WO2002047540A2 - Methods for inhibiting fibrosis - Google Patents

Abstract

A novel strategy of treating fibrosis in a mammal is disclosed. Fibrosis, occurring primary to an autoimmune disease process such as Type I diabetes, or secondary to the implantation of a foreign body, such as a graft containing encapsulated islet cells, is herein treated by administration of TNF-α, a TNF-α inducing substance, or a compound which acts on the TNF-α signaling pathway. The halting, slowing, and/or prevention of fibrosis can be assessed by any of several methods including visualization of fibrosis in tissue samples stained with hematoxylin and eosin, and/or measuring serum levels of PIP. The invention is particularly novel in that it teaches treatment of fibrosis which runs contrary to current modes of treatment which involve the inhibition of the TNF-α signaling pathway.

Description

METHODS FOR INHIBITING FIBROSIS

BACKGROUND

A major problem in clinical medicine is fibrosis in and/or around useful devices such as vehicles containing insulin secreting islet cells, foreign devices such as heart valves, or cardiac stents, infection, or following a surgical or interventional procedure such as carotid endarterectomy or cardiac catheterization. Furthermore, fibrosis is particularly exacerbated in some underlying disease states such as Type I diabetes, and Rheumatoid arthritis. In these clinical settings, fibrosis can occur even in the absence of foreign bodies and can lead to glomerulonephritis (kidney failure), atherosclerosis blindness, stroke, or the need for amputation. Introduction of a foreign body, interventional procedure, or any invasive procedure into an autoimmune setting, such a Type I diabetes, further accentuates the problem. For example, grafts of encapsulated insulin secreting islet cells are commonly rejected within a few days, and those that are not are rapidly rendered inoperable due to the fibrotic scarring that surrounds the graft. To date, the fibrotic complications of autoimmunity have not been remedied, and the disabling fibrosis around foreign bodies, or the fibrosis which occurs following an interventional procedure, or which occurs following infection is not effectively treatable.

SUMMARY OF THE INVENTION

The present invention provides a method of treating fibrosis in a mammal comprising administering to a mammal an amount of TNF-α or a TNF-α inducing substance sufficient to reduce or prevent fibrosis.

In one embodiment, the fibrosis is associated with a disease. i a further embodiment, the disease is selected from the group consisting of: autoimmune disease, type I diabetes, rheumatoid arthritis, and atherosclerosis. In a further embodiment, fibrosis is associated with the implantation of a foreign body.

In a still further embodiment, the fibrosis is associated with a disease and with implantation of a foreign body.

In one embodiment, the foreign body is a cellular encapsulation device, a cardiac stent, a cancer stent, a euglycemic clamp, and an artificial heart valve. In a further embodiment, the foreign body is selected from the group consisting of a cardiac stent, a cancer stent, or a cardiac catheter.

In a still further embodiment, the fibrosis is associated with an interventional procedure.

The present invention further provides a method of identifying an agent that treats fibrosis comprising the steps of: treating a mammal with the agent; determining the level of fibrosis in a mammal; and comparing the level of fibrosis in the mammal to the amount of fibrosis present in a control mammal.

In one embodiment, the level of fibrosis is determined by obtaining at least one tissue from the mammal and observing the tissue with histological or histomorphological techniques. In a further embodiment, the level of fibrosis is determined by obtaining at least one tissue from the mammal, staining at least one tissue from the mammal, and observing the stained tissue by microscopy.

In a further embodiment, the staining is effectuated with a stain suitable for microscopy, selected from the group consisting of: hematoxylin, eosin, Sirus Red, Masson's trichrome, azan, silver, gold, reticulin, and isamine blue.

In a still further embodiment, the level of fibrosis is determined by obtaining at least one tissue from the mammal and quantitating the level of fibrosis using a reverse transcriptase polymerase chain reaction assay.

The methods of the invention provide, for the first time, effective prevention, halting and/or slowing of spontaneously occurring fibrosis associated with disease states, and fibrosis which arises following the implantation of a foreign body.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiment thereof, and from the claims.

DETAILED DESCRIPTION

The present invention provides a method of treating a mammal with TNF-α, or TNF-α inducing agent for the purpose of halting, slowing or preventing fibrosis. The method of the invention describes the treatment of primary fibrosis in normal mammals, in mammals with autoimmune disease, such as type I diabetes, or in mammals possessing a mutation in the lmp2 gene. In another embodiment, the method of the invention describes the treatment of fibrosis secondary to the implantation of a foreign body, or interventional or surgical procedure in a mammal with autoimmune disease, such as type I diabetes or any other disease described herein, or in a mammal possessing a mutation in the lmp2 gene. The present invention also provides a method for assaying fibrosis in a mammal.

The practice of the present invention will employ, unless otherwise indicated, standard techniques of drug administration, tissue biopsy for histological examination, and histological staining. All patents, patent applications, and publications mentioned herein, both supra and infra are hereby incorporated by reference.

Definitions

Fibrosis can occur naturally, or from a disease such as for example, type I diabetes, rheumatoid arthritis, atherosclerosis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjδgren's Syndrome, including keratoconjunctivitis sicca secondary to Sjδgren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens- Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis, or it can develop in response to medical procedures such as, for example, the implantation of a cellular encapsulation device, a cardiac stent, a cancer stent, a euglycemic clamp, or an artificial heart valve, or following an interventional procedure such as cardiac catheterization, or carotid endarterectomy, or it can develop following any surgical procedure, or traumatic event which causes tissue damage.

As used herein, "fibrosis" refers to fibrous tissue formed in fibrotic diseases; that is, the formation of fibrous tissue as a reparative, or reactive process, as opposed to formation of fibrous tissue as a normal constituent of an organ or tissue, wherein fibrous tissue refers to tissue composed of or containing fibroblasts, and also the fibrils and fibers of connective tissue formed by such cells (Steadman's Medical Dictionary. 25^th ed., Edited by William Hensyl, Williams and Wilkins, Baltimore, MD, 1990). According to the invention, a tissue and/or organ is said to demonstrate "fibrosis", if any fibrous tissue that is not present in the normal tissue or organ is detected by one of skill in the art, using any of the methods described herein. For example, a normal organ or tissue sample does not contain fibrotic disease tissue, but it may contain fibrous tissue that is a normal constituent of said organ or tissue. The detection of fibrosis is readily apparent to one skilled in the art using standard histo-mophological assays, such as for example, tissue staining with hematoxylin and eosin (H & E) or Sirius Red F3BA, or other stains, and subsequent microscopic study, or electron microscopy. Active fibroblasts, indicative of fibrosis, are easily distinguished from mature fibroblasts by such indicators as, for example, large rounded nuclei with prominent nucleoli, or extensive cytoplasm with a strongly stained granular appearance evidencing an extensive system of endoplasmic reticulum involved in protein synthesis, or an abundance of secretory vesicles, or a decrease of formed fibers in the extracellular matrix. Assays such as quantitative RT-PCR, provide an even more sensitive method for detecting fibrosis in its early stages. Other such assays for detecting fibrosis as known to one skilled in the art are likewise considered within the scope of this invention. As used herein, "fibrosis" and "fibrotic disease tissue" refers to one or more types of fibrosis including, but not limited to endomyocardial, peritoneal, intrarenal, intraorgan, glomerulonephritis, subcutaneous, intraarterial, idiopathic retroperitoneal, leptomeningeal, mediastinal, nodular subepidermal, pericentral, perimuscular, pipestem, replacement, or subadventitial fibrosis.

Stains useful in evaluating the degree of fibrosis include any substances useful as microscopy stains. This includes, for example, hematoxylin, eosin, isamine blue, Masson's trichrome, Alcian blue, Nan Gieson, reticulin stain, azan, toluidine blue, chrome alum, methelene blue, silver, and gold stains.

As used herein, "halting" or "reducing" refers to preventing additional fibrosis, wherein "additional" refers to a statistically significant increase in fibrosis when compared to the level of fibrosis observed prior to the onset of treatment, or prior to an interventional or surgical procedure for a time period of 1-90 days, preferably 10-60 days, and more, preferably 20-30 days from the onset of treatment, wherein fibrosis is determined by standard histological techniques including, but not limited to, hematoxylin and eosin staining.

