MXPA98008434A - Proteins trifolias intestine - Google Patents

Abstract

The present invention relates to intestinal trifolial proteins and nucleic acids that encode intestinal trifolium factors. The described intestinal trifolium factors are resistant to destruction in the digestive tract and can be used for the treatment of peptic ulcer diseases, inflammatory bowel diseases and other atachias.

Description

INTESTINAL TRIFOLIAS PROTEINS Background The field of the invention is peptides that are useful for the diagnosis, prevention or treatment of wounds, including those that are associated with a gastrointestinal disorder. Jorgensen and others. (Regulatory Peptides 3: 231, 1982) describe a porcine pancreatic peptide, pancreatic spasmolytic peptide (PEP). It was found that PEP inhibits "gastrointestinal motility and secretion of gastric acid in laboratory animals after both parenteral and oral administration". It has been suggested that "if results in animal experiments can be confirmed in man, PEP may have a potential utility in the treatment of gastroduodenal ulcer diseases." SUMMARY OF THE INVENTION In a first aspect, the invention features a purified nucleic acid encoding an intestinal trifolium factor (FTI). In preferred embodiments, the intestinal trifolium factor is the mammalian intestinal trifolium factor, preferably the intestinal trifolium factor of humans, rats, bovines or swine. In another preferred embodiment, the purified nucleic acid encoding an intestinal trifolium factor is present within a vector. In a related aspect, the invention features a cell that includes a vector encoding an intestinal trifolium factor.

In another related aspect, the invention features a substantially pure intestinal trifolio factor. In a preferred embodiment, the polypeptide is detectably labeled. In a related aspect, the invention features a therapeutic composition that includes an intestinal trifolium factor and a pharmacologically acceptable carrier. In another aspect, the invention features a monoclonal antibody that preferentially binds (i.e. forms an immune complex) with an intestinal trifolium factor. In a preferred embodiment, the monoclonal antibody is detestably labeled. In a related aspect, the invention features a method for detecting an intestinal trifolium factor in humans in a human patient. The method includes the steps of contacting a biological sample obtained from the patient with a monoclonal antibody that preferentially binds to the intestinal trifolium factor, and detects immune complexes formed with the monoclonal antibody. In preferred embodiments, the biological sample is an intestinal mucosal fragment, or serum. In a related aspect, the invention features a method for treating digestive disorders in a human patient, which method involves administering to the patient a therapeutic composition that includes an intestinal trifolium factor and a pharmacologically acceptable carrier. Additional disorders that can also be treated are described below. In another aspect, the invention features a method for detecting binding sites for an intestinal trifolium factor in a patient. The method involves contacting a biological sample obtained from the patient with the factor and detecting the factor bound to the biological sample as an indication of the presence of the binding sites in the sample. By "binding sites", as used herein, is meant any antibody or receptor that binds an intestinal trifolio factor, factor, or analog protein. The detection or quantification of binding sites can be a useful reflection of abnormality of the gastrointestinal tract. In another aspect, the invention substantially characterizes a pure trifolium factor. In preferred embodiments, the intestinal trifolio factor is human trifolium factor, of porcine, or bovine. By "intestinal trifolio factor" ("FTI") is meant any protein that is substantially homologous to intestinal trifolio factor (Fig. 2; SEQ ID NO: 2) and that is expressed in the intestine. thick, small intestine or colon to a greater degree that is expressed in tissues other than the small intestine, large intestine or colon. Also included are: allelic variations; natural mutants: induced mutants; proteins encoded by DNA that hybridize under high or low restriction conditions to nucleic acids encoding FTI recovered from material present in nature; and polypeptides or proteins recovered by antiserum to FTI. especially for antisera to the active site or FTI binding domain. The term also includes other chimeric polypeptides that include an FTI.

