Patent Application
20220372544
Inflammatory bowel disease (“IBD”) is the chronic relapsing inflammation of all, or part of, the digestive tract. There are two types of IBD, ulcerative colitis (“UC”) and Crohn's disease (“CD”). Crohn's disease involving only colon is termed as Crohn's colitis (“CC”). When nondefinitive evaluations have been established for criteria for either UC or CC are labeled as “indeterminate colitis (IC)”. UC results in inflammation and ulceration of the mucosal and, to a lesser degree, the submucosal linings of the colon and rectum. CC differs from UC in that it may result in inflammation deeper within all the four colonic layers (transmural inflammation and skip lesions). Furthermore, CC may also affect other organs through fistulation.
UC and CD affect an estimated 2 million people in the US alone with associated annual health care costs of over $6.8 billion. While UC and CD are both types of IBDs, differences between patients having UC or CD has major implications. Currently, clinicians use inexact combined classification for patients having IBD, which include clinical, endoscopy, radiological, and histopathology in an effort to diagnose CD and UC. Nonetheless, differentiating patients having UC or CD among patients suffering from IBD remains challenging, so much so that cases of patients having IBD that are difficult classify as UC or CD are classified as having indeterminate colitis (“IC”). A significant subgroup of IBD patients are misdiagnosed or have a correct diagnosis delayed despite use of a state-of-the-art classification system applying clinical, endoscopic, radiologic and histologic tools. Indeed, it is estimated that 30% of patients suffering from IBD cannot currently be accurately diagnosed as CD or UC.
In addition, 15% of colonic IDB cases that undergo ileal pouch anal anastomosis surgery, as they are diagnosed with UC, will subsequently have their original diagnosis changed to CD based on their postoperative follow-up visits, clinical and histopathology changes, and development of de novo CD in the ileal pouch. Ileal pouch anal anastomosis, a treatment normally suitable for UC but not CD, restores gastrointestinal continuity after surgical removal of the colon and rectum, and involves the creation of a pouch of small intestine to recreate the removed rectum.
Implications of distinguishing cases of UC and CD include choice of medical treatment, timing of surgery, prognosis, whether to offer the patient an ileal pouch anal anastomosis, and lifestyle expectations. For these reasons, there is a need for improving the diagnosis, and subsequent treatment, of subjects having IBD.
It has been discovered that Paneth cells secreted DEFA5 also known as HD5 serve as biomarkers for determining whether a patient suffering from IBD has UC or CD.
In a first aspect, a method of measuring DEFA5 (HD5) in a patient suffering from or at risk of IBD is disclosed, said method comprising: obtaining a sample from the patient; and measuring at least one of the expression of DEFA5 (HD5) and the concentration of DEFA5 (HD5) in the sample.
In a second aspect, a method of treating a patient suffering from or at risk of IBD is disclosed, said method comprising: obtaining a sample from the patient; measuring at least one of the expression of DEFA5 (HD5) and the concentration of DEFA5 (HD5) in the sample; and performing an intervention on the patient to treat one of Crohn's disease and ulcerative colitis.
In a third aspect, a method of measuring MMP-7 in a patient suffering from or at risk of IBD is disclosed, said method comprising: obtaining a sample from the patient; and measuring at least one of the expression of MMP-7 and the concentration of MMP-7 in the sample.
In a fourth aspect, a method of treating a patient suffering from or at risk of IBD is disclosed, said method comprising: obtaining a sample from the patient; measuring at least one of the expression of MMP-7 and the concentration of MMP-7 in the sample; and performing an intervention on the patient to treat one of Crohn's disease and ulcerative colitis.
In a fifth aspect, a method of measuring biomarkers in a patient suffering from or at risk of IBD is disclosed, said method comprising: obtaining a sample from the patient; measuring at least one of the expression of DEFA5 (HD5) and the concentration of DEFA5 (HD5) in the sample; and measuring at least one of the expression of MMP-7 and the concentration of MMP-7 in the sample.
In a sixth aspect, a method of treating a patient suffering from or at risk of IBD is disclosed, said method comprising: obtaining a sample from the patient; measuring at least one of the expression of DEFA5 (HD5) and the concentration of DEFA5 (HD5) in the sample; measuring at least one of the expression of MMP-7 and the concentration of MMP-7 in the sample; and performing an intervention on the patient to treat one of Crohn's disease and ulcerative colitis.
In a seventh aspect, a kit for measuring DEFA5 (HD5) and MMP-7 in a sample is disclosed, the kit comprising: a first assay for measuring at least one of the expression of human DEFA5 (HD5) and the concentration of human DEFA5 (HD5) in a sample; and a second assay for measuring at least one of the expression of human MMP-7 and the concentration of human MMP-7 in a sample.
In an eighth aspect, a method of measuring a biomarker in a patient suffering from or at risk of inflammatory bowel disease (IBD) is disclosed, said method comprising: obtaining a sample from the patient; and measuring a level of the biomarker in the same, the level of the biomarker selected from the group consisting of: the expression of the biomarker, the activity of the biomarker, and the concentration of the biomarker; wherein said biomarker is selected from Table 1.
The above methods may include diagnosing the patient as suffering from CD if the level of DEFA5 (HD5) concentration or DEFA5 (HD5) expression is greater than a given threshold level, diagnosing the patient as suffering from UC if the level of DEFA5 (HD5) concentration or DEFA5 (HD5) expression is below a threshold level, or both.
The above methods may include diagnosing the patient as suffering from CD if the level of MMP-7 concentration or MMP-7 expression is less than a given threshold level, diagnosing the patient as suffering from UC if the level of MMP-7 concentration or MMP-7 expression is above a threshold level, or both.
The above presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key or critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present concepts in a simplified form as a prelude to the more detailed description that is presented later.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art of this disclosure. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and should not be interpreted in an idealized or overly formal sense, unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity or clarity.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The term “consisting essentially of” means that, in addition to the recited elements, what is claimed may also contain other elements (steps, structures, ingredients, components, etc.) that do not adversely affect the operability of what is claimed for its intended purpose as stated in this disclosure. This term excludes such other elements that adversely affect the operability of what is claimed for its intended purpose as stated in this disclosure, even if such other elements might enhance the operability of what is claimed for some other purpose.
The terms “about” and “approximately” shall generally mean an acceptable degree of error or variation for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error or variation are within 20%, preferably within 10%, and more preferably within 5% of a given value or range of values. For biological systems, the term “about” refers to an acceptable standard deviation of error, preferably not more than 2-fold of a given value. Numerical quantities in this detailed description are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
The terms “individual”, “subject”, or “patient” as used herein refer to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans. The terms may specify male or female or both, or exclude male or female.
The terms “treatment”, “treat”, and “treating”, as used herein, refer to a course of action (such as administering a compound or pharmaceutical composition) initiated after the onset of a clinical manifestation of a disease state or condition so as to eliminate or reduce such clinical manifestation of the disease state or condition. Such treating need not be absolute to be useful.
The terms “first”, “second”, and the like are used herein to describe various features or elements, but these features or elements should not be limited by these terms. These terms are only used to distinguish one feature or element from another feature or element. Thus, a first feature or element discussed below could be termed a second feature or element, and similarly, a second feature or element discussed below could be termed a first feature or element without departing from the teachings of the present disclosure.
An assay method of diagnosing UC and CD in a subject, such as a human, suffering from IBD is described. The method measures DEFA5 (HD5) in tissue taken from a subject having IBD. DEFA5 (HD5) is a small, microbicidal innate immune system protein belonging to the alpha defensing family of mammalian defensing peptides. DEFA5 (HD5) is expressed in various tissues and particularly on mucosal surfaces. DEFA5 (HD5) is encoded by the gene DEFA5. DEFA5 (HD)5 is involved in host defense mechanisms, and is highly expressed in secretory granules of Paneth cells of the small intestine (ileum). Like most secreted proteins, HD5 is synthesized as prepro-HD5 (1-94) that undergoes proteolytic processing first, to the inactive pro-HD5s (20-94), HD5 (23-94) and HD5 (29-94). HD5 (23-94) and HD5 (29-94) are found within tissues, while HD5 (20-94) is the predominant intracellular form. The pro-HD5s are then processed to two active or mature forms. HD5 (56-94) and HD5 (63-94) with HD5 (63-94) being the most abundant form. These mature forms of HD5 are cysteine-rich host defense peptides which exert a broad-spectrum antimicrobial activity and contribute to innate immunity in the human gut. As used herein, HD5 may refer to exclusively mature forms or inactive forms of HD5.
Matrix metalloproteinase-7 (MMP-7, encoded by the MMP7 gene) is responsible for cleaving and activating HD5. It is believed that there may be a dysfunction in the activation pathway of HD5 in patients suffering from moderate and severe CD, and thus, an excess amount of inactive form HD5 is a potential mechanism for inflammation in patients suffering from CD. This excessive amount of inactive form HD5 may cause increased damage to the epithelial lining and potentially even a dysregulation in the levels and make-up of gut flora. The canonical structure of human MMP-7 is a 54 residue polypeptide (see Uniprot accession number A5GZ72).
The sample can be taken from any suitable source for measuring HD5 concentration, HD5 expression levels, MMP-7 expression, or MMP-7 concentration, such as the tissue samples from the large intestine or rectum. In this disclosure the term “expression of HD5” should be interpreted to mean the expression of the DEFA5 gene; “levels of HD5” should be interpreted to mean the concentration of HD5; “expression of MMP-7” should be interpreted to mean the expression of the MMP7 gene; “levels of MMP-7” should be interpreted to mean the concentration of matrix metalloproteinase-7.
The sample may be taken from a subject who is suffering from or at risk of IBD. The subject may display one or more symptoms characteristic of IBD, such as severe diarrhea, abdominal pain, fatigue, and weight loss. In some embodiments of the method, the subject displays more than one of said symptoms. In further embodiments the subject displays 2, 3, or 4 of said symptoms.
