KR20070033314A - Methods of Stimulating Innate Immunity Using Cationic Peptides - Google Patents

Abstract

Methods of identifying polynucleotides or patterns of polynucleotides that are regulated by one or more septic or inflammatory inducers and are inhibited by peptides are described. Methods for identifying polynucleotide expression patterns for the inhibition of inflammatory or septic responses are described. The method comprises contacting a cell with LPS, LTA, CpG DNA and / or intact microorganisms or microbial components in the presence or absence of cationic peptides; Detecting a polynucleotide expression pattern for cells in the presence and absence of a peptide, the pattern in the presence of the peptide indicating inhibition of an inflammatory or septic response. Also included are compounds and agents identified by the methods of the present invention. In another aspect, the invention provides methods and compounds for enhancing innate immunity of a subject.

Description

Method of stimulating innate immunity using cationic peptide {METHODS OF STIMULATING INNATE IMMUNITY USING CATIONIC PEPTIDES}

Related Applications Data

This application is incorporated by reference in US Patent Application No. 10 / 308,905, filed December 2, 2002, under 35 USC 120, and US Patent Application No. 60 / 336,632, filed December 3, 2001. It claims priority under 35 USC 119 (e).

The present invention relates generally to peptides, in particular to peptides that are effective as therapeutic agents and that regulate drug development and innate immunity or anti-inflammatory activity associated with pathologies resulting from microbial infection.

Infectious diseases are the leading cause of death worldwide. According to a 1999 World Health Organization survey, 13 million people die from infectious diseases each year. In North America, infectious disease is the third leading cause of death, accounting for 20% of deaths each year and an increase of 50% since 1980. The success of many medical and surgical treatments depends on their ability to control infectious diseases. Antibiotic development and use is one of the greatest achievements of modern medicine. Without antibiotics, doctors would not be able to perform most medical procedures such as surgery, chemotherapy or catheterization.

Current antibiotic sales are $ 26 billion US worldwide. However, the abuse and sometimes improper use of antibiotics have led to the emergence of new antibiotic resistant strains of bacteria. Antibiotic resistance has become part of the medical field. Vancomycin-resistant Enterococcus Cocos (Enterococcus), VRE and Cocos methicillin-resistant Staphylococcus aureus (Staphylococcus aureus) strains and bacteria such as MRSA can not be treated with antibiotics, patients infected with such bacteria are often killed. Antibiotic discovery has been found to be one of the most difficult new drug development areas, and many large pharmaceutical companies have curtailed or completely stopped antibiotic development programs. However, with the rapid increase in antibiotic resistance, including the emergence of incurable infections, it is evident that there is a need for a new class of antimicrobial therapies in the medical field, and agents that affect innate immunity will be agents of this class.

The innate immune system is very effective and leads to a general defense system. Components of innate immunity are always present at low concentrations, but are activated very quickly upon stimulation. Stimulation may include interaction of pattern recognition receptors on the surface of body cells with bacterial signaling molecules or other disease mechanisms. Every day people are exposed to tens of thousands of effective pathogenic microorganisms through the food they eat and water, the air they breathe, and the surfaces they contact, pets and people. The innate immune system prevents pathogens from causing disease. The innate immune system is different from the acquired immune system (antibody and antigen specific B and T lymphocytes) because it is always present, has an immediate effect, and is relatively nonspecific for any given pathogen. The acquired immune system must amplify specific recognition components, and therefore the reaction takes days to weeks. Even if pre-stimulation of immunization with vaccination takes more than three days to respond to the pathogen, innate immunity is available immediately or quickly (several hours). Innate immunity includes various effector functions, including phagocytic cells, complement, and the like, but is generally not fully known. In general, many innate immune responses are "triggered" by the combination of pattern recognition receptors and microbial signaling molecules, called Toll-like receptors, on the surface of a host cell. Many of these effector functions are grouped together as inflammatory responses. However, too much inflammatory response can be harmful to the human body and, in extreme cases, can lead to sepsis and potentially death.

The release of structural components from the infectious agent during the infection causes an inflammatory response that, if not suppressed, can lead to sepsis, a potentially fatal symptom. About 780,000 patients suffer from sepsis every year in North America. Sepsis can occur as a result of infections obtained in some areas, such as pneumonia. Or sepsis may be a complication after trauma, cancer or major surgery treatment. Severe sepsis occurs when the body is overwhelmed by an inflammatory response and the body's organs stop working. Every year, up to 120,000 people die of sepsis in the United States. Sepsis can include bloodborne pathogenic microorganisms or toxins (eg, septicemia), which are the leading cause of human death. Gram-negative bacteria are the most common organisms associated with such diseases. However, there is also an increasing tendency for Gram-positive bacteria to act as infectious agents. Both Gram-negative and Gram-positive bacteria and their components can cause sepsis.

The presence of microbial components leads to the release of pro-inflammatory cytokines, of which tumor necrosis factor-α (TNF-α is very important). TNF-α and other proinflammatory cytokines can trigger the release of other proinflammatory mediators and induce an inflammatory cascade. Gram-negative sepsis is usually caused by the release of lipopolysaccharides (LPS, also called endotoxins), the outer membrane component of bacteria. Endotoxins in the blood, called endotoxins, arise primarily from bacterial infections and can be released during antibiotic treatment. Gram positive sepsis lipoic table Kosan (LTA), peptidoglycan (PG), the rhamnose produced by Streptomyces Cocos (Streptococcus) - bacterial cell walls, such as a capsule polysaccharide produced by the glucose polymer or Staphylococcus Cocos (Staphylococcus) It may be caused by the release of the component. Unlike mammalian DNA, bacteria or other non-mammalian DNA containing unmethylated cytosine-guanosine dimers (CpG DNA) have been shown to induce septic symptoms, including the production of TNF-α. Mammalian DNA often contains a much lower frequency of methylated forms of CpG dinucleotides. In addition to spontaneous release in the course of bacterial infection, antibiotic treatment can also lead to the release of bacterial cell wall components, LPS and LTA, and possibly bacterial DNA. This can make recovery from infection difficult or even cause sepsis.

Although cationic peptides are increasingly recognized as a form of defense against infection, the main effects recognized in the scientific literature are antimicrobial effects (Hancock, REW, and R. Lehrer. 1998. Cationic peptides: a new source of antibiotics. in Biotechnology 16: 82-88). Cationic peptides with antimicrobial activity have been isolated from various organisms. In nature, such peptides provide a defense mechanism against microorganisms such as bacteria and yeast. In general, these cationic peptides are thought to interact with the cytoplasmic membrane and in most cases form channels or lesions to exhibit antimicrobial activity against bacteria. In Gram-negative bacteria, these cationic peptides interact with the LPS to penetrate the outer membrane, thereby facilitating uptake through the outer membrane and making the cytoplasmic membrane accessible. Examples of cationic antimicrobial peptides include indolisidine, defensin, secropin, and margeine.

Recently, there is increasing recognition that such peptides are agonists in other aspects of innate immunity (Hancock, REW and G. Diamond. 2000. The role of cationic peptides in innate host defenses. Trends in Microbiology 8: 402-410; Hancock, REW 2001. Cationic peptides: effectors in innate immunity and novel antimicrobials.Lancet Infectious Diseases 1: 156-164).

Some cationic peptides have a binding affinity for the product of bacteria such as LPS and LTA. Such cationic peptides can inhibit cytokine production in response to LPS and can prevent fatal shock to varying degrees. However, it is not known whether such effects are due to the binding of peptides to LPS and LTA or to the direct interaction of peptides with host cells. In response to stimulation by microorganisms or microbial signaling molecules such as LPS, cationic peptides are induced by regulatory pathways similar to those used by mammalian immune systems (including toll-like receptors and transcription factor NFκB). Thus, cationic peptides appear to play an important role in innate immunity. Mutations that affect the induction of antibacterial peptides can reduce viability in response to bacterial stimulation. In addition, mutations in the toll pathway of drosophila leading to reduced antifungal peptide expression increase the susceptibility to lethal fungal infections. In humans, patients with specific granule deficiency syndrome, which are completely deficient in α-defensin, often suffer from severe bacterial infections. Other evidence includes the induction of some peptides by infectious agents, and very high concentrations recorded at the site of inflammation. In addition, cationic peptides can regulate cell migration to promote the ability of white blood cells to fight bacterial infection. For example, two human α-defensin peptides, HNP-1 and HNP-2, are known to have direct chemotactic activity against rat and human T cells and monocytes, and human β-defensin interacts with CCR6. It appears to act as a chemoattractant for immature dendritic cells and memory T cells. Likewise, the pig cationic peptide PR-39 was found to be chemotactic for neutrophils. However, it is not clear whether peptides of different structure and composition share these properties.

Only one cathelicidine known as derived from human is produced by myeloid precursors, testes, human keratinocytes and airway epithelial cells in inflammatory diseases. A typical feature of cathelicidine peptides is the high level of sequence identity in the N-terminal prepro region called the catelin domain. Cathelicidine peptides are stored as inactive propeptide precursors that are processed into active peptides upon stimulation.

Summary of the Invention

The present invention relates to subject's sepsis based on polynucleotide expression patterns regulated by endotoxin lipopolysaccharides, lipoteichoic acid, CpG DNA or other cellular components (eg, microorganisms or other cellular components) and influenced by cationic peptides. And / or invented discoveries that can screen for novel compounds that block or reduce inflammation. In addition, based on the use of cationic peptides as a tool, one can identify selective enhancers of innate immunity that can block / mitigate inflammatory and / or septic responses without triggering sepsis responses.

Thus, in one embodiment there is provided a method of identifying polynucleotides or patterns of polynucleotides that are regulated by one or more sepsis or inflammation inducing agents and inhibited by cationic peptides. The methods of the present invention comprise contacting the polynucleotide (s) with at least one sepsis or inflammation inducing agent and contacting the polynucleotide (s) with a cationic peptide at the same time or immediately after. Expression differences are detected in the presence and absence of cationic peptides, and changes in expression of upregulation or downregulation indicate polynucleotides or polynucleotide patterns that are modulated by sepsis or inflammation inducers and are inhibited by cationic peptides. In another embodiment, the present invention provides polynucleotide (s) identified by the methods disclosed above. Examples of sepsis or inflammatory modulators include LPS, LTA or CpG DNA or microbial components (or any combination thereof) or related agents.

In another embodiment, the present invention provides a method of identifying an agent that blocks sepsis or inflammation comprising combining a polynucleotide identified by the method disclosed above with an agent, wherein expression of the polynucleotide in the presence of the agent is Compared with and controlled in the absence of the agent, expression control affects the inflammatory or septic response.

In another embodiment, the present invention provides a method for the detection of a polynucleotide expression pattern for a cell in the presence or absence of a cationic peptide and 2) contacting the cell with LPS, LTA and / or CpG DNA. Methods of identifying polynucleotide expression patterns for the inhibition of inflammatory or septic responses are provided. Patterns obtained in the presence of peptides indicate inhibition of inflammatory or septic responses. In another aspect, a pattern obtained in the presence of a peptide is compared to the pattern of a test compound to identify compounds that give a similar pattern. In another aspect, the present invention provides a compound identified by the method described above.

In another embodiment, the present invention provides a method of identifying an agent that enhances innate immunity by contacting a polynucleotide (s) encoding a polypeptide related to innate immunity with the agent, wherein the polynucleotide expression in the presence of the agent Is regulated compared to the expression of polynucleotides in the absence of the agent, and innate immunity is enhanced by expression regulation. Preferably, the formulation does not stimulate the sepsis response of the subject. In one aspect, the agent increases the expression of anti-inflammatory polynucleotides. Non-limiting examples of anti-inflammatory polynucleotides include IL-1R antagonist homolog 1 (AI167887), IL-10R beta (AA486393), IL-10R alpha (U00672) TNF receptor member 1B (AA150416), TNF receptor member 5 ( H98636), TNF receptor member 11b (AA 194983), IK cytokine downregulator of HLA II (R39227), TGF-B-induced early growth response 2 (AI473938), CD2 (AA927710), IL-19 (NM_013371) or IL -10 (M57627) and the like. In one aspect, the agent reduces expression of polynucleotides encoding proteasome subunits involved in NF-κB activation, such as proteasome subunit 26S (NM_013371). In one aspect, the agent may act as an antagonist of protein kinases. In one aspect, the agent is a peptide selected from SEQ ID NOs: 4-54.

In another embodiment, the present invention provides a method of identifying polynucleotide expression patterns to identify compounds that selectively enhance innate immunity. The present invention provides a method for detecting polynucleotide expression patterns for cells in contact with and without cationic peptides, wherein the pattern in the presence of peptides indicates stimulation of innate immunity; Detecting a polynucleotide expression pattern for cells contacted in the presence of a test compound, wherein the pattern in the presence of a test compound similar to the pattern observed in the presence of a cationic peptide indicates a compound that enhances innate immunity. Preferably, the compound does not stimulate the septic response of the subject.

In another embodiment, the invention provides a method of identifying a polynucleotide expression pattern in a nucleic acid sample that is exemplified by increased polynucleotide expression of at least two polynucleotides in Tables 50, 51, and / or 52 compared to an uninfected subject. A method of estimating an infection state of a mammalian subject from a subject's nucleic acid sample is provided. Also included are polynucleotide expression patterns obtained by any of the methods described above.

In another aspect, provided is a cationic peptide that is an antagonist of CXCR-4. According to another aspect, methods are provided for contacting T cells with SDF-1 in the presence or absence of a test peptide to identify cationic peptides that are antagonists of CXCR-4 and to determine chemotaxis. Reduced chemotaxis in the presence of test peptides indicates that the peptide is an antagonist of CXCR-4. Cationic peptides also act to reduce the expression of the SDF-1 receptor polynucleotide (NM_013371).

In all of the methods disclosed above, the compounds or agents of the invention include, but are not limited to, peptides, cationic peptides, peptide analogs, chemical compounds, polypeptides, nucleic acid molecules, and the like.

In another embodiment, the present invention provides isolated cationic peptides. Isolated cationic peptides of the invention are represented by one of the following general formulas using the one letter amino acid code:

X ₁ X ₂ X ₃ IX ₄ PX ₄ IPX ₅ X ₂ X ₁ (SEQ ID NO: 4), where X ₁ is one or two of R, L or K, and X ₂ is one of C, S or A and , X ₃ is one of R or P, X ₄ is one of A or V, and X ₅ is one of V or W;

X ₁ LX ₂ X ₃ KX ₄ X ₂ X ₅ X ₃ PX ₃ X ₁ (SEQ ID NO: 11), where X ₁ is one or two of D, E, S, T or N, and X ₂ is P, G or One or two of D, X ₃ is one of G, A, V, L, I or Y, X ₄ is one of R, K or H, and X ₅ is S, T, C, M or One of R;

X ₁ X ₂ X ₃ X ₄ WX ₄ WX ₄ X ₅ K (SEQ ID NO: 18), wherein X ₁ is 1-4 selected from A, P or R, and X ₂ is 1 or 2 aromatic amino acids (F, Y And W), X ₃ is one of P or K, X ₄ is 0, 1 or 2 selected from A, P, Y or W, and X ₅ is 1 to 3 selected from R or P;

X ₁ X ₂ X ₃ X ₄ X ₁ VX ₃ X ₄ RGX ₄ X ₃ X ₄ X ₁ X ₃ X ₁ (SEQ ID NO: 25), where X ₁ is one or two of R or K, and X ₂ is polar or Charged amino acids (S, T, M, N, Q, D, E, K, R and H), X ₃ is C, S, M, D or A and X ₄ is F, I, V, M or R;

X ₁ X ₂ X ₃ X ₄ X ₁ VX ₅ X ₄ RGX ₄ X ₅ X ₄ X ₁ X ₃ X ₁ (SEQ ID NO: 32), where X ₁ is ₁ of R or K or 2, X ₂ is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X ₃ is one of C, S, M, D or A, X ₄ is one of F, I, V, M or R and X ₅ is one of A, I, S, M, D or R;

KX ₁ KX ₂ FX ₂ KMLMX ₂ ALKKX ₃ (SEQ ID NO: 39), wherein X ₁ is a polar amino acid (C, S, T, M, N and Q) and X ₂ is one of A, L, S or K X ₃ is 1-17 amino acids selected from G, A, V, L, I, P, F, S, T, K and H;

KWKX ₂ X ₁ X ₁ X ₂ X ₂ X ₁ X ₂ X ₂ X ₁ X ₁ X ₂ X ₂ IFHTALKPISS (SEQ ID NO: 46), where X ₁ is a hydrophobic amino acid and X ₂ is a hydrophilic amino acid.

Also according to a further aspect the present invention provides isolated cationic peptides KWKSFLRTFKSPVRTVFHTALKPISS (SEQ ID NO: 53) and KWKSYAHTIMSPVRLVFHTALKPISS (SEQ ID NO: 54).

Also provided are nucleic acid sequences encoding cationic peptides of the invention, vectors comprising such polynucleotides, and host cells comprising the vectors.

In another embodiment, the invention comprises administering to a subject a peptide of the invention, eg, a peptide of SEQ ID NOs: 1-4, 11, 18, 25, 32, 39, 46, 53 or 54 A method of stimulating or enhancing innate immunity of a subject is provided. As shown in the examples herein, innate immunity can be demonstrated, for example, by monocyte activation, proliferation, differentiation or MAP kinase pathway activation. In one aspect, the method further comprises administering to the subject a serum factor such as GM-CSF. The subject is preferably any mammal, more particularly a human subject.

In another embodiment, the present invention provides a method of stimulating innate immunity of a subject with or at risk of an infection comprising administering to the subject a suboptimal concentration of antibiotic in combination with a peptide of the invention. to provide. In one aspect, the peptide is SEQ ID NO: 1 or SEQ ID NO: 7.

1 demonstrates the synergy of SEQ ID NO: 7 with cefepime in treating S. aureus infection. CD-1 mice (8 / group) were IP injected with 1 × 10 ⁷ S. aureus in 5% porcine mucin. Test compounds (50 μg-2.5 mg / kg) were separately IP injected 6 hours after S. aureus. This time, cefepime was also administered at a dose of 0.1 mg / kg. Mice were euthanized after 24 hours, blood removed and plated for survival counts. Mean ± standard error. This experiment was repeated twice.

2 shows that exposure to SEQ ID NO 1 induces phosphorylation of ERK1 / 2 and p38. Eluates in human peripheral blood derived monocytes were exposed to 50 μg / ml of SEQ ID NO 1 for 15 minutes. A) Activation of ERK1 / 2 and p38 was detected using antibodies specific for phosphorylated forms of ERK and p38. All donors tested showed increased phosphorylation of ERK1 / 2 and p38 in response to SEQ ID NO 1 treatment. Representative donor of one of eight. The relative amounts of phosphorylation of ERK (B) and p38 (C) were determined by dividing the intensity of the phosphorylation band by the intensity of the corresponding control band as described in Materials and Methods.

3 shows that LL-37 induced phosphorylation of ERK1 / 2 does not occur in the absence of serum and that the size of phosphorylation depends on the type of serum present. Human blood derived monocytes were treated with 50 μg / ml of LL-37 for 15 minutes. The eluate was run on a 12% acrylamide gel, then transferred to the nitrocellulose membrane and probed with an antibody specific for the phosphorylated (active) form of the kinase. To normalize for protein loading, the blots were probed again with β-actin. Quantification was done with ImageJ software. 3 Implantation demonstrates that LL-37 cannot induce MAPK activation in human monocytes under serum free conditions. Cells were exposed to 50 mg / ml of LL-37 (+) or endotoxin water (-) as vehicle control for 15 minutes. (A) Phosphorylated ERK1 / 2 was detectable after exposure to LL-37 in medium containing 10% fetal bovine serum, but phosphorylation of ERK1 / 2 was not detected in the absence of serum ( n = 3). (B) Elk-1, ERK1 / 2 downstream transcription factors were activated upon exposure to 50 μg / ml of LL-37 in medium containing 10% fetal bovine serum, but not in the absence of serum (n = 2 ).

4 shows that LL-37 induced activation of ERK1 / 2 occurs at lower concentrations and is amplified in the presence of specific cytokines. When fresh isolated monocytes were stimulated in a medium containing both GM-CSF (lOOng / ml) and IL-4 (100 ng / ml), the LL-37 induced phosphorylation of ERK1 / 2 was as much as 5 μg / ml. It is evident even at low concentrations. The synergistic activation of ERK1 / 2 appears to be mainly due to GM-CSF.

5 shows that peptides affect both transcription of various cytokine genes and release of IL-8 in the 16HBE4o-human bronchial epithelial cell line. Cells were grown in saturation on the semipermeable membrane and stimulated at the attachment surface using 50 μg / ml SEQ ID NO 1 for 4 hours. A) The SEQ ID NO 1 treated cells generated significantly more IL-8 than the control as detected by ELISA (but not at the basal surface) in the supernatants collected at the attachment surface. The mean ± standard error of the three independent tests is shown and an asterisk indicates p = 0.002. B) RNA was collected in the above experiments and RT-PCR was performed. Many cytokine genes known to be regulated by ERK1 / 2 or p38 were upregulated upon stimulation with peptides. The mean of two independent experiments is shown.

The present invention provides novel cationic peptides characterized by a group of general formulas that possess the ability to modulate (eg, up and / or downregulate) polynucleotide expression to modulate sepsis and inflammatory responses and / or innate immunity. to provide.

