WAVEl INHIBITION IN THE MEDICAL INTERVENTION OF INFLAMMATORY DISEASES AND/OR INFECTIONS CAUSED BY A

PATHOGEN

The present invention relates to antagonists/inhibitors of WAVEl (Wiskott Aldrich syndrome protein- family verprolin-homologous protein 1) for use in the prevention or treatment of an infection caused by a pathogen and/or in the prevention or treatment of an oxidized phospholipids (OxPL) related inflammatory disease developing in response to an infection caused by a pathogen. Moreover, the present invention relates to the prevention of secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insult leads to elevated level of oxidized phospholipids in vivo. Furthermore, antagonists/inhibitors of WAVEl are comprised that are co-administered with antibiotics, antiviral drugs, antioxidants and/or anti-inflammatory drugs. In a further aspect, the present invention provides for a method for assessing the activity of a candidate molecule suspected of being an antagonist/inhibitor of WAVEl comprising the steps of: (a) optionally pre- incubating a cell, tissue or a non-human animal comprising and expressing WAVEl with OxPL; (b) contacting said cell, tissue or a non-human animal comprising WAVEl with said candidate molecule; (c) detecting a decrease in WAVEl activity; and (d) selecting a candidate molecule that decreases WAVEl activity; wherein a decrease of the WAVEl activity is indicative for the capacity of the selected molecule to ameliorate, prevent or treat an OxPL related to an inflammatory disease developing in response to an infection caused by a pathogen and/or prevent, ameliorate or treat an infection caused by a pathogen and/or prevent an infection subsequent to a physiolgical insult like injury, trauma, surgery or inflammation. In yet another aspect, the present invention provides for a cell, tissue or a non-human animal for screening and/or validation of a compound suspected of being an antagonist/inhibitor of WAVEl . Finally, a kit is provided that is useful for carrying out the method of the invention comprising polynucleotides and/or antibodies capable of detecting the activity of WAVEl . Sepsis is a serious medical condition that is characterized by a whole-body inflammatory state (called a systemic inflammatory response syndrome or SIRS) and the presence of a known or suspected infection. The body may develop this inflammatory response to microbes in the blood, urine, lungs, skin, or other tissues. Invasion of bacteria to otherwise sterile sites like the peritoneal cavity leads to the immediate initiation of an inflammatory response. Integral to this response are oxygen radicals that are primarily generated to kill microbes, but can also damage host structures through the peroxidation of membrane phospholipids (Hampton, M.B., et al. (1998) Blood 92, 3007-3017). It has been showed previously that administration of oxidized phospholipids (OxPL) impaired survival during E. coli peritonitis by inhibiting phagocytosis of bacteria by macrophages (Knapp, S., et al. (2007) J Immunol 178, 993-1001). Bacterial peritonitis is, e.g., a serious infection in which efficient clearance of invading pathogens is essential in preventing overwhelming inflammation, sepsis and death (Holzheimer, R.G., et al. (1991) Infection 19, 447-452; Wickel, D.J., et al. (1997) Ann Surg 225, 744-753; discussion 753-746; Pinheiro da Silva, F., et al. (2007) Nat Med 13, 1368- 1374).

According to the American College of Chest Physicians and the Society of Critical Care Medicine, there are five different levels of sepsis:

1. Systemic inflammatory response syndrome (SIRS). Defined by the presence of two or more of the following findings: Body temperature < 36°C or > 38 C (hypothermia or fever); Heart rate > 90 beats per minute (tachycardia); Respiratory rate > 20 breaths per minute or, on blood gas, a P_aC0₂ less than 32 mm Hg (4.3 kPa) (tachypnea or hypocapnia due to hyperventilation); White blood cell count < 4,000 cells/mm³ or > 12,000 cells/mm^'' (< 4 x 10⁹ or > 12 x 10⁹ cells/L), or greater than 10% band forms (immature white blood cells), (leukopenia, leukocytosis, or bandemia).

2. Sepsis. Defined as SIRS in response to a confirmed infectious process. Infection can be suspected or proven (by culture, stain, or polymerase chain reaction (PCR)), or a clinical syndrome pathognomonic for infection. Specific evidence for infection includes WBCs in normally sterile fluid (such as urine or cerebrospinal fluid (CSF), evidence of a perforated viscus (free air on abdominal x-ray or CT scan, signs of acute peritonitis), abnormal chest x- ray (CXR) consistent with pneumonia (with focal opacification), or petechiae, purpura, or purpura fulminans. 4. Severe sepsis. Defined as sepsis with organ dysfunction, hypoperfusion, or hypotension.

5. Septic shock. Defined as sepsis with refractory arterial hypotension or hypoperfusion abnormalities in spite of adequate fluid resuscitation. Signs of systemic hypoperfusion may be either end-organ dysfunction or serum lactate greater than 4 mmol/dL. Other signs include oliguria and altered mental status. Patients are defined as having septic shock if they have sepsis plus hypotension after aggressive fluid resuscitation (typically upwards of 6 liters or 40 ml/kg of crystalloid).

Sepsis is still one of the main causes of death in intensive care. The incidence is 50-95 cases/100000 and it has increased significantly over the past decades, annually by 9% with women being slightly less affected. Especially patients with an age 65 are affected by an increased incidence and mortality. In Germany, each year about 154.000 people suffer from sepsis, approximately half of all sufferers die.

WAVEl is a A-kinase anchoring protein (AKAP) which directs the local effects of PKA (Protein Kinase A) by subcellular targeting. "WAVEl" relates to "Wiskott Aldrich syndrome protein-family verprolin-homologous protein 1" as described in the art (Scar/WAVE-1 , a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold. Westphal RS, Soderling SH, Alto NM, Langeberg LK, Scott JD. EMBO J. 2000 Sep 1 ; 19(17):4589-600.). WAVEl belongs to the Wiskott-Aldrich syndrome protein (WASP) family that control actin polymerization via the Arp2/3 complex (Takenawa, T. (2007) Nature reviews 8, 37-48). In contrast to all other WASP family members, WAVEl is predominantly known as an AKAP that contributes to the specificity of PKA by tethering it to Arp2/3 (Westphal, R.S., et al. (2000) Embo J 19, 4589-4600). So far, the biological role of WAVEl has been mainly studied in the brain where high expression levels have been detected and knockout mice exhibit altered synaptic transmission, depleted neuronal migration, behavioral deficits and reduced viability (Soderling, S.H., et al. (2003) Proc Natl Acad Sci U S A 100, 1723-1728; Soderling, S.H.. et al. (2007) J Neurosci 27, 355-365). On a molecular level, WAVEl was shown to induce actin polymerization and dendritic spine morphology in neurons (Kim, Y., et al. (2006) Nature 442, 814-817). WAVEl was found expressed in bone marrow derived macrophages, but its function remained unknown (Dinh, H., et al. (2008) J Leukoc Biol 84, 1483-1491). Moreover, it has been demonstrated that overexpression of full- length WAVE1 depressed bacterial invasion of Yersinia pseudotuberculosis relative to untransfected COS-1 cells. Yersinia is an enteropathogen that invades cells and translocates from the intestinal lumen into cells via the bacterial outer membrane protein invasin (Alrutz, M. A., et al., (2001) Molecular Microbiology, 42(3), 689-703). In contrast to the translocation from the intestinal lumen into cells as described for Yersinia, in infections at otherwise sterile sites like the peritoneal cavity the invading bacteria are challenged with phagocytic cells like macrophages.

Present therapies of sepsis rests on antibiotics, surgical drainage of infected fluid collections, fluid replacement and appropriate support for organ dysfunction. This may include hemodialysis in kidney failure, mechanical ventilation in pulmonary dysfunction, transfusion of blood products, and drug and fluid therapy for circulatory failure. Ensuring adequate nutrition preferably by enteral feeding, but if necessary by parenteral nutrition is important during prolonged illness. Most therapies aimed at the inflammation process itself have failed to improve outcome. Despite the increase in therapeutic options, approximately 20-35% of patients with severe sepsis and 40-60% of patients with septic shock die within 30 days. Others die within the ensuing 6 months. Late deaths often result from poorly controlled infection, immunosuppression, complications of intensive care, failure of multiple organs, or the patient's underlying disease.

Consequently, novel biological therapies that specifically influence the efficient clearance of invading pathogens are desired for therapy of inflammatory diseases and/or infections with pathogens as well as SIRS and/or sepsis. Therefore, the technical problem underlying the present invention is the provision of means and methods for the medical intervention of infections and/or inflammatory diseases.

This technical problem is solved by the embodiments provided herein and as characterized in the claims. Specifically and in accordance with the present invention, a solution to this technical problem is achieved by providing an antagonist/inhibitor of WAVE 1 for use in the prevention and/or treatment of an infection caused by a pathogen and/or in the prevention or treatment of an oxidized phospholipids (OxPL) related inflammatory disease developing in response to an infection caused by a pathogen. The present invention relates to an antagonist/inhibitor of WAVE1 for use in the prevention and/or treatment of an infection caused by a pathogen.. Moreover, the present invention relates to the use of an antagonist/inhibitor of WAVE1 for the preparation of a medicament for the prevention and/or treatment of an oxidized phospholipids (OxPL) related inflammatory disease developing in response to a pathogen and/or SIRS and/or sepsis and/or in the prevention and/or treatment of an infection caused by a pathogen. Moreover, the present invention relates to the prevention of secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insult leads to elevated oxidized phospholipids levels in vivo.. It was surprisingly found and illustrated in the appended examples that WAVE1 is involved in bacterial uptake and in phagocytosis. Accordingly, the gist of the present invention is the provision of inhibitors or antagonists of WAVE 1 biological activity or of its expression in the preparation of a medicament for the prevention, amelioration or treatment of an oxidized phospholipids (OxPL) related inflammatory disease developing in response to an infection caused by a pathogen and/or SIRS and/or sepsis. Moreover, the gist of the present invention is the provision of inhibitors or antagonists of the biological activity of WAVE1 (WAVE1- biological activity) or of its expression in the preparation of a medicament for the prevention and/or treatment of an infection caused by a pathogen, in particular in the treatment of sepsis, for example in infections of normally sterile sites, like the peritoneal cavity. Furthermore, inhibitors or antagonists of WAVE 1 -biological activity or of its expression in the preparation of a medicament are provided for the prevention of secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insults lead to elevated oxidized phospholipids..

It was demonstrated that transfected COS-1 cells overexpressing full-length WAVE1 depressed bacterial invasion of Yersinia pseudotuberculosis (normally associated with localized infections in intestinal lymph nodes in mice) relative to untransfected COS-1 cells (Alrutz, M. A., et al , (2001) Molecular Microbiology, 42(3), 689-703). Yersinia is an enteropathogen that invades cells and translocates from the intestinal lumen into cells via the bacterial outer membrane protein invasin. However, WAVE1 has not been associated in the prior art with infections or sepsis caused by invasion of bacteria to otherwise sterile sites like the peritoneal cavity. Yersinia pseudotuberculosis translocates into cells and, subsequently, said bacteria reside inside the cells and escape from cellular defense mechanisms mediated by macrophages. In contrast, in the present invention, it could be shown that invading bacteria are phagocytized by macrophages, cells which are known to be associated with infections at normally sterile sites, like the peritoneal cavity. Accordingly, the present invention relates to the medical intervention of infections at otherwise sterile sites like the peritoneal cavity wherein the invading bacteria are challenged with phagocytic cells like macrophages. Thus, the present invention surprisingly demonstrates that WAVE1 antagonists/inhibitors are in particular useful in the treatment of an infection at normally sterile sites like the peritoneal cavity, and/or the treatment of a sepsis or SIRS. WAVE1 has not been associated in the prior art with neither bacterial uptake nor phagocytosis.

Therefore, it was found in the present invention that WAVE1 antagonists/inhibitors can successfully be used in the medical intervention of infectious diseases like infections with bacteria and/or pathogens, biological fungi, viruses and the like. The present invention demonstrates that WAVE1 antagonists/inhibitors are in particular useful in the treatment or prevention of SIRS and sepsis. Thus, the present invention is particularly useful in the medical intervention of an infection at normally sterile sites like the peritoneal cavity (for example a peritonitis), and/or the treatment of a sepsis and/or SIRS, like peritonitis.

For example, bacterial peritonitis is a serious infection in which efficient clearance of invading pathogens is essential in preventing overwhelming inflammation, sepsis and death (Holzheimer, R.G., et al. (1991) Infection 19, 447-452; Wickel, D.J., et al. (1997) Ann Surg 225, 744-753; discussion 753-746; Pinheiro da Silva, F.. et al. (2007) Nat Med 13, 1 368- 1374). It has been described that oxidized phospholipids (OxPL) are generated early during E. coli peritonitis in vivo, and it has been demonstrated that OxPL potently inhibit phagocytosis of bacteria by macrophages. As demonstrated in the appended examples, the mechanism of action underlying these effects of OxPL has been investigated and it was surprisingly found that OxPL-induced alterations in actin polymerization results in spreading of peritoneal macrophages, with concomitantly diminished uptake of E. coli. Furthermore, OxPL directly activated PKA, and inhibition or silencing of PKA completely abolished OxPL-effects. A- kinase anchoring proteins (AKAPs), which specify the pleiotropic effects of PKA by targeting it to particular sites within the cell (Tasken, K. (2004) A. Physiol Rev 84, 137-167), mediate downstream effects of PICA. Furthermore, it was found that blocking of AKAP-PKA interaction abolished OxPL effects in vitro and prevented enhanced bacterial outgrowth in vivo. More particularly, it was surprisingly found that a particular actin-associated AKAP expressed in macrophages, i.e. WAVEl, was found to mediate OxPL-induced inhibition of phagocytosis. Furthermore, as demonstrated in the appended examples, chimeric WAVEl^{" "} mice were resistant to the detrimental effects of OxPL in vivo. These data demonstrate that WAVEl activity and/or expression itself exerts detrimental effects. Thus, the data provided herein document WAVEl 's influence in innate immunity and provide for WAVEl as an unexpected therapeutic target for inflammation, in particular inflammations relating to severe bacterial infections, such bacterial infections and/or corresponding inflammations are, inter alia, sepsis, acute lung injury, SIRS, head trauma and/or pancreatitis. Thus, it is demonstrated herein that antagonists and/or inhibitors of WAVEl are useful in preventing an enhanced bacterial outgrowth upon inflammation and can, thus, be successfully used in the treatment of oxidized phospholipids (OxPL) related inflammatory diseases developing in response to an infection caused by a pathogen. Moreover, it is shown herein that antagonists and/or inhibitors of WAVEl are useful in the prevention and/or treatment of an infection caused by a pathogen, in particular in the treatment of an infection at normally sterile sites like the peritoneal cavity (for example a peritonitis), and/or the treatment of a sepsis or SIRS. In addition, the antagonists and/or inhibitors of WAVEl are useful in the prevention of secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insults lead to elevated oxidized phospholipids. In this context, WAVEl antagonists/inhibitors are preferably used in the prevention of such secondary infections. The term "elevated oxidized phospholipids" relates to a level of oxidized phospholipids (OxPL) that is higher than the normal level in the non-diseased state and/or the state without insult. The level of oxidized phospholipids can, e.g., be determined as shown in the appended examples; see, e.g, Example 1 , "Measurement of oxidized lipids".

In the context of the present invention, the term "WAVEl" relates to "Wiskott Aldrich syndrome protein- family verprolin-homologous protein 1" as described in the art (Scar/WAVE-1 , a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi- kinase scaffold. Westphal RS, Soderling SH, Alto NM, Langeberg LK, Scott JD. EMBO J. 2000 Sep l ; 19(17):4589-600.) and relates to sequences disclosed herein below. WAVE 1 is a A-kinase anchoring protein (AKAP) which direct the local effects of PKA (Protein Kinase A) by subcellular targeting. As mentioned above, it has been described that oxidized phospholipids (OxPL) are generated early during E. coli peritonitis in vivo, and it has been demonstrated that OxPL potently inhibit phagocytosis of bacteria by macrophages. Phagocytosis requires the active remodeling of actin and actin polymerization was found in response to OxLDL previously. Previous reports revealed that elevated cAMP levels suppressed receptor mediated phagocytosis in marcophages via involvement of PKA (Protein Kinase A). WAVE1 belongs to the Wiskott-Aldrich syndrome protein (WASP) family that control actin polymerization via the Arp2/3 complex (Takenawa, T. (2007) Nature reviews 8, 37-48). In contrast to all other WASP family members, WAVE1 is predominantly known as an AKAP that contributes to the specificity of PKA by tethering it to Arp2/3 (Westphal, R.S., et al. (2000) Embo J 19, 4589-4600). So far, the biological role of WAVE1 has been mainly studied in the brain where high expression levels have been detected and knockout mice exhibit altered synaptic transmission, depleted neuronal migration, behavioral deficits and reduced viability (Soderling, S.H.. et al. (2003) Proc Natl Acad Sci U S A 100, 1723-1728; Soderling, S.H., et al. (2007) J Neurosci 27, 355-365). On a molecular level, WAVE1 was shown to induce actin polymerization and dendritic spine morphology in neurons (Kim, Y., et al. (2006) Nature 442, 814-817). Only recently, WAVE1 was found expressed in bone marrow derived macrophages, but its function remained unknown (Dinh, H., et al. (2008) J Leukoc Biol 84, 1483-1491).

The coding regions of WAVE1 or functional fragments thereof are known in the art and comprise, inter alia, the WAVE1 GenBank entries NMJ303931", "for Homo sapiens, for Mus musculus "NMJD 1877", Rattus norvegicus "NM_001025114" Bos taurus "NM_001034017". The person skilled in the art my easily deduce the relevant coding region of WAVE 1 in these GenBank entries, which may also comprise the entry of genomic DNA as well as mRNA/cDNA (see also

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&terai=wavel%5bgene%5d%20AND%20 alive%5bprop%5d%20NOT%20newentry%5bgene%5d%20&log$=genesensor5&logdbfrom =pubmed&sort=weight) .

There are currently four variants of the human WAVE1 (WASP) cDNA (SEQ ID NOs: 1, 3, 5, 7) (see also http://www cbi.nlm.nih.gov/gene/8936?ordinalpos=l &itool=EntrezSystem2.PEntrez.Gene.G ene_ResultsPanel.Gene_RVDocSum). SEQ ID NO: l is the longest cDNA. Thus, in particular, wild type human WAVE1 may be encoded by the following nucleic acid sequences.