It has been discovered that the genes encoding HD5 and MMP-7 are differentially expressed in subjects having UC and CD; and further, that the concentration of HD5 is significantly higher in tissues of CD patients than in UC patients. Used in this way, HD5 and MMP-7 concentrations, and HD5 expression and MMP-7 expression, can be utilized and measured as biomarkers for distinguishing UC and CD in patients having IBD. This can in turn be used to more effectively treat the subject. For example, as ileal pouch anal anastomosis is clinically much more successfully in patients having UC than in patients suffering from CD, patients identified as having levels of HD5 or MMP-7 levels indicative of UC, or not having CD, may be treated with ileal pouch anal anastomosis. Indeed, as HD5 is produced by Paneth cells only, one would not typically expect to find Paneth cells that secret HD5 in the colon. The inventors have discovered Paneth cells (secreting HD5) are abundantly found in subjects having UC. On the other hand, patients identified as having levels of HD5 or MMP-7 and HD5 or MMP-7 expression indicative of CD may be treated with any suitable treatment for CD. In an embodiment, a diagnosing step, such as diagnosing a subject with UC or CD, is optional.
The methods may include a step of comparing the level of the biomarker in question to a benchmark value. The benchmark value may be a measure of central tendency based on levels observed in one or more populations of subjects that are established to be unafflicted by either of UC or CD. For example, the benchmark value may be a mean level of the gene expression or protein concentration observed in samples from a population of subjects who are unafflicted by UC, unafflicted by CD, or both. The population may be defined by one or more of the patient's geography, age, ethnicity, sex, and medical history. The benchmark value may take into account a measure of variation combined with a measure of central tendency. For example, the benchmark value may be a mean level of the gene expression or protein concentration observed in a given tumor population, plus or minus a margin of error. The benchmark may be based on raw measurements (such as fragments of mRNA or cDNA per kb gene length per million reads) or normalized measurements (such as % of normal expression, or expression compared to a constitutively expressed or widely expressed gene with generally consistent expression, such as β-actin).
The benchmark may also be established by analysis of a control sample that is measured alongside the sample from the subject. Examples of suitable control samples are: a sample from a subject unafflicted with UC, a sample from a subject unafflicted with CD, a sample from a subject afflicted with UC (although unafflicted with CD), a sample from a subject afflicted with CD (although unafflicted with UC), a sample from a subject afflicted with diverticulitis (although unafflicted with either of UC or CD), and a sample from a subject unafflicted from IBD.
In an embodiment, an assay method of differentially diagnosing UC and CD in a patient suffering from IBD includes measuring the level of HD5 or MMP-7 or HD5 or MMP-7 expression present in a sample obtained from the patient. The level of HD5 or MMP-7 concentration or expression in the tissue may be measured by any suitable peptide analysis. For example, the measuring step may include one or more of enzyme-linked immunosorbent assay (ELISA), cation-ion exchange, NMR analysis, genome-wide transcriptome analysis, and mass spectrometry. The method may include comparing the concentration or expression of the biomarker in the sample to the benchmark, and making a diagnosis if the concentration or expression of the biomarker in the sample is significantly less than or significantly greater than the benchmark value. For the example, the method may comprise comparing the concentration or expression of HD5 in the sample to the benchmark, and making a diagnosis of CD if the concentration or expression of HD5 in the sample is significantly greater than the benchmark value. As another example, the method may comprise comparing the concentration or expression of HD5 in the sample to the benchmark, and making a diagnosis of UC if the concentration or expression of HD5 in the sample is not significantly greater than the benchmark value. As another example, the method may comprise comparing the concentration or expression of MMP-7 in the sample to the benchmark, and making a diagnosis of UC if the concentration or expression of MMP-7 in the sample is significantly greater than the benchmark value. As another example, the method may comprise comparing the concentration or expression of MMP-7 in the sample to the benchmark, and making a diagnosis of CD if the concentration or expression of MMP-7 in the sample is not significantly greater than the benchmark value. In a further example, the method comprises measuring the concentration or expression of both MMP-7 and HD5, and making a diagnosis of either: CD if the concentration or expression of MMP-7 in the sample is not significantly greater than the benchmark value and the concentration or expression of HD5 is significantly greater than the benchmark value; or UC if the concentration or expression of MMP-7 in the sample is significantly greater than the benchmark value and the concentration or expression of HD5 is not significantly greater than the benchmark value.
The difference in expression or concentration may be considered significant based on any of a variety of known statistical tests for significance. These are generally based on a collection of measurements made from a sampled population, and are affected by both the population size and the sampling size. Such statistical tests are well known in the art and are not further elaborated upon in this disclosure; outside references can be relied upon to enable those skilled in the art to determine statistical significance, such as Rosener's Fundamentals of Biostatistics, 8th ed. (2015), Cengage Learning, Boston, Mass.
The method may include diagnosing the patient as having UC if HD5 or HD5 expression is at any level that is indicative of a patient not having CD, such as less than 5× normal levels of HD5 (i.e., levels of HD5 typical of a subject unafflicted with CD), less than about 5×-30× normal levels of HD5 or HD5 expression, less than about 31× normal levels, or less than about 118× normal levels. In a further embodiment, the patient is diagnosed as having UC if HD5 expression is at a level of less than 106, 107, 1.9×107, 6×105, or 3×106 HD5 mRNA transcript per 10 ng RNA. In some embodiments of the method the patient may be diagnosed as having CD if the level of HD5 expression is at any level indicative of a patient having CD, such as at least 3×106, 107, 1.9×107, 7×107, 108, 1.2×108, or from about to 3×106 to 1.2×108 HD5 mRNA Transcript per 10 ng RNA. The diagnosing may diagnose the patient as having CD if the patient has a MMP-7 concentration or MMP-7 expression level indicative of a patient having CD, such as up to a threshold limit that is 10× a benchmark value of MMP-7 concentration or MMP-7 expression. In further embodiments, the diagnosis may be CD using a threshold limit of up to 5×, and up to 1× a benchmark value. As used herein, a “normal level” of HD5 or HD5 expression means a level of HD5 or HD5 expression in the digestive tract tissue from a subject not having CD or UC, or a subject suffering from IBD and specifically UC. Normal HD5 expression may refer to from 1×105 to 9×105 HD5 mRNA Transcript per 10 ng RNA, or about 6×105 HD5 mRNA Transcript per 10 ng RNA. As used herein, a “normal level” of MMP-7 or MMP-7 expression means a level of MMP-7 or MMP-7 expression in the digestive tract tissue from a subject not having CD or UC, or a subject suffering from IBD, specifically CD.
In another embodiment, an assay method for differentially diagnosing UC and CD in a patient suffering from, or at risk of, IBD includes measuring the level of MMP-7 or MMP-7 expression present in a sample obtained from the patient. The level of MMP-7 or MMP-7 expression in the tissue may be measured by any suitable peptide analysis. In an embodiment, the method of diagnosing may be performed ex vivo.
In one embodiment, the assay methods involve determining the status of a subject with respect to the activity and/or expression of HD-5 or MMP-7 or the activity and/or expression of a polypeptide regulated by HD-5 or MMP-7. In one embodiment, such methods comprise determining the level of expression or activity of HD-5 or MMP-7 or a polypeptide regulated by HD-5 or MMP-7 in a sample from the subject. The method may further comprise collecting the sample from the subject. As used herein, a biological sample which is subjected to testing is a sample derived from a subject and includes, but is not limited to, any biological material, such as a bodily fluid. Examples of bodily fluids include, but are not limited to, whole blood, serum, saliva, tissue infiltrate, pleural effusions, lung lavage fluid, bronchoalveolar lavage fluid, and the like. The biological fluid may be a cell culture medium or supernatant of cultured cells. For example, the sample can be a blood sample or a serum sample. As another example the sample may be tissue or fluids from the subject's digestive tract. Some embodiments of the method involve a sample of intestinal tissue. In specific embodiments, the biological sample is collected from the colon of a subject (such as colonic tissue) or the ileum of a subject (such as ileal tissue).
Some embodiments of the method comprise measuring the concentration of the biomarker protein by selectively staining or dying the sample form the subject and measuring the signal from the stain. The stain or dye may comprise an antibody or an antibody fragment to recognize the protein. The stain or dye may also comprise a reporter, such a colorimetric group, a radionuclide, a stable isotope, a fluorophore, a chromophore, an enzyme, a magnetic particle, and a quantum dot. The concentration of the protein can then be measured by observing the signal from the reporter, such as by microscopy, colorimetry, radiometry, fluoroscopy, magnetotaxis, or any combination of the foregoing. In a specific embodiment of the method, the concentration of HD5 or MMP-7 is measured by immunostaining the sample with an immunostain that recognizes the biomarker and counting the number of stained cells by microscopy. This approach has the advantage of relative simplicity, and only requires the types of equipment that are already present in typical clinical laboratories. In specific examples in which the biomarker is HD5, a diagnosis can be made based on a threshold number of cells that stain positive, such as 10%, 20%, and 30%. If the number of HD5 stained cells is significantly above the threshold value, than a diagnosis of CD can be made; whereas if the number of HD5 stained cells is significantly below the threshold value, than a diagnosis of UC can be made.
Those subjects in which HD-5 or MMP-7 activity and/or expression differs (increased or decreased) from a control or benchmark value or the activity of a polypeptide regulated by HD-5 or MMP-7 differs as compared to a control or benchmark value are determined to be suffering from or at risk for a disease states and conditions associated with or characterized by increased or decreased HD-5 or MMP-7 activity.
Assay techniques that can be used to determine levels of expression or activity in a sample are known. Such assay methods include, but are not limited to, radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization assays, competitive-binding assays, Western Blot analyses, ELISA assays and proteomic approaches, two-dimensional gel electrophoresis (2D electrophoresis) and non-gel based approaches such as mass spectrometry or protein interaction profiling. Assays also include, but are not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, enzyme immunoassays (EIA), enzyme linked immunosorbent assay (ELISA), sandwich immunoassays, precipitin reactions, gel diffusion reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoresis assays. For examples of immunoassay methods, see U.S. Pat. Nos. 4,845,026 and 5,006,459.
