ZA200404919B - Effectors of innate immunity - Google Patents

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 medical 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 onc 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

E 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 -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 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 (A1167887), 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), TGF-B inducible early growth response 2 (A1473938), 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-xB 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 NO: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:

XX XEIX4PX4IPXsX, X, (SEQ ID NO: 4), where X, is one or two of R, L or

K, X;isone of C, Sor A, X; is one of Ror P, X; is one of A or V and Xs is one of V or W;

X LX2X3K Xa XoXsX PX 3X (SEQ ID NO: 11), where X, is one or two of D,

E,S, TorN,X2isoneortwoof P, Gor DD, Xzisoneof G, A,V,L,lorY, X,is one of R, KorHand Xsisoneof S, TT, C, MorR;

Xi Xo X3XaWX4, WX XsK (SEQ ID NO: 18), where X is one to four chosen from A, P or R, X; 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;

Xi Xo X3XaX; VX3X4RGX 4X 3X X1 X3X, (SEQ ID NO: 25) where X; is one or two of R or K, X; is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H),

X3isC,S,M,DorAand XsisF, I, V,MorR;

X1XoX3X eX VXsX4RGX XXX 1 X3X; (SEQ ID NO: 32), where X| is one or two of R or K, X; is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H),

Xsisoneof C,S,M,D or A, Xsisoneof F, I, V,MorR and Xs isone of A, I, S, M,

D or R; and

KX, KX;FX;KMLMX,ALKK X; (SEQ ID NO: 39), where X; is a polar amino : acid (C, S, T.M, Nand Q); X; isone of A, L, S or K and Xj is 1-17 amino acids chosen from G, A, V,L,I, PF, S, T, K and H;

KWKX,X Xi X2X2X1 XXX: XX, X,IFHTALKPISS (SEQ ID NO: 46), where

X is a hydrophobic amino acid and X; 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

10019] 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 : like.

[0021] As uscd 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 9.0. 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 havc a net positive charge at pH 7.0. 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. 10022] 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, BacZ2A.

LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ

ID NO: 1)

Indolicidin ILPWKWPWWPWRR-NH, (SEQ ID NO: 2)

Bac2A RLARIVVIRVAR-NH; (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 p which have two adjacent cysteines at the N terminus (CC). RANTES, MIP-1a and MIP-18 are members of the B 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 B 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 J. 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 B groups are distinct. While the monomeric structures of the 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 (v) of chemokines (Yoshida

RY!

et al, 1995, FEBS Lett. 360:155-159; and Kelner et al., 1954, Science 266:1395-

[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 : Pharmacol. 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 B chemokine receptors include: CC CKR-1 , which binds RANTES and MIP-1a (Neote et al., 1993, Cell 72: 415-425), CC CKR-4, which binds RANTES, MIP-1a, and MCP-1 (Power et al., 1995, J. Biol. Chem. 270:19495-19500), and CC CKR-5, which binds RANTES, MIP-1q, and MIP-13 (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 p 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 pinding 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 - epithehal 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), TLR5 (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 (A1167887), IL-10 R beta (AA486393), TNF Receptor member 1B (AA150416), TNF receptor member 5 (H98636), TNF receptor member 11b (AA194983), IK cytokine down-regulator of HLA II (R39227), TGF-B inducible early growth response 2 (A1473938), 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,

WO $3/0-18383 PCT/CA02/01830 the agent reduces the expression of TNI-a and/or interleukins including, but not limited to, IL-18, 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, 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. 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 (0 retinoic acid receptor (X06614), G protein-coupled receptors (294155, 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 transiocaics to the nucieus 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 kinascs. Table 21 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 (AI1936909) 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 x 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 (sce 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):

Xi Xo XGIXPX4IPX5X,X, (SEQ ID NO: 4), wherein X, isone or two of R, L or K, X; isone of C, Sor A, Xsisone of R or P, X4 is one of A or V and Xsis 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):

XiLX:X3K Xe X2XsX3PX3X, (SEQ ID NO: 11), wherein X; isoneortwo of D,E,S, T orN, X2isoneortwoof P, Gor D, Xsisoneof G, A, V,L,lorY, Xsisoneof R, K or Hand Xsisoncof 5, 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):

Xi Xo X3XaWX4WX XK (SEQ ID NO: 18) (this formula includes CP12a and CP12d) , wherein X; is one to four chosen from A, P or R, X; is one or two aromatic amino acids (F, Y and W), Xj 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):

X1X2X3X 4X1 VX;3X4RG XX 3X X1 X3X, (SEQ ID NO: 25) wherein X; is one'or two of

R or K, X; is a polar or charged amino acid (S, T,M, N, Q, D, E, K, R and H). Xj; is

C,S,M,DorAand X4isF, I, V, Mor 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):

XX X3XaX; VXsXsRGXsX5 Xa X1X3X; (SEQ ID NO: 32), wherein X; 1s one or two of Ror K, X; is a polar or charged amino acid (S, T, M, N, Q, D, E, K, Rand H), X; : isoneof C,S,M,Dor A, Xsisoneof F,I, V, MorR and Xsisone 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 KX FX, KMLMX,;ALKKX; (SEQ ID NO: 39), wherein X, is a polar amino acid (C,S, T,M,Nand Q); X; isone of A, L, S or K and X;3 is 1-17 amino acids chosen fromG, 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):

KWKX, XX XX Xi XXX 1 Xi XX, IFHTALKPISS (SEQ ID NO: 46), wherein X; is a hydrophobic amino acid and X; 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 “L” 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 in

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 5% 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 celi-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 i 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 CaCl, MgCl, or RbCl 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 meinod 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 doublc- 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. :

EXAMPLE 1

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 10° cells/dish, cultured overnight and then incubated with 100 ng/ml £. coli O111:B4 LPS

(Sigma), without (control) or with 50 pg/mi peptide or medium aione 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 PRHUO04-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 significani 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 1, the peptide, SEQ ID NO: 27 is shown to potently reduce the expression of many of the polynucleotides up-regulated by E. coli O111:B4 LPS as studied by polynucleotide microarrays. Peptide (50 pg/ml) and LPS (0.1 ug/ml) or

LPS alone was incubated with the A549 cells for 4 h and the RNA was isolated. Five ug total RNA was used to make Cy3/Cy5 labeled cDNA probes and hybridized onto

Co 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 O111:B4 LPS

Accession Polynucleotide Control: Ratio: Ratio: LPS+

Number? Gene Function | Mediaonly | LPS/control | ID 27/control

Intensity

ADP- ribosylation

L04510 factor 0.655 213.6 1.4 ring finger hypothetical ee |e | we

Ric -like expressed in

U78166 neurons 0.051 91.7 0.2 mucin 5 subtype

B

AJ001403 | tracheobronchial 0.203 53.4 15.9 serine/threonine protein kinase

AB040057 MASK 0.95 44.3 15.8

Accession | Polynucleotide Control: Ratio: Ratio: LPS+

Number® Gene Function Media only | LPS/control | ID 27/control

Intensity [we

RNA lariat debranching

NM 016216 enzyme 6.151 22.3 | 10.9 hypothetical a I NLC LJ

FEM-1-like death receptor ee Ze growth arrest- cytosolic ovarian carcinoma

AK000353 antigen 1 0.453 13.5 1.0 myeloid/lymphoi d or mixed- lineage leukemia

D14539 | translocated to 1 2033 11.6 3.1 integration site for Epstein-Barr

X76785 virus 0.728 11.6

I LL NL caspase recruitment :

NM_006092 domain 4 0.369 11.0 0.5 integrin_alpha ars | er |e

Accession Polynucleotide Control: Ratio: Ratio: LPS+

Number? Gene Function Media only LPS/control | ID 27/control

Intensity

ADP- ribosylation

NM_001663 factor 6 0.439 9.7 1.7

RAS p21 protein thymidine kinase [am | | a transmembrane 9 superfamily

U94831 member 1 3.265 7.1 1.5 zinc finger

Cn |e Low [0 hypothetical

PE el I I guanine nucleotide

U43083 binding protein 0.841

DKFZp434J181

Pl I IP

ATP-binding cassette sub- family C (CFTR/MRP)

AF085692 member 3 3.175 6.5 2.4 hypothetical protein

AKO001239 FLJ10377 2.204 6.4 1.3

ATPase Na+/K+

RN Fcc PS

Accession | Polynuclestide Control: Ratio: Ratio: LPS+

Number? Gene Function Media only LPS/control | 1D 27/control re ee ewes unactive =

L24804 receptor 3.403 1.1 dual specificity ligase | DNA _ ar [om] on colony stimulating spermidine/ spermine N1-

AL050290 | acetyltransferase 2.724 5.6 1.4

Re ec I WLI retinoic acid ve [So oe | | on putative L-type neutral amino

ABO007896 | acid transporter 0.94 5.3 1.8

DKFZP564B116

Pe Ep hypothetical os] Cn | un | sa

NM_016406 protein 1.314 5.2 1.2 }

Accession Polynucleotide Control: Ratio: Ratio: LPS+

Number® Gene Function | Mediaonly | LPS/control | ID 27/control

Rd = a leukemia trans-

I EE zona pellucida replication initiation region leukemia dentatorubral- pallidoluysian ne

NM_001940 atrophy 2.034 4.7 1.2 cytosolic acyl coenzyme A thioester

U91316 hydrolase 2.043 4.7 1.4 death-associated

KIAAOQ0095 gene monocyte to macrophage differentiation-

X85750 associated 1.01 4.3 1.1

Accession | Polynucleotide Control: Ratio: Ratio: LPS+

Number® Gene Function Media only LPS/control | ID 27/control ee

CD164 antigen_ fibroblast melanoma- associated

U19796 antigen 4.0 hypothetical

PP lO PO hypothetical sterol-C4-methyl

EY I hypothetical ms] hypothetical

RAN member

RAS oncogene :

NM_006325 family 1.242 3.5 1.4 aldehyde

FH1/FH2 domain- .

NM_013241 containing 1.264 3.3 0.6

Accession Polynucleotide Control: Ratio: Ratio: LPS+

Number* Gene Function Media only LPS/control | ID 27/control

Intensity

IE Hc I esterase

D/formylglutathi

AF112219 one hydrolase 1.839 3.3 anaphase- promoting complex subunit

NM _016237 5 2.71 3.2

KIAAQ669 gene hypothetical protein phosphatase 6 proteasome 26S subunit ATPase

AF035309 5 5.628 3.1 1.3

I cc IL NL electron- transfer- flavoprotein alpha

J04058 polypeptide 3.265 3.1 1.2 interleukin 6 galactosidase _ :

Accession Polynuclestide Control: Ratio: Ratio: LPS+

Number® Gene Function Media only | LPS/control | ID 27/control

Intensity

EC 5. 2 mitogen- : activated protein

X80692 Kinase 6 2.463 29 1.3 tumor necrosis factor receptor 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 ug/ml were shown to potently reduce the expression of many of the polynucleotides up-regulated by 100 ng/ml E. coli O111: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 O111:B4 LPS and reduced by Cationic Peptides

Number Media LPS/ LPS+ | LPS+ID | LPS+ID only control | ID 27/ 16/ 22/

Intensity control | control | control oe ribosylation

L04510 factor 213.6 1.4 2.44 3.79 ring finger hypothetical

MHC class Il ve [ae [son [0 [on corns | “en | on | on | ow | ow | on

AK001904 protein 0.03 32.8 593 0.37 0.37 hypothetical thioredoxin- dependent peroxide

L19185 reductase 1 0.06 16.3 0.18 2.15 0.18

Transcobalamin ee | on [on [on [on [en

FEM-1-like death ee] 0 [

Number Media LPS/ LPS+ | LPS+ID | LPS+ID only control | ID 27/ 16/ 22/

Intensity control | control peer] I cytosolic ovarian E ’ smooth muscle

EP pei Pp PY hypothetical

PE RP PN phospholipase C hypothetical retinoic acid putative L-type neutral amino

ABO007896 | acid transporter 0.94 5.3 1.82 2.15 2.41

BAl1-associated ome Tu Ta To Tw oo hypothetical 2 [me] [To To [on on ankyrin 2_ were] | en | [on [on [oe inter-alpha brain and nasopharyngeal carcinoma . susceptibility

Co AF095687 protéin 0.39 21 0.48 0.03 0.98

Number Media LPS/ LPS+ | LPS+ID | LPS+ID only control | ID 27/ 16/ 22/

Intensity control | control | control

NK cell activation inducing ligand

BEREAN

KIAA0981 wi | | an Ta Leo Lon Lor

EXAMPLE 2

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 ng/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 10° cells per well in DMEM and A549 cells were seeded in 24 well plates at a density of 10° cells per well in DMEM and both were incubated at 37°C in 5 % CO; overnight. DMEM was aspirated from cells grown overnight and replaced with fresh medium. In some experiments, blood from volunteer human donors was coliecied (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 of other bacterial products were incubated with the cells for 6-24 hr at 37°C in 5 % CO.

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 ug of E. coli O111: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 pg in 100ul 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 ug E. coli O111: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 AralLAM 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 O111: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

O111: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 murine 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).

