate sampling of FSA-IgA distal disease (25 eosinophils) and proximal disease (6 eosinophils). [Figure 7B] Results are shown for samples taken from different regions of the esophagus, specifically sampled within areas of disease and then sampled in areas without significant disease. Changes in food-specific antibody production were seen when sampling diseased areas compared to disease-free areas, indicating that active esophageal disease itself is likely the source of food-specific antibodies. Figure 7B shows separate sampling of FSA-IgG4 in diseased (25) and disease-free areas (4). [Figure 7C] Results are shown for samples taken from different regions of the esophagus, specifically sampled within diseased areas and then sampled in areas without significant disease. Changes in food-specific antibody production were seen when diseased areas were sampled compared to disease-free areas, indicating that active esophageal disease itself is likely the source of food-specific antibodies. Figure 7C shows separate sampling of FSA-IgG4 in diseased and less diseased areas (22 and 1). [Figure 7D] Results are shown for samples taken from different regions of the esophagus, specifically sampled within diseased areas and then sampled in areas without significant disease. A shift in food-specific antibody production was seen when diseased areas were sampled compared to disease-free areas, indicating that active esophageal disease itself is likely the source of food-specific antibodies. Figure 7D shows separate sampling of FSA-IgA in diseased and less diseased areas (22 vs. 1). [Figure 8A] Figure 8 shows the protein expression of selected biomarkers in esophageal secretions. Figure 8A shows eotaxin. [Figure 8B] Figure 8 shows the protein expression of selected biomarkers in esophageal secretions. Figure 8B shows SCF. [Figure 8C] Protein expression of selected biomarkers in esophageal secretions is shown. Figure 8C shows periostin. [Figure 9A] We show that food-specific antibodies in esophageal secretions are diagnostic of EoE, but not saliva or serum. The wheat:casein:egg response ratios differed in the three patients who had simultaneous sampling of saliva (whole saliva), serum, and esophageal secretions during endoscopy. Figure 9A shows one patient who was unable to complete dietary elimination but was symptomatic with dairy products. As can be seen, esophageal secretions showed high response values ​​to gluten, while serum and saliva had elevated responses to other food antigens, indicating that this participant likely had a local response from diseased tissue. Contamination from serum or saliva should create a similar response pattern in the esophageal collection. This is not seen here. [Figure 9B] This indicates that food-specific antibodies in esophageal secretions are diagnostic of EoE, but not saliva or serum. The wheat:casein:egg response ratios differed in the three patients who had simultaneous sampling of saliva (whole saliva), serum, and esophageal secretions during endoscopy. Figure 9B shows a patient who opted for topical steroids. As can be seen, esophageal secretions showed high response values ​​to gluten, while serum and saliva had elevated responses to other food antigens, indicating that this participant likely had a local response from diseased tissue. Contamination from serum or saliva should create a similar response pattern in the esophageal collection. This is not seen here. [Figure 9C]This shows that food-specific antibodies in esophageal secretions are diagnostic of EoE, but not saliva or serum. The ratio of wheat:casein:egg responses differed in the three patients who underwent simultaneous sampling of saliva (whole saliva), serum, and esophageal secretions during endoscopy. Figure 9C was performed on a patient who eventually underwent a food elimination diet (elimination of six foods). Food reintroduction identified wheat as a trigger food in this patient. In Figure 9C, the patient's EoE was found to be induced by wheat. As can be seen, esophageal secretions showed high response values ​​to gluten, while serum and saliva had elevated responses to other food antigens, indicating that this participant likely had a local response from diseased tissue. Contamination from serum or saliva should create a similar response pattern in the esophageal collection. This is not seen here. [Figure 10] 1 shows small intestine relative values ​​of food-specific IgA for four patients with abdominal pain and discomfort. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention may be understood more readily by reference to the following detailed description of the various aspects of the invention and the Examples contained therein, as well as the drawings and their preceding and following descriptions.

[0014] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods or to specific radiolabeled imaging agents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the disclosed method and composition belongs.Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed method and composition, but particularly useful methods, devices and materials are as described.

[0016] It is understood that the disclosed methods and compositions are not limited to the particular methodology, protocols, and reagents described, which may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the scope of the appended claims.

[0017] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0018] As used herein, the word "or" means any one element of a particular list and also includes any combination of elements of that list.

[0019] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which the event or circumstance occurs as well as instances in which it does not occur.

[0020] As used herein, the term "sample" refers to a tissue or organ from a subject, a cell (either within a subject, taken directly from a subject, or cells maintained in culture or from a cultured cell line), a cell lysate (or lysate fraction) or cell extract, or a solution containing one or more molecules derived from cells or cellular material (e.g., polypeptides or nucleic acids). A sample may also be any bodily fluid or excretion containing cells or cellular components (e.g., but not limited to, blood, urine, stool, saliva, tears, bile), or excretion from a disease state.

[0021] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of "about," it will be understood that the particular value forms another embodiment. It will be further understood that each of the endpoints of a range has significance in relation to the other endpoint, and independently of the other endpoint.

[0022] As used herein, the word "comprise" and variations of this word, such as "comprising" and "comprises," mean "including, but not limited to," and are not intended to exclude, for example, other addenda, components, integers, or steps.

[0023] As used herein, "subject" means an individual. The subject may be a mammal, such as a primate, e.g., a human. The term "subject" also includes domestic animals, such as cats, dogs, and the like, livestock, such as cows, horses, pigs, sheep, goats, and the like, and laboratory animals, such as mice, rabbits, rats, gerbils, opossums, and the like. As used herein, the terms "subject" and "patient" are interchangeable.

[0024] As used herein, the term "patient" refers to a subject suffering from a disease or disorder. The term "patient" includes human and veterinary subjects. In some aspects of the disclosed methods, the "patient" has been diagnosed as in need of treatment, e.g., prior to the administration step.

[0025] As used herein, "mucosal tissue" refers to tissue lining various cavities in the body. Examples of mucosal tissue include, but are not limited to, the nose, paranasal sinuses, bronchi, lungs, conjunctiva, oral cavity, tongue, esophagus, stomach, pylorus, duodenum, jejunum, ileum, ascending colon, cecum, appendix, transverse colon, descending colon, rectum, anus, urethra, dermis, small bowel (small intestine), large bowel (large intestine), biliary and biliary systems, and bladder. Mucosal tissue includes submucosa, which has an epithelial surface, mucus-secreting glandular epithelium, basement membrane, and connective tissue. In some embodiments, the mucosal tissue is from the esophagus of a subject. In some embodiments, the mucosal tissue is from the intestine of a subject. In some embodiments, the mucosal tissue is gastrointestinal mucosal tissue. In some embodiments, the mucosal tissue is gastrointestinal mucosal tissue secretion. In some embodiments, the mucosal tissue is small intestinal fluids from the intestinal lining, hi some embodiments, the mucosal tissue is esophageal mucosal tissue secretions.

[0026] As used herein, "eosinophil granule proteins" are proteins that comprise granules in eosinophils. When eosinophils are activated, granule proteins are released from the cells into the surrounding tissue. The released granule proteins can cause pathological allergic inflammatory responses in the surrounding tissue, for example, esophageal mucosal tissue. Examples of eosinophil granule proteins include, but are not limited to, major basic protein (MBP), major basic protein 1 (MBP-1), major basic protein 2 (MBP-2), eosinophil-derived neurotoxin (EDN, also referred to as RNase2), eosinophil cationic protein (ECP, also referred to as RNase3), and eosinophil peroxidase (EPO). Other examples of eosinophil granule proteins are provided in Kita et al., Biology of Eosinophils, Chapter 19 of Immunology, which is incorporated herein by reference for its teaching of examples of eosinophil granule proteins. In some embodiments, the eosinophil granule protein can be MBP-1.

[0027] As used herein, the term "gene" refers to a region of DNA that codes for a functional RNA or protein. "Functional RNA" refers to an RNA molecule that is not translated into a protein. Generally, gene symbols are shown using italics and protein symbols are shown using non-italics.

[0028] The term "nucleic acid" as used herein refers to a natural or synthetic oligonucleotide or polynucleotide capable of hybridizing to a complementary nucleic acid by Watson-Crick base pairing, whether DNA, RNA, or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense. Nucleic acids of the invention may also include nucleotide analogs, and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). Specifically, nucleic acids may include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0029] "Isolated nucleic acid" or "purified nucleic acid" refers to DNA that is free of the genes that flank it in the native genome of the organism from which the DNA of the invention is derived. Thus, the term includes recombinant DNA that is incorporated into a vector, such as an autonomously replicating plasmid or virus, or that is incorporated into the genomic DNA of a prokaryotic or eukaryotic organism (e.g., a transgene), or that exists as a separate molecule (e.g., cDNA or genomic or cDNA fragments produced by PCR, restriction endonuclease digestion, or chemical or in vitro synthesis). It also includes recombinant DNA that is part of a hybrid gene that codes for additional polypeptide sequences. The term "isolated nucleic acid" also refers to RNA, such as an mRNA molecule encoded by an isolated DNA molecule, or an mRNA molecule that is chemically synthesized, or an mRNA molecule that is separated from or substantially free of at least some cellular components, such as other types of RNA molecules or polypeptide molecules.

[0030] "Specifically binds" means that the antibody recognizes and physically interacts with its cognate antigen and does not significantly recognize or interact with other antigens, and such an antibody may be a polyclonal or monoclonal antibody generated by techniques well known in the art.

[0031] "Probe", "primer", or oligonucleotide refers to a single-stranded DNA or RNA molecule of defined sequence that can base-pair with a second DNA or RNA molecule ("target") that contains a complementary sequence. The stability of the resulting hybrid depends on the degree of base-pairing that occurs. The degree of base-pairing is influenced by parameters such as the degree of complementarity between the probe and target molecule and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is influenced by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to those skilled in the art. Probes or primers specific for nucleic acids (e.g., genes and / or mRNAs) have at least 80%-90% sequence complementarity, preferably at least 91%-95% sequence complementarity, more preferably at least 96%-99% sequence complementarity, and most preferably 100% sequence complementarity, to the region of the nucleic acid to which they hybridize. Probes, primers, and oligonucleotides may be detectably labeled, either radioactively or non-radioactively, by methods well known to those of skill in the art. Probes, primers, and oligonucleotides are used in methods involving nucleic acid hybridization, such as nucleic acid sequencing, reverse transcription and / or nucleic acid amplification by polymerase chain reaction, single-strand conformation polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA).

[0032] "Specifically hybridize" means that a probe, primer, or oligonucleotide recognizes and physically interacts (i.e., base pairs) with a substantially complementary nucleic acid under high stringency conditions and does not substantially base pair with other nucleic acids.

[0033] "High stringency conditions" are 0.5M NaHPO 4High stringency hybridization refers to conditions that allow hybridization equivalent to that obtained by using a DNA probe of at least 40 nucleotides in a buffer containing 7% SDS, 1 mM EDTA, and 1% BSA (V fraction) at a temperature of 65° C., or in a buffer containing 48% formamide, 4.8x SSC, 0.2 M Tris-Cl, pH 7.6, 1x Denhardt's solution, 10% dextran sulfate, and 0.1% SDS at a temperature of 42° C. Other conditions for high stringency hybridization, such as PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well known to those skilled in the art of molecular biology. (See, for example, F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998).

[0034] Eosinophilic esophagitis (EoE) is an immune-mediated disease of the esophagus that is generally triggered by food antigens. EoE was not well characterized before 1993, but is now the most common cause of dysphagia (difficulty swallowing) and bolus obstruction requiring esophageal endoscopy. EoE causes acute bolus obstruction requiring urgent endoscopic removal. Chronic EoE leads to the formation of fibrous strictures and esophageal remodeling, and patients require regular esophageal dilation in order to eat. The mechanism of fibrosis in EoE has not been well studied, and optimal prevention strategies are unknown. EoE is diagnosed by endoscopic biopsy, which is invasive and incurs significant costs to the patient. The food antigens that trigger EoE are determined through a multiyear dietary elimination trial consisting of regular, approximately monthly biopsies.

[0035] The compositions and methods disclosed herein focus on the clinical needs of EoE patients to develop non-invasive diagnostic modalities and rapidly identify trigger antigens. As disclosed herein, we tested whether such antigen footprints reflected recent food intake and whether the disease itself exhibited antigen signals, and determined whether removal of food triggers reduced food-specific signals (i.e., whether the signals were antigen exposure-dependent) despite the disease being ongoing. Using RNA-seq and histopathological validation, we showed that subepithelial immunomodulatory responses are a hallmark of EoE and can induce fibrosis. We also used RNA-seq and quantitative protein assays to identify diagnostic biomarkers in esophageal secretions collected by non-minimally invasive scraping. Also described herein are methods to accurately predict and determine trigger foods using food-specific antibodies in esophageal secretions and responses to active exposure and removal of antigens.

[0036] Eosinophilic esophagitis (EoE) is a severe and increasing immunological cause of esophageal dysphagia and motility disorders (ESDellon; Updated international consensus diagnostic criteria for eosinophilic esophagitis: proceedings of the AGREE conference. Gastroenterology, 155(4):1022-1033. e10, sep 2018). It has become one of the most common causes of dysphagia in children and young adults (ES Dellon; Updated international consensus diagnostic criteria for eosinophilic esophagitis: proceedings of the AGREE conference. Gastroenterology,155(4):1022-1033. e10, sep 2018; JM Spergel,et al. Annals of Allergy, Asthma & Immunology,121(3):281-284, jul 2018, and E. Inage. American Journal of Physiology. Gastrointestinal and Liver Physiology,315(5), sep 2018). 12-23% of dysphagia cases requiring endoscopy are caused by EoE (ES Dellon. Gastroenterology Clinics of North America,43(2):201-218, jun 2014). Recent literature estimates the prevalence of EoE to be 0.5–1 per 1,000 and the incidence of EoE to be 5–10 per 100,000 (ES Delon; Updated international consensus diagnostic criteria for eosinophilic esophagitis: proceedings of the AGREE conference. Gastroenterology, 155(4):1022-1033. e10, sep 2018).The increasing prevalence of EoE is due to the chronic nature of the disease and increased diagnosis as the disease has become more widely recognized (A. Arias and AJ Lucendo. Digestive and Liver Disease, jul 2018, S Hommeida, et al. Diseases of the Esophagus, 31(12), dec 2018, and Jacob Robson, et al. Clinical Gastroenterology and Hepatology, 17(1), jun 2018). Men are 3:1 more likely to suffer from EoE (E Mansoor and GS Cooper. Digestive Diseases and Sciences, 61(10):2928-2934, jun 2016, and CA Liacouras et al. The Journal of Allergy and Clinical Immunology, 128(1):3-20.e6;quiz 21, jul 2011). Whites are three times more likely to develop EoE compared to African-Americans or Asian-Americans (E Mansoor and GS Cooper. Digestive Diseases and Sciences, 61(10):2928-2934, jun 2016, and CA Liacouras et al. The Journal of Allergy and Clinical Immunology, 128(1):3-20.e6; quiz 21, jul 2011). EoE patients are more likely to exhibit atopic disorders (e.g., asthma, allergic rhinitis, and atopic dermatitis) (E Mansoor and GS Cooper. Digestive Diseases and Sciences, 61(10):2928-2934, jun 2016, and CA Liacouras et al. The Journal of Allergy and Clinical Immunology, 128(1):3-20.e6; quiz 21, jul 2011).

