HK1098969A - Methods of stimulating innate immunity using cationic peptides - Google Patents

Abstract

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

Description

Method for stimulating innate immunity with cationic peptides

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Information on related applications

[0001] This application claims priority from U.S. patent application serial No. 10/308,905 filed on 12/2/2002 under 35USC 120, and U.S. patent application serial No. 60/336,632 filed on 12/3/2001 under 35USC 119(e), both of which are incorporated herein by reference in their entirety.

Technical Field

[0002] The present invention relates generally to peptides, and in particular to peptides effective as therapeutic agents and for use in drug discovery involving pathological processes resulting from microbial infection and used to modulate innate immunity or anti-inflammatory activity.

Background

[0003] Infectious diseases are the leading cause of death worldwide. According to the 1999 world health organization survey, over 1 thousand 3 million people die each year from infectious diseases. Infectious diseases are the third leading cause of death in north america, accounting for 20% of deaths each year and increasing by 50% since 1980. The success of many medical and surgical treatments also depends on the control of infectious diseases. The discovery and application of antibiotics is one of the major achievements of modern medicine. Without antibiotics, physicians would be unable to perform complicated surgery, chemotherapy, or most medical interventions such as catheterization.

[0004] Currently worldwide antibiotic sales are $ 260 billion. However, the excessive use and sometimes inappropriate use of antibiotics has led to the development of new antibiotic-resistant bacterial strains. Antibiotic resistance (antibiotic resistance) has become part of the medical phenomenon. Bacteria such as vancomycin-resistant enterococci, VRE and methicillin-resistant staphylococcus aureus (Staphlococcus aureus) and MRSA are strains that cannot be combated with antibiotics, and generally, patients infected with these bacteria die. Antibiotic discovery has proven to be one of the most difficult areas of new drug development, with many large pharmaceutical companies curtailing or completely stopping their antibiotic development programs. However, with the dramatic increase in antibiotic resistance, including the emergence of untreatable infections, there is a clear unmet medical need for new types of antimicrobial therapies (anti-microbial therapeutics) and agents that act on innate immunity would be one such class.

[0005] The innate immune system (lnnate immune system) is a very potent and evolved universal defense system. Components of innate immunity are usually present at low levels and, when stimulated, are activated very rapidly. The stimulus may include the interaction of bacterial signaling molecules with pattern recognition receptors (pattern recognizers) on the surface of body cells or other mechanisms of disease. Every day, humans are exposed to tens of thousands of potentially pathogenic microorganisms through the food and water we ingest, the air we breathe, and the surfaces we contact, pets, and humans. The innate immune system functions to prevent these pathogens from causing disease. The innate immune system differs from the so-called adaptive immunity (which includes antibody and antigen-specific B and T lymphocytes) in that the innate immune system is always present, rapidly produces an effect, and is relatively non-specific to any particular pathogen. The adaptive immune system (adaptive immune system) requires the amplification of specific recognition elements (specific recognition elements) and thus takes days to weeks to respond. Even if adaptive immunity is pre-stimulated with a vaccine, it may take three days or more to respond to the pathogen, however, innate immunity can be acquired immediately or quickly (hours). Innate immunity is involved in a variety of effector (effector) functions, including phagocytes, complement, etc., but is generally poorly understood. In general, many innate immune responses are "triggered" by the binding of microbial signaling molecules to pattern recognition receptors on the surface of host cells called Toll-like receptors. In the inflammatory response (inflammation response), many of these effector functions are grouped together. However, an overly severe inflammatory response can lead to adverse reactions to the body, and in extreme cases sepsis and potential death can occur.

[0006] Structural components (structural components) released from infectious agents during infection cause an inflammatory response that, when uninhibited, can lead to a potentially fatal condition, sepsis (sepsis). Sepsis occurs in approximately 780,000 patients in north america each year. Sepsis may occur as a result of an infectious disease acquired in the community, such as pneumonia, or may be a complication of trauma, cancer or major surgical treatment. When the body is completely unable to control the inflammatory response, severe sepsis can occur and the body organs begin to fail. In the united states, as many as 120,000 deaths occur annually due to sepsis. Sepsis may also involve pathogenic microorganisms or toxins in the blood (e.g., sepsis), which are a major cause of human death. Gram-negative bacteria are the most prevalent organisms associated with such diseases. However, gram-positive bacteria are also increasingly causing infections. Both gram-negative and gram-positive bacteria and their components can cause sepsis.

[0007] The presence of microbial components induces the release of pro-inflammatory cytokines (pro-inflammatory cytokines) of which tumor necrosis factor alpha (TNF-alpha) is of utmost importance. TNF-alpha and other proinflammatory cytokines can in turn cause the release of other proinflammatory mediators (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 known as endotoxin). Endotoxin in blood, known as endotoxemia, is primarily from bacterial infection and can be released during antibiotic therapy. Gram-positive sepsis can be caused by the release of bacterial cell wall components such as lipoteichoic acid (LTA), Peptidoglycan (PG), rhamnose-glucose polymers produced by streptococcus (streptococcus), or capsular polysaccharides produced by staphylococcus (staphyloccci). It has also been shown that septic states, including the production of TNF- α, can be induced by bacterial or other non-mammalian DNA, which typically contains unmethylated cytosine-guanosine dimers (CpG DNA), unlike mammalian DNA. Mammalian DNA contains CpG dinucleotides much less frequently, and they are often in methylated form. In addition to their natural release during bacterial infection, antibiotic treatment may also cause the release of the bacterial cell wall components LPS and LTA, and possibly bacterial DNA. This in turn may prevent recovery from infection, or even cause sepsis.

[0008] Although the main role of cationic peptides is recognized in the scientific and patent literature as antimicrobial effects, cationic peptides are increasingly being considered as a form of defense against infections (Hancock, R.E.W., and R.Lehrer.1998.cationic peptides: a new source of antibiotics. trends in Biotechnology 16: 82-88.). Cationic peptides with antimicrobial activity have been isolated from a wide variety of microorganisms. 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 and lesions. In gram-negative bacteria, they act with LPS to make the outer membrane permeable, thereby leading to self-promoted uptake across the outer membrane and to the cytoplasmic membrane. Examples of cationic antimicrobial peptides include indolicidin (antimicrobial polypeptide from bovine neutrophils), defensins (defensins), cecropins (cecropins) and magainins.

[0009] Although it is not known whether the antimicrobial and effector functions are independent, it has recently become increasingly recognized that this peptide is an effector in other aspects of innate immunity (Hancock, R.E.W.and G.Diamond.2000.the role of cationic peptides in animal microorganisms. trends in microbiology 8: 402; Hancock, R.E.W.2001.cationic peptides: antigens in animal and novel antibodies. Lancet infection Diseases 1: 156. 164).

[0010] Some cationic peptides have an affinity to bind bacterial products such as LPS and LTA. These cationic peptides are able to inhibit cytokine production in response to LPS and thus to varying degrees prevent lethal shock. However, it has not been demonstrated whether this effect is due to binding of the peptide to LPS and LTA, or to direct interaction of the peptide with the host cell. Cationic peptides are induced in response to attack by microorganisms or microbial signalling molecules such as LPS (challenge) through regulatory pathways (involving Toll receptors and transcription factors; NF-. kappa.B) similar to those employed by the mammalian immune system. Cationic peptides thus appear to have an important role in innate immunity. Mutations that affect the induction of antibacterial peptides can reduce survival in response to bacterial challenge. In addition, mutations in the Toll pathway of Drosophila (Drosophila), which result in reduced expression of antifungal peptides, also result in increased susceptibility to fatal fungal infections. In humans, patients with specific granule deficiency syndrome (specific granule deficiency syndrome) are completely deficient in alpha-defensin, suffering from frequent and severe bacterial infections. Other evidence includes that some peptides can be induced by infectious agents, and that the concentrations of these peptides have been found to be quite high at the site where inflammation occurs. Cationic peptides may also regulate cell migration to promote the ability of leukocytes to fight bacterial infection. For example, it has been shown that two human α -defensin peptides, HNP-1 and HNP-2, have direct chemotactic activity on murine and human T cells as well as monocytes and that human β defensin appears to act as a chemoattractant for immature dendritic cells and memory T cells (chemoattractants) by interacting with CCR 6. Similarly, the porcine cationic peptide PR-39 was found to be chemotactic for neutrophils. However, it is unclear as to whether peptides of different structures and compositions share these properties.

[0011] LL-37 is the single known cathelicidin from human (a precursor of antimicrobial peptides with potent membrane activity) produced by bone marrow precursors, testis, human keratinocytes during inflammatory disease and respiratory epithelial cells. Cathelicidin polypeptides are characterized by a high level of sequence identity (sequence identity) in the N-terminal prepro region (prepro region) referred to as the cathelin domain. Cathelicidin polypeptides are stored as unactivated propeptide precursors (propeptide precursors) and upon stimulation, are processed into active peptides.

Summary of The Invention

[0012] The invention is based on the following pioneering findings: novel compounds that block or reduce sepsis and/or inflammation in a subject can be screened based on the pattern of polynucleotide expression that is modulated by endotoxic lipopolysaccharides, lipoteichoic acids, CpG DNA, or other cellular components (e.g., microorganisms or their cellular components), but is affected by the cationic peptide. Furthermore, based on the use of cationic peptides as a tool, selective enhancers of innate immunity can be identified that do not elicit a septic response and are able to block/suppress inflammatory and/or septic responses.

[0013] Thus, in one embodiment, a method is provided for identifying a polynucleotide or pattern of polynucleotides that is modulated by one or more inducers of sepsis or inflammation and that is simultaneously inhibited by a cationic peptide. The methods of the invention comprise contacting one or more polynucleotides with one or more inducers of sepsis or inflammation and simultaneously or immediately subsequently contacting the one or more polynucleotides with a cationic peptide. Detection of differences in expression, either up-regulation or down-regulation, between the presence and absence of the cationic peptide, changes in expression, can be used as an indication of the pattern of polynucleotides or polynucleotides that are regulated by sepsis or inflammatory inducers and simultaneously inhibited by the cationic peptide. In another aspect, the invention provides one or more polynucleotides identified by the above methods. Examples of sepsis or inflammation modulators include LPS, LTA or CpG DNA or microbial components (or any combination thereof), or related agents.

[0014] In another embodiment, the invention provides a method of identifying an agent that can prevent sepsis or inflammation comprising combining a polynucleotide identified by the foregoing method with an agent, wherein the expression of the polynucleotide is modulated in the presence of the agent compared to the expression in the absence of the agent, and such modulation in expression affects the inflammatory or sepsis response.

[0015] In another embodiment, the invention provides a method for identifying a pattern of polynucleotide expression when an inflammatory or septic response is inhibited by 1) contacting a cell with LPS, LTA, and/or CpG DNA in the presence or absence of a cationic peptide, and 2) detecting the pattern of polynucleotide expression of the cell in the presence or absence of the peptide. The pattern obtained in the presence of the peptide represents an inhibition of the inflammatory or septic response. In another aspect, the pattern obtained in the presence of the peptide is compared to the pattern obtained in the presence of a test compound to identify compounds that provide a similar pattern. In another aspect, the invention provides compounds identified by the foregoing methods.

[0016] In another embodiment, the invention provides a method of identifying an agent that potentiates innate immunity comprising contacting one or more polynucleotides encoding polypeptides involved in innate immunity with an agent of interest, wherein expression of the polynucleotide is modulated in the presence of the agent, and the modulated expression results in potentiation of innate immunity, as compared to the absence of the agent. Preferably, the agent does not stimulate a septic response in the 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-1R antagonist homolog 1(AI167887), IL-10 Rbeta (AA486393), IL-10 Ralpha (U00672), TNF receptor member 1B (AA150416), TNF receptor member 5(H98636), TNF receptor member 11B (AA194983), HLA II IK cytokine down-regulator (R39227), TGF-B inducible early growth response protein 2(AI473938), CD2(AA927710), IL-19 (NM-013371), or IL-10 (M57627). In one aspect, the agent reduces expression of a polynucleotide encoding a proteasome subunit involved in NK-kb activation, e.g., proteasome subunit 26S (NM — 013371). In one aspect, the agent can act as an antagonist of a protein kinase. In one aspect, the agent is a nucleic acid sequence selected from SEQ ID NOs: 4-54.

[0017] In another embodiment, the invention provides a method of identifying a polynucleotide expression profile to identify compounds that selectively potentiate innate immunity. The invention includes detecting a polynucleotide expression profile of a cell in the presence and absence of contact with a cationic peptide, wherein the profile in the presence of the peptide represents stimulation of innate immunity; detecting the polynucleotide expression pattern of the cells upon exposure to a test compound, wherein the test compound enhances innate immunity if the pattern using the test compound is similar to the pattern observed in the presence of the cationic peptide. Preferably, the compound does not stimulate a septic response in the individual.

[0018] In another embodiment, the invention provides a method of inferring the infection status of a mammalian subject from a nucleic acid sample from the subject by determining the expression pattern of polynucleotides in the nucleic acid sample, e.g., an increased expression of at least two polynucleotides in tables 50, 51, and/or 52 as compared to an uninfected individual. Also included are polynucleotide expression profiles obtained by any of the above methods.

[0019] In another aspect, cationic peptides are provided that are antagonists of CXCR-4. In yet another aspect, there is provided a method of identifying a cationic peptide that is an antagonist of CXCR-4, comprising contacting a T cell with SDF-1 in the presence or absence of a test peptide, and measuring chemotaxis. A test peptide, if it has reduced chemotaxis in the presence of the peptide, indicates that the peptide is an antagonist of CXCR-4. The cationic peptide may also function to reduce expression of the SDF-1 receptor polynucleotide (NM-013371).

[0020] In all of the above methods, the compounds or agents of the invention include, but are not limited to, peptides (peptides), cationic peptides (cationic peptides), peptidomimetics (peptidomimetics), chemical compounds (chemical compounds), polypeptides (polypetides), nucleic acid molecules (nucleic acid molecules), and the like.

[0021] In yet another aspect, the present invention provides isolated cationic peptides. The isolated cationic peptides of the invention can be represented by any of the following general formulas and one letter amino acid notation:

X₁X₂X₃IX₄PX₄IPX₅X₂X₁(SEQ ID NO: 4) wherein X₁Is one or two of R, L or K, X₂Is C, S or A, X₃Is an R or P, X₄Is an A or V, X₅Is a V or W;

X₁LX₂X₃KX₄X₂X₅X₃PX₃X₁(SEQ ID NO: 11), wherein X₁Is one or two of D, E, S, T or N, X₂Is one or two of P, G or D, X₃Is an G, A, V, L, I or Y, X₄Is an R, K or H, X₅Is an S, T, C, M or R;

X₁X₂X₃X₄WX₄WX₄X₅k (SEQ ID NO: 18), wherein X₁Is one to four amino acids selected from A, P or R, X₂Is one or two aromatic amino acids (F, Y and W), X₃Is a P or K, X₄Is zero to two amino acids selected from A, P, Y or W, X₅Is one to three amino acids selected from R or P;

X₁X₂X₃X₄X₁VX₃X₄RGX₄X₃X₄X₁X₃X₁(SEQ ID NO: 25), whereinX₁Is one or two of R or K, X₂Is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X₃Is C, S, M, D or A, X₄Is F, I, V, M or R;

X₁X₂X₃X₄X₁VX₅X₄RGX₄X₅X₄X₁X₃X₁(SEQ ID NO: 32) wherein X₁Is one or two of R or K, X₂Is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X₃Is C, S, M, D or A, X₄Is an F, I, V, M or R, X₅Is an A, I, S, M, D or R; and

KX₁KX₂FX₂KMLMX₂ALKKX₃(SEQ ID NO: 39) wherein X₁Are polar amino acids (C, S, T, M, N and Q); x₂Is A, L, S or K, X₃Is 1 to 17 amino acids selected from G, A, V, L, I, P, F, S, T, K and H;

KWKX₂X₁X₁X₂X₂X₁X₂X₂X₁X₁X₂X₂IFHTALKPISS (SEQ ID NO: 46), wherein X₁Is a hydrophobic amino acid, X₂Is a hydrophilic amino acid.

[0022] In addition, in another aspect the invention provides isolated cationic peptides KWKSFLRTFKSPVRTVFHTALKPISS (SEQ ID NO: 53) and KWKSYAHTIMSPVRLVFHTALKPISS (SEQ ID NO: 54).

[0023] Also provided are nucleic acid sequences encoding the cationic peptides of the invention, vectors comprising these polynucleotides, and host cells containing these vectors.

[0024] In another embodiment, the invention provides a method of stimulating or enhancing innate immunity in a subject comprising administering to the subject a peptide of the invention, e.g., SEQ ID NO: 1-4, 11, 18, 25, 32, 39, 46, 53, or 54. As shown in the examples herein, innate immunity can be demonstrated by monocyte activation, proliferation, differentiation, or activation of the MAP kinase pathway, which are merely examples. In one aspect, the method comprises further administering to the subject a serum factor, such as GS-CSF. The subject is preferably any mammal, more particularly a human subject.

[0025] In another embodiment, the invention provides a method of stimulating innate immunity in a subject having or at risk of having an infection, comprising administering to the subject a sub-optimal concentration of an antibiotic in combination with a peptide of the invention. In one aspect, the peptide is SEQ ID NO: 1 or SEQ ID NO: 7.

drawings

[0026]FIG. 1 illustrates the Seq ID No: 7 and cefepime in the treatment of staphylococcus aureus (s. CD-1 mice (8/group) containing 1X 10 were administered by Intraperitoneal (IP) injection⁷5% porcine mucin fluid (mucin) of Staphylococcus aureus. Test compounds (50. mu.g-2.5 mg/kg) were administered via a single IP injection 6 hours after administration of Staphylococcus aureus. At this time, cefepime was also administered at a dose of 0.1 mg/kg. 24 hours thereafter, mice were euthanized, blood was drawn and plated for survival counting. Mean ± standard deviation are shown. This experiment was repeated twice.

[0027] Figure 2 shows the exposure to SEQ ID NO: 1, phosphorylation of ERK1/2 and p38 was induced. Lysates from human peripheral blood-derived monocytes were exposed to 50 μ g/ml of SEQ ID NO: 1, exposure for 15 minutes. A) Antibodies specific for the phosphorylated forms of ERK and p38 were used to detect activation of ERK1/2 and p 38. All donors tested showed that phosphorylation of ERK and p38 was responsive to SEQ ID NO: 1 treatment and increases. One representative donor of eight. The relative amounts of phosphorylated erk (b) and p38(C) were determined by dividing the intensity of the phosphorylated bands by the intensity of the corresponding control bands as described in materials and methods.

[0028] FIG. 3 shows that in the absence of serum, LL-37-induced phosphorylation of ERK1/2 did not occur, and that the degree of phosphorylation was dependent on the type of serum present. Human blood derived monocytes (human blood derived monocytes) were treated with 50. mu.g/ml LL-37 for 15 minutes. The lysates were separated on a 12% acrylamide gel, then transferred to nitrocellulose membrane and probed with an antibody specific for the phosphorylated (activated) form of the kinase. To normalize the protein loading, the membrane was probed again with β -actin. Quantification was performed using ImageJ software. The inset in FIG. 3 demonstrates that LL-37 is unable to induce MAPK activation in human monocytes under serum-free conditions. Cells were exposed to 50mg/ml LL-37(+) or endotoxin free water (-) as a vehicle control for 15 minutes. (A) Phosphorylated ERK1/2 could be detected after exposure to LL-37 in medium containing 10% fetal bovine serum, however, in the absence of serum, no phosphorylation of ERK1/2 was detected (n-3). (B) Elk-1 is activated (phosphorylated) after exposure to 50 μ g/ml LL-37 in medium containing 10% fetal bovine serum, Elk-1 is a transcription factor downstream of ERK1/2, but Elk is not activated in the absence of serum (n ═ 2).

[0029] FIG. 4 shows that LL-37-induced activation of ERK1/2 occurs at lower concentrations and is amplified in the presence of some cytokines. LL-37-induced phosphorylation of ERK1/2 was evident at concentrations as low as 5. mu.g/ml when freshly isolated monocytes were stimulated in medium containing GM-CSF (100ng/ml) and IL-4(100 ng/ml). EKR1/2 appears to be primarily due to GM-CSF.

[0030] FIG. 5 shows that in the 16HBE4 o-human bronchial epithelial cell line, peptides affect the transcription of various cytokine genes and the release of IL-8. Cells were grown to confluence on a semi-permeable membrane and plated on the top surface (apicals surface) with 50 μ g/ml of SEQ ID NO: 1 stimulation for 4 hours. A) SEQ ID NO: 1 treated cells produced significantly more IL-8 than the control group as determined by ELISA on supernatants collected on the top, but not the bottom side (basal surface). The mean ± standard deviation (SE) of three independent experiments is shown, with an asterisk denoting p ═ 0.002. B) RNA was collected from the above-mentioned test sample and subjected to RT-PCR. Many cytokine genes known to be regulated by ERK1/2 or p38 are upregulated upon stimulation with peptides. The average of two independent experiments is shown.

Detailed Description

[0031] The present invention provides novel cationic peptides that can be characterized by a set of general formulae that are capable of modulating (e.g., up-regulating and/or down-regulating) the expression of polynucleotides, thereby modulating sepsis and inflammatory responses and/or innate immunity.

[0032] As used herein, "innate immunity" refers to the ability of a organism to defend against pathogen invasion, protecting itself from innate possession. Pathogens or microorganisms as used herein may include, but are not limited to, bacteria, fungi, parasites, and viruses. Innate immunity differs from adaptive/adaptive immunity, in which organisms develop their own defense mechanisms, primarily based on antibodies and/or immune lymphocytes, characterized by specificity, scalability and recognition of self versus non-self. Innate immunity provides extensive nonspecific immunity and has no immunological memory for previous exposure. Innate immunity is characterized by the ability to effectively combat a wide range of potential pathogens, independent of prior exposure to a pathogen, and innate immunity is further characterized by the ability to take effect rapidly (the elicitation of a specific immune response takes days to weeks as compared to a specific immune response). Moreover, innate immunity includes immune responses that affect other diseases such as cancer, inflammatory diseases, multiple sclerosis, various viral infections, and the like.

[0033] As used herein, the term "cationic peptide" refers to an amino acid sequence of about 5 to about 50 amino acids in length. In one aspect, the cationic peptides of the invention are from about 10 to about 35 amino acids in length. A peptide is considered a "cationic peptide" if it has enough 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 are positively charged, such as lysine and arginine. "positively charged" refers to an amino acid residue side chain having a net positive charge at pH 7.0. Examples of naturally occurring cationic antimicrobial peptides include defensins, cathelicidins, magainins, melittin, cecropin, bactenecins (antimicrobial polypeptides from bovine neutrophils), indolicidins, polyphemusins, tacyphesins (antimicrobial polypeptides from Calla cheilosa blood cells), and analogs thereof, which can be recombinantly produced according to the invention. Many organisms produce cationic peptides and use these molecules as part of a non-specific defense mechanism against microorganisms. These isolated peptides are toxic to a wide variety of microorganisms, including bacteria, fungi, and certain enveloped viruses. When cationic peptides exert a resistance against many pathogens, significant abnormalities and varying degrees of toxicity are present. However, this patent discloses other cationic peptides that are not toxic to microorganisms but are capable of infecting by stimulating innate immune defenses, and the invention is not limited to cationic peptides having antimicrobial activity. In fact, many of the peptides used in the present invention do not have antimicrobial activity.

[0034] Cationic peptides known in the art include, for example, human cathelicidin IL-37, the bovine neutrophil polypeptide indolicidin, and variants of bovine bactenecin, Bac 2A.

IL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES(SEQ ID：1)

Indolicidin ILPWKWPWWPWRR-NH₂(SEQ ID NO：2)

Bac2A RLARIVVIRVAR-NH₂(SEQ ID NO：3)

[0035] In innate immunity, the immune response is not antigen-dependent. The innate immune process may include the production of secreted molecules and cellular components as previously set forth. In innate immunity, pathogens are recognized by receptors encoded in the germ line. These Toll-like receptors have a wide range of specificities and are capable of recognizing many pathogens. When cationic peptides are present in the immune response, they assist the host in responding to pathogens. This change in the immune response induces the release of chemokines that cause immune cells to concentrate to the site where the infection occurs.

[0036] Chemokines or chemically induced cytokines belong to a class of immune factors that mediate chemotaxis and other proinflammatory phenomena (see, Schall, 1991, Cytokine 3: 165-183). Chemokines are small molecules of about 70-80 residues in length, generally divided into two subclasses, an alpha subclass with an N-terminal cysteine (CxC) separated by a single amino acid, and a beta subclass with two adjacent cysteines (CCs) at the N-terminal. RANTES, MIP-1 α and MIP-1 β belong to the β subclass (see for review: Horuk, R., 1994, Trends Pharmacol. Sci, 15: 159-. The amino-terminal ends of the beta chemokines RANTES, MCP-1 and MCP-3 are implicated in mediating cell migration and the modulation of inflammatory responses induced by these chemokines. This association is observed experimentally that elimination of 8 residues at the amino terminus of MCP-1, 9 residues at the amino terminus of MCP-3 and 8 residues at the amino terminus of RANTES, as well as the addition of methionine to the amino terminus of RANTES, antagonize chemotaxis, calcium transfer and/or enzyme release stimulated by their natural counterparts (Gong et al, 1996 J.biol.chem.271: 10521-. In addition, chemotactic activity similar to that of the alpha-type chemokine can be introduced into MCP-1 by double mutating Tyr 28 and Arg30 to leucine and valine, suggesting that the internal region of the protein also plays a role in regulating chemotactic activity (Beall et al, 1992, J.biol.chem.267: 3455-.

[0037] All monomeric forms of chemokines that have been characterized to date have significant structural homology, despite differences in the quaternary structure of the α and β forms. The monomeric structures of the beta and alpha chemokines are very similar, but the dimeric structures of these two types are completely different. Another chemokine, lymphotactin (lymphotactin), which has only one N-terminal cysteine, has also been identified and represents a third subfamily of chemokines (. gamma.) (Yoshida et al, 1995, FEBS Lett.360: 155-.

[0038] Receptors for chemokines belong to the large family of receptors (GCR's) with 7 transmembrane domains coupled to G proteins (for review: Horuk, R.1994, Trends Pharmacol. Sci.15: 159-165; and Murphy, P.M.1994, Annu.Rev. Immunol.12: 593-633). Competitive binding and cross-desensitization studies have shown that chemokine receptors have significant promiscuity in binding ligands. Examples of evidence of heterozygosity among beta chemokines include: CC CKR-1 binds RANTES and MIP-1 α (Neote et al, 1993, Cell 72: 415-. Erythrocytes have a receptor (known as the Duffy antigen) which binds to alpha and beta chemokines (Horuk et al, 1994, J.biol.chem.269: 17730-. Thus, significant sequence and structural homology between chemokines and their receptors allows crossover in receptor-ligand interactions.

[0039] In one aspect, the invention provides the use of compounds comprising the cationic peptides of the invention to reduce sepsis and inflammatory responses by acting directly on host cells. In this aspect, methods are provided for identifying a polynucleotide that is modulated by one or more inducers of sepsis or inflammation, wherein the modulation is altered by a cationic peptide. The sepsis or inflammation inducers 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 method relies on contacting the polynucleotide with an inducer of sepsis or inflammation and simultaneously or immediately thereafter with a cationic peptide. Observing the expression of the polynucleotide in the presence and absence of the cationic peptide, wherein a change in expression indicates that the polynucleotide or pattern of polynucleotides is regulated by an inducer of sepsis or inflammation and inhibited by the cationic peptide. In another aspect, the invention provides polynucleotides identified by the method.

[0040] Once identified, such polynucleotides can be used to screen for compounds that can prevent sepsis or inflammation by affecting the expression of the polynucleotide. Such effects on expression may be up regulation or down regulation of expression. The invention also provides methods for identifying innate immunity enhancers by identifying compounds that do not provoke a septic response and compounds that are capable of preventing or inhibiting an inflammatory or septic response. In addition, the present invention provides compounds used and identified in the above methods.

[0041] Candidate compounds are obtained from a wide variety of sources, including libraries of various synthetic and natural compounds. For example, a number of methods can be used to randomly and directionally synthesize various organic compounds and biomolecules, including expression of random oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds (libraries) in the form of bacterial, fungal, plant and animal extracts may be utilised or conveniently produced. In addition, natural or synthetically produced libraries and compounds may be readily modified by conventional chemical, physical and biochemical means, and combinatorial libraries may also be generated using these libraries and compounds. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, and the like, to yield structural analogs. Candidate agents may also be found from biomolecules, including but not limited to: peptides, peptidomimetics, carbohydrates, fatty acids, steroids, purines, pyrimidines, polypeptides, polynucleotides, chemical compounds, derivatives (derivatives), structural analogs, or combinations thereof (combinations).

[0042] The incubation elements (components) of the screening assay include conditions that allow the test compound and the polynucleotide of interest to come into contact with each other. The contacting can be in a liquid phase, a solid phase, or in a cell. To screen multiple compounds, the test compounds can be selected as a combinatorial library. Compounds identified according to the methods of the invention can be further evaluated, detected, cloned, sequenced, etc., in solution or after binding to a solid support, by any method commonly used in detecting compounds.

[0043] Generally, in the methods of the invention, cationic peptides are utilized to detect and localize polynucleotides necessary in the course of sepsis and inflammation. Once identified, the expression profile of the polynucleotide can be obtained by observing its expression in the presence and absence of the cationic peptide. The pattern obtained in the presence of the cationic peptide is also useful for identifying other compounds that inhibit the expression of the polynucleotide and thereby prevent sepsis or inflammation. It is well known to those skilled in the art that non-peptide compounds and peptidomimetics are capable of mimicking the ability of peptides to bind to receptors and enzyme binding sites, and thus can be used to block or elicit biological responses. Where an additional compound of interest provides a similar pattern of polynucleotide expression as in the presence of the cationic peptide, the compound may also be used to modulate sepsis or innate immune response. In this way, the cationic peptides of the invention, while acting as known inhibitors of sepsis and inflammation and known boosters of innate immunity, can also be used as tools to identify other compounds that inhibit sepsis and inflammation and boost innate immunity.

[0044] As will be seen in the examples which follow, the peptides of the invention have the ability to broadly reduce the expression of polynucleotides regulated by LPS. Many of the symptoms seen during severe infection or inflammation, such as fever and elevated white blood cell numbers, are due to high levels of endotoxins in the blood. Endotoxin is a component of the cell wall of gram-negative bacteria and is a pathophysiologically potent initiator of sepsis (trigger). The basic mechanisms of inflammation and sepsis are linked. In example 1, polynucleotide arrays were used to determine the effect of cationic peptides on the transcriptional response of epithelial cells. In particular, effects on more than 14,000 different specific polynucleotide probes induced by LPS were observed. The table shows the changes seen for the peptide-treated cells compared to the control cells. The data obtained indicate that the peptide has the ability to reduce the expression of the polynucleotide induced by LPS.

[0045] Similarly, example 2 demonstrates that the peptides of the invention are capable of neutralizing the stimulation of immune cells by gram-positive and gram-negative bacterial products. In addition, it is noted that certain pro-inflammatory polynucleotides are down-regulated by cationic peptides, as listed 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 β 2(AA977194), IL-18 receptor 1(AA 244889), while anti-inflammatory polynucleotides are up-regulated by cationic polypeptides, as listed in Table 25, such as IL-1R antagonist homolog 1(AI167887), IL-10R β (AA486393), TNF receptor member 1B (AA150416), TNF receptor member 5(H98636), TNF receptor member B (AA 981943), HLA 39II cytokine down-regulator (HLA 39227) Early growth response 2(AI473938) or CD2(AA927710) that can be induced by TGF-B. The applicability and use of these results was demonstrated by in vivo use in mice.

[0046] In another aspect, the invention provides methods of identifying agents that potentiate innate immunity. In this method, a polynucleotide encoding a polypeptide involved in innate immunity is contacted with an agent of interest. Determining the expression of the polynucleotide in the presence and absence of the agent. By comparison of expression, specific modulation of expression indicates that innate immunity is enhanced. On the other hand, the agent does not provoke a septic response, as revealed by the lack of up-regulation (upregulation) of the proinflammatory cytokine TNF- α. In yet another aspect, the agent reduces or blocks an inflammatory or septic response. In yet another aspect, the agent reduces the expression of TNF- α and/or interleukins including, but not limited to, IL-1 β, IL-6, IL-12p40, IL-12p70, and IL-8.

[0047] In another aspect, the invention provides methods of using cationic peptides for direct polynucleotide modulation and the use of compounds, including cationic peptides, in elements (elements) that stimulate innate immunity. In this aspect, the invention provides methods for determining the expression profile of a polynucleotide for the purpose of identifying compounds that potentiate innate immunity. In the method of the invention, cells contacted with and not contacted with the cationic peptide are subjected to an initial detection of the polynucleotide expression pattern. The pattern of polynucleotide expression in the presence of the peptide indicates that innate immunity is stimulated. The expression pattern of the polynucleotide in the presence of the test compound is then determined, where the test compound is used to obtain an expression pattern similar to that obtained in the presence of the cationic peptide, indicating that this is a compound that enhances innate immunity. In another aspect, the invention provides compounds identified in the above methods. In another aspect, the compounds of the invention stimulate the expression of chemokines or chemokine receptors. Chemokines or chemokine receptors may include, but are not limited to CXCR4, CXCR1, CXCR2, CCR2, CCR4, CCR5, CCR6, MIP-1 α, MDC, MIP-3 α, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, and RANTES. In yet another aspect, the compound is a peptide, a peptidomimetic (peptidomimetic), a chemical compound (chemical compounds), or a nucleic acid molecule.

[0048] In yet another aspect, the polynucleotide expression profile comprises expression of a pro-inflammatory polynucleotide. These pro-inflammatory polynucleotides include, but are not limited to, cyclic finger protein 10(D87451), serine/threonine protein kinase MASK (AB040057), KIAA0912 protein (AB020719), KIAA0239 protein (D87076), RAP1, gtpase activator 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), bronchobronchial mucin 5 subtype B (AJ001403), cAMP response element binding protein bpcrea, integrin alpham (03103925), Rho-associated kinase 2(NM _004850), PTD017 protein (AL050361), unknown genes (AK001143, AK034348, 1610450, AL 983), and any combination thereof. In yet another aspect, the polynucleotide expression profile includes expression of cell surface receptors including, but not limited to, retinoic acid receptor (X06614), G protein-coupled receptor (Z94155, X81892, U52219, U22491, AF015257, U66579), chemokine (C-C motif) receptor 7(L31584), tumor necrosis factor receptor superfamily member 17(Z29575), interferon gamma receptor 2(U05875), cytokine receptor-like factor 1(AF059293), class I cytokine receptor (AF053004), lectin II (thrombin) receptor-like 2(U92971), leukemia inhibitory factor receptor (NM _002310), interferon gamma receptor 1(AL 050337).

[0049] In example 4, it can be seen that the cationic peptides of the invention can alter polynucleotide expression in macrophages and epithelial cells. The results of this example demonstrate that pro-inflammatory polynucleotides are down-regulated by cationic peptides (Table 24), while anti-inflammatory polynucleotides are up-regulated by cationic peptides (Table 25).

[0050] As will be illustrated later, e.g., in tables 1-15, the cationic peptides are capable of neutralizing the host response to a signal molecule of an infectious agent and also of altering the transcriptional response of the host cells, primarily by down-regulating the pro-inflammatory response and/or up-regulating the anti-inflammatory response. Example 5 demonstrates that the cationic peptide can assist the host in responding to pathogens by inducing the release of chemokines that promote immune cell aggregation to the site of infection. These results were confirmed by in vivo application in mice.

[0051] As can be seen from the examples that follow, cationic peptides significantly affect the host response to pathogens in that cationic peptides induce a selective pro-inflammatory response, such as a response that promotes the aggregation of immune cells to the site of infection, but do not induce potentially harmful pro-inflammatory cytokines, thereby aiding in the regulation of the host immune response. Sepsis appears to be caused in part by an excessive pro-inflammatory response to an infectious agent. Cationic peptides induce anti-inflammatory responses and inhibit certain potentially harmful pro-inflammatory responses, thereby helping the host produce a "balanced" response to pathogens.

[0052] In example 7, selected MAP kinases were analyzed for activation in order to investigate the basic mechanism by which cationic peptides interact with cells to produce these effects. Macrophages activate MEK/ERK kinase in response to bacterial infection. MEK is a MAP kinase that, when activated, phosphorylates the downstream kinase ERK (extracellular regulatory kinase), which then forms dimers and transfers to the nucleus where it activates transcription factors such as EIK-1, thereby altering expression of the polynucleotide. MEK/ERKK kinase has been shown to impair Salmonella (Salmonella) replication in macrophages. Signal transduction mediated by MEK kinase and NADPH oxidase plays an important role in the innate defense against intracellular pathogens. As shown below, cationic peptides have an effect on bacterial infection by affecting MAP kinases. These cationic peptides can directly affect kinases. Table 21 shows the changes in MAP kinase polynucleotide expression in response to peptides, but is not limited to these. These kinases include MAP kinase 6(H070920), MAP kinase 5 (W698649), MAP kinase 7(H39192), MAP kinase 12(AI936909), and protein kinase 3 activated by MAP kinase (W68281).

[0053] In another method, the methods of the invention can be used in combination to identify agents with multiple characteristics, i.e., peptides that have anti-inflammatory/anti-sepsis activity and are capable of boosting innate immunity in part by inducing chemokines in vivo.

[0054] In another aspect, the invention provides methods of inferring the infection status of a mammalian subject from a nucleic acid sample from the subject, the methods relying on determining the polynucleotide expression pattern in the nucleic acid sample, exemplified by increased polynucleotide expression of at least two polynucleotides in table 55 as compared to an uninfected subject. In another aspect, the invention provides a method of inferring the infection status of a mammalian subject from a nucleic acid sample from the subject, the method relying on determining the polynucleotide expression pattern in the nucleic acid sample, exemplified by the expression of polynucleotides in at least two of the polynucleotides in table 56 or table 57 as compared to an uninfected individual. In one aspect of the invention, the infectious state is due to infectious agents or signal molecules derived therefrom, such as, but not limited to, gram-negative and gram-positive bacteria, viruses, fungi or parasites. In another aspect, the invention provides a polynucleotide expression profile of an infected subject determined according to the above method. Once determined, such polynucleotides will be useful in diagnosing conditions associated with the presence or activity of these infectious agents or signaling molecules.

[0055] This aspect of the invention is shown in example 10 which follows. In particular, table 61 demonstrates that both MEK and NADPH oxidase inhibitors limit bacterial replication (MEK kinase is activated by IFN- γ -induced salmonella typhimurium (s. typhimurium) infection of macrophages). This is an example of how bacterial survival can be affected by altering host cell signaling molecules.

[0056] In yet another aspect of the invention, compounds are presented that inhibit T cell chemotaxis induced by stromal cell derived factor 1 (SDF-1). Compounds that reduce the expression of SDF-1 receptors have also been proposed. These compounds may also be used as antagonists or inhibitors of CXCR-4. In one aspect, the invention provides cationic peptides that are CXCR-4 antagonists. In another aspect, the invention provides methods for identifying cationic peptides as CXCR-4 antagonists. The method comprises contacting a T cell with SDF-1 in the presence and absence of a test peptide and measuring chemotaxis. In the presence of the test peptide, a decrease in chemotaxis indicates that the peptide is an antagonist of CXCR-4. These compounds and methods are useful in the therapeutic application of HIV patients. These types of compounds and their utility have been demonstrated, for example, in example 11 (see also tables 62, 63). In this example, cationic peptides have been shown to inhibit cell migration (cell migration) as well as antiviral activity.

