WO2008154518A2

WO2008154518A2 - Materials and methods for modulating protective pathways in epithelial cells

Info

Publication number: WO2008154518A2
Application number: PCT/US2008/066368
Authority: WO
Inventors: Eugene B. Chang; Mikihiro Fujiya; Mark W. Musch
Original assignee: The University Of Chicago
Priority date: 2007-06-08
Filing date: 2008-06-09
Publication date: 2008-12-18
Also published as: WO2008154518A3

Abstract

The invention provides methods for identifying modulators of an activity of a charged oligopeptide transport molecule of a eukaryotic host cell useful in prophylactic and therapeutic treatments of microbial pathogens, such as virulent intestinal bacteria. In related aspects, the invention provides methods for modulating the activity of Heat Shock Protein 27, Akt, p38 MAP Kinase and/or Ragl.

Description

MATERIALS AND METHODS FOR MODULATING PROTECTIVE PATHWAYS

IN EPITHELIAL CELLS

[0001] This work was supported by the U.S. National Institutes of Health Grant Nos. NIH DK47722 and NIH DK42086.

Background

[0002] The enteric microbiota is a unique ecological niche where microorganisms live normally in the digestive tract in a balanced relationship with other species and the host. The relationship is complex and incompletely understood, often involving bidirectional signals and interactions that not only influence the behavior of microflora, but also host responses essential to the maintenance of intestinal homeostasis (Rakoff-Nahoum et al., 2006). Prime examples of the latter are the host toll-like receptors (TLR) that continuously monitor luminal microbial pattern molecules that are essential for regulation of innate immune responses and epithelial cytoprotection (Pasare and Medzhitov, 2005). Among bacteria, population dynamics are influenced by the secretion of numerous metabolites and effecter molecules that promote species stability, adaptation, and survival within this environment.

[0003] Quorum sensing is one of the most elegant of these processes, providing bacteria with the ability to communicate and change behavior of the same or other species in response to conditions and perturbations of the environment (Bassler and Losick, 2006 and Camilli and Bassler, 2006). Both gram-positive and gram-negative organisms utilize quorum sensing molecules (QSMs) which, in the former, are usually bioactive peptides, whereas in the latter, QSMs include non-peptide molecules such as acyl-homoserine lactone (Bassler and Losick, 2006). QSMs are essential for determining and maintaining microbial composition, as well as for allowing colonic microbes to adapt to perturbations in their environment. They are also critical for biofilm formation, which may be essential in determining how and which microbes can establish "residency" at the mucosal surface. They are therefore critical signals that can be sensed by the host, allowing the host to monitor what is happening in the microbial ecosystem. Because they play a role in determining the diversity and composition of the enteric microbiome, the profile of QSMs at any given time reflects the status or impending changes in the microbiota. Whether eukaryote cells have the ability to detect the complex array of QSMs is unexplored, but such an ability would allow the host to appropriately respond to physiological or pathophysiological perturbations in the microbiota.

[0004] Bacteria use quorum- sen sing molecules (QSMs) to communicate and coordinate population behavior in response to environmental changes, nutrient availability, and resisting other competing or pathogenic microorganisms (Bassler and Losick, 2006 and Camilli and Bassler, 2006). Similarly, pathogenic bacteria use quorum- sensing to co-ordinate their virulence, allowing them to evade immune detection and successfully establish infection.

[0005] Given the role of QSMs in coordinating expression of the virulence phenotype in the intestinal flora, it would be reasonable for host organisms to have developed sensors as a mechanism for detecting and preparing to face the threat of virulence. First-line host cells situated appropriately to detect QSMs elaborated by intestinal flora would be the intestinal epithelia lining the digestive tract. A variety of cell- surface receptors and transporters have been associated with epithelia, including intestinal epithelia. One such class of molecules is the class of transporters of charged peptides. This class of molecules includes OCTNl and OCTN2, which are cationic oligopeptide transporters, as well as MDR-I.

[0006] OCTNl and OCTN2 are members of the organic anion/cation transporter family SLC22 which are expressed in human tissues including kidney, heart, prostate, skeletal muscle as well as small and large intestine. A polynucleotide sequence encoding human OCTN2 is provided in SEQ ID NO:4; the encoded polypeptide has the amino acid sequence of SEQ ID NO:5. Analogously, a polynucleotide sequence encoding human OCTNl is provided in SEQ ID NO: 6; the encoded polypeptide has the amino acid sequence of SEQ ID NOS:7 and 8. Polymorphisms of the OCTNl- andOCTN2-encoding genes, SLC22A4 and SLC22A5, respectively, are within the IBD5 susceptibility locus of Crohn's disease (Peltekova et al., 2004, Noble et al., 2005, Vermeire et al., 2005, Walter et al., 2006 and Leung et al., 2006), although a disease-causing role for these genes has not been established (Trinth et al., 2005). OCTNl and OCTN2 were originally cloned from a rat kidney cDNA library; however, one or both may be expressed in many tissues due to their potential importance in transporting pivotal cell substrates. OCTNl and OCTN2 transport a number of endogenous substrates (e.g., L-carnitine, choline) as well as xenobiotics (e.g., tetraethyl ammonium, quinidine, cimetidine) in either a Na⁺-dependent manner (L-carnitine) or a pH- dependent manner (tetraethyl ammonium). A naturally occurring mutation (autosomal recessive) in the OCTN2 gene in mice resulted in inborn errors of metabolism. The most pronounced changes were metabolic abnormalities that included cardiac or skeletal muscle pathologies, perhaps due to systemic carnitine deficiency. Carnitine is required for mitochondrial β-oxidation of fatty acids and would therefore be anticipated to be an important solute to absorb. OCTN2 may not be the only carrier for carnitine, however. OCTN2 has affinity for carnitine with a Km of approximately 4.3 μM; however, OCTNl may also transport carnitine and has a lower affinity with a Km of approximately 571 μM. Whether other transporters may transport carnitine in intestinal epithelia, where carnitine is absorbed across the apical membrane from the luminal dietary fluids, is presently unknown. Indeed, the regulation of expression of each of the OCTN transporters, including whether expression varies when abnormal conditions are found (e.g., inflammation), has not been adequately investigated.

[0007] Intestinal inflammation is an exemplary disorder that may be induced, or exacerbated, by intestinal pathogens. Interferon-gamma (IFN-γ), a pro-inflammatory cytokine, alters the intestinal epithelial cell expression of a number of proteins such as adhesion molecules, MHC molecules, tight-junction-related proteins, and a number of transport proteins, including sodium/hydrogen exchangers, a sodium-potassium-chloride cotransporter, CFTR, NKCCl, sodium-dependent glucose transporter- 1 and MDR-I (human polynucleotide sequence provided as SEQ ID NO: 9 and encoded amino acid sequence as SEQ ID NOS: 10 and 11). Whether and how changes in the expression levels of these proteins may relate to the pathogenesis of inflammatory bowel diseases is currently not known. TNF-α, another pro-inflammatory cytokine, mediates luminal fluid accumulation and reduction of Na⁺/K⁺ ATPase activity induced by T-cell activation or changes the function of several transporters such as the basal ion regulated transporter (IREG-I) and taurine transporter in Caco-2 cells, thereby affecting the permeability of tight junctions in intestinal epithelia.

[0008] Other receptors, such as the TLR and NOD receptors or molecules, are also known and could be candidates for involvement in host-microflora interactions. The TLR and NOD receptors or molecules are critically important for recognition of microbial-derived cellular or cell wall-derived ligands that are indicative of potential or impending threats by pathogens. As a consequence, innate immune cells can respond rapidly and appropriately to many types of pathogens. Although the immune response plays an important role in defending against pathogenic challenges, the identities and roles of host-cell receptors and/or transporters active in host cells directly interfacing with potential pathogens, such as the intestinal flora, is of great interest as a potential key to host-pathogen interactions leading initially to cell-cell barrier dysfunction that precedes systemic interplay in the form of, e.g., sepsis and the defensive involvement of the immune system. The involvement of the immune system, in turn, implicates the various immune processes and attendant signaling events and pathways in adjusting host responses to microbes, including pathogens. [0009] Thus, a need continues to exist in the art for identifying and exploiting host cell mechanisms for monitoring microbe behavior, such as the elaboration of the virulence phenotype in intestinal pathogens. The results of such identification and exploitation promise new therapeutics for derailing development of the pathogenic phenotype by manipulating pathogenic signaling, as well as new therapeutics for combating the serious consequences of pathogenic attack, such as cell-cell barrier dysfunction of intestinal epithelia and other host cells forming front-line barriers to pathogens. The role of host cell receptors and/or transporters in monitoring the condition of commensal bacteria is of great interest as a potential key to understanding interactions between the host and both beneficial and pathogenic bacteria.

Summary

[0010] The invention disclosed herein satisfies at least one of the aforementioned needs in the art by providing methods for identifying modulators of the activity of eukaryotic transport molecules, such as the cationic oligopeptide transport proteins expressed on the cell surface of intestinal epithelia. An exemplary cationic oligopeptide transport protein is OCTN2; other such transport proteins include OCTNl and MDR-I. Also provided are prophylactic and therapeutic treatment methods to affect the activity of at least one of the group of Heat Shock Proteins, Akt and p38 MAP Kinase, thereby conferring a detectable level of protection to a cell, such as a eukaryotic intestinal epithelial cell, against stress, e.g., inflammatory stress, the stress of microbial virulence.

[0011] One aspect of the invention is drawn to a method for identifying a modulator of Heat Shock Protein 27 activity comprising: (a) contacting a cell comprising a charged oligopeptide transport molecule with a candidate modulator transportable by the molecule (i.e., transportable via the mechanism involving the charged oligopeptide transport molecule); and (b) measuring an activity of Heat Shock Protein 27 of the cell in the presence and absence of the candidate modulator, wherein a different level of Heat Shock Protein 27 activity in the presence and absence of the candidate modulator identifies the candidate modulator as a modulator of Heat Shock Protein 27 activity. The method may further comprise adding a peptide comprising amino acid sequence H₃N-ERGMT-CO₂H (SEQ ID NO:2), such as a Competence and Sporulation Factor (CSF, e.g., a B. subtilis CSF). Exemplifying a CSF precursor is the B. subtilis CSF precursor, which is a peptide of 40 amino acids (SEQ ID NO:1).

[0012] Yet another aspect of the invention is drawn to a method for identifying a modulator of Akt activity comprising: (a) contacting a cell comprising a charged oligopeptide transport molecule with a candidate modulator transportable by the molecule; and (b) measuring an activity of Akt of the cell in the presence and absence of the candidate modulator, wherein a different level of Akt activity in the presence and absence of the candidate modulator identifies the candidate modulator as a modulator of Akt activity. The method may further comprise adding a peptide comprising amino acid sequence H₃N- ERGMT-CO₂H (SEQ ID NO:2).

[0013] Another aspect of the invention is directed to a method for identifying a modulator of p38 MAP Kinase activity comprising: (a) contacting a cell comprising a charged oligopeptide transport molecule with a candidate modulator transportable by the molecule; and (b) measuring an activity of p38 MAP Kinase of the cell in the presence and absence of the candidate modulator, wherein a different level of p38 MAP Kinase activity in the presence and absence of the candidate modulator identifies the candidate modulator as a modulator of p38 MAP Kinase activity. The method may further comprise adding a peptide comprising amino acid sequence H₃N-ERGMT-CO₂H (SEQ ID NO:2).

[0014] In a related aspect, the invention provides a method of modulating the activity of a protein selected from the group consisting of Heat Shock Protein 27, Akt and p38 MAP Kinase comprising delivering an effective amount of Competence and Sporulation Factor to an epithelial cell.

[0015] Another aspect of the invention provides a method for identifying a modulator of a charged oligopeptide transport molecule comprising: (a) contacting a charged oligopeptide transport molecule with a candidate modulator; and (b) measuring an activity of the oligopeptide transport molecule in the presence and absence of the candidate modulator, wherein a different level of oligopeptide transport activity in the presence and absence of the candidate modulator identifies the candidate modulator as a modulator of the oligopeptide transport molecule. Suitable oligopeptide transport molecules include organic cationic oligopeptide transport molecules, such as the OCTN2, OCTNl, or MDR-I polypeptide molecules. In some embodiments, the modulator inhibits the transport activity of the oligopeptide transport molecule. In some embodiments, a eukaryotic cell comprises the oligopeptide transport molecule. Suitable eukaryotic cells include epithelial cells such as mammalian (particularly, human) epithelial cells, as exemplified by an intestinal epithelial cell.

[0016] In a related aspect of the invention, the method described above further comprises adding a peptide comprising amino acid sequence H₃N-ERGMT-CO₂H (SEQ ID NO:2). An exemplary peptide is a B. subtilis Competence and Sporulation Factor.

[0017] Another aspect of the invention is a method for identifying a modulator of Ragl activity comprising: (a) contacting Ragl (Recombination Activating Gene-1) with a candidate modulator; and (b) measuring an activity of Ragl in the cell in the presence and absence of the candidate modulator, wherein a different level of Ragl activity in the presence and absence of the candidate modulator identifies the candidate modulator as a modulator of Ragl activity. In some embodiments, Ragl is within a eukaryotic cell and the method is performed with the cell containing Ragl.

