MXPA06000494A - Agonist polypeptide of receptor for zot and zonulin - Google Patents

Abstract

Agonist polypeptide of a receptor protein has been identified. The agonist can be used to facilitate drug and antigen absorption. Suitable routes of administration include oral, nasal, transdermal, and intravenous. Pharmaceutical formulations may comprise a therapeutic agent or an immunogenic agent in combination with the agonist polypeptide.

Description

ZON AND ZON RECEIVER POLYPETEPID AGENT CAMPO OF THE I NVENTION This application claims the benefit of provisional application serial number 60/487, 889 filed on July 15, 2003, the description of which is expressly incorporated herein.

TECHNICAL FIELD PE LA INVENCIÓN This invention is related to the area of diagnostics, therapeutics, pharmaceuticals, drug discovery and immunotherapy. In particular, it refers to the handling and use of the ZOT / Zonulina receptor system to improve health.

BACKGROUND OF THE INVENTION Intestinal dysfunction of tight joints occurs in a variety of clinical conditions, including food allergies, gastrointestinal tract infections, autoimmune diseases, and inflammatory bowel diseases (42). The healthy, mature intestinal mucosa, with its tight union intact, serves as the main barrier for the passage of macromolecules. During the healthy state, small amounts of immunologically active antigens cross the barrier of the host mucosa. These antigens are absorbed through the mucosa by at least two routes. The vast majority of absorbed proteins (up to 90%) cross the intestinal barrier via the transcellular route, followed by lysosomal degradation that converts proteins into smaller, non-immunogenic peptides. These residual peptides are transported as intact proteins, through the paracellular pathway; it involves a subtle but sophisticated regulation of the intracellular tight junction that leads to antigen tolerance. When the integrity of the tight junction system is compromised, such as with prematurity or after exposure to radiation, chemotherapy and / or toxins, a detrimental immune response to environmental antigens (including autoimmune diseases and food allergies) may be triggered. ). There continues to be a need in the art to diagnose and treat these diseases and conditions. There continues to be a need in the art to identify new drugs for the treatment of these diseases. Several microorganisms exert an irreversible cytopathic effect on the epithelial cells that impact the cytoskeletal organization and the function of the tight junction. These bacteria alter intestinal permeability either directly (ie EPEC) or by toxin elaboration (eg, Clostridium difficile, Bacteroides fragilis) (43). The protein of Zonula Ocludens toxin (ZOT) of phage CXTF of Vibrio cholerae mimics the human protein zonulin, and exploits the physiological mechanisms of regulation of tight junction. The ZOT possesses multiple domains that allow a dual function of the protein as a morphogenetic peptide phage for the CXTF phage of Vibrio cholerae and as an enterotoxin that modulates the narrow intestinal junctions. The action of the ZOT is mediated by a cascade of intracellular events that lead to a PCKa-dependent polymerization of strategically located actin microfilaments to regulate the paracellular pathway (38). The toxin exerts its effect by interacting with the surface of enteric cells. The ZOT connection varies within the intestine, being detectable in the jejunum and in the distal ileum, decreasing along the axis of the hair socket, and is not detectable in the colon (44). This connection distribution coincides with the regional effect of ZOT on intestinal permeability (44) and with the preferential redistribution of F. actin induced by ZOT in mature hair cells (38).

BRIEF DESCRIPTION OF THE INVENTION A first embodiment of the invention is an agonist polypeptide of a human zonulin receptor and phage ZOT protein CXTF of Vibrio cholerae. The agonist polypeptide contains the amino acid sequence FCIGRL (SEQ ID NO: 4). The polypeptide is of less than 100 amino acid residues in length.