18077] 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 pug 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 100 % 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 AralLAM (Table 15). Another peptide, SEQ ID NO: 2, was also found to reduce LTA-induced TNF-a production by

RAW 264.7 cells. Ata concentration of 1 ug/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 ug/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 t+ 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 (ug/mD) | B. cepacia LPS | E. coli LPS S. typhimurium LPS 23.0 + 11.4 36.6 + 7.5 9.8 + 6.6 55.41 8 65.0 + 3.6 311+ 7.0 63.1+8 75.0 + 3.4 374 +75 71.7+5.8 81.0 + 3.5 58.5+10.5 86.7 + 4.3 92.6 +25 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 ug/ml) | Inhibition of TNF-a (%)

SEQID 5 65.6% 1.6

SEQID 6 59.8+1.2

SEQID 7 50.6 + 0.6

SEQID 8 39.3+1.9

50.8 + 1.67 73.3+0.36

[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 O111:B4) for 24 hours. The concentration of IL-8 in the culture supernatants was determined by ELISA. The background levels of IL-8 from cells alone was 0.172 + 0.029 ng/ml. The data is presented as the mean of three experiments + standard error.

IL EU

I EL

[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 O111: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 2 {ug/inl) Inhibition of IL-8 (%)

I EES A

[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 O111: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 (ug/ml) IL-8 (pg/ml)

[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 ug/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 (ug/ml) Production of TNF-a (ng/ml)

IE BL Bc

No LPS control 0.038 | 006

[0085] Table 9: Inhibition of E. coli LPS-induced TNF-a production by delayed addition of SEQ ID 1 to A549 cells. Peptide (20 pg/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 ID 1 Inhibition of TNF-a (%) after LPS (min)

I EE

IE ELS

[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 0111:B4 LPS (3 pg in 0.1 mi PBS). Peptide, SEQ ID 1, (200 pg/mouse = 8mg/kg)

was injected at a separate intraperitoneal siic 15 min after injection of LPS. The mice were monitored for 48 hours and the results were recorded.

D-Galactosamine E. coli Peptide or | Total Survival post : treatment 0111:B4 LPS buffer mice endotoxin shock

[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 ug 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 Treatment | E. coli 0111:B4 [ Number | Survival (%)

LPS added of Mice

SE

Slide nT

[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 ug 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 Treatment E. coli Number of Mice | Survival (%) i i a i NS LL NL

FE EC LN ELA

Fl I SS EO EL

Fl NS INC A i CS NN LA ia LN IR A

Fl ILS IS

Fl NS EL EL

Fl I NN ELA i I I won ew | ow | 0 il I EO A i HN I

Fl IN ICR SL

SEQ ID 29 ng

Ee

Fl I I

Fl IL NN

FC I aL ILS SC EL

[0089] Table 13: Protection against lethal endotoxaemia in BALB/c mice by SEQ

ID 1. BALB/c mice were injected intraperitoneal with 400 pug E. coli 0111:B4 LPS.

Peptide (200 pug/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 Mice Survival (%) an ow | aw [5 [0

IC LT NO EL

[0090] Table 14: Peptide inhibition of TNF-a production induced by S. aureus

LTA. RAW 264.7 mouse macrophage cells were stimulated with 1 ug/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 6 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.

[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 ug/ml AraLAM in the absence and presence of 20 ug/ml peptide or Polymyxin B. The supernatant was collected and tested for levels of TNF- - o 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. ewe [oT

EXAMPLE 3

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, ODg9onm (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 mitochondrial dehydrogenases in viable cells (a non- radioactive alternative to the [*H]-thymidine incorporation assay). In this assay, HBE cells were incubated with 100 ug of peptide for 24 hours, and then 10 ul/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, OD gonm.

18093] The results shown below in Tables 16 and 17 demonstrate thai mosi 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 pg/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.

[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.

EXAMPLE 4

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 10° 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 DNasel (Clontech, Palo Alto, CA) for 1 h at 37°C. After adding termination mix (0.1 M

EDTA [pH 8.0], I 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 B-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). **P-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: B-actin, ubiquitin, ribosomal protein S29, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Ca®* binding protein. When the normalized : hybridization intensity for a given cDNA was less than 20, it was assigned a value of 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 PRHUO03-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 10° 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 cach 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 trcated 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 ug 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 ul 0.1M DTT, and 1 pl

RNaseOUT recombinant ribonuclease inhibitor (40 units/ul) were added and incubated at 42 °C for 2 min, followed by the addition of 1 pl (200 units) of

Superscript 11 (Invitrogen, Burlington, ON). Negative controls for each RNA source were generated using parallel reactions in the absence of Superscript Il. cDNAs were . amplified in the presence of 5’ and 3’ primers (1.0 uM), 0.2 mM dNTP mixture, 1.5 mM MgCl, 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 anneaiing ai 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 B-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 B-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-1R-2 (a putative antagonist of productive IL-1 binding to IL-1R1), PDGF receptor B,

NOTCH4, LIF receptor, LFA-1, TGF 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 Etk1 and Csk demonstrating its widespread effects. It was also discovered from the polynucleotide array studies that SEQ ID NO: 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-1a, oncostatin M and 1L-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, i treatment of RAW macrophage cells”. 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,

converied 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

Co 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 arc presented.

Polynucleotide Polynucleotide Unstimulated Ratio Accession / Protein Function Intensity peptide: Number ! Unstimulated”

Etkl Tyrosine-protein 20 43 M68513

I = Hd

Corticotropin releasing 20 23 X72305

Ih a a ed

NOTCH4 proto- 48 18 MB0456

I Pl ll hed

BMP-1 Bone morpho- 20 i4 L24755 polynucleotidetic protein

Endothelin b Receptor 20 14 U32329 ll Mell c-ret Oncopolynucleotide 20 13 X67812 }

Nc I a

Polynucleotide Polynucleotide Unstimulated Ratio Accession / Protein Function Intensity peptide: Number me

BMP-8a Bone morpho- 20 12 M97017 polynucleotidetic protein

CC cc NO WL LC

MCSF Macrophage colony 85 11 X05010 =r a I

GCSFR Granulocyte colony- 20 M58288 stimulating factor receptor a cc LC IL ILL a I Mi

Cas Crk-associated 31 U48853 re

IL-1B Interleukin precursor 5 M15131 =

SPi2-2 Serine protease 62 5 M64086

NA = I I hd

RE cc WL EO i NC IL Lc pI0inka cdk4 and cdk6 147 2 U19597 i i I I ed

Polynucleotide Polynucleotide Unsiimulated Ratio Accession / Protein Function Intensity peptide: Number

Unstimulated”

GADD45 DNA -damage- 88 3 128177

Re LL LI 8

POLDI1 DNA polymerase 649 2 221848

Vav proto- 613 2 X64361

Te

Cdk7 Cyclin-dependent 475 2 U11822

I rl A hd

MLCiA Myosin light subunit 453 2 M19436 i a al I hd

IFNGR Interferon gamma 308 2 M2R?733 pl =o el I i

Polynucleotide Polynucleotide Unstimulated Ratio Accession / Protein Function Intensity peptide: Number

Unstimulated”

IGF-1Ra Insulin-like growth 218 2 Uo00182

Ne el A fyn Tyrosine-protein 191 2 U70324

Te

BMP-2 Bone morpho- 2 1.25602 polynucleotidetic =

RS Tc I cl

BKLF CACCC Box- binding 138 2 U36340

I dE

Mas Proto- 131 2 X67735

I I I al

[00104] Table 19: Polynucleotides down-regulated by SEQ ID NO: 1 treatment of RAW macrophage cells’. 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. Poiynucieotides with an approximately two fold or greater change in relative expression levels are presented.

Unstimulated Ratio Accession

Polynucleotide Polynucleotide Intensity peptide: Number mm sodium channel Voltage-gated ion 257 0.08 L36179 dl a Ml id

CT cc NLA LI a cc CL Nc

KRT18 Intermediate filament 318 0.28 M11686

NE ll al

RE cc NC NA Rail

Oc IC HO ic

Unstimulated Ratio Accession

Polynucleotide Polynucleotide Intensity peptide: Number e

KRT19 Intermediate filament 622 0.52 M28698 ee

CTLA4 immunoglobin 468 0.57 X05719 =m

MTRP Lysosome-assaciated 498 0.58 U34259

Rl i el Ml hd

[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 $-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.

[00106] Table 21: Polynucleotides up-regulated by peptide treatment of A549 epithelial cells’. The cationic peptides at concentrations of 50 ug/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#PRHUO03-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

Polynucleotide/Protein Intensity (D2 IDI TID [

A I ec I ed

FS 0 I NL iad

SE I cH

NF R superfamily, member

NF R superfamily, member 5 wo Lufoe] | ome

NF R superfamily, member o Joo] interleukin enhancer binding sd PPR oy po oy ps

ON Nl Nh Bl Rial

Accession

Em

Polynucleotide/Protein Intensity (D2 [D3 [IDI IDT al cytokine inducible SH2- AN A AN NN vl RP PE py

K cytokine, down-regulator ytokine inducible SH2-

K cytokine, down-regulator mall inducible cytokine ubfamily A (Cys-Cys), member 21 3.9 2.4 Al922341

GFB inducible early growth rises IP PY PY PY

EN a Ri RA Rc 0% | 0 [39 | 39 | ST | Roan integrin, alpha 2 (CD49B, a alpha 2 subunit of VLA-2. receptor 1.00 2.4 3.6 AA463257 integrin, alpha 3 (antigen

ICD49C, alpha 3 subunit of

LA-3 receptor) 0.94 2.5 1.9 1.1 | AA424695

FE NC cl Wl LI al

Ech NC I lB id

EE NC ll CN id

EN cc IL a Il WE cid ach WO I Wl Wh NA Rid

: Accession

Polynucleotide/Protein Intensity calcium and integrin binding Ea oT disintegrin and : disintegrin and nc IP PY PY PP disintegrin and

Scie PO [PO PP disintegrin and cadherin 1, type 1, E-cadherin seen IPR PP og py adherin 12, type 2 (N-

Ei IVE PP

FC ll Wl Rad protocadherin gamma evil I A catenin (cadherin-associated srvwrigl IPOR P P P aminin R 1 (67kD, ribosomal killer cell lectin-like receptor

Er or ee killer cell lectin-like receptor rived IP PY PR py pe killer cell lectin-like receptor

Finer JPW OY Py poe

FT NO 1 I Ml id I

SL NC I I A dad

Accession

Unstimulated| Ratio Peptide: Unstimulated | Number

IPolynucleotide/Protein Intensity [102 [ID3TIDI] IDL uman proteinase activated

R-2 0.54 1.9 2.2 AA454652 prostaglandin E receptor 3 (subtype EP3) 0.25 4.1 3.8 AA406362 7 Cr growth factor receptor-bound protein 2 0.51 2.2 2.0 2.4 0.3 | AA449831

Mouse Mammary Turmor irus Receptor homolog W93891 prostaglandin E receptor 1 (subtype EP1) 0.65 7.2 1.5 | AA972293 growth factor receptor-bound protein 14 0.34 3.0 6.3 29 R24266 [Epstein-Barr virus induced polynucleotide 2 0.61 1.6 2.4 8.3 | AA037376 omplement component receptor 2 0.22 26 4.5 2.6 18.1 | AA521362 yrosine kinase, non-receptor, 1 0.12 7.8 8.5 10 8.7 Al936324 eceptor tyrosine kinase-like orphan receptor 2 0.40 7.3 5.0 2.5 N94921

Accession me

Polynucleotide/Protein Intensity protein tyrosine phosphatase, ha Rid A

Severna IE PY I PY protein tyrosine phosphatase, : protein tyrosine phosphatase, protein tyrosine phosphatase, sso IPPON PN YY protein tyrosine phosphatase, protein tyrosine phosphatase,

Free [oo en protein tyrosine phosphatase,

Sor EV PP protein tyrosine phosphatase, wri IP AP PI protein tyrosine phosphatase, protein tyrosine phosphatase, protein tyrosine phosphatase, eceptor type, C-associated

Sean ERE

AP kinase-activated protein oe] oo Lor fo

I RL I a NL

Accession

Wg —

Polynucleotide/Protein Intensity

A RM ci

Cc NL FE NL NO Bal regulator of G-protein Ril

EE 4 I egulator of G-protein mr | oo ore

BCL2-interacting killer [ol Lo] Jo aspase 6, apoptosis-related apoplosis-related protein aspase 8, apoptosis-related mp aL Lo

[00107] Table 22: Polynucleotides down-regulated by peptide treatment of

A549 epithelial cells”. 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#PRHUOQ3-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

Polynucleotide/Protein Intensity | 102 [1D3 [ID 19) ID 1

AS SC i i NO NO Rl

NF receptor-associated a

NF receplor-associated rial J

NF receptor superfamily, weil I PE PY

NF R superfamily, member i I I

RAF and TNF receptor-

L-2 receptor, gamma (severe on

Garou J 8 poy eukotriene b4 receptor wrens) IE I PPR small inducible cytokine ubfamily A (Cys-Cys), 2 | or fon] oe mall inducible cytokine ubfamily A (Cys-Cys), small inducible cytokine levee PO

Accession

Polynucleotide/Protein Intensity (D273 [ID1g[ IDI [ a I ) small inducible cytokine AEN NN I subfamily B (Cys-X-Cys), member 6 (granulocyte =n mall inducible cytokine subfamily B (Cys-X-Cys),

Er | ie Loon ee mall inducible cytokine A3 (homologous to mouse Mip-

Sid I EI omplement component Clq cadherin 11, type 2, OB- adherin 3, type 1, P-cadherin ro IPR PF cadherin, EGF LAG seven- pass G-type receptor 2, lamingo (Drosophila) omolog 1.67 0.42 | 041 | 1.21 H39187 cadherin 13, H-cadherin

