DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention provides methods useful for the treatment and prevention of non-allergic inflammatory diseases, including psoriasis, eczema, allergic contact dermatitis, latex dermatitis, and inflammatory bowel disease. These diseases are believed to be caused in part by Th1-mediated immune responses. The methods involve the administration of certain immunostimulatory nucleic acids to subjects that have or are at risk of developing a non-allergic inflammatory disease. Surprisingly, the isolated immunostimulatory nucleic acids useful in the methods for treating and preventing non-allergic inflammatory diseases are known to induce or redirect an immune response toward a Th1-like type of immune response. Since CpG DNA and T-rich oligodeoxynucleotide (ODN) cause Th1-mediated immune activation, it was unexpected that these should be effective in treating these diseases. However, in addition to the Th1 effects, CpG DNA also induces counterregulatory mechanisms, including the generation of IL-10. Redford T W et al. (1998) J Immunol 161:3930-5. Also surprisingly, the isolated immunostimulatory nucleic acids useful in the methods for treating and preventing non-allergic inflammatory diseases are useful when administered systemically to a subject in need of such treatment.

[0045] The present invention also provides methods useful for enhancing a Th1-like immune response to immunostimulatory nucleic acids. It has surprisingly been discovered according to aspects of the present invention that immunostimulatory nucleic acids that induce a Th1-like immune response also induce counter-regulatory molecules, prostaglandins, that may dampen the net Th1 response. The prostaglandins are induced by immunostimulatory nucleic acid-induced up-regulation of expression of prostaglandin synthase, a key enzyme in the synthetic pathway from arachidonic acid to prostaglandin. Thus, inhibition of this pathway and of prostaglandin signaling in the context of immunostimulatory nucleic acids removes the counter-regulatory effect of prostaglandins on Th1-like immune activation.

[0046] Allergy involves the clinically adverse reaction to allergens which reflects the expression of acquired immunologic responsiveness involving allergen-specific antibodies and/or T lymphocytes. Classically, allergy was divided into four types, including type I (immediate hypersensitivity) involving IgE antibodies, and type IV (delayed-type hypersensitivity, DTH) involving sensitized T lymphocytes without any essential role for antibodies. Coombs RRA and Gell PGH (1963) The classification of allergic reactions underlying disease. In: Gell PGH and Coombs RRA, eds. Clinical Aspects of Immunology. Oxford: Blackwell Scientific Publications, pp. 317-37. As used herein, an allergic inflammatory response will refer to IgE-associated immune response that includes the development of a prominent IgE antibody response to an initiating allergen, be the allergen known or unknown. Thus as used herein an allergic inflammatory response corresponds most closely to the classical type I allergic response and does not include the classical type W allergic response (DTH).

[0047] The term “non-allergic inflammatory disease,” as used herein, refers to a disease or disorder of a subject, wherein the disease or disorder is characterized by an inflammatory response essentially independent of an IgE response. By essentially independent of an IgE response is meant that local or systemic levels of IgE, if measured, would not be significantly different from corresponding local or systemic levels in subjects without an inflammatory response. Such non-allergic inflammatory diseases or disorders typically include an inflammatory response to an antigen, which may be known or unknown, that is characterized by infiltration and activation of certain immune cells, including neutrophils, monocytes, macrophages, NK cells, and T cells and by secreted products of those cells, including but not limited to IFN, TNF, IL-1, IL-6, IL-8, and IL-12. The inflammatory response of the non-allergic inflammatory disease may be acute or chronic and it may be intermittent or recurrent. Examples of non-allergic inflammatory diseases include, without limitation, psoriasis, inflammatory bowel disease, eczema, allergic contact dermatitis, latex dermatitis, and autoimmune disorders. In some instances the non-allergic inflammatory disease may encompass chronic allergic inflammation, insofar as this entity is essentially independent of an IgE response.