[0037] EoE is an immune-mediated disease, and its symptoms are induced by antigen exposure (E.S. Dellon; Updated international consensus diagnostic criteria for eosinophilic esophagitis: proceedings of the AGREE conference. Gastroenterology, 155(4):1022-1033.e10, sep 2018; J.M. Spergel, et al. Annals of Allergy, Asthma & Immunology, 121(3):281-284, jul 2018, and E. Inage. American Journal of Physiology. Gastrointestinal and Liver Physiology, 315(5), sep 2018). Its most common inducer is food antigen, and six foods, wheat, dairy products, eggs, soybeans, seafood, and nuts, induce approximately 70% of cases (E.S. Dellon; Updated international consensus diagnostic criteria for eosinophilic esophagitis: proceedings of the AGREE conference. Gastroenterology, 155(4):1022-1033.e10, sep 2018; J.M. Spergel, et al. Annals of Allergy, Asthma & Immunology, 121(3):281-284, jul 2018, and E. Inage. American Journal of Physiology. Gastrointestinal and Liver Physiology, 315(5), sep 2018).Aeroallergens can also induce EoE, and the severity of symptoms can vary with season, pollution, or geography (Matthew I Fogg, et al. The Journal of Allergy and Clinical Immunology, 112(4):796-797, Oct 2003, and Craig C Reed, et al. Annals of Allergy, Asthma & Immunology, 122(3):296-301, Mar 2019).

[0038] EoE has a strong genetic and environmental component. Studies of monozygotic twins have shown that EoE has a genetic inheritance of 14.5%, but 81.0% of the variability is attributable to the shared environment (ES Alexander et al. The Journal of Allergy and Clinical Immunology,134(5):1084-1092.e1,nov 2014). The familial recurrence risk ratio for EoE is 10-64 times, highest among male relatives (ES Alexander et al. The Journal of Allergy and Clinical Immunology,134(5):1084-1092.e1,nov 2014). GWAS studies have identified several genes whose variants are involved in T-helper 2 (Th2) cell signaling that increase the risk of developing EoE (LC Kottyan et al. Nature Genetics,46(8):895-900,aug 2014). RNA-seq studies have also shown increased expression of Th2 cytokines, genes induced by IL-13, in esophageal biopsies from EoE patients (JD Sherrill. Genes and Immunity, 15(6):361-369, sep 2014).

[0039] Downstream effects of Th2 cytokine signaling result in some pathological features of EoE, especially IL-5 and IL-13 signaling (KM O'Shea,et al.,154(2):333-345,2018). In mouse studies, IL-5 was shown to be important for eosinophil maturation and mucosal eosinophilia (F Roufosse.Frontiers in medicine,5:49,apr 2018). In humans, IL-13 expression is increased in EoE patients and induces epithelial expression of eotaxin-3, encoded by CCL26 (C Blanchard,et al.The Journal of Allergy and Clinical Immunology,120(6):1292-1300,dec 2007). In mouse studies, overexpression of IL-13 expression was sufficient to cause esophageal eosinophilia (C Blanchard, et al. Clinical and Experimental Allergy, 35(8):1096-1103, Aug 2005) and could induce eosinophil-independent fibrosis (L Zuo, et al. Journal of Immunology, 185(1):660-669, Jul 2010).

[0040] Acute symptoms of EoE include, but are not limited to, dysphagia, odynophagia, and edema leading to esophageal obstruction by the food bolus (H Philpott, et al. Asia Pacific allergy, 7(1):3-9, jan 2017). Chronically untreated EoE is associated with esophageal remodeling, stiffening, and dysmotility (KM O'Shea, et al. Gastroenterology, 154(2):333-345, 2018). These changes result in esophageal stenosis and impair bolus movement due to lack of coordinated contractions (KM O'Shea, et al. Gastroenterology, 154(2):333-345, 2018). Finally, esophageal dilation may be required to alleviate dysphagia induced by fibrous remodeling (AM Schoepfer,et al. The American Journal of Gastroenterology,105(5):1062-1070,may 2010). Fibrostenosis does not occur in all EoE patients, estimated at 57-90% using ICD-9 codes for dysphagia and bolus obstruction (Evan S Dellon. Gastroenterology Clinics of North America,43(2):201-218,jun 2014, CA Liacouras et al. The Journal of Allergy and Clinical Immunology,128(1):3-20.e6,quiz 21,jul 2011, and Jean P Li-Kim-Moy,et al. Journal of Pediatric Gastroenterology and Nutrition,52(2):147-153,feb 2011).

[0041] The exact mechanism of subepithelial fibrosis in EoE is unclear (Jennifer Armbruster-Lee, et al. Journal of Leukocyte Biology, 104(1):31-40, jul 2018).

[0042] EoE is currently diagnosed by endoscopic biopsy. The current consensus criteria for the diagnosis of EoE is #15 eosinophils per high-power microscopic field in at least one biopsy (ES Dellon et al. Updated international consensus diagnostic criteria for eosinophilic esophagitis: proceedings of the AGREE conference. Gastroenterology, 155(4): 1022-1033. e10, sep 2018). The heterogeneity of eosinophil infiltration in EoE means that more than one biopsy is necessary to avoid false negatives (AJ Lucendo et al. United European gastroenterology journal, 5(3): 335-358, apr 2017). Endoscopic biopsies are also used to assess treatment response (AJ Lucendo et al. United European gastroenterology journal, 5(3): 335-358, apr 2017). Treatment is considered successful if histological resolution is observed by biopsy at follow-up (AJ Lucendo et al. United European gastroenterology journal, 5(3):335-358, apr 2017).

[0043] Dietary elimination trials are performed to identify food triggers that cause EoE in patients (AJ Lucendo et al. United European gastroenterology journal,5(3):335-358,apr 2017). In these trials, a causative food is identified if there is histological resolution after its removal from the patient's diet and disease recurrence after reintroduction of the food into the patient's diet (JB Wechsler,et al. Journal of asthma and allergy,7:85-94,may 2014). Some guidelines recommend the elimination of six foods that trigger the majority of EoE cases by empirical frequency (JB Wechsler,et al. Journal of asthma and allergy,7:85-94,may 2014). In practice, there is considerable variability in the choice and sequence of dietary eliminations due to patients' socioeconomic factors, lifestyle choices, and health literacy (JB Wechsler, et al. Journal of asthma and allergy, 7:85-94, May 2014). Noncompliance with dietary eliminations is frequent, and patient adherence can be difficult to assess (JB Wechsler, et al. Journal of asthma and allergy, 7:85-94, May 2014). The difficulty in assessing patient adherence is further exacerbated by the high prevalence of common EoE antigens in consumer products such as pharmaceutical capsules made from wheat gluten and their confusing use (AR King and August 2012 University of Kansas Drug Information Center Experiential Rotation Students. Gluten content of the top 200 medications: Follow-up to the influence of gluten on a patient's medication choices. Hospital pharmacy, 48(9):736-743, oct 2013).

[0044] Multiple therapies are used to treat EoE. Common first-line therapies include dietary elimination, proton pump inhibitors (PPIs), and topical corticosteroids (TCS) (AJ Lucendo et al. United European gastroenterology journal, 5(3): 335-358, apr 2017). Biologics and targeted therapies are also being explored in clinical trials (KM O'Shea, et al. Gastroenterology, 154(2): 333-345, 2018), but none of them are fully effective in treating EoE, and therefore alternative therapies remain needed.

[0045] Food elimination is a highly effective treatment for EoE, with 74% of patients resolving after elimination of the six most common food triggers (AJ Lucendo et al. United European gastroenterology journal,5(3):335-358,Apr 2017). However, dietary interventions are not always feasible and depend on patient compliance, socio-economic factors, or budgetary constraints (WA Wolf,et al. Dysphagia,31(6):765-770,Aug 2016). Additionally, dietary elimination is not effective for patients with aeroallergen-induced EoE (JB Wechsler,et al. Journal of asthma and allergy,7:85-94,May 2014).

[0046] Proton pump inhibitor therapy results in clinical and histological remission of EoE in approximately one-third of EoE patients (J Molina-Infante,et al.Alimentary Pharmacology&Therapeutics,37(12):1157-1164,jun 2013). Previous guidelines required patients to be non-responsive to PPIs before making a diagnosis of EoE, but patients who recovered with PPIs were classified as PPI-responsive EoE (PPI-REE). Current guidelines do not distinguish between PPI-REE and EoE (ES Dellon et al.Updated international consensus diagnostic criteria for eosinophilic esophagitis:proceedings of the AGREE conference.Gastroenterology,155(4):1022-1033.e10,sep 2018). PPIs are generally well tolerated with relatively mild adverse effects (A Pilotto, et al. World Journal of Gastroenterology, 13(33): 4467-4472, sep 2007). However, in many patients, PPI treatment is not effective in long-term remission of EoE (AJ Lucendo et al. United European gastroenterology journal, 5(3): 335-358, apr 2017). The mechanism of action of PPIs in EoE remains poorly understood (W Asher Wolf and Evan S Dellon. Gastroenterology & hepatology, 10(7): 427-432, jul 2014).

[0047] Topical corticosteroids (TCS) induce clinical and histological details of EoE in some patients (BK Butz, et al. Gastroenterology, 147(2):324-33.e5, aug 2014). TCS is also effective in maintaining long-term remission in some patients (A Straumann, et al. Clinical Gastroenterology and Hepatology, 9(5):400-9.e1, may 2011). However, in 10% of patients, TCS causes esophageal candidiasis due to immunosuppression (A Straumann, et al. Clinical Gastroenterology and Hepatology, 9(5):400-9.e1, may 2011, and DA Andreae et al. The American Journal of Gastroenterology, 111(8):1187-1197, jun 2016).

[0048] Clinical trials of biologics and targeted therapeutics have focused on inhibiting Th2-associated signaling in EoE. Anti-IL5 monoclonal antibodies (i.e., mepolizumab, reslizumab) have shown moderate efficacy in reducing esophageal eosinophilia but had no effect on clinical symptoms (A Straumann, et al. Gut, 59(1):21-30, jan 2010; AH Assa'ad, et al. Gastroenterology, 141(5):1593-1604, nov 2011; and JM Spergel, et al. The Journal of Allergy and Clinical Immunology, 129(2):456-63, 463.e1, feb 2012). Anti-IL13 monoclonal antibodies also reduced esophageal eosinophilia but did not improve clinical symptoms (ME Rothenberg, et al. The Journal of Allergy and Clinical Immunology, 135(2):500-507, feb 2015). A monoclonal antibody targeting the IL-4 receptor is currently being tested in clinical trials for EoE (KM O'Shea, et al. Gastroenterology, 154(2):333-345, 2018). A CRTH2 inhibitor that prevents eosinophil migration reduced esophageal eosinophilia but did not completely resolve it (KM O'Shea, et al. Gastroenterology, 154(2):333-345, 2018).

[0049] Currently, dietary elimination trials are the established method to identify food antigens inducing EoE (N Gonsalves, et al. Gastroenterology, 142(7):1451-9.e1;quiz e14,jun 2012; KA Peterson, et al. The American Journal of Gastroenterology, 108(5):759-766,may 2013; AJ Lucendo, et al. The Journal of Allergy and Clinical Immunology, 131(3):797-804,mar 2013; and J Molina-Infante, et al. The Journal of Allergy and Clinical Immunology, 134(5):1093-9.e1,nov 2014). However, dietary elimination trials often last for years and consist of endoscopic biopsies that must be repeated periodically, often monthly, to assess clinical response (N Gonsalves, et al. Gastroenterology, 142(7):1451-9.e1;quiz e14,jun 2012; KA Peterson, et al. The American Journal of Gastroenterology, 108(5):759-766,may 2013; AJ Lucendo, et al. The Journal of Allergy and Clinical Immunology, 131(3):797-804,mar 2013; and J Molina-Infante, et al. The Journal of Allergy and Clinical Immunology, 134(5):1093-9.e1,nov 2014). These tests entail the financial burden and reduced quality of life imposed by endoscopic biopsy, which may lead to poor patient compliance (JB Wechsler, et al. Journal of asthma and allergy, 7:85-94, May 2014, and WA Wolf, et al. Dysphagia, 31(6):765- 770, Aug 2016).Furthermore, dietary elimination is not effective in patients with aeroallergen-induced EoE (JB Wechsler, et al. Journal of asthma and allergy, 7:85-94, May 2014). Improved methods to identify antigen triggers in EoE are needed.

[0050] Described herein is a method that can be used to identify antibodies in esophageal IgG and IgA obtained by scraping. This method can accurately predict food-specific antibodies to determine EoE food triggers. This method can also be used to monitor treatment response. This method directly addresses the need for improved methods to identify EoE causative antigens.

[0051] method Disclosed herein are methods that can be used to identify food sensitivities and / or food allergies in a subject.

[0052] Disclosed herein is a method for detecting the binding of one or more food-specific immunoglobulin (Ig) antibodies to one or more food antigens in a sample. In some embodiments, the method may include contacting a sample obtained from a subject with one or more food antigens and detecting the binding of one or more food-specific Ig antibodies to the one or more food antigens. In some embodiments, the method further includes comparing the binding of one or more food-specific Ig antibodies to one or more food antigens in a sample obtained before the subject ingests a food containing one or more of the food antigens with a sample obtained after the subject ingests a food containing one or more of the food antigens. In some embodiments, the method further includes diagnosing the subject with EoE. In some embodiments, the method further includes diagnosing the subject with EoE using the method disclosed herein.

[0053] Methods for detecting food-specific immune responses in a sample are disclosed herein. In some embodiments, the method comprises: a) contacting the sample with one or more food antigens; b) determining the presence or level of one or more immunoglobulin (Ig) antibodies bound to the one or more food antigens; and c) comparing the level of one or more Ig antibodies in the sample bound to the one or more food antigens with the level of one or more Ig antibodies in a reference sample bound to the one or more food antigens, whereby a food-specific immune response in the sample is detected when the level of one or more Ig antibodies in the sample bound to the one or more food antigens is higher than the level of one or more Ig antibodies in the reference sample. In some embodiments, the method further comprises diagnosing the subject as having EoE. In some embodiments, the method further comprises diagnosing the subject as having EoE using the methods disclosed herein.

