CA2468907A1 - Effectors of innate immunity - Google Patents

Abstract

A method of identifying a polynucleotide or pattern of polynucleotides regulated by one or more sepsis or inflammatory inducing agents and inhibite d by a peptide is described. A method of identifying a pattern of polynucleoti de expression for inhibition of an inflammatory or septic response. The method includes contacting cells with LPS, LTA, CpG DNA and/or intact microbes or microbial components in the presence or absence of a peptide; detecting a pattern of polynucleotide expression for the cells in the presence and absen ce of the peptide, wherein the pattern in the presence of the peptide represent s inhibition of an inflammatory or septic response. Also included are compound s and agents identified by the methods of the invention. In another aspect, th e invention provides methods and compounds for enhancing innate immunity in a subject.

Description

EFFECTORS OF INNATE IMMUNITY
RELATED APPLICATION DATA
This application claims priority under 35 USC 119(e) to US Patent Application Serial No. 60/336,632, filed December 3, 2001, herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0001] The present invention relates generally to peptides and specifically to peptides effective as therapeutics and for drug discovery related to pathologies resulting from microbial infections and for modulating innate immunity or anti-inflammatory activity.
BACKGROUND OF THE INVENTION

[0002] Infectious diseases are the leading cause of death worldwide. According to a 1999 World Health Organization study, over 13 million people die from infectious diseases each year. Infectious diseases are the third leading cause of death in North America, accounting for 20% of deaths annually and increasing by 50% since 1980.
The success of many rriedical and surgical treatments also hinges on the control of infectious diseases. The discovery and use of antibiotics has been one of the great achievements of modern medicine. Without antibiotics, physicians would be unable to perform complex surgery, chemotherapy or most medical interventions such as catheterization.

[0003] Current sales of antibiotics are US$26 billion worldwide. However,.the overuse and sometimes unwarranted use of antibiotics have resulted in the evolution of new antibiotic-resistant strains of bacteria. Antibiotic resistance has become part of the medical landscape. Bacteria such as vancomycin-resistant Enterococcus, VRE, and methicillin-resistant Staphylococcus aureus and MRSA, strains cannot be treated with antibiotics and often, patients suffering from infections with such bacteria die.

Antibiotic discovery has proven to be one of the most difficult areas for new drug development and many large pharmaceutical companies have cut back or completely halted their antibiotic development programs. However, with the dramatic rise of antibiotic resistance, including the emergence of untreatable infections, there is a clear unmet medical need for novel types of anti-microbial therapies, and agents that impact on innate immunity would be one such class of agents.

[0004] The innate immune system is a highly effective and evolved general defense system. Elements of innate immunity are always present at low levels and are activated very rapidly when stimulated. Stimulation can include interaction of bacterial signaling molecules with pattern recognition receptors on the surface of the body's cells or other mechanisms of disease. Every day, humans are exposed to tens of thousands of potential pathogenic microorganisms through the food and water we ingest, the air we breathe and the surfaces, pets and people that we touch.
The innate immune system acts to prevent these pathogens from causing disease. The innate immune system differs from so-called adaptive immunity (which includes antibodies and antigen-specific B- and T-lymphocytes) because it is always present, effective immediately, and relatively non-specific for any given pathogen. The adaptive immune system requires amplification of specific recognition elements and thus takes days to weeks to respond. Even when adaptive immunity is pre-stimulated by vaccination, it may take three days or more to respond to a pathogen whereas innate immunity is immediately or rapidly (hours) available. Innate immunity involves a variety of effector functions including phagocytic cells, complement, etc, but is generally incompletely understood. Generally speaking many innate immune responses are "triggered" by the binding of microbial signaling molecules with pattern recognition receptors termed Toll-like receptors on the surface of host cells.
Many of these effector functions are grouped together in the inflammatory response.
However too severe an inflammatory response can result in responses that are harmful to the body, and in an extreme case sepsis and potentially death can occur.

[0005] The release of structural components from infectious agents during infection causes an inflammatory response, which when unchecked can lead to the potentially lethal condition, sepsis. Sepsis occurs in approximately 780,000 patients in North America annually. Sepsis may develop as a result of infections acquired in the community such as pneumonia, or it may be a complication of the treatment of trauma, cancer or major surgery. Severe sepsis occurs when the body is overwhelmed by the inflammatory response and body organs begin to fail. Up to 120,000 deaths occur annually in the United Stated due to sepsis. Sepsis may also involve pathogenic microorganisms or toxins in the blood (e.g., septicemia), which is a leading cause of death among humans. Gram-negative bacteria are the organisms most commonly associated with such diseases. However, gram-positive bacteria are an increasing cause of infections. Gram-negative and Gram-positive bacteria and their components can all cause sepsis.

[0006] The presence of microbial components induce the release of pro-inflammatory cytokines of which tumor necrosis factor-a (TNF-a) is of extreme importance. TNF-a and other pro-inflammatory cytokines can then cause the release of other pro-inflammatory mediators and lead to an inflammatory cascade. Gram-negative sepsis is usually caused by the release of the bacterial outer membrane component, lipopolysaccharide (LPS; also referred to as endotoxin). Endotoxin in the blood, called endotoxemia comes primarily from a bacterial infection, and may be released during treatment with antibiotics. Gram-positive sepsis can be caused by the release of bacterial cell wall components such as lipoteichoic acid (LTA), peptidoglycan (PG), rhamnose-glucose polymers made by Streptococci, or capsular polysaccharides made by Staphylococci. Bacterial or other non-mammalian DNA
that, unlike mammalian DNA, frequently contains unmethylated cytosine-guanosine dimers (CpG DNA) has also been shown to induce septic conditions including the production of TNF-a. Mammalian DNA contains CpG dinucleotides at a much lower frequency, often in a methylated form. In addition to their natural release during bacterial infections, antibiotic treatment can also cause release of the bacterial cell wall components LPS and LTA and probably also bacterial DNA. This can then hinder recovery from infection or even cause sepsis.

[0007] Cationic peptides are being increasingly recognized as a form of defense against infection, although the major effects recognized in the scientific and patent literature are the antimicrobial effects (Hancock, R.E.W., and R. Lehrer.
1998.

Cationic peptides: a new source of antibiotics. Trends in Biotechnology 16: 82-88.).
Cationic peptides having antimicrobial activity have been isolated from a wide variety of organisms. In nature, such peptides provide a defense mechanism against microorganisms such as bacteria and yeast. Generally, these cationic peptides are thought to exert their antimicrobial activity on bacteria by interacting with the cytoplasmic membrane, and in most cases, forming channels or lesions. In gram-negative bacteria, they interact with LPS to permeabilize the outer membrane, leading to self promoted uptake across the outer membrane and access to the cytoplasmic membrane. Examples of cationic antimicrobial peptides include indolicidin, defensins, cecropins, and magainins.

[0008] Recently it has been increasingly recognized that such peptides are effectors in other aspects of innate immunity (Hancock, R.E.W. and G. Diamond. 2000. The role of cationic peptides in innate host defenses. Trends in Microbiology 8:402-410.;
Hancock, R.E.W. 2001. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infectious Diseases 1:156-164) although it was not known if the antimicrobial and effector functions are independent.

(0009] Some cationic peptides have an affinity for binding bacterial products such as LPS and LTA. Such cationic peptides can suppress cytokine production in response to LPS, and to varying extents can prevent lethal shock. However it has not been proven as to whether such effects are due to binding of the peptides to LPS
and LTA, or due to a direct interaction of the peptides with host cells. Cationic peptides are induced, in response to challenge by microbes or microbial signaling molecules like LPS, by a regulatory pathway similar to that used by the mammalian immune system (involving Toll like receptors and the transcription factor; NFKB). Cationic peptides therefore appear to have a key role in innate immunity: Mutations that affect the induction of antibacterial peptides can reduce survival in response to bacterial challenge. As well, mutations of the Toll pathway of Drosophila that lead to decreased antifungal peptide expression result in increased susceptibility to lethal fungal infections. In humans, patients with specific granule deficiency syndrome, completely lacking in a-defensins, suffer from frequent and severe bacterial infections. Other evidence includes the inducibility of some peptides by infectious agents, and the very high concentrations that have been recorded at sites of inflammation. Cationic peptides may also regulate cell migration, to promote the ability of leukocytes to combat bacterial infections. For example, two human a-defensin peptides, HNP-1 and HNP-2, have been indicated to have direct chemotactic activity for murine and human T cells and monocytes, and human (3-defensins appear to act as chemoattractants for immature dendritic cells and memory T cells through interaction with CCR6. Similarly, the porcine cationic peptide, PR-39 was found to be chemotactic for neutrophils. It is unclear however as to whether peptides of different structures and compositions share these properties.

[00010] The single known cathelicidin from humans, LL-37, is produced by myeloid precursors, testis, human keratinocytes during inflammatory disorders and airway epithelium. The characteristic feature of cathelicidin peptides is a high level of sequence identity at the N-terminus prepro regions termed the cathelin domain.
Cathelicidin peptides are stored as inactive propeptide precursors that, upon stimulation, are processed into active peptides.
SUMMARY OF THE INVENTION

[00011] The present invention is based on the seminal discovery that based on patterns of polynucleotide expression regulated by endotoxic lipopolysaccharide, lipoteichoic acid, CpG DNA, or other cellular components (e.g., microbes or their cellular components), and affected by cationic peptides, one can screen for novel compounds that block or reduce sepsis and/or inflammation in a subject.
Further, based on the use of cationic peptides as a tool, one can identify selective enhancers of innate immunity that do not trigger the sepsis reaction and that can block/dampen inflammatory and/or septic responses.

[00012] Thus, in one embodiment, a method of identifying a polynucleotide or pattern of polynucleotides regulated by one or more sepsis or inflammatory inducing agents and inhibited by a cationic peptide is provided. The method of the invention includes contacting the polynucleotide or polynucleotides with one or more sepsis or inflammatory inducing agents and contacting the polynucleotide or polynucleotides S

with a cationic peptide either simultaneously or immediately thereafter.
Differences in expression are detected in the presence and absence of the cationic peptide, and a change in expression, either up- or down-regulation, is indicative of a polynucleotide or pattern of polynucleotides that is regulated by a sepsis or inflammatory inducing agent and inhibited by a cationic peptide. In another aspect the invention provides a polynucleotide or polynucleotides identified by the above method. Examples of sepsis or inflammatory regulatory agents include LPS, LTA or CpG DNA or microbial components (or any combination thereof), or related agents.
(0010] In another embodiment, the invention provides a method of identifying an agent that blocks sepsis or inflammation including combining a polynucleotide identified by the method set forth above with an agent wherein expression of the polynucleotide in the presence of the agent is modulated as compared with expression in the absence of the agent and wherein the modulation in expression affects an inflammatory or septic response.
[0011] In another embodiment, the invention provides a method of identifying a pattern of polynucleotide expression for inhibition of an inflammatory or septic response by 1 ) contacting cells with LPS, LTA and/or CpG DNA in the presence or absence of a cationic peptide and 2) detecting a pattern of polynucleotide expression for the cells in the presence and absence of the peptide. The pattern obtained in the presence of the peptide represents inhibition of an inflammatory or septic response.
In another aspect the pattern obtained in the presence of the peptide is compared to the pattern of a test compound to identify a compound that provides a similar pattern.
In another aspect the invention provides a compound identified by the foregoing method.
[0012] In another embodiment, the invention provides a method of identifying an agent that enhances innate immunity by contacting a polynucleotide or polynucleotides that encode a polypeptide involved in innate immunity, with an agent of interest, wherein expression of the polynucleotide in the presence of the agent is modulated as compared with expression of the polynucleotide in the absence of the agent and wherein the modulated expression results in enhancement of innate immunity. Preferably, the agent does not stimulate a sepsis reaction in a subject. In one aspect, the agent increases the expression of an anti-inflammatory polynucleotide.
Exemplary, but non-limiting anti-inflammatory polynucleotides encode proteins such as IL-1 R antagonist homolog 1 (AI167887), IL-10 R beta (AA486393), IL-10 R
alpha (U00672) TNF Receptor member 1B (AA150416), TNF receptor member 5 (H98636), TNF receptor member l lb (AA194983), IK cytokine down-regulator of HLA II (R39227), TGF-B inducible early growth response 2 (AI473938), CD2 (AA927710), IL-19 (NM 013371) or IL-10 (M57627). In one aspect, the agent decreases the expression of polynucleotides encoding proteasome subunits involved in NF-~cB activation such as proteasome subunit 26S (NM 013371). In one aspect, the agent may act as an antagonist of protein kinases. In one aspect, the agent is a peptide selected from SEQ ID N0:4-54.

[0013] In another embodiment, the invention provides a method of identifying a pattern of polynucleotide expression for identification of a compound that selectively enhances innate immunity. The invention includes detecting a pattern of polynucleotide expression for cells contacted in the presence and absence of a cationic peptide, wherein the pattern in the presence of the peptide represents stimulation of innate immunity; detecting a pattern of polynucleotide expression for cells contacted in the presence of a test compound, wherein a pattern with the test compound that is similar to the pattern observed in the presence of the cationic peptide, is indicative of a compound that enhances innate immunity. It is preferred that the compound does not stimulate a septic reaction in a subject.

[0014] In another embodiment, the invention provides a method for inferring a state of infection in a mammalian subject from a nucleic acid sample of the subject by identifying in the nucleic acid sample a polynucleotide expression pattern exemplified by an increase in polynucleotide expression of at least 2 polynucleotides in Table 50, 51 and or 52, as compared to a non-infected subject. Also included is a polynucleotide expression pattern obtained by any of the methods described above.
[00013] In another aspect a cationic peptide that is an antagonist of CXCR-4 is provided. In still another aspect, a method of identifying a cationic peptide that is an antagonist of CXCR-4 by contacting T cells with SDF-1 in the presence of absence of a test peptide and measuring chemotaxis is provided. A decrease in chemotaxis in the presence of the test peptide is indicative of a peptide that is an antagonist of CXCR-4.
Cationic peptide also acts to reduce the expression of the SDF-1 receptor polynucleotide (NM_013371).

[0015] In all of the above described methods, the compounds or agents of the invention include but are not limited to peptides, cationic peptides, peptidomimetics, chemical compounds, polypeptides, nucleic acid molecules and the like.

[0016] In still another aspect the invention provides an isolated cationic peptide. An isolated cationic peptide of the invention is represented by one of the following general formulas and the single letter amino acid code:
X,XZX3IX4PX4IPXSXZX1 (SEQ ID NO: 4), where X~ is one or two of R, L or K, Xz is one of C, S or A, X3 is one of R or P, X4 is one of A or V and X5 is one of V
or W;
X~LXzX3KX4XZX5X3PX3X~ (SEQ ID NO: 11), where X~ is one or two of D, E, S, T or N, X2 is one or two of P, G or D, X3 is one of G, A, V, L, I or Y, X4 is one of R, K or H and X5 is one of S, T, C, M or R;
X~XZX3XqWX4WXqX5K (SEQ ID NO: 18), where X1 is one to four chosen from A, P or R, Xz is one or two aromatic amino acids (F, Y and W), X3 is one of P or K, X4 is one, two or none chosen from A, P, Y or W and X5 is one to three chosen from R or P;
X,XZX3X4X~VX3XqRGX4X3X4XIX3X1 (SEQ ID NO: 25) where X~ is one or two of R or K, XZ is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R
and H), X3 is C, S, M, D or A and X4 is F, I, V, M or R;
X~XZX3X4X~VXSXqRGX4X5X4X~X3X~ (SEQ ID NO: 32), where X~ is one or two of R or K, XZ is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R
and H), X3 is one of C, S, M, D or A, X4 is one of F, I, V, M or R and X5 is one of A, I, S, M, D or R; and KX~KXZFXZKMLMX2ALKKX3 (SEQ ID NO: 39), where X~ is a polar amino acid (C, S, T, M, N and Q); Xz is one of A, L, S or K and X3 is 1-17 amino acids chosen from G, A, V, L, I, P, F, S, T, K and H;
KWKXZX~X,XZXZX~X2XZX,X~XZXzIFHTALKPISS (SEQ ID NO: 46), where X~ is a hydrophobic amino acid and Xz is a hydrophilic amino acid.

[0017] Additionally, in another aspect the invention provides isolated cationic peptides KWKSFLRTFKSPVRTVFHTALKPISS (SEQ ID NO: 53) and KWKSYAHTIMSPVRLVFHTALKPISS (SEQ ID NO: 54).

[0018] Also provided are nucleic acid sequences encoding the cationic peptides of the invention, vectors including such polynucleotides and host cells containing the vectors.
DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides novel cationic peptides, characterized by a group of generic formulas, which have ability to modulate (e.g., up- and/or down regulate) polynucleotide expression, thereby regulating sepsis and inflammatory responses and/or innate immunity.

[0020] "Innate immunity" as used herein refers to the natural ability of an organism to defend itself against invasions by pathogens. Pathogens or microbes as used herein may include, but are not limited to bacteria, fungi, parasites and viruses.
Innate immunity is contrasted with acquired/adaptive immunity in which the organism develops a defensive mechanism based substantially on antibodies and/or immune lymphocytes that is characterized by specificity, amplifiability and self vs.
non-self dsicrimination. With innate immunity, broad, nonspecific immunity is provided and there is no immunologic memory of prior exposure. The hallmarks of innate immunity are effectiveness against a broad variety of potential pathogens, independence of prior exposure to a pathogen, and immediate effectiveness (in contrast to the specific immune response which takes days to weeks to be elicited). In addition, innate immunity includes immune responses that affect other diseases, such as cancer, inflammatory diseases, multiple sclerosis, various viral infections, and the 1 ike.

[0021] As used herein, the term "cationic peptide" refers to a sequence of amino acids from about 5 to about 50 amino acids in length. In one aspect, the cationic peptide of the invention is from about 10 to about 35 amino acids in length. A peptide is "cationic" if it possesses sufficient positively charged amino acids to have a pKa greater than 9Ø Typically, at least two of the amino acid residues of the cationic peptide will be positively charged, for example, lysine or arginine.
"Positively charged" refers to the side chains of the amino acid residues which have a net positive charge at pH 7Ø Examples of naturally occurring cationic antimicrobial peptides which can be recombinantly produced according to the invention include defensins, cathelicidins, magainins, melittin, and cecropins, bactenecins, indolicidins, polyphemusins, tachyplesins, and analogs thereof. A variety of organisms make cationic peptides, molecules used as part of a non-specific defense mechanism against microorganisms. When isolated, these peptides are toxic to a wide variety of microorganisms, including bacteria, fungi, and certain enveloped viruses.
While cationic peptides act against many pathogens, notable exceptions and varying degrees of toxicity exist. However this patent reveals additional cationic peptides with no toxicity towards microorganisms but an ability to protect against infections through stimulation of innate immunity, and this invention is not limited to cationic peptides with antimicrobial activity. In fact, many peptides useful in the present invention do not have antimicrobial activity.

[0022] Cationic peptides known in the art include for example, the human cathelicidin LL-37, and the bovine neutrophil peptide indolicidin and the bovine variant of bactenecin, Bac2A.
LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ
ID NO: 1) Indolicidin ILPWKWPWWPWRR-NHZ (SEQ ID NO: 2) Bac2A RLARIVVIRVAR-NHZ (SEQ ID NO: 3) [0023] In innate immunity, the immune response is not dependent upon antigens.
The innate immunity process may include the production of secretory molecules and cellular components as set forth above. In innate immunity, the pathogens are recognized by receptors encoded in the germline. These Toll-like receptors have broad specificity and are capable of recognizing many pathogens. When cationic peptides are present in the immune response, they aid in the host response to pathogens. This change in the immune response induces the release of chemokines, which promote the recruitment of immune cells to the site of infection.

[0024] Chemokines, or chemoattractant cytokines, are a subgroup of immune factors that mediate chemotactic and other pro-inflammatory phenomena (See, Schall, 1991, Cytokine 3:165-183). Chemokines are small molecules of approximately 70-80 residues in length and can generally be divided into two subgroups, a which have two N-terminal cysteines separated by a single amino acid (CxC) and (3 which have two adjacent cysteines at the N terminus (CC). RANTES, MIP-la and MIP-1(3 are members of the (3 subgroup (reviewed by Horuk, R., 1994, Trends Pharmacol.
Sci, 15:159-165; Murphy, P. M., 1994, Annu. Rev. Immunol., 12:593-633). The amino terminus of the (3 chemokines RANTES, MCP-1, and MCP-3 have been implicated in the mediation of cell migration and inflammation induced by these chemokines.
This involvement is suggested by the observation that the deletion of the amino terminal 8 residues of MCP-1, amino terminal 9 residues of MCP-3, and amino terminal 8 residues of RANTES and the addition of a methionine to the amino terminus of RANTES, antagonize the chemotaxis, calcium mobilization and/or enzyme release stimulated by their native counterparts (Gong et al., 1996 J. Biol. Chem.
271:10521-10527; Proudfoot et al., 1996,1. Biol. Chem. 271:2599-2603). Additionally, a chemokine-like chemotactic activity has been introduced into MCP-1 via a double mutation of Tyr 28 and Arg 30 to leucine and valine, respectively, indicating that internal regions of this protein also play a role in regulating chemotactic activity (Beall et al., 1992, J. Biol. Chem. 267:3455-3459).

[0025] The monomeric forms of all chemokines characterized thus far share significant structural homology, although the quaternary structures of a and (3 groups are distinct. While the monomeric structures of the (3 and a chemokines are very similar, the dimeric structures of the two groups are completely different. An additional chemokine, lymphotactin, which has only one N terminal cysteine has also been identified and may represent an additional subgroup (y) of chemokines (Yoshida et al., 1995, FEBS Lett. 360:155-159; and Kelner et al., 1994, Science 266:1395-1399).

[0026] Receptors for chemokines belong to the large family of G-protein coupled, 7 transmembrane domain receptors (GCR's) (See, reviews by Horuk, R., 1994, Trends Pharntacol. Sci. 15:159-165; and Murphy, P. M., 1994, Annu. Rev. Immunol.
12:593-633). Competition binding and cross-desensitization studies have shown that chemokine receptors exhibit considerable promiscuity in ligand binding.
Examples demonstrating the promiscuity among (3 chemokine receptors include: CC CKR-l, which binds RANTES and MIP-la (Neote et al., 1993, Cell 72: 415-425), CC CKR-4, which binds RANTES, MIP-la, and MCP-1 (Power et al., 1995, J. Biol. Chem.
270:19495-19500), and CC CKR-5, which binds RANTES, MIP-la, and MIP-1(3 (Alkhatib et al., 1996, Science, in press and Dragic et al., 1996, Nature 381:667-674).
Erythrocytes possess a receptor (known as the Duffy antigen) which binds both a and (3 chemokines (Horuk et al., 1994, J. Biol. Chem. 269:17730-17733; Neote et al., 1994, Blood 84:44-52; and Neote et al., 1993, J. Biol. Chem. 268:12247-12249).
Thus the sequence and structural homologies evident among chemokines and their receptors allows some overlap in receptor-ligand interactions.

(0027] In one aspect, the present invention provides the use of compounds including cationic peptides of the invention to reduce sepsis and inflammatory responses by acting directly on host cells. In this aspect, a method of identification of a polynucleotide or polynucleotides that are regulated by one or more sepsis or inflammatory inducing agents is provided, where the regulation is altered by a cationic peptide. Such sepsis or inflammatory inducing agents include, but are not limited to endotoxic lipopolysaccharide (LPS), lipoteichoic acid (LTA) and/or CpG
DNA or intact bacteria or other bacterial components. The identification is performed by contacting the polynucleotide or polynucleotides with the sepsis or inflammatory inducing agents and further contacting with a cationic peptide either simultaneously or immediately after. The expression of the polynucleotide in the presence and absence of the cationic peptide is observed and a change in expression is indicative of a polynucleotide or pattern of polynucleotides that is regulated by a sepsis or inflammatory inducing agent and inhibited by a cationic peptide. In another aspect, the invention provides a polynucleotide identified by the method.

[0028] Once identified, such polynucleotides will be useful in methods of screening for compounds that can block sepsis or inflammation by affecting the expression of the polynucleotide. Such an effect on expression may be either up regulation or down regulation of expression. By identifying compounds that do not trigger the sepsis reaction and that can block or dampen inflammatory or septic responses, the present invention also presents a method of identifying enhancers of innate immunity.
Additionally, the present invention provides compounds that are used or.
identified in the above methods.

[0029] Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, and the like to produce structural analogs.
Candidate agents are also found among biomolecules including, but not limited to:
peptides, peptidiomimetics, saccharides, fatty acids, steroids, purines, pyrimidines, polypeptides, polynucleotides, chemical compounds, derivatives, structural analogs or combinations thereof.

[0030] Incubating components of a screening assay includes conditions which allow contact between the test compound and the polynucleotides of interest.
Contacting includes in solution and in solid phase, or in a cell. The test compound may optionally be a combinatorial library for screening a plurality of compounds.
Compounds identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a compound.

[0031] Generally, in the methods of the invention, a cationic peptide is utilized to detect and locate a polynucleotide that is essential in the process of sepsis or inflammation. Once identified, a pattern of polynucleotide expression may be obtained by observing the expression in the presence and absence of the cationic peptide. The pattern obtained in the presence of the cationic peptide is then useful in identifying additional compounds that can inhibit expression of the polynucleotide and therefore block sepsis or inflammation. It is well known to one of skill in the art that non-peptidic chemicals and peptidomimetics can mimic the ability of peptides to bind to receptors and enzyme binding sites and thus can be used to block or stimulate biological reactions. Where an additional compound of interest provides a pattern of polynucleotide expression similar to that of the expression in the presence of a cationic peptide, that compound is also useful in the modulation of sepsis or an innate immune response. In this manner, the cationic peptides of the invention, which are known inhibitors of sepsis and inflammation and enhancers of innate immunity are useful as tools in the identification of additional compounds that inhibit sepsis and inflammation and enhance innate immunity.

[0032] As can be seen in the Examples below, peptides of the invention have a widespread ability to reduce the expression of polynucleotides regulated by LPS.
High levels of endotoxin in the blood are responsible for many of the symptoms seen during a serious infection or inflammation such as fever and an elevated white blood cell count. Endotoxin is a component of the cell wall of Gram-negative bacteria and is a potent trigger of the pathophysiology of sepsis. The basic mechanisms of inflammation and sepsis are related. In Example 1, polynucleotide arrays were utilized to determine the effect of cationic peptides on the transcriptional response of epithelial cells. Specifically, the effects on over 14,000 different specific polynucleotide probes induced by LPS were observed. The tables show the changes seen with cells treated with peptide compared to control cells. The resulting data indicated that the peptides have the ability to reduce the expression of polynucleotides induced by LPS.

[0033] Example 2, similarly, shows that peptides of the invention are capable of neutralizing the stimulation of immune cells by Gram positive and Gram negative bacterial products. Additionally, it is noted that certain pro-inflammatory polynucleotides are down-regulated by cationic peptides, as set forth in table 24 such as TLR1 (AI339155), TLR2 (T57791), TLRS (N41021), TNF receptor-associated factor 2 (T55353), TNF receptor-associated factor 3 (AA504259), TNF receptor superfamily, member 12 (W71984), TNF receptor superfamily, member 17 (AA987627), small inducible cytokine subfamily B, member 6 (AI889554), IL-12R
beta 2 (AA977194), IL-18 receptor 1 (AA482489), while anti-inflammatory polynucleotides are up-regulated by cationic peptides, as seen in table 25 such as IL-1 R antagonist homolog 1 (AI167887), IL-10 R beta (AA486393), TNF Receptor member 1B (AA150416), TNF receptor member 5 (H98636), TNF receptor member l lb (AA194983), IK cytokine down-regulator of HLA II (R39227), TGF-B
inducible early growth response 2 (AI473938), or CD2 (AA927710). The relevance and application of these results are confirmed by an in vivo application to mice.
Example 3 demonstrates that such peptides do not generally demponstrate toxicity towards the host cells they contact.

[0034] In Example 4 it can be seen that the cationic peptides of the invention alter polynucleotide expression in macrophage and epithelial cells. The results of this example show that pro-inflammatory polynucleotides are down-regulated by cationic peptides (Table 24) whereas anti-inflammatory polynucleotides are up-regulated by cationic peptides (Table 25).

[0035] In another aspect, the invention provides a method of identifying an agent that enhances innate immunity. In the method, a host cell polynucleotide or polynucleotides that encode a polypeptide involved in innate immunity is contacted with an agent of interest. Expression of the polynucleotide is determined, both in the presence and absence of the agent. The expression is compared and of the specific modulation of expression was indicative of an enhancement of innate immunity.
In another aspect, the agent does not stimulate a septic reaction as revealed by the lack of upregulation of the pro-inflammatory cytokine TNF-a. In still another aspect the agent reduces or blocks the inflammatory or septic response. In yet another aspect, the agent reduces the expression of TNF-a and/or interleukins including, but not limited to, IL-1 (3, IL-6, IL-12 p40, IL-12 p70, and IL-8.

[0036] In another aspect, the invention provides methods of direct polynucleotide regulation by cationic peptides and the use of compounds including cationic peptides to stimulate elements of innate immunity. In this aspect, the invention provides a method of identification of a pattern of polynucleotide expression for identification of a compound that enhances innate immunity. In the method of the invention, an initial detection of a pattern of polynucleotide expression for cells contacted in the presence and absence of a cationic peptide is made. The pattern resulting from polynucleotide expression in the presence of the peptide represents stimulation of innate immunity.
A pattern of polynucleotide expression is then detected in the presence of a test compound, where a resulting pattern with the test compound that is similar to the pattern observed in the presence of the cationic peptide is indicative of a compound that enhances innate immunity. In another aspect, the invention provides compounds that are identified in the above methods. In another aspect, the compound of the invention stimulates chemokine or chemokine receptor expression. Chemokine or chemokine receptors may include, but are not limited to CXCR4, CXCRl, CXCR2, CCR2, CCR4, CCRS, CCR6, MIP-1 alpha, MDC, MIP-3 alpha, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, and RANTES. In still another aspect, the compound is a peptide, peptidomimetic, chemical compound, or a nucleic acid molecule.