As used herein, "innate immunity" refers to the natural force that an organism defends itself against invasion by a pathogen. Pathogens or microorganisms used herein include, but are not limited to, bacteria, fungi, parasites and viruses. Innate immunity is in contrast to acquired / acquired immunity, in which an organism forms defense mechanisms based on antibodies and / or immune lymphocytes that are characterized by distinction of specificity, amplification, and autologous versus non-self. Innate immunity provides extensive and nonspecific immunity and does not have immune memory from prior exposure. Characteristics of innate immunity are the effects on various effective pathogens, independence from previous exposure to pathogens, and immediate effects (as opposed to specific immune responses that take days to weeks to induce). Innate immunity also includes immune responses that affect other diseases such as cancer, inflammatory diseases, multiple sclerosis, various viral infections, and the like.

As used herein, the term "cationic peptide" refers to an amino acid sequence derived from amino acids about 5 to about 50 in length. In one aspect, the cationic peptide of the present invention is about 10 to about 35 amino acids in length. When a peptide comprises a sufficiently positively charged amino acid having a pKa greater than 9.0, that peptide is referred to as a "cationic" peptide. Typically two or more of the amino acid residues of the cationic peptide will be positively charged, such as lysine and arginine. "Positively charged" means the side chain of an amino acid residue having a net positive charge at pH 7.0. Examples of natural cationic antimicrobial peptides that can be produced recombinantly in accordance with the present invention include defensins, cathelicidins, marghenins, melittin and cecropins, bactenesin, indolisidine, polyfemucin, tachyflesin And analogs thereof. Various organisms can make cationic peptides into molecules that are used as part of nonspecific defense mechanisms against microorganisms. These peptides, when isolated, are toxic to various microorganisms, including bacteria, fungi and some enveloped viruses. Although cationic peptides work against several pathogens, there are obvious exceptions and varying degrees of toxicity. However, the present application has revealed additional cationic peptides that are not toxic to microorganisms but have the ability to protect against infection through stimulation of innate immunity, and the present invention is not limited to cationic peptides having antimicrobial activity. . Indeed, many of the peptides useful in the present invention do not have antimicrobial activity.

Cationic peptides known in the art include, for example, human cathelicidine LL-37, bovine neutrophil peptide indolidine and bovine variant of Bac2A.

LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 1)

Indolidine ILPWKWPWWPWRR-NH ₂ (SEQ ID NO: 2)

Bac2A RLARIVVIRVAR-NH ₂ (SEQ ID NO: 3)

In innate immunity, the immune response is not antigen dependent. The innate immune process may involve the production of secretory molecules and cellular components as described above. In innate immunity, pathogens are recognized by receptors encoded in the germline. Toll-like receptors have a wide range of specificities and can recognize several pathogens. If cationic peptides are present in the immune response, these peptides aid the host response to the pathogen. Changes in the immune response lead to the release of chemokines that promote the recruitment of immune cells to the site of infection.

Chemokines or chemoattractants cytokines are a subgroup of immune factors that mediate chemotaxis and other proinflammatory phenomena (see Schall, 1991, Cytokine 3: 165-183). Chemokines are small molecules of about 70 to 80 residues in length, and can generally be classified into two subgroups, α and β. α has two N-terminal cysteines (CxC) separated by a single amino acid and β has two adjacent cysteines (CC) at the N terminus. RANTES, MIP-1α and MIP-1β are members of the β subgroup (Horuk, R., 1994, Trends Pharmacol Sci, 15: 159-165; Murphy, PM, 1994, Annu. Rev. Immunol., 12: 593 Reviewed at -633). The amino termini of the β chemokines RANTES, MCP-1, and MCP-3 have been implicated in the mediation of these chemokine-induced cell migration and inflammation. Deletion of 8 amino terminus residues of MCP-1, 9 amino terminus residues of MCP-3 and 8 amino terminus residues of RANTES and addition of methionine to the amino terminus of RANTES resulted in enzyme release stimulated by its natural counterparts. Such an association suggests that antagonism of chemotaxis and / or calcium migration is suggested (Gong et al., 1996 J. Biol. Chem. 271: 10521-10527; Proudfoot et al., 1996 J. Biol) Chem. 271: 2599-2603. In addition, α chemokine-like chemotactic activity is introduced into MCP-1 via double mutation of Tyr28 and Arg30 to leucine and valine, respectively, suggesting that the internal regions of the protein play an important role in regulating chemotactic activity. (Beall et al., 1992, J. Biol. Chem. 267: 3455-3459).

The monomeric form of all chemokines is characterized by a significant share of significant structural homology, but the quaternary structures of the α and β groups are different. The monomer structures of β and α chemokines are very similar, but the dimeric structures of the two groups are completely different. Lymphotatin, an additional chemokine with only one N-terminal cysteine, has been identified and may represent an additional subgroup (γ) of chemokines (Yoshida et al., 1995, FEBS Lett. 360: 155-159; and Kelner et al., 1994, Science 266: 1395-1399).

Receptors for chemokines belong to a large family of G-protein coupled seven transmembrane domain receptors (GCRs) (Horuk, R., 1994, Trends Pharmacol. Sci. 15: 159-165; and Murphy, PM, 1994, Annu Rev. Immunol. 12: 593-633, reviewed literature). Competitive binding and cross-sensitization studies have shown that chemokine receptors exhibit significant indifference in ligand binding. Examples demonstrating indifference between β chemokine receptors include CC CKR-1 (Neote et al., 1993, Cell 72: 415-425), RANTES, MIP-1α, and MCP-1 that bind to RANTES and MIP-1α. CC CKR-4 (Power et al., 1995, J. Biol. Chem. 270: 19495-19500) that binds to, and CC CKR-5 (Alkhatib et al, which binds to RANTES, MIP-1α, and MIP-1β , 1996, Science, and Dragic et al., 1996, Nature 381: 667-674). Erythrocytes possess receptors (known as Duffy antigens) that bind α and β chemokines (Horuk et al., 1994, J. Biol. Chem. 269: 17730-17733; Neote et al., 1994, Blood 84: 44 -52 and Neote et al., 1993, J. Biol. Chem. 268: 12247-12249. Thus, sequence and structural homology events between chemokines and their receptors allow for some overlap in receptor-ligand interactions.

In one aspect, the invention provides the use of a compound, including a peptide of the invention, for use in reducing sepsis and inflammatory responses by acting directly on a host cell. According to this aspect, a method of identifying polynucleotide (s) that is regulated by one or more sepsis or inflammation inducing agents is provided, wherein the regulation is altered by cationic peptides. Non-limiting examples of such sepsis or inflammation inducing agents include endotoxin lipopolysaccharide (LPS), lipoteichoic acid (LTA) and / or CpG DNA or intact bacteria or other bacterial components. Identification is carried out by contacting the polynucleotide (s) with a sepsis or inflammation inducing agent and further contact with a cationic peptide at the same time or immediately after. Polynucleotide expression in the presence and absence of cationic peptide is observed, and expression changes are indicative of a pattern of polynucleotides or polynucleotides that are regulated by sepsis or inflammation inducers and inhibited by cationic peptides. In another aspect, the invention provides a polynucleotide identified by the method.

Once identified, such polynucleotides can be used in methods of screening compounds that can affect the expression of polynucleotides and block sepsis or inflammation. The effect on expression may be up-regulation or down-regulation of expression. The present invention also provides a method for identifying an enhancer of innate immunity by identifying a compound that can block or mitigate an inflammatory or sepsis response without triggering a sepsis response. The present invention also provides a compound used or identified in the method.

Candidate compounds are obtained from various sources, including libraries of synthetic or natural compounds. For example, a number of means are available for the random and directed synthesis of various organic compounds and biomolecules, including the expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds are utilized or readily produced in the form of bacterial, fungal, plant and animal extracts. In addition, libraries and compounds produced naturally or synthetically can be readily modified using conventional chemical, physical and biochemical methods and can be used to generate combinatorial libraries. Structural analogs can be obtained by subjecting known pharmacological agents to designated or random chemical modifications such as acylation, alkylation, esterification, amidation, and the like. Candidate agents are found in biomolecules including, by way of non-limiting example, peptides, peptide analogs, sugars, fatty acids, steroids, purines, pyrimidines, polypeptides, polynucleotides, chemical compounds, derivatives, structural analogs or combinations thereof.

Incubation of the screening assay components includes conditions that enable contact of the test compound with the polynucleotide of interest. Contacting includes contacting in solution, solid phase, or cells. The test compound may optionally be a combinatorial library for screening multiple compounds. Compounds identified in the methods of the present invention can be further assessed, detected, cloned, sequenced, etc., after binding in solution or to a solid support by any method generally applicable to the detection of compounds.

In general, the methods of the present invention utilize cationic peptides to detect and locate polynucleotides essential for sepsis or inflammatory processes. Once identified, expression can be observed in the presence and absence of cationic peptides to obtain polynucleotide expression patterns. Patterns obtained in the presence of cationic peptides are useful for identifying additional compounds that can inhibit the expression of polynucleotides to block sepsis or inflammation. It is known to those skilled in the art that nonpeptidic chemicals and peptide analogs can mimic the ability of peptides to bind to receptors and enzyme binding sites and thus can be used to block or stimulate biological responses. If the corresponding additional compound provides a polynucleotide expression pattern similar to the expression pattern in the presence of a cationic peptide, the compound is also useful for the control of sepsis or an innate immune response. In this way, the cationic peptides of the invention, which are known inhibitors of sepsis and inflammation and enhancers of innate immunity, are useful as tools to identify additional compounds that inhibit sepsis and inflammation and increase innate immunity.

As identified in the examples below, the peptides of the present invention have a wide range of ability to reduce the expression of polynucleotides regulated by LPS. High blood levels of endotoxins cause many of the symptoms found in severe infections or inflammatory processes, such as fever and increased white blood cell counts. Endotoxins are cell wall components of Gram-negative bacteria and are effective triggers for the pathophysiology of sepsis. The underlying mechanisms of inflammation and sepsis are related. In Example 1, the effect of cationic peptides on the transcriptional response of epithelial cells was determined using polynucleotide arrays. Specifically, effects on at least 14,000 other specific polynucleotide probes induced by LPS have been observed. The table shows the changes observed in cells treated with peptides compared to control cells. From the resulting data, it was confirmed that the peptide has the ability to reduce polynucleotide expression induced by LPS.

Similarly, Example 2 shows that the peptides of the invention can neutralize the stimulation of immune cells by gram positive and gram negative bacterial products. In addition, as shown in Table 24, TLR1 (AI339155), TLR2 (T57791), TLR5 (N41021), TNF receptor-related factor 2 (T55353), TNF receptor-related factor 3 (AA504259), TNF receptor phase and member 12 (W71984), some proinflammatory polys such as TNF receptor phase and member 17 (AA987627), small inducible cytokine subfamily B member 6 (AI889554), IL-12R beta 2 (AA977194), IL-18 receptor 1 (AA482489) Nucleotides are downregulated by cationic peptides, IL-1R antagonist homolog 1 (AI167887), IL-10R beta (AA486393), TNF receptor member 1B (AA150416), TNF receptor member 5 (H98636) as shown in Table 25. ), Anti-inflammatory polynucleotides such as TNF receptor member 11b (AA194983), IK cytokine downregulator of HLA II (R39227), TGF-B inducible early growth response factor 2 (AI473938), or CD2 (AA927710) are cationic Upregulated by peptide. The relevance and applicability of these results is confirmed by in vivo application to mice.

In another aspect, the invention provides a method of identifying an agent that enhances innate immunity. In this method, the polynucleotide (s) encoding a polypeptide associated with innate immunity is contacted with the agent. Expression of the polynucleotides in the presence and absence of the agent is determined. Expression is compared and specific expression control indicates increased innate immunity. In another aspect, the agent does not stimulate a septic response, as confirmed by the lack of upregulation of proinflammatory cytokine TNF-α. In another aspect, the agent reduces or blocks an inflammatory or septic response. In another aspect, the agent reduces expression of interleukin and / or TNF-α, including but not limited to IL-1β, IL-6, IL-12 p40, IL-12 p70, and IL-8.

In another aspect, the invention provides methods for direct polynucleotide regulation by cationic peptides and the use of compounds including cationic peptides to stimulate elements of innate immunity. In this aspect, the present invention provides a method of identifying polynucleotide expression patterns for identifying compounds that enhance innate immunity. In the methods of the invention, polynucleotide expression patterns for cells contacted in the presence and absence of cationic peptides can be initially detected. Patterns resulting from polynucleotide expression in the presence of peptides indicate stimulation of innate immunity. The polynucleotide expression pattern is detected in the presence of a test compound, and the pattern in the presence of a test compound similar to the pattern observed in the presence of a cationic peptide indicates that the compound is a compound that enhances innate immunity. In another aspect, the present invention provides a compound identified in the above method. In another aspect, the compounds of the invention stimulate chemokine or chemokine receptor expression. Chemokine or chemokine receptors include CXCR4, CXCR1, CXCR2, CCR2, CCR4, CCR5, CCR6, MIP-1 alpha, MDC, MIP-3 alpha, MCP-1, MCP-2, MCP-3, MCP-4, MCP- 5, and RANTES, but is not limited thereto. In another embodiment, the compound is a peptide, peptide analog, chemical compound or nucleic acid molecule.

In another aspect, the polynucleotide expression pattern includes expression of proinflammatory polynucleotides. Non-limiting examples of such proinflammatory polynucleotides include ring finger protein 10 (D87451), serine / threonine protein kinase MASK (AB040057), KIAA0912 protein (AB020719), KIAA0239 protein (D87076), RAP1, GTPase activating protein 1 (M64788) , FEM-1-like cell death receptor binding protein (AB007856), cathepsin S (M90696), hypothetical protein FLJ20308 (AK000315), pim-1 oncogene (M54915), proteasome subunit beta type 5 (D29011), KIAA0239 Protein (D87076), mucin 5 subtype B bronchus (AJ001403), cAMP response element-binding protein CREBPa, integrin alpha M (J03925), Rho-related kinase 2 (NM_004850), PTD017 protein (AL050361), unknown gene (AK001143) , AK034348, AL049250, AL16199, AL031983) and any combination thereof, but are not limited thereto. In another aspect the polynucleotide expression pattern includes expression of cell surface receptors, and non-limiting examples of such cell surface receptors include retinic acid receptor (X06614), G protein-coupled receptor (Z94155, X81892, U52219, U22491). , AF015257, U66579) chemokine (CC motif) receptor 7 (L31584), tumor necrosis factor receptor superfamily member 17 (Z29575), interferon gamma receptor 2 (U05875), cytokine receptor-like factor 1 (AF059293), class I cytokine receptor (AF053004), coagulation factor II (thrombin) receptor-like 2 (U92971), leukemia inhibitory factor receptor (NM_002310), interferon gamma receptor 1 (AL050337), and the like.

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

For example, cationic peptides can modulate the host cell's transcriptional response as well as neutralize the host response to the signaling molecules of the infectious agent, primarily down-regulating the proinflammatory response and / or upregulating the anti-inflammatory response. It is confirmed in Tables 1-15. Example 5 shows that cationic peptides can assist the host response to a pathogen by inducing the release of chemokines that promote the recruitment of immune cells to the site of infection. This result is confirmed by in vivo application in mice.

Cationic peptides, for example, induce a selective proinflammatory response that promotes the recruitment of immune cells to the site of infection but do not induce potentially harmful proinflammatory cytokines, thereby assisting the regulation of the host immune response to the pathogen. It is confirmed from the examples below that it has a substantial influence on the. Sepsis appears to be caused in part by an overwhelming proinflammatory response to the infectious agent. Cationic peptides induce anti-inflammatory responses and inhibit certain potentially harmful proinflammatory responses to help the host “balance” the pathogen.

In Example 7, the activation of selected MAP kinases was investigated to study the basic mechanisms behind the interaction effect of cells with cationic peptides. Macrophages activate MEK / ERK kinase in response to bacterial infection. MEK is a MAP kinase kinase that phosphorylates downstream kinase ERK (extracellular regulated kinase) upon activation, then dimerizes and translocates to the nucleus where it activates a transcription factor such as Elk-1 to modify polynucleotide expression. MEK / ERK kinases have been shown to impair the replication of Salmonella in macrophages. Signal transduction by MEK kinase and NADPH oxidase may play an important role in innate host defense against intracellular pathogens. Cationic peptides exhibit an effect on bacterial infection by affecting MAP kinases, as described below. Cationic peptides can directly affect kinases. Table 21 demonstrates, but is not limited to, changes in MAP kinase polynucleotide expression in response to peptides. Kinases include MAP kinase kinase 6 (H070920), MAP kinase kinase 5 (W69649), MAP kinase 7 (H39192), MAP kinase 12 (AI936909) and MAP kinase-activated protein kinase 3 (W68281).

In another method, the methods of the present invention can be used together to identify agents with multiple features, i.e., peptides having anti-inflammatory / antiseptic activity and the ability to partially induce chemokines in vivo to enhance innate immunity. .

In another aspect, the invention provides a polynucleotide expression pattern in a nucleic acid sample that is exemplified by an increase in polynucleotide expression of two or more polynucleotides in Table 55 as compared to an uninfected subject, to identify a mammalian subject from a nucleic acid sample of the subject. Provides a method for estimating infection status. In another aspect, the invention provides a polynucleotide expression pattern in a nucleic acid sample that is exemplified by an increase in polynucleotide expression of two or more polynucleotides in Table 56 or Table 57 compared to an uninfected subject, thereby identifying a mammal from the nucleic acid sample of the subject. A method of estimating a subject's infection status is provided. In another aspect of the invention, the infection state is due to an infectious agent or a signaling molecule derived therefrom, and non-limiting examples of infectious agents include gram negative bacteria and gram positive bacteria, viruses, fungi or parasitic preparations, and the like. have. In another aspect, the invention provides a polynucleotide expression pattern of a subject in an infected state identified by said method. Once identified, such polynucleotides are useful in methods of diagnosing symptoms associated with the presence or activity of an infectious agent or signaling molecule.

Example 10 below demonstrates this aspect of the invention. More specifically, Table 61 is the MEK and NADPH oxidase inhibitors demonstrate that it is possible to restrict the replication of bacteria (S. typhimurium infection of macrophages with IFN-γ for priming by (S.typhimurium) is triggered the MEK kinase do). This is an example of how altering host cell signaling molecules affects bacterial survival.

In another embodiment of the invention, compounds that inhibit chemotaxis of T cells induced by epilepsy-derived factor-1 (SDF-1) are provided. Compounds that reduce the expression of the SDF-1 receptor are also shown. Such compounds may also act as antagonists or inhibitors of CXCR-4. According to one aspect of the invention there is provided a cationic peptide that is an antagonist of CXCR-4. According to another aspect of the invention there is provided a method of identifying a cationic peptide that is an antagonist of CXCR-4. The method includes contacting SDF-1 with T cells in the presence or absence of a test peptide and measuring chemotaxis. A decrease in chemotaxis in the presence of test peptides indicates that the peptide is an antagonist of CXCR-4. Such compounds and methods are useful for treatment in HIV patients. These types of compounds and their usefulness are demonstrated, for example, in Example 11 (see also Tables 62 and 63). In this example, the cationic peptide is found to exhibit antiviral activity by inhibiting cell migration.

In one embodiment, the present invention provides an isolated cationic peptide having the amino acid sequence of the following formula (Formula A):

X ₁ X ₂ X ₃ IX ₄ PX ₄ IPX ₅ X ₂ X ₁ (SEQ ID NO: 4), where X ₁ is one or two of R, L or K, X ₂ is one of C, S or A, X ₃ is one of R or P, X ₄ is one of A or V and X ₅ is one of V or W. Non-limiting examples of peptides of the invention include 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). ).

In another embodiment, the present invention provides an isolated linear cationic peptide having the amino acid sequence of the following formula (Formula B):

X ₁ LX ₂ X ₃ KX ₄ X ₂ X ₅ X ₃ PX ₃ X ₁ (SEQ ID NO: 11), where X ₁ is one or two of D, E, S, T or N, and X ₂ is P, G or One or two of D, X ₃ is one of G, A, V, L, I or Y, X ₄ is one of R, K or H, and X ₅ is S, T, C, M or One of R. Non-limiting examples of peptides of the invention include 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). ).

In another embodiment, the present invention provides an isolated linear cationic peptide having the amino acid sequence of the following formula (Formula C):

X ₁ X ₂ X ₃ X ₄ WX ₄ WX ₄ X ₅ K (SEQ ID NO: 18) wherein the formula includes CP12a and CP12d, where X ₁ is 1-4 selected from A, P or R, and X ₂ is One or two aromatic amino acids (F, Y and W), X ₃ is one of P or K, X ₄ is 0, 1 or 2 selected from A, P, Y or W, and X ₅ is R or 1 to 3 selected from P. Non-limiting examples of peptides of the invention include RPRYPWWPWWPYRPRK (SEQ ID NO: 19), RRAWWKAWWARRK (SEQ ID NO: 20), RAPYWPWAWARPRK (SEQ ID NO: 21), RPAWKYWWPWPWPRRK (SEQ ID NO: 22), RAAFKWAWAWWRRK (SEQ ID NO: 23 K) ).