[0018] Yet another aspect of the invention is a method of determining the condition of intestinal flora comprising (a) obtaining a sample of the intestinal contents (e.g., a stool sample); and (b) measuring the level of a cationic peptide bacterial quorum sensing molecule in the sample, thereby obtaining a measure of the condition of intestinal flora. In some embodiments, the cationic peptide bacterial quorum sensing molecule is selected from the group consisting of a peptide comprising the sequence set forth in SEQ ID NO:2 (ERGMT); Plantaricin A and cPD-1. Any known method of measuring the level of a peptide may be used including, e.g., an immunoassay specifically measuring the cationic peptide bacterial quorum sensing molecule.

[0019] A related aspect of the invention is the use of a binding partner specific for a cationic peptide bacterial quorum sensing molecule in the preparation of a medicament for determining the condition of intestinal flora. Any binding partner known in the art or capable of being generated using no more than routine skill is contemplated, including but not limited to any form of naturally occurring or recombinantly generated polyclonal or monoclonal antibody, or fragment, fusion, or variant thereof, as well as the complementing binding partner to a quorum sensing molecule wherein the pair constitute a ligand-receptor pair, a lectin-carbohydrate pair, and any other form of specific binding partners known in the art.

[0020] Still another aspect of the invention is a method of preventing or treating an intestinal disorder (e.g., an inflammatory intestinal disorder, such as Crohn's disease or ulcerative colitis, a pathogenic infection, antibiotic-associated colitis, radiation enteritis, intestinal epithelial cell apoptosis, aberrant cell cycle regulation of an intestinal epithelial cell) comprising administering a therapeutically effective amount of a protein therapeutic selected from the group consisting of Plantaricin A, cPD-1, Competence and Sporulation Factor and variants thereof, wherein each variant is a peptide of at least five amino acids in length and wherein each of the variants comprises an adjacent pair of oppositely charged amino acids at a pH value within the range of pH 5-7.5, and preferably within the range of pH 5-6. The oppositely charged amino acids include one basic and one acidic amino acid at the relevant pH, in either order. Preferably, the oppositely charged pair of amino acids will be at the N-terminus of the five-amino-acid peptide. In some embodiments, the protein therapeutic is Competence and Sporulation Factor, e.g., CSF from Bacillus subtilis. In this aspect and in other aspects, the invention provides methods and uses to prevent or treat intestinal mucosa injuries or insults and provide for improved or maintained intestinal health. A related aspect of the invention is the use of a protein therapeutic selected from the group consisting of Plantaricin A, cPD-1, Competence and Sporulation Factor and variants thereof in the preparation of a medicament for preventing or treating an inflammatory intestinal disorder, wherein each of said variants is a peptide of at least five amino acids in length and wherein each said variant comprises an adjacent pair of oppositely charged amino acids within the range of pH 5-7.5, preferably pH 5-6.

[0021] The invention also provides a method of preventing or treating an intestinal disorder (e.g., an inflammatory intestinal disorder, such as Crohn's disease or ulcerative colitis, a pathogenic infection, antibiotic-associated colitis, radiation enteritis, intestinal epithelial cell apoptosis, aberrant cell cycle regulation of an intestinal epithelial cell) comprising delivering a DNA comprising an expressible coding region for a cationic peptide transporter to an intestinal epithelial cell under conditions suitable for uptake of the DNA by the cell. In an embodiment of the method, the DNA encodes OCTN2, OCTNl or MDR-I. A related aspect of the invention is the use of a DNA comprising an expressible coding region for a cationic peptide transporter in the preparation of a medicament for preventing or treating an intestinal disorder.

[0022] In another aspect, the invention provides a method of preventing or treating an intestinal disorder (e.g., an inflammatory intestinal disorder, a pathogenic infection, antibiotic-associated colitis, radiation enteritis, intestinal epithelial cell apoptosis, aberrant cell cycle regulation of an intestinal epithelial cell) comprising delivering an RNAi or an siRNA of a coding region selected from the group consisting of a cationic peptide transporter coding region and Ragl to an intestinal epithelial cell under conditions suitable for uptake of the RNAi or siRNA by the cell. In some embodiments, the RNAi or siRNA is complementary to an OCTN2 coding region. In a related aspect, the invention provides for the use of an RNAi or an siRNA of a coding region selected from the group consisting of a cationic peptide transporter coding region and Ragl in the preparation of a medicament for preventing or treating an intestinal disorder.

[0023] Further, the invention provides a method of preventing or treating an intestinal disorder (e.g., an inflammatory intestinal disorder, such as Crohn's disease or ulcerative colitis, a pathogenic infection, antibiotic-associated colitis, radiation enteritis, intestinal epithelial cell apoptosis, aberrant cell cycle regulation of an intestinal epithelial cell) comprising delivering an intestinal microorganism to the intestine, wherein the intestinal microorganism secretes a cationic peptide bacterial quorum sensing molecule (QSM). The secreted QSM may be homologous or heterologous and may be expressed at natural levels or may be engineered to provide a non-natural expression pattern of a QSM, such as by expressing an elevated level thereof or by constitutively expressing the QSM, or both. Secretion of the QSM may also be accomplished by natural mechanisms or may be engineered using techniques known in the art. In some embodiments, the cationic peptide bacterial quorum sensing molecule is selected from the group consisting of a peptide comprising the sequence set forth in SEQ ID NO:2 (ERGMT), Plantaricin A (SEQ ID NO: 12) and cPD-1 (SEQ ID NO: 13). A related aspect of the invention provides the use of an intestinal microorganism in the preparation of a medicament for preventing or treating an intestinal disorder, wherein said intestinal microorganism secretes a cationic peptide bacterial quorum sensing molecule.

[0024] In the methods of preventing or treating intestinal disorders, the invention comprehends an intestinal disorder selected from the group consisting of inflammatory disorders of the intestine, pathogenic infection, antibiotic-associated colitis, radiation enteritis, intestinal epithelial cell apoptosis and aberrant cell cycle regulation of an intestinal epithelial cell. Exemplary intestinal disorders are Crohn's disease and ulcerative colitis.

[0025] Other features and advantages of the invention will be better understood by reference to the brief description of the drawing and the detailed description of the invention that follow.

Brief Description of the Drawing

[0026] Figure 1. Bioactive agents in conditioned media from representative Gram- positive bacteria, including the quorum- sensing molecule CSF of Bacillus subtilis, induce heat shock protein 27 (Hsp27) in colonic Caco2bbe cells. A) Conditioned media from most gram-positive (left 5 bars), but not gram-negative (right 3 bars) bacterial species significantly induced Hsp27 protein expression in Caco2bbe cells. Densitometry results are expressed as a percent of the Hsp27 response induced by heat shock in paired control cells. B) Secreted factors in B. subtilis (JH 642, wild type) conditioned media induced Hsp27. Densitometry mean + SEM of Western blots are normalized to unstimulated control values (arbitrarily set at 100 units). No changes in the protein expression of the heat shock cognate, Hsc70, were observed in any of the experimental conditions, including heat shock (HS). C) Bioactive factors from B. subtilis (JH 642, wild type) that induced Hsp27 (shown by Western blot) are less than 3kD (top set of panels), heat-stable, and pepsin-sensitive (lower set of panels). Filtrate and retentate were prepared by passing CM through a 3 kD Centricon filter. D) CM from wild-type B. subtilis JH642, but not CSF-deficient B. subtilis Rsml21 (delta CSF), induced Hsp27 (Western blot). E) CSF (100 nM) also stimulated phosphorylation of Akt and p38 MAPK (shown in Western blots), two additional survival pathways of intestinal epithelial cells. Responses to heat shock (HS) and TNF-alpha (100 ng/ml) stimulation are shown as positive controls. Heat shock cognate, Hsc70, was used as a loading and experimental control. "Con." indicates control cells that were not treated with peptides. Heat shock (HS) control samples are shown, obtained from cells two hours after transient exposure to 41.5⁰C x 23 minutes. * p < 0.05 compared with control at the same time point by analysis of variance.

[0027] Figure 2. A) Caco2bbe uptake of ¹⁴C-labeled CSF (competence and sporulation factor) is enhanced in OCTN2-overexpressed cells and less in OCTN2 siRNA-treated cells (left panel). *p<0.05 compared to mock transfected control cells. B) Caco2bbe cells take up FIT C-labeled CSF, which distributes in the cytoplasm (30 minutes incubation), an effect competed by 10 mM L-carnitine (lower panels). C) CSF-induced Hsp27 (shown by Western blot) is blocked in cells treated with OCTN2-siRNA (without effect on Hsc70 or heat shock (HS) response). Control (Con) cells were not stimulated with CSF. HS samples obtained from cells two hours after transient exposure to 41.5⁰C x 23 minutes. D) Effects of CM of gram-positive (left-most 5 bars) and gram-negative bacteria (3 right bars) on OCTN2 transport, assessed by competition of [³H] -L-carnitine uptake by confluent human colonic epithelial Caco2bbe monolayers. L-carnitine uptake was used to functionally characterize OCTN2 transport activity (Tamai et al., 2000 and Ohashi et al., 2001). Values represent the amount of [³H] -L-carnitine uptake reduced by bacterial CM. * p < 0.05 compared with control at the same time point by analysis of variance.

[0028] Figure 3. Conditioned media (CM) of B. subtilis and quorum-sensing CSF protect intestinal epithelial cells against oxidant- induced cell death and loss of barrier function. A) Pretreatment of Caco2bbe monolayers with wild-type JH642 CM, but not with Rsml21 CM, protected cells against oxidant-induced injury (monochloramine, 0.3 mM). An unrelated quorum- sensing molecule from Enterococcus (cPDl) and a scrambled pentapeptide of CSF (EMTRG; SEQ ID NO:3) had no effects. HBS: HEPES buffered saline. B) Silencing of OCTN2 with siRNA in Caco2 cells inhibited the protective effects of CSF (third set from left) against oxidant-induced stress, as measured by ⁵¹Cr release. Clear bars indicate control cells (no CSF treatment), whereas solid bars indicate CSF-treated cells. Cells treated with a nonsense siRNA still showed protection with CSF (second set from left), indicating specificity of siRNA treatment. In the right panel, inhibitors of the Akt (Ly294002) and p38 MAPK (SB203580) pathways, either alone or together, are shown to not significantly inhibit the CSF protective action against oxidant stress. C) When the agents described above were tested to determine their ability to limit oxidant (monochloramine)-induced increases in barrier function, only CSF and CM of B. subtilis mitigated induced increases in ³H-mannitol flux, a measure of barrier function. ++p<0.05 by ANOVA compared with untreated HBS+ control (n=5 for all). D, E) Silencing of Hsp27 resulted in nearly complete reversal of the CSF- and B. subtilis CM-induced protection of cell viability (Figure 3C) and epithelial barrier function (Figure 3D) against oxidant-induced stress. siRNA to murine Hsp25 (mHsp25), having a polynucleotide sequence unrelated to hHsp27, had no effects, establishing the specificity of Hsp27 silencing. * p≤O.Ol compared with corresponding responses in the absence of CSF or B. subtilis CM, n=4.

[0029] Figure 4. CSF induced Hsp25 and Hsp70 expression and inhibited oxidant- induced alterations in ex vivo intestinal preparations, effects that were blocked by the presence of L-carnitine. A) A Western blot revealing that CSF (10OnM), but not the scrambled peptide (EMTRG; SEQ ID NO:3), induced both Hsp25 and Hsp70 in the mucosa of ex vivo ligated loops of murine small and large intestine (colon, left). B) A Western blot showing that the induction of Hsp25 and Hsp70 by CSF was inhibited by L-carnitine in the small intestine (1OmM, right). "Con." and "Scr" indicate tissues that were not treated and were treated with the scrambled pentapeptide EMTRG (SEQ ID NO:3), respectively. C) CSF, but not the scrambled peptide, protected intestinal barrier function against oxidant- induced stress (NH₂Cl, 0.3 mM). D) CSF-protection of oxidant-induced loss of barrier function (indicated by high mannitol flux) was reversed in the presence of L-carnitine, indicating an effect requiring OCTN2 transport of CSF. No treatment (clear bars) indicates oxidant effects in the absence of either CSF or the scrambled peptide. Permeability was assessed by passive ³H-mannitol flux, calculated by subtracting the value of NH₂Cl-free- from that of NH₂Cl-treated samples. * p≤O.Ol compared with no treatment, n=3.

[0030] Figure 5. OCTN2 and Hsp27 were co-expressed in colonic epithelia. OCTN2 was primarily expressed by surface epithelial cells of the colon (top panel, brown staining) that were in direct contact with the luminal contents and microbes and which exhibited sustained expression of microbial-induced heat shock proteins, Hsp27 (shown, bottom panel) and Hsp70. Hsp27 is the human homolog of murine Hsp25. Higher power magnifications are shown in the insets.