The term FTI also includes analogs of FTI polypeptides present in nature. Analogs may differ from FTI present in nature by differences in amino acid sequences or by modifications that do not affect the sequence, or both. Analogs of the invention will generally exhibit at least 70%, more preferably 80%, more preferably 90%, and even more preferably 95% or even 99%, of homology with all or part of an FTI sequence present in nature. The length of comparison sequences will generally be at least 8 amino acid residues, usually at least 20 amino acid residues, more usually at least 24 amino acid residues, typically 28 amino acid residues, and preferably more than 35 amino acid residues. . Modifications include in vivo or in vitro, chemical derivatization of polypeptides, e.g., acetylation or carboxylation. Also included are glycosylation modifications, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in additional processing steps, e.g., by exposing the polypeptide to enzymes that affect glycosylation derived from cells that normally provide such processing, eg, mammalian glycosylation enzymes. Versions of the same amino acid sequence having phosphorylated amino acid residues, e.g., phosphotyrosine, are also encompassed. phosphoserine, or phosphothreonine. Analogs may differ from FTIs present in nature due to alterations in their primary sequence. These include genetic variants, both natural and induced. Induced mutants can be derived from various techniques, including random mutagenesis of the encoding nucleic acids using irradiation or exposure to ethano methyl sulfate (EMS), or they can incorporate changes produced by site-specific mutagenesis or other molecular biology techniques. See, Sambrook, Fritsch and Maniatis (1989), Molecular Cloning: A Laboratory Manual (2d de.), CSH Press, Cold Spring Harbor, New York. Also included are analogs that include residues other than the L-amino acids present in nature, v. Cg. D-amino acids or amino acids that are not present in nature or synthetic, e.g., β or β? amino acids. In addition to the polypeptides of substantially complete length, the term FTI, as used herein, includes biologically active fragments of the polypeptides. As used herein, the term "fragment", as applied for a polypeptide, will ordinarily be of at least 10 contiguous amino acids, typically of at least 20 contiguous amino acids, more usually of at least 30 contiguous amino acids. usually of at least 40 contiguous amino acids, preferably at least 50 contiguous amino acids, and even more preferably at least 60 or 80 or more contiguous amino acids in length. The FTI fragments can be generated by methods known to those of ordinary skill in the art. The ability of a candidate fragment to exhibit an FTI biological activity can be evaluated by methods known to those skilled in the art. Also included in the term "fragment" are biologically active FTI polypeptides containing amino acids that are normally removed during protein processing, including additional amino acids that are not required for the biological activity of the polypeptide or that include additional amino acids resulting from the division of alternative mRNA or alternative protein processing events. A FTI polypeptide, fragment or analog is biologically active if it exhibits a biological activity of an FTI that is present in nature, e.g., the ability to alter gastrointestinal motility in a mammal. The invention also includes nucleic acid sequences, and purified preparations thereof, which encode the FTI polypeptides described herein, as well as antibodies, preferably monoclonal antibodies, which specifically bind to the FTI polypeptides. As used herein, the term "substantially pure" describes a compound, e.g., a nucleic acid, a protein or a polypeptide, e.g., an FTI protein or polypeptide that is substantially free of the components that accompany him in a natural way. Typically, a compound is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90% and even more preferably at least 99% of the total material (by volume, in wet or dry weight, or in molar percentage or molar fraction) in a sample is the compound of interest. The purity can be measured by any suitable method, e.g., in the case of polypeptides by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. By "isolated DNA" is meant DNA that is free from genes which, in the genome present in the nature of the organism from which the given DNA of the invention is derived, flank the DNA. The term "isolated DNA" therefore encompasses, for example, cDNA, cloned genomic DNA and synthetic DNA. A "purified nucleic acid", as used herein, refers to a nucleic acid sequence that is substantially free of other macromolecules (e.g., other nucleic acids and proteins) with which it occurs naturally within a cell. In preferred embodiments, less than 40% (and more preferably less than 25%) of the purified nucleic acid preparation consists of another macromolecule. "Homologs", as used herein, refers to the similarity of the subunit sequence between two polymeric molecules, e.g., between two nucleic acid molecules, v. Gr. two DNA molecules, or two polypeptide molecules. When a subunit position in both molecules is occupied by the same monomeric subunit, eg, if a position in each of the two DNA molecules is occupied by adenine, then they are homologous in that position. The homology between two sequences is a direct function of the number of equal or homologous positions, eg, if they are half, eg, 5 of 10, of the positions in sequences of two compounds are homologous when the two sequences they are 50% homologous; if 90% of the positions, eg, 9 of 10, are equal or homologous, the two sequences share 90% homology. For example, the DNA sequences 5'-ATTGCC-3 'and 5' -TATGGC-3 'share 50% homology "Substantially homologous" is understood to be largely, but not completely, homologous The FTI proteins of the invention are resistant to destruction in the digestive tract and can be used for treatment of peptic ulcer diseases, inflammatory bowel diseases and for the protection of the intestinal tract from damage caused by attacks such as radiation damage or bacterial infection. An FTI protein, fragment, or analog can also be used to treat neoplastic cancer and as described below, to protect (ie inhibit the formation of injuries) or to treat any part of the body from inflammation or damage such as injury, ulceration, burns or abrasions. Thipfolia proteins, including FTI, are useful for the treatment of disorders and damage to the alimentary canal including the mouth, esophagus, stomach and large and small intestine as well as for the protection and treatment of tissues that lie outside the alimentary canal. The polypeptide can be used either to treat necessary lesions or to inhibit the formation of lesions. The latter tissues include, for example, the external surface of the skin, the surface of the eye, the mucosa of the nasal passages and the respiratory tract and the genitourinary tract. One of the most common bacterial infections is caused by Helicobacter pylori (H. pylori), which leads to chronic gastritis and frequently to associated syndromes such as duodenal ulcer, gastric ulcer, gastric cancer, MALT lymphoma, or Menetrier's syndrome. It has been shown that the eradication of H pylori reduces the recurrence of duodenal and gastric ulcers. In addition, it has been postulated that widespread treatment of H. pylori will reduce the incidence of gastric carcinoma, which is the second leading cause of cancer-related death worldwide. Durable gastritis associated with H pylori infection is often associated with expression of intestinal-like characteristics in the gastric mucosa This condition, termed as intestinal metaplasia (MI), may indicate an increased risk of gastric cancer The etiology of Ml is unclear could represent an adaptation or mutational defense against H. pylori infection. pylori. For example, the metaplastic mucosa can produce mucus and other substances that create an environment that is useful for H. pylori. FTI can be used in the treatment of H. pylori infection and conditions associated with H pylori infection (v gr, ulcers, gastric carcinoma, ulcer-free dyspepsia, gastritis and esophageal lesions associated with gastroesophageal reflux diseases). The FTI is useful for the treatment of these conditions due to its generally protective effect on the gastrointestinal tract. In addition, FTI promotes the maintenance of mucosal integrity. FTI can be used to inhibit adhesion or colonization of the mucosa by H. pylori. In this application, FTI or fragments or variants thereof that inhibit adhesion or colonization of the mucosa by H. pylori are useful. Said molecules can be identified using analyzes known to those skilled in the art, including the analysis of H. pylori binding described below. FTI can also be used to promote the healing of damaged tissues by conditions associated with H. pylori infection. In this regard, it is important that the addition of the trífolias proteins to the wounded monolayers of confluent intestinal epithelial cells increases the rate of migration of epithelial cells in the wound. This effect is increased by the concomitant addition of mucin glycoproteins, the other dominant product of balloon cells. Such as FTI can be used to protect other parts of the gastro-intestinal tract or alimentary canal such as the intestine, can be used to protect the mouth and esophagus from damage caused by radiation therapy or chemotherapy. FTI can also be used to protect against (ie, inhibit the damage caused by) and / or treat damage caused by alcohols or drugs generally. Additional tissues that can be protected or treated by an FTI include those listed above that lie outside the alimentary canal. Members of the trifolia family, including FTI, can be used in the treatments discussed above. Skilled artisans can review these proteins in Sands et al. (1996, Ann. Rev. Physiol. 