JP4852706B2 - H. Method for detecting H. pylori-related idiopathic thrombocytopenic purpura and screening method for prophylactic / therapeutic agent thereof - Google Patents

Description

  The present invention relates to idiopathic thrombocytopenia associated with infection with Helicobacter pylori (hereinafter referred to as “H. pylori” in the present specification and claims) among patients with idiopathic thrombocytopenic purpura. The present invention relates to a method for selectively detecting a patient with purpura (hereinafter referred to as “H. pylori-related idiopathic thrombocytopenic purpura”). The present invention also relates to a method for screening an effective component for the prevention or treatment of the H. pylori-related idiopathic thrombocytopenic purpura.

  It is known that H. pylori infection causes chronic inflammation of the gastric mucosa and is involved in the development of gastroduodenal ulcer, gastric cancer and lymphoma (see Non-Patent Documents 1 to 3). Furthermore, H. pylori has been shown to be associated with the pathogenesis of idiopathic thrombocytopenic purpura (hereinafter also referred to simply as “ITP”) and autoimmune thyroiditis (non-patented) References 4 and 5).

  ITP is one of the most common autoimmune diseases that affect both adults and children (see Non-Patent Documents 6 to 8). Thrombocytopenia in this disease occurs when autoantibodies bind to various platelet membrane receptors, including platelet glycoproteins (GP) such as glycoprotein IIb / IIIa (GPIIb / IIIa) or GPIb / IX complex. (Patent Documents 7 and 8). The binding of autoantibodies to these target antigens results in platelet destruction by the reticuloendothelial system and decreases platelets (see Non-Patent Documents 10 to 13, etc.).

  When H. pylori eradication therapy is performed on patients with chronic ITP (hereinafter also referred to as “cITP”), an increase in platelets is often observed (see Non-Patent Documents 5, 9 and 14). For this reason, ITP is broadly classified into H. pylori-related ITP that is effective for H. pylori eradication therapy and essential ITP that is ineffective for H. pylori eradication therapy. The level of platelet-related immunoglobulin G (PA-IgG) involved in the development of ITP is not as pronounced as that of essential ITP, but also increases in H. pylori-related ITP and decreases with H. pylori eradication (Non-patent document 15).

  Thus, it is clear that there is a group of ITP patients who are effective in H. pylori eradication therapy (H. pylori related ITP), but how H. pylori is involved in the pathology of ITP. It is not clear whether it is.

  Recently, Takahashi et al. Showed that PA-IgG recognizes 100-150 kDa H. pylori-derived CagA protein in H. pylori-related cITP patients (Non-patent Document 15), and Francesco et al. In H. pylori related cITP patients with kDa platelet antigen, it was reported that anti-CagA antibody decreased platelets (Non-patent Document 2). These reports suggest that PA-IgG and / or anti-CagA antibodies are involved in the development of H. pylori-related cITP.

However, anti-H. Pylori antibodies are anti-H. Pylori because the platelet count recovers earlier (within 1-2 weeks) despite the presence in the serum for several months after H. pylori eradication. The direct association between antibodies and H. pylori-related cITP is unclear. Once the mechanism by which H. pylori eradication therapy increases platelets has been elucidated, it is possible to use this mechanism to establish H. pylori-related ITP patients and essentiality in advance without unnecessary H. pylori eradication therapy. It is possible to select ITP patients. In addition, elucidation of the above mechanism not only leads to the development of new treatment methods for H. pylori negative ITP patients and H. pylori eradication therapy invalid ITP patients, but also elucidation of the mechanism of autoimmune diseases other than ITP It is thought to contribute to clinical application.
Cover, TL, et al., 1992. Helicobacterpylori and gastroduodenal disease. Annu. Rev. Med. 43: 135-145. Rauws, EA, et al., 1990. Cure of duodenal ulcer associated with eradication of Helicobacter pylori. Lancet. 335: 1233-1235. Forman, D. 1995. The prevalence of Helicobacter pylori infection in gastric cancer. Aliment. Pharmacol. Ther. 9 Suppl 2: 71-76. Parsonnet, J., et al., 1994. Helicobacterpylori infection and gastric lymphoma. N. Engl. J. Med. 330: 1267-1271. Emilia, G., et al., 2001. Helicobacterpylori eradication can induce platelet recovery in idiopathic thrombocytopenic purpura. Blood. 97: 812-814. Gasbarrini, A., et al., 1999. Autoimmune diseases and Helicobacter pylori infection. Biomed. Pharmacother. 53: 223-226. Harrington, WJ, et al., 1953. Immunologic mechanisms in idiopathic and neonatal thrombocytopenic purpura. Ann. Intern. Med. 38: 433-469. Lusher, JM, et al., 1966. Idiopathic thrombocytopenic purpura in childhood. J. Pediatr. 68: 971-979. McMillan, R. 1981. Chronic idiopathic thrombocytopenic purpura. N. Engl. J. Med. 304: 1135-1147. Beardsley, DS, et al., 1984. Platelet membrane glycoprotein IIIa contains target antigens that bind anti-platelet antibodies in immune thrombocytopenias. J. Clin. Invest. 74: 1701-1707. McMillan, R. 2000. Autoantibodies and autoantigens in chronic immune thrombocytopenia purpura. Semin. Hematol. 37: 239-248. van Leeuwen, EF, et al., 1982. Specificity of autoantibodies in autoimmune thrombocytopenia. Blood. 59: 23-26. Varon, D., et al., 1983. A monoclonal anti-platelet antibody with decreased reactivity for autoimmune thrombocytopenic platelets. Proc. Natl. Acad. Sci. USA. 80: 6992-6995 Kohda, K., et al., 2002. Effect of Helicobacter pylori eradication on platelet recovery in Japanese patients with chronic idiopathic thrombocytopenic purpura and secondary autoimmune thrombocytopenic purpura. Br. J. Haematol. 118: 584-588. Takahashi, et al., 2004. Molecular mimicry by Helicobacter pylori CagA protein may be involved in the pathogenesis of H. pylori-associated chronic idiopathic thrombocytopenic purpura.Br. J. Haematol. 124: 91-96.