A second embodiment of the invention is a pharmaceutical composition for the treatment of a disease. The composition contains a therapeutic agent for treating the disease and an agonist polypeptide of a human zonulin receptor and a ZOT protein from the CXTF phage of Vibrio cholerae. The agonist polypeptide contains the amino acid sequence FC IG RL (I D S ECU ENCE N °: 4). The polypeptide has a length of less than 1000 residues to amino acids. A third embodiment of the invention is a method for delivering a therapeutic agent to a target tissue. A patient with the disease is administered a therapeutic agent to treat a disease and an agonist polypeptide of a zon ulin human receptor and ZOT protein of the CXTF phage of Vibrio cholerae. The agonist polypeptide contains the amino acid sequence FCIG RL (I D SEQUENCE N °: 4). The polypeptide has a length of less than 1000 amino acid residues. A fourth embodiment of the invention is a method for delivering a therapeutic agent to a target tissue. A therapeutic agent for treating a disease is admixed, and an agonist polypeptide of a human zonulin receptor and ZOT protein of CXTF phage from Vibrio cholerae nasally to a patient having the disease. The agonist polypeptide contains the amino acid sequence FCIGRL (I D SEQUENCE N °: 4). The polypeptide has a length of less than 100 amino acid residues. A fifth embodiment of the invention is a method for delivering a therapeutic agent to a target tissue. A therapeutic agent is administered to treat a disease, and an agonist polypeptide of a human zonulin receptor and ZOT protein of the CXTF phage of Vibrio cholerae orally, to a patient having the disease. The agonist polypeptide contains the amino acid sequence FCIGRL (SEQ ID NO: 4). The polypeptide has a length of less than 100 amino acid residues. A sixth embodiment of the invention is a method for delivering a therapeutic agent to a target tissue. A therapeutic agent is administered to treat the disease, and a ZOT protein from the CXTF phage of Vibrio cholerae is passed through the skin of a patient who has the disease. The agonist polypeptide contains the amino acid sequence FCIGRL (I D SEQUENCE N °: 4). The polypeptide has a length of less than 100 amino acid residues. A seventh embodiment of the invention is a method for delivering a therapeutic agent to a target tissue. A therapeutic agent is administered to treat a disease, and an agonist polypeptide of a human zonulin receptor, and a ZOT protein of the CXTF phage of Vibrio cholerae, in the blood of a patient having the disease. The agonist polypeptide contains the amino acid sequence FCIGRL (SEQ ID NO: 4). The polypeptide has a length of less than 100 amino acid residues. An eighth embodiment of the invention is a method for identifying or purifying a human Zonulin receptor and ZOT protein from the CXTF phage of Vibrio cholerae. A sample containing one or more proteins is contacted with an antibody under conditions appropriate for the antibody antigen binding. The antibody increases against the amino acids SLIGKVDGTSHVTG as shown in SEQ ID NO: 5. The proteins in the sample that do not bind to the antibody are removed. The proteins bound to the antibody are identified as a human Zonulin and ZOT receptor, or to form an enriched preparation for said receptor. A ninth embodiment of the invention is a search method for candidate drugs for treating a disease. A first human protein identified by the SAM 1 1 antibody is contacted with a second protein selected from the group consisting of ZOT protein from the CXTF phage of Vibrio cholerae, human Zonulin and MyD88. The contact is made separately in the presence and absence of a test substance. The amount of the first protein that binds to the second protein in the presence of the test substance, it is compared with the amount bound in the absence of the test substance. A test substance is identified as a candidate drug if it decreases the amount of the first protein that binds to the second protein. A tenth embodiment of the invention is a composition for vaccine, intended to induce an immunological response. The vaccine contains an immunogenic agent to induce an immune response and an agonist of a human zonulin receptor and ZOT protein from the CXTF phage of Vibrio cholerae. The agonist polypeptide contains the amino acid sequence FCIGRL (I D SEQUENCE N °: 4). The polypeptide has a length of less than 100 amino acid residues. An eleventh embodiment of the invention is a method for diagnosing an autoimmune disease in a patient. A first body sample of the patient is contacted with an antibody boosted against the amino acids SLIGKVDGTSHVTG, as shown in SEQ ID NO: 5. The amount of antibody bound by the first body sample is compared to a quantity bound by a second body sample of a healthy control that does not have an autoimmune disease. An autoimmune disease is identified in the patient if the first body sample binds more of the antibody than the second. A twelfth embodiment of the invention is a method for treating a patient with increased zonulin expression in relation to a healthy individual control. A boosted antibody against the amino acids SLIGKVDGTSHVTG is administered to a patient as shown in SEQ ID NO: 5. The symptoms of the disease are relieved thereby. A thirteenth embodiment of the invention is an antibody that is potentiated against SLI amino acids GKVDGTSHVTG as shown in SEQ ID NO: 5. The antibody binds to a protein expressed in CaCo2 cells that co-localize with a protein linked by the synthetic inhibitor peptide FZ1 / 0 (as shown in SEQ ID NO: 3). The antibody does not bind to human or rat cells that express a human recombinant PAR-2. The antibody is not SAM 1 1. A fourteenth embodiment of the invention is an antibody that binds to a protein expressed in CaCo2 cells that is co-localized with a protein linked by the synthetic inhibitory peptide FZ1 / 0 (as shown in SEQ ID NO: 3). ). The antibody does not bind to human or rat cells that express a human recombinant PAR-2. The antibody is not SAM 1 1. A fifth embodiment of the invention is an agonist polypeptide of a ZOT protein of the CXTF phage of Vibrio cholerae. The agonist polypeptide comprises a sequence selected from the group consisting of Xaa! Cys lie Gly Arg Leu (ID SEQUENCE N °: 7), Phe Xaa2 l ie Gly Arg Leu (ID SEQUENCE N °: 8), Phe Cys Xaa3 Gly Arg Leu (ID SEQUENCE N °: 9), Phe Cys lie Xaa4 Arg Leu (SEQ ID NO: 10), Phe Cys lie Gly Xaa5 Leu (SEQ ID NO: 1 1), and Phe Cys He Gly Arg Xaa6 (SEQ ID NO.: 12). The polypeptide has a length of less than 100 amino acid residues. Xaa! is selected from the group consisting of Ala, Val, Leu, Lie, Pro, Trp, Tyr, and Met; Xaa2 is selected from the group consisting of Gly, Ser, Thr, Tyr, Asn, and Gln; Xaa3 is selected from the group consisting of Ala, Val, Leu, Lie, Pro, Trp, and Met; Xaa is selected from the group consisting of Gly, Ser, Thr, Tyr, Asn, Ala, and Gln; Xaa5 is selected from the group consisting of Lys and His; Xaa6 is selected from the group consisting of Ala, Val, Leu, Lie, Pro, Trp, and Met. A sixteenth embodiment of the invention is an agonist polypeptide of a human zonulin receptor and ZOT protein of the CXTF phage of Vibrio cholerae. The agonist polypeptide contains a sequence selected from the group consisting of Xaa-, Xaa2 Me Gly Arg Leu (SEQ ID NO: 13), Xaa, Cys Xaa3 Gly Arg Leu (SEQ ID NO: 14), Xaa1 Cys He Xaa4 Arg Leu (ID SEQUENCE N °: 15), Xaa! Cys Me Gly Xaa5 Leu (ID SEQUENCE N °: 16), Xaa! Cys He Gly Arg Xaae (ID SEQUENCE N °: 17), Phe Xaa2 Xaa3 Gly Arg Leu (ID SEQUENCE N °: 18), Phe Xaa2 lie Xaa4 Arg Leu (ID SEQUENCE N °: 19), Phe Xaa2 lie Gly Xaa5 Leu (SEQ ID NO: 20), Phe Xaa2 lie Gly Arg Xaa6 (SEQ ID NO: 21), Phe Cys Xaa3 Xaa4 Arg Leu (ID SEQUENCE N °: 22), Phe Cys Xaa3 Gly Xaa5 Leu (ID SEQUENCE N ° : 23), Phe Cys Xaa3 Gly Arg Xaa6 (SEQ ID NO: 24), Phe Cys lie Xaa4 Xaa5 Leu (SEQ ID NO: 25), Phe Cys Me Xaa4 Arg Xaa6 (SEQ ID NO: 26), and Phe Cys lie Gly Xaa5 Xaa6 (ID SEQUENCE N °: 27). The polypeptide has a length of less than 100 amino acid residues. Xaai is selected from the group consisting of Ala, Val, Leu, Lie, Pro, Trp, Tyr, and Met; Xaa2 is selected from the group consisting of Gly, Ser, Thr, Tyr, Asn, and Gln; Xaa3 is selected from the group consisting of Ala, Val, Leu, Lie, Pro, Trp, and Met; Xaa4 is selected from the group consisting of Gly, Ser, Thr, Tyr, Asn, Ala, and Gln; Xaa5 is selected from the group consisting of Lys and His; Xaa6 is selected from the group consisting of Ala, Val, Leu, He, Pro, Trp, and Met. These and other modalities that will be evident to the experts of the subject when reading the specification, provide the technique with reagents and methods to treat diseases, diagnose diseases and discover drugs.BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and 1B. Comassie immunodetection (Figure 1A) and, Western (Figure 1B) of the six HPLC fractions obtained from used intestinal tissues. The positive fraction of zonulin F5 showed ~ 23kDa that was not present in the other five fractions. Figures 2A and 2B. Immunofluorescence in situ under the microscope, of small intestine of rats exposed to either FZI / 0 (Figure 2A) or fluorescein FZI / 1 (Figure 2B). Note the fluorescence distribution in the upper third of the hairs where the ZOT / zonulin receptor was originally described (see reference 44). Figures 3A to 3C. Co-localization of PAR-2-FZI / 0. Caco 2 cells were immunostained with FITC-FZI / 0 (Figure 3A) or with mouse anti-human PAR2 monoclonal antibodies (Figure 3B). The superposition of the two images (Figure 3C) showed a co-localization of immunofluorescent particles PAR-2 and FZI / 0. Figures 4A-4D. Competitive binding experiments FZI / 0-PAR-2 AP. Caco2 cells exposed to FITC-labeled FZI / O (Figure 4B) showed a significant number of fluorescent particles compared to cells exposed to the control media (Figure 4A). An excess of PAR2-AP (100x) displaced FZI / O (Figure 4C), while 100x of a non-encoded peptide (Figure 4 D). Figures 5A to 5D. Cytoskeletal actin arrangement in Caco2 cells exposed to PAR2-AP (Figure 5A), BSA (Figure 5B), PAR2-AP + FZI / 0 (Figure 5C), or PAR-2 AP + FZI / 1 (Figure 5D). Figures 6A to 6B. Effect of MCP-1 1 (Figure 6A) and PAR-2 AP (Figure 6B) in mouse intestinal TEER. Both MCP-11 (o) and PAR-2 AP (A) induced significant decreases in TEER compared to control tissues (0). These changes were comparable with the changes induced by AG (ß), and were "completely avoided by preincubation with FZI / 0 (x). Figures 7A to 7B. Effect of PAR-2 AP on intestinal TEER in wild type mice (Figure 7A) and MyD88 KO (Figure 7B). In wild-type mice, both PA-2 AP (?) And? G (m) induced significant decreases in TEER compared to control tissues (0) that were completely avoided by preincubation with FZI / 0 ( x) No changes were observed in TEER in MyD88 KO mice under any treatment condition. Figure 8. Proposed activation of the receptor by ZOT and zonulin. As an analog of MCP-I I, zonulin activates the receptor by dividing its N terms, while ZOT binds and activates the receptor by means of its N-terminal homologous motif of PAR-2 AP. The activation of PAR-2 by MCP-1 1 and PAR-2 AP or the activation of the zonulin receptor by zonulin and? G is blocked by the competitive binding inhibitor FZI / 0.