58: 253-273). As stated above, the invention encompasses biologically active fragments of trifolie proteins. Fragments that retain the trifolia structure (ie, the structure of three loops) or that lie within regions of the protein that are highly conserved may prove to be particularly useful. Therefore, such fragments may encompass FTI portions of approximately the same cysteine residue involved in a disulfide bond of the three loop structure around the last cysteine residue involved in the disulfide bond of the three loop structure. Variants of the selected trifolia protein are at least 60%, preferably at least 75%, more preferably at least 90%, and even more preferably 95% identical to the selected trifolia protein, preferably a human trifolia protein, more preferably human FTI. The term "identical", as used herein with reference to the polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. In the case of amino acid sequences that are less than 100% identical to a reference sequence, the non-identical positions preferably, but not necessarily, are conservative substitutions for the reference sequence. Conservative substitutions usually include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid; asparagine, glutamine, serine, threonine, lysine, arginine; and phenylalanine, tyrosine. Sequence identity is usually measured using Sequence Analysis Software such as Sequence Analysis Software Package from Genetics Computer Group at the University of Wisconsin (Biotechnology Center, 1710 University Avenue, Madison, Wl 53705) and the default parameters specified in this . A variant of a selected trif or I protein preferably has the amino acids present in the form present in the nature of the trifolia protein selects the most highly conserved amino acid positions of the protein. Therefore, a human FTI variant is preferably identical to human FTI present in nature in all or almost all of the most highly conserved positions. The conservation of sequences between the trefoil proteins is evident in Table 1 of Sands et al. (Supra) which can be used by those skilled in the art to identify conserved residues. The invention features a method of treating or inhibiting injury formations in the food channel of a patient by administering to the patient at least one trefoil polypeptide, or a biologically active fragment thereof. The lesions usually occur in the mucosa of the alimentary canal and may be present in the patient's mouth, esophagus, stomach or intestine. Injuries can occur in several ways. For example, the patient may receive radiation therapy or chemotherapy for the treatment of cancer. These treatments usually cause injuries to the patient's mouth and esophagus. Skilled artisans will recognize that it may be useful to administer the proteins of the invention to the patient before said treatment is started. Alternatively, injuries can be caused by: (1) any other drug, including alcohol, which damages the alimentary canal, (2) accidental exposure to radiation or a caustic substance; (3) an infection, or (4) a digestive disorder including, but not limited to, ulcer-free dyspepsia, gastritis, peptic or duodenal ulcer, gastric cancer, MALT lymphoma, Menetrier's syndrome, gastro-esophageal reflux disease and disease of Crohn. The tissues that are inside the alimentary canal can also be treated by administering to the patient at least one trifolio peptide, or a biologically active fragment thereof, in the case that the tissues are damaged by inflammation, a lesion, ulcer, abrasion, burn or other injury, or are at risk of being damaged, (ie, the method can be carried out prophylactically).

The peptide that is administered can be any peptide in the family of trifolios, such as intestinal trifolio peptide (TFI), spasmolytic peptide (PE), and pS2. For the treatment of human patients it is expected that the peptide will be expressed by a human gene. However, eukaryotic trefoil peptides, such as those cloned from the genomes of rats and mice, can also be proven to be effective. These peptides can be isolated from a source present in nature or synthesized by recombinant techniques. It is expected that the normal route of administration will be oral. The determination of other routes of administration and the effective dose are within the abilities of the experts and will depend on many factors known to these experts. The trifolie proteins can be administered alone, in combination with one another and / or in combination with glycoprotein preparations. The "treatment of injuries" includes both the inhibition of the formation of injury and the healing of already formed lesions. Biologically active fragments and variants of a trifolia protein, particularly TFI, which promote the healing of lesions or inhibit the formation of lesions, are useful in the treatments of the invention. The polypeptides of the invention can also be used for diagnostic purposes. For example, the polypeptides can be used in an assay to quantify intestinal trifolium factor and related polypeptides, such as fragments and analogues in tissues, serum and other biological samples. In many inflammatory bowel diseases, and potentially many other inflammatory conditions, the expression of TFI is reduced. Therefore, areas where expression is relatively low indicate the presence of damaged tissue. Alternatively, the polypeptides can be linked to a diagnostic marker and administered to a patient. In this circumstance, the polypeptide could facilitate the distribution of the diagnostic marker within any tissue that expresses a receptor for the polypeptide. The diagnostic marker can be any substance that is capable of being detected. Those skilled in the art are aware of numerous imaging agents that can be used in accordance with the present invention. Other aspects and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Brief Description of the Drawings Figure 1 is a description of the nucleotide sequence of rat trefoil factor (SEQ ID NO: 1). Figure 2 is a description of the deduced amino acid sequence of rat trefoil factor (SEQ ID NO: 2). Figure 3 is a description of the amino acid sequences of rat trefoil factor, pS2 protein, and pancreatic spasmolytic (PE) polypeptide. The sequences are aligned to illustrate the homology of amino acid sequences between the proteins. The dashes (-) indicate the insertion of spaces that optimize the alignment. The bars indicate sequence identity. Figure 4 describes the disulfide bond structure proposed by ΔS2 (SEQ ID NO: 15, panel A) and PEP (SEQ ID NO: 16, panel B). Figure 5 is a description of the proposed disulfide bond structure of rat intestinal trifolium factor (SEQ ID NO: 17). Figure 6 is a description of the nucleotide sequence of the human intestinal factor trifolium cDNA and the corresponding deduced amino acid sequence (SEQ ID NO: 3). Figure 7 is a diagram describing the strategy used to mutate the FTI gene in embryonic support cells. Figure 8 is a graph describing survival after administration of Dextran Sulfate Sodium (DSS: 2.5% w / v in drinking water for 9 consecutive days), shown as Kaplan-Meier transformation of probability against days of treatment with DSS. Detailed Description Purification and cloning of FTIr An inhibitor of white agar colony formation by cells BT-20 derived from human breast carcinoma (ATTC HTB79) was isolated from human malignant effusions positive for cytology (Podoisky et al., Cancer Res. 48: 418, 1988). The factor also inhibited the formation of soft agar colony by HCT15 cells derived from human colon carcinoma (ATTC-CCL225). Inhibition was not observed for polyoma and murine sarcoma virus transformed the line of rodent fibroblasts. The isolated factor (cell growth inhibition factor or FIGT) had an apparent molecular weight of 110,000 Da and appeared to consist of two subunits of 55,000 Da bound by sulfhydryl bonds. The purified protein was partially sequenced. The sequence of the 14 amino terminal amino acids was used to produce a group of degenerate oligonucleotide probes to screen a cDNA library of intestinal epithelial cells from rats. The intestinal cDNA library (Lambda ZAP ° II, Stratagene, La Jolla, CA) was produced by standard techniques (Ausubel et al., Eds., In Current Protocols in Molecular Biology, John Wiley &Sons. New York, 1989) using cells purified by the Weisner method (J. Biol. Chem. 248: 2536, 1973). Screening of the cDNA library with the completely degenerate oligonucleotide probe described above resulted in the selection of 21 clones. One of the clones (T3411) included a matrix sequence that encoded a single open reading frame. The nucleotide sequence of the open reading bank and flanking DNA are presented in Figure 1 (SEQ ID NO: 1). The insert present in T3411 was accurately moved (Ausubel et al., Supra) to produce a radioactively labeled probe for Northern blot analysis of poly (A) + rat RNA. Northern analysis showed that the RNA corresponding to the cloned cDNA fragment was expressed in the small intestine, large intestine and kidney; no expression was detected in the lung, vessel, heart, testes, muscle, stomach, pancreas or liver. In the tissues in which the DNA was expressed, the level was comparable with that of actin. The open reading frame of clone T3411 encoded an 81 amino acid peptide (Figure 2, SEQ ID NO: 2). The comparison of the encoded peptide sequence, termed rat intestinal trifolium factor (FTIr), to the protein sequence in the Genbank database revealed significant homology for the peptide associated with human breast cancer (pS2; Jakowlev et al. , Nucleic Acids Res 12: 2861, 1984) and porcine pancreatic spasmolytic peptide (PEP, Thim et al., Biochem Biophys, Acta 827: 410, 1985). Figure 3 illustrates the homology between FTIr and PEP. and pS2. It is thought that both the pancreatic spasmolytic factor (PEP) of porcine and pS2 are bent into a characteristic structure called a trifolio. A trifolia structure consists of three loops formed by three disulfide bonds. It is thought that pS2 includes a trifolium (Figure 4A), and PEP is thought to include two trifolios (Figure 4B). The region of FTIr (nucleotide 114 to nucleotide 230 encoding cys to phe), which is more similar to PEP and pS2, includes six cysteines, all of which are in the same position as the cysteines that form the trifolium in pS2 (Figure 3 ). Five of these six cysteines are in the same position as the cysteines that form the amino terminal trifolium of PEP (Figure 3). Figure 5 describes the proposed disulfide binding configuration of FTIr. Based on the homology of PEP and pS2 (Mori et al., Biochem.