[0031] Figure 6. Competence and Sporulation Factor (CSF) of B. subtilis induced a dose- related increase in Hsp27 expression. CSF induced Hsp 27 in a concentration-dependent manner within the physiological range for CSF (10-100 nM). The bands shown are from Western blots.

[0032] Figure 7. Substances secreted by B. subtilis are selectively taken up by OCTN2- expressing human colonic Caco2bbe cells. Validation of OCTN2 expression and activity in OCTN2 siRNA-transfected cells. A) Differentiated Caco2bbe cells express OCTN2 protein and mRNA, but minimal OCTNl protein or mRNA. The averaged mRNA abundance of OCTNl, OCTN2 and hPepTl were determined by real-time PCR using the comparative threshold cycle method normalized to GAPDH expression. Rabbit polyclonal anti-mouse OCTNl or OCTN2 antisera (Alpha Diagnostic International, San Antonio, TX) were used as primary antibodies for Western blotting (right panel). B) Western blots showing specificity and effectiveness of OCTN2 silencing and overexpression in Caco2bbe colon cells. Villin and hPepTl protein expression were not affected in either case. C) Silencing and overexpression of OCTN2 in Caco2bbe cells results in inhibition and augmentation of endogenous OCTN2 activity (Na-dependent L-carnitine uptake), respectively. *p<0.05 compared to mock-transfected cells (n=3).

[0033] Figure 8. Quorum-sensing CSF competes with L-carnitine uptake by OCTN2 overexpressing Caco2bbe cells. A) OCTN2 protein expression was increased in HSWP cells transfected with CMV-driven OCTN2 cDNA. Note the minimal basal expression of OCTN2 in these cells (mock). The bands shown are from Western blots. B) Na⁺-dependent L- carnitine uptake is significantly increased in OCTN2-transfected (overexpressing) human HSWP cells. In addition, ¹⁴C-CSF uptake by OCTN2-overexpressing HSWP cells was also observed. *p<0.05 compared to mock transfected cells (n=3). C) The scrambled pentapeptide (EMTRG; SEQ ID NO:3) did not affect Na⁺-dependent L-carnitine uptake in Caco2bbe cells, while CSF inhibited the uptake. [³H] L-carnitine uptake was measured over 3 minutes in 10-14-day-post-confluent monolayers in flux buffer containing CSF or scrambled pentapeptide.

[0034] Figure 9. OCTN2 was required for CSF-induced protection of Caco2 cells against oxidant stress. Although the p38 MAPK and Akt pathways were activated by CSF, they appeared to play only a minor role in protection against oxidant stress. LY294002 did not affect CSF protection against oxidant-induced increases in mucosal permeability (assessed by ³H-mannitol flux) in ex vivo small intestinal loops. Additionally, no inhibition of CSF action by SB203580 was observed. *p<0.05 by ANOVA compared to no treatment control (n=5).

[0035] Figure 10. Blot showing CSF (100 nM, left) induced expression of Hsp27 and phosphorylated Akt and p38 MAPK. Inhibitors of the p38 MAPK and Akt pathways (SB 203580 and LY294002) had no effect on CSF-induced Hsp27.

[0036] Figure 11. OCTN2 was expressed in epithelial brush border of the small and large intestine in control mice (A), and Ragl^"7" mouse intestine expressed less OCTN2 as measured by immunohistochemistry (B) and by Western blotting (C). Rabbit anti-mouse OCTN2 polyclonal antibody was used for immunohistochemistry of small and large intestine, and for Western blots of OCTN2. Images shown are representative of 3 separate experiments. Densitometry was performed using NIH Image (Image J), setting the untreated control to 100% at each time point. * p < 0.05 compared with control at the same time point by analysis of variance.

[0037] Figure 12. IFN-γ and TNF-α increased sodium-dependent L-carnitine uptake. Caco-2/bbe cells were treated with OCTN2 siRNA for 24 hours (A) or with the indicated cytokine for 48 hours, all at 50ng/ml. Total and sodium-dependent L-carnitine uptakes were measured by using a [³H] L-carnitine uptake assay. Data are mean+SE for 4 experiments. * p < 0.05 compared with control by analysis of variance using a Bonferroni Correction.

[0038] Figure 13. Both TNF-α and IFN-γ increase L-carnitine uptake in a time- dependent manner. Caco-2/bbe cells were treated with either TNF-α (50ng/ml) or IFN-γ (50ng/ml) for designated times. L-carnitine uptake was measured by [³H] L-carnitine uptake (1 μCi/ml). Data are mean+SE for 4 experiments. * p< 0.05 compared with control at the same time point of analysis of variance.

[0039] Figure 14. IFN-γ increased OCTN2 protein expression in a time- and concentration-dependent manner. Caco-2/bbe cells were treated with IFN-γ (50ng/ml) for the designated times (A) or Caco-2/bbe cells were treated with various concentrations of IFN-γ for a constant time of 48 hours (B). OCTN2 expression was determined by immunoblotting using both total cell homogenates and, separately, extracts enriched for apical membrane proteins by cell-surface protein biotinylation and streptavidin selection. Images shown are representative of 4 separate experiments. Densitometry was performed using NIH image (Image J), setting the untreated control to 100% at each time point. * p< 0.05 compared with control at the same time point of analysis of variance.

[0040] Figure 15. TNF-α increased apical surface, but not total, OCTN2 expression in a time- and concentration-dependent manner. Caco-2/bbe cells were treated with TNF-α (50ng/ml) for designated times (A) or the cells were separately treated with various concentrations of IFN-γfor a constant 48 hours (B). OCTN2 expression was determined by immunoblotting using both total cell homogenates and, separately, extracts enriched for apical membrane proteins by cell-surface protein biotinylation and streptavidin selection. Images shown are representative of 4 separate experiments. Densitometry was performed using NIH image (Image J), setting the untreated control to 100% at each time point. * p< 0.05 compared with control at the same time point of analysis of variance.

[0041] Figure 16. IFN-γ, but not TNF-α, increased OCTN2 mRNA expression in a concentration-dependent manner. cDNA was prepared from total cellular RNA extractions of Caco-2 cells treated with either IFN-γ or TNF-α for 24 hours. cDNA was also prepared from RNA extracted from untreated control Caco-2 cells. OCTN2 mRNA levels were quantitated by real-time PCR. The averaged OCTN2 mRNA expression levels were normalized to GAPDH expression and calculated using the comparative threshold cycle method. Data shown are mean+SE for 3 experiments, and triplicate PCR reactions were performed for each individual test condition. * p< 0.05 compared with control at the same time point of analysis of variance.

[0042] Figure 17. IFN-γ increased sodium-dependent L-carnitine uptake in the proximal colon of mice while TNF-α increased uptake in the small intestine (A; Jejunum, B; Ileum, C; Proximal colon, D; Distal colon). The anti-TNF-α antibody XT 22 decreased sodium- dependent L-carnitine uptake in control mice and negated the effects of IFN-γ and TNF-α on the uptake. Mice were treated with IFN-γ (100 ng) or TNF-α (2 μg) for 48 hours. XT 22 decreased sodium-dependent L-carnitine uptake in all parts of the intestine, even when administered in effective combination with either IFN-γ or TNF-α. Total and sodium- dependent L-carnitine uptakes were measured by [³H] L-carnitine uptake assays. Data are mean+SE for 4 experiments. * p < 0.05 compared with control at the same time point by analysis of variance.

[0043] Figure 18. IFN-γ, but not TNF-α, increased OCTN2 protein expression in the proximal colon of mice treated with IFN-γ (100 ng) or TNF-α (2 μg) for 48 hours. Small and large intestines of mice with or without IFN-γ and/or TNF-α treatment were removed and their luminal surfaces were gently shaved off for Western blotting. (The term "Ilium" recited in the Figure is a reference to the ileum.) Images shown are representative of 4 separate experiments. Densitometry was performed using NIH image (Image J), setting the untreated control to 100% at each time point. * p< 0.05 compared with control at the same time point of analysis of variance.

[0044] Figure 19. IFN-γ, but not TNF-α, increased the mRNA expression of OCTN2 in the proximal colon. cDNA was prepared from total RNA extracted from mouse intestines treated with either IFN-γ or TNF-α for 48 hours. cDNA was also prepared from total RNA extracts of control mouse intestines. OCTN2 mRNA levels were quantitated by real-time PCR. The averaged OCTN2 mRNA expression levels were normalized to GAPDH expression and calculated using the comparative threshold cycle method. Data shown are mean+SE for 4 experiments, and triplicate PCR reactions were performed for each individual test condition. * p< 0.05 compared with control at the same time point of analysis of variance.

[0045] Figure 20. OCTN2 expression was increased in the colon of patients with active Crohn's disease. Specimens were taken from the proximal colon of 3 healthy volunteers and from 6 Crohn's disease patients using a punched biopsy during colonoscopy. Biopsy specimens were processed for Western blotting (A). Images shown are representative of 2 separate experiments. Densitometry was performed using NIH image, setting one healthy volunteer to 1 at each time point (B).

[0046] Figure 21. OCTN2 was strongly expressed in the colonocytes of patients with active Crohn's disease. Immuno staining was performed for colonic specimens taken from 3 healthy volunteers (A) and 6 Crohn's disease patients (B). Rabbit anti-mouse OCTN2 polyclonal antibody was used for immunohistochemistry. Images shown represented 1 healthy volunteer and 1 active Crohn's disease patient.

Detailed Description

[0047] The invention provides materials and methods for identifying a modulator of a charged oligopeptide transport molecule, e.g., OCTN2, OCTNl or MDR-I, as well as methods for identifying modulators of an activity of at least one of the following proteins: Heat Shock Protein 27, Akt, and p38 MAP Kinase. Exemplifying the materials and methods of the invention are materials and methods for identifying a modulator of the transport activity of a charged oligopeptide transport molecule. The invention also provides a therapeutic method for preventing or treating a stressed epithelial cell comprising delivering a prophylactically or therapeutically effective amount of B. subtilis Competence and Sporulation Factor. An exemplary cell is a mammalian intestinal epithelial cell.

[0048] The quorum- sensing pentapeptide, competence and sporulation factor (CSF), of a Gram-positive bacterium Bacillus subtilis, activates key survival pathways, including p38 MAP kinase and protein kinase B (Akt), in intestinal epithelial cells and also induces cytoprotective heat shock proteins (Hsps), the latter preventing oxidant-induced intestinal epithelial cell injury and loss of cell-cell barrier function. These effects of CSF depend on its uptake by an apical membrane organic cation transporter-2 (OCTN2). OCTN2-transported CSF, therefore, serves as an example of a unique mechanism of host-bacterial interactions allowing the host to monitor changes in colonic flora behavior or composition.

[0049] As will be shown in the examples, a QSM pentapeptide, having the amino acid sequence ERGMT (SEQ ID NO:2), also known as Competence and Sporulation Factor (CSF) from Bacillus subtilis, is transported into mammalian intestinal epithelia through a novel cell membrane transporter, organic cation transporter isotype 2 (OCTN2). Once taken up by intestinal epithelial cells, CSF activates key survival pathways, including p38 MAP kinase and protein kinase B (Akt), and CSF also induces cytoprotective heat shock proteins (Hsps), which prevent oxidant-induced intestinal epithelial cell injury and loss of barrier function. The host monitoring of microbiota behavior is exemplified herein as a host response to OCTN2-mediated uptake of CSF. OCTN2, however, as an organic cationic peptide transporter, was also determined to be involved in the eukaryotic cell uptake of other bacterial molecules, such as the bacterial pheromone Plantaricin A (PInA; precursor sequence Of H₃N-MKIQIKGMKQ LSNKEMQKIV GGKSSAYSLQ MGATAIKQVK KLFKKWGW- CO₂H; SEQ ID NO: 10), a bacteriocin-like peptide existing as three cationic peptides of 22, 23 and 26 amino acids in length. Anderssen et al., Appl. Environ. Microbiol. 64:2269-2272 (1998). The 26-amino-acid mature PInA has the sequence of amino acids 23-48 of SEQ ID NO: 10. PInA is secreted by, e.g., Lactobacillus GG. In addition, OCTN2 was determined to transport other bacterial sex pheromones, such as cPDl (H₃N-FLVMFLSG-CO₂H; SEQ ID NO: 11) produced by, e.g., Enterococcus faecalis. Suzuki et al., Science 226:849-850 (1984). The cPDl pheromone is involved in signaling pathways controlling conjugative transfer of bacteriocin-encoding DNAs. Nakayama et al., J. Bacterid. 180:449-456 (1998). Each of the cationic peptides transported by OCTN2, e.g., CSF, PInA and cPDl, are expected to participate with eukaryotic organic cationic peptide transporters such as 0CTN2 in host- microbe interactions providing a basis for host monitoring of microbiota behavior.

[0050] The data disclosed herein establish a physiological role for OCTN2-transported bacterial cationic peptide (e.g., CSF) as a mediator of host-microbial interaction. OCTN2 and other similar pathways for engaging or uptake of QSMs may be essential for the regulation of host responses important for maintenance of intestinal homeostasis. The finding that many gram-positive bacteria, in contrast to gram-negative organisms, produce compounds that compete with L-carnitine uptake (Figure 2D) is interesting because their quorum sensing molecules are typically small peptides, whereas the latter utilize non-peptides (Bassler and Losick, 2006 and Camilli and Bassler, 2006).

