WO2007000046A1

WO2007000046A1 - Methods for diagnosing functional bowel disease

Info

Publication number: WO2007000046A1
Application number: PCT/CA2006/001060
Authority: WO
Inventors: Elizabeth C. Colley; Ronald H. Stead
Original assignee: HOLBURN BIOMEDICAL Corp
Current assignee: HOLBURN BIOMEDICAL Corp
Priority date: 2005-06-27
Filing date: 2006-06-27
Publication date: 2007-01-04
Anticipated expiration: 2007-12-27

Abstract

There is described a method for diagnosing a functional bowel disease in a human patient, wherein the method comprises testing gastrointestinal cells and/or cells circulating in peripheral blood of the patient for the presence of at least one of serotonin and a serotonin receptor.

Description

METHODS FOR DIAGNOSING FUNCTIONAL BOWEL DISEASE

BACKGROUND OF THE INVENTION

1. Field of the Invention [0001] The present invention relates to methods for the diagnosis and treatment of functional bowel diseases, in particular, irritable bowel syndrome, .and for methods to select the appropriate medicament for such treatment.

2. Discussion of Background Information [0002] Mast cells are involved in inflammatory and allergic responses. Thus, mast cells have been considered to be immune response elements, participating in a wide range of defense mechanisms against infectious diseases. Through the expression of immunoglobulin receptors, mast cells can participate in adaptive immune responses (GaIIi et al., 2005); and they can also regulate innate immune responses (Marshall & Jawdat, 2004). Over the last decade, it has become apparent that mast cells are also active participants in homeostatic processes, in part through their interaction with peripheral nerves (Gottwald et al., 1998). In fact, mast cells might act as sentinels, responding to a variety of stimuli by selective release of numerous mediators which they are known to synthesize and release (Stead et al. 2006). Activation of mast cells causes the recruitment of neutrophils and macrophages, as well as eosinophils at a later stage. Thus, stimulation of mast cells initiates a cascade of events known as inflammation (Marshall, 2004). Mast cells are bone marrow derived and migrate through the peripheral blood (generally as precursors) before homing to various tissues and organs (Weller et al., 2005).

[0003] Amongst the mediators released by mast cells immediately after activation is the biogenic amine histamine. It is well established that histamine is synthesized and released by mast cells from numerous tissues and many different species (Xie & He, 2005). Another biogenic amine, serotonin (5- hydroxytryptamine, 5-HT), is also known to be released by rodent mast cells but is generally considered to be absent from human mast cell types (although occasional publications have indicated to the contrary). It has surprisingly been found inter alia that mast cells in gastrointestinal tissues from some, but not all patients contain detectable levels of serotonin.

[0004] Many cell types express receptors for mediators that they release, and such receptors act in inhibitory processes, down-regulating the ongoing release of the mediator (Lippert et ai, 2004). Mast cells express three subtypes of histamine receptor (H 1 , H2 and H4; there is some suggestion that H3 might also be expressed). Thus, mast cells express auto-receptors for the biogenic amine that they release upon stimulation. [0005] Data exist to demonstrate that serotonin can also modulate mast cell activity through different subtypes of serotonin receptors. However, these data are restricted to non-human species, and there is to this point, no published evidence for the expression of serotonin receptors on human mast cells. It has surprisingly been determined, inter alia, that gastrointestinal mast cells express immunoreactivity for 5-HT₄ receptor in mouse, rat and human tissues. It has also been found that in cultured mast cells this receptor is functional, in that after treatment with a serotonin agonist or antagonist the response to a challenge with a mast cell stimulator (e.g., a calcium ionophore such as ionomycin) causes the inhibition of the mediator release. [0006] Functional bowel diseases (also called "functional bowel disorders") are, by definition, disorders with no known underlying pathology (Thompson et ai, 1999). They are generally considered to be collections of symptoms attributable to the gastrointestinal tract. Irritable bowel syndrome (IBS) is one of the functional bowel diseases. Other functional bowel diseases include, by way of non-limiting example, functional abdominal bloating, functional constipation, functional diarrhea, functional abdominal pain, functional dyspepsia, non-cardiac chest pain (NCCP), and chronic abdominal pain. Ethiopathogenic features of functional bowel diseases often include psychosocial disturbances, dysmotility and/or heightened sensitivity. IBS afflicts up to 20% of the adult population and accounts for up to 50% of visits to gastroenterologists. In addition to suffering from abdominal pain and discomfort, IBS sufferers also have altered bowel habits. Some patients suffer from chronic constipation, while others have diarrhea. Patients with diarrhea-predominant IBS benefit from treatment with 5-HT₃ receptor antagonists (De Ponti & Tonini, 2001), such as, e.g., alosetron (Lotronex produced by GSK). Other non-limiting examples of 5-HT₃ receptor antagonists include ondansetron, granisetron, dolasetron, tropisetron, renzapride and zacopride. Constipation-predominant IBS sufferers benefit from treatment with 5- HT₄ receptor agonists (De Ponti & Tonini, 2001 ) such as, e.g., tegaserod (Zelnorm produced by Novartis), renzapride and prucalopride. Other non-limiting examples of 5-HT₄ receptor agonists include 5 methoxytryptamine, and substituted benzamide prokinetics such as, e.g., metoclopramide and cisapride. Various mechanisms of action for these serotonin receptor acting drugs have been proposed, including actions on gastrointestinal nerves and muscle. However, the possibility that at least some of the responses to serotonin receptor-specific agents might be through modulation of other gastrointestinal cell activity and, in particular, mast cell activity has not been considered. In this context, 5HT₃ receptor antagonists and 5HT₄ agonists are used here as examples only. Any serotonin or serotonin receptor ligands might be considered, including full, partial or mixed agonists, antagonists or reverse agonists [0007] Theragnostics refers to diagnostic tests that point to a specific form of therapy. For example, the expression of estrogen receptors by breast cancer cells indicates that the tumor is likely to respond to tamoxifen therapy. In view of the unexpected findings set forth above and the fact that not all patient mast cells contain serotonin and that the numbers of mast cells in the gastrointestinal tract of different patients vary significantly, it is expected that patients expressing serotonin receptors on some gastrointestinal cells and/or cells circulating in peripheral blood and in particular, mast cells will respond to therapy that takes into account the type of receptor expressed and the presence of serotonin in their gastrointestinal (mast) cells and/or circulating cells. [0008] IBS is diagnosed by symptoms using standardized questionnaires. Patients with IBS are generally biopsied during the colonoscopy which is part of the diagnostic process. The biopsies are reviewed in pathology laboratories primarily to exclude other disorders which would indicate that the patients do not have IBS. The choice of drug used to treat these patients is determined by symptoms and is not based upon the likelihood that any given patient will respond because of the relevant ligand or receptor in their gastrointestinal tissues. Using routine patient biopsies, receptors for serotonin, as well as serotonin itself can be localized and detected, respectively, and the extent of expression of the different molecules can be quantified. In other words, the presence and level of expression of serotonin and serotonin receptors such as, e.g., 5-HT₃ receptors and 5-HT₄ receptors can be used as theragnostic markers for the treatment of functional bowel diseases such as, e.g., IBS. Accordingly, the methods of the present invention are expected to be of significant utility in the diagnosis, management and treatment of functional bowel disorders.

SUMMARY OF THE INVENTION [0009] The present invention provides a method for diagnosing a functional bowel disease, in a mammal and, in particular, a human patient. The method comprises the testing of gastrointestinal cells or cells circulating in peripheral blood of the patient for the presence of serotonin and/or at least one serotonin receptor. Non-limiting examples of serotonin receptors include 5-HTi receptor, 5- HT₂ receptor, 5-HT₃ receptor, 5-HT₄ receptor, 5-HT₅ receptor, 5-HT₆ receptor, 5- HT₇ receptor and the various subtypes of these receptors such as, e.g., 5-HT^'i_A receptor, 5-HT₁₈ receptor, 5-HT₁₀ receptor, 5-HTi_E receptor, 5-HT_{1 F} receptor, 5- HT_2A receptor, 5-HT_2B receptor, 5-HT₂₀ receptor, 5-HT_3A receptor, 5-HT_3B receptor, 5-HT_4A receptor, 5-HT₄B receptor, 5-HT₅A receptor, 5-HT_5B receptor, 5- HT₆ receptor and 5-HT₇ receptor.

[0010] In one aspect of the method, the gastrointestinal cells and/or circulating cells are tested for the presence of serotonin and/or a serotonin receptor such as, e.g., 5-HT₃ receptor and/or 5-HT₄ receptor. [0011] In another aspect of the method, the gastrointestinal cells may at least be tested for the presence of serotonin, and/or they may at least be tested for the presence of a serotonin receptor such as, e.g., 5-HT₃ receptor and/or 5-HT₄ receptor. In another aspect, they may be tested for the presence of at least 5-HT₃ receptor and 5-HT₄ receptor. In another aspect, they may be tested for the presence of at least two species, e.g., serotonin and at least one serotonin receptor. In another aspect, they may be tested for three species, e.g., serotonin, 5-HT₃ receptor and 5-HT₄ receptor.

[0012] In another aspect of the method of the present invention, the method may comprise a quantitative determination of serotonin and/or at least one serotonin receptor such as, e.g., 5-HT₃ receptor and 5-HT₄ receptor, for example, a quantitative determination of serotonin and/or a quantitative determination of 5- HT₃ receptor and/or a quantitative determination of 5-HT₄ receptor.

[0013] In yet another aspect of the method, the testing is preferably carried out ex vivo, for example, on a tissue section and/or on cells isolated from tissue samples and/or on cells circulating in peripheral blood.

[0014] In a still further aspect of the embodiments of the present invention, the gastrointestinal cells may comprise one or more of the following types of cells: mast cells, muscle cells, neurons, epithelial cells, lymphocytes, macrophages, granulocytes, dendritic cells, enteroglial cells and enteroendocrine cells. [0015] In a still further aspect of the embodiments of the present invention, the circulating blood cells may comprise one or more of the following types of cells: mast cells, lymphocytes, macrophages, granulocytes, dendritic cells and other circulating cells, particularly leucocytes..

[0016] In another aspect of the method, the functional bowel disease may comprise irritable bowel syndrome.

