WO2008112559A1 - Suppression of galectin-3 for treating an inflammatory condition - Google Patents

Abstract

The present invention relates to the discovery that galectin-3 plays an active role in Th2-mediated inflammatory responses. More specifically, the invention provides for the novel use of a substance that suppresses galectin-3 activity, either by down-regulating galectin-3 expression or by neutralizing galectin-3 protein activity, in the treatment of a Th2-mediated inflammatory condition, such as atopic dermatitis.

Description

SUPPRESSION OF GALECTIN-3 FOR TREATING AN INFLAMMATORY CONDITION

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No.

60/905,937, filed March 9, 2007, the disclosure of which is incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with support under Grant Nos. ROl AI20598 and ROl AI39620 by the National Institutes of Health. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION [0003] Galectins are a family of animal lectins exhibiting a high level of evolutionary conservation, and family members are found in nematodes, insects, and mammals (Barondes et al., Cell (1994) 76:597-598). At least fifteen members have been identified in mammals. Members are defined by shared consensus amino acid sequences in the carbohydrate- recognition domain (CRD) and affinity for β-galactosides (Kasai et al., J Biochem (Tokyo) (1996) 1 19:1-8; Leffler et al, Glycoconj J (2004) 19:433-440). Despite sequence and structural similarities among the CRDs, different galectin CRDs can recognize overlapping but distinct panels of saccharide ligands, which maybe important for the discrete functions of different galectins (Rini et al, Curr Opin Struct Biol (1999) 9:578-584; Hirabayashi et al, Biochim Biophys Acta (2002) 1572:232-254). None of the galectin family members contain a classical signal sequence; however, many studies have demonstrated secretion of these lectins (Hughes, Biochim Biophys Acta (1999) 1473:172-185). A number of galectins have been shown to exert various biological activities through binding to and engaging cell surface glycoconjugates (glycoproteins and glycolipids). They have also been demonstrated to reside inside the cells and function intracellularly (Liu et al, Biochim Biophys Acta (2002) 1572:263-273). [0004] Galectin-3 is the only chimeric galectin that consists of a C-terminal CRD linked to a flexible non-lectin tandem repeats. It is widely distributed in normal and disease tissues. It is abundantly present in the epithelia of several organs, and also expressed by a number of immune and inflammatory cells, including B cells, T cells, neutrophils, dendritic cells, macrophages, mast cells, and eosinophils (Liu, Int Arch Allergy Immunol 2005, 136:385-400; Chen et al., Arch Immunol Ther Exp (Warsz) 2005, 53:497-504). Their expression levels are known to be dependent on cell differentiation and activation (Joo et al, J Leukoc Biol 2001 , 69:555-564; Liu et al, Am J Pathol 1995, 147:1016-1028; Nangia-Makker et al, Cancer Res 1993, 53:5033-5037). Various intracellular and extracellular functions of galectin-3 have been demonstrated in vitro. Extracellular galectin-3 has been shown to activate a variety of cells and play important roles in cell-cell and cell-extracellular matrix interactions, which are mediated by their carbohydrate-binding properties. Galectin-3 also plays a role in regulation of cell growth, cell cycle progression, and apoptosis in a carbohydrate-independent manner (Liu 2002, supra).

[0005] The functions of galectin-3 in various immune cells have been studied by employing recombinant galectin-3 as well as genetic approaches, including the use of transfectants overexpressing the protein, cells in which the protein expression is suppressed, or cells from galectin-3-defϊcient (gal3^~/-) mice. Endogenous galectin-3 has been shown to be anti-apoptotic in T cells (Yang et al, Proc Natl Acad Sci USA 1996, 93:6737-6742). This function has subsequently been confirmed in various cell types against different stimuli and existing data suggest that endogenous galectin-3 mediates this function through interacting with intracellular molecules (Liu 2002, supra). Conversely, exogenously added galectin-3 was found to induce apoptosis in T cells through binding to and engaging cell surface glycoproteins (Fukumori et al, Cancer Res 2003, 63:8302-831 1 ; Stillman et al, J Immunol 2006, 176:778-789). [0006] Extracelluar galectin-3 has been shown to function as a chemoattractant for monocytes, as well as peripheral blood and alveolar macrophages both in vitro and in vivo (Sano et al, J Immunol 2000, 165:2156-2164). More recently, by comparing cells from gal3^+/+ and gal3^{" '"} mice, Sano et al have shown that endogenous galectin-3 plays a critical role in the phagocytic response of macrophages (J Clin Invest 2003, 112:389-397). Galectin- 3 has also been shown to play an important role in the mast cell response, also by functioning intracellularly, in studies of mast cells from gal3-/- mice (Chen et al, J Immunol 2006, 177:4991-4997).

[0007] Atopic dermatitis (AD) is a chronic, relapsing, inflammatory skin disease characterized by pruritic, eczematous skin lesions. The prevalence of AD has increased by two- to three- fold during the past three decades in industrialized countries, where the current prevalence in children is estimated to be 10-20%. Various studies indicate that AD has a complex etiology, with activation of multiple immunologic and inflammatory pathways. Studies of affected skin lesion in AD patients suggest that T cells play a critical role in the pathogenesis of the disease, lmmunohistologic analysis reveals a mononuclear cell infiltrate, predominantly in the dermis, consisting of activated memory CD4⁺ T cells bearing HLA-DR⁺ and CD45RO⁺, as well as macrophages (Leung, Springer Semin Immunopathol 1992, 13:427- 440).

(0008] Following antigen stimulation, CD4⁺ T helper cells (Th) can develop into ThI cells (which generally secrete IFN-γ) or Th2 cells (which generally secrete IL-4, IL-5 and IL-13). It has been proposed that Th2 cells play a key pathogenetic role in AD, and this is supported by the presence of peripheral blood eosinophils and enhanced serum IgE levels in the majority of AD patients. In acute lesions of AD, the number of cells expressing IL-4, IL-5 and IL- 13 mRNA and protein increases substantially, suggesting preferential accumulation of Th2 cells and that Th2 cytokines contribute to the initiation of the inflammatory response. It is generally believed that the Th2 cytokine milieu predominates in initiating stages and acute lesions of AD. A more mixed ThI and Th2 pattern exists in chronic lesions (Grewe et ai, Immunol Today 1998, 19:359-361).

[0009] The present invention is based on the discovery that eliminating the effects of galectin-3 reduces the development of inflammatory responses, such as that observed in atopic dermatitis.

BRIEF SUMMARY OF THE INVENTION

[0010] Accordingly, the invention provides inhibitors of galectin-3 in compositions and methods for the prevention and/ or reduction of inflammatory responses and conditions.

[0011] In some embodiments, the invention provides a method of preventing and/ or treating an inflammatory condition comprising the step of administering to a subject an effective amount of an inhibitor of galectin-3. In some embodiments, the inflammatory condition is a Th-2 mediated inflammatory condition, such as asthma or an allergic reaction. In some embodiments, the inflammatory condition is atopic dermatitis (AD). [0012] In some embodiments, the subject is experiencing AD symptoms. In some embodiments, the subject is at risk of experiencing AD symptoms. For example, the subject may have experienced symptoms of AD in the past, have been exposed to a triggering irritant or event, or have a predisposition for AD. [0013] In some embodiments, the method further comprises administration of an additional therapeutic compound. For example, the galectin-3 inhibitors of the invention can be administered in combination with an anti-inflammatory, pain reliever, or anti-histamine. In some embodiments, the galectin-3 inhibitor is administered at the same time as, or even in the same composition as, the additional therapeutic compound. In some embodiments, the galectin-3 inhibitor is administered at a different time than the additional therapeutic compound, in a separate composition.

[0014] In some embodiments, the galectin-3 inhibitor is an inhibitory nucleic acid. For example, the galectin-3 inhibitor can comprise a sequence complementary to a galectin-3 polynucleotide (e.g., siRNA or antisense). In some embodiments, the inhibitory nucleic acid is complementary to 10-30, 25-50, 50-100, 100-500, or more nucleotides of the galectin-3 polynucleotide. In some embodiments, the galectin-3 inhibitor is an RNA aptamer. [0015] In some embodiments, the galectin-3 inhibitor is an inactivating antibody. In some embodiments, the inactivating antibody is an antibody fragment that specifically binds galectin-3.

[0016] In some embodiments, the galectin-3 inhibitor is a galectin-3 ligand inhibitor. In some embodiments, the galectin-3 ligand inhibitor is a natural ligand, e.g., beta-galactosidase. In other embodiments, the galectin-3 ligand inhibitor is a derivative natural ligand, e.g., a modified version of a natural ligand. In some embodiments, the galectin-3 ligand inhibitor is selected from the group consisting of: galactose, thio-galactoside, thiodi-galactoside, a glycolipid, a glycopeptide, a proteoglycan, a saccharide, a glycodendrimer, N-acetyl lactosamine, C3' amide, or sulfonamide.

[0017] In some embodiments, the galectin-3 inhibitor is a peptide inhibitor. For example, the galectin-3 inhibitor can comprise a galectin-3 peptide, i.e., a subsequence or fragment of the galectin-3 polypeptide. In some embodiments, the peptide inhibitor is a subsequence from the N-terminus, the C-terminus, or the carbohydrate recognition domain of galectin-3. In some embodiments, the peptide inhibitor can be 10-20, 20-30, 30-40, 50-60, 60-75, 75- 100, 100-150, 150-200, or more amino acids of galectin-3. In some embodiments, the galectin-3 peptide inhibitor is not a fragment of galectin-3. [0018] The invention also provides pharmaceutical compositions, said composition comprising an effective amount of an inhibitor of galectin-3 and a pharmaceutically acceptable carrier, wherein the inhibitor is present in an amount effective to suppress galectin-3 expression or activity. [0019] In some embodiments, the composition further comprises an additional therapeutic compound. In some embodiments, the additional therapeutic compound can be an antiinflammatory, pain reliever, or anti-histamine.

[0020] The invention also encompasses kits for treating an inflammatory condition, for example, a kit comprising a pharmaceutical composition comprising a galectin-3 inhibitor. In some embodiments, the kit comprises an additional therapeutic compound. In some embodiments, the kit also comprises instructional material.

[0021] In some embodiments, the invention provides methods of identifying a galectin-3 inhibitor. In some embodiments, the method comprises contacting a sample comprising galectin-3 polypeptide with a test agent, and determining the effect of the test agent on galectin-3 activity. In some embodiments, the method comprises contacting a sample comprising galectin-3 polynucleotide with a test agent, and determining the effect of the test agent on galectin-3 expression. The sample can be an in vitro sample, e.g., a cell culture, in situ tissue sample, or acellular assay, such as an affinity assay or ELISA. In some embodiments, galectin-3 is expressed in a cell or on the cell surface. In some embodiments, the method comprises contacting a test agent with an individual (e.g., human or non-human mammal) in vivo to determine the effect of the test agent on galectin-3 activity or expression.

BRIEF DESCRIPTION OF THE DRAWINGS [0022] The figures display results from mice with either a wild type or knocked-out (null) galectin-3 gene. Wild type mice are designated as either ga!3^{+ +} or WT. Knock-outs are designated as either gal3^" '^' or KO.