Biophys. Res. Comm. 155: 366, 1988; Jakowlew et al., Nucleic Acids Res. 12: 2861, 1984), FTIr includes a putative pro-sequence (I went to ala22) in which 12 of the 22 amino acids have hydrophobic side chains. Production of Anti-FTIr Antibodies A peptide corresponding to the 21 carboxy terminal amino acids of FTIr was synthesized and coupled to bovine serum albumin (BSA). This conjugate (and the unconjugated peptide) is used to form polyclonal antibodies in rabbits. All procedures were normal protocols such as those described in Ausubel et al. (Supra). Anti-FTIr antibodies were used in an indirect immunofluorescence assay for visualization of FTIr in rat tissues. The cryosections of rat tissues were prepared using normal techniques and goat anti-rabbit monoclonal antibody labeled with fluorescence (the labeled antibodies are available from suppliers such as Kirkegaard and Perry Laboratories.

Gaithersberg, MD; and Bioproducts for Science, Inc., Indianapolis, IN) were used to detect the binding of rabbit anti-FTIr antibodies. Through this analysis, it appears that FTIr is present in the cells of the small intestine balloon but not in the stomach or pancreas.

Cloning of Human Intestinal Trifolium Factor The DNA encoding rat intestinal trifolium factor can be used to identify the cDNA colon encoding human intestinal trifolium factor (FTIh). This can be achieved by screening a human colon cDNA library with a FTIr-derived probe or with a probe derived from part of the FTIh gene. The last probe can be obtained from a human intestinal colon cDNA using the polymerase chain reaction to isolate a part of the FITh gene. This probe can serve as a specific probe for the identification of clones that encode the entire FTIh gene. Construction of a cDNA Bank A cDNA bank of human colon or intestinal in? Gt10 or? Gt11, or some other suitable vector is useful for the isolation of FTIh. These banks can be purchased (Clontech Laboratories, Palo Alto, CA. HLI034a, HLI0346b). Alternatively, a bank can be produced using mucosal fragments of colon or human intestine. Briefly, total RNA was isolated from tissue essentially as described by Chirgwin et al. (Biochemistry 1_8: 5294, 1979; see also Ausubel et al., supra). An oligo column (dT) was then used to isolate poly (A t) RNA by the method of Aviv et al. (J. Mol. Biol. 134: 743, 1972; see also Ausubel et al., Supra). The double-stranded cDNA was then produced by reverse transcription using oligo (dT) 12.18 or random hexamer primers (or both). RNAseH and poly DNA from E. coli are then used to replace the RNA strand with a second strand of DNA. In a subsequent step, E. coli DNA ligase and T4 DNA polymerase are used to close gaps in the second DNA strand and create blunt ends. Generally, the created cDNA is then quenched with EcoRI methylase and EcoRI linkers are added (other linkers can be used depending on the vector that will be used). In the subsequent steps the excess linkers are removed by restriction digestion and the cDNA fragments are inserted into the desired vector. See Ausubel et al., Supra and Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSH Laboratory Press, Cold Spring Harbor, NY, 1990) for detailed protocols. Useful vectors include:? Gt11,? Gt10, Lambda ZAP vector = II, Lambda Uni-ZAP ™ XR vector, all available from Stratagene (La Jolla, CA). The cDNA bank must be packed into the phage: this is most easily achieved through the use of commercial in vitro packaging equipment, eg Gigapack ^ ll Gold or Gigapack "II Plus (Stratagene La Jolla, CA). See Ausubel et al., (Supra) for suitable packaging protocols and host strains.The bank preferably is rapidly amplified after packing, this step generates sufficient clones for multiple bank sieving.See Ausubel and others supra or Sambrook and others supra for details of amplification protocols and procedures for storing the amplified library Screening of the cDNA Bank For screening the bank should be placed in an appropriate host strain (e.g., Y1090 or Y1088 for? gt10, C600hflA banks for? gt10 banks) After plating the phage, the plates are transferred to nitrocellulose or nylon filters (see Ausubel and others supra and Sambrook et al., Supra), then the filters are probed with an efficiently translated probe labeled with a32. P derived from FTIr. The probe is preferentially generated using a portion of the FTIr DNA region encoding the trifolia structure (nucleotides 114 to 230 of SEQ ID NO: 1, encoding cys32 to phe71 of SEQ ID NO: 2). This region is conserved between FTIr, pS2 and PEP, and it is likely that this region is conserved between FTIr and FTIh. Once a plate is identified, several plate purification cycles are required to isolate a pure clone that encodes FTIh. Isolation of phage DNA was carried out and the cDNA insert can be subcloned into a suitable vector for mapping and restriction sequencing. If the phage vector is Lambda ZAP = II, co-infection with the helper phage allows rescue and recircularization of the pBluescript SK vector "phagemid" (Stratagene, La Jolla, CA), harboring the cDNA.; alternatively the phage clone is purified and the cDNA insert is subcloned into a suitable vector for mapping and restriction sequencing. If the clone does not contain the entire TFIh gene (as assessed by FTIr homology and the presence of start and strain codons), the bank can be screened again with the original FTIr probe, or preferably with a probe generated from the clone of FTIh obtained. If none of the clones contains the intact gene. it can be reconstructed from clones that have overlapping fragments of FTIh. Direct Isolation of a FTIh Probe by PCR It is possible to isolate part of the FTIh gene directly from the packaged bank or cDNA. To isolate a portion of FTIh directly from the packed bank, a pair of oligonucleotide primers and Taq polymerase are used to amplify the DNA corresponding to the FTIh gene. The primers used could be about 15-20 nucleotides long and correspond in sequence up to 5 'and up to 3' portions of the FTIr coding sequence. Friedman et al., (In PCR Protocols: A Guide to Methods and Applications, Innis et al., Eds. Academic Press, San Diego, CA) describe a procedure for such amplification. In summary, the phage particles are altered by heating; Polymeric Taq, initiators (300 pmol each), dNTPs, and Taq polymerase pH buffer are added; and the mixture is thermally recycled to amplify the DNA. The amplified DNA is isolated by agarose gel electrophoresis. The ends of the fragment are prepared to be ligated into the appropriate vector by making them level with T4 polymerase and, if desired, by adding linkers. Alternatively, a restriction site in the fragment can be treated using primers that have sequence added to their 5 'ends whose sequence will generate an appropriate sticky end when directed. For example, the sequence 5 -GGGCGGCCGC-3 '(SEQ ID NO: 4) can be added to the 5"end of each primer This sequence includes the restriction site? / Ofl flanked at the 5' end by the sequence: GG. The additional nucleotides prevent the 5 'ends from being denaturated and interfere with the subsequent restriction with Notl.The DNA purified from the appropriate size gel is then cloned into a cloning vector for sequencing and restriction mapping.This clone will not have the entire sequence of FTIh, instead it will be a combination of FTIh (the region between the sequences corresponding to the primers) and FTIh (the 5 'and 3' ends corresponding to the primer sequences), however, this DNA can be used to generate a labeled probe, (produced puna precise translation or random initiator labeling) which, since it is the correct FTIh sequence, can be used in a high-restriction screening of the bank from which it is the cDNA was originally. In an alternative approach, cDNA can be used in the above procedure instead of a packed bank. This eliminates the steps of modifying the cDNA for insertion into a vector as well as a cDNA packaging and bank amplification. Ausubel and others, supra. they provide a protocol for the amplification of a particular DNA fragment directly from the cDNA and a protocol for the amplification of poly (A) + RNA. Identification of a Human FTI Clone Assumption An accurately translated probe derived from the FTIr cDNA (corresponding to nucleotides 1 to 431 of SEQ ID NO: 1) was used for Northern blot analysis of poly (A) + RNA derived from human intestinal mucosal scrapings. Probe hybridization and spot washing was carried out according to normal procedures. The probe (5 x 10s cpm / ml hybridization pH buffer) was hybridized to the filter at 45 ° C in 5X SSC with 30% formamide. The filter was then washed at 60 ° C in 5X SSC with 40% formamide. Using this protocol, a band was clearly visible after an overnight exposure of the filter with an intensification screen. This result indicated that there is sufficient homology between FTIr and FTIh to allow the use of probes derived from the FTIr gene sequence for identification in the FTIh gene. A human intestinal cDNA library was obtained from Clontech (Palo Alto, CA). Alternatively, a human intestinal cDNA library can be produced from mucosal scrapings as described above. They were selected four oligonucleotide probes for screening the cDNA library. Two of the probes correspond to the sequences within the FTIr region encoding the trifolium and are referred to as internal probes (5'-GTACATTCTGTCTCTTGCAGA-3 '(SEQ ID NO: 5) and 5'-TAACCCTGCTGCTGCTGGTCCTGG-3- (SEQ ID NO: 6) The other two probes recognize the sequences within FTIr but outside the region encoding the trifolium and are referred to as external probes (5'-GTTTGCGTGCTGCCATGGAGA-3 '(SEQ ID NO: 7) and 5'-CCGCAATTAGAACAGCCTTGT -3 '(SEQ ID NO 8) These probes were tested for their usefulness by using them to screen the rat intestinal cDNA library described above.Each of the four probes could be used to identify a clone that hosts all or part of the FTIr This result indicates that these probes can be used to screen the human intestinal bank for the presence of FTIh.The internal probes were used as described to amplify a cDNA DNA fragment from the human colon bank (Clontech, Palo Alto, CA). The linkers were added to the isolated DNA fragment which was then infected in the pBluescript phagemid vector (Stratagene, La Jolla, CA). The region of this clone corresponding to the human cDNA sequence (ie, which does not include the sequence corresponding to the internal probes) was used to form a radioactively labeled probe by synthesis initiated by random oligonucleotides (Ausubel et al. Supra). This probe was then used to screen the human colon cDNA library. This screening led to the identification of 29 clones. One of these clones (RCPHu-FTI) was compressed to generate a probe for Northern analysis of poly (A) + RNA isolated from human intestinal mucosal scrapings. A single band of almost the same size as the transcript of rats (approximately 0.45 kDa) was observed. Northern analysis of poly (A) + isolated from human tissues indicated that RNA corresponding to this probe was expressed in the small intestine and in the large intestine but not in the stomach or liver. These results indicate that the clone does not encode the human homologue of porcine PEP. Porcine PEP is expressed in the porcine pancreas and was not significantly expressed in the small or large intestine. These results also distinguish the cloned gene from pS2 that is expressed in the stomach. Figure 8 shows the information of nucleic acid sequences for human FTI cDNAs, together with the amino acid sequence deduced in the one letter code (SEQ ID NO: 3). This clone was obtained by the methods described above. Production of FTIh The isolated FTIh gene can be cloned into a mammalian vector for protein expression. Suitable vectors include pMAMneo (Clontech, Palo Alto, CA) which provides an RSV-LTR enhancer linked to a MMTV-LTR promoter inducible by dexamethasone, an SV40 origin of replication (allows replication in COS cells), a neomycin gene, and SV40 cleavage and polyadenylation sites. This vector can be used to express the protein in COS cells, CHO cells or mouse fibroblasts. The gene can also be cloned into a vector for expression in drosophila cells using the bacoluvirus expression system. Purification of Intestinal Trifolium Factor Intestinal trifolium factor can be purified from the intestinal mucosal states of humans, rats or any other species that express FTI (pigs and cows can provide a source of FTI). The purification procedure used for PEP will be useful for the purification of FTI since the proteins are probably homogeneous. Jorgensen et al. Describe a method for the purification of PEP (Regulatory Peptides 3: 207, 1982). The preferred method is the second approach described by Jorgensen et al. (Supra). This method involves chromatography on SP-Sephadex C-25 and QAE Sephadex A-25 columns (Sigma, St. Louis, MO) in acid pH buffer. Monoclonal Antibodies of Trifolium Anti-lntestinal Factor Anti-intestinal trifolium factor monoclonal antibodies can be raised against synthetic peptides whose sequences are based on the amino acid sequence of cloned FTIh (SEQ ID NO: 39.) Most commonly the peptide is based on the 10-20 terminal amino or carboxy amino acids of the protein of interest (here, FTIh) The peptide is usually chemically crosslinked to a carrier molecule such as bovine serum albumin or orbital limpet hemocyanin. In order to generate antibodies that cross-react with the native FTIh, the peptide should correspond to an antigenic region of the peptide of interest, this is achieved by choosing a region of the protein that is (1) exposed on the surface, e.g. , a hydrophobic region or (2) relatively flexible, eg, a region of loops or a region of rotation of ß. , if the peptide that will be coupled to a vehicle must have an amino acid with a side chain capable of participating in the coupling reaction. See Hopp and others (Mo. Immuno., 20:483, 1983; J. Mol. Biol. 157: 105. 1982) for a discussion of the issues involved in the section of antigenic peptides. A second consideration is the presence of an homologous protein for TFIh in the animal that will be immunized. If such a protein exists, it is important to select an FTIh region that is not highly homologous to that homologue. For FTIh, the peptides corresponding to the 15 terminal amino or terminal carboxy amino acids are probably less homologous cross-species and are exposed on the surface (and therefore are antigenic). Therefore, they are preferred for the production of monoclonal antibodies. Purified FTIh can also be used for the generation of antibodies. Genetic Description of a Trifolia Protein Alters Intestinal Mucosa Defense As previously stated, FTI is a member of the family of trifoliate proteins that are specifically and abundantly expressed on the mucosal surface of the gastrointestinal tract.

Other members of this family include pS2, which are expressed almost exclusively by foveal cells of the stomach (Masiakowski et al., Nucí Acids, Res. 1_0: 7896, 1982, Jorgensen et al., Regulatory Peptides 3: 231, 1982), and peptide. pancreatic spasmolytic (PE), which is expressed by the pancreas and the gastric antrum (Jorgensen and others supra). As described above, the expression of these proteins is improved in the vicinity of the damaged intestine. In order to study the role of FTI in vivo, the gene became non-functional by the targeted alteration in mice.

Murine FTI gene isolation and generation of FTI-deficient mice The murine FTI gene was isolated from a phage genomic library using rat FTI cDNA sequence as a probe, and its identity was confirmed by nucleotide sequencing using normal techniques (Mashimo and others Biochem. Biophys.

Res. Comm.210: 31. nineteen ninety five). A vector directed to alter the gene by homologous recombination in embryonic support cells (SE) were designed and constructed as shown in Figure 7. The entire second exon (Ex2) of the murine TFI gene, which is contained within the Xba-EcoRI fragment shown, was replaced with the neomycin resistance gene (neo) roll. As the deleted sequence encodes the majority of the "trefoil domain * ', the ability of any resulting peptide to produce the characteristic of the trefoil restriction loop structure is nullified.A positive-negative selection strategy (Mansour et al. , Nature 336: 348. 1988) was used to enrich homologous recombination events in embryonic (SE) support cells by selecting neo within the homologous DNA and against a herpes simplex virus thymidine kinase (hsv-tk) gene placed at the 3 'end of the vector addressed. The pPNT plasmid (Tybulewicz et al., Cell 65: 1153, 1991) was used to construct the targeted vector.