[0037] In still another aspect the polynucleotide expression pattern includes expression of pro-inflammatory polynucleotides. Such pro-inflammatory polynucleotides may include, but are not limited to, ring finger protein 10 (D87451), serine/threonine protein kinase MASK (AB040057), KIAA0912 protein (AB020719), KIAA0239 protein (D87076), RAP1, GTPase activating protein 1 (M64788), FEM-1-like death receptor binding protein (AB007856), cathepsin S (M90696), hypothetical protein FLJ20308 (AK000315), pim-1 oncogene (M54915), proteasome subunit beta type 5 (D29011), KIAA0239 protein (D87076), mucin 5 subtype B tracheobronchial (AJ001403), cAMP response element-binding protein CREBPa, integrin alpha M
(J03925), Rho-associated kinase 2 (NM 004850), PTD017 protein (AL050361) unknown genes (AK001143, AK034348, AL049250, AL16199, AL031983) and any combination thereof. In still another aspect the polynucleotide expression pattern includes expression of cell surface receptors that may include but is not limited to retinoic acid receptor (X06614), G protein-coupled receptors (Z94155, X81892, U52219, U22491, AF015257, U66579) chemokine (C-C motif) receptor 7 (L31584), tumor necrosis factor receptor superfamily member 17 (Z29575), interferon gamma receptor 2 (U05875), cytokine receptor-like factor 1 (AF059293), class I
cytokine receptor (AF053004), coagulation factor II (thrombin) receptor-like 2 (U92971), leukemia inhibitory factor receptor (NM 002310), interferon gamma receptor 1 (AL050337).

[0038] It is shown below, for example, in tables 1-15, that cationic peptides can neutralize the host response to the signaling molecules of infectious agents as well as modify the transcriptional responses of host cells, mainly by down-regulating the pro-inflammatory response and/or up-regulating the anti-inflammatory response.
Example 5 shows that the cationic peptides can aid in the host response to pathogens by inducing the release of chemokines, which promote the recruitment of immune cells to the site of infection. The results are confirmed by an in vivo application to mice.

[0039] It is seen from the examples below that cationic peptides have a substantial influence on the host response to pathogens in that they assist in regulation of the host immune response by inducing selective pro-inflammatory responses that for example promote the recruitment of immune cells to the site of infection but not inducing potentially harmful pro-inflammatory cytokines. Sepsis appears to be caused in part by an overwhelming pro-inflammatory response to infectious agents. Cationic peptides aid the host in a "balanced" response to pathogens by inducing an anti-inflammatory response and suppressing certain potentially harmful pro-inflammatory responses.

[0040] In Example 7, the activation of selected MAP kinases was examined, to study the basic mechanisms behind the effects of interaction of cationic peptides with cells.
Macrophages activate MEK/ERK kinases in response to bacterial infection. MEK
is a MAP kinase kinase that when activated, phosphorylates the downstream kinase ERK

(extracellular regulated kinase), which then dimerizes and translocates to the nucleus where it activates transcription factors such as Elk-1 to modify polynucleotide expression. MEK/ERK kinases have been shown to impair replication of Salmonella within macrophages. Signal transduction by MEK kinase and NADPH oxidase may play an important role in innate host defense against intracellular pathogens.
By affecting the MAP kinases as shown below the cationic peptides have an effect on bacterial infection. The cationic peptides can directly affect kinases. Table demonstrates but is not limited to MAP kinase polynucleotide expression changes in response to peptide. The kinases include MAP kinase kinase 6 (H070920), MAP
kinase kinase 5 (W69649), MAP kinase 7 (H39192), MAP kinase 12 (AI936909) and MAP kinase-activated protein kinase 3 (W68281).

[0041] In another method, the methods of the invention may be used in combination, to identify an agent with multiple characteristics, i.e. a peptide with anti-inflammatory/anti-sepsis activity, and the ability to enhance innate immunity, in part by inducing chemokines in vivo.

[0042] In another aspect, the invention provides a method for inferring a state of infection in a mammalian subject from a nucleic acid sample of the subject by identifying in the nucleic acid sample a polynucleotide expression pattern exemplified by an increase in polynucleotide expression of at least 2 polynucleotides in Table 55 as compared to a non-infected subject. In another aspect the invention provides a method for inferring a state of infection in a mammalian subject from a nucleic acid sample of the subject by identifying in the nucleic acid sample a polynucleotide expression pattern exemplified by a polynucleotide expression of at least 2 polynucleotides in Table 56 or Table 57 as compared to a non-infected subject.
In one aspect of the invention, the state of infection is due to infectious agents or signaling molecules derived therefrom, such as, but not limited to, Gram negative bacteria and Gram positive bacteria, viral, fungal or parasitic agents. In still another aspect the invention provides a polynucleotide expression pattern of a subject having a state of infection identified by the above method. Once identified, such polynucleotides will be useful in methods of diagnosis of a condition associated with the activity or presence of such infectious agents or signaling molecules.

[0043] Example 10 below demonstrates this aspect of the invention.
Specifically, table 61 demonstrates that both MEK and the NADPH oxidase inhibitors can limit bacterial replication (infection of IFN-y-primed macrophages by S. typhimurium triggers a MEK kinase). This is an example of how bacterial survival can be impacted by changing host cell signaling molecules.

[0044] In still another aspect of the invention, compounds are presented that inhibit stromal derived factor-1 (SDF-1) induced chemotaxis of T cells. . Compounds are also presented which decrease expression of SDF-1 receptor. Such compounds also may act as an antagonist or inhibitor of CXCR-4. In one aspect the invention provides a cationic peptide that is an antagonist of CXCR-4. In another aspect the invention provides a method of identifying a cationic peptide that is an antagonist of CXCR-4. The method includes contacting T cells with SDF-1 in the presence of absence of a test peptide and measuring chemotaxis. A decrease in chemotaxis in the presence of the test peptide is then indicative of a peptide that is an antagonist of CXCR-4. Such compounds and methods are useful in therapeutic applications in HIV
patients. These types of compounds and the utility thereof is demonstrated, for example, in Example 11 (see also Tables 62, 63). In that example, cationic peptides are shown to inhibit cell migration and therefore antiviral activity.

[0045] In one embodiment, the invention provides an isolated cationic peptides having an amino acid sequence of the general formula (Formula A):
X1XZX3IX4PX4IPX5XZX1 (SEQ ID NO: 4), wherein X~ is one or two of R, L or K, XZ
is one of C, S or A, X3 is one of R or P, X4 is one of A or V and XS is one of V or W.
Examples of the peptides of the invention include, but are not limited to:
LLCRIVPVIPWCK (SEQ ID NO: 5), LRCPIAPVIPVCKK (SEQ ID NO: 6), KSRIVPAIPVSLL (SEQ ID NO: 7), KKSPIAPAIPWSR (SEQ ID NO: 8), RRARIVPAIPVARR (SEQ ID NO: 9) and LSRIAPAIPWAKL (SEQ ID NO: 10).

[0046] In another embodiment, the invention provides an isolated linear cationic peptide having an amino acid sequence of the general formula (Formula B):
X~LXZX3KX4XZXSX3PX3X~ (SEQ ID NO: 11), wherein X~ is one or two of D, E, S, T
or N, X2 is one or two of P, G or D, X3 is one of G, A, V, L, I or Y, X4 is one of R, K

or H and XS is one of S, T, C, M or R. Examples of the peptides of the invention include, but are not limited to: DLPAKRGSAPGST (SEQ ID NO: 12), SELPGLKHPCVPGS (SEQ ID NO: 13), TTLGPVKRDSIPGE (SEQ ID NO: 14), SLPIKHDRLPATS (SEQ ID NO: 15), ELPLKRGRVPVE (SEQ ID NO: 16) and NLPDLKKPRVPATS (SEQ ID NO: 17).

[0047] In another embodiment, the invention provides an isolated linear cationic peptide having an amino acid sequence of the general formula (Formula C):
X1XZX3X4WX4WX4X5K (SEQ ID NO: 18) (this formula includes CPl2a and CPl2d) wherein X1 is one to four chosen from A, P or R, XZ is one or two aromatic amino acids (F, Y and W), X3 is one of P or K, X4 is one, two or none chosen from A, P, Y
or W and XS is one to three chosen from R or P. Examples of the peptides of the invention include, but are not limited to: RPRYPWWPWWPYRPRK (SEQ ID NO:
19), RRAWWKAWWARRK (SEQ ID NO: 20), RAPYWPWAWARPRK (SEQ ID
NO: 21), RPAWKYWWPWPWPRRK (SEQ ID NO: 22), RAAFKWAWAWWRRK
(SEQ ID NO: 23) and RRRWKWAWPRRK (SEQ ID NO: 24).

[0048] In another embodiment, the invention provides an isolated hexadecameric cationic peptide having an amino acid sequence of the general formula (Formula D):
X~XZX3X4X~ VX3X4RGX4X3X4X1X3X1 (SEQ ID NO: 25) wherein X1 is one'or two of R or K, XZ is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X3 is C, S, M, D or A and X4 is F, I, V, M or R. Examples of the peptides of the invention include, but are not limited to: RRMCIKVCVRGVCRRKCRK (SEQ ID NO: 26), KRSCFKVSMRGVSRRRCK (SEQ ID NO: 27), KKDAIKKVDIRGMDMRRAR
(SEQ ID NO: 28), RKMVKVDVRGIMIRKDRR (SEQ ID NO: 29), KQCVKVAMRGMALRRCK (SEQ ID NO: 30) and RREAIRRVAMRGRDMKRMRR (SEQ ID NO: 31).

(0049] In still another embodiment, the invention provides an isolated hexadecameric cationic peptide having an amino acid sequence of the general formula (Formula E):
X~XzX3X4X~VXSX4RGX4X5X4X1X3Xl (SEQ ID NO: 32), wherein X~ is one or two of R or K, Xz is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X3 is one of C, S, M, D or A, X4 is one of F, I, V, M or R and XS is one of A, I, S, M, D

or R. Examples of the peptides of the invention include, but are not limited to:
RTCVKRVAMRGIIRKRCR (SEQ ID NO: 33), KKQMMKRVDVRGISVKRKR
(SEQ ID NO: 34), KESIKVIIRGMMVRMKK (SEQ ID NO: 35), RRDCRRVMVRGIDIKAK (SEQ ID NO: 36), KRTAIKKVSRRGMSVKARR (SEQ
ID NO: 37) and RHCIRRVSMRGIIMRRCK (SEQ ID NO: 38).

[0050] In another embodiment, the invention provides an isolated longer cationic peptide having an amino acid sequence of the general formula (Formula F):
KX~KXZFXZKMLMXzALKKX3 (SEQ ID NO: 39), wherein X~ is a polar amino acid (C, S, T, M, N and Q); XZ is one of A, L, S or K and X3 is 1-17 amino acids chosen from G, A, V, L, I, P, F, S, T, K and H. Examples of the peptides of the invention include, but are not limited to: KCKLFKKMLMLALKKVLTTGLPALKLTK (SEQ
ID NO: 40), KSKSFLKMLMKALKKVLTTGLPALIS (SEQ ID NO: 41), KTKKFAKMLMMALKKVVSTAKPLAILS (SEQ ID NO: 42), KMKSFAKMLMLALKKVLKVLTTALTLKAGLPS (SEQ ID NO: 43), KNKAFAKMLMKALKKVTTAAKPLTG (SEQ ID NO: 44) and KQKLFAKMLMSALKKKTLVTTPLAGK (SEQ ID NO: 45).

[0051] In yet another embodiment, the invention provides an isolated longer cationic peptide having an amino acid sequence of the general formula (Formula G):
KWKX2X1X1XzX2XIXzXZXIX~XzXzIFHTALKPISS (SEQ ID NO: 46), wherein X~ is a hydrophobic amino acid and Xz is a hydrophilic amino acid. Examples of the peptides of the invention include, but are not limited to:
KWKSFLRTFKSPVRTIFHTALKPISS (SEQ ID NO: 47), KWKSYAHTIMSPVRLIFHTALKPISS (SEQ ID NO: 48), KWKRGAHRFMKFLSTIFHTALKPISS (SEQ ID NO: 49), KWKKWAHSPRKVLTRIFHTALKPISS (SEQ ID NO: 50), KWKSLVMMFKKPARRIFHTALKPISS (SEQ ID NO: 51) and KWKHALMKAHMLWHMIFHTALKPISS (SEQ ID NO: 52).

[0052] In still another embodiment, the invention provides an isolated cationic peptide having an amino acid sequence of the formula:

KWKSFLRTFKSPVRTVFHTALKPISS (SEQ ID NO: 53) or KWKSYAHTIMSPVRLVFHTALKPISS (SEQ ID NO: 54).

[0053] The term "isolated" as used herein refers to a peptide that is substantially free of other proteins, lipids, and nucleic acids (e.g., cellular components with which an in vivo-produced peptide would naturally be associated). Preferably, the peptide is at least 70%, 80%, or most preferably 90% pure by weight.

[0054] The invention also includes analogs, derivatives, conservative variations, and cationic peptide variants of the enumerated polypeptides, provided that the analog, derivative, conservative variation, or variant has a detectable activity in which it enhances innate immunity or has anti-inflammatory activity. It is not necessary that the analog, derivative, variation, or variant have activity identical to the activity of the peptide from which the analog, derivative, conservative variation, or variant is derived.

[0055] A cationic peptide "variant" is an peptide that is an altered form of a referenced cationic peptide. For example, the term "variant" includes a cationic peptide in which at least one amino acid. of a reference peptide is substituted in an expression library. The term "reference" peptide means any of the cationic peptides of the invention (e.g., as defined in the above formulas), from which a variant, derivative, analog, or conservative variation is derived. Included within the term "derivative" is a hybrid peptide that includes at least a portion of each of two cationic peptides (e.g., 30-80% of each of two cationic peptides). Also included are peptides in which one or more amino acids are deleted from the sequence of a peptide enumerated herein, provided that the derivative has activity in which it enhances innate immunity or has anti-inflammatory activity. This can lead to the development of a smaller active molecule which would also have utility. For example, amino or carboxy terminal amino acids which may not be required for enhancing innate immunity or anti-inflammatory activity of a peptide can be removed. Likewise, additional derivatives can be produced by adding one or a few (e.g., less than 5) amino acids to a cationic peptide without completely inhibiting the activity of the peptide. In addition, C-terminal derivatives, e.g., C-terminal methyl esters, and N-terminal derivatives can be produced and are encompassed by the invention.
Peptides of the invention include any analog, homolog, mutant, isomer or derivative of the peptides disclosed in the present invention, so long as the bioactivity as described herein remains. Also included is the reverse sequence of a peptide encompassed by the general formulas set forth above. Additionally, an amino acid of "D"
configuration may be substituted with an amino acid of "I:" configuration and vice versa. Alternatively the peptide may be cyclized chemically or by the addition of two or more cysteine residues within the sequence and oxidation to form disulphide bonds.

[0056] The invention also includes peptides that are conservative variations of those peptides exemplified herein. The term "conservative variation" as used herein denotes a polypeptide in which at least one amino acid is replaced by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids that can be substituted for one another include asparagine, glutamine, serine and threonine. The term "conservative variation" also encompasses a peptide having a substituted amino acid in place of an unsubstituted parent amino acid. Such substituted amino acids may include amino acids that have been methylated or amidated. Other substitutions will be known to those of skill in the art. In one aspect, antibodies raised to a substituted polypeptide will also specifically bind the unsubstituted polypeptide.

[0057] Peptides of the invention can be synthesized by commonly used methods such as those that include t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise synthesis in which a single amino acid is added at each step starting from the C-terminus of the peptide (See, Coligan, et al., Current Protocols irc Immunology, Wiley Interscience, 1991, Unit 9). Peptides of the invention can also be synthesized by the well known solid phase peptide synthesis methods such as those described by Merrifield, J. Am. Chem. Soc., 85:2149, 1962) and Stewart and Young, Solid Phase Peptides Synthesis, Freeman, San Francisco, 1969, pp.27-62) using a copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer. On completion of chemical synthesis, the peptides can be deprotected and cleaved from the polymer by treatment with liquid HF-10% anisole for about 1/4-1 hours at 0°C.
After evaporation of the reagents, the peptides are extracted from the polymer with a 1% acetic acid solution, which is then lyophilized to yield the crude material. The peptides can be purified by such techniques as gel filtration on Sephadex G-15 using S% acetic acid as a solvent. Lyophilization of appropriate fractions of the column eluate yield homogeneous peptide, which can then be characterized by standard techniques such as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, or measuring solubility. If desired, the peptides can be quantitated by the solid phase Edman degradation.

[0058] The invention also includes isolated nucleic acids (e.g., DNA, cDNA, or RNA) encoding the peptides of the invention. Included are nucleic acids that encode analogs, mutants, conservative variations, and variants of the peptides described herein. The term "isolated" as used herein refers to a nucleic acid that is substantially free of proteins, lipids, and other nucleic acids with which an in vivo-produced nucleic acids naturally associated. Preferably, the nucleic acid is at least 70%, 80%, or preferably 90% pure by weight, and conventional methods for synthesizing nucleic acids in vitro can be used in lieu of in vivo methods. As used herein, "nucleic acid"
refers to.a polymer of deoxyribo-nucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger genetic construct (e.g. , by operably linking a promoter to a nucleic acid encoding a peptide of the invention).
Numerous genetic constructs (e.g., plasmids and other expression vectors) are known in the art and can be used to produce the peptides of the invention in cell-free systems or prokaryotic or eukaryotic (e.g., yeast, insect, or mammalian) cells. By taking into account the degeneracy of the genetic code, one of ordinary skill in the art can readily synthesize nucleic acids encoding the polypeptides of the invention. The nucleic acids of the invention can readily be used in conventional molecular biology methods to produce the peptides of the invention.

[0059] DNA encoding the cationic peptides of the invention can be inserted into an "expression vector." The term "expression vector" refers to a genetic construct such as a plasmid, virus or other vehicle known in the art that can be engineered to contain a nucleic acid encoding a polypeptide of the invention. Such expression vectors are preferably plasmids that contain a promoter sequence that facilitates transcription of the inserted genetic sequence in a host cell. The expression vector typically contains an origin of replication, and a promoter, as well as polynucleotides that allow phenotypic selection of the transformed cells (e.g., an antibiotic resistance polynucleotide). Various promoters, including inducible and constitutive promoters, can be utilized in the invention. Typically, the expression vector contains a replicon site and control sequences that are derived from a species compatible with the host cell.

[0060] Transformation or transfection of a recipient with a nucleic acid of the invention can be carried out using conventional techniques well known to those skilled in the art. For example, where the host cell is E. coli, competent cells that are capable of DNA uptake can be prepared using the CaClz, MgCl2 or RbCI methods known in the art. Alternatively, physical means, such as electroporation or microinjection can be used. Electroporation allows transfer of a nucleic acid into a cell by high voltage electric impulse. Additionally, nucleic acids can be introduced into host cells by protoplast fusion, using methods well known in the art.
Suitable methods for transforming eukaryotic cells, such as electroporation and lipofection, also are known.

[0061] "Host cells" or "Recipient cells" encompassed by of the invention are any cells in which the nucleic acids of the invention can be used to express the polypeptides of the invention. The term also includes any progeny of a recipient or host cell. Preferred recipient or host cells of the invention include E. coli, S. aureus and P. aeruginosa, although other Gram-negative and Gram-positive bacterial, fungal and mammalian cells and organisms known in the art can be utilized as long as the expression vectors contain an origin of replication to permit expression in the host.

[0062] The cationic peptide polynucleotide sequence used according to the method of the invention can be isolated from an organism or synthesized in the laboratory.
Specific DNA sequences encoding the cationic peptide of interest can be obtained by:
1) isolation of a double-stranded DNA sequence from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the cationic peptide of interest; and 3) in vitro synthesis of a double-stranded DNA
sequence by reverse transcription of mRNA isolated from a donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.

[0063] The synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired peptide product is known. In the present invention, the synthesis of a DNA sequence has the advantage of allowing the incorporation of codons which are more likely to be recognized by a~bacterial host, thereby permitting high level expression without difficulties in translation.
In addition, virtually any peptide can be synthesized, including those encoding natural cationic peptides, variants of the same, or synthetic peptides.

[0064] When the entire sequence of the desired peptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the formation of cDNA sequences. Among the standard procedures for'isolating cDNA sequences of interest is the formation of plasmid or phage containing cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the cationic peptide are known, the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single stranded form (Jay, et al., Nuc. Acid Res., 11:2325, 1983).

[0065] The peptide of the invention can be administered parenterally by injection or by gradual infusion over time. The peptide. can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
Preferred methods for delivery of the peptide include orally, by encapsulation in microspheres or proteinoids, by aerosol delivery to the lungs, or transdermally by iontophoresis or transdermal electroporation. Other methods of administration will be known to those skilled in the art.

[0066] Preparations for parenteral administration of a peptide of the invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, sodium acetate, sodium citrate, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0067] The invention will now be described in greater detail by reference to the following non-limiting examples. While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

ANTI-SEPSIS/ANTI-INFLAMMATORY ACTIVITY

[0068] Polynucleotide arrays were utilized to determine the effect of cationic peptides on the transcriptional response of epithelial cells. The A549 human epithelial cell line was maintained in DMEM (Gibco) supplemented with 10 % fetal bovine serum (FBS, Medicorp). The A549 cells were plated in 100 mm tissue culture dishes at 2.5 x cells/dish, cultured overnight and then incubated with 100 ng/ml E. coli 0111:B4 LPS

(Sigma), without (control) or with 50 pg/ml peptide or medium alone for 4 h.
After stimulation, the cells were washed once with diethyl pyrocarbonate-treated phosphate buffered saline (PBS), and detached from the dish using a cell scraper. Total RNA
was isolated using RNAqueous (Ambion, Austin, TX). The RNA pellet was resuspended in RNase-free water containing Superase-In (RNase inhibitor;
Ambion).
DNA contamination was removed with DNA-free kit, Ambion). The quality of the RNA was assessed by gel electrophoresis on a 1% agarose gel.

[0069] The polynucleotide arrays used were the Human Operon arrays (identification number for the genome is PRHU04-S1), which consist of about 14,000 human oligos spotted in duplicate. Probes were prepared from 10 pg of total RNA and labeled with Cy3 or Cy5 labeled dUTP. The probes were purified and hybridized to printed glass slides overnight at 42°-C and washed. After washing, the image was captured using a Perkin Elmer array scanner. The image processing software (Imapolynucleotide 5.0, Marina Del Rey, CA) determines the spot mean intensity, median intensities, and background intensities. A "homemade" program was used to remove background.
The program calculates the bottom 10 % intensity for each subgrid and subtracts this for each grid. Analysis was performed with Genespring software (Redwood City, CA). The intensities for each spot were normalized by taking the median spot intensity value from the population of spot values within a slide and comparing this value to the values of all slides in the experiment. The relative changes seen with cells treated with peptide compared to control cells can be found in Tables 1 and 2.
These tables 2 reflect only those polynucleotides that demonstrated significant changes in expression of the 14,000 polynucleotides that were tested for altered expression. The data indicate that the peptides have a widespread ability to reduce the expression of polynucleotides that were induced by LPS.

[0070] In Table l, the peptide, SEQ ID NO: 27 is shown to potently reduce the expression of many of the polynucleotides up-regulated by E. coli 0111:B4 LPS
as studied by polynucleotide microarrays. Peptide (50 pg/ml) and LPS (0.1 pg/ml) or LPS alone was incubated with the A549 cells for 4 h and the RNA was isolated.
Five pg total RNA was used to make Cy3/Cy5 labeled cDNA probes and hybridized onto Human Operon arrays (PRHU04). The intensity of unstimulated cells is shown in the third column of Table 1. The "Ratio: LPS/control" column refers to the intensity of polynucleotide expression in LPS simulated cells divided by in the intensity of unstimulated cells. The "Ratio: LPS+ ID 27/control" column refers to the intensity of polynucleotide expression in cells stimulated with LPS and peptide divided by unstimulated cells.
Table 1: Reduction, by peptide SEQ ID 27, of A549 human epithelial cell polynucleotide expression up-regulated by E.coli 0111:B4 LPS
Accession PolynucleotideControl: Ratio: Ratio: LPS+

Numbers Gene FunctionMedia only LPS/controlID 27/control Intensity AL031983 Unknown 0.032 302.8 5.1 ADP-ribosylation L04510 factor 0.655 213.6 1.4 ring finger D87451 protein 10 3.896 183.7 2.1 hypothetical AK000869 protein 0.138 120.1 2.3 Ric -like expressed in U78166 neurons 0.051 91.7 0.2 mucin 5 subtype B

AJ001403 tracheobronchial0.203 53.4 15.9 serine/threonine protein kinase AB040057 MASK 0.95 44.3 15.8 299756 Unknown 0.141 35.9 14.0 L42243 interferon 0.163 27.6 5.2 Accession PolynucleotideControl: Ratio: Ratio: LPS+

Number$ Gene FunctionMedia only LPS/controlID 27/control Intensity receptor 2 RNA lariat debranching NM 016216 enzyme 6.151 22.3 10.9 hypothetical AK001589 protein 0.646 19.2 1.3 AL137376 Unknown 1.881 17.3 0.6 FEM-1-like death receptor AB007856 binding protein2.627 15.7 0.6 growth arrest-AB007854 specific 7 0.845 14.8 2.2 cytosolic ovarian carcinoma AK000353 antigen 1 0.453 13.5 1.0 myeloid/lymphoi d or mixed-lineage leukemia D14539 translocated 2.033 11.6 3.1 to 1 integration site for Epstein-Barr X76785 virus 0.728 11.6 1.9 M54915 pim-1 oncogene1.404 11.4 0.6 caspase recruitment NM 006092 domain 4 0.369 11.0 0.5 integrin alpha J03925 M 0.272 9.9 4.2 Accession PolynucleotideControl: Ratio: Ratio: LPS+

Number$ Gene FunctionMedia only LPS/controlID 27/control Intensity ADP-ribosylation NM 001663 factor 6 0.439 9.7 1.7 RAS p21 protein M23379 activator 0.567 9.3 2.8 thymidine kinase K02581 1 soluble 3.099 8.6 3.5 transmembrane 9 superfamily U94831 member 1 3.265 7.1 1.5 zinc finger X70394 protein 146 1.463 6.9 1.7 hypothetical AL137614 protein 0.705 6.8 1.0 guanine nucleotide U43083 binding protein0.841 6.6 1.6 DKFZp434J181 AL137648 3 protein 1.276 6.5 0.8 ATP-binding cassette sub-family C

(CFTR/MRP) AF085692 member 3 3.175 6.5 2.4 hypothetical protein AK001239 FLJ10377 2.204 6.4 1.3 ATPase Na+/K+

NM 001679 transporting 2.402 6.3 0.9 beta Accession PolynucleotideControl: Ratio: Ratio: LPS+

Numbers Gene FunctionMedia only LPS/controlID 27/control Intensity 3 polypeptide unactive progesterone L24804 receptor 3.403 6.1 1.1 dual specificity U15932 phosphatase 0.854 6.1 2.1 ligase I DNA-M36067 ATP-dependent1.354 6.1 2.2 AL161951 Unknown 0.728 5.8 1.9 colony stimulating M59820 factor 3 receptor0.38 5.7 2.0 spermidine/

spermine N1-AL050290 acetyltransferase2.724 5.6 1.4 NM 002291 laminin beta 1.278 5.6 1.8 retinoic acid X06614 receptor- 1.924 5.5 0.8 alpha putative L-type neutral amino AB007896 acid transporter0.94 5.3 1.8 AL050333 protein 1.272 5.3 0.6 hypothetical AK001093 protein 1.729 5.3 2.0 hypothetical NM 016406 protein 1.314 5.2 1.2 M86546 pre-B-cell 1.113 5.2 2.2 Accession PolynucleotideControl: Ratio: Ratio: LPS+

Numbers Gene FunctionMedia only LPS/controlID 27/control Intensity leukemia trans-cription factor zona pellucida X56777 glycoprotein 1.414 5.0 1.4 replication initiation region NM 013400 protein 1.241 4.9 2.0 leukemia NM 002309 inhibitory 1.286 4.8 1.9 factor dentatorubral-pallidoluysian NM 001940 atrophy 2.034 4.7 1.2 cytosolic acyl coenzyme A

thioester U91316 hydrolase 2.043 4.7 1.4 death-associated X76104 protein kinase1.118 4.6 1.8 AF131838 Unknown 1.879 4.6 1.4 AL050348 Unknown 8.502 4.4 1.7 KIAA0095 gene D42085 product 1.323 4.4 1.2 X92896 Unknown 1.675 4.3 1.5 U26648 syntaxin 5A 1.59 4.3 1.4 monocyte to macrophage differentiation-X85750 associated 1.01 4.3 1.1 Accession PolynucleotideControl: Ratio: Ratio: LPS+

Numbers Gene FunctionMedia only LPS/controlID 27/control Intensity CD164 antigen-D14043 sialomucin 1.683 4.2 1.0 fibroblast J04513 growth factor1.281 4.0 0.9 melanoma-associated U19796 antigen 1.618 4.0 0.6 hypothetical AK000087 protein 1.459 3.9 1.0 hypothetical AK001569 protein 1.508 3.9 1.2 AF189009 ubiquilin 1.448 3.8 1.3 sterol-C4-methyl U60205 oxidase-like 1.569 3.7 0.8 hypothetical AK000562 protein 1.166 3.7 0.6 AL096739 Unknown 3.66 3.7 0.5 hypothetical AK000366 protein 15.192 3.5 1.0 RAN member RAS oncogene NM 006325 family 1.242 3.5 1.4 X51688 cyclin A2 1.772 3.3 1.0 aldehyde U34252 dehydrogenase1.264 3.3 1.2 domain-NM 013241 containing 1.264 3.3 0.6 Accession PolynucleotideControl: Ratio: Ratio: LPS+

Number Gene FunctionMedia only LPS/controlID 27/control Intensity protein esterase D/formylglutathi AF112219 one hydrolase1.839 3.3 1.1 anaphase-promoting complex subunit NM 016237 5 2.71 3.2 0.9 KIAA0669 gene AB014569 product 2.762 3.2 0.2 hypothetical AF151047 protein 3.062 3.1 1.0 protein phosphatase X92972 catalytic 2.615 3.1 1.1 subunit proteasome subunit ATPase AF035309 5 5.628 3.1 1.3 U52960 SRB7 homolog 1.391 3.1 0.8 electron-transfer-flavoprotein alpha J04058 polypeptide 3.265 3.1 1.2 interleukin M57230 signal transducer0.793 3.1 1.0 galactosidase_ U78027 alpha 3.519 3.1 1.1 Accession PolynucleotideControl: Ratio: Ratio: LPS+

Numbers Gene FunctionMedia only LPS/controlID 27/control Intensity AK000264 Unknown 2.533 3.0 0.6 mitogen-activated protein X80692 kinase 6 2.463 2.9 1.3 L25931 lamin B receptor2.186 2.7 0.7 X13334 CD14 antigen 0.393 2.5 1.1 tumor necrosis factorreceptor superfamily M32315 member 1B 0.639 2.4 0.4 LPS-induced TNF-alpha NM 004862 factor 6.077 2.3 1.1 interferon gamma receptor AL050337 1 2.064 2.1 1.0 °All Accession Numbers in Table 1 through Table 64 refer to GenBank Accession Numbers.