Sia ranR mE electin L (lymphocyte

ET [eo Jose] [ome vascular cell adhesion il PE YE intercellular adhesion al IEP FP fy

FCA cc i ie

Accession

Unstimulated Number

Polynucleotide/Protein Intensity [02 [D3

Fk Fc lal lil disintegrin and a

EA EP disintegrin-like and metalloprotease with hrombospondin type 1 motif, 3 2.11 0.32 | 0.63 | 0.47 | 0.35 | AA398492 disintegrin-like and metalloprotease with hrombospondin type 1 motif, 1.62 0.39 | 0.42 | 1.02 | 0.62 | AI375048 -cell receptor interacting

CC [ iphtheria toxin receptor (heparin-binding epidermal growth faclor-like growth actor) 1.62 0.49 | 0.85 | 0.62 | 0.15 | R4564C vasoactive intestinal peptide c fragment of IgG, low affinity IIIb, receptor for c fragment of IgG, low affinity IIb, receptor for

Fc fragment of IgE, high

Se EE 0

Accession

Polynucleotide/Protein Intensity (D2 [D3 [ID19| IDI a NN FF leukocyte immunoglobulin- sag J FF py 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), member 4 2.31 0.75 | 0.43 0.40 | AA076350 leukocyte immunoglobulin- wi J PR fo py peroxisome proliferative

Soa FI PF protein tyrosine phosphatase, eceptor type, f polypeptide (PTPRF), interacting protein (liprin), a1 2.19 0.43 0.46 | N49751 protein tyrosine phosphatase, sil PO Po I py protein tyrosine phosphatase, protein tyrosine phosphatase, sna J PO FOP protein tyrosine phosphatase, il PF pp protein tyrosine phosphatase, protein tyrosine phosphatase, re | Jon[olus [ifm

: Accession

Unstimulated Number

Polynucleotide/Protein Intensity EHEC EDN

AP kinase 8 interacting 0.66 | 2.10 | 1.49 | W61116

MAP kinase 8 interacting

SP PF

IMAP kinase kinase kinase

Fl OY

SAA lO li

AP kinase kinase kinase

Sl Fo ed NO il Rl ad ial

Sl Ol a il i Ni

MAP kinase-activated protein

Sl OR Kc OR id

SO OC A Me id ic

SB WO cc a Ol cl egulator of G-protein i png oP egulator of G-protein pg po Fo py oy py BE protein-coupled receptor

BO nl PPO I 4

Accession

Polynucleotide/Protein Intensity (23 pig DI] orphan seven-transmembrane

Sodom PIP apoptosis antagonizing srl I PF pp caspase 1, apoptosis-related ysteine protease (interleukin 1, beta, convertase) 2.83 0.44 0.33 | 0.35 T95052 programmed cell death 8 or fy

[00108] Table 23: Pro-inflammatory polynucleotides up-regulated by peptide treatment of A549 cells. The cationic peptides at concentrations of 50 ug/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#PRHUO3-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 unction Intensity

IL-11 Ra; Receptor for pro- BLA ELAN — inflammatory cytokine, inflammation 0.55 2.39 | 0.98 4.85 1.82 | AA454657

IL-17 R; Receptor for IL-17, an inducer of cytokine production in epithelial cells 0.54 2.05 | 1.97 1.52 1.86 |AW029299

Accession unction Intensity |] mall inducible cytokine Bld subfamily A, member 21; a chemokine 3.88 2.41 Al922341

CD31; Leukocyte and cell to

CCR6; Receptor for integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 eceptor; Adhesion to integrin, alpha 3 (antigen

D49C, alpha 3 subunit of

LA-3 receptor); Leukocyte integrin, beta 4; Leukocyte or

C-type lectin-like receptor- kil [PY PR py

[00109] Table 24: Pro-inflammatory polynucleotides down-regulated by peptide treatment of A549 cells. The cationic peptides at concentrations of 50 ug/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#PRHUQ03-S3. The intensity of polynucleatides 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; Function| Intensity oll-Tike receptor (TLR) 1; lida in Fp Pp

LR 2; Response to gram positive

LR 5; May augment other TLR er a oafun] | eee

LR 7: Putative host defence

NF receptor-associated factor 2:

NF receptor-associated factor 3; oii I PR PP po

NF receptor superfamily, member me wy Jon [an] Joe

NF R superfamily, member 17; weil I I I Pp

RAF and TNF receptor- small inducible cytokine subfamily pani [JP po small inducible cytokine subfamily prvi J Fp oy small inducible cytokine subfamily prc 90 FP small inducible cytokine subfamily

B, member 6 (granulocyte hemotactic protein); Chemokine 3.57 0.11 0.28 | 0.38 | AI889554 mall inducible cytokine subfamily ir Pp mall inducible cytokine A3 mo IY J

Unstim Accession

SR oe a 1L-12 receptor, beta 2; Interleukin HE I NS

Siig poy I electin L (lymphocyte adhesion ascular cell adhesion molecule 1; intercellular adhesion molecule 3; integrin, alpha 1; Leukocyte

Em ve Jean] Joe

[00110] Table 25: Anti-inflammatory polynucleotides up-regulated by peptide treatment of A549 cells. The cationic peptides at concentrations of 50 ug/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#PRHUO03-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 Accession pr [re

IL-1 R antagonist homolog 1; hilid ual rg po po po is

L-10 R beta; Receptor for

Polynucleotide/Protein; Unstim | Ratio Peptide: Unstimulated | Accession

Function Intensity | 02 [ ID3 JIDIS|ID 1] Number

NF R, member 5; Apoptosis (CD40L) 33.71 2.98 0.02 H98636

NF R, member 11b; Apoptosis | 100 1529 | 450 [078] | AA194983

IK cytokine, down-regulator of

HLA 11; Inhibits antigen presentation 0.50 3.11 2.01 1.74 | 3.29 | R39227

GFB inducible early growth esponse 2; anti-inflammatory cytokine 2.38 2.08 | 0.87 Al473938

D2; Adhesion molecule, binds

LFAp3 1.00 2.62 0.87 1.15 | 0.88 | AA927710

[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#PRHUO3-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 Peptide: Unstimulated | Accession

Function Intensity | 102 [ID3 IDIS]ID | Number

[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

Ic FO WL

Ic NJ WL chromosome condensation 1-like eww ow

TERF1 (TRF1)-interacting nuclear

LINE retrotransposable element 1 1-acylglycerol-3-phosphate O-

Vacuolar proton-ATPase, subunit D; V-

potassium voltage-gated channel KQT- histone fold proteinCHRAC17

I LCA LJ pancreatic zymogen granule membrane hypothetical protein FLJ20495

E2F transcription factor 5, p130-binding hypothetical protein FLJ20070 glycoprotein IX (X52997) eukaryotic translation initiation factor

I I NL guanine nucleotide binding protein, gamma transducing activity polypeptide 1 (U41492) 0.80 3.3 glypican 1 (X54232) mucosal vascular addressin cell adhesion i LL translational inhibitor protein p14.5 hypothetical protein FLI20689 protein kinase, CAMP-dependent,

POU domain, class 4, transcription : POU domain, class 2, associating factor ——— matrix metalloproteinase 23A cyclin-dependent kinase inhibitor 1B

Ih WL bone morphogenetic protein 1 (NM_006129) 1.10 :

bone morphogenetic protein receptor, type IB (U89326) 0.50 2.1 interferon regulatory factor 2 (NM 002199) 1.46 2.0 protease, serine 21 (AB03133D

[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 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 (PRHUOQ4). 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 (ALU49263) I CA I XS integrin-linked kinase (U40282)

RTAAO84Z protein (ABOZ0649)

Unknown (ABO37838)

Granulin (AF055008) 8s | 01a glutathione peroxidase 3 (NM_002084)

KIAA0152 gene product (D63486) 09 | 017

TGFB1-induced anti-apoptotic factor 1 (D86970) disintegrin protease (Y 13323) proteasome subunit beta type 7 (D38048) cofactor required for Spl transcriptional activation subunit 3 (AB033042) 0.23

TNF receptor superfamily, member 14 : (U81232) 0.26 proteasome 26S subunit non-ATPase 8 (D38047) 0.28 : proteasome subunit beta type, 4 (D26600)

TNF receptor superfamily member 1B (M32315) 1.7 0.29 cytochrome c oxidase subunit Vic

S100 calcium-binding protein A4 proteasome 26S subunit non-ATPase, 10

MAP Kinase Kinase kinase 2 (NM_006609) | 08 | 032 matrix metalloprotease 14 (Z4848) | 10 | 0.32

MAP kinase-activated protein kinase 2 proteasome 26S subunit, ATPase, 3 interferon alpha-inducible protein CatepnDM633®) | 46 | 036

TGF, bewainduced, 77349) | 18 | 037

TNF receptor superfamily, member 10b nuclear receptor binding protein protease inhibitor 1 alpha-1-antitrypsin proteasome subunit alpha type, 7 oo

LPS-induced TNF-alpha factor proteasome 26S subunit non-ATPase 13

MAP kinase kinase kinase kinase 2 peroxisome proliferative activated receptor defender against cell death 1 (D15057)

TNF superfamily member 10 (U37518) cathepsin H (X16832) protease inhibitor 12 (Z81326) 06 | 048 proteasome subunit alpha type, 4 (D00763) proteasome 26S subunit ATPase, 1 (L02426) 1.8 0.49 proteasome 26S subunit ATPase, 2 (D11094) 2.1

Caspase 7 (U67319) 2a oe matrix metalloproteinase 7 (Z11887)

[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 (PRHUO04). 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 Unstimulated | treated:control cells

AJ000730 solute carrier family | 001 | 282.0 725884 chloride channel 1 oor | 256.2 protein tyrosine phosphatase

M93426 ~ receptor-type,zeta 248.7 olfactory receptor, family 1,

X65857 subfamily D,member 2 0.01 228.7

M55654 TATA box binding protein BEE . AK001411 hypothetical protein

Accession Gene Control: Ratio peptide

Number Unstimulated | treated:control == diphosphooligosaccharide-protein m— tumor necrosis factor,alpha- ci Gc EL WC

SEC24 related gene family, glutamate receptor, ionotropic adenosine monophosphate

IE OC FF unknown | 0.28

Accession Gene Control: Ratio peptide

Number Unstimulated | treated:control = = 5,10-methenyltetrahydrofolate

Pal PY 0 Cc I NL

EC Oc UN a I hepatitis A virus cellular receptor il IV AP

I IL NL

Fl oc CL I cl CC NL LB

NADH dehydrogenase

Krueppel-related zinc finger

Accession Gene Control: Ratio peptide

Number Unstimulated | treated:control - = cc NL NL

TRAF family member-associated acetyl-Coenzyme A ]

UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- mL iT Ec en I

Accession Gene Control: Ratio peptide ~ Number Unstimulated | treated:control cells a disintegrin-Tike and a disintegrin and metalloprotease

[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 (PRHUO4). 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 Unstimulated | NO:1- treated:

Cells control

BH EA

Accession Gene Control: Ratio SEQ iD

Number Unstimulated | NO:1- treated:

Cells control polymerase (RNA) II ras-related C3 botulinum toxin

I J WL a cL IL

Human mRNA for ornithine

HLA-G histocompatibility antigen,

ATP synthase, H+ transporting oe

Homo sapiens TTF-I interacting

Accession Gene Control: Ratio SEQ ID

Number Unstimulated | NO:1- treated: ll I

Oa NO CLA anti-oxidant protein 2 (non- selenium glutathione peroxidase, acidic calctum-independent

D14662 phospholipas 0.19

IE I FL ILA

6-pyruvoyl-tetrahydropterin synthase/dimerization cofactor of

L41559 hepatocyte nuclear factor 1 alpha 9.1 0.20

RN

268 proteasome-associated padl tumor protein, translationally- a purine-rich element binding om | a protein disulfide isomerase-related ae

Accession Gene Control: Ratio SEQ ID

Cells control oe endothelial differentiation-related immediate early response 3 methylene tetrahydrofolate transcriptional intermediary factor solute carrier family 22 member 1- tyrosine 3- monooxy genase/tryptophan 5- - ubiquitin-conjugating enzyme lysosomal-associated membrane

I cc SI

~ [ Accession Gene Control: Ratio SEQ ID

Number Unstimulated | NO:1- treated: fe anti-Mullerian hormone receptor,

Homo sapiens ribosomal protein hypothetical protein glucose phosphate isomerase interferon induced transmembrane cold shock domain protein 6.3 0.26

Accession Gene Control: Ratio SEQ ID

Cells control gonadotropin inducible transcriptn complement component 5 receptor

LL NL cc TO FL LC ec OT FL a OT FO protective protein for beta- 0 cdc NF cc NL NLA

Accession Gene Control: Ratio SEQ ID

Number Unstimulated | NO: 1- treated:

Cells control

CT wee

[00116] Table 31: Up-regulation of Polynucleotide expression in A549 cells induced by Formula A Peptides. The peptides at a concentration of 50 ug/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.