In an ELISA assay, an antibody is prepared, if not readily available from a commercial source, specific to an antigen, such as, for example, HD-5 or MMP-7 or a polypeptide regulated by HD-5 or MMP-7. In addition, a reporter antibody generally is prepared. The reporter antibody is attached to a detectable reagent such as a radioactive, fluorescent, or enzymatic reagent, for example horseradish peroxidase enzyme or alkaline phosphatase. In one embodiment of the ELISA, to carry out the ELISA, antibody specific to the antigen is incubated on a solid support that binds the antibody. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein. Next, the sample to be analyzed is incubated with the solid support, during which time the antigen binds to the specific antibody. Unbound sample is washed out with a buffer. A reporter antibody specifically directed to the antigen and linked to a detectable reagent is introduced resulting in binding of the reporter antibody to any antibody bound to the antigen. Unattached reporter antibody is then washed out. Reagents for detecting the presence of the reporter antibody are then added. The detectable reagent is then determined in order to determine the amount of antigen present. In an alternate embodiment, the antigen is incubated with the solid support, followed by incubation with one or more antibodies, wherein at least one of the antibodies comprises a detectable reagent. Quantitative results may be obtained by reference to a standard curve.
Optionally, a genetic sample from the biological sample can be obtained. The genetic sample comprises a nucleic acid, preferably RNA and/or DNA. For example, in determining the expression of genes mRNA can be obtained from the biological sample, and the mRNA may be reverse transcribed into cDNA for further analysis. Alternatively, the mRNA itself is used in determining the expression of genes. A genetic sample may be obtained from the biological sample using any techniques known in the art (Ausubel et al. Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999); Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984) each of the foregoing being incorporated herein by reference). The nucleic acid may be purified from whole cells using DNA or RNA purification techniques. The genetic sample may also be amplified using PCR or in vivo techniques requiring subcloning. The genetic sample can be obtained by isolating mRNA from the cells of the biological sample and reverse transcribing the RNA into DNA in order to create cDNA (Khan et al. Biochem. Biophys. Acta 1423:17 28, 1999).
Once a genetic sample has been obtained, it can be analyzed. The analysis may be performed using any techniques known in the art including, but not limited to, sequencing, PCR, RT-PCR, quantitative PCR, restriction fragment length polymorphism, hybridization techniques, Northern blot, microarray technology, and similar techniques. In determining the expression level of a gene or genes in a genetic sample, the level of expression may be normalized by comparison to the expression of another gene such as a well-known, well characterized gene or a housekeeping gene (for example, actin). For example, reverse-transcriptase PCR (RT-PCR) can be used to detect the presence of a specific mRNA population in a complex mixture of thousands of other mRNA species. Hybridization to clones or oligonucleotides arrayed on a solid support (e.g., gridding) can be used to both detect the expression of and quantitate the level of expression of that gene. In this approach, a cDNA encoding an antigen is fixed to a substrate. The substrate may be of any suitable type including but not limited to glass, nitrocellulose, nylon, or plastic. At least a portion of the DNA encoding the antigen is attached to the substrate and then incubated with the analyte, which may be RNA or a complementary DNA (cDNA) copy of the RNA, isolated from the sample of interest. Hybridization between the substrate bound DNA and the analyte can be detected and quantitated by several means including but not limited to radioactive labeling or fluorescence labeling of the analyte or a secondary molecule designed to detect the hybrid. Quantitation of the level of gene expression can be done by comparison of the intensity of the signal from the analyte compared with that determined from known standards. The standards can be obtained by in vitro transcription of the target gene, quantifying the yield, and then using that material to generate a standard curve.
The method may include diagnosing the patient as having UC if MMP-7 concentration or MMP-7 expression is at any level indicative of a patient having UC, such as from 2×-100×, 10×, 2×-50×, 5×-15×, or about 10× normal MMP-7 concentration or MMP-7 expression levels. In some embodiments of the method, the patient may be diagnosed as having CD if the MMP-7 concentration or MMP-7 expression is at any level indicative of a patient having CD, such as less than 1×-10× or 2×-5× normal levels of MMP-7 or MMP-7 expression.
A method of treating IBD in a patient suffering from IBD may include: (a) measuring the level of HD5 or HD5 expression present in a sample obtained from the patient, said measuring step optionally comprising one of cation-ion exchange, NMR analysis, genome-wide transcriptome analysis, and mass spectrometry, whereby a level of HD5 or HD5 expression is obtained; (b) if the level of HD5 or HD5 expression is at a level indicative of a patient not having CD, treating the IBD in the patient with a suitable medical treatment for UC; if the level of HD5 or HD5 expression is at a level indicative of a patient having CD, treating the IBD in the patient with a suitable medical treatment for CD. In another embodiment, levels of MMP-7 or MMP-7 expression are measured rather than levels of HD5 to determine whether to treat UC or CD.
Suitable medical treatments for UC include ileal pouch anal anastomosis or the administration of pharmaceutical agents or salts thereof. Suitable pharmaceutical agents may be one or more of: an iron supplement; an oral 5-aminosalicylate, such as mesalamine, balsalazide and olsalazine; an anti-inflammatory; a corticosteroid; an immunosuppressant such as azathioprine, mercaptopurine, methotrexate, and cyclosporine; an anti-TNF-alpha antibody such as infliximab, adalimumab, and golimumab; an anti-α4-integrin antibody such as vedolizumab; and an antibacterial antibiotic, such as ciprofloxacin and metronidazole. Surgeries that are sometimes used to treat UC include a proctocolectomy, and an ileal pouch anal anastomosis. Note that ileal pouch anal anastomosis are recognized as relatively ineffective when used to treat CD, in contrast to UC. It should also be noted that cyclosporine and golimumab, while currently approved for the treatment of UC in the United States, are not currently approved for the treatment of CD. Some embodiments of the method involve performing an intervention that is effective to treat UC, but either ineffective to treat CD or not yet approved by regulatory authorities for the treatment of CD.
Suitable medical treatments for CD include the administration of pharmaceutical agents or salts thereof. Suitable pharmaceutical agents include: an oral 5-aminosalicylate, such as mesalamine; a vitamin supplement, such as a vitamin B-12 supplement and a vitamin D supplement; a mineral supplement, such as a calcium supplement; an anti-inflammatory; a corticosteroid such as prednisone and budesonide; an immunosuppressant such as azathioprine, tacrolimus, methotrexate, and mercaptopurine; an anti-TNF-α antibody, such as infliximab, adalimumab, and certolizumab pegol; an anti-α-4-integrin antibody, such as natalizumab and vedolizumab; an anti-interleukin antibody, such as ustekinumab; and an antibacterial antibiotic, such as metronidazole, and ciprofloxacin. Although certolizumab pegol, methotrexate, and natalizumab are approved in the US for the treatment of CD, they are not currently approved for the treatment of UC. Surgical approaches are sometimes used to treat severe cases of CD. Such surgeries include ostomy, colostomy, ileostomy, bowel resection, colectomy, proctocolectomy, and strictureplasty. In some embodiments of the method, the subject is treated using a diet that is advantageous for the management of CD, but not necessarily advantageous in the management of UC. One such diet is a low fat diet. Some embodiments of the method involve performing an intervention that is effective to treat CD, but either ineffective to treat UC or not yet approved by regulatory authorities for the treatment of UC.
In an embodiment, the level of HD5 or HD5 expression may be elevated above normal levels in patients who are likely to be diagnosed UC but, at the time the HD5 or HD5 expression level is measured, diagnosed as having IC. These patients may be treated with any suitable medical treatments for UC.
A kit is provided for measuring HD5 in a subject. The kit may include a detectable antibody that is capable of binding with HD5. The antibody may be capable of binding with HD5 yet not bind with other defensins, such as HD4 and HD5. The antibody may be a purified HD5-specific monoclonal or polyclonal antibody, such as the HDAC5 Antibody from GenWay Biotech, Inc.
A kit is provided for measuring HD5 and MMP-7 in a subject. The kit may find use in several of the methods provided above, as well as others. The kit may be, for example, used for the diagnosis of inflammatory bowel disease. The kit comprises an assay for measuring at least one of HD5 concentration and HD5 expression; and an assay for measuring at least one of MMP-7 concentration and MMP-7 expression.
Abstract
Inability to distinguish Crohn's colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohn's colitis, p<0.0001. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable differentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohn's colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohn's colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to de novo Crohn's disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohn's colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohn's colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohn's colitis.
Methods
Clinical Samples and Ethical Consideration
In order to carryout tissue profiling of differentially expressed proteins/genes in IBD, we first sought ethical approval from the Meharry Medical College (IRB file #: 100916AM206) and Vanderbilt University Medical Center (IRB file #s: 080898 and 100581) Institutional Review Boards [20]. Informed consent was provided, and patient participation in the study was voluntary. Patient samples comprised of surgical pathology colectomy tissues from adults with definitive UC and CC phenotypes as well as those diagnosed with IC at Vanderbilt University Medical Center (VUMC) between 2000 and 2007. The full thickness surgical samples of colectomy tissue were analyzed by pathology teams at MMC and VUMC, Schools of Medicine following established protocol criteria for IBD subtypes. For each selected sample, medical records data on patient demographics, preoperative variables prior to and after time of ileal pouch-anal anastomosis surgery, surveillance endoscopic and clinical findings, and medical and surgical treatment history were reviewed retrospectively. Samples included in all experiments were taken from various parts of the colon; all inflamed tissue unless otherwise indicated. A condensed list of samples and colon locations are included in Table 3, as shown in
Diagnostic Criteria for Inflammatory Bowel Disease
Pathology teams at MMC and VUMC Schools of Medicine used the following protocol criteria for the final surgical pathology reporting.
