Field of the Invention

The present invention relates to biomarkers of inflammation and particularly, although not exclusively, to biomarkers of inflammatory bowel disease.

Background to the Invention

Inflammatory bowel diseases (IBD) are intestinal immune disorders that cause chronic inflammation in the gut [1]. Crohn's disease (CD) and ulcerative colitis (UC) are the most common forms of IBD, although a number of intermediate forms of colitis also occur. The course of IBD onset usually occurs in early adulthood and is followed by periods of remission punctuated by inflammatory relapses of varying severity [2]. The disease course of IBD is highly unpredictable and the inflammation is often so severe that around 25% of UC patients have their large intestine completely removed and 80% of CD patients require surgical intervention [3].

An estimated 4 million people suffer from IBD; 2.2 million of which live in Europe and between 150-200,000 in the UK [4, 5]. Prevalence in the UK is around 160-240 cases per 100,000 people for UC and 55-140 per 100,000 for CD [6]. The annual cost to the NHS is over £100 million and lifetime care for IBD is comparable in cost to that of other major diseases such as heart disease and cancer [4, 6]. Globally, the incidence of IBD is rising, which suggests that prevalence will also increase as a consequence of the high proportion of young people who are diagnosed with the disease [7]. The treatments and the aims of management for IBD have changed in recent years. Schoepfer et al. (2012) commented that the aims have evolved from relieving symptoms towards mucosal healing. They consider that this shift has been driven by the medications such as the anti-tumour necrosis factor (anti-TNF) drugs. The aim of treatment in active disease is to secure and maintain remission. Management involves diet and lifestyle interventions, drugs and surgery to induce and maintain remission. Drugs include aminosalicylates, corticosteroids, thiopurines, disease-modifying anti-rheumatic drugs (such as methotrexate), immunosuppressants (such as cyclosporin) and anti-TNF drugs (such as infliximab). There is an increased risk of colorectal cancer, so surveillance is part of patient care. Given the complexity of the disease, there is no single "gold standard" diagnostic test or examination to differentiate Crohn's disease and ulcerative colitis, and thus diagnosis is based on a combination of symptoms, clinical examinations, laboratory findings, radiology, and endoscopy with histology, which also is used to assess severity and to predict the outcome of disease. Even when the tests are performed by expert clinicians they can result in diagnostic uncertainty. Moreover, as IBD is a relapsing-remitting disease, constant monitoring is required. Faecal and serologic biomarkers can be used in the diagnosis and management of IBD, including calprotecin, lactoferrin, A100A12, CRP, ESR, serology, 6MP metabolite levels and antibody levels. Faecal markers, such as calprotectin and lactoferrin, have been studied for their ability to identify patients with IBD, assess disease activity, and predict relapse. Antibodies against Saccharomyces cerevisiae and perinuclear antineutrophil cytoplasmic proteins have been used in diagnosis of IBD, to distinguish Crohn's disease from ulcerative colitis, and to predict the risk of complications of Crohn's disease. Similarly, tests for CRP and ESR have been used to assess inflammatory processes and predict the course of IBD progression, while levels of drug metabolites and antibodies against therapeutic agents might be measured to determine why patients do not respond to therapy and to select alternative treatments. That notwithstanding, there is no single biomarker able to adequately diagnose and manage IBD with a high degree of specificity and sensitivity.

Accurately assessing IBD disease activity remains dependent principally on invasive tests, chiefly colonoscopy. Colonoscopy is not only invasive and unpleasant, it is expensive and carries a risk of complications including bowel perforation. It is desirable to have simpler diagnostic tests and biomarkers that can be used for assessment, management and follow up of IBD. Faecal calprotectin was recommended by the National Institute for Health and Care Excellence (NICE) in 2013 to support clinicians with the differential diagnosis of IBD, where faecal calprotectin, as a biomarker, correlates with the level of bowel inflammation, with test results being interpreted in the context of a cut-off value, below which the test is deemed negative and above which is deemed positive. Although calprotectin is now established in the initial screening for IBD versus other noninflammatory bowel disease, it is not adequately useful for accurately monitoring disease activity in individual patients and their response to treatment. Indeed, this was highlighted by NICE as a recommendation for further research on the use and clinical utility of faecal calprotectin testing and the impact on clinical decision-making. The results of calprotectin are highly variable - some patients have relatively low calprotectin levels but have clear disease at colonoscopy; whereas other patients have persistently high calprotectin levels and are asymptomatic, yet potentially subjected to invasive investigations.

There is, therefore, an unmet medical need to identify a suitable test, whether it be a new or existing biomarker, to support clinician decisions for stratification of patients with regards to a) disease course and b) monitoring disease activity and c) treatment response, and is a neglected component of understanding optimal treatment strategies. The ideal biomarker should be simple, easy to perform, noninvasive or microinvasive, cheap, rapid, and reproducible.

Whilst publications have shown that the mucosal expression of RAGE in IBD patients is invariably up-regulated, data on the levels of circulating sRAGE in patients with ulcerative colitis and Crohn's disease is conflicting, as previously demonstrated by Yilmaz et al., Meijer et al. and Ciccocioppo et al. However, the fact that the latter two studies concurred in their findings, suggests that sRAGE levels are significantly lower in ulcerative colitis, both active and inactive, than in controls and Crohn's disease, and inversely proportional with clinical and endoscopic indices of activity in both ulcerative colitis and Crohn's disease.

In a recent study by Meijer et al. (2014), total sRAGE and esRAGE concentrations in patients with IBD were determined and correlated with CRP, endoscopic scores and clinical disease activity scores. Plasma sRAGE concentrations were found to be lower in ulcerative colitis (but not Crohn's disease) than non-IBD subjects (p<0.01 ). Whilst sRAGE concentrations correlated negatively with endoscopic activity in ulcerative colitis (p<0.05), this was not seen in Crohn's disease. In contrast, sRAGE correlated negatively with disease activity in both ulcerative colitis (p=0.002) and Crohn's disease (p=0.0001 ). Furthermore, sRAGE and esRAGE concentrations correlated inversely with CRP values (p<0.0001 ). It was concluded that although total sRAGE varied with activity in ulcerative colitis, sRAGE concentrations correlated inversely with endoscopic disease activity and CRP levels in both ulcerative colitis and Crohn's disease. Summary of the Invention

The inventors have appreciated that the variability in the effectiveness of calprotectin as a marker of inflammatory bowel disease may be due to a lack of awareness of the biology of calprotectin activity. Calprotectin is a product of tissue damage and binds to the cell surface damage receptor RAGE (receptor of advanced glycation end products) to initiate inflammation. However, there is a soluble form of RAGE (sRAGE), which acts as a decoy receptor that binds to calprotectin and prevents its pro-inflammatory action. Accordingly, the inventors propose that the balance between calprotectin and its decoy receptor sRAGE determines whether calprotectin will promote inflammation. Thus, for effective screening and monitoring of patients it is important to measure s-RAGE and calprotectin.

We have recently identified that upregulation of RAGE (receptor for advanced glycation end-products) and associated pathway components are associated with development of colitis in a mouse model. Here, we observed increased expression of RAGE mRNA before the onset of clinical symptoms in colitis-susceptible AKR mice 24 hours after helminth challenge. The T. muris colitis model is not as commonly used as other colitis models, such as DSS-induced colitis, but has been shown to induce a chronic colitis as opposed to acute colitis that clinically, histologically, genetically and immunologically resembles Crohn's disease in susceptible mice [8].

Moreover, the inventors have determined that sRAGE has application as a biomarker of IBD. In particular, sRAGE in serum or in a faecal sample may be detected as a biomarker of inflammation, or active disease. Described herein are methods useful for the clinical management of patients with inflammatory bowel disease (IBD).

Methods disclosed herein involve the detection and/or quantification of sRAGE. In some cases, an increase in the level of sRAGE is indicative of acute resolving inflammation. That is, the level of sRAGE is indicative of a normal inflammatory process that will resolve. In some cases, an absence or change in the level of sRAGE is indicative of chronic inflammation. The increase may be relative to a control, such as the level of sRAGE in a sample from the subject at an earlier time point, or during a period of inactive disease, or minimal or absence of inflammation, such as during remission. Alternatively, the control sample may be a sample obtained when a patient is in relapse. This level may be referred to as the base line level. In some cases, the increase is comparable to a positive control level, such as the level in the subject during a previous period of active disease, such as during relapse. The inventors have further identified that sRAGE may be used in conjunction with calprotectin as an indicator of IBD. In particular the ratio of sRAGE and calprotectin levels may be a powerful biomarker of IBD.

As disclosed herein, the combined analysis of sRAGE and calprotectin may give a good indication of IBD relapse. For example, an individual exhibiting an elevated level of Calprotectin and an elevated level of sRAGE may be determined to have acute resolving inflammation. Alternatively, an individual exhibiting an elevated level of Calprotectin and a normal, insignificantly elevated, or decreased level of sRAGE may be determined to have chronic inflammation.

In some methods disclosed herein, the inflammatory bowel disease is Crohn's disease. In some methods disclosed herein, the inflammatory bowel disease is ulcerative colitis.

In some methods, the level of sRAGE is compared to the level of calprotectin. The level of calprotectin may be determined in the same or a different sample from the patient. Preferable the sample is a faecal sample. In some cases, the level of calprotectin is determined. In some cases the level of S100A12 is determined as an indicator of the level of calprotectin. In some cases a ratio of calprotectin to sRAGE is determined. In some cases, the levels of the biomarkers, such as sRAGE or calprotectin are determined by commercially available assay.

In some cases, the method is a computer implemented method. In some methods disclosed herein, the biomarkers are used in conjunction with other indicators of acute resolving inflammation or chronic inflammation.

Certain methods described herein are useful for monitoring disease progression in a subject. The methods may be useful for making clinical decisions for the patient. For example, where the methods indicate that a patient has chronic inflammation, the patient may be selected for a particular treatment. Alternatively, where the methods indicate that a patient has acute resolving inflammation, they may be selected for an alternative treatment. In some cases, patients who have been diagnosed with inflammatory bowel disease are monitored by the methods of the invention. In some cases, the patient being monitored is asymptomatic. The method maybe used to diagnose inflammatory disease in the patient, in some cases prior to the onset of symptoms. The methods disclosed herein are particularly useful for the early diagnosis of inflammatory disease, and particular for early determination of whether inflammation is likely to resolve (acute resolving inflammation) or will persist (chronic inflammation).

Certain methods described herein may be performed by a clinician such as a doctor or nurse, optionally in a clinical setting such as a doctor's surgery or hospital. In some cases, a sample is obtained from the patient and transferred to a separate testing facility for biomarker analysis.

In other cases, the methods may be performed by the subject, for example using a home testing kit.

In some methods disclosed herein, the level of the biomarker(s) is determined at a plurality of time points. The level of the biomarker(s) may be determined at a first time point (To). The level of the biomarker(s) may then be determined against at a second time point (Τχ). The level may be monitored for a defined period of time, or monitored indefinitely. The level may be determined every day, every two days, once a week, once a fortnight, once a month, once every six months, or once a year. In some cases, the level is determined during relapse. This level is then retained as a baseline level or control for use in the future. In some cases the method involves comparing the level of the biomarker to the baseline level to determine whether it is elevated or unchanged. In some cases the biomarker is sRAGE. In some cases the biomarker is the ratio of sRAGE to calprotectin.

Also disclosed herein is the use of sRAGE, or the ratio of sRAGE to calprotectin as a biomarker of inflammatory disease, and particularly as a biomarker for determining whether a subject has acute resolving inflammation or chronic inflammation. Also disclosed herein is a classifier comprising the level of sRAGE in a sample, preferably a faecal sample, and optionally the level of calprotectin or S1000A12.

In some aspects, provided herein is a kit comprising means for preparation of a faecal sample and means for detecting sRAGE. In some cases, the kit also includes means for detecting calprotectin. The kit may include a lateral flow device. The kit may comprise a positive control and/or a negative control, such as a sample with a known level of sRAGE or calprotectin, or known to be representative of an individual with acute resolving inflammation or chronic inflammation.

In some aspects described herein, there is provided a method of distinguishing between acute resolving inflammation and chronic inflammation in an inflammatory bowel disease patient, said method comprising:

obtaining a faecal sample from a human patient;

detecting the level of sRAGE in the faecal sample by using an immunoassay with a solid support with a hydrophilic surface; and

determining that the patient has acute resolving inflammation if the level of sRAGE in the faecal sample is high, or that the patient has chronic inflammation if the level of sRAGE in the faecal sample is low.

The method may optionally involve the detection of the level of calprotectin in the faecal sample, wherein if the level of calprotectin in the faecal sample is high and the level of sRAGE in the faecal sample is high, then the patient is determined to have active resolving inflammation, and if the level of calprotectin in the faecal sample is high and the level of sRAGE in the faecal sample is low, then the patient is determined to have chronic inflammation.