[0051] At any point in time, the profile of quorum-signaling molecules potentially serves as a composite measure of the status of the colonic microbiota. Thus, the uptake or sampling of QSMs by OCTN2 and potentially other transporters like it is expected to provide the host with the ability to respond or adapt to changes in the microbiome in order to maintain intestinal homeostasis. Furthermore, in vitro study with OCTN2 siRNA disclosed herein below established the relevance of OCTN2 transport in inducing cytoprotective protein Hsps and protecting intestinal epithelial cells by CSF. In this regard, OCTN2 could potentially mediate some of the actions of probiotic microorganisms.

[0052] As demonstrated in the Examples below, the effects of pro- and anti-inflammatory cytokines on the expression and activity of OCTN2 have been examined. OCTN2 expression is shown to be regulated by the acquired immune system because Rag I^"7" mice demonstrate decreased small and large intestinal OCTN2 expression. Further, the pro-inflammatory cytokines IFN-γ and TNF-α increased OCTN2 function in a Caco2/bbe cell line. In vivo, IFN-γ increased OCTN2 mRNA and protein expression as well as activity in the small intestine, while TNF-α increased OCTN2 activity in the large intestine, without changes in protein expression. Similar results were obtained in Caco2/bbe cells where IFN-γ increased OCTN2 mRNA, total protein expression and surface-expressed OCTN2 activity, while TNF- OC did not alter mRNA or total protein expression but increased surface expression and activity. The data establish that OCTN2 is regulated in a manner specific to the longitudinal segment of the intestine and is responsive to inflammatory cytokines.

[0053] The Examples provided herein demonstrate that IFN-γ and TNF-α enhanced the activity of OCTN2 both in vitro and in vivo. Also, OCTN2 expression was decreased in the intestine of Ragl^"7" mice, which essentially lack the ability to develop a mature acquired immune system; 0CTN2 expression was increased in the colonocytes of patients with active Crohn's disease, in which ThI type immune cells are strongly activated. These data indicate that 0CTN2 activity is up-regulated by activated immune cells through the secretions of IFN- γ and TNF-α. Further, 0CTN2, which is activated by IFN-γ and/or TNF-α, is shown to play a role in the pathogenesis of intestinal inflammation, including inflammatory bowel disease. Without wishing to be bound by theory, in inflammatory conditions, IFN-γ and/or TNF-α are believed to increase 0CTN2 function to augment the resistance of epithelial cells to foreign stresses such as microbial infection, inflammation, and oxidant-induced injury, and to maintain intestinal homeostasis. In patients with inflammatory bowel disease, the up- regulation of 0CTN2 function by IFN-γ and/or TNF-α may be inhibited or prevented, e.g., by 0CTN2 gene mutation, by Ragl mutation, or by a factor acting at the level of gene expression (transcription or translation) or at the level of 0CTN2 protein function, resulting in impaired epithelia and/or an excessive immune reaction.

[0054]

In addition to OCTN2-mediated host-microbial interaction, other forms of host-microbe interaction, including pattern recognition receptors or cytoplasmic nucleotide-binding- oligomerization domain (NOD) molecules (Mueller et al. 2005), are expected to contribute to the ongoing interplay between host and microbe. However, the OCTN2 pathway differs from other signaling pathways, such as the TLR and NOD signaling pathways. OCTN2 is primarily expressed by intestinal epithelial cells and to a far lesser extent by innate immune cells. While being a fairly promiscuous transporter capable of taking up many molecules, OCTN2 still requires certain structural features (e.g., small organic cations) that is likely to restrict substrates to particular types or classes of bacterial-derived molecules.

[0055] The experiments disclosed herein indicate that small-peptide, quorum- sensing molecules secreted primarily by gram-positive bacteria are among these molecules. It is also notable that many of these bacteria are not pathogenic in the normal host and, in some cases, have been used as probiotic agents. OCTN2 exemplifies a host mechanism that continuously samples the luminal content for certain microbial constituents within the enteric microbiome, allowing the host to adjust to perturbations or changes that might otherwise affect intestinal homeostasis. Accordingly, it is expected that competitive, non-competitive and uncompetitive inhibitors of a transport activity of OCTN2 will be modulators of OCTN2 suitable for use in methods of adjusting a host response to a bacterial signaling compound. Typically, inhibitory modulators of 0CTN2 activity will be used in methods to modulate 0CTN2 activity under circumstances where host-cell uptake of bacterial compounds associated with elaboration of a virulent phenotype or other deleterious developments is occurring or is likely to occur.

[0056] Stimulatory modulators of OCTN2 activity will be used in methods to modulate OCTN2 activity under circumstances where uptake of, e.g., a compound associated with a probiotic component of the microbiota is occurring or is likely to occur. Modulators of OCTN2 activity include, in addition to the aforementioned stimulators and inhibitors of OCTN2 protein activity, an siRNA or an RNAi of a gene encoding OCTN2, which would reduce the expression of OCTN2 and effectively inhibit the activity of the encoded OCTN2 polypeptide. Additionally contemplated are knockout mutations (deletions, insertions and/or substitutions) eliminating all OCTN2 activity or mutations leading to altered levels of activity, such as a missense mutation leading to reduced OCTN2 protein specific activity or an expression regulatory element mutation leading to enhanced (or inhibited) levels of expression relative to wild-type levels.

[0057] Because OCTN2 function is restricted to particular substrates, we expect other, similar pathways to exist (e.g., OCTNl, MDR-I), allowing the host to survey many constituents of the microbiome. Accordingly, the invention comprehends methods of identifying modulators of OCTNl or MDR-I activity as well as modulators of an activity, e.g., a transport activity, of OCTNl or MDR-I, such as the types of modulators identified above in the context of addressing modulators of 0CTN2. The ability of epithelial cells to sense bacterial QSM and deliver them using the highly specialized transporter molecule 0CTN2 has been demonstrated. Once taken up by intestinal epithelial cells, CSF activates key survival pathways including p38 MAP kinase and protein kinase B (Akt) and induces cytoprotective heat shock proteins, the latter preventing oxidant-induced intestinal epithelial cell injury and loss of barrier function.

[0058] Further consideration of the disclosure of the invention will be facilitated by a consideration of the following express definitions of terms used herein.

[0059] An "abnormal condition" is broadly defined to include mammalian diseases, mammalian disorders and any abnormal state of mammalian health (i.e., a mammalian condition) that is amenable to amelioration or treatment using a protein therapeutic, such as an immunoglobulin-based therapeutic.

[0060] "Administering" is given its ordinary and customary meaning of delivery by any suitable means recognized in the art. Exemplary forms of administering include oral delivery, anal delivery, direct puncture or injection, including intravenous, intraperitoneal, intramuscular, subcutaneous, intratumoral, and other forms of injection, spray (e.g., nebulizing spray), gel or fluid application to an eye, ear, nose, mouth, anus or urethral opening not involving a solid-state carrier such as a microsphere or bead, and cannulation. A preferred mode of administration is injection by syringe, typically a needle-bearing syringe.

[0061] An "animal" is given its conventional meaning of a non-plant, non-protist living being. A preferred animal is a mammal, such as a human.

[0062] "Ameliorating" means reducing the degree or severity of, consistent with its ordinary and customary meaning.

[0063] "Pharmaceutical composition" means a formulation of compounds suitable for therapeutic administration, to a living animal, such as a human patient. Typical pharmaceutical compositions comprise a therapeutic agent such as an immunoglobulin-based therapeutic, in combination with an adjuvant, excipient, carrier, or diluent recognized in the art as compatible with delivery or administration to an animal, e.g., a human. Pharmaceutical compositions do not include therapeutics bound to solid carriers, such as microspheres, beads, ion exchange media and the like.

[0064] "Adjuvants," "excipients," "carriers," and "diluents" are each given the meanings those terms have acquired in the art. An adjuvant is one or more substances that serve to prolong the immunogenicity of a co-administered immunogen. An excipient is an inert substance that serves as a vehicle, and/or diluent, for a therapeutic agent. A carrier is one or more substances that facilitates manipulation of a substance (e.g., a therapeutic), such as by translocation of a substance being carried. A diluent is one or more substances that reduce the concentration of, or dilute, a given substance exposed to the diluent.

[0065] "Media" and "medium" are used to refer to cell culture medium and to cell culture media throughout the application. As used herein, "media" and "medium" may be used interchangeably with respect to number, with the singular or plural number of the nouns becoming apparent upon consideration of the context of each usage. [0066] Having provided a general description of the various aspects of the invention, the following disclosure provides examples illustrative of the invention, wherein Example 1 describes experimental procedures, Example 2 discloses the effect of CSF on Hsps, Akt and p38 MAPK; Example 3 describes the role of OCTN2 in uptake of CSF in intestinal epithelial cells; Example 4 discloses the protective effect of OCTN2-mediated CSF uptake on oxidant stress; and Example 5 confirms the findings of Example 4 in showing the protective effect of CSF uptake by OCTN2 in an ex vivo mouse intestine preparation.

Example 1

Experimental procedures

[0067] Materials. Peptides were purchased from EZ Biolab (Westfield, IN) or Elim Biopharmaceuticals (Hay ward, CA) and [³H] -carnitine, [¹⁴C] -acetic anhydride, [³H]- mannitol, [³⁵S]-EXPRESS and [⁵¹Cr] Cl from Perkin Elmer (Boston, MA). Radiolabeled CSF was made by an exchange method using [1-¹⁴C] acetic anhydride (Moravek Biochemicals, Brea CA). Briefly, 1.9 mg of peptide (CSF (ERGMT; SEQ ID NO:2)) was dissolved in 100 μl water and mixed with 1 ml of sodium acetate saturated water. Two μl of acetic anhydride were added 5 times at 15-minute intervals at O⁰C, and the reaction was stopped by addition of 50 μl ammonium hydroxide. The acetylated peptide was purified by HPLC using a 5μ, 25cm length, 4.6mm diameter Lichro sphere- 100 RP-18 using a solvent gradient: starting at 98% A/2% B (A== 0.1% TFA in water and B= 0.1% TFA in acetonitrile) for 5 minutes and then a linear gradient to 75% A/25% B over 20 minutes at a flow rate of 1 ml/min. Detection was at 220 nm. The fraction containing acetylated ERGMT (SEQ ID NO:2) was analyzed by nuclear magnetic resonance (NMR) spectrometry to confirm structure and identify the acetylated residues (determined to be on amino terminal E).

[0068] Cell culture. Human colonic epithelial Caco2bbe cells were grown in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin and 10 μg/ml transferrin (all from Invitrogen/GIBCO, Grand Island, NY) in a humidified atmosphere of 5% CO₂. Cells were used between passages 55-70. Cells were plated using collagen- coated polycarbonate permeable filter supports (Transwell, 0.4 μm pore size, 24.5 mm diameter, 4.7 cm growth surface, Costar 3412, Cambridge, MA)on 6- or 12-well plates at a density of 10⁵ cells/ cm² and were allowed to differentiate for 10-14 days before use. [0069] Mice. C57B1/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME) or Taconic Labs (Germantown, NY). Small and large intestines, with or without treatments, were removed, rinsed with ice-cold saline, and epithelium was gently sheared off with glass slides for protein or mRNA determinations.

[0070] Ragl^"7" mice (C57B16 background) and age-matched controls were purchased from Jackson Laboratories (Bar Harbor, ME). Small and large intestines of mice, with or without the treatment of IFN -γ or TNF-α, were removed and their luminal surfaces were gently sheared off as described above. To investigate the expression and localization of OCTN2, formalin-fixed specimens of the small intestine and of the large intestine were analyzed by immunohistochemistry.

[0071] Preparation of B. subtilis conditioned media (CM). B. subtilis JH642 was used to generate B. subtilis RSM121. Single colonies of both strains were picked from LB agar, allowed to grow in LB until ODόoonm reached 1.0 (about 3 hours), then pelleted (2000 x g for 15 minutes), and washed three times in minimal medium (S7 minimal salts, 1% wt/vol glucose, 1% wt/vol glutamate and required amino acids (50 μg/ml). Bacteria were then resuspended in 10 ml of minimal medium and grown for an additional 3 hours. Cells were then pelleted and the supernatant (containing secreted products) was filter- sterilized through a O.lμ filter and stored at -8O⁰C until use. For radioactively labeled media, [³⁵S] -methionine and cysteine (³⁵S-EXPRESS labeling reagent) were added to the initial LB growth medium.

[0072] OCTN2 cDNA transfection. Human OCTN2 (SLC22A5) cDNA was subcloned into pDsRED2-Cl (Takara/Clontech, Palo Alto, CA), and used to transfect Caco2bbe and HSWP cells using the polyamine-derived reagent LT-I (Mirus, Madison, WI). Clones were selected on the basis of G418 (600 μg/ml) resistance and individually propagated for flux studies. The degree of OCTN2 transfection of cells was assessed by measuring Na⁺- dependent L-carnitine uptake and OCTN2 immunoblotting.

