WO2017144478A1 - Biomarqueur d'inflammation - Google Patents

Biomarqueur d'inflammation Download PDF

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WO2017144478A1
WO2017144478A1 PCT/EP2017/053956 EP2017053956W WO2017144478A1 WO 2017144478 A1 WO2017144478 A1 WO 2017144478A1 EP 2017053956 W EP2017053956 W EP 2017053956W WO 2017144478 A1 WO2017144478 A1 WO 2017144478A1
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srage
patient
calprotectin
level
inflammation
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Sheena CRUICKSHANK
John Thomas MCLAUGHLIN
Andrew Michael Brass
Michael Lyell BRAMHALL
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University of Manchester
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University of Manchester
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

Definitions

  • the present invention relates to biomarkers of inflammation and particularly, although not exclusively, to biomarkers of inflammatory bowel disease.
  • IBD Inflammatory bowel diseases
  • CD Crohn's disease
  • UC ulcerative colitis
  • 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].
  • anti-tumour necrosis factor 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).
  • Faecal markers such as calprotectin and lactoferrin
  • Faecal markers 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.
  • 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.
  • 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.
  • NICE National Institute for Health and Care Excellence
  • 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.
  • 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.
  • RAGE cell surface damage receptor
  • sRAGE soluble form of RAGE
  • 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.
  • sRAGE has application as a biomarker of IBD.
  • 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).
  • IBD inflammatory bowel disease
  • Methods disclosed herein involve the detection and/or quantification of sRAGE.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ratio of sRAGE and calprotectin levels may be a powerful biomarker of IBD.
  • the combined analysis of sRAGE and calprotectin may give a good indication of IBD relapse.
  • an individual exhibiting an elevated level of Calprotectin and an elevated level of sRAGE may be determined to have acute resolving inflammation.
  • 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.
  • the inflammatory bowel disease is Crohn's disease. In some methods disclosed herein, the inflammatory bowel disease is ulcerative colitis.
  • 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.
  • the method is a computer implemented method.
  • 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.
  • patients who have been diagnosed with inflammatory bowel disease are monitored by the methods of the invention.
  • 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.
  • a sample is obtained from the patient and transferred to a separate testing facility for biomarker analysis.
  • the methods may be performed by the subject, for example using a home testing kit.
  • 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 ( ⁇ ).
  • 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.
  • the method involves comparing the level of the biomarker to the baseline level to determine whether it is elevated or unchanged.
  • the biomarker is sRAGE.
  • the biomarker is the ratio of sRAGE to calprotectin.
  • 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.
  • a classifier comprising the level of sRAGE in a sample, preferably a faecal sample, and optionally the level of calprotectin or S1000A12.
  • kits comprising means for preparation of a faecal sample and means for detecting sRAGE.
  • 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.
  • a method of distinguishing between acute resolving inflammation and chronic inflammation in an inflammatory bowel disease patient comprising:
  • 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.
  • a method of treating IBD in a patient comprising:
  • the anti-IBD therapy may be an antispasmodic, laxative or anti mobility agent, probiotic aminosalicylate, thiopurine, calcineurin inhibitor or anti-TNF therapy.
  • the effective amount of anti-IBD therapy may be a high dose.
  • the present invention includes biomarkers, methods and kits for distinguishing between acute resolving inflammation and chronic inflammation in inflammatory bowel disease patients.
  • the present invention relates to the use of sRAGE as a biomarker, and particularly faecal sRAGE.
  • 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.
  • IBD ulcerative colitis
  • Crohn's disease Crohn's Disease
  • UC Ulcerative Colitis
  • 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 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.
  • 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.
  • 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
  • TSO Trichuris suis ova
  • 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 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.
  • STH soil transmitted helminthiasis
  • Roundworm Ascaris lumbricoides
  • Whipworm Trichuris trichiura
  • Hookworm Ancyclostoma duodenale or Necator americanus
  • Schistosomiasis including parasitism by Schistosoma spp., such as Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi, Schistosoma guineenesis or Schistosoma haemotobium).
  • Lymphatic filariasis including parasitism by Wuchereria bancrofti, Brugia malayi and Brugia timori
  • onchocerciasis Onchocerca volvulus
  • 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 (MectizanTM), 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.
  • RAGE 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- ⁇ 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- ⁇ peptide, ⁇ 2 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 ].
  • DAMPs damage-associated molecular patterns
  • 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].
  • Amyloid/beta sheet fibrils Amyloid- ⁇ peptide
  • Complement receptor Mac-1 (CD1 1 b/CD18, integrin aw integrin fcj
  • RAGE acting as a ligand for CD1 1 b 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.
  • 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].
  • 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.