  Conventionally, treatment for ITP has been determined based on the platelet count and the degree of bleeding symptoms with the goal of improving bleeding symptoms and preventing severe bleeding. In indicated cases, corticosteroids are given first, followed by splenectomy (Cines DB, N Engl J Med 2002,346,995-1008; George JN, et al., Blood 1996, 88, 3-40). On the other hand, cases in which symptoms are not improved by such treatment are called refractory, and various treatment methods such as immunosuppressants and danazol administration have been tried, but there is no established treatment method yet. However, as the existence of H. pylori-related ITP, which has recently been ameliorated by H. pylori eradication therapy, has been clarified, a new treatment guideline has been proposed in which eradication therapy is the first choice in H. pylori-positive cases (Michel M, et al., Blood 2004, 103, 890-896). That is, if ITP is diagnosed, H. pylori is first diagnosed in all cases, and if positive, H. pylori eradication therapy is performed regardless of the platelet count and bleeding symptoms. Ineffective cases in which platelets do not increase even after sterilization start treatment with corticosteroids, etc. according to conventional treatment guidelines together with H. pylori negative cases. By doing so, it is believed that the number of patients who receive conventional treatment such as corticosteroid administration will certainly decrease, contributing to the reduction of side effects and medical costs.

  However, H. pylori eradication therapy has three side effects (gastric acid inhibitor and two antibiotics) for 1 to 2 weeks, so there are no side effects at all. Diarrhea, loose stool, stomach discomfort, Nausea, loss of appetite, etc. can occur. Therefore, if it is known in advance that an ITP patient has H. pylori related ITP, there is no need to receive unnecessary H. pylori diagnosis or sterilization therapy. Can be avoided.

  Therefore, an object of the present invention is to provide an effective method for diagnosing whether an ITP patient is an H. pylori-related ITP that is successfully treated with H. pylori eradication therapy. Furthermore, the present invention relates to a method for screening an active ingredient of the drug in order to develop a drug effective for the treatment of H. pylori related ITP.

  The present inventors thought that elucidating the onset mechanism of H. pylori-related ITP would lead to the solution of the above-mentioned problems, and as a result of extensive studies, platelets were lysed from H. pylori, particularly 17 kDa. Platelet aggregation occurs in response to low molecular weight proteins, and sera from H. pylori-related ITP patients are significantly different from sera from other ITP patients and react strongly to the 17 kDa protein from H. pylori that binds to the platelets (Consequently, it reacts with platelets and the 17kDa protein complex), and the serum specifically contains antibodies against the 17kDa protein and antibodies against small 27kDa and 36kDa proteins. I found it. Furthermore, the H. pylori antibody measurement method developed by the inventors (unlike conventional methods that use only high molecular weight proteins derived from H. pylori as antigens, low to high molecular weight total proteins derived from H. pylori as antigens) Of the antibody titer before and after eradication of H. pylori using only the H. pylori-related ITP patients, the antibody against H. pylori in the serum after eradication of H. pylori As a result, it was confirmed that a low molecular weight protein derived from H. pylori, particularly a 17 kDa low molecular weight protein, was deeply involved in the development of H. pylori-related ITP. And as its onset mechanism, a 17 kDa low molecular weight protein derived from H. pylori binds to platelets to form a complex, and an immune complex formed by further binding an anti-H. Pylori antibody to this, It was thought that the reticuloendothelial system was phagocytosed and destroyed, resulting in thrombocytopenia.

Based on these findings, the present inventors used the presence of an antibody against a 17 kDa low molecular weight protein derived from H. pylori in serum as an index for ITP patients, or otherwise. I was able to identify whether it was ITP (essential ITP), and I was convinced that it would be possible to perform subsequent treatment appropriately depending on the type of ITP.

  In addition, the present inventors prevented the binding of a 17 kDa low molecular weight protein derived from H. pylori and platelets, and more preferably the formation of a three-component immune complex of the low molecular weight protein, platelets and anti-H. Pylori antibody. It was convinced that the substance which has the effect | action which acts is effective in the treatment and prevention of H. pylori related ITP.

  The present invention has been completed based on such findings and includes the following aspects.

(I) Method for detecting H. pylori-related idiopathic thrombocytopenic purpura (H. pylori-related ITP) Item 1. A method for detecting H. pylori-related ITP, comprising detecting an antibody against a protein derived from H. pylori identified as a molecular weight of 17 kDa, 27 kDa, or 36 kDa on a subject by SDS-PAGE.
Item 2. An antibody that reacts with H. pylori cell lysate or a preparation thereof and serum of a subject and reacts with at least one of proteins derived from H. pylori identified by SDS-PAGE as having a molecular weight of 17 kDa, 27 kDa, or 36 kDa Item 2. A detection method according to Item 1, comprising a step of detecting.
Item 3. Item 3. The detection method according to Item 1 or 2, wherein the H. pylori-derived protein identified as having a molecular weight of 17 kDa has an isoelectric point of pH 5-8.