[0054] Methods for identifying food allergies in a subject having active or inactive eosinophilic esophagitis (EoE) in subjects suffering from EoE are disclosed herein. In some embodiments, the method can comprise: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more food antigens in the sample; and b) comparing the level of one or more Ig antibodies bound to one or more food antigens in the sample with the level of one or more Ig antibodies bound to one or more food antigens in a reference sample, wherein the food allergy in the subject is identified by the level of one or more Ig antibodies in the sample bound to one or more food antigens being higher than the level of one or more Ig antibodies in the reference sample bound to one or more food antigens. In some embodiments, the method further comprises diagnosing the subject as having EoE. In some embodiments, the method further comprises diagnosing the subject as having EoE using the methods disclosed herein.

[0055] Disclosed herein is a method for identifying food sensitivity or hypersensitivity of a subject with active or inactive eosinophilic esophagitis (EoE) in a subject suffering from EoE. In some embodiments, the method can include: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more food antigens in a sample; and b) comparing the level of one or more Ig antibodies bound to one or more food antigens in the sample with the level of one or more Ig antibodies bound to one or more food antigens in a reference sample, where the level of one or more Ig antibodies bound to one or more food antigens in the sample is higher than the level of one or more Ig antibodies bound to one or more food antigens in the reference sample, thereby identifying the food sensitivity or hypersensitivity of the subject. In some embodiments, the method further includes diagnosing the subject with EoE. In some embodiments, the method further includes diagnosing the subject with EoE using the method disclosed herein.

[0056] Disclosed herein is a method for identifying the sensitivity or hypersensitivity of a subject with active or inactive eosinophilic esophagitis (EoE) to an antigen in a subject suffering from EoE. In some embodiments, the method can include: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more antigens in the sample; and b) comparing the level of one or more Ig antibodies bound to one or more antigens in the sample with the level of one or more Ig antibodies bound to one or more antigens in a reference sample, where the level of one or more Ig antibodies bound to one or more antigens in the sample is higher than the level of one or more Ig antibodies bound to one or more antigens in the reference sample, thereby identifying the sensitivity or hypersensitivity of the subject to one or more antigens. In some embodiments, the method further includes diagnosing the subject with EoE. In some embodiments, the method further includes diagnosing the subject with EoE using the method disclosed herein.

[0057] A method for identifying food allergy in a subject with irritable bowel syndrome is disclosed herein.In some embodiments, the method can include: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more food antigens in a sample; and b) comparing the level of one or more Ig antibodies bound to one or more food antigens in the sample with the level of one or more Ig antibodies bound to one or more food antigens in a reference sample, where the level of one or more Ig antibodies bound to one or more food antigens in the sample is higher than the level of one or more Ig antibodies bound to one or more food antigens in the reference sample, thereby identifying the food allergy of the subject.In some embodiments, the method further includes diagnosing the subject with EoE.In some embodiments, the method further includes diagnosing the subject with EoE using the method disclosed herein.

[0058] Disclosed herein is a method for treating a subject with active or inactive eosinophilic esophagitis (EoE) in a subject suffering from EoE. In some embodiments, the method can include: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more food antigens in a sample; b) comparing the level of one or more Ig antibodies bound to one or more food antigens in the sample with the level of one or more Ig antibodies bound to one or more food antigens in a reference sample; and c) treating the subject. In some embodiments, the method further includes diagnosing the subject with EoE. In some embodiments, the method further includes diagnosing the subject with EoE using the method disclosed herein.

[0059] Disclosed herein is a method for treating a subject with active eosinophilic esophagitis (EoE) in a subject suffering from EoE, the method comprising: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more food antigens in a sample; b) comparing the level of one or more Ig antibodies bound to one or more food antigens in the sample with the level of one or more Ig antibodies bound to one or more food antigens in a reference sample; and c) treating the subject. In some embodiments, the method further comprises diagnosing the subject with EoE. In some embodiments, the method further comprises diagnosing the subject with EoE using the method disclosed herein.

[0060] Disclosed herein is a method for detecting the cause of symptoms of eosinophilic esophagitis (EoE). In some embodiments, the symptoms of EoE may be caused by an immune response to an antigen in a patient diagnosed with or suffering from EOE. In some embodiments, the method may include obtaining or having obtained an esophageal mucosal sample from a subject. In some embodiments, the esophageal mucosal sample may be obtained from a subject and originates from a site of an immune response. In some embodiments, the esophageal mucosal sample may include one or more immunoglobulin (Ig) antibodies. In some embodiments, the method may include contacting the sample with one or more antigens. In some embodiments, the method may include detecting binding of one or more specific Ig antibodies to the one or more antigens. In some embodiments, the method further includes diagnosing the subject with EoE using a method disclosed herein. Examples of symptoms of EoE include, but are not limited to, difficulty swallowing (dysphagia), food getting stuck in the esophagus after swallowing (obstruction), chest pain (often centrally located and unresponsive to antacids), spitting up undigested food (regurgitation), vomiting, and abdominal pain.

[0061] Disclosed herein is a method for diagnosing a subject with active or inactive EoE. Disclosed herein is a method for diagnosing a subject with active or inactive EoE in a subject suffering from EoE. Disclosed herein is a method for diagnosing and treating a subject with active or inactive EoE. Disclosed herein is a method for diagnosing and treating a subject with active or inactive EoE in a subject suffering from EoE. In some embodiments, the method may include detecting whether the level of IgA, IgE, IgM, IgD, IgG1, IgG2, IgG3, and / or IgG4 antibody (or any combination thereof) is elevated in an esophageal secretion sample obtained from a subject. In some embodiments, the methods may include diagnosing the subject with active EoE when levels of IgA, IgE, IgM, IgD, IgG1, IgG2, IgG3, and / or IgG4 antibodies in the sample are elevated above a predetermined cutoff value, and diagnosing the subject with inactive EoE when levels of IgA, IgE, IgM, IgD, IgG1, IgG2, IgG3, and / or IgG4 antibodies in the sample are below a predetermined cutoff value. In some embodiments, the methods can include treating a subject diagnosed with active EoE, where the subject is diagnosed with inactive EoE when the levels of IgA, IgE, IgM, IgD, IgG1, IgG2, IgG3, and / or IgG4 antibodies are below a predetermined cutoff value, and the subject is diagnosed with active EoE when the levels of IgA, IgE, IgM, IgD, IgG1, IgG2, IgG3, and / or IgG4 antibodies are above a predetermined cutoff value.

[0062] In some aspects, in any of the methods disclosed herein, the level or amount of IgA, IgE, IgM, IgD, IgG1, IgG2, IgG3, and / or IgG4 antibodies (or any combination thereof) can be used to determine or diagnose active EoE. In some aspects, the method can include determining or diagnosing active EoE by comparing the level or amount of IgA, IgE, IgM, IgD, IgG1, IgG2, IgG3, and / or IgG4 antibodies (or any combination thereof) from a sample from a subject to a reference sample or control. In some aspects, the method can include comparing the level or amount of IgA, IgE, IgM, IgD, IgG1, IgG2, IgG3, and / or IgG4 antibodies from a sample from a subject having active EoE to a sample from a subject having inactive EoE. In some aspects, the Ig antibody can be IgG, IgA, or a combination thereof. In some aspects, the Ig antibody can be IgG4, IgA, or a combination thereof. In some aspects, the method can detect whether the level of any of IgA, IgE, IgM, IgD, IgG1, IgG2, IgG3, and / or IgG4 antibodies is elevated (increased) in an esophageal secretion sample obtained from a subject. In some aspects, when the measured IgA, IgE, IgM, IgD, IgG1, IgG2, IgG3, and / or IgG4 antibody level rises above a predetermined cut-off value, the subject can be diagnosed as having EoE or active EoE, or when the IgA, IgE, IgM, IgD, IgG1, IgG2, IgG3, and / or IgG4 antibody level in the sample is below a predetermined cut-off value, the subject can be diagnosed as having inactive EoE. In some aspects, the predetermined cut-off value can be about 25 ng / ml for IgE. In some aspects, when the IgE antibody level is below about 25 ng / ml, the subject is diagnosed as having inactive EoE. In some aspects, the predetermined cut-off value can be about 5000 ng / ml for IgA. In some aspects, when the IgA antibody level is below about 5000 ng / ml, the subject is diagnosed as having inactive EoE.In some embodiments, the predetermined cutoff value may be about 1000ng / ml for IgM. In some embodiments, a subject is diagnosed with inactive EoE when the IgM antibody level is below about 1000ng / ml. In some embodiments, the predetermined cutoff value may be about 200ng / ml for IgG1. In some embodiments, a subject is diagnosed with inactive EoE when the IgG1 antibody level is below about 200ng / ml. In some embodiments, the predetermined cutoff value may be about 50ng / ml for IgG2. In some embodiments, a subject is diagnosed with inactive EoE when the IgG2 antibody level is below about 50ng / ml. In some embodiments, the predetermined cutoff value may be about 4000ng / ml for IgG3. In some embodiments, a subject is diagnosed with inactive EoE when the IgG3 antibody level is below about 4000ng / ml. In some embodiments, the predetermined cutoff value may be about 6000ng / ml for IgG4. In some embodiments, a subject is diagnosed with inactive EoE when IgG4 antibody levels fall below about 6000 ng / ml.

[0063] For example, IgA found along the esophageal mucosal surface can be easily collected and shows reactivity to common trigger foods in EoE. Indeed, IgA was significantly elevated to causative foods in EoE patients. Trigger foods were generally 1.5-fold elevated for IgA than non-trigger foods and routinely showed response values ​​above 340 response units (concentration of 0.01 mg / l at 1:50 dilution). Specific foods such as gluten and dairy showed similar results in their respective categories. Indeed, a positive cutoff of 340 RV (response value, 0.01 mg / l) was over 91% accurate in predicting trigger foods and had both high sensitivity (86%) and specificity (98%).

[0064] Disclosed herein is a method for treating a subject with irritable bowel syndrome.In some embodiments, the method can include: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more food antigens in a sample; b) comparing the level of one or more Ig antibodies bound to one or more food antigens in a sample with the level of one or more Ig antibodies bound to one or more food antigens in a reference sample; and c) treating the subject.In some embodiments, the method can further include diagnosing the subject with EoE.In some embodiments, the method can further include diagnosing the subject with EoE using the method disclosed herein.

[0065] Disclosed herein are methods for diagnosing and treating subjects with active or inactive eosinophilic esophagitis (EoE) in subjects suffering from EoE. In some embodiments, the method can include: a) detecting the presence or level of one or more immunoglobulin (Ig) antibodies bound to one or more food antigens in the sample; b) comparing the level of the one or more Ig antibodies bound to the one or more food antigens in the sample with the level of the one or more Ig antibodies bound to the one or more food antigens in a reference sample; c) diagnosing the subject with active EoE when the level of the one or more Ig antibodies bound to the one or more food antigens in the sample is higher than the level of the one or more Ig antibodies bound to the one or more food antigens in the reference sample, and diagnosing the subject with inactive EoE when the level of the one or more Ig antibodies bound to the one or more food antigens in the sample is lower than the level of the one or more Ig antibodies bound to the one or more food antigens in the reference sample level; and d) treating the subject diagnosed with active EoE, wherein the level of the one or more Ig antibodies bound to the one or more food antigens in the sample is higher than the level of the one or more Ig antibodies bound to the one or more food antigens in the reference sample.

[0066] Also disclosed herein is a method for detecting the binding of one or more specific immunoglobulin (Ig) antibodies to one or more antigens in a sample. In some embodiments, the method may include contacting a sample obtained from a subject with one or more antigens and detecting the binding of one or more specific Ig antibodies to one or more food antigens. In some embodiments, the one or more antigens can be immobilized on a solid support. In some embodiments, the sample can be obtained before the subject is exposed to one or more of the antigens. In some embodiments, the method further includes diagnosing the subject with EoE. In some embodiments, the method further includes diagnosing the subject with EoE using the methods disclosed herein.

[0067] Disclosed herein are methods for diagnosing and / or treating eosinophilic esophagitis in a subject in need thereof. In some embodiments, the method includes the steps of: (i) measuring a gene expression level of at least one gene in an esophageal biopsy sample from a patient, wherein the at least one protein or at least one gene is selected from the group consisting of eosinophil peroxidase, periostin, eotaxin-3, stem cell factor, or CCL26, KITLG, and POSTN, and measuring the protein or gene expression level is performed by a method comprising DNA microarray analysis, polymerase chain reaction analysis, or both; and (ii) measuring a gene expression level of at least one protein or at least one gene in an esophageal biopsy sample from a patient, wherein the at least one protein or at least one gene is selected from the group consisting of eosinophil peroxidase, periostin, eotaxin-3, stem cell factor, or CCL26, KITLG, and POSTN, and measuring the protein or gene expression level is performed by a method comprising DNA microarray analysis, polymerase chain reaction analysis, or both. comparing the protein or gene expression level to its expression level in an esophageal biopsy sample from a normal individual, defined as zero eosinophils per high power field and no basal lamina swelling, and diagnosing eosinophilic esophagitis in the subject if the expression level of at least one gene is increased by more than 10-fold compared to its expression level in an esophageal biopsy sample from the normal individual; and (iv) treating eosinophilic esophagitis in the subject diagnosed according to step (iii) with one or more therapies selected from anti-inflammatory therapy, allergen removal, cytokine inhibitors, immunosuppressants, and complement inhibitors.

[0068] Disclosed herein are methods for diagnosing Eosinophilic Esophagitis (EoE) by detecting one or more biomarkers selected from the group consisting of eosinophil peroxidase, periostin, eotaxin-3, stem cell factor, or CCL26, KITLG, and POSTN.

[0069] In some aspects, the treatment step may include administering to the subject one or more monoclonal agglutinating anti-IgA antibodies, anti-inflammatory therapy, allergen removal, cytokine inhibitors, immunosuppressants, bovine Ig, complement inhibitors, one or more steroids (e.g., corticosteroids), or combinations thereof.

[0070] In some embodiments, the treatment step may include modulating the intake (e.g., dietary change) or cessation of one or more food species or food components from the subject's diet. In some embodiments, the one or more food species or food components can be any food or food component that the subject may ingest that may be correlated with one or more food allergens detected using the methods disclosed herein. In some embodiments, the dietary change or cessation of one or more food species or food components can be based on the detection of one or more Ig antibodies. In some embodiments, the food species can be milk, wheat, soybean, and egg. In some embodiments, the one or more food antigens can be wheat f1, soybean f14, casein f78, egg f245, or combinations thereof. In some embodiments, the treatment step may include cutting off one or more food components from the subject's intake. In some embodiments, the one or more food components can be any food components found in any food species that the subject may ingest. In some aspects, the food ingredient may be casein, whey, emulsifiers, spices, pollutants, aeroallergens, dust mites, and the like.

