COMPOSITION AND METHODS FOR THE TREATMENT OF INFLAMMATORY DISEASES

Abstract: The expression of interleukines can be downregulated through the adminstration of a YopJ protein to the site of inflammation. The present invention discloses pharmaceutical compositions and methods for treatment of inflammatory disease in mammals, in particular for the treatment of inflammatory diseaese in the gastrointestinal tract in humans.

Description: COMPOSITION AND METHODS FOR THE TREATMENT OF INFLAMMATORY DISEASES Field of the invention The present invention concerns the treatment of inflammatory conditions in mammals and specifically an oral microbiological composition for the treatment of inflammatory diseases in the gastrointestinal tract. Prior art It is known that certain bacteria can inhibit inflammatory symptoms but the mode of action has hitherto not been fully elucidated. Yersinia spp. deliver a number of proteins, referred to as Yops, into the cytoplasm of eukaryotic cells via a type in secretion system that is present in several animal and plant pathogens (Cornells and Wolf-Watz, 1997). Up until now, the activity of only one of these cytoplasmically delivered Yops has been determined. YopH, which contains an eukaryotic-like phosphatase domain, likely dephosphorylates phosphotyrosine proteins involved in phagocytosis (Persson et al., 1997). Similarly, the other Yop proteins are presumed to interfere with cellular processes that normally occur in eukaryotic cells upon contact with bacteria. In rodents, Yersinia pestis and Y. pseudotuberculosis are able to colonize visceral organs and submucosal intestinal epithelial tissue, respectively, without eliciting infiltrating leukocytes to the site of infection (Une et al., 1986; Simonet et al., 1990). For both pathogens this ability is dependent on the presence of the virulence plasmide and has been proposed to be due to the ability of Yersinia spp. to inhibit infected cells from releasing of IL-8 (Schulte et al., 1996), a cytokine that serves as a chemoattractant for neutrophils (reviewed in Oppenheim et al., 1991). However, this phenomenon is not fully understood and its practical implications hitherto not disclosed. Simultaneously, the treatment of inflammatory diseases is a source of problems and discomfort for patients and physicians alike. The presently used pharmaceuticals, cortisone and derivatives thereof, such as the glycocorticoides, have severe drawbacks. The systemic administration of steroids can cause changes in the metabolism, such as disorders of the fluid balance, which can lead to pulmonary oedema. Additionally, part of the patients develops resistance or fail to respond to the treatment. The administration of salicylates, another group of compounds used in the treatment of inflammatory diseases, is associated with similar problems. Part of the patients fail to respond to the treatment and others develop a hypersensitivity. The salicylates themselves can also function as irritants of the gastric mucosa. Consequently, the objective of the present invention is to supply a new pharmaceutical composition and method for the treatment of inflammatory diseases, said composition and method being specific, effective and less harmful than the presently known approaches. Summary of the invention It has now been surprisingly found that YopJ affects an eukaryotic signaling pathway involved in the early host immune response. The gene encoding YopJ of Yersinia pseudo-tuberculosis has shown necessary and sufficient to suppress the expression of interleukin (IL)-8 in infected eukaryotic cells. The present invention discloses a new composition and method for the treatment of inflammatory diseases and in particular inflammatory diseases in the gastro-intestinal tract. Further, the invention comprises an oral composition and a method for colonizing the intestinal tract with suitable microorganisms expressing YopJ. The invention specifically discloses the incorporation of an organism expressing YopJ in an oral pharmaceutical composition, such as tablets or suspensions, or in a suitable food stuff, a so-called functional food. Short description of the drawings The present invention will be described in closer detail with reference to the examples and attached drawings, in which Figure 1 shows the message levels of various cytokines in HeLa cells infected with different strains of Y. pseudotuberculosis. Total RNA was isolated from either uninfected or infected cell cultures and analyzed by RT-PCR. Figure 2 shows the NF-κB site-binding activity and NF-κB mRNA and protein levels in HeLa cell extracts following infection with various strains of Y. pseudotuberculosis. Description of the invention The present inventors have surprisingly shown that that the gene encoding YopJ of the enteropathogen Yersinia pseudotuberculosis is necessary and sufficient to downregulate DNA binding activity NF-κB transcription factors following infection. The inventors have also shown that YopJ affects the levels of mRNAs encoding chemoattractant cytokines, whose inducibility have previously been shown to be dependent on NF-κB. Promoter activity of a NF-κB site-containing reporter gene is downregulated in eukaryotic cells infected with wild type Y. pseudotuberculosis compared to the isogenic yopJ mutant strain and furthermore this downregulation is dependent on a phosphorylation-competent form of IκB. The present inventors propose that YopJ, which has been shown to be delivered into the cytoplasm of infected cells by a type III secretion