GettBank: NM 0039

1 atgccgctag tgaaaagaaa catcgatcct aggcacttgt gccacacagc actgcctaga 61 ggcattaaga atgaactgga atgtgtaacc aatatttcct tggcaaatat aattagacaa 121 ctaagtagcc taagtaaata tgctgaagat atatttggag aattattcaa tgaagcacat 181 agtttttcct tcagagtcaa ctcattgcaa gaacgtgtgg accgtttatc tgttagtgtt 241 acacagcttg atccaaagga agaagaattg tctttgcaag atataacaat gaggaaagct 301 ttccgaagtt ctacaattca agaccagcag cttttcgatc gcaagacttt gcctattcca 361 ttacaggaga cgtacgatgt ttgtgaacag cctccacctc tcaatatact cactccttat 421 agagatgatg gtaaagaagg tctgaagttt tataccaatc cttcgtattt ctttgatcta 481 tggaaagaaa aaatgttgca agatacagag gataagagga aggaaaagag gaagcagaag 541 cagaaaaatc tagatcgtcc tcatgaacca gaaaaagtgc caagagcacc tcatgacagg 601 cggcgagaat ggcagaagct ggcccaaggt ccagagctgg ctgaagatga tgctaatctc 661 ttacataagc atattgaagt tgctaatggc ccagcctctc attttgaaac aagacctcag 721 acatacgtgg atcatatgga tggatcttac tcactttctg ccttgccatt tagtcagatg 781 agtgagcttc tgactagagc tgaggaaagg gtattagtca gaccacatga accacctcca 841 cctccaccaa tgcatggagc aggagatgca aaaccgatac ccacctgtat cagttctgct 901 acaggtttga tagaaaatcg ccctcagtca ccagctacag gcagaacacc tgtgtttgtg 961 agccccactc ccccacctcc tccaccacct cttccatctg ccttgtcaac ttcctcatta 1021 agagcttcaa tgacttcaac tcctccccct ccagtacctc ccccacctcc acctccagcc 1081 actgctttgc aagctccagc agtaccacca cctccagctc ctcttcagat tgcccctgga 1141 gttcttcacc cagctcctcc tccaattgca cctcctctag tacagccctc tccaccagta 1201 gctagagctg ccccagtatg tgagactgta ccagttcatc cactcccaca aggtgaagtt 1261 caggggctgc ctccaccccc accaccgcct cctctgcctc cacctggcat tcgaccatca 1321 tcacctgtca cagttacagc tcttgctcat cctccctctg ggctacatcc aactccatct 1381 actgccccag gtccccatgt tccattaatg cctccatctc ctccatcaca agttatacct 1441 gcttctgagc caaagcgcca tccatcaacc ctacctgtaa tcagtgatgc caggagtgtg 1501 ctactggaag caatacgaaa aggtattcag ctacgcaaag tagaagagca gcgtgaacag 1561 gaagctaagc atgaacgcat tgaaaacgat gttgccacca tcctgtctcg ccgtattgct 1621 gttgaatata gtgattcgga agatgattca gaatttgatg aagtagattg gttggagtaa

(SEQ ID NO:l), which corresponds to the following amino acid sequence:

="MPLVKRNIDPRHLCHTALPRGIKNELECVTNISLANIIRQLSSLSKYAEDIFGELFNEAHSFSFRVNSLQERV DRLSVSVTQLDPKEEELSLQDITMRKAFRSSTIQDQQLFDRKTLPIPLQETYDVCEQPPPLNILTPYRDDGKEGL KFYTNPSYFFDLWKEKMLQDTEDKRKEKR QKQK LDRPHEPE VPRAPHDRRRE QKLAQGPELAEDDANLLHK HIEVANGPASHFETRPQTYVDHMDGSYSLSALPFSQMSELLTRAEERVLVRPHEPPPPPPMHGAGDAKPIPTCIS SATGLIENRPQSPATGRTPVFVSPTPPPPPPPLPSALSTSSLRASMTSTPPPPVPPPPPPPATALQAPAVPPPPA PLQIAPGVLHPAPPPIAPPLVQPSPPVARAAPVCETVPVHPLPQGEVQGLPPPPPPPPLPPPGIRPSSPVTVTAL AHPPSGLHPTPSTAPGPHVPLMPPSPPSQVI PASEPKRHPSTLPVI SOARSVLLEAI RKGIQLRKVEEQREQEAK HERIENDVATILSRRIAVEYSDSEDDSEFDEVDWLE"

(SEQ ID NO:2). Genbank: NM 001024934

i atgccgctag tgaaaagaaa catcgatcct aggcacttgt gccacacagc actgcctaga

61 ggcattaaga atgaactgga atgtgtaacc aatatttcct tggcaaatat aattagacaa 121 ctaagtagcc taagtaaata tgctgaagat atatttggag aattattcaa tgaagcacat 181 agtttttcct tcagagtcaa ctcattgcaa gaacgtgtgg accgtttatc tgttagtgtt 241 acacagcttg atccaaagga agaagaattg tctttgcaag atataacaat gaggaaagct 301 ttccgaagtt ctacaattca agaccagcag cttttcgatc gcaagacttt gcctattcca 361 ttacaggaga cgtacgatgt ttgtgaacag cctccacctc tcaatatact cactccttat 421 agagatgatg gtaaagaagg tctgaagttt tataccaatc cttcgtattt ctttgatcta 481 tggaaagaaa aaatgttgca agatacagag gataagagga aggaaaagag gaagcagaag 541 cagaaaaatc tagatcgtcc tcatgaacca gaaaaagtgc caagagcacc tcatgacagg 601 cggcgagaat ggcagaagct ggcccaaggt ccagagctgg ctgaagatga tgctaatctc 661 ttacataagc atattgaagt tgctaatggc ccagcctctc attttgaaac aagacctcag 721 acatacgtgg atcatatgga tggatcttac tcactttctg ccttgccatt tagtcagatg 781 agtgagcttc tgactagagc tgaggaaagg gtattagtca gaccacatga accacctcca 841 cctccaccaa tgcatggagc aggagatgca aaaccgatac ccacctgtat cagttctgct 901 acaggtttga tagaaaatcg ccctcagtca ccagctacag gcagaacacc tgtgtttgtg 961 agccccactc ccccacctcc tccaccacct cttccatctg ccttgtcaac ttcctcatta 1021 agagcttcaa tgacttcaac tcctccccct ccagtacctc ccccacctcc acctccagcc 1081 actgctttgc aagctccagc agtaccacca cctccagctc ctcttcagat tgcccctgga 1141 gttcttcacc cagctcctcc tccaattgca cctcctctag tacagccctc tccaccagta 1201 gctagagctg ccccagtatg tgagactgta ccagttcatc cactcccaca aggtgaagtt 1261 caggggctgc ctccaccccc accaccgcct cctctgcctc cacctggcat tcgaccatca 1321 tcacctgtca cagttacagc tcttgctcat cctccctctg ggctacatcc aactccatct 1381 actgccccag gtccccatgt tccattaatg cctccatctc ctccatcaca agttatacct 1441 gcttctgagc caaagcgcca tccatcaacc ctacctgtaa tcagtgatgc caggagtgtg 1501 ctactggaag caatacgaaa aggtattcag ctacgcaaag tagaagagca gcgtgaacag 1561 gaagctaagc atgaacgcat tgaaaacgat gttgccacca tcctgtctcg ccgtattgct 1621 gttgaatata gtgattcgga agatgattca gaatttgatg aagtagattg gttggagtaa

(SEQ ID NO:3),

which corresponds to the following amino acid sequence:

="MPLVKRNIDPRHLCHTALPRGIKNELECVTNISLANI IRQLSSLSKYAEDIFGELFNEAHSFSFRVNSL

QERVDRLSVSVTQLDPKEEELSLQDITMRKAFRSSTIQDQQLFDRKTLPIPLQETYDVCEQPPPLNILT

PYRDDGKEGLKFYTNPSYFFDLWKEKMLQDTEDKRKEKRKQKQKNLDRPHEPEKVPRAPHDRRRE

WQKLAQGPELAEDDANLLHKHIEVANGPASHFETRPQTYVDHMDGSYSLSALPFSQMSELLTRAEER

VLVRPHEPPPPPPMHGAGDAKPIPTCISSATGLIENRPQSPATGRTPVFVSPTPPPPPPPLPSALSTSS

LRASMTSTPPPPVPPPPPPPATALQAPAVPPPPAPLQIAPGVLHPAPPPIAPPLVQPSPPVARAAPVCE

TVPVHPLPQGEVQGLPPPPPPPPLPPPGIRPSSPVTVTALAHPPSGLHPTPSTAPGPHVPLMPPSPPS

QVIPASEPKRHPSTLPVISDARSVLLEA1RKGIQLRKVEEQREQEAKHERIENDVATILSRRIAVEYSDSE

DDSEFDEVDWLE"

(SEQ ID NO: 4).

Genbank: NM_001024935

1 atgccgctag tgaaaagaaa catcgatcct aggcacttgt gccacacagc actgcctaga 61 ggcattaaga atgaactgga atgtgtaacc aatatttcct tggcaaatat aattagacaa

121 ctaagtagcc taagtaaata tgctgaagat atatttggag aattattcaa tgaagcacat

181 agtttttcct tcagagtcaa ctcattgcaa gaacgtgtgg accgtttatc tgttagtgtt

241 acacagcttg atccaaagga agaagaattg tctttgcaag atataacaat gaggaaagct

301 ttccgaagtt ctacaattca agaccagcag cttttcgatc gcaagacttt gcctattcca

361 ttacaggaga cgtacgatgt ttgtgaacag cctccacctc tcaatatact cactccttat

421 agagatgatg gtaaagaagg tctgaagttt tataccaatc cttcgtattt ctttgatcta

481 tggaaagaaa aaatgttgca agatacagag gataagagga aggaaaagag gaagcagaag

541 cagaaaaatc tagatcgtcc tcatgaacca gaaaaagtgc caagagcacc tcatgacagg

601 cggcgagaat ggcagaagct ggcccaaggt ccagagctgg ctgaagatga gctaatctc

661 ttacataagc atattgaagt tgctaatggc ccagcctctc attttgaaac aagacctcag

721 acatacgtgg atcatatgga tggatcttac tcactttctg ccttgccatt tagtcagatg

781 agtgagcttc tgactagagc tgaggaaagg gtattagtca gaccacatga accacctcca

841 cctccaccaa tgcatggagc aggagatgca aaaccgatac ccacctgtat cagttctgct

901 acaggtttga tagaaaatcg ccctcagtca ccagctacag gcagaacacc tgtgtttgtg

961 agccccactc ccccacctcc tccaccacct cttccatctg ccttgtcaac ttcctcatta

1021 agagcttcaa tgacttcaac tcctccccct ccagtacctc ccccacctcc acctccagcc

1081 actgctttgc aagctccagc agtaccacca cctccagctc ctcttcagat tgcccctgga

1141 gttcttcacc cagctcctcc tccaattgca cctcctctag tacagccctc tccaccagta

1201 gctagagctg ccccagtatg tgagactgta ccagttcatc cactcccaca aggtgaagtt

1261 caggggctgc ctccaccccc accaccgcct cctctgcctc cacctggcat tcgaccatca

1321 tcacctgtca cagttacagc tcttgctcat cctccctctg ggctacatcc aactccatct

1381 actgccccag gtccccatgt tccattaatg cctccatctc ctccatcaca agttatacct

1441 gcttctgagc caaagcgcca tccatcaacc ctacctgtaa tcagtgatgc caggagtgtg

1501 ctactggaag caatacgaaa aggtattcag ctacgcaaag tagaagagca gcgtgaacag

1561 gaagctaagc atgaacgcat tgaaaacgat gttgccacca tcctgtctcg ccgtattgct

1621 gttgaatata gtgattcgga agatgattca gaatttgatg aagtagattg gttggagtaa

(SEQ ID NO: 5),

which corresponds to the following amino acid sequence:

="MPLVKRNIDPRHLCHTALPRGIKNELECVTNISLANIIRQLSSLSKYAEDIFGELFNEAHSFSFRVNSL

QERVDRLSVSVTQLDPKEEELSLQDITMRKAFRSSTIQDQQLFDRKTLPIPLQETYDVCEQPPPLNILT

PYRDDGKEGLKFYTNPSYFFDLWKEKMLQDTEDKRKEKRKQKQKNLDRPHEPEKVPRAPHDRRRE

WQKLAQGPELAEDDANLLHKHIEVANGPASHFETRPQTYVDHMDGSYSLSALPFSQMSELLTRAEER

VLVRPHEPPPPPPMHGAGDAKPIPTCISSATGLIENRPQSPATGRTPVFVSPTPPPPPPPLPSALSTSS

LRASMTSTPPPPVPPPPPPPATALQAPAVPPPPAPLQIAPGVLHPAPPPIAPPLVQPSPPVARAAPVCE

TVPVHPLPQGEVQGLPPPPPPPPLPPPGIRPSSPVTVTALAHPPSGLHPTPSTAPGPHVPLMPPSPPS

QVIPASEPKRHPSTLPVISDARSVLLEAIRKGIQLRKVEEQREQEAKHERIENDVATILSRRIAVEYSDSE

DDSEFDEVDWLE"

(SEQ ID NO:6).

Genbank: NM 001024936

1 atgccgctag tgaaaagaaa catcgatcct aggcacttgt gccacacagc actgcctaga 61 ggcattaaga atgaactgga atgtgtaacc aatatttcct tggcaaatat aattagacaa 121 ctaagtagcc taagtaaata tgctgaagat atatttggag aattattcaa tgaagcacat

181 agtttttcct tcagagtcaa ctcattgcaa gaacgtgtgg accgtttatc tgttagtgtt

241 acacagcttg atccaaagga agaagaattg tctttgcaag ata aacaat gaggaaagct

301 ttccgaagtt ctacaattca agaccagcag cttttcgatc gcaagacttt gcctattcca

361 ttacaggaga cgtacgatgt ttgtgaacag cctccacctc tcaatatact cactccttat

421 agagatgatg gtaaagaagg tctgaagttt tataccaatc cttcgtattt ctttgatcta

481 tggaaagaaa aaatgttgca agatacagag gataagagga aggaaaagag gaagcagaag

541 cagaaaaatc tagatcgtcc tcatgaacca gaaaaagtgc caagagcacc tcatgacagg

601 cggcgagaat ggcagaagct ggcccaaggt ccagagctgg ctgaagatga tgctaatctc

661 ttacataagc atattgaagt tgctaatggc ccagcctctc attttgaaac aagacctcag

721 acatacgtgg atcatatgga tggatcttac tcactttctg ccttgccatt tagtcagatg

781 agtgagcttc tgactagagc tgaggaaagg gtattagtca gaccacatga accacctcca

841 cctccaccaa tgcatggagc aggagatgca aaaccgatac ccacctgtat cagttctgct

901 acaggtttga tagaaaatcg ccctcagtca ccagctacag gcagaacacc tgtgtttgtg

961 agccccactc ccccacctcc tccaccacct cttccatctg ccttgtcaac ttcctcatta

1021 agagcttcaa tgacttcaac tcctccccct ccagtacctc ccccacctcc acctccagcc

1081 actgctttgc aagctccagc agtaccacca cctccagctc ctcttcagat tgcccctgga

1141 gttcttcacc cagctcctcc tccaattgca cctcctctag tacagccctc tccaccagta

1201 gctagagctg ccccagtatg tgagactgta ccagttcatc cactcccaca aggtgaagtt

1261 caggggctgc ctccaccccc accaccgcct cctctgcctc cacctggcat tcgaccatca

1321 tcacctgtca cagttacagc tcttgctcat cctccctctg ggctacatcc aactccatct

1381 actgccccag gtccccatgt tccattaatg cctccatctc ctccatcaca agttatacct

1441 gcttctgagc caaagcgcca tccatcaacc ctacctgtaa tcagtgatgc caggagtgtg

1501 ctactggaag caatacgaaa aggtattcag ctacgcaaag tagaagagca gcgtgaacag

1561 gaagctaagc atgaacgcat tgaaaacgat gttgccacca tcctgtctcg ccgtattgct

1621 gttgaatata gtgattcgga agatgattca gaatttgatg aagtagattg gttggagtaa

(SEQ ID NO: 7), which corresponds to the following amino acid sequence:

- 'MPLVKRNIDPRHLCHTALPRGIKNELECVTNISLANIIRQLSSLSKYAEDIFGELFNEAHSFSFRVNSL

QERVDRLSVSVTQLDPKEEELSLQDITMRKAFRSSTIQDQQLFDRKTLPIPLQETYDVCEQPPPLNILT

PYRDDGKEGLKFYTNPSYFFDLWKEKMLQDTEDKRKEKRKQKQKNLDRPHEPEKVPRAPHDRRRE

WQKLAQGPELAEDDANLLHKHIEVANGPASHFETRPQTYVDHMDGSYSLSALPFSQMSELLTRAEER

VLVRPHEPPPPPPMHGAGDAKPIPTCISSATGLIENRPQSPATGRTPVFVSPTPPPPPPPLPSALSTSS

LRASMTSTPPPPVPPPPPPPATALQAPAVPPPPAPLQIAPGVLHPAPPPIAPPLVQPSPPVARAAPVCE

TVPVHPLPQGEVQGLPPPPPPPPLPPPGIRPSSPVTVTALAHPPSGLHPTPSTAPGPHVPLMPPSPPS

QVIPASEPKRHPSTLPVISDARSVLLEAIRKGIQLRKVEEQREQEAKHERIENDVATILSRRIAVEYSDSE

DDSEFDEVDWLE"

(SEQ ID NO: 8).

Accordingly, the WAVEl molecules to be employed in the context of the present invention comprise, but are not limited to the molecules encoded by the nucleic acid molecules as described herein. Also envisaged are WAVEl orthologs which are at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to nucleic acid sequence as shown in SEQ ID NOs: 1 , 3, 5 or 7. These WAVEl molecules as referred here are defined as molecules that are capable of acting as an AKAP as described herein above and below. These functions and activities include, inter alia, the capability of anchoring PKA to the Arp 2/3 complex as described herein above. For testing the AKAP activity, assays provided herein (like, e.g., measuring the phagocytosis activity or by assaying the physical interaction for WAVEl or PKA as described below; see also Examples I ("Phagocytosis assays") or Example VII) may be used. Furthermore envisaged are WAVEl orthologs which are at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 8 and being capable of and being capable of acting as an AKAP as described herein above and below. . In addition, the term "WAVEl ortholog" comprises molecules which are at least 60%, more preferably at least 80% and most preferably at least 90% homologous to the polypeptide as shown in SEQ ID NO: 2, 4, 6 or 8 and being capable of acting as an AKAP as described herein above and below.

In order to determine whether a nucleic acid sequence has a certain degree of identity to a nucleic acid encoding WAVEl orthologs, the skilled person can use means and methods well known in the art, e.g. alignments, either manually or by using computer programs such as those mentioned herein below in connection with the definition of the term "hybridization" and degrees of homology.

The term "hybridization" or "hybridizes" as used herein may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non- stringent. Said hybridization conditions may be established according to conventional protocols described, e.g., in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as 0.1 x SSC, 0.1 % SDS at 65°C. Non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6 x SSC, 1% SDS at 65 °C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Such fragments may represent nucleic acid sequences which code for WAVEl or a functional fragment thereof which have a length of at least 12 nucleotides, preferably at least 15, more preferably at least 18, more preferably of at least 21 nucleotides, more preferably at least 30 nucleotides, even more preferably at least 40 nucleotides and most preferably at least 60 nucleotides. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules. Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an anti-parallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed). The terms "complementary" or "complementarity" refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G- T" binds to the complementary sequence "T-C-A". Complementarity between two single- stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands. The term "hybridizing sequences" preferably refers to sequences which display a sequence identity of at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, particularly preferred at least 80%, more particularly preferred at least 90%), even more particularly preferred at least 95% and most preferably at least 97% identity with a nucleic acid sequence as described above (i.e. SEQ ID NOs: 1, 3, 5 or 7) encoding WAVEl or a functional fragment thereof and being capable of acting as an AKAP as described herein above and below and tests are described for assaying the AKAP activity (like, e.g., measuring the phagocytosis activity or by assaying the physical interaction for WAVEl or PKA as described below; see also Examples I ("Phagocytosis assays") or Example VII). Moreover, the term "hybridizing sequences" preferably refers to sequences encoding WAVEl or a functional fragment thereof having a sequence identity of at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, particularly preferred at least 80%, more particularly preferred at least 90%, even more particularly preferred at least 95% and most preferably at least 97% identity with an amino acid sequence of the WAVEl sequences as described herein (i.e. SEQ ID NOs: 2, 4, 6 or 8) and being capable of acting as an AKAP as described herein above and below.

