Diagnosis and treatment of inflammation and hyperactive immune conditions Number:7,067,254 from the United States Patent and Trademark Office (PTO) owispatent Ecto-NTPDase function on Langerhans cells is demonstrated to counteract the nucleotide inflammatory response caused by certain types of chemical irritants. The present invention takes advantage of this observation by, first, providing methods for screening of chemicals for irritant potential based on their ability to induce nucleotide release from keratinocytes. Second, methods are provided for the prevention and treatment of inflammation using NTPDase protein or gene therapy. And third, there also are provided methods for screening candidate compounds for NTPDase modulatory activity, thereby identifying possible pro- and anti-inflammatory agents. Additionally, the role of NTPDases and P2 receptors in hyperactive immune conditions such as autoimmune diseases and allergic reactions such as allergic contact dermatitis has been demonstrated. Therefore, the invention also provides methods for the prevention and treatment of hyperactive immune conditions by using NTPDase inhibitors and/or P2 receptor inhibitors. Further provided are methods for screening candidate compounds for modulatory activity of NTPDase-mediated immune conditions, thereby identifying other possible immunotherapeutic agents.<H inflammation, hyperactive, Diagnosis, and, t, 7067254.html, Patent, pto, 7067254

Title: Diagnosis and treatment of inflammation and hyperactive immune conditions

Abstract: Ecto-NTPDase function on Langerhans cells is demonstrated to counteract the nucleotide inflammatory response caused by certain types of chemical irritants. The present invention takes advantage of this observation by, first, providing methods for screening of chemicals for irritant potential based on their ability to induce nucleotide release from keratinocytes. Second, methods are provided for the prevention and treatment of inflammation using NTPDase protein or gene therapy. And third, there also are provided methods for screening candidate compounds for NTPDase modulatory activity, thereby identifying possible pro- and anti-inflammatory agents. Additionally, the role of NTPDases and P2 receptors in hyperactive immune conditions such as autoimmune diseases and allergic reactions such as allergic contact dermatitis has been demonstrated. Therefore, the invention also provides methods for the prevention and treatment of hyperactive immune conditions by using NTPDase inhibitors and/or P2 receptor inhibitors. Further provided are methods for screening candidate compounds for modulatory activity of NTPDase-mediated immune conditions, thereby identifying other possible immunotherapeutic agents.

Patent Number: 7,067,254 Issued on 06/27/2006 to Kumamoto, et al.

Inventors: Kumamoto; Tadashi (Mie, JP); Mizumoto; Norikatsu (Irving, TX); Takashima; Akira (Coppell, TX)
Assignee: Board of Regents, The University of Texas System (Austin, TX)

Appl. No.: 074220

Filed: February 12, 2002

Current U.S. Class: 435/6 ; 435/91.2; 536/23.5; 536/24.31; 536/24.33

Current International Class: C12Q 1/68 (20060101); C07H 21/02 (20060101); C07H 21/04 (20060101); C12P 19/34 (20060101)

Field of Search: 435/6,91.2

Foreign Patent Documents


WO 98/28437	Jul., 1998	WO

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Primary Examiner: Fredman; Jeffrey
Assistant Examiner: Sakelaris; Sally
Attorney, Agent or Firm: Fulbright & Jaworski

Government Interests

The government owns rights in the present invention pursuant to grant numbers R01 AR43777 from the National Institutes of Health.

Parent Case Text

The present application claims priority to co-pending U.S. Provisional Patent Applications Ser. No. 60/273,212 filed Mar. 1, 2001 and 60/334,618 filed Nov. 1, 2001. The entire text of the above-referenced disclosures are specifically incorporated by reference herein without disclaimer.

Claims

What is claimed is:

1. A method for predicting irritant potential of a candidate substance comprising: a) providing a mammalian keratinocyte cell that releases a ATP and/or ADP in response to an inflammatory agent; b) culturing said mammalian keratinocyte cell with a candidate substance; and c) determining ATP and/or ADP release from said mammalian keratinocyte cell, wherein an increase in ATP and/or ADP release from said mammalian keratinocyte cell, as compared to ATP and/or ADP release in the absence of said candidate substance, indicates that said candidate substance is an irritant.

2. The method of claim 1, wherein said cell is a human keratinocyte.

3. The method of claim 1, wherein said cell is a mouse keratinocyte.

4. The method of claim 3, wherein said cell is a PAM 212 cell.

5. The method of claim 1, wherein determining ATP and/or ADP release from said cell comprises measuring nucleotide concentration in the cell culture medium.

6. The method of claim 5, wherein measuring ATP and/or ADP concentration compnses an enzymatic assay.

7. The method of claim 6, wherein said enzymatic assay is a luciferin-luciferase assay.

8. The method of claim 1, wherein said candidate substance is a naturally-occurring compound.

9. The method of claim 1, wherein said candidate substance is a man-made compound.

10. The method of claim 1, further comprising measuring ATP and/or ADP release from said cell in the absence of said candidate substance.

11. The method of claim 1, further comprising a control comprising: a) contacting said cell with a known irritant; and b) measuring ATP and/or ADP release from said cell.

Description

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates to the fields of cell biology and biochemistry generally, and more specifically to the use nucleotide release as a measure of inflammatory action of a chemical. In addition, the present invention relates to the prevention and treatment of inflammation using nucleoside triphosphate diphosphohydrolase (NTPDase) activity, and includes protein-based and gene-based therapies. The invention also provides methods for screening candidate compounds for NTPDase modulatory activity. Furthermore, the invention provides methods for treating and/or preventing hyperactive immune disorders using NTPDase inhibitors or P2-receptor inhibitors. Methods for screening for modulators of NTPDase-mediated immune responses are also provided.

II. Brief Description of the Prior Art

Irritant contact dermatitis, the most frequent type of skin inflammation, results from contact with a substance that chemically damages the skin. As new chemicals are constantly being developed for use in many commercial products, including cosmetics, drugs, clothes, diapers, paints, soaps, shampoos, cleaning solutions, detergents, adhesives, food and food packaging, contraceptives, household cleaners, automobile interiors and parts, recycled paper, garden chemicals and other household products, cases of irritant contact dermatitis are on an exponential rise. Accidental, occupational or consumer-based exposure to such products can result in acute or chronic irritant contact dermatitis--the most common occupational health problem, and the most common skin disorder, in the United States.

Signs and symptoms of contact dermatitis can include skin inflammation characterized by redness, bumps, blisters, scaling, swelling, oozing, crusting, itching, and pain. These symptoms often occur at the site of contact, which is frequently the hands, arms, or legs, or face, but can occur elsewhere on the skin as well.