As used herein, "slowing" refers a decrease in the amount of fibrotic tissue or the rate of formation of fibrotic tissue, i.e., relative to that amount present in the fibrotic state of the same tissue, of at least about 5%, preferably at least about 10%, and more preferably at least about 50%, wherein fibrosis is determined by, for example, standard histological or histomorphological techniques, including but not limited to H & E or other staining, or by a weight reduction in fibrotic tissue, or reduction in the number of cells staining as fibrotic cells, or by other techniques such as RT-PCR, or other such assays known to one skilled in the art.

As used herein, "preventing" is with respect to the onset of fibrosis, such that the level of fibrosis remains substantially nominal when compared to the level of fibrosis detected in a normal individual (e.g., the level of fibrosis as determined the techniques described), and is preferably equivalent to or less than the level of fibrosis detected in a normal individual.

As used herein, "comparing" refers to the evaluation of a statistically significant difference by statistical tests including, but not limited to Students t-test and ANON A, between the treated and untreated mammals, whereby significance is granted to comparisons which generate a P value of at least 0.049, but preferably between 0.049 and 0.001, but most preferably less than 0.001.

As used herein, "treating", or "treatment" refers to the administration to a mammal of one or more agents that act on pathways that cause accelerated cell death of autoimmune cells. Such agents include, for example, TΝF-α, agents that induce TΝF-α expression or activity, TΝF-α agonists, or agents that stimulate TΝF-α signaling. In addition agents, substances, or signaling pathways which interact with, converge with, or otherwise modulate pathways which cause the accelerated cell death of autoimmune cells are included in the group of agents which maybe administered to a mammal during "treatment". For example, there are many methods for stimulating TΝF-α production, including, but not limited to vaccination with killed bacteria or toxoids, e.g. BCG, cholera toxoid, or diptheria toxoid; induction of limited viral infections; administration of LPS, interleukin-1, or UN light; activation of TΝF-α producing cells such as macrophages, B-lymphocytes and some subsets of T-lymphocytes; or administration of the chemotactic peptide fMET-Leu-Phe; CFA-pacellus toxoid, mycobacterium bovis bacillus, TACE (a metalloproteiumas that mediates cellular TΝF-α release), hydrozamates, p38 mitogen activated protein ("MAP") kinase, and viral antigens that activate ΝF-κB transcription factors that normally protect the cells from apoptosis.

Accelerated death of autoimmune cells can also be accomplished by administering agents that act as agonists for the enzyme TNF-α converting enzyme, that cleaves the TNF-α precursor to produce biologically active TNF-α. Autoimmune cells can also be killed by administering agents that disrupt the pathways that normally protect autoimmune cells from cell death, including soluble forms of antigen receptors such as CD28 on autoreactive T cells, CD40 on B cells that are involved in protection of autoimmune cells, and CD59 (i.e., Fes) on T-lymphocytes. Other such agents include 5 p75NTF and lymphotoxin-β receptor.

As used herein, "administration" refers to the introduction of a TNF-α or of a TNF-α inducing substance orally, intravenously, intraarterially, intraperitoneally, subcutaneously, or by direct injection to an organ, or body tissue, in whole or in part.

As used herein, "TNF-α inducing substance" is a substance (administered at a [ 0 dosage of 5ng-500mg/kg body weight) that induces TNF-α expression by 10- 100%, more preferably 30-80% and most preferably 40-60%, or induces TNF-α activity by 10-100%, more preferably 30-80% and most preferably 40-60%. A "TNF-α inducing substance" includes, for example, TNF-α agonists, or agents that stimulate TNF-α signaling, including, but not limited to, complete Freund's adjuvant (CFA), interleukin-1, proteosome inhibitors, NF-κB inhibitors, L 5 sterodial and non-sterodial anti-inflammatory drugs, tissue plasminogen activator, lipopolysaccharide, UV light, vaccination with killed bacteria or toxoids, (e.g., BCG, cholera toxoid, or diptheria toxoid), induction or limited viral infections, activation of TNF-α producing cells such as macrophages, B-lymphocytes, and some subsets of T-lymphocytes, administration of the peptide fMET-Leu-Phe, CFA-pacellus toxoid, Mycobacterium bovis bacillus, TACE (a 20 metalloproteiumas that mediates cellular TNF-α release), hydrozamates, p38 MAP kinase, and viral antigens that activate NF-κB transcription factors that normally protect the cells from apoptosis, or an intracellular mediator of the TNF-α signaling pathway.

As used herein "TNF-α inducing substance" may also refer to agents (administered at a dosage of 2.5 ng to lOOmg/kg body weight) that enhance TNF-α signaling activity by modulating

25 the activity of signaling proteins that associate directly or indirectly with the TNF-α receptors: TNFR1 and TNFR-2 (Hohmann, H. P. et al., J. Biol. Chem. 264 14927-14934 (1989); Brockhaus, M. et al, Proc. Natl. Acad. Sci. USA 87, 3127-3131 (1990)), including but not limited to TRADD (TNFR1 -associated death domain protein; Hsu et al.. (1995) Cell 81,495- 504), RIP (receptor interacting protein; Stanger et al. (1995) Cell 81,513-523), FADD (Fas

30 associated death domain protein; Chinnaiyan et al (1995) Cell 81,505-512) , TRAF (TNF- receptor associated factor; Hsu et al.. Cell. (1996) 84(2):299-308, reviewed by Arch RH et al.Genes Dev. (1998) 12(18):2821-30), SODD (silence of death domains; Jiang, Y, et al (1999) Science 283,543-546) and the caspase family of proteins including caspase 8 (reviewed by Rath PC and Aggarwal BB. J Clin Immunol. (1999) (6):350-64).as well as other TNFR associated proteins containing a caspase-recruiting domain (CARD; Yu PW et al. Curr Biol. (1999) 9(10):539-42, McCarthy et al. J Biol Chem.(1998) 273(27): 16968-75).

As used herein "TNF-α inducing substance" also refers to agents (administered at 7.5ng-

500mg/kg body weight) that enhance the activity of transcription factors that bind directly or indirectly to the TNF-α promoter, including but not limited to the NFkB / Rel and API (Jun,/Fos,/ATF) family of transcription factors. In one embodiment, a "TNF-α inducing substance" refers to agents (administered at a dosage of 2.5ng-100mg kg body weight) that enhance the phosphorylation /ubiquination of IκB by the IκB kinase thus increasing the nuclear translocation and, hence, the activity of NFkB (reviewed by Karin M. Oncogene. (1999) 18(49):6867-74). In another embodiment, "TNF-α inducing substance" may refer to agents (administered at a dosage of Ing-lOOmg/kg body weight) that enhance the activity of the AP-1 transcription complex for instance via activation of the c-jun amino-terminal kinases (JNKs) and p38 MAP kinases (reviewed by Karin et al. (1997) Curr. Opin. Cell Biol. 9, 240-246).

In a preferred embodiment, "TNF-α inducing substance" refers to any agent (administered at a dosage of 5ng to 125mg/kg body weight) that enhances TNF-α induced signaling activity either directly or indirectly by modulating the activity of other signaling pathways and associated kinases including but not limited to MAP/ERK kinase kinase (MEKK1; Baud V. et al (1999) Genes Dev. 13, 1297-1308)), apoptosis signal-stimulating kinase 1 (ASK-1; Ichijo H. et al. (1997) Science, 275:90-94), NFkB-inducing kinase 1 (NIK; reviewed by Karin M. Oncogene. (1999) 18(49):6867-74), phosphatidylinositol 3-OH kinase (PI(3)K)/ Akt/PKB (reviewed in Chan TO. et al. (1999) Ann. Rev. Biochem.68, 965-1014), GSK3β (Hoeflich KP et al. Nature. 2000 Jul 6;406(6791):86-90), atypical protein kinase C (aPKC) and associated proteins (p62) (Sanz L, et al. EMBO J. 1999 18:3044-3053) as well as the N-Sphingomyelinase/ FAN mediated signaling pathway (Klages etal. (1998) J. Leukocyte Biology 63, 678-682)..

As used herein, "TNF-α inducing substance" may also refer to any substance (administered at a dosage of 2.5ng to 475mg/kg body weight) which accelerates the death of autoimmune cells such as agents that act as agonists for the enzyme, TNF-α converting enzyme, that cleaves the TNF-α precursor to produce biologically active TNF-α as well as antagonists of cellular inhibitors of apoptosis (cIAPs; Rothe, M. et al (1995 Cell 83,1243-1252). A "TNF-α inducing substance" may also refer to agents that disrupt the pathways that normally protect autoimmune cells from cell death, including soluble forms of antigen receptors such as CD28 on autoreactive T cells, CD40 on B cells that are involved in protection of autoimmune cells, and CD59 (i.e., Fes) on T-lymphocytes. Other such agents include p75NTF and lymphotoxin-β receptor.