[0023] Figure 1. OVA-sensitized skin sites of gal3^~/- mice showed a lower degree of thickening of the epidermis. Mice were sensitized by three one-week periods of epicutaneous ovalbumin (_OVA) or saline application, each two-weeks apart. Skin sections were obtained from the treated areas 24 h after the third sensitization period. Skin sections were stained with H&E for histological analysis (magnification x 200) and epidermal and dermal thickness was measured. The skin thickness was determined from the average of at least 10 measures for each sample. Bars represent mean ± SEM. *P<0.05. [0024] Figure 2. Eosinophil and mononuclear cell infiltrations were decreased in

OVA sensitized skin sites of gal3^{~ ~} mice. The number of infiltrating cells in sensitized skin sites is indicated. Eosinophils, _mast cells and mononuclear cells in the dermis were counted in 20 high-power fields (HPFs) at x 1000 per mouse. Bars represent mean ± SEM. *P<0.05. [0025] Figure 3. Gal3^~7~ mice exhibited a lower IgE response, but a higher antigen- specific IgG_2a/IgG] index. Mice were bled and sera were collected 24 h after the end of the third epicutaneous sensitization. A: Total IgE and OVA-specific IgE in sera from gal3^{+ +} and gal3^{~ "} mice were determined by ELISA. B: The OVA-specific IgG] and IgG₂₃ were determined by ELISA, and _iatio of OVA-specific IgG_2S to IgGj was calculated. Bars represent mean ± SEM. *P<0.05. C: Real-time PCR was performed to quantify mRNA. The results were normalized to GAPDH. Bars represent mean ± SEM (n = 6 ga!3^{+ +}, n = 5 gal3^{" "})• *P<0.05. [0026] Figure 4. Splenocytes from gal3^~'^~ mice produced higher levels of IL-12 in vitro. A: Single-cell suspensions of spleen cells were prepared 24 h after the third sensitization. Cells were cultured in the presence of OVA₃₂₃_₃₃₉ (50 μg/ml) in vitro, and supernatants were collected after 72 h of culture. IFN -γ, IL-4 and IL- 12 in the supernatants were determined by ELISA. Bars represent mean ± SEM. *P<0.05. B: CD4⁺ T cells from OVA-TCR tg mice were cocultured with bone marrow-derived DCs from gal3^+/+ and gal3^~A~ mice in the presence of OVA_323-339 peptide for 3 days. The same number of T cells (5 X 10⁵) from the two groups were further stimulated with anti-CD3 and anti-CD28 Ab for 48 h. The concentrations of cytokines were measured by ELISA. Results are representative of three experiments. Bars represent mean ± SEM. *P <0.05; **P <0.01. [0027] Figure 5. IFN-γ mRNA expression was increased and IL-4 mRNA expression was decreased in the skin sites of gal3^~7~ mice. Skin biopsies were obtained 24 h after the third sensitization. Total RNA was extracted from each sample and cDNA was synthesized from 10 μg of total RNA. Real-time PCR was performed to quantify mRNA. The results were normalized to GAPDH. The P values are: IL-4, <0.03 ; and IFN-γ, <0.05. [0028] Figure 6. A 1-week epicutaneous sensitization with OVA induced significant thickening of epidermis in recipients of T cells from OVA-TCR transgenic mice. In vz/ro-generated OVA-specific gal3^{+ +} and gal3^~'^~ T cell populations were cultured with APCs in the presence of OVA and IL-2 for 5 days. Gal3^{+ +} and gal3^{~ ~} T cells were transferred into C57BL/6 recipient mice (5 X 10⁶ cells/mouse), and mice were exposed to OVA for 1 week. 24 hours after the sensitization, skin sections were obtained and stained with H&E for histological analysis (magnification X 200). The figure indicates epidermal and dermal thickness measurements. The thickness was determined from the average of at least 10 measures for each sample. Bars represent mean ± SEM.

[0029] Figure 7. OVA-sensitized skin sites of mice that received T cells from gaB^^" /OVA-TCR tg mice had markedly decreased eosinophils in the dermis. Number of infiltrating cells in sensitized skin sites is indicated. Bars represent mean ± SEM (n = 5 animals per group). **P <0.01 ; ***P<0.001. Similar results were obtained in a separate experiment.

[0030] Figure 8. Gal3^+/+ mice that received T cells from gal3 /OVA-TCR tg mice exhibited lower IgE and IgGj but higher IgG₂₃ levels in the serum after exposure to OVA, compared to those receiving T cells from gal3^+/+/OV A-TCR tg mice. Recipient mice were epicutaneously exposed to OVA and bled 24 h after 1-week sensitization. A: Total IgE levels in sera were determined by ELISA. B: The OVA-specific IgG₁ and IgG_2a were determined by ELISA, and ratios of OVA-specific IgG_2a to IgGi were calculated. Bars represent mean ± SEM (n = 5 animals per group). *P<0.05. Similar results were obtained in a separate experiment.

[0031] Figure 9. Splenocytes and draining lymph node cells from mice that received T cells from gaI3^-/7O VA-TCR tg mice secreted less IL-4 and IL-5 but more IFN-γ and IL- 12 following in vitro restimulation with OVA. Recipient mice were epicutaneously exposed to OVA. Spleen and axillary lymph nodes were harvested 24 h after 1 -week sensitization. Splenocytes and lymph node cells were then restimulated in vitro with OVA. Cytokines in culture supernatants were measured at 72 h. Bars represent mean ± SEM. *P<0.05.

DETAILED DESCRIPTION OF THE INVENTION

[0032] As explained above, galectin-3 is a member of a family of β-galactoside-binding animal lectins expressed by a variety of cells and tissues, including activated B and T cells, macrophages, dendritic cells, mast cells, and eosinophils. Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by spongiotic skin lesions that are attributable to Th2-mediated inflammatory responses. The present invention is aimed at prevention and/ or treatment of inflammatory responses such as AD by inhibiting the effects of galectin-3.

A. Definitions

[0033] "Inflammation" or an "inflammatory response" refers to an organism's immune response to irritation, toxic substances, pathogens, or other harmful stimuli. The response can involve innate immune components and/ or adaptive immunity. Inflammation is generally characterized as either chronic or acute. Acute inflammation is characterized by redness, pain, heat, swelling, and/ or loss of function due to infiltration of plasma proteins and leukocytes to the affected area. Chronic inflammation is characterized by persistent inflammation, tissue destruction, and attempts at repair. Monocytes, macrophages, plasma B cells, and other lymphocytes are recruited to the affected area, and angiogenesis and fibrosis occur, often leading to scar tissue.

[0034] An "inflammatory condition" is one characterized by an inflammatory response, as described above. A list of exemplary inflammatory conditions includes: asthma, autoimmune disease, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivities and allergies, skin disorders such as eczema, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, transplant rejection, and vasculitis. [0035] "Th2 -mediated inflammatory conditions" are inflammatory conditions characterized by increased levels of serum IgE, IL-4, IL-5, or IL-13. Peripheral blood eosinophilia is also generally increased in these conditions. Examples of Th2 -mediated inflammatory conditions include atopic dermatitis; allergic rhinitis; type 2 lung granuloma; acute asthmatic inflammation and atopic asthma; ulcerative colitis; inflammation related to helminth infection; and a wide variety of allergen responses, such as allergic airway disease (AAD). [0036] "Atopic dermatitis (AD)," also called "atopic eczema," refers to a chronic inflammatory condition of the skin. AD is characterized by spongiotic skin lesions that are attributable to Th2 -mediated inflammatory responses. The disorder is believed to be hereditary and non-contagious. Outbreaks are triggered abnormally easily, usually by contact with irritants, certain foods, or allergens. Th2 cells are CD4⁺ T cells that secrete IL-4, IL-5 and IL-13, and increase peripheral blood eosinophilia and serum IgE levels.

[0037] "Lectins" are carbohydrate-binding proteins that bind particular sugar moieties with some specificity. Lectins are involved in cell-cell adhesion and interactions, e.g., through binding to membrane-exposed glycolipids, glycoproteins, or the glycocalyx. Lectins are also involved in triggering the complement cascade, e.g., through binding to carbohydrates on the surface of bacteria or other pathogens.

[0038] "Galectins" are a family of proteins with affinity for beta-galactoside. These proteins share an approximately 130 amino acid consensus carbohydrate recognition domain (CRD) responsible for beta-galactoside binding. Various members of the mammalian galectins are active as monomers, hetero- or homo-dimers, or as aggregates. Each of these proteins minimally binds the N-acetyl-lactosamine moiety, with variation among the family members (see, e.g., Camby et al. (2006) Glycobiology 16:137-57). [0039] "Galectin-3" is a 3IkD member of the mammalian galectin family that forms aggregates through its non-CRD domain. As used herein, a "galectin-3 polypeptide" refers to a full length galectin-3 sequence, species homologs, fragments, and variants thereof. A "galectin-3 polynucleotide" refers to a nucleic acid sequence from the galectin-3 gene, including the coding and non-coding regions. "Galectin-3 cDNA," "galectin-3 mRNA," galectin-3 coding sequence," and other such terms refer to a nucleic acid sequence that encodes a galectin-3 polypeptide. [0040] 'inhibitors," "activators," and "modulators" of galectin-3 are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for galectin-3 binding or signaling, e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term "modulator" includes inhibitors and activators. Inhibitors are agents that, e.g., partially or totally block carbohydrate binding, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of galectin-3. In some cases, the inhibitor directly or indirectly binds to galectin-3. Inhibitors, as used herein, are synonymous with inactivators and antagonists. Activators are agents that, e.g., stimulate, increase, facilitate, enhance activation, sensitize or up regulate the activity of galectin-3. Modulators include galectin-3 ligands, including modifications of naturally-occurring ligands and synthetically-designed ligands, antibodies and antibody fragments, antagonists, agonists, small molecules including carbohydrate-containing molecules, siRNAs, RNA aptamers, and the like.

[0041] "Galectin-3 inhibitors" or "galectin-3 inactivators," and "galectin-3 antagonists" include, e.g., galectin-3 ligands or derivatives thereof, transcription-based inhibitors, such as antisense RNA and siRNA, antibodies specific for galectin-3 or galectin-3 targets, peptides corresponding to galectin-3 subsequences, RNA aptamers, or compounds such as those described in Table 1. Assays for galectin-3 inhibitors include, e.g., applying putative inhibitor compounds to a cell expressing galectin-3 and then determining the functional effects on galectin-3 signaling, as described herein. Assays for galectin-3 inhibitors also include cell-free systems, as described herein. Samples or assays comprising galectin-3 treated with a potential inhibitor are compared to control samples without the inhibitor to examine the extent of inhibition. Control samples (not treated with inhibitors) are assigned a relative galectin-3 activity value of 100%. Inhibition of galectin-3 is achieved when the galectin-3 activity or expression level relative to the control is about 80%, 70%, 50%, 20%, 10% or close to 0%.

[0042] A composition "consisting essentially of a galectin-3 inhibitor" is one that includes the galectin-3 inhibitor and no other compounds that contribute significantly to the inhibition of galectin-3. Such compounds may include inactive excipients, e.g., for formulation or stability of a pharmaceutical composition, or active ingredients that do not significantly contribute to the inhibition of galectin-3. Exemplary compounds consisting essentially of a galectin-3 inhibitor include therapeutics, medicaments, andpharmaceutical compositions. [0043] A "galectin-3 ligand" or "galectin-3 target" or "galectin-3 binding moiety" refers to the class of carbohydrate-containing compounds that bind to galectin-3. These embrace galectin-3 ligand inhibitors. Galectin-3 ligand inhibitors include, but are not limited to, galactose, galactoside, glycoconjugates that bind to galectin-3 (e.g., a glycolipid, glycopeptide, or proteoglycan), saccharides (e.g., monosaccharides, di-saccharides, tri- saccharides, polysaccharides, or oligosaccharides such as lactose, tetrasaccharide, beta- gal actosidase, or derivatives thereof), glycodendrimer, N-acetyl lactosamine, or a derivative thereof (e.g., C3' amide, sulfonamide, or urea derivative). Examples of galectin-3 targets are found in Table 1.