In some aspects described herein, there is provided a method of treating IBD in a patient, said method comprising:

obtaining a faecal sample from a human patient;

detecting the level of sRAGE in the faecal sample; and

detecting the level of calprotectin in the faecal sample; and

determining that the patient has acute resolving inflammation if the level of calprotectin in the faecal sample is high and the level of sRAGE in the faecal sample is high, or determining that the patient has chronic inflammation if the level of calprotectin in the faecal sample is high and the level of sRAGE in the faecal sample is low; and

administering an effective amount of anti-IBD therapy to the patient, if the patient is determined to have chronic inflammation.

In these aspects, the anti-IBD therapy may be an antispasmodic, laxative or anti mobility agent, probiotic aminosalicylate, thiopurine, calcineurin inhibitor or anti-TNF therapy.

Where the patient is determined to have chronic inflammation, the effective amount of anti-IBD therapy may be a high dose.

Description

The present invention includes biomarkers, methods and kits for distinguishing between acute resolving inflammation and chronic inflammation in inflammatory bowel disease patients. In particular, the present invention relates to the use of sRAGE as a biomarker, and particularly faecal sRAGE. In some cases, the invention relates to the combination of sRAGE and calprotectin. The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about," it will be understood that the particular value forms another embodiment. Inflammatory Bowel Disease (IBD)

IBD is a term associated with a number of conditions that involve inflammation of the gastrointestinal tract. The two most common forms of IBD are ulcerative colitis and Crohn's disease. These conditions can sometimes have serious complications, including a high risk of surgery and an increased risk of colorectal cancer. In both conditions, some people have active disease but no obvious symptoms. In some aspects described herein, the IBD is Crohn's Disease (CD). In other aspects described herein, the IBD is Ulcerative Colitis (UC).

Crohn's disease is characterised by chronic inflammation in any part of the gastrointestinal tract. Most commonly the terminal ileum or the perianal region are inflamed, and in a non-continuous manner. Histologically, Crohn's disease shows thickened sub-mucosa, transmural inflammation, fissuring ulceration and non-caseating granulomas. Ulcerative colitis, on the other hand, is characterised by inflammation limited to the colon, spreading continuously from the rectum and various distance proximal, and histology shows superficial inflammatory changes limited to the mucosa and sub-mucosa with inflammation of crypts (cryptitis) and crypt abscesses.

Ulcerative colitis is a relapsing and remitting disease characterised by inflammation of the colon, sometimes intense, with bloody diarrhoea, but more often milder. The cause is not known, but some people are more genetically susceptible than others; around 10% of people with ulcerative colitis have a first-degree relative with the condition. There may be an abnormal immune response to the natural bacteria that live in the gut. Sometimes, ulcerative colitis occurs after an episode of gastroenteritis caused by organisms such as Salmonella, Shigella and Campylobacter. However, in this case, the condition is more commonly triggered by resulting changes in the natural gut bacteria than by the direct effects of these organisms. Crohn's disease, on the other hand, can present in different ways, depending on which part of the intestinal tract is affected. Like ulcerative colitis, it is a relapsing and remitting inflammatory disease. However, it can be a much more extensive disease and can affect any part of the gastrointestinal tract. The cause, once again, is unknown, but there is a genetic susceptibility. Like ulcerative colitis, it can occur after infectious gastroenteritis and is associated with disturbances in the natural gut flora. The highest incidence of Crohn's disease is in the 15-30 year age range, although 20 - 30% of people with the condition are younger than 20 years and onset occurs in people younger than 17 years in about 25% of cases. The incidence of Crohn's disease in the general population has been increasing both within the UK and internationally.

Acute Resolving Inflammation and Chronic Inflammation

Inflammatory bowel disease may be associated with acute resolving inflammation and chronic inflammation. Methods disclosed herein may be used to distinguish acute resolving inflammation from chronic inflammation.

Acute resolving inflammation is inflammation that will resolve without treatment. Acute resolving inflammation may resolve, or symptoms may begin to lessen, within two months, 6 weeks, 4 weeks, 2 weeks or 1 week of the onset of symptoms. Resolution may be complete or partial remissions or a lessening of symptoms.

Acute resolving inflammation is indicative of a good prognosis, such as mild disease, or an absence of complications of the disease, such as colon damage. Chronic inflammation is inflammation that will not readily resolve without treatment. In some cases, chronic inflammation is inflammation that will not resolve after one month, 6 weeks, 8 weeks, 10 weeks, 12 weeks or more than 14 weeks after the onset of symptoms. Chronic inflammation is indicative of poor prognosis, such as long term complications from the disease, including colon damage potentially requiring surgery.

Subjects identified are having acute resolving inflammation may be treated or selected for treatment with a less aggressive or less intense management of their disease. In some cases, if a subject is determined to have acute resolving inflammation, it may be determined that no treatment is warranted, or that the treatment is watchful waiting. Subjects determined to have chronic inflammation may be selected for intensive or aggressive treatment, or intensive management. In some cases, such subjects may receive an increased dosage, or an alternative treatment. Remission refers to a period in the disease when symptoms are less severe. Partial remission refers to a remission with the possibility of relapse, or the return of disease activity and/or symptoms. Complete remission refers to permanent loss of disease activity. As used herein, the term "symptoms" is used to describe physiological and biochemical indicators of inflammatory bowel disease. Such symptoms may include abdominal pain or discomfort, bloating, change in a bowel habit, weight loss, anaemia, rectal masses and inflammatory biomarkers. Management of IBD may include watchful waiting, diet and lifestyle interventions, patient education, drugs, behavioural and/or psychological therapies, complementary or alternative therapies. A number of anti-IBD therapies are known in the art.

Anti-IBD therapies include antispasmodic agents, laxatives or anti mobility agents; probiotics (such as E coli Nissle 1917, Mutaflor®, VSL#3, Lactobacillus rhamnosius GC, Bifidobacterium and Saccharomyces boulardii); aminosalicylates (such as 5- aminocalicylic acid (5-ASA), Asacol®, Salofalk®, Ipocol®, Mesren®, Mezavant XL®, sulfasalazine, Salazopyrin®, olsalazine, Dipentum®, balsalazinde, Colazide®); corticosteroids (including prednisolone, prednisone, budesonide, Entocort®, Budenofalk®, hydrocortisone, methylprednisolone, metasulfobenzoate, beclomethasone dipropionate); thiopurines (such as azathioprine (AZA), mercaptopurine (MP)); polyglutamated metabolites of methotrexate (MTX); Calcineurin inhibitors (including ciclosporin (CsA), tacrolimus), and Anti-TNF therapies (such as infliximab (IFX), Adalimumab (ADA)).

Worm Therapy

Administration of Trichuris suis ova (TSO) and/or other parasitic worms has been investigated for the treatment of IBD. However, it is known that some individuals exhibit an inappropriate immune response to worm infection, resulting in chronic inflammation. Methods disclosed herein may be used to determine whether worm therapy is, or is not, appropriate for a particular individual. Thus, in some cases, the present invention provides methods for selecting a patient for worm therapy. Patients with high levels of sRAGE may be selected for worm therapy. Patients who have low or unchanged sRAGE may be determined not to be suitable for worm therapy. Such patients may be considered at risk of chronic inflammation in response to worm therapy. The worm therapy may involve the administration of TSO.

Worm Infection

Methods disclosed herein are also useful for patients with worm infection, and for selecting appropriate treatment for such patients. As used herein, the term "worm infection" encompasses parasitism by worms, and particularly helminth worms. Worm infections particularly contemplated herein relate to parasitism of the gut, but the methods used herein may also be applicable to other parasitic infection of the body by worms. Particularly contemplated are soil transmitted helminthiasis (STH), Roundworm (Ascaris lumbricoides), Whipworm (Trichuris trichiura) and Hookworm (Ancyclostoma duodenale or Necator americanus) and Schistosomiasis (including parasitism by Schistosoma spp., such as Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi, Schistosoma guineenesis or Schistosoma haemotobium). Other worm infections relevant to the present invention include Lymphatic filariasis (including parasitism by Wuchereria bancrofti, Brugia malayi and Brugia timori), onchocerciasis (Onchocerca volvulus), and urogenital schistosomiasis.

In some cases, patients with elevated sRAGE may be considered to expel the worms without requiring further treatment. Patients with negligible or no increase in sRAGE may be selected for treatment, such as anti-helminthic agent.

Agents suitable for the treatment of worm infection include Ivermictin (Mectizan™), albendazole, mebendazole and praziquantel. Methods disclosed herein may be used to select patients for treatment with one or more of these agents. For example, methods disclosed herein may be used to select patients for treatment with such agents, if they exhibit negligible elevation, or normal sRAGE. Such patients may be determined to require therapeutic treatment, as it is unlikely that they will expel the worms of their own accord. RAGE

The methods of the invention relate to the detection or quantification of RAGE, or Receptor for Advanced Glycation End products, and particularly sRAGE (soluble RAGE). RAGE is a 35kDa transmembrane receptor of the immunoglobulin super family.

The interaction between RAGE and its ligands is thought to result in proinflammatory gene activation.

RAGE is a multiligand, transmembrane, cell-surface receptor that initiates diverse proinflammatory signalling cascades including activation of the NF-κΒ signalling pathway [9]. RAGE was discovered as a ligand for AGEs (advanced glycation end-products), which are formed by non-enzymatic protein glycation [10]. AGEs may be present in food but can also be produced by certain bacteria (such as Escherichia coli) and as a consequence of oxidative stress [10]. Identified ligands of RAGE include amyloid-β peptide, β2 integrin Mac-1 (CD1 1 b/CD18), S100 proteins and HMGB1 (high-mobility group box 1 ) (Table 1 ) [9]. The bulk of RAGE ligands can be seen as damage-associated molecular patterns (DAMPs), with release of HMGB1 in particular being associated with tissue necrosis; HMGB1 is usually sequestered in the nucleus and is not released when cells undergo apoptosis [1 1 ]. RAGE ligand-binding and subsequent activation cascade are not well understood, but internalisation of RAGE after ligand binding is required for signal transduction to occur [12].

Ligand Family Examples

Argpyrimidine

Carboxymethyllysine

Carboxyethyllysine

Pronyl-glycine

Oxidised low density lipoprotein-containing AGE epitopes

S100 S100A12 (EN-RAGE)

proteins/Calgranulins S100B

S100A8/9 (ca I protect! n )

AOPPs Advanced oxidation protein products

Amyloid/beta sheet fibrils Amyloid-β peptide

HMGB1 HMGB1

Complement receptor Mac-1 (CD1 1 b/CD18, integrin aw integrin fcj

Table 1. Examples of the different types of ligands that can bind and activate the receptor for advanced glycation end-products. RAGE acting as a ligand for CD1 1 b (Mac-1 ) is interesting as it links RAGE expression with immune cell migration during inflammation. Dendritic cells (DCs) and neutrophils are able to migrate via Mac-1/32 integrin binding, and leukocytes from RAGE ^" ' ^" mice show reduced adherence to peritoneal tissues in caecal ligation and puncture studies [13]. Increased RAGE expression could therefore be linked to a greater influx of immune cells during the onset of colitis. Early migration of DCs has been shown to be protective against colitis in the T. muris model [14], but prolonged accumulation of immune cells in the lamina propria leads to chronic inflammation [15]; highlighting the need for immune cell migration to be tightly regulated, failure of which can lead to pathology. Epithelial cells expressing low levels of RAGE are important in facilitating β2 integrin-mediated migration of neutrophils in colonic tissues during inflammation [16], suggesting that dysregulation of RAGE-mediated immune cell migration could be linked with the onset of colitis. Evidence for the involvement of RAGE in IBD also comes from studies that link RAGE activity to active IBD [17-19] and various other inflammatory diseases including diabetes, Alzheimer's, airway inflammation, cancer and haemorrhagic shock [20-24]. Also, several RAGE ligands have been identified as potential IBD markers, including calprotectin, ENRAGE and HMGB1 , providing further evidence of the pivotal role RAGE may play in IBD and proinflammatory diseases [25-27]. Given that the colitis-susceptible AKR mouse strain showed increased expression of RAGE mRNA in our previous study (data not shown), we hypothesise that this is due to an increase in RAGE expressed on the surface of cells within the lamina propria or epithelial tissues of the colon. sRAGE

The cleavage of the RAGE extracellular domain by several proteases such as ADAM-10 or MMP-9 causes the release of its soluble form (sRAGE), which plays a protective role in inflammation thanks to the ability to capture and block RAGE ligands. Low levels of sRAGE have been found in a number of chronic inflammatory disorders, whilst only limited and contradictory results have been obtained in IBD. The first evidence obtained by Yilmaz et al., (201 1 ) demonstrated a higher serum level of sRAGE, which correlated with disease activity index in adult ulcerative colitis patients in comparison with Crohn's disease and healthy controls, whereas Meijer et al. (2013) observed lower concentrations of plasma (admittedly not serum) sRAGE in patients suffering from ulcerative colitis, which negatively correlated with the endoscopic activity index, with respect to Crohn's disease and non-IBD subjects. In the most recent study conducted, by Ciccocioppo et al. (2015), a cross-sectional study in Crohn's disease, ulcerative colitis and control subjects, disease activity was scored through the clinical, endoscopic and histologic indices of severity and in all cases the levels of serum sRAGE, S100A12, CRP and faecal calprotectin were measured. sRAGE levels were found to be significantly lower in ulcerative colitis, both active and inactive, than in controls and Crohn's disease, and inversely proportional with clinical and endoscopic indices of activity in both I BD groups and with the histologic score in the Crohn's disease group, which fits the findings of Meijer et al. rather than Zilmaz et al. Interestingly, those Crohn's disease patients in the Ciccocioppo et al. study with a penetrating behaviour showed a significant reduction in both sRAGE and S100A12 compared to those with an inflammatory/stricturing pattern.