[0017] In yet another aspect, the method may comprise the incubation of the gastrointestinal cells with one or more antisera for at least one of serotonin and a serotonin receptor such as, e.g., 5-HT₃ receptor and 5-HT₄ receptor, and/or the method may comprise the in situ hybridization of mRNA that is associated with at least one serotonin receptor, e.g., the in situ hybridization of mRNA that is associated with one or both of the 5-HT₃ and 5-HT₄ receptors. By way of non- limiting example, the in situ hybridization may be carried out with at least one biotinylated probe and/or with at least one probe which comprises at least one of the following sequences:

ACCTGCCCAGGGACAGTGTAGTCTATGAAA (SEQ ID NO: 1 )

AGATGCGGTAATAGGCCAGCACCATGAGGA (SEQ ID NO: 2) TAGGGAGAAAAGAAATAAACGTGGGGATGA (SEQ ID NO: 3) GGCACCAAAGGGCATCACCAGCACCGAAAC (SEQ ID NO: 4) AGATCCGCAAAAGCAAGAGATACAATGAAA (SEQ ID NO: 5)

ACACAGCCACCATCACCAGCAGGTTCCCCA (SEQ ID NO: 6)

CGTAGGGCTTGTTGACCATGAAGACACAGT (SEQ ID NO: 7)

CGATGCGCAGAGGGGTCATCTTGTTCCTAT (SEQ ID NO: 8) GCAGATGGCGTAATACCTATCCAGAGAAAT (SEQ ID NO: 9) ATGCACAGGGTCTTGGCTGCTTTGGTCTCT (SEQ ID NO: 10)

AGCACAGGTGAAAAATCGATGCCGTTGTGA (SEQ ID NO: 11 )

AACACCTCCCCATAAATCCAGATGTCTTGA (SEQ ID NO: 12)

CTTATTCAAGAAGGCGTAGAGAAAAGGGTT (SEQ ID NO: 13) TGGATCTGATGGGCATGCTCCTTAGCTGTG (SEQ ID NO: 14)

ACTGACCCGAAACCCTCCTCAGAACTCACA (SEQ ID NO: 15)

ACAGTCTGGCCCAGAATGGAAGGTCTTCGG (SEQ ID NO: 16)

TTGGTGACAAAGAATGGTGCCCAGCAGAGG (SEQ ID NO: 17)

ATGGGATGTAGAAGGCCACCACAGAGCAGG (SEQ ID NO: 18) TGATATAGCCGAGCCAGAGGAAAGCAGTCC (SEQ ID NO: 19) CAATTATGCCAATGTTATTCCAGCCTTGCA (SEQ ID NO: 20)

AGCACCACCTTCTCCACTGACCCGAAACCC (SEQ ID NO: 21 ) GCCGAGCCAGAGGAAAGCAGTCCACACCTG (SEQ ID NO: 22) TGGCCCAGAATGGAAGGTCTTCGGTAGCGC (SEQ ID NO: 23) ACACTGACTCTCCCACTGGCCACCACACTC (SEQ ID NO: 24) GAGCAGGTGATGGCGTAGGGCTTGTTGACC (SEQ ID NO: 25)

GATGAGTGCTATGCTGGTCTGCCGACTGAG (SEQ ID NO: 26)

TGCCGTTGTGAGCAGGACGTCCAGAGATGT (SEQ ID NO: 27)

CTCCTTAGCTGTGACATAGATGCGGTAATA (SEQ ID NO: 28) CACAGCCACCATCACCAGCAGGTTCCCCAA (SEQ ID NO: 29)

CACCAAAGGGCATCACCAGCACCGAAACCA (SEQ ID NO: 30)

5' CTCACACTCCCTGGGCCACCAAGGTTGGGG 3' (SEQ ID NO: 31 )

5' CTCATGTCCCAGCCAGCCTGCCTCTCGTGG 3' (SEQ ID NO: 32) 5' GGAGTGTGGGGAGGAGCAAGGCGAGCAGCG 3' (SEQ ID NO: 33) 5' CTCAGCAGAGCGGGCCTGGTGGTGTTTCGG 3' (SEQ ID NO: 34) 5¹ ACGGTGGTTGGCTTCCTCCAGTCCCTCACG 3' (SEQ ID NO: 35) 5' AGCACCCCCAGCAACTCCCACTCTCCCTGG 3' (SEQ ID NO: 36) 5' GACAGCTCCTGCAGCAGCCCACACACCGCC 3' (SEQ ID NO: 37) 5' CTTGTCCAGCACGGAGCCCACGCGCAGCCA 3' (SEQ ID NO: 38) 5' TCCCCACCTAACCAGGACTGTACCCCCTCC 3' (SEQ ID NO: 39)

5' TGGTGAGCTGCTGGGGGTGGGACAAGGGTG 3' (SEQ ID NO: 40) 5' GCACAGGCGGTTGAAAGCAGTGAAACAAAT 3' (SEQ ID NO: 41 )

5¹ CTTGCTGCTGTGACTGAAATCCTCCTCCCA 3' (SEQ ID NO: 42) 5' TCCCCCTCTGGTTTCGTTGTTATTTTGCTG 3' (SEQ ID NO: 43) 5' TCTGCCGTAGTTGTAGAGTCGTGTTTGAAC 3' (SEQ ID NO: 44) 5' TGAGCGCATACACACATCTGTCCATGTTTG 3' (SEQ ID NO: 45) 5' ACCATGCCAAACACTCAAAAGCCAAAGTTG 3' (SEQ ID NO: 46) and

5¹ CCGTTCCGAACAGTGGTCAGCATGGTTAGT 3'. (SEQ ID NO: 47). Said probe may be labeled with any suitable label, e.g. with a fluorescent label such as fluorescein (FITC), a radiolabel, an enzyme label, a biotin label, digoxygenin (DIG) label, etc, as it is well known in the art.

[0018] In yet another aspect of the method, the gastrointestinal cells may comprise mast cells. In this case, the method may comprise contacting

(challenging) the mast cells with a mast cell stimulator. Non-limiting examples of suitable mast cell stimulators include a calcium ionophore (such as, e.g., ionomycin), IgE and antigen, a neuropeptide (such as, e.g., substance P), a neurotransmitter (such as, e.g., nitric oxide) and other mast cell secretagogues

(such as, e.g., compound 48/80).

[0019] The present invention also provides a method for diagnosing irritable bowel syndrome in a human patient. The method comprises testing gastrointestinal mast cells of the patient for the presence of at least one of serotonin, 5-HT₃ receptor and 5-HT₄ receptor.

[0020] In one aspect of the method, the mast cells may at least be tested for the presence of serotonin and/or they may at least be tested for the presence of 5-HT₃ receptor and/or they may at least be tested for the presence of 5-HT₄ receptor. In another aspect, they may at least be tested for the presence of 5-HT₃ receptor and 5-HT₄ receptor. In another aspect, they may be tested for the presence of serotonin, 5-HT₃ receptor and 5-HT₄ receptor.

[0021] In another aspect of the method, the method may comprise a quantitative determination of one or more of serotonin, 5-HT₃ receptor and 5-HT₄ receptor, i.e., a quantitative determination of serotonin and/or a quantitative determination of 5-HT₃ receptor and/or a quantitative determination of 5-HT₄ receptor.

[0022] In yet another aspect of the method, the testing is preferably carried out ex vivo, optionally on a tissue section or on cells isolated from a tissue or on a peripheral blood sample. [0023] In a still further aspect, the method may comprise the incubation of the mast cells with one or more antisera for at least one of serotonin, 5-HT₃ receptor and 5-HT₄ receptor and/or the method may comprise the in situ hybridization of mRNA that is associated with at least one of the 5-HT₃ and 5-HT₄ receptors. By way of non-limiting example, the in situ hybridization may be carried out with at least one biotinylated probe and/or with at least one probe which comprises at least one of the following sequences:

ACCTGCCCAGGGACAGTGTAGTCTATGAAA (SEQ ID NO: 1)

AGATGCGGTAATAGGCCAGCACCATGAGGA (SEQ ID NO: 2)

TAGGGAGAAAAGAAATAAACGTGGGGATGA (SEQ ID NO: 3) GGCACCAAAGGGCATCACCAGCACCGAAAC (SEQ ID NO: 4) AGATCCGCAAAAGCAAGAGATACAATGAAA (SEQ ID NO: 5)

ACACAGCCACCATCACCAGCAGGTTCCCCA (SEQ ID NO: 6)

CGTAGGGCTTGTTGACCATGAAGACACAGT (SEQ ID NO: 7)

AGCACAGGTGAAAAATCGATGCCGTTGTGA (SEQ ID NO: 11 )

AACACCTCCCCATAAATCCAGATGTCTTGA (SEQ ID NO: 12)

CTTATTCAAGAAGGCGTAGAGAAAAGGGTT (SEQ ID NO: 13) TGGATCTGATGGGCATGCTCCTTAGCTGTG (SEQ ID NO: 14) ACTGACCCGAAACCCTCCTCAGAACTCACA (SEQ ID NO: 15) ACAGTCTGGCCCAGAATGGAAGGTCTTCGG (SEQ ID NO: 16)

TTGGTGACAAAGAATGGTGCCCAGCAGAGG (SEQ ID NO: 17) ATGGGATGTAGAAGGCCACCACAGAGCAGG (SEQ ID NO: 18) TGATATAGCCGAGCCAGAGGAAAGCAGTCC (SEQ ID NO: 19) CAATTATGCCAATGTTATTCCAGCCTTGCA (SEQ ID NO: 20) AGCACCACCTTCTCCACTGACCCGAAACCC (SEQ ID NO: 21 )

GCCGAGCCAGAGGAAAGCAGTCCACACCTG (SEQ ID NO: 22)

TGGCCCAGAATGGAAGGTCTTCGGTAGCGC (SEQ ID NO: 23)

ACACTGACTCTCCCACTGGCCACCACACTC (SEQ ID NO: 24) GAGCAGGTGATGGCGTAGGGCTTGTTGACC (SEQ ID NO: 25)

GATGAGTGCTATGCTGGTCTGCCGACTGAG (SEQ ID NO: 26)

TGCCGTTGTGAGCAGGACGTCCAGAGATGT (SEQ ID NO: 27)

CTCCTTAGCTGTGACATAGATGCGGTAATA (SEQ ID NO: 28)

CACAGCCACCATCACCAGCAGGTTCCCCAA (SEQ ID NO: 29) CACCAAAGGGCATCACCAGCACCGAAACCA (SEQ ID NO: 30)

5' CTCACACTCCCTGGGCCACCAAGGTTGGGG 3' (SEQ ID NO: 31 )

5' CTCATGTCCCAGCCAGCCTGCCTCTCGTGG 3' (SEQ ID NO: 32) 5' GGAGTGTGGGGAGGAGCAAGGCGAGCAGCG 3' (SEQ ID NO: 33) 5' CTCAGCAGAGCGGGCCTGGTGGTGTTTCGG 3' (SEQ ID NO: 34) 5' ACGGTGGTTGGCTTCCTCCAGTCCCTCACG 3' (SEQ ID NO: 35) 5' AGCACCCCCAGCAACTCCCACTCTCCCTGG 3' (SEQ ID NO: 36) 5' GACAGCTCCTGCAGCAGCCCACACACCGCC 3' (SEQ ID NO: 37) 5' CTTGTCCAGCACGGAGCCCACGCGCAGCCA 3' (SEQ ID NO: 38) 5' TCCCCACCTAACCAGGACTGTACCCCCTCC 3' (SEQ ID NO: 39) 5' TGGTGAGCTGCTGGGGGTGGGACAAGGGTG 3' (SEQ ID NO: 40) 5' GCACAGGCGGTTGAAAGCAGTGAAACAAAT 3' (SEQ ID NO: 41 ) 5' CTTGCTGCTGTGACTGAAATCCTCCTCCCA 3' (SEQ ID NO: 42) 5' TCCCCCTCTGGTTTCGTTGTTATTTTGCTG 3' (SEQ ID NO: 43) 5' TCTGCCGTAGTTGTAGAGTCGTGTTTGAAC 3' (SEQ ID NO: 44) 5' TGAGCGCATACACACATCTGTCCATGTTTG 3' (SEQ ID NO: 45) 5' ACCATGCCAAACACTCAAAAGCCAAAGTTG 3' (SEQ ID NO: 46) and 5¹ CCGTTCCGAACAGTGGTCAGCATGGTTAGT 3'. (SEQ ID NO: 47).

[0024] Said probe may be labeled with any suitable label, e.g. with a fluorescent label such as fluorescein (FITC(, a radiolabel, an enzyme label, a biotin label, a digoxygenin (DIG) label, etc, as it is well known in the art.

