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WO1996012965A1 - Methodes de diagnostic de l'helicobacter pylori et necessaires correspondants - Google Patents

Methodes de diagnostic de l'helicobacter pylori et necessaires correspondants Download PDF

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Publication number
WO1996012965A1
WO1996012965A1 PCT/IB1995/001028 IB9501028W WO9612965A1 WO 1996012965 A1 WO1996012965 A1 WO 1996012965A1 IB 9501028 W IB9501028 W IB 9501028W WO 9612965 A1 WO9612965 A1 WO 9612965A1
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WO
WIPO (PCT)
Prior art keywords
helicobacter pylori
solid support
antigen
infection
antibody
Prior art date
Application number
PCT/IB1995/001028
Other languages
English (en)
Inventor
Lily Chan
Randolph Moeckli
Daria Foong Yun Chin
Original Assignee
Genelabs Diagnostics Pte Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genelabs Diagnostics Pte Ltd. filed Critical Genelabs Diagnostics Pte Ltd.
Priority to AU38143/95A priority Critical patent/AU3814395A/en
Publication of WO1996012965A1 publication Critical patent/WO1996012965A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56922—Campylobacter
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)

Definitions

  • T e present invention relates to methods and kits for rapid in vitro detection of Helicobacter pylori infection in mammals. More particularly, the present invention relates to methods of detecting antibodies which are associated with Helicobacter pylori infection and related disease.
  • Helicobacter pylori is a curved Gram-negative bacterium that is commonly present in the human stomach; once acquired, this organism persists for decades (Blaser, M.J.,
  • a non-invasive test is the "Carbon-Labeled Urea Breath Test". Although this test is non-invasive it can be impractical because it utilizes urea labeled with l3 C and l4 C, and reading the test requires either a mass spectre-photometer or a gamma counter, respectively. Serological testing presents an alternative to the invasive tests and urea breath test described above. Individuals infected with H. pylori will mount a significant humoral response to H. pylori proteins (Jones. D.M., et al., J Med Micro (1986) 22:57-62).
  • ELISAs for example, has been a useful measure of the success of antibacterial treatment (Kosunen, T.U., et al.. Lancet (1992) 339:893-5).
  • kits for detecting Helicobacter pylori infection produced by a process of providing a Helicobacter pylori antigen, separating the antigen components, and transferring said antigen components to a solid support.
  • Another embodiment of the invention is a method for detecting infection with
  • the method includes, providing a solid support having Helicobacter pylori proteins bound thereto, reacting biological fluid with the solid support, examining the solid support for the presence of bound antibody and positively identifying infection when bound antibody is found at any two positions on the solid support corresponding to antigens having molecular weights of 19.5kDa, 26.5kDa. and 30kDa, or alternatively, when bound antibody is found at any position on the solid support corresponding to an antigen having a molecular weight of 35kDa, 89kDa, 1 16kDa. or 180kDa.
  • a further embodiment of the invention includes a method for diagnosing disease associated with Helicobacter pylori infection; e.g., gastritis, peptic ulcers, and gastric carcinoma.
  • the method includes providing a solid support having electrophoretically separated Helicobacter pylori antigen bound thereto, reacting biological fluid from the patient with the solid support, examining the solid support for the presence of bound antibody, and comparing the profile of bound antibody to consensus profiles obtained from other patients with confirmed disease conditions.
  • Fig. 1 is a representative Western Blot strip containing separated H. pylori antigen components reacted with a seropositive serum. Molecular weights (in kD) of the various major reactive antigen components are marked, and the putative identification of the major antigen components is provided. Band labelled "Control" is the serum addition control band.
