GELSOLIN USES IN NEUROLOGICAL DISORDERS

GOVERNMENT INTEREST

[0001] This invention was supported, in part, by Grant Number AR 38910 from the NIH. The government may have certain rights in the invention.

FIELD OF INVENTION

[0002] This invention is directed to methods of diagnosis, prognosis, and treatment of a neurological disorder. Specifically, the invention is directed to methods of diagnosis, prognosis, and treatment of a neurological disorder based on measuring changes in the level of gelsolin in cerebrospinal fluid. The invention is also directed to a method for treating, inhibiting, or suppressing a bacterial lipoteichoic acid (LTA)-induced pathogenesis in a subject by contacting the subject with a therapeutically effective amount of gelsolin.

BACKGROUND OF THE INVENTION

[0003] LPS and lipoteichoic acid (LTA) represent the major virulence factors of gram- negative and gram- positive bacteria, respectively. LTA concentrations can reach higher levels at infectious sites compared with LPS. Reported local tissue concentrations of LTA can be as high as 26 μg/ml, which may be associated with the fact that 10 ⁷ gram-positive bacteria contain as much as 1 μg of LTA whereas 10 ⁷ gram- negative bacteria contain only 20 ng of LPS. The primary transmembrane proteins that are activated by proinflammatory bacterial moieties such as LPS and LTA belong to the TLR family. Delivery of bacterial molecules from external fluids to the cell membrane and ultimately to TLR2 or TLR4 (dominant receptors for LTA and LPS, respectively) is complex and involves a number of other factors such as sCD14, LPS-binding protein (LBP), MD2, and moesin. One important factor that determines the toxicity of bacterial products to host cells is the geometry of their aggregation state. Low and high toxicity of LPS was associated with packing into lamellar and hexagonal phases, respectively. LTA and LPS obtained from different bacteria species induce the release of inflammatory cytokines such as TNF-α, IL-lβ, IL-6, and IL-8. All TLR signaling pathways elicit MyD88- or TRIF (TIR domain-containing adaptor-inducing IFN- β) -dependent activation of the transcription factor NF-kB. Those signaling cascades involve recruitment of different proteins such as IL-lR-associated kinase (IRAK), TNFR-associated factor 6 (TRAF6), TGF-β-activated kinase- 1 (TAKl), IKK complex, and MAPK. In RAW 264.7 macrophages, LTA was activates the PDK/ AKT pathway and

p38 MAPK-kinase, which in turn initiates NF-kB activation. TLR2 activation, contrary to earlier observation, was also proposed to function as a serum-independent factor, indicating significant differences between LTA- and LPS-mediated host cell activation. LTA is also a potent pathogenicity factor that causes cardiac dysfunction in gram-positive sepsis, may cause neuronal death, and determines clinical outcome in patients with pneumococcal meningitis. Many of the biological activities of gram-negative bacterial endotoxin are shared by LTA whereas most LPS- or LTA-mediated cellular effects are similar, LPS and LTA share few steps in their activation mechanisms and signal transduction pathways.

[0004] Gelsolin levels are decreased in several pathological inflammatory states, and lowered gelsolin levels have potential for identifying patients at risk for adult respiratory distress syndrome or multiple organ failure in some settings. Repletion of plasma gelsolin was shown to be beneficial in murine hyperoxic lung injury and endotoxemia, and gelsolin was able to attenuate vascular permeability associated with burn injury in rats.

[0005] Infection and sepsis remain a major cause of mortality in the world. A combination of biochemical and cellular studies show that the interaction of gelsolin with LPS and LTA results in both inhibition of gelsolin' s actin-severing activity and the ability of the bacterial toxins to induce an immune response in vitro. Thus, prevention of a decrease in blood gelsolin during sepsis may protect the host from bacterial wall product-mediated increase of cytokines and systemic complications that can lead to septic shock and multiorgan failure, therefore, gelsolin' s interaction with proinflammatory agents highlights its potential as a treatment for subjects with severe infection, or in urgent conditions when gelsolin blood concentration decreases.

[0006] Multiple Sclerosis (MS) is a chronic autoimmune disease that affects the central nervous system. In MS, the body directs antibodies and white blood cells against proteins in the mylein sheath of nerve fibers in the brain and spinal cord. MS affects an estimated 300,000 people in the United States and probably more than 1 million people around world - including twice as many women as men. MS is unpredictable and varies in severity. In some people, MS is a mild illness, but it can lead to permanent disability in others. Current treatments can only slow the course of the disease and relieve pain.

[0007] Cerebrospinal fluid (CSF) is a secretion product of the choroid plexus of the ventricles. Analysis of CSF offers the most direct and innocuous method to assess the chemical and cellular environment of the central nervous system (CNS) in a living patient. The total protein concentration of CSF is about 200 times

lower than that of blood plasma, and the composition of the various extracellular proteins is different. Despite these differences, blood plasma is the origin of most CSF proteins.

[0008] Gelsolin is a multifunctional Ca ²⁺ /polyphosphoinositide-regulated actin-binding protein first described in rabbit macrophages and subsequently in many different vertebrate cells. In addition to being a cytoplasmic regulator of actin organization, gelsolin is also secreted from cells, including neurons, into extracellular fluids. Gelsolin binds G-actin, nucleates actin filament growth, severs actin filaments, and caps their fast-growing ends. In human blood plasma, the gelsolin concentration is 150-300 μg/ml, and muscle is its primary source. Since actin is a major intracellular protein, it would be expected that as a result of tissue destruction, it would be released into the circulation where there are known pathological consequences to the vascular system and non-injured tissue upon actin exposure. Gelsolin binds and disassembles actin filaments, and functions in actin clearance. Gelsolin is also involved in the regulation of inflammatory processes through interactions with lysophosphatidic acid (LPA), platelet-activating factor (PAF) and lipopolysaccharide (LPS). Blood gelsolin levels decrease markedly in a variety of acute clinical conditions such as major trauma, prolonged hyperoxia, acute oxidant lung injury, malaria, sepsis and liver injury.

[0009] Thus, there is a need in the art to evaluate gelsolin levels in neurological diseases, especially in the CSF, which reflects CNS processes, and to determine if it correlates with susceptibility to or manifestation of the neurological diseases, to determine gelsolin' s role in those diseases, and to determine if therapeutic replenishment of gelsolin will abort injurious cascades.

SUMMARY OF THE INVENTION

[00010] In one embodiment, provided herein is a method of diagnosing a neurodegenerative disorder in a subject, comprising the steps of obtaining a cerebrospinal fluid (CSF) sample from the subject; determining the expression level of a gelsolin in the sample; and comparing the expression of a gelsolin to a standard, wherein if the standard is taken from a healthy subject or pool of subjects and the level of gelsolin is different than the standard by at least 50%, the subject has a neurological disorder, or, if the standard is taken from a subject or pool of subjects diagnosed with a neurological disorder and the level of a gelsolin is different than the standard by at least 50%, the subject does not have the neurological disorder.

[00011] In another embodiment, provided herein is a method of determining the risk of developing a neurological disorder in a subject, comprising the steps of obtaining a cerebrospinal fluid (CSF) sample

from the subject; determining the expression level of a gelsolin in the sample; and comparing the expression of a gelsolin to a standard, wherein if the standard is taken from a healthy subject or pool of subjects and the level of gelsolin is different than the standard by at least 50%, the subject is at risk for developing the neurological disorder, or, if the standard is taken from a subject or pool of subjects diagnosed with a neurological disorder and the level of a gelsolin is different than the standard by at least 50%, the subject is at low risk for developing the neurological disorder.

[00012] In another embodiment, provided herein is a method for treating, inhibiting, or suppressing a bacterial lipoteichoic acid (LTA)-induced pathogenesis in a subject resulting from an accumulation of excess bacterial LTA, comprising contacting a cell of said subject with a therapeutically effective amount of gelsolin, or functionally equivalent peptide fragment thereof.

[00013] In another embodiment, provided herein is a method of treating, suppressing, or inhibiting an effect of lipoteichoic acid (LTA) in a subject comprising administering a therapeutically effective dose of gelsolin to said subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[00014] FIGURE 1. In panels A and B, gelsolin was identified using anti-gelsolin and anti-human plasma gelsolin antibody, respectively. The lanes represent: 1 - recombinant human plasma gelsolin (10, ng); 2 - lysed human platelets (4.6 μg of total protein); 3 - human plasma (10 μl of 1:100 dilution); 4, 5, 6, 7 - 7,5 μl of CSF from 4 subjects diagnosed with MS; In panels C and D, lanes 2, 3, 4, 5, 6 - represent purified rabbit muscle actin 5-200 ng; lanes 6, 7, 8, 9, 10 - lysed human platelets (0.1-4.6 μg of total protein); and lanes 11, 12, 13, 14, 15 - 7,5 μl of CSF from 4 subjects diagnosed with MS. Panel E: G-actin nucleation assay. Pyrene fluorescence, proportional to actin polymerization, in control samples (triangles up), and in the presence of recombinant plasma gelsolin (triangles down), CSF obtained from representative subjects diagnosed with MS (squares), ischialgia due to discopathy (diamonds) or SAH (empty circles).

[00015] FIGURE 2. Blood (black column) and CSF were obtained from 25 subjects, and were evaluated by immunoblotting (gray column) or nucleation assay (white column). Error bars indicate that at least 3 samples were obtained from patients suffering from the same conditions. Blood and CSF gelsolin levels are correlated and low in MS patients.

[00016] FIGURE 3. Gelsolin and rhodamine B (PBP) peptide were synthesized based on its phosphatidylinositol bisphosphate (PIP ₂ ) binding sequence.

[00017] FIGURE 4. Lipopolysaccharide (LPS) changes the intrinsic fluorescence of gelsolin (left panel). LPS binds to immobilized rhodamine B-QRLFQVKGRR (PBPlO) in a solid phase asay (right panel).

[00018] FIGURE 5. Lipopolysaccharide (LPS) inhibits severing activity of human blood plasma gelsolin.

[00019] FIGURE 6. Lipopolysaccharide (LPS) effects on astrocyte cytoskeleton and NF-kB are inhibited by plasma gelsolin.

[00020] FIGURE 7. Gelsolin inhibits binding of ¹²⁵ I- lipopolysacharide (LPS) binding protein (LBP) (10 ng) to LPS (1 ng)-coated wells.

[00021] FIGURE 8. Interaction of B-QRLFQVKGRR (PBPlO) and gelsolin with lipoteichoic acid (LTA) from Staphylococcus aureus.

[00022] FIGURE 9. Gelsolin prevents LTA-induced activation of HAEC evaluated by E-selectin expression (A) and human neutrophil adhesion (B). C, Quantification of neutrophil adherence calculated from the fluorescence of calcein-AM. A and B, Data from one representative experiment are shown. C, Error bars represent SD from four measurements. *, Significantly different from LTA (10 μg/ml)- activated samples.