(II) A method for screening an active ingredient for the prevention or treatment of H. pylori-related ITP . A method for screening active ingredients for the prevention or treatment of H. pylori-related idiopathic thrombocytopenic purpura having the following steps:
(1) H. pylori-derived protein (17 kDa protein) and platelets, or 17 kDa protein and platelets, and anti-H. Pylori identified as SDS-PAGE and having a molecular weight of 17 kDa in the presence or absence of the test substance Mixing an antibody or a component containing the same,
(2) Complex production amount (target production amount) generated when 17 kDa protein and platelets, or 17 kDa protein and platelets and anti-H. Pylori antibody or a component containing the same are mixed in the presence of the test substance, and the test substance In the absence of, 17kDa protein and platelets, or the amount of complex produced when mixing 17kDa protein and platelets with anti-H. Pylori antibody or components containing it (control production),
(3) A step of selecting, as an active ingredient of a preventive or therapeutic agent for H. pylori-related idiopathic thrombocytopenic purpura, a test substance when the target production amount is reduced compared to the control production amount.
Item 5. Item 5. The method according to Item 4, wherein the 17 kDa protein has an isoelectric point of pH 5-8.
Item 6. Item 6. The method according to Item 4 or 5, wherein the amount of platelet aggregation is used as the amount of complex produced.
Item 7. Item 7. The method according to any one of Items 4 to 6, wherein the component containing an anti-H. Pylori antibody is serum of a patient suffering from H. pylori-related idiopathic thrombocytopenic purpura.
Item 8. Item 8. The method according to Item 7, wherein the serum of the patient suffering from H. pylori-related idiopathic thrombocytopenic purpura is type B.

  According to the method of the present invention, for patients with idiopathic thrombocytopenic purpura (ITP), the disease ITP is H. pylori-related ITP for which H. pylori eradication therapy is effective, or other essential ITP. As a result, early diagnosis of H. pylori-related ITP is possible (for example, there is no need to wait for the platelet count dynamic effect by eradication), and appropriate treatment guidelines according to the patient It is also possible to build. That is, in the former case, H. pylori eradication therapy can be selected, and in the latter case, for example, medication such as adrenocortical hormone or ITP treatment such as splenectomy can be accurately selected. The right choice of ITP treatment not only reduces the mental, physical or economic burden on the patient, but also reduces medical costs.

  In addition, according to the screening method of the present invention, a component effective for prevention or treatment of H. pylori-related ITP can be obtained.

(I) Detection method of H. pylori-related ITP As mentioned above, idiopathic thrombocytopenic purpura (ITP) is a major effective H. pylori-related ITP and other essential ITP. Can be classified.

In the present invention, H. pylori-related ITP refers to an ITP disease that is positive for H. pylori and is completely or partially ameliorated by I. pylori eradication therapy. It includes H. pylori-positive ITP disease and H. pylori-positive ITP disease that is not ameliorated by I. pylori eradication therapy. ITP is basically determined based on thrombocytopenia (platelet count <120 × 10 ⁹ / l), but PA-IgG level (standard value: 9-25ng / 10 ⁷ platelets), medical history, Physical findings and blood tests (especially platelet rotation tests; in the case of ITP, thrombopoietin is slightly elevated from normal and the ratio of reticulated platelet count is increased) may also be consulted.

Here, the presence or absence of H. pylori infection (whether H. pylori is positive or negative) can be determined by a conventional method such as a ¹³ C-urea breath test. By sterilization therapy, if the platelet count increased to 120 x 10 ⁹ / l or more, it can be said that the ITP was in remission, and the platelet count did not exceed 120 x 10 ⁹ / l, even if the platelet count did not exceed 120 x 10 ⁹ / l. It can be said that the ITP partially remissioned when it increased by 10 ⁹ / l or more.

  The method of the present invention specifically detects a marker component of H. pylori-related ITP in an ITP patient, so that the ITP type of the patient is H. pylori-related ITP or other essential ITP. It is a method of identifying whether there is.

  Here, as a marker component of H. pylori-related ITP, an antibody against a low molecular weight protein derived from H. pylori cells, specifically, a protein having a molecular weight of 17 kDa, 27 kDa or 36 kDa (17 kDa protein, 27 kDa protein or 37 kDa protein) is exemplified. be able to. Here, 17 kDa protein, 27 kDa protein or 37 kDa protein derived from H. pylori cells means sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 12.5 wt% acrylamide and 0.1 wt% sodium dodecyl sulfate (SDS) ( SDS-PAGE) is a protein contained in H. pylori cells identified as having a molecular weight of 17 kDa, 27 kDa or 36 kDa. The protein is prepared by dissolving H. pylori cells in sample buffer (125 mM Tris, 4% SDS, 12% mercaptoethanol, 20% glycerol, and 0.004% BPB), followed by heat treatment at 95 ° C for 10 minutes. The resulting cell lysate is subjected to SDS-PAGE (containing 12.5% by weight acrylamide and 0.1% by weight sodium dodecyl sulfate), corresponding to a molecular weight of about 17 kDa, about 27 kDa, or about 36 kDa compared to a standard marker of molecular weight. Can be prepared by obtaining the fraction to be obtained.

  In the present invention, a protein derived from a cell of H. pylori identified as such a molecular weight of 17 kDa, 27 kDa or 36 kDa by SDS-PAGE is simply referred to as a “17 kDa protein”, a “27 kDa protein” or a “36 kDa protein”. Is sometimes referred to as a “small molecule protein”. The 17 kDa protein targeted by the present invention preferably has an isoelectric point of pH 5-8. As shown in Experimental Example 5, the 17 kDa protein having an isoelectric point of pH 5-8 is obtained by subjecting a cell lysate obtained by crushing and lysing cells of H. pylori by ultrasonic treatment or the like to two-dimensional electrophoresis. (1D: isoelectric focusing, 2D: SDS-PAGE) to obtain a fraction having the isoelectric point (pH 5-8) and corresponding to a molecular weight of about 17 kDa as described above. Can be prepared.

  The method of the present invention detects the presence of an antibody against each of the above low molecular weight proteins in a subject. The detection is performed on the biological sample of the subject. The biological sample may be any one that can contain antibodies against the 17 kDa protein, 27 kDa protein, or 36 kDa protein, and generally those that can serve as specimens in clinical examinations can be widely used. Specifically, blood (including serum), Examples include urine, spinal fluid, amniotic fluid, and saliva. Preferably it is blood (serum).

