Classifications

• • 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
  • G01N33/6875—Nucleoproteins
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the present invention relates to materials and methods for detecting and diagnosing chronic inflammatory bowel diseases (IBD "Inflammatory Bowel Disease") in humans.
• IBD chronic inflammatory bowel diseases
• the invention also comprises the colorimetric kit for implementing such a method .
• HMGB1 High-mobility group box 1
• DAMP molecules prototype DAMP molecules prototype (Damage Associated Molecular Patterns) able to respond to stimuli from tissue damage by inducing an inflammatory response (1).
• HMGB1 is actively secreted by macrophages (2) and enterocytes (3) following pro-inflammatory stimuli such as LPS, T F ⁇ , IL- ⁇ , IL-6, and IL-8 (4) and it is released by necrotic cells, but not apoptotic cells (5) .
• HMGB1 as secreted into the extracellular space, forms highly inflammatory complexes with different molecules: single-stranded DNA, LPS, IL- ⁇ and nucleosomes, which interacting with their respective receptors, such as TLR9, TLR4, IL-1R and TLR2 , activate the innate immunity.
• HMGB1 can bind, without forming complexes, the receptor for glycation end products RAGE (Receptor for Advanced Glucation End products) (6) .
• RAGE Receptor for Advanced Glucation End products
• Extracellular HMGB1 induces the production of inflammatory mediators (4) and may play an important role in the pathogenesis of autoimmune or inflammatory diseases, including rheumatoid arthritis (7), systemic lupus erythematosus (8) and polymyositis (9).
• the invention described in U.S. patent No. 6,303,321 relates to a pharmaceutical composition for the treatment of sepsis.
• the pharmaceutical composition comprises as active substance an effective amount of antagonist or inhibitor of H GB1.
• antagonists of HMGB1 antibodies binding to the HMGB1 protein, antisense sequences of the HMGBl-coding gene, and antagonists of the HMGB1 receptor are used.
• subject of the invention is also a method for treating sepsis comprising administering an effective amount of an antagonist of HMGB1.
• the invention also provides a diagnostic and prognostic method for monitoring the severity of the patient condition and predicting the likely clinical course of sepsis and related conditions for a patient with shock-like symptoms or showing associated symptoms.
• the diagnostic and prognostic method includes measuring the concentration of HMGB1 protein in a sample, particularly in serum or whole blood, comparing it with a standard concentration of HMGB1. Higher levels of HMGB1 are indicative of poor prognosis or likely occurrence of toxic reactions.
• the diagnostic method can also be applied to other tissues or fluids compartments such as cerebrospinal fluid or urine .
• HMGB1 and gastrointestinal tract state of the art
• Signs of stress, tissue damage or microbial antigens in the intestinal mucosa activate cells involved in the innate immune response, such as macrophages and dendritic cells, triggering the inflammatory response.
• HMGB1 The presence of HMGB1, released in the extracellular matrix following inflammatory stimuli, appears to significantly affect the intestinal barrier function by altering the permeability of the epithelial intestinal cells and leading to an increased entry of microbial antigens.
• in vitro and in vivo studies have correlated the presence of HMGB1 secreted by immunostimulated enterocytes, or by other immune cells, and intestinal barrier dysfunctions (10-15).
• HMGB1 due to the inflammatory cytokines release, HMGB1 is also potentially involved in the colon inflammation, as demonstrated in animal models (16, 17), and in necrotizing forms of colitis (18,19).
• HMGB1 The decrease of secreted HMGB1, by anti-HMGBl molecules, appears to correlate with an improvement both in the damage of the intestinal barrier and in the mucosal inflammation (11, 13, 14, 16, 19, 20).
• HMGB1 protein in tissue samples obtained from patients has been already used as a diagnostic and prognostic marker of bowel cancer, particularly of colon and rectum cancers as described in U.S. patent application 2006/0188883.
• the cancer disease is a very different condition from inflammatory bowel disease.
• the object of the patent application is applicable only in the use of biological tissues and no reference is provided in connection with the use of fecal material.
• mice model used in this study is also employing a genetically modified strain of mice with the gene coding for IL-10 deleted, an antiinflammatory cytokine, causing colitis in the mice. This is a rather unlike condition compared to the human disease wherein much more complex contributory factors determine the onset of the disease.
• the environmental pressure in terms of lifestyle (diet, smoking, stress), as well as the use of drugs or the exposure to harmful environmental agents, differs from person to person and also plays a role in the onset of the disease, the composition of the intestinal flora is different from individual to individual as well.
• the mouse model in standard conditions, does not suffer at all, or at least much less, the environmental pressure, also the microbiological profile is much less variable among individuals, receiving the same diet.
• HMGB1 a new antigen of ANCA (Anti- neutrophil cytoplasmic antibody) , as observed in the serum of patients with ulcerative colitis (22).
• ANCA Anti- neutrophil cytoplasmic antibody
• Biological markers represent a non-invasive method to objectively measure the inflammation and may play a primary or secondary role in the evaluation of some diseases (23) , including inflammatory bowel disease (IBD, "Inflammatory Bowel Disease”) .