In another embodiment, the present invention provides an isolated hexameric cationic peptide having the amino acid sequence of the following formula (Formula D):

X ₁ X ₂ X ₃ X ₄ X ₁ VX ₃ X ₄ RGX ₄ X ₃ X ₄ X ₁ X ₃ X ₁ (SEQ ID NO: 25), where X ₁ is one or two of R or K, and X ₂ is polar or Charged amino acids (S, T, M, N, Q, D, E, K, R and H), X ₃ is C, S, M, D or A and X ₄ is F, I, V, M or R. Non-limiting examples of peptides of the present invention include RRMCIKVCVRGVCRRKCRK (SEQ ID NO: 26), KRSCFKVSMRGVSRRRCK (SEQ ID NO: 27), KKDAIKKVDIRGMDMRRAR (SEQ ID NO: 28), RKMVKVDVRGIMIRKDRR (SEQ ID NO: 29), KQCVKVA SEQ ID NO: RRM RMRMRMR (R) ).

In another embodiment, the present invention provides an isolated hexameric cationic peptide having the amino acid sequence of the formula (E):

X ₁ X ₂ X ₃ X ₄ X ₁ VX ₅ X ₄ RGX ₄ X ₅ X ₄ X ₁ X ₃ X ₁ (SEQ ID NO: 32), where X ₁ is ₁ of R or K or 2, X ₂ is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X ₃ is one of C, S, M, D or A, X ₄ is one of F, I, V, M or R, and X ₅ is one of A, I, S, M, D or R. Non-limiting examples of peptides of the invention include RTCVKRVAMRGIIRKRCR (SEQ ID NO: 33), KKQMMKRVDVRGISVKRKR (SEQ ID NO: 34), KESIKVIIRGMMVRMKK (SEQ ID NO: 35), RRDCRRVMVRGIDIKAK (SEQ ID NO: 36), KRTAIKKVSRRGMSVKAS R (SEQ ID NO: 38) ).

In another embodiment, the present invention provides an isolated long chain cationic peptide having the amino acid sequence of the formula (F):

KX ₁ KX ₂ FX ₂ KMLMX ₂ ALKKX ₃ (SEQ ID NO: 39), wherein X ₁ is a polar amino acid (C, S, T, M, N and Q); X ₂ is one of A, L, S or K and X ₃ is 1-17 amino acids selected from G, A, V, L, I, P, F, S, T, K and H. Non-limiting examples of the peptide of the present invention 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 ).

In another embodiment, the present invention provides an isolated long chain cationic peptide having the amino acid sequence of the following formula (Formula G):

KWKX ₂ X ₁ X ₁ X ₂ X ₂ X ₁ X ₂ X ₂ X ₁ X ₁ X ₂ X ₂ IFHTALKPISS (SEQ ID NO: 46), wherein X ₁ is a hydrophobic amino acid and X ₂ is a hydrophilic amino acid. Non-limiting examples of peptides of the invention include KWKSFLRTFKSPVRTIFHTALKPISS (SEQ ID NO: 47), KWKSYAHTIMSPVRLIFHTALKPISS (SEQ ID NO: 48), KWKRGAHRFMKFLSTIFHTALKPISS (SEQ ID NO: 49), KWKKWHSPRKVLTRIFHTALKPKKHKKHHHH, KPH, H, K, K, K, K, K, K, K, K, K ).

In another embodiment, the present 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).

As used herein, the term “isolated” refers to a peptide that is substantially free of other proteins, lipids, and nucleic acids (eg, cellular components that are naturally associated with peptides produced in vivo). Preferably, the peptide is at least 70% by weight, 80% by weight pure, most preferably 90% by weight pure.

The invention also includes analogs, derivatives, conservative variants and cationic peptide variants of the listed polypeptides, provided that the analogs, derivatives, conservative variants or variants have detectable activity or enhance anti-inflammatory activity that enhance innate immunity. Should be. An analog, derivative, variant or variant does not have to have the same activity as that of the peptide from which they are derived.

Cationic peptide "Modifier" means a modified peptide of the reference cationic peptide. For example, the term “variant” includes cationic peptides substituted with one or more amino acids of a reference peptide in an expression library. The term “reference” peptide refers to any cationic peptide of the invention (eg, a peptide as defined above) from which a variant, derivative, analog or conservative variant is derived. The term "derivative" includes hybrid peptides comprising at least a portion of each of the two cationic peptides (eg, 30-80% of each of the two cationic peptides). Also included are peptides in which one or more amino acids are deleted from the peptide sequences listed herein, provided that the derivative has retained activity that enhances innate immunity or has anti-inflammatory activity. Thus, it is possible to produce smaller active molecules that still have utility. For example, one may remove amino or carboxy terminal amino acids that are not necessary to enhance the anti-inflammatory activity or innate immunity of the peptide. Likewise, one or several (eg less than five) amino acids can be added to the cationic peptide that does not completely inhibit the activity of the peptide to produce additional derivatives. It is also possible to produce C-terminal derivatives such as C-terminal methyl esters and N-terminal derivatives, which are included in the present invention. Peptides of the invention include any analogs, homologues, mutants, isomers or derivatives of the peptides disclosed herein as long as they retain the bioactivity as disclosed herein. Also included are the reverse sequences of peptides included in the general formula set forth above. Moreover, "D" type amino acid can be substituted by "L" type amino acid, and vice versa. Alternatively, the peptide can be cyclized by chemically or by adding two or more cysteine residues to the sequence and oxidizing to form disulfide bonds.

The invention also includes peptides that are conservative variants of the peptides exemplified herein. As used herein, the term “conservative variant” means a polypeptide in which one or more amino acids are replaced with another residue that is biologically similar. Examples of conservative variants include substitution of one hydrophobic moiety such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine, or another polar residue Substitution with residues such as substitution of lysine with arginine, substitution of aspartic acid with glutamic acid, substitution of asparagine with glutamine, and the like. Neutral hydrophilic amino acids that may be substituted with one another include asparagine, glutamine, serine and threonine. The term “conservative variant” also includes peptides having substituted amino acids instead of unsubstituted parent amino acids. Such substituted amino acids can include methylated or amidated amino acids. Other substitutions are known to those skilled in the art. In one aspect, the antibody generated against the substituted polypeptide specifically binds to an unsubstituted polypeptide.

The peptide of the present invention can be synthesized by a commonly used method such as a method including t-BOC or FMOC protection of an alpha amino group. Both methods include stepwise synthesis, adding a single amino acid at each step starting from the C-terminus of the peptide (see Colingan, et al., Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). The peptides of the present invention are prepared from Stewart and Young's Solid Phase Peptides Synthesis, Freeman, San Francisco, 1969, pp. Using copolymers of (styrene-divinylbenzene) containing 0.1-1.0 mMol amine per gram of polymer. 27-62 and Merrifield, J. Am. Chem. Soc., 85: 2149, 1962, which can be synthesized by known solid phase peptide synthesis. Once the chemical synthesis is complete, the peptide can be deprotected and degraded from the polymer by treatment with liquid HF-10% anisole at 0 ° C. for about 1/4 to 1 hour. After evaporation of the reagent, the peptide is extracted from the polymer with 1% acetic acid solution and then lyophilized to obtain crude material. The peptide can be purified by the same method as gel filtration on Sephadex G-15 using 5% acetic acid as solvent. Homogeneous peptides can be produced by lyophilization of the appropriate column eluate fractions and then characterized by standard methods such as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar photorotation or solubility determination. If desired, peptides can be quantified by solid Edman degradation.

The invention also encompasses isolated nucleic acids (eg, DNA, cDNA, or RNA) encoding the peptides of the invention. Also included are nucleic acids encoding analogs, mutants, conservative variants and variants of the peptides disclosed herein. As used herein, the term “isolated” 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% by weight, 80% by weight pure, preferably 90% by weight pure, and conventional methods of synthesizing nucleic acids in vitro can be used in place of in vivo methods. As used herein, “nucleic acid” refers to the deoxyribonucleotide or ribonucleotide as a separate fragment form or as a component of a larger gene construct (eg, by operably linking a promoter to a nucleic acid encoding a peptide of the invention). It means a polymer. Many gene constructs (eg, plasmids and other expression vectors) are known in the art and can be used to produce peptides of the invention in acellular systems or in prokaryotic or eukaryotic (eg yeast, insect or mammalian) cells. have. One skilled in the art can readily synthesize nucleic acids encoding polypeptides of the invention in view of the degeneracy of the genetic code. In conventional molecular biology methods, the nucleic acids of the present invention can be readily utilized to produce peptides of the present invention.

DNA encoding the cationic peptide of the present invention can be inserted into an "expression vector." The term “expression vector” refers to a gene construct, such as a plasmid, virus or other vehicle known in the art, which can be engineered to include nucleic acids encoding polypeptides of the invention. Such expression vector is preferably a plasmid comprising a promoter sequence that facilitates transcription of the inserted gene sequence in a host cell. Expression vectors typically include polynucleotides (eg, antibiotic resistant polynucleotides) that allow for the origin of replication, promoters and phenotypic selection of transformed cells. Various promoters can be used in the present invention, including inducible and constitutive promoters. Typically, expression vectors comprise replicon sites and regulatory sequences derived from species suitable for the host cell.

Transformation or transfection of a recipient using the nucleic acid of the present invention can be carried out by conventional methods known to those skilled in the art. For example, if the host cell is E. coli, competent cells capable of absorbing DNA can be prepared using CaCl ₂ , MgCl ₂ or RbCl methods known in the art. Alternatively, physical means such as electroporation or microinjection can be used. Electroporation can deliver nucleic acids to cells by high voltage electric shock. In addition, nucleic acids can be introduced into host cells into plasma fusions using methods known in the art. Suitable eukaryotic cell transformations such as electroporation and lipofection are also known.

"Host cell" or "recipient cell" included in the present invention is any cell capable of expressing a polypeptide of the present invention using a nucleic acid of the present invention. This term includes any progeny of a recipient or host cell. Preferred recipients or host cells of the present invention include E. coli , S. aureus and P. aeruginosa , but expression vectors may be used in the host. Other Gram-negative and Gram-positive bacteria, fungi, and mammalian cells and organisms known in the art can be used as long as they include an origin of replication that allows expression.

Cationic peptide polynucleotide sequences used according to the methods of the present invention can be isolated from an organism or synthesized in the laboratory. Specific DNA sequences encoding the cationic peptides of interest include 1) isolation of a double stranded DNA sequence from genomic DNA; 2) chemical preparation of a DNA sequence providing the necessary codons for the cationic peptide of interest; And 3) in vitro synthesis of double-stranded DNA sequences by reverse transcription of mRNA isolated from donor cells. In the latter case, a double stranded DNA complement of mRNA, commonly called cDNA, is finally formed.

Where the entire sequence of amino acid residues of the desired peptide product is known, the synthesis of DNA sequences is often the method of choice. In the present invention, DNA sequence synthesis has the advantage of introducing a codon which is likely to be recognized by a bacterial host and expressing it at a high concentration without difficulty in translation. In addition, it is possible to synthesize any peptide, including those that eventually encode natural cationic peptides, variants thereof or synthetic peptides.

If the total sequence of the desired peptide is unknown, direct synthesis of the DNA sequence is not possible and the method of selection is to form a cDNA sequence. One standard procedure for isolating cDNA sequences of interest is to form plasmids or phages containing cDNA libraries derived from reverse transcription of mRNA enriched in donor cells expressing genes at high concentrations. When used with the polymerase chain reaction method, very rare expression products can be cloned. If a significant portion of the amino acid sequence of the cationic peptide is known, a labeled single- or double-stranded DNA or RNA probe sequence is generated that replicates the sequence presumed to be present in the target cDNA, thereby cloning the modified cDNA into single-stranded form. Can be used for DNA / DNA hybridization procedures performed on the generated copies (Jay, et al., Nuc. Acid Res., 11: 2325, 1983).

Peptides of the invention can be parenterally administered by gradual infusion over time or by injection. Preferably, the peptide is administered in a therapeutically effective amount to enhance or stimulate the innate immune response. Innate immunity is described herein, and examples of stimulation indicators of innate immunity include, but are not limited to, monocyte activation, proliferation, differentiation or MAP kinase pathway activation.

Peptides can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intranasally or transdermally. Preferred methods for delivering peptides include oral, encapsulation in microspheres or protenoids, aerosol delivery to the lungs or transdermal administration by iontophoretic or transdermal electroporation and the like. Other methods of administration are known in the art.

Formulations for parenteral administration to administer the peptides of the invention include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (eg olive oil), and injectable organic esters (eg ethyl oleate). Aqueous carriers include water, and alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered medium parenteral vehicles, include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or nonvolatile oils. . Intravenous vehicles include bodily fluids and nutrient sources, electrolyte sources (eg, by Ringer's dextrose), and the like. Preservatives and other additives may also be present such as antimicrobial agents, antioxidants, chelating agents and inert gases and the like.

In one embodiment, the present invention provides a method for synergy elevation therapy. For example, the peptides described herein can be used in a synergistic combination with sub-inhibitory concentrations of antibiotics. Examples of specific classes of antibiotics useful in synergistic therapies with the peptides of the invention include aminoglycosides (e.g., tobramycin), penicillins (e.g. piperacillin), cephalosporins (e.g., Ceftazidime), fluoroquinolones (e.g. ciprofloxacin), carbapenems (e.g. imipenem), tetracycline and macrolides (e.g. erythromycin and clarithromycin) Clarithromycin). In addition to the antibiotics listed above, representative antibiotics include aminoglycosides (amikacin, gentamicin, kanamycin, netylmicin, tobramycin, s-treptomycin (s-treptomycin), azithromycin, clarithromycin, erythromycin, erythromycin e-stoleate / ethylsuccinate / glusephate / lactobionate / stearate), beta-lactam, Penicillin (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin, xaxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin , Ticarcillin, carbenicillin, mezlocillin, azlocillin and piperacillin, or cephalosporin (e.g., cephalothin) , Cephazol (cefazolin), cefaclor, c-efamandole, cefoxitin, cefuroxime, cefonicid, cefmetazole, cefmetazole, and cetetane ( cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone , Ceftazidime, cefepime, cefixime, cefpodoxime, and cefsulodin. Other classes of antibiotics include, for example, carbapenem (e.g. imipenem), monobactam (e.g. aztreonam), quinolone (e.g. fleroxacin, nalidixic acid) , Norfloxacin, ciprofloxacin, cilofloxacin, ofloxacin, enoxacin, lomefloxacin and cinoxacin, tetracycline (e.g., doxycycline, Minocycline, tetracycline), and glycopeptides (eg, vancomycin, teicoplanin). Other antibiotics include chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, riamppin, rifampin, mupirocin and cationic peptides. Included.

The efficacy of the peptide was evaluated therapeutically in the infection model, alone and in combination with suboptimal concentrations of antibiotics. S. aureus is an important Gram-positive pathogen and a major cause of antibiotic-resistant infections. Briefly, peptides were tested in the S. aureus infection model for therapeutic efficacy by injection of the peptide alone and in combination with suboptimal doses of antibiotics 6 hours after the start of infection. This will mimic an antibiotic resistant environment that develops during infection, such that the MIC of resistant bacteria is so high that therapy is unsuccessful (ie, the antibiotic dosage applied is suboptimal). This demonstrated that the combination of antibiotics and peptides improved efficacy and suggested the potential for combination therapy (see Example 12).

The invention will now be described in detail with reference to the following non-limiting examples. While the invention has been described in detail with reference to some preferred embodiments, it will be appreciated that modifications and variations are possible within the spirit and scope of the invention as set forth in the claims.

Example  One

Antiseptic Anti-inflammatory activity

Polynucleotide arrays were used to determine the effect of cationic peptides on the transcriptional response of epithelial cells. A549 human epithelial cell line was maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Medicope). A549 cells were placed in a 100 mm tissue culture dish at 2.5 × 10 ⁶ cells / dish, incubated overnight, and then 100 ng / ml E. coli in the presence or absence (control) of 50 μg / ml peptide or medium alone for 4 hours. Incubated with O111: B4 LPS (Sigma). After stimulation, cells were washed once with diethyl pyrocarbonate treated phosphate buffered saline (PBS) and separated from the dish using a cell scraper. Total RNA was isolated using RNAqueous (Ambion, Austin, TX). RNA pellets were resuspended in RNase-free water containing Superase-In (RNase inhibitor; Ambion). DNA contaminants were removed with a DNA kit (Ambion). The quality of RNA was assessed by gel electrophoresis on 1% agarose gel.

The polynucleotide array used was a human operon array (identification number for the genome is PRHU04-S1), consisting of about 14,000 human oligos spotted in duplicate. Probes were prepared from 10 μg total RNA and labeled with Cy3 or Cy5 labeled dUTP. Probes were purified and hybridized and washed overnight at 42 ° C. on printed glass slides. After washing, images were captured using a Perkin Elmer array scanner. Spot average intensity, medium intensity and background intensity were measured with image processing software (Imapolynucleotide 5.0, Marina del Rey, CA). The background was removed using a "homegrown" program. The program calculated the bottom 10% strength for each sublattice and subtracted it for each grating. Analyzes were performed with Genespring software (Redwood City, CA, USA). Intensity for each spot was normalized by taking the median spot intensity value from the population of spot values in the slide and comparing this value to the values of all slides in the experiment. Relative changes seen in cells treated with peptides as compared to control cells can be found in Tables 1 and 2. This Table 2 only reflects polynucleotides that exhibit significant expression changes of 14,000 polynucleotides tested for altered expression. This data shows that the peptide has the general ability to reduce the expression of polynucleotides induced by LPS.

In Table 1, the peptide, SEQ ID NO: 27, was shown to strongly reduce the expression of many polynucleotides upregulated by E. coli O111: B4 LPS as studied by polynucleotide microarrays. Peptides (50 μg / ml) and LPS (0.1 μg / ml) or LPS alone were incubated with A549 cells for 4 hours and RNA was isolated. Cy3 / Cy5 labeled cDNA probes were prepared using 5 μg total RNA and hybridized to human operon array (PRHU04). The intensity of unstimulated cells is shown in the third column of Table 1. The “Ratio: LPS / Control” column relates to the intensity of polynucleotide expression in LPS stimulated cells divided by the strength of unstimulated cells. The “Ratio: LPS + ID 27 / Control” column relates to the intensity of polynucleotide expression in cells stimulated with LPS and peptide divided by unstimulated cells.

TABLE 1Reduction by peptide SEQ ID NO: 27 of A549 human epithelial cell polynucleotide expression upregulated by E. coli O111: B4 LPS

^* All accession numbers in Tables 1-64 relate to GenBank accession numbers.

In Table 2, the 50 μg / ml concentration of cationic peptides strongly reduced the expression of many polynucleotides upregulated by 100 ng / ml E. coli O111: B4 LPS as studied by polynucleotide microarrays. Appeared. Peptides and LPS or LPS alone were incubated with A549 cells for 4 hours and RNA was isolated. Cy3 / Cy5 labeled cDNA probes were prepared using 5 μg total RNA and hybridized to human operon array (PRHU04). The intensity of unstimulated cells is shown in the third column of Table 2. “Ratio: LPS / Control” column refers to the intensity of polynucleotide expression in LPS stimulated cells divided by the intensity of unstimulated cells. Another column represents the intensity of polynucleotide expression in cells stimulated with LPS and peptide divided by unstimulated cells.

TABLE 2 Human A549 epithelial cell polynucleotide expression upregulated by E. coli O111: B4 LPS and reduced by cationic peptides

Example  2

Neutralization of stimulation of immune cells

The ability of compounds to neutralize the stimulation of immune cells by gram negative and gram positive bacterial products was tested. Bacterial products stimulate cells of the immune system to produce inflammatory cytokines, which, if unchecked, can lead to sepsis. Initial experiments were performed on the murine macrophage lines RAW 264.7, human epithelial cells A549, and BALB / c mice (Charles River Laboratories, Wilmington, Mass.) Obtained from the American Type Culture Collection (Manassas, Mass.). Primary macrophages derived from bone marrow were used. Cells from mouse bone marrow were treated with 20% FBS (Sigma Chemical Company, St. Louis, MO) and Dulbecco's denatured eagle medium (DMEM; Life Technologies, Canada) supplemented with 20% L cell condition-regulated medium as a source of M-CSF Cultured in 150 mm plates in Burlington, Ontario). Once the macrophages are 60-80% saturated, they are left out of the L cell conditioned medium for 14-16 hours to render the cells inactive, then 100 ng / ml LPS or 100 ng / ml LPS + 20 μg / ml Treated with peptide for 24 hours. Release of cytokines into the culture supernatants was determined by ELISA (R & D Systems, Minneapolis, MN). Cell lines RAW 264.7 and A549 were maintained in DMEM supplemented with 10% fetal bovine serum. RAW 264.7 cells are seeded in 24 well plates at a density of 10 ⁶ cells per well in DMEM, A549 cells are seeded in 24 well plates at a density of 10 ⁵ cells per well in DMEM, both 37 ° C., 5% CO ₂ Incubated overnight at. DMEM was aspirated from overnight grown cells and replaced with fresh medium. In some experiments, blood from volunteer human donors was collected by venipuncture into tubes containing 14.3 USP unit heparin / ml blood (Becton Dickinson, Franklin Lakes, NJ) (UBC Clinical Research Ethics Committee Certificate C00-0537) Following the procedures allowed by). Blood was mixed with LPS with or without peptide in a polypropylene tube for 6 hours at 37 ° C. Samples were centrifuged at 2000 × g for 5 minutes, plasma was collected and stored at −20 ° C. until analyzed for IL-8 by ELISA (R & D Systems). In experiments with cells, LPS or other bacterial products were incubated with the cells under 5% CO ₂ at 37 ° C. for 6-24 hours. S. typhimurium LPS and E. coli 0111: B4 LPS were purchased from Sigma. Lipoteichoic acid from S. aureus (Sigma) was resuspended in endotoxin-free water (Sigma). A Limulus amoebocyte eluate assay (Sigma) was performed on the LTA preparation to confirm that the lot was not significantly contaminated by endotoxin. Endotoxin contamination was below 1 ng / ml, a concentration that did not cause significant cytokine production in RAW 264.7 cells. Uncapped lipoarabinomannan (AraLAM) was a donation from Dr. John T. Bellisli of Colorado State University. AraLAM from Mycobacterium was filtered sterilized and determined by the Limulus amebosite assay, and found that endotoxin contamination was 3.75 ng per 1.0 mg of LAM. Simultaneously with LPS addition (or later, if specifically described), cationic peptides were added in concentration ranges. The supernatant was removed and tested for cytokine production by ELISA (R & D Systems). All assays were performed three or more times to obtain similar results. To confirm in vivo antiseptic activity, 2-3 μg of E. coli O111: B4 LPS in phosphate buffered saline (PBS; pH 7.2) were injected into galactosamine-sensitized 8-10 week old female CD-1 or BALB / c mice. Intraperitoneal injection induced sepsis. In experiments involving peptides, 200 μg in 100 μl of sterile water were injected into separate intraperitoneal sites within 10 minutes of LPS injection. In another experiment, CD-1 mice were injected with 400 μg E. coli O111: B4 LPS, and 10 minutes later peptide (200 μg) was introduced by intraperitoneal injection. Survival was monitored for 48 hours after injection.