[0073] siRNA. To specifically inhibit expression of OCTN2, the Invitrogen BLOCK-iT RNAi designer (Invitrogen, Carlsbad, CA) was used to select the region of the coding sequence of human OCTN2 (nucleotides 1331-1355 of SEQ ID NO:4) for silencing and to select a nonsense sequence (5'-CCATCTAAGTTGCCCGTGAATCGTT-S' ; SEQ ID NO:12) as a negative control. dsRNA Stealth oligo (Invitrogen) was mixed with siLentfect reagent (Bio-Rad, Hercules, CA; 0.6 μl of reagent per cm² growing surface) in Optimem medium (Invitrogen) and allowed to form complexes for 15 minutes. Sufficient dsRNA was used to obtain a final concentration of 100 nM (Examples 1-5) or 200 nM (Examples 6-12). Complexes were applied when cells were 60% confluent and added for a second time after 2 days. Uptake studies were performed 24-48 hours after the second application.

[0074] Western blotting. Proteins of Caco2bbe cells or mouse intestinal epithelia were analyzed by Western blotting. Twenty to forty μg of each sample were resolved by SDS- PAGE (10-12%) and immediately transferred to a polyvinylidene difluoride (PVDF) membrane using Ix Towbin buffer (25 mM Tris pH 8.8, 192 mM glycine with 15% (vol/vol) methanol). PVDF membranes were incubated in TBS with 0.05% (vol/vol) Tween 20 (T- TBS) containing 3% (wt/vol) BSA for 1 hour at room temperature to block nonspecific binding. Blots were incubated overnight at 4⁰C with the following primary antibodies: anti- mouse Hsp25 antibody (Stressgen, Victoria, British Columbia, Canada), anti-human Hsp27 (Stressgen) or anti-mouse Hsp70 antibodies (Stressgen), anti-total and phosphorylated antibodies to each of the following: Akt, p38 MAP kinase, ERK 1/2 (p44/42), SAPK/JNK (Cell Signaling, Beverly, MA), rabbit polyclonal OCTN2 antiserum (Alpha Diagnostic International, San Antonio, TX), and rabbit polyclonal anti-human PepTl. Blots were washed five times for 10 minutes each in T-TBS at room temperature, incubated for 60 minutes in species-appropriate horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA) T-TBS, washed four times in T-TBS, washed once in TBS, and developed using the Super-Signal West Pico enhanced chemiluminescence system (Pierce Chemical, Rockford, IL).

[0075] For experiments described in Examples 6-12, individual samples each containing a 40 μg quantity of total or biotinylated protein of Caco-2/bbe cells, exposed or not exposed to cytokines, were analyzed by Western blotting. Protein samples were resolved by SDS-PAGE (10%) and immediately transferred to a polyvinylidene difluoride (PVDF) membrane using Ix Towbin buffer. PVDF membranes were incubated in T-TBS containing 3% (wt/vol) BSA for 1 hour at room temperature to block nonspecific binding, as described above. These blots were also incubated with rabbit polyclonal anti-mouse OCTN2 antibodies (Alpha Diagnostic International, San Antonio, TX), overnight at 4⁰C. Blots were washed five times for 10 minutes each in T-TBS and soaked in horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA) for 1 hour at room temperature in T-TBS. After five subsequent washes, immunoreactivity was detected by Super-Signal West Pico enhanced chemiluminescence (Pierce Chemical Company). [0076] Cell viability assay. Caco2bbe cells were grown in 24- well plates until differentiated and then treated with 10% vol/vol of B. subtilis conditioned medium, 100 nM CSF, or other peptides for 24 hours. Cells were loaded with ⁵¹Cr (50 μCi/ml) for 60 minutes, washed, and incubated in media with 0.6 mM monochloramine to induce cell injury. Medium was harvested from the cells after 60 minutes, and the ⁵¹Cr remaining in the cells was extracted with 0.1 wt/vol% SDS. The amounts of ⁵¹Cr in the released and cellular fractions were counted by liquid scintillation spectrometry. The amount of ⁵¹Cr released was calculated as the amount released divided by the total (cellular and released) ⁵¹Cr.

[0077] Ex vivo intestinal loop studies. C57B1/6 mice (18-25 gm) were sacrificed and the small intestine removed beginning at the ligament of Treitz. The first 18 cm were divided into three 6 cm lengths, each end ligated with silk suture and the loops filled with RPMI 1640 medium with 10% vol/vol heat inactivated FBS, with or without peptides (ERGMT (CSF; SEQ ID NO:2) or EMTRG (scrambled; SEQ ID NO:3)) at 10OnM. Loops were filled to moderate distention, about 1 ml per loop. Loops were placed in the outer loop of organ culture dishes (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ), which were filled with 5 ml media as above. Loops were incubated for 2 hours at 37⁰C in a 5% CO2 incubator. A 1 cm segment was removed from the middle and mucosa was scraped off with glass slides and processed for protein analysis. To measure effects on permeability, the two remaining segments were filled with RPMI 1640 medium containing serum with 1 mM mannitol and 1 μCi/ml [³H] -mannitol, with or without 0.3 mM freshly prepared monochloramine. Loops were placed into the middle section of the organ culture dish in 2 ml of RPMI 1640 with serum and without NH₂Cl. Samples were taken at 5, 20, and 35 minutes after contacting the loops with media to determine mannitol flux from lumen to medium outside bathing loops.

[0078] Human colon tissues. Colonic biopsy specimens provided by 3 healthy volunteers were used for immunohistochemistry. For localization of OCTN2 and Hsp27 expression, formalin-fixed specimens of intact small and large intestine were analyzed by immunohistochemistry using the Vector ABC kit per manufacturer's directions.

[0079] Colonic biopsy specimens obtained from 6 Crohn's disease patients and 3 healthy volunteers were used for protein determination. The biopsy specimens of 3 patients with active Crohn's disease were taken from the edge of the ulceration and the other specimens were from the mucosa without obvious inflammation in 3 Crohn's disease patients (inactive Crohn's disease). Proteins were immediately extracted from each sample as described herein. Five colonic specimens surgically resected from Crohn's disease patients and 3 non-inflamed colon specimens were used for immunohistochemistry, in addition to the 3 healthy specimens described above.

[0080] Real-time PCR. RNA was isolated from differentiated Caco2bbe cells using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Two μg of RNA were reverse-transcribed using random primers and Superscript II RT (Invitrogen). Three μl of 1:10 reverse transcription reaction were analyzed using TM iCycler iQ Multicolor Real-Time PCR Detection System (Bio-Rad., Hercules, CA) in triplicate. The averaged OCTNl, 2 or hPepTl mRNA expression levels were normalized to GAPDH expression and calculated using the comparative threshold cycle method.

[0081] Some real-time PCR reactions were performed with OCTN2- specific primers (sense, 5'-TTGACCCGAGTGAGTTACAAGACC-S' (SEQ ID NO:15), anti-sense, 5'- AGCGAAAGCCC AAAATAGCC-3' (SEQ ID NO: 16)) for the analysis of Caco-2/bbe cells, and sense, 5'-ATGGATTGGGGCATCTGTCC-S' (SEQ ID NO:17), anti-sense, 5'- GGAGAGGGAAA AAGACCT-3' (SEQ ID NO:18) for the analysis of mouse intestine) always in triplicate. The averaged OCTN2 mRNA expression levels were normalized to GAPDH expression (GAPDH specific primers; sense, 5'-TCATCTCTGCCCCCTCTGCT-S' (SEQ ID NO:19), anti-sense, 5'-CGACGCCTGCTTCACCACCT-S' (SEQ ID NO:20) for the analysis of human Caco-2/bbe cells and sense, 5'-GGCAAATTCAAGGGCACAGT-S' (SEQ ID NO:21), anti-sense, 5'-AGATGGTGATGGGCTTCCC-S' (SEQ ID NO:22) for the analysis of mouse intestine) and calculated using the comparative threshold cycle method. Each individual assay was performed in triplicate.

[0082] Apical membrane uptake studies. Caco-2/bbe cells were grown on collagen- coated Transwells for 10-14 days before apical membrane uptake studies. Cells were treated with 50μg/ml IFN-γor TNF-α for 0.5, 24, or 48 hours, or the cells were treated with IL- lβ, IL-2, IL-4 or IL-10 for 48 hours before flux measurement. All cytokines were obtained from PeproTech Inc. (Rocky Hill, NJ). Apical membrane L-carnitine uptake was determined in uptake buffer (20 mM NaCl, 5 mM KCl, ImM MgCl₂, 2 mM CaCl₂, 15 mM HEPES, pH7.4 ,and 130 mM N-methyl-D-glucamine) on the basolateral side and on the apical side (140 mM NaCl or 140 mM N-methyl-D-glucamine, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 15 mM HEPES, pH7.4, 1 μCi/ml L- [methyl-³H] -Carnitine hydrochloride (Amersham, Buckinghamshire, UK) yielding a concentration of 12 nM and non-radioactive L-carnitine to yield a final concentration 50 nM of L-carnitine). [0083] Some other transporters, such as OCTNl or ABT °' ⁺, can transport L-carnitine. The majority of Na⁺-dependent L-carnitine uptake is due to 0CTN2 in the presence of sodium ion in Caco-2 cells, however. Na⁺-dependent L-carnitine uptake, which corresponds to 0CTN2 function, was calculated by subtracting Na⁺-independent L-carnitine uptake from total L-carnitine uptake.

[0084] Uptakes were terminated by 4 washes with ice-cold Tris-buffered saline, the filters cut off and solubilized in scintillation liquid, and radioactivity was quantified by liquid scintillation spectroscopy. Protein determination was performed on parallel wells solubilized with 0.1 % (vol/vol) SDS by the modified Lowry method using bicinchoninic acid. Uptakes were expressed as pmoles L-carnitine per 3 minutes per mg protein.

[0085] Surface biotinylation. Proteins on the luminal surface of Caco-2/bbe cell monolayers were labeled using surface biotinylation. Cells were washed with ice-cold phosphate-buffered saline (PBS) and incubated in PBS containing 0.5mg/ml of a cell- impermeant form of biotin (Sulfo-NHS-LC-LC-biotin, Pierce Chemical Company, Rockford, IL) for 30 minutes. Biotinylation was terminated by the addition of 10 μl of 1 M Tris. Cells were scraped, washed once with PBS, and resuspended in 500 μl lysis buffer (1% vol/vol Triton X 100, 20 mM Tris, pH 8, 50 mM NaCl, 5 mM EDTA, 0.2% wt/vol BSA and Complete protease cocktail, Roche Molecular Biochemicals, Indianapolis, IN). Ten μl of lysate were removed for protein determination, 40 μl were removed and mixed with 20 μl 3x Laemmli solution, heated at 95⁰C for 5 minutes and stored at -8O⁰C for analysis of total OCTN2 expression, and to each remainder (450μl), 50 μl of a 50% (wt/vol) slurry of immobilized streptavidin (Pierce Chemical Company) was added and incubated with rotation at 4⁰C overnight. Samples were centrifuged at 5000x g for 1 minute, washed three times with cold lysis buffer, and samples eluted with 50 μl Ix Laemmli solution were heated at 95⁰C for 10 minutes and analyzed for biotinylated-OCTN2 proteins by Western blotting.

[0086] Immunohistochemistry. To detect OCTN2 proteins in mouse intestinal tissues, the avidin-biotin indirect immunoperoxidase method was performed on 4 μm paraffin- embedded sections using the Vectastain Elite ABC kit according to the manufacturer's instruction. Sections were incubated with rabbit polyclonal anti-mouse OCTN2 antibody (described herein) overnight at 4⁰C, then incubated with biotinylated horse anti-mouse IgG for 30 minutes and with streptavidin-biotin-peroxidase complex (Vector, Burlingame, CA, U.S.A.) for 30 minutes at room temperature. Immobilized peroxidase was visualized in diaminobenzidine solution. Sections were counterstained with hematoxylin. Parallel sections were incubated in PBS as a primary antibody for the negative control.

[0087] Intraperitoneal injection of cytokines. Concentrations of cytokines to be injected were IFN-γ (100 ng), TNF-α (2 μg), anti TNF-α monoclonal antibody (XT22, 500 μg) or isotype-matched rat control monoclonal antibody (GLl 13, 500 μg) per mouse, all administered by single intraperitoneal injection. This protocol has been used to investigate the effects of these cytokines on rat intestinal apical sodium/hydrogen exchange, mouse- sodium/potassium- ATPase and mouse-PepTl, where alteration of activity and/or function has been shown to occur. Indicated sections of the intestine were harvested 48 hours later and analyzed by Western blotting and L-carnitine uptake.

