WO2011026081A1 - Chitin-binding proteins as biomarkers - Google Patents

Abstract

Biomarkers specific in the prognosis or diagnosis of different clinical stages of chronic inflammatory disease are identified by proteins associated with phagocytosed chitin microparticles (PAPC), Identification of these biomarkers provide for discovery of new therapies as well as monitoring of a therapy in a patient.

Description

ΗITIN-BINDING PROTEINS AS BM)MARKERS

[0001] This application claims priority to U.S. provisional application No.: 61/238,448 filed August 31, 200^c>, which is incorporated herein by reference in its entirety.

[0002] The invention relates to methods and kits for diagnosing disease states, determining prognoses of immunotherapy, and measuring responses to treatment involving measurement of chitiii binding protein profiles.

[0003] Upon activation and polarization in response to inflammatory stimuli including septic and non-septic components, tissue macrophages (Mø) act as effector cells carrying out a wide variety of pathophysiological and immunorcgulatory functions. Polarization is regulated by the dominant cytokine in a tissue-dependent manner (1, 2). M0 can acquire a "classically activated" phcnotypc (i.e. Ml Mø) with display of anti-bacterial, anti-angiogenic, and antiallergic functions or an '"alternatively-activated"' phenotype (i.e. M2 M0) displaying a pro- angiogenic and pro-allergic phenotype (3). M2 Mø activation, a common feature of the acquired immune response to allergens, helminth parasites and fungi (3), is frequently associated with eosinophil migration in response to Th2 -mediated production of chcmokincs and eicosanoids (4-6),

[0004] A clinically useful ThI adjuvant should stimulate innate immunity and promote acquired cell-mediated immune responses through development of Ml Mø that eliminate intracellular pathogens and down-regulate Th2 -mediated immunological diseases including allergic asthma. However, a satisfactory Th I adjuvant with these properties is not currently available. [0005] This Summary Is provided to present a summary of the invention to briefly indieate the nature and substance of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

Embodiments of the invention are directed to biomarkers which are specific in the prognosis or diagnosis of different clinical stages of chronic inflammatory disease, Methods for identification of these biomarkers comprises, in some embodiment, proteins associated with phagocytosed chitlii microparticlcs (PAPC). Identification of these biomarkers provide for discovery of new therapies as well as monitoring of a therapy in a patient.

10007 J In one embodiment, the PAPC identified markers comprise C-type lectins (mannose receptor [MR], DEC-205, Dectin-1 , SIGN-Rl , MRC2); toll-like receptors CTLR2. TLR4, CD14); phago lysosomal proteins (LAMP-I, LAMP-2); adhesion molecule /chemokine receptor (CD44, L-selcctin [CD62L], CDl 84 [CXCR4]): CHBLl chitolriosidasc (chitinasc-1), acidic mammalian chitinasε (AMCase), chitinase3-like-2 (CHI3L2), oviductal glycoprotein 1 or stabilin-1 interacting chitinasc-like protein (SI-CLP). In some aspects, variants, fragments, alleles and the like are included in the biomarkers,

[0008] Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009J Figure 1 is a graph showing chitin microparticle (CMP)-induced TNFα production by peritonea] macrophages (M0) from mice deficient in TL.R2, TL.R4, MyD88, or CD14.

Peritonea] M0 isolated from indicated knockout mice were stimulated for 4 hrs with CMP at 0 - 50 μg/ml. TNFα levels in the supernatants were measured by ELISA. Mean ± SD, n^:::4.

Figure 2 shows the cell-free TLR2 binding to CMP. To determine whether TLR2 directly bind to CMP in a cell-free condition, 100 μg/ml CMP or chitosan-M P suspended in 20 mM Tris buffer, pH 7.5, containing 10 niM CaCl;, 0.5% BSA and 1% NP40 were mixed with TL.R2 protein (recombinant TLR 2 Fc chimera) at 1 , 5 arid 10 μg/ml as well as saline as vehicle control at 0° for 30 min. Following washing CMP, CMP were stained with fluorochrome- conjugated TLR2 antibody. Results are representative of three independent experiments,

[0011] Figure 3 shows the detection of TLR2 as a PAPC (prolein-associatcd phagocytoscd

CMP). RAW 264.7 macrophages (M 0) were incubated with CMP for 5 rain or 15 rain at 37°C. As a comparison control, RAW cells were treated with cytochalasin D (CylD) at 1 μg/tnl CMP before CMP stimulation. Cellular CMP were recovered by lysis with NP-40, washed with calcium -containing saline, and fixed with 4% paraformaldehyde. Phagocytosed CMP were recovered by centrifugation (1,60Og, 5 min, 4⁰C), washed, fixed in paraformaldehyde, stained with fluorochrome-conjugated anti-TLR2. For FACS analysis, CMP-containing fractions were electronically gated at 1 - 10 μm and their fluorescent intensity was detected.

[0012] Figure 4 shows the kinetics of selected PAPC, MR (mannose receptor), CD62L,

LAMP-I and CHBLl, in RAW 264.7 M0. RAW 264.7 macrophages (M0) (IQ⁶ cells/ml) were incubated with 100 μg/ml of CMP for 5, 15 and 30 min at 37⁰C, then washed and lysεd with NP40. Phagocytosed CMP were recovered by cenlrifugation (1,60Og, 5 min, 4°C), washed, fixed in paraformaldehyde, and stained with fluorochrome-conjugaled antibodies. For FACS analysis, CMP-containing fractions were electronically gated at 1- 10 μm and their fluorescent intensities were detected. The result of CD62L at 15 rain was not shown.

|0013| Figures 5A, 5B show the cytokine-induced Ml and M2 M0 activation and CMP- associated TL.R2 in phagocytosis. RAW 264.7 Mø (5 x 10' cells/ml) were treated with IFNγ (10 ng/tnl), IL-4 (10 ng/ml) and IL- 13 ( 10 ng/ml). Figure 5 A: For TNF α production, each cytokine-treated Mø culture was washed with media and further incubated with 100 μg/ral CMP for 6 h at 37° C. TN Fa in the supernatants was measured by ELISA. Mean ± SD, n=^:4. Figure 5B: For the detection of CMP-associated TLR2 in phagocytosis, the cytokine-treated Mø were incubated with 100 μg/ml CMP for 15 min at 37⁰ C before isolation of CMP/PAPC. Recovered CMP was stained with fluorochrome-conjugated anti-TLR2 and analyzed cytomctrically.

[0014] Figures 6A-6C show results obtained with peritoneal Mø stimulated with CMP and

LCB. Figure 6A: RAW264.7 cells were incubated with 100 μg/ral CMP, LCB (large chit in beads, >50 μm diameter), or medium. After 24 h, proteins were extracted. CHI3L1, iNOS, COX-2 and β-actin levels were determined by Western blot as indicated in the Materials arid Methods. Figure 6B: C57BL/6 mice were given 1 mg LCB, 1 mg CMP, or saline intraperiioneally. One day later peritoneal lavage was performed, Mø were isolated and cellular proteins extracted, Arg T, CMBLl and β-aclin levels were determined by Western blot as indicated in the Materials and Methods, Figure 6C. C57BL/6 mice were given LCB, CMP or saline intraperitoneaily. One day later peritoneal cells were isolated, stained with fluorochrome- conjugatcd monoclonal antibodies listed and analyzed by flow cytometry. For FACS analysis, single peritoneal Mø were electronically gated by forward- and side-scatter, and further gated on F4/8(r ce!l fractions.

[0015] Figure 7 shows the PAPC profiles in M l and M2 M0 activation. Ml, M2 and normal peritoneal Mø were isolated from mice pretreated for 3 days with intraperitoneal CMP inducing an Ml response, LCB (>50 μm) inducing an M2 response, or saline, respectively. Ml and M2 M0 were confirmed by elevated expression of iNOS and Arg !, respectively, on Western blots. For cytometric analysis of PAPC profiles, these Mø ClO⁶ cells/ml) were incubated further with 100 μg/ral of CMP for 15 mm at 37°, then lysed with NP40 detergent. PAPC /CMP were recovered by cεntrifugation (1,60Og, 5 min, 4°C), washed, fixed in paraformaldehyde, stained with selected 14 fluorochrome-tagged antibodies and analyzed by flow cytometry. For FACS analysis, CMP-containing fractions were electronically gated by forward- and sideward-scatter, and further confirmed by analysis of CMP that were stained with FITC-bactcrial chitinasc.

[0016] Figures 8A-8D show chitin particle sizes. Figure 8A, CMP size distribution (red) determined by FACS. References are 1 (blue) and 10 (green) μm latex beads. Figure 8B. Light microscopy of a mixture of LCB, Sephadex G-100 (G-I OO) and 10 μm-iatex beads (10 LB). Figures 8C, 8D. Confocal microscopy of RAW264.7 Mø without phagocytosis of CMP (Figure 8C) or after in vitro phagocytosis of CMP (Figure 8D), stained with FITC-bacterial chitinase (green), Cy3-anti-CHI3Ll (red) antibody and DAPI (blue). Bar = 10 μm.

[0017] Figure 9 shows that intraperitoneal administration of chitin particles induces local εosinophilia in a size-dependent manner. Groups of C57BL/6 mice were given 1 mg LCB, 1 mg CMP, 1 mg Sephadex G-100 beads, 1 rng HK-BCG or saline inlraperitoneally. One day later peritoneal lavage was performed and eosinophils were counted. Mean ± SE, n=^:4. */?<().001 compared to the saline control group. Figures 1OA, 1OB show CRTH2-dependent eosinophil migration induced by non- phagocytosable particles, Figure 1 OA. Groups of Balb/c (WT) and CRTH2^"'^'" mice were given 1 mg LCB, Sephadex G-I OO, CMP or saline intraperitoneal iy. One day later peritonea! lavage was performed and eosinophils were counted. Mean ± SE, n=4, */K0.001 and /j<0.01 compared to the saline control and the corresponding WT group, respectively. Figure 1OB. Groups of C57BL/6 received 3 mg/kg ramatroban orally twice on days -1 and 0, 1 h before intraperitoneal administration of 1 rag LCB or saline. Mean ± SE, n^:::4. */?<().()! and '/;<(), 05 compared to the saline control and the corresponding saline/LCB group,

[0019] Figure 11 shows that intraperitoneal LCB enhances Arg I expression but not

CHOLl . Groups of WT (C57BL/6) and CRTHQ^"'^'" mice were given 1 mg LCB, 1 mg CMP, 1 mg Sephadex G-100 beads, 1 mg HK-BCG or saline intraperitonealiy. One day later peritoneal lavage was performed, M0 were isolated and their cellular proteins were extracted. Arg !, CH (3Ll and β-actin levels were determined by Western blot as indicated in the Materials and Methods. Results are representative of two separate experiments.

[0020] Figure 12 shows the selected surface antigens in peritonea! Mø from mice given intraperitoneal LCB or CMP. C57BL/6 mice were given LCB, CMP or saline intraperitoneal iy. One day later peritoneal cells were isolated, stained with fluorochromc-conjugated monoclonal antibodies listed and analyzed by flow cytometry. For FACS analysis, single peritoneal Mø were electronically gated by forward- and side-scatter, and further gated on F4/80⁺ cell fractions.

[0021 J Figure 13 shows that TN Fa, nitrite and IL-10 levels detected in peritoneal lavage fluid. Groups of C57BL/6 mice were given CMP, LCB, Sephadex G-100, HK-BCG or saline intraperitonealiy, One day later, peritoneal cavities were washed with 1 ml of saline. The supernatants were isolated from peritoneal cells by centrifugation. IL-10 and TN Fa levels in the supernatants were measured by ELISA and nitrite levels by the Griess reagent. Mean ± SE, n=^:4. */K0.01 compared to the cytokine levels of peritoneal lavage fluid isolated from mice given saline intraperitonealiy.

[0022] Figure 14 shows the priming effects of CMP administered intraperitonealiy on ex vivo IL-10 production elicited from peritoneal M0, Peritoneal M0 were isolated from C57BL/6 mice thai previously received intraperitoneal CMP, chitosan-MP, LCB or saline as control.

— D " Isolated peritoneal M0 were further stimulated with CpG or saline in vitro at 37⁰C for 24 h. Mean ± SD, n=3. *,p < 0.01, compared to cells isolated from saline control mice and stimulated with CpG. The data shown are representative of two independent experiments.

[0023] Figure 15 shows the results from CHBL 1 , iNOS and CGX-2 in RAW264.7 M0 exposed to LCB in vitro. RAW264.7 cells were incubated with 100 μg/ml LCB, CMP,

Sephadex G-100, or chitosan-MP and 50 μg/nil HK-BCG or HK-M tuberculosis (Mtb). After 24 h, proteins were extracted. CHDLl, iNGS, COX-2 and β-actin levels were determined by Western blot as indicated in the Materials and Methods. Results arc representative of two separate experiments.

[0024] Figure 16 shows the effects of the addition of CMP at selected time points on CpG- induced in vitro IL-10 production by RAW 264.7 cells. CMP were added to cultures of RAW 264.7 cells at the indicated time points; 0 h, CpG was added to the cultures. Culture supernatants were harvested at 24 h. As comparison controls, 100 μg/ml of chitosan-MP were added as for CMP, Saline alone, CMP alone and chitosan-MP alone all had less than 50 pg/ml IL-10. Mean ± SD, n=3. * p < 0,01, compared to cells stimulated by CpG alone.