[0057]In one embodiment, the present invention provides an isolated cationic peptide having an amino acid sequence of the following general formula (general formula a): x₁X₂X₃IX₄PX₄IPX₅X₂X₁(SEQ ID NO: 4) wherein X is₁Is one or two of R, L or K, X₂Is C, S or A, X₃Is an R or P, X₄Is an A or V, X₅Is a V or W. Examples of 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).

[0058]In another embodiment, the present invention provides an isolated linear cationic peptide having an amino acid sequence of the following general formula (general formula B): x₁LX₂X₃KX₄X₂X₅X₃PX₃X₁(SEQ ID NO: 11), wherein X₁Is one or two of D, E, S, T or N, X₂Is one or two of P, G or D, X₃Is an G, A, V, L, I or Y, X₄Is an R, K or H, X₅Is an S, T, C, M or R. Examples of 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).

[0059]In another embodiment, the present invention provides an isolated linear cationic peptide having an amino acid sequence of the following general formula (general formula C): x₁X₂X₃X₄WX₄WX₄X₅K (SEQ ID NO: 18) (this formula includes CP12a and CP12d), where X₁Is one to four amino acids selected from A, P or R, X₂Is one or two aromatic amino acids (F, Y and W), X₃Is a P or K, X₄Is zero to two amino acids selected from A, P, Y or W, X₅Is one to three amino acids selected from R or P. Examples of 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: 20)SEQ ID NO: 23) and RRRWKWAWPRRK (SEQ ID NO: 24).

[0060]In another embodiment, the present invention provides an isolated hexameric cationic peptide having the amino acid sequence of the following general formula (general formula D): x₁X₂X₃X₄X₁VX₃X₄RGX₄X₃X₄X₁X₃X₁(SEQ ID NO: 25), wherein X₁Is one or two of R or K, X₂Is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X₃Is C, S, M, D or A, X₄Is F, I, V, M or R. Examples of 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).

[0061]In another embodiment, the present invention provides an isolated hexameric cationic peptide having the amino acid sequence of the following general formula (general formula E): x₁X₂X₃X₄X₁VX₅X₄RGX₄X₅X₄X₁X₃X₁(SEQ ID NO: 32) wherein X₁Is one or two of R or K, X₂Is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X₃Is C, S, M, D or A, X₄Is an F, I, V, M or R, X₅Is an A, I, S, M, D or R. Examples of 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).

[0062]In another embodiment, the invention provides isolated longer cations having the amino acid sequence of the following formula (formula F)A sub-peptide: KX₁KX₂FX₂KMLMX₂ALKKX₃(SEQ ID NO: 39), wherein X₁Is a polar amino acid (C, S, T, M, N and Q); x₂Is A, L, S or K, X₃Is 1-17 amino acids selected from G, A, V, L, I, P, F, S, T, K and H. Examples of 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).

[0063]In another embodiment, the invention provides isolated longer cationic peptides having the amino acid sequence of the following general formula (general formula G): KWKX₂X₁X₁X₂X₂X₁X₂X₂X₁X₁X₂X₂IFHTALKPISS (SEQ ID NO: 46), wherein X₁Is a hydrophobic amino acid, X₂Are hydrophilic amino acids. Examples of 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).

[0064] In 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).

[0065] 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 the peptide produced in vivo is naturally associated). Preferably, the peptide is at least 70%, 80%, or most preferably 90% pure by weight.

[0066] The invention also includes analogs (analogs), derivatives (derivitives), conservative variants (conservative variations) and cationic peptide variants (cationic peptide variations) of the enumerated polypeptides, provided that the analogs, derivatives, conservative variants or variants have detectable activity in enhancing innate immunity or anti-inflammatory activity. The activity of an analog, derivative, conservative variant or variant of a peptide need not be exactly the same as the activity of the peptide.

[0067] Cationic peptide "variants" refer to peptides that are variations of the cationic peptide referred to. For example, the term "variant" includes a cationic peptide in which at least one amino acid of a reference peptide is replaced in an expression library. The term "reference" peptide refers to any cationic peptide of the invention (e.g., as defined in the formulas above) from which variants, derivatives, analogs or conservative variations are derived. The term "derivative" includes hybrid peptides that include at least a portion of each of two cationic peptides (e.g., 30-80% of each of the two cationic peptides). Also included are peptides obtained by deleting one or more amino acids from the sequences of the peptides listed herein, as long as the derivatives have an innate immunity-enhancing or anti-inflammatory activity. Smaller reactive molecules can thus be developed which also have utility. For example, amino-terminal amino acids or carboxy-terminal amino acids that are not essential for enhancing innate immunity and anti-inflammatory activity of the peptide may be removed. Likewise, one or a few (e.g., less than 5) amino acids may be added to a cationic peptide without completely inhibiting the activity of the peptide, thus yielding additional derivatives. In addition, C-terminal derivatives, such as C-terminal methyl esters, and N-terminal derivatives may be obtained and are included in the present invention. The peptides of the present invention include any analog, homolog, mutant, isomer or derivative of the disclosed peptides, as long as the biological activity described herein is retained. Also included are the reverse sequences of the peptides encompassed by the previously proposed formulas. Furthermore, amino acids in the "D" configuration may be replaced by amino acids in the "L" configuration, and vice versa. Alternatively, the peptide may be cyclized by chemical means or by adding two or more cysteine residues to its sequence and oxidizing to form a disulfide bond.

[0068] The invention also includes peptides that are conservative variants of those peptides listed herein. The term "conservative variant" as used herein refers to a polypeptide in which at least one amino acid is replaced with another residue having similar biological activity. Examples of conservative variations include the replacement of one hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine, or methionine for another, or the replacement of one polar residue for another, such as the replacement of arginine for lysine, glutamic for aspartic acids, glutamine for asparagine, and the like. Neutral hydrophilic amino acids that may be substituted for each other include asparagine, glutamine, serine, and threonine. "conservative variants" also include peptides obtained by replacing an unsubstituted parent amino acid(s) with a substituted amino acid. These substituted amino acids may include methylated or amidated amino acids. Other alternatives are well known to those skilled in the art. In one aspect, antibodies raised against substituted polypeptides are also capable of specifically binding to unsubstituted polypeptides.

[0069] The peptides of the invention can be synthesized by commonly used methods, for example, which include t-BOC or FMOC protection of the alpha amino group. Both methods involve stepwise synthetic steps in which one amino acid is added to each step starting from the C-terminus of the peptide (see, Coligan et al, Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). The Peptides of the invention may also be synthesised using well known solid phase peptide Synthesis methods, for example as described by Merrifield (J.Am.chem.Soc., 85: 2149, 1962) and Stewart and Young (solid phase Peptides Synthesis, Freeman, San Francisco, 1969, pages 27-62) using a styrene-divinylbenzene copolymer with 0.1-1.0mMol of amine per gram of polymer. After completion of the chemical synthesis, the peptide was deprotected and cleaved from the polymer by treatment with HF-10% anisole at 0 ℃ for 1/4-1 h. After evaporation of the reagents, the peptides were extracted from the polymer with 1% acetic acid solution and then lyophilized to give the crude product. The peptide is purified using techniques such as gel filtration, for example using 5% acetic acid as solvent on Sephadex G-15. Lyophilization of an appropriate fraction of the column eluate can yield a 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 solubility measurements. If desired, the peptide can be quantified using solid phase Edman degradation.

[0070] The invention also includes isolated nucleic acids (e.g., DNA, cDNA, or RNA) encoding the peptides of the invention. Nucleic acids encoding analogs, mutants, conservative variants, and variants of the peptides described herein are also included. The term "isolated" as used herein refers to nucleic acids that are substantially free of proteins, lipids, and other nucleic acids that are naturally associated with nucleic acids produced in vivo. Preferably, the nucleic acid is at least 70%, 80% or preferably 90% pure by weight. Conventional methods for synthesizing nucleic acids in vitro may be used instead of in vivo methods. As used herein, "nucleic acid" refers to a polymer of deoxyribonucleotides or ribonucleotides that may be a separate fragment or part of a large genetic construct (construct) (e.g., a promoter operably linked to a nucleic acid encoding a peptide of the invention). A wide variety of genetic constructs (e.g., plasmids and other expression vectors) are known in the art and can be used to produce the peptides of the invention in cell-free systems or in prokaryotic or eukaryotic (e.g., yeast, insect or mammalian) cells. In view of the degeneracy of the genetic code, a person skilled in the art can readily synthesize a nucleic acid encoding a polypeptide of the invention. The peptides of the invention can be conveniently obtained using conventional molecular biology methods using the nucleic acids of the invention.

[0071] The DNA encoding the cationic peptide of the present invention may be inserted into an "expression vector". The term "expression vector" refers to a genetic construct such as a plasmid, virus, or other vector known in the art that can be designed to contain a nucleic acid encoding a polypeptide of the present invention. These expression vectors are preferably plasmids containing promoter sequences which facilitate transcription of the inserted gene sequences in the host cell. Expression vectors typically contain an origin of replication, a promoter, and a polynucleotide (e.g., an antibiotic resistance polynucleotide) that enables phenotypic selection of the transformed cell. Various promoters can be used in the present invention, including inducible and constitutive promoters. Typically, the expression vector contains a replicon site and control sequences derived from a species compatible with the host cell.

[0072]The nucleic acids of the invention may be transformed or transfected into a recipient (recipient) using conventional techniques well known to those skilled in the art. For example, when the host cell is E.coli, CaCl known in the art may be used₂、MgCl₂Or the RbCl method to prepare competent cells capable of taking up DNA. Alternatively, physical methods such as electroporation or microinjection may be used. Electroporation is the transfer of nucleic acids into cells by high voltage pulses. In addition, the nucleic acid can be introduced into the host cell by protoplast fusion using methods well known in the art. Suitable methods for transforming eukaryotic cells, such as electroporation and lipofection, are also known.

[0073] The invention encompasses "host cell" or "recipient cell" refers to any cell in which a polypeptide of the invention can be expressed using a nucleic acid of the invention. The term also includes any progeny of the recipient cell or host cell. Preferred recipient cells or host cells of the invention include e.coli (e.coli), staphylococcus aureus (s.aureus) and pseudomonas aeruginosa (p.aeruginosa), although other gram-negative and gram-positive bacteria, fungi and mammalian cells and other organisms known in the art may also be utilized, provided that the expression vector contains an origin of replication to allow expression in the host.

[0074] The cationic peptide polynucleotide sequences used in accordance with the methods of the present invention may be isolated from an organism or synthesized in the laboratory. A specific DNA sequence encoding a cationic peptide of interest can be obtained by: 1) isolating double-stranded DNA sequences from the genomic DNA; 2) chemically synthesizing a DNA sequence to provide codons required for the cationic peptide of interest; and 3) in vitro synthesis of double stranded DNA sequences using reverse transcription of mRNA isolated from donor cells. In the latter case, the complementary sequence of the mRNA, which is usually referred to as cDNA, is obtained as double-stranded DNA.

[0075] When the entire sequence of amino acid residues of the desired peptide product is known, synthesis of its DNA sequence is often the method of choice. In the present invention, the synthetic DNA sequence has an advantage of allowing incorporation of codons most likely to be recognized by a bacterial host, thereby allowing high-level expression without translational difficulty. In addition, virtually any peptide can be synthesized, including those that encode natural cationic peptides, variants thereof, or synthetic peptides.

[0076] When the complete sequence of the desired peptide is not known, direct synthesis of the DNA sequence is not possible, and in this case the method of choice is to obtain the cDNA sequence. Standard procedures for isolating cDNA sequences of interest include constructing plasmids or phages containing cDNA libraries obtained by reverse transcription of large amounts of mRNA present in donor cells with high levels of genetic expression. When polymerase chain reaction techniques are used in combination, even rare expression products can be cloned. When a substantial portion of the amino acid sequence of the cationic peptide is known, a labelled single or double stranded DNA or RNA probe sequence can be generated, the same sequence being thought to be present in the target cDNA, and these probes can then be used in DNA/DNA hybridization experiments on copies of cDNA clones which have been denatured to single stranded form (Jay et al, Nuc. acid. Res., 11: 2325, 1983).

[0077] The peptides of the invention may be administered parenterally by injection or by gradual infusion over a period of time. Preferably, the peptide is administered in a therapeutically effective amount to enhance or stimulate the innate immune response. Innate immunity has been described herein, however, examples of an indicator (indicator) that innate immunity is stimulated include, but are not limited to, monocyte activation, proliferation, differentiation, or MAP kinase pathway activation.

[0078] The peptide may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity (intracavitary), or transdermally. Preferred methods of delivering the peptide include oral delivery via microspheres or protein-like capsules, delivery to the lungs in aerosol form, or transdermal delivery using iontophoresis or electroporation. Other methods of administration are known to those skilled in the art.

[0079] Preparations for parenteral administration of the peptides of the invention include sterile aqueous or anhydrous solutions, suspensions and emulsions. Examples of anhydrous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, ethanol/water solutions, emulsions or suspensions, including saline and parenteral vehicles containing buffer media, including sodium chloride solutions, ringer's dextrose solution, dextrose and sodium chloride, lactated ringer's solution or non-volatile oils. Carriers for intravenous administration include fluid and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose solution), and the like. Preservatives and other additives may also be present such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases and the like.

[0080] In one embodiment, the present invention provides a method of synergistic therapy (synergetic therapy). For example, a peptide as described herein may be used in synergistic combination with a sub-inhibitory concentration of antibiotics. Examples of particular types of antibiotics that are applied in conjunction with the peptide of the present invention include aminoglycosides (e.g., tobramycin), penicillins (e.g., piperacillin), cephalosporins (e.g., ceftazidime), fluoroquinolones (e.g., fluqunolones), ciprofloxacin, carbapenems (carbapenems) (e.g., imipenem), tetracyclines (tetracyclines), and macrolides (e.g., erythromycin and clarithromycin). Further to the above antibiotics, typical antibiotics include aminoglycosides (amikacin, gentamicin, kanamycin, netilmicin, tobramycin, streptomycin, azithromycin, clarithromycin, erythromycin estolate/ethylsuccinate/glucoheptonate/lactobionate/stearate), β -lactams such as penicillins (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin, and piperacillin), or cephalosporins (e.g., cephalothin, cefazolin, cefaclor, cefamandole, cefoxitin, cefuroxime, cefonicid, cefmetazole, cefotetan, cefprozil, and piperacillin), or cephalosporins (e.g., cephalothin, cefazolin, cefaclor, cefamandole, cefuroxime, cefixin, cefixime, chlorocarbon, cefetamet, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime, and cefsulodin). Other classes of antibiotics include, for example, carbapenems (e.g., imipenem), monobactams (e.g., aztreonam), quinolones (quinolones) (e.g., fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin, and cinoxacin), tetracyclines (e.g., doxycycline, minocycline, tetracycline), and glycopeptides (glycopeptides) (e.g., vancomycin, teicoplanin). Other antibiotics include chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, mupirocin, and cationic peptides.

[0081] The efficacy of peptides alone and in combination with sub-optimal concentrations of antibiotics was evaluated therapeutically in infection models. Staphylococcus aureus is an important gram-positive pathogen and is a significant cause of antibiotic resistant infections. Briefly, the therapeutic effect of peptides was tested in a model of staphylococcus aureus infection 6 hours after infection had occurred by injecting the peptides alone and in combination with suboptimal concentrations of antibiotics. This would mimic the antibiotic resistant environment that occurs during infection, where the MIC of the resistant bacteria is too high to successfully treat (i.e., the antibiotic dose applied is less than optimal). It was demonstrated that the combination of antibiotics and peptides resulted in improved efficacy, suggesting the potential for combination therapy (see example 12).

[0082] The invention will now be described in more detail with reference to the following non-limiting examples. Although the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations thereof are encompassed within the spirit and scope of the description and claims.

Example 1

Anti-sepsis/anti-inflammatory activity

[0083]Polynucleotide arrays are used to determine the effect of cationic peptides on epithelial transcriptional responses. The a549 human epithelial cell line was maintained in dmem (gibco) and supplemented with 10% fetal bovine serum (FBS, Medicorp). A549 cells were plated in 100mm tissue culture dishes each containing 2.5X 10⁶Cells, cultured overnight, then mixed with 100ng/ml E.coli O111: b4LPS (Sigma) was incubated for 4 hours with 50. mu.g/ml peptide and medium or without peptide and medium alone (as control). After stimulation, cells were washed once with diethylpyrocarbonate-treated Phosphate Buffered Saline (PBS) and scraped with a cell brush. Total RNA was isolated using RNAquesous (Ambion, Austin, TX). The RNA pellet was resuspended In RNase-free water containing Superase-In (RNase inhibitor; Ambion). DNA contamination was removed using a DNA-free kit (Ambion). The quality of the RNA was estimated by electrophoresis on a 1% agarose gel.

[0084] The polynucleotide array used was a Human Operon array (identification number of the genome is PRHU04-S1) consisting of duplicate dots of approximately 14,000 Human oligomers. Probes were prepared with 10. mu.g total RNA and labeled with Cy3 or Cy5 labeled dUTP. The probe was purified and hybridized to a printed glass slide, overnight at 42 ℃ and then washed. After washing, images were taken with a PerkinElmer array scanner. The mean, median and background intensities of the spots were determined using image processing software (Imapolynlucleotide 5.0, Marina DelRey, Calif.). The background was removed using a "homemade" procedure. The program calculates the bottom intensity of each sub-cell (subgrid) as 10% and subtracts this value for each cell (grid). The analysis was performed using Genespring software (Redwood City, Calif.). The intensity value of the middle spot is obtained from a collection of spot values within a slide and compared to the values of all slides in this experiment, thereby normalizing the intensity of each spot. The relative change between the peptide-treated cells and the control cells can be seen in tables 1 and 2. Table 2 shows only those polynucleotides whose expression has changed significantly among the 14,000 polynucleotides tested. This data indicates that the peptide has the ability to broadly reduce LPS-induced polynucleotide expression.

[0085] In table 1, the study of the polynucleotide microarray indicated that SEQ ID NO: 27 effectively reduced the expression of c.coli O111: b4LPS upregulated expression of various polynucleotides. The peptides (50. mu.g/ml) and LPS (0.1. mu.g/ml) were incubated with A549 cells for 4 hours, or LPS alone was incubated with A549 cells for 4 hours, followed by isolation of RNA. Cy3/Cy 5-labeled cDNA probes were prepared using 5. mu.g total RNA and hybridized to a Human Operon array (PRHU 04). The third column of table 1 shows the intensity of the unstimulated cells. "ratio: LPS/control "column refers to the result of dividing the intensity of polynucleotide expression in cells stimulated with LPS by the intensity of unstimulated cells. "ratio: LPS + ID 27/control "column refers to the result of dividing the intensity of polynucleotide expression in cells stimulated with LPS and peptide by the intensity of unstimulated cells.

Table 1: the peptide SEQ ID27 reduced the expression of a polypeptide in a549 human epithelial cells by e.coli O111: b4LPS upregulated polynucleotide expression

Sequence accession numbera	Polynucleotide Gene function	Comparison: medium strength only	The ratio is: LPS/control	The ratio is: LPS + ID 27/control
					AL031983	Is unknown	0.032	302.8	5.1
L04510	ADP-ribosylation factor	0.655	213.6	1.4
					D87451	Ring finger protein 10	3.896	183.7	2.1
AK000869	Hypothetical proteins	0.138	120.1	2.3
					U78166	Ric-like expression in neurons	0.051	91.7	0.2
AJ001403	Tracheal bronchus mucin 5 subtype B	0.203	53.4	15.9
					AB040057	Serine/threonine protein kinase MASK	0.95	44.3	15.8
Z99756	Is unknown	0.141	35.9	14.0
					L42243	Interferon receptor 2	0.163	27.6	5.2
MN_016216	RNA lassoDebranching enzyme	6.151	22.3	10.9
					AK001589	Hypothetical proteins	0.646	19.2	1.3
AL137376	Is unknown	1.881	17.3	0.6
					AB007856	FEM-1-like death receptor binding proteins	2.627	15.7	0.6
AB007854	Growth arrest specific protein 7	0.845	14.8	2.2
					AK000353	Cytoplasmic ovarian cancer antigen 1	0.453	13.5	1.0
D14539	Myeloid/lymphoid or mixed lineage leukemia; is translocated; 1(MLLT1)	2.033	11.6	3.1
					X76785	Integration site of epstein-barr virus	0.728	11.6	1.9
M54915	Pim-1 oncogene	1.404	11.4	0.6
					NM_006092	Caspase recruitment domain 4	0.369	11.0	0.5
J03925	Integrin _ α M	0.272	9.9	4.2
					NM_001663	ADP-ribosylation factor 6	0.439	9.7	1.7
M23379	RAS p21 protein activator	0.567	9.3	2.8
					K02581	Soluble thymidine kinase 1	3.099	8.6	3.5
U94831	Transmembrane 9 superfamily member 1	3.265	7.1	1.5
					X70394	Zinc finger protein 146	1.463	6.9	1.7
AL137614	Hypothetical proteins	0.705	6.8	1.0
					U43083	Guanine nucleotide binding proteins	0.841	6.6	1.6
AL137648	DKFZp434J1813 protein	1.276	6.5	0.8
					AF085692	ATP-binding cassette subfamily C (CFTR/MRP) member 3	3.175	6.5	2.4
AK001239	Hypothetical protein FLJ10377	2.204	6.4	1.3
					NM_001679	ATP enzyme Na+/K+Transporter beta 3 polypeptides	2.402	6.3	0.9

L24804	Inactive progesterone receptors	3.403	6.1	1.1
					U15932	Dual specific phosphatase 5	0.854	6.1	2.1
M36067	ATP-dependent DNA ligase I	1.354	6.1	2.2
					AL161951	Is unknown	0.728	5.8	1.9
M59820	Colony stimulating factor 3 receptor	0.38	5.7	2.0
					AL050290	Spermidine/spermine N1-acyltransferase	2.724	5.6	1.4
NM_002291	Laminin _ beta 1	1.278	5.6	1.8
					X06614	Retinoic acid receptor alpha	1.924	5.5	0.8
AB007896	Putative L-type neutral amino acid transporter	0.94	5.3	1.8
					AL050333	DKFZP564B116 protein	1.272	5.3	0.6
AK001093	Hypothetical proteins	1.729	5.3	2.0
					NM_016406	Hypothetical proteins	1.314	5.2	1.2
M86546	Pre-B cell leukemia transcription factor 1	1.113	5.2	2.2
					X56777	Zona pellucida glycoprotein 3A	1.414	5.0	1.4
NM_013400	Replication initiation region protein	1.241	4.9	2.0
					NM_002309	Leukemia inhibitory factor	1.286	4.8	1.9
NM_001940	Dentate red nucleus globus pallidus atrophy	2.034	4.7	1.2
					U91316	Cytosolic acyl-CoA thioester hydrolase	2.043	4.7	1.4
X76104	Death-related protein kinase 1	1.118	4.6	1.8
					AF131838	Is unknown	1.879	4.6	1.4
AL050348	Is unknown	8.502	4.4	1.7
					D42085	KIAA0095 gene product	1.323	4.4	1.2
X92896	Is unknown	1.675	4.3	1.5
					U26648	Syntaxin 5A	1.59	4.3	1.4
X85750	Is associated with the differentiation of monocytes into macrophages	1.01	4.3	1.1
					D14043	CD164 antigen sialoglycoproteins	1.683	4.2	1.0
J04513	Fibroblast growth factor 2	1.281	4.0	0.9
					U19796	Melanoma associated antigens	1.618	4.0	0.6
AK000087	Hypothetical proteins	1.459	3.9	1.0
					AK001569	Hypothetical proteins	1.508	3.9	1.2
AF189009	Ubiquitin 2	1.448	3.8	1.3
					U60205	sterol-C4-methyl oxidase-like	1.569	3.7	0.8
AK000562	Hypothetical proteins	1.166	3.7	0.6
					AL096739	Is unknown	3.66	3.7	0.5
AK000366	Hypothetical proteins	15.192	3.5	1.0
					NM_006325	RAN Member RAS oncogene superfamily	1.242	3.5	1.4
X51688	Cyclin A2	1.772	3.3	1.0
					U34252	Aldehyde dehydrogenase 9	1.264	3.3	1.2
NM_013241	Egg containing FH1/FH2 domain	1.264	3.3	0.6

	White colour (Bai)
					AF112219	Esterase D/formyl glutathione hydrolase	1.839	3.3	1.1
NM_016237	Interphase promoting complex subunit 5	2.71	3.2	0.9
					AB014569	KIAA0669 Gene product	2.762	3.2	0.2
AF151047	Hypothetical proteins	3.062	3.1	1.0
					X92972	Protein phosphatase 6 catalytic subunit	2.615	3.1	1.1
AF035309	Proteasome 26S subunit ATPase 5	5.628	3.1	1.3
					U52960	SRB7 homologs	1.391	3.1	0.8
J04058	Electron transport flavoprotein alpha polypeptides	3.265	3.1	1.2
					M57230	Interleukin 6 signal transducer	0.793	3.1	1.0
U78027	Alpha galactosidase enzyme	3.519	3.1	1.1
					AK000264	Is unknown	2.533	3.0	0.6
X80692	Mitogen activated protein kinase 6	2.463	2.9	1.3
					L25931	Nuclear lamin B receptor	2.186	2.7	0.7
X13334	CD14 antigen	0.393	2.5	1.1
					M32315	Tumor necrosis factor receptor superfamily member 1B	0.639	2.4	0.4
NM 004862	TNF-alpha factor induced by LPS	6.077	2.3	1.1
					AL050337	Interferon gamma receptor 1	2.064	2.1	1.0

^aAll Accession Numbers in tables 1 through 64 refer to GenBank Accession Numbers (Accession Numbers).

[0086] In table 2, the study of the polynucleotide microarray showed that cationic peptide at a concentration of 50 μ g/ml effectively reduced the expression of c.coli O111: b4 expression of a number of polynucleotides upregulated by LPS. The peptides and LPS were incubated with a549 cells for 4 hours, or LPS alone was incubated with a549 cells for 4 hours, followed by isolation of RNA. Cy3/Cy 5-labeled cDNA probes were prepared using 5. mu.g total RNA and hybridized to a Human Operon array (PRHU 04). The third column of table 2 shows the intensity of the unstimulated cells. "ratio: LPS/control "column refers to the result of dividing the intensity of polynucleotide expression in cells stimulated with LPS by the intensity of unstimulated cells. The other columns refer to the results of dividing the intensity of polynucleotide expression in cells stimulated with LPS and peptide by the intensity of unstimulated cells.

[0087]Table 2: by Escherichia coli O111: b4LPS upregulation and activationIonic peptide reduced human A549 epithelial cell polynucleotide expression

Registration number

Gene

Comparison: medium strength only

The ratio is: LPS/control

The ratio is: LPS + ID 27/control

The ratio is: LPS + ID 16/control

The ratio is: LPS + ID 22/control

AL031983	Is unknown	0.03	302.8	5.06	6.91	0.31
							L04510	ADP-ribosylation factor	0.66	213.6	1.4	2.44	3.79
D87451	Ring finger protein	3.90	183.7	2.1	3.68	4.28
							AK000869	Hypothetical proteins	0.14	120.1	2.34	2.57	2.58
U78166	Ric sample	0.05	91.7	0.20	16.88	21.37
							X03066	Class II MHCDO beta	0.06	36.5	4.90	12.13	0.98
AK001904	Hypothetical proteins	0.03	32.8	5.93	0.37	0.37
							AB037722	Is unknown	0.03	21.4	0.30	0.30	2.36
AK001589	Hypothetical proteins	0.65	19.2	1.26	0.02	0.43
							AL137376	Is unknown	1.88	17.3	0.64	1.30	1.35
L19185	Thioredoxin dependent peroxiredoxin reductase 1	0.06	16.3	0.18	2.15	0.18
							J05068	Transcobalamin protein I	0.04	15.9	1.78	4.34	0.83
AB007856	FEM-1-like death receptor binding proteins	2.63	15.7	0.62	3.38	0.96
							AK000353	Cytoplasmic ovarian cancer antigen 1	0.45	13.5	1.02	1.73	2.33
X16940	Smooth muscle intestinal actin gamma 2	0.21	11.8	3.24	0.05	2.26
							M54915	Pim-1 oncogene	1.40	11.4	0.63	1.25	1.83
AL122111	Hypothetical proteins	0.37	10.9	0.21	1.35	0.03
							M95678	Phospholipase C beta 2	0.22	7.2	2.38	0.05	1.33
AK001239	Hypothetical proteins	2.20	6.4	1.27	1.89	2.25
							AC004849	Is unknown	0.14	6.3	0.07	2.70	0.07
X06614	Retinoic acid receptor alpha	1.92	5.5	0.77	1.43	1.03
							AB007896	Putative L-type neutral amino acid transporter	0.94	5.3	1.82	2.15	2.41
AB010894	BAI1 related protein	0.69	5.0	1.38	1.03	1.80
							U52522	RAC1 mate	1.98	2.9	1.35	0.48	1.38
AK001440	Hypothetical proteins	1.02	2.7	0.43	1.20	0.01
							NM_001148	Ankyrin 2 of neurons	0.26	2.5	0.82	0.04	0.66
X07173	Alpha-inhibitor H2	0.33	2.2	0.44	0.03	0.51
							AF095687	Brain and nasopharyngeal carcinoma susceptible protein	0.39	2.1	0.48	0.03	0.98

NM_016382	NK cell activation-inducing ligand NAIL	0.27	2.1	0.81	0.59	0.04
							AB023198	KIAA0981 protein	0.39	2.0	0.43	0.81	0.92

Example 2

Neutralizing stimulation of immune cells

[0088]Compounds were tested to suppress (neutralize) gram negative bacteria and gramThe ability of positive bacterial products to stimulate immune cells. Bacterial products stimulate cells of the immune system, thereby producing inflammatory cytokines, which when uncompressed, can lead to sepsis. The experiments were initiated using the murine macrophage line RAW 264.7, obtained from the American Type Culture Collection (Manassas, Va.), the human epithelial cell line A549, and primary macrophages from BALB/c murine bone marrow (Charles River Laboratories, Wilmington, Mass.). Cells from murine bone marrow were cultured in 150mm culture plates in Darbeck modified eagle's medium (DMEM; Life Technologies, Burlington, ON) supplemented with 20% FBS (Sigma Chemical Co, St. Louis, Mo.) and 20% L of cell conditioned medium as the M-CSF source. When macrophages were 60-80% confluent, the L cell conditioned medium was removed from the medium, incubated for 14-16 hours until they entered quiescent state, and then treated with 10ng/ml LPS or 100ng/ml LPS + 20. mu.g/ml peptide for 24 hours. By ELISA (R)&D Systems, Minneapolis, MN) determined the cytokine release into the culture supernatant. Cell lines RAW 264.7 and a549 were maintained in DMEM supplemented with 10% fetal calf serum. RAW 264.7 cells at 10 per well⁶The density of individual cells was seeded in 24-well DMEM-containing plates, and A549 cells were plated at 10 per well⁵Individual cell densities were seeded into 24-well plates containing DMEM, both at 37 ℃ in 5% CO₂Culturing overnight. DMEM was aspirated from overnight grown cells and replaced with fresh medium. In some experiments, volunteers' blood was collected by venipuncture (following procedures approved by the ethical committee for UBC clinical studies, certification No. C00-0537) into tubes containing 14.3USP units heparin/ml blood (Becton Dickinson, Franklin Lakes, NJ). The blood was mixed with LPS with or without peptide in a polypropylene tube at 37 ℃ for 6 hours. Samples were centrifuged at 2000 Xg for 5 min, plasma collected and stored at-20 ℃ until ELISA (R)&D Systems) was taken out for IL-8 analysis. In these cell experiments, cells were incubated with LPS or other bacterial products at 37 ℃ in 5% CO₂Incubated for 6-24 hours. Salmonella typhimurium LPS and e.coli O111: b4LPS was purchased from Sigma. From golden grape ballLipoteichoic acid (LTA) from bacteria (Sigma) was resuspended in endotoxin-free water (Sigma). The LTA preparation was subjected to the limulus amebocyte lysate test (Sigma) to confirm that it was not significantly contaminated with endotoxin. Endotoxin contamination was less than 1ng/ml and this concentration did not result in significant cytokine production by RAW 264.7 cells. Capless lipoarabinomannan (AraLAM) was gifted by doctor John t. belisle, ColoradoState University. The AraLAM of Mycobacterium (Mycobacterium) was filter sterilized and endotoxin contamination was determined by limulus amoebocyte assay and found to be 3.75ng/1.0mg LAM. A range of concentrations of cationic peptide is added simultaneously with (or subsequently to) the addition of LPS. The supernatant was removed and ELISA (R) was used&D Systems) were tested for the production of cytokines. All experiments were performed at least three times and similar results were obtained. To demonstrate anti-sepsis activity in vivo, 2 or 3 μ g e.coli O111: b4LPS phosphate buffered saline (PBS; pH 7.2) was injected intraperitoneally into galactosamine sensitized female CD-1 or BALB/c mice for 8-10 weeks, thereby inducing sepsis. In experiments involving peptides, 200 μ g of peptide in 100 μ l sterile water was injected at different intraperitoneal sites within 10 minutes of LPS injection. In other experiments, 400 μ g e.coli O111: b4LPS was injected into CD-1 mice 10 minutes later, and the peptide (200. mu.g) was introduced by intraperitoneal injection. Survival was monitored 48 hours after injection.

[0089] It has been traditionally thought that the production of excess TNF- α is linked to the onset of sepsis. The three types of LPS, LTA or AraLAM used in this example represent products released by gram-negative and gram-positive bacteria. SEQ ID NO: 1 is capable of significantly reducing the level of a peptide derived from salmonella typhimurium, burkholderia cepacia (b.cepacia) and e.coli O111: b4 TNF- α production stimulated by LPS was slightly less affected (Table 3). In the latter two cases it can be seen that peptides at concentrations as low as 1. mu.g/ml (0.25nM) can also lead to a significant reduction in TNF-. alpha.production. A different peptide, SEQ ID NO: 3 did not reduce LPS-induced TNF- α production in RAW macrophages, suggesting that this is a non-uniform and unpredictable property of the cationic peptide. Representative peptides of each formula were also tested for their effect as measured by e.coli O111; b4 ability of LPS to stimulate TNF- α production (table 4). Although many of these peptides reduced TNF- α production by at least 60%, their ability to reduce TNF- α production was differential.

[0090] Certain concentrations of the peptide SEQ ID NO: 1 and SEQ ID NO: 2 can also impair the ability of the bacterial product to stimulate the production of IL-8 by epithelial cell lines. LPS is known to be effective in stimulating IL-8 production by epithelial cells. The peptides were able to suppress the IL-8-induced response of epithelial cells to LPS at low concentrations (1-20. mu.g/ml) (tables 5-7). The peptide SEQ ID2 also inhibited IL-8 production in human whole blood induced by LPS (Table 4). In contrast, high concentrations of the peptide SEQ ID NO: 1 (50-100. mu.g/ml) actually resulted in elevated IL-8 levels (Table 5). This indicates that the peptides have different effects at different concentrations.

[0091] The effect of the peptide on inflammatory stimulation was also demonstrated in primary murine cells, the peptide SEQ ID NO: 1 significantly reduced TNF- α production (> 90%) in bone marrow derived macrophages of BALB/c mice that had been treated with 100ng/ml e.coli O111: b4 was LPS-stimulated (table 8). These experiments were performed in the presence of serum, which contains the LPS Binding Protein (LBP), a protein that mediates the rapid binding of LPS to CD 14. Coli LPS at 100ng/ml one hour after stimulation with SEQ ID NO: 1 delayed addition to the macrophage supernatant still resulted in a significant reduction in TNF-. alpha.production (70%, Table 9).

[0092] SEQ ID NO: 1 are capable of preventing the in vitro induction of TNF- α by LPS, and in accordance with this, certain peptides are also capable of protecting mice from lethal shock induced by high concentrations of LPS. In some experiments, CD-1 mice were made allergic to LPS by pre-injection of galactosamine. Mice sensitized with galactosamine were injected with 3 μ g of e.coli O111: b4 was killed within 4-6 hours after LPS. At 15 minutes after LPS injection, 200 μ g of SEQ ID NO: 1, 50% of the mice survived (Table 10). In other experiments, higher concentrations of LPS were injected into BALB/c mice without prior injections of galactosamine type D, and the protection provided by the peptide was 100%, compared to no survival in the control group (table 13). The other peptides selected were also found to be protective in these models (tables 11, 12).

[0093] Cationic peptides are also capable of attenuating the stimulation of macrophages by products of gram-positive bacteria, such as hatfat-free arabinomannan (AraLAM) of mycobacteria and LTA of staphylococcus aureus (s. For example, SEQ ID NO: 1 inhibited the induction of TNF-. alpha.in RAW 264.7 cells by the gram-positive bacterial products LTA (Table 14) and AraLAM (Table 15), to a lesser extent. Another peptide, SEQ ID NO: 2 reduced LTA induction of TNF- α in RAW 264.7 cells. 1 μ g/ml of SEQ ID NO: 1 was able to significantly reduce (> 75%) the induction of TNF-. alpha.production by 1. mu.g/ml Staphylococcus aureus LTA. In SEQ ID NO: 1 concentration of 20 mug/ml, the inhibition rate of AraLAM induced TNF-alpha is more than 60%. Polymyxin b (pmb) can be introduced as a control to demonstrate that the amino acid sequence shown in SEQ ID NO: 1 inhibition of AraLAM induced TNF- α endotoxin contamination was not a significant factor. These results demonstrate that cationic peptides can attenuate the pro-inflammatory cytokine response of the immune system to bacterial products.

[0094]Table 3: in RAW 264.7 cells SEQ ID NO: 1 reduces TNF- α production induced by LPS. In the presence of the indicated concentrations of SEQ ID1, the strain was tested with 100ng/ml Salmonella typhimurium LPS, 100ng/ml Burkholderia cepacia LPS and 100ng/ml E.coli O111: b4LPS, stimulated RAW 264.7 murine macrophages for 6 hours. The concentration of TNF-. alpha.released into the culture supernatant was determined by ELISA. 100% represents the amount of TNF- α obtained by incubating RAW 264.7 cells with LPS alone for 6 hours (salmonella typhimurium LPS ═ 34.5 ± 3.2ng/ml, burkholderia cepacia LPS ═ 11.6 ± 2.9ng/ml, e.coli O111: B4LPS ═ 30.8 ± 2.4 ng/ml). Background levels of TNF- α production from RAW 264.7 cells cultured for 6 hours without stimulation were 0.037-0.192 ng/ml. Experimental data were derived from two identical samples and are expressed as the mean + standard deviation of three experiments.

Amount of SEQ ID1 (. mu.g/ml)	Inhibition of TNF-alpha (%)*
				Burkholderia cepacia LPS	Escherichia coli LPS	Salmonella typhimurium LPS
	0.1	8.5±2.9	0.0±0.6				0.0±0
1				23.0±11.4	36.6±7.5	9.8±6.6
	5	55.4±8	65.0±3.6				31.1±7.0
10				63.1±8	75.0±3.4	37.4±7.5
	20	71.7±5.8	81.0±3.5				58.5±10.5
50				86.7±4.3	92.6±2.5	73.1±9.1

[0095]Table 4: cationic peptides reduced TNF- α production induced by e.coli LPS in RAW 264.7 cells. Coli O111: b4LPS stimulated RAW 264.7 murine macrophages for 6 hours. The concentration of TNF-. alpha.released into the culture supernatant was determined by ELISA. Background levels of TNF- α production from RAW 264.7 cells cultured for 6 hours without stimulation were 0.037-0.192 ng/ml. Experimental data were derived from two identical samples and are expressed as the mean + standard deviation of three experiments.