[0088] Mouse intestinal uptake study. Two days after cytokine injection, intestinal segments were opened longitudinally and kept in warmed (37 ⁰C) saline. Two separate 0.5 cm segments were minced to small pieces of approximately 0.2 cm and placed into 5 ml pre- warmed and oxygenated apical flux buffers used for the L-carnitine uptake assays of Caco- 2/bbe cells. Tissues in flux buffers were in separate 14-ml polypropylene tubes (Falcon 2059, Becton Dickinson Labware, Franklin Lakes, SA) in a shaking 37⁰C water bath. Pieces were incubated in the flux buffer for 10 minutes, then rapidly (less than 30 seconds) washed 3 times with ice-cold flux buffer without radioactive L-carnitine to reduce extracellular trapped [³H] L-carnitine. Trapped extracellular space was estimated using [³H]-mannitol (100 μmoles/1 , 4400 cpm/nmole), which resulted in 317+48 cpm/mg protein in jejunum (1.8 % of control L-carnitine counts), 261+51 in ileum (1.8 % of control L-carnitine counts), 107+28 in proximal colon (0.9 % of control L-carnitine counts), and 84+29 in distal colon (0.6 % of control L-carnitine counts). Tissues were placed into 10% (wt/vol) trichloroacetic acid, homogenized with a Teflon pestle homogenizer, and allowed to sit on ice for 30 minutes. Precipitated proteins were pelleted (14,000 x g for 10 minutes at 4⁰C) and the supernatant was removed and counted to assess tissue accumulated uptake of [³H] L-carnitine. The precipitated proteins were then solubilized with IN NaOH and quantified using the bicinchoninic acid procedure. Uptakes were expressed as pmoles L-carnitine per 10 minutes per mg protein.

Example 2 CSF induced Hsps and activated a survival pathway (Akt) and a stress signal (p38 MAPK) [0089] Heat shock proteins (Hsps) are essential for maintenance of intestinal homeostasis, rendering colonic epithelial cells less susceptible to injury and stress. Intestinal epithelia express many heat shock proteins, including inducible Hsp27(human)/Hsp25(murine) and Hsp70, as well as constitutively expressing heat shock cognate Hsc70. The nomenclature, Hsp27 and Hsp25, defines the related small heat shock proteins of approximate molecular weight of 27 kDa and 25 kDa of human and murine cells, respectively. Physiological expression of inducible heat shock proteins like Hsp27/25 and Hsp70 is maintained by microbial-derived molecules, including pattern recognition ligands (Rakoff-Nahoum et al., 2004), accounting for their predominant expression in surface colonocytes (see Figure 5. Therefore, the effects of conditioned media (CM) from several representative strains of enteric bacteria in inducing heat shock protein 27 (Hsp27) expression in human colonic epithelial Caco2bbe cells was assessed. As shown in Figure IA, most gram-positive, but not gram-negative, bacteria significantly induced Hsp27 expression in Caco2bbe cells (results expressed as a percent of control Hsp27 response to thermal stress, 41.5⁰C x 23 minutes). In light of this dichotomous response of gram-positive bacteria, the possibility that secreted agents such as quorum sensing peptides might be mediating the actions of this group of microbes was investigated.

[0090] B. subtilis (strain JH 642, wild type) was selected for further study because it is a well-characterized, obligate, gram-positive aerobe that is not only a common soil and water saprophyte, but frequently part of human enteric flora with known probiotic activity (Solomon et al., 1996, Kunst et al., 1997, Lazazzera et al., 1997, Levin et al., 1998 and Tarn et al., 2006). Conditioned medium from the B. subtilis strain JH 642 increased Hsp27 expression to nearly the same extent as heat shock (Figure IB), whereas neither experimental condition altered expression of the constitutively expressed heat shock cognate Hsc70. The latter was anticipated because Hsc70 in most cells is quite stable and less influenced by exogenous stimuli or cell stress. This stability ensures that Hsc70 continues to function in critical processes such as protein folding, chaperone function, and in the formation and stabilization of protein complexes (Morimoto, 1993 and Morimoto, 2002). To further characterize the factor(s) that induced Hsp27, conditioned medium (CM) from B. subtilis JH 642 was size- separated by a 3 kDa molecular mass cutoff filter, with bioactivity largely remaining in the filtrate, indicating a small molecular mass (Figure 1C). Additionally, the Hsp27-inducing bioactivity was heat-stable and pepsin-sensitive. [0091] B. subtilis produces and secretes many bioactive agents, but its competence and sporulation factor (CSF), a QSM, fits the parameters of the above physiochemical characteristics. CSF is a cationic pentapeptide corresponding to the C-terminal 5 amino acids of the 40-amino-acid polypeptide encoded by the phrC gene (Kunst et al., 1997) that functions in quorum-sensing (Lazazzera et al., 1997) with a physiological concentration range between 10-100 nM (Solomon et al., 1996) to alter Bacillus population behavior. To assess CSF' s potential biological role in colonic epithelial cells, CM from wild type (JH 642) cells and the CSF-deficient JH 642-derived B. subtilis strain RSM 121, were added to Caco2bbe cells. CM derived from RSM 121 (delta CSF) failed to induce Hsp27 in Caco2bbe cells, implicating CSF in this effect (Figure ID). To further evaluate this possibility, CSF (ERGMT; SEQ ID NO:2) was chemically synthesized and purified. CSF induced Hsp27 in Caco2bbe cells (Top Western blot of Figure IE) and this induction is concentration dependent and is physiologically relevant (Figure 6). Additionally, the activations of other signaling pathways involved in cell survival were tested and it was found that CSF activated the Akt and p38 MAPK pathways (Lower two sets of Western blots in Figure IE). In intestinal epithelial cells, Akt has been shown to be important in promoting Hsp25 expression (Tao et al., 2006) and p38 MAPK pathways in blocking apoptosis by inhibiting caspase-3 after polyamine depletion (Zhang et al., 2004). In contrast, two other pathways, JNK and ERK, were not influenced by CSF. As a control, a scrambled pentapeptide, EMTRG, did not induce Hsp27 or activate either the Akt or p38 MAPK pathways. Also of note, inhibitors of Akt and p38 MAP Kinase did not inhibit or affect levels of Hsp27 induced by the CM of B. subtilis, e.g., CSF, as shown in Fig. 10.

Example 3

OCTN2 transports CSF and mediates CSF effect on Hsps induction in Caco2bbe cells

[0092] CSF-mediated activation of an early competence promoter (srf A) in B. subtilis cells is dependent on the uptake by a Bacillus oligopeptide transporter. Could a convergent mechanism develop in a eukaryotic host that would mediate a specific uptake of bacterial QSM peptides? In fact, other peptides, such as bacterial chemotactic peptides, can be transported by eukaryote apical membrane oligopeptidyl transporters. Focus was placed on the apical membrane organic cation transporter, OCTN2, as a candidate for CSF uptake because of its transport preference for substrates having physiochemical properties close to CSF (e.g., cationic oligopeptide) (Tamai et al., 2000 and Peltekova et al., 2004). OCTN2 is believed to be the main transporter for dietary carnitine, but its abundant expression in the colon is unexplained, as most carnitine is absorbed in the small intestine. OCTN2 is primarily expressed by surface epithelial cells of the colon that are in direct contact with the luminal contents and microbes and these cells exhibit sustained expression of microbial- induced heat shock proteins (Rakoff-Nahoum et al., 2004) (Figure 5). OCTN2, in contrast to OCTNl, is also expressed in Caco2bbe cells (Figure 7A).

[0093] As shown in Fig. 2A, ¹⁴C-labeled CSF was readily taken up by Caco2bbe cells, an effect that was increased in OCTN2-transfected cells and inhibited in cells with siRNA- silenced OCTN2 expression (Figure 7B and 7C). Similar, but more pronounced, effects were observed in OCTN2-transfected human fibroblast HSWP cells that normally exhibit minimal endogenous expression of OCTN2 (Figure 8 A and 8B). FIT C-labeled CSF was also rapidly taken up by Caco2bbe cells (Figure 2B) and distributed throughout the cytosol within 30 minutes, an effect competed by L-carnitine (10 mM), thereby implicating OCTN2 transport. In addition, while CSF competed with L-carnitine uptake, a scrambled pentapeptide for CSF (EMTRG, SEQ ID NO:3) did not, suggesting the specificity of OCTN-mediated transport of the peptide (FigureδC). Taken together, these results provide compelling evidence for uptake of CSF by OCTN2. CSF induction of Hsp27 was also blocked by inhibiting OCTN2 expression with siRNA, whereas Hsp27 induction by heat shock (41.5⁰C x 23 minutes) was not affected (Figure 2C), showing that OCTN2 is required for CSF to mediate Hsp27 induction. As shown in Figure 2D, conditioned media of other gram-positive, in contrast to gram-negative, bacteria also competed with Na-dependent, L-carnitine uptake in Caco2bbe cells, indicating OCTN2 uptake of soluble molecules derived from these organisms (Table 1).

Table 1

Example 4

OCTN2 mediated-CSF uptake protects epithelial cells from oxidant stress

[0094] To assess the functional role of CSF and OCTN2 in epithelial homeostasis, the capacity of CSF to protect epithelial cells against oxidant (NH₂Cl, monochloramine)-induced injury using ⁵¹Cr release was assessed. Pretreatment of Caco2bbe monolayers with wild-type JH642 CM protected cells against oxidant-induced injury, whereas pretreatment with CM from Rsml21 (ΔCSF) did not (Figure 3A, left panel). When cells were treated with CSF, cPDl, an unrelated quorum- sensing molecule from Enterococcus (Figure 3A, middle panel), or a scrambled pentapeptide of CSF (EMTRG; SEQ ID NO:3) (Figure 3A, right panel), protection against oxidant stress was only seen with CSF. Silencing of OCTN2 with siRNA inhibited the protective effects of CSF against oxidant-induced stress in Caco2bbe cells (Figure 3B).

[0095] Because intestinal barrier function and viability are highly sensitive to inflammation-associated injury, the above agents were tested to determine their ability to limit oxidant (monochloramine)-induced loss or reduction of cell-cell barrier function. Only CSF and CM of B. subtilis mitigated induced increases in ³H-mannitol flux, a measure of barrier function known in the art (Figure 3C). Silencing of Hsp27 resulted in nearly complete reversal of the CSF- and CM-induced protection of cell viability (Figure 3D) and epithelial barrier function (Figure 3E) against oxidant-induced stress. In contrast, treatment of human Caco2bbe cells with siRNA to murine Hsp25 (mHsp25) had no effects, establishing the specificity of Hsp27 silencing.

Example 5

CSF protects intestinal tissues from oxidant stress through 0CTN2 transport in ex vivo preparation of mice

[0096] The effects of CSF (ERGMT; SEQ ID NO:2) and a scrambled pentapeptide molecule (EMTRG; SEQ ID NO:3) on induction of Hsp25 and Hsp70 in ex vivo preparations of murine proximal small intestine and colon were examined. Surgically removed segments of bowel were ligated at both ends and filled with buffer containing CSF or scrambled peptide. After 2 hours, CSF treatment of both small and large intestinal mucosa stimulated a significant increase in Hsp25 and Hsp70 (Figure 4A), best appreciated in small intestine, as basal levels of these proteins were minimal in the small intestine. In contrast, no changes in mucosal Hsc70 (a constitutively expressed heat shock cognate) were noted. Despite the higher basal expression in colonic mucosa due to the presence of enteric flora (Arvans et al., 2004), significant increases in Hsp25 and Hsp70 expression were still observed. In contrast, the scrambled peptide EMTRG (SEQ ID NO:3) slightly induced Hsp25, but not Hsp70, in small intestine. Thus, pentapeptides with similar amino acid sequence similar to CSF may exhibit biological activity in eukaryotic cells. No induction of Hsp25 and Hsp70 was noted in large bowel mucosa exposed to the scrambled peptide. This effect was mediated by OCTN2, as L-carnitine (10 mM) inhibited CSF-induced Hsp25 and Hsp70 induction (Figure 4B).

[0097] To determine the physiological consequences of the CSF effect, transmural ³H- mannitol fluxes were measured in intact small bowel loops to assess intestinal barrier function. As shown in Fig. 4C, increased mucosal permeability in small intestinal loops caused by exposure to oxidant (NH₂Cl, 0.3 mM) was significantly inhibited by luminal CSF (10OnM), but not by the scrambled peptide. This protective action was inhibited when studies were performed with L-carnitine (10 mM), used to competitively inhibit CSF uptake through OCTN2 (Figure 4D). No changes in the basal mannitol permeability were observed with either CSF or the scrambled peptide. Additionally, pretreatment of intestinal loops or Caco2bbe monolayers with inhibitors of the Akt and p38 MAPK pathways (LY294002 and SB203580, respectively) had no significant effects on CSF protection (Figure 9), indicating that induction of heat shock proteins plays a major role in conferring protection against this form of stress. While a contributory role for the Akt and p38 MAPK pathways may exist, other reports have also demonstrated that induced heat shock proteins are particularly effective in protecting cells against oxidant-induced stress (Ropeleski et al., 2003 and Arrigo et al., 2005).