For ulcerative colitis. Characteristic pattern of involvement of colon, worse distally in untreated patients; lack of perianal or fistulizing disease; no granulomas, except in association with ruptured/injured crypts; no transmural lymphoid aggregates or other transmural inflammation; no involvement of terminal ileum, except mild “backwash ileitis” in cases with severe cecal involvement and no pyloric metaplasia in terminal ileum.
For Crohn's disease. Involvement of other sites in the gastrointestinal tract (skip lesions, segmental disease); perianal or fistulizing disease; granulomas, not in association with ruptured/injured crypts and terminal ileum involvement.
For indeterminate colitis. Distribution favors UC, but focal transmural inflammation, or inflammation in ileum more than expected in backwash ileitis and no fistulizing disease.
Vanderbilt Patient Medical Records Database
The availability of a detailed IBD patient database registry at Vanderbilt University Medical Center (VUMC) made chart review and follow-up surveillance possible. Medical records data on patient demographics, preoperative variables prior to and after IPAA surgery, surveillance of endoscopic and clinical findings, and medical and surgical treatment history were retrieved retrospectively.
Indeterminate Colitis Clinical Retrospective Study
A retrospective investigation was conducted to identify a cohort of patients diagnosed with IC and registered in the IBD Center at VUMC. Twenty-one patients, initially classified as IC at the time of diagnosis between years 2000-2007, were identified and reevaluated for disease course in 2014, after a mean surveillance follow-up of 8.7±3.7 (range, 4-14) years, in order to identify the rates of diagnosis resolution to UC or CC. Diagnosis for each patient was determined based on standard clinical and pathologic features as previously described [21,22].
Three gastrointestinal pathologists blinded to clinical diagnosis reconciled and confirmed colitis diagnosis for each patient and represented a consensus among treating physicians. Patients who clinically did not changed and maintained the IC diagnosis were tested via IHC and Nikon Element Advanced Research Analysis Software (NEARAS) for HD5 levels to determine if HD5 could be used to identify CC from UC.
Restorative Proctocolectomy Operated Patients' Retrospective Study
One hundred twenty patients with definitive UC underwent RPC surgery between Apr. 18, 2001 and Jun. 18, 2008. Of the 120 patients, 67 had their diagnosis re-evaluated after a mean follow up of 9.4 (range, 6-13) years of functionally acceptable pouches. Compiled medical records allowed us to re-evaluate a progressive course of UC patients following RPC. Clinical information needed for each of these patients was available in the IBD medical records registry database at VUMC. The aim was to reevaluate patients who underwent RPC operation for definitive UC and had a change in diagnosis to de novo Crohn's ileitis. Patients who had a change in diagnosis should reconcile the molecular biometric test that delineates IC into CC; again using NEARAS for HD5 levels.
cDNA Microarray
We performed a whole-transcriptome microarray with RNA extracted and pooled from human full thickness colon samples from UC and CC patients (n=5/group) (Affymetrix, Santa Clara, Calif.).
NanoString nCounter Human Inflammation Kit Gene Expression
RNA from UC and CCI tissue (and diverticulitis tissue used as a control) was processed by NanoString (NanoString Technologies Inc., Seattle, Wash.) to determine gene expression level according to the manufacturer protocol [23].
Real-Time RT-PCR
Real-Time RT-PCR was used to measure transcript levels of HD5. RNA was extracted from three human colon biopsy samples each from moderate UC and CC, and diverticulitis (DV) as a non-IBD control (RNeasy Miniprep Kit, Qiagen, CA). cDNA was generated using iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif.). Pre-designed TaqMan probes (Thermo Fisher Scientific, Waltham, Mass.) were purchased for HD5 and GAPHD control, and all samples were run in triplicate using a CFX96 qPCR thermocycler (Bio-Rad). Data were analyzed per the ΔΔCt method of analysis.
Western Blot and Immunohistochemistry
Western blot was used to assess any differences in HD5 protein levels. Protein was extracted from a minimum of 10 colon biopsy samples each from mild, moderate, and severe UC; mild, moderate, and severe CC; and non-IBD DV control. Whole cell lysates were extracted from full-thickness colon samples using T-PER (Thermo Fisher Scientific) per manufacturer's protocol. Bradford Assays (Bio-Rad) were run to determine protein concentration, and protein was loaded onto a 4-20% SDS-PAGE tris/glycine gel (Bio-Rad). Proteins were transferred to PVDF (Bio-Rad), and Western blots for HD5 and β-actin loading control were performed with primary and secondary antibodies (Santa Cruz, Dallas, Tex.) per manufacturer's protocol. Blots were visualized with Opti-4CN colorimetric detection kit (Bio-Rad) and imaged with ChemiDoc XRS+imaging system (Bio-Rad). Band intensities were measured and data analysis performed with Image Lab Software (Bio-Rad).
Five colon tissue protein extracts and staining of HD5 per disease by immunohistochemistry (IHC) was done as previously described.24 Quantification of HD5 staining was analyzed manually by microscopy and automatically quantified using Nikon's Eclipse Ti microscope with built-in NEARAS [24,25].
NEARAS Technology for Quantification of Immunohistochemistry Staining
NEARAS (Melville, N.Y.) was used to calculate the number of cells with HD5 staining in IHC tissue. A mean intensity threshold of 20 to 255 intensity units was established to eliminate a false-positive signal from background staining. A circularity parameter of 0.5 to 1 and equivalent diameter of 5-15 micrometer was used to select for cells. All threshold parameters were used in each image to count the number of HD5-positive cells in tissue samples.
Statistical Analysis
The Vanderbilt University Microarray Core Laboratory performed statistical analyses for the microarray. Transcriptome level fold changes and the significance of those changes were calculated using one way ANOVA with Bonferroni's correction for multiple comparisons. Significantly changed transcripts were defined as having >2.0 fold expression change from controls and a Benjamini-Hochberg (BH) false discovery rate corrected ANOVA p-value <0.05. All other statistical analyses were performed using GraphPad Prism v6 software [26]. qRT-PCR and IHC HD5 counts were examined by applying an unpaired two-tailed Student's t-test with the Welch correction, respectively. Western blots were analyzed by ANOVA followed by Fisher's test for multiple comparisons. Chi square tests were utilized for determining relatedness of HD5 levels to CC. For all statistical analyses, p<0.05 indicated a statistical significance.
Dual Staining of Human α-Defensin-5 and Lysozyme
DoubleStain IHC was performed on a Lab Vision autostainer 360 (Thermo fisher) using Abcam's M&R on human tissue (DAB & AP/Red) staining kit (ab210059, Abcam Biotechnology, Cambridge, UK). The manufacture's recommended conditions were used with the following modifications. The mouse anti-α-defensin 5 (sc-53997, Santa Cruz Biotechnology, Inc, Dallas, Tex.) and rabbit anti-lysozyme (ab-2408) were used at a 1:50 dilution in OP Quanto antibody Diluent (Thermo Fisher, Waltham, Wash.). Prior to addition of antibody for 45 minutes, tissues were incubated for 10 min with Utravision hydrogen peroxide block (Thermo Fisher) followed by a 5 min incubation with Ultravisoion Quanto protein block. A single incubation with Permanent Red was used for ileum tissue, whereas two consecutive 10 min permanent Red incubations were performed for colonic tissue. Following hematoxylin counter staining, tissue was exposed to Richard-Allen Scientific Blueing Reagent (Thermo Fisher).
Antigen retrieval was performed in 1 mM EDTA pH 8.4, 0.05% Tween 20 for 20 minutes at 98° C. (60° C. preheat/70° C. cool down) using the Labvision PT Module (Thermo Scioentific). Image color deconvolution was performed with Fiji ImageJ 1.51f (http://imagej.nih.gov/ij) using the Fast Red, Fast Blue and DAB built in stain vector plugin.
Results
Nearly 30% of Indeterminate Colitis Patients Cannot be Delineated into UC or CC
A retrospective investigation was conducted to identify a cohort of patients diagnosed with IC to determine if they could be properly delineated into UC or CC over time. We followed 21 patients who were diagnosed with IC between the years 2000-2007 and reevaluated in 2014. A mean surveillance follow-up period was 8.7±3.7 (range, 4-14) years. Fifteen of the 21 (71.4%) had their original diagnosis changed; 9 to UC (43%) and 6 to CC (28.5%). Six (28.5%) patients remained clinically inconclusive and retained their diagnosis of IC (
Thirty Percent of Restorative Proctocolectomy Operated Crohn's Colitis Patients were Misdiagnosed as Ulcerative Colitis
A retrospective investigation was conducted to identify a cohort of patients that underwent RPC and IPAA surgery for a definitive UC diagnosis to determine if they had been misdiagnosed. We identified 67 such patients. A mean surveillance follow-up period was 9.4 (range, 6-13) years. A change in diagnosis to de novo Crohn's disease of the ileal pouch was clinically observed in 20 (30%) patients (
There is Differential Expression of Human α-Defensin-5 in Inflammatory Bowel Disease
We initially performed whole-transcriptome microarray with RNA extracted and pooled from human full thickness colon samples from UC and CC patients (n=5) using the Affymetrix gene expression array according to the manufacturer's instructions (Affymetrix, Santa Clara, Calif.) Tissues from diverticulitis (DV) were used as control. This analysis showed a total of 484 genes that were upor down-regulated (˜2-fold) between the two diseases. Among the upregulated genes were α-defensin-5, other antimicrobial peptides, and mucins (Table 1). HD5 was increased the most: 31-fold in CC vs. UC (in a previous study HD5 increase by 118-fold in CC versus UC—data not shown). A full list of the microarray results can be found in Table 1.
To replicate these data in a different platform, an independent analysis by PCR array (NanoString Technologies Inc. Seattle, Wash.) was carried out on 5 different human full thickness colon samples from UC and CC patients. Although the NanoString array only specifically targeted inflammatory genes, the only gene to show up in both the microarray and the PCR array was HD5. The NanoString array determined that HD5 was increased 118-fold in CC vs. UC in these human samples, compared to 31-fold in the previous samples analyzed by microarray (Table 2).