Peptide (20. mu.g/ml)	Inhibition of TNF-alpha (%)
		SEQ ID 5	65.6±1.6
SEQ ID 6	59.8±1.2
		SEQ ID 7	50.6±0.6
SEQ ID 8	39.3±1.9
		SEQ ID 9	58.7±0.8
SEQ ID 10	55.5±0.52
		SEQ ID 12	52.1±0.38
SEQ ID 13	62.4±0.85
		SEQ ID 14	50.8±1.67
SEQ ID 15	69.4±0.84
		SEQ ID 16	37.5±0.66
SEQ ID 17	28.3±3.71
		SEQ ID 19	69.9±0.09
SEQ ID 20	66.1±0.78
		SEQ ID 21	67.8±0.6
SEQ ID 22	73.3±0.36
		SEQ ID 23	83.6±0.32
SEQ ID 24	60.5±0.17
		SEQ ID 26	54.9±1.6
SEQ ID 27	51.1±2.8
		SEQ ID 28	56±1.1
SEQ ID 29	58.9±0.005
		SEQ ID 31	60.3±0.6
SEQ ID 33	62.1±0.08
		SEQ ID 34	53.3±0.9
SEQ ID 35	60.7±0.76
		SEQ ID 36	63±0.24
SEQ ID 37	58.9±0.67
		SEQ ID 38	54±1
SEQ ID 40	75±0.45
		SEQ ID 41	86±0.37
SEQ ID 42	80.5±0.76
		SEQ ID 43	88.2±0.65
SEQ ID 44	44.9±1.5
		SEQ ID 45	44.7±0.39
SEQ ID 47	36.9±2.2
		SEQ ID 48	64±0.67
SEQ ID 49	86.9±0.69
		SEQ ID 53	46.5±1.3
SEQ ID 54	64±0.73

[0096]Table 5: SEQ ID1 reduced IL-8 production induced by LPS in a549 cells. A549 cells were stimulated with increasing concentrations of SEQ ID1 for 24 hours in the presence of LPS (100ng/ml e.coli O111: B4). IL-8 concentration in the cultures was determined by ELISA. The background level of IL-8 in individual cells was 0.172. + -. 0.029 ng/ml. Data are presented as mean + standard deviation of three experiments.

SEQ ID 1(μg/ml)	Inhibition of IL-8 (%)
		0.1	1±0.3
1	32±10
		10	60±9
20	47±12
		50	40±13
100	0

[0097]Table 6: SEQ ID2 reduced IL-8 production induced by e.coli LPS in a549 cells. Human a549 epithelial cells were stimulated with increasing concentrations of SEQ ID2 for 24 hours in the presence of LPS (100ng/ml e. Measurement of culture supernatant by ELISAIL-8 concentration of (a). Data are presented as mean + standard deviation of three experiments.

Concentration of SEQ ID2 (. mu.g/ml)	Inhibition of IL-8 (%)
		0.1	6.8±9.6
1	12.8±24.5
		10	29.0±26.0
50	39.8±1.6
		100	45.0±3.5

[0098]Table 7: SEQ ID2 reduces IL-8 induced by e. Coli O111B4LPS and increasing concentrations of peptide were used to stimulate human whole blood for 4 hours. Human whole blood samples were centrifuged, serum removed and IL-8 concentration determined by ELISA. Data are presented as the mean of two donors.

SEQ ID 2(μg/ml)	IL-8(pg/ml)
		0	3205
10	1912
		50	1458

[0099]Table 8: SEQ ID1 reduces TNF- α production induced by e. BALB/c murine bone marrow-derived macrophages were mixed with 100ng/ml E.coli O111: b4LPS was co-cultured for 6 hours or 24 hours. Supernatants were collected and TNF-. alpha.levels were determined by ELISA. Data represent the amount of TNF-. alpha.that was obtained from two culture wells in which bone marrow-derived macrophages were incubated with LPS alone for 6 hours (1.1. + -. 0.09ng/ml) or 24 hours (1.7. + -. 0.2 ng/ml). Background levels of TNF- α were: the concentration was 0.038. + -. 0.008ng/ml at 6 hours, and 0.06. + -. 0.012ng/ml at 24 hours.

SEQ ID 1(μg/ml)	Production of TNF-alpha (ng/ml)
				6 hours	24 hours
LPS Only	1.1	1.7
1	0.02	0.048
			10	0.036	0.08

100	0.033	0.044
			Control without LPS	0.038	0.06

[00100]Table 9: postpone the addition of SEQ ID1 to a549 cells from inhibiting TNF- α production induced by e. At gradually later time points, peptides (20 μ g/ml) were added to the cells already containing a549 human epithelial cells and 100ng/ml e.coli O111: b4LPS culture wells. Supernatants were collected after 6 hours and TNF-. alpha.levels were determined by ELISA. Data are presented as mean + standard deviation of three experiments.

Time to addition of SEQ ID1 after LPS addition (min)	Inhibition of TNF-alpha (%)
		0	98.3±0.3
15	89.3±3.8
		30	83±4.6
60	68±8
		90	53±8

[00101]Table 10: the protection against fatal endotoxemia in the semi-lactosamine sensitized CD-1 mice is performed by SEQ ID 1. CD-1 mice (9 weeks old) were made allergic to endotoxin by three intraperitoneal injections of galactosamine (20mg, dissolved in 0.1ml sterile PBS). Coli O111: b4LPS (3. mu.g in 0.1ml PBS) induced endotoxin shock. 15 minutes after LPS injection, the peptide SEQ ID1 was injected again at a different intraperitoneal site (200 μ g/mouse ═ 8 mg/kg). The mice were monitored for 48 hours and the results were recorded.

D-galactosamine treatment	E.coli O111：B4LPS	Peptides or buffers	Total number of mice	Survival after endotoxic shock
					0	3μg	PBS	5	5(100％)
20mg	3μg	PBS	12	0(0％)
					20mg	3μg	SEQ ID 1	12	6(50％)

[00102]Table 11: protection against fatal endotoxemia in semi-lactosamine-sensitized CD-1 mice is achieved by cationic peptides. CD-1 mice (9 weeks old) were made allergic to endotoxin by intraperitoneal injection of galactosamine (20mg, dissolved in 0.1ml sterile PBS). Coli O111: b4LPS (2. mu.g in 0.1ml PBS) induced endotoxin shock. 15 minutes after LPS injection, the peptide was injected at a different intraperitoneal site (8 mg/kg/200 μ g/mouse). The mice were monitored for 48 hours and the results were recorded.

Peptide treatment	Coli O111 added: b4LPS	Number of mice	Survival (%)
				Control (no peptide)	2μg	5	0
SEQ ID 6	2μg	5	40
				SEQ ID 13	2μg	5	20
SEQ ID 17	2μg	5	40
				SEQ ID 24	2μg	5	0
SEQ ID 27	2μg	5	20

[00103]Table 12: by cationic peptide pairsGalactosamine sensitized BALB/c mice are protected against fatal endotoxemia. BALB/c mice (8 weeks old) were made allergic to endotoxin by intraperitoneal injection of galactosamine (20mg, dissolved in 0.1ml sterile PBS). Coli O111: b4LPS (2. mu.g in 0.1ml PBS) induced endotoxin shock. 15 minutes after LPS injection, the peptide was injected at a different intraperitoneal site (8 mg/kg/200 μ g/mouse). The mice were monitored for 48 hours and the results were recorded.

Peptide treatment	Coli o111 added: b4LPS	Number of mice	Survival (%)
				Without peptides	2μg	10	10
SEQ ID 1	2μg	6	17
				SEQ ID 3	2μg	6	0
SEQ ID 5	2μg	6	17
				SEQ ID 6	2μg	6	17
SEQ ID 12	2μg	6	17
				SEQ ID 13	2μg	6	33
SEQ ID 15	2μg	6	0
				SEQ ID 16	2μg	6	0
SEQ ID 17	2μg	6	17
				SEQ ID 23	2μg	6	0
SEQ ID 24	2μg	6	17
				SEQ ID 26	2μg	6	0
SEQ ID 27	2μg	6	50
				SEQ ID 29	2μg	6	0
SEQ ID 37	2μg	6	0
				SEQ ID 38	2μg	6	0
SEQ ID 41	2μg	6	0
				SEQ ID 44	2μg	6	0
SEQ ID 45	2μg	6	0

[00104]Table 13: BALB/c mice were protected against fatal endotoxemia by SEQ ID 1. Coli O111: b4LPS was injected intraperitoneally into BALB/c mice. Peptides were injected at a different peritoneal site (200 μ g/mouse ═ 8 mg/kg). The mice were monitored for 48 hours and the results were recorded.

Peptide treatment	Coli o111 added: b4LPS	Number of mice	Survival (%)
				Without peptides	400μg	5	0
SEQ ID 1	400μg	5	100

[00105]Table 14: the peptides inhibit TNF- α production induced by LTA of Staphylococcus aureus. RAW 264.7 murine macrophages were stimulated with 1 μ g/ml staphylococcus aureus LTA in the presence and absence of increasing concentrations of peptide. Supernatants were collected and TNF-. alpha.levels were determined by ELISA. Background levels of TNF- α production from RAW 264.7 cells cultured for 6 hours without stimulation were 0.037-0.192 ng/ml. Data are presented as mean + standard deviation of three or more experiments.

Added SEQ ID1(μ g/ml)	Inhibition of TNF-alpha (%)
		0.1	44.5±12.5
1	76.7±6.4
		5	91±1
10	94.5±1.5
		20	96±1

[00106]Table 15: the peptide inhibits TNF-alpha production induced by non-capped lipid arabinomannan of mycobacteria. RAW 264.7 murine macrophages were stimulated with 1. mu.g/ml AraLAM in the presence of 20. mu.g/ml peptide or polymyxin B (Polymyxin B) or without peptide. Supernatants were collected and TNF-. alpha.levels were determined by ELISA. Background levels of TNF- α production from RAW 264.7 cells cultured for 6 hours without stimulation were TNF- α levels of 0.037-0.192 ng/ml. Data are presented as mean + standard deviation of three or more experiments.

Peptide (20. mu.g/ml)	Inhibition of TNF-alpha (%)
		Without peptides	0
SEQ ID 1	64±5.9
		Polymyxin B	15±2

Example 3

Evaluation of toxicity of cationic peptides

[00107]Two methods were used to measure the potential toxicity of the peptides. First, the assay was performed using a Cytotoxicity Detection Kit (Roche) (lactate dehydrogenase-LDH). This is a type of cell death and cell lysisThe solution was subjected to a quantitative colorimetric assay based on measurement of LDH activity released from the cytosol of damaged cells into the supernatant. LDH is a stable cytoplasmic enzyme present in all cells that is released into the supernatant of cell cultures when the plasma membrane is damaged. An increase in the number of dead or plasma membrane-damaged cells leads to an increase in LDH enzyme activity in the culture supernatant, and OD can be measured using an ELISA plate recorder_490nm(the amount of color formed in this assay is directly proportional to the number of lysed cells). In this assay, human bronchial epithelial cells (16HBEo14, HBE) were incubated with 100 μ g of peptide for 24 hours, the supernatant removed and assayed for LDH. Another assay used to measure toxicity of cationic peptides is the WST-1 assay (Roche). The assay is a colorimetric assay for the quantification of cell proliferation and cell viability based on the ability of mitochondrial dehydrogenase in living cells to cleave tetrazolium salt WST-1 (as "para")³H]-substitution of thymidine uptake assay, which is not radioactive). In this assay, HBE cells were incubated with 100. mu.g of peptide for 24 hours, then 10. mu.l of Cell Proliferation Reagent (WST-1) was added per well. Cells were incubated with reagents and OD was measured using an ELISA plate recorder_490nm。

[00108] The results shown below in tables 16 and 17 indicate that most of the peptides are not toxic to the cells tested. However, the results of the two analyses indicated that four of the peptides of formula F (SEQ ID NOS: 40, 41, 42 and 43) indeed caused membrane damage.

[00109]Table 16: toxicity of the cationic peptides was measured by LDH release assay. Human HBE bronchial epithelial cells were incubated with 100. mu.g/ml peptide or polymyxin B for 24 hours. LDH activity was measured in cell culture supernatants. As a control, Triton X-100 was added to release 100% LDH. Data are presented as mean ± standard deviation. Only peptides SEQ ID40, 41, 42 and 43 showed some significant toxicity.

Treatment of	LDH Release (OD)490nm)
		Cell-free controls	0.6±0.1
Control of Triton X-100	4.6±0.1
		Control without peptide	1.0±0.05
SEQ ID 1	1.18±0.05
		SEQ ID 3	1.05±0.04
SEQ ID 6	0.97±0.02
		SEQ ID 7	1.01±0.04
SEQ ID 9	1.6±0.03
		SEQ ID 10	1.04±0.04
SEQ ID 13	0.93±0.06
		SEQ ID 14	0.99±0.05
SEQ ID 16	0.91±0.04
		SEQ ID 17	0.94±0.04
SEQ ID 19	1.08±0.02
		SEQ ID 20	1.05±0.03
SEQ ID 21	1.06±0.04
		SEQ ID 22	1.29±0.12
SEQ ID 23	1.26±0.46
		SEQ ID 24	1.05±0.01
SEQ ID 26	0.93±0.04
		SEQ ID 27	0.91±0.04
SEQ ID 28	0.96±0.06
		SEQ ID 29	0.99±0.02
SEQ ID 31	0.98±0.03
		SEQ ID 33	1.03±0.05
SEQ ID 34	1.02±0.03
		SEQ ID 35	0.88±0.03
SEQ ID 36	0.85±0.04
		SEQ ID 37	0.96±0.04
SEQ ID 38	0.95±0.02
		SEQ ID 40	2.8±0.5
SEQ ID 41	3.3±0.2
		SEQ ID 42	3.4±0.2
SEQ ID 43	4.3±0.2
		SEQ ID 44	0.97±0.03
SEQ ID 45	0.98±0.04
		SEQ ID 47	1.05±0.05
SEQ ID 48	0.95±0.05
		SEQ ID 53	1.03±0.06

Polymyxin B

1.21±0.03

[0100]Table 17: toxicity of the cationic peptides was measured by WST-1 analysis. HBE cells were incubated with 100. mu.g/ml peptide or polymyxin B for 24 hours and cell survival was then measured. Data are presented as mean ± standard deviation. As a control, Triton X-100 was added to release 100% LDH. Only peptides SEQ ID40, 41, 42 and 43 showed some significant toxicity.

Treatment of	OD490nm
		Cell-free controls	0.24±0.01
Control of Triton X-100	0.26±0.01
		Control without peptide	1.63±0.16
SEQ ID 1	1.62±0.34
		SEQ ID 3	1.35±0.12
SEQ ID 10	1.22±0.05
		SEQ ID 6	1.81±0.05
SEQ ID 7	1.78±0.10
		SEQ ID 9	1.69±0.29
SEQ ID 13	1.23±0.11
		SEQ ID 14	1.25±0.02
SEQ ID 16	1.39±0.26
		SEQ ID 17	1.60±0.46
SEQ ID 19	1.42±0.15
		SEQ ID 20	1.61±0.21
SEQ ID 21	1.28±0.07
		SEQ ID 22	1.33±0.07
SEQ ID 23	1.14±0.24
		SEQ ID 24	1.27±0.16
SEQ ID 26	1.42±0.11
		SEQ ID 27	1.63±0.03
SEQ ID 28	1.69±0.03
		SEQ ID 29	1.75±0.09
SEQ ID 31	1.84±0.06
		SEQID 33	1.75±0.21
SEQ ID 34	0.96±0.05
		SEQ ID 35	1.00±0.08
SEQ ID 36	1.58±0.05
		SEQ ID 37	1.67±0.02
SEQ ID 38	1.83±0.03
		SEQ ID 40	0.46±0.06
SEQ ID 41	0.40±0.01
		SEQ ID 42	0.39±0.08
SEQ ID 43	0.46±0.10
		SEQ ID 44	1.49±0.39
SEQ ID 45	1.54±0.35
		SEQ ID 47	1.14±0.23
SEQ ID 48	0.93±0.08
		SEQ ID 53	1.51±0.37

Polymyxin B

1.30±0.13

Example 4

Modulation of polynucleotides by cationic peptides

[0101]Polynucleotide arrays are used to measure the effect of the cationic peptides themselves on the transcriptional response of macrophages and epithelial cells. Murine macrophage RAW 264.7, human bronchial cells (HBE) or A549 human epithelial cells were plated in 150mm tissue culture dishes at a density of 5.6X 10 cells per dish⁶Cells were cultured overnight and then incubated with 50. mu.g/ml peptide for 4 hours or only with the medium. After stimulation, cells were washed once with diethylpyrocarbonate-treated PBS and scraped from the culture dish with a cell brush. Total RNA was isolated using Trizol (GibcoLife technologies). The RNA pellet was resuspended in RNase-free water containing RNase inhibitor (Ambion, Austin, TX). The RNA was treated with DNaseI (Clontech, Palo Alto, Calif.) at 37 ℃ for one hour. Add stop mixture (0.1M EDTA [ pH 8.0 ]]1mg/ml glycogen), the sample is extracted once with phenol/chloroform/isoamyl alcohol (25: 24: 1) and once with chloroform. Then 2.5 volumes of 100% ethanol and 1/10 volumes of sodium acetate were added and the RNA was precipitated at pH 5.2. The RNA was resuspended in RNase-free water containing RNase inhibitor (Ambion) and stored at-70 ℃. The quality of the RNA was estimated by electrophoresis on a 1% agarose gel. Using the isolated RNA as a template, a beta-actin specific primer (5' -GTCCCTGTAT)GCCTCTGGTC-3 ' (SEQ ID NO: 55) and 5'-GATGTCACGCACGATTTCC-3' (SEQ ID NO: 56)) were subjected to PCR amplification to determine whether there was genomic DNA contamination. Agarose gel electrophoresis and ethidium bromide staining confirmed that no amplification product was present after 35 cycles.

[0102]Atlas cDNA expression arrays (Clontech, Palo Alto, Calif.) consisted of 588 via-selected murine cDNAs spotted in duplicate (in duplcate) on a positively charged membrane, which were used in earlier polynucleotide array studies (Table 18, 19). Prepared from 5 mu g of total RNA³²P radiolabeled cDNA probe, which was incubated with the array at 71 ℃ overnight. A flood wash was performed and then exposed to a phospho-screen imaging system (Molecular Dynamics, Sunnyvale, Calif.) for 3 days at 4 ℃. Images were acquired using a molecular dynamics PSI phosphor screen imager (phosphor). Hybridization signals were analyzed using atlas image 1.0 image analysis software (Clontech) and Excel (Microsoft, Redmond, WA). The intensity of each spot was corrected for background levels and normalized for differences between probe labels by using the average of 5 polynucleotides found to have little change between stimulation conditions: beta actin, ubiquitin, ribosomal protein S29, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Ca²⁺Binding proteins. When the normalized hybridization signal for a given cDNA is less than 20, it is assigned a value of 20, from which the ratio and relative expression are calculated.

[0103] The polynucleotide array used in the next step (tables 21-26) is the Resgen Human cDNA array (identification number of the genome is PRHU03-S3), which consists of duplicate 7,458 personal cDNA spots. Probes were prepared with 15-20. mu.g total RNA and labeled with Cy 3-labeled dUTP. The probe was purified and hybridized to a printed glass slide, overnight at 42 ℃ and then washed. After washing, images were taken with a Virtek slide recorder. The mean, median and background intensities of the spots were determined using image processing software (Imagene 4.1, Marina Del Rey, Calif.). The normalization process and analysis were performed using Genespring software (Redwood City, Calif.). The average background intensity was subtracted from the average intensity measured by Imagene to calculate the intensity value. The intensity of each spot was normalized by obtaining the median spot intensity from a set of values for the spot within one slide and comparing that value to the values for all slides in this experiment. The relative change between the peptide-treated cells and the control cells can be seen in the table below.

[0104]Other polynucleotide arrays used (tables 27-35) were the Human Operon array (identification number of the genome is PRHU04-S1), which consists of duplicate 14,000 individual oligo dots. Probes were prepared with 10. mu.g total RNA and labeled with Cy3 or Cy5 labeled dUTP. In these experiments, A549 epithelial cells were plated into 100mm tissue culture dishes at a density of 2.5X 10 per dish⁶And (4) cells. Total RNA was isolated using RNAqueous (Ambion). DNA contamination was removed using a DNA removal kit (Ambion). Probes prepared from total RNA were purified and hybridized to printed glass slides, overnight at 42 ℃ and then washed. After washing, images were taken with a Perkin Elmer array scanner. The mean, median and background intensities of the spots were determined using image processing software (Imagene 5.0, Marina Del Rey, Calif.). The background was removed using a "homemade" procedure. The program calculates the base intensity of each sub-cell as 10% and subtracts this value for each cell. The analysis was performed using Genespring software (Redwood City, Calif.). The intensity of each spot was normalized by obtaining the median spot intensity from a set of values for the spot within one slide and comparing that value to the values for all slides in this experiment. The relative change between the peptide-treated cells and the control cells can be seen in the table below.

[0105]Semi-quantitative RT-PCR was performed to confirm the results of the polynucleotide array. Mu.g of gRNA sample and 1. mu.l of oligo dT (500. mu.g/ml) and 1. mu.l of mixed dNTP stock at a concentration of 1mM were incubated at 65 ℃ for 5 minutes in a reaction volume of 12. mu.l using DEPC treated water in a thermal cycler. Mu.l of 5 XFirst-Strand buffer, 2. mu.l of 0.1M DTT and 1. mu.l of RNaseOUT recombinant ribonuclease inhibitor (40 units/. mu.l) were added, incubated at 42 ℃ for 2 minutes, and then 1. mu.l (200units) of Superscript II (Invitrogen, Burlington, ON) was added. For eachcDNA source, parallel experiments were performed without Superscript II to provide negative controls. Using 5 'and 3' primers (1.0. mu.M), 0.2mM dNTP mix, 1.5mM MgCl₂cDNA was amplified with 1U Taq DNA polymerase (New England Biolabs, Missiauga, ON) and 1 XPCR buffer. Each PCR was performed on a thermal cycler, comprising 30-40 cycles, each cycle comprising denaturation at 94 ℃ for 30 seconds, annealing at 52 ℃ or 55 ℃ for 30 seconds, and extension at 72 ℃ for 40 seconds. The number of cycles of PCR was optimized for each primer and each RNA sample to lie within the linear range of the reaction. To evaluate the extraction step and estimate the amount of RNA, the housekeeping (housekeeping) polynucleotide β actin gene was amplified in each experiment. The reaction products were visualized by electrophoresis and analyzed by densitometry (densitometry), which allows the relative concentration of RNA to be calculated initially with reference to the amplification of the β actin polynucleotide.

[0106] Table 18 shows that, in the selected known polynucleotides, on the small Atlas microarray, the sequences shown in SEQ ID NOs: 1 treatment of RAW 264.7 cells upregulated the expression of more than 30 different polynucleotides therein. The peptide consisting of SEQ ID NO: 1 the upregulated polynucleotides come primarily from two classes: one class includes receptors (growth factors, chemokines, interleukins, interferons, hormones, neurotransmitters), cell surface antigens and cell adhesion, and the other class includes cell-cell communication (growth factors, cytokines, chemokines, interleukins, interferons, hormones), cytoskeleton, cell motility and protein turnover (Turnover). Specific polynucleotides that are upregulated include polynucleotides encoding the following proteins: chemokines MCP-3, anti-inflammatory cytokines IL-10, macrophage colony stimulating factors and receptors such as IL-1R-2 (a putative antagonist of prolific IL-1 that binds to IL-1R 1), PDGF receptor B, NOTCH4, LIF receptor, LFA-1, TGF beta receptor 1, G-CSF receptor, and IFN gamma receptor. The peptides also up-regulate polynucleotides encoding several metalloproteinases and their inhibitors, including bone morphogenetic proteins BMP-1, BMP-2, BMP-8a, TIMP 2 and TIMP 3. Moreover, the peptide upregulates several specific transcription factors, including JunD, as well as YY and LIM-1 transcription factors, and kinases such as Etk1 and Csk, suggesting that it has a broad effect. Polynucleotide array studies have also found that SEQ ID NO: 1 down-regulated at least 20 polynucleotides in RAW 264.7 macrophages (table 19). Polynucleotides downregulated by peptides include DNA repair proteins and several inflammatory mediators such as MIP-1 α, oncostatin M, and IL-12. Many of the effects of peptides on polynucleotide expression were confirmed by RT-PCR (Table 20). Using a medium-sized microarray (7835 polynucleotides), it was also found that the peptide SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 19 and SEQ ID NO: 1, and representative peptides of each formula, alter transcriptional responses in human epithelial cell lines. SEQ ID NO: 1 effects on polynucleotide expression were compared in two human epithelial cell lines, a549 and HBE. Table 21 describes polynucleotides associated with host immune responses upregulated by 2 or more peptides in a 2-fold greater ratio compared to unstimulated cells. Table 22 describes polynucleotides associated with host immune responses down-regulated by 2 or more peptides at a rate of 2-fold more compared to unstimulated cells. Tables 23 and 24 show up-regulated and down-regulated human epithelial pro-inflammatory polynucleotides, respectively. Tables 25 and 26 show anti-inflammatory polynucleotides affected by cationic peptides. One clear trend is that cationic peptides up-regulate the anti-inflammatory response, down-regulate the pro-inflammatory response. It was very difficult to find a polynucleotide that was associated with an anti-inflammatory response, but was down-regulated (Table 26). Proinflammatory polynucleotides that are upregulated by cationic peptides are primarily polynucleotides associated with migration and adhesion. It should be noted that of the proinflammatory polynucleotides that are down-regulated, there are several toll-like receptor (TLR) polynucleotides affected by all cationic peptides, which TLR polynucleotides are important for signaling the host response to infectious agents. An important anti-inflammatory polynucleotide that is upregulated by all peptides is the IL-10 receptor. IL-10 is an important cytokine involved in the regulation of proinflammatory cytokines. For the peptide SEQ ID NO: these effects on polynucleotide expression can also be observed when primary human macrophages are used, as shown in tables 27 and 28. Tables 31-37 below show the effect of representative peptides of each formula on the expression of selected polynucleotides (selected from the 14,000 tested) by human epithelial cells. To test the ability of the peptides to alter the expression of human epithelial polynucleotides, at least 6 peptides were selected for each formula, which did have a broad stimulatory effect. For each formula, typically at least 50 polynucleotides are upregulated by each peptide in the set.

Table 18: peptide SEQ ID NO: 1 treating the upregulated polynucleotide a.

[0107] Cationic peptides at a concentration of 50. mu.g/ml were found to efficiently induce expression of several polynucleotides. Peptides were incubated with RAW cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to Atlas arrays. The intensity of the unstimulated cells is shown in the third column. The column "ratio peptide: unstimulated" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells.

[0108]The intensity of the housekeeping polynucleotides after normalization varies from 0.8 to 1.2 fold, and thus these polynucleotides can be effectively used in the normalization process. When the normalized hybridization intensity of a given cDNA is below 20, the value is assigned to 20, from which the ratio and relative expression are calculated. Array experiments were repeated three times with different RNA preparations, with the average fold-change shown below. Polynucleotides with two-fold or greater changes in relative expression levels are shown.

Polynucleotide/protein	Polynucleotide function	Intensity of non-stimulation	Peptide ratio unstimulated	Sequence accession number
					Etk 1	Tyrosine protein kinase receptors	20	43	M68513
PDGFRB	Growth factor receptors	24	25	X04367
						Corticotropin releasing factor receptors	20	23	X72305
NOTCH4	Proto-cancer polynucleotides	48	18	M80456
					IL-1R2	Interleukin receptors	20	16	X59769
MCP-3	Chemotactic factor	56	14	S71251
					BMP-1	Bone morphogenetic proteins	20	14	L24755
Endothelin b receptor	Receptors	20	14	U32329
					c-ret	Cancer polynucleotide precursors	20	13	X67812
LIFR	Cytokine receptors	20	12	D26177
					BMP-8a	Bone morphogenetic proteins	20	12	M97017
Zfp92	Zinc finger protein 92	87	11	U47104
					MCSF	Macrophage colony stimulating factor 1	85	11	X05010
GCSFR	Granulocyte colony stimulating factor receptor	20	11	M58288
					IL-8RB	Chemokine receptors	112	10	D17630
IL-9R	Interleukin receptors	112	6	M84746
					Cas	Crk-related substrates	31	6	U48853
p58/GTA	Kinase enzymes	254	5	M58633
					CASP2	Caspase (caspase) precursors	129	5	D28492
IL-1 beta precursor	Interleukin precursors	91	5	M15131
					SPI2-2	Serine protease inhibitors	62	5	M64086
C5AR	Chemokine receptors	300	4	S46665
					L-myc	Cancer polynucleotides	208	4	X13945
IL-10	Interleukin	168	4	M37897
					p19ink4	cdk4 and cdk6 inhibitors	147	4	U19597
ATOH2	Non-regulated Gene homolog 2	113	4	U29086
					DNAsel	DNA enzyme	87	4	U00478

CXCR-4	Chemokine receptors	36	4	D87747
					Cyclin D3	Cyclin proteins	327	3	U43844
IL-7Rα	Interleukin receptors	317	3	M29697
					POLA	DNA polymerase alpha	241	3	D17384
Tie-2	Cancer polynucleotides	193	3	S67051
					DNL1	DNA ligase I	140	3	U04674
BAD	Apoptosis proteins	122	3	L37296
					GADD45	DNA damage inducing protein	88	3	L28177
Sik	Scr-related kinase	82	3	U16805
					Integrin 4	Integrins	2324	2	X53176
TGFβR1	Growth factor receptors	1038	2	D25540
					LAMR1	Receptors	1001	2	J02870
Crk	Crk adaptor protein	853	2	S72408
					ZFX	Chromosomal proteins	679	2	M32309
Cyclin E1	Cyclin proteins	671	2	X75888
					POLD1	Subunit of DNA polymerase	649	2	Z21848
Vav	Proto-cancer polynucleotides	613	2	X64361
					YY(NF-E1)	Transcription factor	593	2	L13968
JunD	Transcription factor	534	2	J050205
					Csk	c-src kinase	489	2	U05247
Cdk7	Cyclin dependent kinases	475	2	U11822
					MLC1A	Myosin light subunit isoforms	453	2	M19436
ERBB-3	Receptors	435	2	L47240
					UBF	Transcription factor	405	2	X60831
TRAIL	Apoptosis ligands	364	2	U37522
					LFA-1	Cell adhesion receptors	340	2	X14951
SLAP	Src-like adaptor proteins	315	2	U29056
					IFNGR	Interferon gamma receptors	308	2	M28233
LIM-1	Transcription factor	295	2	Z27410
					ATF-2	Transcription factor	287	2	S76657
FST	Follistatin precursors	275	2	Z29532
					TIMP3	Protease inhibitors	259	2	L19622
RU49	Transcription factor	253	2	U41671
					IGF-1Rα	Insulin-like growth factor receptors	218	2	U00182
Cyclin G2	Cyclin proteins	214	2	U95826
					fyn	Tyrosine protein kinase	191	2	U70324
BMP-2	Bone morphogenetic proteins	186	2	L25602

Brn-3.2POU	Transcription factor	174	2	S68377
					KIF1A	Kinesin family proteins	169	2	D29951
MRC1	Mannose receptor	167	2	Z11974
					PAI2	Protease inhibitors	154	2	X19622
BKLF	CACCC sequence frame binding protein	138	2	U36340
					TIMP2	Protease inhibitors	136	2	X62622
Mas	Proto-cancer polynucleotides	131	2	X67735
					NURR-1	Transcription factor	129	2	S53744

Table 19: the expression of the polypeptide encoded by SEQ ID NO: 1 treating the down-regulated polynucleotide a.

[0109]Cationic peptides at a concentration of 50. mu.g/ml were found to reduce the expression of several polynucleotides. Peptides were incubated with RAW cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to Atlas arrays. The intensity of the unstimulated cells is shown in the third column. The column "ratio peptide: unstimulated" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells. The array experiments were repeated three times with different cells and the mean fold change is shown below. Polynucleotides with approximately two-fold or greater changes in relative expression levels are shown.

Polynucleotide/protein	Polynucleotide function	Intensity of non-stimulation	Peptide ratio unstimulated	Sequence accession number
					Sodium channels	Voltage-gated ion channels	257	0.08	L36179
XRCC1	DNA repair proteins	227	0.09	U02887
					est-2	Cancer polynucleotides	189	0.11	J04103
XPAC	DNA repair proteins	485	0.12	X74351
					EPOR	Receptor precursors	160	0.13	J04843
PEA 3	Ets related proteins	158	0.13	X63190
					Orphan receptors	Nuclear receptors	224	0.2	U11688
N-cadherins	Cell adhesion receptors	238	0.23	M31131
					OCT3	Transcription factor	583	0.24	M34381
PLCβ	Phospholipase enzymes	194	0.26	U43144
					KRT18	Intermediate fibrillar proteins	318	0.28	M11686
THAM	Enzyme	342	0.32	X58384
					CD40L	CD40 ligand	66	0.32	X65453
CD86	T lymphocyte antigens	195	0.36	L25606
					Oncostatin M	Cytokine	1127	0.39	D31942
PMS2DNA	DNA repair proteins	200	0.4	U28724
					IGFBP6	Growth factor	1291	0.41	X81584
MIP-1β	Cytokine	327	0.42	M23503
					ATBF1	AT motif binding factor	83	0.43	D26046
Nuclear binding protein (nucleobin)	Golgi resident proteins	367	0.43	M96823
					bcl-x	Apoptosis proteins	142	0.43	L35049

Uromodulin (uromollin)	Glycoprotein	363	0.47	L33406
					IL-12 p40	Interleukin	601	0.48	M86671
MmRad52	DNA repair proteins	371	0.54	Z32767
					Tob1	Anti-proliferative factor	956	0.5	D78382
Ung1	DNA repair proteins	535	0.51	X99018
					KRT19	Intermediate fibrillar proteins	622	0.52	M28698
PLCγ	Phospholipase enzymes	251	0.52	X95346
					Integrin alpha 6	Cell adhesion receptors	287	0.54	X69902
GLUT1	Glucose transporters	524	0.56	M23384
					CTLA4	Immunoglobulin superfamily	468	0.57	X05719
FRA2	Fos-associated antigens	446	0.57	X83971
					MTRP	Lysosomal associated proteins	498	0.58	U34259

Table 20: in the case of the peptide SEQ ID NO: 1 can be confirmed by RT-PCR.

[0110]RAW 264.7 macrophages were incubated with 50. mu.g/ml peptide for 4 hours or separately with culture medium for 4 hours. Total RNA was isolated and subjected to semi-quantitative RT-PCR. Primer pairs specific for each polynucleotide were used to amplify RNA. Amplification of beta actin was used as a positive control and was used for normalization. Density measurement analysis was performed on the RT-PCR products. The results are expressed as the relative fold change in polynucleotide expression for the peptide-treated cells compared to cells incubated with medium alone. Data are presented as mean ± standard deviation of three experiments.

Polynucleotide	Array ratio-*	RT-PCR-*
			CXCR-4	4.0±1.7	4.1±0.9
IL-8RB	9.5±7.6	7.1±1.4
			MCP-3	13.5±4.4	4.8±0.88
IL-10	4.2±2.1	16.6±6.1
			CD14	0.9±0.1	0.8±0.3
MIP-1B	0.42±0.09	0.11±0.04
			XRCC1	0.12±0.01	0.25±0.093
MCP-1	Without arrays	3.5±1.4

Table 21: polynucleotides upregulated by peptide treatment in A549 epithelial cells^a。

[0111]Cationic peptides at a concentration of 50. mu.g/ml were found to increase the expression of several polynucleotides. The peptides were incubated with human A549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized with human cDNA array ID # PRHU03-S3And (6) carrying out cross-linking. The intensity of the polynucleotide in the unstimulated cells is shown in the second column. The column "ratio peptide: unstimulated" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells.