In the preferred embodiments of the invention, a "TNF-α inducing substance" is one or more of the compounds as described. Likewise, the invention contemplates that a "TNF-α inducing substance" useful in the present invention can also be any substance determined experimentally to have the properties of a "TNF-α inducing substance". Methods of determining TNF-α inducing activity of a candidate substance include ELISA, and/or RIA to measure levels of TNF-α present in a mammal of the invention following treatment with a candidate inducing substance. Additionally, one can determine TNF-α inducing activity in an in vitro assay system, whereby a candidate inducing substance can be said to induce TNF-α if the substance induces an increase in (a) mRNA levels of genes which are known to be regulated by TNF-α including, but not limited to IL-6, p65, NF-κB, p50, TNF-α, or immunoglobulin; and/or (b) the polypeptides encoded by the genes of (a), as determined by techniques routinely used in the art for measuring mRNA and peptide levels. For example, the ability of a substance to stimulate TNF-α may be confirmed by measuring the levels of TNF-α mRNA following treatment with the candidate inducing substance as described in Yang et al. (2000, J. Am. Soc. Nephrol, 11 :2026), and described herein below.

As used herein the term "foreign body" refers to an object inserted into a tissue of an organism. The object may include a medical device, for example, a cardiac stent, a cancer stent, or a stent for biliary or lymphatic or other drainage, or an artificial heart valve, or a euglycemic clamp, or other such device. The term also refers to cells or tissue which may or may not be encapsulated in a cellular encapsulation device.

Also considered within the scope of the term "foreign body" are interventional or surgical procedures which incorporate the insertion of medical devices, either temporarily or permanently, into the body of an organism, such as cardiac catheterization, carotid endarterectomy, or the insertion of any medical device into a blood vessel, organ, or tissue.

As used herein, an "interventional procedure" refers to any surgical procedure.

"Interventional procedures" of the present invention include, but are not limited to carotid endarterectomy, coronary artery bypass, cardiac catheterization, bowel surgery, brain surgery, vascular surgery, laparoscopy, and urogenital surgery.

As used herein, "proteasome inhibitors" refers to compounds which inhibit the proteolytic action of proteasomes by 10-100%, more preferably 30-80% and most preferably 40- 60% including, but not limited to proteasome inhibitor 1, benzyloxycarbonyl-leu-leu- phenylalaninal, MG-262, and lactacystin.

As used herein, "NF-κB inhibitors" refers to substances which directly inhibit the activity of NF-κB, and/or members of the NF-κB family, including, but not limited to NF-κBl (p50/pl05), NF-κB2 (p52/pl00), p65 (RelA), RelB, c-Rel, and or heterodimeric NF-κB complexes comprised of one or more members if the NF-κB family by 10- 100%, more preferably 30-80% and most preferably 40-60%. An "NF-κB inhibitor": also refers to a substance which inhibits components of the NF-κB signaling pathway, or which inhibits signaling pathways which intersect with the NF-κB pathway by 10-100%, more preferably 30- 80%) and most preferably 40-60%. "NF-κB inhibitors" can also refer to substances which inhibit the phosphorylation and degradation of the NF-κB regulatory protein I-κB by 10- 100%, more preferably 30-80% and most preferably 40-60%, including, but not limited to substances which inhibit protein kinase C, protein kinase A, protein kinase R, Raf, casein kinase II, eukaryotic initiation factor-2 kinase, mitogen-activated ribosomal S6 protein kinase, and IκB kinase-α/IκB kinase-β. For example, a "NF-κB inhibitor" could refer to a substance which inhibits the activity of IκB kinase-α, thereby inhibiting the phosphorylation of IκB, thereby inhibiting the degradation of IκB, thereby inhibiting the activation of NF-κB. The ability of a substance to act as an NF-κB inhibitor may be determined by measuring NF-κB DNA binding activity, or by measuring the levels of factors which are downstream from NF-κB in cellular signaling pathways, such as iNOS (Yang et al., (2000) J. Am. Soc. Nephrol. 11 :2017).

As used herein, "inflammatory drugs" refers to any substance which induces an inflammatory response, such as TNF-α, CFA, LPS, or TNF-α agonists which act on the TNF-α receptor. "Inflammatory drugs" can also mean any drug which acts opposite to anti-TNF-α agonists, anti-TNF-α antibodies, TNFR2 fusion proteins (Immunex), Embrel, anti IL-1 therapies, TNF-α convertase inhibitors, p38 MAP kinase inhibitors, phosphodiesterase inhibitors, thalidomide analogs and adenosine receptor agonists. "Inflammatory drugs" can also mean drugs which stimulate the IL-1 pathway, or drugs which interact with converging pathways such as Fas, FasL, TACI, ATAR, RANK, DR5, DR5, DCR2, DCR1, DR3, etc. As used herein, "tissue plasminogen activator" refers to a serine protease which is endogenously synthesized by a variety of tissues, or can be introduced into a mammal exogenously, which cleaves plasminogen to plasmin, thereby stimulating the process of fibrinolysis, by which fibrin is degraded.

As used herein, "lipopolysaccharide" refers to a group of glycolipids of gram-negative bacteria, which act as surface antigens when present on the bacterial cell surface, and which, if shed from the bacterial cell, will behave as a toxin upon introduction to a mammal, inducing inflammation and fever.

As used herein, "intracellular mediator of the TNF-α signaling pathway" refers to any agent which induces TNF-α expression or activity, TNF-α agonists, agents that stimulate TNF-α signaling, or agents which are involved in the TNF-α signaling pathway including, but not limited to TNF-RI (p55), TNF-RII (p75), TNF-RIII (TNF-RP), Ras, Raf, mitogen activated protein kinase, c-Jun N-terminal kinase, c-Jun, NIK, IKKα, IKKβ, IκB, NF-κB, AP-1, TRAF1, TRAF2, TRADD, RIP, Fas, Daxx, FADD, and caspase-8.

As used herein , "a mutation in the lmp2 gene", or "a mutation in the lmp2 gene or equivalent thereof refers to a cell that has a defect, such that it is abnormally susceptible to apoptotic cell death, for example, a cell that has an ablation at a critical point in an apoptotic cell death pathway. In another aspect, "a mutation in the lmp2 gene" means that a cell has a mutation in the lmp2 gene or a gene that encodes a protein that carries out a function that is the same as or similar to the function of the protein product of the lmp2 gene (i.e., a gene encoding a proteasome subunit). Alternatively, the phrase "a mutation in the lmp2 gene" can be used to refer to a cell that has a mutation in a gene that encodes a regulator of the lmp2 gene or a gene encoding another component of the proteasome complex. For example, a human homolog of the murine lmp2 gene is an equivalent of the lmp2 gene according to the present invention. As another example, a gene that encodes a protein that carries out the same or a similar function as the protein product of the lmp2 gene, but that has a low amino acid sequence similarity, would also be considered as an equivalent of the lmp2 gene according to the present invention.

As used herein, "implantation of a foreign body" refers to the placement, using surgical techniques, of a foreign object into a whole organ, or any part thereof, subcapsular space, or any body cavity, pleural space, or peritoneal space. As used herein, "cellular encapsulation device" refers to a hydrogel-based microcapsule fabricated from alginate and low-molecular mass poly L-lysine, which can contain a syngenic and or allogenic population of cells including, but not limited to pancreatic B-cells and or splenocytes.

As used herein, "euglycemic clamp" refers to a cellular encapsulation device in which the encapsulated cells whose properties allow for short term glycemic control of insulin exchange, but prevents direct cell-cell contact, wherein the encapsulated cells are normal pancreatic islet cells which express MHC class I and have few associated passenger lymphocytes that express both MHC class I and class II molecules.