[0044] A "galectin-3 inactivating antibody" is an antibody or antibody fragment (e.g., an Fab fragment) that binds specifically to galectin-3 and interferes with, reduces, or inhibits the activity of galectin-3 as compared to the sample without the galectin-3 inactivating antibody. An example of such an antibody is B2C 10.

[0045] As used herein, an "effective amount" or a "therapeutically effective amount" means the amount of a compound that, when administered to a subject or patient for treating a disorder, is sufficient to prevent, reduce the frequency of, or alleviate the symptoms of the disorder. The effective amount will vary depending on a variety of the factors, such as a particular compound used, the disease and its severity, the age, weight, and other factors of the subject to be treated. Amelioration of a symptom of a particular condition by administration of a pharmaceutical composition described herein refers to any lessening, whether permanent or temporary, that can be associated with the administration of the pharmaceutical composition. For example, the amount of galectin-3 inhibitor is considered therapeutically effective for treating AD when treatment results in reduced severity, delayed onset, or frequency of symptoms, such as discomfort, irritation, inflammation, and Th2- mediated effects.

[0046] "Subject," or "subject in need of treatment," as used herein, includes individuals who seek medical attention due to risk of, or actual suffering from, a condition, such as an inflammatory condition. Subjects also include individuals currently undergoing therapy that seek manipulation of the therapeutic regimen. The term subject also includes animals and humans. Subjects or individuals in need of treatment include those that demonstrate symptoms of the condition or are at risk of suffering from these symptoms. For example, in the case of AD, a subject in need of treatment includes individuals with a genetic predisposition for the condition, those that have suffered AD symptoms in the past, those that have been exposed to a triggering substance or event, as well as those suffering from chronic or acute symptoms of the condition.

[0047] The term "nucleic acid" or "polynucleotide^"' refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605- 2608 (1985); and Cassol et al. (1992); Rossolini et al., MoI. Cell. Probes 8:91-98 (1994)). The terms nucleic acid and polynucleotide are used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0048] The term "target nucleic acid" refers to a nucleic acid (often derived from a biological sample) to which a nucleic acid probe or inhibitory nucleic acid is designed to specifically hybridize. The target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding probe directed to the target. The term target nucleic acid may refer to the specific subsequence of a larger nucleic acid to which the inhibitory nucleic acid or probe is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to target. The difference in usage will be apparent from context. For example, a galectin-3 target sequence can comprise a portion of the coding sequence, a portion of non-coding sequence, or the entire galectin-3 gene. [0049] The terms "polypeptide,'^" "peptide'^" and "protein'^" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds. [0050] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. [0051] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0052] An "antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. [0053] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively. [0054] Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'_2, a dimer of Fab which itself is a light chain joined to V_H-C_HI by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'₂ dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, NY (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies {e.g., single chain Fv). These antibody fragments are also useful for methods requiring antigen recognition. [0055] Chimeric antibodies combine the antigen binding regions (variable regions) of an antibody from one animal with the constant regions of an antibody from another animal. Generally, the antigen binding regions are derived from a non-human animal, while the constant regions are drawn from human antibodies. The presence of the human constant regions reduces the likelihood that the antibody will be rejected as foreign by a human recipient.

[0056] "Humanized" antibodies combine an even smaller portion of the non-human antibody with human components. Generally, a humanized antibody comprises the hypervariable regions, or complementarity determining regions (CDR), of a non-human antibody grafted onto the appropriate framework regions of a human antibody. Antigen binding sites may be wild type or modified by one or more amino acid substitutions, e.g., modified to resemble human immunoglobulin more closely. Both chimeric and humanized antibodies are made using recombinant techniques, which are well-known in the art (see, e.g., Jones et al. (1986) Nature 321 :522-525). [0057] The phrase "specifically (or selectively) binds to an antibody" or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein, e.g., galectin-3. For example, antibodies raised against galectin-3 can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins, except for polymorphic variants, e.g., proteins at least 80%, 85%, 90%, 95%, or 99% identical to galectin-3 or a fragment thereof, e.g., a domain or unique subsequence. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically, a specific or selective reaction will be at least twice the background signal or noise and more typically more than 10 to 100 times background.

B. Galectin-3 Inhibitors 1. Inhibitory nucleic acids

[0058] Inhibition of galectin-3 gene expression can be achieved through the use of inhibitory nucleic acids. Inhibitory nucleic acids can be single-stranded nucleic acids or oligonucleotides that can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex or triplex is formed. These nucleic acids are often termed "antisense" because they are usually complementary to the sense or coding strand of the gene, although recently approaches for use of "sense" nucleic acids have also been developed. The term "inhibitory nucleic acids" as used herein, refers to both "sense" and "antisense" nucleic acids. [0059] In one embodiment, the inhibitory nucleic acid can specifically bind to a target galectin-3 polynucleotide. Administration of such inhibitory nucleic acids can inhibit Th2- mediated immune responses by reducing or eliminating the effects of galectin-3. Nucleotide sequences encoding galectin-3 are known for several species, including the human cDNA (see Genbank Ace. No. NM 005567). One can derive a suitable inhibitory nucleic acid from the human galectin-3, species homologs, and variants of these sequences. [0060] By binding to the target nucleic acid, the inhibitory nucleic acid can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking DNA transcription, processing or poly(A) addition to mRNA, DNA replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradation. Inhibitory nucleic acid methods therefore encompass a number of different approaches to altering expression of specific genes that operate by different mechanisms. These different types of inhibitory nucleic acid technology are described in Helene and Toulme (1990) Biochim. Biophys. Acta., 1049:99-125. [0061] Inhibitory nucleic acid therapy approaches can be classified into those that target DNA sequences, those that target RNA sequences (including pre-mRNA and mRNA), those that target proteins (sense strand approaches), and those that cause cleavage or chemical modification of the target nucleic acids. [0062] Approaches targeting DNA fall into several categories. Nucleic acids can be designed to bind to the major groove of the duplex DNA to form a triple helical or "triplex" structure. Alternatively, inhibitory nucleic acids are designed to bind to regions of single stranded DNA resulting from the opening of the duplex DNA during replication or transcription. See Helene and Toulme, supra. [0063] More commonly, inhibitory nucleic acids are designed to bind to mRNA or mRNA precursors. Inhibitory nucleic acids are used to prevent maturation of pre-mRNA. Inhibitory nucleic acids may be designed to interfere with RNA processing, splicing or translation. The inhibitory nucleic acids are often targeted to mRNA. In this approach, the inhibitory nucleic acids are designed to specifically block translation of the encoded protein. Using this approach, the inhibitory nucleic acid can be used to selectively suppress certain cellular functions by inhibition of translation of mRNA encoding critical proteins. For example, an inhibitory antisense nucleic acid complementary to regions of a target mRNA inhibits protein expression (see, e.g., Wickstrom et al. (1988) Proc. Natl. Acad. ScL USA 85:1028-1032 and Harel-Bellan et al. (1988) Exp. Med., 168:2309-2318). As described in Helene and Toulme, supra, inhibitory nucleic acids targeting mRNA have been shown to work by several different mechanisms in order to inhibit translation of the encoded protein(s).

[0064] The inhibitory nucleic acids introduced into the cell can also encompass the "sense" strand of the gene or mRNA to trap or compete for the enzymes or binding proteins involved in mRNA translation. See Helene and Toulme, supra. [0065] The inhibitory nucleic acids can also be used to induce chemical inactivation or cleavage of the target genes or mRNA. Chemical inactivation can occur by the induction of crosslinks between the inhibitory nucleic acid and the target nucleic acid within the cell. Alternatively, irreversible photochemical reactions can be induced in the target nucleic acid by means of a photoactive group attached to the inhibitory nucleic acid. Other chemical modifications of the target nucleic acids induced by appropriately derivatized inhibitory nucleic acids may also be used.

[0066] Cleavage, and therefore inactivation, of the target nucleic acids can be effected by attaching to the inhibitory nucleic acid a substituent that can be activated to induce cleavage reactions. The substituent can be one that effects either chemical, photochemical or enzymatic cleavage. For example, one can contact an mRNA: an ti sense oligonucleotide hybrid with a nuclease which digests mRNA:DNA hybrids. Alternatively cleavage can be induced by the use of ribozymes or catalytic RNA. In this approach, the inhibitory nucleic acids would comprise either naturally occurring RNA (ribozymes) or synthetic nucleic acids with catalytic activity.

[0067] Inhibitory nucleic acids can also include RNA aptamers, which are short, synthetic oligonucleotide sequences that bind to proteins (see, e.g., Li et al. (2006) Nuc. Acids Res. 34: 6416-24). They are notable for both high affinity and specificity for the targeted molecule, and have the additional advantage of being smaller than antibodies (usually less than 6kD). RNA aptamers with a desired specificity are generally selected from a combinatorial library, and can be modified to reduce vulnerability to ribonucl eases, using methods known in the art.

2. Peptide inhibitors

[0068] Galectin-3 activity can be inhibited using peptide antagonists. For example, peptides comprising a subsequence of the full length galectin-3 polypeptide have been shown to inhibit galectin-3 activity {see U.S. Patent Publ. No. 2006/0148712). Such peptide subsequences have from about 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, or more amino acid residues. One of skill can derive an inhibitory peptide from human galectin-3 (e.g., Genbank Ace. No. NM 005567), or from species homologs or variants of these sequences. [0069] Galectin-3 inhibitory peptides can be drawn, e.g., from N-terminal or C-terminal fragments, or the CRD of galectin-3. For example, the following peptide sequences can be used to inhibit galectin-3: SMEPALPDWWWKMFK; DKPTAFVSVYLKTAL; PQNSKIPGPTFLDPH; APRPGPWLWSNADSV; GVTDSSTSNLDMPHW; PKMTLQRSNIRPSMP; PQNSKIPGPTFLDPH; LYPLHTYTPLSLPLF; LTGTCLQYQSRCGNTR; AYTKCSRQWRTCMTTH; ANTPCGPYTHDCPVKR; NISRCTHPFMACGKQS; and PRNICSRRDPTCWTTY.

[0070] Galectin-3 peptide antagonists also include peptides that do not correspond to galectin-3 sequences. For example, peptides selected from combinatorial libraries can serve to inhibit galectin-3 activity.

3. Inactivating antibodies

[0071] Inhibition of galectin-3 activity can be achieved with an inactivating antibody. An inactivating antibody can comprise an antibody or antibody fragment that specifically binds to galectin-3. Inactivating antibody fragments include, e.g., Fab fragments, heavy or light chain variable regions, single complementary determining regions (CDRs), or combinations of CRDs with galectin-3 binding activity.

[0072] Any type of antibody agonist may be used according to the methods of the invention. Generally, the antibodies used are monoclonal antibodies. Monoclonal antibodies can be generated by any method known in the art (e.g., using hybridomas, recombinant expression and/or phage display).