WO (3/048383 PCT/CA02/01830

Ss £ < ~ 0 oN wos |e — c 0 w= at on |o6 [oo 0 : [an] °o — — — — .. © > £5 a 2 “ ~ NE pe a = < a) <t ™ Nz ™m = 8 . © ® L = 0 oN = = =z wn a ~ r~ < wv ae TN (SN oe 2 3 “w B ~ 5 wy Ae i 0 a = <r wn jon fi r~ = 8 . © © E wn 2 © x No |= — a = on <r o~ o Oo Iv | O \O - ° (3) .w © } v5 2 0 |= 3 “ pt © (=) =~] «® ~~ — — wn — ~N = 5 1] 2S uh <r > on ON ~~ <tr

ES A = 8 N Q = RNS = g Q fe) Oo |o o o oo |e o

[5]

L] ° To) To)

ES == = 21 2 g QO o |e o oS o

[3] ! } .S [ wv I) E 9 |o = ' ! © 21% 552 BIE —|8 ¢ S15 IE %

S15 = ES 2 als >| 3 |3 (3 |S = = Ele FE |= © = Eg |€ |e (8 2

S 2 |= J |= (0 ¥ie Elo |Z 2 |g E £ |° cS ig cs |e S aS [30] 8 gv 5 8 IS = S R= a. 3 = Q Qa. = EE Jo —~ £ |0 N gE 2 ] °0 vn 5 |” c aN |e =) = oS 2 o O° =] ~ on < 0 : gd El © S 3 El | IRE| R g 3 pA NSIS] K 8 S leo © Eo) < on) x |< >< D oD | |< =

< °

SE Soo © - |e 0 = a £ ~ |e~ ~ ~ |r~ O Na) —- 3 . © ok SI: - o |e [7 |~ S

EE Agr a § << |< ~~ eT (VI bot [oe 8 .. © © £ “| NA <M oy = : a sg NSN a Oo | ~ fo 1%) . - ~ £ NW: Se < 5 S a £ ~ {en ~ =I Lg wy = ws 1%) . o

SC NN o Ne [2 oy 0 2 2 £ eT [a] w NTR [Ro A No ~ = — & . wv CZ on |n = — [ov [nn jo o ~ a £ ~~ | - NN [|e ~ r~

J— 15] —

S un a | <r Oo NN 00 = — |N N Nn oN = s © Oo |o o oc lo lo |o o (5) — e = - hog 0 IN [N10 00

EES A AN IN j= [N < 5g © o o Oo lo Io lo =) [¥] |/M < {0 m 2 le IB = . 8 . [9] |= . ‘aS lo Q = 4 > |E NIE TIE 82a lE|8 |g © © cE Q le = |e © | |= = |g =

S EI S212 28 = lg|¥|g|2 lg © = £ ££ Ble 8 28|l2 vs |e lE |S 2 ica © w

S | x |s |= Q jv (X |Oo = == — vo © (a © =] © 5B |d o (> Ss Lele |E |& & la © = ©T 3 |Z E © 0 a i= i |3 |= 3 © a | = 28 |= = cE 3 |& |< o

Ss — oo ~~ ~t

LS w 3 |x EO ~ ~ 2 £ bell Fy har A “J MR = Q =

Pu g E << [Mm 0 NB |= hn 2 = : 3 oo wn NT QO A <e ce Z CO A a nn © Mm NY RY << |< jun) = |X |< |< < <

Ss © -_ 5 |= ™~ ~ EEE SHEERS AJ ~M . a g O wn wn VaR [YOR [Vo wn Te) “w o . os £ \ on 7) 0 |\O |= ™~ <r a og | ISN] © A it | ™m oJ = [=] - -

S oe ° O ~~ a g [= — ~ NL SN [oN] o~ ~ .. © ~ Ew o ™~ aN en |r < ~ 2 |= eA ~ ~ je je< ™M t~ aE “vw © on © I lo | © r~ LN on ~ a 5 on ol <r hn | STN wv CF o » © ~r [oN] \O = ~ [nN NO ol oS) a S N ~N — — g un w on © [fn | 00 00 b=E ~™m - i Ai DA << ~N g Q o o o lo © ’ o o

Oo

A }

SC eo | un re) INES) <x 00 - Pn — ha NNN ~N ~N £ Q |c =) f=) SS lo lo o o

[5]

QQ 1

Q - = I= [= fos ol —- aa [e) g [=f = N pe = 3 ~My ~~ - o ‘= fo 3 © 2 c QL oe T= Q : oc @ -— [04 E [«] ~~ Q oo <S oD = [2] = x |e 8 alk 8 20 Cl I © 22 5 © - [9] — = I [3 [53 = 0 an Z © E I) BO o — a oO I = (= > 0 = Je) Oo [o}

ES 0 oF 8&8 ¢ jc aE |EBIE g &|5 2 5 - a. =) E 20 3 > « BR [] on

CN) @ 4 S = eS 5 00 ~~ r~ ~™ \O =] » Ve) Ve) oo ™ wv 7 Lis MM — o In un < 2 2 Elo wn oN oo | |~ — 8 5 I S S| SEB = g < Z |i tn us| wv | jo N < < < x << (xX fan] <

= © : —- = N= = oy 1°] oo ~ ~ I a E |v |» wn << I~ ~ <r <r ~ |= ~~ 9 : . © > Em |m o Tle ol; © 2 + |T a £ | |< SN Toh — uw o CNN -_— OQ [¥} .. © © Eo |= r~ 0 00 he. ~ 00 a 5 Im |v a = NN [ae] on J = NA 1% © co [I~ ~N ~ Coe foo — x d 5 wv ~ < ) ! _ ) ] , 82 § |© |N <r — |= — Ne) Oo wn : bat “w B® — o

Ee | ~ s on : oN a ££ =~ a « © joo wv ~~ x < _— 9 © . . ° i= (=) Lp 9 Qn N < ~ wv a & |» | Ne) aes Ww ~ ™ ~~ —_— Q

[5] —

Sn |v ~ < > [= = ™ Te) < |v = = (A (N ~ <= - — — |= § OO lo |o = oo o c o o |e

Q ra en |n |w ~ © [me on 00 on — f=

EE rl - - — AN N jn a <o co |o o o o o lo

Q

Yo) = 3 g => ° = ce |e (© < c |S ZT ~ [8 3 IS — |= = => a £ | | [8&8 & +o 5 © |= = |% 5 |8 [2d oc rT Id © LO OY = = lag ©

Q © |E In lo |lE EE B12 |e 88g = |= c |E|~ BF 8 lg le 5 o < 5 | 5 |@

X |x lop = [Bd 2 [XIE e E oO ®(g 2 (oo = |Tv £ jE |e & IN al ia 5 £ | ££ |& 218 <= a 3 |3 8 [2% Se a EE |5 8B |= E lo hv CS « o£ |= a = o° 2 3

SE oo |= Ne)

Ss 5 |a I ] NR N (h ~ nw L | lon un << {on ies o << I oo 2 E |= lo <r ~ |o 3 = ~ wo IS ] Qo IN — — jun D | on \O i . gS 2 [ee jo oo Ea [= aN — aN < |= < < |< < Z < = |Z

.e ° . [=] . —_ £ <t < on aN AN | — fe . a § <t ~r ~ <r < |< < |x 3S ee S ~ . 8 E ~ uN ~ 00 0 | < 5] .. 8 © L N=) N wv — ~~ Joo on =) g IN N ~t <t J lo ~ & . E ~ £ ™ hd oy — NR T jn

Q WN a ~~ ~ ~ |< To IT)

[3] : .. ©

No £ r~ © 0 ™ Jo =~ 8 § ~N <r \o ™ BU [0 J |= 9 .. © pi = — ~ < < |e < (mm 2 5 ~ 0 mM <Q Mm (N — e un O oy oe) £ > oe: = ~ SN Q ~ g o oc o = o =) @

LJ

3 oC a e on ~~

E> Q ~ S [= ~ |=

S o o o lo oo & oP 3? 2 © lo 5 50 = oc = S =]

QB 5 € Cig =|- 8 0 [8 c |E | =

Eg 2p 2 £18 a8 2 5 (8 5 (2 |x = slo Bl CS BE vw|g ls 21218 o |B = la oO Z Bl 5 | 2» EE oo IE 0 2 2 HS) bs oo = o o o 7 [e) [3°] o [ = = hot 2 |= & | 9 8 |5 = 0 a |a a 3] = e |5 ~E @ p = 2 E 3 8 | 2 5 = 3 a

S Q. o

SS r— <r Oo

Sg 8 2 Sos sR w = \O wv |S 17} [= — o = 2 0 [00 $ E <i N S 3 S IB S I3

Q Z s oO <Q — oR = KR > . < <3 4 nn | TR [=

Zz < << = < |< jo J je)

sS ©

R - 0 |x ™~ = = ~ MM = on 3 3 =) S 0 [ap] [ag] on on [ag] [ag] [3 . . © :

SE ~ |= b ~N Q lll 8 5 < |v ~t No) oo PERRIN >] — .e [=] ® 5 SY = — =< i: a aN ~ Ise) oN Te)

Tt [=4 [>] .. © ~ k no ~N a S ™ = i 8 5 < | ve) < ~ on < i

[3] . ©

R= nN ™ n — — — a c 00 |™M on <r a} r~ Vo) [=] [= : [3 . © vos “|e oN 3 o ~~ “ c NN 5 : 2 5 5 ~N a < & — e wy 0 {e~ ~N on oN) <r — = NM AN — ~N — —

FS ® o lo oS oS o o f=

S : — © = mT oN <r r~ <t l= = RAD ™ < n “a NS g © o |e = =] o Lo | o |e

[3] [} 9] 2] - LM =~ D — 3) [=

Mo hE ST o £2 SS ££ 3 |= = 5le |S 7 £ o |[@ 3 a 2 = 12 = £13

E 28 |S 5 2&8 BIZ ~ | 8" 8 Bis

Ss EES 18 8 EE [© 2 I= Zs © £ ZF = 17] Q ‘ah esd o-— < by a. [=% 2 Q s a. £2 Qo |2 I= te 73 = 2 lo = 3 2 E jo 2 5 ee > a Do a |= on £ 2 — ony < @ = 9c = s

S un Te) ~ ~~ 2 3 o- [5 50 ¢| 3 ¥ 2 E ~ |= 3 < 0 S S |e @ © |o oa — © S 8 |©

S = [oN < [en] ~™M [a=] oS © . & Z = | vy 5s o 3 |X = |< PS < = < D |< oe °

S 5 ~ a ~ - — S SS . a =] ag) on (ag) ag} 2g) (20) on - 8 . © . o£ o — <1 — ~ 00 5) a = — ~ <r (ae) ~ (eo <r fo [=] (3) wv © ® © © ~N wn ~ ™

I] Vo) 0 wn ™ —- 5 2 § — ©

[5] . O° ~- k ° N ~ A o < a = on r~ 00 (V2) <r wy [=] [=

S

“ © © Ek ™ nn AN io) ~ a a a \O (=) ~r (oV] 0 on o~ [==] [=]

Q

. © “

SAE = DA : ~ “ «© N a Ee ~ <r a ~ < < IN

So [=] oO . . £ wn Ig) ~ m= < on a) = — —- ~ N — - — . S ®] o o o oS o ) o

Oo vo =m <x 0 ~ un v— oN fo = ~ ~N - Mm a] ™ ~N £ @) o fe) o o o lo) o @ [V9] «© [+0] = c = 2 wo I+ 8 EE |= < 5 o EE oo [8 a |+ cs © |o = Z O ja >» |la S

[3] oO — Cc LQ c 4 Prt re pal = I] > Pot [@] = -o m — c— Q a. o. © Q — £

S 6 BR Els SDD Lis iF = tle 9 5 = = 3 |@ < o> 818 9 |v & 8 = [3] [3] c= = |= O < = [5 LQ |e 3 [3] N= a © Q Is 9 (XM — ad a a, ££ 2 315 8% S$ <|E EE E = alo Q |g hY4 [@] > =

E ono |© hs = oa |< o [3] © es ~

Se 3 & A ™ 1] E — <r 1% [eo] [@, r~

Oo = NR) tn -— AN O — on 0 © Nl < b= wn < © . < Z {, — ~ m oo 00 wn ] < < x < [) x io]

SS © . —_ = < a a io EE EEA ~N : a £ 4 ~~ |e aN al aN |e « — Q ’ i: © on fe

SAE] < : : wy on © | iN 0 a £ 0 ho <r aN Nea [es O

A) . ° ww “a < lm o ™M 00 0 hat se El | $p| 2] = C= o

[5] . © ~ ~ £ > a © N = | ht a ae} = 3 ~ ~ < | <

Po

[5] .. © - © 5 - 0 RQ ed) AT Ln A wn a g = on <t o\] non in on — bs} : . © nb 0 on © wn \O Und LB on [an] g ~ ~ ~ on fo NE 1s oT [200 3]

L bet -_

S un ) wv {oo — ~ — It lo wn = - i AN oN DA Ap he - : g Q o co |o =) =) co lo lo o 8 ’ — em 00 ~ wn <t O [00 [mM fo = Ao < Oo XN i hs g Oo o So o f=) co (lo jo =) (3) on TN : LY © ~ le 2 2 = ® s |, 2 |e a .