In spite of the rise in the number of cases of irritant contact dermatitis, efforts to develop assays which accurately predict the irritant potential of various chemicals have not been successful. Thus, there remains a need to develop accurate and sensitive assays for predicting irritant potential, as well as methods for the treatment thereof.

In addition to irritant contact dermatitis, diseases that are caused by hyperactivity of the immune system also are of concern. These diseases include a spectrum of allergic and autoimmune conditions that are associated with immunologically-mediated damage to the host tissue. Allergic contact dermatitis is another form of dermatitis which is one of the leading occupational hazards in the United States. Other allergic conditions include atopic dermatitis, hay fever, asthma and the like. Examples of autoimmune diseases include multiple sclerosis, rheumatoid arthritis, Crohn's disease, systemic lupus eryrthmatosus (SLE), etc. These diseases afflict many individuals and cause significant morbidity and mortality. Treatments for autoimmune diseases generally include immune suppression. Unfortunately, generalized immune suppression often results in increased incidence of infections and malignancies. Therefore, treatment of patients with an immunological disorder is at the cost of placing the patient at a risk of developing other, possibly life threatening, diseases.

What is required in the art is the identification of molecular targets of the immune system that play a role in hyperactive immune conditions and the development of screening methods to identify agents that can modulate these molecular targets. These agents can then be used therapeutically to treat/prevent immune hyperactivity.

SUMMARY OF THE INVENTION

The present invention overcomes these and other defects in the art and demonstrates that an ecto-NTPDase function on dendritic Langerhans cells is required to counteract nucleotide mediated inflammatory response caused by certain types of chemical irritants. The invention provides methods for screening of chemicals for irritant potential based on their ability to induce nucleotide release from cells. The invention also provides methods for the prevention and treatment of inflammation using NTPDase protein-based and/or gene-based therapies. In addition, the invention also provides methods for screening candidate compounds for NTPDase modulatory activity, thereby identifying pro- and anti-inflammatory agents.

Therefore, provided is a method for predicting the irritant potential of a candidate substance comprising: (a) providing a mammalian cell that releases a nucleotide in response to an inflammatory agent; (b) culturing the cell with a candidate substance; and (c) determining nucleotide release from the cell, wherein an increase in nucleotide release from the cell, as compared to nucleotide release in the absence of the candidate substance, indicates that the candidate substance is an irritant.

In one embodiment of this method, the cell is a fibroblast. In another embodiment, the cell is a keratinocyte. In some specific embodiments, the keratinocyte is a human keratinocyte. In other specific embodiments, the keratinocyte is a mouse keratinocyte. In yet another embodiment of the method, the cell is a PAM 212 cell. The skilled artisan will recognize that the cell can be any cell that is capable of releasing a nucleotide in response to an inflammatory agent or to a chemical irritant and that the practice of this invention is not limited to the examples described above.

The nucleotide can be a nucleotide triphosphate (NTP), a nucleotide diphosphate (NDP), or a nucleotide monophosphate (NMP). In one embodiment, the nucleotide is ATP and/or ADP and/or AMP. In another embodiment, the nucleotide is UTP and/or UDP and/or UMP. In yet another embodiment, the nucleotide is CTP and/or CDP and/or CMP. In still other embodiments, the nucleotide is GTP and/or GDP and/or GMP. In yet other embodiments, the nucleotide is TTP and/or TDP and/or TMP.

In one embodiment, determining nucleotide release from the cell comprises measuring nucleotide concentration in the cell culture medium. Thus, for example, the ATP and/or ADP and/or AMP release can be measured by measuring the ATP and/or ADP and/or AMP concentration in the cell medium; the UTP and/or UDP and/or UMP release can be measured by measuring the UTP and/or UDP and/or UMP concentration in the cell medium, and likewise for other nucleotides. In another embodiment, measuring nucleotide concentration comprises an enzymatic assay. The enzymatic assay can be a luciferin-luciferase assay and/or a pyruvate kinase assay. In an alternative embodiment, measuring nucleotide concentration comprises chromatographic measurements and includes any chromatographic procedure including, for example, thin layer chromatography and liquid chromatography.

In some aspects, the candidate substance with irritant potential is a naturally-occurring compound. In other aspects the candidate substance is a man-made compound.

In one embodiment the method further comprises measuring nucleotide release from the cell in the absence of the candidate substance as a control. The method described can also further comprise another control which comprises: (i) contacting the cell with a known irritant; and (ii) measuring nucleotide release from the cell.

The invention also provides a method for preventing an inflammatory response in a subject comprising administering to the subject a composition comprising a NTPDase. The NTPDase can be an ATPase and/or an ADPase; a UTPase and/or a UDPase; a CTPase and/or a CDPase; a GTPase and/or a GDPase; or a TTPase and/or a TDPase. In some embodiments of this method, the NTPDase is selected from the group consisting of CD39, CD39L1, CD39L2, CD39L3, CD39L4, Golgi-associated ecto-ATPase and ecto-uridine diphosphatase (UDPase), lysosomal ecto-apryase LALP70, hepatic canalicular ecto-apyrase, (.alpha.-sarcoglycan and potato apyrase.

In other embodiments of this method, the inflammatory response is chemical skin irritation, and the NTPDase is applied as a topical formulation. In yet other aspects, the NTPDase is applied as an oral, intranasal, intratracheal, intraesophageal, intrabronchial, intra-vaginal or rectal formulation.

The inflammatory response can be caused by a pro-inflammatory leukocyte or by a soluble pro-inflammatory factor. In one aspect of this embodiment, the soluble pro-inflammatory factor is a cytokine, a prostaglandin, or a histamine.

The invention also provides methods for treating an inflammatory response in a subject which comprise administering to the subject a composition comprising a NTPDase.

The invention also provides a method for preventing an inflammatory response in a subject comprising administering to the subject an expression construct comprising a DNA segment encoding a NTPDase under the control of a promoter active in cells of the subject. In one embodiment of the method, the expression construct is a viral expression construct. In a specific embodiment, the viral expression construct is selected from the group consisting of a retrovirus, an adenovirus, an adeno-associated virus, a herpesvirus, a polyoma virus, and a vaccinia virus.

In another embodiment, the expression construct is a non-viral expression construct. In one aspect, the non-viral expression construct can be administered as a naked DNA. In another aspect, the non-viral expression construct is administered in a liposomal formulation.

In one embodiment of this method, the NTPDase encoded by the expression construct is selected from the group consisting of CD39, CD39L1, CD39L2, CD39L3, CD39L4, Golgi-associated ecto-ATPase and ecto-uridine diphosphatase (UDPase), lysosomal ecto-apryase LALP70, hepatic canalicular ecto-apyrase, .alpha.-sarcoglycan and potato apyrase.