DETAILED DESCRIPTION OF THE INVENTION The inventors have developed an agonist polypeptide of a human zonulin receptor and ZOT protein from the CXTF phage of Vibrio cholerae. The polypeptide contains the amino acid sequence FCIGRL (I D SEQUENCE N °: 4). The polypeptide is less than 100 amino acid residues, or less than 50, 40, 30, 20, 10 or 8 amino acid residues. The polypeptide may contain only the six amino acids FCIGRL (SEQ ID NO: 4), or may have additional amino acids. The other amino acids can provide other functions (e.g., antigen tags, to facilitate purification) The agonist polypeptide can be used to facilitate the absorption of therapeutic or immunogenic agents The agonist polypeptide facilitates absorption through the intestine, the blood barrier Brain, skin, and nasal mucosa Thus, the agonist polypeptide can be formulated or co-administered with a therapeutic or immunogenic agent directed to the gut, brain, skin, nose A pharmaceutical composition according to the present invention does not need to be premixed prior to administration, but may be formed in vivo when two agents are administered within 24 hours of each other., the two agents are administered within 12, 8, 4, 2 or 1 hour of each other. The therapeutic agents according to the invention are any that can be used to treat a human or other mammal. The agent can be for example, an antibody or an antibody fragment (such as a Fab, F (ab ') 2, a single chain antibody (ScFv)), an anti-cancer drug, an antibiotic, a hormone or a cytokine The therapeutic agent can be one that acts on any organ of the body, such as the heart, brain, intestine, or kidneys. Diseases that can be treated according to the invention include food allergies, gastrointestinal infection, autoimmune disease, inflammatory bowel disease, celiac disease, gastrointestinal inflammation, but are not limited thereto. Compositions in intravenous doses for delivery to the brain are well known in the art. These compositions in intravenous doses generally contain a physiological diluent, for example distilled water, or 0.9% (weight / volume) of NaCl. A composition for "nasal" delivery differs from a composition for "intestinal" delivery in that the latter has to have gastroresistant properties in order to avoid acid degradation of the active agents (eg, zonulin receptor agonist and therapeutic agent ) in the stomach, although the former generally contains water-soluble polymers with a diameter of approximately 50 μm in order to reduce the mucociliary space, and achieve reproducible bioavailability of the nasally administered agents. A composition for supply "intravenous" differs from the com positions for "nasal" and "intestinal" delivery in that there is no need for gastroresistant or water soluble polymers in the composition for "intravenous" delivery. The form of administration is not critical to the present. The form of administration can be oral, for intestinal supply; intranasal, for nasal delivery, and intravenous for delivery through the blood-brain barrier. Other forms of administration may be used as are known in the art, including, without limitation, intrathecal, intramuscular, intrabronchial, intrarectal, infraocular, and intravaginal delivery. Compositions in oral doses for delivery to the small intestine are well known in the art. These compositions for oral doses may include gastroresistant tablets or capsules (Remington's Pharmaceutical Sciences, 16th edition., Eds. Osol, Mack Publishing Co., Chapter 89 (1980); Digenis and co-authors, J. Pharm. Sci., 83: 915. -921 (1994), Vantini and coauthors, Clínica Terapéutica, 145: 445-451 (1993), Yoshitomi and co-authors, Chem. Pharm. Bull., 40: 1902-1905 (1992), Thoma and co-authors, Pharmazie, 46: 331-336 (1991), Morishita and co-authors, Drug Design and Delivery, 7: 309-319 (1991), and Lin and co-authors, Pharmaceutical Res., 8: 919-924 (1991)); each of which is incorporated in the present application by reference in its entirety). The tablets are made gastroresistant by the addition, for example, of cellulose acetate phthalate or cellulose acetate terephthalate. Capsules are solid dosage forms in which the biologically active ingredient or ingredients are enclosed in a soluble or soft gelatin shell or reservoir. The gelatin used in the manufacture of the capsules is obtained from collagen material by hydrolysis. There are two types of gelatin. Kind A, derived from pig skins by acid processing, and type B, obtained from animal bones and skins by alkaline processing. The use of hard gelatin capsules allows a choice by prescribing a single biologically active ingredient or a combination of them at the exact dose level considered best for the individual. The hard gelatin capsule typically consists of two sections, one sliding over the other, thus completely surrounding the biologically active ingredient, or gastroresistant beads containing the biologically active ingredient, at the longer end of the capsule, and then sliding the cap . Hard gelatine capsules are made primarily of gelatin, FD and C dyes, and sometimes of an opacifying agent, such as titanium dioxide. The United States Pharmacopeia (USP) convention allows gelatin for this purpose to contain 0.15% (weight / volume) of sulfur dioxide to prevent decomposition during manufacture. Compositions for oral doses for delivery to the small intestine also include liquid compositions which may optionally contain aqueous regulating agents that prevent the therapeutic agent and the agonist polypeptide from being inactivated significantly by gastric fluids in the stomach, thereby allowing the biologically active ingredient and the polypeptide agonist reach the small intestines in an active form. Examples of these aqueous regulating agents that may be employed in the present invention include bicarbonate buffer (pH 5.5 to 8.7, preferably about pH 7.4). When the composition for oral dose is a liquid composition, it is preferable that the composition be prepared just before the administration, in order to minimize stability problems. In this case, the liquid composition can be prepared by dissolving the lyophilized therapeutic agent and the agonist polypeptide in the aqueous regulatory agent. The compositions for nasal doses, for nasal delivery, they are well known in the field. These compositions for nasal doses generally comprise water-soluble polymers that have been widely used to prepare pharmaceutical dosage forms (Martin and co-authors, in: Physical Chemical Principles of Pharmaceutical Sciences, 3rd Ed., Pages 592-638 (1983)) which can serve as carriers for peptides for nasal administration (Davis, In: Delivery Systems for Peptide Drugs, 125: 1-21 (1986)). It has been shown that nasal uptake of peptides embedded in polymer matrices is improved by delaying mucociliary compensation (Illum et al., J. Pharm., 46: 261-265 (1988)). Other possible enhancement mechanisms include an increased concentration gradient or a decreased diffusion path for peptide uptake (Ting and co-authors, Pharm. Res., 9: 1330-1335 (1992)). However, it has been predicted that the reduction in the mucociliary compensation rate is a good approach for the achievement or for the reproducible bioavailability of systemic drugs administered nasally (Gonda et al., Pharm. Res., 7: 69-75 (1990). ). Microparticles with a diameter of approximately 50 μm are expected to be deposited in the nasal cavity (Bjork and co-authors, Int. J. Pharm., 62: 187-192 (1990)); and lllum and coauthors, Int. J. Pharm., 39: 189-199 (1987), whereas microparticles with a diameter below 10 μm can escape from the filtering system of the nose and deposit in the lower respiratory tract. Microparticles as large as 200 μm in diameter will not be retained in the matrix after nasal administration (Lewis and co-authors, Proc. Int. Symp. Control Reí. Bioact. Mater., 17: 280-290 (1990)) . The particular water soluble polymer employed is not critical to the present invention and can be selected from any of the well known water soluble polymers employed for nasal dosage forms. A typical example of a water-soluble polymer useful for nasal delivery is polyvinyl alcohol (PVA). This material is a hydrophilic polymer that increases in volume with water, whose physical properties depend on the molecular weight, degree of hydrolysis, cross-linking density, and crystallinity (Peppas and co-authors, in: Hydrogels in Medicine and Pharmacy, 3: 109- 131 (1987)). PVA can be used in the coating of dispersed materials by phase separation, spray drying, spray inlay and spray densification (Ting and coauthors, supra). A skin supply composition may include in addition to a therapeutic or immunogenic agent, fragrance, creams, ointments, colorants, and other compounds so long as the added component does not detrimentally affect the transdermal delivery of the therapeutic or immunogenic agent. It is also possible to add conventional emulsifiers, surfactants, suspending agents, antioxidants, osmotic enhancers, extenders, diluents and preservatives acceptable for pharmaceutical use.