  Specifically, the method of the present invention comprises the presence or absence of an antigen-antibody reaction (complex) by reacting the biological sample (preferably serum) of a subject with each of the low molecular weight proteins (17 kDa, 27 kDa or 36 kDa protein) described above. Can be carried out by determining the presence or absence of an antibody against each of the low molecular weight proteins in the biological sample, using as an index. More specifically, (i) H. pylori cell lysate and the biological sample (preferably serum) of the subject are mixed, and each low molecular protein and biological sample component contained in the H. pylori cell lysate are mixed. (Ii) each H. pylori cell lysate fraction containing low molecular weight proteins and the subject's biological sample (preferably serum) diffused in an agar gel and sedimented Method of detecting presence or absence of reaction (double immunodiffusion method), (iii) H. pylori cell lysate was separated by electrophoresis and then reacted with the above biological sample (preferably serum) of the subject, A method for detecting the binding (complex) of a biological sample component to a band corresponding to a molecular protein can be mentioned. The method (iii) is preferred.

  In the method (iii), a biological sample of a subject is reacted with an isolate of H. pylori cell lysate, and then (a) a secondary antibody bound to an enzyme is further reacted, ELISA method for detecting color development, (b) Fluorescent immunoassay method (FIA) for measuring fluorescence color development by further reacting a secondary antibody conjugated with a fluorescent dye, or (c) Secondary antibody conjugated with a chemiluminescent substance And chemiluminescence immunoassay (CLIA) for measuring chemiluminescence. Such a measuring method is known, and known enzymes, fluorescent dyes, and chemiluminescent substances used as labeling agents can be used without discrimination. For example, typical examples include alkaline phosphatase and peroxidase as enzymes, and fluorescent dyes such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (RITC).

  Examples of secondary antibodies bound to these labeling agents include anti-human immunoglobulin antibodies that can bind to the antibody to be measured (antibody against 17 kDa protein) (immunoglobulin). The anti-human immunoglobulin antibody may be either a polyclonal antibody or a monoclonal antibody, and commercially available antibodies can also be used.

  The presence or absence of the antibody to be measured (antibody against the 17 kDa protein) in the biological sample of the subject, or its content, is determined according to a conventional method, and the labeling activity corresponding to the type of labeling agent used for labeling the secondary antibody is measured. The antibody content can be calculated as an antibody titer converted from the obtained measured value.

  Thus, when the presence of an antibody against the 17 kDa protein is observed in the biological sample of the subject, the subject can be diagnosed with H. pylori related ITP. Conversely, when the presence of an antibody against the 17 kDa protein is not observed, the subject can be diagnosed with an ITP (essential ITP) other than H. pylori-related ITP.

(II) Method for screening active ingredient for prevention or treatment of H. pylori-related ITP As described above, the present inventors have described that H. pylori-related ITP is a 17 kDa derived from the aforementioned H. pylori in platelets. It appears that the immune complex opsonized by the anti-H. Pylori antibody binding to the complex bound to the low molecular weight protein (17kDa protein) is engulfed and destroyed in the reticuloendothelial system. I found it. The 17 kDa protein preferably has an isoelectric point of pH 5-8.

  This prevents and ameliorates the development of H. pylori-related ITP by blocking the binding of platelets to the 17 kDa protein or the binding of platelets to the 17 kDa protein and anti-H. Pylori antibodies. Means that you can. That is, it is considered that a substance having an action of inhibiting any of these bindings can be an effective medicinal ingredient for preventing or treating H. pylori related ITP. The screening method of the present invention is based on this idea.

The method can be carried out by the following steps:
(1) 17 kDa protein (a component) and platelet (b component) derived from H. pylori or 17 kDa protein (a component) derived from H. pylori in the presence or absence of the test substance A step of mixing platelets (component b) and anti-H. Pylori antibody or a component containing the antibody (component c),
(2) In the presence of the test substance, in the absence of the test substance, the amount of compound produced (target production amount) generated when the a component and b component or the a component, b component and c component are mixed. , A component and b component, or a composite product amount (control product amount) produced when a component, b component and c component are mixed,
(3) A step of selecting a test substance when the target production amount is reduced as compared with the control production amount as an active ingredient of a prophylactic or therapeutic agent for H. pylori-related ITP.

  Examples of components containing anti-H. Pylori antibodies include sera of patients suffering from H. pylori-related ITP. Preferably, it is a serum of a patient whose blood group suffering from H. pylori-related ITP is type B.

  Although it does not restrict | limit as a test substance, Any of a nucleic acid (a polynucleotide is included), a peptide (a polynucleotide is included), an organic compound, an inorganic compound, etc. may be sufficient. Specifically, the screening is performed by subjecting the test substance or a sample containing the test substance (test sample) to a mixture of 17 kDa protein (component a) and platelet (component b), or 17 kDa protein (component a), platelet (component b). ) And an anti-H. Pylori antibody or a mixture of the antibody-containing component (component c). Such test samples include cell extracts, gene library expression products, plant and animal natural product extracts, and the like.

  In screening, the conditions for bringing the test substance into contact with each component are not particularly limited as long as the components can be combined to form a complex in the absence of the test substance.

  The formation of a complex of 17 kDa protein and platelets, and the formation of a complex of 17 kDa protein and platelets with anti-H. It can be judged by measuring according to a standard method.

  In step (3), by comparing the target production amount (platelet aggregation amount) obtained in step (2) with the control production amount (platelet aggregation amount) measured without the presence of the test substance, the 17 kDa protein It is possible to select a substance that inhibits the formation of a complex of phospholipid and platelet, or has an action of inhibiting the formation of a complex of 17 kDa protein and platelet with an anti-H. Pylori antibody or a component containing the same. That is, when the target production amount obtained in (2) is smaller than the control production amount, the test substance can be selected as an effective substance for preventing or treating H. pylori-related ITP. it can.