As used herein, "measuring" as it refers to fibrosis, refers to the use of one or more methods to directly and/or indirectly and either qualitatively or quantitatively assess the degree of fibrosis including, but not limited to visualization of fibrosis, or fibrous tissue in H&E stained tissue samples, and/or measuring serum levels of carboxyterminal propeptide of procollagen (PIP) as a marker of collagen type I synthesis and deposition; collagen type I is the principal type of collagen synthesized by fibroblasts, and a primary component of fibrotic tissue. In a further embodiment of the invention, "measuring" refers to the semi-quantitative assessment of type I collagen and TGF-β mRNA, wherein an increase in type I collagen and TGF-β mRNA is indicative of an increase in fibrosis.

As it refers to the assessment of fibrosis in blood vessels, "measuring" of fibrosis may be conducted using imaging techniques, known to those of skill in the art, useful in visualizing or quantifying blood flow in a mammal. Such techniques include, but are not limited to MRI, PET scan, or CAT scan. Data obtained from such measurements of blood flow in a mammal may be compared with data obtained from a similar (control) mammal, or may be compared with data obtained from the same mammal from an earlier time point. Alternatively, the blood flow of a mammal may be "measured" before and after treatment with one or more substances that cause accelerated death of autoimmune cells. In all blood flow measurements, an increase in blood flow is indicative of a decrease in fibrosis.

As it refers to the assessment of fibrosis following the implantation of encapsulated insulin-producing cells, fibrosis may be "measured" by quantifying the level of insulin being produced by the encapsulated cells. Insulin levels may be measured by any method known to those of skill in the art, wherein an increase in insulin production is indicative of the viability of the encapsulated cells, and is thus indicative of the prevention of, or slowed progression of fibrosis. In addition, in mammals which depend on implanted encapsulated islet cells for blood sugar maintenance, blood sugar levels may be monitored, wherein homeostatic regulation of blood sugar is indicative of a decrease or prevention of fibrosis.

As used herein, "hematoxylin-eosin staining of tissue" refers to the use of colored dyes which bind to negatively charged molecules in the case of hematoxylin and positively charged molecules in the case of eosin to allow for the visualization of and distinction between tissue type, and cell type in a particular tissue sample.

As used herein, "mammal" refers to any organism of the phylogenetic class Mamallia, including, but not limited to rodents, ungulates, feline, canine, etc. In preferred embodiments, a mammal is a human.

I. Autoimmune Disease and Type I Diabetes

The present invention provides a method of halting, slowing or preventing fibrosis in mammals, occurring primary to a disease process, or secondary to surgically implanted foreign bodies, or interventional procedures by treating with one or more agents that cause accelerated cell death of autoimmune cells, such as TNF-α or a TNF-α inducing substance, i.e., the induction of apoptosis in fibrosis inducing cells. In preferred embodiments, this method is used to treat fibrosis in mammals with an autoimmune disease, such as type I diabetes where fibrosis occurs spontaneously, or is enhanced following the implantation of a foreign body. Such autoimmune diseases can also include, without limitation, atherosclerosis, multiple sclerosis, premature ovarian failure, scleroderm, Sjogren's disease, lupus, vilelego, alopecia (baldness), polyglandular failure, Grave's disease, hypothyroidism, polymyosititis pempligus, Chron's disease, colititis, autoimmune hepatitis, hypopituitansm, myocardititis, Addison's disease, autoimmune skin diseases, uveititis, prericious anemia, hypoparathyroidism, and rheumatoid arthritis. The invention provides a novel therapeutic approach to treat fibrosis, which is in contrast to the current standard of care for treating fibrotic lesions.

Type I diabetes results from destruction of the cells of the Islet of Langerhans of the pancreas via a severe autoimmune process. The goal for treatment of Type I diabetic mammals is to permanently halt the autoimmune process so that pancreatic islets are preserved. Alternatively, in cases where islet destruction from autoimmunity is complete, the goal is to provide a method of replacing islet cells, or allowing them to regenerate. It has been previously shown that one can increase or maintain the number of functional cells of a predetermined type for treatment of established cases of diabetes mellitus by implanting encapsulated Islet cells and treating with TNF-α or TNF-α inducing substance, where existing autoimmunity is reversed. A major complication in the current islet replacement therapies is the development of fibrosis around the implant, leading to graft rejection. The present invention thus provides a method for attenuating fibrosis by inducing the death of cells responsible for generating such lesions.

II. TNF-α

Tumor necrosis factor (TNF, also referred to as TNF-α) is a potent cytokine produced mainly by activated macrophages and a few other cell types, including eosinophils. The large number of biological effects elicited by TNF include hemorrhagic necrosis of transplanted tumors, cytotoxicity, a role in endotoxin shock, inflammatory, immunoregulatory, proliferative, and antiviral responses (reviewed in Goeddel, D. N. et al., Cold Spring Harbor Symposia on Quantitative Biology 51, 597-609 (1986); Beutler, B. and Cerami, A., Ann. Rev. Biochem. 57, 505-518 (1988); Old, L. J., Sci. Am. 258(5), 59-75 (1988); Fiers, W. FEBS Lett. 285(2), 199-212 (1991)). The literature has reported that TΝF and other cytokines such as IL-1 may protect against the deleterious effects of ionizing radiation produced during the course of radiotherapy, such as denaturation of enzymes, lipid peroxidation, and DΝA damage (Νeta et al., J. Immunol. 136(7): 2483, (1987); Νeta et al., Fed. Proc. 46: 1200 (abstract), (1987); Urbascheket al., Lymphokine Res. 6: 179 (1987); U.S. Pat. No. 4,861,587; Neta et al., J. Immunol. 140: 108 (1988)).

The first step in the induction of the various cellular responses mediated by TNF is the binding to specific cell surface receptors. Two distinct TNF receptors of approximately 55-kDa (TNF-R1) and 75-kDa (TNF-R2) have been identified (Hohmann, H. P. et al., J. Biol. Chem. 264 14927-14934 (1989); Brockhaus, M. et al., Proc. Natl. Acad. Sci. USA 87, 3127-3131 (1990)), and human and mouse cDNAs corresponding to both receptor types have been isolated and characterized (Loetscher, H. et al., Cell 61, 351 (1990); Schall, T. J. et al., Cell 61, 361 (1990); Smith, C. A. et al., Science 248, 1019 (1990); Lewis, M. et al., Proc. Natl. Acad. Sci. USA 88, 2830-2834 (1991); Goodwin, R. G. et al., Mol. Cell. Biol. 11, 3020-3026 (1991)). Both TNF-Rs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions. The extracellular portions of both receptors are found naturally also as soluble TNF-binding proteins (Nophar, Y. et al., EMBO J. 9, 3269 (1990); and Kohno, T. et al, Proc. Natl. Acad. Sci. U.S.A. 87 8331 (1990)). Although not yet systematically investigated, the majority of cell types and tissues appear to express both TNF receptors. It has been observed that both polyclonal and monoclonal antibodies directed against TNF-Rl can act as specific agonists for this receptor and elicit several TNF activities such as cytotoxicity, fibroblast proliferation, resistance to chlamydiae, and synthesis of prostaglandin E (Engelmann, H. et al., J. Biol. Chem. 265. 14497-14504 (1990); Espevik, T. et al., J. Exp. Med. 171, 415-426 (1990); Shalaby, M. R. et al., J. Exp. Med. 172, 1517-1520 (1990)).

Monoclonal antibodies against human TNF-Rs that block the binding of TNF to TNF-R and antagonize several of the effects of TNF and TNF agonists (such as cytotoxicity and activation of NF-kB) have been described (Espevik, T. et al., Supra; Shalaby, M. R. et al, Supra; Naume, B. et al., J. Immunol. 146 3035-3048 (1991); Hohmann, H. P. et al., J. Biol. Chem. 265, 22409-22417 (1990)).