Water soluble polymers can also be used as carriers. The particular therapeutic or immunogenic agent employed is not critical to the present invention, and may be, for example, any pharmacological compound, biologically active peptide, vaccine, or any other portion that is not absorbed through the transcellular route, regardless of the size or load. Examples of compounds with drugs that can be employed in the present invention include drugs that act on the cardiovascular system, drugs that act on the central nervous system, antineoplastic drugs and antibiotics. Examples of drugs that act on the cardiovascular system, which may be employed in the present invention, include lidocaine, adenosine, dobutamine, dopamine, epinephrine, norepinephrine and phentolamine. It is also possible to use others known in the art. Examples of drugs that act on the central nervous system, which may be employed in the present invention, include doxapran, alfentanil, dezocin, nalbuphine, buprenorphine, naloxone, ketrolac, midazolam, propofol, metacurin, mivacurium and succinylcholine. It is also possible to use others known in the art. Examples of antineoplastic drugs that can be employed in the present invention include cytarabine, mitomycin, doxorubicin, vincristine and vinblastine. It is also possible to use others known in the art. Examples of antibiotics that can be employed in the present invention include methicillin, mezlocillin, piperacillin, ketoxytin, cefonicid, cefmetazole and aztreonam. It is also possible to use others known in the art. Examples of biologically active peptides that can be employed in the present invention include testosterone, nandrolene, menotropins, insulin and urofoltropin. It is also possible to use others known in the art. When the biologically active ingredient is insulin, the oral dose composition of the present invention is useful for the treatment of diabetes. Examples of lymphokines that can be employed in the present invention include interferon α, interferon β, interferon α, interleukin 1, interleukin 2, interleukin 4 and interleukin 8. Examples of globulins that can be employed in the present invention include globulins, β globulins and? globulins (immunoglobulin). Examples of immunoglobulins that can be employed in the present invention include polyvalent IgG or specific IgG, IgA and IgM, for example, anti-tetanus antibodies. An example of albumin that can be employed in the present invention is human serum albumin. It is also possible to use others known in the art. Examples of vaccines that can be employed in the present invention include peptide antigens and microorganisms and attenuated viruses. Examples of peptide antigens that can be employed in the present invention include subunit B of enterotoxin of heat-laden enterotoxigenic E. coli, subunit B of cholera toxin, capsular antigens of enteric pathogens, fimbria or pili of enteric pathogens , surface antigens of HIV, dust allergens and mite allergens. It is also possible to use others known in the art.

Examples of microorganisms and attenuated viruses that can be employed in the present invention include those of enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, Vibrio cholerae, Shigella flexneri, Salmonella typhi and rotavirus (Fasano and co-authors, In: Le Vaccinazion i in Pediatrics, Eds Vierucci and co-editors, CSH, Milan, pages 109- 121 (1991), Guandalini and co-authors, in: Management of Digestive and Liver Disorders in Infants and Children, Elsevior, Eds. Butz and coauthors, Amsterdam, Chapter 25 (1993); Levine and co-authors, Sem. Ped. Infect. Dis., 5: 243-250 (1994); and Kaper and co-authors, Clin. Micrbiol. Rev., 8: 48-86 (1995), each of which is incorporated to the present application by reference in its entirety). The amount of therapeutic or immunogenic agent employed is not critical to the present invention and will vary depending on the particular ingredient selected, the disease or condition that is being treated, as well as the age, weight and sex of the subject being treated. The amount of the ZOT receptor agonist polypeptide employed is also not critical to the present invention, and will vary depending on the age, weight and sex of the subject being treated. Generally, the final concentration of the ZOT receptor agonist polypeptide employed in the present invention to improve absorption of the biologically active ingredient by the intestine is on the scale from about 10-5 M up to 10-10 M, preferably from about 10-6 M up to 5.0 x 10- 5 M. To achieve this final concentration in the intestine, the amount of ZOT receptor agonist polypeptide in a composition of the invention in a single oral dose will generally be from about 4.0 ng to 1000 mg, preferably from about 40 ng to 80 ng. The proportion of therapeutic or immunogenic agent relative to the ZOT receptor agonist polypeptide employed or is critical to the present invention, and will vary depending on the amount of biologically active ingredient that is to be delivered within the selected time period. Generally, the weight ratio of therapeutic or immunogenic agent to the ZOT receptor agonist polypeptide employed in the present invention is in the range of from about 1: 10 to 3: 1, preferably from about 1: 5 to 2: 1. Antibodies that bind to the protein identified by an antibody enhanced against the amino acids SLIGKVDGTSHVTG (SEQ ID NO: 5), can be used diagnostically, or therapeutically, and as a research tool. One of these antibodies is SAM1 1, which is available from Zymed Laboratories, South San Francisco, California. Other of these antibodies can be easily made using standard techniques to enhance monoclonal or polyclonal antibodies. The up-regulation of zonulin receptors in diseases can be detected using SAM1 1 antibodies or other antibodies that bind to the same human protein. The antibodies can be conjugated with a therapeutic or toxic agent, including radionuclides, antineoplastic agents, etc. The identification of binding partners for the zonulin receptor and ZOT receptor protein allows substances that interrupt the binding to be tested. The binding partners identified to date include MyD88, zonulin, ZOT and? G. Any test for the binding of two proteins can be used. These can be in vitro or in vivo tests. The tests may employ antibodies or solid phase binding substrates. Any of these tests can be used as is known in the art. Conservative substitutions may be made, in which the amino acid is exchanged for another having similar properties, in the agonist polypeptide having the sequence of SEQ ID NO: 4. Examples of conservative substitutions include, without limitation, Gly <; - Ala, Val < - »Leu, Asp- -Glu, Lys < - »Arg, Asn- ^ Gln, and Phe- -Trp < ? -Tyr? Conservative amino acid substitutions typically fall in the range from about 1 to 2 amino acid residues. Guidance can be found to determine which amino acid residues can be substituted without eliminating biological or immunological activity using computer programs well known in the art, such as DNASTAR software, or Dayhoff and co-authors (1978) in Atlas of Protein Sequence and Structure ( Nati, Biomed, Res. Found., Washington, DC). The amino acid substitutions are defined as amino acid replacements one by one. They are conservative in nature when the substituted amino acid has similar structural and / or chemical properties. Examples of conservative replacements are the substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Preferred oligopeptide analogs include substitutions that are conservative in nature, i.e. those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into families: (1) acid: aspartate and glutamate; (2) basic: lysine, arginine, histidine; (3) non-polar: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) uncharged polar: glycine, asparagine, glutamine, cystine, threonine serine, and tyrosine; and (5) aromatic amino acids: phenylalanine, tryptophan, and tyrosine. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological activity. Any test known in the art can be used to determine the biological activity of the ZOT receptor agonist. For example, the test may involve (1) assays to determine the decrease in tissue resistance (Rt) of Leo mounted on Ussing chambers as described by Fasano and coinvestigators, Proc. Nati Acad. Sci., USA, 8: 5242-5246 (1991); (2) assays to determine the decrease in tissue resistance (Rt) of monolayers of intestinal epithelial cells in Ussing chambers, as described below; or (3) assays for determining the improvement of intestinal or nasal absorption of a therapeutic or immunogenic agent, as described in WO 96/37196; U.S. Patent Application No. 08 / 443,864, filed May 24, 1995; U.S. Patent Application No. 08 / 598,852, filed February 9, 1996; and U.S. Patent Application No. 08/781, 057, filed January 9, 1997. The receptor ZOT receptor agonists will rapidly open the tight junctions in a reversible and reproducible manner, and thus can be used as absorption enhancers. of a therapeutic or immunogenic agent in the same manner in which ZOT is used as a nasal or intestinal absorption enhancer, as described in WO 96/37196; in U.S. Patent Application No. 08/443, 864, filed May 24, 1995; in U.S. Patent Application No. 08 / 598,852, filed on February 9, 1996; and in U.S. Patent Application No. 08/781, 057, filed January 9, 1997.

