KR20130136964A - Use of hmgb1 as a biological marker of bowel inflammatory conditions, non-invasive method for its detection in fecal samples and kit thereof - Google Patents

Abstract

The present invention includes an assay protocol for detecting the presence of HMGB1 in feces via Western blot assays or ELISA assays using appropriate antigen-antibodies, the presence of HMGB1 in fecal extracts and chronic inflammatory bowel disease, especially Crohn In the pathogenesis of disease (CD) and ulcerative colitis (UC), the involvement of the protein provides a noninvasive method for measuring human intestinal inflammatory conditions.
The present invention also includes colorimetric kits for practicing this method.

Description

Use of HMGB1 as a Biological Marker of Bowel Inflammatory Conditions, Non-Invasive Method for Its Detection in Fecal Samples and Kit Thereof}

The present invention relates to materials and methods for detecting and diagnosing chronic inflammatory bowel disease (IBD) in humans. In particular, the present invention relates to human intestinal inflammation through the presence of HMGB1 in fecal extract and through the involvement of the protein in the pathogenesis of chronic inflammatory bowel disease, particularly Crohn's disease (CD) and ulcerative colitis (UC). Describe a noninvasive method for measuring a condition. The invention also includes a colorimetric kit for carrying out this method.

High-mobility group box 1 (HMGB1) is a non-histone nuclear protein associated with non-histone chromatin generated as a Damage Associated Molecular Patterns (DAMP) molecular prototype that can respond to stimuli from tissue damage by inducing an inflammatory response ( One). HMGB1 is present in macrophages (2) and enterocytes (3), following pro-inflammatory stimulants such as LPS, TNFα, IL-β, IL-6, and IL-8 (4). Is actively secreted and released by necrotic cells rather than apoptotic cells 5. HMGB1 secreted into the extracellular space forms a highly inflammatory complex with different molecules as follows: activating innate immunity, interacting with their respective receptors, such as TLR9, TLR4, IL-1R and TLR2 Single-stranded DNA, LPS, IL-Iβ and nucleosomes. HMGB1, on the other hand, can bind a receptor for glycosylation target product RAGE (receptor for higher glycosylation target product) without forming a complex (6).

Extracellular HMGB1 causes the production of inflammatory mediators (4) and plays an important role in the pathogenesis of autoimmune or inflammatory diseases, including rheumatoid arthritis (7), systemic lupus erythematosus (8) and polymyositis (9). can do. The invention described in US Pat. No. 6,303,321 relates to a pharmaceutical composition for treating sepsis. The pharmaceutical composition comprises an effective amount of an HMGB1 antagonist or inhibitor as the active substance. Preferably, among the antagonists of HMGB1, antibodies that bind to HMGB1 protein, antisense sequences of the HMGB1-encoding gene, and antagonists of the HMGB1 receptor are used. Therefore, it is an object of the patent invention to provide a method for treating sepsis, comprising administering an antagonist effective amount of HMGB1. The invention also provides diagnostic and prognostic methods for monitoring the severity of patient conditions and for predicting similar clinical processes of sepsis and related conditions for patients exhibiting shock or related symptoms. Diagnostic and prognostic methods include measuring the concentration of HMGB1 protein in a sample, particularly serum or whole blood, and comparing the concentration to a standard concentration of HMGB1. Higher amounts of HMGB1 indicate a poor prognosis or the likelihood of toxic reactions occurring. Diagnostic methods can also be used in other tissues or fluids compartments such as cerebrospinal fluid or urine.

HMGB1 and Gastrointestinal Tract: Technology

Signs of stress, tissue damage or microbial antigens in the membranes activate inflammatory responses by activating cells associated with the innate immune response, such as phagocytic and branched cells.

The presence of HMGB1 released from the inflammatory stimuli following the extracellular matrix appears to significantly influence intestinal barrier function by altering epithelial intestinal cell permeability and causing increased influx of microbial antigens. In fact, in vitro and in vivo studies have shown that the presence of HMGB1 secreted by immunostimulated enterocytes or other immune cells correlates with intestinal barrier dysfunction (10-15). In addition, due to inflammatory cytokine release, HMGB1 is also likely associated with necrotic forms of colon inflammation and colitis (18, 19), as demonstrated in animal models (16, 17).