Polynucleotide/protein	Strength of not being stimulated	Peptide ratio unstimulated				Registration number
							ID 2	ID 3	ID 19	ID 1
		IL-1R antagonist homologues	0.00	3086	1856						870		AI167887

Object 1
							IL-10Rβ	0.53	2.5	1.6	1.9	3.1	AA486393
IL-11Rα	0.55	2.4	1.0	4.9	1.8	AA454657
							IL-17R	0.54	2.1	2.0	1.5	1.9	AW029299
TNF R superfamily, member 1B	0.28	18	3.0	15	3.6	AA150416
							TNF R superfamily, member 5(CD40LR)	33.71	3.0	0.02			H98636
TNF R superfamily, member 11b	1.00	5.3	4.50	0.8		AA194983
							IL-8	0.55	3.6	17	1.8	1.1	AA102526
Interleukin enhancer binding factor 2	0.75	1.3	2.3	0.8	4.6	AA894687
							Interleukin enhancer binding factor 1	0.41	2.7		5.3	2.5	R56553
Protein containing SH2 and capable of being induced by cell factor	0.03	33	44	39	46	AA427521
							Down-regulators of IK cytokines, HLAII	0.50	3.1	2.0	1.7	3.3	R39227
Can be made of cellsFactor-induced SH 2-containing protein	0.03	33	44	39	46	AA427521
							Down-regulators of IK cytokines, HLAII	0.50	3.1	2.0	1.7	3.3	R39227
Small inducible cytokine subfamily A (Cys-Cys), member 21	1.00	3.9			2.4	AI922341
							TGFB-induced early growth response 2	0.90	2.4	2.1	0.9	1.1	AI473938
NK cell R	1.02	2.5	0.7	0.3	1.0	AA463248
							CCR6	0.14	4.5	7.8	6.9	7.8	N57964
Cell adhesion molecules	0.25	4.0	3.9	3.9	5.1	R40400
							Melanoma adhesion molecules	0.05	7.9	20	43	29.1	AA497002
CD31	0.59	2.7	3.1	1.0	1.7	R22412
							Integrin, α 2(CD49B, the α 2 subunit of the VLA-2 receptor)	1.00	0.9	2.4	3.6	0.9	AA463257
Integrin, α 3(CD49C, the α 3 subunit of the VLA-3 receptor)	0.94	0.8	2.5	1.9	1.1	AA424695
							Integrins, α E	0.01	180	120	28	81	AA425451
Integrins, beta 1	0.47	2.1	2.1	7.0	2.6	W67174
							Integrins, beta 3	0.55	2.7	2.8	1.8	1.0	AA037229
Integrins, beta 3	0.57	2.6	1.4	1.8	2.0	AA666269
							Integrin,. beta.4	0.65	0.8	2.2	4.9	1.5	AA485668

Integrin beta 4 binding proteins	0.20	1.7	5.0	6.6	5.3	AI017019
							Calcium and integrin binding proteins	0.21	2.8	4.7	9.7	6.7	AA487575
Disintegrin and metalloprotease domain 8	0.46	3.1		2.2	3.8	AA279188
							Disintegrin and metalloprotease domains 9	0.94	1.1	2.3	3.6	0.5	H59231
Disintegrin and metalloprotease domains 10	0.49	1.5	2.1	3.3	2.2	AA043347
							Disintegrin and metalloprotease domains 23	0.44	1.9	2.3	2.5	4.6	H11006
Cadherin type 1, 1E-Calleonectin (epithelium)	0.42	8.1	2.2	2.4	7.3	H97778
							Cadherin type 12, 2 (N-cadherin 2)	0.11	13	26	9.5		AI740827
Procadherin 12	0.09	14.8	11.5	2.6	12.4	AI652584
							Procadherin gamma subfamily C, 3	0.34	3.0	2.5	4.5	9.9	R89615
Catenin (cadherin-related protein), delta 1	0.86	1.2	2.2	2.4		AA025276
							Laminin R1(67kD, ribosomal protein SA)	0.50	0.4	2.0	4.4	3.0	AA629897
Killer cell lectin-like receptor subfamily C, member 2	0.11	9.7	9.0	4.1	13.4	AA190627
							Killer cell lectin-like receptor subfamily C, member 3	1.00	3.2	1.0	0.9	1.3	W93370
Killer cell lectin-like receptor subfamily G, member 1	0.95	2.3	1.7	0.7	1.1	AI433079
							C-type lectin-like receptor 2	0.45	2.1	8.0	2.2	5.3	H70491
CSF 3R	0.40	1.9	2.5	3.5	4.0	AA458507
							Macrophage stimulation of 1R	1.00	1.7	2.3	0.4	0.7	AA173454
BMP type R IA	0.72	1.9	2.8	0.3	1.4	W15390
							Formyl peptide receptor 1	1.00	3.1	1.4	0.4		AA425767
CD2	1.00	2.6	0.9	1.2	0.9	AA927710
							CD36	0.18	8.2	5.5	6.2	2.5	N39161
Vitamin DR	0.78	2.5	1.3	1.1	1.4	AA485226
							Human protease activation of R-2	0.54	6.1	1.9	2.2		AA454652

Prostaglandin E receptor 3(EP3 subtype)	0.25	4.1	4.9	3.8	4.9	AA406362
							PDGFR beta polypeptides	1.03	2.5	1.0	0.5	0.8	R56211
VIP R2	1.00	3.1			2.0	AI057229
							Growth factor receptor binding protein 2	0.51	2.2	2.0	2.4	0.3	AA449831
Murine mammary tumor virus receptor homologs	1.00	6.9		16		W93891
							Adenosine A2a R	0.41	3.1	1.8	4.0	2.5	N57553
Adenosine A3R	0.83	2.0	2.3	1.0	1.2	AA863086
							T cell R delta locus	0.77	2.7	1.3		1.8	AA670107
Prostaglandin E receptor 1(EP1 subtype)	0.65	7.2		6.0	1.5	AA972293
							Growth factor receptor binding protein 14	0.34		3.0	6.3	2.9	R24266
Epstein-Barr Virus induced Polynucleotide 2	0.61	1.6	2.4		8.3	AA037376
							Complement component receptor 2	0.22	26	4.5	2.6	18.1	AA521362
Endotoxin receptor type A	0.07	12	14	14	16	AA450009
							v-SNARE R	0.56	11	12	1.8		AA704511
Tyrosine kinases, non-receptor, 1	0.12	7.8	8.5	10	8.7	AI936324
							Receptor tyrosine kinase-like orphan receptor 2	0.40	7.3	5.0	1.6	2.5	N94921
Protein tyrosine phosphatase, non-receptor type 3	1.02	1.0	13.2	0.5	0.8	AA682684
							Protein tyrosine phosphatase, non-receptor type 9	0.28	2.5	4.0	0.9	5.3	AA434420
Protein tyrosine phosphatase, non-receptor type 11	0.42	2.9	2.4	2.2	3.0	AA995560
							Protein tyrosine phosphatase, non-receptor type 12	1.00	2.3	2.2	0.8	0.5	AA446259
A protein-tyrosine-phosphatase (PTP) enzyme,non-receptor type 13	0.58	1.7	2.4	3.6	1.7	AA679180
							Protein tyrosine phosphatase, non-receptor type 18	0.52	3.2	0.9	1.9	6.5	AI668897
Protein tyrosine phosphatases, receptor types, A	0.25	4.0	2.4	16.8	12.8	H82419
							Protein tyrosine phosphatases, receptor types, J	0.60	3.6	3.2	1.6	1.0	AA045326
Protein tyrosine phosphatases, receptor types, T	0.73	1.2	2.8	3.0	1.4	R52794
							Protein tyrosine phosphatase, receptor type, U	0.20	6.1	1.2	5.6	5.0	AA644448

Protein tyrosine phosphatases, receptor types, C-related proteins	1.00	5.1			2.4	AA481547
							Phospholipase A2 receptor 1	0.45	2.8	2.2	1.9	2.2	AA086038
MAP kinase activated protein kinase 3	0.52	2.1	2.7	1.1	1.9	W68281
							MAP kinase 6	0.10	18	9.6		32	H07920
MAP kinase 5	1.00	3.0	5.2	0.8	0.2	W69649
							MAP kinase 7	0.09		11.5	12	33	H39192
MAP kinase 12	0.49	2.1	1.7	2.2	2.0	AI936909
							G protein coupled receptor 4	0.40	3.7	3.0	2.4	2.5	AI719098
G protein coupled receptor 49	0.05		19	19	27	AA460530
							G protein coupled receptor 55	0.08	19	15	12		N58443
G protein coupled receptor 75	0.26	5.2	3.1	7.1	3.9	H84878
							G protein coupled receptor 85	0.20	6.8	5.4	4.9	5.0	N62306
Regulatory protein 20 for G protein signaling	0.02	48	137	82		AI264190
							Regulatory protein 6 for G protein signaling	0.27		3.7	8.9	10.6	R39932
Killer protein (inducing apoptosis) acting with BCL2	1.00	1.9		5.2		AA291323
							Inhibitor of apoptosis 5	0.56	2.8	1.6	2.4	1.8	AI972925
caspase 6, apoptosis-related cysteine protease	0.79	0.7	2.6	1.3	2.8	W45688
							Apoptosis-related protein PNAS-1	0.46	2.2	1.4	2.3	2.9	AA521316
caspase 8, apoptosis-related cysteine protease	0.95	2.2	1.0	0.6	2.0	AA448468

Table 22: polynucleotides downregulated by peptide treatment in A549 epithelial cells^a。

[0112]Cationic peptides at a concentration of 50. mu.g/ml were found to reduce the expression of several polynucleotides. The peptides were incubated with human A549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to human cDNA array ID # PRHU 03-S3. The intensity of the polynucleotide in the unstimulated cells is shown in the second column. The column "ratio peptide: unstimulated" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells.

Polynucleotide/protein	Strength of not being stimulated	Peptide ratio unstimulated				Registration number
							ID 2	ID 3	ID 19	ID 1
		TLR 1	3.22	0.35	0.31						0.14	0.19	AI339155
TLR 2	2.09					0.52	0.31	0.48	0.24	T57791
		TLR 5	8.01	0.12	0.39								N41021
TLR 7	5.03					0.13	0.11	0.20	0.40	N30597

TNF receptor associated factor 2	0.82	1.22	0.45	2.50	2.64	T55353
							TNF receptor associated factor 3	3.15	0.15		0.72	0.32	AA504259
TNF receptor superfamily, member 12	4.17	0.59	0.24		0.02	W71984
							TNF R superfamily, member 17	2.62		0.38	0.55	0.34	AA987627
TRAF and TNF receptor associated proteins	1.33	0.75	0.22	0.67	0.80	AA488650
							IL-1 receptor, type I	1.39	0.34	0.72	1.19	0.34	AA464526
IL-2 receptor, alpha	2.46	0.41	0.33	0.58		AA903183
							IL-2 receptor, gamma (severe combined immunodeficiency)	3.34	0.30	0.24		0.48	N54821
IL-12 receptor, beta 2	4.58	0.67	0.22			AA977194
							IL-18 receptor 1	1.78	0.50	0.42	0.92	0.56	AA482489
TGF-beta receptor III	2.42	0.91	0.24	0.41	0.41	H62473
							Leukotriene b4 receptor (chemokine receptor-like-1)	1.00		1.38	4.13	0.88	AI982606
Small inducible cytokine subfamily A (Cys-Cys), member 18	2.26	0.32		0.44	1.26	AA495985
							Small inducible cytokine subfamily A (Cys-Cys), member 20	2.22	0.19	0.38	0.45	0.90	AI285199
Small inducible cytokine subfamily A (Cys-Cys), member 23	2.64	0.38	0.31	1.53		AA916836
							Small inducible cytokine subfamily B (Cys-X-Cys), member 6 (granulocyte chemotactic protein 2)	3.57	0.11	0.06	0.28	0.38	AI889554
Small inducible cytokine subfamily B (Cys-X-Cys), member 10	2.02	0.50	1.07	0.29	0.40	AA878880
							Small inducible cytokine A3 (homologous to murine Mip-1 a)	2.84	1.79	0.32	0.35		AA677522
Cytokine-induced kinase	2.70	0.41	0.37	0.37	0.34	AA489234
							Complement component Clq receptors	1.94	0.46	0.58	0.51	0.13	AI761788
Calnexin type 11, 2, OB-calnexin (osteoblasts)	2.00	0.23	0.57	0.30	0.50	AA136983

Cadherin type 3, 1, P-cadherin (placenta)	2.11	0.43	0.53	0.10	0.47	AA425217
							Cadherin, EGFLAG seven-pass G receptor 2, flumingo homolog	1.67	0.42	0.41	1.21	0.60	H39187
Calyosin 13, H-calyosin (heart)	1.78	0.37	0.40	0.56	0.68	R41787
							Lectin L (lymphocyte adhesion molecule 1)	4.43	0.03	0.23	0.61		H00662
Vascular cell adhesion molecule 1	1.40	0.20	0.72	0.77	0.40	H16591
							Intermolecular adhesion molecule 3	1.00	0.12	0.31	2.04	1.57	AA479188
Integrins, alpha 1	2.42	0.41	0.26		0.56	AA450324
							Integrins, alpha 7	2.53	0.57	0.39	0.22	0.31	AA055979
Integrins, alpha 9	1.16	0.86	0.05	0.01	2.55	AA865557
							Integrins, alpha 10	1.00	0.33	0.18	1.33	2.55	AA460959
Integrins, beta 5	1.00	0.32	1.52	1.90	0.06	AA434397
							Integrins, beta 8	3.27	0.10	1.14	0.31	0.24	W56754
Disintegrin and metalloprotease domains 18	2.50	0.40	0.29	0.57	0.17	AI205675
							Disintegrin-like and metalloproteases having the platelet binding protein 1 motif, 3	2.11	0.32	0.63	0.47	0.35	AA398492
Disintegrin-like and metalloproteases having the platelet binding protein 1 motif, 5	1.62	0.39	0.42	1.02	0.62	AI375048
							T cell receptor effector molecules	1.00	0.41	1.24	1.41	0.45	AI453185
Diphtheria toxin receptor (heparin binding epidermal growth factor-like growth factor)	1.62	0.49	0.85	0.62	0.15	R45640
							Vasoactive intestinal peptide receptor 1	2.31	0.43	0.31	0.23	0.54	H73241
Fc fragment of IgG, Low affinity IIIb, receptor-directed (CD16)	3.85	-0.20	0.26	0.76	0.02	H20822
							Fc fragment of IgG, Low affinity IIb, receptor-directed (CD32)	1.63	0.27	0.06	1.21	0.62	R68106
Fc fragment of IgE, high affinity I, receptor pair; alpha polypeptides	1.78	0.43	0.00	0.56	0.84	AI676097

Leukocyte immunoglobulin-like receptor, subfamily A	2.25	0.44	0.05	0.38	0.99	N63398
							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	0.19	0.40	AA076350
							Leukocyte immunoglobulin-like receptor, subfamily B	1.67	0.35	0.60	0.18	0.90	H54023
Peroxisome proliferator activated receptor, alpha	1.18	0.38	0.85	0.87	0.26	AI739498
							Protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha 1	2.19	0.43		1.06	0.46	N49751
Protein tyrosine phosphatases, receptor types, C	1.55	0.44	0.64	0.30	0.81	H74265
							Protein tyrosine phosphatases, receptor types, E	2.08	0.23	0.37	0.56	0.48	AA464542
Protein tyrosine phosphatases, receptor types, N polypeptide 2	2.27	0.02	0.44		0.64	AA464590
							Protein tyrosine phosphatase, receptor type, H	2.34	0.11	0.43	0.24	0.89	AI924306
Protein tyrosine phosphatase, receptor type, Z polypeptide 1	1.59	0.63	0.34	0.72	0.35	AA476461
							Protein tyrosine phosphatase, non-receptor type 21	1.07	0.94	0.43	0.25	1.13	H03504
MAP kinase 8 interacting protein 2	1.70	0.07	0.85	0.47	0.59	AA418293
							MAP kinase 4	1.27	0.37	0.79	1.59	-5.28	AA402447
MAP kinase 14	1.00	0.34	0.66	2.10	1.49	W61116
							MAP kinase 8 interacting protein 2	2.90	0.16	0.35	0.24	0.55	AI202738
MAP kinase 12	1.48	0.20	0.91	0.58	0.68	AA053674
							MAP kinase 3	2.21	0.45	0.20	1.03	0.41	AA043537
MAP kinase 6	2.62	0.37	0.38		0.70	AW084649
							MAP kinase	1.04	0.96	0.09	0.29	2.79	AA417711

Kinase 4
							MAP kinase 11	1.53	0.65	0.41	0.99	0.44	R80779
MAP kinase 10	1.32	1.23	0.27	0.50	0.76	H01340
							MAP kinase 9	2.54	0.57	0.39	0.16	0.38	AA157286
MAP kinase 1	1.23	0.61	0.42	0.81	1.07	AI538525
							MAP kinase 8	0.66	1.52	1.82	9.50	0.59	W56266
MAP kinase activated protein kinase 3	0.52	2.13	2.68	1.13	1.93	W68281
							MAP kinase 2	0.84	1.20	3.35	0.02	1.31	AA425826
MAP kinase 7	1.00	0.97		1.62	7.46	AA460969
							MAP kinase 7	0.09		11.45	11.80	33.43	H39192
MAP kinase 6	0.10	17.83	9.61		32.30	H07920
							Regulatory protein 5 for G protein signaling	3.7397	0.27	0.06	0.68	0.18	AA668470
Regulatory protein 13 of G protein signaling	1.8564	0.54	0.45	0.07	1.09	H70047
							G protein coupleLinked receptors	1.04	1.84	0.16	0.09	0.96	R91916
G protein-coupled receptor 17	1.78	0.32	0.56	0.39	0.77	AI953187
							G protein-coupled receptor kinase 7	2.62		0.34	0.91	0.38	AA488413
Seven transmembrane receptors in solitary son, associated with chemokines	7.16	1.06	0.10	0.11	0.14	AI131555
							Apoptosis-antagonistic transcription factors	1.00	0.28	2.50	1.28	0.19	AI439571
caspase 1, apoptosis-related cysteine protease (interleukin 1, beta, convertase)	2.83	0.44		0.33	0.35	T95052
							Programmed cell death 8 (factor inducing apoptosis)	1.00	1.07	0.35	1.94	0.08	AA496348

Table 23: a549 proinflammatory polynucleotide upregulated by peptide treatment in cells.

[0113]Cationic peptide concentrations of 50 μ g/ml were found to increase the expression of certain pro-inflammatory polynucleotides (data is part of table 21).The peptides were incubated with human A549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to human cDNA array ID # PRHU 03-S3. The intensity of the polynucleotide in the unstimulated cells is shown in the second column. The column "ratio peptide: unstimulated" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells.

Polynucleotide/protein and function	Strength of not being stimulated	Peptide ratio unstimulated				Registration number
							ID 2	ID 3	ID 19	ID 1
		IL-11R α; receptor of proinflammatory cytokines, inflammation	0.55	2.39	0.98						4.85	1.82	AA454657
IL-17R; IL-17 receptor, inducer of cytokine production in epithelial cells	0.54					2.05	1.97	1.52	1.86	AW029299
		Small inducible cytokine subfamily a, member 21; chemotactic factor	1.00	3.88								2.41	AI922341
CD 31; leukocyte and cell-to-cell adhesion (PECAM)	0.59					2.71	3.13	1.01	1.68	R22412
		CCR 6; receptors for the cytokine MIP-3 alpha	0.14	4.51	7.75						6.92	7.79	N57964
Integrin, α 2(CD49B, the α 2 subunit of the VLA-2 receptor); adhesion to leukocytes	1.00					0.89	2.44	3.62	0.88	AA463257
		Integrin, α 3(CD49C, the α 3 subunit of the VLA-3 receptor); adhesion to leukocytes	0.94	0.79	2.51						1.88	1.07	AA424695
Integrins, α E; adhesion	0.01					179.33	120.12	28.48	81.37	AA425451
		Integrin,. beta.4; leukocyte adhesion	0.65	0.79	2.17						4.94	1.55	AA485668
C-type lectin-like receptor 2; leukocyte adhesion	0.45					2.09	7.92	2.24	5.29	H70491

Table 24: a549 proinflammatory polynucleotide downregulated by peptide treatment in a cell.

[0114]Cationic peptide concentrations of 50 μ g/ml were found to reduce the expression of certain pro-inflammatory polynucleotides (data is part of table 22). The peptides were incubated with human A549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to human cDNA array ID # PRHU 03-S3. The intensity of the polynucleotide in the unstimulated cells is shown in the second column. The column "ratio peptide: unstimulated" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells.

A polynucleotide/protein; function(s)	Strength of not being stimulated	Peptide ratio unstimulated				Registration number
							ID 2	ID 3	ID 19	ID 1
		Toll-like receptor (TLR) 1; response to gram-positive bacteria	3.22	0.35	0.31						0.14	0.19	AI339155
TLR 2; response to gram-Positive bacteria and Yeast	2.09					0.52	0.31	0.48	0.24	T57791
		TLR 5; can enhance the responsiveness of other TLRs to flagellin	8.01	0.12	0.39								N41021
TLR 7; putative host defense mechanisms	5.03					0.13	0.11	0.20	0.40	N30597
		TNF receptor-related factor 2; inflammation(s)	0.82	1.22	0.45						2.50	2.64	T55353
TNF receptor-related factor 3; inflammation(s)	3.15					0.15		0.72	0.32	AA504259
		TNF receptor superfamily, member 12; inflammation(s)	4.17	0.59	0.24							0.02	W71984
TNF receptor superfamily, member 17; inflammation(s)	2.62						0.38	0.55	0.34	AA987627
		TRAF and TNF receptor associated proteins; TNF signaling	1.33	0.75	0.22						0.67	0.80	AA488650
Small inducible cytokine subfamily a, member 18; chemotactic factor	2.26					0.32		0.44	1.26	AA495985
		Small inducible cytokine subfamily a, member 20; chemotactic factor	2.22	0.19	0.38						0.45	0.90	AI285199
Small inducible cytokine subfamily a, member 23; chemotactic factor	2.64					0.38	0.31	1.53		AA916836
		Small inducible cytokine subfamily B, member 6; (granulocyte chemotactic protein); chemotactic factor	3.57	0.11	0.06						0.28	0.38	AI889554
Small inducible cytokine subfamily B, member 10; chemotactic factor	2.02					0.50	1.07	0.29	0.40	AA878880
		Small inducible cytokine A3 (mouse and rat)	2.84	1.79	0.32						0.35		AA677522

Mip-1a homology); chemotactic factor
							IL-12 receptor, β 2; interleukin and interferon receptors	4.58	0.67	0.22			AA977194
IL-18 receptor 1; induction of INF-gamma	1.78	0.50	0.42	0.92	0.56	AA482489
							Lectin L (leukocyte adhesion molecule 1); leukocyte adhesion	4.43	0.03	0.23	0.61		H00662
Vascular cell adhesion molecule 1; leukocyte adhesion	1.40	0.20	0.72	0.77	0.40	H16591
							Intercellular adhesion molecule 3; leukocyte adhesion	1.00	0.12	0.31	2.04	1.57	AA479188
Integrin, α 1; leukocyte adhesion	2.42	0.41	0.26		0.56	AA450324

Table 25: an anti-inflammatory polynucleotide that is upregulated by peptide treatment in a549 cells.

[0115]Cationic peptides at a concentration of 50 μ g/ml were found to increase the expression of certain anti-inflammatory polynucleotides (data is part of table 21). The peptides were incubated with human A549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to human cDNA array ID # PRHU 03-S3. The intensity of the polynucleotide in the unstimulated cells is shown in the second column. The column "ratio peptide: unstimulated" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells.

A polynucleotide/protein; function(s)	Strength of not being stimulated	Peptide ratio unstimulated				Registration number
							ID 2	ID 3	ID 19	ID 1
		IL-1R antagonist homolog 1; inhibitors of septic shock	0.00	3085.96	1855.90						869.57		AI167887
IL-10R β; receptors for inhibitors of cytokine synthesis	0.53					2.51	1.56	1.88	3.10	AA486393
		TNF R, member 1B; apoptosis of cells	0.28	17.09	3.01						14.93	3.60	AA150416
TNF R, member 5; apoptosis (CD40L)	33.71					2.98	0.02			H98636
		TNF R, member 11 b; apoptosis of cells	1.00	5.29	4.50						0.78		AA194983
IK cytokines, down regulators of HLA II; suppression of	0.50					3.11	2.01	1.74	3.29	R39227

Antigen presentation
							TGFB inducible early growth response protein 2; anti-inflammatory cytokines	0.90	2.38	2.08	0.87	1.11	AI473938
CD 2; adhesion molecules, binding to LFAp3	1.00	2.62	0.87	1.15	0.88	AA927710

Table 26: an anti-inflammatory polynucleotide that is down-regulated by peptide treatment in a549 cells.

[0116]Cationic peptides at a concentration of 50 μ g/ml were found to reduce the expression of certain anti-inflammatory polynucleotides (data is part of table 21). The peptides were incubated with human A549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to human cDNA array ID # PRHU 03-S3. The intensity of the polynucleotide in the unstimulated cells is shown in the second column. The column "ratio peptide: unstimulated" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells.

A polynucleotide/protein; function(s)	Strength of not being stimulated	Peptide ratio unstimulated				Registration number
							ID 2	ID 3	ID 19	ID 1
		MAP kinase 9	2.54	0.57	0.39						0.16	0.38	AA157286

Table 27: primary human macrophages are encoded by SEQ ID NO: 6 an up-regulated polynucleotide.

[0117]The peptide SEQ ID NO: 6 increases the expression of a number of polynucleotides. Peptides were incubated with Human macrophages for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a Human Operon array (PRHU 04). The intensity of the polynucleotide in the unstimulated cells is shown in the second column. The column "ratio peptide treatment: control" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells.

Gene (registration number)	Comparison: unstimulated cells	Ratiometric peptide treatment to control
			Proteoglycan 2(Z26248)	0.69	9.3
Unknown (AK001843)	26.3	8.2
			Phosphorylase kinase alpha 1(X73874)	0.65	7.1
Actin, α 3(M86407)	0.93	6.9
			DKFZP586B2420 protein (AL050143)	0.84	5.9
Unknown (AL109678)	0.55	5.6
			Transcription factor 21(AF047419)	0.55	5.4
Unknown (A433612)	0.62	5.0
			Chromosome condensation 1-sample (AF060219)	0.69	4.8
Unknown (AL137715)	0.66	4.4
			Inhibitor of apoptosis 4(U75285)	0.55	4.2
Nuclear factor 2 (NM-012461) acting with TERF1(TRF1)	0.73	4.2
			LINE reverse rotation seat element 1(M22333)	6.21	4.0

1-acylglycerol-3-phosphate O-acyltransferase 1(U56417)	0.89	4.0
			Vacuolar protic atpase, subunit D; v-ATPase, subunit D (X71490)	1.74	4.0
KIAA0592 protein (AB011164)	0.70	4.0
			KQT-like subfamily member 4 of voltage-gated potassium channel (AF105202)	0.59	3.9
CDC14 homolog A (AF000367)	0.87	3.8
			Histone folding protein CHRAC17(AF070640)	0.63	3.8
Cryptogamic pigment 1(D83702)	0.69	3.8
			Pancreatic zymogen granule membrane associated protein (AB035541)	0.71	3.7
Sp3 transcription factor (X68560)	0.67	3.6
			Hypothetical protein FLJ20495(AK000502)	0.67	3.5
E2F transcription factor 5, p130 binding (U31556)	0.56	3.5
			Hypothetical protein FLJ20070(AK000077)	1.35	3.4
Glycoprotein IX (X52997)	0.68	3.4
			KIAA1013 protein (AB023230)	0.80	3.4
Eukaryotic translation initiation factor 4A, isoform 2(AL137681)	2.02	3.4
			FYN binding protein (AF198052)	1.04	3.3
Guanylate binding protein, gamma-transducing active polypeptide 1(U41492)	0.80	3.3
			Glypican 1(X54232)	0.74	3.2
Mucosal vascular addressen cell adhesion molecule 1(U43628)	0.65	3.2
			Lymphocyte antigen (M38056)	0./0	3.2
H1 Histone family, member 4(M60748)	0.81	3.0
			Translation inhibiting protein p14.5(X95384)	0.78	3.0
Hypothetical protein FLJ20689(AB032978)	1.03	2.9
			KIAA1278 protein (AB03104)	0.80	2.9
Unknown (AL031864)	0.95	2.9
			Chymotrypsin-like protease (X71877)	3.39	2.9
Network cavity calcium binding protein (NM _001219)	2.08	2.9
			Protein kinase, cAMP dependent, regulatory, type I, beta (M65066)	7.16	2.9
POU Domain, class 4, transcription factor 2(U06233)	0.79	2.8
			POU Domain, class 2, correlation factor 1(Z49194)	1.09	2.8
KIAA0532 protein (AB011104)	0.84	2.8
			Not known (AF068289)	1.01	2.8
Unknown (AL117643)	0.86	2.7
			Cathepsin E (M84424)	15.33	2.7
Interstitial metalloprotease 23A (AF056200)	0.73	2.7
			Interferon receptor 2(L42243)	0.70	2.5
MAP kinase 1(L11284)	0.61	2.4
			Protein kinase C, alpha (X52479)	0.76	2.4
c-Cb1 action protein (AF230904)	0.95	2.4
			c-fos inducible growth factor (Y12864)	0.67	2.3

Cyclin-dependent kinase inhibitor 1B (S76988)	0.89	2.2
			Zinc finger protein 266(X78924)	1.67	2.2
MAP kinase 14(L35263)	1.21	2.2
			KIAA0922 protein (AB023139)	0.96	2.1
Bone morphogenetic protein 1(NM _006129)	1.10	2.1
			NADH dehydrogenase 1. alpha. sub-complex, 10(AF087661)	1.47	2.1
Bone morphogenetic protein receptor, type 1B (U89326)	0.50	2.1
			Interferon regulatory factor 2(NM002199)	1.46	2.0
Protease, serine, 21(AB031331)	0.89	2.0

Table 28: primary human macrophages are encoded by SEQ ID NO: 6 down-regulated polynucleotides.

[0118]The peptide SEQ ID NO: 6 reduces the expression of many polynucleotides. Peptides were incubated with Human macrophages for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a Human Operon array (PRHU 04). The intensity of the polynucleotide in the unstimulated cells is shown in the second column. The column "ratio peptide treatment: control" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells.

Gene (registration number)	Comparison: unstimulated cells	Ratiometric peptide treatment to control
			Unknown (AL049263)	17	0.06
Integrin associated kinase (U40282)	2.0	0.13
			KIAA0842 protein (AB020649)	1.1	0.13
Unknown (AB037838)	13	0.14
			Granulin (AF055008)	8.6	0.14
Glutathione peroxidase 3(NM _002084)	1.2	0.15
			KIAA0152 Gene product (D63486)	0.9	0.17
TGFBI-inducible anti-apoptotic factor 1(D86790)	0.9	0.19
			Disintegrin protease (Y13323)	1.5	0.21
Proteasome subunit beta type 7(D38048)	0.7	0.22
			Sp1 accessory factor required for transcriptional activation of subunit 3 (AB033042)	0.9	0.23
TNF receptor superfamily, member 14(U81232)	0.8	0.26
			Proteasome 26S subunit non-ATP enzyme 8(D38047)	1.1	0.28
Proteasome subunit beta type, 4(D26600)	0.7	0.29
			TNF receptor superfamily member 1B (M32315)	1.7	0.29
Cytochrome c oxidase subunit Vic (X13238)	3.3	0.30
			S100 calcium binding protein A4(M80563)	3.8	0.31
Proteasome subunit alpha type, 6(X59417)	2.9	0.31
			Proteasome 26S subunit non-ATP enzyme, 10(AL031177)	1.0	0.32
MAP kinase 2(NM _006609)	0.8	0.32
			Ribosomal protein L11(X79234)	5.5	0.32
Interstitial metalloprotease 14(Z48481)	1.0	0.32
			Proteasome subunit beta type, 5(D29011)	1.5	0.33
MAP kinase activated protein kinase 2(U12779)	1.5	0.34

caspase 3(U13737)	0.5	0.35
			jun D protooncogene (X56681)	3.0	0.35
Proteasome 26S subunit, ATPase, 3(M34079)	1.3	0.35
			IL-1 receptor like 1(AB012701)	0.7	0.35
Interferon alpha inducibility protein (AB019565)	13	0.35
			SDF receptor 1 (NM-012428)	1.6	0.35
Cathepsin D (M63138)	46	0.36
			MAP kinase 3(D87116)	7.4	0.37
TGF, beta inducibility (M77349)	1.8	0.37
			TNF receptor superfamily, member 10b (AF016266)	1.1	0.37
Proteasome subunit beta type, 6(M34079)	1.3	0.38
			Nuclear receptor binding protein (NM _013392)	5.2	0.38
Unknown (AL050370)	1.3	0.38
			Protease inhibitor 1 alpha-1-antitrypsin (X01683)	0.7	0.40
Proteasome subunit alpha type, 7(AF054185)	5.6	0.40
			TNF-alpha factor induced by LPS (NM-004862)	5.3	0.41
Transferrin receptor (X01060)	14	0.42
			Proteasome 26S subunit non-ATP enzymes13(AB009398)	1.8	0.44
MAP kinase 5(U25265)	1.3	0.44
			Cathepsin L (X12451)	15	0.44
IL-1 receptor associated kinase 1(L76191)	1.7	0.45
			MAP kinase 2(U07349)	1.1	0.46
Peroxisome proliferator activated receptor delta (AL022721)	2.2	0.46
			TNF superfamily, member 15(AF039390)	16	0.46
Cell death defense 1(D15057)	3.9	0.46
			TNFSuperfamily member 10(U37518)	287	0.46
Cathepsin H (X16832)	14	0.47
			Protease inhibitor 12(Z81326)	0.6	0.48
Proteasome subunit alpha type, 4(D00763)	2.6	0.49
			Proteasome 26S subunit ATPase, 1(L02426)	1.8	0.49
Proteasome 26S subunit ATPase, 2(D11094)	2.1	0.49
			caspase 7(U67319)	2.4	0.49
Interstitial metalloprotease 7(Z11887)	2.5	0.49

Table 29: HBE cells are encoded by SEQ ID NO: 1 an up-regulated polynucleotide.

[0119]The peptide SEQ ID NO: 1 increases the expression of a number of polynucleotides. Peptides were incubated with Human HBE epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a Human Operon array (PRHU 04). The intensity of the polynucleotide in unstimulated cells is shown in the third column. The column "ratio peptide treatment: control" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells.

Registration number

Gene

Comparison: is not stimulated

At ratiometric peptide position

		Of (2) cells	Comparison and treatment
				AL110161	Is unknown	0.22	5218.3
AF131842	Is unknown	0.01	573.1
				AJ000730	Family of solute carriers	0.01	282.0
Z25884	Chloride ion channel 1	0.01	256.2
				M93426	Protein tyrosine phosphatase receptor-type, zeta	0.01	248.7
X65857	Olfactory receptor, family 1, subfamily D, member 2	0.01	228.7
				M55654	TATA sequence box binding proteins	0.21	81.9
AK001411	Hypothetical proteins	0.19	56.1
				D29643	Polyterpene diphosphoglucooligosaccharide-protein glycosyltransferase	1.56	55.4
AF006822	Myelin transcription factor 2	0.07	55.3
				AL117601	Is unknown	0.05	53.8
AL117629	DKFZP434C245 protein	0.38	45.8
				M59465	Tumor necrosis factor, alpha-inducing protein 3	0.50	45.1
AB013456	Aquaporin 8	0.06	41.3
				AJ131244	SEC 24-related Gene family, Member A	0.56	25.1
AL110179	Is unknown	0.87	24.8
				AB037844	Is unknown	1.47	20.6
Z47727	Polymerase II polypeptide K	0.11	20.5
				AL035694	Is unknown	0.81	20.4
X68994	Human CREB gene	0.13	19.3
				AJ238379	Hypothetical proteins	1.39	18.5
NM_003519	H2B histone family member	0.13	18.3
				U16126	Glutamate receptor, ionotropic kainate 2	0.13	17.9
U29926	Adenosine monophosphate deaminase	0.16	16.3
				AK001160	Hypothetical proteins	0.39	14.4
U18018	ets variant gene 4	0.21	12.9
				D80006	KIAA0184 protein	0.21	12.6
AK000768	Hypothetical proteins	0.30	12.3
				X99894	Insulin promoter factor 1	0.26	12.0
AL031177	Is unknown	1.09	11.2
				AF052091	Is unknown	0.28	10.9
L38928	5, 10-methylene tetrahydrofolate synthetase	0.22	10.6
				AL117421	Is unknown	0.89	10.1
AL133606	Hypothetical proteins	0.89	9.8
				NM_016227	Membrane protein CH1	0.28	9.6
NM_006594	Connexin-related protein complex 4	0.39	9.3
				U54996	ZW10 homologue, protein	0.59	9.3
AJ007557	Potassium channels	0.28	9.0
				AF043938	Muscle RAS oncogene	1.24	8.8
AK001607	Is unknown	2.74	8.7
				AL031320	Peroxisome biogenesis factor 30	0.31	8.4

D38024	Is unknown	0.31	8.3
				AF059575	LIM homeobox TF	2.08	8.2
AF043724	Hepatitis a virus cell receptor 1	0.39	8.1
				AK002062	Hypothetical proteins	2.03	8.0
L13436	Natriuretic peptide receptors	0.53	7.8
				U33749	Thyroid transcription factor 1	0.36	7.6
AF011792	Cell cycle process 2 protein	0.31	7.6
				AK000193	Hypothetical proteins	1.18	6.8
AF039022	Exportin (tRNA)	0.35	6.8
				M17017	Interleukin 8	0.50	6.7
AF044958	NADH dehydrogenase	0.97	6.5
				U35246	Vesicle protein sorting	0.48	6.5
AK001326	Tetratransmembrane protein 3	1.59	6.5
				M55422	Kruepe-related zinc finger proteins	0.34	6.4
U44772	Palmitoyl-protein thioesterases	1.17	6.3
				AL117485	Hypothetical proteins	0.67	5.9
AB037776	Is unknown	0.75	5.7
				AF131827	Is unknown	0.69	5.6
AL137560	Is unknown	0.48	5.2
				X05908	Annexin A1	0.81	5.1
X68264	Melanoma adhesion molecules	0.64	5.0
				AL161995	neurturin	0.86	4.9
AF037372	Cytochrome c oxidase	0.48	4.8
				NM_016187	Bridged integrin 2	0.65	4.8
AL137758	Is unknown	0.57	4.8
				U59863	TRAF family member-associated NFKB activators	0.46	4.7
Z30643	Chloride ion channel Ka	0.70	4.7
				D16294	acetyl-CoA acyltransferase 2	1.07	4.6
AJ132592	Zinc finger protein 281	0.55	4.6
				X82324	POU Domain TF	1.73	4.5
NM_016047	CGI-110 protein	1.95	4.5
				AK001371	Hypothetical proteins	0.49	4.5
M60746	H3 Histone family member D	3.05	4.5
				AB033071	Hypothetical proteins	4.47	4.4
AB002305	KIAA0307 gene product	1.37	4.4
				X92689	UDP-N-acetyl- α -D-galactosamine: polypeptide N-acetylgalactosamine aminotransferase	0.99	4.4
AL049543	Glutathione peroxidase 5	1.62	4.3
				U43148	PTCH(patched homolog)	0.96	4.3
M67439	Dopamine receptor D5	2.61	4.2
				U09850	Zinc finger protein 143	0.56	4.2
L20316	Glucagon receptor	0.75	4.2
				AB037767	Disintegrin-like and metalloproteases	0.69	4.2

NM_017433	Myosin IIIA	99.20	4.2
				D26579	Disintegrin and metalloprotease domain 8	0.59	4.1
L10333	reticulon 1	1.81	4.1
				AK000761	Is unknown	1.87	4.1
U91540	NK homeobox family 3, A	0.80	4.1
				Z17227	Interleukin 10 receptor, beta	0.75	4.0

Table 30: HBE cells were encoded by the peptide SEQ ID NO: 1 (50. mu.g/ml) down-regulated polynucleotide.

[0120]The peptide SEQ ID NO: 1 reduces the expression of a plurality of polynucleotides. Peptides were incubated with Human HBE epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a Human Operon array (PRHU 04). The intensity of the polynucleotide in unstimulated cells is shown in the third column. The column "ratio peptide treatment: control" refers to the intensity of polynucleotide expression in cells stimulated with peptide divided by the intensity of unstimulated cells.