Overproduction of TNF-α has been customarily associated with the development of sepsis. Three types of LPS, LTA or AraLAM used in this example represent products released by gram negative and gram positive bacteria. Peptide, SEQ ID NO: 1, could significantly reduce TNF-α production stimulated by S. typhimurium, B. sephacia, and E. coli O111: B4 LPS, the former being somewhat less affected (Table 3). At concentrations as low as 1 μg / ml peptide (0.25 nM), a substantial decrease in TNF-α production was observed in the latter two cases. The different peptide, SEQ ID NO: 3, did not reduce the LPS induced production of TNF-α in RAW macrophages, indicating that this is not a uniform and predictable property of cationic peptides. Representative peptides from each mode were also tested for their ability to affect TNF-α production stimulated by E. coli O111: B4 LPS (Table 4). Peptides varied in their ability to reduce TNF-α production, but most of them lowered TNF-α by 60% or more.

In addition, at certain concentrations, peptide SEQ ID NO: 1 and SEQ ID NO: 2 could reduce the ability of bacterial products to stimulate production of IL-8 by epithelial cell lines. LPS is a known powerful stimulator of IL-8 by epithelial cells. At low concentrations (1-20 μg / ml), the peptide neutralizes the IL-8 induced response to LPS of epithelial cells (Tables 5-7). In addition, peptide SEQ ID NO: 2 inhibited LPS-induced production of IL-8 in human whole blood (Table 4). In contrast, high concentrations of peptide SEQ ID NO: 1 (50-100 μg / ml) actually increased the levels of IL-8 (Table 5). This suggests that peptides have different effects at different concentrations.

In addition, the effect of peptides on inflammatory stimuli was seen in primary murine cells, where peptide SEQ ID NO 1 was derived from bone marrow from BALB / c mice stimulated with 100 ng / ml E. coli O111: B4 LPS. TNF-α production by macrophages was significantly reduced (> 90%) (Table 8). This experiment was performed in the presence of serum containing LPS binding protein (LBP), a protein that can mediate the rapid binding of LPS to CD14. One hour after stimulation with 100 ng / ml E. coli LPS, delayed addition of SEQ ID NO: 1 to the supernatant of macrophages still substantially reduced (70%) TNF-α production (Table 9).

Consistent with the ability of SEQ ID NO: 1 to prevent LPS induced production of TNF-α in vitro, certain peptides also protected mice against lethal shock induced by high concentrations of LPS. In some experiments, CD-1 mice were sensitized for LPS by prior injection of galactosamine. Galactosamine sensitized mice injected with 3 μg E. coli O111: B4 LPS were all sacrificed within 4 to 6 hours. When 200 μg of SEQ ID NO: 1 was injected 15 minutes after LPS, 50% of mice survived (Table 10). In another experiment, when high concentrations of LPS were injected into BALB / c mice without D-galactosamine, the peptides were 100% protected compared to the control without survival (Table 13). In addition, other peptides were found to be protective in these models (Tables 11, 12).

In addition, cationic peptides were able to reduce the stimulation of macrophages by Gram-positive bacterial products such as Mycobacterium uncapped lipoarabinomannan (AraLAM) and S. aureus LTA. For example, SEQ ID NO: 1 inhibited the induction of TNF-α in RAW 264.7 cells by Gram positive bacterial products, LTA (Table 14) and to a lesser extent AraLAM (Table 15). Another peptide, SEQ ID NO: 2, has also been found to reduce LTA induced TNF-α production by RAW 264.7 cells. At a concentration of 1 μg / ml, SEQ ID NO: 1 could substantially reduce (> 75%) 1 μg / ml of induction of TNF-α production. In 20 μg / ml SEQ ID NO: 1, AraLAM induced TNF-α was inhibited> 60%. Polymyxin B (PMB) was included as a control to demonstrate that endotoxin contamination was not a significant factor in inhibition of SEQ ID NO: 1 of AraLAM induced TNF-α. These results indicate that cationic peptides can reduce the proinflammatory cytokine response of the immune system to bacterial products.

TABLE 3 Reduction by SEQ ID NO: 1 of LPS induced TNF-α production in RAW 264.7 cells.

RAW 264.7 mouse macrophages were treated with 100 ng / ml S. typhimurium LPS, 100 ng / ml B. Sephacia LPS and 100 ng / ml E. coli O111: B4 LPS for 6 hours in the presence of the predetermined concentration of SEQ ID NO: 1. Stimulated. The concentration of TNF-α released into the culture supernatant was measured by ELISA. 100% represents the amount of TNF-α produced from RAW 264.7 cells incubated with LPS alone for 6 hours (S. typhimurium LPS = 34.5 ± 3.2 ng / ml, B. Sephacia LPS = 11.6 ± 2.9 ng / Ml and E. coli O111: B4 LPS = 30.8 ± 2.4 ng / ml). Background levels of TNF-α production by RAW 264.7 cells cultured without stimulation for 6 hours resulted in TNF-α levels ranging from 0.037 to 0.192 ng / ml. Data was obtained from two sets of samples and provided as mean + standard error of three experiments.

TABLE 4 Reduction by E. coli LPS induced TNF-α producing cationic peptide in RAW 264.7 cells.

RAW 264.7 mouse macrophages were stimulated with 100 ng / ml E. coli O111: B4 LPS in the presence of a predetermined concentration of cationic peptide for 6 hours. The concentration of TNF-α released into the culture supernatant was measured by ELISA. Background levels of TNF-α production by RAW 264.7 cells cultured without stimulation for 6 hours resulted in TNF-α levels ranging from 0.037 to 0.192 ng / ml. Data was obtained from two sets of samples and provided as mean + standard error of three experiments.

TABLE 5 Reduction by SEQ ID NO: 1 of LPS induced IL-8 production in A549 cells.

A549 cells were stimulated by increasing the concentration of SEQ ID NO: 1 in the presence of LPS (100 ng / ml E. coli O111: B4) for 24 hours. The concentration of IL-8 in the culture supernatant was measured by ELISA. The background level of IL-8 from the cells alone was 0.172 ± 0.029 ng / ml. Data is given as mean + standard error of 3 experiments.

TABLE 6 Reduction by SEQ ID NO: 2 of E. coli LPS induced IL-8 production in A549 cells.

Human A549 epithelial cells were stimulated by increasing the concentration of SEQ ID NO: 2 in the presence of LPS (100 ng / ml E. coli O111: B4) for 24 hours. The concentration of IL-8 in the culture supernatant was measured by ELISA. Data is given as mean + standard error of three experiments.

TABLE 7 Reduction by SEQ ID NO: 2 of E. coli LPS induced IL-8 in human blood.

Human whole blood was stimulated by increasing concentrations of peptide and E. coli O111: B4 LPS for 4 hours. Human blood samples were centrifuged, serum removed and tested for IL-8 by ELISA. Data is provided as the average of two donors.

TABLE 8 Reduction by SEQ ID NO: 1 of E. coli LPS induced TNF-α production in rat bone marrow macrophages.

Macrophages derived from BALB / c mouse bone marrow were incubated with 100 ng / ml E. coli O111: B4 LPS for 6 hours or 24 hours in the presence or absence of 20 μg / ml peptide. Supernatants were collected and tested for levels of TNF-α by ELISA. The data show the amount of TNF-α produced from two wells of bone marrow-derived macrophages incubated for 6 hours (1.1 ± 0.09 ng / ml) or 24 hours (1.7 ± 0.2 ng / ml) with LPS alone. The background level of TNF-α was 0.038 ± 0.008 ng / ml for 6 hours and 0.06 ± 0.012 ng / ml for 24 hours.

TABLE 9 Inhibition of E. coli LPS induced TNF-α production by delayed addition to SEQ ID NO: 1 A549 cells.

Peptides (20 μg / ml) were added to wells containing A549 human epithelial cells and 100 ng / ml E. coli O111: B4 LPS beforehand at increasing time points. Supernatants were collected after 6 hours and tested for levels of TNF-α by ELISA. Data are given as mean + standard error of three experiments.

TABLE 10 Protection against lethal endotoxemia in galactosamine sensitized CD-1 mice by SEQ ID NO: 1.

CD-1 mice (9 weeks old) were sensitized to endotoxin by three intraperitoneal injections of galactosamine (20 mg in 0.1 ml sterile PBS). Endotoxin shock was then induced by intraperitoneal injection of E. coli O111: B4 LPS (3 μg in 0.1 mL PBS). Peptide, SEQ ID NO: 1 (200 μg / mouse = 8 mg / kg) was injected into a separate intraperitoneal site 15 minutes after LPS injection. Mice were monitored for 48 hours and results recorded.

TABLE 11 Protection against lethal endotoxemia in galactosamine sensitized CD-1 mice by cationic peptides.

CD-1 mice (9 weeks old) were sensitized to endotoxin by three intraperitoneal injections of galactosamine (20 mg in 0.1 ml sterile PBS). Endotoxin shock was then induced by intraperitoneal injection of E. coli O111: B4 LPS (2 μg in 0.1 mL PBS). Peptide, SEQ ID NO: 1 (200 μg / mouse = 8 mg / kg) was injected into a separate intraperitoneal site 15 minutes after LPS injection. Mice were monitored for 48 hours and results recorded.

TABLE 12 Protection against lethal endotoxemia in galactosamine sensitized BALB / c mice by cationic peptides.

BALB / c mice (8 weeks old) were sensitized to endotoxin by three intraperitoneal injections of galactosamine (20 mg in 0.1 ml sterile PBS). Endotoxin shock was then induced by intraperitoneal injection of E. coli O111: B4 LPS (2 μg in 0.1 mL PBS). Peptide, SEQ ID NO: 1 (200 μg / mouse = 8 mg / kg) was injected into a separate intraperitoneal site 15 minutes after LPS injection. Mice were monitored for 48 hours and results recorded.

TABLE 13 Protection against lethal endotoxemia in BALB / c mice by SEQ ID NO: 1.

BALB / c mice were injected intraperitoneally with 400 μg E. coli O111: B4 LPS. Peptides (200 μg / mouse = 8 mg / kg) were injected into separate intraperitoneal sites and mice were monitored for 48 hours and results recorded.

TABLE 14 Peptide inhibition of TNF-α production induced by S. aureus LTA.

RAW 264.7 mouse macrophages were stimulated with 1 μg / ml S. aureus LTA in the absence and presence of increased concentrations of peptide. Supernatants were collected and tested for levels of TNF-α by ELISA. Background levels of TNF-α production by RAW 264.7 cells cultured without stimulation for 6 hours resulted in TNF-α levels ranging from 0.037 to 0.192 ng / ml. Data are given as mean + standard error of three or more experiments.

TABLE 15 Peptide inhibition of TNF-α production induced by Mycobacterium uncapped lipoarabinomannans.

RAW 264.7 mouse macrophages were stimulated with 1 μg / ml AraLAM in the absence and presence of 20 μg / ml peptide or Polymyxin B. Supernatants were collected and tested for levels of TNF-α by ELISA. Background levels of TNF-α production by RAW 264.7 cells incubated without stimulation for 6 hours resulted in TNF-α levels ranging from 0.037 to 0.192 ng / ml. Data are given as mean + standard error of three or more experiments.

Example  3

Cationic  Evaluation of Toxicity of Peptides

The potential toxicity of the peptide was measured in two ways. First, a cytotoxicity detection kit (Roche) (lactate dehydrogenase-LDH) assay was used. This is a colorimetric assay for the quantification of cell death and cell elution based on the measurement of LDH activity released by the cytosol of damaged cells into the supernatant. LDH is a stable cytoplasmic enzyme present in all cells, which is released into the cell culture supernatant upon cytoplasmic membrane damage. Increasing the amount of killed or cytoplasmic membrane damaged cells results in an increase in LDH enzyme activity in the culture supernatant as measured by ELISA plate reader, OD ₄₉₀ nm (the amount of color formed in the assay is proportional to the number of cells eluted). In this assay, human bronchial epithelial cells (16HBEo14, HBE) cells were incubated with 100 μg of peptide for 24 hours, the supernatant was removed and tested for LDH. Another assay used to determine the toxicity of cationic peptides was the WST-1 assay (Roche). This assay is a colorimetric assay for the quantification of cell proliferation and cell viability, based on the degradation of tetrazolium salt WST-1 by viable intracellular mitochondrial dehydrogenase ([ ³ H] -thymidine transduction assay) Non-radioactive method instead. In this assay, HBE cells were incubated with 100 μg peptide for 24 hours, then 10 μl / well cell proliferation reagent WST-1 was added. Cells were incubated with this reagent and then plates were measured with an ELISA plate reader, OD ₄₉₀ nm.

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

TABLE 16 Toxicity of cationic peptides as measured by LDH release assay.

Human HBE bronchial epithelial cells were incubated with 100 μg / ml peptide or polymyxin B for 24 hours. LDH activity was analyzed in the supernatant of cell culture. As a control for 100% LDH release, Triton X-100 was added. Data are given as mean ± standard deviation. Only peptide SEQ ID NOs: 40, 41, 42 and 43 showed any significant toxicity.

TABLE 17 Toxicity of Cationic Peptides as Measured by WST-1 Assay.

HBE cells were incubated with 100 μg / ml peptide or polymyxin B for 24 hours and cell viability was tested. Data are given as mean ± standard deviation. As a control for 100% LDH release, Triton X-100 was added. Only peptide SEQ ID NOs: 40, 41, 42 and 43 showed any significant toxicity.

Example  4

Cationic  Polynucleotide Regulation by Peptides

Polynucleotide assays were used to determine the effect of cationic peptides on the transcriptional response of macrophages and epithelial cells. Mouse macrophage RAW 264.7, human bronchial cells (HBE) or A549 human epithelial cells were placed in a 150 nm tissue culture dish at 5.6 × 10 ⁶ cells / dish, incubated overnight, followed by 50 μg / ml peptide, or medium alone. Incubated for 4 hours. After stimulation, cells were washed once with diethyl pyrocarbonate treated PBS and removed from the dish using a cell scraper. Total RNA was isolated using Trizol (Gibco Life Technologies). RNA pellets were resuspended in RNase-free water (Ambion, Austin, TX) containing RNase inhibitors. RNA was treated with DNaseI (Clontech, Palo Alto, CA, USA) for 1 hour at 37 ° C. After addition of a termination mix (0.1 M EDTA [pH 8.0], 1 mg / ml glycogen), the sample was extracted once with phenol: chloroform: isoamyl alcohol (25: 24: 1) and once with chloroform. . RNA was then precipitated by adding 2.5 volumes of 100% ethanol and 1/10 volume of sodium acetate (pH 5.2). RNA was resuspended in RNase-free water (Ambion) with RNase inhibitor and stored at -70 ° C. The quality of RNA was assessed by gel electrophoresis on 1% agarose gel. Genomic DNA contamination by using isolated RNA as a template for PCR amplification with β-actin specific primers (5'-GTCCCTGTATGCCTCTGGTC-3 '(SEQ ID NO: 55) and 5'-GATGTCACGCACGATTTCC-3' (SEQ ID NO: 56)) Was assessed to be absent. Agarose gel electrophoresis and ethidium bromide staining confirmed the absence of amplicons after 35 cycles.

Atlas cDNA expression assay (Clontech, Palo Alto, CA, USA) consisting of 588 selected mouse cDNAs spotted on two positively charged membranes was used for the initial polynucleotide array study (Tables 18, 19). ³² P-radiolabeled cDNA probes prepared from 5 μg total RNA were incubated at 71 ° C. overnight with arrays. The filter was washed roughly and then exposed to a phosphoimizer screen (Molecular Dynamics, Sunnyvale, Calif.) For 3 days at 4 ° C. Images were captured using a Molecular Dynamics PSI photoimager. Hybridization signals were analyzed using AtlasImage 1.0 image analysis software (Clontech) and Excel (Microsoft, Redmond, Washington, USA). Corrected for the background level of the intensity for each spot, and stimulation conditions: slightly changed between the β- actin, ubiquitin, ribosomal protein S29, glyceraldehyde-3-phosphate dehydrogenase claim dehydrogenase by Sergio aldehyde (GAPDH) and Ca ^{2 +} binding protein Means for the five polynucleotides observed to be used were normalized to probe labeling differences. If the normalized hybridization intensity for a given cDNA was less than 20, a value of 20 was assigned to calculate ratio and relative expression.

The following used polynucleotide arrays (Tables 21-26) were Resgen human cDNA arrays consisting of 7,458 human cDNAs spotted in duplicates (identification number for the genome is PRHU03-S3). Probes were prepared from 15-20 μg total RNA and labeled with Cy3 labeled dUTP. The probe was purified and hybridized to glass slides printed at 42 ° C. overnight and washed. After washing, images were captured using a Virtek slide reader. Spot average intensity, medium intensity and background intensity were measured with image processing software (Imagene 4.1, Marina del Rey, CA). Normalization and analysis was performed with Genespring software (Redwood City, CA, USA). Intensity values were calculated by subtracting the average background intensity from the average intensity value measured by Imagene. Intensity for each spot was normalized by taking the median spot intensity value from the population of spot values in the slide and comparing this value to the values of all slides in the experiment. Relative changes seen in cells treated with peptides as compared to control cells can be found in the table below.

Another polynucleotide array used (Tables 27-35) was a human operon array consisting of about 14,000 human oligos spotted in duplicates (identification number for the genome is PRHU04-S1). Probes were prepared from 10 μg total RNA and labeled with Cy3 or Cy5 labeled dUTP. In these experiments, A549 epithelial cells were placed in 100 mm tissue culture dishes at 2.5 × 10 ⁶ cells / plate. Total RNA was isolated using RNAqueous (Ambion). DNA contamination was removed with a DNA free kit (Ambion). Probes prepared from total RNA were purified, hybridized to glass slides printed at 42 ° C. overnight, and washed. After washing, images were captured using a Perkin Elmer array scanner. Spot average intensity, medium intensity and background intensity were measured with image processing software (Imagene 5.0, Marina del Rey, CA). The background was removed using a "homegrown" program. The program calculated the bottom 10% strength for each sublattice and subtracted it for each grating. Analyzes were performed with Genespring software (Redwood City, CA, USA). Intensity for each spot was normalized by taking the median spot intensity value from the population of spot values in the slide and comparing this value to the values of all slides in the experiment. Relative changes seen in cells treated with peptides as compared to control cells can be found in the table below.

Semiquantitative RT-PCR was performed to confirm the polynucleotide array results. 1 μg RNA samples were incubated with 1 μl oligodT (500 μg / ml) and 1 μl mixed dNTP stock at 1 μm in 12 μl volume with DEPC treated water at 65 ° C. for 5 minutes in a thermocycler. 4 μl 5X first strand buffer, 2 μl 0.1 M DTT and 1 μl RNaseOUT recombinant ribonuculease inhibitor (40 units / μl) were added and incubated at 42 ° C. for 2 minutes before Superscript II (Invitrogen, Canada 1 μl (200 units) of Burlington, Ontario) was added. Negative controls for each RNA source were generated using a parallel reaction in the absence of Superscript II. cDNA was amplified in the presence of 5 ′ and 3 ′ primers (1.0 μM), 0.2 mM dNTP mixture, 1.5 mM MgCl, Taq DNA polymerase (New England Biolabs, Mississauga, Ontario, Canada) 1 U and 1 × PCR buffer. . Each PCR was performed with a thermal cycler by using 30-40 cycles consisting of 94 ° C denaturation 30 seconds, 52 ° C or 55 ° C annealing 30 seconds, and 72 ° C extension 40 seconds. The cycle number of the PCR was optimized to be in the linear phase of the reaction for each primer and set of RNA samples. Housekeeping polynucleotide β-actin was amplified in each experiment to evaluate the extraction procedure and to assess the amount of RNA. Reaction products were visualized by electrophoresis, analyzed by densitometry, and relative starting RNA concentrations were calculated as a reference for β-actin amplification.