  As described above, the substance selected in (1), (2) and (3) can be further subjected to other pharmacological tests, clinical tests and toxicity tests as necessary, and thus more suitable for humans. It can be an active ingredient of an effective and safe preventive or therapeutic agent (pharmaceutical or health food) of H. pylori related ITP. The substance thus obtained can be provided as a prophylactic or therapeutic agent (pharmaceutical or health food) for H. pylori-related ITP by formulation and formulation by a known method.

  In addition, according to the components found by the above method, it is possible to analyze and search for a platelet binding site to which a low molecular weight protein (17 kDa, 27 kDa or 36 kDa) derived from H. pylori binds. It can also contribute to identification and elucidation of the onset mechanism of other autoimmune diseases.

Hereinafter, the present invention will be described in more detail with reference to experimental examples, but the scope of the present invention is not limited to these experimental examples. In addition, the preparation method of the patient used in the following experimental examples, and the bacteria, bacterial lysate and platelets used in the experiment is as follows:
(1) Patient
Fifteen Japanese adult cITP patients (2 men, 13 women; average age 49 years) were included. cITP was determined based on thrombocytopenia (platelet count <120 × 10 ⁹ / l). These cITP patients were classified into 3 groups according to the following criteria:
(i) H. pylori negative group (n = 3) (hereinafter also referred to as “HP-N group”)
(ii) H. pylori positive and complete or partial remission with H. pylori eradication therapy (n = 7) (hereinafter also referred to as “CR group”)
(iii) A group positive for H. pylori and not ameliorated by H. pylori eradication therapy (n = 5) (hereinafter also referred to as “NR group”).

The ¹³ C-urea breath test was used to determine the presence of H. pylori infection. The presence or absence of H. pylori eradication by H. pylori eradication was evaluated 8 weeks after the H. pylori eradication treatment using the above test. Clinical response to H. pylori eradication (variation in platelet count) was assessed 4 months after eradication. In clinical response, the case where the number of platelets increased to more than 120 × 10 ⁹ / l, "cITP complete response", 20 × 10 ⁹ / l than before treatment until platelet count is not more than 120 × 10 ⁹ / l The case where it increased above was defined as “cITP partial remission”. Patient serum samples were stored at −80 ° C. until use.

(2) Bacteria
Campylobacter jejuni (hereinafter referred to as “C. jejuni”) is a clinical isolate derived from a Japanese patient suffering from infectious enteritis, a clinical isolate derived from a Japanese or Westerner suffering from gastric ulcer or gastric cancer as H. pylori Isolates were used. Specifically, C.jejuni and H. pylori KMT3 (patients with stomach cancer) were both Kochi University Medical Hospital, and H. pylori HPK5 (gastric ulcer patient) was a clinical isolate isolated at Yamaguchi University Medical Hospital. It is. H. pylori NCTC11637 (gastric ulcer patients, USA) and 26695 (gastric ulcer patients, Europe) are standard foreign clinical isolates that are widely used worldwide.

  PCR confirmed that all H. pylori strains (HPK5, NCTC11637, 26695) except KMT3 have ureA, vacA and cagA genes, and KMT3 has ureA and vacA genes other than cagA gene .

(3) Preparation of bacterial cell lysate
H. pylori and C. jejuni strains are cultured for 3 days at 37 ° C. under microaerobic conditions on Brucella-broth (Becton Dickinson) agar supplemented with 10% horse serum. Further, E. coli strain (ATCC 25922) is cultured on Mueller Hinton II agar (Becton Dickinson) medium at 37 ° C. for 1 day under aerobic conditions. These bacteria are then collected, washed twice with PBS, and suspended in PBS or sterile pure water. The resulting suspension is sonicated on ice using a tip type UP 50H sonicator (Dr. Hielscher GmbH) until clear and then centrifuged at 8,000g for 15 minutes at 4 ° C. The supernatant is collected as a bacterial cell lysate. The prepared bacterial cell lysate was stored at −80 ° C. until use.

(4) Preparation of platelets Platelets were prepared from healthy subjects. Using a sterilized venous puncture (21 gauge needle), collect whole blood from a healthy person who has not undergone drug treatment, and use 1/9 volume of 3.8% sodium citrate aqueous solution to prevent blood clotting. Diluted with By centrifugation at 150 g for 20 minutes at room temperature, plasma containing a large amount of platelets (hereinafter also referred to as “platelet-rich plasma” or “PRP”) was obtained. On the other hand, plasma containing little platelets (hereinafter also referred to as “low platelet plasma” or “PPP”) was prepared by centrifuging the PRP at 2,000 g for 15 minutes at room temperature. All cITP patients in the CR and NR groups have confirmed that there are no abnormalities in the platelets themselves by blood tests on whole blood and bone marrow.

Experimental Example 1 Platelet Aggregation Test (1) To 200 μl of PRP (platelet-rich plasma) containing about 250 × 10 ⁹ / l platelets, various bacteria [H. pylori (NCTC11637, 26695, HPK5 and KMT3), E. coli, C .Jejuni] cell lysate was added and mixed, and the amount of platelet aggregation was measured. Platelet aggregation was measured using an automated platelet aggregometer (MCM Hema Tracer 212 MC Medical, Japan). Specifically, for platelet aggregation, the light transmittance of PRP before addition of cell lysate was set to 0%, and the light transmittance of PPP (low platelet plasma) was set to 100%, and bacterial cell lysis in PRP The percentage of light transmission after the addition of the product was evaluated by recording at 10 minute intervals.

In addition, PBS or sterilized pure water (dH ₂ O) was used instead of the bacterial cell lysate as a negative control, and adenosine diphosphate (ADP) that causes platelet aggregation in PRP was used as a positive control. Thus, the amount of platelet aggregation was measured. In addition, each test was performed 3 times using platelets derived from 3 different healthy subjects. The test was completed within 2 hours after blood collection.