[0071] In Table 2, the cationic peptides at a concentration of 50 pg/ml were shown to potently reduce the expression of many of the polynucleotides up-regulated by ng/ml E. coli 0111:B4 LPS as studied by polynucleotide microarrays. Peptide and LPS or LPS alone was incubated with the A549 cells for 4 h and the RNA was isolated. 5 pg total RNA was used to make Cy3/Cy5 labeled cDNA probes and hybridized onto Human Operon arrays (PRHU04). The intensity of unstimulated cells is shown in the third column of Table 2. The "Ratio: LPS/control" column refers to the intensity of polynucleotide expression in LPS-simulated cells divided by in the intensity of unstimulated cells. The other columns refer to the intensity of polynucleotide expression in cells stimulated with LPS and peptide divided by unstimulated cells.

[0072] Table 2: Human A549 Epithelial Cell Polynucleotide Expression up-regulated by E.coli 0111:B4 LPS and reduced by Cationic Peptides AccessionGene Control:Ratio: Ratio: Ratio: Ratio:

Number Media LPS/ LPS+ LPS+ID LPS+ID

only controlID 27/ 16/ 22/

Intensity controlcontrolcontrol AL031983 Unknown 0.03 302.8 5.06 6.91 0.31 ADP-ribosylation L04510 factor 0.66 213.6 1.4 2.44 3.79 ring finger D87451 protein 3.90 183.7 2.1 3.68 4.28 hypothetical AK000869 protein 0.14 120.1 2.34 2.57 2.58 U78166 Ric like 0.05 91.7 0.20 16.88 21.37 MHC class II

X03066 DO beta 0.06 36.5 4.90 12.13 0.98 hypothetical AK001904 protein 0.03 32.8 5.93 0.37 0.37 AB037722 Unknown 0.03 21.4 0.30 0.30 2.36 hypothetical AK001589 protein 0.65 19.2 1.26 0.02 0.43 AL137376-Unknown 1.88 17.3 0.64 1.30 1.35 thioredoxin-dependent peroxide L19185 reductase 0.06 16.3 0.18 2.15 0.18 Transcobalamin J05068 I 0.04 15.9 1.78 4.34 0.83 FEM-1-like death AB007856 receptor 2.63 15.7 0.62 3.38 0.96 binding AccessionGene Control:Ratio: Ratio: Ratio: Ratio:

Number Media LPS/ LPS+ LPS+ID LPS+ID

only controlID 27/ 16/ 22/

Intensity controlcontrolcontrol protein cytosolic ovarian AK000353 carcinoma 0.45 13.5 1.02 1.73 2.33 ag 1 smooth muscle X16940 enteric actin0.21 11.8 3.24 0.05 2.26 y2 M54915 pim-1 oncogene1.40 11.4 0.63 1.25 1.83 hypothetical AL122111 protein 0.37 10.9 0.21 1.35 0.03 phospholipase C

M95678 beta 2 0.22 7.2 2.38 0.05 1.33 hypothetical AK001239 protein 2.20 6.4 1.27 1.89 2.25 AC004849 Unknown 0.14 6.3 0.07 2.70 0.07 retinoic acid X06614 receptor- 1.92 5.5 0.77 1.43 1.03 alpha putative L-type neutral amino AB007896 acid transporter0.94 5.3 1.82 2.15 2.41 BAIL-associated AB010894 protein 0.69 5.0 1.38 1.03 1.80 U52522 partner of 1.98 2.9 1.35 0.48 1.38 hypothetical AK001440 protein 1.02 2.7 0.43 1.20 0.01 ankyrin 2_ NM 001148neuronal 0.26 2.5 0.82 0.04 0.66 inter-alpha X07173 inhibitor 0.33 2.2 0.44 0.03 0.51 brain and nasopharyngeal carcinoma susceptibility AF095687 protein 0.39 2.1 0.48 0.03 0.98 AccessionGene Control:Ratio: Ratio: Ratio: Ratio:
Number Media LPS/ LPS+ LPS+ID LPS+ID
only controlID 27/ 16/ 22/
Intensity controlcontrolcontrol NK cell activation inducing ligand NM 016382NAIL 0.27 2.1 0.81 0.59 0.04 .

AB023198 protein 0.39 2.0 0.43 0.81 0.92 NEUTRALIZATION OF THE STIMULATION OF IMMUNE CELLS

[0073] The ability of compounds to neutralize the stimulation of immune cells by both Gram-negative and Gram-positive bacterial products was tested. Bacterial products stimulate cells of the immune system to produce inflammatory cytokines and when unchecked this can lead to sepsis. Initial experiments utilized the murine macrophage cell line RAW 264.7, which was obtained from the American Type Culture Collection, (Manassas, VA), the human epithelial cell line, A549, and primary macrophages derived from the bone marrow of BALB/c mice (Charles River Laboratories, Wilmington, MA). The cells from mouse bone marrow were cultured in 150-mm plates in Dulbecco's modified Eagle medium (DMEM; Life Technologies, Burlington, ON) supplemented with 20 % FBS (Sigma Chemical Co,St. Louis, MO) and 20 % L cell-conditioned medium as a source of M-CSF. Once macrophages were 60-80 % confluent, they were deprived of L cell-conditioned medium for 14-16 h to render the cells quiescent and then were subjected to treatments with 100 rig/ml LPS
or 100 ng/ml LPS + 20 pg/ml peptide for 24 hours. The release of cytokines into the culture supernatant was determined by ELISA (R&D Systems, Minneapolis, MN).
The cell lines, RAW 264.7 and A549, were maintained in DMEM supplemented with % fetal calf serum. RAW 264.7 cells were seeded in 24 well plates at a density of 106 cells per well in DMEM and A549 cells were seeded in 24 well plates at a density of 105 cells per well in DMEM and both were incubated at 37°C in 5 %
COZ
overnight. DMEM was aspirated from cells grown overnight and replaced with fresh medium. In some experiments, blood from volunteer human donors was collected (according to procedures accepted by UBC Clinical Research Ethics Board, certificate C00-0537) by venipuncture into tubes (Becton Dickinson, Franklin Lakes, NJ) containing 14.3 USP units heparin/ml blood. The blood was mixed with LPS with or without peptide in polypropylene tubes at 37°C for 6 h. The samples were centrifuged for 5 min at 2000 x g, the plasma was collected and then stored at -20°C until being analyzed for IL-8 by ELISA (R&D Systems). In the experiments with cells, LPS
or other bacterial products were incubated with the cells for 6-24 hr at 37°C in 5 % CO2.
S. typhimurium LPS and E. coli 0111:B4 LPS were purchased from Sigma.
Lipoteichoic acid (LTA) from S. aureus (Sigma) was resuspended in endotoxin free water (Sigma). The Limulus amoebocyte lysate assay (Sigma) was performed on LTA preparations to confirm that lots were not significantly contaminated by endotoxin. Endotoxin contamination was less than 1 ng/ml, a concentration that did not cause significant cytokine production in the RAW 264.7 cells. Non-capped lipoarabinomannan (AraLAM ) was a gift from Dr. John T. Belisle of Colorado State University. The AraLAM from Mycobacterium was filter sterilized and the endotoxin contamination was found to be 3.75 ng per 1.0 mg of LAM as determined by Limulus Amebocyte assay. At the same time as LPS addition (or later where specifically described), cationic peptides were added at a range of concentrations. The supernatants were removed and tested for cytokine production by ELISA (R&D
Systems). All assays were performed at least three times with similar results.
To confirm the anti-sepsis activity in vivo, sepsis was induced by intraperitoneal injection of 2 or 3 pg of E. coli 0111:B4 LPS in phosphate-buffered saline (PBS; pH 7.2) into galactosamine-sensitized 8- to 10- week-old female CD-1 or BALB/c mice. In experiments involving peptides, 200 ~g in 1001 of sterile water was injected at separate intraperitoneal sites within 10 min of LPS injection. In other experiments, CD-1 mice were injected with 400 ~g E. coli 0111:B4 LPS and 10 min later peptide (200 pg) was introduced by intraperitoneal injection. Survival was monitored for 48 hours post injection.

[0074] Hyperproduction of TNF-a has been classically linked to development of sepsis. The three types of LPS, LTA or AraLAM used in this example represented products released by both Gram-negative and Gram-positive bacteria. Peptide, SEQ
ID NO: 1, was able to significantly reduce TNF-a production stimulated by S.
typhimurium, B. cepacia, and E. coli 0111:B4 LPS, with the former being affected to a somewhat lesser extent (Table 3). At concentrations as low as 1 pg/ml of peptide (0.25 nM) substantial reduction of TNF-a production was observed in the latter two cases. A different peptide, SEQ ID NO: 3 did not reduce LPS-induced production of TNF-a in RAW macrophage cells, demonstrating that this is not a uniform and predictable property of cationic peptides. Representative peptides from each Formula were also tested for their ability to affect TNF-a production stimulated by E.
coli 0111:B4 LPS (Table 4). The peptides had a varied ability to reduce TNF-a production although many of them lowered TNF-a by at least 60%.

[0075] At certain concentrations peptides SEQ ID NO: 1 and SEQ ID NO: 2, could also reduce the ability of bacterial products to stimulate the production of IL-8 by an epithelial cell line. LPS is a known potent stimulus of IL-8 production by epithelial cells. Peptides, at low concentrations (1-20 pg/ml), neutralized the IL-8 induction responses of epithelial cells to LPS (Table 5-7). Peptide SEQ ID 2 also inhibited LPS-induced production of IL-8 in whole human blood (Table 4). Conversely, high concentrations of peptide SEQ ID NO: 1 (50 to 100 pg/ml) actually resulted in increased levels of IL-8 (Table 5). This suggests that the peptides have different effects at different concentrations.

[0076] The effect of peptides on inflammatory stimuli was also demonstrated in primary marine cells, in that peptide SEQ ID NO: 1 significantly reduced TNF-a production (>90 %) by bone marrow-derived macrophages from BALB/c mice that had been stimulated with 100 ng/ml E. coli 0111:B4 LPS (Table 8). These experiments were performed in the presence of serum, which contains LPS-binding protein (LBP), a protein that can mediate the rapid binding of LPS to CD14.
Delayed addition of SEQ ID NO: 1 to the supernatants of macrophages one hour after stimulation with 100 ng/ml E. coli LPS still resulted in substantial reduction (70 %) of TNF-a production (Table 9).

[0077] Consistent with the ability of SEQ ID NO: 1 to prevent LPS-induced production of TNF-a in vitro, certain peptides also protected mice against lethal shock induced by high concentrations of LPS. In some experiments, CD-1 mice were sensitized to LPS with a prior injection of galactosamine. Galactosamine-sensitized mice that were injected with 3 pg of E. coli 0111:B4 LPS were all killed within 4-6 hours. When 200 pg of SEQ ID NO: 1 was injected 15 min after the LPS, 50 % of the mice survived (Table 10). In other experiments when a higher concentration of LPS was injected into BALB/c mice with no D-galactosamine, peptide protected % compared to the control group in which there was no survival (Table 13).
Selected other peptides were also found to be protective in these models (Tables 11,12).

[0078] Cationic peptides were also able to lower the stimulation of macrophages by Gram-positive bacterial products such as Mycobacterium non-capped lipoarabinomannan (AraLAM) and S. aureus LTA. For example, SEQ ID NO: 1 inhibited induction of TNF-a in RAW 264.7 cells by the Gram-positive bacterial products, LTA (Table 14) and to a lesser extent AraLAM (Table 15). Another peptide, SEQ ID NO: 2, was also found to reduce LTA-induced TNF-a production by RAW 264.7 cells. At a concentration of 1 pg/ml SEQ ID NO: 1 was able to substantially reduce (>75 %) the induction of TNF-a production by 1 pg/ml S.
aureus LTA. At 20 pg/ml SEQ ID NO: 1, there was >60 % inhibition of AraLAM induced TNF-a. Polymyxin B (PMB) was included as a control to demonstrate that contaminating endotoxin was not a significant factor in the inhibition by SEQ
ID NO:
1 of AraLAM induced TNF-a. These results demonstrate that cationic peptides can reduce the pro-inflammatory cytokine response of the immune system to bacterial products.

[0079] Table 3: Reduction by SEQ ID 1 of LPS induced TNF-a production in RAW 264.7 cells. RAW 264.7 mouse macrophage cells were stimulated with 100 ng/ml S. typhimurium LPS, 100 ng/ml B. cepacia LPS and 100 ng/ml E. coli 0111:B4 LPS in the presence of the indicated concentrations of SEQ ID 1 for 6 hr. The concentrations of TNF-a released into the culture supernatants were determined by ELISA. 100 % represents the amount of TNF-a resulting from RAW 264.7 cells incubated with LPS alone for 6 hours (S. typhimurium LPS = 34.5 ~ 3.2 ng/ml, B.
cepacia LPS = 11.6 ~ 2.9 ng/ml, and E. coli 0111:B4 LPS = 30.8 ~ 2.4 ng/ml).
Background levels of TNF-a production by the RAW 264.7 cells cultured with no stimuli for 6 hours resulted in TNF-a levels ranging from 0.037 - 0.192 ng/ml.
The data is from duplicate samples and presented as the mean of three experiments +
standard error.
Amount of Inhibition of TNF-a (%)~

SEQ ID 1 (p,g/ml)B. cepacia E. coli LPS S. typhimurium LPS LPS

0.1 8.5 + 2.9 0.0 + 0.6 0.0 + 0 1 23.0+11.4 36.6+7.5 9.8+6.6 55.4+8 65.0+3.6 31.1+7.0 63.1+8 75.0+3.4 37.4+7.5 71.7+5.8 81.0+3.5 58.5+10.5 50 ~ 86.7+4.3 92.6+2.5 73.1+9.1 ~

(0080] Table 4: Reduction by Cationic Peptides of E. coli LPS induced TNF-a production in RAW 264.7 cells. RAW 264.7 mouse macrophage cells were stimulated with 100 ng/ml E. coli 0111:B4 LPS in the presence of the indicated concentrations of cationic peptides for 6 h. The concentrations of TNF-a released into the culture supernatants were determined by ELISA. Background levels of TNF-a production by the RAW 264.7 cells cultured with no stimuli for 6 hours resulted in TNF-a levels ranging from 0.037 - 0.192 ng/ml. The data is from duplicate samples and presented as the mean of three experiments + standard deviation.
Peptide (20 Inhibition of TNF-a (%) ~ug/ml) SEQ ID 5 65.6 1.6 SEQ ID 6 59.8 1.2 SEQ ID 7 50.6 0.6 SEQ ID 8 39.3 1.9 Peptide (20 ~ug/ml)Inhibition of TNF-a (%) SEQ ID 9 58.7 0.8 SEQ ID 10 55.5 0.52 SEQ ID 12 . 52.1 0.38 SEQ ID 13 62.4 0.85 SEQ ID 14 50.8 1.67 SEQ ID 15 69.4 0.84 SEQ ID 16 37.5 0.66 SEQ ID 17 28.3 3.71 SEQ ID 19 69.9 0.09 SEQ ID 20 66.1 0.78 SEQ ID 21 I 67.8 0.6 SEQ ID 22 7.3.3 0.36 SEQ ID 23 83.6 0.32 SEQ ID 24 60.5 0.17 SEQ ID 26 54.9 1.6 SEQ ID 27 51.1 2.8 SEQ ID 28 56 1.1 SEQ ID 29 58.9 0.005 SEQ ID 31 60.3 0.6 SEQ ID 33 62.1 0.08 SEQ ID 34 53.3 0.9 SEQ ID 35 60.7 0.76 SEQ ID 36 63 0.24 SEQ ID 37 58.9 0.67 SEQ ID 40 75 0.45 SEQ ID 41 86 0.37 SEQ ID 42 80.5 0.76 SEQ ID 43 88.2 0.65 SEQ ID 44 44.9 1.5 Peptide (20 ~ug/ml)Inhibition of TNF-a (%) SEQ ID 45 44.7 0.39 SEQ ID 47 36.9 2.2 SEQ ID 48 64 0.67 SEQ ID 49 86.9 0.69 SEQ ID 53 46.5 1.3 SEQ ID 54 64 0.73 [0081] Table 5: Reduction by SEQ ID 1 of LPS induced IL-8 production in A549 cells. A549 cells were stimulated with increasing concentrations of SEQ ID 1 in the presence of LPS (100 ng/ml E. coli 0111:B4) for 24 hours. The concentration of in the culture supernatants was determined by ELISA. The background levels of from cells alone was 0.172 ~ 0.029 ng/ml. The data is presented as the mean of three experiments + standard error.
SEQ ID 1 (pg/ml) Inhibition of IL-8 (%) 0.1 1 + 0.3 1 32 + 10 60+9 47 + 12 50 40 + 13 [0082] Table 6: Reduction by SEQ ID 2 of E. coli LPS induced IL-8 production in A549 cells. Human A549 epithelial cells were stimulated with increasing concentrations of SEQ ID 2 in the presence of LPS (100 ng/ml E. coli 0111:B4) for 24 hours. The concentration of IL-8 in the culture supernatants was determined by ELISA. The data is presented as the mean of three experiments + standard error.

Concentration of SEQ ID Inhibition of IL-8 (%) 2 (p,g/ml) 0.1 6.8 + 9.6 1 12.8 + 24.5 29.0 + 26.0 50 39.8 + 1.6 100 45.0 + 3.5 [0083] Table 7: Reduction by SEQ ID 2 of E. coli. LPS induced IL-8 in human blood. Whole human blood was stimulated with increasing concentrations of peptide and E.coli Ol 11:B4 LPS for 4 hr. The human blood samples were centrifuged and the serum was removed and tested for IL-8 by ELISA. The data is presented as the average of 2 donors.
SEQ ID 2 (p,g/ml) IL-8 (pg/ml) 10 ~ 1912 [0084] Table 8: Reduction by SEQ ID 1 of E. coli LPS induced TNF-a production in murine bone marrow macrophages. BALB/c Mouse bone marrow-derived macrophages were cultured for either 6 h or 24 h with 100 ng/ml E.
coli 0111:B4 LPS in the presence or absence of 20 pg/ml of peptide. The supernatant was collected and tested for levels of TNF-a by ELISA. The data represents the amount of TNF-a resulting from duplicate wells of bone marrow-derived macrophages incubated with LPS alone for 6 h (1.1 ~ 0.09 ng/ml) or 24 h (1.7 ~ 0.2 ng/ml).
Background levels of TNF-a were 0.038 ~ 0.008 ng/ml for 6 h and 0.06 ~ 0.012 ng/ml for 24h.

SEQ ID 1 (pg/ml) Production of TNF-a (ng/ml) , 6 hours 24 hours LPS alone 1.1 1.7 1 0.02 0.048 0.036 0.08 100 0.033 0.044 No LPS control 0.038 0.06 (0085] Table 9: Inhibition of E. coli LPS-induced TNF-a production by delayed addition of SEQ ID 1 to A549 cells. Peptide (20 p.g/ml) was added at increasing time points to wells already containing A549 human epithelial cells and 100 ng/ml E.
coli 0111:B4 LPS. The supernatant was collected after 6 hours and tested for levels of TNF-a by ELISA. The data is presented as the mean of three experiments +
standard error.
Time of addition of SEQ Inhibition of TNF-a ID 1 (%) after LPS (min) 0 98.3 + 0.3 15- - g9.3+3.8 30 83 + 4.6 60 68 + 8 9 -. -~ 53+8 [0086] Table 10: Protection against lethal endotoxaemia in galactosamine-sensitized CD-1 mice by SEQ ID 1. CD-1 mice (9 weeks-old) were sensitized to endotoxin by three intraperitoneal injections of galactosamine (20 mg in 0.1 ml sterile PBS). Then endotoxic shock was induced by intraperitoneal injection of E. coli O111:B4 LPS (3 pg in 0.1 ml PBS). Peptide, SEQ ID 1, (200 pg/mouse = 8mg/kg) was injected at a separate intraperitoneal site 15 min after injection of LPS.
The mice were monitored for 48 hours and the results were recorded.
D-GalactosamineE. coli Peptide Total Survival post treatment 0111:B4 or mice endotoxin shock LPS buffer 0 3 ~g PBS 5 5 (100%) 20 mg 3 pg PBS 12 0 (0%) 20 mg 3 ~g SEQ ID 12 6 (50%) [0087] Table 11: Protection against lethal endotoxaemia in galactosamine-sensitized CD-1 mice by Cationic Peptides. CD-1 mice (9 weeks-old) were sensitized to endotoxin by intraperitoneal injection of galactosamine (20 mg in 0.1 ml sterile PBS). Then endotoxic shock was induced by intraperitoneal injection of E. coli 0111:B4 LPS (2 pg in 0.1 ml PBS). Peptide (200 ~ug/mouse = 8mg/kg) was injected at a separate intraperitoneal site 15 min after injection of LPS. The mice were monitored for 48 hours and the results were recorded.
Peptide TreatmentE. coli 0111:B4Number Survival (%) LPS added of Mice Control (no 2 pg 5 0 peptide) SEQ ID 6 2 ~g 5 40 SEQ ID 13 . 2 pg 5 20 SEQ ID 17 2 pg 5 40 SEQ ID 24 2 pg 5 0 SEQ ID 27 2 pg 5 20 [0088] Table 12: Protection against lethal endotoxaemia in galactosamine-sensitized BALB/c mice by Cationic Peptides. BALB/c mice (8 weeks-old) were sensitized to endotoxin by intraperitoneal injection of galactosamine (20 mg in 0.1 ml sterile PBS). Then endotoxic shock was induced by intraperitoneal injection of E. coli 0111:B4 LPS (2 pg in 0.1 ml PBS). Peptide (200 pg/mouse = 8mg/kg) was injected at a separate intraperitoneal site 15 min after injection of LPS. The mice were monitored for 48 hours and the results were recorded.
Peptide TreatmentE. coli Number of Survival 0111:B4 LPS Mice (%) added No peptide 2 pg 10 10 SEQ ID 1 2 pg 6 17 SEQ ID 3 2 pg 6 0 SEQ ID 5 2 pg 6 17 SEQ ID 6 2 ~g 6 17 SEQ ID 12 2 pg 6 17 SEQ ID 13 2 ~g 6 33 SEQ ID 15 2 dug 6 0 SEQ ID 16 2 pg 6 0 SEQ ID 17 2 pg 6 17 SEQ ID 23 2 pg 6 0 SEQ ID 24 2 pg 6 17 SEQ ID 26 2 dug 6 0 SEQ ID 27 2 pg 6 50 SEQ ID 29 2 pg 6 0 SEQ ID 37 2 pg 6 0 SEQ ID 38 2 pg 6 0 SEQ ID 41 2 pg 6 0 .

SEQ ID 44 2 pg 6 0 SEQ ID 45 2 pg 6 0 [0089] Table 13: Protection against lethal endotoxaemia in BALB/c mice by SEQ
ID 1. BALB/c mice were injected intraperitoneal with 400 pg E. coli 0111:B4 LPS.
Peptide (200 ~g/mouse = 8mg/kg) was injected at a separate intraperitoneal site and the mice were monitored for 48 hours and the results were recorded.
Peptide E. coli Number of MiceSurvival (%) Treatment 0111:B4 LPS

No peptide 400 pg 5 0 SEQ ID 1 400 pg 5 100 [0090] Table 14: Peptide inhibition of TNF-a production induced by S. aureus LTA. RAW 264.7 mouse macrophage cells were stimulated with 1 pg/ml S. aureus LTA in the absence and presence of increasing concentrations of peptide. The supernatant was collected and tested for levels of TNF-a by ELISA. Background levels of TNF-a production by the RAW 264.7 cells cultured with no stimuli for hours resulted in TNF-a levels ranging from 0.037 - 0.192 ng/ml. The data is presented as the mean of three or more experiments + standard error.
SEQ ID 1 added (~g/ml)Inhibition of TNF-a (%) 0.1 44.5 + 12.5 1 76.7 + 6.4 91+1 94.5 + 1.5 96 + 1 [0091] Table 15: Peptide inhibition of TNF-a production induced by Mycobacterium non-capped lipoarabinomannan. RAW 264.7 mouse macrophage cells were stimulated with 1 pg/ml AraLAM in the absence and presence of 20 ~g/ml peptide or Polymyxin B. The supernatant was collected and tested for levels of TNF-a by ELISA. Background levels of TNF-a production by the RAW 264.7 cells cultured with no stimuli for 6 hours resulted in TNF-a levels ranging from 0.037 -0.192 ng/ml. The data is presented as the mean inhibition of three or more experiments + standard error.
Peptide (20 ~g/ml)Inhibition of TNF-a (%) No peptide 0 SEQ ID 1 64 + 5.9 Polymyxin B 15 + 2 ASSESSMENT OF TOXICITY OF THE CATIONIC PEPTIDES

[0092] The potential toxicity of the peptides was measured in two ways. First, the Cytotoxicity Detection Kit (Roche) (Lactate dehydrogenase -LDH) Assay was used.
It is a colorimetric assay for the quantification of cell death and cell lysis, based on the measurement of LDH activity released from the cytosol of damaged cells into the supernatant. LDH is a stable cytoplasmic enzyme present in all cells and it is released into the cell culture supernatant upon damage of the plasma membrane. An increase in the amount of dead or plasma membrane-damaged cells results in an increase of the LDH enzyme activity in the culture supernatant as measured with an ELISA plate reader, OD49onm (the amount of color formed in the assay is proportional to the number of lysed cells). In this assay, human bronchial epithelial cells (16HBEo14, HBE) cells were incubated with 100 pg of peptide for 24 hours, the supernatant removed and tested for LDH. The other assay used to measure toxicity of the cationic peptides was the WST-1 assay (Roche). This assay is a colorimetric assay for the quantification of cell proliferation and cell viability, based on the cleavage of the tetrazolium salt WST-1 by mitochondria) dehydrogenases in viable cells (a non-radioactive alternative to the [3H]-thymidine incorporation assay). In this assay, HBE
cells were incubated with 100 pg of peptide for 24 hours, and then 10 pl/well Cell Proliferation Reagent WST-1 was added. The cells are incubated with the reagent and the plate is then measured with an ELISA plate reader, OD49onm.

[0093] The results shown below in Tables 16 and 17 demonstrate that most of the peptides are not toxic to the cells tested. However, four of the peptides from Formula F (SEQ ID NOS: 40, 41, 42 and 43) did induce membrane damage as measured by both assays.

[0094] Table 16: Toxicity of the Cationic Peptides as Measured by the LDH
Release Assay. Human HBE bronchial epithelial cells were incubated with 100 p.g/ml peptide or Polymyxin B for 24 hours. LDH activity was assayed in the supernatant of the cell cultures. As a control for 100% LDH release, Triton X-100 was added. The data is presented as the mean ~ standard deviation. Only peptides SEQ ID
40,41,42 and 43 showed any significant toxicity.
Treatment LDH Release (OD49o nm) No cells Control0.6 0.1 Triton X-100 4.6 0.1 Control No peptide control1.0 0.05 SEQ ID 1 1.18 0.05 SEQ ID 3 1.05 0.04 SEQ ID 6 . 0.97 0.02 SEQ ID 7 1.01 0.04 SEQ ID 9 1.6 0.03 SEQ ID 10 1.04 0.04 SEQ ID 13 0.93 0.06 SEQ ID 14 0.99 0.05 SEQ ID 16 0.91 0.04 SEQ ID 17 0.94 0.04 SEQ ID 19 1.08 0.02 SEQ ID 20 1.05 0.03 SEQ ID 21 1.06 0.04 SEQ ID 22 1.29 0.12 SEQ ID 23 1.26 0.46 SEQ ID 24 1.05 0.01 Treatment LDH Release (OD49o nm) SEQ ID 26 0.93 0.04 SEQ ID 27 0.91 0.04 SEQ ID 28 0.96 0.06 SEQ ID 29 0.99 0.02 SEQ ID 31 0.98 0.03 SEQ ID 33 1.03 0.05 SEQ ID 34 1.02 0.03 SEQ ID 35 0.88 0.03 SEQ ID 36 0.85 0.04 SEQ ID 37 0.96 0.04 SEQ ID 38 0.95 0.02 SEQ ID 40 2.8 0.5 SEQID41 3.30.2 SEQ ID 42 3.4 0.2 SEQ ID 43 4.3 0.2 SEQ ID 44 0.97 0.03 SEQ ID 45 0.98 0.04 SEQ ID 47 1.05 0.05 SEQ ID 48 0.95 0.05 SEQ ID 53 1.03 0.06 Polymyxin B 1.21 0.03 [0095] Table 17: Toxicity of the Cationic Peptides as Measured by the WST-1 Assay. HBE cells were incubated with 100 pg/ml peptide or Polymyxin B for 24 hours and cell viability was tested. The data is presented as the mean ~
standard deviation. As a control for 100% LDH release, Triton X-100 was added. Only peptides SEQ ID 40,41,42 and 43 showed any significant toxicity.