In some cases, the methods disclosed herein involve the detection of esRAGE (endogenous secretory RAGE). esRAGE is a splice variant of RAGE that can be secreted.

In some aspects described herein, the level of sRAGE in the faecal sample is determined to be low if it is less than 100 pg/ml, less than 95 pg/ml, less than 90 pg/ml, less than 85 pg/ml, less than 80 pg/ml, less than 77pg/ml, less than 75 pg/ml or less than 70 pg/ml.

In some aspects described herein, the level of sRAGE in the faecal sample is determined to be high if it is more than 90pg/ml, more than 95pg/ml, more than 100 pg/ml , more than 105 pg/ml, more than 1 10 pg/ml, more than 1 15 pg/ml, more than 130 pg/ml, more than 130 pg/ml, more than 135 pg/ml, more than 140 pg/ml, more than 145 pg/ml, more than 150 pg/ml, more than 154 pg/ml, more than 155 pg/ml, more than 160 pg/ml or more than 165 pg/ml.

In some aspects described herein, the level of sRAGE in the serum sample is determined to be low if it is less than 10000 pg/ml, less than 9000 pg/ml, less than 8000 pg/ml, less than 7000 pg/ml, less than 6000 pg/ml, less than 5000pg/ml, less than 4000 pg/ml or less than 3000 pg/ml.

In some aspects described herein, the level of sRAGE in the serum sample is determined to be high if it is more than 10000/ml, more than 1 1 ,000pg/ml, more than 12,000pg/ml , more than 13,000pg/ml, more than 14,000pg/ml, more than 15,000pg/ml, more than 16,000pg/ml, more than 17,000pg/ml, more than 18,000pg/ml, more than 19,000pg/ml, more than 20,000pg/ml, more than 21 ,000pg/ml, more than 22,000pg/ml, more than 23,000pg/ml, more than 24,000pg/ml, more than 25,000pg/ml, more than 26,000pg/ml, or more than 27,000pg/ml.

Calprotectin

Calprotectin is a 36 kDa calcium- and zinc-binding protein that represents 60% of cytosolic proteins in granulocytes. Calprotectin is a heterodimer of proteins S100A8 and S100A9. It is stable in faeces when stored at room temperature for up to one week (Roseth et al., 1992). The concentration of calprotectin in faeces is an indirect measure of neutrophil infiltrate in the bowel mucosa. Numerous studies have addressed whether faecal calprotectin could be used to select patients with symptoms of IBD that warrant endoscopic or radiologic evaluation. Von Roon et al. (2007) summarised data from 30 studies that included 5,983 patients (1210 had IBD). The estimated sensitivity and specificity values for the identification of patients with IBD, compared to those without, were 89% and 81 %, respectively, in studies that used a threshold faecal calprotectin concentration of 50 μg g; and 98% and 91 % in studies that used a threshold faecal calprotectin concentration of 100 μg g.

However, these estimates come from combinations of different studies, rather than tests of different threshold levels in a single study. In fact, it is implausible for the faecal calprotectin assay to have higher sensitivity when using a higher threshold to define a positive test. Therefore, these data cannot be used to select an optimal cut-off point. van Rheenen et al. (2010) performed a similar analysis that was limited to studies that included only patients suspected to have IBD based on signs and symptoms. Among the six studies, the sensitivity and specificity for identification of IBD in adults were 93% and 96%, respectively. In children, the test's sensitivity was 92%, but the specificity was only 76%. The authors conclude that using these tests to choose what patients require further testing reduces the need for endoscopy or radiology tests in a large portion of patients, but would delay the diagnosis of IBD in 6% and 8% of the adults and children with disease, respectively. Calprotectin vs sRAGE

As disclosed herein, the inventors have determined that the balance between calprotectin and sRAGE is a critical factor as to whether calprotectin is promoting inflammation. Thus, if both sRAGE and calprotectin are high, calprotectin would be bound by sRAGE and not switch on the pro-inflammatory pathways. In contrast, if calprotectin were high but sRAGE was low, pro-inflammatory pathways would be initiated. Methods described herein may involve the comparison of the level of calprotectin with the level of sRAGE. The comparison may indicate the likelihood of inflammation resolving, or may indicate that the inflammation is chronic. In some cases, the result may indicate the likelihood of a return of symptoms of IBD, or a return to an active disease state.

The relationship between calprotectin and sRAGE may be expressed as a ratio. The ratio may be calculated directly from the levels of calprotectin and sRAGE. Alternatively, the value for the level may be processed prior to calculation of the level. For example, through the calculation of the log value, or the calculation of mean values from a plurality of samples or measurements. The value for the ratio may differ depending on the data used to calculate the ratio, or the protocol used to obtain the value for the level. Therefore, the ratio may be interpreted in view of ratios calculated using the same protocol for obtaining the value for the level and/or the same processing prior to calculation of the level.

As described herein, a high ratio may be indicative of acute resolving inflammation, and a low ratio may be indicative of chronic inflammation. S100A12

S100A12 is a S100 protein that is similar to calprotectin. It is sometimes referred to as Calgranulin C. In a study of children, (de Jong et al., 2006) faecal levels of S100A12 greater than 10 mg/kg identified IBD with a sensitivity of 96% and a specificity 92%. In a subsequent study of adults (Kaiser et al., 2007), S100A12 distinguished patients with IBD from those with irritable bowel syndrome with sensitivity and specificity values of 86% and 96%, respectively. S100A12 can be measured in serum. Although serum levels are increased in patients with IBD, this test does not distinguish IBD from IBS with the same levels of sensitivity and specificity as the faecal assay (Monolakis et al., 2010). Manolakis et al., (201 1 ) quote a 96% to 97% sensitivity and a 92% to 100% specificity for S100A12 to differentiate IBD from normal gut as well as slightly lower numbers (86%- 97% sensitivity and 92%-97% specificity) to differentiate IBD from IBS. Endoscopically based studies would bring these numbers to a lower range, with sensitivities of 24% to 97% and specificities of 94% to 97% for distinguishing IBD from non-IBD in colonic disease.

To date, five biopsy-matched studies have evaluated the role of S100A12 in determining disease activity (Vrabie & Kane, 2014). There is growing consensus regarding cut-off values for active IBD vs no IBD, with 75 ng/mL to 82 ng/mL in serum or 0.06 mg/kg to 1 .2 mg/kg in stool as the upper limit of normal. The cut-off values for active vs inactive IBD, although more clinically relevant, are less well established. There is also substantial agreement between the studies that S100A12 is a good marker for colonic disease, although perhaps less so for small bowel inflammation.

Interesting histologic data have also been presented, demonstrating that S100A12 is preferentially detected at sites of active inflammation (eg, Crohn's disease granulomas and ulcerative colitis crypt abscesses). Leach et al., (2007) evaluated serum and mucosal S100A12 levels in a paediatric population. Serum levels of calprotectin and S100A12 correlated well with each other (r=0.746; P<.0001 ), but the mucosal levels did not. On a histologic level, this protein was not only expressed in the lamina propria of non-inflamed tissue but was also abundant in the epithelium of inflamed specimens. Serum S100A12 was also found to be the most specific of the markers measured in this study but had lower sensitivity. (Calprotectin, platelets, CRP, ESR, and albumin measures were all more sensitive.)

Kaiser et al., (2007) demonstrated that faecal S100A12 was the most accurate marker of inflammation of all the markers employed in the study (S100A12, CRP level, ESR, platelet count, white blood cell count, and haemoglobin level). The authors found similarly low faecal S100A12 values in patients with IBS and healthy control subjects and equally elevated levels in patients with Crohn's disease and those with ulcerative colitis. Values were also elevated in active vs inactive disease. In adults, the faecal S100A12 values did correlate in a weak but statistically significant fashion with other markers of inflammation, such as histology inflammation score (A=0.44; P<.01 ), ESR (A=0.77; P<.01 ), CRP level (A=0.396; P<.01 ), platelet count (r=0.418; P<.01 ), white blood cell count (A=0.287; P<.05), and haemoglobin level (r=-0.512; P<.001 ).

Sidler et al., (2008) studied S100A12 in a paediatric population and were able to demonstrate that, in this cohort, faecal S100A12 level had a positive predictive value of 97% and a negative predictive value of 97% and was, therefore, more specific than faecal calprotectin for detecting active disease (97% vs 67%, respectively). This marker did not correlate with calprotectin level, Paediatric CDAI score, ESR, CRP level, or platelet count and only weakly correlated with albumin level (r=0.3917; P=.03), perhaps indicating its unique role in paralleling disease activity in this patient group.

Sipponen et al., (2012) demonstrated poor correlation of S100A12 with capsule endoscopy in terms of detecting ileal disease, with a positive predictive value of 38% and a better negative predictive value of 82%. These findings might be expected, as faecal markers in general have been thought of as more illustrative of colonic disease, although there are some encouraging data for calprotectin as a marker of small bowel Crohn's disease, as mentioned earlier (Jensen et al., 201 1 ).

A strength of this marker is its high specificity for active disease (especially compared with other markers) as well as the fact that it can be measured in both serum and faeces. Limitations include that S100A12 is nonspecific to IBD - with levels also being elevated due to other causes, such as infection (viral or bacterial, including diverticulitis), polyposis (colon cancer and adenomas), other autoimmune disorders (celiac disease and immunodeficiency), increased age, obesity, and physical inactivity - and that S100A12 is decreased with more fibre consumption. Another limitation is the weak ability of S100A12 to measure small bowel disease, according to current data (Manolakis et al., 201 1 ).

Subjects

Subjects to which the present methods may be applied may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. Therapeutic uses may be in human or animals (veterinary use).

The subject may be a patient. That is to say that the subject may be supervised by a physician or medical professional in relation to the disease or disorder to which the invention pertains. Thus, in some cases the subject has inflammatory bowel disease, and the subject may be an inflammatory bowel disease patient or otherwise be an individual who has previously received a diagnosis of IBD. In some cases, the subject or patient is experiencing symptoms of disease. In other cases, the subject or patient is in remission. In certain cases, the subject or patient is normal, indicating that they have a previous diagnosis of IBD, but are in an extended period of remission. Subject Selection

Methods disclosed herein relate to the selection of subjects. As used herein, subjects who are considered suitable for treatment are those subjects who are expected to benefit from, or respond to, the treatment. Particular methods described herein are useful for the identification of subjects undergoing a relapse of inflammatory bowel disease, and particularly for distinguishing subjects with resolving inflammatory for those with chronic inflammation. Patients with chronic inflammation may be selected for more aggressive therapy, or more intensive management than those with resolving inflammation.

Detection and Quantification

Methods disclosed herein involve the detection and/or quantification of biomarkers. Detection, as used herein, refers to measurement of biomarkers without quantification. Methods for detection and quantification of biomarkers are well known in the art and will be readily appreciated by a skilled person. In particularly preferred methods, the biomarkers to be detected are sRAGE, calprotectin and/or S100A12.

Methods according to the present invention may be performed in vitro or ex vivo. The term "in vitro" is intended to encompass experiments with materials, biological substances, cells and/or tissues in laboratory conditions or in culture. "Ex vivo" refers to something present or taking place outside an organism, e.g. outside the human or animal body, which may be on tissue (e.g. whole organs) or cells taken from the organism.

The methods disclosed herein particularly relate to the determination of protein expression. Protein expression can be measured by quantifying the amount of protein in a cell, tissue or sample, or by observing the localization of the protein within cells and tissues.

Protein, for example, may be detected or quantified by immunoassay. Immunoassay methods are well known in the art and will generally comprise: (a) providing a polypeptide comprising an epitope bindable by an antibody against said protein; (b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said polypeptide is formed. Immunoassay methods include western blotting and ELISA.