  The results of tracking platelet aggregation over time are shown in FIG. The horizontal axis of each graph in FIG. 1 represents the reaction time (minutes) between PRP and cell lysate, and the vertical axis represents the amount of platelet aggregation (%) calculated from the ratio of light transmittance. As can be seen from FIG. 1, platelets showed clear platelet aggregation in response to all cell lysates of the four H. pylori (NCTC11637, 26695, HPK5 and KMT3) (in FIG. 1, 26695 and KMT3). Only the results are shown). On the other hand, it did not aggregate with the lysate of E.coli and C.jejuni. From these results, it was considered that the ability to aggregate with platelets is unique only to cell lysates of H. pylori. Moreover, there is no difference in the degree of platelet aggregation between H. pylori strains lacking the cagA gene (KMT3) and non-cag strains (NCTC11637, 26695, HPK5), and clinical isolation isolated from Japanese and foreigners. There was no difference in the degree of platelet aggregation between the objects. From this, it was judged that the platelet aggregation reaction to H. pylori was independent of the geographical distribution of H. pylori and elements based on the cagA gene.

  (2) Platelet aggregation due to the reaction of the above PRP with various bacterial cell lysates was also confirmed by visual observation using an optical microscope. Specifically, a part of each sample after the reaction was smeared on a glass slide and dried at room temperature. In order to stain, Wright stain (Muto Chemical Co., Ltd.) was dropped onto the sample of each glass slide after drying, and incubated for 5 minutes. Next, an equivalent amount of PBS was added dropwise, mixed gently, further washed with PBS, dried at room temperature, and then observed with an optical microscope. FIG. 2 shows the state of platelet aggregation due to the reaction of PRP with cell lysates of various bacteria [H. pylori (26695, KMT3), E. coli]. In the figure, the result shown by PBS is an optical microscope image of a mixture of PRP and PBS (control). The upper figure shows the unstained optical microscope image, and the lower figure shows the stained optical microscope image stained with the Wright solution. As can be seen from FIG. 2, platelet aggregation (indicated by arrows in the figure) was observed only in the cell lysate of H. pylori. In the figure, the scale bar is 20 μm.

Experimental Example 2 Immunoblotting In order to identify how the reactivity (antibody profile) of antibodies to H. pylori antigens differs among cITP patients (HP-N, CR and NR groups), the serum of cITP patients ( Antibody) and various proteins (antigens) contained in H. pylori cell lysate were examined.

  Specifically, H. H. pylori cells were dissolved in a sample buffer (125 mM Tris, 4% SDS, 12% mercaptoethanol, 20% glycerol, and 0.004% BPB), and then heat-treated at 95 ° C. for 10 minutes. This was size fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (containing 12.5 wt% acrylamide and 0.1 wt% sodium dodecyl sulfate) and blotted onto a nitrocellulose membrane. . The membrane was blocked with Tris-buffered saline (TBS-T) containing 0.1% Tween 20, cITP patient serum as the first antibody, and horseradish peroxidase-conjugated goat anti-human IgG polyclonal antibody (hereinafter referred to as the second antibody). And “HRP-anti-human IgG antibody”) (Invitrogen). The membrane was then washed 4 times with TBS-T, incubated with ECL + PLUS reagent (RPN 2132; Amersham Pharmacia Biotech AB) for 5 minutes at room temperature and exposed to photographic X-ray film (50NIF, Fujifilm) for visualization did.

The results regarding cITP patients in each group (HP-N group: 3 people, NR group: 5 people, CR group: 7 people) are shown in FIG. As shown in FIG. 3, the sera of patients in the CR group (H. pylori positive / disinfecting) have a high molecular weight protein of 50 kDa or more and a relatively low molecular weight protein of 36, 27 and 17 kDa. Reacted. In addition, these molecular weights are molecular weight markers (proteins of known molecular weight) having molecular weights of 10 kDa, 15 kDa, 20 kDa, 25 kDa, 37 kDa, 50 kDa, and 75 kDa (Precision Plus Protein ^™ All Blue Stabdards: Bio -Rad (Catalog No.161-0373)) was identified by comparing with the band position.

  These results are summarized in Table 1. The results show the number of specimens in the denominator and the number of specimens that reacted with each low molecular weight protein in the numerator.

  As shown in Table 1, the low molecular weight protein (17,27,36 kDa) is 100% (7 out of 7) of cITP patients in the CR group (H. pylori positive / disinfecting) Combined. On the other hand, these proteins bind only to 40% (2 out of 5) of sera from cITP patients in the NR group (H. pylori positive / no sterilization effect), and the HP-N group (H It did not react at all with sera from cITP patients with.

Experimental Example 3 ELISA analysis IgG antibody amount (titer) against H. pylori cell lysate (total protein) contained in serum before and after eradication of H. pylori in each cITP patient (HPN group, CR group or NR group) Was evaluated by the following ELISA protocol.

Specifically, 50 μl (1.6 μg / ml) of H. pylori cell lysate or E. coli cell lysate dispersed in a coating buffer (20 mM carbonate) was added to a 96-well flat bottom plate (Sumitomo Bakelite) And incubated overnight at 4 ° C. to coat plates (wells) with these cell lysates. The plates are then washed 5 times with Tris-buffered saline (TBS-T) containing 0.1% Tween 20, and then 250 μl blocking buffer (TBS-T containing 2% skim milk to prevent non-specific reactions). ) Was added. The resulting mixture was incubated for 1 hour at room temperature and washed 5 times with TBS-T. 50 μl of a 1/5000 dilution of individual cITP patient serum was added to each well and incubated for 1 hour at 37 ° C. and finally washed 5 times with TBS-T. 50 μl of a 1/2000 dilution of HRP-anti-human IgG antibody (Invitrogen) was added to each well, incubated for 1 hour at 37 ° C., and washed 5 times with TBS-T. 50 μl of 3,3 ′, 5,5′-tetramethylbenzidine substrate (BioLegend) was added to each well and incubated for 30 minutes at room temperature. The reaction was stopped by adding 50 μl stop buffer (2MH ₂ SO ₄ ) and analyzed using a plate reader with 450 nm filtering (Multiscan JX, Termo electron corporation). The antibody titer was shown as absorbance at a wavelength of 450 nm.