[0073] Antibodies can be derived from any appropriate organism, e.g., mouse, rat, rabbit, gibbon, goat, horse, sheep, etc. The galectin-3 inactivating antibody can be a chimeric {e.g., mouse/ human ) antibody made up of regions from an non-human anti-galectin-3 antibody together with regions of human antibodies. For example, a chimeric H chain can comprise the antigen binding region of a heavy chain variable region, or at least parts thereof, such as a CDR, of the non-human antibody linked to at least a portion of a human heavy chain constant region. This humanized or chimeric heavy chain may be combined with a chimeric L chain that comprises the antigen binding region of a light chain variable region, or at least parts thereof, such as a CDR, of the non-human antibody linked to at least a portion of the human light chain constant region. In some embodiments, the heavy chain constant region can be an IgM, IgG, or IgA antibody.

[0074] The chimeric antibodies of the invention may be monovalent, divalent, or polyvalent immunoglobulins. For example, a monovalent chimeric antibody is a dimer (HL) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain, as noted above. A divalent chimeric antibody is a tetramer (H₂ L₂) formed by two HL dimers associated through at least one disulfide bridge. A polyvalent chimeric antibody is based on an aggregation of chains. [0075] In some embodiments, galectin-3 inactivating antibodies of the invention bind to the galectin-3 CRD or interfere with galectin-3 ligand binding. In some embodiments, the galectin-3 inactivating antibodies of the invention do not bind to the CRD and/or interfere with galectin-3 ligand binding. In some embodiments, the galectin-3 inactivating antibody is B2C10.

4. Galectin-3 ligand inhibitors [0076] Galectin-3 inhibitors also include compounds that are derived from natural ligands of galectin-3 and that bind to galectin-3. These compounds include natural ligands of galectin-3, as well as naturally-occurring or synthetic derivatives thereof. Galectin-3 ligand inhibitors also include synthetic compounds that are designed to specifically target galectin-3, e.g., to interfere with galectin-3 binding to a natural ligand.

[0077] A galectin-3 binding inhibitor can include galactose, isomers, and derivatives thereof. These include galactosides such as thio-galactoside or thiodi-galactoside. |0078) A galectin-3 binding inhibitor can also include a glycoconjugate or its derivative. Examples of glycoconjugates include glycolipids, glycopeptides, and proteoglycans. [0079] Galectin-3 binding inhibitors also include saccharides. Saccharides in any form can be used, e.g., monosaccharides, disaccharides, trisaccharides, polysaccharides, and oligosaccharides. Examples include lactose, tetrasaccharide, beta-galactoside, or an analog or derivative thereof.

[0080] Galectin-3 binding inhibitors can comprise a glycodendrimer. Glycodendrimers are highly-ordered, branched molecules that display a carbohydrate group, and are known in the art. N-acetyl lactosamine and derivatives thereof are also effective galectin-3 inhibitors, as are C3' amide, sulfonamide, and urea derivatives. [0081] Additional galectin-3 binding inhibitors are found in Table 1. Table 1: Galectin-3 Inhibitors

Kd

Linear & cyclic peptides

Andre S. et al. (2005) BioorgMed Chem. 13(2):563-73.

~1 mM Arnusch CJ et al. (2004) BioorgMed Chem Lett 14: 1437-1440 Andre S. et al. (2006) Bioorg Med Chem Lett. 17: 793.

Derivatized thiodiglycosides mM Andre S. et al. (2006). BioorgMed Chem. 14(18):6314-6326.

33 nM Cumpstey I. et al. (2005) Angewante Chemie Int Ed 44: 2. -150 μM Cumpstey I. et al. (2005) Org Biomol Chem. 3(10):1922-32. 107 μM Salameh BA et al. (2005) BioorgMed Chem Lett. 15(14):3344-46.

Derivatized glycans

0.1 μM Aplander K. et al. (2006) Carbohydr Res. 341 (10): 1363-69. 3^?-naphthamido-LacNAc fluorescein

Oberg CT. et al. (2003) Bioconjug Chem. 14(6): 1289-97. ~1 μM Sorme P. et al. (2004) Anal Biochem. 334(1 ):36-47. ~4 μM Sorme P. et al. (2002) Chembiochem. 3(2-3): 183-89. 180 μM Tejler J. et al. (2005) Bioorg Med Chem Lett. 15(9):2343-45.

Modified thiodiglycosides

Cumpstey (2005) Angew Chem Int Ed 44:51 10-12 Modified glycans

Sorme P. et al. (2002) Chembiochem. 3(2-3): 183-99.

[0082] One of skill will appreciate that such galactin-3 binding inhibitors can be used alone or in combination. In some cases, a single galectin-3 binding inhibitor, e.g., with a high affinity for galectin-3, can be used. In some cases, it can be advantageous to target a wide range of binding sites, e.g., with a combination of such compounds. Most of these compounds are readily available from commercial sources (e.g., Sigma (St. Louis, MO), Aldrich (St. Louis, MO), etc.). Alternatively, standard organic chemical methods can be used to derive such compounds, or attach additional functional groups.

5. Identification of galectin-3 inhibitors [0083] One can identify compounds that are therapeutically effective galectin-3 modulators by screening a variety of compounds and mixtures of compounds for their ability to modulate galectin-3 activity, or which bind to galectin-3 or a galectin-3 ligand, e.g., to prevent galectin- 3 ligand binding. The testing can be performed using a minimal region or subsequence of galectin-3, or a full length polypeptide. [0084] An important aspect of the present invention is directed to methods for screening compounds for galectin-3 modulating activity. Such compounds can be in a mixture of suitable galectin-3 modulators, or each in substantially isolated form. An example of an in vitro binding assay can comprise galectin-3 polypeptides or fragments thereof; a test binding compound attached to a matrix; and a detectable ligand for galectin-3. Another typical sample comprises a mixture of synthetically produced or naturally occurring compounds, such as a cell culture broth. Suitable cells include any cultured cells such as mammalian, insect, microbial (e.g., bacterial, yeast, fungal) or plant cells.

[0085] In addition to assaying for an effect on galectin-3 ligand binding to identify suitable modulators, one can test directly for an effect on inflammation. Animal models for inflammation are known in the art, and can be established to assess the efficacy of modulator compounds. One example is the OVA₃₂₃-₃₃₉-specific T cell receptor transgenic mouse, described in a later section of this application. Sensitization with the OVA peptide causes a Th2 -mediated inflammatory response in these mice, which allows one to determine the effect of candidate modulators on various aspects of the response. For example, the effect of the modulator can be determined by observing: acanthosis, eosinophil and mononuclear cell infiltration to the sensitized site, IgE levels, or the levels of IL- 12, IFN -γ, IL-4, IL-5, or IL- 13.

[0086] In preferred embodiments, the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays).

[0087] As noted, the invention provides in vitro assays for galectin-3 activity in a high throughput format. For each of the assay formats described, "no modulator" control reactions which do not include a modulator provide a background level of galectin-3 activity. In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay many different plates per day; assay screens for up to about 6,000-20,000, and even up to about 100,000-1 ,000,000 different compounds is possible using the integrated systems of the invention. [0088] In some assays it will be desirable to have positive controls to ensure that the components of the assays are working properly. For example, a known modulator of galectin-3 activity can be incubated with one sample of the assay, and the resulting increase or decrease in signal determined according to the methods herein. [0089] Essentially any chemical compound can be tested as a potential modulator of galectin-3 activity for use in the methods of the invention. Most preferred are generally compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. It will be appreciated that there are many suppliers of chemical compounds, such as Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), and Fluka Chemika-Biochemica Analytika (Buchs Switzerland). [0090] Modulators of galectin- 3 activity or binding can be identified by screening a combinatorial library containing a large number of potential therapeutic compounds

(potential modulator compounds). Such "combinatorial chemical libraries" can be screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

[0091] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al, Nature 354:84-88 (1991)) and carbohydrate libraries {see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No. WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication No. WO 92/00091 ), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. ScL USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc. 1 14:6568 (1992)), nonpeptidal peptidomimetics with β-D-glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al, J. Amer. Chem. Soc. 1 16:2661 (1994)), oligocarbamates (Cho et al, Science 261 : 1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see, Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; and benzodiazepines, U.S. Patent 5,288,514). [0092] Alternatively, one can identify compounds that are suitable galectin-3 modulators by screening a variety of compounds and mixtures of compounds for their ability to inhibit or enhance galectin-3 expression. Methods of detecting expression levels are well known in the art, and include both protein- and nucleic acid-based methods. [0093] For example, a test agent can be contacted in vitro with cells expressing galectin-3. An agent that modulates galectin-3 is one that results in an increase or decrease in the level of galectin-3 polypeptide or transcript, as measured by any appropriate assay common in the art (e.g., Northern blot, RT-PCR, Western blot, or other hybridization or affinity assays), when compared to expression without the test agent. In some embodiments, a test nucleic acid inhibitor can be introduced into a cell, e.g., using standard transfection or transduction techniques, and the level of galectin-3 expression detected.

C. Inflammatory Responses and Conditions

[0094] Identification and diagnosis of inflammation, as well as methods of monitoring the effectiveness of a therapeutic regimen as described herein, are included in the present invention. As explained above, inflammation is generally characterized by redness, swelling, pain, and occasional loss of function. However, symptoms vary between tissues, so that some inflammatory conditions are not easily detectable (e.g., atherosclerosis). [0095] Although the inflammatory response can play a role in the healing process by destroying, diluting, and isolating injurious agents and stimulating repair of the affected tissue, inflammatory responses can also be harmful. For example, inflammation results in leakage of plasma from the blood vessels. Although this leakage can have beneficial effects, it causes pain and when uncontrolled can lead to loss of function and death (such as adult respiratory distress syndrome). Anaphylactic shock, arthritis, and gout are among the conditions that are characterized by uncontrolled or inappropriate inflammation.

[0096] On a cellular level, an inflammatory response is typically initiated by endothelial cells producing molecules that attract and detain inflammatory cells (e.g., myeloid cells such as neutrophils, eosinophils, and basophils) at the site of injury or irritation. The inflammatory cells then are transported through the endothelial barrier into the surrounding tissue. The result is accumulation of inflammatory cells, in particular neutrophils. Such accumulation is easily detectable by one of skill.

[0097] Adaptive immune cells (T and B cells) are often involved in inflammatory conditions. As explained above, these cells release cytokines and antibodies in response to the source of the irritation. Thus, an inflammatory response can also be detected by detecting a change in the level of inflammatory cytokines, e.g., in a localized region of irritation or in the serum or plasma of an individual.

[0098] In the case of atopic dermatitis (AD), symptoms are often chronic and relapsing, and usually appear early in life. As explained above, the disease is characterized by eczema-like skin lesions and discomfort. However, the symptoms vary substantially between individuals. The skin of AD patients is unusually sensitive, and recurrence is usually triggered by an irritant or food substance. Aside from the skin lesions, AD can be detected by immunohistologic analysis, e.g., to detect a mononuclear cell infiltrate, predominantly in the dermis at the affected site. In acute episodes, Th2 cytokines are present at a high level, while in more chronic stages, both ThI and Th2 cytokines are present. Major symptoms of AD include intense itching; characteristic rashes, especially on the arms, cheeks, and legs; and a family history of allergic reactions and AD. Additional symptoms include high serum IgE levels, dry skin, ichthyosis, hyperlinear palms, keratosis pilaris, and cheilitis. [0099] It will be appreciated by those of skill in the art that each of these symptoms can be detected in an individual for the purposes of diagnosis. Further, a subject undergoing therapy for an inflammatory condition such as AD can be monitored, e.g., to determine lessening of symptoms.