Treatment OD49o nm No cells Control 0.24 0.01 Triton X-100 Control 0.26 0.01 No peptide control 1.63 0.16 SEQ ID 1 1.62 0.34 SEQ ID 3 1.35 0.12 SEQ ID 10 1.22 0.05 SEQ ID 6 1.81 0.05 SEQ ID 7 1.78 0.10 SEQ ID 9 1.69 0.29 SEQ ID 13 1.23 0.11 SEQ ID 14 1.25 0.02 SEQ ID 16 1.39 0.26 SEQ ID 17 1.60 0.46 SEQ ID 19 1.42 0.15 SEQ ID 20 1.61 0.21 SEQ ID 21 1.28 0.07 SEQ ID 22 1.33 0.07 SEQ ID 23 1.14 0.24 SEQ ID 24 1.27 0.16 SEQ ID 26 1.42 0.11 SEQ ID 27 1.63 0.03 SEQ ID 28 1.69 0.03 SEQ ID 29 1.75 0.09 SEQ ID 31 1.84 0.06 SEQ ID 33 1.75 0.21 SEQ ID 34 0.96 0.05 SEQ ID 35 1.00 0.08 SEQ ID 36 1.58 0.05 SEQ ID 37 1.67 0.02 SEQ ID 38 1.83 0.03 Treatment OD49o nm SEQ ID 40 0.46 0.06 SEQ ID 41 0.40 0.01 SEQ ID 42 0.39 0.08 SEQ ID 43 0.46 0.10 SEQ ID 44 1.49 0.39 SEQ ID 45 1.54 0.35 SEQ ID 47 1.14 0.23 SEQ ID 48 0.93 0.08 SEQ ID 53 1.51 0.37 Polymyxin B 1.30 0.13 POLYNUCLEOTIDE REGULATION BY CATIONIC PEPTIDES

[0096] Polynucleotide arrays were utilized to determine the effect of cationic peptides by themselves on the transcriptional response of macrophages and epithelial cells.
Mouse macrophage RAW 264.7, Human Bronchial cells (HBE), or A549 human epithelial cells were plated in 150 mm tissue culture dishes at 5.6 x 106 cells/dish, cultured overnight and then incubated with 50 pg/ml peptide or medium alone for 4 h.
After stimulation, the cells were washed once with diethyl pyrocarbonate-treated PBS, and detached from the dish using a cell scraper. Total RNA was isolated using Trizol (Gibco Life Technologies). The RNA pellet was resuspended in RNase-free water containing RNase inhibitor (Ambion, Austin, TX). The RNA was treated with DNaseI
(Clontech, Palo Alto, CA) for 1 h at 37°C. After adding termination mix (0.1 M
EDTA [pH 8.0], 1 mg/ml glycogen), the samples were extracted once with phenol:
chloroform: isoamyl alcohol (25:24:1), and once with chloroform. The RNA was then precipitated by adding 2.5 volumes of 100% ethanol and 1/10'" volume sodium acetate, pH 5.2. The RNA was resuspended in RNase-free water with RNase inhibitor (Ambion) and stored at -70°C. The quality of the RNA was assessed by gel electrophoresis on a 1% agarose gel. Lack of genomic DNA contamination was assessed by using the isolated RNA as a template for PCR amplification with (3-actin-specific primers (5'-GTCCCTGTATGCCTCTGGTC-3' (SEQ ID NO: 55) and 5'-GATGTCACGCACGATTTCC-3' (SEQ ID NO: 56)). Agarose gel electrophoresis and ethidium bromide staining confirmed the absence of an amplicon after 35 cycles.

[0097] Atlas cDNA Expression Arrays (Clontech, Palo Alto, CA), which consist of 588 selected mouse cDNAs spotted in duplicate on positively charged membranes were used for early polynucleotide array studies (Tables 18,19) 32P-radiolabeled cDNA probes prepared from 5 pg total RNA were incubated with the arrays overnight at 71°C. The filters were washed extensively and then exposed to a phosphoimager screen (Molecular Dynamics, Sunnyvale, CA) for 3 days at 4°C. The image was captured using a Molecular Dynamics PSI phosphoimager. The hybridization signals were analyzed using AtlasImage 1.0 Image Analysis software (Clontech) and Excel (Microsoft, Redmond, WA). The intensities for each spot were corrected for background levels and normalized for differences in probe labeling using the average values for 5 polynucleotides observed to vary little between the stimulation conditions: (3-actin, ubiquitin, ribosomal protein 529, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and CaZ+ binding protein. When the normalized hybridization intensity for a given cDNA was less than 20, it was assigned a value of 20 to calculate the ratios and relative expression.

[0098] The next polynucleotide arrays used (Tables 21-26) were the Resgen Human cDNA arrays (identification number for the genome is PRHU03-S3), which consist of 7,458 human cDNAs spotted in duplicate. Probes were prepared from 15-20 pg of total RNA and labeled with Cy3 labeled dUTP. The probes were purified and hybridized to printed glass slides overnight at 42°-C and washed. After washing, the image was captured using a Virtek slide reader: The image processing software (Imagene 4.1, Marina Del Rey, CA) determines the spot mean intensity, median intensities, and background intensities. Normalization and analysis was performed with Genespring software (Redwood City, CA). Intensity values were calculated by subtracting the mean background intensity from the mean intensity value determined by Imagene. The intensities for each spot were normalized by taking the median spot intensity value from the population of spot values within a slide and comparing this value to the values of all slides in the experiment. The relative changes seen with cells treated with peptide compared to control cells can be found in the Tables below.

[0099] The other polynucleotide arrays used (Tables 27-35) were the Human Operon arrays (identification number for the genome is PRHU04-S1), which consist of about 14,000 human oligos spotted in duplicate. Probes were prepared from 10 pg of total RNA and labeled with Cy3 or Cy5 labeled dUTP. In these experiments, A549 epithelial cells were plated in 100 mm tissue culture dishes at 2.5 x 106 cells/dish.
Total RNA was isolated using RNAqueous (Ambion). DNA contamination was removed with DNA-free kit (Ambion). The probes prepared from total RNA were purified and hybridized to printed glass slides overnight at 42°-C and washed. After washing, the image was captured using a Perkin Elmer array scanner. The image processing software (Imagene 5.0, Marina Del Rey, CA) determines the spot mean intensity, median intensities, and background intensities. An "in house"
program was used to remove background. The program calculates the bottom 10% intensity for each subgrid and subtracts this for each grid. Analysis was performed with Genespring software (Redwood City, CA). The intensities for each spot were normalized by taking the median spot intensity value from the population of spot values within a slide and comparing this value to the values of all slides in the experiment. The relative changes seen with cells treated with peptide compared to control cells can be found in the Tables below.

[00100] Semi-quantitative RT-PCR was performed to confirm polynucleotide array results. 1 pg RNA samples were incubated with 1 pl oligodT (500 pg/ml) and 1 pl mixed dNTP stock at 1 mM, in a 12 pl volume with DEPC treated water at 65°C for 5 min in a thermocycler. 4 pl 5X First Strand buffer, 2 pl O.1M DTT, and 1 pl RNaseOUT recombinant ribonuclease inhibitor (40 units/pl) were added and incubated at 42 °C for 2 min, followed by the addition of 1 pl (200 units) of Superscript II (Invitrogen, Burlington, ON). Negative controls for each RNA
source were generated using parallel reactions in the absence of Superscript II.
cDNAs were amplified in the presence of 5' and 3' primers (1.0 pM), 0.2 mM dNTP mixture, 1.5 mM MgCI, 1 U of Tag DNA polymerase (New England Biolabs, Missisauga, ON), and 1X PCR buffer. Each PCR was performed with a thermal cycler by using 30-40 cycles consisting of 30s of denaturation at 94 °C, 30s of annealing at either 52 °C or 55 °C and 40s of extension at 72 °C. The number of cycles of PCR
was optimized to lie in the linear phase of the reaction for each primer and set of RNA samples. A
housekeeping polynucleotide (3-actin was amplified in each experiment to evaluate extraction procedure and to estimate the amount of RNA. The reaction product was visualized by electrophoresis and analyzed by densitometry, with relative starting RNA concentrations calculated with reference to ~3-actin amplification.

[00101] Table 18 demonstrates that SEQ ID NO: 1 treatment of RAW 264.7 cells up-regulated the expression of more than 30 different polynucleotides on small Atlas microarrays with selected known polynucleotides. The polynucleotides up-regulated by peptide, SEQ ID NO: 1, were mainly from two categories: one that includes receptors (growth, chemokine, interleukin, interferon, hormone, neurotransmitter), cell surface antigens and cell adhesion and another one that includes cell-cell communication (growth factors, cytokines, chemokines, interleukin, interferons, hormones), cytoskeleton, motility, and protein turnover. The specific polynucleotides up-regulated included those encoding chemokine MCP-3, the anti-inflammatory cytokine IL-10, macrophage colony stimulating factor, and receptors such as IL-(a putative antagonist of productive IL-1 binding to IL-1R1), PDGF receptor B, NOTCH4, LIF receptor, LFA-l, TGF(3 receptor 1, G-CSF receptor, and IFNy receptor. The peptide also up-regulated polynucleotides encoding several metalloproteinases, and inhibitors thereof, including the bone morphogenetic proteins BMP-1, BMP-2, BMP-8a, TIMP2 and TIMP3. As well, the peptide up-regulated specific transcription factors, including JunD, and the YY and LIM-1 transcription factors, and kinases such as Etkl and Csk demonstrating its widespread effects. It was also discovered from the polynucleotide array studies that SEQ ID N0: 1 down-regulated at least 20 polynucleotides in RAW 264.7 macrophage cells (Table 19). The polynucleotides down-regulated by peptide included DNA repair proteins and several inflammatory mediators such as MIP-la, oncostatin M and IL-12. A number of the effects of peptide on polynucleotide expression were confirmed by RT-PCR
(Table 20). The peptides, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 19, and SEQ ID NO:
1, and representative peptides from each of the formulas also altered the transcriptional responses in a human epithelial cell line using mid-sized microarrays (7835 polynucleotides). The effect of SEQ ID NO: 1 on polynucleotide expression was compared in 2 human epithelial cell lines, A549 and HBE. Polynucleotides related to the host immune response that were up-regulated by 2 peptides or more by a ratio of 2-fold more than unstimulated cells are described in Table 21.
Polynucleotides that were down-regulated by 2 peptides or more by a ratio of 2-fold more than unstimulated cells are described in Table 22. In Table 23 and Table 24, the human epithelial pro-inflammatory polynucleotides that are up- and down-regulated respectively are shown. In Table 25 and Table 26 the anti-inflammatory polynucleotides affected by cationic peptides are shown. The trend becomes clear that the cationic peptides up-regulate the anti-inflammatory response and down-regulate the pro-inflammatory response. It was very difficult to find a polynucleotide related to the anti-inflammatory response that was down-regulated (Table 26). The pro-inflammatory polynucleotides upregulated by cationic peptides were mainly polynucleotides related to migration and adhesion. Of the down-regulated pro-inflammatory polynucleotides, it should be noted that all the cationic peptides affected several toll-like receptor (TLR) polynucleotides, which are very important in signaling the host response to infectious agents. An important anti-inflammatory polynucleotide that was up-regulated by all the peptides is the IL-10 receptor. IL-10 is an important cytokine involved in regulating the pro-inflammatory cytokines.
These polynucleotide expression effects were also observed using primary human macrophages as observed for peptide SEQ ID NO: 6 in Tables 27 and 28. The effect of representative peptides from each of the formulas on human epithelial cell expression of selected polynucleotides (out of 14,000 examined) is shown in Tables 31-37 below. At least 6 peptides from each formula were tested for their ability to alter human epithelial polynucleotide expression and indeed they had a wide range of stimulatory effects. In each of the formulas there were at least 50 polynucleotides commonly up-regulated by each of the peptides in the group.

[00102] Table 18: Polynucleotides up-regulated by peptide, SEQ ID NO: 1, treatment of RAW macrophage cellsa. The cationic peptides ~at a concentration of 50 pg/ml were shown to potently induce the expression of several polynucleotides.
Peptide was incubated with the RAW cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Atlas arrays. The intensity of unstimulated cells is shown in the third column. The "Ratio Peptide:
Unstimulated"
column refers to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.

[00103] The changes in the normalized intensities of the housekeeping polynucleotides ranged from 0.8-1.2 fold, validating the use of these polynucleotides for normalization. When the normalized hybridization intensity for a given cDNA was less than 20, it was assigned a value of 20 to calculate the ratios and relative expression. The array experiments were repeated 3 times with different RNA
preparations and the average fold change is shown above. Polynucleotides with a two fold or greater change in relative expression levels are presented.
PolynucleotidePolynucleotide UnstimulatedRatio Accession / Protein Function Intensity peptide: Number Unstimulatedb Etkl Tyrosine-protein20 43 M68513 kinase receptor PDGFRB Growth factor 24 25 X04367 receptor Corticotropin 20 23 X72305 releasing factorreceptor NOTCH4 proto- 48 18 M80456 oncopolynucleotide IL-1R2 Interleukin 20 16 X59769 receptor MCP-3 Chemokine 56 14 S71251 BMP-1 Bone morpho- 20 14 L24755 polynucleotidetic protein Endothelin Receptor 20 14 U32329 b receptor c-ret Oncopolynucleotide20 13 X67812 precursor LIFR Cytokine receptor20 12 D26177 PolynucleotidePolynucleotide UnstimulatedRatio Accession / Protein Function Intensity peptide: Number Unstimulatedb BMP-8a Bone morpho- 20 12 M97017 polynucleotidetic protein Zfp92 Zinc finger protein87 11 U47104 MCSF Macrophage colony85 11 X05010 stimulating factor GCSFR Granulocyte colony-20 11 M58288 stimulating factor receptor IL-8RB Chemokine receptor112 10 D17630 IL-9R Interleukin receptor112 6 M84746 Cas Crk-associated 31 6 U48853 substrate p58/GTA Kinase 254 5 M58633 CASP2 Caspase precursor129 5 D28492 IL-1(3 Interleukin precursor91 5 M15131 precursor SPI2-2 Serine protease 62 5 M64086 inhibitor CSAR Chemokine receptor300 4 546665 L-myc Oncopolynucleotide208 4 X13945 IL-10 Interleukin 168 4 M37897 pl9ink4 cdk4 and cdk6 147 4 U19597 inhibitor ATOH2 Atonal homolog 113 4 U29086 DNAseI DNase 87 4 U00478 CXCR-4 Chemokine receptor36 4 D87747 Cyclin D3 Cyclin 327 3 U43844 IL-7Ra Interleukin receptor317 3 M29697 POLA DNA polymerasen 241 3 D17384 Tie-2 ' Oncopolynucleotide193 3 S67051 PolynucleotidePolynucleotide UnstimulatedRatio Accession / Protein Function Intensity peptide: Number Unstimulatedb DNL1 DNA ligase I 140 3 U04674 BAD Apoptosis protein122 3 L37296 GADD45 DNA-damage- 88 3 L28177 inducible protein Sik Src-related kinase82 3 U16805 integrina4 Integrin 2324 2 X53176 TGF(3R1 Growth factor 1038 2 D25540 receptor LAMR1 Receptor 1001 2 J02870 Crk Crk adaptor protein853 2 572408 ZFX Chromosomal protein679 2 M32309 Cyclin E1 Cylcin 671 2 X75888 POLD1 DNA polymerase 649 2 221848 subunit Vav proto- 613 2 X64361 oncopolynucleotide YY (NF-E1) Transcription 593 2 L13968 factor JunD Transcription 534 2 J050205 factor Csk c-src kinase 489 2 U05247 Cdk7 Cyclin-dependent475 2 U11822 kinase MLClA Myosin light 453 2 M19436 subunit isoform ERBB-3 Receptor 435 2 L47240 UBF Transcription 405 2 X60831 factor TRAIL Apoptosis ligand364 2 U37522 LFA-1 Cell adhesion 340 2 X14951 receptor SLAP Src-like adaptor315 2 U29056 protein IFNGR Interferon gamma308 2 M28233 receptor LIM-1 Transcription 295 2 227410 factor ATF2 Transcription 287 2 576657 factor PolynucleotidePolynucleotide UnstimulatedRatio Accession / Protein Function Intensity peptide: Number Unstimulatedb FST Follistatin precursor275 2 229532 TIMP3 Protease inhibitor259 2 L19622 RU49 Transcription 253 2 U41671 factor IGF-1Ra Insulin-like 218 2 U00182 growth factorreceptor Cyclin G2 Cyclin 214 2 U95826 fyn Tyrosine-protein191 2 U70324 kinase BMP-2 Bone morpho- 186 2 L25602 polynucleotidetic protein Brn-3.2 Transcription 174 2 S68377 POU factor KIF1A Kinesin family 169 2 D29951 protein MRC1 Mannose receptor167 2 211974 PAI2 Protease inhibitor154 2 X19622 BKLF CACCC Box- binding138 2 U36340 protein .TIMP2 Protease inhibitor136 2 X62622 Mas Proto- 131 2 X67735 oncopolynucleotide NURR-1 Transcription 129 2 553744 factor [00104] Table 19: Polynucleotides down-regulated by SEQ ID NO: 1 treatment of RAW macrophage cellsa. The cationic peptides at a concentration of 50 pg/ml were shown to reduce the expression of several polynucleotides. Peptide was incubated with the RAW cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Atlas arrays. The intensity of unstimulated cells is shown in the third column. The "Ratio Peptide: Unstimulated" column refers to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells. The array experiments were repeated 3 times with different cells and the average fold change is shown below. Polynucleotides with an approximately two fold or greater change in relative expression levels are presented.
UnstimulatedRatio Accession PolynucleotidePolynucleotide Intensity peptide: Number / Protein Function Unstimulated sodium channelVoltage-gated 257 0.08 L36179 ion channel XRCC1 DNA repair protein227 0.09 U02887 ets-2 Oncopolynucleotide189 0.11 J04103 XPAC DNA repair protein485 0.12 X74351 EPOR Receptor precursor160 0.13 J04843 PEA 3 Ets-related protein158 0.13 X63190 orphan receptorNuclear receptor 224 0.2 U11688 N-cadherin Cell adhesion 238 0.23 M31131 receptor OCT3 Transcription 583 0.24 M34381 factor PLC(3 phospholipase 194 0.26 U43144 KRT18 Intermediate filament318 0.28 M11686 proteins THAM Enzyme 342 0.32 X58384 CD40L CD401igand 66 0.32 X65453 CD86 T-lymphocyte antigen195 0.36 L25606 oncostatin Cytokine 1127 0.39 D31942 M

PMS2 DNA DNA repair protein200 0.4 U28724 IGFBP6 Growth factor 1291 0.41 X81584 MIP-1(3 Cytokine 327 0.42 M23503 ATBFl AT motif-binding 83 0.43 D26046 factor nucleobindinGolgi resident 367 0.43 M96823 protein bcl-x Apoptosis protein142 0.43 L35049 uromodulin glycoprotein 363 0.47 L33406 IL-12 p40 Interleukin 601 0.48 M86671 MmRad52 DNA repair protein371 0.54 232767 UnstimulatedRatio Accession PolynucleotidePolynucleotide Intensity peptide: Number / Protein Function Unstimulated Tobl Antiproliferative956 0.5 D78382 factor Ungl DNA repair protein535 0.51 ~ X99018 KRT19 Intermediate filament622 0.52 M28698 proteins PLCy phospholipase 251 0.52 X95346 Integrin Cell adhesion 287 0.54 X69902 ab receptor GLUTl Glucose transporter524 0.56 M23384 CTLA4 immunoglobin 468 0.57 X05719 superfamily FRA2 Fos-related antigen446 0.57 X83971 MTRP Lysosome-associated498 0.58 U34259 protein [00105] Table 20: Polynucleotide Expression changes in response to peptide, SEQ ID NO: 1, could be confirmed by RT-PCR. RAW 264.7 macrophage cells were incubated with 50 pg/ml of peptide or media only for 4 hours and total RNA
isolated and subjected to semi-quantitative RT-PCR. Specific primer pairs for each polynucleotide were used for amplification of RNA. Amplification of [3-actin was used as a positive control and for standardization. Densitometric analysis of RT-PCR
products was used. The results refer to the relative fold change in polynucleotide expression of peptide treated cells compared to cells incubated with media alone. The data is presented as the mean ~ standard error of three experiments.
PolynucleotideArray Ratio-'~ RT-PCR Ratio -CXCR-4 4.0 1.7 4.1 0.9 IL-8RB 9.5 7.6 7.1 1.4 MCP-3 13.5 4.4 4.8 0.88 IL-10 4.22.1 16.66.1 PolynucleotideArray Ratio-'~ RT-PCR Ratio -~

CD14 0.90.1 0.80.3 MIP-1B 0.42 0.09 0.11 0.04 XRCC1 0.12 0.01 0.25 0.093 MCP-1 Not on array 3.5 1.4 [00106] Table 21: Polynucleotides up-regulated by peptide treatment of A549 epithelial cellsa. The cationic peptides at concentrations of 50 pg/ml were shown to increase the expression of several polynucleotides. Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The "Ratio Peptide: Unstimulated" columns refers to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
Accession UnstimulatedRatio Number Peptide:
Unstimulated Polynucleotide/ProteinIntensity ID ID ID ID

L-1 R antagonist 0.00 3086 1856 870 AI167887 homolog 1 L-10 R beta 0.53 2.5 1.6 1.9 3.1 AA486393 L-11 R alpha 0.55 2.4 1.0 4.9 1.8 AA454657 L-17 R 0.54 2.1 2.0 1.5 1.9 AW029299 NF R superfamily, member 0.28 18 3.0 15 3.6 AA150416 NF R superfamily, member 33.71 3.0 0.02 H98636 (CD40LR) NF R superfamily, member 1.00 5.3 4.50 0.8 AA194983 llb L-8 0.55 3.6 17 1.8 1.1 AA102526 interleukin enhancer binding 0.75 1.3 2.3 0.8 4.6 AA894687 factor 2 interleukin enhancer0.41 2.7 5.3 2.5 856553 binding Accession UnstimulatedRatio Number Peptide:
Unstimulated olynucleotide/ProteinIntensity ID ID ID ID

factor 1 cytokine inducible SH2- 0.03 33 44 39 46 AA427521 containing protein K cytokine, down-regulator of HLA II 0.50 3.1 2.0 1.7 3.3 839227 cytokine inducible SH2- 0.03 33 44 39 46 AA427521 containing protein K cytokine, down-regulator of HLA II 0.50 3.1 2.0 1.7 3.3 839227 small inducible cytokine 1.00 3.9 2.4 AI922341 subfamily A (Cys-Cys), ember 21 GFB inducible early growth 0.90 2.4 2.1 0.9 1.1 AI473938 espouse 2 K cell R 1.02 2.5 0.7 0.3 1.0 AA463248 CCR6 0.14 4.5 7.8 6.9 7.8 N57964 cell adhesion molecule0.25 4.0 3.9 3.9 5.1 840400 elanoma adhesion 0.05 7. 20 43 29.1 AA497002 molecule 9 CD31 0.59 2.7 3.1 1.0 1.7 822412 integrin, alpha 2 (CD49B, 1.00 0.9 2.4 3.6 0.9 AA463257 alpha 2 subunit of VLA-2.
receptor integrin, alpha 3 (antigen 0.94 0.8 2.5 1.9 1.1 AA424695 CD49C, alpha 3 subunit of VLA-3 receptor) integrin, alpha 0.01 180 120 28 81 AA425451 E

integrin, beta 0.47 2.1 2.1 7.0 2.6 W67174 integrin, beta 0.55 2.7 2.8 1.8 1.0 AA037229 integrin, beta 0.57 2.6 1.4 1.8 2.0 AA666269 integrin, beta 0.65 0.8 2.2 4.9 1.5 AA485668 integrin beta 4 0.20 1.7 5.0 6.6 5.3 AI017019 binding protein Accession UnstimulatedRatio Number Peptide:
Unstimulated olynucleotide/ProteinIntensity ID ID ID ID

calcium and integrin binding rotein 0.21 2.8 4.7 9.7 6.7 AA487575 disintegrin and etalloproteinase 0.46 3.1 2.2 3.8 AA279188 domain 8 disintegrin and etalloproteinase 0.94 1.1 2.3 3.6 0.5 H59231 domain 9 disintegrin and metalloproteinase 0.49 1.5 2.1 3.3 2.2 AA043347 domain 10 disintegrin and etalloproteinase 0.44 1.9 2.3 2..5 4.6 H11006 domain 23 cadherin 1, type 1, E-cadherin (epithelial) 0.42 8.1 2.2 2.4 7.3 H97778 cadherin 12, type 2 (N-cadherin 2) 0.11 13 26 9.5 AI740827 rotocadherin 12 0.09 14.8 11.5 2.6 12.4 AI652584 rotocadherin gamma subfamily C, 3 0.34 3.0 2.5 4.5 9.9 889615 catenin (cadherin-associated rotein), delta 0.86 1.2 2.2 2.4 AA025276 laminin R 1 (67kD, ribosomal rotein SA) 0.50 0.4 2.0 4.4 3.0 AA629897 filler cell lectin-like receptor subfamily C, member0.11 9.7 9.0 4.1 13.4 AA190627 filler cell lectin-like receptor subfamily C, member1.00 3.2 1.0 0.9 1.3 W93370 filler cell lectin-like receptor subfamily G, member0.95 2.3 1.7 0.7 1.1 AI433079 C-type lectin-like0.45 2.1 8.0 2.2 5.3 H70491 receptor-2 CSF 3 R 0.40 1.9 2.5 3.5 4.0 AA458507 acrophage stimulating1.00 1.7 2.3 0.4 0.7 AA173454 MP R type IA 0.72 1.9 2.8 0.3 1.4 W 15390 Accession UnstimulatedRatio Number Peptide:
Unstimulated olynucleotide/ProteinIntensityID, ID ID ID

formyl peptide receptor1.00 3.1 1.4 0.4 AA425767 CD2 1.00 2.6 0.9 1.2 0.9 AA927710 CD36 0.18 8.2 5.5 6.2 2.5 N39161 vitamin D R 0.78 2.5 1.3 1.1 1.4 AA485226 uman proteinase activated 0.54 6.1 1.9 2.2 AA454652 rostaglandin E receptor 3 0.25 4.1 4.9 3.8 4.9 AA406362 (subtype EP3) DGF R beta polypeptide1.03 2.5 1.0 0.5 0.8 856211.

IP R 2 1.00 3.1 2.0 AI057229 growth factor receptor-bound rotein 2 0.51 2.2 2.0 2.4 0.3 AA449831 ouse Mammary Turmor Virus Receptor homolog1.00 6.9 16 W93891 adenosine A2a R 0.41 3.1 1.8 4.0 2.5 N57553 adenosine A3 R 0.83 2.0 2.3 1.0 1.2 AA863086 cell R delta locus 0.77 2.7 1.3 1.8 AA670107 rostaglandin E receptor 1 0.65 7.2 6.0 1.5 AA972293 (subtype EP1) growth factor receptor-bound rotein 14 0.34 3.0 6.3 2.9 824266 pstein-Barr virus induced 0.61 1.6 2.4 8.3 AA037376 olynucleotide 2 complement component eceptor 2 0.22 26 4.5 2.6 18.1 AA521362 endothelin receptor0.07 12 14 14 16 AA450009 type A

v-SNARE R 0.56 11 12 1.8 AA704511 yrosine kinase, non-receptor, 0.12 7.8 8.5 10 8.7 AI936324 eceptor tyrosine kinase-like 0.40 7.3 5.0 1.6 2.5 N94921 orphan receptor Accession UnstimulatedRatio Number Peptide:
Unstimulated olynucleotide/ProteinIntensity ID ID ID ID

rotein tyrosine phosphatase, on-receptor type 1.02 1.0 13.2 0.5 0.8 AA682684 rotein tyrosine phosphatase, on-receptor type 0.28 3.5 4.0 0.9 5.3 AA434420 rotein tyrosine phosphatase, non-receptor type 0.42 2.9 2.4 2.2 3.0 AA995560 rotein tyrosine phosphatase, non-receptor type 1.00 2.3 2.2 0.8 0.5 AA446259 rotein tyrosine phosphatase, on-receptor type 0.58 1.7 2.4 3.6 1.7 AA679180 rotein tyrosine phosphatase, on-receptor type 0.52 3.2 0.9 1.9 6.5 AI668897 rotein tyrosine phosphatase, receptor type, 0.25 4.0 2.4 16.8 12.8 H82419 A

rotein tyrosine phosphatase, receptor type, 0.60 3.6 3.2 1.6 1.0 AA045326 J

rotein tyrosine phosphatase, eceptor type, T 0.73 1.2 2.8 3.0 1.4 852794 rotein tyrosine phosphatase, eceptor type, U 0.20 6.1 1.2 5.6 5.0 AA644448 rotein tyrosine phosphatase, receptor type, C-associated rotein 1.00 5.1 2.4 AA481547 hospholipase A2 0.45 2.8 2.2 1.9 2.2 AA086038 receptor 1 AP kinase-activated protein inase 3 0.52 2.1 2.7 1.1 1.9 W68281 MAP kinase kinase 0.10 18 9.6 32 H07920 AP kinase kinase 1.00 3.0 5.2 0.8 0.2 W69649 AP kinase 7 0.09 11.5 12 33 H39192 AP kinase 12 0.49 2.1 1.7 2.2 2.0 AI936909 rG protein-coupled0.40 3.7 3.0 2.4 ~ 2.5 AI719098 receptor 4 ~

Accession UnstimulatedRatio Number Peptide:
Unstimulated olynucleotide/ProteinIntensity ID ID ID ID

G protein-coupled 0.05 19 19 27 AA460530 receptor 49 G protein-coupled 0.08 19 15 12 N58443 receptor 55 G protein-coupled 0.26 5.2 3.1 7.1 3.9 H84878 receptor 75 G protein-coupled 0.20 '6.8 5.4 4.9 5.0 N62306 receptor 85 regulator of G-protein signalling 20 0.02 48 137 82 AI264190 regulator of G-protein signalling 6 0.27 3.7 8.9 10.6 839932 BCL2-interacting killer 1.00 1.9 5.2 AA291323 (apoptosis-inducing) apoptosis inhibitor0.56 2.8 1.6 2.4 1.8 AI972925 caspase 6, apoptosis-related cysteine protease 0.79 0.7 2.6 1.3 2.8 W45688 apoptosis-related protein 0.46 2.2 1.4 2.3 2.9 AA521316 caspase 8, apoptosis-related cysteine protease 0.95 2.2 1.0 0.6 2.0 AA448468 [00107] Table 22: Polynucleotides down-regulated by peptide treatment of A549 epithelial cellsa. The cationic peptides at concentrations of 50 pg/ml were shown to decrease the expression of several polynucleotides. Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The "Ratio Peptide: Unstimulated" columns refers to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.