system, inhibits expression of chemoattractant cytokines primarily through the NF-κB signaling pathway and that this activity accounts for the lack of an inflammatory host response observed in experimental yersiniosis. The amino acid sequence of YopJ is attached to this application as Sequence No. 1 and the corresponding nucleotide sequence as Sequence No. 2. Both can readily be retrieved from public databases, e.g. under the accession number L33833. In screening Y. pseudotuberculosis strains deficient in various known virulence genes, the present inventors observed that HeLa cells infected with the yopJ mutant (Galyov et al. , 1994) released significantly more IL-8 compared to cells infected with the isogenic wildtype strain (118 ng/ml and 18 ng/ml, respectively). HeLa cells infected with the yopJ mutant containing a yopJ-encoding plasmide had IL-8 levels (20 ng/ml) similar to that observed in wildtype-infected cells. The inhibition of NF-κB-mediated signaling, either by targeted disruption of NF-κB- encoding genes (Sha et al., 1995) or by exposure to glucocorticoids (Scheinman et al., 1995) or NF-κB-specifιc antigens oligonucleotides (Neurath et al., 1996), result in a decreased inflammatory response. Likewise, YopJ apparently is able to block the various signals that feed into the NF-κB-mediated signaling pathway that are activated during a bacterial infection. In the absence of YopJ, it may be that the activities of the other cytoplasmically-delivered Yops activate NF-κB. It is interesting to note that yopJ shares some degree of sequence similarity to genes found in two species of bacteria, one a plant pathogen and the other a plant symbioant (Whalen et al., 1993; Freiberg et al., 1997). The latter, the nitrogen-fixing Rhizobium, which from sequence analysis may contain a type in secretion system, possess an ORF predicted to encode a protein 43% similar to YopJ (Freiberg et al, 1997) suggesting that a plant symbioant and an animal pathogen may perform similar functions during their interactions with eukaryotic cells. The term "inflammatory diseases" is, in this context, meant to include inflammatory diseases in general and inflammatory diseases in the gastrointestinal tract in particular. The former comprises e.g. rheumatoid arthritis, localized neuritis and sepsis. The latter comprises Crohn's disease (regional ileitis), colitis, ulcerative colitis and proctitis. Further examples of gastrointestinal diseases with an inflammatory etiology or symptoms are e.g. celiac disease and colon irritabile. The YopJ protein can, according to the present invention, be administered to the site of inflammation in many ways. The mode of administration is governed by the nature of the inflammatory disease, its location in the mammal to be treated and by other factors, apparent to the treating physician. On one hand, die YopJ protein can be administered as such, incorporated in suitable carriers, generally approved for pharmaceutical use, such as liquids, gels and solid carriers. Examples of carriers include sterile water for injection or saline, oils and fats, oil-in- water emulsions and conventional tabletting aids. On the other hand, the YopJ protein can be expressed by a suitable microorganism, colonizing or merely passing the site of inflammation. According to a preferred embodiment of the invention, a non-pathogenic microorganism expressing YopJ is administered to the site of inflammation. This non- pathogenic microorganism can be an existing microorganism or a transgenic organism, developed using conventional technology of genetic engineering. An organism of choice is naturally one listed as GRAS (Generally Regarded As Safe). It is specially preferred that the yopJ gene is cloned into a microorganism, presently accepted for use in foodstuffs. Preferably said non-pathogenic microorganism expressing YopJ is administered orally to a patient, in a composition so formulated, that it retains its viability in the passage through the ventriculus and intestines and accordingly can express and release YopJ protein in the gastrointestinal tract. Said microorganism or culture of microorganisms can be incorporated in a pharmaceutical composition as such or after freezing, drying, lyophilization, or similar techniques, making the composition more easy to store awaiting use. Suitable techniques also include microencapsulation, following which the encapsulated microorganism can be incorporated in optional food stuff or pharmaceutical composition. When the yopJ gene is cloned into a microorganism, filling the GRAS-requirements, the resulting transgenic micro-organism can be incorporated in or used for the production of a foodstuff without the destruction of the functionality of the yopJ gene or the YopJ protein, encoded by said gene. Examples of suitable foodstuffs include, but are not limited to, fermented foodstuffs such as fermented dairy products, e.g. yogurt, sour milk, kefir and similar products, and fermented cereal products, such as fermented oatmeal gruel and similar products. Examples of suitable host microorganisms, presently used in foodstuffs, include the Lactobacillus species, e.g. L. plantarum, L. lactis, L. cremoris, L. diacetylactis, L. bulgaricus, L. acidophilus, Leuconostoc cremoris, Streptococcus thermophilus, Candida kefir, the Saccaromyces species and related organisms. Naturally, also other microorganisms can be used. In the case that the chosen microorganism lacks the property of externalizing the YopJ protein, this property should be