[00023] FIGURE 10. Lysophosphatidic acid (LPA), lipoteichoic acid (LTA) and sphingosine 1 -phosphate (SlP) inhibit gelsolin' s severing activity where percent severing activity of gelsolin drops following the addition of LPA, LTA , or SlP.

[00024] FIGURE 11. A dose-response curve demonstrates that the actin severing activity of gelsolin is markedly inhibited by increasing concentrations of phosphatidylinositol bisphosphate (PIP2) and sphingosy- phosphoryl-choline (SPC).

[00025] FIGURE 12. Neutrophil morphology following a 2-h exposure to LPS (100 ng/ml) or LTA (10 μg/ml) with or without 2 μM rhGSN. Results are shown from one experiment performed in triplicate.

[00026] FIGURE 13. Gelsolin prevents lipopolysaccharide (LPS) internalization in human aortic endothelial cells (HAEC).

[00027] FIGURE 14. A, Purified LTA from S. aureus -induced IL- 8 release from human neutrophils in a concentration-dependent manner. Gelsolin prevents IL- 8 release from neutrophils treated with purified LTA (B), LPS (E. colϊ) or nonpurified-LTA (S. aureus) (C), and neutrophils treated with heat-inactivated gram- negative P. aeruginosa PAOl or gram-positive B. subtilis ATCC 6051 (lμl of each) (D). Error bars represent SDs from three measurements performed in duplicate. *, significantly different from control neutrophil samples or those treated with LPS, LTA, or heat-inactivated bacteria.

[00028] FIGURE 15. Bactericidal activity of LL37 against B. subtilis (ATC6051) and P. aeruginosa (PAOl) alone or in the presence of 2 μM rhGSN. Error bars represent SDs from three measurements (A). LL37 MIC value (μg/ml) for B. subtilis (ATC 6051) evaluated in Mueller-Hinton broth (MH) did not change in the presence of rhGSN (B).

[00029] FIGURE 16. The size distribution (filled symbols) and light scattering intensity (open symbols) (A) with progressive dilutions of LPS (squares) and LTA (triangles) in PBS (EC, E. coli; PA, P. aeruginosa, KP, K. pneumoniae; SA, S. aureus; SF, S. faecalis). DLS evaluations are shown of 1 mM LPS (B) and 0.5 mM LTA (C) aggregation states in PBS solution without rhGSN (open column) and 5 min after rhGSN addition (62 μM, gray column).

[00030] FIGURE 17. Binding of lipopolysaccharide (LPS) to gelsolin inhibits gelsolin' s actin binding activity. Gelsolin is also involved in the regulation of inflammatory processes through interactions with lysophosphatidic acid (LPA), platelet-activating factor (PAF), and LPS, because binding of gelsolin to LPS inhibits some, but not all, effects of LPS and lipoteichoic acid (LTA) on cells in vitro. Gelsolin also inhibits binding of LPS to LPS binding protein (LBP) and the effect of LPS on astrocyte cytoskeleton is inhibited by plasma gelsolin. Plasma gelsolin and secreted Gc-globulin act to depolymerize and remove the actin filaments released from damaged cells.

[00031] FIGURE 18. Using an antibody that recognized recombinant human gelsolin (rhGSN), the presence of gelsolin was detected in saliva, bile, blood and CSF.

DETAILED DESCRIPTION OF THE INVENTION

[00032] In one embodiment, provided herein is a composition for treating a neurodegenerative disorder in a subject, comprising an agent capable of modulating actin polymerization. In one embodiment, said agent is a recombinant protein or functional fragment thereof. In one embodiment, said neurological disorder is multiple sclerosis (MS), Spinocerebellar Ataxia Type 2 (SCA2), Parkinson's Disease (PD), Alzheimer's Disease (AD), Schizophrenia, Amyotrophic lateral sclerosis (ALS), or Huntington's Disease (HD).

[00033] In one embodiment, an agent capable of modulating actin polymerization is gelsolin, its peptidomimetic molecule or their combination. In another embodiment, said agent is a administered in the form of a liquid, a capsule, a patch or their combination. In another embodiment, said agent further comprising a pharmaceutically acceptable carrier, further comprising a protease inhibitor cocktail, and diluents.

[00034] It is to be understood any of the methods of the present invention described herein as requiring gelsolin may instead use a peptidomimetic molecule or an a biologically active fragment of gelsolin, as described herein.

[00035] In one embodiment, a peptidomimetic molecule is a small protein-like chain designed to mimic a peptide. In one embodiment, a peptidomimetic molecule arises from modification of an existing peptide in order to alter the molecule's properties, which in one embodiment is the molecule's stability and, in another embodiment, is the molecule's biological activity. In one embodiment, these modifications involve changes to the peptide such as altered backbones and the incorporation of nonnatural amino acids.

[00036] In one embodiment, a gelsolin peptide may be used in place of gelsolin in the compositions and methods of the present invention, hi one embodiment, the gelsolin peptide is QRLFQVKGRR (gelsolin residues 160-169, SEQ ID NO: 1).

[00037] In one embodiment, gelsolin used or detected in the methods of the present invention have an amino acid sequence as set forth in Genbank Accession No: NP_000168.1, NPJ)Ol 121134.1, NPJ)01121135.1, NPJ)Ol 121136.1, NP_001121137.1, NP_001121138.1, NP_001121139.1, or NP337895.1. In another embodiment, the gelsolin has any gelsolin amino acid sequence known in the art. In another embodiment, the gelsolin is a homologue of a sequence from one of the above GenBank entries. In another embodiment,

the gelsolin is a variant of a sequence from one of the above GenBank entries. In another embodiment, the gelsolin is an isoform of a sequence from one of the above GenBank entries. In another embodiment, the gelsolin is a fragment of a sequence from one of the above GenBank entries. Each possibility represents a separate embodiment of the present invention.

[00038] In one embodiment, a "homolog" refers to any protein or peptide, or any sequence, whether amino acid or nucleotide sequence, in whcih a percentage of amino acid residues or nucleic acid residues, as appropriate, in the candidate sequence that are identical with the residues of a corresponding native polypeptide or nucleic acid, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for the alignment are well known in the art.

[00039] Homology is, in another embodiment, determined by computer algorithm for sequence alignment, by methods well described in the art. For example, computer algorithm analysis of nucleic acid sequence homology can include the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.

[00040] In another embodiment, "homology" refers to the identity between two sequences being greater than 70%. In another embodiment, "homology" refers to the identity between two sequences being greater than 72%. In another embodiment, "homology" refers to the identity between two sequences being greater than 75%. In another embodiment, "homology" refers to the identity between two sequences being greater than 78%. In another embodiment, "homology" refers to the identity between two sequences being greater than 80%. In another embodiment, "homology" refers to the identity between two sequences being greater than 82%. In another embodiment, "homology" refers to the identity between two sequences being greater than 83%. In another embodiment, "homology" refers to the identity between two sequences being greater than 85%. In another embodiment, "homology" refers to the identity between two sequences being greater than 87%. In another embodiment, "homology" refers to the identity between two sequences being greater than 88%. In another embodiment, "homology" refers to the identity between two sequences being greater than 90%. In another embodiment, "homology" refers to the identity between two sequences being greater than 92%. In another embodiment, "homology" refers to the identity between two sequences being greater than 93%. In another embodiment, "homology" refers to the identity between two sequences being greater than 95%. In another embodiment, "homology" refers to the identity between two sequences being greater than 96%. In another embodiment, "homology" refers to the identity between two sequences being greater than 97%. In another embodiment, "homology" refers to the identity between two sequences being greater than

98%. In another embodiment, "homology" refers to the identity between two sequences being greater than 99%. In another embodiment, "homology" refers to the identity between two sequences being 100%.

[00041] In another embodiment, homology is determined via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N. Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y). In other embodiments, methods of hybridization are carried out under moderate to stringent conditions, to the complement of a DNA encoding a native caspase peptide. Hybridization conditions being, for example, overnight incubation at 42°C in a solution comprising: 10-20 % formamide, 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7. 6), 5 X Denhardt's solution, 10 % dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.

[00042] Homology for any amino acid or nucleic acid sequence listed herein is determined, in another embodiment, by methods well described in the art, including immunoblot analysis, or via computer algorithm analysis of amino acid sequences, utilizing any of a number of software packages available, via established methods. Some of these packages include the FASTA, BLAST, MPsrch or Scanps packages, and, in another embodiment, employ the use of the Smith and Waterman algorithms, and/or global/local or BLOCKS alignments for analysis, for example. Each method of determining homology represents a separate embodiment of the present invention.

[00043] In one embodiment, "fragment" refers to a portion of a larger polypeptide or polynucleotide. In one embodiment, a fragment retains one or more particular functions as the larger molecule from which it was derived. In one embodiment, a fragment may maintain a functional domain, which in one embodiment, is a nuclear localization signal (NLS), a glycosylation site, a cleavage site, a binding site, a DNA contact site or residues, a G-actin binding site or residues, a metal binding site or residues, a Sph chimera fusion site, a catalytic His, a Hi hydorgen-bond pair, or a combination thereof. In one embodiment, a fragment may be approximately 50% of the length of the source polypeptide or polynucleotide.

[00044] In another embodiment, a fragment may be approximately 90% of the length of the source polypeptide or polynucleotide. In another embodiment, a fragment may be approximately 75% of the length of the source polypeptide or polynucleotide. In another embodiment, a fragment may be approximately 70% of the length of the source polypeptide or polynucleotide. In another embodiment, a fragment may be approximately 60% of the length of the source polypeptide or polynucleotide. In another embodiment, a

fragment may be approximately 40% of the length of the source polypeptide or polynucleotide. In another embodiment, a fragment may be approximately 30% of the length of the source polypeptide or polynucleotide. In another embodiment, a fragment may be approximately 25% of the length of the source polypeptide or polynucleotide. In another embodiment, a fragment may be approximately 10% of the length of the source polypeptide or polynucleotide. In another embodiment, a fragment may be approximately 5% of the length of the source polypeptide or polynucleotide.

[00045] In another embodiment, a fragment is approximately 150 amino acids. In another embodiment, a fragment is approximately 180 amino acids. In another embodiment, a fragment is approximately 200 amino acids. In another embodiment, a fragment is 100-200 amino acids. In another embodiment, a fragment is 125-175 amino acids. In another embodiment, a fragment is 140-160 amino acids. In another embodiment, a fragment is 50-150 amino acids. In another embodiment, a fragment is the equivalent number of nucleic acids required to encode an amino acid as described, as would be understood by a skilled artisan.

[00046] In one embodiment, "isoform" refers to a version of a molecule, for example, a protein, with only slight differences to another isoform of the same protein. In one embodiment, isoforms may be produced from different but related genes, or in another embodiment, may arise from the same gene by alternative splicing. In another embodiment, isoforms are caused by single nucleotide polymorphisms.