I ‘D R= © [= QL be > oS a. — L 2 hd c |= | Lz |2 «| 50 5 21] |8 xg & - sje 5a 8 2l< 2 5(2|2|& E 8

Oo a |e c c j= BV Ie) od [~~ Q >> | [= Zz So oO |= © | (Oo = D2 oo ol = £ = [od — jo > C— c po = [5] |[& (= 2 [5 IE © E 25 © 8 ie la a 9 phony iba 1 = = -

E 2 |8 & Nn © is 2 |e > la |p 2 E

Q |= o — ! E oh SS

I] —_ <t © — on = ~ 00 3 © In Sls IB 2 <£ nf ~ — PA = Ne

[7] E — — — 0 \O

Q — I~ po —t \O o =. [eal ~~ [Z] = O o [ng aN I | <r < o — ~M = < |« - — Zz |< |Z = on =]

NN = c © a << — ]S a cS o = gg 2 =

Ar “v= =

ZS En — ~ — 2 2 538 8 = © 2 . a § | ~ oN ~ © o = = 2 >! a — ve = £ 5 a = ss Ex we © EE = 9 wu ow a £5 2] 2 4 g 2 5 E 3 8 § [m 2g] ~ — © © = 5 = 1] 2 85 & © § . smo - 8 g oo 2 — ¥ 3 5 [@)

A 4 A 2 BZ © E |n < S 00 mE = a ev on <r 00 s «© 2 = 3 _— Oo I EAT. —~ B= <Q = a 2 2 9 5 J

E oo N = . 3B EO 5 8 ~ leo 00 a ~ € 8 ££ § = t |< ‘ . . a 5 Oo = a <t t~ 00 < ou LY => ao ©

SE 2 AZ 2g

T 82 EE — tt ww © ¢ 2 « 2

JE ~~ on oo 3 = » £5 a < . on TT § 9 ~ 5 82 5 © — ~ —_ £3 8 z 2 © 2 2 a2 §& p= T 5 < @ w £ . °S 3 Zz 0 4 a & | ~ & > oo «»u E —_ 0 ve = = 3 bs) < 8s © © = v oo Oo £ E353 5 © = ce © | 8 =

Eg wn |v ~ LC eg — = 2

EJS S 2 2 2 EE

SS o oo @ = E 8 5] I SRS & TY,

S23 EG 3 : $ Fg 5 2 8 ~ » © 9 =

S eo ln en Ny T eo gg 9° TF 2 = n(n ™ ha ES gg = ou = [=] 2 LY LL 0 51 © @ © © YE 2 @ = 8 23 ££ 8 = 2 Ue « 2 ££ 9 & FZ 3 on | 2 = r= 4 © TT 2 wv Ea c |S 3 SU |g 3 & 2 98 g 0° = Js © 2 |S = Som S$ oa x

TIE = € |B RIE 2 Se £ z 2 a = £ |lE § © 58 © se 5 2 3% ¢ 3 oi? £ ale gj = wm ce un @Q gE 8 Fle lz s cS wv Z Zu = 5 | = gE SC 3g SS p ® 50 =

Lc por} So © @Q ° wn ° Bo 2 2 2 2 2 ¥ ZZ o = =

I: EB >on & £ ov lo S58 E>

LC 9 IB x= 2 YY 9 2 2 8 nn ~ a oo + 2 gg BB gg E NY <r 00 NR = ££ =z oS 3 |= AR va) °S TT wv 3B [1 [en] ~~ oy Neo] Ew Sg ° «< Z | =H) <t — 2 2 ou ££ 2 = ¢ < = a a gE 8 3 FE =3 2 0 = £ ©

EC w= XT = — £3 2 38 :

E53 2 o« 8 - oo £ LL 7 3 : 2S § 5 ££ a E : co : : 2 38 oT & Boa

& OP —_ - 5 SE AS Ld ng It < x |= ONIN . a E ho A J f= NI (V=) 2) ~ 5 lN (FoR [NI IV) = 3 . - © ; ; Oo : Nn vg a £ NZS To i 6 |N jw 0 |N - 9 wv ® ~t oo — - ££ IN : | | 1\Q : QI ~~ N FI Loli Ly I Lp a § |v [Tv |v em = < | [VaR oT hemi SN LOW (Ve - 3 $F I~ = - E OT ed a «Q ~ |® AN (I~ joo (je Oo a £8 [x jm |Z |n < a NJ [OV I [oN I [oN I (NI Fo =~ 3 4 2 < |o wv Yo) ~~ ~ - 5 | s IY IN oo |= ees 9 a S|] [2 |v |~ |X XQ |= Nn en (8 |e — © a E oO joo |J— joo << - & : g : : ; ~ w= nN IS a 5 ac BL BL S = oo 00 r~ |= |~ ho - 3 — eg un r~ 0 | o> ~ © 0 jon foo ln un loo - — So < i — | [N= (SQ = |®) oS co lo o oc lo oo lo lo lo |e [3 —

S mn [on |x 0 0 te on ~ No f~ : ce Ae |= [N — N = | — Sn 8 O | |o |o o o o |e o oc lo lo |o

[5] > = ’ £ Ele |Z = ce ¥ £ ‘DS

Q vo |v |2 g = <r ] Ss |=

I< 3 J a. 3 = I) ° c Oo [= = = c ht [= W 3 00 [| = 8 = [=] edt Fr a £ 3 |[& 12 [|e |8 5 5 z [© £2 lz |&|5 [= |& 2 lo [gx |E I? 8% oio | 2 | |x | |® sc | |e |& Sc a |k c = |e [oo SENET] o0 [o] — —- [an} [e] < [34] 4 a 2 | |x = ls 9 |& x |g 2 |x | = ec © le [.€ [€ | © (oO cs |3 7 | © (© |e 2

Sj (3 | [9 [0 2B leo Ss lg > |3 |< a | |» < © [8 |&a = 2 < [0 | |& = ec |X o = E 3 —_ S |Z = [94

WY, ~N 2 ES RY = |= — = | 9 Te} 0 wn O |N ~

S 3 8 |= |N 3 00 QR | o 2 vw LIF Y= =v ~ o [© © (No lo 2 IR 7) E t~ | |= oS O fo —_ 1S (aT [= = J Pe NE = Io . ¥ 3 |v» 9 |= EA A co — IT 2 [M2 [un

S —_ oO |e <r ~ Oo |@ Oo 0 IQ I 9 |e 2 Z [0 ®» 32 |Z IN ~ |X 2 mo |¥¢ < |< |< [Zz [= = oD |< < |< {€ X |< (< se 2B

Lo ve 0 |n SN NEE oy a E « < © < | NOC Nm 0) . - =} 2 = ae ~~ fo ol Pa i Te BO VET gl = £ 0) «| < 12 2 @ in RN |< |=

Q 3 on BE 51 so 8 = & \0 o |o ENE I NE a a £ N=) a | < JN [0 NT [8 [2m [2 fen 2 ° _- = BS

IE oy — | Nn |e [© | [= t= Jon jo [9 |o ce To) aN No JN [To RNR INCU Foo [oN TR FOS FS CN I FN I TN 2 3 — J = on [aN I 8 0 [a] — ~ B ~ < lo Testi MINGIz =] ve) LU < |< © |v |» =m hn Ye a 3 . -J

I=) = E r ~~ |m o lov ja |r 0 |= = |= c Oo O° 0 (oS [VoRE (Vo NN [VSS (VOR [Vo FoR [Yo 2 [an BE 5]

LJ

2° FS CSUR Fo NO Ll [= NUN [SS a ES £2 N RN AREER RERKN EIS g © o oo oc lo ole lo lo lo lo lo lo

[3] . ° 0 lm ho fs js |= Joo

ER 2 FRI ZRRRERE MRR © oS I= [= oc lo Io lo io lo ole lo

[3] [y] 2 |E £12 |5 2 ¢ = IE z 28 Es g = |3 § |% £10 212 5205/5185 |g 5 12 8 | Zid Slo 28 818 lo |B |S g |= 9 |g 2 o [2 & |= 8 2

Lo |= = LE |€ oo [8B I c |e c |3 0 a 8 = = 18 jon Ix | Ix |X |= [2 |S

E |S w= [© c BB = | © |g nN [| loc i2 le £ lm 3 |e © 8 | [© |m [3 |3 |< [3 |«

EE |= = - OO | 1 < © | a «a [0 |€ |x ©» o£ |E «|S Iv] a | <a M 4 < = of g 5 = B 1 = 3 som — — 5 2 1 2 12 Nn IR mE Rl INQ I= = £ ~~ wn [oN BI [A oO on ’

Q J IY | VR a ae Bl [= fo

Oo 3 2 pt Ng o |! S |= 5 | [=

Sz 2 a |Z ~~ RERELDRBEOR < BS = |< Ala le jz a |< |< alc |D

<8 - = No No Eh Se Yn a & Noe jn hn STEERS ~ | oT (NI fe NE TON . = S g ? % . EE - nN RS > |N aN pn I hE a c aN nn | | via SNe | | |e a 3s aN be [3]

Ww © - = N Mm Mm | x [= NN Mm a E IN] < [oo wv oo nen I a WR [Vo [0 1) — 9 © - 5 — Nn =e a | SN «0 NN c SI [SSE ER LN Mm ~ al ~ aN |e a so — 1] - © - 5 Qe Ne x |™M Mm nN QI NN a £ < |v |N <r a |oo Mm ~ NT |» - 3 a4 © —- E Qe vm A oy [oe og joo | Ts c Tol (VaR AS SA <t |< |e aT (ao I aa Bl [a2 2 3 — 9 — eS un 0 [~~ [ZT {Ny on lo ~ [wo ~~ f= = - ANY [NN NM <Q |= — NNN

Q oo lo lo io oc (Oo oc lo Co lo leo |e [®] -— © eo aa J Sn [= NF Oo (wn on 0 IT [> |= - Ne | No Mm | N xT [= mM 5 @)] o lo lo jo <o lo o o jlo lo |e ’ (3) > 9 |S RS 2 Iz g 3 »n (WN S |N Is} 2 < oO = oi = — —

E12 c £15 | QB ce |alg « |= S |& |S 3 \O .— Ih) .— =] [5 Q ° £ [© a jo (oo |= | 9 (9 ls 5 |o [2 © [© [> an 7 |g (E88 |v J | |» = |€ JE IE [720 (= Pol |X ji fe 9} hl (© QL | I] ~ (00 [=]

CIN ale je | IN alo | 8 |e |o ce [2 |& 5 | 3 13 {'® [| < 5 S lo s [«€ 15 2 = > X < |3 El < |o 0 i= ray

Si pV 2 ne - NN

I. o ~ FoR a wv Oo

Le 9 a RIS p=, — a J («J pt w £2 N= (00 D |= 0 Isr 00 |X |X = — I~ a |Z oS |m <+ 2 o° Qe 3 . g = © |= [© ln (= [= SIF NI [= [= 4 Z AN NE I a ¥ 1M a |S ~ [@ |@ |= < | |<€ |X < |< < |X DC |< |Z 4

NC © - £ ~ NEE EE Al A : ~oNe ~N wv a E oo aa TI (VT fo NR EN py Ns [em ~ ws - 3 . & © o <r <r © - = Js 2 L S 2S | © — 1 [Sy ) ) . a § ha aVINN RLS [NO Po = at TI i A aa} - 3 os Ir)

LE i Dt S |= Mn f= \n 0 ~ E oN pS PEN NO | ~ ~ = 3 ¥ 2 - Oo vo [NY © Sy [00 |= N wn a £ < < jn [NN w << |N 00 ol = 3

AP — - kL : un <n : —- N ™~ oC a £ = aN ~ |= S ~ r~ o ~ - 9 4 © -_ & © Qo (n(n <. Mm ™M N — ao 1] (ag) [a2 TI [a TI [ao NN [4.0 (a2) on jen en on on = — eS wn 0 AN fe fe oN IIA I BC RN Nn = = < | N < <= [=e N AN

Q oS oS lo Ss = Cc lo |o <> to)

Oo —

Eg = 00 ~ | |= |= oN ~ [00 |e ~ ~ = < = NN [pn N NINN — AN

Ss Q o oo jo jo = o [lo | o o

[3] = Pe) 2 = “ % 2 |g ~ 5 N E S 2 |2 8 3) 2 12 |8 o |B |< E Ee 818 id = © oO. =f = e R= QQ. = | ry 5 Ec x [3 |o0 e = 2 |g —~ |9Q ©

Bs — ~ 3} oo Oo c Q £4 — = oO = 2 [8 |= jo [8 |2 |E ol BIS (88 = |a &

SZ le 8 wv lo £2 5 2 |= E = {9 © «© = y— o Q - 1 — ~~ by = = s [9] [3] 3 10 ec |= wn SS QL [i wn La [oR

So |S [2 @ £ |S | << 3s js |e pe = [8 |= 8 (& (38 e E 0 (© jo) 5 = a | en wv |= E 3 © | oO a {> on) Q [=I IS = = ¥ < A . x

Ss IB EN ~ R13 © » LL o ~ 2 lo (= Ne) = l= | =) 4 E N - 5 |Z lo 132) 8 [NI F-4 = < Z v4 ~ |= |[O |2 a S Ma — 5s) < x [Zz |< [< = < |< |< N <

Y

A = <n a < TS a — wn = fond ~ 9 oO © 0 .E —- = aN OAT DE el ST DE TE 2 = g

A & nN nn mn jn | Ss 3 FT © O° . [=] oo = = £8 2 _-— a 2 s&s = § — Q = © 5 a

E © “ £E S 2 «= x . - 5 A SAE PE SE A A LT A . 7 ® a © £ ~ |< YN [NN w 3 >)

Q 2 ¥ 5 © Em - 0 2 2 c 2 38 = 8 = 0° cc © OO © 3 & Ss E O° 3 = £ Jim [wn Jeo |v ~ ~ oo 2 2 2 a § © iN NN IN] O £ ZTE = = 9 E 8 ZS vw g = x 3 < [>= Bd .e = E ~ o + 9 Y = © - & No ho [oj [Ny wn 5 2 8 gg => a E a NON [Ns Joo ~ = a § 8 3 = LQ 5 Oo -C ZF ~ 2 a 8 hw oo uc es = TT § gg £ = a 2 o 0 ¥ = 8 = w - = 2 mS — | jn Ss = e ££