Reduced secretion of HMGB1 by anti-HMGBl molecules appears to correlate with improving both intestinal barrier damage and mucosal inflammation (11, 13, 14, 16, 19, 20).

The presence and amount of HMGB1 protein in tissue samples taken from patients has already been used as a diagnostic and prognostic marker for intestinal cancers, particularly colon and rectal cancers, as described in US Patent Application 2006/0188883. However, it is known that cancer disease is a very different condition from inflammatory bowel disease. In addition, the present invention is applicable only to biological tissues, and there is no reference regarding the use of feces.

In a recent work by Dave et al. (16), results have been associated with the use of anti-inflammatory agents, such as ethyl pyruvate, in a mouse model of chronic colitis to reduce HMGB1 secretion. Tests conducted on stool samples showed that the HMGB1 content in the stool was reduced after administration of ethyl pyruvate.

However, the experiments conducted in Dave's study of colitis are only relevant in mouse models, and the results obtained in those experiments do not always apply naturally to humans and their diseases, and in practice the results obtained by animal models are based on molecular markers. In some cases, it is well known that there is no coincidence with the corresponding human disease in the clinical course of the disease and in response to specific treatment.

In addition, genetically modified strains of mice, along with genetic coding for IL-10-removed anti-inflammatory cytokines in the mouse model used in this study, cause colitis in mice. This is rather a condition in which a much more complex contributing factor may be different from the human disease that determines the onset of the disease.

In fact, it is known that the genetic and environmental diversity that characterizes humans can never be reproduced in research animal models. In particular, inflammatory bowel disease is a multifactorial disease in which genetic and environmental diversity plays an important role in the development and progression of the disease.

Indeed, to date, more than 30 susceptibility loci have been identified for CD (less in the case of CUs), and also not all individual sites affecting express the same gene mutation, It does not necessarily mean that the disease progresses. In other words, unlike most mouse models, genetic homogeneity has a large genetic variation among people with inflammatory diseases.

In addition, environmental pressures in lifestyle (diet, smoking, stress), as well as the use of drugs or exposure to hazardous environmental drugs vary from person to person, and also play a role in the development of disease, and the composition of intestinal flora Of course it varies from individual to individual. In this regard, recent research conducted by important national and international organizations highlights the important role of commensal bacterial flora in inflammatory bowel disease, which is virtually altered by influences compared to healthy individuals. It is important to remember. In other words, the mouse model in a standard state is not subjected to environmental burdens or at least much less, and the microbiological profile can also change much less among individuals when eating the same food.

FIG. 1 shows HMGBl protein in stool samples detected by Western blot analysis using anti-HMGBl monoclonal antibodies of the R & D system.
Figure 2 is a graph showing the results of comparing the content of HMGBl protein detected in the stool and HMGBl protein content of fecal calprotectin.

Role of HMGB1 in Human Intestinal Inflammation

There is very little research on the role of HMGB1 in human intestinal inflammation: recent publications indicate ligands of RAGE, including HMGB1, as potential "biomarkers" of pathologies such as arthritis and colitis (21), second edition. In E. coli, HMGB1 is identified as a new antigen of the anti-neutrophil cytoplasmic antibody (ANCA), as observed in the serum of ulcerative colitis 22 patients.

Protein used as marker of intestinal inflammation

Biological markers represent a non-invasive method of objectively measuring inflammation and may play a primary or secondary role in the evaluation of some diseases 23, including Inflammatory Bowel Disease (IBD).

Such markers can be identified as serum or stool, diagnose a particular process, classify the disease into different subtypes, assess its activity, development and prognosis, or respond to treatment or relapse 24 Can be used to predict

Serum markers available for several inflammatory diseases, including IBD, include erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), anti-neutrophil cytoplasmic antibody (ANCA), and anti-saccharomyces cerevisiae. (Saccharomyces cerevisiae) antibody (ASCA) 24. However, they exhibit low sensitivity and specificity for intestinal inflammation and are low in correlation with the symptoms and indices of disease activity 24.