  • 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.
  • the methods disclosed herein involve the detection of esRAGE (endogenous secretory RAGE).
  • esRAGE endogenous secretory RAGE
  • esRAGE is a splice variant of RAGE that can be secreted.
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • 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.
  • a high ratio may be indicative of acute resolving inflammation, and a low ratio may be indicative of chronic inflammation.
  • S100A12 is a S100 protein that is similar to calprotectin. It is sometimes referred to as Calgranulin C.
  • 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%.
  • 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.
  • 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).
  • S100A12 CRP level
  • ESR platelet count
  • white blood cell count a marker of inflammation of all the markers employed in the study.
  • 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.
  • 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 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.
  • the subject has inflammatory bowel disease
  • the subject may be an inflammatory bowel disease patient or otherwise be an individual who has previously received a diagnosis of IBD.
  • the subject or patient is experiencing symptoms of disease.
  • the subject or patient is in remission.
  • the subject or patient is normal, indicating that they have a previous diagnosis of IBD, but are in an extended period of remission.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ELISA Enzyme-linked immunosorbent assay
  • lateral flow test latex agglutination
  • latex agglutination other forms of immunochromatography
  • western blot and/or magnetic immunoassay.
  • Protein may also be detected or quantified using mass spectrometry.
  • mass spectrometry using electrospray ionization (ESI) or matrix-assisted laser desorption/ionisation (MALDI).
  • ESI electrospray ionization
  • MALDI matrix-assisted laser desorption/ionisation
  • Other methods of protein quantification include spectroscopy based methods. Such methods may involve colorimetric assays or spectrophotometric assays.
  • 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.
  • 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.
  • an aptamer is used instead of, or together with, the antibody.
  • Immunoassays include immunohistochemistry, ELISA, immunoblotting and flow cytometry.
  • 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.
  • 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.
  • the solid support is a multiwall or microtiter plate.
  • 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.
  • 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.
  • the method involves detection and/or quantification of the biomarker using qPCR (quantitative PCR).
  • qPCR quantitative PCR
  • 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.
  • 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.
  • 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.
  • 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.
  • the target is detected by immunoblotting, or western blotting.
  • 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 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.
  • 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.
  • FACS Fluorescence Activated Cell Sorting
  • Immunohistochemistry 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.
  • a sample is formalin fixed and paraffin embedded (FFPE).
  • 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.
  • 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.
  • the sample is incubated with the antibody for 32 minutes at 37°C.
  • the samples may additionally be counter-stained to facilitate analysis.
  • 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 VentanaTM BenchMark ULTRATM. Alternatively, slide staining may be performed using a VentanaTM BenchMark XTTM, VentanaTM BenchMark GXTM, Dako OmnisTM, Dako Autostainerl_ink48TM, LeicaTM BOND RXTM, LeicaTM BOND-INTM or LeicaTM BOND MAXTM Following incubation of the sample with the labelled antibody, they may be analysed using a microscope.
  • 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.
  • a target antigen generally has numerous binding sites, also called epitopes, recognized by Complementarity Determining Regions (CDRs) on multiple antibodies.
  • CDRs Complementarity Determining Regions
  • 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.
  • monoclonal antibody 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.
  • 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).
  • the method involves comparing biomarker levels in a sample from a patient with biomarker levels in one or more control samples.
  • control samples and subjects will be appreciated by those of skill in the art.
  • the control is a sample previously obtained from the same patient, at an earlier point in time.
  • 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.
  • 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.
  • the sample in a faecal or stool sample in particularly preferred methods described herein.
  • sRAGE and optionally calprotectin is detected and/or quantified in a faecal sample.
  • both sRAGE and calprotectin are detected and/or quantified in the same sample.
  • both the sRAGE and calprotectin levels are determined in a faecal sample.
  • 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.
  • the sample may be taken from any tissue or bodily fluid.
  • 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, prognosing and prognose refer to estimating the risk of future outcomes in an individual based on their clinical and non-clinical characteristics.
  • 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.
  • 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.
  • a poor prognosis may indicate chronic inflammation, or that inflammation is occurring, and may indicate that symptoms of disease will return or persist.
  • 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.
  • 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.
  • a level of 50 ug/g or more may be indicative of IBD relapse.
  • 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.
  • Methods disclosed herein may involve the detection of elevated levels of biomarkers.