Antibodies to the zot / zonulin receptor can be used as an anti-inflammatory agent for the treatment of gastrointestinal inflammation that provides increased intestinal permeability. Thus, the antibodies of the present invention are useful, for example, for the treatment of intestinal conditions that cause enteropathy with protein loss. The enteropathy with protein loss may appear due to: infection, for example infection with C. difficile, enterocolitis , shigellosis, viral gastroenteritis, infestation by parasites, bacterial overgrowth, Whipple's disease, diseases with erosion or mucosal ulcerations, for example, gastritis, gastric cancer, collagen colitis, inflammatory bowel disease, diseases marked by lymphatic obstruction, for example lymphangiectasia congenital intestinal, sarcoidosis-lymphoma, mesenteric tuberculosis, and post-surgical correction of congenital heart disease with Fontan operation; mucosal diseases with ulceration, for example, Menetrier's disease, celiac disease, eosinophilic gastroenteritis; and diseases of the immune system, for example, systemic lupus erythematosus or food allergies, primarily to milk (see also Table 40-2 of Pediatric Gastrointestinal Disease Pathophysiology Diagnosis Management, Eds. Wyllie and co-publishers, Saun Co. (1993) , pages 536-543, which is incorporated in the present application by reference in its entirety). Antibodies can be administered to patients with cancer, autoimmune disease, vascular disease, bacterial infection, celiac disease, asthma and irritable bowel syndrome. The foregoing description generally describes the present invention. All references described herein are expressly incorporated by reference. A more complete untanding may be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE 1 Intestinal tissues of the small intestine of rats were analyzed by a combination of gel filtration chromatography and ELISA with zonulin. Rat intestine homogenates were loaded onto a Sephacryl column (length 90 cm, diameter 2.6 cm, calibrated as standard molecular weight markers) and fractions were collected and analyzed by ELISA with zonulin to determine zonulin concentrations. Of the six fractions (F1-F6) tested, F5 contained the highest zonulin concentrations. Each fraction was resolved by SDS-PAGE, transferred, and immunodetected with anti-ZOT antibodies immunoreactive to zonulin (Figure 1B). Western analysis revealed two main bands that migrated with an apparent mann receptor (Mr) of approximately 24,000 and 23,000 in the zonulin-positive fraction, F5, whereas the zonulin-negative fractions, FI-4.6, each revealed only one immuno-reactive band (-24 kDa). Accordingly, the ~ 23 kDa band of F5 (see arrow in Fig. 1 B) was removed from a Blue-stained Comassie gel and subjected to matrix-assisted ionization desorption-based mass spectrometry (MALDI). The search using Profound search engine for protein matches (domain number 129.85.19.192, profound directory drawer, WebProFound.exe program? FORM = 1) revealed a high similarity of this protein (estimated Z score of 1.58) with the protease II 1 of rat mast cells). Rat mast cell proteases are serine proteinases contained in mast cell granules with properties similar to trypsin (tryptase) and chymotrypsin (chymase) (46). Mucosal mast cells (MMC) contain predominantly protease I I (MDP-I I), whereas mast cells of connective tissues contain mainly protease I (46). MCP-H is particularly abundant in the pulmonary and gastrointestinal mucosa (47). In the gastrointestinal tract, one of the best characterized bioactivities of the MCP-II is the modulation of the permeability of the epithelial mucosa after the infestation with nematodes (48). In vitro studies suggest that MCP-I I opens the epithelial barrier by interrupting the tight junction complex. Therefore, our proposed hypothetical model for the zonulin system and the established functions of MCP-II are clearly compatible.

However, we detected greater differences between zonulin and MCP-I I, including its sources (zonulin is present in enterocytes (49) and macrophages) and stimuli for its release (intestinal parasite infestation for MCP-II). [48] and bacteria and gliadin [49] for zonulin). We carried out experiments in micro-receptacles under pressure in WBB6 / F1-W / Wv mice that have pleiotropic defects in germ cells, RBC and mucosal mast cells, and therefore, lack MCP-I I (55). Tissues that were mounted in the micro-receptacle system under pressure and were exposed at increasing time intervals (up to 3 hours) to zonulin-releasing stimuli, showed a decrease in TEER (-1 70 ± 15.8 Ohms / cm2 vs. -43). ± 1 1 of untreated tissues) and a parallel increase in the release of zonulin (10.0 ± 0.08 ng / mg of protein versus 0.2 + 0.7 in untreated tissues) in a manner similar to that seen in wild-type animals ( -120 ± 20 and 15.1 ± 3.1, respectively). Taken together, our data suggest that zonulin is distinct from MCP-I I and may represent one of several physiological activators of PAR-2 or a variant of PAR-2. Pancreatic trypsin is the most efficient activator of PAR-2, but there is a discrepancy between the availability of pancreatic trypsin and the distribution of PAR-2 (47). Biologically active trypsin is present in the lumen of the small intestine, where it can activate PAR-2 in the apical membrane of enterocytes (47), but PAR-2 is also found in many tissues where it has to be activated by an already identified physiological activator (50). Zonulin represents a strong candidate for this activator of PAR-2 and can reconcile this apparent discrepancy, given that it has been isolated in intestinal and extraintestinal tissues (51).

EXAMPLE 2 We have previously shown that ZOT binds to the surface of the rabbit intestinal epithelium, and that it varies along different regions of the intestine (44). The distribution of the link coincides with the regional effect of ZOT on intestinal permeability and with the preferential redistribution of F-actin induced by ZOT in mature hair cells (38,44). To further characterize the ZOT receptor, we perform the following experiments.

A. LACE EXPERIMENTS The binding experiments were performed with several epithelial cell lines, including endothelial cells I EC6 (rat, intestine), CaCo2 (human, hair-like enterocytes), T84 (human, cavity-like enterocytes), MCDK (canine, kidney) , and bovine pulmonary artery (B PA). For the immunofluorescence analysis, confluent monolayers (2.0 x 105) were incubated on glass plates to increase the time intervals (5 min, 30 min, 60 min), at 4 ° C or at 27 ° C with 5 x 10" 9 M ZOT or a negative control After incubation of the monolayers with ZOT (0.2 μM) for 15 minutes at 37 ° C, the cells were washed 10 times with cold PBS, suspended and used. were resolved by SDS-PAGE, transferred to PVDF membranes, and tested with anti-ZOT antibodies.To establish the specificity of the ZOT-binding, radiolabeled ZOT was used.These experiments were performed in the absence or in the presence of 10 or 50-fold molar excess of unlabeled cold ZOT When incubated with ZOT protein to increase time intervals, intestinal epithelial cells CaCo2 and I EC6, as well as endothelial cells, showed binding on the cell surface, compared with the c cells exposed to negative control. by contrast; no staining was observed neither in T84 cells nor in MDCK after incubation for up to 60 minutes with His-ZOT. The cellular specificity of the ZOT binding was confirmed by immunodetection analysis. ZOT was found for IEC6, CaCo2 and BPA, but not for T84 and MCDK cells.

B. PURIFICATION OF ENZZING PROTEIN WITH ZOT A column with His-ZOT affinity was prepared by immobilizing overnight, at room temperature, 1.0 mg of purified His-ZOT for a pre-activated gel (aminolink, Pierce). The column was washed with PBS, and then loaded with a used crude cell obtained using 1 06 of EC6 cells I or of CaC02 cells. After a incubation of 90 m inutes at room temperature, the column was washed five times with 14 ml of PBS, and the proteins that bound to His-ZOT were eluted from the column with 4.0 ml of 50 μM of glycine. Na (pH 2.5), 1 50 mM NaCl and 0.1% (volume / volume) of Triton X-1 00. The p H of the 0.1 ml fractions eluted was immediately neutralized with 1.0 N. of NaOH. The fractions collected were subjected to SDS-PAGE gradient from 6.0% to 15.0% (weight / volume) under reducing conditions. The resolved proteins were transferred to a nitrocellulose membrane and subjected to an N-terminal N-terminal sequencing using a Perkin elmer Applied Biosystems model 494. Eluted fractions obtained from I EC6 and CaCo2 cells contained a single band of proteins with a 66 kDa, as observed by SDS-PAGE under reducing conditions. Treatment with neuraminidase reduced the size of the supposed ZOT receptor to 35 kDa, suggesting that this receptor is sialylated (51).