The targeting vector was linearized with the restriction enzyme NotI and electrophoresed in J1 pluripotent cells (Li et al., Cell 69: 915. 1992) under conditions previously described (Strittmatter et al., Cell 80: 445, 1995). The alteration of the FTI gene in the SE cells after homologous recombination was distinguished from the random integration of the vector directed by Southern blot analysis of the genomic DNA of individual clones of cells digested with the Xhol restriction enzyme. The pFTI2 probe identified a 19 kb "wild-type" fragment and a 23 kb "null" fragment created by introducing an Xhol site on the homologous insertion of the targeted vector. Approximately 10% of SE clones resistant to neomycin were found to have undergone homologous FTI recombination using this method. The polymerase chain reaction (PCR) was used to confirm the directed mutation in the following manner. A 200 bp region of DNA was amplified using primers that expanded FTI exon 2 (5'-GCAGTGTAACAACCGTGGTTGCTRGC-3 '(SEQ ID NO: 9) and 5'-TGACCCTGTGTCATCACCCTGGC-3' (SEQ ID NO: 10)); and a 400 bp region of the neo gene was amplified with a second set of primers (5'-CGGCTGCTCTGATGGCCGCC-3 '(SEQ ID NO: 11) and 5'-GCCGGCCACAGTCGATGAATC-3' (SEQ ID NO.12)). The DNA standard for the PCR reaction was obtained from tail tissue Approximately 0.5 cm of the tail was cut from each animal and the samples were digested with proteinase-K (200 μl at 0.5 mg / ml in 50 mM Tris-HCl pH 8.0 and 0.5% Triton X-100; Sigma, St. Louis, MO) at 55 ° C overnight. One μl of this mixture was added directly to a 25 μl PCR reaction (by Stratagene, Menosha, Wl). The reaction was started with a "warm start" (incubation at 96 ° C for 10 minutes), and the next cycle was repeated 30 times: 72 ° C for 120 seconds (hybridization and elongation) and 96 ° C for 30 seconds (denaturation) ). Ten μl of each reaction mixture was electrophoresed on a 2% agarose gel. Wild type animals were identified by presence of a fragment of 200 bp, corresponding to an FTI gene, heterozygous animals were identified by the presence of this band and, in addition, a fragment of 400 bp produced by amplification of the neo gene, and deficient FTI animals (of nullification) were identified by the presence of only the fragment corresponding to the neo gene. Two SE clones, which arise independently, were used to derive two FTI lines lacking mice. These mice were screened by analysis of Southern genomic spots as described by SE clones, or by PCR. Expression Analysis of Trifolium Peptide in Wild-type and Mutant Mice Although the expression of FTI is abolished in mutant mice, the expression of other trefoil genes is conserved. Northern blot analysis was performed using cDNA probes for FTI (Suemori et al, Proc Nati, Acad Sci USA 88: 11017, 1991), PE (Jeffrey et al., Gastroenterology 106: 336, 1994), and , as a positive control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The nucleic acid probe for murine pS2 was performed by reverse transcription polymerase chain reaction (RCP-TI) using the oligonucleotide pairs: 5'-GAGAGGTTGCTGTTTTGATGACA-3 '(SEQ ID NO: 13) and 5'-GCCAAGTCTTTGATGTAGCGAGTT -3"(SEQ ID NO: 14), which were synthesized based on the pS2 cDNA sequence of published mice (GenBank Accession Number: Z21858). The RNA PCR kit from GenAmp (Perkin Elmer) was used. according to the manufacturer's instructions, as well as the cloning vector pCR ™ ll (Invitrogen) .The RNA was extracted from the following tissues of both wild-type and FTI-deficient mice (knockdown): stomach, duodenum, ileum terminal, right colon, appendix, transverse colon, left and right colon, Fifteen μg of total RNA from each sample, were electrophoresed on a 1% agarose gel and transferred to nitrocellulose paper, after hybridization, washing and autoradiography the mice of wild type exhibited a tissue expression pattern considered normal. FTI was expressed in small intestine and colon which is the same expression pattern seen for FTI in rats and humans. The analysis of mutant mice confirmed the lack of expression of FTI in the gastrointestinal tract. In contrast, the expression of other trefoil proteins PE and pS2, are not altered in the gastrointestinal tract of mutant mice. PE was expressed in the stomach and. at lower levels, in the duodenum of both wild type and mutant mice. Similarly, pS2 was expressed in the stomach of both wild and FTI deficient mice. Immunochemistry reveals that FTI is not expressed in the mouse colon. FTI deficient In order to confirm that the FTI protein was not expressed by FTI elimination mice, the immunochemistry was carried out in the following manner. Colon and small intestine tissue were fixed in the infusion course, immersed in 4% paraformaldehyde (McLean et al., J. Histochem. Cytochem. 22: 1077, 1974) and imbibed in paraffin. Sections were recovered and stained with a polyclonal antibody raised against a synthetic peptide predicted by the 18 terminal amino acids in murine FTI carboxy or a monoclonal antibody against colonic mucin (Podoisky et al., J. Clin. Invest. 77_: 1263 , 1986). The binding of primary antibodies was visualized with a biotinylated secondary antibody, Avidin DH, biotinylated horseradish peroxidase H and diaminobenzidine tetrachloride reagents according to the manufacturer's instructions. After immunocytochemistry, the sections were stained for hematoxylin counting and observed.

The balloon cells in the colon of wild type mice were Immunoreactive with both antibodies, staining positively for FTI and mucin. In contrast, balloon cells in the FTI-deficient mouse colon lacked detectable FTI but continued to express colonic mucin. Induction of Moderate Colonic Epithelial Damage with Dextran Sodium Sulfate The FTI deficient mice derived from each SE clone appeared to develop normally and were indistinguishable in large part from the heterozygous and wild type siblings. Their development did not slow down and they reached maturity without obvious diarrhea or hidden fecal blood loss. However, the colon of mice deficient in FTI may be more prone to damage than the colon of wild type mice. To investigate this hypothesis, sodium dextran sulfate (DSS), which reproducibly creates colonic epithelial damage derived with ulceration in mice (Kim et al., Scand. J Gastroent, 27.:529, 1992; Wells et al. J Acquired Immune Deficiency Syndrome 3_: 361, 1990; Okayasu et al., Gastroenterology 98694, 1990) was administered in the drinking water of the animals. After the normalization of DSS effects in comparable wild type mice, a group of 20 wild type mice was treated and 20 mice deficient in FTI (siblings of heterozygous crosses, weighing more than 20 grams each) with 2 5% of DSS in their drinking water for nine days Although 85% of wild type mice and 100% of mice deficient in FTI treated with DSS demonstrate occult blood (using Hemoculture Smith Kline Diagnostics, San Jose, CA) in their evacuation during the treatment period, the mice deficient in FTI were notoriously more sensitive to the effects Detrimental to DSS Fifteen percent of FTI-deficient mice developed bloody diarrhea and died (Figure 8) In contrast, only 10% of wild-type mice treated similarly exhibited bloody diarrhea and only 5% died. Weight loss it was also significantly more pronounced in FTI deficient mice than wild-type mice receiving DSS. FTI Deficient Mice Treated with Dextran Sulfate Sodium (DSS) Develop Severe Colonic Erosions. After seven days of treatment with DSS (2.5% w / v) the colon of wild type mice and mice deficient in TFI were examined histologically. The left colon transections were fixed in 4% paraformaldehyde, mounted in paraffin and stained with hematoxylin and eosin. Multiple sites of obvious ulceration and hemorrhage were present in the colon of TFI deficient mice, while the colon of most of the wild type mice were indistinguishable in large part from those of untreated mice. Histological examination of FTI deficient colon treated with DSS confirmed the presence of multiple erosions and intense inflammatory changes including crypt abscesses. The damage was more pronounced in the distal colon, that is, colon descendant, sigmoid colon and rectum, which contained large and wide areas of mucosal ulceration. When similarly inspected, mucosal erosions could be observed in the tissue of 80% of wild-type mice treated with DSS, but most were small lesions that also appeared to be cured, with complete re-epithelialization of most lesions. There was no evidence of re-epithelialization in the colon of mice deficient in FTI exposed to DSS.