  • Fig. 2 shows Reactivity with Clinical Samples - a Western Blot strip containing separated H. pylori antigen components reacted with four serum samples from patients which were histology, culture, and ELISA positive (Lanes 1-4) and four serum samples from patients which were histology, culture, and ELISA negative (Lanes 5-8).
  • Antigen is used in its broadest sense and includes Helicobacter pylori whole cells or homogeneous, near homogeneous or heterogeneous extracts from H. pylori and which is capable of binding to specific antibody in a biological fluid.
  • Antigen components contemplated by the present invention include protein, polysaccharide or lipid or any combination thereof.
  • the antigen is protein, lipopolysaccharide or cell extract of H. pylori prepared by, for example, sonication, pressure disintegration, detergent extraction or fractionation.
  • a “biological fluid” is any fluid derived from the body of a mammal. Biological fluids may be, for example, blood, serum, urine, mucous, gastric secretions or saliva.
  • a “reporter molecule” means a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen- bound antibody.
  • Helicobacter pylori related disease means any disease caused in whole or in part by infection with H. pylori. Such disease may be, for example, gastritis, peptic ulceration, and gastric adenocarcinoma.
  • an individual's "antibody profile” is the pattern created by antibodies contained in the individual's bodily fluid that are reactive with separated H. pylori antigen components.
  • an individual's antibody profile may be represented, for example, by the pattern of antigen component bands which are detected in a western blot assay.
  • a "consensus profile” is an artificially created antibody response profile based a compilation of the most common antibody responses within a particular H. pylori associated disease.
  • Helicobacter pylori may be obtained from the American Type Culture Collection (ATCC, Rockville, Md.). The preferred embodiment contemplates the use of strain designation NCTC11916. This strain is an ulcer producing strain which contains all the proteins associated with pathogenesis and immunologically important antigen components, including CagA, vacuolating protein, and the various Urease subunits. Other strains which contain these proteins are known in the art and can be utilized as well.
  • the antigen preparation was obtained as follows and as detailed in
  • Example 1 Briefly, bacteria were cultured in broth base medium to a suitable density. These were harvested by centrifugation and washed several times in PBS. The pellet was weighed, gently sonicated and resuspended in PBS to a concentration of 100 mg/ml of wet weight pellet. At this point the sonicate may be stored in small aliquots at -70 degrees C for future use. Alternatively, an H. pylori antigen preparation is available commercially from Microbix Biosystems (Ontario, Canada).
  • Antigen components are separated by size according to the present invention. Such size fractionation is preferentially accomplished electrophoretically but may be accomplished by other methods known to those skilled in the art; e.g., chromatography.
  • H. pylori antigenic components are separated as follows, and detailed in Example 1.
  • the H. pylori antigen preparation is loaded on a poly acrylam ide gel which may or may not contain individual wells. Preferably about .66-10 ug antigen preparation is loaded on the gel per centimeter of well width. More preferably about 5 ug cm well width is loaded.
  • Prior to loading the antigen preparation is boiled in order to denature the antigen components, preferably for about 5 minutes.
  • the poly aery lam ide gel can be any concentration determined to give adequate resolution of the desired antigen components described infra.
  • the polyacrylamide gel consists of two parts; a resolving gel, preferably between 10 and 13% acrylamide and a stacking gel, preferably between 4 and 8% acrylamide. In the most preferable embodiment the polyacrylamide gel has an 11.5% resolving section and a 6% stacking section. Electrophoresis is conducted until the antigen components have separated sufficiently to discriminate between them. It is preferable to start electrophoresis with a relatively low current until the pyronin Y had migrated into the resolving gel at which point the current is increased until the Pyronin Y has migrated to between about 9 and 12 cm into the resolving gel.