Registration number	Gene	Comparison: unstimulated cells	Ratiometric peptide treatment to control
				AC004908	Is unknown	32.4	0.09
S70622	G1 stage specific gene	43.1	0.10
				Z97056	DEAD/H box polypeptide	12.8	0.11
AK002056	Hypothetical proteins	11.4	0.12
				L33930	CD24 antigen	28.7	0.13
X77584	Thioredoxin	11.7	0.13
				NM_014106	PRO1914 protein	25.0	0.14
M37583	H2A histone family member	22.2	0.14
				U89387	Polymerase (RNA) II polypeptide D	10.2	0.14
D25274	ras-related C3 botulinum toxin substrate 1	10.3	0.15
				J04173	Phosphoglyceride mutase 1	11.4	0.15
U19765	Zinc finger protein 9	8.9	0.16
				X67951	Proliferation-related gene A	14.1	0.16
AL096719	Actin inhibitory protein 2	20.0	0.16
				AF165217	Promyoglobin 4	14.6	0.16
NM_014341	Mitochondrial Carrier protein homolog 1	11.1	0.16
				AL022068	Is unknown	73.6	0.17
X69150	Ribosomal protein S18	42.8	0.17
				AL031577	Is unknown	35.0	0.17
AL031281	Is unknown	8.9	0.17
				AF090094	Human ornithine decarboxylase-resistant mRNA	10.3	0.17
AL022723	The "HLA-G histocompatibility antigen, class I, G	20.6	0.18
				U09813	ATP synthase, H + transporting mitochondrial F0 complex	9.8	0.18

AF000560	Human TTF-1 interacting peptides 20	20.2	0.19
				NM_016094	HSPC042 protein	67.2	0.19
AF047183	NADH dehydrogenase	7.5	0.19
				D14662	Antioxidant protein 2 (non-selenium glutathione peroxidase, acid calcium dependent phospholipase))	8.1	0.19
X16662	Annexin A8	8.5	0.19
				U14588	Pilin	11.3	0.19
AL117654	DKFZP586D0624 protein	12.6	0.20
				AK001962	Hypothetical proteins	7.7	0.20
L41559	6-pyruvoyl tetrahydropterin synthase/hepatocyte nuclear factor 1 alpha dimerizing cofactor	9.1	0.20
				NM_016139	16.7Kd protein	21.0	0.21
NM_016080	CGI-150 protein	10.7	0.21
				U86782	26S proteasome-associated pad1 homolog	6.7	0.21
AJ400717	Tumor protein, translation control protein 1	9.8	0.21
				X07495	Homology box C4	31.0	0.21
AL034410	Is unknown	7.3	0.22
				X14787	Platelet binding protein 1	26.2	0.22
AF081192	Purine element-rich binding protein B	6.8	0.22
				D49489	Protein disulfide isomerase-related protein	11.0	0.22
NM_014051	PTD011 protein	9.3	0.22
				AK001536	Is unknown	98.0	0.22
X62534	2 group of high-speed swimming proteins	9.5	0.22
				AJ005259	Endothelial differentiation related factor 1	6.7	0.22
NM_000120	Ring (C)Oxide hydrolase 1, microsomal	10.0	0.22
				M38591	S100 calcium binding protein A10	23.9	0.23
AF071596	Immediate early response protein 2	11.5	0.23
				X16396	Methylenetetrahydrofolate dehydrogenase	8.3	0.23
AK000934	ATP enzyme inhibitor precursors	7.6	0.23
				AL117612	Is unknown	10.7	0.23
AF119043	Transcription intermediate factor 1 gamma	7.3	0.23
				AF037066	Solute carrier family 22 member 1-like antisense	7.6	0.23
AF134406	Cytochrome c oxidase subunit	13.3	0.23
				AE000661	Is unknown	9.2	0.24
AL157424	synaptojanin 2	7.2	0.24
				X56468	Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein	7.2	0.24
U39318	Ubiquitin conjugating enzyme E2D3	10.7	0.24
				AL034348	Is unknown	24.4	0.24
D26600	Proteasome subunit beta type 4	11.4	0.24
				AB032987	Is unknown	16.7	0.24
J04182	Lysosomal associated membrane protein 1	7.4	0.24
				X78925	Zinc finger protein 267	16.1	0.25
NM_000805	Gastrin	38.1	0.25

U29700	Anti-muir hormone receptor, type II	12.0	0.25
				Z98200	Is unknown	13.4	0.25
U07857	Signal recognition particles	10.3	0.25
				L05096	Human ribosomal protein L39	25.3	0.25
AK001443	Hypothetical proteins	7.5	0.25
				K03515	Phosphoglucose isomerase	6.2	0.25
X57352	Interferon-inducible transmembrane protein 3	7.5	0.26
				J02883	Pancreatic co-lipase	5.7	0.26
M24069	Cold shock domain proteins	6.3	0.26
				AJ269537	Chondroitin-4-sulfotransferase	60.5	0.26
AL137555	Is unknown	8.5	0.26
				U89505	RNA binding motif protein 4	5.5	0.26
U82938	CD27 binding proteins	7.5	0.26
				X99584	SMT3 homolog 1	12.8	0.26
AK000847	Is unknown	35.8	0.27
				NM_014463	Lsm3 protein	7.8	0.27
AL133645	Is unknown	50.8	0.27
				X78924	Zinc finger protein 266	13.6	0.27
NM_004304	Malignant lymphoma kinase	15.0	0.27
				X57958	Ribosomal protein L7	27.9	0.27
U63542	Is unknown	12.3	0.27
				AK000086	Hypothetical proteins	8.3	0.27
X57138	H2A histidine family member N	32.0	0.27
				AB023206	KIAA0989 protein	6.5	0.27
AB021641	Gonadotropin-induced transcription repressor 1	5.5	0.28
				AF050639	NADH dehydrogenase	5.5	0.28
M62505	Complement component 5 receptor 1	7.5	0.28
				X64364	basigin	5.8	0.28
AJ224082	Is unknown	22.5	0.28
				AF042165	Cytochrome c oxidase	20.4	0.28
AK001472	anillin	10.9	0.28
				X86428	Protein phosphatase 2A subunit	12.7	0.28
AF227132	Candidate taste receptor T2R5	5.1	0.28
				Z98751	Is unknown	5.3	0.28
D21260	Clathrin heavy polypeptide	8.3	0.28
				AF041474	Actin-like 6	15.1	0.28
NM_005258	GTP cyclohydrolase I protein	7.6	0.28
				L20859	Solute carrier family 20	9.6	0.29
Z80783	H2B histone family member	9.0	0.29
				AB011105	Laminin alpha 5	7.1	0.29
AL008726	Beta-galactosidase protective protein	5.2	0.29
				D29012	Proteasome subunits	12.6	0.29
X63629	Cadherin 3P-cadherin	6.8	0.29
				X02419	Plasminogen activator urokinase	12.9	0.29

X13238	Cytochrome c oxidase	8.0	0.29
				X59798	Cyclin D1	12.7	0.30
D78151	Proteasome 26S subunit	7.6	0.31
				AF054185	Proteasome subunits	18.8	0.31
J03890	Lung-associated surfactant protein C	5.5	0.32
				M34079	Proteasome 26S subunit	5.2	0.33

Table 31: upregulation of polynucleotide expression induced by the peptide of formula a in a549 cells.

[0121]A peptide concentration of 50. mu.g/ml was found to increase the expression of many polynucleotides. The peptides were incubated with human a549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a human operon array (PRHU 04). The intensities of the polynucleotides in the unstimulated control cells are shown in the third and fourth columns, which correspond to the cDNAs labeled with the dyes Cy3 and Cy5, respectively. The column "ID #: control" refers to the intensity of polynucleotide expression in peptide-stimulated cells divided by the intensity of unstimulated cells.

Registration number	Gene	control-Cy 3	control-Cy 5	ID 5: control	ID 6: control	ID 7: control	ID 8: control	ID 9: control	ID 10: control
										U12472	Glutathione S-transferase	0.09	0.31	13.0	3.5	4.5	7.0	4.3	16.4
X66403	Cholinergic receptors	0.17	0.19	7.8	9.9	6.0	6.4	5.0	15.7
										AK001932	Is unknown	0.11	0.25	19.4	4.6	9.9	7.6	8.1	14.5
X58079	S100 calcium binding protein	0.14	0.24	12.2	7.6	8.1	4.3	4.5	13.2
										U18244	Solute carrier family 1	0.19	0.20	6.1	9.7	11.9	5.0	3.7	10.6
U20648	Zinc finger proteins	0.16	0.13	5.3	6.2	5.6	3.1	6.8	9.5
										AB037832	Is unknown	0.10	0.29	9.0	4.2	9.4	3.1	2.6	8.7
AC002542	Is unknown	0.15	0.07	10.5	15.7	7.8	10.1	11.7	8.2
										M89796	Transmembrane 4-structureDomain, subfamily A	0.15	0.14	2.6	6.1	7.6	3.5	13.3	8.1
AF042163	Cytochrome c oxidase	0.09	0.19	3.9	3.2	7.6	6.3	4.9	7.9
										AL032821	Vanin 2	0.41	0.23	2.5	5.2	3.2	2.1	4.0	7.9
U25341	Melatonin receptor 1B	0.04	0.24	33.1	5.1	23.3	6.6	4.1	7.6
										U52219	G protein-coupled receptors	0.28	0.20	2.1	6.2	6.9	2.4	3.9	7.1
X04506	Apolipoprotein B	0.29	0.32	7.9	3.4	3.3	4.8	2.6	7.0
										AB011138	Type IV ATPase	0.12	0.07	3.5	12.9	6.6	6.4	21.3	6.9
AF055018	Is unknown	0.28	0.22	3.8	6.9	5.0	2.3	3.1	6.8
										AK002037	Hypothetical proteins	0.08	0.08	2.9	7.9	14.1	7.9	20.1	6.5
AK001024	Guanylic acid binding protein	0.16	0.11	7.7	11.9	5.0	10.3	6.0	6.3
										AF240467	TLR-7	0.11	0.10	20.4	9.0	3.4	9.4	12.9	6.1
AF105367	Glucagon-like polypeptide 2 receptor	0.15	0.35	23.2	2.6	3.0	10.6	2.9	5.7
										AL009183	TNFR superfamily, member 9	0.46	0.19	10.6	4.7	3.7	2.8	6.5	5.7
X54380	Pregnancy zone proteins	0.23	0.08	4.7	11.9	7.2	12.7	3.8	5.5
										AL137736	Is unknown	0.22	0.15	2.1	7.2	3.3	7.1	4.6	5.5
X05615	Thyroglobulin	0.28	0.42	6.3	2.7	7.7	2.4	3.1	5.4

D28114	Myelin-associated proteins	0.24	0.08	2.5	15.9	13.0	7.1	13.7	5.4
										AK000358	Microfibrillar associated protein 3	0.28	0.28	8.7	4.2	7.2	3.2	2.4	5.3
AK001351	Is unknown	0.12	0.22	3.9	7.6	8.7	3.9	2.3	5.2
										U79289	Is unknown	0.14	0.27	2.5	2.7	2.8	2.0	4.3	5.1
AB014546	Zinc finger proteins	0.12	0.34	6.8	2.4	4.1	2.7	2.0	5.0
										AL117428	DKFZP434A236 protein	0.10	0.07	2.8	16.1	12.8	9.7	14.2	4.9
AL050378	Is unknown	0.41	0.14	3.5	8.7	11.7	3.5	7.0	4.9
										AJ250562	Transmembrane 4 superfamily member 2	0.13	0.10	5.2	5.7	14.2	3.8	10.3	4.8
NM_001756	Corticosteroid hormone binding globulin	0.28	0.13	4.0	7.9	6.5	14.9	5.6	4.8
										AL13747I	Hypothetical proteins	0.29	0.05	3.7	18.0	6.2	7.2	16.3	4.7
M19684	Protease inhibitor 1	0.41	0.14	3.5	4.6	5.4	2.8	9.4	4.7
										NM_001963	Epidermal growth factor	0.57	0.05	3.4	6.2	1.8	32.9	14.7	4.4
NM_000910	Neuropeptide Y receptors	0.62	0.36	3.1	2.7	2.3	2.6	3.1	4.4
										AF022212	Rho GTP enzyme activator protein 6	0.19	0.02	9.0	45.7	25.6	12.4	72.2	4.4
AK001674	Cofactor required for Sp1	0.11	0.13	8.4	6.5	7.9	4.5	7.4	4.3
										U51920	Signal recognition particles	0.23	0.27	3.4	3.8	2.1	4.1	8.8	4.2
AK000576	Hypothetical proteins	0.27	0.06	4.4	14.7	7.4	14.1	8.6	4.2
										AL080073	Is unknown	0.17	0.20	21.6	3.9	4.3	8.8	2.6	4.1
U59628	Paired cassette Gene 9	0.34	0.06	3.4	14.1	5.4	7.9	4.9	4.1
										U90548	Lactophagous protein, subfamily 3, member A3	0.4I	0.31	2.3	4.7	5.5	6.8	3.4	4.1
M19673	Caspase inhibitors SA	0.43	0.26	2.3	8.5	4.5	2.5	4.1	3.8
										AL161972	ICAM 2	0.44	0.37	2.0	3.6	2.0	2.7	5.5	3.8
X54938	Inositol 1, 4, 5-trisphosphate 3-kinase A	0.32	0.22	3.9	3.3	6.2	3.1	4.4	3.7
										AB014575	KIAA0675 gene product	0.04	0.13	46.2	4.5	10.2	8.0	6.2	3.4
M83664	MHC II，DP β1	0.57	0.29	2.9	2.1	2.0	3.1	6.6	3.4
										AK000043	Hypothetical proteins	0.34	0.14	2.7	7.1	3.7	9.4	8.8	3.3

U60666	Testis-specific leucine-rich repeat protein	0.21	0.11	9.9	9.0	4.1	5.5	13.0	3.3
										AK000337	Hypothetical proteins	0.49	0.19	4.3	5.1	4.7	10.6	7.1	3.3
AF050198	Inferred as mitochondrial space protein	0.34	0.15	7.0	6.3	3.6	5.6	11.9	3.3
										AJ251029	Odour binding protein 2A	0.28	0.12	4.4	9.4	7.2	8.8	7.1	3.2
X74142	Forked head type frame G1B	0.12	0.33	19.5	4.5	8.4	6.4	4.4	3.2
										AB029033	KIAA1110 protein	0.35	0.24	3.1	2.2	5.6	5.2	3.1	3.1
D85606	Cholecystokinin A receptors	0.51	0.14	4.3	3.9	4.6	3.5	7.2	3.1
										X84195	Acylphosphatase 2 muscle type	0.32	0.19	4.8	3.7	5.0	11.2	9.8	3.0
U57971	ATP enzyme calcium ion transport cytoplasmic Membrane 3	0.29	0.13	2.2	7.9	1.8	6.3	4.8	3.0
										J02611	Apolipoprotein D	0.28	0.10	2.8	11.0	3.7	10.3	8.4	3.0
AF071510	Lecithin retinol acyltransferase	0.07	0.05	7.9	3.8	11.7	46.0	16.3	3.0
										AF131757	Is unknown	0.10	0.08	4.8	9.0	44.3	9.3	10.7	3.0
L10717	IL 2-inducible T cell kinase	0.45	0.21	2.5	4.9	2.8	10.9	4.5	2.9
										L32961	4-aminobutyrate aminotransferase	0.64	0.32	3.6	2.9	3.2	5.3	2.3	2.9

Table 32: upregulation of polynucleotide expression in a549 cells induced by the peptide of formula B.

[0122]A peptide concentration of 50. mu.g/ml was found to increase the expression of many polynucleotides. The peptides were incubated with Human a549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a Human Operon array (PRHU 04). The intensities of the polynucleotides in the unstimulated control cells are shown in the third and fourth columns, which correspond to the cDNAs labeled with the dyes Cy3 and Cy5, respectively. The column "ID #: control" refers to the intensity of polynucleotide expression in peptide-stimulated cells divided by the intensity of unstimulated cells.

Registration number	Gene	control-Cy 3	control-Cy 5	ID 12: control	ID 13: control	ID 14: control	ID15∶Control	ID 16: control	ID 17: control
										AL157466	Unknown-0	0.05	0.06	18.0	21.4	16.7	5.2	6.8	8.6
AB023215	KIAA0998 protein	0.19	0.07	14.8	10.6	7.9	14.4	6.6	16.1
										AL031121	Is unknown	0.24	0.09	14.1	5.7	3.8	5.5	2.8	4.6

NM_016331	Zinc finger proteins	0.16	0.08	12.8	7.2	11.0	5.3	11.2	9.7
										M14565	Cytochrome P450	0.16	0.12	10.6	12.5	5.0	3.6	10.1	6.3
U22492	G protein-coupled receptor 8	0.28	0.07	10.4	8.9	4.8	10.8	6.6	3.6
										U76010	Solute carrier family 30	0.14	0.07	9.7	18.6	3.7	4.8	5.6	8.9
AK000685	Is unknown	0.51	0.10	9.0	3.1	2.8	3.9	15.3	3.0
										AF013620	Immunoglobulin heavy chain variable region 4-4	0.19	0.18	8.5	2.6	6.2	5.7	8.2	3.8
AL049296	Is unknown	0.61	0.89	8.1	3.2	2.7	3.2	2.7	2.0
										AB006622	KIAA0284 protein	0.47	0.28	7.5	5.0	2.8	11.1	5.5	4.6
X04391	CD5 antigen	0.22	0.13	7.2	16.7	2.7	7.7	6.1	5.9
										AK000067	Hypothetical proteins	0.80	0.35	7.1	4.6	2.1	3.2	8.5	2.2
AF053712	TNF superfamily member 11	0.17	0.08	6.9	17.7	3.0	6.2	12.3	5.2
										X58079	S100 calcium binding protein A1	0.14	0.24	6.7	6.7	5.9	6.5	5.3	2.5
M91036	Hemoglobin _ Gamma A	0.48	0.36	6.7	14.2	2.1	2.9	2.7	4.8
										AF055018	Is unknown	0.28	0.22	6.3	10.7	2.7	2.6	4.6	6.5
L17325	Pre-T/NK cell related proteins	0.19	0.29	6.1	4.4	6.5	4.7	4.0	4.0
										D45399	Phosphodiesterase enzyme	0.21	0.18	6.1	4.6	5.0	2.8	10.8	4.0
AB023188	KIAA0971 protein	0.29	0.13	5.9	10.6	3.6	3.4	10.6	7.2
										NM_012177	F-box protein	0.26	0.31	5.9	5.5	3.8	2.8	3.0	6.8
D38550	E2F TF3	0.43	0.39	5.8	3.4	2.1	4.5	2.5	2.4
										AL050219	Is unknown	0.26	0.04	5.7	17.0	3.1	9.2	30.3	16.1
AL137540	Is unknown	0.67	0.79	5.5	3.2	3.9	10.9	2.9	2.3
										D50926	KIAA0136 protein	0.57	0.21	5.4	5.6	2.0	3.3	4.4	3.2
AL137658	Is unknown	0.31	0.07	5.4	12.1	2.6	10.8	3.9	8.6
										U21931	Fructose bisphosphatase 1	0.48	0.14	5.4	4.1	2.9	3.6	6.0	3.2
AK001230	DKFZP586D211 protein	0.43	0.26	5.0	4.6	2.1	2.2	2.5	2.7
										AL137728	Is unknown	0.67	0.47	5.0	5.9	2.2	6.8	5.9	2.1

AB022847	Is unknown	0.39	0.24	4.5	2.2	3.5	4.3	3.8	3.7
										X75311	Mevalonate kinase	0.67	0.22	4.3	4.0	2.0	8.3	4.0	5.1
AK000946	DKFZP566C243 protein	0.36	0.29	4.1	3.8	3.9	5.4	25.8	2.7
										AB023197	KIAA0980 protein	0.25	0.30	4.0	8.3	2.1	8.8	2.2	4.9
AB014615	Fibroblast growth factor 8	0.19	0.07	3.9	3.3	7.0	3.4	2.2	7.7
										X04014	Is unknown	0.29	0.16	3.8	2.5	2.2	3.0	5.5	3.1
U76368	Solute carrier family 7	0.46	0.17	3.8	3.8	2.8	3.2	4.2	3.0
										AB032436	Is unknown	0.14	0.21	3.8	2.7	6.1	3.2	4.5	2.6
AB020683	KIAA0876 protein	0.37	0.21	3.7	4.2	2.2	5.3	2.9	9.4
										NM_012126	Carbohydrate sulfotransferase 5	0.31	0.20	3.7	5.2	3.2	3.4	3.9	2.5
AK002037	Hypothetical proteins	0.08	0.08	3.7	17.1	4.6	12.3	11.0	8.7
										X78712	Glycerol kinase pseudogene 2	0.17	0.19	3.6	2.5	4.5	5.3	2.2	3.3
NM_014178	HSPC156 protein	0.23	0.12	3.5	8.4	2.9	6.9	14.4	5.5
										AC004079	Homology box A2	0.31	0.11	3.5	7.0	2.1	2.0	7.3	9.1
AL080182	Is unknown	0.51	0.21	3.4	3.5	2.2	2.1	2.9	2.4
										M91036	Hemoglobin gamma G	0.22	0.02	3.4	26.3	5.8	6.8	30.4	21.6
AJ000512	Serum/glucocorticoid-regulated kinase	0.27	0.43	3.3	2.1	4.9	2.3	3.9	2.7
										AK002140	Hypothetical proteins	0.28	0.14	3.3	9.9	2.8	2.1	16.6	7.2
AL137284	Is unknown	0.22	0.04	3.3	7.2	4.1	6.0	12.2	3.7
										Z11898	POU Domain _ type 5TF 1	0.12	0.29	3.2	3.7	8.2	2.5	6.6	2.2
AB017016	Brain specific proteins	0.27	0.29	3.1	2.8	2.5	2.8	3.3	5.5
										X54673	Solute carrier family 6	0.34	0.08	2.9	12.0	2.2	10.4	7.4	5.9
AL033377	Is unknown	0.40	0.22	2.6	2.6	2.6	2.3	4.5	2.2
										X85740	CCR4	0.34	0.05	2.6	2.3	2.6	2.5	12.5	5.2
AB010419	Core binding proteins	0.59	0.20	2.5	12.8	2.0	2.8	2.9	5.9
										AL109726	Is unknown	0.14	0.15	2.3	9.0	4.3	4.4	2.6	3.7

NM_012450	Sulfate Transporter 1	0.15	0.10	2.2	3.1	8.2	9.9	4.7	5.9
										J04599	Biglycan proteoglycan	0.39	0.30	2.1	3.3	6.6	2.2	2.7	5.4
AK000266	Hypothetical proteins	0.49	0.35	2.1	3.5	3.5	6.6	4.3	4.0

Table 33: upregulation of polynucleotide expression induced by the peptide of formula C in a549 cells.

[0123]A peptide concentration of 50. mu.g/ml was found to increase the expression of many polynucleotides. The peptides were incubated with Human a549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a Human Operon array (PRHU 04). The intensities of the polynucleotides in the unstimulated control cells are shown in the third and fourth columns, which correspond to the cDNAs labeled with the dyes Cy3 and Cy5, respectively. The column "ID #: control" refers to the intensity of polynucleotide expression in peptide-stimulated cells divided by the intensity of unstimulated cells.

Registration number	Gene	control-Cy 3	control-Cy 5	ID 19: control	ID 20: control	ID 21: control	ID 22: control	ID 23: control	ID 24: control
										AF030555	Fatty acid coenzyme A ligase	0.10	0.39	8.7	2.2	11.3	9.9	3.0	2.1
AL050125	Is unknown	0.23	0.07	8.6	14.3	5.2	2.8	18.7	8.3
										AB011096	KIAA0524 protein	0.21	0.08	8.5	24.4	4.7	6.8	10.4	7.5
J03068	N-acylaminoacyl-peptide hydrolases	0.54	0.21	8.3	2.4	2.2	4.1	3.0	6.0
										M33906	Class II MHC, DQ α 1	0.14	0.08	7.6	4.5	15.2	6.1	7.5	7.9
AJ272265	Secretion of phosphoproteins	0.21	0.09	7.6	9.0	3.3	4.9	18.8	14.5
										J00210	Interferon alpha 13	0.41	0.07	7.2	15.0	2.8	3.1	11.0	4.3
AK001952	Hypothetical proteins	0.42	0.21	6.9	4.9	2.5	3.1	7.6	4.5
										X54131	Protein tyrosine phosphatases, receptor types	0.09	0.20	6.4	6.5	7.7	15.0	5.6	4.1
AF064493	LIM binding Domain 2	0.46	0.14	5.9	5.6	2.2	2.9	8.5	5.8
										AL117567	DKFZP566O084 protein	0.44	0.22	5.8	3.3	2.9	2.3	5.7	14.9
L40933	Phosphoglucomutase 5	0.16	0.03	5.6	11.0	4.8	3.5	8.5	76.3
										M27190	Pancreatic stone protein (regenerating islet-derived 1 alpha)	0.19	0.28	5.3	3.0	3.8	3.6	5.8	3.6

AL031121	Is unknown	0.24	0.09	5.3	3.8	3.2	3.9	3.0	27.9
										U27655	Modulators of G protein signaling	0.24	0.29	5.0	9.0	4.5	8.3	4.2	4.5
AB037786	Is unknown	0.12	0.03	4.7	54.1	2.8	2.3	2.2	11.0
										X73113	Myosin binding protein C	0.29	0.13	4.7	6.5	6.0	2.4	6.7	6.3
AB010962	Matrix metalloproteinases	0.08	0.12	4.7	6.2	2.4	4.7	10.9	4.2
										AL096729	Is unknown	0.36	0.13	4.7	7.7	3.2	2.4	6.3	6.2
AB018320	Arg/Abl interacting protein	0.16	0.18	4.6	7.1	3.0	3.3	5.8	8.9
										AK001024	Guanylic acid binding protein	0.16	0.11	4.6	2.0	9.8	2.6	7.6	14.1
AJ275355	Is unknown	0.15	0.08	4.6	17.3	5.4	9.2	5.1	5.5
										U21931	Fructose bisphosphatase 1	0.48	0.14	4.6	4.3	2.6	2.1	8.4	9.6
X66403	Cholinergic receptors	0.17	0.19	4.4	9.0	10.9	9.3	5.1	6.7
										X67734	contactin 2	0.25	0.09	4.3	6.8	3.1	5.8	7.9	8.4
U92981	Is unknown	0.20	0.23	4.3	3.2	4.8	5.6	5.4	6.3
										X68879	Air door homolog 1	0.05	0.08	4.3	2.0	12.3	2.7	5.6	4.7
AL137362	Is unknown	0.22	0.22	4.2	4.1	2.7	4.1	9.3	4.2
										NM_001756	Corticosteroid hormone binding globulin	0.28	0.13	4.4	10.6	3.9	2.7	10.3	5.5
U80770	Is unknown	0.31	0.14	4.1	4.1	23.3	2.7	7.0	10.1
										AL109792	Is unknown	0.16	0.19	4.0	4.5	4.3	8.8	8.7	3.9
X65962	Cytochrome P450	0.33	0.05	3.8	25.3	5.7	5.1	19.8	12.0
										AK001856	Is unknown	0.40	0.21	3.8	7.0	2.6	3.1	2.9	7.8
AL022723	MHC, class I, F	0.55	0.18	3.7	5.7	4.4	2.3	3.3	5.2
										D38449	Putative G protein-coupled receptors	0.18	0.09	3.5	11.1	13.3	5.8	4.8	5.2
AL137489	Is unknown	0.74	0.26	3.3	2.9	2.6	3.3	2.5	5.4
										AB000887	Small inducible cytokine subfamily A	0.76	0.18	3.3	5.0	2.6	2.4	5.9	10.3
NM_012450	Sulfate Transporter 1	0.15	0.10	3.3	9.0	10.0	10.9	4.6	8.7
										U86529	Glutathione S-transferase ζ 1	0.55	0.15	3.2	6.8	4.4	2.3	9.3	5.1

AK001244	Is unknown	0.79	0.31	3.2	5.5	2.3	2.3	3.9	2.8
										AL133602	Is unknown	0.16	0.21	3.1	7.8	8.7	2.6	4.1	5.6
AB033080	Cell cycle process 8 protein	0.31	0.31	3.1	4.6	3.0	3.5	2.2	4.2
										AF023466	Putative glycine-N-acyltransferase	0.27	0.18	3.1	5.0	4.2	7.4	10.1	3.8
AL117457	Actin (cofilin)2	0.68	0.53	3.0	4.6	3.3	2.4	7.4	3.4
										AC007059	Is unknown	0.37	0.35	3.0	5.7	3.1	2.4	2.6	2.4
U60179	Growth hormone receptors	0.34	0.21	2.9	3.5	2.3	3.1	8.0	4.7
										M37238	Phospholipase C, gamma 2	0.60	0.36	2.9	2.0	3.2	2.1	2.9	4.6
L22569	Cathepsin B	0.32	0.12	2.9	2.1	6.2	3.0	13.1	16.7
										M80359	MAP/microtubule affinity regulated kinase 3	0.37	0.76	2.9	3.1	6.1	7.6	2.1	3.3
S70348	Integrin beta 3	0.58	0.31	2.6	4.8	4.1	2.6	2.6	2.6
										L13720	Growth retardation specific protein 6	0.36	0.26	2.4	2.5	6.8	4.8	3.9	3.7
AL049423	Is unknown	0.33	0.30	2.4	3.7	3.8	2.8	2.9	3.4
										AL050201	Is unknown	0.68	0.29	2.2	3.1	3.7	3.0	3.0	2.2
AF050078	Growth retardation specific protein 11	0.87	0.33	2.1	8.4	2.5	2.2	2.6	4.4
										AK001753	Hypothetical proteins	0.53	0.28	2.1	5.0	2.2	2.8	3.6	4.6
X05323	Is unknown	0.39	0.13	2.1	7.8	2.6	2.4	21.5	3.5
										AB014548	KIAA0648 protein	0.61	0.30	2.0	2.4	4.8	3.4	4.9	3.9

Table 34: upregulation of polynucleotide expression in a549 cells induced by the peptide of formula D.

[0124]A peptide concentration of 50. mu.g/ml was found to increase the expression of many polynucleotides. The peptides were incubated with Human a549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a Human Operon array (PRHU 04). The intensities of the polynucleotides in the unstimulated control cells are shown in the third and fourth columns, which correspond to the cDNAs labeled with the dyes Cy3 and Cy5, respectively. The column "ID #: control" refers to the intensity of polynucleotide expression in peptide-stimulated cells divided by the intensity of unstimulated cells.

Registration number	Gene	control-Cy 3	control-Cy 5	ID 26: control	ID 27: control	ID 28: control	ID 29: control	ID 30: control	ID 31: control
										U68018	MAD homolog 2	0.13	0.71	11.2	2.2	8.0	2.3	6.7	25.6
NM_016015	CGI-68 protein	0.92	1.59	2.3	2.3	3.5	3.7	3.4	22.9
										AF071510	Lecithin retinol acyltransferase	0.07	0.05	15.4	10.3	5.3	44.1	2.1	21.2
AC005154	Is unknown	0.17	1.13	2.7	7.2	12.6	6.4	3.3	20.6
										M81933	Cell division cycle 25A	0.13	0.21	4.3	3.1	3.2	4.3	5.6	18.2
AF124735	LIM HOX Gene 2	0.17	0.21	2.1	4.4	5.9	5.2	7.6	17.0
										AL110125	Is unknown	0.30	0.08	5.0	2.7	6.8	10.2	2.8	12.0
NM_004732	Voltage gated potassium channels	0.15	0.16	7.6	4.0	3.4	2.2	2.9	11.4
										AF030555	Fatty acid coenzyme A ligase _ Long chain 4	0.10	0.39	10.5	2.2	6.4	3.0	5.1	10.7
AF000237	1-acylglycerol-3-phosphate O-acyltransferase 2	1.80	2.37	3.4	2.5	2.4	2.1	3.7	9.9
										AL031588	Hypothetical proteins	0.40	0.26	5.8	20.2	2.8	4.7	5.6	9.1
AL080077	Is unknown	0.15	0.21	2.4	2.0	11.9	3.8	2.3	8.7
										NM_014366	Presumed to be nucleotide binding protein estradiol inducibility	0.90	2.52	2.4	4.3	2.4	2.6	3.0	8.6
AB002359	Phosphoribosyl formyl glycinamidine synthetase	0.81	2.12	3.2	2.7	5.5	2.5	2.8	6.9
										U33547	MHC class II antigen HLA-DRB6 mRNA	0.14	0.16	2.5	5.3	4.5	5.0	3.1	6.6
AL133051	Is unknown	0.09	0.07	7.7	6.3	5.4	23.1	5.4	6.5
										AK000576	Hypothetical proteins	0.27	0.06	7.1	9.3	5.0	6.9	2.9	6.2
AF042378	Spindle polar body protein	0.36	0.39	3.3	3.0	9.5	4.5	3.4	6.2
										AF093265	Homer neuron immediate early gene _3	0.67	0.53	2.7	13.3	6.5	5.0	2.9	6.2
D80000	Mitotic chromosome segregation 1	1.01	1.56	3.6	2.5	4.9	3.2	6.3	6.1
										AF035309	Proteasome 26S subunit ATPase 5	3.61	4.71	2.7	6.6	5.2	4.9	2.7	6.0
M34175	Connexin-related protein complex 2 beta 1 subunit	4.57	5.13	3.2	3.1	4.0	4.6	2.7	6.0
										AB020659	KIAA0852 protein	0.18	0.37	4.1	7.6	5.7	4.8	2.5	5.7
NM_004862	TNF alpha factor induced by LPS	2.61	3.36	3.8	4.8	4.1	4.9	3.2	5.6

U00115	Zinc finger protein 51	0.51	0.07	18.9	2.2	3.5	7.2	21.2	5.6
										AF088868	fibrousheathin II	0.45	0.20	4.7	10.0	3.2	6.4	6.0	5.6
AK001890	Is unknown	0.42	0.55	2.4	3.5	3.6	2.3	2.2	5.6
										AL137268	KIAA0759 protein	0.49	0.34	3.8	2.3	5.0	3.5	3.3	5.4
X63563	Polymerase II polypeptide B	1.25	1.68	2.5	8.1	3.4	4.8	5.2	5.4
										D12676	CD36 antigen	0.35	0.39	2.9	3.4	2.6	2.2	3.5	5.3
AK000161	Hypothetical proteins	1.06	0.55	3.4	8.7	2.1	6.7	2.9	5.1
										AF052138	Is unknown	0.64	0.51	2.9	2.8	2.7	5.2	3.6	5.0
AL096803	Is unknown	0.36	0.03	20.1	18.3	3.7	19.3	16.1	4.9
										S49953	DNA binding transcriptional activator	0.70	0.15	3.7	4.0	2.1	6.6	4.0	4.8
X89399	RAS p21 protein activator	0.25	0.10	8.5	14.9	4.8	18.6	4.3	4.8
										AJ005273	Antigenic determinants of recA protein	0.70	0.10	7.6	11.1	2.8	9.9	12.0	4.6
AK001154	Hypothetical proteins	1.70	0.96	2.4	4.4	2.9	8.9	2.4	4.5
										AL133605	Is unknown	0.26	0.15	12.4	4.2	4.4	3.3	3.3	4.1
U71092	G protein-coupled receptor 24	0.53	0.06	19.0	9.1	2.2	12.0	3.3	4.1
										AF074723	RNA polymer II transcription regulatory medium	0.67	0.54	4.0	3.2	3.1	3.4	6.0	4.0
AL137577	Is unknown	0.32	0.12	31.4	6.2	5.3	10.1	25.3	3.9
										AF151043	Hypothetical proteins	0.48	0.35	2.6	2.2	2.0	3.3	2.2	3.8
AF131831	Is unknown	0.67	0.81	2.1	7.0	3.5	3.2	3.9	3.7
										D50405	Histidine deacetylase 1	1.52	1.62	3.1	7.2	2.9	4.1	2.8	3.7
U78305	Protein phosphatase 1D	1.21	0.20	4.7	13.0	3.5	5.9	4.2	3.7
										AL035562	Paired box gene 1	0.24	0.01	30.2	81.9	5.6	82.3	6.2	3.7
U67156	Mitogen-activated protein kinase 5	1.15	0.30	6.6	3.0	2.2	2.3	2.5	3.6
										AL031121	Is unknown	0.24	0.09	5.2	3.7	2.3	6.5	9.1	3.6
U13666	G protein-coupled receptor 1	0.34	0.14	3.8	5.4	3.1	3.3	2.8	3.6
										AB018285	KIAA0742 protein	0.53	0.13	14.9	13.9	5.9	18.5	15.2	3.5

D42053	Site 1 protease	0.63	0.40	2.6	7.1	5.6	9.2	2.6	3.5
										AK001135	Sec23 interacting protein p125	0.29	0.53	5.7	4.5	3.4	2.6	11.3	3.4
AL137461	Is unknown	0.25	0.02	23.8	9.0	2.7	59.2	12.5	3.3
										NM_006963	Zinc finger protein 22	0.10	0.08	3.2	7.6	3.7	7.9	11.2	3.2
AL137540	Is unknown	0.67	0.79	3.9	2.6	5.6	4.2	3.5	3.1
										AL137718	Is unknown	0.95	0.18	4.7	8.0	4.0	13.3	3.0	3.1
AF012086	RNA binding protein 2-like 1	1.20	0.59	4.6	4.0	2.0	4.6	3.6	3.1
										S57296	HER2/neu receptor	0.59	0.17	7.3	12.1	2.3	20.0	22.2	3.0
NM_013329	GC sequence-rich DNA binding factor candidates	0.16	0.08	6.9	14.3	9.7	3.3	7.2	3.0
										AF038664	UDP-Gal: beta GlcN Ac beta 1_ 4-galactosyltransferase	0.15	0.03	13.4	22.2	5.4	15.8	17.6	3.0
AF080579	Human integral membrane protein	0.34	1.03	3.3	3.0	6.7	2.1	2.9	2.9
										AK001075	Hypothetical proteins	0.67	0.10	2.1	2.6	2.6	8.9	2.2	2.9
AB011124	KIAA0552 Gene products	0.46	0.04	9.6	72.0	6.0	33.9	13.6	2.9
										J03068	N-acylaminoacyl-peptide hydrolases	0.54	0.21	2.2	5.0	2.4	5.2	3.6	2.8
D87120	Osteoblast proteins	0.87	0.87	2.2	2.0	4.7	2.3	2.0	2.8
										AB006537	IL-1R accessory proteins	0.17	0.07	2.9	7.0	14.5	5.3	6.6	2.8
L34587	Transcriptional elongation factor B	2.49	1.23	2.2	16.3	5.0	15.8	5.5	2.7
										D31891	SET Domain _ fork _1	1.02	0.29	3.9	6.0	4.3	4.9	6.6	2.7
D00760	Proteasome subunit alpha type 2	4.97	4.94	4.1	2.6	2.0	2.8	2.7	2.7
										AC004774	digital-less homology box 5	0.25	0.12	2.3	6.3	3.8	5.2	5.2	2.6
AL024493	Is unknown	1.46	0.54	4.8	13.5	2.1	11.6	6.8	2.6
										AB014536	copine III	1.80	1.29	3.2	9.5	3.8	6.8	2.6	2.6
X59770	IL-1R type II	0.59	0.16	9.6	4.7	3.9	3.2	4.9	2.5
										AF052183	Is unknown	0.65	0.76	4.0	3.7	2.3	5.0	3.0	2.5
AK000541	Hypothetical proteins	0.92	0.27	4.5	13.9	3.6	18.1	4.3	2.5
										U88528	cAMP response element binding proteins	1.37	0.86	3.1	5.4	2.1	2.8	2.1	2.4

M97925	Defensin alpha 5 paneth cell specificity	0.33	0.07	4.6	35.9	2.0	7.8	6.5	2.4
										NM_013393	Cell division protein FtsJ	1.38	0.94	3.1	5.8	2.1	4.2	2.6	2.3
X62744	Class II MHC DM alpha	0.86	0.32	4.0	4.7	2.3	2.9	6.1	2.3
										AF251040	Presumed to be a nuclear protein	0.64	0.30	6.7	3.4	2.9	3.9	5.7	2.2
AK000227	Hypothetical proteins	1.49	0.43	3.4	7.1	2.3	3.3	9.1	2.1
										U88666	SFRS protein kinase 2	1.78	0.37	3.4	5.9	2.6	8.4	6.1	2.0

Table 35: upregulation of polynucleotide expression induced by a peptide of formula E in a549 cells.

[0125]A peptide concentration of 50. mu.g/ml was found to increase the expression of many polynucleotides. The peptides were incubated with Human a549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a Human Operon array (PRHU 04). The intensities of the polynucleotides in the unstimulated control cells are shown in the third and fourth columns, which correspond to the cDNAs labeled with the dyes Cy3 and Cy5, respectively. The column "ID #: control" refers to the intensity of polynucleotide expression in peptide-stimulated cells divided by the intensity of unstimulated cells.