In another embodiment of this method, the inflammatory response is chemical skin irritation, and the NTPDase is applied as a topical formulation. In specific aspects the inflammatory response is mucosal irritation, and the NTPDase is applied as an oral, intranasal, intratracheal, intraesophageal, intrabronchial, intra-vaginal or rectal formulation. In other specific aspects, the inflammatory response is caused by a pro-inflammatory leukocyte. In yet other aspects, the inflammatory response is caused by a soluble pro-inflammatory factor, such as a cytokine, a prostaglandin, or a histamine.

The invention also provides a method for treating an inflammatory response in a subject comprising administering to the subject an expression construct comprising a DNA segment encoding a NTPDase under the control of a promoter active in cells of the subject.

The invention further provides methods of screening for modulators of inflammation comprising: (a) providing a cell that expresses a NTPDase; (b) contacting the cell with a candidate substance; and (c) determining the effect of the candidate substance on the NTPDase expression in the cell, wherein a change in the expression of a NTPDase in the cell, as compared to NTPDase expression in the absence of the candidate substance, indicates that the candidate substance is a modulator of a NTPDase expression, and therefore a modulator of inflammation. In one embodiment, the modulator is an inhibitor of inflammation. In another embodiment, the modulator is a promoter of inflammation.

In one aspect of this method the cell is a dendritic cell. In specific aspects, the dendritic cell is a Langerhans cell or cell line and is exemplified by the XS52 or the XS106 cells.

In one embodiment, the cell comprises an expression construct comprising a DNA segment encoding the NTPDase under the control of a promoter active in the cell. In one aspect, the determining comprises measuring NTPDase levels in the cell. In another aspect, the measuring comprises measuring a NTPDase on the surface of the cell. In yet another aspect, the determining comprises measuring the NTPDase activity of the cell.

The NTPDase activity can be measured by methods that measure the surface enzyme activity. Concentrations of nucleotides, released as a result of the surface enzyme activity, can be measured to measure the NTPDase activity. The NTPDase activity can also be measured by methods comprising using an enzyme linked immunoassay using an enzyme-labeled anti-NTPDase antibody. In another aspect of the method, fluorescent activated cell sorting of cells using a fluorescent-labeled anti-NTPDase antibody is used. In yet another aspect, the determining comprises measuring NTPDase mRNA levels in the cell. In such embodiments, the method can comprise Northern blotting and/or quantitative RT-PCR.

In specific examples of this embodiment, the NTPDase can be a ATPase and/or ADPase; an UTPase and/or an UDPase; etc.

The invention additionally provides a method of screening for modulators of inflammation comprising: (a) providing a cell that comprises an expression construct comprising a DNA segment encoding a screenable marker under the control of a promoter for a NTPDase; (b) contacting the cell with a candidate substance; and (c) determining the effect of the candidate substance on expression of the selectable marker, wherein a change in the expression of the selectable marker, as compared to selectable marker expression in the absence of the candidate substance, indicates that the candidate substance is a modulator of NTPDase promoter expression, and therefore a modulator of inflammation. The modulator can be either an inhibitor of inflammation or a promoter of inflammation.

In specific aspects of this method, the NTPDase can be selected from the group consisting of CD39, CD39L1, CD39L2, CD39L3, CD39L4, Golgi-associated ecto-ATPase and ecto-uridine diphosphatase (UDPase), lysosomal ecto-apryase LALP70, hepatic canalicular ecto-apyrase, .alpha.-sarcoglycan and potato apyrase.

In one embodiment, the screenable marker is an enzyme and determining comprises measuring enzyme activity. Examples of such enzymes include urease, alkaline phosphatase or peroxidase for which colorimetric indicator substrates can be employed to provide a detection means visible to the human eye or by spectroscopy.

In one embodiment, the invention also provides methods of screening for a modulator of inflammation comprising: (a) providing a cell that expresses a membrane bound NTPDase, or ecto-NTPDase in a culture solution comprising a nucleotide; (b) contacting the cell with a candidate substance; and (c) determining the effect of the candidate substance on NTP, NDP and/or NMP levels in the culture solution, wherein a change in the NTP, NDP and/or NMP levels in the culture solution, as compared to NTP, NDP and/or NMP levels in the absence of the candidate substance, indicates that the candidate substance is a modulator of NTPDase activity, and therefore a modulator of inflammation.

In one aspect of this method, the cell expresses an NTPDase. In another aspect the cell expresses an NTPDase. The modulator can be either an inhibitor of inflammation or a promoter of inflammation.

In some aspects, the NMP levels, such as AMP, UMP, GMP, CMP and/or TMP, are detected by chromatographic methods, including thin layer chromatography, liquid chromatography, high-performance liquid chromatography and cation-exchange chromatography.

The present inventors have also identified that NTPDases are involved in certain hyperactive immune responses such as autoimmune conditions and allergic reactions. Therefore, the present invention also provides methods for treating a hyperactive immune response in a subject comprising administering to the subject a composition comprising a NTPDase inhibitor.

The NTPDase inhibitor may be an ATPase inhibitor and/or ADPase inhibitor, an UTPase inhibitor and/or UDPase inhibitor, a CTPase inhibitor and/or CDPase inhibitor, a TTPase inhibitor and/or TDPase inhibitor, or a GTPase inhibitor and/or GDPase inhibitor.

In one embodiment of the invention, the NTPDase inhibitor is an NTPDase antagonist, an anti-NTPDase antibody, an antisense oligonucleotide, or a chemical substance.

In one specific embodiment, the NTPDase antagonist is Azide, Evans Blue, Suramin, PPADS, DEPC, P-CMPS, P-HMB, NP-40, FSBA. Concentrations of the NTPDase antagonists are approximately in the range of 10 20 .mu.M for Azide, 100 .mu.M of Evans Blue, 100 .mu.M of Suramin, 100 .mu.M of PPADS, 1 mM of DEPC, 40 .mu.M of P-CMPS, 40 .mu.M of P-HMB, 10:1, w/w of NP-40, and 2 mM of FSBA.

In another specific embodiment of the invention, the anti-NTPDase antibody is an anti-ATPase and/or anti-ADPase antibody, an anti-UTPase and/or anti-UDPase antibody, a anti-CTPase and/or anti-CDPase antibody, a anti-TTPase and/or anti-TDPase antibody, or a anti-GTPase and/or anti-GDPase antibody.

In yet another specific embodiment of the invention, the antisense oligonucleotide comprises a nucleic acid that is complementary to a nucleic acid sequence encoding an ATPase and/or ADPase, an UTPase and/or UDPase, a CTPase and/or CDPase, a TTPase and/or TDPase, or a GTPase and/or GDPase, or a fragment thereof.