C. CHARACTERIZATION OF THE REC EPTOR D E ZOT / ZONULINA Our recent data suggesting that zonulin is structurally similar to mast cell protease (MCP) -II has led us to construct the hypothesis that the ZOT / zonulin receptor could be similar, if not identical, to the receptor. MCP-II activated by proteinase (PAR-2). PAR-2 has several characteristics in common with those described for the ZOT / zonulin receptor. Specifically, mature PAR-2 is a 68 to 80 kDa glycoprotein that is reduced to 36-40 kDa by deglycosylation (47). Similarly, the receiver of ZOT / zonulin has a molecular mass of 66 kDa that is reductive by deglycosylation at 35 kDa (51). The distribution of PAR-2 within the gastrointestinal tract (47) coincides with the distribution of the ZOT / zonulin receptor in the intestine (44). Intracellular PAR-2 signaling involves the activation of phospholipase C (PLC), protein kinase C (PCK) (52), and actin polymerization leading to cytoskeletal rearrangement (53). Zot and zonulin activate these same intracellular signaling pathways through a common intestinal surface receptor (38). Similar to the effect of ZOT / zonulin in the intestine, activation of intestinal PAR-2 results in increased intestinal permeability (54). finally, PAR-2 is activated by division of its extracellular domain by trypsin, creating a new N-terminus that acts as a "bound ligand". The exogenous addition of the SLIGRL peptide (PAR-2 AP), which corresponds to the newly created N term, proteolytically activated, also activates PAR-2 regardless of the division of the receptor (52). The N-terminus of the biologically active 12 kDa ZOT fragment (? G) encompasses the extracellular domain of ZOT (residues aa 288-399) that are proteolytically divided by Vibrio cholerae in the host's intestinal tract. The N-terminus? G contains a peptide (amino acids FCIGRL 288-293), which is structurally similar to the agonist ligand motif of PAR-2. To more precisely define the structural requirements to engage and activate the target receptor, two? G mutants were synthesized by mutating the binding motif with PAR-2 (? G291) or the region just downstream of the ligand motif (? G298). These peptides were compared with? G for their ability to bind to EC-6 cells, as well as for their biological activity in rat small intestine mounted on Ussing chambers. EC6 I cells incubated with? G291 (G291 V) showed reduced binding to IEC6 cells, compared to cells incubated with? G, while no binding was observed in the cells incubated with the peptide? G298 (G298V). Biological tests in the Chambers of Ussing showed that "G291 had a residual effect, but not statistically significant on the disassembly of tj, while" G298 failed to obtain any detectable penetration effect. These results were parallel to the effects obtained with these two mutants in the binding test and suggested that residue F at position 291, and more importantly, residue G at 298 can play crucial roles in the binding of? G and in the activation of its target receptor, possibly through changes in the configuration of the protein. Currently, one of the main limitations in the study of the functions of PAR-2 under physiological and pathological circumstances is the absence of specific inhibitors of PAR-2 (52). Based on our structure-function analyzes, we designed a synthetic octapeptide (corresponding to the amino acid residues of ZOT 291 -298) that encompasses the two G residues to which we point by mutagenesis, but lacks the first 3 amino acid residues (288 -290) of the supposed ligand motive. This synthetic peptide, FZI / 0, was tested in loyal tissue mounted in Ussing chambers, either alone or in combination with ZOT,? G or zonulin. No changes were observed in the Rt tissues exposed to FZI / 0 or the encoded octapeptide (FZI / 1). The treatment of loyal tissues with FZI / 0 before the study and during the study period, prevented the changes in Rt in response to ZOT,? G, and zonulin where the permeant effect of the three proteins was affected by the treatment previous co FZI / 1. These data strengthen our hypothesis that ZOT and zonulin point to the same receptor and suggest that FZI / 0 can exert its inhibitory effect by binding, but not activating, this receptor. To test this last hypothesis, we performed in situ binding experiments using rat small intestine incubated with FZI / 0 or fluoresceinized FZ1 / 1. The tissue exposed to FZI / 0 showed numerous fluorescent particles, although no signal was detected in tissues incubated with FZI / 1.

EXAMPLE 3 The synthetic ZOT / zonulin inhibitor FZI / 0 binds to PAR-2. To establish if the FZI / 0 is linked to PAR-2, microscopic unofluorescence was performed with co-localization with double label, in Caco2 cell monolayers. Briefly, the cells were incubated to increase the time intervals either with FITC-FZI / 0 or with mouse anti-human PAR-2 monoclonal antibodies, followed by incubation with rhodamine-labeled anti-mouse IgG antibodies. The cells were then washed 3 times with PBS, fixed in 3.7% formaldehyde in PBS (pH 7.4) for 15 minutes at room temperature, the slide covers were mounted with glycerol-PBS (1: 1) with a pH of 8.0 and they were analyzed with fluorescence microscopy (ZEISS). Immunofluorescent particles were observed in cells exposed to FITC-FZI / 0 and anti-PAR-2 antibodies (Figure 3). The superposition of the two images showed that the co-localization of the receptor of PAR-2 and FZI / 0 was evident (Figure 3).

EXAMPLE 4 COMPETITIVE LINK EXPERIMENTS OF FZI / 0 - PAR-2 AP The activation of PAR-2 requires the link of its portion N-terminal divided, generated by tryptase, or of the equivalent synthetic peptide PAR-2 AP TO THE EXTRACELLULAR LOOP 2 OF THE RECEIVER (ecl2) (47) ,. To establish whether the FZI / 0 does not bind to the same receptor domain, competitive binding experiments were performed on Caco2 cells. Cell monolayers were incubated with FITC-FZI / O (2x10"8 M)), either in the presence of an excess of PAR-2 AP (10" 6 M), or a peptide encoded and then analyzed by fluorescence af microscope . Cells exposed to an excess of PAR-2 AP showed a significant reduction of immunofluorescent particles of FZI / 0 compared to the monolayers exposed to the encoded peptide (Figure 4), suggesting that FZI / 0 binds to PAR-2 and it can be competitively displaced by PAR-2 AP.

EFFECT OF THE ZOT / ZONULIN INHIBITOR FZI / 0 ON THE ACTAC REACOMODO I NDUCIO BY PAR-2 AP It has recently been reported that activation of the PAR-2 receptor by PAR-2 AP promotes cytoskeletal reorganization (53). To establish whether this effect can be avoided by the ZOT / zonulin peptide inhibitor, FZI / 0, immunofluorescence studies were performed on monolayers of Caco2 cells. Cells exposed to 10"6M of PAR-2 AP (Figure 5A), showed fiber solution in tension where the monolayers treated with BSA did not (Figure 5B) .These cytoskeletal changes were blocked by the previous incubation with 2x10" 6M of FZI / 0 (Figure 5C), but not by the encoded peptide FZI / 1 (Figure 5D). Consequently, PAR-2 AP and FZI / 0 appeared to bind to the identical structure in the enterocytes.

EXAMPLE 5 EFFECT OF PAR-2 AP AND MCP-H ON INTESTINAL LAPERMEABILITY PAR-2 is highly expressed in the apical membrane of enterocytes, and presumably regulates one or more enteric functions (52). We asked if one of these functions could be the regulation of intestinal permeability mediated by zonulin in response to bacterial colonization. To explore this hypothesis, we tested the effect of treatment with MCP-II and with PAR-2 AP on the small intestine of the mouse in the test with micro-receptacles under pressure. The addition of 10 ~ 6 M of PAR-2 AP or of MCP-II (10 ~ 8 M) to the luminal aspect of the intestine decreased the TEER compared to untreated tissues, and this PAR-2 dependent decrease was completely avoided by the previous treatment with FZI / 0 (figure 6). These results provide a further line of evidence to support the hypothesis that PAR-2 is the target receptor for both ZOT and zonulin, and suggest that this receptor is also involved in the regulation of tight intercellular junctions.