  The results are shown in FIG. In the figure, black bars indicate antibody values before H. pylori eradication therapy, and white bars indicate antibody values after H. pylori eradication therapy. As shown in FIG. 4, the amount of antibody against H. pylori cell lysate was significantly decreased only in the CR group, and no significant difference was observed in the decrease in the NR group.

Experimental Example 4
200 μg of cell lysate of H. pylori (26695 strain) was added to PRP (containing 5 × 10 ⁷ platelets) and incubated for 15 minutes at 37 ° C. The complex of 17 kDa protein derived from H. pylori and platelets was recovered as a pellet by centrifugation (10,000 rpm, 10 minutes), and this was washed 3 times with PBS. Then, 1 ml of lysis buffer [50 mM Tris-HCl, pH 7.4; 1% Nonidet-P-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM ethylene glycol-bis (β-aminoethyl) -N, N , N ′, N′-tetraacetoxymethyl ester; 1 mM sodium fluoride; 100 μM sodium orthovanadate; 100 μM phenylmethylsulfonyl fluoride; protase inhibitor (Roche Diagnostics)), incubated on ice for 30 minutes, 4 The supernatant was obtained by centrifugation at 15 ° C. for 15 minutes. 1 ml of this supernatant was reacted with rabbit anti-human platelet polyclonal antibody (5 μg) (Cortex Biochem) for 2 hours at 4 ° C., and then immunoprecipitation was promoted with protein G PLUS-agarose (Santa Cruz Biotechnology).

  The resulting immunoprecipitate was washed 5 times with lysis buffer, subjected to SDS-PAGE, separated and transferred to a nitrocellulose membrane. This was immunoblotted with serum from cITP patients (CR group, NR group).

  As a result, it was confirmed that, among H. pylori low molecular weight proteins, a low molecular weight protein of 17 kDa mainly binds to platelets, and the 17 kDa protein reacted strongly with the serum of cITP patients in the CR group (Fig. 5). As shown in Experimental Example 2, since antibodies against the 17 kDa protein are specifically present in the serum of cITP patients in the CR group, the 17 kDa protein, platelets and 17 There is a possibility that an immune complex is formed in which antibodies against the kDa protein are bound to each other.

Experimental Example 5
In order to identify the 17 kDa protein that was determined to form a complex with platelets in Experimental Example 4 above, H. pylori lysate was subjected to two-dimensional electrophoresis (one-dimensional is agarose IEF (isoelectric focusing), then the sample is developed and separated by SDS-PAGE (two-dimensional electrophoresis)) (The conceptual diagram is shown in FIG. 6).

  Specifically, as shown below, Two gels were prepared by subjecting the H. pylori lysate to two-dimensional electrophoresis (agarose IEF, SDS-PAGE). One of the gels was transferred to a PVDF membrane and then treated in the same manner as in Experimental Example 2 (in patients with H. pylori-infected ITP patients whose platelet count was recovered by H. pylori eradication, so-called H. pylori). Western blotting was performed on a related ITP patient), and a spot where a reaction was observed around a molecular weight of 17 kDa was confirmed. Using another gel (silver staining to visualize the protein), the protein separated at the same position as the spot was cut out from the gel (see FIG. 6B). This was subjected to protein identification mass analysis (TOF MS: requested by Shimadzu Corporation) to identify a 17 kDa protein.

<Two-dimensional electrophoresis and protein identification mass analysis>
(1) H. H. pylori cells are dissolved in sample buffer (60 mM Tris, 5 M urea, 1 M thiourea, 1% CHAPS, 1% Triton X-100, 1% DTT and complete ^TM mini EDTA-free (add 1 tablet per 10 ml)) And then centrifuged at 15,000 rpm for 30 minutes at 4 ° C. Furthermore, add 1/10 volume of 1M acrylamide solution of the sample solution, mix, and overlay on the agarose gel (pH 3-10) packed in the electrophoresis column of the sample solution that was allowed to stand for 10 minutes. Point electrophoresis was performed.
(2) The gel after electrophoresis was fixed with trichloroacetic acid, washed, and equilibrated with SDS equilibration solution.
(3) The obtained gel was further subjected to second-dimensional electrophoresis (SDS-PAGE using 12.5 wt% acrylamide and 0.1 wt% sodium dodecyl sulfate).
(4) After electrophoresis of the gel after silver staining, a spot with a molecular weight of about 17 kDa cut out from the gel was subjected to PMF (Peptide Mass Fingerprint) analysis (requested from Shimadzu Corporation).
(5) PMF analysis was performed by subjecting the sample to enzyme digestion with trypsin, desalting and purification, and directly attaching the eluted sample to a MALDI plate. This was air-dried, then subjected to air-drying using a matrix solution, and then subjected to mass spectrometry using an AXIMA-CFR laser ionization time-of-flight mass spectrometer (MALDI-TOFMS).

  From the above two-dimensional electrophoresis and Western blotting analysis results, it was found that the 17 kDa protein has a molecular weight of 17 ± 1-2 kDa and an isoelectric point in the range of pH 5-8. In addition, from the results of protein identification mass analysis, one of the 17 kDa proteins is the invA protein of H. pylori (in terms of functional classification, cellular processes (proteins involved in cell synthesis and formation processes) and hydrolase activity (proteins involved in hydrolysis). )].