[0071] In some embodiments, the methods disclosed herein may further include detecting EoE in a subject prior to the detection step. In some embodiments, EoE can be detected in a subject by detecting eosinophil granule protein in esophageal mucosal tissue in the subject. In some embodiments, the method may include administering to the subject radiolabeled heparin under conditions in which the radiolabeled heparin binds to eosinophil granule protein to form a radiolabeled heparin / eosinophil granule protein complex, and detecting the radiolabeled heparin / eosinophil granule protein complex in the esophageal mucosal tissue, thereby detecting eosinophilic esophagitis in the subject by detecting the radiolabeled heparin / eosinophil granule protein complex in the esophageal mucosal tissue.

[0072] In some embodiments, the method may be specific to a single antigen or a single food (e.g., milk) or food component. A single food is an antigenically complex mixture (e.g., milk contains many antigens). In some embodiments, the method may detect or identify a specific single antigen or a specific single food antigen on a bead (or other carrier), or detect or identify two or more (or multiple) antigens on one bead, or detect or identify one or more (or multiple) antigens or food antigens on two or more beads. For example, a Luminex device can analyze 100 or antigens in one test. In some embodiments, the method may be specific to two or more single antigens or a single food antigen. In some embodiments, the single antigen may be a combination of food antigens and environmental antigens.

[0073] In some embodiments, the antigen can be a food antigen. In some embodiments, the antigen can be an environmental antigen. In some embodiments, the environmental antigen can be pollen, fungus, dander (e.g., cat dander), dust mites, animal shedding, insect shedding, etc. In some embodiments, the antigen can be an aeroallergen. In some embodiments, the aeroallergen can be pollen, spores, mold, animal dander, or insect-derived antigen. In some embodiments, the antigen can be an antigen component. For example, the antigen can be a component of milk (e.g., casein, but not whey, can be detected as a protein in dairy products that is responsible for the increase in immunoglobulins detected). In some embodiments, the antigen can be a causative antigen. In some embodiments, the causative antigen can be a food antigen, an environmental antigen, an aeroallergen, or an antigen component.

[0074] In some embodiments, the level of one or more specific Ig antibodies bound to the one or more antigens may be higher in the sample compared to the level of one or more specific Ig antibodies bound to the one or more antigens in a reference sample, thereby indicating a specific immune response to the antigen.

[0075] In some embodiments, the level of one or more food-specific Ig antibodies bound to one or more food antigens may be higher in the sample compared to the level of one or more food-specific Ig antibodies bound to one or more food antigens in a reference sample, thereby indicating a food-specific immune response.

[0076] In some embodiments, one or more antigens can be immobilized on a solid support. In some embodiments, one or more food antigens can be immobilized on a solid support. In some embodiments, one or more environmental antigens can be immobilized on a solid support.

[0077] An array is a form of solid support. Array detectors are also a form of solid support to which a plurality of different capture or detection compounds are attached in an array, grid, or other organized pattern.

[0078] Solid substrates for use in solid supports can include, for example, any solid material to which molecules can be attached. Examples of such materials include acrylamide, agarose, cellulose, nitrocellulose, glass, polystyrene, polyethylenevinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicate, polycarbonate, Teflon, fluorocarbon, nylon, silicone rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids. Solid substrates can have any useful form, including thin films, membranes, bottles, dishes, fibers, woven fibers, molded polymers, particles, beads, microparticles, or any combination thereof. Solid substrates and solid supports can be porous or non-porous. One example of a solid substrate is a microtiter dish (e.g., standard 96-well type). Multi-well glass slides can also be used. For example, those containing one array per well can be used to allow for better control of assay reproducibility, increased throughput and sample processing, and easy automation.

[0079] Different compounds and components disclosed herein can be used together as a set. A set can be used as a mixture of all or a subset of compounds used separately in separate reactions, or immobilized in an array. Compounds used separately or as a mixture can be physically separated, for example, through association with or immobilization to a solid support. An array can include multiple compounds immobilized at a specific or predefined location on the array. Each predefined location on the array generally has one type of component (i.e., all components at that location are the same). Each location can have multiple copies of a component. The spatial separation of different components in the array allows separate detection and identification of the polynucleotides or polypeptides disclosed herein.

[0080] A given array does not have to be a single unit or structure. A set of compounds can be distributed across any number of solid supports. For example, each compound can be immobilized in a separate reaction tube or container, or on a separate bead or microparticle. Different aspects of the disclosed methods and uses of gene expression panels or arrays or diagnostic devices can be carried out with different components (e.g., different compounds specific for different proteins) immobilized on solid supports.

[0081] Some solid supports may have a capture compound, such as an antibody, attached to the solid substrate. Such a capture compound may be specific to a mineralized nanoparticle or a protein on the mineralized nanoparticle. The captured mineralized nanoparticle or protein can then be detected by binding of a second detection compound, such as an antibody. The detection compound may be specific to the same or a different protein on the mineralized nanoparticle.

[0082] Methods for immobilizing nucleic acids, peptides, or antibodies (and other proteins) to solid substrates are well established. Immobilization can be achieved, for example, by attachment to aminated, carboxylated, or hydroxylated surfaces using standard immobilization chemistries. Examples of attachment agents are cyanogen bromide, succinimide, aldehydes, tosyl chloride, avidin biotin, photocrosslinkers, epoxides, maleimides, and N-[y-maleimidobutyryloxy]succinimide ester (GMBS), and heterobifunctional crosslinkers. Antibodies can be attached to substrates by chemically crosslinking free amino groups on the antibodies to reactive side groups present in the solid substrate. Antibodies can be chemically crosslinked to substrates containing free amino, carboxyl, or sulfur groups, for example, using glutaraldehyde, carbodiimide, or GMBS as crosslinkers, respectively. In this method, an aqueous solution containing free antibodies can be incubated with the solid substrate in the presence of glutaraldehyde or carbodiimide.

[0083] A method for attaching antibodies or other proteins to solid substrates is to functionalize the substrate with aminosilanes or thiolsilanes, followed by activation of the functionalized substrate with a homobifunctional crosslinker such as bis-sulfo-succinimidyl suberate (BS3) or a heterobifunctional crosslinker such as GMBS. For crosslinking with GMBS, glass substrates can be chemically functionalized by immersion in a solution of mercaptopropyltrimethoxysilane (1% volume / volume in 95% ethanol, pH 5.5) for 1 hour, rinsing in 95% ethanol, and heating at 120° C. for 4 hours. Thiol-derivatized slides can be activated by immersion in a solution of 0.5 mg / ml GMBS in 1% dimethylformamide, 99% ethanol for 1 hour at room temperature. Antibodies or proteins can be added directly to the activated substrate, which can be blocked with a solution containing an agent such as 2% bovine serum albumin and air-dried. Other standard immobilization chemistries are known to those skilled in the art.

[0084] Each of the components (e.g., compounds) immobilized on the solid support can be located in different predefined regions of the solid support. Each of the different predefined regions can be physically separated from each other. The distance between the different predefined regions of the solid support can be constant or variable. For example, in an array, each of the components can be located at a constant distance from each other, while the components associated with beads will not be in a constant spatial relationship. The variable distance can be obtained by using multiple solid support units (e.g., multiple beads).

[0085] Components can be associated or immobilized on a solid support at any density. Components can be immobilized on a solid support at a density of more than 400 different components per cubic centimeter. An array of components can have any number of components. For example, an array can have at least 1,000 different components immobilized on a solid support, at least 10,000 different components immobilized on a solid support, at least 100,000 different components immobilized on a solid support, or at least 1,000,000 different components immobilized on a solid support.

[0086] In some embodiments, the sample can be obtained before the subject is exposed to a particular antigen (e.g., a food antigen or an environmental antigen). In some embodiments, the sample can include one or more of the one or more antigen-specific Ig antibodies. In some embodiments, the sample can be obtained before the subject ingests a food that includes one or more of the food antigens. In some embodiments, the method can further include obtaining or having obtained the sample from the subject. In some embodiments, the sample can include one or more of the one or more food-specific Ig antibodies. In some embodiments, the Ig antibodies can be IgA, IgD, IgE, IgG, IgM, or combinations thereof. In some embodiments, the IgA antibodies can be IgA1, IgA2, or combinations thereof. In some embodiments, the IgG antibodies can be IgG1, IgG2, IgG3, IgG4, or combinations thereof. In some embodiments, the sample can be a mucosal sample. In some embodiments, the mucosal sample can be esophageal secretions. In some embodiments, the sample can be mucosal fluid. In some embodiments, the sample can be discharge from an esophageal disease. In some aspects, the sample may be an output from the small intestine.

[0087] In some embodiments, the subject has active eosinophilic esophagitis. In some embodiments, the subject has resolved eosinophilic esophagitis. In some embodiments, the subject has at least one symptom of eosinophilic esophagitis. In some embodiments, the subject has irritable bowel syndrome. In some embodiments, the subject has at least one symptom of irritable bowel syndrome.

[0088] In some embodiments, the one or more food specific Ig antibodies may be IgG, IgA, IgD, IgE, IgM, or a combination thereof. In some embodiments, the one or more food specific Ig antibodies may be IgG, IgA, IgE, or a combination thereof. In some embodiments, the one or more food specific Ig antibodies may be IgG, IgA, or a combination thereof. In some embodiments, the one or more food specific Ig antibodies may be IgG4, IgA, or a combination thereof.

[0089] As used herein, the term "expression" when used in the context of determining or detecting the expression or expression level of one or more genes can refer to determining or detecting the transcription of a gene (i.e., determining the mRNA level) and / or determining or detecting the translation of a gene (e.g., determining or detecting the protein produced). Determining the expression level of a gene means determining whether the gene is expressed, and if expressed, to what relative degree it is expressed.

[0090] The expression level of one or more genes disclosed herein can be determined directly (e.g., immunoassay, mass spectrometry) or indirectly (e.g., determining the mRNA expression of protein or peptide). Examples of mass spectrometry include ionization sources such as EI, CI, MALDI, ESI, and analysis such as Quad, ion trap, TOF, FT, or combinations thereof, spectroscopy, isotope ratio mass spectrometry (IRMS), thermal ionization mass spectrometry (TIMS), spark source mass spectrometry, multiple reaction monitoring (MRM), or SRM. Any of these techniques can also be performed in combination with pre-fractionation or enrichment methods. Examples of immunoassays include immunoblots, Western blots, enzyme-linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), and radioimmunoassays.

[0091] Immunoassay methods use antibodies to detect and determine the levels of antigens are known in the art. The antibody or antigen can be immobilized on a solid support such as a stick, plate, bead, microbead, or array.

[0092] The expression level of one or more of the genes described herein can also be determined indirectly by determining the mRNA expression for one or more of the genes in tissue samples.RNA expression methods include, but are not limited to, extracting cellular mRNA and Northern blot using a labeled probe that hybridizes to the transcript that codes for all or part of the gene, amplifying mRNA using gene-specific primers, polymerase chain reaction (PCR), and reverse transcription polymerase chain reaction (RT-PCR), followed by quantitative detection of gene products by various methods; extracting RNA from cells, then labeling, and then using it to probe cDNA or oligonucleotides that code for the gene, in situ hybridization, and detecting reporter genes.

[0093] Methods for measuring protein expression levels include, but are not limited to, Western blot, immunoblot, ELISA, radioimmunoassay, immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemistry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry. The methods may also include specific protein property-based assays, including, but not limited to, enzymatic activity or enzyme interaction with other protein partners. Binding assays may also be used and are well known in the art. For example, a BIAcore machine may be used to determine the binding constant of a complex between two proteins. Other suitable assays for determining or detecting the binding of one protein to another include immunoassays such as ELISA and radioimmunoassays. Determination of binding by monitoring changes in spectroscopy may be used, or the optical properties of the protein may be determined via fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Alternatively, immunoassays using specific antibodies may be used to detect the expression of a particular protein in a sample.

[0094] Reference mRNA expression level reference. As used herein, the terms "reference", "reference expression", "reference sample", "reference value", "control", "control sample" and the like, when used in the context of a sample or expression level of one or more genes or proteins, refer to a reference standard that is expressed at a constant level among different tissues (i.e., not the same tissue, but multiple tissues), is not affected by experimental conditions, and indicates the level in a sample of a given disease state (e.g., not suffering from EoE or active EoE, IBS, or immune response after exposure to a specific antigen). The reference value can be a predetermined standard value or a range of predetermined standard values ​​that does not represent a disease, or a disease of a given type or severity.

[0095] The reference expression can be the level of one or more genes described herein in a reference sample derived from a subject or a pool of subjects not suffering from EoE or active EoE, IBS, or immune response after exposure to a specific antigen, or a given severity or type of EoE, IBS, or immune response after exposure to a specific antigen. In some embodiments, the reference value is the level of one or more genes disclosed herein in a tissue (or a sample from a subject) of a subject(s), and the subject(s) is not suffering from EoE or active EoE, IBS, or immune response after exposure to a specific antigen.

[0096] Comparison of the expression level of one or more genes disclosed herein.For example, the expression level of one or more of CCL26, KITLG and POSTN can be compared with the reference expression level of, for example, CCL26, KITLG and POSTN to determine the active EoE in a subject.

[0097] Determining the expression level of one or more genes disclosed herein may include determining whether the gene is upregulated or increased compared to a control or reference sample, downregulated or decreased compared to a control or reference sample, or unchanged compared to a control or reference sample.As used herein, the terms "upregulated" and "increased expression level" or "increased level of expression" refer to the sequence corresponding to one or more genes disclosed herein, and the measure of the amount of the sequence represents an increased level of expression when compared to a reference sample or "normal" control.In some embodiments, a normal control may refer to a subject who does not have EoE.In some embodiments, a normal control may refer to a subject who has resolved EoE. For example, the terms "upregulated" and "increased expression level" or "increased level of expression" refer to a sequence corresponding to one or more genes disclosed herein, where the measure of the amount of the sequence represents an increased level of expression of one or more of eosinophil peroxidase, periostin, eotaxin-3, and stem cell factor proteins and / or mRNAs as compared to the expression of the same mRNAs from a reference sample or "normal" control. An "increased expression level" refers to an increase in expression of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more, such as 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90%, or more, or more than 1-fold, up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more. As used herein, the terms "downregulated," "reduced level of expression," or "reduced expression level" refer to a sequence corresponding to one or more genes disclosed herein that is expressed, and a measure of the amount of the sequence exhibits a reduced level of expression when compared to a reference sample or "normal" control.For example, the terms "downregulated," "reduced level of expression," or "reduced expression level" refer to a sequence corresponding to one or more genes disclosed herein, where the measure of the amount of the sequence represents a reduced level of expression of one or more of eosinophil peroxidase, periostin, eotaxin-3, and stem cell factor proteins and / or mRNAs as compared to the expression of the same mRNAs from a reference sample or "normal" control. A "reduced level of expression" refers to a decrease in expression of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more, such as 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90%, or more, or more than 1-fold, up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more.