Nd | A~7 lAs S| (en Pa = r=] a £ ARMS [od J Xo I pl ag} °T 3 2 = o - gE 5 © 2 °C = 0° £2 2 a = & a 9 TT = a 29 - 8 Oy No eo nn IN © 3 Zz © 4 a E NNN NN IN NN @ a x Ss €

S wn E — © wR 2 = 5 < 3 BD 0 =

LV oo O 3 gE E gg 5 © eS wn © |N jv [oo (In oO wn ge © © 8 I

E = =a = I I le: S ¢c — = 8 gE © Co lo lo jo |e oc |o ‘2 5 © 3 = 3 $ 2 EE o 8 vo 5 © 2 x ao L ££ 3H ow — EE EE = e en So iv | |v jv | Jo @ — =

ER SRRRBRRERERER ® 2 z BT 2

EO ooo lo lo jo jlo jo ES g 2 2 = = © 3 833 CE 2 9 2 23 = - — = E oo 8 . oO & = e = D > cc © 8 = 3 = 3 2 3 © 2 T° 8 «< < < = 154 & 2, = gg © hal [= Se QO la ia [eo] = w= © a > 3] 2 I |® 2 |F |S |x © z 2 a = 2 eo |= |g [3 [2 [2 (S ce 2 oc YU BZ

NO te 5 = SQ « 32 “- c = OQ |B |g Is |= |= e = 3 Z|C |E |= |S |®|3 cE wz £5 9 2 8 |= Lo |= = : Pl RU IEINRB] 385% 238s =] p = a Bw 2 2 T° = —_ Io) = 2 ££ « © ££ =} 3] 2 Z » = © « @ ” ft EE 20 2B & = 8 = > 5 co r~ AN \O \O Wig Oo wv T

Le ow ~ = 8 | O me TT eg ez nL on <t I~ [oN] v= = = 17} [aa] o ~~ lon [© © O © ©

SE TRIFERPRBRE| £35223 T $2 SBRLERLRE| 2 ozé 22 es Z wy |= joo [@ | S 1M ~ © ww . ZZ & x |< (KX |< |< |Z |= |< ST 2g = 3 —_ Ek 8 © 2 3 > - - : 25832 << EO = uv £ LL ZF 3 2 § EE 3 E . E 5 2 9° 3 ev BS gM]

QE ~ i: SI ST DAC ET En 0 | |m a 5 = — ACTIN CN SV I hon hemi NO CE aS NI _— 9 vat - = ~ o ~N TN LN I [TR

TE &| 8 SIEBER ENES ol ht : a § [aV] — —_ |" LE PCT MVE SO PN <n - & °F © jo «a ~ TIN ee Te TT SEIN a § on od a NER N=TN oN [ NT LOU [YoU [FoR AR HE EN _-

LA < o lo

IN wn I OT Sc LST (I AS Lo Cle © a § 3] lo |» [oo |v [of HN DT (VN

Ld

[3] & 2 ~N = << on <t

NR Go| 5 3s ~ 2% —y

O

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Dy oJ —_—

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SE Sle E|= £ (3 (2 (82 | |&|a |Z |e 2 |u o Sx 2 |2 2 oo fo 8 |5 lo [8 [2 < |2 |8 lo | © IE |g |g 18 YB 2 |B vic (8

E Io = | 8 |¥ |[¥ | 2 |¥|8 IZ |g N |< 5 «© a |8a ale |e |e (2 |& |= |e lz [=EN(e 4 = = |< [>] S13 “© lols |° j= J = - FANE 2 |o & & 8 ~ [00 \O 0

SC ou bars a A I EN =I 0 |X nw LQ on TT == 9 |8 =< 2 Mm NIK 2 E = pe sq [J [SVN (SA TE [= DG Pon [= PA Lod IN 43 oS = < [© hin [20 12 |< [<8 foo f=

S 3 LN I —_ OS |S lo |S S = [25 es Z > 00 COR NT A = I EA PR PR ® |= pd >< < |€ [€ (€ |< |Z |Z |< 2 1<C (<< a 5 — |" | a cM on ~ [00 a © a lo el ~~ RUE IE ~ . I) bt 9 - © . a = o | [T © 7) x 12 lo Noli Vo) s Mm Re a} ~~ 0 I= f~ wi |oo a H pu — — — — [3] oo °

NJ © —- — oy [= |= 2 eo = ~ <r Ne) < | i RR [oV] a =) ~— onl 5} = 3 ~ a k AD SE Lh ~N : Mm (x (nn ~ NN a § =o |e |e IN wv ~ oN |e ~N — [3

S 8 a |= o x 5 ~ ! . < wn A= wn NO

J ASA a <t n= © jn a S ~N — ~ — _- 0 o © —- & ~ wn i] YS Oo NOY BER (5) [= oe} oo [oe} r~ ~ |~ [Wo On a 3 - 2 -— gE wn [> Joo — 0 on rs | o |= = mM <Q |e NN = <Q |e |N aN g Q o lo |e o f=) oc lo (jo oc lo (5) . —

S = oO ln |e <r <r — {m= | Ne) es — NN wn - NYY = £ Oo oc lo lo o o co lo lo o [¥) oh) = £ |. o m LE = 3 zz 8% ES (8 | ; |E

NOR 3 | = ‘Ds |= |0o |E © 9 [5 j= co bast «< O lt = |= = 7 I] a |S ~ 4 4 a |C =~ 3 [@) Q |= |O « > = lo 9g (2 ec © |, o£ |= I= | = & (5 = 5 |= [5 3 DoJ J = SE BI A= = SE e Q 2012 NIE 22 ££ |2 © |c | 12 < 5 |S alg = |¥ |v |= 5 2 |g Sloe le F 2° a |= ce © [= lo 52 a |& |g | 2 2 |e = oo < - 8S |la » jo | | © ww [|= © | 3 1 |S = 0 6 le |= 2 ££ 9 |B = v A le) oO Q a |. Lo 5] [ST fa » = oO bet bd = bot ' Q o Part Q. a py g MZ a |= gE |= = — = £5 wn ho ol

Le 9 AR SI = 3 wn a wn LD wn |= |O \O ~ DN — 2 g So lo |= % = N= ~ iT ed [a2 J LY A TE ho) \O [oN ~ [= oS —_ NO : g = o © |e oo oN a N o [© < |< |< = p= < 8 |< x |<

-« S a on on © a = < NSS ell woz ~” oN IN a £ hi 2 = IN < |= Ne) <

I [5] 4 & © a & ~~ wn 0 I< NIN ™~ ™ |Q a S [Ve 0 wn {en < IN 0 On : — [5] a4 ©

INE ~™ wn Qo 1% Mm |™M I. Ln A a a g ~N ol eT a} 0 | (N] ot IN |e buat [5] —

RL a & 0) Q © [N noe < NN © a g a\] ~t len ~ iN Ne) > I 2 BR 3s)

J

(®] = I Oo —

R= “2 ll od \ Ney |= a £ or - 5 |™ NS Nel ON bt [5] & © - 5 00 0 “a [™M a A ™~ ~ ie a £ 7) Ww nn wn [= <t < |= |< bt [3 —

Ag) ~ oN 0 a |m on a | oo = ~N QS N NS —- = ®} = et o oS © o o oo ©

[5] — . 2) a |= < |e fo 0 ho ho £2 3 = IN NR: Q Es |= g Q (=) SS jo oc © fa) Co lo lo

[3] @ on

[72] ] Q > £ TS © 2 on 2 5 8 1B BE. E. El. [8

Q e |S ww ® [3 |6 E [3 | Ol. 8 (E|5 © 3 |S £E ~|ojx wig [8 EE © |g |g

Nn = =n fe c (8 & le i [SI = e |= =) << & 2] oc oe |S Q. i a, 2 loo CE oO |x |= % IE © |g © [¥€ |8

N [= Re] [) > c |= 5 [=] wn = = ce |e

I £ c = |3 [3 © |3 |e & ws |Z |< 4 > 8 9 v O Fo D > a g |¥ “5 |B E | |Z i oe o = \O [3 I [= NI [=] 4 by 5 ~ 00 NTE PC (oN =) 2 © | r~ A j~

QO 3 = o ~ 2 RP = Ss 18 < Z = F oN om ~ na |R < = = |< oD << ES < |< |<

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S © ~~ No)

SE o 2 on ~ OS fm ) f= nn

E aN < os Nt SFI R (a) [=] — — oN - J -— = Ai A heal A eI Mm iN |e a E < |< < (|< |= < |= << |< |< - 3 —

Se wn — [oo + [on on 0 | m= 7) - A — |= — |= N < NY — | =

FS @) oc lo = J (=) =) o lo o |o o [3%] — e =m To) 0 |v nn |© nN 0 |(— {Oo [=m = on —- RE hl A BA <Q | AN = g @ cS oS Io lo lo oS |o co lo |e [*] ©

Q — = = [Up]

Ss 2. |, 28 Nie | — |e |3 2 le |e la

Q [~~]

Eis 21318 32 Ez [ESE 2iz|8 © 3 (2 |e &jo lg = lo |B lela = |e l2 (2 [2 |E 212 wit |S & 5 18 |E |@ © |g |& c |e 2 | oo | 8 | X |O °0 jx |x [© gle E212 9oi5 |g |g (> E [¥ = EE |= [E |&8

EE BU |3|« ££ [93a I5 ig 5 [3 |53 [oc = a |.= =} = [e) § 5 2 5 E o£ ht > ire) ro) >= od 5 9) - hs 9 < [oN] [SN]

Lg 9 NI 0 = fo » 0 (en) on oo Th on IN o © I iN 2 E = |» mm lo |7 leo ~ | S | I wo g 3 SIN SA VS IN 2 |= LS ER —_ & Zz x 2 a [© ho a O |= = lo | |o < |< nT EO FO x |< Z [D0 |<< [XX

<+ E Ml

S lr~ [nn To (Vo) ES |e won in < |en 2 3° . bt [%] “- © a So |™ wn Se REE ha NT . £ [nN (mM oN not a [Mm [f nL

Q I=) — - J - a 5 |= ™ © IM ~ Mm Mm |e nos £ lm (nN nm ~ aa ed oes a s - = © on a Eo | JRE Oo A NE I IN a § [NT MEI [OV IN < | Joo <r p— [3] v3

QS = o ~ — lo =] REE) cl a & |[~ Wn — | wn woe <n . 8 - QO os © - 8 | | nM Mm iM NN = - g [7 [mm (aa BI [ae [aT (ae! (a aT (a on {en e 3 i uv |= 0 = oR I — loo < > YN AN —- = [7 |N Mn

Qo o o o lo |e o Jo

Q

-— =m lo |v 0 |= On non — = ° Yn ~~ ~ = wn ~™ (NN

E Uo © o |e o |S c lo co lo o -— 1] j= = -— wg 8 2 FE S|: i z - | B a. ££ 5 Ln oS w £ |, i= © |g 5 & e le ¥ |g |g |& § _ |= : 2 lag loo 9 2 |53 29 Sola z2 12 lo 2 Et [9 ols lo € |g [8 2 £18 Zia lely 2 © |=

SRE EE © CIS [2 8 |[E |g |0 2 35 |=

XX [Tle PB {¥2 I= a SE 5s |2 2 |= & 2 io ec 0 J|lz2 2 |e j= © Ss & le |s | wall

Slr EE 21° is = iz wv [3 |3 jo © 5 2 2 E S | S$ Qo. I>] = 3 8 a 2 5 = 3 . (=% o 2 id Oo.