In one embodiment of the method, the NTPDase inhibitor is an inhibitor of CD39, CD39L1, CD39L2, CD39L3, CD39L4, Golgi-associated ecto-ATPase and ecto-uridine diphosphatase (UDPase), lysosomal ecto-apryase LALP70, hepatic canalicular ecto-apyrase, .alpha.-sarcoglycan or potato apyrase.

In one embodiment, the hyperactive immune response can be an allergic reaction and includes, but is not limited to conditions such as allergic contact dermatitis, atopic dermatitis, allergic rhinitis (hay fever), bronchial asthma, and the like.

In another embodiment, the hyperactive immune response is an autoimmune disease and is exemplified in non-limiting examples by Addison's disease, alopecia, ankylosing spondylitis, antiphospholipid syndrome, Behcet's disease, chronic fatigue syndrome, Crohn's disease, ulcerative colitis, diabetes, fibromyalgia, Goodpasture syndrome, Graves' disease, idiopathic thrombocytopenic purpura, lupus, Meniere's multiple sclerosis, myasthenia gravis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, vitiligo, or Wegener's granulomatosis.

The NTPDase inhibitor may be administered by topical, oral, intranasal, intratracheal, intraesophageal, intrabronchial, intravenous, intraarterial, intramuscular, subcutaneous, intra-vaginal or rectal routes. The route of choice for administration will be decided on the nature and location of the hyperactive immune being treated.

The invention also provides methods for preventing a hyperactive immune response in a subject comprising administering to the subject a composition comprising a NTPDase inhibitor.

Additionally, methods for treating or preventing a hyperactive immune response in a subject comprising administering to the subject an expression construct comprising a DNA segment encoding a NTPDase inhibitor under the control of a promoter active in cells of the subject are provided.

In one embodiment of this method, the NTPDase inhibitor is an ATPase inhibitor and/or ADPase inhibitor, an UTPase inhibitor and/or UDPase inhibitor, a CTPase inhibitor and/or CDPase inhibitor, a TTPase inhibitor and/or TDPase inhibitor, or a GTPase inhibitor and/or GDPase inhibitor.

In another aspect of the method, the NTPDase inhibitor is an inhibitor of CD39, CD39L1, CD39L2, CD39L3, CD39L4, Golgi-associated ecto-ATPase and ecto-uridine diphosphatase (UDPase), lysosomal ecto-apryase LALP70, hepatic canalicular ecto-apyrase, .alpha.-sarcoglycan or potato apyrase.

In yet another aspect of the method, the NTPDase inhibitor is an antisense oligonucleotide. The antisense oligonucleotide comprises a nucleic acid that is complementary to the nucleic acid sequence encoding an ATPase and/or ADPase, an UTPase and/or UDPase, a CTPase and/or CDPase, a TTPase and/or TDPase, or a GTPase and/or GDPase or a fragment thereof.

In some embodiments of this method, the expression construct is a viral expression construct and may be a retroviral construct, an adenoviral construct, an adeno-associated viral construct, a herpesviral construct, a polyoma viral construct, and a vaccinia viral construct.

In other embodiments of the method, the expression construct is a non-viral expression construct. The non-viral expression construct may be administered either as a naked DNA construct or in a liposomal formulation.

The invention also provides methods of screening for modulators of NTPDase-mediated immune responses comprising: a) providing a cell that expresses a membrane bound NTPDase or an ecto-NTPDase; b) contacting the cell with a candidate substance; and c) determining the effect of the candidate substance on the NTPDase level in the cell, wherein a change in the level of a NTPDase in the cell, as compared to NTPDase level in the absence of the candidate substance, indicates that the candidate substance is a modulator of NTPDase level, and therefore a modulator of NTPDase-mediated immune responses.

In one embodiment of the invention, the cell is a dendritic cell. The dendritic cell can be a Langerhans cell or cell line. Examples of dendritic cell lines include the XS52 or the XS106 cell lines.

In another embodiment, the NTPDase is an ATPase and/or ADPase, an UTPase and/or UDPase, a CTPase and/or CDPase, a TTPase and/or TDPase, or a GTPase and/or GDPase.

In yet another embodiment of the method, the cell expresses an ATPase, an ADPase, an UTPase or an UDPase.

In another aspect of the invention, the cell comprises an expression construct comprising a DNA segment encoding the NTPDase under the control of a promoter active in the cell.

In one embodiment of the method, the determining comprises measuring NTPDase activity of the cell. The NTPDase activity can be measured by: a) culturing the cell in a culture solution comprising a nucleotide; b) determining the effect of the candidate substance on nucleotide levels in the culture solution; and c) measuring a change in the nucleotide levels in the culture solution as compared to nucleotide levels in the absence of the candidate substance.

The nucleotide is ATP and/or ADP and/or AMP, UTP and/or UDP and/or UMP, CTP and/or CDP and/or CMP, TTP and/or TDP and/or TMP, or GTP and/or GDP and/or GMP.

In some aspects, the AMP levels may be detected by chromatographic methods.

In other embodiments of the invention, the measuring comprises measuring the NTPDase levels on the surface of the cell. Such methods may comprise an enzyme linked immunoassay using an enzyme-labeled anti-NTPDase antibody. Some examples of anti-NTPDase antibodies that may be used include an anti-ATPase and/or anti-ADPase antibody, an anti-UTPase and/or anti-UDPase antibody, a anti-CTPase and/or anti-CDPase antibody, a anti-TTPase and/or anti-TDPase antibody, or a anti-GTPase and/or anti-GDPase antibody.

In another embodiment of the method, the measuring comprises fluorescent activated cell sorting of cells using a fluorescent-labeled anti-NTPDase antibody.

In one embodiment of the method, the determining comprises measuring the NTPDase mRNA level in the cell. In another embodiment of the method, the measuring comprises Northern blotting. In yet another embodiment of the method, the measuring comprises quantitative RT-PCR.

The invention also provides methods of screening for modulators of NTPDase-mediated immune responses comprising: a) providing a cell that comprises an expression construct comprising a DNA segment encoding a screenable marker under the control of a promoter for a NTPDase; b) contacting the cell with a candidate substance; and c) determining the effect of the candidate substance on expression of the selectable marker, wherein a change in the expression of the selectable marker, as compared to selectable marker expression in the absence of the candidate substance, indicates that the candidate substance is a modulator of NTPDase promoter expression, and therefore a modulator of NTPDase-mediated immune responses.

In one embodiment of this method, the NTPDase-mediated immune responses is an immune response mediated by an NTPDase such as CD39, CD39L1, CD39L2, CD39L3, CD39L4, Golgi-associated ecto-ATPase and ecto-uridine diphosphatase (UDPase), lysosomal ecto-apryase LALP70, hepatic canalicular ecto-apyrase, .alpha.-sarcoglycan or potato apyrase.