D. Pharmaceutical Compositions 1. Formulations

[0100] Compounds of the present invention are useful in the manufacture of a pharmaceutical composition or a medicament. A pharmaceutical composition or medicament can be administered to a subject for the treatment of, for example, an inflammatory condition or disease as described herein. [0101] Compounds of the present invention, e.g., galectin-3 inhibitors, and compounds and agents identified by the methods disclosed herein, are useful in the manufacture of a pharmaceutical composition or a medicament comprising an effective amount thereof in conjunction or mixture with excipients or carriers suitable for either enteral or parenteral application. [0102] A preferred pharmaceutical composition for inhibiting galectin-3 comprises (i) a galectin-3 inhibitor as described herein or a compound obtained or obtainable according to a screening method described herein, and (ii) a pharmaceutically acceptable excipient or carrier. The terms pharmaceutically-acceptable and physiologically-acceptable are used synonymously herein. The galectin-3 inhibitor may be provided in a therapeutically effective dose for use in a method for treatment as described herein.

[0103] A galectin-3 inhibitor of the invention can be administered via liposomes, which serve to target the conjugates to a particular tissue, as well as increase the half-life of the composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the inhibitor to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among the targeted cells {e.g., skin cells), or with other therapeutic or immunogenic compositions. Thus, liposomes filled with a desired inhibitor of the invention can be directed to the site of inflammation, where the liposomes then deliver the selected inhibitor compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al. (\980) Ann. Rev. Biophys. Bϊoeng. 9: 467, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028.

[0104} Pharmaceutical compositions or medicaments for use in the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and in "Remington's

Pharmaceutical Sciences" by E.W. Martin. Compounds and agents of the present invention and their physiologically acceptable salts and solvates can be formulated for administration by any suitable route, including via inhalation, topically, nasally, orally, parenterally, or rectally. [0105] Typical formulations for topical administration include creams, ointments, sprays, lotions, and patches. The pharmaceutical composition can, however, be formulated for any type of administration, e.g., intradermal, subdermal, intravenous, intramuscular, intranasal, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary or intratumoral injection, with a syringe or other devices. Formulation for administration by inhalation (e.g., aerosol), or for oral, rectal, or vaginal administration is also contemplated.

2. Routes of administration

[0106] Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0107] Suitable formulations for transdermal application include an effective amount of a compound or agent of the present invention with carrier. Preferred carriers include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used. [0108] For oral administration, a pharmaceutical composition or a medicament can take the form of, for example, a tablet or a capsule prepared by conventional means with a pharmaceutically acceptable excipient. Preferred are tablets and gelatin capsules comprising the active ingredient, i.e., a compound of the present invention, together with (a) diluents or fillers, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose {e.g., ethyl cellulose, microcrystalline cellulose), glycine, pectin, polyacrylates and/or calcium hydrogen phosphate, calcium sulfate, (b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt, metallic stearates, colloidal silicon dioxide, hydrogenated vegetable oil, corn starch, sodium benzoate, sodium acetate and/or polyethyleneglycol; for tablets also (c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and/or hydroxypropyl methylcellulose; if desired (d) disintegrants, e.g., starches {e.g., potato starch or sodium starch), glycolate, agar, alginic acid or its sodium salt, or effervescent mixtures; (e) wetting agents, e.g., sodium lauryl sulphate, and/or (f) absorbents, colorants, flavors and sweeteners. [0109] Tablets may be either film coated or enteric coated according to methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, for example, suspending agents, for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats; emulsifying agents, for example, lecithin or acacia; non-aqueous vehicles, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, for example, methyl or propyl -p-hydroxybenzoates or sorbic acid. The preparations can also contain buffer salts, flavoring, coloring, and/or sweetening agents as appropriate. If desired, preparations for oral administration can be suitably formulated to give controlled release of the active compound.

[0110] Compounds and agents of the present invention can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

[01111 F°^r administration by inhalation, the compounds and agents, may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, for example, lactose or starch. [0112] The compounds and agents can also be formulated in rectal compositions, for example, suppositories or retention enemas, for example, containing conventional suppository bases, for example, cocoa butter or other glycerides.

[0113] Furthermore, the compounds and agents can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0114] A pharmaceutical composition or medicament of the present invention comprises (i) an effective amount of a compound as described herein that inhibits the level or activity of galectin-3 and (ii) another therapeutic agent. When used with a compound of the present invention, such therapeutic agent may be used individually, sequentially, or in combination with one or more other such therapeutic agents (e.g., a first therapeutic agent, a second therapeutic agent, and a compound of the present invention). Administration may be by the same or different route of administration or together in the same pharmaceutical formulation.

3. Dosage

[0115] Pharmaceutical compositions or medicaments can be administered to a subject, preferably a human or a non-human animal, at a therapeutically effective dose to prevent, treat, or control an inflammatory condition or disease as described herein. The pharmaceutical composition or medicament is administered to a subject in an amount sufficient to elicit an effective therapeutic response in the subject.

[0116] The dosage of active agents administered is dependent on the species of mammal, the body weight, age, individual condition, surface area or volume of the area to be treated and on the form of administration. The size of the dose also will be determined by the existence, nature, and extent of any adverse effects that accompany the administration of a particular compound in a particular subject. For example, each type of galectin-3 inhibitor will likely have a unique dosage. A unit dosage for oral administration to a mammal of about 50 to 70 kg may contain between about 5 and 500 mg of the active ingredient. Typically, a dosage of the active compounds of the present invention, is a dosage that is sufficient to achieve the desired effect. Optimal dosing schedules can be calculated from measurements of agent accumulation in the body of a subject. In general, dosage may be given once or more daily, weekly, or monthly. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates. [0117] To achieve the desired therapeutic effect, compounds or agents may be administered for multiple days at the therapeutically effective daily dose. Thus, therapeutically effective administration of compounds to treat an inflammatory condition or disease described herein in a subject requires periodic (e.g., daily) administration that continues for a period ranging from three days to two weeks or longer. Typically, agents will be administered for at least three consecutive days, often for at least five consecutive days, more often for at least ten, and sometimes for 20, 30, 40 or more consecutive days. While consecutive daily doses are a preferred route to achieve a therapeutically effective dose, a therapeutically beneficial effect can be achieved even if the agents are not administered daily, so long as the administration is repeated frequently enough to maintain a therapeutically effective concentration of the agents in the subject. For example, one can administer the agents every other day, every third day, or, if higher dose ranges are employed and tolerated by the subject, once a week. [0118] Optimum dosages, toxicity, and therapeutic efficacy of such compounds or agents may vary depending on the relative potency of individual compounds or agents and can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio, LD₅₀/ED₅₀. Agents that exhibit large therapeutic indices are preferred. While agents that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue to minimize potential damage to normal cells and, thereby, reduce side effects.

[0119] The data obtained from, for example, cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration. For any agents used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (the concentration of the agent that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography (HPLC). In general, the dose equivalent of agents is from about 1 ng/kg to 100 mg/kg for a typical subject. [0120] Exemplary dosages for some of the galectin-3 inhibitors described herein are provided. Dosage for an inhibitory nucleic acid, such as an anti-angiogenic aptamer, can be between 0.1- 0.5 mg/eye, with intravitreous administration (e.g., 5- 30 mg/kg). Small organic compounds can be administered orally at between 5-1000 mg, or by intravenous infusion at between 10- 500 mg/ml. Monoclonal antibody inhibitors can be administered by intravenous injection or infusion at 50- 500 mg/ml (over 120 minutes); 1-500 mg/kg (over 60 minutes); or 1-100 mg/kg (bolus) five times weekly. Peptide inhibitors can be administered subcutaneously at 10- 500 mg; 0.1- 500 mg/kg intravenously twice daily, or about 50 mg once weekly, or 25 mg twice weekly. [0121] Pharmaceutical compositions of the present invention can be administered alone or in combination with at least one additional therapeutic compound. Exemplary advantageous therapeutic compounds include systemic and topical antiinflammatories, pain relievers, antihistamines, anesthetic compounds, and the like. The additional therapeutic compound can be administered at the same time as, or even in the same composition with, galectin-3 inhibitor. The additional therapeutic compound can also be administered separately, in a separate composition, or a different dosage form than the galectin-3 inhibitor. Some doses of the galectin-3 inhibitors of the invention can be administered at the same time as the additional therapeutic compound, while others are administered separately, depending on the particular symptoms and characteristics of the individual. [0122] The dosage of a pharmaceutical composition of the invention can be adjusted throughout treatment, depending on severity of symptoms, frequency of recurrence, and physiological response to the therapeutic regimen. Those of skill in the art commonly engage in such adjustments in therapeutic regimen.

F. Kits

[0123] For use in the screening and therapeutic applications described above, kits are also provided by the present invention. In the screening applications such kits may include any one or all of the following: assay reagents, enzymes, buffers, a galectin-3 polypeptide or fragment thereof, a galectin-3 nucleic acid, an anti -galectin-3 antibody, a galectin-3 ligand, hybridization probes and/or primers detecting a galectin-3 nucleic acid, a galectin-3 expression construct, or any other compound or composition described herein. In addition to a galectin-3 inhibitor, a therapeutic kit can include: a device for administration, an additional therapeutic composition, instruction material, sterile saline or another pharmaceutically acceptable emulsion or suspension base. [0124] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of the same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which they are presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed.

[0125] Typically, the components of a kit are provided in separate containers. In a preferred embodiment of the present invention, a kit for inhibiting the level or activity of galectin-3 comprises a container containing a galectin-3 inhibitor as described herein or a pharmaceutical composition comprising a galectin-3 inhibitor.

[0126] In addition, a kit may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. The instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. [0127] A wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user. [0128] In a preferred embodiment of the present invention, the kit is a pharmaceutical kit and comprises a pharmaceutical composition comprising (i) a galectin-3 inhibitor as described herein, and (ii) a pharmaceutically acceptable carrier. Pharmaceutical kits optionally comprise an instruction stating that the pharmaceutical composition can or should be used for treating an inflammatory condition, e.g., AD.

G. Examples

[0129] The following examples are provided for the purposes of illustration only. It will be readily apparent to one of ordinary skill in the art, in light of the teachings of this invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims

1. Introduction

[0130] To establish the role of galectin-3 in the development of AD, we used a mouse model of allergic skin inflammation induced by repeated epi cutaneous sensitization with ovalbumin (OVA), which displays many of the features of human AD. OVA-treated gal3^{~ "} mice exhibited less marked epidermal thickening, lower eosinophil and mast cell infiltrations in the dermis, and lower total and antigen-specific IgE levels but higher antigen-specific IgG2a/IgGl ratio in the serum, compared to similarly treated gal3^+/+ mice. In addition, the former had lower IL-4 mRNA expression, but higher IFN-γ mRNA expression than the latter in the OVA-treated skin sites, indicative of a ThI response. Moreover, following in vitro restimulation with OVA, splenocytes and draining lymph node cells from OVA-sensitized gal3^{~ ~} mice secreted less IL-4, but more IFN-γ and IL- 12 than those from similarly treated gal3^+/+ mice. Finally, gal3^{+ +} mice that received T cells from gal3^""^ mice backcrossed to OVA-specific TCR transgenic (gal3^~7O VA-TCR tg) mice developed a markedly lower Th2 response and significantly less pronounced dermatitis after exposure to OVA, compared to those receiving T cells from gal3^+/+/OVA-TCR tg mice. The results indicate that endogenous galectin-3 in T cells regulates Thl/Th2 polarization, and that galectin-3 plays a key role in atopic dermatitis.