[0098] Determining active EoE. As described herein, a sample from a subject can be compared with a reference sample to determine the expression ratio and determine whether the subject has active EoE. The reference sample can be from a subject whose level of one or more of the following genes is "normal": CCL26, KITLG, and POSTN. Suitable statistical and other analyses can be carried out to identify changes (e.g., increased expression or higher levels) in one or more of CCL26, KITLG, and POSTN compared to in the reference sample, where the ratio of the sample expression level of one or more of CCL26, KITLG, and POSTN to the reference expression level of one or more of CCL26, KITLG, and POSTN indicates that the expression level of one or more of CCL26, KITLG, and POSTN in the sample is higher. In some embodiments, the ratio of the sample expression levels of two or more of CCL26, KITLG, and POSTN to the reference expression levels of two or more of CCL26, KITLG, and POSTN indicates that the expression levels of two or more of CCL26, KITLG, and POSTN in the sample are higher, indicating that the subject has active EoE.

[0099] When compared to the reference expression levels of CCL26, KITLG, and POSTN, higher or increased expression levels of one or more of CCL26, KITLG, and POSTN may indicate active EoE. A characteristic pattern of increased (higher) or decreased (lower) sample expression levels of one or more of CCL26, KITLG, and POSTN when compared to the reference expression levels of one or more of CCL26, KITLG, and POSTN can be observed, thereby indicating active EoE in the subject.

[0100] The expression level of one or more genes described herein can be, for example, the measured value of one or more genes per unit weight or unit volume. In some embodiments, the expression level can be a ratio (e.g., the amount of one or more genes in a sample relative to the amount of one or more markers of a reference value).

[0101] In some embodiments, a sample from a subject can be compared to a reference sample to determine a rate of change and to determine whether the subject has active EoE. In other words, the expression level can be expressed as a percentage. For example, if the expression level of one (or two) or more of CCCL26, KITLG, and POSTN is increased (or higher) by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the reference expression levels of CCL26, KITLG, and POSTN, the rate of change of the expression level of one or more genes indicates active EoE. Alternatively, the rate of change of the expression level of one or more genes can be decreased (or lower) by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the reference expression level.

[0102] In some embodiments, an increase or decrease, or a combination thereof, in the expression levels of genes or proteins other than those disclosed herein may indicate active or resolved EoE, or a diagnosis of EoE or active EoE in a subject. In some embodiments, a characteristic pattern of increase or decrease in the expression levels of one or more of the genes or proteins disclosed herein is indicative.

[0103] Diagnostic Devices Disclosed herein is a diagnostic device for diagnosing eosinophilic esophagitis in a subject (e.g., human).In some embodiments, a sample of mucosa or esophageal secretion can be obtained from the subject, and the level or expression level in the sample can be compared with a reference value.

[0104] The diagnostic device may include one or more biomarkers. In some embodiments, the biomarker may be an antigen or an antibody. In some embodiments, the biomarker may bind or hybridize to one or more genes, RNA products, or peptides disclosed herein. As used herein, the term "marker" or "biomarker" refers to a detectable or measurable substance (e.g., an antigen, antibody, gene, gene product, protein, etc.) in a sample that may indicate a biological state, disease, condition, prediction of clinical outcome, etc. In some embodiments, the biomarker may be eosinophil peroxidase, periostin, eotaxin-3, stem cell factor, or CCL26, KITLG, and POSTN, or fragments thereof, or an antibody or fragment thereof that binds to one or more of the biomarkers. The diagnostic device may be incorporated into a kit for diagnosing EoE or active EoE in a subject.

[0105] Protein Arrays Polypeptide or protein arrays are disclosed herein. In some aspects, protein arrays can comprise probes that contain antigens, antibodies, aptamers and other cognate binding ligands specific to the components of the gene panels disclosed herein. Protein arrays and methods for constructing protein arrays are well known to those skilled in the art.

[0106] One type of protein array that may be suitable uses immobilized "capture antibodies". Polypeptides are attached to a solid substrate (e.g., glass) using a treated surface (e.g., aminosilane) or via biotin-streptavidin conjugate. The array is then incubated with a solution containing a probe that can bind to the capture antibody in a manner that depends on the time, buffer composition, and recognition specificity. The probe can then be visualized directly if previously labeled, or can be bound to a secondary labeling reagent (e.g., another antibody). The amount of probe bound to the capture antibody that is visualized can depend on the labeling method used, and generally a CCD imager or laser scanner can be used that uses a filter set suitable for exciting and detecting the emission of the label. The imager converts the amount of detected photons into an electronic signal (often 8-bit or 16-bit scale), which can be analyzed using commercially available software packages.

[0107] The substrate of the array can be organic or inorganic, biological or non-biological, or any combination of these materials.The substrate can be transparent or translucent.Examples of materials suitable for use as substrate in array include silicon, silica, quartz, glass, controlled pore glass, carbon, alumina, titanium dioxide, germanium, silicon nitride, zeolite, and gallium arsenide, as well as metals including gold, platinum, aluminum, copper, titanium, and their alloys.Ceramic and polymer can also be used as substrate. Suitable polymers include, but are not limited to, polystyrene, poly(tetra)fluoroethylene, (poly)vinylidene difluoride, polycarbonate, polymethyl methacrylate, polyvinylethylene, polyethyleneimine, poly(ether ether) ketone, polyoxymethylene (POM), polypolyphenol, polylactide, polymethacrylimide (PMI), polyalkene sulfone (PAS), polyhydroxyethyl methacrylate, polydimethylsiloxane, polyacrylamide, polyimide, coblock polymer, and Eupergit®. Photoresists, polymerized Langmuir-Blodgett films, and LIGA structures can also function as substrates.

[0108] The array may further include a coating that may be formed on or applied to the substrate. The substrate may be modified with the coating using thin film techniques based on either physical vapor deposition (PVD) or plasma enhanced chemical vapor deposition (PECVD). Alternatively, plasma exposure may be used to directly activate the substrate. For example, a plasma etching procedure may be used to oxidize the polymer surface (i.e., polystyrene or polyethylene to expose polar functional groups such as hydroxyl, carboxylic acid, aldehyde, etc.).

[0109] The coating may include a metal film. Examples of metal films include aluminum, chromium, titanium, nickel stainless steel zinc, lead, iron, magnesium, manganese, cadmium, tungsten, cobalt, and alloys or oxides thereof. In some embodiments, the metal film may be a noble metal film. Examples of noble metals that may be used in the coating include, but are not limited to, gold, platinum, silver, copper, and palladium. In some embodiments, the coating includes gold or a gold alloy. Electron beam evaporation may be used to provide a thin coating of gold on the surface. In some embodiments, the metal film may be about 50 nm to about 500 nm thick.

[0110] Alternatively, the coating may be silicon, silicon oxide, silicon nitride, silicon hydride, indium tin oxide, magnesium oxide, alumina, glass, hydroxide surfaces, and polymers.

[0111] The arrays described herein may comprise a collection of addressable elements. Such elements may be spatially addressable, such as an array contained within a microtiter plate, or an array printed on a flat surface where each element may reside at a distinct X and Y coordinate. Alternatively, the elements may be addressable based on tags, beads, nanoparticles, or physical properties. Microarrays can be prepared according to methods known to those skilled in the art. The term "array" as used herein may refer to any biological assay having multiple addressable elements. In some embodiments, the addressable elements may be polypeptides (e.g., antibodies or fragments thereof) or nucleic acid probes. As used herein, "elements" refers to any probe (polypeptide or nucleic acid based) to which a transcript encoding a polypeptide, such as an organ-specific polypeptide, a polypeptide fragment, or one related or associated with any of the genes or proteins disclosed herein, may bind. Molecules may be, but are not limited to, proteins, polypeptides, peptides, RNA, DNA, lipids, glycosylated molecules, carbohydrates, polypeptides with phosphorylation modifications, and polypeptides with citrulline modifications, aptamers, oxidized molecules, and other molecules.

[0112] With respect to the elements described herein, "addressable" refers to a location, position, tag, cleavable tag or marker, identifier, spectral property, electrophoretic property, or other physical property that allows for the identification of the element. An example of addressability, also known as coding, is spatial addressability, where the location of a molecule is fixed and the location correlates with its identity. Spatial arrays of this kind can generally be synthesized or spotted on planar substrates, for example, where many different molecules are densely placed in a small area (e.g., 1 cm 2 Each cell contains at least about 400 different sequences. 2 1000 arrays per cm 2This produces microarrays (which may contain as many as 5000 sequences per cm2, or more). Lower density arrays (e.g., ELISA or RIA plates), where the wells in the plate each contain a distinct probe, can contain from about 96 sequences per plate, up to about 100 sequences per cm2, the density of a microarray. Other spatial arrays utilize optical fibers, where distinct probes are attached to the fibers, which can be formed into bundles for binding and analysis. Methods for the production and use of spatial arrays of polypeptides are known in the art.

[0113] An alternative to this type of spatially coded array is the use of molecular "tags", where target probes can be attached to detectable labels or tags that can provide coded information about the sequence of the probe. These tags can be cleaved from the elements and then detected to identify the elements. In some embodiments, a set of probes can be synthesized or attached to a set of coded beads, where each bead can be linked to a separate probe, and the beads can be coded in a manner that allows for the identification of the attached probe. In this type of "tag array", flow cytometry can be used to detect binding. For example, microspheres with fluorescent codes can identify specific microspheres. Probes can be covalently linked to "color-coded" entities. Labeled target polypeptides can be detected by flow cytometry, and the coding on the microspheres can be used to identify the bound probes (e.g., immunoglobulins, antigen-binding fragments of immunoglobulins, or ligands).

[0114] In some embodiments, the array may be an array that includes one or more antigens (e.g., environmental or food antigens or allergens). In some embodiments, the array may be an immunoglobulin array (e.g., an array that includes antibodies or antigen-binding fragments thereof). As used herein, "immunoglobulin array" refers to a set of spatially separated distinct molecular entities that can bind to target polypeptides arranged in a manner that allows identification of the polypeptides contained within a sample. In some embodiments, the array may include one or more of proteins, polypeptides, peptides, RNA, DNA, lipids, glycosylated molecules, polypeptides with phosphorylation modifications, and polypeptides with citrulline modifications, aptamers, and other molecules.

[0115] kit In some embodiments, kits are provided for measuring one or more immunoglobulins in a sample. In some embodiments, kits are provided for measuring the RNA (e.g., RNA products) of one or more biomarkers disclosed herein. The kits can include materials and reagents that can be used to measure the expression of the RNA of one or more biomarkers. Examples of suitable kits include RT-PCR or microarrays. These kits can include the reagents required to perform the measurement of RNA expression levels. Alternatively, the kits can further include additional materials and reagents. For example, the kits can include the materials and reagents required to measure the RNA expression levels of any number of genes, up to 1, 2, 3, 4, 5, 10, or more, that are not biomarkers disclosed herein.

[0116] Gene Expression Panels Disclosed herein are gene expression panels and arrays for diagnosing EoE or active EoE in a subject (e.g., human), which consist of primers or probes capable of detecting one or more genes disclosed herein.The disclosed gene expression panels or arrays may include any number of genes disclosed herein.For example, the gene expression panels or arrays may be used to detect one or more of CCL26, KITLG, and POSTN.In some embodiments, the gene expression panels or arrays may include CCL26, KITLG, and POSTN.

[0117] In some embodiments, the sample may be an esophageal biopsy, a mucosal sample, or an esophageal secretion.

[0118] A gene expression panel or array disclosed herein may consist of primers or probes capable of detecting or amplifying any number of genes disclosed herein. A gene expression panel or array disclosed herein may further include primers or probes capable of detecting or amplifying any number of genes not disclosed herein. For example, the primers or probes may detect or amplify between 1-5, 5-10, 10-100, or more, or any variation therebetween.

[0119] The gene expression panels or arrays disclosed herein may be an independent method for evaluating EoE in a subject, or may be combined with one or more other gene expression panels or arrays not disclosed herein. They may be used with one or more diagnostic tests. In some embodiments, the gene expression panels or arrays may further comprise a second diagnostic test. The gene expression panels or arrays disclosed herein may also be used in a method for generating a specific profile. The profile may be provided in the form of a heat map or a box plot.

[0120] The profile of gene expression levels can be used to calculate a statistically significant value based on the differential expression of one or more genes disclosed herein, where the calculated value correlates with the diagnosis of EoE subtype.The variance in the obtained profile of the expression levels of the selected genes or gene expression products can be either up-regulated or down-regulated in subjects with increased susceptibility compared to reference subjects or controls.Additional details are provided in the Examples section.For example, if the expression levels of one or more of CCL26, KITLG, and POSTN are up-regulated, EoE or active EoE is indicated.As described herein, a person skilled in the art can use any combination of genes disclosed herein to form a profile that can be used later to evaluate EoE or active EoE, or to determine (and diagnose) whether a subject has EoE or active EoE.

[0121] Disclosed herein are methods for diagnosing EoE using the gene expression panels or arrays described herein.

[0122] In some embodiments, the gene expression panels or arrays disclosed herein can be used to determine or assess EoE or active EoE in a subject, where the expression level of CCL26, KITLG, or POSTN in a sample is compared to a reference expression level of CCL26, KITLG, or POSTN. In some embodiments, the gene expression panels or arrays disclosed herein can be used to determine or assess EoE or active EoE in a subject, where the ratio (or percent change) of the sample expression level of one or more of CCL26, KITLG, or POSTN to the reference expression level of one or more of CCL26, KITLG, or POSTN indicates a higher expression level of one or more of CCL26, KITLG, or POSTN in the sample. In some embodiments, the ratio (or percent change) of the sample expression levels of two or more of CCL26, KITLG, or POSTN to the reference expression levels of two or more of CCL26, KITLG, or POSTN indicates that the expression levels of two or more of CCL26, KITLG, or POSTN are higher in the sample, which indicates that the subject has EoE or active EoE. Suitable statistical and other analyses can be performed to confirm the change (e.g., increased expression or higher level) in one or more of CCL26, KITLG, or POSTN when compared to the reference sample.

[0123] Gene expression panel or array can be composed of primers or probes that can detect, amplify or otherwise measure the presence or expression of one or more genes disclosed herein.For example, the specific primers that can be used in the methods disclosed herein include, but are not limited to, the primers suitable for use in standard exon arrays from the Affymetrix website listed at http: / / www.affYmetrix.com.In some embodiments, the gene expression panel or array disclosed herein can be used to determine or evaluate EoE or active EoE in a subject, where CCL26, KITLG, or POSTN RNA expression levels are detected in a sample.