To further validate these data, we assessed the expression of HD5 by semi-quantitative RT-PCR using RNA extracted from moderate CC and moderate UC tissues (n=3). This analysis also showed a significant increase in transcript levels of HD5 in CC compared to UC (
Table 1 shows a list of targets from an affymetrix cDNA microarray. A total of 484 genes were highlighted in the microarray as potential markers for distinguishing UC from CC. The gene showing the largest fold change between the two diseases was Human Defensin 5 (HD5).
Table 2 shows a full list of targets from NanoString Human Inflammation PCR array. 16 inflammatory genes were changed in this subset of samples. HD5 was the only gene to appear in both the microarray and the NanoString PCR array.
Human α-Defensin-5 Levels are Aberrant in Indeterminate Colitis and Restorative Proctocolectomy Operated Patients
In order to determine if HD5 could be used to assess whether IC patients could be delineated into a diagnosis of either UC or CC, we assessed levels of HD5 in surgical pathology colectomy samples via IHC in patients described in
Further, RPC and IPAA-operated patients described in
These images can be compared to normal ileum control (
Aberrantly Regulated Human α-Defensin-5 in Crohn's Colitis Patients May be Caused by Ectopic Colonic Crypt Paneth Cells HD5 is a Paneth cell product; therefore, we wanted to determine if Paneth cells were present in the colon crypt of Crohn's colitis patients. All 20 UC RPC operated patients with de novo Crohn's showed pools of ectopic crypt PCs in the colectomy samples, as demonstrated by H&E representative photomicrography (
Human α-Defensin-5 (DEFA5) is a Better Candidate Biomarker than Paneth Cells for Crohn's Colitis.
Finally, we sought to determine if HD5 and Paneth cells were both upregulated in the normal, adjacent tissue of CC patients compared to UC patients (
Studies of MMP-7
Semi-quantitative real-time PCR (qPCR) was used to measure transcript levels of MMP-7. To do this, RNA was extracted from three human colon biopsy samples per condition; three each from moderate UC and CC, and from DV biopsy samples as a non-IBD control using the Qiagen RNeasy Miniprep Kit, (Valencia, Calif.). cDNA was generated using the iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif.), then used in the qPCR reactions using pre-designed TaqMan probes for MMP-7, and GAPHD control, and universal PCR master mix (Thermo Fisher Scientific, Waltham, Mass.). The reactions were run in triplicate using a CFX96 real-time PCR thermocycler (Bio-Rad). Data were analyzed according to the ΔΔCt method.
To assess any differences in the expression of HD5 and MMP-7 at the protein level, colon surgical resections (n=10) were used from mild, moderate, and severe UC; mild, moderate, and severe CC; and non-IBD DV control. Whole tissue protein extracts were prepared from full-thickness colon samples (n=10) using T-PER protein extraction kit according to manufacturer's protocol (Thermo Fisher Scientific). Bradford Assays (Bio-Rad) were used to determine protein concentration, and equal amounts of protein were separated in 4-20% SDS-PAGE tris/glycine gel (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) (Bio-Rad), then transferred to PVDF (polyvinylidene difluoride) membranes (Bio-Rad). The membranes were probed with antibodies against HD5, MMP-7, and β-actin loading control according to manufacturer's protocol. Blots were visualized with Opti-4CN colorimetric detection kit (Bio-Rad) and imaged with ChemiDoc XRS+imaging system (Bio-Rad). Band intensities were measured and data analysis performed with Image Lab Software (Bio-Rad).
Discussion
Colectomy surgical pathology samples of patients with unambiguous CC and UC undergoing colectomy in connection with RPC and IPAA were analyzed [6,7]. We those protein profiles which had the necessary (i) specificity; (ii) sensitivity; (iii) discriminatory; and (iv) predictive capacity to determine the heterogeneity of IBD [6,7] were identified and compared. It was possible to molecularly delineate UC and CC with molecular signatures of HD5 using IHC and quantified by NEARAS. Alpha-Defensins HD5 and HD6 are PC products and their altered expression has been linked to IBD pathogenesis.
It was not expected that one could not visualize PCs in these tissues even though one could detect HD5 in the same tissue (
To date, there is no diagnostic gold standard tool for IBD. Differentiating UC and CC among patients with IC has remained painstaking and is a major challenge in endoscopic medicine and colorectal surgery [1,12,28,29]. Clinicians use an inexact classification system of clinical, endoscopy, radiologic, and histopathology findings in order to diagnose CC and UC [21,30,31]. Even with a combination of these diagnostic modalities, up to 15% of IBD patients are labeled as IC when no definitive evaluations can be made [13,30,32]. In addition, CC is mistakenly diagnosed and RPC and IPAA-operated as definitive UC in another 15% of IBD patients because of overlap in the clinical, endoscopic, radiological and histologic findings [12,33-36]. Further, most IC patients who undergo RPC and IPAA surgery for presumed UC are subsequently found to develop a recurrent de novo Crohn's disease in the ileal pouch [1,12,33]. This is a serious consequence that may hinder the restoration of intestinal continuity and its intractable nature leads to pouch failure, often requiring pouch diversion or excision with a permanent terminal-ileostomy, resulting in negative psycho-sociological implications and poorer quality of life [1,29,31,36-41]. Curative treatment for UC is often surgical [42].
Success of RPC and IPAA surgery is largely dependent on careful patient selection combined with meticulous surgical technique and diagnostic accuracy [9-11,13]. Available clinical presentations and experience suggest that it is difficult to identify patients with CC who are likely to have a successful outcome after RPC and IPAA surgery [10,13,34,43]. However, in highly selected patients with CC, RPC and IPAA has been indicated [44-47]. Thus, RPC operation may be considered and should remain a careful option for certain subgroup of patients with CC, but an acceptable care option for patients with UC and for those IC patients predicted to develop UC [9,42].
These studies of HD5 as a candidate biomarker for CC suggest it could be a diagnostic signature to efficiently distinguish CC from UC. Newly published data shows that patients with small bowel Crohn's disease (Crohn's ileitis) are characterized with a deficiency of HD5, as shown by a reduced expression and secretion of the Paneth cell HD5, a fundamental feature of Crohn's ileitis [48-51]. Based on this study, in CC, the reverse is true. It was found that Paneth cell HD5 is a predominantly expressed antimicrobial peptide. This indicates that definitive CC and Crohn's ileitis may have distinct etiologies and mechanisms. In these studies, all IC patient samples have been reconciled into UC and CC using molecular biomarker, HD5, and verified the reconciliation by patient outcomes (
Accurately distinguishing CC from UC is of utmost importance when determining the candidacy of a patient for RPC [1,42]. Early diagnostic accuracy of IBD will lead to timely appropriate medical options. This study confirms that HD5 can differentiate CC and UC and reclassify IC into CC. In addition to distinguishing the colitides, HD5 could objectively be used to evaluate biophysiological processes and therapeutic outcomes and potentially play a pivotal role in IBD clinics as an attractive, non-invasive avenue [52,53].
Thus, this working example shows that tissue samples taken from patients suffering from CD have levels of HD5 significantly higher than the HD5 levels in samples from patients suffering from UC. Additionally, this working example shows that samples taken from patients suffering from CD have levels of MMP-7 significantly lower than the MMP-7 levels in samples of patients suffering from UC.
The following references were cited in the above working example. Such citation is not to be construed as an admission that any reference meets the legal definition of “prior art” in any country, nor as an admission that any reference is relevant to the patentability of anything claimed. Any such reference shall be incorporated herein by reference only to the extent it is necessary for a person of ordinary skill in the art to make and use anything claimed.
In addition to anything described above or currently claimed, it is specifically contemplated that any of the following embodiments may be claimed:
Emb. 1: A method of measuring HD5 in a patient suffering from or at risk of inflammatory bowel disease (IBD), said method comprising: obtaining a sample from the patient; and measuring at least one of the expression of HD5 and the concentration of HD5 in the sample.
Emb. 2: A method of treating a patient suffering from or at risk of inflammatory bowel disease (IBD), said method comprising: performing the method of measuring HD5 in the patient according to embodiment 1; and performing an intervention on the patient to treat Crohn's disease.
Emb. 3: Any one of the methods of embodiments 1-2, comprising: comparing the expression of HD5 or the concentration of HD5 in the sample to a benchmark value that is typical of a subject not suffering from Crohn's disease;
and diagnosing Crohn's disease if the expression of HD5 or the concentration of HD5 in the sample significantly exceeds the benchmark value.
Emb. 4: Any one of the methods of embodiments 1-3, wherein the expression of HD5 or the concentration of HD5 in the sample exceeds a benchmark value that is typical of a subject not suffering from Crohn's disease.
Emb. 5: Any one of the methods of embodiments 1-4, wherein the expression of HD5 is measured to be significantly greater in the sample than in a control sample from a subject not suffering from Crohn's disease.
Emb. 6: Any one of the methods of embodiments 1-5, wherein the expression of HD5 is measured to be at least about 31 times greater in the sample than in a control sample from a subject not suffering from Crohn's disease.
Emb. 7: Any one of the methods of embodiments 1-6, wherein the expression of HD5 is measured to be at least about 118 times greater in the sample than in a control sample from a subject not suffering from Crohn's disease.
Emb. 8: Any one of the methods of embodiments 1-7, wherein the expression of HD5 is measured to be greater than about 106 HD5 mRNA transcripts per 10 ng RNA.
Emb. 9: Any one of the methods of embodiments 1-8, wherein the expression of HD5 is measured to be greater than 107 HD5 mRNA transcripts per 10 ng RNA.
Emb. 10: Any one of the methods of embodiments 1-9, wherein the expression of HD5 is measured to be greater than 1.9×107 HD5 mRNA transcripts per 10 ng RNA.