Table 18 shows that SEQ ID NO 1 treatment of RAW 264.7 cells upregulated the expression of at least 30 different polynucleotides in small atlas microarrays using known polynucleotides selected. Polynucleotides upregulated by peptide, SEQ ID NO: 1, are mainly comprised of two categories: receptors (growth, chemokines, interleukins, interferons, hormones, neurotransmitters), cell surface antigens and cell adhesion and cell-cell exchange ( Growth factors, cytokines, chemokines, interleukins, interferons, hormones), cytoskeleton, motility and protein conversion. Specific polynucleotides upregulated include chemokine MCP-3, anti-inflammatory cytokine IL-10, macrophage colony stimulating factors and receptors such as IL-1R-2 (presumed antagonist of productivity IL-1 that binds IL-1R1), PDGF receptor B, NOTCH4, LIF receptor, LFA-1, TGFβ receptor 1, G-CSF receptor and those encoding IFNγ receptors were included. In addition, the peptides upregulated polynucleotides encoding several metalloproteinases and their inhibitors, including the bone morphogenic proteins BMP-1, BMP-2, BMP-8a, TIMP2 and TIMP3. In addition, the peptides upregulated specific transcription factors, including JunD, and YY and LIM-1 transcription factors, and kinases such as Etk1 and Csk, which exhibit broad effects. In addition, polynucleotide array studies have found that SEQ ID NO: 1 downregulates 20 or more polynucleotides in RAW 264.7 macrophages (Table 19). Polynucleotides downregulated by peptides included DNA repair proteins and several inflammatory mediators such as MIP-1α, oncostatin M and IL-12. Multiple effects of peptides on polynucleotide expression were confirmed by RT-PCR (Table 20). In addition, peptides, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 19 and SEQ ID NO: 1, and representative peptides from each formula, altered the transcriptional response of human epithelial cell lines using medium size microarrays (7835 polynucleotides). I was. The effect of SEQ ID NO: 1 on polynucleotide expression was compared in 2 human epithelial cell lines, A549 and HBE. The polynucleotides involved in the host immune response up-regulated by at least two peptides at a rate two times greater than unstimulated cells are listed in Table 21. Polypeptides downregulated by at least 2 peptides at a rate more than 2 times greater than unstimulated cells are listed in Table 22. Table 23 and Table 24 show up- and down-regulated human epithelial proinflammatory polynucleotides, respectively. In Tables 25 and 26, anti-inflammatory polynucleotides affected by cationic peptides are shown. It is clear that cationic peptides upregulate anti-inflammatory responses and down regulate pro-inflammatory responses. It is very difficult to find polynucleotides involved in down regulated anti-inflammatory responses (Table 26). Proinflammatory polynucleotides upregulated by cationic peptides were polynucleotides primarily involved in migration and adhesion. Of the down-regulated proinflammatory polynucleotides, it should be noted that all cationic peptides affected several toll-like receptor (TLR) polynucleotides, which is very important for signaling host response to infectious agents. An important anti-inflammatory polynucleotide upregulated by all peptides is the IL-10 receptor. IL-10 is an important cytokine involved in regulating proinflammatory cytokines. In addition, these polynucleotide expression effects were observed using primary human macrophages as observed for peptide SEQ ID NO: 6 in Tables 27 and 28. The effect of representative peptides from each formula on human epithelial cell expression of selected polynucleotides (out of 14,000 investigated) is shown in Tables 31-37 below. Six or more peptides from each formula were tested for the ability to alter human epithelial polynucleotide expression, which had a wide range of stimulatory effects. In each formula, there were at least 50 polynucleotides that were typically upregulated by each peptide in the group.

TABLE 18 Polynucleotides upregulated by peptide sequence number 1 treatment of RAW macrophages ^a .

Cationic peptides at a concentration of 50 μg / ml have been shown to strongly induce 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 unstimulated cells is shown in the third column. "Ratio: Peptide Stimulation: Unstimulated" column refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the strength of unstimulated cells.

Normalized intensity changes of housekeeping polynucleotides ranged from 0.8 to 1.2 fold, and the use of such polynucleotides for normalization was effective. If the normalized hybridization intensity for a given cDNA was less than 20, a value of 20 was assigned to calculate ratio and relative expression. Array experiments were repeated three times with different RNA preparations and the mean magnification change is shown above. Provided are polynucleotides whose relative expression levels have changed more than twofold.

TABLE 19 Polynucleotides Down-regulated by Sequence Number 1 Treatment of RAW Macrophages ^a .

Cationic peptides at a concentration of 50 μg / ml have been shown 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 unstimulated cells is shown in the third column. “Ratio: Peptide Stimulation: Unstimulated” column refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the strength of unstimulated cells. Array experiments were repeated three times with different cells and the mean magnification change is shown below. Provided are polynucleotides whose relative expression levels have changed more than twofold.

TABLE 20 Polynucleotide expression changes that differ in the peptide, SEQ ID NO: 1, can be confirmed by RT-PCR.

RAW 264.7 macrophages were incubated with 50 μg / ml peptide or medium alone for 4 hours, total RNA was isolated, and semiquantitative RT-PCR was performed. Primer pairs specific for each polynucleotide were used for amplification of RNA. Amplification of β-actin was used for normalization as a positive control. Density analysis of the RT-PCR product was used. The results relate to the relative magnification change in polynucleotide expression of peptide treated cells compared to cells incubated with medium alone. Data are given as mean ± standard error of 3 experiments.

TABLE 21 Polynucleotides Upregulated by Peptide Treatment of A549 Epithelial Cells ^a .

Cationic peptides at a concentration of 50 μg / ml have been shown to increase the expression of several polynucleotides. 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 # PRHU03-S3. The intensity of unstimulated intracellular polynucleotides is shown in the second column. "Ratio peptide stimulation: non-stimulation" refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the strength of unstimulated cells.

TABLE 22 Polynucleotides Down-regulated by Peptide Treatment of A549 Epithelial Cells ^a .

Cationic peptides at a concentration of 50 μg / ml have been shown to reduce the expression of several polynucleotides. 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 # PRHU03-S3. The intensity of unstimulated intracellular polynucleotides is shown in the second column. "Ratio: Peptide Stimulation: Non-Stimulation" means the intensity of polynucleotide expression in peptide stimulated cells divided by the strength of unstimulated cells.

TABLE 23 Pro-inflammatory polynucleotides upregulated by peptide treatment of A549 cells.

Cationic peptides at a concentration of 50 μg / ml have been shown to increase expression of certain proinflammatory polynucleotides (data shown in the small column of Table 21). 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 # PRHU03-S3. The intensity of unstimulated intracellular polynucleotides is shown in the second column. "Ratio peptide stimulation: non-stimulation" refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the strength of unstimulated cells.

TABLE 24 Pro-inflammatory polynucleotides down regulated by peptide treatment of A549 cells.

Cationic peptides at a concentration of 50 μg / ml have been shown to increase the expression of certain proinflammatory polynucleotides (data shown in the small column of Table 22). 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 # PRHU03-S3. The intensity of unstimulated intracellular polynucleotides is shown in the second column. "Ratio peptide stimulation: non-stimulation" refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the strength of unstimulated cells.

TABLE 25 Upregulated anti-inflammatory polynucleotides by peptide treatment of A549 cells

Cationic peptides at a concentration of 50 μg / ml have been shown to increase the expression of certain anti-inflammatory polynucleotides. (See data in small columns in Table 21). Peptides were incubated for 4 hours using human A549 epithelial cells, RNA was isolated, converted to labeled cDNA probes, and hybridized to human cDNA array ID # PRHU03-S3. The strength of polynucleotides in unstimulated cells is described in the second column. A "peptide stimulated: non-stimulated ratio" column refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the intensity of unstimulated cells.

TABLE 26 Anti-inflammatory polynucleotides downregulated by peptide treatment of A549 cells

Cationic peptides at a concentration of 50 μg / ml have been shown to increase the expression of certain anti-inflammatory polynucleotides. (See data in small columns in Table 21). Peptides were incubated for 4 hours using human A549 epithelial cells, RNA was isolated, converted to labeled cDNA probes, and hybridized to human cDNA array ID # PRHU03-S3. The strength of polynucleotides in unstimulated cells is described in the second column. A "peptide stimulated: non-stimulated ratio" column refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the intensity of unstimulated cells.

TABLE 27 Polynucleotides Upregulated by SEQ ID NO: 6 in Primary Human Macrophages

Peptide SEQ ID NO: 6 at a concentration of 50 μg / ml was shown to increase the expression of multiple polynucleotides. Peptides were incubated for 4 hours using human macrophages, RNA was isolated, converted to labeled cDNA probes, and hybridized to human operon array (PRHU04). The strength of polynucleotides in unstimulated cells is described in the second column. A “treated peptide: control ratio” column refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the intensity of unstimulated cells.

TABLE 28 Polynucleotides Downregulated by SEQ ID NO: 6 in Primary Human Macrophages

Peptide SEQ ID NO: 6 at a concentration of 50 μg / ml was shown to increase the expression of multiple polynucleotides. Peptides were incubated for 4 hours using human macrophages, RNA was isolated, converted to labeled cDNA probes, and hybridized to human operon array (PRHU04). The strength of polynucleotides in unstimulated cells is described in the second column. A “treated peptide: control ratio” column refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the intensity of unstimulated cells.

TABLE 29 Polynucleotides Upregulated by SEQ ID NO: 1 in HBE Cells

Peptide SEQ ID NO: 1 at a concentration of 50 μg / ml was shown to increase the expression of multiple polynucleotides. Peptides were incubated for 4 hours using human HBE epithelial cells, RNA was isolated, converted to labeled cDNA probes, and hybridized to human operon array (PRHU04). The strength of polynucleotides in unstimulated cells is described in the second column. A “treated peptide: control ratio” column refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the intensity of unstimulated cells.

TABLE 30 Polynucleotides Downregulated by Peptides in HBE Cells (50 μg / mL), SEQ ID NO: 1

Peptide SEQ ID NO: 1 at a concentration of 50 μg / ml was shown to increase the expression of multiple polynucleotides. Peptides were incubated for 4 hours using human A549 epithelial cells, RNA was isolated, converted to labeled cDNA probes, and hybridized to human operon array (PRHU04). The intensity of the polynucleotides in the non-irritating cells is described in the third column. A “treated peptide: control ratio” column refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the intensity of unstimulated cells.

TABLE 31 Upregulation of Polynucleotide Expression in A549 Cells Induced by Formula A Peptides

Peptides at a concentration of 50 μg / ml have been shown to increase the expression of many polynucleotides. Peptides were incubated for 4 hours using human A549 epithelial cells, RNA was isolated, converted to labeled cDNA probes, and hybridized to human operon array (PRHU04). The intensity of the polynucleotides in control unstimulated cells is described in the second and third columns for labeling cDNA with dyes Cy3 and Cy5, respectively. "ID #: Control" column refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the intensity of unstimulated cells.

TABLE 32 Upregulation of Polynucleotide Expression in A549 Cells Induced by Formula B Peptides

Peptides at a concentration of 50 μg / ml have been shown to increase the expression of many polynucleotides. Peptides were incubated for 4 hours using human A549 epithelial cells, RNA was isolated, converted to labeled cDNA probes, and hybridized to human operon array (PRHU04). The intensity of polynucleotides in control unstimulated cells is described in the second and third columns for labeling cDNA with dyes Cy3 and Cy5, respectively. "ID #: Control" column refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the intensity of unstimulated cells.

TABLE 33 Upregulation of Polynucleotide Expression in A549 Cells Induced by Formula C Peptides

Peptides at a concentration of 50 μg / ml have been shown to increase the expression of many polynucleotides. Peptides were incubated for 4 hours using human A549 epithelial cells, RNA was isolated, converted to labeled cDNA probes, and hybridized to human operon array (PRHU04). The intensity of the polynucleotides in control unstimulated cells is described in the second and third columns for labeling cDNA with dyes Cy3 and Cy5, respectively. "ID #: Control" column refers to the intensity of polynucleotide expression in peptide stimulated cells divided by the intensity of unstimulated cells.

TABLE 34 Upregulation of Polynucleotide Expression in A549 Cells Induced by Peptides of Formula D

Peptides at a concentration of 50 μg / ml have been shown to increase the expression of many polynucleotides. Peptides were incubated with A549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes and hybridized to human operon array (PRHU04). The intensity of the polynucleotides in the control non-stimulatory cells is shown in the second and third columns for cDNA labeled with dyes Cy3 and Cy5, respectively. The “SEQ ID NO: Control” column is the intensity of polynucleotide expression in peptide-stimulated cells divided by the intensity of unstimulated cells.

TABLE 35 Upregulation of Polynucleotide Expression in A549 Cells Induced by Peptides of Formula E

Peptides at a concentration of 50 μg / ml have been shown to increase the expression of many polynucleotides. Peptides were incubated with human A549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes and hybridized to human operon array (PRHU04). The intensity of the polynucleotides in the control unstimulated cells is shown in the second and third columns for cDNA labeling using dyes Cy3 and Cy5, respectively. The “SEQ ID NO: Control” column is the intensity of polynucleotide expression in peptide-stimulated cells divided by the intensity of unstimulated cells.

TABLE 36 Upregulation of Polynucleotide Expression in A549 Cells Induced by Peptides of Formula F

Peptides at a concentration of 50 μg / ml have been shown to increase the expression of many polynucleotides. Peptides were incubated with human A549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes and hybridized to human operon array (PRHU04). The intensity of the polynucleotides in the control non-stimulatory cells is shown in the second and third columns for cDNA labeled with dyes Cy3 and Cy5, respectively. The “SEQ ID NO: Control Ratio” column is the intensity of polynucleotide expression in peptide-stimulated cells divided by the intensity of unstimulated cells.

TABLE 37 Upregulation of A549 Intracellular Polynucleotide Expression Induced by Expression G and Additional Peptides

Peptides at a concentration of 50 μg / ml have been shown to increase the expression of many polynucleotides. Peptides were incubated with human A549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes and hybridized to human operon array (PRHU04). The intensity of the polynucleotides in the control non-stimulatory cells is shown in the second and third columns for cDNA labeled with dyes Cy3 and Cy5, respectively. The “SEQ ID NO: Control Ratio” column is the intensity of polynucleotide expression in peptide-stimulated cells divided by the intensity of unstimulated cells. Accession numbers and gene names include U00115, zinc finger proteins; M91036, hemoglobin gamma G; K000070, hypothetical protein; AF055899, solute carrier family 27; AK001490, a hypothetical protein; X97674, nuclear receptor co-activating factor 2; AB022847, unknown; AJ275986, transcription factor; D10495, protein kinase C, delta; L36642, EphA7; M31166, pentaxin-related gene; AF176012, unknown; AF072756, A kinase anchor protein 4; NM_014439, IL-1 phase and z; AJ271351, putative transcriptional regulator; AK000576, hypothetical protein; AJ272265, phosphoprotein 2 secreted; AL122038, a hypothetical protein; AK000307, hypothetical protein; AB029001, KIAA1078 protein; U62437 cholinergic receptor; AF064854, unknown; AL031588, a hypothetical protein; X89399, RAS p21 protein activating factor; D45399, phosphodiesterase; AB037716, hypothetical protein; X79981, kadherin 5; AF034208, RIG-like 7-1; AL133355, Chlorophyll 21 open reading frame 53; NM_016281, STE20-like kinase; AF023614, dural activating factor and CAML interactor; AF056717, Ash2-like; AB029039, KIAA1116 protein; J03634, inhibin, beta A; U80764, unknown; AB032963, unknown; X82835, sodium channel, voltage-gate, type IX.

Example  5

Cell line human whole blood  And by peptides in mice Chemokine  Judo

Rat macrophage lines RAW 264.7, THP-1 cells (human monocytes), human epithelial cell line (A549), human bronchial epithelial cells (16HBEo14) and human whole blood were used. HBE cells were grown in MEM containing Earle's. THP-1 cells were grown and maintained in RPMI 1640 medium. RAW and A549 cell lines were maintained in DMEM supplemented with 10% fetal bovine serum. These cells were seeded in 24 well plates at a density of 10 ⁶ cells per well in DMEM (see above) and A549 cells were seeded in 24 well plates at a density of 10 ⁵ cells per well in DMEM (see above) and both 5 Incubated at 37 ° C. overnight in% CO ₂ . DMEM was aspirated from overnight grown cells and replaced with fresh medium. After incubating the cells with peptides, the release of chemokines into the culture supernatants was measured by ELISA (R & D Systems, Minneapolis, Minn.).

Animal studies were approved by the UBC Animal Care Association (UBC ACC # A01-0008). BALB / c mice were purchased from Charles River Laboratories and raised in standard animal facilities. Mature mice of age, sex and weight were intraperitoneally using 200 μl of 10% body weight of avertin (4.4 mM 2-2-2-tribromoethanol, 2.5% 2-methyl-2-butanol in distilled water) Anesthetized by my injection. Infusions were made using the nonsurgical intratracheal injection method adopted by Ho and Furst in 1973. Briefly, the anesthetized mouse is placed and the upper teeth of the anesthetized mouse are hooked on the wire at the top of the support frame, the jaw is opened and the spring is pushed into the throat to face the pharynx, larynx and trachea in a vertical line. The air pathway was irradiated from the outside and an intubation catheter was inserted into the clearly irradiated tracheal lumen. 20 μl of peptide suspension or sterile water was placed in the well at the proximal end of the catheter and 200 μl of air was slowly injected into the trachea. After infusion, the animals were held in an upright position for 2 minutes to allow fluid to drain into the respirator. 300 mg / kg of pentobarbital was euthanized by intraperitoneal injection. The trachea was exposed and the intravenous catheter passed through the proximal trachea and secured with a suture chamber. Washing was performed by introducing 0.75 ml of sterile PBS into the lungs through the tracheal cannula and fluid was withdrawn a few seconds later. This was repeated three times with the same PBS sample. The wash was placed in a tube on ice and the total recovery volume per mouse was about 0.5 ml. Alveolar lavage (BAL) solution was centrifuged at 1200 rpm for 10 minutes and then clear supernatant was tested for TNF-α and MCP-1 by ELISA.

Upregulation of chemokines by cationic peptides has been identified in several different systems. The homologues of murine MCP-1, human MCP-1 are members of the β (C-C) chemokine family. MCP-1 has been validated to recruit monocytes, NK cells and some T lymphocytes. When RAW 264.7 macrophages and human whole blood (obtained from three donors) were stimulated by increasing the concentration of peptide of SEQ ID NO: 1, significant levels of MCP-1 were generated in the supernatant as determined by ELISA (Table 36). . RAW 264.7 cells stimulated with peptides in the 20-50 μg / ml concentration range for 24 hours produced significant levels of MCP-1 (200-400 pg / ml, background above). High levels of MCP-1 were produced when cells (24 hours) and human whole blood (4 hours) were stimulated with 100 μg / ml of LL-37.

In addition, the effect of cationic peptides on chemokine induction was also investigated in completely different cell lines, A549 human epithelial cells. Interestingly, these cells produced MCP-1 in response to LPS and this reaction was antagonized by the peptide, but there was no production of MCP-1 by A549 cells in a direct response to the peptide of SEQ ID NO: 1. However, high concentrations of the peptide of SEQ ID NO: 1 induced the production of IL-8, neutrophil specific chemokines (Table 37). Thus, SEQ ID 1 can elicit different response spectra at different cell types and at different concentrations. Multiple peptides from each group were tested for the ability to induce IL-8 in A549 cells (Table 38). Many of these peptides induced IL-8 above background levels at low concentrations, 10 μg / ml. At high concentrations (100 μg / ml), SEQ ID NO 13 has been shown to induce IL-8 in human whole blood (Table 39). The peptide of SEQ ID NO: 2 also significantly induced IL-8 in HBE cells (Table 40) and undifferentiated THP-1 cells (Table 41).

BALB / c mice were subjected to endotracheal injection of SEQ ID NO: 1 or endotoxin-free water and examined for levels of MCP-1 and TNF-α in alveolar lavage after 3-4 hours. Mice treated with 50 μl / ml peptide of SEQ ID NO: 1 were found to produce significantly increased levels of MCP-1 compared to mice receiving only water or anesthetics (Table 42). Since the peptide did not induce significantly more TNF-α than mice that received only water or anesthetics, this was not a proinflammatory response to the peptide of SEQ ID NO: 1. The peptide of SEQ ID NO: 1 was also found to not significantly induce TNF-α production by bone marrow-derived macrophages and RAW 264.7 cells treated with the peptide of SEQ ID NO: 1 (100 μg / ml or less) (Table 43 ). Thus, the peptide of SEQ ID NO: 1 selectively induces the production of chemokines without inducing the production of inflammatory mediators such as TNF-α. This demonstrates the dual role of the peptide of SEQ ID NO: 1 as a factor capable of blocking the bacterial product-induced inflammation while aiding the recruitment of phagocytes that can cure infection.

TABLE 38 MCP-1 Induction in RAW 264.7 Cells and Human Whole Blood

RAW 264.7 mouse macrophages or human whole blood were stimulated with LL-37 at increased concentration for 4 hours. Human blood samples were centrifuged and serum separated and tested for MCP-1 by ELISA with supernatants obtained from RAW 264.7 cells. RAW cell data present is mean ± standard error of at least 3 experiments and human blood data is mean ± standard error from 3 individual donors.

TABLE 39 Induction of IL-8 in A549 Cells and Human Whole Blood

A549 cells or human whole blood were stimulated with increased concentrations of peptides for 24 and 4 hours, respectively. Human blood samples were centrifuged and serum isolated and tested for IL-8 by ELISA with supernatants obtained from A549 cells. A549 cell data present is mean ± standard error of at least 3 experiments and human blood data is mean ± standard error from 3 individual donors.