A. Determining if a substance is a TNF-α inducing substance

The present invention provides a method of inhibiting fibrosis comprising treating a mammal with one or more substances which induce TNF-α. The induction of TNF-α may be determined by any method known to those of skill in the art. One potential technique for measuring TNF-α levels in a mammal following treatment with a TNF-α inducing substance, is to measure changes in TNF-α mRNA levels as described in Yang et al. (2000) J. Am. Soc. Nephrol. 11:2017. Briefly, following treatment with a TNF-α inducing substance by any means described herein, cells may be obtained from the mammal that received treatment by any means known to those of skill in the art (e.g., biopsy) for mRNA analysis. Total RNA may then be extracted from the cells using, for example, the guanidinium thiocyanate-phenol-chloroform methods using RNA-zol (Cinna/Biotecx Laboratories International Inc., Friendswood,. TX). The total RNA is then treated with RNase-free DNase I (Boehringer Mannheim, Manneheim, Germany) at 37° C for 30 min. Approximately 1 μg of RNA is then reverse transcribed with avian myeloblastosis virus reverse transcriptase (RT AMN, Boehringer Mannheim) at 42 ° C for 60 min. cDΝA is then amplified for 30 to 42 cycles in a 100 μl reaction volume containing 50 mM KC1, 20 mM Tris-HCl (pH 8.4), 10 mM dTΝP, 1.5 to 3.0 mM MgCl₂, 1 unit Taq polymerase, and 10 pmol of TΝF-α-specific PCR primers. The reaction is then thermally cycled according to the following parameters: 94° C for 1 min., 60°C for 1 min., and 72° C for 3 min. Amplified products are then separated on a 4% agarose gel with ethidium bromide. TΝF-α- specific PCR primers which may be used to determine TΝF-α mRΝA levels are as follows: 5'- agtagcacagaaagcatgatccgc-3' (sense), and 5'-ccaaagtagacctgcccggactc-3' (antisense), which will yield an approximate 692-bp PCR product. To quantitate the levels of TNF-α mRNA following treatment with a TNF-α inducing substance, quantitative PCR may be performed as described in Yang et al. (1998), Clin Exp fmmunol 113:258 and Yang et al. (1997) Nephron 77:290. According to the present invention, a TNF-α inducing substance preferably will stimulate an increase in TNF-α mRNA levels by at least 2-fold compared to TNF-α mRNA levels measured in the absence of a TNF-α inducing substance, preferably 3 -fold, more preferably 4-fold, and still more preferably, at least a 5-fold increase in TNF-α mRNA.

B. Determining if a substance inhibits NF-κB

One potential method to determine whether a substance inhibits the production or activity of NF-κB, is to conduct a nuclear binding assay. NF-κB is a nuclear transcription factor, and thus decreased binding of NF-κB, extracted from the nuclei of cells treated with aNF-κB inhibiting substance, to a DNA sample indicates decreased NF-κB activation.

Nuclear proteins may be prepared according to Satriano and Schlondorff (1994, J. Clin. fnvest., 94:1629) with modifications. Cells which have been treated with an NF-κB inhibitor according to the invention may be harvested by any means known in the art (e.g., biopsy), treated with trypsin and pelleted after centrifugation. Pellets from cells are washed with ice-cold phosphate-buffered saline and resuspended with buffer A containing 10 mM Hepes (pH 7.9), 1.5 mM MgCl₂, 10 mM KC1, 3.5 mM dithiothreitol (DTT), H₂O, and protease inhibitor (Complete™, Boehringer Mannheim). Cells are then incubated on ice for 10 min and centrifuged for 5 min at 650 X g. The pellets are resuspended with the same buffer A containing 0.5%)

Nonidet P-40, lysed by vortexing, and allowed to swell on ice for 20 min. The nuclear fractions are then pelleted for 5 min at 6,000 x g and resuspended with buffer B containing 5 mM Hepes (pH 7.9), 26% glyceral, 1.5 mM MgCl₂, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM DTT, 0.4 M NaCl, H ₂O, and complete. After incubation on ice for 30 min, the nuclear fractions are centrifuged for 10 min at 12,000 x g. The supernatants containing nuclear protein can be divided into aliquots and stored at -80°C for subsequent use. Protein concentrations may be determined by a Bradford assay using the Bio-Rad protein assay.

To determine whether a substance is an NF-κB inhibitor, whole-cell nuclear extracts may be subjected to assays for NF-κB binding activity using aNF-κB consensus oligonucleotide (5'- AGT TGA GGG GAC TTT AGG C-3*; Promega, Madison, WI) radiolabeled with [γ-³²P] dATP by T polynucleotide kinase 3 (Amersham Pharmacia Biotech, Uppsala, Sweden). A total of 10 μg of nuclear protein is incubated with 70 to 80 kcpm of P-labeled NF-κB oligonucleotide in a binding mixture (50 mM Hepes [pH 7.9], 20% glycerol, 5 mM MgCl₂, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 0.5 μgpoly dl-dC [Pharmacia Biotech], and H ₂O), to a final volume of 15 μl. After incubation at room temperature for 20 min, the protein-DNA complexes are resolved on native 4% polyacrylamide gel in 0.5x Tris-borate-EDTA buffer system and run at 200 V for 1.'5 h in a 4°C cold room. Gels are then transferred to Whatmann paper, dried, and exposed to Kodak XR5 film (Rochester, NY) in film holders for 4 to 16 h at -80°C.

Specific competition control of unlabeled oligonucleotide at 100-fold excess can be added to the binding reaction mixture for 10 min before the addition of the labeled NF-κB probe. To confirm the specificity of binding reaction, a 100-fold unrelated unlabeled oligonucleotide can be added to the binding reaction mixture 10 min before the addition of the labeled NF-κB probe.

Radiolabeled bands correspond to DNA binding of the NF-κB transcriptional factor. Accordingly, a decrease in the signal following treatment with an NF-κB inhibitor indicated a decrease in NF-κB activity. According to the invention, a substance which acts as an NF- B inhibitor will preferably decrease radiolabeled DNA binding by at lease 1-fold compared to binding observed following treatment with an known NF-κB activator such as INFα/β (Yang et al., (2000) Proc Natl Acad Sci USA 97:13631), preferably 2-fold, more preferably 3-fold, more preferably 4-fold, and still more preferably 5-fold.

An alternative method to determine whether a substance is an NF-κB inhibitor is to examine the mRNA levels of downstream targets of NF-κB. For example iNOS mRNA levels are generally increased as a result of NF-κB activation. Accordingly iNOS mRNA levels may be measured by a method similar to that described above for the reverse transcription PCR-based measurement of TNF-α mRNA described above, with the exception that the primers used to amplify iNOS cDNA are as follows: (sense) 5'-gtgttccaccaggagatgttg-3'; (antisense) 5'- tctggtcgatgtcatgagcaaagg-3', which should yield an approximate 508-bp PCR product (Yang et al., (2000) J. Am. Soc. Nephrol. 11:2017). According to the present invention, aNF-κB inhibitor preferably will induce a decrease in iNOS mRNA levels by at least 2-fold compared to iNOS mRNA levels obtained in the presence of a known NF-κB activator such as INFα/β (Yang et al., (2000) Proc Natl Acad Sci USA 97: 13631), preferably 3-fold, more preferably 4-fold, and still more preferably, at least a 5 -fold decrease in iNOS mRNA. III. Imp2 Gene and Gene Defects

Genetic and functional studies have identified mutations in the lmp2 gene in non-obese diabetic (NOD) diabetic mice, a murine model for human type I diabetes (Li et al, Proc. Natl. Acad. Sci., USA (1994) 91:11128-32; Yan et al., J. Immunol. (1997) 159:3068-80; Fu et al., Annals of the New York Academy of Sciences (1998) 842: 138-55; Hayashi et al., Molec. Cell. Biol. (1999) 19:8646-59). Lmp 2 is an essential subunit of the proteasome, a multi-subunit particle responsible for processing a large number of intracellular proteins. The pronounced proteasome defect in lmp2 results in defective production and activation of the transcription factor NF-κB through impaired proteolytic processing of NF-KB to generate NF-κB subunits p50 and p52 and impaired degradation of the NF-κB inhibitory protein, I-κB. NF-κB plays an important role in immune and inflammatory responses as well as in preventing apoptosis induced by TNF-α. Autoreactive lymphoid cells expressing the lmp2 defect are selectively eliminated by treatment with TNF-α, or any TNF-α inducing agent, such as complete Freund's adjuvant (CFA), or an agent that acts on a pathway required for cell death protection, for example, any pathway converging on the defective apoptotic activation mechanism. This is well illustrated by faulty apoptosis protection in the NOD mouse which lacks formation of protective NFKB complexes.