10025 J Figure 17 shows the effect of cytochaiasin D on in vitro IL- 10 production by

RAW264.7 cells stimulated with CpG and CMP. RAW264.7 cells were incubated with 1 μg/ml CpG and/or 100 μg/ml CMP for 24 h. When indicated, groups of RAW cell cultures were also treated with 1 μg/ml cytochaiasin D. IL-10 levels in the culture supernatants were measured by ELISA. Mean ± SD, n^:::3. */?<().01 and ⁴p < 0.01, compared to cells stimulated CpG alone and Medium control with CpG stimulation, respectively

[0026] Figure 18 shows the effects of in vitro addition of CMP on the levels of CpG- induced IL-10 rnRNA. RAW cells were stimulated with CpG in the presence of 100 μg/ml CMP or chitosan-MP for 6 and 24 h, RNA extraction and RT-PCR were done as described in the Materials and Methods section. rnRNA levels of IL-10 and GAPDH were shown. The data shown are representative of three independent experiments. DBl AlLBD DESCRIPl SON

[0027] Embodiments described herein are dirceted Io biomarkers, methods and kits for measuring proteins associated with phagocytosed chilm raicroparticles (PAPC) and associating the measurcment(s) with clinical disease stages. Assays and diagnostic kits arc used to measure human CBP profiles as PAPC (proteins associated with phagocytosed CMP), for example in immunocompromised patients who are receiving immunotherapies as well as those who are not. In some embodiments, the PAPC profiles of immunocompromised patients receiving treatment (e.g., immunotherapy) are compared to the PAPC profiles of immunocompromised patients who are not reaching treatment. The methods and kits described herein can be used for determining a diagnosis or a prognosis of an immunocompromised individual (e.g., human patient) receiving immunotherapy (e.g., chitin microparticlc therapy), for example.

[0028] Further embodiments of the invention comprise are methods to identify and quantify selected PAPC including c hi tmases, calcium (C) -type lectins, toll-like receptor 2 (TLR2) and phagosomal antigens, which are involved during phagocytosis of chitin microparticles (CMP) and activation of macrophages (M0), By digesting M0 with a mild detergent for isolation of phagocytosed CMP, the selected PΛPC associated with CMP can be cytometrically measured. The PAPC profiles are directly measure states of Ml activation which are probably polari/ed in chronic inflammatory diseases including allergic asthma, food allergy, allergic dermatitis, infections, cancers and autoimmune diseases. Current methods available for characterization measuring 'classical' and 'alternative' macrophage activation states needed to measure individual key phenotypes take at least 2 - 3 days (Babu et ah, J. Infect Dis. Vol. 199:1827-1837;

MacKinnon et ah, J. Immunol. Vol. 180:2650-2658). In contrast, the methods described herein using multicolor cytometric detection of CMP-associatcd PAPC profiles take only a few hours.

[0029] The present invention is described with reference to the attached figures, wherein like reference numerals are used throughout the figures to designate similar or equivalent elements. The figures arc not drawn to scale and they are provided merely to illustrate the instant invention. Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods, The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events arc required to implement a methodology in accordance with the present invention.

[0030] All genes, gene names, and gene products disclosed herein are intended to correspond to homoiogs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but riot limited to other mammals, fish, amphibians, reptiles, and birds, In preferred embodiments, the genes or nucleic acid sequences are human, [0031] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Definitions

[0032] The terminology used herein is for the purpose of describing particular

embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including",

''includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term

"comprising."

[0033] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about'' can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, ""about'" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value, Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0034] As used herein, "classically activated" macrophages (Ml M0) develop in ThI immune responses and kill intracellular bacteria, whereas Th2 responses lead to ""alternatively activated" macrophages (M2 M0) that kill extracellular parasites and the mediation of humoral immunity (ThI vs. Th2 immune profiles). For example, immunocompromised conditions develop "alternative activation" of macrophages. As further shown herein, treatment with chitin microparticles (CMP) results in "classical activation" of macrophages.

[0035] As used herein, "substance" refers to any substance to which an immune response may be directed, and includes antigens and pathogens.

[0036] An "immunological response" or "immune response" against a selected agent, antigen or a composition of interest is the development in an individual of a humoral and/or a cellular immune response to molecules (e.g., antigen) present in the agent or composition of interest.

[0037] The term "induces or enhances an immune response" is meant causing a statistically measurable induction or increase in an immune response over a control sample Io which the peptide, polypeptide or protein has not been administered. Preferably the induction or enhancement of the immune response results in a prophylactic or therapeutic response in a subject. Examples of immune responses are increased production of type 1 IFN, increased resistance to viral and other types of infection by alternate pathogens. The enhancement of immune responses to tumors (anti-tumor responses), or the development of vaccines to prevent tumors or eliminate existing tumors,

[0038] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

10039 J As used herein, the term ""safe and effective amount"' refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. By "therapeutically effective amount" is meant an amount of a compound of the present invention effective to yield the desired therapeutic response. For example, an amount effective to delay the growth of or to cause a cancer, either a sarcoma or lymphoma, or to shrink the cancer or prevent metastasis. The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

""Cells of the immune system" or 'immune cells", is meant to include any cells of the immune system that may be assayed, including, but not limited to, B lymphocytes, also called B cells, T lymphocytes, also called T cells, natural killer (NK) cells, natural killer T (NK) cells, lymphokinc-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor-infiltrating (TlL) cells, gene modified immune cells including hybridomas, drug modified immune cells, and derivatives, precursors or progenitors of the above cell types.

[0041] "Immune effector cells'^" refers to cells capable of binding an antigen and which mediate an immune response selective for the antigen. These cells include, but are not limited to, T cells (T lymphocytes), B cells (B lymphocytes), monocytes, macrophages, natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL. clones, and CTLs from tumor, inflammatory, or other infiltrates,

[0042] "Immune related molecules" refers to any molecule identified in any immune cell. whether in a resting ("non-stimulated") or activated state, and includes any receptor, ligand, cell surface molecules, nucleic acid molecules, polypeptides, variants and fragments thereof. 'T cells" or "T lymphocytes" arc a subset of lymphocytes originating in the thymus and having heterodimeric receptors associated with proteins of the CD3 complex (e.g., a rearranged T cell receptor, the heterodimeric protein on the T cell surfaces responsible for antigcn/MHC specificity of the cells), T cell responses may be delected by assays for their effects on other cells (e.g., target cell killing, activation of other immune cells, such as B-eells) or for the cytokines they produce.

[0044] The term "biomolecule" or "marker" refers to DNA, RNA (including mRNA, rRNA, tRNA and tmRNA), nucleotides, nucleosides, analogs, polynucleotides, peptides, polypeptides, proteins and any combinations thereof. Preferably, the markers arc peptides or proteins. These markers in the context of the present invention refers to a polypeptide, which is differentially present in a sample taken from patients having for example, an inflammatory disease or is immune compromised, as compared to a comparable sample taken from control subjects (e.g., a person with a negative diagnosis or undetectable inflammation, normal or healthy subject).

[0045] The term "expression" is used herein to mean the process by which a polypeptide is produced from DNA. The process involves the transcription of the gene into mRNA and the translation of this mRNA into a polypeptide. Depending on the context in which used,

''expression'^" may refer to the production of RNA, protein or both.

[0046J The term "measuring" means methods which include detecting the presence or absence of markεr(s) in the sample, quantifying the amount of marker(s) in the sample, and/or qualifying the type of biomarker. Measuring can be accomplished by methods known in the art and those further described herein. See, for example, the examples section which follows. Any suitable methods can be used to detect and measure one or more of the markers described herein. These methods include, without limitation, immunoassay, mass spectrometry (e.g., laser dcsorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasraon resonance, cUipsometry and atomic force microscopy.

[0047 J The phrase '"differentially present" refers to differences in the quantity and/or the frequency of a marker present in a sample taken from patients with classically activated macrophages as compared to a control subject or alternatively activated macrophages.

Furthermore, a marker can be a polypeptide, which is detected at a higher frequency or at a lower

I ! frequency in samples of patients compared to samples of control subjects. A marker can be differentially present in terms of quantity, frequency or both,

10048 J As used herein, a ''molecular profile"' or ''marker profile'¹ or "expression profile'" or

"molecular signature" are used interchangeably herein and refers to the biomolecules or markers which are indicative of die activation pathway (classical vs. alternative activation) of

macrophages. For example, different markers may be present in one pathway, the markers may be increased, decreased, or expressed at the same levels as compared to a normal control See, for example, Table 1. As more biomolecules are discovered, each newly identified biomolecules can be assigned to any one or more biomarker or molecular profile. For example, any additional molecules that bind to chitin microparlicles which can be detected using the methods described herein, for example, including microbial chitinascs that are released in the hosts during infections. Each biomolecule can also be removed, reassigned or reallocated to a molecular signature. Mammalian markers are preferred, with human markers the most preferred.

[0049] A "'test amount" of a marker refers to an amount of a marker present in a sample being tested. A test amount can be either in absolute amount (e.g., μg/tnl) or a relative amount (e.g., relative intensity of signals).

[0050] A '"diagnostic amount" of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of, for example, inflammatory disease. A diagnostic amount can be either in absolute amount (e.g., 1 μg/ml) or a relative amount (e.g., relative intensity of signals).

[0051] A "control amount" of a marker can be any amount or a range of amount, which is to be compared against a test amount of a marker. For example, a control amount of a marker can be the amount of a marker in a person without a disease or condition, such as for example, chronic inflammatory disease. A control amount can be either in absolute amount (e.g., μg/ml) or a relative amount (e.g., relative intensity of signals).

[0052] "Sample" is used herein in its broadest sense. A sample comprising

polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDN A, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like,

10053 J The terms "patient", "subject^"' or "individual'" are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates,

10054 J "Diagnostic^"' or "diagnosed'" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The

"'sensitivity'" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives^'"). Diseased individuals not detected by the assay are "false negatives," Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "'false positive"' rate is defined as the proportion of those without the disease who lest positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication thai aids in diagnosis.

[0055] "Treatment"' is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented, In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy, As used herein, "ameliorated'^" or ''treatment" refers to a symptom which is approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests. Biomarkers

[0056] 1 - 10 μm chitin micropariiclcs (CMP) induce "classically" activated (Ml) Mø that produce IL-12, IL- 18 and TNFα at levels comparable to those stimulated by bacterial

components including zymosan, LPS and CpG (10-12). In a preferred embodiment, CMP is a ThI adjuvant. Chitin, N-acetyiglucosaminc polymer, is the second most abundant

polysaccharide in nature occurring as a structural component of crustaceans, fungi, helmirithes and insects; importantly it is not antigenic or allergenic. Supplementing food with CMP reduces Th2 immunological disorders including allergic asthma, seasonal rhinitis and ulcerative colitis in mice and humans (^", 8, 13), Furthermore. CMP down-regulate inflammatory Mø producing excess amounts of immunosuppressive interlcukin 10 (lL-10), an inhibitor of Ml M0 activation, that also enhances M2 (alternative macrophage activation) activation (3, 14-17).

[0057] Mechanisms of Mø activation by CMP uniquely require phagocytosis, mimicking entry of intracellular pathogens (18, 19). CMP-induced Mø activation is sensitive to

cytochalasiii D, an inhibitor for actin polymerization and phagocytosis, and distinct from those induced by the above bacterial agonists which stimulate Mø through transmembrane signals, insensitive to cytochalasiii D. Neither soluble chitin nor large chitin beads (LCB, >50 μm) that Mø cannot phagocytose induce classically activated macrophage (Ml Mø) activation (10, 16, 17), further supporting the requirement of chitin phagocytosis. Without wishing to be bound by theory, it was hypothesized that activation of the NFKB pathway leading to M 1 activation of Mø occurs within 20 minutes through sequential steps of phagocytosis of CMP including (a) ligation of CMP to chitin-binding proteins (CBP) in Mø plasma membranes, (b) internalization

(phagocytosis) through binding to intracellular CBP, and (c) activation of TLR2-mcdiated phosphorylation of MAPK (p38, Erkl/2 arid JNK) within 20 min, leading to NF-κB pathway of increased Ml M0 activation. Depletion of cellular cholesterol in Mø by methy-β cytodextrin, a cholesterol removal agent, accelerates arid enhances Mø MAPK activation by CMP but not a TL.R2 ligand Mycobacterium bovis BCG (17). In particular, after 20 minutes there was more phosphoryiatcd MAPR in the lysosomal associated membrane protein- 1 [LAMP-I]^"" phagosomes associated with CMP from Mø treated with methy-β cytodextrin than saline-treated controls, suggesting that cholesterol plays a role in regulating M0 activation during phagosome formation, 10058 J A number of markers of activated M0 have been previously identified: M2 Mø preferentially metabolize L-arginine using a M2 marker arginase 1 (Arg-1). which degrades arginine, instead of inducible nitric oxide synthase (iNOS), an Ml marker (3). Ml M0 express more T LR 2 than unstimulated Mø, whereas M2 Mø express increased levels of lectins such as intclectin. chitinases, and chitinase-like proteins (which bind chilin, but lack en/ymatic activity to iyse chitin), C -type lectins and intclectin compared to those expressed by normal or M 1 macrophages (3, 20-22). All of these receptors are potentially involved in phagocytosis of CMP, and regulation of inflammatory process by shifting M2-to-Ml Mø (8, 23).