TABLE 40 Induction of IL-8 in A549 Cells by Cationic Peptides

A549 human epithelial cells were stimulated with 10 μg peptide for 24 hours. Supernatants were separated and tested for IL-8 by ELISA.

TABLE 41 Induction of IL-8 by Peptides in Human Blood

Human whole blood was stimulated with increasing concentrations of peptide for 4 hours. Human blood samples were centrifuged to separate serum and tested for IL-8 by ELISA. Data is shown on average 2 donors.

TABLE 42 Induction of IL-8 in HBE Cells

Increasing concentrations of peptides were incubated with HBE cells for 8 hours, and the supernatants were separated and tested for IL-8. Data presented are mean ± standard error of 3 or more experiments.

TABLE 43 Induction of IL-8 in Undifferentiated THP-1

Human monocyte THP-1 cells were incubated with the indicated concentrations of peptides for 8 hours. The supernatant was separated and tested for IL-8 by ELISA.

TABLE 44 Induction of MCP-1 by Peptide of SEQ ID NO: 1 in Mouse Airways

BALB / c mice were anesthetized with avertin and did not or did not infuse peptide or water (traditional). Mice were monitored for 4 hours, anesthetized, BAL fluid was isolated and analyzed for MCP-1 and TNF-α concentrations by ELISA. The data described are each condition mean ± standard error of 4 or 5 mice.

TABLE 45 Significant TNF-α Induced Deficiency by Cationic Peptides

RAW 264.7 macrophages were incubated with the indicated peptides (40 μg / ml) for 6 hours. Supernatants were harvested and tested for levels of TNF-α by ELISA. Data is shown as mean ± standard error of three or more experiments.

Example  6

Cationic  Peptides Chemokines  Increases surface expression of receptors

To analyze cell surface expression of IL-8RB, CXCR-4, CCR2 and LFA-1, RAW macrophages were stained with 10 μg / ml of appropriate primary antibody (Santa Cruz Biotechnology) followed by FITC-conjugated goat anti -Rabbit IgG [IL-8RB and CXCR-4 (Jackson ImmunoResearch Laboratories, West Grove, Pa.) Or FITC-conjugated donkey anti-goat IgG (Santa Cruz). Cells were analyzed using FACscan to count 10,000 results and gated on anterior and lateral scatters to remove cell fragments.

Polynucleotide array data suggested that some peptides upregulated the expression of chemokine receptors IL-8RB, CXCR-4 and CCR2 10, 4 and 1.4 fold for these non-stimulatory cells, respectively. Surface expression was examined by flow cytometry of these receptors on RAW cells stimulated with peptides for 4 hours to confirm polynucleotide array data. When incubated 50 μg / ml of peptide with RAW cells for 4 hours, IL-8RB is upregulated 2.4-fold on average of the non-irritating cells, CXCR-4 is 1.6-fold upregulation of the non-irritating cells, CCR2 was upregulated on average 1.8-fold of the non-irritating cells (Table 46). Control CEMA has been shown to cause similar upregulation. Bac2A was the only peptide showing significant upregulation of LFA-1 (3.8 times higher than control cells).

TABLE 46 Increasing surface expression of CXCR-4, IL-8RB and CCR2 in response to peptides

RAW macrophages were stimulated with peptides for 4 hours. Cells were washed and stained with appropriate primary and FITC-labeled secondary antibodies. Data presented represent mean (fold change of RAW cells stimulated with peptide from medium) ± standard error.

Example  7

Cationic  MAP by peptide Kinase Phosphorylation

Cells were seeded at 2.5 × 10 ⁵ to 5 × 10 ⁵ cells / ml and left overnight. They were washed once in the medium and serum fasted in the morning (serum-free medium-4 hours). The medium was separated, replaced with PBS, then placed at 37 ° C. for 15 minutes and returned to room temperature for 15 minutes. Peptides were added (concentration Ol μg / ml to 50 μg / ml) or H ₂ 0 was added and incubated for 10 minutes. PBS was removed very quickly and replaced with ice cold radioimmunoprecipitates (RIPA) buffer containing inhibitors (NaF, B-glycerophosphate, MOL, vanadate, PMSF, leupetin aprotinin). The plate was shaken on ice for 10-15 minutes or until the cells eluted and the eluate was collected. The procedure for THP-1 cells was somewhat different and more cells (2 × 10 ⁶ ) were used. They fasted serum overnight and added 1 ml of ice cold PBS to stop the reaction, then left on ice for 5-10 minutes, spin down and resuspended in RIPA. Protein concentration was measured using protein analysis (Pierce, Rockford, Ill.). Cell eluate (20 μg protein) was separated by SDS-PAGE and transferred to nitrocellulose filter. The filter was blocked for 1 hour with 10 mM Tris-HCl, pH 7.5, 150 mM NaCl (TBS) / 5% skim milk powder for 1 hour and then cold overnight with primary antibody in TBS / 0.05% Tween 20. Incubated. After washing for 30 minutes with TBS / 0.05% Tween 20, the filter was incubated with 1 μg / ml secondary antibody in TBS for 1 hour at room temperature. The filter was washed with TBS / 0.05% Tween 20 for 30 minutes and then incubated with horseradish peroxidase-conjugated amount of anti-mouse IgG (1: 10,000, in TBS / 0.05% Tween 20) for 1 hour at room temperature. It was. After washing the filter with TBS / 0.1% Tween 20 for 30 minutes, the immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection. Peripheral blood (50-100 ml) was collected from all subjects in experiments with peripheral blood mononuclear cells. Monocytes were isolated from peripheral blood by density gradient centrifugation on Ficoll-Hypaque. Hepatic cells (monocytes) were harvested and washed and then resuspended in 10% fetal bovine serum (FCS) and 1% L-glutamine in the recommended cell culture primary medium (RPMI-1640). Cells were added to 6 well culture plates at 4 × 10 ⁶ cells / well and allowed to attach at 37 ° C. in 5% CO ₂ atmosphere for 1 hour. Supernatant medium and non-adherent cells were washed away and appropriate medium containing peptides was added. The newly harvested cells consumed trypan blue, so surviving at all times surpassed 99%. After stimulation with the peptide, the eluate was harvested by eluting the cells in RIPA buffer in the presence of various phosphatase- and kinase-inhibitors. Protein content was analyzed and about 30 μg of each sample was loaded onto a 12% SDS-PAGE gel. Gels were blotted onto nitrocellulose and blocked with 5% skim milk powder in Tris buffered saline (TBS) containing 1% of Triton X 100 for 1 hour. Phosphorylation was detected with phosphorylation-specific antibodies.

The results of peptide-induced phosphorylation are summarized in Table 46. SEQ ID NO 2 has been found to cause dose dependent phosphorylation of p38 and ERK1 / 2 in mouse macrophage RAW cell lines and HBE cells. SEQ ID NO 3 resulted in phosphorylation of MAP kinase in THP-1 human monocyte cell line and phosphorylation of ERK1 / 2 in mouse RAW cell line.

TABLE 47 Phosphorylation of MAP Kinase in Response to Peptides

TABLE 48 Peptide phosphorylation of MAP kinase in human blood monocytes.

50 μg / ml of SEQ ID NO: 1 was used to promote phosphorylation.

Example  8

Cationic  Peptides have an immune response By reinforcement  Prevents bacterial infection

BALB / c mice were injected with intraperitoneal injection of 1 × 10 ⁵ Salmonella and cationic peptides (200 μg). Mice were monitored for 24 hours at their euthanasia point, spleens were isolated, homogenized, resuspended in PBS and plated in Luria nutrient medium agar plates containing canamycin (50 μg / ml). Plates were incubated overnight at 37 ° C. and viable bacteria were counted (Tables 49 and 50). CD-1 mice were injected with 1 × 10 ⁸ S, S. aureus and cationic peptide (200 μg) in 5% porcine mucin by intraperitoneal injection (Table 51). Mice were monitored for 3 days at the time of their euthanasia and blood was separated and plated for survival counts. CD-1 male mice received 5.8 × 10 ⁶ CFU EHEC bacteria and cationic peptides (200 μg) by intraperitoneal (IP) injection and monitored for 3 days (Table 52). In each of these animal models, subsets of peptides have been shown to prevent infection. Most of the protective peptides in the Salmonella model demonstrated the ability to induce a common subset of genes in epithelial cells when comparing the results of the protection assays of Tables 50 and 51 to the gene expression results of Tables 31-37 (Table 53). This clearly indicates that there is a pattern of gene expression consistent with the peptide's ability to exhibit protection. Many cationic peptides have not been shown to be direct antimicrobial when tested by minimum inhibitory concentration (MIC) analysis (Table 54). This demonstrates that the peptide's ability to prevent infection depends on the peptide's ability to stimulate the host's innate immunity rather than its direct antimicrobial activity.

Table 49 Effect of Cationic Peptides on Salmonella Infection in BALB / c Mice

After 24 hours of IP injection of Salmonella and peptides into BALB / c mice, the animals were euthanized and the spleens were isolated, homogenized, diluted in PBS and counted on plates to determine bacterial viability.

Table 50 Effect of Cationic Peptides on Salmonella Infection in BALB / c Mice

After 24 hours of IP injection of Salmonella and peptides into BALB / c mice, the animals were euthanized and the spleens were isolated, homogenized, diluted in PBS and counted on plates to determine bacterial viability.

TABLE 51 Influence of Cationic Peptides on Rat S. aureus Infection Model

CD-1 mice were injected with 1 × 10 ⁸ bacteria in 5% porcine mucin via intraperitoneal (IP) injection. Cationic peptide (200 μg) was fed via separate IP injection. Mice were monitored for 3 days at the time of their euthanasia and blood was separated and plated for survival counts. The peptide of SEQ ID NO: 48, SEQ ID NO: 26 is S. a. It was not effective in controlling Aureus infection.

Table 52 Influence of peptides on rat EHEC infection model

CD-1 male mice (5 weeks old) were injected with 5.8 × 10 ⁶ CFU EHEC via intraperitoneal (IP) injection. Cationic peptide (200 μg) was fed via separate IP injection. Mice were monitored for 3 days.

TABLE 53 Upregulation of gene expression patterns in A549 epithelial cells induced by in vivo active peptides

The peptides of SEQ ID NO: 30, SEQ ID NO: 7 and SEQ ID NO: 13 were found to have increased expression of the gene pattern after 4 hours of treatment at concentrations of 50 μg / ml, respectively. Peptides were incubated with human A549 epithelial cells for 4 hours, RNA was isolated, converted to labeled cDNA probes, and hybridized to human operon array (PRHU04). The intensity of the non-irritating intracellular polynucleotide as a control is shown in the second column (average of Cy3 and Cy5) for labeling of cDNA. The fold upregulation column shows the intensity of polynucleotide expression in peptide-stimulated cells divided by the strength of unstimulated cells. The peptide of SEQ ID NO: 37 was included as a non-active negative control in a rat infection model.

TABLE 54

Most cationic peptides studied here, particularly cationic peptides that are effective in infection models, are not significantly antimicrobial. Serial dilution peptides were incubated overnight with the indicated bacteria in 96-well plates. The lowest concentration of peptide that killed the bacteria was used as the MIC. The symbol> indicates that the MIC is too large to measure. MICs of 8 μg / ml or less are considered to be clinically meaningful activity. The abbreviations are as follows: E. coli , Escherichia coli ); s. S. aureus , Staphylococcus aureus ; blood. P. aerug , Pseudomonas Pseudomonas aeruginosa ); s. S. Typhim , Salmonella Enteritidis S.P. Typhimurium (Salmonella enteritidis ssp typhimurium.); Seed. Rod ( C. rhod ), Chitobacter rhodensis ; EHEC, Enterohaemorrhagic E. coli.

Example  9

Bacteria in Diagnosis / Screening Signaling  Use of Molecule Induced Polynucleotides

S. typhimurium LPS and E. coli 0111: B4 LPS were purchased from Sigma Chemical Co. (St. Louis, MO). LTA (Sigma) derived from S. aureus was resuspended in water (Sigma) containing no endotoxin. Limulus amoebocyte elution assay (Sigma) was performed with LTA preparations so that the aliquots were not significantly contaminated by endotoxins [ie, do not produce cytokines significantly in RAW cell assays. Concentration, <1 ng / ml]. CpG oligodeoxynucleotides were synthesized using Applied Biosystems Inc.'s Model 392 DNA / RNA Synthesizer (Mississauga, Otario), and then purified and resuspended in water containing no endotoxin (Sigma). The following sequences were used: CpG: 5'-TCATGACGTTCCTGACGTT-3 '(SEQ ID NO: 57) and non-CpG: 5'-TTCAGGACTTTCCTCAGGTT-3' (SEQ ID NO: 58). The non-CpG oligos were tested for their cytokine production stimulating ability and found little production of TNF-α or IL-6, which were considered negative controls. Medium alone for 4 hours, and 100 ng / ml of S. typhimurium LPS, 1 μg / ml of S. aureus LTA or 1 μM of CpG [These concentrations were determined by tumor necrosis factor (TNF-α) in RAW cells. RNA is isolated from RAW 264.7 cells incubated with The RNA was used to make cDNA probes hybridized to Clontech Atlas polynucleotide array filters into polynucleotides as described above. Hybridization of cDNA probes to each immobilized DNA was visualized by radiograph and quantified using a phosphoimizer. The results from 2-3 independent experiments are summarized in Tables 55-59. Treatment of RAW 264.7 cells with LPS results in polynucleotides encoding inflammatory proteins such as IL-1β, inducible nitric oxide synthase (iNOS), MIP-1α, MIP-1β, MIP-2α, CD40 and various transcription factors. Increased expression of polynucleotides, including at least 60-fold. When comparing the changes in polynucleotide expression induced by LPS, LTA, and CpG DNA, all three of these bacterial products are proinflammatory polynucleotides such as iNOS, MIP-1α, MIP-2α, IL-1β, IL- It was found that the expression rates of 15, TNFR1 and NF-κB were raised to a similar degree (see Table 57). Table 57 describes that 19 polynucleotides upregulated to a similar extent by bacterial products differ by less than 1.5 times between the three bacterial products in stimulation rate. There were also several polynucleotides downregulated to a similar degree by LPS, LTA and CpG. In addition, the inventors also found that there are a number of polynucleotides that are sequentially regulated in response to the three bacterial products, including a plurality of polynucleotides that differ by at least 1.5 times the expression level between one or more bacterial products (Table 58). ). LTA treatment affects the expression rate of the largest subset of polynucleotides, including hyperstimulation of Jun-D, Jun-B, Elk-1 and cyclin G2 and A1 expression, sequentially compared to LPS or CpG. There are also a few polynucleotides whose expression is further altered by LPS or CpG treatment. Polynucleotides whose expression is further increased due to LPS treatment than LTA or CpG treatment include cAMP response element DNA-binding protein 1 (CRE-BP1), interferon inducible protein 1 and CACCC box-binding protein BKLF. Polynucleotides whose expression is further increased after CpG treatment than during LPS or LTA treatment include leukemia inhibitory factor (LIF) and protease nexin 1 (PN-1). These results suggest that, although LPS, LTA and CpG DNA stimulate a large overlap of polynucleotide expression responses, they also show the ability to sequentially regulate specific subsets of polynucleotides.

Other polynucleotide arrays used include human operon arrays (genome identification number PRHU04-S1), which consists of about 14,000 human oligos distributed in duplicate. Probes were prepared from 5 μg total RNA and labeled with Cy3 or Cy5 labeled dUTP. In this experiment, A549 epithelial cells were plated in a 100 mm tissue culture dish at a concentration of 2.5 × 10 ⁶ cells / dish and incubated overnight, followed by 100 ng / ml of E. coli O111: B4 for 4 hours. Stimulated with LPS. Total RNA was isolated using RNAqueous (Ambion). DNA contamination was removed with a DNA-free kit (Ambion). Probes prepared from total RNA were purified and hybridized onto glass slides printed overnight at 42 ° C. and washed. After washing, images were captured using a Perkin Elmer array scanner. Image processing software (Imapolynucleotide 5.0, Marina Del Rey, Calif.) Measures the mean intensity, medium intensity and background intensity of a spot. The background was removed using a self-made program. The program calculates a baseline 10% intensity for each subgrid and subtracts it for each grid. Analysis was performed using Polynucleotidespring software (Redwood City, Calif.). The intensity for each spot was normalized by taking the median spot intensity value from the value of the in-slide spot cluster and comparing this value to all slide values from the experiment. Relative changes for control cells, which appear in LPS treated cells, are presented in the table below. A number of previously unreported changes useful for diagnosing infections are presented in Table 60.

To confirm and evaluate the functional significance of these changes, selected mRNA and protein levels were assessed and quantified by density measurement. Northern blot using CD14, non-mentin and tristetraproline-specific probes showed similar expression rates after stimulation with all three bacterial products (Table 60). Similarly, the enzyme activity of nitric oxide synthase, iNOS, was measured with Griess reagent to determine the level of NO, an inflammation mediator, and the results showed that the NO levels produced after 24 hours were almost consistent with similar upregulation of iNOS expression. It was found (Table 59). Western blot analysis confirmed that CpG stimulated more leukemia inhibitory factor (LIF, a member of the IL-6 family of cytokines) (Table 59). Through other confirmatory experiments, LPS upregulates the expression of TNF-α and IL-6 (as assessed by ELISA), upregulates the expression of MIP-2α and IL-1β mRNA, and DP-1 And cyclin D mRNA downregulation, as assessed by Northern blot analysis. This assay extended to clinically more relevant extracellular systems by observing the ability of bacterial elements to stimulate proinflammatory cytokine production in human whole blood. This shows that E. coli LPS, S. typhimurium LPS and S. aureus LTA all stimulated similar amounts of serum TNF-α and IL-1β. CpG also stimulated the production of these cytokines, albeit at low levels, which in part confirmed the cell line data.

TABLE 55 Polynucleotides Upregulated by E. coli O111: B4 LPS in A549 Epithelial Cells

E. coli O111: B4 LPS (100 ng / ml) increased the expression of multiple polynucleotides in A549 cells as studied by polynucleotide microarrays. LPS was incubated with A549 cells for 4 hours to separate RNA. Cy3 / Cy5 labeled cDNA probes were prepared using 5 μg total RNA, which was also hybridized onto the human operon array (PRHU04). The strength of unstimulated cells is shown in the second column of Table 55. "LPS / control ratio" column refers to the polynucleotide expression intensity of LPS stimulating cells divided by the intensity of non-stimulatory cells.