Accession UnstimulatedRatio Number Peptide:
Unstimulated olynucleotide/ProteinIntensity ID ID ID ID

LR 1 3.22 0.35 0.31 0.14 0.19 AI339155 LR 2 2.09 0.52 0.31 0.48 0.24 T57791 LR 5 8.01 0.12 0.39 N41021 LR 7 5.03 0.13 0.11 0.20 0.40 N30597 NF receptor-associated factor 2 0.82 1.22 0.45 2.50 2.64 T55353 NF receptor-associated actor 3 3.15 0.15 0.72 0.32 AA504259 NF receptor superfamily, ember 12 4.17 0.59 0.24 0.02 W71984 NF R superfamily, member 2.62 0.38 0.55 0.34 AA987627 RAF and TNF receptor-associated protein1.33 0.75 0.22 0.67 0.80 AA488650 L-1 receptor, type1.39 0.34 0.72 1.19 0.34 AA464526 I

L-2 receptor, alpha2.46 0.41 0.33 0.58 AA903183 L-2 receptor, gamma (severe 3.34 0.30 0.24 0.48 N54821 combined immunodeficiency) L-12 receptor, 4.58 0.67 0.22 AA977194 beta 2 L-18 receptor 1 1.78 0.50 0.42 0.92 0.56 AA482489 GF beta receptor 2.42 0.91 0.24 0.41 0.41 H62473 III

leukotriene b4 receptor 1.00 1.38 4.13 0.88 AI982606 (chemokine receptor-like 1) small inducible cytokine 2.26 0.32 0.44 1.26 AA495985 subfamily A (Cys-Cys), ember 18 small inducible cytokine 2.22 0.19 0.38 0.45 0.90 AI285199 subfamily A (Cys-Cys), ember 20 small inducible cytokine 2.64 0.38 0.31 1.53 AA916836 subfamily A (Cys-Cys), Accession UnstimulatedRatio Number Peptide:
Unstimulated olynucleotide/ProteinIntensity ID ID ID ID

ember 23 small inducible cytokine subfamily B (Cys-X-Cys), ember 6 (granulocyte chemotactic protein3.57 0.11 0.06 0.28 0.38 AI889554 2) small inducible cytokine subfamily B (Cys-X-Cys), ember 10 2.02 0.50 1.07 0.29 0.40 AA878880 small inducible cytokine A3 (homologous to mouse Mip-la) 2.84 1.79 0.32 0.35 AA677522 cytokine-inducible2.70 0.41 0.37 0.37 0.34 AA489234 kinase complement component Clq eceptor 1.94 0.46 0.58 0.51 0.13 AI761788 cadherin 11, type 2, OB-cadherin (osteoblast)2.00 0.23 0.57 0.30 0.50 AA136983 cadherin 3, type 1, P-cadherin (placental) 2.11 0.43 0.53 0.10 0.47 AA425217 cadherin, EGF LAG
seven-ass G-type receptor 2, lamingo (Drosophila) omolog 1.67 0.42 0.41 1.21 0.60 H39187 cadherin 13, H-cadherin (heart) 1.78 0.37 0.40 0.56 0.68 841787 selectin L (lymphocyte adhesion molecule 4.43 0.03 0.23 0.61 H00662 1) vascular cell adhesion olecule 1 1.40 0.20 0.72 0.77 0.40 H16591 intercellular adhesion olecule 3 1.00 0.12 0.31 2.04 1:57 AA479188 integrin, alpha 2.42 0.41 0.26 0.56 AA450324 Accession UnstimulatedRatio Number Peptide:
Unstimulated olynucleotide/ProteinIntensity ID ID ID ID

integrin, alpha 2.53 0.57 0.39 0.22 0.31 AA055979 integrin, alpha 1.16 0.86 0.05 0.01 2.55 AA865557 integrin, alpha 1.00 0.33 0.18 1.33 2.25 AA460959 integrin, beta 1.00 0.32 1.52 1.90 0.06 AA434397 integrin, beta 3.27 0.10 1.14 0.31 0.24 W56754 disintegrin and etalloproteinase 2.50 0.40 0.29 0.57 0.17 AI205675 domain 18 disintegrin-like and etalloprotease with hrombospondin type 1 motif, 3 2.11 0.32 0.63 0.47 0.35 AA398492 disintegrin-like and etalloprotease with hrombospondin type 1 motif, 5 1.62 0.39 0.42 1.02 0.62 AI375048 -cell receptor interacting olecule 1.00 0.41 1.24 1.41 0.45 AI453185 diphtheria toxin receptor (heparin-binding epidermal growth factor-like growth actor) 1.62 0.49 0.85 0.62 0.15 845640 vasoactive intestinal peptide receptor 1 2.31 0.43 0.31 0.23 0.54 H73241 c fragment of IgG, low affinity IIIb, receptor for (CD16) 3.85 -0.200.26 0.76 0.02 H20822 c fragment of IgG, low affinity IIb, receptor for (CD32) 1.63 0.27 0.06 1.21 0.62 868106 c fragment of IgE, high affinity I, receptor1.78 0.43 0.00 0.56 0.84 AI676097 for; alpha Accession UnstimulatedRatio Number Peptide:
Unstimulated olynucleotide/ProteinIntensity ID ID ID ID

olypeptide leukocyte immunoglobulin-like receptor, 2.25 0.44 0.05 0.38 0.99 N63398 subfamily A

leukocyte immunoglobulin-like receptor, subfamily B

(with TM and ITIM
domains), member 3 14.21 1.10 0.07 AI815229 leukocyte immunoglobulin-like receptor, subfamily B

(with TM and ITIM
domains), ember 4 2.31 0.75 0.43 0.19 0.40 AA076350 leukocyte immunoglobulin-ike receptor, subfamily1.67 0.35 0.60 0.18 0.90 H54023 B

eroxisome proliferative activated receptor,1.18 0.38 0.85 0.87 0.26 AI739498 alpha rotein tyrosine phosphacase, eceptor type, f polypeptide (PTPRF), interacting protein (liprin), al 2.19 0.43 1.06 0.46 N49751 rotein tyrosine phosphatase, eceptor type, C 1.55 0.44 0.64 0.30 0.81 H74265 rotein tyrosine phosphatase, eceptor type, E 2.08 0.23 0.37 0..560.48 AA464542 rotein tyrosine phosphatase, receptor type, 2.27 0.02 0.44 0.64 AA464590 N polypeptide rotein tyrosine phosphatase, receptor type, 2:34 0.11 0.43 0.24 0.89 AI924306 H

rotein tyrosine phosphatase, receptor-type, 1.59 0.63 0.34 0.72 0.35 AA476461 Z polypeptide rotein tyrosine phosphatase, on-receptor type 1.07 0.94 0.43 0.25 1.13 . H03504 Accession UnstimulatedRatio Number Peptide:
Unstimulated olynucleotide/ProteinIntensity ID ID ID ID

AP kinase 8 interacting rotein 2 1.70 0.07 0.85 0.47 0.59 AA418293 AP kinase kinase 1.27 0.37 0.79 1.59 -5.28AA402447 kinase 4 MAP kinase kinase 1.00 0.34 0.66 2.10 1.49 W61116 kinase 14 AP kinase 8 interacting rotein 2 2.90 0.16 0.35 0.24 0.55 AI202738 AP kinase kinase 1.48 0.20 0.91 0.58 0.68 AA053674 kinase 12 AP kinase kinase kinase 2.21 0.45 0.20 1.03 0.41 AA043537 inase 3 MAP kinase kinase 2.62 0.37 0.38 0.70 AW084649 kinase 6 AP kinase kinase kinase 1.04 0.96 0.09 0.29 2.79 AA417711 inase 4 MAP kinase kinase 1.53 0.65 0.41 0.99 0.44 880779 kinase 11 MAP kinase kinase 1.32 1.23 0.27 0.50 0.76 H01340 kinase 10 AP kinase 9 2.54 0.57 0.39 0.16 0.38 AA157286 AP kinase kinase 1.23 0.61 0.42 0.81 1.07 AI538525 kinase 1 MAP kinase kinase 0.66 1.52 1.82 9.50 0.59 W56266 kinase 8 MAP kinase-activated protein 0.52 2.13 2.68 1.13 1.93 W68281 inase 3 AP kinase kinase 0.84 1.20 3.35 0.02 1.31 AA425826 MAP kinase kinase 1.00 0.97 1.62 7.46 AA460969 kinase 7 AP kinase 7 0.09 11.4511.8033.43H39192 MAP kinase kinase 0.10 17.839.61 32.30H07920 egulator of G-protein signalling 5 3.7397 0.27 0.06 0.68 0.18 AA668470 egulator of G-protein signalling 13 1.8564 0.54 0.45 0.07 1.09 H70047 G protein-coupled 1.04 1.84 0.16 0.09 0.96 891916 receptor G protein-coupled 1.78 0.32 0.56 0.39 0.77 AI953187 receptor 17 G protein-coupled receptor 2.62 0.34 0.91 0.38 AA488413 inase 7 Accession UnstimulatedRatio Number Peptide:
Unstimulated olynucleotide/ProteinIntensity ID ID ID ID

orphan seven-transmembrane receptor, chemokine7.16 1.06 0.10 0.11 0.14 AI131555 related apoptosis antagonizing transcription factor1.00 0.28 2.50 1.28 0.19 AI439571 caspase 1, apoptosis-related cysteine protease (interleukin l, beta, convertase)2.83 0.44 0.33 0.35 T95052 rogrammed cell death 8 (apoptosis-inducing1.00 1.07 0.35 1.94 0.08 AA496348 factor) [00108] Table 23: Pro-inflammatory polynucleotides up-regulated by peptide treatment of A549 cells. The cationic peptides at concentrations of 50 ~tg/ml were shown to increase the expression of certain pro-inflammatory polynucleotides (data is a subset of Table 21). Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The "Ratio Peptide:
Unstimulated"
columns refers to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
Accession olynucleotide/ProteinUnstim.Ratio Number and Peptide:
Unstimulated unction IntensityID ID ID ID 1 IL-11 Ra; Receptor for pro-inflammatory cytokine, inflammation 0.55 2.39 0.98 4.8.5 1.82 AA454657 L-17 R; Receptor for IL-17, an inducer of cytokine roduction in epithelial0.54 2.05 1.97 1.52 1.86 AW029299 cells Accession olynucleotide/ProteinUnstim.Ratio Number and Peptide:
Unstimulated unction IntensityID ID ID ID 1 small inducible cytokine subfamily A, member 21; a chemokine 1.00 3.88 2.41 AI922341 CD31; Leukocyte and cell to cell adhesion (PECAM)0.59 2.71 3.13 1.01 1.68 822412 , CCR6; Receptor for chemokine MIP-3a 0.14 4.51 7.75 6.92 7.79 N57964 integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 eceptor; Adhesion to leukocytes 1.00 0.89 2.44 3.62 0.88 AA463257 integrin, alpha 3 (antigen CD49C, alpha 3 subunit of LA-3 receptor);
Leukocyte dhesion 0.94 0.79 2.51 1.88 1.07 AA424695 integrin, alpha 0.01 179.33120.1228.48 81.37 AA425451 E; Adhesion integrin, beta 4; Leukocyte adhesion 0.65 0.79 2.17 4.94 1.55 AA485668 C-type lectin-like receptor-2;Leukocyte adhesion0.45 2.09 7.92 2.24 5.29 H70491 [00109] Table 24: Pro-inflammatory polynucleotides down-regulated by peptide treatment of A549 cells. The cationic peptides at concentrations of 50 pg/ml were shown to decrease the expression of certain pro-inflammatory polynucleotides (data is a subset of Table 22). Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The "Ratio Peptide:
Unstimulated" columns refers to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.

Unstim Ratio Accession Peptide:Unstimulated olynucleotide/Protein;IntensityID ID ID ID Number Function 2 3 19 1 oll-like receptor (TLR) 1;

esponse to gram positive3.22 0.35 0.31 0.14 0.19 AI339155 bacteria LR 2; Response to gram positive acteria and yeast 2.09 0.52 0.31 0.48 0.24 T57791 LR 5; May augment other TLR

esponses, Responsive 8.01 0.12 0.39 N41021 to flagellin LR 7: Putative host defence echanism 5.03 0.13 0.11 0.20 0.40 N30597 NF receptor-associated factor 2;

nflammation 0.82 1.22 0.45 2.50 2.64 T55353 NF receptor-associated factor 3;

nflammation 3.15 0.15 0.72 0.32 AA504259 NF receptor superfamily, member 12; Inflammation 4.17 0.59 0.24 0.02 W71984 NF R superfamily, member 17;

Inflammation 2.62 0.38 0.55 0.34 AA987627 RAF and TNF receptor-associated protein; 1.33 0.75 0.22 0.67 0.80 AA488650 TNF signalling small inducible cytokine subfamily A, member 18; Chemokine2.26 0.32 0.44 1.26 AA495985 small inducible cytokine subfamily A, member 20; Chemokine2.22 0.19 0.38 0.45 0.90 AI285199 small inducible cytokine subfamily member 23; Chemokine 2.64 0.38 0.31 1.53 AA916836 small inducible cytokine subfamily member 6 (granulocyte chemotactic protein);3.57 0.11 0.06 0.28 0.38 AI889554 Chemokine small inducible cytokine subfamily member 10; Chemokine 2.02 0.50 1.07 0.29 0.40 AA878880 small inducible cytokine (homologous to mouse 2.84 1.79 0.32 0.35 AA677522 Mip-la);

Unstim Ratio Accession Peptide:Unstimulated olynucleotide/Protein;IntensityID ID ID ID Number Function 2 3 19 1 Chemokine IL-12 receptor, beta 2; Interleukin 4.58 0.67 0.22 AA977194 and Interferon receptor IL-18 receptor 1; 1.78 0.50 0.42 0.92 0.56 AA482489 Induces 1FN-y selectin L (lymphocyte adhesion 4.43 0.03 0.23 0.61 H00662 molecule 1); Leukocyte adhesion vascular cell adhesion molecule 1; 1.40 0.20 0.72 0.77 0.40 H16591 eukocyte adhesion intercellular adhesion molecule 3; 1.00 0.12 0.31 2.04 1.57 AA479188 eukocyte adhesion integrin, alpha 1;
Leukocyte 2.42 0.41 0.26 0.56 AA450324 adhesion [00110] Table 25: Anti-inflammatory polynucleotides up-regulated by peptide treatment of A549 cells. The cationic peptides at concentrations of 50 pg/ml were shown to increase the expression of certain anti-inflammatory polynucleotides (data is a subset of Table 21). Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The "Ratio Peptide:
Unstimulated"
columns refers to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
Polynucleotide/Protein;Unstim Ratio Accession Peptide:
Unstimulated unction IntensityID 2 ID ID ID Number L-1 R antagonist homolog 1; 0.00 3085.961855.90869.57 AI167887 nhibitor of septic shock L-10 R beta; Receptor for 0.53 2.51 1.56 1.88 3.10AA486393 cytokine synthesis inhibitor NF R, member 1B; 0.28 17.09 3.01 14.933.60AA150416 Apoptosis ~ ~ ~ ~ ~

olynucleotide/Protein;Unstim Ratio Accession Peptide:
Unstimulated unction IntensityID 2 ID ID ID Number NF R, member 5; Apoptosis (CD40L) 33.71 2.98 0.02 H98636 NF R, member 1 lb; 1.00 5.29 4.50 0.78 AA194983 Apoptosis K cytokine, down-regulator of LA II; Inhibits antigen resentation 0.50 3.11 2.01 1.74 3.29839227 GFB inducible early growth response 2; anti-inflammatory cytokine 0.90 2.38 2.08 0.87 1.11AI473938 CD2; Adhesion molecule, binds LFAp3 1.00 2.62 0.87 1.15 0.88AA927710 [00111] Table 26: Anti-inflammatory polynucleotides down-regulated by peptide treatment of A549 cells. The cationic peptides at concentrations of 50 pg/ml were shown to increase the expression of certain anti-inflammatory polynucleotides (data is a subset of Table 21). Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The "Ratio Peptide:
Unstimulated" columns refers to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
olynucleotide/Protein; Unstim Ratio Accession Peptide:
Unstimulated unction IntensityID 2 ID ID ID Number AP kinase 9 2.54 0.57 0.39 0.16 0.38AA157286 [00112] Table 27: Polynucleotides up-regulated by SEQ ID NO: 6, in primary human macrophages. The peptide SEQ ID NO: 6 at a concentration of 50 ~ug/ml was shown to increase the expression of many polynucleotides. Peptide was incubated with the human macrophages for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in unstimulated cells is shown in the second column. The "Ratio peptide treated : Control" columns refer to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
Gene (Accession Number) Control: Ratio peptide Unstimulated treated:control cells proteoglycan 2 (Z26248) 0.69 9.3 Unknown (AK001843) 26.3 8.2 phosphorylase kinase alpha 0.65 7.1 1 (X73874) actinin, alpha 3 (M86407) 0.93 6.9 DKFZP586B2420 protein (AL050143)0.84 5.9 Unknown (AL109678) 0.55 5.6 transcription factor 21 (AF047419)0.55 . 5.4 Unknown (A433612) 0.62 5.0 chromosome condensation 1-like (AF060219) 0.69 4.8 Unknown (AL137715) 0.66 4.4 apoptosis inhibitor 4 (U75285)0.55 4.2 TERF1 (TRFl)-interacting nuclear 0.73 4.2 factor 2 (NM 012461) LINE retrotransposable element 1 6.21 4.0 (M22333) 1-acylglycerol-3-phosphate O- 0.89 4.0 acyltransferase 1 (U56417) Vacuolar proton-ATPase, subunit D; V- 1.74 4.0 ATPase, subunit D (X71490) KIAA0592 protein (AB011164) 0.70 4.0 potassium voltage-gated channel KQT- 0.59 3.9 like subfamily member 4 (AF105202) CDC14 homolog A (AF000367) 0.87 3.8 histone fold proteinCHRAC17 (AF070640) 0.63 3.8 Cryptochrome 1 (D83702) 0.69 3.8 pancreatic zymogen granule membrane 0.71 3.7 associated protein (AB035541) Sp3 transcription factor 0.67 3.6 (X68560) hypothetical protein FLJ20495 (AK000502) 0.67 3.5 E2F transcription factor 5, p130-binding 0.56 3.5 (U31556) hypothetical protein FLJ20070 (AK000077) 1.35 3.4 glycoprotein IX (X52997) 0.68 3.4 KIAA1013 protein (AB023230) 0.80 3.4 eukaryotic translation initiation factor 2.02 3.4 4A, isoform 2 (AL137681) FYN-binding protein (AF198052)1.04 3.3 guanine nucleotide binding protein, 0.80 3.3 gamma transducing activity polypeptide 1 (U41492) glypican 1 (X54232) 0.74 3.2 mucosal vascular addressin cell adhesion 0.65 3.2 molecule 1 (U43628) lymphocyte antigen (M38056) 0.70 3.2 H1 histone family, member 0.81 3.0 4 (M60748) translational inhibitor protein p14.5 0.78 3.0 (X95384) hypothetical protein FLJ20689 (AB032978) 1.03 2.9 KIAA1278 protein (AB03104) 0.80 2.9 unknown (AL031864) 0.95 2.9 chymotrypsin-like protease 3.39 2.9 (X71877) calumenin (NM 001219) 2.08 2.9 protein kinase, cAMP-dependent, regulatory, type I, beta 7.16 2.9 (M65066) POU domain, class 4, transcription factor 2 (U06233) 0.79 2.8 POU domain, class 2, associating factor 1.09 2.8 1 (Z49194) KIAA0532 protein (AB011104) 0.84 2.8 unknown (AF068289) 1.01 2.8 unknown (AL117643) 0.86 2.7 cathepsin E (M84424) 15.33 2.7 matrix metalloproteinase 23A 0.73 2.7 (AF056200) interferon receptor 2 (L42243)0.70 2.5 MAP kinase kinase 1 (L11284)0.61 2.4 protein kinase C, alpha (X52479)0.76 2.4 c-Cbl-interacting protein 0.95 2.4 (AF230904) c-fos induced growth factor 0.67 2.3 (Y12864) cyclin-dependent kinase inhibitor 1B 0.89 2.2 (S76988) zinc finger protein 266 (X78924)1.67 2.2 MAP kinase 14 (L35263) 1.21 2.2 KIAA0922 protein (AB023139) 0.96 2.1 bone morphogenetic protein 1 1.10 2.1 (NM 006129) NADH dehydrogenase 1 alpha 1.47 2.1 subcomplex, 10 (AF087661) bone morphogenetic protein receptor, 0.50 2.1 type IB (U89326) interferon regulatory factor 2 (NM 1.46 2.0 002199) protease, serine, 21 (AB031331)0.89 2.0 ~

[00113] Table 28: Polynucleotides down-regulated by SEQ ID NO: 6, in primary human macrophages. The peptide SEQ ID NO: 6 at a concentration of 50 ~g/ml was shown to increase the expression of many polynucleotides. Peptide was incubated with the human macrophages for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in unstimulated cells is shown in the second column. The "Ratio of Peptide: Control" columns refer to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
Gene (Accession Number) Control: Ratio peptide Unstimulated treated:control cells Unknown (AL049263) 17 0.06 integrin-linked kiriase (U40282)2.0 0.13 KIAA0842 protein (AB020649) 1.1 0.13 Unknown (AB037838) 13 0.14 Granulin (AF055008) 8.6 0.14 glutathione peroxidase 3 (NM 1.2 0.15 002084) KIAA0152 gene product (D63486)0.9 0.17 TGFB1-induced anti-apoptotic factor 1 0.9 0.19 (D86970) disintegrin protease (Y13323) 1.5 0.21 proteasome subunit beta type 0.7 0.22 7 (D38048) cofactor required for Spl transcriptional activation subunit 3 (AB033042)0.9 0.23 TNF receptor superfamily, member 14 0.8 0.26 (U81232) proteasome 26S subunit non-ATPase 8 1.1 0.28 (D38047) proteasome subunit beta type, 0.7 0.29 4 (D26600) TNF receptor superfamily member 1B 1.7 0.29 (M32315) cytochrome c oxidase subunit Vic 3.3 0.30 (X13238) ~

S100.calcium-binding protein A4 3.8 0.31 (M80563) proteasome subunit alpha type,2.9 0.31 6 (X59417) proteasome 26S subunit non-ATPase, 1.0 0.32 (AL031177) MAP kinase kinase kinase 2 0.8 0.32 (NM 006609) ribosomal protein L11 (X79234)5.5 0.32 matrix metalloproteinase 14 1.0 0.32 (Z48481) proteasome subunit beta type, 1.5 0.33 5 (D29011) MAP kinase-activated protein kinase 2 1.5 0.34 (U 12779) caspase 3 (U13737) 0.5 0.35 jun D proto-oncogene (X56681) 3.0 0.35 proteasome 26S subunit, ATPase, 3 1.3 0.35 (M34079) IL-1 receptor-like 1 (AB012701)0.7 0.35 interferon alpha-inducible protein 13 0.35 (AB019565) SDF receptor 1 (NM 012428) 1.6 0.35 Cathepsin D (M63138) 46 0.36 MAP kinase kinase 3 (D87116) 7.4 0.37 TGF, beta-induced, (M77349) 1.8 0.37 TNF receptor superfamily, member lOb 1.1 0.37 (AF016266) proteasome subunit beta type, 1.3 0.38 6 (M34079) nuclear receptor binding protein (NM 013392) 5.2 0.38 Unknown (AL050370) 1.3 0.38 protease inhibitor 1 alpha-1-antitrypsin (X01683) 0.7 0.40 proteasome subunit alpha type, 7 5.6 0.40 (AF054185) LPS-induced TNF-alpha factor (NM 004862) 5.3 0.41 transferrin receptor (X01060) 14 0.42 proteasome 26S subunit non-ATPase 13 1.8 0.44 (AB009398) MAP kinase kinase 5 (U25265) 1.3 0.44 Cathepsin L (X12451) 15 0.44 IL-1 receptor-associated kinase1.7 0.45 1 (L76191) MAP kinase kinase kinase kinase 2 1.1 0.46 (U07349) peroxisome proliferative activated receptor 2.2 0.46 delta (AL022721) TNF superfamily, member 15 16 0.46 (AF039390) defender against cell death 3.9 0.46 1 (D15057) TNF superfamily member 10 (U37518)287 0.46 cathepsin H (X16832) 14 0.47 protease inhibitor 12 (281326)0.6 0.48 proteasome subunit alpha type,2.6 0.49 4 (D00763) proteasome 26S subunit ATPase, 1 1.8 0.49 (L02426) proteasome 26S subunit ATPase, 2 2.1 0.49 (D 11094) caspase 7 (U67319) 2.4 0.49 matrix metalloproteinase 7 2.5 0.49 (211887) [00114] Table 29: Polynucleotides up-regulated by SEQ ID NO: 1, in HBE
cells. The peptide SEQ ID NO: 1 at a concentration of 50 pg/ml was shown to increase the expression of many polynucleotides. Peptide was incubated with the human HBE epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in unstimulated cells is shown in the second column. The "Ratio Peptide: Control" columns refer to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
Accession Gene Control: Ratio peptide Number Unstimulatedtreated:control cells ALl 10161 Unknown 0.22 5218.3 AF131842 Unknown 0.01 573.1 AJ000730 solute carrier family 0.01 282.0 225884 chloride channel 1 0.01 256.2 protein tyrosine phosphatase M93426 receptor-type,zeta 0.01 248.7 olfactory receptor, family X65857 1, 0.01 228.7 subfamily D,member 2 M55654 TATA box binding protein 0.21 81.9 AK001411 hypothetical protein 0.19 56.1 D29643 dolichyl- 1.56 55.4 Accession Gene Control: Ratio peptide Number Unstimulatedtreated:control cells diphosphooligosaccharide-protein glycosyltransferase AF006822 myelin transcription factor0.07 55.3 AL 117601 Unknown 0.05 53.8 AL117629 DKFZP434C245 protein 0.38 45.8 tumor necrosis factor,alpha-M59465 induced protein 3 0.50 45.1 AB013456 aquaporin 8 0.06 41.3 SEC24 related gene family, AJ131244 member A 0.56 25.1 AL 110179 Unknown 0.87 24.8 AB037844 Unknwon 1.47 20.6 247727 polymerise II polypeptide0.11 20.5 K

AL035694 Unknown 0.81 20.4 X68994 H.sapiens CREB gene 0.13 19.3 AJ238379 hypothetical protein 1.39 18.5 NM 003519 H2B histone family member0.13 18.3 glutamate receptor, ionotropic U16126 kainate 2 0.13 17.9 adenosine monophosphate U29926 deaminase 0.16 16.3 .