In another embodiment of this method, the screenable marker is an enzyme and the determining comprises measuring enzyme activity.

The invention also provides a method for treating a hyperactive immune response in a subject comprising administering to the subject a composition comprising a P2-receptor inhibitor.

In one embodiment of the invention, the P2-receptor inhibitor is an P2-receptor antagonist, an anti-P2-receptor antibody, an antisense P2-receptor oligonucleotide, or a chemical substance that inhibits a P2 receptor.

The P2-receptor inhibitor may be an inhibitor of any P2 receptor such as, but not limited to, P2X.sub.1, P2X.sub.4, P2X.sub.5, P2X.sub.7, P2X.sub.1, P2Y.sub.1, P2Y.sub.1, P2Y.sub.2, P2Y.sub.4, P2Y.sub.5, P2Y.sub.6, P2Y.sub.10, or P2Y.sub.11.

In one embodiment of the method, the P2-receptor inhibitor is an P2-receptor antagonist and is exemplified in non-limiting examples by suramin, KN-62, MRS2179, TNP-ATP, TNP-GTP, oxidized ATP, PPADS, Reactive Blue2.

In another embodiment, the P2-receptor inhibitor is an antisense oligonucleotide composition and comprises a nucleic acid that is complementary to a nucleic acid sequence encoding a P2X.sub.1, P2X.sub.4, P2X.sub.5, P2X.sub.7, P2X.sub.1, P2Y.sub.1, P2Y.sub.1, P2Y.sub.2, P2Y.sub.4, P2Y.sub.5, P2Y.sub.6, P2Y.sub.10, or P2Y.sub.11 receptor, or to a fragment thereof.

In one embodiment of the method, the hyperactive immune response is an allergic reaction and is exemplified by allergic contact dermatitis, atopic dermatitis, allergic rhinitis (hay fever), bronchial asthma and the like.

In another embodiment of the method, the hyperactive immune response is an autoimmune disease and is exemplified by Addison's disease, alopecia, ankylosing spondylitis, antiphospholipid syndrome, Behcet's disease, chronic fatigue syndrome, Crohn's disease, ulcerative colitis, diabetes, fibromyalgia, Goodpasture syndrome, Graves' disease, idiopathic thrombocytopenic purpura, lupus, Meniere's multiple sclerosis, myasthenia gravis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, vitiligo, or Wegener's granulomatosis, etc.

It is contemplated that the P2-receptor inhibitor can be administered by topical, oral, intranasal, intratracheal, intraesophageal, intrabronchial, intra-vaginal, rectal intravenous, intraarterial, subcutaneous, or intramuscular routes.

The invention also provides a method for preventing a hyperactive immune response in a subject comprising administering to the subject a composition comprising a P2-receptor inhibitor.

Also provided is a method for treating or preventing a hyperactive immune response in a subject comprising administering to the subject an expression construct comprising a DNA segment encoding a P2-receptor inhibitor under the control of a promoter active in cells of the subject.

In one such embodiment, the P2-receptor inhibitor is an inhibitor of the P2X.sub.1, P2X.sub.4, P2X.sub.5, P2X.sub.7, P2X.sub.1, P2Y.sub.1, P2Y.sub.1, P2Y.sub.2, P2Y.sub.4, P2Y.sub.5, P2Y.sub.6, P2Y.sub.10, or P2Y.sub.11 receptor. In another such embodiment, the P2-receptor inhibitor is an antisense oligonucleotide. The antisense oligonucleotide comprises a nucleic acid that is complementary to a nucleic acid sequence encoding a P2X.sub.1, P2X.sub.4, P2X.sub.5, P2X.sub.7, P2X.sub.1, P2Y.sub.1, P2Y.sub.1, P2Y.sub.2, P2Y.sub.4, P2Y.sub.5, P2Y.sub.6, P2Y.sub.10, or P2Y.sub.11 receptor, or a fragment thereof.

In some aspects, the expression construct is a viral expression construct and may be selected from the group consisting of a retrovirus, an adenovirus, an adeno-associated virus, a herpesvirus, a polyoma virus, and a vaccinia virus.

In other aspects, the expression construct is a non-viral expression construct. The non-viral expression construct may be administered as a naked DNA or in a liposomal formulation.

The exact doses and routes of administration will be decided at the time of therapy by a trained physician depending on factors such as site of lesion or disease, health of the patient and other related factors.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A, 1B, & 1C. Lack of LC-associated ecto-NTPDase activities in CD39-deficient mice. (FIG. 1A), Epidermal sheets prepared from the ear samples of CD39.sup.+/+, CD39.sup.+/-, or CD39.sup.-/- mice were stained for the indicated markers. The data shown are representative fields from three independent experiments (original magnification: .times.200). (FIG. 1B), Surface densities of IA-, DEC205-, or V.gamma.3-positive epidermal cells were counted under a fluorescence microscope using an image analysis program. Data shown are the mean.+-.SD (n=3) in a representative experiment. (FIG. 1C), Epidermal sheets prepared from the ear samples of CD39.sup.+/+, CD39.sup.+/-, or CD39.sup.-/- mice that were subjected to histo-enzymatic staining for ecto-NTPDase activities with the ATP or ADP substrate. The data shown are representative fields from three independent experiments after "overexposure" to detect any residual enzymatic activities.

FIGS. 2A, 2B, 2C, & 2D. CD39 mRNA and protein expression by LC. (FIG. 2A), The indicated cell lines were subjected to histo-enzymatic staining for ecto-NTPDase activities with the ATP or ADP substrate. (FIG. 2B), The indicated cell lines were examined for CD39 mRNA expression by RT-PCR (30 cycles) (Enjyoji et al., 1999). (FIG. 2C), Membrane fractions isolated from the indicated cell lines were examined for CD39 protein expression by Western blot using polyclonal anti-CD39 antibodies (Enjyoji et al., 1999). (FIG. 2D), RNA isolated from the whole skin of CD39.sup.+/+ mice (lane 1) or CD39.sup.-/- mice (lane 2) were examined for CD39 mRNA expression by RT-PCR. In parallel, epidermal cells isolated from CD39.sup.+/+ mice were examined for CD39 mRNA expression before (lane 3) or after LC depletion with magnet beads coated with anti-IA antibody (lane 4). All data shown in this figure are representative of at least three independent experiments.

