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  Compositions and methods for activating toll-like receptor 4USPTO Application #: 20060089305Title: Compositions and methods for activating toll-like receptor 4 Abstract: Methods for activating Toll-like receptor 4 via cholesterol-dependent cytolysins isolated from a Gram-positive bacteria are provided. In addition compositions containing an isolated cholesterol-dependent cytolysin or a fragment thereof or a mimetic of the cytolysin or fragment thereof and methods for use of such composition in inhibiting binding and/or interaction of Toll-like receptor 4 with endotoxin are provided. Methods for identifying modulators of Toll-like receptor 4 activation by a cholesterol-dependent cytolysin and use of such modulators in treatment of septicemia and/or septic shock are also provided. (end of abstract) Agent: Licatla & Tyrrell P.C. - Marlton, NJ, US Inventors: Richard Rest, Michael Karin, Jin Mo Park USPTO Applicaton #: 20060089305 - Class: 514012000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 25 Or More Peptide Repeating Units In Known Peptide Chain Structure The Patent Description & Claims data below is from USPTO Patent Application 20060089305. Brief Patent Description - Full Patent Description - Patent Application Claims
INTRODUCTION [0001] This application claims the benefit of priority from U.S. Provisional Application Ser. No. 60/617,894 filed Oct. 12, 2004, which is herein incorporated by reference in its entirety. FIELD OF THE INVENTION [0003] The present invention relates to compositions comprising a member of the cholesterol-dependent cytolysin (CDC) family and methods of using these compositions to activate Toll-like receptor 4 and to identify modulators of Toll-like receptor 4 activation. Activation of Toll-like receptor 4 by endotoxin or lipopolysaccharide (LPS) can lead to septic shock or septicemia. Compounds identified as modulators, and more particularly inhibitors, of Toll-like receptor 4 activation by a member of the cholesterol-dependent cytolysin (CDC) family are expected to useful in the treatment of septic shock and/or septicemia. It is believed that isolated cholesterol-dependent cytolysins or fragments or mimetics thereof may inhibit the interaction of Toll-like receptor 4 and endotoxin or LPS as well. BACKGROUND OF THE INVENTION [0004] Toll-like receptors play pivotal roles in recognizing and resisting microbial infection (Kopp, E. and Medzhitov, R. Curr. Opin. Immunol. 2003 15:396-401; Beutler, B. and Rietschel, E. T. Nat. Rev. Immunol. 2003 3:169-176). Among the immediate outcomes of the TLR-dependent immune response is the production of cytokines by inflammatory cells such as macrophages. The production and release of such cytokines is responsible for the inflammatory response that accompanies bacterial infection. Toll-like receptor 4 (TLR4) activation by endotoxin or lipolysaccharide (LPS) produced by gram-negative bacteria leads to septic shock (Cohen, J. Nature 2002 420:885-891). [0005] Infection of bone marrow derived macrophages with live B. anthracis (Sterne strain) has also been demonstrated to result in extensive apoptosis dependant on signaling from the LPS-responsive Toll-like receptor TLR4, which activates the proapoptotic double stranded (ds) RNA-dependent protein kinase PKR (Hsu et al. Nature 2004 428:341-345). However, B. anthracis is a Gram-positive bacterium that does not produce LPS. [0006] In addition, the interaction of TLR4 with the cholesterol-dependent cytolysin pneumolysin was recently reported to be critically involved in the innate immune response to gram-positive pneumococcus (Malley et al. Proc. Natl Acad. Sci 2003 100(4):1966-1971). In particular, experiments were performed demonstrating that the inflammatory response of macrophages to pneumolysin was dependent on TLR4. Further, experiments with mutant mice lacking functional TLR4 revealed the mutant mice to be significantly more susceptible to invasive disease and death after exposure to pneumolysin-producing pneumococci when compared to control mice. Malley et al. thus concluded that the interaction of the pneumolysin with TLR4 is an important protective component of the host response to pneumococcus (Malley et al. Proc. Natl Acad. Sci 2003 100(4):1966-1971). [0007] Cholesterol-dependent cytolysins (CDCs) are known to be major virulence factors in Gram-positive bacterial infections (Portnoy et al. Infect. Immun. 1992 60:1263-1267; Paton, J. C. Trends Microbiol. 1996 4:103-106; Rood, J. I. Annu. Rev. Microbiol. 1998 52:333-360). The mechanisms by which CDCs contribute to pathogenesis in Gram-positive infections have so far been attributed to their cytolytic activity and their role in regulating intracellular compartmentalization of pathogenic bacteria (Decatur, A. L. and Portnoy, D. A. Science 2000 290:992-995). For example, Listeriolysin O (LLO) from Listeria monocytogenes and Streptolysin O (SLO) from Streptococcus pyogenes have also been shown to activate macrophages and mast cells to produce proinflammatory cytokines and chemokines (Tsukada et al. Cell. Immunol. 1992 140:21-30; Stassen et al. Infect. Immun. 2003 71:6171-6177). In addition, Streptolysin O (SLO) from Streptococcus pyogenes has been shown to mediate vectorial transport of streptococcal proteins into the cytoplasm of host cells (Madden et al. Cell 2001 104:143-152; Meehl, M. A. and Caparon, M. G. Mol. Microbiol. 