  • elevated biomarker levels are those which are statistically significantly increase relative to control levels.
  • elevated expression is used interchangeably with increased expression, high expression or high level.
  • the methods disclosed herein involve the detection of the biomarkers sRAGE and calprotectin.
  • 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
  • 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.
  • sample preparation buffers for example, sample preparation buffers.
  • the kit may include reagents for preparation of a faecal sample.
  • the kit may include one or more controls.
  • 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.
  • the lateral flow device provides a qualitative indication of the presence of the biomarker.
  • 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.
  • 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 ⁇ 8 ⁇ ) in naive mice and at 1 , 21 and 31 days post infection.
  • D Mean muscle wall thickness (in ⁇ ) 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.
  • FIG. 1 Dendritic cell and macrophage infiltration in the colonic lamina intestinal 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 +/” ).
  • FIG. 3 Monocyte and neutrophil infiltration in the lamina intestinal 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.
  • 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.
  • 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.
  • Figure 10. Flow cytometry gating strategy for epithelial cell identification (CD326 + ) and RAGE expression of epithelial subpopulations from the colonic lamina limbal of naive and Trichuris muris infected male AKR and BALB/c mice (aged 6-8 weeks).
  • FIG. 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 ⁇ ).
  • B Mean goblet cells (per cryptiSD).
  • 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
  • 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.
  • SCID Severe combined immunodeficient 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.
  • 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].
  • 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. 5 ⁇ sections were then dewaxed, rehydrated and stained using a standard Haematoxylin & Eosin (H&E) stain or Alcian blue/Periodic Acid Schiff's stain [37].
  • NAF neutral buffered formalin
  • PBS neutral buffered formalin
  • H&E Haematoxylin & Eosin
  • Alcian blue/Periodic Acid Schiff's stain [37].
  • Moderate crypt distortion 2 Moderate increase in 2: Unequivocal erosion and/or moderate cryptitis mixed inflammatory cells
  • 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
  • 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.
  • 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, Arlington, 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).
  • 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 70 ⁇ 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.
  • CasyR 1 Scharfe System, Reutlingen Germany
  • FACS buffer PBS containing 0.5% BSA and 0.1 % NalS ; Sigma-Aldrich
  • MLN cells were prepared by forcing MLNs through a 70 ⁇ 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 2 ⁇ g 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).
  • 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.
  • the colitis scores in BALB/c mice peaked at 21 days post infection and had begun to recover by 31 days post infection.
  • mice initiate a diverse immune response to T. muris challenge within 24 hours of infection
  • colonic lamina limbal 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).
  • BALB/c mice had a robust early and resolving immune response.
  • 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.
  • 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 .
  • Colonic epithelial cells express RAGE dynamically during Trichuris muris challenge in AKR and BALB/c mice
  • 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).
  • 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 ).
  • Faecal S100A8 remained relatively unchanged in naive and T. muris infected mice of both AKR and BALB/c strains (Figure 5D).
  • 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).
  • S100A8 levels were highly variable in both AKR and BALB/c mice at all time points investigated.
  • 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.
  • 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.
  • 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).
  • Figure 5F The majority, 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
  • RAGE is a receptor for DAMPs associated with cell death
  • 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
  • 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.
  • 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.
  • sRAGE may occur as a result of splice variants either as a truncated RAGE molecule or a modified and secreted decoy receptor [45, 46].
  • 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.
  • 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.
  • 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.
  • calprotectin and sRAGE screening would improve the efficacy of calprotectin and provide a more accurate diagnosis and prognosis for patients.
  • 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].
  • 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.
  • AGEs Advanced Glycation Endpoints

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Abstract

La présente invention concerne des biomarqueurs d'inflammation, notamment, mais non exclusivement, des biomarqueurs de maladie intestinale inflammatoire. L'invention concerne également des méthodes utiles à la gestion clinique de patients atteints de maladie intestinale inflammatoire (IBD) et, particulièrement, des méthodes permettant de distinguer une inflammation de résolution aiguë d'une inflammation chronique, sur la base de la présence, de l'absence ou de l'élévation d'un biomarqueur.
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CN114288386A (zh) * 2022-01-25 2022-04-08 华中科技大学同济医学院附属协和医院 Del-1作为炎症性肠病新的生物标志物及治疗药物应用

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CN114288386A (zh) * 2022-01-25 2022-04-08 华中科技大学同济医学院附属协和医院 Del-1作为炎症性肠病新的生物标志物及治疗药物应用
CN114288386B (zh) * 2022-01-25 2023-12-12 华中科技大学同济医学院附属协和医院 Del-1作为炎症性肠病新的生物标志物及治疗药物应用

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