FIGS. 3A, 3B, & 3C. Release of ATP and ADP from Pam 212 keratinocytes following in vitro treatment with skin irritant chemicals. (FIG. 3A), Pam 212 keratinocytes were treated with croton oil (CO), benzalkonium chloride (BAC), or ethyl phenylpropiolate (EPP) at the indicated concentrations or exposed to UVB irradiation at the indicated doses. Culture supernatants were examined for ATP concentrations by the luciferin-luciferase assay and for LDH using the CytoTox 96 assay kit (Promega). (FIG. 3B), Pam 212 keratinocytes were incubated for 10 min with the indicated chemicals and examined for release of ATP and ADP into culture media. ADP concentrations were measured after pyruvate kinase-mediated conversion to ATP. (FIG. 3C), Pam 212 keratinocytes and XS52 LC were co-cultured at the indicated cell densities and treated for 10 min with PBS alone or with 0.0015% BAC. All data shown in this figure are representative sets of results (mean.+-.SD, n=3) from three independent experiments. * p<0.05; ** p<0.01 (two-tailed Student's t-test).

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, & 4H. Irritant contact hypersensitivity responses in CD39-deficient mice. CD39.sup.-/- mice (circles), CD39.sup.+/- mice (triangles) or CD39.sup.+/+ mice (squares) received topical application of 1% croton oil (FIG. 4A), 10% BAC (FIG. 4B), or 30% EPP (FIG. 4C) on the right ear and vehicle alone on the left ear. The data shown are the mean.+-.SEM (n=10) of the swelling responses (the right ear thickness minus the left ear thickness) at the indicated time points, being displayed in two different time scales. Statistical significant differences compared to the CD39.sup.+/+ mice or to the CD39.sup.+/- mice are indicated by asterisks or sharps, respectively (*.sup./# p<0.05 and **.sup./## p<0.01; two-tailed Student's t test). FIG. 4D, Ear skin samples were harvested from CD39.sup.+/+ or CD39.sup.-/- mice 5 days after topical application of vehicle alone or 1% CO and processed to histological analyses after H&E staining (original magnification: .times.250). FIG. 4E, CD39.sup.-/- mice (n=6) or C57BL/6 wild-type mice (n=10) (FIG. 4F) received local injections of apyrase on the right ear (closed circles) or PBS alone on the left ear (open circles) before and after topical application of 1% CO on both ears. The data shown are the mean.+-.SEM of the swelling responses (the ear thickness after CO application minus the baseline thickness before application) at the indicated time points. Statistical significant differences (one-tailed Student's t-test) are indicated by asterisks (*p<0.05 and **p<0.01). FIG. 4G, CD39.sup.-/- mice (circles), CD39.sup.+/- mice (triangles) or CD39.sup.+/+ mice (squares) received a single dorsal irradiation of 1,500 J/m.sup.2 of UVB radiation and examined for inflammatory responses. The data shown are the mean.+-.SEM (n=10) of the swelling responses (the thickness after irradiation minus the baseline thickness before irradiation). FIG. 4H, CD39.sup.-/- mice (circles), CD39.sup.+/- mice (triangles) or CD39.sup.+/+ mice (squares) were examined for allergic contact hypersensitivity responses to oxazolone (OX). The data shown are the mean.+-.SEM (n=10) of the swelling responses (the thickness of OX-challenged right ear minus the thickness of vehicle-challenged left ear). All data shown in this figure are representative of at least two independent experiments.

FIGS. 5A, 5B, 5C, 5D, 5E, 5F, 5G, & 5H. Attenuated immune responses in CD39-deficient mice. CD39.sup.-/- mice (circles), CD39.sup.+/- mice (triangles), or CD39.sup.+/+ mice (squares) were examined for acute "sunburn" reactions (FIG. 5A) and for allergic contact hypersensitivity responses to OX (FIG. 5B). The data shown are the means.+-.s.e.m. (n=10) of the swelling responses. Statistically significant differences compared to the CD39.sup.+/+ panel or to the CD39.sup.+/- panel are indicated by asterisks or sharps, respectively (*.sup./# P<0.05 and **.sup./## P<0.01). (FIG. 5C) Mice received local injection of TNF.alpha. and examined for surface densities of IA.sup.+ epidermal cells (means.+-.s.d., n=3). At 24 hr after topical application of FITC, the DLN were examined for the numbers of migratory LC (means.+-.s.d., n=3) (FIG. 5D) localization of FITC.sup.+ migratory LC after counter-staining of the B cell areas with PE-conjugated B220 mAb (FIG. 5E), and for the surface phenotype of FITC.sup.+ migratory LC (FIG. 5F). Closed and open histograms represent the staining profiles with the indicated mAb and control IgG, respectively. Numbers in the parentheses indicate the mean fluorescence intensities (MFI). (FIG. 5G), Migratory LC isolated from OX-painted CD39.sup.-/- mice (circles) or CD39.sup.+/+ mice (squares) were co-cultured with OX-reactive, wild-type T-cells. The data shown are the means.+-.s.d. (n=3) of interferon-.gamma. (IFN-.gamma.) concentrations in the culture supernatants (* P<0.05). (FIG. 5H), Bone marrow-derived DC generated from CD39.sup.-/- mice (circles) or CD39.sup.+/+ mice (squares) were pulsed with TNBS and injected into CD39.sup.+/+ mice. The recipient animals were challenged with TNCB and examined for swelling responses (means.+-.s.e.m., n=10) (* P<0.05 and ** P<0.01 between CD39.sup.-/- DC and CD39.sup.+/+ DC).

FIGS. 6A, 6B, & 6C. P2Y receptor desensitization in CD39.sup.-/- DC. (FIG. 6A), T-cells isolated from CD39.sup.+/+ mice were stimulated with the indicated mAb in immobilized forms and examined for pericellular ATP concentrations (means.+-.s.d., n=5) (* P<0.05 and ** P<0.01). (FIG. 6B), Bone marrow-derived DC generated from CD39.sup.-/- mice (circles) or CD39.sup.+/+ mice (squares) were incubated for 60 min in the presence of the indicated concentrations of ATP and then examined for cell viability (means.+-.s.d., n=3) (** P<0.01 compared to the baseline cell viability). (FIG. 6C), CD39.sup.-/- or CD39.sup.+/+ DC were pre-treated with apyrase or buffer alone and then examined for ATP-mediated activation responses. Data shown are the means.+-.s.d. (n=3) of cell viability (left), IL-6 production (middle), and LY uptake (right). Statistically, significant differences are indicated by asterisks (** P<0.01). All data shown in this figure are representative of at least two independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

Langerhans cells (LC) are skin-specific members of the dendritic cell family of antigen presenting cells. Wolff & Winkelmann discovered, over thirty years ago, that LC defined under electron microscopy by the inclusion of Birbeck granules could be identified at the light microscopy level by ATPase staining (Wolff & Winkelmann, 1967). In this histo-enzymatic method, the ATP substrate is hydrolyzed in the presence of lead to form lead phosphate, and then converted to lead sulfide with ammonium sulfide. Deposition of lead sulfide granules observed along the plasma membrane at the exterior surface, indicates that LC express ecto-ATPase activity. Subsequently, LC were found to hydrolyze not only ATP, but also the ADP substrate in a divalent cation-dependent manner (Chaker et al., 1984).