• IBD inflammatory bowel disease
• Such markers can be identified as serological or fecal and be used to diagnose a specific process, to classify the disease into different subtypes, to evaluate its activity, evolution and prognosis, to predict the response to a therapeutic treatment or a recurrence (24 ) .
• IBDs erythrocyte sedimentation rate
• CRP C-reactive protein
• ANCA anti-neutrophil cytoplasmic antibodies
• ASCA anti-Saccharomyces cerevisiae antibodies
• fecal markers show greater specificity for the diagnosis of gastrointestinal diseases, such as IBD, because their levels do not increase in diseases not involving the digestive system (25, 26) ; furthermore they have the advantage of not necessarily requiring the endoscopic analysis to assess the disease activity (26, 27).
• Lactoferrin and calprotectin are at the time the most used fecal markers for bowel inflammation (24, 25, 28, 29). In fact, the presence of these proteins in the stool is a reasonably accurate measure of disease activity, recurrence prediction, and identification of high-risk groups among patients with severe colitis and monitoring on the effects of medical therapies.
• HMGB1 Given the well-known ability of HMGB1 to release signals directed to the recruitment of cell inflammatory repertoire and to activate the immune response due to exogenous or endogenous stimuli, the inventors have proposed to investigate the possible involvement of this protein in the pathogenesis of human inflammatory bowel diseases, more particularly CD and UC.
• the CD is characterized by transmural inflammation that can affect any section of the digestive tract, from mouth to anus. Typically there is the involvement of more sections in a discontinuous way.
• the inflammation involves the entire wall of the affected section and often spreads to nearby mesentery and lymph nodes. Most frequently it involves the terminal ileum and colon.
• UC ulcerative colitis
• the involvement of the rectum is constant and can be accompanied by the involvement of a variable upstream section of the colon .
• HMGBl protein were subjected to densitometric analysis performed by using the ImageQuant software (GE Healthcare Life Sciences, Uppsala, Sweden) ; thus it was possible to assign a numerical value to range of HMGBl levels.
• the diagnosis of IBD in patients was performed according to endoscopic and histological criteria widely recognized and shared (30) .
• the activity of the CD was measured by the "pediatric Crohn's disease activity index" (PCDA) , a measure based on clinical and laboratory parameters (31) : the disease is considered inactive if the value is ⁇ 10, mild to moderate if the value is > 10-30 and severe if the value is > 30.
• PCDA pediatric Crohn's disease activity index
• the activity of the UC was ranked according to the "pediatric ulcerative colitis activity index" (PUCAI) (32): the latter is a multi-parametric method recently validated, non-invasive, according to which the disease is considered in remission (score lower than 10), mild disease (score between 10 and 34), moderate (score between 35 and 64) and severe disease (score between 65 and 85) .
• PUCAI pediatric ulcerative colitis activity index
• the endoscopic score was determined using the SES- CD (33) and Matts' score (34) for ulcerative colitis.
• SES-CD the bowel was divided into five segments (ileum, left colon, transverse colon, right colon, rectum) , and to the degree of disease activity in each segment was assigned a value ranging from 0 to 12 (total value range: 0-60).
• Matts' score the intestine was divided into six segments (blind, ascending colon, transverse colon, descending colon, sigmoid colon, rectum) , and in each segment a value that ranges from 1 to 4 (total value range: 6-24) was assigned to the degree of disease activity . Based on these indices in the enrolled patients in the study the disease has been found severe in 13 IBD (8 CD and 5 UC) , mild to moderate in 11 patients (3 CD and 8 UC) and inactive in 16 (8 CD and 8 UC) (Table 1) .
• Table 1 lists the patients, divided according to disease type and severity, enrolled in the clinical trial.
• Fecal samples were obtained from pediatric patients affected by IBD (Table 1), with varying degrees of severity of illness, and from healthy controls, recruited at the Department of Pediatrics, Pediatric Gastroenterology and Hepatology Unit, University of Rome "La Sapienza", directed by Professor Salvatore Cucchiara.
• Each sample (equivalent to the contents of the spoon inside a standard container for feces) was removed with a sterile tip from the container, put in a 1.5 ml eppendorf tube and weighed using a digital scale.
• the sample was resuspended in extraction buffer (phosphate buffered saline solution PBS pH 7.2) marketed by the company ScheBo Biotech containing detergent and sodium azide, to obtain a final concentration of 500 mg/ml.
• extraction buffer phosphate buffered saline solution PBS pH 7.2
• the sample was vortexed for one minute at room temperature (RT) and then placed in an orbital shaker at room temperature for about one hour. Following centrifugation for 5 minutes at 10000 rpm at 4 °C, the supernatant, defined extracted fecal, was collected and the protein concentration was measured by Bradford assay (Biolabs) .
• the sample obtained can be immediately analyzed by Western blot assay or stored at -80 °C and subsequently analyzed.