Immunoassays include, but are not limited to, Enzyme-linked immunosorbent assay (ELISA), lateral flow test, latex agglutination, other forms of immunochromatography, western blot, and/or magnetic immunoassay.

Protein may also be detected or quantified using mass spectrometry. For example, mass spectrometry using electrospray ionization (ESI) or matrix-assisted laser desorption/ionisation (MALDI). Other methods of protein quantification include spectroscopy based methods. Such methods may involve colorimetric assays or spectrophotometric assays.

Particularly preferred methods disclosed herein involve the use of immunoassays. Immunoassays are used to detect the target in a sample from the subject. Immunoassays use antibodies with specific affinity for the target molecule in conjunction with a detectable molecule. In some cases, the antibody is conjugated to the detectable molecule. The detectable molecule may be referred to as a label. The detectable molecule produces a detectable signal when the antibody is bound to the target molecule. The detectable signal may be a quantifiable signal. In some cases, an aptamer is used instead of, or together with, the antibody. Immunoassays include immunohistochemistry, ELISA, immunoblotting and flow cytometry. In certain aspects described herein, the assay is an immunohistochemistry assay. Such assays commonly use antibodies, although other target specific molecules such as aptamers or other ligands may be used. In certain aspects, the method uses a solid support with a hydrophilic surface. The solid support may have been pretreated to render the surface hydrophilic, or may be constructed at least partially from a hydrophilic material. In some cases, the solid support is a multiwall or microtiter plate. In some cases, the solid support is a Maxisorp Nunc plate. The solid support may have a surface of approximately 2.5atom% oxygen. The solid support may have a surface of at least 1 . 5atom% oxygen, at least 2atom% oxygen, or at least 2.5atom% oxygen. The surface may comprise polystyrene, or treated polystyrene.

The method may be approved for use by a regulatory agency. The method may be an FDA approved method.

Methods described herein may involve the step of determining the level of a biomarker. In some cases, determining the level of the biomarker is not an active step of the method. The steps of determining the level of the biomarker, and interpretation of the results, are not necessarily undertaken by the same party, or as part of a continuous process. That is, the level may have already been determined, either with the intention of performing a method as described, or the method may be performed on a level previously determined for another purpose. The level may have been previously determined by the party performing the classification or diagnosis of the patient. Alternatively, the level may have been determined by a third party. In some cases, therefore, a value for level of the biomarker may be obtained.

In certain aspects, the method involves detection and/or quantification of the biomarker using qPCR (quantitative PCR). ELISA

In some cases, the target may be detected by ELISA (enzyme-linked immunosorbent assay). Target molecules from a sample are attached to a surface and detected using a specific antibody. The target may be attached to the surface non-specifically (via adsorption to the surface) or specifically (using a specific capture agent such as an antibody). ELISA may be used to quantify target in a sample. ELISA is particularly suited to the analysis of liquid samples, such as serum, urine or saliva or pre-pared faecal samples. Commercially available ELISA assays are available for the detection and/or quantification of sRAGE, calprotectin and/or S100A12 and may be used with the methods of the invention. Several sRAGE tests are available. These include Abeam Anti-RAGE antibody (ab361 1 ) for fluorescent staining and flow cytometry, Sigma Mouse RAGE/AGER ELISA Kit (RAB0008) for first ELISAs and R&D Mouse RAGE DuoSet ELISA (DY1 179) for second lot of ELISAs. Other suppliers include Merck Millipore, Thermo Fisher Scientific, Sigma Aldrich, Nordic BioSite and MyBioSource.

Several faecal calprotectin tests are available, including fully quantitative laboratory- based technologies, fully quantitative rapid tests and semi-quantitative point of care tests. Information on such tests may be found on the world wide web, such as at nice.org.uk/guidance/dg1 1/chapter/4-the-diagnostic-tests. These include:

Because faecal calprotectin correlates with the level of bowel inflammation, test results need to be interpreted in the context of a cut-off value, below which the test is deemed negative and above which is deemed positive. For example, CalDetect reports 1 of 4 results when the test runs correctly: negative - faecal calprotectin is not detectable; negative - faecal calprotectin level is equal to or less than 15 micrograms/g; positive - faecal calprotectin level is 16-60 micrograms/g; and positive - faecal calprotectin level is more than 60 micrograms/g. Users might apply local cut-offs for interpreting the results of POCTs; for example, a cut-off of 60 micrograms/g might be applied, test results below which are deemed negative and above which are deemed positive. In particularly preferred cases, the cut-off is 50 micrograms/g.

Immunoblotting

In some aspects, the target is detected by immunoblotting, or western blotting. In such methods, proteins in a sample are separated based on their electrical charge or size. They may be separated by an electrophoresis based method. The separated proteins are transferred to a membrane, where they are stained with an antibody that is specific to the target. The antibody is then detected, either directly by virtue of the antibody being conjugated to a detectable label, or indirectly, by adding a labelled secondary antibody.

Flow Cytometry

Flow cytometry based biomarker detection may be used to detect cells expressing a biomarker of interest, such as sRAGE. Cells from the sample are suspended in a stream of fluid and directed past an electronic detection apparatus. The cells may be labelled with an antibody that is specific to the biomarker of interest. In particular, the cells may be labelled with a fluorescent antibody. Cells that express the biomarker of interest may be detected and quantified, based on the fluorescent signal from the label.

A type of flow cytometry useful in the methods disclosed herein is Fluorescence Activated Cell Sorting (FACS). Using FACS, cells may be separated into two or more vessels, based on the presence or absence of the fluorescent label on the cell.

Immunohistochemistry

Immunohistochemistry (IHC) is broadly used and well established as a diagnostic test methodology particularly in oncology indications and provides highly accurate results if used under standardized conditions (Demidova, Barinov et al., 2014).

IHC refers to the process of detecting targets in cells of a tissue section by exploiting the principle of antibodies binding specifically to the target in biological tissues. IHC is widely used in the diagnosis of abnormal cells, such as those found in cancerous tumors. Visualizing an antibody-target interaction can be accomplished in a number of ways. Commonly, an antibody is conjugated to label. Alternatively, the antibody is detected by a secondary antibody, which is itself labelled. Detection of the label is thus indicative of the presence of target. IHC can be used to determine the cellular localization of a target and the amount of target present. IHC may be qualitative or semi-quantitative. Immunohistochemistry methods are known in the art and are suitable for use as described herein. IHC methods commonly involve the fixation of a sample so that the sample is preserved from degradation. In certain aspects, a sample is formalin fixed and paraffin embedded (FFPE). In other aspects, IHC is performed on frozen samples. Prepared samples may be sectioned prior to analysis.

The sample may undergo pre-treatment, such as with Ventana CC1 (Cell Conditioning 1 ) solution. Where the sample is a FFPE sample, the method may involve deparaffinization of the sample. Prepared samples are incubated with an antibody that is specific to the target. The samples may be incubated with an anti-biomarker antibody. The conditions and duration of incubation will depend on the particular antibody used. In some cases, the sample is incubated for between 10 minutes and 60 minutes, between 20 minutes and 45 minutes, or between 25 minutes and 35 minutes. In some cases, the sample may be incubated with the antibody for around 30 minutes, such as for 32 minutes. Incubation may occur at room temperature, or between about 20°C and 50°C, between 30°C and 40°C, or around 35°C, such as 37°C. Preferably the sample is incubated with the antibody for 32 minutes at 37°C. The samples may additionally be counter-stained to facilitate analysis. For example, the sample may be stained with haematoxylin and eosin (H&E) stained.

The methods disclosed herein may be performed manually or automatically. Preferably, the methods are at least partially automated. For example, slide staining steps may be automated. Slide staining may be performed using a Ventana™ BenchMark ULTRA™. Alternatively, slide staining may be performed using a Ventana™ BenchMark XT™, Ventana™ BenchMark GX™, Dako Omnis™, Dako Autostainerl_ink48™, Leica™ BOND RX™, Leica™ BOND-IN™ or Leica™ BOND MAX™ Following incubation of the sample with the labelled antibody, they may be analysed using a microscope.

Antibodies

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies {e.g., bispecific antibodies), intact antibodies (also described as "full-length" antibodies) and antibody fragments, so long as they exhibit the desired biological activity, for example, the ability to bind sRAGE. Antibodies may be murine, human, humanized, chimeric, or derived from other species such as rabbit, goat, sheep, horse or camel.

An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C, Travers, P., Walport, M., Shlomchik (2001 ) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by Complementarity Determining Regions (CDRs) on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody may comprise a full- length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass, or allotype (e.g. human G1 m1 , G1 m2, G1 m3, non-G1 m1 [that, is any allotype other than G1 m1 ], G1 m17, G2m23, G3m21 , G3m28, G3m1 1 , G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1 , A2m2, Km1 , Km2 and Km3) of immunoglobulin molecule. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin.

"Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, US 4816567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991 ) Nature, 352:624-628; Marks et al (1991 ) J. Mol. Biol., 222:581 -597 or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459).

Control

In some cases, the method involves comparing biomarker levels in a sample from a patient with biomarker levels in one or more control samples.

Suitable control samples and subjects will be appreciated by those of skill in the art. In particularly preferred aspects disclosed herein, the control is a sample previously obtained from the same patient, at an earlier point in time. For example, in a sample obtained from a subject before the onset of IBD symptoms, or before undergoing treatment for IBD. In some cases, the control is obtained from an individual who is known to be undergoing acute resolving inflammation or chronic inflammation. The comparison may not require the analysis of the control sample to be simultaneously or sequentially performed with the analysis of the sample from the patient. Instead, the comparison may be made with results previously obtained from a control sample, such as results stored in a database.

The control sample may be a sample obtained from the patient prior to the onset of symptoms, or from an earlier time point when the patient experienced acute resolving inflammation or chronic inflammation. The control sample may be a sample obtained from another individual. The individual may be matched to the patient according to one or more characteristics, for example, sex, age, medical history, ethnicity, weight or expression of a particular marker.

Samples

The methods of the invention may involve determining the level of a biomarker in a sample. Methods described herein may be performed on a sample that has been obtained from a patient. Such methods may thus be performed ex vivo. They may be performed in vitro. Where the method involves the detection of more than one biomarker, the biomarkers may be quantified and/or detected in the same or different samples.

In particularly preferred methods described herein, the sample in a faecal or stool sample. In some methods described herein, sRAGE and optionally calprotectin is detected and/or quantified in a faecal sample. In some cases, both sRAGE and calprotectin are detected and/or quantified in the same sample. In some cases, both the sRAGE and calprotectin levels are determined in a faecal sample. In some cases, one of the sRAGE and calprotectin levels is determined in a faecal sample, and the level of the other is determined in a plasma or serum sample.

Alternatively, the sample may be taken from any tissue or bodily fluid. For example, the sample may be derived from: a quantity of blood; a quantity of serum derived from the individual's blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells, a quantity of plasma, or cells derived from a blood sample. The sample may be a sample taken from a bodily fluid, such as a fluid that circulates through the body. Accordingly, the sample may be a blood sample or lymph sample. In some cases, the sample is not a plasma or serum sample. Prognosis

Prognosis, prognosing and prognose refer to estimating the risk of future outcomes in an individual based on their clinical and non-clinical characteristics. In particular, a method of determining the prognosis as used herein refers to the prediction of the outcome of, or future course of, an individual's or subject's cancer and, in particular, whether the subject is likely to respond to endocrine therapy. Prognosis includes the prediction of patient's survival. Prognosis may be useful for determining an appropriate therapeutic treatment. Prognostic testing may be undertaken with (e.g. at the same time as) the diagnosis of a previously undiagnosed cancerous condition, or may relate to an existing (previously diagnosed) condition.

As used herein, a good prognosis may indicate that inflammation in the bowel is decreasing or will decrease. A good prognosis may indicate that the symptoms of disease will decrease in severity, or are or may become absent.

A poor prognosis is a prediction that a disease, will not respond to therapy, and may recur or worsen. In the present case, a poor prognosis may indicate chronic inflammation, or that inflammation is occurring, and may indicate that symptoms of disease will return or persist.

In some aspects described herein, a method of prognosis is provided. The method is for determining the prognosis of a patient with IBD, and comprises obtaining a value for the level of sRAGE in a sample from the patient, and obtaining a value for the level of calprotectin in a sample from the patient, wherein if the value of calprotectin is high, and the value of sRAGE is high, then the patient is determined to have a good prognosis, whereas if the value of calprotectin is high but the value of sRAGE is low, then the patient is determined to have a poor prognosis. Detection of Calprotectin

Certain methods described herein involve the detection and/or quantification of calprotectin. Methods for the detection of calprotectin are known in the art, and the skilled practitioner will readily comprehend an appropriate test for use herein. Particularly preferred methods involve the detection and/or quantification of faecal calprotectin. Methods may involve a chromatographic immunoassay. The method may involve anti-calprotectin antibodies, particularly anti-calprotectin monoclonal antibodies. In particular, methods for detecting and/or quantifying calprotectin that are suitable for use in the methods disclosed herein include those set above.