Deposit number gene Control group: strength when containing only medium LPS / Control of Rain D87451 Ring Finger Protein 10 715.8 183.7 AF061261 C3H-Type Zinc Finger Protein 565.9 36.7 D17793 Aldo-Keto Reductase Family 1, Member C3 220.1 35.9 M14630 Prothymosin, alpha 168.2 31.3 AL049975 Unknown 145.6 62.3 L04510 ADP-ribosylation factor domain protein 1, 64 kD 139.9 213.6 U10991 G2 protein 101.7 170.3 U39067 Eukaryotic Translation Initiation Factor 3, Subunit 2 61.0 15.9 X03342 Ribosome Protein L32 52.6 10.5 NM_004850 Rho-related, coiled coils containing protein kinase 2 48.1 11.8 AK000942 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 cell death receptor binding protein 41.2 15.7 J02783 Procollagen-proline, 2-oxoglutarate 4-dioxygenase 36.1 14.1 AL137376 Unknown 32.5 17.3 AL137730 Unknown 29.4 11.9

Deposit number gene Control group: strength when containing only medium LPS / Control of Rain D25328 Phosphofructokinase, platelets 27.3 8.5 AF047470 Maleate dehydrogenase 2, NAD 25.2 8.2 M86752 Stress-induced-phosphoprotein 1 22.9 5.9 M90696 Cathepsin S 19.6 6.8 AK001143 Unknown 19.1 6.4 AF038406 NADH dehydrogenase 17.7 71.5 AK000315 Hypothetical protein FLJ20308 17.3 17.4 M54915 pim-1 cancer gene 16.0 11.4 D29011 Proteasome Subunit, Beta Type, 5 15.3 41.1 AK000237 Membrane Proteins of Cholinergic Synaptic Vesicles 15.1 9.4 AL034348 Unknown 15.1 15.8 AL161991 Unknown 14.2 8.1 AL049250 Unknown 12.7 5.6 AL050361 PTD017 protein 12.6 13.0 U74324 RAB interaction factor 12.3 5.2 M22538 NADH dehydrogenase 12.3 7.6 D87076 KIAA0239 Protein 11.6 6.5 NM_006327 Translocase of the Internal Mitochondrial Membrane 23 (Yeast) Homolog 11.5 10.0 AK001083 Unknown 11.1 8.6 AJ001403 Mucin 5, subtype B, bronchus 10.8 53.4

Deposit number gene Control group: strength when containing only medium LPS / Control of Rain M64788 RAP1, GTPase Activating Protein 1 10.7 7.6 X06614 Retinoic Acid Receptor, Alpha 10.7 5.5 U85611 Calcium and Integrating Binding Proteins 10.3 8.1 U23942 Cytochrome P450,51 10.1 10.2 AL031983 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 Ribophorin 1 9.3 35.7 L05515 cAMP response element-binding protein CRE-BPa 8.9 6.2 X83368 Phosphoinositide-3-kinase, Catalytic, Gamma Polypeptides 8.7 27.1 M30269 Nidogen (Inactin) 8.7 5.5 M91083 Chromosome 11 open reading frame 13 8.2 6.6 D29833 Saliva Proline-Rich Protein 7.7 5.8 AB024536 Immunoglobulins Containing Leucine-Rich Repeats 7.6 8.0 U39400 Chromosome 11 open reading frame 4 7.4 7.3 AF028789 unc119 (C. elegance) homologue 7.4 27.0 NM_003144 Signal Sequence Receptor, Alpha (Translocon-Related Protein Alpha) 7.3 5.9

Deposit number gene Control group: strength when containing only medium LPS / Control of Rain X52195 Arachidonate 5-lipoxygenase-activated protein 7.3 13.1 U43895 Human Growth Factor-Regulatory Tyrosine Kinase Substrate 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 Pre-Stylated Alanine-Rich Protein Kinase C Substrate 6.1 5.8 NM_014442 Sialic Acid Bonded Ig-Like Lectin 8 6.1 7.6 U81375 Solute Carrier Family 29 6.0 6.4 AF041410 Malignant Tumor-Related Proteins 5.9 5.3 U24077 Killer Cell Immunoglobulin-Like Receptors 5.8 14.4 AL137614 Virtual protein 4.8 6.8 NM_002406 Mannosyl (alpha-1,3) -glycoprotein beta-1,2-N-acetylglucosaminyl transferase 4.7 5.3 AB002348 KIAA0350 Protein 4.7 7.6 AF165217 Tropomodulin 4 (Muscle) 4.6 12.3 Z14093 Branched Chain Ketosan Dehydrogenase E1, Alpha Polypeptides 4.6 5.4 U82671 Caltratin 3.8 44.5

Deposit number gene Control group: strength when containing only medium LPS / Control of Rain AL050136 Unknown 3.6 5.0 NM_005135 Solute Carrier Family 12 3.6 5.0 AK001961 Hypothetical protein FLJ11099 3.6 5.9 AL034410 Unknown 3.2 21.3 S74728 Antiquitin 1 3.1 9.2 AL049714 Ribosome Protein L34 G2 Gene 3.0 19.5 NM_014075 PRO0593 Protein 2.9 11.5 AF189279 Phospholipase A2, IIE group 2.8 37.8 J03925 Integrin, alpha M 2.7 9.9 NM_012177 F-box protein Fbx5 2.6 26.2 NM_004519 Potassium Voltage-Gated Channels, KQT-Like Subfamily, Member 3 2.6 21.1 M28825 CD1A antigen, polypeptide 2.6 16.8 X16940 Actin, gamma 2, intestinal smooth muscle 2.4 11.8 X03066 Primary histocompatibility complex, class II, DO beta 2.2 36.5 AK001237 Hypothetical protein FLJ10375 2.1 18.4 AB028971 KIAA1048 Protein 2.0 9.4 AL137665 Unknown 2.0 7.3

TABLE 56 Polynucleotides Downregulated by E. coli O111: B4 LPS in A549 Epithelial Cells

As studied by polynucleotide microarrays, E. coli O111: B4 LPS (100 ng / ml) reduced the expression of multiple polynucleotides in A549 cells. LPS was incubated with A549 cells for 4 hours before RNA was isolated. Cy3 / Cy5 labeled cDNA probes were prepared using 5 μg total RNA, which was also hybridized onto the human operon array (PRHU04). The strength of unstimulated cells is shown in the second column of the table below. "LPS / control ratio" column refers to the polynucleotide expression intensity in LPS stimulating cells divided by the intensity of unstimulated cells.

Deposit number gene Control group: strength when containing only medium LPS / Control of Rain 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 Virtual protein 602.8 0.05 M96843 Inhibitors of DNA Binding 2, Negative Helix-Loop-Helix Protein 30.7 0.05 S79854 Diiodinase, Iodothyronine, Type III 39.4 0.06 AB018266 Matryn 3 15.7 0.08 M33374 NADH dehydrogenase 107.8 0.09 AF005220 Homo sapiens mRNA for NUP98-HOXD13 fusion protein, partial cds 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 (Stomach) 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 X-ray repair complementarity defect repair in Chinese hamster cells 5 11.3 0.16 L36055 Eukaryotic Translation Initiation Factor 4E 182.5 0.16

Deposit number gene Control group: strength when containing only medium LPS / Control of Rain Binding protein 1 Z80779 H2B Histone Family, member G 54.3 0.16 AF226869 5 (3) -deoxyribonucleotidase; RB-related KRAB inhibitor 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-Related Atogenogen 5 6.2 0.19 X57138 H2A Histone Family, member N 58.3 0.20 AF081281 Lysophospholipase Ⅰ 7.2 0.22 U96759 Saw Hippel-Lindau Binding Protein 1 6.6 0.22 U85977 Human ribosomal protein L12 gastric, partial cds 342.6 0.22 D13315 Glyoxalase I 7.5 0.22 AC003007 Unknown 218.2 0.22 AB032980 RU2S 246.6 0.22 U40282 Integrin-binding kinase 10.1 0.22 U81984 Epithelial PAS Domain Protein 1 4.7 0.23 X91788 Chloride channel, nucleotide-sensitive, 1A 9.6 0.23 AF018081 Collagen, Type Soap, Alpha 1 6.9 0.24 L31881 Nuclear phase I / VIII (CCAAT-binding transcription factor) 13.6 0.24 X61123 B-cell translocation gene 1, anti-proliferative 5.3 0.24 L32976 Mitogen-activated protein kinases 6.3 0.24

Deposit number gene Control group: strength when containing only medium LPS / Control of Rain Kinase 11 M27749 Immunoglobulin lambda-like polypeptide 3 5.5 0.24 X57128 H3 Histone Family, member C 9.0 0.25 X80907 Phosphoinositide-3-kinase, regulatory subunit, polypeptide 2 5.8 0.25 Z34282 H. sapiens (MAR11) MUC5AC mRNA for mucin (part) 100.6 0.26 X00089 H2A Histone Family, member M 4.7 0.26 AL035252 CD39-Like 2 4.6 0.26 X95289 PERB11 family member in MHC class I domain 27.5 0.26 AJ001340 U3 snoRNP-related 55 kDa protein 4.0 0.26 NM_014161 HSPC071 Protein 10.6 0.27 U60873 Unknown 6.4 0.27 X91247 Thioridoxin reductase 1 84.4 0.27 AK001284 Virtual Protein FLJ 10422 4.2 0.27 U90840 Synovial Sarcoma, X Rupture Point 3 6.6 0.27 X53777 Ribosome Protein L17 39.9 0.27 AL035067 Unknown 10.0 0.28 AL117665 DKFZP586M1824 Protein 3.9 0.28 L14561 ATPase, Ca2 + transport, plasma membrane 1 5.3 0.28 L19779 H2A Histone Family, Member O 30.6 0.28 AL049782 Unknown 285.3 0.28 X00734 Tublin, Beta 5 39.7 0.29 AK001761 Retinoic Acid Induction 3 23.7 0.29 U72661 Ninh Jurin 1 4.4 0.29

Deposit number gene Control group: strength when containing only medium LPS / Control of Rain S48220 Diiodinase, Iodothyronine, Type I 1,296.1 0.29 AF025304 EphB2 4.5 0.30 S82198 Chymotrypsin C 4.1 0.30 Z80782 H2B Histone Family, member K 31.9 0.30 X68194 Synaptophycin-like protein 7.9 0.30 AB028869 Unknown 4.2 0.30 AK000761 Unknown 4.3 0.30

TABLE 57 Polynucleotides expressed to a similar degree after stimulation with bacterial products LPS, LTA and CpG DNA

Bacterial products (100 ng / ml S. typhimurium LPS, 1 μg / ml S. aureus LTA or 1 μM CpG) have been shown to potently induce the expression of several polynucleotides. Peptides were incubated with RAW cells for 4 hours, RNA was isolated and then converted to labeled cDNA probes to hybridize to Atlas arrays. The intensity of the control cells, unstimulated cells, is shown in the second column. “LPS / LTA / CpG: Control Ratio” column refers to the intensity of polynucleotide expression in bacterial product-stimulated cells divided by the strength of unstimulated cells.

TABLE 58Polynucleotides sequentially regulated by bacterial products LPS, LTA and CpG DNA

Bacterial products (100 ng / ml S. typhimurium LPS, 1 μg / ml S. aureus LTA or 1 μM CpG) have been shown to potently induce the expression of several polynucleotides. Peptides were incubated with RAW cells for 4 hours, RNA was isolated and then converted to labeled cDNA probes to hybridize to Atlas arrays. The intensity of the control cells, unstimulated cells, is shown in the second column. “LPS / LTA / CpG: Control Ratio” column refers to the intensity of polynucleotide expression in bacterial product-stimulated cells divided by the intensity of polynucleotide expression in unstimulated cells.

TABLE 59 IDENTIFICATION OF TABLE 57 AND 58 ARRAY DATA

a) Isolating total RNA from unstimulated RAW macrophages, the cells were combined with 100 ng / ml S. typhimurium LPS, 1 μg / ml S. aureus LTA, 1 μM CpG DNA for 4 hours, Or media alone, followed by Northern blot [as described above, the membrane probed for GAPDH, CD14, bimentin and tristetraproline; Scott et al.]. Comparing the hybridization strength of the Northern blot with GAPDH, we found a loading mismatch. This experiment was repeated three or more times, and the data shown below are the average relative level (measured by a density meter) ± standard error of each condition compared to the medium.

b) RAW 264.7 cells were stimulated by incubation with 100 ng / ml S. typhimurium LPS, 1 μg / ml S. aureus LTA, 1 μM CpG DNA, or with medium alone for 24 hours. The protein eluate was prepared and then run on an SDS PAGE gel and western blots were performed to detect LIF (R & D Systems). This experiment was repeated three or more times, and the data shown below are relative levels of LIF (measured by a density meter) ± standard error compared to the medium.

c) Supernatants were collected from RAW macrophages treated with 100 ng / ml S. typhimurium LPS, 1 μg / ml S. aureus LTA, 1 μM CpG DNA, or media alone for 24 hours. This was tested with Griess reagent for the amount of NO produced in the supernatant, measured from the accumulation of stable NO metabolite nitrite as described above [Scott et al.]. The data presented here are the mean ± standard error of three experimental values.

TABLE 60 Gene Expression Patterns in A549 Human Epithelial Cells Up-Regulated by Bacterial Signaling Molecule (LPS)

E. coli O111: B4 LPS (100 ng / ml) increased the expression of multiple polynucleotides in A549 cells, as studied by polynucleotide microarrays. LPS was incubated with A549 cells for 4 hours before RNA was isolated. Cy3 / Cy5 labeled cDNA probes were prepared using 5 μg total RNA, which was also hybridized onto human operon array (PRHU04). For example of changes in polynucleotide expression in LPS stimulated cells, changes in intensity levels of LPS treated cells derived from untreated cells are more than twofold.

Example  10

For protection against bacterial infections Signaling  change

Salmonella typhimurium strain SL1344 was obtained from the US Bacterial Culture Collection (ATCC, Manor, Va.) And grown in Luria-Butani (LB) broth. For macrophage infection, 10 ml of LB obtained from frozen glycerol stock was inoculated into 125 ml flasks and incubated overnight on 37 ° C. in stationary phase with shaking. RAW 264.7 cells (1 × 10 ⁵ cells / well) were seeded in 24 well plates. The bacteria were diluted in culture medium to give a nominal multiplicity of infection (MOI) of about 100, and the bacteria were centrifuged at 1000 rpm for 10 minutes to synchronize infection on a monolayer and also at 37 ° C., 5% CO _2. Infection was allowed to proceed for 20 minutes in the incubator. Cells were washed three times with PBS to remove extracellular bacteria, which were then incubated in 10% FBS (Sigma, St. Louis, MO) containing DMEM + 100 μg / ml of gentamicin. Re-infection was prevented by killing extra bacteria. After 2 hours, the gentamycin concentration was lowered to 10 μg / ml and maintained at this concentration throughout the assay. Cells were pretreated with inhibitor for 30 minutes and then infected at the following concentrations: 50 μM PD 98059 (Calbiochem), 50 μM U 0126 (Promega), 2 mM diphenylyodonium (DPI), 250 μM acetobar Nilon (aposinine, Aldrich), 1 mM ascorbic acid (Sigma), 30 mM N-acetyl cysteine (Sigma), and 2 mM N ^G -L-monomethyl arginine (L-NMMA, Molecular Probes) or 2 mM N ^G -D-monomethyl arginine (D-NMMA, Molecular Probes). Efficacy was confirmed by the addition of fresh inhibitors 2 hours and 6-8 hours after infection. Control cells were treated with eastern blood dimethylsulfoxide (DMSO) per ml of medium. As mentioned above, the intracellular survival / replicate rate of S. typhimurium SL1344 was measured using a Gentamicin-resistant assay. In summary, cells were washed twice with PBS to remove gentamicin and eluted with 1% Triton X-100 / 0.1% SDS in PBS at 2 and 24 hours post infection and then from colonies on LB agar plates. The number of intracellular bacteria was counted. As assessed by standard plate counting, under these infection conditions, macrophages contained an average of one bacterium per cell, allowing analysis of macrophages 24 hours after infection. Bacteria filamentation is associated with bacterial stress. NADPH oxidase and iNOS can be activated by MEK / ERK signaling. The results (Table 61) clearly demonstrated that altering cell signaling is a way to solve the problem of intracellular Salmonella infection. Thus, bacteria upregulate many genes in human cells, so the technique of blocking signaling represents a common treatment for infection.

TABLE 61 Effect of signaling molecule MEK on intracellular bacteria in IFN-γ-primed RAW cells

Example  11

Antiviral activity

SDF-1, ie CXC chemokine, is a natural ligand for HIV-1 co-receptor-CXCR4. Chemokine receptors CXCR4 and CCR5 are considered potential targets for inhibition of HIV-1 replication. The crystal structure of SDF-1 exhibits a form that plays an important role in binding to the antiparallel β-sheet and the positively charged surface, the negatively charged extracellular loop of CXCR4. These findings suggest that chemokine derivatives, small CXCR4 antagonists or agonists that simulate the structure or ionicity of chemokines may be useful as agents for treating X4 HIV-1 infection. Cationic peptides have been found to inhibit SDF-1 inducible T-cell migration, suggesting that the peptides can act as CXCR4 antagonists. Migration analysis was performed as follows. Human Jurkat T-cells were resuspended in chemotaxis medium [RPMI 1640/10 mM Hepes / 0.5% BSA] at a concentration of 5 × 10 ⁶ / ml. Migration assays were performed in 24 well plates using a 5 μm polycarbonate Transwell insert (Costar). In summary, peptides or controls were diluted in chemotactic media, placed in the lower chamber, and 0.1 ml of cells (5 × 10 ⁶ / ml) were added to the upper chamber. After 3 hours at 37 ° C., the number of cells moved to the lower chamber by flow cytometry was measured. The medium obtained from the lower chamber was passed through a FACscan for 30 seconds and gated with an omnidirectional and lateral scatter to exclude cellular debris. The number of viable cells was compared to the “100% migration control” where the viable cells were pipetted directly into the lower chamber with 5 × 10 ⁵ / ml cells and counted on FACscan for 30 seconds. The results inhibited the migration of human Jurkat T-cells (Table 62) due to affecting CXCR4 expression (Tables 63 and 64).

TABLE 62 Peptides Inhibit Migration of Human Jurkat-T Cells:

% Movement Experiment Positive control SDF-1 (100 ng / ml) SDF-1 + SEQ ID NO: 1 (50 μg / ml) Negative control One 100 32 0 <0.01 2 100 40 0 0

TABLE 63 Polynucleotide Array Data Corresponding to Table 56

Polynucleotide / protein Polynucleotide function Strength of unstimulated cells Peptides: Non-irritating Cells Deposit number CXCR-4 Chemokine receptor 36 4 D87747

TABLE 64 FACs Data Corresponding to Tables 62 and 63

Peptide Concentration (μg / ml) Increase in Folding in Protein Expression CXCR-4 SEQ ID NO: 1 10 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

Cooperative Combination

Method and material

S. aureus was prepared at a final expected concentration of 1-4 × 10 ⁷ CFU / mL in phosphate buffer solution (PBS) and 5% porcine mucin (Sigma). 100 μl of S. aureus (mixed with 5% porcine mucin) was injected intraperitoneally (IP) into each CD-1 mouse (6-8 week old female, 20-25 g in weight) (Charles River). Six hours after the start of infection, 100 μl of peptide was injected IP with 0.1 mg / kg Cefepime (50-200 μg total). After 24 hours, animals were sacrificed and 100 ul of blood was removed through the heart. The blood was diluted in 1 ml PBS containing heparin. It was then diluted further and plated for viable colony counts on Mueller-Hinton agar plates (10 ⁻¹ , 10 ⁻² , 10 ⁻³ , & 10 ⁻⁴ ). After 24 hours, viable colonies, colony forming units (CFUs) were counted. Each experiment was performed at least three times. Data are shown as mean CFU ± standard error per treatment group (8-10 mice / group).

Experiments were performed with peptides and the next dose of cefepime 6 hours after the start of systemic S. aureus infection (FIG. 1). The data in FIG. 1 are shown as mean ± standard error of survival counts from blood taken from mice 24 hours after the start of infection. Subsequent doses of the antibiotic (cefepime) and the combination of SEQ ID NO: 7 improved the efficacy of the treatment. An important finding is the ability of peptides to work in combination with suboptimal concentrations of antibiotics in rat infection models. This suggests the potential for reducing the incidence of antibiotic resistance and prolonging antibiotic life in clinics.

For example, SEQ ID 1 induces activation and phosphorylation of mitogen activated protein kinases, ERK 1/2 and p38, in human peripheral blood derived monocytes and human bronchial epithelial cell lines, but not in B- or T-lymphocytes. . Phosphorylation is not dependent on the G-protein coupled receptor, FPRL-1, which has previously been suggested as a receptor for SEQ ID NO 1 induced chemotaxis in human monocytes and T cells. Activation of ERK 1/2 and p38 was markedly increased by the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) but not macrophage-colony stimulating factor (M-CSF). In addition, exposure to SEQ ID NO: 1 activated Elk-1, a downstream transcription factor activated by phosphorylated ERK 1/2, and upregulated various Elk-1 control genes. The ability of SEQ ID NO: 1 to signal through these pathways broadly implies immunity, monocyte activation, proliferation and differentiation.

Method and material

SEQ ID NO: 1 (SEQ ID NO: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was synthesized by Fmoc [(N- (9-fluorenyl) methoxycarbonyl)] chemistry in 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 List Biological Laboratories Inc. (Campbell, CA, USA).

Blood monocytes were prepared using standard techniques. Briefly, 100 ml of fresh human venous blood was collected from a volunteer in a sodium heparin vacutainer collection tube (Becton Dickinson, Mississauga, ON, Canada) according to the UBC Clinical Research Ethics Committee Certificate Protocol C02-0091. RPMI 1640 medium [10% v / v fetal bovine serum (FBS), 1% L in endotoxin-free bottles washed with E-toxa-clean (Sigma- Aldrich, Oakville, ON, Canada) -Glutamine, supplemented with 1 nM sodium pyruvate] was used to mix the blood in a 1: 1 ratio. PBMC was isolated at room temperature using Ficoll-Paque Plus (Amersham Pharmacia Biotech, Baie D'Urfe, PQ, Canada) and washed with phosphate buffered saline (PBS). Monocyte is neuraminidase ( Vibrio to Vibrio cholera T cells were removed by repeating with fresh sheep red blood cells (UBC animal care unit) pretreated with cholerae neuraminidase) (Calbiochem Biosciences Inc., La Jolla, CA, USA) and repeated isolation by Ficoll Paque Plus Was strengthened. The enhanced monocytes were washed with PBS, then incubated at 37 ° C. for 1 hour (approximately 2-3 × 10 ⁶ per well) and then non-adherent cells were removed; Monocytes were> 95% pure as determined by flow cytometry (data not shown). B-lymphocytes were isolated by removing non-adherent cells and adding them to a new plate at 37 ° C. for 1 hour. This was repeated three times in total. Any remaining monocytes and residual non-adherent cells attached to the plate were predominantly B cells. Cells were cultured in Falcon tissue culture 6 well plates (Becton Dickinson, Mississauga, ON, Canada). The attached monocytes were incubated at 37 ° C. in 1 ml medium added with SEQ ID NO: 1 and / or cytokines dissolved in endotoxin free water (Sigma-Aldrich, Oakville, ON, Canada). Endotoxin free water was added as vehicle control. For the study using pertussis toxin, the medium was replaced with 1 ml fresh medium containing 100 ng / ml of toxin and incubated at 37 ° C. for 60 minutes. SEQ ID NO: 1 and cytokines were added directly to the medium containing pertussis toxin. For isolation of T lymphocytes, rosetted T cells and sheep erythrocytes were resuspended in 20 ml PBS and both erythrocytes were eluted by the addition of 10 ml distilled water. The cells were then centrifuged at 1000 rpm for 5 minutes, then the supernatant was removed. Pelletized T cells were immediately washed with PBS and increasing amounts of water were added until all both red blood cells eluted. Residual T cells were washed once with PBS and viability was confirmed using 0.4% Trypan Blue solution. Incubating primary human blood monocytes and T cells in RPMI 1640 (GIBCO Invitrogen Corporation, Burlington, ON, Canada) supplemented with 10% v / v heat inactivated FBS, 1% v / v L-glutamine, 1 nM sodium pyruvate It was. 2 to 8 donors were used for each experiment.