Emb. 11: Any one of the methods of embodiments 1-10, wherein the expression of HD5 is measured to be greater than 7×107 HD5 mRNA transcripts per 10 ng RNA.
Emb. 12: Any one of the methods of embodiments 1-11, wherein the expression of HD5 is measured by qRT-PCR, wherein the method comprises measuring the expression of HD5 mRNA in a control sample from a subject not suffering from Crohn's disease, and wherein the expression of HD5 mRNA in the sample is significantly greater than the expression of HD5 mRNA in the control sample.
Emb. 13: Any one of the methods of embodiments 1-12, wherein the sample is intestinal tissue, and comprising measuring the concentration of HD5 by: immunostaining the sample with an anti-HD5 immunostaining agent; and measuring the percentage of cells in the sample that stain positive; wherein the percentage of cells in the sample that stain positive is at least 10%.
Emb. 14: Any one of the methods of embodiments 1-13, wherein the sample is intestinal tissue, and comprising measuring the concentration of HD5 by: immunostaining the sample with an anti-HD5 immunostaining agent; and measuring the percentage of cells in the sample that stain positive; wherein the percentage of cells in the sample that stain positive is at least 20%.
Emb. 15: Any one of the methods of embodiments 1-14, wherein the sample is intestinal tissue, and comprising measuring the concentration of HD5 by: immunostaining the sample with an anti-HD5 immunostaining agent; and measuring the percentage of cells in the sample that stain positive; wherein the percentage of cells in the sample that stain positive is at least about 30%.
Emb. 16: Any one of the methods of embodiments 1-15, wherein the intervention is not effective to treat ulcerative colitis.
Emb. 17: Any one of the methods of embodiments 1-16, wherein the intervention is a surgery.
Emb. 18: Any one of the methods of embodiments 1-17, wherein the intervention is a surgery selected from the group consisting of: ostomy, colostomy, ileostomy, bowel resection, colectomy, proctocolectomy, and strictureplasty.
Emb. 19: Any one of the methods of embodiments 1-18, wherein the intervention is administration of a drug.
Emb. 20: Any one of the methods of embodiments 1-19, wherein the intervention is administration of a drug, to the exclusion of a surgery.
Emb. 21: Any one of the methods of embodiments 1-20, wherein the intervention is administration of a drug selected from the group consisting of: a vitamin supplement, vitamin B12, vitamin D, a mineral supplement, calcium, an anti-inflammatory, a corticosteroid, a 5-aminosalicylate, an immunosuppressant, azathioprine, mercaptopurine, an anti-TNF-alpha antibody, infliximab, adalimumab, certolizumab pegol, methotrexate, an anti-α4-integrin antibody, natalizumab, vedolizumab, an anti-interleukin antibody, ustekinumab, an antibacterial antibiotic, ciprofloxacin, and metronidazole.
Emb. 22: Any one of the methods of embodiments 1-21, wherein the intervention is administration of a drug selected from the group consisting of: certolizumab pegol, methotrexate, and natalizumab.
Emb. 23: Any one of the methods of embodiments 1-22, wherein the intervention is placement of the subject on a low fat diet.
Emb. 24: A method of treating a patient suffering from or at risk of inflammatory bowel disease (IBD), said method comprising:
performing the method of measuring HD5 in the patient according to embodiment 1; and; and
performing an intervention on the patient to treat ulcerative colitis.
Emb. 25: The method of embodiment 24, comprising: comparing the expression of HD5 or the concentration of HD5 in the sample to a benchmark value that is typical of a subject not suffering from Crohn's disease; and diagnosing ulcerative colitis if the expression of HD5 or the concentration of HD5 in the sample does not significantly exceed the benchmark value.
Emb. 26: The method of any one of embodiments 24-25, wherein the expression of HD5 or the concentration of HD5 in the sample is below a benchmark value that is typical of a subject suffering from Crohn's disease.
Emb. 27: The method of any one of embodiments 24-25, wherein the expression or concentration of HD5 in the sample is measured to be significantly less than in a control sample from a subject suffering from Crohn's disease.
Emb. 28: The method of any one of embodiments 24-27, wherein the expression of HD5 is measured in the sample is no more than about 1/31 of expression of HD5 measured in a control sample from a subject not suffering from Crohn's disease.
Emb. 29: The method of any one of embodiments 24-28, wherein the expression of HD5 is measured in the sample is no more than about 1/118 of expression of HD5 measured in a control sample from a subject not suffering from Crohn's disease.
Emb. 30: The method of any one of embodiments 24-29, wherein the expression of HD5 is measured to be less than 106 HD5 mRNA transcripts per 10 ng RNA.
Emb. 31: The method of any one of embodiments 24-30, wherein the expression of HD5 is measured to be less than 107 HD5 mRNA transcripts per 10 ng RNA.
Emb. 32: The method of any one of embodiments 24-31, wherein the expression of HD5 is measured to be less than 1.9×107 HD5 mRNA transcripts per 10 ng RNA.
Emb. 33: The method of any one of embodiments 24-32, wherein the expression of HD5 is measured to be less than 6×105 HD5 mRNA transcripts per 10 ng RNA.
Emb. 34: The method of any one of embodiments 24-33, wherein the expression of HD5 is measured by qRT-PCR, wherein the method comprises measuring the expression of HD5 in a control sample from a subject suffering from Crohn's disease, and wherein the expression of HD5 mRNA in the sample is significantly less than the expression of HD5 in the control sample.
Emb. 35: The method of any one of embodiments 24-34, wherein the sample is intestinal tissue, and comprising measuring the concentration of HD5 by: immunostaining the sample with an anti-HD5 immunostaining agent; and measuring the percentage of cells in the sample that stain positive; wherein the percentage of cells in the sample that stain positive is less than 10%.
Emb. 36: The method of any one of embodiments 24-35, wherein the sample is intestinal tissue, and comprising measuring the concentration of HD5 by: immunostaining the sample with an anti-HD5 immunostaining agent; and measuring the percentage of cells in the sample that stain positive; wherein the percentage of cells in the sample that stain positive is less than 20%.
Emb. 37: The method of any one of embodiments 24-36, wherein the sample is intestinal tissue, and comprising measuring the concentration of HD5 by: immunostaining the sample with an anti-HD5 immunostaining agent; and measuring the percentage of cells in the sample that stain positive; wherein the percentage of cells in the sample that stain positive is less than about 30%.
Emb. 38: The method of any one of embodiments 24-37, wherein the intervention is not effective to treat Crohn's disease.
Emb. 39: The method of any one of embodiments 24-38, wherein the intervention is a surgery.
Emb. 40: The method of any one of embodiments 24-39, wherein the intervention is a surgery combined with the administration of a drug.
Emb. 41: The method of any one of embodiments 24-40, wherein the intervention is a surgery selected from the group consisting of: a proctocolectomy, and an ileal pouch anal anastomosis.
Emb. 42: The method of any one of embodiments 24-41, wherein the intervention is administration of a drug selected from the group consisting of: an iron supplement, an anti-inflammatory, a corticosteroid, a 5-aminosalicylate, an immunosuppressant, azathioprine, mercaptopurine, cyclosporine, an anti-TNF-alpha antibody, infliximab, adalimumab, golimumab, methotrexate, an anti-α4-integrin antibody, vedolizumab, an antibacterial antibiotic, ciprofloxacin, and metronidazole.
Emb. 43: The method of any one of embodiments 24-42, wherein the intervention is administration of a drug selected from the group consisting of: cyclosporine, and golimumab.
Emb. 44: The method of any one of embodiments 1-43, wherein the expression of HD5 is measured.
Emb. 45: The method of any one of embodiments 1-44, wherein the concentration of HD5 is measured.
Emb. 46: The method of any one of embodiments 1-45, wherein the sample is intestinal tissue.
Emb. 47: The method of any one of embodiments 1-46, wherein the sample is from the subject's large intestine.
Emb. 48: The method of any one of embodiments 1-47, wherein the sample is colonic tissue.
Emb. 49: The method of any one of embodiments 1-48, wherein the sample is ileal tissue.
Emb. 50: The method of any one of embodiments 1-49, wherein the expression of HD5 or the concentration of HD5 is measured in the sample ex vivo.
Emb. 51: The method of any one of embodiments 1-50, wherein HD5 expression is measured by a technique selected from the group consisting of: whole transcriptome analysis, whole-transcriptome microarray, Northern blot, DNA microarray, PCR, sequencing PCR, RT-PCR, quantitative PCR, restriction fragment length polymorphism, in situ hybridization assay, and a competitive-binding assay.
Emb. 52: The method of any one of embodiments 1-51, wherein HD5 concentration is measured by a technique selected from the group consisting of: Western blot, ELISA, two-dimensional gel electrophoresis, mass spectrometry, protein interaction profiling, a competitive binding assay, a non-competitive binding assay, a radioimmunoassay, an enzyme immunoassays, an enzyme linked immunosorbent assay (ELISA), a sandwich immunoassay, a precipitation reaction, a gel diffusion reaction, an immunodiffusion assay, an agglutination assay, a complement-fixation assay, an immunoradiometric assay, a fluorescent immunoassay, a protein A immunoassay, NMR analysis, and an immunoelectrophoresis assay.
Emb. 53: The method of any one of embodiments 1-52, wherein the patient is suffering from IBD.
Emb. 54: The method of any one of embodiments 1-53, wherein the patient displays a symptom selected from the group consisting of: severe diarrhea, abdominal pain, fatigue, and weight loss.
Emb. 55: The method of any one of embodiments 1-54, wherein the patient displays severe diarrhea, abdominal pain, fatigue, and weight loss.
Emb. 56: The method of any one of embodiments 1-55, wherein the expression of HD5 is measured.
Emb. 57: The method of any one of embodiments 1-56, wherein the concentration of HD5 is measured.
Emb. 58: The method of any one of embodiments 1-57, further comprising measuring at least one of the expression of MMP-7 and the concentration of MMP-7 in the sample.