Extracellularly released nucleotides, for example, ATP and ADP, are known to regulate many different forms of intercellular communication via binding to the ligand gated ion channel P2X receptors and G-protein coupled P2Y receptors (Williams and Jarvis, 2000). For example, ATP acts as a fast excitatory neurotransmitter in nervous tissue, with P2X and P2Y receptors being widely distributed on neurons, astroglia, microglia, and oligodendroglia. ATP and ADP are released from endothelial cells by mechanical shear forces, stretch, changes in osmolarity, oxidative stress, and microbial products (e.g., LPS), thereby exerting apoptotic, inflammatory, and thrombotic effects in the vascular system.

With respect to purinergic signaling in skin, in vitro treatment of keratinocytes with ATP has been reported to mobilize intracellular Ca.sup.2+, inhibit terminal differentiation, affect proliferation, and induce apoptosis (Pillai and Bikle, 1992; Pillai, 1995; Sutter et al., 1991; Girolomoni, 1993). In 1999, five groups reported independently that dendritic cells (DC) express mRNA for a variety of P2 receptors (P2X.sub.1, P2X.sub.4, P2X.sub.5, P2X.sub.7, P2X.sub.1, P2Y.sub.1, P2Y.sub.1, P2Y.sub.2, P2Y.sub.4, P2Y.sub.5, P2Y.sub.6,P2Y.sub.10, and P2Y.sub.11) and that ATP triggers Ca.sup.2+ influx, phenotypic maturation, chemotactic migration, and apoptosis of DC, whereas ADP (as well as UTP and UDP) induces production of IL-1.beta., IL-6, IL-10, and IL-12 (p40) by DC (Mutini et al., 1999; Liu et al., 1999; Coutinho-Silva et al., 1999; Berchtold et al., 1999; Marriott et al., 1999).

The present inventors have determined that CD39, a vascular ecto-NTPDase, is responsible for the ADPase and ATPase functions of LC. This was accomplished by creating CD39 knockout mice that lacked one or both functional alleles for CD39. A comparison of the relative inflammatory responses of these animals to croton oil demonstrated that the double-knock out animals (i.e., the CD39.sup.-/- mice), had much stronger inflammatory responses than did normal animals, with the heterozygote (i.e., the CD39.sup.+/- mice), showing intermediate inflammation. Interestingly, all the animals were comparable in their response to UVB radiation-induced inflammation, which involves a non-ADP/ATP inflammatory pathway. Together, these data not only identify CD39 as responsible for the ADPase/ATPase function of LC, but indicate that nucleotides such as ADP/ATP (or UTP and UDP) act as specific mediators of chemically induced inflammation, and that CD39 is responsible for protection of animals from this response. Moreover, hydrolysis of extracellular ADP and ATP by LC-associated CD39 represents an important regulatory mechanism controlling the magnitude of inflammation in the skin.

The present inventors also examined allergic contact hypersensitivity responses, wherein hapten-pulsed LC play a pathogenic role by activating hapten-reactive T-cells. CD39.sup.-/-, CD39.sup.+/-, and CD39.sup.+/+ mice were sensitized by topical application of oxazolone (OX), challenged 5 days later with the same hapten on ear skin, and ear swelling responses were measured. In marked contrast to the heightened inflammatory responses to irritant chemicals, immune responses to OX were severely attenuated in CD39.sup.-/- mice and the CD39.sup.+/- mice showed intermediate responses. These results demonstrate that LC-associated CD39 plays a unique role in cellular immune responses and reduces hyperactive immune responses.

Thus, the present inventors have also demonstrated that immune responses to reactive haptens are severely attenuated in CD39.sup.-/- mice and that T-cells increase pericellular ATP concentrations in response to CD3/TCR-mediated co-stimulatory signals. Furthermore, CD39.sup.-/- DC were found to be functionally impaired in their antigen presenting capacity. Based on these findings, the inventors contemplate that T-cell-derived ATP acts as a signaling molecule during antigen presentation and that this signaling pathway is regulated by CD39-mediated hydrolysis of excess amounts of ATP. This is further supported by the recent reports documenting that DC express mRNA for a variety of P2 receptors (e.g., P2X.sub.1, P2X.sub.4, P2X.sub.5, P2X.sub.7, P2Y.sub.1, P2Y.sub.2, P2Y.sub.4, P2Y.sub.5, P2Y.sub.6, P2Y.sub.10, and P2Y.sub.11), and that DC respond to nucleotide stimulation by pore formation, Ca.sup.2+ influx, phenotypic maturation, chemotactic migration, apoptosis, and cytokine production (Mutini et al., 1999; Liu et al., 1999; Coutinho-Silva et al., 1999; Berchtold et al., 1999; Marriott et al., 1999; Nihei et al., 2000; Ferrari et al., 2000).

Although, the mechanisms underlying the impaired antigen presenting capacity of CD39.sup.-/- DC remain to be fully elucidated, the inventors determined that CD39.sup.-/- DC are unresponsive to ATP-induced death and IL-6 production, while exhibiting intact pore formation after ATP stimulation. This indicates that one or more P2Y receptors, but not pore forming P2X receptors, are desensitized in CD39.sup.-/- DC. This is supported by reports that have documented a unique, P2Y.sub.1 receptor-mediated apoptosis pathway and the involvement of a P2Y.sub.6 receptor in nucleotide-induced IL-8 production (Sellers et al., 2001; Warny et al., 2001). The inventors are currently investigating the identity of the P2 receptor(s) that are desensitized in CD39.sup.-/- DC and the identity of the P2 receptors that are functionally involved in extracellular nucleotide-mediated DC:T-cell communication.

Thus, CD39 plays important roles in cellular immunity responses of DC and is required for proper antigen presentation and primary stimulation of hapten-reactive T-cells. Thus, the inventors contemplate that CD39 and other NTPDase inhibitors will be useful in preventing and/or treating hyperactive immune conditions such as allergic reactions, autoimmune conditions.

As P2-receptors are involved in nucleotide mediated signaling in dendritic cells, another embodiment of this invention provides P2-receptor inhibitors as therapeutics for NTPDase-mediated hyperactive immune responses.

Thus, the present invention describes the diverse roles of CD39 in regulating extracellular nucleotide-mediated signaling in inflammatory responses to environmental insults and in intercellular communication between DC and T-cells in the immune system.