During the normal course of growth and development, the intestinal epithelial cells originate from the supporting cells in the intestinal crypts and progress rapidly up the crypt and hair that will be extruded from the tip of the hair within five days. After intestinal damage, the epithelial envelope is repopulated by cells that appear to generate signals to heal the lesion by modulation of epithelial and mensecal cell development and matrix formation (Poulsom et al., J. Clin. Gastroenterol. 1_7: S78, 1993). In vitro evidence suggests that trifolie proteins play a key role in the restoration of mucosal integrity after damage. Despite the normal restriction of PE and pS2 expression to the proximal gastrointestinal tract. These trifoliate and FTI proteins are abundantly expressed at sites of colonic damage and repaired. The DSS model described above provides a system for testing the protective effects of FTIs other than trefoil peptides, or fragments of active polypeptides or variants thereof. Someone can administer a molecule that will be tested for mice tested with DSS, whether they are wild-type or FTI-deficient mice, they determine if the molecule has therapeutic effects by carrying out the analyzes described above. In addition to the use of DSS, any chemical compound that is known to damage the mucosa that covers the digestive tract can be used to analyze the proteins of the invention. These compounds include, but are not limited to, alcohol, indomethacin methotrexate. For example, methotrexate (MTX) can be administered intraperitoneally to mice at a dose of 40 mg / kg. A group of animals treated with MTX could also be given the protein in question. Various parameters, such as body weight, the presence of lesions in the digestive tract, and mortality of these animals could then be compared with equivalent measurements taken from animals that were not treated with the protein. Binding Analysis of H. pylori In Situ A method to determine whether a given protein (or protein fragment or variant) is useful in the prevention or treatment of diseases associated with H. pylori infection for examination in the context of an animal model established infection by H. pylori. One such model was recently developed by Falk et al. (Proc. Nati, Acad. Sci. USA 92: 1515-1519, 1995). This model involves the use of transgenic mice that express the enzyme a-1, 3/4-fucosyltransferase and, as a consequence, expresses Le on the surface of mucosal cells that bind to clinical isolates of H. pylori. If the addition of a protein, such as FTI, for this system reduces the level of H. pylori binding to the mucosal cell, the protein could be considered as an inhibitor of H. pylori. More specifically, the analysis could be carried out in the following manner. H. pylori is obtained, for example, from patients with gastric ulcers or chronic active gastritis, develops in the stationary phase, and marked, for example with isothiocyanate ae digosigenin or fluorescence (FITC). The labeled bacteria are then exposed, together with the protein of interest, to freeze prepared sections of the stomach, duodenum, ileum or liver of adult transgenic mice (as described above). As a control, the experiment could be carried out in parallel-use tissue from a wild-type sibling. The sections are fixed with ice-cooled methanol for 5 minutes, washed three times with washing buffer (TBS); 0.1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2; 10 minutes / cycle), and treated with a buffer blocking solution (Boehringer Mannheim, see also Falk supra). The bacteria are diluted to an OD 600 of 0.05 with dilution buffer [TBS; 1 mM MnCl2, 1 mM MgCl2 containing leopeptin (11 μg / ml), aprotinin (1 μg / ml), [1-p-tosylamido-2-phenylethyl chloromethyl ketone (100 μg / ml), phenylmethylsulfonyl fluoride (100 μg / ml), and pepstatin A (1 μg / ml)] and spread over the sections for 2 hours at room temperature in a humidified chamber. The cuts are then washed twice in washing buffer on a rotating platform (5 minutes / cycle at room temperature). The digoxigenin-labeled bacteria are visualized on the cuts washed with sheep anti-digoxigenin imunoglobulin conjugated with FITC (Boehringer Mannheim) diluted with 1 100 in histoblock buffer. The nuclei were stained with bisbenzimide (Sigma). For blocking controls, stationary phase bacteria conjugated with digoxigenin can be suspended in dilution pH buffer at OD60o DE 0.05 and shaken with or without Leb-HSA or Lea-HSA (final concentration 50 μg / ml. reaction mixture, 200 μl) for 1 hour at room temperature. The suspension is then placed on frozen sections fixed in methanol. Use In the practice of the present invention, FTI can be administered as described above for the treatment of diseases of peptic ulcers, inflammatory bowel diseases, for protection of the intestinal tract from damage caused by bacterial infection, radiation damage or other evils. Tissues that are not part of the alimentary canal can also be treated. These tissues include the skin, the corneal surface of the eye, and tissues within the respiratory and genitourinary tracts. The mode of administration, dosage and formulation of FTI will depend on the condition being treated. Next, additional guidelines are given regarding treatment regimens. In addition, treatment may begin before an injury has occurred because it is thought that the polypeptides and compositions of the invention exert a protective effect. Other Modalities Antibody Production FTI can be used to produce monoclonal antibodies for the detection of FTI in intestinal tissue or blood serum by means of an indirect immunoassay. FTI can be detectably labeled and used in an in situ hybridization analysis for the detection of FTI binding sites. The labels may include, but are not limited to, fluorescein or a radioactive ligand. The FTI can be used to protect and stabilize other proteins. This protection is achieved by forming a hybrid molecule, in which all or part of the FTI is fused to the carboxy terminus or amino terminus (or both) of the protein of interest. Because FTI is resistant to degradation in the digestive system, it will protect the protein of interest from such degradation. As a consequence, the protein of interest probably remains active in the digestive system and / or will be more readily absorbed in an intact form. The stabilized dimerized protein trifolia can be used in the methods of the invention. Said molecules can be prepared by stably interlacing trifolium monomers or by expressing a gene encoding a repeat followed by a trifolia protein (e.g., FTI) or a portion thereof (e.g., a portion capable of forming a protein structure). three loops characteristic of the trifoliate proteins). Also in the method of the invention, the trifolie proteins produced by chemical synthesis are useful. The invention also encompasses antibodies that bind to the polypeptides of the invention, i.e., trefoil polypeptides such as FTI. Antibodies that specifically recognize one or more epitopes of these polypeptides, or fragments thereof, are also encompassed by the invention. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab, F (ab ') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic antibodies (anti-ld) and epitope binding fragments of any of the foregoing. The antibodies of the invention can be used, for example, in the detection of FTI in a biological sample and therefore, can be used as part of a diagnostic or prognostic technique whereby patients can be tested for abnormal amounts of FTI. Normally, FTI expression is downregulated in the immediate vicinity of an injury, such as that caused by inflammatory bowel diseases (e.g., colitis). For the production of antibodies, several host animals can be immunized by injection with a peptide having a sequence that is present, for example, in FTI. Such host animals may include but are not limited to rabbits, mice and rats, to name a few. Several auxiliaries can be used to increase the immune response, depending on the host species, including but not limited to those of Freund (complete and incomplete), mineral gels such as aluminum hydroxide, active surface substances such as lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, hemocyanin Orifice limiter, dinitrophenol and potentially useful human auxiliaries such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the serum of immunized animals. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the Kohler and Milstein hybridoma technique (Nature 256: 495-497, 1974).; and Patent of E.U.A. No. 4,376,110), the hybrid technique of human B cells (Kosbor et al., Immunology Today 4:72, 1983; Cole et al., Proc. Nati, Acad. Sci. USA 80: 2026-2030, 1983), and the Hybridoma EBV technique (Cole and others, "Monoclonal Antibodies And Cancer Therapy," Alana R. Liss, Inc., pp. 77-96, 1985). Said antibodies can be any kind of immunoglobulin including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing mAb of this invention can be cultured in vitro or in vivo. The production of high titers of mAbs in vivo makes this the currently preferred method of production. In addition, techniques developed for the production of "chimeric antibodies" can be used (Morrison et al., Proc. Nati, Acad. Sci. USA, 81: 6851-6855, 1984, Neuberger et al., Nature, 312: 604-608). 1984; Takeda et al., Nature, 314: 452-454, 1985) by dividing the genes of a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those that have a variable region derived from a murine mAb and a human immunoglobulin constant. Alternatively, the techniques described for the production of single chain antibodies (U.S. Patent 4,946,778; Bird, Science 242: 423-426, 1988; Huston et al., Proc. Nati, Acad. Sci. USA 85: 5879-5883, 1988; and Ward et al., 1989, Nature 334: 544-546, 1989) can be adapted to produce single chain antibodies against trefoil polypeptides such as FTI. The single chain antibodies are formed by the binding of heavy and light chain fragments of the Fv region via an amino acid, resulting in a single chain polypeptide. Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include but are not limited to: the fragments of F (ab ') 2 that can be produced by digestion of pepsin from the antibody molecule and the Fab fragments that can be generated by reducing the disulphide bridges of the fragments of F (ab ') 2. Alternatively, Fab expression libraries can be constructed (Huse et al., Science, 246: 1275-1281, 1989) for rapid and easy identification of monoclonal Fab fragments with the desired specificity.

These antibodies, in turn, can be used to generate anti-idiotypic antibodies that "mimic" FTI, using techniques well known to those skilled in the art. (See, for example, Greenspan and Bona, FASEB J. 7: 437-444, 1993, and Nissinoff, J. Immunol., 147: 2429-2438, 1991). Such anti-idiotypic or neutralizing Fab fragments of said anti-idiotypic ones can be used in diagnostic regimes to detect disorders associated with death of apoptotic cells. The antibodies can be humanized by methods known in the art. For example, monoclonal antibodies with a desired binding specificity can be humanized commercially (Scotgene, Scotland, Oxford Molecular, Palo Alto, CA). Whole human antibodies, such as those expressed in transgenic animals are also aspects of the invention (Green et al., Nature Genetics 7: 13-21, 1994; see also U.S. Patent Nos. 5,545,806 and 5,569,825, both of which are incorporated herein) by reference FTI Administration to Protect or Treat Tissues Outside the Food Channel The polypeptides of the invention, including FTI, analogs and fragments thereof, as well as other trifolium factors, such as PE (spasmolytic polypeptide) and PS2, can be used to protect or treat tissues that are not within the alimentary canal Polypeptides can be used, for example, to treat any kind of wound, such as a lesion, an ulcer, a burn or an abrasion of the skin, the surface of the eye (ie, the cornea) or within the respiratory or genitourinary tracts.The exact nature of the damage and the cause of the injury do not need to be get in with pressure. Regardless of the location of the lesion (ie, regardless of whether the lesion is within the alimentary canal), the toxicity and therapeutic efficacy of a compound can be determined by normal pharmaceutical procedures, using cells in culture or experimental animals to determine the LD50 (the lethal dose to 50% of the population) and the ED50 (the therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the DL5o / DE5o ratio. Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care must be taken to design a delivery system that directs such compounds to the affected tissue site in order to minimize potential damage to unaffected cells, thereby reducing side effects. The sustained data from cell culture analyzes and animal studies can be used in formulations on a dose scale for use in humans. The dosage of said compounds preferably lies within a range of circulating concentrations that include ED50 with little or no toxicity. The dose may vary within this scale depending on the dosage form used and the route of administration used. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from the cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration scale that includes Cl50 (ie, the concentration of the test compounds that achieves the maximum mean inhibition of symptoms) as determined in cell culture. This information can be used to determine more precisely the useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography. The pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. The pharmaceutical compositions may also contain mucin glycoproteins. Thus, the compounds and their physiologically acceptable salts and solvates can be formulated for administration by inhalation or insufflation (either through the mouth or nose) or oral, buccal, parenteral or rectal administration. Since the trefoil polypeptides are not degraded within the digestive tract, it is expected that the route of administration will be oral. The polypeptide could be administered, for example, in the form of a tablet, capsule, or pill, or it could be suspended in a solution such as a syrup, which the patient ingests.