A level of more than 15 ug/gn or more, 20 ug or more, 25 ug or more, 30 ug or more, 35 ug or more, 40 ug or more, 45 ug or more, 50 ug or more, 55 ug or more, 60 ug or more, 65 ug or more, 70 ug or more 75 ug or more may be indicative of IBD relapse. In particular a level of 50 ug/g or more may be indicative of IBD relapse.

Preparation of a faecal sample

Certain methods disclosed herein involve the detection of a biomarker in a faecal sample. Methods for detection may involve preparation of the faecal sample by dissolving in a solution, such as, but not limited to, water, or isotonic buffer. Biomarker detection may then be performed on the resulting solution.

Biomarker levels

Methods disclosed herein may involve the detection of elevated levels of biomarkers. As used herein, elevated biomarker levels are those which are statistically significantly increase relative to control levels. As used herein, elevated expression is used interchangeably with increased expression, high expression or high level. In particular, the methods disclosed herein involve the detection of the biomarkers sRAGE and calprotectin.

In some cases described herein, calprotectin levels are considered elevated, if calprotectin is present in a faecal sample at more than 15 ug/g, more than 20 ug or more, 25 ug or more, 30 ug or more, 35 ug or more, 40 ug or more, 45 ug or more, 50 ug or more, 55 ug or more, 60 ug or more, 65 ug or more, 70 ug or more 75 ug or more may be indicative of IBD relapse. In particular a level of 50 ug/g or more may be indicative of IBD relapse. Kits

In a particularly preferred aspect, the present disclosure provides a kit. The kit may include one or more diagnostic reagents. The diagnostic reagent may include an antibody that is specific to the biomarker. The kit may include a labelling agent for detection of the biomarker, or detection of the diagnostic reagent.

The kit may include one or more reagents for preparation of the sample. For example, sample preparation buffers. In particular embodiments, the kit may include reagents for preparation of a faecal sample.

In some cases, the kit may include one or more controls. For example, the kit may include a positive control and/or a negative control.

The kit may include instructions for use, such as instructions for preparation of a sample, or instructions for detection of the biomarker.

The kit may be a kit for home testing, point of care testing, or laboratory use.

The kit may comprise or consist of a lateral flow device for detection of the biomarker. Lateral flow devices may use immunochromatography to indicate the presence of the biomarker. In some cases, the lateral flow device provides a qualitative indication of the presence of the biomarker. In some cases, the lateral flow device provides a quantitative indication of the level of biomarker. Lateral flow devices involve a series of capillary beds with the capacity to transport a fluid sample into the device so as to contact the sample with an agent capable of detecting the target biomarker. The agent may be an anti- biomarker antibody. The agent may be labelled with a detectable label, or the device may contain a further agent for detecting biomarker immobilised on the agent, for example a second agent that is capable of detecting biomarker bound by the agent. The agent may be immobilised within the device. The lateral flow kit may comprise buffers and reagents to allow or facilitate the reaction between the agent and biomarker in the sample. In some cases, the buffers and reagents are present in a dried form, such as freeze dried or lyophilised form. The lateral flow device may involve a sandwich or a competitive assay. Brief Description of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which: Figure 1. Colitis-susceptible AKR mice show delayed expulsion of Trichuris muris worms at 21 days and increased evidence of colitis at 31 days post infection. (A) Mean body weight (±SD) as % of starting weight up to 31 days post infection with 200 T. muris eggs. (B) Mean worm burden (±SD) at 21 days post infection. (C) Mean colonic crypt length (in μΓη±8ϋ) in naive mice and at 1 , 21 and 31 days post infection. (D) Mean muscle wall thickness (in μηΊΐεϋ) in naive mice and at 1 , 21 and 31 days post infection. (E) Cumulative colitis score (0-9) based on the grading of histological changes including active inflammation, immune cell infiltration and surface ulceration. (F) Representative images of haematoxylin and eosin stained proximal colon sections from naive mice and at 31 days post infection; note the high levels of immune cell infiltration and loss of goblet cells in the colonic tissues of AKR mice at 31 days post-infection. Β3Γ=50μηΊ. N=3-5 mice per time point. Analysis by Student's T test or two-way ANOVA followed by Sidak's multiple comparisons test where appropriate. ^* P<0.05.

Figure 2. Dendritic cell and macrophage infiltration in the colonic lamina propria of naive and Trichuris muris infected male AKR and BALB/c mice (aged 6-8 weeks) at 1 , 7, 14, 21 and 31 days post infection with 200 T. muris eggs. (A) Flow cytometry gating strategy for identification of dendritic cells (CD45 ⁺ MHCII ⁺ CD1 1 c ⁺ F4/80 ^" CD103 ^+/" CD1 1 b ^+/" ) and macrophages (CD45 ⁺ MHCIT F4/80 ⁺ CD1 1 c ^+/" ). (B-F) Mean populations of identified cell types as proportion of CD45 ⁺ cells (±SD) in naive mice and during T. muris challenge. n=3 mice per time point. Analysis by two-way ANOVA with Sidak's multiple comparisons post hoc test. ^*** P<0.001 , ^** P<0.01 , ^* P<0.05.

Figure 3. Monocyte and neutrophil infiltration in the lamina propria of naive and Trichuris muris infected male AKR and BALB/c mice (aged 6-8 weeks) at 1 , 7, 14, 21 and 31 days post infection with 200 T. muris eggs. (A) Flow cytometry gating strategy for identification of resident monocytes (CD45 ⁺ Ly6G ^" CD1 1 b ⁺ CD1 15 ⁺ ), inflammatory monocytes (CD45 ⁺ Ly6G ⁺ CD1 1 b ⁺ CD1 15 ⁺ ) and neutrophils (CD45 ⁺ Ly6G ⁺ CD1 1 b ⁺ CD1 15 ^" ). (B-D) Mean populations of identified cells as proportion of CD45 ⁺ cells (±SD) in naive mice and during T. muris challenge. n=3 mice per time point. Analysis by two-way ANOVA with Sidak's multiple comparisons post hoc test. ^*** P<0.001 , ^** P<0.01 , ^* P<0.05. Figure 4. Colonic epithelial expression of the receptor for advanced glycation end- products (RAGE) in male AKR and BALB/c mice (aged 6-8 weeks) by flow cytometry and immunohistochemistry. (A-B) Proportion of epithelial cells (CD326 ⁺ ) expressing high or low RAGE respectively. (C-D) Median fluorescence of RAGE high or low epithelial cells respectively. (E) Representative images of colon sections stained for RAGE (FITC; green) and nuclei (DAPI; blue) in naive mice and at 7 days post-infection (Bar = Ι ΟΟμηη; inset bar = 22μη"ΐ). n=3 mice per time point. Analysis by two-way ANOVA with Sidak's post hoc test.

Figure 5. Circulating and faecal sRAGE and S100A8 in naive and Trichuris muris infected 6-8 week old male AKR and BALB/c mice (A-D) and IL-10 ^" ' ^" and C56BL6 mice (E and F) at 1 , 7 and 21 days post infection (±SD) measured by ELISA. n=3-5 mice per time point. Results are representative of two separate experiments. Analysis by two-way ANOVA with Sidak's multiple comparisons post hoc test. ^**** P<0.0001 , ^** P<0.01 . Figure 6. (A) Apoptosis assessed by TUNEL assay in naive male AKR and BALB/c mice (aged 6-8 weeks) and 1 day post infection with Trichuris muris. (B) Mean apoptotic cells counted per crypt (±SD). Scale bar = Ι ΟΟμηη. n=2-3 mice per time point. Analysis by two- way ANOVA with Sidak's multiple comparisons post hoc test. Figure 7. Membrane-bound ADAM10 expression in the proximal colon. Representative images of proximal colon from naive and Trichuris muris infected AKR and BALB/c mice at 21 days post infection. Sections are stained for nuclei (DAPI; blue), epithelial cytokeratin (FITC; green) and ADAM 10 (AF555; red). Inset area shows high ADAM 10 expression in the immune cells of the lamina propria. Bar=100^m, ίη8βί=50μη"ΐ ² . Figure 8. Immune cells present in the mesenteric lymph nodes of naive and Trichuris muris infected male AKR and BALB/c mice (aged 6-8 weeks) at 1 , 7, 14, 21 and 31 days post infection. Flow cytometry gating as per Figure 2 and Figure 3. (A-H) Populations of dendritic cells (CD45 ⁺ MHCII ⁺ CD1 1 c ⁺ F4/80 ^" CD103 ^+/" CD1 1 b ^+/ -), macrophages (CD45 ⁺ MHCi F4/80 ⁺ CD1 1 c ^+/ -), resident monocytes (CD45 ⁺ Ly6G ^" CD1 1 b ⁺ CD1 15 ⁺ ), inflammatory monocytes (CD45 ⁺ Ly6G ⁺ CD1 1 b ⁺ CD1 15 ⁺ ) and neutrophils (CD45 ⁺ Ly6G ⁺ CD1 1 b ⁺ CD1 15 ^" ) as proportion of CD45 ⁺ cells (±SD) in naive mice and during T. muris challenge. n=3 mice per time point. Analysis by two-way ANOVA with Sidak's multiple comparisons post hoc test. ^*** P<0.001 , ^** P<0.01 , ^* P<0.05.

Figure 9. Median RAGE expression on neutrophils (CD45 ⁺ Ly6G ⁺ CD1 1 b ⁺ CD1 15 ^" ) of the colonic lamina propria of naive and Trichuris muris infected male AKR and BALB/c mice (aged 6-8 weeks) at 1 , 7, 14, 21 and 31 days post infection. n=3 mice per time point. Figure 10. Flow cytometry gating strategy for epithelial cell identification (CD326 ⁺ ) and RAGE expression of epithelial subpopulations from the colonic lamina propria of naive and Trichuris muris infected male AKR and BALB/c mice (aged 6-8 weeks).

Figure 11. Crypt lengthening, goblet cell hyperplasia and changes in neutrophil number in naive Trichuris muris infected male IL-10 ^" ' ^" and C57BL6 mice (aged 4-8 weeks) and at 1 , 7 and 21 days post infection. (A) Mean colonic crypt length (in μηΊΐεϋ). (B) Mean goblet cells (per cryptiSD). (C) Mean neutrophils per field of view at 40x magnification. n=2-4 mice per time point. Analysis by two-way ANOVA followed by Sidak's multiple comparisons test. ^*** P<0.001 , ^** P<0.01 .

Figure 12: Plot of Log faecal calprotectin vs, log faecal sRAGE in human samples. N: normal, I: IBS, R: Remission, D: disease. Log is used to compensate for large range of Calprotectin values. Examples

Example 1 : Materials and Methods

Mice

All animal procedures used in this project were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986. 6-8 week old male BALB/c and AKR mice (Harlan UK, Bicester, UK), or C57BL6 and IL-10 ^" ' ^" mice (bred in house) were used for all experiments. Mice purchased from external suppliers were left to acclimate for 7 days prior to the experiment start. Severe combined immunodeficient (SCID) mice were bred in house and used for parasite maintenance only. Mice were housed in groups of 3-5 mice in individually ventilated cages with nesting material and were maintained under constant 12h light-dark cycle at 21 -23 °C with free access to water and standard chow (Beekay Rat and Mouse Diet, Bantin & Kingham, Hull, UK). Euthanasia was carried out by schedule 1 procedure of CO2 asphyxiation followed by exsanguination. 2-5 mice were used per strain, per time point studied.

Parasites

Professor Kathryn Else, The University of Manchester, kindly provided eggs of Trichuris muris Edinburgh (E) isolate for use in all infection studies. Egg infectivity and maintenance of parasite stocks were carried out using SCID mice as described by Wakelin, 1967 [35]. Egg infectivity was used to correct for the number of eggs administered in experimental infections. Experimental mice were infected with 200 embryonated eggs in 200μΙ of ultra-pure distilled water via oral gavage.

Worm burden assessment

T. muris infection provides a highly naturalistic model for triggering the onset of chronic inflammation via epithelial barrier interference. T. muris larvae burrow into the epithelium of the caecum and proximal colon within 24 hours of infection, causing localised tissue damage and potentially allowing bacteria to come into direct contact with the epithelium [28]. Bacterial interaction with the gut is an important precursor to the onset of intestinal inflammation evidenced by numerous studies that demonstrate mice susceptible to spontaneous colitis (e.g. IL-10 ^" ' ^" , mdrl a ^" ' ^" ) do not develop colitis when reared in germ-free conditions [29, 30]. In addition, clinical pathology in the T. muris model does not occur very early during infection [28], unlike more robust challenges such as the commonly used DSS (Dextran Sulfate Sodium induced) colitis model that quickly causes acute changes to mucosal barrier and alters the structural integrity of the epithelium of the gut [31 ]. Due to DSS causing clinical changes at day 1 post administration [32], it is not well suited to assessing preclinical changes that preclude chronic colitis. Worm burden was assessed at day 21 post infection. Caecum and proximal colon were harvested at autopsy to determine the infectivity of each mouse at the end of each experiment as described by Else et al., 1990 [36].