Emb. 59: A method of measuring MMP-7 in a patient suffering from or at risk of inflammatory bowel disease (IBD), said method comprising: obtaining a sample from the patient; and measuring at least one of the expression of MMP-7 and the concentration of MMP-7 in the sample.
Emb. 60: A method of treating a patient suffering from or at risk of inflammatory bowel disease (IBD), said method comprising:
performing the method of measuring MMP-7 in the patient according to any one of embodiments 58-59; and
performing an intervention on the patient to treat Crohn's disease.
Emb. 61: The method of any one of embodiments 58-60, comprising: comparing the expression of MMP-7 or the concentration of MMP-7 in the sample to a benchmark value that is typical of a subject not suffering from ulcerative colitis; and diagnosing Crohn's disease if the expression of MMP-7 or the concentration of MMP-7 in the sample does not significantly exceed the benchmark value.
Emb. 62: The method of any one of embodiments 58-61, wherein the expression of MMP-7 or the concentration of MMP-7 in the sample is below a benchmark value that is typical of a subject suffering from ulcerative colitis.
Emb. 63: The method of any one of embodiments 58-61, wherein the expression of MMP-7 is measured to be significantly less in the sample than in a control sample from a subject suffering from ulcerative colitis.
Emb. 64: The method of any one of embodiments 58-63, wherein the expression of MMP-7 is measured in the sample to be at most about 1/10 of the expression of MMP-7 measured in a control sample from a subject not suffering from ulcerative colitis.
Emb. 65: The method of any one of embodiments 58-64, wherein the expression of MMP-7 is measured by qRT-PCR, wherein the method comprises measuring the expression of MMP-7 in a control sample from a subject suffering from ulcerative colitis, and wherein the expression of MMP-7 in the sample is significantly less than the expression of MMP-7 in the control sample.
Emb. 66: The method of any one of embodiments 58-65, wherein the sample is intestinal tissue, and comprising measuring the concentration of MMP-7 by: immunostaining the sample with an antiMMP-7 immunostaining agent; and measuring the percentage of cells in the sample that stain positive.
Emb. 67: The method of any one of embodiments 58-66, wherein the intervention is not effective to treat ulcerative colitis.
Emb. 68: The method of any one of embodiments 58-67, wherein the intervention is a surgery.
Emb. 69: The method of any one of embodiments 58-68, wherein the intervention is a surgery selected from the group consisting of: ostomy, colostomy, ileostomy, bowel resection, colectomy, proctocolectomy, and strictureplasty.
Emb. 70: The method of any one of embodiments 58-69, wherein the intervention is administration of a drug.
Emb. 71: The method of any one of embodiments 58-70, wherein the intervention is administration of a drug, to the exclusion of a surgery.
Emb. 72: The method of any one of embodiments 58-71, wherein the intervention is administration of a drug selected from the group consisting of: a vitamin supplement, vitamin B12, vitamin D, a mineral supplement, calcium, an anti-inflammatory, a corticosteroid, a 5-aminosalicylate, an immunosuppressant, azathioprine, mercaptopurine, an anti-TNF-alpha antibody, infliximab, adalimumab, certolizumab pegol, methotrexate, an anti-α4-integrin antibody, natalizumab, vedolizumab, an anti-interleukin antibody, ustekinumab, an antibacterial antibiotic, ciprofloxacin, and metronidazole.
Emb. 73: The method of any one of embodiments 58-72, wherein the intervention is administration of a drug selected from the group consisting of: certolizumab pegol, methotrexate, and natalizumab.
Emb. 74: The method of any one of embodiments 58-73, wherein the intervention is placement of the subject on a low fat diet.
Emb. 75: A method of treating a patient suffering from or at risk of inflammatory bowel disease (IBD), said method comprising:
performing the method of measuring MMP-7 in the patient according to embodiment 58; and
performing an intervention on the patient to treat ulcerative colitis.
Emb. 76: The method of any one of embodiments 58, 59 and 75, comprising: comparing the expression of MMP-7 or the concentration of MMP-7 in the sample to a benchmark value that is typical of a subject not suffering from ulcerative colitis; and diagnosing ulcerative colitis if the expression of MMP-7 or the concentration of MMP-7 in the sample significantly exceeds the benchmark value.
Emb. 77: The method of any one of embodiments 58, 59 and 75-76, wherein the expression of MMP-7 or the concentration of MMP-7 in the sample is above a benchmark value that is typical of a subject not suffering from ulcerative colitis.
Emb. 78: The method of any one of embodiments 58, 59 and 75-76, wherein the expression or concentration of MMP-7 in the sample is measured to be significantly greater than in a control sample from a subject not suffering from ulcerative colitis.
Emb. 79: The method of any one of embodiments 58, 59 and 75-78, wherein the expression of MMP-7 measured in the sample is at least about 5 times the expression of MMP-7 measured in a control sample from a subject not suffering from ulcerative colitis.
Emb. 80: The method of any one of embodiments 58, 59 and 75-79, wherein the expression of MMP-7 measured in the sample is at least about 10 times the expression of MMP-7 measured in a control sample from a subject not suffering from ulcerative colitis.
Emb. 81: The method of any one of embodiments 58, 59 and 75-80, wherein the expression of MMP-7 is measured by qRT-PCR, wherein the method comprises measuring the expression of MMP-7 in a control sample from a subject not suffering from ulcerative colitis, and wherein the expression of MMP-7 in the sample is significantly greater than the expression of MMP-7 in the control sample.
Emb. 82: The method of any one of embodiments 58, 59 and 75-81, wherein the sample is intestinal tissue, and comprising measuring the concentration of MMP-7 by: immunostaining the sample with an antiMMP-7 immunostaining agent; and measuring the percentage of cells in the sample that stain positive.
Emb. 83: The method of any one of embodiments 58, 59 and 75-82, wherein the intervention is not effective to treat Crohn's disease.
Emb. 84: The method of any one of embodiments 58, 59 and 75-83, wherein the intervention is a surgery.
Emb. 85: The method of any one of embodiments 58, 59 and 75-84, wherein the intervention is a surgery combined with the administration of a drug.
Emb. 86: The method of any one of embodiments 58, 59 and 75-85, wherein the intervention is a surgery selected from the group consisting of: a proctocolectomy, and an ileal pouch anal anastomosis.
Emb. 87: The method of any one of embodiments 58, 59 and 75-86, wherein the intervention is administration of a drug selected from the group consisting of: an iron supplement, an anti-inflammatory, a corticosteroid, a 5-aminosalicylate, an immunosuppressant, azathioprine, mercaptopurine, cyclosporine, an anti-TNF-alpha antibody, infliximab, adalimumab, golimumab, methotrexate, an anti-α4-integrin antibody, vedolizumab, an antibacterial antibiotic, ciprofloxacin, and metronidazole.
Emb. 88: The method of any one of embodiments 58, 59 and 75-87, wherein the intervention is administration of a drug selected from the group consisting of: cyclosporine, and golimumab.
Emb. 89: The method of any one of embodiments 58-88, wherein the expression of MMP-7 or the concentration of MMP-7 is measured in the sample ex vivo.
Emb. 90: The method of any one of embodiments 58-89, wherein MMP-7 expression is measured by a technique selected from the group consisting of: whole transcriptome analysis, whole-transcriptome microarray, Northern blot, DNA microarray, PCR, sequencing PCR, RT-PCR, quantitative PCR, restriction fragment length polymorphism, in situ hybridization assay, and a competitive-binding assay.
Emb. 91: The method of any one of embodiments 58-90, wherein MMP-7 concentration is measured by a technique selected from the group consisting of: Western blot, ELISA, two-dimensional gel electrophoresis, mass spectrometry, protein interaction profiling, a competitive binding assay, a non-competitive binding assay, a radioimmunoassay, an enzyme immunoassays, an enzyme linked immunosorbent assay (ELISA), a sandwich immunoassay, a precipitation reaction, a gel diffusion reaction, an immunodiffusion assay, an agglutination assay, a complement-fixation assay, an immunoradiometric assay, a fluorescent immunoassay, a protein A immunoassay, NMR analysis, and an immunoelectrophoresis assay.
Emb. 92: The method of any one of embodiments 58-91, wherein the patient is suffering from IBD.
Emb. 93: The method of any one of embodiments 58-92, wherein the patient displays a symptom selected from the group consisting of: severe diarrhea, abdominal pain, fatigue, and weight loss.
Emb. 94: The method of any one of embodiments 58-93, wherein the patient displays severe diarrhea, abdominal pain, fatigue, and weight loss.
Emb. 95: The method of any one of embodiments 58-94, wherein the sample is intestinal tissue.
Emb. 96: The method of any one of embodiments 58-95, wherein the sample is from the subject's large intestine.
Emb. 97: The method of any one of embodiments 58-96, wherein the sample is colonic tissue.
Emb. 98: The method of any one of embodiments 58-97, wherein the sample is ileal tissue.
Emb. 99: A kit for measuring HD5 and MMP-7 in a sample, the kit comprising: a first assay for measuring at least one of the expression of human HD5 and the concentration of human HD5 in a sample; and a second assay for measuring at least one of the expression of human MMP-7 and the concentration of human MMP-7 in a sample.
Emb. 100: The kit of embodiment 99, wherein said kit is for the diagnosis of inflammatory bowel disease.
Emb. 101: The kit of any one of embodiments 99-100, wherein the first assay comprises an antibody that recognizes human HD5; and wherein the second assay comprises antibody that recognizes human MMP-7.
Emb. 102: The kit of any one of embodiments 99-101, wherein the first assay comprises an oligonucleotide probe that binds to human HD5 cDNA; and wherein the second assay comprises an oligonucleotide probe that binds to human MMP-7 cDNA.
Emb. 103: The kit of any one of embodiments 99-102, wherein the first assay comprises a pair of primers complementary to a region of human HD5 cDNA; and wherein the second assay comprises a pair of primers complementary to a region of human MMP-7 cDNA.