[0048] The non-allergic inflammatory disease in certain preferred embodiments involves an epithelium. An epithelium is a tissue composed of closely aggregated cells that are in apposition over a large part of their surface and so form a continuous layer of cells covering and defining an external or internal surface. An epithelium may include more than one layer and more than one kind of cell. Certain tissues and organs have specialized forms of epithelium, e.g., simple, stratified and pseudostratified; squamous, cuboidal, columnar, and transitional. Epithelia may also have associated specialized structures and functions, such as microvilli, cilia, and glands. The surface of the skin, or epidermis, is a stratified squamous epithelium. In addition, the epithelium lining the gastrointestinal tract is termed a mucosal epithelium, consisting of an epithelial lining (associated and in communication with mucosal and submucosal glands) overlying a loose connective tissue layer (lamina propria) and a thin layer of smooth muscle (muscularis mucosa). Thus in certain preferred embodiments the non-allergic inflammatory disease involves the skin, and in certain preferred embodiments the non-allergic inflammatory disease involves the mucosa lining the gastrointestinal tract.

[0049] Psoriasis is a chronic inflammatory disease of the skin that affects 1-3% of the Caucasian population worldwide. Barker J N W N (1994) Bailliere's Clin Rheumatol 8:429-37. This complex disease is characterized by alterations in a variety of different cells of the skin. These include epidermal keratinocyte hyperproliferation and altered differentiation indicated by focal parakeratosis (cell nuclei in stratum corneum), aberrant expression of the hyperproliferation-associated keratin pair 6/16 (Stoler A et al. (1988) J Cell Biol 107:427-46; Weiss R A et al. (1984) J Cell Biol 98:1397-1406), involucrin and filaggrin (Bernard B A et al. (1986) Br J Dermatol 114:279-83; Dover R et al. (1987) J Invest Dermatol 89:349-52; Ishida-Yamamoto A et al. (1995) J Invest Dermatol 104:391-5), and integrin adhesion molecules (VLA-3, -5 and -6, α ₆ β ₄ ) (Hertle M D et al. (1992) J Clin Invest 89:1892-1901; Kellner I et al. (1991) Br J Dermatol 125:211-6). In addition, de novo expression of major histocompatibility complex (MHC) class II and intercellular adhesion molecule-1 (ICAM-1, CD54) by keratinocytes is observed (Barker J N W N et al. (1990) J Clin Invest 85:605-8; Gottlieb A B et al. (1986) J Exp Med 164:1013-28; Griffiths C E M et al. (1989) J Am Acad Dermatol 20:617-29; Nickoloff B J et al. (1990) J Invest Dermatol 94:151S-157S; Veale D et al. (1995) Br J Dermatol 132:32-8). Endothelial cells also are hyperproliferative, resulting in angiogenesis and dilation (Detmar M et al. (1994) J Exp Med 180:1141-6; Goodfield M et al. (1994) Br J Dermatol 131:808-13; Malhotra R et al. (1989) Lab Invest 61:162-8; Mordovtsev V N et al. (1989) Am J Dermatopathol 11:33-42) and express increased levels of ICAM-1, E-selectin (CD62E) and vascular cell adhesion molecule-1 (VCAM-1, CD106) (Das P K et al. (1994) Acta Derm Venereol Suppl 186:21-2) as well as MHC class II (Bjerke J R et al. (1988) Acta Derm Venereol 68:306-11). Finally, a mixed leukocytic infiltrate is seen, composed of activated T lymphocytes which produce inflammatory cytokines (Ramirez-Bosca A et al. (1988) Br J Dermatol 119:587-95; Schlaak J F et al. (1994) J Invest Dermatol 102:145-9), neutrophils within the dermis and forming Munro's microabscesses in the epidermis (Christophers E and Sterry W (1993) Psoriasis. In: Dermatology in General Medicine, T B Fitzpatrick, A Z Eisen, K Wolff, I M Freedberg and K F Austen, eds. (New York: McGraw-Hill, Inc.), pp. 489-514), and an increased number of dermal mast cells (Brody I (1986) Ups J Med Sci 91:1-16; Brody I (1984) J Invest Dermatol 82:460-4; Rothe M J et al. (1990) J Am Acad Dermatol 23:615-24; Schubert C et al. (1985) Arch Dermatol Res 277:352-8; Toruniowa B et al. (1988) Arch Dermatol Res 280:189-93; van de Kerkhof P C et al. (1995) Skin Pharmacol 8:25-9).