Immunohistochemistry

Histology

Proximal colon snips were fixed in neutral buffered formalin (NBF; 10% neutral buffered formalin in PBS) for 24 hours, processed (Shandon Citadel 2000; ThermoShandon, Runcorn, UK) and embedded in paraffin wax. 5μηι sections were then dewaxed, rehydrated and stained using a standard Haematoxylin & Eosin (H&E) stain or Alcian blue/Periodic Acid Schiff's stain [37]. Images were acquired using a 20x/0.80 Plan Apo objective using the 3D Histech Pannoramic 250 Flash II slide scanner and samples were measured for crypt hyperplasia, immune cell infiltration, immune cell location, goblet cell loss and muscle wall thickening using 3D Histech Pannoramic Viewer software. All slides were measured and counted blind and in a randomised order. Colitis scoring was carried out following a methodology described previously [38]. Briefly, inflammation, lamina propria cellularity and surface ulceration were blindly graded from 0 (normal) to 3 (most severe colitis) (Table 1 ). The resulting combined score indicates the colitis severity, where 0=normal and 9=severe colitis.

Active inflammation Lamina propria cellularity Surface ulceration

0: Normal 0: Normal 0: No ulceration with intact surface epithelium

1 : Mild crypt distortion and 1 : Mild but unequivocal 1 : Probable erosion with loss and/or mild cryptitis increase in mixed focally stripped epithelium (<5% of crypts infiltrated by inflammatory cells

neutrophils) and/or mild

goblet cell depletion

2: Moderate crypt distortion 2: Moderate increase in 2: Unequivocal erosion and/or moderate cryptitis mixed inflammatory cells

(<50% of crypts infiltrated

by neutrophils) with mild

crypt abscess formation

3: Severe crypt distortion 3: Severe and diffuse 3: Surface ulceration and and loss with widespread increase in mixed granulation tissue formation and diffuse cryptitis (>50% inflammatory cells

of crypts involved) and

diffuse goblet cell depletion

Table 1. Histological scoring system for mucosal inflammation. Immunofluorescence

Antibodies specific to RAGE (Abeam, Cambridge, UK), ADAM 10 (R&D Systems Europe, Abingdon, UK) and cytokeratin (Sigma-Aldrich, Poole, UK) were used to detect RAGE expression, ADAM10 protein expression and epithelial cells. Apoptotic cells were stained using the In Situ Cell Death Detection Kit (Fluorescein; Roche, Burgess Hill, UK) as per the manufacturer's instructions.

5μηι sections of proximal colon were fixed in 4% paraformaldehyde and blocked using the tyramide blocking kit (Perkin Elmer, Cambridge, UK). Sections were then incubated with the primary antibodies for 60 minutes. After washing, samples were incubated with fluorescently labelled secondary antibodies for 60 minutes in the dark. Slides were mounted using Vectashield pro-long anti-fade reagent containing the nuclear counter- stain 4', 6'-diamidino-2-phenylindole (DAPI; Vector Laboratories).

Negative controls for the TUNEL stain were produced by omitting the enzyme solution in the TUNEL reaction mixture. Positive controls were produced by incubating slides with DNase I recombinant for 10 min at room temperature to induce DNA strand breaks, prior to the labelling procedures. Apoptotic cells were counted per crypt and slides were blinded for both mouse strain and time point.

Fluorescence imaging was carried out using an Olympus BX51 microscope using either a 10x/0.30 UPlanFLN objective or a 40x/0.75 UPlanFLN objective with DAPI, FITC and Cy3 filters, coupled with a CoolSNAP EZ camera (Photometries, Tucson, USA). Specific band pass filter sets for DAPI, FITC and Cy3 were used to prevent bleed through from one channel to the next. Images were first captured on MetaVue (Molecular Devices, Sunnyvale, USA) and then corrected using lmageJ64 v1 .44o (National Institute for Health) with 'ImageJ for Microscopy' plugins (McMaster Biophotonics Facility, Hamilton, Canada).

Flow cytometry

Caecum and colon were harvested at autopsy and digested in RPMI-1640 containing 5% L-glutamine, 5% penicillin/streptomycin, 10% foetal bovine serum, collagenase (1 mg/ml; type VIII from Clostridium histolyticum; Sigma-Aldrich) and dispase (0.5mg/ml). Cells were forced through a 70μηι nylon cell strainer (Beckton Dickinson), spun and resuspended in 40% Percoll solution, which was overlaid onto an 80% Percoll solution. Suspended cells were then spun in the Percoll gradient and those at the interface were harvested, counted using an automated cell counter (CasyR 1 ; Scharfe System, Reutlingen Germany) and resuspended at 1 x10 ⁶ cells/ml in FACS buffer (PBS containing 0.5% BSA and 0.1 % NalS ; Sigma-Aldrich) prior to staining and flow cytometry acquisition.

MLN cells were prepared by forcing MLNs through a 70μηι nylon cell strainer (Beckton Dickinson). Cells were counted and adjusted to a concentration of 5x10 ⁶ cells/ml and resuspended in FACS buffer prior to staining and flow cytometry acquisition.

Fc receptors were blocked using anti-CD16/32 2μg ml (eBioscience, Hatfield, UK) and immune cells were stained with antibodies specific to CD1 15, CD1 1 b, CD45, Ly6G, RAGE, CD1 1 c, F4/80, MHCII, CD103 and CD326 (EpCAM). Cells were acquired by flow cytometry using an LSRII (Becton Dickinson). Data was analysed using FlowJo v10 flow cytometry software (Tree Star, Oregon, USA).

Enzyme-linked immunosorbant assay

At autopsy, blood was collected by cardiac puncture and centrifuged at 12000g for 10 min to separate out the serum. The serum supernatant was diluted in ELISA reagent diluent (1 % BSA in PBS, 0.22μηι filtered) prior to ELISA analysis. Faecal proteins were extracted using stool preparation tube (Bioserv diagnostics, Rostock, Germany) according to the manufacturer's protocol. Soluble RAGE (sRAGE) and S100A8 present in serum and faecal samples were assessed using the mouse RAGE DuoSet and mouse S100A8 DuoSet kits (R&D Systems) as per the manufacturer's instructions.

Statistics

Where statistics are quoted, experimental groups were compared using a Student's T test or two-way analysis of variance (ANOVA) test followed by Sidak's post hoc multiple comparisons test where appropriate. P values <0.05 were considered significant. Data are presented as mean ± standard deviation. Statistical analyses were carried out using GraphPad Prism 6.05 for Windows or 6. Of for Macintosh (GraphPad Software, La Jolla, California, USA; www.graphpad.com). Example 2: Results and Discussion

Impaired worm expulsion and increased colitis in AKR mice compared to BALB/c mice

Following challenge with Trichuris muris, we observed no reduction in body weight in either AKR or BALB/c mice (Figure 1A). However, while BALB/c mice expelled most or all of the worms by 21 days post infection (mean=3.6±4.51 ), AKR mice were unable to expel all worms and remained infected with a significantly higher worm burden (P=0.03; mean=49.25±38.62) (Figure 1 Error! Reference source not found. B). In addition, histological assessment of the colons of both AKR and BALB/c mice showed significant changes in crypt length over time (P=0.0002), and while these changes did not differ significantly between mouse strains, at 21 days post infection crypt length of AKR mice was longer than that seen in BALB/c mice and this remained the case at 31 days post infection (AKR mean=143.82Mm±30.27 and BALB/c mean=1 15.64μηι±1 1 .13) (Figure 1 C). This is consistent with early physiological changes during the onset of colitis, which is characterised by lengthening of the colonic crypts.

Colonic muscle wall thickness was significantly different between AKR and BALB/c mice (P=0.017) but did not differ significantly when assessed by time point post-infection. However, at 31 days post infection there was a trend towards AKR mice having thicker muscle walls .23μΓη±22.01 ) than BALB/c mice

(Figure 1 D), which are characteristic of colonic inflammation. Representative images of haematoxylin and eosin stained proximal colon sections in naive mice and at 31 days post infection are shown in Figure 1 F.

Colitis scoring revealed an increase in histological changes associated with inflammation in both AKR and BALB/c mice after infection (Figure 1 E). These changes included influx of immune cells, presence of immune cells in the submucosa, crypt hyperplasia and goblet cell loss. In agreement with previously shown data, the colitis scores in BALB/c mice peaked at 21 days post infection and had begun to recover by 31 days post infection. As expected, colitis scores in AKR mice rose after infection and peaked at 31 days post infection where the colitis score was significantly greater than that of the BALB/c mice (P=0.022; Figure 1 Error! Reference source not found. E). Collectively these results indicate that, in correlation with previously published research in the AKR/BALB/c infection model [8] the BALB/c mice initiate an acute, resolving inflammation after T. muris challenge whereas AKR mice develop a chronic inflammation phenotype due to a failure to expel worms.

BALB/c mice initiate a diverse immune response to T. muris challenge within 24 hours of infection

To investigate immune cell recruitment during onset of inflammation, colonic lamina propria immune cells were assessed by flow cytometry at 1 , 7, 14, 21 and 31 days post infection as per the gating strategy shown in Figure 2A (for DC and macrophage subtypes) and Figure 3A (for neutrophils, resident monocytes and inflammatory monocytes).

Despite having a lower inflammatory score at the onset of colitis (D31 ), BALB/c mice had a robust early and resolving immune response. At 24 hours post infection, BALB/c mice respond rapidly to T. muris challenge, with higher proportions of CD1 1 c ⁺ CD103 ⁺ CD1 1 b ^" DCs (AKR=0.481 % ±0.109, BALB/c=2.267% ±0.169; P<0.001 ) and F4/80 ⁺ CD1 1 c ^" macrophages (AKR=4.183% ±3.234, BALB/c=13.733% ±1 .106; P<0.001 ) present in colonic lamina propria tissues than observed in AKR mice (Figure 2D and Figure 2F). BALB/c mice also had a slight increase in CD1 1 c ⁺ CD103 ^" CD1 1 b ⁺ DCs at 24 hours post infection (AKR=0.138% ±0.017, BALB/c=0.267 ±0.059; not significant) compared to AKR mice but this was not significant (Figure 2C). BALB/c mice also showed greater increases in the proportions of DCs, macrophages and neutrophils in the mesenteric lymph nodes at 24 hours post infection in comparison to AKR mice (Figure 8).

Neutrophil (Ly6G ⁺ CD1 1 b ⁺ CD1 15 ^" ) numbers were seen to increase in similar proportions in both AKR and BALB/c mice at 24 hours post infection and these cells made up a large proportion of the immune cells present for both mouse strains (Figure 3D). Additionally, there was an increase in neutrophil RAGE expression (as measured by median fluorescence intensity) at 24 hours post infection in both AKR and BALB/c mice (Figure 9). However, while BALB/c mice showed a greater increase in neutrophil RAGE expression at 24 hours post infection, there were no significant differences in neutrophil RAGE expression or early neutrophil migration between mouse strains. At 14 and 21 days post infection BALB/c mice showed greater proportions of numerous immune cell types when compared to AKR mice. These changes included increases in all DC and macrophage cell types: CD1 1 c ⁺ CD103 ⁺ CD1 1 b ⁺ DCs (AKR=0.046% ±0.013, BALB/c=1 .042% ±0.520; PO.001 ; Figure 2B), CD1 1 c ⁺ CD103 ^" CD1 1 b ⁺ DCs (AKR=0.092% ±0.009, BALB/c=5.337% ±5.601 ; P<0.01 ; Figure 2C), CD1 1 c ⁺ CD103 ⁺ CD1 1 b ^" DCs (AKR=0.063% ±0.012, BALB/c=0.278% ±0.106; P<0.05; Figure 2D), CD1 1 c ⁺ macrophages (AKR=0.932% ±0.431 , BALB/c=27.000% ±7.374; P<0.001 ; Figure 2E) and CD1 1 c ^" macrophages (AKR=2.073% ±0.435, BALB/c=1.599% ±1 .002; P<0.01 ; Figure 2F) were all increased in BALB/c mice compared to AKR mice at 14 days post infection.