[0025] In yet another aspect of the method, the method may comprise contacting (challenging) the mast cells with a stimulator such as, e.g., a calcium ionophore, for example ionomycin. [0026] The present invention also provides a method for diagnosing a serotonin-related gastrointestinal disorder in a human patient that may be a functional bowel disease, e.g. irritable bowel syndrome. The method comprises testing gastrointestinal cells or cells circulating in peripheral blood of the patient for the presence of serotonin and/or a serotonin receptor such as, e.g., 5-HT₃ receptor and/or 5-HT₄ receptor. Aspects of this method include those which have been set forth above in connection with the method for diagnosing a functional bowel disease in a mammal.

[0027] The present invention also provides a method for the treatment of a functional bowel disease in a mammal, preferably a human patient, whose gastrointestinal cells and/or cells circulating in peripheral blood have been determined to comprise (express) one or more serotonin receptors. The method comprises the administration to the mammal of at least one serotonin receptor antagonist and/or at least one serotonin receptor agonist in an effective amount for alleviating the symptoms of the functional bowel disease. [0028] In one aspect of the method, the functional bowel disease may comprise irritable bowel syndrome. In another aspect of the method, the gastrointestinal cells may comprise mast cells. [0029] The present invention also provides a method for the treatment of a functional bowel disease in a mammal, preferably a human patient, whose gastrointestinal cells have been determined to comprise (express) 5-HT₃ receptor. The method comprises the administration to the mammal of a 5-HT₃ receptor antagonist in an effective amount for alleviating the symptoms of the functional bowel disease. [0030] In one aspect of the method, the functional bowel disease may comprise irritable bowel syndrome. In another aspect of the method, the gastrointestinal cells may comprise mast cells. In yet another aspect, the 5-HT₃ receptor antagonist may comprise alosetron. [0031] The present invention also provides a method for the treatment of a functional bowel disease in a mammal, preferably a human patient, whose gastrointestinal cells and/or cells circulating in peripheral blood have been determined to comprise (express) 5-HT₄ receptor. The method comprises the administration to the mammal of a 5-HT₄ receptor agonist in an effective amount for alleviating the symptoms of the functional bowel disease. Furthermore, the present invention provides a method for determining which medicament out of the marketed medicaments for treating functional bowel disease is suitable for a particular individual.

[0032] In one aspect of the methods, the functional bowel disease may comprise irritable bowel syndrome. In another aspect of the methods, the gastrointestinal cells may comprise mast cells. In yet another aspect, the 5-HT₄ receptor agonist may comprise tegaserod.

[0033] The present invention further provides a medicament that comprises a serotonin receptor antagonist and/or a serotonin receptor agonist and is associated with instructions to administer the medicament to a patient whose gastrointestinal cells and/or cells circulating in peripheral blood comprise (express) the serotonin receptor. The present invention also provides a label or leaflet on or in a packing of a medicament for treating functional bowel disease indicating the use thereof according to the type of HT receptor found on gastrointestinal cells and/or cells circulating in peripheral blood of the patient. [0034] In one aspect of the medicament or the label or leaflet, the gastrointestinal cells and/or cells circulating in peripheral blood may comprise mast cells. In another aspect, the medicament may be indicated for treating a functional bowel disease such as, e.g., irritable bowel syndrome. [0035] The present invention further provides a medicament that comprises a 5-HT₃ receptor antagonist and is associated with instructions to administer the medicament to a patient whose gastrointestinal cells and/or cells circulating in peripheral blood comprise (express) 5-HT₃ receptor.

[0036] In one aspect of the medicament, the gastrointestinal cells and/or cells circulating in peripheral blood may comprise mast cells. In another aspect, the medicament may be indicated for treating a functional bowel disease such as, e.g., irritable bowel syndrome.

[0037] The present invention also provides a medicament that comprises a 5- HT₄ receptor agonist and is associated with instructions to administer the medicament to a patient whose gastrointestinal cells and/or cells circulating in peripheral blood comprise (express) 5-HT₄ receptor. [0038] In one aspect of the medicament, the gastrointestinal cells and/or cells circulating in peripheral blood may comprise mast cells. In another aspect, the medicament may be indicated for treating a functional bowel disease such as, e.g., irritable bowel syndrome. [0039] The present invention also provides a probe for the hybridization of mRNA that is associated with at least one 5-HT receptor as well as a diagnostic kit comprising the probe and the use of the diagnostic kit. The probe comprises at least one of the following sequences:

ACCTGCCCAGGGACAGTGTAGTCTATGAAA (SEQ ID NO: 1 )

AGATGCGGTAATAGGCCAGCACCATGAGGA (SEQ ID NO: 2) TAGGGAGAAAAGAAATAAACGTGGGGATGA (SEQ ID NO: 3) GGCACCAAAGGGCATCACCAGCACCGAAAC (SEQ ID NO: 4) AGATCCGCAAAAGCAAGAGATACAATGAAA (SEQ ID NO: 5) ACACAGCCACCATCACCAGCAGGTTCCCCA (SEQ ID NO: 6)

CGTAGGGCTTGTTGACCATGAAGACACAGT (SEQ ID NO: 7)

CGATGCGCAGAGGGGTCATCTTGTTCCTAT (SEQ ID NO: 8)

GCAGATGGCGTAATACCTATCCAGAGAAAT (SEQ ID NO: 9) ATGCACAGGGTCTTGGCTGCTTTGGTCTCT (SEQ ID NO: 10) AGCACAGGTGAAAAATCGATGCCGTTGTGA (SEQ ID NO: 11 ) AACACCTCCCCATAAATCCAGATGTCTTGA (SEQ ID NO: 12)

TTGGTGACAAAGAATGGTGCCCAGCAGAGG (SEQ ID NO: 17)

ATGGGATGTAGAAGGCCACCACAGAGCAGG (SEQ ID NO: 18)

TGATATAGCCGAGCCAGAGGAAAGCAGTCC (SEQ ID NO: 19) CAATTATGCCAATGTTATTCCAGCCTTGCA (SEQ ID NO: 20) AGCACCACCTTCTCCACTGACCCGAAACCC (SEQ ID NO: 21 )

GCCGAGCCAGAGGAAAGCAGTCCACACCTG (SEQ ID NO: 22)

TGGCCCAGAATGGAAGGTCTTCGGTAGCGC (SEQ ID NO: 23)

ACACTGACTCTCCCACTGGCCACCACACTC (SEQ ID NO: 24) GAGCAGGTGATGGCGTAGGGCTTGTTGACC (SEQ ID NO: 25) GATGAGTGCTATGCTGGTCTGCCGACTGAG (SEQ ID NO: 26) TGCCGTTGTGAGCAGGACGTCCAGAGATGT (SEQ ID NO: 27) CTCCTTAGCTGTGACATAGATGCGGTAATA (SEQ ID NO: 28) CACAGCCACCATCACCAGCAGGTTCCCCAA (SEQ ID NO: 29)

CACCAAAGGGCATCACCAGCACCGAAACCA (SEQ ID NO: 30) 5' CTCACACTCCCTGGGCCACCAAGGTTGGGG 3' (SEQ ID NO: 31 )

5' CTCATGTCCCAGCCAGCCTGCCTCTCGTGG 3' (SEQ ID NO: 32) 5' GGAGTGTGGGGAGGAGCAAGGCGAGCAGCG 3' (SEQ ID NO: 33) 5' CTCAGCAGAGCGGGCCTGGTGGTGTTTCGG 3' (SEQ ID NO: 34) 5' ACGGTGGTTGGCTTCCTCCAGTCCCTCACG 3' (SEQ ID NO: 35) δ' AGCACCCCCAGCAACTCCCACTCTCCCTGG S' (SEQ ID NO: 36) 5' GACAGCTCCTGCAGCAGCCCACACACCGCC 3' (SEQ ID NO: 37) 5' CTTGTCCAGCACGGAGCCCACGCGCAGCCA 3' (SEQ ID NO: 38) 5' TCCCCACCTAACCAGGACTGTACCCCCTCC 3' (SEQ ID NO: 39)

5' CTTGCTGCTGTGACTGAAATCCTCCTCCCA 3' (SEQ ID NO: 42) 5' TCCCCCTCTGGTTTCGTTGTTATTTTGCTG 3' (SEQ ID NO: 43) 5' TCTGCCGTAGTTGTAGAGTCGTGTTTGAAC 3' (SEQ ID NO: 44) 5' TGAGCGCATACACACATCTGTCCATGTTTG 3' (SEQ ID NO: 45) 5' ACCATGCCAAACACTCAAAAGCCAAAGTTG 3' (SEQ ID NO: 46) and 5' CCGTTCCGAACAGTGGTCAGCATGGTTAGT 3'. (SEQ ID NO: 47). Said probe may be labeled with any suitable label, e.g. with a fluorescent label such as fluorescein (FITC), a radiolabel, an enzyme label, a biotin label, a digoxygenin (DIG) label, etc, as it is well known in the art.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Gel Electrophoresis confirmed the size of the plasmids for 5- HT_2B and 5-HT₄.

Figure 2: Serotonin immunoreactivity in surgical resections of human bowel (A - F), rat jejunum (G) and mouse jejunum (H). Figure 3: 5-HT₃ immunoreactivity in surgical resections of human bowel.

Figure 4: 5-HT4-immunoreactivity in human gut. Figure 5: 5-HT4- immunoreactivity in rat gut. Figure 6: Localization of mRNA for 5-HT4 in human gut. Figure 7: 5HT4-immunoreactivity in human, rat and mouse cell lines. Figure 8: 5HT4-immunoreactivity in 5HT4b-transfected CHO cells.

Figure 9: The 5-HT₄ receptor partial agonist, RS 67506, augments ionomycin-induced calcium mobilization in 5-HT_4B-transfected CHO cells.

Figure 10: The 5-HT₄ receptor partial agonist, RS 67506, attenuates ionomycin-induced calcium mobilization in BMMC. Figure 1 1 : The 5-HT₄ receptor partial agonist, RS 67506, attenuates ionomycin-induced calcium mobilization in RBL (A) and KU812 (B) cells.

Figure 12: The 5-HT₄ receptor partial agonist, RS 67506, attenuates ionomycin-induced TNFα release from RBL cells (A) and BMMC (B).

Figure 13: The 5-HT₄ receptor partial agonist, RS 67506, attenuates ionomycin-induced β-hexosaminidase release from KU812 cells.

Figure 14: Variable expression of 5-HT₄ in the lamina propria of terminal ileum endoscopic biopsies from six different patients (A - 007, B - 068, C - 075, D - 077, E - 095, F - 102).

Figure 15: Variable expression of 5-HT₄ in the lamina propria of rectal endoscopic biopsies from six different patients (A - 007, B - 068, C - 075, D - 077, E - 095, F - 102). Figure 16: 5-HT₄ immunoreactive cell counts in the lamina propria of terminal ileum and rectal endoscopic biopsies from patients illustrated in Figures 9 and 10.

Figure 17: 5-HT₄-immunoreactivity in the lamina propria of IBS patients in dependence of clinical symptoms.

DETAILED DESCRIPTION OF THE PRESENT INVENTION [0041] The particulars shown herein are by way of example and for purposes of illustrative discussion of the embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the present invention. In this regard, no attempt is made to show elements of the present invention in more detail than is necessary for the fundamental understanding of the present invention, the description making apparent to those skilled in the art how the several forms of the present invention may be embodied in practice.

1. MATERIALS AND METHODS

Bone Marrow Mast Cells (BMMC)

[0042] Bone marrow mast cells were obtained from BALB/c mice and cultured in DMEM medium containing 10% FCS, penicillin/streptomycin with mouse rlL-3 and mouse rSCF (both at concentration of 10 ng/ml) in a 25 cm³ flask at a cell density of 2 x 10⁵/ml. Prior to testing the cultures were maintained for a minimum of 3 weeks at 37⁰C and 5% CO₂ and the medium was changed weekly.