[0050] There is strong evidence that psoriasis is characterized by Th1 and proinflammatory cytokines. IL-2 and IFN-γ are predominant within skin lesions, while IL-4 and IL-10 are scant or absent. Uyemura K et al. (1993) J Invest Dermatol 101:701-5; Schlaak J F et al. (1994) J Invest Dermatol 102:145-9. The proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α are also present in psoriatic lesions. Ohta Y et al. (1991) Arch Dermatol Res 283:351-6; Lemster B H et al. (1995) Clin Exp Immunol 99:148-54; Nickoloff, B J et al. (1991) Am J Pathol 138:129-40; Ettehadi P et al. (1994) Clin Exp Immunol 96:146-51. Intracutaneous secretion of cytokines is thought to mediate some or all of the tissue alterations seen in psoriasis. These cytokines include TNF-α and IL-1 (Kupper T S (1990) J Clin Invest 86:1783-6); IFN-γ (Barker J N W N et al. (1991) J Dermatol Sci 2:106-11; Gottlieb A B et al. (1988) J Exp Med 167:670-5; Livden J K et al. (1989) Arch Dermatol Res 281:392-7); IL-6 (Castells-Rodellas A et al. (1992) Acta Derm Venereol 72:165-8; Grossman R M et al. (1989) Proc Natl Acad Sci USA 86:6367-71; Neuner P et al. (1991) J Invest Dermatol 97:27-33); IL-8 (Barker J N et al. (1991) Am J Pathol 139:869-76), vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) (Detmar M et al. (1994) J Exp Med 180:1141-6), and TGF-α (Elder J T et al. (1989) Science 243:811-4; Gottlieb A B et al. (1988) J Exp Med 167:670-5; Prinz J C et al. (1994) Eur J Immunol 24:593-8).

[0051] Over the past decade, research into the pathophysiology of psoriasis has focused primarily on immunologic mechanisms. Evidence is accumulating that this disease has an immunological basis. However, it has not been convincingly determined if the primary defect that results in psoriasis is an immunologic disorder or resides within the epithelium (Barker J N W N (1994) Bailliere's Clin Rheumatol 8:429-38; Christophers E and Sterry W (1993) Psoriasis. In: Dermatology in General Medicine, T B Fitzpatrick, A Z Eisen, K Wolff, I M Freedberg and K F Austen, eds. (New York: McGraw-Hill, Inc.), pp. 489-514). Abnormal immune regulation is suggested by the frequent association of psoriasis with the expression of certain MHC alleles including HLA B13, B17, Bw57 and Cw6 (Russell T J et al. (1972) N Engl J Med 287:738-40; Tiilikainen A et al. (1980) Br J Dermatol 102:179-84; Watson W et al. (1972) Arch Dermatol 105:197-207; White S H et al. (1972) N Engl J Med 287:740-3), the improvement of psoriatic lesions by treatment with immunosuppressive agents such as cyclosporine A (Ellis C N et al. (1986) JAMA 256:3110-6; Mueller W et al. (1979) N Engl J Med 301:555) and the lymphocyte-specific fusion toxin DAB ₃₈₉ IL-2 (Gottlieb J L et al. (1995) Nat Med 1:442-7), the possible linkage of a psoriasis susceptibility gene with a gene involved in IL-2 regulation (Tomfohrde J et al. (1994) Science 264:1141-5), and the failure of psoriasis to recur after bone marrow transplantation (Eedy D J et al. (1990) Br Med J 300:908; Jowitt S N et al. (1990) Br Med J 300:1398-9). However, underlying epidermal and/or dermal defects are suggested by altered keratinocyte cell cycle and differentiation (Gelfant S (1982) Cell Tissue Kinet 15:393-7; Weinstein G D et al. (1985) J Invest Dermatol 84:579-83), by aberrant expression of adhesion molecules by keratinocytes and endothelial cells (Das P K et al. (1994) Acta Derm Venereol Suppl 186:21-2; Nickoloff B J et al. (1990) J Invest Dermatol 94:151S-157S; Petzelbauer P et al. (1994) J Invest Dermatol 103:300-5; Veale D et al. (1995) Br J Dermatol 132:32-8; Wakita H et al. (1994) Arch Dermatol 130:457-63), and by the abnormal expression of protooncogenes within keratinocytes (Elder J T et al. (1990) J Invest Dermatol 94:19-25).