  • the gel is removed from the glass plates and the upper gel and the bottom portion of the resolving gel from the tracking dye downward is cut off. Separation conducted under the preferred conditions described above provides optimum separation of particularly useful antigen components as will be seen below.
  • molecular weight markers can be run adjacent to the H. pylori antigen. After electrophoresis, the position of the molecular weight markers may be determined by techniques known in the art for example, coomassie blue staining the polyacrylamide gel or ponceau S stain after transfer to the solid support.
  • the antigen preparation will for convenience and preference be bound to a solid support.
  • Suitable solid supports include a nitrocellulose membrane, glass or a polymer.
  • the most commonly used polymers for this purpose are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene, but the invention is not limited to them.
  • the solid support is preferentially in the form of strips, but may also be tubes, beads, discs, or microplates, or any other surface suitable for conducting an immunoassay.
  • Antigen components of H. pylori useful in this invention may be either covalently or non-covalently ("passively") bound to the solid surface.
  • Suitable binding processes are well known in the art and generally consist of crosslinking, covalently binding or physically adsorbing the antigen to the solid support.
  • Example 1 a piece of nitrocellulose membrane which has been presoaked in a Tris-based buffer, is layered onto a piece of wet blotting paper, and the trimmed gel is laid onto the nitrocellulose paper. Another piece of wet blotting paper is laid over the trimmed gel and this sandwich is placed into a transfer tank and transferred by applying a voltage.
  • the membrane is removed from the transfer apparatus after the transfer process and placed in IX PBS until optionally slotting on of additional proteins such as anti-human IgG, anti-human IgA, or recombinantly or synthetically produced peptide Helicobacter pylori antigens.
  • Such additional proteins are useful, for example, as positive or negative controls suitable for the type of biological fluid being tested.
  • H. pylori peptide antigens which encompass specific eptiopes can be useful as positive controls and for confirming infection. Slotting techniques are well known to those of skill in the art.
  • the membrane is removed from the slotting apparatus and blocked, preferably in PBS buffer containing about 5% non-fat dry milk at room temperature on a rocker platform.
  • the nitrocellulose is rinsed in PBS-Tween buffer and the sheets are then air dried on paper towels, and left in a 37 degree C incubator overnight for drying. Sheets are cut into convenient sized strips, preferably about 3mm wide and kept in dry containers until used in an assay.
  • the invention is directed to a method of diagnosing individuals with Helicobacter pylori infection wherein a solid support with separated antigen components bound thereto is used to examine an individual's biological fluid for the presence of H. /> / ⁇ ri-specific antibodies.
  • Biological fluid to be tested for H. pylori-specific antibodies are contacted with the solid support described above.
  • the formation of antigen/antibody complex is then detected by conventional techniques, generally by reaction with a reporter-labelled secondary antibody which is specific for the type of biological fluid tested.
  • reporter molecules in this assay are either enzymes, fluorophores or radionuclide containing molecules (i.e., radioisotopes).
  • an enzyme is conjugated to the secondary antibody, generally by means of glutaraldehyde or periodate.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, Beta-galactosidase and alkaline phosphatase, among others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change.
  • the serum samples described above were reacted with the solid support prepared above by western blot assay. Briefly, the strips with electrophoretically separated H. pylori antigenic components bound thereto were placed into reaction trays, one strip per well. The strips were pre- wetted in wash buffer (Tris buffer containing "Tween 20TM” detergent). After 10 minutes, the buffer was aspirated, and blotting buffer, containing Tris, inactivated goat serum, and 5% non-fat dry milk, was added. Human serum samples were added to individual wells to a final dilution of 1/100 and incubated for one hour at room temperature.