At 21 days post infection, the DC response in BALB/c mice was reduced in magnitude compared with D1 and 14 post infection, although CD1 1 c ⁺ CD103 ⁺ CD1 1 b ⁺ DCs still remain moderately increased when compared to AKR mice (AKR=0.046% ±0.034, BALB/c=0.617% ±0.161 ; not significant; Figure 2B). However, CD1 1 c ⁺ macrophages (AKR=1.040% ±0.852, BALB/c=23.433% ±2.940; PO.001 ; Figure 2E) and CD1 1 c ^" macrophages (AKR=3.930% ±1 .105, BALB/c=16.500% ±0.557; PO.001 ; Figure 2F) were present in greater numbers in BALB/c mice compared to AKR mice at 21 days post infection.

Neutrophils, resident monocytes and inflammatory monocytes were all observed in significantly greater numbers in BALB/c mice than AKR mice at both D14 and D21 post infection. Neutrophils in particular made up a very large proportion of the CD45 ⁺ cells present in BALB/c mice (D14 AKR=3.673% ±0.937, BALB/c=22.167% ±5.534; D21 AKR=5.123% ±1 .884, BALB/c=27.367% ±0.231 ; Figure 3D). Monocyte numbers were also significantly lower in AKR mice compared to BALB/c mice at D14 and D21 post infection, with resident monocytes (D14 AKR=0.841 % ±0.347, BALB/c=5.590 ±1.274; D21 AKR=0.979% ±0.222, BALB/c=4.867% ±0.474; Figure 3B) and inflammatory monocytes (D14 AKR=0.918% ±0.602, BALB/c=29.700% ±12.569; D21 AKR=1.675% ±1 .168, BALB/c=32.767% ±2.730; Figure 3C) showing significantly greater increases (PO.001 ) in BALB/c mice compared to AKR mice at both time points. Delayed immune response in AKR mice occurs when BALB/c immune response resolves

At 31 days post infection, BALB/c mice showed a reduction in immune cells whereas AKR mice showed increased proportions of almost all immune cells in the lamina propria of the colon. Dendritic cells of the CD1 1 c ⁺ CD103 ⁺ CD1 1 b ⁺ (AKR=2.303% ±0.753, BALB/c=0.214% ±0.092; PO.001 ; Figure 2B) and CD1 1 c ⁺ CD103 ^" CD1 1 b ⁺ subtypes (AKR=2.237% ±0.894, BALB/c=0.454% ±0.074; not significant; Figure 2C) were increased in AKR mice compared to BALB/c mice at D31 post infection.

Proportions of macrophages were also greater in AKR mice than BALB/c mice at D31 , where the BALB/c mice had reduced proportions of macrophages when compared to D21 . CD1 1 c ⁺ macrophages (AKR=30.033% ±4.994, BALB/c=4.330% ±3.427; PO.001 ; Figure 2E) and CD1 1 c ^" macrophages (AKR=9.990% ±1 .518, BALB/c=2.700% ±0.289; P<0.001 ; Figure 2F) were proportionally higher in AKR mice than BALB/c mice.

Neutrophils (AKR=21 .167% ±4.903, BALB/c=2.933% ±1.534; PO.001 ; Figure 3D), resident monocytes (AKR=3.997% ±0.316, BALB/c=1.206% ±0.572; P .001 ; Figure 3B) and inflammatory monocytes (AKR=15.133% ±4.620, BALB/c=1 .735% ±1 .796; PO.01 ; Figure 3C) were all significantly increased in AKR mice at 31 days post infection, whereas the proportions of these cell types returned to baseline levels in BALB/c mice.

Colonic epithelial cells express RAGE dynamically during Trichuris muris challenge in AKR and BALB/c mice

To account for the increase in RAGE mRNA in the AKR mice observed in our microarray data (data not shown), we expected to see a greater proportion of RAGE-expressing immune cell infiltration (e.g. neutrophils) during early infection in AKR mice when compared to BALB/c mice. However we saw no significant difference in the degree of cellular influx between the two mouse strains that would account for the post- transcriptional changes observed in our previous study (Figure 2). Therefore, we investigated whether the colonic epithelial cells were expressing extracellular RAGE via immunohistochemistry and flow cytometry. Given the large number of epithelial cells present in the colon, even a relatively small transcriptional upregulation of RAGE could result in large amounts of RAGE mRNA present. Also, as T. muris burrows into the epithelium within 24 hours of infection, these cells are very rapidly exposed to mechanical damage from burrowing worms and are therefore the cells that will first respond to the infection.

Colonic epithelial cells were identified as CD326 ⁺ cells via flow cytometry (Figure 10). Over 90% of epithelial cells expressed RAGE (data not shown) and they fell into two distinct groups, expressing either low (RAGE ¹⁰ ) or high (RAGE ^hi ) levels of RAGE. The proportion of RAGE ¹⁰ to RAGE ^hi cells was similar in both naive AKR (mean RAGE ^hi =58.5%±3.87; mean RAGE'°=41 .5%±3.87) and BALB/c mice (mean RAGE ^hi =54.7%±2.35; mean RAGE'°=45.3%±2.35) (Figure 4A and Figure 4B). However, there were significant changes to the proportion of RAGE' ⁰ to RAGE ^hi observed 2 days after T. muris infection (P<0.0001 ), where a decrease in the proportion of RAGE ^hi epithelial cells was observed in both AKR (mean RAGE ^hi =43.5%±2.44; mean RAGE'°=56.5%±2.44) and BALB/c mice (mean RAGE ^hi =45.77%±3.37; mean RAGE'°=54.23%±3.37) (Figure 4A and Figure 4B).

Median fluorescence intensity of RAGE in RAGE ^hi and RAGE' ⁰ CD326 ⁺ epithelial cells was measured in order to determine changes within these populations during T. muris infection (Figure 4C and Figure 4D). Whilst a drop in median fluorescence intensity was observed in the RAGE ^hi population 2 days after infection in both AKR and BALB/c mice this data was quite variable between samples and no significant changes occurred (Figure 4C). However, median fluorescence intensity in the RAGE' ⁰ epithelial cells demonstrated significant differences between mouse strain (P=0.014) and between time points (P<0.0001 ) (Figure 4D). Median fluorescence intensity of RAGE' ⁰ expression on CD326 ⁺ epithelial cells increased in both AKR and BALB/c mice during the first 7 days post infection, with AKR mice having consistently higher median fluorescence of RAGE (Figure 4D).

Immunohistochemistry was used to confirm expression of RAGE in epithelial cells (Figure 4E). Akin to the flow data we saw high expression of RAGE throughout the colonic epithelium, with high expression observed at the base of the crypts and minimal fluorescence seen in the lamina propria where immune cells responding to the T. muris stimulus were present; suggesting that epithelial cells express greater amounts of membrane-bound RAGE than much of the immune cell infiltrate and were more likely to be the source of the RAGE mRNA increase observed in our previous study (data not shown). The reduced intensity of RAGE staining at 7 days post infection by immunohistochemistry (Figure 4D) compared to naive mice also appears to correlate with the measured shift in proportions of epithelial cells from RAGE ^hi to RAGE ¹⁰ cells measured by flow cytometry. Faecal and serum sRAGE increased in BALB/c mice in response to T. muris infection

RAGE levels observed by immunohistochemistry and flow cytometry may be affected by internalisation of the extracellular portion of the RAGE receptor after ligand binding [39]. However, RAGE may also be detached from the cell membrane and released as sRAGE via enzymatic shedding, a function of the matrix metalloproteinase ADAM10 [40]. To investigate whether RAGE was being shed as sRAGE and entering the circulation as a decoy receptor we assessed circulating sRAGE levels in serum and sRAGE released in the faeces by ELISA. Serum sRAGE levels in AKR mice remained at zero prior to infection and until 21 days post infection, where sRAGE was detected at 104.61 1 pg/ml ±209.2 (Figure 5A). Conversely, serum sRAGE levels in BALB/c mice went from low levels in naive mice (287.86pg/ml ±438.2) to a peak of 20789.78pg/ml ±14919.04 at 7 days post infection (Figure 5A). Although variable, serum sRAGE was significantly higher in BALB/c mice during infection (P<0.01 ). sRAGE was also detectable in faeces, with BALB/c mice having significantly higher levels at 24 hours post infection (AKR=383.316pg/ml ±200.079, BALB/c=1528.055pg/ml ±665.703; PO.01 ) and at 21 days post infection (AKR=103.422pg/ml ±15.020, BALB/c=2714.939pg/ml ±366.816; Figure 5C). As we saw elevated sRAGE in BALB/c mice we then investigated levels of the RAGE ligand S100A8 (one part of the calprotectin protein) in serum and faeces as an indicator of whether sRAGE might be quenching the effects of circulating RAGE ligands. Serum S100A8 increased during infection in both AKR and BALB/c mice, but no statistical differences were observed between the two strains. At 21 days post infection BALB/c mice had greater levels of circulating S100A8 than AKR mice (AKR=634.692pg/ml ±194.570, BALB/c=1258.151 pg/ml ±497.217; not significant; Figure 5B). Faecal S100A8 remained relatively unchanged in naive and T. muris infected mice of both AKR and BALB/c strains (Figure 5D). As with serum S100A8, faecal S100A8 was raised in BALB/c mice at 21 days post infection (1846.412pg/ml ±945.029) compared to AKR mice (1 131.462pg/ml ±233.996) but this was not significant (Figure 5D). However, S100A8 levels were highly variable in both AKR and BALB/c mice at all time points investigated.

To compare systemic and faecal sRAGE levels with another mouse model of colitis we conducted ELISA assessment of sRAGE in T. mt/r/ ^' s-infected IL-10 ^" ' ^" and C57BL6 controls (Figure 5E and Figure 5F). IL10 ^" ' ^" mice are a well-established model to study colitis and have previously been shown to develop colitis by 21 days post infection with T. muris ([33]). As expected, we observed evidence for the onset of early colitis in IL-10 ^" ' ^" mice at 21 days post infection through significant lengthening of the colonic crypts, goblet cell hyperplasia and influx of neutrophils in comparison to C57BL6 mice (Figure 1 1 ). C57BL6 mice that get a resolving inflammation akin to the BALB/c mice showed a similar range of serum sRAGE levels as BALB/c mice, with sRAGE increasing during the course of infection and dropping again by 31 days post infection. However, both C57BL6 and IL-10 ^" ' ^" had much higher levels of serum sRAGE prior to infection (C57BL6=21031 .30pg/ml ±9804.12; IL-10 ^" ' ^" =28191.47pg/ml ±8054.63; not significant) than that seen in AKR and BALB/c mice (Figure 5E). Serum sRAGE levels were similar in IL-10 ^" ' ^" mice and C57BL6 controls but the amounts present were more variable than those seen in the AKR/BALB/c animal model. However, like the AKR mice, IL-10 ^" ' ^" mice did show a reduction in serum sRAGE at 21 days post-infection (8281 .84pg/ml ±3703.83) compared to C57BL6 mice (20687.66pg/ml ±7640.63), correlating with failure to expel worms and an increase in colitic pathology. However, the difference in serum sRAGE between IL-10 ^" ' ^" and C57BL6 mice at 21 days post infection was not significant.

Faecal sRAGE levels in colitis-susceptible IL-10 ^" ' ^" mice increased slightly at 24 hours post infection (378.31 pg/ml ±131 .39) and reduced to no detectable sRAGE at 21 days post infection (Figure 5F). Faecal sRAGE in C57BL6 mice had a much higher mean at 21 days post infection (546.61 pg/ml ±548.21 ), but this was highly variable (Figure 5F). Overall, there were no significant differences in serum of faecal sRAGE levels between IL-10 ^" ' ^" and C57BL6 mice at any point pre or post infection.

Apoptosis remains unchanged in AKR and BALB/c mice during the first three weeks of T. muris infection

As RAGE is a receptor for DAMPs associated with cell death, we assessed the extent of apoptosis during T. muris infection via TUNEL stain. Also, epithelial cell apoptosis is associated with chronic gut infection with nematodes so this would provide additional evidence for mouse worm burden, although increases in apoptosis are typically observed at around 6 weeks post infection [28]. Apoptosis in the crypts of the proximal colon was quantified in naive mice and at 1 , 7 and 21 days post infection with T. muris (Figure 6). No significant increases in apoptosis were observed during T. muris challenge in either strain.

Membrane-bound ADAM10 is present in both naive and T. muris infected AKR and BALB/c mice

As we had observed differences in the quantities of sRAGE present in AKR and BALB/c mice at various time points post infection we begun a preliminary investigation into the presence of ADAM10 in colonic tissues during the course of infection with T. muris. We stained for the presence of membrane-bound ADAM10 in the epithelium and lamina propria of proximal colon sections in naive mice and at 21 days post infection with T. muris (Figure 7). ADAM10 was detectable throughout the colonic tissues and was present in the epithelium. ADAM10 was also highly expressed by immune cells present in the lamina propria.