incorporated in the microorganism during the genetic engineering of the same. The present invention further comprises a method of treating inflammatory diseases, in particular inflammatory diseases in the gastrointestinal tract in humans, characterized in that the intestinal tract is more or less colonized with microorganisms expressing a YopJ-type protein. By colonization in this context is meant that the microorganism passes through the gastrointestinal tract with unimpaired viability and with its YopJ expressing function intact, and that this passage is slow enough for the expressed YopJ to have die desired effect on the site of inflammation. The colonization is achieved by administering to the patient a viable culture of microorganisms at intervals and in amounts, sufficient to ensure a fairly stabile level of YopJ at the site of inflammation. The exact dosage as to amounts and intervals can be determined by the treating physician. Most preferably, the transgenic microorganism according to the invention is incorporated in a so-called functional foodstuff, a foodstuff exhibiting therapeutic properties to a certain degree, without being classified as a drug. An example can be a fermented cereal product, e.g. on oatmeal basis, comprising a viable culture of microorganisms expressing YopJ. Such a product could ttien be ingested by patients suffering from inflammatory diseases in the gastrointestinal tract. The microorganism would then colonize the intestines of said patients and the YopJ protein expressed in me intestinal lumen could help to alleviate the symptoms of, or possibly cure the inflammatory disease without the systemic effects and side effects of presently used pharmaceutical regimens. A viable culture of microorganisms, meeting the GRAS requirements and expressing a protein of YopJ type, can also be incorporated in tablets, suspensions and other forms, suitable for oral ingestion. Microencapsulated microorganisms could, e.g. be provided in a dry, particulate composition or powder to be mixed with a suitable liquid before ingestion. Preferably said powder further includes conventional additives, such as compounds increasing the storage time, anti caking agents, coloring and flavoring agents etc. The present invention can naturally also find application in the local treatment of inflammatory diseases outside the intestinal tract. A pharmaceutical composition comprising a YopJ protein can be applied locally, for example topically to inflammated areas. A suitable vehicle is e.g. an oil-in- water emulsion or gel. It is also contemplated, that a protein of YopJ type can find utility in the systemic treatment of serious inflammatory diseases, such as sepsis. A composition according to the present invention can also find utility in the treatment of other diseases that are accompanied by increased local cytokine expression levels, regardless of their cause, i.e. bacterial infections and macrophage triggered reactions. Different bacteria is often very opportune to colonize inflammated sites, in particular ulcers in the gastrointestinal tract and skin lesions associated with psoriasis, mentioning only two, non-limiting examples. The administration of a protein of YopJ type through an expression system, i.e. a GRAS classified organism expressing the desired protein, will have the added positive effect of balancing or competing with and potentially eradicate the pathogen or unwanted organisms. It has further been shown that the YopJ protein can inhibit phosphorylation of the kinase p38. This inhibition is highly specific. It is therefore suggested by the present inventors to use pure YopJ and/or coupled to a delivery substance or cassette, such as a DNA expression cassette or adeno virus vector, as a probe for discrimination or identification of different signal transduction pathways. YopJ would thus function as a specific instrument for mapping signal transduction pathways. Examples Example 1. Mutant strain of Y. pseudotuberculosis containing deletions in vopM. vopE. vopK. vpkA. and vopj To determine whether YopJ affects IL-8 levels in the absence of the other known Y. Pseudotuberculosis ' Yop virulence genes, the present inventors utilized a mutant strain of Y. pseudotuberculosis containing deletions in yopM, yopE, yopK, ypkA, and yopJ, designated as MYM (Hakansson et al., 1996a). The present inventors observed a more than four-fold decrease in the amount of IL-8 secreted by HeLa cells infected with the MYM strain containing a YopJ-expressing plasmide compared to HeLa cells infected with the parental MYM strain (34 ng/ml and 9 ng/ml, respectively). HeLa cells infected with a MYM strain containing a plasmide encoding YopM (Leung et al, 1990) secreted levels of IL-8 (30 ng/ml) that were similar to the parental MYM strain. The present inventors cannot account for the differences in the levels of IL-8 secreted between HeLa cells infected with the yopJ strain and the MEKA strain. These data indicate that yopJ encodes a product that affects the amount of IL-8 secreted by infected HeLa cells. Example 2. RT-PCR analysis of RNA from infected and uninfected HeLa cells To determine whether YopJ affected steady-state IL-8 mRNA levels, total RNA was isolated from either uninfected HeLa cells or cells infected with various Y. pseudotuberculosis strains and analyzed by RT-PCR (fig. 1). The differences in IL-8 mRNA levels between uninfected- and wildtype-infected HeLa cells were probably not significant due to differences in the amounts of input RNA between the