[00047] In one embodiment, "variant" refers to an amino acid or nucleic acid sequence (or in other embodiments, an organism or tissue) that is different from the majority of the population but is still sufficiently similar to the common mode to be considered to be one of them, for example splice variants. In one embodiment, the variant may a sequence conservative variant, while in another embodiment, the variant may be a functional conservative variant. In one embodiment, a variant may comprise an addition, deletion or substitution of 1 amino acid. In one embodiment, a variant may comprise an addition, deletion, substitution, or combination thereof of 2 amino acids. In one embodiment, a variant may comprise an addition, deletion or substitution, or combination thereof of 3 amino acids. In one embodiment, a variant may comprise an addition, deletion or substitution, or combination thereof of 4 amino acids. In one embodiment, a variant may comprise an addition, deletion or substitution, or combination thereof of 5 amino acids. In one embodiment, a variant may comprise an addition, deletion or substitution, or combination thereof of 7 amino acids. In one embodiment, a variant may comprise an addition, deletion or substitution, or combination thereof of 10 amino acids. In one embodiment, a variant may comprise an addition, deletion or substitution, or combination thereof of 2-15 amino acids, hi one embodiment, a variant may comprise an addition, deletion or substitution, or combination thereof of 3-20 amino acids. In one

embodiment, a variant may comprise an addition, deletion or substitution, or combination thereof of 4-25 amino acids.

[00048] In one embodiment, gelsolin is a serum protein that has a cytoplasmic isoform. In another embodiment, gelsolin is a serum protein that has a secreted isoform. In another embodiment, the secreted isoform is derived by alternative splicing of the message from a single gene. In another embodiment cytoplasmic gelsolin exists as an abundant secreted isoform of nearly identical structure. In another embodiment, cytoplasmic gelsolin is a product of the same gene. In another embodiment, gelsolin has a sixfold sequence repeat structure that is highly conserved among gelsolins of vertebrate species. In another embodiment, the six-fold sequence repeat structure is characteristic of a large family of gelsolin-related proteins. In another embodiment, gelsolin is secreted by many tissues. In another embodiment, the major source of human plasma gelsolin is striated muscle. In yet another embodiment, gelsolin is distributed throughout extracellular fluids and has a typical residence time in plasma. In another embodment, in human blood plasma, the gelsolin concentration is 150-300 μg/ml. In another embodiment, in CSF the gelsolin concentration is about 5.9 μg/ml.

[00049] In another embodiment, plasma gelsolin depletion precedes and predicts complications of severe injury, such as respiratory failure and death in animals including humans. In another embodiment, a drop in plasma gelsolin levels to <50% is a strong predictor of adverse clinical outcomes associated with massive inflammation. In one embodiment, in healthy subjects, blood gelsolin is present at much higher concentrations than the other high-affinity ligands, and, unlike LBP and CD14, gelsolin is not an acute- phase protein.

[00050] In another embodiment, in response to acute trauma and/or infection, abundant but normally intracellular G-actin (monomelic actin) is released into extracellular spaces from damaged or dying cells circulating in body fluid and blood. In another embodiment, in response to acute trauma and/or infection, abundant but normally intracellular G-actin (monomeric actin) is released into tissues. In another embodiment, once released, G-actin has a strong tendency to polymerize to F-actin. In another embodiment, the persistance of filaments of F-actin in the microvasculature of mammals can result in venous obstruction, pulmonary microthrombii or endothelial injury. In another emdobiment, the persistence of filaments of F- actin in the microvasculature of mammals, induces or enhances platelet agglutination in the blood. In another embodiment, the persistence of filaments of F-actin in the microvasculature of mammals triggers thrombus development. In another embodiment, these effects alter the characteristics of normal vascular flow in mammals. In another embodiment, these effects can result in actin toxicity diseases. In another

embodiment, these effects can contribute to the pathogenesis of organ injury at sites removed from the primary insult.

[00051] In another embodiment, provided herein is a method of modulating the state of actin polymerization in blood and/or cerebral spinal fluid of a subject that is afflicted with a neurodegenerative disorder, comprising the step of contacting the subject with a gelsolin. In another embodiment, the contacting step will increase the gelsolin level in CSF by at least 50%. In another embodiment, the contacting step will up- regulate the gelsolin level in CSF of the subject as described herein. In one embodiment, said contacting with gelsolin will be directly into the CSF of the subject.

[00052] In some embodiments, LPS and LTA represent the major virulence factors of gram-negative and gram-positive bacteria, wherein LTA concentrations can reach higher levels at infectious sites compared to LPS. In other embodiments, gelsolin is involved in both LTA and LPS presentation to (Toll-like receptor 4) TLR4 and TLR2, whereby it acts as a scavenger or delivery promoter of immunogenic bacterial wall components.

[00053] hi one embodiment, LTA and/or LPS are potent mediators of the innate immune response, where efficacy of the host recognition may determine survival during bacterial infection. In another embodiment, bacterial wall molecules LPS and LTA are major targets for bactericidal activity.

[00054] In another embodiment, the binding of gelsolin to LTA or LPS determines their interaction with other proteins such as LBP, CD 14, or MD2 and factors such as lipoprotein involved in host cell detection and elimination of bacterial products, where in some embodiments, gelsolin has a buffering effect on the availability of LTA/LPS for these targets and LPS and LTA binding to gelsolin determines the immune response at different steps of the signaling pathway. In one embodiment, LTA, LPS, LPA, and PAF bind to at least one common site within the gelsolin molecule indicating the possibility for competition among these lipids.

[00055] In one embodiment, extracellular gelsolin binds LTA from different gram-positive bacteria strains, where in other embodiments, the result of this binding is the inhibition of gelsolin's F-actin depolymerizing activity and compromised ability of LTA to activate endothelial cells, as measured by E-selectin expression, activation of the transcription factor NF- kB, and neutrophil adhesion. In other embodiments, gelsolin

inhibits the release of IL-8 from human neutrophils subjected to LTA, LPS, and heat- inactivated bacteria treatment. In yet another embodiment, gelsolin is an anti-inflammatory agent.

[00056] In one embodiment, gelsolin modulates LTA/LPS interaction with its ligands in bodily fluids, such as blood or CSF. hi another embodiment, the term "bodily fluids" refers to fluids such as saliva, urine, cerebro- spinal fluid, blood, plasma, sera, sperm, mucous, or any other fluid or combination thereof known in the art to originate from or derived from a subject as provided herein.

[00057] In other embodiments, provided herein is a method of inhibiting the severing activity of gelsoin comprising the step of adding an effective amount of LPA, LTA or sphingosine 1-phosphate (SlP).

[00058] In one embodiment, various actin-regulating proteins contribute to the reversible conversion of filaments ("gel") and monomers (liquid "sol"). In another embodiment, changes occur depending on extracellular stimuli. In another embodiment, plasma gelsolin and secreted Gc-globulin act in a coordinated manner, representing an "actin- scavenger system," to depolymerize and remove the actin filaments released from damaged cells.

[00059] In another embodiment, gelsolin binds to both monomeric and filamentous actin. In another embodiment, following injury, gelsolin preferably binds to and severs the actin filaments to promote rapid depolymerization, whereas Gc-globulin binds to the actin monomers to shift the actin monomer/polymer equilibrium back toward depolymerization and prevents repolymerization. In another embodiment, this binding requires the presence of micromolar concentrations of calcium (Ca ²⁺ ), which may include added calcium or endogenously available Ca ²⁺ in the patient. In yet another embodiment, the binding is very tight, having a dissociation constant in the nanomolar range.

[00060] In another embodiment, when gelsolin binds actin filaments in the presence of calcium, it ruptures or severs the filaments at the binding site by breaking the noncovalent bonds holding actin monomers together within the polymer. In another embodiment, following the actin severing reaction, gelsolin remains tightly bound to another end of the polarized actin filament, the end conventionally defined as "barbed" and this is also the end that rapidly exchanges monomers.

[00061] In another embodiment, removing calcium by chelation does not dissociate gelsolin from the barbed ends of the actin filaments. In another embodiment, phosphoinositides (also referred to as phosphorylated inositol phospholipids, or "PPIs"), effect this separation at the plasma membrane.

[00062] In another embodiment, gelsolin binds with high affinity and selectivity to PPI and to lysophosphatidic acid (LPA) In another embodiment, PPIs, regulate the intracellular actin-binding function of gelsolin. In another embodiment, a reciprocal relationship, between calcium transients and membrane phosphoinositide synthesis and degradation, regulates gelsolin and cellular actin remodeling responses. In another embodiment, gelsolin binding to LPA, modulates its receptor-mediated biological effects. In another embodiment, gelsolin acts as a carrier of LPA to some cellular receptors, and buffers bioactive inflammatory lipid mediators.

[00063] In another embodiment, calcium and phosphoinositides control the actin-binding functions of plasma gelsolin in vitro. In another embodiment, the PPI regulatory site of gelsolin resides within a 20 residue linear sequence that connects the first and second folded domains of the protein, hi yet another embodiment, biochemical and mutational studies have implicated 10 strategically organized basic and hydrophobic amino acids ( QRLFQVKGRR, SEQ ID NO:1, residues 160-169) in the 684-residue plasma gelsolin molecule that accommodate tight binding to the negatively-charged phosphomonoesters and hydrophobic acyl chains of anionic phospholipids. Further, and in another embodiment, synthetic peptides of this sequence have a PPI binding affinity similar to that of intact gelsolin.

[00064] In one embodiment, provided herein is a method for diagnosing a neurological disorder in a subject, comprising the steps of obtaining a biological sample from the subject and analyzing the level of a gelsolin in the biological sample. In another embodiment, the expression of a gelsolin is compared to a standard. In another embodiment, the standard is taken from an apparently healthy subject or pool of subjects. In another embodiment, the standard is the expression profile in a subject or pool of subject correctly diagnosed as having neurological disorder. In another embodiment, the standard is taken from the mean of the pool of subjects. In another embodiment, the standard is taken from the average of the pool of subjects. In another embodiment, the standard is taken from the median of the pool of subjects, hi another embodiment, if the level of gelsolin is different than the standard by more than a predetermined threshold, the subject has a neurological disorder. In another embodiment, if the level of a gelsolin is different than the standard by more than a predetermined threshold, the subject does not have the neurological disorder. In another embodiment, downregulation of gelsolin expression is associated with the etiology of a neurological

disorder. In another embodiment, the gelsolin expression is downregulated by about 65%. In another embodiment, the biological sample is cerebral spinal fluid. In another embodiment, upregulation of gelsolin expression is associated with the etiology of a neurological disorder.

[00065] In another embodiment, methods of the present invention determine both gelsolin expression levels and gelsolin function. In another embodiment, methods of the present invention determine gelsolin expression levels, in one embodiment, via immunoblotting techniques. Other assays for determining gelsolin expression levels are known in the art. In another embodiment, methods of the present invention determine gelsolin function, in one embodiment, via a fluorometric actin polymerization assay. Other assays for determining gelsolin function are known in the art.