[0043] For experiments BMMC were washed and re-suspended in DMEM medium at a concentration of 10⁶AnI and placed in 3 ml sterile tubes. Cells were pre-incubated with tested compounds for 15 minutes, followed by activation with the calcium ionophore ionomycin (1 μM). After a 4 hour incubation cells were spun down, the supernatant was removed and frozen at -2O⁰C for further analysis (released mediators). Tyrode's buffer was added to cell pellets, followed by 2 cycles of freezing and boiling after which supernatants were collected (total mediators) and frozen for further analysis.

[0044] Relevant SOP # 030-0700-043; 010-0300-274; 010-0300-275; 030- 0900-002.

Cultured cells

[0045] Rat basophilic cells RBL-2H3 and human KU812 cells were cultured in RPMI medium containing 10% FCS, penicillin/streptomycin in 75 cm³ flasks at a cell density of 2 x 10⁶/ml. Cultures were maintained at 37⁰C and 5% CO₂ and the medium was changed as necessary.

[0046] For experiments cultured cells were treated similarly to BMMC with the exception that ionomycin was used at 2 μM concentration.

[0047] Relevant SOP # 030-0900-003; 030-0900-015.

Transfection of CHO cells with 5HT_4B

An open reading frame clone for human 5HT₄ (B slice variant; ORF clone IOH35666, Invitrogen, Burlington) was cloned into pENTR221 and recombined with pcDNA3.1/nV5-DEST, using Gateway Technology. Plasmids were prepared using Concert Maxiprep kits (Invitrogen) and linearized with CIa I. A second serotonin receptor, 5HT_2B, was cloned in parallel (ORF clone IOH40708) and linearized with BgI II. Gel Electrophoresis was used to confirm the size of the resulting plasmids (Figure 1 ). Each plasmid contained a V5 tag (epitope from SV5) and a neomycin resistance gene.

Chinese Hamster ovary (CHO) cells were transfected with plasmid using Lipofectamine (Invitrogen) and successfully transfected cells were selected using Geneticin (G418). Cell cultures, cell smears and NBF-fixed cell blocks were prepared, as required.

Figure 1: Gel Electrophoresis confirmed the size of the plasmids for 5-HT_2B and 5-HT₄. 5-HT₂B located in lane 6 is 6651 basepairs (bp). 5-HT₄ located in lane 8 had two bands of 2262bp and 4668 bp. Lanes 5 and 7 are supercoiled DNA.

TNF- α Assay

[0048] The TNF-α assay may be used to assess the response to ionomycin. BD OptEIA Set Mouse TNF-α ELISA or BD OptEIA Rat TNF-α ELISA kit was obtained from BD Biosciences (# 558874 and 558870 respectively). Briefly, plates were coated with 100 μl of capture antibody and incubated overnight at 4⁰C. Wells were washed (3x) with washing buffer and blocked with 300 μl of 2% BSA in PBS. After washing again (3x), standard or 100 μl of sample (cell supernatant) was loaded and incubated for 2 hr at room temperature. After washing detection antibody was added and incubation continued for 1 hr. At that time (after 5 x wash) detection antibody was added followed up by wash (7x) and enzyme conjugate (Avidin-HRP). After a 30-minute incubation in darkness, the reaction was stopped by adding 50 μl of 1 N HCI. Absorbance was read at 450 nm using an ELISA plate reader and the concentration was determined using the KCJunior program (Bio-Tech Instrument).

[0049] Relevant SOP # 010-0230-206; 010-0230-205; 030-1000-002.

β-Hexosaminidase Assay

β-Hexosaminidase assay is used to measure mast cell degranulation. Briefly, cells at density ~ 10⁶AnI are pre-incubated with tested substances for 15 minutes at 37⁰C after which ionomycin is added and incubation continued for additional

30 minutes. Equal volume of ice cold HEPES Tyrode buffer (pH 7.35) is added and cells are spin at 1000 rpm for 10 minutes. Supernatant is collected (released) and cell pellet is re-suspended in 1 ml of HTB. Cells in the pellet are disrupted by repeated freezing/thawing or sonication and cell debris removed by ultracenthfugation for 1 minute. Supernatant from pellet (total) is collected.

Fifty μl_ of supernatant (released/total) is added to 96-well microplate to which 50 μl_ of 1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide in 0.1 M citric buffer (pH4.5) is added. After 1 hr incubation at 37⁰C reaction is terminated by adding 200 μ- L/well of 0.1 M carbonate buffer (pH 10.5). Plate is read at 405 nm and release ratio calculated using the formula:

% release = P.P. supernatant - P.P. control x 100

(P.P. supernatant - P.P. control) + (P.P. pellet - P.P. control)

Calcium Fluorescence Method

[0050] BMMC cells (1 *10⁵ per ml), RBL-2H3 cells (1 ^χ10⁵ per ml), KU812 cells (1 ^χ10⁵ per ml) or CHP (wild type or 5-HT₄ transfected) (1 *10⁵ per ml) were grown overnight on poly-P-lysine coated glass-bottom culture dishes (Mattek Corp., MA, USA). The cells were loaded with fura-2/AM (final concentration 1-5 μM, Invitrogen, PN, Canada) for 30 minutes at 37°C. Pishes were then placed on the stage of a Leica PRIRB inverted fluorescence microscope (Leica, PN, Canada) and perfused with Krebs solution (composition in mM: NaCI 118.0, KCI 4.7, NaH₂PP₄ 1.0, NaHCP₃ 25.0, MgSP₄ 1.2, CaCI₂ 2.5, P-Glucose 11.1 , with pH adjusted to 7.3 by using NaPH) for 10-30 minutes (4.5 ml/min) to wash away background fura-2 fluorescence and any unattached cells. [0051] Fura-2 signals were monitored using an lonoptix imaging system (lonoptix, MA, USA) under a 4Ox Leica FLUPSTAR lens. Fura-2 excitation was achieved using a mercury arc lamp shuttered between 340 nm and 380 nm excitation filters and emission signals were passed through a 510 nm cutoff emission filter and collected by a Myocam CCP video camera (lonoptix, MA, USA). Data was acquired at 5 or 33 Hz and analyzed using IonWizard software (V4.4.13).

[0052] Cells were perfused with Krebs solution or Krebs solution containing one of the following compounds: 5-HT (10 μM, Sigma, ON, Canada); RS 67506 (2-20 μM, Tocris, MO, USA; 5-HT₄ agonist). Baseline fura-2 fluorescence was monitored and then the response of cells was observed following administration of ionomycin (perfusion of 2 μM for 30 sec or application of 0.03 μM ionomycin directly onto the cells via micropipette, Sigma, ON, Canada). . [0053] The fura-2 fluorescence was averaged every 10 sec over a 10 min period. Responses to ionomycin under different conditions were compared using a 1 -way ANOVA.

[0054] Relevant SOP # 010-0000-194; 010-0000-197; 010-0210-087; 010- 0300-384.

Tissue Preparation for lmmunohistochemistry

[0055] Patients undergoing surgery (including endoscopy) for a variety of conditions consented to having tissues removed and used for research purposes.

Consent and use of tissues was conducted using ethically appropriate guidelines.

Specimens were promptly removed and routinely fixed in 10% neutral buffered formalin for 48 hours and processed to paraffin. Samples from rats and mice were handled similarly. Cultured cells were pelleted in 2% agarose and processed in parallel.

lmmunohistochemistry [0056] Sections from each paraffin block were cut at 2 μm on a Leica microtome. Tissue sections were placed on microslides and left to dry overnight.

[0057] Sections were then deparaffinized with xylene, hydrated in alcohol and then immersed in 3% H₂O₂to remove endogenous peroxidase activity.

[0058] In order to expose the antigens, which were potentially masked by formalin fixation, sections were subjected to no pre-treatment, enzyme digestion

(slides were incubated in 0.5% trypsin in 5% CaCI₂ for 60 minutes at 37⁰C) or heat induced antigen retrieval (sections were placed in plastic coplin jars filled with citrate buffer, pH 6.0; each jar contained 9 slides which were incubated in a household steamer for 30 min at 100⁰C). The slides were then rinsed with Tris buffer to allow slides to cool to room temperature (approximately 15 min) and then immunostained. For sections requiring no pre-treatment, following peroxidase block slides were rinsed with Tris buffer before continuing with immunohistochemical procedures.

[0059] Sections were incubated in 20% normal goat serum for 20 min to block non-specific binding sites. Details of the three commercial primary antisera are presented in Table 1 below. One hour incubation in primary antisera at room temperature was employed, with the exception of 5-HT₃ receptor which required an overnight incubation at room temperature. For negative controls, primary antiserum was replaced by non-immune serum (Dako X0902 for whole serum antisera and Dako X0903 for purified antisera). [0060] Following antisera incubation, slides were washed with Tris buffer containing 0.05% Tween twice for 10 minutes each.

[0061] Sites of antibody were detected in human sections using Zymed's non- biotin amplification (NBA) system. A FITC conjugated secondary antibody and HRP conjugated tertiary antibody were both supplied and used neat. The reagents were applied for 10 min at room temperature. As an alternative, comparable detection systems may be substituted for the NBA system (e.g. Spring Biosciences goat polyvalent and streptavidin peroxidase). Following incubations in secondary and tertiary antibodies sections were washed in Tris buffer containing 0.05% Tween for 5 minutes. The sections were developed using chromagen AEC for 15 minutes, followed by counter staining in Harris Haemotoxylin. The slides were mounted with an aqueous medium following standard protocols.

[0062] Relevant SOP # 030-0000-006; 030-0100-030; 030-0200-001 ; 030- 0200-002; 030-0200-008; 030-0210-086; 010-0230-238; 010-0230-007; 010- 0230-024; 030-0200-005; 030-0200-017; 030-0100-021 ; 010-0230-188; 030- 0100-017; 030-1300-009; 010-0230-160. Table 1

Tissue Preparation for in situ Hybridization

[0063] Patients undergoing surgery for a variety of conditions consented to having tissues removed and used for research purposes. Consent and use of tissues was conducted using ethically appropriate guidelines. Specimens were promptly removed and routinely fixed in 10% neutral buffered formalin for 48 hours and processed to paraffin. Tissues from rats and mice were handled similarly. Cultured cells were pelleted in 2% agarose and processed in parallel.

In situ Hybridization

[0064] Sections from each block were cut at 2 μm on a Leica microtome.

Tissue sections were placed on microslides and left to dry overnight on a hotplate set to 60⁰C.

[0065] Sections were deparaffinized with xylene, three changes of 100% alcohol were used to clear the xylene. Sections were then hydrated in 70, 50 and

20% alcohol and rinsed in distilled water.

[0066] In order to expose mRNA, which was potentially masked by formalin fixation, sections were subjected to proteinase K enzyme digestion. Briefly, sections were placed in coplin jars containing warm proteinase K buffer in a water bath set to 37⁰C for 20 minutes. Slides were then digested in a solution of

0.01% proteinase K (with an activity of 44 units/mg) for 8 minutes. The slides were removed and rinsed twice in 37°C proteinase K buffer, followed by a 5 minute incubation in glycine buffer at room temperature. [0067] In preparation for probe application, slides were rinsed in two changes of distilled water, followed by dehydration in 70, 90 and 100% alcohol. Sections were allowed to air dry for two to three minutes prior to probe application. [0068] 5-HT₃ and 5-HT₄ 3'-biotinylated probes were prepared to a concentration of 1 ng/μl in hybridization buffer containing 4x SSC and 1.5% dextran sulfate without formamide. Slides were covered with glass coverslips and sealed with rubber cement and allowed to dry.