two samples as indicated by differences in beta-actin mRNA levels. IL-8 message levels were significantly higher in cells infected with the yopJ strain compared to either the wildtype or the complemented yopJ strains (compare lanes 3 with 2 and 4). Increased IL-8 mRNA levels were also observed in cells infected with the yopB strain (lane 5). The yopB gene product has been shown to be required for d e delivery of Yop virulence factors into infected eukaryotic cells (Hakansson et al., 1996b). Two other cytokines that have been reported to be chemoattractants for neutrophils, IL-1 (and MGSA (Dinarello et al., 1991; Richmond et al., 1985), had similar patterns of expression as IL-8 in HeLa cells infected widi the various Y. pseudotuberculosis strains (fig. lc and Id). The YopJ-mediated effect on IL-8 message levels was not dependent on the bacterium invading the host cell since this effect was also observed in HeLa cells pretreated widi cytochalasin D (not shown) which prevents the uptake of bacteria into eukaryotic cells (Finlay and Falkow, 1988). Example 3. Transient transfection of HeLa cells To investigate whether YopJ affected IL-8 promoter activity, HeLa cells were transiently transfected widi a reporter gene under the control of the IL-8 promoter and then infected with the various bacterial strains. HeLa cells infected with wildtype Y. pseudotuberculosis had lower levels of luciferase activity compared to me uninfected control cells (table I). In contrast, HeLa cells infected wid the yopJ strain had higher levels of luciferase activity than cells infected wid the wildtype strain. The YopJ-mediated effect on IL-8 promoter activity mimics the pattern observed in IL-8 secretion and mRNA levels and suggest mat YopJ affects IL-8 expression at the transcriptional level. The IL-8 promoter region contains a number of binding sites specific for various families of transcription factors (Mukaida et al., 1989). Inducibility of IL-8 promoter activity, as well as the promoters of IL-1 (and MGSA, have been shown to be dependent on their respective NF- B binding site (Kunsch and Rosen, 1993; Matsusaka et al., 1993; IL-1 and MGSA refs). To determine whemer YopJ modulates me activity of NF-κB transcription factors, HeLa cells were transiently transfected with a reporter gene under die control of the TK promoter containing two NF-κB binding sites and were infected widi various strains of Y. pseudotuberculosis. The expression pattern of the NF-κB-TK promoter in uninfected, wildtype infected, and yopJ infected HeLa cells was similar to the expression pattern observed in HeLa cells transfected widi me IL-8 promoter-containing reporter gene (table I) suggesting that NF-KB site-binding transcription factors are involved in me YopJ-mediated inhibition of IL-8 promoter activity. Example 4. Influence of phosphorylation-competent IκB NF-κB site-binding transcription factors are retained in d e cytoplasm of uninduced cells by IκB proteins. Upon the appropriate stimulus, IκB is phosphorylated and degraded thereby unmasking NF-κB's nuclear localization signals and allowing NF-κB to translocate to the nucleus (Baeuerle and Baltimore, 1988). To test whether the effects on the NF-κB site-containing promoter described above were dependent on a phosphorylation-competent IκB, the present inventors cotransfected the NF-κB-TKp-luciferase plasmide widi a plasmide encoding eid er a wildtype or a dominant-negative mutant (i.e. lacking the phosphorylation sites) form of IκB followed by infecting the transformants with either YopJ-t- or YopJ- strains of Y. pseudotuberculosis. Luciferase activity in extracts prepared from wildtype IκB transformants infected with the YopJ- strain were two-fold higher compared to levels observed in extracts prepared from wildtype IκB transformants infected widi YopJ+ bacteria (table I). In contrast, levels of luciferase activity in extracts prepared from cells transformed with the mutated IκB were not higher following infection with a YopJ- strain compared to a YopJ+ strain. These data support a model in which YopJ blocks NF-κB-mediated signaling upstream of IκB phosphorylation. Example 5. Binding activity in cells infected with various Y. pseudotuberculosis strains NF-κB site-binding transcription factors are activated by various stress-related stimuli including exposure to bacterial padiogens such as Listeria monocytogenes and Shigella flexneri (Hauf et al, 1994; Dyer et al., 1993). NF-κB site-binding activity was measured in nuclear extracts prepared from HeLa cells infected wid various Y. pseudotuberculosis strains in order to determine whether YopJ affects the activation of NF- SUBSTΓΓUTE SHEET (RULE 26) KB site transcription factors. There is a several-fold increase in binding activity in cells infected widi the yopB strain (see above) of Y. pseudotuberculosis compared to uninfected cells (fig. 2a) suggesting tiiat a functional type in secretion system is required for YopJ to block the activation of NF- B. The level of binding activity in extracts prepared from cells infected widi eitiier the wildtype or the yopH strains (Persson et al., 1995) of Y. pseudotuberculosis were similar to uninfected controls in contrast to the several-fold increase in binding activity observed in extracts prepared from cells infected wid the YopJ- strain. Wildtype-like binding activity could be restored to extracts prepared from cells infected with the trans-complemented YopJ- strain (compare lanes 3 and 6). Differences in NF-κB site-binding activity between extracts