Alternatively, the solution containing the polypeptide can be administered as a gastric lavage. The polypeptide can also be included in a solution that is administered as an enema, or it can be administered as a suppository. For oral administration, which can be used to treat damaged tissue within the alimentary canal, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (for example, lactose, microcrystalline cellulose or calcium acid phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (e.g., potato starch glycolate or sodium starch); or wetting agents (e.g., sodium lauryl sulfate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can be separated by conventional means with pharmaceutically acceptable additives such as suspending agents, for example, (eg, sorbitol syrup, cellulose derivatives or hydrogenated edible fats): emulsifying agents (eg, lecithin or acacia); non-aqueous agents (eg, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl- or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain regulatory salts, flavors, colorants and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration, which can be used to treat tissue within the mouth, throat or upper esophagus, the compositions can take the form of tablets or troscismus formulated in a conventional manner. For administration by inhalation, which can be used to treat damaged tissue within the respiratory tract, the compounds to be used in accordance with the present invention are conveniently supplied in the form of an aerosol spray presentation of pressurized packets or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane. trichlorofluoromethane, dichlorotetrafluroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dose unit can be determined by providing a valve to supply a measured quantity. Capsules and cartridges, for example. Gelatin for use in an inhaler or an ipsuflator can be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch. The compositions containing the polypeptides of the invention can also be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be present in unit dosage form, for example, in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use. The compositions may also be formulated in rectal compositions such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example in an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The compositions, if desired, may be present in a pack or dispenser device that may contain one or more dosage unit forms containing the active ingredient. The package for example may comprise metal or plastic foil, such as a pack of ampoule. The package or dispenser device may be accompanied by instructions for its administration. The therapeutic compositions of the invention may also contain an excipient carrier, many of which are known to those skilled in the art. The excipients that may be employed include regulatory solutions (eg, citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g. serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol and glycerol. The nucleic acids, polypeptides, antibodies or modulator compounds of the invention can be administered by any normal route of administration. For example, administration can be parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, transmucosal or oral. The modulator compound can be formulated in various ways, according to the corresponding route of administration. For example, liquid solutions may be made for ingestion or injection; Gels or powders can be produced for ingestion, inhalation or topical application. Methods for forming such formulations are well known and can be found in, for example, "Reminton's Pharmaceutical Sciences." The preferred route of administration is expected to be oral. In medical technology, it is well known that the doses for any patient depend on many factors, including the general health, sex, weight, body surface area and age of the patient, as well as the particular compound to be administered, time and route of administration and other drugs that are administered concurrently. The doses for the polypeptides and antibodies of the invention will vary. For oral administration, the polypeptide can be administered in doses of about 10 mg to about 500 mg. For example 10, 50, 100, 200, 250, 300, 400 or 500 mg can be administered. These doses can be administered on a periodic basis. For example, a dose can be taken four times a day. For topical administration, the polypeptide can be administered in doses of about 1 to about 10 mg / ml within an ointment or cream. This composition can also be administered periodically, if necessary. For other routes of administration, doses will also vary, for example, from about 0.1 to 1,000 mg per application. The determination of the correct dose within a given therapeutic regimen is within the capabilities for someone skilled in the pharmacology technique. In order to determine the efficacy of the polypeptide in the treatment for treating a particular disorder, those skilled in the art can perform routine studies using any of several well-known injury models. For example, the efficacy of a polypeptide for treating damage to the cornea can be carried out using an in vitro model for curing corneal wounds described by Collin et al. (Current Eye Res. 1_4: 331-339, 1995). In this model system, corneas that were not suitable for transplantation were obtained from human organ donors and used within 5 days post-mortem. A cautery tip was used to create a linear non-perforating thermal burn approximately 5mm in length on the cornea. Wounded corneas were dissected immediately and placed in an air / liquid organ culture system (as described by Richard et al., Curr. Eye Res. 1_0: 739-749, 1991; and Anderson et al., Ophthalmol. Sci. 3: 442-449, 1993). Therefore, to determine the efficacy of a polypeptide of the invention for the purpose of treating said lesion, the polypeptide could be simplified to the injured cornea, for example by placing the polypeptide in the tissue culture medium and evaluating the effect of the polypeptide on wound healing in injured corneas versus non-injured ones. If additional guidance is required to assess the wound, experts can again consult Collin et al. (Supra), who also describes the histochemical analyzes of cornea wounds. Protocols for a wound closure model using cultured rabbit corneal endothelial cells can also be used (e.g., see Joyce et al., Invest. Ophthalmol, Vis. Sci 31: 1816-1826, 1990) Alternatively, to evaluate the efficacy of a polypeptide in the context of a physical wound to the cornea, the induced injury can be used as described by Kessler (Curr. Eye Res. 14: 985-992, 1995). Similarly, there are numerous models available in which the effectiveness of a polypeptide is tested in order to avoid or cure a wound to the epidermis. Those of ordinary skill in the art are aware of these models and can carry out the procedures described in the art without conducting undue experimentation. Other embodiments are within the following claims.