In accordance with the present invention, the term "identical" or "percent identity" in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably, 70-95%> identity, more preferably at least 95% identity with the nucleic acid sequences of, e.g., SEQ ID NOs: 1 , 3, 5 or 7 or with the amino acid sequence of, e.g., SEQ ID NOs: 2, 4, 6 or 8 and being capable of acting as an AKAP as described herein above and below), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably the described identity exists over a region that is at least about 15 to 25 amino acids or nucleotides in length, more preferably, over a region that is about 50 to 100 amino acids or nucleotides in length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.

Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389-3402; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul (1990) J. Mol. Biol. 215:403-410). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 1 1 , an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring matrix (Henikoff (1989) PNAS 89: 10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

Moreover, the present invention also relates to nucleic acid molecules whose sequence is being degenerate in comparison with the sequence of an above-described hybridizing molecule. When used in accordance with the present invention the term "being degenerate as a result of the genetic code" means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid.

In order to determine whether an amino acid residue or nucleotide residue in a nucleic acid sequence corresponds to a certain position in the amino acid sequence of, e.g., SEQ ID NOs: SEQ ID NOs: 2, 4, 6 or 8 or nucleotide sequence of e.g. SEQ ID NOs: 1 , 3, 5 or 7, the skilled person can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as those mentioned further down below in connection with the definition of the term "hybridization" and degrees of homology.

For example, BLAST 2.0, which stands for Basic Local Alignment Search Tool BLAST (Altschul (1997), loc. cit; Altschul (1993), loc. cit; Altschul (1990), loc. cit), can be used to search for local sequence alignments. BLAST, as discussed above, produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying similar sequences. The fundamental unit of BLAST algorithm output is the High- scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cut-off score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score which is defined as:

% sequence identity x % maximum BLAST score

100 and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules. Another example for a program capable of generating sequence alignments is the CLUSTALW computer program (Thompson (1994) Nucl. Acids Res. 2:4673-4680) or FASTDB (Brutlag (1990) Comp. App. Biosci. 6:237-245), as known in the art.

The term "antagonist" or "inhibitor" as used herein is known in the art and relates to a compound/sub stance capable of fully or partially preventing or reducing the physiologic activity of (a) specific protein(s). In the context of the present invention said antagonist, therefore, may prevent or reduce or inhibit or inactivate the physiological activity of a protein such as WAVE1 upon binding of said compound/substance to said protein. Binding of an "antagonist/inhibitor" to a given protein, e.g. WAVE1 , may compete with or prevent the binding of an endogenous activating molecules binding to said protein. As used herein, accordingly, the term "antagonist" also encompasses competitive antagonists, (reversible) non-competitive antagonists or irreversible antagonist, as described, inter alia, in Mutschler, "Arzneimittelwirkungen" (1986), Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Germany. In addition thereto, however, an "antagonist" or "inhibitor" of WAVEl in the context of the present invention may also be capable of preventing the function of a given protein, such as WAVEl , by preventing/reducing the expression of the nucleic acid molecule encoding for said protein. Thus, an antagonist/inhibitor of WAVEl may lead to a decreased expression level of WAVEl (e.g. decreased level of WAVEl mRNA, WAVEl protein) which is reflected in an decreased activity of WAVEl . This decreased activity can be measured/detected by the herein described methods. An inhibitor of WAVEl in the context of the present invention, accordingly, may also encompass transcriptional repressors of WAVEl expression that are capable of reducing WAVEl function. As described herein below in detail, the decreased expression and/or activity of WAVEl by an antagonist/inhibitor of WAVEl leads to an increased activity (and/or expression) of WAVEl , thereby increasing the phagocytosis activity in overwhelming inflammation and preventing an enhanced bacterial outgrowth upon inflammation.

The term "inflammatory disease" as used herein is known in the art and relate to any kind of inflammation is the complex biological response of tissues to harmful stimuli, such as pathogens, damaged cells, or irritants Inflammation is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. Inflammation is not a synonym for infection. Even in cases where inflammation is caused by infection, the two are not synonymous: infection is caused by an exogenous pathogen, while inflammation is one of the responses of the organism to the pathogen, or other irritants and damaged cells. Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the activation of immune cells followed by the release of various mediators and increased movement of leukocytes from the blood into the injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells which are present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.

Sepsis as used herein is well-known in the art and relate to serious medical conditions as already mentioned in the introductory part. The present invention is particularly useful in the medical intervention of an infection at normally sterile sites like the peritoneal cavity (for example a peritonitis), and/or the treatment of a sepsis and/or SIRS, like peritonitis.

Accordingly, the infection to be treated with WAVE1 antagonists and/or inhibitors in accordance with the present invention may be sepsis and/or SIRS or an infection that develops subsequent to insults leading to elevated oxidized phospholipids like, without being limiting, trauma, injury, surgery and/or inflammation. Thus, the infection may develop subsequent to injury, trauma or inflammation wherein said insults may lead to detrimental effects of oxidized phospholipids (OxPL) that are explained in the context of the present invention in more detail in the following.

An "effect of oxidized phospholipids (OxPL)" is known in the art and relates to altered host responses through the effects of peroxidized phospholipids originating from membranes, lipoproteins or microparticles. The generation of oxygen radicals is part of the immediate inflammatory response to the invasion of pathogens to otherwise sterile sites of the body or to any injurious event such as sterile inflammation or chronic inflammation. During infections, oxygen radicals are primarily generated to this response to kill microbes, but also damage host structures as mentioned above. It has to be noted that oxygen radicals are generated during any kind of inflammation, i.e., e.g., during any sterile inflammation or chronic inflammation like, e.g., arteriosclerosis. It has been shown that the administration of oxidized phospholipids impair survival during E.coli peritonitis by inhibiting phagocytosis of bacteria by marcrophages or neutrophils (Knapp, S., et al. (2007) J Immunol 178, 993-1001). It has been described that the inhibitory action of OxPL is not restricted to phagocytosis of E. coli. Considering the multitude of endocytotic pathways elicited by macrophages, it has been found that OxPL not only impair phagocytosis of bacteria but also polystyrene particles as well as receptor-mediated, fluid-phase endocytosis and macropinocytosis) (Knapp, S., et al. (2007) J Immunol 178. 993-1001). Hence, these data underline the broad and harmful impact of oxidation products generated during bacterial infections or chronic inflammation in vivo. Accordingly, the effect of oxidized phospholipids may only relate to the effect of oxidized phospholipids in general, but in particular to detrimental effects of oxidation products generated at sites of inflammation where the innate immune response to bacterial infections is impaired by OxPL inhibition of the phagocytosing capacity of cells involved in innate immunity.

An inflammatory disease that develops in response to an infection caused by a pathogen or that develops subsequent to insults leading to elevated oxidized phospholipids may be detectable preceding, during or following the inflammation causing disease or event. The inflammatory disease may develop in response to an infection like a pathogen infection, for example a bacterial infection. In another embodiment, said infection is caused by a pathogen. Said infection may be sepsis and/or SIRS. The present invention relates in particular to the medical intervention of of a pathogen infection, like an infection with parasites, fungi, viruses and/or bacteria. In yet another embodiment, said infection is a bacterial infection. The bacterial infection may be caused by a gram positive or a gram negative bacterium. In another embodiment, secondary infections may develop subsequent insults like trauma, injury, surgery or inflammation. Said injury or trauma may be acute lung injury or head trauma. Said inflammation may be pancreatitis. Also these secondary infections and/or inflammations may be treated or prevented by the use of WAVE 1 inhibitors/antagonists.

The oxidized phospholipids (OxPL) related inflammatory disease developing in response to an infection caused by a pathogen or the infection caused by a pathogen or the secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insults lead to elevated oxidized phospholipids may be sepsis. The inflammatory diseases include all inflammatory diseases that increase the risk of secondary infections by parasite, fungus, virus and, preferably, bacteria. Hence, said oxidized phospholipids (OxPL) related inflammatory disease developing in response to an infection caused by a pathogen or the infection caused by a pathogen or the secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insults lead to elevated oxidized phospholipids may be SIRS and/or sepsis. Said secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insults lead to elevated oxidized phospholipids may be selected from the group consisting of, but not limited to, acute lung injury, head trauma and/or pancreatitis. As already mentioned above, the term "elevated oxidized phospholipids" relates to a level of oxidized phospholipids (OxPL) that is higher than the normal level in the non-diseased state and/or the state without insult.

The present invention relates to the treatment of oxidized phospholipids (OxPL) related inflammatory diseases developing in response to an infection caused by a pathogen or to infections caused by a pathogen, wherein said inflammatory disease is preferably an inflammation of the peritoneum with WAVEl antagonists/inhibitors. Said inflammatory disease may be selected from the group consisting of an inflammation of the lung, brain, liver, kidney, heart, joint and/or intestine.

Without being bound by theory, it is believed that inhibitors/antagonists of WAVEl interfere with WAVEl as described herein below and exert thereby their effect on the stimulation of phagocytosis via its interaction with the actin cytoskeleton, thereby improving the bacterial uptake and increasing the phagocytosis activity in overwhelming inflammation. It is, therefore, envisaged that WAVEl antagonists/inhibitors prevent, inter alia, an enhanced bacterial outgrowth upon inflammation and can, thus, successfully used in the treatment or prevention of inflammatory diseases related to the effect of oxidized phospholipids (OxPL) and/or infectious disorders.

As disclosed herein, WAVEl mediates antiphagocytic properties of OxPL in vitro and in vivo and silencing of WAVEl prevents OxPL associated cell spread and inhibition of phagocytosis.

The successful use of WAVEl antagonists/inhibitors disclosed herein is also illustrated by the herein described experiments, i.e. reduced WAVEl expression completely abolished OxPL- associated actin spread and inhibition of phagocytosis. Furthermore, the effect of WAVEl is reflected in the finding that primary WAVEL ^" peritoneal macrophages exhibited neither spread nor impaired bacterial uptake upon OxPL treatment.

As illustrated in the appended examples, the function of WAVEl during E. coli peritonitis in vivo was demonstrated, determining its critical role in phagocytosis and host defense. In this context, the successful use of antagonists of WAVEl disclosed herein is, therefore, also illustrated by the herein described mouse model, i.e. the WAVEl^7" deficient mice, wherein both alleles of the WAVE1 gene are knocked out, which reflects the action of WAVE1 antagonists/inhibitors on WAVE1 activity. Following i.p. injection of OxPL, said mice were infected with E.coli and examined their ability to contain bacterial dissemination. OxPL treatment led to enhanced bacterial outgrowth in mice that received WT bone marrow. However, in contrast, chimeric mice with peritoneal macrophages are resistant to the effects of OxPL. Furthermore, the absence of WAVE1 in peritoneal macrophages prevented the OxPL-associated impairment of survival during E.coli peritonitis. These data are indicative of a role of WAVE 1 in mediating the inhibition of phagocytosis caused by OxPL in vitro and in vivo.

In summary, as illustrated in the appended examples, it is demonstrated herein that antagonists and/or inhibitors of WAVE1 are useful in treating and/or preventing an enhanced bacterial outgrowth upon inflammation and can, thus, be successfully used in the treatment of oxidized phospholipids (OxPL) related inflammatory diseases developing in response to an infection caused by a pathogen and/or in the treatment of an infection caused by a pathogen and/or (systemic) infections related to the effect of oxidized phospholipids (OxPL) and developing in response to an infection with a pathogen and/or an infection developing subsequent to injury, trauma or inflammation. Thus, the present invention is particularly useful in the medical intervention of an infection at normally sterile sites like the peritoneal cavity (for example a peritonitis), and/or the treatment of a sepsis and/or SIRS, like peritonitis.

The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of a disease in a subject and includes: (a) preventing and ameliorating an inflammatory disease related to the effects of oxidized phospholipids (OxPL) from occurring in a subject which may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development like the inhibition of an infection like sepsis and/or SIRS; or (c) relieving the disease, i.e. causing regression of the disease, like the repression of an infection of sepsis, SIRS and the like. In accordance with the present invention, the term "prevention" or "preventing" of an infection/infectious disease means the infection per se can be hindered of developing or to develop into an even worse situation. Accordingly, it is one of the advantages of the present invention that a WAVEl inhibitor/antagonist can be employed in avoidance of a (secondary or additional infection with a pathogen, like a bacterium, or in avoidance of a sepsis after an insult occurs or will occur. Such an insult may trauma (like head trauma), injury (like acute lung injury) , surgery or even an inflammation, like pancreatitis. Therefore and in accordance with the present invention, WAVEl inhibitors/antagonists may also be employed before an infection and/or a sepsis or an inflammation develops.

However, as disclosed and provided for herein, WAVEl inhibitors/antagonist may also be employed in the amelioration and/or treatment of disorders wherein the diseased status has already developed, i.e. in the treatment of an existing infection caused by a pathogen, or in the treatment of an OxPL related inflammatory disorder developing in response to an infection with a pathogen. Accordingly, the term "treatment" as used herein relates to medical intervention of an already manifested disorder, like the treatment of an already defined and manifested infection, sepsis, SIRS and the like. Thus, the present invention is particularly useful in the medical intervention of an infection at normally sterile sites like the peritoneal cavity (for example a peritonitis), and/or the treatment of a sepsis and/or SIRS, like peritonitis.

A "patient" or "subject" for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.

The compounds capable of reducing WAVEl function or (a) fragment(s) thereof, are expected to be very beneficial as agents in pharmaceutical settings disclosed herein and to be used for medical purposes, in particular, in the treatment of inflammatory diseases related to effects of oxidized phospholipids (OxPL). Said antagonist/inhibitor of WAVEl may be selected from the group consisting of A AP inhibitory peptide, small binding molecules, RNAi, anti-WAVEl antisense molecules, intracellular binding-partners of WAVE1, aptamers or intramers specifically directed against WAVE1.

Compounds which may function as specific an "antagonist" or "inhibitor" of WAVE1 may comprise small binding molecules such as small (organic) compounds or ligands for WAVE1. The term "small molecule" in the context of drug discovery is known in the art and relates to medical compounds having a molecular weight of less than 2,500 Daltons, preferably less than 1 ,000 Daltons, more preferably between 50 and 350 daltons. (Small) binding molecules comprise natural as well as synthetic compounds. The term "compound" in context of this invention comprises single substances or a plurality of substances. Said compound/binding molecules may be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms. Furthermore, said compound(s) may be known in the art but hitherto not known to be capable of (negatively) influencing the activity WAVE1 or not known to be capable of influencing the expression of the nucleic acid molecule encoding for WAVE1, respectively. The plurality of compounds may be, e.g., added to a sample in vitro, to the culture medium or injected into the cell.

Yet it is also envisaged in the context of the present invention that compounds including, inter alia, peptides, proteins, nucleic acids including cDNA expression libraries, small organic compounds, ligands, PNAs and the like can be used as an antagonist of WAVE1 function. Said compounds can also be functional derivatives or analogues. Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, "Handbook of Organic Chemistry", Springer Edition New York, or in "Organic Synthesis", Wiley, New York. Furthermore, said derivatives and analogues can be tested for their effects, i.e. their antagonistic effects of WAVE1 function according to methods known in the art. Furthermore, peptidomimetics and/or computer aided design of appropriate antagonists or inhibitors of WAVE 1 can be used. Appropriate computer systems for the computer aided design of, e.g., proteins and peptides are described in the prior art, for example, in Berry (1994) Biochem. Soc. Trans. 22: 1033-1036; Wodak (1987) , Ann. N. Y. Acad. Sci. 501 : 1-13; Pabo (1986) , Biochemistry 25:5987-5991. The results obtained from the above-described computer analysis can be used in combination with the method of the invention for, e.g., optimizing known compounds, substances or molecules. Appropriate compounds can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e.g., according to the methods described herein. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh (1996) Methods in Enzymology 267:220-234 and Dorner (1996) Bioorg. Med. Chem. 4:709- 715. Furthermore, the three-dimensional and/or crystallographic structure of antagonists of WAVE1 can be used for the design of (peptidomimetic) antagonists of WAVE 1.

The RNAi-approach is also envisaged in context of this invention for use in the preparation of a pharmaceutical composition for the treatment of diseases/disorders related to an insufficient immune response as disclosed herein.

The term "RNA interference" or "inhibiting RNA" (RNAi/iRNA) describes the use of double- stranded RNA to target specific mRNAs for degradation, thereby silencing their expression. Preferred inhibiting RNA molecules may be selected from the group consisting of double- stranded RNA (dsRNA), RNAi, siRNA, shRNA and stRNA. dsRNA matching a gene sequence is synthesized in vitro and introduced into a cell. The dsRNA may also be introduced into a cell in form of a vector expressing a target gene sequence in sense and antisense orientation, for example in form of a hairpin mRNA. The sense and antisense sequences may also be expressed from separate vectors, whereby the individual antisense and sense molecules form double-stranded RNA upon their expression. It is known in the art that in some occasions the expression of a sequence in sense orientation or even of a promoter sequence suffices to give rise to dsRNA and subsequently to siRNA due to internal amplification mechanisms in a cell. Accordingly, all means and methods which result in a decrease in activity (which may be reflected in a lower expression of WAVE1), in particular by taking advantage of WAVE 1 -specific siRNAs (i.e. siRNAs that target specifically WAVE1 mRNA or a functional fragment thereof) are to be used in accordance with the present invention. For example sense constructs, antisense constructs, hairpin constructs, sense and antisense molecules and combinations thereof can be used to generate/introduce these siRNAs. The dsRNA feeds into a natural, but only partially understood process including the highly conserved nuclease dicer which cleaves dsRNA precursor molecules into short interfering RNAs (siRNAs). The generation and preparation of siRNA(s) as well as the method for inhibiting the expression of a target gene is, inter alia, described in WO 02/055693, Wei (2000) Dev. Biol. 15:239-255; La Count (2000) Biochem. Paras. 111 :67-76; Baker (2000) Curr. Biol. 10: 1071-1074; Svoboda (2000) Development 127:4147- 4156 or Marie (2000) Curr. Biol. 10:289-292. These siRNAs built then the sequence specific part of an RNA-induced silencing complex (RISC), a multicomplex nuclease that destroys messenger RNAs homologous to the silencing trigger). Elbashir (2001) EMBO J. 20:6877- 6888 showed that duplexes of 21 nucleotide RNAs may be used in cell culture to interfere with gene expression in mammalian cells. It is already known that RNAi is mediated very efficiently by siRNA in mammalian cells but the generation of stable cell lines or non-human transgenic animals was limited. However, new generations of vectors may be employed in order to stably express, e.g. short hairpin RNAs (shRNAs). Stable expression of siRNAs in Mammalian Cells is inter alia shown in Brummelkamp (2002) Science 296:550-553. Also Paul (2002) Nat. Biotechnol. 20:505-508 documented the effective expression of small interfering RNA in human cells. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells was also shown by Yu (2002) PNAS 99:6047-6052. The shRNA approach for gene silencing is well known in the art and may comprise the use of st (small temporal) RNAs; see, inter alia, Paddison (2002) Genes Dev. 16:948-958. These approaches may be vector-based, e.g. the pSUPER vector, or RNA polIII vectors may be employed as illustrated, inter alia, in Yu (2002), loc. cit.; Miyagishi (2002), loc. cit. or Brummelkamp (2002), loc. cit. It is envisaged that the regulatory sequences of the present invention are used in similar fashion as the systems based on pSUPER or RNA polIII vectors.