In contrast, stool markers show greater specificity in the diagnosis of gastrointestinal disorders such as IBD because the levels of these markers do not increase in diseases not involving the digestive system (25, 26). . This marker also has the advantage of not necessarily requiring endoscopy to assess disease activity 26, 27. Lactoferrin and calprotectin are most frequently used as stool markers for intestinal inflammation (24, 25, 28, 29). Indeed, the presence of these proteins in feces can accurately measure disease activity and recurrence predictions, identify high-risk groups among patients with severe colitis, and confirm the effectiveness of medical treatment.

There is an increasing need to identify methods for detecting non-invasive, more sensitive, specific, and economically gastrointestinal inflammation, and as a result, there is a continuing interest in research to identify new molecules that match these characteristics.

Objective and Preliminary Results

Since it is well known that HMGB1 has a function of generating signals on the recruitment of the cellular inflammation list and activating immune responses due to exogenous or endogenous stimulation, the inventors of the human inflammatory bowel It was proposed to investigate the possible involvement of these proteins in the pathogenesis of disease, particularly CD and UC.

CD is characterized by transmural inflammation that can affect any part of the digestive tract from the mouth to the anus. Usually, there are discontinuities in more parts. Inflammation includes all walls of the affected area and often spreads to nearby mesentery and lymph nodes. Most often, inflammation includes terminal ileum and colon.

In UC, the inflammatory process is limited to the colon and affects only the mucosal layer. Recurrences of the rectum are constant and can accompany the incidences of the various upward portions of the colon.

In recent Western countries (Europe and the United States), the incidence of these diseases is about 70-150 cases per 100,000 for UC and about 20-40 cases per 100,000 for CD. They are predominantly diseases of late adolescents and young adults, peaking incidence between 15 and 35 years of age.

In this regard, the inventors have found that HMGBl is found in the stool of an IBD pediatric patient because it is known that when proteins are secreted from the extracellular matrix it is inflammatory and accurately indicates that feces are produced and removed from the intestines. I think the most important. The data obtained were compared with that of the control group.

It was surprisingly found that the amount of HMGBl observed in the stool of IBD patients was significantly increased than that of healthy controls (FIG. 1). Thus it was established that the measurement of HMGBl in the stool of a patient can be used as a marker of intestinal inflammation. In addition, it has been evident that patients suffering from moderately severe disease due to treatment (groups with disease index PCDAI / PUCAI <25/60) exhibited a decrease in HMGBl compared to patients with severe disease. Therefore, this protein will also be a good indicator of response to treatment, in addition to good markers of inflammation (FIG. 1). The method developed for this purpose is described below.

Sampling

Stool samples collected from 40 IBD pediatric patients (19 patients with Crohn's disease (CD) and 21 patients with ulcerative colitis (UC)), and 13 controls, were evaluated by Western blot to assess the presence of HMGBl. To be analyzed. The condition of Western blots has been specifically developed for the purpose of using two specific antibodies for the detection of HMGBl.

Bands highlighted in relation to the presence of HMGBl protein were introduced into densitometric assays conducted using ImageQuant software (GE Healthcare Life Sciences, Uppsala, Sweden) and numerical values in the HMGBl content range could be obtained.

Diagnosis of IBD in patients was performed according to widely recognized and shared endoscopy and histological criteria (30). CD activity was measured by the "pediatric Crohn's disease activity index" (PCDA), a measure based on clinical and empirical parameters (31): inactive disease when the index is 10 or less, weak on 10-30, or Common disease, and more than 30 cases are classified as severe. The activity of UC has been graded according to the Pediatric Ulcerative Colitis Activity Index (PUCAI) (32): the latter is a recently proven non-invasive multiparameter method, thus relieving disease (less than score 10), mild disease (Scores 10 to 34), moderate disease (scores 35 to 64) and severe disease (scores 65 to 85).

Endoscopic scores were measured using SES-CD (33) and Matts score (34) for ulcerative colitis. To calculate the SES-CD, separate the intestines into five parts (the intestines, left colon, transverse colon, right colon, rectum), and in each part the values in the range 0-12 (total numerical range: 0-60). It was used for this disease activity. To calculate the Matts score, the intestines were divided into 6 parts (blind, ascending, transverse, descending, twisting, rectal), and in each part, values ranging from 1 to 4 (total range: 6 to 24). Used for disease activity.