I. NTPDases

NTPDases are a group of extracellular nucleotidase enzymes that are often membrane-bound. These enzymes have their active nucleotide hydrolysis sites facing the exterior of the cell. The NTPDases possess other unique characteristics besides an extracellular active site. They have a broad nucleotide substrate range (e.g. ATP and ADP, or UTP and UDP, etc. depending on the specific types of nucleotides hydrolyzed and therefore such enzymes are also called ATPase and/or ADPases; or UTPase and/or UDPase; GTPase and/or GDPases; CTPase and/or CDPases; TTPase and/or TDPases), a requirement for calcium or magnesium for activity, and insensitivity to many of the classic inhibitors effective against the mitochondrial, lysosomal, and plasma membrane ATPases. They are also present in cells in extremely low amounts, making the isolation and definitive identification of them very difficult until recently.

Some non-limiting examples of the NTPDases include CD39, CD39L1, CD39L2, CD39L3, CD39L4, Golgi-associated ecto-ATPase and ecto-uridine diphosphatase (UDPase), lysosomal ecto-apryase LALP70, hepatic canalicular ecto-apyrase, .alpha.-sarcoglycan and potato apyrase.

The NTPDases are involved in a large number of physiological functions. Several NTPDases are found in vascular endothelial cells, smooth muscle cells, and in the heart (Yeung et al., 2000). It is believed that these enzymes inactivate extracellular nucleotide signals in both, the cardiovascular system and associated smooth muscle. Extracellular nucleotides like ATP are effective regulators of blood vessel vascular tone by their ability to bind to specific purine receptors, causing blood vessels to dilate in some instances and to constrict in other cases. This directly modulates blood pressure. The NTPDase ectonucleotidases are involved in the control of vascular tone by regulating the level of circulating ATP.

NTPDase 1, also called, CD39, has been shown to play an important role in maintaining blood hemostasis and preventing thrombosis. NTPDase activity hydrolyzes ADP, which is an important coagulant of blood platelets, thereby modulating blood clotting. Soluble forms of NTPDases have been found in the salivary glands of blood-feeding insects such as mosquitoes, ticks, bedbugs etc.

NTPDases are also found in the brain. Extracellular nucleotides interact with and activate nucleotide type 2 (P2) receptors in the brain that initiate a wide variety of signaling pathways, important for functional associations between neurons and glial cells and for the regulation of blood flow, haemostatic and inflammatory reactions in the brain.

Ecto-NTPDase activities have also been widely used as markers of epidermal Langerhans cells, which are skin-specific members of the dendritic cell family of antigen presenting cells.

Some known NTPDase antagonists include Azide, Evans Blue, Suramin, PPADS, DEPC, P-CMPS, P-HMB, NP-40, FSBA (Knowles et al., 1999; Heine et al., 1999; Zinchuk et al., 1999; Dumbrowski et al., 1998).

(i) ADPases and ATPase

ADPases and ATPases are NTPDase enzymes that convert the substrates adenosine di-phosphate (ADP) or adenosine tri-phosphate (ATP) to adenosine mono-phosphate (AMP) by a hydrolysis reaction. Of particular interest in the present invention is the ecto-ATPase CD39. CD39 also has UTPase and/or UDPase activity and the present inventors envision the use of this activity in relation to this invention as well.

CD39 was originally identified as an obscure activation marker expressed on B cells, T-cells, natural killer cells, dendritic cells and endothelial cells (Maliszewski et al., 1994; Kansas et al., 1991). More recently, it has been found to be responsible for the ecto-NTPDase activity on endothelial cells (Kaczmarek et al., 1996; Wang & Guidotti, 1996).

Other CD39-like transcripts have been characterized since and include, CD39L1, CD39L2, CD39L3, and CD39L4, all of which share extensive amino acid homology with other nucleotide triphosphatases in vertebrates, invertebrates, as well as plants, indicating that these genes also encode proteins with ecto-nucleotidase activity (Chadwick et al., 1998). The expression pattern of five human members of this gene family maps these genes to a region associated with audiogenic seizure susceptibility in mouse. CD39 family members have also been shown to be expressed in microglia and in the cerebrovascular endothelial and smooth muscle cells (Braun et al., 2000), where they are believed to regulate P2 receptor-mediated functions of microglia and blood flow and thrombogenesis. CD39L2 proteins are found in the heart muscle and capillary endothelial cells (Yeung et al., 2000).

In relation to the present invention, any NTPDase may be involved or used in the either the assay; and/or the screening methods; and/or the therapies described above, including CD39, CD39L1, CD39L2, CD39L3, CD39L4, Golgi-associated ecto-ATPase and ecto-uridine diphosphatase (UDPase), lysosomal ecto-apryase LALP70, hepatic canalicular ecto-apyrase, .alpha.-sarcoglycan and potato apyrase as some non-limiting examples.

II. P2 Receptors

Membrane-bound P2-receptors mediate the actions of extracellular nucleotides in cell-to-cell signaling. Two main families of P2 receptors are known, the P2X-receptors which are ligand-gated ion channels, and the P2Y-receptors which are G-protein-coupled receptors. To date, eight subtypes of the P2Y family have been cloned and functionally characterized.

DC are known to express mRNA for a variety of P2 receptors including the P2X.sub.1, P2X.sub.4, P2X.sub.5, P2X.sub.7, P2Y.sub.1, P2Y.sub.2, P2Y.sub.4, P2Y.sub.5, P2Y.sub.6, P2Y.sub.10, and the P2Y.sub.11 receptors. Furthermore, DC respond to nucleotide stimulation by pore formation, Ca.sup.2+ influx, phenotypic maturation, chemotactic migration, apoptosis, and cytokine production which are the characteristic responses of P2 receptors (Mutini et al., 1999; Liu et al., 1999; Coutinho-Silva et al., 1999; Berchtold et al., 1999; Marriott et al., 1999; Nihei et al., 2000; Ferrari et al., 2000).

The present inventors have shown that P2 receptors are involved in DC and T-cell communication and have found that the P2Y receptors are desensitized in CD39 knockout mice.

Some of the known P2 receptor antagonists include suramin, KN-62, MRS2179, TNP-ATP, TNP-GTP, oxidized ATP, PPADS, Reactive Blue2 (Williams et al., 2000; Ralevic et al., 1998).

III. Screening for Nucleotide Release

Irritant contact dermatitis is the most frequently observed occupational health problem among factory workers. No in vitro tests are currently available to predict relative skin irritant potentials of industrial and environmental chemicals. Many attempts to create such assays by companies have, in fact, failed. This may be due, at least in part, to the lack of information on the patho-physiology of irritant contact dermatitis.

In some embodiments, the present inventors have developed an assay to