was not due to differences in levels of invasion among the various Yersinia strains since an identical pattern of binding activity was observed in the presence of cytochalasin D (not shown). A similar pattern of NF-κB site- binding activity was observed in die B cell line K46 infected widi either YopJ+ or YopJ- strains of Y. pseudo-tuberculosis (not shown). Increased NF-κB site-binding activity in YopJ—infected nuclear extracts were not accompanied with increased mRNA levels of the NF-κB transcription factors p65 and pi 05 nor widi changes in p65 protein levels (fig. 2b and 2c, respectively). Taken together, these data suggest tiiat the role of YopJ during die infection process is to block the translocation of NF-κB transcription factors to the nucleus, which normally occurs in eukaryotic cells following contact with pathogenic bacteria. Example 6. Determination whether the reporter protein was exported from the bacterial cell The dependence of me YopJ-mediated effect on IL-8 mRNA levels and NF-κB site- binding activity on yopB, whose gene product has been demonstrated to be required for die delivery of other Yops into d e cytoplasm of infected eukaryotic cells (Hakansson et al., 1996b), suggest that YopJ exerts its activity from within the eukaryotic cell. The present inventors constructed a reporter gene containing yopJ fused to die adenylate cyclase- encoding domain of d e cyclolysin gene of Bordetella pertussis (Sory and Cornells, 1994). The activity of the resulting fusion protein is activated by calmodulin, an intracellular eukaryotic protein. The subcellular location of YopJ-Cya was compared to YopE-Cya and YopB-Cya; me former has been shown to be both exported from the bacterial cell and delivered into d e cytoplasm of infected eukaryotic cells (Sory et al., 1995) while YopB-Cya has been shown to be exported from die bacterial cell but not delivered into eukaryotic cells (Hikansson et al., 1996b). YopE has been shown to cause a disruption of actin microfilaments resulting in an inability of die infected cell to undergo the cytoskeletal rearrangements necessary for phagocytosis (Rosqvist et al., 1991). To determine whether the reporter protein was exported from the bacterial cell, intact cells of Y. pseudotuberculosis containing the YopJ-Cya-encoding plasmide were assayed for calmodulin-dependent cyclase activity (Schesser et al, 1996). Low but significant levels of cyclase activity were detected in yopJ-cya-containing Y. pseudotuberculosis cells while in cells containing either a yopE-cya- or a yopB-cya-containing plasmides had significantiy higher levels of cyclase activity. Extracts prepared from HeLa cells infected widi the yopJ- cya-containing MYM strain (Hakansson et al., 1996a) had cAMP levels similar to the levels observed in extracts prepared from HeLa cells infected with the yopE-cya-containing MYM strain. In contrast, cAMP was not detected in uninfected HeLa cell extracts or in extracts prepared from HeLa cells infected widi a yopB-cya-containing MYM strain. These data suggest that YopJ-Cya is similar to YopE-Cya in terms of its delivery into the cytoplasm of eukaryotic cells during infection. Table 1. Luciferase activity in HeLa cells transiently transfected with NF-κB site-containing reporter genes and infected widi various strains of Y. pseudotuberculosis. Cells were transfected wid 26 (g of eitiier IL-8p-luc or NF-κB-TKp-luc with eittier none or 5 (g of the IκB-encoding plasmides as indicated. Two days following transfection cells were subcultured onto four separate dishes and subsequently infected widi d e indicated Y. pseudotuberculosis strains as described (Schesser et al., 1996). The data is representative of at least three separate experiments. Relative Luciferase Activity Reporter gene(s) Uninfected Wildtvpe YopJ- YopJ-/vopJ+ IL-8p-luc 1.0 0.5 1.2 0.7 NF-KB-TKp-luc 1.0 0.6 1.3 0.9 NF-κB-TKp-luc + CMVp- - 1.0 2.3 - wtIκB NF-KB-TKp-luc + CMVp- - 1.0 1.2 - mutIκB Table 2. Export of various Yop-Cva hybrid proteins bv Y. pseudotuberculosis and their subsequent delivery into die cytoplasm of infected HeLa cells. Data is reported as d e mean of diree measurements of a representative experiment (+ the standard deviation). Whole Cell Cyclase Activity " (pmol cAMP/minODml) Relative genotypes Infections b -calmodulin + calmodulin (pmol cAMP/well) yopJ-cya 18 (1) 39 (2) 0.53 (0.03) yopE-cya 49 (4) 502 (40) 0.61 (0.02) yopB-cya 14 (4) 557 (36) < 0.05 'Bacteria were collected after being induced for Yop expression, washed, and resuspended in cyclase activity assay buffer either in die absence or presence of calmodulin as described (Schesser et al., 1996). "Overnight bacterial cultures were diluted in antibiotic-free tissue culture medium and induced for Yop expression before being added to HeLa cells. Four hours following d e addition of bacteria HeLa cells were washed and whole cell lysates were prepared and measured for cAMP as described (Schesser et al., 1996). Although d e invention has been described widi regard to its preferred embodiments, which constitute die best mode presently known to die inventors, it should be understood tiiat various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from d e scope of d e invention which is set forth in the claims appended hereto. SUBSTTTUTE SHEET (RULE 26) References Anisowicz, A., M. Messineo, S. Lee, and R. Sager. 1991. An NF-kappa B-like transcription factor mediates IL-1/TNF-alpha induction of gro in human fibroblasts. J. Immunol. 147:520-527. Baeuele, P., and D. Baltimore. 