[0052] Conventional treatments for psoriasis include topical corticosteroids (hydrocortisone, betamethasone, triamcinolone, fluocinolone acetonide, fluocinonide), tar preparations, ultraviolet light (UVB, 295-310 nm, with or without tar; or UVA, 320-400 nm, with or without psoralen), vitamin A analogs (etretinate, acitretin), retinoids (tazarotene), vitamin D analogs (calcipotriol), and immunosuppressive regimens employing potentially toxic agents cyclosporine or methotrexate. Immunosuppressive agents which dampen cell-mediated immunity, including cyclosporine, methotrexate, and lymphocyte-selective toxins, are effective but often are expensive and/or associated with significant risks related to potential side effects.

[0053] At least with respect to psoriasis, the foregoing agents, both conventional and experimental, are included among what are herein termed non-allergic inflammatory disease medicaments.

[0054] Despite its name, “allergic contact dermatitis” as used herein is a non-allergic inflammatory disease because it involves a cell-mediated, delayed-type hypersensitivity (DTH) reaction rather than an IgE-associated reaction. It is to be distinguished from irritant contact dermatitis which is caused by exposure to substances that directly cause physical, mechanical, or chemical irritation of the skin. Antigens commonly involved in allergic contact dermatitis include plant oleoresins found in poison ivy, poison oak, and poison sumac; certain topical medications, including topical hydrocortisone, topical antibiotics (e.g., neomycin and bacitracin), and benzocaine; nickel in jewelry; fragrances in perfumes, cosmetics, and washing agents; wool alcohols (lanolin); components of rubber (including latex), for example associated with rubber and latex glove use; preservatives (e.g., thimerosal, formaldehyde, quaternium-15); and nail polish. Latex dermatitis, as used herein, refers to delayed-type hypersensitivity involving contact of skin or mucosa with latex. Latex dermatitis occurs commonly but not exclusively in the context of use of latex gloves, for example by health care providers and food service workers. Allergic contact dermatitis in its afferent phase involves presentation of processed antigen by Langerhans cells, professional antigen-presenting cells resident within the dermis, to CD4+T cells. The afferent phase results in the expansion of an antigen-specific T-cell clone that is primed to interact with the triggering antigen upon re-exposure. Upon their encounter with the triggering antigen, sensitized T cells that have migrated to the skin release a cascade of cytokines that leads to inflammation characteristic of allergic contact dermatitis. Conventional medicaments for allergic contact dermatitis include topical corticosteroids, topical antihistamines, and, for severe cases, systemic corticosteroids.

[0055] Inflammatory bowel disease, as used herein, includes Crohn's disease and ulcerative colitis. While the precise causes of Crohn's disease and ulcerative colitis remain uncertain, these are well described diseases with partially overlapping but nonetheless distinct clinical and pathologic features. For a review, see Glickman R M, Inflammatory bowel disease: ulcerative colitis and Crohn's disease, in Harrison's Principles of Internal Medicine, 14th Edition, A S Fauci et al. (eds.), New York, McGraw-Hill, 1998. The estimated prevalence of these diseases in the United States is about 70-150 per 100,000 for ulcerative colitis and 20-40 per 100,000 for Crohn's disease.

[0056] Ulcerative colitis, which involves primarily the colonic mucosa, with rectal involvement in nearly all cases, is a chronic and recurrent disease clinically characterized by bloody diarrhea and abdominal pain. It may be complicated by anemia, dehydration and electrolyte abnormalities, weight loss, extraintestinal manifestations including arthritis, as well as life-threatening dilation and perforation of the colon. Approximately 25 percent of patients require colectomy at some point in their disease. Patients with longstanding ulcerative colitis are also at increased risk of having or developing cancer of the colon. Characteristically, ulcerative colitis most commonly has uniform, continuous, nontransmural involvement of the colon with loss of surface epithelial cells in involved areas.

[0057] Crohn's disease may involve any portion of the gastrointestinal tract, but most commonly it involves the distal small bowel and/or the colon. It too is a chronic disease and, while its symptoms are more variable due to the potential to involve any portion of the gastrointestinal tract, Crohn's disease is more likely than ulcerative colitis to have complications and to require hospitalization. Over two-thirds of patients require surgery at some point in their disease. The bowel inflammation in Crohn's disease characteristically involves the full thickness of the bowel wall. The inflammation extends to involve the mesentery and regional lymph nodes, and this intense inflammatory process results in bowel obstruction in 20 to 30 percent of patients at some point in their disease, as well as fistula and abscess formation in many patients. Also unlike ulcerative colitis, the lesions of Crohn's disease characteristically are discontinuous along the length of the bowel.