Methods to deduce and construct siRNAs are known in the art and are described in Elbashir (2002) Methods 26: 199-213, at the internet web sites of commercial vendors of siRNA, e.g. Qiagen GmbH (https://wwwl .qiagen.com/GeneGlobe/Default.aspx); Dharmacon (www.dharmacon.com); Xeragon Inc. (http://www.dharmacon.com/Default.aspx), and Ambion (www.ambion.com), or at the web site of the research group of Tom Tuschl (http://www.rockefeller.edu/labheads/tuschl/sima.html). In addition, programs are available online to deduce siRNAs from a given mRNA sequence (e.g. http://www.ambion.com/techlib/misc/siRNA_fmder.html or http://katahdin.cshl. org:9331/RNAi/html/rnai.html). Uridine residues in the 2-nt 3 ' overhang can be replaced by 2'deoxythymidine without loss of activity, which significantly reduces costs of RNA synthesis and may also enhance resistance of siRNA duplexes when applied to mammalian cells (Elbashir (2001) loc. cit). The siRNAs may also be sythesized enzymatically using T7 or other RNA polymerases (Donze (2002) Nucleic Acids Res 30:e46). Short RNA duplexes that mediate effective RNA interference (esiRNA) may also be produced by hydrolysis with Escherichia coli RNase III (Yang (2002) PNAS 99:9942-9947). Furthermore, expression vectors have been developed to express double stranded siRNAs connected by small hairpin RNA loops in eukaryotic cells (e.g. (Brummelkamp (2002) Science 296:550- 553). All of these constructs may be developed with the help of the programs named above. In addition, commercially available sequence prediction tools incorporated in sequence analysis programs or sold separately, e.g. the siRNA Design Tool offered by www.oligoEngine.com (Seattle,WA) may be used for siRNA sequence prediction.

Accordingly, specific interfering RNAs can be used in accordance with the present invention as antagonists (inhibitors) of WAVE1 (expression and/or function). These siRNAs are formed by an antisense and a sense strand, whereby the antisense/sense strand preferably comprises at least 10, more preferably at least 12, more preferably at least 14, more preferably at least 16, more preferably at least 18, more preferably at least 19, 20, 21 or 22 nucleotides.

The use of the following shRNA is particularly preferred in context of the present invention: shRNA (a) : ACCGGACCG ATTGTCTGTT AGTTTCAAG AG A ACT AAC AG AC AATCGGTCCTTTTTC (SEQ ID No: 9)

It is preferred herein that the 5' end of the shRNAs (in particular of SEQ ID NO 9) is phosphorylated. If the 5' end is phosphorylated, the respective sequences can also be depicted as follows:

5 ' -PACCGG ACCGATTGTCTGTT AGTTTCAAG AGAACTAACAG ACAATCGGTCCTTTTTC (SEQ ID No: 10). wherein "5'-P" reflects the phosphorylated 5' end.

As mentioned above, methods for preparing siRNAs to be used in accordance with the present invention are well known in the art. Based on the teaching provided herein, a skilled person in the art is easily in the position not only to prepare such siRNAs but also to assess whether a siRNA is capable of antagonizing/inhibiting WAVE1. It is envisaged herein that the above described siRNAs lead to a degradation of WAVE 1 mRNA and thus to a decreased protein level of WAVE 1. In other words, siRNAs lead to a pronounced decrease in mRNA and/or protein levels of WAVE1 (i.e. to a reduced expression of WAVE1). This decrease in expression may be reflected in a decreased activity of WAVE1. For example, WAVE1 -specific siRNAs may lead to a decreased capacity of WAVE1 to inhibit phagocytosis in overwhelming inflammation. Hence, the use of potent antagonists/inhibitors of WAVE 1 (such as the herein described siRNAs) will lead to a higher phagocytosis activity and improved bacterial uptake which will in turn result in a potential prevention or amelioration of inflammatory diseases related to the effects of oxidized phospholipids. An exemplary effect of the silencing of WAVE1 in macrophages using shRNA is shown in the appended example. As demonstrated therein, the shRNA silencing of WAVE1 reduced WAVE1 expression and completely abolished OxPL-associated actin spread and inhibition of phagocytosis.

As used herein the term "small interfering RNA" (siRNA), sometimes known as short interfering RNA or silencing RNA, refers to a class of generally short and double-stranded RNA molecules that play a variety of roles in biology and, to an increasing extent, in treatment of a variety of diseases and conditions. As mentioned above, siRNAs are involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene (see, e.g. Zamore Nat Struct Biol 2001, 8(9):746-50; Tuschl T. CHEMBIOCHEM. 2001, 2:239-245; Scherr and Eder, Cell Cycle. 2007 Feb;6(4):444-9; Leung and Whittaker, Pharmacol Ther. 2005 Aug;107(2):222-39; de Fougerolles et al., Nat. Rev. Drug Discov. 2007, 6: 443-453).

Such siRNAs are generally 18-27 nt long, generally comprising a short (usually 19-21-nt) double-strand of RNA (dsRNA) with or without 2-nt 3' overhangs on either end. Each strand can have a 5' phosphate group and a 3' hydroxyl (-OH) group or the phosphate group can be absent on one or both strands. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. siRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. In this context, other structures than those described above are also envisaged, provided they are capable of interfering with gene expression. Preferably, the double-stranded part has a length of about 12 to about 50 base pairs, more preferably 16 to 30, more preferably 18 to 25, more preferably 19 to 21 in length. Most preferably, the double- stranded part has a langth of 19 base pairs. The siRNA of the invention may either have overhanging sequences of up to 10 bases, preferably not more than 5 bases in length at either end or at one end, or may be blunt-ended. Also preferred is that the complementarity to the target gene extends over the entire length of the double-stranded part. The region which is complementary to the target gene is at least 12 bases, preferably at least 15, 16, 17, 18, 19, 20, 21, 22, 23 or more bases in length. The siRNA of the invention may be fully complementary to the target gene. Alternatively, the siRNA may comprise up to 5%, 10%, 20% or 30% mismatches to the target gene. Furthermore, siRNAs and also antisense RNAs can be chemically modified e.g. on the backbone including the sugar residues. Preferred modifications of the siRNA molecules of he invention include linkers connecting the two strands of the siRNA molecule. Chemical modifications serve inter alia to improve the pharmacological properties of siRNAs and antisense RNAs such as in vivo stability and/or delivery to the target site within an organism. The skilled person is aware of such modified siRNAs as well as of means and methods of obtaining them, see, for example, Zhang et al, Curr Top Med Chem. 2006;6(9):893-900; Manoharan, Curr Opin Chem Biol. 2004 Dec;8(6):570-9.

Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. This has made siRNAs an important tool for gene function and drug target validation studies as well as for therapeutic intervention which is envisaged here. The siRNAs disclosed herein are capable of reducing or blocking the expression of WAVEl .

It is envisaged that antisense molecules inhibit the expression or function of WAVEl , in particular of human WAVEl and interact with WAVEl as expressed by the coding regions, mRNAs/cDNAs as defined herein above as well as with WAVEl as expressed by isoforms and variants of said WAVEl . Said isoforms or variants may, inter alia, comprise allelic variants or splice variants. Furthermore, it is also envisaged that the antisense molecules to be used in accordance with the present invention against WAVEl expression or function interfere specifically with regulatory sequences of WAVEl as defined herein below. The term "variant" means in this context that the WAVEl nucleotide sequence and the encoded WAVEl amino acid sequence, respectively, differs from the distinct sequences available under the above-identified GenBank Accession numbers, by mutations, e.g. deletion, additions, substitutions, inversions etc.

Therefore, the antisense-molecule to be employed in accordance with the present invention specifically interacts with/hybridizes to one or more nucleic acid molecules encoding WAVEl . Preferably said nucleic acid molecule is RNA, i.e. pre m-RNA or mRNA. The term "specifically interacts with/hybridizes to one or more nucleic acid molecules encoding WAVEl " relates, in context of this invention, to antisense molecules which are capable of interfering with the expression of WAVEl . Yet, highly mutated anti- WAVEl antisense constructs, which are not capable of hybridizing to or specifically interacting with WAVE1- coding nucleic acid molecules are not to be employed in the context of the present invention. The person skilled in the art can easily deduce whether an antisense construct specifically interacts with/hybridizes to WAVEl encoding sequences. These tests comprise, but are not limited to hybridization assays, RNAse protection assays, Northern Blots, North-western blots, nuclear magnetic resonance and fluorescence binding assays, dot blots, micro- and macroarrays and quantitative PGR. In addition, such a screening may not be restricted to WAVEl mRNA molecules, but may also include WAVEl mRN A/protein (RNP) complexes (Hermann (2000) Angew Chem Int Ed Engl 39: 1890-1904; DeJong (2002) Curr Trop Med Chem 2:289-302). Furthermore, functional tests including Western blots, immunohistochemistry, immunoprecipitation assay, and bioassays based on WAVE1 - responsive promoters are envisaged for testing whether a particular antisense construct is capable of specifically interacting with/hybridizing to the WAVEl encoding nucleic acid molecules.

The term "antisense-molecule" as used herein comprises in particular antisense oligonucleotides. Said antisense oligonucleotides may also comprise modified nucleotides as well as modified intemucleoside-linkage, as, inter alia, described in US 6,159,697.

Most preferably, the antisense oligonucleotides of the present invention comprise at least 8, more preferably at least 10, more preferably at least 12, more preferably at least 14, more preferably at least 16 nucleotides. The deduction as well as the preparation of antisense molecules is very well known in the art. The deduction of antisense molecules is, inter alia, described in Smith, 2000. Usual methods are "gene walking", Rnase H mapping, RNase L mapping (Leaman (1999) Meth Enzymol 18:252-265), combinatorial oligonucleotide arrays on solid support, determination of secondary structure analysis by computational methods (Walton (2000) Biotechnol Bioeng, 65:1-9), aptamer oligonucleotides targeted to structured nucleic acids (aptastruc), thetered oligonucleotide probes, foldback triplex-forming oligonucleotides (FTFOs) (Kandimalla (1994) Gene 149: 115-121) and selection of sequences with minimized non-specific binding (Han (1994) Antisense Res Dev 4:53-65).

The antisense molecules of the present invention may be stabilized against degradation. Such stabilization methods are known in the art and, inter alia, described in US 6,159,697. Further methods described to protect oligonucleotides from degradation include oligonucleotides bridged by linkers (Vorobjev (2001) Antisense Nucleic Acid Drug Dev, 1 1 :77-85), minimally modified molecules according to cell nuclease activity (Samani (2001) Antisense Nucleic Acid Drug Dev, 11 : 129-136), 2'0-DMAOE oligonucleotides (Prakash (2001) Nucleosides Nucleotides Nucleic Acids 20:829-832), 3'5'-Dipeptidyl oligonucleotides (Schwope (1999) J Org Chem 64:4749-4761), 3'methylene thymidine and 5-methyluridine/cytidine h- phosphonates and phosphonamidites (An (2001) J Org Chem, 66:2789-2801), as well as anionic liposome (De Oliveira (2000) Life Sci 67: 1625-1637) or ionizable aminolipid (Semple (2001) Biochim Biophys Acta, 10:152-166) encapsulation.

In addition thereto, the antagonist/inhibitor of WAVE1 expression or function may also comprise intracellular binding partners of WAVE 1. As used herein, the term "intracellular binding partner" relates to intracellular molecules capable of preventing or reducing WAVE1 activity. Such intracellular binding partners of WAVE 1, inter alia, may relate to endogenous inhibitor/repressor proteins of WAVE1. In another embodiment of the invention the intracellular binding partner is an intracellular antibody. Intracellular antibodies are known in the art and can be used to modulate or inhibit the functional activity of the target molecule. This therapeutic approach is based on intracellular expression of recombinant antibody fragments, either Fab or single chain Fv, targeted to the desired cell compartment using appropriate targeting sequences (Teillaud (1999) Pathol Biol 47:771-775). The antagonist/inhibitor of WAVE 1 may also be an AKAP inhibitory peptide. As described above and exemplified in the examples, A-kinase anchoring proteins (AKAP) spatially and temporally control the specificity of cAMP downstream effects by targeting PKA to particular subcellular regions. As illustrated in the examples, the cell-permeable AKAP inhibitory peptide "stearated Ht-31", that specifically prevents the association of the regulatory subunit RII of PKA with AKAPs, abrogated the change in cell shape caused by OxPL. In addition, as demonstrated in the examples, administration of Ht-31 together with OxPL at the onset of E. coli peritonitis in mice prevented the increase in bacterial loads caused by OxPL in vivo. Furthermore, survival analysis corroborated these findings, as Ht-31 treatment was able to reverse the detrimental effects of OxPL during E. coli peritonitis in vivo. Hence, the data presented in the appended examples clearly indicate that the inhibition of WAVE 1 by AKAP inhibitory peptides increase phagocytosis of bacteria in vitro and in vivo. Accordingly, also antagonists and/or inhibitors of WAVE1 that may also be AKAP inhibitory peptides are useful in preventing an enhanced bacterial outgrowth upon inflammation and can, thus, successfully used in the treatment of inflammatory diseases related to the effect of oxidized phospholipids (OxPL).

Accordingly, specific AKAP inhibitory peptides can be used in accordance with the present invention as antagonists (inhibitors) of WAVE1 (expression and/or function). In particular, the AKAP inhibitory peptide to be used in accordance with this invention is Ht-31 with the following amino acid sequence:

Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys-Ala-Ala- Tyr

(SEQ ID NO: 11)

(Association of the type II cAMP-dependent protein kinase with a human thyroid RII- anchoring protein. Cloning and characterization of the Rll-binding domain. Carr DW, Hausken ZE, Fraser ID, Stofko-Hahn RE, Scott JD. J Biol Chem. 1992 Jul 5;267(19): 13376- 82). The AKAP inhibitory peptide to be used in accordance with this invention is not limited to the molecule (Ht-31) of SEQ ID NO: l l . Also envisaged are molecules that are at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:l 1 and having the activity to inhibit AKAP. It is also envisaged to use other AKAP inhibitory peptides in accordance with this invention. Such AKAP inhibitory peptides are well known to the person skilled in the art. Some AKAP inhibitory peptides are exemplarily described in the following documents: Alto, N. M., Soderling, S. H., Hoshi, N., Langeberg, L. K., Fayos, R., Jennings, P. A. and Scott, J. D. (2003) Bioinformatic design of A-kinase anchoring protein-m silico: a potent and selective peptide antagonist of type II protein kinase A anchoring. Proc. Natl. Acad. Sci. U.S.A. 100, 4445-4450. Burns-Hamuro, L. L., Ma, Y., Kammerer, S., Reineke, U., Self, C, Cook, C, Olson, G. L,, Cantor, C. R., Braun, A. and Taylor, S. S. (2003). Designing isoform-specific peptide disruptors of protein kinase A localization. Proc. Natl. Acad. Sci. U.S.A. 100, 4072- 4077. Carlson, C. R., Lygren, B., Berge, T., Hoshi, N., Wong, W., Tasken, and Scott, J. D. (2006) Delineation of type I protein kinase a selective signaling events using an RI anchoring disruptor (RIAD). J. Biol. Chem. 281, 21535-21545. Hundsrucker, C, Krause, G., Beyermann, M., Prinz, A., Zimmermann, B., Diekmann, O., Lorenz, D., Stefan, E., Nedvetsky, P., Dathe, M. et al. (2006) High-affinity AKAP75-protein kinase A interaction yields novel protein kinase A anchoring disruptor peptides. Biochem. J. 396, 297-306. Whereas Ht-31 is a RII alpha inhibitor of PKA, inhibitors of another PKA isoform (RI) are described in Biochem J. 2006 Dec 15;400(3):493-9. Characterization of A-kinase-anchoring disruptors using a solution-based assay. Stokka AJ, Gesellchen F, Carlson CR, Scott JD, Herberg FW, Tasken K.

The AKAP inhibitory peptides of the present invention are not restricted to the ones described above. Variants of such proteins/polypeptides are also envisaged wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs as well as other than the 20 gene-encoded amino acids, such as selenocysteine. Peptides, oligopeptides and proteins may be tenxied polypeptides. The terms polypeptide and protein are often used interchangeably herein. The term polypeptide also refers to, and does not exclude, modifications of the polypeptide, e.g., glycosylation, acetylation, phosphorylation and the like. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.

As mentioned herein above, the antagonist/inhibitor of WAVE! expression or function may also comprise an aptamer. In the context of the present invention, the term "aptamer" comprises nucleic acids such as RNA, ssDNA (ss = single stranded), modified RNA, modified ssDNA or PNAs which bind a plurality of target sequences having a high specificity and affinity. Aptamers are well known in the art and, inter alia, described in Famulok (1998) Curr. Op. Chem. Biol. 2:320-327. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold (1995) Ann. Rev. Biochem. 64:763-797).

Accordingly, aptamers are oligonucleotides derived from an in vitro evolution process called SELEX (systematic evolution of ligands by exponential enrichment). Pools of randomized RNA or single stranded DNA sequences are selected against certain targets. The sequences of tighter binding with the targets are isolated and amplified. The selection is repeated using the enriched pool derived from the first round selection. Several rounds of this process lead to winning sequences that are called "aptamers". Aptamers have been evolved to bind proteins which are associated with a number of disease states. Using this method, many powerful antagonists of such proteins can be found. In order for these antagonists to work in animal models of disease and in humans, it is normally necessary to modify the aptamers. First of all, sugar modifications of nucleoside triphosphates are necessary to render the resulting aptamers resistant to nucleases found in serum. Changing the 2ΌΗ groups of ribose to 2'F or 2'NH2 groups yields aptamers which are long lived in blood. The relatively low molecular weight of aptamers (8000-12000) leads to rapid clearance from the blood. Aptamers can be kept in the circulation from hours to days by conjugating them to higher molecular weight vehicles. When modified, conjugated aptamers are injected into animals, they inhibit physiological functions known to be associated with their target proteins. Aptamers may be applied systemically in animals and humans to treat organ specific diseases (Ostendorf (2001) J Am Soc Nephrol. 12:909-918). The first aptamer that has proceeded to phase I clinical studies is NX-1838, an injectable angiogenesis inhibitor that can be potentially used to treat macular degeneration-induced blindness. (Sun (2000) Curr Opin Mol Ther 2: 100-105). Cytoplasmatic expression of aptamers ("intramers") may be used to bind intracellular targets (Blind (1999) PNAS 96:3606-3610; Mayer (2001) PNAS 98:4961-4965). Said intramers are also envisaged to be employed in context of this invention.

As used herein, the term "nucleic acid sequence" relates to the sequence of bases comprising purine- and pyrimidine bases which are comprised by nucleic acid molecules, whereby said bases represent the primary structure of a nucleic acid molecule. Nucleic acid sequences include DNA, cDNA, genomic DNA, NA, synthetic forms and mixed polymers, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Preferably, the term "WAVE1" when used in the context of expressing WAVE1 refers to the nucleic acid molecule encoding WAVE1 protein, or a functional fragment thereof. Exemplary nucleic acid sequences are known in the art and also disclosed herein.

As used herein, the term "polypeptide" relates to a peptide, a protein, or a polypeptide which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds. However, peptidomimetics of such proteins/polypeptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention as well as other than the 20 gene-encoded amino acids, such as selenocysteine. Peptides, oligopeptides and proteins may be termed polypeptides. The terms polypeptide and protein are often used interchangeably herein. The term polypeptide also refers to, and does not exclude, modifications of the polypeptide, e.g., glycosylation, acetylation, phosphorylation and the like. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Preferably, the term "WAVE1", particularly when used in context of "activity of WAVE 1", refers to the protein/polypeptide having the specific WAVE1 activity as disclosed herein.