1988. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-κB transcription factor. Cell 53:211-217. Beauparlant, P., and J. Hiscott. 1996. Biological and biochemical inhibitors of the NF- kappa B/Rel proteins and cytokine synthesis. Cytokine Growth Factor Rev. 7:175-190. Cornells, G., and H. Wolf-Watz. 1997. Yop review in Trends in Microbiology. Dinarello, C. 1991. Interleukin-1 and interleukin-1 antagonism. Blood 77:1627-1652. Dyer R., C. CoUaco, D. Niesel, and N. Herzog. 1993. Shigella flexneri invasion of HeLa cells induces NF-κB DNA-binding activity. Infect. Immun. 61:4427-4433. Eckmann, L., M. Kagnoff, and J. Fierer. 1993. Epitiielial cells secrete die chemokine interleukin-8 in response to bacterial entry. Infect. Immun. 61:4569-4574. Finaly, B., and S. Falkow. 1988. Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication. Biochimie 70:1089-1099. Galyov, E., S. Hakansson, and H. Wolf-Watz. 1994. Characterization of the operon encoding die YpkA ser/thr protein kinase and die YopJ protein of Yersinia pseudotuberculosis. J. Bact. 176:4543-4548. Hakansson, S., E. Galyov, R. Rosqvist, and H. Wolf-Watz. 1996a. The Yersinia YpkA ser/thr kinase is translocated and subsequently targeted to die inner surface of die HeLa cell plasma membrane. Mol. Microbiol. 20:593-603. Hakansson, S., K. Schesser, C. Persson, E. Galyov, R. Rosqvist, F. Homble, and H. Wolf-Watz. 1996b. The YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across die target cell plasma membrane and displays a contact-dependent membrane-disrupting activity. EMBO J. 15:5812-5823. Hauf, N., W. Goebel, E. Serfling, and M. Kuhn. 1994. Listeria monocytogenes infection enhances transcription factor NF-κB in P388D1 macrophage-like cells. Infect. Immun. 62:2740-2747. Characterization of a functional NF-kappa B site in the human interleukin 1 beta promoter: evidence for a positive autoregulatory loop. AU: Hiscott-J; Marois-J; Garoufalis-J; D'Addario-M; Roulston-A; Kwan-I; Pepin- N; Lacoste-J; Nguyen-H; Bensi-G; et-al SO: Mol-Cell-Biol. 1993 Oct; 13(10): 6231-40 Kunsch, C, and C. Rosen. 1993. NF-κB subunit-specific regulation of the interleukin-8 promoter. Mole. Cell. Biol. 13:6137-6146. Leung, K., B. Reisner, and S. Straley. 1990. YopM inhibits platelet aggregation and is necessary for virulence of Yersinia pestis in mice. Infect. Immun. 58:3262-3271. Matsusaka, T., K. Fujikawa, Y. Nishio, N. Mukaida, K. Matsushima, T. Kishimoto, and S. Akira. 1993. Transcription factors NF-IL6 and NF-κB synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc. Natl. Acad. Sci. USA 90:10193-10197. Mukaida, N., M. Shiroo, and K. Matsushima. 1989. Genomic structure of die human monocyte-derived neutrophil chemotactic factor IL-8. J. Immunol. 143:1366-1371. Oppenheim, J., C. Zachariae, N. Mukaida, and K. Matsushima. 1991. Properties of the novel proinflammatory supergene 'intercrine' cytokine family. Ann. Rev. Immunol. 9:617- 634. Persson, C, R. Nordfelth, A. Holmstrδm, S. Hakansson, R. Rosqvist, and H. Wolf-Watz. 1995. Cell-surface-bound Yersinia translocate the protein tyrosine phosphatase YopH by a polarized mechanism into the target cell. Mol. Microbiol. 18:135-150. Ray, A., and K. Prefontain. 1994. Physical association and functional antagonism between the p65 subunit of transcription factor NF-κB and die glucocorticoid receptor. Proc. Natl. Acad. Sci. USA 91:752-756. Richmond A., D. Lawson, D. Nixon, and R. Chawla. 1985. Characterization of autostimulatory and transforming growth factors from human melanoma cells. Cancer Res. 45:6390-6394. Rosqvist, R., A. Forsberg, and H. Wolf-Watz. 1991. Intracellular targeting of the Yersinia YopE cytotoxin in mammalian cells induces actin microfilament disruption. Infect. Immun. 59:4562-4569. Rossi, A., G. Elia, and M. Santoro. 1997. Inhibition of nuclear factor KB by prostaglandin Al: An effect associated widi heat shock transcription factor activation. Proc. Natl. Acad. Sci. USA 94:746-750. Scheinman, R., P. Cogswell, A. Lofquist, and A. Baldwin. 1995. Role of transcriptional activation of IκB α in mediation of immunosuppression by glucocorticoids. Science 270:283-286. Schesser, K., E. Frithz-Lindsten, and H. Wolf-Watz. 1996. Delineation and mutational analysis of the Yersinia pseudotuberculosis YopE domains which mediate translocation across bacterial and eukaryotic cellular membranes. J. Bact. 178:7227-7233. Schulte, R., P. Wattiau, E. Hartland, R. Robins-Browne, and G. Cornells. 1996. Differential secretion of interleukin-8 by human epithelial cell lines upon entry of virulent or nonvirulent Yersinia enterocolitica. Infect. Immun. 64:2106-2113. Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses. AU: Sha-WC; Liou-HC; Tuomanen-EI; Baltimore-D SO: Cell. 1995 Jan 27; 80(2): 321-30 Simonet, M., S. Richard, and P. Berche. 1990. Electron microscopic evidence for in vivo extracellular localization of Yersinia pseudomberculosis harboring the pYV plasmide. Infect. Immun. 58:841-845. Sory, M.-P., and G. Cornells. 1994. Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol. Microbiol. 14:583-594. Sory, M.-P, A. Boland, I. Lambermont, and G. Cornells. 1995. Identification of d e YopE and YopH domains required for secretion and internalization into the cytosol of macrophages. Proc. Natl. Acad. Sci. USA 92:11998-12002. HMGI(Y) and Spl in addition to NF-kappa B regulate transcription of the MGSA/GRO alpha gene. AU: Wood-LD; Farmer-AA; Richmond-A SO: Nucleic-Acids-Res. 1995 Oct 25; 23(20): 4210-9 Une, T., R. Nakajima, and R. Brubaker. 