[0058] Conventional medical approaches to treatment of inflammatory bowel disease include therapy based on sulfasalazine (AZULFIDINE®, Pharmacia & Upjohn), of which 5-aminosalicylic acid is believed to be the active component, and corticosteroids. Related 5-aminosalicylic acid products used in the treatment of ulcerative colitis include olsalazine (DIPENTUM®, Pharmacia & Upjohn) and mesalamine (ROWASA®, Solvay). Corticosteroids include prednisone and prednisolone. Other agents sometimes used in the treatment of ulcerative colitis include immunosuppressive agents cyclosporine A (SANDIMMUNE® and NEORAL®, Novartis), tacrolimus (FK506, PROGRAF®, Fujisawa), and azathioprine (IMURAN®, Faro). At least with respect to inflammatory bowel disease, these agents, as well as experimental agents mentioned elsewhere herein (other than the immunostimulatory nucleic acids of the invention), are included among what are herein termed non-allergic inflammatory disease medicaments.

[0059] Th1-like immune activation refers to induction of immune response with a preponderance of Th1 character. For example, Th1-like activation may involve induction of lymphocytes to secrete Th1-like cytokines (e.g., IFN-γ, IL-2, IL-12, IL-18, and TNF) and antibodies (e.g., IgG2a in mice). Th1-like immune activation may also involve activation and/or proliferation of NK cells, CTLs, and macrophages.

[0060] A subject in need of Th1-like immune activation is a subject that has or is at risk of developing a disease, disorder, or condition that would benefit from an immune response skewed toward Th1. Such a subject may have or be at risk of having a Th2-mediated disorder that is susceptible to Th1-mediated cross-regulation or suppression. Such disorders include, for example, certain organ-specific autoimmune diseases. Alternatively, such a subject may have or be at risk of having a Th1-deficient state. Such disorders include, for example, tumors, infections with intracellular pathogens, and AIDS. In addition, according to the present invention, such disorders also include non-allergic inflammatory disorders in which further Th1 activation is beneficial in controlling or treating the non-allergic inflammatory disorders.

[0061] An immune cell as used herein refers to a cell belonging to the immune system. Such cells include, but are not restricted to, T- and B-lymphocytes, macrophages, monocytes, neutrophils, NK cells, professional antigen-presenting cells, dendritic cells, and their precursors.

[0062] The combination of immunostimulatory nucleic acids together with NSAIDs gives a surprising degree of synergy in terms of inducing stronger Th1 responses than would have been expected from additive effects. It has been discovered according to the invention that immunostimulatory nucleic acids such as CpG ODN may cause immune cells to produce Th1 counter-regulatory molecules such as PGE ₂ through activating the synthesis of COX-2. NSAIDs inhibit COX-2 activity, removing the negative effects of PGE ₂ on CpG-induced immunity. As a result, CpG DNA induces a much stronger Th1-like response in the presence of NSAIDs than in their absence. The compounds do not have to be administered at the same time, and often pre-treatment with NSAIDs may be desirable prior to injection with CpG ODN. NSAID therapy may be continued after CpG therapy for several weeks to several months, depending upon the desired duration of Th1 enhancement. The combination of NSAIDs with CpG therapy is compatible with CpG monotherapy, but also immunization or combinations with monoclonal antibodies, chemotherapy, radiation therapy, and other treatments.

[0063] CpG DNA may induce the secretion of Th1 cytokines such as IL-12 and IFN-γ, as well as proinflammatory cytokines including tumor necrosis factor TNF-α, IL-6 and type I IFN. Klinman D M et al. (1996) Proc Natl Acad Sci USA 93:2879-83. In addition, CpG DNA activates NK cells to secrete IFN-γ and enhances their lytic activity. Ballas Z K et al. (1996) J Immunol 157:1840-45; Cowdery J S et al. (1996) J Immunol 156:4570-75; Chace J H et al. (1997) Clin Immunol Immunopathol 84:185-93. These studies demonstrate that CpG DNA is able to induce multiple protein mediators of the immune and inflammatory response.