EXAMPLE 6 INVOLVING MYD88 IN PAR-2 SIGNALING Many microbial structures, such as bacterial lipopolysaccharide or the respiratory syncytial virus fusion protein, as well as certain endogenous proteins, activate the cells of the innate immune system through intracytoplasmic signaling that is initiated by receptors similar to Toll (TLR; 55). To date, ten TLRs have been identified in mammals. Within the intracytoplasmic domains of the TLRs and the interleukin-1 and interleukin-18 receptors, there is a region of homology that is termed the "IL1 Toll Receptor", or "TIR" domain. The RI R domain is responsible for joining critical adapter molecules, such as Myeloid Differentiation Factor 88 (MyD88). The striking similarity of the signaling pathways coupled by the activation of PAR-2 and the ones labeled by the TLR, for example, NF-? B, etc .; 52) leads us to construct the hypothesis that zot / zonulin could be coupled with a member of the TLR family or with a closely related protein. Therefore, we tested the ability of ZOT and PAR-2 AP to induce changes in transepithelial intestinal electrical resistance (TEER) in wild type mice and in mice that have an objective mutation ("knockout", KO) in the MyD88 gene (figure 7A and 7B). The data in Figure 7A indicate that both AP-AP 2 and ΔG induce a comparable drop in intestinal resistance over time in wild-type tissues, which was reversed by fa previous incubation with the zot inhibitor peptide, FZI. / 0. In contrast, intestinal tissues derived from MyD88 knockout mice failed to respond to any stimulus to show a decrease in TEER. Taken together, these results suggest that ZOT and zonulin activate the same receptor (a variant or homolog of PAR-2), possibly through two different mechanisms (Figure 8). Our data support the notion that the ZOT binds directly to PAR-2 (variant or homolog) ECL2 and activates receptor signaling, whereas zonulin, as an analogue of MCPII, can activate the target receptor by dividing it into its terminus. N (figure 8). Furthermore, we propose that PAR-2 (variant or homolog) can be directly coupled to MyD88 through a domain similar to TI R.

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Claims (80)