[00066] In another embodiment, the biological sample is bile, blood, sera, plasma, saliva, sperm, urine, mucous, cerebrospinal fluid, or their combination.

[00067] In one embodiment, the lower gelsolin concentration observed in subjects with a neurological disorder in plasma gelsolin levels is from disturbed gelsolin-actin interaction, binding of gelsolin to cellular mediators, modulation of gelsolin synthesis in response to actin release or their combination.

[00068] In one embodiment, low gelsolin concentration in plasma or CSF is causally related to MS pathogenesis. In another embodiment, low gelsolin concentration in plasma or CSF is predictive of future MS pathogenesis. In another embodiment, a neurological disorder is treated by administration of recombinant gelsolin protein.

[00069] In another embodiment, a gelsolin protein or biologically active fragment thereof is downregulated in a neurological disorder. In one embodiment, "downregulated" is a level is at least about 5-10% less than the measured mean, average or median level for a given population of subjects. In another embodiment, "downregulated" is a level is at least about 11-20% less than the measured mean, average or median level for a given population of subjects. In another embodiment, "downregulated" is a level is at least about 21- 30% less than the measured mean, average or median level for a given population of subjects. In another embodiment, "downregulated" is a level is at least about 31-40% less than the measured mean, average or median level for a given population of subjects. In another embodiment, "downregulated" is a level is at least about 41-50% less than the measured mean, average or median level for a given population of subjects. In another embodiment, "downregulated" is a level is at least about 51-60% less than the measured mean,

average or median level for a given population of subjects. In another embodiment, "downregulated" is a level is at least about 61-65% less than the measured mean, average or median level for a given population of subjects. In another embodiment, "downregulated" is a level is at least about 66-70% less than the measured mean, average or median level for a given population of subjects. In another embodiment, "downregulated" is a level is at least about 71-75%. In another embodiment, "upregulated" is a level is at least about 5-10% less than the measured mean, average or median level for a given population of subjects. In another embodiment, "upregulated" is a level at least about 11-20% more than the measured mean, average or median level for a given population of subjects. In another embodiment, "upregulated" is a level at least about 21-30% more than the measured mean, average or median level for a given population of subjects. In another embodiment, "upregulated" is a level at least about 31-40% more than the measured mean, average or median level for a given population of subjects. In another embodiment, "upregulated" is a level at least about 41-50% more than the measured mean, average or median level for a given population of subjects. In another embodiment, "upregulated" is a level at least about 51-60% more than the measured mean, average or median level for a given population of subjects.

[00070] In another embodiment, levels of gelsolin are upregulated in a patient or in a patient population with a neurological disorder. In one embodiment, "upregulated" is a level is at least about 61-65% less than the measured mean, average or median level for a given population of subjects. In another embodiment, "upregulated" is a level is at least about 66-70% In another embodiment, "upregulated" is a level at least about 71-90% more than the measured mean, average or median level for a given population of subjects. In another embodiment, "upregulated" is a level at least about 91-100% more than the measured mean, average or median level for a given population of subjects. In another embodiment, the mean gelsolin level can depend upon the particular population of subjects. In yet another embodiment, an apparently healthy population will have a different "normal" range of gelsolin than will a population of subjects which have had a prior infection or other condition.

[00071] hi one embodiment, the term "predetermined threshold" refers to a gelsolin level that differs by at least 1% compared to the measured mean level for a given population of subjects or a standard. In another embodiment, the gelsolin level differs at by least 5-10% compared to the measured mean level for a given population of subjects or a standard. In another embodiment, the gelsolin level differs by at least 11-20% compared to the measured mean level for a given population of subjects or a standard. In another embodiment, the gelsolin level differs by at least 21% compared to the measured mean level for a given population of subjects or a standard. In yet another, the gelsolin level differs by at least 22-30%, 31-40%,

41-50%, 51-60%, 61-70%, 71-75%, when compared to the measured mean level for a given population of subjects or a standard. In another embodiments, the gelsolin level is below about 15.0 μg/mL (micrograms/milliliter) of cerebrospinal fluid.

[00072] In one embodiment, provided herein is a method of treating a neurodegenerative disease in a subject, comprising the step of administering into the subject's CSF a composition comprising gelsolin, thereby reducing vulnerability to excitotoxicity in neurons.

[00073] In one embodiment, provided herein is a method of treating a neurodegenerative disease in a subject, comprising the step of contacting the subject's CSF with a composition comprising gelsolin, thereby reducing vulnerability to excitotoxicity in neurons.

[00074] In one embodiment, said step of contacting or administering as described herein increases CSF gelsolin expression levels by greater than 50%. In another embodiment, said step of contacting or administering as described herein upregulates CSF gelsolin expression levels by a specific percentage or range as described hereinabove.

[00075] In one embodiment, methods of the present invention may be used to treat, inhibit, suppress, ameliorate symptoms of a neurological or neurodegenerative disease, while in another embodiment, methods of the present invention may be used to treat, inhibit, suppress, ameliorate symptoms of a neurological or neurodegenerative disorder.

[00076] In provided herein is a method of treating an inflammatory disease in a subject, comprising the step of administering a subject with a composition comprising a gelsolin thereby reducing vulnerability to excitotoxicity in neurons. In another embodiment, is a method of preventing, inhibiting or suppressing a disease in a subject, comprising the step of administering a subject with a composition comprising gelsolin. In another embodiment, is a method of treating, preventing, inhibiting or suppressing a disease in a subject, comprising the step of administering administering a subject with a composition comprising a gelsolin.

[00077] In one embodiment, provided herein is a method of determining the progression of an inflammatory disease in a subject, comprising the steps of obtaining a cerebrospinal fluid (CSF) sample from the subject and analyzing the level of a gelsolin in the cerebrospinal fluid (CSF) sample. In another embodiment, the expression of a gelsolin is compared to a standard. In another embodiment, the standard is taken from an

apparently healthy subject or pool of subjects. In another embodiment, the standard is the expression profile in a subject or pool of subjects correctly diagnosed as having a neurological disorder. In another embodiment, the standard is taken from the mean of the pool of subjects. In another embodiment, the standard is taken from the average of the pool of subjects. In another embodiment, the standard is taken from the median of the pool of subjects. In another embodiment, if the level of gelsolin is different than the standard by more than a predetermined threshold, the progression of a neurological disorder in a subject is determined.

[00078] In one embodiment, the inflammatory disease includes but is not limited to, arthritis, rheumatoid arthritis, asthma, inflammatory bowel disease (Crohn's disease or ulcerative colitis), chronic obstructive pulmonary disease (COPD), allergic rhinitis, vasculitis (polyarteritis nodosa, temporal arteritis, Wegener's granulomatosus, Takayasu's arteritis, or Behcet syndrome), inflammatory neuropathy, psoriasis, systemic lupus erythematos s (SLE), chronic thyroiditis, Hashimoto's thyroiditis, Addison's disease, polymyalgia rheumatica, Sjogren's syndrome, or Churg-Strauss syndrome. In some important embodiments, the inflammatory disease is rheumatoid arthritis.

[00079] In another embodiment, provided herein is a method of inhibiting or suppressing a neurodegenerative disease in a subject, comprising the step of administering into the subject's CSF, a composition comprising gelsolin, thereby reducing vulnerability to excitotoxicity in neurons.

[00080] In another embodiment, provided herein is a method of inhibiting or suppressing a neurodegenerative disease in a subject, comprising the step of contacting the subject's CSF with a composition comprising gelsolin, thereby reducing vulnerability to excitotoxicity in neurons.

[00081] In one embodiment, provided herein is a method of ameliorating symptoms associated with a neurodegenerative disorder in a subject, comprising the step of administering into the subject's CSF a composition comprising gelsolin, thereby reducing vulnerability to excitotoxicity in neurons. In another embodiment, administering is via oral, or parenteral administration. In another embodiment, the composition further comprises beta-inteferon, copaxon, corticosteroids, natalizumab or their combination.

[00082] In one embodiment, provided herein is a method of ameliorating symptoms associated with a neurodegenerative disorder in a subject, comprising the step of contacting the subject's CSF with a composition comprising gelsolin, thereby reducing vulnerability to excitotoxicity in neurons. In another

embodiment, administering is via oral, or parenteral administration. In another embodiment, the composition further comprises beta-inteferon, copaxon, corticosteroids, natalizumab or their combination.

[00083] hi one embodiment, provided herein is a method for determining the prognosis of a subject developing a neurological disorder comprising the steps of obtaining a cerebrospinal fluid (CSF) sample from the subject and analyzing the level of a gelsolin in the cerebrospinal fluid (CSF) sample. In another embodiment, the expression of a gelsolin is compared to a standard. In another embodiment, the standard is taken from a apparently healthy subject or pool of subjects. In another embodiment, the standard is the expression profile in a subject or pool of subject correctly diagnosed as having neurological disorder. In another embodiment, the standard is taken from the mean of the pool of subjects. In another embodiment, the standard is taken from the average of the pool of subjects. In another embodiment, the standard is taken from the median of the pool of subjects. In another embodiment, if the level of gelsolin is different than the standard by more than a predetermined threshold, the prognosis of aneurological disorder is determined.

[00084] In one embodiment, provided herein is a method for determining the progression of a neurological disorder in a subject comprising the steps of obtaining a cerebrospinal fluid (CSF) sample from the subject and analyzing the level of a gelsolin in the cerebrospinal fluid (CSF) sample. In another embodiment, the expression of a gelsolin is compared to a standard. In another embodiment, the standard is taken from an apparently healthy subject or pool of subjects. In another embodiment, the standard is the expression profile in a subject or pool of subjects correctly diagnosed as having a neurological disorder. In another embodiment, the standard is taken from the mean of the pool of subjects. In another embodiment, the standard is taken from the average of the pool of subjects. In another embodiment, the standard is taken from the median of the pool of subjects. In another embodiment, if the level of gelsolin is different than the standard by more than a predetermined threshold, the progression of a neurological disorder in a subject is determined.

[00085] In one embodiment, provided herein is a kit for diagnosing or providing prognosis for a subject developing a neurodegenerative disorder, comprising equipment, reagents, standards and instructions for analyzing the expression level of a gelsolin in a cerebrospinal fluid (CSF) sample of the subject.

[00086] In one embodiment, measuring levels of gelsolin in CSF is used to diagnose a neurological disorder. In another embodiment, measuring levels of gelsolin in CSF is used to treat a neurological disorder. In another embodiment, measuring levels of gelsolin in CSF is used to determine treatment for a neurological

disorder. In another embodiment, measuring levels of gelsolin in CSF is used to determine the prognosis of a neurological disorder.

[00087] In one embodiment levels of gelsolin drop in CSF or plasma drop when inflammation or sepsis occurs. In another embodiment the levels of gelsolin drop in CSF or plasma drop when a subject is afflicted with a neurological disorder.