R

E wu |O r~ T | S ho eS 3 |v IN 2 | © I NS) ® |S nw LL [90 I~ SN 0 |. on |N jo <Q |T 2 OE [= | << (~ =I Pa a [im ™M [on @ j= SIN 3 |= =] i wn | |m Qo 8 = [<M Po Pere) — oO O oO — [4 [eo] < Z |X |A a |= [|= © [M1 fn |= < |< a |< < |Z 2 |< |< < |<

¥ © ~

SE EN N 5 “ho Non |e an E ole < ha a Ils] a a BE [SV IR . 2) _— 9

A) a = < [© o | —~ | o ov [© Iwo <3 ~ | a (2 NEN a NE EE GN

Q 3

J

I)

N 5 < |= — —~ 2 © [© © lo |o |N = NER Ie) a | ~ | o NN 2 3 0

Ar) ~ 5 |= “ NN — |= © joo I~ jn = en IN NS Oi OY [Mm | lw

Qe lan BEN 5) ~ ©

SE © rs ny S |= ~ loo nfs = < | I) NEN Moi am 2 o _ o © - = =a =] CO a |S So |e EL LN I Ae ec on ton [aN] al AN) [aN IE [aN] al IN iN {Nl a _- — . e un mn — O (a \O |e Ov lo Joon Im

Es = VM N |= r~ |™ NM Nm g © oo o oo co |e Oo jo jo |o

[3] . []

E =m 00 [r= N oN r~ loo 0 Mm feo |r = = OM = on ™ |v AEE NE NS ns! sg © co lo o o cS lo co lo lo jo

[3]

[0] Ty 2 0

L c Q 32 £ Moje =

Q —t

Sines 28 «B12 2 ww |81]8 © cic |®

L 2 {ee 9 |&8 « |[E | 2 co (2 2 |5 ws LE |& a l= €£ |2 0 2 = |o |o g I= |B |1€ & |= a 12 ¥ « |E l= 5 IE |E |E

SE lx |e 90/2 EI2I|E ZL [E15 8 |¥ |% |3

E89 ET 2a @|EI& 2 2 |0|z2 als (2 = |° |3 Iz @ 0 |® ja 2 CE -ER ~ 5} Q =} Oo |£ E Cc |x on =] = = Ge -— | 20 o0 a = = so fo oe) 00

Zz 2 N= o 2 lo A lo EEE

SS E ~ ls ~ NEN = [IX Nx [Bd |B 3 = - 8 = ~ [0 Je] Soe je

SZ ao 3 «oq 2 2 cll eT Ne 4 < |< oo) = = |» a |< |< |<

QL fo = <r —t «un = < Ss ° $ 6 = eo = a 5 © hn jon TT EE vw = g = < [3 [en = 3 3» T © —_ a 3 8a = £ oo 3 -— © . = 9 a © 5 = 9 cn = Q © —-— - uv 5 8 cs |Q o— = ES oo a Q 3 Ia <3 £ 9 «& x -Q en I [Ve = = = Q [a IEEw oO | Re’ 2] = 2 $s ZZ 2 £ ww _ a 8 oN g 5 O E T Ss © = Qo = 2 ¢ 32 § a EI 2 8 8 © © £ |o = SS ES 3 2 ¢g [= ¥ 5 [3 —_— oa 2 [9 3 CO 3 a kB © (X= 2 + = g a § aN es Q £ 35 = > 3 = 9 £ 3 5 = = Q& Em 2 2 oo § < a ££ [x ve E oo n o - ® - 2 = Oo = © (3) = = a §| N° rl TR % B - 2a Za 3 aN 5 : vo E c

S © T 8&2 = gs

Q& E SQ jo |w gE SS £ 2 a § aE A © 8.8 = o - = 3 £35’ zo N En

[3] 3 2 2 2% £3 a § © eo 9 © > gL vv 2 =- 9

Lan = <e ©“ 95 Zz LL qu = [SNE [SION a fA s © —

Qe 3S TT A g = - 3 nw Hn 9 2 © fla [ed —_— 3 NN =] << << Tv OT = c |= ge EE 5 2 8 2 ¢ |— — = vO T - 0 un 0 Mm |o ce © ££ 8

Ed a a 2 & 332 5 ow |=

S ¢ 2 E E o EE» ge ¢ 9g 2 gE Ve * 3 EE A- N , £2 Le Te 2 2 TS “ |m g 0 cS lo |o ES g £ 2 = EE = 2 o 2 2 - 8 = g EQ c 5 8 ¥ © . E S 2 © 8 = > = ec = ® = 5 vu jo c 9 = ,, YY cc ‘DS — 2 € 9 § 2 3 3 $ £2 22% os — | -— ~~ ¥} = |& |g |B « = 8 § & = g 2 215 3 |e cs 2 235 8 3 3

BERENS Ett: PP oo |[€ |S SS eZ Zo» ou £ « = = “15 1° |Z S g ® 5g O 2

J uu Qo Q a — Wz £ £ £5 > iv; TIC B= BE <= TET 2m 2 5 = & = § Z > 3 Ss 2

[72] e 5 wy <Q +t oo + & gg = [TIE Na [a 1a 2 r~ 0 © own yg E> 2 g Z |8 2 EB —~ Iq |= ¥ «w & 7 To < Oo 5 5 8 In |B 2° un = 8 a - : < S = 2 8 . = 8 < >< FF Fg —_ 5 8 © 2 3 : oO © = DD < ) - vu Q < = - © <S OL > gE SS pg Z 2 § 2 8 a E

= £ oy No ~N So < ™~ b= IN — | 00 ~ jen — o = I Ia! EY —_— — fem - = ny 1 :

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EN S — Nd a = r~ A on ~ : oN (= = ~ I |S ~ wo IN ~ (=) I=) <r —

Pt [¥] © © Ne)

N= wn IN ~ oy |oQ <, ~ = on wn (YN oA no ~ Ne) a =) — a>)

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SQ £5 on Te ~ — lo Ne) ! ~Nuad . wy - . . . . a) c oJ IN <r aN (nn ~ ot a o —

OD

— [= wy oN wy [ap] — — [vo] [oN

Eg = rp |= IN NS 5) g @] — [<= o o | o <Q

SA) IN ~ [~ ~ ~ | wn = 2» on S| — — |™ — gE Q I= oS (lo S oc |e oS ° ° [2] - ° < . pe Q ~ | Eg c [€ = [X cE 8 5 (® @ on c |p & nw wn O00 ™N = 3S wo Q I'S £ @ oO [¥ T= © 21s 8 FF oo |o [3 2 cE |8 = 5 = 2 cE o |2 |Z o c |e | ou €|T x

LQ © |g ~ 2 0 S |» N

Oo £ Oo ¢ © = |© py Lv |X S c 0 & |S = = 9 Ww [€ |g 8 ES © = S015 2M S 18 = 9 |=

Lc 3) > [+03 [SIN | a oo “— Q 0 9] 3 > Q —_— 3+] c 2 =

So - <tr [Vol (Vol 0) 2 9 3 Sw» IN S$ ve) [=] ht w= ~N ~ |e Py ve $ E o = 8 2 32 S = 9 35 S18 = N= 3 — — < Z = = |O © |= =

Zz < |< = < |< Zz <

= °

S 8 % 5 o ve ° . & E ~ 0m SS — [5] 3 a

EI — ™~ jo0 0

Qa 5 ~ < (™ ~ 3 0 a 5 0 , a H a [a\) [oN - 9 ~ [=] ~ 5 Lg S ~” a 5 ™~ QIN <t ( , P=

S 5 © |v < a § 9 Tol ON IN - —— enn r~ Oem 3] s = “ NN 0 2 |) ~ Cc | ~

So od pel o o ln = . — [I 51 1 [] [7] — 00 = 2 © Po] :

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Oo ¢ = od id 2 EE |¥1|5 © £ 2 &£ T

S [3] = = = e a 5 c 3 oO a $= = Ss ™ Q = = [Vo] es by ~ 00 jr~ 2 ‘a a on 0 |r~ ~<t @ [a}] wn [a I — $g E = — |S b=)

[3] = QO oo) o0

[3] ya < QO < i he I es pS < < |< Z oe ir) - £ 0 ~ ~N N a f=] \O \O \O \O =) — 3}

J a EE x — I So << S a £ ~ ~|n ~ ~ ~

LL] & .e r=)

Q for Te) fT A uw S bt . . . . = ~ TI IAG <r wn 2 3 ~

[5] © ©

NE wn wv S wn nn a £ Ve) < (un wi oN Ve) — (¥) oe ° 8 E r~ mn mM o ™ : ) ) ) > a £ ~ Tee) fo ae) ™m — [¥} e ©

N= N nr — AE o~ a S ~ «rs r~ ~ IN —

[5] — © 0 > fo on £2 = — |e ie wn

S Oo ol oo |© o o

Q

= ~N ” XO

Q Ss = S 15} oo — ! — : on

[72] Py [3] = o Q << <Q -— o — _ [5 I > LU wv IE |O °o 9 - @ 2 ®E 28 FQ «lz Ela g|8 8 3 2 5 Ss = 2 sc C2 Z| 22 BIE 53 9 < = 2 Oo IF =} [= [F) x |x ° 3 © cESSR HF EREERE 8 E = oc Lo = @© = = a oO = 5 = ZS = > 3 cs | [o,3 — \O [o's] wn

Lv in wn r~ ~ ve) wn Oo on ~~ 2 wn on a ¢ E = 3 |= | § x bt o wv | <Q << =)

So 3 = |= S So

Pe [4 o'] |= 4 a , < 2 |< < < <

TRC

-~ E — ™~ © |e : cs \O wy uy [Va a 3 : bt Q

S © ~ a = ~M ~ ~ wv NO a 5 O [aV} [aN] [aV] ~™ ~ bt [3 3 & = ~ o 0 a IN [= on < << < [r~ 2 pt >) oS ] E ~ S r ~ |n = un <r un <t on

Qe sg - 0

NI

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ED RA S — ” NE g @) — ~ (Va) o mo o e [ag] — -— ~~ o0 — — = = Qo wn — on g Q — i) ~r oc ao

Oo

Ct [od — © 0 ov = ‘© EN BT © — £ E g € vw, 2 9 5 c 18 = 9 © LQ 5 Sd 2 ww | © xX o lo E& |3 a wn 2 = 8B a a 2 218 5 LL 3 & 3 | 5 8 |E

S T OO OO =~ |g 3 8 ln. TB 9 la 2 {cE ES SL = = Eg wu 9 Mg g OQ IT mm & lo o E 3 5 wn > TT 8 5 ZL aw Zz «= |8 on = =O A SSE [V) a ‘N ££ [o8 — Q hb] [3 aN e pe ~ a > [@)3

LS we 2 in 2 & E S te ~ = = 8 £ 2 S|] 2

S 3 S o <x <Q oo < Zz el fr, ~ m PRIS a < = < Zz |=

= ~ E © [© ~ TM ~ je |= © [- [Yo RN (Vo [Tp] [So [Va] wv jn | <T wT 2 3 - = & E ~N “ Nn a [o |Z = a € IN 5 wo |e an [2 ~f - 8

A

Q E T | I © |N oN (2 2 a E NBII] 3 < | NSN Vo 5) «©

ES) - 9 © 2

NE N Ig |e — |r — : a £ nen un oN [a VI [QV IE oo N= o am lee ©

NE 2 |n = —- |x ~ leo (2 SH c S im IN 0 |m 0 (| < |Z a 3 — -Q

R=)

SE |= Q n joy Toy |T 0 = < | a aed Re (IS eo loo

Q s&s -

LJ

Se wn o jun <r fo wn fe |e no £ > Nn ae none = |= 5s © oo oS oI ole jo = [=]

S

ES om 0 je on nv jun o |v vo © |in << EE DA =, Nn Se mM ~ oN sg S |o = - lo — lo |e oS lo 3] [= — [J 50 “© — |= =e 1A EP EA cle IE _ 4 ® © |g 0 £8 222 =z 22 2 ¢ = ec |@ oc I© © |= = | jo © lo lols & s |= 3] Tle =SlE|€ 82 [SIE BEE |8 © = |» ] So |2 Z1< EE alg 5 |¥ |X |g 3 2 |< o Cc [= als SX [7 ia ajc |c a Q

I~ 3 = =" 10 13 3 |3 jz & © |x

Q. 15 A © © iy & el oa a |O a =

E o 00 — loo [m © 9 R® Ia O OR |o i -| 0 | ~N 0 ~~ |= [60 rod ¢ E 0 [= r~ o I= S (vv [Yo no

S 3 0 | Ia) no SL un on™

Q oS |S —_ MN i == (= < Z EN — O |r RV [oa S$ < |< < xX 1a < |<€ |<€ vn IX

WO (33/048383 PCT/CA02/91830 - . - E 0 no - oS ov

A E ~ < |< ~ < |= = 3 .e ir) oe NOE “ SM a E N aes 3 vo 4 = 3 3 £ on < <x

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Claims (24)

1. 24-11-2003 CA0201830 « WHAT IS CLAIMED IS: IY 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 polynucieotide or polynucleotides with one or more sepsis or inflammatory inducing agents, contacting the polynucleotide or polynucleotides witha 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. J
2. 2. The method of claim 1, wherein the sepsis or inflamniatory inducing agent is LPS, LTA or CpG DNA, bacterial components or whole cells, or related agents. oo
3. 3. The method of claim 1, wherein the determining a change in expression comprises determining the level of expression of the polynucleotide prior to and following contacting with the sepsis or inflammatory inducing agent.
4. 4. A polynucleotide or polynucleotide pattern identified by the method of claim 1.
5. 5. A polynucleotide of claim 4, wherein the polynucleotide encodes a polypeptide involved in an inflammatory or septic response. : - .
6. 6. + Amethod 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 of expression affects the inflammatory or septic response. :
7. 7. The method of claim 6, wherein the effect to the inflammatory or septic response is inhibition of the inflammatory or septic response. —
8. 8. An agent identified by the method of claim 6.
9. 9. The agent of claim 8, wherein the agent is a peptide, peptidomimetic, chemical compound, nucleic acid molecule or 2 polypeptide.
10. 10. The agent of claim 8, wherein the peptide is selected from SEQ ID NO:4-54. Empfangsaeit 24.Nov. 20:41 191 AMENDED SHEET
11. 24-11-2003 CA0201830 . 11. A method of identifying a pattem 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 patter 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. 12. A method of identifying a compound that inhibits an inflammatory or septic response, 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 a o polynucleotide expression similar to a pattem obtained in thie method of claim 11 with a cationic peptide that inhibits an inflammatory or septic response.
13. 13. A compound identified by the method of claim 12,
14. 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. 15. The method of claim 14, wherein the agent does not stimulate a septic reaction.
16. 16. The method of claim 14, wherein the agent inhibits the inflammatory or septic response.
17. 17. The method of claim 14, wherein the agent blocks the inflammatory or septic response.
18. 18. Tbe method as in claim 16 or 17, wherein the agent increases the expression of a polynucleotide encoding an anti-inflammatory protein. Eppfangszeit 24.Nov. 20:41 192 AMENDED SHEET
19. 24-11-2003 CA0201830 . 19. The method of claim 18, wherein the anti-inflammatory protein is selected from a subset that includes IL-1 R antagonist homolog 1 (A1167887), IL-10 R beta (AA486393), IL- ~ 10Ralpha (000672), TNF Receptor member 1B (AAL 50416), TNF receptor member 5 (98636), TNF receptor member 110 (AA194983), IK cytokine down-regulator of HLA IL (R39227), TGFB inducible early growth response 2 (A1473938), CD2 (AA927710), glucocorticoid-related polynucleotides (AK000892), or 11-10 (M5762720.
20. 20. The method of claim 18, wherein the agent inhibits the expression of TNF-alpha.
21. 51. The method of claim 18, wherein the agent inhibits the expression of interleukins.
22. 72. The method of claim 21, wherein the interleukin is IL-8.
23. 53. The method of claim 16, wherein the agent is a peptide. I
24. 24. The method of claim 93, wherein the peptide is selected from SEQ ID NO:4-54.