[0124] In some embodiments, diagnostic kits are disclosed that contain one or more probes or primers capable of detecting, amplifying, or measuring the presence or expression of one or more genes disclosed herein.

[0125] Disclosed herein is a solid support that comprises one or more primers, probes, polypeptides, or antibodies that can hybridize or bind to one or more of the genes disclosed herein. A solid support is a solid substrate or support with which molecules such as analytes and analyte-binding molecules can be associated. Analytes (e.g., mineralized nanoparticles and proteins) can be directly or indirectly associated with a solid support. For example, analytes can be directly immobilized on a solid support. Analyte capture agents (e.g., capture compounds) can also be immobilized on a solid support.

[0126] As mentioned herein, those skilled in the art can determine the expression level of one or more genes (or proteins) disclosed herein by any method.To detect or quantify the level of the RNA product of biomarker in a sample, arrays, such as microarrays, RT-PCR (including quantitative RT-PCR), nuclease protection assay, and Northern blot analysis can be used.Thus, in some embodiments, biomarker expression level can be determined using arrays, microarrays, RT-PCR, quantitative RT-PCR, nuclease protection assay, or Northern blot analysis.

[0127] In addition, the genes described herein can also be used as markers (i.e., biomarkers) for EoE or susceptibility to active EoE or the presence or progression of the same. The methods and assays described herein can be performed over time to assess changes in the levels of the markers. For example, assays can be performed every 24-72 hours for a period of 6 months to 1 year, and then as needed. Assays can also be completed before, during, or after a treatment protocol. Collectively, the genes disclosed herein can be used to profile an individual's EoE status. As used within this context, the term "differentially expressed" or "differential expression" refers to differences in the expression levels of the biomarkers disclosed herein, which can be assayed by measuring the levels of expression of the product (e.g., RNA or gene product) of the biomarker, such as a messenger RNA transcript or a portion thereof, or a difference in the level of the expressed protein of the biomarker. In some embodiments, the differences are significantly different.

[0128] To improve sensitivity, more than one gene disclosed herein can be assayed in a given sample.Different proteins, antibodies, nucleic acid specific binding agents provided herein can be combined in a single assay.Furthermore, multiple primers or probes can be used simultaneously.To support such assays, certain biomarkers can assist the specificity of such tests.

[0129] Expression levels can be measured at the transcriptional and / or translational levels. At the translational level, expression of any of the genes described herein can be measured using immunoassays, including immunohistochemical staining, Western blotting, ELISA, etc., with antibodies that selectively bind to the corresponding gene or a fragment thereof. Detection of proteins using protein-specific antibodies in immunoassays is known in the art. At the transcriptional level, mRNA can be detected, for example, by amplification (e.g., PCR, LCR), or hybridization assays (e.g., Northern hybridization, RNAse protection, or dot blotting). Protein or mRNA levels can be detected, for example, by using directly or indirectly labeled detection agents (e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzyme-labeled antibodies). Changes in transcription levels (e.g., increases or decreases) can also be measured using promoter-reporter gene fusion constructs. For example, the promoter region of a gene encoding any of the genes disclosed herein can be fused (i.e., operably linked) to a coding sequence of a polypeptide that generates a detectable signal. Reporter constructs are well known in the art. Examples of reporter sequences include fluorescent proteins (eg, green, red, yellow), phosphorescent proteins (eg, luciferase), antibiotic resistance proteins (eg, beta-lactamase), enzymes (eg, alkaline phosphatase). EXAMPLES

[0130] Example 1: Food-specific antibodies in esophageal secretions: Association with trigger foods in eosinophilic esophagitis (EoE) Objective: To examine whether antibodies against foods that induce eosinophilic esophagitis are secreted into the esophageal lumen, where they can be collected by esophageal scraping.

[0131] METHODS: Food-specific immune responses were assessed in scrapings of 68 patients (12 controls, 13 with resolved eosinophilic esophagitis, and 43 with active eosinophilic esophagitis) undergoing endoscopy. Trigger foods in 17 participants were identified via food elimination diets. Immunoglobulin A and immunoglobulin G4 antibodies to the four most common food triggers of eosinophilic esophagitis were measured in esophageal scrapings using the ImmunoCAP assay. Food-specific antibody values ​​were compared between active eosinophilic esophagitis, resolved eosinophilic esophagitis, and controls.

[0132] RESULTS: Patients with active eosinophilic esophagitis (>15 eosinophils / hpf) showed increased IgA and IgG4 levels to common eosinophilic esophagitis triggers compared with controls (327±380 vs. 150±130 for IgA, and 1534±3346 vs. 178±123 for IgG4, p<0.003). Specific trigger foods were associated with elevated IgA and IgG4 responses compared with foods that did not induce esophageal eosinophilia (733±469 vs. 142±64 for IgA, p<0.001, and 2620±3228 vs. 526±1050 for IgG4, p<0.001).

[0133] CONCLUSIONS: Food-specific antibodies were collected along the esophageal lumen of patients with eosinophilic esophagitis.

[0134] Introduction: Eosinophilic esophagitis (EoE) is increasingly recognized as a common cause of reduced quality of life, dysphagia, and bolus obstruction in all age groups (Holbreich M. Allergy Asthma Proc 2019;40:198-203; Peiris CD, Tarbox JA. JAMA 2019;321:1418; Reed CC, Dellon ES. Med Clin North Am 2019;103:29-42, and Lucendo AJ, et al. Aliment Pharmacol Ther 2017;46:401-409). Although an immunological response to food is involved, allergy testing (skin prick testing, immunoglobulin E antibody testing, or atopy patch testing) does not identify the food causing the disease (Aceves SS. Allergy Testing in Patients with Eosinophilic Esophagitis. Gastroenterol Hepatol (NY) 2016;12:516-518, Anyane-Yeboa A, Wang W, Kavitt RT. The Role of Allergy Testing in Eosinophilic Esophagitis. Gastroenterol Hepatol (NY) 2018;14:463-469, and Philpott H,et al. Aliment Pharmacol Ther 2016;44:223-33).However, food antigens have been implicated in the pathogenesis and exacerbation of EoE in both pediatric and adult populations, as evidenced by resolution of disease following an elimination diet (Philpott H,et al. Aliment Pharmacol Ther 2016;44:223-33; Chehade M,Sher E. Allergy Asthma Proc 2017;38:170-176; Fahey LM,et al. Clin Transl Gastroenterol 2018;9:139; de Bortoli N,Penagini R,Savarino E,et al.Dig Liver Dis 2017;49:254-260; and Philpott H,Thien F.The Role of Allergy Testing in Eosinophilic Esophagitis:an Update of the Evidence.Curr Treat Options Gastroenterol 2017;15:26-34). Immunoglobulin E is elevated in the mucosa of EoE patients, but clinical trials evaluating the accuracy of IgE-based skin prick tests to identify causative foods have not shown satisfactory results (Philpott H, Thien F. Curr Treat Options Gastroenterol 2017;15:26-34; Kamdar TA,et al. Clin Mol Allergy 2010;8:16; Gottlieb SJ,et al. J Allergy Clin Immunol 2013;131:242-3; Aceves SS. Clin Gastroenterol Hepatol 2014;12:1216-23; Paquet B, Begin P, Paradis L,et al. J Allergy Clin Immunol 2013;131:613; Erwin EA,et al. J Allergy Clin Immunol Pract 2015;3:896-904 e3, and Turnbull JL, et al. Aliment Pharmacol Ther 2015;41:3-25).Similarly, clinical trials of omalizumab failed to reduce eosinophil infiltration in the esophagus of patients with EoE (Clayton F, et al. Gastroenterology 2014;147:602-9). Others have evaluated the sensitivity and specificity of atopy patch testing, hypothesizing that a cell-mediated response to food leads to esophageal eosinophilia, but with poor results (Aceves SS. Allergy Testing in Patients with Eosinophilic Esophagitis. Gastroenterol Hepatol (NY) 2016;12:516-518; Anyane-Yeboa A,et al. The Role of Allergy Testing in Eosinophilic Esophagitis. Gastroenterol Hepatol (NY) 2018;14:463-469; Bahna SL. Allergy Asthma Proc 2008;29:447-52; and Blanchard C,et al. J Intern Med 2017;281:448-457). In EoE, inflammation is localized to the esophagus, allowing the disease to be characterized as the result of a local, esophageal-specific immune response. For example, Clayton et al. identified increased levels of total immunoglobulin G4 in tissue homogenates from EoE, suggesting that a local immune response occurs (Clayton F,et al.Gastroenterology 2014;147:602-9). Forceps and scraping biopsies revealed that immunoglobulins (immunoglobulin G4 and immunoglobulin E) are increased in EoE tissue (Ramaswamy AT,et al.Int Forum Allergy Rhinol 2019;9:870-875). Although not specifically studied in EoE, immunoglobulin A is a known mucosal clearance antibody secreted to the gastrointestinal surface to protect the body from possible threats such as bacteria and viruses (Carlier FM,et al.Clin Exp Allergy 2016;46:1372-1388).Local responses in diseased tissues may also involve either secretory or immune-mediated immunoglobulin A production, as seen in celiac disease (Koninckx CR, et al. J Pediatr Gastroenterol Nutr 1984;3:676-82, and Lamm ME, et al. APMIS 1995;103:241-6). Immunoglobulin A levels and responses are increasingly being studied in food allergy (Ahrens N, et al. Clin Exp Immunol 2008;151:455-8, and Andre C. Letter: Allergy, tolerance, and immunoglobulin A. Lancet 1974;2:782). It is plausible that immunoglobulin A may be secreted in response to food mediating antigen responses in the esophagus. We performed studies to determine whether immunoglobulin G4 and immunoglobulin A are produced in the esophagus of EoE and to discover foods that may cause eosinophilic esophageal inflammation.

[0135] Methods: Study design. This study was designed to test whether EoE-inducing foods also stimulate immunoglobulin A and immunoglobulin G4 antibody production. Patients consented to one-time sampling of the esophagus when attending for upper endoscopy for dysphagia or monitoring for known EoE. Patients were invited to enroll if they met one of two criteria: 1) no previous diagnosis of EoE but dysphagia requiring endoscopic evaluation, or 2) previously diagnosed EoE participating in follow-up endoscopic biopsy to assess disease status. Patients consented to allow the investigator to follow the patient's clinical course and biopsy results during routine clinical care for follow-up. Patients diagnosed with a coexisting immune disorder, including autoimmune esophagitis (i.e., lichen planus), Crohn's disease, systemic sclerosis, or those requiring systemic immunosuppression, were excluded from further analysis. In 68 patients, both immunoglobulin A food-specific and immunoglobulin G4 food-specific antibodies were measured by esophageal brushings and included in the final analysis. The three cohorts analyzed were active EoE, resolved EoE, and controls. Patients were diagnosed with EoE by standard criteria (Dellon ES, et al. Updated International Consensus Diagnostic Criteria for Eosinophilic Esophagitis: Proceedings of the AGREE Conference. Gastroenterology 2018;155:1022-1033 e10), at least one esophageal biopsy and 15 eosinophils per high power field (HPF) for concomitant esophageal symptoms. Patients with a history of EoE who had <15 eosinophils / HPF in their most recent biopsy (and whose symptoms resolved) were considered as resolved EoE patients. Those presenting with dysphagia without a history of esophageal diagnosis who had normal histopathology in their esophageal biopsy were considered as controls.

[0136] Collection of Esophageal Secretions. Esophageal scrapings were obtained during upper endoscopy prior to esophageal biopsy. A Cook medical cytobrush (Cook Medical, Indianapolis, IN) was applied through the endoscope to the lumen of the esophagus and withdrawn from the distal 5 cm of the esophagus into the upper esophagus (approximately 15-20 cm from the esophagus). The brush was then removed and flash frozen at -70°C until further evaluation.

[0137] Clinical course of patients on elimination diets. Patients with EoE who chose a food elimination diet as part of their clinical care were monitored and food triggers were recorded. Food triggers were recorded as a drop in eosinophilia to <15 eosinophils / HPF with elimination (patients undergoing single food elimination) or an esophageal eosinophilia >15 eosinophils / HPF after resolution with reintroduction (multiple food eliminations). Standard care includes a 4-week reintroduction of specific foods before repeat endoscopy to identify trigger foods (Gonsalves N. Gastrointest Endosc Clin N Am 2018;28:89-96, and Gonsalves N,et al. Gastroenterology 2012;142:1451-9 el). Symptoms alone were not sufficient to classify patients as reactive to specific foods. For patients who had multiple foods eliminated, foods that did not increase eosinophil counts above 15 eosinophils / HPF after reintroduction following resolution were identified as true negatives. Patients who completed reintroduction were included in the final analysis.

[0138] Food-specific antibody testing. Brushes were thawed and equilibrated with 1 ml of Phadia diluent (product no. 10-9498-01, Kalamazoo, MI) for approximately 30 minutes with occasional shaking. In a separate experiment, the amount of secretion per brush was measured by centrifugation at 10,000 RPM for 1 minute, yielding approximately 21-23 microliters of fluid per brush. Extracted esophageal fluid in Phadia diluent at approximately 1:50 dilution was analyzed for immunoglobulin G4 and immunoglobulin A antibodies to four food allergens, wheat f1, soybean f14, casein f78, and egg f245, using Phadia ImmunoCap reagent. Casein and gluten were selected for testing because they are specific food proteins believed to be the primary reactants to dairy and wheat, respectively. Briefly, 40 μL of diluted secretions were added to a food-specific solid-phase antigen called CAP, followed by washing and adding the immunoglobulin A / immunoglobulin G4 antibody conjugate, repeated washing, and finally adding the development solution according to the protocol. Stop solution was added and the resulting fluorescent signal (response value) was measured. Due to the limitations of concentration measurement from a 1:50 dilution, response values ​​were measured and reported. Results are listed as response values. For assay validation, low and high range control samples were included in each run. Participants were assayed for food-specific antibodies against gluten, casein, soy, and egg with both immunoglobulin G4 and immunoglobulin A assays and report response values ​​detected using the Immunocap system.

[0139] Statistical analysis. Comparisons between groups for response values ​​were performed via ANOVA and Mann-Whitney U-tests depending on the group evaluated. To assess food triggers, for patients who underwent food elimination tests, foods that induced EoE (true positives) and foods that did not induced EoE (true negatives) were determined. To take into account that collected volumes differed between individuals and to maintain valid comparisons afterwards, immunoglobulin G4 and immunoglobulin A response values ​​were standardized within individuals (newly scaled to have a mean of zero and a standard deviation of one). To identify food triggers for each individual, heat maps were used for the standardized immunoglobulin G4 and immunoglobulin A response values ​​for gluten, soy, casein, and egg, and the results were confirmed by a post hoc probabilistic logistic regression model. The statistical significance of differences in categorical and quantitative variables between groups was evaluated using Fisher's exact test and Mann-Whitney U-test, respectively.