Discussion

We have identified a mechanism by which soluble or membrane-bound forms of RAGE are associated with susceptibility and resistance to chronic inflammatory responses during helminth challenge in a mouse model of colitis. We previously described how an upregulation of RAGE mRNA was associated with susceptibility to infection (data not shown), but now we have additionally identified a link between a lack of circulating sRAGE with susceptibility to chronic helminth infection and colitis in AKR mice. Rapid production of sRAGE and a robust, early immune response was associated with helminth expulsion and an acute, resolving inflammation in resistant BALB/c mice. Interestingly, day 21 post infection has previously been shown to be the point at which T. muris infection enters chronicity in IL-10 ^" ' ^" mice [33] and the time point at which resistant mice, such as C57BL6, mice expel worms and begin to resolve inflammation [41]. Our results show a similarly robust colitis phenotype in IL-10 ^" ' ^" mice at 21 days post infection and the inclusion of later time points in the IL-10 ^" ' ^" model may have yielded significant differences in sRAGE and S100A8 levels. However, the focus of this study was to investigate pre- colitic changes as predictors of colitis onset and after 21 days infection with T. muris IL- 10 ^" ' ^" suffer high morbidity and mortality [34], hence the shorter infection time course selected. Levison et al. [42] have shown that Trichuris muris infection in AKR mice causes colitis that correlates phenotypically and transcriptionally with the profile of human CD, suggesting that these novel findings in this animal model may translate succesfully to human IBD patients. Cruickshank et al. previously identified an early influx of DCs as being protective against chronic inflammation in high dose T. muris infections in BALB/c mice and we have replicated these findings here [14]. As expected, AKR mice did not initiate early DC migration and by 31 days post infection showed histological changes to the colon indicative of early colitis. Conversely, DCs numbers rapidly increased in BALB/c mice and by day 31 the parasites were expelled and the mice had begun to resolve the inflammation with minimal adverse affects to colonic tissues. However, cell death levels were similar in AKR and BALB/c mice during the first three weeks of T. muris infection suggesting that the damage and colits was not extreme, even in the susceptible AKR mice. These observations are constent with previous studies showing apoptosis peaking at 42 days post infection in the AKR/BALB/c T. muris infection model [28].

The role of RAGE in facilitating the early DC migration in the T. muris model is unclear. RAGE-mediated leukocyte migration via Mac-1 is likely to play a role in the successful migration of immune cells to the site of injury and has been linked to successful homing of DCs to the lymph nodes [43], but while we observed differences in DC migration we saw no differences between AKR and BALB/c mice in surface expression of RAGE in the epithelium during the course of infection. Our results suggest that although RAGE expression on the epithelial surface may facilitate immune cell migration in both colitis- resistant and colitis-susceptible mice, it is difficult to determine whether RAGE plays a role in the delay in immune cell recruitment in AKR mice.

Neutrophils have been identified as expressing large amounts of RAGE [44]. We carried out flow cytometry to look at RAGE expression in multiple cell types present in and around the lamina propria and crypts of the colon to determine whether neutrophils were responsible for increased RAGE expression in AKR mice. Whilst neutrophils did express RAGE, we observed only modest neutrophil infiltration in the first 24 hours post infection, although this was associated with an increase in neutrophil RAGE expression. However, there was also no difference in early neutrophil migration between colitis-susceptible or colitis-resistant mice. Therefore, it was unclear to what extent neutrophils were responsible for increased RAGE expression following T. muris infection. Following this, we investigated epithelial RAGE expression as a possible route for differential expression of RAGE in the colon.

Our observation of two distinct RAGE-expressing epithelial cell populations (RAGE ^hi and RAGE ¹⁰ ) that changed dynamically during the course of infection is interesting and could account for the RAGE mRNA changes from our previous study (data not shown). Small changes to mRNA expression could result in large changes in the quantities of RAGE protein present in the colon given the vast numbers of epithelial cells present. Further analysis to determine epithelial subtypes that make up the RAGE ^hi and RAGE ¹⁰ populations would provide a greater understanding of their function in the initiation of intestinal immune responses. Previous studies have shown that epithelial cells not only express RAGE, but also upregulate RAGE expression during colonic inflammation [16]. Changes observed in the levels of RAGE expressed by these epithelial cells suggest that RAGE expression is actively involved in the host response to T. muris infection.

Reduction in the amount of RAGE present at the cell membrane immediately following T. muris infection suggests either internahsation of activated RAGE-ligand complexes or ADAMI O-mediated shedding to produce sRAGE [12, 40]. It is unclear from our results whether ADAMI O-mediated shedding is the primary driver of sRAGE increases seen in BALB/c mice. Membrane-bound ADAM10 is clearly present in both naive and T. muris infected AKR and BALB/c mice but the extent to which ADAM10 is physiologically relevent in this model needs more investigation. Additionally, sRAGE may occur as a result of splice variants either as a truncated RAGE molecule or a modified and secreted decoy receptor [45, 46]. As a result, the function of ADAM10 in this model may not be relevant in the onset or prevention of chronic colitis as alternative processes may account for increased sRAGE. However, the large amounts of ADAM10 detected on infiltrating immune cells, linked with more robust, early immune cell infiltration in BALB/c mice following T. muris infection may result in greater amounts of available ADAM10 to facilitate early RAGE shedding in BALBC/c mice. The process by which epithelial cells may undergo RAGE shedding represents an important distinction in the course of gut immunity and homeostasis, and may be an essential component in dictating whether inflammation enters chronicity or promotes tolerance.

We have discovered striking differences in the levels of faecal and systemic sRAGE between colitis-resistant and colitis-susceptible mice, with BALB/c mice rapidly producing sRAGE in response to T. muris infection. We identified a large increase in serum sRAGE in the resistant mice, which could explain their ability to prevent initiation of chronic inflammation in this model. Circulating proinflammatory ligands released by tissue damage may bind to sRAGE and be prevented from activating membrane-bound RAGE [47]. Levels of sRAGE have been shown to be reduced in patients suffering from chronic inflammatory diseases [48]. The reduction in epithelial RAGE expression followed by increases in circulating sRAGE in colitis-resistant mice suggests shedding of RAGE to form sRAGE as a protective process against chronic inflammation. Colitis-susceptible AKR mice show the same reduction in epithelial RAGE expression but did not produce sRAGE in the same quantities, suggesting internalisation and activation of RAGE and subsequent proinflammatory pathways after T. muris infection.

In contrast to our data with sRAGE, the RAGE ligand calprotectin (S100A8) did not provide a consistent signal. As S100A8 is released as a result of heavy inflammation [49], it may be that as colitis progresses in susceptible mice then the signal from S100A8 becomes clearer at time points beyond those covered in this study. A longer time course study would be useful in confirming this and a combination of S100A8 and sRAGE in faeces and serum may be a more reliable marker of uncontrolled and damaging inflammation. Diagnosis of IBD usually involves an assessment of clinical history and physical examination, with endoscopy and histology considered to be the gold standard tools [50]. Accurately assessing disease activity remains dependent on colonoscopy [51 - 53]. The invasiveness of current diagnostic methods is not ideal and recent work has aimed to identify serum or faecal biomarkers that can reliably identify active disease [54, 55]. The number of potential IBD biomarkers is high, there remains a lack of reliable and reproducible biomarkers for use in clinical practice [49]. Efficacy of current therapies is also variable, with risks of sometimes serious side effects, especially infection meaning there is considerable interest for new biomarkers and new therapeutics [56].

Calprotectin has recently entered clinical practice as an IBD biomarker to aid clinical diagnosis non-invasively, but measurements of faecal calprotectin are variable and there is little agreement about what should be considered a normal baseline level in healthy patients [57]. Calprotectin is a product of tissue damage and binds to RAGE to promote inflammation, but this action will be reduced in the presence of the decoy receptor sRAGE. Based on our experimental work we propose that the balance between calprotectin and its decoy receptor sRAGE determines whether calprotectin will promote inflammation. For effective screening and monitoring of patients it is therefore important to measure sRAGE and calprotectin simultaneously. A combination of calprotectin and sRAGE screening would improve the efficacy of calprotectin and provide a more accurate diagnosis and prognosis for patients. Given that sRAGE is also an anti-inflammatory agent, it may also be clinically useful as a therapy for IBD. Drugs to reduce RAGE expression would also confer similar benefits and telmisartan is already available for this purpose. Telmisartan suppresses RAGE expression via peroxisome proliferator-activated receptor-gamma activation [58].

We have now identified that sRAGE is highly expressed in the serum and faeces of animals with resolving pathology but absent or low in actively colitic mice. Analogous to human data [59], calprotectin was more variable. Importantly, high sRAGE predicted recovery regardless of whether calprotectin was also upregulated. Clinically, a cumulative evaluation of calprotectin with sRAGE could improve significantly on the performance of calprotectin alone. The tests could be run in parallel using routine samples taken at clinic visits or home, making the tests cheap, convenient and further reduce the need for invasive tests such as colonoscopy. We propose that our additional test will improve the accuracy of the current approach and reduce the need for invasive tests and associated costs.

Example 3

Optimization of ELISA for human sRAGE samples in stool and serum

Having determined that sRAGE was highly expressed in the serum and faeces of mice with resolving pathology, but absent in low or actively colitic mice, we were interested to determine whether sRAGE was also highly expressed in the serum and faeces of human individuals with IBD. We tested human faecal samples using the same protocol that we had used in our mouse studies. Specifically, for detection of sRAGE in the mouse stool and serum samples we used ThermoScientific plates where we coat the plate with coating antibodies (according to manufacturer's protocol, Mouse RAGE DuoSet ELISA, Cat. No. DY1 179) overnight followed by washing. These plates were further blocked with blocking buffer and washing following manufacturer's protocol. These plates were then ready for the ELISA of the test samples.

We found difficulty in detecting sRAGE in human samples using the same protocol but with human kit (Human RAGE DuoSet ELISA, Cat. No. DY1 145). Therefore was therefore a need to modify the protocol to improve detection of the proteins in the samples.

The basic principle behind binding is that when capture antibodies are coated on the Polystyrene plates they are usually passively adsorped. This process is commonly called coating. Most proteins adsorb to plastic surfaces, probably as a result of hydrophobic interactions between nonpolar protein substructures and the plastic matrix. The interactions are independent of the net charge of the protein, and thus each protein has a different binding constant. The hydrophobicity of the plastic/protein interaction can be exploited to increase binding since most of proteins' hydrophilic residues are at the outside and most of the hydrophobic residues orientated towards the inside. This is where we further optimized checking the characteristics of different plates.

We found that if we use a Maxisorp Nunc plate that have been pre-treated to make it hydrophilic, the plates increase the binding of the capture antibody. This therefore works well with immobilization of the test proteins on the plate and hence leads to test protein detection. This principle is also supported in manufacture's datasheet that describes the anionic content of a non-treated polystyrene microplate surfaces to contain only about 0.5 atom% oxygen, whereas that of high binding microplates is approximately 2.5 atom%.

Moreover, sRAGE in the patient samples may be bound to AGEs (Advanced Glycation Endpoints) that makes them more bulky and may result in desorption or leaching that may also account for non-detectable levels with human samples while the previous protocol was used.

Therefore, it was absolutely necessary to change the protocol and use high binding plates in order to determine the level of sRAGE in human samples. Example 4

We obtained faecal samples from 13 human patients attending gastroenterology clinic for bowel associated symptoms - either to assess if they had IBD (some patients had irritable bowel syndrome and not IBD) or to monitor their IBD status to confirm if they were healthy or relapsed. The patients were routinely screened for calprotectin in their faeces and may have been providing blood samples and then undergoing endoscopy for future investigation. Sample leftover from this clinical work was used in our study, and was analysed to determine levels of calprotectin and sRAGE.

One patient had ischaemia and was excluded from subsequent analysis as being neither IBD nor normal healthy.

Table 3: human patient data. N: normal remission, I: Irritable bowel syndrome R:

remission, D: disease. Note that 0.33pg/ml is the limit of detection for Calprotectin. As we expected, based on our earlier results in mouse, diseased patients presently in remission exhibited elevated calprotectin as compared to currently healthy IBD patients. These patients also exhibited elevated sRAGE levels, meaning that the ratio between faecal levels of Calprotectin and sRAGE levels was also low (below 1 ). For currently healthy patients the ratio was very low (0.3-0.4).

In contrast, for patients experience active disease (diseased and unwell), the ratios were very high. Despite the higher sRAGE in the 2 ^nd patient, the calpro/SRAGE ratio is >2 These data were plotted, as shown in Figure 12. This revealed data clusters, with the normal and IBS (non-inflammatory bowel disease) more tightly grouped than the "remission" and "diseased." Note one patient in "remission group" is far from normal and IBS group which correlates with the patient with creeping calpro and low sRAGE, and who may be at risk of a return to active disease.

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