Based on their indices in patients enrolled in this study, the disease is severe in 13 IBD patients (8 CD and 5 UC), 11 patients (3 CD and 8 UC) are weak or moderate, and Sixteen patients (8 CD and 8 UC) were found to be inactive (Table 1).

Table 1 lists patients enrolled in clinical trials and separated by disease type and severity.

Table 1. Demographic characteristics and indices of disease activity of IBD in the studied patient population. Pediatric Crohn's Disease Activity Index (PCDAI): "pediatric Crohn's disease activity index"; Pediatric Ulcerative Colitis Activity Index (PUCAI): "Child Ulcerative Colitis Activity Index"

Preparation of Stool Samples

A pediatric, pediatric gastroenterology and pediatric unit, supervised by Professor Salvatore Cucchiara, recruited from the University of Rome "La Sapienza", IBD pediatric patients (Table 1) and healthy depending on the severity of the condition Stool samples were obtained from control persons.

Samples collected in stool sterile containers were stored at temperatures between -20 ° C and -80 ° C until molecular analysis.

Basis weight of fecal samples and suspension in buffer

Each sample (corresponding to the amount of spoon in a standard fecal container) was removed from the vessel with a sterile tip, then placed in a 1.5 ml eppendorf tube and weighed with a digital balance. The sample was resuspended in extraction buffer (phosphate buffered saline solution PBS, pH 7.2) containing detergent and sodium azide (ScheBo Biotech) to obtain a final concentration of 500 mg / ml.

Homogenization and Extraction of Feces

The sample was stirred for 1 minute at room temperature (RT) and then placed in an orbital shaker for 30 minutes at room temperature. After centrifugation at 4 ° C. and 10,000 rpm for 5 minutes, the supernatant of the extracted feces was collected and the protein concentration was measured by Bradford assay (Biolabs). The obtained samples were immediately analyzed by Western blot analysis or stored at -80 ° C and then analyzed.

Analysis of Fecal Extract by Western Blot

To 20 μg of fecal protein extract add the same volume of 2X sample buffer (100 mM Tris-Cl, pH 6.8, 10% β-mercaptoethanol, 4% SDS, 20% glycerol, 0.2% bromophenol blue) The sample was boiled for 5 minutes and then briefly centrifuged before analysis of the extract by Western blot (WB). Fecal protein extracts were separated using a 12% SDS-polyacrylamide gel and then transferred to a PVDF filter (Amersham) by an electronic converter for 1 hour at 70 volts. Nonspecific sites on the filter were blocked by incubating for 1 hour at room temperature with blocking buffer (0.02M Tris-Cl, pH 7.6, 0.137 M NaCl, 5% nonfat dry milk), and then the filter was blocked with antibody buffer (0.02). Anti-HMGBl polyclonal antibody (Catalog No. H9593, Sigma) diluted 1: 1000 in M Tris-Cl, pH 7.6, 0.137 M NaCl, 3% nonfat dry milk or 1: 500 in antibody buffer. Incubated with anti-H GBl monoclonal antibody (Catalog number MAB 1690, R & D Systems, Minneapolis, USA) at 4 ° C. for 16 hours. After washing three times for 5 minutes each with TBS-T 0.1% Tween (0.02M Tris-Cl, pH 7.6, 0.137 M NaCl, 0.1% Tween), when using anti-HMGBl (manufactured by Sigma), the filter was Anti-mouse secondary when incubated with rabbit secondary antibody for 1 hour at room temperature and in combination with anti-HMGBl antibody R & D system conjugated to peroxidase (Santacruz) diluted at a ratio of 1: 4,000 in antibody buffer. Antibodies were used. After three more washes for 5 minutes with TBS-T + 0.1% Tween, chemiluminescent signals were detected using ECLplus (Amersham) and autoradiographic film (Kodak).