[0140] Results: Patient characteristics. Sixty-eight patients were enrolled at the time of endoscopy and successfully underwent esophageal brushing prior to esophageal biopsy. Demographics are shown in Table 1. Twelve control participants had dysphagia but no concomitant systemic autoimmune or allergic disease and normal histopathology on biopsy. Thirteen patients with resolved EoE showed <15 eosinophils / HPF versus >15 eosinophils / HPF on previous biopsy, and resolved patients were treated with topical steroids (n=3), proton pump inhibitors (n=3), or food elimination diets (n=7). The remaining enrolled patients met criteria for active EoE.

[0141] Table 1: Demographics of the total cohort. Forty-three patients with active eosinophilic esophagitis (EoE: >15 eosinophils / HPF and esophageal symptoms) were enrolled, 17 completed the food elimination and reintroduction diets. Thirteen patients were treated and had <15 eosinophils / HPF with resolution of symptoms and are patients with resolved eosinophilic esophagitis (resolved EoE). Twelve patients had dysphagia but normal esophageal biopsies, these patients are controls. [Table 1]

[0142] Active EoE patients show food-specific immunoglobulin A and immunoglobulin G4. Analysis of esophageal secretions revealed the presence of immunoglobulin A responses and immunoglobulin G4 responses, as shown in Figures 1 and 2 respectively. Overall, EoE patients had increased levels of immunoglobulin A responses and immunoglobulin G4 responses over the course of the examination when compared to controls (for immunoglobulin A, 327±380 vs. 150±130, and for immunoglobulin G4, 1534±3346 vs. 178±123, both p<0.003). When comparing among individual populations, active EoE patients had significantly higher values of immunoglobulin A responses to casein (p<0.001), gluten (p<0.001), soybean (p<0.001), and egg (p<0.008) on average compared to resolved EoE patients. Participants with active EoE had higher values of immunoglobulin A responses to gluten (p<0.001) and casein (p<0.001) compared to controls, but the response values for soybean (p = 0.223) and egg (p = 0.379) were not significantly different from those of controls. Participants with active EoE showed higher levels of immunoglobulin G4 responses to all foods when compared to resolved EoE and controls (Table 2).

Table 2

[0143] With an elimination diet, provoking foods were identified in 17 patients. A total of 27 patients tried a food elimination diet. Seven patients failed the selected elimination diet and did not proceed to additional food elimination (3 failed 6 (or more) food elimination diets, 2 failed 2 food eliminations, and 2 failed 1 food elimination). Three patients responded to 6 food elimination diets but failed the reintroduction of all foods.

[0144] A total of 17 patients completed all selected dietary trials. Five patients underwent single food elimination and had resolution of esophageal eosinophilia with the single excluded food as the trigger food. The remaining 12 patients chose to eliminate multiple foods from the diet. A trigger food was identified when symptoms and esophageal eosinophilia returned after reintroduction of that one particular food group after initial resolution of disease. Before food elimination, eosinophil counts averaged 60.5 eosinophils / HPF (normal 32.1), whereas after, eosinophil counts averaged 6.2 eosinophils / HPF (normal 4.1) (p<0.001) (Figure 3). In addition, the food elimination diet improved endoscopic reference scores assessing edema, rings, exudates, grooves, and strictures, changing from a high of 4.5 (normal 1.9) before the diet to 2 (normal 1.6) after the diet (p<0.001). Strictures were unlikely to resolve with a diet. Food triggers were either wheat (n=9), dairy products (n=4), or both (n=4). One patient eliminated pork and beef prior to testing due to a "reaction." Another patient responded to reintroduction of chicken and rice with esophageal eosinophilia of >15 eosinophils / HPF. Finally, an additional patient felt ill upon reintroduction of eggs but did not develop esophageal eosinophilia (8 eosinophils / HPF).

[0145] Immunoglobulin A and Immunoglobulin G4 food-specific antibody reactivity is increased to trigger foods. Immunoglobulin A and Immunoglobulin G4 antibody levels in 17 patients who successfully completed food elimination trials are shown in Table 3 and Figures 4 and 5. Foods known to be triggers of EoE (trigger foods) had higher IgA antibody levels than non-trigger foods (733±469 vs. 142±64, p<0.001). Participants with wheat as a trigger food had higher IgA antibody levels to gluten than patients without wheat as a trigger food (707±430 vs. 185±80, p<0.004). Participants with dairy as a trigger food had higher IgA antibody levels to casein than those without dairy as a trigger food (859±515 vs. 197±94, p<0.021).

[0146] Table 3. Immunoglobulin A and Immunoglobulin G4 antibody levels to gluten, soy, casein, and egg in 17 patients who completed food elimination and reintroduction diets. Most trigger foods showed high levels for Immunoglobulin A and Immunoglobulin G4. Variability in Immunoglobulin G4 within and between patients made interpretation difficult for the entire cohort. Immunoglobulin A and Immunoglobulin G4 antibody levels to trigger foods are in bold in the table. *Measurements are based on immunofluorescence reactivity (i.e., response values). [Table 3]

[0147] Trigger foods also produced greater immunoglobulin G4 response values ​​than non-trigger foods (2620±3228 vs. 526±1050, p<0.001). However, when comparing immunoglobulin G4 antibody levels specifically to gluten, participants with a wheat trigger did not differ significantly from participants for whom wheat was not a trigger food (2988±4097 vs. 936±908, p<0.25). Participants with a dairy trigger did not have a significantly different response to casein than participants without a dairy trigger (2349±2000 vs. 976±1818, p<0.49), which may be due to the high variability seen in the immunoglobulin G4 response.

[0148] Foods implicated as triggers by elevated IgA and IgG4 responses. A total of 21 food triggers were identified in 17 patients (Table 2). Although significant variation in response values ​​occurred between patients, food triggers were almost always identified by higher antibody responses for IgA and IgG4. For IgA responses, in 19 of 21 cases, the food trigger gave the highest response value (or the top two highest response values ​​in the case of two food triggers). For IgG4 responses, although there was considerable variation across and between patients, 20 of 21 food triggers gave the top response value. Thus, most food triggers were identified by positive responses to both IgA and IgG4. IgA worked most accurately in identifying the causative food causing EoE. Post-hoc logistic regression analysis of the heatmaps (Figures 4b and 4d) confirmed that in accordance with the immunoglobulin A results, 1 out of 17 misclassifications were found for wheat as a trigger food (i.e., 94.1% correct classification), 2 for dairy (i.e., 88.2% correct classification), and 2 for wheat / dairy (i.e., 88.2% correct classification), indicating high classification ability.

[0149] Saliva, serum, and esophageal secretions were collected and examined from three patients during upper endoscopy. Immunoglobulin response ratios differed among the three different sources, indicating that the esophageal collections were not significantly affected by blood or saliva contamination (Figure 9).

[0150] The follow-up of patients in the active EoE group was recorded when they attended for follow-up endoscopies regarding diet, and both immunoglobulin A and immunoglobulin G4 tests were performed on patient scrapings. In the three cases examined, food-specific antibodies appeared to decrease in parallel with the resolution of the disease (Table 4).

[0151] Table 4. Three patients underwent esophageal sampling before and after a food elimination diet when eosinophilia had resolved (<15 eosinophils / HPF). Food antibody response values ​​appeared to decrease with resolution of disease and / or elimination of foods. Patient 1 was sampled after undergoing wheat and dairy elimination. Patient 2 had repeat sampling (based on skin IgE testing) after elimination of dairy, peas, legumes, fruits, and vegetables. Patient 3 underwent dairy elimination before the second sampling. Patient 4 was using topical esophageal steroids at the time of the second sampling. [Table 4]

[0152] Discussion: Currently, accurate testing for food triggers in Eosinophilic Esophagitis is not available, leading to patient and physician frustration (Anyane-Yeboa A,et al.The Role of Allergy Testing in Eosinophilic Esophagitis. Gastroenterol Hepatol(NY)2018;14:463-469, Philpott H,et al.Aliment Pharmacol Ther 2016;44:223-33, Aceves SS.Clin Gastroenterol Hepatol 2014;12:1216-23, and Assa'ad A.Ann Allergy Asthma Immunol 2005;95:309-11). Skin prick testing has been found to be less than 20% accurate in well-performed dietary elimination trials (Gonsalves N. Gastrointest Endosc Clin N Am 2018;28:89-96, and Gonsalves N,et al. Gastroenterology 2012;142:1451-9). It has been speculated that the esophagus may develop a local immune response by B-cell switching (Turnbull JL,et al. Aliment Pharmacol Ther 2015;41:3-25). This theory may explain why skin prick testing may not reflect esophageal triggers. Warners et al. tested this hypothesis by performing a prick test on food in the esophagus of patients with EoE during endoscopy. This study demonstrated that the esophagus is capable of immediate responses to specific antigens (Warners MJ, et al. Gastroenterology 2018;154:57-60 e2, and Warners MJ, et al. Am J Physiol Gastrointest Liver Physiol 2017;313:G230-G238). Such esophageal responses, potentially related to immunoglobulin E, were distinct from those identified by skin prick testing.However, it remains unclear whether this localized test has the ability to identify causative triggers (Blanchard C, et al. J Intern Med 2017;281:448-457). Previous studies treating EoE patients with omalizumab (Xolair) have failed to reduce EoE esophageal eosinophilia, undermining the idea that IgE responsiveness is important in EoE pathophysiology (Philpott H, et al. Aliment Pharmacol Ther 2016;44:223-33). IgG4 is highly upregulated in EoE esophageal mucosa and may reflect a causative response to disease-inducing antigens (Clayton F, et al. Gastroenterology 2014;147:602-9). However, several studies have found that esophageal tissue homogenates and sera tested for food-specific immunoglobulin G4 did not accurately predict the causative antigen (Wright BL,et al. J Allergy Clin Immunol 2016;138:1190-1192, Bjorksten B,et al. Allergy 1983;38:119-24, Guhsl EE,et al. Allergy 2015;70:59-66, McGowan EC,et al. Ann Allergy Asthma Immunol 2019, Pope AE,et al. J Pediatr Gastroenterol Nutr 2019;68:689-694, and Schuyler AJ,et al. J Allergy Clin Immunol 2018;142:139-148). A recent study combined peripheral CD4+ T cell proliferation with esophageal tissue immunoglobulin G4 food-specific detection to manage food elimination. This resulted in patient improvement, but only modest resolution (McGowan EC, et al. Ann Allergy Asthma Immunol 2019). These results may have been influenced by the patchy nature of immunoglobulin G4 in the mucosa in EoE. The presence of immunoglobulin G4 is variable throughout the esophageal mucosa and may not be accurately reflected in homogenates from a single biopsy.However, immunoglobulin testing may provide answers to food triggers in EoE, especially in light of recent data identifying food allergens in the mucosa in EoE (Philpott H, Dellon ES. Gastroenterology 2017;153:605-606).

[0153] When scrapings obtained from the affected esophagus of patients with EoE were analyzed to perform more extensive surface sampling, the results suggest that the levels of immunoglobulin A and immunoglobulin G4 against specific foods were elevated in esophageal secretions obtained along the esophageal surface. Furthermore, these food-specific immunoglobulins identified food triggers in EoE patients with a high classification ability ranging from 88.2% to 94.1%. In addition, the results show that immunoglobulin A and immunoglobulin G4 antibodies are not substantially elevated in normal mucosal secretions in resolved EoE and control patients, but rather appear to be closely associated with the active eosinophilic disease itself. Food-specific antibodies are not easily detected after the disease has resolved (by topical steroids or diet). Note that the results in Figures 1 and 2 between active and resolved EoE show minimal immunoglobulin A and immunoglobulin G4 antibody levels for food antigens in resolved patients. In addition, locally produced food-specific antibodies do not appear to be significantly contaminated by plasma or saliva. For this reason, esophageal scrapings from affected esophagi in EoE may contain valuable information and be useful in identifying the causative antigen. Three patients were followed before and after dietary therapy, and these data suggest that food-specific antibodies appear to decrease when the disease is controlled with diet. However, it remains unclear whether the expression of food-specific antibodies depends on active food intake and / or active disease.

[0154] Immunoglobulin A, a food-specific antibody found along the affected esophageal lumen, appears to distinguish trigger foods (foods that cause esophageal inflammation) from non-trigger foods better than immunoglobulin G4 responses. This may be due to the high variability of immunoglobulin G4 responses in relatively small cohorts. In addition, immunoglobulin G4 to foods (especially dairy products) is commonly elevated in the serum of both food-allergic and non-allergic cohorts (Schuyler AJ, et al. J Allergy Clin Immunol 2018;142:139-148). For this reason, it is plausible that immunoglobulin G4 responses can occur without the indication of a trigger food. Interestingly, however, immunoglobulin G4 food-specific antibody responses were most markedly elevated to trigger foods compared to other foods. Overall, immunoglobulin A response values ​​were less variable at the time of sampling and were significantly higher to foods implicated in the causation of EoE compared to foods that were not causative of EoE.

[0155] Contamination of esophageal collections from plasma or saliva was also examined (Figure 9). The inflamed esophageal lumen may contain proteins derived from plasma extravasation during inflammation, as well as contamination from salivary secretions. Saliva contains abundant immunoglobulin A, which may interfere with the assay. Food-specific immunoglobulin expression decreased in the food lumen after resolution of the disease, regardless of therapy modality (proton pump inhibitors, topical steroids, elimination diet), arguing against salivary contamination.

[0156] Although the study described herein is limited by its small sample size, the results show that patients with wheat- and dairy-induced EoE are present in this study in sufficient numbers to allow for meaningful calculation of diagnostic sensitivity. Furthermore, the study was limited to food-specific immunoglobulin A and immunoglobulin G4, and no other immunoglobulins were tested. Total immunoglobulin A was tested, and no distinction was made between immunoglobulin A1 and immunoglobulin A2. Nevertheless, the initial assay appears to distinguish between wheat and dairy triggers and identify them as important. EoE patients generally appeared to be more immune responsive than controls, even to foods that do not cause EoE. The immunoglobulin A response values ​​to soybeans and eggs (non-trigger foods) in EoE patients were not significantly different from controls. However, the immunoglobulin G4 response to these same non-trigger foods was increased in EoE compared to controls. This may be related to the high variability of immunoglobulin G4 responses seen among EoE patients themselves. IgG4 testing was highly variable within and between patients, making overall comparisons between patients of IgG4 responses compared with IgA difficult.