Example 6

0CTN2 expression decreased in Ragl^'Λ mice

[0098] To assess intestinal expression of OCTN2 and to determine if immune cells affect OCTN2 expression in an in vivo environment, immunohistochemical staining was performed on small and large intestinal tissues of control and recombination activating gene Y^1'

(Ragl ⁷ ) mice. Ragl^"7" mice, which lack Ragl, lack mature T and B cells. The immunohistochemistry was performed as described in Example 1. The results showed that, in control mice, OCTN2 was expressed at the highest level in the brush border, and faintly in the cytosol of epithelial cells nearest the intestinal lumen in both colon and small intestine. OCTN2 was more strongly expressed in the colon compared to the small intestine. Decreased OCTN2 staining was found in both the small and large intestines of Ragl^"7" mice. As immunohistochemistry is poorly quantitative (but allows cellular location), intestinal epithelia homogenizations were analyzed by Western blotting. Western blots of control and Ragl^"7" mice intestines demonstrated that OCTN2 expression is decreased, particularly in the colon of Ragl^"7" mice (Fig. 11). These data indicate that mature T cells or B cells affect OCTN2 expression in intestinal epithelial cells, possibly through mediators such as cytokines. Further, modulators that affect the activity of Ragl are expected to be useful in effectively reducing the activity of charged oligopeptide transport molecules, such as OCTN2, OCTNl or MDR-I.

Example 7

Modulation of OCTN2-mediated transport

[0099] To confirm that the sodium-dependent L-carnitine uptake was mainly due to OCTN2-mediated transport, Caco-2/bbe cells were treated with OCTN2 siRNA 24 hours before uptake measurements. The siRNA experiment was conducted in accordance with the description provided in Example 1. Results revealed that Na⁺-dependent L-carnitine uptake in Caco-2/bbe cells untreated or treated with OCTN2 siRNA were 0.88 + 0.10 or 0.16 + 0.06 pmol/mg protein*3 minutes, respectively, demonstrating that more than 80% Of Na⁺- dependent L-carnitine uptake was due to 0CTN2-mediated transport in Caco-2/bbe cells (Fig. 12a).

[0100] To determine if immune cell-derived cytokines affected 0CTN2 activity, Caco- 2/bbe cells were treated with 50 ng/ml of pro-inflammatory (IFN-γ, TNF-α, IL-Ib, IL-2, IL- 4) or anti-inflammatory (IL-10) cytokines for 48 hours before apical membrane carnitine uptake. These cytokine concentrations were known in the art. Further experimental detail is provided in Example 1. Results showed that both IFN- γ and TNF- α significantly increased the Na⁺-dependent L-carnitine uptake that had been demonstrated to be positively correlated to OCTN2 activity and, thus, useful as a measure thereof. In contrast, other cytokines had no effect (Fig. 12b).

[0101] The time dependence of the effects of IFN-γ or TNF-α on Na⁺-dependent L- carnitine uptake was next determined. Cells were treated for periods from 0.5 to 48 hours with either 50ng/ml IFN-γ or 50ng/ml TNF-α before apical carnitine uptake assay was initiated (see Example 1 for experimental details). The results shown in Fig. 13 establish that IFN-γ increased the Na⁺-dependent L-carnitine uptake at 24 hours or greater, while TNFα increased Na⁺-dependent L-carnitine uptake in 30 minutes and significantly increased that uptake in 24 hours.

[0102] It is thus contemplated that either IFN-γ, TNF-α, both compounds, or one or more agonists thereof are contemplated as useful in elevating a charged oligopeptide transport level (e.g., an OCTN2 level) in methods of treating intestinal disorders by affecting the activity levels of any one or more of Hsp27, Akt, or p38 MAP Kinase. Also contemplated is the use of any inhibitor or antagonist of either IFN-γ (e.g. an anti-IFN-γ antibody), TNF-α (e.g., an anti-TNF-α antibody), or both to reduce the level of activity of a charged oligopeptide transport molecule (e.g., OCTN2) in methods of treating intestinal disorders by affecting the activity levels of any one or more of Hsp27, Akt, or p38 MAP Kinase.

Example 8

Increased OCTN2 expression mediated by pro-inflammatory cytokines

[0103] To determine whether the pro-inflammatory cytokines IFN-γ or TNF-α. alone or in combination, elevated OCTN2 activity by stimulating or increasing the expression of OCTN2, an expression study using the methods described in Example 1 was conducted. In brief, Caco2/bbe cells were treated for various times with various concentrations of IFN-γ or TNF-oc. The results revealed that IFN-γ increased both total and apical surface 0CTN2 expression at 24 hours after addition. This effect was observed at concentration as low as 10 ng/ml. In contrast, TNF-α increased expression of apical membrane OCTN2, which was observed at 30 minutes after addition and had become a significant effect at 24 hours after addition. TNF-α, unlike IFN-γ, exhibited no effect on the total level of OCTN2 expression (Figure 14). This pattern was observed at concentrations of 25 ng/ml TNFα or greater.

[0104] The results were consistent with the findings reported in Example 7 and serve to underscore the aspects of the invention drawn to the use of one or more pro-inflammatory cytokines (e.g., IFN-γ, TNF-α, both, or agonists of at least one pro-inflammatory cytokine) to elevate the activity of a charged oligopeptide transport molecule such as OCTN2 and thereby treat or prevent an intestinal disorder (e.g., IBD, a pathogenic infection, antibiotic-associated colitis, radiation enteritis, intestinal epithelial cell apoptosis, aberrant cell cycle regulation of an intestinal epithelial cell), as well as methods to reduce the activity of a charged oligopeptide transport molecule (e.g., OCTN2) by administering an inhibitor or antagonist of one or more pro-inflammatory cytokines such as IFN-γ, TNF-α, or both, thereby treating an intestinal disorder such as IBD, a pathogenic infection, antibiotic-associated colitis, radiation enteritis, intestinal epithelial cell apoptosis, aberrant cell cycle regulation of an intestinal epithelial cell.

Example 9

Cytokine effect on 0CTN2 transcription

[0105] Beyond the expression studies reported in Example 8, an investigation was undertaken to determine whether a pro-inflammatory cytokine was modulating a charged oligopeptide transport molecule (OCTN2) by affecting expression at the transcriptional level. To determine if IFN-γ or TNF-α increased OCTN2 mRNA expression, real-time PCR analysis was used (see Example 1 for further experimental details). IFN-γ increased OCTN2 mRNA expression in a concentration-dependent manner at 24 hours, indicating that IFN-γ treatment increased either OCTN2 gene transcription or OCTN2 mRNA stability. In contrast, TNF-α had no effect on OCTN2 mRNA expression. These results are consistent with the change of OCTN2 protein expression by IFNγor TNFα (Fig. 16).

[0106] This in vitro study showed that IFNγ increased OCTN2 activity and expression by affecting gene transcription. In addition to the classical Janus activated kinase (JAK)- signal transduction and the signal transducer and activator of transcription (STAT)- signaling pathways, IFN-γ activates several other signaling cascades, such as the phosphatidylinositol 3-kinase (PDK)-signaling pathway, and the c-cbl protooncogene product, CRKL, which regulates the activation of the guanine exchange factor C3G. According to GenBank (location; 5q31, Gene ID; 6548), there are five regions which are believed to be targets for STATl binding (i.e., TTN5AA, where N represents any nucleotides) within 1000 base pairs upstream of the 0CTN2 gene. IFN-γ may augment 0CTN2 mRNA production through the JAK-STATs signal pathway.

[0107] In contrast, TNF-α increased the apical protein expression of OCTN2 24 hours after addition, but didn't increase total protein expression of OCTN2 in Caco-2/bbe cells. Thus, TNF-α changed the distribution of OCTN2 from the cytosol to the cell membrane. Recent iron transport studies have shown that TNF-α induces relocation of the basal ion regulated transporter (IREG-I) 24 hours after addition, but not earlier, consistent with a role for TNF-α as a regulator of transporter functions through alterations in transporter distribution. These observations, which do not limit the claimed subject matter, are consistent with reports that TNF-α, acting on neuronal TNF-Rl receptors, increases the exocytosis of AMPARs through a PI3 kinase-dependent pathway.

[0108] The in vivo studies demonstrated that each of IFN-γ and TNF-α increased OCTN2 function. Further, neutralizing TNF-α by an anti-TNF-α monoclonal antibody decreased OCTN2 function in all parts of the intestine, indicating that TNF-α is a pivotal cytokine for regulating OCTN2 function. However, injected TNF-α increased OCTN2 function in the small intestine, but not in the colon. In the colonic mucosa, however, TNF-α is constitutively secreted because of stimulation by commensal bacteria, diets and xenobiotics. Because endogenous TNF-α constitutively up-regulated OCTN2 function, injected TNF-α could not augment OCTN2 activity in the colon. Although IFN-γ increased transcription of the OCTN2 gene as well as its activity, neutralizing TNF-α diminished the effects of IFN-γ on OCTN2 function, indicating that the effects of IFN-γ on OCTN2 function were mediated by TNF-α in vivo.

[0109] The results presented in this Example provide a basis for preventing or treating intestinal disorders by altering the basal level of activity of a charged oligopeptide transport molecule such as OCTN2. The relatively high levels of charged oligopeptide transport molecule (e.g., OCTN2) activity in the colon attributable, at least in part, to the high levels of circulating TNF-α in the colon, may be inhibited or reduced for a period of time by administering an antagonist to TNF-α, thereby reducing the activity of the transporter. Based on the disclosure herein, the reduced transporter activity will inhibit or retard chemical signaling to the colonic epithelia, leading to altered levels of at least one of Hsp27, Akt or p38 MAP Kinase, thereby affecting the development and/or course of an intestinal disorder, exemplified by IBD (e.g., Crohn's disease, ulcerative colitis), a pathogenic infection, antibiotic-associated colitis, radiation enteritis, intestinal epithelial cell apoptosis, or aberrant cell cycle regulation of an intestinal epithelial cell.

Example 10

Cytokine effects on 0CTN2 uptake in mouse intestine

[0110] OCTN2 uptake activity in vivo was also determined. To characterize OCTN2 in functional terms in vivo, mice were treated with IFN-γ (100 ng/mouse), TNF-α (2 μg/mouse) or anti-TNF-α antibody XT22 (500 μg/mouse) for 48 hours before flux assays (further experimental detail is provided in Example 1). Results demonstrated that IFN-γ significantly increased sodium-dependent L-carnitine uptake in the proximal colon of mice, but not in the jejunum, ileum or distal colon. In contrast, TNF-α increased sodium-dependent L-carnitine uptake in the jejunum as well as the ileum, but not in the colon. However, the XT 22 antibody decreased sodium-dependent L-carnitine uptake in all parts of the intestine, indicating that TNF-α is necessary for the function of OCTN2 in the intestine. The effect of TNF-α on the function of OCTN2 seemed to be negated by the naturally occurring TNF-α quantity found in the colon (Fig. 17). On the other hand, XT22 negated the effect of IFN-γ on sodium-dependent L-carnitine uptake in the proximal colon. Thus, IFN-γ appears to increase the function of OCTN2 through a TNF pathway.

[0111] The results presented in this Example establish that the in vitro culture experiments accurately reflect the biology found in vivo. The data thereby confirm that modulators identified by methods according to the invention, including modulators of Hsp27 expression and/or activity, Akt expression and/or activity, p38 MAP Kinase expression and/or activity, and charged oligonucleotide transport molecule (e.g., OCTN2) expression and/or activity, along with modulators of Ragl expression or activity, will be useful in preventing or treating intestinal disorders such as IBD.

Example 11

Cytokine effects on OCTN2 in vivo [0112] To identify the effects of IFN-γ and TNF-α on OCTN2 expression in intestinal epithelial cells in an in vivo environment, mice were treated with IFN-γ or TNF-α, as provided in the description of Fig. 17. Treatment of the mice with IFN-γ increased the expression of OCTN2 in the colon. No significant changes in OCTN2 expression were observed in either the jejunum or the ileum 48 hours after cytokine injection. With TNF- OC, no effect on the expression of OCTN2 was observed in either the small or the large intestine of mice (Fig. 18).

[0113] To determine if IFN-γ or TNF-α increases OCTN2 gene transcription, OCTN2 mRNA expression was quantitated in intestinal epithelia of mice treated with IFN-γ or TNF- α with Real-time PCR (Figure 9). The control C_T values were 22.71 + 0.59 for jejunum, 22.28 + 1.13 for ileum, 24.16 + 0.28 for proximal colon, and 24.80 + 0.87 for distal colon (n=4). IFN-γ, but not TNF-α, increased the OCTN2 mRNA expression in the proximal and the distal colon. These data are consistent with in vitro data, indicating that OCTN2 activity is increased by IFN-γ and TNF-α by different mechanisms. IFN-γ increased the OCTN2 function through the enhancement of OCTN2 gene transcription. In contrast, TNF-α changed the distribution of OCTN2 from cytosol to membrane, resulting in a stimulation of OCTN2 activity.