  • wash buffer Tris buffer containing "Tween 20TM” detergent
  • Negative 4 12 4 1 1 2 0 6/110
  • antigen components which typically react have approximate molecular weights of 116kDa (possibly the CagA protein), 89kDa (possibly the Vacuolating protein), 60kDa (possibly the ureB protein or the hsp60 heat shock protein), 35kDa (unknown), 30kDa (possibly the ureH protein), 26.5kDa (possibly the ureA protein), and 19.5kDa (possibly the ferritin like protein).
  • Figure 1 is a Western Blot strip which shows a reactivity profile including all of the above mentioned antigen components.
  • pylori infection if it is reactive with any two antigen components corresponding to molecular weights of 19.5kDa, 26.5kDa, or 30kDa, or alternatively, if it is reactive with any one antigen component corresponding to a molecular weight of 35kDa, 89kDa, 116kDa, or 180kDa.
  • Sensitivity of the assay may be defined as a fraction equal to the number of positive serum samples which were actually western blot positive divided by the number of patients which were expected to be positive.
  • specificity of the assay may be defined as a fraction equal to the number of serum samples which were accurately diagnosed divided by the total number of samples tested.
  • specificity of the assay is 96% sensitive (49/51) and 95% specific (153/161) for Helicobacter pylori infection.
  • the antigen specific criteria for a positive diagnosis may be altered; e.g., additional antigen components may be added to the assay, currently evaluated antigen components may be deleted from the assay, and different combinations may be required for a positive test result.
  • the invention is directed to a method of diagnosing a Helicobacter pylori associated disease by examining that individual's biological fluids for antibodies to Helicobacter pylori antigen components and comparing the profile of antibody reactivity to a consensus profile.
  • H. pylori may have different clinical outcomes ranging from gastritis to peptic ulcers and gastric cancer. It is as yet unclear why a particular individual's only clinical outcome is gastritis while another individual will develop more serious disease. Without being bound to a particular theory, these clinical outcomes may represent progressive stages of disease, certain individuals may be predisposed to peptic ulcers and gastric carcinoma, and/or environmental factors may influence disease progression. It is further possible that predisposition to a clinical outcome may be related to immunologic factors.
  • results obtained with the solid phase assay, described above and shown in Figure 2 indicate that there is a heterogeneous antibody response to infection with H. pylori; i.e., different infected individuals generate antibodies to different antigen components. It has been discovered that while the infected population produces a heterogeneous antibody response, subpopulations have homogeneous antibody responses. Furthermore, these subpopulations of reactivity profiles may be linked to clinical outcome. For example, individuals who suffer from H. pylori induced gastritis are likely to show a similar antibody responses which would be different than an individual suffering from H. pylori induced gastric adenocarcinoma.
  • a library of antibody response profiles is produced, according to the invention, by first diagnosing individuals for H. pylori related disease with standard methods known in the art (e.g., culture, histology, and ELISA) and subsequently obtaining antibody response profiles from them. The library of antibody response profiles is then used to determine the highest correlation between disease and antibody response, thereby forming a consensus profile for each disease.
  • standard methods known in the art e.g., culture, histology, and ELISA
  • Diagnosis of a test individual is then made by testing the individual's biological fluid in order to obtain an antibody response profile and then comparing the proflle to the consensus profiles for each disease. If criteria for a match is met for any one of the diseases, a positive diagnosis is made.
  • Helicobacter pylori strain NCTC 11916 is obtained from the American Type Culture Collection (Rockville, MD).