[00088] In another embodiment, provided herein is a method of preventing LPS-mediated cell adhesion comprising the step of contacting the cells of a subject with a gelsolin. In another embodiment, provided herein is a method of preventing neutrophil activation comprising the step of contacting the cells of a subject with a gelsolin. In other embodiments, provided herein is a method of preventing LPS/LTA-induced release of IL-8 from human neutrophils and inhibiting IL-8 secretion after addition of bacterial wall components to human neutrophils the method comprising the step of contacting neutrophils with a gelsolin.

[00089] In one embodiment, lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria. Reported local tissue concentrations of LTA can be as high as 26 μg/ml, which may be associated with the fact that 10 ⁷ gram-positive bacteria contain as much as 1 μg of LTA. The primary transmembrane proteins that are activated by proinflammatory bacterial moieties such as LPS and LTA belong to the TLR family. Delivery of bacterial molecules from external fluids to the cell membrane and ultimately to TLR2 (dominant receptor for LTA) is complex and involves a number of other factors such as sCD14, LPS- binding protein (LBP), MD2, and moesin. One important factor that determines the toxicity of bacterial products to host cells is the geometry of their aggregation state. LTA obtained from different bacteria species induce the release of inflammatory cytokines such as TNF-α, IL-lβ, IL-6, and IL-8. All TLR signaling pathways elicit MyD88- or TRIF (TIR domain-containing adaptor-inducing IFN-β)-dependent activation of the transcription factor NF-KB. Those signaling cascades involve recruitment of different proteins such as IL-lR-associated kinase (IRAK), TNFR- associated factor 6 (TRAF6), TGF-β-activated kinase-1 (TAKl), IKK complex, and MAPK. In RAW 264.7 macrophages, LTA was also found to activate the PI3K/ AKT pathway and p38 MAPK-kinase, which in turn initiates NF-KB activation. TLR2 activation functions as a serum- independent factor, indicating significant differences between LTA- and LPS-mediated host cell activation. LTA is also a potent pathogenicity factor that causes cardiac dysfunction in gram- positive sepsis, may cause neuronal death, and determines clinical outcome in patients with pneumococcal meningitis.

[00090] In one embodiment, the presence of gelsolin in CSF combined with the observed decrease in chronic immune-inflammatory diseases such as multiple sclerosis and the ability of LTA to induce neuronal cell death and microglial cell activation suggest the potential for gelsolin' s involvement in modulating LTA/LPS interaction with its ligands in the CNS. In one embodiment, gelsolin-dependent severing activity of CSF is decreased by LTA or LPS .

[00091] In one embodiment, the present invention demonstrates that extracellular gelsolin binds LTA from different gram-positive bacteria strains. In one embodiment, the result of this binding is the inhibition of gelsolin's F-actin depolymerizing activity and compromised ability of LTA to activate endothelial cells, as measured by E-selectin expression, activation of the transcription factor NF-KB, and neutrophil adhesion. In another embodiment, gelsolin was also found to inhibit the release of IL-8 from human neutrophils subjected to LTA, LPS, and heat-inactivated bacteria treatment.

[00092] In one embodiment, provided herein is a method of compromising the ability of LTA to activate endothelial cells comprising the step of contacting a cell with a gelsolin. hi one embodiment, provided herein is a method of preventing NF-KB translocation to the nucleus of a cell comprising the step of contacting a cell with a gelsolin. In another embodiment, provided herein is a method of inhibiting E- selectin expression in a cell comprising the step of contacting a cell with a gelsolin. In another embodiment, provided herein is a method of decreasing neutrophil adhesion comprising the step of contacting a cell with a gelsolin. In another embodiment, provided herein is a method of decreasing IL-8 release from neutrophils comprising the step of contacting a cell with a gelsolin. In one embodiment, the effects of gelsolin are via inhibition of LTA.

[00093] In another embodiment, the present invention provides a method for blocking, reducing, ameliorating or preventing bacterial LTA -induced pathogenesis in a subject resulting from an accumulation of excess bacterial LTA triggered thereby, whrerein the method comprises administering under conditions suitable for gelsolin binding, a therapeutically effective amount of gelsolin, or functionally equivalent peptide fragment thereof, that is sufficient to block, reduce, ameliorate or prevent said pathogenesis.

[00094] hi another embodiment, the present invention provides a method for treating, inhibiting, or suppressing a bacterial LTA-induced pathogenesis in a subject resulting from an accumulation of excess bacterial LTA, comprising contacting a cell of said subject with a therapeutically effective amount of gelsolin, or functionally equivalent peptide fragment thereof.

[00095] In another embodiment, the present invention provides a method for treating a subject having or at risk of developing a gram-positive bacterial infection comprising administering a therapeutically effective dose of gelsolin to said subject.

[00096] In another embodiment, the present invention provides a method of treating, suppressing, or inhibiting the effects of lipoteichoic acid (LTA) in a subject comprising administering a therapeutically effective dose of gelsolin to said subject.

[00097] It is a further object to provide methods for decreasing the concentration of gram-positive bacterial LTA in blood or extracellular fluid of a patient, thereby preventing, neutralizing or reducing LTA-induced septic shock in a patient in vitro or in vivo, wherein the patient is subject to or susceptible to gram positive bacterial infection, said method comprising administering a therapeutically effective amount of gelsolin, or functionally equivalent peptide fragment thereof, to decrease the concentration of bacterial LTA and protect the patient from LTA-induced sepsis. Consequently, the method further blocks, reduces or ameliorates bacterial LTA-induced disruption of mammalian cellular responses or formation of toxic structures in vitro or in vivo. It also blocks, reduces or ameliorates the inhibition of fibrinolysis by excess, extracellular free actin in blood or extracellular fluid of a patient in vitro or in vivo. In addition it restores or maintains normal aggregation of platelets in a patient, wherein the patient is subject to or susceptible to LTA-induced generalized coagulation dysfunction.

[00098] It is an additional object that each embodied method be useful in vitro or in the actual gelsolin replacement therapy of a patient.

[00099] When such methods are used in vivo, it is a further object that the method effectively inhibits, ameliorates or prevents secondary tissue injury in the patient resulting from an accumulation of excess bacterial LTA, said method comprising administering a therapeutically effective amount of gelsolin, or functionally equivalent peptide fragment thereof. Moreover, the methods are effective even when the secondary tissue injury in the patient is remote from the site of primary infection or trauma.

[000100] It is yet another object to provide method for predicting adverse clinical outcome associated with massive inflammation in a patient susceptible to inflammatory shock or LTA-induced sepsis, said method comprising measuring the circulating gelsolin concentration in the patient, wherein a decrease of normal,

pre-trauma or pre-infection gelsolin levels predicts such adverse outcome and predicts a need for gelsolin therapy. Methods for producing gelsolin or active fragment thereof for use in the methods of the present invention are well known in the art.

[000101] In one embodiment, LTA-induced pathogeneis is induced arthritis, nephritis, uveitis, encephalomyelitis, meningeal inflammation, periodontal lesions, or triggered cascades resulting in septic shock and/or multiorgan failure.

[000 ' 02] In another embodiment, said composition further comprises a pharmaceutically acceptable carrier. In another embodiment, said composition further comprises a protease inhibitor cocktail. In another embodiment, said composition further comprises diluents. In another embodiment, said composition further comprises a second agent for use in the methods provided herein, wherein said agent is co-administered or administered prior to or after administering gelsolin.

[000103] In one embodiment, the term "second agent" include but are not limited to Alclofenac, Alclometasone Dipropionate, Algestone Acetonide, Alpha Amylase, Amcinafal, Amcinafide, Amfenac Sodium, Amiprilose Hydrochloride, Anakinra, Anirolac, Anitrazafen, Apazone, Balsalazide Disodium, Bendazac, Benoxaprofen, Benzydamine Hydrochloride, Bromelains, Broperamole, Budesonide, Carprofen, Cicloprofen, Cintazone, Cliprofen, Clobetasol Propionate, Clobetasone Butyrate, Clopirac, Cloticasone Propionate, Cormethasone Acetate, Cortodoxone, Cyclooxygenase-2 (COX-2) inhibitor, Deflazacort, Desonide, Desoximetasone, Dexamethasone Dipropionate, Diclofenac Potassium, Diclofenac Sodium, Diflorasone Diacetate, Diflumidone Sodium, Diflunisal, Difluprednate, Diftalone, Dimethyl Sulfoxide, Drocinonide, Endrysone, Enlimomab, Enolicam Sodium, Epirizole, Etodolac, Etofenamate, Felbinac, Fenamole, Fenbufen, Fenclofenac, Fenclorac, Fendosal, Fenpipalone, Fentiazac, Flazalone, Fluazacort, Flufenamic Acid, Flumizole, Flunisolide Acetate, Flunixin, Flunixin Meglumine, Fluocortin Butyl, Fluorometholone Acetate, Fluquazone, Flurbiprofen, Fluretofen, Fluticasone Propionate, Furaprofen, Furobufen, Halcinonide, Halobetasol Propionate, Halopredone Acetate, Ibufenac, Ibuprofen, Ibuprofen Aluminum, Ibuprofen Piconol, Ilonidap, Indomethacin, Indomethacin Sodium, Indoprofen, Indoxole, Intrazole, Isoflupredone Acetate, Isoxepac, Isoxicam, Ketoprofen, Lofemizole Hydrochloride, Lornoxicam, Loteprednol Etabonate, Meclofenamate Sodium, Meclofenamic Acid, Meclorisone Dibutyrate, Mefenamic Acid, Mesalamine, Meseclazone, Methylprednisolone Suleptanate, Morniflumate, Nabumetone, Naproxen, Naproxen Sodium, Naproxol, Nimazone, Olsalazine Sodium, Orgotein, Orpanoxin, Oxaprozin, Oxyphenbutazone, Paranyline Hydrochloride, Pentosan Polysulfate Sodium, Phenbutazone Sodium

Glycerate, Pirfenidone, Piroxicam, Piroxicam Cinnamate, Piroxicam Olamine, Pirprofen, Prednazate, Prifelone, Prodolic Acid, Proquazone, Proxazole, Proxazole Citrate, Rimexolone, Romazarit, Salcolex, Salnacedin, Salsalate, Sanguinarium Chloride, Seclazone, Sermetacin, Sudoxicam, Sulindac, Suprofen, Talmetacin, Talniflumate, Talosalate, Tebufelone, Tenidap, Tenidap Sodium, Tenoxicam, Tesicam, Tesimide, Tetrydamine, Tiopinac, Tixocortol Pivalate, Tolmetin, Tolmetin Sodium, Triclonide, Triflumidate, Zidometacin, cyclooxygenase-2 (COX-2) inhibitors or Zomepirac Sodium.

[000104] In some embodiments, gelsolin has no effect on either the growth of gram-positive and gram- negative bacterial strains or on the antibacterial activity of antimicrobial agents known in the art, such as but not limited to, cathelicidin-derived LL37.