The lmp2 gene is genetically linked to the MHC locus (Hayashi et al., supra). Antigen presenting cells of NOD mice cease production of LMP2 protein at approximately 5-6 weeks, a process that terminates the proper processing of endogenous peptides for display in the context of MHC class I on the cell surface. Surface display of endogenous peptide in the context of MHC class I molecules is essential for the selective elimination of T cells reactive to self antigens (Faustman et al., Science (1991) 254:1756-61; Ashton-Rickardt et al., Cell (1993) 73:1041-9; Aldrich et al., Proc. Natl. Acad. Sci. USA (1994) 91(14):6525-8; Glas et al., J. Exp. Med. (1994) 179:661-72). Current theory suggests that interruption of endogenous peptide presentation via MHC class I prevents proper T cell education and is responsible for a diverse array of autoimmune diseases (Faustman et al., supra; Fu et al., J. Clin. Invest. (1993) 91:2301- 7). These data are also consistent with the clear sex-, tissue-, and age-specific differences in the expression of this error which parallel the initiation and disease course of insulin-dependent (type I) diabetes. It is hypothesized that the trigger for the initiation of autoimmunity is the tissue- and developmental-specific disregulation of the proteasome (or MHC class I) in islet cells, as opposed to lymphocytes. As mentioned above, it is possible that this defect triggers a pathologic T cell response to islet cells via interruption of proper T cell education (Hayashi et al., supra). As mentioned above, autoreactive lymphoid cells expressing the lmp2 defect are selectively eliminated, for example, by treatment with TNF-α, or any TNF-α inducing agent, such as complete Freund's adjuvant (CFA). Although the specific gene defect has not been identified in human autoimmune mammals, it is known that human splenocytes in the human diabetic mammal, like murine splenocytes in the NOD mouse, have defects in resistance to TNF- α induced apoptosis (Hayashi et al., supra). Specific cells in human autoimmune mammals might express a genetic defect, similar to the proteasome defect in mice, that increases susceptibility to TNF-α induced apoptosis or an analogous apoptotic cell death pathway. Therefore, in mammals expressing the genetic defect, only the autoimmune cells are killed.

IN. Treatment of Fibrosis with Agents That Cause Accelerated Cell Death of

Autoimmune Cells

According to the present non-limiting theory of the invention, multiple cell death pathways exist in a cell, and any one or more of these cell death-related pathways may be defective, accentuating the sensitivity of these cells to cell death. For example, susceptibility to TΝF-α induced apoptosis could occur via a failed cell death inhibition pathway (e.g., by defective production and activation of the transcription factor ΝF B, as in type I diabetes). Further, it is well known that there are two different TΝF-α receptors. Defective signaling through either receptor could render autoimmune cells susceptible to TΝF-α induced apoptosis. As but another example, defective cell signaling through surface receptors that stimulate pathways that interact with the cell death pathway, i.e., LPS, IL-1, TPA, UN light etc., could render autoimmune cells susceptible to apoptosis according to the theory of the present invention. Therefore, methods of the present invention that are beneficial in the treatment of fibrosis in mammals with autoimmune disease are applicable to any autoimmune mammal that has a defect in a cell death pathway.

Current therapies for fibrosis are directed toward decreasing the inflammatory reaction that is thought to be responsible for the attendant increase in collagen deposition. Given that TΝF-α is proinflammatory mediator of this response, many of the major approved therapies for treating autoimmune, or inflammatory disease involve the administration of anti-inflammatory drugs that inhibit the production of TΝF-α, including COX-2 inhibitors, and TΝF-α antagoninsts. However, studies have shown that these conventional therapies are actually deleterious, in that they bring about expansion of the population of harmful autoimmune cells in a mammal, increasing the number and severity of autoimmune lesions and autoreactive infiltrates. In addition, many anti-inflammatory drug therapies cause severe rebound disease after discontinuation. Thus, according to the present theory, induction of an inflammatory response, rather than inhibition of an inflammatory response, is the preferred method of treating fibrosis in an autoimmune individual.

It is possible that TNF-α is inducing a cytokine, toxoid, or other related molecule that participates in the inflammatory response that is the responsible for the benefit of TNF-α treatment. If so, induction of inflammation via TNF-α treatment is still in agreement with the theory of the invention. In a preferred embodiment, induction of inflammation via TNF-α treatment induces mediators of autoimmune cell death which target selectively, / p2-deficient lymphocytic cell types responsible for fibrosis.

Similarly, a variety of well known methods can be used in the present invention to accomplish ablation of autoimmune cells. One preferred treatment is the administration of TNF- α, which is available from Genetech Corporation, South San Franscisco, CA; Roche; Boehringer Ingleheim; Asahi Chemical Industry; and Sigma Chemicals. The administration intraperitoneally of TNF-α in diabetes-prone mice is described in Rabinovitch et al., J. Autoimmunity 8: 357-366 (1995), hereby incorporated by reference.

The present invention also features a drug combination that includes two or more TNF-α inducing agents. One particularly preferred combined TNF-α treatment is the combination of TNF-α and IL-1. Other preferred treatments can utilize other TNF-α inducing substances such as CFA, NF-κB inhibitors, LPS, TPA, UN light, or other intracellular mediator of the TΝF signaling pathway. This treatment strategy goes against the current dogma surrounding treatment of fibrosis. The treatment of the present invention discloses induction of inflammation, which is the opposite of the treatment believed to be effective by those skilled in the art, that is, suppression of inflammation. In the current treatment, according to the invention, inflammation is prevented form occurring because the inflammatory cells actually die prior to arriving at the site of potential inflammation or are killed at the site of potential inflammation.

The present invention is not limited to a combined TΝF inducing therapy that includes only the combination of TΝF-α and IL1, but includes any combination of TΝF-α-inducing therapies, e.g., vaccination with BCG etc., viral infection, LPS, activation of cells that normally produce TΝF-α (i.e., macrophages, B cells, T, cells), the chemotactic peptide fMet-Leu-Phe, bacterial and viral proteins that activate ΝF-κB, agents that induce signaling pathways involved in adaptive immune responses (i.e., antigen receptors on B and T cells, CD28 on T cells, CD40 on B cells), agents that stimulate specific auto-reactive cell death receptors (i.e., TNF, Fas, CD95), CD40, p75NF, and lymphotoxin β-receptor (LtβR), and drugs that stimulate TNF-α converting enzyme (TACE) which cleaves the TNF-α precursor (i.e., to provide biological activity capable of stimulating enhanced production or enhanced cytokine life after secretion).

Accelerated cell death of autoimmune cells may further be stimulated by agents that disrupt the pathways that normally protect autoimmune cells from cell death, including soluble forms of antigen receptors such as CD28 on autoreactive T cells, CD40 on B cells that are involved in protection of autoimmune cells, and CD59 (i.e., Fes) on T-lymphocytes. Other such agents include p75NTF and lymphotoxin-β receptor.

Identification of Inflammation-Inducing Agents

h preferred embodiments, the present invention provides inflammatory agents for the treatment of autoimmune associated fibrosis that are counter to the anti-inflammatories used to treat fibrosis today. For example, current methods for treating autoimmune disease associated fibrosis include TNF-α antagonists. Thus, the present invention provides TNF-α agonists (i.e., chemicals, peptides, or antibodies) that act on a TNF-α receptor. Other preferred treatments could fall under the categories of drugs that act opposite to anti-TNF-α agonists, anti-TNF-α antibodies, TNFR2 fusion proteins (Immunex), Embrel, anti-ILl therapies, TNF-α convertase inhibitors, p38 MAP kinase inhibitors, phophodiesterase inhibitors, thalidomide analogs and adenosine receptor agonists. In another preferred embodiment, the invention allows for the identification of drugs that induce cell death or selectively hamper the autoimmune cells by binding to cell surface receptors or interacting with intracellular proteins. For example, drugs that stimulate the IL-I pathway or drugs that interact with converging pathways such as Fas, FasL, TACI, ATAR, RANK, DR5, DR4, DCR2, DCR1, DR3, etc. The drugs of the present invention can be characterized in that they only kill autoimmune cells having a selective defect in a cell death pathway which can be characterized by two distinct phenotypes, 1) defects in antigen presentation for T cell education and 2) susceptibility to apoptosis.