⁾J Some cells of the immune system, including macrophages and dendritic cells (DC), function as part of the adaptive immune system as well by taking up foreign antigens through pattern recognition receptors, combining peptide fragments of these antigens with major histocompatibility complex (MHC) class S and class SI molecules, and stimulating naive CDS^{^} and CD4^f T cells respectively (Banchereau and Steinman, supra; Holmskov el al, Immunol. Today. 1994, 15:67; ΪJlevitch and Tobias Arum. Rev. Immunol. 1995, 13:437). Professional antigen-presenting cells (APCs) communicate with these T cells, leading to the differentiation of naive CD4⁺ T cells into T-helper 1 (ThI) or T-helper 2 (Th2) lymphocytes that mediate cellular and humoral immunity, respectively (Trinchicri Annu. Rev. Immunol. 1995, 13:251: Howard and O'Garra, Immunol. Today. 1992, 13:198; Abbas el al, Nature. 1996, 383:787; Okamura eϊ ai, Adv. Immunol. 1998. 70:281; Mosmann and Sad, Immunol Today. 1996, 17:138; O^'Garra Immunity. 1998, 8:275).

10060 J A secondary immune response or adaptive immune response may be active or passive, and may be humoral (antibody based) or cellular thai is established during the life of an animal, is specific for an inducing antigen, and is marked by an enhanced immune response on repeated encounters with said antigen. A key feature of the T lymphocytes of the adaptive immune system is their ability to detect minute concentrations of pathogen-derived peptides presented by MHC molecules on the cell surface. Upon activation, naϊve CD4 T cells differentiate into one of at least two cell types, ThI cells and Th2 cells, each type being characterized by the cytokines it produces. "ThI cells" are primarily involved in activating macrophages with respect to cellular immunity and the inflammatory response, whereas "Th2 cells'^" or "helper T cells'^" arc primarily involved in stimulating B cells to produce antibodies (humoral immunity). CD4 is the receptor for the human immunodeficiency vims (HIV). Effector molecules for ThI cells include, but arc not limited to, IFN-γ, GM-CSF, TNF-α, CD40 ligand. Fas ligand. IL-J, TNF-β, and IL-2. Effector molecules for Th2 cells include, but arc not limited to. IL-4, IL -5, CD40 ligand, 1L-3, GS-CSF, IL-IO, IL-13, TGF-β, and cotaxin.

Activation of the ThI type cytokine response can suppress the Th2 type cytokine response, and reciprocally, activation of the Th2 type cytokine response can suppress the ThI type response. Thus, the immune response is "polarized^'" toward a ThI or Th2 response.

[0061] In adaptive immunity, adaptive T and B cell immune responses work together with innate immune responses. The basis of the adaptive immune response is that of clonal recognition and response. An antigen selects the clones of cell which recognize it, and the first element of a specific immune response must be rapid proliferation of the specific lymphocytes. This is followed by further differentiation of the responding cells as the effector phase of the immune response develops. In T-eell mediated non-infective inflammatory diseases and conditions, immunosuppressive drugs inhibit T-cell proliferation and block their differentiation and effector functions.

[0062] Appropriate differentiation of T_H cells into effector subsets best suited for host defense against an invading pathogen is of critical importance to the immune system. CD4 T cells differentiate into at least four known subsets, three effector subsets (T_RI , T_H2 and T_R 17) and one T regulatory subset (Treg). Based on the cytokines that they produce, T cells were historically divided into T_HI and Tπ2 cells, and this has provided a framework to understand how specific cytokine milieus produced by cells of the innate immune system guide the development of adaptive immunity. T_HI cells, which are potently induced by dendritic cells (DC) secreting IL-12, are characterized by the expression of the lineage-specific transcription factor T-bet (T box 21) and the production of IFN-γ. Tπ2 cells, which depend on IL-4 during differentiation and lack of IL- 12, produce IL-4, IL -5, IL-9, and IL-13 and are characterized by the expression of the transcription factor GATA-3.

[0Θ63J In preferred embodiments, the classical (Ml) or alternative (M2) activation of macrophages (Mø) is identified by the biomarkcrs herein. These biomarkers arc important in many prophylactic therapy and also for the treatment of inflammatory diseases as the treatment can be monitored by the biomarker profiles. For example, in the examples section which follows, selected CBP that associated with CMP during phagocytosis by M0 w^rerc characterized. Briefly, employing RAW264.7 Mø-likc cells and mouse peritoneal Mø, the approach taken was to determine whether the proteins associated with phagocytosed CMP (PAPC) arc dependent on the M 1/M2 polarization state of Mø, The results evidence that the profiles of PA PC can provide diagnostic arid prognostic biomarkers to aide in staging chronic inflammatory diseases and evaluating efficacy of immunotherapy.

|0064| In a preferred embodiment, proteins associated with phagocytosed CMP (PAPC) provide diagnostic and prognostic biornarkers to aide in staging chronic inflammatory diseases and evaluating efficacy of immunotherapy.

[0065] In another preferred embodiment, the proteins associated with phagocytosed CMP

(PAPC) provide diagnostic and prognostic biornarkers of inflammatory /antiinflammatory activity and/or immunomodulatory activity. Examples of various medical conditions include: cutaneous T-cell lymphoma, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, glaucoma, diabetes, sepsis, shock, sarcoidosis, idiopathic pulmonary fibrosis, bronchopulmonary dysplasia, retinal disease, scleroderma, osteoporosis, renal ischemia, myocardial infarction, cerebral stroke, cerebral ischemia, nephritis, hepatitis, glomerulonephritis, cryptogenic fibrosing aveolitis, psoriasis, transplant rejection, atopic dermatitis, vasculitis, allergy, seasonal allergic rhinitis, Crohn's disease, inflammatory bowel disease, reversible airway obstruction, adult respiratory distress syndrome, asthma, chronic obstructive pulmonary disease (COPD) or bronchitis. Examples of diseases or disorders associated with immune compromised patients include diseases caused by microbial infections, including bacterial infections, fungal infections and viral infections, particularly among vulnerable patient groups such as the elderly, premature babies, infants, transplantation patients, immunosupprcssed patients such as chemotherapy patients, hospital patients at risk of opportunistic infection, patients on ventilators, cystic fibrosis patients and patients with AIDS.

[0066] In one preferred embodiment, the PAPC markers comprise C -type lectins (mannosc receptor [MRj, DEC-205, Dcctin-1 , SIGN-Rl . MRC2); toll-like receptors (TLR2, TLR4, CD14); phagolysosomal proteins (LAMP-I, LAMP-2); adhesion molecule /chemokine receptor (CD44. L-selectin [CD62L.J, CDl 84 [CXCR4J): CH13L1 chitolriosidasc (chitinase-1), acidic mammalian chitinasc (AMCase). chitinase3-like-2 (CHI3L2), oviductal glycoprotein 1 or stabilin-1 interacting chitinase-like protein (SI-CLP). ! ι7 , ] In another preferred embodiment, further biomarkers comprise any additional molecules that bind to chitiri niicroparticles, including microbial chilinases that are released in the hosts during infections.

10068 J Assessment of the expression of the markers is described in the examples which follow. However, expression of a marker of the invention may be assessed by any of a wide variety of well known methods for detecting expression of a transcribed molecule or its corresponding protein. Non-limiting examples of such methods include immunological methods for detection of secreted proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods. In a preferred embodiment, expression of a marker gene is assessed using an antibody (e.g. a radio-labeled, chromophore-labeled, fluorophorc-labelcd, or enzyme-labeled antibody), an antibody derivative (e.g. an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair {e.g. biotin-streptavidin}), or an antibody fragment (e.g. a single-chain antibody, an isolated antibody hypervariable domain, etc) which binds specifically with a protein corresponding to the marker gene, such as the protein encoded by the open reading frame corresponding to the marker gene or such a protein which has undergone all or a portion of its normal post-translational modification. In another embodiment, expression of a marker gene is assessed by preparing mRNA/cDNA (i.e. a transcribed polynucleotide) from cells in a patient sample, and by hybridizing the niRNA/cDNA with a reference polynucleotide which is a complement of a polynucleotide comprising the marker gene, and fragments thereof. cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide; preferably, it is not amplified.

[0069] In another preferred embodiment, the detection in a sample of the biomarkers is diagnostic of chronic inflammatory diseases, inflammatory diseases, autoimmune diseases, or associated disorders thereof. Preferably, the markers are modulated at levels by at least between 1%, 2%, 5%, 10% in a patient sample as compared to levels in a normal sample; more preferably, the markers are modulated by about 50% in a patient sample as compared to levels in a normal sample; more preferably, the markers are modulated by about 75% in a patient sample as compared to levels in a normal sample. The term "modulated" refers to an increase or decrease in level, concentration, amount etc, as compared to a normal cell or normal healthy subject, The term can also be applied as "differential expression" wherein one or more markers are increased, decreased or remain at baseline levels relative to each other arid baseline normal controls. Examples of diseases or disorders include without limitation: rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, glaucoma, diabetes, osteoporosis, renal ischemia, cerebral stroke, cerebral ischemia, nephritis, psoriasis, allergy, inflammatory disorders of the lungs and gastrointestinal tract such as Crohn's disease, and respiratory tract disorders such as reversible airway obstruction, asthma, chronic obstructive pulmonary disease (COPD) and bronchitis.

Chitin Mieropartides (CMP)

In another preferred embodiment, the chitin microparticles arc between about 0.1 μm to about 15 μm in diameter, preferably, the chitin microparticles are about 1 μm up to 10 μm in diameter. Chitin microparticles (CMP) can be used as a diagnostic probe to evaluate disease states of macrophages, When macrophages phagocytose CMP, proteins associated with phagocytosed chitin microparticles (PAPC) in macrophages are detected. In a typical embodiment of determining or measuring a subject's PAPC profile, the following steps are perfoπned. In test tubes, macrophages (blood monocytes) isolated from immunocompromised patients are mixed with CMP for 15 minutes or so at 37°C. Immediately after the incubation, macrophages are washed to remove unphagocytosed CMP, Then, macrophages are digested with a lysis buffer containing a detergent to recover intracellular CMP from macrophages. After washing, PAPC which are associated with CMP are detected by multi-color cytometric analysis using selected antibodies recognizing CMP. Proteins associated with phagocytosed chitin microparticles (PAPC) profiles in immunocompromised conditions arc distinct from those in n orm al/c ontro 1 gro ups .

[0071] This diagnostic approach potentially applies to individuals having any abnormal condition including malnourished individuals, neonates, the elderly, those with chronic inflammatory diseases (e.g. cancer, infections, autoimmune diseases, diabetes atherosclerosis), and who are likely to develop persistent activation of macrophages populations. It is also applicable to patients with allergic asthma, food allergy, allergic dermatitis, rhinitis, and colitis. [0072] This approach is also useful for prognosis of therapeutic and prophylactic treatments including CMP treatment. A diagnostic kit, including CMP, fluorochrome-conjugated antibodies, and lysis buffer, can be used for measuring PAPC profiles, diagnosing a disease state, and monitoring a subject's response to treatment (e.g., immunotherapy). In addition to CMP, chemically synthetic microparticles with appropriate carbohydrates can be used for in the methods and kits described herein.

As discussed above, chitiii is a polymer of N-acctyl-D-glucosaminc and has a similar structure to cellulose. It is an abundant polysaccharide in nature, comprising the horny substance in the cxoskelctons of crab, shrimp, lobster, cuttlefish, and insects as well as fungi. Any of these or other sources of chitin are suitable for the preparation of CMP preparations for use according to the present invention. In addition to chitin, chitin derivatives can be used in the preparation of microparticles. One such derivative known in the art is chitosan. Chitosan is a de-acεtylatεd form of chitin and occurs naturally in some fungi, and, like chitin, is insoluble in water, see, for example, U.S. Pat, No. 6,638,918, the contents of which are hereby incorporated herein bv reference.

There are currently many methods known in the art for extracting natural chitin from the exoskeletons of marine crustaceans and oilier natural sources, and any such method can be used in accordance with the methods of the present invention. References illustrative of the prior art methods of separating, extracting and purifying the chitin from shellfish include: Chang & Tsai, 1997, ""Response Surface Optimization and Kinetics of Isolating Chitin from Pink Shrimp Shell Waste'^', J. Agric. Food Chem., Vol. 45, pgs 1900-1904; Kawaguti, '62, "Electron Microscopy of the Integumental Structure and its Calcification during Molting in a Crayfish", Biol. J. Okayama, Univ., Vol. 8, pp. 43-58; No & Myers, 1995, "Preparation and

Characterization of Chitin and Chitosan— A Review, J. Aquatic Food Product Tedmol. Vol. 4, pp 27-41; Roer & Dillamen, 1984, 'The Structure and Calcification of the Crustacean Cuticle", Amer. ZooL, Vol. 24, pp 893-909; Waterman, T. Ii. '60, '"Metabolism and Growth", Vol. 1 in "The Physiology of Crustacea", Acad. Press, p. 449, as cited in U.S. Pat. No. 4,199,496, CoL 3.; and U.S. Pat. Nos. 4,066,735, 4,199,496, 4,293,098, 5,053,113, 5,210,186, the contents of which are hereby incorporated herein by reference. If the chitin to be used is derived from a natural source, the chitin may be effectively separated from other non-chitinous materials located therein such as lipids, protein, and calcium carbonate. Since chitin is insoluble in virtually everything except highly concentrated acidic solutions, one conventional practice known in the art is to remove the unwanted non-chitinous contaminants from the insoluble chitin matrix by using strong acidic solutions. Examples of methods to accomplish the separation of chitin from non-chitinous material include are described in U.S. Pat. Nos. 5,210,186, 5,053,1 13, 4,066,735, and 4,199,496, the contents of which are incorporated herein by reference.