[0114] The data disclosed herein provide a flexible approach to the prevention and/or treatment of intestinal disorders such as inflammatory bowel disease. IFN-γ has been shown to increase the activity of a charged oligopeptide transport molecule (OCTN2) in epithelial cells, and to do so at least in part by elevating transcription of the OCTN2 gene. Another pro-inflammatory cytokine, TNF-α, also elevates the activity of a charged oligopeptide transport molecule (OCTN2) in epithelial cells, but this cytokine does not affect the level of transcription of the OCTN2 gene. Rather, TNF-α re-distributes the transporter to the apical membrane. Notwithstanding these mechanistic distinctions, however, the data herein establish that IFN-γ exerts its effects through a TNF-α pathway insofar as an anti-TNF-α antibody counters the OCTN2 effects of each of these pro-inflammatory cytokines (i.e., TNF- α and IFN-γ) (see Example 10, below). Armed with this information, it will be apparent to one of skill in the art that any number of effectors (e.g., stimulators or inhibitors) of a proinflammatory cytokine such as TNF-α or IFN-γ would be useful in modulating the activity of a charged oligopeptide transport molecule such as OCTN2, whether by modulating expression at the transcriptional level, at the translational level, at the post-translational level or by affecting the distribution of the expressed and processed gene product.

Example 12

Expression of 0CTN2 in Crohn 's disease in humans

[0115] To assess levels of 0CTN2 in the colonocytes of Crohn's disease patients, 0CTN2 proteins extracted from human biopsy specimens were analyzed using Western blotting. Colonic 0CTN2 expression in active Crohn's disease patients was increased in comparison to the colonic OCTN2 expression in healthy volunteers. There is no change of colonic OCTN2 expression between patients with inactive Crohn's disease and healthy volunteers (Fig. 20). Immunohistochemistry showed that OCTN2 was strongly expressed in both the apical membrane and in the cytosol of colonocytes found in the upper half crypt in active Crohn's disease patients, while OCTN2 expression was limited in the apical membrane of the surface colonocytes in non-inflamed mucosa (Fig. 21).

[0116] Strong expression of OCTN2 in the surface area of mice colonocytes was demonstrated by both immunohistochemistry and Western blot analyses, as disclosed in the preceding Examples. The data establish that the host-bacterial interaction occurs, in part, by chemical communication through transporters such as OCTN2, and those communications are key for the regulation of intestinal inflammation. Consistently, another transporter, hPepTl, transports muramyl dipeptide, which stimulates the translocation of NF-kB and the secretion of IL-8 and MCP-I, and OCTN2 transport of Competence and Sporulation Factor induced heat shock proteins 27 and 70 and activated both the Akt and p38 MAPK pathways. Additionally, hPepTl has been reported to transport fMLP, which is a major peptide neutrophil chemotactic factor produced by E. coli. Epithelial membrane transport is expected to contribute to the regulation of intestinal inflammation through the absorption (transport) of some factors produced by bacteria. The data disclosed herein also lead to the expectation that alteration of certain membrane transporters will result in abnormal bacterial-host interaction, thereby contributing to the development of IBD.

[0117] The disclosure provides, for the first time, that changes in OCTN2 expression are associated with IBD. Colonic OCTN2 expression was increased in all three patients with active Crohn's disease. Activated T-lymphocytes secrete abundant IFN -γ and TNF-α in intestinal tissues of active Crohn's disease patients. It is expected that augmented IFN-γ and/or TNF-α lead to an increase in colonic OCTN2 expression in Crohn's disease. Further, the data disclosed in this Example confirm and are entirely consistent with the data obtained from the in vivo mouse studies disclosed in previous Examples. Accordingly, the mouse model is useful in modeling events associated with human intestinal disorders, such as humans with Crohn's disease.

[0118] The experiments disclosed herein establish that pivotal pro-inflammatory cytokines, IFN-γ and TNF-α, increased the function of an organic cation transporter, OCTN2. Furthermore, IFN-γ increased the transcription of the OCTN2 gene and TNF-α promoted the membrane insertion of OCTN2 in vitro and both of these cytokines increased OCTN2 function in vivo. OCTN2, which is strongly expressed in colonocytes that are themselves in direct contact with commensal bacteria, is expected to play an important role in intestinal inflammation through host-bacterial interaction. In an analogous manner, other transporter molecules, such as OCTNl, MDR-I, hPepTl, and the like, are expected to be involved in such epithelial disorders and diseases as inflammatory bowel disease and other stress- producing situations involving the intestinal flora.

References

Arrigo, et al., Antioxid Redox Signal. 7, 414-22 (2005).

Arvans, et al., Am. J. Physiol. Gastrointest. Liver Physiol. 288, G696-704 (2004).

Bassler, et al., Cell 121, 125(2):237-46 (2006).

Camilli, et al., Science 311, 1113-1116 (2006).

Charrier, et al., Lab. Invest. 86, 490-503 (2006).

Kunst, et al., Nature 390, 249-256 (1997).

Lazazzera, et al., Cell 89, 917-925 (1997).

Leung, et al., Immunol Cell Biol. 84, 233-6 (2006).

Levin, et al., Curr. Opin. Microbiol. 1, 630-635 (1998).

Morimoto, Science. 259, 1409-1410 (1993).

Morimoto, Cell. 110, 281-284 (2002).

Mueller, et al., Curr. Opin. Gastroenterol. 21, 419-425 (2005).

Noble, et al., Gastroenterology 129, 1854-1864 (2005).

Ohashi, et al., MoI. Pharmacol. 59, 358-366 (2001). Pasare, et al., Adv Exp Med Biol. 560, 11-8 (2005).

Peltekova, et al., Nat. Genet. 36, 471-475 (2004).

Rakoff-Nahoum, et al., Cell 118, 229-241 (2004).

Rakoff-Nahoum, et al., Curr. Top. Microbiol. Immunol. 308, 1-18 (2006).

Ropeleski, et al., Gastroenterol. 124, 1358- 1368 (2003).

Solomon, et al., Genes. Dev. 10, 2014-2024 (1996).

Tarn, et al., J. Bacterid. 188, 2692-2700 (2006).

Tamai, et al., J. Biol. Chem. 275, 40064-40072 (2000).

Tao, et al., Am. J. Physiol. 290, C1018-1030 (2006).

Trinh, et al., Gastroenterol. 129, 2106-10 (2006).

Vermeire, et al., Gastroenterol. 129, 1845-1853 (2005).

Waller, et al., Gut 55, 809-14 (2006).

Zhang, et al., J. Biol. Chem. 279, 22539-22547 (2004).

[0119] Numerous modifications and variations of the invention are possible in view of the above teachings and are within the scope of the invention. The entire disclosures of all publications cited herein are hereby incorporated by reference.

Claims

ClaimsWhat is claimed is:

1. A method for identifying a modulator of Heat Shock Protein 27 activity comprising:

(a) contacting a cell comprising a charged oligopeptide transport molecule with a candidate modulator transportable by said molecule; and

(b) measuring an activity of Heat Shock Protein 27 of said cell in the presence and absence of said candidate modulator, wherein a different level of Heat Shock Protein 27 activity in the presence and absence of said candidate modulator identifies said candidate modulator as a modulator of Heat Shock Protein 27 activity.

2. The method according to claim 1 further comprising adding a peptide comprising the amino acid sequence set forth in SEQ ID NO:2.

3. A method for identifying a modulator of Akt activity comprising:

(b) measuring an activity of Akt of said cell in the presence and absence of said candidate modulator, wherein a different level of Akt activity in the presence and absence of said candidate modulator identifies said candidate modulator as a modulator of Akt activity.

4. The method according to claim 3 further comprising adding a peptide comprising the amino acid sequence set forth in SEQ ID NO:2.

5. A method for identifying a modulator of p38 MAP Kinase activity comprising:

(b) measuring an activity of p38 MAP Kinase of said cell in the presence and absence of said candidate modulator, wherein a different level of p38 MAP Kinase activity in the presence and absence of said candidate modulator identifies said candidate modulator as a modulator of p38 MAP Kinase activity.

6. The method according to claim 5 further comprising adding a peptide comprising the amino acid sequence set forth in SEQ ID NO:2.

7. A method of modulating the activity of a protein selected from the group consisting of Heat Shock Protein 27, Akt and p38 MAP Kinase comprising delivering an effective amount of Competence and Sporulation Factor to an epithelial cell.

8. A method for identifying a modulator of a charged oligopeptide transport molecule comprising:

(a) contacting a charged oligopeptide transport molecule with a candidate modulator; and

(b) measuring an activity of said oligopeptide transport molecule in the presence and absence of said candidate modulator, wherein a different level of oligopeptide transport activity in the presence and absence of said candidate modulator identifies said candidate modulator as a modulator of said oligopeptide transport molecule.

9. The method according to claim 8 wherein the oligopeptide transport molecule is an organic cationic oligopeptide transport molecule.

10. The method according to claim 8 wherein the oligopeptide transport molecule is selected from the group consisting of OCTN2, OCTNl and MDR-I.

11. The method according to claim 10 wherein said oligopeptide transport molecule is OCTN2.

12. The method according to claim 8 wherein said modulator inhibits the transport activity of said oligopeptide transport molecule.

13. The method according to claim 8 wherein a mammalian intestinal epithelial cell comprises said oligopeptide transport molecule.

14. The method according to claim 8 further comprising adding a peptide selected from the group consisting of a Competence and Sporulation Factor, Plantaricin A and cPD-1.

15. The method according to claim 14 wherein said peptide comprises the amino acid sequence set forth in SEQ ID NO:2.

16. A method of determining the condition of intestinal flora comprising

(a) obtaining a sample of the intestinal contents; and

(b) measuring the level of a cationic peptide bacterial quorum sensing molecule in said sample, thereby obtaining a measure of the condition of intestinal flora.

17. The method according to claim 16 wherein the cationic peptide bacterial quorum sensing molecule is selected from the group consisting of a peptide comprising the sequence set forth in SEQ ID NO:2, Plantaricin A and cPD-1.

18. The method according to claim 16 wherein the measuring comprises performing an immunoassay specifically measuring the cationic peptide bacterial quorum sensing molecule.

19. Use of a binding partner specific for a cationic peptide bacterial quorum sensing molecule in the preparation of a medicament for determining the condition of intestinal flora.

20. A method of preventing or treating an intestinal disorder comprising administering a therapeutically effective amount of a protein therapeutic selected from the group consisting of Plantaricin A, cPD-1, Competence and Sporulation Factor and variants thereof, wherein each of said variants is a peptide of at least five amino acids in length and wherein each said variant comprises an adjacent pair of oppositely charged amino acids at pH 7.

21. The method according to claim 20 wherein the intestinal disorder is an inflammatory intestinal disorder.

22. The method according to claim 20 wherein the protein therapeutic is Competence and Sporulation Factor.

23. Use of a protein therapeutic selected from the group consisting of Plantaricin A, cPD-1, Competence and Sporulation Factor and variants thereof in the preparation of a medicament for preventing or treating an inflammatory intestinal disorder, wherein each of said variants is a peptide of at least five amino acids in length and wherein each said variant comprises an adjacent pair of oppositely charged amino acids at pH 7.

24. A method of preventing or treating an intestinal disorder comprising delivering a DNA comprising an expressible coding region for a cationic peptide transporter to an intestinal epithelial cell under conditions suitable for uptake of said DNA by said cell.

25. The method according to claim 24 wherein the intestinal disorder is an inflammatory intestinal disorder.

26. The method according to claim 24 wherein said DNA encodes 0CTN2.

27. Use of a DNA comprising an expressible coding region for a cationic peptide transporter in the preparation of a medicament for preventing or treating an intestinal disorder.

28. A method of preventing or treating an intestinal disorder comprising delivering an RNAi or an siRNA of a coding region selected from the group consisting of a cationic peptide transporter coding region and Ragl to an intestinal epithelial cell under conditions suitable for uptake of said RNAi or siRNA by said cell.

29. The method according to claim 28 wherein the intestinal disorder is an inflammatory intestinal disorder.

30. The method according to claim 28 wherein said RNAi or siRNA is complementary to an OCTN2 coding region.

31. Use of an RNAi or an siRNA of a coding region selected from the group consisting of a cationic peptide transporter coding region and Ragl in the preparation of a medicament for preventing or treating an intestinal disorder.

32. A method of preventing or treating an intestinal disorder comprising delivering an intestinal microorganism to the intestine, wherein said intestinal microorganism secretes a cationic peptide bacterial quorum sensing molecule.

33. The method according to claim 32 wherein the intestinal disorder is an inflammatory intestinal disorder.

34. The method according to claim 32 wherein said cationic peptide bacterial quorum sensing molecule is selected from the group consisting of a peptide comprising the sequence set forth in SEQ ID NO:2, Plantaricin A and cPD-1.

35. Use of an intestinal microorganism in the preparation of a medicament for preventing or treating an intestinal disorder, wherein said intestinal microorganism secretes a cationic peptide bacterial quorum sensing molecule.

36. The method according to claim 24, claim 28 or claim 32, wherein the intestinal disorder is selected from the group consisting of inflammatory disorders of the intestine, pathogenic infection, antibiotic-associated colitis, radiation enteritis, intestinal epithelial cell apoptosis and aberrant cell cycle regulation of an intestinal epithelial cell.

37. The method according to claim 36 wherein the intestinal disorder is Crohn's disease.