V. Inflammation and Fibrosis

Inflammation is a complex cellular and chemical reaction, evolutionarily conserved, in which numerous factors contribute to the biological response to injury. The inflammatory response is intimately associated with processes of repair, in that, while the inflammation serves to destroy dead, necrotic, or injured tissue, repair processes begin to occur early in the inflammatory process, often triggered by the same chemical mediators that responded to the initial insult, and ultimately result in the generation of fibroblastic tissue (scarring/fibrosis). J_n processes of chronic inflammation, whether associated with the surgical implantation of a foreign body, or disease such as atherosclerosis, one of the central cellular mediators is the macrophage (Cotran et al. Robbins Pathologic Basis of Disease, 50-107 (1999)). In response to chemical cues, macrophages can become activated, thus enhancing their phagocytic properties. As mentioned previously, a necessary part of inflammation is the process of repair, leading to fibrosis. In addition to phagocytosis, activated macrophages secrete chemical factors, including PDGF, TGF-β, and bFGF, which stimulate fibroblast migration and proliferation at the site of inflammation; fibroblast migration is also stimulated by TNF-α and IL-6. Fibroblasts, in turn, stimulated by macrophage released PDGF, TGF-β, bFGF, and TNF-α. elaborate type I collagen and other extracellular matrix proteins resulting in fibrosis. A key feature of this cascade, as it pertains to the present invention, is that the initiation of macrophage activation is induced by activated T-cells. In autoimmune diseases, such as type I diabetes, atherosclerosis, or diseases of chronic inflammation, such as rheumatoid arthritis, activated, or autoreactive T-cells predominate. The present invention, thus discloses a method of selectively eliminating such T- cells, by taking advantage of their lmp2 deficiency, through treatment with TNF-α, or TNF-α inducing substance, thus blocking an early step in the generation of fibrosis, by attenuating the T-cell induced activation of macrophages.

Measuring fibrosis

The present invention provides a method of preventing, slowing, or halting fibrosis resulting from autoimmune disease such a atherosclerosis in type I diabetes, or resulting from the implantation of a foreign body such as a cellular encapsulation device, or from an interventional procedure such as carotid endarterectomy comprising treating a mammal with one or more agents which accelerate the cell death of autoimmune cells, such as TNF-α or a TNF-α inducing substance, followed by measurement of fibrosis. According to the invention, fibrosis can be measured in several ways.

In certain embodiments of the present invention, a mammal is treated with one or more substances to accelerate the cell death of autoimmune cells, such as TNF-α or a TNF-α inducing substance to treat fibrosis associated with atherosclerosis, or any fibrotic condition leading to impaired blood flow. In such an embodiment, a direct assessment of fibrosis by H&E staining is not feasible, however alternative means of measuring fibrosis may be employed. Given that atherosclerosis, particularly of major vessels such as the aorta, carotid arteries, and coronary arteries, leads to impaired circulation and thus compromised cardiac function, tests which measure or visualize overall cardiac and circulatory function may be used, according to the invention, to indirectly assess fibrosis. Such tests include, but are not limited to MRI, exercise stress test, ultrasonagraphy, organ function tests, echocardiogram, and Doppler ultrasonagraphy, wherein increased blood flow, or enhanced stress test performance in indicative of a decrease in fibrosis.

In embodiments comprising implanting a foreign body in a mammal, including, but not limited to a euglycemic clamp, or wherein a foreign body is inserted into a mammal during an interventional procedure, subsequent to treatment with TNF-α or a TNF-α inducing substance, the foreign body can be removed and examined grossly for evidence of fibrosis or fibrous tissue adherent to the foreign body.

In any embodiments of the invention including that of the preceding paragraph, tissue samples may be taken from a mammal that had undergone treatment with one or more substances which accelerate the cell death of autoimmune cells, such as TNF-α or a TNF-α inducing substance, or a mammal that has not received TNF-α treatment, and examined for fibrosis following H&E staining. H&E staining of tissue is a standard technique, routinely used in histological analysis. The technique, useful in the invention, comprises removing a tissue sample from a mammal of the invention, cutting the tissue into thin (10-50 μm) sections using any sectioning technique known in the art including, but not limited to cryosection, microtome, freezing microtome, vibratome, etc., placing the tissue section on a glass microscope slide, and fixing the tissue using any fixative known to those of skill in the art including, but not limited to neutral buffered formalin, formaldehyde, paraformaldehyde, glutaraldehyde, Bouin's solution, mercuric chloride, or zinc formalin. The slides are then immersed in a solution of Harris Hematoxylin, rinsed in water, immersed in a solution of Eosin, rinsed in water, and dehydrated in ascending alcohol solutions. The tissue sections are then cleared in xylenes and coverslips are mounted over the tissue using Permount, or other suitable organic mounting medium. The tissue sections are then examined under a microscope at low and/or high power. In H&E stained tissue, nuclei and other basophilic structures stain blue, whereas cytoplasm and other acidophilic structures, such as collagen fibers, stain light to dark red (Sheehan and Hrapchak, Theory and Practice of Histotechnology, 2^nd Edition, Battelle Memorial Institute, Columbus, OH (1987)). Fibrosis is deemed present if any fibrous tissue is observed in tissue samples of which fibrous tissue is not a normal component. In tissues where fibrous tissue is a natural component, fibrosis is deemed present if the quantity or extent of fibrous tissue in the sample is at all greater that that found in a normal sample of the same tissue type. In tissue samples in which fibrosis is observed, fibrosis will be deemed decreased if the extent or degree of fibrosis is reduced by at least 5% upon subsequent examination, preferably following treatment with TNF-α or a TNF-α inducing substance.

In embodiments of the present invention relating to the assessment of fibrosis following the implantation of encapsulated insulin-producing cells, fibrosis may be measured by quantifying the level of insulin being produced by the encapsulated cells. Insulin levels may be measured by any method known to those of skill in the art, wherein an increase in insulin production is indicative of the viability of the encapsulated cells, and is thus indicative of the prevention of, or slowed progression of fibrosis. In addition, in mammals which depend on implanted encapsulated islet cells for blood sugar maintenance, blood sugar levels may be monitored, wherein homeostatic regulation of blood sugar is indicative of a decrease or prevention of fibrosis.

Histological examination of H&E stained tissue following treatment with TNF-α or a

TNF-α inducing substance gives a direct measure of fibrosis. Alternative embodiments of the invention provide indirect methods of assessing fibrosis before and after treatment with TNF-α or a TNF-α inducing substance. For example, in certain embodiments fibrosis may be assessed by measuring the serum levels of the carboxyterminal propeptide of procollagen type I (PIP). During collagen type I synthesis, the carboxyterminal portion of the propeptide is cleaved off, thus detection of PIP in serum, or moreover, the detection of increased PIP levels in serum is indicative of increased collagen type I synthesis, and suggests increased collagen deposition and fibrosis (Diez et al., Circulation 93: 1026-1032, 1996).

In a further embodiment of the invention, fibrosis can be indirectly measured by examining the levels of mRNA for type I collagen and TGF-β 1. Type I collagen is the principle collagen species elaborated by fibroblasts and is the primary collagen species in the fibrous tissue of fibrotic lesions; TGF-βl is a major profibrogneic cytokine. Thus increased mRNA levels for type I collagen and TGF-βl are indicative of increased fibrosis. mRNA samples may be isolated from tissue samples obtained from mammals before and/or after treatment with TNF- α or a TNF-α inducing substance, using techniques routinely employed in the art (Ausubel et al., Short Protocols in Molecular Biology, John Wiley and Sons, 1992), and evaluated, for example, using semi-quantitative reverse transcription polymerase chain reaction assay (Paradis et al., J. Clin. Pathol, 49: 998-1004, 1996)

Dosage and Administration

Agents which accelerate the cell death of autoimmune cells, such as TNF-α or a TNF-α inducing substance may be administered in a pharmaceutical formulation, which comprises TNF-α or a TNF-α inducing substance mixed in a physiologically acceptable diluent such as water, phosphate buffered saline, or saline. Administration may be intravenous, intraperitoneal, nasal, etc. In embodiments of the invention comprising the treatment of fibrosis associated with the implantation of a foreign body, TNF-α or a TNF-α inducing substance may be administered prior to, concurrent with, and/or following surgical implantation of the foreign body.

The dosage of TNF-α according to the invention will depend upon the area of administration and the route of administration, but should be generally between 1-250 μg/m², preferably from about 1-10 μg/m², and most preferably 10 μg/m². Dosages of other agents which accelerate the cell death of autoimmune cells are known to those of skill in the art, but, in general, should be sufficient to ablate TNF-α-sensitive cells.