Registration number	Gene	control-Cy 3	control-Cy 5	ID 33: control	ID 34: control	ID 35: control	ID 36: control	ID 37: control	ID 38: control
										AL049689	Novel human mRNA	0.25	0.05	2.7	26.5	3.3	21.7	5.4	37.9
AK000576	Hypothetical proteins	0.27	0.06	3.0	19.1	3.9	23.0	3.1	28.3
										X74837	Mannosidase, alpha class 1A member 1	0.10	0.07	5.6	10.0	10.8	12.3	12.0	19.9
AK000258	Hypothetical proteins	0.27	0.07	14.0	11.1	7.9	16.1	6.2	18.9
										X89067	Transient receptors	0.20	0.14	3.7	2.2	2.4	2.6	8.0	18.1
AL137619	Is unknown	0.16	0.08	6.3	6.7	10.8	10.5	7.9	16.5
										NM_003445	Zinc finger proteins	0.17	0.07	4.0	23.6	2.9	13.6	4.3	14.4
X03084	Complement component 1	0.36	0.15	2.4	3.1	2.9	7.7	3.4	13.7
										U27330	Fucosyltransferase 5	0.39	0.08	2.4	2.5	2.6	12.1	3.5	13.0
AF070549	Is unknown	0.16	0.09	2.7	4.7	7.9	10.3	4.2	12.6
										AB020335	sel-1 sample	0.19	0.24	2.9	2.6	2.0	7.3	4.7	12.4

M26901	Renin	0.09	0.12	14.9	2.2	7.3	12.0	20.8	12.0
										Y07828	Ring finger protein	0.09	0.06	9.0	26.6	8.9	16.0	3.6	11.6
AK001848	Hypothetical proteins	0.21	0.07	6.2	8.2	2.7	5.2	5.5	10.9
										NM_016331	Zinc finger proteins	0.16	0.08	7.6	5.1	7.0	25.5	5.5	10.9
U75330	Neural cell adhesion molecule 2	0.42	0.08	2.5	3.6	2.0	5.8	6.2	9.9
										AB037826	Is unknown	0.16	0.11	3.8	6.0	3.4	13.4	6.0	9.8
M34041	Adrenergic alpha-2B receptors	0.30	0.13	4.5	4.5	3.7	8.6	5.6	9.8
										D38449	Putative G protein-coupled receptors	0.18	0.09	2.3	25.8	11.7	2.3	3.2	9.5
AJ250562	Transmembrane 4 superfamily member 2	0.13	0.10	10.0	8.4	2.2	8.1	16.3	9.1
										AK001807	Hypothetical proteins	0.18	0.12	4.2	5.3	4.6	3.2	4.0	8.3
AL133051	Is unknown	0.09	0.07	5.1	13.6	6.0	9.1	2.2	8.2
										U43843	Nerve-d 4 homolog	0.61	0.10	2.0	6.4	2.3	16.6	2.2	8.1
NM_013227	Cartilage aggrecan 1	0.28	0.15	7.5	3.1	2.5	6.9	8.5	7.8
										AF226728	Somatostatin receptor interacting proteins	0.23	0.17	7.0	3.6	3.1	5.5	3.5	7.7
AK001024	Guanylic acid binding protein	0.16	0.11	0.39	12.3	2.7	7.4	3.3	7.0
										AC002302	Is unknown	0.13	0.14	16.1	5.8	5.8	2.6	9.6	6.2
AB007958	Is unknown	0.17	0.27	2.0	2.3	11.3	3.3	3.0	6.1
										AF059293	Cytokine receptor-like factor 1	0.19	0.22	3.6	2.5	10.2	3.8	2.7	5.9
V01512	v-fos	0.27	0.21	6.7	3.7	13.7	9.3	3.7	5.4
										U82762	Sialyltransferase 8	0.23	0.15	3.2	6.5	2.7	9.2	5.7	5.4
U44059	Thyroid stimulating embryonic factor	0.05	0.13	22.9	7.1	12.5	7.4	9.7	5.4
										X05323	Antigens confirmed by monoclonal antibodies	0.39	0.13	4.3	2.5	2.2	7.4	2.8	5.1
U72671	ICAM 5，	0.25	0.14	5.3	2.7	3.7	10.0	3.2	4.8
										AL133626	Hypothetical proteins	0.26	0.25	2.2	4.2	2.9	3.0	2.6	4.7
X96401	MAX binding proteins	0.31	0.29	6.9	2.3	4.9	3.1	2.9	4.6
										AL117533	Is unknown	0.05	0.26	8.2	2.7	11.1	2.5	11.9	4.5

AK001550	Hypothetical proteins	0.10	0.30	8.0	2.0	4.9	2.1	7.8	4.5
										AB032436	Human BNPI mRNA	0.14	0.21	5.1	2.2	9.1	4.5	6.4	4.4
AL035447	Hypothetical proteins	0.28	0.23	4.3	3.7	8.7	5.2	3.7	4.2
										U09414	Zinc finger proteins	0.28	0.25	4.0	2.2	4.7	3.3	7.2	4.2
AK001256	Is unknown	0.09	0.08	5.3	6.5	31.1	12.7	6.4	4.1
										L14813	Carboxylate ligase like	0.64	0.21	2.7	6.2	3.1	2.1	3.4	3.9
AF038181	Is unknown	0.06	0.18	34.1	6.4	4.5	8.7	11.3	3.9
										NM_001486	Glucokinase	0.21	0.08	3.0	2.2	6.5	12.4	5.7	3.9
AB033000	Hypothetical proteins	0.24	0.22	3.4	3.3	7.1	5.5	4.5	3.8
										AL117567	DKFZP566O084 protein	0.44	0.22	2.2	2.7	3.9	4.0	4.5	3.7
NM_012126	Carbohydrate sulfotransferase 5	0.31	0.20	5.5	5.4	3.8	5.5	2.6	3.5
										AL031687	Is unknown	0.16	0.27	5.9	2.6	3.4	2.3	4.9	3.5
X04506	Apolipoprotein B	0.29	0.32	5.4	4.4	6.9	5.5	2.1	3.5
										NM_006641	CCR9	0.35	0.11	3.3	3.3	2.2	16.5	2.3	3.5
Y00970	Acrosin	0.12	0.14	8.2	8.8	3.1	6.2	17.5	3.4
										X67098	rTS beta protein	0.19	0.26	2.4	3.1	7.8	3.5	4.4	3.3
U51990	Pre-mRNA splicing factor	0.56	0.19	2.2	3.0	2.8	13.7	2.9	3.0
										AF030555	Fatty acid coenzyme A	0.10	0.39	3.5	6.9	13.3	4.4	7.5	2.9
AL009183	TNFR superfamily, member 9	0.46	0.19	6.0	4.1	2.8	8.6	2.6	2.8
										AF045941	sciellin	0.16	0.21	11.6	2.4	2.8	2.2	4.1	2.8
AF072756	Kinase ankyrin 4	0.33	0.07	2.5	5.3	3.9	32.7	2.3	2.7
										X78678	Hexanone kinase	0.10	0.20	18.0	3.5	4.1	2.5	14.6	2.6
AL031734	Is unknown	0.03	0.39	43.7	2.3	41.7	4.0	10.8	2.5
										D87717	KIAA0013 Gene product	0.35	0.42	4.2	2.3	3.6	2.6	2.9	2.5
U01824	Solute carrier family 1	0.42	0.29	4.8	2.3	4.2	7.1	4.2	2.4
										AF055899	Solute carrier family 27	0.14	0.31	9.5	12.3	7.4	4.7	6.6	2.3

U22526	Lanosterol synthetase	0.09	0.45	4.1	3.4	10.4	2.2	17.9	2.3
										AB032963	Is unknown	0.19	0.34	6.3	6.1	2.9	2.1	5.7	2.2
NM_015974	Lambda-crystallins	0.17	0.25	11.4	2.8	5.9	2.4	5.8	2.2
										X82200	Stimulated trans-acting factor	0.23	0.15	8.2	3.4	3.0	2.8	11.3	2.2
AL137522	Is unknown	0.12	0.26	12.1	3.7	12.6	6.9	4.3	2.2
										Z99916	Crystallin, beta B3	0.28	0.65	2.5	2.1	3.6	2.2	2.6	2.1
AF233442	Ubiquitin-specific protease 21	0.41	0.31	2.6	3.6	3.6	4.5	3.4	2.1
										AK001927	Hypothetical proteins	0.24	0.52	7.6	5.6	5.0	2.5	4.1	2.0

Table 36: upregulation of polynucleotide expression induced by the peptide of formula F in a549 cells.

[0126]A peptide concentration of 50. mu.g/ml was found to increase the expression of many polynucleotides. The peptides were incubated with Human a549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a Human Operon array (PRHU 04). The intensities of the polynucleotides in the unstimulated control cells are shown in the third and fourth columns, which correspond to the cDNAs labeled with the dyes Cy3 and Cy5, respectively. The column "ratio ID #: control" refers to the intensity of polynucleotide expression in peptide-stimulated cells divided by the intensity of unstimulated cells.

Registration number	Gene	control-Cy 3	control-Cy 5	Ratio ID40 comparison	Ratio ID42 comparison	Ratio ID43 comparison	Ratio ID44 comparison	Ratio ID45 comparison
									AF025840	Polymerase epsilon 2	0.34	0.96	3.4	2.0	2.0	2.1	4.3
AF132495	CGI-133 protein	0.83	0.67	3.0	2.2	2.6	2.8	5.1
									AL137682	Hypothetical proteins	0.73	0.40	2.0	5.3	4.8	2.9	8.2
U70426	Modulators of G protein signaling 16	0.23	0.25	3.1	3.0	5.3	3.1	12.2
									AK001135	Sec23 interacting protein p125	0.29	0.53	3.2	2.6	3.3	14.4	5.2
AB023155	KIAA0938 protein	0.47	0.21	2.7	4.8	8.1	4.2	10.4
									AB033080	Cell cycle process 8 protein	0.31	0.31	4.4	2.2	5.9	4.3	6.9
AF061836	Ras-associated domain family 1	0.29	0.31	3.2	2.5	11.1	18.8	6.8

AK000298	Hypothetical proteins	0.48	0.27	3.3	2.2	7.1	5.6	7.7
									L75847	Zinc finger proteins	0.35	0.52	3.2	3.0	4.0	3.0	3.9
X97267	Protein tyrosine phosphatase	0.19	0.24	4.1	9.3	2.4	4.2	8.3
									Z11933	POU Domain class 3TF 2	0.09	0.23	8.7	2.5	3.6	4.3	8.2
AB037744	Is unknown	0.37	0.57	2.6	2.9	2.7	3.0	3.1
									U90908	Is unknown	0.12	0.16	11.8	7.7	3.4	7.8	11.2
AL050139	Is unknown	0.29	0.60	5.2	2.4	3.3	3.0	2.8
									AB014615	Fibroblast growth factor 8	0.19	0.07	5.4	3.5	8.5	3.2	22.7
M28825	CD1A antigen	0.51	0.36	4.1	2.6	2.0	4.6	4.4
									U27330	Fucosyltransferase 5	0.39	0.08	3.3	2.1	24.5	8.2	19.3
NM_006963	Zinc finger proteins	0.10	0.08	10.4	12.6	12.3	29.2	20.5
									AF093670	Peroxisome biogenesis factor	0.44	0.53	4.0	2.6	2.6	4.3	2.9
AK000191	Hypothetical proteins	0.50	0.18	2.3	3.6	4.4	2.2	8.2
									AB022847	Is unknown	0.39	0.24	2.1	6.9	4.5	2.8	6.2
AK000358	Microfibrillar associated protein 3	0.28	0.28	5.7	2.0	3.5	5.2	5.2
									X74837	Mannanase _ α _ class 1A	0.10	0.07	13.1	18.4	23.6	16.3	20.8
AF053712	TNF superfamily member 11	0.17	0.08	11.3	9.3	13.4	10.6	16.6
									AL133114	DKFZP586P2421 protein	0.11	0.32	8.5	3.4	4.9	5.3	4.3
AF049703	E74-like factor 5	0.22	0.24	5.1	6.0	3.3	2.7	5.4
									AL137471	Hypothetical proteins	0.29	0.05	4.0	15.0	10.1	2.7	25.3
AL035397	Is unknown	0.33	0.14	2.3	2.8	10.6	4.6	9.3
									AL035447	Hypothetical proteins	0.28	0.23	3.8	6.8	2.7	3.0	5.7
X55740	CD73	0.41	0.61	2.1	3.3	2.9	3.2	2.1
									NM_004909	Paclitaxel resistance related gene 3	0.20	0.22	3.9	2.9	6.5	3.2	5.6
AF233442	Ubiquitin-specific protease	0.41	0.31	2.9	4.7	2.7	3.5	3.9
									U92980	Is unknown	0.83	0.38	4.2	4.1	4.8	2.3	3.1

AF105424	Myosin heavy polypeptide-like	0.30	0.22	2.8	3.3	4.4	2.3	5.3
									M26665	histatin 3	0.29	0.26	7.9	3.5	4.6	3.5	4.5
AF083898	Ventral antigen 2 of neural tumor	0.20	0.34	18.7	3.8	2.2	3.6	3.5
									AJ009771	ariadne Drosophila homolog	0.33	0.06	2.3	17.6	15.9	2.5	20.3
AL022393	Hypothetical protein P1	0.05	0.33	32.9	2.4	3.0	69.4	3.4
									AF039400	Calcium-activated chloride channel family member 1	0.11	0.19	8.4	2.9	5.1	18.1	5.9
AJ012008	Dimethyl arginine dimethyl amino hydrolase	0.42	0.43	5.1	3.3	3.2	6.2	2.6
									AK000542	Hypothetical proteins	0.61	0.24	2.1	4.5	5.0	3.7	4.4
AL133654	Is unknown	0.27	0.40	2.8	2.1	2.5	2.5	2.6
									AL137513	Is unknown	0.43	0.43	6.4	3.2	3.8	2.3	2.3
U05227	GTP-binding proteins	0.38	0.36	5.0	3.1	3.1	2.2	2.8
									D38449	Putative G protein-coupled receptors	0.18	0.09	5.8	6.7	6.7	9.1	10.4
U80770	Is unknown	0.31	0.14	3.9	3.8	6.6	3.1	6.8
									X61177	IL-5Rα	0.40	0.27	2.6	4.4	9.8	8.1	3.6
U35246	Vesicle sorting protein 45A	0.15	0.42	5.8	2.8	2.6	4.5	2.2
									AB017016	Brain specific protein p25 alpha	0.27	0.29	6.0	2.6	3.4	3.1	3.1
X82153	Cathepsin K	0.45	0.20	4.2	5.2	4.8	4.4	4.6
									AC005162	Most likely a carboxypeptidase precursor	0.12	0.28	11.9	3.4	6.8	18.7	3.2
AL137502	Is unknown	0.22	0.16	3.9	4.9	7.3	3.9	5.3
									U66669	3-hydroxyisobutyryl-CoA hydrolase	0.30	0.40	10.3	3.5	5.2	2.3	2.1
AK000102	Is unknown	0.39	0.30	2.8	5.3	5.2	4.1	2.8
									AF034970	Doxogenin 2	0.28	0.05	3.3	8.5	15.7	4.0	17.3
AK000534	Hypothetical proteins	0.13	0.29	6.8	2.3	4.0	20.6	2.9
									J04599	Biglycan proteoglycan	0.39	0.30	4.0	3.7	4.0	4.8	2.8
AL133612	Is unknown	0.62	0.33	2.7	3.4	5.2	3.0	2.5
									D10495	Protein kinase C delta	0.18	0.10	12.0	20.7	8.7	6.8	8.1

X58467	Cytochrome P450	0.07	0.24	15.4	4.7	7.9	34.4	3.4
									AF131806	Is unknown	0.31	0.25	2.6	3.4	5.7	7.0	3.2
AK000351	Hypothetical proteins	0.34	0.13	4.0	6.9	5.5	2.8	6.3
									AF075050	Hypothetical proteins	0.55	0.09	2.7	17.8	5.1	2.2	8.3
AK000566	Hypothetical protein is unknown	0.15	0.35	6.7	2.2	6.8	6.4	2.1
									U43328	Cartilage connexin 1	0.44	0.19	2.5	6.2	6.9	7.8	3.8
AF045941	sciellin	0.16	0.21	6.8	7.5	4.8	6.9	3.4
									U27655	Modulators of G protein signaling 3	0.24	0.29	5.5	4.9	2.9	4.9	2.4
AK000058	Hypothetical proteins	0.25	0.15	5.0	9.7	16.4	2.7	4.5
									AL035364	Hypothetical proteins	0.32	0.26	4.4	4.2	7.3	2.8	2.6
AK001864	Is unknown	0.40	0.25	3.7	3.7	4.6	3.2	2.6
									AB015349	Is unknown	0.14	0.24	10.5	2.8	3.7	8.0	2.7
V00522	Class II MHC DR beta 3	0.62	0.22	4.8	3.9	4.7	2.5	3.0
									U75330	Neural cell adhesion molecule 2	0.42	0.08	2.1	9.6	13.2	3.3	7.8
NM_007199	IL-1R-related kinase M	0.15	0.25	8.7	7.8	8.6	16.1	2.5
									D30742	Calcium/calmodulin-dependent protein kinase IV	0.28	0.09	6.2	28.7	7.4	2.4	6.8
X05978	Cystatin A (cystatin A)	0.63	0.17	2.7	4.8	9.4	2.2	3.6
									AF240467	TLR-7	0.11	0.10	13.8	13.3	4.7	7.7	4.9

Table 37: upregulation of polynucleotide expression in a549 cells induced by peptides of formula G and other peptides.

[0127]A peptide concentration of 50. mu.g/ml was found to increase the expression of many polynucleotides. Co-incubation of peptides with human A549 epithelial cells4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a Human Operon array (PRHU 04). The intensities of the polynucleotides in the unstimulated control cells are shown in the second and third columns, which correspond to the cDNAs labeled with the dyes Cy3 and Cy5, respectively. The column "ratio ID #: control" refers to the intensity of polynucleotide expression in peptide-stimulated cells divided by the intensity of unstimulated cells. Accession numbers and genes are expressed as: u00115, zinc finger protein; m91036, hemoglobin γ G; k000070, hypothetical protein; AF055899, solute carrier family 27; AK001490, hypothetical protein; x97674, nuclear receptor coactivator 2; AB022847, unknown; AJ275986, a transcription factor; d10495, protein kinase C, δ; l36642, EphA 7; m31166, pentaxin-related gene; AF176012, unknown; AF072756, ankyrin 4; NM-014439, IL-1 superfamily z; AJ271351, a putative transcriptional regulator; AK000576, hypothetical protein; AJ272265, phosphoprotein 2 secretion; AL122038, hypothetical protein; AK000307, hypothetical protein; AB029001, KIAA1078 protein; u62437, cholinergic receptors; AF064854, unknown; AL031588, hypothetical protein; x89388, RAS p21 protein activator; d45399, phosphodiesterase; AB037716, hypothetical protein; x79981, cadherin 5; AF034208, RIG-like 7-1; AL133355, chromosome 21 open reading frame 53; NM — 016281, STE 20-like kinase; AF023614, transmembrane activator and CAML interacting protein; AF056717, as ash 2; AB029039, KIAA1116 protein; j03634, inhibin, β a; u80764, unknown; AB032963, unknown; x82835, type IX voltage-gated sodium ion channels.

Registration number	control-Cy 3	control-Cy 5	ID 53: control	ID 54: control	ID 47: control	ID 48: control	ID 49: control	ID 50: control	ID 51: control	ID 52: control
											U00115	0.51	0.07	27.4	7.3	2.4	3.1	4.8	8.3	3.5	20.0
M91036	0.22	0.02	39.1	32.5	5.2	2.2	37.0	6.0	16.2	18.0
											AK000070	0.36	0.18	3.8	7.6	2.6	15.1	12.2	9.9	17.2	15.3
AF055899	0.14	0.31	6.7	3.7	9.7	10.0	2.2	16.7	5.4	14.8
											AK001490	0.05	0.02	14.1	35.8	3.2	28.6	25.0	20.2	56.5	14.1
X97674	0.28	0.28	3.2	3.7	4.0	10.7	3.3	3.1	4.0	13.2
											AB022847	0.39	0.24	4.1	4.4	4.5	2.7	3.7	10.4	5.0	11.3
AJ275986	0.26	0.35	5.8	2.3	5.7	2.2	2.5	9.7	4.3	11.1
											D10495	0.18	0.10	8.0	3.4	4.6	2.0	6.9	2.5	12.7	10.3
L36642	0.26	0.06	5.8	14.2	2.6	4.1	8.9	3.4	6.5	6.6
											M31166	0.31	0.12	4.8	3.8	12.0	3.6	9.8	2.4	8.8	6.4
AF176012	0.45	0.26	3.1	2.9	2.8	2.6	2.3	6.9	3.0	5.8
											AF072756	0.33	0.07	9.9	9.3	4.4	4.3	3.2	4.9	11.9	5.4
NM_014439	0.47	0.07	12.0	7.1	3.3	3.3	4.7	5.9	5.0	5.4
											AJ271351	0.46	0.12	3.4	3.5	2.3	4.7	2.3	2.7	6.9	5.2
AK000576	0.27	0.06	7.4	15.7	2.9	4.7	9.0	2.4	8.2	5.1
											AJ272265	0.21	0.09	6.2	7.9	2.3	3.7	10.3	4.5	4.6	4.7
AL122038	0.46	0.06	6.7	4.5	2.6	4.3	16.4	6.5	26.6	4.6

AK000307	0.23	0.09	3.7	4.0	4.3	3.2	5.3	2.9	13.1	4.4
											AB029001	0.52	0.21	14.4	4.3	4.6	4.4	4.8	21.9	3.2	4.2
U62437	0.38	0.13	12.6	6.5	4.2	6.7	2.2	3.7	4.8	3.9
											AF064854	0.15	0.16	2.6	2.9	6.2	8.9	14.4	5.0	9.1	3.9
AL031588	0.40	0.26	8.3	5.2	2.8	3.3	5.3	9.0	5.6	3.4
											X89388	0.25	0.10	15.8	12.8	7.4	4.2	16.7	6.9	12.7	3.3
D45399	0.21	0.18	3.0	4.7	3.3	4.4	8.7	5.3	5.1	3.3
											AB037716	0.36	0.40	5.1	7.5	2.6	2.1	3.5	3.1	2.4	2.8
X79981	0.34	0.10	4.7	7.2	3.2	4.6	6.5	5.1	5.8	2.7
											AF034208	0.45	0.24	2.7	10.9	2.1	3.7	2.3	5.9	2.2	2.5
AL133355	0.22	0.23	2.3	3.4	7.3	2.7	3.3	4.3	2.8	2.5
											NM_016281	0.40	0.19	6.6	10.6	2.1	2.8	5.0	11.2	10.6	2.5
AF023614	0.11	0.42	2.2	2.2	6.0	7.5	5.0	2.7	2.0	2.4
											AF056717	0.43	0.62	4.3	3.2	5.1	4.0	4.6	9.7	3.1	2.2
AB029039	0.79	0.49	2.7	3.3	3.7	2.0	2.3	2.4	4.8	2.2
											J03634	0.40	0.12	3.7	2.3	2.3	4.0	10.5	4.1	9.1	2.2
U80764	0.31	0.18	2.3	7.4	4.2	2.3	5.1	3.3	8.8	2.1
											AB032963	0.19	0.34	4.0	7.3	5.0	3.0	2.9	6.7	3.8	2.1
X82835	0.25	0.38	2.0	2.7	2.9	7.7	3.3	3.1	3.5	2.0

Example 5

Chemokine induction with peptides in cell lines, human whole blood and mice

[0128]The murine macrophage line RAW 264.7, THP-1 cells (human monocytes), the human epithelial cell line (a549), the human bronchial epithelial cells (16HBEo14) and human whole blood were used. HBE cells were grown in MEM containing Erer's solution. THP-1 cells were grown and maintained in RPMI1640 medium. RAW and a549 cell lines were maintained in DMEM supplemented with 10% fetal bovine serum. These cells were seeded into 24-well plates with DMEM at a density of 10 per well⁶Individual cells (see above), a549 cells were seeded into 24-well plates with DMEM at a density of 10 per well⁵Cells (see above), all at 37 ℃ in 5% CO₂Incubate overnight at medium temperature. DMEM was aspirated from overnight grown cells and replaced with fresh medium. After incubation of these cells with peptides, ELISA (R) was used&D Systems, Minneapolis, MN) to determine the chemokine release into the culture supernatant.

[0129] Animal studies were approved by the UBC animal management Committee (UBCACC # A01-0008). BALB/c mice were purchased from Charles River Laboratories and fed on standard animal facilities. Age, sex and weight matched adult mice were anesthetized by intraperitoneal injection of avermectin (4.4mM 2-2-2-tribromoethanol, 2.5% 2-methyl-2-butanol in distilled water) at a dose of 200 μ l per 10g body weight. Instillation was performed using a non-surgical intratracheal instillation method modified from Ho and Furst 1973. Briefly, the upper maxillary teeth of anesthetized rats were hooked on a wire at the top of a support frame to open their jaws and the chest was pushed with a spring to place their pharynx, larynx and trachea in a vertical line. The trachea is illuminated from the outside and an insertion catheter is inserted into the lumen of the trachea which is clearly illuminated. Mu.l of peptide suspension or sterile water was placed in a small hole at the proximal end of the cannula and slowly instilled into the trachea with 200. mu.l of air. After instillation, the animals were held in an upright position for 2 minutes to allow the fluid to flow into the respiratory tree. After 4 hours, these mice were euthanized by intraperitoneal injection of 300mg/kg pentobarbital. The trachea is exposed, an intravenous catheter is inserted into the proximal end of the trachea, and tied appropriately with sutures. Performing lavage; 0.75 ml of sterile PBS was introduced into the lungs through the tracheal cannula and after a few seconds the fluid was aspirated. This procedure was repeated three times with the same PBS sample. The lavage solution was placed in tubes and on ice, with a total recovery volume of approximately 0.5ml per mouse. This bronchoalveolar lavage (BAL) solution was centrifuged at 1200rpm for 10 minutes at high speed, the supernatant was removed, and TNF-. alpha.and MCP-1 were detected by ELISA.

[0130] The upregulation of chemokines by cationic peptides has been demonstrated in a number of different systems. Murine MCP-1 is a homolog of human MCP-1, which is a member of the β (C-C) chemokine family. MCP-1 has been shown to recruit monocytes, NK cells and some T lymphocytes. When RAW 264.7 macrophages and human whole blood from 3 donors are enriched with increasing concentrations of the peptide SEQ ID NO: 1 stimulation, ELISA showed that they produced significant amounts of MCP-1 in their supernatant (Table 36). Stimulation of 24 h RAW 264.7 cells with peptides at concentrations ranging from 20-50. mu.g/ml produced significant amounts of MCP-1 (200-400 pg/ml above background). When these cells (24 hours) and whole blood (4 hours) were stimulated with 100. mu.g/ml LL-37, high levels of MCP-1 were produced.

[0131] The effect of cationic peptides on chemokine induction was also tested in a completely different cell system, a549 human upper grade cells. Interestingly, although these cells produce MCP-1 in response to LPS, and this response is antagonized by the peptide; however, a549 cells respond directly to the peptide SEQ ID NO: at 1, no MCP-1 is produced. However, high concentrations of the peptide SEQ ID NO: 1 did induce the production of the neutrophil-specific chemokine IL-8 (table 37). Thus, SEQ ID NO: 1 are capable of inducing different patterns of response at different concentrations and in different cell types. A number of peptides corresponding to each formula were tested for their ability to induce IL-8 in A549 cells (Table 38). Many of these peptides induced IL-8 above background levels at low concentrations of 10. mu.g/ml. High concentrations (100. mu.g/ml) of SEQ ID NO: 13 IL-8 was induced in human whole blood (Table 39). Peptides of SEQ ID NO: 2 also induced IL-8 significantly.

[0132] BALB/c mice were given by intratracheal instillation SEQ ID NO: MCP-1 and TNF-alpha levels in bronchoalveolar lavage fluid were measured after 3-4 hours in water with or without endotoxin. We found that the peptide SEQ ID NO: 1 the former produced significantly higher levels of MCP-1 than mice given either water or anesthetic alone (Table 42). For the peptide SEQ ID NO: 1 did not have a pro-inflammatory response because the peptide did not induce significantly more TNF-alpha than mice given water or anesthetic alone. It was also found that the peptide SEQ ID NO: 1 (up to 100 μ g/ml) treated RAW 264.7 cells and bone marrow derived macrophages, peptide SEQ ID NO: 1 did not significantly induce TNF-. alpha.production (Table 43). Thus, the peptide SEQ ID NO: 1 selectively induces the production of chemokines without inducing the production of inflammatory mediators such as TNF-alpha. This demonstrates that the peptide SEQ ID NO: 1 has a dual role, both as a factor that can prevent the bacterial products from inducing inflammation, and to help recruit phagocytic cells that can clear infection.

Table 38: induction of MCP-1 in RAW 264.7 cells and human whole blood.

[0133]RAW 264.7 murine macrophages and human whole blood were stimulated with increasing concentrations of LL-37 for 4 hours. Mixing human whole bloodThe samples were centrifuged and the serum was removed and MCP-1 was detected by ELISA, while MCP-1 was detected in the supernatant of RAW 264.7 cells by ELISA. Data for RAW cells are expressed as mean ± standard deviation of three or more experiments, and data for human whole blood are expressed as mean ± standard deviation from three different donors.

Peptide, SEQ ID NO: 1(μ g/ml)	Monocyte chemistry inducing protein (MCP) -1(pg/ml)*
				RAW cells	Whole blood
0	135.3±16.3	112.7±43.3
			10	165.7±18.2	239.3±113.3
50	367±11.5	371±105
			100	571±17.4	596±248.1

Table 39: induction of IL-8 in a549 cells and human whole blood.

[0134]A549 cells and human whole blood were stimulated with increasing concentrations of peptide for 24 hours and 4 hours, respectively. Human whole blood samples were centrifuged, the serum removed and IL-8 in the supernatants of A549 cells was assayed by ELISA simultaneously with IL-8 in the serum. Data for a549 cells are expressed as mean ± standard deviation of three or more experiments, and data for human whole blood are expressed as mean ± standard deviation from three different donors.

Peptide, SEQ ID NO: 1(μ g/ml)	IL-8(pg/ml)
				A549 cell	Whole blood
0	172±29.1	660.7±126.6

1	206.7±46.1
			10	283.3±28.4	945.3±279.9
20	392±31.7
			50	542.3±66.2	1160.3±192.4
100	1175.3±188.3

Table 40: the cationic peptide in A549 cells induced IL-8.

[0135]A549 human epithelial cells were stimulated with 10 μ g of peptide for 24 hours. The supernatant was removed and IL-8 was detected by ELISA.

Peptide (10. mu.g/ml)	IL-8(ng/ml)
		Without peptides	0.164
LPS, peptide free	0.26
		SEQ ID NO：1	0.278
SEQ ID NO：6	0.181
		SEQ ID NO：7	0.161
SEQ ID NO：9	0.21
		SEQ ID NO：10	0.297
SEQ ID NO：13	0.293
		SEQ ID NO：14	0.148
SEQ ID NO：16	0.236
		SEQ ID NO：17	0.15
SEQ ID NO：19	0.161
		SEQ ID NO：20	0.151
SEQ ID NO：21	0.275
		SEQ ID NO：22	0.314
SEQ ID NO：23	0.284
		SEQ ID NO：24	0.139
SEQ ID NO：26	0.201
		SEQ ID NO：27	0.346
SEQ ID NO：28	0.192
		SEQ ID NO：29	0.188
SEQ ID NO：30	0.284
		SEQ ID NO：31	0.168
SEQ ID NO：33	0.328
		SEQ ID NO：34	0.315
SEQ ID NO：35	0.301
		SEQ ID NO：36	0.166
SEQ ID NO：37	0.269
		SEQ ID NO：38	0.171
SEQ ID NO：40	0.478
		SEQ ID NO：41	0.371
SEQ ID NO：42	0.422
		SEQ ID NO：43	0.552
SEQ ID NO：44	0.265
		SEQ ID NO：45	0.266
SEQ ID NO：47	0.383
		SEQ ID NO：48	0.262

SEQ ID NO：49	0.301
		SEQ ID NO：50	0.141
SEQ ID NO：51	0.255
		SEQ ID NO：52	0.207
SEQ ID NO：53	0.377
		SEQ ID NO：54	0.133

Table 41: peptides in human blood induce IL-8.

[0136]Human whole blood was stimulated with increasing concentrations of peptide for 4 hours. Human blood samples were centrifuged, the serum removed, and IL-8 detected by ELISA. Data are from the mean of two donors.

SEQ ID NO：3(μg/ml)	IL-8(pg/ml)
		0	85
10	70
		100	323

Table 42: induction of IL-8 in HBE cells.

[0137]Increasing concentrations of the peptide were incubated with HBE cells for 8 hours, the supernatant removed, and IL-8 detected. Data are presented as mean ± standard deviation of three or more experiments.

SEQ ID NO：2(μg/ml)	IL-8(pg/ml)
		0	552±90
0.1	670±155
		1	712±205
10	941±15
		50	1490±715

Table 43: induction of IL-8 in undifferentiated THP-1 cells.

[0138]The peptides at the indicated concentrations were incubated with human monocyte THP-1 cells for 8 hours, the supernatant removed, and IL-8 detected by ELISA.

SEQ ID NO：3(μg/ml)	IL-8(pg/ml)
		0	10.6
10	17.2
		50	123.7

Table 44: in the mouse airway the peptide SEQ ID NO: 1 induces MCP-1.

[0139]BALB/c mice were anesthetized with Avertin and either instilled intratracheally with peptide or water or without instillation (no treatment). The mice were monitored for 4 hours, anesthetized, and BAL fluid was isolated and analyzed for MCP-1 and TNF-. alpha.concentrations by ELISA. Data are presented as mean ± standard deviation of four or five mice under various conditions.

Condition	MCP-1(pg/ml)	TNF-α(pg/ml)
			Water (W)	16.5±5	664±107
Peptides	111±30	734±210
			Avermectin (Avermectin)	6.5±0.5	393±129

Table 45: the cationic peptide did not significantly induce TNF- α.

[0140]The indicated peptide (40. mu.g/ml) was macrophage-reacted with RAW 246.7Cells were incubated for 6 hours. Supernatants were collected and assayed for TNF-. alpha.levels by ELISA. Data are presented as mean ± standard deviation of three or more experiments.

Peptide treatment	TNF-α(pg/ml)
		Background of the culture Medium	56±8
LPS treatment, no peptide	15207±186
		SEQ ID NO：1	274±15
SEQ ID NO：5	223±45
		SEQ ID NO：6	297±32
SEQ ID NO：7	270±42
		SEQ ID NO：8	166±23
SEQ ID NO：9	171±33
		SEQ ID NO：10	288±30
SEQ ID NO：12	299±65
		SEQ ID NO：13	216±42
SEQ ID NO：14	226±41
		SEQ ID NO：15	346±41
SEQ ID NO：16	341±68
		SEQ ID NO：17	249±49
SEQ ID NO：19	397±86
		SEQ ID NO：20	285±56
SEQ ID NO：21	263±8
		SEQ ID NO：22	195±42
SEQ ID NO：23	254±58
		SEQ ID NO：24	231±32
SEQ ID NO：26	281±34
		SEQ IDNO：27	203±42
SEQ ID NO：28	192±26
		SEQ ID NO：29	242±40
SEQ ID NO：31	307±71
		SEQ ID NO：33	196±42
SEQ ID NO：34	204±51
		SEQ ID NO：35	274±76
SEQ ID NO：37	323±41
		SEQ ID NO：38	199±38
SEQ ID NO：43	947±197
		SEQ ID NO：44	441±145
SEQ ID NO：45	398±90
		SEQ ID NO：48	253±33
SEQ ID NO：49	324±38
		SEQ ID NO：50	311±144
SEQ ID NO：53	263±40
		SEQ ID NO：54	346±86

Example 6

Cationic peptides increase surface expression of chemokine receptors

[0141] To analyze cell surface expression of IL-8RB, CXCR-4, CCR2 and LFA-1, RAW macrophages were stained with 10 μ g/ml of appropriate primary antibody (Santa Cruz Biotechnology) followed by FITC-conjugated goat anti-rabbit IgG [ IL-8RB and CXCR-4(Jackson ImmunoResearch Laboratories, WestGrove, PA) - ] or FITC-conjugated donkey anti-goat IgG (Santa Cruz ]. Cells were analyzed by FACScan, counted 10,000 times and front and side diffuser (forward and side scatter) was turned on to exclude cell debris.

[0142] Polynucleotide array data indicate that some peptides upregulate expression of the chemokine receptors IL-8RB, CXCR-4, and CCR2 by 10, 4, and 1.4 fold, respectively, compared to unstimulated cells. To confirm the polynucleotide array data, the surface expression of the receptor on RAW cells stimulated with the peptide for 4 hours was detected by flow cytometry. When 50 μ g/ml of peptide was incubated with RAW cells for 4 hours, IL-8RB was upregulated on average 2.4-fold above unstimulated cells, CXCR-4 was upregulated on average 1.6-fold above unstimulated cells, and CCR2 was upregulated 1.8-fold above unstimulated cells (Table 46). As a control, CEMA was shown to result in similar up-regulation. Bac2A was the only peptide that could significantly up-regulate LFA-1 (3.8 fold higher than control cells). Table 46: in response to the peptide, the surface expression of CXCR-4, IL-8RB and CCR2 was increased.

[0143]RAW macrophages were stimulated with the peptide for 4 hours. The cells were washed and stained with appropriate primary and FITC-labeled secondary antibodies. Data shown are expressed as mean (fold change in RAW cells stimulated with peptide) ± standard deviation.

Peptides	Concentration (μ g/ml)	Fold increase in protein expression
					IL-8RB	CXCR-4	CCR2
		SEQ ID NO：1	10	1.0				1.0	1.0
SEQ ID NO：1	50				1.3±0.05	1.3±0.03	1.3±0.03
		SEQ ID NO：1	100	2.4±0.6				1.6±0.23	1.8±0.15
SEQ ID NO：3	100				2.0±0.6	Do not make	4.5
		CEMA	50	1.6±0.1				1.5±0.2	1.5±0.15
	100				3.6±0.8	Do not make	4.7±1.1

Example 7

Cationic peptide induced phosphorylation of MAP kinase

[0144]At 2.5X 10⁵-5×10⁵Cells were seeded at a density of one cell/ml and allowed to stand overnight. In the morning, cells were washed once with serum-free medium (serum-free medium-4 hours). The medium was removed and replaced with PBS, and then left at 37 ℃ for 15 minutes and at room temperature for 15 minutes. Peptides (at a concentration of 0.1. mu.g/ml to 50. mu.g/ml) or water were added and incubated for 10 minutes. PBS was removed quickly and replaced with ice-cold Radioimmunoprecipitation (RIPA) buffer and inhibitors (NaF, B-glycerophosphate, MOL, vanadate, PMSF, leupeptin aprotinin). The plates were shaken on ice for 10-15 minutes or until the cells were lysed and the lysate was collected. For THP-1 cells, the procedure was slightly different; more cells (2X 10) were used⁶). They were left overnight in the absence of serum, 1ml of ice-cold PBS was added to stop the reaction, then placed on ice for 5-10 minutes, spun down, and then resuspended with RIPA. Protein concentration was determined using protein assay (Pierce, Rockford, IL.). Cell lysates (20. mu.g protein) were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was blocked with 10mM Tris-HCl, pH7.5, 150mM NaCl (TBS)/5% skim milk powder for 1 hour, then incubated overnight in cold TBS containing primary antibody/0.05% Tween 20. After washing with TBS/0.05% Tween 20 for 30 minutes, the membranes were incubated with TBS containing 1. mu.g/ml secondary antibody for 1 hour at room temperature. The membrane was washed with TBS/0.05% Tween 20 for 30 minutes and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1: 10,000 in TBS/0.05% Tween 20) for 1 hour at room temperature. Immunoreactive bands were visualized using Enhanced Chemiluminescence (ECL) detection after washing the membranes with TBS/0.1% Tween 20 for 30 minutes. For experiments using peripheral blood mononuclear cells: peripheral blood (50-100ml) was collected from all subjects. Monocytes were isolated from peripheral blood by density gradient centrifugation on Ficoll-Hypaque. Interphase cells (monocytes) were recovered, washed, and then resuspended in the recommended primary medium for cell culture (RPMI-1640) containing 10% Fetal Calf Serum (FCS) and 1% L-glutamine. At each hole 4×10⁶Density of Individual cells were loaded into 6-well plates at 37 ℃ in 5% CO₂The mixture was left in the air for 1 hour to allow adhesion to occur. The surface-floating medium and non-adherent cells are washed away and an appropriate medium containing the peptide is added. It was estimated from their ability to exclude trypan blue that > 99% of freshly harvested cells should survive. After stimulation with the peptide, the cells were lysed with RIPA buffer in the presence of various phosphatase inhibitors and kinase inhibitors, and the lysates were collected. Protein content was analyzed and approximately 30. mu.g of each sample was loaded onto a 12% SDS-PAGE gel. Proteins in the gel were transferred to nitrocellulose and blocked with Tris Buffered Saline (TBS) containing 5% skim milk powder and 1% Triton X100 for 1 hour. Phosphorylation was detected with phosphorylation-specific antibodies.