[0076] Chitin may also be produced in vitro using chitin synthase enzymes that catalyze the polymerization of N-acetylglucosaminyl residues into chitin from uridine 5'-diphospho-N- acetylglucosamine. Such methods are known in the art, see for example, published U.S. Patent Application No, 20030166235 Al .

10077] In addition to the above methods, chitin can be obtained in pre-purified form from any commercial source. In one embodiment, chitin is produced by physically reducing it, e.g. by soni cation or milling, to particles having a diameter of less than 50 μm, more preferably less than 40 μm, more preferably less than 20 μm, more preferably less than 10 μm and preferably less than 5 μm. As shown in the examples section, that the effects caused by chitin microparticles are size dependent, it is preferred that the chitin microparticles have average diameters which are less than 10 μrn, An upper limit of chitin particles size may be functionally defined by macrophages not recognizing the particles. The lower size limit is less important, but preferably the particles are at least 0.5 μm in diameter. Particles size and size distribution can readily be determined by the skilled person for example using flow cytometry or a microscope. Methods suitable for the detection of particles and the determination of particle size by flow-cytomctry arc known in the art, see for example, U.S. Pat, Nos. 4,765,737, 5,444,527, and 6,549,275, the contents of which are hereby incorporated herein by reference. Alternatively or additionally, the chitin microparticles can be made by coating earner particles, e.g. formed from a biocompatible material such as polystyrene or latex, with N-Acetyl-D-Glucosamine, chitin or a fragment thereof, to form particles having the sizes as defined above, and these compositions are included within the term chitin microparticles (CMP) as used herein.

_ ">l _ It should be recognized that in a composition, the chitin microparticies may have a distribution of sizes, typically a norma] distribution. However, within a population of chitin microparticies forming a CMP preparation, preferably at least 60%, more preferably at least 75%, more preferably at least 90%, and more preferably 95% and most preferably at least 99%, of the chitin particles have a size distribution within the limits set out above.

Drug Discovery

[0079] The present invention also comprises methods to screen for candidate compounds useful in the treatment of inflammation, treatment of immune compromised patients, monitoring of therapy, and the like. Examples include autoimmune diseases such as diabetes, chronic rheumatoid arthritis, SLE and pulmonary fibrosis; inflammatory diseases such as hepatitis, hepatic cirrhosis and inflammatory bowel diseases, gastrointestinal inflammatory diseases; and allergic diseases such as hypersensitive interstitial pneumonia, asthma, atopic cutaneous inflammatory diseases, sarcoidosis, etc. Examples of inflammatory gastrointestinal diseases include hepatitis, hepatic cirrhosis and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease.

[0080 J In other preferred embodiments, the markers are useful for the identification of new drugs in the treatment of inflammatory diseases and disorders.

|0081 ] In another preferred embodiment, markers would verify whether a patient's treatment is progressing. For example, the markers may change during the course of treatment and reflect normal controls.

In one embodiment, the invention comprises a method to screen for a candidate compound useful to treat inflammatory diseases comprising: (i) identifying a candidate compound which regulates the expression of one or more markers comprising: C -type lectins (mannosε receptor [MR], DEC-205, Dεctin-1, SlGN-Rl, MRC2); toll-like receptors (TLR2, TL.R4, CD14); phagolysosome! proteins (LAMP-I , L.AMP-2); adhesion molecule /cheraokine receptor (CD44, L-selεctin [CD62L], CDl 84 [CXCR4]); CHBLl chitotriosidase (chitinase-1), acidic mammalian chitinasc (AMCase), chithiase3-like-2 (CHI3L2), oviductal glycoprotein 1 or

„ 01 . stabilin-1 interacting chitinase-like protein (SI-CLP) markers; and (ii) determining whether such candidate compound is effective to treat an inflammatory disease,

10083] As used herein, the terra "'compound^*' refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, conditions, or disorder of bodily function. Compounds comprise both known and potential therapeutic compounds. A compound can be determined to be therapeutic by screening using the screening methods of the present invention. Examples of test compounds include, but are not limited to peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, and combinations thereof.

[0084] Small Molecules: Small molecule test compounds or candidate therapeutic compounds can initially be members of an organic or inorganic chemical library. As used herein, "small molecules'^" refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. The small molecules can be natural products or members of a combinatorial chemistry library. A set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity. Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Wεight Compound Libraries,

Pergamon-Elsevier Science Limited (1998), and include those such as the "'split and pool'" or ''parallel'" synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem, Bio,, 1:60 (1997). In addition, a number of small molecule libraries are commercially available.

Particular screening applications of this invention relate to the testing of pharmaceutical compounds in drug research. The reader is referred generally to the standard textbook ''Li riiro Methods in Pharmaceutical Research^'", Academic Press, 1997, and U.S. Pat. No. 5,030,015). Assessment of the activity of candidate pharmaceutical compounds generally involves administering a candidate compound, determining any change in the morphology, marker phεnotypε and expression, or metabolic activity of the cells and function of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change.

- °3 - [0086 J The screening may be done, for example, either because the compound is designed to have a pharmacological effect on certain cell types, or because a compound designed to have effects elsewhere may have unintended side effects. Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially). Io detect possible drug— drug interaction effects. In some applications, compounds are screened initially for potential toxicity (Castcll et a/., pp. 375-410 in "//? vitro Methods in Pharmaceutical

Research,^"1' Academic Press, 1997), Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and expression or release of certain markers, receptors or enzymes. Effects of a drug on chromosomal DNA can be determined by measuring DNA synthesis or repair. ['Hjthymidine or BrdU incorporation, especially at unscheduled times in the cell cycle, or above the level required for cell replication, is consistent with a drug effect. Unwanted effects can also include unusual rates of sister chromatid exchange, determined by metaphasc spread. The reader is referred to A. Vickcrs (PP 375-410 in "'In vitro Methods in Pharmaceutical Research,'" Academic Press, 1997) for further elaboration.

In one embodiment of the invention, a method of identifying a candidate agent is provided said method comprising: (a) contacting a biological sample from a patient with the candidate agent and determining the level of expression of one or more markers described herein; (b) determining the level of expression of a corresponding marker or markers in an aliquot of the biological sample not contacted with the candidate agent; (c) observing the effect of the candidate agent by comparing the level of expression of the marker or markers in the aliquot of the biological sample contacted with the candidate agent and the level of expression of the corresponding marker or markers in the aliquot of the biological sample not contacted with the candidate agent; and (d) identifying said agent from said observed effect, wherein an at least 1%, 2%, 5%, 10% difference between the level of expression of the marker or combination of markers in the aliquot of the biological sample contacted with the candidate agent and the level of expression of the corresponding marker or combination of markers in the aliquot of the biological sample not contacted with the candidate agent is an indication of an effect of the candidate agent,

[0088 J In preferred embodiments, the effects of the drag are correlated with the expression of the markers associated with a classical or alternative activation of macrophages.

. 7Λ . [0089J In another embodiment of the invention, a candidate agent derived by the method according to the invention is provided.

[0090J In another embodiment of die invention, a pharmaceutical preparation comprising an agent according to the invention is provided.

[0091] In another preferred embodiment of the invention, a method of producing a drug comprising the steps of the method according to the invention (i) synthesizing the candidate agent identified in step (c) above or an analog or derivative thereof in an amount sufficient to provide said drug in a therapeutically effective amount to a subject; and/or (ii) combining the drug candidate the candidate agent identified in step (c) above or an analog or derivative thereof with a pharmaceutically acceptable carrier,

[0092] Vectors, Cells: In some embodiments it is desirable to express the biomolecules that comprise a biomarker, in a vector and in cells. The applications of such combinations are unlimited. The vectors and cells expressing the one or more biomolecules can be used in assays, kits, drug discovery, diagnostics, prognostics and the like. The cells can be stem cells isolated from the bone marrow as a progenitor cell, or cells obtained from any other source, such as for example, ATCC.

[0093 J In a preferred embodiment, a method of identifying candidate therapeutic compounds comprises cuhuring cells expressing at least one or more markers comprising: C-lype lectins (mannose receptor [MR], DEC-205, Dectin-1, SIGN-Rl, MRC2): toll-like receptors (TLR2, TLR4, CD14); phagolysosome 1 proteins (LAMP-I, LAMP-2); adhesion molecule /chemokine receptor (CD44, L-selectin [CD62L], CD184 [CXCR4]}; CHI 3 Ll chitotriosidase (chiiinase-1), acidic mammalian chitinasc (AMCase), chitinasc3-like-2 (CHI3L2), oviductal glycoprotein 1 or stabilin-1 interacting chilinase-like protein (SI-CLP) markers.

[0094] Such compounds are useful, e.g., as candidate therapeutic compounds for the treatment of inflammatory disorders and conditions thereof, immunomodulatory drugs, etc. Thus, included herein are methods for screening for candidate therapeutic compounds for the treatment of, for example., cutaneous T-cell lymphoma, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, glaucoma, diabetes, sepsis, shock, sarcoidosis, idiopathic pulmonary fibrosis, bronchopulmonary dysplasia, retinal disease, scleroderma, osteoporosis, renal ischemia, myocardial infarction, cerebral stroke, cerebral ischemia, nephritis, hepatitis, glomerulonephritis, cryptogenic fibrosing aveolitis, psoriasis, transplant rejection, atopic dermatitis, vasculitis, allergy, seasonal allergic rhinitis, Crohn's disease, inflammatory bowel disease, reversible airway obstruction, adult respiratory distress syndrome, asthma, chronic obstructive pulmonary disease (COPD), bronchitis and the like, It is contemplated that a compound of this invention may be useful in treating one or more of such diseases.

[0095J The methods include administering the compound to a model of the condition, e.g., contacting a cell (in vitro) model with the compound, or administering the compound to an animal model of the condition, e.g., an animal model of a condition associated with an inflammatory disease. The model is then evaluated for an effect of the candidate compound on the clinical outcome in the model and can be considered a candidate therapeutic compound for the treatment of the condition. Such effects can include clinically relevant effects, decreased pain; increased life span; and so on. Such effects can be determined on a macroscopic or microscopic scale. Candidate therapeutic compounds identified by these methods can be further verified, e.g., by administration to human subjects in a clinical trial.

[0096] The biomolecules can be expressed from one or more vectors. A "vector'"

(sometimes referred to as gene delivery or gene transfer "vehicle") refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. The polynucleotide to be delivered may comprise a coding sequence of interest in gene therapy. Vectors include, for example, viral vectors (such as adenoviruses ("Ad'"), adeno- associated viruses (AAV), and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell. Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells. Other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide. Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. Other vectors include those described by Chen et al; BioTeclwiques, 34: 167-171 (2003). Large varieties of such vectors are known in the art and are generally available.

[0097] In another preferred embodiment, a vector expresses one or more one or more markers comprising: C-type lectins (mannosc receptor [MR], DEC-205, Dcctin-1, SlGN-Rl, MRC2); toll-like receptors (TLR2, TLR4, CD14); phagolysosomal proteins (LAMP-I, LAMP- 2); adhesion molecule /chemokine receptor (CD44, L-selectin [CD62L], CDl 84 [CXCR4]); CHBLl chitotriosidase (chitinase-1), acidic mammalian chitinase (AMCase), chitinase3-like-2 (CMI3L.2), oviductal glycoprotein 1 or stabiiin-1 interacting chitinase-iike protein (Sl-CLP) markers.

Kits

[0098] In another preferred embodiment, a kit is provided comprising any one or more markers comprising: C-type lectins (mannose receptor [MR], DEC-205, Dectin-1, S(GN-Rl, MRC2); toll-like receptors (TLR2, TLR4, CD14); phagolysosomal proteins (LAMP-I, LAMP- 2); adhesion molecule /cheraokinc receptor (CD44, L-sclectin [CD62L], CD 184 [CXCR4]); CHI3L1 chitotriosidase (chitinase-1), acidic mammalian chitinase (AMCase), chitinase3-like-2 (CMI3L2), oviducta] glycoprotein 1 or stabilin-1 interacting chitinase-iike protein (Sl-CLP) markers.

[0099] For use in the applications described or suggested above, kits or articles of manufacture are also provided by the invention. Such kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a probe that is or can be detectably labeled. Where the kit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin- binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.

_ ¹J" _ The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable frorn a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for cither in vivo or in vitro use, such as those described above.

jOlOli The kits of the invention have a number of embodiments. A typical embodiment is a kit comprising a container, a label on said container, and a composition contained within said container; wherein the composition includes a primary antibody that binds to the biomolecules of each biomarker and instructions for using the antibody for evaluating the presence of

biornolecules in at least one type of mammalian cell. The kit can further comprise a set of instructions and materials for preparing a tissue sample and applying antibody and probe to the same section of a tissue sample, The kit may include both a primary and secondary antibody, wherein the secondary antibody is conjugated to a label, e.g., an enzymatic label.

[0102] Another embodiment is a kit comprising a container, a label on said container, and a composition contained within said container; wherein the composition includes a polynucleotide that hybridizes to a complement of the polynucleotides under stringent conditions, the label on said container indicates that the composition can be used to evaluate the presence of a molecular signature in at least one type of mammalian cell, and instructions for using the polynucleotide for evaluating the presence of biornolecule RNA or DNA in at least one type of mammalian cell. |0103| Other optional components in the kit include, microarrays, one or more buffers (e.g., block buffer, wash buffer, substrate buffer, etc), other reagents such as substrate (e.g., chromogen) which is chemically altered by an enzymatic label, epitope retrieval solution, control samples (positive and/or negative controls), control slide(s) etc.