AK001160 hypothetical protein 0.39 14.4 U18018 ets variant gene 4 0.21 12.9 D80006 KIAA0184 protein 0.21 12.6 AK000768 hypothetical protein 0.30 12.3 X99894 insulin promoter factor 0.26 12.0 1, AL031177 Unknown 1.09 11.2 AF052091 unknown 0.28 10.9 Accession Gene Control: Ratio peptide Number Unstimulatedtreated:control cells 5,10-methenyltetrahydrofolate L38928 synthetase 0.22 10.6 AL117421 unknown . 0.89 10.1 AL133606 hypothetical protein 0.89 9.8 NM 016227 membrane protein CH1 0.28 9.6 NM 006594 adaptor-related protein 0.39 9.3 complex 4 U54996 ZW10 homolog,protein 0.59 9.3 AJ007557 potassium channel, 0.28 9.0 AF043938 muscle RAS oncogene 1.24 8.8 AK001607 unknown 2.74 8.7 AL031320 peroxisomal biogenesis 0.31 8.4 factor 3 D38024 unknown ~ 0.31 8.3 AF059575 LIM homeobox TF 2.08 8.2 hepatitis A virus cellular AF043724 receptor 0.39 8.1 AK002062 hypothetical protein 2.03 8.0 L13436 natriuretic peptide receptor0.53 7.8 U33749 thyroid transcription 0.36 7.6 factor 1 AF011792 cell cycle progression 0.31 7.6 2 protein AK000193 hypothetical protein 1.18 6.8 AF039022 exportin, tRNA 0.35 6.8 M17017 interleukin 8 0.50 6.7 AF044958 NADH dehydrogenase 0.97 6.5 U35246 vacuolar protein sorting 0.48 6.5 AK001326 tetraspan 3 1.59 6.5 Krueppel-related zinc M55422 finger 0.34 6.4 protein U44772 palmitoyl-protein thioesterase1.17 6.3 Accession Gene Control: Ratio peptide Number Unstimulatedtreated:control cells AL117485 hypothetical protein 0.67 5.9 AB037776 unknown 0.75 5.7 AF131827 unknown 0.69 5.6 AL137560 unknown 0.48 5.2 X05908 annexin A 1 0.81 5.1 X68264 melanoma adhesion molecule0.64 5.0 AL161995 neurturin 0.86 4.9 AF037372 cytochrome c oxidase 0.48 4.8 NM 016187 bridging integrator 2 0.65 4.8 AL137758 unknown 0.57 4.8 TRAF family member-associated U59863 NFKB activator 0.46 4.7 230643 chloride channel Ka 0.70 4.7 acetyl-Coenzyme A
D16294 acyltransferase 2 1.07 4.6 AJ132592 zinc finger protein 281 0.55 4.6 X82324 POU domain TF 1.73 4.5 NM 016047 CGI-110 protein 1.95 4.5 AK001371 hypothetical protein 0.49 ~ 4.5 M60746 H3 histone family member 3.05 4.5 D

AB033071 hypothetical-protein 4.47 4.4 AB002305 KIAA0307 gene product 1.37 4.4 UDP-N-acetyl-alpha-D-X92689 galactosamine:polypeptide0.99 4.4 N-acetylgalactosaminyltransferase AL049543 glutathione peroxidase 1.62 4.3 U43148 patched homolog 0.96 4.3 M67439 dopamine receptor D5 2.61 4.2 Accession Gene Control: Ratio peptide Number Unstimulatedtreated:control cells U09850 zinc finger protein 143 0.56 4.2 L20316 glucagon receptor 0.75 4.2 a disintegrin-like and AB037767 metalloprotease 0.69 4.2 NM 017433 myosin IIIA 99.20 4.2 a disintegrin and metalloprotease D26579 domain 8 0.59 4.1 L10333 reticulon 1 1.81 4.1 AK000761 unknown 1.87 4.1 -U91540 NK homeobox family 3, 0.80 4.1 A

217227 interleukin 10 receptor, 0.75 4.0 beta [00115] Table 30: Polynucleotides down-regulated by Peptide (50 pg/ml), SEQ
ID NO: 1, in HBE cells. The peptide SEQ ID NO: 1 at a concentration of 50 pg/ml was shown to decrease the expression of many polynucleotides. Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in unstimulated cells is shown in the third column. The "Ratio Peptide: Control" columns refer to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
Accession Gene Control: Ratio SEQ
ID

Number UnstimulatedNO:1- treated:

Cells control AC004908 Unknown 32.4 0.09 570622 G1 phase-specific gene 43.1 0.10 Accession Gene Control: Ratio SEQ
Number UnstimulatedID
Cells NO:1- treated:
control 297056 DEAD/H box polypeptide 12.8 0.11 AK002056 hypothetical protein 11.4 0.12 L33930 CD24 antigen 28.7 0.13 X77584 thioredoxin 11.7 0.13 NM 014106 PR01914 protein 25.0 0.14 M37583 H2A histone family member22.2 0.14 , polymerise (RNA) II
U89387 polypeptide D 10.2 0.14 ras-related C3 botulinum D25274 toxin 10.3 0.15 substrate 1 J04173 phosphoglycerate mutase 11.4 0.15 U19765 zinc finger protein 9 8.9 0.16 X67951 proliferation-associated 14.1 0.16 gene A

AL096719 profilin 2 20.0 0.16 AF165217 tropomodulin 4 14.6 0.16 NM 014341 mitochondria) carrier 11.1 0.16 homolog 1 AL022068 Unknown 73.6 0.17 X69150 ribosomal protein S 18 42.8 0.17 AL031577 Unknown 35.0 0.17 AL031281 Unknown 8.9 0.17 Human mRNA for ornithine AF090094 decarboxylase antizyme, 10.3 0.17 HLA-G histocompatibility AL022723 antigen, 20.6 0.18 class I, G

ATP synthase, H+ transporting U09813 mitochondria) FO complex 9.8 0.18 Homo sapiens TTF-I interacting AF000560 peptide 20 20.2 0.19 Accession Gene Control: Ratio SEQ
Number UnstimulatedID
Cells NO:1- treated:
control NM 016094 ~ HSPC042 protein 67.2 0.19 AF047183 NADH dehydrogenase 7.5 0.19 anti-oxidant protein 2 (non-D14662 selenium glutathione peroxidase,8.1 0.19 acidic calcium-independent phospholipas X16662 annexin A8 8.5 0.19 U14588 paxillin 11.3 0.19 AL117654 DKFZP586D0624 protein 12.6 0.20 AK001962 hypothetical protein 7.7 0.20 6-pyruvoyl-tetrahydropterin L41559 synthase/dimerization 9.1 0.20 cofactor of hepatocyte nuclear factor 1 alpha NM 016139 16.7Kd protein 21.0 0.21 NM 016080 CGI-150 protein 10.7 0.21 26S proteasome-associated U86782 padl 6.7 0.21 homolog tumor protein, translationally-AJ400717 controlled 1 9.8 0.21 X07495 homeo box C4 31.0 0.21 AL034410 Unknown 7.3 0.22 X14787 thrombospondin 1 26.2 0.22 purine-rich element binding AF081192 protein B 6.8 '0.22 protein disulfide isomerase-related D49489 protein 11.0 0.22 NM 014051 PTDO11 protein 9.3 0.22 AK001536 Unknown 98.0 0.22 Accession Gene Control: Ratio SEQ
Number UnstimulatedID
Cells NO:1- treated:
control X62534 high-mobility group protein9.5 0.22 endothelial differentiation-related AJ005259 factor 1 6.7 0.22 NM 000120 epoxide hydrolase 1, microsomal10.0 0.22 M38591 5100 calcium-binding protein23.9 0.23 AF071596 immediate early response 11.5 0.23 methylene tetrahydrofolate X16396 dehydrogenase 8.3 0.23 AK000934 ATPase inhibitor precursor7.6 0.23 AL117612 Unknown 10.7 0.23 transcriptional intermediary AF119043 factor 7.3 0.23 1 gamma solute carrier family AF037066 22 member 1- 7.6 0.23 like antisense AF134406 cytochrome c oxidase subunit13.3 0.23 AE000661 Unknown 9.2 0.24 AL157424 synaptojanin 2 7.2 0.24 tyrosine 3-X56468 monooxygenase/tryptophan 7.2 0.24 monooxygenase activation protein, ubiquitin-conjugating U39318 enzyme 10.7 0.24 AL034348 Unknown 24.4 0.24 D26600 proteasome subunit beta 11.4 0.24 type 4 AB032987 Unknown 16.7 0.24 lysosomal-associated membrane J04182 protein 1 7.4 0.24 X78925 zinc finger protein 267 16.1 0.25 Accession Gene Control: Ratio SEQ
Number UnstimulatedID
Cells NO:1- treated:
control NM 000805 gastrin 38.1 0.25 anti-Mullerian hormone U29700 receptor, 12.0 0.25 type II

298200 Unknown 13.4 0.25 U07857 ~ signal recognition particle10.3 0.25 Homo Sapiens ribosomal L05096 protein 25.3 0.25 AK001443 hypothetical protein 7.5 0.25 K03515 glucose phosphate isomerase6.2 0.25 interferon induced transmembrane X57352 protein 3 7.5 0.26 J02883 colipase pancreatic 5.7 0.26 M24069 cold shock domain protein6.3 0.26 AJ269537 chondroitin-4-sulfotransferase60.5 0.26 AL137555 Unknown 8.5 0.26 U89505 RNA binding motif protein5.5 0.26 U82938 CD27-binding protein 7.5 0.26 X99584 SMT3 homolog 1 12.8 0.26 AK000847 Unknown 35.8 0.27 NM 014463 Lsm3 protein 7.8 0.27 AL133645 Unknown 50.8 0.27 X78924 zinc finger protein 266 13.6 0.27 NM 004304 anaplastic lymphoma kinase15.0 0.27 X57958 ribosomal protein L7 27.9 0.27 U63542 Unknown 12.3 0.27 AK000086 hypothetical protein 8.3 0.27 X57138 H2A histone family member32.0 0.27 N

AB023206 KIAA0989 protein 6.5 0.27 Accession Gene Control: Ratio SEQ
Number UnstimulatedID
Cells NO:l- treated:
control gonadotropin inducible AB021641 transcriptn 5.5 0.28 repressor-1 AF050639 NADH dehydrogenase 5.5 0.28 complement component 5 M62505 receptor 7.5 0.28 X64364 basigin 5.8 0.28 AJ224082 Unknown 22.5 0.28 AF042165 cytochrome c oxidase 20.4 0.28 AK001472 anillin 10.9 0.28 X86428 protein phosphatase 2A 12.7 0.28 subunit AF227132 candidate taste receptor 5.1 0.28 298751 Unknown 5.3 0.28 D21260 clathrin heavy polypeptide8.3 0.28 AF041474 actin-like 6 15.1 0.28 NM 005258 GTP cyclohydrolase I protein7.6 0.28 L20859 solute carrier family 9.6 0.29 280783 H2B histone family member9.0 0.29 AB011105 laminin alpha 5 7.1 0.29 protective protein for AL008726 beta- 5.2 0.29 galactosidase D29012 proteasome subunit 12.6 0.29 X63629 cadherin 3 P-cadherin 6.8 0.29 X02419 plasminogen activator 12.9 0.29 urokinase X13238 cytochrome c oxidase 8.0 0.29 X59798 cyclin D1 12.7 0.30 D78151 proteasome 26S subunit 7.6 0.31 AF054185 proteasome subunit 18.8 0.31 J03890 surfactant pulmonary-associated5.5 0.32 Accession Gene Control: Ratio SEQ
ID

Number UnstimulatedNO:1- treated:

Cells control protein C

M34079 proteasome 26S subunit, 5.2 0.33 [00116] Table 31: Up-regulation of Polynucleotide expression in A549 cells induced by Formula A Peptides. The peptides at a concentration of SO pg/ml were shown to increase the expression of many polynucleotides. Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in control, unstimulated cells are shown in the second and third columns for labeling of cDNA with the dyes Cy3 and Cy5 respectively. The "ID#: Control" columns refer to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.

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z Iso EXAMPLE S
INDUCTION OF CHEMOK>CNES IN CELL LINES, WHOLE HUMAN
BLOOD, AND IN MICE BY PEPTIDES
[00123] The murine macrophage cell line RAW 264.7, THP-1 cells (human monocytes), a human epithelial cell line (A549), human bronchial epithelial cells (16HBEo14), and whole human blood were used. HBE cells were grown in MEM
with Earle's. THP-1 cells were grown and maintained in RPMI 1640 medium. The RAW and A549 cell lines were maintained in DMEM supplemented with 10% fetal calf serum. The cells were seeded in 24 well plates at a density of 106 cells per well in DMEM (see above) and A549 cells were seeded in 24 well plates at a density of cells per well in DMEM (see above) and both were incubated at 37°C in 5 % COZ
overnight. DMEM was aspirated from cells grown overnight and replaced with fresh medium. After incubation of the cells with peptide, the release of chemokines into the culture supernatant was determined by ELISA (R&D Systems, Minneapolis, MN).
[00124] Animal studies were approved by the UBC Animal Care Committee (UBC
ACC # A01-0008). BALB/c mice were purchased from Charles River Laboratories and housed in standard animal facilities. Age, sex and weight matched adult mice were anaesthetized with an intraperitoneal injection of Avertin (4.4 mM 2-2-2-tribromoethanol, 2.5% 2-methyl-2-butanol, in distilled water), using 200 pl per 10 g body weight. The instillation was performed using a non-surgical, intratracheal instillation method adapted from Ho and Furst 1973. Briefly, the anaesthetized mouse was placed with its upper teeth hooked over a wire at the top of a support frame with its jaw held open and a spring pushing the thorax forward to position the pharynx, larynx and trachea in a vertical straight line. The airway was illuminated externally and an intubation catheter was inserted into the clearly illuminated tracheal lumen.
Twenty-~1 of peptide suspension or sterile water was placed in a well at the proximal end of the catheter and gently instilled into the trachea with 200 ~1 of air.
The animals were maintained in an upright position for 2 minutes after instillation to allow the fluid to drain into the respiratory tree. After 4 hours the mice were euthanaised by intraperitoneal injection of 300 mg/kg of pentobarbital. The trachea was exposed; an intravenous catheter was passed into the proximal trachea and tied in place with suture thread. Lavage was performed by introducing 0.75 ml sterile PBS into the lungs via the tracheal cannula and then after a few seconds, withdrawing the fluid.
This was repeated 3 times with the same sample of PBS. The lavage fluid was placed in a tube on ice and the total recovery volume per mouse was approximately 0.5 ml.
The bronchoalveolar lavage (BAL) fluid was centrifuged at 1200 rpm for 10 min, the clear supernatant removed and tested for TNF-a and MCP-1 by ELISA.
[00125] The up-regulation of chemokines by cationic peptides was confirmed in several different systems. The murine MCP-1, a homologue of the human MCP-1, is a member of the (3(C-C) chemokine family. MCP-1 has been demonstrated to recruit monocytes, NK cells and some T lymphocytes. When RAW 264.7 macrophage cells and whole human blood from 3 donors were stimulated with increasing concentrations of peptide, SEQ ID NO: 1, they produced significant levels of in their supernatant, as judged by ELISA (Table 36). RAW 264.7 cells stimulated with peptide concentrations ranging from 20-50 pg/ml for 24 hr produced significant levels of MCP-1 (200-400 pg/ml above background). When the cells (24h) and whole blood (4h) were stimulated with 100 pg/ml of LL-37, high levels of MCP-1 were produced.
[00126] The effect of cationic peptides on chemokine induction was also examined in a completely different cell system, A549 human epithelial cells.
Interestingly, although these cells produce MCP-1 in response to LPS, and this response could be antagonized by peptide; there was no production of MCP-1 by A549 cells in direct response to peptide, SEQ ID NO: 1. Peptide SEQ ID NO: 1 at high concentrations, did however induce production of IL-8, a neutrophil specific chemokine (Table 37).
Thus, SEQ ID NO: 1 can induce a different spectrum of responses from different cell types and at different concentrations. A number of peptides from each of the formula groups were tested for their ability to induce IL-8 in A549 cells (Table 38).
Many of these peptides at a low concentration, 10 pg/ml induced IL-8 above background levels. At high concentrations (100 ~g/ml) SEQ ID NO: 13 was also found to induce IL-8 in whole human blood (Table 39). Peptide SEQ ID NO: 2 also significantly induced IL-8 in HBE cells (Table 40) and undifferentiated THP-1 cells (Table 41).
[00127] BALB/c mice were given SEQ ID NO: 1 or endotoxin-free water by intratracheal instillation and the levels of MCP-1 and TNF-a examined in the bronchioalveolar lavage fluid after 3-4 hr. It was found that the mice treated with 50 pg/ml peptide, SEQ ID NO: 1 produced significantly increased levels of MCP-1 over mice given water or anesthetic alone (Table 42). This was not a pro-inflammatory response to peptide, SEQ ID NO: 1 since peptide did not significantly induce more TNF-a than mice given water or anesthetic alone. peptide, SEQ ID NO: 1 was also found not to significantly induce TNF-a production by RAW 264.7 cells and bone marrow-derived macrophages treated with peptide, SEQ ID NO: 1 (up to 100 pg/ml) (Table 43). Thus, peptide, SEQ ID NO: 1 selectively induces the production of chemokines without inducing the production of inflammatory mediators such as TNF-a. This illustrates the dual role of peptide, SEQ ID NO: 1 as a factor that can block bacterial product-induced inflammation while helping to recruit phagocytes that can clear infections.
[00128] Table 38: Induction of MCP-1 in RAW 264.7 cells and whole human blood. RAW 264.7 mouse macrophage cells or whole human blood were stimulated with increasing concentrations of LL-37 for 4 hr. The human blood samples were centrifuged and the serum was removed and tested for MCP-1 by ELISA along with the supernatants from the RAW 264.7 cells. The RAW cell data presented is the mean of three or more experiments ~ standard error and the human blood data represents the mean ~ standard error from three separate donors.
Peptide, SEQ ID Monocyte chemoattractant NO: 1 protein (MCP)-1 (pg/ml) (pg/ml)~

RAW cells Whole blood 0 135.3+16.3 112.7+43.3 165.7+18.2 239.3+113.3 Peptide, SEQ ID Monocyte chemoattractant NO: 1 protein (MCP)-1 (pg/ml) (pg/ml)'~

RAW cells Whole blood 50 367 + 11.5 371 + 105 100 571 + 17.4 596 + 248.1 [00129] Table 39: Induction of IL-8 in A549 cells and whole human blood.
A549 cells or whole human blood were stimulated with increasing concentrations of peptide for 24 and 4 hr respectively. The human blood samples were centrifuged and the serum was removed and tested for IL-8 by ELISA along with the supernatants from the A549 cells. The A549 cell data presented is the mean of three or more experiments ~ standard error and the human blood data represents the mean ~
standard error from three separate donors.
Peptide, SEQ ID IL-8 (pg/ml) NO: 1 (p,g/ml) A549 cells Whole blood 0 I72 + 29.1 660.7 + 126.6 1 206.7 + 46.1 ' 283.3 + 28.4 945.3 + 279.9 392 + 31.7 50 542.3 + 66.2 1160.3 + 192.4 100 1175.3 + 188.3 [00130] Table 40: Induction of IL-8 in A549 cells by Cationic peptides. A549 human epithelial cells were stimulated with 10 pg of peptide for 24 hr. The supernatant was removed and tested for IL-8 by ELISA.
Peptide (10 ug/ml)IL-8 (ng/ml) No peptide 0.164 Peptide (10 ug/ml)IL-8 (ng/ml) LPS, no peptide 0.26 SEQ ID NO: 1 0.278 SEQ ID NO: 6 0.181 SEQ ID NO: 7 0.161 SEQ ID NO: 9 0.21 SEQ ID NO: 10 0.297 SEQ ID NO: 13 0.293 SEQ ID NO: 14 0.148 SEQ ID NO: 16 0.236 SEQ ID NO: 17 0.15 SEQ ID NO: 19 0.161 SEQ ID NO: 20 0.151 SEQ ID NO: 21 0.275 SEQ ID NO: 22 0.314 SEQ ID NO: 23 0.284 SEQ ID NO: 24 0.139 SEQ ID NO: 26 0.201 SEQ ID NO: 27 0.346 SEQ ID NO: 28 0.192 SEQ ID NO: 29 0.188 SEQ ID NO: 30 0.284 SEQ ID NO: 31 0.168 SEQ ID NO: 33 0.328 SEQ ID NO: 34 0.315 SEQ ID NO: 35 0.301 SEQ ID NO: 36 0.166 SEQ ID NO: 37 0.269 SEQ ID NO: 38 0.171 SEQ ID NO: 40 0.478 SEQ ID NO: 41 0.371 Peptide (10 ug/ml)IL-8 (ng/ml) SEQ ID NO: 42 0.422 SEQ ID NO: 43 0.552 SEQ ID NO: 44 0.265 SEQ ID NO: 45 0.266 SEQ ID NO: 47 0.383 SEQ ID NO: 48 0.262 SEQ ID NO: 49 0.301 SEQ ID NO: 50 0.141 SEQ ID NO: 51 0.255 SEQ ID NO: 52 0.207 SEQ ID NO: 53 0.377 SEQ ID NO: 54 ~ 0.133 [00131] Table 41: Induction by Peptide of IL-8 in human blood. Whole human blood was stimulated with increasing concentrations of peptide for 4 hr . The human blood samples were centrifuged and the serum was removed and tested for IL-8 by ELISA. The data shown is the average 2 donors.
SEQ ID NO: 3 (~g/ml)IL-8 (pg/ml) [00132] Table 42: Induction of IL-8 in HBE cells. Increasing concentrations of the peptide were incubated with HBE cells for 8 h, the supernantant removed and tested for IL-8. The data is presented as the mean of three or more experiments ~
standard error.

SEQ ID NO: 2 IL-8 (pg/ml) (pg/ml) 0 552 + 90 0.1 670 + 155 1 712 + 205 941 + 15 50 1490 + 715 [00133] Table 43: Induction of IL-8 in undifferentiated THP-1 cells. The human monocyte THP-1 cells were incubated with indicated concentrations of peptide for 8 hr. The supernatant was removed and tested for IL-8 by ELISA.
SEQ ID NO: 3 IL-8 (pg/ml) (~g/ml) 0 10.6 10 17.2 50 123.7 [00134] Table 44: Induction of MCP-1 by Peptide, SEQ ID NO: 1 in mouse airway. BALB/c mice were anaesthetised with avertin and given intratracheal instillation of peptide or water or no instillation (no treatment). The mice were monitored for 4 hours, anaesthetised and the BAL fluid was isolated and analyzed for MCP-1 and TNF-a concentrations by ELISA. The data shown is the mean of 4 or 5 mice for each condition + standard error.
Condition MCP-1 (pg/ml) TNF-a (pg/ml) Water 16.5 + 5 664 + 107 peptide 111 + 30 734 + 210 Avertin 6.5 + 0.5 393 + 129 [00135] Table 45: Lack of Significant TNF-a induction by the Cationic Peptides. RAW 264.7 macrophage cells were incubated with indicated peptides (40 pg/ml) for 6 hours. The supernatant was collected and tested for levels of TNF-a by ELISA. The data is presented as the mean of three or more experiments +
standard error.
Peptide Treatment TNF-a (pg/ml) Media background 56 8 LPS treatment, No 15207 186 peptide SEQ ID NO: 1 274 15 SEQ ID NO: 5 223 45 SEQ ID NO: 6 297 32 SEQ ID NO: 7 270 42 SEQ ID NO: 8 166 23 SEQ ID NO: 9 171 33 SEQ ID NO: 10 288 30 SEQ ID NO: 12 ~ 299 65 SEQ ID NO: 13 216 42 SEQ ID NO: 14 226 41 SEQ ID NO: 15 346 41 SEQ ID NO: 16 341 68 SEQ ID NO: 17 249 49 SEQ ID NO: 19 397 86 SEQ ID NO: 20 285 56 SEQ ID NO: 21 263 8 SEQ ID NO: 22 195 42 SEQ ID NO: 23 254 58 SEQ ID NO: 24 231 32 SEQ ID NO: 26 281 34 SEQ ID NO: 27 203 42 Peptide Treatment TNF-a (pg/ml) SEQ ID NO: 28 192 26 SEQ ID NO: 29 242 40 SEQ ID NO: 31 307 71 SEQ ID NO: 33 196 42 SEQ ID NO: 34 204 51 SEQ ID NO: 35 274 76 SEQ ID NO: 37 323 41 SEQ ID NO: 38 199 38 SEQ ID NO: 43 947 197 SEQ ID NO: 44 441 145 SEQ ID NO: 45 398 90 SEQ ID NO: 48 253 33 SEQ ID NO: 49 324 38 SEQ ID NO: 50 311 144 SEQ ID NO: 53 263 40 SEQ ID NO: 54 346 86 CATIONIC PEPTIDES INCREASE SURFACE EXPRESSION
OF CHEMOKINE RECEPTORS
[00136] To analyze cell surface expression of IL-8RB, CXCR-4, CCR2, and LFA-1, RAW macrophage cells were stained with 10 pg/ml of the appropriate primary antibody (Santa Cruz Biotechnology) followed by FITC-conjugated goat anti-rabbit IgG [IL-8RB and CXCR-4 (Jackson ImmunoResearch Laboratories, West Grove, PA)] or FITC-conjugated donkey anti-goat IgG (Santa Cruz). The cells were analyzed using a FACscan, counting 10,000 events and gating on forward and side scatter to exclude cell debris.
[00137] The polynucleotide array data suggested that some peptides up-regulate the expression of the chemokine receptors IL-8RB, CXCR-4 and CCR2 by 10, 4 and 1.4 fold above unstimulated cells respectively. To confirm the polynucleotide array data, the surface expression was examined by flow cytometry of these receptors on RAW
cells stimulated with peptide for 4 hr. When 50 pg/ml of peptide was incubated with RAW cells for 4 hr, IL-8RB was upregu(ated an average of 2.4-fold above unstimulated cells, CXCR-4 was up-regulated an average of 1.6-fold above unstimulated cells and CCR2 was up-regulated 1.8-fold above unstimulated cells (Table 46). As a control CEMA was demonstrated to cause similar up-regulation.
Bac2A was the only peptide to show significant up-regulation of LFA-1 (3.8 fold higher than control cells).
[00138] Table 46: Increased surface expression of CXCR-4, IL-8RB and CCR2 in response to peptides. RAW macrophage cells were stimulated with peptide for hr. The cells were washed and stained with the appropriate primary and FITC-labeled secondary antibodies. The data shown represents the average (fold change of RAW
cells stimulated with peptide from media) + standard error.
ConcentrationFold Increase in Protein Expression Peptide (pg/ml) IL-8RB CXCR-4 CCR2 SEQ ID 10 1.0 1.0 1.0 NO: 1 SEQ ID 50 1.3 + 0.05 1.3 + 0.03 1.3 + 0.03 NO:1 SEQID 100 2.4+0.6 1.6+0.23 1.8+0.15 NO:1 SEQ ID 100 2.0 + 0.6 Not Done 4.5 NO: 3 CEMA 50 1.6+0.1 1.5+0.2 1.5+0.15 100 3.6 + 0.8 Not Done 4.7 + 1.1 PHOSPHORYLATION OF MAP KINASES BY CATIONIC PEPTIDES
[00139] The cells were seeded at 2.5x105 - 5 x 105 cells/ml and left overnight. They were washed once in media, serum starved in the morning (serum free media -4hrs).
The media was removed and replaced with PBS, then sat at 37°C for 15 minutes and then brought to room temp for 15 minutes. Peptide was added (concentrations O.lug/ml - 50ug/ml) or H20 and incubated 10 min. The PBS was very quickly removed and replaced with ice-cold radioimmunoprecipitation (RIPA) buffer with inhibitors (NaF, B-glycerophosphate, MOL, Vanadate, PMSF, Leupeptin Aprotinin).
The plates were shaken on ice for 10-15 min or until the cells were lysed and the lysates collected. The procedure for THP-1 cells was slightly different; more cells (2x106) were used. They were serum starved overnight, and to stop the reaction lml of ice-cold PBS was added then they sat on ice 5-10 min, were spun down then resuspended iri RIPA. Protein concentrations were determined using a protein assay (Pierce, Rockford, IL.). Cell lysates (20 p.g of protein) were separated by SDS-PAGE
and transferred to nitrocellulose filters. The filters were blocked for 1 h with 10 mM
Tris-HCI, pH 7.5, 150 mM NaCI (TBS)/5% skim milk powder and then incubated overnight in the cold with primary antibody in TBS/0.05% Tween 20. After washing for 30 min with TBS/0.05% Tween 20, the filters were incubated for 1 h at room temperature with 1 pg/ml secondary antibody in TBS. The filters were washed for 30 min with TBS/0.05% Tween 20 and then incubated 1 h at room temperature with horseradish peroxidase-conjugated sheep anti-mouse IgG (1:10,000 in TBS/0.05%
Tween 20). After washing the filters for 30 min with TBS/0.1% Tween 20, immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection. For experiments with peripheral blood mononuclear cells: The peripheral blood (50-100m1) was collected from all subjects. Mononuclear cells were isolated from the peripheral blood by density gradient centrifugation on Ficoll-Hypaque.
Interphase cells (mononuclear cells) were recovered, washed and then resuspended in recommended primary medium for cell culture (RPMI-1640) with 10% fetal calf serum (FCS) and 1% L-glutamine. Cells were added to 6 well culture plates at 4x106 cells/well and were allowed to adhere at 37° C in 5% COZ atmosphere for 1 hour. The supernatant medium and non-adherent cells were washed off and the appropriate media with peptide was added. The freshly harvested cells were consistently >99%
viable as assessed by their ability to exclude trypan blue. After stimulation with peptide, lysates were collected by lysing the cells in RIPA buffer in the presence of various phosphatase- and kinase-inhibitors. Protein content was analyzed and approximately 30 pg of each sample was loaded in a 12% SDS-PAGE gel. The gels were blotted onto nitrocellulose, blocked for 1 hour with 5% skim milk powder in Tris buffered saline (TBS) with 1% Triton X 100. Phosphorylation was detected with phosphorylation-specific antibodies.
[00140] The results of peptide-induced phosphorylation are summarized in Table 46. SEQ ID NO: 2 was found to cause dose dependent phosphorylation of p38 and ERK1/2 in the mouse macrophage RAW cell line and the HBE cells. SEQ ID NO: 3 caused phosphorylation of MAP kinases in THP-1 human monocyte cell line and phosphorylation of ERK1/2 in the mouse RAW cell line.
[00141 ] Table 47: Phosphorylation of MAP kinases in response to peptides.
Cell Line Peptide MAP kinase phosphorylated p38 ERK1/2 RAW 264.7 SEQ ID NO: 3 - +

SEQ ID NO: 2 + +

HBE SEQ ID NO: 3 +

SEQ ID NO: 2 + +

THP-1 SEQ ID NO: 3 + +

SEQ ID NO: 2 (00142] Table 48: Peptide Phosphorylation of MAP kinases in human blood monocytes. SEQ ID NO: I at 50 pg/ml) was used to promote phosphorylation.
p38 phosphorylation ERK1/2 phosphorylation 15 minutes60 minutes 15 minutes 60 minutes + - + +

CATIONIC PEPTIDES PROTECT AGAINST BACTERIAL INFECTION
BY ENHANCING THE IMMUNE RESPONSE
[00143] BALB/c mice were given lx 105 Salmonella and cationic peptide (200 pg) by intraperitoneal injection. The mice were monitored for 24 hours at which point they were euthanized, the spleen removed, homogenized and resuspended in PBS
and plated on Luria Broth agar plates with Kanamycin (50 pg/ml). The plates were incubated overnight at 37°C and counted for viable bacteria (Table 49 and 50). CD-1 mice were, given 1 x 108 S. aureus in 5 % porcine mucin and cationic peptide (200 fig) by intraperitoneal injection (Table 51). The mice were monitored for 3 days at which point they were euthanized, blood removed and plated for viable counts. CD-1 male mice were given 5.8 x 106 CFU EHEC bacteria and cationic peptide (200 fig) by intraperitoneal (IP) injection and monitored for 3 days (Table 52). In each of these animal models a subset of the peptides demonstrated protection against infections.
The most protective peptides in the Salmonella model demonstrated an ability to induce a common subset of genes in epithelial cells (Table 53) when comparing the protection assay results in Tables 50 and 51 to the gene expression results in Tables 31-37. This clearly indicates that there is a pattern of gene expression that is consistent with the ability of a peptide to demonstrate protection. Many of the cationic peptides were shown not to be directly antimicrobial as tested by the Minimum Inhibitory Concentration (MIC) assay (Table 54). This demonstrates that the ability of peptides to protect against infection relies on the ability of the peptide to stimulate host innate immunity rather than on direct antimicrobial activity.