SEQUENCE LIST (1) GENERAL INFORMATION (I) APPLICANT: The General Hospital Corporation (ii) TITLE OF THE INVENTION: INTESTINAL TRIFOLYTE PROTEINS (iii) NUMBER OF SEQUENCES: 18 (iv) ADDRESS OF CORRESPONDENCE (TO) RECIPIENT: Fish & Richardson, P.C. (B) STREET: 225 Franklin Street (C) CITY: Boston (D) STATE: MA (E) COUNTRY: E.U.A. (F) ZP: 02110-2804 (v) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIA: Floppy disk (B) COMPUTER: compatible with IBM (C) OPERATING SYSTEM: Windows 95 (D) SOFTWARE: FastSEQ for Windows Version 2.0 (vi) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: PCT / US97 / - - (B) DATE OF SUBMISSION: 11-APRIL-1997 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER : 08 / 631,469 (B) DATE OF SUBMISSION: 12-APR I L-1996 (A) APPLICATION NUMBER: 08 / 191,352 (B) DATE OF SUBMISSION: 02-FEBRUARY-1994 (A) APPLICATION NUMBER: 08 / 037,741 (B) DATE OF SUBMISSION: MARCH 25, 1993 (A) APPLICATION NUMBER: 07 / 837,192 (B) DATE OF SUBMISSION: FEBRUARY 13, 1992 (A) APPLICATION NUMBER: 07 / 655,965 (B) DATE OF SUBMISSION: FEBRUARY 14, 1991 (viii) EMPLOYEE / AGENT INFORMATION: (A) NAME: Meiklejohn, Ph.D., Anita L. (B) NUMBER OF REGISTRATION: 35,283 (C) REFERENCE / CASE NUMBER: 00786 / 322WO1 (¡x) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 617-542-5070 (B) TELEFAX: 617-542-8906 (C) TELEX: 200154 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 431 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: genomic DNA (ix) FEATURE: (A) NAME / KEY: Coding sequence (B) LOCATION: 18 ... 260 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAAGTTTGCG TGCTGCC ATG GAG ACC AGA GCC TTC TGG ATA ACC CTG CTG 50 Met Glu Thr Arg Wing Phe Trp He Thr Leu Leu 1 5 10 CTG GTC CTG GTT GCT GGG TCC TCC TGC AAA GCC CAG GAA TTT GTT GGC 98 Leu Val Leu Val Ala Gly Ser Ser Cys Lys Wing Gln Glu Phe Val Gly 15 20 25 CTA TCT CCA AGC CAA TGT ATG GCG CCA ACA AAT GTC AGG GTG GAC TGT 146 Leu Ser Pro Ser Gln Cys Met Ala Pro Thr Asn Val Arg Val Asp Cys 30 35 40 AAC TAC CCC ACT GTC ACA TCA GAG CAG TGT AAC AAC CGT GGT TGC TGT 194 Asn Tyr Pro Thr Val Thr Ser Glu Gln Cys Asn Asn Arg Gly Cys Cys 45 50 55 TTT GAC TCC AGC ATC CCA AAT GTG CCC TGG TGC TTC AAA CCT CTG CAA 242 Phe Asp Ser Ser He Pro Asn Val Pro Trp Cys Phe Lys Pro Leu Gln 60 65 70 75 GAG ACA GAA TGT ACA TTT TGAAGCTGTC CAGGCTCCAG GAAGGGAGCT CCACACCC 298 Glu Thr Glu Cys Thr Phe 80 TGGACTCTTG CTGATGGTAG TGGCCCAGGG TAACACTCAC CCCTGATCTG CTCCCTCGCG 358 CCGGCCAATA TAGGAGCTGG GAGTCCAGAA GAATAAAGAC CTTACAGTCA GCACAAGGCT 418 GTTCTAATTG CGG 431 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 81 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2: Met Glu Thr Arg Wing Phe Trp He Thr Leu Leu Leu Val Leu Val Wing 1 5 10 15 Gly Ser Ser Cys Lys Wing Gln Glu Phe Val Gly Leu Ser Pro Ser Gln 20 25 30 Cys Met Wing Pro Thr Asn Val Arg Val Asp Cys Asn Tyr Pro Thr Val 35 40 45 Thr Ser Glu Gln Cys Asn Asn Arg Gly Cys Cys Phe Asp Ser Ser lie 50 55 60 Pro Asn Val Pro Trp Cys Phe Lys Pro Leu Gln Glu Thr Glu Cys Thr 65 70 75 80 Phe (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 403 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0.3: GATGCTGGGG CTGGTCCTGG CCTTGCTGTC CTCCAGCTCT GCTGAGGAGT ACGTGGGCCT 60 GTCTGCAAAC CAGTGTGCCG TGCCGGCCAA GGACAGGGTG GACTGCGGCT ACCCCCATGT 120 CACCCCCAAG GAGTGCAACA ACCGGGGCTG CTGCTTTGAC TCCAGGATCC CTGGAGTGCC 180 TTGGTGTTTC AAGCCCCTGA CTAGGAAGAC AGAATGCACC TTCTGAGGCA CCTCCAGCTG 240 CCCCTGGGAT GCAGGCTGAG CACCCTTGCC CGGCTGTGAT TGCTGCCAGG CACTGTTCAT 300 CTCAGTTTTT CTGTCCCTTT GCTCCCGGCA AGCTTTCTGC TGAAAGTTCA TATCTGGAGC 360 CTGATGTCTT AACGAATAAA GGTCCCATGC TCCACCCGAA AAA 403 (2) INFORMATION FOR SEQ ID N0: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4: GGGCGGCCGC 10 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GTACATTCTG TCTCTTGCAG A 21 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO.6: TAACCCTGCT GCTGCTGGTC CTGG 24 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 21 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GTTTGCGTGC TGCCATGGAG A 21 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY : linear (i) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO 8: CCGCAATTAG AACAGCCTTG T 21 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GCAGTGTAAC AACCGTGGTT GCTGC 25 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 23 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (¡i) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TGACCCTGTG TCATCACCCT GGC 23 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CGGCTGCTCT GATGGCCGCC 20 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GCCGGCCACA GTCGATGAAT C 21 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GAGAGGTTGC TGTTTTGATG ACA 23 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GCCAAGTCTT GATGTAGCCA GTT 23 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS. (A) LENGTH: 60 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Glu Ala Gln Thr Glu Thr Cys Thr Val Ala Pro Arg Glu Arg Gln Asn 1 5 10 15 Cys Gly Phe Pro Gly Val Thr Pro Ser Gln Cys Wing Asn Lys Gly Cys 20 25 30 Cys Phe Asp Asp Thr Val Arg Gly Val Pro Trp Cys Phe Tyr Pro Asn 35 40 45 Thr He Asp Val Pro Pro Glu Glu Glu Cys Glu Phe 50 55 60 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 62 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: Protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID N0: 16: Glu Lys Pro Wing Wing Cys Arg Cys Ser Arg Gln Asp Pro Lys Asn Arg 1 5 10 15 Val Asn Cye Gly Phe Pro Gly He Thr Be Asp Gln Cys Phe Thr be 20 25 30 Gly Cys Cys Phe Asp Ser Gln Val Pro Gly Val Pro Trp Cys Phe Lys 35 40 45 Pro Leu Pro Wing Gln Glu Ser Glu Glu Cys Val Met Glu Val 50 55 60 (2) INFORMATION FOR SEQ ID N0: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 17: Gln Glu Phe Val Gly Leu Ser Pro Ser Gln Cys Met Ala Pro Thr Asn 1 5 10 15 Val Arg Val Asp Cys Asn Tyr Pro Thr Val Thr Ser Glu Gln Cys Asn 20 25 30 Asn Arg Gly Cys Cys Phe Asp Ser Ser He Pro Asn Val Pro Trp Cys 35 40 45 Phe Lys Pro Leu Gln Glu Thr Glu Cys Thr Phe 50 55 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 74 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID N0: 18: Met Leu Gly Leu Val Leu Wing Leu Leu Being Being Ser Wing Glu Glu 1 5 10 15 Tyr Val Gly Leu Ser Wing Asn Gln Cys Wing Val Pro Wing Lvs Asp Arg 20 25 30 Val Asp Cys Gly Tyr Pro His Val Thr Pro Lvs Glu Cys Asn Asn Arg 35 40"45 Gly Cys Cys Phe Asp Ser Arg He Pro Gly Val Pro Trp Cvs Phe Lys 50 55 60 Pro Leu Thr Arg Lys Thr Glu Cys Thr Phe 65 70

Claims (33)

1. CLAIMS I. A polypeptide comprising a trefoil polypeptide, or a biologically active fragment thereof, for treating or inhibiting the formation of lesions.
2. 2. The polypeptide of claim 1, wherein the trifolio polypeptide is a human trifolio polypeptide.
3. 3. The polypeptide of claim 1, wherein the trifolio polypeptide is an intestinal trifolio polypeptide (FTI).
4. 4. The polypeptide of claim 1, wherein the trefoil polypeptide is a spasmolytic polypeptide (PE).
5. 5. The polypeptide of claim 1, wherein the trifolio polypeptide is PS2.
6. 6. The polypeptide of claim 1, wherein the lesion is within the alimentary canal.
7. 7. The polypeptide of claim 6, wherein the lesion is inside the patient's mouth.
8. 8. The polypeptide of claim 6, wherein the lesion is in the esophagus of the patient.
9. 9. The polypeptide of claim 6, wherein the lesion is in the patient's stomach.
10. 10. The polypeptide of claim 6, wherein the lesion is in the patient's intestine.
11. II. The polypeptide of claim 6, wherein the patient receives radiation therapy for the treatment of cancer.
12. 12. The polypeptide of claim 6, wherein the patient receives chemotherapy for the treatment of cancer.
13. 13. The polypeptide of claim 6, wherein the patient receives a drug that damages the alimentary canal.
14. 14. The polypeptide of claim 6, wherein the patient suffers from a digestive disorder.
15. 15. The polypeptide of claim 14, wherein the digestive disorder is a dyspepsia without ulcers.
16. 16. The polypeptide of claim 14, wherein the digestive disorder is gastritis.
17. 17. The polypeptide of claim 14, wherein the digestive disorder is a gastro-esophageal reflux disease.
18. 18. The polypeptide of claim 14, wherein the digestive disorder is a peptic ulcer or a duodenal ulcer.
19. 19. The polypeptide of claim 6, wherein the administration is oral administration.
20. 20. The polypeptide of claim 6, wherein the oral administration comprises administration of about 10 milligrams to about 100 milligrams of the polypeptide.
21. 21. The polypeptide of claim 1, wherein the lesion is within a tissue other than a tissue in the alimentary canal.
22. 22. The polypeptide of claim 21, wherein the tissue is the skin.
23. 23. The polypeptide of claim 22, wherein the administration is topical administration.
24. 24. The polypeptide of claim 23, wherein the administration comprises administering ointment containing about 1 mg / ml to about 10 mg / ml of the polypeptide.
25. 25. The polypeptide of claim 21, wherein the tissue comprises the corneal surface of the eye.
26. 26. The polypeptide of claim 21, wherein the tissue comprises a tissue in the respiratory tract.
27. 27. The polypeptide of claim 21, wherein the tissue comprises a tissue in the genitourinary tract.
28. 28. A composition comprising a trefoil polypeptide for treating or inhibiting the formation of lesions in the tissue of a patient.
29. 29. The use of the composition of claim 28, in the manufacture of a medicament for the treatment of tissue injuries of a patient.
30. 30. The trifolio polypeptide of claim 1, further the polypeptide comprises a label.
31. 31. The trefoil polypeptide of claim 30, wherein the marker comprises an imaging agent.
32. 32. A method for detecting an FTI receptor in a tissue, the method comprising contacting the tissue with the detectably labeled trifolium polypeptide and measuring the level of detectable trifolium polypeptide bound to the tissue.
33. 33. The method for detecting a trefoil polypeptide in a tissue, said method comprising contacting the tissue with an antibody that specifically binds to the trefoil polypeptide.