[000105] In another embodiment as used herein an "apparently healthy subject" is a subject who has no signs and/or symptoms of a disease. In one embodiment, the composition further comprises a carrier, excipient, lubricant, flow aid, processing aid or diluent, wherein said carrier, excipient, lubricant, flow aid, processing aid or diluent is a gum, starch, a sugar, a cellulosic material, an acrylate, calcium carbonate, magnesium oxide, talc, lactose monohydrate, magnesium stearate, colloidal silicone dioxide or mixtures thereof. In another embodiment, the compositions described herein are specifically formulated to be deliverd intraspinally, or to be effective in the cerebrospinal fluid.

[000106] In one embodiment, the term "cerebrospinal fluid" or "CSF" comprises whole cerebrospinal fluid or derivatives or fractions thereof well known to those of skill in the art. In another embodiment, the term "cerebrospinal fluid" refers to the fluid, secreted by the choroid plexuses of the ventricles of the brain, that fills the ventricles and the subarachnoid cavities of the brain and spinal cord. Thus a cerebrospinal fluid sample can include various fractionated forms of cerebrospinal fluid or can include various diluents as may be added to facilitate storage or processing in a particular assay. Such diluents are well known to those of skill in the art and include various buffers, preservatives and the like that become a part of the compositions described herein.

[000107] In another embodiment, the composition further comprises a binder, a disintegrant, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetner, a film forming agent, or any combination thereof.3

[000108] In one embodiment, the composition is a particulate composition coated with a polymer (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral. In one embodiment the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intraspinally, subcutaneously, intraperitonealy, intraventricularly, or intracranially.

[000109] In one embodiment, the compositions of this invention may be in the form of a pellet, a tablet, a capsule, a solution, a suspension, a dispersion, an emulsion, an elixir, a gel, an ointment, a cream, or a suppository.

[000110] In another embodiment, the composition is in a form suitable for oral, intravenous, intraaorterial, intraspinal, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, or topical administration. In one embodiment the composition is a controlled release composition. In another embodiment, the composition is an immediate release composition, hi one embodiment, the composition is a liquid dosage form. In another embodiment, the composition is a solid dosage form.

[000111] The compounds utilized in the methods and compositions of the present invention may be present in the form of free bases in one embodiment or pharmaceutically acceptable acid addition salts thereof in another embodiment. In one embodiment, the term "pharmaceutically- acceptable salts" embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically- acceptable acid addition salts of compounds described herein are prepared in another embodiment, from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylamino sulfonic, stearic, algenic, b- hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable pharmaceutically-acceptable base

addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding compound by reacting, in another embodiment, the appropriate acid or base with the compound.

[000112] In one embodiment, the term "pharmaceutically acceptable carriers" refers to 0.01-0. IM and preferably 0.05M phosphate buffer, or in another embodiment 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be in another embodiment 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.

[000113] In one embodiment, the compounds of this invention may include compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987). Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity may be achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound.

[000114] The pharmaceutical preparations of the invention can be prepared by known dissolving, mixing, granulating, or tablet-forming processes. For oral administration, the active ingredients, or their physiologically tolerated derivatives in another embodiment, such as salts, esters, N-oxides, and the like are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions. Examples of suitable inert vehicles are conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders such as acacia,

cornstarch, gelatin, with disintegrating agents such as cornstarch, potato starch, alginic acid, or with a lubricant such as stearic acid or magnesium stearate.

[000115] Examples of suitable oily vehicles or solvents are vegetable or animal oils such as sunflower oil or fish-liver oil. Preparations can be effected both as dry and as wet granules. For parenteral administration (subcutaneous, intravenous, intraarterial, or intramuscular injection), the active ingredients or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are converted into a solution, suspension, or emulsion, if desired with the substances customary and suitable for this purpose, for example, solubilizers or other auxiliaries. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

[000116] In addition, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient. An active component can be formulated into the composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[000117] In one embodiment, the term "administering" or "contacting" refers to bringing a subject in contact with the compositions provided herein. For example, in one embodiment, the compositions provided herein are suitable for oral, cerebrospinal, intracranial or epidural administration. Other examples of administering may include parenterally, paracancerally, transmuco sally, transdermally, intramuscularly, intravenously, intradermally, intraspinally, subcutaneously, intraperitonealy, intraventricularly, or intracranially. In another embodiment, bringing the subject in contact with the composition comprises ingesting the composition. A person skilled in the art would readily recognize that the methods of bringing the subject in contact with the compositions provided herein, will depend on many variables such as, without any intention to limit the

modes of administration; the cardiovascular disorder treated, age, pre-existing conditions, other agents administered to the subject, the severity of symptoms, location of the affected area and the like, hi one embodiment, provided herein are embodiments of methods for administering the compounds of the present invention to a subject, through any appropriate route, as will be appreciated by one skilled in the art.

[000118] Alternatively, targeting therapies may be used in another embodiment, to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable in one embodiment, for a variety of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.

[000119] The compositions of the present invention are formulated in one embodiment for oral delivery, wherein the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. In addition, the active compounds may be incorporated into sustained-release, pulsed release, controlled release or postponed release preparations and formulations.

[000120] Controlled or sustained release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g. poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors.

[000121] In one embodiment, the composition can be delivered in a controlled release system. For example, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment, polymeric materials can be used. In another embodiment, a controlled release system can be placed in proximity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990).

[000122] Such compositions are in one embodiment liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCL, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention incorporate particulate forms, protective coatings, protease inhibitors, or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, and oral.

[000123] In another embodiment, the compositions of this invention comprise one or more, pharmaceutically acceptable carrier materials. In one embodiment, the carriers for use within such compositions are biocompatible, and in another embodiment, biodegradable. In other embodiments, the formulation may provide a relatively constant level of release of one active component, hi other embodiments, however, a more rapid rate of release immediately upon administration may be desired. In other embodiments, release of active compounds may be event-triggered. The events triggering the release of the active compounds may be the same in one embodiment, or different in another embodiment. Events

triggering the release of the active components may be exposure to moisture in one embodiment, lower pH in another embodiment, or temperature threshold in another embodiment. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative postponed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as phospholipids. The amount of active compound contained in one embodiment, within a sustained release formulation depends upon the site of administration, the rate and expected duration of release and the nature of the condition to be treated suppressed or inhibited.

[000124] In another embodiment, recombinant human gelsolin (rhGSN) is produced in E. coli. In another embodiment, rhGSN produced in E. coli differs from natural human plasma gelsolin by a disulfide bond that is present in the natural protein. In another embodiment, rhGSN is properly oxidized after purification. In another embodiment rhGSN' s structure is indistinguishable from purified human plasma gelsolin. In another embodiment rhGSN' s function is indistinguishable from purified human plasma gelsolin.

[000125] In another embodiment, lysophosphatidic acid (LPA) and PIP2 (phosphatidylinositol-4,5- bisphosphate) binding to gelsolin is strongest when the lipid is in micelles or putative lipid clusters within bilayer vesicles.

[000126] In another embodiment, natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as phenylglycine, TIC, naphthylelanine (NoI), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. In another embodiment, the peptides of the present invention may also include another or more modified amino acids or another or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).

[000127] In another embodiment, the term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phospho threonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids.

[000128] The term "treatment" or "treating" is intended to include prophylaxis, amelioration, prevention or cure of infections. "Treating" or "treatment" embraces in another embodiment, the amelioration of an existing condition. The skilled artisan would understand that treatment does not necessarily result in the complete absence or removal of symptoms. Treatment also embraces palliative effects: that is, those that reduce the likelihood of a subsequent medical condition. The alleviation of a condition that results in a more serious condition is encompassed by this term.

[000129] The term "about" as used herein means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.

[000130] The term "subject" refers in another embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans. The term "subject" does not exclude an individual that is normal in all respects.

[000131] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES Materials and Methods:

Materials

[000132] (L4015), and Bacillus subtilis (L3265) were purchased from Sigma-Aldrich. According to the manufacturer's quality control, the preparation of LTA contained <1 ng of LPS/ 1 mg of LTA, and therefore LPS would contribute <10 pg/ml culture medium in the highest LTA concentration used. Purified LTA from S. aureus was prepared as described previously. To calculate molar concentrations of LPS and LTA, the lowest range of their reported molecular mass in buffer without divalent cations (stock solution was made in H ₂ O) was used. Recombinant human plasma gelsolin (rhGSN) was obtained from Biogen Idee. Solution of human albumin was from Baxter Healthcare. Human aortic endothelial cells (HAECs) were obtained from BioWhittaker. ELISA kit for IL- 8 determination was from BioLegend. Heat-inactivated P. aeruginosa (PAOl) and B. subtilis (American Type Culture Collection (ATCC) 6051) were obtained by autoclaving their suspension (10 CFU/ml) in PBS for 1 h at ~120°C. Monomeric G-actin was prepared from an acetone

powder of rabbit skeletal muscle. Recombinant human plasma gelsolin (rhGSN) was obtained from Biogen- Idec, Inc (Cambridge, MA, USA). Mouse monoclonal anti-human gelsolin (G4896), anti α-muscle actin (A2547) and anti β-actin (A5451) antibodies were from Sigma (St Louis, MO, USA). Rabbit polyclonal antibody specific for human plasma gelsolin was a kind gift from Dr. Po-Shun Lee (Harvard Medical School, Boston, MA, USA).

[000133] Patients and samples preparation. CSF and blood were obtained from individuals admitted to the Department of Neurology at the Medical University of Bialystok and undergoing lumbar puncture for diagnostic purposes. Samples of CSF and anticoagulated blood were centrifuged (2000 x g, 20 min), and the supernatants were subjected to total protein analysis and frozen. The study was approved by the Medical University of Bialystok Ethics Committee for Research on Humans and Animals and written consent was obtained from all subjects. The degree of neurodegenerative impairment in patients diagnosed with MS, from whom CSF was obtained, was evaluated using the EDSS scale. All evaluations rated between 1-2 points, which indicates the early stages of MS. All MS patients suffered from the relapsing-remitting form of the disease and were not treated with any disease-modifying drugs (β-interferon, Natalizumab) or glucocorticosteroids at the time of lumber puncture. All CSF samples were clear, with the average number of lymphocytes equal to 8, and average Cl ^" and glucose concentrations of 135 mEq and 57 mg %, respectively.