10104 J The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope of the invention. The following non-limiting examples are illustrative of the invention.

10105 J All documents mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "'prior art"' Io their invention.

[0106] The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

[0107] Example 1: Toll-like receptor 2 as a protein associated with phagocytosis of chitin microparticles (CMP) and signaling in Ml activated macrophages

[0108] Materials and Methods

[0109] Reagents and antibodies: Chitiri microparticles (1 - 10 μm, CMP) and cm^'losari microparticles (chitosan-MP) were prepared as described previously ( 10. 16, 24). Large chitin beads (>50 μm. LCB) were purchased from New England Biolab (Ipswich. MA).

[01 10] Mice: Non -pregnant female C57BL./6, 8 - 14 weeks old, were purchased from

Jackson Laboratory (Bar Harbor, ME) and maintained in barrier-filtered cages and fed Purina laboratory chow and tap water ad libitum. Experimental protocols employed in this study were approved by the IACUC of Florida Atlantic University and Scripps Research Institute { La Jolla, CA).

[OHi] Peritoneal Mø preparation following intraperitoneal administration of CMP, LCB, or saline: Groups of WT mice (4 - 5/grouρ) received 1 mg CMP, 1 mg LCB, or 0.1 ml saline intraperitoncally (i.p.) on day 0. Peritoneal lavage with 5 ml saline was performed on day 1 (25), Nucleated peritoneal cells were counted with a Coulter counter (Model Zl, Beckraan Coulter, Hialeah, FL). Differential cell counts were performed manually on cytospin preparations (Shandon Southern Instruments Co., Sewickley, PA) stained with Dlff-Quik. To enrich plastic- adherent Mø, 10⁶ peritoneal cells/ml were suspended in RPMl 1640 plus 5% heat-inactivated fetal bovine serum (FBS) and incubated in culture dishes for 2 h. Non-adherent cells

(lymphocytes) were removed by washing with warmed media. Adherent Mø were cultured with 100 μg/ml CMP for an additional 15 min.

- OQ - [0112] Cell culture: RAW 264,7 cells (American Type Culture Collection, Manassas, VA) were grown arid maintained in RPMI 1640 containing 5% FBS and stimulated with CMP at various doses as described previously (16, 17). For TNFα production, culture supernatants were isolated within 4 h. TNFα levels in the supernatants were measured by two-site ELISA specific for mouse TNFα (BD Biosciencε, San Diego, CA).

|0ll3 J For the study of CM P-induced TNFα production by peritonea! Mø, 1 ml of thiogiycollate broth was given intraperiloneally in C57BL/6 background mouse strains that were WT or individually deficient in TLR2, CD14, Tirap, MydδS, Irak4, TLRl, TLR3, TLR4, TLR6, TLR7, TLR9, CD36, TRAM, or Trif. Thiogly col late-elicited peritoneal Mø were isolated by peritoneal lavage with 5 ml saline. Plastic adherent Mø at 10° cells/ml were stimulated with CMP for 4 h. TNFα in the supernatants were measure by specific ELISA. The studies were performed at the Scripps Research Institute, La JoIIa, CA.

[0114] Western blot analysis: Peritoneal Mø prepared as described above, were washed 3 times with cold saline. Washed cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 4 mM EDTA, 0,1% SDS, 1:500 protease inhibitor cocktail [P8340, Sigma Aldrich], 1 % Nonidet P-40, 0.1% sodium deoxycholate, 10 mM NaF, and 2 mM Na₃VO₄).

Debris was eliminated by centrifugation (10 min, 10,000 g) and protein concentration in the Iy sate was measured by bicinchoninic acid assay (Pierce, Rockford, IL) with bovine serum albumin as standard. Equal amounts (5 - 10 μg) of protein from each sample were separated by SDS-PAGE. Proteins were then transferred to a polyvirrySidene di fluoride (PVDF) membrane (Millipore, Bedford, MA). The membrane was blocked with 10% nonfat dry milk, and incubated with antibodies fanli-Arg I, 1:200 [BD Transduction Laboratories, San Diego, CA] at 22°C; and at 4°C for anti-chilinase 3-like 1 (CPtULl), 1 :5,000 (26), anli-iNOS, 1:4,000 [Upstate,

Temecula, CA], anti-cyclooxygenase 2 (COX-2). 1:800 [Cayman Chemical] and anti-β actiri, 1 :5,000 [Sigma] for detection of β-aclin as constilutively expressed protein control) in 7,5% nonfat dry milk, overnight. Following incubation with peroxidase-conjugaled goat anti-rabbit IgG for CHI3L1, iNOS and COX-2 detection, or donkey anti-mouse IgG for Arg 1 and β-actin (both 1:10,000; Jackson Immunollesearch, West Grove, PA), stained proteins were detected by cherai luminescence (ECL plus, Amersham, Piscataway, NJ) following the manufacturer's instructions. [0115] Cytometric detection of surface antigens: Standard methodology was used for the direct and indirect immunofluorescent characterization of peritoneal leukocytes., In brief, 10° PC were incubated with Fc Block (BD Pharmingen, Franklin Lakes, NJ ) in 50 μl of P13S for 5 minutes to block non-specific binding of antibodies to cellular Fc receptors. Subsequently, optima! concentrations of Ph ycoerythrin (PE)-conjugated F4/80 antibody and single other fluorochromc-conjugated antibodies recognizing surface antigens (TLR2. MR, CD205, SIGN-Rl, Dectin-1) purchased from BioLegend, San Diego, CA, and BD PharMingen were added in 50 μl of FACS buffer for dual staining. After 30 minutes of incubation on ice. the cells were washed three times with FACS buffer and fixed in 200 μl of 1 % paraformaldehyde, Fluorescence of stained cells was quantified with a FACSCalibur flow cytomcter (Becton Dickinson, Franklin Lakes, NJ) using the FlowJo program (Tree Star, Ashland, OR). M0 fractions were

electronically gated by forward- and side-scatter, and positive expression of F4/80.

[0116] Recovery of proteins associated with phagocytosed CMP (PAPC) and FACS analysis: Peritoneal Mø and RAW 264.7 M0 both at 10" cells/ml in RPMl 1640 + 5⁰Z⁷O FBS incubated with 100 μg/ml CMP for 5 - 30 min at 37°C. Following washing, cells were lyscd with ice-cold lysis buffer containing 20 mM Tris, pH 7.5, 1% NP-40, 10 niM CaCl;., and protease inhibitors (Sigma). PAPC/CMP recovered were washed 3 times with calcium - containing saline, fixed with 4% paraformaldehyde, and stained with antibodies listed. Using references of 1 and 10 μm latex beads (Sigraa-Aldrich), PAPC/CMP isolated from Mø were electronically gated at 1 - 10 μm for cytometric analyses of protein profiles.

[01 17] TLR2 binding to CMP in a cell-free condition: To determine whether TL.R2 directly bind to CMP in a cell-free condition, 100 μg/ml CMP or chitosan-MP as a control were mixed with 0.1 - 5 μg/ml TL.R2 protein (recombinant TLR2 Fc chimera, 1530-TR, R&D System, Minneapolis, MN) in 20 mM Tris buffer, pH 7.5, containing 10 mM CaCl₂, 0.5% BSA and 1% NP40 at 0° for 30 min. CMP alone without TLR2 protein was also prepared as control,

Following washing CMP with the buffer 3 times at 800g cenfri {ligation, the precipitated CMP were stained with Alexa Fluor 647-conjugated TLR2 antibody (BioLegend).

[0118] Statistics: Data were analyzed by one way analysis of variance. For cell culture studies. tissues isolated from at least 4 mice were pooled unless indicated; cells were cultured in at least triplicate in each group. P <0.05 was considered statistically significant. [0119] Results:

[0120] TLR2 -mediated Ml M0 activation: CMP-induced macrophage (M0) activation is independent of TLR4 (10), TLR2 is involved in CMP-induced Mø activation (9). To confirm whether Mø activation requires a co-receptor of TLR2 (CD 14), major adaptor proteins of TLR2 (Tirap [toll-intcrleukin-l receptor domain containing adaptor protein], Myd88 [myeloid differentiation factor 88], Irak4 [interleukin-1 receptor-associated kinase 4]), or TLRl and TLR6 which form hcterodimers with TLR2 for recognition of bacterial agonists, peritoneal Mø obtained from mice deficient in each of the listed genes were stimulated with CMP in vitro, Ml activation was determined by measurement of TN Fa in the supernatants. Figure 1 shows that all Mø from mice deficient in CD14, Tirap, Myd88, or Irak4 showed little to no production of TNFα in response to CMP. However, CMP did induce Ml activation in Mø from VVT and TLR4~deficient mice ( Figure 1), as well as in Mø deficient in TLR3, TLR7, TL.R9, CD3(\ TRlF (toll-interleukin 1 receptor domain-containing adaptor including interferon- β), or TRAM (TRIF-related adaptor molecule) as well as TLRl and TLliό, indicating that these gene products do not contribute to the Ml Mø activation.

[012Ϊ] Cell-free binding of soluble TLR2 and CMP: To determine whether TLR2 binds directly to CMP, recombinant TLR2-Fc and CMP were incubated in a cell-free condition and the binding was measured cytomelricaily. As shown in Figure 2, TLR2 protein bound to CMP, but not chitosan-microparticles, indicating chemical selectivity.

[0122] Proteins associated with phagocytosed CMP (PAPC) in RA W264.7 Mø: M APK activation by CMP phagocytosis can be detected within 20 min, indicating that proteins associated with phagocytosed CMP (PAPC) at the earlier stages of phagocytosis contribute to the Ml M0 activation. To characterize TLR2 behavior as a PAPC, RAW264.7 M0 were incubated with CMP for 5, 15 and 30 min, before isolating CMP by homogeni/alion with mild NP40 detergent and cytometric detection of PAPC? as previously described (22, 27).

[0123] TLR2 associated with CMP in the first 5 minutes of phagocytosis, and further increased by 15 minutes (Figure 3) before plaleauing. When phagocytosis was inhibited by pre-trcatment with cytochalasin D, TLR2 levels binding to CMP were detected comparably at 5 min but at 15 min were not increased (Figure 3), The results indicate that TLR2 binding to CMP is further increased during internalization of CMP,

[0124] As shown in Figure 4, MR (marmose receptor), LAMP-I, and CHBLl were detected as CAPP within 15 min, although the kinetics varied depending on the specific PAPC. CD62L (L-selectin) was detected within 5 min and increased by 30 rain. These PAPC levels were reduced in the presence of EDTA, suggesting Ca^"" dependent binding to CMP.

[0125] PAPC in RA W264.7 M0 treated with IFNγor IL4/IL13: The ThI cytokine IFNγ and Th2 cytokines IL-4/IL-13 induce Ml and M2 Mø activation, respectively. To determine whether Ml and M2 activated Mø exhibit distinct PAPC profiles, RAW cells were treated with 10 ng/ml IFNγ and 10 ng/ml IL-13 (or IL-4) for 24 hrs. The treatments modulated CMP- induced TNF α production. As shown in Figure 3, although IFNγ prc -treatment had no effect on CMP-induced production oi^'TNFα, prctreatmeiit with IL-13 or IL-4 reduced TNFo;

production.

[0126] PAPC were recovered from the eytokmε-treated RAW cells after phagocytosis of CMP for 15 minutes. The number of phagocytosεd CMP recovered from M0 was almost comparable among untreated and cytokine-treatεd Mø groups. Figures 5 A, 5 B shows that TLR2 levels were increased by IFNγ-treated M0 and reduced in IL-4 or IL-13-treated M0, evidencing differential TLR2 detections between M l and M2 activation stages,

[0127] Chitin particle-induced M1/M2 peritoneal M0 and their PAPC profiles:

Intraperitoneal administration of chitin particles (CMP and LCB) results in the induction of Ml and M2 activated peritoneal Mø, respectively, in a size-dependent manner (7, 8, 1 1, 27). There was a greater expression of Arg I, a marker of M2 Mø, in peritoneal M0 isolated from mice treated with intraperitoneal LCB than in those from mice treated with saline (Figures 6A-6C), Selected surface antigens expressed by F4/80^f peritoneal Mø were determined cytometrically. TLR2, CHI3L1, MR, CD205, Dectin-1 and SIGN-Rl were expressed by normal peritoneal M0. Mø activated by LCB had increased mean fluorescent intensity (FI) values for Dectin-1, decreased values for TLR2, CHI3L1, CD205 and SIGN-Rl, and almost for unchanged MR (Figures 6A-6C). In contrast, intraperitoneal CMP resulted in increased mean fluorescent intensity (FI) values for Dectin-1, decreased values for MR, CD205 and SIGN-Rl, and almost unchanged for TLR2 and CHI3L1 (Figures 6A-6C). Interestingly. FI ratios of selected antigens reflected normal. Ml and M2 M0 populations. For example, the FI ratios of TLR2:MR in normal/M l (CMP)/M2 (LCB) peritonea] M0 were 1/2/0.5.