[0069] Slides were then placed in a 90°C oven for 10 minutes for probe denaturation/melt. For 5-HT₃ receptor, slides were then moved to a 50⁰C incubator and allowed to hybridize overnight. For 5-HT₄ receptor, slides were moved to a 50⁰C incubator and allowed to hybridize for one hour. [0070] Following the appropriate hybridization time, slides were immediately submerged in 0.5x SSC at 50°C to remove coverslips. Slides were then washed in two changes of 0.5x SSC for 10 minutes, with 10 agitations. The final stringency wash was with 0.1x SSC at 50⁰C for 10 minutes, with 10 agitations. Slides were then washed well in Tris buffer containing 0.05% Tween for 5 minutes at room temperature.

[0071] Sites of mRNA probe binding were detected with Dako anti-biotin alkaline phosphatase secondary antibody prepared in Tris buffer containing 0.05% Tween. Sections were incubated at room temperature in a humidity chamber for 45 minutes. Slides were washed twice in Tris buffer containing 0.05% Tween for 5 minutes at room temperature. Slides were then washed in alkaline phosphatase buffer for two minutes at room temperature. [0072] The sections were developed using chromagen Nitro Blue Tetrazolium Chloride and 5-Bromo, 4-Chloro, 3-lndolyl Phosphate (NBT/BCIP) diluted in alkaline phosphatase buffer. Sections were incubated at room temperature in a humidity chamber for 30 - 45 minutes. Sections were checked microscopically to ensure proper color development. [0073] Following appropriate color development, slides were washed in three changes of distilled water. The slides were mounted with an aqueous medium following standard protocols. [0074] The probes used were 30mer oligonucleotides synthesized commercially. These were 3'-biotinylated using Biotin-11-dUTP, dATP and TdT, using standard methodology before purification using spin-columns. [0075] Relevant SOP # 030-0100-027; 030-0300-010; 030-0100-028; 010- 0230-320; 010-0230-350; 010-0230-248; 030-0300-014; 030-0300-011 ; 030- 0300-012; 030-0300-007. Specifically, the probes used included the following 3'- biotinylated probes:

ACCTGCCCAGGGACAGTGTAGTCTATGAAA (SEQ ID NO: 1 )

AGATGCGGTAATAGGCCAGCACCATGAGGA (SEQ ID NO: 2)

ACACAGCCACCATCACCAGCAGGTTCCCCA (SEQ ID NO: 6)

CGTAGGGCTTGTTGACCATGAAGACACAGT (SEQ ID NO: 7) CGATGCGCAGAGGGGTCATCTTGTTCCTAT (SEQ ID NO: 8) GCAGATGGCGTAATACCTATCCAGAGAAAT (SEQ ID NO: 9) ATGCACAGGGTCTTGGCTGCTTTGGTCTCT (SEQ ID NO: 10)

AGCACAGGTGAAAAATCGATGCCGTTGTGA (SEQ ID NO: 11 )

AACACCTCCCCATAAATCCAGATGTCTTGA (SEQ ID NO: 12)

ACTGACCCGAAACCCTCCTCAGAACTCACA (SEQ ID NO: 15)

ACAGTCTGGCCCAGAATGGAAGGTCTTCGG (SEQ ID NO: 16)

GCCGAGCCAGAGGAAAGCAGTCCACACCTG (SEQ ID NO: 22)

TGGCCCAGAATGGAAGGTCTTCGGTAGCGC (SEQ ID NO: 23)

GATGAGTGCTATGCTGGTCTGCCGACTGAG (SEQ ID NO: 26)

TGCCGTTGTGAGCAGGACGTCCAGAGATGT (SEQ ID NO: 27)

CTCCTTAGCTGTGACATAGATGCGGTAATA (SEQ ID NO: 28)

5' CTCACACTCCCTGGGCCACCAAGGTTGGGG 3' (SEQ ID NO: 31 ) 5' CTCATGTCCCAGCCAGCCTGCCTCTCGTGG 3' (SEQ ID NO: 32) 5' GGAGTGTGGGGAGGAGCAAGGCGAGCAGCG 3' (SEQ ID NO: 33) 5' CTCAGCAGAGCGGGCCTGGTGGTGTTTCGG 3' (SEQ ID NO: 34) 5' ACGGTGGTTGGCTTCCTCCAGTCCCTCACG 3' (SEQ ID NO: 35) 5' AGCACCCCCAGCAACTCCCACTCTCCCTGG 3' (SEQ ID NO: 36) 5' GACAGCTCCTGCAGCAGCCCACACACCGCC 3' (SEQ ID NO: 37) 5' CTTGTCCAGCACGGAGCCCACGCGCAGCCA 3' (SEQ ID NO: 38) 5' TCCCCACCTAACCAGGACTGTACCCCCTCC 3' (SEQ ID NO: 39) 5' TGGTGAGCTGCTGGGGGTGGGACAAGGGTG 3' (SEQ ID NO: 40) 5' GCACAGGCGGTTGAAAGCAGTGAAACAAAT 3' (SEQ ID NO: 41 ) 5' CTTGCTGCTGTGACTGAAATCCTCCTCCCA 3' (SEQ ID NO: 42) 5' TCCCCCTCTGGTTTCGTTGTTATTTTGCTG 3¹ (SEQ ID NO: 43) 5' TCTGCCGTAGTTGTAGAGTCGTGTTTGAAC 3' (SEQ ID NO: 44) 5' TGAGCGCATACACACATCTGTCCATGTTTG 3' (SEQ ID NO: 45) 5' ACCATGCCAAACACTCAAAAGCCAAAGTTG 3' (SEQ ID NO: 46) and 5' CCGTTCCGAACAGTGGTCAGCATGGTTAGT 3'. (SEQ ID NO: 47).

[0076] Probe SEQ ID NOS. 1-30 are particularly suitable for 5-HT₄ receptor. Probe SEQ ID NOS. 31-40 are particularly suitable for 5-HT₃ receptor. Probe SEQ ID NOS. 41-47 are particularly suitable for 5-HT₂B receptor.

2. EXAMPLES Example 1

Localization of 5-HT and 5-HT receptors in human and rodent intestine [0077] Immunoreactivity was observed in both human and rat intestinal tissue using antibodies against 5-HT, 5-HT₃ receptors and 5-HT₄ receptors. In addition, in situ hybridization was used to detect mRNA for 5-HT₄ receptors in human intestinal tissue.

Figure legends

[0078] Figure 2: Serotonin immunoreactivity in surgical resections of human bowel (A - F), rat jejunum (G) and mouse jejunum (H). Prominent serotonin immunoreactivity is apparent in enterochromaffin cells (in A and ► in B, C, D, G and H). Mast cells are clearly serotonin immunoreactive in all rodent gut samples (* in G and H) and in approximately 1 in 5 human samples (* in C, D, E and F). [0079] Figure 3: 5-HT₃ immunoreactivity in surgical resections of human bowel. All samples are non-involved tissues from cancer resections. Note clear enteroendocrine cell staining (A - C) and minimal labelling in the lamina propria (B, C and D).

[0080] Figure 4: 5-HT₄-immunoreactivity in human gut. 5-HT₄ receptors are localized to mast cells in the lamina propria (A), enteroendocrine cells (B) muscle (muscularis propria; C) and in a punctuate pattern in the lamina propria (D), at all levels of the human gastrointestinal tract.

[0081] Figure 5: 5-HT₄- immunoreactivity in rat gut. Mast cells in the submucosa (A) and mucosa in normal rat gut are 5-HT₄ -immunoreactive. Following infection with Nippostrongylus brasiliensis, many more mucosal mast cells express strong 5-HT4-immunoreactivity (B).

[0082] Figure 6: Localization of mRNA for 5-HT₄ in human gut. A submucosal mast cell expressing message for 5-HT₄ (antisense probe 36LA) is evident in (B) but mast cells do not bind the message sense probe (A). Similarly, muscle cells express 5-HT₄ mRNA (D) but do not label with message sense probes (C).

Example 2

Localization of 5-HT₄ receptor immunoreactivity in human and rodent mast cells lines

[0083] As immunoreactivity was visualized in mast cells in human and rodent tissues, 5-HT₄ receptor immunohistochemistry was performed on human and rodent mast cell lines in order to attempt to verify this observation. 5-HT₄ receptor immunoreactivity was observed on the human mast cell line KU812, primary mouse cultured bone marrow mast cells and rat basophil leukaemia cells. Figure legends

[0084] Figure 7: 5HT₄ -immunoreactivity in human, rat and mouse cell lines KU812 (human mast cell/basophil) cells express strong 5HT₄-immunoreactivity (A); but staining is not observed using an equivalent concentration of normal rat immunoglobulins (negative control; B). Both primary mouse cultured bone marrow mast cells (C) and rat basophil leukaemia cells (mucosal mast cell like; RBL; D) also stain intensely for 5HT₄.

Example 3 Localization of 5-HT₄ receptor immunoreactivity in 5-HT_4b-transfected CHO cells

[0085] In order to verify that the 5-HT₄ receptor antibody being used was interacting with and hence staining 5-HT₄ receptors, immunohistochemistry was performed on wild type CHO cells and CHO cells transfected with 5-HT_4b receptor. 5-HT₄ receptor immunoreactivity was only observed in 5-HT_4b- transfected CHO cells.

Figure legends

[0086] Figure 8: 5HT4-immunoreactivity in 5HT_4b-transfected CHO cells. Smears of wild type (A & E) and 5HT₄ -transfected (B & F) CHO cells and paraffin processed cell pellets of wild type (C & G) and transfected (D & H) cells are illustrated, lmmunostaining for the V5 tag labels a small proportion of only the transfected cells (B & D) but not the wild type cells (A & C). Anti-5HT₄ antibodies detect a similar population of transfected CHO cells (F & H) but not wild type cells (E & G).

Example 4

Evidence for functional 5-HT₄ receptors on mast cells - calcium imaging [0087] In order to determine whether the 5-HT₄ receptors observed on mast cells using immunohistochemistry were functional 5-HT₄ receptors, various mast cell lines and 5-HT_4b-transfected CHO cells were activated by a calcium ionophore in the presence or absence of 5-HT or the 5-HT₄ receptor agonist RS67506 whilst the intracellular calcium concentration was monitored. Using this functional assay 5-HT₄ agonism was shown to inhibit mast cell activation, but augment calcium mobilization in 5-HT_4b-transfected CHO cells. This differential effect of 5-HT₄ agonism maybe due to different downstream coupling of 5-HT₄ receptors in the two different cell types. Interestingly, earlier work on 5HT₄ transfected HEK cells showed the opposite effect on cAMP levels to natively 5- HT₄ expressing cells (Bockaert et al. 2004)

Figure legends

[0088] Figure 9: The 5-HT₄ receptor partial agonist, RS 67506, augments ionomycin-induced calcium mobilization in 5-HT_4B-transfected CHO cells. In wild type CHO cells ionomycin (2 μM) resulted in a reproducible elevation of intracellular calcium as measured with Fura-2. Pre-incubation with 20 μM RS had no impact on calcium elevation. Stimulation of the 5-HT₄ transfected CHO cells with ionomycin resulted in an elevation of intracellular calcium, similar to the levels observed in wild type cells. However, pre-incubation with RS resulted in a significant increase in the Fura-2 ratio, indicating an enhanced elevation of intracellular calcium. ANOVA p < 0.0001. Data are expressed as percentages of the mean peak responses ± SEM.