The simian virus 40-transformed immortalized 16HBE4o-bronchial epithelial cell line was obtained from Dr. D. Gruenert (University of California, San Francisco, CA). Cells were routinely cultured at 37 ° C., 100% humidity and 5% CO ₂ . They were grown in Minimal Essential medium using Earles' salts (GIBCO Invitrogen Corporation, Burlington, ON, Canada) containing 10% FBS (Hyclone), 2 mM L-glutamine. For the experiment, cells were grown on Costar Transwell inserts (pore size 3 μm, Fischer Scientific) in 24-well plates. 0.95 mL of medium was added to the bottom of the well and seeded with 5 × 10 ⁴ cells per 0.25 mL of medium on top of the insert while incubating the cells at 37 ° C., 5% CO ₂ . Intermembrane resistance was measured daily using a Millipore voltohmeter and when the resistance was 500-700 ohms, the inserts were typically used for experiments after 8-10 days. Cells were used between 8 and 20 passages.

Weston Immunoblotting (Western Immunoblotting )

After stimulation, cells were washed with ice cold PBS containing 1 mM vanadate (Sigma). Next, 125 μl of RIPA buffer (50 mM Tris-HCl, pH 7.4, NP-40 1%, sodium deoxycholate 0.25%, NaCl 150 mM, EDTA 1 mM, PMSF 1 mM, Aprotinin, Peptin, pepstatin 1 μg / ml each, sodium orthovanadate 1 mM, NaF 1 mM) were added and cells were incubated on ice until fully eluted as assessed visually. Eluates were quantified using BCA analysis (Pierce). 30 μg of eluate was loaded into a 1.5 mm thick gel and developed at 100 volts for approximately 2 hours. The protein was transferred to a nitrocellulose filter at 70 V for 75 minutes. The filter was blocked in TBST (10 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% Tween-20) using 5% skim milk for 2 hours at room temperature. The filter was then incubated at 4 ° C. overnight with anti-ERK1 / 2-P or anti-p38-P (Cell Signaling Technology, Ma) monoclonal antibody. Immunoreactive bands were detected using horseradish peroxidase-conjugated amounts of anti-mouse IgG antibody (Amersham Pharmacia, New Jersey) and chemiluminescent detection (Sigma, Mo). In order to quantify the band, the film was scanned and quantified by a density meter using a software program ImageJ. Blots were probed again with β-actin antibody (ICN Biomedical Incorporated, Ohio) and corrected for protein loading using a density meter.

Kinase  analysis

ERK1 / 2 activity assays were performed using a non-radioactive kit (Cell Signaling Technology). Briefly, cells were treated for 15 minutes and eluted in elution buffer. The same amount of protein was immunoprecipitated with an immobilized phospho-ERK1 / 2 antibody that reacts only with the phosphorylated (ie active) form of ERK1 / 2. The immobilized precipitating enzyme was then used for kinase analysis using Elk-1, followed by Western blot analysis using an antibody that allowed the detection and quantification of phosphorylated substrates.

IL- 8 of  Quantification

Human IL-8 from the supernatant of 16HBE40-cells was measured using a commercial enzyme-linked immunosorbent assay kit (Biosource) according to the manufacturer's instructions.

Semiquantitative  RT- PCR

Total RNA from two independent experiments was isolated from 16HBE4o cells using RNaqueous (Ambion) as described by the manufacturer. After DNase treatment of the samples, cDNA synthesis was achieved using a first strand cDNA synthesis kit (Gibco). The resulting cDNAs were subjected to various cytokine genes MCP-1 (5 * -TCATAGCAGCCACCTTCATTC-3 '(SEQ ID NO: 59), 5'-TAGCGCAGATTCTTGGGTTG-3 (SEQ ID NO: 60)), MCP-3, (5 * -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 carried out at least in duplicate. The results were analyzed by linear amplification phase and normalized to control glyceraldehyde 3-phosphate dehydrogenase. The response was demonstrated for RNA amplification by including a control without reverse transcriptase.

result

A. Peptides induce ERK1 / 2 and p38 phosphorylation in monocytes derived from peripheral blood.

To determine whether the peptide induces activation of MAP kinases, ERK1 / 2 and / or p38, peripheral blood derived monocytes were treated with 50 μg / ml SEQ ID NO 1 or water (as vehicle control) for 15 minutes. In order to visualize the activating (phosphorylated) form of the kinase, Western using antibodies specific for the double phosphorylated form of kinase (phosphorylated to Thr202 + Tyr204 and Thr180 + Tyr182 for ERK1 / 2 and p38, respectively) Blots were performed. The gel was probed again using antibodies against β-actin to normalize for loading differences. In all, an increase in phosphorylation of ERK1 / 2 (n = 8) and p38 (n = 4) was observed corresponding to SEQ ID NO 1 treatment (FIG. 2).

2 shows that exposure to SEQ ID NO 1 induces phosphorylation of ERK1 / 2 and p38. Eluate from human peripheral blood derived monocytes was exposed to 50 μg / ml of SEQ ID NO 1 for 15 minutes. A) Activation of ERK1 / 2 and p38 was detected using antibodies specific for phosphorylated forms of ERK and p38. All donors tested showed increased phosphorylation of ERK1 / 2 and p38 in response to SEQ ID NO 1 treatment. Representative donor of one of eight. The relative amounts of phosphorylation of ERK (B) and p38 (C) were determined by dividing the intensity of the phosphorylation band by the intensity of the corresponding control band as described in Materials and Methods.

B. Peptide induced activation of ERK1 / 2 is greater in human serum than fetal bovine serum.

The LL-37 induced phosphorylation of ERK1 / 2 does not occur in the absence of serum and the magnitude of phosphorylation may result in that activation of ERK1 / 2 is much superior to human serum (HS) than to fetal bovine serum (FBS). It could be proved to depend on the type of serum present.

3 shows that LL-37 induced phosphorylation of ERK1 / 2 does not occur in the absence of serum and that the size of phosphorylation depends on the type of serum present. Human blood derived monocytes were treated with 50 μg / ml of LL-37 for 15 minutes. The eluate was run on a 12% acrylamide gel, then transferred to the nitrocellulose membrane and probed with an antibody specific for the phosphorylated (active) form of the kinase. To normalize for protein loading, the blots were probed again with β-actin. Quantification was done with ImageJ software. 3 Implantation demonstrates that LL-37 cannot induce MAPK activation in human monocytes under serum free conditions. Cells were exposed to 50 mg / ml of LL-37 (+) or endotoxin water (-) as vehicle control for 15 minutes. (A) Phosphorylated ERK1 / 2 was detectable after exposure to LL-37 in medium containing 10% fetal bovine serum, but phosphorylation of ERK1 / 2 was not detected in the absence of serum ( n = 3). (B) Elk-1, ERK1 / 2 downstream transcription factors were activated upon exposure to 50 μg / ml of LL-37 in medium containing 10% fetal bovine serum, but not in the absence of serum (n = 2 ).

C. Peptide-induced activation of ERK1 / 2 and p38 is dose dependent and demonstrates synergy with GM-CSF.

GM-CSF, IL-4, or M-CSF (at 100 ng / ml each) was added simultaneously with SEQ ID NO: 1 and phosphorylation of ERK1 / 2 was measured in fresh isolated human blood monocytes. ERK1 / 2 phosphorylation is seen when cells are treated with 50 μg / ml of SEQ ID NO 1 (8.3 times greater than untreated, n = 9) but not at lower concentrations (n = 2). In the presence of 100 ng / ml GM-CSF, SEQ ID NO: 1 induced ERK1 / 2 phosphorylation was significantly increased (58 times greater than untreated, n = 5). In addition, in the presence of GM-CSF, activation of ERK1 / 2 occurred corresponding to SEQ ID NO 1 concentrations of 5 and 10 μg / ml, respectively, for the two donors tested (FIG. 4). This demonstrates that SEQ ID NO 1 induced activation of ERK1 / 2 occurs in the lower reverse in the presence of GM-CSF, a cytokine found locally at the site of infection.

4 shows that LL-37 induced activation of ERK1 / 2 occurs at lower concentrations and is amplified in the presence of specific cytokines. When fresh isolated monocytes were stimulated in a medium containing both GM-CSF (lOOng / ml) and IL-4 (100 ng / ml), the LL-37 induced phosphorylation of ERK1 / 2 was as much as 5 μg / ml. It is evident even at low concentrations. The synergistic activation of ERK1 / 2 appears to be mainly due to GM-CSF.

D. Activation of ERK1 / 2 causes transcription of Elk-1 control genes and secretion of IL-8.

IL-8 release is at least partly governed by the activation of ERK1 / 2 and p38 kinases. To determine whether the peptide can induce IL-8 secretion, the human bronchial cell line, 16HBE4o-, was grown in a transwell filter in saturation, which allowed for polarization of cells with clear attachment and basal surface development. Allow. When cells were stimulated with 50 μg / ml of SEQ ID NO 1 at the attachment surface for 4 hours, it was detected that the amount of IL-8 released into the attachment supernatant was statistically significantly increased (FIG. 5). To determine the downstream transcriptional effect of peptide induced MAP kinase activation, expression of genes known to be regulated by ERK1 / 2 or p38 was assessed by RT-PCR. RT-PCR was performed on RNA isolated from 16HBE4o-cells from two independent experiments and treated for 4 hours using 50 μg / ml SEQ ID NO 1 in the presence of serum. MCP-1 and IL-8 are under transcriptional control of both ERK1 / 2 and p38, and they have been demonstrated to be upregulated by 2.4 and 4.3 fold, respectively. Transcription of MCP-3 is affected by the activation of mitogen activated protein kinases, and accordingly, it has not been previously demonstrated that expression is not affected by peptide treatment (FIG. 5). These data are consistent with the assumption that activation of the ERK1 / 2 and p38 signaling pathways has a functional effect on the transcription of cytokine genes with immunomodulatory functions. Insertion of FIG. 3B also demonstrates that the peptide induces phosphorylation of the transcription factor Elk-1 in a serum dependent manner.

5 shows that peptides affect both transcription of various cytokine genes and release of IL-8 in the 16HBE4o-human bronchial epithelial cell line. Cells were grown in saturation on the semipermeable membrane and stimulated at the attachment surface using 50 μg / ml SEQ ID NO 1 for 4 hours. A) The SEQ ID NO 1 treated cells generated significantly more IL-8 than the control as detected by ELISA (but not at the basal surface) in the supernatants collected at the attachment surface. The mean ± standard error of the three independent tests is shown and an asterisk indicates p = 0.002. B) RNA was collected in the above experiments and RT-PCR was performed. Many cytokine genes known to be regulated by ERK1 / 2 or p38 were upregulated upon stimulation with peptides. The mean of two independent experiments is shown.

While the present invention has been described with reference to preferred embodiments, it will be understood that various modifications are possible without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

                         SEQUENCE LISTING <110> THE UNIVERSITY OF BRITISH COLUMBIA       HANCOCK, Robert E. W.       FINLAY, B. Brett       SCOTT, Monisha Gough       BOWDISH, Dawn       ROSENBERGER, Carrie Melissa       POWERS, Jon-Paul STEVEN <120> EFFECTORS OF INNATE IMMUNITY DETERMINATION <130> UBC1180-2KR <150> PCT / CA2004 / 001602 <150> 2004-09-10 <150> US 10 / 661,471 <151> 2003-09-12 <150> US 10 / 308,905 <151> 2002-12-02 <160> 66 <170> PatentIn version 3.1 <210> 1 <211> 37 <212> PRT <213> Homo sapiens <400> 1 Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5 10 15 Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val             20 25 30 Pro Arg Thr Glu Ser         35 <210> 2 <211> 13 <212> PRT <213> Bovine <400> 2 Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg 1 5 10 <210> 3 <211> 12 <212> PRT <213> Bovine <400> 3 Arg Leu Ala Arg Ile Val Val Ile Arg Val Ala Arg 1 5 10 <210> 4 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Cationic peptide <220> <221> MISC_FEATURE (222) (1) .. (2) <223> Xaa is independently R, L or K and one or both may be present <220> <221> MISC_FEATURE (222) (3) .. (3) <223> Xaa is one of C, S or A <220> <221> MISC_FEATURE (222) (4) .. (4) <223> Xaa is one of R or P <220> <221> MISC_FEATURE (222) (6) .. (6) <223> Xaa is one of A or V <220> <221> MISC_FEATURE (222) (8) .. (8) <223> Xaa is one of A or V <220> <221> MISC_FEATURE <222> (11) .. (11) <223> Xaa is one of V or W <220> <221> MISC_FEATURE (222) (12) .. (12) <223> Xaa is one of C, S or A <220> <221> MISC_FEATURE (222) (13) .. (14) <223> Xaa is independently R, L or K and one or both may be present <400> 4 Xaa Xaa Xaa Xaa Ile Xaa Pro Xaa Ile Pro Xaa Xaa Xaa Xaa 1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Cationic peptide <400> 5 Leu Leu Cys Arg Ile Val Pro Val Ile Pro Trp Cys Lys 1 5 10 <210> 6 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Cationic peptide <400> 6 Leu Arg Cys Pro Ile Ala Pro Val Ile Pro Val Cys Lys Lys 1 5 10 <210> 7 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Cationic peptide <400> 7 Lys Ser Arg Ile Val Pro Ala Ile Pro Val Ser Leu Leu 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Cationic peptide <400> 8 Lys Lys Ser Pro Ile Ala Pro Ala Ile Pro Trp Ser Arg 1 5 10 <210> 9 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Cationic peptide <400> 9 Arg Arg Ala Arg Ile Val Pro Ala Ile Pro Val Ala Arg Arg 1 5 10 <210> 10 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Cationic peptide <400> 10 Leu Ser Arg Ile Ala Pro Ala Ile Pro Trp Ala Lys Leu 1 5 10 <210> 11 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Cationic peptide <220> <221> MISC_FEATURE (222) (1) .. (2) <223> Xaa is independently D, E, S, T or N and one or both may be        present <220> <221> MISC_FEATURE (222) (4) .. (5) <223> Xaa is P, G or D and one or both may be present <220> <221> MISC_FEATURE (222) (6) .. (6) <223> Xaa is one of G, A, V, L, I or Y <220> <221> MISC_FEATURE (222) (8) .. (8) <223> Xaa is one of R, K or H <220> <221> MISC_FEATURE (222) (9) .. (10) <223> Xaa is P, G or D and one or both may be present <220> <221> MISC_FEATURE <222> (11) .. (11) <223> Xaa is one of S, T, C, M or R <220> <221> MISC_FEATURE (222) (12) .. (12) <223> Xaa is one of G, A, V, L, I or Y <220> <221> MISC_FEATURE (222) (14) .. (14) <223> Xaa is one of G, A, V, L, I or Y <220> <221> MISC_FEATURE <222> (15) .. (16) <223> Xaa is D, E, S, T or N and one or both may be present <400> 11 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 chosen from A, P or R and one, two, three or all four may        be present <220> <221> MISC_FEATURE (222) (5) .. (6) X223 is an aromatic amino acid (F, Y and W) <220> <221> MISC_FEATURE (222) (7) .. (7) <223> Xaa is one of P or K <220> <221> MISC_FEATURE (222) (8) .. (9) <223> Xaa is chosen from A, P, Y or W and one, both or none may be        present <220> <221> MISC_FEATURE (222) (11) .. (12) <223> Xaa is chosen from A, P, Y or W and one, both or none may be        present <220> <221> MISC_FEATURE (222) (14) .. (15) <223> Xaa is chosen from A, P, Y or W and one, both or none may be        present <220> <221> MISC_FEATURE (222) (16) .. (18) <223> Xaa is chosen from R or P and one, two or three may be present <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 one or both may be present <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 one or both may be present <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 one or both may be present <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 one or both may be present <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 one of C, S, M, D or A <220> <221> MISC_FEATURE (222) (4) .. (4) <223> Xaa is one of F, I, V, M or R <220> <221> MISC_FEATURE (222) (5) .. (6) <223> Xaa is R or K and one or both may be present <220> <221> MISC_FEATURE (222) (8) .. (8) <223> Xaa is one of A, I, S, M, D or R <220> <221> MISC_FEATURE (222) (9) .. (9) <223> Xaa is one of F, I, V, M or R <220> <221> MISC_FEATURE (222) (12) .. (12) <223> Xaa is one of F, I, V, M or R <220> <221> MISC_FEATURE (222) (13) .. (13) <223> Xaa is one of A, I, S, M, D or R <220> <221> MISC_FEATURE (222) (14) .. (14) <223> Xaa is one of 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 one of 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) Xaa is a polar amino acid (C, S, T, M, N and Q) <220> <221> MISC_FEATURE (222) (4) .. (4) <223> Xaa is one of A, L, S or K <220> <221> MISC_FEATURE (222) (6) .. (6) <223> Xaa is one of A, L, S or K <220> <221> MISC_FEATURE <222> (11) .. (11) <223> Xaa is one of A, L, S or K <220> <221> MISC_FEATURE <222> (16) .. (16) <223> Xaa is one of A, L, S or K <220> <221> MISC_FEATURE (222) (16) .. (31) <223> Xaa is amino acids chosen from G, A, V, L, I, P, F, S, T, K and H         and one to seventeen may be present <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 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 Ser 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 at residues 4, 7, 8, 10, 11,14, 15 is a hydrophobic amino        acid <220> <221> MISC_FEATURE (222) (1) .. (26) <223> Xaa at residues 5, 6, 9, 12, 13 is a hydrophilic amino acid <400> 46 Lys Trp Lys Xaa 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> PCR amplification primer <400> 55 gtccctgtat gcctctggtc 20 <210> 56 <211> 19 <212> DNA <213> Artificial sequence <220> PCR amplification primer <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> nonCpG oligonucleotide <400> 58 ttcaggactt tcctcaggtt 20 <210> 59 <211> 21 <212> DNA <213> Homo sapiens <400> 59 tcatagcagc caccttcatt c 21 <210> 60 <211> 20 <212> DNA <213> Homo sapiens <400> 60 tagcgcagat tcttgggttg 20 <210> 61 <211> 22 <212> DNA <213> Homo sapiens <400> 61 tgtcctttct cagagtggtt ct 22 <210> 62 <211> 21 <212> DNA <213> Homo sapiens <400> 62 tgcttccata gggacatcat a 21 <210> 63 <211> 21 <212> DNA <213> Homo sapiens <400> 63 acctgaacct tccaaagatg g 21 <210> 64 <211> 20 <212> DNA <213> Homo sapiens <400> 64 gcgcagaatg agatgagttg 20 <210> 65 <211> 21 <212> DNA <213> Homo sapiens <400> 65 gtgcagaggg ttgtggagaa g 21 <210> 66 <211> 21 <212> DNA <213> Homo sapiens <400> 66 ttctcccgtg caatatctag g 21

Claims (22)

Innate immunity of a subject, comprising stimulating an immune response by administering to the subject a therapeutically effective amount of a peptide as set forth in SEQ ID NOs: 1-4, 11, 18, 25, 32, 39, 46, 53, or 54 How to stimulate. The method of claim 1, wherein innate immunity is demonstrated by host immune cell activation, proliferation, differentiation, or MAP kinase pathway activation. The method of claim 2, wherein the MAP kinase is MEK and / or ERK. The method of claim 1, further comprising administering GM-CSF to the subject. Infectious or at risk of infection, comprising administering the antibiotic to a subject in combination with a peptide as shown in SEQ ID NOs: 1-4, 7, 11, 18, 25, 32, 39, 46, 53, or 54 How to stimulate innate immunity of a subject. The method of claim 1, wherein the peptide comprises one or more amino acids that are D-enantiomers. The method of claim 1, wherein the peptide is cyclic. The method of claim 1 wherein the peptide sequence is reversed. The method of claim 1, further comprising administering an antibiotic to the subject. The method of claim 9 wherein the antibiotic is aminoglycoside, penicillin, cephalosporin, cerbacephem, cephamycin, cephamycin, chloramphenicol, glycyl Glycylcycline, licosamide, aminocyclitol, cationic antimicrobial peptides, lipopeptides, poymyxin, streptogramin, oxazoladinone, lincosamide (lincosamide), fluoroquinolone, carbapenem, tetracycline, macrolide, macrolide, beta-lactam carbapenem, monoobtam, quinolone, quinolone, tetra Method selected from tracycline, or glycopeptide. The method of claim 5, wherein the peptide has anti-inflammatory activity. The method of claim 5, wherein the peptide has antiseptic activity. The method of claim 5, wherein the peptide comprises one or more amino acids that are D-enantiomers. The method of claim 5, wherein the peptide is cyclic. The method of claim 5, wherein the peptide sequence is reversed. The method of claim 5 wherein the antibiotic is aminoglycoside, penicillin, cephalosporin, cervacefem, cephamycin, chloramphenicol, glycylcycline, lycosamide, aminocyclitol, cationic antimicrobial peptide, lipopeptides, In pomimicin, streptogramamine, oxazoladinone, lincosamide, fluoroquinolone, carbapenem, tetracycline, macrolide, beta-lactam carbapenem, monobactam, quinolone, tetracycline, or glycopeptide The method chosen. Infection or risk of infection comprising administering GM-CSF to a subject in combination with a peptide as set forth in SEQ ID NOs: 1-4, 7, 11, 18, 25, 32, 39, 46, 53, or 54 To stimulate innate immunity of a subject. The method of claim 17, wherein the peptide has anti-inflammatory activity. The method of claim 17, wherein the peptide has antiseptic activity. The method of claim 17, wherein the peptide comprises one or more amino acids that are D-enantiomers. The method of claim 17, wherein the peptide is cyclic. The method of claim 17, wherein the peptide sequence is reversed.

Images