[0064] Prostaglandins (PGs) are lipid mediators that are also key effectors of acute and chronic inflammation. Needleman P et al. (1997) J Rheumatol 24:6-8; Portanova J P et al. (1996) J Exp Med 184:883-91; Anderson G D et al. (1996) J Clin Invest 97:2672-79; Amin A R et al. (1999) Curr Opin Rheumatol 11:202-209; MacDermott R P (1994) Med Clin North Am 78:1207-31. Moreover, PGs are important regulators of cell-mediated immune responses. For example, PGE ₂ is a potent inhibitor of Th1-type T cell responses (Betz M et al. (1991) J Immunol 146:108-13), inhibiting IFN-γ production as well as IL-12 and IL-12 receptor expression (Betz M et al. (1991) J Immunol 146:108-13; Wu C Y et al. (1998) J Immunol 161:2723-30). Exogenous PGE ₂ is also a potent inhibitor of macrophage-derived inflammatory mediators, including TNF-α production (Kunkel S L et al. (1988) J Biol Chem 263:5380-84) and nitric oxide production (Corraliza I M et al. (1995) Biochem Biophys Res Commun 206:667-73).

[0065] Prostaglandins are synthesized from arachidonic acid by prostaglandin G/H synthase, also known as PG endoperoxide synthase and cyclooxygenase (COX). Prostaglandin G/H synthase exists in two isoforms, now termed cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). COX-1 is constitutively expressed in cultured endothelial cells and vascular smooth muscle cells. COX-2 is inducibly expressed in response to cytokines, growth factors, phorbol esters, lipopolysaccharide, injury and stress. The generation of other products of the arachidonic acid cascade (besides cyclooxygenase-produced metabolites) is inhibited neither by non-selective nor by COX-2 selective NSAIDs.

[0066] There are three broad classes of cyclooxygenase inhibitors: salicylates like aspirin (salicylic acid); nonselective COX inhibitors like indomethacin and other NSAIDs, and selective inhibitors of COX-2 catalytic activity (coxibs).

[0067] NSAIDs include, without limitation, diclofenac, diflunisal, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, naproxen, olsalazine, oxaprozin, piroxicam, sulfasalazine, sulindac, tolmetin, salicylates, coxibs, and nitric oxide (NO)-releasing NSAIDs.

[0068] Coxibs include, but are not limited to, celecoxib (SC-58635); rofecoxib; valdecoxib; etoricoxib; nimesulide; meloxicam; NS-398; L-745,337; SC236; SC-58125; SC-58236; C-phycocyanin; BMS-279652, BMS-279654, and BMS-279655 (Zhu H et al. (2002) Proc Natl Acad Sci USA 99:3932-37), wogonin (5,7-dihydroxy-8-methoxyflavone; Wakabayashi I et al. (2000) Eur J Pharmacol 406:477-81); and nabumetone.

[0069] Inducers of COX-2 expression include TGF-β1 (Sheng H et al. (2000) J Biol Chem 275:6628-35); Ras (Sheng H et al. (1998) J Biol Chem 273:22120-27); IL-1α (Ristimaki A et al. (1994) J Biol Chem 269:11769-75); IL-1β (O'Banion M K et al. (1992) Proc Natl Acad Sci USA 89:4888-92); src oncogene product (Xie W et al. (1991) Proc Natl Acad Sci USA 88:2692-96); peroxynitrite (ONOO ⁻ ; Migita K et al. (2002) Clin Exp Rheumatol 20:59-62); lipopolysaccharide (Lee S H et al. (1992) J Biol Chem 267:25934-38); epidermal growth factor (EGF, Saha D et al. (1999) Neoplasia 1:508-17); and TNF-α (Diaz A et al. (1998) Exp Cell Res 241:222-29).

[0070] COX-2 expression inhibitors include wogonin (Wakabayashi I et al. (2000) Eur J Pharmacol 406:477-81); anti-TGF-β1 antibody (Sheng H et al. (2000) J Biol Chem 275:6628-35); dexamethasone (Ristimaki A et al. (1994) J Biol Chem 269:11769-75; Ristimaki A et al. (1996) Biochem J 318:325-31); IL-4, IL-10, and IL-13 (Endo T et al. (1996) J Immunol 156:2240-46); and natriuretic peptides (Kiemer A K et al. (2002) Endocrinology 143:846-52).