[0089] Figure 10: The 5-HT₄ receptor partial agonist, RS 67506, attenuates ionomycin-induced calcium mobilization in BMMC. In control BMMC ionomycin resulted in a reproducible elevation of intracellular calcium as measured with Fura-2. However, pre-incubation with RS resulted in a significant decrease in the Fura-2 ratio, suggesting that stimulation of the with the 5-HT₄ receptor partial agonist resulted in a decrease in the stimulated elevation of intracellular calcium.

[0090] Figure 11: The 5-HT₄ receptor partial agonist, RS 67506, attenuates ionomycin-induced calcium mobilization in RBL (A) and KU812 (B) cells. In control RBL cells, ionomycin resulted in a reproducible elevation of intracellular calcium as measured with Fura-2. In addition, stimulation of the KU812 cells with ionomycin caused a reproducible elevation of intracellular calcium. Preincubation of the RBL cells with 20 μM RS resulted in a significant decrease in the Fura-2 ratio. Similarly, pre-incubation of the KU812 cells with RS (2 - 20 μM) resulted in a dose-dependent decrease in the Fura-2 ratio. Furthermore, pre- incubating the KU812 cells with 10 μM 5-HT resulted in a decrease in Fura-2 ratio (B), similar to that observed with the higher concentrations of RS. ANOVA for RBL: p < 0.001 and for KU812: p = 0.118. All data are expressed as a percentage of the control response.

Example 5

Evidence for functional 5-HT₄ receptors on mast cells - release assays [0091] In order to determine whether the 5-HT₄ receptors observed on mast cells using immunohistochemistry were functional 5-HT₄ receptors, various mast cell lines were activated by the calcium ionophore in the presence or absence of 5-HT or the 5-HT₄ receptor agonist RS67506 and the activation status of the mast cells was recorded using two different ELISA release assays. Using this functional assay 5-HT₄ agonism was shown to inhibit mast cell activation.

Figure legends

[0092] Figure 12: The 5-HT₄ receptor partial agonist, RS 67506, attenuates ionomycin-induced TNFα release from RBL cells (A) and BMMC (B). Stimulating either cell type resulted in a significant increase in the amount of TNFα released into the culture medium. Stimulation with 5-HT had no significant impact on the amount of TNF release in either RBL or BMMC cell types. Similarly, stimulation with the 5-HT₄ receptor antagonist caused no significant changes in the amount of TNF release. In contrast, stimulation of the RBL cells with RS resulted in a significant 72% decrease in TNF release. A similar trend was observed in the BMMC; however, this failed to reach statistical significance. TNF levels were measured using species specific ELISA. ANOVA for RBL: p < 0.001 and for BMMC p = 0.26. [0093] Figure 13: The 5-HT₄ receptor partial agonist, RS 67506, attenuates ionomycin-induced β-hexosaminidase release from KU812 cells. Stimulating KU812 cells with 2 μM ionomycin resulted in a significant increase in the amount of β-hex release. Pre-incubating these cells with 20 μM RS resulted in a significant decrease in β-hex release. ANOVA p = 0.11. Data are expressed as β-hex released as a percentage of total ± SEM.

Example 6

Evidence for variable expression of 5-HT₄ receptors in the lamina propria of human gut biopsies

[0094] As functional 5-HT₄ receptors appear to be expressed in human intestine, especially on mast cells, and mast cell numbers and relationships with nerves are reported to change in irritable bowel syndrome (Barabara et al. 2006), this raises the possiblity that 5-HT₄ receptors could be used as a theragnostic tool to determine the most appropriate treatment for patients suffering from functional bowel disorders such as irritable bowel syndrome. Hence the levels of 5-HT₄ receptor expression was determined in patients suffering from irritable bowel syndrome and controls. Quantitation of the 5HT4 immunoreactivity in the lamina propria of rectal biopsies using image analysis software revealed numerical differences in the amount of staining in different patients. The number of mast cells (and other cell types) expressing 5-HT₄ receptors varied over a wide range (>20 fold differences). Quantitative analysis of integrated optical densities of 5-HT₄-immunoreactivity in these samples (illustrated in Figures 14 and 15) is ongoing and the preliminary data show differences that reveal a trend that 5-HT₄ receptor expression varies according to the type of irritable bowel syndrome (diarrhea, constipation or alternator) with constipation patients showing a 5-HT₄ receptor expression close to the control (Figure 17) (close is intended to mean within about 20 % deviation, preferably about 10 % deviation with respect to the control). These data indicate that there is heterogeneity in 5-HT₄ receptor expression in mast cells in different IBS patient groups and suggests that this could be an important indicator of either the nature of the functional bowel disorder and/or the type of treatment that would be most effϊcaceous for each patient. There was, however, a large within group (constipation, diarrhea or alternator) variability. It may be that the heterogeneity of expression within the different patient groups reflects the variability in response to treatment. Additional studies are required to determine this.

[0095] Preliminary results for the 5-HT₃ receptor on gastrointestinal cells show a similar tendency.

Figure legends

[0096] Figure 14: Variable expression of 5-HT₄ in the lamina propria of terminal ileum endoscopic biopsies from six different patients (A - 007, B - 068, C - 075, D - 077, E - 095, F - 102). Compare with Figure 10.

[0097] Figure 15: Variable expression of 5-HT₄ in the lamina propria of rectal endoscopic biopsies from six different patients (A - 007, B - 068, C - 075, D - 077, E - 095, F - 102). Compare with Figure 9.

[0098] Figure 16: 5-HT₄ immunoreactive cell counts in the lamina propria of terminal ileum and rectal endoscopic biopsies from patients illustrated in Figures 9 and 10.

[0099] Figure 17: Quantitation of the 5-HT₄-immunoreactivity in the lamina propria of rectal biopsies . Using imaging software numerical differences in the amount of staining in different patients was revealed. The classification according to patient group show a trend that in patients suffering from IBS with constipation, the level of 5-HT₄ receptor expression is close to healthy controls, whereas the the level of 5-HT₄ receptor expression in patients suffering from IBS with diarrhea or alternating constipation and diarrhea is lower. [0100] It is noted that the foregoing examples have been provided merely for the purpose of explanation and are in no way to be construed as limiting of the present invention. While the present invention has been described with reference to exemplary embodiments, it is understood that the words which have been used herein are words of description and illustration, rather than words of limitation. Changes may be made, within the purview of the appended claims, as presently stated and as amended, without departing from the scope and spirit of the present invention in its aspects. Although the present invention has been described herein with reference to particular means, materials and embodiments, the present invention is not intended to be limited to the particulars disclosed herein; rather, the present invention extends to all functionally equivalent structures, methods and uses, such as are within the scope of the appended claims.

Reference List

Barbara, G., Stanghellini, V., De Giorgio, R., & Corinaldesi, R. (2006). Functional gastrointestinal disorders and mast cells: implications for therapy. Neurogastroenterol.Motil. 18, 6-17.

Bockaert, J., Claeysen, S., Compan, V., & Dumuis, A. (2004). 5-HT4 receptors. Cυrr.Drug Targets.CNS.Neυrol.Disord. 3, 39-51.

De Ponti, F. & Tonini, M. (2001). Irritable bowel syndrome: new agents targeting serotonin receptor subtypes. Drugs 61 , 317-332.

GaIIi₁ S. J., Nakae, S., & Tsai, M. (2005). Mast cells in the development of adaptive immune responses. Nat.lmmunol. 6, 135-142.

Gottwald, T., Coerper, S., Schaffer, M., Koveker, G., & Stead, R. H. (1998). The mast cell-nerve axis in wound healing: a hypothesis. Wound. Repair Regen. 6, 8- 20.

Lippert, U., Artuc, M., Grutzkau, A., Babina, M., Guhl, S., Haase, I., Blaschke, V., Zachmann, K., Knosalla, M., Middel, P., Kruger-Krasagakis, S., & Henz, B. M. (2004). Human skin mast cells express H2 and H4, but not H3 receptors. J. Invest Dermatol. 123, 116-123.

Marshall, J. S. (2004). Mast-cell responses to pathogens. Nat.Rev. Immunol. 4, 787-799.

Marshall, J. S. & Jawdat, D. M. (2004). Mast cells in innate immunity. J.AIIergy Clin. Immunol. 114, 21-27.

Stead, R. H., Colley, E. C, Wang, B., Partosoedarso, E., Lin, J., Stanisz, A., & Hillsley, K. (2006). Vagal influences over mast cells. Auton.Neurosci. 125, 53-61.

Thompson, W. G., Longstreth, G. F., Drossman, D. A., Heaton, K. W., Irvine, E. J., & Muller-Lissner, S. A. (1999). Functional bowel disorders and functional abdominal pain. Gut 45 Suppl 2, II43-II47. Weller, C. L., Collington, S. J., Brown, J. K., Miller, H. R., Al Kashi, A., Clark, P., Jose, P. J., Hartnell, A., & Williams, T. J. (2005). Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors. J. Exp. Med.

Xie, H. & He, S. H. (2005). Roles of histamine and its receptors in allergic and inflammatory bowel diseases. World J.Gastroenterol. 11 , 2851-2857.

Claims

WHAT IS CLAIMED IS:

1. A method for diagnosing a functional bowel disease in a human patient, wherein the method comprises testing gastrointestinal cells and/or cells circulating in peripheral blood of the patient for presence of at least one of serotonin and a serotonin receptor.

2. The method of claim 1 , wherein the serotonin receptor comprises at least one of 5-HT₃ receptor and 5-HT₄ receptor.

3. The method of claim 1 , wherein the gastrointestinal cells and/or cells circulating in peripheral blood are tested at least for the presence of serotonin.

4. The method of claim 1 , wherein the gastrointestinal cells and/or cells circulating in peripheral blood are tested at least for the presence of a serotonin receptor.

5. The method of claim 4, wherein the gastrointestinal cells and/or cells circulating in peripheral blood are tested at least for the presence of 5-HT₃ receptor.

6. The method of claim 4, wherein the gastrointestinal cells and/or cells circulating in peripheral blood are tested at least for the presence of 5-HT₄ receptor.

7. The method of claim 4, wherein the gastrointestinal cells and/or cells circulating in peripheral blood are tested at least for the presence of 5-HT₃ receptor and 5-HT₄ receptor.

8. The method of claim 1 , wherein the gastrointestinal cells and/or cells circulating in peripheral blood are tested for the presence of serotonin, 5-HT₃ receptor and 5-HT₄ receptor.

9. The method of any one of claims 1 to 8, wherein the method comprises a quantitative determination of at least one of serotonin and a serotonin receptor.

10. The method of any one of claims 1 to 9, wherein testing is carried out ex vivo.

11. The method of any one of claims 1 to 10, wherein the gastrointestinal cells are selected from one or more of mast cells, muscle cells, neurons, epithelial cells, lymphocytes, macrophages, granulocytes, dendritic cells, enteroglial cells and enteroendocrine cells and the cells circulating in peripheral blood are selected from one or more of mast cells, lymphocytes, macrophages, granulocytes, dendritic cells and other cells.

12. The method of any one of claims 1 to 11 , wherein the functional bowel disease comprises irritable bowel syndrome.

13. The method of any one of claims 1 to 12, wherein the method comprises incubation of the cells with an antiserum for at least one of serotonin and a serotonin receptor.

14. The method of any one of claims 1 to 13, wherein the method comprises in situ hybridization of mRNA associated with at least one of serotonin receptor.

15. The method of claim 13, wherein the in situ hybridization is carried out with at least one biotinylated probe.

16. The method of any one of claims 1 to 15, wherein the gastrointestinal cells and/or cells circulating in peripheral blood comprise mast cells.