As used herein, a "functional fragment" of a protein which displays a specific biological activity relates to fragments of said protein having a sufficient length to display said activity. Accordingly, a functional fragment of a protein showing e.g. a specific (enzymatic) activity may relate to a polypeptide which corresponds to a fragment of said protein which is still capable of showing said (enzymatic) activity. For example, a functional fragment of WAVE 1 in the context of the protein binding activity of WAVE1 may correspond to the protein- binding domain of WAVE1 as defined herein below. Methods for determining whether a certain fragment of a protein is a functional fragment are known in the art. For example, test for determining whether a fragment of WAVE1 is still capable of binding a protein are described herein below. Preferably, a functional fragment of WAVE1 has substantially the same biological activity as WAVE1 itself. Furthermore, a person skilled in the art will be aware that the (biological) activity as described herein often correlates with the expression level, preferably the protein or mRNA level. The term "expression" as used herein refers to the expression of a nucleic acid molecule encoding a polypeptide/protein, whereas "activity" refers to the activity of said polypeptide/protein, which can be determined as outlined herein. The explanations given herein above and below in respect of the activity of "WAVE1" also apply, mutatis mutandis, to (a) "functional fragment(s) of WAVE1". In other words, a "functional fragment of WAVE1" has essentially the same activity as WAVE1 as defined herein. Accordingly, also inhibitors/antagonists of functional fragments of WAVE1 are disclosed and provided herein. As mentioned, methods/assays for determining the activity of "WAVEl" and "functional fragment of WAVE1" are well known in the art and also described herein above and below. Preferably, the functional fragment has at least 60 %, more preferably at least 70 %, 75 %, 80 %, 85 %, 90 % and even more preferably at least 95 % or 99 % of WAVEl .

WAVEl antagonists/inhibitors of WAVEl function may be deduced by methods in the art. Such methods are described herein and, inter alia, may comprise, but are not limited to methods where a collection of substances is tested for interaction with WAVEl or with (a) fragment(s) thereof and where substances which test positive for interaction in a corresponding readout system are further tested in vivo, in vitro or in silico for their inhibiting effects on WAVEl expression or function.

Said "test for WAVEl interaction" of the above described method may be carried out by specific immunological, molecular biological and/or biochemical assays which are well known in the art and which comprise, e.g., homogenous and heterogenous assays as described herein below. The natural endogenous ligand(s) of WAVEl remain(s) to be identified. Yet, WAVEl ligands capable of inhibiting WAVEl function may be identified by screening large compound libraries based on their capacity to interact with the WAVEl protein. In a preferred embodiment, such antagonists or inhibitors of WAVEl function are capable of binding the protein binding domain of WAVE 1.

Besides molecules capable of binding to WAVEl , antagonists or inhibitors of WAVEl function may be capable of preventing/reducing the expression of the nucleic acid molecule encoding the WAVEl protein. The skilled person is readily capable of identifying regulatory sequences (such as promoter sequences, enhancer sequences, replication origins and other regulatory elements) of WAVE1 expression e.g. by using in silico gene prediction methods and experimental validation of functional sites (Elnitski (2006) Genome Res 16: 1455-64).

In a further aspect, the present invention relates to a pharmaceutical composition comprising the antagonist/inhibitor of WAVE1 as described herein, optionally further comprising a pharmaceutical carrier. The (pharmaceutical) compositions of the invention may be in solid or liquid form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). Furthermore, it is envisaged that the medicament of the invention might comprise further biologically active agents, depending on the intended use of the pharmaceutical composition.

Administration of the suitable (pharmaceutical) compositions may be effected by different ways, e.g., by parenteral, subcutaneous, intraperitoneal, topical, intrabronchial, intrapulmonary and intranasal administration and, if desired for local treatment, intralesional administration. Parenteral administrations include intraperitoneal, intramuscular, intradermal, subcutaneous intravenous or intraarterial administration. The compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like a specifically effected organ.

Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. Suitable carriers may comprise any material which, when combined with the biologically active protein of the invention, retains the biological activity of the comprised antagonist/inhibitor of WAVE 1 (see Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed). Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions). Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present including, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition of the present invention might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin.

These pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Pharmaceutically active matter may be present in amounts between 1 μg and 20 mg/kg body weight per dose, e.g. between 0.1 mg to 10 mg/kg body weight, e.g. between 0.5 mg to 5 mg/kg body weight. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg per kilogram of body weight per minute. Yet, doses below or above the indicated exemplary ranges also are envisioned, especially considering the aforementioned factors.

Furthermore, it is envisaged that the pharmaceutical composition of the invention might comprise further biologically active agents, depending on the intended use of the pharmaceutical composition. These further biologically active agents may be e.g. antibodies, antibody fragments, hormones, growth factors, enzymes, binding molecules, cytokines, chemokines, nucleic acid molecules and drugs. In a preferred embodiment, the pharmaceutical composition of the present invention is to be co-administered with other known antibiotics, antifungal drugs and/or antiviral drugs or treatments. In another preferred embodiment, the pharmaceutical composition of the invention may be co-administered with drugs that reduce oxidative stress. In a preferred embodiment, such a drug that reduces oxidative stress is an antioxidant. In another embodiment, the pharmaceutical composition of the invention may be co-administered with other conventional anti-inflammatory drugs.

Furthermore, the present invention relates to a screening method for assessing the activity of a candidate molecule suspected of being an antagonist/inhibitor of WAVE 1 which comprises the measurement of the activity of WAVE 1. Accordingly, screening methods for antagonists/inhibitors of WAVEl in cells, tissue and/or a non-human animal are provided. Also identification methods for antagonists of WAVEl are provided. These methods are highly useful in identifying/screening (a) candidate molecule(s) suspected of being inhibitors of WAVEl activity. Potent inhibitors identified/screened by these methods can be used in the medical intervention of an oxidized phospholipids (OxPL) related inflammatory disease developing in response to an infection caused by a pathogen and/or in the medical intervention of an infection caused by a pathogen and/or in the prevention of (systemic) infections related effects of oxidized phospholipids (OxPL) developing in response to an infection with a pathogen and/or in the intervention subsequent to injury, trauma or inflammation that causes detrimental effects of oxidized phospholipids (OxPL). In accordance with the present invention, a candidate molecule that may be suspected of being an antagonist of WAVEl can, in principle, be obtained from any source as defined herein. The candidate molecule(s) may be (a) naturally occurring substance(s) or (a) substance(s) produced by a transgenic organism and optionally purified to a certain degree and/or further modified as described herein. Practically, the candidate molecule may be taken from a compound library as they are routinely applied for screening processes.

Accordingly, the present invention relates to a method for assessing the activity comprising the steps of:

(a) optionally pre-incubating a cell, tissue or a non-human animal comprising and expressing WAVEl with OxPL;

(b) contacting said cell, tissue or a non-human animal comprising WAVEl with said candidate molecule;

(c) detecting a decrease in WAVEl activity; and

(d) selecting a candidate molecule that decreases WAVEl activity;

wherein a decrease of the WAVEl activity is indicative for the capacity of the selected molecule to ameliorate or treat OxPL related inflammatory disease developing in response to an infection caused by a pathogen and/or ameliorate or treat an infection caused by a pathogen and/or prevent an infection subsequent to an insult, like injury, trauma, surgery or inflammation.

It is to be understood that the detected activity of WAVEl in the above method is compared to a standard or reference value of WAVEl activity. The standard/reference value may be detected in a cell, tissue, or non-human animal as defined herein, which has not been contacted with a potential WAVEl inhibitor or prior to the above contacting step. The decrease in the activity of WAVEl may also be compared to the decrease in WAVEl activity by (a) routinely used reference compound(s). A skilled person is easily in the position to determine/assess whether the activity and/or expression of WAVEl is (preferably statistically significant) increased.

In accordance with this invention, in particular the screening or identifying methods described herein, a cell, tissue or non-human animal to be contacted with a candidate molecule comprises and expresses WAVEl . For example said cell, tissue or non-human animal may express a WAVEl gene, in particular also (an) additional (copy) copies of a WAVEl gene, (a) WAVEl mutated gene(s), a recombinant WAVEl gene construct and the like. As explained herein below, the capability of a candidate molecule to inhibit/antagonize WAVEl may, accordingly, be detected by measuring the expression level of such gene products of WAVEl or of corresponding gene constructs (e.g. mRNA or protein), wherein a low expression level (compared to a standard or reference value) is indicative for the capability of the candidate molecule to act as inhibitor/antagonist.

The term "candidate molecule" as used herein refers to a molecule or substance or compound or composition or agent or any combination thereof to be tested by one or more screening method(s) of the invention as a putative antagonist or inhibitor of WAVEl function, activity or expression. A test compound can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate, a lipid, or a combination thereof or any of the compounds, compositions or agents described herein. It is to be understood that the term "candidate molecule" when used in the context of the present invention is interchangeable with the terms "test compound", "test molecule", "test substance", "potential candidate", "candidate" or the terms mentioned herein above.

Also preferred potential candidate molecules or candidate mixtures of molecules to be used when contacting a cell expressing/comprising WAVEl as defined and described herein may be, inter alia, substances, compounds or compositions which are of chemical or biological origin, which are naturally occurring and/or which are synthetically, recombinantly and/or chemically produced. Thus, candidate molecules may be proteins, protein-fragments, peptides, amino acids and/or derivatives thereof or other compounds as defined herein, which bind to and/or interact with WAVEl , regulatory proteins/sequences of WAVEl function or functional fragments thereof. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.) are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means. Results obtained from deorphanisation programs based on phylogenetic analysis methods may aid to find natural ligands for WAVEl and, e.g., will allow in silico profiling of potential ligands for WAVEl .

The generation of chemical libraries with potential ligands for WAVEl is well known in the art. For example, combinatorial chemistry is used to generate a library of compounds to be screened in the assays described herein. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building block" reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining amino acids in every possible combination to yield peptides of a given length. Millions of chemical compounds can theoretically be synthesized through such combinatorial mixings of chemical building blocks. For example, one commentator observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds. (Gallop, Journal of Medicinal Chemistry, Vol. 37, No. 9,1233-1250 (1994)). Other chemical libraries known to those in the art may also be used, including natural product libraries. Once generated, combinatorial libraries are screened for compounds that possess desirable biological properties. For example, compounds which may be useful as drugs or to develop drugs would likely have the ability to bind to the target protein identified, expressed and purified as described herein.

In the context of the present invention, libraries of compounds are screened to identify compounds that function as an antagonist or inhibitor of WAVEl . First, a library of small molecules is generated using methods of combinatorial library formation well known in the art. US 5,463,564 and US 5,574,656 are two such teachings. Then the library compounds are screened to identify those compounds that possess desired structural and functional properties. US 5,684,711 , discusses a method for screening libraries. To illustrate the screening process, the target cell or gene product and chemical compounds of the library are combined and permitted to interact with one another. A labelled substrate is added to the incubation. The label on the substrate is such that a detectable signal is emitted from metabolized substrate molecules. The emission of this signal permits one to measure the effect of the combinatorial library compounds on the enzymatic activity of target enzymes/activity of target protein by comparing it to the signal emitted in the absence of combinatorial library compounds. The characteristics of each library compound are encoded so that compounds demonstrating activity against the cell/enzyme/target protein can be analyzed and features common to the various compounds identified can be isolated and combined into future iterations of libraries. Once a library of compounds is screened, subsequent libraries are generated using those chemical building blocks that possess the features shown in the first round of screen to have activity against the target protein. Using this method, subsequent iterations of candidate compounds will possess more and more of those structural and functional features required to inhibit the target protein, until a group of antagonists/inhibitors with high specificity for the protein can be found. These compounds can then be further tested for their safety and efficacy as an WAVE1 inhibitor/antagonist agent for use in animals, such as mammals. It will be readily appreciated that this particular screening methodology is exemplary only. Other methods are well known to those skilled in the art. For example, a wide variety of screening techniques are known for a large number of naturally-occurring targets when the biochemical function of the target protein is known. For example, some techniques involve the generation and use of small peptides to probe and analyze target proteins both biochemically and genetically in order to identify and develop drug leads. Such techniques include the methods described in WO 99/35494, WO 98/19162, WO 99/54728.

Preferably, candidate agents to be tested encompass numerous chemical classes, though typically they are organic compounds, preferably small (organic) molecules as defined herein above. Candidate agents may also comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise carbocyclic or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

Exemplary classes of candidate agents may include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxylic terminus, e.g. for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like. Other methods of stabilization may include encapsulation, for example, in liposomes, etc.

As mentioned above, candidate agents are also found among other biomolecules including amino acids, fatty acids, purines, pyrimidines, nucleic acids and derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

Accordingly, assessing the activity of a candidate molecule suspected of being an antagonist/inhibitor of WAVE 1 may comprise a first step of pre-incubating a cell, tissue or non-human animal comprising and expressing WAVE1 with OxPL. Step (b) of the screening method as defined herein above may be accomplished by contacting, e.g., the cell(s), tissue(s), or non-human-animal comprising and expressing WAVE1 with (a) candidate molecule(s) to be tested and it is measured whether said candidate molecule(s) lead(s) to a decrease in the activity of WAVE 1. Such a change/increase is indicative for the capacity of the candidate molecule to enhance phagocytosis and improve bacterial uptake in overwhelming inflammation. In other words, the activity of the candidate molecule(s) as inhibitors/antagonists of WAVE1 is assessed based on their capacity to decrease the activity of WAVE1 wherein a decrease of the WAVE1 activity is indicative for the capacity of the selected molecule to ameliorate an inflammatory disease related to detrimental effects of oxidized phospholipids (OxPL). In particular the use of (a) (transgenic) cell(s), tissue(s), or non-human animal(s) overexpressing WAVE1 is envisaged, since these may allow a more sensitive/easier detection of a decrease of WAVE 1 activity.

It is to be understood that in a high throughput screening routinely, many (often thousands of candidate molecules) are screened simultaneously. Accordingly, in a (first) screen candidate molecules are selected, which decrease WAVE1 activity.

Step (b) of the screening methods of the present invention, i.e. the "contacting step" may be accomplished by adding a candidate molecule or a (biological) sample or composition containing said candidate molecule or a plurality of candidate molecules (i.e. various different candidate molecules) to (a) cell(s)/tissue(s)/non-human animal comprising WAVE1 or a functional fragment thereof.

The term "contacting" may also refer to the addition of a candidate molecule a cell, tissue, non-human animal comprising WAVEl in a way that the candidate molecule may become effective to the cell at the cell surface or upon cellular uptake and thereby exert its inhibitory function on WAVEl -dependent responses.

Generally, the candidate molecule(s) or a composition comprising/containing the candidate molecule(s) may for example be added to a cell, tissue or non-human animal comprising WAVEl . As defined and disclosed herein, the term "comprising WAVEl" refers not only to the WAVEl gene(s) or proteins known in the art and described herein, but also to reporter constructs comprising a reporter as described in detail below. Exemplary reporters (preferably associated with the reporter signals disclosed herein) are luciferase and fluorescent proteins, like GFP, FP and the like. Also reporter constructs comprising a promoter and/or enhancer region of WAVEl can be used in the screening/identifying methods. Accordingly, the cell(s), tissue(s) and/or non-human animals used in the context of the present invention, in particular in context of the screening/identifying methods can be stably or transiently transfected with the reporter constructs disclosed herein.

In particular the identification/assessment of candidate molecules which are capable of inhibiting/antagonizing WAVEl may be, inter alia, performed by transfecting an appropriate host with a nucleic acid molecule encoding WAVEl (or a functional fragment thereof) and contacting said host with (a) candidate molecule(s). The host (cell, tissue, non-human animal) can also be transfected. The host cell may comprise, but is not limited to, CHO-cell, HE 293, HeLa, Cos 7, PC 12 or NIH3T3 cell, frog oocytes or primary cells like primary cardiomyocytes, fibroblasts, muscle, endothelial or embryonic stem cells. Preferrably, the host cell may comprise cells that are capable of phagocytosis, i.e., e.g., RAW264.7, MH-S, U937, HL60, THP-1. Alternatively, it is also possible to use cell lines stably transfected with a nucleic acid molecule encoding WAVEl or a functional fragment thereof. In a preferred embodiment of the present invention, the cell or the cell line used for stable transfection or transiently transfected with a nucleic acid molecule encoding WAVEl or a functional fragment thereof is a Jurkat T cell or a cell line comprising or derived from Jurkat T cells. The explanations given herein above in respect of "cells" also apply to tissues/non-human animals comprising or derived from these cells.

The (biological) sample or composition, comprising a plurality of candidate molecules are usually subject to a first screen. The samples/compositions tested positive in the first screen are often subject to subsequent screens in order to verify the previous findings and to select the most potent inhibitors/antagonists of WAVEl . Upon multiple screening and selection rounds those candidate molecules will be selected which show a pronounced capacity to inhibit/antagonize WAVEl as defined and disclosed herein. For example, batches (i.e. compositions/samples) containing many candidate molecules will be rescreened and batches with no or insufficient inhibitory activity of candidate molecules be discarded without re- testing. For example, if a (biological) sample or composition with many different candidate molecules is tested and one (biological) sample or composition is tested positive, then it is either possible in a second screening to screen, preferably after purification, the individual molecule(s) of the (biological) sample or composition. It may also be possible to screen subgroups of the (biological) sample or composition of the first screen in (a) subsequent screen(s). The screening of compositions with subgroups of those candidate molecules tested in previous screening rounds will thus narrow in on (an) potential potent WAVE1 inhibitor(s). This may facilitate and accelerate the screening process in particular when a large number of molecules is screened. Accordingly, the cycle number of screening rounds is reduced compared to testing each and every individual candidate molecule in (a) first (and subsequent) screen(s) (which is, of course, also possible). Thus, depending on the complexity or the number of the candidate molecules, the steps of the screening method described herein can be performed several times until the (biological) sample or composition to be screened comprises a limited number, preferably only one substance which is indicative for the capacity of inhibiting WAVE1 or decreasing the WAVE1 activity wherein a decrease of the WAVE1 activity is indicative for the capacity of the selected molecule to ameliorate, prevent or treat an oxidized phospholipids (OxPL) related inflammatory disease developing in response to an infection caused by a pathogen or to ameliorate, prevent or treat an infection caused by a pathogen as well as to prevent secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insult leads to elevated oxidized phospholipids.

The term "decrease in WAVE1 activity" in step (c) of the screening method as described above means that the "activity of WAVE 1" is reduced upon contacting the cell, tissue, or non- human animal comprising WAVE1 with the candidate molecule, preferably in comparison to a (control) standard or reference value, as defined herein wherein a decrease of the WAVE1 activity is indicative for the capacity of the selected molecule to ameliorate, prevent or treat an oxidized phospholipids (OxPL) related inflammatory disease developing in response to an infection caused by a pathogen or to ameliorate, prevent or treat an infection caused by a pathogen as well as to prevent secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insult leads to elevated oxidized phospholipids..

As defined and disclosed herein, the term "comprising WAVE1" refers not only to the WAVE1 gene(s) or proteins known in the art and described herein. Also reporter constructs comprising a promoter and/or enhancer region of WAVEl can be used in the screening/identifying methods. Accordingly, the cell(s), tissue(s) and/or non-human animals used in the context of the present invention, in particular in context of the screening/identifying methods can comprise the reporter constructs disclosed herein and described below.

The activity of WAVEl can be quantified in cells, tissue or non-human animals, wherein the cells comprise, but are not limited to any cells that are capable of phagocytosis. Hence, said cells may comprise, but are not limited to primary macrophages, RAW 264.7 macrophages, primary neutrophils, murine macrophage cell-lines (such as MH-S cells, AMJ2-C1 1), human macrophage cell lines (THP. l, Mono Mac, U937, 2MAC, HL-60) primary microglia and microglia cell-lines (such as HM06).