[0145] The results of peptide-induced phosphorylation are summarized in table 46. It was found that in murine macrophage RAW cell line and HBE cells, SEQ ID NO: 2 caused dose-dependent phosphorylation of p38 and ERK 1/2. In the THP-1 human monocyte cell line, SEQ ID NO: 3 leads to phosphorylation of MAP kinase, in murine RAW cell line, seq id NO: 3 leads to phosphorylation of ERK 1/2.

Table 47: phosphorylation of MAP kinase in response to peptides.

Cell lines	Peptides	MAP kinase phosphorylated
				p38	ERK1/2
		RAW 264.7	SEQ ID NO：3			-	+
SEQ ID NO：2	+			+
			HBE		SEQ ID NO：3		+
SEQ ID NO：2	+	+
				THP-1	SEQ ID NO：3	+	+
SEQ ID NO：2

Table 48:

[0146]peptide phosphorylation of MAP kinase in human blood monocytes SEQ ID NO: 1, (50. mu.g/ml) was used to promote phosphorylation.

phosphorylation of p38		ERK1/2 phosphorylation
				15 minutes	60 minutes	15 minutes	60 minutes
+	-	+	+

Example 8

Cationic peptides defend against bacterial infections by boosting immune responses

[0147]BALB/c mice were given 1X 10 injections intraperitoneally⁵Salmonella and cationic peptide (200 μ g). After 24 hours monitoring of the mice, they were sacrificed, spleens removed, homogenized, resuspended in PBS, and plated on Luria Broth agar plates containing kanamycin (50. mu.g/ml). The plates were incubated overnight at 37 ℃ and the surviving bacteria were counted (tables 49 and 50). CD-1 mice were administered by intraperitoneal injection containing 1X 10⁸5% porcine mucin fluid of Staphylococcus aureus and cationic peptide (200. mu.g) (Table 51). After 3 days of monitoring, the mice were sacrificed, blood was removed, plated, and the number of viable cells was calculated. CD-1 male mice were administered 5.8X 10 by Intraperitoneal (IP) injection⁶CFU EHEC bacteria and cationic peptide (200 μ g) were monitored for 3 days (table 52). In each of these animal models a fraction of the peptides showed protection against infection. When the results of the defense analysis in tables 50 and 51 were compared with the results of gene expression in tables 31-37, it was found that the most defensive peptides in the salmonella model were able to induce a common subset of genes in epithelial cells (table 53). This clearly indicates that the pattern of gene expression is consistent with the ability of the peptide to display defense. Minimum of inhibitionThe results of the limiting concentration (MIC) tests (table 54) indicate that many of these cationic peptides are not directly resistant to microorganisms. This suggests that the ability of a peptide to defend against infection is dependent on the ability of the peptide to stimulate the host's innate immunity, rather than on direct antimicrobial activity.

Table 49: effect of cationic peptide on Salmonella infection in BALB/c mice.

[0148]Salmonella and peptide were injected intraperitoneally into BALB/c mice, and 24 hours later, the animals were euthanized, spleens were removed, homogenized, diluted with PBS, plates counted, and bacterial survival was determined.

Peptide treatment	Viable bacteria in the spleen (CFU/ml)	Statistical significance (p value)
			Control	2.70±0.84×105
SEQ ID NO：1	1.50±0.26×105	0.12
			SEQ ID NO：6	2.57±0.72×104	0.03
SEQ ID NO：13	3.80±0.97×104	0.04
			SEQ ID NO：17	4.79±1.27×104	0.04
SEQ ID NO：27	1.01±0.26×105	0.06

Table 50: effect of cationic peptide on Salmonella infection in BALB/c mice.

[0149]Salmonella and peptide were injected intraperitoneally into BALB/c mice, and 24 hours later, the animals were euthanized, spleens were removed, homogenized, diluted with PBS, plates counted, and bacterial survival was determined.

Peptide treatment	Viable bacteria in the spleen (CFU/ml)
		Control	1.88±0.16×104
SEQ ID NO：48	1.98±0.18×104
		SEQ ID NO：26	7.1±1.37×104
SEQ ID NO：30	5.79±0.43×103
		SEQ ID NO：37	1.57±0.44×104
SEQ ID NO：5	2.75±0.59×104
		SEQ ID NO：7	5.4±0.28×103
SEQ ID NO：9	1.23±0.87×104
		SEQ ID NO：14	2.11±0.23×103
SEQ ID NO：20	2.78±0.22×104
		SEQ ID NO：23	6.16±0.32×104

Table 51: effect of cationic peptide in murine model of Staphylococcus aureus infection.

[0150]Will contain 1X 10⁸Bacterial 5% porcine mucin solution was injected Intraperitoneally (IP) into CD-1 mice. Cationic peptide (200 μ g) was administered via additional intraperitoneal injections. The monitoring is carried out for 3 days, the mice are euthanized, blood is removed and plated to count the number of survivals. The following peptides were not effective in controlling staphylococcus aureus infections: SEQ ID NO: 48, SEQ ID NO: 26.

treatment of	CFU/ml (blood)	# surviving mice (3 days)/Total number of mice in this group
			Without peptides	7.61±1.7×103	6/8
SEQ ID NO：1	0	4/4
			SEQ ID NO：27	2.25±0.1×102	3/4
SEQ ID NO：30	1.29±0.04×102	4/4
			SEQ ID NO：37	9.65±0.41×102	4/4
SEQ ID NO：5	3.28±1.7×103	4/4
			SEQ ID NO：6	1.98±0.05×102	3/4
SEQ ID NO：7	3.8±0.24×103	4/4
			SEQ ID NO：9	2.97±0.25×102	4/4
SEQ ID NO：13	4.83±0.92×103	3/4
			SEQ ID NO：17	9.6±0.41×102	4/4
SEQ ID NO：20	3.41±1.6×103	4/4
			SEQ ID NO：23	4.39±2.0×103	4/4

Table 52: effect of peptides in murine EHEC infection model.

[0151]Mixing 5.8X 10⁶CFU EHEC bacteria were injected Intraperitoneally (IP) into CD-1 males (5 weeks old). The cationic peptide (200 μ g) was administered via one additional intraperitoneal injection. These mice were monitored for 3 days.

Treatment of	Peptides	Survival (%)
			Control	Is free of	25
SEQ ID NO：23	200μg	100

Table 53: peptides active in vivo induce upregulation of gene expression patterns in a549 epithelial cells.

[0152]It was found that the peptide of SEQ ID NO: 30. SEQ ID NO: 7 and SEQ ID NO13 increased expression of a set of genes (a patterns of genes) for each peptide after 4 hours of treatment. The peptides were incubated with Human a549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to a Human Operon array (PRHU 04). The intensity of the polynucleotide in unstimulated control cells is shown in the second column (average of both cases of labeling cDNAs with Cy3 and Cy 5). The fold-up column refers to the intensity of polynucleotide expression in cells stimulated with the peptide divided by the intensity of non-stimulated cells. The table also includes peptides SEQ ID NO: 37, which is not active in a murine infection model.

Target (registration number)	Unstimulated cell strength	Fold-up of upregulation of Gene expression relative to untreated cells
						SEQ IDNO：30	SEQ IDNO：7	SEQ IDNO：13	SEQ IDNO：37
		Zinc finger protein (AF061261)	13	2.6	9.4					9.4	1.0

Cell cycle gene (S70622)	1.62	8.5	3.2	3.2	0.7
						IL-10 receptor (U00672)	0.2	2.6	9	4.3	0.5
Transferase (AF038664)	0.09	12.3	9.7	9.7	0.1
						Homeobox protein (AC004774)	0.38	3.2	2.5	2.5	1.7
Bifurcate head type protein (AF042832)	0.17	14.1	3.5	3.5	0.9
						Unknown (AL096803)	0.12	4.8	4.3	4.3	0.6
KIAA0284 protein (AB006622)	0.47	3.4	2.1	2.1	1.3
						Hypothetical protein (AL022393)	0.12	4.4	4.0	4.0	0.4
Receptor (AF112461)	0.16	2.4	10.0	10.0	1.9
						Hypothetical protein (AK002104)	0.51	4.7	2.6	2.6	1.0
Protein (AL050261)	0.26	3.3	2.8	2.8	1.0
						Polypeptide (AF105424)	0.26	2.5	5.3	5.3	1.0
SPR1 protein (AB031480)	0.73	3.0	2.7	2.7	1.3
						Dehydrogenase (D17793)	4.38	2.3	2.2	2.2	0.9
Transferase (M63509)	0.55	2.7	2.1	2.1	1.0
						Peroxisome factor (AB013818)	0.37	3.4	2.9	2.9	1.4

Table 54:

[0153]most cationic peptides studied here, and in particular those effective in infection models, are not significantly antimicrobial. Serial dilutions of peptides were incubated with the indicated bacteria in 96-well plates overnight. The lowest concentration of peptide that killed the bacteria was expressed as the MIC. Symbol > indicates that the MIC is too large to measure. MICs of 8. mu.g/ml or less are considered clinically significant activities. Abbreviations: coli, escherichia coli (escherichia coli); s. aureus, Staphylococcus aureus (Staphylococcus aureus); p. aerug, Pseudomonas aeruginosa (Pseudomonas aeruginosa); typhim, salmonella enteritidis, typhimurium, c.rhod, citrobacter rhodens; EHEC, enterohemorrhagic escherichia coli (Enterohaemorrhagic e.

Peptides	MIC(μg/ml)
							e.coli	S.aureus	P.aerug	S.typhim	C.rhod	EHEC
	Polymyxin	0.25	16	0.25	0.5	0.25							0.5
Gentamicin							0.25	0.25	0.25	0.25	0.25	0.5
	SEQ ID NO：1	32	＞	96	64	8							4
SEQ ID NO：5							128	＞	＞	＞	64	64
	SEQ ID NO：6	128	＞	＞	128	64							64
SEQ ID NO：7							＞	＞	＞	＞	＞	＞
	SEQ ID NO：8	＞	＞	＞	＞	＞							＞
SEQ ID NO：9							＞	＞	＞	＞	＞	＞
	SEQ ID NO：10	＞	＞	＞	＞	＞							64
SEQ ID NO：12							＞	＞	＞	＞	＞	＞
	SEQ ID NO：13	＞	＞	＞	＞	＞							＞
SEQ ID NO：14							＞	＞	＞	＞	＞	＞
	SEQ ID NO：15	128	＞	＞	＞	128							64
SEQ ID NO：16							＞	＞	＞	＞	＞	＞

SEQ ID NO：17	＞	＞	＞	＞	＞	＞
							SEQ ID NO：19	8	16	16	64	4	4
SEQ ID NO：2	4	16	32	16	64
							SEQ ID NO：20	8	8	8	8	16	8
SEQ ID NO：21	64	64	96	64	32	32
							SEQ ID NO：22	8	12	24	8	4	4
SEQ ID NO：23	4	8	8	16	4	4
							SEQ ID NO：24	16	16	4	16	16	4
SEQ ID NO：26	0.5	32	64	2	2	0.5
							SEQ ID NO：27	8	64	64	16	2	4
SEQ ID NO：28	＞	＞	＞	64	64	128
							SEQ ID NO：29	2	＞	＞	16	32	4
SEQ ID NO：30	16	＞	128	16	16	4
							SEQ ID NO：31	＞	＞	128	＞	＞	64
SEQ ID NO：33	16	32	＞	16	64	8
							SEQ ID NO：34	8	＞	＞	32	64	8
SEQ ID NO：35	4	128	64	8	8	4
							SEQ ID NO：36	32	＞	＞	32	32	16
SEQ ID NO：37	＞	＞	＞	＞	＞	＞
							SEQ ID NO：38	0.5	32	64	4	8	4
SEQ ID NO：40	4	32	8	4	4	2
							SEQ ID NO：41	4	64	8	8	2	2
SEQ ID NO：42	1.5	64	4	2	2	1
							SEQ ID NO：43	8	128	16	16	8	4
SEQ ID NO：44	8	＞	128	128	64	64
							SEQ ID NO：45	8	＞	128	128	16	16
SEQ ID NO：47	4	＞	16	16	4	4
							SEQ ID NO：48	16	＞	128	16	1	2
SEQ ID NO：49	4	＞	16	8	4	4
							SEQ ID NO：50	8	＞	16	16	16	8
SEQ ID NO：51	4	＞	8	32	4	8
							SEQ ID NO：52	8	＞	32	8	2	2
SEQ ID NO：53	4	＞	8	8	16	8
							SEQ ID NO：54	64	＞	16	64	16	32

Example 9

Use of polynucleotides induced by bacterial signaling molecules in diagnosis/screening

[0154] Salmonella typhimurium LPS and E coli O111: b4LPS was purchased from Sigma Chemical Co, (st. LTA (Sigma) from Staphylococcus aureus was resuspended in endotoxin-free water (Sigma). The Limulus amoebocyte lysate test (Sigma) was performed on LTA preparations to confirm that they were not significantly contaminated with endotoxin (i.e., less than 1ng/ml, which concentration did not result in significant cytokine production by RAW cells). CpG oligodeoxynucleotides were synthesized using a Model 392DNA/RNA synthesizer from Applied biosystems (Applied biosystems, Missisaga, ON.), purified and resuspended in endotoxin-free water (Sigma). The following sequences CpG were used: 5'-TCATGACGTTCCTGACGTT-3' (SEQ ID NO: 57) and non-CpG: 5'-TTCAGGACTTTCCTCAGGTT-3' (SEQ ID NO: 58). The ability of the non-CpG oligomer to stimulate cytokine production was tested and found to not result in significant production of TNF- α or IL-6, and thus can be considered a negative control. RAW 264.7 cells were incubated with medium alone, 100ng/ml Salmonella typhimurium LPS, 1. mu.g/ml Staphylococcus aureus LTA or 1. mu.M CpG for 4 hours (these concentrations optimally induced tumor necrosis factor (TNF-. alpha.) production by RAW cells), and RNA was isolated from these cells. Polynucleotide cDNA probes were prepared using this RNA and hybridized to Clontech Atlas polynucleotide array filters as described previously. Hybridization of the cDNA probes to the respective immobilized DNAs can be visualized by autoradiography, and can be quantified using a phosphorescence imaging system. Tables 55-59 summarize the results of at least 2-3 independent experiments. It was found that treatment of RAW 264.7 cells with LPS resulted in increased expression of over 60 polynucleotides encoding proteins including inflammatory proteins such as IL-1 β, Induced Nitric Oxide Synthase (iNOS), MIP-1 α, MIP-1 β, MIP-2 α, CD40 and various transcription factors. Comparison of the changes in polynucleotide expression induced by LPS, LTA and CpG DNA revealed that all three bacterial products increased the expression of pro-inflammatory polynucleotides to similar extents, such as iNOS, MIP-1 α, MIP-2 α, IL-1 β, IL-15, TNFR1 and NF- κ B (Table 57). Table 57 describes 19 polynucleotides that are up-regulated to a similar extent by bacterial products whose stimulation ratios do not differ more than 1.5-fold between the three bacterial products. Several polynucleotides are also down-regulated to a similar extent by LPS, LTA and CpG. It was also found that in response to these three bacterial products, many polynucleotides were differentially regulated (Table 58), including many polynucleotides whose expression levels differed by more than 1.5-fold between one or more bacterial products. LTA treatment differentially affected the expression of the largest subset of polynucleotides compared to LPS or CpG, including over-stimulation of Jun-D, Jun-B, Elk-1 and cyclins G2 and A1. Only some of the polynucleotides were more altered in expression by LPS or CpG treatment. LPS treatment was more able to increase the expression of several polynucleotides, including cAMP response element DNA binding protein (CRE-BP), interferon inducible protein 1, and CACCC frame binding protein BKLF, compared to LTA or CpG treatment. CpG treatment is more capable of increasing the expression of several polynucleotides, including Leukemia Inhibitory Factor (LIF) and protease connexin 1(PN-1), than LPS or LTA treatment. These results indicate that although the polynucleotides in response to LPS, LTA and CpG DNA stimulation of expression overlap to a large extent, they also exhibit different abilities to modulate certain of them.

[0155]Other polynucleotide arrays that may be used are the Human Operon array (the identification number of this genome is PRHU04-S1), which consists of approximately 14,000 individual oligomer spots spotted in duplicate. Probes were prepared with 5. mu.g total RNA and labeled with Cy3 or Cy5 labeled dUTP. In these experiments, A549 epithelial cells were plated into 100mm tissue culture dishes at a density of 2.5X 10 per dish⁶Cells, incubated overnight, then incubated with 100ng/ml e.coli O111: b4LPS was stimulated for 4 hours. Total RNA was isolated using RNAqueous (Ambion). DNA contamination was removed using a DNA removal kit (Ambion). Probes prepared from total RNA were purified and hybridized to printed glass slides, overnight at 42 ℃ and then washed. After washing, images were taken with a Perkin Elmer array scanner. The mean, median and background intensities of the spots were determined using image processing software (Imapolynlucleotide 5.0, Marina Del Rey, Calif.). The background was removed using a "homemade" procedure. The program calculates the base intensity of each sub-cell as 10% and subtracts this value for each cell. Analysis was performed using polynuceotidization software (Redwood City, CA). Intermediate point intensities are obtained from a set of values for points within a slide and the values are comparedThe intensity of each spot was normalized by comparison with the values of all slides in this experiment. The relative change between LPS treated cells and control cells can be seen in the table below. Table 60 describes a number of previously unreported changes that would be useful in diagnosing infection.

[0156] To confirm and assess the functional significance of these changes, the levels of selected mRNA and protein were estimated and quantified by densitometry. Northern blot hybridization using CD14, vimentin, and triple tetraprolin-specific probes demonstrated similar expression following stimulation with all three bacterial products (Table 60). Similarly, the enzymatic activity of the nitric oxide synthase iNOS was determined by assessing the level of inflammatory mediator NO using Griess' reagent, demonstrating that the levels of NO produced after 24 hours were comparable, consistent with a similar upregulation of iNOS expression (table 59). Western blot analysis demonstrated that CpG more preferentially stimulates leukemia inhibitory factor (LIF, a member of the cytokine IL-6 family) (Table 59). Additional confirmatory experiments demonstrated that LPS upregulation regulates TNF- α and IL-6 expression, as a result of ELISA assays; also up-regulated was the expression of MIP-2 α and IL-1 β mRNA and down-regulated was the expression of DP-1 and cyclin D mRNA as a result of Northern blot analysis. This assay can be extended to an ex vivo system more clinically relevant by testing the ability of bacterial components to stimulate the production of proinflammatory cytokines in human whole blood. Coli LPS, Salmonella typhimurium LPS and Staphylococcus aureus LTA were found to all stimulate similar amounts of serum TNF- α and IL-1 β. CpG also stimulates the production of these cytokines, although at much lower levels, partially supporting the data for cell lines.

Table 55: a549 epithelial cells are differentiated by E coli O111: b4LPS upregulated polynucleotide.

[0157]Studies of polynucleotide microarrays have shown that E coli O111: b4LPS (100ng/ml) increased the expression of many polynucleotides. LPS was incubated with a549 cells for 4 hours and RNA was isolated. cDNA probes labeled with Cy3/Cy5 were prepared from 5. mu.g of total RNA and ligated with HumanOperon array (PRHU 04). The intensity in unstimulated cells is shown in the third column of table 55. "ratio: LPS/control "column refers to the result of dividing the intensity of polynucleotide expression in LPS-stimulated cells by the intensity of non-stimulated cells.

Registration number	Gene	Comparison: medium strength only	The ratio is: LPS/control
				D87451	Ring finger protein 10	715.8	183.7
AF061261	C3H type zinc finger protein	565.9	36.7
				D17793	Aldehyde-ketone reductase family 1, member C3	220.1	35.9
M14630	Thymosin alpha	168.2	31.3
				AL049975	Is unknown	145.6	62.3
L04510	ADP-ribosylation factor domain protein 1, 64kD	139.9	213.6
				U10991	G2 protein	101.7	170.3
U39067	Eukaryotic translation initiation factor 3, subunit 2	61.0	15.9
				X03342	Ribosomal protein L32	52.6	10.5

NM_004850	Rho-associated, coiled-coil-containing protein kinase 2	48.1	11.8
				AK000942	Is unknown	46.9	8.4
AB040057	Serine/threonine protein kinase MASK	42.1	44.3
				AB020719	KIAA0912 protein	41.8	9.4
AB007856	FEM-1-like death receptor binding proteins	41.2	16.7
				J02783	Procollagen-proline, 2-oxoglutarate 4-dioxygenase	36.1	14.1
AL137376	Is unknown	32.5	17.3
				AL137730	Is unknown	29.4	11.9
D25328	Phosphofructokinase, platelet	27.3	8.5
				AF047470	Malate dehydrogenase 2, NAD	25.2	8.2
M86752	Stress-inducible phosphoprotein 1	22.9	5.9
				M90696	Cathepsin S	19.6	6.8
AK001143	Is unknown	19.1	6.4
				AF038406	NADH dehydrogenase	17.7	71.5
AK000315	Hypothetical protein FLJ20308	17.3	17.4
				M54915	Pim-1 oncogene	16.0	11.4
D29011	Proteasome subunit, beta form, 5	15.3	41.1
				AK000237	Membrane proteins of cholinergic synaptic vesicles	15.1	9.4
AL034348	Is unknown	15.1	15.8
				AL161991	Is unknown	14.2	8.1
AL049250	Is unknown	12.7	5.6
				AL050361	PTD017 protein	12.6	13.0
U74324	RAB interactivity factor	12.3	5.2
				M22538	NADH dehydrogenase	12.3	7.6
D87076	KIAA0239 protein	11.6	6.5
				NM_006327	(Yeast) mitochondrial inner Membrane ectopase 23 homologs	11.5	10.0
AK001083	Is unknown	11.1	8.6
				AJ001403	Mucin 5, subtype B, of the tracheobronchial type	10.8	53.4
M64788	RAP1, GTPase activating protein 1	10.7	7.6
				X06614	Retinoic acid receptor, alpha	10.7	5.5
U85611	Calcium and integrin binding proteins	10.3	8.1
				U23942	Cytochrome P450, 51	10.1	10.2
AL031983	Is unknown	9.7	302.8
				NM_007171	protein-O-mannosyltransferase 1	9.5	6.5
AK000403	Hypothetical protein FLJ20396	9.5	66.6
				NM_002950	Ribosomal receptor protein I	9.3	35.7
L05515	cAMP response element binding protein CRE-BPa	8.9	6.2
				X83368	Phosphoinositide 3-kinases, catalytic, gamma polypeptides	8.7	27.1
M30269	Nestin (enactin)	8.7	5.5
				M91083	Chromosome 11 open reading frame 13	8.2	6.6
D29833	Proline-rich proteins of saliva	7.7	5.8

AB024536	Immunoglobulin superfamily containing leucine-rich repeats	7.6	8.0
				U39400	Chromosome 11 open reading frame 4	7.4	7.3
AF028789	unc119(C.elegans) homologue	7.4	27.0
				NM_003144	Signal sequence receptor, alpha (translocon-related protein alpha)	7.3	5.9
X52195	Arachidonic acid 5-lipoxygenase activating protein	7.3	13.1
				U43895	Human growth factor-modulating tyrosine kinase substrates	6.9	6.9
L25876	Cyclin-dependent kinase inhibitor 3	6.7	10.3
				L04490	NADH dehydrogenase	6.6	11.1
Z18948	S100 calcium binding protein	6.3	11.0
				D10522	Myristoylated alanine-rich protein kinase C substrates	6.1	5.8
NM_014442	Sialic acid binding to Ig-like lectin 8	6.1	7.6
				U81375	Solute carrier family 29	6.0	6.4
AF041410	Malignant tumor associated protein	5.9	5.3
				U24077	Killer cell immunoglobulin-like receptors	5.8	14.4
AL137614	Hypothetical proteins	4.8	6.8
				NM_002406	Mannosyl (alpha-1, 3) -glycoprotein beta-1, 2-N-acetylglucosaminyltransferase	4.7	5.3
AB002348	KIAA0350 protein	4.7	7.6
				AF165217	Protoglobin regulatory protein 4 (muscle)	4.6	12.3
Z14093	Branched-chain ketoacid dehydrogenase E1, alpha Polypeptides	4.6	5.4
				U82671	Kallikrein	3.8	44.5
AL050136	Is unknown	3.6	5.0
				NM_005135	Solute carrier family 12	3.6	5.0
AK001961	Hypothetical protein FLJ11099	3.6	5.9
				AL034410	Is unknown	3.2	21.3
S74728	antiquitin 1	3.1	9.2
				AL049714	Ribosomal protein L34 pseudogene 2	3.0	19.5
NM_014075	PRO0593 protein	2.9	11.5
				AF189279	Phospholipase A2, class IIE	2.8	37.8
J03925	Integrins, α M	2.7	9.9
				NM_012177	F-box protein Fbx5	2.6	26.2
NM_004519	Voltage-gated potassium channel, KQT-like subfamily, member 3	2.6	21.1
				M28825	CD1A antigen, polypeptide	2.6	16.8
X16940	Actin, gamma 2, smooth intestinal muscle	2.4	11.8
				X03066	Class II major histocompatibility complex, DO beta	2.2	36.5
AK001237	Hypothetical protein FLJ10375	2.1	18.4
				AB028971	KIAA1048 protein	2.0	9.4
AL137665	Is unknown	2.0	7.3

Table 56: a549 epithelial cells are differentiated by E coli O111: b4 polynucleotide for LPS down-regulation.

[0158]Studies of polynucleotide microarrays have shown that E coli O111: b4LPS (100ng/ml) reduced the expression of many polynucleotides in a549 cells. LPS was incubated with a549 cells for 4 hours and RNA was isolated. cDNA probes labeled with Cy3/Cy5 were prepared from 5. mu.g total RNA and hybridized to a Human Operon array (PRHU 04). The intensity in the unstimulated cells is shown in the third column of the table. "ratio: LPS/control "column refers to the result of dividing the intensity of polynucleotide expression in LPS-stimulated cells by the intensity of non-stimulated cells.

Registration number	Gene	Comparison: medium strength only	The ratio is: LPS/control
				NM_017433	Myosin IIIA	167.8	0.03
X60484	H4 Histone family member E	36.2	0.04
				X60483	H4 Histone family member D	36.9	0.05
AF151079	Hypothetical proteins	602.8	0.05
				M96843	DNA-binding arrestin 2, dominant negative helix-loop-helix protein	30.7	0.05
S79854	Deiodinase, iodothyronine, type III	39.4	0.06
				AB018266	matrin 3	15.7	0.08
M33374	NADH dehydrogenase	107.8	0.09
				AF005220	mRNA, partial cds of human NUP98-HOXD13 fusion protein	105.2	0.09
Z80783	H2B Histone family, member L	20.5	0.10
				Z46261	H3 Histone family, member A	9.7	0.12
Z80780	H2B Histone family, member H	35.3	0.12
				U33931	Erythrocyte membrane protein band 7.2(stomatin)	18.9	0.13
M60750	H2B Histone family, member A	35.8	0.14
				Z83738	H2B Histone family, member E	19.3	0.15
Y14690	Collagen, type V, alpha 2	7.5	0.15
				M30938	XRCC5, X-ray repair, supplementation of defective repair function of Chinese hamster cells	11.3	0.16
L36055	Eukaryotic translation initiation factor4E binding protein 1	182.5	0.16
				Z80779	H2B Histone family, member G	54.3	0.16
AF226869	5(3) -deoxyribonuclease; RB-related KRAB repressors	7.1	0.18
				D50924	KIAA0134 gene product	91.0	0.18
AL133415	Vimentin	78.1	0.19
				AL050179	Tropomyosin 1 (. alpha.)	41.6	0.19
AJ005579	RD element	5.4	0.19
				M80899	AHNAK nucleoprotein	11.6	0.19
NM_004873	BCL2 relevance athanogene 5	6.2	0.19
				X57138	H2A Histone family, member N	58.3	0.20
AF081281	Lysophospholipase I	7.2	0.22

U96759	von Hippel-Linau binding protein I	6.6	0.22
				U85977	Human ribosomal protein L12 pseudogene, part of cds	342.6	0.22
D13315	Glyoxalases I	7.5	0.22
				AC003007	Is unknown	218.2	0.22
AB032980	RU2S	246.6	0.22
				U40282	Integrin-associated kinases	10.1	0.22
U81984	Endothelial PAS domain protein 1	4.7	0.23
				X91788	Chloride channels, nucleotide sensitive, 1A	9.6	0.23
AF018081	Collagen, type XVIII, alpha 1	6.9	0.24
				L31881	Nuclear factor I/X (CCAAT-binding transcription factor)	13.6	0.24
X61123	B cell translocation Gene 1, antiproliferation	5.3	0.24
				L32976	Mitogen-activated protein kinase 11	6.3	0.24
M27749	Immunoglobulin lambda-like polypeptide 3	5.5	0.24
				X57128	H3 Histone family, member C	9.0	0.25
X80907	Phosphoinositide-3-kinases, regulatory subunits, polypeptide 2	5.8	0.25
				Z34282	Human mucin (MAR11) MUC5ACmRNA (part)	100.6	0.26
X00089	H2A Histone family, member M	4.7	0.26
				AL035252	CD 39-like 2	4.6	0.26
X95289	PERB11 family members of the MHC class I region	27.5	0.26
				AJ001340	U3 snoRNP-related 55kDa protein	4.0	0.26
NM_014161	HSPC071 protein	10.6	0.27
				U60873	Is unknown	6.4	0.27
X91247	Thioredoxin reductase 1	84.4	0.27
				AK001284	Hypothetical protein FLJ10422	4.2	0.27
U90840	Synovial sarcoma, X breakpoint 3	6.6	0.27
				X53777	Ribosomal protein L17	39.9	0.27
AL035067	Is unknown	10.0	0.28
				AL117665	DKFZP586M1824 protein	3.9	0.28
L14561	ATP enzyme, calcium ion transport, plasma Membrane 1	5.3	0.28
				L19779	H2A Histone family, member O	30.6	0.28
AL049782	Is unknown	285.3	0.28
				X00734	Tubulin, beta, 5	39.7	0.29
AK001761	Retinoic acid inducibility 3	23.7	0.29
				U72661	ninjurin 1	4.4	0.29
S48220	Deiodinase, iodothyronine, type I	1,296.1	0.29
				AF025304	EphB2	4.5	0.30
S82189	Chymotrypsin C	4.1	0.30
				Z80782	H2B Histone family, member K	31.9	0.30
X68194	Synaptic vesicle protein-like proteins	7.9	0.30
				AB028869	Is unknown	4.2	0.30

AK000761

Is unknown

4.3

0.30

Table 57: polynucleotides expressed to a similar extent after stimulation by the bacterial products LPS, LTA and CpG DNA.

[0159]Bacterial products (100ng/ml Salmonella typhimurium LPS, 1. mu.g/ml Staphylococcus aureus LTA or 1. mu.M CpG) were found to efficiently induce expression of several polynucleotides. After incubation with RAW cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to Atlas arrays. The intensity in control cells that were not stimulated is shown in the second column. "ratio: the LPS/LTA/CpG: control column refers to the intensity of polynucleotide expression in cells stimulated by bacterial product divided by the intensity of unstimulated cells.

Registration number	Control non-stimulated intensity	Ratio LPS to control	Ratio LTA: control	Ratio CpG: control	Protein/polynucleotide
						M15131	20	82	80	55	IL-1β
M57422	20	77	64	90	Triple tetra proline basic protein
						X53798	20	73	77	78	MIP-2α
M35590	188	50	48	58	MIP-1B
						L28095	20	49	57	50	ICE
M87039	20	37	38	45	iNOS
						X57413	20	34	40	28	TGFβ
X15842	20	20	21	15	c-rel proto-oncogenic polynucleotides
						X12531	489	19	20	26	MIP-1α
U14332	20	14	15	12	IL-15
						M59378	580	10	13	11	TNFR1
U37522	151	6	6	6	TRAIL
						M57999	172	3.8	3.5	3.4	NF-κB
U36277	402	3.2	3.5	2.7	I-kappa B (alpha subunit)
						X76850	194	3	3.8	2.5	MAPKAP-2
U06924	858	2.4	3	3.2	Stat 1
						X14951	592	2	2	2	CD18
X60671	543	1.9	2.4	2.8	NF-2
						M34510	5970	1.6	2	1.4	CD14
X51438	2702	1.3	2.2	2.0	Vimentin
						X68932	4455	0.5	0.7	0.5	c-Fms
Z21848	352	0.5	0.6	0.6	DNA polymerase
						X70472	614	0.4	0.6	0.5	B-myb

Table 58: polynucleotides differentially regulated by the bacterial products LPS, LTA and CpG DNA.

[0160]Bacterial products (100ng/ml Salmonella typhimurium LPS, 1. mu.g/ml Staphylococcus aureus LTA or 1. mu.M CpG) were found to efficiently induce expression of several polynucleotides. After incubation with RAW cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to Atlas arrays. The intensity in control cells that were not stimulated is shown in the second column. "ratio: the LPS/LTA/CpG: control column refers to the intensity of polynucleotide expression in cells stimulated by bacterial product divided by the intensity of unstimulated cells.

Registration number

Control

Ratio of

Ratio of

Ratio of

Protein/polynucleotide

	Intensity of non-stimulation	LPS control	LTA: control	CpG: control
						X72307	20	1.0	23	1.0	Hepatocyte growth factor
L38847	20	1.0	21	1.0	Transmembrane kinase ligand for liver cancer
						L34169	393	0.3	3	0.5	Thrombopoietin
J04113	289	1	4	3	Nur77
						Z50013	20	7	21	5	H-ras proto-oncogene polynucleotide
X84311	20	4	12	2	Cyclin A1
						U95826	20	5	14	2	Cyclin G2
X87257	123	2	4	1	Elk-1
						J05205	20	18	39	20	Jun-D
J03236	20	11	19	14	Jun-B
						M83649	20	71	80	42	Fas 1 receptor
M83312	20	69	91	57	CD40L receptor
						X52264	20	17	23	9	ICAM-1
M13945	573	2	3	2	Pim-1
						U60530	193	2	3	3	Mad-related proteins
D10329	570	2	3	2	CD7
						X06381	20	55	59	102	Leukemia Inhibitory Factor (LIF)
X70296	20	6.9	13	22	Protease connexin 1(PN-1)
						U36340	20	38	7	7	CACCC frame binding protein BKLF
S76657	20	11	6	7	CRE-BPI
						U19119	272	10	4	4	Interferon inducible protein 1

Table 59: validation of the array data of tables 57 and 58.

[0161] a) Total RNA was isolated from unstimulated RAW macrophages and cells treated with 100ng/ml Salmonella typhimurium LPS, 1. mu.g/ml Staphylococcus aureus LTA, 1. mu.M CpG DNA or medium alone for 4 hours were subjected to Northern blot hybridization to probe for GAPDH, CD14, vimentin and triply tetraprolin on the membrane as described previously [ Scott et al ]. The hybridization intensity of Northern blots was compared to GAPDH to find inconsistencies in the load. These experiments were repeated at least three times, and the data presented were the mean levels of the various conditions (measured using densitometry) versus the medium ± standard deviation.

[0162] b) RAW 264.7 cells were stimulated with 100ng/ml Salmonella typhimurium LPS, 1. mu.g/ml Staphylococcus aureus LTA, 1. mu.M CpGDNA or medium alone for 24 hours. Protein lysates were prepared, separated on SDS-PAGE gels, and Western blot hybridization was performed to detect LIF (R & D system). These experiments were repeated at least three times, and the data presented were the level of LIF relative to the medium (measured using densitometry) ± standard deviation.

[0163]c) Stimulating RAW macrophages with 100ng/ml Salmonella typhimurium LPS, 1. mu.g/ml Staphylococcus aureus LTA, 1. mu.M CpGDNA or medium alone for 24 hours, collecting the cell supernatant, and detecting the amount of NO formed in the supernatant using Griess reagent, the amount of NO being estimated by the accumulation of nitrite, a stable NO metabolite, as described previously [ Scott et al]. Data presented are mean ± standard deviation of three experiments.

Product of

Relative horizontal

	Untreated	LPS	LTA	CpG
					CD14a	1.0	2.2±0.4	1.8±0.2	1.5±0.3
Vimentina	1.0	1.2±0.07	1.5±0.05	1.3±0.07
					Triple tetra proline basic proteina	1.0	5.5±0.5	5.5±1.5	9.5±1.5
LIFb	1.0	2.8±1.2	2.7±0.6	5.1±1.6
					NOc	8±1.5	47±2.5	20±3	21±1.5

Table 60: a549 gene expression pattern upregulated by bacterial signaling molecules (LPS) in human epithelial cells.

[0164]Studies of polynucleotide microarrays have shown that e.coli O111: b4LPS (100ng/ml) increased the expression of many polynucleotides in A549 cells. LPS was incubated with a549 cells for 4 hours and RNA was isolated. Cy3/Cy5 labeled cDNA probes were prepared with 5. mu.g total RNA and hybridized to a Human Operon array (PRHU 04). These examples of changes in polynucleotide expression in LPS-stimulated cells represent greater than 2-fold changes in intensity levels compared to untreated cells.

Registration number	Gene
		AL050337	Interferon gamma receptor 1
U05875	Interferon gamma receptor 2
		NM_002310	Leukemia inhibitory factor receptors
U92971	Coagulation factor II (thrombin) receptor like 2
		Z29575	Tumor necrosis factor receptor superfamily member 17
L31584	Chemokine receptor 7
		J03925	cAMP response element binding proteins
M64788	RAP1, GTPase activating protein
		NM_004850	Rho-associated kinase 2
D87451	Ring finger protein 10
		AL049975	Is unknown
U39067	Eukaryotic translation initiation factor 3, subunit 2
		AK000942	Is unknown
AB040057	Serine/threonine protein kinase MASK
		AB020719	KIAA0912 protein
AB007856	FEM-1-like death receptor binding proteins
		AL137376	Is unknown
AL137730	Is unknown
		M90696	Cathepsin S
AK001143	Is unknown
		AF038406	NADH dehydrogenase
AK000315	Hypothetical protein FLJ20308
		M54915	Pim-1 oncogene
D29011	Proteasome subunit, beta form, 5
		AL034348	Is unknown
D87076	KIAA0239 protein
		AJ001403	Tracheal bronchial mucin 5, subtype B,
J03925	integrins, α M

Example 10

Altering signaling to protect against bacterial infection

[0165]Salmonella typhimurium strain SL1344 was obtained from the American type culture Collection (ATCC; Manassas, Va.) and cultured in Luria-Bertani (LB) medium. To effect macrophage infection, a frozen glycerol stock was inoculated into 10ml LB in a 125ml shake flask and cultured overnight at 37 ℃ with shaking to a stable phase. RAW 264.7 cells (1X 10)⁵Individual cells/well) were seeded into 24-well plates. The bacteria were diluted with medium until a nominal multiplicity of infection (MOI) of approximately 100 was achieved, centrifuged onto the monolayer by centrifugation at 1000rpm for 10 minutes to synchronize infection, and allowed to infect at 37 ℃ in 5% CO₂The incubator was continued for 20 minutes. Cells were washed 3 times with PBS to remove extracellular bacteria, and then incubated in DMEM + 10% FBS containing 100 μ g/ml gentamicin (Sigma, st. louis, MO) to kill any residual extracellular bacteria, avoiding reinfection. After two hours, the gentamicin concentration was reduced to 10 μ g/ml and maintained at this value throughout the analysis. Cells were pretreated for 30 min with inhibitors with the following concentrations before infection: 50 μ MPD98059(Calbiochem), 50 μ M U0126 (Promega), 2mM Diiodo (DPI), 250 μ M acetovanidone (apocynin, Aldrich), 1mM ascorbic acid (Sigma), 30mM N-acetylcysteine (Sigma) and 2mM N^GL-monomethyl arginine (L-NMMA, Molecular Probe) or 2mMN^GD-monomethyl arginine (D-NMMA, Molecular Probe). Fresh inhibitor was added immediately, 2 hours and 6-8 hours after infection to ensure efficacy. Control detailsCells were treated with equal volumes of dimethyl sulfoxide (DMSO) per ml of medium. The intracellular survival/replication of salmonella typhimurium SL1344 was determined using the gentamicin resistance assay, as described previously. Briefly, at 2 and 24 hours post-infection, cells were washed twice with PBS to remove gentamicin, lysed with PBS containing 1% Triton X-100/0.1% SDS, and the number of bacteria inside the cells was calculated from the number of colonies on LB agar plates. Under these infection conditions, macrophages contain an average of one bacterium per cell as estimated by standard plate counts, and thus can be analyzed 24 hours after infection. Bacterial fibrillation (filamentation) is associated with bacterial stress (stress). NADPH oxidase and iNOS can be activated by MEK/ERK signaling. The results (Table 61) clearly demonstrate that altering cell signaling is one way by which Salmonella intracellularis infection can be addressed. Since bacteria upregulate multiple genes in human cells, this strategy of blocking signal transduction represents a common approach to anti-infective therapy.