17. The method of claim 16, wherein the method comprises contacting the mast cells with a mast cell stimulator.

18. The method of claim 17, wherein the mast cell stimulator comprises a calcium ionophore.

19. The method of claim 18, wherein the calcium ionophore comprises ionomycin.

20. The method of claim 14, wherein the in situ hybridization is carried out with at least one probe that comprises at least one of the following sequences:

ACCTGCCCAGGGACAGTGTAGTCTATGAAA (SEQ ID NO: 1 ) AGATGCGGTAATAGGCCAGCACCATGAGGA (SEQ ID NO: 2) TAGGGAGAAAAGAAATAAACGTGGGGATGA (SEQ ID NO: 3) GGCACCAAAGGGCATCACCAGCACCGAAAC (SEQ ID NO: 4)

AGATCCGCAAAAGCAAGAGATACAATGAAA (SEQ ID NO: 5)

ACACAGCCACCATCACCAGCAGGTTCCCCA (SEQ ID NO: 6)

CGTAGGGCTTGTTGACCATGAAGACACAGT (SEQ ID NO: 7) CGATGCGCAGAGGGGTCATCTTGTTCCTAT (SEQ ID NO: 8) GCAGATGGCGTAATACCTATCCAGAGAAAT (SEQ ID NO: 9)

ATGCACAGGGTCTTGGCTGCTTTGGTCTCT (SEQ ID NO: 10)

AGCACAGGTGAAAAATCGATGCCGTTGTGA (SEQ ID NO: 11 )

AACACCTCCCCATAAATCCAGATGTCTTGA (SEQ ID NO: 12) CTTATTCAAGAAGGCGTAGAGAAAAGGGTT (SEQ ID NO: 13) TGGATCTGATGGGCATGCTCCTTAGCTGTG (SEQ ID NO: 14) ACTGACCCGAAACCCTCCTCAGAACTCACA (SEQ ID NO: 15) ACAGTCTGGCCCAGAATGGAAGGTCTTCGG (SEQ ID NO: 16)

TTGGTGACAAAGAATGGTGCCCAGCAGAGG (SEQ ID NO: 17)

ATGGGATGTAGAAGGCCACCACAGAGCAGG (SEQ ID NO: 18)

GCCGAGCCAGAGGAAAGCAGTCCACACCTG (SEQ ID NO: 22)

TGGCCCAGAATGGAAGGTCTTCGGTAGCGC (SEQ ID NO: 23)

GATGAGTGCTATGCTGGTCTGCCGACTGAG (SEQ ID NO: 26)

TGCCGTTGTGAGCAGGACGTCCAGAGATGT (SEQ ID NO: 27)

CTCCTTAGCTGTGACATAGATGCGGTAATA (SEQ ID NO: 28)

CACAGCCACCATCACCAGCAGGTTCCCCAA (SEQ ID NO: 29) and CACCAAAGGGCATCACCAGCACCGAAACCA (SEQ ID NO: 30)

21. The method of claim 14, wherein the in situ hybridization is carried out with at least one probe that comprises at least one of the following sequences:

5' CTCACACTCCCTGGGCCACCAAGGTTGGGG 3' (SEQ ID NO: 31) 5' CTCATGTCCCAGCCAGCCTGCCTCTCGTGG 3' (SEQ ID NO: 32) 5' GGAGTGTGGGGAGGAGCAAGGCGAGCAGCG 3' (SEQ ID NO: 33) 5' CTCAGCAGAGCGGGCCTGGTGGTGTTTCGG 3' (SEQ ID NO: 34) 5' ACGGTGGTTGGCTTCCTCCAGTCCCTCACG 3' (SEQ ID NO: 35) 5' AGCACCCCCAGCAACTCCCACTCTCCCTGG 3' (SEQ ID NO: 36) 5' GACAGCTCCTGCAGCAGCCCACACACCGCC 3' (SEQ ID NO: 37) 5" CTTGTCCAGCACGGAGCCCACGCGCAGCCA 3' (SEQ ID NO: 38) 5' TCCCCACCTAACCAGGACTGTACCCCCTCC 3¹ (SEQ ID NO: 39) and 5' TGGTGAGCTGCTGGGGGTGGGACAAGGGTG 3' (SEQ ID NO: 40)

22. The method of claim 14, wherein the in situ hybridization is carried out with at least one probe that comprises at least one of the following sequences:

5' GCACAGGCGGTTGAAAGCAGTGAAACAAAT 3' (SEQ ID NO: 41 ) 5' CTTGCTGCTGTGACTGAAATCCTCCTCCCA 3' (SEQ ID NO: 42) 5' TCCCCCTCTGGTTTCGTTGTTATTTTGCTG 3' (SEQ ID NO: 43) 5' TCTGCCGTAGTTGTAGAGTCGTGTTTGAAC 3' (SEQ ID NO: 44) 5' TGAGCGCATACACACATCTGTCCATGTTTG 3' (SEQ ID NO: 45) 5' ACCATGCCAAACACTCAAAAGCCAAAGTTG 3' (SEQ ID NO: 46) and 5' CCGTTCCGAACAGTGGTCAGCATGGTTAGT 3'. (SEQ ID NO: 47).

23. The method of any one of claims 1 to 22, wherein the testing is carried out on tissue sections or on cells isolated from a tissue or on a peripheral blood sample.

24. A method for diagnosing irritable bowel syndrome in a human patient, wherein the method comprises testing gastrointestinal mast cells and/or mast cells circulating in peripheral blood of the patient for presence of at least one of serotonin, 5-HT₃ receptor and 5-HT₄ receptor.

25. The method of claim 24, wherein the mast cells are tested at least for the presence of serotonin.

26. The method of claim 24, wherein the mast cells are tested at least for the presence of 5-HT₃ receptor.

27. The method of claim 24, wherein the mast cells are tested at least for the presence of 5-HT₄ receptor.

28. The method of claim 27, wherein the mast cells are tested at least for the presence of 5-HT₃ receptor and 5-HT₄ receptor.

29. The method of claim 24, wherein the mast cells are tested for the presence of serotonin, 5-HT₃ receptor and 5-HT₄ receptor.

30. The method of any one of claims 24 to 29, wherein the method comprises a quantitative determination of at least one of serotonin, 5-HT₃ receptor and 5-

HT₄ receptor.

31. The method of any one of claims 24 to 30, wherein testing is carried out ex vivo.

32. The method of any one of claims 24 to 31 , wherein the method comprises incubation of the mast cells with an antiserum for at least one of serotonin, 5-HT₃ receptor and 5-HT₄ receptor.

33. The method of any one of claims 24 to 32, wherein the method comprises in situ hybridization of mRNA associated with at least one of the 5-HT₃ and 5-HT₄ receptors.

34. The method of any one of claims 24 to 33, wherein the method comprises contacting the mast cells with a mast cell stimulator.

35. The method of claim 34, wherein the mast cell stimulator comprises a calcium ionophore.

36. The method of claim 35, wherein the calcium ionophore comprises ionomycin.

37. The method of claim 33, wherein the in situ hybridization is carried out with at least one probe that comprises at least one of the following sequences:

ACCTGCCCAGGGACAGTGTAGTCTATGAAA (SEQ ID NO: 1 )

AGATGCGGTAATAGGCCAGCACCATGAGGA (SEQ ID NO: 2)

TAGGGAGAAAAGAAATAAACGTGGGGATGA (SEQ ID NO: 3)

GGCACCAAAGGGCATCACCAGCACCGAAAC (SEQ ID NO: 4) AGATCCGCAAAAGCAAGAGATACAATGAAA (SEQ ID NO: 5) ACACAGCCACCATCACCAGCAGGTTCCCCA (SEQ ID NO: 6)

CGTAGGGCTTGTTGACCATGAAGACACAGT (SEQ ID NO: 7)

CGATGCGCAGAGGGGTCATCTTGTTCCTAT (SEQ ID NO: 8)

GCAGATGGCGTAATACCTATCCAGAGAAAT (SEQ ID NO: 9) ATGCACAGGGTCTTGGCTGCTTTGGTCTCT (SEQ ID NO: 10) AGCACAGGTGAAAAATCGATGCCGTTGTGA (SEQ ID NO: 11 ) AACACCTCCCCATAAATCCAGATGTCTTGA (SEQ ID NO: 12) CTTATTCAAGAAGGCGTAGAGAAAAGGGTT (SEQ ID NO: 13)

TGGATCTGATGGGCATGCTCCTTAGCTGTG (SEQ ID NO: 14)

ACTGACCCGAAACCCTCCTCAGAACTCACA (SEQ ID NO: 15)

ACAGTCTGGCCCAGAATGGAAGGTCTTCGG (SEQ ID NO: 16) TTGGTGACAAAGAATGGTGCCCAGCAGAGG (SEQ ID NO: 17)

ATGGGATGTAGAAGGCCACCACAGAGCAGG (SEQ ID NO: 18)

TGATATAGCCGAGCCAGAGGAAAGCAGTCC (SEQ ID NO: 19)

CAATTATGCCAATGTTATTCCAGCCTTGCA (SEQ ID NO: 20) AGCACCACCTTCTCCACTGACCCGAAACCC (SEQ ID NO: 21 ) GCCGAGCCAGAGGAAAGCAGTCCACACCTG (SEQ ID NO: 22) TGGCCCAGAATGGAAGGTCTTCGGTAGCGC (SEQ ID NO: 23)

ACACTGACTCTCCCACTGGCCACCACACTC (SEQ ID NO: 24)

GAGCAGGTGATGGCGTAGGGCTTGTTGACC (SEQ ID NO: 25)

GATGAGTGCTATGCTGGTCTGCCGACTGAG (SEQ ID NO: 26) TGCCGTTGTGAGCAGGACGTCCAGAGATGT (SEQ ID NO: 27) CTCCTTAGCTGTGACATAGATGCGGTAATA (SEQ ID NO: 28)

CACAGCCACCATCACCAGCAGGTTCCCCAA (SEQ ID NO: 29) and

CACCAAAGGGCATCACCAGCACCGAAACCA (SEQ ID NO: 30)

38. The method of claim 33, wherein the in situ hybridization is carried out with at least one probe that comprises at least one of the following sequences:

5^! CTCACACTCCCTGGGCCACCAAGGTTGGGG 3' (SEQ ID NO: 31) 5' CTCATGTCCCAGCCAGCCTGCCTCTCGTGG 3' (SEQ ID NO: 32) 5' GGAGTGTGGGGAGGAGCAAGGCGAGCAGCG 3' (SEQ ID NO: 33) 5' CTCAGCAGAGCGGGCCTGGTGGTGTTTCGG 3' (SEQ ID NO: 34) 5' ACGGTGGTTGGCTTCCTCCAGTCCCTCACG 3' (SEQ ID NO: 35) 5' AGCACCCCCAGCAACTCCCACTCTCCCTGG 3' (SEQ ID NO: 36) 5' GACAGCTCCTGCAGCAGCCCACACACCGCC 3' (SEQ ID NO: 37) 5' CTTGTCCAGCACGGAGCCCACGCGCAGCCA 3' (SEQ ID NO: 38) 5' TCCCCACCTAACCAGGACTGTACCCCCTCC 3' (SEQ ID NO: 39) and 5¹ TGGTGAGCTGCTGGGGGTGGGACAAGGGTG 3' (SEQ ID NO: 40)

39. The method of any one of claims 24 to 38, wherein the testing is carried out on tissue sections or on mast cells isolated from a tissue or on mast cells of a peripheral blood sample.

40. A method for diagnosing a serotonin-related gastrointestinal disorder in a human patient, wherein the method comprises testing gastrointestinal cells and/or cells circulating in peripheral blood of the patient for presence of at least one of serotonin and a serotonin receptor.