The method for assessing the activity of a candidate molecule suspected of being an antagonist/inhibitor of WAVEl can be accomplished by determining a decrease in the activity of WAVEl , wherein the decrease in WAVEl activity can be detected with polynucleotides capable of hybridizing the WAVEl sense molecule. Alternatively, the decrease in WAVEl activity can be detected with antibodies capable of binding the WAVEl protein. Therefore, the activity of WAVE l can be quantified by measuring, for example, the level of gene products (e.g. mRNA and/or protein of WAVEl) by any of the herein described methods, activities, the WAVEl concentration or other cellular functions, like inter alia, the phagocytosis activity. As mentioned herein above the candidate compound to be tested may lead to a modified activity of WAVEl and a decrease in the WAVEl activity is indicative for the capacity to antagonize WAVEl and thus to ameliorate, prevent or treat an oxidized phospholipids (OxPL) related inflammatory disease developing in response to an infection caused by a pathogen or to ameliorate, prevent or treat an infection caused by a pathogen as well as to prevent secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insult leads to elevated oxidized phospholipids..

As mentioned, a "decreased WAVEl activity" and, accordingly, a decreased concentration/amount of WAVEl proteins in a sample may be reflected in a decreased expression of the corresponding gene(s) encoding the WAVEl protein(s). Therefore, a quantitative assessment of the gene product (e.g. protein or spliced, unspliced or partially spliced mRNA) can be performed in order to evaluate decreased expression of the corresponding gene(s) encoding the WAVEl protein(s). Also here, a person skilled in the art is aware of standard methods to be used in this context or may deduce these methods from standard textbooks (e.g. Sambrook, 2001 , loc. cit). For example, quantitative data on the respective concentration/amounts of mRNA from WAVEl can be obtained by Northern Blot, Real Time PCR and the like. Preferably, the concentration/amount of the gene product (e.g. the herein above described WAVEl mRNA or WAVEl protein) may be decreased by at least about 10 %, 20 %, 30 %, 40 %, preferably by at least 50 %, 60 %, 70 %, 80 %, 90 %, or 100 % compared to a control sample. It is preferred herein that WAVEl proteins are (biologically) active or functional. Methods for determining the activity of WAVEl are described herein above. Since the WAVEl proteins are preferably (biologically) active/functional (wherein it is preferred that at least 70 %, 75 %, preferably at least 80%, 85 %, 90 %, 95 %, 96, %, 97%, 98 % and most preferably, at least 99 % of WAVEl proteins of a sample a (biologically) active/functional), an decreased concentration/amount of WAVEl proteins in a sample reflects a decreased (biological) activity of the WAVEl protein.

As mentioned, a person skilled in the art is aware of standard methods to be used for determining or quantitating expression of a nucleic acid molecule encoding, for example, the WAVEl (or fragments thereof). For example, the expression can be determined on the protein level by taking advantage of immunoagglutination, immunoprecipitation (e.g. immunodiffusion, immunelectrophoresis, immune fixation), western blotting techniques (e.g. (in situ) immuno histochemistry, (in situ) immuno cytochemistry, affmitychromatography, enzyme immunoassays), and the like. Amounts of purified polypeptide in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular polypeptide in a mixture rely on specific binding, e.g of antibodies. Specific detection and quantitation methods exploiting the specificity of antibodies comprise for example immunohistochemistry (in situ). For example, concentration/amount of WAVEl proteins in a cell, tissue or a non-human animal can be determined by enzyme linked-immunosorbent assay (ELISA). Alternatively, Western Blot analysis or i m munohi s tochcmi ca 1 staining can be performed. Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies. Electrophoresis may be multi-dimensional such as 2D electrophoresis. Usually, polypeptides are separated in 2D electrophoresis by their apparent molecular weight along one dimension and by their isoelectric point along the other direction. Expression can also be determined on the nucleic acid level (e.g. if the gene product/product of the coding nucleic acid sequence is an unspliced/partially spliced/spliced mRNA) by taking advantage of Northern blotting techniques or PCR techniques, like in-situ PCR or Real time PCR. Quantitative determination of mRNA can be performed by taking advantage of northern blotting techniques, hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for mRNA transcripts or PCR techniques referred to above, like, for example, quantitative PCR techniques, such as Real time PCR.

These and other suitable methods for detection and/or determination of the concentration/amount of (specific) mRNA or protein(s)/polypeptide(s) are well known in the art and are, for example, described in Sambrook (2001 ), loc. cit).

A skilled person is capable of determining the amount of mRNA or polypeptides/proteins, in particular the gene products described herein above, by taking advantage of a correlation, preferably a linear correlation, between the intensity of a detection signal and the amount of, for example, the mRNA or polypeptides/proteins to be determined.

The difference, as disclosed herein is statistically significant and a candidate molecule(s) is (are) selected, if the WAVEl activity (or of a corresponding reporter signal) is strongly decreased, preferably is very low or non-detectable. For example, the WAVEl activity (or of a corresponding reporter signal) may be decreased by at least 50%, 60%, 70%, 80%>, more preferably by at least 90% compared to the (control) standard value. In a cell based method the cells can be transfected with one or more constructs encoding WAVEl or a functional fragment thereof.

Preferably, the selected compound has a high WAVEl inhibiting/antagonizing activity. This can be reflected in the capacity of the WAVEl antagonist/inhibitor to potently decrease the activity of WAVEl .

The above detected difference between the activity of WAVEl or the activity of a functional fragment of WAVEl in a cell, tissue or a non-human animal contacted with said candidate molecule and the activity in the (control) standard value (measured e.g. in the absence of said candidate molecule) may be reflected by the presence, the absence, the increase or the decrease of a specific signal in the readout system.

Genetic readout systems are also envisaged. Analogously, the activity of WAVE1 or of a functional fragment thereof may be quantified by any molecular biological method as described herein. A skilled person is also aware of standard methods to be used in determining the amount/concentration of WAVE1 expression products (in particular the protein and the nucleic acid level of WAVE1) in a sample or may deduce corresponding methods from standard textbooks (e.g. Sambrook, 2001).

Also in this context, reporter constructs comprising a promoter and/or enhancer region of WAVE1 can be used in the screening/identifying methods. Exemplary reporters (preferably associated with the reporter signals disclosed herein) are luciferase and fluorescent proteins, like GFP, RFP and the like. It is preferred that a promoter and/or enhancer element/region of WAVE1 is used in this context and is fused to a reporter.

Exemplary reporter signals, reporters and reporter constructs are described herein below. Interesting reporters, namely reporter gene products, which can be used in the screening and identifying methods of the invention like luciferase, (green/red) fluorescent protein and variants thereof, EGFP (enhanced green fluorescent protein), RFP (red fluorescent protein, like DsRed or DsRed2), CFP (cyan fluorescent protein), BFP (blue green fluorescent protein), YFP (yellow fluorescent protein), β-galactosidase or chloramphenicol acetyltransferase as well as methods for their detection are also described herein below in detail. Luciferase is a well known reporter; see, for example, Jeffrey (1987) Mol. Cell. Biol. 7(2), 725-737. A person skilled in the art can easily deduce further luciferase nucleic and amino acid sequences to be used in context of the present invention from corresponding databases and standard text books/review.

Further exemplary reporter constructs to be employed in context of the present invention, in particular the screening and identifying methods, comprise promoter(s) (and/or (a) enhancer region(s)) of WAVE1 , wherein the (initiation/enhancement of the) expression of the reporter(s) is under control of the promoter and/or enhancer of WAVE 1. A skilled person may easily retrieve these and other well-known sequences from databases (like NCBI) and use these sequences in the generation of reporter constructs to be employed herein. For example, the WAVEl promoter sequence the corresponding database.

These reporter constructs comprising a reporter and a promoter (and/or enhancer) as defined above, are particularly useful in screening methods and assays, since the reporter signal associated with the reporter can easily be detected. A change in the reporter signal is indicative for the capacity of a candidate molecule tested to act as antagonist/inhibitor of WAVEl . For example, an antagonist of WAVEl will lead to a decrease of a reporter signal/activity of a reporter under control of the WAVEl promoter region and, hence, to ameliorate an inflammatory disease related to detrimental effects of oxidized phospholipids. In particular, a reporter construct may comprise a luciferase gene and a promoter of WAVEl. A person skilled in the art is easily in the position to generate this and other reporter constructs using routine techniques. Inter alia, vectors such as the pRL-T RENILLA Vector and other well known vectors may be employed in the generation of the reporter constructs.

Apparently, decreased expression of the reporter gene/activity of the reporter gene product will reflect a decreased WAVEl activity, in particular a decreased concentration/amount of WAVEl protein. Alternatively, the effect of the antagonist/inhibitor on the expression of (a) reporter gene(s) may be evaluated by determining the amount/concentration of the gene product of the reporter gene(s) (e.g. protein or spliced, unspliced or partially spliced mRNA). Further methods to be used in the assessment of mRNA expression of a reporter gene are within the scope of a skilled person and also described herein below.

As mentioned above, it is preferred that a promoter and/or enhancer element/region of WAVEl is used in this context and is fused to a reporter. As used herein, the term "reporter construct for WAVEl -inhibition" relates to any biotechnologically engineered construct allowing the detection of WAVEl inhibition. Accordingly, said reporter construct may allow the detection of WAVEl -inhibition by inducing a change in the signal strength of a detectable signal. Said detectable signal may be selected from the group consisting of, but not limited to a fluorescence resonance energy transfer (FRET) signal, a fluorescence polarization (FP) signal and a scintillation proximity (SP) signal. In a further embodiment, said detectable signal may be associated with a reporter gene product. Examples of reporter gene products include luciferase, (green/red) fluorescent protein and variants thereof, like EGFP (enhanced green fluorescent protein), RFP (red fluorescent protein, like DsRed or DsRed2), CFP (cyan fluorescent protein), BFP (blue green fluorescent protein), YFP (yellow fluorescent protein), β-galactosidase or chloramphenicol acetyltransferase, and the like. For example, GFP can be derived from Aequorea victoria (US 5,491,084). A plasmid encoding the GFP of Aequorea victoria is available from the ATCC Accession No. 87451. Other mutated forms of this GFP including, but not limited to, pRSGFP, EGFP, RFP/DsRed, DSRed2, and EYFP, BFP, YFP, among others, are commercially available from, inter alia, Clontech Laboratories, Inc. (Palo Alto, California).

In another preferred embodiment, the non-human animal comprising said reporter construct for detecting WAVE1 inhibition is a transgenic non-human animal. The non-human organism to be used in the described screening assays is preferably selected from the group consisting of C. elcgans, yeast, drosophila, zebrafish, guinea pig, rat and mouse. The generation of such a transgenic animal is within the skill of a skilled artisan. Corresponding techniques are, inter alia, described in "Current Protocols in Neuroscience" (2001), John Wiley&Sons, Chapter 3.16. Accordingly, the invention also relates to a method for the generation of a non-human transgenic animal comprising the step of introducing a reporter construct for detecting WAVE1 inhibition as disclosed herein into an ES-cell or a germ cell. The non-human transgenic animal provided and described herein is particular useful in screening methods and pharmacological tests described herein above. In particular the non-human transgenic animal described herein may be employed in drug screening assays as well as in scientific and medical studies wherein antagonists/inhibitors of WAVE1 for the treatment of a disease related to an insufficient immune response are tracked, selected and/or isolated.

The trans genic/genetically engineered cell(s), tissue(s), and/or non-human animals to be used in context of the present invention, in particular, the screening/identifying methods, preferably comprise the herein described and defined reporter constructs. Hence, in this context, reporter constructs may comprise a promoter and/or enhancer region of WAVE1 as defined herein. Exemplary reporters (preferably associated with the reporter signals disclosed herein) are luciferase and fluorescent proteins, like GFP, RFP and the like. Exemplary, non-limiting constructs to be used may comprise a luciferase reporter under control of a (human) WAVE1 promoter and/or enhancer region. Exemplary reporters are luciferase and fluorescent proteins, like GFP, RFP and the like. The method for assessing the activity of a candidate molecule suspected of being an antagonist/inhibitor of WAVE 1 can be accomplished by determining a decrease in the activity of WAVE 1 , wherein the decrease in WAVE1 activity can be detected with polynucleotides capable of hybridizing the WAVE1 sense molecule.

In a preferred embodiment, the method for assessing the activity of a candidate molecule suspected of being an antagonist/inhibitor of WAVE 1 can be accomplished by determining a decrease in the activity of WAVE 1, wherein the decrease in WAVE1 activity can be detected by monitoring the phagocytosis activity of a cell, wherein an increase of the phagocytosis activity is indicative for the decrease of the WAVE1 activity and, therefore, indicative for the capacity of the selected molecule to ameliorate, prevent or treat an oxidized phospholipids (OxPL) related inflammatory disease developing in response to an infection caused by a pathogen or to ameliorate, prevent or treat an infection caused by a pathogen as well as to prevent secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insult leads to elevated oxidized phospholipids. A cell to be used is a cell that comprises and expresses WAVE1. The phagocytosis activity can be quantified in cells, tissue or non-human animals, wherein the cells comprise, but are not limited to any cells that are capable of phagocytosis. In a preferred embodiment of the present invention, the used cells are primary macrophages, RAW 264.7 macrophages, primary neutrophils, other murine macrophage cell-lines (such as MH-S cells, AMJ2-C11), human macrophage cell lines (such as THP- 1 , Mono Mac, U937, 2MAC, HL-60), primary microglia and microglia cells (e.g. HM06). The phagocytosis activity can be assayed as described in the appended examples. Hence, phagocytosis activity of FITC-labeled E. coli can be measured by FACS after we pre- incubated cells with carrier (NaCl), DMPC or OxPAPC for 15 min. To assess phagocytosis, 0.5x10 (Knapp, S., et al. (2007) J Immunol 178, 993-1001) adherent cells can be incubated, washed, and subsequently incubated for 15 min with phospholipids or saline as indicated in RPMI. Then FITC-labeled heat-killed E. coli (018:K1) at a MOI of 100 are added for 1 hour at 37°C or 4°C, respectively, washed with PBS and treated with Proteinkinase K at 50μg ml for 15 min. Immediately thereafter the cells were placed on ice, washed, and fluorescence was analyzed using a FACScan (BectonDickinson). The phagocytosis index of each sample was analyzed as: (mean fluorescence of positive cell x % positive cells at 37°C) minus (mean fluorescence of positive cells x % positive cells at 4°C). 'Positive cells' are considered those that ingested FITC and thus display a fluorescence above control cells that were not incubated with FITC-labelled bacteria (on average displaying a fluorescence >10).

In a preferred embodiment, the method for assessing the activity of a candidate molecule suspected of being an antagonist/inhibitor of WAVE 1 can be accomplished by determining the physical interaction/binding of candidate molecules with WAVE1 or PKA. Interaction methods are known in the art and, for the identification of AKAP inhibitors, numerous methods have been described (see, e.g., Alto, N. M., Soderling, S. H., Hoshi, N., Langeberg, L. K., Fayos, R., Jennings, P. A. and Scott, J. D. (2003) Bioinformatic design of A-kinase anchoring protein- m silico a potent and selective peptide antagonist of type II protein kinase A anchoring. Proc. Natl. Acad. Sci. U.S.A. 100, 4445^1450. Burns-Hamuro, L. L., Ma, Y., Kammerer, S., Reineke, U., Self, C, Cook, C, Olson, G. L., Cantor, C. R., Braun, A. and Taylor, S. S. (2003); Biochem J. 2006 Dec 15;400(3):493-9. Characterization of A-kinase- anchoring disruptors using a solution-based assay. Stokka AJ, Gesellchen F, Carlson CR, Scott JD, Herberg FW, Tasken K.) Interaction assays employing read-out systems are well known in the art and comprise, inter alia, two hybrid screenings (as, described, inter alia, in EP-0 963 376, WO 98/25947, WO 00/02911), GST-pull-down columns, co-precipitation assays from cell extracts as described, inter alia, in Kasus-Jacobi (2000) Oncogene 19:2052- 2059, "interaction-trap" systems (as described, inter alia, in US 6,004,746) expression cloning (e.g. lamda gtll), phage display (as described, inter alia, in US 5,541,109), in vitro binding assays and the like. Further interaction assay methods and corresponding read out systems are, inter alia, described in US 5,525,490, WO 99/51741, WO 00/17221, WO 00/14271 , WO 00/05410 or Yeast Four hybrid assays as described in Sandrok (2001) JBC 276:35328- 35333.

Said interaction assays for WAVE1 or PKA also comprise assays for FRET-assays, TR- FRETs (in "A homogenius time resolved fluorescence method for drug discovery" in: High throughput screening: the discovery of bioactive substances. Kolb (1997) J.Devlin. NY, Marcel Dekker 345-360) or commercially available assays, like "Amplified Luminescent Proximity Homogenous Assay", BioSignal Packard. Furthermore, the yeast-2-hybrid (Y2H) system may be employed to elucidate further particular and specific interaction, association partners of WAVE 1 or PKA. Said interaction/association partners are suspected of being an antagonist/inhibitor of WAVE1 and are further screened for their antagonistic/inhibiting effects as described above. As mentioned herein above the candidate compound that interacts with WAVEl or PKA may lead to a modified activity of WAVEl and a decrease in the WAVEl activity is indicative for the capacity to antagonize WAVEl and thus to ameliorate, prevent or treat an oxidized phospholipids (OxPL) related inflammatory disease developing in response to an infection caused by a pathogen or to ameliorate, prevent or treat an infection caused by a pathogen as well as to prevent secondary infections subsequent to an insult, like injury, surgery, trauma or inflammation wherein said insult leads to elevated oxidized phospholipids..

Similarly, interacting molecules (for example) (polypeptides may be deduced by cell-based techniques well known in the art. These assays comprise, inter alia, the expression of reporter gene constructs or "knock-in" assays, as described, for, e.g., the identification of drugs/small compounds influencing the (gene) expression of WAVEl . Said "knock-in" assays may comprise "knock-in" of WAVEl (or (a) fragment(s) thereof) in tissue culture cells, as well as in (transgenic) animals. Examples for successful "knock-ins" are known in the art (see, inter alia, Tanaka (1999) Neurobiol. 41 :524-539 or Monroe (1999) Immunity 1 1 :201-212). Furthermore, biochemical assays may be employed which comprise, but are not limited to, binding of the WAVEl (or (a) fragment(s) thereof) to other molecules/( polypeptides, peptides or binding of the WAVEl (or (a) fragment(s) thereof) to itself (themselves) (dimerizations, oligomerizations, multimerizations) and assaying said interactions by, inter alia, scintillation proximity assay (SPA) or homogenous time-resolved fluorescence assay (HTRFA).

Said "testing of interaction" may also comprise the measurement of a complex formation. The measurement of a complex formation is well known in the art and comprises, inter alia, heterogeneous and homogeneous assays. Homogeneous assays comprise assays wherein the binding partners remain in solution and comprise assays, like agglutination assays. Heterogeneous assays comprise assays like, inter alia, immuno assays, for example, ELISAs, RIAs, IRMAs, FIAs, CLIAs or ECLs.

The interaction of the antagonistic molecules of WAVEl mRNA and WAVEl protein or fragments thereof may also be tested by molecular biological methods, like two-, three- or four-hybrid-assays, RNA protection assays, Northern blots, Western blots, micro-, macro- and protein- or antibody arrays, dot blot assays, in situ hybridization and immunohistochemistry, quantitative PCR, coprecipitation, far western blotting, phage based expression cloning, surface plasmon resonance measurements, yeast one hybrid screening, DNAse I, footprint analysis, mobility shift DNA-binding assays, gel filtration chromatography, affinity chromatography, immunoprecipitation, one- or two dimensional gel electrophoresis, aptamer technologies, as well as high throughput synthesis and screening methods.