  • Anti-human IgG can be obtained from the Berkeley Antibody Co. (Berkeley, CA).
  • the "Mighty SmallTM” transfer apparatus may be obtained from Hoeffer Scientific Instruments (San Francisco, CA).
  • EXAMPLE 1 Preparation of Helicobacter pylori Antigen A. Antigen Preparation
  • the Helicobacter pylori strain (NCTC 11916) obtained from the ATCC is grown in broth-based media to a suitable density. Specific ingredients are listed in Table 2 below.
  • the bacteria are harvested by centrifugation and washed several times in Phosphate Buffered Saline (PBS). The pellet is weighed, gently sonicated and resuspended in PBS to a concentration of 100 mg/ml of wet pellet.
  • PBS Phosphate Buffered Saline
  • the protein concentration of the antigen is determined by the Bradford protein assay. Approximately 80 ug of the antigen preparation is mixed in a volume of 800 ul with sample buffer containing glycerol, 2-mercaptoethanol, and pyronin Y. This mixture is boiled for 5 minutes and then layered in the well of a polyacrylamide gel having a 6% stacking portion and an 11.5% resolving portion.
  • molecular weight markers comprised of: Myosin 200 kD, Phosphorylase b 97.4 kD, Bovin Serum Albumin 69 kD, Ovalbumin 46 kD, Carbonic Anhydrase 30 kD, Trypsin inhibitor 21.5 kD, and Lysozyme 14.3 kD; are added in an adjacent well.
  • Polyacrylamide gel electrophoresis (PAGE) was conducted at 15 mAmps overnight and was increased to 30 mAmps. Pyronin Y runs at the buffer front and electrophoresis is allowed to continue until the pyronin Y has migrated 10.5 cm into the resolving gel.
  • the polyacrylamide gel is removed from the glass plates and the stacking gel and lower portion of the resolving gel below the pyronin Y is cut off.
  • a piece of nitrocellulose measuring 12.8 cm by 10.5 cm which has been presoaked in a Tris-based buffer (pH 8.3), is layered onto a piece of wet blotting paper, and the trimmed polyacrylamide gel is laid onto the nitrocellulose paper.
  • Another piece of wet nitrocellulose blotting paper is laid over the trimmed gel and this sandwich is placed into the transfer apparatus (Mighty SmallTM) and transblotted by applying a voltage of 30 V for 30 minutes and 100 V for 1.4 hours.
  • the membrane is removed from the transfer apparatus and stained with ponceau S. The position of each molecular weight marker is marked on the filter with indelible ink.
  • Commercially prepared anti-human IgG (Berkeley Antibody Co.) is diluted to a concentration of about 10 ug/ml in 0.1 M Carbonate buffer, pH 9.6. and slotted at approximately 1.6 cm from the bottom of the membrane, which is the area of the low molecular weight proteins.
  • the membrane is placed in blocking solution containing 5% non-fat dry milk in PBS pH 7.5 for 45 minutes on a platform rocker. The nitrocellulose is then washed for 15 minutes in PBS-0.05% polyoxyethylene (20) monolaurate "Tween 20TM" (Sigma).
  • the sheets are then air dried on paper towels, and left in a 37 degree C incubator overnight for drying. Sheets are cut into strips according to the width of each lane and the length of the gel from top to bottom and kept in dry containers for use.
  • Example 2 The strips processed in Example 1 were placed into reaction trays, one strip per well, on a platform rocker. The strips are pre-wetted in wash buffer (Tris buffer containing 0.05% Tween 20TM) for 10 minutes. The buffer is aspirated, and 2ml blotting buffer (Tris pH 7.4, 5% inactivated goat serum, and 5% non-fat dry milk) is added. To the blotting buffer is added 20 ul of patient serum (final dilution 1/100) and this is incubated for one hour at room temperature. The fluid is removed by aspiration and the strips are washed 3 times with wash buffer for 5 minutes each.
  • wash buffer Tris buffer containing 0.05% Tween 20TM
  • 2ml blotting buffer Tris pH 7.4, 5% inactivated goat serum, and 5% non-fat dry milk
  • Goat anti-human IgG (Berkeley Antibody Co., Berkeley, CA) conjugated to alkaline phosphatase diluted 1/1,000 with blotting buffer is added to the strips and incubated for one hour at room temperature. The strips are washed 3 times with wash buffer for 5 minutes each.
  • Helicobacter pylori antigen component bands reactive with patient serum are visualized after the addition of 2 ml of a nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate solution.