1 shows HMGBl protein in stool samples detected by Western blot analysis using anti-HMGBl monoclonal antibodies of the R & D system. In particular, Panel A shows the results of the Western blot, Panel B shows the graph of the density measurements of the bands highlighted by the Western blot in the patient, and Panel C shows Western in patients separated into groups according to the severity of the disease. A graph of the density measurements of the bands highlighted by the blot is shown.

Density analysis of the highlighted bands by Western blot was performed to obtain values related to the content of HMGBl present in the stool sample. In particular, when expressed in Arbitrary Densitometric Units (ADUs), a healthy person represents 1,000 to 3,000, an average of 1200 ADUs; CD patients exhibited values ranging from 20,000 to 380,000 ADU, with an average of 190,000 ADU; All UC patients, on the other hand, were found to exhibit 6,000-280,000 ADUs, an average of 120,000 ADUs, as values associated with the HMGBl content in the analyzed fecal samples (FIG. 1 -B). In the figure, asterisk means statistical significance assessed through the Mann-Whitney statistical test, where * p <0.05, ** p <0.01, *** p <0.001.

As a result, HMGBl protein expression was significantly increased in the feces of IBD patients compared to the control group, which was undetectable (p <0.001) (FIG. 1). These results indicate that the presence of HMGBl protein detected in feces is a marker of inflammation in the human intestine. The presence of HMGBl protein was also detected in the stool of 16 patients with disease who were limited to inactive based on the PCDAI and PUCAI indicators. However, upon evaluation of the endoscopy score, these patients had some degree of intestinal inflammation consistent with the detection of HMGBl in their feces.

In particular, the median SES-CD and Matts scores in active CD and UC patients were 23.0 (range: 14-34) and 18.0 (range: 8-24), respectively, and in patients with inactive CD and UC, SES-CD and Matts The centers of scores were 7.5 (range: 0-15) and 11.5 (range: 6-18), respectively. These indices are very sensitive to such inactive inflammatory conditions because there is an intestinal inflammation pitch in such defined inactive patients according to the PCDAI and PUCAI indices, and HMGBl can also provide an indication of the inactive state of the disease. Indicates that it is considered a marker. The content of HMGBl protein detected in feces was compared to the HMGBl protein content of fecal calprotectin, which has recently been considered a reliable and biomarker for the diagnosis of intestinal inflammation by ELISA (29, 35). The results of this analysis are shown in FIG. 2, where the asterisks show statistical significance according to Mann-Whitney's statistical tests, * p <0.05, ** p <0.01, *** p <0.001. Both proteins were significantly increased (p <0.001) in the stool of the patient compared to healthy control persons (FIGS. 1-B, FIG. 2-A). However, inactive CD and UC groups showed low levels of calprotectin, but the stool content of HMGBl was significantly increased compared to control humans (p <0.01, in CD and UC) (FIG. 1-C, FIG. 2-B). ). In summary, the comparison showed a significant correlation between the content of 2 proteins in the stool samples of all diagnosed active inflammatory disease patients (CD and UC) (r: 0.77, CD, r: 0.70, UC, p <0.01). (Where r = rank is the correlation coefficient according to the Spearman test). This correlation disappeared when only patients with inactive inflammatory disease were considered (r: 0.22, CD, r: 0.18, UC, no symptoms). In fact, despite indicating the extent of inflammation according to the endoscope score, HMGB1 increased significantly in all 16 patients with inactive disease as defined above according to the index of PCDAI and PUCAI, whereas calprotectin was found in two of them. Only rose to. This indicates that HMGB1 is a very sensitive marker of chronic intestinal inflammatory patients with clinically inactive diseases, as indicated by the conventional index of disease activity. However, the latter does not always correlate with intestinal inflammation detected by endoscopy, given the clinical and laboratory mix.

Therefore, it is possible to infer the use of HMGBl protein as a potential molecular prognostic parameter for relapse in patients with apparently alleviated disease. In Figure 1, IBD patients show a significant increase in HMGBl in feces compared to healthy controls. There is also a direct correlation between the HMGBl content and the severity of the disease. In conclusion, HMGBl can be used as a marker for therapeutic response, in addition to being a good marker of inflammation, it also provides a good indicator of the severity of the disease.