[00144] Table 49: Effect of Cationic Peptides on Salmonella Infection in BALB/c mice. The BALB/c mice were injected IP with Salmonella and Peptide, and 24 h later the animals were euthanized, the spleen removed, homogenized, diluted in PBS and plate counts were done to determine bacteria viability.
Peptide Viable Bacteria in the Statistical Significance Treatment Spleen (p value) (CFU/ml) Control 2.70 0.84 X 10' SEQ ID NO: 1.50 0.26 X 10' 0.12 SEQ ID NO: 2.57 0.72 X 10" 0.03 SEQ ID NO: 3.80 0.97 X 10'' 0.04 SEQ ID NO: 4.79 1.27 X 10'' 0.04 SEQ ID NO: 1.01 0.26 X 10' 0.06 [00145] Table S0: Effect of Cationic Peptides on Salmonella Infection in BALB/c mice. The BALB/c mice were injected intraperitoneally with Salmonella and Peptide, and 24 h later the animals were euthanized, the spleen removed, homogenized, diluted in PBS and plate counts were done to determine bacteria viability.
Peptide Treatment Viable Bacteria in the Spleen (CFU/ml) Control 1.88 0.16 X 10 SEQ ID NO: 48 _ 1.98 0.18 X 10 SEQ ID NO: 26 7.1 1.37 X 10"

SEQ ID NO: 30 5.79 0.43 X 10' SEQ ID NO: 37 1.57 0.44 X 10"

SEQ ID NO: 5 2.75 0.59 X 10'' SEQ ID NO: 7 5.4 0.28 X 10' SEQ ID NO: 9 1.23 0.87 X 10 SEQ ID NO: 14 2.11 0.23 X 10' SEQ ID NO: 20 2.78 0.22 X 10"

SEQ ID NO: 23 6.16 0.32 X 10 [00146] Table 51. Effect of Cationic Peptides in a Murine S. aureus infection model. CD-1 mice were given 1 x 10g bacteria in 5 % porcine mucin via intraperitoneal (IP) injection. Cationic peptide (200 pg) was given via a separate IP
injection. The mice were monitored for 3 days at which point they were euthanized, blood removed and plated for viable counts. The following peptides were not effective in controlling S. aureus infection: SEQ ID NO: 48, SEQ ID NO: 26 Treatment CFU/ml (blood) # Mice Survived (3 days)/
Total mice in group No Peptide 7.61 1.7 x 10' 6 / 8 SEQ ID NO: 1 0 4 / 4 SEQ ID NO: 27 2.25 0.1 X 10' 3 / 4 SEQ ID NO: 30 1.29 0.04 X 10' 4 / 4 SEQ ID NO: 37 9.65 0.41 X 10' 4 / 4 SEQIDNO:S 3.281.7x10' 4/4 SEQ ID NO: 6 1.98 0.05 X 10 3 / 4 SEQ ID NO: 7 3.8 0.24 x 10' 4 / 4 SEQ ID NO: 9 2.97 0.25 X 10' 4 / 4 SEQ ID NO: 13 4.83 0.92 x 10' 3 / 4 SEQIDN0:17 9.60.41X10' 4/4 SEQ ID NO: 20 3.41 1.6 x 10' 4 / 4 SEQ ID NO: 23 4.39 2.0 x 10' 4 / 4 [00147] Table 52 Effect of Peptide in a Murine EHEC infection model. CD-1 male mice ( 5 weeks old) were given 5.8 x 106 CFU EHEC bacteria via intraperitoneal (IP) injection. Cationic peptide (200 pg) was given via a separate IP
injection. The mice were monitored for 3 days.
Treatment PeptideSurvival (%) control none 25 SEQ ID NO: 200pg 100 [00148) Table 53. Up-regulation of patterns of gene expression in A549 epithelial cells induced by peptides that are active in vivo. The peptides SEQ
ID
NO: 30, SEQ ID NO: 7 and SEQ ID NO: 13 at concentrations of 50 pg/ml were each shown to increase the expression of a pattern of genes after 4 h treatment.
Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labelled cDNA probes and hybridised to Human Operon arrays (PRHU04). The intensity of polynucleotides in control, unstimulated cells are shown in the second columns for labelling of cDNA (average of Cy3 and Cy5). The Fold Up regulation column refers to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells. The SEQ ID NO:

peptide was included as a negative control that was not active in the murine infection models.
Fold Up regulation of Gene Target (Accession UnstimulatedExpression relative to Untreated number) Cell IntensityCells SEQ SEQ SEQ SEQ ID
ID ID ID

N0:30 N0:7 N0:13 N0:37 Zinc finger protein (AF061261 ) 13 2.6 9.4 9.4 1.0 Cell cycle gene 1.62 8.5 3.2 3.2 0.7 (S70622) IL-10 Receptor (U00672)0.2 2.6 9 4.3 0.5 Transferase (AF038664)0.09 12.3 9.7 9.7 0.1 Homeobox protein (AC004774) 0.38 3.2 2.5 2.5 1.7 Forkhead protein (AF042832) 0.17 14.1 3.5 3.5 0.9 Unknown (AL096803) 0.12 4.8 4.3 4.3 0.6 KIAA0284 Protein (AB006622) 0.47 3.4 2.1 2.1 1.3 Hypothetical Protein (AL022393) 0.12 4.4 4.0 4.0 0.4 Receptor (AF112461) 0.16 2.4 10.0 10.0 1.9 Hypothetical Protein (AK002104) 0.51 4.7 2.6 2.6 1.0 Protein (AL050261) 0.26 3.3 2.8 2.8 1.0 Polypeptide (AF105424)0.26 2.5 5.3 5.3 1.0 SPR1 protein (AB031480)0.73 3.0 2.7 2.7 1.3 Dehydrogenase (D17793)4.38 2.3 2.2 2.2 0.9 Transferase (M63509)0.55 2.7 2.1 2.1 1.0 Peroxisome factor (AB013818) 0.37 3.4 2.9 2.9 1.4 [00149] Table 54: Most cationic peptides studied here and especially the cationic peptides effective in infection models are not significantly antimicrobial. A
dilution series of peptide was incubated with the indicated bacteria overnight in a 96-well plate. The lowest concentration of peptide that killed the bacteria was used as the MIC. The symbol > indicates the MIC is too large to measure. An MIC of 8 pg/ml or less was considered clinically meaningful activity. Abbreviations: E.coli, Escherichia coli; S.aureus, Staphylococcus aureus; P.aerug, Pseudomonas aeruginosa;
S.Typhim, Salmonella enteritidis ssp. typhimurium; C. rhod, Citobacter rhodensis; EHEC, Enterohaemorrhagic E. coli.
MIC
(wg/ml) Peptide E. coliS.aureusP. aerug.S.typhim.C. rhod.EHEC

Polymyxin 0.25 16 0.25 0.5 0.25 0.5 Gentamicin 0.25 0.25 0.25 0.25 0.25 0.5 SEQ ID NO: 32 > 96 64 8 4 SEQ ID NO: 128 > > > 64 64 SEQ ID NO: 128 > > 128 64 64 .

SEQ ID NO: > > > > > >

SEQ ID NO: > > > > > >

SEQ ID NO: > > > > > >

SEQ ID NO: > > > > > 64 SEQ ID NO: > > > > > >

SEQ ID NO: > > > > > >

SEQ ID NO: > > > > > >

SEQ ID NO: 128 > > > 128 64 SEQ ID NO: > > > > > >

SEQ ID NO: > > > > > >

SEQ ID NO: 8 16 16 64 4 4 SEQ ID NO: 4 16 32 16 64 SEQ ID NO: 8 8 8 8 16 8 SEQ ID NO: 64 64 96 64 32 32 SEQ ID NO: 8 12 24 8 4 4 SEQ ID NO: 4 8 8 16 4 4 SEQ ID NO: 16 16 4 16 16 4 SEQ ID NO: 0.5 32 64 2 2 0.5 SEQ ID NO: 8 64 64 16 2 4 SEQ ID NO: > > > 64 64 128 MIC
(~.g/ml) Peptide E. coliS.aureusP. aerug.S.typhim.C. rhod.EHEC

SEQ ID NO: 2 > > 16 32 4 SEQ ID NO: 16 > 128 16 16 4 SEQ ID NO: > > 128 > > 64 SEQ ID NO: 16 32 > 16 64 8 SEQ ID NO: 8 > > 32 64 8 SEQ ID NO: 4 128 64 8 8 4 SEQ ID NO: 32 > > 32 32 16 SEQ ID NO: > > > > > >

SEQ ID NO: 0.5 32 64 4 8 4 SEQ ID NO: 4 32 8 4 4 2 SEQ ID NO: 4 64 8 8 2 2 SEQ ID NO: 1.5 64 4 2 2 1 SEQ ID NO: 8 128 16 16 8 4 SEQ ID NO: 8 > 128 128 64 64 SEQ ID NO: 8 > 128 128 16 16 SEQ ID NO: 4 > 16 16 4 4 SEQ ID NO: 16 > 128 16 1 2 SEQ ID NO: 4 > 16 8 4 4 SEQ ID NO: 8 > 16 16 16 8 SEQ ID NO: 4 > 8 32 4 8 SEQ ID NO: 8 > 32 8 2 2 SEQ ID NO: 4 > 8 8 16 8 SEQ ID NO: 64 > 16 64 16 32 USE OF POLYNUCLEOTIDES INDUCED BY BACTERIAL SIGNALLING
MOLECULES IN DIAGNOSTIC/SCREENING
[00150] S. typhimurium LPS and E. coli Ol 11:B4 LPS were purchased from Sigma Chemical Co. (St. Louis, MO). LTA (Sigma) from S. aureus, was resuspended in endotoxin free water (Sigma). The Limulus amoebocyte lysate assay (Sigma) was performed on LTA preparations to confirm that lots were not significantly contaminated by endotoxin (i.e. <1 ng/ml, a concentration that did not cause significant cytokine production in the RAW cell assay). The CpG
oligodeoxynucleotides were synthesized with an Applied Biosystems Inc., Model DNA/RNA Synthesizer, Mississauga, ON., then purified and resuspended in endotoxin-free water (Sigma). The following sequences were used CpG: 5'-TCATGACGTTCCTGACGTT-3' (SEQ ID NO: 57) and nonCpG: 5'-TTCAGGACTTTCCTCAGGTT-3' (SEQ ID NO: 58). The nonCpG oligo was tested for its ability to stimulate production of cytokines and was found to cause no significant production of TNF-a or IL-6 and therefore was considered as a negative control. RNA was isolated from RAW 264.7 cells that had been incubated for 4h with medium alone, 100 ng/ml S. typhimurium LPS, 1 ~g/ml S. aureus LTA, or 1 pM CpG
(concentrations that led to optimal induction of tumor necrosis factor (TNF-a) in RAW cells). The RNA was used to polynucleotiderate cDNA probes that were hybridized to Clontech Atlas polynucleotide array filters, as described above.
The hybridization of the cDNA probes to each immobilized DNA was visualized by autoradiography and quantified using a phosphorimager. Results from at least 2 to 3 independent experiments are summarized in Tables 55-59. It was found that LPS
treatment of RAW 264.7 cells resulted in increased expression of more than 60 polynucleotides including polynucleotides encoding inflammatory proteins such as IL-1(3, inducible nitric oxide synthase (iNOS), MIP-la, MIP-1(3, MIP-2a, CD40, and a variety of transcription factors. When the changes in polynucleotide expression induced by LPS, LTA, and CpG DNA were compared, it was found that all three of these bacterial products increased the expression of pro-inflammatory polynucleotides such as iNOS, MIP-la, MIP-2a, IL-1(3, IL-15, TNFRl and NF-KB to a similar extent (Table 57). Table 57 describes 19 polynucleotides that were up-regulated by the bacterial products to similar extents in that their stimulation ratios differed by less than 1.5 fold between the three bacterial products. There were also several polynucleotides that were down-regulated by LPS, LTA and CpG to a similar extent.
It was also found that there were a number of polynucleotides that were differentially regulated in response to the three bacterial products (Table 58), which includes many of these polynucleotides that differed in expression levels by more than 1.5 fold between one or more bacterial products). LTA treatment differentially influenced expression of the largest subset of polynucleotides compared to LPS or CpG, including hyperstimulation of expression of Jun-D, Jun-B, Elk-1 and cyclins G2 and A1. There were only a few polynucleotides whose expression was altered more by LPS or CpG treatment. Polynucleotides that had preferentially increased expression due to LPS treatment compared to LTA or CpG treatment included the cAMP
response element DNA-binding protein 1 (CRE-BPI), interferon inducible protein and CACCC Box-binding protein BKLF. Polynucleotides that had preferentially increased expression after CpG treatment compared to LPS or LTA treatment included leukemia inhibitory factor (LIF) and protease nexin 1 (PN-1). These results indicate that although LPS, LTA, and CpG DNA stimulate largely overlapping polynucleotide expression responses, they also exhibit differential abilities to regulate certain subsets of polynucleotides.
[00151] The other polynucleotide arrays used are the Human Operon arrays (identification number for the genome is PRHU04-S1), which consist of about 14,000 human oligos spotted in duplicate. Probes were prepared from 5 pg of total RNA
and labeled with Cy3 or Cy5 labeled dUTP. In these experiments, A549 epithelial cells were plated in 100 mm tissue culture dishes at 2.5 x 106 cells/dish, incubated overnight and then stimulated with 100 ng/ml E. coli O111:B4 LPS for 4 h.
Total RNA was isolated using RNAqueous (Ambion). DNA contamination was removed with DNA-free kit (Ambion). The probes prepared from total RNA were purified and hybridized to printed glass slides overnight at 42°-C and washed. After washing, the image was captured using a Perkin Elmer array scanner. The image processing software (Imapolynucleotide 5.0, Marina Del Rey, CA) determines the spot mean intensity, median intensities, and background intensities. An "in house"
program was used to remove background. The program calculates the bottom 10 % intensity for each subgrid and subtracts this for each grid. Analysis was performed with Polynucleotidespring software (Redwood City, CA). The intensities for each spot were normalized by taking the median spot intensity value from the population of spot values within a slide and comparing this value to the values of all slides in the experiment. The relative changes seen with cells treated with LPS compared to control cells can be found in the Tables below. A number of previously unreported changes that would be useful in diagnosing infection are described in Table 60.
[00152] To confirm and assess the functional significance of these changes, the levels of selected mRNAs and proteins were assessed and quantified by densitometry.
Northern blots using a CD14, vimentin, and tristetraprolin-specific probe confirmed similar expression after stimulation with all 3 bacterial products (Table 60).
Similarly measurement of the enzymatic activity of nitric oxide synthetase, iNOS, using Griess reagent to assess levels of the inflammatory mediator NO, demonstrated comparable levels of NO produced after 24 h, consistent with the similar up-regulation of iNOS
expression (Table 59). Western blot analysis confirmed the preferential stimulation of leukaemia inhibitory factor (LIF, a member of the IL-6 family of cytokines) by CpG
(Table 59). Other confirmatory experiments demonstrated that LPS up-regulated the expression of TNF-a and IL-6 as assessed by ELISA, and the up-regulated expression of MIP-2a, and IL-1(3 mRNA and down-regulation of DP-1 and cyclin D mRNA as assessed by Northern blot analysis. The analysis was expanded to a more clinically relevant ex vivo system, by examining the ability of the bacterial elements to stimulate pro-inflammatory cytokine production in whole human blood. It was found that E.
coli LPS, S. typhimurium LPS, and S. aureus LTA all stimulated similar amounts of serum TNF-a, and IL-1 (3. CpG also stimulated production of these cytokines, albeit to much lower levels, confirming in part the cell line data.
[00153] Table 55: Polynucleotides Up-regulated by E. coli 0111:B4 LPS in A549 Epithelial Cells. E. coli 0111:B4 LPS (100 ng/ml) increased the expression of many polynucleotides in A549 cells as studied by polynucleotide microarrays.
LPS
was incubated with the A549 cells for 4 h and the RNA was isolated. 5 pg total RNA
was used to make Cy3/Cy5 labelled cDNA probes and hybridised onto Human Operon arrays (PRHU04). The intensity of unstimulated cells is shown in the second column of Table 55. The "Ratio: LPS/control" column refers to the intensity of polynucleotide expression in LPS simulated cells divided by in the intensity of unstimulated cells.
Accession Gene Control: Ratio:
Number Media only LPS/control Intensity D87451 ring finger protein 715.8 183.7 AF061261 C3H-type zinc finger 565.9 36.7 protein aldo-keto reductase D17793 family 1, ~ 220.1 35.9 member C3 M14630 prothymosin, alpha 168.2 31.3 AL049975 Unknown 145.6 62.3 ADP-ribosylation factor L04510 domain protein 1, 139.9 213.6 64kD

U10991 G2 protein 101.7 170.3 eukaryotic translation U39067 initiation factor 61.0 15.9 3, subunit 2 X03342 ribosomal protein 52.6 10.5 Rho-associated, coiled-coil NM 004850 containing protein 48.1 11.8 kinase 2 AK000942 Unknown 46.9 8.4 serine/threonine protein AB040057 kinase MASK 42.1 44.3 AB020719 KIAA0912 protein 41.8 9.4 FEM-1-like death receptor AB007856 binding protein 41.2 15.7 procollagen-proline, J02783 2- 36.1 14.1 oxoglutarate 4-dioxygenase AL137376 Unknown 32.5 17.3 AL137730 ~ Unknown 29.4 11.9 Accession Gene Control: Ratio:
Number Media only LPS/control Intensity D25328 phosphofructokinase, 27.3 8.5 platelet malate dehydrogenase AF047470 2, 25.2 8.2 NAD

stress-induced-M86752 phosphoprotein 1 22.9 5.9 M90696 cathepsin S 19.6 6.8 AK001143 Unknown 19.1 6.4 AF038406 NADH dehydrogenase 17.7 71.5 hypothetical protein AK000315 FLJ20308 17.3 17.4 M54915 pim-1 oncogene 16.0 11.4 proteasome subunit, D29011 beta 15.3 41.1 type, 5 membrane protein of AK000237 cholinergic synaptic 15.1 9.4 vesicles AL034348 Unknown 15.1 15.8 AL161991 Unknown 14.2 8.1 AL049250 Unknown 12.7 5.6 AL050361 PTD017 protein 12.6 13.0 U74324 RAB interacting factor12.3 5.2 M22538 NADH dehydrogenase 12.3 7.6 D87076 KIAA0239 protein 11.6 6.5 translocase of inner NM 006327 mitochondrial membrane11.5 10.0 (yeast) homolog AK001083 Unknown 11.1 8.6 mucin 5, subtype B, AJ001403 tracheobronchial 10.8 53.4 Accession Gene Control: Ratio:

Number Media only LPS/control Intensity RAP1, GTPase activating M64788 protein 1 10.7 7.6 X06614 retinoic acid receptor,10.7 5.5 alpha calcium and integring binding U85611 protein 10.3 8.1 U23942 cytochrome P450, 51 10.1 , 10.2 AL031983 Unknown 9.7 302.8 protein-O-NM 007171 mannosyltransferase 9.5 6.5 hypothetical protein AK000403 FLJ20396 9.5 66.6 NM 002950 ribophorin I 9.3 35.7 cAMP response element-L05515 binding protein CRE-BPa8.9 6.2 phosphoinositide-3-kinase, X83368 catalytic, gamma polypeptide8.7 27.1 M30269 nidogen (enactin) 8.7 5.5 chromosome 11 open reading M91083 frame 13 8.2 6.6 D29833 salivary proline-rich7.7 5.8 protein immunoglobulin superfamily AB024536 containing leucine-rich7.6 8.0 repeat chromosome 11 open reading U39400 frame 4 7.4 7.3 AF028789 unc119 (C.elegans) 7.4 27.0 homolog signal sequence receptor, alpha (translocon-associated NM 003144 protein alpha) 7.3 5.9 Accession Gene Control: Ratio:

Number Media only LPS/control Intensity arachidonate 5-lipoxygenase-X52195 activating protein 7.3 13.1 human growth factor-regulated tyrosine kinase U43895 substrate 6.9 6.9 cyclin-dependent kinase L25876 inhibitor 3 6.7 10.3 L04490 NADH dehydrogenase 6.6 11.1 218948 5100 calcium-binding 6.3 11.0 protein myristoylated alanine-rich D10522 protein kinase C substrate6.1 5.8 sialic acid binding Ig-like NM 014442 lectin 8 6.1 7.6 U81375 solute carrier family6.0 6.4 malignancy-associated AF041410 protein 5.9 5.3 killer cell immunoglobulin-U24077 like receptor 5.8 14.4 AL137614 hypothetical protein 4.8 6.8 mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-NM 002406 acetylglucosaminyltransferase4.7 5.3 AB002348 KIAA0350 protein 4.7 7.6 AF165217 tropomodulin 4 (muscle)4.6 12.3 branched chain keto acid dehydrogenase E1, alpha 214093 polypeptide 4.6 5.4 U82671 caltractin 3.8 44.5 Accession Gene Control: Ratio:
Number Media only LPS/control Intensity AL050136 Unknown 3.6 5.0 NM 005135 solute carrier family3.6 5.0 hypothetical protein AK001961 FLJ 11099 3.6 5.9 AL034410 Unknown 3.2 21.3 S74728 antiquitin 1 3.1 9.2 ribosomal protein AL049714 L34 3.0 19.5 pseudogene 2 NM 014075 PR00593 protein 2.9 11.5 AF189279 phospholipase A2, 2.8 37.8 group IIE

J03925 integrin, alpha M 2.7 9.9 NM 012177 F-box protein FbxS 2.6 26.2 potassium voltage-gated NM 004519 channel, KQT-like 2.6 21.1 subfamily, member 3 M28825 CD1A antigen, a polypeptide2.6 16.8 actin, gamma 2, smooth X16940 muscle, enteric 2.4 11.8 major histocompatibility X03066 complex, class II, 2.2 36.5 DO beta hypothetical protein AK001237 FLJ10375 2.1 18.4 AB028971 KIAA1048 protein 2.0 9.4 AL137665 Unknown 2.0 7.3 [00154] Table 56: Polynucleotides Down-regulated by E. coli 0111:B4 LPS in A549 Epithelial Cells. E. coli 0111:B4 LPS (100 ng/ml) decreased the expression of many polynucleotides in A549 cells as studied by polynucleotide microarrays.
LPS

was incubated with the A549 cells for 4 h and the RNA was isolated. 5 pg total RNA
was used to make Cy3/Cy5 labeled cDNA probes and hybridized onto Human Operon arrays (PRHU04). The intensity of unstimulated cells is shown in the second column of the Table. The "Ratio: LPS/control" column refers to the intensity of polynucleotide expression in LPS simulated cells divided by in the intensity of unstimulated cells.
Accession Gene Control: Ratio:
Number Media onlyLPS/control Intensity NM 017433 myosin IIIA 167.8 0.03 X60484 H4 histone family member E 36.2 0.04 X60483 H4 histone family member D 36.9 0.05 AF151079 hypothetical protein 602.8 0.05 inhibitor of DNA binding 2, M96843 dominant 30.7 0.05 negative helix-loop-helix protein 579854 deiodinase, iodothyronine, 39.4 0.06 type III

AB018266 matrin 3 15.7 0.08 M33374 NADH dehydrogenase 107.8 0.09 Homo sapiens mRNA for NUP98-AF005220 HOXD13 fusion protein, partial105.2 0.09 cds 280783 H2B histone family, member 20.5 0.10 L

246261 H3 histone family, member 9.7 0.12 A

280780 H2B histone family, member 35.3 0.12 H

erythrocyte membrane protein U33931 band 7.2 18.9 0.13 (stomatin) M60750 H2B histone family, member 35.8 0.14 A

283738 H2B histone family, member 19.3 0.15 E

Y14690 collagen, type V, alpha 2 7.5 0.15 X-ray repair complementing M30938 defective 11.3 0.16 repair in Chinese hamster cells 5 L36055 eukaryotic translation initiation182.5 0.16 factor 4E

Accession Gene Control: Ratio:
Number Media onlyLPS/control Intensity binding protein 1 280779 H2B histone family, member 54.3 0.16 G

5(3)-deoxyribonucleotidase;
AF226869 RB- 7.1 0.18 associated KRAB repressor D50924 KIAA0134 gene product 91.0 0.18 AL133415 vimentin 78.1 0.19 AL050179 tropomyosin 1 (alpha) 41.6 0.19 AJ005579 RD element 5.4 0.19 M80899 AHNAK nucleoprotein 11.6 0.19 NM 004873 BCL2-associated athanogene 6.2 0.19 X57138 H2A histone family, member 58.3 0.20 N

AF081281 lysophospholipase I 7.2 0.22 U96759 von Hippel-Lindau binding 6.6 0.22 protein 1 Human ribosomal protein L12 U85977 pseudogene, partial cds 342.6 0.22 D13315 glyoxalase I 7.5 0.22 AC003007 Unknown 218.2 0.22 AB032980 RU2S 246.6 0.22 U40282 integrin-linked kinase 10.1 0.22 U81984 endothelial PAS domain protein4.7 0.23 chloride channel, nucleotide-sensitive, X91788 1 A 9.6 0.23 AF018081 collagen, type XVIII, alpha 6.9 0.24 nuclear factor I/X (CCAAT-binding L31881 transcription factor) 13.6 0.24 B-cell translocation gene X61123 l, anti- 5.3 0.24 proliferative L32976 mitogen-activated protein 6.3 0.24 kinase kinase Accession Gene Control: .Ratio:
Number Media onlyLPS/control Intensity kinase 11 immunoglobulin lambda-like M27749 polypeptide 3 5.5 0.24 X57128 H3 histone family, member 9.0 0.25 C

phosphoinositide-3-kinase, X80907 regulatory 5.8 0.25 subunit, polypeptide 2 H.sapiens (MAR11) MUCSAC mRNA
234282 for mucin (partial) 100.6 0.26 X00089 H2A histone family, member 4.7 0.26 M

AL035252 CD39-like 2 4.6 0.26 PERB11 family member in MHC
X95289 class I 27.5 0.26 region AJ001340 U3 snoRNP-associated 55-kDa 4.0 0.26 protein NM 014161 HSPC071 protein 10.6 0.27 U60873 Unknown 6.4 0.27 X91247 thioredoxin reductase 1 84.4 0.27 AK001284 hypothetical protein FLJ104224.2 0.27 U90840 synovial sarcoma, X breakpoint6.6 0.27 X53777 ribosomal protein L17 39.9 0.27 AL035067 Unknown 10.0 0.28 AL117665 DKFZP586M1824 protein 3.9 0.28 ATPase, Ca++ transporting, L14561 plasma 5.3 0.28 membrane 1 L19779 H2A histone family, member 30.6 0.28 O

AL049782 Unknown 285.3 0.28 X00734 tubulin, beta, 5 39.7 0.29 AK001761 retinoic acid induced 3 23.7 0.29 U72661 ninjurin 1 4.4 0.29 Accession Gene Control: Ratio:
Number Media onlyLPS/control Intensity S48220 deiodinase, iodothyronine, 1,296.1 0.29 type I , AF025304 EphB2 4.5 0.30 S82198 chymotrypsin C 4.1 0.30 280782 H2B histone family, member 31.9 0.30 K

X68194 synaptophysin-like protein 7.9 0.30 AB028869 Unknown 4.2 0.30 AK000761 Unknown 4.3 0.30 [00155] Table 57: Polynucleotides expressed to similar extents after stimulation by the bacterial products LPS, LTA, and CpG DNA. Bacterial products (100 ng/ml S. typhimurium LPS, l~g/ml S. aureus LTA or 1 pM CpG) were shown to potently induce the expression of several polynucleotides. Peptide was incubated with the RAW cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Atlas arrays. The intensity of control, unstimulated cells is shown in the second column. The "Ratio LPS/LTA/CpG:
Control" column refers to the intensity of polynucleotide expression in bacterial product-simulated cells divided by the intensity of unstimulated cells.
ccessionControlRatio Ratio Ratio Protein/polynucleotide number nstim. LPS: TA: CpG:
IntensityControlControlControl 15131 20 82 80 55 IL-1(3 M57422 20 77 64 90 tristetraprolin 53798 20 73 77 78 MIP-2a 35590 188 50 8 58 MIP-1[3 ccessionControlRatio Ratio Ratio Protein/polynucleotide number nstim. LPS: TA: CpG:
IntensityControlControlControl M87039 20 37 38 5 iNOS

X57413 20 34 0 28 TGF(3 15842 20 20 21 15 c-rel proto-oncopolynucleotide X12531 89 19 20 26 MIP-loc 57999 172 3.8 3.5 3.4 NF-KB

U36277 02 3.2 3.5 2.7 I-KB (alpha subunit) 76850 194 3 3.8 2.5 MAPKAP-2 06924 858 2.4 3 3.2 Stat 1 X60671 543 1.9 2.4 2.8 NF-2 34510 5970 1.6 2 1.4 CD14 X51438 2702 1.3 2.2 2.0 vimentin X68932 455 0.5 0.7 0.5 c-Fms 221848 352 0.5 0.6 0.6 DNA polymerase X70472 614 0.4 0.6 0.5 B-myb [00156] Table 58: Polynucleotides that were differentially regulated by the bacterial products LPS, LTA, and CpG DNA. Bacterial products (100 ng/ml S.
typhimurium LPS, 1 ~g/ml S. aureus LTA or 1 ~M CpG) were shown to potently induce the expression of several polynucleotides. Peptide was incubated with the RAW cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Atlas arrays. The intensity of control, unstimulated cells is shown in the second column. The "Ratio LPS/LTA/CpG: Control" column refers to the intensity of polynucleotide expression in bacterial product-simulated cells divided by the intensity of unstimulated cells.
ccessionUnstim. Ratio Ratio Ratio Protein/polynucleotide number Control PS: TA: CpG:
IntensityControlControlControl 72307 20 1.0 23 1.0 hepatocyte growth factor 38847 20 1.0 21 1.0 hepatoma transmembrane inase ligand 34169 393 0.3 3 0.5 thrombopoietin 04113 289 1 3 Nur77 250013 20 7 21 5 H-ras proto-oncopolynucleotide X84311 20 12 2 Cyclin A1 U95826 20 5 14 2 Cyclin G2 87257 123 2 1 Elk-1 05205 20 18 39 20 Jun-D