    95. 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 jnnate 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 inimumnity; ’ detecting a pattern of polynucleotide expression for cells contacted in the presence of a test compound, wherein a pattern with the test compound that js similar to the pattern observed in the presence of the cationic peptide, is indicative of a compound that enhances

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

    29. The method of claim 27, wherein the compound does not stimulate a septic reaction. Enpfangszeit 24.Nov. 20:41 193 AMENDED SHEET }

    24-11-2003 CA0201830 . 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 (D8745 1), serine/threonine protein kinase MASK (AB040057), KIAA0912 protein (AB020719), KIAA0239 protein (D87076), RAF], GTPase activating protein 1 (M64788), FEM-1-like death receptor binding protein (AB007856), cathepsin S (M90696), hypothetical protein FLI20308 (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 CREBP3, integrin alpha M (J 03925), - Rho-associated kinase 2 (NM_004850), PTDO17 protein (AL050361) unknown genes : (AK001143, AK034348, AL049250, AL16199, AL031983), retinoic acid receptor Co (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 (729575), interferon garama receptor 2 (U05875), cytokine receptor like factor 1 (AF059293), class 1 cytokine receptor (AF 053004), coagulation factor I (thrombin) receptor-like 2 (U9297 1), leukemia inhibitory factor receptor WM_002310), interferon gamma receptor 1 (AL050337) or any combination thereof.

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

    33. The method of claim 27, wherein the expression pattem 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 conprising: i 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 : Eppfangszeit 24.Nov. 20:41 194 AMENDED SHEET

    24-11-2003 CA0201830 . 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 36, wherein 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, CCRS5, 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 2 polypeptide capable of suppressing inflammatory or septic responses and ii) a polynucleotide or polynucleotides that encode a polypeptide involved in immate brumwmity, 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 & mammalian subject from a nucleic acid sample of the subject comprising identifying in the nucleic acid sample a polynucleotide - expression pattern exemplified by an mcrease in polynucleotide expression of at least 2 polynucleotides in Table 55 as compared to a pon-infected subject. ‘

    43. A method for infewing 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. : . Enpfangszeit 24.Nov. 20:41 195 AMENDED SHEET

    24-11-2003 Co CA0201830 . 44, Amethod 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 2 non-infected subject. : no © 45. The method of any of claims 42, 43 or 44, wherein the state of infectionisduetoa bacteria, virus, fungus or parasitic agent. : oo

    46. The method of any of claims 42, 43 or 44, wherein the state of infection is due 10 2 Gram positive or Gram negative bacteria. oo \

    47. A polynucleotide expression pattern of a subject having a state of infection identified by the method of claim 43. 3 a. Cee vn

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

    49. A method of identifying a cationic peptide that is am 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 XXX IKP KAP XXX (SEQ TD NO: 4), wherein X; is one or two of R, Lor K, X; is one of C, Sor A, X3isoneof R or P, Xs isons of Aor V and Xsis one of Vor W. CL Ce

    51. The cationic peptide of claim 50, wherein the peptide is selected from the group consisting of: LLCRIVPVEPWCK (SEQ ID NO: 3), LRCPIAPVIPVCKK (SEQ ID NO: 6), KSRIVPAIPVSLL (SEQ ID NO: 7), KKSPIAPAIPWSR (SEQ ID NO: 8), RRARIVPAIPVARR (SEQ ID NO: 9) and LSRIAPATPWAKL (SEQ ID NO: 10).

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

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

    54. Anisolated cationic peptide comprising the general formula X, LX; XK X Xo Xs XP XX (SEQ ID NO: 11), wherein X is one or wo of D, E, S,TorN, X2 is one or two of P, Gor D, Xzisone of G, AV, L,IorY,Xsisone of R, KorH and Xs js one of S, T, C,MorR. Eppfansszeit 24-Nov. 20:41 196 AMENDED SHEET

    24+11-20083 CA0201830 _ 55. The cationic peptide of claim 54, wherein the peptide is selected from the group + consisting of: DILPAKRGSAPGST (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 54, wherein the peptide has anti-inflammatory activity. :

    57. The peptide of claim 34, wherein the peptide has anti-sepsis activity.

    58. An isolated cationic peptide comprising the general formula XXX Xs WX WX XK (SEQ ID NO: 18), wherein X is one to four chosen from A, P or R, Xj is one or two aromatic amino acids (F, Y and W), Xa is one of P or K, Xa is one, two or none chosen from } : we A, P,Y or Wand Xs is one to three chosen romRorP. ... Tans -

    59. The cationic peptide of claim 58, wherein the peptide is selected from the group consisting of: RPRYPWWPWWPYRPRXK (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 58, wherein the peptide has anti-inflammatory activity.

    61. The peptide of claim 58, wherein the peptide has anti-sepsis activity. : oe An isolated cationic peptide comprising the general formula . oT XXX XX: VX XR GX Xa Xa Xa Xa Xy (SEQ ID NO: 25) wherein X, is one or two of RorK, X, is a polar or charged amino acid (S, T,M,N,Q,D, E,K, Rand H), X;3 isC,S,M,DorA and XaisF, LL, V,MorR. '

    63. The cationic peptide of claim 62, wherein the peptide is selected from the group consisting of: RRMCIKVCVRGVCRRKCRK (SEQ ID NO: 26), KRSCFKVSMRGVSRRRCK (SEQ ID NO: 27), KKDAIKKVDIRGMDMRRAR (SEQID NO: 28), REMVKVDVRGIMIRKDRR (SEQ P NO: 29), KQCVKVAMRGMALRRCK (SEQ ID NO: 30) and REEARRVAIRORDIR (SEQ ID NO: 31). ;

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

    65. The peptide of claim 62, wherein the ii has anti-sepsis activity. : Eapfangszeit 24.Nov. 20:41 197 : AMENDED SHEET

    24-11-2003 . | : CA0201830 . 66. Anisolated cationic peptide comprising the general formula X XXX Xa VX SUR GX XXX Xa Xi (SEQ ID NO: 32), wherein X, is one or two of R or K, X; is a polar or charged amino acid (S, T, M,N, Q, D, E,K, Rand H), X; is one of C, 8, .. M,DorA Xsis one of F, I, V,M or R and Xs isoneof AL, S,M,DorR.

    67. The cationic peptide of claim 66, wherein the peptide is selected from the group consisting of: RTCVKRVAMRGIRKRCR (SEQ ID NO: 33), KK OMMKRVDVRGISVKRKR (SEQ ID NO: 34), KESIKVIIRGMMVRMKK (SEQID NO: 35), RRDCRRVMVRGIDIKAK (SEQID NO: 36), KRTAIKKVSRRGMSVKARR . (SEQ ID NO: 37) and RHCIRRVSMRGIIMRRCK (SEQ ID NO: 38). . \

    68. The peptide of claim 66, wherein the peptide has anti-inflammatory activity.

    69. The peptide of claim 66, wherein the peptide has anti-sepsis activity. -

    70. Anisolated cationic peptide comprisingjthe general formula KX KX FX KMLMX,ALKKX 3 (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 PFS, T,Kand H. .

    71. The cationic peptide of claim 70, wherein the peptide is selected from the group consisting of: KCKLFKKMIMLALKKVLTTGLPALKLTK (SEQ ID NO: 40), KSKSFLKMLMKALKKVLTTGLPALIS (SEQ ID NO: 41), © KTKKFAKMLMMALKKVVSTAKPLAILS (SEQ ID NO: 42), SR i KMKSFARMLMLALKKVLKVLTTALTLKAGLPS (SEQ ID NO: 43), KNKAFAKMLMKALKKVTTAAKPLTG (SEQ ID NO: 44) and oo KQKLFAKMLMSALKKKTLVTTPLAGK (SEQ ID NO: 45). | © :

    72. The peptide of claim 70, wherein the peptide has anti-inflammatory activity. : 43. The peptide of claim 70, wherein the peptide has anti-sepsis activity. .

    24. An isolated cationic peptide comprising the general formula oo KWKXX) Xa XXX XoXo Xi Xa XoXo IFHT ALKPISS (SEQ ID NO: 46), wherein X, 15 a hydrophobic amino acid and X, is a hydrophilic amino acid. + So Empfangszeit 24.Nov. 20:41 198 AMENDED SHEET :

    24-11-2003 : CA0201830 [] : " . 75. The cationic peptide of claim 74, wherein the peptide is selected from the group consisting of: KWKSFLRTFKSPVRTIFHTALKPISS (SEQ ID NO: 47), oo : KWKSYAHTIMSPVRLIFHTALKPISS (SEQ ID NO: 48), KWKRGAHRFMKFLSTIFHTALKPISS (SEQ ID NO: 49), Co KWKKWAHSPRKVLTRIFHTALKPISS (SEQ ID NO: 50), KWKSLVMMFKKPARRIFHTALKPISS (SEQ ID NO: 51) and Co KWKHALMKAEMLWHMIFHTALKPISS (SEQ ID NO: 52).

    76. The peptide of claim 74, wherein the peptide has anti-inflammatory activity. :

    77. The peptide of claim 74, wherein the peptide has anti-sepsis activity.

    78. . Anisolated cationic peptide comprising the sequence. Co : KWKSFLRTFKSPVRTVFHTALKPISS (SEQ ID NO: 53).

    79. Anisolated cationic peptide coraprising the sequence KWKSYAHTIMSPVRLVFHTALKPISS (SEQ ID NO: 54). . 80. The method of claim 14, wherein the agent is a Zinc finger protein (AF061261); Cell cycle gene (S70622); IL-10 Receptor U00672); Transferase (AF038664); Homeobox protein . (AC004774); Forkhead protein (AF 042832); Unknown (AL096803); KIAA0284 Protein (AB006622); Hypothetical Protein (AL022393); Receptor (AF112461); Hypothetical Protein (AK002104); Protein (AL050261); Polypeptide (AF105424); SPR1 protein (AB031480); : i: Dehydrogenase (017793); Transferase (M63509); and Peroxisome factor (ABO13818). :

    81. The polynucleotide expression pattern of a subject having a state of infection identified by claim 42, wherein the genes upregulated are Accession number D87451 -ring finger protein 10; Accession number AL049975, Unknown; Accession number U39067, eukaryotic translation initiation factor 3 subunit 2; Accession number AK000942, Unknown; Accession nuraber AB040057, serine/threonine protein kinase MASK; Accession number AB020719, KIAA0912 protein; Accession number AB007856, FEM-1 -like dedth receptor binding protein; Accession number AL137376, Unknown; Accession number AL137730, Unknown; Accession number M90696, cathepsin S; Accession number AK001 143, Unknown; Accession number AF038406, NADH dehydrogenase; Accession number : AK000315, hypothetical protein FLJ20308; Accession number M54913, pim-1 oncogene; Accession number D29011, proteasome subunit beta type 5; Accession number AL034348, Enpfangszeit 24.Nov. 20:4] 199 oo AMENDED SHEET

    24-11-2003 CA0201830 «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), PTDO17 protein (AL050361) unknown genes

    (AK.001143, AK034348, AL049250, AL16199, AL031983), retinoic acid receptor (X06614), G protein-coupled receptors (Z94155, X81852, U52219, U22491, AF015257, U66579) chemokine (C-C motif) receptor 7 (L31584), tumor necrosis factor receptor superfamily member 17 (229575), interferon gamma receptor 2 (U05875), cytokine receptor- like factor 1 (AF059293), class I cytokine receptor (AF 053004), coagulation factor II (thrombin) receptor-like 2 (092971), leukemia inhibitory factor receptor (NM_002310), interferon gamma receptor 1 (AL050337), or any combination thercof. i} 82. The method of claim 32, wherein the chemokines include CXCR4, CXCR1, CXCR2, . : CCR2, CCR4, CCRS, 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 include TGFp inducible early growth response 2 (A1473938), 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 (807920), MAP kinase kinase 5 (W69649), MAP kinase 7 (139192), MAP kinase 12 (AI936909), MAP kinase-activated protein kinase 3 (W68281), or MAP kinase kinase 1 (1.11284). Co

    86. An agent of claim 25, wherein the agent decreases proteasome subunit expression.

    87. An agent of claim 86, wherein the proteasome subunit includes polynucleotides with accession numbers D11094, 1.02426, D00763, AB009398, AF054185, M34079, M34079, or AI031177. :

    88. Anisolated cationic peptide that reduces polynucleotide expression of SDF-1 receptor. Empfangszeit 24.Nov. 20:41 200 AMENDED SHEET