1. CLAIMS 1. An agonist polypeptide of a ZOT protein from the CXTF phage of Vibrio cholerae, said agonist polypeptide contains FCIFRL (SEQ ID NO: 4), further characterized in that said polypeptide has a length less than 100 amino acid residues.
2. 2. The agonist polypeptide of claim 1, further characterized in that said polypeptide contains the amino acid residues FCIFRL (SEQ ID NO: 4) 3. The agonist polypeptide of claim 1, further characterized in that said polypeptide does not contain residues 294-298 of SEQ ID NO: 1. 4. The agonist polypeptide of claim 1, further characterized in that said polypeptide has a length of less than 50 amino acid residues. 5. The agonist polypeptide of claim 1, further characterized in that said polypeptide has a length of less than 40 amino acid residues. 6. The agonist polypeptide of claim 1, further characterized in that said polypeptide has a length of less than 30 amino acid residues. 7. The agonist polypeptide of claim 1, further characterized in that said polypeptide has a length of less than 20 amino acid residues. 8. The agonist polypeptide of claim 1, further characterized in that said polypeptide has a length of less than 10 amino acid residues. 9. The agonist polypeptide of claim 1, further characterized in that said polypeptide has a length of less than 8 amino acid residues. 10. A pharmaceutical composition for treating a disease, which contains: a therapeutic agent for the treatment of the disease; and the agonist polypeptide of claim 1. eleven . A pharmaceutical composition for the treatment of a disease, which contains: a therapeutic agent to treat the disease; and the agonist polypeptide of claim 2. 12. The pharmaceutical composition of claim 10, further characterized in that the disease is a food allergy. The pharmaceutical composition of claim 10, further characterized in that the disease is a gastrointestinal infection 14. The pharmaceutical composition of claim 10, further characterized in that the disease is an autoimmune disease. 15. The pharmaceutical composition of claim 10, further characterized in that the disease is inflammatory bowel disease l. 1 6. The pharmaceutical composition of claim 1 0, further characterized because the disease is celiac disease. 7. The pharmaceutical composition of claim 10, further characterized in that the disease is gastrointestinal inflammation. 8. The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is a drug. 9. The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is a biologically active peptide. 20. The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is an antibody. twenty-one . The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is an antibody fragment. 22. The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is a single chain antibody (ScFv). 23. The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is an anti-cancer drug. 24. The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is an antibiotic. 25. The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is a hormone. 26. The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is a cytokine. 27. The pharmaceutical composition of claim 10, further characterized in that the ratio of said therapeutic agent to said agonist polypeptide is from 1: 10 to 5: 1. 28. The pharmaceutical composition of claim 10, further characterized in that the ratio of said therapeutic agent to said agonist polypeptide is from 1: 5 to 2: 1. 29. The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is directed to the heart. 30. The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is directed to the brain. 31 The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is directed to the intestine. 32. The pharmaceutical composition of claim 10, further characterized in that the therapeutic agent is directed to the kidney. 33. The pharmaceutical composition of claim 10, further characterized in that the agonist polypeptide is present in an amount sufficient to improve the absorption of said therapeutic agent in the heart. 34. The pharmaceutic composition of claim 10, further characterized in that the agonist polypeptide is present in an amount sufficient to improve the absorption of said therapeutic agent in the brain. 35. The pharmaceutical composition of claim 10, further characterized in that the agonist polypeptide is present in an amount sufficient to improve the a b sorption of said therapeutic agent in the intestine. 36. The pharmaceutical composition of claim 10, further characterized in that the agonist polypeptide is present in an amount sufficient to improve the a b sorption of said therapeutic agent in the kidney. 37. A method for supplying a therapeutic agent to a target tissue, comprising: administering the composition of claim 12 to a patient with said disease. 38. A method for delivering a therapeutic agent to a target tissue, comprising: administering the composition of claim 13 to a patient with said disease. 39. A method for delivering a therapeutic agent to a target tissue, comprising: administering the composition of claim 14 to a patient with said disease. 40. A method for delivering a therapeutic agent to an objective tissue, comprising: administering the composition of claim 15 to a patient with said disease. 41. A method for delivering a therapeutic agent to a target tissue, comprising: administering the composition of claim 16 to a patient with said disease. 42. A method for delivering a therapeutic agent to a target tissue, comprising: administering the composition of claim 17 to a patient with said disease. 43. A method for delivering a therapeutic agent to a target tissue, comprising: administering the composition of claim 33 to a patient with said disease. 44. A method for delivering a therapeutic agent to a target tissue, comprising: administering the composition of claim 34 to a patient with said disease. 45. A method for delivering a therapeutic agent to a target tissue, comprising: administering the composition of claim 35 to a patient with said disease. 46. A method for delivering a therapeutic agent to a target tissue, comprising: administering the composition of claim 36 to a patient with said disease. 47. A method for delivering a therapeutic agent to a target tissue, comprising: administering the composition of claim 10 to a patient with said disease, nasally. 48. A method for delivering a therapeutic agent to an objective tissue, comprising: administering the composition of claim 10 to a patient with said disease, orally. 49. A method for delivering a therapeutic agent to a target tissue, comprising: administering the composition of claim 10 to a patient with said disease, through the skin of the patient. 50. A method for delivering a therapeutic agent to a target tissue, comprising: administering the composition of claim 10 to a patient with said disease, through the blood of the patient. 51. A method for identifying or purifying a human Zonulin receptor and ZOT protein from the CXTF phage of Vibrio cholerae, comprising: contacting a sample containing one or more proteins with an antibody that is enhanced against the amino acids SLIGKVDGTSHVTG as show in SEQ ID NO: 5, under conditions appropriate for the binding of antibody antigens; extracting proteins in the sample not linked to the antibody, characterized in that the proteins that bind to the antibody are identified as receptors for human zonulin and ZOT, or formers of an enriched preparation for said receptor. 52. The method of claim 51, further comprising the step of: immunizing a mammal with the preparation enriched for said receptor; collecting antibodies from the mammal to form a preparation of antibodies that bind to said receptor. 53. The method of claim 51, further characterized in that the sample is selected from the group consisting of intestinal cells, enterocytes, endothelial cells, macrophages, lymphocytes, and used thereof. 54. A method for identifying candidates for drugs for the treatment of a disease, comprising: contacting a first human protein identified by the SAM 1 1 antibody with a second protein selected from the group consisting of the ZOT protein of the CXTF phage of Vibrio cholerae, human Zonulin, and MyD88, characterized in that the contact is carried out separately in the presence and absence of a test substance; comparing the amount of the first protein in loop to the second protein, in the presence of the test substance with respect to the amount bound in the absence of the test substance; identifying a test substance as a drug candidate reduces the quality of the first protein linked to the second protein. 55. The method of claim 54, further characterized in that the second protein is ZOT. 56. The method of claim 54, further characterized in that the second protein is Zonulin. 57. The method of claim 54, further characterized in that the second protein is MyD88. 58. The method of claim 54, further characterized in that the second protein is the? G fragment of ZOT (I D SEQUENCE N °: 6). 59. A vaccine composition for inducing an immunological response, which contains: an immunogenic agent to induce an immune response; and the agonist polypeptide of claim 1. 60. A method for diagnosing an autoimmune disease in a patient, comprising: contacting a first body sample of the patient with an antibody boosted against the amino acids SLIG KVDGTSHVTG as shown in I D SEQUENCE No. 5; compare the amount of antibody bound by the first body sample, with an amount bound by a second body sample from a healthy control that does not have an autoimmune disease; identify an autoimmune disease if the first body sample binds more of the antibody than the second. 61 A method for treating a patient with increased expression of zonulin relative to a healthy control individual, comprising: administering to the patient an antibody enhanced against the amino acids SLIGKVDGTSHVTG as shown in SEQ ID NO: 5, by which the symptoms of the disease are relieved. 62. The method of claim 61, further characterized in that the disease is cancer. 63. The method of claim 61, further characterized in that the disease is autoimmune. 64. The method of claim 61, further characterized in that the disease is vascular. 65. The method of claim 61, further characterized in that the disease is a bacterial infection. 66. The method of claim 61, further characterized in that the disease is gastritis. 67. The method of claim 61, further characterized in that the disease is gastric cancer. 68. The method of claim 61, further characterized in that the disease is collagenous colitis. 69. The method of claim 61, further characterized in that the disease is inflammatory bowel disease. 70. The method of claim 61, further characterized in that the disease is celiac disease. 71. The method of claim 61, further characterized in that the disease is systemic lupus erythematosus. 72. The method of claim 61, further characterized in that the disease is food allergy. 73. The method of claim 61, further characterized in that the disease is asthma. 74. The method of claim 61, further characterized in that the disease is irritable bowel syndrome. 75. An antibody that is boosted against the amino acids SLIGKVDGTSHVTG as shown in SEQ ID NO: 5, binds to a protein expressed in CaCo2 cells that co-localize with a protein linkage by the synthetic inhibitor peptide FZ1 / 0 (as shown in SEQ ID NO: 3), and does not bind to human or rat cells expressing a human recombinant PAR-2, further characterized in that the antibody is not SAM11. 76. An antibody that binds to a protein expressed in CaCo2 cells that are co-localized with a protein linkage by the synthetic inhibitor peptide FZ1 / 0 (as shown in SEQ ID NO: 3), and not it binds to human or rat cells expressing a human recombinant PAR-2, further characterized in that the antibody is not SAM 1 1. 77. An antagonist polypeptide of a human zonulin receptor and ZOT protein of the CXTF phage of Vibrio cholerae, said agonist polypeptide contains a sequence selected from the group consisting of Xaa! Cys l ie Gly Arg Leu (ID SECU ENCE N °: 7), Phe Xaa2 He Gly Arg Leu (ID SEQUENCE N °: 8), Phe Cys Xaa3 Gly Arg Leu (ID SEQUENCE N °: 9), Phe Cys lie Xaa4 Arg Leu (SEQ ID NO: 10), Phe Cys lie Gly Xaa5 Leu (SEQ ID NO: 1 1), Phe Cys Me Gly Arg Xaa6 (SEQ ID NO: 12), further characterized in that said polypeptide has a shorter length to 100 amino acid residues, further characterized in that Xaai is selected from the group consisting of Ala, Val, Leu, Lie, Pro, Trp, Tyr, and Met; further characterized in that Xaa2 is selected from the group consisting of Gly, Ser, Thr, Tyr, Asn, and Gln; Xaa3 is selected from the group consisting of Ala, Val, Leu, Lie, Pro, Trp, and Met; Xaa is selected from the group consisting of Gly, Ser, Thr, Tyr, Asn, Ala, and Gln; Xaa5 is selected from the group consisting of Lys and His; Xaa6 is selected from the group consisting of Ala, Val, Leu, Lie, Pro, Trp, and Met. 78. The agonist polypeptide of claim 77, further characterized in that said polypeptide consists of six amino acid residues. 79. An agonist polypeptide of a human zonulin receptor and ZOT protein of the CXTF phage of Vibrio cholerae, said agonist polypeptide contains a sequence selected from the group consisting of: Xaa! Xaa2 lie Gly Arg Leu (ID SEQUENCE N °: 13), Xaa! Cys Xaa3 Gly Arg Leu (ID SEQUENCE N °: 14), Xaa! Cys He Xaa4 Arg Leu (ID SEQUENCE N °: 15), Xaa! Cys Be Gly Xaa5 Leu (ID SEQUENCE N °: 16), Xaa-, Cys He Gly Arg Xaa6 (ID SEQUENCE N °: 17), Phe Xaa2 Xaa3 Gly Arg Leu (ID SEQUENCE N °: 18), Phe Xaa2 He Xaa4 Arg Leu (ID SEQUENCE N °: 19), Phe Xaa2 He Gly Xaa5 Leu (ID SEQUENCE N °: 20), Phe Xaa2 lie Gly Arg Xaa6 (ID SEQUENCE N °: 21), Phe Cys Xaa3 Xaa4 Arg Leu (ID SEQUENCE N °: 22), Phe Cys Xaa3 Gly Xaa5 Leu (ID SEQUENCE N °: 23), Phe Cys Xaa3 Gly Arg Xaa6 (ID SEQUENCE N °: 24), Phe Cys lie Xaa4 Xaa5 Leu (ID SEQUENCE N °: 25) , Phe Cys He Xaa4 Arg Xaa6 (SEQ ID NO: 26), and Phe Cys lie Gly Xaa5 Xaa6 (SEQ ID NO: 27), further characterized in that said polypeptide has a length of less than 100 amino acid residues, further characterized by Xaai is selected from the group consisting of Ala, Val, Leu, Me, Pro, Trp, Tyr, and Met; further characterized in that Xaa2 is selected from the group consisting of Gly, Ser, Thr, Tyr, Asn, and Gln; Xaa3 is selected from the group consisting of Ala, Val, Leu, He, Pro, Trp, and Met; Xaa is selected from the group consisting of Gly, Ser, Thr, Tyr, Asn, Ala, and Gln; Xaa5 is selected from the group consisting of Lys and His; Xaa6 is selected from the group consisting of Ala, Val, Leu, He, Pro, Trp, and Met. 80. The agonist polypeptide of claim 79, further characterized in that said polypeptide consists of six amino acid residues.