[0157] Immunoglobulin G4 is a known antibody recently identified in EoE. Immunoglobulin G4 has been shown to correlate with histological features in EoE (Pope AE, et al. J Pediatr Gastroenterol Nutr 2019;68:689-694, and Rosenberg CE, et al. Allergy 2018;73:1892-1901). Immunoglobulin A is the most abundant immunoglobulin in mucosa, where it acts as an active barrier through immune clearance of ingested antigens. Its role in allergy remains unclear. Immunoglobulin A may act as a protective mechanism in EoE and bind to allergens before infiltrating tissues (Turnbull JL, et al. Aliment Pharmacol Ther 2015;41:3-25). However, immunoglobulin A may also contribute to disease pathogenesis as it has been shown to activate eosinophils and induce degranulation (Motegi Y, et al. Int Arch Allergy Immunol 2000;122 Suppl 1:25-7, and Muraki M, et al. Int Arch Allergy Immunol 2011;154:119-27).

[0158] In summary, these findings support the conclusion that the measurement of antibodies against food in scrapings from eosinophilic esophagitis can be used to identify food triggers of eosinophilic esophagitis.Furthermore, low antibody response values ​​against food can serve as a marker of successful EoE treatment.The results described herein suggest that the esophagus secretes detectable local immunoglobulins against antigens involved in the disease process, and that the methods disclosed herein can be used as an immunological test for patients with EoE.

[0159] Example 2: Specific production of antibodies against antigens arises from the disease itself 2. Background Art Eosinophilic esophagitis (EoE) is increasingly recognized as a common cause of reduced quality of life, dysphagia, and bolus obstruction in all age groups. Food antigens have been implicated in the pathogenesis and exacerbation of EoE in both pediatric and adult populations, as evidenced by resolution of disease after an elimination diet.

[0160] Food-specific antibodies have been shown to be expressed from affected esophagi in eosinophilic esophagitis and to be able to identify foods causing inflammation in the esophagus. It remains unclear whether food-specific antibodies are easily reproducible in diseased areas. In addition, it has not been determined whether food-specific antibodies were different between diseased and disease-free areas, arguing against local production of antibodies due to the disease itself. Two tests were performed on samples along the affected esophageal surface (to demonstrate that the results could be replicated along the affected surface). Other tests included comparison of responsiveness to foods and magnitude of response (>15 eosinophils / HPF in biopsy) depending on whether active disease was present in the tissue.

[0161] Methods. This study was designed to test whether food-specific immunoglobulin A and immunoglobulin G4 antibody production (food-specific antibody (FSA)-IgA and FSA-IgG4) differed between areas of active and inactive eosinophilic disease. Patients consented to a one-time sampling of the esophagus when attending for upper endoscopy for dysphagia or monitoring for known EoE. Esophageal scrapings were obtained during upper endoscopy and prior to esophageal biopsy. A Cook medical cytobrush (Cook Medical, Indianapolis, IN) was applied through the endoscope to the lumen of the esophagus and scrapings were made over areas of endoscopically active disease followed by areas of inactive disease if different degrees of disease were seen in the esophagus. If the entire esophagus was involved, two scrapings were applied to active disease to determine whether food-specific antibody measurements rose and fell within disease or produced similar reproducible results. The brushes were then removed and flash frozen at -70°C until further evaluation. Histopathology (ie, eosinophil counts) was recorded for each area sampled.

[0162] Collection of Esophageal Secretions. Esophageal scrapings were obtained during upper endoscopy prior to esophageal biopsy. A Cook medical cytobrush (Cook Medical, Indianapolis, IN) was applied through the endoscope to the lumen of the esophagus and withdrawn from the distal 5 cm of the esophagus into the upper esophagus (approximately 15-20 cm from the esophagus). The brush was then removed and flash frozen at -70°C until further evaluation.

[0163] Food-specific antibody testing. Food-specific antibody testing was performed as described herein. Briefly, brushes were thawed and equilibrated with 1 ml of Phadia dilution solution (product no. 10-9498-01, Kalamazoo, MI) for approximately 30 minutes with occasional shaking. Briefly, 40 μL of diluted secretions were added to a food-specific solid-phase antigen called CAP, followed by washing and adding Immunoglobulin A / Immunoglobulin G4 antibody conjugate, repeated washing, and finally adding development solution according to the protocol. Stop solution was added and the resulting fluorescent signal (response value) was measured. Due to the limitations of concentration measurement from a 1:50 dilution, response values ​​were measured and reported. Results are listed as response values. Low and high range control samples were included in each analysis for assay validation.

[0164] Results. Figures 6 and 7 represent both food-specific IgA and IgG4 ingested along diseased areas in the esophagus. The graphs represent the results of two different scrapings sampled in areas of active disease in the esophagus. As shown in the graph in Figure 6, the food antibody ratios for food antigens appear to be similar between each scraping per patient. The food-specific antibody ratios are relatively stable when ingested along areas of disease. In Figure 7, two scrapings from each patient were taken in different areas: (1) areas of histologically positive disease (>15 eosinophils / HPF) and (2) areas of less disease (<7 eosinophils / HPF). In Figure 7A, the ratio of food-specific IgA (FSA-IgA) for casein vs. other foods is much greater in areas of disease (left) compared to areas of less disease (right).

[0165] In Figure 7B, IgG4 appears to change in its affinity for food antigens between diseased (25 eosinophils) and non-diseased areas (4 eosinophils), suggesting that this may be directly produced by the disease itself.

[0166] Figure 7B shows a dramatic increase in IgG4 for casein and eggs in diseased areas (22 eosinophils) compared to non-diseased areas (1 eosinophil). Figure 7D shows some variation in immunoglobulin A eggs between diseased areas (22 eosinophils) and non-diseased areas (1 eosinophil). IgA appears to change little in its affinity for food antigens, but still changes in its presentation depending on whether disease is present or absent.

[0167] These results suggest that IgA may be more indicative of food triggers, even in disease-free areas. IgG4 appears to vary significantly with the presence or absence of disease. IgA also goes up and down in strength of response, indicating that immunoglobulins may be produced by the disease itself and not by the mucosa.

[0168] In conclusion, food-specific antibody tests are reproducible when sampled directly from diseased areas in patients with eosinophilic esophagitis. However, results from both immunoglobulin A and immunoglobulin G4 antigen tests appear to vary depending on whether sampling occurs in diseased or healthy tissue. This suggests that the specific production of antibodies to antigens emerges from the disease itself and does not reflect a normal mucosal response. To obtain accurate food antigen tests, food-specific antibodies must be reliably obtained from diseased tissue.

[0169] Example 3: Protein biomarkers in diseased esophageal secretions are a feasible low-cost and low-invasive diagnostic modality for eosinophilic esophagitis Noninvasive diagnostic biomarkers are needed for EoE.

[0170] Introduction. Eosinophilic esophagitis (EoE) is a chronic inflammatory condition dominated by a form of non-IgE hypersensitivity. Recent literature indicates that the incidence of EoE in children and adults is rapidly increasing (J Robson, et al. Clinical Gastroenterology and Hepatology, 17(1):107-114.e1, January 2019). Although EoE symptoms often improve with treatment, disease activity persists and may contribute to esophageal fibrosis (ES Dellon, et al. Gastrointestinal Endoscopy, 79(4):577-585.e4, 2014).

[0171] Noninvasive biomarkers to track EoE activity in response to treatment are not currently applied in clinical practice. Thus, patients consent to frequent endoscopies with biopsies. Endoscopies require sedation, carry procedural risks, and contribute to an estimated $1.4 billion in annual EoE-attributable healthcare costs in the United States (ET Jensen, et al. The American journal of gastroenterology, 110(5):626-632, May 2015). The burden of endoscopy likely leads to substantial nonadherence to recommended EoE disease surveillance. Inexpensive, less invasive EoE diagnostic and follow-up modalities are needed.

[0172] Recent studies have used RNA sequencing and machine learning to create EoE diagnostic tools and identify specific EoE phenotypes (BF Sallis, et al. The Journal of allergy and clinical immunology, 141(4):1354-1364.e9, April 2018). Data are described herein that identify EoE genes using machine learning algorithms that specifically target EoE genes using protein transcripts. Esophageal secretions can be reliably obtained in the awake state of the patient at a significantly lower cost and risk to the patient compared to endoscopy (H Saffari, et al. The American journal of gastroenterology, 111(7):933-939, July 2016). It was evaluated whether expressed proteins could be collected in esophageal luminal secretions and used to distinguish between active EoE, treatment-resolved EoE, and control patients.

[0173] Methods. Identification of potential biomarkers via transcriptome analysis. To discover potential biomarkers for EoE in a high-throughput manner, we analyzed RNA-seq data from 14 EoE patients and 14 controls. [6] A decision tree, a type of machine learning algorithm, was fitted to gene expression values ​​measured in transcripts per million reads (TPM). Five-fold cross-validation was used to assess the diagnostic accuracy of each gene. Genes were designated as potential biomarkers if the criteria that diagnostic accuracy was 100% and the gene encodes a secreted protein product that can be measured using a commercially available assay were met.

[0174] Validation of biomarkers in esophageal secretions. The diagnostic accuracy of the candidate biomarkers was evaluated at the protein level in esophageal secretions. Esophageal secretions from five patients with active EoE (EoE), six patients with EoE resolved with treatment (resolved), and six controls (controls) were obtained using an endoscopic cytobrush. Protein expression was assayed for each sample using a Luminex 23-cytokine panel.

[0175] Results: Genes encoding protein biomarkers were identified through transcriptome analysis, including CCL26, encoding eotaxin-3, KITLG, encoding stem cell factor (SCF), and POSTN, encoding periostin (Figures 8A-8C). Eotaxin-3 was highly expressed in active EoE cases compared to controls, and more highly expressed in active EoE compared to treatment-resolved EoE.

[0176] Example 4: Food-specific antibodies in the small intestine: Association with trigger foods in eosinophilic esophagitis (EoE) Small intestinal relative values ​​of food-specific IgA for four patients with abdominal pain and discomfort are shown in FIG. 10. Samples were obtained from patients with irritable bowel syndrome (IBS). More specifically, samples were obtained from the small intestine. Two with resolved eosinophilic esophagitis appeared to have a lower response in the small intestine. One with resolved EoE (far left) noticed that his abdominal pain returned when he added eggs as part of an elimination diet. This patient's intestinal sample suggests that eggs are a stronger reactant. Another (far right) noticed that 80% of his abdominal pain resolved when he removed milk from his diet. This patient's small intestine showed higher values ​​of food-specific IgA to casein. The two in the middle have active EOE and abdominal pain (one with right upper quadrant pain, the other with non-specific "IBS"). Both had increased food-specific antibodies to foods compared to the patient with resolved EOE, and both showed high levels of IgA to gluten. Other patients without abdominal discomfort have even lower values ​​with resolved EOE.

Claims

1. 1. A method for detecting binding of one or more food-specific immunoglobulin A (IgA) antibodies to one or more food antigens in a sample, the method comprising contacting an esophageal secretion sample obtained from a subject with eosinophilic esophagitis with one or more food antigens, and detecting binding of the one or more food-specific IgA antibodies to the one or more food antigens.

2. The method of claim 1 , wherein the one or more food antigens are immobilized on a solid support.

3. 3. The method of claim 1 or 2, wherein the esophageal secretion sample is obtained before the subject ingests a food containing one or more of the food antigens.

4. 3. The method of claim 1 or 2, wherein the esophageal secretion sample is obtained after the subject has ingested a food containing one or more of the food antigens.

5. 3. The method of claim 1 or 2, further comprising comparing binding of the one or more food-specific IgA antibodies to the one or more food antigens in the esophageal secretion sample obtained before the subject ingests the food containing the one or more of the food antigens with binding of the one or more food-specific IgA antibodies to the one or more food antigens in the esophageal secretion sample obtained after the subject ingests the food containing the one or more of the food antigens.

6. A method for detecting a food-specific immune response in a sample obtained from a subject having eosinophilic esophagitis, the method comprising: a) contacting said esophageal secretion sample with one or more food antigens; b) determining the presence or level of one or more immunoglobulin A (IgA) antibodies bound to said one or more food antigens; and c) comparing the level of the one or more IgA antibodies in the esophageal secretion sample bound to the one or more food antigens of b) with the level of the one or more IgA antibodies bound to the one or more food antigens in a reference sample; This method detects the food-specific immune response in the esophageal secretion sample when the level of the one or more IgA antibodies bound to the one or more food antigens in the esophageal secretion sample is higher than the level of the one or more IgA antibodies in a reference sample.

7. 1. A method for determining food allergy in a subject with active or inactive eosinophilic esophagitis (EoE), said method comprising: a) detecting the presence or level of one or more immunoglobulin A (IgA) antibodies bound to one or more food antigens in an esophageal secretion sample obtained from said subject; b) comparing the level of the one or more IgA antibodies bound to the one or more food antigens in the esophageal secretion sample with the level of the one or more IgA antibodies bound to the one or more food antigens in a reference sample, wherein the level of the one or more IgA antibodies bound to the one or more food antigens in the esophageal secretion sample is higher than the level of the one or more IgA antibodies bound to the one or more food antigens in the reference sample, determining a food allergy in the subject.

8. The method of any one of claims 1 to 7, wherein the subject has active eosinophilic esophagitis.

9. The method of any one of claims 1 to 7, wherein the subject has resolved eosinophilic esophagitis.

10. The method of any one of claims 1 to 7, wherein the subject has at least one symptom of esophagitis.

11. The method of any one of claims 1 to 7, wherein the subject has irritable bowel syndrome.

12. The method of any one of claims 1 to 11, wherein the sample contains one or more food-specific IgA antibodies.

13. 8. The method of any one of claims 1 to 7, wherein the one or more food antigens are wheat f1, soybean f14, casein f78, egg f245, or a combination thereof.

14. 2. The method of claim 1, wherein the level of the one or more food specific IgA antibodies bound to the one or more food antigens is higher in the sample compared to the level of the one or more food specific IgA antibodies bound to the one or more food antigens in a reference sample, indicating a food specific immune response.

15. 1. A method for detecting the cause of eosinophilic esophagitis (EoE) symptoms, the EoE symptoms being caused by an immune response to an antigen in a patient diagnosed with or suffering from EOE, the method comprising: obtaining an esophageal mucosal sample from a subject, the esophageal mucosal sample obtained from the subject being derived from a site of the immune response, the esophageal mucosal sample comprising one or more immunoglobulin A (IgA) antibodies; contacting the sample with one or more food antigens; detecting binding of said one or more specific IgA antibodies to said one or more antigens.

16. The method of claim 1, further comprising detecting eosinophilic esophagitis (EoE) in the subject prior to the detecting step.

17. The method of claim 16, wherein EoE is detected in a subject by detecting eosinophil granule protein in mucosal tissue of the subject's esophagus, comprising administering to the subject radiolabeled heparin under conditions such that the radiolabeled heparin binds to eosinophil granule protein to form a radiolabeled heparin / eosinophil granule protein complex, and detecting the radiolabeled heparin / eosinophil granule protein complex in the mucosal tissue of the esophagus.

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