The duration of treatment will extend through the course of the disease symptoms and signs (clinical features), possibly continuously. Monitoring of fibrosis is performed at any time during the course of treatment. The number of doses will depend upon disease delivery vehicle and efficacy data from clinical trials. Symptoms for fibrosis are indicated by the conventional clinical description of the disease, in conjunction with data obtained from measuring fibrosis by the methods of the invention, and will be selected for monitoring by the physician treating the disease. When clinical symptoms are assessed, the physician monitors the symptoms and evaluates whether the symptoms are getting worse or better as the disease progresses or recedes, respectively. One such example is monitoring the reduction of fibrosis as an indicator of the success of treatment.

Examples

There are both spontaneous and induced animal models of diabetes to test drugs for inhibiting fibrosis. Spontaneous animal models of autoimmune diabetes include the NOD mouse (Makino et al, (1980) Jikken Dobutsu 29:1; Kataoka et al., (1983) Diabetes 32:247) and BB rat (Nakhooda et al., (1978) Diabetologia 14:199). These well established animal models mimic Type I diabetes and are commercially available throughout the world from commercial animal vendors (Charles River Laboratories, ME).

Example 1. Treatment Of Fibrosis In A Mouse Model

Adult, female NOD mice were implanted with alginate encapsulated B6 islet cells

(euglycemic clamp) in the abdominal cavity. 24 hours prior to implantation the mice were given up to two bilateral foot-pad injections of CFA (Difco, Detroit, Michigan); a single injection of CFA results in an increase of serum TNF-α that is maintained for several days (Rabinovitch et al., J. Immunol. 159: 6298-6303, 1997). The islet transplants were removed from different populations of mice at 40 to 42 days following surgery. At the indicated times, the islet grafts were removed. The encapsulated islet grafts themselves were examined grossly and histologically for evidence of fibrosis. Samples of the underlying kidney tissue were snap-frozen in liquid nitrogen upon removal, cryosectioned at 20 μm onto gelatin-coated slides, and fixed in 10% neutral buffered formalin. The sections were stained with H&E, and examined by bright- field microscopy at both low and high power for the presence of fibrosis. Furthermore, since the mice were dependent on the encapsulated islet cells for blood sugar maintenance, blood sugar levels were monitored to determine islet function, wherein homeostatic blood sugar levels were indicative of a decrease or prevention of fibrosis.

Example 2. Other Models for Testing Fibrotic Complications

Diabetes can also be induced in diverse rat and murine strains by single injection IN of streptozotocin (Junod et al., 1967 Proc Soc Exp Biol Med 126:201). This chemical selectively destroys the insulin secreting beta cells and these rodents also become diabetic. This mouse model is different from the NOD mouse described above as the NOD mouse displays a spontaneous phenotype, versus the induced diabetic phenotype achieved with straptozotocin injections. Like humans with Type I diabetes, spontaneous and induced animal models of diabetes, develop fibrosis associated with cardiac disease, renal disease, retinal disease, etc. The natural progression of fibrosis can be studied in these models and the halt of fibrosis can similarly be studied in these models after drug interventions.

Briefly, streptozotocin-induced mildly diabetic rats may be produced by injecting a streptozotocin solution in saline adjusted to pH 4.5 with 0.01 M citric acid (at a dose of 20mg/2 ml/kg) into the tail vein of 8 week old male rats of the Wistar strain which have been fasted for 24 hours. 24 hours prior to the administration of streptozotocin animals may be given up to two bilateral foot pad injections of CFA (Difco, Detroit, Michigan); a single injection of CFA results in an increase of serum TNF-α that is maintained for several days; animals may be given additional injections of CFA in 5 day intervals following the onset of treatment. Animals are monitored for signs of fibrosis, including atherosclerosis, and collagen type I mRNA levels as described herein above, at intervals of 2 weeks, four weeks, 8 weeks, and 16 weeks following streptozotocin injections. The degree of fibrosis (e.g., collagen type I mRNA levels) measured in animals treated with CFA may be compared to measurements of fibrosis in animals which received streptozotocin injections but were not treated with CFA. It is expected that the animals treated with CFA will demonstrate significantly lower levels of fibrosis as measured by any index described herein, compared with animals which did not receive CFA treatment.

Example 3. Injury Animal Models

Fibrotic reactions can also be studied in animal "injury" models, both in inbred and outbred strains of rats and mice from diverse animal vendors (Charles River Laboratories, Wilmington, MA). For example, it is possible to obtain animal models of common carotid artery catheterization (Order code: CARART, Charles River Laboratories, Wilmington, MA), External carotid artery catheterization (Order code: XCARART, Charles River Laboratories, Wilmington, MA), and Femoral artery catheterization (Order code: FEMART, Charles River Laboratories, Wilmington, MA) In these models, typically a large vessel is damaged by placement of a cardiac stent, s/p cardiac balloon injury or after a wire injury (See, for example Kim and Iwao, (1997) J. Hypertens. Suppl. 15:S3). Again, interventions to prevent the injured vessel fibrosis can be administered to the rat or mouse and the degree of vessel fibrotic injury can be studied either by comparing fibrosis in an injured vs. non-injured animal, or if testing of a drug is performed, a comparison can be made of fibrosis in the presence and absence of the administered drug.

For example, a mouse model of carotid artery injury induced by catheterization may be obtained from Charles River Laboratories (Wilmington, MA). Upon receipt of the surgically manipulated animals, animals may be given up to two bilateral foot pad injections of CFA (Difco, Detroit, Michigan); a single injection of CFA results in an increase of serum TNF-α that is maintained for several days. Animals are monitored for signs of fibrosis, including atherosclerosis, and collagen type I mRNA levels as described herein above, at intervals of 2 weeks, four weeks, 8 weeks, and 16 weeks following CFA injections. Alternatively,, animals may be sacrificed, and the catheterized carotid artery removed and examined grossly and histologically with H&E for signs of fibrosis. The degree of fibrosis (e.g., collagen type I mRNA levels, histological findings) measured in animals treated with CFA may be compared to measurements of fibrosis in animals which received carotid artery catheterization but were not treated with CFA. In addition, animals may be subjected to unilateral carotid artery catheterization, and thus, fibrosis measured after treatment may be compared between the catheterized artery and the contralateral control artery. It is expected that the animals treated with CFA will demonstrate significantly lower levels of fibrosis, and atherosclerosis as measured by any index described herein, compared with animals which did not receive CFA treatment.

OTHER EMBODIMENTS

Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing detailed description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention are not limited to the above examples, but are encompassed by the following claims.

Claims

Claims:

1. A method of treating fibrosis in a mammal comprising administering to said mammal an amount of TNF-α or a TNF-α inducing substance sufficient to reduce or prevent fibrosis.

2. The method of claim 1, wherein said fibrosis is associated with a disease.

3. The method of claim 2, wherein said disease is selected from the group consisting of: autoimmune disease, type I diabetes, rheumatoid arthritis, and atherosclerosis.

4. The method of claim 1 , wherein said fibrosis is associated with the implantation of a foreign body.

5. The method of claim 2 or claim 4, wherein said fibrosis is associated with a disease and with implantation of a foreign body.

6. The method of claim 5 or 6, wherein said foreign body is a cellular encapsulation device, a cardiac stent, a cancer stent, a euglycemic clamp, and an artificial heart valve.

7. The method of claim 5 or 6, wherein said foreign body is selected from the group consisting of a cardiac stent, a cancer stent, or a cardiac catheter.

8. The method of claim 1, wherein said fibrosis is associated with an interventional procedure.

9. A method of identifying an agent that treats fibrosis comprising the steps of: (a) treating a mammal with said agent; (b) determining the level of fibrosis in said mammal; and

(c) comparing the level of fibrosis in said mammal to the amount of fibrosis present in a control mammal.

10. The method of claim 10, wherein the level of fibrosis is determined by obtaining at least one tissue from said mammal and observing the tissue with histological or histomorphological techniques.

11. The method of claim 10, wherein the level of fibrosis is determined by obtaining at least one tissue from said mammal, staining at least one tissue from said mammal, and observing the stained tissue by microscopy.

12. The method of claim 11, wherein the staining is effectuated with a stain suitable for microscopy, selected from the group consisting of: hematoxylin, eosin, Sirus Red, Masson's trichrome, azan, silver, gold, reticulin, and isamine blue.

13. The method of claim 10, wherein the level of fibrosis is determined by obtaining at least one tissue from said mammal and quantitating the level of fibrosis using a reverse transcriptase polymerase chain reaction assay.