• Non-specific sites on the filter were blocked by incubation for 1 hour at room temperature with Blocking Buffer (0.02M Tris-Cl pH 7.6, 0.137M NaCl, 5% fat-free milk powder) , then the filter was incubated for 16 hours at 4 °C with anti-HMGBl polyclonal antibody (Cat. No. H9593, Sigma), diluted 1:1000 in Antibody Buffer (0.02M Tris-Cl pH 7.6, 0.137M NaCl, 3% fat-free milk powder) or with anti-H GBl monoclonal antibody (Cat. No. MAB 1690, R&D Systems, Minneapolis, USA) diluted 1:500 in Antibody Buffer.
• Blocking Buffer 0.02M Tris-Cl pH 7.6, 0.137M NaCl, 5% fat-free milk powder
• Figure 1 shows the HMGBl protein in stool samples, detected by Western blot analysis using anti-HMGBl monoclonal antibody of R&D System.
• panel A shows the result of Western blot
• panel B shows a graph of the densitometric values of the highlighted bands by Western blot in patients
• panel C shows a graph of the densitometric values of the highlighted bands by Western blot in patients divided in groups according to the severity of the disease.
• the densitometric analysis of the highlighted bands by Western blot has allowed to obtain a numerical value related to the level of HMGBl present in the fecal sample.
• ADU Arbitrary Densitometric Units
• subjects with CD show a numeric value ranging from 20000 to 380000 ADU, average value 190000 ADU
• all patients affected by UC show a numeric value related to the level of HMGBl present in analyzed fecal samples ranging from 6000 to 280000 ADU, with average value equal to 120000 ADU (Fig. 1-B) .
• the asterisks refer to statistical significance evaluated through the Mann- Whitney statistical test: where *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0, 001.
• HMGBl protein expression is significantly increased in the feces of IBD patients compared to controls, where is not detectable (p ⁇ 0.001) ( Figure 1).
• This result indicates that the presence of HMGBl protein detected in the feces is a marker of inflammation in the human gut.
• the presence of HMGBl protein has been also detected in the feces of 16 patients with disease defined inactive on the basis of the PCDAI and PUCAI indices; however, according to the assessment of the endoscopic score, these patients had some extent of intestinal inflammation, in agreement with the detectio ' n of HMGBl in their feces.
• the medians of the SES-CD and of the Matts' scores were respectively 23.0 (range values: 14-34) and 18.0 (range of values: 8-24), and in patients with inactive CD and UC the medians of the SES-CD and of the Matts' scores were respectively 7.5 (range of values: 0-15) and 11.5 (range of values: 6-18).
• These indices show that even in the so defined inactive patients according to the PCDAI and PUCAI indices there is a pitch of intestinal inflammation, and that HMGBl may also provide an indication of the inactive state of the disease and therefore regarded as very sensitive marker for such inactive inflammatory conditions.
• HMGBl protein levels detected in the feces were compared with those of fecal calprotectin, which is currently considered a biomarker of choice and reliable for the diagnosis of intestinal inflammation by ELISA (29, 35) .
• the results of this analysis are shown in Figure 2, where the asterisks indicate statistical significance according to the statistical test of Mann-Whitney, where *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0,001. Both proteins resulted to be strongly increased in the feces of patients (p ⁇ 0.001) compared with healthy controls (Figure 1-B, Figure 2- A) .
• H GB1 is significantly elevated in all 16 patients with so defined inactive disease according to the indices of PCDAI and PUCAI, despite they still show a degree of inflammation in .accordance with the endoscopic score, while calprotectin was elevated in only two of them.
• HMGB1 is a very sensitive marker of persistent intestinal inflammation in patients with clinically quiescent disease, as revealed by classical indices of disease activity. The latter, however, being a mixture of the clinical and laboratory features, are not always correlated with intestinal inflammation detected by endoscopy.
• HMGBl protein as a potential molecular prognostic parameter for recurrence in patients with disease in apparent remission.
• patients with IBD show a significant increase of HMGBl in the feces compared to healthy controls.
• HMGBl levels and severity of the disease there is a direct correlation between HMGBl levels and severity of the disease.
• HMGBl beside being a good marker of inflammation, seems also to provide a good indicator of the severity of the disease and, therefore, could be used as a marker of response to therapy.
• HMGBl as a biological marker and the method to detect its presence in stool samples is a significant step forward in order to diagnose in a safe and non-invasive manner the presence and the level of a human intestinal inflammation, avoiding the often repeated imaging studies that are highly traumatic for most patients.
• protein expression levels can be used as prognostic marker of recurrence of disease and as a marker of response to therapy.
• the choice to provide an ELISA protocol, in addition to the Western blot, is dictated by the fact that this technique is simple and moreover allows to better quantify the reaction, in fact, the colour intensity of the ELISA plate is proportional to the number of antigen-antibody complexes (primary) and thus to the concentration of antigen (capable of binding the primary antibody) in the analyzed sample.

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• 2010
  • 2010-08-05 IT ITRM2010A000442A patent/IT1406051B1/it active
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