2. Materials and Methods

[0131] Animals. Gal3^{~ ~} mice were developed as described (Hsu et al., Am J Pathol 2000, 156:1073- 1083) and were backcrossed to BALB/c or C57BL/6 mice for nine generations. OVA₃₂₃_₃₃₉-specific and I-A^b restricted T cell receptor transgenic mice on C57BL/6 background (OT-II mice) were obtained from The Jackson Laboratory (Bar Harbor, ME). Gal3^→~ mice in the C57BL/6 background were backcrossed to OT-II mice, also congenic in C57BL/6 (gal3^~7OVA-TCR tg mice). Age- and sex -matched wild-type (gal3^+/+) mice were used as control in all experiments. All mice were kept in a pathogen-free environment. All experiments with mice were approved by the Institutional Animal Care and Use Committee of the University of California-Davis (Sacramento, CA).

[0132] Sensitization protocols . Epicutaneous sensitization of 6- to 10-week-old female BALB/c mice was performed as described previously (Spergel et al., J Clin Invest 1998, 101 :1614-1622). Briefly, an area on the trunk of anesthetized mice was shaved with an electric razor and tape-stripped six times. One hundred microgram of OVA (Grade V; Sigma, St. Louis, MO) in lOOμl of saline or lOOμl of saline alone was placed on a patch of sterile gauze (1 X 1 cm). The gauze was secured to the back with Tegaderm (3M Health Care Ltd, St. Paul, MN), which was reinforced with BAND-AID (Johnson & Johnson Medical Inc, Arlington, TX). The patches were placed for a one-week period and then removed. Two weeks later, an identical patch was reapplied to the same skin sites. Each mouse had a total of three one-week exposures to the antigen separated from each other by two- week intervals. [0133] In vivo T cell transfer experiments. Spleens were harvested from gal3^~7OVA-TCR tg mice and gal3⁺ VOVA-TCR tg mice, and single-cell suspensions of splenocytes were prepared (1 X 10⁶ cells/ml). The cells were then cultured together with antigen presenting cells (irradiated syngeneic spleen cells, I X l O⁶ cells/ml) in the presence of OVA (100 μg/ml) and IL-2 (10 ng/ml) in RPMI 1640 containing 10% fetal bovine serum (FBS) at 37°C for 5 days. The purity of the CD4⁺ cell population was determined by FACS analysis and was 80- 85%. The resulting cells were injected intravenously into wild-type C57BL/6 mice (5 X 10⁶ cells per mouse). Twenty-four hours after the injection, an area on the trunk of the recipients was shaved, tape stripped and sensitized with OVA (100 μg in 100 μl of PBS) or 100 μl of PBS placed on a patch of sterile gauze, as described above. The patches were placed for a one-week period and then removed. [0134] The experiment was repeated using a more purified T cell population. In brief, CD4+ T cells were separated from total splenocytes using EasySep magnetic selection kit (StemCell Technologies). Puritiy was >90%, as confirmed by FACS with a CD4 antibody. [0135] Histology. For histological examination, specimens were obtained 24 h after removal of the skin patch. Specimens were embedded in Tissue Tek oxacalcitriol (OCT) compound (Sakura Finetek, Torrance, CA) on dry ice. Multiple 4 μm sections were stained with hematoxylin and eosin (H&E). To detect eosinophils and mast cells, tissue sections were stained with Giemsa. The numbers of inflammatory cells were counted in a blind fashion in 20 HPF at a magnification of XlOOO and are expressed as mean cell numbers per HPF ± SEM. [0136] The sections were also processed for immunohistochemical analysis. The samples were stained for galectin-3 using anti-galectin-3 monoclonal antibody A3A12 as described previously (Liu et al. (1996) Biochemistry 35:6073-79; Hsu et al. (1999) Int. J. Cancer 81 :519-26). Bound antibody was detected by an avidin-biotin-immunoperoxidase method according to the manufacturer's instructions (DakoCyotmation, Inc. Carpinteria, CA).

[0137] Quantitation of IgE, IgG; and IgG _2a in the serum. Mice were bled and sera were collected 24 hours after the last epicutaneous sensitizations. The standard protocol for sandwich ELISA was used to quantify the total amount of antibodies in the serum. Total IgE levels were measured by ELISA using anti-IgE as capture antibody and horseradish peroxidase-conjugated rat anti-mouse IgE (BD Biosciences, San Jose, CA) for detection as described previously (Liu et al, J Immunol 1980, 124:2728-2737). OVA-specific IgGl and IgG2a antibodies were measured by ELISA using OVA-coated plates and horseradish peroxidase-conjugated anti-mouse IgGi and IgG₂₃, antibodies (Zymed Laboratories, San Francisco, CA) as described previously (Zuberi et al, Am J Pathol 2004, 165:2045-2053). [0138] Since IgE represents a small amount of the total antibody response to specific antigen, OVA-specific IgE was quantitated by a different method, using rat anti-IgE as capture antibody, and horseradish peroxidase-conjugated rabbit anti-OVA antibodies (United States Biological, Massachusetts, MA) for detection (Spergel et al. 1998, supra). [0139] Analysis of in vitro cytokine synthesis. Spleen and draining (axillary) lymph nodes were harvested at 24 hours after the last epicutaneous sensitizations. Single-cell suspensions of these organs were prepared in complete RPMI 1640 supplemented with 10% FBS. Cells were cultured in the above medium at 4 x 10⁶ cells/ml in 24-well plates in the presence of OVA (50 μg/ml). After 72 h, supernatants were collected and assayed for cytokines by standard sandwich ELISA. The antibody pairs for IL-4, IFN-γ and IL-5 ELISAs were from eBiosciences as follows. Coating antibody: clones 1 IBl 1, AN-18 and TRFK5; detection antibody clones: BVD6-24G2, R4-6A2 and TRFK4, respectively. Antibody pairs for IL- 12 were from BD Pharmingen, and were 9A5 and C 17.8 for coating and detection, respectively. The assays were performed according to the protocols provided by the manufacturers. [0140] RNA preparation, cDNA synthesis, and real-time PCR. Skin biopsy specimens were obtained 24 h after the patch for the third sensitization was removed and immediately immersed in RNAlater (Ambion, Austin, TX). To extract RNA, the samples were homogenized in Trizol (GIBCO BRL, Gaithersburg, MD), following the manufacturer^'s instruction. cDNA was synthesized from 10 μg of total RNA in a 40 μl reaction mix through use of Superscript II (Invitrogen, Carlsbad, CA) for 120 min at 42°C. Real-time PCR was performed using the TaqMan probes for mouse IL-4, IFN -γ and GAPDH (Overbergh et a!., J Biomol Tech 2003, 14:33-43) and the iCycler ϊQ system (Bio Rad laboratories, Hercules, CA).

[0141] Dendritic cells derived from bone marrow. DCs were generated from bone marrow cells as described . Cells were cultured in RPMI medium supplemented with 20 ng/ml GM- CSF. Cells were harvested on day 10.

[0142] Cytokine secretion by T cells from OVA-specific TCR transgenic mice. CD4 T cells from spleens fo OVA-TCR tg mice were purified as described above. DCs from gal3-/- and gal+/+ mice were pulsed with OVA₃₂₃-₃₃₉ peptide (10 μg/ml) for 3h. Ova-pulsed DCs wehre harvested, washed an dmixed with purified naϊve T cells from OVA-TCR tg mice, suspended in RPMI containing 10% FBS and 50 mM 2-ME DCs (2 x 10⁴) and T cells (2 x 10⁵) were co-cultured for 72 h. Expanded T cells (5 x 10⁵) were restimulated with plate- bound anti-CD3 (10 μg/ml) and anti-CD28 Ab (2 μg/ml) for an additional 48 h. Supernatants were collected before and after stimulation and assayed for IFN-gamma and IL-4. [0143] Statistical analysis. Statistical analysis was accomplished by Student's t test using the software GraphPad Prism version 4. Values of p<0.05 were considered significant.

3. Example 1: GaB^{' ~} mice develop a lower degree of acanthosis after epicutanous sensitization.

[0144] To examine the role of galectin-3 in the development of the inflammatory response in the skin, we compared sensitized skin patches in gal3^~7~ and wild type mice. Biopsy samples from OVA- and saline-sensitized skin sites were harvested 24 h after the completion of the third sensitization period in both groups. Epicutaneous sensitization with OVA induced significant thickening of epidermis and dermis in both g&\y'^~ and gal3^+/+ mice. However, the degree of thickening was significantly lower in gal3^{~ ~} mice compared to gal3^{+ +} mice (Fig. 1). The results suggest that galectin-3 contributes to acanthosis of the epidermis in a mouse model of AD.

4. Example 2: Gal3 mice develop a lower amount of eosinophil and mononuclear cell infiltrations at the OVA-sensitized skin sites.

[0145] Dermal infiltration with eosinophils is an important feature of atopic dermatitis. Eosinophils were barely detectable in saline-sensitized skin sites, but their numbers dramatically increased following epicutaneous sensitization with OVA in gal3^{+ +} mice. In contrast, significantly lower amounts of eosinophils were detected at the similarly treated skin sites of gal3^{~ ~} mice (Fig. 2). In addition, the number of dermal mononuclear cells were lower in OVA-sensitized skin sites of gal3^{" ~} mice. There was no statistically significant difference in the number of circulating eosinophils and mononuclear cells between gal3 -/- and gal3 >+/+ mice. These results suggest that galectin-3 is critical for eosinophil and mononuclear cell infiltration of the skin following epi cutaneous sensitization with antigen.

5. Example 3: Gal 3 mice mount a lower IgE response after epicutaneous OVA sensitization.

[0146] The mouse model of atopic dermatitis we employed is characterized by a Th2- dominated systemic response including elevated IgE levels in the serum. We compared the total and antigen-specific IgE response between gal3^+/+ and gal3^~'^~ mice. As shown in Figure 3A, gaB^"7" mice sensitized with OVA exhibited strikingly lower serum IgE levels compared to similarly treated gal3^+/+ mice. In addition, the levels of OVA-specific IgE in the serum were also significantly lower in gal3^{~ ~} mice (Fig. 3A).

6. Example 4: Gal3 ^μ mice exhibit a lower Th2 response and a higher ThI response to epicutaneous OVA sensitization. [0147] Th2 cytokines play a critical role in isotype switching to IgG], while ThI cytokines play a role in isotype switching to IgG_2a. We measured the OVA-specific IgG] and OVA- specific IgG_2S levels in the serum, and calculated the ratio. After epicutaneous OVA sensitization, gal3^~; mice exhibited a trend toward lower OVA-specific IgGi but higher OVA-specific IgG_2a levels, and showed significantly higher IgG_2a / IgGi ratio compared to gal3^+/+ mice (Fig. 3B). These results suggest that gaB^"7" mice have lower Th2 but higher ThI responses compared to gal3^{+ +} mice.