In sum, the present invention provides for the first time methods for identifying, and characterizing (a) candidate molecule(s) or (a) compound(s) which are capable of inhibiting/antagonizing WAVEl whereby said inhibition may lead to an increase in phagocytosis activity and improved bacterial uptake inflammatory diseases related to oxidized phospholipids. Therefore the present invention provides for screening as well as identification methods for antagonists of WAVEl. As also disclosed herein above, the term "antagonist" relates to molecules or compounds that bind to WAVEl or a functional fragment thereof, thereby inhibiting and/or reducing WAVEl activity, wherein these WAVEl antagonists are capable of augmenting, ameliorating and/or preventing an inflammatory disease related to (detrimental effects) of oxidized phospholipids.

In a further embodiment, the present invention relates to the use of a cell, tissue or non-human animal for screening and/or validation of a compound suspected of being an antagonist/inhibitor of WAVEl . The term "cell" as used in this context may also comprise a plurality of cells as well as cells comprised in a tissue. A cell to be used is a cell that comprises and expresses WAVEl . In a preferred embodiment of the present invention, the used cells are primary macrophages, RAW 264.7 macrophages, primary neutrophils, other murine macrophage cell-lines (such as MH-S cells, AMJ2-C11), human macrophage cell lines (such as THP-1, Mono Mac, U937, 2MAC, HL-60), primary microglia and microglia cells (e.g. HM06). Since WAVEl leads to a decrease in phagocytosis and in an elimination of bacteria as described above, a cell or a cell culture with a high WAVEl expression will usually have a low activity in phagocytosis. Accordingly, these cells are highly useful in detecting an increase in the phagocytosis activity. Therefore, the use of cell(s), tissues or non- human animal is particularly envisaged, if these cells have a high WAVEl expression (reflected in a high protein or mRNA level). Cells, tissues and non-human animals to be used in accordance with the present invention are also described herein above. The used non-human animal or cell may be transgenic or non transgenic. In this context the term "transgenic" particularly means that at least one of the WAVEl gene as described herein is overexpressed, thus the WAVEl activity in the non-human transgenic animal or a transgenic animal cell is enhanced. Generally, it is preferred herein that WAVEl is highly expressed in (a) cell(s), tissue(s), non-human animal to be used in the screening methods as described above.

The term "transgenic non-human-animal", "transgenic cell" or "transgenic tissue" as used herein refers to an non-human animal, tissue or cell, not being a human that comprises different genetic material of a corresponding wild-type animal, tissue or cell. The term "genetic material" in this context may be any kind of a nucleic acid molecule, or analogues thereof, for example a nucleic acid molecule, or analogues thereof as defined herein. The term "different" means that additional or fewer genetic material in comparison to the genome of the wild type animal or animal cell. An overview of different expression systems to be used for generating transgenic cell/animal refers for example to Methods in Enzymology 153 (1987), 385-516, in Bitter et al (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al, (Methods in Molecular Biology 75 (1997), 427-440).

In a preferred embodiment, the (transgenic) non-human animal or (transgenic) cell is or is derived from a mammal. Non-limiting examples of the (transgenic) non-human animal or derived (transgenic) cell are selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig and Drosophila.

Generally, the (transgenic) cell may be a eukaryotic cell. For example, the (transgenic) cell in accordance with the present invention may be but is not limited to yeast, fungus, plant or animal cell. In general, the transformation or genetically engineering of a cell with a nucleic acid construct or a vector can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.

The invention also relates to a kit useful for carrying out the methods as described herein comprising the polynucleotides and/or antibodies capable of detecting the activity of WAVEl as characterized above. The embodiments disclosed in this context with the method of the present application apply, mutatis mutandis, to the kit of the present invention.

Advantageously, the kit of the present invention further comprises, optionally (a) reaction buffers, storage solutions, wash solutions and/or remaining reagents or material required in the pharmacological and drug screening assays or the like as described herein. Furthermore, parts of the kit of the invention can be packed individually in vials or bottles or in combination in containers or multicontainer units.

In a preferred embodiment of the present invention, the kit may be advantageously used for carrying out the method for detecting the WAVEl activity or changes in the WAVEl activity as described herein. Additionally, the kit of the present invention may contain means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.

Similarly, kits are provided which comprise the candidate molecule as described herein, the nucleic acid molecule, the cell, tissue or non-human animal of the invention. These kits provided herein are particularly useful in the methods of the present invention and in particular in the determination of the WAVE l activity or changes in the WAVEl activity. These kits as well as the methods provided herein are also useful in pharmacological screenings, also comprising "high-throughput" screening.

In addition, the invention relates to a method of treating an inflammatory disease related to effects, preferably detrimental effects, of exidized phospholipids (OxPL), comprising administering an effective amount of an antagonist/inhibitor of WAVEl to a patient in need thereof. Preferably, said patient is a human.

The following figures show and illustrate the present invention: Figure 1. Oxidation of lipids occurs in E. coli peritonitis in vivo, and leads to an actin- dependent change in cell shape in vitro: (a) RAW 267.4 cells were incubated with indicated doses of OxPAPC or DMPC (5(^g/ml) for 15 min, before phagocytosis of FITC-labeled E. coli was assessed after 60 or 120 min (representative of 3 independent experiments), (b) FACS-histogram showing uptake of FITC-labeled E. coli by resident peritoneal macrophages pretreated with of OxPAPC or DMPC after 60m in. (c) Mice were infected with 10⁴ CFU E. coli i.p. and treated with either control lipids (DMPC) or OxPAPC, respectively. Peritoneal CFU counts were enumerated lOh after infection, (d) Endogenous oxidized phosphatidylcholine levels were measured in PLF of healthy mice (n=3) and lOh following induction of bacterial peritonitis (n=8) using the E06 niAb. Data are presented as mean ± SEM; * indicates p < 0.05, and ** indicates p < 0.01 versus respective control groups, (e) RAW 264.7 cells were incubated with carrier, DMPC, or OxPAPC at ΙΟμ^πά for 30 min, or preincubated with Cytochalasin D at 2μΜ for 30 min prior to carrier, DMPC, or OxPAPC. Cells were subsequently fixed and stained for F-actin using Alexa Fluor 488-labelled phalloidin (green) and propidium-iodide (PI) for nuclei (red). Cells were visualized using a LSM 510 confocal laserscarming microscope; magnification: x 800.

Figure 2: PKA activation mediates OxPAPC-associated ceil spread and inhibition of phagocytosis: (a) RAW 264.7 cells were treated with carrier, DMPC, or OxPAPC at 10μg/ml for 30 min alone, or following preincubation with H89 (10μΜ) or PKA amid (Bryn, T., et al. (2006) J Immunol 176, 7361-7370; Carr, D.W., et al. (1992) The Journal of biological chemistry 267, 13376-13382; Wong, W. et al. (2004) Nature reviews 5, 959-970; Soderling, S.H.. et al. (2003) Proc Natl Acad Sci U S A 100, 1723-1728; Murch, A.R., et al. (1984) J Pathol 144, 81-87; Binder, C.I.. et al. (2002) Nat Med 8, 1218-1226; Hansson. G.K. et al. (2006) Nat Rev Immunol 6, 508-519; Yoshimi, N., et al. (2005) Lung 183, 109-121 ; Nakamura, T., et al. (1998) Anal Biochem 262, 23-32) (20μΜ) for 30 min. Cells were subsequently fixed and stained for F-actin using Alexa Fluor 488-labelled phalloidin (green) and PI for nuclei (red); magnification: x 800. (b and c) Phagocytosis assays were performed using RAW 264.7 cells that were preincubated with carrier or indicated phospholipids (5μg/ml; for 15 min) alone, or after pretreatment with (b) H89 (10μΜ), or (c) PKA amid (Bryn, T., et al. (2006) J Immunol 176, 7361-7370; Carr, D.W., et al. (1992) The Journal of biological chemistry 267, 13376-13382; Wong, W. et al. (2004) Nature reviews 5, 959-970; Soderling, S.H., et al. (2003) Proc Natl Acad Sci U S A 100, 1723-1728; Murch, A.R., et al. (1984) J Pathol 144, 81 -87; Binder, C.J., et al. (2002) Nat Med 8, 1218-1226; Hansson, G.K. et al. (2006) Nat Rev Immunol 6, 508-519; Yoshimi, N., et al. (2005) Lung 183, 109-121 ; Nakamura, T., et al. (1998) Anal Biochem 262, 23-32) (20μΜ), respectively. Uptake of FITC- labeled E. coli was evaluated after 60 min and is expressed relative to carrier (data are representative of 3 independent experiments). Data are duplicates and shown as mean ± SEM * indicates p < 0.05, and ** p < 0.01 versus corresponding carrier, (d) RAW 264.7 cells were transfected with shRNA to the a-isoform of PKAc and silencing was verified by Western blot, (e) Control (vector or scrambled control, respectively) and shRNA transfected cells were preincubated with carrier, DMPC or OxPAPC {5 iglm\) for 15 min and phagocytosis of FITC- labeled E. coli was examined after 60 min by FACS analysis. Uptake of bacteria is expressed relative to carrier. Representative data of 3 independent experiments in triplicate are shown. Data depicted are mean ± SEM; * indicates p < 0.05, and *** p < 0.001 versus corresponding carrier control, (f) For confocal microscopy studies, vector, scrambled-control or shRNA (to PKAc) cells were incubated with carrier, DMPC or OxPAPC at 10μg/ml for 30 min after which cells were fixed and stained with phalloidin-Alexa 488 and PI; magnification x 800 (representative images of 3 independent experiments are shown).

Figure 3: AKAP-inhibition prevents detrimental effects of OxPAPC in vitro and in vivo:

(a) RAW 264.7 cells were treated with carrier, DMPC or OxPAPC (K^g/ml) for 30 min alone, or after preincubation with ΙΟΟμΜ Ht-31. Cells were subsequently fixed and stained using phalloidin Alexa 488 (green) and PI (red) and analyzed; magnification: x 800. (b) RAW 264.7 cells were treated with carrier or phospholipids (5μξ/ηύ) for 15 min alone, or after preincubation with Ht-31 (ΙΟΟμΜ) for 30 min. Phagocytosis of FITC-labeled E. coli after 60 min was analyzed using FACS. Uptake is expressed relative to carrier and ** indicates p < 0.01. Data shown are representative mean ± SEM of 3 independent experiments in duplicate, (c) Mice received i.p. carrier, 2.5 mg/kg OxPAPC, and/or ΙΟΟμΜ of Ht-31, immediately before infection with 10 (Tasken, K. et al. (2004) A. Physiol Rev 84, 137-167) CFU E. coli. At t=10h PLF was harvested and bacterial CFUs enumerated. Data are mean ± SEM of 2 independent experiments from n=7-9 mice/group; ** indicates p < 0.01 versus carrier, (d) Mice received 2.5 mg/kg DMPC or OxPAPC i.p. followed by i.p. injection of vehicle or Ht- 31 (OxPAPC-Ht-31), after which they were infected with 10⁴ CFU E. coli. Survival was monitored every 2 h; n=12 mice/group, p value of each experiment is indicated.

Figure 4: WAVE1 mediates antiphagocytic properties of OxPAPC in vitro and in vivo:

(a) Constitutive expression of AKAP-Lbc (150bp), WAVE1 (1 16bp) and Gravin (136bp) on niRNA level in RAW 264.7 or (b) primary peritoneal macrophages; GAPDH (372bp) was used as a loading control, (c) mRNA expression of WAVE1 in WT and WAVE1^{" "} primary peritoneal macrophages, (d) Western blot analysis of WAVE1 in primary peritoneal macrophages of WT and WAVEl^{" ~} mice, actin (39kD) was used as a loading control, (e) Primary peritoneal macrophages of WT and WAVE ^" mice were stimulated with carrier, DMPC or OxPAPC (2(^g/ml) for 30 min. After this cells were fixed and stained with phalloidin Alexa 488 (green) and PI (red); magnification: x 800. (f) Phagocytosis was assessed using primary peritoneal macrophages from WT and WAVEl^{" "} mice; uptake of FITC-labeled E.coli was analyzed 60 min after prior incubation with DMPC or OxPAPC (10μg/ml, 15 min). Data are in triplicate and expressed as mean ± SEM of 2 independent experiments; * p < 0.01 versus corresponding DMPC control, (g-i) WT and chimeric WAVET^7" mice were treated with DMPC or OxPAPC (2.5mg/kg) i.p. and infected with 10 (Hampton, M.B., et al. (1998) Blood 92, 3007-3017) CFU E. coli i.p. (g) Peritoneal, (h) liver and (i) blood CFU counts were enumerated lOh after infection. Data are from n=9-l l mice/group and presented as mean ± SEM; * p < 0.05 versus corresponding DMPC control, (j) WT and chimeric WAVEr ^" mice received 2.5 mg/kg OxPAPC i.p. and were infected with 10 (Tasken, K. et al. (2004) A. Physiol Rev 84, 137-167) CFU E. coli. Survival was monitored every 2 h; n=T2 mice/group, p value is indicated.

Figure 5: OxPAPC induced ceil spread and inhibition of phagocytosis is not mediated by CD36 (a) Primary peritoneal macrophages of WT and CD36^obl mice were incubated with carrier, DMPC, or OxPAPC at 20μg/ml for 30 min. Cells were subsequently fixed and stained for F-actin using Alexa Fluor 488-labelled phalloidin (green) and propidium-iodide (PI) for nuclei (red). Cells were visualized using a LSM 510 confocal laserscanning microscope; magnification: x 800 (b) WT and CD36^obl peritoneal macrophages were incubated with carrier, DMPC or OxPAPC at \Q igl \ for 15 min, and phagocytosis of FITC-labeled E. coli was assayed after 60 min. Uptake is expressed relative to carrier. Data are representative of two independent experiments performed in triplicates; mean ± SEM; * p < 0.05 versus carrier.

Figure 6: OxPAPC-mediated inhibition of phagocytosis is reversible. RAW 264.7 cells were incubated with DMPC or OxPAPC at 50μg/ml for 15 min. In indicated samples OxPAPC was removed after 15min by thoroughly washing cells with PBS (OxPAPC washed). Cells were then incubated with FITC-labeled E. coli and uptake was analyzed by FACS after 60, 120 and 180 min, respectively. Date are presented as phagocytosis-index as described in the Methods section. Depicted data are in duplicate and expressed as mean ± SEM; * p < 0.05 compared to DMPC.

Figure 7: OxPAPC but not PKA activators induce actin polymerization. Confocal microscopy images showing RAW 264.7 cells treated with carrier, 6-Bnz-cAMP (specific PKA activator; ΙΟΟμΜ), forskolin (ΙΟΟμΜ), or OxPAPC (^g/ml) for 30 min. Cells were subsequently fixed and stained for F-actin using Alexa Fluor 488-labelled phalloidin (green) and PI for nuclei (red). Magnification: x 800, representative images of 3 independent experiments are shown.

Figure 8: Silencing of WAVE1 prevents OxPAPC induced cell spread and inhibition of phagocytosis. RAW 264.7 cells were transfected with shRNA to WAVE1 , or scrambled control. Stable cell lines were generated as described in the method section, (a) Silencing of WAVE1 was verified by western blot, (b) Confocal microscopy images: scrambled-control and shRNA (targeting WAVE1) cells were incubated with carrier, DMPC, or OxPAPC (ΙΟμ^πύ), respectively, for 30 min. Cells were subsequently fixed and stained for F-actin using Alexa Fluor 488-labelled phalloidin (green) and PI for nuclei (red); magnification: x 800. (c) Scrambled-control and shRNA-WAVEl cells were preincubated with carrier, DMPC or OxPAPC ^g/ml) for 15 min, and phagocytosis was assayed 60min after addition of FITC-labeled E. coli. Bacterial uptake is expressed relative to carrier. Data are in triplicate and depicted as mean ± SEM, * p < 0.05 versus corresponding carrier. Figure 9: Quantitative WAVE1 mRNA transcript levels in resident peritoneal macrophages of chimeric mice reconstituted with WT or WAVE1^{_ "} bone marrow cells. Mice were randomly picked (n=3 mice/group); data depicted are mean +/- SEM.

The following non-limiting examples illustrate the invention:

Example I: Materials and Methods as employed in the following

Mice

We purchased pathogen free C57BL/6 mice from Charles River. CD36 mutant oblivious (CD36^0bl) C57BL/6 mice were kindly provided by Bruce Beutler via the Mutant Mouse Regional Resource Centers (MMRC) (Hoebe, K.. et al. (2005) Nature 433, 523-527). WAVEl^7" mice were generated as described (Soderling, S.H., et al. (2003) Proc Natl Acad Sci U S A 100, 1723-1728) and backcrossed 10 times to a C57BL/6 background. The local animal care committee of the Medical University of Vienna and Ministry of Sciences approved all experiments.

Phospholipids

We obtained l-palmitoyl-2-arachidonoyl~OT-glycero-3-phosphorylcholine (PAPC) and dimyristoyl-phosphatodyl-choline (DMPC) from Sigma. We used DMPC as control lipid as it lacks unsaturated fatty acids and thus cannot be oxidized. We generated oxidized PAPC (OxPAPC) as described (Knapp, S., et al. (2007) J Immunol 178, 993-1001 ; Bochkov, V.N., et al. (2002) Nature 419, 77-81). We obtained l-palmitoyl-2-arachidonoyl-s7z-glycero-3- phosphorylcholine (PAPC) and dimyristoyl-phosphatodyl-choline (DMPC) from Sigma. We used DMPC as control lipid as it lacks unsaturated fatty acids and thus cannot be oxidized. We generated oxidized PAPC (OxPAPC) by air oxidation, and confirmed the extent of oxidation by electrospray ionization-mass spectrometry. We used only preparations showing a reproducible pattern of lipid oxidation products, and after testing for biological activity and exclusion of LPS contamination using the Limulus assay. Induction of peritonitis, enumeration of bacteria and monitoring survival

We induced peritonitis by i.p. injection of 200μ1 saline containing 10 (Tasken, K. et al. (2004) A. Physiol Rev 84, 137-167)-10 (Hampton, M.B., et al. (1998) Blood 92, 3007-3017) CFUs E.coli 018: 1 that were harvested at mid-log phase (Knapp, S., et al. (2007) J Immunol 178, 993-1001), and administered OxPAPC or DMPC (both at 2.5mg/kg) i.p. immediately before. We injected ΙΟΟμΜ St-Ht-31 (Promega) i.p. immediately before administering lipids and bacteria. In survival studies, 12 mice/group were inoculated with E. coli, and mortality was assessed every 2h. For quantification of bacteria, peritoneal lavage fluid (PLF) and organs were harvested lOh after infection, and processed for bacterial quantification as described ((Knapp, S., et al. (2007) J Immunol 178, 993-1001). Serial dilutions of the homogenate or PLF were then plated on blood agar plates.

Measurement of oxidized lipids

We collected PLF 1 Oh after induction of E. coli peritonitis and measured oxidized lipids using the E06 Ab (kindly provided by J. L. Witztum, University of California San Diego) as described (Imai, Y.. et al. (2008) Cell 133, 235-249). We adjusted PLF samples and cleared tissue homogenates to 100 μ^ηιΐ protein concentration in PBS containing 0.27 mM EDTA applied to 96-well MicroFluor microtiter plates (ThermoLabsystems) overnight at 4°C . We then incubated with isotype control Ab, E06 or LR04 Ab, respectively, (kindly provided by J. L. Witztum UCSD) for 2h at room temperature, followed by a goat-anti-mouse IgM-AP labelled secondary antibody (Sigma; at 1 :35,000). For development we added 25 μΐ of 33% LumiPhos Plus solution (Lumigen) and measured light emissions as relative light units (RLU) on a WALLAC VIKTOR II luminometer (Perkin Elmer).