1986. Roles of V antigen in promoting virulence in Yersinia. Contrib. Microbiol. Immunol. 9:179-185. SEQUENCE ID NO: 1 YopJ SEQUENCE TYPE: amino acid SOURCE: Yersinia pseudotuberculosis l MIGPISQINISGGLSEKETSSLISNEELKNΠTQLETDIS 41 DGSWFHKNYSRMDVEVMPALVIQANNKYPEMNLNLVTSPL 8i DLSIEIKNVIENGVRSSRFΠNMGEGGIHFSVIDYKHING 121 KTSLILFEPANFNSMGPAMLAIRTKTAIERYQLPDCHFSM 161 VEMDIQRSSSECGIFSFALAKKLYIERDSLLKIHEDNIKG 201 ILSDGENPLPHDKLDPYLPVTFYKHTQGKKRLNEYLNTNP 221 QELVLLLTKKMKPSLIDLITINPL SEQUENCE ID NO: 2 SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 4963 STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: genomic DNA ORIGINAL SOURCE: Yersinia pseudotuberculosis 1 aatcactaaa aatcagtggc tggaagtgat atattctggg acggacgggg tttaatcaat 61 gatagatatc accgtaaatc agattaaaga gcttttgtgt gattaacacc acctttactg 121 agttactccc aaaaatagca agtcactttg gattagataa attgagccaa gatgaatatg 181 gcttgtgtga gcttatcctc aacgaccgag tcgttattat gctgagggct gatgaaatat 241 tgaatcgatt gactctgttg gggccaatct taggattttc tggaccagag gcgcgcagcg 301 ccgctagtca gctttttttc tgttatagca tcaatgcctt gaataaggac ggcccttgtt 361 tcgcttggag tgaagaactg gggctgatcg cattcaagca cctttctctc gacgagctga 421 atgttgagaa cgttagcaag gagatagcga acttttacga ctggttgagc ttggtcagtt 481 taccagcaga aactcagcag gaactgcccc ttcatactca atctactcaa tcggttaaat 541 ggggatgagt aaagcatgaa aagcgtgaaa atcatgggaa ctatgccacc gtcgatctcc 601 ctcgccaaag ctcatgagcg catcagccaa cattggcaaa atcctgtcgg tgagctcaat 661 atcggaggaa aacggtatag aattatcgat aatcaagtgt tgcgcttgaa cccccacagt 721 ggtttttctc tctttcgaga aggggttggt aagatctttt cggggaagat gtttaacttt 781 tcaattgctc gtaaccttac tgacacactc catgcggccc agaaaacgac ttcgcaggag 841 ctaaggtctg atatccccaa tgctctcagt aatctctttg gagccaagcc acagaccgaa 901 ctgccgctgg gttggaaagg ggagcccttg tcaggagctc cggatcttga agggatgcga 961 gtggctgaaa ccgataagtt tgccgagggc gaaagccata ttagtataat agaaactaag 1021 gataagcagc ggttggtagc taagattgaa cgctccattg ccgaggggca tttgttcgca 1081 gaactggagg cttataaaca catctataaa accgcgggca aacatcctaa tcttgccaat 1141 gttcatggca tggctgtggt gccatacggt aaccgtaagg aggaagcatt gctgatggat 1201 gaggtggatg gttggcgttg ttctgacaca ctaagaaccc tcgccgatag ctggaagcaa 1261 ggaaagatca atagtgaagc ctactgggga acgatcaagt ttattgccca tcggctatta 1321 gatgtaacca atcaccttgc caaggcaggg gtagtacata acgatatcaa acccggtaat 1381 gtggtatttg accgcgctag cggagagccc gttgttattg atctaggatt acactctcgt 1441 tcaggggaac aacctaaggg gtttacagaa tccttcaaag cgccggagct tggagtagga 1501 aacctaggcg catcagaaaa gagcgatgtt tttctcgtag tgtcaaccct tctacattgt 1561 atcgaaggtt ttgagaaaaa tccggagata aagcctaatc aaggactgag attcattacc 1621 tcagaaccag cgcacgtaat ggatgagaat ggttatccaa tccatcgacc tggtatagct 1681 ggagtcgaga cagcctatac acgcttcatc acagacatcc ttggcgtttc cgctgactca 1741 agacctgatt ccaacgaagc cagactccac gagttcttga gcgacggaac tatcgacgag 1801 gagtcggcca agcagatcct aaaagatacc ctaaccggag aaatgagccc attatctact 1861 gatgtaaggc ggataacacc caagaagctt cgggagctat ctgatttgct taggacgcat 1921 ttgagcagtg cagcaactaa gcaattggat atgggggggg ttttgtcgga tcttgatacc 1981 atgttggtgg cactcgacaa ggccgaacgc gaggggggag tagacaagga tcagttgaag 2041 agttttaaca gtttgattct gaagacttac agagtgattg aagactatgt caaaggcaga 2101 gaaggggata ccaagaattc cagtacggaa gtatccccct atcatcgcag taactttatg 2161 ctatcgatcg tcgaaccttc actgcagagg atccagaagc atctggacca gacacactct 2221 ttttctgata tcggttcact agtgcgcgcc aataagcacc tggaaacgct tttagaggtc 2281 ttagtcacct tgtcacagca agggcagccc gtgtcctctg aaacctacgg cttcctgaat 2341 cgattagctg aggctaagat caccttgtcg cagcaattga acactctcca gcagcagcag 2401 gagagtgcga aagcgcaact atctattctg attaatcgtt caggttcttg ggccgatgtt 2461 gctcgtcagt ccctgcagcg ttttgacagt acccggcctg tagtgaaatt cggcactgag 2521 cagtataccg caattcaccg tcagatgatg gcggcccatg cagctattac gctacaggag 2581 gtatcggagt ttactgatga tatgcgaaac tttacagtgg actctattcc actactgatt 2641 caacttggac gaagcagttt aatggatgag catttggttg aacagagaga aaagttgcga 2701 gagctgacga ccatcgccga gcgactgaac cggttggagc gggaatggat gtgacaagtg 2761 ccccctaagc cttgagttga tatatccgag aataggttaa gatttggcaa ttgcttaaca 2821 ataattattt tcttattaaa aatacgtaac acaaaaaata cgttatatat acaaatgaaa 2881 attccagtat taatctcaac aagtttctct accggagaat attaatctgg aatgtgtaat 2941 agagaaaatt tttgatgcta tcaaattttc ctttttcgca gaaatatcca atgtataggt 3001 atgatagaag ttattgggaa tttttgttcg agtgctgccc gtctgttcct ggttcaccca 3061 tcaacgattg aacgtcttat tgcaatgtac cgtttatctg gaatataaaa ttcataccgc 3121 tgttaattcc ctgaataagg ataaataaat gatcggacca atatcacaaa taaatatctc 3181 cggtggctta tcagaaaaag agaccagttc tttaatcagt aatgaagagc ttaaaaatat 3241 cataacacag ttggaaactg atatatcgga tggatcctgg ttccataaaa attattcacg 3301 tatggatgta gaagtcatgc ccgcattggt aatccaggcg aacaataaat atccggaaat 3361 gaatcttaat cttgttacat ctccattgga cctttcaata gaaataaaaa acgtcataga 3421 aaatggagtt agatcttccc gcttcataat taacatgggg gaaggtggaa tacatttcag 3481 tgtaattgat tacaaacata taaatgggaa aacatctctg atattgtttg aaccagcaaa 3541 ctttaacagt atggggccag cgatgctggc aataaggaca aaaacggcta ttgaacgtta 3601 tcaattacct gattgccatt tctccatggt ggaaatggat attcagcgaa gctcatctga 3661 atgtggtatt tttagttttg cactggcaaa aaaactttac atcgagagag atagcctgtt 3721 gaaaatacat gaagataata taaaaggtat attaagtgat ggtgaaaatc ctttacccca 3781 cgataagttg gacccgtatc tcccggtaac tttttacaaa catactcaag gtaaaaaacg 3841 tcttaatgaa tatttaaata ctaacccgca ggagttggta ctgttgttaa caaaaaaaat 3901 gaaaccatcg ttaatagatt tgataacaat aaatccattg tagatggaaa ggaattatca 3961 gtttcggtac ataaaaagag aatagctgaa tataaaacac ttctcaaagt ataatgtatt 4021 ttggaaatct tgctccagta tgggaatacg gttcagttct ttctggctca tggtcaccaa 4081 catagacgct tcggattgcc tgcctgtgaa gaaacagatt aactggggtt ctacgccgga 4141 atcccagatt tttccgtcac