[0148] Skin lesions of AD are characterized by increased expression of IL-4 mRNA, although IFN-γ mRNA tends to increase in chronic lesions. We next examined the expression of IL-4 and IFN-γ mRNA at the skin sites. Low levels of IL-4 and IFN-γ mRNA were detected in the PBS-sensitized skin sites and the levels were comparable between gaB- /- and ga!3 +/+ mice. Expression of IL-4 mRNA, but not IFN-γ mRNA markedly increased at the OVA-sensitized sites of gal3 +/+ mice, suggesting the presence of Th2 cells in the skin. In contrast, IL-4 mRNA did not significantly increase in OVA-sensitized skin of gaB -/- mice, whereas IFN-γ mRNA was significantly upregulated (Fig. 3C). These results suggest that following epicutaneous sensitization, galectin-3 deficiency skews the cytokine profile of infiltrating T cells towards ThI and away from Th2. 7. Example 5: APCs secrete more IL-12 and induce a ThI -polarized response [0149] To further examine the role of galectin-3 in the systemic Th2 response, we also examined cytokine production in response to in vitro restimulation of splenocytes with OVA. OVA stimulation of splenocytes from epicutaneously sensitized ga\3^~'^~ mice induced the secretion of IL-4 in amounts comparable to those secreted by ga!3^+/+ mice. The treatment caused higher IFN-γ production in gal3^{^ ~}mice compared to gal3^{+ +} mice, but the difference was not statistically significant (Fig. 4A).

[0150] IL-12 is a potent immunoregulatory cytokine that promotes ThI differentiation and is known to induce IFN-γ production from T cells as well as NK cells. Splenocytes from gal3^{~ ~}mice secreted significantly more IL-12 than those from gal3^+/+ mice (Fig. 4A). We examined the effect of galectin-3 deficiency on the ability of DCs to drive Th cell differentiation in vitro. Presentation of OVA_323-339 peptide by gal3^{^}'^" DCs to T cells from OVA-TCR tg mice resulted in significantly higher IFN-γ secretion but lower IL-4 secretion, compared to presentation by gal3^{+ +} (Fig. 4B). These results suggest that gal3 plays an important role in the development of the systemic Th2 response to epicutaneously-introduced protein antigen.

8. Example 6: Gal3^{~ ~} mice demonstrate lower IL-4 mRNA expression but higher IFN- γ expression at the OVA-sensitized skin sites.

[0151] AD lesions are characterized by increased expression of IL-4 mRNA, although IFN- γ mRNA tends to increase in chronic lesions (Leung, J Allergy Clin Immunol 1999, 104:S99- 108). We next examined the expression of IL-4 and IFN-γ mRNA in sensitized skin sites using real-time PCR. Low and comparable levels of IL-4 and IFN-γ mRNA were detected in the saline-sensitized skin from both gal3^{~ ~} and gal3^{+ +} mice. After sensitization, expression of IL-4 mRNA, but not IFN-γ mRNA markedly increased at the skin sites of gal3^{+ +} mice, suggesting the presence of Th2 cells in the skin. In contrast, IL-4 mRNA did not significantly increase in OVA-sensitized skin of gal3^{~ ~} mice, while IFN-γ mRNA was significantly upregulated (Fig. 5). These results suggest that following epicutaneous sensitization, galectin-3 deficiency skews the cytokine profile of infiltrating T cells towards ThI and away from Th2.

9. Example 7: Mice receiving T cells from gal3^{~ '} mice develop lower levels of dermatitis after exposure to OVA.

[0152] T cells are known to play a key role in the development of human AD and mouse model of AD. It has been shown that RAG2 mice, which lack both B and T cells, failed to display dermatitis, while IgH ' mice, which lack mature B cells, show dermatitis equivalent to those observed in wild-type controls after epicutaneous exposure to OVA (Woodward et al, J Allergy Clin Immunol 2001 , 107:359-366). The data suggest that T cells, but not B cells, are required to develop skin allergic inflammation in mouse AD model. [0153] To further determine the role of galectin-3 in regulation of the immune response in AD, we adoptively transferred gal3^{+ +} or gal3^{~ ~} T cells from mice of the OT-Il mice background, which are transgenic for the TCR recognizing POVA₃₂₃-₃₃₉. Splenocytes from gal3 ' /OVA-TCR tg or gal3^+/+/OVA-TCR tg mice were activated with APC, OVA and IL-2 in vitro, then injected intravenously into recipient gal3^{+ +} mice on C57BL/6 background. Flow cytometric analysis indicated that over 80% of the cells transferred were CD4⁺ T cells. One day after injection, mice were epicutaneously sensitized with OVA, and biopsies were taken from the OVA-sensitized skin sites one day after completion of the 1 -week sensitization. Interestingly, similar to the established model with three 1-week sensitizations, histological examination indicated that one-week exposure to OVA in splenocyte recipients induced epidermal and dermal thickening with a dense dermal infiltration. Skin inflammation was not observed in control mice which were engrafted with splenocytes and treated with saline alone. The epidermis of OVA-sensitized skin sites exhibited focal acanthosis and spongiosis. Recipients that received T cells from gal3^~ 7 OVA-TCR tg mice exhibited lower inflammation in the skin compared to those receiving T cells from gal3⁺ /OVA-TCR tg mice. However, the difference was not statistically significant (Fig. 6). To rule out the possible effects of other cell types {e.g., APCs) transferred, we transferred purified CD4⁺ T cells (90%), and obtained nearly identical results.

10. Example 8: Mice receiving T cells from gal3 mice show significantly lower eosinophil infiltration at OVA-sensitized skin sites. [0154] We next quantified the dermal cellular infiltrates in this new AD model. We observed a dense dermal infiltration in recipients after the 1-week sensitization. Eosinophil infiltration was significantly lower in OVA-sensitized mice that received T cells from gal3^{~ ~} mice. There was no significant difference in mononuclear cell infiltration between gal3^{~ ~} and gal3^+/+ T cell recipients (Fig. 7A). Nearly identical results were obtained when purified CD4⁺ T cells were transferred (Fig. 7B). These results suggest that endogenous galectin-3 in T cells is critical for the development of local inflammation in the skin. 11. Example 9: Mice receiving T cells from gaB mice exhibit a markedly lower Th2 response but an exaggerated ThI response.

[0155] We measured the serum IgE levels in recipients after the 1-week sensitization. Serum IgE levels did not increase after one- week sensitization in the model of repeated epicutaneous OVA exposure (Wang et al, J Immunol 1996, 156:4077-4082). In the AD model comprising T cell engraftment, however, significantly higher levels of total IgE antibodies were detected in gaI3^+/+ T cell recipients after one-week sensitization. In contrast, there was no increase in the serum IgE levels in mice that received gal3^{~ ~} T cells. Serum IgE levels after OVA sensitizations were significantly higher in gal3^{+ +} T cell recipients than in gal3^~7~ T cell recipients (Fig. 8A). OVA-specific IgE levels were below the limit of detection in all groups (data not shown).

[0156] Furthermore, recipients mounted OVA-specific IgGj and IgG₂₃ antibodies following 1 -week epicutaneous sensitization. We demonstrated that mice that received T cells from g&\y'^~ mice mounted significantly lower levels of OVA-specific IgG] antibodies, but significantly higher IgG_2a antibodies than those receiving gal3^{+ +} T cells. Thus, gal3^~7~ T cell recipients showed significantly higher IgG₂₃ / IgGi ratio (Fig. 8B).

[0157] We examined cytokine production by splenocytes or draining (axillary) lymph node cells stimulated with OVA antigen in vitro. We found that those cells from mice that received T cells from gal3^~'7 OVA-TCR tg mice secreted less IL-4 and IL-5 but more IFN-γ and IL-12 (Fig. 9 and Table 2). Taken together, these results suggest that galectin-3 plays an important role in the development of the systemic Th2 response to epicutaneously-introduced protein antigen.

Table 2: Analysis of in vitro cytokine synthesis from recipient mice

Donor cells: In vitro expanded T cells

Spleen DLN gal3 +/+ gal3 -/- gal3 +/+ gal -/-

IL-4 (ng/ml) 0.251 ± 0.006 0.219 ± 0.011 0.210 ± 0.006 0.186 ± 0.003

IFN-γ (ng/ml) 78.1 ± 9.1 178.1 ± 29.4 154.5 ± 19.3 217.3 ± 9.1

IL-5 (ng/ml) 1.13 ± 0.16 0.54 ± 0.16 1.09 ± 0.07 0.49 ± 0.11

Donor cells: Purified CD4+ T cells

Spleen DLN gal3 +/+ gaB -/- gal3 +/+ ga!3 -/-

IL-4 (ng/ml) 0.22 ± 0.009 0.191 ± 0.009 0.248 ± 0.013 0.212 ± 0.004

IFN-γ (ng/ml) 72.8 ± 1 1.4 169.9 ± 23.9 122.6 ± 17.9 222.4 ± 29.4

IL-5 (ng/ml) 0.98 ± 0.19 0.46 ± 0.12 1.29 ± 0.17 0.63 ± 0.13

Enriched populations (upper panel) of OVA-specific gal3^{+ +} and gal3 T cells generated in vitro or purified CD4⁺ T cells (lower panel) were adoptively transferred into wild-type mice (5 x 10⁶ cells/mouse) and recipient mice were exposed to OVA for 1 week. Spleen and draining lymph nodes (DLN) were harvested from recipient mice, and restimulated as described in Fig 3. Mean value ± SEM are shown (n = 5 animals per group). *P <0.05; **P <0.01 vs similarly treated gal3^+/+ controls. Similar results were obtained in a separate experiment.

12. Discussion

[0158] These results demonstrate that galectin-3 contributes significantly to allergic skin inflammation in a mouse model of AD. First, gal3^~'^~ mice manifest significantly reduced allergic skin inflammation, as measured by the thickness of epidermis and the number of eosinophils and mononuclear cells in the dermis, compared to gal3^+/+ mice. Second, gal3^{~ ~} mice exhibit a lower Th2 response and a higher ThI response to epi cutaneous OVA sensitization. Third, mice receiving T cells from gal3 ^"'^" mice develop a markedly lower Th2 response and appreciably reduced dermatitis after exposure to OVA, compared to those receiving T cells from gal3^+/+ mice. The results suggest that endogenous galectin-3 contributes to allergic skin inflammation by directing the immune response toward Th2 and exerts the effects at least in part through regulating the T cell function.

[0159] Another remarkable aspect of the present study is the development of a novel mouse AD model in which Th2-mediated dermatitis is induced by epicutaneous sensitization in mice following transfer of in vitro activated T cells bearing OVA-specific TCR. In contrast to the existing model in which three one-week periods of sensitization are required, dermatitis develops after just one week of antigen exposure in the new model. In addition, the degrees of cutaneous eosinophil infiltration, and local as well as systemic Th2 response developed in this model are comparable to the existing model. A number of immunological events need to be elicited for development of dermatitis by epicutaneous sensitization. Adoptive transfer of activated T cells bearing antigen-specific TCR allows some of the events to be bypassed, such as antigen uptake by dendritic cells, migration of dendritic cells from epidermis to the draining lymph nodes, T cell activation by dendritic cells at regional lymph node, and clonal expansion of antigen-specific T cells. We envision that in vitro activated OVA-specific effector T cells quickly accumulate in the area of sensitization and develop local inflammation through cytokine production. Our findings indicate that T cells play a key role in the development of human AD and mouse model of AD.