[000134] Immunoblotting analysis. After being thawed, gel sample buffer was added to CSF and plasma samples that were then boiled and subjected to electrophoresis on 10% polyacrylamide gels in the presence of SDS. Recombinant human plasma gelsolin (rhpGSN) was loaded as a standard in each gel in a concentration range comparable to the gelsolin concentration in the samples. After electrophoresis, proteins were transferred to PVDF membranes (Amersham, Biosciences Little Chalfont, UK), which were blocked by incubation in 5% (w/v) non-fat dry milk dissolved in TBS-T (150 mM NaCl, 50 mM TRIS, 0.05% Tween 20, pH = 7.4). Following transfer, proteins were probed with a monoclonal anti-human gelsolin antibody that reacts with both plasma and cytoplasmic gelsolin or a specific anti-human plasma gelsolin antibody that reacts only with the plasma form of gelsolin. Both antibodies were used at 1:10000 dilution in TBS-T. HRP-conjugated secondary antibodies were used at 1:20000 dilution in TBS-T. Immunoblots were developed with the Fuji Film LAS-300 system using an ECL Plus HRP-targeted chemiluminescent substrate (Amersham, Biosciences Little Chalfont, UK). Densitometry analysis was performed using Image Gauge (version 4.22) software (Fuji Photo Film Co, USA). Proteins transferred to PVDF membrane were also tested with anti-α-actin (1: 1000) and anti-β-actin (1:10000) antibodies. The limits of actin detection (50 ng

and 3 ng with anti-α-actin and anti-β-actin, respectively) were established with purified rabbit muscle actin and lysed purified human platelets assuming that actin constituted 20% of total platelet protein. With at least 10 μg of total protein applied for each lane of SDS electrophoresis gels, α and β-actin were not present at a detectable level (Figure 1).

[000135] G-actin preparation and nucleation activity of gelsolin. Monomeric G-actin was prepared from acetone powder of rabbit skeletal muscle and labeled with pyrene-iodoacetamide according to previously published methods. Nucleation activity was measured in 4 μM G-actin samples (containing 40% pyrene- labeled G-actin) after addition of gelsolin (standard) or CSF (50-100 μl). In this system G-actin polymerization was induced by adding 150 mM KCl and 2 mM MgCl ₂ and the fluorescence intensity change was monitored for 5 minutes as described previously. Nucleation activity was calculated from the initial slope of the fluorescence increase.

[000136] Interaction of LPS and LTA with B-QRLF QVKGRR (PBPlO) and Gelsolin. Fifteen minutes after addition of various concentrations of to To determine potential effects of LTA and LPS, PIP ₂ , LPA and PS on the structure of gelsolin, OD at 280 nm was measured in solutions containing varying concentrations of LTA (from S. aureus) and Malp-2 added to 0.1 mg/ml of rhGSN in PBS. OD was also measured with PIP2 and phosphatidylcholine as positive and negative controls, respectively. A decrease in tyrosine and tryptophan fluorescence, due to decreased absorbance, has been previously documented as an assay for PIP2 binding to gelsolin. The fluorescence of rhodamine B-QRLFQVKGRR (PBPlO) or rhodamine B (RhB)-QRL (λex 565 nm, λem 590 nm) was measured 15 min after addition of various concentrations of PIP2 or LTA from different bacterial strains to 2 μM peptide solutions (PBPlO or RhB- QRL) in buffer A (10 mM Tris, 10 mM MES (pH 7.0)). The expectation was that if peptides bound to lipids, their surface concentration would become much higher than their bulk concentration, thereby resulting in either changes of rhodamine B fluorescence or formation of pyrene-excimer with a shift of fluorescence emission.

[000137] Cell culture. Rat primary astrocytes were obtained from prenatal rats and maintained for 14 days in culture before use. Cells were grown in Neurobasal medium supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 50 μg/ml steptomycin and 50 u/ml penicillin. In all experiments, the medium was changed to serum-free medium 6-12h prior to LPS, TNF-α, or gelsolin addition. Astrocyte cultures were incubated for 10 min in medium containing 10 μg/ml LPS, 10 μg/ml of LTA alone or LPS that had been pre-incubated with 0.16 mg/ml of human gelsolin. Cultures were fixed with ice-cold methanol and stained

with FITC labeled phalloidin. NF-κB translocation was manipulated by a 2 hour incubation in serum-free medium containing either 10ng/ml TNF-α, 10 μg/ml LPS alone or LPS that had been preincubated with 0.16 mg/ml human gelsolin. Location of NF-κB was observed using a monoclonal antibody to NF-κB and cell nuclei were detected by counterstaining with 4',6-diamidino-2-phenylindole dihydrochloride.

[000138] F-actin preparation and severing and nucleation activity of gelsolin. The nonpolymerizing solution contained 2mM TRIS, 0.2 mM CaCl ₂ , 0.5 mM ATP, 0.2 mM DTT, pH 7.4. Actin was polymerized by adding 150 mM KCl and 2 mM MgC^ to G-actin solutions and incubating for 1 hr at room temperature (RT). Recombinant human gelsolin (rhGLS) and blood serum severing activity was measured in 0.4 μM Pyrene labeled F-actin samples after adding gelsolin/serum or their combination with either LPS, PIP _2, LPA, Lipid A or PS. The fluorescence intensity of F-pyrene actin was monitored for 10 minutes, and the calculation of severing activity based fluorescence decrease. Gelsolin nucleation activity was measured in 4 μM G-actin samples after addition of gelsolin (standard) or CSF (50-100 μl). In this system, G-actin polymerization was induced by adding 150 mM KCl and 2 mM MgCl ₂ , and the fluorescence intensity change was monitored for 5 minutes. Nucleation activity was calculated from the initial slope of the fluorescence increase.

[000139] Adhesion of neutrophils to LTA-activated HAEC. To determine if gelsolin is able to affect adhesivity on neutrophils to LTA stimulated HAECs, the adhesion of calcein-AM labeled neutrophils to confluent cultures of HAEC treated with LTA without or with 2 μM gelsolin was measured.

[000140] Microscopy. Neutrophils treated with E. coli LPS (100 ng/ml) or S. aureus LTA (5 μg/ml) with or without 2 μM gelsolin after a 2 h incubation were viewed using a Leica microscope with a X40 objective.

[000141] Determination of IL-8 concentration in the cell supernatant. Neutrophils (5 x 10 ⁶ cells/ml) suspended in RPMI 1640 buffer containing 2% human albumin were activated with highly purified LTA from S. aureus (0.1-10 μg/ml), LPS from E. coli (10 ng/ml), conventionally purified LTA from S. aureus (5 μg/ml), or dilutions of autoclaved bacterial suspension (1 μl/ml) with or without rhGSN (0.5-4 μM) or LBP peptide (10 μM). Cell-free neutrophil supernatants were collected by centrifugation at 5000 μ g for 5 min and stored at -8O ⁰ C until cytokine determination. IL-8 was measured using a sandwich ELISA, according to the manufacturer's instructions. The detection limit was 30 pg/ml.

[000142] Antimicrobial activity. To test the hypothesis that binding of gelsolin to the bacterial wall components LPS or LTA will prevent LL37 membrane insertion, we evaluated LL37 antibacterial activity

in the presence of rhGSN. The bactericidal activities of the LL37 peptide against gram-negative kanamycin- resistant P. aeruginosa (PAOl) and gram-positive B. subtilis (ATCC 6051) was measured. Bacteria were grown to mid- log phase at 37 ⁰ C (controlled by the evaluation of OD at 600 nm) and resuspended in PBS. The bacteria suspensions were then diluted in 100 μl of solutions containing antibacterial agents by themselves or with 2 μM rhGSN. After a 1-h incubation at 37 ⁰ C, the suspensions were placed on ice and diluted 10- to 1000-fold. Then, 10 μl aliquots of each dilution were spotted on P. aeruginosa isolation agar or Luria-Bertani agar plates for overnight culture at 37 ⁰ C. The number of colonies at each dilution was counted the following morning. The CFU per milliliter of the individual samples were determined using the dilution factor.

[000143] Evaluation of minimal inhibitory concentration (MIC.) The MIC of the LL37 peptide was determined by a microbroth dilution method with Mueller-Hinton broth (MH) or MH supplemented with 2 mM MgC12 with or without the addition of rhGSN. A series of 2-fold dilutions of LL37 in 0.25x MH broth were prepared from a stock solution and placed in 96-well plates to which dilutions of B. subtilis bacteria were then added. After incubation for 18 h at 37°C, the bacterial concentration was measured as the OD at 595 nm, and the MIC was read as the lowest concentration resulting in inhibition of detectable bacterial growth.

[000144] Evaluation of gelsolin effect on LPS and LTA aggregation state by dynamic light scattering

(DLS). LPS and LTA molecules are amphipathic and form aggregates of varying sizes. These aggregates can be evaluated using DLS spectroscopy. In the absence of surface- active agents and divalent cations, LPS and LTA self-assemble into micellar structures. The LPS or LTA aggregate size (hydrodynamic diameter) was determined using a DynaPro 99 DLS instrument. The method measures the diffusion constant of the aggregates from the autocorrelation function of scattered light intensity. The diameter is calculated from the relation D= kT/6πηRh, where D is the translational diffusion constant, η is the solvent viscosity, k is Boltzman's constant, and Rh is the hydrodynamic radius. To determine whether gelsolin affects the aggregation state of LPS and LTA, solutions of bacterial wall products were evaluated before and after 20 min of incubation with either gelsolin or BSA (62 μM of each).

[000145] Statistical analysis. Data are reported as means +/- SD from three to six experiments. Differences between means were evaluated using the unpaired Student's t test, with p < 0.05 being taken as the level of significance.

RESULTS

EXAMPLE 1: GELSQLIN CONCENTRATION IN CSF AND BLOOD IN HEALTY SUBJECTS AND SUBJECTS AFFLICTED WITH A NEUROLOGICAL DISORDER

[000146] CSF protein composition and concentration were examined to identify individual proteins or protein combinations that can be used as diagnostic markers. Using an antibody that recognized recombinant human gelsolin (rhGSN), the presence of gelsolin was detected in saliva, bile, blood and CSF (Fig. 16).

[000147] To evaluate gelsolin concentration in CSF, quantitavite immunoblotting, where gelsolin was identified and quantitified in various samples using anti-gelsolin and anti-human plasma gelsolin antibody (Fig. IA-C) and a functional assay of gelsolin' s ability to accelerate actin polarization in vitro (Fig. IE) were carried out. Using these tools, the concentration of gelsolin was determined in a limited number of CSF samples (n=25), where the range of gelsolin concentration was 1.29-15.9 ug/ml and 0.61-9.97 ug/ml (Fig. 2 and Table 1). Using quantitative immunoblotting, the average gelsolin concentration in CSF samples obtained from patients suffering from idiopathic cephalgia, ischialgia due to discopathy, or idiopathic (Bell's) facial nerve palsy or entrapment radial neuropathy (conditions that do not affect the results of conventional CSF analyses) was within the range of 7.2+4.3 μg/ml. However, CSF gelsolin concentration from three subjects diagnosed with ischialgia due to discopathy (13.1±2.9 μg/ml) was 2.5 times higher than that detected in patients diagnosed with idiopathic cephalgia (5.8+2.1 μg/ml) or idiopathic (Bell's) facial nerve palsy (4.9+1.2). Comparing all samples, the lowest gelsolin concentrations were observed in patients diagnosed with multiple sclerosis (2.1+0.7 μg/ml) or recovering from SAH (1.95+2.9 μg/ml). The low level of CSF gelsolin in patients with multiple sclerosis was accompanied by a depletion of gelsolin in plasma.