[0128] For PAPC analysis, peritoneal Mø were harvested three days after the

intraperitoneal CMP administration. These studies showed that the M0 isolated from CMP- treated mice contained no CMP. Fourteen macrophage (Mø) proteins were selected as major PAPC: C-type lectins (mannose receptor [MR]. DEC-205. Dectin-1. SIGN-Rl, MRC2); toll-like receptors (TLR2, TLI14, CD 14); phago lysosomal proteins (LAMP-I, LAMP-2); adhesion molecule /chcmokinc receptor (CD44, L-seleclin [CD62L]. CD 184 [CXCR4]); and CHDLl (Figure 7). For Ml- and M2-dcpcndcnl changes in PAPC profiles, the mean florescent intensity (Fl) of each PAPC was obtained from the histogram analysis by the FlowJo program, indicating that increased FI values observed for Ml and M2 were TLR2/LAMP1 and

MR/CD62L/CD205/S SGN-Rl. respectively. In contrast, decreased Fl values for M 1 and M2 were observed in MR/CD62L/CTII3L1 and TLR2/LAMP1 , respectively. Other markers were unchanged among normal. Ml and M2 M0 populations. The PAPC analysis is summarized in Table 1. Interestingly, FI ratios of selected PAPC preferentially reflected specific Mø populations. Thus, the respective FI ratios for normal/Ml/M2 Mø populations were

0.5/1.3/0.03 for TLR2:MR, 0.3/3.7/0.01 for TLR2:CD62L, 0.25/1.1/0.07 for LAMP-I :MR and 0.13/3/0.03 for LAMP-I :CD62L.

Table 1. Proteins-associated with phagocytosed CMP ( PAPC) in peritoneal Mø isolated from mice given intraperitoneal CMP or LCB.

- 54 -

As indicated in Figure 7, peritoneal macrophages (Mø) were isolated from mice prctreated for 3 days with intraperitoneal administration of CMP, LCB or saline. PAPC were isolated and quantified cytometricaljy as indicated in Figure 7. t, Detection; -, no detection; T^- >.

increased, decreased and unchanged compared to that of saline control, respectively,

Discussion

Phagocytosis of CMP, a ThI adjuvant, by macrophages (Mø) results in development of TLR2-dcpcndcnt innate immunity (7-9) and promotion of acquired immune responses that mediate ThI cellular activation along with down-regulation of Th2 allergic responses (7, 8). In this study, the requirement of TLR2 for CM P-induced M0 activation was confirmed and the results ( 10, 12, 16, 17), summarized that: (i) M0 recognize and phagocytosc CMP through interactions with cellular chitin binding lectins— designated as proteins associated with phagocylosed CMP (PAPC)— which include TLR2, mannose receptor (MR), and chitinase-like protein (CFl 13Ll). The consequent Ml activation results in production of ThI cytokines: IL-12. IL- 18 and TNFcc. (ii) CMP stimulation occurs without inducing Ml activation of M0 in mice deficient in TLR2, its co-receptor (CD 14) or its major signaling molecules (Tirap. MydSS, and Irak4). (iii) CMP induce Ml activation of Mø in WT mice and in those deficient in TLRl, TLR3, TLR4. TLR6, TLR7, TLR9, CD36, TRAM, or Trif. (iv) TL.R2 binds to CMP in a cell-free condition, (v) During the first 15 minutes of exposure and ingestion in phagocytosis, cellular TLR2 binds to CMP maximally with activation of the TLR2/MyD88 pathway resulting in MAPK/NFκB-medialed ThI cytokine production, (vi) Production of the Th I cytokine TNFα following phagocytosis of CMP is significantly reduced by M2 Mø compared to those produced by norma] [steady-state] and M 1 M0.

[0132] In general, TLR2 is a cell surface receptor that senses a diversity of structures including lipids, acylated sugars and proteins, and certain polysaccharides derived from not only bacteria, fungi and virus but also host inflammatory components, although bacterial lipoproteins are the most potent TLR.2 agonists. TLR2 needs to form hetcrodimers with either TLR 1 or TLR6 to generate signals and these TLR2/1 and TLR2/6 complexes discriminate different microbial soluble products. For example, triaeylated bacterial lipoproteins (mimicked by the lipopeptide Pam3CSK4) and diacylated lipoproteins (including the lipopeplide MALP-2, from mycoplasma) activate cells through TLR2/1 and TLR2/6 hetcrodimers, respectively (33, 34). The results herein, indicate that neither TLRl nor TLR 6 is required for CMP-induced TNFα production. It is possible that TLRl O, which recognizes triaeylated lipopeptides (35, 36), would cooperate with TLR2 in the recognition of CMP. Furthermore, TLR2 functionally interacts with structurally unrelated co-receptors like Dectin-1 , CD 14 and CD36 which serve to increase the repertoire of agonists for TLR2 (30, 31, 37. 38). The study herein indicates that CD 14 is involved in the recognition/signaling of CMP-induced Mø activation. Neither CD14 nor Dectin-1 directly binds to CMP during phagocytosis. Further studies will confirm whether co- receptors cooperated for CMP-induced Mø activation are distinct from those that mediate activation by soluble TLR2 agonists, which require cither TLRl or TLR6.

[0133] Intraperitoneal administration of CMP and LCB induces Ml and M2 activation of mouse peritoneal M0 depending on particle sizes. Using the M0 activation model, PAPC profiles were characterized in these two polarized peritoneal M0 populations. These results indicate that PAPC profiles differ among Mø activation stages, indicating that the PAPC profile could bε a useful alternative for experimental determination of Mø activation stages. Current methods available for characterization of Ml and M2 Mø activation states measure individual key phenotypes and take at least 2 - 3 days ( 14, 39), whereas this method using multicolor cytometric detection of PAPC profiles lakes only a few hours. Further studies should establish a reliable method to conventionally monitor PAPC in clinical samples including blood monocytes from patients with chronic inflammatory diseases.

[0134] The evolutionarily conserved chilin-binding 18-glycosyl-hydrolase family contains true chitinases and chitinase-likε proteins that bind chitin, but lack enzymatic activity to lyse chitin. Both types of chilinase, such as acidic mammalian chitiriase (AMCase) and chilinase-like proteins such as chitinase 3-like 1 (CH 13 LI, also called breast regression protein 39 [BRP39], and a human homologue YKL-40) are increased in Th2 -mediated allergic inflammatory disease and are pro-allergic in mice (40-42). Mouse CHI3L1 is also expressed in exaggerated quantities correlating with disease activity in cancers and colitis (26, 43). Studies of mice deficient in CHI3L1 also show that this chitinase induces 'alternative' Mø activation, enhances allergen sensitization and IgE induction, and is a therapeutic target in Th2- and Mø-mediated disorders (41). Interestingly, patients with inflammatory_' bowl diseases as well as colorectal cancer frequently express increased serum chitinases and chitinase-like proteins (44). CH13L1 is increased in Mø and epithelial cells of the inflamed intestines of dextran sulfate sodium-induced colitis in mice, probably enhancing the adhesion and internalization of commensal microbes into colonic epithelial cells where the microbes act as "surrogate pathogens" to stimulate a chronic inflammatory response (26). Interestingly, oral administration of CMP potently reduces intestinal CHI3L1 decreasing bacterial adhesion and invasion into colonic epithelial cells and Mø (13, 26). Therefore, PAPC profiles in inflammatory M0 provide potential prognostic biomarkers for close monitoring of treatments with immunotherapy (including oral CMP treatments) in chronic inflammatory diseases such as allergic asthma, colitis and colon cancer.

Example 2: EosinophiUa and alternative macrophage activation in response to chitin are particle size dependent

jOOSj Reagents and antibodies: Chitin microparlicles (1^■- 10 μm, CMP) and chitosan microparticlcs (chitosan-MP) were prepared as described above. Large chitin beads (>50 μm, LCB) and Scphadex G-IOO beads were purchased from New England Biolabs ( NEB, Ipswich, MA) and GE Healthcare Biosciences (Pittsburgh, PA), respectively, CpG-ODN (5' TCC ATG ACG TTC CTG ACG TT 3'; untnethylated) with phosphorothioate backbone was purchased from TriLink (Sorrento Mesa, CA), HK-M bovis BCG and HK-M tuberculosis (HK-Mtb) were prepared as described previously (Shinohara, T., et al. 2009. Am J Respir Cell MoI Biol 41 : 1 Ab- 154).

[0136] Mice: Non-pregnant female C57BL/6 and Balb/^'c mice, 8 - 14 weeks old, were obtained from Jackson Laboratory (Bar Harbor, ME). Homozygous CRTH2"^" breeders with C57BL/6 or Balb/c backgrounds were previously described (Satoh, T., et al. 2006. J Immunol 177:2621-2629) and bred at Florida Atlantic University. Mice were maintained in barrier- filtered cages and fed Purina laboratory chow and tap water ad libitum. Experimental protocols employed in this, study were approved by the !ACUC of Florida Atlantic University.

[0137] Peritoneal Mø preparation following in vivo treatment with CMP, LCB, cJύtosan-

MP, HK-BCG or saline; Groups of mice (4 - 5 /group) received 1 mg CMP, 1 rag LCB, 1 nig chitosan-MP, 1 mg HK-BCG or 0.1 ml saline intrapcritoneally (i.p.) on day 0. In some groups, mice were given 3 mg/^'kg ratnatroban (Cayman Chemical, Ann Arbor, MI), a CRTFI2 antagonist, twice on days (-) 1 and 0 before intraperitoneal particle treatment. Peritoneal lavage with 5 ml saline was performed on day 1. Nucleated peritoneal cells, (PC) were counted with a Coulter counter ( Model Zl, Beckman Coulter, Hialcah, FL). Differential cell counts were performed manually on cytospin preparations (Shandon Southern Instruments Co,, Scvvickley, PA) stained with Diff-Quik, To enrich plastic-adherent Mø, following washing with serum- free RPMI 1640. 2 x 10⁶ peritonea! cells/ml were suspended in RPMI 1640 plus 5% heat-inactivated fetal bovine serum (FBS) and incubated in culture dishes for 2 h. Non-adherent cells (lymphocytes) were removed by washing with warmed media. Adherent Mø were cultured with 100 μg/ml CMP, LCB or HK-BCG for an additional 6 or 24 h.

RA W 264.7 cell culture: RAW 264.7 cells (American Type Culture Collection, Maπassas, VA) were grown arid maintained in RPMI 1640 containing 5% FBS.

[0139] Western blot analysis: Peritoneal Mø prepared as described above, were washed 3 times with cold saline. Washed cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 4 mM EDTA, 0.1% SDS, 1:500 protease inhibitor cocktail [P8340, Sigma Aldrich], 1% Nonidct P-40, 0.1% sodium deoxycholatc, 10 niM NaF, and 2 mM NaWO₄). Debris, was eliminated by ccnlrifugation (10 niin, 10,000 g) and protein concentration in the lysate was measured by bicinchoninic acid assay (Pierce, fioekford, IL) with bovine serum albumin as standard. Equal amounts (2 - 5 μg) of protein from each sample were separated by SDS-PAGE. Proteins were then transferred to a polyvinylidene difiuoride ( PVDF) membrane (Millipore, Bedford, MA). The membrane was blocked with 10% nonfat dry milk, and incubated with antibodies (anti-Arg I, 1 :200 [BD Transduction Laboratories, San Diego, CA] at 22°C; and at 4°C for anti-chitinase 3-like 1 (CHDLI), 1:5,000 (30), anti-iNOS, 1 :4,000 [Upstate,

Temecula, CA], anti-cyclooxygenase 2 (COX-2), 1 :800 [Cayman Chemical] and anti-β actin, 1 :5,000 [Sigma] for detection of β-actin as constitute vely expressed protein control) in 7.5% nonfat dry milk, overnight. Following incubation with peroxidase-conjugated goat anti-rabbit IgG for CHBLl, iNOS and COX-2 detection, or donkey anti-mouse IgG for Arg I and β-actin (both 1:10,000; Jackson ImmunoRcscarch. West Grove. PA), stained proteins were detected by chemiluminescence ( ECL plus, Amersham, Piscataway, NJ) following the manufacturer's instructions.

Measurement of mediators: CpG (1 μg/nil) or saline were added to cultures of RAW cells (5 x 10⁵ /mi) or PC (2 x I O⁶ /ml) at 0 h and incubated at 37°C for 6 h (tor TN Fa) and 24 h (for IL-10). CMP or LCB at 100 μg/ml or saline were added to cultures at 0 h unless specified. TNFα and IL-10 levels in culture supernatants were measured by two-site ELlSA specific for the respective cytokines (BD Biosciencc, San Diego, CA). Nitrite as an indicator of nitric oxide (NQ) was measured by the Griess Reagent.

[0141] Cytometric detection of surface antigens: Standard methodology was used for the direct and indirect immunofluorescent characterization of peritoneal leukocytes. In brief, 10⁶ PC were incubated with Fc Block (BD PharMingen, Franklin Lakes, NJ) in 50 μl of PBS for 5 minutes to block non-specific binding of antibodies to cellular Fc receptors. Subsequently, optimal concentrations of Phycoerylhrin (PE)-conjugated F4/80 antibody and single oilier tluorochromc- conjugated antibodies recognizing surface antigens (TLR2, MR, CD205, SIGN-RI, Dectin-1 ) purchased from BioLegend, San Diego. CA, and BD PharMingen were added in 50 μl of FACS buffer for dual staining. After 30 minutes incubation on ice, the cells were washed three times with FACS buffer and fixed in 200 μl of 1% paraformaldehyde. Fluorescence of stained cells was quantified with a FACSCalibur flow cytomctcr (Bccton Dickinson, Franklin Lakes, NJ) using the Flow Jo program (Tree Star, Ashlaπd, OR), M0 fractions were electronically gated by forward- and side-scatter, and positive expression of F4/80.