[0160] Kootiratrakarn et al. observed epidermal hyperplasia and dermal infiltration after one week of epicutaneous sensitization with OVA, three weeks after intraperitoneal priming in BALB/c mice {Eur J Immunol 2005, 35:3277-3286). Thus, transfer of T cells achieves the effect rendered by systemic sensitization. In their model, mice were primed with OV A/alum three times at one- week intervals before one week of sensitization. In our model, epicutaneous sensitization is rendered one day after T cells are transferred, and required only a total of 13 days.

[0161] The lower levels of dermatitis in gal3^{~ ~} mice may be explained by the deviation of the immune response to ThI . The results are consistent with our previous findings in the studies of a mouse model of asthma, where gaB^ mice exhibited reduced airway inflammation and mounted a lower Th2 response, but a higher ThI response compared to gal3^+/+ mice (Zuberi et ah, Am J Pathol 2004, 165:2045-2053). Antigen-presenting cells are known to play an important role in Th cell polarization. We noted that splenocytes from gal3 ^~~'^~ mice produce higher amounts of IL- 12, which is known to be a potent inducer of ThI response, and is mainly produced by macrophages and dendritic cells, after epicutaneous sensitization. The data are consistent with the recent report that galectin-3-deficient dendritic cells produce larger amounts of IL- 12 (Bernardes et al, Am J Pathol 2006, 168:1910-1920). These results suggest that galectin-3 may regulate the immune response by influencing IL-12 production by antigen-presenting cells.

[0162] However, the results from our T cell transfer experiment demonstrate that the action of galectin-3 in skin inflammation can be exerted at the level of the T cells, and that endogenous galectin-3 in T cells has the potential to promote the differentiation of naϊve T cells into Th2 cells. The exact mechanism for galectin-3 's regulation of Thl/Th2 polarization remains to be elucidated. It has been demonstrated that mice deficient in UDP-7V-acetyl- glucosaminyltransferase V (Mgat5) show inefficient formation of multivalent lattices of galectin-3 and 7V-glycans in the TCR complexes, resulting in increased TCR activation, enhanced ThI response, and susceptibility to autoimmune diseases (Demetriou et al, Nature 2001 , 409:733-739). These data suggest that galectin-3 interacts with TCR, and influences the threshold of TCR signaling. Thus, galectin-3-deficiency may result in increased sensitivity of ThI cells to antigen and higher production of ThI cytokines. [0163] We have shown previously that galectin-3 is anti-apoptotic when transfected into the Jurkat human T cell line (Yang et al. , Proc Natl Acad Sci USA \ 996, 93 :6737-6742).

Subsequent studies established that endogenous galectin-3 exerts this function intracellularly by interacting with molecules in the apoptosis regulation pathways (Hsu et al., Am J Pathol 2000, 156:1073-1083; Kim et al, Cancer Res 1999, 59:4148-4154; Matarrese et al, IntJ Cancer 2000, 85:545-554; Moon et al, Am J Pathol 2001, 159:1055-1060; Yoshii et al, J Biol Chem 2002, 277:6852-6857; Takenaka et al., MoI Cell Biol 2004, 24:4395-4406). On the other hand, more recently, Stillman et al. {J Immunol 2006, 176:778-789) reported that exogenously added galectin-3 can induce apoptosis in T cells, probably through binding to cell surface glycoproteins, such as CD29, CD43, CD45 and CD71 , all of which were shown to bind to immobilized galectin-3. Thus, a possibility exists that galectin-3 regulates apoptosis in ThI and Th2 differentially, and its expression favors the higher Th2/Thl ratio observed.

[0164] We found a significantly decreased dermal mononuclear cell infiltration in gal3 ^{^} mice in a model with three sensitizations. On the other hand, mononuclear cell infiltration in the dermis is not decreased in skin from mice receiving gal3^{~ ~} T cells. These results indicate that lack of endogenous galectin-3 in T cells does not affect T cell migratory activity. The recruitment of T cells to the skin is dependent on several factors, including the production of chemokines and cytokines by resident skin cells. Our result suggests that lack of endogenous galectin-3 in resident or newly recruited skin cells, including keratinocytes, dendritic cells, macrophages, mast cells, and eosinophils, may affect the local production of chemoattractants involved in T cell trafficking. It may also be explained by the anti-apoptotic activity of galectin-3 mentioned above. Upon activation, galectin-3-deficient T cells may be more susceptible to apoptosis than wild-type T cells in vivo. The difference of viability between gal3^{~ ~} and gal3^{+ +} T cells may not be evident in a one-week sensitization, becoming apparent only in more long-term, repeated sensitizations.

[0165] Infiltration of the skin with eosinophils is a hallmark of human AD and mouse models of AD. Skin from gal3^{~ ~} mice showed impaired eosinophil infiltration following epicutaneous sensitization with OVA. This is likely not due to poor survival of galectin-3- deficient eosinophils, since eosinophil infiltration is also significantly impaired in skin from gal3^{~ ~} T cell recipient mice in our T cell transfer experiment. Instead, our results indicate that T cells may be an essential source of chemoattractants that is important for eosinophil migration in these models.

[0166] We show that both splenocytes and draining lymph node cells from ga!3 ^~!~ T cell recipient mice have a decreased capacity to produce IL-5. IL-5 is known to prolong survival, differentiation, and activation of eosinophils. IL-5 stimulates the maturation of eosinophils from CD34⁺ precursor cells in the bone marrow and their release into the circulation (Gleich, J Allergy Clin Immunol 2000, 105:651-663). IL-5 also primes eosinophils for responsiveness to chemotactic factors (van de Rijn et al., J Allergy Clin Immunol 1998, 102:65-74). A previous report has shown that eosinophils were virtually absent in OVA-sensitized skin sites of IL-5^"7" mice (Spergel et al, J Clin Invest 1999, 103:1103-1 1 11). Therefore, the decreased capacity to produce IL-5 in galectin-3 -deficient T cells may be, at least in part, involved in the lower eosinophil infiltrations in gal3^{~ ~} mice. On the other hand, exogenously added galectin-3 has been shown to suppress the production of IL-5 in eosinophils and T cells (Cortegano et al. , J Immunol 1998, 161 :385-389). The apparent contradictory effects of administration of exogenous galectin-3 versus ablation of the galectin-3 gene illustrate its ability to function both outside and inside the cell, as mentioned above with regard to T cell survival. [0167] Galectin-3 could contribute to the development of dermatitis through other cells involved in the development of the skin inflammation in the model we used. Galectin-3 is expressed by keratinocytes as well as leukocytes present in the skin, including B cells, T cells, neutrophils, dendritic cells, macrophages, mast cells and eosinophils (Liu, Int Arch Allergy Immunol 2005, 136:385-400). Several differences of the results between two different models, the one with three sensitizations and the T cell engraftment model, provide several insights into the regulatory roles of galectin-3 in AD. We note that the gal3^{" ""} mice develop a markedly lower degree of acanthosis compared to gal3^+/+ mice in the model with three sensitizations. In contrast, the difference in the degree of acanthosis between mice receiving gal3^{~ ~} T cells and control group was not statistically significant in T cell transfer experiment. These results suggest that galectin-3 in other cell types may also affect the development of acanthosis.

[0168] Extracellular galectin-3 has been reported to function as a chemoattractant for monocytes and macrophages (Sano et al, J Immunol 2000, 165:2156-2164). In addition, we have shown that galectin-3 plays a critical role in phagocytosis by macrophage through an intracellular mechanism (Sano et al, J Clin Invest 2003, 1 12:389-397). We found that galectin-3- deficient dendritic cells exhibited significantly impaired migratory activity through an intracellular mechanism. Other investigators using the same mouse AD model have demonstrated that the D prostanoid receptor 1 agonist, a potent inhibitor of epidermal Langerhans cell emigration, dramatically enhanced the epidermal thickening at the OVA- sensitized site (Angeli et al, J Immunol 2004, 172:3822-3829). The data suggest that migratory activity of Langerhans cells is important for the development of acanthosis. Thus, impaired function of skin dendritic cells in gal3^{~ ~} mice may also contribute to the decreased epidermal thickening in the mouse model of AD we employed.

[0169] Another possibility is the contribution of mast cells. Mast cells are well-known to be key effector cells in IgE-mediated immediate hypersensitivity reactions. In a model of allergen-induced airway inflammation, mast cell-deficient mice sensitized intraperitoneally with OVA had lower number of eosinophils in bronchoalveolar lavage fluid and lungs after inhaled-allergen challenge (Nagai et al., Clin Exp Allergy 1996, 26:642-647). Recently we have demonstrated that galectin-3 -deficient mast cells exhibit impaired degranulation and diminished IL-4 production (Chen et al, J Immunol 2006, 177:4991-4997). The impaired function of galectin-3 -deficient mast cells could be involved in the development of acanthosis in gaB^"^ mice.

[0170] In summary, endogenous galectin-3 is a proinflammatory molecule and a potentiator for skin inflammation in a mouse model of AD. Galectin-3 can contribute to the allergic skin inflammation by directing the immune response toward Th2.

[0171] All patents, patent applications, Genbank disclosures, and other publications cited in this application, including published amino acid or polynucleotide sequences, are incorporated by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:

L A method for treating an inflammatory condition, comprising the step of administering to a subject an effective amount of an inhibitor of galectin-3.

2. The method of claim 1 , wherein the inhibitor is an inactivating antibody of galectin-3.

3. The method of claim 1 , wherein the inhibitor is an inhibitory nucleic acid comprising a sequence complementary to a galectin-3 polynucleotide.

4. The method of claim 1 , wherein the inhibitor is a peptide inhibitor of galectin-3.

5. The method of claim I , wherein the inhibitor is an RNA aptamer specific for galectin-3.

6. The method of claim 1 , wherein the inflammatory condition is a Th-2 mediated inflammatory condition.

7. The method of claim 1 , wherein the inflammatory condition is atopic dermatitis.

8. The method of claim 1 , further comprising treatment with an additional therapeutic compound.

9 . The method of claim 8, wherein the additional therapeutic compound and the inhibitor of galectin-3 are administered at different times.

10. The method of claim 8, wherein the additional therapeutic compound and the inhibitor of galectin-3 are administered at the same time.

I L A composition comprising an effective amount of an inhibitor of galectin-3 and a pharmaceutically acceptable carrier, wherein the inhibitor is present in an amount effective to suppress galectin-3 expression or activity.

12. The composition of claim 11 , wherein the inhibitor is an inactivating antibody of gal ectin-3.

13. The composition of claim 1 1 , wherein the inhibitor is an inhibitory nucleic acid comprising a sequence complementary to a galectin-3 polynucleotide.

14. The composition of claim 11 , wherein the inhibitor is a peptide inhibitor of galectin-3.

15. The composition of claim 1 1 , wherein the inhibitor is an RNA aptamer specific for galectin-3.

16. The composition of claim 11, further comprising an additional therapeutic compound.

17. A kit for treating an inflammatory condition, said kit comprising the composition of claim 11.

18. The kit of claim 17, wherein the inhibitor is an inactivating antibody of galectin-3.

19. The kit of claim 17, wherein the inhibitor is a polynucleotide comprising a sequence complementary to a galectin-3 polynucleotide.

20. The kit of claim 17, wherein the inhibitor is a peptide inhibitor of galectin-3.

21. The kit of claim 17, wherein the inhibitor is an RNA aptamer specific for galectin-3.

22. The kit of claim 17, further comprising an additional therapeutic compound.