[0071] At least four subtypes of PGE ₂ (E-prostanoid) receptors have been reported. These include EP1, EP2, EP3, and EP4. Stimulation of the EP1 receptor results in activation of phosphatidylinositol hydrolysis and in elevation of intracellular calcium concentration. Funk C D et al. (1993) J Biol Chem 268:26767-72. EP2 and EP4 receptors increase intracellular cAMP concentration through activation of adenylate cyclase. Regan J W et al. (1994) Mol Pharmacol 46:213-20; Bastien L et al. (1994) J Biol Chem 269:11873-77. The EP3 receptor inhibits adenylate cyclase leading to a decrease of cAMP concentration. Furthermore, the EP3 receptor exists as multiple isoforms. Kotani M et al. (1995) Mol Pharmacol 48:869-79; An S et al. (1994) Biochemistry 33:14496-502; Schmid A et al. (1995) J Biochem 228:23-30.

[0072] Agents that inhibit PGE ₂ signaling through its receptor include, but are not limited to, antibodies specific for PGE ₂ , antibodies specific for the receptors, antisense nucleic acids specific for the receptors, and small molecule receptor antagonists. As used herein, “agents that inhibit PGE ₂ signaling through its receptor” does not include cyclooxygenase inhibitors (described above). Polyclonal and monoclonal antibodies have been raised against PGE ₂ . Mnich S J et al. (1995) J Immunol 155:4437-44. Antibodies have also been raised against each of the four subtypes of PGE ₂ receptors. Morath R et al. 1999) J AM Soc Nephrol 10:1851-60. In addition, a number of EP antagonists have been described, including SC-19220 (EP1), ZM325802 (EP1), AH6809 (EP1 and EP2), and AH23848B (EP4). Sylvia V L et al. (2001) J Steroid Biochem Mol Biol 78:261-74; Santangelo S et al. (2000) J Trauma 48:826-30. AH6809 is 6-isopropoxy-9-oxaxanthene-2-carboxylic acid. AH23848B is [1 alpha(z), 2beta5alpha]-(+/−)-7-[5-[[(1,1′-biphenyl)-4-yl]methoxy]- 2-(4-morpholinyl)-3-oxo-cyclopentyl]-4-heptenoic acid.

[0073] An “immunostimulatory nucleic acid” as used herein is any nucleic acid containing an immunostimulatory motif or backbone that induces a Th1 immune response and/or suppresses a Th2 immune response. Immunostimulatory motifs include, but are not limited to, CpG motifs, poly-G motifs, and T-rich motifs. In one embodiment immunostimulatory motifs include CpG motifs and T-rich motifs. Immunostimulatory backbones include, but are not limited to, phosphate modified backbones, such as phosphorothioate backbones. Immunostimulatory nucleic acids have been described extensively in the prior art and a brief summary of these nucleic acids is presented below.

[0074] The terms “nucleic acid” and “oligonucleotide” are used interchangeably to mean multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). As used herein, the terms refer to oligoribonucleotides as well as oligodeoxyribonucleotides. The terms shall also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base-containing polymer. Nucleic acids include vectors, e.g., plasmids as well as oligonucleotides. Nucleic acid molecules may be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic (e.g., produced by oligonucleotide synthesis).

[0075] The terms nucleic acid and oligonucleotide also encompass nucleic acids or oligonucleotides with substitutions or modifications, such as in the bases and/or sugars. For example, they include nucleic acids having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3′ position and other than a phosphate group at the 5′ position. Thus modified nucleic acids may include a 2′-O-alkylated ribose group. In addition, modified nucleic acids may include sugars such as arabinose instead of ribose. Thus the nucleic acids may be heterogeneous in backbone composition thereby containing any possible combination of polymer units linked together such as peptide-nucleic acids (which have amino acid backbone with nucleic acid bases). In some embodiments, the nucleic acids are homogeneous in backbone composition. Nucleic acids also include substituted purines and pyrimidines such as C-5 propyne-modified bases. Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties. Other such modifications are well known to those of skill in the art.

[0076] Exemplary immunostimulatory nucleic acids as those described herein as well as various control nucleic acids include but are not limited to those presented in Table 1. 1