cccagtttca gcgctgctag agtacgggtg gtatgagccg 4201 ctagcagaag ctctaaatag taacttcttc caatggccga aaaagaaagc gttaaaaaat 4261 cacagtacgg gcatttctcg ggtttacgtt atttgtgcag aacgcacaaa tcaggttatt 4321 agatattatt gcttatgaac gggtagtatt cagcgaaata cagctcctaa ataactgcac 4381 caaatagtag atcactgagg gaactcaatc cggtttaagc gatctgatca atcgctgaat 4441 atcccaaatc accacaaccg gactgagtta tgccgatcat agcaccgata cccagaaata 4501 aacgacatca gatggaaaaa attgtccata aaacagcaga caaaaaccat tccagacatc 4561 tcatcgctga tccctcccca atatccgtac caggctaaat cagagatccg gacctttttg 4621 atgacttcgg gcaaattctg ccggagtcag gttatttaac gaagaatgcg gacgaaaatg 4681 attatattct tgccgccatt gttcaatttt ctcctgagca tcttccagag aaaggaaccc 4741 gtgcacgttc agacattcat ccctcagact gccattaaat gactcgataa aggcattatc 4801 tgtaggcttt ccggggcgtg aacagtccat cgtgaccctg ttttcatacg cccatcggtc 4861 catcgacttc gagatgaatt cgctgccgtt atctgtctgc ag

Claims: Claims 1. Pharmaceutical composition, characterized in that said composition contains effective amounts of a YopJ-type protein. 2. Pharmaceutical composition for treatment of inflammatory diseases in mammals, in particular for the downregulation of interleukine expression, characterized in that said composition contains effective amounts of a YopJ protein. 3. Pharmaceutical composition for treatment of inflammatory diseases in die gastrointestinal tract in humans, characterized in that said composition contains effective amounts of a YopJ protein. 4. Pharmaceutical composition according to any one of claims 1 to 3, characterized in tiiat said YopJ protein has the following amino acid sequence: SEQUENCE ID NO: 1 5. Pharmaceutical composition for the treatment of inflammatory diseases in the gastrointestinal tract in humans, characterized in that said composition contains at least one non-pathological microorganism with a functional gene expressing a YopJ protein. 6. Pharmaceutical composition according to claim 5, characterized in that said non-pad ological microorganism or microorganisms is/are chosen from the following group: Lactobacillus plantarum, L. lactis, L. cremoris, L. diacetylactis, L. bulgaricus, L. acidophilus, Leuconostoc cremoris, Streptococcus thermophilus, Candida kefir and related organisms or a combination thereof. 7. Method for the treatment of inflammatory diseases in mammals, in particular for the downregulation of interleukine expression, characterized in that a composition containing effective amounts of a YopJ protein is administered to die site of the inflammation. 8. Method for the treatment of treatment of inflammatory diseases in the gastrointestinal tract in humans, characterized in that a non-patiiological microorganism with a functional gene expressing a YopJ protein is admimstered near and/or at a site of inflammation. 9. Method for die treatment of treatment of inflammatory diseases in the gastrointestinal tract in humans, characterized in that a non-pathological microorganism with a functional gene expressing a YopJ protein is administered orally to a patient in a manner, allowing the microorganism to colonize parts of the gastrointestinal tract of said patient. 10. Metiiod according to claim 9, characterized in that said non-patiiological microorganism or microorganisms is/are chosen from the following group: Lactobacillus plantarum, L. lactis, L. cremoris, L. diacetylactis, L. bulgaricus, L. acidophilus, Leuconostoc cremoris, Streptococcus thermophilus, Candida kefir and related organisms or a combination thereof. 11. Use of a YopJ protein for the manufacture of a pharmaceutical composition. 12. Use of a YopJ protein for the manufacmre of a composition for the treatment of inflammatory diseases in mammals. 13. Use of a YopJ protein for the manufacmre of a composition for the treatment of inflammatory diseases in the gastrointestinal tract of humans. 14. Use of a YopJ protein for the manufacmre of a composition for the treatment of a disease chosen from the following group: rheumatoid arthritis, localized neuritis, sepsis, Crohn's disease (regional ileitis), colitis, ulcerative colitis, proctitis, celiac disease and colon irritabile or combinations thereof. 15. A transgenic non-pathological microorganism, characterized in that said microorganism contains a gene encoding a YopJ protein and said protein is expressed by the microorganism. 16. Use of a transgenic microorganism according to claim 15 for the treatment of inflammatory diseases in d e gastrointestinal tract in humans. 17. Use of a transgenic microorganism according to claim 15 for the manufacmre of a composition for the treatment of inflammatory diseases in the gastrointestinal tract of humans. 18. Use of a transgenic microorgamsm according to claim 15 for the manufacmre of a composition for die treatment of a disease chosen from the following group: rheumatoid arthritis, localized neuritis, sepsis, Crohn's disease (regional ileitis), colitis, ulcerative colitis, proctitis, celiac disease and colon irritabile or combinations thereof. 19. Use of a YopJ protein or fragments thereof as a probe. 20. Use of a YopJ protein or fragments tiiereof in die mapping of signal transduction patiiways. 21. A probe for mapping of signal transduction pathways, wherein said probe comprises a YopJ protein or fragments thereof.

Inventors: PETTERSSON SVEN, WOLF-WATZ HANS, PETTERSSON, SVEN, WOLF-WATZ, HANS
Applicants: KAROLINSKA INNOVATIONS AB, PETTERSSON SVEN, WOLF WATZ HANS, PETTERSSON, SVEN, WOLF-WATZ, HANS
Application year: 1998
Main classification: C07K14
IPC codes: A61K38, A61K48, A61P29, C07K14