41. The method of claim 39, wherein the gastrointestinal cells and/or cells circulating in peripheral blood are tested for at least one of 5-HT₃ receptor and 5-

HT₄ receptor.

42. A method for treating a functional bowel disease in a human patient, wherein gastrointestinal cells and/or cells circulating in peripheral blood of the patient have been determined to comprise a serotonin receptor and wherein the method comprises administering to the patient at least one of an agonist and an antagonist of the receptor.

43. The method of claim 42, wherein the functional bowel disease comprises irritable bowel syndrome.

44. The method of any one of claims 42 and 43, wherein the gastrointestinal cells and/or cells circulating in peripheral blood comprise mast cells.

45. A method for treating a functional bowel disease in a human patient, wherein gastrointestinal cells and/or cells circulating in peripheral blood of the patient have been determined to comprise 5-HT₃ receptor and wherein the method comprises administering to the patient a 5-HT₃ receptor antagonist.

46. The method of claim 45, wherein the functional bowel disease comprises irritable bowel syndrome.

47. The method of any one of claims 45 and 46, wherein the gastrointestinal cells and/or cells circulating in peripheral blood comprise mast cells.

48. The method of any one of claims 45 to 47, wherein the 5-HT₃ receptor antagonist comprises alosetron.

49. A method for treating a functional bowel disease in a human patient, wherein gastrointestinal cells and/or cells circulating in peripheral blood of the patient have been determined to comprise 5-HT₄ receptor and wherein the method comprises administering to the patient a 5-HT₄ receptor agonist.

50. The method of claim 49, wherein the functional bowel disease comprises irritable bowel syndrome.

51. The method of any one of claims 49 and 50, wherein the gastrointestinal cells and/or cells circulating in peripheral blood comprise mast cells.

52. The method of any one of claims 49 to 51 , wherein the 5-HT₄ receptor agonist comprises tegaserod.

53. A medicament comprising at least one of a serotonin receptor agonist and a serotonin receptor antagonist, wherein the medicament is associated with instructions to administer the medicament to a human patient whose gastrointestinal cells and/or cells circulating in peripheral blood comprise the serotonin receptor.

54. The medicament of claim 53, wherein the gastrointestinal cells and/or cells circulating in peripheral blood comprise mast cells.

55. The medicament of any one of claims 53 and 54, wherein the medicament is for treating a functional bowel disease.

56. The medicament of any one of claims 53 to 55, wherein the medicament is for treating irritable bowel syndrome.

57. A medicament comprising a 5-HT₃ receptor antagonist, wherein the medicament is associated with instructions to administer the medicament to a human patient whose gastrointestinal cells and/or cells circulating in peripheral blood comprise 5-HT₃ receptor.

58. The medicament of claim 57, wherein the gastrointestinal cells and/or cells circulating in peripheral blood comprise mast cells.

59. The medicament of any one of claims 57 and 58, wherein the medicament is for treating a functional bowel disease.

60. The medicament of any one of claims 57 to 59, wherein the medicament is for treating irritable bowel syndrome.

61. A medicament comprising a 5-HT₄ receptor agonist, wherein the medicament is associated with instructions to administer the medicament to a human patient whose gastrointestinal cells and/or cells circulating in peripheral blood comprise 5-HT₄ receptor.

62. The medicament of claim 61 , wherein the gastrointestinal cells and/or cells circulating in peripheral blood comprise mast cells.

63. The medicament of any one of claims 61 and 62, wherein the medicament is for treating a functional bowel disease.

64. The medicament of any one of claims 61 to 63, wherein the medicament is for treating irritable bowel syndrome.

65. A probe for hybridization of mRNA associated with at least one 5-HT receptor, wherein the probe that may be labeled comprises at least one of the following sequences:

ACCTGCCCAGGGACAGTGTAGTCTATGAAA (SEQ ID NO: 1 )

ACACAGCCACCATCACCAGCAGGTTCCCCA (SEQ ID NO: 6)

CGTAGGGCTTGTTGACCATGAAGACACAGT (SEQ ID NO: 7)

CGATGCGCAGAGGGGTCATCTTGTTCCTAT (SEQ ID NO: 8) GCAGATGGCGTAATACCTATCCAGAGAAAT (SEQ ID NO: 9) ATGCACAGGGTCTTGGCTGCTTTGGTCTCT (SEQ ID NO: 10) AGCACAGGTGAAAAATCGATGCCGTTGTGA (SEQ ID NO: 11 ) AACACCTCCCCATAAATCCAGATGTCTTGA (SEQ ID NO: 12) CTTATTCAAGAAGGCGTAGAGAAAAGGGTT (SEQ ID NO: 13) TGGATCTGATGGGCATGCTCCTTAGCTGTG (SEQ ID NO: 14)

ACTGACCCGAAACCCTCCTCAGAACTCACA (SEQ ID NO: 15)

ACAGTCTGGCCCAGAATGGAAGGTCTTCGG (SEQ ID NO: 16)

TTGGTGACAAAGAATGGTGCCCAGCAGAGG (SEQ ID NO: 17) ATGGGATGTAGAAGGCCACCACAGAGCAGG (SEQ ID NO: 18) TGATATAGCCGAGCCAGAGGAAAGCAGTCC (SEQ ID NO: 19) CAATTATGCCAATGTTATTCCAGCCTTGCA (SEQ ID NO: 20)

AGCACCACCTTCTCCACTGACCCGAAACCC (SEQ ID NO: 21 ) GCCGAGCCAGAGGAAAGCAGTCCACACCTG (SEQ ID NO: 22) TGGCCCAGAATGGAAGGTCTTCGGTAGCGC (SEQ ID NO: 23) ACACTGACTCTCCCACTGGCCACCACACTC (SEQ ID NO: 24)

GAGCAGGTGATGGCGTAGGGCTTGTTGACC (SEQ ID NO: 25)

GATGAGTGCTATGCTGGTCTGCCGACTGAG (SEQ ID NO: 26)

TGCCGTTGTGAGCAGGACGTCCAGAGATGT (SEQ ID NO: 27) CTCCTTAGCTGTGACATAGATGCGGTAATA (SEQ ID NO: 28)

CACAGCCACCATCACCAGCAGGTTCCCCAA (SEQ ID NO: 29) and

CACCAAAGGGCATCACCAGCACCGAAACCA (SEQ ID NO: 30)

66. A probe for hybridization of mRNA associated with at least one 5-HT receptor, wherein the probe that may be labeled comprises at least one of the following sequences: 5' CTCACACTCCCTGGGCCACCAAGGTTGGGG 3' (SEQ ID NO: 31 ) 5' CTCATGTCCCAGCCAGCCTGCCTCTCGTGG 3' (SEQ ID NO: 32) 5' GGAGTGTGGGGAGGAGCAAGGCGAGCAGCG 3' (SEQ ID NO: 33) 5' CTCAGCAGAGCGGGCCTGGTGGTGTTTCGG 3' (SEQ ID NO: 34) 5' ACGGTGGTTGGCTTCCTCCAGTCCCTCACG 3' (SEQ ID NO: 35) 5' AGCACCCCCAGCAACTCCCACTCTCCCTGG 3¹ (SEQ ID NO: 36) 5' GACAGCTCCTGCAGCAGCCCACACACCGCC 3' (SEQ ID NO: 37) 5' CTTGTCCAGCACGGAGCCCACGCGCAGCCA 3' (SEQ ID NO: 38) 5' TCCCCACCTAACCAGGACTGTACCCCCTCC 3' (SEQ ID NO: 39) and 5' TGGTGAGCTGCTGGGGGTGGGACAAGGGTG 3' (SEQ ID NO: 40)

67. A probe for hybridization of mRNA associated with at least one 5-HT receptor, wherein the probe that may be labeled comprises at least one of the following sequences:

5' GCACAGGCGGTTGAAAGCAGTGAAACAAAT 3' (SEQ ID NO: 41 ) 5' CTTGCTGCTGTGACTGAAATCCTCCTCCCA 3' (SEQ ID NO: 42) 5' TCCCCCTCTGGTTTCGTTGTTATTTTGCTG 3' (SEQ ID NO: 43) 5' TCTGCCGTAGTTGTAGAGTCGTGTTTGAAC 3' (SEQ ID NO: 44) 5' TGAGCGCATACACACATCTGTCCATGTTTG 3' (SEQ ID NO: 45)

5' ACCATGCCAAACACTCAAAAGCCAAAGTTG 3' (SEQ ID NO: 46) and 5' CCGTTCCGAACAGTGGTCAGCATGGTTAGT 3'. (SEQ ID NO: 47).

68. A diagnostic kit for the detection or quantification of at least one 5-HT receptor, wherein the kit comprises the probe of claim 65.

69. A diagnostic kit for the detection or quantification of at least one 5-HT receptor, wherein the kit comprises the probe of claim 66.

70. A diagnostic kit for the detection or quantification of at least one 5-HT receptor, wherein the kit comprises the probe of claim 67.

71. Use of the diagnostic kit of any of claims 68 to 70 for the detection or quantification of at least one 5-HT receptor.

72. A method for determining whether a medicament for treating functional bowel disease in a mammal is suitable for a particular individual, wherein gastrointestinal cells and/or cells circulating in peripheral blood of said individual are tested for the level of expression of a serotonin receptor preferably selected from 5-HT₃ receptor and 5-HT₄ receptor or both; and a 5-HT₃ receptor antagonist is determined to be the suitable medicament for said individual, if the level of expression of 5-HT₃ receptor on the gastrointestinal cells and/or cells circulating in peripheral blood of said individual is close to or higher than said level of a healthy control; and a 5-HT₄ receptor agonist is determined to be the suitable medicament for said individual, if the level of expression of 5-HT₄ receptor on the gastrointestinal cells and/or cells circulating in peripheral blood of said individual is close to or higher than said level of a healthy control;

73. The method of claim 72, wherein the functional bowel disease comprises irritable bowel syndrome.

74. The method of claim 72 or 73, wherein the gastrointestinal cells and/or cells circulating in peripheral blood of said individual comprise mast cells.

75. The method of any of claims 72 to 75, wherein the 5-HT₃ receptor antagonist comprises alosetron.

76. The method of any of claims 72 to 75, wherein the 5-HT₄ receptor agonist comprises tegaserod.

77. The method of any of claims 72 to 76, wherein said mammal is a human.

78. A combination of a medicament for treating functional bowel disease in a mammal and a label or a package insert, which indicates that, if said medicament is a 5-HT₃ receptor antagonist, it is a suitable medicament for said mammal, if the level of expression of 5-HT₃ receptor on the gastrointestinal cells and/or cells circulating in peripheral blood of said mammal is close to or higher than said level of expression in healthy controls; and if said medicament is a 5-HT₄ receptor agonist, it is a suitable medicament for said mammal, if the level of expression of 5-HT₄ receptor on the gastrointestinal cells and/or cells circulating in peripheral blood of said mammal is close to or higher than said level in normal healthy controls.

79. The label or leaflet of claim 78, wherein said mammal is a human.

80. The label or leaflet of claim 78 or 79, wherein said functional bowel disease comprises irritable bowel syndrome.

81. The label or leaflet of any of claims 78 to 80, wherein said gastrointestinal cells and/or cells circulating in peripheral blood comprise mast cells.

82. The label or leaflet of any of claims 78 to 81 , wherein the 5-HT₃ receptor antagonist comprises alosetron.

83. The label or leaflet of any of claims 78 to 81 , wherein the 5-HT₄ receptor agonist comprises tegaserod.