Table 1: Blood and CSF Gelsolin levels.

[000148] This study shows for the first time that the gelsolin levels in CSF can be measured using two relatively simple and cheap techniques: immunoblotting and fluorometric actin polymerization assays. The higher gelsolin: total protein ratio in CSF compared plasma suggests that, in addition to blood, CNS cells might be a significant source of CSF gelsolin. The differences between gelsolin concentrations obtained in the same samples using the two techniques might result from the fact that antibody recognition of gelsolin separated in denaturing/reducing conditions is unlikely to be affected by gelsolin interactions with other molecules, whereas gelsolin nucleation activity in unmodified CSF can be affected by the binding of gelsolin to lipid or protein mediators.

[000149] Using an antibody designed to recognize only plasma gelsolin based on its unique N-terminal epitope, we found that in CSF samples obtained from MS subjects, the vast majority of gelsolin is in its plasma isoform. The lack of detectable actin in the CSF samples indicates that they were not significantly contaminated by cytosolic proteins and that loss of gelsolin does not lead to significantly increased circulating actin in CSF even in conditions like MS where cellular destruction is significant.

[000150] Gelsolin concentration in CSF and plasma is measured in biological samples collected from a pool of healthy subjects and compared to a pool of subjects afflicted with a neurological disorder such as multiple sclerosis (MS), Spinocerebellar Ataxia Type 2 (SCA2), Parkinson's Disease (PD), Alzheimer's Disease (AD), Schizophrenia (SZ), Amyotrophic lateral sclerosis (ALS), or Huntington's Disease (HD). It is

found that gelsolin level decrease is more sensitive and specific for each particular neurological disorder when measured in CSF as compared to plasma.

EXAMPLE 2: CHARACTERIZATION OF GELSOLIN

[000151] The PPI regulatory site of gelsolin resides within a 20 residue linear sequence that connects the first and second folded domains of the protein where biochemical and mutational studies have implicated 10 strategically organized basic and hydrophobic amino acids (QRLFQ VKGRR, SEQ K) No:l, amino acids 160-169, in the 684-residue plasma gelsolin molecule that accommodate tight binding to the negatively- charged phosphomonoesters and hydrophobic acyl chains of anionic phospholipids. Further, synthetic peptides of this sequence, such as phosphoinositide-binding peptide 10 (PBPlO), have a PPI binding affinity similar to that of intact gelsolin (Figure 3).

[000152] The binding of LPS to gelsolin and its synthetic peptide PBPlO was determined. In this experiment, increasing concentrations of LPS were used to determine binding to gelsolin. The results demonstrated a decrease in optical density of gelsolin as a result of a change in gelsolin' s intrinsic fluorescence due to LPS binding (Fig. 4, left panel).

[000153] The severing activity of gelsolin in the presence of LPS, PIP2, LPA, Lipid A or PS was determined (Fig. 5). As evidenced by the decrease in fluorescence intensity of pyrene-labeled F- actin as a function of time, gelsolin's severing activity of F-actin (standard and serum, left and right hand panels, respectively) was inhibited in the presence of LPS. Likewise, Fig. 10 and Fig. 11 show a similar inhibition in the presence of LTA and Sphingosyl phosphoryl choline (SPC) (respectively).

[000154] Further, gelsolin inhibits the toxic effect of LPS on astrocytes, where addition of gelsolin prevented LPS-induced cell damage (Fig. 6 D-E). Moreover, the effects of LPS on NF-kB translocation were also prevented by the addition of gelsolin where LPS-induced nuclear translocation of NF-kB was prevented (Fig. 6 G-J) as can be seen when overlapped with panels K-N on Fig. 6 showing DAPI stained nuclei. Gelsolin also inhibits the binding of ¹²⁵ I- lipopolysacharide (LPS) binding protein (LBP) (10 ng) to LPS (1 ng)-coated wells (Fig. 7).

EXAMPLE 3: GELSOLIN INTERACTS WITH LTA

[000155] The effect of LTA on the fluorescence of the gelsolin-derived PBPlO peptide is shown in Fig. 8. Upon interaction of PBPlO with LPS, there is an initial decrease in fluorescence at low LPS/peptide ratios; as the amount of LPS increased, so did the level of peptide fluorescence, indicating insertion of the peptide- bound rhodamine B into a more hydrophobic environment. At the molar ratios tested, only the first stage of decreased fluorescence was seen with LTA or PIP2. LTA from different gram-positive bacteria, including purified LTA from S. aureus, had similar effects on PBPlO fluorescence. There was no significant fluorescence change after adding LTA to a control peptide with the sequence RhB-QRL (data not shown). Binding of LTA to intact gelsolin was evident from a change in UV absorbance shown in Fig. 8, right hand panel. Purified LTA decreases the absorbance of gelsolin. This result indicates that in addition to gelsolin' s ability to bind LPS from gram-negative bacteria, gelsolin is also able to interact with LTA, a gram-positive bacterial wall component.

EXAMPLE 4: EXPRESSION OF E-SELECTIN ON HAECs

[000156] We detected an increase in E-selectin expression on HAECs after activation with LTA. As shown in Fig. 9A, 2 μM gelsolin partially prevents LTA-mediated E-selectin expression on HAECs. Fluorescence quantification revealed that the average intensity of E-selectin markers was 180 +/- 75, 250 +/- 95, 355 +/- 180, and 260 +/- 120 for control, TNF-α, LTA, and LTA + gelsolin samples, respectively.

[000157] Similar to HUVECs and human lung microvascular endothelial cells, treatment of HAECs with LTA increases neutrophil adhesion to the cell surface. The adhesion of neutrophils to LTA (1-10 μg/ml)- treated HAECs for 8 h is shown in Fig. 9B and 9C. Recombinant plasma gelsolin effectively prevents LTA- induced activation of HAECs that translates to a decrease in neutrophil adhesion. Quantification of fluorescence from calcein-AM-labeled neutrophils documents a significant decrease in neutrophil adhesion to HAEC activated with 10 μg/ml LTA in the presence of 2 μM rhGSN.

EXAMPLE 5: EXPOSURE OF NEUTROPHILS TO LTA OR LPS RESULTS IN CHANGES IN

CELL MORPHOLOGY

[000158] Neutrophil activation is associated with marked changes in cellular morphology. Typically, shortly after activation by LPS or LTA, neutrophils adopt an elongated shape with a rough surface and form protrusions and aggregates (Fig. 12, upper panels). After 2 h of incubation with LPS or LTA in the presence of rhGSN, the morphological predictors of neutrophil activation were less pronounced and were limited to a

lower population of cells (Fig. 12, lower panels). This result suggests that gelsolin may modulate neutrophil activation by sequestering bacterial wall products and buffering their interaction with TLRs. [000159] Gelsolin also prevents LPS internalization in HAEC (Fig 13).

EXAMPLE 6: GELSOLIN PARTIALLY PREVENTS IL-8 RELEASE FROM LPS, LTA QR LYSED BACTERIA-ACTIVATED NEUTROPHILS

[000160] Unstimulated human neutrophils produce very low, but detectable, amounts of cytokines, and after activation they produce and release several cytokines, including IL-8, TNF-α, and G-CSF at levels 10-50 times higher compared with the resting state. Although IL-8 is produced by a variety of cell types, neutrophils are the major source of this proinflammatory cytokine. In the presence of LTA, time-dependent induction of IL-8 release was observed, with a maximum reached at 24 h (data not shown). IL-8 secretion induced by addition of purified LTA was also concentration dependent. The amount of IL-8 released after exposure to 10 ng/ml of LPS was comparable to that observed after neutrophil activation with 5-10 μg/ml of LTA or 1 μl/ml of heat-inactivated bacteria (Fig. 14). Neutrophils coincubated with LPS, purified and nonpurified LTA, or lysed bacteria in the presence of rhGSN released significantly lower amounts of IL-8. In the case of activation with purified LTA, we observed a partial but progressive decrease of released IL-8 when an increased concentration of rhGSN was added. Gelsolin may function as an inhibitor of LTA/LPS- induced IL-8 synthesis. The inhibition of IL-8 was also observed after LPS addition in the presence of LBP peptide (aa 86 -99) that binds lipid A and neutralizes LPS. Gelsolin addition to neutrophil samples 30 min after LPS or LTA treatment did not prevent IL-8 release to the same extent as when gelsolin was added together with the stimuli (data not shown). This result supports the hypothesis that gelsolin acts as an LTA/LPS buffer preventing their agonistic effect on TLRs, although gelsolin may also interfere with other mediators involved in the regulation of neutrophil synthesis or release of IL-8.

EXAMPLE 7: GELSOLIN DOES NOT INTERFERE WITH BACTERIAL KILLING BY LL37

[000161] rhGSN by itself had no effect on bacterial growth or the ability of the synthetic human cathelicidin-derived antibacterial peptide LL37 to kill B. subtilis or P. aeruginosa (Fig. 15A). The MIC value for the LL37 peptide with or without gelsolin (Fig. 15B) was unchanged. These data suggest that gelsolin is unable to interact with LTA molecules within the intact bacterial wall in the same way as takes place upon LTA release from dividing or dying bacteria.

EXAMPLE 8: GELSOLIN AFFECTS AGGREGATION STAGE OF LPS AND LTA MOLECULES

[000162] Amphiphilic molecules such as LPS, lipid A, and LTA form aggregates in aqueous environments above a critical micellar concentration. The actual structure of these aggregates is not a constant, but depends on concentration, solvent conditions, and the effects of other solutes that can co-assemble in the micelles. Accordingly, the average size of LPS and LTA molecules was observed to decrease as their solutions were diluted (Fig. 16A). Previous analyses of aqueous LPS suspensions by negative staining and platinum shadowing revealed the presence of small globular aggregates (diameter 20-80 nm) and short filaments. Upon the addition of serum proteins, mainly albumin, LPS aggregates become larger (200 nm) and form structures several micrometers in length. A decrease in LPS and LTA aggregate size (Fig. 16) in the presence of purified gelsolin, was observed. This effect is consistent with a gelsolin-specific interaction with LPS and LTA.

[000163] Extracellular gelsolin binds LTA from different gram-positive bacteria strains. In one embodiment, the result of this binding is the inhibition of gelsolin' s F-actin depolymerizing activity and compromised ability of LTA to activate endothelial cells, as measured by E-selectin expression, activation of the transcription factor NF-KB, and neutrophil adhesion. Gelsolin was also found to inhibit the release of IL-8 from human neutrophils subjected to LTA, LPS, and heat-inactivated bacteria treatment.