<Discussion>
As shown in FIG. 3 and Table 1, sera from cITP patients belonging to the CR group reacted significantly with small molecules of H. pylori cell lysate (36, 27 and 17 kDa). This indicates that H. pylori low molecular weight protein is involved in the development of H. pylori-related cITP (Experimental Example 2). In addition, antibodies against low molecular weight proteins (36, 27 and 17 kDa) were not detected in H. pylori-infected persons who were not cITP and in H. pylori non-infected persons including healthy persons. The result of Experimental Example 4 shows that among these low molecular weight proteins, a 17 kDa protein (isoelectric point pH 5-8) binds to platelets, and this complex is an antibody (IgG antibody) in the serum of cITP patients belonging to the CR group. It shows that it reacts remarkably. From these facts, it is considered that an immune complex composed of platelets, H. pylori 17 kDa protein (antigen), and an antibody against the 17 kDa protein (antigen) plays an important role in the development of H. pylori-related cITP.

It is a figure which shows the amount of platelet aggregation over time after adding cell lysate of various bacteria [H. pylori (NCTC11637, 26695, HPK5 and KMT3), E. coli, C. jejuni] to PRP (experimental example) . For H. pylori, only 26695 and KMT3 results are shown. The results of using sterilized pure water (dH ₂ O) instead of the negative control and cell lysate, and adenosine diphosphate (ADP) as the positive control are also shown. The horizontal axis of each graph shows the reaction time (minutes) between PRP and cell lysate (or dH ₂ O, ADP), and the vertical axis shows the amount of platelet aggregation (%) calculated from the ratio of light transmittance. The optical microscope image which shows the mode of platelet aggregation by reaction with PRP shown in FIG. 1 and the cell lysate of various bacteria [H. pylori (26695, KMT3), E. coli] is shown. In the figure, the result shown by PBS is an optical microscope image of a mixture of PRP and PBS (control). The upper figure shows an unstained optical microscope image, and the lower figure shows a stained optical microscope image stained with Wright solution. In the figure, the arrow indicates platelet aggregation, and the scale bar indicates 20 μm. The result of immunoblotting the H. pylori cell lysate with the serum of cITP patients (HP-N group, CR group and NR group) is shown. In the figure, A shows the results after H. pylori eradication therapy and B shows the results before H. pylori eradication therapy (Experimental Example 2). The amount of IgG antibody against the H. pylori cell lysate contained in the serum before (black bars) and after (open bars) H. pylori eradication therapy for each cITP patient (HPN group, CR group or NR group) (Experimental Example 3) is shown. O.D. 450 nm on the vertical axis means the absorbance at 450 nm corresponding to the amount of IgG antibody. Results are expressed as mean ± SE. *: p <0.05, **: p <0.01. Figure A shows, from above, the 17 kDa protein in the cell lysate of H. pylori strain (26695), the immunoprecipitation component of only platelets (middle), and the immunoprecipitation after reaction of platelets with H. pylori strain (26695) cell lysate. The result of immunoblotting the component (17 kDa protein) (bottom) and the serum of each cITP patient in the CR group is shown. FIG. B shows, from the top, the 17 kDa protein (upper) in the cell lysate of H. pylori strain (26695), the immunoprecipitation component of only platelets (middle), and the immunoprecipitation after reaction of platelets with H. pylori (26695) cell lysate. The result of immunoblotting the component (17 kDa protein) (bottom) and the serum of each cITP patient in the NR group is shown. FIG. A shows a conceptual diagram of two-dimensional electrophoresis (separation / development by isoelectric point and separation / development by molecular weight) performed in Experimental Example 5. FIG. B shows the result of two-dimensional electrophoresis (silver staining) performed in Experimental Example 5.

Claims (8)

A method for detecting H. pylori-related idiopathic thrombocytopenic purpura, comprising detecting an antibody against a protein derived from H. pylori identified as a molecular weight of 17 kDa by SDS-PAGE. SDS-PAGE contained in H. pylori cell lysate or its preparation is reacted with H. pylori-derived protein identified as having a molecular weight of 17 kDa and subject's serum, and the presence or absence of antigen-antibody reaction is used as an index. a step of detecting antibodies that react to the protein from H. pylori which is identified as the molecular weight 17kDa with -PAGE, detection method of claim 1, wherein.   The detection method according to claim 1 or 2, wherein the H. pylori-derived protein identified as having a molecular weight of 17 kDa has an isoelectric point of pH 5-8. A method for screening active ingredients for the prevention or treatment of H. pylori-related idiopathic thrombocytopenic purpura having the following steps:
(1) H. pylori-derived protein (17 kDa protein) and platelets, or antibodies against 17 kDa protein, platelets and 17 kDa protein, identified as SDS-PAGE with a molecular weight of 17 kDa in the presence or absence of the test substance Or a step of mixing components containing the same,
(2) Complex production amount (target production amount) generated when 17 kDa protein and platelets, or 17 kDa protein and platelets and antibodies against 17 kDa protein or components containing the same are mixed in the presence of the test substance, In the absence, the 17 kDa protein and platelets, or the complex production amount (control production amount) that occurs when the 17 kDa protein and platelets are mixed with antibodies against the 17 kDa protein or components containing the same, are compared.
(3) A step of selecting, as an active ingredient of a preventive or therapeutic agent for H. pylori-related idiopathic thrombocytopenic purpura, a test substance when the target production amount is reduced compared to the control production amount.   The method according to claim 4, wherein the 17 kDa protein has an isoelectric point of pH 5-8.   The method according to claim 4 or 5, wherein the amount of platelet aggregation is used as the amount of complex production. The method according to any one of claims 4 to 6, wherein the component containing an antibody against the 17 kDa protein is serum of a patient suffering from H. pylori-related idiopathic thrombocytopenic purpura.   8. The method of claim 7, wherein the serum of a patient suffering from H. pylori-related idiopathic thrombocytopenic purpura is type B.

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