[0142] Confocal microscopy of intracellular CMP: R AW264.7 cells that phagocytosed

CMP were fixed in 4% paraformaldehyde in PBS, pH 7.5, for 40 min. The fixed cells were perraeabilized with 1% Triton X-100 in PBS for 15 rain and incubated in blocking buffer consisting of PBS with 10% FBS 2 h at 22⁰C prior to incubation with FITC-chitin binding protein (1:500, Bacterial chitinase, NSB) and anli-CHDLl , 1 :1 ,000 in blocking buffer, overnight at 22⁰C. Subsequently, cells were washed with PBS three times and incubated with Cy3- conjugaled donkey anti-rabbit IgG (1:500; Jackson ImmunoRcsearch, West Glove. PA) for 1 h at 22°C. After washing three times, nuclei were stained with 4", 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingamc, CA) at 1.5 μg/'ml. Cells were examined with a laser scanning confocal microscope (LSM700, ZEISS, Thorawood, NY).

[0143] IL-IO rnRNΛ detection by RT-PCR: IL-10 expression was analyzed by RT-PCR as described previously (Nishiyama. A., et al. 2008. Am J Physio! Cell Physiol 295:C341-349). Briefly, after stimulation of RAW cells (5 ^x 1 C /ml) with CpG or saline for 6 or 24 h in the presence or absence of CMP, RNA extraction with TRizol reagent (Invitrogen) and reverse transcription with Superscript™ First-Strand Synthesis System for RT-PCR (Invitrogen) with oligo-(dT) primer were performed. PCR primers used in this study were: IL-10 (forward: 5^"- GGT TGC CAA GCC TTA TCG GA-3'. SEQ ID NO: 1; reverse: 5'-ACC TGC TCC ACT GCC TTG CT-3', SEQ ID NO: 2) and GAPDH as constitute vely expressed control (forward: 5'-TTC ACC ACC ATG GAG AAG GC-3\ SEQ ID NO: 3: reverse: 5'-GGC ATG GAC TGT GGT CAT GA-3' SEQ ID NO: 4).

[0144] Endotoxin removal: Endotoxin was removed from soluble materials for culture by filtration and sterilization through a 0.22-μm Zctapore membrane (AMF-Cuno; Cuno, Mcriden, CT). The final preparations were monitored for endotoxin by the Limidus amebocyte assay (Sigma).

[0145J Statistics: Data were analyzed by one way analysis of variance. For cell culture studies, tissues isolated from at least 4 mice were pooled unless indicated; cells were cultured in at least triplicate in each group. P <0.05 was considered statistically significant. [0146] Realty.

[0147] Sizes of CMP and LCB: Cliitin microparticlcs (CMP) used in this study were sized cylometrically with 1 and 10 μrn latex beads as standards. Over 96% of particles were 1 - 10 μm diameter (Figure 8A), Figure 8B shows that both typical large chitin beads (LCB; obtained from NEB) and Sephadex G-100 beads are >50 μm although the majority of LCB are slightly bigger than the Sephadex beads. Figures SC and 8D show that RAW264.7 Mø expressed CHB Ll constitutively in the cytosol and nuclear region. When CMP were phagocytosed, intracellular CMP were detected by FITC -bacterial chitinase (Figure 8D). The distribution and appearance of cellular CF113L1 (Figure 8C) was not significantly altered by phagocytosis of CMP (Figure 8D). Phagocytosis of CMP is required for Ml Mø activation, which docs not occur following phagocytosis of latex beads or chitosan-MP.

[0148] Size-dependent particle induction of non-all er genie eosinophilia. To compare the effects of particle size and composition, mice were given intraperitoneal injections of 1 mg LCB, 1 mg Sephadex beads, 1 mg CMP. 1 mg HK-BCG or saline. Eosinophils in the peritoneal fluid were counted and the results are shown in Figure 9. These results indicate that eosinophilia occurs only in response to LCB and Sephadex beads. In contrast, the smaller phagocytosable sizes of CMP and HK-BCG did not induce eosinophil migration to the peritoneal cavity (Figure *^■>).

[0149] PGD₂, a major prostanoid produced by activated mast cells, has long been implicated as an essential Th2 mediator up-regulating allergic disease and attracting eosinophils through CRTH2, a PGD₂ receptor. To determine whether Sephadex beads and LCB induce local eosinophilia in a CRTH2-depcndent manner, CRTH2^"'" and WT mice were given the two non- phagoeytosabie particles inlraperiloneally. As shown in Figure 1 OA there was a significant decrease in eosinophils in CRTH2^"'^" mice. To further confirm the role of CRTH2, groups of C57BL/6 mice were treated orally with the CRTH2 antagonist ramatroban (3 mg/kg, Cayman), before intraperitoneal administration of LCB. which also resulted in significantly reduced LCB- induced eosinoplύlia (Figure 10B), These results indicate that these large, nori-phagocytosable beads induce local eosinophilia that is independent of the specific carbohydrate composition of the particles, and that migration is regulated, at least in part, by CRTH2 and endogenous PGD;, possibly derived from LTB₄-slimulaled mast cells. LCB-indιιced eosinophilia and the role of CRTH2 were observed in both Balb/c and C57BL/6 mouse strains. Chitin particles induce M2 Mø activation in a size-dependent manner. As shown in Figure 11, there was a greater expression of Arg I, a marker of M2 Mø, in peritoneal Mø isolated from WT mice treated with intraperitoneal LCB than in those from mice treated with saline, CMP or HK-BCG. Neither CMP nor LCB administration resulted in increased

CHOL 1 expression in peritoneal Mø. Unlike eosinophiiia, which was dependent on CRTH2 (Figures 1OA, 10B), Arg I expression induced by LCB was independent of CRTH2 (Figure 1 i).

[0151] Selected surface antigens expressed by F4/80^" peritoneal Mø were determined cytomctrically. As shown in Figure 12, TLR2, CHBLl, maniiosc receptor (MR), CD205, Dectin-1 and SIGN-R l were expressed by normal peritoneal Mø. Intraperitoneal CMP resulted in increased mean fluorescent intensity (FI) values for Declin-1 , decreased for MR, CD205 and SlGN-Rl, and almost unchanged for TLR2 and CHBLl. In contrast, Mø activated by LCB had decreased FI values for TLR2, CH13L1, CD205 and SlGN-Rl , and almost unchanged for MR (Figure 12), Dectin-1 expression by F4/80-positive M0 in response to intraperitoneal LCB was biphasic with predominantly decreased FI relative to saline control cells while for other Mø the Dectin-1 F! was increased relative to the controls (Figure 12). In accord with the changes in antigen expression, Fl ratios of selected antigens reflected normal. Ml and M2 Mø populations. For example, the FI ratios of TLR2:MR in normal/Ml (CMP)/M2 (LCB) peritoneal M0 were 1/2/0.5.

[0152] Neither CMP nor LCB induces IL-10 production in vitro. To determine whether peritoneal administration of these particles induces local production of IL-10, TNFα and nitrite levels in peritoneal lavage were measured. Figure 13 shows that neither CMP nor larger particles (LCB and Scphadcx G-IOO) induced local IL-10 production, although CMP induced TNFα and nitrite. HK-BCG, as a positive control, increased all three mediators (Figure 13). However, iNOS was not detected by western blotting in peritoneal M0 isolated from mice given intraperitoneal saline (control), CMP or LCB, indicating the possibility of another NOS enzyme or another cell type as a source of NO.

[0153] IL-10 production ex vivo by peritoneal Mø: It was also investigated whether peritoneal M0 are activated by i.p. CMP, LCB or chitosan-MP produce IL-10 when further stimulated ex vivo with CpG. Mø agonists derived from bacteria including CpG, LPS, FIK-BCG and FIK-Mtb induce IL-IO production, and from these, CpG was chosen as an agonist for the present study. As shown in Figure 14, CMP-elϊcited peritonea] M0 decreased !L-IO production, However, there was no effect of pre-treatmerit of mice with LCB or chitosan-MP on IL-I O production in response to CpG when compared with saline prc-treatment (Figure 14). The results also have shown that splenic Mø isolated from C57BL/6 mice given intraperitoneal HK-BCG produced a large amount of IL-IO ex vivo whereas the amounts were significantly reduced for splenic Mø from mice treated with a mixture of BCG/CMP. These results evidence that CMP reduces IL-IO levels under inflammatory conditions that have elevated IL-10.

[0154] In vitro effects of chiiin particles on Mø activation: RAW264.7 Mø-likc cells treated in vivo with LCB or Scphadcx G-100 beads, did not express iNOS or COX-2 (Figure 15). In contrast, RAW cell activation induced by CMP, HK-BCG or HK-Mtb, resulted in the expression of LNOS and COX-2 (Figure 15), despite the results noted above for the undetectable iNOS expression by peritoneal Mø treated with CMP or LCB in vivo. RAW cells expressed CHBLl constitutively which was unchanged by any of die treatments (Figure 15). Although Arg I was detected for 25 μg/lane protein samples from RAW⁷ cells that were treated with a mixture of IL- 4, IL- 13 and cholera toxin, protein samples from the LCB-stimulated Mø group at 50 μg/lane for western blotting showed no Arg I bands for any of the treatments listed. These results demonstrate that the in vivo and in vitro effects of LCB are distinct for these markers of Mø polarization.

|0155i OW/³ reduces in vitro CpG-induced IL- 10 production by RA W 264.7 M0-like cells: To further characterize the CMP-induccd decrease in IL-10 production following exposure to CpG, RAW cells were employed. Like peritoneal and splenic Mø from normal mice, RAW⁷ cells produce IL-10 in response to CpG, but not CMP. In this study, R AW^r cells were stimulated with CpG accompanied by pre- or post- treatment with CMP. As shown in Figure 16, IL-10 production was inhibited by not only pretrcatment with CMP, but also up to 6 h post-treatment. Similar inhibitory effects of CMP were also observed for HK-BCG-induced IL-10 production. When the culture supematants isolated from RAW cell cultures treated with CMP were added to CpG-stimulated RAW cell culture, IL-10 production was not reduced evidencing that the suppression of IL-10 production by CMP is probably not associated with an endocrine or paracrine soluble mediator. [0156] To determine whether phagocytosis of CMP is required for the down-regulation of IL- 10 production, RAW cells were pre-treated with cytochalasin D, an aclin polymerization inhibitor, before stimulation with CMP plus CpG. Cytochalasin D is known to inhibit CM P-induced MAPK activation and ThI cytokine production but docs not inhibit CpG-induccd MAPK activation. Although cytochalasin D slightly inhibited IL- 10 production by CpG alone, our results (Figure 17) indicate thai there is no further inhibitory effect of CMP on IL-IO production in the presence of cytochalasin D, which is consistent with MAPK -mediated suppression of IL- 10 production in response to CMP. As shown in Figure 18, CMP did not alter CpG-induced IL- 10 raRNA levels at 6 h and 24 h, suggesting post-lranscriplional regulation of IL-10 production by CMP.

[0157] Discussion:

[0158] Chit in microparticles (CMP) have been studied as a potential treatment for allergic disease. The results presented here indicate that intraperitoneal administration of CMP induces Ml activation in peritoneal M0, without inducing eosinophil migration. In contrast, non- phagocytosablc LCB purchased from NEB induce eosinophil migration within 24 h after intraperitoneal administration. Intraperitoneal administration of Sephadex G-100 (cross-linked dcxtran) beads in this study (Figure 9) or Scpharose (agarose) beads (galactose polymer, 40 -- 145 μrn, Sigma) also indicated induction of peritoneal eosinophilia, Thus, the results confirm that Ml Mø activation by chitin particles requires both a phagocytosablc size and the chitin chemical structure. It is further shown that peritoneal eosinophilia induced by LCB is particle- size dependent but carbohydrate composition independent (Figure 9).

[0159] Non-allergenic eosinophil io and M2 M 0 activation by non-phagocyiosahle sizes of particles. In addition to peritonea! eosinophilia, lung eosinophilia and granuloma formation are also induced in experimental animals by non-allergenic stimuli such as intravenously or intratracheal Iy administered Sephadex beads. The palbohistologic changes induced by Sephadex beads are associated with non-spccifically increased Th2 cytokines such as IL-4, IL-5, IL-13 and colaxin in the lungs (Maddad el, B., et a3. 2002. J Immunol 168:3004-3016) and airway hyperresponsiveness ( Laycock, S. M., et al. 1987. Im Arch Allergy App I Immunol 82:347-348). Taken together, the results herein, evidence that exposure to non-phagocytosablc. carbohydrate particles of either chitin or dcxtran induces local eosinophilia along with Th2/M2-mediated i nil animal ion.

In vitro studies of CM P-induced Ml M0 activation includes phagocytosis and results in MAPK activation within 20 min followed by ThI cytokine production. TLR2 and MR are binding proteins in Ml Mø activation by CMP. When M0 are treated in vitro with LCB, neither Ml nor M2 Mø activation occurs which demonstrates that LCB-induccd M2 M0 activation requires additional in vivo factors which may include additional cells, humoral factors or both.