2004 52:1665-1676). SUMMARY OF THE INVENTION [0008] An aspect of the present invention relates to a method for activating Toll-like receptor 4 which comprises contacting Toll-like receptor 4 with a cholesterol-dependent cytolysin isolated from a Gram-positive bacteria. [0009] Another aspect of the present invention relates to a method for identifying modulators of Toll-like receptor 4 activation which comprises measuring activation of Toll-like receptor 4 by a cholesterol-dependent cytolysin in the presence and absence of a test agent, wherein a change in activation of Toll-like receptor 4 in the presence of the test agent is indicative of the test agent being a modulator of Toll-like receptor 4 activation. [0010] Another aspect of the present invention relates to compositions comprising an isolated cholesterol-dependent cytolysin or a fragment or mimetic thereof which inhibits binding and/or interaction of Toll-like receptor 4 with endotoxin. [0011] Another aspect of the present invention relates to a method for treating septicemia or septic shock in a subject comprising administering to the subject a composition comprising an isolated cholesterol-dependent cytolysin or a fragment or mimetic thereof which inhibits binding and/or interaction of Toll-like receptor 4 with endotoxin. [0012] Another aspect of the present invention relates to a method for treating septicemia or septic shock in a subject comprising administering to the subject an agent which inhibits activation of Toll-like receptor 4 activity by a cholesterol-dependent cytolysin. [0013] Yet another aspect of the present invention relates to compositions for treatment of septicemia or septic shock comprising an agent which inhibits Toll-like receptor 4 activation by a cholesterol-dependent cytolysin. DETAILED DESCRIPTION OF THE INVENTION [0014] It has now been found that cholesterol-dependent cytolysins induce inflammatory and apoptotic responses in host immune cells via Toll-like receptor 4 (TLR4), a major contributor to Gram-positive induced septic shock. [0015] Identification of a cholesterol-dependent cytolysin as a TLR4 agonist was first performed in B. anthracis. [0016] In these experiments, B. anthracis cell wall preparations and culture supernatants were first tested for their ability to stimulate TNF-alpha gene expression and induce apoptosis of bone marrow-derived macrophages (BMDMs) in the presence of the p38 inhibitor SB202190. Treatment of BMDMs with a crude, commercially available, B. anthracis cell wall preparation did not strongly induce TNF-alpha mRNA expression or apoptosis. In contrast, the B. anthracis culture supernatant induced both TNF-alpha mRNA and apoptosis under the same conditions. The TNF-alpha- and apoptosis-inducing activity in the culture supernatant was sensitive to proteinase K digestion, indicating that a proteinaceous component is responsible for both activities. As only TLR4 agonists, but not agonists for other TLRs, can strongly potentiate macrophage apoptosis in the presence of SB202190 (Park et al. Science 2002 297:2048-2051; Hsu et al. Nature 2004 428:341-345), this protein component was expected to act as a TLR4 agonist. [0017] To identify this protein, the B. anthracis culture supernatant was sequentially purified through DEAE-Sepharose, Mono S, and phenyl-Sepharose chromatography columns. On the phenyl-Sepharose column, the TNF-alpha- and apoptosis-inducing activities cofractionated as a single peak centered at fraction 26. Analysis of the protein composition of the different column fractions revealed that a 63-kDa polypeptide copurified with both activities. Among the numerous secreted proteins predicted by the B. anthracis genome sequence to be present was anthrolysin O, a cholesterol-dependent cytolysin (CDC) encoded by the BA3355 gene (Shannon et al. Infect. Immun. 2003 71:3183-3189). The anthrolysin O polypeptide consists of 512 amino acids with the N-terminal 35 residues coding for a signal peptide, a size consistent with the 63-kDa band described above. [0018] The phenyl-Sepharose fractions were thus analyzed by immunoblotting with anti-anthrolysin O antibody. It was found that anthrolysin O indeed co-purified with the 63-kDa protein, as well as the macrophage-stimulating and apoptosis-inducing activities. [0019] To directly test whether anthrolysin O stimulated macrophages in a TLR4-dependent manner, pure recombinant anthrolysin O was prepared by expression in E. coli. Treatment of BMDMs with recombinant anthrolysin O or other TLR agonists with different receptor specificities resulted in a strong induction of TNF-alpha mRNA. To compare the gene induction specificity of anthrolysin O to those of other TLR agonists, the mRNA levels of other cytokines including interleukin (IL)-1alpha, IL-1.beta., and IL-6 were examined. Whereas TNF-alpha was induced to similar extents by anthrolysin O and the different TLR agonists, the IL-1alpha and IL-6 genes were most strongly induced by anthrolysin O and LPS, but were less responsive to other TLR agonists. Furthermore, treatment of BMDMs with either anthrolysin O or the different TLR agonists in conjunction with SB202190 revealed that only anthrolysin O and LPS were able to cause a robust apoptotic response. [0020] To specifically examine the role of TLR4 in the response to anthrolysin O, BMDMs were prepared from wild type (C3H/OuJ) and TLR4 mutant (C3H/HeJ) mice and their responses to anthrolysin O treatment were compared. Anthrolysin O induced activation of p38 MAPK and degradation of I.kappa.B.alpha. in TLR4 wild type, but not in TLR4 mutant BMDMs. The TLR4 mutant BMDMs showed no defect in their response to the TLR2 agonist synthetic bacterial lipopeptide. Anthrolysin O also failed to induce TNF-alpha and IL-6 gene expression in TLR mutant BMDMs. These observations indicate that anthrolysin O activates macrophages via TLR4. Continue reading... Full patent description for Compositions and methods for activating toll-like receptor 4 Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Compositions and methods for activating toll-like receptor 4 patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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