Emb. 104: The kit of any one of embodiments 99-103, wherein: the first assay comprises a means for detecting HD5 protein; and the second assay comprises a means for detecting MMP-7 protein.
Emb. 105: The kit of embodiment 104, wherein: the means for detecting the HD5 protein is a first probe comprising a first ligand group that specifically binds to HD5 protein; and the means for detecting MMP-7 protein is a second probe comprising a second ligand group that specifically binds to MMP-7 protein.
Emb. 106: The kit of embodiment 105, wherein the first ligand group is an immunoglobulin.
Emb. 107: The kit of any one of embodiments 105-106, wherein the second ligand group is an immunoglobulin.
Emb. 108: The kit of any one of embodiments 105-107, wherein the first probe and the second probe are immobilized to a surface.
Emb. 109: The kit of any one of embodiments 99-108, wherein: the assay for measuring the expression of HD5 detects a first target sequence of at least 15 bp that is present in a first cDNA or mRNA of HD5; and the assay for measuring the expression of MMP-7 detects a second target sequence of at least 15 bp that is present in a second cDNA or mRNA of MMP-7.
Emb. 110: The kit of embodiment 109, wherein: the assay for detecting the first target sequence comprises a first probe comprising a first polynucleotide of at least 15 bp that hybridizes under highly stringent conditions with the first target sequence of at least 15 bp that is present in the first cDNA or mRNA of HD5; and the assay for detecting the second target sequence comprises a second probe comprising a second polynucleotide of at least 15 bp that hybridizes under highly stringent conditions with the second target sequence of at least 15 bp that is present in the second cDNA or mRNA of MMP-7.
Emb. 111: The kit of any one of embodiments 109-110, comprising a container of a reverse transcriptase.
Emb. 112: The kit of any one of embodiments 102-111, wherein the first probe comprises a first reporter, and the second probe comprises a second reporter.
Emb. 113: The kit of embodiment 112, wherein the first reporter is selected from the group consisting of: a radionuclide, a stable isotope, a fluorophore, a chromophore, an enzyme, a magnetic particle, and a quantum dot; and the second reporter selected from the group consisting of: a radionuclide, a fluorophore, a chromophore, an enzyme, a magnetic particle, and a quantum dot.
Emb. 114: The kit of any one of embodiments 110-113, wherein the first polynucleotide is single stranded DNA; and wherein the second polynucleotide is single stranded DNA.
Emb. 115: The kit of any one of embodiments 102-114, wherein the first probe and the second probe are components of a DNA array.
Emb. 116: The kit of any one of embodiments 102-115, wherein the first probe and the second probe are components of a DNA microarray.
Emb. 117: The kit of any one of embodiments 110-116, wherein the first polynucleotide is at least 20 bp and the second polynucleotide is at least 20 bp.
Emb. 118: The kit of any one of embodiments 110-117, wherein the first polynucleotide is at least 25 bp and the second polynucleotide is at least 25 bp.
Emb. 119: A method of diagnosing and treating Crohn's disease in a subject suffering from inflammatory bowel disease, the method comprising:
obtaining a sample from the patient;
measuring at least one of the expression of HD5 and the concentration of HD5 in the sample;
comparing the expression of HD5 or the concentration of HD5 in the sample to a benchmark value that is typical of a subject not suffering from Crohn's disease;
diagnosing Crohn's disease if the expression of HD5 or the concentration of HD5 in the sample significantly exceeds the benchmark value; and treating the subject for Crohn's disease by way of a non-surgical intervention.
Emb. 120: A method of diagnosing Crohn's disease in a subject suffering from inflammatory bowel disease, comprising: measuring the level of HD5 or HD5 expression in a sample from the subject, wherein the measuring is selected from the group consisting of radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization assays, competitive-binding assays, Western Blot analyses, ELISA assays and proteomic approaches, cation-ion exchange, NMR analysis, genome-wide transcriptome analysis, mass spectrometry, and combinations thereof; and diagnosing the subject as suffering from Crohn's disease if the level of HD5 is indicative of a subject having Crohn's disease.
Emb. 121: The method of embodiment 120, wherein the subject is diagnosed as suffering from Crohn's disease if the level of HD5 or HD5 expression is from about 1.9×107 HD5 mRNA Transcript per 10 ng RNA to about 7×107 HD5 mRNA Transcript per 10 ng RNA.
Emb. 122: A method of diagnosing ulcerative colitis in a subject suffering from inflammatory bowel disease, comprising: measuring the level of HD5 or HD5 expression in a sample from the subject, wherein the measuring is selected from the group consisting of radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization assays, competitive-binding assays, Western Blot analyses, ELISA assays and proteomic approaches, cation-ion exchange, NMR analysis, genome-wide transcriptome analysis, mass spectrometry, and combinations thereof; and diagnosing the subject as suffering from ulcerative colitis if the level of HD5 or HD5 expression is indicative of a subject having ulcerative colitis.
Emb. 123: The method of embodiment 122, wherein the subject is diagnosed as suffering from ulcerative colitis if the level of HD5 or HD5 expression is from about 6×105 HD5 mRNA Transcript per 10 ng RNA to about 1.8×107 HD5 mRNA Transcript per 10 ng RNA.
Emb. 124: A method of treating inflammatory bowel disease in a subject, comprising: measuring the level of HD5 or HD5 expression present in a sample obtained from the subject, said measuring step wherein the measuring is selected from the group consisting of radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization assays, competitive-binding assays, Western Blot analyses, ELISA assays and proteomic approaches, cation-ion exchange, NMR analysis, genome-wide transcriptome analysis, mass spectrometry, and combinations thereof, whereby a level of HD5 or HD5 expression is obtained; and if the level of HD5 or HD5 expression is at a level indicative of a subject not having Crohn's disease, treating the inflammatory bowel disease in the subject with a suitable medical treatment for ulcerative colitis; if the level of HD5 or HD5 expression is at a level indicative of a subject having Crohn's disease, treating the inflammatory bowel disease in the subject with a suitable medical treatment for Crohn's disease.
Emb. 125: The method according to embodiment 122, wherein the suitable medical treatment for ulcerative colitis comprises performing ileal pouch anal anastomosis in the subject.
Emb. 126: The method according to embodiment 122, wherein the suitable medical treatment for Crohn's disease comprises the administration of one or more of 5aminosalicylate, a corticosteroid, and an immunosuppressant to the subject.
Emb. 127: The method according to embodiment 122, wherein the sample is collected from the large intestine, and the subject is human.
Emb. 128: An assay for detecting elevated levels of HD5, comprising an HD5 antibody capable of binding with HD5.
Emb. 129: The assay according to embodiment 128, wherein the assay is provided in a kit.
Emb. 130: The novel and non-obvious embodiments and features disclosed herein.
Emb. 131: A method of measuring a biomarker in a patient suffering from or at risk of inflammatory bowel disease (IBD), said method comprising: obtaining a sample from the patient; and measuring a level of the biomarker in the same, the level of the biomarker selected from the group consisting of: the expression of the biomarker, the activity of the biomarker, and the concentration of the biomarker; wherein said biomarker is selected from Table 1.
Emb. 132: A method of treating a patient suffering from or at risk of inflammatory bowel disease (IBD), said method comprising: performing the method of measuring the level of the biomarker in the patient according to embodiment 131; and performing an intervention on the patient to treat Crohn's disease.
Emb. 133: Any one of the methods of embodiments 131-132, comprising: comparing the level of the biomarker in the sample to a benchmark value that is typical of a subject not suffering from Crohn's disease; and diagnosing Crohn's disease if the expression of the biomarker in the sample significantly differs from the benchmark value.
It is to be understood that any given elements of the disclosed embodiments of the invention may be embodied in a single structure, a single step, a single substance, or the like. Similarly, a given element of the disclosed embodiment may be embodied in multiple structures, steps, substances, or the like.
The foregoing description illustrates and describes the processes, machines, manufactures, compositions of matter, and other teachings of the present disclosure. Additionally, the disclosure shows and describes only certain embodiments of the processes, machines, manufactures, compositions of matter, and other teachings disclosed, but as mentioned above, it is to be understood that the teachings of the present disclosure are capable of use in various other combinations, modifications and environments and are capable of changes or modifications within the scope of the teachings as expressed herein, commensurate with the skill and/or knowledge of a person having ordinary skill in the relevant art. The embodiments described hereinabove are further intended to explain certain best modes known of practicing the processes, machines, manufactures, compositions of matter, and other teachings of the present disclosure and to enable others skilled in the art to utilize the teachings of the present disclosure in such, or other, embodiments and with the various modifications required by the particular applications or uses. Accordingly, the processes, machines, manufactures, compositions of matter, and other teachings of the present disclosure are not intended to limit the exact embodiments and examples disclosed herein. Any section headings herein are provided only for consistency with the suggestions of 37 C.F.R. § 1.77, or otherwise to provide organizational queues. These headings shall not limit or characterize the invention(s) set forth herein.
This application is a divisional of U.S. patent application Ser. No. 16/571,034, filed Sep. 13, 2019, which is a continuation of PCT/US2018/024069 filed Mar. 23, 2018, which claims the benefit of U.S. Provisional Patent Application No. 62/475,506, filed Mar. 23, 2017, each of which is relied upon for priority and incorporated by reference herein in its entirety.
This invention was made with government support under grant numbers R21DK095186; U54CA091408-09S1; U54CA091408-0952; U54RR026140; U54MD007593; UL1RR024975; UL1TR000445; G12MD007586; U54CA163069; R24 DA036420; and S10RR0254970 awarded by the National Institute of Health. The government has certain rights in the invention. In this context “government” refers to the government of the United States of America.
| Number | Date | Country | |
|---|---|---|---|
| 62475506 | Mar 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16571034 | Sep 2019 | US |
| Child | 17872674 | US | |
| Parent | PCT/US2018/024069 | Mar 2018 | US |
| Child | 16571034 | US |