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Abstract

L'invention décrit un dosage aux fins de la détection d'une infection due à l'Helicobacter pylori. Ce dosage est destiné à la détection de ladite infection et à la surveillance de l'état infectieux faisant suite au traitement. Ce dosage, qui donne lieu à une immunoempreinte pour des prélèvements de fluide biologique, fait appel à un nécessaire, par le truchement duquel un antigène d'Helicobacter pylori est immobilisé sur un support de membrane. L'invention fait également état d'une méthode de diagnostic de maladies associées à une infection causée par l'Helicobacter pylori.
PCT/IB1995/001028 1994-10-20 1995-10-19 Methodes de diagnostic de l'helicobacter pylori et necessaires correspondants WO1996012965A1 (fr)

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AU38143/95A AU3814395A (en) 1994-10-20 1995-10-19 Helicobacter pylori diagnostic methods and kits

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US32663894A 1994-10-20 1994-10-20
US08/326,638 1994-10-20

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998012562A1 (fr) * 1996-09-20 1998-03-26 Cortecs International Limited Adhesines obtenues d'heliobacter pylori et leurs utilisations therapeutiques et diagnostiques
WO2000020862A1 (fr) * 1998-10-02 2000-04-13 Abp Diagnostics Ltd. Procede et appareil destines a la detection in vitro de substances multiples
WO2000070348A1 (fr) * 1999-05-14 2000-11-23 Onco Alert Pty Ltd Methodes permettant de prevoir et/ou diagnostiquer les risques de cancers gastriques
CN102967705A (zh) * 2012-11-26 2013-03-13 深圳市伯劳特生物制品有限公司 一种幽门螺杆菌分型检测的试剂盒

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0329570A2 (fr) * 1988-02-18 1989-08-23 Martin J. Blaser Compositions antigéniques composées de fragments de Campylobacter pylori et procédés pour leur production et utilisation
JPH03127735A (ja) * 1989-10-09 1991-05-30 Takeda Chem Ind Ltd 抗菌剤
WO1992019970A1 (fr) * 1991-04-26 1992-11-12 Emanuel Calenoff Methode de detection et de traitement des maladies causees par des allergenes bacteriens
WO1993022682A1 (fr) * 1992-04-29 1993-11-11 Auspharm International Limited Test in vitro pour helicobacter pylori
WO1995017677A1 (fr) * 1993-12-20 1995-06-29 Calenoff Emanuel J Methodes et preparations pour le diagnostic et le traitement d'affections dues aux antigenes de micro-organismes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0329570A2 (fr) * 1988-02-18 1989-08-23 Martin J. Blaser Compositions antigéniques composées de fragments de Campylobacter pylori et procédés pour leur production et utilisation
JPH03127735A (ja) * 1989-10-09 1991-05-30 Takeda Chem Ind Ltd 抗菌剤
WO1992019970A1 (fr) * 1991-04-26 1992-11-12 Emanuel Calenoff Methode de detection et de traitement des maladies causees par des allergenes bacteriens
WO1993022682A1 (fr) * 1992-04-29 1993-11-11 Auspharm International Limited Test in vitro pour helicobacter pylori
WO1995017677A1 (fr) * 1993-12-20 1995-06-29 Calenoff Emanuel J Methodes et preparations pour le diagnostic et le traitement d'affections dues aux antigenes de micro-organismes

Non-Patent Citations (1)

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Title
CHEMICAL ABSTRACTS, vol. 116, no. 10, Columbus, Ohio, US; abstract no. 91382d, T. IWANIKI ET AL.: "Bactericides containing pyridothiadiazinobenzimidazoles against Campylobacter." page 508; *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998012562A1 (fr) * 1996-09-20 1998-03-26 Cortecs International Limited Adhesines obtenues d'heliobacter pylori et leurs utilisations therapeutiques et diagnostiques
WO2000020862A1 (fr) * 1998-10-02 2000-04-13 Abp Diagnostics Ltd. Procede et appareil destines a la detection in vitro de substances multiples
WO2000070348A1 (fr) * 1999-05-14 2000-11-23 Onco Alert Pty Ltd Methodes permettant de prevoir et/ou diagnostiquer les risques de cancers gastriques
CN102967705A (zh) * 2012-11-26 2013-03-13 深圳市伯劳特生物制品有限公司 一种幽门螺杆菌分型检测的试剂盒

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