03236 20 11 19 14 Jun-B

83649 20 71 80 2 Fas 1 receptor 83312 20 69 91 57 CD40L receptor 13945 573 2 3 2 Pim-1 U60530 193 2 3 3 Mad related protein X06381 20 55 59 102 Leukemia inhibitory factor (LIF) X70296 20 6.9 13 22 Protease nexin 1 (PN-1) U36340 20 38 7 7 CACCC Box- binding protein KLF

U19119 272 10 interferon inducible protein 1 [00157] Table 59: Confirmation of Table 57 and 58 Array Data. a) Total RNA
was isolated from unstimulated RAW macrophage cells and cells treated for 4 hr with 100 ng/ml S. typhimurium LPS, 1 pg/ml S. aureus LTA, 1 ~tM CpG DNA or media alone and Northern blots were performed the membrane was probed for GAPDH, CD14, vimentin, and tristetraprolin as described previously [Scott et al]. The hybridization intensities of the Northern blots were compared to GAPDH to look for inconsistencies in loading. These experiments were repeated at least three times and the data shown is the average relative levels of each condition compared to media (as measured by densitometry) + standard error.
b) RAW 264.7 cells were stimulated with 100 ng/ml S. typhimurium LPS, 1 pg/ml S.
aureus LTA, 1 pM CpG DNA or media alone for 24 hours. Protein lysates were prepared, run on SDS PAGE gels and western blots were performed to detect LIF
(R&D Systems). These experiments were repeated at least three times and the data shown is the relative levels of LIF compared to media (as measured by densitometry) + standard error.
c) Supernatant was collected from RAW macrophage cells treated with 100 ng/ml S.
typhimurium LPS, 1 pg/ml S. aureus LTA, 1 pM CpG DNA, or media alone for 24 hours and tested for the amount of NO formed in the supernatant as estimated from the accumulation of the stable NO metabolite nitrite with the Griess reagent as described previously [Scott, et al]. The data shown is the average of three experiments + standard error.
Relative levels Product UntreatedLPS LTA CpG

CDl4a 1.0 2.2+0.4 1.8+0.2 1.5+0.3 Vimentina 1.0 1.2 + 0.071.5 + 0.05 1.3 + 0.07 Tristetraprolina1.0 5.5 + 0.5 5.5 + 1.5 9.5 + 1.5 LIF 1.0 2.8 + 1.2 2.7 + 0.6 5.1 + 1.6 NO' 8+1.5 47+2.5 20+3 21+1.5 [00158] Table 60. Pattern of Gene expression in A549 Human Epithelial cells up-regulated by bacterial signalling molecules (LPS). E. coli O111:B4 LPS (100 ng/ml) increased the expression of many polynucleotides in A549 cells as studied by polynucleotide microarrays. LPS was incubated with the A549 cells for 4 h and the RNA was isolated. 5 pg total RNA was used to make Cy3/Cy5 labelled cDNA probes and hybridised onto Human Operon arrays (PRHU04). The examples of polynucleotide expression changes in LPS simulated cells represent a greater than 2-fold intensity level change of LPS treated cells from untreated cells.
Accession NumberGene AL050337 interferon gamma receptor 1 U05875 interferon gamma receptor 2 NM 002310 leukemia inhibitory factor receptor U92971 coagulation factor II (thrombin) receptor-like 229575 tumor necrosis factor receptor superfamily member 17 L31584 Chemokine receptor 7 J03925 cAMP response element-binding protein M64788 RAP1, GTPase activating protein NM 004850 Rho-associated kinase 2 D87451 ring finger protein 10 AL049975 Unknown U39067 eukaryotic translation initiation factor 3, subunit 2 AK000942 Unknown AB040057 serine/threonine protein kinase MASK

AB020719 KIAA0912 protein AB007856 FEM-1-like death receptor binding protein AL137376 Unknown AL137730 Unknown M90696 cathepsin S

AK001143 Unknown AF038406 NADH dehydrogenase AK000315 hypothetical protein FLJ20308 M54915 pim-1 oncogene D29011 proteasome subunit, beta type, 5 AL034348 Unknown D87076 KIAA0239 protein AJ001403 mucin 5, subtype B, tracheobronchial J03925 integrin, alpha M

ALTERING SIGNALING TO PROTECT
AGAINST BACTERIAL INFECTIONS
[00159] The Salmonella Typhimurium strain SL1344 was obtained from the American Type Culture Collection (ATCC; Manassas, VA) and grown in Luria-Bertani (LB) broth. For macrophage infections, 10 ml LB in a 125 mL flask was inoculated from a frozen glycerol stock and cultured overnight with shaking at 37°C
to stationary phase. RAW 264.7 cells (1x105 cells/well) were seeded in 24 well plates.
Bacteria were diluted in culture medium to give a nominal multiplicity of infection (MOI) of approximately 100, bacteria were centrifuged onto the monolayer at rpm for 10 minutes to synchronize infection, and the infection was allowed to proceed for 20 min in a 37°C, 5% COZ incubator. Cells were washed 3 times with PBS to remove extracellular bacteria and then incubated in DMEM + 10% FBS containing 100 pg/ml gentamicin (Sigma, St. Louis, MO) to kill any remaining extracellular bacteria and prevent re-infection. After 2 h, the gentamicin concentration was lowered to 10 pg/ml and maintained throughout the assay. Cells were pretreated with inhibitors for 30 min prior to infection at the following concentrations: 50 pM PD
98059 (Calbiochem), 50 ~M U 0126 (Promega), 2 mM diphenyliodonium (DPI), 250 pM acetovanillone (apocynin, Aldrich), 1 mM ascorbic acid (Sigma), 30 mM N-acetyl cysteine (Sigma), and 2 mM N~-L-monomethyl arginine (L-NMMA, Molecular Probes) or 2 mM N~-D-monomethyl arginine (D-NMMA, Molecular Probes). Fresh inhibitors were added immediately after infection, at 2 h, and 6-8 h post-infection to ensure potency. Control cells were treated with equivalent volumes of dimethylsulfoxide (DMSO) per mL of media. Intracellular survival/replication of S. Typhimurium SL1344 was determined using the gentamicin-resistance assay, as previously described. Briefly, cells were washed twice with PBS to remove gentamicin, lysed with 1% Triton X-100/0.1% SDS in PBS at 2 h and 24 h post-infection, and numbers of intracellular bacteria calculated from colony counts on LB
agar plates. Under these infection conditions, macrophages contained an average of 1 bacterium per cell as assessed by standard plate counts, which permitted analysis of macrophages at 24 h post-infection. Bacterial filiamentation is related to bacterial stress. NADPH oxidase and iNOS can be activated by MEK/ERK signaling. The results (Table 61) clearly demonstrate that the alteration of cell signaling is a method whereby intracellular Salmonella infections can be resolved. Thus since bacteria to up-regulate multiple genes in human cells, this strategy of blocking signaling represents a general method of therapy against infection.
[00160] Table 61: Effect of the Signaling Molecule MEK on Intracellular Bacteria in IFN-y-primed RAW cells.
Treatments Effect 0 None MEK inhibitor U 0126 Decrease bacterial filamentation (bacterial stress) Increase in the number of intracellular S.

Typhimurium MEK inhibitor PD 98059 Decrease bacterial filamentation (bacterial stress) Increase in the number of intracellular S.

Typhimurium Treatments Effect NADPH oxidase inhibitor Decrease bacterial filamentation (bacterial stress) Increase in the number of intracellular S.

Typhimurium ANTI-VIRAL ACTIVITY
[00161] SDF-1, a C-X-C chemokine is a natural ligand for HIV-1 coreceptor-CXCR4. The chemokine receptors CXCR4 and CCRS are considered to be potential targets for the inhibition of HIV-1 replication. The crystal structure of SDF-1 exhibits antiparallel (3-sheets and a positively charged surface, features that are critical in binding to the negatively charged extracellular loops of CXCR4. These findings suggest that chemokine derivatives, small-size CXCR4 antagonists, or agonists mimicking the structure or ionic property of chemokines may be useful agents for the treatment of X4 HIV-1 infection. It was found that the cationic peptides inhibited SDF-1 induced T-cell migration suggesting that the peptides may act as CXCR4 antagonists. The migration assays were performed as follows. Human Jurkat T
cells were resuspended to 5 x 106 / ml in chemotaxis medium (RPMI 1640 / lOmM Hepes /
0.5 % BSA). Migration assays were performed in 24 well plates using 5 pm polycarbonate Transwell inserts (Costar). Briefly, peptide or controls were diluted in chemotaxis medium and placed in the lower chamber while 0.1 ml cells (5 x 106 / ml) was added to the upper chamber. After 3 hr at 37°C, the number of cells that had migrated into the lower chamber was determined using flow cytometry. The medium from the lower chamber was passed through a FACscan for 30 seconds, gating on forward and side scatter to exclude cell debris. The number of live cells was compared to a "100 % migration control" in which 5 x 105 /ml cells had been pipetted directly into the lower chamber and then counted on the FACscan for 30 seconds.
The results demonstrate that the addition of peptide results in an inhibition of the migration of Human Jurkat T-cells (Table 62) probably by influencing CXCR4 expression (Tables 63 and 64).

[00162] Table 62: Peptide inhibits the migration of human Jurkat-T cells:
Migration (%) ExperimentPositive SDF-1 SDF-1 + Negative control (100 ng/ml)SEQ ID 1 control (50 pg/ml) 1 100 % 32 % 0 % <0.01 %

2 100% 40% 0% 0%

[00163) Table 63: Corresponding polynucleotide array data to Table 56:
UnstimulatedRatio Accession PolynuclPolynucleotide Intensity peptide: Number eotide Function Unstimulated /

Protein CXCR-4 Chemokine receptor36 4 D87747 ~

[00164] Table 64: Corresponding FACs data to Tables 62 and 63:
Concentration Fold Increase in Protein Peptide (pg/ml) Expression SEQ ID NO: 10 No change SEQ ID NO:l50 1.3 + 0.03 SEQ ID NO:1100 1.6 + 0.23 SEQ ID NO: 100 1.5 + 0.2 [00165] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims (88)

WHAT IS CLAIMED IS:

1. A method of identifying a polynucleotide or pattern of polynucleotides regulated by one or more sepsis or inflammatory inducing agents and inhibited by a cationic peptide comprising contacting the polynucleotide or polynucleotides with one or more sepsis or inflammatory inducing agents, contacting the polynucleotide or polynucleotides with a cationic peptide either simultaneously or immediately thereafter, and determining a change in expression, wherein a change is indicative of a polynucleotide or pattern of polynucleotides that is regulated by a sepsis or inflammatory inducing agent and reduced by a cationic peptide.

2. The method of claim 1, wherein the sepsis or inflammatory inducing agent is LPS, LTA or CpG DNA, bacterial components or whole cells, or related agents.

3. The method of claim 1, comprising determining the level of expression of the polynucleotide prior to and following contacting with the sepsis or inflammatory inducing agent.

4. A polynucleotide or polynucleotide pattern identified by the method of claim 1.

5. A polynucleotide of claim 3, wherein the polynucleotide encodes a polypeptide involved in an inflammatory or septic response.

6. A method of identifying an agent that blocks sepsis or inflammation comprising combining a polynucleotide of claim 5 with an agent, wherein expression of the polynucleotide in the presence of the agent is modulated as compared with expression in the absence of the agent and wherein the modulation in expression affects the inflammatory or septic response.

7. The method of claim 6, wherein the effect is inhibition of the inflammatory or septic response.

8. An agent identified by the method of claim 6.

9. The agent of claim 8, wherein the agent is a peptide, peptidomimetic, chemical compound, nucleic acid molecule or a polypeptide.

10. The agent of claim 8, wherein the peptide is selected from SEQ ID NO:4-54.

11. A method of identifying a pattern of polynucleotide expression for inhibition of an inflammatory or septic response comprising:
contacting cells with LPS, LTA, CpG DNA and/or intact bacteria or bacterial components in the presence or absence of a cationic peptide;
detecting a pattern of polynucleotide expression for the cells in the presence and absence of the peptide, wherein the pattern in the presence of the peptide represents inhibition of an inflammatory or septic response.

12. The method of claim 11, further comprising contacting cells with one or more compounds suspected of inhibiting an inflammatory or septic response and identifying a compound that provides a pattern of polynucleotide expression similar to a pattern obtained with a cationic peptide that inhibits an inflammatory or septic response.

13. A compound identified by the method of claim 11.

14. A method of identifying an agent that enhances innate immunity comprising:
contacting a polynucleotide or polynucleotides that encode a polypeptide involved in innate immunity, with an agent of interest, wherein expression of the polynucleotide in the presence of the agent is modulated as compared with expression of the polynucleotide in the absence of the agent and wherein the modulated expression results in enhancement of innate immunity.

15. The method of claim 14, wherein the agent does not stimulate a septic reaction.

16. The method of claim 14, wherein the agent inhibits the inflammatory or septic response.

17. The method of claim 14, wherein the agent blocks the inflammatory or septic response.

18. The method as in any of claims 16 or 17, wherein the agent increases the expression of an anti-inflammatory encoding polynucleotide.

19. The method of claim 18, wherein the anti-inflammatory gene is selected from a subset that includes IL-1 R antagonist homolog 1 (AI167887), IL-10 R beta (AA486393), IL-10 R alpha (U00672), TNF Receptor member 1B (AA150416), TNF
receptor member 5 (H98636), TNF receptor member 11b (AA194983), IK cytokine down-regulator of HLA II (R39227), TGFB inducible early growth response 2 (AI473938), CD2 (AA927710), glucocorticoid-related polynucleotides (AK000892), or IL-10 (M5762720.

20. The method of claim 19, wherein the agent inhibits the expression of TNF-alpha.

21. The method of claim 19, wherein the agent inhibits the expression of interleukins.

22. The method of claim 23, wherein the interleukin is IL-8.

23. The method of claim 16, wherein the agent is a peptide.

24. The method of claim 23, wherein the peptide is selected from SEQ ID NO:4-54.

25. An agent identified by the method of claim 14.

26. An agent of claim 25, wherein the agent is a peptide, peptidomimetic, chemical compound, or a nucleic acid molecule.

27. A method of identifying a pattern of polynucleotide expression for identification of a compound that selectively enhances innate immunity comprising:

detecting a pattern of polynucleotide expression for cells contacted in the presence and absence of a cationic peptide, wherein the pattern in the presence of the peptide represents stimulation of innate immunity;
detecting a pattern of polynucleotide expression for cells contacted in the presence of a test compound, wherein a pattern with the test compound that is similar to the pattern observed in the presence of the cationic peptide, is indicative of a compound that enhances innate immunity.

28. A compound identified by the method of claim 27.

29. The method of claim 27, wherein the compound does not stimulate a septic reaction.

30. The method of claim 27, wherein the polynucleotide expression pattern includes expression of pro-inflammatory polynucleotides.

31. The method of claim 30, wherein the pro-inflammatory polynucleotides include ring finger protein 10 (D87451), serine/threonine protein kinase MASK
(AB040057), KIAA0912 protein (AB020719), KIAA0239 protein (D87076), RAP1, GTPase activating protein 1 (M64788), FEM-1-like death receptor binding protein (AB007856), cathepsin S (M90696), hypothetical protein FLJ20308 (AK000315), pim-1 oncogene (M54915), proteasome subunit beta type 5 (D29011), KIAA0239 protein (D87076), mucin 5 subtype B tracheobronchial (AJ001403), cAMP response element-binding protein CREBPa, integrin alpha M (J03925), Rho-associated kinase 2 (NM_004850), PTD017 protein (AL050361) unknown genes (AK001143, AK034348, AL049250, AL16199, AL031983), retinoic acid receptor (X06614), G
protein-coupled receptors (Z94155, X81892, U52219, U22491, AF015257, U66579) chemokine (C-C motif) receptor 7 (L31584), tumor necrosis factor receptor superfamily member 17 (Z29575), interferon gamma receptor 2 (U05875), cytokine receptor-like factor 1 (AF059293), class I cytokine receptor (AF053004), coagulation factor II (thrombin) receptor-like 2 (U92971), leukemia inhibitory factor receptor (NM_002310), interferon gamma receptor 1 (AL050337) or any combination thereof.

32. The method of claim 27, wherein the expression pattern includes expression of polynucleotides encoding chemokines.

33. The method of claim 27, wherein the expression pattern includes expression of cell differentiation factors.

34. The method of claim 27, wherein the polynucleotide expression pattern includes expression of cell surface receptors.

35. The method of claim 34, wherein the cell surface receptors include chemokine receptors or integrin receptors.

36. A method of identifying an agent that is capable of selectively enhancing innate immunity comprising:
contacting a cell containing a polynucleotide or polynucleotides that encode a polypeptide involved in innate immunity, with an agent of interest, wherein expression of the polynucleotide or polynucleotides in the presence of the agent is modulated as compared with expression in the absence of the agent and wherein the modulated expression results in enhancement of innate immunity.

37. The method of claim 26 in which the pattern of expression is utilized in screening for compounds that enhance innate immunity.

38. A compound of claim 28, wherein the compound stimulates chemokine or chemokine receptor expression.

39. A compound of claim 38, wherein the chemokine or chemokine receptor is CXCR4, CCR5, CCR2, CCR6, MIP-1 alpha, IL-8, MCP-1, MCP-2, MCP-3, MCP-4, or MCP-5.

40. A compound of claim 28, wherein the compound is a peptide, peptidomimetic, chemical compound, or a nucleic acid molecule.

41. A method of identifying an agent that is capable of both suppressing or blocking septic or inflammatory responses and enhancing innate immunity comprising:
contacting a cell containing i) a polynucleotide or polynucleotides that encode a polypeptide capable of suppressing inflammatory or septic responses and ii) a polynucleotide or polynucleotides that encode a polypeptide involved in innate immunity, with an agent of interest, wherein expression of in the presence of the agent is modulated as compared with expression of the polynucleotide or polynucleotides in the absence of the agent and wherein the modulated expression results in suppression of inflammatory or septic responses and enhancement of innate immunity.

42. A method for inferring a state of infection in a mammalian subject from a nucleic acid sample of the subject comprising identifying in the nucleic acid sample a polynucleotide expression pattern exemplified by an increase in polynucleotide expression of at least 2 polynucleotides in Table 55 as compared to a non-infected subject.

43. A method for inferring a state of infection in a mammalian subject from a nucleic acid sample of the subject comprising identifying in the nucleic acid sample a polynucleotide expression pattern exemplified by a decrease in polynucleotide expression of at least 2 polynucleotides in Table 56 as compared to a non-infected subject.

44. A method for inferring a state of infection in a mammalian subject from a nucleic acid sample of the subject comprising identifying in the nucleic acid sample a polynucleotide expression pattern exemplified by a polynucleotide expression of at least 2 polynucleotides in Table 57 as compared to a non-infected subject.

45. The method of any of claims 30, 31 or 32, wherein the state of infection is due to a bacteria, virus, fungus or parasitic agent.

46. The method of any of claims 30, 31 or 32, wherein the state of infection is due to a Gram positive or Gram negative bacteria.

47. A polynucleotide expression pattern of a subject having a state of infection identified by the method of claim 31.

48. A cationic peptide that is an antagonist of CXCR-4.

49. A method of identifying a cationic peptide that is an antagonist of CXCR-4 comprising contacting T cells with SDF-1 in the presence of absence of a test peptide and measuring chemotaxis, wherein a decrease in chemotaxis in the presence of the test peptide is indicative of a peptide that is an antagonist of CXCR-4.

50. An isolated cationic peptide comprising the general formula X1X2X3IX4PX4IPX5X2X1 (SEQ ID NO: 4), wherein X1 is one or two of R, L or K, X2 is one of C, S or A, X3 is one of R or P, X4 is one of A or V and X5 is one of V or W.

51. The cationic peptide of claim 38, wherein the peptide is selected from the group consisting of: LLCRIVPVIPWCK (SEQ ID NO: 5), LRCPIAPVIPVCKK
(SEQ ID NO: 6), KSRIVPAIPVSLL (SEQ ID NO: 7), KKSPIAPAIPWSR (SEQ ID
NO: 8), RRARIVPAIPVARR (SEQ ID NO: 9) and LSRIAPAIPWAKL (SEQ
ID NO: 10).

52. The peptide of claim 38, wherein the peptide has anti-inflammatory activity.

53. The peptide of claim 38, wherein the peptide has anti-sepsis activity.

54. An isolated cationic peptide comprising the general formula X1LX2X3KX4X2X5X3PX3X1 (SEQ ID NO: 11), wherein X1 is one or two of D, E, S, T
or N, X2 is one or two of P, G or D, X3 is one of G, A, V, L, I or Y, X4 is one of R, K
or H and X5 is one of S, T, C, M or R.

55. The cationic peptide of claim 42, wherein the peptide is selected from the group consisting of: DLPAKRGSAPGST (SEQ ID NO: 12), SELPGLKHPCVPGS
(SEQ ID NO: 13), TTLGPVKRDSIPGE (SEQ ID NO: 14), SLPIKHDRLPATS (SEQ
ID NO: 15), ELPLKRGRVPVE (SEQ ID NO: 16) and NLPDLKKPRVPATS (SEQ
ID NO: 17).

56. The peptide of claim 42, wherein the peptide has anti-inflammatory activity.

57. The peptide of claim 42, wherein the peptide has anti-sepsis activity.

58. An isolated cationic peptide comprising the general formula X1X2X3X4WX4WX4X5K (SEQ ID NO: 18), wherein X1 is one to four chosen from A, P or R, X2 is one or two aromatic amino acids (F, Y and W), X3 is one of P or K, X4 is one, two or none chosen from A, P, Y or W and X5 is one to three chosen from R
or P.

59. The cationic peptide of claim 46, wherein the peptide is selected from the group consisting of: RPRYPWWPWWPYRPRK (SEQ ID NO: 19), RRAWWKAWWARRK (SEQ ID NO: 20), RAPYWPWAWARPRK (SEQ ID NO:
21), RPAWKYWWPWPWPRRK (SEQ ID NO: 22), RAAFKWAWAWWRRK
(SEQ ID NO: 23) and RRRWKWAWPRRK (SEQ ID NO: 24).

60. The peptide of claim 46, wherein the peptide has anti-inflammatory activity.

61. The peptide of claim 46, wherein the peptide has anti-sepsis activity.

62. An isolated cationic peptide comprising the general formula X1X2X3X4X1VX3X4RGX4X3X4X1X3X1 (SEQ ID NO: 25) wherein X1 is one or two of R or K, X2 is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X3 is C, S, M, D or A and X4 is F, I, V, M or R.

63. The cationic peptide of claim 50, wherein the peptide is selected from the group consisting of: RRMCIKVCVRGVCRRKCRK (SEQ ID NO: 26), KRSCFKVSMRGVSRRRCK (SEQ ID NO: 27), KKDAIKKVDIRGMDMRRAR
(SEQ ID NO: 28), RKMVKVDVRGIMIRKDRR (SEQ ID NO: 29), KQCVKVAMRGMALRRCK (SEQ ID NO: 30) and RREAIRRVAMRGRDMKRMRR (SEQ ID NO: 31).

64. The peptide of claim 50, wherein the peptide has anti-inflammatory activity.

65. The peptide of claim 50, wherein the peptide has anti-sepsis activity.

66. An isolated cationic peptide comprising the general formula X1X2X3X4X1VX5X4RGX4X5X4X1X3X1 (SEQ ID NO: 32), wherein X1 is one or two of R or K, X2 is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X3 is one of C, S, M, D or A, X4 is one of F, I, V, M or R and X5 is one of A, I, S, M, D
or R.

67. The cationic peptide of claim 54, wherein the peptide is selected from the group consisting of: RTCVKRVAMRGIIRKRCR (SEQ ID NO: 33), KKQMMKRVDVRGISVKRKR (SEQ ID NO: 34), KESIKVIIRGMMVRMKK
(SEQ ID NO: 35), RRDCRRVMVRGIDIKAK (SEQ ID NO: 36), KRTAIKKVSRRGMSVKARR (SEQ ID NO: 37) and RHCIRRVSMRGIIMRRCK
(SEQ ID NO: 38).

68. The peptide of claim 54, wherein the peptide has anti-inflammatory activity.

69. The peptide of claim 54, wherein the peptide has anti-sepsis activity.

70. An isolated cationic peptide comprising the general formula KX1KX2FX2KMLMX2ALKKX3 (SEQ ID NO: 39), wherein X1 is a polar amino acid (C, S, T, M, N and Q); X2 is one of A, L, S or K and X3 is 1-17 amino acids chosen from G, A, V, L, I, P, F, S, T, K and H.

71. The cationic peptide of claim 58, wherein the peptide is selected from the group consisting of: KCKLFKKMLMLALKKVLTTGLPALKLTK (SEQ ID NO:
40), KSKSFLKMLMKALKKVLTTGLPALIS (SEQ ID NO: 41), KTKKFAKMLMMALKKVVSTAKPLAILS (SEQ ID NO: 42), KMKSFAKMLMLALKKVLKVLTTALTLKAGLPS (SEQ ID NO: 43), KNKAFAKMLMKALKKVTTAAKPLTG (SEQ ID NO: 44) and KQKLFAKMLMSALKKKTLVTTPLAGK (SEQ ID NO: 45).

72. The peptide of claim 58, wherein the peptide has anti-inflammatory activity.

73. The peptide of claim 58, wherein the peptide has anti-sepsis activity.

74. An isolated cationic peptide comprising the general formula KWKX2X1X1X2X2X1X2X2X1X1X2X2IFHTALKPISS (SEQ ID NO: 46), wherein X1 is a hydrophobic amino acid and X2 is a hydrophilic amino acid.

75. The cationic peptide of claim 62, wherein the peptide is selected from the group consisting of: KWKSFLRTFKSPVRTIFHTALKPISS (SEQ ID NO: 47), KWKSYAHTIMSPVRLIFHTALKPISS (SEQ ID NO: 48), KWKRGAHRFMKFLSTIFHTALKPISS (SEQ ID NO: 49), KWKKWAHSPRKVLTRIFHTALKPISS (SEQ ID NO: 50), KWKSLVMMFKKPARRIFHTALKPISS (SEQ ID NO: 51) and KWKHALMKAHMLWHMIFHTALKPISS (SEQ ID NO: 52).

76. The peptide of claim 62, wherein the peptide has anti-inflammatory activity.

77. The peptide of claim 62, wherein the peptide has anti-sepsis activity.

78. An isolated cationic peptide comprising the sequence KWKSFLRTFKSPVRTVFHTALKPISS (SEQ ID NO: 53).

79. An isolated cationic peptide comprising the sequence KWKSYAHTIMSPVRLVFHTALKPISS (SEQ ID NO: 54).

80. The method of claim 28, wherein the agent is a Zinc finger protein (AF061261); Cell cycle gene (570622); IL-10 Receptor U00672); Transferase (AF038664); Homeobox protein (AC004774); Forkhead protein (AF042832);
Unknown (AL096803); KIAA0284 Protein (AB006622); Hypothetical Protein (AL022393); Receptor (AF112461); Hypothetical Protein (AK002104); Protein (AL050261); Polypeptide (AF105424); SPR1 protein (AB031480); Dehydrogenase (D17793); Transferase (M63509); and Peroxisome factor (AB013818).

81. The polynucleotide expression pattern of a subject having a state of infection identified by claim 56 wherein the genes upregulated are Accession number ring finger protein 10; Accession number AL049975, Unknown; Accession number U39067, eukaryotic translation initiation factor 3 subunit 2; Accession number AK000942, Unknown; Accession number AB040057, serine/threonine protein kinase MASK; Accession number AB020719, KIAA0912 protein; Accession number AB007856, FEM-1-like death receptor binding protein; Accession number AL137376, Unknown; Accession number AL137730, Unknown; Accession number M90696, cathepsin S; Accession number AK001143, Unknown; Accession number AF038406, NADH dehydrogenase; Accession number AK000315, hypothetical protein FLJ20308; Accession number M54915, pim-1 oncogene; Accession number D29011, proteasome subunit beta type 5; Accession number AL034348, Unknown;
Accession number D87076, KIAA0239 protein; Accession number AJ001403, tracheobronchial mucin 5 subtype B; Accession number J03925, integrin alpha M, Rho-associated kinase 2 (NM_004850), PTD017 protein (AL050361) unknown genes (AK001143, AK034348, AL049250, AL16199, AL031983), retinoic acid receptor (X06614), G protein-coupled receptors (Z94155, X81892, U52219, U22491, AF015257, U66579) chemokine (C-C motif) receptor 7 (L31584), tumor necrosis factor receptor superfamily member 17 (Z29575), interferon gamma receptor 2 (U05875), cytokine receptor-like factor 1 (AF059293), class I cytokine receptor (AF053004), coagulation factor II (thrombin) receptor-like 2 (U92971), leukemia inhibitory factor receptor (NM_002310), interferon gamma receptor 1 (AL050337), or any combination thereof.

82. The method of claim 32, wherein the chemokines include CXCR4, CXCR1, CXCR2, CCR2, CCR4, CCR5, CCR6, MIP-1 alpha, MDC, MIP-3 alpha, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, and RANTES.

83. The method of claim 33, wherein the cell differentiation factors includeTGF.beta.
inducible early growth response 2 (AI473938), zinc finger proteins (AF061261, U00115, X78924), and transcription factors (U31556, AL137681, X68560).

84. A compound of claim 38, wherein the compound modifies kinase activity.

85. A compound of claim 84, wherein the kinase is selected from MAP kinase kinase 3 (D87116), MAP kinase kinase 6 (H07920), MAP kinase kinase 5 (W69649), MAP kinase 7 (H39192), MAP kinase 12 (AI936909), MAP kinase-activated protein kinase 3 (W68281), or MAP kinase kinase 1 (L11284).

86. A compound of claim 21, wherein the compound decreases proteasome subunit expression.

87. A compound of claim 86, wherein the proteasome subunit includes polynucleotides with accession numbers D11094, L02426, D00763, AB009398, AF054185, M34079, M34079, or AL031177.

88. An isolated cationic peptide that reduces polynucleotide expression of SDF-receptor.