From what has been described so far, the importance of the present invention is evident as follows: The use of HMGBl as a biological marker and the method of detecting its presence in a stool sample are highly traumatic and frequently repeated images for most patients. While avoiding research, it is an important step to diagnose the level and presence of human intestinal inflammation in a safe and non-invasive way.

In addition, protein expression levels can be used as prognostic markers of disease recurrence and as markers of response to treatment.

It should be noted that not only Western blot analysis can be used for the analysis of fecal extracts. We have developed an assay protocol for detecting the presence of HMGB1 in stool by ELISA using the same antibodies used in Western blot assays that provide more than acceptable results in the case of specificity and sensitivity to the target protein. Attention was paid. Therefore, we want to prepare an ELISA kit using two antibodies already used for detection of protein by WB in stool samples. The choice to provide an ELISA protocol in addition to the Western blot is easily made from the fact that this technique is simple and further quantify the response, in fact the color saturation of the ELISA plate is proportional to the number of antigen-antibody complexes (primary) Therefore, it is proportional to the concentration of antigen (which can bind primary antibody) in the sample analyzed.

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Claims (12)

A non-invasive method for detecting, diagnosing, and prognosing intestinal inflammatory conditions in the same patient, characterized by detecting the content of HMGB1 in a stool sample of a human patient.
A method wherein the decrease in the content of HMGB1 in the stool sample is used as a response marker for a particular treatment.
2. The method of claim 1, wherein said bowel inflammatory condition is selected from the group consisting of chronic inflammatory bowel disease (IBD), particularly Crohn's disease (CD) and ulcerative colitis (CU).
The method of claim 1 or 2, wherein the human patient is a pediatric patient suffering from IBD.
The method according to any one of claims 1 to 4, comprising the following steps:
Weighing the stool sample and suspending it in PBS extraction buffer;
Homogenizing the stool and extracting after centrifugation of the stool extract supernatant;
Assessing protein concentration by Bradford assay; And
Analyzing fecal extract by Western blot.
The method according to any one of claims 1 to 5, wherein the fecal extract transferred to the PVDF filter is incubated with an anti-HMGBl polyclonal or monoclonal antibody during analysis of the extract by Western blot.
The anti-HMGBl polyclonal antibody according to any one of claims 1 to 6, wherein the anti-HMGBl polyclonal antibody is generated in a rabbit using a synthetic peptide corresponding to amino acids 165-180 of human HMGBl as an immunogen, and the anti-HMGBl monoclonal antibody And cloned to clone 115603 of hybridoma obtained from the fusion of B-cell obtained from mouse immunized with purified E. coli-derived recombinant human HMGBl protein with mouse myeloma.
The method of claim 5 wherein the analysis of the stool extract is carried out by ELISA assay.
9. The method of claim 1, wherein the same antibody used in the Western Blot Assay is used as an antibody to perform the ELISA assay.
The anti-HMGBl polyclonal antibody produced in a rabbit according to any one of claims 1 to 9, wherein the anti-HMGBl antibody used is a synthetic peptide corresponding to amino acids 165-180 of human HMGBl as an immunogen, and purification. And anti-HMGBl monoclonal antibody corresponding to clone 115603 of hybridoma obtained from fusion of mouse B myeloma with B-cell obtained from mouse immunized with recombinant E. coli-derived recombinant human HMGBl protein.
A colorimetric kit for detecting HMGBl protein in a human stool sample according to any one of claims 1 to 10 based on an antigen-antibody specific reaction, wherein the antibody is an anti-HMGBl polyclonal antibody or an anti-HMGBl monoclonal. Colorimetric kit for detecting HMGBl protein in human stool samples, characterized in that the antibody.
The anti-HMGBl polyclonal antibody produced in rabbits using a synthetic peptide corresponding to amino acids 165-180 of human HMGB1 as an immunogen, and purified E. coli derived recombinant human HMGBl protein. A colorimetric kit, characterized in that it is an anti-HMGBl monoclonal antibody corresponding to clone 115603 of hybridoma obtained from fusion of B cells obtained from mice immunized with mouse myeloma.

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