Table 61: effect of the signalling molecule MEK on intracellular bacteria in IFN- γ sensitized (primary) RAW cells.

Treatment ofa	Effectb
		0	Is free of
MEK inhibitor U0126	Reducing bacterial fibrillation (bacterial stress)cIncreasing the number of Salmonella typhimurium in cells
		MEK inhibitor PD98059	Reducing bacterial fibrillation (bacterial stress)cIncreasing the number of Salmonella typhimurium in cells
NADPH oxidase inhibitorsd	Reducing bacterial fibrillation (bacterial stress)cIncreasing the number of Salmonella typhimurium in cells

Example 11

Antiviral activity

[0166]SDF-1 is a C-X-C chemokine, which is a natural ligand of the HIV-1 co-receptor CXCR-4. In order to inhibit the replication of HIV-1, the chemokine receptors CXCR-4 and CCR5 are considered as possible targets. The crystal structure of SDF-1 exhibits antiparallel beta-sheets and a positively charged surface, characteristics that are important for its binding to the negatively charged extracellular loops of CXCR-4. These findings suggest that chemokine derivatives, small CXCR4 antagonists, or agonists that mimic the structural or ionic properties of chemokines may be useful agents for the treatment of X4HIV-1 infection. The cationic peptides were found to inhibit SDF-1 induced T cell migration, suggesting that these peptides may act as CXCR-4 antagonists. Migration analysis was performed as follows. Human Jurkat T cells were resuspended to 5X 10 with chemotactic medium (RPMI1640/10mM Hepes/0.5% BSA)⁶Individual cells/ml. Migration analysis was performed in 24-well plates using a5 μm polycarbonate Transwell insert (Costar). Briefly, peptides or controls were diluted with chemotactic medium and placed in the lower chamber while 0.1ml of cells (5X 10)⁶Pieces/ml) was added to the upper chamber. After 3 hours at 37 ℃, the number of cells migrating into the lower chamber was determined by flow cytometry. The culture in the lower chamber took 30 seconds to traverse the FACscan, opening the front and side diffusers to expel cell debris. The number of viable cells was compared with the "100% migration control", for which 5X 10⁵Cells per ml were pipetted directly into the lower chamber and then counted for 30 seconds using a FACscan. The results indicate that the addition of the peptide results inMigration of human Jurkat T cells was inhibited (table 62), probably because CXCR-4 expression was affected (tables 63 and 64).

Table 62: the peptides inhibit migration of human Jurkat T cells:

	migration (%)
					Experiment of	Positive control	SDF-1(100ng/ml)	SDF-1+SEQ ID 1(50μg/ml)	Negative control
1	100％	32％	0％	＜0.01％
					2	100％	40％	0％	0％

Table 63: polynucleotide array data corresponding to table 56:

polynucleotide/protein	Polynucleotide function	Intensity of non-stimulation	Peptide ratio unstimulated	Registration number
					CXCR-4	Chemokine receptors	36	4	D87747

Table 64: FACs data corresponding to tables 62 and 63:

peptides	Concentration (μ g/ml)	Fold increase of protein expression CXCR-4
			SEQ ID NO：1	10	Has no change
SEQ ID NO：1	50	1.3±0.03
			SEQ ID NO：1	100	1.6±0.23
SEQ ID NO：3	100	1.5±0.2

Example 12

Synergistic combinations (syntactical combinations)

[0167] Method and material

[0168]Staphylococcus aureus was prepared in Phosphate Buffered Saline (PBS) and 5% porcine mucin (Sigma) to the final desired concentration of 1-4X 10⁷CFU/ml. 100 μ l of Staphylococcus aureus (mixed with 5% porcine mucin) was injected intraperitoneally into each CD-1 mouse (6-8 weeks old, female, 20-25g in weight (Charles river)). 6 hours after infection had occurred, 100. mu.l of peptide (50-200. mu.g total) were injected IP together with 0.1mg/kg Cefepime (Cefepime). After 24 hours, the animals were sacrificed, cardiac puncture was performed, and 100. mu.l of blood was taken out. Blood was diluted with 1ml PBS containing heparin. Then, it was further diluted and plated on Mueller-Hinton agar plates (10)^-1、10^-2、10^-3And 10^-4) Plating and counting surviving colonies. Surviving colonies, i.e. Colony Forming Units (CFU), were counted after 24 hours. Each test was performed a minimum of three times. Data are expressed as mean CFU ± standard deviation of each treatment group (8-10 mice per group).

[0169] The experiment was performed 6 hours after systemic staphylococcus aureus infections had developed hair, with the administration of peptide and suboptimal (sub-optimal) cefepime (figure 1). The data in figure 1 are expressed as mean ± standard deviation of the number of surviving colonies from blood taken from mice 24 hours after infection occurred. Suboptimal antibiotic (cefepime) doses were compared to SEQ ID NO: 7, improved therapeutic effect is obtained as a result. The ability of peptides to work in combination with suboptimal antibiotics in murine infection models is an important finding. It suggests the potential to extend the life of antibiotics and reduce the incidence of antibiotic resistance in the clinic.

[0170] SEQ ID NO: 1 As an example, induction of phosphorylation and activation of mitogen-activated protein kinases, ERK1/2 and p38, occurs in human peripheral blood mononuclear cells and human bronchial epithelial cell lines, but not in B or T lymphocytes. Phosphorylation was not dependent on the G protein-coupled receptor FPRL-1, which was previously thought to be involved on human monocytes and T cells as shown by SEQ ID NO: 1 receptor of induced chemotaxis. The presence of granulocyte macrophage colony stimulating factor (GM-CSF), but not macrophage colony stimulating factor (M-CSF), significantly enhanced the activation of ERK1/2 and p 38. Exposure to SEQ ID NO: 1 also leads to activation of Elk-1 and up-regulation of various genes regulated by Elk-1, a transcription factor activated by ERK1/2 located downstream of ERK1/2 and phosphorylated. SEQ ID NO: 1 the ability to signal transduction through these pathways has broad implications in immunity, monocyte activation, proliferation and differentiation.

[0171] Method and material

[0172] SEQ ID NO: 1 (seq. No. LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was synthesized by Fmoc [ (N- (9-fluorenyl) methoxycarbonyl) ] chemistry at the Nucleic Acid/Protein Synthesis (NAPS) Unit of UBC. Human recombinant granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4), and macrophage colony stimulating factor (M-CSF) were purchased from research diagnostics Inc. (Flanders, NJ, USA). Pertussis toxin is supplied by List Biological Laboratories Inc. (Campbell, Calif., USA).

[0173]Blood mononuclear cells are prepared using standard techniques. Briefly, 100ml of freshHuman venous blood was collected in sodium heparin vacuum collection tubes (Becton Dickinson, Mississauga, ON, Canada) from volunteers according to UBCCLinical Research Ethics Board protocol C02-0091. In E-toxa-clean (Sigma-Aldrich, Oakville, ON, Canada) endotoxin-free flasks, blood was mixed with RPMI1640 medium [ 10% v/v supplemented Fetal Bovine Serum (FBS), 1% L-glutamine, 1nM sodium pyruvate)]And (4) mixing. PBMCs were separated using Ficoll-Paque Plus (Amersham Pharmacia Biotech, Baied' Urfe, PQ, Canada) at room temperature and washed with Phosphate Buffered Saline (PBS). Monocytes were enriched by removing T cells by rosetting with fresh sheep red blood cells (UBC animal care unit) pretreated with Vibrio cholerae (Vibrio cholerae) neuraminidase (Calbiochem Biosciences Inc., La Jolla, Calif., USA) and repeatedly isolated by Ficoll-Paque Plus. The enriched monocytes were washed with PBS followed by incubation at 37 ℃ for 1 hour (approximately 2-3X 10 per well)⁶) Subsequently removing non-adherent cells; the purity of the monocytes was greater than 95% as determined by flow cytometry analysis (data not shown). B lymphocytes were isolated by removing non-adherent cells and adding them to a new plate at 37 ℃ for 1 hour. This process was repeated 3 times in total. Any remaining monocytes, and remaining non-adherent cells, that are attached to the plate are essentially B cells. Cells were cultured in Falcon 6-well tissue culture plates (Becton Dickinson, Mississauga, ON, Canada). Adherent monocytes were cultured at 37 ℃ in 1ml of a medium to which was added the nucleotide sequence of SEQ ID NO: 1 and/or a cytokine. Endotoxin-free water was added as a vehicle control. For studies using pertussis toxin, the medium was replaced with 1ml of fresh medium containing 100ng/ml toxin and incubated at 37 ℃ for 60 minutes. Converting SEQ ID NO: 1 and cytokines were added directly to the pertussis toxin-containing medium. To isolate T lymphocytes, rosette-forming T cells and sheep red blood cells were resuspended in 20ml PBS and 10ml distilled water was added to lyse the sheep red blood cells. Then, the cells were centrifuged at 1000rpm for 5 minutes, followed by removing the supernatant. The precipitated T cells were washed rapidly in PBSAn incremental amount of water was added until all sheep red blood cells were lysed. Residual T cells were washed once in PBS and their survival was confirmed with 0.4% trypan blue solution. Primary human blood monocytes and T cells were cultured in RPMI1640 supplemented with 10% v/v heat-inactivated FBS, 1% v/v L-glutamine, 1nM sodium pyruvate (GIBCO Invitrogen Corporation, Burlington, ON, Canada). For each experiment, 2 to 8 donors were used.

[0174]The simian virus 40-transformed immortalized 16HBE4 o-bronchial epithelial cell line was donated by dr.d. gruenert (University of California, San Francisco, CA). At 37 deg.C, 100% humidity and 5% CO as usual₂Cells were cultured until confluent. Cells were grown in minimal essential medium with Earles' salts (GIBCO Invitrogen Corporation, Burlington, ON, Canada) containing 10% fbs (hyclone), 2mM L-glutamine. For the experiments, cells were grown on Costar Transwell inserts (inserts) (3 μm pore size, Fischer Scientific) in 24-well plates. At 5X 10 per 0.25ml of medium⁴Individual cells were seeded onto the top of the insert and 0.95ml of media was added to the bottom of the well at 37 ℃ and 5% CO₂And (5) culturing. Transmembrane resistance was measured daily using a Millipore voltage resistance meter, and typically after 8 to 10 days, the inserts were used for the test at a resistance of 500-. Cells between passage 8 and 20 (betaenpassages 8 and 20) were used.

[0175] Western immunoblotting

[0176] After stimulation, cells were washed with ice cold PBS containing 1mM vanadate (Sigma). Then 125. mu.l of RIPA buffer (50mM Tris-HCl, pH 7.4, NP-401%, sodium deoxycholate 0.25%, NaCl150mM, EDTA 1mM, PMSF 1mM, aprotinin, leupeptin, pepstatin each 1. mu.g/ml, sodium orthovanadate 1mM, NaF 1mM) was added and the cells were incubated on ice until they were visually observed to be completely lysed. Lysates were quantified using BCA assay (Pierce). Mu.g of lysate were loaded onto a 1.5mm thick gel and the sample was treated at 100 volts for approximately 2 hours. Proteins were transferred to nitrocellulose membranes for 75 min at 70V. Membranes were blocked with TBST (10mM Tris-HCl pH 8, 150mM NaCl, 0.1% Tween 20) containing 5% skim milk powder for 2 hours at room temperature. The membranes were then incubated with monoclonal antibodies against ERK1/2-P or against P38-P (Cell Signaling technology, Ma) overnight at 4 ℃. Immunoreactive bands were detected with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Amersham Pharmacia, New Jersey) and chemiluminescent detection (Sigma, Mo). To quantify the bands, the membrane was scanned and subsequently quantified by densitometry using the software program ImageJ. The dot blots were again probed with β -actin antibody (ICN biological Incorporated, Ohio) and densitometric to correct for protein loading.

[0177] Kinase assays

[0178] ERK1/2 activity assays were performed using a non-radioactive kit (Cell Signaling Technology). Briefly, cells were treated for 15 minutes and lysed in lysis buffer. Equal amounts of protein were immunoprecipitated with immobilized phospho-ERK 1/2 antibody that reacted only with phosphorylated (i.e., activated) form of ERK 1/2. The immobilized precipitated enzyme was then used in a kinase assay using Elk-1 followed by western blot analysis with antibody, whereby phosphorylated substrates could be detected and quantified.

[0179] Quantification of IL-8

[0180] Human IL-8 in the supernatant of I6HBE 40-cells was assayed using a commercially available enzyme-linked immunosorbent assay kit (Biosource) according to the manufacturer's instructions.

[0181] Semi-quantitative RT-PCR

[0182] Total RNA from two independent experiments was isolated from 16HBE4 o-cells using RNAqueous (Ambion) as described by the manufacturer. The sample was treated with DNase, and then cDNA synthesis was completed using first strand cDNA (first-strand cDNA) synthesis kit (Gibco). The resulting cDNAs are used as templates in PCRs for the synthesis of various cytokine genes: MCP-1 (5X-TCATAGCAGCCACCTTCATTC-3 ' (SEQ ID NO: 59), 5 ' -TAGCGCAGATTCTTGGGTTG-3(SEQ ID NO: 60)), MCP-3 (5X-TGTCCTTTCTCAGAGTGGTTCT-3 ' (SEQ ID NO: 61), 5'-TGCTTCCATAGGGACATCATA-3' (SEQ ID NO: 62)) IL-6, (5'-ACCTGAACCTTCCAAAGATGG-3' (SEQ ID NO: 63), 5'-GCGCAGAATGAGATGAGTTG-3' (SEQ ID NO: 64)), and IL-8, (5'-GTGCAGAGGGTTGTGGAGAAG-3' (SEQ ID NO: 65), 5'-TTCTCCCGTGCAATATCTAGG-3' (SEQ ID NO: 66)). Each RT-PCR reaction was performed at least twice. The results of the linear amplification phase were analyzed and normalized against a housekeeping control, glyceraldehyde-3-phosphate dehydrogenase. The RNA amplification reaction was confirmed by inclusion into a control without reverse transcriptase addition.

[0183] Results

[0184] Peptides induced phosphorylation of ERK1/2 and p38 in peripheral blood-derived monocytes.

[0185] To determine whether the peptides induced MAP kinase, ERK1/2 and/or p38 activation, the peptide was purified using 50 μ g/ml of seq id NO: peripheral blood-derived monocytes were treated for 15 minutes with 1 or water (as vehicle control). To show the activated (phosphorylated) form of the kinase, western blots were performed with antibodies specific for the doubly phosphorylated form of the kinase (phosphorylated on Thr202+ Tyr204 and Thr180+ Tyr182, respectively, for ERK1/2 and p 38). The gel was re-probed with the β -actin antibody to normalize loading differences. In summary, in the case of SEQ ID NO: in response to treatment 1, increased phosphorylation of ERK1/2 (n-8) and p38 (n-4) was observed (fig. 2).

[0186] FIG. 2 shows, for SQE ID NO: exposure to 1 induced phosphorylation of ERK1/2 and p 38. Lysates from human peripheral blood-derived monocytes were exposed to 50 μ g/ml SQE ID NO: 1, time is 15 minutes. A) Activation of ERK1/2 and p38 was detected with antibodies specific for phosphorylated forms of ERK and p 38. All donors tested showed that the response to SEQ ID NO: 1 treatment, increased phosphorylation of ERK1/2 and p 38. One representative donor of eight. The relative amounts of phosphorylation of erk (b) and p38(C) were determined as described in materials and methods, by dividing the intensity of the phosphorylated bands by the intensity of the corresponding control bands.

[0187] Peptide-induced activation of ERK1/2 in human serum was greater than peptide-induced activation of ERK1/2 in fetal bovine serum.

[0188] We have been able to demonstrate that in the absence of serum, LL-37 induced phosphorylation of ERK1/2 does not occur, and that the amount of phosphorylation depends on the type of serum present, so that ERK1/2 activation is much higher in Human Serum (HS) than in Fetal Bovine Serum (FBS).

[0189] FIG. 3 shows that in the absence of serum, LL-37-induced phosphorylation of ERK1/2 did not occur, and the amount of phosphorylation was dependent on the type of serum present. Human blood-derived monocytes were treated with 50. mu.g/ml LL-37 for 15 min. The lysates were separated on a 12% acrylamide gel, then transferred to nitrocellulose membrane and hybridized with an antibody specific for the phosphorylated (activated) form of the kinase. To standardize the protein loading, the blot was rehybridized with β -actin. Quantification was performed using ImageJ software. FIG. 3 is an inset showing that LL-37 is unable to induce MAPK activation in human monocytes under serum-free conditions. Cells were exposed to either LL-37(+) at 50mg/ml, or endotoxin-free water (-) as a vehicle control for 15 minutes. (A) Phosphorylated ERK1/2 was detectable after exposure to LL-37 in medium containing 10% fetal bovine serum, however, no phosphorylation of ERK1/2 was detected in the absence of serum (n-3). (B) Elk-1 is a transcription factor downstream of ERK1/2, and Elk-1 is activated (phosphorylated) after exposure to 50 μ g/ml LL-37 in medium containing 10% fetal bovine serum, but Elk-1 is not activated in the absence of serum (n ═ 2).

[0191] Peptide-induced activation of ERK1/2 and p38 was dose-dependent and demonstrated synergy with GM-CSF.

[0192] In freshly isolated human blood mononuclear cells, GM-CSF, IL-4 or M-CSF (all 100ng/ml) was combined with the amino acid sequence of SEQ ID NO: 1 was co-added and the phosphorylation of ERK1/2 was measured. The DNA fragment was purified with 50. mu.g/ml of SEQ ID NO: phosphorylation of ERK1/2 was evident in 1 treated cells (8.3 fold higher than in untreated cells, n-9), but this did not occur at lower concentrations (n-2). In the presence of 100ng/ml GM-CSF, the amino acid sequence of SEQ ID NO: 1-induced phosphorylation of ERK1/2 was significantly increased (58-fold higher than untreated cells, n-5). Furthermore, in the presence of GM-CSF, in the two donors tested, the response was determined in response to the concentrations of SEQ ID NO: 1, activation of ERK1/2 occurred (FIG. 4). This demonstrates that, in the presence of GM-CSF, the amino acid sequence of SEQ ID NO: 1-induced activation of ERK1/2 occurred at a lower threshold, and GM-CSF is a cytokine found locally at the site of infection.

[0193] FIG. 4 shows that LL-37-induced activation of ERK1/2 occurs at lower concentrations and is amplified in the presence of some cytokines. LL-37-induced phosphorylation of ERK1/2 was also evident at concentrations as low as 5. mu.g/ml when freshly isolated monocytes were stimulated in medium containing GM-CSF (100ng/ml) and IL-4(100 ng/ml). This synergistic activation of ERK1/2 appears to be primarily due to GM-CSF.

[0194] Activation of ERK1/2 leads to transcription of Elk-1 controlled genes and secretion of IL-8

[0195] The release of IL-8 is at least partially governed by the activation of ERK1/2 and p38 kinase. To determine whether the peptide was able to induce IL-8 secretion, the human bronchial epithelial cell line 16HBE4 o-was grown to confluence in Transwell membranes (Transwell filters), which allowed cellular polarization to occur, resulting in different apical (apical) and basal (basal) surfaces. The DNA fragment was purified with 50. mu.g/ml of SEQ ID NO: 1 cells on the apical surface were stimulated for 4 hours, and a statistically significant increase in the amount of IL-8 released into the apical supernatant was detected (FIG. 5). To determine the downstream transcriptional effects of peptide-induced MAP kinase activation, RT-PCR was used to assess the expression of genes known to be regulated by ERK1/2 or p 38. RT-PCR was performed on RNA isolated from the serum in the presence of 50 μ g/ml of SEQ ID NO: 1 treatment of 4 hours of 16HBE4 o-cells from two independent experiments. MCP-1 and IL-8 have been shown to be under transcriptional control of ERK1/2 and p38, consistent with being up-regulated by 2.4-fold and 4.3-fold, respectively. It was not previously demonstrated that MCP-3 transcription is affected by activation of mitogen-activated protein kinase, consistent with expression not being affected by peptide treatment (FIG. 5). These data are consistent with the following assumptions: activation of the ERK1/2 and p38 signaling pathways has a functional effect on transcription of cytokine genes with immune regulatory functions. The inset in FIG. 3B also demonstrates that the peptide induces phosphorylation of the transcription factor Elk-1 in a serum-dependent manner.

[0196] FIG. 5 shows that in the 16HBE4 o-human bronchial epithelial cell line, the peptides affect both transcription of various cytokine genes and release of IL-8. Cells were grown to confluence on a semi-permeable membrane and incubated with 50. mu.g/ml of SEQ ID NO: 1 stimulation on the top surface for 4 hours. A) SEQ ID NO: 1 treated cells produced significantly more IL-8 than control cells, this increase was detected by ELISA in supernatants collected from the apical, but not basolateral (basal surface). The mean ± standard deviation of three independent experiments is shown, with an asterisk representing p ═ 0.002. B) RNA was collected from the above assay and subjected to RT-PCR. Many cytokine genes known to be regulated by ERK1/2 or p38 are upregulated after stimulation with peptides. The average of two independent experiments is shown.

[0197] While the invention has been described with respect to a presently preferred embodiment, it will be understood that various modifications may be made without departing from the spirit of the invention. Accordingly, the invention is not limited except as by the appended claims.

Sequence listing

<110> university of British Columbia

R, E, W, Hancock

B, Fenli

M, G, Scott

D, rebaudioside

C.M.Rosenberg

J-P.S.palls

<120> Effector of innate immunity decision (Determination)

<130>581-304PCT

<140>US 10/661,471

<141>2003-09-12

<150>US 10/308,905

<151>2002-12-02

<150>US 60/336,632

<151>2001-12-03

<160>66

<170>PatentIn version 3.1

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Pro Arg Thr Glu Ser

35

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Xaa Xaa Leu Xaa Xaa Xaa Lys Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa

1 5 10 15

<210>12

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>12

Asp Leu Pro Ala Lys Arg Gly Ser Ala Pro Gly Ser Thr

1 5 10

<210>13

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>13

Ser Glu Leu Pro Gly Leu Lys His Pro Cys Val Pro Gly Ser

1 5 10

<210>14

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>14

Thr Thr Leu Gly Pro Val Lys Arg Asp Ser Ile Pro Gly Glu

1 5 10

<210>15

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>15

Ser Leu Pro Ile Lys His Asp Arg Leu Pro Ala Thr Ser

1 5 10

<210>16

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>16

Glu Leu Pro Leu Lys Arg Gly Arg Val Pro Val Glu

1 5 10

<210>17

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>17

Asn Leu Pro Asp Leu Lys Lys Pro Arg Val Pro Ala Thr Ser

1 5 10

<210>18

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<220>

<221>MISC_FEATURE

<222>(1)..(4)

<223> Xaa is selected from A, P or R and one, two, three or all four may be present

<220>

<221>MISC_FEATURE

<222>(5)..(6)

<223> Xaa is an aromatic amino acid (F, Y and W)

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223> Xaa is a P or K

<220>

<221>MISC_FEATURE

<222>(8)..(9)

<223> Xaa is selected from A, P, Y or W and one or two may or may not be present

<220>

<221>MISC_FEATURE

<222>(11)..(12)

<223> Xaa is selected from A, P, Y or W and one or two may or may not be present

<220>

<221>MISC_FEATURE

<222>(14)..(15)

<223> Xaa is selected from A, P, Y or W and one or two may or may not be present

<220>

<221>MISC_FEATURE

<222>(16)..(18)

<223> Xaa is selected from R or P and there may be one, two or three

<400>18

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Trp Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Lys

<210>19

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>19

Arg Pro Arg Tyr Pro Trp Trp Pro Trp Trp Pro Tyr Arg Pro Arg Lys

1 5 10 15

<210>20

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>20

Arg Arg Ala Trp Trp Lys Ala Trp Trp Ala Arg Arg Lys

1 5 10

<210>21

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>21

Arg Ala Pro Tyr Trp Pro Trp Ala Trp Ala Arg Pro Arg Lys

1 5 10

<210>22

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>22

Arg Pro Ala Trp Lys Tyr Trp Trp Pro Trp Pro Trp Pro Arg Arg Lys

1 5 10 15

<210>23

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>23

Arg Ala Ala Phe Lys Trp Ala Trp Ala Trp Trp Arg Arg Lys

1 5 10

<210>24

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>24

Arg Arg Arg Trp Lys Trp Ala Trp Pro Arg Arg Lys

1 5 10

<210>25

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<220>

<221>MISC_FEATURE

<222>(1)..(2)

<223> Xaa is R or K and there may be one or two

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223> Xaa is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H)

<220>

<221>MISC_FEATURE

<222>(4)..(4)

<223> Xaa is C, S, M, D or A

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223> Xaa is F, I, V, M or R

<220>

<221>MISC_FEATURE

<222>(6)..(7)

<223> Xaa is R or K and there may be one or two

<220>

<221>MISC_FEATURE

<222>(9)..(9)

<223> Xaa is C, S, M, D or A

<220>

<221>MISC_FEATURE

<222>(10)..(10)

<223> Xaa is F, I, V, M or R

<220>

<221>MISC_FEATURE

<222>(13)..(13)

<223> Xaa is F, I, V, M or R

<220>

<221>MISC_FEATURE

<222>(14)..(14)

<223> Xaa is C, S, M, D or A

<220>

<221>MISC_FEATURE

<222>(15)..(15)

<223> Xaa is F, I, V, M or R

<220>

<221>MISC_FEATURE

<222>(16)..(17)

<223> Xaa is R or K and there may be one or two

<220>

<221>MISC_FEATURE

<222>(18)..(18)

<223> Xaa is C, S, M, D or A

<220>

<221>MISC_FEATURE

<222>(19)..(20)

<223> Xaa is R or K and there may be one or two

<400>25

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa Arg Gly Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa

20

<210>26

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>26

Arg Arg Met Cys Ile Lys Val Cys Val Arg Gly Val Cys Arg Arg Lys

1 5 10 15

Cys Arg Lys

<210>27

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>27

Lys Arg Ser Cys Phe Lys Val Ser Met Arg Gly Val Ser Arg Arg Arg

1 5 10 15

Cys Lys

<210>28

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>28

Lys Lys Asp Ala Ile Lys Lys Val Asp Ile Arg Gly Met Asp Met Arg

1 5 10 15

Arg Ala Arg

<210>29

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>29

Arg Lys Met Val Lys Val Asp Val Arg Gly Ile Met Ile Arg Lys Asp

1 5 10 15

Arg Arg

<210>30

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>30

Lys Gln Cys Val Lys Val Ala Met Arg Gly Met Ala Leu Arg Arg Cys

1 5 10 15

Lys

<210>31

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>31

Arg Arg Glu Ala Ile Arg Arg Val Ala Met Arg Gly Arg Asp Met Lys

1 5 10 15

Arg Met Arg Arg

20

<210>32

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> Xaa is R or K

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223> Xaa is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H)

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223> Xaa is a C, S, M, D or A

<220>

<221>MISC_FEATURE

<222>(4)..(4)

<223> Xaa is F, I, V, M or R

<220>

<221>MISC_FEATURE

<222>(5)..(6)

<223> Xaa is R or K and there may be one or two

<220>

<221>MISC_FEATURE

<222>(8)..(8)

<223> Xaa is A, I, S, M, D or R

<220>

<221>MISC_FEATURE

<222>(9)..(9)

<223> Xaa is F, I, V, M or R

<220>

<221>MISC_FEATURE

<222>(12)..(12)

<223> Xaa is F, I, V, M or R

<220>

<221>MISC_FEATURE

<222>(13)..(13)

<223> Xaa is A, I, S, M, D or R

<220>

<221>MISC_FEATURE

<222>(14)..(14)

<223> Xaa is F, I, V, M or R

<220>

<221>MISC_FEATURE

<222>(15)..(15)

<223> Xaa is R or K

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> Xaa is a C, S, M, D or A

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223> Xaa is R or K

<400>32

Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa Arg Gly Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa

<210>33

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>33

Arg Thr Cys Val Lys Arg Val Ala Met Arg Gly Ile Ile Arg Lys Arg

1 5 10 15

Cys Arg

<210>34

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>34

Lys Lys Gln Met Met Lys Arg Val Asp Val Arg Gly Ile Ser Val Lys

1 5 10 15

Arg Lys Arg

<210>35

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>35

Lys Glu Ser Ile Lys Val Ile Ile Arg Gly Met Met Val Arg Met Lys

1 5 10 15

Lys

<210>36

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>36

Arg Arg Asp Cys Arg Arg Val Met Val Arg Gly Ile Asp Ile Lys Ala

1 5 10 15

Lys

<210>37

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>37

Lys Arg Thr Ala Ile Lys Lys Val Ser Arg Arg Gly Met Ser Val Lys

1 5 10 15

Ala Arg Arg

<210>38

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>38

Arg His Cys Ile Arg Arg Val Ser Met Arg Gly Ile Ile Met Arg Arg

1 5 10 15

Cys Lys

<210>39

<211>31

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223> Xaa is a polar amino acid (C, S, T, M, N and Q)

<220>

<221>MISC_FEATURE

<222>(4)..(4)

<223> Xaa is A, L, S or K

<220>

<221>MISC_FEATURE

<222>(6)..(6)

<223> Xaa is A, L, S or K

<220>

<221>MISC_FEATURE

<222>(11)..(11)

<223> Xaa is A, L, S or K

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> Xaa is A, L, S or K

<220>

<221>MISC_FEATURE

<222>(16)..(31)

<223> Xaa is an amino acid selected from the group consisting of: g, A, V, L, I, P, F, S, T, K and H and may be present in 1 to 17

<400>39

Lys Xaa Lys Xaa Phe Xaa Lys Met Leu Met Xaa Ala Leu Lys Lys Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

20 25 30

<210>40

<211>28

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>40

Lys Cys Lys Leu Phe Lys Lys Met Leu Met Leu Ala Leu Lys Lys Val

1 5 10 15

Leu Thr Thr Gly Leu Pro Ala Leu Lys Leu Thr Lys

20 25

<210>41

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>41

Lys Sar Lys Ser Phe Leu Lys Met Leu Met Lys Ala Leu Lys Lys Val

1 5 10 15

Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser

20 25

<210>42

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>42

Lys Thr Lys Lys Phe Ala Lys Met Leu Met Met Ala Leu Lys Lys Val

1 5 10 15

Val Ser Thr Ala Lys Pro Leu Ala Ile Leu Ser

20 25

<210>43

<211>32

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>43

Lys Met Lys Ser Phe Ala Lys Met Leu Met Leu Ala Leu Lys Lys Val

1 5 10 15

Leu Lys Val Leu Thr Thr Ala Leu Thr Leu Lys Ala Gly Leu Pro Ser

20 25 30

<210>44

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>44

Lys Asn Lys Ala Phe Ala Lys Met Leu Met Lys Ala Leu Lys Lys Val

1 5 10 15

Thr Thr Ala Ala Lys Pro Leu Thr Gly

20 25

<210>45

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>45

Lys Gln Lys Leu Phe Ala Lys Met Leu Met Ser Ala Leu Lys Lys Lys

1 5 10 15

Thr Leu Val Thr Thr Pro Leu Ala Gly Lys

20 25

<210>46

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<220>

<221>MISC_FEATURE

<222>(1)..(26)

<223> Xaa is a hydrophobic amino acid at residue 4, 7, 8, 10, 11, 14, 15

<220>

<221>MISC_FEATURE

<222>(1)..(26)

<223> Xaa is a hydrophilic amino acid at residues 5, 6, 9, 12, 13

<400>46

Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile

1 5 10 15

Phe His Thr Ala Leu Lys Pro Ile Ser Ser

20 25

<210>47

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>47

Lys Trp Lys Ser Phe Leu Arg Thr Phe Lys Ser Pro Val Arg Thr Ile

1 5 10 15

Phe His Thr Ala Leu Lys Pro Ile Ser Ser

20 25

<210>48

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>48

Lys Trp Lys Ser Tyr Ala His Thr Ile Met Ser Pro Val Arg Leu Ile

1 5 10 15

Phe His Thr Ala Leu Lys Pro Ile Ser Ser

20 25

<210>49

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>49

Lys Trp Lys Arg Gly Ala His Arg Phe Met Lys Phe Leu Ser Thr Ile

1 5 10 15

Phe His Thr Ala Leu Lys Pro Ile Ser Ser

20 25

<210>50

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>50

Lys Trp Lys Lys Trp Ala His Ser Pro Arg Lys Val Leu Thr Arg Ile

1 5 10 15

Phe His Thr Ala Leu Lys Pro Ile Ser Ser

20 25

<210>51

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>51

Lys Trp Lys Ser Leu Val Met Met Phe Lys Lys Pro Ala Arg Arg Ile

1 5 10 15

Phe His Thr Ala Leu Lys Pro Ile Ser Ser

20 25

<210>52

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>52

Lys Trp Lys His Ala Leu Met Lys Ala His Met Leu Trp His Met Ile

1 5 10 15

Phe His Thr Ala Leu Lys Pro Ile Ser Ser

20 25

<210>53

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>53

Lys Trp Lys Ser Phe Leu Arg Thr Phe Lys Ser Pro Val Arg Thr Val

1 5 10 15

Phe His Thr Ala Leu Lys Pro Ile Ser Ser

20 25

<210>54

<211>26

<212>PRT

<213> Artificial sequence

<220>

<223> cationic peptide

<400>54

Lys Trp Lys Ser Tyr Ala His Thr Ile Met Ser Pro Val Arg Leu Val

1 5 10 15

Phe His Thr Ala Leu Lys Pro Ile Ser Ser

20 25

<210>55

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> PCR amplification primers

<400>55

gtccctgtat gcctctggtc 20

<210>56

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> PCR amplification primers

<400>56

gatgtcacgc acgatttcc 19

<210>57

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> CpG oligonucleotide

<400>57

tcatgacgtt cctgacgtt 19

<210>58

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> non-CpG oligonucleotide

<400>58

ttcaggactt tcctcaggtt 20

<210>59

<211>21

<212>DNA

<213> human

<400>59

tcatagcagc caccttcatt c 21

<210>60

<211>20

<212>DNA

<213> human

<400>60

tagcgcagat tcttgggttg 20

<210>61

<211>22

<212>DNA

<213> human

<400>61

tgtcctttct cagagtggtt ct 22

<210>62

<211>21

<212>DNA

<213> human

<400>62

tgcttccata gggacatcat a 21

<210>63

<211>21

<212>DNA

<213> human

<400>63

acctgaacct tccaaagatg g 21

<210>64

<211>20

<212>DNA

<213> human

<400>64

gcgcagaatg agatgagttg 20

<210>65

<211>21

<212>DNA

<213> human

<400>65

gtgcagaggg ttgtggagaa g 21

<210>66

<211>21

<212>DNA

<213> human

<400>66

ttctcccgtg caatatctag g 21

Claims (22)

1. A method of stimulating innate immunity in a subject comprising: administering to the subject a therapeutically effective amount of the polypeptide of SEQ id no: 1-4, 11, 18, 25, 32, 39, 46, 53, or 54, thereby stimulating an immune response.

2. The method of claim 1, wherein the innate immunity is evidenced by host immune cell activation, proliferation, differentiation, or MAP kinase pathway activation.

3. The method of claim 2, wherein the MAP kinase is MEK and/or ERK.

4. The method of claim 1, further comprising administering GM-CSF to the subject.

5. A method of stimulating innate immunity in a subject having or at risk of having an infection, comprising: administering to the subject an antibiotic in combination with the polypeptide of SEQ ID NO: 1-4, 7, 11, 18, 25, 32, 39, 46, 53, or 54.

6. The method of claim 1, wherein the peptide comprises at least one amino acid that is the D-enantiomer.

7. The method of claim 1, wherein the peptide is cyclic.

8. The method of claim 1, wherein the peptide sequence is inverted.

9. The method of claim 1, further comprising administering an antibiotic to the subject.

10. The method of claim 9, wherein the antibiotic is selected from the group consisting of aminoglycosides, penicillins, cephalosporins, cerbacephems, cephamycins, chloramphenicol, glycylcyclines, lipoamides, aminocyclitols, cationic antimicrobial peptides, lipopeptides, poxyxins, streptogramins, oxazolidinones, lincosamides, fluoroquinolones, carbapenems, tetracyclines, macrolides, β -lactam carbapenems, monobactams, quinolones, tetracyclines, or glycopeptides.

11. The method of claim 5, wherein the peptide has anti-inflammatory activity.

12. The method of claim 5, wherein the peptide has anti-sepsis activity.

13. The method of claim 5, wherein the peptide contains at least one amino acid that is the D-enantiomer.

14. The method of claim 5, wherein the peptide is cyclic.

15. The method of claim 5, wherein the peptide sequence is inverted.

16. The method of claim 5, wherein the antibiotic is selected from the group consisting of aminoglycosides, penicillins, cephalosporins, cerbacephems, cephamycins, chloramphenicol, glycylcyclines, lipoamides, aminocyclines, cationic antimicrobial peptides, lipopeptides, poxyxins, streptogramins, oxazolidinones, lincosamines, fluoroquinolones, carbapenems, tetracyclines, macrolides, β -lactam carbapenems, monobactams, quinolones, tetracyclines, or glycopeptides.

17. A method of stimulating innate immunity in a subject having or at risk of having an infection, comprising: administering to the subject GM-CSF in combination with the polypeptide of SEQ ID NO: 1-4, 7, 11, 18, 25, 32, 39, 46, 53, or 54.

18. The method of claim 17, wherein the peptide has anti-inflammatory activity.

19. The method of claim 17, wherein the peptide has anti-sepsis activity.

20. The method of claim 17, wherein the peptide contains at least one amino acid that is a D-enantiomer.

21. The method of claim 17, wherein the peptide is cyclic.

22. The method of claim 17, wherein the peptide sequence is inverted.