Bone marrow transplantation

We ablated recipient bone marrow with a single dose of radiation (9 Gy) using a Cobalt 60 irradiator (MDS Nordion) followed by injection of 2x10 (Knapp, S., et al. (2007) J Immunol 178, 993-1001) bone marrow cells via the retro-orbital sinus as described (Pawlinski, R., et al. (2004) Blood 103, 1342-1347). To verify lethal irradiation one mouse from each group did not receive bone marrow and was followed over approximately lOd, after which all of them succumbed. After 9 weeks we verified successful reconstitution of peritoneal macrophages with donor cells by checking for WAVE1 transcripts. Confocal microscopy

We performed stainings after blocking steps using PBS/ 1% BSA for 30 min using Alexa- Fluor 488 labelled phalloidin (Invitrogen). We used propidium iodide (Sigma) in the presence of 0.1% Triton X-100 for nuclear staining. We visualized cells using a LSM 510 Confocal Laser scanning Microscope (Zeiss). Pre-treatment with carrier (NaCl), phospholipids, H89, PKA-inhibitor amide (Bryn, T.. et al. (2006) J Immunol 176, 7361-7370; Carr, D.W., et al. (1992) The Journal of biological chemistry 267, 13376-13382; Wong, W. et al. (2004) Nature reviews 5, 959-970; Soderling, S.H., et al. (2003) Proc Natl Acad Sci U S A 100, 1723-1728; Murch, A.R., et al. (1984) J Pathol 144, 81-87; Binder, C.J.. et al. (2002) Nat Med 8, 1218- 1226; Hansson, G.K. et al. (2006) Nat Rev Immunol 6, 508-519; Yoshimi, N., et al. (2005) Lung 183, 109-121 ; Nakamura, T., et al. (1998) Anal Biochem 262, 23-32) (Calbiochem), Forskolin (Sigma), or specific PKA-activator N6-benzoyladenosine-3 '5 ^'-cAMP (Biolog) was performed as indicated.

Phagocytosis assays

We assessed phagocytosis of FITC-labeled /·,^'. coli by FACS after we pre-incubated cells with carrier (NaCl), DMPC or OxPAPC for 15 min, exactly as described (Knapp, S., et al. (2007) J Immunol 178, 993-1001). To assess phagocytosis, we incubated 0.5x10 (Knapp, S., et al. (2007) J Immunol 178, 993-1001) adherent cells, washed them, and subsequently incubated for 15 min with phospholipids or saline as indicated in RPMI. Then we added FITC-labeled heat-killed E. coli (018:K1) at a MOI of 100 for 1 hour at 37°C or 4°C, respectively, washed with PBS and treated with Proteinkinase K at 50μg/ml for 15 min. Immediately thereafter we placed the cells on ice, washed them, and analyzed fluorescence using a FACScan ( BectonDickinson). We analyzed the phagocytosis index of each sample as: (mean fluorescence x % positive cells at 37°C) minus (mean fluorescence x % positive cells at 4°C).

PKA kinase assay

We treated adherent RAW 264.7 cells as indicated and performed a PKA kinase assay (PepTag, Promega) according to the manufacturer's instructions. We treated adherent RAW 264.7 cells as indicated, performed the PKA kinase assay (PepTag, Promega) according to the manufacturer's instructions, and loaded equal amounts of protein of each sample (adjusted by Bradford) and controls on a 0.8% agarose gel. Chemiluminescence was recorded with a Bio- Rad U V -transil lum i nator. Generation of PKAc, WAVE-1 and AKAP-13 shRNA cell lines

We carried out PKAc and WAVEl gene silencing by designing short hairpins using the siRNA target designer (Promega) to nucleotide regions; 930-948 (PKAc, Genbank accession no: NM_008854), 219-237 (WAVEl, Genbank accession no: NM_031877). As a control we used scrambled nucleotide sequences comprising the shRNA to each transcript (Table 1). We then annealed nucleotides and ligated them into the Pstl site of the psiSTRIKE vector (Promega). We transfected 2μg of purified DNA into 2 x 10 (Knapp, S., et al. (2007) J Immunol 178, 993-1001) RAW 264.7 cells using amaxa cell line kit V (Amaxa). We selected for transfected cells with 7μg/ml puromycin and thereby generated stable cell lines.

Western blotting

We washed cells and lysed them as described (Lagler, H., et al. (2009) J Immunol 183, 2027- 2036) after which we separated 25 g of supernatant by electrophoresis on a 10% SDS polyacrylamide gel and transferred the gels to polyvinylidene difluoride (PVDF) membranes. We used antibodies specific for PKAca (Santa Cruz) and WAVEl (Sigma) at a dilution of 1 : 1000, and β-actin antibody (Sigma) at 1 :500 to detect immunoreactive proteins by enhanced chemiluminescent protocol (GE Healthcare).

Evaluation of mRNA expression in peritoneal macrophages

We used Qiagens RNEasy kit for RNA extraction, which included a DNase step, and converted to cDNA using the Superscript III first strand synthesis system as described by the supplier (Invitrogen). We conducted RT-PCR according to the LightCycler FastStart DNA MasterPLUS SYBR Green I system using the Roche Light cycler II sequence detector (Roche Applied Science), (sequences are listed in Table 2).

Statistical analysis

Data are presented as the mean ± SEM. Comparisons between groups were assessed with either Mann- Whitney U test or ANOVA followed by Bonferroni^'s multiple comparisons analysis for more than 2 groups, survival analysis was carried out with Gehan-Breslow- Wilcoxon test using GraphPad Prism Software. Differences were considered significant if p- vaiues were < 0.05. Table 1

Sequences of nucleotides used for generation of shRNA or scrambled control plasmids (sense orientation only).

Table 2

Sequences of primers used for RT-PCR

Example II: Oxidation of lipids occurs in E. coli^' peritonitis in vivo, and leads to an actin-dependent change in cell shape in vitro

Invasion of bacteria to otherwise sterile sites like the peritoneal cavity leads to the immediate initiation of an inflammatory response. Integral to this response are oxygen radicals that are primarily generated to kill microbes, but can also damage host structures through the peroxidation of membrane phospholipids (Hampton, M.B., et al. (1998) Blood 92, 3007- 3017). We showed previously that administration of oxidized phospholipids (OxPL) impaired survival during /·.^'. coli peritonitis by inhibiting phagocytosis of bacteria by macrophages (Knapp, S., et al. (2007) J Immunol 178, 993-1001). Indeed, in detailed analyses we now found that OxPL dose-dependently diminished the uptake of bacteria by peritoneal macrophages (Fig. la,b). Consequently, administration of OxPL but not native phospholipids to mice that were infected with E. coli led to enhanced bacterial loads in the peritoneal cavity (Fig. lc). Importantly, we found that OxPL are generated endogenously during E. coli peritonitis in vivo, indicating their role as biologically relevant modulators in bacterial infections. Ten h after infection with E. coli, levels of OxPL-epitopes in the peritoneal lavage fluid (PLF) were significantly increased compared to the PLF of healthy mice, as measured with a monoclonal antibody that specifically recognizes, the phosphocholine headgroup of OxPL (Fig.ld) (Friedman, P., et al. (2002) The Journal of biological chemistry 277, 7010- 7020).

Because phagocytosis requires the active remodeling of actin (Kaksonen, M., et al. (2006) Nature reviews 7, 404-414) and actin polymerization was found in response to OxLDL previously (Miller, Y.I., et al. (2003) Mol Biol Cell 14, 4196-4206), we tested if OxPL themselves have any direct effect on the arrangement of the actin cytoskeleton. Using confocal imaging we observed that RAW 264.7 macrophages treated with OxPL showed a dramatic change in cell shape that could not be seen upon incubation with unoxidized phospholipids (Fig. le). This 'spreading' of cells was prevented by cytochalasin D, which inhibits actin polymerization.

Example III: OxPAPC induced cell spread and inhibition is not mediated by CD36

A similar actin-spread in macrophages upon OxLDL treatment that depended on the presence of CD36 was recently described in the context of atherogenesis (Park, Y.M., et al. (2009) J Clin Invest 119, 136-145) and CD36 has been shown to be a receptor for OxPL (Boullier, A., et al. (2000) The Journal of biological chemistry 275, 9163-9169). Thus, we assessed the role of this scavenger receptor in mediating these OxPL-effects. Interestingly, we could not discern an important role for CD36 in cell spreading, as CD36^mut peritoneal macrophages appeared indistinguishable from WT cells treated with OxPL (Fig. 5a). Regarding bacterial uptake we found that although the absence of functional CD36 affected phagocytosis of E. coli, OxPL still impaired phagocytosis to the same extent as seen in WT macrophages (Fig. 5b). Example IV: OxPAPC-mediated inhibition of phagocytosis is reversible

To exclude the possibility of toxic effects exerted by OxPL, we performed phagocytosis experiments in which phospholipids were removed after pre-incubation, and prior to addition of bacteria. We observed that the inhibitory effects of OxPL were fully reversible over time (Fig. 6). These data argue against toxic effects or competitive receptor-antagonism between E. coli and OxPL, and rather suggest downstream signaling events to account for OxPL- mediated effects on phagocytosis and cell spreading.

Example V: PKA activation mediates OxPAPC-associated cell spread and inhibition of phagocytosis and O PAPC activates PKA distinctly, and thereby induces actin polymerization

To identify the downstream signaling pathways involved in OxPL-iriduced effects on macrophages we first considered published reports indicating an important role for I3K and small GTPases in phagocytosis and actin polymerization (Underbill, D.M. et al. (2002) Annu Rev Immunol 20. 825-852), the involvement of small GTPases in actin remodeling caused by OxPL in endothelial cells (Birukov, K.G., et al. (2004) Ore Res 95, 892-901), and activation of PI3K by oxidized lipids (Miller, Y.I., et al. (2003) Mol Biol Cell 14, 4196-4206). Inhibitor studies revealed that neither PI3K (using wortmannin and LY294002) nor the small GTPases Rho and Rac (using Rho kinase inhibitor Y27632, and toxin B. which inhibits Rho, Rac and Cdc42) had any impact on actin polymerization or inhibition of phagocytosis caused by O PL (data not shown). Because a previous report revealed that elevated cAMP levels suppressed receptor-mediated phagocytosis in macrophages via involvement of PKA (Bryn, T., et al. (2006) J Immunol 176, 7361 -7370) we investigated the role of PKA in OxPL mediated effects. Pre -treatment with pharmacological PKA-inhibitors such as H89 or PKA amid (Bryn, T., et al. (2006) J Immunol 176, 7361-7370; Carr, D.W., et al. (1992) The Journal of biological chemistry 267, 13376- 13382; Wong, W. et al. (2004) Nature reviews 5, 959-970; Soderling. S.H.. et al. (2003) Proc Natl Acad Sci U SA 100, 1723-1728; Murch, A.R.. (1984) J Pathol 144. 81-87; Binder. C.J.. et al. (2002) Nat Med 8. 1218-1226; Hansson. G.K. (2006) Nat Rev Immunol 6. 508-519; Yoshimi. N., et al. (2005) Lung 183, 109-121; Nakamura, T., et al. (1998) Anal Biochem 262, 23-32), respectively, abolished OxPL-induced actin spread (Fig. 2a) and completely abrogated the inhibition of phagocytosis (Fig. 2b,c). To rule out confounding effects of PKA-inhibitors and to strengthen the evidence for PKA-involvement, we silenced the a-isoform of the catalytic subunit of PKA using sh NA (Fig. 2d). PKA- silencing successfully reversed OxPL-associated inhibition of phagocytosis (Fig. 2e) and reduced OxPL-induced spreading (Fig. 2f). These findings led us to hypothesize that OxPL itself activates PKA. Indeed, incubation with OxPL but not native phospholipids (DMPC) increased PKA activity (Fig. 2g). Thus, OxPL activates PKA, which is associated with actin spread and inhibition of phagocytosis. Unexpectedly however, incubating macrophages with the adenylyl cyclase activator forskolin, which activates PKA and Epac-1 , or N6- benzoyladenosine-3 '5 ^'-cAMP that selectively activates PKA, had no inhibitory effect on phagocytosis (Fig. 2h) or actin fiber formation (Fig. 7).

Example VI: AKAP-inhibition prevents detrimental effects of OxPAPC in vitro and in vivo

Knowing that PKA is involved in a large number of cellular processes, we speculated that OxPL-induced PKA-effects might underlie distinctive control mechanisms. A-kinase anchoring proteins (AKAPs) spatially and temporally control the specificity of cAMP downstream effects by targeting PKA to particular subcellular regions (Tasken, K. & et al. (2004) A. Physiol Rev 84, 137-167). To investigate if PKA-AKAP interaction is required for OxPL-induced effects, we exploited a cell-permeable AKAP inhibitory peptide (stearated Ht- 31) that specifically prevents the association of the regulatory subunit RII of PKA with AKAPs (Carr, D.W., et al. (1992) The Journal of biological chemistry 267, 13376-13382). Pre-incubating macrophages with Ht-31 abrogated the change in cell shape caused by OxPL (Fig. 3a) and concomitantly prevented OxPL-associated inhibition of phagocytosis (Fig. 3b). Notably, administration of Ht-31 together with OxPL at the onset of E. coli peritonitis in mice prevented the increase in bacterial loads caused by OxPL in vivo (Fig. 3c). Survival analysis corroborated these findings, as Ht-31 treatment was able to reverse the detrimental effects of OxPL during E.coli peritonitis in vivo (Fig. 3d-e). These data indicate that AKAPs specifically target OxPL-induced PKA activation resulting in diminished phagocytosis of bacteria in vitro and in vivo.

Example VII: WAVE1 mediates antiphagocytic properties of OxPAPC in vitro and in vivo and silencing of WAVE1 prevents OxPAPC associated cell spread and inhibition of phagocytosis

Among the 50 AKAPs discovered thus far, only Gravin, AKAP-Lbc, and Wiskott Aldrich syndrome protein-family verprolin-homologous protein 1 (WAVE1) have been described to bind to the actin cytoskeleton (Wong, W. et al. (2004) Nature reviews 5, 959-970). By investigating their specific expression patterns in primary and RAW 264.7 macrophages, we found only AKAP-Lbc and WAVE1 to be expressed (Fig. 4a,b). To analyze the functional role of AKAP-Lbc and WAVE1 we silenced either protein in macrophages using shRNA. While silencing of AKAP-Lbc did not affect actin spread or phagocytosis (data not shown), reduced WAVE1 expression completely abolished OxPL-associated actin spread and inhibition of phagocytosis (Fig. 8a-c). Furthermore, primary WAVE L ^" peritoneal macrophages (Soderling, S.H., et al. (2003) Proc Natl Acad Sci U S A 100, 1723- 1728). (Fig. 4c-d) exhibited neither spread nor impaired bacterial uptake upon OxPL treatment (Fig. 4c-f).

Since phagocytosis critically determines host defense against bacteria we finally aimed at corroborating the function of WAVEl during E. coli peritonitis in vivo. To exclude the potential influence of the altered size of homozygous WAVEL'^" mice, we generated chimeric mice on a C57BL/6 background and ensured complete reconstitution of donor peritoneal macrophages (Fig. 9) (Murch, A.R., (1984) J Pathol 144, 81 -87). Following i.p. injection of either DMPC or OxPL, we infected mice with E. coli i.p. and examined their ability to contain bacterial dissemination. OxPL treatment led to enhanced bacterial outgrowth in mice that received WT bone marrow (Fig. 4g-i). In contrast, chimeric mice with WAVEl^{" "} peritoneal macrophages appeared resistant to the effects of OxPL, as illustrated by identical CFU counts in PLF, liver and blood as observed in mice that received unoxidized lipids. Moreover, the absence of WAVEl in peritoneal macrophages prevented the OxPL-associated impairment of survival during E. coli peritonitis (Fig. 4j). These data confirm that WAVEl mediates the inhibition of phagocytosis caused by OxPL in vitro and in vivo.

In this study we investigated the mechanism by which OxPL impact phagocytosis during E. coli peritonitis and found a previously unrecognized role of WAVEl in macrophages. Our data emphasize the impact of modified lipids during infectious diseases and thus extend previous reports that documented a role for OxPL in various inflammatory conditions such as atherosclerosis (Binder, C.J., et al. (2002) Nat Med 8, 1218-1226; Hansson, G.K. et al. (2006) Nat Rev Immunol 6, 508-519) lung inflammation (Yoshimi, N., et al. (2005) Lung 183, 109- 121 ; Nakamura, T., et al. (1998) Anal Biochem 262, 23-32; Imai, Y.. et al. (2008) Cell 133, 235-249; Matt, U., et al. (2009) American journal of respiratory and critical care medicine), or inflammatory brain lesions (Newcombe, J., et al. (1994) Neuropathol Appl Neurobiol 20, 152-162; Dei, R., et al. (2002) Acta Neuropathol 104, 113-122). The precise contribution of OxPL to these diseases is still not fully understood, with some reports postulating a proinflammatory role (Imai, Y., et al. (2008) Cell 133, 235-249), while others describe antiinflammatory properties (Bochkov, V.N., et al. (2002) Nature 419, 77-81). In the peritonitis model discussed in this report, we primarily observed that OxPL diminish bacterial clearance, which could be prevented by interfering with PKA-downstream pathways that blocked WAVEl activity.

WAVEl belongs to the Wiskott-Aldrich syndrome protein (WASP) family that control actin polymerization via the Arp2/3 complex (Takenawa, T. et al. (2007) Nature reviews 8, 37-48). In contrast to all other WASP family members, WAVEl is predominantly known as an AKAP that contributes to the specificity of PKA by tethering it to Arp2/3 (Westphal, R.S., et al. (2000) Embo J 19, 4589-4600). So far, the biological role of WAVEl has been mainly studied in the brain where high expression levels have been detected and knockout mice exhibit altered synaptic transmission, depleted neuronal migration, behavioral deficits and reduced viability (Soderling, S.H.. et al. (2003) Proc Natl Acad Sci U S A 100. 1723-1728; Soderling, S.H., et al. (2007) J Neurosci 27, 355-365). On a molecular level, WAVEl was shown to induce actin polymerization and dendritic spine morphology in neurons (Kim, Y.. et al. (2006) Nature 442, 814-817). Only recently, WAVEl was found expressed in bone marrow derived macrophages, but its function remained unknown (Dinh, H., et al. (2008) J Leukoc Biol 84, 1483-1491). We hereby confirmed the expression of WAVEl in macrophages and found a crucial role for WAVEl in innate immunity, as WAVEl^"'^" peritoneal macrophages retained their phagocytic properties upon OxPL challenge in vitro as well as in vivo. Importantly though, WAVEl affected phagocytosis only in the presence of OxPL, thus indicating the requirement for oxidative stress as seen during serious inflammatory diseases or infections.

Considering the established importance of phagocytosis to combat bacterial infections (Pinheiro da Silva, F., et al. (2007) Nat Med 13, 1368-1374; Underbill, D.M. et al. (2002) Annu Rev Immunol 20, 825-852) and the lack of effective therapies in sepsis (Riedemann, N.C., et al. (2003) J Clin Invest 112, 460-467), targeting OxPL's negative impact on bacterial phagocytosis by WAVEl inhibitors prevents mortality from sepsis.