[0161 ] Eosinophilia but not Ml M '0 ^'activation is regulated by CRTHl: PGD₂ acts via the

G-protein-coupled receptors, D-prostanoid receptor (DP) and CRTFI2. The expression patterns and signaling pathways for DP and CRTH2 are different, indicating that they have distinct roles in allergic responses. It appears that signals via DP promote eosinophil survival, whereas signals via CRTH2 mediate shape change, chcmotaxis, and degranulation of eosinophils. In human nasal mucosa, CRTH2 is expressed on eosinophils and a subset of T cells. Eosinophil infiltration into mucosal sites appears to be regulated by mucosal CRTH2. but not DP. These studies clearly indicate that non-allcrgcnic eosinophilia induced by LCB is, at least in part, dependent on CRTH2. However, M2 Mø activation by LCB, indicated by Arg I expression (Figure 1 1), is independent of CRTH2. consistent with induction of eosinophil migration in response to local activation of M2 Mø.

|0162j IL- 10 production: M0 from immunocompromised populations (infected, aged, neonates, tumor-bearers, atherosderotics, and diabetics) frequently express high levels of TL-H). which potentially impact host defense and reduce the efficacy of immunotherapy. Recent studies have demonstrated that Mø rather than T cells are the major source of IL-I O in tuberculosis and Mø-derived IL-IO triggers aspects of M2 activation including enhanced Arg-I and promotion of M tuberculosis reactivation (Schreiber. T., et al, 2009. J Immunol 183:1301-1312). The suppression of excessive IL-10 production, therefore, could be beneficial for

immunocompromised populations .

[0163] A further provocative finding from the studies herein, is that on-going IL-10 production by Mø in vivo can be down-regulated by prc- or post-treatment with CMP (Figure 9), indicating

- 4S - a therapeutic potential for CMP in the down-regulation of immunosuppressive IL- 10 in inflammation arid infections, IL-IO production by MO may be regulated transcriptionally arid post-transcriptionaily. Activation of transcription factor SpI via p38 induces transcription of the IL-IO gene in LPS-stimulaicd THP-I cells. In addition, the 3 ^"untranslated region of IL-IO mRNA plays a repressive role in IL-K) translation, indicating translationai regulation. In apparent contrast to results reported here, Da Suva ct al, (Da Silva. C. A., ct al. 2009. J

Immunol 182:3573-3582) found that <40 μm (largely 2 - 10 μm) chitiri particles, but not 40 - 70 μm chitin particles, induce IL-10 production by thioglycollatc-clicitcd peritoneal Mø in a manner dependent on TLR2 and Dcctin-1 but independent of phagocytosis.

[0164] The results herein show that, compared to Mø from control mice, some peritoneal Mø from mice treated intrapcritoneally not only with LCB but also with CMP express higher levels of Dectin-1, a β-glucan receptor (Figure 12). Although Dectin-1 is known to augment yeast zymosan-stimulated IL-10 production, neither CMP-induced Ml nor LCB-induced M2 M0 activation resulted in increased IL-10 production. Further studies will be conducted to define the regulation of IL-10 production following CMP phagocytosis by different tissue Mø isolated from mice with normal and inflammatory conditions, and to examine possible differences in thiogiycollate-elicited peritoneal M0.

5] Chilin and CIlBLl in Mø activation, Chitinase 3-like-l (CTII3L1; also known as

YKL -40. breast regression protein 39, or human cartilage glycoprotein 39), which binds chitin, but lacks enzymatic activity to lyse microbial cell walls, is expressed primarily by epithelial cells and Mø of inflamed intestines in dextran sulfale-induced colitis. CHI3L1 , which can be uprcgulatcd after pro-inflammatory cytokine stimulation, enhances the adhesion arid

internalization of commensal microbes into colonic epithelial cells, where the intracellular microbes as "surrogate pathogens" stimulate a chronic inflammatory response. Further, in vivo neutralization of CHI3L1 significantly suppresses the development of dextran sulfate-induced colitis by decreasing bacterial adhesion and invasion into colonic epithelial cells and M0. ThI and Th2 cytokines enhance CHI3L1 production in vitro. Studies of CHBLl '^" mice (Lee, C. G., εt al. 2009. J Exp Med 206:1 149-1 166) demonstrated that CHI3L.1 induces M2 Mø in vitro and regulates lung eosinophilia in mice with allergen-induced Th2 responses. In the current study, peritoneal Mø and RAW264.7 cells constitutively express CHI3L1. The total cellular CHI3L1 levels are unchanged during CMP phagocytosis/Ml activation or M2 activation by treatment with LCB (Figures 1 1 and 15), although the surface CBlBLl level seems Io be slightly decreased in peritoneal M0 treated in vivo with LCB but not CMP (Figure 12), It was also found that prε- treatment of RAW cells with the neutralizing anti-CHBLl prior to CMP exposure did not affect the stimulation of TNFα. It seems unlikely that CMBLl regulates 'acute/innate' Mø activation by CMP, LCB, or mycobacteria, or probably non-allergenic eosinophil migration in these models.

[0166] In conclusion, a clinically useful ThI adjuvant should stimulate innate immunity and promote acquired cell-mediated immune responses through development of Ml M0, eliminate intracellular pathogens and down-regulate allergic/Th2 responses. However, a satisfactory Th 1 adjuvant with these properties is riot heretofore available. It is concluded that CMP induce Ml Mø while reducing inflammatory IL- 10 production. The recent contradictory report (Reese, T. A., et al. 2007. Nature 447:92-96) indicating the induction of M2 M0 activation and eosinophilia in response to chitin particles, negative features of ThI adjuvants, appears to be a consequence of using much larger, non-phagocytosable chitin beads (LCB); the data herein clearly demonstrate the size-dependence of the response to chitin. While the features of cellular responses to CMP in these studies were confirmed and its properties found to be suitable for a ThI adjuvant, the results also emphasize the importance of careful quality control in the production of phagocytosablc particles without introducing undesirable (Th2 responses with eosinophilia) effects. Therefore, the oral administration of phagocytosable sizes of CMP continues to be a potential therapeutic option as a Th 1 adjuvant in various Th2-mediated immunological diseases. Further studies directly comparing CMP and LCB would also show that the differential effects on local Mø activation of CMP and LCB given orally are comparable to those following intraperitoneal or intranasal administration.

10167 J Although the invention has been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired arid advantageous for any given or particular application. The Abstract of the disclosure will allow the reader to quickly ascertain the nature of the technical disclosure. It is, submitted with the understanding thai it will not be used to interpret or limit the scope or meaning of the following claims.

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" D l ™

Claims

What is claimed is:

1. A bioniarker for the prognoses or for diagnosing chronic inflammatory diseases comprising: C-type lectins frnannose receptor [MRj, DEC-205, Dectin-1. SIGN-Rl, MRC2); toll-like receptors (TLR2, TLR4, CD14): phagolysosomal proteins (LAMP-I, LAMP-2);

adhesion molecule /chemokinc receptor (CD44, L-sclectin [CD62L], CD 184 [CXCR4]);

CHBL l chitotriosidasc (chitinasc- 1 ), acidic mammalian chitinasc (AMCasc), chitinase3-likc-2 (CHI3L2), oviductal glycoprotein 1 or stabilin-1 interacting chitinase-likc protein (SI-CLP) markers.

The bioniarker of claim I, wherein detection of at least one marker or combinations thereof is prognostic or diagnostic of different clinical stages of chronic inflammatory disease.

3. The bioniarker of claim 2, wherein early stage chronic inflammatory disease is characterized by marker profiles comprising: mannose receptor { MR, CD206), DEC-205 (CD2051 β-glucan receptor (Dectin-1), SIGN-Rl (ER-TR9), Mannose receptor C type 2 (Endol80), TLR2, TLR4, CDl 4, LAMP-I (CDl 07a), LAMP-2 (CD107b, Mac-3), CD44 (phagocytic glycoprotein 1), CD62L (L selcclin), CD 184 (CXCR4) and CHI3L1.

4. The biomarker of claim 3, wherein expression of TL.R2 is increased with respect to a normal control and CD206, CD205. CD62L and CHI3L1 expression is decreased with respect to a normal control

5. The biomarker of claim 2, wherein late chronic stage chronic inflammatory diseases is characterized by detection of increased expression of markers comprising: mannose receptor (MR, CD206), DEC-205 (CD205), SIGN-Rl (ER-TR9), CD62L (L selectin), CD 184 (CXCR4) or CMDLl : and, decreased expression of markers comprising: TLR2 and LAMP-I as compared to a normal control.

6. A biomarker for evaluating therapy of a disease comprising: C-typc lectins (raannose receptor [MRJ, DEC-205, Dectin-1, SlGN-Rl, MRC2); toll-like receptors, (TLR2, TLR4, CD14); phagolysosonial proteins (LAMP-I, LAM P-2); adhesion molecule /chemokine receptor (CD44, L-sclectin [CD62L], CD 184 [CXCR4]); CHBLl chitotriosidase (chiiinase-1), acidic mammalian chitinase ( AM Case), chitinase3-like-2 (CHI3L2), oviductal glycoprotein 1 or stabilin-1 interacting chitinase-like protein (SI-CLP) markers.

7. A method of diagnosing a disease state in a subject, the method comprising the s of:

(a) obtaining a biological sample from the subject;

(b) detecting proteins associated with phagocytosed chitiii microparticles (PAPC)in the biological sample; and

(c) correlating the measured chitin binding protein profiles with a particular disease and obtaining a diagnosis,

8. The method of claim 7, wherein the subject is immunocompromised and correlating the detected proteins associated with phagocytosed chitin microparticles (PAPC) with a particular disease and obtaining a diagnosis comprises comparing the proteins associated with phagocytosed chitin microparticles (PAPC) to proteins associated with phagocytosed chitin rnicToparticles (PAPC) from an individual that is not immunocompromised or a population of individuals that are not immunocompromised.

9, The method of claim 7, wherein proteins associated with phagocytosed chit in microparticles (PAPC) comprises: C-type lectins (mannose receptor [Mil], DEC -205, Dectin-1 , SIGN-Rl, MRC2); toll-like receptors (TLR2, TLR4, CD14): phagolysosomal proteins (LAMP- 1 , LAMP-2); adhesion molecule /chemokine receptor (CD44, L-seleclin [CD62L], CD 184

[CXCR4]); CHI3L1 chitotriosidase (chitinasc-1), acidic mammalian chitinase (AMCasc), dύtinasc3-Kke-2 (CTI 13L2), oviductal glycoprotein 1 or stabilin-1 interacting chitinase-like protein ( SI-CLP): or combinations thereof.

10. A method of monitoring a subject's response to therapeutic treatment comprising the steps of:

(a) obtaining a first biological sample from the subject at a first time point;

(b) detecting proteins associated with phagocytosed chitin microparticles (PAPC) in the first biological sample;

(c) obtaining a second biological sample from the subject at a second time point, wherein the subject receives a therapeutic treatment after the first time point but before the second time point;

(d) detecting proteins associated with phagocytosed chitin microparticles (PAPC) in the second biological sample; and

(c) correlating a difference between the proteins associated with phagocytosed chitin microparticles (PAPC) in the first biological sample and the proteins associated with

phagocytosed chitin microparticles (PAPC) in the second biological sample with a response to the therapeutic treatment.

11. The method of claim 10, wherein proteins associated with phagocytosed chitin microparticles (PAPC) comprises: C-type lectins (marmose receptor [MRj, DEC -205, Dectin-1, SlGN-Rl, MRC2); toll-like receptors (TLR2, TLR4, CD14): phagolysosomal proteins (LAMP- 1, LAMP-2); adhesion molecule /chcmokinc receptor (CD44, L-sclcctin [CD62L], CD 184

[CXCR4]); CHBLl chilotriosidase (chitinase-1), acidic mammalian chitiπase (AMCasε), chitinasc3-like-2 (CHI3L2), oviductal glycoprotein 1 or stabilhi-i interacting chitinase-likc protein (SI-CLP); or combinations thereof.

12. The method of claim 10, wherein the therapeutic treatment is immunotherapy.

13. The method of claim 12, wherein the immunotherapy comprises administration of chitin microparticles. IA. A kit for measuring proteins associated with phagocytosed chitin microparticlcs (PAPC) in a biological sample from a subject, the kit comprising:

(a) chitin microparticles;

(b) at least one fluorochromc-conjugated antibody;

(c) lysis buffer; and

(d) instructions for use.

15. A method of preventing or treating eosinophilia hi vivo, comprising:

administering to a patient a therapeutically effective amount of chitin microparticlcs wherein the chitin microparticles have a diameter of between about 0.5 μm to about 15 μra; and,

preventing or treating eosinophilia in vivo.

16. The method of claim 15, wherein the diameter of the chitin microparticles is about I μrn up to 10 μm.

1 ^". The method of claim 15, wherein phagocytosis of the chitin microparticlcs by mononuclear cells express a protein profile comprising: C-type lectins (marmose receptor [MRj, DEC-205, Dcctin-1, SlGN-Rl, MRC2); toll-like receptors (TLR2, TLR4, CD 14);

phagolysosomal proteins (LAMP-I, LAMP-2); adhesion molecule /chcmokine receptor (CD44, L-selectin [CD62LJ, CD 184 [CXCR4]); CH13L1 chitotriosidase (chitinase-1), acidic mammalian chitiiiase (AMCase), chitinasc3-like-2 (CHI3L2), oviductal glycoprotein 1 or stabilin-1 interacting chitinase-like protein ( SI-CLP); or combinations thereof.

18. The method of claim 15, wherein the chitin microparticles induce classical activation (Ml ) of macrophages in vivo.

19. The method of claim 15, wherein the markers further comprise molecules which bind to chilin microparticles arid microbial chitinases that are released in the hosts during infections.